The cholesterol-complexing agent digitonin modulates ligand binding of the bovine hippocampal serotonin 1A receptor

The serotonin(1A) (5-HT(1A)) receptor is an important member of the superfamily of seven transmembrane domain G-protein-coupled receptors. We have examined the modulatory role of cholesterol on the ligand binding of the bovine hippocampal 5-HT(1A) receptor by cholesterol complexation in native membranes using digitonin. Complexation of cholesterol from bovine hippocampal membranes using digitonin results in a concentration-dependent reduction in specific binding of the agonist 8-OH-DPAT and antagonist p-MPPF to 5-HT(1A) receptors. The corresponding changes in membrane order were monitored by analysis of fluorescence polarization data of the membrane depth-specific probes, DPH and TMA-DPH. Taken together, our results point out the important role of membrane cholesterol in maintaining the function of the 5-HT(1A) receptor. An important aspect of these results is that non-availability of free cholesterol in the membrane due to complexation with digitonin rather than physical depletion is sufficient to significantly reduce the 5-HT(1A) receptor function. These results provide a comprehensive understanding of the effects of the sterol-complexing agent digitonin in particular, and the role of membrane cholesterol in general, on the 5-HT(1A) receptor function.     

Versatility and commercial status of microbial keratinases: a review

The world’s increasing population and shortage of food and feed is creating an urgently for us to look for new protein sources from waste products like keratinous waste. Poor management of these wastes has made them one of the major recalcitrant pollutants in nature. Microbial keratinases offers an economic and eco-friendly alternative for degrading and recycling keratinous waste into valuable byproducts. Diverse groups of microorganisms viz., bacteria, fungi and actinomycetes have the ability to degrade recalcitrant keratin by producing keratinase enzyme. Microbial keratinases exhibits great diversity in its biochemical properties with respect to activity and stability in various pH and temperature ranges as well as in the range of recalcitrant proteins it degrades like those present in feathers, hairs, nails, hooves etc. Owing to diverse properties and multifarious biotechnological implications, keratinases can be considered as promising biocatalysts for preparation of animal nutrients, protein supplements, leather processing, fiber modification, detergent formulation, feather meal processing for feed and fertilizer, the pharmaceutical, cosmetic and biomedical industries, and waste management. This review article presents an overview of keratin structure and composition, mechanism of microbial keratinolysis, diversity of keratinolytic microorganisms, and their potential applications in various fields.     

Molecular biology of C4 phosphoenolpyruvate carboxylase: Structure,regulation and genetic engineering

Three to four families of nuclear genes encode different isoforms of phosphoenolpyruvate (PEP) carboxylase (PEPC): C₄-specific, C₃ or etiolated, CAM and root forms. C₄ leaf PEPC is encoded by a single gene (ppc) in sorghum and maize, but multiple genes in the C₄-dicot Flaveria trinervia. Selective expression of ppc in only C₄-mesophyll cells is proposed to be due to nuclear factors, DNA methylation and a distinct gene promoter. Deduced amino acid sequences of C₄-PEPC pinpoint the phosphorylatable serine near the N-terminus, C₄-specific valine and serine residues near the C-terminus, conserved cysteine, lysine and histidine residues and PEP binding/catalytic sites. During the PEPC reaction, PEP and bicarbonate are first converted into carboxyphosphate and the enolate of pyruvate. Carboxyphosphate decomposes within the active site into Pi and CO₂, the latter combining with the enolate to form oxalacetate. Besides carboxylation, PEPC catalyzes a HCO₃ ^--dependent hydrolysis of PEP to yield pyruvate and Pi. Post-translational regulation of PEPC occurs by a phosphorylation/dephosphorylation cascade in vivo and by reversible enzyme oligomerization in vitro. The interrelation between phosphorylation and oligomerization of the enzyme is not clear. PEPC-protein kinase (PEPC-PK), the enzyme responsible for phosphorylation of PEPC, has been studied extensively while only limited information is available on the protein phosphatase 2A capable of dephosphorylating PEPC. The C₄ ppc was cloned and expressed in Escherichia coli as well as tobacco. The transformed E. coli produced a functional/phosphorylatable C₄ PEPC and the transgenic tobacco plants expressed both C₃ and C₄ isoforms. Site-directed mutagenesis of ppc indicates the importance of His¹³⁸, His⁵⁷⁹ and Arg⁵⁸⁷ in catalysis and/or substrate-binding by the E. coli enzyme, Ser⁸ in the regulation of sorghum PEPC. Important areas for further research on C₄ PEPC are: mechanism of transduction of light signal during photoactivation of PEPC-PK and PEPC in leaves, extensive use of site-directed mutagenesis to precisely identify other key amino acid residues, changes in quarternary structure of PEPC in vivo, a high-resolution crystal structure, and hormonal regulation of PEPC expression.Abbreviations OAA oxalacetate - PEP phosphoenolpyruvate - PEPC PEP carboxylase - PEPC-PK PEPC-protein kinase - PPDK pyruvate, orthophosphate dikinase - Rubisco ribulose 1,5-bis-phosphate carboxylase/oxygenase - CAM Crassulacean acid metabolism     

