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Biomedical subjects

S Harada

Publications and source records attributed to S Harada.

At least 235 records · Page 13Linked to original sources
PubMed1996

Orderly disposition of heterogeneous small subunits in D-ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach.

We determined the crystal structure of spinach ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) by x-ray diffraction at 1.8-A resolution and found that the enzyme contained two kinds of S, SI and SII, present in equal number and disposed in an orderly way within the Rubisco holoenzyme. The electron density maps suggested that leucine was at residue 56 in SI, although histidine was at that position in SII. There were other residue differences. Thus, spinach Rubisco has a L8SI4SII4 subunit structure. The orderly disposition of the heterogeneous small subunits in the Rubisco holoenzyme provides accounts of a multigene family of S in plants.

Crystallography, X-Ray↗
PubMed1996

Identification of glycyrrhizin-binding protein kinase as casein kinase II and characterization of its associated phosphate acceptors in mouse liver.

Two forms (G-I and G-II kinases) of casein kinase II(CK-II) in a partially purified CK-II fraction (Mono Q fraction) of mouse liver were separated by means of glycyrrhizin (GL)-affinity column chromatography. Biochemical characterization revealed that these two GL-binding kinases were identical to CK-II. Two phosphate acceptors [p99 (pI 7.0) and p56] copurified with CK-II were identified as ERp99 (Hsp-90-family protein) and calreticulin, respectively. Another protein [p100 (pI 9.0)], which crossreacted with anti-serum against human glucocorticoid receptor (GR), was associated with ERp99. Phosphorylation of p99 [a hetero-complex of p99 (pI 7.0) and p100 (pI 9.0)] and p56 by CK-II in vitro was stimulated significantly by low levels (1-3 microM) of GL, but inhibited significantly at doses above 20 microM. However, no effect of GL on autophosphorylation of ERp99 was detected. The data provided here suggest that GL can regulate CK-II-mediated phosphorylation involved in the GL-induced biological effects in mammalian cells.

Animals↗
PubMed1996

The expression of and insulin binding to cellular thyroid hormone binding protein, but not insulin degrading enzyme, is increased during 3T3-L1 adipocytes differentiation.

Specific insulin binding to several proteins (cytosolic insulin binding proteins; CIBPs) in the isolated cytoplasm of numerous cell types has been demonstrated. CIBPs include insulin degrading enzyme (IDE), CIBP p55 (identified as cellular thyroid hormone binding protein (CTHBP), which is also known as protein disulfide isomerase, or glutathione insulin transhydrogenase). To assess the possible role of CIBP p55/CTHBP in insulin action, we compared 125I-insulin binding to CIBP in cytosol isolated from 3T3-L1 cells at various time points during differentiation of the adipocytes. Insulin did not bind to CTHBP in fibroblasts, but the labeling was markedly increased during adipocyte differentiation. In contrast, insulin binding to IDE did not change during differentiation. Protein expression level of CTHBP in the cytosol fraction increased gradually during the differentiation of adipocytes, whereas that of IDE did not change throughout the period. These data indicate that CTHBP, but not IDE, was up-regulated during differentiation of the adipocytes, suggesting that CIBP p55/CTHBP may play a role in regulating some insulin action, especially the counter regulation between insulin and other hormones during adipocyte differentiation.

3T3 Cells↗
PubMed1996

Advanced glycosylation end products stimulate the growth but inhibit the prostacyclin-producing ability of endothelial cells through interactions with their receptors.

The influence of advanced glycosylation end products (AGE) on endothelial cells was investigated. When human umbilical endothelial cells were cultured with AGE-bovine serum albumin, viable cell number as well as DNA synthesis was significantly stimulated, whereas prostacyclin production by the endothelial cells was decreased. Antisense oligodeoxyribonucleotides against mRNA coding for AGE receptor were found to reverse both the AGE-induced growth stimulation and the inhibition of prostacyclin production in endothelial cells. These results thus suggest that AGE ligand-receptor interactions in endothelial cells can promote angiogenesis and thrombogenesis, leading to the development of diabetic vascular complications.

