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Biomedical subjects

K Sekiguchi

Publications and source records attributed to K Sekiguchi.

At least 109 records · Page 6Linked to original sources
PubMed1994

Cell-adhesive activity and receptor-binding specificity of the laminin-derived YIGSR sequence grafted onto Staphylococcal protein A.

Laminin contains multiple oligopeptide motifs to promote cell adhesion and migration. One of these motifs is YIGSR within the B1 chain. We reconstituted the cell-adhesive activity of YIGSR motif by grafting it onto a truncated form of the Staphylococcal protein A (designated tSPA) via cassette mutagenesis. When coated on a polystyrene surface, the YIGSR-grafted tSPA (YIGSR-tSPA) promoted attachment and spreading of mouse melanoma and human rhabdomyosarcoma cells, but not of hamster fibroblasts. The cell-adhesive activity of YIGSR-tSPA was abolished by amino acid substitution or scrambling of the inserted YIGSR sequence. Divalent cations Mn2+ and Mg2+, but not Ca2+, promoted the cell adhesion to YIGSR-tSPA. Interestingly, the YIGSR-tSPA-mediated cell adhesion was barely inhibited by the linear peptide CDPGYIGSR-NH2, but was strongly inhibited by the cyclic peptide CDPGYIGSRC and another peptide PEILDVPST, which is a specific inhibitor for integrin alpha 4 beta 1. Among various anti-integrin antibodies, anti-alpha 4 and anti-beta 1 antibodies specifically inhibited the cell adhesion to YIGSR-tSPA. In support of these observations, adhesion of rhabdomyosarcoma cells to intact laminin was also partially inhibited by synthetic PEILDVPST peptide and anti-alpha 4 antibody. These results, taken together, indicate that the YIGSR motif exerts its cell-adhesive activity through interaction with integrin alpha 4 beta 1.

Amino Acid Sequence↗
PubMed1994

Targeting of the immunoglobulin-binding domain of protein A to the extracellular matrix using a minifibronectin expression vector.

A truncated form of fibronectin consisting of the N-terminal 70-kDa and C-terminal 37-kDa regions, referred to as "minifibronectin," retains the ability to assemble into the extracellular matrix, even though it lacks the central approximately 120-kDa region containing most of the type III modules (Ichihara-Tanaka, K., Titani, K., and Sekiguchi, K., FEBS Lett. 299, 155-158, 1992). Taking advantage of the matrix assembly activity of minifibronectin, we developed a novel method to target non-matrix proteins to the extracellular matrix by inserting them between the N-terminal 70-kDa and the C-terminal 37-kDa regions of minifibronectin. Using the immunoglobulin-binding domain of Staphylococcal protein A as a model, we demonstrated that the bacterial protein expressed in mouse L cells as a chimeric protein with minifibronectin is secreted as disulfide-bonded dimers and successfully deposited onto the extracellular matrix of transfected cells. The chimeric protein retained the immunoglobulin-binding activity not only in solution but also after deposition at the matrix. This targeting strategy we developed will provide a means to manipulate the biological functions of the extracellular matrix through targeting of a wide variety of non-matrix proteins.

Animals↗
PubMed1994

[Recent advances in radiotherapy for breast cancer].

Radiotherapy for breast cancer has been performed as adjuvant lymph node irradiation after radical operation, irradiation for chest wall relapse, and palliative treatment for bone, lung and brain metastases. However, attention has been paid to irradiation of the breast as a conservative treatment. The role of radiotherapy in breast conservative treatment is to reduce local relapse after shrinkage of operation, and to enhance the cosmetic outcome. The indications of this treatment, technique of irradiation, effects and side effects of irradiation, and risk of radiation-induced cancer were discussed. In Japan, we have experience with this treatment. Breast conservation treatment and modified mastectomy showed the same results in local control and survival. Sometimes the cure and most cases of relief of complaints are attained for recurrent or advanced breast cancer by radiotherapy. High dose rate brachytherapy by 192-Ir showed high local control of uncontrollable tumor by external irradiation. And hyperthermia combined with radiotherapy is also effective for superficial (less than 3 cm in depth) tumor. Even in patients with lung and or brain metastases, radiotherapy sometimes showed complete remission for years. The standard method of radiotherapy for bone and brain metastases is 30 Gy/2 wks, and the symptom relief rate is over 80%. Oncologists should be made aware of the high complaint relief rate by radiotherapy.

Brachytherapy↗
PubMed1994

[Neonatal brain tumor, a report of three cases].

