Evaluating the effects of vanadyl sulfate on biomarkers of oxidative stress and inflammation in renal tissue of rats with diabetes type 2

Vanadyl sulfate (VS) is an ingredient in some food supplements and experimental drugs. This study was designed to assay the effects of VS on biomarkers of oxidative stress and inflammation in renal tissue of rats with diabetes type 2. 30 male Wistar rats were divided into three equal groups as follow: non-diabetics, non-treated diabetics and VS-treated diabetics. Diabetes type 2 has been induced through high fat diet and fructose in the animals. Diabetic rats were treated with 25 mg/kgBW of VS in water for 12 weeks. At the end of study, glucose and insulin were measured using commercially available kits in serum and biomarkers of oxidative stress and inflammation in renal homogenates of animals were measured by related methods. Compared to controls, glucose and insulin were increased significantly in non-treated diabetic rats (p-value <0.05) that showed the induction of diabetes type 2 in rats. The results showed that in VS-treated diabetic rats compared to the non-treated diabetic group, vanadyl sulfate significantly reduced the glucose and insulin secretion and changed renal inflammatory and oxidative markers, except protein carbonyl so that we couldn't find any significant changes. Our study showed that vanadyl supplementation had positive effects on oxidative stress and inflammation biomarkers in kidney of diabetic rats

Development of a novel resin with antimicrobial properties for dental application

The adhesion of biofilm on dental prostheses is a prerequisite for the occurrence of oral diseases. Objective: To assess the antimicrobial activity and the mechanical properties of an acrylic resin embedded with nanostructured silver vanadate (β-AgVO3). Material and Methods: The minimum inhibitory concentration (MIC) of β-AgVO3 was studied in relation to the species Staphylococcus aureus ATCC 25923, Streptococcus mutans ATCC 25175, Pseudomonas aeruginosa ATCC 27853, and Candida albicans ATCC 10231. The halo zone of inhibition method was performed in triplicate to determine the inhibitory effect of the modified self-curing acrylic resin Dencor Lay - Clássico®. The surface hardness and compressive strength were examined. The specimens were prepared according to the percentage of β-AgVO3 (0%-control, 0.5%, 1%, 2.5%, 5%, and 10%), with a sample size of 9x2 mm for surface hardness and antimicrobial activity tests, and 8x4 mm for the compression test. The values of the microbiologic analysis were compared and evaluated using the Kruskal-Wallis test (α=0.05); the mechanical analysis used the Shapiro-Wilk's tests, Levene's test, ANOVA (one-way), and Tukey's test (α=0.05). Results: The addition of 10% β-AgVO3 promoted antimicrobial activity against all strains. The antimicrobial effect was observed at a minimum concentration of 1% for P. aeruginosa, 2.5% for S. aureus, 5% for C. albicans, and 10% for S. mutans. Surface hardness and compressive strength increased significantly with the addition of 0.5% β-AgVO3 (p<0.05). Higher rates of the nanomaterial did not alter the mechanical properties of the resin in comparison with the control group (p>0.05). Conclusions: The incorporation of β-AgVO3 has the potential to promote antimicrobial activity in the acrylic resin. At reduced rates, it improves the mechanical properties, and, at higher rates, it does not promote changes in the control. .

Inhibitory and stimulating effect of magnesium on vanadate-induced lipid peroxidation under in vitro conditions.

The behaviour of Mg related to vanadium(V)-induced lipid peroxidation (LPO) under in vitro conditions was examined. The studies performed on the liver supernatants (LS) obtained from control, sodium metavanadate-intoxicated, and sodium metavanadate-magnesium sulphate-administered male Wistar rats revealed and confirmed the pro-oxidative potential of V. Simultaneously, they indicated that the improved Mg status may be one of the mechanisms by which the treatment with this element may contribute to reduction of oxidative stress under the conditions of vanadate exposure. On the other hand, the results confirmed that Mg may also stimulate LPO and demonstrated that the incubation conditions and the experimental treatment of the rats from which the liver supernatants were obtained affect the intensity of the examined free radical process.

Emergence of oxyl radicals as selective oxidants.

