Applications of β-gal-III isozyme from Bacillus coagulans RCS3, in lactose hydrolysis

Bacillus coagulans RCS3 isolated from hot water springs secreted five isozymes i.e. β-gal I-V of β-galactosidase. β-gal III isozyme was purified using DEAE cellulose and Sephadex G 100 column chromatography. Its molecular weight characterization showed a single band at 315 kD in Native PAGE, while two subunits of 50.1 and 53.7 kD in SDS PAGE. β-Gal III had pH optima in the range of 6-7 and temperature optima at 65 °C. It preferred nitro-aryl-β-d-galactoside as substrate having K_m of 4.16 mM with ONPG. More than 85% and 80% hydrolysis of lactose (1-5%, w/v) was recorded within 48 h of incubation at 55 °C and 50 °C respectively and pH range of 6-7. About 78-86% hydrolysis of lactose in various brands of standardized milk was recorded at incubation temperature of 50 °C. These results marked the applications of β-gal III in processing of milk/whey industry.     

Human coronary heart disease: importance of blood cellular miR-2909 RNomics

The characterization of atherosclerosis as a chronic inflammatory disease has triggered extensive research worldwide to dissect the pro- and anti-inflammatory, cellular as well as molecular mechanisms governing the pathogenesis of this dreadful disease. Though several microRNAs have been shown to play crucial role in regulating lipid metabolism and inflammation, we are far from resolving the role of epigenomic signals in etiology of coronary heart disease (CHD). The present study was addressed to understand the role of a novel microRNA, miR-2909, in the regulation of genes involved in the initiation and progression of human coronary occlusion. Peripheral blood mononuclear cells were isolated from human CHD subjects at various stages of coronary occlusion (n = 80) and their corresponding normal healthy counterparts (n = 20). Various experimental strategies involving gene expression and silencing, reporter plasmid assays, and flow cytometric analysis were blend together to address the current problem. The present study shows for the first time that the blood cellular miR-2909 expression increases with the severity of coronary occlusion, exhibiting a strong positive correlation (r = 0.943 at p < 0.01). Further, miR-2909 was shown to regulate genes involved in inflammation, immunity, and oxLDL uptake, thereby contributing significantly to the initiation and progression of CHD patho-physiological process. Based upon these results, we propose that miR-2909 RNomics may be a step forward in understanding human CHD at the epigenomic level and can be exploited for designing new therapeutic strategies as well as diagnostic and prognostic markers for this disease in future.     

Caspase Cleavage Sites in the Human Proteome: CaspDB,a Database of Predicted Substrates

Caspases are enzymes belonging to a conserved family of cysteine-dependent aspartic-specific proteases that are involved in vital cellular processes and play a prominent role in apoptosis and inflammation. Determining all relevant protein substrates of caspases remains a challenging task. Over 1500 caspase substrates have been discovered in the human proteome according to published data and new substrates are discovered on a daily basis. To aid the discovery process we developed a caspase cleavage prediction method using the recently published curated MerCASBA database of experimentally determined caspase substrates and a Random Forest classification method. On both internal and external test sets, the ranking of predicted cleavage positions is superior to all previously developed prediction methods. The in silico predicted caspase cleavage positions in human proteins are available from a relational database: CaspDB. Our database provides information about potential cleavage sites in a verified set of all human proteins collected in Uniprot and their orthologs, allowing for tracing of cleavage motif conservation. It also provides information about the positions of disease-annotated single nucleotide polymorphisms, and posttranslational modifications that may modulate the caspase cleaving efficiency.     

Context-dependent conformation of diethylglycine residues in peptides.

Diethylglycine (Deg) residues incorporated into peptides can stabilize fully extended (C5) or helical conformations. The conformations of three tetrapeptides Boc-Xxx-Deg-Xxx-Deg-OMe (Xxx=Gly, GD4; Leu, LD4 and Pro, PD4) have been investigated by NMR. In the Gly and Leu peptides, NOE data suggest that the local conformations at the Deg residues are fully extended. Low temperature coefficients for the Deg(2) and Deg(4) NH groups are consistent with their inaccessibility to solvent, in a C5 conformation. NMR evidence supports a folded beta-turn conformation involving Deg(2)-Gly(3), stabilized by a 4-->1 intramolecular hydrogen bond between Pro(1) CO and Deg(4) NH in the proline containing peptide (PD4). The crystal structure of GD4 reveals a hydrated multiple turn conformation with Gly(1)-Deg(2) adopting a distorted type II/II' conformation, while the Deg(2)-Pro(3) segment adopts a type III/III' structure. A lone water molecule is inserted into the potential 4-->1 hydrogen bond of the Gly(1)-Deg(2) beta-turn.     

Occipital hair whorl as a racial criterion: a critical assessment

Reports on the incidence of single clockwise occipital whorls as opposed to double and counterclockwise whorls reveal no consistent geographical pattern. Earlier workers arrived at different conclusions. While Bernstein ('25b) and Schwartzburg ('27) suggested a dominant inheritance of counter-clockwise whorls, others like Winchester ('58) and Montagu ('63) believed the clockwise condition to be dominant. In the light of these and some other studies, Scheinfeld's ('56) remarks that chance factor in prenatal development may influence dominance, seem appropriate. The results of present study indicate that clockwise and anti-clockwise conditions show inconsistent variation which may be the result of change element rather than any specific mode of distribution. Two groups from India: 164 Pandits of Kashmir, pre-dominantly Caucasian and 95 Tibetan refugees, predominantly Mongoloid are contrasted. The incidence of a single clockwise whorl among Pandits is 67%, whereas among Tibetans it is 78%. A review of other populations studied shows greater variance within than between the so-called major racial stocks.     

The deduced protein sequence of the human carboxypeptidase N high molecular weight subunit reveals the presence of leucine-rich tandem repeats

Human plasma carboxypeptidase N is a 280-kDa tetramer with two high molecular mass (83-kDa) glycosylated subunits which protect the two 50-kDa catalytic subunits and keep them in the circulation. An initial clone for the 83-kDa subunit was obtained by screening two lambda gt11 human liver cDNA expression libraries with antiserum specific for carboxypeptidase N or the 83-kDa subunit. The libraries were rescreened with the labeled cloned cDNA, and the largest clone obtained (2536-base pair insert) was completely sequenced. The deduced protein sequence matched the sequence of several tryptic peptides from the 83-kDa subunit but did not contain the NH2-terminal sequence. The remaining portion of the protein coding sequence was synthesized by the polymerase chain reaction, cloned, and sequenced. The composite cDNA sequence is 2870 base pairs long with an open reading frame of 1608 base pair coding for a protein of 536 amino acids (Mr = 58,762). The protein sequence contains seven potential N-linked glycosylation sites and a threonine/serine-rich region which is a potential site for attachment of O-linked carbohydrate. The most striking feature is a region (residues 68-355) containing 12 leucine-rich tandem repeats of 24 residues with the following consensus sequence: P-X-X-alpha-F-X-X-L-X-X-L-X-X-L-X-L-X-X-N-X-L-X-X-L (X = any amino acid and alpha = aliphatic amino acids, I, L, or V). This repeating pattern is found in the leucine-rich alpha 2-glycoprotein and in other proteins where it might mediate interactions with macromolecules. This region also contains five sequences with heptad repeating leucine residues comprising a leucine zipper motif. The leucine-rich domain likely constitutes an important structural or functional element in the interactions of the 83- and 50-kDa subunits to form the active tetramer of carboxypeptidase N.