Biomedical subjects

M Kataoka

Publications and source records attributed to M Kataoka.

At least 181 records · Page 10Linked to original sources

PubMed1997

Direct interaction of the calcium sensor protein synaptotagmin I with a cytoplasmic domain of the alpha1A subunit of the P/Q-type calcium channel.

Synaptotagmins are synaptic vesicle proteins containing two calcium-binding C2 domains which are involved in coupling calcium influx through voltage-gated channels to vesicle fusion and exocytosis of neurotransmitters. The interaction of synaptotagmins with native P/Q-type calcium channels was studied in solubilized synaptosomes from rat cerebellum. Antibodies against synaptotagmins I and II, but not IV co-immunoprecipitated [125I]omega-conotoxin MVIIC-labelled calcium channels. Direct interactions were studied between in vitro-translated [35S]synaptotagmin I and fusion proteins containing cytoplasmic loops of the alpha1A subunit (BI isoform). Gel overlay revealed the association of synaptotagmin I with a single region (residues 780-969) located in the intracellular loop connecting homologous domains II and III. Saturable calcium-independent binding occurred with equilibrium dissociation constants of 70 nM and 340 nM at 4 degrees C and pH 7.4, and association was blocked by addition of excess recombinant synaptotagmin I. Direct synaptotagmin binding to the pore-forming subunit of the P/Q-type channel may optimally locate the calcium-binding sites that initiate exocytosis within a zone of voltage-gated calcium entry.

Amino Acid Sequence↗

PubMed1997

Structure of the murine CD156 gene, characterization of its promoter, and chromosomal location.

The murine cell surface antigen mCD156 is a glycoprotein that is expressed in monocytic cell lines and consists of a metalloprotease domain, a disintegrin domain, a cysteine-rich domain, and an epidermal growth factor-like domain in the extracellular region. The mCD156 gene is composed of 24 exons and 23 introns and spans approximately 14 kilobases. The first exon encodes most of the signal peptide sequence, and the transmembrane region is encoded by a single exon (19). In contrast, the other regions are composed of multiple exons. Of these, exons 7-12 and 12-15 encode a metalloprotease domain and a disintegrin domain, respectively. Sequence analysis of the 5'-flanking DNA revealed many potential regulatory motifs. Chloramphenicol acetyltransferase analysis demonstrated that nucleotides at positions -183, -334, and -623 contained cis-acting enhancing elements in a mouse monocytic cell line, aHINS-B3. Nucleotides at positions -183 and -390 contained elements responsible for lipopolysaccharide (LPS) inducibility, although several other 5'-flanking regions were also involved in LPS responsiveness. Regions -202, -507, and -659 play a role in interferon-gamma inducibility. Some of the potential regulatory motifs and other unknown cis elements may be involved in the constitutive expression, and LPS and interferon-gamma inducibilities. The mCD156 gene was mapped to chromosome 7, region F3-F4.

ADAM Proteins↗

PubMed1997

Purification and characterization of L-allo-threonine aldolase from Aeromonas jandaei DK-39.

L-allo-Threonine aldolase (L-allo-threonine acetaldehyde-lyase), which exhibited specificity for L-allo-threonine but not for L-threonine, was purified from a cell-free extract of Aeromonas jandaei DK-39. The purified enzyme catalyzed the aldol cleavage reaction of L-allo-threonine (K(m) = 1.45 mM, Vmax = 45.2 mumol min-1 mg-1). The activity of the enzyme was inhibited by carbonyl reagents, which suggests that pyridoxal-5'-phosphate participates in the enzymatic reaction. The enzyme does not act on either L-serine or L-threonine, and thus it can be distinguished from serine hydroxy-methyltransferase (L-serine:tetrahydrofolate 5,10-hydroxy-methyltransferase, EC 2.1.2.1) or L-threonine aldolase (EC 4.1.2.5).

Aeromonas↗

PubMed1997

Application of the variable region in 16S rDNA to create an index for rapid species identification in the genus Streptomyces.

