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N Yoshikawa

Publications and source records attributed to N Yoshikawa.

At least 19 recordsLinked to original sources
PubMed2000

FK506 augments glucocorticoid-mediated cyclooxygenase-2 down-regulation in human rheumatoid synovial fibroblasts.

Prostaglandins (PG) formed by cyclooxygenase (COX) enzymes are important mediators of inflammation in rheumatoid arthritis. The contribution of the inducible COX-2 to inflammation in the rheumatoid synovium is well documented. We examined the regulation of COX-2 mRNA and protein expression in response to both glucocorticoids (GC) and FK506 using rheumatoid synovial fibroblasts. Combined treatment of FK506 and a low concentration of dexamethasone (DEX) (10(-9) M) down-regulated synovial COX-2 mRNA and protein expression. In contrast, neither FK506 nor DEX (10(-9) M) alone influenced COX-2 expression. Immunocytochemical studies showed that pretreatment with FK506 enhanced the nuclear translocation of the glucocorticoid receptor (GR) in synovial fibroblasts in the presence of low concentrations of DEX (10(-9) M). Transient transfection experiments showed that treatment of cells with FK506 enhanced the expression of glucocorticoid-responsive gene reporter in the presence of DEX (10(-9) M). NF-kappaB is known to mediate the transcriptional activation of the COX-2 gene. Electrophoretic mobility shift assay demonstrated that DNA-binding activity of NF-KB was suppressed more profoundly by FK506 plus DEX (10(-9) M) treatment with those of DEX (10(-9)M) alone in IL-1beta-stimulated synovial cells. Our results indicated that FK506-induced potentiation of GR-mediated repression of synovial COX-2 gene transcription is the result of increased translocation of GR to the nucleus and subsequent repression of NF-kappaB transactivation. Our results also suggest that FK506 may exert anti-inflammatory effects in the rheumatoid synovium by potentiating GR-mediated signal transduction.

Arthritis, Rheumatoid↗
PubMed2000

A multicenter trial of mizoribine compared with placebo in children with frequently relapsing nephrotic syndrome.

BACKGROUND: The use of corticosteroids or cytotoxic/immunosuppressive agents such as cyclophosphamide, chlorambucil, and cyclosporine for the treatment of frequently relapsing nephrotic syndrome (FRNS) is limited because of their adverse effects. This study was conducted to evaluate the efficacy and safety of mizoribine, a relatively new immunosuppressive drug developed in Japan, in children with FRNS. METHODS: A double-blind, placebo-controlled, multicenter trial was carried out in children, from 2 to 19 years old, with FRNS. At relapse, patients were treated with prednisolone. According to a dynamic allocation, mizoribine or a placebo was concurrently administered to each patient. Prednisolone was gradually tapered and discontinued within 12 weeks. The test drug was maintained for 48 weeks. The primary end point was the relapse rate (the total number of relapses/the total treatment days for all patients). Analyses were performed according to the intention-to-treat principle. RESULTS: The primary analysis was conducted on 99 mizoribine- and 98 placebo-treated patients. The relapse rate was lower in the mizoribine group than in the placebo group (0.0055 vs. 0.0067; ratio 0.81, 95% CI, 0.61 to 1.05, P = 0.12). The hazard ratio of the cumulative remission rate between the two groups was 0.79 (95% CI, 0. 57 to 1.08). In the subgroups consisting of patients 10 years old or younger, the relapse rate ratio between the mizoribine subgroup (54 patients) and the placebo subgroup (57 patients) was 0.66 (95% CI, 0. 44 to 0.94, P = 0.017). The hazard ratio of the cumulative remission rate between the two subgroups was 0.56 (95% CI, 0.37 to 0.85, P = 0. 007). Hyperuricemia was the most common adverse event with mizoribine (16%), but was transient. CONCLUSIONS: Compared with the placebo, mizoribine significantly decreased the relapse rate and prolonged the remission period in the subgroup consisting of patients 10 years old or younger. This drug may be useful in young children with FRNS who generally relapse more frequently than older children.

Adolescent↗
PubMed2000

Platelet-activating factor acetylhydrolase gene mutation in Japanese children with Escherichia coli O157-associated hemolytic uremic syndrome.

