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Biomedical subjects

Y Morikawa

Publications and source records attributed to Y Morikawa.

At least 127 records · Page 7Linked to original sources
PubMed1996

Complete inhibition of human immunodeficiency virus Gag myristoylation is necessary for inhibition of particle budding.

Myristoylation of human immunodeficiency virus (HIV) Gag protein is essential for virus particle budding. Two reactions are involved; activation of free myristate to myristoyl-CoA and transfer of the myristoyl residue to the Gag N-terminal glycine. We have investigated the effects of triacsin C, an inhibitor of long chain acyl-CoA synthetase, on release of HIV Gag virus-like particle (VLP) produced using the recombinant baculovirus system. First, inhibition of acyl-CoA formation by triacsin C was confirmed using the membrane fractions of insect Sf9 cells as an enzyme source. Second, when HIV Gag protein was expressed in the presence of triacsin C (0-48 microM), Gag myristoylation was inhibited in a dose-dependent manner. Budding of Gag VLP, however, did not follow similar inhibition kinetics but appeared unaffected up to 24 microM, yet was completely abolished at 48 microM when the myristoylation of Gag protein was also completely inhibited. The "all-or-none" inhibition of Gag VLP budding suggests that although inhibition of acyl-CoA synthetase blocks the production of myristoylated Gag protein, only complete inhibition of Gag myristoylation prevents VLP budding. Thus, relatively few myristoylated Gag molecules are sufficient for plasma membrane targeting and VLP budding.

Acyl Coenzyme A↗
PubMed1996

Nodular lymphoid hyperplasia of the terminal ileum: report of a case and the findings of an immunological analysis.

The immunological and immunohistochemical characteristics of a patient with nodular lymphoid hyperplasia (NLH) of the terminal ileum were investigated pre- and post-operatively. The patient presented with diarrhea, lower abdominal pain, and weight loss, and an abdominal X-ray following barium enema revealed multiple small nodules in the terminal ileum which were subsequently confirmed to be NLH by histological examination. Preoperatively, although the serum immunoglobulin levels were normal, the T-cell responses in the peripheral blood, including the phytohemagglutinin (PHA)-blastogenesis and the T4/T8 ratio, were impaired. However, following an ileocecal resection, the PHA-blastogenesis became augmented and the T4/T8 ratio recovered to within the normal range. The immunohistochemical findings revealed a more marked accumulation of T cells in the interstitium around the hyperplastic follicles than in the interstitium of the normal intestine.

CD4-CD8 Ratio↗
PubMed1996

Baculovirus vectors for expression in insect cells.

Recombinant baculoviruses now represent a mature technology in which vector development, particularly for the control of expression level, has reached a plateau. However, other aspects of expression, such as the production of multiple proteins, improved product purification or maximizing protein processing, remain areas for novel vector and host cell development. This year has seen these topics come to the fore in descriptions of new expression systems.

Animals↗
PubMed1996

Gag-Gag interactions in the C-terminal domain of human immunodeficiency virus type 1 p24 capsid antigen are essential for Gag particle assembly.

Seven internal deletions within the p24 domain of the human immunodeficiency virus type 1 Gag precursor have been assessed for their effect on Gag particle formation following their expression using recombinant baculoviruses. In addition, each deleted molecule was assessed for its ability to bind soluble p24 antigen in vitro. The mutants fell into three different phenotypic groups: (i) three mutants that had no effect on either p24 binding or Gag particle assembly, (ii) three mutants that abolished both features and (iii) one mutant that bound p24 in vitro but failed to assemble particles. Mutations that abolished both in vitro p24 binding and particle assembly mapped to the C terminus of p24 confirming this region as critical for virion assembly. We suggest the division of virion assembly into at least two distinct phases and suggest a model in which the critical sequences mapped to date are combined with available structural information.

Animals↗
PubMed1996

Two types of novel dipeptidyl aminopeptidases from Pseudomonas sp. strain WO24.