Neonatal brain tumor is rare, but is so important a disease when considering the pathogenesis of the brain tumor that numerous review articles have been described. Today, the availability of noninvasive imaging procedures such as CT and MRI make it easy to diagnose, and some successful operative cases have been reported. We report three cases of neonatal brain tumors presented clinically within the first 2 weeks. Case 1: A full term boy admitted with projectile vomiting, enlarged head and left peripheral type facial palsy at the age of 12 days. CT scan revealed a large heterogeneous mixed-density mass in the left cerebellar hemisphere. Partially removed surgical specimen consisted of primitive glial cells differentiating with the ependymal cell immunohistochemically and electron microscopically, and diagnosed as ependymoblastoma. He had whole brain irradiation postoperatively, but died from respiratory distress 7 months later without tumor regrowth indicated on CT. Case 2: A full term boy admitted with progressive enlarging of the head at the age of 10 days. CT scan revealed a high-density mass in the cerebellar vermis and an obstructive hydrocephalus. Partially removed surgical specimen, diagnosed as medulloblastoma. He was irradiated throughout the whole brain and spinal cord, but died from intracranial dissemination 5 months later. Case 3: A 32-year-old female multipara was diagnosed as hydramnion during 28 weeks gestation. The concentration of AFP was very high in the amniotic fluid. A premature female fetus, weighing 1,650 g, was delivered by cesarean section for premature separation of the placenta during an estimated 30 weeks gestation.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain Neoplasms↗
PubMed1993

Hepatocyte growth factor may act as a pulmotrophic factor on lung regeneration after acute lung injury.

Hepatocyte growth factor (HGF) has been shown to have hepatotrophic and renotropic functions for regeneration of the liver and kidney through its mitogenic, motogenic, and morphogenic properties. To examine the involvement of HGF in lung regeneration after acute injury, we analyzed changes of HGF mRNA, HGF activity, and HGF receptors in the rat lung after lung insult and measured HGF concentration in sera of patients with various lung diseases. Following the onset of acute lung injury induced by intratracheal hydrochloride injection, a compensatory DNA synthesis occurred in the bronchial epithelium with a peak at 24 h and in the alveolar epithelium with a peak at 48 h. Expression of HGF mRNA in the rat lung remarkably increased only 3 h after the treatment and HGF activity in the lung also increased to about 3-fold at 6 h later. HGF receptors in the lung but not in the other noninjured organs were down-regulated 12 h later. These marked increases in HGF mRNA and HGF activity and the concomitant down-regulation of HGF receptor occurred before the marked compensatory DNA synthesis in bronchial and alveolar epithelial cells. HGF concentration in sera of patients with various lung diseases, as measured by radioimmunoassay, was much higher than that in healthy donors. These results suggest that HGF is newly produced in the lung after acute lung injury and may have a role in regeneration of the lung.

Animals↗
PubMed1993

An amino-terminal fibronectin fragment stimulates the differentiation of ST-13 preadipocytes.

Differentiation of ST-13 preadipocytes into adipocytes was inhibited almost completely by addition of rat plasma fibronectin (FN) (approximately 100 micrograms/mL), but was reversed by GRGDSP cell recognition peptide (1.5 mM) and anti-alpha 5 beta 1. On the contrary, the thermolysin digest of FN stimulated adipocyte differentiation in a dose-dependent manner, in which remarkable increases in the values of the differentiation indexes, the number of adipocytes (8-fold above the control), glycerophosphate dehydrogenase (GPD) activity (12-fold), and triacylglycerol content (5-fold), were observed by inclusion of the thermolysin digest (100 micrograms/mL). The increase in GPD activity by the thermolysin digest was inhibited remarkably (about 70% inhibition) by an antibody directed to the amino-terminal fibrin-binding (Fib 1) domain of FN and slightly (about 15%) by an antibody directed to the central cell-binding (Cell) domain, but not by anti-gelatin-binding domain and anti-carboxy-terminal fibrin-binding domain. Treatment of ST-13 cells by a purified 24K fragment (100 micrograms/mL) derived from the Fib 1 domain caused an over 20-fold augmentation of the GPD activity, accounting for a major part of the differentiation stimulatory activity of the thermolysin digest. The differentiation stimulatory effect of the 24K Fib 1 fragment was not affected by either GRGDSP peptide or anti-alpha 5 beta 1. Thus, FN can regulate adipose development of ST-13 cells by its two antipodal, inhibitory and stimulatory, activities, the latter of which is expressed only upon fragmentation. Proteolytic cleavage of FN may play an important role in controlling the action of FN on adipocyte differentiation.

Adipose Tissue↗
PubMed1993

Structural and functional analyses of the Arg-Gly-Asp sequence introduced into human lysozyme.

