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Biomedical subjects

M Shirai

Publications and source records attributed to M Shirai.

At least 19 recordsLinked to original sources
PubMed1999

Molecular cloning of a novel 130-kDa cytoplasmic protein, Ankhzn, containing Ankyrin repeats hooked to a zinc finger motif.

A novel gene was trapped in mouse embryonic stem cells with a promoterless gene trap vector. Fused transcripts were isolated from the embryos by rapid amplification of cDNA ends, which were used for full-length cDNA cloning. The protein predicted from the cDNA consisting of 7143 nucleotides comprises 1184 amino acids, which was confirmed by in vitro transcription/translation assaying. An antibody against the synthesized peptide reacted with an approximate 130-kDa protein on SDS-PAGE. A search of available databases revealed that this protein is a novel protein composed of 17 ankyrin repeats hooked to a zinc finger motif, which we named Ankhzn. Ankhzn was observed on the endosomal membrane on immunoelectron microscopic analysis. Ankhzn belongs to a new subgroup of double zinc finger proteins which may be involved in vesicle or protein transport. Ankhzn mRNA and its protein were expressed ubiquitously from embryonic day 10.5 to adulthood.

Amino Acid Sequence↗
PubMed1999

Light-dependent and rhythmic psbA transcripts in homologous/heterologous cyanobacterial cells.

The psbA2 gene exhibits light-dependent and rhythmic expression in a unicellular cyanobacterium, Microcystis aeruginosa (Synechocystis) K-81. To further understand the psbA2 expression, biological analyses were performed in homologous and heterologous cyanobacterial cells. The results of the experiments using the K-81 cells revealed that (i) the light-dependent expression appeared on transcriptional and/or post-transcriptional level(s) under light/dark cycles, (ii) circadian-rhythmic transcripts were also observed under the control of an endogenous clock. To assess whether light-dependent and rhythmic psbA2 expression occurs in heterologous cyanobacterium, Synechococcus sp. strain PCC 7942, the K-81 psbA2 5'-upstream region of which the promoter and its around sequences share with those of PCC 7942 psbAII, was fused to the bacterial lacZ reporter gene, introduced into the genome of PCC 7942 and the psbA2 transcripts were directly investigated by primer extension. The K-81 psbA2 specific transcripts were also light-dependent and rhythmic in PCC 7942, strongly demonstrating that a common regulatory mechanism exists per se for the psbA2 expression in both strains. Furthermore, psbA2 expression in the recombinant PCC 7942 strain, AG400 in which the region from -404 to +111 of psbA2 is fused to lacZ, exhibited clear rhythmicity, while very little or no rhythmicity was observed in AG429 (-38 to +14, the only promoter region), suggesting that the region(s) around the promoter was essentially required for clear rhythmic expression.

Cyanobacteria↗
PubMed1999

T cell recognition of hypervariable region-1 from hepatitis C virus envelope protein with multiple class II MHC molecules in mice and humans: preferential help for induction of antibodies to the hypervariable region.

Hypervariable region-1 (HVR1) from the hepatitis C virus (HCV) envelope protein is thought to be a target for neutralizing Abs. To explore HVR1 recognition by helper T cells, and their role in Ab responses, we attempted to generate helper T cells specific for HVR1 in mice of three MHC types, and with PBMC from HCV-infected HLA-diverse humans. In both species, HVR1 was presented by >1 class II MHC molecule to CD4+ helper T cells and showed surprising interisolate cross-reactivity. The epitope for two DR4+ patients was mapped to a more conserved C-terminal sequence containing a DR4 binding motif, possibly accounting for cross-reactivity. Strikingly, Abs to patients' own HVR1 sequences were found only in patients with T cell responses to HVR1, even though all had Abs to envelope protein, suggesting that induction of Abs to HVR1 depends on helper T cells specific for a sequence proximal to the Ab epitope. Thus, helper T cells specific for HVR1 may be functionally important in inducing neutralizing Abs to HCV. These results may be the first example of "T-B reciprocity," in which proximity of a helper T cell epitope determines Ab epitope specificity, in a human disease setting.

Adult↗
PubMed1999

Reduced C-terminal Src kinase (Csk) activities in hepatocellular carcinoma.

