Production and purification of Clostridium perfringens alpha-toxin using a protein-hyperproducing strain, Bacillus brevis 47

Abstract Clostridium perfringens alpha-toxin was produced in a protein-hyperproducing strain, Bacillus brevis 47, by cloning the gene into the constructed expression-secretion vector which has the multiple promoters and the signal peptide coding region of an outer cell wall protein gene. The amount of alpha-toxin produced by the B. brevis 47 transformant carrying the gene was approximately 10 times greater than that produced by a B. subtilis transformant carrying the toxin gene. Biological activities and the N-terminal amino acid sequence of the toxin secreted by the B. brevis 47 transformant were identical to those of wild-type alpha-toxin.     

Effect of Clostridium perfringens Beta Toxin on Blood Pressure of Rats

Guanethidine treatment or adrenal medullectomy significantly inhibited the elevation in blood pressure induced by Clostridium perfringens beta toxin, and the combination of the two drastically reduced the pressure rise, to less than 19% of that in control rats. When rats were pretreated with tetrodotoxin or hexamethonium, the toxin-evoked rise was significantly inhibited. Elevation in blood pressure induced by the toxin in spinal rats tended to be less than that in control rats. When investigated by a microscopical technique, arteriolar constriction in the mesenteric vasculature was observed after the blood pressure elevation induced by the toxin reached a maximum. Blood flow in the skin decreased with an increase in blood pressure following intravenous injection of the toxin. It is concluded that beta toxin acts on the autonomic nervous system and produces arterial constriction.     

Molecular Structure of Xanthocidin

In order to establish industrial production of 5′-inosinic acid (5′-IMP), a permeability mutant, KY13171, of Brevibacterium ammoniagenes, which accumulated 7 to 8 grams of 5′-IMP per liter and 4 to 6 grams of hypoxanthine (Hx) per liter (calculated as 5′-IMP), was improved by a genetical procedure. Further improved mutants were selected stepwise through repeating mutational work. The finally selected mutant. KY13369, accumulated 20 to 27 grams of 5′-IMP per liter, but not Hx.

Increased productivity of 5′-IMP and decreased productivity of Hx were not caused by the changes in 5′-IMP degrading activity, because these activities were not significantly different among the mutants. These results appear to indicate that the increased accumulation of 5′-IMP may be caused by the improvement in membrane permeability for 5′-IMP. However, the changes in phospholipid and fatty acid compositions were not enough to explain the increased permeability.     

Presence of rhodopsin-like proteins in the planarian head

The presence of rhodopsin-like protein was detected in the head of the freshwater planarian Dugesia japonica japonica Ichikawa et Kawakatsu by use of anti-frog-rhodopsin rabbit IgG. Two membrane proteins of molecular weights 65000 and 62000 were separated by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis and found to react with the anti-rhodopsin IgG. The antibody may be useful for monitoring regeneration of the planarian eye.     

Physiological assay of liposome-mediated transport of a drug across Xenopus intestine: cell-liposome interaction

(1) The antagonistic effect of atropine methyl bromide entrapped in liposomes on contraction of Xenopus intestine in vitro induced by acetylcholine was studied. The results provided some insight into cell-liposome interaction. (2) Acetylcholine (0.1 mM) was added to the medium in the bath (serosal solution), while liposomes containing atropine methyl bromide in their internal and external phases were added on the mucosal side of the intestine. Large multilamellar liposomes were prepared from egg lecithin (phosphatidylcholine, PC) and cholesterol in various molar ratios. Atropine methyl bromide had most effect in liposomes composed of PC and cholesterol in a ratio of 7:3, less in those with a ratio of 4:5, and none in those with a ratio of 9:1. These effects were parallel with the sizes of these liposomes, determined by quasi-elastic light-scattering; that is, the larger the liposomes, the greater was their effect. Addition (to the liposomes) of phosphatidic acid, the negative charge of which increases the distance between the lamellar layers, increased the effect, indicating that atropine methyl bromide in the space between lamellar layers was effective. Another type of liposomes in which atropine methyl bromide was present only in the external phase of liposomes was as effective as liposomes in which atropine methyl bromide was present in both the internal and external phases. (3) From these results the following new model for liposome-mediated stimulation of transport of atropine methyl bromide is proposed. Large multilamellar liposomes have structural defects in their external lamellae through which atropine methyl bromide in the mucosal solution can penetrate into the space between the external lamellar layers and move into intestinal cells through regions of fusion between the outermost layers of the liposomes and the cell membrane.     

Tumour necrosis factor and the lysosomal enzymes of macrophages or macrophage-like cell line

Summary The relationship between tumour necrosis factor (TNF) and macrophages or macrophage-like cell line, especially the lysosomal enzymes was investigated. The serum lysosomal enzymes and LDH activities were increased in proportion to the TNF production even in different strains of mice. Lysosomal enzymes and TNF activity were released into the supernatant of the culture medium of macrophage-enriched peritoneal exudate cells (PEC) or spleen cells derived from Propionibacterium acnes-primed mice after addition of lipopolysaccharide (LPS). After passage through a Sephadex G-10 column, TNF activity could not be detected in the supernatant of these spleen cells after addition of LPS. Also TNF activity could not be detected in the supernatant following destruction of PEC. These results suggest that TNF producibility is strongly related to the degree of activation of macrophages, especially the lysosomal enzymes. The murine macrophage-like cell line, J 774, also released TNF activity and lysosomal enzymes after addition of LPS.     

Specific cleavages of arginyl peptide bonds at basic amino acid pairs by a serine proteinase from the microsomal membranes of rat liver

The specificity of action of a serine proteinase from the microsomal membranes of rat liver was investigated at pH 7.5 and 37 degrees C using various peptides as substrates. HPLC analyses of the peptides produced followed by their amino acid analyses have revealed that the enzyme is a unique endopeptidase specifically cleaving arginyl peptide bonds at paired basic amino acid residues. Thus, the enzyme is suggested to be a kind of processing proteinase involved in the conversion of proproteins to their mature forms. Indeed, the enzyme cleaved specifically the NH2-terminal 20-residue peptide of proalbumin at the Arg-Arg sequence.     

Induction of Neurite Outgrowth through Contactin and Nr-CAM by Extracellular Regions of Glial Receptor Tyrosine Phosphatase β

Receptor protein tyrosine phosphatase β (RPTPβ) is expressed as soluble and receptor forms with common extracellular regions consisting of a carbonic anhydrase domain (C), a fibronectin type III repeat (F), and a unique region called S. We showed previously that a recombinant Fc fusion protein with the C domain (βC) binds to contactin and supports neuronal adhesion and neurite growth. As a substrate, βCFS was less effective in supporting cell adhesion, but it was a more effective promoter of neurite outgrowth than βCF. βS had no effect by itself, but it potentiated neurite growth when mixed with βCF. Neurite outgrowth induced by βCFS was inhibited by antibodies against Nr-CAM and contactin, and these cell adhesion molecules formed a complex that bound βCFS. NIH3T3 cells transfected to express βCFS on their surfaces induced neuronal differentiation in culture. These results suggest that binding of glial RPTPβ to the contactin/Nr-CAM complex is important for neurite growth and neuronal differentiation.