Connective tissue growth factor.
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Amino Acid Sequence↗
Biomedical subjects
A Igarashi
Publications and source records attributed to A Igarashi.
Production of dihydrofolate reductase by cloned Escherichia coli and its application to asymmetric synthesis of l-leucovorin.
We have investigated culture conditions for production of dihydrofolate reductase by Escherichia coli harboring a high expression plasmid, pTP64-1. Sorbitol addition and pH control were effective for the production of the enzyme in a jar fermentor. The enzyme was purified from a cell-free extract by column chromatographies on DEAE-Cellulofine and Superose Prep12 and showed a single band on SDS-polyacrylamide gel electrophoresis. The reduction of 200 mM dihydrofolate to 6(S)-tetrahydrofolate, an intermediate for l-leucovorin synthesis, was complete in 2 hr under anaerobic conditions, using 1.5 units/ml of the purified enzyme.
Chromatography, DEAE-Cellulose↗
Chemical identification of catfish growth hormone and prolactin.
Isolation and primary structure of growth hormone (GH) and prolactin (PRL) from the pituitary gland of catfish (Ictalurus punctatus) are described. Alkaline extract of the pituitary glands was fractionated by gel filtration on Sephadex G-75, ion-exchange chromatography on DEAE-cellulose, and reversed-phase high-performance liquid chromatography on Octadecyl silica ODS. Catfish GH and PRL were identified by Western blotting with antisera against chum salmon GH and PRL. The catfish GH consists of 178 residues and is the most similar to carp GH, with sequence identity of 77%, although there is an uninterrupted deletion of 10 amino acid residues that corresponds to carp GH (90-99). The PRL is composed of 187 residues, which also exhibits the highest identity (79%) with carp PRL. Sequence identity between catfish GH and PRL is only 27%.
Amino Acid Sequence↗
[Structure of Japanese encephalitis virus genome and its detection by using polymerase chain reaction].
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Base Sequence↗
[Genomic structure of togavirus and flavivirus].
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Flavivirus↗
Epidemiology and control of Japanese encephalitis.
Japanese encephalitis (JE) remains endemo-epidemic in several countries in East, South-East and South Asia. The disease has been under control in Japan since the 1970s owing to mass immunization using mouse-brain-derived inactivated vaccine and to reduced vector mosquito populations. The vector density which was once reduced by wide spraying of insecticides in rice fields showed an increasing trend after the 1980s as a result of mosquito resistance. In the Republic of Korea, the number of JE cases showed a significant decrease after 1983 also because of mass immunization using mouse-brain-derived vaccine. On the other hand, large outbreaks of JE continued to occur in China, Viet Nam, Thailand, India, Nepal and Sri Lanka. In China, a hamster-kidney cell-derived vaccine was developed and used for human immunization. Besides human JE, the fatal outcome of equine JE is an economic problem in China. Current JE vaccine derived from mouse brain is highly purified and its safety and efficacy have been proved by field-tests as well as laboratory experiments. In spite of slight antigenic differences among JE virus isolates, JE vaccine produced by a classical Nakayama strain was effective in preventing overt JE in a field study in Thailand. The technology of mouse-brain-derived inactivated JE vaccine production was transferred from Japan to India, Thailand and Viet Nam. The production of JE vaccine in these countries is still on a pilot scale and insufficient for mass-immunization of susceptible target populations.(ABSTRACT TRUNCATED AT 250 WORDS)
Encephalitis Virus, Japanese↗
Purification, crystallization and characterization of N-acetylneuraminate lyase from Escherichia coli.
N-Acetylneuraminate lyase produced by Escherichia coli was purified and crystallized from a genetically engineered strain (E. coli SF8/pNAL1). The enzyme showed apparent molecular masses of 105,000 Da on gel filtration and 35,000 Da on SDS/PAGE, suggesting that the enzyme is a trimer. The apparent optimum pH and temperature were found to be 6.5-7.0 and 80 degrees C respectively. The Km values for N-acetylneuraminate and N-glycollylneuraminate were 3.3 and 3.3 mM respectively. The enzyme was inhibited by reduction with NaBH4 in the presence of the substrate, indicating that the enzyme belongs to the Schiff-base-forming Class I aldolases. The enzyme was strongly inhibited by Cu2+ ions, p-chloromercuribenzoate and N-bromosuccinimide, and also inhibited competitively by the reaction product, pyruvate, and its structurally related compounds, dihydroxyacetone and DL-glyceraldehyde.
