Caspases are a group of cysteine proteases involved in apoptosis and inflammation. A multiparametric homogeneous assay capable of measuring activity of three different caspases in a single well of a microtiter plate is described. Different fluorescent europium, samarium, terbium, and dysprosium chelates were coupled to a caspase substrate peptide, their luminescence properties, were analyzed, and their function in a time-resolved fluorescence quenching-based caspase 3 assay was studied. Substrates for caspases 1, 2, 3, 6, and 8 and granzyme B were also synthesized and their specificities for different caspases were determined. By selecting suitable lanthanide chelates and substrates we developed a multiparametric homogeneous time-resolved fluorescence quenching-based assay for caspases 1, 3, and 6. The assay was capable of measuring the activity of both single caspases and a mixture of three caspases mixed in the same well. 相似文献
An access to brain cell cultures from fish would enable screening of possible neurotoxic chemicals contaminating the aquatic environment. In the present study, a protocol for a successful routine isolation and culturing of brain cells from juvenile rainbow trout was worked out. The coating material was shown to be of importance for cell proliferation. Cells grow better on a surface coated with laminin than on those coated with poly-L-lysine (PLL), poly-D-lysin (PDL) or poly-L-ornithine (PLO). The best cell growth was obtained on double-coated surfaces (PLL, PDL or PLO plus laminin). On such a culture substrate and with a seeding density of 1 x 10(7) cells/cm(2) confluence was obtained within 3-4 weeks at an incubation temperature of 18 degrees C. Approximately 95% of the cells were identified as astrocytes on the basis of a positive staining with antibodies against the astrocyte specific glial protein (GFAP). No oligodendrocytes or fibroblasts were identified in the cultures, and despite several efforts, neurons did not grow under the culture conditions used. When challenged with ligands known to awake a calcium transient in mammalian astrocytes, 44% of the cells responded to ATP with an increase in [Ca 2+](i), 38% to norepinephrine, 27% to 5-hydroxytryptamine, 7% to histamine and 6% to glutamate. Kainate, quisqualate and gamma-aminobutyric acid did not awake a calcium transient in the cells. Using a proper protocol, it is thus quite easy to get an almost pure culture of astrocyte, whereas neurones proved to very difficult to culture. 相似文献
The VHS (Vps-27, Hrs and STAM) domain is a 140 residue long domain present in the very NH2-terminus of at least 60 proteins. Based on their functional characteristics and on recent data on the involvement of VHS in cargo recognition in trans-Golgi, VHS domains are considered to have a general membrane targeting/cargo recognition role in vesicular trafficking. Structurally, VHS is a right-handed superhelix of eight helices with charged surface patches probably serving as sites of protein-protein recognition and docking. 相似文献
To study the relation between steroid receptor concentrations and biological response, we measured cytosol and nuclear progesterone receptors from rabbit uterus under different experimental conditions, and compared receptor values with induction of uteroglobin, a progesterone-regulated protein. A 5-day progesterone treatment (1 mg/kg per day) reduced the nuclear receptor content by 40%, slightly elevated cytosol receptor levels and increased uteroglobin content 3000-fold. Estradiol and tamoxifen altered progesterone-induced changes in the receptor and uteroglobin values: cytosol and nuclear receptors rose significantly, but uteroglobin induction declined markedly, when progesterone treatment was supplemented with estradiol or tamoxifen. A 50% inhibition of progesterone action on uteroglobin synthesis was achieved with 1 μg/kg of estradiol per day. Thus under certain conditions, there is a clear disparity between steroid receptor levels and biological response. 相似文献
Summary Production and release of cellulolytic enzymes by Trichoderma reesei QM 9414 were studied under induced and non-induced conditions. For that purpose, a method was developmed to produce cellulases by Trichoderma reesei QM 9414 using the soluble inducer, cellobiose, as the only carbon source. The production was based on continuous feeding of cellobiose to a batch culture. For optimum production, the cellobiose supply had to be adjusted according to the consumption so that cellobiose was not accumulated in the culture. With a proper feeding program the repression and/or inactivation by cellobiose could be avoided and the cellulase production by Trichoderma reesei QM 9414 was at least equally as high as with cellulose as the carbon source.During the cultivation, specific activities against filter paper, carboxymethyl cellulose (CMC) and p-nitrophenyl glucoside were analyzed from the culture medium as well as from the cytosol and the cell debris fractions. There was a base level of cell debris bound hydrolytic activity against filter paper and p-nitrophenyl glucoside even in T. reesei grown non-induced on glycerol. T. reesei grown on cellobiose was induced to produce large amounts of extracellular filter paper and CMC hydrolyzing enzymes, which were actively released into the medium even in the early stages of cultivation. -Glucosidase was mainly detected in the cell debris and was not released unless the cells were autolyzing. 相似文献
Experimental data suggest that sex steroids have a role in the development of breast and prostate cancers. The biological activity of sex steroid hormones in target tissues is regulated by several enzymes, including 17β-hydroxysteroid dehydrogenases (17HSD). Changes in the expression patterns of these enzymes may significantly modulate the intracellular steroid content and play a pathophysiological role in malignant transformation. To further clarify the role of 17HSDs in breast cancer, we analyzed the mRNA expressions of the 17HSD type 1, 2, and 5 enzymes in 794 breast carcinoma specimens. Both 17HSD type 1 and 2 mRNAs were detected in normal breast tissue from premenopausal women but not in specimens from postmenopausal women. Of the breast cancer specimens, 16% showed signals for 17HSD type 1 mRNA, 25% for type 2, and 65% for type 5. No association between the 17HSD type 1, 2, and 5 expressions was detected. The patients with tumors expressing 17HSD type 1 mRNA or protein had significantly shorter overall and disease-free survival than the other patients. The expression of 17HSD type 5 was significantly higher in breast tumor specimens than in normal tissue. The group with 17HSD type 5 overexpression had a worse prognosis than the other patients. Cox multivariate analyses showed that 17HSD type 1 mRNA, tumor size, and ER had independent prognostic significance.
