The structure elucidation and biological activities of high molecular weight algal toxins: maitotoxin, prymnesins and zooxanthellatoxins.
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Lipoproteins↗
Biomedical subjects
M Murata
Publications and source records attributed to M Murata.
The CitST two-component system regulates the expression of the Mg-citrate transporter in Bacillus subtilis.
citS and citT genes encoding a new two-component system were identified in the 71 degrees region between the pel and citM loci on the Bacillus subtilis chromosome. citS- and citT-deficient strains were unable to grow on minimal plates including citrate as a sole carbon source. In addition, a strain deficient in citM, which encodes the secondary transporter of the Mg-citrate complex, exhibited the same phenotype on this medium. Northern blot analysis revealed that citM was polycistronically transcribed with the downstream yflN gene, and that CitS and CitT were necessary for transcription of the citM-yflN operon. Upon addition of 2 mM citrate to DSM, this operon was strongly induced after the middle of the exponential growth phase in the wild type, but not in the citST double null mutant. Moreover, the transcription of this operon was completely repressed in the presence of 1% glucose. We found a sequence exhibiting homology to a catabolite-responsive element (cre) in the citM promoter region. Glucose repression was lost in ccpA and citM-cre mutants. From the result of a citM-promoter deletion experiment, putative CitT target sequences were found to be located around two regions, from -62 to -74 and from -149 to -189, relative to the citM start point. Furthermore, DNase I footprinting assays revealed that these two CitT target regions extended maximally from -36 to -84 and from -168 to -194. From these findings, we concluded that the expression of citM is positively regulated by the CitST system and negatively regulated by CcpA.
Amino Acid Sequence↗
Germ cell-specific heat shock protein 105 binds to p53 in a temperature-sensitive manner in rat testis.
Heat shock protein (HSP)105 is a testis-specific and HSP90-related protein. The aim of this study was to explore the functions of HSP105 in the rat testis. Signals of HSP105 were detected immunohistochemically in the germ cells and translocated from the cytoplasm to the nucleus at 2 days after experimental induction of cryptorchidism. In cultured testicular germ cells, a significant increase in the expression of HSP105 in response to heat stress (37 degrees C) was detected in the insoluble protein fractions. Several binding proteins were isolated from rat testis using a HSP105 antibody immunoaffinity column, and p53, the tumor suppressor gene product, was copurified with these. Furthermore, immunoprecipitation using antibodies to p53 led to coprecipitation of HSP105 together with p53 after culturing germ cells at 32.5 degrees C, but not at 37 or 42 degrees C. In conclusion, HSP105 is specifically localized in the germ cells and may translocate into the nucleus after heat shock. HSP105 is suggested to form a complex with p53 at the scrotal temperature, and dissociate from it at suprascrotal temperatures. At scrotal temperature, HSP105 may thus contribute to the stabilization of p53 proteins in the cytoplasm of the germ cells, preventing the potential induction of apoptosis by p53.
Animals↗
18O-Labelling pattern of okadaic acid from H218O in dinoflagellate Prorocentrum lima elucidated by tandem mass spectrometry.
Okadaic acid is a metabolite of the unicellular algae dinoflagellate. Its biosynthesis has attracted considerable attention since the skeletal structure was shown to be synthesized via an unprecedented route. However, its relevant intermediates or enzymes are unknown. In the course of our previous investigations on the oxygen source of okadaic acid by tandem mass spectrometry (CID MS/MS), we determined the level of 18O incorporation for each oxygen site from 18O2 and [18O2]acetate. In the present study, we examined H218O-labelling patterns of okadaic acid from dinoflagellates in comparison with salinomycin from actinomycetes and has provided intriguing information regarding biosynthesis. Unexpectedly, oxygen atoms originating from acetate were not labelled from H218O; this can not be accounted for by the usual metabolic route where acetyl-CoA is biosynthesized via pyruvate. Similar experiments for salinomycin revealed that all of its oxygen atoms derived from acetate or propionate were labelled by H218O. Another interesting feature is that two oxygen sites were derived from both O2 and H2O while the others were labelled only from O2. These results imply that an oxidation mechanism other than those in actinomycetes polyethers may be involved in the biosynthesis of okadaic acid.
Animals↗
Superparamagnetic iron oxide-enhanced magnetic resonance images of hepatocellular carcinoma: correlation with histological grading.
