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  • 1
    ISSN: 1432-203X
    Keywords: Key words Rubber tree ; Agrobacterium tumefaciens ; calcium ; gene transfer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The influence of CaCl2 was investigated on Agrobacterium tumefaciens-mediated gene transfer in Hevea brasiliensis friable calli which are usually proliferated on maintenance medium (MM) containing 9 mM CaCl2.Five A. tumefaciens strains (C58pMP90, C58pGV2260, AGL1, LBA4404 and EHA 105) and two binary vectors (pGIN and pCAMBIA2301) were tested and the strain EHA105pC2301 was selected to conduct further experiments. The calli were precultured on MM containing a range of CaCl2 concentrations, then inoculated with Agrobacterium suspension. Transfer of friable calli from MM containing 9 mM CaCl2 to calcium-free medium significantly enhanced the transient β-glucuronidase activity. Interestingly, the use of calcium-free Agrobacterium resuspension medium to inoculate friable calli again dramatically increased the transformation efficiency. Induction of Agrobacterium's virulence with acetosyringone remained an important factor to stimulate transformation.
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    GeoJournal 50 (2000), S. 97-100 
    ISSN: 1572-9893
    Keywords: disease ; health ; morbidity ; mortality ; pollution ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Geography
    Notes: Abstract In general, the health of Poles improved markedly in the thirty years after the Second World War, but there was some deterioration after 1989 before improvement resumed. Only in the case of cancer is there an upward trend and so Poles are now healthier than they have been at any time in the past. However there are sharp regional variations well exemplified by the incidence of tuberculosis, where there appears to be some correlation with poorer housing and atmospheric pollution. High death rates in Lodz (consistently the highest in the country at the voivodship level between 1989 and 1996), may also be linked with environmental pollution as well as the ageing of the population. Variations between town and country are small, but Poland shows up in a poor light when compared with other European countries. These are important issues for the administration and financing of the welfare services.
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    GeoJournal 52 (2000), S. 253-262 
    ISSN: 1572-9893
    Keywords: map sheets ; GIS ; seamless spatial database ; transformation ; computational procedure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Geography
    Notes: Abstract Map sheets have been often used as a basic spatial unit for managing spatial data produced from paper maps. This often results in incompatibility between adjacent map sheets, because spatial objects do not cross the boundaries smoothly and even the boundaries themselves do not match their neighbors exactly. To solve the problem this paper proposes a computational procedure for joining separate map sheets to obtain seamless spatial data. Line objects digitized separately in different map sheets are considered, which are frequently used to represent road networks, gas pipelines, and boundaries of polygon objects. The procedure consists of three steps: (1) extraction of end nodes, (2) detection of matching nodes, and (3) transformation of the map sheet. Each step goes interactively so that unexpected errors can be avoided by human observation. To test the validity of the procedure, map sheets are combined containing the road network data of Tokyo 23-ku area, Japan.
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    GeoJournal 50 (2000), S. 151-155 
    ISSN: 1572-9893
    Keywords: demography ; experts ; rural ; Romania ; rural development ; social issues ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Geography
    Notes: Abstract There is no standard model of transformation for post- socialist countries and each country encounters specific problems rooted in the geographical characteristics of the areas concerned. The human resources are of the greatest importance because it really matters how people (especially the decision-makers) perceive system change and continually reformulate their expectations and strategies; so investigations into the views of people caught up in the transformation can provide a deeper understanding of the background to structural change. Working the national, regional level and local levels in Romania, experts were asked to consider the advantages and disadvantages arising out of the transformation, the most important problems and constraints for future rural development and the policies needed. The paper examines the responses on demographic and social issues. It emerges the most detailed responses were supplied by local-level representatives while respondents at the regional level steered a middle course between the need to address local problems and the prime importance of stimulating the Romanian economy so as to generate resources for welfare programmes (with the latter issue the overriding concern of interviewees at national level). There was general agreement on the importance of foreign investment and European integration for economic development, with local actors taking only small steps in line with the existing opportunities.
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Antonie van Leeuwenhoek 78 (2000), S. 323-329 
    ISSN: 1572-9699
    Keywords: gene transfer ; plasmids ; Streptomyces
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Studies were made of naturally occurring plasmids hosted in Streptomyces strains isolated from two different terrestrial ecosystems: an agricultural field and a protected forest area. Six out of the 147 screened isolates contained plasmids. The strains containing these plasmids were all isolated from the agricultural soil. Plasmids were not found among the strains isolated from the forest area. Cross hybridization of the six newly isolated plasmids revealed very high similarities between four of them. However, no similarities were found between the six newly isolated plasmids and well studied streptomycete plasmids such as pIJ101 and SCP2*. The host strains of the four similar plasmids belonged to three different species S. anulatus, S. rochei and S. diastaticus. This implies a possible conjugative transfer of these plasmids within the streptomycete population in the agricultural area. The reason for the absence of streptomycete plasmids from the populations derived from the forest area is discussed.
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  • 6
    ISSN: 1573-904X
    Keywords: gene transfer ; lipid emulsions ; poly(ethylene glycol) ; transfection ; cationic lipids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. To develop a non-viral gene delivery system in the form ofan oil-in-water (o/w) lipid emulsionMethod. Cationic lipid emulsions were formulated with soybean oil,1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane (DOTAP) as acationic emulsifier and other co-emulsifiers. The physicalcharacteristics of the lipid emulsion and the emulsion/DNA complex weredetermined. The in vitro transfection efficiency of the emulsion/DNAcomplex was determined in the presence of up to 90% serum. Results. The average droplet size and zeta potential of emulsions wereca. 180 nm and ca. +50 mV, respectively. Among the emulsions, astable formulation was selected to form a complex with a plasmidDNA encoding chloramphenicol acetyltransferase. By increasing theratio of emulsion to DNA, zeta-potential of the emulsion/DNA complexincreased monotonously from negative to positive without any changesin the complex size. The complex was stable against DNase I digestionand an anionic poly-l-aspartic acid (PLAA). The complex deliveredDNA into the cells successfully, and the transfection efficiency wasnot affected by complex formation time from 20 min to 2 h. Moreimportantly, the cationic lipid emulsion facilitated the transfer of DNAin the presence of up to 90% serum. Conclusions. The cationic lipid emulsion/DNA complex has physicalstability and serum resistant properties for gene transfer.
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Chemistry of natural compounds 36 (2000), S. 137-139 
    ISSN: 1573-8388
    Keywords: 1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-5,5′-diisopropyl-2,2′-dinaphthylidene-8,8′-dibarbituric acid ; transformation ; DMSO
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Transformations of 1,1′,6,6′,7,7′-hexahydroxy-3,3′-dimethyl-5,5′-diisopropyl-2,2′-dinaphthylidene-8,8′-dibarbituric acid (batridene) in DMSO are studied.
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  • 8
    ISSN: 1573-904X
    Keywords: liposomes ; cancer vaccines ; cytokines ; immunotherapy ; interferon gamma ; gene transfer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. Liposomal systems may be useful as a cytokine supplementin tumor cell vaccines by providing a cytokine reservoir at the antigenpresentation site. Here, we examined the effect of liposomeincorporation of mIFNγ on its potency as adjuvant in an established tumor cellvaccination protocol in the murine B16 melanoma model. Adjuvanticityof the mIFNγ-liposomes was compared to that achieved bymIFNγ-gene transfection of the B16 tumor cells. Furthermore, we studiedwhether liposomal incorporation of mIFNγ indeed increases theresidence time of the cytokine at the vaccination site. Methods. C57B1/6 mice were immunized with i) irradiated IFNγ-genetransfected B16 melanoma cells or ii) irradiated wild type B16 cellssupplemented with (liposomal) mIFNγ, followed by a challenge withviable B16 cells. The residence time of the (liposomal) cytokine at thesubcutaneous (s.c.) vaccination site was monitored using radiolabeledmIFNγ and liposomes. Results. Immunization with irradiated tumor cells admixed withliposomal mIFNγ generated comparable protection against B16 challenge asimmunization with mIFNγ-gene modified tumor cells. Irradiated tumorcells admixed with soluble mIFNγ did not generate any protectiveresponses. Radiolabeling studies indicated that free mIFNγ rapidlycleared from the s.c. injection site. Association of [125I]-mIFNγwith liposomes increased the local residence time substantially: liposomalassociation of mIFNγ resulted in a prolonged local residence time ofthe cytokine as reflected by a 4-fold increase of the area under thecurve. The amount of released cytokine in the optimal dose rangecorresponds to the amount released by the gene-transfected cells. Moderate but significant CTL-activity against B16 cells was found for miceimmunized with irradiated cells supplemented with mIFNγ-liposomescompared to untreated control animals. Conclusions. Prolonged presence of mIFNγ at the site of antigenpresentation is crucial for the generation of systemic immune responsesin the B16 melanoma model. These studies show that liposomalencapsulation of cytokines is an attractive strategy for paracrine cytokinedelivery in tumor vaccine development.
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    World journal of microbiology and biotechnology 16 (2000), S. 177-182 
    ISSN: 1573-0972
    Keywords: Conjugation ; metal resistance ; plasmid DNA ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Heavy metal concentrations in soil treated with industrial wastewater of Aligarh City (U.P.), India were determined. The analysis of test samples revealed high levels of Fe, Zn, Ni and Cu. A total of 45 Pseudomonas spp. were isolated from soil and were characterized on the basis of morphological, cultural and biochemical characteristics. MICs of Hg2+, Cd2+, Cu2+, Cr3+, and Zn2+ for each isolate were determined. Eighty percent of the strains isolated from soil harboured resistance to copper, whereas 73.3% of the isolates exhibited resistance to cadmium, 71.1% to chromium and zinc and 48.8% to mercury. A maximum MIC of 200 μg/ml for mercury and 1600 μg/ml for other metals was observed. Metal resistance was found to be plasmid mediated as evidenced by transformation studies. Further, the transmissible nature of chromium resistance was confirmed by conjugation. Agarose gel electrophoresis using the miniprep method for plasmid isolation revealed that these isolates harboured plasmids of molecular weights (45 & 47 kb) using EcoRI and HindIII digests of λDNA and undigested λDNA as standard markers.
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  • 10
    ISSN: 1573-5060
    Keywords: Barley ; Hordeum vulgare ; transformation ; particle bombardment ; particle gun
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Immature embryos of the spring barley variety GoldenPromise, were bombarded with three different particledelivery systems and both transient and stabletransformation examined. In addition, a range oftechniques for the preparation of the DNA coated goldparticles was examined. Fertile transgenic barleyplants were obtained using three particle preparationtechniques which differed in the amount of gold andDNA used for each bombardment. However, only one ofthe particle delivery systems, the PDS 1000/He device,appeared to be effective in yielding transformedbarley plants.
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  • 11
    ISSN: 1573-5060
    Keywords: cell suspension ; monocotyledon ; selection ; somatic embryogenesis ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The successful application of plant biotechnology to Alstroemeria improvement will largely depend on the availability of an efficient regeneration/transformation system. Regeneration in Alstroemeria is accomplished from nodular embryogenic callus initiated from zygotic embryos. Histological studies of embryogenic callus initiation from 4-weeks old cultured ovules revealed that the outermost layers of the protoderm of the embryogenic nodules divided to form either a new nodule or aproembryo. Transient gene expression after particle bombardment of nodular embryogenic callus was optimized using DNA of pAHC25. The highest β-glucuronidase expression was found when the GUS gene was under control of the maize ubiquitin promoter, the target tissue was placed 5 cm below the microcarrier launch assembly and when the rupture disc-breakage point was between 650–900 psi. Kanamycin blocked regeneration of somatic embryos, however, did not block growth of nodular embryogenic callus. With phosphinothricin both callus growth and regeneration were blocked. Bombardment of nodular embryogenic callus with DNA of pAHC25 combined with selection on medium containing phosphinothricin resulted in putative transgenic chimeric. Friable calli were selected from nodular embryogenic callus and used to initiate suspensions. These cell suspensions were subjected to transformation by particle bombardment using DNA of pAHC25 and resulted in a stable transformed friable callus line after selection based on luciferase activity. Even after 2 years of maintenance this callus line was luciferase positive and the Polymerase Chain Reaction analysis demonstrated the presence of the introduced gene in this friable callus line.
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  • 12
    ISSN: 1573-5087
    Keywords: alfalfa ; cell division cycle ; somatic embryogenesis ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Two simple, rapid and efficient protocols for theregeneration of transformed tetraploid lines ofalfalfa (Medicago falcata L.) have beendeveloped and compared. Leaf explants fromembryogenic lines 47/1-150 and 47/1-5 were inoculatedwith Agrobacterium tumefaciens containingconstructs carrying the nptII selectable markergene and promoter:gusA gene fusions under thecontrol of the CaMV 35S or Arabidopsis cdc2a,CycB1 and CycA2 promoters. In the firstregeneration system (the MSH system), inoculated leafexplants were incubated on MS medium supplemented with2,4-D and kinetin and then subcultured onto plantgrowth regulator-free MS medium in order to inducedirect somatic embryogenesis. In the secondregeneration system (the B5h system), the inoculatedexplants were incubated on B5h medium to induceindirect production of somatic embryos viaembryogenic callus. In both systems, an effectivekanamycin selection regime was employed and wasmaintained when the embryos were subcultured onto arecovery medium (Boi2Y) to promote further embryodevelopment. The use of Boi2Y medium was particularlyimportant for shortening the regeneration time andpromoting a higher frequency of healthy plantletproduction from the somatic embryos. The maturesomatic embryos were finally transferred to plantgrowth regulator-free MS medium for plantletformation. Transgenic plantlets were produced within10–14 weeks in the MSH system and 12–16 weeks in theB5h system. The MSH system appears to be the fastesttransformation system reported for leguminous speciesto date. Confirmation of transformation was obtainedusing a re-callusing assay on kanamycin and subsequentSouthern blot hybridisation and PCR analysis. Theability to induce expression of GUS activity in leafexplants containing the cell division cycle genepromoter:gusA constructs by 2,4-D treatment alsoproved to be a reliable indicator of transformation.
