Biomedical subjects

K Sekiguchi

Publications and source records attributed to K Sekiguchi.

At least 91 records · Page 5Linked to original sources

PubMed1996

Distinct structural requirements for interaction of the integrins alpha 5 beta 1, alpha v beta 5, and alpha v beta 6 with the central cell binding domain in fibronectin.

At least 10 different members of the integrin family have been reported to bind to fibronectin, and eight of these interact with the arginine-glycine-aspartic acid (RGD) site in the tenth type III repeat. However, studies utilizing recombinant fibronectin fragments have shown that for three of these, alpha 5 beta 1, alpha IIb beta 3, and alpha v beta 3, the structural requirements for binding to fibronectin differ. In the present study, we report that two additional integrins, alpha v beta 6, and alpha v beta 5 also demonstrate unique requirements for interaction with recombinant fibronectin fragments, alpha v beta 6, like alpha v beta 3, can support cell adhesion to the RGD-containing tenth repeat alone, and does not require the presence of a synergy site in the adjacent ninth repeat. In the cells used in this study, alpha v beta 5 only minimally supported adhesion to intact fibronectin, but did support adhesion to fragments composed of the eighth, ninth and tenth repeats or the tenth repeat, alone. Mutant fragments in which the eighth and tenth repeats were adjacent to one another enhanced adhesion mediated by alpha v beta 5, as well as adhesion mediated by alpha v beta 6. alpha v beta 5 and alpha v beta 6-mediated adhesion to all fibronectin fragments required interaction with the RGD site, as inferred by inhibition of adhesion with an RGD-containing peptide. These data suggest that each integrin that interacts with the RGD site in fibronectin has unique structural requirements for this interaction.

Antigens, Neoplasm↗

PubMed1996

Positive inotropic and chronotropic effects of toborinone and its electrophysiological properties in the isolated hearts of rabbits and guinea-pigs.

The inotropic and chronotropic actions of toborinone ((+/-)-6-[3-(3,4-dimethoxybenzylamino)-2-hydroxy-propoxy]-2(1H)-qu inolinone CAS 128667-95-8, OPC-18790) in the isolated right atrial and papillary muscles of rabbit and guinea-pig were examined and compared with those of milrinone (CAS 78415-72-2), a pure cyclic GMP-inhibited phosphodiesterase inhibitor. OPC-18790 (10(-7)-10(-4) mol/l) and milrinone (10(-3)-10(-4) mol/l) exerted concentration-dependent increases of contraction in both atrial and papillary muscles isolated from rabbits and guinea-pigs. In the isolated right atrium of rabbits and guinea-pigs, OPC-18790 showed limited increase in heart rate, while milrinone increased heart rate concentration-dependently. OPC-18790 (10(-6) and 10(-5) mol/l in guinea-pigs and 10(-7)-10(-4) mol/l in rabbits) prolonged the action potential duration in the isolated papillary muscles, while milrinone exerted no such change. In whole-cell voltage clamp experiments using isolated cardiac myocytes of guinea-pigs, OPC-18790(10(-5) mol/l) increased the L-type calcium current and inhibited outward potassium currents such as inward rectifying currents and delayed rectifier currents. OPC-18790 and milrinone (10(-6), 3 x 10(-5) mol/l) increased intracellular cyclic AMP levels with an increase in developed tension in isolated right ventricular muscles of guinea-pigs. From the above results, OPC-18790 was shown to be a positive inotropic agent with limited chronotropic effect and electrophysiologically properties different from those of milrinone. It was suggested that the prolongation of action potential duration, which is due to the inhibition of potassium currents, may be involved in OPC-18790's mechanism of positive inotropic action with limited chronotropic effect in addition to the inhibition of cyclic GMP-inhibited phosphodiesterase.

Action Potentials↗

PubMed1995

Peptide ligands for integrin alpha v beta 3 selected from random phage display libraries.

The integrin alpha v beta 3 binds promiscuously to cell-adhesive proteins: vitronectin, fibronectin, and several others containing the RGD motif. We have explored molecular recognition by alpha v beta 3 through selection of ligands from large random libraries of peptides displayed on phage. Ligands bound by alpha beta 3 consisted primarily of RGD peptides; however, these peptides showed considerable heterogeneity with respect to the identities of amino acids flanking RGD. The tolerance of alpha v beta 3 for RGD peptides of diverse composition is consistent with its role in vivo as a versatile receptor for RGD-containing extracellular matrix proteins. Peptide ligands for alpha v beta 3 also included a novel binding sequence, identical to a tetrapeptide found in vitronectin, which is a candidate for a synergistic site in this adhesive protein that may act in concert with RGD to promote molecular recognition.