Superparamagnetic iron oxide (SPIO)-enhanced magnetic resonance (MR) imaging has been used for the detection of hepatic tumors. However, little is known about this technique in relation to hepatocellular carcinoma (HCC). The aim of this study was to investigate whether SPIO-enhanced MR imaging can be useful in assessing histological grades of HCC. The authors studied histologically proven tumors including 31 HCCs and 6 dysplastic nodules. The ratio of the Kupffer-cell count in the tumorous tissue relative to that in the nontumorous tissue (Kupffer-cell-count ratio) decreased as HCCs became less well differentiated. The ratio of the intensity of the tumorous lesion to that of the nontumorous area on SPIO-enhanced MR images (SPIO intensity ratio) correlated inversely with Kupffer-cell-count ratio in HCCs and dysplastic nodules (r = -.826, P <.001) and increased as the degree of differentiation of HCCs decreased, indicating that the uptake of SPIO in HCCs decreased as the degree of differentiation of HCCs declined. All of the dysplastic nodules and some well-differentiated HCCs showed hypointense or isointense enhancement, relative to the surrounding liver parenchyma, indicating greater or similar uptake of SPIO in the tumor when compared with nontumorous areas. These results suggest that SPIO-enhanced MR imaging reflects Kupffer-cell numbers in HCCs and dysplastic nodules, and is useful for estimation of histological grading in HCCs, although uncertainties persist in differentiating dysplastic nodules from well-differentiated HCCs.
Aged↗
Electrophysiologic findings of a patient with inappropriate sinus tachycardia cured by selective radiofrequency catheter ablation.
Radiofrequency catheter ablation (RFCA) for inappropriate sinus tachycardia (IST) is associated with a high recurrence rate and sometimes requires pacemaker implantation, especially after extensive ablation. We report a patient with drug-refractory IST who was successfully treated by selective RFCA to the 2 earliest activation sites. During tachycardia, the earliest atrial activation preceded the surface P wave by 50 ms or more, whereas it was only 27 ms for the rest of the right atrium after ablation. Our patient had the longest activation period during tachycardia among the reported patients. In IST patients, a longer activation time at the site of the earliest atrial activation may imply that the abnormality is confined to a small area within the sinus node and may predict the efficacy of selective RFCA.
Adult↗
Localization of endothelial nitric oxide synthase messenger ribonucleic acid by in situ hybridization in ectopic endometrial tissue in patients with adenomyosis.
This study was undertaken to determine the expression of endothelial nitric oxide synthase (NOS) mRNA in ectopic endometrium in adenomyosis. Endothelial NOS mRNA expression was investigated by in situ hybridization using a human endothelial NOS RNA probe. The subjects comprised six cases of adenomyosis, and 14 women with normal fecundity as controls. Specific expression of endothelial NOS mRNA was found in glandular and surface epithelial cells in eutopic endometrium throughout the menstrual cycle in all groups. It is noteworthy that the expression of endothelial NOS mRNA was moderately positive in ectopic endometrial tissues in three of the six cases of adenomyosis. In conclusion, the endothelial NOS mRNA expression seen in the ectopic endometrium suggests a possible relationship with the various pathologies associated with adenomyosis.
Adult↗
Copper-dependent DNA damage induced by hydrazobenzene, an azobenzene metabolite.
Hydrazobenzene is carcinogenic to rats and mice and azobenzene is carcinogenic to rats. Hydrazobenzene is a metabolic intermediate of azobenzene. To clarify the mechanism of carcinogenesis by azobenzene and hydrazobenzene, we investigated DNA damage induced by hydrazobenzene, using 32P-5'-end-labeled DNA fragments obtained from the c-Ha-ras-1 protooncogene and the p53 tumor suppressor gene. Hydrazobenzene caused DNA damage in the presence of Cu(II). Piperidine treatment enhanced the DNA damage greatly, suggesting that hydrazobenzene caused base modification and liberation. However, azobenzene did not cause DNA damage even in the presence of Cu(II). Hydrazobenzene plus Cu(II) caused DNA damage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited Cu(II)-mediated DNA damage by hydrazobenzene. Typical *OH scavengers did not inhibit the DNA damage. The main active species is probably a metal oxygen complex, such as Cu(I)-OOH. Formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine was increased by hydrazobenzene in the presence of Cu(II). Oxygen consumption and UV-Visible spectroscopic measurements have shown that hydrazobenzene is autoxidized to azobenzene with H2O2 formation. It is considered that the metal-mediated DNA damage by hydrazobenzene through H2O2 generation may be relevant for the expression of carcinogenicity of azobenzene and hydrazobenzene.
