Biomedical subjects

S Kozaki

Publications and source records attributed to S Kozaki.

At least 55 records · Page 3Linked to original sources

PubMed1995

Immunological characterization of the neurotoxin produced by Clostridium botulinum type A associated with infant botulism in Japan.

The neurotoxin associated with type A infant botulism in Japan shows different antigenic properties from those produced by authentic strains. The monoclonal antibodies recognizing the light chain reacted to both neurotoxins, whereas half the antibodies recognizing the heavy chain reacted specifically to the respective neurotoxin. Each neurotoxin showed its own manner of binding to brain synaptosomes. These results indicate that the distinguishable characteristics are ascribable to the heavy chain but not to the light chain. In both neurotoxins, an epitope recognized by the monoclonal antibody that reacts to the light chain and neutralizes the toxin was found to be very close to the amino-terminal half (H-1 fragment) of the heavy chain. This may support the hypothesis that the H-1 fragment functions in the transport of the light chain in the target cell.

Amino Acid Sequence↗

PubMed1995

Variation in the primary structure of waxy proteins (granule-bound starch synthase) in diploid cereals.

The molecular weights of waxy proteins, by SDS-PAGE, and the N-terminal amino acid sequences of mature protein and of V8 protease-induced fragments were determined in diploid cereals. The homology of the primary structure was relatively high among cereals examined here, and there appeared to be a common sequence, V-F-V-G-A-E-M-A, in the vicinity of the N terminus. Based on the amino acid sequences, these cereals could be divided into two groups, including corn and rice in one and diploid wheat, four Aegilops species, rye, and barley in the other. In diploid wheat and Aegilops species there were substitutions of amino acids in the primary structure. Variations of this sort suggest that the primary structure of waxy proteins would provide clues to the phylogenetic relations in the wheat group.

Amino Acid Sequence↗

PubMed1994

The light chain of botulinum neurotoxin forms channels in a lipid membrane.

The ability of botulinum neurotoxin and its isolated subunits, the heavy and light chains, to bind to a lipid membrane and to form channels in the membrane was examined. At pH 4.0, the neurotoxin caused aggregation of calcein-containing liposomes, providing evidence of binding of the neurotoxin to the surface of the outer lipid membrane. Aggregation was followed by the release of calcein, as a result of the formation of channels. The heavy chain evoked the same responses as those of the neurotoxin. The light chain did not cause aggregation of the liposomes but did evoke the release of calcein. The channel-forming ability of the light chain appeared to be higher than that of the neurotoxin or the heavy chain. This novel property of the light chain may help us to understand the mechanism of action of botulinum neurotoxin.

Botulinum Toxins↗

PubMed1994

Identification of protein receptor for Clostridium botulinum type B neurotoxin in rat brain synaptosomes.

The protein receptor for Clostridium botulinum type B neurotoxin was purified 340-fold from rat synaptosomes by successive chromatography on DEAE-Sepharose, phenyl-Toyopearl, and heparin-Toyopearl columns. 125I-Labeled neurotoxin bound to lipid vesicles containing the protein receptor and ganglioside GT1b or GD1a. The reconstituted receptor showed the same affinities as the native receptor on synaptosomes. Chemical cross-linking of 125I-toxin to the receptor in the presence of gangliosides resulted in formation of a cross-linked product of 161 kDa under reducing conditions. Cross-linking was specific, as it was inhibited by the presence of excess unlabeled toxin. A monoclonal antibody against the purified 58-kDa receptor protein and a monoclonal antibody against the heavy chain (103 kDa) of the neurotoxin reacted with the cross-linked product of 161 kDa in immunoblotting experiments. We determined partial amino acid sequences of the 58-kDa protein, which were identical to synaptotagmin, a synaptic vesicle membrane protein. In addition, the monoclonal antibody against the 58-kDa receptor protein recognized recombinant rat synaptotagmin. These results suggest that synaptotagmin in association with ganglioside GT1b or GD1a may be a natural receptor for C. botulinum type B neurotoxin at the nerve terminals.

Amino Acid Sequence↗

PubMed1994

Involvement of phospholipids in the intoxication mechanism of botulinum neurotoxin.