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  • 13
    Electronic Resource
    Electronic Resource
    Springer
    Biochemical genetics 38 (2000), S. 41-55 
    ISSN: 1573-4927
    Keywords: gene transfer ; liposome ; IGF-I ; wound healing ; rat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Our hypothesis is that gene transfer of an IGF-I CMV-cDNA with cholesterol containing cationic liposomes is an efficient tool for transient transfection of growth factors in vitro and in vivo. In vitro, we transiently cotransfected IGF-I cDNA with a CMV construct and a Lac Z CHKβCHK-galactosidase cDNA/CMV construct using cholesterol containing cationic liposomes and measured CHKβCHK-galactosidase and IGF-I mRNA and protein. In vivo, we subcutaneously injected 3-month-old male Sprague–Dawley rats with IGF-I cDNA and CHKβCHK-galactosidase cDNA into rat skin. After IGF-I and CHKβCHK-galactosidase were cotransfected into PC12 cells, Northern blot analysis showed that the peak time of IGF-I expression was 2 days for mRNA and 5 days for protein. In vivo, a cDNA/liposome ratio of 1:2 was most effective. IGF-I protein expression in IGF-I-transfected skin resulted in significant transfection from day 5 to day 7. In situ determination of CHKβCHK-galactosidase activity confirmed that transfections resulted in a restricted expression area.
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  • 14
    Electronic Resource
    Electronic Resource
    Springer
    Bioscience reports 20 (2000), S. 419-432 
    ISSN: 1573-4935
    Keywords: liposomes ; gene therapy ; gene transfer ; nonviral vectors ; polylysine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Synthetic gene transfer vectors based on polyplexes complexed to anionic liposomes (LPDII vectors) were characterized for their transfection efficiency in cultured mammalian cells. The effects of polycation to DNA ratio, lipid to DNA ratio, choice of polycation and lipid composition were systematically evaluated in human oral carcinoma KB cells, using a luciferase reporter gene. For LPDII formulations containing poly-L-lysine and dioeoylphosphatidylethanolamine/cholesteryl hemisuccinate (DOPE/CHEMS) anionic liposomes, at a constant lipid to DNA ratio, an increase in the polycation/DNA (N/P) ratio resulted in an increase in transfection activity. Meanwhile, the optimal lipid to DNA ratio for efficient gene delivery was influenced by the N/P ratio used, and was increased at higher N/P ratios. For the DNA condensing agent, poly-L-lysine could be replaced by polyethylenimine (PEI) as the DNA condensing agent in the formulations. For the lipidic components, CHEMS could be replaced by other anioniclipids including oleic acid, dicetylphosphate and phosphatidylserine, but DOPE, a fusogenic helper lipid, could not be replaced by dioleolyphosphatidylcholine. LPDII formulation showed significantly less cytotoxicity compared to the commonly used cationic lipsomes or PEI mediated transfection and several cell lines were transfected with high efficiency. LPDII vectors avoid the use of toxic cationic lipids and may have potential application in gene therapy.
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  • 15
    ISSN: 1573-4935
    Keywords: fusogenic peptides ; HA2 influenza virus hemagglutinin ; polylysine ; transfection ; gene transfer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Amphiphilic anionic peptides have been used to enhance the efficiency of transfection by helping plasmids to escape from endosomes to the cytosol. It has been shown that efficiency of an eicosamers containing five glutamyl residues (E5), can be considerably enhanced either by transforming it into a dimer or by adding a tripeptide WYG in a C-terminal position (E5WYG). The dimerization of the peptide E5WYG leads to a more efficient tool when the dimerization device includes the tripeptide WYG unit and a longer spacer arm made of Gly-βAla-βAla residues, but to a 10-fold less efficient tool when the dimerization device includes a shorter spacer, a glycyl residue. Both dimers are taken up by the cells to a similar extent. Both dimers seem to be surrounded similarly as far as the environmental pH is concerned. In contrast, we found a correlation between the propensity of the peptides to adopt a helical structure at neutral pH and the gene transfer efficiency.
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  • 16
    ISSN: 1573-4889
    Keywords: Fe–Cr–Al alloy ; oxidation ; molybdenum ; breakaway oxidation ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Mechanical Engineering, Materials Science, Production Engineering, Mining and Metallurgy, Traffic Engineering, Precision Mechanics
    Notes: Abstract Thin foils of Fe–20Cr–5Al alloys are susceptible to breakawayoxidation once the aluminum content of the substrate has fallen below somecritical value. The combined addition of 0.1 wt.% lanthanum and 0, 1, or 2wt% molybdenum has a beneficial effect on the high-temperature oxidation ofsuch foils. Lanthanum has the well-known reactive-element effect on adhesionof the protective alumina scale, thereby increasing the time to onset ofbreakaway oxidation, while, for alloys containing molybdenum, breakawayoxide spreads relatively slowly over the specimen in comparison to alloysthat contain no molybdenum. In particular, molybdenum-containing alloys areable to develop a protective Cr2O3 layer at the breakawayoxide–substrate interface. Conversely, molybdenum-free alloys form aninternal-oxide zone in the substrate adjacent to this interface, rather thana Cr2O3 layer, so breakaway oxide spreads rapidly. A martensitic phase isobserved in the substrate adjacent to the breakaway oxide formed on Fe–20Cr–5Al–La specimens, which means that theα-phase has transferred to the γ -phase at the temperature ofthe oxidation test (1150°C). Conversely, α-phase is retained inthe molybdenum-containing alloy, even after breakaway takes place, sincemolybdenum, which is a strong ferrite former, is enriched in the alloyadjacent to areas of breakaway oxide. The diffusion rate of chromium isslower in the γ than in the α-phase so a continuouschromium-rich oxide layer, which is effective in inhibiting breakawayoxide from spreading, cannot be established at the breakawayoxide–substrate interface for the molybdenum-free alloys.
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  • 17
    ISSN: 1573-5060
    Keywords: conifers ; salgareño pine ; tissue culture ; transformation ; transient gene expression ; uidA expression ; vir gene
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Cotyledons from dissected sterile embryos of salgareño pine (Pinus nigra Arn. ssp. salzmannii (Dunal) Franco) were inoculated with different disarmed Agrobacterium tumefaciens strains harbouring the binary vector p35SGUSint. The transient expression of a β-glucuronidase gene (uidA) was studied, using a histochemical staining procedure. Nineteen days after inoculation, the activity of β-glucuronidase was detected in epidermal and subepidermal layers of cotyledonary explants. The EHA105 strain harbouring a disarmed agropine-type Ti-plasmid (pTiBO542) was the most effective for gene transfer of the uidA gene. The effects of exudates and extracts from 0-day-old embryos on induction of vir gene expression in A. tumefaciens were also examined. The results of this study showed that salgarño pine embryo exudates contain a substance(s) that induce vir gene expression, in similar way to that observed with 100 μM acetosyringone (AS).All these findings suggest that T-DNA processing and transfer might take place when Agrobacterium infects suitable tissues of salgareño pine.
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  • 18
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    Springer
    Reviews in endocrine & metabolic disorders 1 (2000), S. 205-215 
    ISSN: 1573-2606
    Keywords: Na+/I- symporter ; expression ; promoter ; gene transfer ; radioiodide therapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 19
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    Springer
    Pituitary 3 (2000), S. 117-122 
    ISSN: 1573-7403
    Keywords: Cushing's disease ; silent ; pituitary ; tumour ; macroadenoma ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Silent pituitary adenomas occur in 25–40% of all clinically apparent pituitary tumours. However, the subsequent development of florid Cushing's disease in a patient with a previous non-functioning tumour is extremely rare. We describe a 47 year-old woman presenting initially with a large, invasive and recurrent, non-functioning pituitary tumour. Histopathologic study of the initial tissue did not stain for any hormones. Six years after the initial presentation, she manifested florid ACTH dependent Cushing's syndrome. A recurrent invasive pituitary macroadenoma necessitated a third transphenoidal surgery. The resected specimen, in this instance, revealed positive staining for ACTH, FSH, prolactin, and growth hormone on immunocytochemistry. An incomplete response to surgical, radiation and medical therapy necessitated a bilateral adrenalectomy to control the hypercortisolism, which resulted in remarkable clinical improvement. We also review five previous case reports from the revision literature of similar transformations from non-secreting pituitary adenomas to Cushing's disease. This subset of patients may represent yet another entity in the widening spectrum of Cushing's syndrome.
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  • 20
    Electronic Resource
    Electronic Resource
    Springer
    Transgenic research 9 (2000), S. 471-486 
    ISSN: 1573-9368
    Keywords: Arabidopsis thaliana ; Agrobacterium tumefaciens ; floral spray ; SOD ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In this study, floral spray and floral dip were used to replace the vacuum step in the Agrobacterium-mediated transformation of a superoxide dismutase (SOD) gene into Arabidopsis. The transgene was constructed by using a CaMV 35S promoter to drive a rice cytosolic CuZnSOD coding sequence in Arabidopsis. The transgene construct was developed in binary vectors and mobilized into Agrobacterium. When Arabidopsis plants started to initiate flower buds, the primary inflorescence shoots were removed and then transformed by floral spray or floral dip. More than 300 transgenic plants were generated to assess the feasibility of floral spray used in the in planta transformation. The result indicates that the floral spray method of Agrobacterium can achieve rates of in planta transformation comparable to the vacuum-infiltration and floral dip methods. The floral spray method opens up the possibility of in planta transformation of plant species which are too large for dipping or vacuum infiltration.
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  • 21
    Electronic Resource
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    Springer
    Transgenic research 9 (2000), S. 405-415 
    ISSN: 1573-9368
    Keywords: Manihot esculenta ; transformation ; Agrobacterium tumefaciens ; mannose ; hygromycin ; selection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In order to improve the efficiency of cassava (Manihot esculenta Crantz) transformation, two different selection systems were assessed, a positive one based on the use of mannose as the selective agent, and a negative one based on hygromycin resistance encoded by an intron-containing hph gene. Transgenic plants selected on mannose or hygromycin were regenerated for the first time from embryogenic suspensions cocultivated with Agrobacterium. After the initial selection using mannose and hygromycin, 82.6% and 100% of the respective developing embryogenic callus lines were transgenic. A system allowing plant regeneration from only transgenic lines was designed by combining chemical selection with histochemical GUS assays. In total, 12 morphologically normal transgenic plant lines were produced, five using mannose and seven using hygromycin. The stable integration of the transgenes into the nuclear genome was verified using PCR and Southern analysis. RT-PCR and northern analyses confirmed the transgene expression in the regenerated plants. A rooting test on mannose containing medium was developed as an alternative to GUS assays in order to eliminate escapes from the positive selection system. Our results show that transgenic cassava plants can be obtained by using either antibiotic resistance genes that are not expressed in the micro-organisms or an antibiotic-free positive selection system.
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  • 22
    Electronic Resource
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    Springer
    Applied mathematics and mechanics 21 (2000), S. 1423-1431 
    ISSN: 1573-2754
    Keywords: nonlinear differential equations ; transformation ; algorithm ; analytical solution ; O175.29
    Source: Springer Online Journal Archives 1860-2000
    Topics: Mechanical Engineering, Materials Science, Production Engineering, Mining and Metallurgy, Traffic Engineering, Precision Mechanics , Mathematics , Physics
    Notes: Abstract The idea of AC=BD was applied to solve the nonlinear differential equations. Suppose that Au=0 is a given equation to be solved and Dv=0 is an equation to be easily solved. If the transformation u=Cv is obtained so that v satisfies Dv=0, then the solutions for Au=0 can be found. In order to illustrate this approach, several examples about the transformation C are given.
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  • 23
    ISSN: 1573-9368
    Keywords: inheritance ; Rubisco small subunit promoters ; sonication ; sulphur nutrition ; sunflower albumin ; transformation ; white clover
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract With the aim of increasing the rumen-protected level of the sulphur amino acids cysteine and methionine in Trifolium repens, we introduced the coding sequence of the sunflower seed albumin (SSA) into T. repens by Agrobacterium tumefaciens-mediated transformation. The SSA gene was modified such that the protein would be localised to the endoplasmic reticulum (ER). Four different T-DNA constructions all containing the SSA gene driven by either the promoter of a gene encoding the small subunit of ribulose bisphosphate carboxylase (Rubisco) from Arabidopsis thaliana (A ssu), the promoter of the gene encoding the small subunit of Rubisco of Medicago sativa (L ssu), or the Cauliflower Mosaic Virus 35S promoter (CaMV35S), were transferred to T. repens cv. Haifa. Transgenic T 0-plants and inter-transgenic hybrids were analysed for the level of SSA accumulation in the leaves by western blotting. The highest observed level of SSA accumulation was 0.1% of total extractable leaf protein. We observed that the promoter had a substantive effect on the level of SSA accumulation with A ssu〉CaMV35S〉L ssu. Results from the inter-transgenic hybrids showed that the capacity to synthesise SSA was inherited. However the level of SSA accumulation in the leaves generally appears not to be additive with extra transgenic loci. During this work, we attempted to improve the efficiency of A. tumefaciens-mediated transformation of T. repens using the SAAT-method (Sonication Assisted Agrobacterium-mediated Transformation) on cotyledons of T. repens. T-DNA transfer was in general not enhanced by sonication compared to traditional A. tumefaciens-mediated transformation. Furthermore, Southern blot analyses of plants regenerated from the same cotyledon after A. tumefaciens treatment and under selection, indicated that multiple shoots were usually derived from the same transformation event. We concluded from these results that only one plant from each A. tumefaciens-treated cotyledon should be taken to avoid transgenic clones.
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  • 24
    ISSN: 1573-9368
    Keywords: Hyoscyamus muticus ; particle bombardment ; transformation ; tropane alkaloids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We report an efficient whole plant transformation system for Hyoscyamus muticus, an important medicinal plant of the Solanaceous family. We developed a system using a plasmid carrying the nptII and gusA genes, which was delivered into leaf explants by particle bombardment. Ten percent of bombarded leaf explants formed kanamycin-resistant callus, from which putative transgenic plants were recovered. The nptII gene conferring kanamycin resistance was found to be incorporated into the genome of all transgenic plants screened. Over 50% of the kanamycin resistant plants showed strong expression of the non-selected gusA gene. The majority of transgenic plants reached maturity, could be self pollinated, and produced fertile seed. A simple and efficient whole plant transformation system for this medicinal plant is an important step in furthering our understanding of tropane alkaloid production in plants.
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  • 25
    ISSN: 1573-6776
    Keywords: casein hydrolysate ; Coleus forskohlii ; forskolin ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Casein hydrolysate at 2.0 g l−1 significantly enhanced forskolin content (2.3 mg g−1 cell dry wt) in a rhizogenic tumourous line, GCO-RCH-2 of Coleus forskohlii. In rooty teratoma line, RC-ST-2/4, forskolin content enhanced to 1.7 mg g−1 cell dry wt in presence of 2.5 g l−1 casein hydrolysate. Unlike untransformed calli and rhizogenic/root cultures, all the forskolin yielding transformed cultures of C. forskohlii have been maintained for over 5 years.