To determine the functional conformation of the Arg-Gly-Asp (RGD) sequence, we have constructed mutant proteins by inserting 4-12 amino acid residues from the RGD region of human fibronectin between Val74 and Asn75 of human lysozyme. RGDS-, GRGDSP-, TGRGDSPA-, VTGRGDSPAS-, and AVTGRGDS-PASS-introduced mutant lysozymes were expressed in yeast, purified, and designated as RGD4, -6, -8, -10, and -12, respectively. Using baby hamster kidney cells, RGD8, RGD10, and RGD12 were shown to possess high cell adhesion activity nearly equal to 10% of human vitronectin activity. RGD4 and RGD6 exhibited somewhat lower cell adhesion activity. The activities of these mutant proteins were inhibited by the addition of either GRGDSP peptide or polyclonal antibody against vitronectin receptor, as was the case for the vitronectin activity. The results suggest that the cell adhesion signals are transduced to cells through the interaction with the vitronectin receptor. The three-dimensional structures of RGD4 and RGD8 were determined at 1.8-A resolution by x-ray crystallography. A model of the inserted region in RGD4 could be built in the electron density map, but the positions of the preceding residues, Ala73-Val74, were uncertain. The inserted region in RGD8 did not demonstrate continuous electron densities. The results suggest that these RGD sequence-containing regions are highly flexible and that such flexibility could allow the conformation of the RGD regions to be induced to fit into the binding pocket of the integrin receptor.

Amino Acid Sequence↗
PubMed1993

Coordinate oncodevelopmental modulation of alternative splicing of fibronectin pre-messenger RNA at ED-A, ED-B, and CS1 regions in human liver tumors.

The molecular diversity of fibronectin arises from alternative RNA splicing at regions termed ED-A, ED-B, and IIICS. We investigated the splicing patterns of fibronectin pre-mRNA at both ED-B and IIICS regions in various human liver tissues with an emphasis on the expression of the alternative cell adhesive site CS1 within the IIICS region. The relative abundance of the fibronectin mRNA containing the CS1 sequence was significantly increased in both fetal and cancerous liver tissues, although it was not affected in nonmalignant tissues with chronic hepatitis and cirrhosis. Similarly, the relative abundance of the fibronectin mRNA containing the ED-B region was also increased in both fetal liver and liver tumors, showing a close parallelism with the splicing pattern at the ED-A region. Immunohistochemical examination of cancerous liver tissues with monoclonal antibodies directed to the ED-A and ED-B segments revealed that the fibronectin isoforms containing these extra peptide segments were specifically deposited in the tumor nodules. Other genes encoding kininogen, gamma chain of fibrinogen, and beta-amyloid protein precursor, all of which had been shown to be alternatively processed, did not show any significant alteration in the splicing pattern in cancerous liver tissues. These results indicate that the alternative splicing of fibronectin pre-mRNA at the ED-A, ED-B, and IIICS regions is coordinately modulated in both fetal and cancerous liver tissues toward inclusion of the extra peptide segments and that not all but only selected genes are susceptible for "fine tuning" of alternative RNA splicing in cancerous liver tissues.

Alternative Splicing↗
PubMed1993

Chloroplast envelope protein encoded by chloroplast genome.

The gene product of an open reading frame of chloroplast genome, ORF 231 in pea, was immunochemically detected in chloroplast and etioplast envelopes. This is the first protein of a Chloroplast Envelope Membrane encoded by a chloroplast genome. It was named CEM A and the gene, cem A. CEM A is an acidic protein having an apparent molecular mass of 34 kDa on SDS-PAGE, and a minor component detected in the fractionated inner envelope.

Amino Acid Sequence↗
PubMed1993

Electropherotypes, serotypes, and subgroups of equine rotaviruses isolated in Japan.

Electropherotypes (ET), serotypes, and subgroups of equine rotaviruses isolated from foals in Japan were determined. The ETs of 136 isolates from 1981 through to 1991 were divided into six groups: ET-A-ET-F. The ET-A, -B, -C, -D, -E, and -F were present in 3, 1, 121, 9, 1, and 1 strains, respectively. Representative viruses of ET-A, -B, -C, and -D were identified as serotype G3. Viruses of ET-E and -F were identified as serotypes G 10 and G 5, respectively. The four representative viruses of serotype G 3 did not belong to either subgroup I or II. The two viruses of serotypes G 5 and G 10 belonged to subgroup I. Serotype G 3 strains possessing ET-C were prevalent among the foals throughout the study period.

Animals↗
PubMed1993

Ribonuclease activity of sialic acid-binding lectin from Rana catesbeiana eggs.

Sialic acid-binding lectin (SBL) isolated from Rana catesbeiana eggs is a basic protein which agglutinates a large variety of tumour cells and has an amino acid sequence homologous to that of human angiogenin and pancreatic ribonuclease (RNase). Although SBL and angiogenin lack the Cys-65-Cys-72 disulphide bond of pancreatic RNase, the locations of the other three disulphide bonds are similar among the three molecules. SBL was found to exhibit RNase activity, as well as catalytic properties resembling those of bovine RNase A in some respects. For example, SBL hydrolyses poly(uridylic acid) and poly(cytidylic acid) as substrates, and prefers the former. RNase A and angiogenin are strongly inhibited by human placental RNase inhibitor, whereas the RNase activity and tumour cell agglutination activity of SBL are not affected by this inhibitor.

