Bacterial cellulases and saccharification of lignocellulosic materials

Five locally isolated bacterial strains produced extracellular cellulase enzymes, primarily CMCase, when grown on different natural and commercial cellulosic substrates. Extracellular CMCase and avicelase activity was higher with the strain CLS-32, a Cytophaga sp., compared to four other strains. The whole-cell preparations of these isolates were found to saccharify cellulosic substrates to reducing sugars. Maximum release of reducing sugar (5.75 mg ml⁻¹) was obtained with CLS-32 using sugar cane bagasse as growth and hydrolysis substrates.     

Modulation of sialic acid-binding proteins of rat uterus in response to changing hormonal milieu

A group of sialic acid binding (SAS) agglutinins has been isolated from the rat uteri at different stages [Proestrus (P), estrus (E) and diestrus (D)] of estrous cycle. Studies of biochemical properties indicate that SAS agglutinins are glycoprotein in nature having molecular weights between 28–31 Kd and microheterogenous pI. Function-based characterization revealed that inspite of the fact that all three proteins exhibit sialic acid binding property, the sialic acid binding affinities, calculated from Scatchard analysis, using 4-methylumbelliferyl sialic acid as a ligand, varied in stage specific manner (Ka:D-SAS-9.03×10⁵ M^–1, P-SAS-2.33×10⁵ M^–1, E-SAS-2.13×10⁵ M^–1). Circular dichroism spectra of these three agglutinins suggested that differences exist in the secondary structures of the proteins isolated from different stages. Removal of carbohydrate moiety by trifluoromethane sulfonic acid treatment and CNBr cleavage studies showed some homology between these proteins, however, the variation in the carbohydrate moiety was apparent from the sugar analysis data. Functionally and immunologically these proteins can be grouped as estrogenic and progestogenic SAS agglutinins.     

A Puzzle Unsolved: Failure to Observe Different Effects of God and Religion Primes on Intergroup Attitudes

Religious priming has been found to have both positive and negative consequences, and recent research suggests that the activation of God-related and community-related religious cognitions may cause outgroup prosociality and outgroup derogation respectively. The present research sought to examine whether reminders of God and religion have different effects on attitudes towards ingroup and outgroup members. Over two studies, little evidence was found for different effects of these two types of religious primes. In study 1, individuals primed with the words “religion”, “God” and a neutral control word evaluated both ingroup and outgroup members similarly, although a marginal tendency towards more negative evaluations of outgroup members by females exposed to religion primes was observed. In study 2, no significant differences in attitudes towards an outgroup member were observed between the God, religion, and neutral priming conditions. Furthermore, the gender effect observed in study 1 did not replicate in this second study. Possible explanations for these null effects are discussed.     

Identification and characterization of the herpes simplex virus type 2 gene encoding the essential capsid protein ICP32/VP19c.

We describe the characterization of the herpes simplex virus type 2 (HSV-2) gene encoding infected cell protein 32 (ICP32) and virion protein 19c (VP19c). We also demonstrate that the HSV-1 UL38/ORF.553 open reading frame (ORF), which has been shown to specify a viral protein essential for capsid formation (B. Pertuiset, M. Boccara, J. Cebrian, N. Berthelot, S. Chousterman, F. Puvian-Dutilleul, J. Sisman, and P. Sheldrick, J. Virol. 63: 2169-2179, 1989), must encode the cognate HSV type 1 (HSV-1) ICP32/VP19c protein. The region of the HSV-2 genome deduced to contain the gene specifying ICP32/VP19c was isolated and subcloned, and the nucleotide sequence of 2,158 base pairs of HSV-2 DNA mapping immediately upstream of the gene encoding the large subunit of the viral ribonucleotide reductase was determined. This region of the HSV-2 genome contains a large ORF capable of encoding two related 50,538- and 49,472-molecular-weight polypeptides. Direct evidence that this ORF encodes HSV-2 ICP32/VP19c was provided by immunoblotting experiments that utilized antisera directed against synthetic oligopeptides corresponding to internal portions of the predicted polypeptides encoded by the HSV-2 ORF or antisera directed against a TrpE/HSV-2 ORF fusion protein. The type-common immunoreactivity of the two antisera and comparison of the primary amino acid sequences of the predicted products of the HSV-2 ORF and the equivalent genomic region of HSV-1 provided evidence that the HSV-1 UL38 ORF encodes the HSV-1 ICP32/VP19c. Analysis of the expression of the HSV-1 and HSV-2 ICP32/VP19c cognate proteins indicated that there may be differences in their modes of synthesis. Comparison of the predicted structure of the HSV-2 ICP32/VP19c protein with the structures of related proteins encoded by other herpes viruses suggested that the internal capsid architecture of the herpes family of viruses varies substantially.     

Regulation of ketogenesis, gluconeogenesis and the mitochondrial redox state by dexamethasone in hepatocyte monolayer cultures.