Platelet-activating factor (PAF) may be involved in the pathogenesis of Escherichia coli O157-associated hemolytic uremic syndrome (HUS). PAF is degraded to inactive products by PAF acetylhydrolase. In this study, we investigated whether a PAF acetylhydrolase gene mutation (G-->T transversion at position 994) is involved in HUS in Japanese children. A point mutation in the PAF acetylhydrolase gene (G994T) was identified using polymerase chain reaction in 50 Japanese children with E coli O157-associated HUS and 100 healthy Japanese. We then determined the relationship between the PAF acetylhydrolase G994T gene mutation and clinical features of HUS. There was no difference in genotype and allele frequencies between patients with HUS and healthy controls. The mean duration of oligoanuria was significantly longer in patients with the GT genotype than in those with the GG genotype (P = 0.012). Although 11 of 15 patients (73%) heterozygous for the mutant allele (GT) required dialysis, only 13 of the 35 wild-type homozygotes (GG; 37%) required dialysis (P = 0. 030). Mean plasma PAF acetylhydrolase activity was significantly less in patients with the GT genotype than in those with the GG genotype (P < 0.0001). In conclusion, we have shown an association between the G994T PAF acetylhydrolase gene mutation and the severity of renal damage in E coli O157-associated HUS. Our study suggests that analysis of the PAF acetylhydrolase gene mutation in Japanese children with E coli O157-associated HUS may allow the prediction of the severity of HUS.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed2000

Haemophilus parainfluenzae antigen and antibody in children with IgA nephropathy and Henoch-Schönlein nephritis.

Although the pathogenesis of immunoglobulin A (IgA) nephropathy and Henoch-Schönlein nephritis (HSN) remains uncertain, there is substantial evidence that they are immune complex-mediated diseases. Recently, Haemophilus parainfluenzae antigens were shown in the glomerular mesangium of adult patients with IgA nephropathy, and greater levels of IgA antibody against H parainfluenzae were also shown in the sera of adult patients with IgA nephropathy. The present study was performed to detect H parainfluenzae antigens and antibody against H parainfluenzae in children with IgA nephropathy and HSN. H parainfluenzae antigens in the mesangium were examined by indirect immunofluorescence, and antibody against H parainfluenzae was examined by enzyme-linked immunosorbent assay. Diffuse global staining of the mesangium with rabbit antisera against H parainfluenzae was shown in 10 of the 32 patients (31%) with IgA nephropathy and 12 of the 34 patients (35%) with HSN. Conversely, only 2 of the 47 patients (4%) with other renal diseases showed staining of glomeruli with rabbit antisera against H parainfluenzae (IgA nephropathy versus other renal diseases, P = 0.003; HSN versus other renal diseases, P = 0.0006). Patients with IgA nephropathy and those with HSN showed significantly greater levels of plasma IgA1 antibody against H parainfluenzae than patients with other renal diseases (IgA nephropathy versus other renal diseases, P = 0.008; HSN versus other renal diseases, P = 0.025). These findings suggest that H parainfluenzae has a role in the cause of these two conditions in a subset of patients.

Adolescent↗
PubMed2000

Nucleotide sequence and genome organization of apple latent spherical virus: a new virus classified into the family Comoviridae.

A virus with isometric virus particles (ca. 25 nm) was isolated from an apple tree and named Apple latent spherical virus (ALSV). Virus particles purified from infected Chenopodium quinoa formed two bands with densities of 1.41 and 1.43 g/cm(3) in CsCl equilibrium density-gradient centrifugation, indicating that the virus is composed of two components. The virus had two ssRNA species (RNA1 and RNA2) and three capsid proteins (Vp25, Vp24 and Vp20). The complete nucleotide sequences of RNA1 and RNA2 were determined to be 6815 nt and 3384 nt excluding the 3' poly(A) tail, respectively. RNA1 contains two partially overlapping ORFs encoding polypeptides of molecular mass 23 kDa ('23K'; ORF1) and 235 kDa ('235K'; ORF2); RNA2 has a single ORF encoding a polypeptide of 108 kDa ('108K'). The 235K protein has, in order, consensus motifs of the protease cofactor, the NTP-binding helicase, the cysteine protease and the RNA polymerase, in good agreement with the gene arrangement of viruses in the COMOVIRIDAE: The 108K protein contains an LPL movement protein (MP) motif near the N terminus. Direct sequencing of the N-terminal amino acids of the three capsid proteins showed that Vp25, Vp20 and Vp24 are located in this order in the C-terminal region of the 108K protein. The cleavage sites of the 108K polyprotein were Q/G (MP/Vp25 and Vp25/Vp20) and E/G (Vp20/Vp24). Phylogenetic analysis of the ALSV RNA polymerase domain showed that ALSV falls into a cluster different from the nepo-, como- and fabavirus lineages.

