Transport and signaling through the phosphate-binding site of the yeast Pho84 phosphate transceptor

A novel concept in eukaryotic signal transduction is the use of nutrient transporters and closely related proteins as nutrient sensors. The action mechanism of these “transceptors” is unclear. The Pho84 phosphate transceptor in yeast transports phosphate and mediates rapid phosphate activation of the protein kinase A (PKA) pathway during growth induction. We have now identified several phosphate-containing compounds that act as nontransported signaling agonists of Pho84. This indicates that signaling does not require complete transport of the substrate. For the nontransported agonist glycerol-3-phosphate (Gly3P), we show that it is transported by two other carriers, Git1 and Pho91, without triggering signaling. Gly3P is a competitive inhibitor of transport through Pho84, indicating direct interaction with its phosphate-binding site. We also identified phosphonoacetic acid as a competitive inhibitor of transport without agonist function for signaling. This indicates that binding of a compound into the phosphate-binding site of Pho84 is not enough to trigger signaling. Apparently, signaling requires a specific conformational change that may be part of, but does not require, the complete transport cycle. Using Substituted Cysteine Accessibility Method (SCAM) we identified Phe¹⁶⁰ in TMD IV and Val³⁹² in TMD VIII as residues exposed with their side chain into the phosphate-binding site of Pho84. Inhibition of both transport and signaling by covalent modification of Pho84^F160C or Pho84^V392C showed that the same binding site is used for transport of phosphate and for signaling with both phosphate and Gly3P. Our results provide to the best of our knowledge the first insight into the molecular mechanism of a phosphate transceptor.     

Osteonecrosis with renal damage in HIV patients undergoing HAART

Rheumatoid arthritis in HIV patients undergoing HAART is associated with increased risk of side effect. Elevation of uric acid (UA) is important in tissue damage, deposition of crystal in joints leads to the development of rheumatoid arthritis in the HAART complaint group. This study was carried out to investigate the relationship of uric acid, RA factor, ANA, ESR, cystatin C, urea and creatinine in the HAART complaint group. Moreover; the ratio of uric acid/cystatin C, uric acid/urea and uric acid/creatinine were also studied. To analyze the progression of HIV, the immunological parameters were correlated with uric acid. Our result showed a statistically high significant increase in uric acid, RA factor, ANA, ESR, cystatin C, urea and creatinine in the HAART complaint group when compared to HAART non-complaint group, early stage and control. The ratio of uric acid/cystatin C, uric acid/urea, uric acid/creatinine were significantly increased in the HAART complaint group. Statistically significant positive correlation was observed between uric acid and cystatin C, urea, creatinine, absolute CD4 and CD8 count. The increased level of uric acid, RA factor, ANA, ESR, cystatin C and increased ratio of uric acid/cystatin C in the HAART complaint group might conclude the mechanism underlying the increased risk for rheumatoid arthritis in the HAART complaint group which may relate to the combined effects of low-grade inflammation and renal dysfunction.     

Hepatitis B virus BCP,precore/core,X gene mutations/genotypes and the risk of hepatocellular carcinoma in India

The study aims to characterize mutations of the HBV genome involving BCP, Precore/core and X regions and also defines HBV genotypes in patients of hepatocellular carcinoma (HCC). The study involved 150 HBV‐related HCC cases and 136 HBV‐related chronic liver disease patients without HCC as controls. HBV DNA was subjected to mutational analysis using SSCP technique, genotyping by RFLP, and direct nucleotide sequencing. HBV DNA was found in 58.7% (88/150) of the HCC cases and 74.3% (101/136) of controls. HBV mutants were observed in 44.3% of HCC cases and 43.2% of controls. HBV/D was prevalent amongst the patients and controls, followed by HBV/A. The prevalence of the TT1504 mutation in the X gene, the V1753 and T1762/A1764 mutations in the BCP region, and G1914 mutation in the core gene were significantly higher in the HCC group than in the non‐HCC group. Multivariate analyses showed that the TT1504, V1753, A1762T/G1764A, and the G1914 mutations and the patient's age, sex, and HBeAg status increased the risk of HCC development significantly. Also, patients with HCC had lower levels of serum albumin, viral load, and platelet counts but higher values of alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, bilirubin, and Alpha feto‐protein than those of controls (P < 0.001 for all comparisons). HBV/D was the predominant genotype associated with HCC cases seen in India. The presence of different types of HBV mutations, age, sex, HBeAg status, and viral load was found to increase significantly the risk of HCC development in India. J. Med. Virol. 82: 1115–1125, 2010. © 2010 Wiley‐Liss, Inc.     