The proto-oncogene product pp60(c-src) is the cellular homologue of the Rous sarcoma transforming gene, and it is a non-receptor-linked and membrane-associated tyrosine kinase. There is a close correlation between elevated pp60(c-src) activity and cell transformation. We have recently reported that pp60(c-src) was activated in hepatocellular carcinoma (HCC) of human and Long-Evans cinnamon (LEC) rats. However, the mechanisms involved in this process remain unknown. C-terminal Src kinase (Csk) is a novel cytoplasmic protein tyrosine kinase that inactivates the members of the Src family protein tyrosine kinase in vitro. We investigated the role of Csk in hepatocarcinogenesis by analyzing the location, amount of Csk, and its kinase activity levels in nontumorous cirrhotic and tumorous sections of HCC of patients and an animal model of LEC rats. Csk tyrosine kinase activity was significantly reduced in tumorous tissues compared with nontumorous sections of patients as well as LEC rats. A single immunoreactive band at 50 kd was detected with Csk antibody in normal liver (NL), chronic hepatitis (CH), and nontumorous cirrhotic (NTC) segments of HCC of patients and LEC rats. In human tumorous tissues, Western blot revealed a 53-kd immunoreactive band, which was slightly larger than the usual 50-kd band of Csk. These results suggest that the reduced activity of tyrosine kinase of Csk may play an important role in the malignant transformation of hepatocytes in human and LEC rat, and the appearance of 53-kd Csk-related protein may be closely involved in the progression of cirrhosis to HCC in humans, and that 50-kd Csk may act as an antioncogene through the negative regulation of pp60(c-src) in the development of human HCC.

Aged↗
PubMed1999

An intrinsic DNA curvature found in the cyanobacterium Microcystis aeruginosa K-81 affects the promoter activity of rpoD1 encoding a principal sigma factor.

The rpoD1 gene in the unicellular cyanobacterium Microcystis aeruginosa K-81 encodes a principal sigma factor of RNA polymerase and is transcribed under light and dark conditions to produce multiple monocistronic transcripts. In the 5'-upstream region from rpoD1 Promoter 2, which has a sequence of Escherichia coli type, we found a sequence-directed DNA curvature with an AT-rich sequence. Insertions of 2 to 21 base pairs introduced into the curved center changed a gross geometry of the original curved DNA structure. The rpoD1 promoter activities assayed in vivo by using transcriptional lacZ fusions were correlated with the change in the gross geometry in not only a cyanobacterium but also E. coli. In addition, RNA polymerase binding to the rpoD1 promoter region and the efficiency of the mRNA synthesis from the rpoD1 Promoter 2 were also affected in vitro by the change in the geometry. These results suggest that the tertiary structure of the curved DNA is important for the rpoD1 transcription. The deletion of the center region of the curvature resulted in a considerable reduction of the transcription from Promoter 2 in the cyanobacterium. This report demonstrates that a curved DNA plays a significant role in transcription in cyanobacteria, and that this functional curvature is located in the 5'-upstream region from the rpoD gene, which encodes a principal sigma factor in eubacteria.

Bacterial Proteins↗
PubMed1998

Specific recognition of the cyanobacterial psbA promoter by RNA polymerases containing principal sigma factors.

The psbA2 gene of a unicellular cyanobacterium, Microcystis aeruginosa K-81, encodes a D1 protein homolog in the reaction center of photosynthetic Photosystem II. To clarify the promoter recognition by a sigma factor of RNA polymerase, in vivo and in vitro analyses were performed for the photosynthetic gene. Although the specific transcript from the psbA2 promoter, whose sequence is of Escherichia coli consensus type, was observed in both cyanobacterium K-81 and E. coli cells, the expression was light-dependent in K-81 whereas it was constitutive in E. coli under the conditions of light and darkness (L/D). The specific psbA2-dependent transcripts were also detected in vitro by RNA polymerases containing the principal sigma factors, E. coli sigma70 and K-81 sigmaA1 (constitutively exists in K-81 grown under L/D cycles). Furthermore, a series of promoter fragments were constructed to confirm minimal cis elements for the in vitro psbA2 transcription. A -80 to +6 or -38 to +46 region, the sequences of which consisted of a core promoter (-38 to +6), was identified as the potential minimal cis element using the RNA polymerase fraction (*EsigmaA1) containing sigmaA1 partially purified from K-81. These results suggest that the psbA2 transcription with the minimal sequence was induced by the RNA polymerase (EsigmaA1) containing the principal sigma factor, sigmaA1, under both light and dark conditions in K-81.

