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S Kotani

Publications and source records attributed to S Kotani.

At least 73 records · Page 4Linked to original sources
PubMed1990

Microtubule assembly inhibitor protein consists of a rigid globule essential for its activity and highly mobile coils.

The structure of microtubule assembly inhibitor protein (MIP) was studied by proton nuclear magnetic resonance (NMR) and limited proteolysis. Spin-diffusion experiments revealed that MIP has a tightly folded structure, a "rigid globule." This globule was irreversibly denatured by heat treatment at 80 degrees C, and the denatured MIP showed little ability to inhibit microtubule assembly. This indicates that the native globular structure is essential for the activity. By spin-diffusion and spin-echo experiments, the other part of the molecule was found to be highly mobile. We termed the region "highly mobile coils." Most of the acidic residues appeared to be clustered in the highly mobile coils. By limited proteolysis using subtilisin, the mobile region was digested into shorter pieces, and a single 17-kDa fragment remained. Proton NMR spectrum of this fragment was much the same as the spin-diffusion subspectra of the rigid globule in the intact MIP molecule. Furthermore, the 17-kDa fragment was found to retain the activity to inhibit the microtubule assembly. These results indicate that MIP consists of two moieties; one domain forms a rigid globule which is essential for its activity to inhibit microtubule assembly, and the other acidic one is highly mobile and tails from the globule. The tertiary structures of these two domains appear to be independent from each other. These domains may be responsible for two different functions of MIP, the interaction with the cytoskeleton and the interaction with, for example, nuclear components.

Amino Acids↗
PubMed1990

Lack of response of murine peritoneal macrophages to in vitro activation by muramyl dipeptide (MDP). I. Macrophage activation by MDP is species dependent.

Peritoneal exudate macrophages from mice, rats, and guinea pigs were assessed using six different parameters of macrophage activation to determine whether the cells were stimulated under similar experimental conditions. Peritoneal exudate macrophages from mice, irrespective of strain, were far less responsive to a variety of in vitro stimulatory effects of N-acetylmuramyl-L-alanyl-D-isoglutamine than those from rats or guinea pigs, while no significant differences were noted with the reactivity to stimulation by endotoxic lipopolysaccharide. We conclude that macrophage activation by MDP in vitro is species dependent.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1990

Comparison of two kinds of cell packets of Micrococcus luteus by scanning electron microscopy: outermost layer maintaining the packet structure.

Two kinds of cell packets of Micrococcus luteus, one having teichuronic acids (TUA) in the cell wall and the other lacking TUA, have been independently reported by two groups of workers. A comparison by scanning electron microscopy of these packets provided a possibly consistent interpretation for the seemingly conflicting opinions whether TUA were involved in packet induction. It was strongly suggested that the packets having TUA in the wall were rigidly maintained by a bridging structure of the outermost layer of the peripheral wall, while the packets lacking TUA showed low contribution of the outermost layer to the bridging structure probably due to the absence of TUA.

Cell Wall↗
PubMed1990

Macrophages are stimulated by muramyl dipeptide to induce polymorphonuclear leukocyte accumulation in the peritoneal cavities of guinea pigs.

N-Acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide [MDP]) injected intraperitoneally significantly increased the number of cells entering the peritoneal cavity of guinea pigs primed with liquid paraffin or thioglycollate. There was a close relationship between peritoneal polymorphonuclear leukocyte (PMN) accumulation and the uptake of glucosamine by macrophages in guinea pigs treated with a variety of bacterial cell surface components such as cell wall peptidoglycan subunits and bacterial or synthetic lipid A. The PMN accumulation was also facilitated by the intraperitoneal transfer of the peritoneal macrophages that had been stimulated by MDP in vitro. Furthermore, cell-free lavage fluids taken from the peritoneum of MDP-treated guinea pigs also initiated the influx of PMNs when introduced into the peritoneal cavities of liquid paraffin-pretreated guinea pigs. These results suggest that a soluble factor which attracts neutrophils is produced by MDP-treated macrophages. Partial characterization of the factor is described.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1990

[Studies of antigens related to metabolic and growth inhibitions of Staphylococcus aureus L-form].