Amino Acid Sequence↗

PubMed1995

The modified method of two-step differential extraction of sperm and vaginal epithelial cell DNA from vaginal fluid mixed with semen.

This investigation was undertaken as an efficient method for isolating sperm DNA from a mixed fluid sample which contains vaginal epithelial cells in a greater amount. The modified method of the two-step differential extraction procedure was found to be suitable for separating sperm DNA and vaginal epithelial cell DNA from the mixed stains. As the first step of digestion, vaginal epithelial cells in the mixed stains were lysed with Proteinase K and SDS, and sperm heads remaining in the lysed solution were collected by centrifugation. As the second step digestion, the sperm heads were lysed with the buffer containing Proteinase K, SDS and DTT as reducing agent. DNA fractions extracted from the two lysed solutions were enriched, one with sperm DNA and the other with vaginal epithelial cell DNA. MCT118(D1S80), ApoB VNTR and HLADQ alpha types of sperm DNA were detected and were confirmed by matching with corresponding male blood DNA. In the case of vaginal secretion mixed with semen of two males, the mixture of MCT118 types of the two males was detected in sperm DNA fraction.

Base Sequence↗

PubMed1995

Novel thiazole derivatives as inhibitors of superoxide production by human neutrophils: synthesis and structure-activity relationships.

Neutrophils have an important role in the self-defense systems of organisms through the production of superoxide. On the other hand, it has been proposed that abnormal amounts of superoxide produced by neutrophils are a serious factor in tissue injury. A series of novel thiazole derivatives was prepared and evaluated inhibitory effect on superoxide production by human neutrophils in vitro. Among these compounds, 6-[2-(3,4-diethoxyphenyl)thiazol-4-yl]-pyridine-2-carboxylic acid (OPC-6535) was selected as one of the most promising compounds. The synthesis and structure-activity relationships of these compounds are reported herein.

Adult↗

PubMed1995

EDA-containing fibronectin is synthesized from rheumatoid synovial fibroblast-like cells.

OBJECTIVE: To identify the cells that synthesize EDA-containing fibronectin (FN) and examine the role of EDA+FN in the pathogenesis of rheumatoid joint lesions. METHODS: Localization of EDA+FN and c-Fos protein in rheumatoid joints was studied immunohistochemically by utilizing antibodies for EDA+FN and c-Fos. Expression of EDA+FN was studied by immunoelectron microscopy and in situ hybridization. The amount of EDA+FN was measured by enzyme-linked immunosorbent assay. RESULTS: EDA+FN was specifically localized in the synovial lining layer of synovium with active rheumatoid arthritis (RA) (n = 17), but not in that with osteoarthritis (n = 4) or with inactive fibrous RA (n = 2). EDA+FN messenger RNA was localized in the synovial lining layer. EDA+FN was immunoelectron microscopically localized in the synovial lining fibroblast-like (type B) cells. EDA+FN was also detected at the cartilage-pannus junction and on the surface of RA cartilage. Double staining showed that EDA+FN colocalized with c-Fos protein in the rheumatoid synovial lining layer. Quantification of EDA+FN showed that it was highly concentrated in rheumatoid synovial fluids. CONCLUSION: EDA+FN is synthesized by the synovial lining fibroblast-like (type B) cells in situ in rheumatoid synovium, and appears to be expressed in association with activated or transformed states of synovium.

Animals↗

PubMed1995

Up-regulation of integrin alpha 5 beta 1 expression by interleukin-6 in rabbit corneal epithelial cells.

Interleukin-6 (IL-6) has been shown to promote the attachment of rabbit corneal epithelial cells to fibronectin-coated substratum and ex vivo migration of the cells on the corneal stroma. To examine whether IL-6 promotes cell attachment through up-regulation of expression of integrin alpha 5 beta 1, i.e., the major cell surface fibronectin receptor, we quantified the levels of both alpha 5 and beta 1 subunit transcripts by reverse transcription-polymerase chain reaction in cultured rabbit corneal epithelial cells pretreated with various concentrations of IL-6. The levels of both alpha 5 and beta 1 mRNAs were dose-dependently elevated by IL-6, attaining 1.5- and 1.8-fold increases, respectively, at 10 ng/ml. The stimulatory effect of IL-6 was transient; the levels of both subunit mRNAs reached a maximum 1 h after the addition of IL-6 and returned to the basal levels after 6 h. The IL-6-induced up-regulation of integrin alpha 5 and beta 1 mRNAs was also confirmed by Northern blot analysis. These results indicate that the increased attachment of corneal epithelial cells to fibronectin and enhanced ex vivo migration on corneal stroma by IL-6 is, at least in part, due to the temporal up-regulation of integrin alpha 5 beta 1 expression in corneal epithelial cells.