Phospholipids were examined for their potential to interact with botulinum neurotoxin by an in vivo toxin-inactivation assay and a direct binding assay on a thin layer plate. Type E neurotoxin was inactivated by negatively charged phospholipids, phosphatidylserine (PS) and phosphatidylinositol (PI). The toxicity of the neurotoxin was not affected by phosphatidylcholine (PC) without an electric charge or phosphatidylethanolamine (PE) with a positive electric charge. The neurotoxin bound directly to PS and PI but not to PC or PE. These results suggest that the negatively charged phospholipids in the cell membranes are involved in the intoxication mechanism of botulinum neurotoxin. The phospholipids PS and PI were tested for their potential to interact within three domains [L, H-1, and H-2] which compose the neurotoxin. All three domains bound to PS; whereas, PI specifically accepted the binding of the H-1 domain relative to the penetration of the neurotoxin into the lipid membrane. In this paper, we discuss the interaction between the neurotoxin and the lipid membrane in the intoxication mechanism.

Animals↗

PubMed1994

Morphological effects, rate of incorporation, and the enzymatic action of botulinum ADP-ribosyltransferase, known as C3 exoenzyme, on human neuroblastoma GOTO cells.

The susceptibility of various lines of cultured cells to botulinum ADP-ribosyltransferase, known as C3 exoenzyme, was examined. Human neuroblastoma GOTO cells were most sensitive. The C3 exoenzyme caused a change in cell shape that involved extension of neurites. The exoenzyme evoked the outgrowth of neurites from chick ganglion as effectively as nerve growth factor, suggesting that C3 exoenzyme possesses neurotropic activity. Experiments with 125I-labeled enzyme revealed that C3 exoenzyme was rapidly incorporated into cells but the number of incorporated enzyme molecules was small. Once C3 exoenzyme had been incorporated, ADP-ribosylation of the substrate (Rho protein) in GOTO cells occurred immediately and rapidly reached a maximum level. However, some of Rho proteins remained unmodified even after induction of the change in morphology. These findings suggest that ADP-ribosylation by C3 exoenzyme is directly associated with the differentiation of GOTO cells but that other events may also participate in this process.

ADP Ribose Transferases↗

PubMed1993

Solubilization and characterization of the acceptor for Clostridium botulinum type B neurotoxin from rat brain synaptic membranes.

The acceptor for Clostridium botulinum type B neurotoxin was solubilized from rat brain synaptic membrane with nonionic detergent, nonanoyl-N-methylglucamide (MEGA-9). The solubilized acceptor was assayed for the binding activity by precipitating the acceptor with acetone in the presence of phosphatidylcholine. 125Ilabeled neurotoxin specifically bound to the lipid vesicles having incorporated the acceptor together with gangliosides. The lipid vesicles having incorporated either the acceptor or gangliosides alone showed extremely low binding activity. The treatment of the solubilized acceptor with lysyl endopeptidase and glycopeptidase F but not with sialidase resulted in decreased toxin binding, indicating that the putative acceptor is a glycoprotein accompanying an N-linked carbohydrate moiety. The observations suggest also that a protein acceptor/ganglioside complex may be required to form the functional toxin receptor.

Animals↗

PubMed1993

Purification and characterization of the ganglioside-binding fragment of Clostridium botulinum type E neurotoxin.

A way of fragmentation of Clostridium botulinum neurotoxin was carried out to elucidate the structure-function relationship of neurotoxin. The hitherto only plausible fragment was isolated from the trypsin-treated heavy chain of botulinum type E neurotoxin. In the presence of 4 M urea, one protein peak emerged from QAE-Sephadex column loaded with the heavy chain mildly treated with trypsin by elution with 0.1 M sodium chloride. Although many protein bands were detected in SDS-PAGE of the treated heavy chain, the eluted protein migrated in a single band to the position of 41,000 Da. The recovery of the 41,000-Da fragment was 28.6%, but with a 2 M urea-containing buffer as eluant, the recovery was less than 12%. The 41,000-Da fragment bound to gangliosides GD1a, GT1b, and GQ1b, to which neurotoxin and the heavy chain bound. The 41,000-Da fragment partially interfered with the binding of 125I-labeled neurotoxin to mouse brain synaptosomes. We have proposed a three-fragment structure (L.H-1.H-2) for botulinum type E neurotoxin. The characters of the 41,000-Da fragment described in this paper seem to substantiated our proposal that type E neurotoxin consists of three fragments, L.H-1.H-2, and that the ganglioside-binding fragment is H-2.