Metabolic and growth inhibiting activities in immunoglobulin (of anti-S. aureus L-form serum and anti-S. aureus coccal form serum) could be absorbed by cell membranes of S. aureus L-form and its coccal form, respectively. These activities could not be absorbed by cell membrane of Micrococcus luteus, Streptococcus pyogenes or Actinomyces viscosus. These findings suggested the existence of species-specific antigens of cell membrane. The membrane antigens of L-form related to the metabolic and growth inhibiting activities were stable to trypsin, heating and periodate, and were not solubilized by trypsin. A large part of the antigen in a typsin-insoluble membrane precipitate of L-form could be extracted by acetone and the subsequent use of chloroform-methanol (2: 1). A fractionation study of chloroform-methanol extract by using silicic acid calum indicated that more than two components were involved in metabolic and growth inhibiting activities.

Antibodies, Bacterial↗
PubMed1989

A common amino acid sequence in 190-kDa microtubule-associated protein and tau for the promotion of microtubule assembly.

Previously we reported that chymotryptic fragments of bovine adrenal 190-kDa microtubule-associated proteins (27-kDa fragment) and bovine brain tau (14-kDa fragment) contained microtubule-binding domain (Aizawa, H., Murofushi, H., Kotani, Hisanaga, S., Hirokawa, N., and Sakai, H. (1987) J. Biol. Chem. 262, 3782-3787; Aizawa, H., Kawasaki, H., Murofushi, H., Kotani, S., Suzuki, K., and Sakai, H. (1988) J. Biol. Chem. 263, 7703-7707). In order to study the structure of microtubule-binding domain of the two microtubule-associated proteins, we analyzed the amino acid sequence of the 27-kDa fragment and compared the sequence with that of the 14-kDa fragment. This revealed that 190-kDa microtubule-associated protein and tau contained at least one common sequence of 20 amino acid residues in their microtubule-binding domains. A synthetic polypeptide corresponding to the common sequence (Lys-Asn-Val-Arg-Ser-Lys-Val-Gly-Ser-Thr-Glu-Asn-Ile-Lys- His-Gln-Pro-Gly-Gly-Gly-Arg-Ala-Lys) was bound to microtubules competitively with the 190-kDa MAP. The apparent dissociation constant (KD) for the binding of the polypeptide to microtubules was estimated to be 1.8 x 10(-4) M, and the maximum binding reached 1.2 mol of the synthetic polypeptide/mol of tubulin dimer. This synthetic polypeptide increased the rate and extent of tubulin polymerization and decreased the critical concentration of tubulin for polymerization. The polypeptide-induced tubulin polymers were morphologically normal microtubules and were disassembled by cold treatment. The common sequence (termed assembly-promoting sequence) was thus identified as the active site of 190-kDa microtubule-associated protein and tau for the promotion of microtubule assembly. The reconstitution system of microtubules with this synthetic polypeptide with assembly-promoting sequence may be useful to elucidate detailed molecular mechanism of the promotion of microtubule assembly by microtubule-associated proteins.

Adrenal Glands↗
PubMed1989

T cell line specific for bacterial peptidoglycan subunit: possible role of the COOH-terminal amino acid of the disaccharide tetrapeptide in binding to the T cell receptor.

The T cell line specific for a bacterial cell wall peptidoglycan subunit, disaccharide tetrapeptide of diaminopimelic acid type, was examined for epitope specificity in elicitation of delayed-type hypersensitivity (DTH) in X-irradiated Lewis rats, using pairs of analogs different in optical configuration of the COOH-terminal amino acid. The test cell line induced DTH against analogs with the COOH-terminal D-amino acid but not against those with the L-amino acid at the COOH terminus. A close correlation was found between the T cell line-induced DTH reaction in vivo and the proliferative response in vitro, in terms of clear discrimination of the optical configuration of COOH-terminal amino acid of disaccharide tetrapeptide. The L-isomers (non-stimulatory analogs of T cell proliferation) competitively inhibited the proliferation of the T cell line by the corresponding D-isomers. Thus the L-isomers appear to interact with Ia molecules on antigen-presenting cells. We conclude that COOH-terminal D-amino acid of the disaccharide tetrapeptide could be involved in binding to the T cell receptor, induction of T cell proliferation, and elicitation of DTH.

