An Inhibitor of the UDP-N-Acetylgalactosamine:GM3, N-Acetylgalactosaminyltransferase: Purification and Properties, and Preparation of an Antibody to This Inhibitor

An inhibitor of the UDP-N-acetylgalactosamine:GM3, N-acetylgalactosaminyltransferase (EC 2.4.1.92) has been purified close to 100-fold from chicken blood serum. The method of purification includes heating, dialysis, passage through a column of DEAE-Sephadex, filtration through Amicon XM 100, and passage through Sepharose 6B. The molecular weight determined by Sepharose 6B was 200,000, but on sodium dodecyl sulfate-polyacrylamide gel electrophoresis it appears as if the compound dissociated into components of 68,000. The inhibitor was not active on other glycosyl transferases and lost its inhibitory activity following treatment with pronase and trypsin. alpha-Chymotrypsin did not affect the inhibitor. An antibody to this inhibitor was prepared which decreased its inhibitory capability and precipitated with it in a radial double immunodiffusion experiment.     

Effect of hyperoxia acclimation on catalase and glutathione peroxidase activities and in vivo peroxidation products in various tissues of the frog Rana ridibunda perezi

Among vertebrates, adult amphibians are known to be especially tolerant to exposure to high environmental oxygen tensions. To clarify the basis for this high O2 tolerance, adult Rana ridibunda perezi frogs were acclimated for 15 days to water-air phases with either 149 mm Hg O2 (normoxia) or 710 mm Hg O2 (hyperoxia). At the end of the acclimation, various morphometric and biochemical parameters related to oxidative stress were measured in seven organs and tissues. Hyperoxia acclimation did not change either the total weight of the animals or the total and relative wet weights of the organs studied, except for the brain, which showed weight increases in the hyperoxic group. In vivo tissue peroxidation increased in the kidney; decreased in the skeletal muscle and skin; and did not change in the liver, lung, brain, and heart after hyperoxic exposures. Whereas liver, lung, and skin showed glutathione peroxidase (GSH-Px) activities with both cumene hydroperoxide (cumene-OOH) and H2O2 as substrates, skeletal muscle only showed H2O2 GSH-Px activity. Hyperoxia acclimation did not change either catalase (CAT) or GSH-Px activities in any organ, except for the liver in which CAT activity was induced by hyperoxia. Thus hyperoxia tolerance in this species does not need the induction of H2O2-detoxifying enzymes in the majority of the organs. It is suggested that the high O2 tolerance of this amphibian species is related to its comparatively high constitutive GSH-Px activities.     

The functional domains of coagulation factor VIII:C

A lack of factor VIII:C, manifested as a bleeding disorder due to the absence of clot formation, is known as hemophilia A, an X chromosome-linked inherited disease afflicting 1-2 males/10,000. To determine the minimum functional domain(s) essential for factor VIII:C activity, we have expressed the amino-terminal (92-kDa) and carboxyl-terminal (80-kDa) proteolytic cleavage products as individual, secreted polypeptides in monkey cells without the 909-residue central region. We have found that neither terminal domain alone is able to promote coagulation in factor VIII:C-deficient plasma. However, when the 92- and 80-kDa peptides are co-expressed, clotting activity is readily detected. Thus, these two chains alone constitute an active or activatable complex. The central domain is required neither for activity nor for the assembly of an active complex from two chains expressed in trans. These results suggest that a truncated derivative of factor VIII:C may be useful in coagulation therapy.     

Separation and kinetic properties of the molecular forms of chicken liver cytoplasmic aspartate aminotransferase

A method is proposed for the separation of the five molecular forms, alpha, beta, gamma, delta and epsilon, of chicken liver cytoplasmic aspartate aminotransferase free from lactate dehydrogenase activity. These molecular forms varied in isoelectric point, but no differences were observed either in their Michaelis constants or in the degree of their inhibition by excess of 2-oxoglutarate or L-aspartate.     

Anti-oxidant defences and peroxidation in liver and brain of aged rats.

Old rats (28 months), when compared with young adults (9 months), did not show differences in activities of superoxide dismutase (SOD) or selenium-dependent and -independent glutathione peroxidases (GPx), or in levels of GSH, GSSG, GSSG/GSH and endogenous peroxidation in liver and brain. Rates of stimulated peroxidation in vitro were decreased in the livers of old rats. Old animals showed decreased levels of hepatic catalase and glutathione reductase. Nevertheless, when enzyme activities were referred to cytochrome oxidase activity these decreases disappeared, and GPx and SOD (brain) were even increased in old rats.     

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

Investigation of subpopulation heterogeneity and plasmid stability in recombinant escherichia coli via a simple segregated model

Many microbial and cell cultures exhibit phenomena that can best be described using a segregated modeling approach. Heterogeneties are more marked in recombinant cell cultures because subpopulations, which often exhibit different growth and productivity characteristics, are more easily identified by selective markers. A simple segregated mathematical model that simulates the growth of recombinant Escherichia coli cells is developed. Subpopulations of different growth rate, plasmid replication rate, and plasmid segregation probability are explicitly considered. Results indicate that a third mechanism of plasmid instability, referred to here as a "downward selective pressure," is significant when describing plasmid loss in batch and chemostat cultures. Also, the model agrees well with experimental data from cultures under antibiotic selective pressure. Finally, model simulations of chemostat cultures reveal the importance of initial conditions on culture stability and the possible presence of nonrandom partitioning functions. (c) 1993 John Wiley & Sons, Inc.