Biomedical subjects

Yutaka Ito

Publications and source records attributed to Yutaka Ito.

28 records · Page 2Linked to original sources

PubMed2003

An NMR study on the interaction of Escherichia coli DinI with RecA-ssDNA complexes.

The SOS response, a set of cellular phenomena exhibited by eubacteria, is initiated by various causes that include DNA damage-induced replication arrest, and is positively regulated by the co- protease activity of RecA. Escherichia coli DinI, a LexA-regulated SOS gene product, shuts off the initiation of the SOS response when overexpressed in vivo. Biochemical and genetic studies indicated that DinI physically interacts with RecA to inhibit its co-protease activity. Using nuclear magnetic resonance (NMR) spectroscopy, we show that DinI tightly binds to the central region of RecA (between the N- and C-terminal domains) and that this interaction is enhanced upon the oligomerisation of RecA. On the other hand, DinI did not inhibit the interaction between 4mer single-stranded (ss)DNA and RecA- ATPgammaS, but had a slight effect on the structure of ssDNA-RecA-ATPgammaS complexes involving 8mer and 12mer ssDNA. We hypothesise that prevention of repressor binding to the intermolecular cleft region of RecA protomers by DinI, with the possibility of a slight conformational change induced in the DinI-bound ssDNA-RecA-ATPgammaS complex, together function to inhibit the co-protease activity of RecA.

Adenosine Triphosphate↗

PubMed2003

Results of surgical treatment for lumbar canal stenosis due to degenerative spondylolisthesis: enlargement of the lumbar spinal canal.

The purpose of this retrospective study was to analyze clinical results and radiographic findings in patients who underwent surgical enlargement of the lumbar spinal canal combined with resection of the posterosuperior margin underneath the slipping vertebral body for the treatment of lumbar canal stenosis due to degenerative spondylolisthesis. A series of 64 patients who were observed for 3 years or more after operation were examined. The mean age at the time of operation was 64.2 years. The follow-up period was 3-17 years. The Japanese Orthopaedic Association (JOA) score increased from 14.9 points before operation to 25.4 points at the time of the study on average. The general improvement rate was 75.6%. The height and range of motion of the enlarged intervertebral disc were mildly to moderately decreased, and it was found there was a small effect on the adjacent intervertebral disc. On computed tomography, the total level of the enlarged region of the posterosuperior margin increased from 184.4 mm(2) to 339.1 mm(2) on average, but the area of the resected region was 163.3 mm(2) and accounted for 48% of the postoperative area of the spinal canal in the posterosuperior margin. This enlargement of the spinal canal was maintained along the dural canal, and physiological morphology was established. By surgically enlarging the lumbar spinal canal combined with resecting the posterosuperior margin underneath the slipping vertebral body, concomitant repositioning or spinal fixation was unnecessary.

Adult↗

PubMed2003

A novel method for the biosynthesis of deuterated proteins with selective protonation at the aromatic rings of Phe, Tyr and Trp.

A novel biosynthetic strategy is described for the preparation of deuterated proteins containing protons at the ring carbons of Phe, Tyr and Trp, using the aromatic amino acid precursor shikimic acid. Specific protonation at aromatic side chains, with complete deuteration at C(alpha/beta) positions was achieved in proteins overexpressed in bacteria grown in shikimate-supplemented D2O medium. Co-expression of a shikimate transporter in prototrophic bacteria resulted in protonation levels of 62-79%, whereas complete labeling was accomplished using shikimate auxotrophic bacteria. Our labeling protocol permits the measurement of important aromatic side chain derived distance restraints in perdeuterated proteins that could be utilized to enhance the accuracy of NMR structures calculated using low densities of NOEs from methyl selectively protonated samples.

Bacterial Proteins↗

PubMed2003

Seeligeriolysin O, a cholesterol-dependent cytolysin of Listeria seeligeri, induces gamma interferon from spleen cells of mice.

Seeligeriolysin O (LSO), one of the cholesterol-dependent cytolysins produced by Listeria seeligeri, shows 80% homology to listeriolysin O (LLO) produced by Listeria monocytogenes at the amino acid sequence level. In addition to cytolytic activity, LLO has been shown to exhibit cytokine-inducing activity. In order to determine whether LSO is also capable of exhibiting these two different activities, we constructed a recombinant full-length LSO (rLSO530) and a noncytolytic truncated derivative with a C-terminal deletion (rLSO483) and compared these molecules with recombinant LLO. The cytolytic rLSO530 molecule could induce gamma interferon (IFN-gamma) production in spleen cells when the cytolytic activity was blocked by treatment with cholesterol. The noncytolytic truncated rLSO483 molecule also induced IFN-gamma production. Anti-LLO polyclonal antibody inhibited not only LLO-induced IFN-gamma production but also LSO-induced IFN-gamma production. Both NK cells and CD11b(+) cells were required for LSO-induced IFN-gamma production. Among the various cytokines expressed in CD11b(+) cells, interleukin-12 (IL-12) and IL-18 appeared to be essential. We concluded that LSO exhibits the same biological activity as LLO.

