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  • 1995-1999  (713)
  • 1985-1989
  • 1999  (713)
  • Industrial Chemistry  (469)
  • chemotherapy  (86)
  • crystal structure  (79)
  • gene expression  (79)
Material
Years
  • 1995-1999  (713)
  • 1985-1989
Year
Keywords
  • 101
    Electronic Resource
    Electronic Resource
    Springer
    Annals of oncology 10 (1999), S. 305-307 
    ISSN: 1569-8041
    Keywords: biliay tract cancer ; chemotherapy ; 5-fluorouracil ; gemcitabine ; intra-arterial chemotherapy ; pancreatic cancer ; regional chemotherapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Chemotherapy of carcinomas of the pancreas and biliary tract has always been of limited value. Pancreatic cancer is well known for its aggressive nature, poor prognosis and resistance to antineoplastic agents which are effective in other solid tumors. 5-Fluorouracil has long been the mainstay of the treatment of pancreatic cancer, although the response rate to this agent is 〈10% and the influence on survival and quality of life is neglegible. Combination chemotherapy in pancreatic cancer adds to the side effects of treatment, but has had no proven effect on effectiveness. The only new anticancer drug of which an improvement in clinical benefit has been indicated on the basis of randomized clinical research, is gemcitabine, although the magnitude of improvement is limited. Due to the rarity of tumors of the biliary tract, the data on the effect of chemotherapy in this disease is sparse but does not suggest that it leads to superior results than supportive care alone. Likewise, no literature exists supporting the routine application of regional chemotherapy infusion in these type of tumors.
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  • 102
    Electronic Resource
    Electronic Resource
    Springer
    Annals of oncology 10 (1999), S. 139-146 
    ISSN: 1569-8041
    Keywords: anthracycline ; breast cancer ; chemotherapy ; HER-2 antibody ; N,N-diethyl-2[4-(phenylmethyl)-phenoxy] ethanamine.HCl (DPPE, BMS-217380-01) ; paclitaxel
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Anthracyclines and taxanes are the two most active classes of chemotherapy for the treatment of advanced breast cancer. Recent studies have investigated combination therapy including doxorubicin (Dox) and paclitaxel. The efficacy of this combination has been established in a phase III study conducted by ECOG, comparing Dox/paclitaxel versus Dox versus paclitaxel. The combination is superior to Dox or paclitaxel with respect to response rate and time to disease progression, indicating that the combination provides a new standard for the first line treatment of metastatic breast cancer [1]. Phase II studies using higher doses of Dox and using shorter infusions of paclitaxel have suggested the combination can be further optimised; Gianni reported a 94% objective response rate using Dox 60 mg/m2 followed by paclitaxel 175 mg/m2 given over three hours [2]. The more active regimens are associated with enhanced cardiotoxicity; this toxicity can be avoided, however, by limiting the exposure to doxorubicin. The newer regimens have now been moved into phase III studies. Future progress for this disease will depend on the introduction of new agents. Two novel drugs are currently being investigated in randomised phase III trials as potentiators of Dox and/or paclitaxel. One is a monoclonal antibody from Genentech (Herceptin, trastuzumab) directed at the HER-2/neu oncogene, which is overexpressed in 〉25% of breast cancers [3]. Recent results indicate that Herceptin in combination with paclitaxel (or with a Dox plus cyclophosphamide regimen) induces a higher response rate (RR) and prolongs the time to disease progression when compared to chemotherapy alone. The second agent N,N-diethyl-2[4-(phenylmethyl)-phenoxyl] ethanamine.HCl (DPPE, BMS-217380-01), when combined with Dox, was associated with a higher RR than previously observed with Dox alone [4]. A randomised trial of Dox versus Dox plus DPPE is ongoing. The possible mechanisms underlying chemo-potentiation by these agents are discussed. As new anthracycline/taxane combinations establish themselves in earlier stages of the disease, the need for effective, non-cross resistant salvage regimens will emerge.
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  • 103
    ISSN: 1569-8041
    Keywords: chemotherapy ; cisplatin ; long-term toxicity ; testicular cancer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Background: The prognosis of advanced testicular cancer has improved considerably after the introduction of cisplatin-based combination chemotherapy. The improved prognosis of testicular cancer has brought the long-term toxicity of the treatment into focus. Patients and methods: Long-term toxicity was investigated prospectively intil more than 10 years after after treatment in a group of 22 patients treated with six series of cisplatin based chemotherapy (PVB) for testicular cancer. We have focused on nephro-, neuro-, pulmonary-, and gonadal toxicity. Results: Glomerular filtration rate (GFR) decreased significantly during treatment but increased during follow-up and all the patients had normal values of GFR 10–15 years after treatment. Carbon monoxide diffusion capacity (TLco) decreased during PVB treatment in smokers. TLco remained unchanged during the first years after PVB treatment, but improvement of TLco was seen in some patients more than 43 months after treatment. Paresthesia was reported by 83% of the patients immidiately after treatment, 50% at follow-up 4–9 years after chemotherapy and 14% prevalence 11–15 hears after treatment. The reported decline in neurotoxicity was verified by normalisation of vibration perception. Gonadal toxicity was severe and persistent although improvement was seen in a few patients even many years after treatment. Conclusions: The patients treated with PVB were physically and socially well-being at follow-up investigation 11–15 years after treatment. Improvements in pulmonary- and renal function, and recovery from neurotoxicity was seen during the long-term follow-up periode. Gonadal toxicity was severe and persistent.
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  • 104
    ISSN: 1569-8041
    Keywords: chromosomal aberrations ; gene expression ; oncogenes ; pancreatic cancer ; tumor suppressor genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Aim: The present review summarizes our strategies aimed at identifying and characterizing genetic alterations occuring at the transcriptional and chromosomal level in pancreatic cancer. Methods: To study transcriptional alterations we have used a number of techniques including modified versions of differential hybridizations and cDNA-RDA (representational difference analysis). Comparative genomic hybridization (CGH) was used to study chromosomal aberrations occuring in pancreatic cancer tissues. Results: The study of transcriptional alterations led to the identification of more than 500 genes with differential expression in pancreatic cancer. The sum of these alterations represented the first expression profile characteristic for pancreatic tumors. The CGH analysis allowed the identification of a number of chromosomal regions containing putative tumor suppressor genes or oncogenes. These regions are presently being characterized at the molecular level. In a first approach the myb-oncogene was identified as the relevant oncogene of an amplification on 6q occurring in up to 10% of pancreatic cancer patients. Conclusions: Genes isolated in both approaches represent potential new disease genes for pancreatic cancer and are at present being characterized by individual or serial analysis.
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  • 105
    Electronic Resource
    Electronic Resource
    Springer
    Annals of oncology 10 (1999), S. 157-161 
    ISSN: 1569-8041
    Keywords: chemotherapy ; 5-fluorouracil ; gemcitabine ; pancreatic cancer ; pancreaticobiliary cancer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract At time of presentation the majority of patients with pancreaticobiliary cancer have locally advanced or metastatic disease which makes them unamenable for curative surgery. In these patients chemotherapy is an option which has gained more support over the past few years. Special problems faced in chemotherapeutic treatment are the patient's poor condition and the difficulties faced in evaluating response. 5-FU has been the only drug with some efficacy for a long time, but more recently gemcitabine appeared to be more efficient. In locally advanced pancreatic cancer the combination of chemotherapy with radiotherapy has not gained much support. However, studies are implicating better local control with combined treatment and recurrences appear more often at distant sides. In some cases irresectable tumors became resectable. Because of the poor survival after surgery with curative intent, adjuvant and neoadjuvant therapy are becoming important issues. Although studies of adjuvant therapy suggest benefit, research is seriously hampered by poor patient accrual due to the morbidity of pancreaticoduodenectomy. Neoadjuvant treatment may overcome this problem. Until now there has been only modest improvement in the treatment of pancreatic cancer. Hopefully, new treatment modalities such as immunotherapy, gene therapy and antiangiogenic therapy will alter this dismal picture. In biliary cancer the role of chemotherapy is less well defined, since only few studies with low patients numbers have been performed.
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  • 106
    Electronic Resource
    Electronic Resource
    Springer
    Annals of oncology 10 (1999), S. 234-238 
    ISSN: 1569-8041
    Keywords: chemotherapy ; 5-fluorouracil ; gemcitabine ; pancreatic carcinoma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The current role of chemotherapy in pancreatic cancer is limited. Chemotherapy usually consists of 5-fluorouracil (5FU) and gemcitabine either as a single agent or in combinations. However, response rates are below 15% with minor effects on overall survival. Due to the aggressive behavior of the disease, current emphasis of new experimental chemotherapy is also focusing on clinical benefit: improvement of pain, performance status or weight. The results with gemcitabine indicated that evaluation of new chemotherapeutic agents in pancreatic cancer should not be limited to the evaluation of response rates; single agent gemcitabine not only showed higher response rates than 5FU, but also resulted in clinical benefit for the patients. Several new agents have been introduced into the clinic for treatment of various gastro-intestinal malignancies, whereas novel agents with different types of targets, such as marimastat deserve further attention. Several oral formulations of 5FU, such as capecitabine, UFT, and eniluracil with 5FU, aim to simulate long-term continuous infusion. Response rates of these formulations are comparable to those of 5FU continuous infusion and 5FU bolus injections. However, the convenience of oral administration with reliable plasma drug concentrations makes these agents very attractive as a replacement of traditional 5FU administration. Since 5FU acts by inhibition of thymidylate synthase (TS), resulting in inhibition of DNA synthesis, several new antifolates, directed towards TS, have been developed. However, these agents, such as ZD1694 (Tomudex, Raltitrexed) and LY231514 (MTA, multitargetted antifolate) showed only limited efficacy. Other new agents active in colorectal cancer, e.g. the topoisomerase I inhibitors topotecan and CPT-11, showed only minor activity. The same was observed for the taxanes. Combinations of gemcitabine (cisplatin, 5FU, epirubicin, marimastat) show promising activities, not only regarding response but also with respect to clinical benefit. The effects were better than that for each agent separately. Thus, despite limited activity of single agents, novel combinations especially with gemcitabine are promising, with emphasis on improvement of the clinical benefit of patients.
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  • 107
    Electronic Resource
    Electronic Resource
    Springer
    Annals of oncology 10 (1999), S. 77-82 
    ISSN: 1569-8041
    Keywords: chemotherapy ; non-small cell lung cancer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Until recently the role of chemotherapy in NSCLC has generally been questioned. Major concerns included marginal activity, considerable toxicity and high cost of this treatment. There has, however, been increasing evidence from individual studies and meta-analyses that chemotherapy in advanced NSCLC is able to increase survival and improve quality of life. In the past few years a series of active drugs (paclitaxel, docetaxel, gemcitabine, vinorelbine, topotecan and irinotecan) with novel mechanisms of action and favourable toxicity profiles have been developed. These agents appear to hold the promise of added therapeutic benefit. In consequence, chemotherapy has currently been considered an important part of the standard treatment in selected patients with advanced NSCLC. Despite recent developments, treatment outcomes in advanced NSCLC remain far from satisfactory, and new effective means are desperately needed if more patients are to enjoy the prospects of long-term survival.
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  • 108
    ISSN: 1569-8041
    Keywords: chemotherapy ; osteosarcoma ; relative dose intensity ; survival ; tumour response
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Background: Despite advances in the treatment of primary limb osteosarcoma, the outcome of patients with primary metastatic and axial skeletal disease remains poor. The European Osteosarcoma Intergroup have assessed a combination chemotherapy regimen consisting of ifosfamide (IFOS) 3 g/m2/d1–2, doxorubicin (DOX) 25 mg/m2/d1–3 i.v. bolus and cisplatin (CDDP) 100 mg/m2/d1. Patients and methods: One hundred nine previously untreated patients with primary osteosarcoma were registered. Eligibility was confirmed in 103. At presentation, 45 eligible patients had metastatic disease, 15 axial skeletal primary tumours and 43 non-metastatic limb tumours. Results: The major toxicities were myelosuppression (90%, grade 3 or 4) and nausea and vomiting (74%, grade 3 or 4). Overall mean relative dose intensity (RDI) was 80% (88% CDDP, 75% IFOS, 81% DOX). Clinical response as measured by reduction in tumour volume occurred in 36% (95% confidence interval (95% CI): 27%–47%) of primary tumours. Response of pulmonary metastases to chemotherapy was seen in 33% (95% CI: 19%–49%). Good histological response (≥90% necrosis of the tumour) occurred in 33% (95% CI: 22%–45%) of resected tumours. Five-year survival was 62% in limb-non-metastatic, 41% in axial skeletal and 16% in limb metastatic patients. Conclusions: This regimen is active in osteosarcoma but does not appear to be more active than the two-drug CDDP–DOX regimen currently recommended by EOI.
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  • 109
    ISSN: 1569-8041
    Keywords: chemotherapy ; drugs ; generics ; prioritization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The WHO has previously produced recommendations on the essential drugs required for cancer therapy. Over the last five years several new anti cancer drugs have been aggressively marketed. Most of these are costly and produce only limited benefits. We have divided currently available anti-cancer drugs into three priority groups. Curable cancers and those cancers where the cost-benefit ratio clearly favours drug treatment can be managed appropriately with regimens based on only 17 drugs. All of these are available, at relatively low cost, as generic preparations. The wide availability of these drugs should be the first priority. The second group of drugs may have some advantages in certain clinical situations. Based on current evidence, drugs in the third group are judged as currently not essential for the effective delivery of cancer care. Adequate supportive care programmes with the widespread availability of effective drugs for pain control are of considerably greater importance. The adoption of these priorities will help to optimise the effectiveness and efficiency of chemotherapy and ensure equitable access to essential drugs especially in low resource environments. Clearly this paper represents the views of its contributors. The WHO welcomes feedback from all oncologists so that the advice it gives to governments in prioritising the procurement of anti cancer drugs can be as comprehensive as possible.
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  • 110
    ISSN: 1569-8041
    Keywords: chemotherapy ; hematopoietic growth-factor support ; high-dose chemotherapy ; peripheral blood stem-cell transplantation ; small-cell lung cancer ; treatment toxicity and mortality
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Background: We conducted a phase I–II trial to assess the feasibility and activity of a combination chemotherapy regimen with etoposide, ifosfamide, cisplatin or carboplatin, and epirubicin in limited-disease (LD, stages I–IIIB) and extensive-stage (ED, stage IV) small-cell lung cancer (SCLC). Patients and methods: Standard-dose chemotherapy (SDC) consisting of etoposide (500 mg/m2), ifosfamide (4000 mg/m2), cisplatin (50 mg/m2) and epirubicin (50 mg/m2) (VIP-E), followed by granulocyte colony-stimulating factor (G-CSF), was given to 100 patients with SCLC. Thirty patients with qualifying responses to VIP-E proceeded to high-dose chemotherapy (HDC) with autologous peripheral blood stem-cell transplantation (PBSCT) after etoposide (1,500 mg/m2), ifosfamide (12,000 mg/m2), carboplatin (750 mg/m2) and epirubicin (150 mg/m2) (VIC-E) conditioning. Results of standard-dose VIP-E: Ninety-seven patients were evaluable for response. The objective response rate was 81% in LD SCLC (33% CR, 48% PR; excluding patients in surgical CR) and 77% in ED SCLC (18% CR, 58% PR). The treatment-related mortality (TRM) of SDC was 2%. Two additional patients in CR from their SCLC developed secondary non-small-cell lung cancers (NSCLC), and both were cured by surgery. The median survival was 19 months in LD SCLC and 6 months in ED SCLC. The five-year survivals were 36% in LD and 0% in ED SCLC. Results of high-dose VIC-E: HDC was feasible in 16% of ED-, and 58% of LD-patients. All HDC patients (n = 30) improved or maintained prior responses. Four patients died of early treatment-related complications (TRM 13%). Two additional patients in CR from their SCLC developed secondary malignancies (esophageal cancer, secondary chronic myelogenous leukemia). The median survivals were 26 months in LD SCLC, and 8 months in ED SCLC. The five-year survival was 50% in LD and 0% in ED SCLC. Conclusions: Despite high response rates, survival after VIP-E SDC and VIC-E HDC in patients with ED SCLC is not superior to that achieved with less toxic traditional regimens. The high five-year survival rates achieved with these protocols in LD SCLC probably reflect both patient selection (high proportion of patients with prior surgical resection) and the high activity of our chemotherapy regimen in combination with radiotherapy. A study comparing protocols using simultaneous radiation therapy and chemotherapy, and other dose-escalated forms of SDC with HDC is needed to further define the role of this treatment modality in SCLC. Given the high rate of secondary malignancies observed in patients in CR 〉2 years in our study, close follow-up and early treatment of these neoplasms may contribute to maintaining overall survival in patients with SCLC.
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  • 111
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    Electronic Resource
    Springer
    Annals of oncology 10 (1999), S. 231-233 
    ISSN: 1569-8041
    Keywords: chemoradiation ; chemotherapy ; 5FU ; gemcitabine ; radiotherapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Chemoradiation for gastrointestinal cancers is actively under study in the Radiation Therapy Oncology Group (RTOG) and consists of external irradiation combined with simultaneously administered chemotherapy given to provide radiation sensitization and to attack micro metastatic disease. Two national protocols for the treatment of patients with pancreatic and biliary cancers are now active. RTOG 97-04 is a phase III post-operative combined modality program for patients with resected pancreatic cancer. All patients receive protracted infusional 5-fluorouracil (5-FU) combined with 50.4 Gy given in 28 fractions. Prior to and after chemoradiation all patients are randomized to receive multiple cycles of either infusional 5-FU or Gemcitabine to determine the effect on survival. In the other study (RTOG 98-12) patients with unresectable pancreatic cancer are given 50.4 Gy combined with weekly Paclitaxel (50 mg/m2) to examine the efficacy of this active combination in a phase II trial in a multi-institutional setting. Both of these trials have recently been opened to accrual. A third RTOG study for patients with biliary cancer will examine the efficacy of giving pre-operative chronomodulated infusional 5-FU chemoradiation. The background and the rationale for these studies is based on the long history of 5-FU radiation sensitization in the treatment of cancers of these anatomic sites and will be summarized. A brief review of recently published trials using chemoradiation in conjunction with new irradiation treatment techniques with "3D" conformal therapy for these diseases will be discussed.