EFFECTS OF DIETARY PROTEINS ON FETAL BRAIN PROTEIN AND GLUTAMIC ACID METABOLISM IN RAT

Abstract— The effects of feeding dietary wheat and Bengal gram proteins to pregnant rats on brain protein and glutamic acid metabolism in 15-, 17- and 19-day fetuses were investigated. Wheat and Bengal gram diets resulted in loss of brain weight with decreased DNA, RNA, protein, free x amino N and deficits in the activities of brain glutamine synthetase, glutaminase I. glutaminase II and glutamate decarboxylase at all the gestational ages studied without any change in glutamine transferase activity. The concentrations of the amino acids alanine, glutamic acid, glutamine and GABA were found to be significantly lower on wheat and Bengal gram diets than the control on a 10% casein diet. The wheat with lysine and Bengal gram with methionine, cystine and tryptophan resulted in similar mean values of all the characteristics studied to the mean values observed in rats on the control diet. However, glutaminase I activity remained significantly low on lysine fortified wheat diet, and aspartic acid content was found to increase on both fortified and unfortified wheat and Bengal gram diets. A 20% casein diet showed increased brain weight, DNA. RNA. protein and free x amino N concentrations as compared with the 10% casein diet, while the other parameters remained unchanged.     

The heterotrophic eubacterial and archaeal co-inhabitants of the halophilic Dunaliella salina in solar salterns fed by Bay of Bengal along south eastern coast of India

Halophilic microbes are studied to understand the metabolic pathways adopted by organisms in such extreme environment and for their biotechnological exploitation. In thallosohaline environments worldwide, the autotrophic alga Dunaliella salina Teodoresco is omnipresent, but it is being recently realised that the heterotrophic components vary in different regions. The unexplored eastern coastline of India abutted by Bay of Bengal was investigated for the heterotrophic halophilic microbes in this region. The waters in the salterns – replicas of natural hyper-saline water bodies of that region, were collected at four sites along 650 km of the coastal belt. In cultures set up from these waters, green and pink colonies were observed. The green colonies were found to be those of D. salina while the pink colonies were of heterotrophs. To identify the heterotrophic microbes, light microscopy, 16S rRNA typing and pigment profiling through spectrophotometry and HPLC were done. The cells in pink colonies were rod shaped. 16S rRNA typing of cells in these colonies detected the presence of Halomonas sp. – a eubacterium. The pigment profile of cells in pink cultures matched that of the archaea – Halobacterium; bacterioruberin derivatives were found. Thus, it was concluded that Halomonas and Halobacterium spp. are among the co-inhabitant heterotrophs of D. salina. Cultures of D. salina established from these salterns showed the typical three colours seen in the ponds of different sub-plots of salterns. They were green until 30 days, turning dark orange by 60 days and pink when 90 day old. In the 90 day old cultures, innumerable rod shaped cells were found. These cells were similar to the cells of the waters from the ponds of pink sub-plots of salterns and the pink colonies established from saltern waters in the laboratory. In the old (90 days) laboratory cultures of D. salina, the glycerol and proteins released from degenerating cells and the increase in salt concentration to super saturation levels due to evaporation of water in the medium led to the gregarious appearance of the heterotrophs – the co-inhabitants in natural environment.     