Amino Acid Sequence↗

PubMed1995

Detection of equine arteritis virus (EAV) by polymerase chain reaction (PCR) and differentiation of EAV strains by restriction enzyme analysis of PCR products.

A polymerase chain reaction (PCR) based assay capable of detecting and differentiating seven strains of equine arteritis virus (EAV) from around the world was developed. The primers for the PCR were chosen from the ORF6 gene encoding the unglycosylated membrane protein (M). Viral RNA from cell culture fluids infected with each of the seven EAV strains and RNA from the live vaccine, Arvac, was detected by PCR using four sets of primers. The sensitivity of detection was increased from 100 to 1,000 times by performing nested PCR enabling the detection of RNA at a level of 0.5-5 PFU. Differentiation among the virus strains and the live vaccine was achieved by cutting the PCR-amplified products from three sets of primers with six restriction endonucleases. Using this procedure it was possible to distinguish among the seven EAV strains used.

Base Sequence↗

PubMed1995

DNA strand breaks induced through active oxygen radicals by fragrant component 4-hydroxy-2-hydroxymethyl-5-methyl-3(2H)-furanone in Maillard reaction of hexose/amino acid.

The Maillard reaction of glucose/amino acid produces components that induce strand breakage of supercoiled DNA. This study was designed to elucidate the structure of the active components and the mechanisms of their DNA strand breakage. When an aqueous mixture of glucose/glycine heated under reflux for 4 hr was extracted with ethyl acetate, the extract showed mutagenicity to Salmonella typhimurium TA100 and strand-breaking activity for supercoiled DNA. One of the components with DNA strand-breaking activity was isolated by repeated HPLC using a reverse phase column. The component was identified as 4-hydroxy-2-hydroxymethyl-5-methyl-3(2H)-furanone (HHMF), a fragrant component in the Maillard reaction mixture. HHMF was similarly produced in the reaction of glucose/alanine and fructose/glycine. DNA strand-breaking activity of the component at pH 7.4 increased with increasing dose of the component and with increasing incubation time. The strand-breaking activity of the component was greater at pH 7.4 than at pH 4.4 and 9.4; it was inhibited in the presence of superoxide dismutase, catalase, hydroxyl radical scavengers, spin-trapping agents, thiol compounds and metal chelators, and also by removal of dissolved oxygen from the reaction mixture. The strand-breaking activity was enhanced in the presence of ionic iron. Incubation of HHMF with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) gave electron spin resonance signals characteristics of the DMPO-OH adduct, indicating generation of the hydroxyl radical. HHMF generated superoxide, hydrogen peroxide and then hydroxyl radical with the aid of a trace amount of metal ions, which effectively cleaved the DNA strand.

Amino Acids↗

PubMed1995

Restricted expression of Xenopus midkine gene during early development.

Midkine (MK) is a heparin-binding growth factor and forms a novel protein family together with another member, pleiotrophin (PTN)/heparin-binding growth-associated molecule (HB-GAM). A cDNA clone isolated from Xenopus laevis specifies for the Xenopus counterpart of MK (XMK), and the mode of XMK expression was studied by in situ hybridization and Northern blot analysis. XMK was first expressed at stage 11 (middle gastrula) and was located in the neural anlage at stage 12 (late gastrula). Through stage 13 to 15 (early neurula), XMK expression was restricted to the neural folds. At stage 23 (tailbud stage), XMK was predominantly localized in the brain and neural tube. At the larva stage, XMK expression was again restrictedly observed in the brain, the optic vesicles, the otic vesicle, and the spinal cord, all of which are derivatives of the neural tube, as well as in the branchial arches, derivatives of the cranial neural crest. Comparing the mode of MK expression between Xenopus and the mouse, we propose that MK plays evolutionally conserved roles in neurogenesis and development of the craniofacial architecture of ectomesenchymal origin. We also found that XMK was expressed in various adult organs; strong expression was observed in the brain, the eye and the spinal cord, all of which showed intense MK expression at the larva stage.