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  • 112
    Electronic Resource
    Electronic Resource
    Springer
    Annals of oncology 10 (1999), S. 753-759 
    ISSN: 1569-8041
    Keywords: chemotherapy ; brain metastases ; lung cancer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In lung cancer patients brain metastases develop with a high frequency. For years radiotherapy has been the standard treatment for these patients. Here we review the experience with chemotherapy for brain metastases in lung cancer patients. The concept of the brain as pharmacological sanctuary site when brain metastases are present is challenged and it is argued that chemotherapy does play a role in this situation. Recent clinical trials indicate that the combination of chemotherapy and radiotherapy may become the standard treatment for lung cancer patients with brain metastases. It is unclear whether for micrometastatic disease to the brain, blood brain barrier function is of importance for the outcome of chemotherapy in lung cancer patients with respect to the development of overt brain metastases. Areas of improvement of delivery of cytotoxic agents to the brain when brain metastases have not yet developed are discussed.
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  • 113
    Electronic Resource
    Electronic Resource
    Springer
    Annals of oncology 10 (1999), S. 891-898 
    ISSN: 1569-8041
    Keywords: brachytherapy ; chemotherapy ; hormonal therapy ; prostate cancer ; recent advances
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract As new evidence for prostate cancer treatment has emerged in the last few years, longstanding controversies in the treatment of prostate cancer have resurfaced. A number of long-held tenets of prostate cancer therapy have been revisited, sometimes with surprising and challenging results. Although neoadjuvant hormonal therapy prior to radical prostatectomy decreases positive surgical margin rates, longer follow-up is needed to support survival improvement of this combined modality therapy. Androgen deprivation combined with radiation therapy appears to improve disease-free survival (and survival in one series) in patients with locally advanced cancer. Another approach to locally advanced prostate cancer using three-dimensional conformal radiation therapy may improve long term outcome. The data are currently insufficient to conclude that interstitial low dose rate brachytherapy is equivalent to conventional treatments: patients with small tumor volumes and low Gleason grade seem to obtain more benefit, whereas for large tumors with higher gleason grades this approach seems inferior to conventional treatments. In advanced prostate cancer recent data suggest that immediate hormonal therapy improves survival. In this group of patients the use of maximum androgen blockade remains controversial but may adversely affect quality of life compared to orchiectomy alone. Intermittent hormonal therapy may improve quality of life, although effect upon survival is unknown. Chemotherapy in combination with androgen deprivation is currently being studied as front-line therapy in advanced prostate cancer. Palliative benefit of chemotherapy for hormone refractory prostate cancer remains an important endpoint; survival advantage has not been seen in any randomized trials. Suramin may delay disease progression in hormone refractory prostate cancer. Many aspects of prostate cancer treatment will remain controversial until results of large, randomized trials with longer follow-up are available.
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  • 114
    Electronic Resource
    Electronic Resource
    Springer
    Journal of thermal analysis and calorimetry 55 (1999), S. 797-806 
    ISSN: 1572-8943
    Keywords: cadmium coordination ; crystal structure ; enthalpy of solution ; lattice energy ; lattice enthalpy ; NTO salt ; preparation ; standard enthalpy of dehydration ; standard enthalpy of formation ; thermal decomposition mechanism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract [Cd(NTO)4Cd(H2O)6]·4H2O was prepared by mixing the aqueous solution of 3-nitro-1,2,4-triazol-5-one and cadmium carbonate in excess. The single crystal structure was determined by a four-circle X-ray diffractometer. The crystal is monoclinic, space group C2/c with crystal parameters of a=2.1229(3) nm, b=0.6261(8) nm, c=2.1165(3) nm, β=90.602(7)°, V=2.977(6) nm3, Z=4, Dc=2.055 g·cm−3, μ=15.45 cm−1, F(000)=1824, λ(MoKα)=0.071073 nm. The final R is 0.0282. Based on the results of thermal analysis, the thermal decomposition mechanism of [Cd(NTO)4Cd(H2O)6]·4H2O was derived. From measurements of the enthalpy of solution of [Cd(NTO)4Cd(H2O)6]·4H2O in water at 298.15 K, the standard enthalpy of formation, lattice energy, lattice enthalpy and standard enthalpy of dehydration have been determined as -(1747.8±4.8), -2394, -2414 and 313.6 kJ mol−1 respectively.
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  • 115
    ISSN: 1572-9001
    Keywords: Tetrahydrotetrol ; polycyclic aromatic hydrocarbon derivative ; carcinogen ; crystal structure ; structure-activity relationship
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The molecular structures of two racemic trans-tetrahydrotetrols formed by hydrolysis of the (±)anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro derivatives of the strong carcinogen benzo[a]pyrene and the weak carcinogen chrysene have been determined by X-ray crystallographic methods.3 Focusing on the (+)isomers, the stereochemical features of the two tetrols are discussed in detail to determine structural differences, which can be related to the different biological activity of their parent hydrocarbons, since they provide a model for the hydrocarbon moiety in the major PAH-DNA adduct. As a result of this study, a tentative correlation can be stated between biological activity of quasi-planar bay-region PAHs and the presence of structural features, which can decrease the steric hindrance of the aromatic skeleton and the other hydroxy groups on the pseudo-axial O4, which mimics the position of the covalent bond to DNA. The significant structural features seem to be an out-of-plane distortion with a negative torsion angle at the bay region and a half chair conformation of the saturated ring distorted toward the envelope with C8 at the tip as in BPT, in addition to the axial and pseudo-axial conformations of the hydroxy groups at C9 and C10 due to the near bay region. They may be considered generated by trans-opening of the oxirane ring in an anti-diol epoxide biologically active because it is highly strained. The strains, derived from the activation of bay-region PAHs with a negative torsion angle, seem greater in an anti-diol epoxide relative to the syn-isomer. Thus, when the strains are released, the derived trans-tetrol occurs with structural features, which seem to better fit the target. As a consequence, the presence of a methyl group at a bay region should increase the biological activity of a PAH, imposing greater strains to the structure of the anti-diol epoxide.
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  • 116
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    Journal of thermal analysis and calorimetry 57 (1999), S. 807-821 
    ISSN: 1572-8943
    Keywords: bibliography ; critical temperature ; crystal structure ; CuRh2S4 ; CuRh2Se4 ; survey of chalcogenide superconductors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The thread that runs through all research in the field of superconductivity is new physics through discovery of new materials. The knowledge of superconducting materials has become voluminous and complex. The comprehensive review of the superconducting materials is of particular importance. The main purpose of this report is to present the results of classification for chalcogenide superconductors. Superconducting critical temperature Tc, crystal-structure type and the references proper to these compounds are summarized. Brief survey of the superconductivity in chalcogen elements is also given. Furthermore, as representative sulfide and selenide, superconducting characteristics of CuRh2S4 and CuRh2Se4 will be shown.
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  • 117
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    Journal of thermal analysis and calorimetry 58 (1999), S. 257-267 
    ISSN: 1572-8943
    Keywords: crystal structure ; lattice energy ; lattice enthalpy ; lead salt of NTO ; preparation ; quantum chemical calculation ; thermal decomposition mechanism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The single crystal of lead salt of 3-nitro-1,2,4-triazol-5-one (NTO), [Pb(NTO)2(H2O)] was prepared and its structure was determined by a four-circle X-ray diffractometer. The crystal is monoclinic, its space group is P21/n with crystal parameters of a=0.7262(1) nm, b=1.2129(2) nm, c=1.2268(3) nm, β=90.38(2)°, V=1.0806(2) nm3, Z=4, Dc=2.97 g cm−3, µ=157.83cm−1, F(000)=888. The final R is 0.027. By using SCF-PM3-MO method we obtained optimized geometry for [Pb(NTO)2⋅H2O] and particularly positions for hydrogen atoms. Through the analyses of MO levels and bond orders it is found that Pb atom bond to ligands mainly with its 6pz and 6py AOs. The thermal decomposition experiments are elucidated when [Pb(NTO)2⋅H2O] is heated, ligand water is dissociated first and NO2 group has priority of leaving. Based on the thermal analysis, the thermal decomposition mechanism of [Pb(NTO)2⋅H2O] has been derived. The lattice enthalpy and its lattice energy were also estimated.
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  • 118
    ISSN: 1572-9001
    Keywords: Nickel complex ; dithiocarbimate ; sulfonamide ; crystal structure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Potassium N-4-methylphenylsulfonyldithiocarbimate, K2(4-CH3C6H4SO2N=CS2), reacted with nickel(II) chloride hexahydrate and tetrabutylammonium bromide to form the bis(N-4-methylphenylsulfonyldithiocarbimate)nickelate(II) tetrabutylammonium salt (Bu4N)2[Ni(4-CH3C6H4S2C=NSO2)2]. The elemental analyses, IR and UV-Vis data obtained were consistent with the formation of a $${\text{nickel}}{\kern 1pt} - {\kern 1pt} {\text{sulfur}}$$ diamagnetic planar complex. The 1H NMR and the 13C NMR spectra showed the expected signals for the dithiocarbimate moiety and the tetrabutylammonium cation. The single-crystal structure analysis showed that this substance crystallizes in the triclinic $${P\bar 1}$$ space group with a = 10.474(3) Å, b = 10.767(3) Å, c = 13.657(3) Å and α = 81.54(2)°, β = 80.44(2)°, γ = 67.63(2)°, V = 1398.5(8) Å3, and Z = 2. The nickel atom is coordinated to four sulfur atoms.
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  • 119
    ISSN: 1572-9001
    Keywords: Nicotine-derived nitrosaminoketone ; nitrosamine ; crystal structure ; molecular dimensions
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The three-dimensional structure of the carcinogenic nicotine-derived nitrosaminoketone, 4-(methyl-nitrosamino)-l-(3-pyridyl)-l-butanone, has been determined by X-ray crystallographic techniques. The molecule is essentially planar except for the methylnitrosamine group which is oriented at a dihedral angle of 68.7° to the pyridine ring. Molecules pack by way of —H⋯O interactions that involve the —NNO group.
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  • 120
    ISSN: 1573-4919
    Keywords: microbodies ; diabetes mellitus ; steroid hormone receptor ; β-oxidation ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract To determine whether the increased fatty acid β-oxidation in the peroxisomes of diabetic rat liver is mediated by a common peroxisome proliferation mechanism, we measured the activation of long-chain (LC) and very long chain (VLC) fatty acids catalyzed by palmitoyl CoA ligase (PAL) and lignoceryl CoA ligase and oxidation of LC (palmitic acid) and VLC (lignoceric acid) fatty acids by isotopic methods. Immunoblot analysis of acyl-CoA oxidase (ACO), and Northern blot analysis of peroxisome proliferator-activated receptor (PPAR-α), ACO, and PAL were also performed. The PAL activity increased in peroxisomes and mitochondria from the liver of diabetic rats by 2.6-fold and 2.1-fold, respectively. The lignoceroyl-CoA ligase activity increased by 2.6-fold in diabetic peroxisomes. Palmitic acid oxidation increased in the diabetic peroxisomes and mitochondria by 2.5-fold and 2.7-fold, respectively, while lignoceric acid oxidation increased by 2.0-fold in the peroxisomes. Immunoreactive ACO protein increased by 2-fold in the diabetic group. The mRNA levels for PPAR-α, ACO and PAL increased 2.9-, 2.8- and 1.6-fold, respectively, in the diabetic group. These results suggest that the increased supply of fatty acids to liver in diabetic state stimulates the expression of PPAR-α and its target genes responsible for the metabolism of fatty acids.
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  • 121
    ISSN: 1573-4919
    Keywords: regucalcin ; Ca2+-binding protein ; protein kinase C ; Ca2+signaling ; gene expression ; H4-II-E hepatoma cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The expression of hepatic Ca2+-binding protein regucalcin in the cloned rat hepatoma cells (H4-II-E) was investigated. The change in regucalcin mRNA levels was analyzed by Northern blotting using rat liver regucalcin complementary DNA (0.9 kb of open reading frame). Regucalcin mRNA was expressed in H4-II-E hepatoma cells. This expression was clearly stimulated in the presence of serum (10% fetal bovine serum). Bay K 8644 (2. 5 × 10-6 M), a Ca2+ channel agonist, significantly stimulated regucalcin mRNA expression in the absence or presence of 10% serum. Dibutyryl cyclic AMP (10-3 M) did not have a stimulatory effect on the regucalcin mRNA expression. The presence of phorbol 12-myristate 13-acetate (PMA; 10-6 M) or estrogen (10-8 M) caused a significant increase in regucalcin mRNA levels in the hepatoma cells cultured in serum-free medium, while insulin (5 × 10-9 M) or dexamethasone (10-6 M) had no effect. Bay K 8644-stimulated regucalcin mRNA expression in the hepatoma cells was completely blocked in the presence of trifluoperazine (10-5 M), an antagonist of calmodulin, or staurosporine (10-7 M), an inhibitor of protein kinase C. The stimulatory effect of PMA was clearly inhibited in the presence of stauroporine. The present study demonstrates that regucalcin mRNA is expressed in the transformed H4-II-E hepatoma cells, and that the expression is stimulated through Ca2+-dependent signaling factors.
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  • 122
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    Molecular and cellular biochemistry 199 (1999), S. 189-200 
    ISSN: 1573-4919
    Keywords: lung ; cancer ; urokinase ; receptor ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The urokinase-type plasminogen activator (uPA) interacts with its receptor (uPAR) to promote proteolysis as well as cell proliferation and migration. These functions contribute to the pathogenesis of neoplastic growth and invasiveness. Expression of uPAR in tumor extracts also inversely correlates with prognosis in many forms of cancer. In this study, we sought to determine if differences in uPAR expression were distinguishable between cultured human lung carcinoma and malignant mesothelioma subtypes. We also sought to determine if, as in malignant mesothelioma cells, uPAR expression is regulated at the posttranscriptional level in cultured malignant lung carcinoma cells. Using 125I-uPA binding and ligand blotting techniques, uPAR was expressed by phenotypically diverse lung carcinoma cell lines, including the H460, H157 and H1395 non-small cell lines and the H146 small cell lung carcinoma line. Increased uPAR expression was also detected in spindle-shaped (M33K) and epithelioid (M9K and MS-1) malignant mesothelioma cells. Selected mediators, including TGF-β, TNF-α, LPS and PMA, uniformly enhanced uPAR expression in each of the tumor cell lines. Steady state uPAR mRNA expression was determined by RNase protection assay and correlated directly with the changes in cell surface uPAR expression. By gel mobility shift and UV-cross linking assays, a uPAR mRNA binding protein (uPAR mRNABp) implicated in the posttranscriptional control of message stability, was identified in each of the cell lines. Expression of uPAR and its message in cultured lung carcinoma and malignant mesothelioma cells is similarly influenced by effectors present in the tumor microenvironment. Regulation of the uPAR message occurs at the posttranscriptional level in cultured small and non-small cell lung carcinoma cells as well as spindle-shaped and fibrous malignant mesothelioma cell lines. Posttranscriptional regulation of uPAR in all these cells involves the interaction of the uPAR mRNABp with uPAR mRNA, which promotes uPAR mRNA destabilization.
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  • 123
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    Molecular and cellular biochemistry 201 (1999), S. 111-123 
    ISSN: 1573-4919
    Keywords: complement factor I ; TPA ; protein kinase C ; gene expression ; Hep G2 cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract This study examined the role of the protein kinase C (PKC) signalling pathway in the regulation of expression of human complement factor I (CFI) gene. The production of CFI by Hep G2 cells was enhanced in a dose- and time-dependent fashion by 12-O-tetradecanoyl-1,2-phorbol 13-acetate (TPA), a potent PKC activator. 4α-phorbol didecanoate, an inactive phorbol ester, had no effect on CFI synthesis. The TPA-dependent increase in CFI secretion was correlated with an increase in CFI mRNA levels. Forskolin, a cAMP-inducing agent, augmented the TPA response. W7, an inhibitor of protein kinase A and genistein, an inhibitor of protein tyrosine kinase(s) both did not prevent the increase in CFI expression mediated by TPA. However, calphostin C, a specific inhibitor of PKC, abolished the TPA-induced increase in CFI mRNA levels. Down regulation of intracellular PKC levels by prior exposure of Hep G2 cells to a high concentration of TPA also blocked the increase in CFI mRNA levels induced by TPA suggesting that the TPA effects were mediated via activation of PKC. mRNA decay studies indicated that the half-life of CFI mRNA in TPA-induced cells was not significantly different from control. Nuclear run-on transcriptional assays on the other hand demonstrated that whereas the CFI gene is transcribed under basal conditions in Hep G2 cells, TPA induced a 3-4 fold increase in the transcription rate of CFI gene in 24 h. The transcription rate of GAPDH gene did not change, indicating that the effects were not general on gene transcription. Transient transfections of Hep G2 cells with chloramphenicol acetyltransferase reporter gene (CAT) constructs containing a series of sequential 5′ deletions of the CFI promoter and CAT assays showed that the sequence between -136 and -130, containing an AP-1 consensus sequence (TGAGTCA) was required for the TPA response. This observation was substantiated by the finding that mutation of this AP-1 site to TttaTCA or TtAtcCA abolished the TPA responsiveness. The enhancement of the activity of transfected chimeric CAT constructs by TPA was abrogated by calphostin C and by pyrrolidine dithiocarbamate (an inhibitor of NF-κB and AP-1 transactivation). These results indicate that TPA regulation of CFI gene requires PKC signalling and is mediated by via a TPA response element (TRE) in the CFI promoter region located at -136/-130 and involves the transactivation of AP-1 and NF-κB transcription factors. We suggest that PKC may be one of the intracellular pathways that control CFI gene expression and that cellular processes (involving growth factors, hormones, cytokines etc.) that activate PKC may upregulate the expression of the CFI gene.