Two kinds of dipeptidyl aminopeptidase I (DAP I [cathepsin C])-like activities which hydrolyze Gly-Phe-p-nitroanilide (Gly-Phe-pNA) were detected in Pseudomonas sp. strain WO24. They were purified and characterized. The isolated enzymes, named DAP BII and DAP BIII, were revealed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. DAP BII was estimated to have a molecular mass of 150,000 Da by gel filtration and a subunit size of 73,000 Da by SDS-PAGE, indicating it to be a homodimer. The molecular mass of DAP BIII was evaluated to be approximately 60,000 Da by gel filtration and 69,000 Da by SDS-PAGE, indicating that it is monomeric. The isoelectric points of DAP BII and DAP BIII were 6.1 and 5.0, and their optimal pHs were 8.0 and 8.5 to 9.0, respectively. The result of peptide mapping for DAP BII and DAP BIII showed that these enzymes consist of different components. Both enzymes were completely inhibited by diisopropylphosphofluoride but not by general thiol inhibitors, indicating that they are serine proteases. DAP BII and DAP BIII hydrolyzed Gly-Phe-pNA but not Gly-Arg-pNA, both of which are model substrates for mammalian DAP I. Despite these shared activities toward DAP I, DAP BII released dipeptides from Ala-Ala-pNA and Lys-Ala-4-methylcoumarinamide (a substrate for DAP II), whereas DAP BIII did not hydrolyze either of these compounds and was presumed to prefer substrates composed of bulky, hydrophobic amino acids at P1 and P1' positions. In addition, DAP BII showed no endopeptidase activity, whereas DAP BIII possessed the activity on N-terminally blocked peptide derivatives besides exopeptidase activity. Assays performed with bioactive peptides such as angiotensin I and neuromedin N as substrates indicate that DAP BII has a considerably broader substrate specificity than DAP BIII and is able to hydrolyze an X-Pro bond, an imido bond that few peptidases and no known DAPs can cleave. These characteristics, namely, substrate specificities, molecular mass, pI, peptide mapping, pH optimum, and effect of inhibitors, suggested that the two DAPs purified in this work are distinct enzymes and do not belong to any of the previously reported DAP classes.

Bacterial Proteins↗
PubMed1996

A novel dipeptidyl aminopeptidase from Pseudomonas sp. strain WO24.

An activity similar to that of dipeptidyl aminopeptidase I (DAP I) which releases dipeptide from Gly-Arg-p-nitroanilide (Gly-Arg-pNA) was detected in a Pseudomonas sp. An enzyme was isolated and purified about 400-fold by a series of column chromatographies. The enzyme, named DAP BI (DAP from bacteria, type I), was revealed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The molecular mass was estimated to be 82 kDa by SDS-PAGE and 65 kDa by gel filtration, suggesting that the enzyme may be a monomer. The enzyme had an isoelectric point of 4.7. It is optimally active at pH 9.0. The Km and Vmax of the enzyme for Gly-Arg-pNA were 0.25 mM and 195 micromol/min/mg, respectively. The purified enzyme did not hydrolyze Gly-Phe-pNA, which was also a substrate for DAP I, whereas it hydrolyzed Arg-Arg-4-methoxy-beta-naphthylamide (Arg-Arg-MNA), a model substrate for DAP III. The Km and Vmax for Arg-Arg-MNA were 0.019 mM and 145 micromol/min/mg, respectively. This purified enzyme can also catalyze the removal of Asp-Arg from the N termini of angiotensins I and II. The enzyme activity was completely inhibited by Zn(II) (0.5 mM), tosyl-L-Lys-chloromethyl ketone (0.1 mM), and leupeptin (0.1 mM) and partially inhibited by Co(II) (0.5 mM) and chymostatin (0.1 mM), whereas the enzyme was not affected by general serine protease inhibitors (phenylmethylsulfonyl fluoride and diisopropylfluorophosphate) and thiol protease inhibitors. The substrate specificity, classification of catalytic site, and other enzymatic properties demonstrate that this enzyme is distinct from the previously described mammalian DAPs I and III and Saccharomyces cerevisiae DAP III. These results indicate that DAP BI may be a new type of the DAP family.

Amino Acid Sequence↗
PubMed1996

Dipeptidyl aminopeptidase IV from Pseudomonas sp. WO24.

Dipeptidyl aminopeptidase IV from Pseudomonas sp. WO24 was purified as two molecular forms of 84 and 82-kDa by SDS-PAGE. Peptide mapping and N-terminal sequence analyses indicated that both proteins might be derived from the same protein, and that the 82-kDa molecule might be a truncated form from the 84-kDa molecule at least at the N-terminus. The DAP IV gene of Pseudomonas sp. WO24 was cloned and expressed in E. coli. The enzyme expressed in E. coli JM109 harboring a hybrid plasmid, pYO-6A, with about a 3-kbp fragment containing the DAP IV gene, was purified with an activity recovery of 24%. The recombinant enzyme also had the same two molecular forms, though the ratio of the two forms (about 1:1) was different from that of the native ones (about 1:4). The native and recombinant enzyme preparations had similar specific activities, suggesting that the 84 and 82-kDa molecules are in an active form and have almost the same specific activity. The molecular mass, the subunit number, the substrate specificity, and the effects of various inhibitors of the native enzyme indicated that this enzyme was a typical DAP IV and had properties similar to those of Flavobacterium meningosepticum rather than others.

Amino Acid Sequence↗
PubMed1996

Effects of maternal uninephrectomy on the development of the proximal tubule in fetal rat kidney: morphometrical study.

