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Biomedical subjects

K Morikawa

Publications and source records attributed to K Morikawa.

At least 109 records · Page 6Linked to original sources
PubMed1996

Structure of a replication-terminator protein complexed with DNA.

The crystal structure of the Escherichia coli replication-terminator protein (Tus) bound to terminus-site (Ter) DNA has been determined at 2.7 A resolution. The Tus protein folds into a previously undescribed architecture divided into two domains by a central basic cleft. This cleft accommodates locally deformed B-form Ter DNA and makes extensive contacts with the major groove, mainly through two interdomain beta-strands. The unusual structural features of this complex may explain how the replication fork is halted in only one direction.

Amino Acid Sequence↗
PubMed1996

Immunomodulatory effect of recombinant human superoxide dismutase (SOD) on human B lymphocyte function in vitro.

Recent investigations demonstrate that B lymphocytes possess an oxygen-generating system which is similar to the phagocytic NADPH-oxidase system. Reduction of nitroblue tetrazolium by stimulated tonsillar B cells is inhibited by superoxide dismutase (SOD). However, the biological significance of the superoxide-generating property of B cells remains to be explored. In this study, we examined the immunomodulatory effect of a recombinant human SOD (rh-SOD) on the activation of human B lymphocytes in vitro. A supplement of rh-SOD in the B cell culture increased the proliferation of unstimulated B cells in the presence of SAC, but not of SAC-preactivated B cells in the presence of cytokines such as IL-2 or IL-4. In addition, rh-SOD enhanced the immunoglobulin generation by B cells at the terminal stage of differentiation. Inactivation of the enzymatic activity of SOD by treatment with anti-SOD antibody abrogated the enhancing effects. These data suggest that the superoxide-generating system in B cells may be involved in the cellular activation process.

Antibodies↗
PubMed1996

Potent inhibition of spontaneous rhythmic contraction by a novel beta 2-adrenoceptor agonist, HSR-81, in pregnant rat uterus.

We examined the effect of HSR-81 ((-)-(R)-alpha-[(tert-butylamino)methyl]-2-chloro-4-hydroxybenzyl alcohol L-tartrate), a newly developed, potent and selective beta 2-adrenoceptor agonist, as well as ritodrine and isoproterenol, on the spontaneous rhythmic contraction in uteri isolated from late pregnant, middle pregnant and non-pregnant (dioestrous and oestrous) rats. The three agonists inhibited the spontaneous rhythmic contraction at all the stages in a concentration-dependent manner. The pD2 value for HSR-81 was greater in late pregnancy than in dioestrus and oestrus. In the uterine preparations of late pregnancy and dioestrus, ICI-118,551 (1-(7-methylindan-4-yloxy)-3-isopropyl-aminobutan-2-ol , a selective beta 2-adrenoceptor antagonist) and atenolol (a selective beta 1-adrenoceptor antagonist) produced a parallel rightward shift of the concentration-response curves for HSR-81. The pKB values for ICI-118,551 and atenolol suggest that the inhibitory effect of HSR-81 was mediated through beta 2-adrenoceptors in the two stages. In the membranes prepared from rat uteri in late pregnancy and dioestrus, the equilibrium dissociation constant for [125I]iodocyanopindolol binding was not significantly different between the two stages. The three beta-adrenoceptor agonists and the two antagonists competed for the specific [125I]iodocyanopindolol binding and the pKi values were not significantly different between the two stages. However, the maximum number of binding sites was significantly greater in late pregnancy than in dioestrus. The configuration of the competition curves and the pKi values for the two antagonists confirmed the fact that these membranes contain predominantly beta 2-adrenoceptor subtype. These results indicate that the potent inhibition of the spontaneous rhythmic contraction by HSR-81 in the pregnant uterus may be due to the increased number of beta 2-adrenoceptors.

Adrenergic beta-2 Receptor Agonists↗
PubMed1996

Spermidine-preferential uptake system in Escherichia coli. Identification of amino acids involved in polyamine binding in PotD protein.