Animals↗

PubMed1993

Prevention and control of nosocomial infection caused by methicillin-resistant Staphylococcus aureus in a premature infant ward--preventive effect of a povidone-iodine wipe of neonatal skin.

In early 1983 we experienced a small scale epidemic of Staphylococcus aureus coagulase type IV in the premature infants unit. Children had bacteraemia or impetigo. The microorganism was resistant to methicillin, erythromycin and lincomycin and was susceptible to tetracycline, chloramphenicol and cefmetazole. The results of coagulase typing and antimicrobial sensitivities indicated that these cases represented nosocomial infection with MRSA. The source and route of the infection were investigated, and measures were taken to prevent bacterial spread from carriers and to keep instruments and environments clean. As the source of infection was not identified, we tried wiping the body surface of the premature infants with a diluted IsodineR solution (10% povidone-iodine; 1:100 dilution) in order to prevent colonization of the microorganism on the body surface. As a result, no additional MRSA infection occurred in the premature infant unit. During the subsequent 6 years of frequent surveys of carriers and wiping the appropriate body surface with diluted IsodineR solution we have had no recurrence of MRSA. None of the premature infants wiped with IsodineR solution showed any objective abnormalities, although laboratory testing disclosed an elevated blood iodine level and a transient mild reduction of T4 in some infants.

Administration, Topical↗

PubMed1993

ADP-ribosylation of the rhoA gene product by botulinum C3 exoenzyme causes Swiss 3T3 cells to accumulate in the G1 phase of the cell cycle.

Using botulinum C3 exoenzyme, which specifically ADP-ribosylates the rho gene products (rho proteins), we examined the role of these proteins in cell cycle progression in Swiss 3T3 cells. Incubation of cell lysates with C3 exoenzyme revealed a single [32P]ADP-ribosylated protein with an M(r) of 23K. This protein was identified as rhoA protein by isoelectric focusing and peptide mapping. When C3 exoenzyme was added to the culture, it ADP-ribosylated the substrate protein in the cells and reduced their growth rate and saturation density. The reduction was dependent on the amount of C3 exoenzyme and on the extent of ADP-ribosylation of the rho protein in the cells. Flow cytometric analysis of logarithmically growing cells showed that the enzyme treatment concentration-dependently accumulated the cells in the G1 phase of the cell cycle. When G1-enriched cells were treated with C3 exoenzyme and cell cycle progression initiated by the addition of serum was monitored, inhibition of G1-S transition was clearly observed. These results suggest that the rhoA gene product plays a critical role in G1-S progression in cultured Swiss 3T3 cells and that the ADP-ribosylation abolishes this activity and causes the cells to accumulate in G1 phase.

3T3 Cells↗

PubMed1992

A rho gene product in human blood platelets. II. Effects of the ADP-ribosylation by botulinum C3 ADP-ribosyltransferase on platelet aggregation.

In the accompanying paper (Nemoto, Y., Namba, T., Teru-uchi, T., Ushikubi, F., Morii, N., and Narumiya, S. (1992) J. Biol. Chem. 267, 20916-20920), we have identified rhoA protein as the sole substrate protein for botulinum C3 ADP-ribosyltransferase (C3 exoenzyme) in human blood platelets. Here we examined the role of rhoA protein in platelet functions. C3 exoenzyme added to washed platelets dose- and time-dependently ADP-ribosylated rhoA protein in situ in the cells. Concomitant with this modification, inhibition of thrombin-induced platelet aggregation was observed. This inhibition was not reversed by washing the treated platelets, but was not found when C3 exoenzyme was pretreated with mouse monoclonal anti-C3 exoenzyme antibody. C3 exoenzyme treatment did not affect thrombin-induced inositol 1,4,5-trisphosphate production. Secretion of preloaded [14C]serotonin was delayed by the enzyme treatment, but the extent of the secretion was not influenced. In addition, the enzyme treatment did not change the expression of the glycoprotein IIb-IIIa complex on the platelet surface. The enzyme treatment also suppressed platelet aggregation induced by phorbol myristate acetate. These results suggest that rhoA protein plays a role mainly in the aggregation process downstream from receptor-phospholipase C coupling. This, together with the previous finding that rhoA protein modulates stress fiber formation in cultured fibroblasts (Paterson, H. F., Self, A. J., Garrett, M. D., Just, I., Aktories, K., and Hall, A. (1990) J. Cell Biol. 111, 1001-1007), suggests that rhoA protein regulates the assembly of actin filaments and the avidity of the platelet integrin (glycoprotein IIb-IIIa) in the aggregation process.