The kidneys of fetal rats from uninephrectomized mothers were investigated morphometrically on days 18, 20, and 22 of gestation. Maternal uninephrectomy was performed on day 5. On days 20 and 22, the proximal tubular cell height in fetuses from uninephrectomized mothers was significantly lower than that in fetuses from sham-operated mothers. The luminal diameter, length and total volume of the proximal tubule were significantly larger in fetuses from uninephrectomized mothers than in those from sham-operated mothers. The total glomerular volume of mature type in fetuses from uninephrectomized mothers was significantly larger than that in fetuses from sham-operated mothers. On day 18, maternal uninephrectomy caused no significant difference in any kind of parameters used for the proximal tubule, but induced an increase in the total glomerular volume of immature type. These results suggest that maternal uninephrectomy stimulates morphological development of the proximal tubule in the fetal kidney.

Animals↗
PubMed1996

Blood pressure and urinary C-peptide excretion in subjects with varying degrees of glucose tolerance.

To determine whether blood pressure is associated with 24-h urinary C-peptide excretion in subjects with varying degrees of glucose tolerance, we studied 247 Japanese men aged 30-69 who had never been treated with antihypertensive medications or with insulin. Plasma glucose and insulin responses during a 75-g oral glucose tolerance test, blood pressure and body mass index were obtained and urinary C-peptide excretion, in total and per kg body weight, were examined by 24-h urine collection. In monovariate analyses, urinary C-peptide excretion per kg body weight increased significantly as the blood pressure level rose (p < 0.05). After adjustment for age and body mass index by analysis of covariance, this relationship remained significant (p < 0.05), where adjusted mean values (+/- SEM) of urinary C-peptide per kg body weight were 1.56 +/- 0.05 microgram/24h/kg in the normotensive group and 2.04 +/- 0.17 microgram/24h/kg in the hypertensive (stage 2-4) group. When stratified simultaneously by glucose tolerance status and blood pressure level, adjusted mean values of urinary C-peptide per kg body weight were significantly higher in diabetic hypertensives than in diabetic normotensives. These results suggest that increase in 24-h urinary C-peptide excretion, i.e. 24-h insulin secretion, might contribute to an elevation of blood pressure both in normal and diabetic individuals.

Adult↗
PubMed1996

Induction of experimental bone metastasis in mice by transfection of integrin alpha 4 beta 1 into tumor cells.

Cell adhesion receptors (eg, integrins and CD44) play an important role in invasion and metastasis during tumor progression. The increase in integrin alpha 4 beta 1 expression on primary melanomas has been reported to significantly correlate with the development of metastases. alpha 4 beta 1 is a cell surface heterodimer that mediates cell-cell and cell-extracellular matrix interactions through adhesion to vascular cell adhesion molecule (VCAM)-1 and to the IIICS region of fibronectin. To test the effects of alpha 4 beta 1 expression on tumor cell metastasis, Chinese hamster ovary cells were transfected with human alpha 4 cDNA. Whereas alpha 4-negative Chinese hamster ovary cells developed only pulmonary metastasis, alpha 4-positive Chinese hamster ovary cells developed bone and pulmonary metastasis in 3 to 4 weeks when injected intravenously into nude mice. Bone metastasis was inhibited by antibody against alpha 4 or VCAM-1. Expression of alpha 3 beta 1, alpha 6 beta 1, or alpha V beta 1 did not induce bone metastasis. Expression of alpha 4 beta 1 also induced bone metastasis in K562 human erythroleukemia cells injected into SCID mice. These results demonstrate that alpha 4 beta 1 can induce tumor cell trafficking to bone, probably via interaction with VCAM-1 that is constitutively expressed on bone marrow stromal cells.

Animals↗
PubMed1996

Expression of the fibroblast growth factor receptor-1 in human normal tissues and tumors determined by a new monoclonal antibody.

OBJECTIVE: To characterize the distribution of the receptor for the fibroblast growth factor in human normal tissues and tumors using a new monoclonal antibody. DESIGN: Monoclonal antibodies for a kinase-insert region of fibroblast growth factor receptor-1 (FGFR-1) were generated. We conducted an immunohistological analysis of FGFR-1 using a highly specific antibody we generated, and we examined the distribution of this receptor in normal human tissues and in tumors. RESULTS: Intense positivity of FGFR-1 was observed in astrocytes in the brain, smooth muscles in the uterus, cardiac myocytes, respiratory epithelium in the lung, tubular epithelium in the kidney, acinar cells in the pancreas, follicular cells in the thyroid, and ductal and lobular epithelium in the breast. We also observed FGFR-1 expression in the fibroblasts and the tissue microvasculature. In addition to some nonepithelial tumors, some epithelial tumors expressed FGFR-1, including pancreatic adenocarcinomas, thyroid papillary carcinomas, invasive ductal carcinomas of the breast, lung adenocarcinomas, renal cell carcinomas, and colonic adenocarcinomas. Although FGFR-1 was expressed in colonic adenocarcinomas, which have invasive potential, tubular adenomas, which are noninvasive, did not express FGFR-1. CONCLUSION: We have been able to define the distribution of FGFR-1 in human normal tissues and tumors. Especially in colonic tumors, FGFR-1 expression may lead adenoma cells to invade and grow in the surrounding tissue.