Animals↗

PubMed2003

Differences in gamma interferon production induced by listeriolysin O and ivanolysin O result in different levels of protective immunity in mice infected with Listeria monocytogenes and Listeria ivanovii.

Two pathogenic species in the genus Listeria, Listeria monocytogenes and Listeria ivanovii, are characterized by the production of hemolysins belonging to cholesterol-dependent cytolysins, listeriolysin O (LLO) and ivanolysin O (ILO), respectively. LLO, produced by L. monocytogenes, is able to induce gamma interferon (IFN-gamma) production and contributes to the generation of Th1-dependent protective immunity. On the other hand, nothing is known about the role of ILO, produced by L. ivanovii, in this regard. In this study, we immunized mice with 0.1 50% lethal dose (LD(50)) of L. monocytogenes and L. ivanovii. Protective immunity against a challenge with 10 LD(50) was generated in mice infected with L. monocytogenes, whereas L. ivanovii infection did not induce protection. After immunization, the level of IFN-gamma in serum samples was increased in mice given L. monocytogenes but not in those given L. ivanovii. To determine the IFN-gamma-inducing activity of cytolysins, recombinant protein was constructed. Recombinant ILO exhibited significantly lower IFN-gamma-inducing activity than LLO. By comparing the IFN-gamma-inducing activity of a chimera incorporating LLO and ILO, it was found that domains 1 to 3 of LLO were critical for IFN-gamma-inducing activity while the counterpart in ILO was unable to induce cytokine production. These results suggested that the weak ability of ILO to induce IFN-gamma production is responsible for the failure of L. ivanovii to generate effective protective immunity.

Amino Acid Sequence↗

PubMed2002

A novel approach for the sequential backbone assignment of larger proteins: selective intra-HNCA and DQ-HNCA.

Sequential assignment of backbone resonances in larger proteins can be achieved by recording two or more complementary triple-resonance NMR spectra of deuterated proteins. For such proteins, higher fields and experiments based on the TROSY method provide the needed resolution and sensitivity. However, increasingly rapid carbonyl relaxation at the high magnetic field strengths required by TROSY techniques renders assignment strategies that rely on sequential HN(CO)CA-type experiments much less efficient for proteins >40 kDa. Here we present two complementary new experiments, which allow backbone assignments with good sensitivity for larger deuterated proteins. A 3D intra-HNCA experiment provides uniquely the intraresidue connection, while a 3D DQ-HNCA experiment, which detects a (13)C(alpha)(i)()(13)C(alpha)(i-1)() double-quantum (DQ) coherence, contains the sequential information. The experiments work well at high magnetic fields, and their utility is demonstrated on a protein with a correlation time of 28 ns ( approximately 60 kDa). For larger proteins the sensitivity is predicted through simulations which suggest that the approach should work for proteins with correlation times >50 ns.

Carbon Isotopes↗

PubMed2002

Induction of gamma interferon and nitric oxide by truncated pneumolysin that lacks pore-forming activity.

Pneumolysin (PLY), an important virulence factor of Streptococcus pneumoniae, is known to exert various effects on the host immune cells, including cytokine induction, in addition to its known cytolytic activity as a member of the thiol-activated cytolysins. It is of interest to determine whether cytolytic activity is involved in triggering the cytokine production. In this study, we constructed full-length recombinant PLY and noncytolytic truncated PLYs with C-terminal deletions to examine the response of spleen cells to these PLY preparations. When cytolytic activity was blocked by treatment with cholesterol, full-length PLY was capable of inducing gamma interferon (IFN-gamma) production. Truncated PLYs that originally exhibited no cytolytic activity were also active in IFN-gamma induction. Therefore, the IFN-gamma-inducing ability of PLY appeared to be independent of the cytolytic activity. Furthermore, IFN-gamma-inducing preparations were also capable of inducing nitric oxide synthase expression and nitric oxide (NO) production, and the addition of neutralizing antibody to IFN-gamma abolished the NO production. These results clearly demonstrated that PLY is capable of inducing IFN-gamma production in spleen cells by a mechanism different from pore formation and that the induced IFN-gamma stimulates NO production. These findings were discussed with reference to the contribution of PLY to the virulence of S. pneumoniae in vivo.

