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  • 1
    ISSN: 1573-4919
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Summary The influence of dexamethasone on the isozyme patterns of ATP-hexose phosphotransferases, aldolase and pyruvate kinase of adult rat hepatocytes maintained in primary cultures has been studied. A progressive loss of the typical adult liver isozymes glucokinase, pyruvate kinase L and aldolase B, with a simultaneous increase of both pyruvate kinase A and hexokinase activities, was observed in hepatocytes cultured in the absence of added glucocorticoid. When the culture medium was supplemented with 10−7 M dexamethasone, the adult liver patterns of pyruvate kinase and aldolase were preserved for at least seven days of culture, the initial level of glucokinase was maintained for three days, and the rise of hexokinase activity was delayed and partially blocked. These results are discussed in relation to the known beneficial effect of glucocorticoids on the survival of cultured hepatocytes.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1435-702X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract • Purpose: To investigate the effect of diclofenac sodium salt and cyclosporin A (CsA) on human lens epithelial cell (HLEC) growth in culture. • Methods: Cultures of HLEC were obtained from anterior capsules from extracapsular cataract surgery. Third-passage cells were seeded in 96-well plates in 0.1 ml culture medium. Cytotoxicity was estimated by the tetrazolium test in confluent monolayers after 24 h exposure to a wide range of concentrations of diclofenac and CsA. The effect of subcytotoxic concentrations of diclofenac and CsA on HLEC proliferation in subconfluent cultures was evaluated after 24 and 72 h of exposure. To investigate the relationship between PGEZ synthesis and the inhibitory effect of these drugs, after 24 h of exposure to diclofenac and CsA the production of PGE2 was measured by radioimmunoassay. We also tested the effect of exogenous PGE2 addition to diclofenac 72-h-treated cultures. • Results: Diclofenac and CsA (at concentrations ≥65 μM and ≥2.5 μM, respectively) inhibited the proliferation of subconfluent cultures of HLEC in a dose-dependent fashion. Diclofenac inhibits PGE2 synthesis, while CsA at high doses stimulates PGE2 synthesis of cultured HLEC. Exogenous PGE2 addition reversed in part the inhibitory effect of diclofenac. • Conclusions: Diclofenac and CsA at appropriate doses are effective in inhibiting cultured HLEC proliferation. This could be of interest to prevent posterior capsule opacification. Further in vivo experimental studies seem worthwhile.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1573-0603
    Keywords: intracellular lactate dehydrogenase ; tetrazolium salt ; micromethod
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The present paper describes a rapid, easy, sensitive, and automated spectrophotometric assay for intracellular lactate dehydrogenase (LDH) measurement in 96-well plates of adherent cells for cytotoxicity studies. The procedure involves “in situ” homogenization of cells, followed by measurement of LDH activity with a colorimetric method based on the reduction of a tetrazolium salt to a violet formazan by the NADH formed by LDH. Color intensity can be measured in conventional ELISA readers, and the data can be fed to an “on line” computer for rapid processing. The color absorbance measured is time- and enzyme-concentration dependent. LDH activity measured with this micromethod is coincident with that measured in larger culture plates after individual homogenization and conventional LDH measurement. The advantages of this method are the smaller number of cells required, easy automation, drastic reduction of time for processing individual wells, and the possibility of examining multiple variables in the same experiment.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1573-0778
    Keywords: endothelial cells ; prostacyclin ; thromboxane A2
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Human umbilical endothelial cells in culture retain differentiated morphological and functional characterization in primary culture and even in the early subcultures, after which they begin to degenerate. We have studied the morphological and biochemical characterization (ability to produce prostacyclin, prostaglandin E2 and thromboxane A2 in culture) of endothelial cells in the first seven subcultures. In addition the influence of serum and endothelial cell growth factor added to the culture medium have been evaluated. With 20% normal human serum, cell proliferation is faster than with the same concentration of human fetal or bovine fetal serum. After the 3rd passage, morphological and growth alterations become observable in the endothelial cells. However, prostacyclin, prostaglandin E2 and thromboxane A2 production showed no variations during the study.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Digestive diseases and sciences 33 (1988), S. 833-838 