Hydroxyl radicals (HO·) are derived in Fenton reaction with ferrous salt and H2O2 in acid medium, and at neutral pH, metal-oxyl radicals (M-O·) predominate. Evidence is accumulating that M-O· radicals are also active in oxidation reactions, in addition to metal-oxo (M=O) now shown in many publications. Reactivity of these radicals gives selective oxidized products useful in cellular activities, in contrast to purported indiscriminate cell damage by hydroxyl radicals. Reactions with vanadium compounds, such as diperoxovanadate, peroxo-bridged mixed valency divanadate, vanadium-oxyl radical, tetravalent vanadyl and decavanadate illustrates selective gain in oxidative capacity of oxo- and oxyl- species. Occurrence of ESR signals typical of hydroxyl radicals is demonstrated in cell homogenates and tissue perfusates treated with spin trap agents. It is known for a long time lipid peroxides are formed in tissue microsomal systems exclusively in presence of salts of iron, among many metals tested. Oxygen and a reducing agent, ascorbate (non-enzymic) or NADPH (enzymic) are required to produce 'ferryl', the chelated Fe=O active form (possibly Fe-O· and Fe-O-O-Fe ?) for the crucial step of H-atom abstraction. Yet literature is replete with unsupported affirmations that hydroxyl radicals initiate lipid peroxidation, an unexplained fixation of mindset. The best-known ·OH generator, a mixture of ferrous salt and H2O2, does not promote lipid peroxidation, nor do the many hydroxyl radical quenching agents stop it. The availability of oxo and oxyl-radical forms with transition metals, and also with non metals, P, S, N and V, calls for expansion of vision beyond superoxide and hydroxyl radicals and explore functions of multiple oxygen radicals for their biological relevance.

Evaluation of the interaction of vanadium with glutathione in human blood components

Metallo-elements including Vanadium [V] have strong affinity for sulfhydryl [-SH] groups in biological molecules including Glutathione [GSH] in tissues. Because of this fact it was of interest to further investigate the interaction of Ammonium Vanadate [NH[4]VO[3]] with Glutathione as a biomarker of toxicity and the role of Glutathione in the detoxification and conjugation processes in whole blood components including plasma and cytosolic fraction. Effects of different concentrations of Ammonium Vanadate [NH[4]VO[3]] on the level of reduced Glutathione in whole blood components [Plasma and Cytosolic fraction] were examined. GSH depletion in plasma and cytosolic fraction was Ammonium Vanadate's concentration-dependent. Depleted GSH level was more pronounced with more incubation time period. These findings show that changes in the GSH status produced by Ammonium Vanadate could be due to either by adduct formation of Vanadium and glutathione i.e. [V-SG] or by increased production of oxidized Glutathione [2GSH +V[+5] - GSSG]. This change in GSH metabolic status provides some information regarding the mechanism of toxicity by Ammonium Vanadate and the protective role of glutathione

High-throughput screening of human soluble protein tyrosine phosphatase 1B inhibitors / 药学学报

To screen potential human soluble protein tyrosine phosphatase 1B (PTP1B) inhibitors, a high-throughput screening (HTS) model in 384-well microplate with total volume of 50 microL was established. Recombinant PTP1B was cloned and expressed in E. coli. with its specific substrate 4-nitrophenyl phosphate disodium salt hexahydrate (PNPP). The HTS model was based on enzyme reaction rate with enhanced sensitivity and specificity (Z' = 0.78). A total of 24,240 samples were screened, among them 80 samples with inhibition greater than 70% were selected for further rescreening. Finally, six compounds with high inhibitory activity were identified, whose IC50 values were 21.58, 18.39, 15.37, 11.92, 37.27, and 36.61 microg x mL(-1), separately. The results indicated that the method was stable, sensitive, reproducible and also suitable for high-throughput screening.

Triptolide Inhibits the Proliferation of Immortalized HT22 Hippocampal Cells Via Persistent Activation of Extracellular Signal-Regulated Kinase-1/2 by Down-Regulating Mitogen-Activated Protein Kinase Phosphatase-1 Expression