Partial nucleotide sequences (120 bp) of the 16S rRNA gene (rDNA) containing a variable alpha region were compared in 89 strains of the genus Streptomyces belonging to eight major clusters of category I in Bergey's Manual of Systematic Bacteriology. Fifty-seven kinds of partial 16S rDNA sequences were observed among the 89 strains. Forty-three of the strains were grouped into 11 'identity groups', based on the fact that the strains in each group shared an identical sequence in the 120-bp region. The results of a phylogenetic analysis based on the 16S rDNA 120-bp sequences revealed that 60 of the 89 strains could be categorized into seven clusters, each consisting of four or more strains. Based on these observations it was concluded that short nucleotide sequences bearing the variable alpha region are useful for Streptomyces species identification.

DNA, Ribosomal↗

PubMed1997

Evidence for proton transfer from Glu-46 to the chromophore during the photocycle of photoactive yellow protein.

Photoactive yellow protein (PYP) belongs to the novel group of eubacterial photoreceptor proteins. To fully understand its light signal transduction mechanisms, elucidation of the intramolecular pathway of the internal proton is indispensable because it closely correlates with the changes in the hydrogen-bonding network, which is likely to induce the conformational changes. For this purpose, the vibrational modes of PYP and its photoproduct were studied by Fourier transform infrared spectroscopy at -40 degrees C. The vibrational modes characteristic for the anionic p-coumaryl chromophore (Kim, M., Mathies, R. A., Hoff, W. D., and Hellingwerf, K. J. (1995) Biochemistry 34, 12669-12672) were observed at 1482, 1437, and 1163 cm-1 for PYP. However, the bands corresponding to these modes were not observed for PYPM, the blue-shifted intermediate, but the 1175 cm-1 band characteristic of the neutral p-coumaryl chromophore was observed, indicating that the phenolic oxygen of the chromophore is protonated in PYPM. A 1736 cm-1 band was observed for PYP, but the corresponding band for PYPM was not. Because it disappeared in the Glu-46 --> Gln mutant of PYP, this band was assigned to the C=O stretching mode of the COOH group of Glu-46. These results strongly suggest that the proton at Glu-46 is transferred to the chromophore during the photoconversion from PYP to PYPM.

Bacterial Proteins↗

PubMed1997

A local electrostatic change is the cause of the large-scale protein conformation shift in bacteriorhodopsin.

During light-driven proton transport bacteriorhodopsin shuttles between two protein conformations. A large-scale structural change similar to that in the photochemical cycle is produced in the D85N mutant upon raising the pH, even without illumination. We report here that (i) the pKa values for the change in crystallographic parameters and for deprotonation of the retinal Schiff base are the same, (ii) the retinal isomeric configuration is nearly unaffected by the protein conformation, and (iii) preventing rotation of the C13-C14 double bond by replacing the retinal with an all-trans locked analogue makes little difference to the Schiff base pKa. We conclude that the direct cause of the conformational shift is destabilization of the structure upon loss of interaction of the positively charged Schiff base with anionic residues that form its counter-ion.

Bacteriorhodopsins↗

PubMed1997

Cysteine string proteins associated with secretory granules of the rat neurohypophysis.

The properties and subcellular distribution of cysteine string proteins (csps) were analyzed in peptidergic nerve terminals of the rat neurohypophysis. Polyclonal antibodies raised against recombinant rat brain csp recognized a 36 kDa protein in isolated neurosecretosomes from the post-pituitary. After chemical deacylation, a single 27 kDa form was detected that displayed identical properties to csps in a whole-brain synaptosomal fraction. Immunoisolation demonstrated that synaptophysin and csps were located in the same vesicles. Density gradient centrifugation of postsynaptosomal supernatants of neurohypophysial homogenates revealed that csps and VAMP were present in two distinct vesicle populations. Synaptophysin was only detected in the slowly migrating population corresponding to small synaptic vesicles, whereas arginine vasopressin was present in the more rapidly sedimenting population indicating that it contains large dense core vesicles (LDCVs). Immobilized antibodies against csp, synaptotagmin, or VAMP captured vesicular arginine vasopressin confirming the association of these proteins with LDCVs. Co-immunoprecipitation assays with proteins solubilized from neurohypophysial or whole-brain nerve terminals failed to reveal complexes containing csp and [125I]omegaGVIA receptors. These results indicate that csps in the CNS are associated with both small synaptic vesicles and LDCVs. However, they do not provide support for the hypothesis that protein complexes implicated in exocytosis, which interact with presynaptic N-type calcium channels, contain csps.

Animals↗

PubMed1997

CD156 (human ADAM8): expression, primary amino acid sequence, and gene location.

