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K Namba

Publications and source records attributed to K Namba.

At least 37 records · Page 2Linked to original sources
PubMed2000

The bacterial flagellar cap as the rotary promoter of flagellin self-assembly.

The growth of the bacterial flagellar filament occurs at its distal end by self-assembly of flagellin transported from the cytoplasm through the narrow central channel. The cap at the growing end is essential for its growth, remaining stably attached while permitting the flagellin insertion. In order to understand the assembly mechanism, we used electron microscopy to study the structures of the cap-filament complex and isolated cap dimer. Five leg-like anchor domains of the pentameric cap flexibly adjusted their conformations to keep just one flagellin binding site open, indicating a cap rotation mechanism to promote the flagellin self-assembly. This represents one of the most dynamic movements in protein structures.

Bacteria↗
PubMed2000

Amelioration of experimental autoimmune uveoretinitis by pretreatment with a pathogenic peptide in liposome and anti-CD40 ligand monoclonal antibody.

We have defined a peptide K2 (ADKDVVVLTSSRTGGV) that corresponds to residues 201-216 of bovine interphotoreceptor retinoid-binding protein and induces experimental autoimmune uveoretinitis (EAU)4 in H-2Ak-carrying mice (H-2Ak mice). In this study, we attempted to ameliorate EAU in the H-2Ak mice without nonspecific suppression of T cell responses. Preceding s.c. administration of liposomes including K2 (liposomal K2) specifically inhibited subsequent generation of T cell response to K2. The same result was obtained with a combination of OVA323-339 peptide and the OVA-specific TCR-transgenic T cells. It was suggested that the inhibition was mainly attributed to peripheral anergy induction of T cells specific for the peptide Ag, although specific cell death might also be involved in the inhibition. Pretreatment with liposomal K2 also considerably abolished IFN-gamma production but not IL-4 production. The specific inhibitory effect of the pretreatment with liposomal peptide was augmented by a simultaneous administration of anti-CD40 ligand (anti-CD40L) mAb. Moreover, it was shown that the pretreatment with liposomal K2 reduced both the incidence and severity of the subsequent K2-induced EAU, and the simultaneous administration of anti-CD40L mAb augmented this preventive effect by liposomal K2. Our findings demonstrate that the s.c. administration of liposomal pathogenic peptide and anti-CD40L mAb can be applied to preventing autoimmune diseases without detrimental nonspecific suppression of T cell responses.

Amino Acid Sequence↗
PubMed2000

The process of amyloid-like fibril formation by methionine aminopeptidase from a hyperthermophile, Pyrococcus furiosus.

Amyloid is associated with serious diseases including Alzheimer's disease and senile-systemic amyloidosis due to misfolded proteins. In the course of study of the denaturation process of methionine aminopeptidase (MAP) from the hyperthermophile P. furiosus, we found that MAP forms amyloid-like fibrils, and we then investigated the mechanism of amyloid fibril formation. The kinetic experiments on denaturation monitored by CD at 222 nm indicated that MAP in the presence of 3.37 M GuHCl at pH 3.31 changed to a conformation containing a considerable content of beta-sheet structure after the destruction of the alpha-helical structure. MAP in this beta-rich conformation was highly associated, and its stability was remarkably high: the midpoint of the GuHCl denaturation curve was 4.82 M at pH 3.0, and a thermal transition was not observed up to 125 degrees C by calorimetry. The amyloid-like fibril formation of MAP was confirmed by Congo red staining with a typical peak at 542 nm in the difference spectrum, showing a cross-beta X-ray diffraction pattern with a clear sharp reflection at 4.7 A and a characteristic unbranched fibrillar appearance with a length of about 1000 A and a diameter of about 70 A in the electron micrographs. Present results indicate that the amyloid-like form of MAP appears just after the protein is almost completely denatured, and even highly stable proteins can also form amyloid-like conformation under conditions where the denatured state of the protein is abundantly populated.

Aminopeptidases↗
PubMed2000

Indispensable role of the transcription factor PEBP2/CBF in angiogenic activity of a murine endothelial cell MSS31.