Amino Acid Sequence↗
Differential binding, biological and biochemical actions of recombinant PDGF AA, AB, and BB molecules on connective tissue cells.
We have compared the biological and biochemical properties of recombinant PDGF AA, AB, and BB using three types of fibroblastic cells: NIH/3T3, human skin fibroblast, and fetal bovine aortic smooth muscle. PDGF binding, receptor autophosphorylation, phosphatidyl inositol hydrolysis, as well as chemotactic and mitogenic responses of the cells were analyzed. PDGF-AB and PDGF-BB showed similar receptor binding, receptor autophosphorylation, and potent biological activity for all three of the cell types tested. In contrast, PDGF-AA was biologically active only for the NIH/3T3 cells in which binding sites for PDGF-AA were abundant, but was inactive for bovine aortic smooth muscle cells and human skin fibroblasts in which binding sites for PDGF-AA were absent. PDGF-AA could not induce any biochemical changes in the human skin fibroblasts or smooth muscle cells. Western blot studies with anti-Type alpha and beta PDGF receptor antibodies indicate that the NIH/3T3 cells contained both PDGF alpha and beta receptors, whereas the human skin fibroblasts and bovine smooth muscle cells contained only detectable levels of beta receptors. These results indicate that cells possessing high levels of PDGF beta receptors only are capable of responding equally well to either PDGF AB or BB.
3T3 Cells↗
Response of scleroderma fibroblasts to various growth factors.
Abnormal growth regulation in lesional skin fibroblasts may be related to scleroderma pathogenesis. We report on the abnormal response of cultured fibroblasts derived from sclerotic lesions to various growth factors. We investigated the responses of skin fibroblasts (10 strains) and normal fibroblasts (9 strains) to the growth factors as PDGF, TGF-beta 1, EGF and basic FGF. Experiments were conducted during the proliferation and confluent stages. PDGF, EGF and basic FGF stimulated fibroblast growth during the proliferation and confluent stages, but the response of scleroderma fibroblasts was significantly lower than that of normal fibroblasts. TGF-beta 1 slightly stimulated confluent fibroblast growth and inhibited proliferating fibroblasts, and the response of scleroderma fibroblasts exceeded that of normal fibroblasts. The decreased response to growth-stimulating factors observed in scleroderma fibroblasts suggests that cultured fibroblasts derived from scleroderma lesions were already senescent because they have been activated by growth-stimulating factors and repeatedly divided in vivo. Thus, abnormal growth regulation of skin fibroblasts may be partially related to the pathogenesis of scleroderma.
Adolescent↗
Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10.
Human umbilical vein endothelial (HUVE) cells have been previously reported to express the genes for the A and B chains of PDGF and to secrete PDGF-related factors into culture media. Antihuman PDGF IgG affinity chromatography was used to purify PDGF-related activity from HUVE cell-conditioned media. Immunoblot analysis of the affinity-purified proteins with anti-PDGF IgG and antibodies specific for the A or B chain peptides of PDGF combined with chemotactic and mitogenic assays revealed that the major PDGF immunorelated molecule secreted by HUVE cells is a monomer of approximately 36-38 kD and that less than 10% of the purified biologically active molecules are PDGF A or B chain peptides. Screening of an HUVE cell cDNA library in the expression vector lambda gtl 1 with the anti-PDGF antibody resulted in the cloning and sequencing of a cDNA with an open reading frame encoding a 38-kD cysteine-rich secreted protein which we show to be the major PDGF-related mitogen secreted by human vascular endothelial cells. The protein has a 45% overall homology to the translation product of the v-src-induced CEF-10 mRNA from chick embryo fibroblasts. We have termed this new mitogen connective tissue growth factor.
Amino Acid Sequence↗
Japanese encephalitis virus fusion protein with protein A expressed in Escherichia coli confers protective immunity in mice.
A complementary DNA (cDNA) that codes C-terminal, one-third of envelope glycoprotein (E) and N-terminal 65 amino acids of NS1 protein of Japanese encephalitis (JE) virus was inserted into Escherichia coli expression vector pRIT2T. The inserted gene was expressed as a fusion protein with protein A, and the expressed protein was intraperitoneally injected into mice. The immunized mice produced anti-JE antibodies measured by the hemagglutination-inhibition and neutralization tests as well as ELISA and were protected from the lethal challenge of JE virus by intraperitoneal inoculation.