Amino Acid Sequence↗
PubMed1993

Engineering of artificial cell-adhesive proteins by grafting EILDVPST sequence derived from fibronectin.

Fibronectin contains at least two distinct oligopeptide sequences serving as signals for the interaction with cell surface adhesion receptors termed integrins. One of these sequences, Arg-Gly-Asp-Ser (RGDS) tetrapeptide, was shown to be transferred to a truncated form of Staphylococcal IgG-binding protein (hereafter referred to as tSPA) with retention of its cell-adhesive activity [Maeda, T. et al. (1989) J. Biol. Chem. 264, 15165-15168]. We have extended the observation to another cell-adhesive sequence, Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr (referred to as "CS1" sequence), to demonstrate that: i) the tSPA grafted with the sequence mediated adhesion of human lymphoma and rhabdomyosarcoma cells, mouse melanoma cells, but not of hamster fibroblasts; ii) antibodies against integrin alpha 4 and beta 1 subunits specifically inhibited cell adhesion mediated by the CS1-grafted tSPA; iii) a heterodivalent tSPA grafted with both RGDS and CS1 sequences at different sites was more potent in promoting cell adhesion than the monovalent tSPAs grafted with either sequence alone. These results indicate that not only the RGDS but also the CS1 sequence can be transferred to tSPA with retention of its cell-adhesive activity as well as its cell-type specificity, and that the grafted CS1 sequence is recognized by the same integrin isotype as the authentic sequence within intact fibronectin.

Amino Acid Sequence↗
PubMed1993

Cellular fibronectin in plasma: its implications in fibrinogen-associated cryoprecipitation and other related reactions.

Elimination of cryoprecipitable plasma components (cryogels) by cryofiltration from the circulating blood of patients with drug-resistant rheumatoid arthritis (RA) alleviates clinical symptoms including morning stiffness and arthralgia. The cryogels thus isolated from the blood were found to consist mainly of fibrinogen (Fbg) and fibronectin (FN). Analysis by immunoblotting with an anti-cellular FN monoclonal antibody revealed that cellular FN (cFN) co-existed with plasma FN (pFN) in the cryogels derived from the patients. Using an ELISA, we assessed cFN together with the total FN (pFN+cFN) in plasmas and cryogels derived from the patients. The cFN/total FN ratio was distinctly higher in the cryogels than in the plasmas, suggesting that cFN was more readily precipitated than pFN in association with Fbg under cold conditions.

Arthritis, Rheumatoid↗
PubMed1992

Matrix assembly of recombinant fibronectin polypeptide consisting of amino-terminal 70 kDa and carboxyl-terminal 37 kDa regions.

Three different forms of recombinant human fibronectin polypeptides consisting of the amino-terminal 70 kDa region, the carboxyl-terminal 37 kDa region, or both, were expressed in mouse L cells. Although either the amino-terminal or the carboxyl-terminal region alone was only poorly incorporated into the extracellular matrix, the fused form of the polypeptide was highly capable of assembling into the matrix. These results indicate that matrix assembly of fibronectin requires both regions and can proceed in the absence of the type III repeats including the one containing the cell adhesive Arg-Gly-Asp sequence.

Animals↗
PubMed1992

Immune response to GOR, a marker for non-A, non-B hepatitis and its correlation with hepatitis C virus infection.

Recently, identification and molecular cloning of a host cellular gene designated GOR from chimpanzees experimentally infected with non-A, non-B hepatitis (NANBH) agent was reported. It was further demonstrated that there is a close association between the immune response to an antigenic peptide of GOR (GOR2) and NANBH. In order to define the specificity of the immune response, in the present study we have identified an additional epitope in the GOR gene sequence, upstream from GOR2, and studied its correlation with the immune response to hepatitis C virus (HCV) in NANBH patients. An enzyme-linked immunoassay (EIA) was developed which utilizes synthetic peptides designated spGOR346 and spGOR2 as the serological target for the detection of anti-GOR antibodies in patient serum samples from various hepatic and non-hepatic disease categories. GOR peptides identified 80-90% of the NANBH samples that were positive for HCV C100-3 and about 70% of the NANBH samples that were positive by Abbott prototype second-generation HCV antibody assay. Among a normal donor population(s), only 2-3% of the samples were positive for antibodies to GOR sequences, whereas from the patient categories unrelated to viral hepatitis as well as various nonhepatic diseases, the immune response to both GOR peptides was closely associated with the presence of antibodies to HCV. The data indicate that antibodies to GOR is a marker associated with NANBH.

Amino Acid Sequence↗