Base Sequence↗
PubMed1998

Do obesity and non-insulin dependent diabetes mellitus aggravate exercise-induced microproteinuria?

We evaluated the role of obesity in proteinuria by treadmill exercising of simple obese subjects and non-obese subjects with non-insulin dependent diabetes mellitus in whom the albumin excretion rate at rest was within normal range. Non-obese healthy volunteers were studied as the controls. The fractional renal clearances of four endogenous proteins, albumin, IgG, IgG4, and beta2-microglobulin were measured before, during, and after treadmill exercise in 17 simple obese and 15 non-obese diabetic subjects, and in 21 normal subjects. Exercise increased the fractional albumin clearance in all groups. In diabetic subjects, the fractional IgG4 clearance also increased: fractional beta2-microglobulin clearance increased in normal controls and in diabetics. In obese subjects, the fractional clearances of albumin, IgG, and IgG4 were similar to those in normal controls, but fractional beta2-microglobulin clearance was significantly lower. These results suggest that enhanced microalbuminuria in obese subjects is probably of glomerular origin. In normal subjects and diabetics, exercise-induced microproteinuria is probably of both glomerular and tubular origin. Defect in the charge-selective barrier of the glomerular capillary wall has been implicated in diabetics. Thus some additional factors relevant to obesity must be taken into account in the consideration of the mechanism of microalbuminuria in diabetics with obesity.

Adolescent↗
PubMed1998

Synthesis of novel antifungal agents (2).

Synthesis of novel cyclohexyl analogs of restricticin using intramolecular radical cyclization and their in vitro and in vivo antifungal activities are described.

Animals↗
PubMed1998

Sodium alkyl ether sulfate preparative electrophoresis for the preparation of reaction centers without H-subunit from Rhodopseudomonas viridis.

Sodium alkyl ether sulfate (AES), an analog of sodium dodecyl sulfate (SDS) was used in polyacrylamide gel electrophoresis (PAGE) for the partial decomposition of the photosynthetic reaction center (RC) of Rhodopseudomonas viridis. Unlike SDS, AES did not completely dissociate RC into its subunits but selectively detached H-subunit from RC to give RC(-H) without losing the spectroscopic nature of RC. For the denaturation of RC(-H), AES was found to be as mild as 3-[3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS).

Electrophoresis, Polyacrylamide Gel↗
PubMed1998

Outbreak of severe infection due to adenovirus type 7 in a paediatric ward in Japan.

Between November 1996 and January 1997, 14 patients were diagnosed as having infection caused by adenovirus type 7 in a paediatric ward of Asahikawa Kosei Hospital. The age range of the patients was from two months to five years. Their diseases and abnormal laboratory findings were pneumonia in all 14, leukocytopenia in 10, myositis in nine, gastroenteritis in eight, encephalitis in five, liver dysfunction in three, pleuritis in two, inappropriate secretion of antidiuretic hormone syndrome in two, and thrombocytopenia in two. The infected patients, except for the first had been hospitalized in the paediatric ward for treatment of another disease and re-admitted because of high fever and coughing a few days after improvement or discharge. It is thought that the cause of the outbreak was hospital-acquired infection.

Adenovirus Infections, Human↗
PubMed1998

Dipyridamole and dilazep suppress oxygen radicals in puromycin aminonucleoside nephrosis rats.

BACKGROUND: Reactive oxygen species (ROS) are involved in the pathophysiology of puromycin aminonucleoside (PAN) nephrosis. To elucidate further the role of radicals in PAN nephrosis and the to determine the particular radical species scavenged by dipyridamole (DPM) and dilazep (DZ), we applied chemiluminescence and electron spin resonance (ESR) techniques. METHODS: Chemiluminescence of glomeruli, which were isolated on day 7 from rats injected with 100 mg kg-1 PAN, was measured with or without scavengers. The inhibitory effects of DPM and DZ on hydroxyl radical adduct formation in the Fenton's reaction were evaluated using ESR. RESULTS: Chemiluminescence was greater in glomeruli from rats with PAN nephrosis than in the the glomeruli of control rats. This increase was suppressed by superoxide dismutase, catalase, dimethylthiourea and also by DPM and DZ. ESR indicated that DPM and DZ inhibited hydroxyl radical adduct formation with a second-order rate constant of 2.9 x 10(10) and 1.6 x 10(10) (mol L(-1) s(-1) respectively, similar to that of dimethylthiourea. CONCLUSION: DPM and DZ scavenge hydroxyl radicals, thereby alleviating PAN nephrosis.