Spermidine-binding sites on PotD protein, substrate-binding protein in periplasm, in the spermidine-preferential uptake system in Escherichia coli were studied by measuring polyamine transport activities of right-side-out membrane vesicles with mutated PotD proteins prepared by site-directed mutagenesis of the potD gene and by measuring polyamine binding activities of these mutated PotD proteins. Polyamine transport activities of the mutated PotD proteins paralleled their polyamine binding activities. It was found that Trp-34, Thr-35, Glu-36, Tyr-37, Ser-83, Tyr-85, Asp-168, Glu-171, Trp-229, Trp-255, Asp-257, Tyr-293, and Gln-327 of PotD protein were involved in the binding to spermidine. When spermidine uptake activities were measured in intact cells expressing the mutated PotD proteins, it was found that Glu-171, Trp-255, and Asp-257 were more strongly involved in the binding of spermidine to PotD protein than the other amino acids listed above. The dissociation constants of spermidine for the mutated PotD proteins at Glu-171, Trp-255, and Asp-257 increased greatly in comparison with those for the other mutated PotD proteins. Since these three amino acids clearly interact with the diaminopropane moiety of spermidine, the results are in accordance with the finding that PotD protein has a higher affinity for spermidine than for putrescine. Putrescine was found to bind at the position of the diaminobutane moiety of spermidine.

Amino Acid Sequence↗
PubMed1996

Crystal structure of PotD, the primary receptor of the polyamine transport system in Escherichia coli.

PotD protein is a periplasmic binding protein and the primary receptor of the polyamine transport system, which regulates the polyamine content in Escherichia coli. The crystal structure of PotD in complex with spermidine has been solved at 2.5-A resolution. The PotD protein consists of two domains with an alternating beta-alpha-beta topology. The polyamine binding site is in a central cleft lying in the interface between the domains. In the cleft, four acidic residues recognize the three positively charged nitrogen atoms of spermidine, while five aromatic side chains anchor the methylene backbone by van der Waals interactions. The overall fold of PotD is similar to that of other periplasmic binding proteins, and in particular to the maltodextrin-binding protein from E. coli, despite the fact that sequence identity is as low as 20%. The comparison of the PotD structure with the two maltodextrin-binding protein structures, determined in the presence and absence of the substrate, suggests that spermidine binding rearranges the relative orientation of the PotD domains to create a more compact structure.

ATP-Binding Cassette Transporters↗
PubMed1996

Crystallization and preliminary X-ray analysis of the primary receptor (PotD) of the polyamine transport system in Escherichia coli.

The primary receptor (potD, M(r) = 39 000) of the polyamine transport system in Escherichia coli has been crystallized by the vapor-diffusion method. Two crystal forms were obtained in the presence of spermidine, and were examined by X-ray analysis. Form I crystals, which diffract to 2.5 A resolution, belong to the space group P2(1), with unit-cell dimensions a = 145.3, b = 69.1, c = 72.5 A and beta = 107.6 degrees. Four molecules are contained in an asymmetric unit. These form two dimers that are related to each other by a local translation of about half of the unit cell along the a axis. The two protein molecules in each dimer are similarly related by a local dyad. Form II crystals diffract to 1.8 A resolution and belong to the space group I4(1), with unit-cell dimensions a = b = 130.3 and c = 38.7 A. They contain one molecule per asymmetric unit.

Journal Article↗
PubMed1996

Identification of a damaged-DNA binding domain of the XPA protein.

The XPA (xeroderma pigmentosum group A) protein is a zinc metalloprotein consisting of 273 amino acids which binds preferentially to UV- or chemical carcinogen-damaged DNA, suggesting that it is involved in the recognition of several types of DNA damage during nucleotide excision repair processes. Here we identify a DNA binding domain of the XPA protein. The region of the XPA protein responsible for preferential binding to DNA damaged by UV or cis-diammine-dichloroplatinum(II) (cisplatin) is contained within a truncated derivative of the XPA protein, MF122, consisting of 122 amino acids and containing a C4 type zinc finger motif. CD (circular dichroism) measurements of the MF122 protein showed that it has a helix-rich secondary structure, suggesting that it is a discretely folded, functional mini-domain. The MF122 protein should be useful for structural investigation of the XPA protein and of its interaction with damaged DNA.

Base Sequence↗
PubMed1996

Crystallization and preliminary X-ray analysis of the Escherichia coli replication terminator protein complexed with DNA.

Crystals of the Escherichia coli replication terminator protein (Tus) complexed with its binding site DNA were obtained by a microdialysis method using PEG 4000. They belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 with the unit cell parameter: a = 68.1 A, c = 230.7 A and contain one protein-DNA complex in an asymmetric unit. The native data set has been collected to 2.7 A resolution.

Bacterial Proteins↗
PubMed1996

The 1.8-A X-ray structure of the Escherichia coli PotD protein complexed with spermidine and the mechanism of polyamine binding.