6-Ketoprostaglandin F1 alpha↗
PubMed1996

Molecular cloning of human eotaxin, an eosinophil-selective CC chemokine, and identification of a specific eosinophil eotaxin receptor, CC chemokine receptor 3.

The CC chemokine eotaxin is a selective chemoattractant for guinea pig eosinophils, first purified from bronchoalveolar lavage fluid in a guinea pig model of allergic airway inflammation. We have now isolated the gene and cDNA for a human counterpart of eotaxin. The gene maps to chromosome 17 and is expressed constitutively at high levels in small intestine and colon, and at lower levels in various other tissues. The deduced mature protein sequence is 66% identical to human monocyte chemoattractant protein-1, and 60% identical to guinea pig eotaxin. Recombinant human eotaxin produced in insect cells induced a calcium flux response in normal human eosinophils, but not in neutrophils or monocytes. The response could not be desensitized by pretreatment of eosinophils with other CC chemokines, suggesting a unique receptor. In this regard, we show that human eotaxin is a potent and highly specific agonist for CC chemokine receptor 3, a G protein-coupled receptor selectively expressed in human eosinophils. Thus eotaxin and CC chemokine receptor 3 may be host factors highly specialized for eosinophil recruitment in inflammation, and may be good targets for the development of selective drugs for inflammatory diseases where eosinophils contribute to pathogenesis, such as asthma.

Amino Acid Sequence↗
PubMed1996

Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.

An efficient method of constructing recombinant adenoviruses (Ads) has been established. The expression unit to be introduced into recombinant Ad was first inserted into the unique Swa I site of the full-length Ad genome cloned in a cassette cosmid. The cassette bearing the expression unit was then cotransfected into human embryonic kidney 293 cells together with the Ad DNA-terminal protein complex digested at several sites with Eco T22I or Ase I/EcoRI. The use of the parent Ad DNA-terminal protein complex instead of the deproteinized Ad genome DNA allowed very efficient recovery of the desired recombinant Ad, and the above restriction digestion drastically reduced regeneration of the parent virus. Several hundred virus clones were readily obtained in each experiment, and about 70% of the clones were the desired recombinant viruses. Furthermore, because the cassette contained the full-length Ad genome, any position of the genome could be easily modified to develop a new vector design. We established construction systems for two types of Ad vectors, the E1-substitution type and the E4-insertion type. This method may greatly facilitate the application of recombinant Ads and should be useful for further improvement of Ad vectors.

Adenovirus E4 Proteins↗
PubMed1996

Dexamethasone inhibits insulin binding to insulin-degrading enzyme and cytosolic insulin-binding protein p82.

We recently demonstrated that insulin specifically binds to several cytosolic insulin-binding proteins (CIBPs) including insulin-degrading enzyme (IDE) and CIBP p82 in cytosol isolated from H35 rat hepatoma cells. Insulin binding to these CIBPs was regulated by culture conditions, such as serum or insulin. In the present study, we examined the effect of dexamethasone on insulin binding to CIBPs in H35 cells. When the cells were treated with 100 nM dexamethasone for 24 hrs, insulin binding to IDE and CIBP p82 was decreased by about 50% without decreasing the expression level of IDE. Insulin added with the dexamethasone prevented the steroid's effect. Furthermore, dexamethasone directly blocked insulin binding to CIBPs in isolated cytosol. These results suggest that dexamethasone, directly or as a complex with other proteins, binds to IDE and CIBP p82 and changes their ability to bind insulin, possibly by inducing a conformational change or by blocking insulin binding sites. IDE was recently identified as a receptor accessory factor for androgen and glucocorticoid receptors and plays an important role in the regulation of gene transcriptional responses. Combined with previous reports, our findings suggest IDE and other CIBPs such as CIBP p82 may play a role in the cross-talk between insulin and the signal transduction pathways of steroid hormones.

Animals↗
PubMed1996

Hepatocyte growth factor in nephronophthisis-medullary cystic disease complex.

A 13-year-old Japanese girl presented with severe anemia and renal dysfunction. The nephronophthisis-medullary cystic disease complex was diagnosed from the results of renal biopsy and a family study. Immunohistochemical detection of hepatocyte growth factor in the epithelial cells of dilated renal tubules suggested that it may have a role in the development of the tubular cystic changes which are characteristic of this disease.

