Biomedical subjects

S Goda

Publications and source records attributed to S Goda.

At least 19 recordsLinked to original sources

PubMed2004

Hyper-production of an isomalto-dextranase of an Arthrobacter sp. by a proteases-deficient Bacillus subtilis: sequencing, properties, and crystallization of the recombinant enzyme.

Arthrobacter globiformis T6 is unique in that it produces an enzyme yielding only isomaltose from dextran. In the present study, the organism was re-identified and its classification as a new species of the genus Arthrobacter, A. dextranlyticum, was proposed. The high G+C gene (66.8 mol%) for the isomalto-dextranase was sequenced. The deduced amino acid sequence, with a calculated molecular mass of 65,993 Da (603 amino acids), was confirmed by nanoscale capillary liquid chromatography coupled to tandem mass spectrometry, which covered 71.1% of the amino acid residues of the entire sequence. The enzyme was grouped into glycoside hydrolase family 27, and the C-terminal domain has homology to carbohydrate-binding module family 6. Hyper-exoproduction of the recombinant enzyme was achieved at a level corresponding to approximately 4.6 g l(-1) of culture broth when proteases-deficient Bacillus subtilis cells were used as the host. The purified enzyme (65.5 kDa) had an optimal pH and temperature for activity of 3.5 and 60 degrees C, respectively. It was crystallized using the sitting-drop vapor-diffusion method at 293 K.

Amino Acid Sequence↗

PubMed2004

Enzymatic properties and nucleotide and amino acid sequences of a thermostable beta-agarase from a novel species of deep-sea Microbulbifer.

An agar-degrading bacterium, strain JAMB-A7, was isolated from the sediment in Sagami Bay, Japan, at a depth of 1,174 m and identified as a novel species of the genus Microbulbifer. The gene for a novel beta-agarase from the isolate was cloned and sequenced. It encodes a protein of 441 amino acids with a calculated molecular mass of 48,989 Da. The deduced amino acid sequence showed similarity to those of known beta-agarases in glycoside hydrolase family 16, with only 34-55% identity. A sequence similar to a carbohydrate-binding module was found in the C-terminal region of the enzyme. The recombinant agarase was hyper-produced extracellularly using Bacillus subtilis as the host, and the enzyme purified to homogeneity had a specific activity of 398 U (mg protein)(-1) at pH 7.0 and 50 degrees C. It was thermostable, with a half-life of 502 min at 50 degrees C. The optimal pH and temperature for activity were around 7 and 50 degrees C, respectively. The pattern of agarose hydrolysis showed that the enzyme was an endo-type beta-agarase, and the final main product was neoagarotetraose. The activity was not inhibited by NaCl, EDTA, and various surfactants at high concentrations. In particular, sodium dodecyl sulfate had no inhibitory effect up to 2%.

Alteromonadaceae↗

PubMed2001

Fractalkine, a CX3C-chemokine, functions predominantly as an adhesion molecule in monocytic cell line THP-1.

A newly identified CX3C-chemokine, fractalkine, expressed on activated endothelial cells plays an important role in leucocyte adhesion and migration. Co-immobilized fractalkine with fibronectin or intercellular adhesion molecule-1 enhanced adhesion of THP-1 cells, which express the fractalkine receptor (CX3CR1), compared with that observed for each alone. That adherence was fractalkine-dependent and was confirmed in blocking studies. However, soluble fractalkine induced little chemotaxis in THP-1 cells in comparison to monocyte chemotactic protein-1 (MCP-1), which induced a strong chemotactic response. Moreover, the membrane form of fractalkine expressed on ECV304 cells reduced MCP-1 mediated chemotaxis of THP-1 cells. These results indicate that fractalkine may function as an adhesion molecule between monocytes and endothelial cells rather than as a chemotactic factor.

Animals↗

PubMed2000

Fractalkine-mediated endothelial cell injury by NK cells.