OBJECTIVE: Triptolide (TP) has been reported to suppress the expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), of which main function is to inactivate the extracellular signal-regulated kinase-1/2 (ERK-1/2), the p38 MAPK and the c-Jun N-terminal kinase-1/2 (JNK-1/2), and to exert antiproliferative and pro-apoptotic activities. However, the mechanisms underlying antiproliferative and pro-apoptotic activities of TP are not fully understood. The purpose of this study was to examine whether the down-regulation of MKP-1 expression by TP would account for antiproliferative activity of TP in immortalized HT22 hippocampal cells. METHODS: MKP-1 expression and MAPK phosphorylation were analyzed by Western blot. Cell proliferation was assessed by 3H-thymidine incorporation. Small interfering RNA (siRNA) against MKP-1, vanadate (a phosphatase inhibitor), U0126 (a specific inhibitor for ERK-1/2), SB203580 (a specific inhibitor for p38 MAPK), and SP600125 (a specific inhibitor for JNK-1/2) were employed to evaluate a possible mechanism of antiproliferative action of TP. RESULTS: At its non-cytotoxic dose, TP suppressed MKP-1 expression, reduced cell growth, and induced persistent ERK-1/2 activation. Similar growth inhibition and ERK-1/2 activation were observed when MKP-1 expression was blocked by MKP-1 siRNA and its activity was inhibited by vanadate. The antiproliferative effects of TP, MKP-1 siRNA, and vanadate were significantly abolished by U0126, but not by SB203580 or SP600125. CONCLUSION: Our findings suggest that TP inhibits the growth of immortalized HT22 hippocampal cells via persistent ERK-1/2 activation by suppressing MKP-1 expression. Additionally, this study provides evidence supporting that MKP-1 may play an important role in regulation of neuronal cell growth.

Effect of resveratrol on baroreceptor activity of carotid sinus in anesthetized male rats / 药学学报

This study is to evaluate the effect of resveratrol on carotid baroreceptor activity (CBA). The functional curve of carotid baroreceptor (FCCB) was constructed and the functional parameters of carotid baroreceptor were measured by recording sinus nerve afferent discharge in anesthetized male rats with perfused isolated carotid sinus. Resveratrol (30, 60 and 120 micromol x L(-1)) inhibited CBA, which shifted FCCB to the right and downward. There was a marked decrease in peak slope (PS) and peak integral value (PIV) of carotid sinus nerve charge in a concentration-dependent manner. Pretreatment with N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 micromol x L(-1)), an inhibitor of nitric oxide synthase (NOS), eliminated the inhibitory effect of resveratrol. Pretreatment with Bay K8644 (an agonist of L-type calcium channel, 500 nmol x L(-1)) abolished the effect of resveratrol on CBA. A potent inhibitor of tyrosine phosphatase (sodium orthovanadate, 1 mmol x L(-1)) did not influence the effect of resveratrol on CBA. Resveratrol inhibits carotid baroreceptor activity, which may be mediated by the locally released NO and decreased calcium influx. Several studies have showed a cardioprotective effect of resveratrol, with the penetrating study of resveratrol, it may show a potential value in the clinical treatment of cardiovascular disease as an alternative medicine.

[Vanadyl sulphate and its regenerative and trophic effects on beta cells of pancreas of STZ-induced diabetic mellitus rats]

Vanadyl sulfate [vanadium] has insulin like activity and trophic effects on the pancreatic beta cells of experimental-induced partial diabetic mellitus rats. In this study we investigated the trophic and regenerative effects of vanadium on pancreatic beta cells in conjunction with its insulin like actions in moderate diabetic rats. Moderate diabetic hyperglycemia was induced by IV injection of 40 mg/kg streptozotocin [STZ]. Diabetic animals with blood glucose levels [BG] of 500-600 mg/dl were randomly divided into three groups and treated as follows: group I [n=9], remained untreated [diabetic] whereas normoglycemia was induced in group II by daily IP injection of NPH insulin [n=11]; and group III [n=10] used fluid containing 1mg/ml vanadium. Blood samples were taken at specified times during the two months of treatment by nicking the tip of the tail to measure BG. Finally the rats were deeply anesthetized and sacrificed for histological evaluation of their pancreas. BG remained high in group I [552 +/- 7mg/dl], whereas group II were euglycemic and in group III, vanadium reduced BG level to 320 +/- 33mg/dl. Comparison of histological slides obtained from the pancreases of the three groups, with the exception of group III, revealed small and scarce islets of the pancreas, whereas, in group III, vanadium increased the size and the number of these islets looking like of normal rats. Amelioration of hyperglycemia in conjunction with increases in the size and the number of beta cells of group III seems to indicate that vanadium has regenerative and trophic effects on degenerated beta cells of moderate diabetic rats, and therefore, seems to cure diabetes by improving the activity beta cells of diabetic rats when degeneration of beta cells was not complete

