Protein kinase B/Akt mediates cAMP- and cell swelling-stimulated Na+/taurocholate cotransport and Ntcp translocation

Cyclic AMP and cell swelling stimulate hepatic Na+/TC cotransport and Ntcp translocation via the phosphoinositide 3-kinase signaling pathway. To determine the downstream target of the phosphoinositide 3-kinase action, we examined the role of protein kinase B (PKB)/Akt using SB203580 in hepatocytes as well as by transfection with a dominant negative (DN-PKB) or a constitutively active (CA-PKB) form of PKB in HuH-Ntcp cells. Both cAMP and cell swelling stimulated p38 mitogen-activated protein (MAP) kinase as well as PKB activity. Although 100 microm SB203580 inhibited cell swelling- and 8-chlorophenylthio-cAMP-induced activation of both p38 MAP kinase and PKB, 1 microm SB203580 inhibited activation of p38 MAP kinase, but not of PKB, in hepatocytes. 100 microm, but not 1 microm SB203580, inhibited cell swelling- and cAMP-induced increases in taurocholate (TC) uptake and Ntcp translocation in hepatocytes. TC uptake in HuH-Ntcp cells was more than 90% dependent on extracellular Na+. Cyclic AMP and cell swelling increased TC uptake by 50-100% and PKB activity 2-4-fold in HuH-Ntcp cells transfected with the empty vector and failed to increase PKB activity, TC uptake, and Ntcp translocation in DN-PKB-transfected HuH-Ntcp cells. Transfection with CA-PKB increased PKB activity, TC uptake, and Ntcp translocation in HuH-Ntcp cells compared with cells transfected with the empty vector. In contrast, transfection with DN-PKB did not affect basal PKB activity, TC uptake, or Ntcp translocation. Taken together, these results strongly suggest that cell swelling and cAMP-mediated stimulation of hepatic Na+/TC cotransport and Ntcp translocation requires activation of PKB and is mediated at least in part via a phosphoinositide 3-kinase/PKB-signaling pathway.     

Acephate induced oxidative damage in erythrocytes

The effect of oral administration of acephate (360 mg/kg body weight), for 15 days, daily, was investigated on the erythrocytes of male rats. Activities of acetyl cholinesterase and glucose-6-phosphate dehydrogenase decreased, while those of glutathione-s-transferase and glutathione reductase increased. Decreased glutathione content and increased lipid peroxidation suggest that there was increased oxidative stress in the erythrocytes of treated animals. Increased cholesterol/phospholipid ratio in the erythrocyte membranes and morphological changes in RBCs (scanning electron microscopy studies) were observed in acephate treated animals. The results clearly suggest that acephate induced oxidative stress in erythrocytes leads to morphological changes.     

Effect of UV radiation on thermotolerance,ethanol tolerance and osmotolerance of Saccharomyces cerevisiae VS1 and VS3 strains

After a previous mass screening and enrichment programme for the isolation of thermotolerant yeasts, VS1, VS2, VS3 and VS4 strains isolated from soil samples, collected within the hot regions of Kothagudem Thermal Power Plant, AP, India, had a better thermotolerance, osmotolerance and ethanol tolerance than the other isolates. Among these isolates VS1 and VS3 were best performers. Efforts were made to further improve their osmotolerance, thermotolerance and ethanol tolerance by treating them with UV radiation. Mutants of VS1 and VS3 produced more biomass and ethanol than the parent strains at high temperature and glucose concentrations. The amount of biomass produced by VS1 and VS3 mutants was 0.25 and 0.20 g l(-1) more than the parent strains at 42 degrees C using 2% glucose. At high glucose concentrations VS1 and VS3 mutants produced biomass which was 0.70 and 0.30 g l(-1) at 30 degrees C and 0.10 and 0.20 g l(-1) at 40 degrees C more than the parent strains. The amount of ethanol produced by the mutants (VS1 and VS3) was 8.20 and 1.20 g l(-1) more than the parent strains at 42 degrees C using 150 g l(-1) glucose. More ethanol was produced by mutants (VS1 and VS3) than the parents at high glucose concentrations of 5.0 and 6.0 g l(-1) at 30 degrees C and 13.0 and 3.0 g l(-1) at 42 degrees C, respectively. These results indicated that UV mutagenesis can be used for improving thermotolerance, ethanol tolerance and osmotolerance in VS1 and VS3 yeast strains.     