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  • 124
    ISSN: 1573-4919
    Keywords: regucalcin ; calcium-binding protein ; gene expression ; Ca2+-ATPase ; brain microsomes ; aging
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The expression of calcium-binding protein regucalcin and its effect on the microsomal Ca2+-ATPase activity in rat brain tissues was investigated. The expression of regucalcin mRNA was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) analysis in brain tissues using rat regucalcin-specific primers. Regucalcin concentration in the brain tissues was about 5 × 10-9 M as measured using enzyme-linked immunoadsorbent assay (ELISA), and this level was lowered with increasing age (50 weeks old). The presence of regucalcin (10-9 to 10-7 M) in the enzyme reaction mixture caused a significant decrease in Ca2+-ATPase activity in the brain microsomes of young rats (5 weeks old). Meanwhile, the enzyme activity was not significantly altered by the addition of calmodulin (1 or 50 μg/ml), calbindin (1 or 10 μg/ml), and S-100 A protein (5 or 25 μg/ml), which are other Ca2+-binding proteins in rat brain. The effect of regucalcin to inhibit microsomal Ca2+-ATPase activity was weakened in the brain of rats with increasing age (50 weeks old). The present study demonstrates that regucalcin is expressed in the brain, and that it can uniquely inhibit Ca2+-ATPase activity in the brain microsomes of rats. The findings suggest that regucalcin plays a role in the regulation of microsomal Ca2+-ATPase activity in rat brain tissues.
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  • 125
    ISSN: 1573-4919
    Keywords: nitrosourea ; chemotherapy ; anemia ; leukopenia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Most chemotherapy agents function by causing damage to the DNA of rapidly dividing cells, such as those in the bone marrow, leading to anemia and leukopenia during chemotherapy and the development of secondary leukemias in the years following recovery from the original disease. We created an animal model of nitrosourea-based chemotherapy using ethylnitrosourea (ENU) to investigate the effect of niacin deficiency on the side effects of chemotherapy. Weanling Long-Evans rats were fed diets containing various levels of niacin for a period of 4 weeks. ENU treatment started after 1 week of feeding and consisted of 12 doses delivered by gavage, every other day. Cancer incidence was also monitored in the following months. ENU treatment caused many of the acute symptoms seen in human chemotherapy patients, including anemia and neutropenia. Niacin deficiency (ND) had several interesting effects, alone and in combination with ENU. Niacin deficiency alone caused a modest anemia, while in combination with ENU it induced a severe anemia. Niacin deficiency alone caused a 4-fold increase in circulating neutrophil numbers, and this population was drastically reduced by ENU-treatment. In the long term, macin deficiency caused an increased incidence of cancer, especially chronic granulocytic leukemias.
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  • 126
    ISSN: 1573-4919
    Keywords: mechanical stretch ; smooth muscle cells ; differential display ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Physical forces induce profound changes in cell phenotype, shape and behavior. These changes can occur in vascular structures as a result of pressure overload and their effects can be seen in atherosclerotic vessels in which smooth muscle cells have undergone hyperplastic and hypertrophic changes. At the molecular level, mechanical stimuli are converted into chemical ones and lead to modulation of gene expression and/or the activation of a new repertoire of genes whose encoded proteins help the cells to adapt to their microenvironment. In this study, we have used a two primer-based mRNA differential display technique to identify candidate mechano-responsive genes in pulmonary artery smooth muscle cells. As compared to the original method described by Liang and Pardee, this technique uses two arbitrary primers instead of an anchored oligo(dt) plus an arbitrary primer in the polymerase chain reaction. The chief advantages of these modifications are an increase in the efficiency of the amplification and in the identification of differentially expressed clones. Using this approach, we compared the pattern of expressed genes in cells cultured under static conditions with those in cells that were mechanically stretched (1 Hz) for 24 h in a well-defined in vitro mechanical system. Three candidate genes that showed reproducible differences were chosen for further characterization and cloning. One clone was under expressed in stretched cells and had a DNA sequence with 90% homology to the human fibronectin gene. Two other clones were highly expressed in stretched cells and had a 92% and a 83% sequence homology with human platelet-activating factor (PAF) receptor and rat insulin-like growth factor-I (IGF-I) genes respectively. Northern blot analysis confirmed low levels of fibronectin mRNA transcripts in stretched cells. In contrast, accumulation of PAF receptor mRNA occurred 30 min after mechanical stretch was initiated whereas IGF-I mRNA levels peaked at 8 h. Both mRNA levels were sustained for up to 24 h of mechanical stretching. These results demonstrate the usefulness of the two primer-based mRNA differential display that enabled us to identify and characterize alterations at the level of gene expression among matrix proteins, G-protein coupled receptors and growth factors, each of whose response to mechanical strain is different. A more complete understanding of these responses will provide further insight into the pathologic processes associated with hypertension and atherosclerosis.
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  • 127
    ISSN: 1871-4528
    Keywords: Solanum tuberosum L. ; tuberisation ; extensin ; acyl carrier protein thioesterase ; high mobility group protein ; gene expression ; plant development
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary In screening to isolate a full-length copy of a previously isolated cDNA clone, a further three cDNAs were also isolated from a library prepared from sub-apical swelling-stolon tissue of potato (Solanum tuberosum L.). Sequence analysis showed these clones to be similar to extensin-like protein genes, acyl carrier protein thioesterase genes and high mobility group protein genes, respectively. A further cDNA, isolated by subtractive hybridisation, was similar to a tomato cDNA previously isolated on the basis of its down-regulation following nematode infection. While all the newly isolated genes were expressed in swelling stolons, for most, maximal expression was seen to be in stem tissue. Possible roles for these genes in the development of potato plants are discussed, as is the significance of gene expression in stems and stolons to the process of tuberisation.
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  • 128
    ISSN: 1573-9368
    Keywords: transgenic mice ; prolactin ; mammary gland ; gene expression ; Stat5 ; β-globin insulator
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In order to establish a possible correlation between in vitro prolactin induction and the transcriptional activity of mammary gene promoters in transgenic mice, a functional Stat5-binding site was created by means of site-directed mutagenesis at position −70 on a 560 bp murine α-lactalbumin promotor linked to a CAT reporter gene. Surprisingly, the wild-type promoter was constitutively active in vitro and could not be induced by prolactin. Introducing the proximal Stat5 site abolished this constitutive activity and resulted in prolactin dependence in both CHO-K1- and HC11-transfected cells. In transgenic mice, both the frequency of lines expressing the transgene and the prevalence of mid to late pregnancy expression were increased.
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  • 129
    ISSN: 1573-0646
    Keywords: chemotherapy ; platinum (IV) analogues ; cisplatin resistance ; pharmacodynamics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology , Medicine
    Notes: Abstract Ormaplatin (NSC 363812, tetraplatin) is a stable platinum (IV) analog which has exhibited activity against cisplatin-resistant cell lines. A phase I trial of ormaplatin administered as a 1-h infusion every 4 weeks was performed. Forty-one patients received 101 cycles of drug over the dose range 4–128 mg/m2. The dose-limiting toxicity was reversible thrombocytopenia and granulocytopenia. Minimal myelosuppression was observed at dose levels ≤ 78 mg/m2, while grade 3 or 4 myelosuppression (thrombocytopenia and/or granulocytopenia) was seen in 4/8 patients at 98 mg/m2 and 4/5 patients at 123 mg/m2. Nausea and vomiting was observed at all dose levels but was controlled with antiemetic premedication. Neurotoxicity was observed in 5/41 patients and the incidence appeared related to cumulative dose rather than to dose level or drug clearance. Platinum was measured by furnace atomic absorption spectrophotometry. Ormaplatin-derived plasma ultrafilterable platinum (UF-Pt) exhibited linear pharmacokinetics over the dose range studied. The mean total body clearance of UF-Pt was 135 ml/min/m2 and the mean elimination half-life (t1/2β) was 13.6 h. Ormaplatin exhibited a high degree of protein binding, with more than 70% of platinum protein bound by the end of the infusion. Urinary excretion of platinum accounted for 37% of the total dose of ormaplatin in 24 hours. A phase II dose of 98 mg/m2 is recommended for testing in a patient population with cisplatin-refractory disease.
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  • 130
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    Investigational new drugs 17 (1999), S. 313-320 
    ISSN: 1573-0646
    Keywords: cell cycle ; flavopiridol ; cyclin-dependent kinases ; cyclin D1 ; flavonoids ; chemotherapy ; protein kinases ; signal transduction ; clinical trials
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology , Medicine
    Notes: Abstract The discovery and cloning of the cyclin-dependent kinases (cdks), main regulators of cell cycle progression, allowed several investigators to design novel modulators of cdk activity. Flavopiridol (HMR 1275, L86-8275), a flavonoid derived from an indigenous plant from India, demonstrated potent and specific in vitro inhibition of all cdks tested (cdks 1, 2, 4 and 7) with clear block in cell cycle progression at the G1/S and G2/M boundaries. Moreover, preclinical studies demonstrated the capacity of flavopiridol to induce programmed cell death, promote differentiation, inhibit angiogenic processes and modulate transcriptional events. The relationship between the latter effects and cdk inhibition is still unclear. Initial testing in early clinical human trials with infusional flavopiridol showed activity in some patients with non-Hodgkin's lymphoma, renal, prostate, colon and gastric carcinomas. Main side effects were secretory diarrhea and a pro-inflammatory syndrome associated with hypotension. Biologically active plasma concentrations of flavopiridol (∼300–500 nM) are easily achievable in patients receiving infusional flavopiridol. Phase 2 trials with infusional flavopiridol in several tumor types, other schedules and combination with standard chemotherapies are being assessed. In conclusion, flavopiridol is the first cdk inhibitor to be tested in clinical trials. Although important questions remain to be answered, this positive experience will stimulate the development of novel cdk modulators for cancer therapy.
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  • 131
    ISSN: 1573-0778
    Keywords: CHO cells ; gene expression ; kinetic model ; protein secretion ; transcription ; translation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The elevation of expression levels for secreted glycoproteins by gene amplification in mammalian cells shows a saturation behavior at high levels of gene amplification. At high expression levels a drop in the secretion efficiency for the recombinant protein occurs (Schröder and Friedl, 1997), coinciding with the appearance of misfolded protein in the cell. In this communication we investigated whether additional limitations exist at the levels of transcription and translation. Four Chinese hamster ovary (CHO) cell lines expressing different amounts of human antithrombin III (ATIII) were used as a model system. A tenfold increase in the ATIII cDNA copy number from the lowest to the highest producing cell line coincided with a 38-fold increase in ATIII mRNA levels, and an 80-fold increase in the amount of intracellular ATIII levels. The data was analyzed using a simple kinetic model. The following conclusions were derived: I. The transcriptional activity for the recombinant protein is not saturated. II. Translation itself is not saturated either, but may be downregulated as secretion efficiency drops. III. Two explanations for the previously reported drop in secretion efficiency for the recombinant protein with increasing expression level are possible: A. Protein degradation is an alternative fate for translated ATIII and the fraction of ATIII degraded after translation increases as expression level is increased. B. Translation is downregulated as the secretory apparatus becomes exhausted to maintain cell viability.
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  • 132
    ISSN: 1573-0778
    Keywords: cardiogenesis ; cell differentiation ; gene expression ; mouse embryonic stem cells ; myogenesis ; neurogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Embryonic stem cells, totipotent cells of the early mouse embryo, were established as permanent cell lines of undifferentiated cells. ES cells provide an important cellular system in developmental biology for the manipulation of preselected genes in mice by using the gene targeting technology. Embryonic stem cells, when cultivated as embryo-like aggregates, so-called ‘embryoid bodies’, are able to differentiate in vitro into derivatives of all three primary germ layers, the endoderm, ectoderm and mesoderm. We established differentiation protocols for the in vitro development of undifferentiated embryonic stem cells into differentiated cardiomyocytes, skeletal muscle, neuronal, epithelial and vascular smooth muscle cells. During differentiation, tissue-specific genes, proteins, ion channels, receptors and action potentials were expressed in a developmentally controlled pattern. This pattern closely recapitulates the developmental pattern during embryogenesis in the living organism. In vitro, the controlled developmental pattern was found to be influenced by differentiation and growth factor molecules or by xenobiotics. Furthermore, the differentiation system has been used for genetic analyses by ‘gain of function’ and ‘loss of function’ approaches in vitro.
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  • 133
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    Cytotechnology 30 (1999), S. 71-83 
    ISSN: 1573-0778
    Keywords: gene expression ; HEK293(EBNA) cells ; serum-free ; transient transfection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract In order to establish a simple and scaleable transfection system we have used the cationic polymer polyethylenimine (PEI) to study transient transfection in HEK293 and 293(EBNA) cells grown in serum-free suspension culture. The transfection complexes were made directly within the cell culture by consecutively adding plasmid and PEI (direct method). Alternatively, the DNA-PEI transfection complexes were prepared in fresh medium (1/10 culture volume) and then added to the cells (indirect method). The results of this study clearly show that the ratio of PEI nitrogen to DNA phosphate is very important for high expression levels. The precise ratio is dependent on the DNA concentration. For example, using 1 μg/ml DNA by the indirect method, the ratio of optimal PEI:DNA was about 10–13:1. However, the ratio increases to 33:1 for 0.1–0.2 μg/ml DNA. By testing several different molecular weights of the polycationic polymer we could show that the highest transfection efficiency was obtained with the PEI 25 kDa. Using PEI 25 kDa the indirect method is superior to the direct addition because significantly lower DNA concentrations are needed. The expression levels of the soluble human TNF receptor p55 are even higher at low DNA compared to 1 μg/ml plasmid. The EBV-based pREP vectors gave better transient gene expression when used in 293(EBNA) cells compared to HEK293 cells in suspension culture. No differences in expression levels in the two cell lines were observed when the pC1 (CMV)-TNFR was used. In conclusion, PEI is a low-toxic transfection agent which provides high levels of transient gene expression in 293(EBNA) cells grown in serum-free suspension culture. This system allows highly reproducible, cost-effective production of milligram amounts of recombinant proteins in 2–5 l spinner culture scale within 3–5 days. Fermentor scale experiments, however, are less efficient because the PEI-mediated transient tranfection is inhibited by conditioned medium.
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  • 134
    ISSN: 1573-0778
    Keywords: cell cycle ; CHO ; flow cytometry ; gene expression ; synchronisation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Understanding the relationships between cell cycle and protein expression is critical to the optimisation of media and environmental conditions for successful commercial operation of animal cell culture processes. Using flow cytometry for the analysis of the early phases of synchronised batch cultures, the dependency of product expression on cell cycle related events has been evaluated in a recombinant CHO cell line. Although the production of recombinant protein is initially found to be cell cycle related, the maximum specific protein productivity is only achieved at a later stage of the exponential phase which also sees a maximum in the intracellular protein concentration. Subsequent work suggests that it is the batch phase/medium composition of cultures which is the major determinant of maximum specific productivity in this cell line. Furthermore the effect of the positive association between S phase and specific productivity is subordinate to the effect of batch phase/medium composition on the specific productivity of batch cultures.
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  • 135
    ISSN: 1573-7276
    Keywords: gene expression ; immunohistochemistry ; mRNA DD ; neoplasia metastasis ; RAB5A
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract For the sake of better understanding the molecular mechanism of neoplasia, we have used the mRNA differential display technique to analyze two human lung adenocarcinoma cell lines, AGZY83-a and Anip973. Anip973 was isolated from AGZY83-a, but manifested much higher metastatic potential than the parent line. We found that a significant differential cDNA fragment in Anip973 was over-expressed, then over-expressed cDNA fragment was cloned and sequenced. It showed that the over-expressed cDNA in Anip973 was RAB5A cDNA. And the RAB5A cDNA sequence was corresponding between the two cells. To determine whether RAB5A may be differentially expressed in the two human lung adenocarcinoma cells at protein level, we further detected RAB5A protein in the two cells by using immunofluorescent method. RAB5A protein was upregulated in highly metastatic Anip973. We also detected the difference in RAB5A gene expression at RNA level in human non-small cell lung carcinoma by RT-PCR. Using immunohistochemical staining, we also examined RAB5A change at protein level in 45 cases human non-small cell lung carcinoma paraffin sections. The results proved the evidence of upregulation of RAB5A in malignant tumor, indicated over-expression of RAB5A gene was correlated with the malignant degree and metastatic potential of lung cancer(χ2 test, p 〈0.01). The RAB5A gene is a member of RAS superfamily, which can transcribe GTP-binding protein that plays an important role in signal transduction of protein trafficking at the cell surface and GDP/GTP cycle in the regulation of endocytotic membrane traffic. Thus our results indicated that over-expression of the RAB5A gene was involved in the process of transformation from AGZY83-a to the higher metastatic cell line Anip973. The result may be a powerful experimental evidence that over-expression of RAB5A gene associated with neoplasia metastasis.
