Inhibitory effect of cortisone acetate on the stimulation of rat liver cytosol l-serine Pyruvate aminotransferase by dibutyryl adenosine 3′,5′-monophosphate

An injection of cortisone acetate at a dose of 5 mg/100 g body weight concomitant with dibutryl cyclic AMP prevents the increase in the activity of rat liver cytosol serine aminotransferase (L-serine: pyruvate aminotransferase, EC 2.6.1.51) elicited by the nucleotide with a lag of about 2 h. If the glucocorticoid is given 2 h prior to the nucleotide inducer, the lag disappears. The inhibitory effect of cortisone acetate gradually decays and is no longer detectable 12 h following its administration. Theophylline, insulin and glucose at doses which affect significantly the level of tyrosine aminotransferase, have no effect on the level of serine aminotransferase and on the cortisone inhibition. The inhibitory effect of the glucocorticoid on the dibutyryl cyclic AMP-mediated increase in serine aminotransferase diminishes with the age of animals. Increase in the enzyme activity by a single dose of glucagon can also be inhibited by cortisone acetate and actinomycin D as in the case with dibutyrl cyclic AMP as an inducer. The possibility of the existence of a specific inhibitory factor which is formed in response to cortisone acetate is discussed.     

Improving solubility of catalytic domain of human beta-1,4-galactosyltransferase 1 through rationally designed amino acid replacements.

beta-1,4-galactosyltransferase 1 (beta4gal-T1, EC 2.4.1.38) transfers galactose from UDP-galactose to free N-acetyl-D-glucosamine or bound N-acetyl-D-glucosamine-R. Soluble beta4gal-T1, purified from human milk has been refractory to structural studies by X-ray or NMR. In a previous study (Malissard et al. 1996, Eur. J. Biochem. 239, 340-348) we produced in the yeast Saccaromyces cerevisiae an N-deglycosylated form of soluble beta4gal-T1 that was much more homogeneous than the human enzyme, as it displayed only two isoforms when analysed by IEF as compared to 13 isoforms for the native beta4gal-T1. The propensity of recombinant beta4gal-T1 to aggregate at concentrations > 1 mg.mL(-1) prevented structural and biophysical studies. In an attempt to produce a beta4gal-T1 form suitable for structural studies, we combined site-directed mutagenesis and heterologous expression in Escherichia coli. We produced a mutated form of the catalytic domain of beta4gal-T1 (sfbeta4gal-T1mut) in which seven mutations were introduced at nonconserved sites (A155E, N160K, M163T, A168T, T242N, N255D and A259T). Sfbeta4gal-T1mut was shown to be much more soluble than beta4gal-T1 expressed in S. cerevisiae (8.5 mg.mL(-1) vs. 1 mg.mL(-1)). Catalytic activity and kinetic parameters of sfbeta4gal-T1mut produced in E. coli were shown not to differ to any significant extent from those of the native enzyme.     

The Question of a Vomer in Brachiopterygian Fish

Abstract The term vomer was coined over four centuries ago to denote one of the 23 bones normally comprising an adult human skull. As time went on more and more craniates were designated vomerate. To date, however, the issue as to whether brachiopterygian fish are or are not vomerate has remained unresolved. Those who consider these fish to be vomerate have suggested at least three different palatal bones as the homologue of the human vomer. Examination of embryological material of Polypterus senegalus shows that this brachiopterygian actually develops a vomer which eventually becomes united indistinguishably with the anterior part of the parasphenoid. The vomer is suggested to have arisen through the fusion of dental plates belonging to a supplementary set of buccopharyngeal denticulate bones. The other subethmoidal bones of the brachiopterygian palate seem likely to be derivatives of the tooth plates which originally were applied to the first two pairs of infrapharyngeals. When these endoskeletal elements were put to use for encapsulation of the organs of smell early in craniate phylogeny, some of the tooth plates that covered their inner surfaces fused to provide anterior palatal bones. Identified at one time or another as vomers, these bones are here termed glochinals and dacnils.     

Plant regeneration from protoplasts of the monocotyledonous Haworthia magnifica v. Poelln.

Calli were initiated from flower buds, gynoecia and inflorescence segments of Haworthia magnifica v. Poelln. and subcultured on solid medium. Two liquid culture steps were necessary to prepare the calli for the isolation of protoplasts capable of sustained cell divisions. Plants were regenerated from protoplast-derived calli. The influence of both the osmolality of the culture media and exudates on the viability of protoplasts and protoplast-derived cell colonies is briefly discussed.     

Semicontinuous penicillin production by two Penicillium chrysogenum strains immobilized in calcium alginate beads

Summary The growth rates of immobilized Penicillium chrysogenum strains are important in their application to semicontinuous penicillin production. Immobilized P. chrysogenum strains produced about 10–15% less biomass but about 1–2 times more penicillin than free suspended mycelia.In a chemically defined medium an industrial P. chrysogenum strain, S₁, produced about 10–12 times more penicillin than strain ATCC 12690. In a complex medium the immobilized P. chrysogenum S₁ produced about 12% penicillin more than in shaken cultures. In bubble column fermentations, penicillin production was 163% higher in the complex medium than in the chemically defined medium.     

Integration of hepatitis B virus: analysis of unoccupied sites.