Amino Acid Sequence↗
PubMed2000

Spontaneous and cytokine regulated c-fos gene expression in rheumatoid synovial cells: resistance to cytokine stimulation when the c-fos gene is overexpressed.

OBJECTIVE: To study the effect of cytokines on the transactivation of the c-fos gene in relation to the contribution of overexpression of c-fos/AP-1 in rheumatoid joint destruction. METHODS: The promoter region (-447 to +109) of the human c-fos gene was integrated upstream of the chloramphenicol acetyltransferase (CAT) reporter gene, and the effect of cytokines on the expression of the c-fos gene was studied in the rheumatoid synovial cells of early (3-4) or late (14-18) passages, in the presence or absence of cytokines, by the transient transfection assay. RESULTS: Expression of c-fos gene was enhanced by tumour necrosis factor alpha (TNF alpha) and interleukin 6 (IL6) in the synovial cells of early passage, whereas it was not enhanced in the synovial cells of late passage. The c-fos gene expression was also enhanced by 13-O-tetradecanoyl phorbol-13-acetate (TPA) in early passage but was somewhat suppressed in the late passage. It was found that the c-fos gene and c-Fos protein were both increased in the synovial cells of late passage. Similarly, c-fos gene expression was also not increased by TPA or cytokine stimulation in the stable c-fos transformants (fos-pH8) or H-ras transformed NIH3T3 cells (NIH H-ras cells) that constitutively expressed c-fos genes. CONCLUSIONS: Although TNF alpha and IL6 augmented c-fos gene expression of rheumatoid synovial cells, transactivation of c-fos gene became resistant against cytokine stimulation under prolonged expression of c-fos gene, which may impart a tumour-like characteristic to rheumatoid synovial cells.

Arthritis, Rheumatoid↗
PubMed2000

The RING finger protein SNURF modulates nuclear trafficking of the androgen receptor.

The androgen receptor (AR) is a transcription factor that mediates androgen action. We have used the green fluorescent protein (GFP) technique to investigate dynamics of nuclear trafficking of human AR in living cells. In the absence of ligand, the GFP-AR fusion protein is distributed between cytoplasm and nuclei. Androgen exposure leads to a rapid and complete import of GFP-AR to nuclei of CV-1 cells (>=90% nuclear in 30 minutes), whereas a pure antiandrogen, Casodex, elicits a slower (<40% nuclear in 30 minutes) and incomplete transfer. Unliganded ARs with mutations in the basic amino acids of the bipartite nuclear localization signal (NLS) within the second zinc finger and the hinge region are predominantly cytoplasmic and their androgen-dependent nuclear import is severely compromised ((3/4)20% nuclear in 30 minutes). Interestingly, substitutions of the Leu residues flanking the bipartite NLS lead to inefficient nuclear transfer in response to androgen ((3/4)20% nuclear in 30 minutes). The ligand-binding domain of AR, which represses bipartite NLS activity, contains an agonist-specific NLS. The small nuclear RING finger protein SNURF, which interacts with AR through a region overlapping with the bipartite NLS, facilitates AR import to nuclei and retards its export on hormone withdrawal. More AR is associated with the nuclear matrix in the presence than absence of coexpressed SNURF. We suggest that the SNURF-mediated tethering of AR in nuclei represents a novel mechanism for activating steroid receptor functions.