Animals↗
PubMed1998

Altered anionic GBM components in monoclonal antibody against slit diaphragm-injected proteinuric rats.

BACKGROUND: We previously reported that monoclonal antibody (mAb) 5-1-6 bound to renal filtration slits induces massive proteinuria without causing ultrastructural changes in the glomerulus. This study evaluated the underlying mechanisms of the increase in glomerular permeability. METHODS: The distribution of endogenous albumin and IgG in the glomerular basement membrane (GBM) was studied in in situ drip-fixed glomeruli of Munich-Wistar rats by use of immunogold immunocytochemistry in the presence and absence of mAb 5-1-6. The density of foot process glycocalyx components was estimated by labeling with Limax fluvus lectin- or Helix pomatia lectin-gold complexes. Anionic sites in the GBM were examined by labeling with cationic gold at pH 2.0 or 7.4. Carboxyl groups, which also furnish an anionic charge to the GBM, were examined by specific biotinylation and colloidal gold probe methods. In addition, the infusion-staining of anionic sites was performed by use of ruthenium red in both Munich-Wistar and Wistar rats. RESULTS: The urinary excretion of albumin and IgG was increased markedly in the treated rats, indicating a non-selective barrier defect. In the control rats, albumin and IgG molecules were mainly located along the inner half of the GBM, and to a lesser degree in the lamina rara externa. In the treated rats, the albumin and IgG moieties were more equally distributed throughout the width of the GBM. Newly appearing, small dense peaks at the outer side of the GBM were evident, indicating a barrier function of outer zone of the GBM and/or epithelial cell layer. No intergroup differences in the density of lectin binding sites on foot processes were seen. The reduction in the number of ruthenium red-positive anionic sites and cationic gold (pH 2. 0)-labeled anionic sites in the lamina rara externa was significant in the treated rats at day 3, indicating a possible alteration of charged proteoglycan in the lamina rara externa. No such changes were seen with cationic gold (pH 7.4)-labeled anionic sites in the GBM. The density of labeled carboxyl groups was significantly reduced in the treated rats relative to the controls. CONCLUSIONS: These results show that the injection of mAb 5-1-6 induced a perturbation of the charge- and probably the size-selective glomerular filtration barrier. The observed reduction in the levels of various negatively charged substances resulted in massive proteinuria, implying that alteration of target antigens can affect the integrity of the GBM constituents maintaining the normal barrier function.

Albuminuria↗
PubMed1998

Persistent infection by Helicobacter pylori down-modulates virus-specific CD8+ cytotoxic T cell response and prolongs viral infection.

To determine whether Helicobacter pylori infection affects clearance of a concomitant viral infection and cytotoxic T lymphocyte (CTL) and cytokine response to that infection, H. pylori-infected BALB/c mice were challenged with a recombinant vaccinia virus expressing human immunodeficiency virus type 1 gp160. Two H. pylori strains, a colonizing clinical isolate (KS612) and an established standard noncolonizing strain (NCTC11637), were compared. Clearance of recombinant vaccinia virus was reduced in KS612-infected mice compared with NCTC11637-infected and control mice. As a potential mechanism, in contrast to control or NCTC11637-infected mice, the H. pylori clinical isolate KS612 diminished gp160-specific and vaccinia virus-specific CTL activity, even in the presence of exogenous interleukin-2. Furthermore, KS612-infected mice had reduced Th1 cytokine responses to gp120 in vitro compared with control or NCTC11637-infected mice. These results have implications for possible effects of prevalent H. pylori infection on other human diseases.

Animals↗
PubMed1998

Modification and application of self-trimming hairpin ribozymes to targeting a transcribed RNA in vitro.

The three-domain hairpin ribozyme was improved by increasing the number of linker bases connecting domain I' with domain II from six to seven, and a new ribozyme was designed to release a trans-acting ribozyme that is able to bind with target RNAs by three hybridizing arms. The trans-cleavage activities of the trimmed ribozyme were used to cleave a long target RNA, with 85 bases transcribed from a synthetic gene encoding induced nitric oxide synthase (iNOS). Although the ribozyme with three-hybridizing arms did not efficiently cleave the long target RNA, the overall activity, from self-trimming to the trans-cleavage reaction of the target RNA, was higher than that of the ribozyme with two hybridizing arms.