Amino Acid Sequence↗

PubMed1995

Detection of methicillin-resistant Staphylococcus aureus (MRSA) with antibodies against synthetic peptides derived from penicillin-binding protein 2'.

Ten kinds of peptides (21 to 32 amino acids in length) were synthesized based on the reported amino acid sequences of the penicillin-binding protein 2' (PBP2') of methicillin-resistant Staphylococcus aureus (MRSA). Antibodies against these synthetic peptides (SPs) were generated by immunizing rabbits. The antibodies raised against all the peptides except for one reacted to PBP2' of MRSA and to SPs used for immunization but not to any other protein of MRSA or methicillin-susceptible S. aureus (MSSA) tested by ELISA and Western blotting. A sandwich immunoradiometric assay (IRMA) for the detection of PBP2' was developed using these antibodies. The method could detect PBP2' extracted from as few as 3 x 10(4) cells of a clinical MRSA isolate, and a good correlation between cell number and signal radio-count was observed. IRMA was positive for all 51 methicillin-resistant staphylococci isolated from patients, and was negative for all the 28 methicillin-susceptible ones and 19 strains of other bacterial species. IRMA could be a simple and reliable method for MRSA detection in the clinical bacterial laboratory.

Amino Acid Sequence↗

PubMed1995

[Species and serovar-distribution, and drug-resistance of Shigella strains isolated from imported and domestic cases from 1990 through 1994 in Tokyo].

A total of 446 Shigella strains consisting of 336 imported and 110 domestic strains isolated in Tokyo from 1990 to 1994 were examined for their species and serovar-distribution, and their drug-resistance. In both imported and domestic strains, S. sonnei was found to be the most prevalent species, followed by S. flexneri. In imported strains, however, the isolation frequency of S. flexneri, S. boydii and S. dysenteriae were higher than that of domestic strains, and the serovar of each species was distributed in a wider range than that of the domestic strains. Provisional new serovar Shigella strains were isolated from 8 imported cases and 2 domestic cases. The drug-resistance-test using 9 drugs (CP, TC, SM, KM, ABPC, ST, NA, FOM, and NFLX) showed that 82.7% of the imported strains and 89.1% of the domestic strains were resistant to any drugs examined. Drugs of a high resistant rate were SM, TC, ST for the both groups. Drug-resistance-patterns of the resistant isolates varied to 21 types. Among those, a triple drug-resistance-type with TC SM ST was found the most frequently in both groups. None of the strains were resistant to FOM or NFLX.

Anti-Bacterial Agents↗

PubMed1995

[Regarding the Shigella flexneri 4 strains isolated in recent years].

A total of 38 strains of Shigella flexneri 4, 14 of which were subserotype 4a but 24 of which were of an undecided subserotype, isolated in recent years, were studied for their biochemical and serological characteristics, and drug susceptibility. Among the biochemical characteristics examined, 21 of the 38 strains (55.3%) were mannitol negative biotype and utilized sodium acetate, meanwhile, 17 of the mannitol positive strains did not utilize sodium acetate. The results of serological tests showed that 22 of the 24 strains, were of an undecided subserotype, basically subserotype 4a. Drug susceptibility was tested for nine drugs (CP, TC, SM, KM, ABPC, ST, NA, FOM and NFLX). Fifteen of the 17 mannitol positive strains (88.2%) and 12 of the 21 mannitol negative ones (57.1%), were found to be resistant to the 5 drugs, such as CP, TC, SM, ABPC or ST. None of the strains were resistant to KM, NA, FOM or NFLX.

Anti-Bacterial Agents↗

PubMed1995

Immunological detection of penicillin-binding protein 2' of methicillin-resistant staphylococci by using monoclonal antibodies prepared from synthetic peptides.

Two synthetic peptides 31 and 32 amino acids in length were prepared as deduced from a known amino acid sequence of penicillin-binding protein 2' (PBP2') of methicillin-resistant Staphylococcus aureus. Two monoclonal antibodies were generated from fused cells of myeloma cells and splenic cells of mice immunized with the synthetic peptides. Western blot (immunoblot) analysis demonstrated specific binding of the antibodies to PBP2' of a methicillin-resistant S. aureus strain. An immunoradiometric assay was developed by using these antibodies for simple detection of PBP2'.

Amino Acid Sequence↗

PubMed1995

Role of the carboxyl-terminal Fib2 domain in fibronectin matrix assembly.