Amino Acids↗
PubMed1989

Enhancement of humoral immune responses against viral vaccines by a non-pyrogenic 6-O-acylmuramyldipeptide and synthetic low toxicity analogues of lipid A.

6-O-Acyl derivatives of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) and synthetic, low toxicity lipid-A analogues were examined for their ability to enhance the potency of current viral vaccines. 6-O-(2-Tetradecylhexadecanoyl)-MDP (B30-MDP) in non-irritative vehicles such as physiological saline, phosphate-buffered saline (PBS), squalene-PBS emulsion, Intralipid or liposomes, significantly stimulated the primary and secondary antibody production of guinea-pigs against influenza split or subunit vaccine and inactivated the hepatitis B virus surface (HBs) antigen. Mice seemed less responsive to the adjuvanticity of B30-MDP than guinea-pigs. Two low toxicity lipid A analogues, acylated beta(1-6)-D-glucosamine disaccharide bisphosphates (which do not have amide-bound or ester-bound 3-acyloxyacyl groups unlike fully toxic Escherichia coli-type lipid A), caused significantly enhanced antibody responses, primary or secondary, when administered to mice by incorporation into liposomes with inactivated HBs antigen.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1989

Somnogenic activities of synthetic lipid A.

Bacterial infections and various immune response modifiers, including endotoxin and its lipid A moiety, alter sleep duration. The purpose of this study is to amplify our understanding of lipid A structure-somnogenic-pyrogenic activity relationships. Four synthetic disaccharide analogs of lipid A (LA-15-PP, LA-15-PH, LA-16-PH, and LA-18-PP) and a biosynthetic monosaccharide analog of lipid A (lipid X) were tested in rabbits for their effects on slow-wave sleep, rapid-eye-movement sleep, electroencephalographic slow-wave (0.5- to 4.0-Hz) amplitudes, and brain-colonic temperatures. Substances were injected intravenously and intracerebroventricularly. Compound LA-15-PP was the most potent; it significantly increased slow-wave sleep, delta electroencephalographic amplitudes, and brain-colonic temperatures while reducing rapid-eye-movement sleep. Compound LA-15-PH (monophosphoryl analog of LA-15-PP) induced effects similar to those of LA-15-PP, although the responses were weaker. Compound LA-18-PP induced increases in slow-wave sleep and delta amplitudes only after high doses, whereas compound LA-16-PH was devoid of these activities at the doses tested. Intracerebroventricular, but not intravenous, injections of lipid X induced small but significant increases in both slow-wave sleep and rapid-eye-movement sleep without affecting delta amplitudes or brain-colonic temperatures. These data suggest that the somnogenic actions of these lipid A analogs depend on the acylation or phosphorylation pattern and backbone structures of the molecules. Their soporific activities parallel their relative behaviors in other biological assays.

Animals↗
PubMed1989

Structural requirements of lipid A for endotoxicity and other biological activities.

