The effect of temperature on catalytic function of glyceraldehyde-3-phosphate dehydrogenase from muscle of pig and carp Cyprinus carpio

1. The temperature dependence of the kinetics of glyceraldehyde-3-phosphate dehydrogenase from white muscle of carp and skeletal muscle of pig was examined. 2. The Km values of carp muscle enzyme were stable over the temperature range 5-35 degrees C, but increased for pig muscle enzyme with increasing temperature. 3. The Arrhenius plot for pig muscle enzyme is linear but non-linear for carp muscle enzyme. 4. The differences indicate that glyceraldehyde-3-phosphate dehydrogenase from white carp muscle may contribute to the adaptive mechanism of carp to varied temperature conditions.     

Evading apoptosis by calcitriol-differentiated human leukemic HL-60 cells is not mediated by changes in CD95 receptor system but by increased sensitivity of these cells to insulin

Previous studies revealed that 1,25-dihydroxyvitamin D(3) (calcitriol)-induced differentiation of human promyelocytic leukemia cells leads to an increased resistance of the cells to apoptosis-inducing agents. However many attempts were made to explain it, the mechanism underlying this effect still remains unclear. Our results suggest that the acquired resistance to apoptosis-inducing agents in HL-60 cells is not mediated by the CD95 receptor/ligand system. The expression of CD95 on the surface of HL-60 cells is very low and does not change during the calcitriol-induced differentiation of HL-60 cells. Studies presented here provide a strong indication that this receptor is unable to transmit the death signal in either differentiated or undifferentiated HL-60 cells. We therefore asked if evading apoptosis by differentiated human leukemia HL-60 cells may be caused by their increased sensitivity to growth factors contained in fetal calf serum. This study demonstrates that HL-60 promyelocytic leukemia cells, differentiated by exposure to calcitriol, undergo apoptosis in serum-free conditions. As low as 1% of fetal calf serum is enough to prevent cell death of differentiated HL-60 cells. The ability of 1% fetal calf serum to prevent apoptosis can be blocked by the specific inhibitor of phosphatidylinositol 3-kinase, LY294002. We then tried to find out which component of fetal calf serum may be able to prevent serum-free cell death of differentiated cells. It appeared that serum-free cell death of differentiated HL-60 cells is reversed by addition of 10 microM insulin to the culture medium. The antiapoptotic activity of insulin can be inhibited by LY294002. Moreover, insulin increases the viability of differentiated, but not of undifferentiated, HL-60 cells.     

Impaired nuclear localization of vitamin D receptor in leukemia cells resistant to calcitriol-induced differentiation

Calcitriol, the hormonal form of vitamin D₃, induces differentiation of monocytic leukemia cell lines in vitro, without inducing cytotoxicity of the cells. Besides this broad in vitro activity, a clinical implementation of calcitriol, or its analogs, as agents for differentiation therapy has been unsuccessful until now. A better understanding of cellular activities of calcitriol necessary for completion of cell differentiation program could help find better solutions for differentiation therapy of myeloid leukemias. In this paper we describe work carried on subline of acute monocytic leukemia, THP-1 resistant to calcitriol induced differentiation. This resistance correlates with impaired nuclear localization of vitamin D receptor, but not with its total expression in the cells. It also correlates with the resistance to calcitriol-induced growth inhibition, and to phorbol myristate acetate (PMA)-induced cell differentiation.     

Visfatin levels do not change after the oral glucose tolerance test and after a dexamethasone-induced increase in insulin resistance in humans