Efeito do sulfato de vanadil sobre o comprometimento metabólico muscular induzido pela imobilização de membro posterior de ratos / Efecto del sulfato de vanadil sobre comprometimiento metabólico muscular inducido por la inmovilización del miembro posterior en ratones / Effect of the vanadyl sulphate on the muscular metabolic compromising induced by immobilization of posterior limb of rats

A proposta deste trabalho foi avaliar o efeito do sulfato de vanadil (SV) no perfil metabólico muscular de membro posterior imobilizado de ratos. Ratos Wistar foram divididos nos grupos (n = 6): controle (C), imobilizado em posição neutra do tornozelo (I), tratado com sulfato de vanadil (SV, 0,25mM, VO) e imobilizado tratado com SV (I + SV) durante sete dias. Após o período experimental, foram avaliadas as reservas de glicogênio (RG) dos músculos sóleo (S), gastrocnêmio branco (GB) e vermelho (GV), tibial anterior (TA) e extensor longo dos dedos (ELD), além do peso do S e ELD. A análise estatística foi realizada pela ANOVA seguida pelo teste de Tukey (p < 0,05). No grupo SV, os resultados mostraram elevação significativa nas RG (S 110 por cento, GB 71 por cento, GV 85 por cento, TA 125 por cento, EDL 108 por cento) e no peso (S 9 por cento, EDL 11 por cento). A imobilização reduziu significativamente as RG (S 31,6 por cento, GB 56,6 por cento, GV 39,1 por cento, ELD 41,7 por cento, TA 45,2 por cento) e peso (S 34,2 por cento e ELD 27 por cento); já no grupo I + SV, houve o aumento das RG em todos os músculos (S 211 por cento, GB 115 por cento, GV 148 por cento, ELD 161,9 por cento, TA 147 por cento), além de impedir a perda de peso do S (75 por cento) e ELD (46 por cento). O tratamento com sulfato de vanadil promoveu elevação nas reservas de glicogênio do grupo controle e imobilizado, além de impedir a perda de peso, demonstrando que seu efeito insulino-mimético é representado pela ação glicogênica associado a uma possível ação anticatabólica.

The purpose of this study was to evaluate the metabolic performance of immobilized skeletal muscle in rats treated with vanadyl sulphate. Male Wistar rats were divided in groups (n = 6): control (C), immobilized (I), treated with vanadyl sulphate (VS, 0,25 mM) and immobilized treated with vanadyl sulphate (I + VS) during seven days. The concentration of vanadyl sulphate diluted in water was 0,25 mM. After experimental stage, the glycogen content (GC) was evaluated in soleus (S), white gastrocnemius (WG), red gastrocnemius (RG), tibialis anterior (TA) and extensor digitorum longus (EDL) muscles, besides S and EDL weight. The statistical analysis was realized by the ANOVA followed by Tukey test (p < 0,05). In VS group, the results showed a significant increase in GC (S 110 percent, WG 71 percent, RG 85 percent, TA 125 percent, EDL 108 percent) and in the weight (S 9 percent, EDL 11 percent). The immobilization reduced significantly the GC (S 31.6 percent, WG 56.6 percent, RG 39.1 percent, EDL 41.7 percent, TA 45.2 percent) and weight (S 34.2 percent and ELD 27 percent), and in I + VS group, there was a increase of the GC in all muscles (S 211 percent, WG 115 percent, RG 148 percent, EDL 161.9 percent, TA 147 percent), besides hindering the weight loss in S (75 percent) and EDL (46 percent). The vanadyl sulphate treatment promoted an increase in the glycogen content of control and immobilized groups, besides hindering the weight loss, showing that the insulino-mimetic effect is represented by glycogenic action associate to a possible anti-catabolic action.

