Teruo Ueno

Isolation and structure of cecropins, inducible antibacterial peptides, from the silkworm, Bombyx mori

1. Cecropins, inducible antibacterial peptides, were purified by simple two step chromatography from immunized larval hemolymph of the silkworm, Bombyx mori. 2. The cecropins were further separated into four major and two minor forms by reverse-phase HPLC. 3. The four major cecropins were sequenced and divided into two types, A and B, whose structures were quite similar to cecropins A and B of Hyalophora cecropia. 4. Three of them contained an unusual amino acid, delta-hydroxylysine. 5. No remarkable difference in antibacterial activity against E. coli was detected among these cecropins.

Bacterial peptidoglycan as elicitor of antibacterial protein synthesis in larvae of the silkworm, Bombyx mori

Various bacteria were tested for elicitor of anti-bacterial protein synthesis in Bombyx mori larvae by injecting formalin-killed cells into the hemocoel. All of Gram-negative and some species of Gram-positive bacteria were effective elicitor, but Micrococcus luteus and most of Lactobacilli were ineffective. Peptidoglycan purified from Escherichia coli or Bacillus megaterium and lipopolysaccharide from Escherichia coli were also effective elicitors. The peptidoglycans from Escherichia coli and Bacillus megaterium were hydrolyzed with lysozyme and fractionated by gel-filtration. The isolated fragments larger than four sugar units were good elicitor, but similar preparations from Micrococcus luteus had poor elicitor activity. The results suggest that Bombyx mori larva recognizes some definite structure of peptidoglycan for induction of antibacterial proteins, and the recognition system is independent of that for prophenoloxidase activation.

Metabolism of n-alkylcyclohexanes with an even number of carbon atoms in the side chain by Micrococcus sp. RCO-4M

The metabolism of n-alkyleyclohexanes with an even number of carbon atoms (6, 8, 10, 12, 14) in the side chain by Micrococcus sp. RCO-4M was investigated. Evidence for the formation of cyclohexanecarboxylic acid (I), cyclohexaneacetic acid (II), 1-cyclohexenecarboxylic acid (III), 6-hydroxyhexanoic acid (IV), adipic acid (V), and trans-4-hydroxycyclohexaneacetic acid (VI) is presented. The presence of products (II), (IV) and (V) especially represent the complete degradation of n-alkylcyclohexanes with an even number of carbon atoms in the side chain by a single organism. The occurrence of a newly-identified product (VI) suggests that a new metabolic pathway for n-alkylcyclohexanes with even-carbon-number side chain operates in this organism.

Bioconversion of cyclohexaneacetic acid to monohydroxycyclohexaneacetic acids by Chlorella pyrenoidosa chick

Abstract Algal conversion of cyclohexaneacetic acid was studied with five axenic strains of Chlorella ; in two Chlorella pyrenoidosa strains, cyclohexaneacetic acid was transformed to three monohydroxycyclohexaneacetic acids. As biotransformation products, cis - and trans -3-hydroxycyclohexaneacetic acid and trans -4-hydroxycyclohexaneacetic acid were identified by gas chromatography-mass spectrometry.

Purification and properties of calmodulin-dependent cyclic nucleotide phosphodiesterase in silkworm fat body

Abstract Calmodulin-dependent cyclic nucleotide phosphodiesterase (EC 3.1.4.17) was purified from silkworm pupal fat body by DEAE-Sepharose and calmodulin-Sepharose column chromatographies. The calmodulin-dependent activity accounted for 3–5% of the total cytosol phosphodiesterase activity, and was purified approx. 5000-fold from the fat body extract. The purified phosphodiesterase readily lost sensitivity to calmodulin during storage at 4°C or limited digestion by trypsin. The enzyme could hydrolyze both cyclic AMP and cyclic GMP, and was activated 2- to 4-fold by calmodulin and Ca 2+ . The molecular weight of the enzyme was calculated from the sedimentation constant and Stokes radius to be 115,000. Kinetic analyses suggested the enzyme molecule had at least two distinct sites for the cyclic nucleotides.