Endothelial cells (ECs) are primary targets of immunological attack, and their injury can lead to vasculopathy and organ dysfunction in vascular leak syndrome and in rejection of allografts or xenografts. A newly identified CX3C-chemokine, fractalkine, expressed on activated ECs plays an important role in leukocyte adhesion and migration. In this study we examined the functional roles of fractalkine on NK cell activity and NK cell-mediated endothelial cell injury. Freshly separated NK cells expressed the fractalkine receptor (CX3CR1) determined by FACS analysis and efficiently adhered to immobilized full-length fractalkine, but not to the truncated forms of the chemokine domain or mucin domain, suggesting that fractalkine functions as an adhesion molecule on the interaction between NK cells and ECs. Soluble fractalkine enhanced NK cell cytolytic activity against K562 target cells in a dose- and time-dependent manner. This enhancement correlated well with increased granular exocytosis from NK cells, which was completely inhibited by the G protein inhibitor, pertussis toxin. Transfection of fractalkine cDNA into ECV304 cells or HUVECs resulted in increased adhesion of NK cells and susceptibility to NK cell-mediated cytolysis compared with control transfection. Moreover, both enhanced adhesion and susceptibility of fractalkine-transfected cells were markedly suppressed by soluble fractalkine or anti-CX3CR1 Ab. Our results suggest that fractalkine plays an important role not only in the binding of NK cells to endothelial cells, but also in NK cell-mediated endothelium damage, which may result in vascular injury.

Adjuvants, Immunologic↗

PubMed2000

CX3C-chemokine, fractalkine-enhanced adhesion of THP-1 cells to endothelial cells through integrin-dependent and -independent mechanisms.

Leukocyte adhesion and trafficking at the endothelium requires both cellular adhesion molecules and chemotactic factors. A newly identified CX3C chemokine, fractalkine, expressed on activated endothelial cells, plays an important role in leukocyte adhesion and migration. We examined the functional effects of fractalkine on beta1 and beta2 integrin-mediated adhesion using a macrophage-like cell line, THP-1 cells. In this study, we report that THP-1 cells express mRNA encoding a receptor for fractalkine, CX3CR1, determined by Northern blotting. Scatchard analysis using fractalkine-SEAP (secreted form of placental alkaline phosphatase) chimeric proteins revealed that THP-1 cells express a single class of CX3CR1 with a dissociation constant of 30 pM and a mean expression of 440 sites per cell. THP-1 cells efficiently adhered, in a fractalkine-dependent manner, to full-length of fractalkine immobilized onto plastic and to the membrane-bound form of fractalkine expressed on ECV304 cells or TNF-alpha-activated HUVECs. Moreover, soluble-fractalkine enhanced adhesion of THP-1 cells to fibronectin and ICAM-1 in a dose-dependent manner. Pertussis toxin, an inhibitor of Gi, inhibited the fractalkine-mediated enhancement of THP-1 cell adhesion to fibronectin and ICAM-1. Finally, we found that soluble-fractalkine also enhanced adhesion of freshly separated monocytes to fibronectin and ICAM-1. These results indicate that fractalkine may induce firm adhesion between monocytes and endothelial cells not only through an intrinsic adhesion function itself, but also through activation of integrin avidity for their ligands.

Adjuvants, Immunologic↗

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

Effect of extra N-terminal residues on the stability and folding of human lysozyme expressed in Pichia pastoris.

A human lysozyme expression system by Pichia pastoris was constructed with the expression vector of pPIC9, which contains the alpha-factor signal peptide known for high secretion efficiency. P. pastoris expressed the human lysozyme at about 300 mg/l broth, but four extra residues (Glu(-4)-Ala(-3)-Glu(-2)-Ala(-1)-) were added at the N-terminus of the expressed protein (EAEA-lysozyme). To determine the effect of the four extra residues on the stability, structures and folding of the protein, calorimetry, X-ray crystal analysis and GuHCl denaturation experiments were performed. The calorimetric studies showed that the EAEA-lysozyme was destabilized by 9.6 kJ/mol at pH 2.7 compared with the wild-type protein, mainly caused by the substantial decrease in the enthalpy change (DeltaH). On the basis of structural information on the EAEA-lysozyme, thermodynamic analyses show that (1) the addition of the four residues slightly affected the conformation in other parts far from the N-terminus, (2) the large decrease in the enthalpy change due to the conformational changes would be almost compensated by the decrease in the entropy change and (3) the decrease in the Gibbs energy change between the EAEA and wild-type human lysozymes could be explained by the summation of each Gibbs energy change contributing to the stabilizing factors concerning the extra residues.