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  • 26
    ISSN: 1573-5028
    Keywords: Agrobacterium tumefaciens ; sorghum ; transformation ; transgenic plants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Agrobacterium tumefaciens was used to genetically transform sorghum. Immature embryos of a public (P898012) and a commercial line (PHI391) of sorghum were used as the target explants. The Agrobacterium strain used was LBA4404 carrying a `Super-binary' vector with a bar gene as a selectable marker for herbicide resistance in the plant cells. A series of parameter tests was used to establish a baseline for conditions to be used in stable transformation experiments. A number of different transformation conditions were tested and a total of 131 stably transformed events were produced from 6175 embryos in these two sorghum lines. Statistical analysis showed that the source of the embryos had a very significant impact on transformation efficiency, with field-grown embryos producing a higher transformation frequency than greenhouse-grown embryos. Southern blot analysis of DNA from leaf tissues of T0 plants confirmed the integration of the T-DNA into the sorghum genome. Mendelian segregation in the T1 generation was confirmed by herbicide resistance screening. This is the first report of successful use of Agrobacterium for production of stably transformed sorghum plants. The Agrobacterium method we used yields a higher frequency of stable transformation that other methods reported previously.
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  • 27
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    Journal of applied psychoanalytic studies 2 (2000), S. 109-115 
    ISSN: 1573-3459
    Keywords: James Joyce ; “The Dead” ; fiction ; reading ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Psychology
    Notes: Abstract The author offers a personal reading of James Joyce's “The Dead.” She focuses on how the sounds of the language are used to portray the main character's deadness and his beginning to come to life once the barriers to inner self and self-knowledge are broken.
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  • 28
    ISSN: 1573-4986
    Keywords: Cancer ; caveolae ; caveolin ; cholesterol ; glucosylceramide ; multidrug resistance ; rafts ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The carcinogenic process involves a complex series of genetic and biochemical changes that enables transformed cells to proliferate, migrate to secondary sites and, in some cases, acquire mechanisms that make cancer cells resistant to chemotherapy. This phenomenon in its most common form is known as multidrug resistance (MDR). It is usually mediated by overexpression of P-glycoprotein (P-gp) or other plasma membrane ATPases that export cytotoxic drugs used in chemotherapy, thereby reducing their efficacy. However, additional adaptive changes are likely to be required in order to confer a full MDR phenotype. Recent studies have shown that acquisition of MDR is accompanied by up-regulation of lipids and proteins that constitute lipid rafts and caveolar membranes, notably glucosylceramide and caveolin. These changes may be related to the fact that in MDR cells a significant fraction of cellular P-gp is associated with caveolin-rich membrane domains, they may be involved in drug transport and they could have an impact on drug-induced apoptosis and on the phenotypic transformation of MDR cancer cells.
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  • 29
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    Molecular breeding 6 (2000), S. 185-194 
    ISSN: 1572-9788
    Keywords: Agrobacterium ; transformation ; pea ; Pisum sativum L. ; PCR analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Factors influencing the efficiency of Agrobacterium-mediated transformation of pea were tested using highly efficient, direct regeneration system. The virulence of three Agrobacterium strains (octopine LBA 4404, nopaline C58C1 and succinamopine, hypervirulent EHA 105) clearly varied giving 1 transgenic plant per 100 explants for LBA 4404, 2.2 for C58C1 and 8.2 for EHA 105. To test the efficacy of selection agents we used the hypervirulent EHA 105 strain carrying pGPTV binary vector with one of four different selection genes: nptII, hpt, dhfr or bar. The mean number of transgenic, kanamycin-resistant plants for two cultivars tested was 4.2 per 100 explants and was slightly higher than the number of phosphinothricin-resistant plants (3.6 plants per 100 explants). The proportion of transgenics among kanamycin-selected plants was also higher than among phosphinothricin-resistant plants (35% and 28% respectively). There was no regeneration on hygromycin or methotrexate media (transformation with hpt and dhfr genes). Acetosyringone had no apparent influence on efficiency of transformation with hypervirulent EHA 105 strain, however it did affect the rate of transformation when moderately virulent C58C1 was used. Recovery of transgenic plants was enhanced after application of 5-azacytidine. The presence of integrated T-DNA was checked by PCR and confirmed by Southern hybridization. T-DNA was stably transmitted to the next generation.
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  • 30
    ISSN: 1572-9788
    Keywords: antisense DNA ; co-transformation ; nucleocapsid gene ; pathogen-derived resistance ; somatic embryogenesis ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Peanut (Arachis hypogaea L.) lines transgenic for the antisense nucleocapsid (N) gene of a Tomato spotted wilt virus (TSWV) strain isolated from peanut were generated by microprojectile-mediated transformation of repetitive somatic embryos of cultivars VC1 and AT120. The selectable marker (hygromycin resistance) and the N gene were on separate plasmids. A total of 207 VC1 and 120 AT120 hygromycin-resistant lines were produced. Of all the VC1 plants recovered 71% were cotransformed with the N gene (N+), but all plants were sterile. For AT120, 48 of the transgenic cell lines converted into plants. Polymerase chain reaction (PCR) screening showed 15 of the lines were transgenic for the N gene (N+), and two of these lines were fertile. A field test was conducted in 1998 at Ashburn, GA, using seeds from each fertile line, along with segregated and non-transgenic controls. Plants from four randomly selected field plots were examined for symptoms and analyzed by double-antibody sandwich enzyme-linked immunoabsorbent assay and PCR at 10 and 14 weeks after planting. At 14 weeks, 76% of the N+ plants were symptomless, while 2% were severely symptomatic or dead. In contrast, only 42% of the plants lacking the N gene were symptomless and 50% were severely symptomatic or dead. Northern blot analysis of selected field-resistant plants detected transgene RNA, and the transcript level appeared undiminished after viral exposure.
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  • 31
    ISSN: 1572-9788
    Keywords: bar ; cry3A ; Eucalyptus ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Transgenic Eucalyptus camaldulensis containing both the insecticidal cry3A gene and the bar gene (conferring tolerance to the herbicide glufosinate ammonium) have been produced by Agrobacterium tumefaciens-mediated transformation of seedling explants. Transgenic plants from two lines tested were resistant to first instars of chrysomelid beetles that are important pests of commercial Australian eucalypt plantations. Both lines also exhibit tolerance to the broad-spectrum herbicide Liberty® at 6 l/ha (1.2 kg active ingredient per hectare), twice the field application rate. Transgenic insect- and herbicide-resistant eucalypts like these are likely to provide better insect and weed control options in plantations, particularly during the vulnerable establishment phase, provided that any adverse ecological impacts of releasing transgenic trees into the environment can be assessed and minimized.
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  • 32
    ISSN: 1572-9788
    Keywords: choline oxidase ; glycinebetaine ; Japanese persimmon ; salt tolerance ; transformation ; woody plant
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract This report describes the first successful genetic engineering of tolerance to salt in an agriculturally important species of woody plants by Agrobacterium-mediated transformation with the codA gene of Arthrobacter globiformis. This gene encodes choline oxidase, which catalyzes the oxidation of choline to glycinebetaine. The binary plasmid vector pGC95.091, containing a kanamycin-resistance gene (nptII), a gene for β-glucuronidase (gusA) and the codA gene in its T-DNA region, was used with a disarmed strain of Agrobacterium tumefaciens, EHA101, to transform Japanese persimmon (Diospyros kaki Thunb. `Jiro') by the leaf disk transformation method. The pRS95.101 plasmid that included only nptII and gusA in the T-DNA region was used as a control. We selected eight transgenic lines with one or two copies of the T-DNA after transformation with pGC95.091 (PC lines) and three lines after transformation with pRS95.101 (PR lines). The eight PC lines produced choline oxidase and glycinebetaine whereas neither was found in untransformed `Jiro' and in the control PR lines. Transgenic plants grew normally, resembling wild-type plants both in vitro and ex vitro. The activity of photosystem II in leaves of the transgenic Japanese persimmon plants under NaCl stress was determined in terms of the ratio of the variable (F v) to the maximum (F m) fluorescence of chlorophyll (F v/F m). The rate of decline in (F v/F m under NaCl stress was lower in the PC lines than in the control PR lines. These results demonstrated that genetic engineering of Japanese persimmon, which allowed it to accumulate glycinebetaine, enhanced the tolerance to salt stress of this plant.
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  • 33
    ISSN: 1572-9788
    Keywords: heterologous protein accumulation ; phytate phosphorus digestibility ; phytase ; phytic acid ; transformation ; Triticum aestivum L.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The Aspergillus niger phytase-encoding gene (phyA) has been constitutively expressed in wheat. Transgenic wheat lines were generated by microprojectile bombardment of immature embryos, using the bar-Bialaphos selection system. The bar and the phyA gene expression were controlled by the maize ubiquitin-1 promoter. To ensure secretion and glycosylation of the microbial phytase, an expression cassette was designed (Ubi-SP-Phy) where an α-amylase signal peptide sequence was inserted between the promoter and the phytase coding region. A similar cassette was constructed without the signal peptide sequence (Ubi-Phy). Five lines of fertile wheat transformed with the Ubi-SP-Phy were generated and two lines with the Ubi-Phy construct. The inheritance of the phyA gene was monitored through three generations. Western blotting of leaf and seed derived protein revealed the presence of an immunoreacting polypeptide of the size expected for the Aspergillus phytase. Up to 25 days after pollination, the heterologous phytase was exclusively present in the pericarp-seed coat-aleurone fraction. Thereafter, it accumulated in the endosperm in amounts exceeding that found in the seed coat and aleurone. The phyA mRNA and derived protein could at no stage be detected in the embryo. The Ubi-SP-Phy transgenic seeds exhibited up to 4-fold increase of phytase activity while up to 56% increase was found in Ubi-Phy plants. It is concluded that a functional Aspergillus phytase can be produced in significant amounts in wheat grains. This may be of relevance for improving the phytate-phosphorus digestibility when wheat grains are used for non-ruminant animal feed.
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  • 34
    ISSN: 1572-9788
    Keywords: Choline oxidase ; glycinebetaine ; transformation ; Brassica juncea ; salt stress
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The codA gene for biosynthesis of glycinebetaine from Arthrobacter globiformis was used for transforming Brassica juncea cv. Pusa Jaikisan (which lack any means to synthesize glycinebetaine) through Agrobacterium mediated transformation. The stable insertion of the codA gene in the shoots obtained on medium with kanamycin and hygromycin was confirmed by PCR analysis of the nptII gene. Southern hybridization with a codA probe further demonstrated its successful integration. Immunoblot analysis revealed the presence of choline oxidase demonstrating that the bacterial codA gene had been successfully transcribed and translated. The seeds of transgenic lines showed enhanced capacity to germinate under salt stress as compared to that of the wild type. Further, the seedlings of transgenic plants that expressed codA gene showed significantly higher growth than that of the wild type under salt stress conditions. These results demonstrated that the introduction of a biosynthetic pathway for glycinebetaine into Brassica juncea significantly enhanced their salt tolerance. Hence, homozygous genotypes of selected transformed lines can be exploited for improving the salt tolerance of the desirable cultivars of Brassica juncea through breeding programmes.
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  • 35
    ISSN: 1573-9368
    Keywords: gene transfer ; selectable marker ; adenosine analogues ; transgenic maize ; adenosine deaminase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A murine adenosine deaminase (ADA) gene, driven by the maize ubi-1 promoter and intron region, was transformed into embryogenic maize callus, along with a bar and gusA gene-containing plasmid, using microparticle bombardment. Selection in the presence of either the herbicide Basta® or the adenosine analogue 2′-deoxyadenosine resulted in transgenic cultures that expressed GUS and accumulated a 41kD protein that immunoprecipated with an ADA-specific polyclonal antibody. ADA enzyme activity was observed in extracts from transgenic callus as well as regenerated plants and progeny. Cultures expressing ADA grew in the presence of 200mg/l 2′-deoxyadenosine, a concentration which completely inhibited the growth of non-transgenic cultures. ADA activity appeared to segregate in progeny of regenerated plants as a single, dominant Mendelian trait. These results suggest that ADA, in combination with adenosine analogue selection, represents a potentially viable selectable marker system for transgenic maize production.
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  • 36
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    Systemic practice and action research 13 (2000), S. 475-501 
    ISSN: 1573-9295
    Keywords: reflection ; transformation ; self-society dynamics ; critical systems thinking ; systemic intervention ; critical self-reflection ; ideology-critique ; critical appreciation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Sociology
    Notes: Abstract This paper sets out to uncover some relationships between reflection, discourse and action. By challenging and synthesizing some polemical arguments concerning the creation, maintenance, and transformation of self and society, a model of self-society dynamics that operates through reflection, discourse, and action is developed. The model of self-society dynamics brings together aspects of self-reflection and ideology-critique (explored in the paper), which it is suggested are required for any intervention (transformative action) to be grounded in locally meaningful ways. By creating a dialog community in which self- and group assumptions can be subjected to validation through discourse, it is proposed that a dynamic balance between individual needs and broader societal aims may be achieved. If individuals can be open to such discourse (i.e., they can become critically self-reflective), then it is argued that possibilities for achieving sustainable change will be significantly enhanced.
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  • 37
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    Journal of thermal analysis and calorimetry 56 (1999), S. 1133-1140 
    ISSN: 1572-8943
    Keywords: branched diamine ; melting ; polyamides ; polymorphism ; transformation ; WXRD
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Our X-ray work of Dytek®-A, 2-methyl-pentamethylenediamine, containing polyamides shows polymorphism, whereas the polyamides with linear diamines do not. The polyamide of Dytek®-A and dodecanedioic acid, MPMD-12, is singled out for discussion and compared with the unbranched analogs of polyamides 6,12 and 5,12. Due to the presence of the -CH3 side group in the 2-position of the diamine, the polyamide MPMD-12 exhibits two stable crystal conformations. The new δ polymorph is not seen in linear polyamides 6,12 and 5,12. Studies by DSC polyamide MPMD-12 clearly illustrates at least two crystal forms, γ and δ, coexisting over a wide temperature range, and the isolation of each phase is possible by controlling temperature and time. The DMA modulus in the temperature region between the glass transition (or alpha relaxation) and melting transition shows strong dependence on the thermal history as demonstrated in a study of crystallization kinetics.