Adolescent↗
PubMed1996

Determination of bioconcentration potential of tetrachloroethylene in marine algae by 13C.

The use of stable isotope of organic-carbon, organic-13C, as a tracer for the determination of the concentration of tetrachloroethylene (PCE), CA, in Heterosigma akashiwo and Skeletonema costatum was examined. CA determined by the 13C and GC methods showed good agreement with each other. This suggests that it is reasonable and reliable to determine the bioconcentration potential of PCE in marine algae. Fitting values of bioconcentration potential parameters, including uptake rate constant k1, elimination rate constant k2 and bioconcentration factor on the basis of dry weight BCFD, were done not only to the time course for PCE uptake by the algae with the bioconcentration model, but also to experimental data for "percent inhibition(%) approximately exposure concentration of PCE approximately time" with the combined bioconcentration and probability model. The values obtained from the bioconcentration model were consistent with those from the combined bioconcentration and probability model. With the parameters (such as k1, k2, growth rate constant kG, critical concentration of HOCs in the organism resulting in growth inhibition CA* and spread factor S) the variability in toxicity (such as EC10, EC50, EC70) can be estimated from the combined bioconcentration and probability model, which fits well with the experimental observations.

Carbon Radioisotopes↗
PubMed1996

Corticotropin-releasing factor up-regulates its own receptor mRNA in the paraventricular nucleus of the hypothalamus.

We investigated the role of CRF in regulating receptor expression in the paraventricular nucleus (PVN). First, to clarify the effect of exogenously administered CRF, 1 microgram of ovine CRF was injected into rat lateral ventricle and changes in concentration of the CRF type 1 receptor (CRF1-R) and CRF mRNA in the PVN were semiquantified after in situ hybridization. Second, we determined the effect of stress, as a stimulant of endogenous CRF secretion, on mRNA accumulation. While CRF1-R mRNA expression was low to be undetectable in the PVN of controls, both intracerebroventricular administration of CRF and restraint significantly increased CRF1-R and CRF signals in the parvocellular PVN. Thus CRF may modulate CRF production and release from the PVN, by regulating CRF1-R expression.

Animals↗
PubMed1996

Trichoblastic fibroma. A case report and an immunohistochemical study of cytokeratin expression.

A 47-year-old woman noticed a nodule on her right shoulder that had been gradually increasing in size without symptoms. Histologic features of the biopsied nodule included round to irregularly shaped epithelial lobules demarcated by abundant sclerotic stroma located within the lower dermis and extending to the subcutis. The epithelial lobules consisted of cuboidal to columnar basaloid cells and were frequently arranged in narrow strands with many bifurcations and branching. Cystic structures containing lamellar keratinous material were occasionally found in connection with the lobules. The histologic findings were interpreted as trichoblastic fibroma. Immunohistochemical studies with antibodies directed against cytokeratins (CK) and involucrin revealed positive staining in most of the tumor cells with RCK102 and 34 beta E12 antikeratin antibodies, whereas the epithelial cords and the peripheral cells of the cystic structures stained with 170.2.14, 4.1.18, and CAM 5.2 antikeratin antibodies. However, CK1 or simple epithelial cytokeratins were not detected in any neoplastic elements. Based on comparative immunohistochemical findings in normal hair follicles, we propose that trichoblastic fibroma may first differentiate toward the outermost cell layer of the outer root sheath between the lower permanent portion and the upper transient portion and then into various other parts of the hair follicle.

Epithelium↗
PubMed1996

Retention of HIV-1 inside infected MOLT-4 cells in association with adhesion-induced cytoskeleton reorganization.