8-Hydroxy-2'-Deoxyguanosine↗
Seasonal variation in heart rate variability in asthmatic children.
Asthma is a "seasonal disease" with symptoms either aggravated by environmental changes during specific seasons or prevalent at certain times of the year for other reasons. We examined whether the heart rate variability (HRV) of asthmatic children changes by season. The HRV during a portion of one night (00:00-04:00) and day (12:00-16:00) and the entire 24h period (00:00-24:00) during each of the four seasons was analyzed. The data of 95 children with asthma and 106 healthy children, as controls, were assessed. In children with asthma during the 24h period, seasonal variation in the low-frequency (LF) band (0.04-0.15 Hz) and the high-frequency (HF) band (0.15-0.4 Hz) were detected (HF: F=6.81, p=.0003; LF: F=4.18, p= .008). The HF value in the summer was significantly higher than in autumn and spring (Scheffe test: autumn vs. summer, s = 4.46, p < .001: spring vs. summer, s = 2.86, p < .05), while the LF value in autumn was significantly lower than in summer (s = 3.42, p < .01). In the control group, no seasonal variation in HF, LF, or LF/HF was detected. The findings infer the HRV, a surrogate measure of autonomic nervous system function, of asthmatic children is more susceptible to seasonal changes brought about by either endogenous annual rhythms or environmental weather phenomena.
Adolescent↗
Vascular endothelial growth factor (VEGF) enhances the expression of receptors and activates mitogen-activated protein (MAP) kinase of dog retinal capillary endothelial cells.
Since the galactose-fed dog is an animal model that develops the advanced stage of proliferative retinopathy, the effects of vascular endothelial growth factor (VEGF) on cell growth, receptor expression and the activation of mitogen-activated protein (MAP) kinase pathway of dog retinal capillary endothelial cells were investigated. Dog retinal endothelial cells were cultured at 37 degrees C under 5% carbon dioxide atmosphere in CS-C medium supplemented with endothelial cell growth factor (ECGF). VEGF receptor expression was examined by RT-PCR, and activation of MAP kinase was examined with antibody against phospho-Elk-1 (Ser383). When growth factors were removed from the culture medium, cell survival of dog endothelial cells was significantly reduced. Addition of VEGF protected these cells from cell death induced by growth factor starvation. VEGF also enhanced tube formation in dog endothelial cells and increased the expression of two VEGF receptors, Flt-1 and KDR/Flk-1. Cells treated with VEGF also displayed the phosphorylation of the transcription factor, Elk-1. Addition of the tyrosine kinase inhibitor, genistein, eliminated VEGF-induced cell growth and Elk-1 phosphorylation. These data confirm that cell growth and tube formation of dog retinal capillary endothelial cells are stimulated by VEGF. VEGF also increases the expression of the receptors, KDR and Flt-1, and activates the p44/42 MAP kinase pathway.
Animals↗
Reconstitution of brefeldin A-induced golgi tubulation and fusion with the endoplasmic reticulum in semi-intact chinese hamster ovary cells.
The fungal metabolite brefeldin A (BFA) induces the disassembly of the Golgi complex in mammalian cells. The drug seems to accentuate tubule formation and causes the subsequent fusion with the endoplasmic reticulum (ER). To investigate the biochemical requirements and kinetics of BFA-induced Golgi disassembly, we have reconstituted the process of green fluorescent protein-tagged Golgi complex disassembly in streptolysin O-permeabilized semi-intact Chinese hamster ovary cells. For quantitative analysis of the morphological changes to the Golgi complex in semi-intact cells, we developed a novel morphometric analysis. Based on this analysis, we have dissected the BFA-induced Golgi disassembly process biochemically into two processes, Golgi tubule formation and fusion with the ER, and found that the formation is induced by only ATP and the residual factors in the cells and that the subsequent fusion is mediated in an N-ethylmaleimide-sensitive factor-dependent manner via Golgi tubules. Tubulation occurs by two pathways that depend on either microtubule integrity or exogenously added cytosol. In the presence of GTPgammaS, coat protein I inhibited the Golgi tubule fusion with the ER but showed no apparent effect on tubulation. Additionally, we analyzed the kinetics of tubulation and fusion independently in nocodazole-treated and -untreated semi-intact cells and found that tubulation is a rate-limiting step of the Golgi disassembly.