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  • 38
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    Plant molecular biology reporter 17 (1999), S. 323-331 
    ISSN: 1572-9818
    Keywords: Agrobacterium ; modular vector ; transformation ; wheat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Wheat (cv Chinese Spring) tissues were transformed using Agrobacterium tumefasciens and a new plasmid modular vector, pMVTBP. We constructed pMVTBP with unique restriction sites connecting (1) the CaMV 35S promoter, (2) a Kozak sequence, (3) the FLAG epitope, (4) the (His)6 epitope, (5) a coding region (for wheat TATA Binding Protein, wTBP) and (6) the CaMV 35S 3′UTR. This vector thus allows easy exchange of different regulatory or coding sequences. Explants of either germinating mature seeds, or immature embryos, were induced to callus for up to two weeks, treated with virulence-induced bacteria for one hour, then regenerated into plantlets. Transient expression of a GUS reporter gene, assayed at about one week, occurred in 10–12% of calluses. Expression of the FLAG-tagged wTBP was also detected, by immunostaining. Stable expression, by selective growth on geneticin, and by GUS expression at about six weeks, occurred in 1–2% of calluses, quite comparable to that achieved by other methods.
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  • 39
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    GeoJournal 49 (1999), S. 43-51 
    ISSN: 1572-9893
    Keywords: housing market ; suburbanisation ; transformation ; urban development ; urban renewal ; East Germany
    Source: Springer Online Journal Archives 1860-2000
    Topics: Geography
    Notes: Abstract The aim of this paper is to analyse the main characteristics of post-socialist urban development in East Germany, especially the differences compared to urban development in other East and Central European countries. In spite of the many similar problems and processes in urban development, specific features of East Germany are characterised by the rapid growth of suburbia, especially in the first phase of transition, by the proceeding activities of urban renewal and revitalisation, and by a lower level of social polarisation and socio-spatial segregation as compared to other post-socialist countries. Important conditions for urban development in East Germany exist in special support programmes, high subsidies and other financial transfers as well as in engaged planning conceptions of the local authorities.
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  • 40
    ISSN: 1573-904X
    Keywords: gene transfer ; cytotoxicity ; polyethylenimine ; polyfection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. Low molecular weight branched polyethylenimine (LMW-PEI) was synthesized and studied as a DNA carrier for gene delivery with regard to physico-chemical properties, cytotoxicity, and transfection efficiency. Methods. The architecture of LMW-PEI, synthesized by acid catalyzed ring-opening polymerization of aziridine was characterized by size exclusion chromatography in combination with laser light scattering and 13C-NMR-spectroscopy. In vitro cytotoxic effects were quantified by LDH and MTT assay and visualized by transmission electron microscopy. The potential for transgene expression was monitored in ECV304 cells using luciferase driven by a SV40 promoter as reporter gene system. Results. LMW-PEI (Mw 11′900 D) with a low degree of branching was synthesized as a DNA carrier for gene delivery. In contrast to high molecular weight polyethylenimines (HMW-PEI; Mw l′616′OOO D), the polymer described here showed a different degree of branching and was less cytotoxic in a broad range of concentrations. As demonstrated by transmission electron microscopy the LMW-PEI formed only small aggregates which were efficiently taken up by different cells in the presence of serum, most likely by an endocytic pathway. LMW-PEI yielded transfection efficiencies measured via expression of the reporter gene luciferase which were up to two orders of magnitude higher than those obtained with HMW-PEI. The reporter gene expression was concentration dependent, but in contrast to lipofection independent of serum addition. Conclusions. The LMW-PEI described here is a new, highly efficient, and non-cytotoxic vector with a favorable efficiency/toxicity profile for gene therapeutic applications.
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  • 41
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    Cancer and metastasis reviews 18 (1999), S. 215-230 
    ISSN: 1573-7233
    Keywords: transformation ; tumour ; Frizzled ; Dishevelled ; glycogen synthase kinase-3β
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Wnt signalling is involved in a variety of mammalian developmental processes, including cell proliferation, differentiation and epithelial–mesenchymal interactions, through which they contribute to the development of tissues and organs such as the limbs, the brain, the reproductive tract and the kidney. Wnts are secreted ligands that control cell processes via at least two pathways, one of which, the ‘canonical’ Wnt signalling pathway, operates through the cytosolic stabilisation of a transcriptional co-factor, β-catenin. This is achieved by downregulating the activity of a β-catenin turnover complex. Evidence from tumour expression studies, transgenic animals and in vitro experiments suggests that inappropriate activation of the canonical Wnt signalling pathway is a major feature in human neoplasia and that oncogenic activation of this pathway can occur at many levels. Inappropriate expression of the Wnt ligand and Wnt binding proteins have been found in a variety of human tumours. Further downstream, dysregulation of the β-catenin turnover complex, by loss of the Adenomatous Polyposis Coli or Protein Phosphatase 2A proteins, or by activating mutations of β-catenin, has been found in several tumour types, and is believed to be a key step in neoplastic progression. Transcriptional targets of the Wnt pathway include the cellular oncogenes cyclin D1 and c-myc. Activation of the Wnt signalling pathway by various means can therefore be a primary cause in oncogenesis, affecting cell proliferation, morphology and contact inhibition, as well as co-operating with other oncogenes in multistep tumour progression.
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  • 42
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    Journal of assisted reproduction and genetics 16 (1999), S. 546-550 
    ISSN: 1573-7330
    Keywords: breast cancer ; mycoplasma ; Ureaplasma urealyticum ; foreign DNA ; gene transfer ; transgenic
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Purpose: Biological vectors for cell transfection are mainly viral in origin, with inherent shortcomings. Mycoplasmas are ubiquitous organisms that traverse cells easily. The objective was to determine if Ureaplasma urealyticum (T-mycoplasma) would vector exogenous BRCA1 DNA into blastocysts. Methods: Hatching mouse blastocysts (N = 70) were incubated in the presence of either viable or dead Ureaplasma urealyticum at 37°C for 1 hr. The blastocysts were exposed to human BRCA1 DNA lacking homology in the mouse genome for 2 hr, followed by DNase-I treatment and wash. Polymerase chain reaction and agarose gel electrophoresis analysis of amplified products were performed. Results: The BRCA1 gene was detected in the blastocysts only when viable Ureaplasma was present. PCR analyses of control Ureaplasma and untreated blastocysts were negative. Conclusion: Viable Ureaplasma organisms were shown to mediate the uptake of DNA fragments into blastocysts, resulting in transgenic mouse blastocysts with a normal human BRCA1 exon 11 gene.
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  • 43
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    European journal of plant pathology 105 (1999), S. 221-229 
    ISSN: 1573-8469
    Keywords: mutagenesis ; transformation ; plant disease ; recombination ; plant pathogenic fungi
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Development of molecular techniques for phytopathogenic fungi aims at the identification of fungal genes whose products are essential for successful infection of the host plant. Initial approaches have relied on isolating candidate genes and generating null-mutations by homologous recombination. Unfortunately, the results of this strategy have not been overly successful. This has led to a search for alternatives which allow an unbiased identification of pathogenicity genes. One method, which has proved successful in several systems, is a tagging mutagenesis procedure termed restriction enzyme mediated integration (REMI). In this mini-review we describe this procedure and review its features and results of its use when applied to the identification of fungal genes required for disease development in planta.
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  • 44
    ISSN: 1572-9729
    Keywords: abiotic ; biological ; cell-free extract ; chloroethane ; dechlorination ; 1,1-dichloroethane ; 1,1-dichloroethene ; digester ; methanogenic ; transformation ; 1,1,1-trichloroethane
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Energy, Environment Protection, Nuclear Power Engineering , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Anaerobic transformations of 1,1,1-trichloroethane (TCA), 1,1-dichloroethane (DCA), and chloroethane (CA) were studied with sludge from a lab-scale, municipal wastewater sludge digester. TCA was biologically transformed to DCA and CA and further to ethane by reductive dechlorination. TCA was also converted to acetic acid and 1,1-dichloroethene (11DCE) by cell-free extract. 11DCE was further biologically converted to ethene. This pathway was confirmed by transformation tests of TCA, DCA and CA, by tests with cell-free extract, and by chloride release during TCA degradation. With cell-free extract, acetic acid accounted for approximately 90% of the TCA transformed; tests with live cells indicate that the fraction of TCA transformed by this pathway decreased with lower biomass. The dechlorination of DCA to CA and CA to ethane was not stoichiometric. A high rate of TCA removal was observed under the experimental conditions. The results indicate that removal of TCA in anaerobic digestion should be complete, but DCA and CA could persist in a normally operating digester.
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  • 45
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    World journal of microbiology and biotechnology 15 (1999), S. 411-415 
    ISSN: 1573-0972
    Keywords: Electroporation ; Micrococcus species ; steroid biotransformation ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A steroid-biotransforming strain RJ6 was identified as Micrococcus roseus. This bacterium has a 10 kb plasmid pMQV10. Curing mediated through cultivation of the culture with a low concentration (200 ng/ml) of mitomycin C is described. Loss of cholesterol degradation (chol+) and streptomycin resistance (Smr) phenotypes as a consequence of the loss of plasmid indicate the extrachromosomal location of these two genes in this strain. An electroporation procedure was developed for transformation of cured strain of Micrococcus (RJC6) by plasmids. Frequency of greater than 105 transformants/μg DNA was achieved, which is 100-fold higher than the standard transformation procedure that yielded 5.3×103 transformants/μg DNA in the same strain.
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  • 46
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    World journal of microbiology and biotechnology 15 (1999), S. 1-6 
    ISSN: 1573-0972
    Keywords: Bacteria ; conjugation ; DNA ; evolution ; gene transfer ; transduction ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The transfer of genetic information by transformation, conjugation and transduction in bacteria occurs frequently in nature. These diverse gene transfer mechanisms in bacteria are the result of evolution and are not linked to reproduction as in eukaryotic organisms. In this review, gene transfer in bacteria will be considered from an evolutionary perspective.
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  • 47
    ISSN: 1573-5028
    Keywords: conditional lethal dominant gene ; Cre/loxP ; Nicotiana tabacum ; site-specific recombinase ; transformation ; transient expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transgenic tobacco plants were produced that contained single-copy pART54 T-DNA, with a 35S-uidA gene linked to loxP-flanked kanamycin resistance (nptII) and cytosine deaminase (codA) genes. Retransformation of these plants with pCre1 (containing 35S transcribed cre recombinase and hygromycin (hpt) resistance genes) resulted in excision of the loxP-flanked genes from the genome. Phenotypes of progeny from selfed-retransformed plants confirmed nptII and codA excision and integration of the cre-linked hpt gene. To avoid integration of the hpt gene, and thereby generate plants totally free of marker genes, we attempted to transiently express the cre recombinase. Agrobacterium tumefaciens (pCre1) was cocultivated with leaf discs of two pART54-transformed lines and shoots were regenerated in the absence of hygromycin selection. Nineteen of 773 (0.25%) shoots showed tolerance to 5-fluorocytosine (5-fc) which is converted to the toxic 5-fluorouracil by cytosine deaminase. 5-fc tolerance in six shoots was found to be due to excision of the loxP-flanked region of the pART54 T-DNA. In four of these shoots excision could be attributed to cre expression from integrated pCre1 T-DNA, whereas in two shoots excision appeared to be a consequence of transient cre expression from pCre1 T-DNA molecules which had been transferred to the plant cells but not integrated into the genome. The absence of selectable marker genes was confirmed by the phenotype of the T1 progeny. Therefore, through transient cre expression, marker-free transgenic plants were produced without sexual crossing. This approach could be applicable to the elimination of marker genes from transgenic crops which must be vegetatively propagated to maintain their elite genotype.
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  • 48
    ISSN: 1573-5044
    Keywords: ß-glucuronidase ; dendrobium ; hygromycin phosphotransferase ; orchid ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protocorms of orchid (Dendrobium hybrid) were transformed by microprojectile bombardment with a helium-pressured PDS 1000 particle gun. Gold particles coated with plasmid DNA containing ß-glucuronidase (GUS) and hygromycin phosphotransferase (Hpt) marker genes were used. Potentially transformed tissues were identified by active growth on MS medium supplemented with 50mg l-1 hygromycin. After 4–6 months of continuous selection, 15 hygromycin-resistant lines were recovered. Integration of transgenes into the genome of the transformed protocorms and plantlets were confirmed by GUS histochemical assay and Southern blot hybridization. The transgenic protocorms have gone through propagation for more than 8 months and maintained their transgenic characters. These results indicate that we have established a system for orchid transformation in a relatively high frequency and the transgenes are stably expressed in the transgenic plants.
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  • 49
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    Plant cell, tissue and organ culture 58 (1999), S. 171-176 
    ISSN: 1573-5044
    Keywords: Bacillus thuringiensis Brassicaceae ; gene transfer ; insect resistance ; plant regeneration ; Rorippa nasturtium-aquaticum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Adventitious shoot regeneration could be obtained from more than 80% of the calluses initiated from stem explants of watercress (Rorippa nasturtium-aquaticum) by using an induction medium and a shoot regeneration medium. The induction medium contained 1.15 μM 2,4-dichlorophenoxyacetic acid and 5 μM thidiazuron; the shoot regeneration medium was composed of 0.5 μM thidiazuron and 2.25 μM 6-benzylaminopurine. This regeneration procedure was incorporated into an Agrobacterium-mediated transformation procedure for gene transfer into watercress. Factors affecting transformation included preculture, selection agents, use of tobacco nurse cells, and the length of coculture. A transgenic line of watercress transformed with a wild-type Bacillus thuringiensis insecticidal gene, cry1Ia3, was not toxic to larvae of the diamondback moth (Plutella xylostella), presumably due to premature polyadenylation of the transcript encoded by this gene in the plant.
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  • 50
    ISSN: 1573-4935
    Keywords: Oligonucleotides ; gene transfer ; routing ; membrane lectins ; glycoconjugates
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Nucleic acids (plasmids as well as oligonucleotides) used to specifically express or modulate the expression of a gene, must reach the cytosol and/or the nucleus. Several systems have been developed to increase their uptake and their efficiency. Glycosylated polylysines have been shown to specifically help nucleic acids to be taken up in cells expressing a given cell surface membrane lectin. However, it appeared that the efficiency of the imported nucleic acid was not directly related to the extent of the uptake. Indeed, some glycosylated polylysines bearing sugar moities which are poor ligands of the cell surface lectins of a given cell were found to be more efficient than those bearing better sugar ligands. The interpretation of this paradoxal result is discussed with regards to the nature of the compartment allowing the nucleic acid to cross the membrane and to be delivered in the cytosol on the one hand, and to the presence of intracellular lectins on the other hand.
