Glucose regulation of pre-steady state kinetics of ATP hydrolysis by Na,K-ATPase

The effect of glucose and 2-deoxy-D-glucose on pre-steady state kinetics of ATP hydrolysis by Na,K-ATPase has been investigated by following pH transients in a stopped-flow spectrophotometer. A typical pre-steady state signal showed an initial decrease then subsequent increase in acidity. Under optimal Na^＋ （120 mM） and K^＋ （30 mM） concentrations, magnitudes of both H^＋ release and H^＋ absorption were found to be approximately 1.0/ATPase molecule. The presence of 1 mM glucose significantly decreased H^＋ absorption at high Na^＋ concentrations, whereas it was ineffective at low Na^＋. H^＋ release was decreased significantly in the presence of 1 mM glucose at Na^＋ concentrations ranging from 30 mM to 120 mM. Similar to the control, K^＋ did not show any effect on either H^＋ release or H^＋ absorption at all tested combinations of Na^＋ and K^＋ concentrations. Pre-steady state H^＋ signal obtained in the presence of 2-deoxy-D-glucose did not vary significantly as compared with glucose. Delayed addition of K^＋ （by 30 ms） to the mixture （enzyme＋ 120 mM Na^＋ATP＋glucose） showed that only small fractions of population absorb H^＋ in the absence of K^＋. No H^＋ absorption was observed in the absence of Na^＋. Delayed mixing of Na^＋ or K^＋ did not have any effect on H^＋ release. Effect of 2-deoxy-D-glucose on H^ absorption and release was almost the same as that of glucose at all combinations of Na^＋ and K^＋ concentrations. Results obtained have been discussed in terms of an extended kinetic scheme which shows that, in the presence of either glucose or 2-deoxy-D-glucose, significantly fewer enzyme molecules reache the E-P（3Na＋） stage and that K^ plays an important role in the conversion of E1 .ADP.P（3Na^＋） to H^＋.E1-（3Na^＋） complex.     

Axenization and optimization of in vitro growth of clonal cultures of Tetratrichomonas gallinarum and Trichomonas gallinae

A rapid and simple procedure was established to obtain clonal axenic cultures of Tetratrichomonas gallinarum and Trichomonas gallinae and to optimize their in vitro growth conditions. Medium 199 was used for axenization of two genetically different clones of T. gallinarum and T. gallinae. Six different media were used to optimize the growth behaviour of axenically grown parasites: Medium 199, TYM, TYI-S-33, Hollander fluid (HF), Trichomonas vaginalis (TV) and modified TV media. The highest cell yields for both axenic clones of T. gallinarum were obtained in modified TV medium without antibiotics. The maximum numbers of trophozoites of T. gallinae were obtained in an optimized HF medium. This study demonstrated that axenic cultures for T. gallinarum and T. gallinae could be obtained avoiding the migration technique through a V-tube. Following axenization and optimization, both clones of T. gallinarum and T. gallinae could be propagated both aerobically and anaerobically.     

SHP-2-Erk signaling regulates concanavalin A-dependent production of TIMP-2

To search for the signaling critical for the production of tissue inhibitor of metalloproteinase-2 (TIMP-2), we investigated the role of SHP-2 in TIMP-2 production with Concanavalin A (Con A)-treated cells. In wild-type fibroblasts, Con A-treatment dramatically activated TIMP-2 production. In contrast, production of TIMP-2 in response to Con A-treatment was severely impaired in cells expressing mutant SHP-2 whose 65 amino acids in the SH2-N domain were deleted. Con A-treatment activated dual signaling pathways, Erk and p38, in a SHP-2-dependent manner. Pretreatment of wild-type cells with U0126, a potent inhibitor of MEK1, significantly inhibited the production of TIMP-2, whereas SB203580, a specific inhibitor for p38, could not. Finally, expression of exogenous wild-type SHP-2 in SHP-2 mutant cells clearly rescued Erk activation and TIMP-2 production in response to Con A-treatment. Taken together, our results strongly suggest that SHP-2 plays a critical role as a positive modulator for the production of TIMP-2 via MEK1-Erk signaling in fibroblasts.     

Use of cloud-point preconcentration for spectrophotometric determination of trace amounts of antimony in biological and environmental samples

This work presents a cloud-point extraction process using the micelle-mediated extraction method for simultaneous preconcentration and determination of Sb(III) and Sb(V) species in biological and environmental samples as a prior preconcentration step to their spectrophotometric determination. The analytical system is based on the selective reaction between Sb(III) and 3-dichloro-6-(3-carboxy-2-hydroxy-1-naphthylazo)quinoxaline (DCHNAQ) in the presence of cetyltrimethylammonium bromide (CTAB) and potassium iodide at pH 4.5. Total Sb concentration was determined after reduction of Sb(V) to Sb(III) in the presence of potassium iodide and ascorbic acid. The optimal reaction conditions and extraction were studied, and the analytical characteristics of the method (e.g., limits of detection and quantification, linear range, preconcentration, improvement factors) were obtained. Linearity for Sb(III) was obeyed in the range of 0.2–20 ng ml⁻¹. The detection and quantification limits for the determination of Sb(III) were 0.055 and 0.185 ng ml⁻¹, respectively. The method has a lower detection limit and wider linear range, inexpensive instrument, and low cost, and is more sensitive compared with most other methods. The interference effect of some anions and cations was also studied. The method was applied to the determination of Sb(III) in the presence of Sb(V) and total antimony in blood plasma, urine, biological, and water samples.     