Calorimetry, Differential Scanning↗

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↗

PubMed1997

Importance of van der Waals contact between Glu 35 and Trp 109 to the catalytic action of human lysozyme.

The importance of van der Waals contact between Glu 35 and Trp 109 to the active-site structure and the catalytic properties of human lysozyme (HL) has been investigated by site-directed mutagenesis. The X-ray analysis of mutant HLs revealed that both the replacement of Glu 35 by Asp or Ala, and the replacement of Trp 109 by Phe or Ala resulted in a significant but localized change in the active-site cleft geometry. A prominent movement of the backbone structure was detected in the region of residues 110 to 120 and in the region of residues 100 to 115 for the mutations concerning Glu 35 and Trp 109, respectively. Accompanied by the displacement of the main-chain atoms with a maximal deviation of C alpha atom position ranging from 0.7 A to 1.0 A, the mutant HLs showed a remarkable change in the catalytic properties against Micrococcus luteus cell substrate as compared with native HL. Although the replacement of Glu 35 by Ala completely abolished the lytic activity, HL-Asp 35 mutant retained a weak but a certain lytic activity, showing the possible involvement of the side-chain carboxylate group of Asp 35 in the catalytic action. The kinetic consequence derived from the replacement of Trp 109 by Phe or Ala together with the result of the structural change suggested that the structural detail of the cleft lobe composed of the residues 100 to 115 centered at Ala 108 was responsible for the turnover in the reaction of HL against the bacterial cell wall substrate. The results revealed that the van der Waals contact between Glu 35 and Trp 109 was an essential determinant in the catalytic action of HL.

Catalysis↗

PubMed1995

Comparison of the phosphorylation of myelin-associated glycoprotein in cultured oligodendrocytes and Schwann cells.

Phosphorylation of the large and small isoforms of myelin-associated glycoprotein (L- and S-MAG) was investigated in primary oligodendrocyte cultures and in immortalized Schwann cells by incubating the cells with inorganic [32P]phosphate and immunoprecipitating MAG. In oligodendrocytes, both L- and S-MAG were phosphorylated, but L-MAG was much more heavily labeled. In Schwann cells, most of the phosphorylation was in S-MAG, which is the predominant isoform expressed by these cells. In both types of cells, the principal phosphorylated amino acid in MAG was serine. Radioactive phosphothreonine and phosphotyrosine were also detected in the MAG from oligodendrocytes. In Schwann cells, there was less phosphorylation of threonine and labeled phosphotyrosine was not detected. In both oligodendrocytes and Schwann cells, the phosphorylation of MAG was stimulated by phorbol ester and a calcium ionophore, but not by forskolin. The results indicate that the phosphorylation of MAG is catalyzed by protein kinase C and possibly other calcium-activated kinases in both types of myelinating cells, but not by cAMP-activated kinase. An inhibitor of tyrosine phosphatase, ammonium vanadate, increased the amount of radioactive phosphate in MAG several fold in both oligodendrocytes and Schwann cells. However, even in the presence of vanadate, the great majority of radioactivity in MAG was in phosphoserine and only a small amount was in phosphotyrosine, suggesting that tyrosine phosphorylation of other proteins may indirectly increase the phosphorylation of MAG. The current status of our understanding of MAG phosphorylation is reviewed in the context of similarities and differences between our results and other reports in the literature.

Animals↗

PubMed1994

Inhibition of the proliferation of cultured immortalized Schwann cells by forskolin with a decreased basal level of diacylglycerol.

The repetitive passages of a Schwann cell culture results in the appearance of immortalized cells. In order to investigate the direct effects of cyclic AMP (cAMP) on Schwann cell proliferation, we used the immortalized Schwann cells because the responses of a short-term Schwann cell culture to agents increasing the intracellular cAMP are more complicated and it does not seem that all of them are due to the direct effects of cAMP. By adding up to 200 microM of forskolin, an adenylate cyclase activator, to the culture medium, Schwann cell proliferation was inhibited and the intracellular 1,2-diacylglycerol (DG) level was decreased in a dose-dependent manner to 44 and 53% of the control values, respectively. The protein phosphorylation activity in the cytosol from the cell treated with 100 microM forskolin, assayed with myelin basic protein as the acceptor, decreased to 78% and this inhibition was then reversed by the addition of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeable DG, to the assay mixture. The cell proliferation inhibited by forskolin was also restored by the addition of OAG. These data suggest that cAMP inhibits both the activity of protein kinase C (PKC) and consequently cell proliferation through suppression of intracellular DG level, an activator of PKC. Since the inositol 1,4,5-triphosphate level and the hydrolysis of phosphatidylcholine to DG and phosphorylcholine were not affected, forskolin therefore appears to suppress the de novo synthesis of DG.

