Regulation of protein kinases and coregulatory interplay of S-100beta and serotonin transporter on serotonin levels in diabetic rat brain.

Protein kinases are critical component in the regulation of signal transduction pathways, including neurotransmitters. Our previous studies have shown that serotonin (5-HT) altered under diabetic condition was accompanied by alterations of protein kinase C-alpha (PKC-alpha) and CaMKII, and those alterations were reversed after insulin administration. The current study showed that alloxan-induced diabetic animals revealed hyperglycemia and was associated with an increase in the content of 5-HT, PKC-alpha expression and PKC activity (P < 0.05) simultaneously in striatum (ST), midbrain (MB), pons medulla (PM), cerebellum (CB), and cerebral cortex (CCX) from 7 days to 60 days. Although the 5-HT levels in hippocampus (HC) and hypothalamus (HT) were not altered, the PKC-alpha expression and PKC activity showed increases (P < 0.05) in level in HC. Insulin administration reversed all these changes to a normal level. In contrast, the in vitro study has shown that the 5-HT levels correlated with PKC-alpha expressions as well as PKC activity (P < 0.05) only in ST, MB, and CB either after induction with phorbol 12-myristate 13-acetate (PMA) or blocking with chelerythrine, whereas PM and CCX remained elevated (P < 0.05), implying a regulatory role for PKC-alpha only in ST, MB, and CB. However, our consecutive studies have shown that the 5-HT level in PM was regulated by p38-mitogen-activated protein kinase (p38-MAPK) both in vivo and in vitro, whereas the 5-HT level in CCX was coregulated by S-100beta by protein-protein interaction with serotonin transporter (SERT) via 8-bromoadenosine 3',5'-cyclic monophosphate sodium salt (8-Br-cAMP)-induced cAMP/PKAII pathway(s).

Oxidative stress evoked damages on rat sperm and attenuated antioxidant status on consumption of aspartame.

Although several studies on toxic effect of aspartame metabolite have been studied, controversial reports over the use of aspartame owing to the fact that it releases methanol as one of its metabolite during metabolism exist. This present study is proposed to investigate whether aspartame (40 mg kg-1 b.wt) administration for 90 days could induce oxidative stress and alter antioxidant status of epididymal sperm in Wistar strain male albino rats. To mimic the human methanol metabolism, methotrexate (MTX)-treated rats were included to study the effects of aspartame. Oral intubations of FDA approved 40 mg kg-1 b.wt aspartame were given daily for 90 days to Wistar strain male albino rats and studied along with controls and MTX-treated controls. Sperm count, viability, morphology, morphometry and motility were assessed. A significant decrease in sperm function of aspartame treated animals was observed when compared with the control and MTX control. The free radical generation were observed in epididymal sperm by assessing the scavenging enzymes, enzymatic and non-enzymatic antioxidants. Result suggest that there was a significant increase glutathione-s-transferase (GST), with a significant decrease in reduced glutathione (GSH), superoxide dismutase activity (SOD), glutathione peroxidase levels (GPx), catalase activity (CAT) and glutathione reductase concentration. The increase in free radicals generation could have ultimately caused the lipid peroxidation mediated damages on the testis. Aspartame treated animals also revealed the reduced space in seminiferous tubules, which resulted in reduced Leydig cells when compared with control in histopathology. These findings demonstrate that aspartame metabolites could be a contributing factor for development of oxidative stress in the epididymal sperm.

Modification of allergenicity and immunogenicity of formate dehydrogenase by conjugation with linear mono methoxy poly ethylene glycol: improvement in detoxification of formate in methanol poisoning.

BACKGROUND: Single bolus intravenous infusion of native formate dehydrogenase (FD), isolated from Candida boidinii was found to eliminate formate, a highly toxic metabolite in methanol poisoning. In order to prevent immunological reactions which might be produced by multiple dosing of formate dehydrogenase and to prolong the serum half life of the enzyme, the N-hydroxysuccinimidyl ester of methoxy polyethylene glycol propionic acid (mPEG-SPA 5000) was conjugated to native formate dehydrogenase. METHOD: PEGylation reactions were run at 20 degrees C for 30 min in a reaction buffer (0.2 mol/l sodium phosphate buffer, pH 8.3). The PEGylated molecules were purified from unreacted PEG with Amicon Ultra-4 (10 K) and by Sephacryl S-300 HR gel-filtration chromatography. Unreacted formate dehydrogenase molecules were removed by DEAE Sepharose FF anion-exchange chromatography. PEG-FD enzyme molecules obtained from reacting ratio of FD/PEG of 1/40 had an enzyme activity of 68% of unmodified enzyme. Immunogenicity of PEGylated and native enzyme was evaluated by ELISA. Allergenicity was evaluated by active systemic anaphylaxis and passive cutaneous anaphylaxis tests. In vivo efficacy of PEG-FD or native FD was comparatively evaluated by single intravenous administration of PEG-FD or native FD in folate deficient methanol intoxicated albino rats along with Carbicarb buffer infusion. Methanol and formate were estimated at specific time points respectively with HPLC and fluorescence spectrophotometer. RESULT: PEG-FD had comparatively longer half life and lower immunogenicity than native FD. PEG-FD had better in vivo efficacy than native FD in eliminating the formate. CONCLUSION: Conjugation of mPEG-SPA 5000 with native FD reduces its immunogenicity and increases its efficacy in detoxification of formate in methanol poisoning.

An acute hyperglycemia or acidosis-induced changes of indolamines level correlates with PKC-alpha expression in rat brain.