The PotD protein from Escherichia coli is one of the components of the polyamine transport system present in the periplasm. This component specifically binds either spermidine or putrescine. The crystal structure of the E. coli PotD protein complexed with spermidine was solved at 1.8 A resolution and revealed the detailed substrate-binding mechanism. The structure provided the detailed conformation of the bound spermidine. Furthermore, a water molecule was clearly identified in the binding site lying between the amino-terminal domain and carboxyl-terminal domain. Through this water molecule, the bound spermidine molecule forms two hydrogen bonds with Thr 35 and Ser 211. Another periplasmic component of polyamine transport, the PotF protein, exhibits 35% sequence identity with the PotD protein, and it binds only putrescine, not spermidine. To understand these different substrate specificities, model building of the PotF protein was performed on the basis of the PotD crystal structure. The hypothetical structure suggests that the side chain of Lys 349 in PotF inhibits spermidine binding because of the repulsive forces between its positive charge and spermidine. On the other hand, putrescine could be accommodated into the binding site without any steric hindrance because its molecular size is much smaller than that of spermidine, and the positively charged amino group is relatively distant from Lys 349.

Amino Acid Sequence↗
PubMed1996

Constitutive expression of granulocyte-colony stimulating factor receptor on a human B-lymphoblastoid cell line.

The present study demonstrated that a human B-cell line derived from non-Hodgkin's lymphoma. HCF-MLpN. constitutively expressed G-CSF receptor on the cell surface. G-CSF binding to the cell surface was shown by immunofluorescence staining using biotinylated G-CSF preparation and analysed by flow cytometry. Specific binding of G-CSF to the cells was shown by pretreatment with unlabelled G-CSF. In the radioreceptor assay and Scatchard plot analysis using radiolabelled ligand, MLpN cells revealed a single species of binding site with an equilibrium dissociation constant of 167 (153-182) pM and a maximal binding site per cell of 1076 (1044-1116). The G-CSF receptor mRNA transcript was exhibited in the RNA from MLpN cells by reverse transcriptase polymerase chain reaction procedure. [3H]thymidine incorporation and trypan blue exclusion showed that the G-CSF receptor was capable of transducing the growth signal to HCF-MLpN cells. A small fraction of fresh B blasts from six patients with B-cell lymphoma and leukaemia displayed G-CSF binding by two-colour immunofluorescence staining. In contrast, a panel of seven B-cell lines was negative for the binding to biotinylated G-CSF preparation. These results suggest that the phenotype of G-CSF binding may be lost during the culture. The expression of G-CSF receptor in HCF-MLpN cells appeared to be exceptional.

B-Lymphocytes↗
PubMed1996

Implications of the zinc-finger motif found in the DNA-binding domain of the human XPA protein.

BACKGROUND: The XPA (xeroderma pigmentosum group A) protein specifically recognizes the UV-or chemically damaged DNA lesions, and triggers the nucleotide excision repair process. This XPA protein contains the functional domain which is crucial to the recognition of damaged DNA. Its primary structure suggests that this DNA binding domain may contain a zinc-finger motif. To gain a more detailed insight into this zinc-finger motif, we have measured the 113Cd-NMR spectra of the DNA binding domains derived from the wild-type and mutant XPA proteins. RESULTS: 113Cd-NMR analysis, combined with atomic absorption and site-directed mutagenesis, revealed that the DNA binding domain contains one zinc ion, coordinated with four cysteine residues (Cys105, Cys108, Cys126, and Cys129), that is essential for correct protein folding in vivo and in vitro. CONCLUSIONS: The four ligand cysteine residues form a Cys-X2-Cys-X17-Cys-X2-Cys motif, which is reminiscent of the (Cys)4 type zinc-finger motif found in numerous transcriptional regulatory proteins. However, the secondary structure prediction and the 3D-1D compatibility analysis demonstrate that there is no structural similarity in the vicinity of the zinc-finger motif between the XPA protein and other zinc-finger containing proteins. We conclude that the XPA protein contains a new type of zinc-finger motif.

Amino Acid Sequence↗
PubMed1996

Proposal for new catalytic roles for two invariant residues in Escherichia coli ribonuclease HI.

Three mutants of Escherichia coli ribonuclease HI, in which an invariant acidic residue Asp134 was replaced, were crystallized, and their three-dimensional structures were determined by X-ray crystallography. The D134A mutant is completely inactive, whereas the other two mutants, D134H and D134N, retain 59 and 90% activities relative to the wild-type, respectively. The overall structures of these three mutant proteins are identical with that of the wild-type enzyme, except for local conformational changes of the flexible loops. The ribonuclease H family has a common active site, which is composed of four invariant acidic residues (Asp10, Glu48, Asp70 and Asp134 in E.coli ribonuclease HI), and their relative positions in the mutants, even including the side-chain atoms, are almost the same as those in the wild-type. The positions of the delta-polar atoms at residue 134 in the wild-type, as well as D134H and D134N, coincide well with each other. They are located near the imidazole side chain of His124, which is assumed to participate in the catalytic reaction, in addition to the four invariant acidic residues. Combined with the pH profiles of the enzymatic activities of the two other mutants, H124A and H124A/D134N, the crystallographic results allow us to propose a new catalytic mechanism of ribonuclease H, which includes the roles for Asp134 and His124.