Animals↗
Secular trends in growth and changes in eating patterns of Japanese children.
Changes in the daily macronutrient intake of Japanese children have been studied over the past half century to determine the effects of such changes on a child's healthy growth and development. Data from government and other reports show that in 5-y-old boys in the Tokyo metropolitan area, the intake of fat as a percentage of total energy intake was 12.6%, 20.9%, 28.4%, 33.8%, and 33.2% in 1952, 1960, 1970, 1982, and 1994, respectively. The prevalence of obesity in school-aged children is increasing; recently, nearly 10% of the children in this age group were considered obese. About 5% of obese children experience some adverse effects caused by obesity, eg, hypertension and hyperlipidemia. Nationwide surveys on serum cholesterol concentrations conducted in 1960, 1970, 1980, and 1990 showed that the cholesterol concentrations in 10-19-y-old males and females increased year after year. The height of boys and girls rapidly increased during the 15-y period from 1945 to 1960, and after 1970 the adult height in Japan was thought to have reached its maximum. The factors that contribute to the health problems facing today's Japanese children include their sedentary lifestyle, irregular intake of meals (especially skipping breakfast), and the increasing daily ratio of fat to total energy intake. Presently, changing the sedentary lifestyle is the most significant issue to be resolved.
Adolescent↗
Substrate specificity of bovine cathepsin B and its inhibition by CA074, based on crystal structure refinement of the complex.
The crystal structure of the bovine spleen cathepsin B (BSCB)-CA074 complex was refined to R = 0.152 using X-ray diffraction data up to 2.18 A resolution. BSCB is characterized by an extra Cys148-Cys252 disulfide bridge, as compared with rat and human CBs. Although the crystal structures of these enzymes showed similar overall folding, a difference was observed in the occluding loop, a structural element specific only to CB. Comparison of the torsion angles indicated the different flexibilities of their loop structures. The oxirane C6 atom of CA074 was covalently bonded to the Cys29 S(gamma) atom (C3-S(gamma)=1.81 A), where the S-configuration was transformed to the R-form. Concerning the oxirane carbon atom that participates in the covalent bonding with the Cys residue, an acceptable rule has been proposed. The substrate specificities at the Sn (n = 1-3) and Sn' (n=1 and 2) subsites of CB, together with the interaction features as to CA074, have been discussed in comparison with the crystal structure of the papain-CA028 (a CA074-related inhibitor) complex.
Amino Acid Sequence↗
Mitochondrial localization of AtOXA1, an arabidopsis homologue of yeast Oxa1p involved in the insertion and assembly of protein complexes in mitochondrial inner membrane.
Components of some protein complexes present in the inner membrane of mitochondria are encoded in both nuclear and mitochondrial genomes, and correct sorting and assembly of these proteins is necessary for proper respiratory function. Recent studies in yeast suggest that Oxa1p, a protein conserved between prokaryotes and eukaryotes, is an essential factor for protein sorting and assembly into membranes. We previously identified AtOXA1, an Arabidopsis homologue of OXA1 by functional complementation of a yeast oxa1- mutant. In this study, we investigated the genomic organization of AtOXA1 and localization of the AtOXA1 protein. Characterization of the AtOXA1 genomic region indicated that the gene consists of 10 exons and is located on chromosome V. A database search also revealed another gene coding for a putative protein homologous to AtOXA1 on chromosome II. Transient expression of a green fluorescent protein (GFP) fusion in suspension-cultured tobacco cells showed that AtOXA1 is targeted into mitochondria by its N-terminal presequence. Antibodies raised against AtOXA1 recognized a 38-kDa intrinsic protein of the inner mitochondrial membrane. Thus, localization of AtOXA1 in the mitochondrial inner membrane, together with our previous complementation experiment in yeast, suggested that it is a functional homologue of Oxa1p.
Arabidopsis↗
The YELLOW VARIEGATED (VAR2) locus encodes a homologue of FtsH, an ATP-dependent protease in Arabidopsis.
