N-Acetylornithine-δ-aminotransferase-deficient and N-Acetylglutamokinase-deficient Arginine Auxotrophs of Corynebacterium glutamicum

An N-acetylglutamokinase-deficient arginine-requiring mutant, KY9390 and an N-acetylornithine aminotransferase-deficient arginine-requiring mutant, AA-7 were derived from a wild-type strain and an l-arginine-producing mutant of Corynebacterium glutamicum, respectively. KY 9390 accumulated 7.5 mg per ml of N-acetylglutamic acid and AA-7 accumulated 2 mg per ml of N-acetylglutamate-γ-semialdehyde, intermediates of arginine biosynthesis, in a culture medium.

The production of N-acetylglutamate-γ-semialdehyde by AA-7 was not affected by the concentration of l-arginine in the medium, whereas that of N-acetylglutamic acid by KY 9390 was inhibited by the addition of l-arginine in the medium.     

An Arabinogalactan-protein from Rape Leaves

A l-fucose-containing arabinogalactan-protein that strongly inhibited hemagglutination by eel anti-H agglutinin of human O erythrocytes was purified from hot phosphate-buffered saline extracts of mature leaves of rape, Brassica campestris. The purified glycoconjugate consisted of 90% of the polysaccharide moiety comprising l-fucose, l-arabinose, d-galactose, 4-O-methyl-d-glucuronic acid, and d-glucuronic acid, and 4% of the hydroxyproline-rich protein portion. Upon methylation, periodate oxidation, and enzymatic degradation, we found that consecutive β-(→3)-linked d-galactopyranosyl residues constituted a backbone chain of the polysaccharide moiety, to which the side chains of β-(→6)-linked d-galactopyranosyl residues were attached through O-6. Most of l-arabinofuranosyl residues were linked as single units through 0-3 to the side chains while a small quantity of the sugar was present as (1→2)-, (1→3)-, or (1→5)-linked inter-chain residues. Single residues of α-l-fucopyranose, apparently attached to (1→2)-linked l-arabinofuranosyl residues, reacted with eel anti-H precipitin and Aleuria aurantia l-fucose-specific lectin, and were assumed to be crucial in the expression of the H-like activity. The uronosyl residues were also located at the non-reducing terminal ends. Reductive alkaline degradation of the arabinogalactan-protein provided indications that the polysaccharide chains were mainly conjugated through serine-O-glycosidic linkages to the polypeptide core. In an immunoprecipitation test, the rape leaf arabinogalactan-protein cross-reacted with antisera raised against radish leaf arabinogalactan-protein, indicating that these cruciferous arabinogalactan-proteins share common immunodeterminant(s) in their molecules.     

Role of Some Hormones in Protein Sparing Action of Dietary Fat

Feeding rats with a fat meal caused marked reduction in the level of plasma urea and urinary output of urea and total nitrogen. Experiments were carried out to examine the possible intervention of some hormones in these phenomena. Protein sparing action of fat was exerted even in the alloxan-diabetic, adrenalectomized, hypophysectomized and thyroidectomized rats. Feeding rats with a fat meal caused no appreciable change in the level of cyclic AMP in liver and gastrocnemius muscle. The overall results obtained here are through! to suggest that the action of fat may be exerted independently of any hormones examined; insulin, glucocorticoids, cyclic AMP and other hormones excreted from adrenal, hypophysis and thyroid gland.     

Epiderstatin and Its Related Glutarimide Antibiotics Inhibit the Cell Growth Induced by Mitogen Stimulation

To investigate the immunosuppressive effects of glutarimide antibiotics including a new antibiotic named epiderstatin, we tested these antibiotics for inhibition of the blastogenesis of mouse spleen cells induced by mitogen stimulation (concanavalin A or lipopolysaccharide). The inhibitory activity was measured by colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Among the glutarimide antibiotics tested, epiderstatin and acetoxycycloheximide especially strongly inhibited the blastogenesis of mouse spleen cells induced by concanavalin A and lipopolysaccharide, however, selectivity between T and B lymphocytes was not observed.     

Studies on l-Threonine Fermentation

Fifteen strains of bacteria were treated with ultraviolet light or N-methyl-N′-nitro-N-nitrosoguanidine to derive auxotrophic mutants, which were screened for their ability to produce l-threonine. A number of auxotrophs were derived from each strain. Among them, those which produced a large amount of l-threonine were found in Aerobacter aerogenes, Serratia marcescens and Escherichia coli, the members of the family Enterobacteriaceae. Nutritional requirements of these threonine producers were proved to be methionine, lysine, or α, ε-diaminopimelic acid (DAP).

In A. aerogenes and E. coli, double and triple auxotrophs were derived with futher mutational treatment. As a, rule, imposition of additional block led to the increase of l-threonine production. In E. coli, many triple auxotrophs (DAP^?, Met^?, He^?) and their isoleucine revertants were screened for their ability to produce l-threonine. Enhancement of l-threonine production was achieved with these mutants.

One of the isoleucine revertants, KY8280, was used to investigate some cultural conditions. As a result, l-threonine accumulation reached to a level of 13.8 mg/ml with the medium containing 7.5% fructose.     

Fatty Acids as Inhibitors of Microbial Cell Wall Synthesis

The Maillard reaction of DNA with ketoses was investigated. Several days of incubation of d-fructose 6-phosphate with deoxyribonucleotides or with polymer DNA in an aqueous buffer resulted in the formation of chromophores and fluorophores. Aminoguanidine and sodium cvanoborohydride inhibited the formation of fluorophores. Transition metal ions such as Cu²⁺, Fe³⁺, Fe²⁺, or Mn^{2 +} promoted the formation of chromophores and fluorophores. Metal-chelating agents such as DETAPAC, citrate, and Desferal inhibited the formation of fluorophores. Superoxide dismutase and catalase also inhibited the formation of fluorophores. The transition metal ion-catalyzed autoxidation of d-fructose 6-phosphate or of the Heyns rearrangement products were to be partially involved in the glycation of DNA and subsequent formation of chromophores and of fluorophores.     

Antitumor Lipopolysaccharide from Heptoseless Mutant of Proteus mirabilis

Studies on lipopolysaccharide (LPS) from the cells of Proteus mirabilis RMS-203 were focused upon reduction of lethal toxicity and of pyrogenicity by biological and chemical modification. A heptoseless mutant, strain N-434, was isolated by the use of phage resistancy as a tool. LPS from that heptoseless mutant was completely deficient in neutral sugars and mainly composed of 2-keto-deoxy-octonic acid (KDO), glucosamine and fatty acids. It revealed almost the same antitumor activity as LPS of the wild type but it was less toxic and less pyrogenic.

Hydroxylaminolysis and reduction with LiAlH₄ resulted in removal of fatty acids from LPS accompanied with decrease in lethal toxicity and antitumor acitivity but not in pyrogenicity.

Lipid A fractions showed almost the same antitumor activity as intact LPS but less lethality and less pyrogenicity.