Polyacrylamide grafted Agar: Synthesis and applications of conventional and microwave assisted technique

Polyacrylamide grafted Agar (Ag-g-PAM) has been successfully synthesized by conventional method and microwave assisted method. The former method employs ceric ammonium nitrate (CAN) as the free radical initiator while the latter uses the combination of ceric ammonium nitrate (CAN) and microwave irradiation. The synthesized graft copolymers have been characterized by elemental analysis (C, H, N, O and S), FTIR spectroscopy, intrinsic viscosity measurement and scanning electron micrograph (SEM); taking agar as a reference. Flocculation efficacy of synthesized graft copolymers was studied in kaolin suspension and in waste water through 'Jar test' procedure. In the present investigation, we have observed that polyacrylamide grafted agar synthesized by microwave assisted technique shows superior properties than conventional technique. These properties are reported in terms of intrinsic viscosity, flocculation efficacy and pollutant load reduction of waste water.     

Acetoxy drug: protein transacetylase of buffalo liver-characterization and mass spectrometry of the acetylated protein product

The purification and characterization of the buffalo liver microsomal transacetylase (TAase) catalyzing the transfer of acetyl groups from a model acetoxy drug: 7,8-diacetoxy-4-methylcoumarin (DAMC) to GST3-3 has been described here. The enzyme was routinely assayed using DAMC and cytosolic GST as the substrates and was partially purified from microsomes of the buffalo liver. The enzyme was found to have approximate molecular of weight 65 kDa. The action of TAase and DAMC on liver cytosolic GST resulted in the formation of monoacetoxymonohydroxy-4-methylcoumarin (MAMHC) and 7,8-dihydroxy-4-methylcoumarin (DHMC), although the former was the major metabolite. The buffalo liver microsomal TAase exhibited hyperbolic kinetics and yielded K(m) (1667 microM) and V(max) (192 units) when the concentration of DAMC was varied keeping the concentration of GST constant. After having characterized the nature of the substrates and a product of the TAase-catalyzed reaction, we set out to identify the acetylated protein which is another product of the reaction. GST3-3 was used as a model protein substrate for the action of TAase using DAMC as the acetyl donor. The subunit of control and modified GST3-3 were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and digested with trypsin. The tryptic peptides were extracted from the gel pieces and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOFMS). The data search for calibrated and labeled mass peaks of peptides was performed on the Matrix Science Server using the search engine Mascot. The peptide maps so obtained covered 97% of the GST3-3 sequence. On comparison of MALDI peptide maps of modified and control GST, seven new peaks were recognized corresponding to the potentially acetylated peptides in peptide map. The mass value of each of them was 42 Da higher than the theoretical mass of a non-modified GST3-3 tryptic peptide, strongly suggesting acetylation. By examining the fragmentation patterns and by comparing experimental and predicted values for MS/MS daughter ions, the identity of the seven acetylated GST tryptic peptides could be confirmed by the application of LC/MS/MS. In the modified GST, N-terminal proline and six lysines (Lys(51), Lys(82), Lys(123), Lsy(181), Lys(191) and Lys(210)) were found to be acetylated. The structure of acetylated GST revealed that the lysines that underwent acetylation were peripheral in positions.     

Effect of bromocriptine,a dopamine receptor agonist,on the experimentally induced gastric ulcers in albino rats

The effect of bromocriptine, a dopamine receptor agonist, has been studied on the aspirin, phenylbutazone and reserpine induced gastric ulcers in rats. A single dose of bromocriptine 4 mg/kg s.c. produced a significant exacerbation of gastric ulcers induced by all the three ulcerogenic drugs, whereas in the same dose administered once daily for 5 consecutive days, it produced a marked protective effect in all the models. A review of the literature shows that different mechanisms may be involved in the opposite effects of acutely and chronically administered bromocriptine observed in this study. The study also points towards a role of dopamine in the pathogenesis of gastroduodenal ulceration.     

Galanin-Like Immunoreactivity Is Unchanged in Alzheimer''s Disease and Parkinson''s Disease Dementia Cerebral Cortex

Galanin is a recently isolated neuropeptide that is of particular interest in dementing disorders because of its known colocalization with choline acetyltransferase in magnocellular neurons of the basal nucleus of Meynert. These neurons degenerate in Alzheimer's disease, and there is a corresponding deficiency of cortical choline acetyltransferase activity. In the present study, galanin-like immunoreactivity was measured in the postmortem cerebral cortex and hippocampus of 10 controls and 14 patients who had had Alzheimer's disease. Significant reductions of choline acetyltransferase activity (50-60%) were found in all regions examined; however, there was no significant effect on concentrations of galanin-like immunoreactivity. Similar measurements were made in postmortem tissues of 12 control and 13 demented Parkinsonian patients who had had Alzheimer-type cortical pathology. Choline acetyltransferase activity was again significantly decreased in all regions examined but there were no significant reductions in galanin-like immunoreactivity. Experimental lesions of the fornix in rats produced parallel significantly correlated reductions of both choline acetyltransferase activity and galanin-like immunoreactivity in the hippocampus. Galanin-like immunoreactivity in the human hypothalamus consisted of two molecular-weight species on gel-permeation chromatography, and two forms were resolved by reverse-phase HPLC. The paradoxical preservation of galanin-like immunoreactivity, despite depletion of the activity of choline acetyltransferase, with which it is colocalized, is as yet unexplained. Recent studies have shown that galanin inhibits both acetylcholine release in the hippocampus and memory acquisition; therefore, preserved galanin may exacerbate the cholinergic and cognitive deficits that accompany dementia.     