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  • 136
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    Journal of neuro-oncology 41 (1999), S. 235-246 
    ISSN: 1573-7373
    Keywords: brain tumor ; intra-arterial administration ; chemotherapy ; drug delivery ; drug streaming
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The influences of the flow rate on the concentration and distribution of drug in the rat brains and brain tumors after intra-arterial (intra-carotid) administration of [3H]SarCNU (sarcosinamide chloroethyl-1-nitrosourea) were examined. Results obtained at three flow rates via intra-carotid route were compared to those obtained with intravenous administrations. Adult female Wistar rats bearing C6 brain tumor were randomized into four-groups. Groups 1 (G.1) to 3 (G.3) received intra-arterial injection and Group 4 (G.4) received intravenous administration of [3H]SarCNU. G.1 (slow infusion rate) was administered 1 ml of [3H]SarCNU solution over 60 min (0.017 ml/min), Group 2 (G.2; medium infusion rate): 0.2 ml over 5 min (0.04 ml/min), G.3 (fast infusion rate): 1 ml over 5 min (0.2 ml/min), and G.4 (intravenous infusion): 1 ml intravenously over 5 min. Quantitative autoradiographic method was used to measure the concentration and the distribution of [3H]SarCNU in the brain and the brain tumors. The tissue uptake constant of SarCNU in both viable (tumor tissue excluding necrosis) and peak regions (the area of tumor containing top 20% of the tracer concentration) of the intra-arterial injection groups were significantly higher (p〈0.0001) than those in the intravenous group. The mean concentrations of the viable tumor in the intra-arterial groups were 2.92 (G.1), 16.06 (G.2), and 20.8 (G.3) times higher than those of intravenous group. Between the intra-arterial groups, the mean concentration in the viable tumors of G.1 (slow flow rate) was significantly (p〈0.0001) lower than in G.2 and G.3. However, there was no significant difference between G.2 and G.3. In three intra-arterial groups the mean concentration delivery ratios of the brain tumors were high and ranged from 3.07 (G.3) to 3.87 (G.2), but there was no significant difference between them. Only G.4, intravenous group, showed significantly (p〈0.005) lower concentration delivery ratio, 1.26. These results suggest that higher infusion rate in the intra-arterial chemotherapy could have an effect not only on the streaming phenomenon which results in the brain toxicities, but also on the increase in the concentration and the sufficient distribution of a drug in tumors. By finding chemotherapeutic agents to which tumors show high sensitivity and using intra-arterial administration of these agents at more effective flow rate, better clinical results could be achieved in the treatment of patients with malignant brain tumors.
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  • 137
    ISSN: 1573-7373
    Keywords: glioma ; chemotherapy ; cisplatin ; amifostine ; DNA adducts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The chemoprotective effect of amifostine (WR2721) was studied in a BDIX rat model with intracerebral BT4C glioma implants. Twenty-one rats were given cisplatin 5 mg/kg i.p., 21 were given amifostine 200 mg/kg i.p.+cisplatin 5 mg/kg i.p. Ten rats served as untreated controls. An immunohistochemical method for analysis of cisplatin–DNA adducts was used to elucidate the adduct formation in tumor, normal brain and kidney. Tumor volume and serum creatinine level were analysed 10 days after treatment. In animals pretreated with amifostine there was a delayed adduct formation rate in the normal brain, and in the kidney cortex the number of tubular cells with extremely high adduct level was reduced. No difference in adduct formation was seen in tumors. Tumor volume was significantly larger following amifostine+cisplatin (66% of controls) compared to cisplatin alone (38% of controls). Weight loss was, however, severe in rats given cisplatin alone. In the tumor growth study only 3 out of 11 rats treated with cisplatin 5 mg/kg alone survived until time of sacrifice at 10 days, whereas all those pretreated with amifostine survived. Mean serum creatinine was 48 µmol/l (controls), 146 µmol/l (cisplatin) and 59 µmol/l (amifostine+cisplatin). A marked reduction of histopathological renal changes was found when amifostine was added. Amifostine thus significantly reduced general and renal toxicity of cisplatin. The tumor growth retardation was stronger when cisplatin was given alone but this is probably related to general toxicity and malnutrition indirectly supported by the fact that amifostine did not significantly reduce cisplatin–DNA adduct formation in tumors. The results of the present study suggest that amifostine may have a role in increasing the therapeutic ratio of cisplatin, also in the treatment of malignant glioma.
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  • 138
    ISSN: 1573-7373
    Keywords: malignant glioma ; chemotherapy ; anthracyclines ; KRN8602(MX2) ; phase II study
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract KRN8602(MX2) is a newly developed morpholino-anthracycline that has been found to cross the blood–brain barrier and be distributed in brain tissue after intravenous administration and to be effective against human glioma cells and the intracerebrally transplanted tumors in vivo. In order to confirm these promising preclinical observations clinically, we performed a phase II trial of KRN8602 in patients with recurrent malignant glioma. The 44 patients enrolled received at least 2 cycles of KRN8602 35 mg/m2/day at 3–4 week intervals by intravenous bolus. Of the 44 patients, 37 could be evaluated for response, and 39 for toxicity. One patient with anaplastic astrocytoma had a complete response (1/37, 3%), and 2 patients with anaplastic astrocytoma and 1 with brain stem glioma had a partial response (3/37, 8%). The overall response rate was 11% (4/37). All patients who responded had received prior chemotherapy that included nitrosoureas. No response was observed in the patients with glioblastoma. Myelosuppression was moderately severe, with 72% of patients developing grade 3 or 4 leukopenia. Severe nausea/vomiting was observed in 31% of the patients. No severe cardiotoxicity was observed. The results indicate that KRN8602 has modest activity against recurrent malignant glioma with relatively severe, but manageable toxicity. It seems to be worthwhile to further assess the efficacy and toxicity of KRN8602 against malignant glioma, which is generally less sensitive to chemotherapy.
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  • 139
    ISSN: 1573-7373
    Keywords: anaplastic oligodendroglioma ; surgery ; radiotherapy ; chemotherapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Purpose: To investigate feasibility, toxicity and antitumor activity of combined surgery, postoperative radiation therapy (RT) and adjuvant chemotherapy (CHT) in adult patients with pure anaplastic oligodendroglioma (PAO) or mixed anaplastic oligoastrocytoma (MAO). Methods: Between January 1988, and June 1993, 23 patients entered into a phase II study. After surgery, post-operative RT was administered with 60 Gy in 30 daily fractions in 30 treatment days in 6 weeks. Two weeks after RT, adjuvant ‘modified’ PCV (mPCV) (Procarbazine, 60 mg/m2, days 1–14; CCNU, 100 mg/m2, day 1; and vincristine, 1.4 mg/m2 (max. 2 mg), days 1 and 8) was administered every six weeks up to six cycles or until progression occurred. Results: Median survival time is not attained yet, while 1–5 year survival rates are 100%, 100%, 78%, 61%, and 52%, respectively. Median time to tumor progression is not attained yet, while 1–5 year progression-free survival rates are 100%, 100%, 70%, 52%, and 52%, respectively. On univariate analysis of potential prognostic factors, sex, tumor location (frontal versus other), and histology (pure versus mixed anaplastic oligodendroglioma) were not found to influence survival. Age of 〈50 years carried improved prognosis as well as Karnofsky performance status (KPS) 90–100 when compared to KPS of 70–80. Patients having tumors ≤4 cm did better than those with tumors 〉4 cm as well as those with total tumor resection when compared to those with subtotal tumor resection or biopsy only. Acute high-grade (≥3) CHT-related toxicity was mainly hematological with only 3 (13%) patients experiencing acute grade 4 toxicity. Conclusions: Combined treatment modality consisting of surgery, postoperative high-dose RT and mPCV chemotherapy for patients with anaplastic oligodendroglioma was effective with acceptable toxicity. Further studies are needed with more patients and longer follow-up to verify these results in this rare disease.
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  • 140
    ISSN: 1573-7373
    Keywords: brain tumor ; chemotherapy ; stem cell rescue ; medulloblastoma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract High dose chemotherapy (HDCT) with autologous (bone marrow or peripheral blood) stem cell rescue (ASCR) has had success in the treatment of some malignant pediatric brain tumors. We report a series of adults enrolled in one of three HDCT and ASCR protocols for malignant primary brain tumors. Overall toxic mortality was 18%; chemotherapy regimen, tumor type, and prior treatment did not predict transplant-related mortality. Patients over the age of 30 had a higher rate of toxic mortality. Patients with recurrent medulloblastoma had a significant improvement in long-term survival (median: 34 months) as compared with historical reports; two patients with glioblastoma survive beyond four years without progression, but overall, a significant improvement in long-term survival could not be demonstrated for malignant gliomas.
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  • 141
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    Journal of neuro-oncology 45 (1999), S. 37-46 
    ISSN: 1573-7373
    Keywords: topoisomerase ; drug resistance ; chemotherapy ; glioma ; phosphorylation ; MRP
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The efficacy of the epipodophyllotoxins VP-16 and VM-26 is limited by the occurrence of drug resistance in the tumor cell population. Cellular insensitivity to drugs that stabilize the cleavable complex is frequently expressed as multidrug resistance (MDR). In some cell lines, overexpression of MDR-1/P-glycoprotein or the multidrug resistance associated protein (MRP) has been demonstrated and implicated as the mechanism of resistance. Typically, these cells have reduced drug accumulation, secondary to increased drug efflux. In other cell lines, an atypical MDR phenotype has been identified, with the predominant mechanism of resistance shown to be qualitative and/or quantitative changes in the levels and activity of topoisomerase II. For VP-16, increased expression of MDR-1 or MRP and alterations in topoisomerase II α have been shown to confer tolerance. To further understand resistance to VP-16, T98G-VP(1000) was initially isolated as a single clone from parental cell, T98G, by exposure to VP-16. Subsequently, a population of cells from this subline was exposed to three-fold higher drug concentration allowing stable sublines to be established at higher extracellular drug concentration. Characterization of the resistant sublines demonstrates the adaptation that occurs with advancing drug concentrations during in vitro selections. Reduced topoisomerase II mRNA and protein levels were observed in the initial isolate. This reduction was accompanied by a decrease in topoisomerase II activity and cellular growth rate and was associated with 47-fold resistance to topoisomerase II poisons. With advancing resistance, MRP expression increased, with increased VP-16 efflux and reduced accumulation. This adaptation allowed for partial restoration of topoisomerase II activity secondary to increased expression and hyperphosphorylation, with a resultant increase in growth rate. In this cell line, hyperphosphorylation coincided with increased casein kinase II mRNA protein levels, without increased PKC protein levels, suggesting a role for this kinase in the acquired hyperphosphorylation. In this cell line, hyperphosphorylation mediated the increased activity despite a fall in topoisomerase IIα protein levels secondary to an acquired 615 bp deletion in one topoisomerase IIα allele, which resulted in reduced protein levels. In this subline, high levels of resistance were attained as a result of synergism between the reduced topoisomerase IIα levels and MRP overexpression. These studies demonstrate how cellular adaptation to increasing drug pressure occurs and how more than one mechanism can contribute to the resistant phenotype when increasing selecting pressure is applied. Reduced expression of topoisomerase II is sufficient to confer substantial resistance early in the selection process, with synergy from additional mechanisms helping to confer high levels of resistance.
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  • 142
    ISSN: 1573-7365
    Keywords: glutamine synthetase ; gene expression ; portacaval anastomosis ; hepatic encephalopathy ; liver ; skeletal muscle ; ammonia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The effects of chronic liver insufficiency resulting from end-to-side portacaval anastomosis (PCA) on glutamine synthetase (GS) activities, protein and gene expression were studied in brain, liver and skeletal muscle of male adult rats. Four weeks following PCA, activities of GS in cerebral cortex and cerebellum were reduced by 32% and 37% (p〈0.05) respectively whereas GS activities in muscle were increased by 52% (p〈0.05). GS activities in liver were decreased by up to 90% (p〈0.01), a finding which undoubtedly reflects the loss of GS-rich perivenous hepatocytes following portal-systemic shunting. Immunoblotting techniques revealed no change in GS protein content of brain regions or muscle but a significant loss in liver of PCA rats. GS mRNA determined by semi-quantitative RT-PCR was also significantly decreased in the livers of PCA rats compared to sham-operated controls. These findings demonstrate that PCA results in a loss of GS gene expression in the liver and that brain does not show a compensatory induction of enzyme activity, rendering it particularly sensitive to increases in ammonia in chronic liver failure. The finding of a post-translational increase of GS in muscle following portacaval shunting suggests that, in chronic liver failure, muscle becomes the major organ responsible for the removal of excess blood-borne ammonia.
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  • 143
    ISSN: 1573-7373
    Keywords: non-Hodgkin's lymphoma ; diabetes insipidus ; hypophyseal localization ; chemotherapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We report the case of a 64 year old male patient with a history of ischemic heart disease who underwent surgery for an abdominal mass. The histological diagnosis was highly malignant non-Hodgkin's lymphoma. After surgery the patient was admitted to our Department and received 6 courses of chemotherapy according to the COP schedule, followed by radiotherapy to the left upper abdominal region and ipsilateral lung base. The patient achieved partial remission. One month later he began to complain of left axillary lymphadenomegaly, polydipsia and polyuria. A NMR brain scan showed a hypophyseal mass. The patient was treated with DDAVP and chemotherapy with the PRO-MACE protocol; the polyuria and lymphadenomegaly disappeared and the size of the hypophyseal mass reduced markedly. The clinical picture was, therefore, attributed to a hypophyseal localization of the non-Hodgkin's lymphoma, which is a very rare manifestation of lymphomatous spread to the central nervous system. Our case is also interesting because it shows that a favorable outcome can be obtained with chemotherapy, provided that the latter is sufficiently aggressive. This is not necessarily the case with radiotherapy which may also be followed by late and severe neurologic sequelae.
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  • 144
    ISSN: 1573-7373
    Keywords: brain neoplasm ; lymphoma ; CNS ; radiotherapy ; chemotherapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We treated 23 patients with primary central nervous system lymphoma with a protocol of conventional radiation up to 55±5 Gy followed by 4 to 6 cycles of intravenous doxorubicin (30 mg/m2), vincristine (1 mg/m2) and cyclophosphamide (350 mg/m2), and oral prednisolone (8–30 mg/m2) (VEPA chemotherapy) repeated at 2-week intervals. The median age of the 23 patients was 59 years, and the median World Health Organization performance status score was 2. Seventeen patients received 4 or more courses of the chemotherapy, but 6 received only 1 or 2 courses for various reasons. The median survival time for all 23 patients was 25.5 months and their 5-year survival rate was 23%. These values were 34 months and 32%, respectively, for the 17 patients who received 4–6 courses of chemotherapy. After treatment, decline in performance status unaccompanied with tumor recurrence was observed in 44% of the patients; the incidence was apparently higher in older than in younger patients. The survival results obtained with this combined radiochemotherapy regimen appear to be better than those reported in most previous studies of patients treated with radiation alone. Post-irradiation VEPA chemotherapy appears to be worthy of further evaluation.
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  • 145
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    Journal of neuro-oncology 45 (1999), S. 61-67 
    ISSN: 1573-7373
    Keywords: ependymoma ; chemotherapy ; CNS ; advanced
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Background. The role of chemotherapy in recurrent ependymoma is poorly defined. This study was performed to help clarify the benefits of chemotherapy in this setting. Patients and methods. We retrospectively reviewed the charts of patients with advanced ependymoma of the CNS who received chemotherapy in our institution between 1974 and 1993, inclusive. Sixteen consecutive patients were treated with regimens containing either nitrosourea, platinum, or other combinations exclusive of nitrosourea or platinum. No patient received nitrosourea and platinum concurrently. Two methods were used to define response. The first was a direct comparison of radiographic images before and after chemotherapy more than one month apart. A second broader definition of response that employed four other criteria in addition to imaging studies (symptoms, signs, performance status, and neurologic functional status) was also used. Results. Results were as follows (response rate by imaging studies followed by response rate by scoring in parenthesis): Platinum-based chemotherapy resulted in a 67% (83%) response rate with 33% (0%) remaining stable. Nitrosourea-based regimens resulted in a 25% (60%) response rate with 50% (10%) remaining stable. When combinations other than platinum or nitrosourea were used, 11% (22%) responded and 56% (44%) remained stable. Relative differences in response rates between chemotherapy regimens persisted when the data were analyzed by grade. Median time to progression was 6, 10, and 3 months, respectively. Conclusion. Platinum-based chemotherapy regimens appear to result in higher response rates with lower rates of progression than nitrosourea-based regimens. Other regimens that do not include cisplatinum or nitrosourea appear to be even less effective.
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  • 146
    ISSN: 1573-7373
    Keywords: high risk medulloblastoma ; chemotherapy ; carboplatin ; oral etoposide
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We report a case of high risk medulloblastoma with leptomeningeal intracranial and spinal metastasis in a 10-year-old girl treated successfully with conventional prolonged chemotherapy without radiotherapy. This is a particular case of medulloblastoma that at onset did not receive standard therapy for medulloblastoma i.e. neither surgery nor craniospinal irradiation. This 10-year-old Chinese girl affected with localized medulloblastoma was previously treated at a medical department in China only with radiotherapy on the posterior fossa. When the child arrived in Italy with progressed metastatic medulloblastoma, she was treated with carboplatin/etoposide association i.v. followed by oral etoposide and partial surgery of the primitive mass. The schedule of chemotherapy was etoposide 300 mg/sqm followed by carboplatin 1000 mg/sqm in one day every 21–28 days for the first six courses, then etoposide 200 mg/sqm and carboplatin 600 mg/sqm in one day every 28–35 days for further 11 courses and oral etoposide 50 mg/sqm/day for ten consecutive days and one week interval between two cycles for one year. At present the girl is alive and disease-free, and has been off-therapy for 31 months. Interestingly, in this case a long-lasting complete remission was obtained without radiotherapy and without myeloablative chemotherapy. Oral etoposide played an important role in achieving a complete remission.
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  • 147
    ISSN: 1573-7365
    Keywords: Oligodendrocyte cultures ; glucose ; gene expression ; malic enzyme ; membrane synthesis ; myelination ; undernutrition
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We have previously demonstrated that the developmental upregulation of myelin-specific genes in mixed glial cultures is strongly attenuated by hypoglycemia. The present study was designed to evaluate the effect of hypoglycemia on differentiation-dependent upregulation of myelin genes in purified oligodendrocyte cultures. The expression of major myelin protein genes, i.e., proteolipid protein (PLP), basic protein (BP) and myelin associated glycoprotein (MAG) were monitored by Northern blot analysis. In control cultures maintained at 6 mg/ml of glucose, the expression of all the genes upregulated rapidly, and plateaued at approximately day 4. A similar pattern of differentiation-dependent upregulation was observed for the gene encoding a lipogenic enzyme, i.e., malic enzyme (ME). In contrast to mixed glial cultures, however, this developmental gene upregulation was not significantly affected by severe hypoglycemia (approximately 0.02 mg/ml). The results indicate that the effect of glucose deprivation on oligodendrocyte genes observed in mixed glial cultures is mediated by other cells. The upregulation of the genes in differentiating oligodendrocytes was accompanied by the production of myelin-related membrane that was isolated by density gradient fractionation. In contrast to the effect on gene expression, this anabolic activity was highly dependent on glucose, as seen from a profound suppression by severe hypoglycemia.