La propuesta de este trabajo ha sido la de evaluar el efecto del sulfato de vanadil (SV) en el perfil metabólico muscular de miembro posterior inmovilizado de ratones. Ratones Wistar fueron divididos en grupos (n = 6): control (C), inmovilizado en posición neutra de tobillo (I), tratado con sulfato de vanadil (SV, 0,25mM, VO) e inmovilizado tratado con SV (I + SV) durante 7 días. Después del periodo experimental, fueron evaluadas las reservas de glicógeno (RG) de los músculos soleo (S), gastrocnemio blanco (GB) y colorado (GV), tibial anterior (TA) y extensor largo de los dedos (ELD), además del peso de S y ELD. El análisis estadístico fue realizado por ANOVA seguido del test de Tukey (p < 0,05). En el grupo SV, los resultados mostraron elevación significativa en las RG (S 110 por ciento, GB 71 por ciento, GV 85 por ciento, TA 125 por ciento, EDL 108 por ciento) y en el peso (S 9 por ciento, EDL 11 por ciento). La inmovilización redujo significativamente las RG (S 31,6 por ciento, GB 56,6 por ciento, GV 39,1 por ciento, ELD 41,7 por ciento, TA 45,2 por ciento) y peso (S 34,2 por ciento e ELD 27 por ciento), por otro lado en el grupo I + SV, hubo aumento de las RG en todos los músculos (S 211 por ciento, GB 115 por ciento, GV 148 por ciento, ELD 161,9 por ciento, TA 147 por ciento), además de impedir la pérdida de peso de S (75 por ciento) y ELD (46 por ciento). El tratamiento con sulfato de vanadil promovió una elevación en las reservas de glicógeno del grupo control e inmovilizado, además de impedir la pérdida de peso, lo que demuestra que su efecto insulina mimético está representado por la acción glicogénica asociado a una posible acción anticatabólica.

Hypoglycemic effects of vanadium on alloxan monohydrate induced diabetic dogs

The hypoglycemic effects after oral administration of vanadium have been studied previously in many species such as rats, mice and even humans. However, there has been no prior report on the glucose lowering effect of vanadium on diabetic dogs. Therefore, the purpose of this study was to evaluate the hypoglycemic effects of oral vanadium on diabetic dogs. Diabetes mellitus in the dogs studied was induced by alloxan monohydrate intravenous injection. The dogs were divided into two groups, one was the diabetic control (DC) group (n = 4) and the other was the vanadium treated (DV) group (n = 6). Fresh water was supplied to the dogs in the DC group, but sodium metavanadate solution (0.1~0.2 mg/ml) was given to the dogs in DV group from one week after the alloxan injection. The fasting glucose levels, fructosamine and serum chemistry profiles were compared between the two groups weekly for three weeks. The fasting blood glucose levels in DV group were significantly lower than those in the DC group (p < 0.01). Fructosamine levels in the DV group were also lower than those in the DC group (p < 0.05). The serum chemistry profiles were not significantly different in comparisons between the two groups. However, the cholesterol levels were significantly lower in the DV group compared to the DC group (p < 0.05). Our findings showed that oral vanadium administration had a hypoglycemic effect on chemically induced diabetic dogs.

Tyrosine phosphatase and cytochrome P450 activity are critical in regulating store-operated calcium channels in human fibroblasts

Diverse signaling pathways have been proposed to regulate store-operated calcium entry (SOCE) in a wide variety of cell types. However, it still needs to be determined if all of these known pathways operate in a single cell type. In this study, we examined involvement of various signaling molecules in SOCE using human fibroblast cells (HSWP). Bradykinin (BK)-stimulated Ca2+ entry, previously shown to be via SOCE, is enhanced by the addition of vanadate, an inhibitor of tyrosine phosphatases. Furthermore, SOCE is regulated by cytochrome P-450, as demonstrated by the fact that the products of cytochrome P-450 activity (14,15 EET) stimulated SOCE while econazole, an inhibitor of cytochrome P450, suppressed BK-stimulated Ca2+ entry. In contrast, Ca2+ entry was unaffected by the guanylate cyclase inhibitor LY83583, or the membrane permeant cyclic GMP analog 8-bromo-cyclic GMP (8-Br-cGMP). Neither nitric oxide donors nor phorbol esters affected BK-stimulated Ca2+ entry. SOCE in HSWP cells is primarily regulated by tyrosine phosphorylation and the cytochrome P-450 pathway, but not by cyclic GMP, nitric oxide, or protein kinase C. Thus, multiple pathways do operate in a single cell type leading to the activation of Ca2+ entry and some of these signaling pathways are more prominently involved in regulating calcium entry in different cell types.

