Analysis of tryptophan‐rich region in Clostridium septicum alpha‐toxin involved with binding to glycosylphosphatidylinositol‐anchored proteins

Clostridium septicum alpha‐toxin has a unique tryptophan‐rich region (³⁰²NGYSEWDWKWV³¹²) that consists of 11 amino acid residues near the C‐terminus. Using mutant toxins, the contribution of individual amino acids in the tryptophan‐rich region to cytotoxicity and binding to glycosylphosphatidylinositol (GPI)‐anchored proteins was examined. For retention of maximum cytotoxic activity, W307 and W311 are essential residues and residue 309 has to be hydrophobic and possess an aromatic side chain, such as tryptophan or phenylalanine. When residue 308, which lies between tryptophans (W307 and W309) is changed from an acidic to a basic amino acid, the cytotoxic activity of the mutant is reduced to less than that of the wild type. It was shown by a toxin overlay assay that the cytotoxic activity of each mutant toxin correlates closely with affinity to GPI‐anchored proteins. These findings indicate that the WDW_W sequence in the tryptophan‐rich region plays an important role in the cytotoxic mechanism of alpha‐toxin, especially in the binding to GPI‐anchored proteins as cell receptors.     

Elucidation of biosynthetic pathway of a plant hormone abscisic acid in phytopathogenic fungi

ABSTRACT

Abscisic acid (ABA) is one of the plant hormones that regulates physiological functions in various organisms, including plants, sponges, and humans. The biosynthetic machinery in plants is firmly established, while that in fungi is still unclear. Here, we elucidated the functions of the four biosynthetic genes, bcABA1-bcABA4, found in Botrytis cinerea by performing biotransformation experiments and in vitro enzymatic reactions with putative biosynthetic intermediates. The first-committed step is the cyclization of farnesyl diphosphate to give α-ionylideneethane catalyzed by a novel sesquiterpene synthase, BcABA3, which exhibits low amino acid sequence identities with sesquiterpene synthases. Subsequently, two cytochrome P450s, BcABA1 and BcABA2, mediate oxidative modifications of the cyclized product to afford 1?,4?-trans-dihydroxy-α-ionylideneacetic acid, which undergoes alcohol oxidation to furnish ABA. Our results demonstrated that production of ABA does not depend on the nucleotide sequence of bcABA genes. The present study set the stage to investigate the role of ABA in infections.     

An Acidic Polysaccharide Isolated from the Midrib of Leaves of Nicotiana tabacum

An acidic polysaccharide (APS-H) purified from the hemicellulosic fraction of the midrib of Nicotiana tabacum was composed of d-galacturonic acid, l-rhamnose, l-arabinose and d-galactose in a molar ratio of 31.8: 15.4: 9.9: 42.9. Its molecular weight was estimated to be 90,000 by gel filtration chromatography. APS-H had a pectin-like structure in which the rhamnogalacturonan backbone was composed of (1 → 2)-linked l-rhamnopyranosyl and (1 → 4)-linked d-galacturonosyl residues in a ratio of approximately 1: 2.1. It also contained (1 → 4)-linked d-galactan and (1 → 5)-linked l-arabinofuranosyl moieties as the side chains. Branch points occurred mainly at C-4 of (1 → 2)-linked l-rhamnosyl residues in the backbone and at C-6 of (1 → 4)-linked d-galactosyl residues in the side chains.     

Translocation domain of botulinum neurotoxin A subtype 2 potently induces entry into neuronal cells

Botulinum neurotoxin (BoNT) is the causative agent of botulism in humans and animals. Only BoNT serotype A subtype 1 (BoNT/A1) is used clinically because of its high potency and long duration of action. BoNT/A1 and BoNT/A subtype 2 (BoNT/A2) have a high degree of amino acid sequence similarity in the light chain (LC) (96%), whereas their N-and C-terminal heavy chain (H_N and H_C) differ by 13%. The LC acts as a zinc-dependent endopeptidase, H_N as the translocation domain, and H_C as the receptor-binding domain. BoNT/A2 and BoNT/A1 had similar potency in the mouse bioassay, but BoNT/A2 entered faster and more efficiently into neuronal cells. To identify the domains responsible for these characteristics, H_N of BoNT/A1 and BoNT/A2 was exchanged to construct chimeric BoNT/A121 and BoNT/A212. After expression in Escherichia coli, chimeric and wild-type BoNT/As were purified as single-chain proteins and activated by conversion to disulfide-linked dichains. The toxicities of recombinant wild-type and chimeric BoNT/As were similar, but dropped to 60% compared with the values of native BoNT/As. The relative orders of SNAP-25 cleavage activity in neuronal cells and toxicity differed. BoNT/A121 and recombinant BoNT/A2 have similar SNAP-25 cleavage activity. BoNT/A2 H_N is possibly responsible for the higher potency of BoNT/A2 than BoNT/A1.     