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  • 148
    ISSN: 1573-7373
    Keywords: malignant glioma ; gossypol ; lactate dehydrogenase isoenzymes ; chemotherapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Gossypol, a polyphenolic compound which depletes cellular energy by inhibition of several intracellular dehydrogenases, has been shown to have antiproliferative activity against human glial tumor cell lines in vitro and in nude mouse xenografts. Human trials of gossypol as a male contraceptive have demonstrated safety of long-term administration. We studied the activity of Gossypol 10 mg PO bid in 27 patients with pathologically confirmed glial tumors which had recurred after radiation therapy. Fifteen patients had glioblastoma, 11 patients anaplastic astrocytoma, 1 patient relapsed low grade glioma. Response was assessed every 8 weeks using CT/MRI scan and clinical criteria including decadron requirement. Treatment was continued until disease progression. Two patients had partial response (PR); 4 had stable disease for 8 weeks or more. One patient maintained a PR with improved KPS for 78 weeks. The other had a PR lasting 8 weeks. Toxicity was mild: 2 heavily pretreated patients had mild thrombocytopenia, 5 patients developed hypokalemia, 3 patients developed grade 2 hepatic toxicity and peripheral edema. Gossypol levels measured by HPLC did not correlate with response or toxicity in this study. We conclude that gossypol is well tolerated and has a low, but measurable, response rate in a heavily pretreated, poor-prognosis group of patients with recurrent glioma. The presumed novel mechanism of action, lack of significant myelosuppression, and activity in patients with advance glioma support further study of gossypol as an antineoplastic agent.
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  • 149
    ISSN: 1573-7373
    Keywords: malignant melanoma ; brain metastases ; chemotherapy ; fotemustine ; DTIC ; radiation therapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We report results of a conservative treatment for brain metastases from malignant melanoma with a combination of irradiation and chemotherapy (fotemustine and/or DTIC). To date, 12 patients have been treated. There was a complete remission of the brain metastases in four patients. In two patients a partial remission was observed. The mean survival of the responder was 8.2 months (95% confidence interval 3.8–12.6 months). The most common side effects were thrombocytopenia, leukopenia, and alopecia. Altogether, the treatment was well tolerated. As the outcome of patients with brain metastases from malignant melanoma is generally poor, this combined chemo- and radiation therapy may provide improved care for such patients.
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  • 150
    ISSN: 1573-7373
    Keywords: radiotherapy ; chemotherapy ; paclitaxel ; brain tumors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The present study was conducted to evaluate the activity and toxicity profile of radiation (RT) and concomitant chemotherapy in patients with glioblastoma multiforme (GBM). Thirty-nine patients were treated postoperatively with RT and concomitant administration of paclitaxel. Cranial irradiation was initiated 2–3 weeks postoperatively and was administered in 2.0 fractions, one fraction per day, for 5 consecutive days per week, to a total of 60 Gy. Paclitaxel was delivered at a dose of 100 mg/m2 over 3-h once weekly for 6 weeks. Thirty-three patients received all 6 cycles of paclitaxel according to the protocol. Totally, 217 cycles were delivered all of them at full dose. The median relative dose intensity of paclitaxel was 1 (range 0.88–1.1). Three (7.5%) patients achieved complete and 9 (23%) partial response, while 12 (30.5%) patients demonstrated stabilization of the disease. Side effects from combined chemoradiotherapy were mainly mild. Grade III toxicity included infection (7.5%) and alopecia (5%). Median time to progression was 6 (range 0.9–27) months and median survival 10.7 (range 0.9–39.5+) months. The present study has clearly shown that 100 mg/m2 of paclitaxel in 1-h infusion weekly can be safely given concomitantly with RT in patients with GBM with manageable toxicity. However, the efficacy of this combined modality treatment does not appear to be superior to that of RT alone.
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  • 151
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    Journal of neuro-oncology 41 (1999), S. 159-166 
    ISSN: 1573-7373
    Keywords: neuroblastoma ; chemotherapy ; apoptosis ; dopamine receptors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract While neuroscientists are often involved in the assessment and care of patients with central nervous system tumors, they are only rarely involved in the case of peripheral nervous system neoplasia. Neuroblastoma is a childhood tumor of the primitive sympathetic nervous system. It is at once one of the most common and one of the most deadly tumors of childhood. The prognosis for children with this tumor has not changed in the past two decades. Clearly, a fresh approach to neuroblastoma is needed. The neuroscientist has much to add to our understanding and treatment of neuroblastoma and its sequelae. Conversely, neuroblastoma has much to teach us regarding the normal development of the neural crest and the aberrant loss of neurons in this lineage. A neuroscientist's approach to neuroblastoma, its biology and clinical features, is presented herein.
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  • 152
    ISSN: 1573-7373
    Keywords: chondrosarcoma ; cranial base ; chemotherapy ; ifosfamide ; doxorubicin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The case of a 46-year old woman with recurrent chondrosarcoma of the cranial base, refractory to neurosurgical intervention and external radiotherapy is reported. She received five cycles of systemic chemotherapy utilizing ifosfamide and doxorubicin which resulted in a durable clinical and radiographic response lasting 52+ months. A review of the management options for recurrent chondrosarcoma of the cranial base is also presented.
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  • 153
    ISSN: 1573-7373
    Keywords: apoptosis ; chemotherapy ; human cell lines ; lovastatin ; medulloblastoma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Medulloblastoma is a malignant paediatric central nervous system tumor with a poor prognosis, stimulating the evaluation of improved treatment strategies. Lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, is currently used to treat patients with hypercholesterolemia. This compound also inhibits the production of non-steroidal mevalonate derivatives that are implicated in the control of cellular proliferation, and can induce cell-cycle arrest in vitro. We recently showed that lovastatin inhibited growth and promoted apoptosis of neuroblastoma, the peripheral nervous system ‘cousin’ of medulloblastoma. Therefore the potential of lovastatin as a possible anticancer drug against medulloblastoma was evaluated in vitro. Four medulloblastoma cell lines, Daoy, UW228, D341 Med and D283 Med, were treated with 1–40 µM of lovastatin in vitro. Analysis of cell morphologic changes, cell viability, DNA fragmentation and flow cytometry in all four cell lines showed growth inhibition and induction of apoptosis with lovastatin treatment. As little as 10 µM of lovastatin was sufficient to cause a marked reduction in cell numbers, and more than 20 µM of lovastatin induced 〉90% cells to undergo apoptosis, after intervals ranging between 36 and 96 h, depending on the cell line. Lovastatin induced apoptosis in these cell lines was concomitant with cell cycle arrest in G1. The attached cell lines UW228 and Daoy were more sensitive to lovastatin than D283 Med and D341 Med. Daoy cells which survived several cycles of lovastatin treatment could still be induced to undergo apoptosis after longer treatment times. The efficient induction of apoptosis by lovastatin favours this drug as a potential new avenue of therapeutic intervention for medulloablastoma.
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  • 154
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    Journal of neuro-oncology 43 (1999), S. 249-257 
    ISSN: 1573-7373
    Keywords: primary CNS lymphoma ; radiotherapy ; chemotherapy ; neurotoxicity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Primary central nervous system lymphoma (PCNSL) is a relatively uncommon primary brain tumor, but it has become the focus of many clinical trials because of its rising incidence and unique sensitivity to systemic chemotherapeutic agents. Radiotherapy can achieve high response rates and remissions in most patients, but survival is usually only 12–18 months because disease recurs. The addition of systemic chemotherapy, particularly intravenous methotrexate, had markedly improved disease control and many patients can achieve a durable remission and occasionally cure of their disease. Conventional systemic lymphoma drug combinations such as cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) are ineffective. High-dose methotrexate is the single most active and important agent in the treatment of this disease. Whether improved disease control can be accomplished by adding other drugs to high-dose methotrexate or whether it is sufficient as a single agent has yet to be answered. High-dose methotrexate combined with cranial irradiation yields a median survival of at least 40 months and five year survival rates of 22%. However, neurotoxicity is substantial in a significant proportion of patients, particularly those over the age of 60 at the time of treatment. As many as 50% of such patients develop severe dementia. This is particularly important in a disease where approximately half of patients above the age of 60 had presentation. Efforts are now being directed towards not only improving disease control but also minimizing late neurotoxicity. Most efforts are currently directed towards using chemotherapy as the sole modality in the treatment of PCNSL, but both an optimal chemotherapy regimen, and the role of radiotherapy remain to be determined.
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  • 155
    ISSN: 1573-7373
    Keywords: malignant gliomas ; elderly ; radiotherapy ; chemotherapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The benefit of standard treatment of malignant glioma in older patients is debated. In order to assess the effect of a combination of surgery, radiotherapy and chemotherapy on survival of elderly patients with high grade gliomas, 30 consecutive patients older than 70 years with malignant supratentorial gliomas were studied between 9/93 and 9/96. Median age was 73 years (70–79). The mean Karnofsky performance status (KPS) was 66 (30–100). Patients underwent maximum possible surgery, followed by a course of radiotherapy (45 Gy/25 fractions/5 weeks) with 3 or 4 orthogonal beams and a 2 cm margin around the tumor bed. The administration of chemotherapy was left at the discretion of the responsible physician and 12 patients received reduced dose nitrosourea-based chemotherapy. The overall median survival was 36 weeks. The median time to progression was 26 weeks. Three months after surgery, 26 patients were alive, 5 were in complete response, 2 in partial response and 10 were stabilized. Pre-radiotherapy KPS was the only significant prognostic factor with a median survival of 40 weeks in patients with KPS ≥70 and 25 weeks when KPS was 〉70 (logrank test, p=0.05). In responding and stable patients (57% of the group) the median KPS was 68 and 66 at 1 and 3 months after the completion of radiotherapy. There was no case of radiotherapy-induced dementia with this regimen. Four out of 12 patients who received chemotherapy, experienced WHO grade 3/4 hematotoxicity. This study suggest that some patients older than 70 years with KPS ≥70 may benefit from the treatment of malignant gliomas with surgery followed by reduced dose of limited field radiotherapy. Further studies are needed to define the most appropriate dose of radiotherapy and to evaluate further the risk/benefit ratio of a reduced dose chemotherapy in this population.
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  • 156
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    Journal of neuro-oncology 43 (1999), S. 287-291 
    ISSN: 1573-7373
    Keywords: primary central nervous system lymphoma ; chemotherapy ; radiotherapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The optimal treatment for primary central nervous system lymphoma (PCNSL) remains undefined. In this paper, we review the main multi-institutional ongoing protocols for PCNSL. Most of the current protocols evaluate the efficacy of combination high-dose methotrexate-based chemotherapy and brain irradiation in phase II studies. Some trials focus on chemotherapy alone as initial treatment, in order to minimize long-term cognitive sequellae. Because old age appears as a major predisposing factor for combined RT and CT neurotoxicity some specific protocols have been proposed to this particular population.
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  • 157
    ISSN: 1573-7373
    Keywords: recurrent ; glioma ; astrocytoma ; chemotherapy ; cisplatin ; etoposide
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Background. In most patients with recurrent glioma chemotherapy is the only remaining treatment option. In general results of chemotherapy in these patients are poor, and trials on new regimens are indicated. Because relatively good results have been achieved with combinations of platin compounds and etoposide, we investigated a dose-intensified cisplatin regimen with oral etoposide. Methods. Eligible patients, with recurrent glioma after surgery and radiation therapy were treated with two four week-cycles with cisplatin 70 mg/m2 on days 1, 8 and 15, combined with oral etoposide 50 mg daily on days 1–15. In responding or stabilized patients, treatment was continued with six four week-cycles of oral etoposide 50 mg/m2 on days 1–21. Toxicity was assessed using the NCI Common Toxicity Criteria, a 50% decrease in contrast enhancing area on MRI scan was considered a partial response. Time to progression was measured from the start of chemotherapy. Results. Sixteen patients were included, 11 were progressive during or immediately after the induction cycles. Two patients achieved a partial response with a time to progression of 42 and 58 weeks. Three patients were stable for 11, 14 and 15 weeks respectively. Toxicity was modest. Discussion. This dose-intensified cisplatin regimen did not result in a significant number of objective responses and even the number of ‘stable disease’ was small. Given the low response rate of this intensive treatment, we consider this intensive regimen inappropriate for these patients.
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  • 158
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    Journal of neuro-oncology 44 (1999), S. 163-168 
    ISSN: 1573-7373
    Keywords: meningeal neoplasm ; glioma ; meningeal gliomatosis ; chemotherapy ; radiation therapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract To evaluate whether vigorous treatment is beneficial for patients with meningeal gliomatosis (MG) we reviewed the case records of 20 consecutive patients treated for a symptomatic MG in our center. All received systemic or intrathecal chemotherapy and six received additional cranial or spinal radiotherapy. Six patients (30%) achieved a partial response (one low-grade astrocytoma, two anaplastic astrocytomas, one anaplastic oligodendroglioma and two glioblastomas). In these cases, clinical improvement was associated with radiological improvement on CT scan or MRI in five and with a major cerebrospinal fluid improvement in three. Three patients (15%) were stable for 3 months or more and 11 (55%) had progressive disease. Median survival was longer for the responding patients (10 months) than for the other patients (2 months). This study suggests that some patients with MG may benefit from a treatment combining radiotherapy to symptomatic areas and chemotherapy with agents that cross the blood–brain barrier or are delivered directly into the CSF.
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  • 159
    ISSN: 1573-7373
    Keywords: brain neoplasm ; germinoma ; CNS ; recurrence ; radiotherapy ; chemotherapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Systemic chemotherapy has been increasingly used in the primary treatment of intracranial germinoma. However, the recurrence rate seems to be very high after treatment with chemotherapy alone. We used radiation to treat 5 patients harboring intracranial germinoma that recurred following primary chemotherapy. They had received systemic chemotherapy (4 with cisplatin plus etoposide and 1 with adriamycin, vincristine, cyclophosphamide, prednisolone, and cisplatin) 7–24 months before referral. All patients were treated with conventional radiotherapy directed to the primary tumor site or the craniospinal axis with a dose to the primary site ranging from 39.6 to 47.0 Gy (mean, 42.6 Gy). Response to radiation of all the recurrent tumors was good and all tumors disappeared on diagnostic imaging below the dose of 24 Gy. All patients are alive without further recurrence at 61–129 months after salvage radiotherapy. Germinomas recurring after primary chemotherapy do not seem to have acquired cross resistance to radiotherapy. They can usually be cured by standard radiation therapy with 40–47 Gy.
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  • 160
    ISSN: 1573-7373
    Keywords: PCNSL ; lymphoma ; brain tumor ; chemotherapy ; methotrexate (MTX) ; QOL
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In anticipation of a consortium study of methotrexate (MTX) therapy provided to patients with primary central nervous system lymphoma (PCNSL) we have provided intravenous MTX without irradiation therapy to 31 non-immunosuppressed individuals. Twenty (65%) achieved complete response and 11 (35%) partial response to therapy. For the 31 patients the median survival was 30.43 months with an actuarial median follow up time of 30.69 months. The 2+ year survival was 63% for all patients and 90% for complete responders. Of 375 drug cycles, grade 3 leukopenia was identified in 3 cycles, mucositis in 6 cycles and delayed drug clearance in 47 cycles. Recurrences included brain (9/20) and/or spinal fluid (2/20). The median Karnofsky scale improved from 40 (10–80) prior to therapy to 90 after treatment. Eleven patients, in complete response for a median of 22+ months after diagnosis were evaluated using 4 instruments that assess Quality of Life Functional Assessment of Cancer Therapy – Brain (FACT-BR) modified, Symptom Questionnaire, Social Adjustment Scale-Self-Report and Problem Solving Inventory. Their psychosocial adjustment, well-being and stress coping abilities were comparable to the normative groups. Further there was no evidence of any MTX-induced, Magnetic Resonance Imaging (MRI)-detected encephalopathy in these individuals and there was preservation of clinical cognition and memory. We conclude that therapy with MTX, without radiation can be used in PCNSL patients without limitations of age or pretreatment Karnofsky scores. Further rates of response and median survival approach those of therapies using multiple drugs and radiation, but with a less likely risk of dementia.
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  • 161
    ISSN: 1573-7373
    Keywords: BCNU ; tamoxifen ; chemotherapy ; glioblastomas
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract From May 1990 to November 1994, 70 consecutive patients suffering from glioblastoma multiforme were treated following surgery with conventional radiotherapy and adjuvant IV BCNU administered alone or in combination with tamoxifen. Twenty-five patients received BCNU alone (control group A) while 24 patients also received 40 mg of tamoxifen (TMX) PO daily (group B) and 21 received 100 mg of TMX PO daily (group C). There were no significant differences between the 3 groups concerning age, type of resection and median post-operative Karnofsky performance status (KPS). Blood toxicity over grade II occurred in 33.5% of patients receiving TMX versus 12% of patients treated with BCNU alone (p〈0.05). Deep venous thrombosis complications were observed in 4 patients of each TMX group, whereas they were not observed in the control group (p〈0.04). Median time to tumor progression (MTTP) was 35 weeks in the control group and 27 weeks in both TMX groups B and C. Median survival time (MST) was 56, 66 and 51 weeks, respectively. These results suggest that the addition of TMX to standard treatment of glioblastomas does not affect the time to tumor progression and overall survival but may increase the risk of deep venous thrombosis or nitrosourea-induced blood toxicity.