A truncated form of fibronectin consisting of the N-terminal 70 kDa and C-terminal 37 kDa regions, designated r70F2, retained the ability to assemble into the extracellular matrix when expressed in cultured fibroblasts (Ichihara-Tanaka et al. (1992) FEBS Lett. 299, 155-158). To elucidate the role of the C-terminal 37 kDa region in fibronectin matrix assembly, we expressed a panel of mutant forms of r70F2 with various deletions and amino acid substitutions in mouse L cells. Although substitution of Ser for two Cys residues in the C-terminal dimerforming segment led to a marked reduction in the matrix assembly activity of r70F2, the resulting monomeric r70F2 still retained a low, but significant activity to assemble into the matrix. Neither the N-terminal 70 kDa nor the C-terminal 37 kDa regions, when expressed as monomeric forms, exhibited any residual activity, suggesting that the core domain of the 37 kDa region consisting of III15 and I10 through I12 modules, termed Fib2 domain, is actively involved in the matrix assembly of r70F2. In support of the role of Fib2 domain, the proteolytic fragment derived from the 37 kDa region inhibited the assembly of r70F2. Furthermore, en bloc deletion of the Fib2 domain or deletion of the I10 through I12 modules from r70F2 resulted in a marked decrease of the matrix assembly activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Cranioplasty with split-thickness calvarial bone.

Cranioplasty with autogenous split-thickness calvarial bone was performed in 10 patients. Follow-up for a mean of 19 months (range 2-43 mos) showed satisfactory protection of the brain and cosmetic reconstruction. No serious complication was seen except in one patient with postoperative epidural abscess. Split-thickness calvarial bone graft is recommended in patients with previous infection or high risk of infection, in poorly vascularized recipient sites resulting from multiple operations or irradiation, and in younger patients aged more than 7 years.

Adolescent↗

PubMed1994

[Prognostic factors of radiation therapy for patients with non-operative stage III non-small cell lung cancer].

Records of patients with non-operative stage III non-small cell lung cancer registered at the Department of Radiotherapy, Cancer Institute Hospital, from January 1978 to June 1980 were examined in order to present prognostic information on survival. The data were derived from 100 carefully staged patients and include results from the patients' history, physical examination, pretreatment laboratory tests, radiological findings, nature of radiotherapy and response to treatment. Serum albumin, tumor site, performance status (PS), hemoglobin, TDF and response were identified as important prognostic factors in the univariate analyses. On the other hand, tumor site, PS and serum albumin were significantly related with survival in the multivariate analyses. However it was found that TDF and response significantly affected survival when posttreatment variables were taken into account. These patients were classified into low, intermediate and high risk groups according to those three pretreatment factors (tumor site, PS and serum albumin), and the median survival times for the groups were 10.3 months, 10.4 months and 2.5 months, respectively. These results suggest that high risk patients with stage III non-small cell cancer generally might be treated palliatively.

Adult↗

PubMed1994

Site-specific O-glycosylation of cell adhesive lysozyme in yeast.

The cell adhesive protein RGD8 has been constructed using a yeast expression system by inserting eight amino acid residues (TGRGDSPA) between Val74 and Asn75 of human lysozyme [Yamada et al. (1993) J. Biol. Chem. 268, 10588-10592]. Purified RGD8 from yeast culture supernatant was found to contain glycosylated variants, in addition to the unglycosylated form. Peptide mapping analyses suggested that the glycosylation occurred at the inserted Thr residue in the RGD8 molecule. Electrospray ionization mass spectrometric analysis demonstrated the presence of four or five hexose residues in the glycosylated variants. Only mannose was detected in the sugar analysis of the oligosaccharide mixture obtained by mild alkaline treatment of the variants, and the structures of these carbohydrate chains were identified as Man alpha 1-3Man alpha 1-2Man alpha 1-2Man alpha and Man alpha 1-3Man alpha 1-3Man alpha 1-2Man alpha 1-2Man alpha by 1H-NMR spectroscopy. No other glycosylation was found, although the RGD8 molecule possesses a total of 13 Thr and Ser residues. In addition, no O-glycosylation was observed when the RGD8 protein was expressed in mouse L-cells. Thus, this O-glycosylation looks specific for yeast and the site of the Thr residue. The O-glycosylated variants of RGD8 exhibited a high level of adhesion activity to baby hamster kidney cells, which was almost comparable to that of the unglycosylated form.

Amino Acid Sequence↗