Using an LNCaP prostate cancer cell line, we developed a cell model to study the progression of prostate cancer. In this model, androgen-sensitive LNCaP cells are transformed in culture conditions into more aggressive, androgen-independent cells. The model was used to study androgen and estrogen metabolism during the transformation process. Our results indicate that substantial changes in androgen and estrogen metabolism occur in the cells during the process. A remarkable decrease in oxidative 17HSD activity was seen, whereas reductive activity seemed to increase. Since local steroid metabolism controls the bioavailability of active steroid hormones of target tissues, the variations in steroid-metabolizing enzymes during cancer progression may be crucial in the regulation of the growth and function of organs. 相似文献
Overconsumption of fructose, as a highly lipogenic sugar, may profoundly affect hepatic metabolism and has been associated with many components of the metabolic syndrome, particularly with insulin resistance and Type 2 diabetes. In this study, we proposed that high fructose diet may enhance lipogenesis and decrease insulin sensitivity in the liver through dysregulation of glucocorticoid signaling. Therefore, we examined the effects of long-term consumption of 10% fructose solution on triglyceridemia, liver histology and intracellular corticosterone level, as well as on 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) and hexose-6-phosphate dehydrogenase (H6PDH) mRNA and protein levels in the rat liver. Glucocorticoid action was assessed by glucocorticoid receptor (GR) expression and intracellular redistribution. We also analyzed the expression of enzymes involved in gluconeogenesis and lipogenesis, phosphoenolpyruvate carboxykinase (PEPCK) and lipin-1. The results have shown that fructose-rich diet led to increase in 11βHSD1 and H6PDH protein levels, while hepatic corticosterone concentration remained unchanged. Concomitantly, GR was increasingly accumulated in the cytoplasm, whereas its nuclear level was unchanged and accompanied by diminished PEPCK mRNA level. Elevation of lipin-1 in the liver microsomes suggested that fructose diet led to an increase in lipogenesis and consequently to hypertriglyceridemia. The observed increase of insulin receptor supstrate-1 phosphorylation on Ser307 represents a hallmark of impaired insulin signaling in the liver of fructose-fed rat and probably is a consequence of the alterations in 11βHSD1 and lipin-1 levels. Overall, our findings suggest that fructose-rich diet may perturb hepatic prereceptor glucocorticoid metabolism and lipogenesis, resulting in hypertriglyceridemia and attenuated hepatic insulin sensitivity. 相似文献
In some animal populations, immigrants have lower survival than philopatric individuals. Costs of dispersal or low phenotypic quality of dispersers may explain the pattern. However, apparent adult survival estimates, which describe real survival combined with site fidelity cannot be separated from permanent emigration. Thus, heterogeneity in breeding dispersal propensities of immigrants and philopatrics can bias fitness correlates of dispersal. Differences in breeding dispersal propensities may be caused by different strategies in response to environmental cues inducing dispersal, such as reproductive success. In such cases, the reported differences between immigrants and philopatric individuals may not reflect true variation in survival. We studied whether dispersal status specific apparent adult survival is associated with reproductive success in a Temminck's stint Calidris temminckii population. We analysed two long term capture–recapture datasets characterised by low and high nest predation levels. Philopatric individuals had higher apparent adult survival than immigrants in both datasets and the difference was highlighted during the high nest predation period. By contrasting return rates between successful and unsuccessful breeders as a proxy for dispersal, we found that unsuccessful immigrants breeding for the first time dispersed more likely than successful immigrants, but such a pattern was not found among philopatric individuals. Our results support the hypothesis that immigrant and philopatric individuals have different breeding dispersal strategies following reproductive failure and that their apparent adult survival differences are at least partly explained by different breeding dispersal propensities. Our results also suggest that the recent decline of the study population reflects a multiple response to increased nest predation through decreased local recruitment and increased emigration. 相似文献