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  • 162
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    European journal of plant pathology 105 (1999), S. 519-533 
    ISSN: 1573-8469
    Keywords: genome ; gene expression ; mollicute ; recombination ; transposition ; virus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Spiroplasmas are members of the Class Mollicutes, wall-less prokaryotes having a high adenosine–thymidine content in their small genomes. Spiroplasma citri is a plant pathogen that inhabits phloem. Like other phytopathogenic spiroplasmas and the related phytoplasmas, it is transmitted from plant to plant by phloem-feeding leafhoppers that serve as alternate hosts for the spiroplasma as well as vectors. Genetic information in spiroplasmas is carried on a circular chromosome, on plasmids and/or in virus genomes. A picture emerging from recent research on the S. citri genome is one of frequent and often extensive variation, resulting from a number of different mechanisms. Expansion and contraction events must continually be occurring in about equal proportions so that the net genome size varies within defined boundaries. Particularly impressive are large changes in genome size that can occur in only a few generations. As with most organisms, genetic variation in S. citri results from variation in extrachromosomal DNA content, changes due to DNA replication and repair processes and changes due to recombination. The implied flux of genetic information into and out of the S. citri genome should be beneficial to the bacterium, allowing it, with its small genome size, to adapt to new environments.
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  • 163
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    Cancer and metastasis reviews 18 (1999), S. 427-436 
    ISSN: 1573-7233
    Keywords: EGF ; receptor ; monoclonal antibodies ; cancer ; chemotherapy ; radiation therapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Recent technological advances, together with the discovery of the important role many growth factors play in modulating cell proliferation and differentiation, have led to the development of novel therapeutic agents for the treatment of cancer. In particular, advances in hybridoma technology and molecular engineering have permitted the development of humanized or chimeric monoclonal antibodies capable of interfering with growth factor signaling pathways. One promising target of interest is the epidermal growth factor receptor (EGFr), which is activated by the ligands EGF and TGF-α. This ligand receptor interaction plays a crucial role in the growth and survival of many human cancers. A chimeric (human/mouse) monoclonal antibody cetuximab (IMC-C225) targets the EGFr and has potential clinical value as an anticancer agent.
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  • 164
    ISSN: 1573-4935
    Keywords: Mucin ; lung cancer ; gene expression ; secretion ; lung adenocarcinoma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Mucins comprise an important class of tumor-associated antigens. The objectives of the present study were (a) to establish an in vitro model system using human non-small cell lung adenocarcinoma cell lines NCIH650 and NCIH2077 (b) provide evidence that these cell lines secrete mucin in culture conditions and (c) investigate the effects of select secretagogues on mucin secretion. The cell lines were established in ACL-4 medium containing several growth factors and retinoic acid and 5% fetal calf serum. The high molecular weight glycoconjugates secreted in the culture medium were purified by ammonium sulfate precipitation and Superose 6 and Superose 12 FPLC chromatography. The purified high molecular weight glycoconjugate fraction and the carcinoma cells were shown to have mucin by dot blot, Western blot and immunohistochemical analysis, respectively, using specific antibodies to purified major mucin, HTM-1. Also, incorporation experiments with mucin precursor 3H-glucosamine demonstrated that the cells indeed synthesize high molecular weight mucins. The effects of secretagogues such as, 8-bromocyclic AMP, ionomycin, phorbol-12-myristate-13-acetate and neutrophil elastase on mucin secretion were also investigated. Only 8-bromocyclic AMP and neutrophil elastase influenced mucin secretion. These studies provided strong evidence that the lung adenocarcinoma cell lines secrete high molecular weight mucins in culture conditions and only two of the four tested secretagogues significantly increased mucin secretion. Thus, this in vitro model system may be useful in determining alterations in mucin structure, if any, in lung adenocarcinomas as well as in studying the regulation of mucin gene expression.
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  • 165
    ISSN: 1573-904X
    Keywords: adriamycin ; doxorubicin ; HPMA copolymer ; apoptosis, multidrug resistance ; gene expression ; signal transduction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. To study peculiarities and the mechanism of the anticancer effect of free and HPMA copolymer-bound ADR in sensitive and resistant human ovarian carcinoma cells. Methods. Sensitive A2780 and ADR resistant A2780/AD cells were exposed to different doses of drugs during 12, 24, 36, 48, 60, and 72 hours. Cell viability, drug accumulation, apoptosis, cellular metabolism, lipid peroxidation, DNA content and gene expression were studied. Results. HPMA copolymer-bound ADR (P(GFLG)-ADR) possessed a comparable cytotoxicity to free ADR when comparison was based on intracellular concentrations. While free ADR up-regulated genes encoding ATP driven efflux pumps (MDR1, MRP), P(GFLG)-ADR overcame existing pumps and down regulated the MRP gene. Free ADR also activated cell metabolism and expression of genes responsible for detoxification and DNA repair. P(GFLG)-ADR down-regulated HSP-70, GSr-π, BUDP, Topo-IIα, β, and TK-1 genes. Apoptosis, lipid peroxidation and DNA damage were significantly higher after exposure to P(GFLG)-ADR, as reflected by simultaneous activation of p53, c-fos in A2780 cells) or c-jun (A2780/AD) signaling pathways and inhibition of the bcl-2 gene. Differences between free ADR and P(GFLG)-ADR increased with the time of incubation and drug concentration. Conclusions. P(GFLG)-ADR overcame drug efflux pumps, more significantly induced apoptosis and lipid peroxidation, inhibited DNA repair, replication, and biosynthesis when compared to free ADR.
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  • 166
    ISSN: 1573-5028
    Keywords: UV-B ; soybean ; chalcone synthase ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract By developing gene-specific RT-PCR and using filters to allow transmission down to 290 nm (UV-B+) or blocking all radiation below 320 nm (UV-B−), the effect of UV-B+ and UV-B− light on expression of each of the presently known seven members of soybean chalcone synthase (CHS) gene family in dark-grown seedlings was analyzed. Dark expression was detectable already in 18 h dark-germinating embryos, with progressive increases on successive days, suggesting that chs belongs to a class of genes expressed very early during germination, and that the expression at this stage is either constitutive or induced by non-light-dependent factors present in the seed or made available following imbibition. Exposure of 18 h dark-germinating embryos to UV-B− or to UV-B+ light did not lead to an increase in chs signal. However, the 24 h dark-germinating embryos showed a distinct effect of UV-B+, interestingly coinciding with the stage when the head of seedlings was in the process of being pushed up above ground by stem elongation, suggesting the possibility of a developmental switch modulating the appearance of UV-B response. The response to UV-B− was most prominent in chs1 and almost silent in chs2, while the up-regulation by UV-B+ was most prominent in chs5 and chs6 and much less so in chs2. Interestingly, chs2 was noted to be the only member of the Gmchs gene family devoid of H-box, raising the possibility that the H-box may be a good indicator of the photo-inducibility of a chs gene.
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  • 167
    ISSN: 1573-5028
    Keywords: amphidiploid genome structure ; gene expression ; glutamine synthetase ; multigene family ; nitrogen assimilation ; oilseed rape
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In the amphidiploid genome of oilseed rape (Brassica napus) the diploid ancestral genomes of B. campestris and B. oleracea have been merged. As a result of this crossing event, all gene loci, gene families, or multigene families of the A and C genome types encoding a certain protein are now combined in one plant genome. In the case of the multigene family for glutamine synthetase, the key enzyme of nitrogen assimilation, six different cDNA sequences were isolated from leaf and root specific libraries. One sequence pair (BnGSL1/BnGSL2) was characterized by the presence of amino- terminal transit peptides, a typical feature of all nuclear encoded chloroplast proteins. Two other cDNA pairs (BnGSR1-1/BnGSR1-2 and BnGSR2-1/BnGSR2-2) with very high homology between each other were found in a root specific cDNA library and represent protein subunits for cytosolic glutamine synthetase isoforms. Comparative PCR amplifications of genomic DNA isolated from B. napus, B. campestris and B. oleracea followed by sequence–specific restriction analyses of the PCR products permitted the assignment of the cDNA sequences to either the A genome type (BnGSL1/BnGSR1- 1/BnGSR2-1) or the C genome type (BnGSL2/BnGSR1-2/BnGSR2-2). Consequently, the ancestral GS genes of B. campestris and B. oleracea are expressed simultaneously in oilseed rape. This result was also confirmed by RFLP (restriction fragment length polymorphism) analysis of RT-PCR products. In addition, the different GS genes showed tissue specific expression patterns which are correlated with the state of development of the plant material. Especially for the GS genes encoding the cytosolic GS isoform BnGSR2, a marked increase of expression could be observed after the onset of leaf senescence.
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  • 168
    ISSN: 1573-5028
    Keywords: defense ; gene expression ; leaf senescence ; nitrilase ; pathogen-free ; salicylic acid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract To determine the range of gene activities associated with leaf senescence, we have identified genes that show preferential transcript accumulation during this developmental stage. The mRNA levels of a diverse array of gene products increases during leaf senescence, including a protease, a ribosomal protein, two cinnamyl alcohol dehydrogenases, a nitrilase and glyoxalase II. Two of the genes identified are known to be pathogen-induced. The senescence specificity of each gene was determined by characterization of transcript accumulation during leaf development and in different tissues. The increased expression of nitrilase in senescent leaves is paralleled by an increase in free indole-3-acetic acid (IAA) levels. Additionally, we have demonstrated that the induction of defense-related genes during leaf senescence is pathogen-independent and that salicylic acid accumulation is not essential for this induction. Our data indicate that the induction of certain genes involved in plant defense responses is a component of the leaf senescence program.
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  • 169
    ISSN: 1573-5028
    Keywords: gene expression ; gibberellin ; H1 histone ; H2B histones ; leaf ; Lycopersicon esculentum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract After differential screening we isolated cDNA clones encoding a histone H1 (leH1) and three variants of histone H2B (leH2B-1, -2 and -3) from the gibberellin (GA)-deficient mutant of tomato (gib-1). The deduced polypeptide of leH1 is 271 amino acids long and exhibits the typical tripartite structure of histones H1. The full-length cDNA clone leH2B-1 encodes for a protein of 142 amino residues and shows the tripartite organization of histones H2B. The histones leH1 and leH2B, which show no tissue specificity, are developmentally expressed in the leaf. The mRNA accumulation was higher in organs which contain meristematic tissue and/or which have a high proportion of actively cycling cells. In the leaf of the gib-1 mutant we demonstrated GA-enhanced histone leH1 and leH2B expression which was not observed in the wild type. GAs of the early-13-hydroxylated pathway (GA1 and GA3) caused most enhanced transcription compared to GAs of the early-non-hydroxylation pathway (GA4 and GA9). Application of GA to the mutant increased histone expression that could correlate with enhanced DNA replication in leaf tissue. Increased chromosome replication may indicate that there is a higher rate of cell division and/or increase of endopolyploidy which both may be dependent on cell elongation induced by GAs.
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  • 170
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    Plant molecular biology 39 (1999), S. 933-943 
    ISSN: 1573-5028
    Keywords: cloning ; fruit development ; gene expression ; pea ; polyamine ; spermidine synthase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Two cDNAs from young pea fruits coding for functional spermidine synthases (EC 2.5.1.16) were isolated. The corresponding genes were named psSPDSYN1 and psSPDSYN2. Both cDNAs complemented spe3Δ gene when introduced into the Y480 strain of Saccharomyces cerevisiae, which is a null mutant for the spermidine synthase gene. psSPDSYN1 and psSPDSYN2 are regulated differentially. psSPDSYN1 is up-regulated early after fruit set whereas psSPDSYN2 is expressed later. Spermidine synthase activity was detected in pea ovaries, and correlates with the pattern of expression of psSPDSYN1. In the pea plant, psSPDSYN1 is highly expressed in actively growing tissues, whereas the highest level of psSPDSYN2 mRNA was detected in fully elongated stem.
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  • 171
    ISSN: 1573-5028
    Keywords: chitin oligomer ; chitinase ; elicitor ; gene expression ; rice ; signal transduction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Expression patterns of chitinase transcripts induced by N-acetylchitooligosaccharide elicitor were analyzed by northern blot hybridization in order to reveal a signal transduction pathway leading to the activation of class I chitinase genes (Cht-1 and Cht-3), which may play an important role in producing N-acetylchitooligosaccharide elicitor. The transcription level of both genes was enhanced in response to N-acetylchitooligosaccharides larger than pentaose at subnanomolar concentrations. These structure and dose dependencies were consistent not only with those for a 75 kDa high-affinity binding protein for N-acetylchitooligosaccharide elicitor in the plasma membrane, but also with other series of cellular responses including phytoalexin production and the expression of elicitor-responsive genes (EL2, EL3). Therefore, the elicitor signal to evoke these cellular responses including the activation of the chitinase genes could be common and transmitted into cells through the 75 kDa protein. However, the signal transduction pathway for the activation of the chitinase gene appeared to diverge from those for the other elicitor-responsive genes shortly after the signal perception. It was shown that the induction of chitinase expression by N-acetylchitooligosaccharide would require protein phosphorylation, but not de novo protein synthesis. The oxidative burst was demonstrated not to be necessary for transcriptional induction of the all four elicitor-responsive genes (Cht, PAL, EL2, EL3) by N-acetylchitooligosaccharide.
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  • 172
    ISSN: 1573-5028
    Keywords: cytochrome b5 ; fruit and flower development ; gene expression ; in situ hybridisation ; olive
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We report the characterisation of two cytochrome b5 genes and their spatial and temporal patterns of expression during development in olive, Olea europaea. A PCR-generated probe, based on a tobacco cytochrome b5 sequence, was used to isolate two full-length cDNA clones (cytochrome b5-15 and cytochrome b5-38) from a library derived from 13 WAF olive fruits. The cDNAs encoded proteins of 17.0 and 17.7 kDa, which contained all the characteristic motifs of cytochromes b5 from other organisms and exhibited 63% identity and 85% similarity with each other. The olive cytochrome b5-15 cDNA was then used as a probe for more detailed analysis. Southern blotting revealed a gene family of at least 4–6 members while northern blotting and in situ hybridisation showed a highly specific pattern of gene expression. Very low levels of cytochrome b5 mRNA were detected in tissues characterised by high rates of lipid accumulation, such as young expanding leaves, maturing seeds and ripening mesocarp. The cytochrome b5 genes were not induced at 6 °C and their response to ABA was relatively slow compared with fatty acid desaturase genes. In contrast, high levels of cytochrome b5 gene expression were found in young fruits at the pattern formation (globular/heart) stage of embryogenesis and in vascular and transmitting tissues of male and female reproductive organs. The data are consistent with a major role for cytochrome b5 in developmental processes related to plant reproduction in addition to being an electron donor to microsomal desaturases.
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  • 173
    ISSN: 1573-5028
    Keywords: Amaranthus hypochondriacus) ; gene expression ; trypsin inhibitor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We previously isolated and sequenced the major trypsin inhibitor from Amaranthus hypochondriacus seeds. This amaranth trypsin inhibitor (AmTI) is a 69 amino acid protein with high homology to members of the potato-1 inhibitor family. This paper describes the cloning and expression of a cDNA encoding this trypsin inhibitor in various vegetative tissues of the amaranth plant during seed development and imbibition, and investigates the possible induction of AmTI expression by wounding. We obtained a 393 bp cDNA sequence with an open reading frame corresponding to a polypeptide with 76 amino acid residues. With the exception of one residue (Ser-41), the polypeptide agrees with the amino acid sequence previously reported, plus 7 more residues at the N-terminus. These N-terminal residues are thought to be part of the signal used for intracellular sorting. The organ specificity of AmTI gene expression was investigated by northern analysis, showing that mRNA corresponding to AmTI genes was present in stems of plants growing under normal conditions. The kinetics of accumulation of the AmTI-mRNA, protein, and inhibitory activity during seed development and imbibition was determined. AmTI-mRNA accumulation reached a maximum at 14 days after anthesis (daa) and then gradually decreased, being barely detectable 36 daa. The AmTI protein accumulation followed the same profile as the inhibitory activity, both were delayed with respect to the mRNA. The maximum level was observed 22 daa, and then gradually decreased until a steady state was reached as seed maturation proceeded. Upon imbibition, a gradual decrease in AmTI protein and inhibitory activity was shown; however, an AmTI transcript was detected 24 h after imbibition. In contrast to representative members of the potato I family, this inhibitor was not inducible by wounding of leaves.
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  • 174
    ISSN: 1573-5028
    Keywords: embryo ; gene expression ; Glycine max ; oxidoreductase ; seed coat ; testa
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The soybean Ep gene encodes an anionic peroxidase enzyme that accumulates in large amounts in seed coat tissues. We have isolated a second peroxidase gene, Prx2, that is also highly expressed in developing seed coat tissues. Sequence analysis of Prx2 cDNA indicates that this transcript encodes a cationic peroxidase isozyme that is far removed from Ep in peroxidase phylogeny. To determine the expression patterns for these two peroxidases in developing seeds, the abundance and localization of the Ep and Prx2 transcripts were compared by in situ hybridization. Results show the expression of Ep begins in a small number of cells flanking the vascular bundle in the seed coat, spreads to encircle the seed, and then migrates to the hourglass cells as they develop. Expression of Prx2 occurs throughout development in all cell layers of the seed coat, and is also evident in the pericarp and embryo. Nonetheless, the Ep-encoded enzyme accounts for virtually all of the peroxidase activity detected in mature seed coats. The Prx2 enzyme is either insoluble in a catalytically inactive form, or is subject to degradation during seed maturation.
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  • 175
    ISSN: 1573-5028
    Keywords: ABRE ; embryogenesis ; G-box ; gene expression ; maize ; protein-DNA interaction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transcription of the rab28 gene from maize is induced in late embryo development and in response to abscisic acid. We have studied the regulation of the activity of the rab28 promoter in embryos. Two abscisic acid-responsive elements (ABREs) were necessary for expression in embryos of transgenic Arabidopsis and in transient transformation in maize embryos. In vivo footprinting showed that there was protein binding to the ABREs and to other cis elements in the promoter in young embryos before expression of rab28. This shows that the rab28 promoter is in an open chromatin structure before developmental activation. The ABREs are important for the induction and have protein binding in young embryos. Nuclear proteins extracted from embryos before activation of rab28 bound to the ABREs in band shift assays. A complex with different mobility was formed between nuclear proteins and the ABREs after induction of rab28 suggesting a modification of the ABRE-binding factor or an exchange of proteins. The footprints on the ABREs were unaltered by induction with abscisic acid or during developmental activation of rab28. These results indicate that constitutive binding of transcription factor(s) on the ABRE is central in embryonic regulation of the rab28 gene.