Aspartic Acid↗
PubMed1996

Modulatory effect of antibiotics on cytokine production by human monocytes in vitro.

Some antimicrobial agents have been reported to modify the host immune and inflammatory responses both in vivo and in vitro. Fosfomycin (FOM) and clarithromycin (CAM) have immunomodulatory activity on human lymphocyte function. In the present study, we examined the effects of FOM and CAM on cytokine synthesis by lipopolysaccharide (LPS)-stimulated human monocytes in comparison with that of dexamethasone in vitro. The three drugs demonstrated positive or negative effects on the synthesis of various cytokines by LPS-primed monocytes. They suppressed the synthesis of tumor necrosis factor alpha, interleukin 1 alpha (IL-1 alpha), IL-1 beta, the IL-1 receptor antagonist, and granulocyte-macrophage colony-stimulating factor in a concentration-dependent manner at concentrations between 1.6 and 40 micrograms/ml. On the contrary, the drugs showed different actions on the synthesis of IL-6 and IL-10. Namely, FOM enhanced both IL-6 and IL-10 synthesis, CAM enhanced only IL-10 synthesis, but dexamethasone deeply suppressed the synthesis of both cytokines. These data indicate that antibacterial agents may modify acute-phase inflammatory responses through their effects on cytokine synthesis by monocytes.

Anti-Bacterial Agents↗
PubMed1996

Gastroprokinetic effect of a new benzamide derivative itopride and its action mechanisms in conscious dogs.

The novel benzamide derivative itopride was assayed for its effect on gastrointestinal motility in conscious dogs when it was administered intraduodenally (i.d.). Gastrointestinal motility was measured by means of chronically implanted force transducers, and itopride at a dose of 10 mg/kg, i.d. or more increased the gastric contractile force during the digestive state. Intraduodenal cisapride, domperidone and metoclopramide also stimulated gastric motility, and their threshold doses were 1, 3 and 1 mg/kg, respectively. Dopamine infusion (1 mg/kg/hr, i.v.) caused the postprandial gastric motility to disappear, but it was immediately restored by itopride at a dose of 3 mg/kg, i.d. With itopride at 1 and 3 mg/kg, i.d., acetylcholine (0.05 mg/kg/min)-induced contractions were greatly enhanced. In addition to its gastric stimulation, itopride at doses of 10-100 mg/kg, p.o. inhibited apomorphine (0.1 mg/kg, s.c.)-induced vomiting in dogs. In conclusion, intraduodenal itopride stimulates gastric motility through both anti-dopaminergic and anti-acetylcholinesterase actions. Its gastroprokinetic threshold dose was as large as 3-10 times those of cisapride, domperidone and metoclopramide. These findings suggest that itopride is an orally active gastroprokinetic with a moderate anti-emetic action.

Animals↗
PubMed1996

The beta-prism: a new folding motif.

A new protein fold with internal symmetry has been observed in two proteins: vitelline membrane outer layer protein I (VMO-I) and delta-endotoxin. Despite lacking any discernible sequence similarity, both proteins have similar three-dimensional structures as well as a carbohydrate-binding site in the top region of the common fold.

Amino Acid Sequence↗
PubMed1995

Atomic model of a pyrimidine dimer excision repair enzyme complexed with a DNA substrate: structural basis for damaged DNA recognition.

T4 endonuclease V is a DNA repair enzyme from bacteriophage T4 that catalyzes the first reaction step of the pyrimidine dimer-specific base excision repair pathway. The crystal structure of this enzyme complexed with a duplex DNA substrate, containing a thymine dimer, has been determined at 2.75 A resolution. The atomic structure of the complex reveals the unique conformation of the DNA duplex, which exhibits a sharp kink with a 60 degree inclination at the central thymine dimer. The adenine base complementary to the 5' side of the thymine dimer is completely flipped out of the DNA duplex and trapped in a cavity on the protein surface. These structural features allow an understanding of the catalytic mechanism and implicate a general mechanism of how other repair enzymes recognize damaged DNA duplexes.

Adenine↗