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  • 51
    ISSN: 1573-5079
    Keywords: Chlamydomonas reinhardtii ; LHC II phosphorylation ; mutagenesis ; Photosystem II redox control ; state 2 to state 1 transition ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Oxygenic photosynthetic organisms adapt to varying light conditions by changing the distribution of light energy between Photosystem II (PS II) and photosystem I (PS I) during so-called state transitions. To identify the genes involved in this process, we have exploited a simple chlorophyll fluorescence video-imaging technique to screen a library of nuclear mutants of Chlamydomonas reinhardtii for colonies grown on agar plates that are disturbed in their ability to regulate light energy distribution between PS I and PS II. Subsequent modulated fluorescence measurements at room temperature and 77 K fluorescence emission spectra confirmed that 5 mutants (0.025% of total number screened) were defective in state transitions. [32P]orthophosphate phosphorylation experiments in vivo revealed that in one of these mutants, designated stm1, the level of LHC II polypeptide phosphorylation was drastically reduced compared with wild type. Despite WT levels of PS I and PS II, stm1 grew photoautotrophically at reduced rates, compared with WT especially under low light conditions, which is consistent with an important physiological role for state transitions. Our results highlight the feasibility of video imaging in tandem with mutagenesis as a means of identifying the genes involved in controlling state transitions in eukaryotic photosynthetic organisms.
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  • 52
    ISSN: 1573-5028
    Keywords: cytochrome c biogenesis ; gene transfer ; mitochondria ; pea ; ribosomal protein ; soybean
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The pea mitochondrial genome contains a truncated rps7 gene lacking ca. 40 codons at its 5′ terminus. This single-copy sequence is immediately downstream of and slightly overlapping an actively transcribed and edited reading frame of 744 bp (designated ccb248) homologous to the bacterial helC gene which encodes a subunit of the ABC-type heme transporter involved in cytochrome c biogenesis. This region of mitochondrial DNA appears recombinogenic, and the carboxy-termini of helC-type proteins are predicted to vary in sequence and length among plants. Sequences corresponding to the 5′ coding region of rps7 were not detected elsewhere in the pea mitochondrial genome using wheat rps7 probes, and only a very short internal rps7 segment was observed in soybean mitochondrial DNA. The presence of rps7-homologous sequences in the nuclear genomes of pea and soybean is consistent with the recent transfer of a functional mitochondrial rps7 gene to the nucleus in certain plant lineages.
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  • 53
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    Plant molecular biology 40 (1999), S. 711-717 
    ISSN: 1573-5028
    Keywords: Agrobacterium tumefaciens ; Arabidopsis thaliana ; binary vector ; T-DNA ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A streamlined mini binary vector was constructed that is less than 1/2 the size of the pBIN19 backbone (3.5 kb). This was accomplished by eliminating over 5 kb of non-T-DNA sequences from the pBIN19 vector. The vector still retains all the essential elements required for a binary vector. These include a RK2 replication origin, the nptIII gene conferring kanamycin resistance in bacteria, both the right and left T-DNA borders, and a multiple cloning site (MCS) in between the T-DNA borders to facilitate cloning. Due to the reduced size, more unique restriction sites are available in the MCS, thus allowing more versatile cloning. Since the traF region was not included, it is not possible to mobilize this binary vector into Agrobacterium by triparental mating. This problem can be easily resolved by direct transformation. The mini binary vector has been demonstrated to successfully transform Arabidopsis plants. Based on this mini binary vector, a series of binary vectors were constructed for plant transformation.
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  • 54
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    Plant cell, tissue and organ culture 57 (1999), S. 207-210 
    ISSN: 1573-5044
    Keywords: biolistics ; gene expression ; haploid ; transformation ; Triticum aestivum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Using the PDS-1000/He Biolistic® Particle Delivery System, the microprojectile travel distance, rupture disk pressure and DNA/gold particle concentrations were assessed in order to optimise short and longer-term β-glucuronidase reporter gene expression in microspore-derived embryos of wheat. The effects were also evaluated of using sterile filter paper to support explants and treatment with a high osmoticum medium (0.2 M mannitol/0.2 M sorbitol or 0.4 M maltose). In the optimised procedure, wheat microspore-derived embryos (MDEs), were placed on filter paper and incubated on medium containing 0.4 M maltose, for 4 h pre- and 45 h post-bombardment. Five μl pAHC25 (0.75 mg ml-1 in TE buffer) was precipitated onto 25 μl gold particles (60 mg ml-1 in sterile water), using 20 μl spermidine (0.1 M) and 50 μl CaCl2 (2.5 M). The particles were centrifuged and resuspended in 75 μl absolute ethanol prior to the preparation of 6 macrocarriers. A microprojectile travel distance of 70 mm, a rupture pressure of 1300 p.s.i., and a vacuum of 29′′ Hg were employed. Maltose at 0.4 M in the support medium was the most important factor influencing GUS activity in bombarded tissues. GUS activity, 1 day post-bombardment, reached 52 ± 17 GUS-positive foci/MDE (mean ± s.e.m, n=3), with 17 ± 4 foci/MDE at 15 days, giving a 3.0-fold increase (p〈0.05) compared to expression in MDEs bombarded on medium without a high osmoticum treatment.
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  • 55
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    Water, air & soil pollution 110 (1999), S. 57-66 
    ISSN: 1573-2932
    Keywords: DDT ; kinetic ; organic pollutant ; sediment ; sorption ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Energy, Environment Protection, Nuclear Power Engineering
    Notes: Abstract The overall objective of this study was to investigate the sorption kinetics of DDT in sediment under similar experimental conditions employed in corresponding toxicity studies for bentic organisms. A batch of aerated Schoonrewoerdse Wiel sediment, initially spiked with DDT, was sampled over a period of seven days. Concentrations of DDT, DDD and DDE were determined in both the solid and the solution phase in the sediment/water system after separation by centrifugation. It was found that the extractable amount of DDT decreased with increasing contact time. This can partly be explained in terms of transformation of DDT into DDD. Furthermore, the present applied extraction procedure seems to be less effective with increasing contact time, indicating an increase in binding strength of DDT with the sediment material. Finally, on the basis of DDT, DDE and DDD concentrations in both the solid phase and the solution phase, partition coefficients were calculated, which appeared to be independent of the contact time. This points at a very rapid equilibrating between DDT in pore water and in the extractable forms adsorbed at the solid phase.
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  • 56
    ISSN: 1573-2932
    Keywords: atmospheric fate ; atmospheric transport ; deposition ; emission ; long-range transport ; pesticides ; registration ; remote area ; risk assessment ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Energy, Environment Protection, Nuclear Power Engineering
    Notes: Abstract The Health Council of the Netherlands organised an international workshop on the fate of pesticides in the atmosphere and possible approaches for their regulatory environmental risk assessment. Approximately forty experts discussed what is currently known about the atmospheric fate of pesticides and major gaps in our understanding were identified. They favoured a tiered approach for assessing the environmental risks of atmospheric dispersion of these chemicals. In the first tier a pesticide's potential for emission during application, as well as its volatilisation potential should be assessed. Estimates of the former should be based on the application method and the formulation, estimates of the latter on a compound's solubility in water, saturated vapour pressure and octanol/water partition coefficient. Where a pesticide's potential for becoming airborne exceeds critical values, it should be subjected to a more rigorous second tier evaluation which considers its toxicity to organisms in non-target areas. This evaluation can be achieved by calculating and comparing a predicted environmental concentration (PEC) and a predicted no-effect concentration (PNEC). By applying an extra uncertainty factor the PNEC can be provisionally derived from standard toxicity data that is already required for the registration of pesticides. Depending on the distance between the source and the reception area, the PEC can be estimated for remote areas using simple dispersion, trajectory type models and for nearby areas using common dispersion models and standard scenarios of pesticide use. A pesticide's atmospheric transport potential is based on factors such as its reaction rate with OH radicals. It should be used to discriminate between those compounds for which only the risks to nearby ecosystems have to be assessed, and those for which the risks to remote ecosystems also have to be determined. The participants were of the opinion that this approach is, in principle, scientifically feasible, although the remaining uncertainties are substantial. Further field and laboratory research is necessary to gain more reliable estimates of the physico-chemical properties of pesticides, to validate and improve environmental fate models and to validate the applicability of standard toxicity data. This will increase both the accuracy of and our confidence in the outcome of the risk assessment.
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  • 57
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    Higher education 38 (1999), S. 275-290 
    ISSN: 1573-174X
    Keywords: academic staff ; curriculum change ; equity ; governance ; staff development ; student needs ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Nature of Science, Research, Systems of Higher Education, Museum Science
    Notes: Abstract In South Africa the restructuring of the higher education system and the transformation of higher education institutions are located within the country's broad political and socio-economic transition to democracy. This paper focuses particularly on institutional transformation, and pays attention to the implications of the process of transformation for academic staff. The following five interlinked and interdependent issues characterizing institutional transformation in South African higher education are identified: democratising the governance structures of institutions increasing access for educationally and financially disadvantaged students restructuring the curriculum focusing on developmental needs in research and community service redressing inequalities in terms of race and gender. Although the overall effect of institutional transformation is experienced rather negatively by many academic staff members, the paper concludes that academics have to be empowered by means of staff development to remain active partners in the transformation process.
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  • 58
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    Journal of engineering mathematics 36 (1999), S. 241-254 
    ISSN: 1573-2703
    Keywords: flows in porous media ; transformation ; heat transfer ; drying bins ; conformal mapping.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Mathematics , Technology
    Notes: Abstract The design of a drying or cooling store aims to provide an even airflow distribution, when aerated, for preservation purposes. The airflow in some curved bottom bins are studied in this paper. The flow is modelled, using Darcy's law. A generalized Schwarz-Christoffel transformation is employed to reduce the problem of computing streamlines and isobars of airflow to solving a single nonlinear equation for the flow angle along the wall. Corresponding to different bin shapes, a few computed streamlines and isobars of airflow are presented, showing the effect of changing bottom geometries on the air flow. Heat transfer in such bins is also investigated. Based on an analysis of the far field of airflow, finite-height bins are considered. Analytical solutions of the heat conduction equation in terms of streamlines and isobars are obtained.
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  • 59
    ISSN: 1573-2932
    Keywords: arid-zone soils ; field capacity ; fractionation ; heavy metals ; kinetics ; redistribution ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Energy, Environment Protection, Nuclear Power Engineering
    Notes: Abstract Solid-phase transformation of added Cd, Cu, Cr, Ni, Pb and Zn, in two arid-zone soils incubated in the field capacity moisture regime for one year, were studied. The heavy metals were fractionated into six empirically defined fractions using a selective sequential dissolution (SSD) protocol optimized for arid-zone soils. Each of these fractions was named based on the major soil component targeted for dissolution during the specific SSD step, but it is not assumed that they are mineralogically and chemically totally specific. The transformations of the metals in the two soils incubated at the field capacity regime were compared with those at the moisture saturation regime (Han and Banin, 1997). An initial fast stage of transformation of the soluble metals from the exchangeable (EXC) fraction to the less labile fractions (the carbonate (CARB) fraction for Cd, Pb, Zn, Ni and Cu, and the organic matter (OM) fraction for Cr, and to some extent Cu and Ni) occurred during the fractionation and within one hour after addition. This was followed by a second stage, involving long-term transformation processes of all metals: added Cd was transferred from the EXC into the CARB fraction; added Cr was transferred from the CARB to the OM fraction and Pb was transferred very slowly to the easily reducible oxide (ERO) fraction. Added Cu, Ni and Zn were transferred from the EXC and CARB fractions into the ERO fraction and to some extent OM and RO fractions. In Part I of this series, we reported that during incubation in the saturated moisture regime, Zn and Ni were transferred mainly into the RO and OM fractions. Cadmium, Cr and Pb underwent the same transformation pathways during the slow long-term process, with slightly different rates, in both water regimes. At low levels of addition, the incubated soils moved over one year towards a distribution similar to that of the native soil. At higher levels, the soils still remained removed from the quasi-equilibrium which characterized the native soil, even at the end of one year of incubation.
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  • 60
    ISSN: 1573-7373
    Keywords: adenovirus ; dominant negative ; fibroblast growth factor receptor ; gene transfer ; glioma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Basic fibroblast growth factor (FGF-2) and high affinity FGF receptor (FGFR) have been detected in the nucleus as well as the cytoplasm of many human gliomas, and are known to stimulate cellular proliferation and angiogenesis in the tumors. To investigate the effects of inactivation of FGFR on the growth of malignant gliomas, we constructed a replication-deficient recombinant adenovirus vector encoding a truncated form of chicken FGFR1 (AxCA Δ FR). AxCA Δ FR-infected cells were confirmed to express truncated FGFR protein by immunoblotting and FGF-2-dependent clonogenicity of NIH3T3 cells was suppressed by infection with this virus vector. Then human malignant glioma cell lines U-251MG and T98G, both of which have been reported to express FGF-2 and FGFR, were infected with AxCA Δ FR. These infected cells showed nuclear as well as cytoplasmic expression of a truncated FGFR protein. Proliferation rate and the ability to form colonies in soft agar of the cells infected with this virus vector were significantly suppressed compared with those of uninfected and lacZ-expressing adenovirus-infected cells. Moreover, intratumoral injection of AxCA Δ FR significantly suppressed the subcutaneous tumor growth of the glioma cells in nude mice. We concluded that inactivation of the cytoplasmic and nuclear FGFR using this truncated FGFR-expressing adenovirus vector can inhibit the growth of malignant gliomas both in vitro and in vivo.
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  • 61
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    Computer supported cooperative work 8 (1999), S. 63-93 
    ISSN: 1573-7551
    Keywords: activity theory ; action ; transformation ; expansive learning ; intervention ; visibilization ; health care ; medical records
    Source: Springer Online Journal Archives 1860-2000
    Topics: Computer Science
    Notes: Abstract Work is commonly made visible along two dimensions: the linear and the socio-spatial. Both are limited to depicting work in terms of relatively discrete actions. Activity theory introduces the crucial distinction between collective activity systems and individual actions. Expansive visibilization of collective activity systems offers a powerful intervention methodology for dealing with major transformations of work. The linear and the socio-spatial dimensions of work actions are seen in the broader perspective of a third, developmental dimension of work activity. Four steps are identified in a cycle of expansive visibilization, combining activity-level visions and action-level concretizations. The cycle is examined in detail as it unfolded in an intervention study at a children's hospital in Finland. It is concluded that expansive visibilization, driven by contradictions and seeking to reconceptualize the object and motive of work, is not a straightforward process which can be neatly controlled from above. Coherent analytical explanation and goal-setting may come only after the creation and practical implementation of innovative solutions.