Amino Acid Sequence↗
PubMed2000

Genetics of immunoglobulin A nephropathy.

INTRODUCTION: Immunoglobulin (Ig) A nephropathy is the most common primary glomerulonephritis in the world and about 20% to 50% of patients with it develop progressive renal failure. There is considerable evidence to show that IgA nephropathy is influenced by genetic factors. The purpose of this review is to provide useful information concerning genetics of IgA nephropathy. METHODS AND RESULTS: Epidemiological, familial clustering, human leukocyte antigen and IgA immune system (immunoglobulin class switch gene, I alpha 1 germ-line transcript regulatory region gene) studies have led to the hypothesis of genetic susceptibility to IgA nephropathy. Moreover, research on renin angiotensin system, platelet activating factor acetylhydrolase, neuropeptide Y Y1 receptor and others genes has demonstrated that genetic factors influence the pathological severity and natural course of IgA nephropathy. CONCLUSIONS: The evidence presented in this review strongly supports the role of genetic factors in IgA nephropathy. Detection of genetic risk factors for IgA nephropathy will allow us to study further the pathogenesis of IgA nephropathy and devise effective therapy.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1999

Expression, abundance, and RNA polymerase binding properties of the delta factor of Bacillus subtilis.

The delta protein is a dispensable subunit of Bacillus subtilis RNA polymerase (RNAP) that has major effects on the biochemical properties of the purified enzyme. In the presence of delta, RNAP displays an increased specificity of transcription, a decreased affinity for nucleic acids, and an increased efficiency of RNA synthesis because of enhanced recycling. Despite these profound effects, a strain containing a deletion of the delta gene (rpoE) is viable and shows no major alterations in gene expression. Quantitative immunoblotting experiments demonstrate that delta is present in molar excess relative to RNAP in both vegetative cells and spores. Expression of rpoE initiates from a single, sigmaA-dependent promoter and is maximal in transition phase. A rpoE mutant strain has an altered morphology and is delayed in the exit from stationary phase. For biochemical analyses we have created derivatives of delta and sigmaA that can be radiolabeled with protein kinase A. Using electrophoretic mobility shift assays, we demonstrate that delta binds core RNAP with an apparent affinity of 2.5 x 10(6) M-1, but we are unable to demonstrate the formation of a ternary complex containing core enzyme, delta, and sigmaA.

Bacillus subtilis↗
PubMed1999

Redox-dependent regulation of nuclear import of the glucocorticoid receptor.

A number of transcription factors including the glucocorticoid receptor (GR) are regulated in a redox-dependent fashion. We have previously reported that the functional activity of the GR is suppressed under oxidative conditions and restored in the presence of reducing reagents. In the present study, we have used a chimeric human GR fused to the Aequorea green fluorescent protein and demonstrated that both ligand-dependent and -independent nuclear translocation of the GR is impaired under oxidative conditions in living cells. Substitution of Cys-481 for Ser within NL1 of the human GR resulted in reduction of sensitivity to oxidative treatment, strongly indicating that Cys-481 is one of the target amino acids for redox regulation of the receptor. Taken together, we may conclude that redox-dependent regulation of nuclear translocation of the GR constitutes an important mechanism for modulation of glucocorticoid-dependent signal transduction.

Amino Acid Sequence↗
PubMed1999

Direct association with thioredoxin allows redox regulation of glucocorticoid receptor function.

The glucocorticoid receptor (GR) is considered to belong to a class of transcription factors, the functions of which are exposed to redox regulation. We have recently demonstrated that thioredoxin (TRX), a cellular reducing catalyst, plays an important role in restoration of GR function in vivo under oxidative conditions. Although both the ligand binding domain and other domains of the GR have been suggested to be modulated by TRX, the molecular mechanism of the interaction is largely unknown. In the present study, we hypothesized that the DNA binding domain (DBD) of the GR, which is highly conserved among the nuclear receptors, is also responsible for communication with TRX in vivo. Mammalian two-hybrid assay and glutathione S-transferase pull-down assay revealed the direct association between TRX and the GR DBD. Moreover, analysis of subcellular localization of TRX and the chimeric protein harboring herpes simplex viral protein 16 transactivation domain and the GR DBD indicated that the interaction might take place in the nucleus under oxidative conditions. Together these observations indicate that TRX, via a direct association with the conserved DBD motif, may represent a key mediator operating in interplay between cellular redox signaling and nuclear receptor-mediated signal transduction.