Stimulatory Effect of Acetate and Propionate on Aspergillic Acid Formation by Aspergillus oryzae A 21

Abstract The stimulatory effect of acetate and propionate on the formation of aspergillic acid (AA) by Aspergillus oryzae A 21 was investigated. 14C labeled AA with a high specific activity was biosynthesized from [2-14C]propionate or [1-14C]acetate in a medium without leucine and isoleucine, precursors in the biosynthesis of AA. Incorporation of three intact acetate units derived from [1,2-13C]acetate into AA via leucine and isoleucine was demonstrated by 13C NMR analyses. Furthermore, a depressive effect of propionate on the catabolism of leucine and isoleucine was assumed to bring about the increase in the formation of AA.

Microbial degradation of aspergillic acid by Trichoderma koningii M 102

Abstract A microorganism, strain M 102, capable of degrading aspergillic acid (AA), was first isolated from a soil sample in a drainage ditch and was identified as Trichoderma koningii Oudemans. This fungus degraded AA, but not hydroxyaspergillic acid (HAA) or deoxyaspergillic acid (DAA). The AA-degrading ability of M 102 was induced by incubation with AA but not with HAA or DAA. AA-degradation activity was found in a crude enzyme prepared from the mycelia induced by AA; this AA degradation reaction required NAD(P)H and oxygen.

Metabolism of n-alkyl-substituted cyclohexanes with an odd number of carbon atoms in the side chain by Micrococcus sp. RCO-4M

The metabolism of n-alkyl-substituted cyclohexanes (n-ACH) with an odd number of carbon atoms (7, 9, 11) in the side chain by Micrococcus sp. RCO-4M was investigated. Evidence for the formation of cyclohexanecarboxylic acid (C6CA), 1-cyclohexenecarboxylic acid (1-ene-C6CA), and trans-4-hydroxycyclohexanecarboxylic acid (t-4-OH-C6CA) is presented. A large amount of C6CA, 1-ene-C6CA and the small quantity of t-4-OH-C6CA were accumulated in culture broth. It is suggested that Micrococcus sp. RCO-4M metabolized n-ACH with an odd number of carbon atoms in the side chain to C6CA by beta-oxidation of the side chain and furthermore degraded C6CA by two pathways involving 1-ene-C6CA and t-4-OH-C6CA as key intermediates.

Structural and kinetic properties of glycogen phosphorylase a from fat body of the silkworm, Bombyx mori.

Abstract 1. 1. Glycogen phosphorylase b was isolated from the pupal fat body of Bombyx mori, and phosphorylated to the a form by phosphorylase kinase purified from the same tissue. 2. 2.|The structural and kinetic properties of the two forms of fat body phosphorylase were compared. 3. 3. The mol. wts of phosphorylase a estimated by gel filtration HPLC and SDS-gel electrophoresis were found to be 95,000 and 90,000 respectively, indicating that the enzyme is a monomeric protein (as is the b form). 4. 4. The S20 (sedimentation coefficient) value of phosphorylase a obtained by a sucrose density gradient centrifugation was 7.8, which is somewhat larger than 6.9 of the b form, suggesting some conformational changes have occurred during the phosphorylation. 5. 5. The a form has 2- to 3-fold higher affinity for both substrates, glycogen and inorganic phosphate (Pi), compared to the b form.

Identification of the degradation products of aspergillic acid by Trichoderma koningii M 102

Abstract Isolation and identification of the degradation products of aspergillic acid, a secondary metabolite of fungi, by Trichoderma koningii M 102 were undertaken. 14 C-labeling experiments indicated that aspergillic acid was broken-down to a water-soluble degradation product and a chloroform-soluble one. Consequently, leucine and a new microbial metabolite, 2-hydroxyimino-3-methyl-1-pentanol, were isolated and identified as the degradation products formed by cleavage of the pyrazine ring of aspergillic acid by T. koningii M 102.