Animals↗

PubMed1994

Biochemical and cellular properties of three immortalized Schwann cell lines expressing different levels of the myelin-associated glycoprotein.

Biochemical and cellular properties of three immortalized Schwann cell lines expressing different levels of the myelin-associated glycoprotein (MAG) were compared. The S16 line generated by repetitive passaging was described previously and expresses a level of MAG comparable to that in adult sciatic nerve. The S42 line was generated independently by the same procedure, divides more slowly than the S16 line, and expresses an even higher level of MAG. The S16Y line arose spontaneously from a passage of the S16 cells, divides much more rapidly, and does not express MAG. The levels of MAG expression in the three lines are inversely related to their rates of proliferation, and MAG mRNA levels parallel the amounts of MAG. The S16 and S42 lines consist mainly of flat cells at low density and develop many processes at high density, whereas most of the S16Y cells are spindle-shaped, resembling primary Schwann cells in appearance. Surface immunostaining with the O4 antibody was positive for the S16 and S42 cells and negative for the S16Y cells, but all three lines were negative for surface staining with the O1 antibody. The overall protein compositions of the three lines are very similar, but the S16 and S42 cells express larger amounts of several glycoproteins than the S16Y cells, including the adhesion proteins, neural cell adhesion molecule, L1, and laminin. S16 and S42 cells (but not S16Y cells) also express P0 glycoprotein, galactocerebroside, and sulfatide, but, unlike MAG, these other myelin-related components were present at much lower levels than in adult nerve. Myelin basic protein and proteolipid protein were not detected in any of the lines, although all three lines contained proteolipid protein mRNA. 2',3'-Cyclic nucleotide 3'-phosphodiesterase and glial fibrillary acidic protein were present in all three lines. Conditions have not yet been found in which any of the lines will myelinate dorsal root ganglion neurons in vitro, but the S16 and S42 cells differ from the S16Y cells by clustering around neurons after 1 week in coculture. In many respects, the S16 and S42 cells biochemically resemble Schwann cells at an early stage in their preparation to myelinate and should be useful for investigating the cell biology of MAG and other myelin-related components.

Animals↗

PubMed1994

Micrographia due to focal cerebral lesions as seen in the dysarthria-clumsy hand syndrome.

Five patients with dysarthria-clumsy hand syndrome developed micrographia. Four had lacunar infarctions involving the putamen or the genu of the internal capsule; the fifth patient had a small hemorrhage in the putamen. Micrographia may be a sign of focal cerebral disease, and basal ganglia dysfunction may be involved in the supratentorial dysarthria-clumsy hand syndrome.

Aged↗

PubMed1993

Involvement of protein kinase C in the proliferation of cultured Schwann cells.

We investigated the signal transduction system when the proliferation of cultured Schwann cells was activated by glial growth factor (GGF). When 10 micrograms/ml of GGF was added to the culture medium, Schwann cell division was activated by about 3-fold, while protein kinase C (PKC) activity was translocated from the cytosol to the particulate fraction, and diacylglycerol (DG) production was increased in the cells. PKC activity in the particulate fraction and intracellular DG content increased to 167% and 158%, respectively. However, intracellular cyclic AMP levels remained unchanged. The GGF-induced proliferation of Schwann cells was significantly inhibited by staurosporine, a PKC inhibitor, in a dose-dependent manner, while H-8, a specific inhibitor for cyclic nucleotide-dependent protein kinases, did not show an inhibitory effect even at a high concentration. These data suggest that the signal transduction system through PKC is involved in the proliferation of Schwann cells when treated with GGF.

Animals↗

PubMed1991

Expression of the myelin-associated glycoprotein in cultures of immortalized Schwann cells.