Animals↗
PubMed1999

The molecular mechanism of inhibition of interleukin-1beta-induced cyclooxygenase-2 expression in human synovial cells by Tripterygium wilfordii Hook F extract.

OBJECTIVE: Several extracts of Tripterygium wilfordii Hook F (TWHF) have been reported to be effective in patients with rheumatoid arthritis. We investigated the effect of multi-glycosides ofTWHF (GTW), a TWHF extract, on interleukin (IL)-1beta stimulated human rheumatoid synovial cells. MATERIALS AND METHODS: IL-1beta-stimulated synovial cells were used to detect the effects of GTW on cyclooxygenase (COX)-1 and COX-2 activities, expression of COX protein and mRNA, and nuclear transcription factors in experiments using respective reporter plasmids. RESULTS: GTW inhibited prostaglandin E2 production by IL-1beta-stimulated synovial cells in a concentration-dependent manner, and also inhibited COX-2 protein and mRNA expression in a similar fashion to dexamethasone. However, GTW did not act as a glucocorticoid agonist. GTW repressed IL-1beta-induced nuclear factor-kappaB activity, but did not have a significant influence on activating protein-1 activity. CONCLUSION: The anti-rheumatic effect of GTW or TWHF may be partly mediated through the inhibition of prostaglandin E2 production in human synovial cells due to suppression of COX-2 mRNA, possibly via inhibition of nuclear factor-kappaB activity.

Animals↗
PubMed1999

Two-year cyclosporin treatment in children with steroid-dependent nephrotic syndrome.

We describe a prospective study of 2-year moderate-dose cyclosporin (CS) treatment in 13 children with steroid-dependent minimal change nephrotic syndrome (MCNS). CS treatment was commenced at 100-150 mg/m2 per day after remission was attained with prednisolone therapy, was adjusted to a target trough level of 100 ng/ml, and was administered for 2 years. The number of relapses during CS treatment significantly decreased compared with before CS treatment, all patients were able to discontinue prednisolone therapy, and steroid toxicity was reduced; 54% of patients remained in remission during CS treatment. Renal biopsies performed before CS treatment all showed MCNS without tubulointerstitial lesions. Creatinine clearance and urinary beta 2-microglobulin levels during CS treatment were normal in all patients, but renal biopsies performed after CS treatment revealed chronic CS nephrotoxicity in 7 patients. Clinical data, including CS dose and CS trough blood levels, were not significantly different between patients with and without nephrotoxicity. In conclusion, 2-year moderate-dose CS treatment in children with steroid-dependent MCNS is effective in preventing relapse and decreasing steroid toxicity. This treatment can, however, result in a high incidence of chronic nephrotoxicity. Renal function is not a reliable indicator of chronic CS nephrotoxicity. Renal biopsy is therefore necessary to monitor chronic CS nephrotoxicity.

Adolescent↗
PubMed1999

Relationship between Helicobacter pylori infection and histologic features of gastritis in biopsy specimens in gastroduodenal diseases, including evaluation of diagnosis by polymerase chain reaction assay.

We investigated the relationship between Helicobacter pylori infection and the histologic features of gastritis in gastroduodenal disease, and evaluated the diagnostic usefulness of the polymerase chain reaction (PCR) assay for the detection of H. pylori before and after eradication therapy. Endoscopic biopsy specimens from 81 patients with gastroduodenal disease were examined for the presence of H. pylori by culture and histologic examination. Histologic features of gastritis were classified according to the updated Sydney System, and results of the PCR assay were compared with those of histologic examination, using histologic scores. The density of H. pylori was significantly correlated with polymorphonuclear neutrophil activity and chronic inflammation. These findings suggest that the grades of infiltration of polymorphonuclear neutrophil cells and chronic inflammatory cells correspond to the density of H. pylori infection assessed by the updated Sydney System. Patients with positive results on PCR assay and negative results on histologic examination may have a low density of H. pylori because of severe atrophy in the gastric mucosa. Differences in results for the PCR assay and histologic examination were found in 2 of 12 patients in the detection of H. pylori after eradication therapy. According to the results of the PCR assay and histologic features before and after eradication, the gastric tissue-based PCR assay for H. pylori after eradication may be too sensitive to judge successful eradication of H. pylori.

