Genetic analysis of type E botulinum toxin-producing Clostridium butyricum strains

Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of the bont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene. Nucleotide sequences of the bont/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined. The nucleotide sequences of the bont/E genes of 11 C. butyricum isolates from China were identical. The deduced amino acid sequence of BoNT/E from the Chinese isolates showed 95.0 and 96.9% identity with those of BoNT/E from C. butyricum BL 6340 and Clostridium botulinum type E, respectively. The BoNT/E-producing C. butyricum strains were divided into the following three clusters based on the results of RAPD assay, PFGE profiles of genomic DNA digested with SmaI or XhoI, and Southern blot hybridization: strains associated with infant botulism in Italy, strains associated with food-borne botulism in China, and isolates from soil specimens of the Weishan lake area in China. A DNA probe for the bont/E gene hybridized with the nondigested chromosomal DNA of all toxigenic strains tested, indicating chromosomal localization of the bont/E gene in C. butyricum. The present results suggest that BoNT/E-producing C. butyricum is clonally distributed over a vast area.     

Cloning and nucleotide sequence of a novel 28-kDa protein from the mantle muscle of the squid Todarodes pacificus with homology to tropomyosin

In recent studies, we found autodegradation of collagen from the mantle muscle of the squid Todarodes pacificus and also that the 28- and 25-kDa proteins are closely related to this phenomenon [Connect. Tissue Res. 45 (2004) 109-121]. We obtained partial sequences of three internal portions of this protein, which suggested that 25-kDa protein is a partially degraded form of the 28-kDa protein. We determined the full cDNA sequence of this protein by the degenerate polymerase chain reaction (PCR) using the information of amino acid sequences. The deduced amino acid sequence corresponding to the 212-bp cDNA contained all of the amino acid identified from the 28-kDa protein. Rapid amplification of cDNA ends (RACE) and squid mantle muscle RNA allowed cloning of the full 522-bp sequence, corresponding to a protein of 174 amino acids. A database search indicated that this is a new protein that shares 27-34% identity with tropomyosins from various animals. Structural prediction suggested that it possesses heptad repeats that form coiled-coil structures. We expressed a recombinant protein encoded by the 212-bp cDNA in Escherichia coli and used it to generate a polyclonal antibody. Western blotting with this antibody showed that the 28-kDa protein is expressed in fin, tentacle, and mantle muscle, but not in liver.     

Relationship between sex-role identity and functional cerebral lateralization

The present study examined the relationship between sex-role identity (SRI) and functional cerebral lateralization (FCL) in right-handed males. Two tasks (figure task and location task) were used to assess FCL. The figure task required the identification of shape stimuli, while the location task involved identification of the position of stimuli. SRI was assessed by the Bem Sex Role Inventory (BSRI). Males with higher masculine scores in the BSRI indicated greater differences in the reaction time between right and left visual-fields in the location task. This finding suggests that males with higher masculinity in SRI might have greater FCL.     

Kinetic and molecular analysis of 5-epiaristolochene 1,3-dihydroxylase, a cytochrome P450 enzyme catalyzing successive hydroxylations of sesquiterpenes