Sensitive spectrophotometry methods for quantitative determination of chlorprothixene in pharmaceutical dosage form

Simple and sensitive UV-VIS spectrophotometric methods for the determination of chlorprothixene hydrochloride have been developed. One of them is based on the oxidation of chlorprothixene [CPT] by ammonium metavanadate with the formation of colourless product. The second method involves the formation of ion-pair between the drug under investigation and inorganic complexes of titanium [IV] thiocyanate followed by its extraction with mixture of butanol-chloroform [1:9, v/v]. The optimum conditions for the oxidation of CPT or ion-pair formation are established. The studies are examined by UV-VIS, IR or NMR spectroscopy. The methods permit the determination of CPT over the concentration range of 2.5-25 micro g/ml and 4-35 microg/ml using ammonium metavanadate or the titanium [IV] thiocyanate complex, respectively. The methods are rapid, highly reproducible and accurate with +/- 0.8%. The methods are applicable to the assay of the drug under investigation in different dosage forms and the results are in good agreement with those obtained by the official methods. Common excipients used as additives to active ingredient in pharmaceutical preparations do not interfere in the proposed methods. The extractive spectrophotometric method can be applied to the determination of chlorprothixene hydrochloride in tablets after solid phase extraction [SPE]

Evaluation of photodynamic activity of octaethylporphyrin and vanadyl octaethylporphyrin

Objective: in this work we have investigated the photodynamic efficiency of octaethylporphyrin (OEP) and vanadyl octaethylporphyrin(VOOEP). Methods: this study was performed by the evaluation of photophysical parameters of these porphyrins, the photooxidationrate constants (kf) of the biomolecules (tryptophan -Trp and bovine serum albumin - BSA) and the erythrocytes photodestructionpercentage. Results: photophysical parameters value such as singlet oxygen quantum yield (Õ∆) and triplet state lifetime (ôT)indicated that OEP (Õ∆= 0.64 ±0.02, tT= 0.91 ± 0.02 ms) is more efficient than VOOEP (Õ∆= 0.26 ±0.02, tT= 0.22 ± 0.03ms). The values of kf/10-4 s-1for Trp and BSA photoxidation demonstrated that OEP (Trp= 2.80 ± 0.05 and BSA= 2.50 ± 0.1)is more efficient than VOOEP (Trp= 0.81 ± 0.08 and BSA= 0.62 ± 0.04). The photodestruction percentage of erythrocytesrevealed that the photodynamic activity of OEP is more pronounced than photoactivity of VOOEP. These results indicated thatdifferences observed in the photodynamic activity between the porphyrins could be associated with differences in their molecularstructures. Conclusion: photophysical parameters, photooxidation of biomolecules, and photodestruction of erythrocytes clearlyindicate that the vanadyl group (V=O) interferes in the photoactivity of OEP, causing a considerable reduction in its efficiency.

Amelioration of altered antioxidant status and membrane linked functions by vanadium and Trigonella in alloxan diabetic rat brains.

Trigonella foenum graecum seed powder (TSP) and sodium orthovanadate (SOV) have been reported to have antidiabetic effects. However, SOV exerts hypoglycemic effects at relatively high doses with several toxic effects. We used low doses of vanadate in combination with TSP and evaluated their antidiabetic effects on anti-oxidant enzymes and membrane-linked functions in diabetic rat brains. In rats, diabetes was induced by alloxan monohydrate (15 mg/100 g body wt.) and they were treated with 2 IU insulin, 0.6 mg/ml SOV, 5% TSP and a combination of 0.2 mg/ml SOV with 5% TSP for 21 days. Blood glucose levels, activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), Na+/K+ ATPase, membrane lipid peroxidation and fluidity were determined in different fractions of whole brain after 21 days of treatment. Diabetic rats showed high blood glucose (P less than 0.001), decreased activities of SOD, catalase and Na+/K+ ATPase (P less than 0.01, P less than 0.001 and P less than 0.01), increased levels of GPx and MDA (P less than 0.01 and P less than 0.001) and decreased membrane fluidity (P less than 0.01). Treatment with different antidiabetic compounds restored the above-altered parameters. Combined dose of Trigonella and vanadate was found to be the most effective treatment in normalizing these alterations. Lower doses of vanadate could be used in combination with TSP to effectively counter diabetic alterations without any toxic effects.