Optimization of laccase production by Pleurotus ostreatus IMI 395545 using the Taguchi DOE methodology

Production of laccase using a submerged culture of Pleurotus orstreatus IMI 395545 was optimized by the Taguchi orthogonal array (OA) design of experiments (DOE) methodology. This approach facilitates the study of the interactions of a large number of variables spanned by factors and their settings, with a small number of experiments, leading to considerable savings in time and cost for process optimization. This methodology optimizes the number of impact factors and enables to calculate their interaction in the production of industrial enzymes. Eight factors, viz. glucose, yeast extract, malt extract, inoculum, mineral solution, inducer (1 mM CuSO₄) and amino acid (l‐asparagine) at three levels and pH at two levels, with an OA layout of L18 (2¹ × 3⁷) were selected for the proposed experimental design. The laccase yield obtained from the 18 sets of fermentation experiments performed with the selected factors and levels was further processed with Qualitek‐4 software. The optimized conditions shared an enhanced laccase expression of 86.8% (from 485.0 to 906.3 U). The combination of factors was further validated for laccase production and reactive blue 221 decolorization. The results revealed an enhanced laccase yield of 32.6% and dye decolorization up to 84.6%. This methodology allows the complete evaluation of main and interaction factors. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Genetic Polymorphism of Glutathione S Transferases M1 and T1 in Indian Patients with Hepatocellular Carcinoma

Objective: Our aim was to evaluate whether the association of GSTM1/T1 gene polymorphisms modifies the risk of Hepatocellular carcinoma (HCC) and what is its correlation with other predisposing risk factors like alcohol intake, cigarette smoking and hepatitis B and C infections. Study design/setting: It was a case-control study, included 254 HCC cases compared with 525 hospital-based age and sex matched cases of chronic liver disease without HCC as controls from Indian population. The GSTM1 and GSTT1 genotypes were detected using conventional multiplex PCR method. Results: In this case-control study, we observed a positive correlation between age, HBV and HCV infection, smoking habit of > 20 packs/year, alcohol consumption of > 100 g/day and risk of liver cancer. We found significantly increased risk associated with GSTM1 null genotype (OR = 3.49; 95% CI = 2.52–4.84) as well as GSTT1 null genotype (OR = 3.12; 95% CI = 2.19–4.45), respectively. However, an increased risk of HCC was observed among heavy drinkers with GSTM1 (OR = 2.01; 95% CI = 1.11–3.66). Further, cigarette smoking showed a non-significant association with GSTT1 (OR = 1.49; CI = 0.69–3.25). Conclusion: Our results suggest that the variants in low penetrance gene such as GSTM1 and GSTT1 are associated with an increased liver cancer risk. Further, an influence of GSTM1/T1 null genotypes may contribute in the etiology of HCC in patients with higher cigarette and alcohol consumption.     