Mice lacking the AML1/PEBP2alphaB/CBFa2 gene or PEBP2beta/CBFb gene exhibit a defect in definitive hematopoiesis and die in utero because of hemorrhage in the central nervous system. Hematopoiesis in the embryo is considered to be tightly associated with vascular development. Here we examined whether PEBP2/CBF plays any role in angiogenesis besides that in definitive hematopoiesis. We found that AML1/PEBP2alphaB/CBFa2, PEBP2alphaA/CBFa1, and PEBP2beta/CBFb were expressed in a murine endothelial cell line MSS31. The expression of these molecules as well as the DNA binding activity of PEBP2/CBF were augmented by angiogenic growth factors such as bFGF and VEGF. Moreover, the expression of PEBP2 alpha/CBFa protein in endothelial cells was confirmed at the site of angiogenesis in vivo. To further clarify the role of PEBP2/CBF in angiogenesis, we established permanent transfectants of PEBP2 beta-MYH11 gene, one that interacts with the runt domain of the alpha subunit and deregulates PEBP2/CBF in a dominant interfering manner. Proliferation, migration, and tube formation of the PEBP2 beta-MYH11 transfectants were significantly reduced in comparison with those activities of the mock transfectants. These results suggest that transcription factor PEBP2/CBF plays an important role in angiogenesis.

Animals↗
PubMed2000

Participation of leukocyte function-associated antigen-1 and NK cells in the homing of thymic CD8+NKT cells to the liver.

A distinct CD8+NKT cell population expressing TCRalpha/beta or TCRgamma/delta has been identified in liver and thymus. We wondered whether cell adhesion molecules play a role in the homing of CD8+NKT cells to the liver. The number of liver CD8+NKT cells was markedly reduced in leukocyte function-associated antigen (LFA)-1-/- mice compared with wild-type (WT) mice. The reduction was restricted to the liver only and no measurable alterations were found in other organs. In the liver of SCID mice reconstituted with thymocytes from LFA-1-/- or WT mice, the number of donor-derived CD8+NKT cells was comparable and the vast majority of these cells expressed TCRalpha/beta. In a reciprocal radiation thymocyte reconstitution system with LFA-1-/- and WT mice, LFA-1 expressed on liver cells other than CD8+NKT cells appeared to be required for the homing of thymic CD8+NKT cells to the liver. The accumulation of donor thymocyte-derived CD8+NKT cells in the liver of SCID mice was severely impaired by in vivo depletion of NK cells, but not of Kupffer cells. These results not only indicate that thymus provides a source for CD8+NKT cells expressing TCRalpha/beta in the liver, but also suggest that LFA-1 expressed on NK cells is involved in the homing of thymic CD8+NKT cells to the liver.

Animals↗
PubMed2000

A new purification method for overproduced proteins sensitive to endogenous proteases.

Proteolysisis a major problem in purification of overproduced proteins for structural studies. We developed a new method to avoid proteolysis of the products even in cases where popular protease inhibitors do not work effectively. When we cloned FlgF, a flagellar rod protein, from Salmonella typhimurium and overproduced it in Escherichia coli, FlgF was highly susceptible to cleavage by endogenous proteases after cell disruption even in the presence of various protease inhibitors. However, FlgF was not digested when the cells were disrupted in the presence of urea, which allowed us to develop the following new purification procedure. After cell disruption in the presence of urea and removal of the cell debris, the supernatant was passed through tandem-connected cation- and anion-exchange columns. Proteases were trapped in the cation-exchange column, and protease-free FlgF was eluted from the disconnected anion-exchange column. This gave a stable full-length product suitable for crystallization trials. The key procedures are cell disruption in the presence of urea and linked ion-exchange chromatography to quickly remove proteases as well as urea. This fast and simple method can be applied to purification of other overproduced proteins that are very sensitive to proteolysis.

Bacterial Outer Membrane Proteins↗
PubMed2000

Crystallization of the F41 fragment of flagellin and data collection from extremely thin crystals.

Flagellin, which constructs supercoiled filaments of the bacterial flagellum, is very difficult to crystallize because of its strong tendency to polymerize. We therefore crystallized the F41 fragment of flagellin, which does not polymerize because terminal regions that play important roles in polymerization are cleaved off. F41 was crystallized by the hanging drop vapor diffusion method in a mixture of polyethylene glycol, glycerol, and isopropanol, with a reservoir solution covered with silicon oil. The two key factors for success in growing sufficiently large crystals were isopropanol and silicon oil, which worked well to reduce the otherwise very high nucleation rate that resulted in hundreds of tiny crystals. The crystals were grown to very thin plates with thickness less than 10 microm, which made the collection of diffraction data very difficult. Freezing and annealing of the crystals and irradiation at synchrotron beamlines had to be carried out by specific methods and under specific conditions for its structure analysis at 2.0-A resolution.

