Nitrate respiratory metabolism in an obligately autotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6

Hydrogenobacter thermophilus strain TK-6 was observed to grow anaerobically on nitrate as an electron acceptor when molecular hydrogen was used as an energy source. Nitrite was detected as the product of a respiratory reaction. 15NO, 15N2O, and 15N2 were detected with Na15NO3 as an electron acceptor. Western immunoblot analysis showed that cell-free extracts from cells grown on nitrate reacted with antibodies against heme cd1-type nitrite reductase from Pseudomonas aeruginosa. The positive bands, which had molecular masses similar to that of the heme cd1-type nitrite reductase, were also stained by heme staining. These results indicate that nitrite reductase of strain TK-6 is a heme cd1-type enzyme. Activity of ATP:citrate lyase, one of the key enzymes of the reductive TCA cycle, was detected in cell-free extract of cells cultivated on nitrate, which indicates that the cycle operates during anaerobic growth.     

A method for preparing 2- to 50-µm-thick fresh-frozen sections of large samples and undecalcified hard tissues

This article describes a method for preparing 2- to 50-micron-thick fresh-frozen sections from large samples and completely calcified tissue samples. In order to perform the more routine work involved, a tungsten carbide disposable blade was installed to a heavy-duty sledge cryomicrotome. An entire 10-day-old rat and bone and tooth samples from a 7-month-old rat were rapidly frozen. The frozen samples were attached to the cryomicrotome stage. The cutting surface of the samples was covered with a polyvinylidene chloride film coated with synthetic rubber cement and cut at -25 degrees C. The soft tissues and the hard tissues were satisfactorily preserved and all tissue cells were easily identifiable. Enzymatic activity in the fresh sections was much stronger than that in chemically fixed and/or decalcified sections. The sections permitted histological and histochemical studies without trouble. In addition, the sections can be used for multiple experiments such as immunohistochemistry, in situ hybridization, and electron microprobe X-ray micro-analysis. This method can be used with conventional cryomicrotome equipment.     

Identification of a gene disrupted by inv(11)(q13.5;q25) in a patient with left-right axis malformation

An inv(11)(q13.5;q25) inversion was previously identified in a 9-month-old male patient with complex cyanotic heart defects, altered lung lobation, symmetric liver, and abnormally lobulated spleen (polysplenia). This chromosomal rearrangement was inherited from the phenotypically normal father. We termed these regions DHTX-A (disrupted in heterotaxy)-- A at 11q13.5 and DHTX-B at 11q25. Here, we report the isolation and characterization of the inversion breakpoints and the gene that is disrupted by the DHTX-A breakpoint. The putative DHTX is identical to the UVRAG gene, which was originally identified as a gene that complements the UV sensitivity of xeroderma pigmentosum complementation group C. The 4-kb mRNA was found to be encoded by a large gene, at least 300 kb long, composed of 15 exons. The function of the gene product remains largely unknown. However, the near central portion of the UVRAG protein is predicted to contain a coiled-coil domain, which has been implicated in mediating protein-protein interactions. Southern analyses and fluorescence in situ hybridization (FISH) revealed that the DHTX-A breakpoint in the patient and his father lies within the intron between exons 6 and 7 of UVRAG. Northern blot analysis indicated strong expression in human fetal and adult tissues and in mouse embryonic day-7 and adult tissues, respectively. Whole mount in situ hybridization also showed that the Uvrag gene is expressed in the presomite-stage embryo. Several hypotheses are discussed to explain the relationship between the chromosomal inversion and the accompanying phenotypes.     

Effect of a New Variety of Apis mellifera Propolis onMutans Streptococci

The effects of a new variety of propolis, from Northeastern Brazil (BA), on growth of mutans streptococci, cell adherence, and water-insoluble glucan (WIG) synthesis were evaluated. Propolis from Southeastern (MG) and Southern (RS) Brazil were also tested as an extension of our previous work. Ethanolic extracts of propolis (EEP) were prepared and analyzed by reversed-phase HPLC. For the antibacterial activity assays, minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of EEPs against Streptococcus mutans, S. sobrinus, and S. cricetus were determined. Cell adherence of S. mutans and S. sobrinus to a glass surface was measured spectrophotometrically at 550 nm. WIG synthesized from sucrose by glucosyltransferase (Gtf) was extracted and quantified by the phenol-sulfuric method. The HPLC profile of the new variety of propolis was entirely different from Southeastern and Southern propolis. Neither flavonoid aglycones nor p-coumaric acid were detected in EEP BA. All EEPs demonstrated biological activities against mutans streptococci; EEP BA showed the highest potency in all in vitro parameters evaluated in this study. The ranges of MIC values were 50 (EEP BA)–400 μg/ml (MG), for S. mutans; and 25 (BA)–400 μg/ml (MG), for S. sobrinus and S. cricetus. The bactericidal concentration of EEPs was four to eight times the MIC values. The adherence of S. mutans and S. sobrinus cells and WIG synthesis were markedly inhibited by EEPs, demonstrating significant inhibition at all concentrations compared with the control (80% ethanol) (p < 0.05). EEP BA showed 80% inhibition of cell adherence and WIG synthesis at concentrations as low as 12.5 and 7.8 μg/ml, respectively. The results show that the new variety of propolis was exceptionally effective in all in vitro parameters tested against mutans streptococci; biological effects of propolis are likely not to be due solely to flavonoids and (hydroxy)cinnamic acid derivatives. Received: 14 February 2000 / Accepted: 8 May 2000     