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  • 176
    ISSN: 1573-5028
    Keywords: alanine aminotransferase ; gene expression ; GUS expression ; promoter
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cDNA clone encoding alanine aminotransferase (AlaAT) has isolated from randomly sequenced clones derived from a cDNA library of maturing rice seeds by comparison to previously identified genes. The deduced amino acid sequence was 88% and 91% homologous to those of the enzymes from barley and broomcorn millet (Panicum miliaceum), respectively. Using this cDNA as a probe, we isolated and sequenced the corresponding genomic clone. Comparison of the sequences of the cDNA and the genomic gene revealed that the coding region of the gene was interrupted by 14 introns 66 to 1547 bp long. Northern and western blotting analyses showed that the gene was expressed at high levels in developing seeds. When the 5′-flanking region between −930 and +85 from the site of initiation of transcription was fused to a reporter gene for β-glucuronidase (GUS) and then introduced into the rice genome, histochemical staining revealed strong GUS activity in the inner endosperm tissue of developing seeds and weak activity in root tips. Similar tissue-specific expression was also detected by in situ hybridization. These results suggest that AlaAT is involved in nitrogen metabolism during the maturation of rice seed.
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  • 177
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    Plant molecular biology 39 (1999), S. 161-169 
    ISSN: 1573-5028
    Keywords: expansin ; fruit growth ; fruit softening ; gene expression ; Lycopersicon esculentum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract cDNA clones encoding homologues of expansins, a class of cell wall proteins involved in cell wall modification, were isolated from various stages of growing and ripening fruit of tomato (Lycopersicon esculentum). cDNAs derived from five unique expansin genes were obtained, termed tomato Exp3 to Exp7, in addition to the previously described ripening-specific tomato Exp1 (Rose et al. (1997) Proc Natl Acad Sci USA 94: 5955–5960). Deduced amino acid sequences of tomato Exp1, Exp4 and Exp6 were highly related, whereas Exp3, Exp5 and Exp7 were more divergent. Each of the five expansin genes showed a different and characteristic pattern of mRNA expression. mRNA of Exp3 was present throughout fruit growth and ripening, with highest accumulation in green expanding and maturing fruit, and lower, declining levels during ripening. Exp4 mRNA was present only in green expanding fruit, whereas Exp5 mRNA was present in expanding fruit but had highest levels in full-size maturing green fruit and declined during the early stages of ripening. mRNAs from each of these genes were also detected in leaves, stems and flowers but not in roots. Exp6 and Exp7 mRNAs were present at much lower levels than mRNAs of the other expansin genes, and were detected only in expanding or mature green fruit. The results indicate the presence of a large and complex expansin gene family in tomato, and suggest that while the expression of several expansin genes may contribute to green fruit development, only Exp1 mRNA is present at high levels during fruit ripening.
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  • 178
    ISSN: 1573-5028
    Keywords: cDNA cloning ; fruit ripening ; gene expression ; non-climacteric fruit ; wild strawberry (Fragaria vesca L.)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Wild strawberry (Fragaria vesca L.) is an attactive model system for studying ripening in non-climacteric fruit, because of its small diploid genome, its short reproductive cycle, and its capacity for transformation. We have isolated eight ripening-induced cDNAs from this species after differential screening of a cDNA library. The predicted polypeptides of seven of the clones exhibit similarity to database protein sequences, including acyl carrier protein, caffeoyl- CoA 3-O-methyltransferase, sesquiterpene cyclase, major latex protein, cystathionine γ-synthase, dehydrin and an auxin- induced gene. A ninth cDNA clone that was constitutively expressed is predicted to encode a metallothionein-like protein. None of these proteins appear to be directly related to events generally associated with ripening such as cell wall metabolism or the accumulation of sugars and pigments, rather, their putative functions are indicative of the wide range of processes upregulated during fruit ripening.
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  • 179
    ISSN: 1573-5028
    Keywords: cowpea (Vigna unguiculata L.) ; drought ; gene expression ; lipid degradation ; phospholipase D
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Phospholipase D, a major lipid-degrading enzyme in plants, was studied in two cultivars of Vigna unguiculata L.Walp, differing in their tolerance to drought (cv. EPACE-1, drought-tolerant, and cv. 1183, drought-susceptible). Enzymatic activities, measured with 14C-PC as substrate, increased when plants were submitted to water stress, the increase being much higher in the drought-sensitive cultivar. A 2911 bp cDNA encoding a putative phospholipase D (VuPLD1) was isolated from a cDNA library prepared from V. unguiculata leaves. The deduced amino acid sequence (809 residues) shows 85.5% identity and 91.3% similarity to that of PLD from Ricinus communis. The expression of the VuPLD1 gene in the leaves is differently modulated by water deficit, depending on the intensity of stress and the tolerance or sensitivity of the plants. In the drought-susceptible V. unguiculata cv. 1183, it readily increased under water stress, reaching maximum values at mild water deficit (−1.5 MPa). In the drought-tolerant cv. EPACE-1, VuPLD1 mRNA remained low throughout the whole drought treatment. Dehydration of leaves led to a dramatic increase in transcript level in both cultivars. Changes in protein amounts semi-quantified by immunoblotting correlated well with variations in transcript steady-state level. Taken together, these results showed that phospholipase D in cowpea plants is essentially regulated at the transcriptional level, and that gene expression is strongly stimulated even by moderate water deficit in the drought-sensitive plant. On the contrary, the drought-tolerant plant presents a remarkable stability of PLD gene expression in conditions of water stress.
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  • 180
    ISSN: 1573-5028
    Keywords: reproductive development ; gene expression ; subtractive hybridization ; cauliflower
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Using the meristems of the cauliflower curd as a source of tissue and a series of subtractive hybridizations and amplification reactions, we have constructed a cDNA library highly enriched in cDNAs expressed in reproductive meristems. The analysis of a sample of 250 clones from this library identified 22 cDNA clones corresponding to genes specifically expressed in these cauliflower meristems. Apart from two clones that corresponded to APETALA1, and two other ones showing similarity to different aminoacyl-tRNA synthetases, the remaining clones showed no similarity to any sequence in the databases and may correspond to novel genes. One of these clones, BoREM1, was further characterized and found to correspond to a gene encoding a protein with features of regulatory proteins that follows a expression pattern very similar to the LEAFY transcripts.
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  • 181
    ISSN: 1573-5028
    Keywords: ammonium ; gene expression ; glutamine synthetase ; nodules ; positive element ; promoter
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In order to identify important promoter elements controlling the ammonium-regulated expression of the soybean gene GS15 encoding cytosolic glutamine synthetase, a series of 5′ promoter deletions were fused to the GUS reporter gene. To allow the detection of positive and negative regulatory elements, a series of 3′ deletions were fused to a −90 CaMV 35S promoter fragment placed upstream of the GUS gene. Both types of construct were introduced into Lotus corniculatus plants and soybean roots via Agrobacterium rhizogenes-mediated transformation. Both spectrophotometric enzymatic analysis and histochemical localization of GUS activity in roots, root nodules and shoots of transgenic plants revealed that a strong constitutive positive element (SCPE) of 400 bp, located in the promoter distal region is indispensable for the ammonium- regulated expression of GS15. Interestingly, this SCPE was able to direct constitutive expression in both a legume and non- legume background to a level similar to that driven by the CaMV 35S full-length promoter. In addition, results showed that separate proximal elements, located in the first 727 bp relative to the transcription start site, are essential for root- and root nodule-specific expression. This proximal region contains an AAAGAT and two TATTTAT consensus sequences characteristic of nodulin or nodule-enhanced gene promoters. A putative silencer region containing the same TATTTAT consensus sequence was identified between the SCPE and the organ-specific elements. The presence of positive, negative and organ-specific elements together with the three TATTTAT consensus sequences within the promoter strongly suggest that these multiple promoter fragments act in a cooperative manner, depending on the spatial conformation of the DNA for trans-acting factor accessibility.
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  • 182
    ISSN: 1573-5028
    Keywords: embryo-abundant cDNAs ; gene expression ; gymnosperm ; Picea glauca ; somatic embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Six somatic embryogenesis-associated cDNAs (PgEMB2, 6, 7, 8, 24 and 34) from white spruce (Picea glauca (Moench) Voss) somatic embryos have been characterized. Transcript accumulation during somatic embryo development and subsequent germination related to these genes, indicated that they were developmentally regulated. The transcripts related to clones PgEMB2, 6, 24 and 34 were also detected during zygotic embryo development, but transcripts of clones PgEMB7 and 8 were not. PgEMB24 had a similar gene expression pattern to spruce Em-like late embryo abundant (lea) gene, but other clones had no similarities in gene expression to either spruce lea-like or storage protein genes. Abscisic acid, a stimulator for spruce somatic embryo maturation, did not obviously affect gene expression corresponding to these cDNAs. The predicted proteins are distinguishable from known LEA proteins based on analyses of hydropathy plots, amino acid compositions and deduced protein structures. The similarities of the spruce cDNAs, and protein sequences predicted from these cDNAs, to other sequence data are described.
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  • 183
    ISSN: 1573-5028
    Keywords: gene expression ; GT-1 ; PR-1a ; PR proteins ; salicylic acid-induced ; transcription factors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Infection of Nicotiana tabacum Samsun NN with tobacco mosaic virus (TMV) results in a hypersensitive plant response and leads to systemic acquired resistance (SAR). The induction of SAR is mediated by the plant hormone salicylic acid (SA) and is accompanied by the induced expression of a number of genes including the pathogenesis-related (PR) gene 1a. Previously, it has been found that TMV infection and SA treatment resulted in a reduction of binding of nuclear protein GT-1 to far-upstream regions (−902 to −656) of the PR-1a gene. To test if GT-1 is a negative regulator of PR-1a gene expression, the effects of mutations in the seven putative GT-1 binding sites in this region were studied in vitro using dimethyl sulfate interference footprinting and band shift assays. This showed that at least one of the seven sites is indeed a GT-1 binding site. However, when tested in transgenic plants, the mutations did not result in constitutive expression of the chimeric PR-1a/GUS transgene, while inducible expression after SA treatment was decreased. The results suggest that binding of GT-1-like proteins to far-upstream PR-1a promoter regions indeed influences gene expression. A possible model for GT-1's mode of action in PR-1a gene expression is discussed.
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  • 184
    ISSN: 1573-5028
    Keywords: aquaporin ; gene expression ; growth ; Oryza sativa ; plasma membrane intrinsic protein (PIP) ; rice
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Membrane intrinsic proteins facilitate movement of small molecules often times functioning as water channels. We have identified two genes from rice which encode proteins with characteristic features of plasma membrane intrinsic proteins (PIP). They possess six membrane-spanning domains, an NPA repeat, overall high sequence homologies and characteristic C- and N-terminal hallmark motifs which allowed assignment of OsPIP1a to the PIP1 subfamily and of OsPIP2a to the PIP2 subfamily. OsPIP1a and OsPIP2a showed similar but not identical expression patterns. The two genes were expressed at higher levels in seedlings than in adult plants and expression in the primary root was regulated by light. In internodes of deepwater rice plants which were induced to grow rapidly by submergence, transcript levels were slightly induced in the intercalary meristem (IM) and slightly reduced in the elongation zone (EZ) after 18 h. In internodes of GA-induced excised stem sections transcript levels transiently declined in the IM and EZ after 1 h and subsequently recovered to elevated levels after 18 h. GA also induced OsPIP expression in non-growing tissue after 18 h. In the IM of submergence-induced stem sections transcript levels remained constitutive. The different growth-promoting treatments showed no direct correlation between growth rate and OsPIP gene expression in dividing or expanding cells. In fact, treatment of excised stem sections with ABA or drought stress induced similar changes in OsPIP expression in the growing zone during the first 6 h as GA did. We conclude that regulation of OsPIP1a and OsPIP2a expression is not primarily controlled by growth. GA-induced growth may however change the water status of cells which in turn results in altered PIP abundance.
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  • 185
    ISSN: 1573-5028
    Keywords: ACC synthase ; chilling ; Citrus sinensis ; ethylene ; gene expression ; peel
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Diurnal change in the temperature below or above 12.5 °C hastens the degreening of citrus peel and elicits the phytohormone ethylene production in citrus fruit. Ethylene triggers the degradation of chlorophyll and synthesis of carotenoids in citrus peel. To investigate if ethylene is required for the degreening of citrus peel elicited by low temperatures, we studied the chilling-regulated gene expression of ACC synthase, one of the key enzymes catalyzing ethylene biosynthesis. We isolated and characterized a chilling-inducible 1-aminocyclopropane-1-carboxylate synthase (ACC synthase) gene, CS-ACS1, and a chilling-repressible gene, CS-ACS2, from citrus peel. The CS-ACS1 transcript 1.7 kb in length encodes a polypeptide of 483 amino acids (M r 54 115, pI 6.63), whereas the CS-ACS2 transcript of 1.8 kb encodes a polypeptide of 477 amino acids (M r 53 291, pI 6.72). Both genes showed a rapid but transient induction (within 2.4 h) of transcripts upon rewarming after the chilling (4 °C) treatment. After 24 h of incubation at room temperature, CS-ACS1 mRNA diminished to an undetectable level, whereas the CS-ACS2 mRNA regained its basal level of expression attained prior to the chilling treatment. Chilling-induced ethylene production and ACC accumulation were also observed upon rewarming. Both genes were also induced by the wound stress (excision). The protein synthesis inhibitor cycloheximide super-enhances the accumulation of both ACS transcripts at room temperature. Molecular analysis of the 3.3 kb genomic DNA of CS-ACS1 revealed that this gene consists of three introns and four exons. The intron 3 is exceptionally large (1.2 kb) and shares significant homology with mitochondrial DNA, supporting the intron-late theory.
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  • 186
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    Plant molecular biology 41 (1999), S. 351-361 
    ISSN: 1573-5028
    Keywords: chromatin ; gene expression ; high-mobility-group protein HMG1 ; HMGe ; protein stability ; Zea mays
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The nuclear HMG1 proteins of higher plants are small non-histone proteins that have DNA-bending activity and are considered architectural factors in chromatin. The occurrence of the chromosomal HMG1 proteins, HMGa, HMGc1/2 and HMGd, in various maize tissues was analyzed, and in the course of these studies a novel HMG1 protein, now termed HMGe, was identified. Purification and characterization of HMGe (Mr 13 655) and cloning of the corresponding cDNA revealed that it displays only moderate similarity to other members of the plant HMG1 protein family. The five maize HMG1 proteins could be detected in kernels, leaves, roots and suspension culture cells, indicating that these proteins can be expressed simultaneously and occur relatively ubiquitously. However, the various HMG1 proteins are present in significantly different quantities with HMGa and HMGc1/2 being the most abundant HMG1 proteins in all tissues tested. Furthermore, the relative amounts of the various HMG1 proteins differ among the tissues examined. The HMG1 proteins were found to be relatively stable proteins in vivo, with HMGc1/2, HMGd and HMGe having a half-life of ca. 50 h in cultured cells, while the half-life of the HMGa protein is ca. 65 h. Collectively, these findings are compatible with the concept that the different plant HMG1 proteins might act as general architectural proteins in concert with site-specific factors in the assembly of certain nucleoprotein structures involved in various biological processes.
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  • 187
    ISSN: 1573-5028
    Keywords: alkaloids ; gene expression ; Nicotiana tabacum ; nicotine ; putrescine N-methyltransferase ; tobacco gene evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The structure and nuclear genomic organization of the gene family encoding putrescine N-methyltransferase (PMT), the key enzyme in diverting polyamine metabolism towards the biosynthesis of nicotine and related alkaloids, was examined in Nicotiana tabacum. Five genes encoding PMT are present in the N. tabacum genome and all are expressed. The complete coding region and immediate 5′- and 3′- flanking regions were characterized for four members of the gene family and the Exon 1 region of the fifth member of the family was determined. Comparison of the nucleotide and deduced amino acid sequences of the N. tabacum PMT genes with those of presumed progenitor species, N. sylvestris, N. tomentosiformis and N. otophora, revealed that three members of the N. tabacum PMT gene family were most similar to the three genes present in N. sylvestris, whereas the two remaining PMT genes were similar to PMT genes present in N. tomentosiformis and N. otophora genomes, respectively. These data are consistent with an evolutionary origin of N. tabacum resulting from a cross involving N. sylvestris and an introgressed hybrid between N. tomentosiformis and N. otophora. The five PMT genes present in N. tabacum are expressed in the roots of wild-type plants, but not in other organs. The steady-state level of all five PMT transcripts is transiently increased in roots following topping (removal of the floral meristem), although the maximum level of induction for the individual transcripts varies considerably. In contrast to wild-type plants, no increase in PMT transcript levels was observed in a low-alkaloid (nic1nic2) mutant of Burley 21. These data support a role for nic1 and nic2 in the global regulation of alkaloid formation in tobacco and provide for the first time molecular confirmation of the presumed origin of cultivated tobacco.
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  • 188
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    Plant molecular biology 41 (1999), S. 425-433 
    ISSN: 1573-5028
    Keywords: D1 protein ; gene expression ; psbA genes ; redox regulation ; Synechococcus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Three psbA genes encode two different forms of the photosystem II reaction centre protein D1 in Synechococcus sp. PCC 7942. The psbAI gene encoding D1 protein form I (D1:1) is mainly expressed under low growth light conditions while the psbAII and psbAIII genes, encoding D1 protein form II (D1:2), are induced under stress conditions (e.g. high light or low temperature). In this paper we show that psbAII/III genes can be rapidly induced even under low growth light conditions by adding the thiol reductant (DTTred) to Synechococcus cell culture, at a concentration that does not affect cell growth or photosynthetic activity. Similar induction of psbAII/III genes was obtained by illuminating the cells with photosystem I light. In both instances psbAI gene down-regulation coincided with the up-regulation of psbAII/III genes. DTTred-induced exchange in transcript pools was subsequently followed by an exchange of D1:1 for D1:2 at the protein level. Thiol oxidants, iodosobenzoic acid or diamide, reverted the effects of DTTred on psbA gene expression. Thiol oxidants and the thiol-modifying agent N-ethylmaleimide also totally prevented high-light induction of psbAII/III genes. These data strongly suggest that the up-regulation of psbAII/III genes that occurs under stress conditions is mediated by production of thiol reductants, whereas the expression of the psbAI gene is sustained by the more oxidizing conditions that prevail during the steady-state growth of cells.