The murine cell-surface antigen MS2 [ADAM 8; mouse CD156 (mCD156)] is a transmembrane glycoprotein that is highly expressed in monocytic lineages. mCD156 consists of a long exterior region containing domains strikingly similar to those of hemorrhagic snake venom proteins. cDNA for human CD156 was isolated from cDNA libraries from the human macrophage cell line THP-1 and from human granulocytes. The CD156 cDNA detected mRNA from human macrophage cell lines, granulocytes, monocytes, and B cell but not T cell lines. The nucleotide sequence of the CD156 cDNA showed 65.6% homology with that of mCD156, and its amino acid sequence had high homology with hemorrhagic snake venom proteins and other related mammalian proteins. The CD156 gene was mapped to human chromosome 10q26.3.

ADAM Proteins↗

PubMed1997

A role for macrophage scavenger receptors in atherosclerosis and susceptibility to infection.

Macrophage type-I and type-II class-A scavenger receptors (MSR-A) are implicated in the pathological deposition of cholesterol during atherogenesis as a result of receptor-mediated uptake of modified low-density lipoproteins (mLDL). MSR-A can bind an extraordinarily wide range of ligands, including bacterial pathogens, and also mediates cation-independent macrophage adhesion in vitro. Here we show that targeted disruption of the MSR-A gene in mice results in a reduction in the size of atherosclerotic lesions in an animal deficient in apolipoprotein E. Macrophages from MSR-A-deficient mice show a marked decrease in mLDL uptake in vitro, whereas mLDL clearance from plasma occurs at a normal rate, indicating that there may be alternative mechanisms for removing mLDL from the circulation. In addition, MSR-A-knockout mice show an increased susceptibility to infection with Listeria monocytogenes or herpes simplex virus type-1, indicating that MSR-A may play a part in host defence against pathogens.

Animals↗

PubMed1997

Primary structure of rat CD14 and characteristics of rat CD14, cytokine, and NO synthase mRNA expression in mononuclear phagocyte system cells in response to LPS.

Rat CD14 cDNA clones were isolated. The predicted protein sequence exhibits 82, 61.6, and 64% identity with the mouse, rabbit, and human CD14, respectively. The levels of rat CD14 mRNA expression in resident peritoneal macrophages (PM), alveolar macrophages (AM), and peripheral blood monocytes (BM) were constitutively high, whereas that in Kupffer cells (KC) was low. On intravenous injection with lipopolysaccharide (LPS), the expression of rat CD14 mRNA in KC increased markedly, whereas the increases in PM, AM, and BM were mild. Similar features of expression of rat CD14 in these cells were observed after stimulation with LPS in vitro. The level of tumor necrosis factor alpha (TNF-alpha) mRNA expression in KC after stimulation with LPS in vivo was comparable to that in PM, AM, and BM, whereas that of TNF-alpha mRNA expression in KC and PM after stimulation with LPS in vitro was lower than that in AM and BM. Interleukin (IL)-1 beta and iNOS mRNA expressions in KC after stimulation with LPS in vivo and in vitro were low, whereas those in PM, AM, and BM were high. Little or no expression of IL-6 was observed in KC after stimulation with LPS in vivo and in vitro, whereas higher expression was observed in PM, AM, and BM than in KC.

Amino Acid Sequence↗

PubMed1997

Structural characterization of the molten globule of alpha-lactalbumin by solution X-ray scattering.

A compact denatured state is often observed under a mild denaturation condition for various proteins. A typical example is the alpha-lactalbumin molten globule. Although the molecular compactness and shape are the essential properties for defining the molten globule, there have been ambiguities of these properties for the molten globule of alpha-lactalbumin. Using solution X-ray scattering, we have examined the structural properties of two types of molten globule of alpha-lactalbumin, the apo-protein at neutral pH and the acid molten globule. The radius of gyration for the native holo-protein was 15.7 A, but the two different molten globules both had a radius of gyration of 17.2 A. The maximum dimension of the molecule was also increased from 50 A for the native state to 60 A for the molten globule. These values clearly indicate that the molten globule is not as compact as the native state. The increment in the radius of gyration was less than 10% for the alpha-lactalbumin molten globule, compared with up to 30% for the molten globules of other globular proteins. Intramolecular disulfide bonds restrict the molecular expansion of the molten globule. The distance distribution function of the alpha-lactalbumin molten globule is composed of a single peak suggesting a globular shape, which is simply swollen from the native state. The scattering profile in the high Q region of the molten globule indicates the presence of a significant amount of tertiary fold. Based on the structural properties obtained by solution X-ray scattering, general and conceptual structural images for the molten globules of various proteins are described and compared with the individual, detailed structural model obtained by nuclear magnetic resonance.