Hepatitis B virus subgenotype A1 predominates in liver disease patients from Kerala,India

AIM:To molecularly characterize hepatitis B virus(HBV)isolates from Kerala and to relate them to the clinical manifestation of infection.METHODS:Sera and clinical data were collected from91 patients diagnosed with chronic HBV infection and HBV-related hepatocellular carcinoma(HCC).HBV from44 HCC,22 cirrhotic and 25 chronic hepatitis patients were genotyped by sequencing of the complete S region or by restriction fragment length polymorphism assays.The basic core promoter/precore region was sequenced.The complete surface DNA sequences were assembled and aligned manually,and then compared with the sequences of HBV of genotypes(A-J)from GenBank.The evolutionary history was inferred using the Neighbor-Joining method and the evolutionary distances computed using the Kimura 2-parameter method.Bootstrapping was performed using 1000 replicates.The TaqMan BS-1 probe was used to quantify HBV DNA at a lower detection limit of approximately20 IU/mL.Continuous variables were compared using an independent Student’s t test.Theχ2test or Fisher’s exact test was used to compare categorical variables.The differences were considered statistically significant at P<0.05.RESULTS:Irrespective of disease status,the predominant genotype was A(72%);95%belonging to subgenotype A1,followed by genotypes D(27%)and C(1%).HCC patients infected with subgenotype A1were significantly younger than those infected with D.Mutation A1762T/G1764A was significantly associated with HCC in both genotypes A and D.Mutation G1862T was more frequent in subgenotype A1(P<0.0001),and in combination with A1762T/G1764A,it was significantly associated with HBV from HCC patients.Mutation C1766T/T1768A was significantly associated with genotype A(P=0.05)and HCC(P=0.03).The preS2start codon M1T/I mutation was unique to genotype A strains(15.6%)from all disease groups and occurred at a higher frequency in isolates from HCC patients(P=0.076).A higher frequency of preS deletion mutants(33.3%)was observed in genotype A from HCC compared with non-HCC patients,but     

Inhibitory effect of Gymnema Montanum leaves on α-glucosidase activity and α-amylase activity and their relationship with polyphenolic content

The present study was attempted to investigate the effect of G. montanum leaf extract on inhibition of α-glucosidase and α-amylase activity. The ethanol extract of G. montanum (GLEt) at various concentrations (1–10 μg/ml) was tested for its inhibition pattern against α-glucosidase and α-amylase activity in vitro and compared with the commercially available α-glucosidase inhibitor, acarbose. The GLEt showed competitive inhibition against yeast α-glucosidase and noncompetitive inhibition against salivary α-amylase in a concentration-dependent manner. Further, we investigated the effect of extract on levels of blood glucose and plasma insulin in neonatal streptozotocin-induced type 2 diabetic rats. Long-term administration (12 weeks) of the GLEt effectively reduced the blood glucose level and also increased the insulin level. In a preliminary phytochemical analysis, G. montanum leaves were rich in phenolic composition and found to be positively correlated with the inhibitory effect of α-glucosidase and α-amylase activity. These results suggest that the GLEt might exert its antidiabetic effect by suppressing carbohydrate absorption from the intestine and thereby reducing hyperglycemia.     

Hepatitis C virus related hepatocellular carcinoma: a case control study from India

Infection with hepatitis C virus (HCV) is a major risk factor for hepatocellular carcinoma (HCC) in India. The study was designed to investigate the clinical and molecular profiles of HCV related HCC cases in Indian patients. In a prospective study, 68 HCV related HCC, 55 HCV related chronic hepatitis, and 68 HCV related patients with cirrhosis were included. Glutathione S‐transferase gene polymorphism was analyzed in all the cases. The sex ratios were 5.18:1, 1.39:1, and 0.83:1 with mean age of 50.57 ± 12.47, 39.41 ± 13.34, and 46.08 ± 15.06 years, respectively, in three groups. Amongst the HCV related HCC cases seen in India, 49.2% (30 out of 68) were with Okuda stage I while 34.4% (21 out of 68) cases were classified as stage II. Older age, poor standards of living, HCV genotype 4, smoking, and null genotypes of GST were the risk factors associated significantly with the development of HCC. In 55.9% cases (38 out of 68) the size of the tumor was ≥5 cm while in 38.2% cases (26 out of 68) the size was between 2 and 5 cm, indicating an advanced stage of the disease at presentation. J. Med. Virol. 84: 1009–1017, 2012. © 2012 Wiley Periodicals, Inc.