Effect of dynein inhibitor on mouse oocyte in vitro maturation and its cyclin B1 mRNA level / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To evaluate the effect of dynein inhibitor on mouse oocyte in vitro maturation and its cyclin B1 transcription level.</p><p><b>METHODS</b>Immature mouse oocytes were cultured in vitro with a known dynein ATPase activity inhibitor-sodium orthovanadate (SOV) to detect the changes of maturation rate, and semi-quantitative RT-PCR and single cell RT-PCR were performed to detect the changes of cyclin B1 mRNA level.</p><p><b>RESULTS</b>In dose-dependent experiments, the maturation rates of oocytes were significantly different between 5 micromol/L SOV and control groups (P < 0.05), and decreased with SOV increasing doses. However, the elevation of cyclin B1 mRNA level of immatured oocytes cultured for 12 h depended on SOV concentrations ranging from 50 to 500 micromol/L. In incontinuity exposed SOV experiments, the maturation rates of oocytes markedly reduced after the first incubation with 400 micromol/L SOV at least for 1 h and were first cultured in SOV-free medium for 4 h or 8 h before exposure to SOV (P < 0.05). In time-course experiment, the opposite changes of cyclin B1 mRNA level in oocytes between SOV and control groups were observed.</p><p><b>CONCLUSION</b>Dynein inhibitor might delay oocytes meiosis process, and cause ectopic expression of cyclin B1 in oocytes. Most Oocytes incubated with SOV blocked at germinal vesicles (GV) stage or M I to anaphase transition due to dynein dysfunction and ectopic transcription level of cyclin B1.</p>

Effects of genistein on intracellular free-calcium concentration in guinea pig ventricular myocytes / 生理学报

The effects of genistein (GST) on intracellular calcium concentration ([Ca(2+)](i)) were investigated in guinea pig ventricular myocytes. [Ca(2+)](i) was detected by confocal microscopy and represented by relative fluorescent intensity (FI-F(0)) /FI(0), %). The results showed that GST (10-40 micromol/L) reduced [Ca(2+)](i) in normal Tyrode's solution, Ca(2+)-free Tyrode's solution and normal Tyrode's solution containing 3 mmol/L EGTA in a concentration-dependent manner. The effects of GST on [Ca(2+)](i) in normal Tyrode's solution were partially inhibited by pretreatment with sodium orthovanadate, a potent inhibitor of protein tyrosine phosphatase, or L-type Ca(2+) channel agonist Bay K8644. GST also markedly inhibited the ryanodine-induced [Ca(2+)](i) responses in Ca(2+)-free Tyrode's solution. When Ca(2+) waves were produced by increasing extracellular Ca(2+) concentration from 1 to 10 mmol/L, GST (40 micromol/L) could block the propagating waves of elevated [Ca(2+)](i), and reduce the velocity and duration of propagating waves. These results suggest that GST may reduce the [Ca(2+)](i) in isolated guinea pig ventricular myocytes. The inhibition of voltage-dependent Ca(2+) channel, tyrosine kinase inhibition, and alleviation of Ca(2+) release from SR are possibly involved in the GST effect.

Phospholipase D is involved in oxidative stress-induced migration of vascular smooth muscle cells via tyrosine phosphorylation and protein kinase C

Oxidative stress has been implicated in mediation of vascular disorders. In the presence of vanadate, H2O2 induced tyrosine phosphorylation of PLD1, protein kinase C-a (PKC-a), and other unidentified proteins in rat vascular smooth muscle cells (VSMCs). Interestingly, PLD1 was found to be constitutively associated with PKC-a in VSMCs. Stimulation of the cells by H2O2 and vanadate showed a concentration-dependent tyrosine phosphorylation of the proteins in PLD1 immunoprecipitates and activation of PLD. Pretreatment of the cells with the protein tyrosine kinase inhibitor, genistein resulted in a dose-dependent inhibition of H2O2-induced PLD activation. PKC inhibitor and down-regulation of PKC abolished H2O2-stimulated PLD activation. The cells stimulated by oxidative stress (H2O2) caused increased cell migration. This effect was prevented by the pretreatment of cells with tyrosine kinase inhibitors, PKC inhibitors, and 1-butanol, but not 3-butanol. Taken together, these results suggest that PLD might be involved in oxidative stress-induced migration of VSMCs, possibly via tyrosine phosphorylation and PKC activation.