INTRODUCTION, MATERIAL AND METHODS: Visfatin is a cytokine, mainly expressed in visceral fat, that exerts insulin-mimicking effects in rodents through activation of an insulin receptor, although the binding-site is distinct from that of insulin. However, the mechanisms that regulate visfatin synthesis are still not fully understood. In particular, it is not clear whether short-term glucose-induced hyperglycaemia and hyperinsulinaemia as well as a glucocorticoid-induced increase in insulin resistance are reflected in appreciable alterations in serum visfatin levels in humans. In order to investigate this we measured serum visfatin, glucose and insulin concentrations during a 75.0 gram oral glucose tolerance test (OGTT) [Study 1], as well as before and after oral administration of dexamethasone [Study 2]. Study 1 included 17 subjects (2 males), aged 35.7 +/- 15.6 (mean +/- SD) years of BMI 35.2 +/- 9.3 kg/m(2). Blood samples were taken before (0 minutes) and at 60 and 120 minutes after glucose administration. Study 2 included 20 subjects (4 males, 5 subjects with type 2 diabetes), aged 42.1 +/- 17.2 years of BMI 36.7 +/- 8.38 kg/m(2) who underwent screening for Cushing's disease/syndrome. Dexamethasone was administered at a dose of 0.5 mg every 6 hours for 48 hours. Fasting serum concentrations of visfatin, glucose and insulin were assessed before (D0) and after 48 hours of dexamethasone administration (D2). Insulin resistance was assessed according to the HOMA method in non-diabetic individuals (n = 15). RESULTS: In Study 1 two subjects were found to have impaired glucose tolerance and one subject was found to have diabetes mellitus. Glucose administration resulted in a highly significant increase in insulin (from 11.4 +/- 7.2 microU/mL at 0 min to 98.9 +/- 68.6 microU/mL at 60 min and 72.6 +/- 45.1 microU/mL at 120 minute of OGTT, p < 0.001 for 60 and 120 minutes in comparison to baseline). However, there was no change in serum visfatin concentrations (84.6 +/- 11.6 ng/mL at 0 minutes, 82.6 +/- 12.7 ng/mL at 60 minutes and 81.1 +/- 14.5 ng/mL at 120 minutes of OGTT, p = ns). All subjects in Study 2 achieved suppression of cortisol concentrations below 50 nmo/l. Dexamethasone administration resulted in an increase in fasting insulin (from 11.5 +/- 6.9 to 16.9 +/- 7.6 microU/mL; p = 0.011) and an increase in HOMA (from 2.73 +/- 1.74 to 4.02 +/- 2.27; p = 0.015), albeit without a significant change in serum visfatin concentrations (61.1 +/- 19.8 vs. 68.3 +/- 19.4 ng/mL, p = ns). In neither Study 1 nor Study 2 was there any significant correlation between serum visfatin and age, BMI or HOMA. CONCLUSIONS: There is a striking difference between the marked rise in insulin concentrations and the lack of change in visfatin concentrations during the oral glucose tolerance test. This implies that it is highly unlikely that visfatin is involved in the short-term regulation of glucose homeostasis in human subjects. Dexamethasone administration (4 mg/48 hours) induces an increase in insulin resistance, although without significant change in serum visfatin concentrations. Therefore in contrast to the in vitro data, short term glucocorticoid administration does not result in appreciable changes in serum levels of this adipocytokine. Furthermore, the results of our study do not support the notion that glucocorticoid-induced insulin resistance is likely to be related to changes in serum concentrations of visfatin.     

Association analysis of vitamin D receptor gene polymorphisms with bone mineral density in young women with Graves' disease

Graves' (GD) hyperthyroidism induces accelerated bone turnover that leads to decreased bone mineral density (BMD). The role of the VDR gene in predisposition to primary osteoporosis has been recognized. Recent studies show associations between the VDR gene polymorphisms and susceptibility to autoimmune diseases. Here we analyzed if VDR gene polymorphisms: BsmI, ApaI, TaqI, and FokI may predispose women with Graves' hyperthyroidism to BMD reduction or to disease development. The subjects were 75 premenopausal female Polish patients with GD and 163 healthy women. The genotyping was performed by the use of the restriction fragment length polymorphism analysis (RFLP). We studied the association of the VDR polymorphisms and their haplotypes with patients' BMD and also SNPs and haplotypes association with Graves' disease. We found a strong linkage disequilibrium for the BsmI, ApaI, and TaqI polymorphims that formed three most frequent haplotypes in Graves' women: baT (47.9%), BAt (34.9%), and bAT (16.4%). We did not show statistically significant association of analyzed VDR polymorphisms or haplotypes with decreased bone mineral density in Graves' patients. However, the presence of F allele had a weak tendency to be associated with Graves' disease (with OR=1.93; 95% CI: 0.97-3.84; p=0.058). In conclusion: VDR gene polymorphisms do not predict the risk of decreased BMD in Polish women with Graves'. It may be speculated that the F allele carriers of the VDR-FokI polymorphism are predisposed to Graves' disease development.     

Synthesis and biological evaluation of N-arylpiperazine derivatives of 4,4-dimethylisoquinoline-1,3(2H,4H)-dione as potential antiplatelet agents

Despite the substantial clinical success of aspirin and clopidogrel in secondary prevention of ischemic stroke, up to 40% of patients remain resistant to the available antiplatelet treatment. Therefore, there is an urgent clinical need to develop novel antiplatelet agents with a novel mechanism of action. Recent studies revealed that potent alpha 2B-adrenergic receptor (alpha 2B-ARs) antagonists could constitute alternative antiplatelet therapy. We have synthesized a series of N-arylpiperazine derivatives of 4,4-dimethylisoquinoline-1,3(2H,4H)-dione as potential alpha 2B receptor antagonists. The most potent compound 3, effectively inhibited the platelet-aggregation induced both by collagen and ADP/adrenaline with IC₅₀ of 26.9?μM and 20.5?μM respectively. Our study confirmed that the alpha 2B-AR antagonists remain an interesting target for the development of novel antiplatelet agents with an alternative mechanism of action.