For the past ten years, several groups were engaged in synthetic studies of lipid A, namely the lipid portion of bacterial lipopolysaccharides (LPS) that has been assumed to be the bioactive center of LPS, but has not been unanimously approved. Among them, Shiba, Kusumoto, and colleagues, Osaka, Japan have synthesized most energetically and successfully a variety of counterparts of lipid As, biosynthetic lipid A precursors, and their analogs. The endotoxic and related bioactivities of these synthetic compounds were studied by Japanese and German groups, including ours. In 1985, one of the compounds, having an acylation and phosphorylation pattern in beta(1-6)-D-glucosamine disaccharide which was proposed for Escherichia coli F515 lipid A was found to be exhibit full endotoxic and related bioactivities identical to those of the bacterial product. The study was extended by synthesis and examination of bioactivities of variously acylated D-glucosamine di- and monosaccharide phosphates, which correspond to structural components of lipid As, and their analogs or derivatives. Thus, structural requirements have been fairly well elucidated. In this article, first we will review the progress of synthetic and biological studies, with particular emphasis on chemical structure--bioactivities relationships of lipid As, and then we will discuss possible usefulness of some less or nontoxic lipid A-related synthetic compounds in clinical and preventive medicine.

Adjuvants, Immunologic↗
PubMed1989

[Seizure and tremor occurring in acute leukemia patients treated with imipenem/cilastatin].

We report here four patients with acute leukemia, who developed seizure or tremor following treatment with imipenem, a new broad-spectrum antibiotic. All four patients had no renal dysfunction and recovered after discontinuation of the drug. Two patients who developed seizure had a past history of cerebral hemorrhage with symptoms of meningitis in one and the other had received frequent intrathecal injections of methotrexate. Seizure also occurred in another patient who was given multiple intrathecal injections of methotrexate. The remaining old patient developed tremor after the first administration of imipenem which did not progress to convulsion. Cerebral hemorrhage or meningitis is known to predispose patients for convulsion following imipenem treatment. In addition, the present study suggests that central nervous system damage related with intrathecal injections of methotrexate may be a predisposing factor. Thus, imipenem should be given with caution to acute leukemia patients who often have risk factors for developing imipenem-related complications.

Acute Disease↗
PubMed1988

Microtubule-assembly inhibitor protein. Its distribution, localization and physicochemical properties.

Microtubule-assembly inhibitor protein (MIP) is an acidic protein with Mr 33,000 which inhibits microtubule assembly in vitro [Kotani, S., Murofushi, H., Nishida, E. & Sakai, H. (1984) J. Biochem. (Tokyo) 96, 959-969]. Anti-MIP antibody was affinity-purified from rabbit anti-MIP sera raised against chemically modified MIP. MIP was localized in the nucleus in interphase culture cells as revealed by immunofluorescent light microscopy. Immunoblotting experiments showed that MIP exists in a variety of mammalian cells and tissues. Kidney appeared to be a better source of MIP than brain, the original source. Kidney MIP was isolated by the same procedure as for brain MIP and proved to be indistinguishable from brain MIP in the inhibitory activity of microtubule assembly, molecular mass, immunoreactivity, and one-dimensional peptide mapping. Physico-chemical characteristics of MIP were studied using the kidney protein. It contained 20% aspartic acid and 25% glutamic acid, accounting for its acidic nature. Hydrodynamically, MIP was a monomer with S20,w = 1.9 S and Mr = 30,000. The frictional ratio, f/fo = 1.7, indicated that MIP is not a globular molecule but has either an elongated or an expanded structure. Circular dichroic results showed a low content of alpha-helix or beta-sheet structure for MIP. Proton nuclear magnetic resonance analysis provided evidence that MIP consists mainly of very flexible structures (random-coil-like structures), but still contains a hydrophobic core structure below 60 degrees C.

Amino Acids↗
PubMed1988

Purification and characterization of kinesin from bovine adrenal medulla.

Kinesin was purified from bovine adrenal medulla. The sedimentation coefficient was 8.8 S. Sedimentation equilibrium ultracentrifugation studies showed the molecular weight of kinesin to be 300,000. The calculated axial ratio was 1:16. The Stokes radius was estimated to be 8.9 nm by gel filtration. Circular dichroism showed the alpha-helix content to be about 50%. Purified kinesin preparation contained a major polypeptide with a molecular weight of 120,000 and minor ones with molecular weights of 71,000, 68,000, and 65,000. Bovine adrenal kinesin had an ATPase activity which was stimulated severalfold by microtubules to a specific activity of about 0.1 mumol/min.mg. Kinesin molecules adsorbed to a glass slide promoted the movement of microtubules on the glass surface at a rate of about 0.5 micron/s. Immunostaining of EBTr (bovine embryonic trachea fibroblast) cells and bovine adrenal chromaffin cells in interphase with an affinity-purified antibody against the major polypeptide of kinesin showed that some kinesin was located on microtubules and the rest distributed throughout the cytoplasm in a diffuse manner. EBTr cells in mitotic phase gave a staining pattern showing that kinesin was present throughout the cytoplasm with higher concentration in the region of mitotic apparatus.