Insulin-dependent Stimulation of a Subtype of p38Map Kinases and Its Role in Insulin's Antiapoptotic Activity / 대한내분비학회지

BACKGROUND: The p38 mitogen-activated protein kinases (p38Map kinases) are a family of prolinedirected serine/threonine kinases. At least four isoforms of p38Map kinases have been identified; however, their physiological significances remain to be understood. Recently, the role of p38Map kinase in insulin-stimulated glucose uptake has been suggested. The present study aimed to investigate which isoform(s) were responsive to insulin stimulation. In addition, the activities of p38 Map kinase isoforms that may participate in the insulin's antiapoptotic function in CHO-IR cells were also determined. METHODS: Chinese hamster ovary cells, expressing wild- or mutated human insulin receptors (CHO-IR cells), were used to investigate whether insulin can stimulate any of the isoform(s) of the p38Map kinases. The p38Map kinase activity was determined by measuring the degree of 32P-labelling of ATF-2 protein, a specific substrate of p38Map kinase. A DNA laddering assay was performed to examine the degree of apoptosis and a RT-PCR analysis to determine which isoform(s) of the p38Map kinases were expressed in response to insulin. RESULTS: p38Map kinase activation by insulin was sharply suppressed in only the CHO-IR/A1018K cells, which lack the intrinsic tyrosine kinase activity of insulin receptors. Insulin stimulation of p38Map kinase was insensitive to SB203580, an inhibitor of the alpha(alpha)-and beta(beta)-isoforms of p38Map kinases. Moreover, orthovanadate, known as a specific stimulator of the gamma(gamma)-and delta(delta-) isoforms, stimulated the p38Map kinase activity in CHO-IR cells. Insulin increased the degree of mRNA expression of the delta-isoform, but not that of the alpha-isoform p38Map kinase. Interestingly, PD98059, an inhibitor of ERK, suppressed p38Map kinase stimulation, as well as the antiapoptotic protection of cells by insulin. As insulin was found to still protect ERK-lacking cells (CHO-IR/ SOS) from apoptosis, any substantial role(s) of ERK might be excluded. CONCLUSION: Our data suggest that insulin may stimulate the activity and expression of the-isoform of p38Map kinase in a MEK1/2-dependent manner. The involvement of the delta-isoform of p38Map kinase in insulin's antiapoptotic protection was also suggested, but remains to be investigated further to clarify the nature of its mechanism of action

Insulin-dependent Stimulation of a Subtype of p38Map Kinases and Its Role in Insulin's Antiapoptotic Activity / 대한내분비학회지

BACKGROUND: The p38 mitogen-activated protein kinases (p38Map kinases) are a family of prolinedirected serine/threonine kinases. At least four isoforms of p38Map kinases have been identified; however, their physiological significances remain to be understood. Recently, the role of p38Map kinase in insulin-stimulated glucose uptake has been suggested. The present study aimed to investigate which isoform(s) were responsive to insulin stimulation. In addition, the activities of p38 Map kinase isoforms that may participate in the insulin's antiapoptotic function in CHO-IR cells were also determined. METHODS: Chinese hamster ovary cells, expressing wild- or mutated human insulin receptors (CHO-IR cells), were used to investigate whether insulin can stimulate any of the isoform(s) of the p38Map kinases. The p38Map kinase activity was determined by measuring the degree of 32P-labelling of ATF-2 protein, a specific substrate of p38Map kinase. A DNA laddering assay was performed to examine the degree of apoptosis and a RT-PCR analysis to determine which isoform(s) of the p38Map kinases were expressed in response to insulin. RESULTS: p38Map kinase activation by insulin was sharply suppressed in only the CHO-IR/A1018K cells, which lack the intrinsic tyrosine kinase activity of insulin receptors. Insulin stimulation of p38Map kinase was insensitive to SB203580, an inhibitor of the alpha(alpha)-and beta(beta)-isoforms of p38Map kinases. Moreover, orthovanadate, known as a specific stimulator of the gamma(gamma)-and delta(delta-) isoforms, stimulated the p38Map kinase activity in CHO-IR cells. Insulin increased the degree of mRNA expression of the delta-isoform, but not that of the alpha-isoform p38Map kinase. Interestingly, PD98059, an inhibitor of ERK, suppressed p38Map kinase stimulation, as well as the antiapoptotic protection of cells by insulin. As insulin was found to still protect ERK-lacking cells (CHO-IR/ SOS) from apoptosis, any substantial role(s) of ERK might be excluded. CONCLUSION: Our data suggest that insulin may stimulate the activity and expression of the-isoform of p38Map kinase in a MEK1/2-dependent manner. The involvement of the delta-isoform of p38Map kinase in insulin's antiapoptotic protection was also suggested, but remains to be investigated further to clarify the nature of its mechanism of action