OBJECTIVE: To study the mechanism of the suppression of HIV release during cell-to-cell adhesion. DESIGN AND METHODS: To investigate the effects of cell-to-cell adhesion on HIV release in association with cytoskeletal elements, chronically HIV-infected T cells were cocultured with different adherent cell lines, cultured on a fibronectin-coated surface, or treated with cytochalasin D. The amount of viral protein released in the culture supernatant and retained inside the cells was monitored by a p24 enzyme-linked immunosorbent assay and Western blotting. For F-actin staining, cells were stained with FITC-labelled phalloidine and examined by immunofluorescence microscopy. RESULTS: Cocultivation resulted in a reduced amount of virus in the culture supernatant and induced the retention of viral protein inside the infected cells. On adhesion to cells, the F-actin of the infected cells was polarized towards the cell periphery from a diffuse cytoplasmic distribution. Similar data were obtained when the infected cells were treated with cytochalasin D or adhered to fibronectin. CONCLUSION: Cell-to-cell adhesion induced polarization of F-actin, which might facilitate HIV retention inside infected T cells.

Actins↗
PubMed1996

The N-terminal half of EBNA2, except for seven prolines, is not essential for primary B-lymphocyte growth transformation.

Previous molecular genetic analyses of Epstein-Barr virus nuclear protein 2 (EBNA2) identified a negative effect of deletion of codons 19 to 33 on transformation and gene transactivation, while deletion of codons 19 to 110 was a null mutation for transformation and gene transactivation. We here report the surprising finding that codons 2 to 88, which encode the highly conserved unique N terminus (amino acids 1 to 58) and most of the polyproline repeat (amino acids 59 to 95), can be deleted with only minimal effects on transformation. Codons 97 to 122 can also be deleted with only minimal effects on transformation. However, deletion of 35 of the 37 prolines (amino acids 59 to 93) or deletion of codons 2 to 95 results in a null transforming phenotype. Although EBNA2 from which codons 59 to 93 were deleted was a null mutation for transformation, it was similar to some transforming mutants of EBNA2 in abundance, in interaction with RBPJK, and in transactivation of the LMP1 promoter in transient transfection assays. These data indicate that between three and seven prolines are critical for EBNA2 structure or for intermolecular interaction. Aside from these seven prolines, codons encoding the rest of the N-terminal half (amino acids 2 to 230) of EBNA2 are nonessential for primary B-lymphocyte growth transformation.

Antigens, Viral↗
PubMed1996

Plasma thrombomodulin: usefulness as a blood access failure marker in hemodialysis patients.

In 144 patients on hemodialysis (76 males and 68 females, median age 55.7 +/- 14.1 years, mean period on dialysis 44.1 +/- 33.3 months), thrombomodulin was determined by enzyme immunoassay prior to initiation of hemodialysis. The results showed that the mean thrombomodulin value of hemodialysis patients was 13.59 +/- 3.63 ng/ml which was significantly higher than the control value (3.20 +/- 0.90 ng/ml). The thrombomodulin values were significantly higher in patients with blood access failure (15.27 +/- 4.45 ng/ml) than in those without (13.11 +/- 3.31 ng/ml), and the rate of blood access failures was also significantly higher in those with thrombomodulin values of 15.0 ng/ml or higher than in those with values < 15.0 ng/ml. It was evident that there is a higher risk of blood access failure in patients with severe systemic vascular endothelial injury, and thrombomodulin is a useful marker of such an injury.

Adult↗
PubMed1996

Different expression of immediate-early genes in the rat paraventricular nucleus induced by stress: relation to corticotropin-releasing factor gene transcription.

Stressful stimuli induce the transcriptional activation of the corticotropin-releasing factor (CRF) gene as well as that of various immediate-early genes (IEGs). Among them, members of the fos/jun families play an important role in the regulation of gene expression. The possible relation between stress-induced IEG expression and CRF gene transcription was investigated by analysis of stress-induced changes in the abundance of c-fos, c-jun, jun-B, jun-D, and NGFI-B mRNA as well as CRF heteronuclear RNA (hnRNA), which reflects gene transcription, in the rat paraventricular nucleus (PVN) by in situ hybridization. Restraint stress induced rapid (within 5 min) and transient increases in the level of c-fos mRNA and CRF hnRNA in the PVN, with peak expression apparent 30 min after the onset of stress. The induction of jun-B and NGFI-B gene expression was also rapid and transient, but was delayed relative to that of c-fos mRNA and the CRF hnRNA. In contrast, both jun-D and c-jun were constitutively expressed in the PVN, and the amount of the corresponding transcripts increased only slightly in response to stress. These results reveal differential patterns of expression of IEGs in the PVN in response to stress. The observation that the level of mRNAs corresponding to these IEGs did not increase before that of CRF hnRNA may suggest that the products of these genes do not seem to directly mediate the stress-induced increase in CRF gene transcription in the PVN.