Animals↗
Kyrle disease associated with sarcoidosis and renal failure.
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Female↗
Rapid identification of dengue virus serotypes by using polymerase chain reaction.
Four primer pairs were selected on the basis of the published sequence data of four dengue virus serotypes so that each unique target sequence size could be amplified for each serotype by polymerase chain reaction. The procedure consists of (i) RNA preparation, (ii) reverse transcription, and (iii) polymerase chain reaction, all of which could be completed within 2 h in a single tube for each specimen. The amplified sequence size revealed by ethidium bromide-stained agarose gel electrophoresis was unique for each serotype, using infected culture fluid of isolates from dengue fever or dengue hemorrhagic fever patients in Thailand, Indonesia, and the Philippines as well as from prototype viruses, thus facilitating simultaneous identification and typing.
Animals↗
Partial sequence analysis of type 1 dengue virus coding for the nonstructural hydrophilic protein NS-3.
A complementary DNA copy of DEN-1 RNA was synthesized using reverse transcriptase and a random primer. The double-stranded DNA copy was cloned at the Sma I site of the pUC13 vector and was used to transform Escherichia coli JM83. Among the transformants selected for characterization by nucleotide sequence analysis, we report here one that codes for a region of nonstructural hydrophilic protein, NS-3. Computer analysis of this sequence (967 bp) showed about 87% conservation at the amino acid level and 79% at the nucleotide level when compared with dengue serotypes 2, 3 and 4 and Japanese encephalitis virus. This suggests an important function which is common to all four serotypes. Comparison of the cloned region with sequences of the above strains also suggested conservation of hydrophobic regions.
Amino Acid Sequence↗
[Anesthetic management of a patient with Chubby Puffer syndrome].
Chubby Puffer syndrome produces symptoms such as sleep apnea, cor pulmonale and upper airway obstruction due to adenotonsillar enlargement. We gave anesthesia for adenotonsillectomy in a 6-year-old boy with this syndrome. The child was massively obese. Anesthesia was induced with thiamylal, nitrous oxide and enflurane by monitoring SaO2. Tracheostomy was performed following orotracheal intubation because of possible difficult postoperative course. At the beginning of operation arterial blood studies showed hypoxemia. Positive end-expiratory pressure ventilation was effective to improve oxygenation. After adenotonsillectomy the symptoms were relieved.
Adenoidectomy↗
Chemo-enzymatic synthesis of optically pure l-leucovorin, an augmentor of 5-fluorouracil cytotoxicity against cancer.
Optically pure l-leucovorin was synthesized on a large scale by the combination of chemical and enzymatic processes. After reduction of folate with zinc, dihydrofolate was reduced asymmetrically to (6)-tetra-hydrofolate by use of dihydrofolate reductase from E. coli C600/pTP600, with simultaneous NADPH cofactor recycling using glucose dehydrogenase from Gluconobacter scleroideus KY3613. Calcium l-leucovorin.4H2O (113 g) was obtained from (6S)-tetrahydrofolate via 5,10-methyenyltetrahydrofolate by formylation, reflux, addition of calcium ions and floricil column chromatography, with an overall yield of 50% based on folate. The l-leucovorin showed optical purity of 99.9% de as (6S)-form.
Escherichia coli↗
The effects of scleroderma sera on endothelial cell survival in vitro.
The effects of sera and of platelet-poor plasma from patients with scleroderma on endothelial cell survival in vitro were studied. The survival ratio of rat heart endothelial cells was studied both in 10% test serum and in 10% platelet-poor plasma. Sera from patients with scleroderma decreased the survival ratio significantly when compared with sera from normal controls. In contrast, there was no significant difference between platelet-poor plasma from patients with scleroderma and that from normal controls. Our data indicate that platelets in the patients with scleroderma may cause vascular damage by affecting endothelial cell survival.
Adult↗
Dipyridamole specifically decreases platelet-derived growth factor release from platelets.
The authors have previously reported that dipyridamole decreased platelet-derived growth factor levels in human serum by lowering the release of this factor during blood clotting. In the present study, we have shown that this effect is specific to dipyridamole, and does not occur with other antiplatelet drugs such as aspirin, trapidil or ticlopidine. In addition, dipyridamole has been shown to decrease the PDGF level selectively, but not the levels of other factors from alpha granules in platelets (beta-thromboglobulin and platelet factor 4). These data indicate that dipyridamole may be an effective drug for the prevention of PDGF-related disorders.
Blood Platelets↗