Antigens, Bacterial↗
PubMed2000

Laminectomy performed using a micro bone saw.

In 154 clinical cases requiring laminectomy at any part of the spinal column, the laminae were removed using a micro bone saw. Immediately after the laminectomy two surgeons using a microscope examined the field for any damage to the dura, the spinal cord, the nerve roots and the epidural vessels. No injury to the spinal cord or the nerve roots was observed. In 5 patients the dura were lacerated for a length of up to 10 mm, and in all the patients adhesions were present due to previous operations. Epidural vessels were severed in 18 cases. The method is useful and less time-consuming than conventional laminectomy.

Adolescent↗
PubMed2000

Incomplete replication of human parainfluenza virus type 4 in LLC-MK2 cells and in L929 cells.

Human parainfluenza virus type 4A (hPIV-4A) and type 4B (hPIV-4B) were tested for their ability to replicate in the monkey kidney LLC-MK2 cell line (MK2 cells) and the murine L929 cell line (L929 cells). These cells are normally non-permissive for replication of hPIV-4; however, treatment with acetylated trypsin led to virus replication in MK2 cells, but was less effective for L929 cells. Endogenously produced interferon (IFN) played no role in virus replication in L929 cells. Synthesis of virus-specific polypeptides was suppressed in L929 cells. Whereas NP-mRNA and HN-mRNA were detected in MK2 cells, no HN-mRNA was detected in L929 cells. These results indicate that hPIV-4 can infect both MK2 cells and L929 cells. In MK2 cells, when protease exists in the extracellular medium, hPIV-4 exhibits multistep growth. In L929 cells, however, the cause of incomplete replication might be lack of other unknown factors.

Animals↗
PubMed2000

Effective priming of cytotoxic T lymphocyte precursors by subcutaneous administration of peptide antigens in liposomes accompanied by anti-CD40 and anti-CTLA-4 antibodies.

Recently it has been shown that modulation of CD40 molecules on antigen (Ag) carrying dendritic cells (DC) can bypass T cell help, resulting in priming cytotoxic T lymphocytes (CTL) specific for the Ag. In the present study we attempted to prime peptide Ag-specific CTL by a new method in which a peptide Ag in liposome (liposomal peptide), consisting of phosphatidylserine and phosphatidylcholine (3:7), was administrated subcutaneously with anti-CD40 and/or CTLA-4 monoclonal antibodies (mAb) to mice. We found that the subcutaneous administration of the liposomal peptide with both anti-CD40 and anti-CTLA-4 mAb enhanced CTL responses comparing with those induced by the liposomal peptide alone or the liposomal peptide plus each mAb. It was shown that liposomes were critical for induction of the CTL activity. Flow cytometry analysis of a peptide-bearing DC in lymph nodes (LN) and measurement of serum IL-12 indicated that anti-CD40 mAb promoted migration of DC to the LN, where DC might differentiate and acquire ability of priming CTL. These findings provide a possibility that our procedure is applicable to cancer patients.

Abatacept↗
PubMed2000

Different influence of macrophage migration inhibitory factor (MIF) in signal transduction pathway of various T cell subsets.

It has been shown that macrophage migration inhibitory factor (MIF) modulates not only macrophage functions, but also T cell functions. However, detailed analysis of the MIF function on responses of various T cell subpopulations remained to be elucidated. In this report, using a neutralizing anti-MIF monoclonal antibody (mAb) we examined MIF functions on various T cell lineages. It was shown that anti-MIF mAb inhibited antigen-specific responses of both IFN-gamma producing and IL-4 producing T cells. The inhibition appeared to be related to blockade of the signal pathway via T cell receptor (TCR) but not that via IL-2 receptor (IL-2R). However, the anti-MIF mAb showed no inhibitory effect on NK-T cell responses stimulated through TCR. These results suggest that MIF is involved in the signal pathway via TCR in mainstream T cells but not in NK-T cells.

Amino Acid Sequence↗
PubMed2000

Amyloid protofilament formation of hen egg lysozyme in highly concentrated ethanol solution.