Pharmacokinetics of the active antifungal enantiomer, SCH 42427 (RR), and evaluation of its chiral inversion in animals following its oral administration and the oral administration of its racemate genaconazole (RR/SS)

Genaconazole (SCH 39304) is a potent triazole antifungal agent that is active both orally and topically. Genaconazole is a racemic mixture which contains 50% of the RR (SCH 42427) and 50% of the SS (SCH 42426) enantiomers. The RR isomer accounts for most of the antifungal activity of genaconazole. Serum concentrations of the RR and SS enantiomers were analyzed by a chiral HPLC method which involved extraction of serum with organic solvent followed by separation on a Cyclobond I column and quantification by UV absorbance at 205 nm. The bioavailability and pharmacokinetic profiles of the two enantiomers after oral administration of the racemate (genaconazole) were very similar in cynomolgus monkeys. In rats following dosing with genaconazole, the RR enantiomer had a lower C(max) and a longer t(1/2) than the SS enantiomer, while the AUC(I) values of the two enantiomers were similar. Based on chiral HPLC analysis, there was no evidence for the inversion of the RR to the SR isomer, or of the SS to the SR isomer, indicating that there was no chiral inversion of the RR or SS enantiomers in either species. Genaconazole at 20 mg/kg and the RR (SCH 42427) enantiomer at 10 mg/kg had very similar serum concentration-time profiles and C(max), AUC(I), and t(1/2) values for the RR enantiomer in both rats and monkeys, indicating that the two treatments were equivalent with respect to the bioavailability of the RR enantiomer.     

Radioprotective effect of Curcuma longa extract on γ-irradiation-induced oxidative stress in rats

This study was conducted to evaluate the modulatory effect of aqueous extract of Curcuma longa (L.) against γ-irradiation (GR), which induces biochemical disorders in male rats. The sublethal dose of GR was determined in primary hepatocytes. Also, the effect of C.?longa extract was examined for its activity against GR. In rats, C.?longa extract was administered daily (200?mg/kg body mass) for 21?days before, and 7?days after GR exposure (6.5 Gy). The lipid profile and antioxidant status, as well as levels of transaminases, interleukin-6 (IL-6), and tumour necrosis factor?α (TNFα) were assessed. The results showed that in hepatocytes, the aqueous extract exhibited radioprotective activity against exposure to GR. Exposure of untreated rats to GR resulted in transaminase disorders, lipid abnormalities, elevation of lipid peroxidation, trace element alterations, release of IL-6 and TNF, and decrease in glutathione and protein level of superoxide dismutase-1 (SOD-1) and peroxiredoxin-1 (PRDX-1). However, treatment of rats with this extract before and after GR exposure improved antioxidant status and minimized the radiation-induced increase in inflammatory cytokines. Changes occurred in the tissue levels of trace elements, and the protein levels of SOD-1 and PRDX-1 were also modulated by C.?longa extract. Overall, C.?longa exerted a beneficial radioprotective effect against radiation-induced oxidative stress in male rats by alleviating pathological disorders and modulating antioxidant enzymes.     

The ?4817 G>A (rs2238136) variant of the vitamin D receptor gene: a probable risk factor for colorectal cancer

Vitamin D appears to have anti-tumor activities in the large bowel. Our aim was to investigate whether -4817 G>A (rs2238136) polymorphism located at 5'-untranslated region (5'-UTR) of the human vitamin D receptor (VDR) gene was associated with colorectal cancer (CRC) risk. We conducted a case-control study and VDR genotypes, determined by Bpu10I restriction endonuclease digestion of PCR-amplified DNA, were performed on 327 cases with CRC and 327 controls. The distribution of VDR -4817 G>A genotypes and alleles differed significantly between cases with CRC and controls even after adjustment for confounding factors such as age, BMI, sex, and smoking status. Individuals carrying the "AA" genotype had a 2.09-fold increased risk compared with those with "GG" genotype (P?=?0.016, OR?=?2.09, 95% CI?=?1.15-3.78) and a 1.87-fold increased risk compared with those with "GG and GA" genotypes (P?=?0.033, OR?=?1.87, 95% CI?=?1.05-3.33) for CRC. Furthermore, the VDR "A" allele was significantly overrepresented in cases with CRC than controls (P?=?0.044; OR?=?1.28, 95% CI?=?1.01-1.63). Interestingly, the analysis of the SNP revealed that all these associations were stronger for women subjects than for all subjects combined. These data indicated for the first time a direct association between "AA" genotype of VDR gene -4817 G>A polymorphism and CRC, with a stronger association for female subjects. However, our findings remain to be confirmed in other populations.