Hyperglycemia and ketoacidosis are the two most serious factors in acute metabolic complications of both type 1 and type 2 diabetes. Dysfunction of the central nervous system is a well-documented complication of diabetes. We and others have previously reported that acute or chronic diabetes in animal's results in altered brain neurotransmitter levels. In this study, we investigated the effects of acute (7 days) glucose-induced hyperglycemia and sodium acetoacetate (NaAcAc) or ammonium chloride (NH4Cl) induced acidosis on the level of indolamines (5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA)) as well as PKC-alpha expression/activity in discrete areas of rat brain. Glucose-induced (500 mg/kg, bw) hyperglycemic ( approximately 249 mg%) rats showed significant (p<0.05) increase in 5-HT levels in mid brain (MB), pons medulla (PM) and cerebellum (CB), respectively. 5-HIAA level increased in hippocampus (HC) (p<0.05) as compared to control. The rats treated with sodium acetoacetate (NaAcAc) for 7 days (60 mg/kg, bw) showed significant decrease (p<0.05) of 5-HT level in hypothalamus (HT). Whereas, the 5-HIAA level increased in MB (p<0.05). Similarly, the PKC-alpha expression as well as the enzyme activity showed significant increase in HC, MB, PM and CB under glucose-induced hyperglycemia and that changes correlated the changes of indolamines, suggesting that the hyperglycemia may be the major metabolic disorder in diabetic complications.

PKC-alpha mediated alterations of indoleamine contents in diabetic rat brain.

We previously have reported that acute or chronic diabetes in animals resulted in altered neurotransmitter levels. In this study, we investigated the concentrations of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in discrete areas of brain viz. striatum (ST), hippocampus (HC), hypothalamus (HT), midbrain (MB), pons medulla (PM), cerebellum (CB) and cerebral cortex (CCX) of control, untreated diabetic and insulin treated diabetic rats after 30 days. Alloxan (45 mg/kg) diabetic untreated rats, which showed hyperglycemia (>250 mg%), revealed significant increases of 5-HT level in ST, MB, PM, CB and CCX and the 5-HIAA level found to be increased significantly in ST, HC and MB. Whereas the insulin treated rats, which was maintained under normal glucose level (80-110 mg%), showed no significant changes in any of the areas studied. The expressions of PKC-alpha studied by immunoblotting also showed significant changes in ST, HC, MB, PM, CB and CCX that is identical to the changes of both 5-HT and 5-HIAA under similar condition, suggesting that the PKC-alpha may regulate the synthesis and release of indoleamines in diabetic animals.

Evaluation of the growth inhibitory activities of Triphala against common bacterial isolates from HIV infected patients.

The isolation of microbial agents less susceptible to regular antibiotics and the rising trend in the recovery rates of resistant bacteria highlights the need for newer alternative principles. Triphala has been used in traditional medicine practice against certain diseases such as jaundice, fever, cough, eye diseases etc. In the present study phytochemical (phenolic, flavonoid and carotenoid) and antibacterial activities of aqueous and ethanol extracts of Triphala and its individual components (Terminalia chebula, Terminalia belerica and Emblica officinalis) were tested against certain bacterial isolates (Pseudomonas aeruginosa, Klebsiella pneumoniae, Shigella sonnei, S. flexneri, Staphylococcus aureus, Vibrio cholerae, Salmonella paratyphi-B, Escherichia coli, Enterococcus faecalis, Salmonella typhi) obtained from HIV infected patients using Kirby-Bauer's disk diffusion and minimum inhibitory concentration (MIC) methods. T. chebula was found to possess high phytochemical content followed by T. belerica and E. officinalis in both aqueous and ethanol extracts. Further, most of the bacterial isolates were inhibited by the ethanol and aqueous extracts of T. chebula followed by T. belerica and E. officinalis by both disk diffusion and MIC methods. The present study revealed that both individual and combined aqueous and ethanol extracts of Triphala have antibacterial activity against the bacterial isolates tested.

Involvement of Ca2+/calmodulin-dependent protein kinase II in the modulation of indolamines in diabetic and hyperglycemic rats.

Hyperglycemia and acidosis are the key factors in diabetic complications. It has been shown that acute or chronic diabetes alters serotonin levels in brain. However, the mechanism of hyperglycemia- or acidosis-induced changes in serotonin levels remains poorly understood. Because Ca2+-dependent protein kinases play a major role in the regulation of serotonin synthesis and release, we investigated the effect of diabetes, hyperglycemia, and acidosis on the level of indolamines [5-hydroxytryptamine (5-HT) and/or 5-hydroxyindoleacetic acid (5-HIAA)] and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) enzyme activity or protein expression in different brain regions. Alloxan-induced (45 mg/kg bw) diabetic rats (30 days) showed increased level of 5-HT in striatum (ST; 183%), midbrain (MB; 199%), pons medulla (PM; 151%), cerebellum (CB; 214%), and cerebral cortex (CCX; 162%) compared with control (P < 0.05), and these changes were reversed after insulin administration. Rats treated with glucose (500 mg/kg bw) for 30 days showed a 146%, 183%, 208%, and 177% (P < 0.05) increase in 5-HT levels in ST, PM, CB, and CCX, respectively. 5-HIAA level increased in hippocampus (HC; 172%) and in MB (145%; P < 0.05). In addition, rats treated with sodium acetoacetate (NaAcAc) for 30 days (60 mg/kg bw) showed significant increases (P < 0.05) of 5-HT level in ST (152%) and MB (174%). However, the levels of 5-HIAA increased only in MB (151%, P < 0.05). Rats treated with NH4Cl, which induced acidosis (150 mg/kg bw), showed an increased level of 5-HT only in HC (165%, P < 0.05). The increased activity and protein expression of CaMKII in ST, MB, PM, CB, and CCX under diabetic conditions were correlated with the levels of indolamines changes during diabetic, hyperglycemic, or acidotic conditions. These results suggest that CaMKII may be involved in the regulation of indolamines in diabetic animals.