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  • 62
    ISSN: 1573-0778
    Keywords: culture ; dog ; Duchenne dystrophy ; gene transfer ; satellite cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We have developed and characterized cultures of healthy and dystrophic canine myoblasts for the evaluation of various gene transfer protocols. The number of desmin-positive myoblasts was elevated (〉〉80%) in cultures of myoblasts obtained from different muscle territories, the diaphragm muscle giving rise to the purest cultures. Myoblasts from dogs turned out to be a very convenient source of well transfectable and transducible cells. Transfection with plasmid DNA allowed efficient transgene expression (50% of β-galactosidase positive cells and about 375 ng luciferase/mg protein after transfection with a calcium phosphate-precipitated plasmid). Infection with high concentrations of adenoviral and retroviral vectors allowed transgene (β-galactosidase or mini-dystrophin) detection in about 75 to 90% of the canine cells. Therefore, primary dog myoblast cultures represent a useful in vitro model for viral and non-viral gene delivery, as well as for functional evaluation and cell grafting with applications in genetic diseases, vaccination or production of circulating therapeutic proteins.
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  • 63
    ISSN: 1572-9788
    Keywords: alternative oxidase ; antisense ; male-sterility ; tapetum-specific promoter ; tobacco ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The alternative oxidase of plant mitochondria is the terminal oxidase of the cyanide-insensitive respiratory pathway and is encoded by a nuclear gene. A 1 kb genomic fragment including exon 3 of the alternative oxidase was amplified by PCR from the genome of Arabidopsis thaliana. This fragment was connected to a tapetum-specific promoter in the antisense orientation and then introduced into tobacco. The pollen viability in three transgenic plants ranged from 2% to 60%. The reduced pollen viability cosegregated with the transgene in a selfed progeny. Immunolocalization of alternative oxidase protein in the immature flower bud section indicated that expression of alternative oxidase protein in tapetum of the transgenic plant was much lower than that of the non-transformant. The histological observation and protein gel-blot analysis showed that the development of pollen grains in the transgenic plant did not progress after the degradation of the tapetum, and the amount of alternative oxidase in pollen grains of the transgenic plant became lower than that of the non-transformant. These results suggested that the alternative oxidase activity in the tapetum has a significant effect on the pollen development.
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  • 64
    ISSN: 1572-9788
    Keywords: Cyamopsis tetragonoloba ; β-lactamase inhibitor ; sulbactam ; transformation ; transgene stability ; transgenic guar
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract A procedure for transformation of the large-seeded endospermous legume guar (Cyamopsis tetragonoloba L.) and a study on transmission of the transgenes to offspring generations are presented. Using Agrobacterium tumefaciens with a T-DNA construct harbouring a β-glucuronidase gene (uidA) and a neomycin phosphotransferase gene (nptII), maximum transformation frequencies of cotyledonary explants were obtained using 145 mg/l kanamycin sulfate as selective agent. Carbenicillin and cefotaxime, used for the elimination of Agrobacterium after co-culture, displayed considerable toxicity to guar tissues but replacing most of these β-lactams by the non-phytotoxic β-lactamase inhibitor sulbactam as well as addition of thidiazuron and silver thiosulfate increased transformation frequencies up to 10-fold in total. The presence of the transgenes in the primary transformants was demonstrated by genomic DNA analysis of GUS-positive shoots. Chimaeric plants (5–10%) were identified by GUS analysis at the flowering stage and were discarded. Analysis of the R1 offspring from 17 independent transformants showed that in 41% of those, the uidA gene(s) was expressed and stably inherited consistent with Mendelian genetics. This was also found for the R2 and R3 generations of single copy transformants. On the other hand, a large proportion (47%) of the primary transformants gave R1 offspring in which 100% of the plants were GUS-negative. Analysis of these plants by PCR revealed that, at least, most of the transgene sequences were absent, suggesting that they had not been transmitted from the parent transformants. This occurred at similar high frequencies (40–50%) irrespective of the estimated copy number of the transgenes. Thus, major parts of the transgenes, even when present in multiple copies, displayed aberrant transmission, at a high frequency, in the process of going from the primary transformants to the first offspring generation.
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  • 65
    ISSN: 1573-9368
    Keywords: Lactuca sativa ; Agrobacterium tumefaciens ; bialaphos ; phosphinothricin acetyltransferase ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Resistance to bialaphos, a broad-spectrum herbicide, was introduced into Lactuca sativa cv. Evola by Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strains 0310 and 1310, both carrying the bialaphos resistance (bar) and neomycin phosphotransferase (nptII) genes, were used for transformation. Primary transformants were selected on kanamycin sulphate-supplemented shoot regeneration medium. Integration of both transgenes was confirmed by non-radioactive Southern hybridisation. The hypervirulent plasmid ToK47 in A. tumefaciens strain 1310 generated multiple insertions of T-DNA in some transgenic plants; the absence of pToK47 (strain 0310) resulted in single gene inserts in all plants tested. Resistance to glufosinate ammonium was observed in axenic seedlings grown on medium supplemented with the herbicide at 5 mg l−1 and in glasshouse-grown plants sprayed with the compound at 300 mg l−1. Stable expression of the bar gene was observed in R2 generation plants. The kanamycin resistance of R1 seedlings was observed by germinating seeds on medium supplemented with 200 mg l−1 kanamycin sulphate. The presence of NPTII protein and PAT enzyme activity were demonstrated by ELISA and PAT enzyme assay respectively. Transgenes segregated in a Mendelian fashion in some plant lines in the R1 generation; herbicide resistance also segregated in the expected ratio in the R2 generation in most transgenic lines. This study confirmed that an agronomically important transgene can be integrated and stably expressed over several generations in lettuce.
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  • 66
    ISSN: 1573-9368
    Keywords: Agrobacterium tumefaciens ; ß‐glucuronidase ; lamiaceae ; lavandin ; neomycin phosphotransferase II ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Lavandin (Lavandula x Emeric ex Loiseleur) is an aromatic plant, the essential oil of which is widely used in the perfume, cosmetic, flavouring and pharmaceutical industries. The qualitative or quantitative modification of its terpenes‐containing essential oil by genetic engineering could have important scientific and commercial applications. In this study, we report the first Agrobacterium tumefaciens‐mediated gene transfer into lavandin. The transformation protocol was optimized by lengthening precultivation and cocultivation periods and by testing five different bacterial strains. We obtained transformed callus lines at a frequency of 40–70 with strains AGL1/GI, EHA105/GI and C58/GI. Transgenic shoots were regenerated from these kanamycin resistant calli and rooted on selective medium with 150 mg l-1 kanamycin. The final percentage of transgenic plants obtained varied from 3 to 9, according to the strain used, within 6 months of culture. The presence of the introduced β‐glucuronidase and neomycin phosphotransferase II genes was shown both by PCR and Southern blot analysis. Transgene expression was investigated using histoenzymatic β‐glucuronidase assays, leaf callus assays and RT‐PCR. Results showed that both β‐glucuronidase and neomycin phosphotransferase II genes were expressed at a high level in at least 41 of the transgenic plants regenerated. This efficient transformation strategy could be used to modify some genetic traits of lavandin (flower colour, pathogens resistance) and to study the biosynthesis of the major monoterpene components of its essential oil (linalool, linalyl acetate, camphor and 1,8‐cineole).
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  • 67
    ISSN: 1573-6784
    Keywords: Bacillus ; plasmids ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A simple and easy method for the introduction of plasmid DNA into different species of Bacillus was developed. The method involves the suspension in a transformation buffer of nutrient agar grown cells in their late exponential phase and the addition of unpurified plasmid DNA. Transformants were obtained at a frequency of about 103 to 105 stable transformants per μg of plasmid DNA.
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  • 68
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    Cell biology and toxicology 15 (1999), S. 193-202 
    ISSN: 1573-6822
    Keywords: DNA ; gene transfer ; importin ; nuclear import ; nuclear localization signal
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract One of the major steps limiting nonviral gene transfer efficiency is the entry of plasmid DNA from the cytoplasm into the nucleus of the transfected cells. The nuclear localization signal (NLS) of the SV40 large T antigen is known to efficiently induce nuclear targeting of proteins. We have developed two chemical strategies for covalent coupling of NLS peptides to plasmid DNA. One method involves a site-specific labeling of plasmid DNA by formation of a triple helix with an oligonucleotide–NLS peptide conjugate. After such modification with one NLS peptide per plasmid molecule, plasmid DNA remained fully active in cationic lipid-mediated transfection. In the other method, we randomly coupled 5–115 p-azidotetrafluorobenzyllissamine–NLS peptide molecules per plasmid DNA by photoactivation. Oligonucleotide–NLS and plasmid–lissamine–NLS conjugates interacted specifically with the NLS-receptor importin α. Plasmid–lissamine–NLS conjugates were not detected in the nucleus, after cytoplasmic microinjection. Plasmids did not diffuse from the site of injection and plasmid–lissamine–NLS conjugates appeared to be progressively degraded in the cytoplasm. The process of plasmid DNA sequestration/degradation stressed in this study might be as important in limiting the efficiency of nonviral gene transfer as the generally recognized entry step of plasmid DNA from the cytoplasm into the nucleus
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  • 69
    ISSN: 1573-5028
    Keywords: constitutive expression ; GFP ; GUS ; Musa ; ScBV ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A 1369 bp DNA fragment (Sc) was isolated from a full-length clone of sugarcane bacilliform badnavirus (ScBV) and was shown to have promoter activity in transient expression assays using monocot (banana, maize, millet and sorghum) and dicot plant species (tobacco, sunflower, canola and Nicotiana benthamiana). This promoter was also tested for stable expression in transgenic banana and tobacco plants. These experiments showed that this promoter could drive high-level expression of the β-glucuronidase (GUS) reporter gene in most plant cells. The expression level was comparable to the maize ubiquitin promoter in standardised transient assays in maize. In transgenic banana plants the expression levels were variable for different transgenic lines but was generally comparable with the activities of both the maize ubiquitin promoter and the enhanced cauliflower mosaic virus (CaMV) 35S promoter. The Sc promoter appears to express in a near-constitutive manner in transgenic banana and tobacco plants. The promoter from sugarcane bacilliform virus represents a useful tool for the high-level expression of foreign genes in both monocot and dicot transgenic plants that could be used similarly to the CaMV 35S or maize polyubiquitin promoter.
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  • 70
    ISSN: 1573-8221
    Keywords: human papilloma virus ; transformation ; actin ; fibronectin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Transfection of rat embryonic fibroblasts with E7 gene of type 16 human papilloma virus changed the cytoskeleton and cell-cell and cell-matrix interactions in two clones of transformed cells. Cell morphology and substrate-dependent proliferation were also changed.
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  • 71
    ISSN: 1572-9788
    Keywords: Triticum turgidum L. var. durum ; pasta wheat ; transformation ; seed protein modification ; flour quality improvement
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Particle bombardment has been used to transform three cultivars (L35, Ofanto, Svevo) and one breeding line (Latino × Lira) of durum wheat (Triticum turgidum L. var. durum). These varieties were co-transformed with plasmids containing selectable and scorable marker genes (bar and uidA) and plasmids containing one of two high-molecular-weight (HMW) glutenin subunit genes (encoding subunits 1Ax1 or 1Dx5). Ten independent transgenic lines were recovered from 1683 bombarded scutella (transformation efficiency thus 0.6%). Five lines expressed either subunit 1Dx5 or 1Ax1 at levels similar to those of endogenous subunits encoded on chromosome 1B. To identify the effects of the transgenes on the functional properties of grain, three lines showing segregation for transgene expression were used to isolate sibling T2 plants which were null or positive for the transgene product. Analysis of these plants using a small-scale mixograph showed that expression of the additional subunits resulted in increased dough strength and stability, demonstrating that transformation can be used to modify the quality of durum wheat for bread and pasta making.
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  • 72
    ISSN: 1573-9368
    Keywords: gene construct ; gene transfer ; heritability ; marker gene ; pigmentation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transgenic mice provide a valuable tool in all fields of basic and applied biological and medical research. In this study, we describe the fate of integrated transgenes in the mammalian host genome over a large number of generations. The stability of the germ-line transmission of integrated tyrosinase transgene copies was monitored up to generation F20 in a large number of individuals from seven transgenic mouse lines. Phenotypic and molecular genetic analysis of the offspring both within the different lines and in cross-breeding experiments revealed the high stability of the transgene integration sites in mice. Only very few individuals were affected by a transgene copy loss. These results indicate that, once homozygous transgenic lines are established, breeding programs can be continued to a high number of generations without further stringent molecular genetic analysis.