The complete sequence and expression patterns of the atrial myosin heavy chain in the developing chick

We have earlier reported partial cloning of a cDNA of a chick atrial myosin heavy chain (MHC) gene, CCSV2 and its expression pattern in embryonic chick hearts (Oana et al (1995) Eur J Cell Biol 67, 42-49). In this study, five overlapping cDNA clones (including CCSV2) which together encode the entire open reading frame of the chick atrial MHC gene were characterized, and both the entire nucleotide sequence consisting of 5825 bases and the deduced amino acid sequence consisting of 1931 amino acids determined. Reinvestigation of the nucleotide sequence of the previously reported and presumably different chick atrial specific MHC cDNA clone, AMHC1 (Yutzey et al (1994) Development 120, 871-883), revealed that our clone and AMHC1 encoded the same MHC. The chick atrial MHC gene was strongly expressed in developing chick atria from a very early stage (Hamburger and Hamilton stage 9, 29-33 h) to the adult stage. This gene was also expressed, although weakly, in the ventricle, somite (the precursor to skeletal muscle) and skeletal muscle during embryonic stages but not in adults.     

Assessment of the Annual Additional Effective Doses amongst Minamisoma Children during the Second Year after the Fukushima Daiichi Nuclear Power Plant Disaster

An assessment of the external and internal radiation exposure levels, which includes calculation of effective doses from chronic radiation exposure and assessment of long-term radiation-related health risks, has become mandatory for residents living near the nuclear power plant in Fukushima, Japan. Data for all primary and secondary children in Minamisoma who participated in both external and internal screening programs were employed to assess the annual additional effective dose acquired due to the Fukushima Daiichi nuclear power plant disaster. In total, 881 children took part in both internal and external radiation exposure screening programs between 1^st April 2012 to 31^st March 2013. The level of additional effective doses ranged from 0.025 to 3.49 mSv/year with the median of 0.70 mSv/year. While 99.7% of the children (n = 878) were not detected with internal contamination, 90.3% of the additional effective doses was the result of external radiation exposure. This finding is relatively consistent with the doses estimated by the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR). The present study showed that the level of annual additional effective doses among children in Minamisoma has been low, even after the inter-individual differences were taken into account. The dose from internal radiation exposure was negligible presumably due to the success of contaminated food control.     

Extracellular Matrix and Growth Factors Improve the Efficacy of Intramuscular Islet Transplantation

BackgroundThe efficacy of intramuscular islet transplantation is poor despite being technically simple, safe, and associated with reduced rates of severe complications. We evaluated the efficacy of combined treatment with extracellular matrix (ECM) and growth factors in intramuscular islet transplantation.MethodsMale BALB/C mice were used for the in vitro and transplantation studies. The following three groups were evaluated: islets without treatment (islets-only group), islets embedded in ECM with growth factors (Matrigel group), and islets embedded in ECM without growth factors [growth factor-reduced (GFR) Matrigel group]. The viability and insulin-releasing function of islets cultured for 96 h were significantly improved in Matrigel and GFR Matrigel groups compared with the islets-only group.ResultsBlood glucose and serum insulin levels immediately following transplantation were significantly improved in the Matrigel and GFR Matrigel groups and remained significantly improved in the Matrigel group at postoperative day (POD) 28. On histological examination, significantly decreased numbers of TdT-mediated deoxyuridine triphosphate-biotin nick end labeling-positive islet cells and significantly increased numbers of Ki67-positive cells were observed in the Matrigel and GFR Matrigel groups at POD 3. Peri-islet revascularization was most prominent in the Matrigel group at POD 14.ConclusionsThe efficacy of intramuscular islet transplantation was improved by combination treatment with ECM and growth factors through the inhibition of apoptosis, increased proliferation of islet cells, and promotion of revascularization.     

Peptide substrates for G protein-coupled receptor kinase 2

G protein-coupled receptor kinases (GRKs) control the signaling and activation of G protein-coupled receptors through phosphorylation. In this study, consensus substrate motifs for GRK2 were identified from the sequences of GRK2 protein substrates, and 17 candidate peptides were synthesized to identify peptide substrates with high affinity for GRK2. GRK2 appears to require an acidic amino acid at the −2, −3, or −4 positions and its consensus phosphorylation site motifs were identified as (D/E)X_1–3(S/T), (D/E)X_1–3(S/T)(D/E), or (D/E)X_0–2(D/E)(S/T). Among the 17 peptide substrates examined, a 13-amino-acid peptide fragment of β-tubulin (DEMEFTEAESNMN) showed the highest affinity for GRK2 (K_m, 33.9 μM; V_max, 0.35 pmol min⁻¹ mg⁻¹), but very low affinity for GRK5. This peptide may be a useful tool for investigating cellular signaling pathways regulated by GRK2.