Rad23 promotes the targeting of proteolytic substrates to the proteasome

Rad23 contains a ubiquitin-like domain (UbL(R23)) that interacts with catalytically active proteasomes and two ubiquitin (Ub)-associated (UBA) sequences that bind Ub. The UBA domains can bind Ub in vitro, although the significance of this interaction in vivo is poorly understood. Rad23 can interfere with the assembly of multi-Ub chains in vitro, and high-level expression caused stabilization of proteolytic substrates in vivo. We report here that Rad23 interacts with ubiquitinated cellular proteins through the synergistic action of its UBA domains. Rad23 plays an overlapping role with Rpn10, a proteasome-associated multi-Ub chain binding protein. Mutations in the UBA domains prevent efficient interaction with ubiquitinated proteins and result in poor suppression of the growth and proteolytic defects of a rad23 Delta rpn10 Delta mutant. High-level expression of Rad23 revealed, for the first time, an interaction between ubiquitinated proteins and the proteasome. This increase was not observed in rpn10 Delta mutants, suggesting that Rpn10 participates in the recognition of proteolytic substrates that are delivered by Rad23. Overexpression of UbL(R23) caused stabilization of a model substrate, indicating that an unregulated UbL(R23)-proteasome interaction can interfere with the efficient delivery of proteolytic substrates by Rad23. Because the suppression of a rad23 Delta rpn10 Delta mutant phenotype required both UbL(R23) and UBA domains, our findings support the hypothesis that Rad23 encodes a novel regulatory factor that translocates ubiquitinated substrates to the proteasome.     

Thermodynamic effects of replacements of Pro residues in helix interiors of maltose-binding protein

Introduction of Pro residues into helix interiors results in protein destabilization. It is currently unclear if the converse substitution (i.e., replacement of Pro residues that naturally occur in helix interiors would be stabilizing). Maltose-binding protein is a large 370-amino acid protein that contains 21 Pro residues. Of these, three nonconserved residues (P48, P133, and P159) occur at helix interiors. Each of the residues was replaced with Ala and Ser. Stabilities were characterized by differential scanning calorimetry (DSC) as a function of pH and by isothermal urea denaturation studies as a function of temperature. The P48S and P48A mutants were found to be marginally more stable than the wild-type protein. In the pH range of 5-9, there is an average increase in T(m) values of P48A and P48S of 0.4 degrees C and 0.2 degrees C, respectively, relative to the wild-type protein. The other mutants are less stable than the wild type. Analysis of the effects of such Pro substitutions in MBP and in three other proteins studied to date suggests that substitutions are more likely to be stabilizing if the carbonyl group i-3 or i-4 to the mutation site is not hydrogen bonded in the wild-type protein.     

From biological databases to platforms for biomedical discovery

The use of high-throughput DNA sequencing and proteomic methods has led to an unprecedented increase in the amount of genomic and proteomic data. Application of computing technologies and development of computational tools to analyze and present these data has not kept pace with the accumulation of information. Here, we discuss the use of different database systems to store biological information and mention some of the key emerging computing technologies that are likely to have a key role in the future of bioinformatics.     

Interaction between the small-nuclear-RNA cap hypermethylase and the spinal muscular atrophy protein,survival of motor neuron

The biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) requires the cytoplasmic assembly of the Sm-core complex, followed by the hypermethylation of the small nuclear RNA (snRNA) 5′ cap. Both the Sm-core complex and the snRNA trimethylguanosine cap are required for the efficient nuclear import of snRNPs. Here, we show that trimethylguanosine synthase 1 (TGS1), the human homologue of the yeast snRNA cap hypermethylase, interacts directly with the survival of motor neuron (SMN) protein. Both proteins are similarly distributed, localizing in the cytoplasm and in nuclear Cajal bodies. The interaction between TGS1 and SMN is disrupted by a mutation in SMN that mimics the predominant isoform of the protein that is expressed in patients with the neurodegenerative disease, spinal muscular atrophy. These data indicate that, in addition to its function in cytoplasmic Sm-core assembly, the SMN protein also functions in the recruitment of the snRNA cap hypermethylase.     

Genomic structures and population histories of linguistically distinct tribal groups of India

There are various conflicting hypotheses regarding the origins of the tribal groups of India, who belong to three major language groups--Austro-Asiatic, Dravidian and Tibeto-Burman. To test some of the major hypotheses we designed a genetic study in which we sampled tribal populations belonging to all the three language groups. We used a set of autosomal DNA markers, mtDNA restriction-site polymorphisms (RSPs) and mtDNA hypervariable segment-1 (HVS-1) sequence polymorphisms in this study. Using the unlinked autosomal markers we found that there is a fair correspondence between linguistic and genomic affinities among the Indian tribal groups. We reconstructed mtDNA RSP haplotypes and found that there is extensive haplotype sharing among all tribal populations. However, there is very little sharing of mtDNA HVS-1 sequences across populations, and none across language groups. Haplogroup M is ubiquitous, and the subcluster U2i of haplogroup U occurs in a high frequency. Our analyses of haplogroup and HVS-1 sequence data provides evidence in support of the hypothesis that the Austro-Asiatic speakers are the most ancient inhabitants of India. Our data also support the earlier finding that some of the western Eurasian haplogroups found in India may have been present in India prior to the entry of Aryan speakers. However, we do not find compelling evidence to support the theory that haplogroup M was brought into India on an "out of Africa" wave of migration through a southern exit route from Ethiopia. On the contrary, our data raise the possibility that this haplogroup arose in India and was later carried to East Africa from India.