Animals↗

PubMed1997

Urea-induced conformational changes in cold- and heat-denatured states of a protein, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor (SSI) is known to exist in at least two distinct denatured states, cold-denatured (D') and heat-denatured (D) under acidic conditions. In the present work, we investigated the manner how increasing urea concentration from 0 to 8 M changes the polypeptide chain conformation of SSI that exists initially in the D' and D states as well as in the native state (N), in terms of the secondary structure, the tertiary structure, and the chain form, based on the results of the experiments using circular dichroism (CD), small-angle X-ray scattering (SAXS) and 1H-NMR spectroscopy. Our results indicate that the urea-induced conformational transitions of SSI under typical conditions of D' (pH 1.8, 3 degrees C) occur at least in two steps. In the urea concentration range of 0-2 M (step 1), a cooperative destruction of the tertiary structure occurs, resulting in a mildly denatured state (DU), which may still contain a little amount of secondary structures. In the concentration range of 2-4 M urea (step 2), the DU state gradually loses its residual secondary structure, and increases the radius of gyration nearly to a maximum value. At 4 M urea, the polypeptide chain is highly disordered with highly mobile side chains. Increasing the urea concentration up to 8 M probably results in the more highly denatured or alternatively the stiffer chain conformations. The conformational transition starting from the N state proceeds essentially the same way as in the above scheme in which D' is replaced with N. The conformational transition starting from the D state lacks step 1 because the D state contains no tertiary structures and is similar to the DU state. The fact that similar conformations are reached at urea concentrations above 2 M from different conformations of D', D, and N indicates that the effect of urea dominates in determining the polypeptide conformation of SSI in the denatured states rather than the pH and temperature.

Bacterial Proteins↗

PubMed1997

Three single-strand origins located on both strands of the Streptomyces rolling circle plasmid pSN22.

pSN22 is an 11-kbp, high-copy-number Streptomyces plasmid which replicates via a single-stranded intermediate by the rolling circle replication (RCR) mechanism. We identified an unidirectional single-strand origin (SSO) of pSN22, sso1, where the initiation of second-strand synthesis takes place, located between the spdA and traR genes in a noncoding region which is functional in its natural orientation. The nucleotide sequence of sso1 is similar over 170 bp to the SSOs of the Streptomyces plasmids pIJ101 and pJV1. A previous report described that a 548-bp BglII-SmaI fragment has an SSO activity (ori2; Kataoka et al., Mol. Gen. Genet. 242, 130-136, 1994). To our surprise, we discovered that on pSN22, the SSO in the BglII-SmaI fragment is in the wrong, inactive, orientation and thus cannot be involved in the conversion of the single-stranded pSN22 replication intermediate to the double-stranded form of the plasmid. We revealed that this BglII-SmaI fragment contains two SSO fragments. Secondary structure analysis of these two SSOs showed similarity to the consensus TAGCGT which is conserved in SSOs of RCR plasmids from Staphylococcus and the other several Gram-positive bacteria. Deletion of these hexanucleotide sequences caused loss of SSO activities. Our result shows that two types of SSOs, Streptomyces type and Staphylococcus-like type, can function in Streptomyces lividans.

Base Sequence↗

PubMed1997

Multiple ulcers of the ileum due to Cytomegalovirus infection in a patient who showed no evidence of an immunocompromised state.

68-year-old woman presented with abdominal pain and vomiting. After initial conservative therapy, laparotomy showed multiple ulcers of the ileum, one of which had perforated and adhered to the uterus. The affected segment of the ileum was resected. Numerous cytomegalic cells, corresponding to endothelia and macrophages, with intranuclear inclusion bodies, were found in microscopic sections of the ulcerated lesions. These findings were consistent with cytomegalic vasculitis and enteritis. Cytomegalovirus infections of the alimentary tract have been reported mainly in severely immunocompromised patients or those with predisposing disorders such as ulcerative colitis; their prognosis is usually poor. In our patient, there was no obvious immunocompromised state or other gastrointestinal disorders. The postoperative course has been uneventful for 2 years after surgery. The prognosis of Cytomegalovirus-associated lesions in the alimentary tract may be quite good in the immunocompetent patient.