ADP Ribose Transferases↗

PubMed1992

NAD+ binding site of Clostridium botulinum C3 ADP-ribosyltransferase. Identification of peptide in the adenine ring binding domain using 2-azido NAD.

C3 ADP-ribosyltransferase is an exoenzyme produced by certain strains of Clostridium botulinum types C and D, which specifically ADP-ribosylates rho proteins in eukaryotic cells. Using the photoaffinity probe [alpha-32P]nicotinamide-2-azidoadenine dinucleotide, we have identified the adenine ring binding domain of the NAD+ binding site. The specificity of labeling was demonstrated by saturation effects and protection by the natural compound at physiologically relevant concentrations. Saturation of labeling was observed at 50 microM. Protection experiments indicated an 80% protection of labeling by 100 microM NAD+ when protein was photolyzed in the presence of 10 microM probe. Trypsin or Staphylococcus aureus V8 protease digestion of the photolabeled protein, along with boronate affinity chromatography and immobilized metal affinity chromatography, was used to specifically isolate the peptide region photolabeled with the probe. The peptide corresponded to Phe9-Gly19 near the N terminus.

ADP Ribose Transferases↗

PubMed1992

Nucleotide sequences and characterization of haemolysin genes from Aeromonas hydrophila and Aeromonas sobria.

Extracellular haemolysin is thought to be one of the important virulence factors in Aeromonas infection. Two extracellular haemolysin genes (AHH3 and AHH4) from Aeromonas hydrophila strain 28SA, one (AHH5) from A. hydrophila strain AH-1 and one (ASA1) from Aeromonas sobria strain 33 were cloned into cosmid and plasmid vector DNA in Escherichia coli. The nucleotide sequences of the open reading frames of AHH3 and AHH4 are both 1476 basepairs (bp), whereas AHH5 and ASA1 are 1455 and 1467 bp in length, respectively. The deduced amino acid sequences of AHH3, AHH4, AHH5 and the previously reported aerolysin from A. hydrophila showed a significant degree of sequence homology of over 90% each. The amino acid identity of the ASA1 haemolysin and those from A. hydrophila and Aeromonas trota aerolysins ranged from 58-68%. From DNA hybridization analysis using our cloned haemolysin genes as probes, we found that the AHH5 and ASA1 DNA probes hybridized with about 31 and 75% strains of motile Aeromonas species, respectively. The activity of haemolysins of cloned genes were different in medium agar containing various erythrocytes.

Aeromonas↗

PubMed1991

Clostridium botulinum C3 ADP-ribosyltransferase gene. Cloning, sequencing, and expression of a functional protein in Escherichia coli.

C3 ADP-ribosyltransferase is an exoenzyme produced by certain strains of Clostridium botulinum types C and D, which specifically ADP-ribosylates rho and rac proteins in eukaryotic cells. The enzyme was purified from a culture filtrate of C. botulinum type C strain 003-9, and the amino acid sequence from the amino-terminal Ser to Asn192 was determined by Edman degradation. Using a set of degenerate primers based on the sequence, we amplified a part of the gene for this enzyme by polymerase chain reaction. A 2.1-kilobase pair HincII fragment of C. botulinum DNA containing the whole structural gene was then identified by Southern analysis with the polymerase chain reaction product as a probe, and the complete nucleotide structure of the gene together with flanking regions was determined by cloning and DNA sequencing the HincII fragment. The gene encodes a protein of 244 amino acids with a Mr of 27,362 which begins with a putative signal peptide of 40 amino acids. Escherichia coli carrying this gene produced the active enzyme, and about 60% of it was found in the culture medium. Immunoblot analysis with antiserum against the enzyme revealed the presence of two immunoreactive proteins of 27 and 23 kDa in the cytoplasmic/membrane fraction and only the 23-kDa protein in the periplasm and the medium, suggesting that the enzyme expressed is processed in the E. coli, exported into the periplasm and released into the culture medium.

