Biomedical subjects

Chikara Kaito

Publications and source records attributed to Chikara Kaito.

13 recordsLinked to original sources

PubMed2006

Colony spreading in Staphylococcus aureus.

Wild-type Staphylococcus aureus rapidly expands on the surface of soft agar plates. The rates of expansion and the shapes of the resultant giant colonies were distinct for different strains of laboratory stocks and clinical isolates. The colony spreading abilities did not correlate with the biofilm-forming abilities in these strains. Insertional disruption of the dltABCD operon, which functions at the step of D-alanine addition to teichoic acids, and of the tagO gene, which is responsible for the synthesis of wall teichoic acids, decreased the colony spreading ability. The results indicate that wall teichoic acids and D-alanylation of teichoic acids are required for colony spreading.

Agar↗

PubMed2006

Use of silkworm larvae to study pathogenic bacterial toxins.

Injection of stationary phase culture-supernatants of Staphylococcus aureus and Pseudomonas aeruginosa into the hemolymph of silkworm larvae caused their death, whereas a culture-supernatant of a non-pathogenic strain of Escherichia coli did not. A culture-supernatant of a mutant of agr, a global virulence regulator of S. aureus that is required for exotoxin production, was much less toxic to silkworm larvae. A culture-supernatant of a disruption mutant of the S. aureus beta-toxin gene did not kill larvae, whereas one of a deletion mutant of alpha-toxin, gamma-toxin, or aureolysin killed larvae, indicating that the beta-toxin gene is required for staphylococcal supernatant-mediated killing of silkworm larvae. The 50% lethal doses (LD50) of staphylococcal alpha-toxin and beta-toxin, Pseudomonas exotoxin A and diphtheria toxin were 12 microg/g, 9 microg/g, 0.14 microg/g and 1.1 microg/g, respectively. As the purified toxins killed the larvae, silkworm larvae could be used as a model to study the actions of pathogenic bacterial toxins in animal bodies.

Animals↗

PubMed2006

Cleavage and survival of Xenopus embryos exposed to 8 T static magnetic fields in a rotating clinostat.

In this study, we examined cleavage and survival of fertilized Xenopus embryos exposed to 8 T static magnetic fields (SMFs). We investigated fertilized Xenopus embryos exposed to magnetic field either in static chamber or in a rotating culture system. Our results showed that the exposure to the strong magnetic field of 8 T changed the third cleavage furrow from the usual horizontal one to a perpendicular one; however, when the direction of gravity was randomized by exposing embryos to magnetic field in a rotating culture system, the third cleavage furrow were formed horizontally, a finding which suggests that the observed distortion of the third cleavage furrow in magnetism-exposed embryos was accomplished by altering gravity effects which were elicited by diamagnetic force due to high gradient magnetic field. Our results also showed that the exposure to the strong magnetic field did not damage survival. These results demonstrate that SMF and altering gravity cause distortion of the third cleavage furrow and show that effects of exposing cleavage embryos to magnetic field were transient and did not affect the post-cleavage development. We also showed that strong magnetic field is not hazardous to the cleavage and blastula-gastrula transition of developing embryonic cells.

Animals↗

PubMed2006

Novel DNA binding protein SarZ contributes to virulence in Staphylococcus aureus.

We previously reported that the cvfA gene is a virulence regulatory gene in Staphylococcus aureus. Here, we identified a novel gene named sarZ that acts as a multicopy suppressor of decreased haemolysin production in the cvfA deletion mutant. The amount of sarZ transcripts was decreased in the cvfA mutant. The sarZ-deletion mutant produced less haemolysin and attenuated virulence in a silkworm-infection model and a mouse-infection model. The amino acid sequence of the sarZ gene product had 19% identity with the transcription factor MarR in Escherichia coli, and the internal region contained a winged helix-turn-helix motif (wHTH), a known DNA binding domain. Purified recombinant SarZ protein had binding affinity for the promoter region of the hla gene that encodes alpha-haemolysin. SarZ mutant proteins with an amino acid substitution in the N-terminal region or in the wHTH motif had significantly decreased DNA binding. The mutated sarZ genes encoding SarZ mutant proteins with a low affinity for DNA did not complement the decreased haemolysin production or the attenuated killing ability against silkworms in the sarZ mutant. These results suggest that the DNA binding activity of the SarZ protein is required for virulence in S. aureus.

Amino Acid Sequence↗

PubMed2005

Occurrence of pre-MBT synthesis of caspase-8 mRNA and activation of caspase-8 prior to execution of SAMDC (S-adenosylmethionine decarboxylase)-induced, but not p53-induced, apoptosis in Xenopus late blastulae.