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  • 73
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    Diabetologia 41 (1998), S. 1401-1409 
    ISSN: 1432-0428
    Keywords: Keywords Beta-cell lines ; conditional transformation ; gene transfer ; glucose-regulated promoters ; immunomodulation ; insulin biosynthesis ; insulin secretion ; islet regeneration ; proinsulin processing ; transplantation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The application of gene therapy to Type I (insulin-dependent) diabetes mellitus awaits improvements in gene transfer technologies and the development of better tools for accurate diagnosis of pre-diabetic people. Identification of the most promising candidate genes for gene transfer requires further elucidation of the molecular events involved in beta-cell autoimmune destruction, islet ontogeny and differentiation, and beta-cell function. This review outlines a number of possible targets for gene therapy in Type I diabetes, which could help prevent the autoimmune damage to islets, induce islet regeneration, and restore insulin production through engineering of self non-beta cells or beta-cell transplantation. It also evaluates their potential merits and drawbacks. [Diabetologia (1998) 41: 1401–1409]
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  • 74
    ISSN: 1432-0428
    Keywords: Keywords Lentiviral vector ; retrovirus ; human islet beta-cell ; gene transfer ; transplantation.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Pancreatic islet cells are terminally differentiated endocrine cells and are refractory to stable infection by retroviral vectors, which require the breakdown of the nuclear membrane during cell division in order to insert the transgene into the host cell genome. Thus, attempts to render beta-cell allografts less immunogenic have had to rely on stable transfection of surrogate cells. Similarly, this problem has precluded the development of conditionally immortalized human beta cells for clinical allotransplantation. In this report, we demonstrate that adult human islet beta cells can be transduced by a new three-plasmid integrating lentiviral vector with an efficiency of 62 ± 1.8 % at a multiplicity of infection (MOI) of 2.5 in vitro. This work makes genetic engineering of adult human pancreatic beta cells possible for the first time, allowing strategies to render beta-cell allografts non-immunogenic to be optimized and to creating conditionally immortalized human beta cells for clinical transplantation. [Diabetalogia (1998) 41: 736–739]
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  • 75
    ISSN: 1569-8041
    Keywords: cytotoxic T lymphocyte (CTL) ; dendritic cell ; Epstein-Barr virus (EBV) ; EBV-associated lymphoproliferative disease (EBV-LPD) ; gene-marking ; gene transfer ; Hodgkin's disease ; immunotherapy ; latent membrane protein 2 (LMP2)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Donor-derived Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) are successful in the prevention and treatment of Epstein-Barr virus (EBV)-associated lymphoproliferative disease (LPD) in allogeneic bone marrow transplant (BMT) recipients [1, 2]. This finding prompted us to use a similar approach to the treatment of relapsed EBV-positive Hodgkin's disease [3]. Autologous EBV-specific CTL lines could be generated on the first or second attempt from 11 of 15 patients with Hodgkin's disease. Peripheral blood TCR ζ-chain levels were low, but increased in the activated CTL lines. Three patients have received gene-marked autologous CTL. The first two patients experienced alleviation of stage B symptoms and a drop in peripheral blood EBV load. However, this situation reversed between 6 and 12 weeks after infusion, when chemotherapy and radiation were reinstated. Both patients eventually progressed and died. The third patient had a pleural effusion, which increased after CTL infusion. Analysis of the pleural effusion revealed both tumor cells and levels of marker gene over 100 fold greater than in peripheral blood. The infused CTL line showed activity against LMP2. The patient initially improved and then remained stable for over eight months after CTL infusion, but now has progressive disease. We currently are evaluating methods for introducing the LMP2 gene into dendritic cells and using these to present LMP2 to autologous T cells. Using both retrovirus and herpesvirus vectors to express LMP2 in dendritic cells, LMP2-specific CTL were successfully generated from individuals who were EBV-seronegative or who were non-responsive to LMP2 when presented on autologous LCL. In future protocols, LMP2-specific CTL will be used for treatment.
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  • 76
    ISSN: 1572-994X
    Keywords: xenotropic endogenous MuLV ; gene transfer ; Bxv-1
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Development of methods for gene transfer into specific cell types or tissues is important for experimental research as well as clinical therapeutical approaches. We report here the cloning and characterization of the envelope (env) gene and the U3 region of a retrovirus from an infected human Small Cell Lung Cancer (SCLC) cell line. The replication of this murine retrovirus is also fully supported by other lung cancer cell lines of different histological origin. We present evidence that a long terminal repeat (LTR)-β-galactosidase (β-Gal) reporter construct performed as well as an analogous cytomegalovirus (CMV) promoter β-Gal construct in the human lung epithelial cell line A549 and in the human larynx carcinoma cell line HEp2.
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  • 77
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    Plant molecular biology reporter 16 (1998), S. 129-131 
    ISSN: 1572-9818
    Keywords: Agrobacterium tumefaciens ; binary vector ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We report the construction of a binary vector for Agrobacterium tumefaciens-mediated transformation, pBIN20, which contains a superlinker region located between the left and right Ti border sequences. This vector, derived from pBI121, simplifies the cloning of plant expression cassettes and has been used in our laboratory to create lines of transgenic BY-2 tobacco cells. This new vector contains more than 20 unique restriction sites as well as the nptII selectable marker gene within the Ti-DNA borders.
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  • 78
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    GeoJournal 44 (1998), S. 215-224 
    ISSN: 1572-9893
    Keywords: cross-border region ; transformation ; regional economic development ; Poland ; Germany
    Source: Springer Online Journal Archives 1860-2000
    Topics: Geography
    Notes: Abstract One of the major means to foster European integration is the establishment of border spanning regions (‘Euroregions’). This is particularly important on the Eastern borders of the EU, e.g. in Eastern Germany. There, however, a double transformation to post-socialist society is taking place, both inside and outside the EU. Tensions arise between objectives on local and higher political levels, intensified by totally different economic structures and access to EU funds on both sides of the border. This is particularly true for the case of the emerging Euroregion Viadrina. Problems in preserving old industrialised localities in East Germany (e.g. steel) and attempts to resurrect the urban fair place Frankfurt/Oder, clash with transition in agriculture and consumer industries and with new concepts in tourism development and environmental protection in the Polish border zone. In region building, political, economic and ideological goals compete with each other. Local initiatives and higher political governance may both support and hamper each other. The same holds true for the interdependence of cultural integration and economic development. The paper concludes that regional economic development can only be expected if, via the building of the Euroregion, the interplay of these factors leads to compromise and harmonization between the different parties involved.
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  • 79
    ISSN: 1572-9893
    Keywords: chamber of commerce ; economy ; experts ; institutions ; local government ; Romania ; rural ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Geography
    Notes: Abstract This study evaluates some aspects of the socio-economic transformation of rural Romania with reference to the views of representative organisations (at national, regional and local levels) and other experts. Interviews conducted in ten communes of nine Romanian counties (‘judete’) focus attention on the advantages and disadvantages of system change experienced since 1989; the most important problems and constraints for future socio-economic change; and appropriate policies and perspectives for development in the immediate future. Wherever appropriate the claims of interviewees are substantiated through reference to statistics, drawn in many cases from Chambers of Commerce & Industry (CCI). Local level representatives presented much more negative views on recent change than their national and regional level counterparts, but all agreed on the crucial problem of capital shortage. Thus while specific programmes to assist rural areas are justified, they cannot fully succeed until the national economy is able to grow more rapidly and attract greater foreign investment.
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  • 80
    ISSN: 1572-9893
    Keywords: agriculture ; border region ; communities ; commuting ; conservation ; Romania ; tourism ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Geography
    Notes: Abstract A most distinctive feature of the settlement pattern of the Brasov area is the extreme dispersal of mixed farming encountered in the western extreme of the county to the north and south of Zarnesti: the Bran and Poiana Marului areas. Here a system of peasant subsistence farming developed in a political borderland between the Habsburg and Ottoman Empires. Despite feudal pressures, the peasantry took all available opportunities to extend their independence including elaborate transhumance systems. And after seeing transfrontier commerce as a source of plunder, in the tradition of Balkan highway robbery within relatively unregulated spaces, the peasantry has profited through employment in factories, particularly during the communist period. However, the current recession in manufacturing is throwing the rural population back on limited land resources. Although farming assumes an important subsistence role which contributes to stability, the long-term survival of these communities will depend on new sources of income. Rural tourism has considerable potential and a promising start has been made in Bran. There are, however, constraints on the further development of the business and great attention will have to be given to the conservation of the environment in both the Bucegi Mountains and the Piatra Craiului where national park status is proposed.
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  • 81
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    GeoJournal 46 (1998), S. 263-269 
    ISSN: 1572-9893
    Keywords: Croatia ; decentralisation ; diversification ; industry ; innovation ; rural ; transformation ; urbanisation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Geography
    Notes: Abstract Rural diversification in Croatia is well advanced because many rural families have been able to find work in secondary and tertiary activities without the need to migrate to the towns. Many rural settlements have now attained an urban character although there are regional variations, including a contrast between the continental zone with a relatively high level of commitment to agriculture and the coastal areas, with pronounced ‘deagrarisation’ where the ports and tourist resorts are well developed and the natural resource conditions for agriculture are poor. These variations are examined at the municipality level with reference to two key indicators: the share of nonagricultural population and the share of workers in the total active population. Four categories of socio-economic transformation are recognised: more urbanised, urbanised, less urbanised and rural. The main regional differences between the continental and coastal areas are confirmed with the latter showing a relatively high level of socio-economic transformation through the prominence of more highly urbanised municipalities.
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  • 82
    ISSN: 1573-904X
    Keywords: gene therapy ; gene transfer ; cationic polymer ; chitosan ; polyethylenimine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. Chitosan, a natural cationic polysaccharide, is a candidate non-viral vector for gene delivery. With the aim of developing this system, various biophysical characteristics of chitosan-condensed DNA complexes were measured, and transfections were performed. Methods. Transmission electronic microscopy (TEM) visualizations, sedimentation experiments, dynamic light scattering (DLS), and zeta potential measurements were realized. Transfections were made by using the luciferase reporter gene. Results. In defined conditions, plasmid DNA formulated with chitosan produced homogenous populations of complexes which were stable and had a diameter of approximately 50−100 nm. Discrete particles of nicely condensed DNA had a donut, rod, or even pretzel shape. Chitosan/DNA complexes efficiently transfected HeLa cells, independently of the presence of 10% serum, and did not require an added endosomolytic agent. In addition, gene expression gradually increased over time, from 24 to 96 hours, whereas in the same conditions the efficacy of polyethylenimine-mediated transfection dropped by two orders of magnitude. At 96 hours, chitosan was found to be 10 times more efficient than PEI. However, chitosan-mediated transfection depended on the cell type. This dependency is discussed here. Conclusions. Chitosan presents some characteristics favorable for gene delivery, such as the ability to condense DNA and form small discrete particles in defined conditions.
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  • 83
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    Neurochemical research 23 (1998), S. 421-426 
    ISSN: 1573-6903
    Keywords: Oligodendrocytes ; electroporation ; gene transfer ; transfection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The transient transfection of transgenes into oligodendrocytes offers an important tool for studying the function of proteins during myelin formation. Currently established procedures, however, have generally resulted in low survival rates and low levels of uptake of the transgene into primary oligodendrocyte progenitors. We describe an electroporation method which yields transient transfection of oligodendrocyte progenitors of up to 10–15% of the surviving cells, and provides approximately 104 surviving, transfected cells per electroporation reaction. In recent applications transgene expression persisted as the transfected progenitors progressed through subsequent stages of the oligodendrocyte lineage. This technique is expected to facilitate the study of the function of key proteins and lipids during the development of primary cultured oligodendrocytes.
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  • 84
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    Breast cancer research and treatment 47 (1998), S. 197-199 
    ISSN: 1573-7217
    Keywords: breast cancer ; insulin-like growth factor system ; IGF-I receptor ; IGF-II receptor ; binding proteins ; prognosis ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In 1992, a special issue of Breast Cancer Research and Treatment was devoted to the insulin-like growth factors and breast cancer. In that issue, identification of the key components of the IGF system was reviewed and their potential role in breast cancer growth was described. In this issue, we revisit the IGF system with particular attention to data that further supports their role in the growth regulation of breast cancer. Several new facets of the IGF system are described, and several laboratories have more clearly defined how each individual component of the IGF system may influence breast cancer biology.
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  • 85
    ISSN: 1573-904X
    Keywords: gene transfer ; colon 26 ; monocyte chemotactic and activating factor ; chemokine ; biological response modulater
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. In order to evaluate the possibility of synergistic antitumor gene therapy by the gene delivery of monocyte chemotactant protein-1 (MCP-1/MCAF/IE), the effect of a biological response modulater for macrophages on tumor progression of gene transfected tumor cells was studied. Methods. Cachexia-inducing adenocarcinoma cells (cell line colon 26, clone 20) were transfected with either a control plasmid or MCP-1 cDNA. Results. The production of MCP-1 reached 70-80 ng/ml in vitro when transfectant cells were cultured at a cell density of 1 × 105 cells/ml for 3 days. Transfection of MCP-1 cDNA did not affect the growth ratein vitro. Also, MCP-1-transfectants formed tumors after intra-footpad inoculation similar in size to the parental cells. The number of infiltrating macrophages in the primary tumor of the transfectant rapidly increased from the 3rd to 5th day after inoculation as revealed by immunohistochemical staining using an antibody against mouse macrophages. An earlier, greater, but no longer-lasting increase in tumor-infiltrating macrophages was induced in tumors by MCP-1 transfection was compared to that induced by the parent cells. On the 10th day after the inoculation, the tumor-infiltrating macrophages in mice inoculated MCP-1 transfectants were decreased to a level similar to that of the parent cells. Groups of mice were treated intraperitoneally with LPS at different times after the inoculation. Tumor cells producing high levels of MCP-1 were significantly lysed by macrophages treated with LPS, whereas parental or control transfected cells were not. Conclusions. Combination immunotherapy can provide a rationale for the application of MCP-1 treatment to increase immunological responses to cancer.
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  • 86
    ISSN: 1573-904X
    Keywords: gene transfer ; airways ; cationic lipids ; surface charge ; co-lipid content ; topology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. Cationic lipids are capable of transferring foreign genes to the pulmonary epithelium in vivo. It is becoming increasingly clear that factors other than lipid molecular structure also influence efficiency of delivery using cationic lipid systems. This study is aimed at evaluating the effect of formulation variables such as cationic lipid structure, cationic lipid/DNA ratio, particle size, co-lipid content and plasmid topology on transgene expression in the lung. Methods. The effect of varying the surface and colloidal properties of cationic lipid-based gene delivery systems was assessed by intratracheal instillation into rats. An expression plasmid encoding chloramphenicol acetyl transferase (CAT) was used to measure transgene expression. Results. Cationic lipid structure, cationic lipid/DNA ratio, particle size, co-lipid content and topology of the plasmid, were found to significantly affect transgene expression. Complexation with lipids was found to have a protective effect on DNA integrity in bronchoalveolar lavage fluid (BALF). DNA complexed with lipid showed enhanced persistence in rat lungs as measured by quantitative polymerase chain reaction. Conclusions. Fluorescence microscopy analysis indicated that the instilled formulation reaches the lower airways and alveolar region. Data also suggests cationic lipid-mediated gene expression is primarily localized in the lung parenchyma and not infiltrating cells isolated from the BALF.
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  • 87
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    Antonie van Leeuwenhoek 73 (1998), S. 69-77 
    ISSN: 1572-9699
    Keywords: spermosphere ; rhizosphere ; bulk soil ; gene transfer ; seed coating
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transfer of plasmid RP4 to indigenous bacteria in bulk soil could only be detected in soil with nutrient amendment. Lack of physiological active donor and recipient cells was apparently one of the limiting factors in un-amended bulk soil. Plasmid transfer was detected both in the spermosphere and rhizosphere of barley seedlings. Transfer occured from seed coated donor bacteria (i) to introduced recipient bacteria and (ii) to indigenous bacteria present in soil. Plasmid transfer was also detected from donor bacteria introduced to the soil to seed coated recipient bacteria. Transfer efficiencies in the rhizosphere were significantly below the transfer efficiencies obtained in the spermosphere. The transfer efficiencies detected in the barley spermosphere were among the highest reported from any natural environment.