Association of genetic polymorphisms in glutathione S-transferases and susceptibility to head and neck cancer

Polymorphism in glutathione S-transferase (GST) genes (GSTM1, GSTT1 and GSTP1) and interaction with environmental factors such as tobacco (smoking or chewing) and alcohol on susceptibility to head and neck squamous cell carcinoma (HNSCC) was studied in a case-control study. The study group consisted of 175 patients suffering from HNSCC and 200 age matched healthy controls. Statistical analysis showed an increase in risk to HNSCC in the patients with null genotype of GSTM1 (OR: 2.02; 95% CI: 1.32-3.10; P=0.001) or GSTT1 (OR: 1.66; 95% CI: 1.02-2.69; P=0.04), though the risk was not found to be significant when adjusted for age, sex, smoking, tobacco chewing or alcohol use by multivariate logistic regression model. Our data further showed that combination of deletion genotypes of GST (GSTM1 and GSTT1) confer an even higher risk of HNSCC. Interestingly, GSTP1 wild type genotype in combination with GSTM1 null or GSTT1 null genotype increased susceptibility for HNSCC (OR: 2.49 and 2.75, respectively). Likewise a much greater risk for HNSCC was observed in the patients carrying a genotype combination of GSTM1 null, GSTT1 null and GSTP1 (Ile/Ile) (OR: 4.47; 95% CI: 1.62-12.31; P=0.002). Our data have further provided evidence that tobacco chewing and alcohol consumption are the important risk factors for HNSCC. The interaction between tobacco chewing and null genotype of GSTM1 or GSTT1 resulted in about 3.5- and 2.2-fold increase in the risk respectively in the patients when compared to those not chewing tobacco. Alcohol use resulted in more than 4-fold increase in the risk in the patients with null genotype of GSTM1 as compared to those who are non-drinkers. Alcohol consumption also increased the risk (approx. 3-fold) in the cases with null genotype of GSTT1, though the association was not found to be significant when compared to non-drinkers. Our data have provided evidence that GST polymorphism modifies the susceptibility to HNSCC and have further demonstrated importance of gene-environment interaction in modulating the risk to HNSCC.     

Proteomic screening and identification of differentially distributed membrane proteins in Escherichia coli

Bacteria show asymmetric subcellular distribution of many proteins involved in diverse cellular processes such as chemotaxis, motility, actin polymerization, chromosome partitioning and cell division. In many cases, the specific subcellular localization of these proteins is critical for proper regulation and function. Although cellular organization of the bacterial cell clearly plays an important role in cell physiology, systematic studies to uncover asymmetrically distributed proteins have not been reported previously. In this study, we undertook a proteomics approach to uncover polar membrane proteins in Escherichia coli. We identified membrane proteins enriched in E. coli minicells using a combination of two-dimensional electrophoresis and mass spectrometry. Among a total of 173 membrane protein spots that were consistently detected, 36 spots were enriched in minicell membranes, whereas 15 spots were more abundant in rod cell membranes. The minicell-enriched proteins included the inner membrane proteins MCPs, AtpA, AtpB, YiaF and AcrA, the membrane-associated FtsZ protein and the outer membrane proteins YbhC, OmpW, Tsx, Pal, FadL, OmpT and BtuB. We immunolocalized two of the minicell-enriched proteins, OmpW and YiaF, and showed that OmpW is a bona fide polar protein whereas YiaF displays a patchy membrane distribution with a polar and septal bias.     

PicU, a second serine protease autotransporter of uropathogenic Escherichia coli

Escherichia coli is the major aetiological agent of urinary tract infections (UTI). Like diarrhoeagenic strains of E. coli, uropathogenic isolates possess virulence determinants that distinguish them from commensal strains and allow them to produce the clinical manifestations associated with UTI. Several autotransporter proteins have been associated with the ability of E. coli, and other Gram-negative bacteria, to cause disease. Recently, we described the existence within uropathogenic E. coli (UPEC) strains of Sat, a toxin of the serine protease autotransporter of Enterobacteriaceae (SPATE) subfamily. Using features common to proteins secreted via the autotransporter pathway we have identified nine additional autotransporter proteins from the genomic sequence data of UPEC CFT073. Surprisingly, two additional members of the SPATE subfamily were identified. One protein, designated PicU, was homologous to the Pic protein identified in Shigella flexneri and enteroaggregative E. coli. The PicU protein was expressed and investigated for functional activity.     

Coupled in vitro import of U snRNPs and SMN, the spinal muscular atrophy protein

Cytoplasmic assembly of Sm-class small nuclear ribonucleoproteins (snRNPs) is a central process in eukaryotic gene expression. A large macromolecular complex containing the survival of motor neurons (SMN) protein is required for proper snRNP assembly in vivo. Defects in SMN function lead to a human neuromuscular disorder, spinal muscular atrophy (SMA). SMN protein localizes to both nuclear and cytoplasmic compartments, and a reduction in nuclear levels of SMN is correlated with the disease. The mechanism of SMN nuclear import, however, is unknown. Using digitonin-permeabilized cells, we show that SMN import depends on the presence of Sm snRNPs. Conversely, import of labeled U1 snRNPs was SMN complex dependent. Thus, import of SMN and U snRNPs are coupled in vitro. Furthermore, we identify nuclear import defects in SMA patient-derived SMN mutants, uncovering a potential mechanism for SMN dysfunction.     

Library on a slide for bacterial comparative genomics

Background  

We describe a novel application of microarray technology for comparative genomics of bacteria in which libraries of entire genomes rather than the sequence of a single genome or sets of genes are arrayed on the slide and then probed for the presence or absence of specific genes and/or gene alleles.