Determinants of expression variability

The amount of tissue-specific expression variability (EV) across individuals is an essential characteristic of a gene and believed to have evolved, in part, under functional constraints. However, the determinants and functional implications of EV are only beginning to be investigated. Our analyses based on multiple expression profiles in 41 primary human tissues show that a gene’s EV is significantly correlated with a number of features pertaining to the genomic, epigenomic, regulatory, polymorphic, functional, structural and network characteristics of the gene. We found that (i) EV of a gene is encoded, in part, by its genomic context and is further influenced by the epigenome; (ii) strong promoters induce less variable expression; (iii) less variable gene loci evolve under purifying selection against copy number polymorphisms; (iv) genes that encode inherently disordered or highly interacting proteins exhibit lower variability; and (v) genes with less variable expression are enriched for house-keeping functions, while genes with highly variable expression tend to function in development and extra-cellular response and are associated with human diseases. Thus, our analysis reveals a number of potential mediators as well as functional and evolutionary correlates of EV, and provides new insights into the inherent variability in eukaryotic gene expression.     

(2')3',5'-Bisphosphate nucleotidase

(2')3',5'-Bisphosphate nucleotidase has been prepared in electrophoretically homogeneous form from guinea pig liver. The enzyme catalyzes the hydrolysis of the 2'- or 3'-phosphate from the appropriate nucleoside 2',5'- and 3',5'-bisphosphates and is active with 3'-phosphoadenosine 5'-phosphosulfate and with coenzyme A but not with ATP. The 40,000-dalton protein is a monomer that requires Mg2+ for activity.     

Chromium(III)-insulin derivatives and their implication in glucose metabolism

Insulin has been reacted with five chromium(III) complexes that are capable of relatively facile substitution of aquo ligands. The new Cr(III) insulin derivatives have been characterized by means of electronic and infrared spectra, and evidence for major changes in the protein structure, including the state of aggregation, has been presented. Supporting evidence for the arguments favoring the beneficiary role of chromium(III) in glucose metabolism has been obtained using in vivo studies, and it has been shown that insulin derived with Cr(salen) (H2O)2+ is capable of reversing the blood sugar, serum cholesterol, and phospholipids levels to those of normal rats. The results emphasize the dependence of biopotency on the structure of Cr(III) complexes used for derivation of insulin and discount the postulates that Cr(III) serves to assemble insulin and receptor units through metal-sulphur bonding. The influence of Cr(III) on the structural stability and state of aggregation of insulin and their possible role in glucose metabolism is discussed.     

Chymotrypsin-catalyzed hydrolysis of chromium (III) derivatives of insulin: evidence for stabilization of the protein through interactions with metal ions

We investigated the chymotrypsin-promoted hydrolysis of a series of chromium(III)-insulin complexes containing chelating or macrocyclic ligands. It has been shown that Cr(III) stabilizes insulin against the chymotrypsin-promoted hydrolysis of the protein. The molecular weights of Cr(III) containing peptides have been estimated to be of the order of 2,700-3,700 daltons. The Cr(III) containing peptides are richer in glutamic acid than the intact insulin and are devoid of any isoleucine. High molecular weights and the observed glutamic acid/histidine ratios in Cr(III) containing peptides have been rationalized in terms of Cr(III) being associated with insulin aggregates rather than the monomer of the protein. The chymotrypsin hydrolysis of Cr(III) insulin derivatives is influenced markedly by the nature, charge, and type of Cr(III) complex with which the protein has been reacted. Arguments have been advanced that chymotrypsin-promoted hydrolysis of insulin Cr(III) derivatives does not lead to cleavages at or near every tyrosine residue.     