ADP Ribose Transferases↗

PubMed1991

Distinction between Clostridium botulinum type A strains associated with food-borne botulism and those with infant botulism in Japan in intraintestinal toxin production in infant mice and some other properties.

Two strains of Clostridium botulinum type A associated with food-borne botulism and six strains associated with infant botulism in Japan were compared in intraintestinal toxin production in infant mice, in vitro toxin and hemagglutinin production, molecular sizes of the toxins, and some other properties. The infant botulism-associated strains, producing M toxin (Mr 300 kDa) but no hemagglutinin, showed significantly lower 50% infective doses in infant mouse intestines. The antigenicities of the toxin differed between the two groups, while the biochemical properties of the cultures did not. Besides infant botulism-associated strains, this set of properties were found only in a strain isolated from honey of South American origin.

Animals↗

PubMed1991

Properties of a protease-sensitive acceptor component in mouse brain synaptosomes for Clostridium botulinum type B neurotoxin.

To characterize an acceptor for Clostridium botulinum type B neurotoxin, its binding kinetics were examined with mouse brain synaptosomes treated with various enzymes. The amount of 125I-labelled neurotoxin bound to synaptosomes decreased upon treatment with lysyl endopeptidase, neuraminidase, or phospholipase C. The binding of the neurotoxin was partially recovered by incubation of neuraminidase-treated synaptosomes with ganglioside GT1b or GD1a. Gangliosides incorporated into untreated, lysyl endopeptidase-treated, and phospholipase C-treated synaptosomes had no effect on the binding of the neurotoxin. These results may suggest that type B neurotoxin binds to gangliosides in cooperation with a certain protease-sensitive substance on the neural membranes.

Animals↗

PubMed1991

[Isolation of enteropathogenic microorganism from patients with infection of the digestive tract during 1976 to 1988 in Tenri Hospital].

Enteropathogenic microorganisms isolated from feces of 9,393 patients with diarrhea or enteritis in our hospital between 1976 and 1988 were analyzed. As the result of the examination of 5,443 outpatients, 1,811 strains of pathogens were isolated from 1,686 cases (31.0%). Several species including Salmonella spp., Escherichia coli serotype, Vibrio parahaemolyticus, were isolated before 1978, and the incidence of pathogens was low (14.8%). For the 10-year period since 1979, the incidence markedly increased to 34.4%, and the number of pathogens isolated also increased to about twice that before 1978. The main cause of the increase was Campylobacter species. The major pathogens detected since 1979 were Campylobacter spp., E. coli serotype, Salmonella spp., V. parahaemolyticus, etc., but Rota virus, Clostridium difficile, Aeromonas spp., Vibrio fluvialis, etc. have also been detected, showing an increase in the number and diversity of the detected pathogens. As the result of the examination of 3,950 inpatients, 835 strains of pathogens were isolated from 800 cases (20.3%). The incidence of C. difficile was the highest, 423 of 800, followed by E. coli serotype, Salmonella spp., Campylobacter spp., V. parahaemolyticus and Aeromonas spp., in that order. All the inpatients from whom C. difficile was isolated manifested diarrhea or enteritis after administration of antimicrobial agents. The pathogens causing communicable disease were Salmonella spp. serovar Typhi, Salmonella spp. serovar Paratyphi A, Shigella flexneri, Shigella sonnei and Entamoeba histolytica, which were isolated from 5, 1, 3, 2 and inpatients, respectively.

Adolescent↗

PubMed1991

Immunological characterization of Clostridium butyricum neurotoxin and its trypsin-induced fragment by use of monoclonal antibodies against Clostridium botulinum type E neurotoxin.

We examined the reactivities of Clostridium butyricum neurotoxin to nine monoclonal antibodies against Clostridium botulinum type E neurotoxin which recognize the light chain or the amino-terminal half (H-1 fragment) or the carboxyl-terminal half (H-2 fragment) of the heavy chain of botulinum neurotoxin. Butyricum neurotoxin and its derived chains did not react to two of four monoclonal antibodies recognizing the light chain, one of three recognizing the H-1 fragment, and one of two recognizing the H-2 fragment. The results indicate that the immunological difference between the two neurotoxins is not attributable to a particular portion of the toxin molecule. The fragment of butyricum neurotoxin obtained by prolonged tryptic treatment was found to comprise the light chain and H-1 fragment linked together by a disulfide bond.

Animals↗