High pressure conditions stimulate expression of chloramphenicol acetyltransferase regulated by the lac promoter in Escherichia coli

Abstract Recombinant plasmids with the chloramphenicol acetyltransferase (CAT) structural gene behind several kinds of promoters were tested for expression in Escherichia coli during growth at atmospheric pressure (0.1 MPa) and at high pressure (30 MPa). Expression of the CAT gene from the lac promoter was remarkably activated (approx. 78-fold) by high pressure in the absence of the inducer isopropyl-β-d-thiogalactopyranoside (IPTG). The stimulation of the CAT activity by the lac promoter at high pressure did not simply result from an increased plasmid copy number, because the CAT activities from the other promoters and β-lactamase activities were unaffected at high pressure.     

A two-stage bioreactor system for the production of recombinant proteins using a genetically engineered baculovirus/insect cell system

A two-stage bioreactor scheme was developed for the large-scale production of recombinant proteins using a genetically engineered baculovirus/insect cell system. The first bioreactor was employed for cell growth and the second for cell infection. Silkworm Bm5 cells were infected with a recombinant baculovirus, BmNPV/P5.cat, containing a bacterial chloramphenicol acetyltransferase (CAT) gene under the control of the polyhedrin gene promoter of Bombyx mori nuclear polyhedrosis virus (BmNPV). This recombinant baculovirus has been used as an expression vector for the production of recombinant CAT enzyme. A specific productivity of 82 to 90 mug CAT/(10(6) cells) was obtained using the BmNPV/Bm5 expression system, a yield similar to that achieved using the AcNPV/Sf expression system. Repeated infection of high-density cell cultures did not reduce the specific productivity of the CAT enzyme. Most importantly, the problems associated with the infection of high-density cell cultures were resolved by means of controlled infection conditions and appropriate replenishment of spent culture medium following infection. The glucose uptake rate by the cells following infection was 50% higher than that by the cells before infection. Not only did the infection of high-density cell cultures result in consistent yields of 250 mg/L of CAT enzyme, but also the two-stage bioreactor system was proven to be reliable for a long-term operation beyond 600 h. (c) 1993 John Wiley & Sons, Inc.     

The role of nickel in acetyl-CoA synthesis by the bifunctional enzyme CO dehydrogenase/acetyl-CoA synthase: enzymology and model chemistry

 CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) is one of the four known nickel enzymes. It is a bifunctional protein that catalyzes the oxidation of CO to CO₂ at a nickel iron-sulfur cluster (Cluster C) and a remarkable condensation reaction between a methyl group (donated from a methylated corrinoid iron-sulfur protein), carbon monoxide, and coenzyme A to form acetyl-CoA at a separate nickel iron-sulfur cluster (Cluster A). This review focuses on the current understanding of the structure and function of Cluster A and on related model chemistry. It describes studies that uncovered the first example of a biological organometallic reaction sequence. The mechanism of acetyl-CoA synthesis includes enzymebound methylnickel, iron-carbonyl, and acylmetal intermediates. Discovery of the methylnickel species constituted the first example of an alkylnickel species in biology and unveiled a new biological role for nickel. Received: 10 April 1996 / Accepted: 4 July 1996     

Sympathetic Sprouting: Time Course of Changes of Noradrenergic, Cholinergic, and Serotonergic Markers in the Denervated Rat Hippocampus

Abstract: As a first step for experiments investigating the presynaptic characteristics of sympathetic fibers grown into the denervated hippocampus, we studied the time course of changes of neurochemical markers in the rat hippocampus, subsequent to aspiration lesions of the fimbria-fornix and the overlying callosal and cortical structures. At various postsurgical delays (1, 2, 8, 24, and 40 weeks), the activity of choline acetyltransferase, the high-affinity synaptosomal uptake of choline and noradrenaline, and the concentrations of noradrenaline, serotonin, and 5-hydroxyindoleacetic acid were measured in a dorsal, an intermediate, and a ventral part of the hippocampus. Levels of all markers were significantly reduced shortly (1–2 weeks) after the lesions. However, whereas the cholinergic (choline uptake and choline acetyltransferase activity) and the serotonergic (concentrations of serotonin and 5-hydroxyindoleacetic acid) markers remained significantly reduced for up to 40 weeks, both noradrenergic markers recovered to near-normal (noradrenaline uptake) or even supranormal (noradrenaline concentration) levels, although with clear-cut differences in the time course and the regional characteristics. The noradrenaline content reached control levels already 8 weeks after lesion surgery and was about two to three times higher 40 weeks later, with the most dramatic effects in the ventral hippocampus. In contrast, high-affinity noradrenaline uptake reached control values only 24 weeks after lesion and exceeded them only in the ventral hippocampus 40 weeks after surgery. It is concluded (a) that hippocampal noradrenaline concentration is a more sensitive marker for sympathetic sprouting than high-affinity noradrenaline uptake and (b) that functional in vitro studies on hippocampal sympathetic ingrowth appear to fit optimal conditions in the ventral hippocampus at a delay of at least 40 weeks after surgery.     

Amino acid concentrations and selected enzyme activities in rat auditory,olfactory, and visual systems

Homogenates of specific brain regions of three sensory systems (auditory, olfactory, and visual) were prepared from pigmented Long-Evans Hooded rats and assayed for amino acid concentrations and activities of glutaminase, aspartate aminotransferase (total, cytosolic, and, by difference, mitochondrial), malate dehydrogenase, lactate dehydrogenase, and choline acetyltransferase. Comparing the quantitative distributions among regions revealed significant correlations between AAT and aspartate, between glutaminase and glutamate, between glutamate and glutamine, and between AAT plus glutaminase, or glutaminase alone, and the sum of aspartate, glutamate, and GABA, suggesting a metabolic pathway involving the synthesis of a glutamate pool as precursor to aspartate and GABA. Of the inhibitory transmitter amino acids, GABA concentrations routinely exceeded those of glycine, but glycine concentrations were relatively high in brainstem auditory structures.     

On the origin of mitochondria: a reexamination of the molecular structure and kinetic properties of pyruvate dehydrogenase complex from brewer''s yeast

45Ca2+ incorporated in response to glucose was selectively mobilized from the beta-cell-rich pancreatic islets of ob/ob-mice after raising the intracellular Na+ by removal of K+ or addition of ouabain or veratridine. Also studies of insulin release indicated opposite effects of glucose and Na+ on the intracellular sequestration of calcium. The fact that glucose inhibits insulin release induced by raised intracellular Na+ indicates that this sugar can lower the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+] might well explain previous observations of an inhibitory component in the glucose action on the 45Ca2+ efflux.