Adolescent↗
PubMed1999

Apple chlorotic leaf spot virus 50 kDa protein is targeted to plasmodesmata and accumulates in sieve elements in transgenic plant leaves.

We investigated the in situ localization of the 50 kDa protein encoded by ORF2 of Apple chlorotic leaf spot virus (ACLSV) genome which is thought to be a movement protein. In immunogold electron microscopy of ACLSV-infected Chenopodium quinoa leaves, the 50 kDa protein was localized on plasmodesmata and nearby cytoplasm. Observation of transgenic Nicotiana occidentalis leaves expressing the 50 kDa protein fused to enhanced green fluorescent protein (EGFP) by fluorescence and confocal laser scanning microscopes revealed that green fluorescence was observed as spots on the cell wall or strands passing through the cell wall of several cell types, i.e., epidermal, palisade and spongy mesophyll and collenchyma cells. In transverse and longitudinal sections of leaf veins of transgenic plants showed that the 50K-EGFP fusion accumulated in sieve elements and formed an extensive interconnecting network of threadlike structure. These results indicated that ACLSV 50 kDa protein can target plasmodesmata and traffic into sieve elements.

Chenopodiaceae↗
PubMed1999

Gradients in cytoplasmic calcium concentration ([Ca2+]i) in migrating human umbilical vein endothelial cells (HUVECs) stimulated by shear-stress.

Using a parallel-plate flow-chamber and confocal laser scanning microscopy (CLSM), we studied the distribution and temporal changes in intracellular Ca2+ concentration ([Ca2+]i) in migrating HUVECs stimulated by shear-stress. In the presence or absence of ATP, shear-stress (10 dyne/cm2) caused morphological change and migration of individual HUVECs in the random direction. After 120 minute exposure to shear-stress, 70% of the cells migrated in the direction of flow, whereas, as many as 30% of the cells migrated to the upstream against flow. A nonspecific plasma membrane Ca2+ channel blocker, Ni2+, abolished such responses markedly, suggesting that Ca2+ influx may be essential for shear-stress dependent morphological change and migration of HUVECs. Analysis of [Ca2+]i distribution revealed the appearance of localized [Ca2+]i elevation inside lamellipodium formed in the direction of cell migration. The localized rise in [Ca2+]i might be closely related with morphological change to regulate the direction of cell migration induced by shear-stress.

Adenosine Triphosphate↗
PubMed1999

Detection of mutations in the COL4A5 gene in over 90% of male patients with X-linked Alport's syndrome by RT-PCR and direct sequencing.

X-linked Alport's syndrome is caused by mutations in the COL4A5 gene encoding the type IV collagen alpha5 chain (alpha5[IV]). Polymerase chain reaction-single-str and conformation polymorphism (PCR-SSCP) on genomic DNA has previously been used to screen for mutations in the COL4A5 gene, but this method was relatively insensitive, with mutations detected in less than 50% of patients. Here, we report a systematic analysis of the entire coding region of the COL4A5 gene, using nested reverse-transcription-polymerase chain reaction (RT-PCR) and the direct sequence method using leukocytes. This study examines twenty-two unrelated Japanese patients with X-linked Alport's syndrome showing abnormal expression of alpha5(IV) in the glomerular or epidermal basement membranes. Mutations that were predicted to be pathogenic were identified in 12 of the 13 male patients (92%) and five of the nine female patients (56%). Six patients had missense mutations, four had out-of-frame deletion mutations, three had nonsense mutations, and three had mutations causing exon loss of the transcript. The current study shows that nested RT-PCR and the direct sequence method using leukocytes are highly sensitive and offer a useful approach for systematic gene analysis in patients with X-linked Alport's syndrome.

Adolescent↗