Mutant human lysozymes (Ile56Thr & Asp67His) have been reported to form amyloid deposits in the viscera. From the standpoint of understanding the mechanism of amyloid formation, we searched for conditions of amyloid formation in vitro using hen egg lysozyme, which has been extensively studied from a physicochemical standpoint. It was found that the circular dichroism spectra in the far-ultraviolet region of the hen egg lysozyme changed to those characteristic of a beta-structure from the native alpha-helix rich spectrum in 90% ethanol solution. When the concentration of protein was increased to 10 mg/mL, the protein solution formed a gel in the presence of 90% ethanol, and precipitated on further addition of 10 mM NaCl. The precipitates were examined by electron microscopy, their ability to bind Congo red, and X-ray diffraction to determine whether amyloid fibrils were formed in the precipitates. Electron micrographs displayed unbranched protofilament with a diameter of approximately 70 A. The peak point of the difference spectrum for the Congo red binding assay was 541 nm, which is characteristic of amyloid fibrils. The X-ray diffraction pattern showed a sharp and intense diffraction ring at 4.7 A, a reflection that arises from the interstrand spacing in beta-sheets. These results indicate that the precipitates of hen egg lysozyme are amyloid protofilament, and that the amyloid protofilament formation of hen egg lysozyme closely follows upon the destruction of the helical and tertiary structures.

Amyloid↗
PubMed2000

Neuropeptide regulation of immunity. The immunosuppressive activity of alpha-melanocyte-stimulating hormone (alpha-MSH).

The ocular microenvironment is an extreme example of regional immunity. Within its microenvironment, expression of delayed type hypersensitivity (DTH) is suppressed. This immunosuppression is mediated in part by the constitutive expression of alpha-MSH. Previously we have found that alpha-MSH suppresses the production of IFN-gamma by activated effector T cells. Recently we have found that alpha-MSH can mediate induction of TGF-beta-producing T cells that act as regulatory T cells. This has encouraged us to further examine the potential for alpha-MSH to suppress T cell-mediated inflammation (autoimmune disease) and to regulate lymphokine production by effector T cells. When alpha-MSH was injected i.v. into mice at the time of peak retinal inflammation, the severity of experimental autoimmune uveitis (EAU) was significantly suppressed. Effector T cells activated in vitro in the presence of alpha-MSH proliferated and produced IL-4 and enhanced levels of TGF-beta while their IFN-gamma and IL-10 production was suppressed. The alpha-MSH-treated T cells functioned as regulatory T cells by suppressing in vitro IFN-gamma production by other inflammatory T cells. This regulatory activity was the function of alpha-MSH-treated CD4+ CD25+ T cells. Therefore, alpha-MSH mediates immunosuppression by inducing a differential expression of lymphokine production and by inducing activation of regulatory functions in T cells. This implies that alpha-MSH may take part in regional mechanisms of immunosuppression and possibly peripheral tolerance. Thus, alpha-MSH can be used to suppress autoimmune disease and possibly reestablish tolerance to autoantigens.

Animals↗
PubMed2000

Antimycobacterial activities of novel levofloxacin analogues.

In order to investigate structure-activity relationships between antimycobacterial activities and basic substituents at the C-10 position of levofloxacin (LVFX), we synthesized a series of pyridobenzoxazine derivatives by replacement of the N-methylpiperazinyl group of LVFX with various basic substituents. A compound with a 3-aminopyrrolidinyl group had one-half the activity of LVFX against Mycobacterium avium, M. intracellulare, and M. tuberculosis. Mono- and dimethylation of the 3-amino moiety of the pyrrolidinyl group increased the activities against M. avium and M. intracellulare but not those against M. tuberculosis. On the other hand, dialkylation at the C-4 position of the 3-aminopyrrolidinyl group enhanced the activities against M. avium, M. intracellulare, and M. tuberculosis. Thus, introduction of an N-alkyl or a C-alkyl group(s) into the 3-aminopyrrolidinyl group may contribute to an increase in potency against M. avium, M. intracellulare, and/or M. tuberculosis, probably through elevation of the lipophilicity. However, among the compounds synthesized, compound VII, which was a 2,8-diazabicyclo[4.3.0]nonanyl derivative with relatively low lipophilicity, showed the most potent activity against mycobacterial species: the activity was 4- to 32-fold more potent than that of LVFX and two to four times as potent as that of gatifloxacin. These results suggested that an increase in the lipophilicity of LVFX analogues in part contributed to enhancement of antimycobacterial activities but that lipophilicity of the compound was not a critical factor affecting the potency.