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xenopus fertilized eggs activates caspase-9 and executes maternal program of apoptosis shortly after midblastula transition (MBT). We find that overexpression of caspase-8 and p53, like that of SAMDC, induces apoptosis in Xenopus late blastulae. The apoptosis induced by p53 was abolished by injection of mRNA for xdm-2, a negative regulator of p53, and by injection of a peptide inhibitor or a dominant-negative type mutant of caspase-9, but not caspase-8. The apoptosis induced by SAMDC was not abolished by injection of xdm-2 mRNA, but was abolished by injection of a peptide inhibitor or a dominant-negative type mutant mRNA of both caspase-9 and caspase-8. Unlike caspase-9 mRNA, caspase-8 mRNA did not occur as a maternal mRNA rather induced to be expressed during cleavage stage (pre-MBT stage) by overexpression of SAMDC but not p53. Furthermore, while activities to process procaspase-8 and procaspase-9 appeared in SAMDC-overexpressed apoptotic embryos, the activity to process procaspase-8 did not appear in p53-overexpressed apoptotic embryos. We conclude there are at least two pathways in the execution of the maternal program of apoptosis in Xenopus embryos; one being through do novo expression of caspase-8 gene during cleavage stage, and the other without involvement of caspase-8.

Adenosylmethionine Decarboxylase↗

PubMed2005

Participation of Rho-dependent transcription termination in oxidative stress sensitivity caused by an rpoB mutation.

The role of transcription termination process for gene expression regulation is poorly understood. Either a multicopy supply of the rof gene or bicyclomycin, both of which inhibit the transcription termination Rho factor, suppressed the increased sensitivity to oxidative stress of the rifampicin-resistant rpoB mutation in Escherichia coli. Multi-copy supply of the rnk gene also suppressed oxidative stress sensitivity, coincident with the recovery of the reduced concentration of nucleoside triphosphates in the mutant cells, which is one of the factors that affects transcription termination efficiency in vitro. Thus, an appropriate, nonexcessive termination frequency at Rho-dependent transcription terminators might contribute to oxidative stress survival. Clinical application of oxidative stress against drug resistant bacteria is also discussed.

DNA-Directed RNA Polymerases↗

PubMed2005

Silkworm pathogenic bacteria infection model for identification of novel virulence genes.

Silkworms are killed by injection of pathogenic bacteria, such as Staphylococcus aureus and Streptococcus pyogenes, into the haemolymph. Gene disruption mutants of S. aureus whose open reading frames were previously uncharacterized and that are conserved among bacteria were examined for their virulence in silkworms. Of these 100 genes, three genes named cvfA, cvfB, and cvfC were required for full virulence of S. aureus in silkworms. Haemolysin production was decreased in these mutants. The cvfA and cvfC mutants also had attenuated virulence in mice. S. pyogenes cvfA-disrupted mutants produced less exotoxin and had attenuated virulence in both silkworms and mice. These results indicate that the silkworm-infection model is useful for identifying bacterial virulence genes.

Animals↗

PubMed2004

Quantitative evaluation of the therapeutic effects of antibiotics using silkworms infected with human pathogenic microorganisms.

The injection of bacteria (Staphylococcus aureus, Stenotrophomonas maltophilia) or true fungi (Candida albicans, Candida tropicalis) that are pathogenic to humans into the silkworm hemolymph leads to death of the larvae within 2 days. Antibiotics used for clinical purposes have therapeutic effects on silkworms infected with these pathogens. The 50% effective doses obtained by injection into the silkworm hemolymph are consistent with those reported for mice. Injection of vancomycin and kanamycin into the silkworm hemolymph was effective, but oral administration was not. Chloramphenicol, which is effective by oral administration, appeared in the silkworm hemolymph soon after injection into the midgut, whereas vancomycin did not. Isolated midgut membranes were impermeable to vancomycin. Thus, the ineffectiveness of oral administration of vancomycin to silkworms is due to a lack of intestinal absorption.

Administration, Oral↗

PubMed2003

Inhibitory effects of basic or neutral phospholipid on acidic phospholipid-mediated dissociation of adenine nucleotide bound to DnaA protein, the initiator of chromosomal DNA replication.