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  • 189
    ISSN: 1573-5036
    Keywords: ammonium assimilation ; Amanita muscaria ; carbon allocation ; ectomycorrhiza ; gene expression ; Picea abies ; sugar transport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The symbiosis (ectomycorrhiza, ECM) between roots of trees and shrubs of boreal and temperate forest ecosystems and soil fungi is essential for water and nutrient acquisition of the plants. The functionality of ECM is largely dependent on the ability of the host plant to supply photoassimilates to the fungus via the symbiotic interface. Based on sterile in vitro and non-sterile pot experiments, we review data which gives evidence that hexoses are supplied to the fungus by the host plant (mainly glucose and fructose), and that these sugars, at least in part, control development and function of ECM by interfering with fungal gene expression. We further show that any factor which reduces hexose allocation to the host–fungus interface will adversely affect ECM development. As an example, we address the impact of increased supply of nitrogen on the biochemistry of plant–fungus interaction and discuss potential consequences on host performance.
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  • 190
    ISSN: 1573-5028
    Keywords: bundle sheath cell ; C4 photosynthesis ; gene expression ; PEP carboxykinase ; prokaryotic expression ; Zea mays
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We isolated a full-length cDNA that encodes ATP-dependent phosphoenolpyruvate carboxykinase (EC 4.1.1.49, PCK) from leaves of maize, an NADP-malic enzyme type C4 plant. The mRNA was specifically and rather abundantly expressed in bundle sheath cells in accordance with the recent finding of cell-type-specific localization of PCK protein in maize, which has been detected with antibodies against cucumber PCK protein. The predicted protein had an N-terminal extension, which is characteristic of plant PCKs. The transcript level was much higher in the daytime than at night in 14-day old seedlings. However, in 42-day old plants the extent of diurnal change decreased. The maize PCK was expressed in Escherichia coli with the pET32 plasmid and purified to homogeneity. Through digestion with enterokinase, two types of enzyme were prepared; one with an intact N-terminus and the other lacking its N-terminal 77 amino acid residues due to over-digestion. The truncated protein had about 2-fold higher specific activity than the intact one, and was inhibited by 3-phosphoglycerate (3-PGA) with an I0.5 of 17.5 mM. In contrast, the intact protein was almost insensitive to 3-PGA. These results strongly suggest that the intact N-terminal extension may be involved in the regulation of PCK activity in vivo through some modification such as reversible phosphorylation.
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  • 191
    ISSN: 1573-5028
    Keywords: calcium-dependent protein kinase ; gene expression ; immunoassays ; light regulation ; rice ; seed development
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We investigated the spatial and temporal expression patterns of two rice calcium-dependent protein kinases (CDPKs), OsCDPK2 and OSCDPK11, using isoform-specific antisera. Bands of the expected molecular sizes for OsCDPK2 (59 kDa) and OsCDPK11 (61 kDa) were detected on western blots. OsCDPK2 and OsCDPK11 mRNA and protein levels increased in unison during flower development. However, at the onset of seed development, the protein expression profiles diverged significantly. OsCDPK2 protein was expressed at low levels during early seed development, but increased to high levels that were maintained in later stages (20 days after fertilisation, DAF). Conversely, OsCDPK11 protein levels were high at the beginning of seed development, but fell rapidly from 10 DAF onwards. This decrease in the level of OsCDPK11 protein was associated with the abundant synthesis of a truncated mRNA species. OsCDPK2 expression was also closely associated with light perception. OsCDPK2 protein was barely detectable in green leaves exposed to light, but levels increased sharply when plants were shifted to darkness. Initially, this increase reflected a rapid elevation in the levels of OsCDPK2 mRNA, which was normally located in the mesophyll. Conversely, OsCDPK11 mRNA and protein levels were unaffected by light. These data strongly indicate that two rice CDPK isoforms have different functions in seed development and in response to light in leaves.
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  • 192
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    Euphytica 106 (1999), S. 117-123 
    ISSN: 1573-5060
    Keywords: differential display ; gene expression ; heterosis ; hybrid wheat ; seedling leaf
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Differential display of mRNA was used to analyze the differences of gene expression in seedling leaves between heterotic hybrid/nonheterotic hybrid and their parental inbreds in order to study the molecular basis of heterosis in wheat. The results indicated that patterns of gene expression in hybrids differ significantly from their parents. Both quantitative and qualitative differences were observed. The quantitative differences include gene over-expression, gene under-expression in hybrid and dominant expression of highly-expressed parental genes in hybrids. The qualitative differences include silencing in hybrids of genes expressed either in male or female parent, and silencing in hybrids of genes expressed in both parents. Expression in hybrid of genes only expressed either in male or female parent was also observed. It was also found that some genes expressed at high level in heterotic hybrid were underexpressed or expressed at low level in nonheterotic hybrid. One differentially expressed cDNA fragment 4B was cloned and sequenced after being confirmed through Northern blot analysis. Homology search in GenBank proved that the cDNA fragment is a new sequence. The selection of primers for differential RNA display in wheat and the relationship between wheat heterosis and alteration of gene expression in hybrids as compared to their parental inbreds were also discussed.
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  • 193
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    Plant growth regulation 29 (1999), S. 47-76 
    ISSN: 1573-5087
    Keywords: cold ; chill ; freezing ; gene expression ; signal transduction pathways
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Temperature is expected to affect all plant processes. Consistent with this, expression of a large number of specific mRNAs and proteins is up-regulated during cold-acclimation. Their possible functions are outlined, encompassing a wide range of processes, some possibly related to other winter stresses besides cold. Some of the cold-responsive sequences are associated with constitutive or stress-related metabolism, others are probably protective, some may influence freezing, and many are of as yet unknown function. While many of the sequences code for intracellular proteins, a significant number code for apoplastic proteins with a variety of possible functions. Transient influx of calcium into the cytosol appears to be a key step in the response to cold, and also to many other stresses and signals. Similarly, the cold-responsive promoter element identified so far is also responsive to drought and salt. However, how cold is sensed, and any cold-specific aspects of the cold signal transduction pathway, are, so far, unknown. What is clear is that, at least in the model plant Arabidopsis thaliana, cold-, desiccation-, salt- and ABA-triggered signal transduction pathways, and possibly others, run partly in parallel and partly intersect. This may be partly explained by a need for an integrated winter-response. The total number of genes which are cold-responsive and the quantity of resources which this implies are used in this way, indicate that many must have a positive role in acclimation. Experiments which modify membrane lipid unsaturation or solute accumulation, achieved by transformation of plants to express exotic or heterologous genes or by other means, confirm that these factors affect chill- or freezing-tolerance. A transgenic test has shown that one cold-up-regulated gene of previously unknown function contributes to freezing-tolerance, but the small effect re-emphasises the probably cumulative nature of the contributions of many cold-up-regulated sequences to acclimation. On the other hand, mutant analysis indicates some genes may make a comparatively larger contribution. Transformation of alfalfa to overexpress a superoxide dismutase gene increased cold-tolerance and drought-resistance and demonstrated that improvements in field-survival of stresses is possible by transgenic means. Over-expression of a transcription factor, CBF1, conferred freezing-tolerance on Arabidopsis, showing that manipulation of the signal transduction pathway could be an important method for modifying cold-tolerance.
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  • 194
    ISSN: 1573-5168
    Keywords: fish ; gene expression ; GH ; GtH ; gonad ; growth factors ; RT-PCR ; salmonid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In this study, Northern blot analysis of RNA from trout testis revealed a single transcript of insulin-like growth factor II (IGF-II) around 4.7 kb. The cellular distribution of IGF-II mRNA was studied and quantified in different testicular cells enriched populations by RT-PCR. IGF-II mRNA appears to be expressed in all cellular types tested: spermatogonia A and B, primary spermatocytes, spermatids and secondary spermatocytes and Sertoli cells. A significantly higher expression of IGF-II was found in premeiotic germ cells. The levels of IGF-II mRNA appear to be higher than those of IGF-I in immature trout testis, as judged from the semi-quantitative RT-PCR results. These data suggest that in addition to IGF-I, IGF-II may play a role in testicular physiology in fish. The hormonal regulation of IGF-I and IGF-II gene expression was investigated both in vitro and in vivo using RT-PCR approach. Gonadotropin (GtH) added to testicular explants increased IGF-II mRNA levels but had no effect on IGF-I. No statistically significant effect was observed with androgens. In vivo, GH and pituitary extracts resulted in an 8 fold and 2-3 fold increase in both IGF-I and IGF-II mRNA levels, respectively. Taken together, our study suggests that IGF-I and IGF-II may act as local mediators of GH and GtHs in fish testis. Moreover, our results imply that in fish testicular cells, IGFs are potential paracrine/autocrine regulators inside the spermatogenic compartment and can act directly on germ cells to stimulate their proliferation.
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  • 195
    ISSN: 1573-6903
    Keywords: Tubulin ; glial fibrillary acidic protein ; gene expression ; human fetal brain
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Developmental alterations in the expression of glial fibrillary acidic protein (GFAP) and α-tubulin were examined at the level of mRNA and protein in human fetal brain between weeks 13–23 of gestation. Except for a transient increase at week 15, GFAP expression in the cytoskeletal (CSK) fraction was low until week 17, when it increased steadily to week 23, corresponding to the phase of glial proliferation. The developmental profile of α-tubulin in the CSK fraction displayed a biphasic pattern, with an initial rise between weeks 13–16 coinciding with the early phase of neuroblast multiplication, and a second rise between weeks 17–23 corresponding to the phase of glial proliferation. No significant difference in the spatial distribution of α-tubulin was found in different region of brain but GFAP expression varied with a higher level in cerebellum than that in cerebrum at late midgestation.
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  • 196
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    Journal of bioenergetics and biomembranes 31 (1999), S. 467-473 
    ISSN: 1573-6881
    Keywords: Uncoupling proteins ; fatty acids ; skeletal muscle ; brown adipose tissue ; obesity ; thermogenesis ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract The recently discovered uncoupling protein 3 (UCP3) is highly homologous to the mitochondrialinner membrane protein UCP1, which generates heat by uncoupling the respiratory chainfrom oxidative phosphorylation. The thermogenic function of UCP1 protects against cold andregulates the energy balance in rodents. We review in vitro studies investigating the uncouplingactivity of UCP3 and in vivo studies, which address UCP3 gene expression in brown adiposetissue and skeletal muscle under various metabolic conditions. The data presented are, for themost, consistent with an uncoupling role for UCP3 in regulatory thermogenesis. We alsodiscuss mediators of UCP3 regulation and propose a potential role for intracellular fatty acidsin the mechanism of UCP3 modulation. Finally, we hypothesize a role for UCP3 in themetabolic adaptation of the mitochondria to the degradation of fatty acids.
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  • 197
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    Neurochemical research 24 (1999), S. 1233-1240 
    ISSN: 1573-6903
    Keywords: Anandamide ; AMPA GluR3 receptor subunit ; cannabinoid CB1 receptor ; cAMP ; gene expression ; cannabinoid antagonist ; SR141716A ; Xenopus oocytes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The discovery of endocannabinoids such as anandamide and the wide spread localization of cannabinoid receptors in the brain and peripheral tissues, suggests that the cannabinoid system represents a previously unrecognized ubiquitous net work in the nervous system, whose physiology and function is unfolding. In this study, we tested the hypothesis that some of the actions of anandamide are independent of a cannabinoid receptor mechanism. This was accomplished by the use of cannabinoid agonist and antagonist interaction in an in-vitro and in-vivo test systems. In-vitro, we used Xenopus laevis oocytes expression system and two-voltage clamp technique in combination with differential display polymerase chain reaction to determine whether the differential display of genes following treatment with anandamide may be linked to AMPA glutamate receptor. The differential expression of genes in vivo after the sub-acute administration of anandamide could not be directly linked with the AMPA glutamate receptor. In the voltage clamp studies we investigated the effects of anandamide on recombinant AMPA GluR3 sub-unit currents generated by kainic acid in oocytes expressing the AMPA glutamate receptor. In the in-vitro studies, we present evidence that anandamide inhibited the kainate activated currents in oocytes expressing AMPA glutamate receptor involves cAMP transduction via a cannabinoid receptor independent mechanism. In the in-vivo studies, SR141716A, the CB1 antagonist, induced anxiolysis, that was dependent on the mouse strain used in the anxiety model and blocked the anxiogenic effects of anandamide or methanandamide whereas SR141716A had no effect on the anandamide inhibition of kainate activated currents in-vitro.
    Type of Medium: Electronic Resource
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  • 198
    ISSN: 1573-6784
    Keywords: Brevibacillus ; cyclodextrin ; gene expression ; glycosyltransferase ; mannitol ; polyol
    Source: Springer Online Journal Archives 1860-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Expression of Brevibacillus brevis CD162 cyclodextrin glycosyltransferase (CGTase) gene using pET22b(+) vector in Escherichia coli BL21(DE3) resulted in the formation of inactive inclusion bodies under the usual induction conditions. However, by lowering the induction temperature to 30 °C and/or adding 0.5 M mannitol as an osmolyte, the formation of insoluble aggregates was prevented and about a 34-fold increase (8.51 U ml−1) in biologically active soluble form was achieved after 6 h induction. The active CGTase enzyme was estimated to comprise as much as 24% of the total soluble proteins. In addition, other polyols such as glycerol, erythritol, xylitol, sorbitol, and arabitol showed similar effects with mannitol on the production of active CGTase enzyme.
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  • 199
    ISSN: 1573-7217
    Keywords: apoptosis ; Bax ; Bcl‐2 ; breast cancer ; chemotherapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Optimizing chemotherapeutic drug delivery strategies relies, in part, on identification of the most clinically effective sequence, dose, and duration of drug exposure. The combination of dose intensive etoposide (VP‐16) followed by cyclophosphamide has clinical efficacy in the treatment of advanced breast cancer. However, molecular mechanisms that underlie the effectiveness of this combination of chemotherapeutic agents have not been investigated. In this study we investigated regulation of BAX and BCL‐2 expression by VP‐16 and cyclophosphamide as a potential mechanism for the induction of breast cancer cell death induced by this regimen. There was a dose and time dependent increase in BAX expression in the breast cancer cell lines MCF‐7, MDA‐MB‐435S, and MDA‐MB‐A231 following in vitro treatment with 50–100 μM VP‐16. Elevation of BAX protein expression in the presence of VP‐16 alone did not correlate with reduced viability or induction of apoptosis in MCF‐7, MDA‐MB‐435S, or MDA‐MB‐A231. VP‐16 did effectively block the breast cancer cell lines evaluated (MCF‐7 and MDA‐MB‐435S) at G2/M phase of the cell cycle, confirming activity of the drug in vitro. MCF‐7 and MDA‐MB‐435S cells that were pre‐treated with VP‐16 and subsequently exposed to 1.0–12.0 μg/m1 4‐hydroperoxycyclophosphamide (4HC), an active metabolite of cyclophosphamide, had markedly reduced viability when compared to matched controls treated with either VP‐16 or 4HC individually. Consistent with this loss of viability, exposure of all three cell lines to the combination of VP‐16 and 4HC resulted in higher BAX protein levels than those observed following treatment with either single agent. This combination of chemotherapeutic agents also resulted in reduced BCL‐2 expression. These observations suggest that combination chemotherapy may derive its efficacy, in part, through coordinated regulation of specific gene products associated with apoptosis. Characterization of molecular events that underlie susceptibility of specific tumor cells to combination chemotherapeutic regimens may lead to additional improvements in treatment strategies for this disease.
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  • 200
    ISSN: 1573-7217
    Keywords: advanced breast cancer ; chemotherapy ; gemcitabine ; vinorelbine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Purpose. A phase II trial was performed to investigate the efficacy and tolerance of gemcitabine, vinorelbine, and recombinant human granulocyte colony‐stimulating factor (G‐CSF) in advanced breast cancer. Patients and methods. Between April 96 and August 97, 60 patients entered this trial. Forty‐five patients were previously untreated and 15 patients had failed previous palliative chemotherapy with (n = 10) or without anthracyclines (n = 5). Therapy consisted of gemcitabine 1000 mg/m2 on days 1 + 15 + 21 and vinorelbine 40 mg/m2 on days 1 + 21, both diluted in 250 ml saline and infused over 30 min. G‐CSF was administered at 5 μg/kg/day subcutaneously from days 2–6 and 22–26. Courses were repeated every 5 weeks. Treatment was continued in case of response or stable disease until a total of six courses. Results. The overall response rate was 55.5% for patients who had not received prior palliative chemotherapy (95% confidence interval, 40%–70.3%), including 5 CR (11.1%) and 20 PR (44.4%) 12 patients (27%) had stable disease (SD), and 8 (18%) progressed. Second‐line treatment with this regimen resulted in 6/15 (40%) objective remissions, 5 had SD, and 4 PD. The median time to progression was 9.5 months (range, 1.5–28) in previously untreated patients, and 7.0 months (range, 2–23) in those who had failed prior chemotherapy. After a median follow‐up time of 15 months, 44 patients (73%) are still alive with metastatic disease. Myelosuppression was commonly observed, though WHO 3 and 4 neutropenia occured in only 9 (l5%) and 2 patients (3%), and was never complicated by septicaemia; grade 3 anemia was noted in 2 patients. Severe (WHO grade 3) nonhematologic toxicity was rarely observed, and included nausea/emesis in 3 and constipation in 2 patients. Conclusions. Our data suggest that gemcitabine and vinorelbine plus G‐CSF is an effective and tolerable first‐ as well as second‐line combination regimen for treatment of advanced breast cancer.
    Type of Medium: Electronic Resource
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