Animals↗

PubMed2002

Essential role of interleukin-12 (IL-12) and IL-18 for gamma interferon production induced by listeriolysin O in mouse spleen cells.

The mechanism of gamma interferon (IFN-gamma) production induced by listeriolysin O (LLO), a cytolytic virulence factor of Listeria monocytogenes, was analyzed with special reference to the involvement of macrophage-derived cytokines in spleen cells of mice. LLO purified from the culture supernatant of L. monocytogenes was capable of inducing a high level of IFN-gamma when its cytolytic activity was blocked by cholesterol treatment. The IFN-gamma-inducing ability of LLO was not dependent on possibly contaminating lipopolysaccharide. Depletion of CD11b(+) cells resulted in a profound decrease in IFN-gamma production in response to LLO stimulation. Negative selection also suggested the contribution of DX5(+) cells in IFN-gamma production. Reverse transcription-PCR revealed that expression of interleukin-12 (IL-12) p35 and p40 was induced by LLO but that the IL-18 mRNA level in the CD11b(+) fraction of spleen cells was unchanged. There was no change in the expression of the IFN-gamma-inducing cytokine genes in the CD11b(-) fraction. Neutralization of IL-12 and IL-18 in culture abolished the IFN-gamma production almost completely. Spleen cells from IL-12- or IL-18-deficient mice never produced IFN-gamma after stimulation with LLO. These results clearly indicated that LLO, a well-known virulence factor of L. monocytogenes, is capable of inducing IFN-gamma from NK cells through induction of IL-12 and IL-18 from macrophages. LLO appeared to play essential roles, not only as a bacterial virulence factor but also as a bacterial modulin in the immune response of the host.

Animals↗

PubMed2002

Dissociated linkage of cytokine-inducing activity and cytotoxicity to different domains of listeriolysin O from Listeria monocytogenes.

Listeriolysin O (LLO), a cholesterol-binding cytolysin of Listeria monocytogenes, exhibits cytokine-inducing and cytolytic activities. Because the cytolytic activity was abolished by cholesterol treatment but the cytokine-inducing activity was not, these activities appeared to be linked to different domains of the LLO molecule. In this study, we constructed recombinant full-length LLO (rLLO529) and various truncated derivatives and examined their cytolytic, cholesterol-binding, and gamma interferon (IFN-gamma)-inducing activities. rLLO529 exhibited both IFN-gamma-inducing and cytolytic activities. Four truncated rLLOs possessing different C termini, which did not exert either cytolytic or cholesterol-binding activity, stimulated IFN-gamma production in normal spleen cells. However, a truncated rLLO corresponding to domain 4 (rLLO416-529) did not exhibit IFN-gamma-inducing activity, whereas it did bind to immobilized cholesterol. In addition, though the hemolysis induced by rLLO529 was inhibited by rLLO416-529, such inhibition was not detected upon rLLO529-induced IFN-gamma production. These data indicated that domain 4 was responsible for binding of LLO to membrane cholesterol followed by oligomerization and pore formation by the entire LLO molecule. In contrast, the other part of LLO, corresponding to domain 1-3, was essential for IFN-gamma-inducing activity. These findings implied a novel aspect of the function of LLO as a bacterial modulin.

Animals↗

PubMed2002

Effect of denture cleaner using ozone against methicillin-resistant Staphylococcus aureus and E. coli T1 phage.

We examined the bactericidal and virucidal effectiveness of a denture cleaner that uses ozone (ozone concentration, 10 ppm) against methicillin-resistant Staphylococcus aureus (MRSA) and T1 phage, respectively. In the bactericidal activity test, with the ozone supply turned on, the number of bacteria was 3.1 x 10(3) CFU/mL at the beginning of the experiment, fell to 1.0 x 10(0) CFU/mL 10 min later, and was 1.0 x 10(0) CFU/mL or less afterwards. In contrast, when the ozone supply was cut off (air bubble only), the number of bacteria was 3.4 x 10(3) CFU/mL at the beginning of the experiment, and had fallen to 3.0 x 10(3) CFU/mL 60 min later (no statistically significant difference). In the virucidal activity test, the number of phages was 1.2 x 10(6) PFU/mL before ozone treatment, fell to about 1/10 of that number 10 min later, and was 6.1 x 10(0) PFU/mL 40 min later. These results indicate that the use of ozone in this denture cleaner is effective against MRSA and viruses.

Anti-Bacterial Agents↗