DnaA protein activity, the initiator of chromosomal DNA replication in bacteria, is regulated by acidic phospholipids such as phosphatidylglycerol (PG) or cardiolipin (CL) via facilitation of the exchange reaction of bound adenine nucleotide. Total lipid isolated from exponentially growing Staphylococcus aureus cells facilitated the release of ATP bound to S. aureus DnaA protein, whereas that from stationary phase cells was inert. Fractionation of total lipid from stationary phase cells revealed that the basic phospholipid, lysylphosphatidylglycerol (LPG), inhibited PG- or CL-facilitated release of ATP from DnaA protein. There was an increase in LPG concentration during the stationary phase. A fraction of the total lipid from stationary phase cells of an integrational deletion mprF mutant, in which LPG was lost, facilitated the release of ATP from DnaA protein. A zwitterionic phospholipid, phosphatidylethanolamine, also inhibited PG-facilitated ATP release. These results indicate that interaction of DnaA protein with acidic phospholipids might be regulated by changes in the phospholipid composition of the cell membrane at different growth stages. In addition, the mprF mutant exhibited an increased amount of origin per cell in vivo, suggesting that LPG is involved in regulating the cell cycle event(s).

Adenine Nucleotides↗

PubMed2003

Isolation and mutation site determination of the temperature-sensitive murB mutants of Staphylococcus aureus.

The murB gene encodes UDP-N-acetylenolpyruvylglucosamine reductase and functions in bacterial peptidoglycan biosynthesis. A plasmid carrying the murB gene restored the temperature-sensitive growth of six Staphylococcus aureus mutants, in which peptidoglycan biosynthesis stopped at a restrictive temperature. Specific activity of UDP-N-acetylenolpyruvylglucosamine reductase in extracts from the mutants was lower than that from wild-type cells. Nucleotide sequence determination revealed that each mutant had a single amino acid substitution in the murB gene and five of six mutations were located within domain 3, where the proposed substrate binding site is located. These results suggest that the murB gene is essential for growth of S. aureus and that domain 3 is important for the MurB activity.

Amino Acid Sequence↗

PubMed2003

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xenopus embryos activates maternal program of apoptosis as a "fail-safe" mechanism of early embryogenesis.

In Xenopus, injection of S-adenosylmethionine decarboxylase (SAMDC) mRNA into fertilized eggs or 2-cell stage embryos induces massive cell dissociation and embryo-lysis at the early gastrula stage due to activation of the maternal program of apoptosis. We injected SAMDC mRNA into only one of the animal side blastomeres of embryos at different stages of cleavage, and examined the timing of the onset of the apoptotic reaction. In the injection at 4- and 8-cell stages, a considerable number of embryos developed into tadpoles and in the injection at 16- and 32-cell stages, all the embryos became tadpoles, although tadpoles obtained were sometimes abnormal. However, using GFP as a lineage tracer, we found that descendant cells of the blastomere injected with SAMDC mRNA at 8- to 32-cell stages are confined within the blastocoel at the early gastrula stage and undergo apoptotic cell death within the blastocoel, in spite of the continued development of the injected embryos. These results indicate that cells overexpressed with SAMDC undergo apoptotic cell death consistently at the early gastrula stage, irrespective of the timing of the mRNA injection. We assume that apoptosis is executed in Xenopus early gastrulae as a "fail-safe" mechanism to eliminate physiologically-severely damaged cells to save the rest of the embryo.

Adenosylmethionine Decarboxylase↗

PubMed2002

Isolation and characterization of temperature-sensitive mutants of the Staphylococcus aureus dnaC gene.

A protein encoded by the Staphylococcus aureus dnaC gene has 44% and 58% homology with Escherichia coli DnaB and Bacillus subtilis DnaC replicative DNA helicases, respectively. We identified five mutant strains whose temperature-sensitive colony formation phenotypes were complemented by the dnaC gene. DNA replication in these mutants has a fast-stop phenotype, indicating that the S. aureus dnaC gene encodes the replicative DNA helicase required for the elongation step. These mutants were also sensitive to UV irradiation, suggesting that the dnaC gene is involved in DNA repair. The number of viable mutant cells decreased at a non-permissive temperature, suggesting that S. aureus DnaC helicase is a promising target for antibiotics providing bactericidal effects.

Amino Acid Sequence↗

PubMed2002

Silkworm larvae as an animal model of bacterial infection pathogenic to humans.

Silkworm larvae, Bombyx mori, were examined as an animal model of human infection with pathogenic bacteria. When 3 x 10(7) cells of Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa, or Vibrio cholerae were injected into the blood of fifth instar silkworm larvae, over 90% of the larvae died within 2 days, whereas over 90% survived for 5 days after injection of the same amount of Escherichia coli. Growth of S. aureus was observed in larvae blood and tissues. Immunostaining analysis revealed that S. aureus proliferated at the surface of the midgut. Infection of silkworm larvae by methicillin-sensitive S. aureus was cured by ampicillin, oxacillin, and vancomycin, whereas infection by methicillin-resistant S. aureus was not cured by ampicillin or oxacillin, although vancomycin was effective. Disinfectants were not effective because of toxicity against the larvae. Thus, silkworm larvae are useful for evaluating antibiotics for pathogenic bacterial infection in humans.

Animals↗