Saburo Hara

Amino acid sequence of a heat-stable enterotoxin isolated from enterotoxigenic Escherichia coli strain 18D

A heat‐stable enterotoxin produced by a strain of enterotoxigenic Escherichia coli 18D was purified by ion‐exchange and reversed‐phase high‐pressure liquid chromatography. The amino acid sequence of the purified toxin was determined by Edman‐degradation and a combination of fast atom bombardment mass spectrometry and carboxypeptidase digestion to be Asn‐Thr‐Phe‐Tyr‐Cys‐Cys‐Glu‐Leu‐Cys‐Cys‐Asn‐Pro‐Ala‐Cys‐Ala‐Gly‐Cys‐Tyr.

Essential structure for full enterotoxigenic activity of heat-stable enterotoxin produced by enterotoxigenic Escherichia coli.

Several analogues of heat‐stable enterotoxins (STh and STP) produced by enterotoxigenic Escherichia coli were synthesized. Peptides (STh[6–18] and STP[5–17]) consisting of 13 amino acid residues from the Cys residue near the N‐terminus to the Cys residue near the C‐terminus and linked by three disulfide bonds had the same biological and immunological properties as native STh and STP, respectively. The results indicated that the sequence with the 13 amino acid residues and three disulfide linkages is essential for full biological activity of ST.

Primary structure of heat-stable enterotoxin produced by Yersinia enterocolitica

A heat-stable enterotoxin was isolated and purified from the culture supernatant of Yersinia enterocolitica by reversed-phase high-performance liquid chromatography. The amino acid sequence of the purified toxin was determined to be as follows: (sequence; see text) (X: not determined). The C-terminal sequence containing 6 half-cystine residues was highly homologous to that of heat-stable enterotoxin of enterotoxigenic Escherichia coli.

Cloning and sequence analysis of a cDNA for cellulase (FI-CMCase) from Aspergillus aculeatus

SummaryWa have cloned and characterized the cDNA coding for a major component of cellulase, endoglucanase (FI-CMCase), produced by Aspergillus aculeatus. The cDNA was isolated from a A. aculeatus cDNA library using synthetic oligonuceotide mixtures that correspond to the internal amino acid sequence of the mature FI-CMCase protein. Nucleotide sequence analysis of the cloned cDNA insert revealed a 711 bp open reading frame that encoded a protein of 237 amino acid residues. The primary structure of FI-CMCase deduced from the nucleotide sequence of cDNA agreed with that found by amino acid sequencing of peptide fragments obtained by digestion with several proteinases and cyanogen bromide cleavage. There may be a signal peptide sequence of 16 amino acid residues at the N-terminus. The molecular mass of the mature protein calculated from the cDNA is 24002 daltons, which compares favorably with molecular mass estimates of purified FI-CMCase obtained from SDS-PAGE (25000 Da). No distinct homology was found between the amino acid sequence of FI-CMCase and known cellulase sequences of other microorganisms. This study is the first example of cDNA cloning of an endoglucanase from the genus Aspergillus.

Mulberry leaf extract prevents amyloid beta-peptide fibril formation and neurotoxicity

Mulberry leaf has been reported to possess medicinal properties, including hypoglycemic, hypotensive and diuretic effects. Little is known, however, about its medicinal properties for central nervous system disorders, including Alzheimers disease. Accumulating evidence suggests that amyloid &bgr;-peptide (1–42) plays an important role in the etiology of Alzheimers disease. Here we show that mulberry leaf extract inhibits the amyloid &bgr;-peptide (1–42) fibril formation by both the thioflavin T fluorescence assay and atomic force microscopy. Furthermore, mulberry leaf extract protected hippocampal neurons against amyloid &bgr;-peptide (1–42)-induced cell death in a concentration-dependent manner. These results suggest that mulberry leaf extract provides a viable treatment for Alzheimers disease through the inhibition of amyloid &bgr;-peptide (1–42) fibril formation and attenuation of amyloid &bgr;-peptide (1–42)-induced neurotoxicity.