Aged↗

PubMed1997

Screening of novel microbial enzymes for the production of biologically and chemically useful compounds.

Enzymes have been generally accepted as superior catalysts in organic synthesis. Micro-organisms in particular have been regarded as treasure sources of useful enzymes. The synthetic technology using microbial enzymes or micro-organisms themselves is called microbial transformation. In designing a microbial transformation process, one of the most important points is to find a suitable enzyme for the reaction of interest. Various kinds of novel enzymes for specific transformations have been discovered in micro-organisms and their potential characteristics revealed. This article reviews our current results on the discovery of novel enzymes for the production of biologically and chemically useful compounds, and emphasizes the importance of screening enzymes in a diverse microbial world.

Alcohol Oxidoreductases↗

PubMed1997

Enzymatic production of ethyl (R)-4-chloro-3-hydroxybutanoate: asymmetric reduction of ethyl 4-chloro-3-oxobutanoate by an Escherichia coli transformant expressing the aldehyde reductase gene from yeast.

The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate (CHBE) using Escherichia coli JM109 (pKAR) cells expressing the aldehyde reductase gene from Sporobolomyces salmonicolor AKU4429 as a catalyst was studied. The reduction required NADP+, glucose and glucose dehydrogenase for NADPH regeneration. In an aqueous system, the substrate was unstable, and inhibition of the reaction by the substrate was also observed. Efficient conversion of COBE to (R)-CHBE with a satisfactory enantiomeric excess (ee) was attained on incubation with transformant cells in an n-butyl acetate/water two-phase system containing the above NADPH-regeneration system. Under the optimized conditions, with the periodical addition of COBE, glucose and glucose dehydrogenase, the (R)-CHBE yield reached 1530 mM (255 mg/ml) in the organic phase, with a molar conversion yield of 91.1% and an optical purity of 91% ee. The calculated turnover of NADP+, based on the amounts of NADP+ added and CHBE formed, was about 5100 mol/mol.

Aldehyde Reductase↗

PubMed1997

Functional expression and site-directed mutagenesis of photoactive yellow protein.

The gene encoding photoactive yellow protein (PYP) was isolated from Ectothiorhodospira halophila, and a high-level expression system for PYP was constructed in Escherichia coli. The molecular weight and the absorption spectrum of PYP expressed in E. coli were identical with those of the native PYP isolated from E. halophila. The amino acid residues which might interact with the chromophore (Tyr42, Glu46, Thr50, Arg52, and Cys69) were mutated by site-directed mutagenesis and the absorption spectra of these mutants were examined to study the chromophore/protein interaction in PYP. The former three substitutions (Y42F, E46Q, and T50V) brought about red-shifts of the absorption spectra, but the substitution of Arg52 (R52Q) brought about no change and that of Cys69 (C69S) led to no formation of pigments. These results suggest that Tyr42, Glu46, and Thr50 strongly interact with the chromophore, while Arg52 does not contribute the color tuning of PYP.

Amino Acid Sequence↗

PubMed1997

X-ray diffraction studies of bacteriorhodopsin. Determination of the positions of mercury label at several engineered cysteine residues.

The single cysteine-containing bacteriorhodopsin mutants F27C, L100C, T170C, F171C and I222C were labeled with p-chloromercuribenzoic acid, which specifically reacts with sulfhydryl groups. These cysteines should be located at the cytoplasmic ends of the transmembrane helices A, C, F or G. We determined the positions of the bound mercury atoms by X-ray diffraction of purple membrane films, with better than 1 A accuracy. The determined mercury positions were compared with the structural model from cryoelectron microscopy (N. Grigorieff, T. A. Ceska, K. H. Downing, J. M. Baldwin and R. Henderson, J. Mol. Biol. 259, 393-421, 1996). Given that the distance between the mercury and the C alpha atom of the cysteine in the xy plane must be shorter than 4.5 A and that the mercury atom is located at the delta position, the positions obtained for the mercury labels agree with their expected positions from the structural model. The present results give a rationale for detecting structural changes upon illumination as shifts occur in the mercury label position.

Amino Acid Sequence↗