The final step of capsidiol biosynthesis is catalyzed by 5-epiaristolochene dihydroxylase (EAH), a cytochrome P450 enzyme that catalyzes the regio- and stereospecific insertion of two hydroxyl moieties into the bicyclic sesquiterpene 5-epiaristolochene (EA). Detailed kinetic studies using EA and the two possible monohydroxylated intermediates demonstrated the release of 1beta-hydroxy-EA ((OH)EA) at high EA concentrations and a 10-fold catalytic preference for 1beta(OH)EA versus 3alpha(OH)EA, indicative of a preferred reaction order of hydroxylation at C-1, followed by that at C-3. Sequence alignments and homology modeling identified active-site residues tested for their contribution to substrate specificity and overall enzymatic activity. Mutants EAH-S368C and EAH-S368V exhibited wild-type catalytic efficiencies for 1beta(OH)EA biosynthesis, but were devoid of the successive hydroxylation activity for capsidiol biosynthesis. In contrast to EAH-S368C, EAH-S368V catalyzed the relative equal biosynthesis of 1beta(OH)EA, 2beta(OH)EA, and 3beta(OH)EA from EA with wild-type efficiency. Moreover, EAH-S368V converted approximately 1.5% of these monohydroxylated products to their respective ketone forms. Alanine and threonine mutations at position 368 were significantly compromised in their conversion rates of EA to capsidiol and correlated with 3.6- and 5.7-fold increases in their Km values for the 1beta(OH)EA intermediate, respectively. A role for Ile486 in the successive hydroxylations of EA was also suggested by the EAH-I468A mutant, which produced significant amounts 1beta(OH)EA, but negligible amounts of capsidiol from EA. The altered product profile of the EAH-I486A mutant correlated with a 3.6-fold higher Km for EA and a 4.4-fold slower turnover rate (kcat) for 1beta(OH)EA. These kinetic and mutational studies were correlated with substrate docking predictions to suggest how Ser368 and Ile486 might contribute to active-site topology, substrate binding, and substrate presentation to the oxo-Fe-heme reaction center.     

Effects of dietary polyamine deficiency on Trypanosoma gambiense infection in rats

Nishimura, K., Araki, N., Ohnishi, Y., and Kozaki, S. 2001. Effects of dietary polyamine deficiency on Trypanosoma gambiense infection in rats. Experimental Parasitology 97, 95-101. A diet deficient in polyamines decreases the availability of dietary polyamines. We used rats infected with the Wellcome strain of Trypanosoma gambiense to examine the effects of polyamine-deficient chow (PDC) on trypanosome proliferation and symptoms of infection. Rats fed PDC showed limited increase of trypanosome and symptoms of infection and limited loss of body weight and anemia. Survival in these rats was prolonged. Before infection, the heparinized plasma concentration of spermidine in the PDC-fed rats was lower than that in control rats fed with standard chow. After infection, the content of spermidine in red blood cells increased in the control rats, but was only slightly increased in PDC-fed rats. The content of spermidine in the trypanosomes after infection was low in the PDC-fed rats. Decreases in the polyamine content of trypanosomes limited their increase. These observations suggest that a reduction in dietary polyamines may help in the regulation of trypanosome infection.     

Structure-Function Analyses of Cytochrome P450revI Involved in Reveromycin A Biosynthesis and Evaluation of the Biological Activity of Its Substrate,Reveromycin T

Numerous cytochrome P450s are involved in secondary metabolite biosynthesis. The biosynthetic gene cluster for reveromycin A (RM-A), which is a promising lead compound with anti-osteoclastic activity, also includes a P450 gene, revI. To understand the roles of P450revI, we comprehensively characterized the enzyme by genetic, kinetic, and structural studies. The revI gene disruptants (ΔrevI) resulted in accumulation of reveromycin T (RM-T), and revI gene complementation restored RM-A production, indicating that the physiological substrate of P450revI is RM-T. Indeed, the purified P450revI catalyzed the C18-hydroxylation of RM-T more efficiently than the other RM derivatives tested. Moreover, the 1.4 Å resolution co-crystal structure of P450revI with RM-T revealed that the substrate binds the enzyme with a folded compact conformation for C18-hydroxylation. To address the structure-enzyme activity relationship, site-directed mutagenesis was performed in P450revI. R190A and R81A mutations, which abolished salt bridge formation with C1 and C24 carboxyl groups of RM-T, respectively, resulted in significant loss of enzyme activity. The interaction between Arg¹⁹⁰ and the C1 carboxyl group of RM-T elucidated why P450revI was unable to catalyze both RM-T 1-methyl ester and RM-T 1-ethyl ester. Moreover, the accumulation of RM-T in ΔrevI mutants enabled us to characterize its biological activity. Our results show that RM-T had stronger anticancer activity and isoleucyl-tRNA synthetase inhibition than RM-A. However, RM-T showed much less anti-osteoclastic activity than RM-A, indicating that hemisuccinate moiety is important for the activity. Structure-based P450revI engineering for novel hydroxylation and subsequent hemisuccinylation will help facilitate the development of RM derivatives with anti-osteoclast activity.