Base Sequence↗
PubMed1998

Chlamydia pneumoniae in coronary and iliac arteries of Japanese patients with atherosclerotic cardiovascular diseases.

Recent studies suggest the association of atherosclerotic cardiovascular diseases with Chlamydia pneumoniae infection in western populations. It is of great interest whether such an association exists in Asians with their distinct genetic background. Symptomatic patients with coronary heart disease (29) or arteriosclerosis obliterans (10) who underwent directional endo-atherectomy were studied. Atherectomy specimens of coronary and iliac arteries were examined for C. pneumoniae by culture, nested PCR and immunohistochemical stain (IHC) with one Chlamydia genus-specific, two C. pneumoniae species-specific, and two C. trachomatis species-specific monoclonal antibodies. Among the 29 patients with coronary artery disease, C. pneumoniae was detected in the coronary arteries of 13 by IHC, 16 by PCR and 20 by IHC or PCR, or both. C. pneumoniae was also found in the iliac arteries of four patients by IHC, three by PCR and five by IHC or PCR, or both, of the 10 patients with arteriosclerosis obliterans. Attempts to isolate C. pneumoniae by culture were unsuccessful. The re-stenotic rate after atherectomy was higher in the C. pneumoniae-positive group than in the negative group, but not significantly so. These findings support the high incidence of C. pneumoniae in atherosclerotic lesions of symptomatic patients with coronary heart disease and arteriosclerosis obliterans in Asians.

Aged↗
PubMed1998

Characterization of anti-histone antibodies in patients with type 1 autoimmune hepatitis.

We have recently found that antibodies to total histones are common in a group of American patients with type 1 autoimmune hepatitis (AIH). In an attempt to determine the profile and clinical association of anti-histone antibody (AHA), 45 Japanese AIH patients were studied for serum isotypic reactivity with individual histones (H1, H2A, H2B, H3, H4) by enzyme-linked immunosorbent assay and western blotting. The results revealed that 40% of sera had reactivities with at least one of individual histones and that the antibodies were detected in all three classes of immunoglobulins (IgG, IgM, IgA). Immunoglobulin G type anti-H3 showed the dominant reactivity and it characterized 72% of sera with AHA. The titre of anti-H3 decreased significantly (P < 0.0075) after steroid therapy and the index of decrease for anti-H3 was correlated in individuals with that for serum aminotransferase. In general, patients with AHA showed higher serum level of alanine aminotransferase (P < 0.05), immunoglobulin G (P < 0.025), and higher frequency of A2-DR4 haplotype (53 vs 17%) than their seronegative counterparts. However, the titre of AHA was low in this disease condition and histone class-specific antibodies did not distinguish patients with distinctive clinical features, although patients with anti-H3 tended to be younger than those without AHA.

Adolescent↗
PubMed1998

Nucleotide sequence and characterization of cdrA, a cell division-related gene of Helicobacter pylori.

We identified cell division-related gene cdrA in Helicobacter pylori HPK5. The putative gene product, CdrA, is a 367-amino-acid polypeptide that exhibited a high level of homology to conserved hypothetical ATP-binding protein HP0066 of H. pylori 26695, except in the N-terminal region, and showed some similarity to the FtsK/SpoIIIE family proteins. We isolated a cdrA-disrupted mutant by allelic exchange mutagenesis. Because of the low transformation frequency, the possibility that a suppressing mutation would be found in the obtained cdrA mutant was discussed. A repressive role for CdrA on cell division was suggested by the observations that the wild-type strain formed filamentous cells in a high-salt level medium at early stationary phase, while a cdrA-disrupted mutant did not show such an abnormality. In addition, the wild-type strain adopted coccoid forms in the stationary phase, whereas the cdrA-disrupted mutant remained mostly as short rods. Furthermore, the cdrA-disrupted mutant regained the filamentation phenotype when the intact cdrA gene was introduced by allelic exchange. Taken together, these observations show that the cdrA gene plays an important role in the cell growth of H. pylori.

Amino Acid Sequence↗