Animals↗
PubMed1996

Cerebellar abscess associated with pulmonary arteriovenous fistula and hereditary hemorrhagic telangiectasia--case report.

A 57-year-old male with a past history of bilateral pulmonary arteriovenous fistulas (PAVFs) experienced a sudden onset of headache and gait disturbance. There was a family history of PAVF and recurrent epistaxis. He had diffuse telangiectasia of the tongue, and hereditary hemorrhagic telangiectasia was diagnosed. Neuroimaging revealed a brain abscess in the right cerebellar hemisphere, which was successfully aspirated under ultrasound guidance. The PAVFs were resected afterwards in two-staged operation. No recurrence of the abscess has been observed. Cerebral abscesses complicated by PAVF are usually supratentorial. Complete eradication of PAVF is essential because the brain abscess will sometimes recur if the PAVF is left untreated.

Arteriovenous Fistula↗
PubMed1996

Intracellular ionic changes induced by bullous pemphigoid IgG subclasses.

To ascertain whether membrane signal transduction is induced by bullous pemphigoid (BP) antibody and whether cell lysis is induced by its complement activation, we assessed the intracellular Ca2+ concentration ([Ca2+]i), intracellular pH, membrane potential and morphology of living cells by following the time course of fluorescence intensity of Fluo-3/AM, Snaff-1/AM, Dioc-5 and Luciffer yellow, respectively. A transient increase of Fluo-3 fluorescence intensity in DJM-1 cells (a squamous cell carcinoma line) was revealed when the cells were incubated with 2 of five IgG1 BP antibodies. However, no transient increase of Fluo-3 fluorescence intensity was revealed when the cells were incubated with IgG2 and IgG4 BP antibodies. A transient increase of Fluo-3 fluorescence intensity was revealed in DJM-1 cells incubated with 3 of seven IgG1 and 1 of four IgG2 BP antibodies in an EGTA-containing low-Ca2+ medium. On the other hand, the Dioc-5 fluorescence intensity did not change significantly, though the increase of Fluo-3 fluorescence intensity was observed. The increase of Snarf-1 fluorescence intensity was revealed in DJM-1 cells incubated with 2 of five IgG1 BP antibodies, but was not revealed in the cells incubated with IgG2 or IgG4 of BP antibodies. Study of complement activation by BP IgG1 showed a transient increase of Fluo-3 fluorescence intensity of with 3 of five IgG1 BP antibodies when DJM-1 cells were incubated with complement-supplemented normal-Ca2+ medium. At the same time, however, endocytosis and cell lysis were not observed with 2 IgG1 BP antibodies which did induce an increase of Fluo-3 fluorescence intensity when Lucifer-yellow-loaded DJM-1 cells were incubated with complement-supplemented normal-Ca2+ medium. We examined next whether anti-180 kD BP antigen monoclonal antibodies (mAbs R-223 and 233) induce an increase of Fluo-3 fluorescence intensity. MAb R-223 did not induce any increase of Fluo-3 fluorescence intensity in DJM-1 cells, when incubated with normal- and low-Ca2+ media However, mAb R-223 induced a transient increase of Fluo-3 fluorescence intensity in DJM-1 cells when incubated with complement-supplemented normal-Ca2+ medium. MAb 233 did not induced an increase of Fluo-3 fluorescence intensity in DJM-1 cells when incubated with normal- and low-Ca2+ media. These results suggest that the BP IgG1 induces Ca2+ release from intracellular storage sites, however, the complement activated by BP IgG1 does not induce cell lysis. It could not be confirmed that anti-180 kD BP antigen antibody induced Ca2+ release from intracellular storage sites.

Calcium↗