Chromatin-bound PCNA as S-phase marker in mononuclear blood cells of patients with acute lymphoblastic leukaemia or multiple myeloma

Objectives: Proliferating cell nuclear antigen (PCNA) has often been used as a marker to aid assessment of tumour growth fraction. This paper addresses the question of whether it can be used as an S‐phase marker, when the non‐chromatin‐bound form of the protein is removed by pepsin treatment. Materials and methods: Cytofluorometric measurements were carried out after immunofluorescence staining of PCNA and counterstaining of DNA. S‐phase fraction was determined with the help of windows on PCNA versus DNA scattergrams, or mathematically from DNA histograms. Results: S‐phase fractions obtained using the two methods correlated well, but did not always agree, exact discrepancies depending on the mathematical model used for histogram analysis. Conclusions: Determination of S‐phase fractions with the help of PCNA immunofluorescence staining is possible, and probably more reliable than calculation of S‐fractions from DNA histograms. It thus offers an alternative to assays involving BrdU labelling in vivo.     

Structure-guided affinity maturation of a single-chain variable fragment antibody against the Fu-bc epitope of the dengue virus envelope protein

Dengue is one of the most dominant arthropod-borne viral diseases, infecting at least 390 million people every year throughout the world. Despite this, there is no effective treatment against dengue, and the only available vaccine has already been withdrawn owing to the significant adverse effects. Therefore, passive immunotherapy using monoclonal antibodies is now being sought as a therapeutic option. To date, many dengue monoclonal antibodies have been identified, most of which are serotype-specific, and only a few of which are cross-reactive. Furthermore, antibodies that cross-react within serotypes are weakly neutralizing and frequently induce antibody-dependent enhancement, which promotes viral entry and replication. Therefore, broadly neutralizing antibodies with no risk of antibody-dependent enhancement are required for the treatment of dengue. Here, we developed a single-chain variable fragment (scFv) antibody from an anti-fusion loop E53 antibody (PDB: 2IGF). We introduced previously predicted favorable complementarity-determining region (CDR) mutations into the gene encoding the scFv antibody for affinity maturation, and the resultant variants were tested in vitro against the highly conserved fusion and bc epitope of the dengue virus envelope protein. We show some of these scFv variants with two to three substitution mutations in three different CDRs possess affinity constants (K_D) ranging from 20 to 200 nM. The scFv-mutant15, containing D31L, Y105W, and S227W substitutions, showed the lowest affinity constant, (K_D = 24 ± 7 nM), approximately 100-fold lower than its parental construct. We propose that the scFv-derivative antibody may be a good candidate for the development of an effective and safe immunotherapy.     

Growth promotion activity and biological control for the management of leaf blight incited by Alternaria helianthi

The Pseudomonas fluorescens (Pf1), Bacillus subtilis (Bs1) are the major potential biocontrol agents against foliar pathogens. MPf1 and MBs1 were found to be the most effective in inhibiting the mycelial growth of Alternaria helianthi. These biocontrol agents have the maximum capacity in controlling the spore germination, and data showing the growth-promoting effect of biocontrol agents and inhibition of seed-borne fungi are available. Seed-borne infections of A. helianthi are controlled by seed treatment with P. fluorescens, which showed least seed infection. The root length and shoot length has also been increased.