Amino Acid Sequence↗
PubMed1996

Applications of retrovirus-mediated expression cloning.

We have recently established a novel expression cloning system using retroviral vectors. The system is based on a high-efficiency packaging cell line, BOSC23, and a simplified retroviral vector, pBabeX, carrying no selection marker. cDNA libraries, constructed in the pBabeX vector, are transiently transfected into BOSC23 cells. The supernatant contains more than 3X10(6)/mL, which would cover large complexities of cDNA libraries. The retrovirus stock gave 100% infection efficiency in NIH3T3 cells and 5-40% infection efficiency in various hematopoietic cell lines. In contrast to the conventional expression cloning system, in which it is necessary to transfect cDNA libraries transiently into particular cell types such as COS cells, retrovirus-mediated expression cloning allows us to transduce cDNAs into a wide variety of cell types. This method therefore makes it possible to select cells expressing a cDNA of interest by various functional assays. When combined with polymerase chain reaction (PCR)-driven random mutagenesis, this system is also useful in searching for mutations of various molecules that will result in alterations of their functions.

3T3 Cells↗
PubMed1996

[A case of genitourinary double cancers detected by health screening].

A 58-year-old male with left renal cell carcinoma and prostatic carcinoma occurring synchronously, is reported. He visited our hospital, because of the high level of serum prostate-specific antigen (PSA) pointed out in a health screening by his company. Prostatic cancer was detected in both lobes of the prostate by needle biopsy specimens and histopathology represented moderately > poorly differentiated adenocarcinoma. Magnetic resonance imaging (MRI) and computed tomography (CT) revealed no cancer invasion beyond the prostate and no lymph node metastasis. Bone scintigram showed no abnormal RI accumulation to bone. Therefore, his prostatic cancer was considered to be at stage B2. Abdominal ultrasound echogram showed the mass lesion in the left kidney. CT and angiogram also demonstrated a left renal tumor. Left radical nephrectomy was performed and histopathology showed a mixed subtype of renal cell carcinoma (stage: pT2b, pN0, pM0). Although 94 cases of double cancers associated with genitourinary organs have been reported in the Japanese literature, only 4 cases of double cancers of renal cell carcinoma and prostatic cancer have been reported.

Adenocarcinoma↗
PubMed1996

[Polyorchidism: a case report and review of the literature].

An 11-year-old boy visited our department complaining of left scrotal painless mass from 6 months ago. The small mass was palpable beside the left testis. Ultrasonography shows isoechoic round shaped mass 5 mm in diameter. At surgery, the mass was connected with the left testis and epididymal head and sharply excised from them. Histological examination showed normal testicular architecture, but spermatogenesis was not found. We report the 18th case of polyorchidism in Japan and made some discussion about polyorchidism.

Child↗
PubMed1996

Prosthetic management for edentulous glossectomy patients.

The prosthetic treatment of edentulous glossectomy patients is very difficult. The tongue defect results in oral dysfunction and affects the stability of the mandibular denture. The appropriate denture form and occlusion can be established by using the modified functional impression technique. Through the use of x-ray television and an image-processing system in clinical experiments, it was shown that a wide buccolingual table, an occlusal table height matched to that of the tongue body, and close adherence of the tongue and the lingual flange are effective means of preventing food from dropping to the oral floor, keeping the food on the occlusal table, and crushing the food.

Denture Bases↗
PubMed1995

Molecular cloning and functional characterization of a novel member of the C-C chemokine family.

Chemokines play an important role in immune and inflammatory responses by inducing migration and adhesion of leukocytes. We have isolated a novel chemokine cDNA, designated CCF18, from a cDNA library of an IL-3-dependent murine pro-B cell line, Ba/F3. The cDNA encodes a protein structurally related to the C-C chemokine members. Among this family, C10 shows the highest homology to CCF18, and MIP-1 alpha also has a significant homology but to a lesser extent. CCF18 produced from COS cells induced chemotaxis and Ca2+ flux in CD4+ T cell clones. Moreover, prior administration of MIP-1 alpha desensitized the cells to CCF18. The CCF18 gene (Scya10) was mapped to a middle region of murine chromosome 11, where other genes for several C-C chemokine members are localized. These results clearly indicate that CCF18 is a new member of the C-C chemokine family. Since a high level of CCF18 mRNA is constitutively expressed in macrophage and myeloid cell lines, CCF18 may play a role in inflammatory processes.

Amino Acid Sequence↗