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  • 88
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    Antonie van Leeuwenhoek 73 (1998), S. 147-153 
    ISSN: 1572-9699
    Keywords: transformation ; plasmid integration ; Phaffia rhodozyma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Stable red astaxanthin-producing transformants were obtained after genetic transformation of two Phaffia rhodozyma mutants. A yellow mutant, accumulating β-carotene, and an albino mutant, accumulating phytoene, from P. rhodozyma were transformed using a genomic library of wild-type strain UCD 67-385 in the pBluescript vector. Hybridization assays, using the pBluescript DNA as a radioactive probe, indicate integration of vector sequences into the genome of the transformants. Transformants DNA was digested with restriction endonucleases, ligated with T4 DNA ligase and then used to transform E. coli. Ampicillin resistant plasmids, containing 0.1, 0.2, and 2.5 kb DNA inserts of P. rhodozyma, were rescued from the yeast red transformants. The molecular analysis indicate that transformation has occurred by an integration event of donor DNA into the genome of the host strains.
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  • 89
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    Euphytica 103 (1998), S. 95-102 
    ISSN: 1573-5060
    Keywords: gene transfer ; physical mapping ; RFLPs ; wheat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract A physical deletion map of the Lr19 translocated chromosome segment was extended by mapping three additional Thinopyrum RFLP loci. The relative locations of the marker loci on the translocated segment were determined as: centromere, Sd1, Xpsr165, Xpsr105, Xpsr129, XcsIH81-1, Xwg380, Xmwg2062, Lr19, Wsp-D1, Sr25/Y. Various recombinants, putative recombinats and mutants of the Lr19 segment were also characterised with respect to the additional markers.
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  • 90
    ISSN: 1573-4919
    Keywords: FGF-2 ; transcription ; gene transfer ; HSV-thymidine kinase promoter
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract We have cloned the rat fibroblast growth factor-2 (FGF-2) promoter region including 1058 base pairs (bp) of 5′-flanking DNA. Complete sequencing of this promoter region revealed a 74 bp domain between nucleotides -793 and -720 that was greater than 97% A/G-rich. A repeat of the sequence 5′-AGGGAGGG-3′ separated by 11 bp was located at the core of this domain. A 37 bp A/G-rich oligonucleotide containing these AGGG-repeat sequences was synthesised, and tested for function on a minimal herpes simplex virus thymidine kinase (TK) promoter, fused to the firefly luciferase gene (TKp.luc), in transiently transfected neonatal rat cardiac myocytes. Promoter activity was stimulated ~3 fold in the presence of AGGG-repeat sequences. This effect was neither tissue or species-specific since TK promoter activity was increased ~11 fold in both rat and human glial tumor cells. Four specific complexes (C14) were detected between neonatal rat heart nuclear proteins and the 37 bp A/G-rich oligonucleotide by gel mobility shift assay. Competition with excess unlabelled 37 bp A/G-rich oligonucleotide revealed that two complexes represented very high affinity/specificity interactions (C2 〉 C4) while C1 and C3 were of lower affinity. As a result, competition with up to a 25 fold molar excess of 37 bp A/G-rich oligonucleotide led to the loss of C2 and C4, and a corresponding and transient increase in the levels of C1 and C3, which themselves were reduced with more competitor oligonucleotide. The AGGG-repeat resembles the 5′-gGGGAGGG-3′ sequence previously implicated in the response of the atrial natriuretic factor promoter to the α-adrenergic agonist, phenylephrine. Although an additional 1.5 fold increase in TK promoter activity was detected in the presence of the 37 bp A/G-rich oligonucleotide with phenylephrine treatment of transfected myocytes, this effect was not statistically significant. Furthermore, there was no difference in the gel mobility shift (C14) pattern obtained with the 37 bp A/G-rich oligonucleotide and nuclear protein isolated from neonatal rat cardiac myocytes grown in the presence or absence of norepinephrine. These data suggest that the A/G rich sequences in the rat FGF-2 gene 5′-flanking DNA, including the AGGG-repeat, are able to confer stimulatory activity on a promoter in a tissue- and species-independent manner, but alone are not able to induce a significant phenylephrine response in neonatal rat cardiac myocytes.
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  • 91
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    Euphytica 100 (1998), S. 219-223 
    ISSN: 1573-5060
    Keywords: cereals ; wheat ; transformation ; genetic modification ; transgenic plants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract A method for efficient genetic transformation of wheat has been developed using immature embryos as targets for microprojectile-mediated gene transfer and a helium driven particle delivery system. Screening and selection of transgenic cells, somatic embryos and regenerated plants are performed with the gus-gene and the phosphinothricin acetyl transferase (bar) gene coding for Basta-resistance as the selectable marker. On average, one fertile transgenic plant can be obtained from about 100 microprojectile treated, immature embryos. The number of integrated copies of the transferred gene ranges from 1 up to about 10. Stable integrated genes are inherited in most of the transgenic lines in a normal mendelian fashion segregating 3:1 in the F2. Homozygous, as well as heterozygous, lines have been followed and analysed genetically at the molecular level and up to F5. Apart from normal stable gene expression, examples have also been found which showed a loss of gene activity or unexpected segregation pattern. For applied aspects, different genes are transferred aiming for improved disease resistance, modification of quality, or other characteristics. First results from these transgenic lines are reported, and problems still existing with the production of stable transgenic wheat lines are discussed.
    Type of Medium: Electronic Resource
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  • 92
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    Plant cell, tissue and organ culture 55 (1998), S. 175-181 
    ISSN: 1573-5044
    Keywords: Conifer ; transformation ; virulence genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract As a preliminary step in efforts to develop a successful protocol for Agrobacterium-mediated transformation of cotyledonary explants of Pinus pinea L. embryos, we tested the ability of embrionary exudates of this species to induce the expression of the virulence genes virA, virB, virC, virD, virE and virG in Agrobacterium tumefaciens containing vir: lacZ fusion constructs. The results obtained in the vir induction assay indicated the absence of bactericidal or bacteriostatic plant compounds affecting A. tumefaciens growth, and showed that cotyledonary and embrionary exudates of P. pinea are able to induce all virulence genes studied, except virG. The data suggest that A. tumefaciens can be used for gene transfer into this important forest and fruit species.
    Type of Medium: Electronic Resource
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  • 93
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    Environmental and ecological statistics 5 (1998), S. 197-222 
    ISSN: 1573-3009
    Keywords: kriging ; non-separable space-time correlation ; spatial scale ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Energy, Environment Protection, Nuclear Power Engineering
    Notes: Abstract We present an approach to estimate hourly grid-cell surface ozone concentrations based on observations from point monitoring sites in space, for comparison with grid-based results from the SARMAP photochemical air-quality model for a region of northern California. Statistical estimation is carried out on a transformed (square root) scale, followed by back-transforming to the original scale of ozone in parts per billion, adjusting for bias and variance. We estimate a spatially-varying diurnal mean structure and a non-separable space-time correlation structure on the transformed scale. Temporal pre-whitening is followed by modelling of a spatially non-stationary, diurnally-varying spatial correlation structure using a spatial deformation approach. Comparisons of SARMAP model results with the estimated grid-cell ozone levels are presented.
    Type of Medium: Electronic Resource
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  • 94
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    Electronic Resource
    Bognor Regis [u.a.] : Wiley-Blackwell
    Journal of Polymer Science Part A: Polymer Chemistry 36 (1998), S. 2209-2214 
    ISSN: 0887-624X
    Keywords: cationic polymerization ; anionic polymerization ; transformation ; samarium ; block copolymerization ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Transformation of the cationic growing center of living poly(tetrahydrofuran) [poly(THF)] into an anionic one was achieved in high efficiency (62%) by the end-capping of living poly(THF) with potassium iodide followed by the reduction with bis(pentamethylcyclopentadienyl)samarium (Cp*2Sm), whereas the direct reduction with Cp*2Sm without the end-capping resulted in the formation of poly(THF) with pentamethylcyclopentadienyl group at the terminal. The increase in the molecular weight of poly(THF) after the reduction was observed, which indicates the presence of the dimerization of poly(THF) during the reduction. The polymerization of a variety of electrophilic monomers including δ-valerolactone, 2-oxo-1,3-dioxane, and alkyl methacrylates with the macroanion provided good yields of the corresponding block copolymers consisting of both cationically and anionically polymerizable monomers. © 1998 John Wiley & Sons, Inc. J. Polym. Sci. A Polym. Chem. 36: 2209-2214, 1998
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
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  • 95
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 272-281 
    ISSN: 0006-3592
    Keywords: gene transfer ; retrovirus ; cell cycle ; intracellular stability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Recombinant retroviruses are currently used as gene delivery vehicles for the purpose of gene therapy. It is generally believed that the efficiency of retroviral transduction depends on the cell cycle status of the target cells. However, it has been reported that this is not the case for the transduction of human and murine fibroblasts, in contrast to other cell types such as lymphocytes. The predictions of a mathematical model that we constructed, offer an explanation of this contradiction, based on the dynamics of the underlying processes of target cell growth and the intracellular decay of retroviral vectors. The model suggests that the utility of synchronization experiments, that are usually employed to study cell cycle specificity, is severely limited when the time scales of the above kinetic events are comparable to each other. The predictions of the model also suggest the use of retroviral vectors as cell cycle markers, as an alternative way to detect cell cycle dependence of retroviral transduction. This method obviates the need for cell synchronization and therefore, it does not perturb the cell cycle or interfere with the life cycle of retroviral vectors. Moreover, it does not depend on the intracellular stability of retroviral vectors. Our results show that in contrast to previously reported results, transduction of murine fibroblasts is cell cycle dependent, and they are consistent with the current notion that mitosis is the phase that confers transduction susceptibility. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:272-281, 1998.
    Additional Material: 7 Ill.
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  • 96
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    Social indicators research 43 (1998), S. 197-209 
    ISSN: 1573-0921
    Keywords: transformation ; anomie ; social integration ; state ; quality of life
    Source: Springer Online Journal Archives 1860-2000
    Topics: Sociology
    Notes: Abstract The weakening of social integration and anomie are unavoidable in the transformation of societies. The effect is a decrease of quality of life accompanied by disenchantments, aggressiveness and escapism. In some countries in Eastern Europe like Bulgaria the anomie effects of transformation became particularly strong. The major reason is the political instability. The dissolution of the previous state-centered over-integration of society developed into a dissolution of major mechanisms of political integration. The prospects for improvement of quality of life are focused on the balance of economic, political and cultural re-integration of Bulgarian society.
    Type of Medium: Electronic Resource
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  • 97
    ISSN: 1572-9788
    Keywords: antisense ; chalcone synthase ; flower colour ; lisianthus ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Three cultivars of lisianthus (Eustoma grandiflorum (Grise.)) were transformed with a homologous antisense CHS cDNA via Agrobacterium-mediated transformation. Over 50% of the transgenics derived from the purple flowering lines exhibited an altered flower colour pattern ranging from small streaks of white on the wild-type purple background through to completely white flowers. A significant portion of the transgenic lines showed unstable phenotypes. Northern and biochemical analysis showed that the altered flower patterns were associated with a loss of CHS gene transcript and a corresponding loss of CHS enzyme activity. In the white flowering line the level of total flavonoids was reduced to ca. 2.0% of the wild-type level. Some of the transgenic plants also exhibited alterations in flower form such as the formation of frilled petal tips and reduced flower opening. Several of the new patterned lines are being evaluated for stability and possible commercial release.
    Type of Medium: Electronic Resource
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  • 98
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    Molecular breeding 4 (1998), S. 531-541 
    ISSN: 1572-9788
    Keywords: Agrobacterium ; Brassica oleracea ; cauliflower ; regeneration ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract We have developed an efficient and simpler method for genetic transformation and regeneration of cauliflower, Brassica oleracea var. botrytis plants. Explants from 4-day old seedlings were inoculated and cocultivated with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector with the neomycin phosphotransferase-II gene under the regulatory control of nopaline synthase promoter and terminator sequences, permitting transformed shoots to be selected on kanamycin containing medium. After three months rooted transformed plantlets were successfully transferred and grown under glasshouse conditions. Higher numbers of transformed plants were obtained from cotyledon than hypocotyl explants, presumably indicating cotyledons of cauliflower are more amenable to genetic transformation. Integration and expression of the introduced transgene were analysed by DNA gel blot and PCR analysis and NPT-II expression assay. Factors influencing transformation efficiency include explant age, concentration of bacterium used for infection, duration of infection and cocultivation with Agrobacterium. Transgenic plants of three commercial genotypes of cauliflower were produced using this method. We also show that introduction of antisense Bcp1 (pollen-specific gene) linked to a pollen-specific promoter (Lat52) resulted in the expected sterility of 50% pollen carrying this transgenic construct.
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  • 99
    ISSN: 1573-9368
    Keywords: Saccharum ; Agrobacterium ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract This is the first successful report of the recovery of morphologically normal transgenic sugarcane plants from co-cultivation of calluses with Agrobacterium tumefaciens. Transformation frequencies (total of transgenic plants/number of cell clusters) were between 9.4 × 10−3 and 1.15 × 10−2. In our experiments, both LBA4404 (pTOK233) and EHA101 (pMTCA3IG), carrying a super-binary vector or supervirulent strain, respectively, were successful for sugarcane transformation. We found that three main factors: (1) the use of young regenerable calluses as target explants; (2) induction and/or improvement of the A. tumefaciens virulence system with sugarcane cell cultures and (3) pre-induction of organogenesis or somatic-embryogenesis-like sexual embryos, seem to be crucial in order to increase the cells competence for T-DNA transfer process. Patterns generated by Southern hybridization confirmed that T-DNAs were randomly integrated into sugarcane genome without th e persistence of A. tumefaciens in the transgenic plants
    Type of Medium: Electronic Resource
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  • 100
    ISSN: 1573-9368
    Keywords: Oryza sativa ; particle bombardment ; transformation ; transgenic rice
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We developed a practical and efficient gene transfer system for indica rice utilizing mature-seed derived explants and a simple bombardment device which uses compressed helium for accelerating DNA-coated metal particles. Unlike instruments which have been described in the literature previously, this new bombardment device, which is an improvement of the particle inflow concept, does not require vacuum. This attribute simplifies the transformation procedure significantly and it makes rice transformation technology accessible to laboratories which may not have the resources to invest in more expensive particle bombardment instruments. We determined experimentally that we could recover transgenic rice plants utilizing three different particle bombardment instruments at comparable frequencies.
    Type of Medium: Electronic Resource
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