Uptake dynamics of scrapie agent in the intestinal villous epithelium of suckling and weanling Syrian hamsters

In mice, the number of intestinal villous columnar epithelium cells that incorporate abnormal prion protein (PrP(Sc) ) decreases significantly after weaning. In this study, the dynamics of PrP(Sc) uptake during the growth of hamsters were investigated by inoculating scrapie 263K agent orally into suckling and weanling Syrian hamsters and estimating the number of PrP(Sc) -positive villous epithelium cells immunohistochemically. The number of PrP(Sc) -positive cells declined significantly as the hamsters aged. The present results suggest that a tendency toward decline of PrP(Sc) -positive cells with increasing age might be a common phenomenon among the superfamily Muridae.     

Colored floral organs influence pollinator behavior and pollen transfer in Commelina communis (Commelinaceae)

Visual floral guides such as colored anthers, lines, dots, and UV-absorption patterns on petals are commonly observed in insect-pollinated angiosperms. Floral guides that are known to enhance foraging efficiency of visitors on flowers thus promote return visits (foraging facilitation hypothesis, which predicts that visitors will discriminate against flowers with inferior floral guides). In this study, we experimentally examined the hypothesis that floral guides also prevent pollen-theft behavior by floral visitors (theft prevention hypothesis), which has rarely been tested. Nectarless flowers of Commelina communis have three types of brightly colored floral organs: large blue petals, rewarding yellow anthers, and nonrewarding yellow anthers. Colored floral organs were removed artificially from plants in two natural populations of C. communis. Removal of the nonrewarding yellow anthers diminished hoverings in front of flowers and tended to reduce the number of total floral visitor landings, supporting the foraging facilitation hypothesis. Additionally, removal of the rewarding yellow anther decreased the frequency of legitimate landings on flowers and the legitimate landing-to-total landing ratio, which is consistent with the theft prevention hypothesis. The nonrewarding anthers and the rewarding yellow anthers were shown to play an important role in increasing visitor landings and orienting floral visitors toward a landing point appropriate for pollination, respectively. We also showed that the absence of yellow anthers decreased both pollen dispatch from brown anthers and receipt by stigmas in C. communis. These findings support both the foraging facilitation hypothesis and the theft prevention hypothesis.     

Botulinum neurotoxin A subtype 2 reduces pathological behaviors more effectively than subtype 1 in a rat Parkinson’s disease model

Recent reports indicate that interruption of acetylcholine release by intrastriatal injection of botulinum neurotoxin type A (BoNT/A) in a rat Parkinson’s disease model reduces pathogenic behavior without adverse side effects such as memory dysfunction. Current knowledge suggests that BoNT/A subtype 1 (BoNT/A1) and BoNT/A subtype 2 (BoNT/A2) exert different effects. In the present study, we compared the effects of BoNT/A1 and BoNT/A2 on rotation behavior and in vivo cleavage of presynaptic protein SNAP-25 in a rat unilateral 6-hydroxydopamine-induced Parkinson’s disease model. BoNT/A2 more effectively reduced pathogenic behavior by efficiently cleaving SNAP-25 in the striatum compared with that of BoNT/A1. Our results suggest that BoNT/A2 has greater clinical therapeutic value for treating subjects with Parkinson’s disease compared to that of BoNT/A1.     

Stability of toxigenicity in proteolytic Clostridium botulinum type B upon serial passage

Proteolytic Clostridium botulinum type B strains were investigated for stability of toxigenicity and bont/b gene upon serial passage. Strains with bont/b gene located on their plasmids showed loss or decrease of toxigenicity during serial passage. Some strains lost the bont/b gene-encoding plasmid. The stability of the plasmids varied between strains.