Variegated leaves are often caused by a nuclear recessive mutation in higher plants. Characterization of the gene responsible for variegation has shown to provide several pathways involved in plastid differentiation. Here we describe an Arabidopsis variegated mutant isolated by T-DNA tagging. The mutant displayed green and yellow sectors in all green tissues except for cotyledons. Cells in the yellow sector of the mutant contained both normal-appearing and mutant chloroplasts. The isolated mutant was shown to be an allele of the previously reported mutant, yellow variegated (var2). Cloning and molecular characterization of the VAR2 locus revealed that it potentially encodes a chloroplastic homologue of FtsH, an ATP-dependent metalloprotease that belongs to a large protein family involved in various cellular functions. ftsH-like genes appear to comprise a small gene family in Arabidopsis genome, since at least six homologues were found in addition to VAR2. Dispensability of VAR2 was therefore explained by the redundancy of genes coding for FstHs. In the yellow regions of the mutant leaves, accumulation of photosynthetic protein components in the thylakoid membrane appeared to be impaired. Based on the role of FtsH in a protein degradation pathway in plastids, we propose a possibility that VAR2 is required for plastid differentiation by avoiding partial photooxidation of developing chloroplasts.
ATP-Dependent Proteases↗
Crystallization and preliminary X-ray analysis of cephalosporin C acylase from Pseudomonas sp. strain N176.
Cephalosporin C acylase from Pseudomonas sp. strain N176, a heterodimer of 25 and 58 kDa, has been crystallized using polyethylene glycol 6000 as precipitant. The crystals are orthorhombic and have unit-cell parameters a = 141.41, b = 192.10, c = 80.75 A. They belong to space group P2(1)2(1)2(1) and diffract to at least 2.7 A resolution. Calculations indicate that there are two heterodimers in the asymmetric unit. The structure is being solved by molecular replacement using penicillin G acylase from Escherichia coli as a search model and by multiple isomorphous replacement.
Crystallization↗
Apoptotic cytotoxic effects of a histone deacetylase inhibitor, FK228, on malignant lymphoid cells.
Histone deacetylases are promising targets for cancer treatment. Here we studied the in vitro effects of a potent histone deacetylase inhibitor, FK228 (formerly FR901228), on human leukemia / lymphoma cells and cell lines compared with normal hematopoietic cells. In a lymphoma cell line, Raji, a nanomolar concentration of FK228 induced G1 arrest and / or apoptotic cell death, depending on the concentration and exposure time. Growth of lymphoid cell lines including Raji (N = 13) was inhibited by 50% (IC(50)) after 2-day treatment at concentrations of 0.83 to 1.87 ng / ml. Viability of clinical samples from patients with acute lymphoblastic leukemia was decreased by 50% at 0.78 +/- 0.46 ng / ml, whereas the IC(50) values for normal mononuclear cells from peripheral blood and bone marrow were 2.3 +/- 0.96 and 7.8 +/- 1.0 ng / ml, respectively. The IC(50) values for normal progenitor cells were 3.1, 4.4 and 7.8 ng / ml for BFU-E, CFU-GM and CFU-Mix, respectively. Expression levels of HDAC-1 and HDAC-3 proteins, which varied among cell lines, but were stable during the treatment with FK228, did not correlate with the sensitivity to FK288. This novel agent might be useful in the treatment of lymphoid malignancies, because the above concentrations are clinically achievable in vivo according to a recent clinical study.
Anti-Bacterial Agents↗
Molecular and biological analysis of carcinoma of the small intestine: beta-catenin gene mutation by interstitial deletion involving exon 3 and replication error phenotype.
The genetic mechanisms of carcinomas of the small intestine are not well understood. We report the results of analysis of genetic alterations in a case of small intestinal carcinoma. A tumor in the terminal ileum was resected in a 59-yr-old woman. Histologically, the tumor was classified as well-differentiated adenocarcinoma. We screened for genetic alterations in adenomatous polyposis coli (APC), beta-catenin, K-ras, and p53 genes, as well as microsatellite instability, which are known to be involved in colorectal tumorigenesis. The tumor exhibited somatic interstitial deletion of 425-bp, which included the entire exon 3 in beta-catenin gene. Immunohistochemical staining confirmed accumulation of aberrant beta-catenin protein in the cytoplasm and nuclei of the malignant tissue. Furthermore, a frameshift mutation in the transforming growth factor beta receptor type II gene with replication error phenotype was detected in the tumor DNA. In contrast, no genetic alterations were found in the APC, K-ras, and p53 genes. Our results suggested that both beta-catenin gene mutation and replication error phenotype might contribute to carcinogenesis of the small intestinal tumor in our case. This is the first report that activation of beta-catenin gene by somatic gene mutation is involved in the development of carcinoma of the small intestine.
Amino Acid Sequence↗