Adenosine Triphosphatases↗
PubMed1988

Structural requirements of lipid A species in activation of clotting enzymes from the horseshoe crab, and the human complement cascade.

The structure/activity relationship of lipid A, a bioactive center of endotoxic lipopolysaccharides, in the activation of the clotting enzyme cascade of a horseshoe crab amoebocyte lysate (Limulus activity) and the complement system in human serum, was examined using synthetic lipids A and related compounds. Regarding Limulus activity, a newly developed colorimetric method, which utilizes a mixture of recombined clotting factors and a chromogenic substance, was much more sensitive for detecting changes in the chemical structure of test compounds than the conventional gelation method using the amoebocyte whole lysate. (beta 1-6)-D-Glucosamine disaccharide bisphosphates, which had neither 3-hydroxyacyl nor 3-acyloxyacyl groups, and acylglucosamine phosphates, which in structure correspond or are analogous to the non-reducing or reducing moieties of lipids A and biosynthetic disaccharide lipid A precursors showed only negligible activity in the colorimetric tests, but they exhibited a distinct though much weaker gelation activity than the parent disaccharide molecules. The assay results obtained by the colorimetric Limulus test correlate better with the pyrogenicity of the test synthetic compounds than those given by the gelation method, although the dependence of pyrogenicity on chemical structure is greater. The presence of 3-hydroxyacyl groups on the bisphosphorylated (beta 1-6)-D-glucosamine disaccharide backbone is the prerequisite for effective activation of the clotting enzyme cascade of horseshoe crab amoebocyte lysate, while the presence of an adequate number (one or two) of 3-acyloxyacyl groups on the disaccharide bisphosphate backbone is needed for full pyrogenicity. Complement activation, on the other hand, showed structural requirements quite different from those for the colorimetric Limulus activity and the pyrogenicity. The disaccharide compounds that had only non-hydroxylated acyl groups, acylated glucosamine phosphates that had the structure of the non-reducing portion of lipids A and biosynthetic disaccharide precursors, which were scarcely active in the colorimetric Limulus test, caused complement activation comparable to or sometimes stronger than that of the parent disaccharide molecules. Acylglucosamine phosphates, corresponding in structure to the reducing moiety of disaccharide compounds, however, showed little activity.

Animals↗
PubMed1988

Microtubule-binding domain of tau proteins.

Limited chymotryptic digestion of whole tau proteins produced a fragment of Mr 14,000 (CT14), which was able to bind to microtubules reconstituted from tubulin alone in the presence of taxol. This fragment was also found to persist in microtubules when microtubules consisting of tau proteins and tubulin were digested by chymotrypsin. Analysis of amino acid composition revealed that CT14 was rich in lysine and proline residues, suggesting unique structure of microtubule-binding domain of tau proteins. Amino-terminal sequence of CT14 was determined to be Ser-Ser-Pro-Gly-Ser-Pro-Gly-Thr-Pro-Gly-Ser-Arg-Ser-Arg-X-Pro-Ser-Leu-Pr o. No heterogeneity was detected in this amino-terminal sequence of 19 residues. Five species of polypeptides consisting of tau proteins were separated from each other by gel electrophoresis and subjected to chymotryptic digestion. CT14 was produced from each of the tau polypeptides by chymotryptic digestion, indicating that all tau polypeptides have a common microtubule-binding domain.

Amino Acids↗