The effects of the glucocorticoid dexamethasone on fatty acid and pyruvate metabolism were studied in rat hepatocyte cultures. Parenchymal hepatocytes were cultured for 24 h with nanomolar concentrations of dexamethasone in either the absence or the presence of insulin (10 nM) or dibutyryl cyclic AMP (1 microM BcAMP). Dexamethasone (1-100 nM) increased the rate of formation of ketone bodies from 0.5 mM-palmitate in both the absence and the presence of BcAMP, but inhibited ketogenesis in the presence of insulin. Dexamethasone increased the proportion of the palmitate metabolized that was partitioned towards oxidation to ketone bodies, and decreased the cellular [glycerol 3-phosphate]. The latter suggests that the increased partitioning of palmitate to ketone bodies may be associated with decreased esterification to glycerolipid. The Vmax. of carnitine palmitoyltransferase (CPT) and the affinity of CPT for palmitoyl-CoA were not affected by dexamethasone, indicating that the increased ketogenesis was not due to an increase in enzymic capacity for long-chain acylcarnitine formation. Dexamethasone and BcAMP, separately and in combination, increased gluconeogenesis. In the presence of insulin, however, dexamethasone inhibited gluconeogenesis. Changes in gluconeogenesis thus paralleled changes in ketogenesis. Dexamethasone decreased the [3-hydroxybutyrate]/[acetoacetate] ratio, despite increasing the rate of ketogenesis and presumably the mitochondrial production of reducing equivalents. The more oxidized mitochondrial NADH/NAD+ redox couple with dexamethasone is probably due either to an increased rate of electron transport or to increased transfer of mitochondrial reducing equivalents to the cytoplasm.     

Production of endothelial progenitor cells obtained from human Wharton's jelly using different culture conditions

Endothelial progenitor cells (EPC) participate in revascularization and angiogenesis. EPC can be cultured in vitro from mononuclear cells of peripheral blood, umbilical cord blood or bone marrow; they also can be transdifferentiated from mesenchymal stem cells (MSC). We isolated EPCs from Wharton's jelly (WJ) using two methods. The first method was by obtaining MSC from WJ and characterizing them by flow cytometry and their adipogenic and osteogenic differentiation, then applying endothelial growth differentiating media. The second method was by direct culture of cells derived from WJ into endothelial differentiating media. EPCs were characterized by morphology, Dil-LDL uptake/UEA-1 immunostaining and testing the expression of endothelial markers by flow cytometry and RT-PCR. We found that MSC derived from WJ differentiated into endothelial-like cells using simple culture conditions with endothelium induction agents in the medium.     

Improving antibody affinity by mimicking somatic hypermutation in vitro.

In vivo affinity maturation of antibodies involves mutation of hot spots in the DNA encoding the variable regions. We have used this information to develop a strategy to improve antibody affinity in vitro using phage display technology. In our experiment with the antimesothelin scFv, SS(scFv), we identified DNA sequences in the variable regions that are naturally prone to hypermutations, selected a few hot spots encoding nonconserved amino acids, and introduced random mutations to make libraries with a size requirement between 10(3) and 10(4) independent clones. Panning of the hot spot libraries yielded several mutants with a 15- to 55-fold increase in affinity compared with a single clone with a fourfold increased affinity from a library in which mutagenesis was done outside the hot spots. The strategy should be generally applicable for the rapid isolation of higher-affinity mutants of Fvs, Fabs, and other recombinant antibodies from antibody phage libraries that are small in size.     

Implications of Using Steady-State Conditions in Estimating Dermal Uptake of Volatile Compounds in Municipal Drinking Water: An Example of THMs

Dermal exposure to volatile compounds (VC) in municipal water while showering is typically estimated using a steady-state condition between VC in water impacting on skin and skin exposed to water. The lag times to achieve steady-state between VC and skin can vary in the range of 7.5–218.3 min, while shower duration is often less than these values. Estimates of dermal exposure to VC using steady-state while showering may misinterpret exposure. This study developed models and estimated exposure to some disinfection byproducts (DBPs) through dermal pathway by considering lag times while showering. Dermal uptakes of VC were compared using different approaches. In the proposed approach, uptakes of trihalomethanes were estimated between 9.55 × 10^?10–1.43 × 10^?8 mg/cm² of skin during the lag times from exposure to water with trihalomethanes of 50 μg/L. These values were higher than the steady-state estimates (1.37 × 10^?10–4.34 × 10^?9 mg/cm²), and lower than the average exposure analysis (4.12 × 10^-8–1.93 × 10^?6 mg/cm²). Using the Drinking Water Surveillance Program data in Ontario, chronic daily intakes of trihalomethanes were estimated to be 9.40 × 10^?7 (1.85 × 10^?7–1.65 × 10^?6), 3.89 × 10^?6 (7.11 × 10^?7–2.33 × 10^?5), and 1.40 × 10^?6 (4.0 × 10^?7–1.77 × 10^?6) mg/kg/day in Toronto, Ottawa, and Hamilton, respectively. The findings can be useful in understanding THMs exposure and risk through dermal pathway.