Although the myelin-associated glycoprotein (MAG) cannot be detected in primary cultures of rat Schwann cells in the absence of neurons, MAG expression was demonstrated in some lines of cultured Schwann cells that had been immortalized by repetitive passaging. Radioimmunoassay of one such Schwann cell line, S-16, showed a remarkably high MAG concentration of about 1 ng/microgram of total protein, a level that is comparable to the MAG concentration in adult sciatic nerve. The S-16 cells divide very rapidly, are rounder than normal Schwann cells, and elaborate many processes after reaching high density. The cells are galactocerebroside positive, but express little or no P0 glycoprotein or myelin basic protein. As in nerve, the MAG synthesized by the cultured cells is primarily the shorter isoform (S-MAG). Furthermore, the posttranslational processing resembles that occurring in vivo including a similar degree of glycosylation, sulfation of oligosaccharides, and phosphorylation of the polypeptide. The sensitivity of MAG to treatment of the intact cells with trypsin or neuraminidase, as well as surface labeling with [3H]borohydride reduction after periodate oxidation, demonstrated that most of the MAG expressed by the S-16 cells is located on the cell surface. This line of immortalized Schwann cells expressing a remarkably high level of MAG should be useful for investigating the cell biology and function of this glycoprotein.

Animals↗

PubMed1989

Myelination in the jimpy mouse in the absence of proteolipid protein.

A point mutation in the gene for proteolipid protein (PLP) has been suggested to account for the dysmyelination seen in the jimpy mouse mutant. Despite the absence of PLP, the major integral membrane protein of central nervous system (CNS) myelin, this study shows that there are many scattered myelinated fibers present in the spinal cord of this murine mutant which are immunocytochemically positive for myelin basic protein (MBP), yet negative for PLP. This lack of PLP results in an abnormal compaction of the extracellular leaflets of the myelin sheath and the formation of an abnormal intraperiod line. These results are similar to those seen in another X-linked myelin mutant, the myelin-deficient rat (Duncan et al.: Proc. Natl. Acad. Sci. U.S.A., 84:6287-6291, 1987), and show that a multilamellar membrane can be formed in the absence of its major integral membrane protein.

Animals↗

PubMed1987

Hydrolysis of galactosylsphingosine and lactosylsphingosine by beta-galactosidases in human brain and cultured fibroblasts.

Enzymatic properties of beta-galactosidases with galactosylsphingosine (psychosine) and lactosylsphingosine as the substrates were examined. Although bile salts were stimulatory on the hydrolysis of the glycolipids in normal brain and cultured fibroblasts, the hydrolytic activities could be readily assayed, without detergents. The in vitro hydrolysis of lactosylsphingosine in cultured fibroblast homogenates was catalyzed by two enzymes, as is the case with the hydrolysis of galactosylceramide and lactosylceramide. Lactosylsphingosine beta-galactosidase activities assayed in the absence and the presence of taurocholate (probably lactosylceramidase I) were deficient in fibroblasts from patients with globoid cell leukodystrophy, while the activity assayed with sodium cholate (probably lactosylceramidase II) was deficient in GM1 gangliosidosis fibroblasts. In contrast, galactosylsphingosine beta-galactosidase was not activated by cholate and the enzyme activities assayed with the no-additive and taurocholate systems were deficient in brain and fibroblasts from patients with globoid cell leukodystrophy, thereby indicating that the hydrolysis of galactosylsphingosine is catalyzed by one enzyme, galactosylceramidase I. Exogenous lipids and an activator protein purified from normal spleen activated galactosylsphingosine beta-galactosidase but they were inhibitory to lactosylsphingosine beta-galactosidase. Because the Km values of lactosylsphingosine beta-galactosidase assayed with cholate were several magnitude higher than those obtained with the no-additive system and because lactosylsphingosine is readily hydrolyzed with the no-additive system in vitro, it is likely that the in vivo hydrolysis of the lipid is catalyzed by only one enzyme, lactosylceramidase I.

Bile Acids and Salts↗

PubMed1987

Clinical improvement after administration of coenzyme Q10 in a patient with mitochondrial encephalomyopathy.

In a patient with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes [MELAS] who had normal mitochondrial enzyme activity, high doses of coenzyme Q10 (CoQ) were administered. Clinical improvement with decreased serum lactate and pyruvate levels was observed. Though the mechanism of action of CoQ is not known, a trial is worthwhile in patients with MELAS.

Acidosis, Lactic↗