Concurrence of agaroid and carrageenan chains in funoran from the red seaweed Gloiopeltis furcata Post. et Ruprecht (Cryptonemiales, Rhodophyta)

Abstract Funoran extracted from the red seaweed Gloiopeltis furcata Post. et Ruprecht was fractionated into four polysaccharide fractions in terms of differences in solubility of their cetylpyridinium salt in potassium chloride solution. Besides the main fraction constructed by an agarose sulphate structure with negative optical rotation, minor polysaccharide fractions with positive optical rotation were obtained. One of the minor polysaccharide fractions contained d - and l -galactose (Gal), 6-O-methyl- d -Gal, 3,6-anhydro- l -Gal and sulphate. Partial hydrolysis and partial methanolysis studies of this fraction led to identification of oligosaccharides attributable to both agaroid and carrageenan backbones, i.e. [ → 3) d -Gal(1 → 4) l -Gal(1 → ][→ 3) d -Gal(1 → 4)3,6-and [ → 3)6- O -methyl- d -Gal(1 → 4)D-Gal(l → ]. Methylation analysis and alkaline treatment study of this fraction revealed that the sulphate groups were located at O-6 of all the (1 → 4)-linked l - and a part of the (1 → 4)-linked d -Gal residues, O-6 of a part of the (1 → 3)-linked d -Gal residue, and both O-2 and O-4 of the (1 → 3)-linked 6-O-methyl- d -Gal residue and the other (1 → 3)-linked d -Gal residue.

A novel regioselective desulfation of polysaccharide sulfates: specific 6-O-desulfation with N,O-bis(trimethylsilyl)acetamide

Abstract Treatment of the pyridinium salts of glycosaminoglycans and galactan sulfates with N,O -bis(trimethylsilyl)acetamide (BTSA) in pyridine for 2 h at 60 °C caused specific 6- O -desulfation without depolymerisation or other chemical changes.

Pectinolytic enzymes from Pseudomonas marginalis MAFF 03-01173.

Two pectinolytic enzymes were purified from the culture broth of Pseudomonas marginalis pv. marginalis MAFF 03-01173 with total 33% recovery of the initial activity. From the substrate specificities against pectin and polygalacturonic acid, the requirement of calcium ion for the enzymatic activity, and the N-terminal sequences, the enzymes were identified as pectin lyase and pectate lyase. The M,s of pectin lyase and pectate lyase were estimated to be 34,000 and 43,000, respectively, by SDS polyacrylamide gel electrophoresis. Both enzymes showed almost the same pH dependent activity curves with the highest activity at pH 8.3

Funoran from the red seaweed, Gloiopeltis complanata : polysaccharides with sulphated agarose structure and their precursor structure

Abstract Funoran extracted from the red seaweed, Gloiopeltis complanata, was fractionated based on the solubility of its cetylpyridinium salt in KCl solution to give the fractions PSl and PS2. PS2 was further fractionated into PS2G that formed a gel in 0.5 n KCl, and PS2S that did not form a gel in saturated KCl. PSl, PS2G and PS2S were different in composition and properties showing that the funoran was heterogeneous. On the basis of the results from composition analyses, partial methanolysis studies, methylation studies, 13C-NMR spectrum measurements, and regioselective desulphation studies employing a silylating reagent, N,O-bis(trimethylsilyl)acetamide, PS2G was shown to consist chiefly of 6-O-sulphated agarose, repeats of →3)6-SO3-β- d -Gal(1→4)3,6-anhydro-α- l -Gal(1→; and PS2S to contain repeats of →3)6-SO3-β- d -Gal(1→4)6-SO3-α- l -Gal(1→ as well as smaller amounts of →3)2,6-di-SO3-β- d -Gal(1→ and →3)2-SO3-β- d -Gal(1→ residues.

Analysis of neutral sugars as glycamines using an amino acid analyzer.

Abstract A new method of the analysis of neutral sugars was developed based on the separation of their corresponding glycamines. Sugars and ammonia were combined by means of the reduction with sodlum cyanoborohydride. The glycamines thus obtained were quantitatively analyzed by an automatic amino acid analyzer. Satisfactery results were obtained in the analyses of the constituent sugars of several polysaccharides and glycoconjugates.