Anti-Infective Agents↗
PubMed2000

Species differences in oral bioavailability of methotrexate between rats and monkeys.

The contributions of incomplete absorption and a first-pass effect to the low bioavailability (BA) of methotrexate (MTX) were evaluated pharmacokinetically in rats and monkeys which respectively have a lower and higher aldehyde oxidase (AO) activity than humans. Plasma concentration profiles of MTX in rats showed linear and nonlinear pharmacokinetics respectively after intravenous (i.v.) and oral dosing of 0.1, 0.5 or 2.5 mg/kg MTX. In rats, most of the dose was excreted as the parent compound into bile and urine after i.v. dosing of 0.5 mg/kg MTX, while the radioactivity was largely eliminated in expired air after oral dosing of 0.5 mg/kg 14C-MTX. Elimination in expired air fell markedly following antibiotics treatment. 7-Hydroxymethotrexate (7-OH-MTX), formed from MTX by AO, was detected in monkey plasma after i.v. and oral dosing of 0.5 mg/kg MTX, but not in rat plasma. The ratio of the cumulative urinary excretion of 7-OH-MTX to MTX in monkeys was higher after oral dosing than after i.v. dosing. The low BA in rats (10% at 0.5 mg/kg) was shown to be mainly due to incomplete absorption, including limited absorption and degradation to 2,4-diamino-N10-methylpteroic acid (DAMPA) and glutamic acid (Glu) by the carboxypeptidase of intestinal bacteria. The low BA in monkeys (5% at 0.5 mg/kg) was shown to be mainly due to the extensive first-pass effect, including metabolism to 7-OH-MTX.

Animals↗
PubMed2000

Inhibition of experimental autoimmune uveoretinitis with anti-macrophage migration inhibitory factor antibodies.

PURPOSE: The role of macrophage migration inhibitory factor (MIF) in the regulation of ocular autoimmune disease was studied in experimental autoimmune uveoretinitis (EAU) in rats following immunization with a retinal antigen (Ag), interphotoreceptor retinoid-binding protein (IRBP). METHODS: LEW rats were immunized with a single injection of IRBP derived peptide, R16(ADGSSWEGVGVVPDV). A neutralizing monoclonal antibody (mAb, IgM) to MIF was injected intraperitoneally every second day from day 0 to day 6 (group A), or from day 8 to day 14 (group B). Control rats were treated with unrelated mouse IgM or PBS. T cell proliferative responses were measured 12 days after immunization. The occurrence and severity of EAU were observed and compared among experimental and control groups. RESULTS: T cell proliferative responses against R16 were inhibited in rats treated with anti-MIF mAb compared with the control rats. The development of EAU was delayed in the rats of group A in comparison with those of group B and the control group. The mean histological EAU score on day 18 in group A was 1.11 +/- 0. 11 and significantly lower than those of the group B (1.29 +/- 0.19) and the control (1.67 +/- 0.19). CONCLUSIONS: The present result suggests that MIF plays an important role in induction of EAU.

Amino Acid Sequence↗
PubMed1999

Folding energetics of a multidomain protein, flagellin.

Thermodynamic investigations of flagellin from Salmonella typhimurium and its proteolytic fragments were conducted by differential scanning calorimetry (DSC) and circular dichroism (CD) melting measurements. A new method of analysis for a multi-state transition based on our original theoretical treatment of thermodynamic equations has been developed to analyze those data. The analysis of DSC curves confirmed the three thermodynamic domains of flagellin. The thermodynamic parameters of each domain were revised from those previously reported and the new values of the parameters have a good correlation to the apparent molecular masses of the morphological domains. CD melting measurements at far and near-UV wavelengths showed sequential unfolding of the domains. Therefore, we could reasonably assign the thermodynamically identified domains to the morphological domains. Further analysis of both DSC and CD data provided insights into the folding energetics of the multidomain structure of flagellin. An inner domain (Df1) of flagellin in the filament unfolds through a relatively broad transition, while the two outer domains unfold cooperatively and show sharp transitions. This indicates that the interdomain interactions between Df1 and D2 has different characteristics from the apparently more intimate interactions between D2 and D3. These characteristics suggest that flagellin is organized with relatively flexible domains and rigid domains, which appears to be responsible for the well-regulated assembly mechanism of the bacterial flagellar filament.

Calorimetry, Differential Scanning↗