    ISSN: 1573-2568
    Keywords: ascitic fluid ; cholesterol ; fibronectin ; cirrhosis ; malignancy ; peritoneal metastases ; hepatocellular carcinoma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The ascitic fluid concentrations of cholesterol and fibronectin and the serum-ascites albumin difference were compared with two conventional tests of ascitic fluid, total protein and LDH, in their diagnostic ability for detection of malignancy in ascitic samples from 69 patients with ascites: 54 with ascites due to liver disease and 15 whose ascites was caused by peritoneal metastases. Sixteen cirrhotic patients with superimposed hepatocellular carcinoma in whom ascites was of uncertain etiology were considered separately. The mean ascitic fluid total protein, LDH, cholesterol, and fibronectin values in the peritoneal metastases group were 3.70±1.20 g/dl, 247.26±148.14 units/liter, 109. 06±29.85 mg/dl, and 91.57±41.52 μg/ml, respectively, and all were significantly higher than the corresponding values in the liver disease group (P〈0.001), which were 1.37±0.59 g/dl, 75.40±110.70 units/liter, 23.75±11.22 mg/dl, and 31.86±10.51 μg/ml,respectively. Mean serum-ascites albumin difference in the peritoneal metastases group was 0.62±0.38 g/dl, which was significantly different from the corresponding value in the liver disease group (1.92±0.41 g/dl, P 〈0.001). Both ascitic cholesterol above 46 mg/dl and an ascitic fibronectin concentration 〉50 μg/mlhad high diagnostic accuracy (97%) for malignancy, being higher than that achieved using a serum-ascites albumin difference under 1.1 g/dl and an ascitic total protein above 2.5 g/dl, which had accuracies of 94% and 93%, respectively. Ascitic fluid LDH was the least reliable test. No differences in the ascitic fluid analysis were found between cirrhotic patients with and without hepatocellular carcinoma. We conclude that both ascitic cholesterol and ascitic fibronectin are clinically more accurate than the serum-ascites albumin difference, ascitic total protein,and ascitic LDH in the diagnosis of malignant ascites. Of these tests, the determination of ascitic cholesterol may be the preferred one because of its simplicity and cost effectiveness.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Hepatocytes entrapped in collagen gel and cultured in serum-free conditions survived longer than cells cultured on plastic (5 days vs. 3 weeks), showed fewer signs of early cell senescence (no increase in c-fos oncoprotein expression), and maintained the expression of differentiated hepatic metabolic functions over a longer period of time. Cells cultured in collagen gels retained their ability to respond to hormones. The insulin-stimulated glycogen synthesis rate remained fairly constant during 18 days in culture (between 5.4 ± 0.37 and 9 ± 2.7 nmol glucose/h/μg DNA). Collagen-cultured hepatocytes recovered glycogen stores to levels similar to those found in liver, or in hepatocytes isolated from fed rats. Urea synthesis from ammonia remained stable for more than 2 weeks (average value, 23 ± 4 nmol urea/h/μg DNA). The rate of albumin synthesis in collagen-entrapped cells was maintained above the day-1 level during 18 days in culture. Cells showed high levels of glutathione (GSH) (1,278 ± 152 pmol/μg DNA). Biotransformation activities CYP4501A1, CYP4502A2, CYP4502B1, and CYP4503A1 remained fairly stable in collagen-cultured hepatocytes. CYP4502E1 and CYP4502C11 decreased but were still measurable after 18 days. After 4 days in culture, GST activity returned to levels observed in isolated hepatocytes. In contrast with plastic cultures, cells responded to CYP450 inducers (methylcholanthrene for CYP4501A1, CYP4501A2, and gluthatione-transferase, and ethanol for CYP4502E1) for more than 2 weeks. CYP4501A1, CYP4501A2, and glutathione-transferase A2 (GST A2) induction was preceded by an increase in specific mRNA, while the effects on CYP4502E1 seemed to be at a posttranslational level. Analysis of the expression of relevant hepatic genes by reverse Northern and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that culturing hepatocytes in collagen gels results in a sustained higher expression of key liver transcription factor genes DBP, C/EBP-α and -β, and HNF-1 and -4, as well as specific liver enzyme genes (phosphoenol pyryvate carboxykinase, and carbamoylphosphate-synthetase I). J Cell Physiol 177:553-562, 1998. © 1998 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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