Hepatitis B virus (HBV) sequences integrated in the PLC/PRF/5 cell line (Alexander cells), which was derived from a human primary liver carcinoma, were previously extensively studied. Here we describe the analysis of the unoccupied sites of two linearly integrated forms of HBV DNA, AL-14 and AL-26, that were characterized previously. No major cellular DNA rearrangements were seen at the integration sites except for small deletions of host sequences: 2 kilobases of DNA in AL-14 and 17 base pairs (bp) in AL-26. The unoccupied site of AL-26 was found to be missing 182 bp, which previously mapped next to the right end of the integration sites of several independent clones. These were believed to be of cellular origin, but we show here that these 182 bp are in fact from unusual HBV sequences. Surprisingly, a region of this newly detected HBV DNA sequence is more homologous to that of woodchuck HBV DNA. Our analysis shows that the normal counterparts of both AL-14 and AL-26 contain minisatellite-like repetitive sequences. Based on the data presented here and our previous finding of HBV DNA integration at satellite sequences, we propose that genomic simple repetitive sequences are hot spots for HBV DNA integration.     

The involvement of biological quinones in the formation of hydroxyl radicals via the Haber-Weiss reaction

The present investigation was made to evaluate biologically relevant quinones as possible catalysts in the generation of hydroxyl radicals from hydrogen peroxide and superoxide radicals. ESR spectra demonstrated that menadione, plastoquinone, and ubiquinone derivatives could all be reduced to their semiquinone forms by electron transfer from superoxide radicals. Reductive homolytic cleavage of H₂O₂ was observed to be dependent upon the presence of semiquinones in the reaction medium. Our data strongly support the concept that quinones normally involved in physiological processes may also play a role as catalysts of the Haber-Weiss reaction in the biological generation of hydroxyl radicals.     

Developmental changes of nerve growth factor and its mRNA in the rat hippocampus: Comparison with choline acetyltransferase

Previous experiments have demonstrated that in the septo-hippocampal system choline acetyltransferase (ChAT) is induced by nerve growth factor (NGF) (Gnahn et al. (1983) Dev. Brain Res. 9, 45-52) and that hippocampal NGF and mRNANGF levels are correlated with the density of cholinergic innervation (Korsching et al. (1985) EMBO J. 4, 1389-1393). In the present investigation we have compared the developmental changes of ChAT, NGF, and mRNANGF levels in this system. During the postnatal development of the hippocampus the time courses of NGF and ChAT were well correlated including the most rapid increase between P12 and P14. This increase in hippocampal NGF was preceded by a corresponding increase in mRNANGF. The developmental changes in hippocampal NGF levels were also closely reflected by corresponding changes in the septum. This, together with previous observations (Korsching et al., 1985) that the adult septum, in spite of relatively high NGF levels, does not contain measurable quantities of mRNANGF, suggests that the NGF levels in the septum are determined by the quantity of NGF transported retrogradely from the field of innervation rather than by local synthesis. During the prenatal period hippocampal NGF levels were relatively high, whereas the mRNANGF was below the level of detection. Since the ingrowth of septal fibers, and with that also the removal of NGF by retrograde transport, begins around birth, the relatively high prenatal NGF levels probably result from an accumulation produced by a small copy number of mRNANGF prior to the removal of NGF by retrograde axonal transport. It is concluded that the correlation of the developmental changes in NGF and mRNANGF with the ChAT activity in the hippocampus further supports the concept of a physiological role of NGF in the central nervous system.     

Pyridine nucleotide analog interference with metabolic processes in mitogen-stimulated human T lymphocytes

The differential metabolic effects of three nicotinamide analogs, 6-aminonicotinamide, 3-aminobenzamide, and 5-methylnicotinamide, were analyzed in mitogen-stimulated preparations of human T lymphocytes. Mitogen stimulation with the phorbol ester TPA and a monoclonal antibody to the T3 cell surface antigen caused an increase in cellular NAD and ATP levels and a marked increase in glucose metabolism as demonstrated by an increase in cellular levels of glucose 6-phosphate and a sevenfold increase in radioactive CO2 formation from [l-14C]glucose. 6-Aminonicotinamide had drastic inhibitory effects on the mitogen-stimulated increases in NAD and ATP levels as well as on the metabolism of glucose. Treatment of the mitogen-stimulated cells with 6-aminonicotinamide also caused a marked increase in cellular levels of 6-phosphogluconate, suggesting inhibition of the hexose monophosphate shunt at 6-phosphogluconate dehydrogenase. Radioactive CO2 formation from [6-14C]glucose showed that metabolism through the tricarboxylic acid cycle was not used to compensate for the inhibition of the hexose monophosphate shunt pathway. Treatment of cells with 3-aminobenzamide had the opposite effect of 6-aminonicotinamide in that cellular NAD levels increased, presumable due to inhibition of poly(ADP-ribose) polymerase. 3-Aminobenzamide did not interfere with ATP or glucose 6-phosphate levels and did not cause significant elevations of 6-phosphogluconate. Thus, 6-aminonicotinamide appears to have direct inhibitory effects on the synthesis of both pyridine nucleotides and poly(ADP-ribose), whereas 3-aminobenzamide has its major inhibitory effect on poly(ADP-ribose) synthesis. 5-Methylnicotinamide also interferes with the mitogen-stimulated increase in NAD levels but not as effectively as 6-aminonicotinamide. The alterations in pyridine nucleotide metabolism resulting from treatment with these nicotinamide analogs can produce drastic and diverse alterations in pathways of glucose utilization and energy generation.