Characteristics of tripeptide transport in human jejunal brush-border membrane vesicles

These studies are aimed at characterizing the transport of the tripeptide, glycylglycyl-L-proline (GlyGlyPro) across human jejunal brush-border membrane vesicles. GlyGlyPro (0.65 mM) was hydrolyzed by brush-border membrane vesicles with the extent of hydrolysis per mg protein being 23% at 0.5 min, 57% at 1 min and complete hydrolysis at 60 min. Treatment of the membrane vesicles with gel-complexed papain (to remove membrane peptidases) resulted in minimal hydrolysis of GlyGlyPro up to 10 min of incubation. Measurement of GlyGlyPro influx with papain-treated vesicles in the presence of increasing medium osmolarity showed that uptake occurred into an osmotically reactive intravesicular space. Transport of GlyGlyPro with normal and papain-treated membrane vesicles was similar in the presence of an inward Na+ or K+ gradient. No overshoot phenomenon was observed in the presence of an inward proton gradient (extravesicular pH 5.5; intravesicular pH 7.5). An interior negative membrane potential induced by a K+ diffusion potential in the presence of valinomycin stimulated the uptake of the peptide. The effect of increasing concentrations on initial rates of GlyGlyPro uptake revealed the presence of a saturable component as well as a diffusional component. Preloading the membrane vesicles with 20 mM glycylsarcosylsarcosine stimulated uptake by 4-fold. Uptake of GlyGlyPro was inhibited greater than 50% by dipeptides and tripeptides and less than 15% by free amino acids. These results indicate that GlyGlyPro uptake in jejunal brush-border membrane vesicles is not energized by a Na+ or proton gradient and that transport occurs by carrier-mediated and diffusional processes.     

Prevalence of Muellerius capillaris in free-ranging spotted deer (Cervus axis) in India and its experimental cross-transmission to goats

A survey to assess the prevalence of parasitic infections among axis deer (Cervus axis) in three National Parks in India revealed infections with the lungworm Muellerius capillaris. Clinical signs were not evident in infected animals. Therefore, it is suggested that C. axis is probably a carrier of the infection. Under laboratory conditions, terrestrial molluscs (Macrochalamys sp.) were infected with first stage larvae of M. capillaris collected from fecal pellets of C. axis. Feeding of third stage larvae collected from these snails on day 14 post exposure produced patent infections in goats. On day 31 post infection, adult M. capillaris could be collected from the lungs of infected goats. This study establishes the possibility of cross-transmission of M. capillaris between wild and domestic animals in India.     

Partial purification and some properties of a latent CO2 reductase from green potato tuber chloroplasts

We have partially purified the CO2 reductase, present in green potato tuber chloroplasts, as a latent form. Illumination of the chloroplasts in the absence of substrate, bicarbonate, activated the enzyme, which could then be obtained in soluble forms. Purification of the enzyme was achieved by (NH4)2SO4 fractionation (0-30%) and adsorption and elution from a DEAE-Sephadex A-50 column. The final preparation showed 15-fold purification and 50% recovery of the activity. The pH optimum for CO2 reductase was 8.0. Hepes and Tricine buffers showed maximum activity whereas Tris/phosphate or borate failed to show any activity. The enzyme reaction was sensitive to the presence of metal ions like Fe3+, Hg2+, Cu2+, Mo6+ and Zn2+, however, a threefold activation was observed with Fe2+. The metal requirement for CO2 reductase was evident from the observed inhibition by metal chelators like o-phenanthroline, alpha, alpha'-dipyridyl, bathocuproine, 8-hydroxyquinoline etc. Out of these o-phenanthroline was the strongest inhibitor and its concentration for 50% inhibition was 40 microM. The presence of Fe2+ ions in the reaction mixture protected the enzyme from heat denaturation upto 50 degrees C. Maximum enzyme activity was observed at 15 degrees C. The enzyme activity showed a 30-s lag period and the maximum was reached in 90 s. Supplementation of sodium dithionite in the reaction activated enzyme activity threefold, suggesting involvement of dithiol groups in the catalytic activity. There was strong inhibition by -SH inhibitors like 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide and -SH reagents like dithiothreitol, 2-mercaptoethanol and cysteine. Various nucleotide coenzyme tried inhibited the enzyme strongly.