Emergence of Pathogenic Coronaviruses in Cats by Homologous Recombination between Feline and Canine Coronaviruses

Type II feline coronavirus (FCoV) emerged via double recombination between type I FCoV and type II canine coronavirus (CCoV). In this study, two type I FCoVs, three type II FCoVs and ten type II CCoVs were genetically compared. The results showed that three Japanese type II FCoVs, M91-267, KUK-H/L and Tokyo/cat/130627, also emerged by homologous recombination between type I FCoV and type II CCoV and their parent viruses were genetically different from one another. In addition, the 3′-terminal recombination sites of M91-267, KUK-H/L and Tokyo/cat/130627 were different from one another within the genes encoding membrane and spike proteins, and the 5′-terminal recombination sites were also located at different regions of ORF1. These results indicate that at least three Japanese type II FCoVs emerged independently. Sera from a cat experimentally infected with type I FCoV was unable to neutralize type II CCoV infection, indicating that cats persistently infected with type I FCoV may be superinfected with type II CCoV. Our previous study reported that few Japanese cats have antibody against type II FCoV. All of these observations suggest that type II FCoV emerged inside the cat body and is unable to readily spread among cats, indicating that these recombination events for emergence of pathogenic coronaviruses occur frequently.     

Influence of hydraulic retention time on anaerobic digestion of pretreated sludge

Continuous anaerobic digestion of waste activated sludge pretreated at low temperatures below 100°C increased methane generation by 30%. pH values of the digestion mixture increased, approximately from 0.3 to 0.55 by pretreatment, although its volatile fatty acids concentration was greater than the control. An abrupt increase in propionate : acetate ratio in digestion stage (e.g. from less than 1:1 to over 3.5 :1), provided a reliable indicator for impending failure.This revised version was published online in October 2005 with corrections to the Cover Date.     

Conformation of guanosine 5'-diphosphate as bound to a human c-Ha-ras mutant protein: a nuclear Overhauser effect study

1H NMR spectra of a GDP/GTP-binding domain of human c-Ha-ras gene product (residues 1-171) in which glutamine-61 was replaced by leucine [ras(L61/1-171) protein] were analyzed. By one-dimensional and two-dimensional homonuclear Hartmann-Hahn spectroscopy and nuclear Overhauser effect (NOE) spectroscopy of the complex of the ras(L61/1-171) protein and GDP, the ribose H1', H2', H3', and H4' proton resonances of the bound GDP were identified. The guanine H8 proton resonance of the bound GDP was identified by substituting [8-2H]GDP for GDP. The dependences of the H1' and H8 proton resonance intensities on the duration of irradiation of the H1', H2', H3', and H8 protons were measured. By numerical simulation of these time-dependent NOE profiles, the conformation of the protein-bound GDP was elucidated; the guanosine moiety takes the anti form about the N-glycosidic bond with a dihedral angle of chi = -124 +/- 2 degrees and the ribose ring takes the C2'-endo form. Such an analysis of the conformation of a guanine nucleotide as bound to a GTP-binding protein will be useful for further studies on the molecular mechanism of the conformational activation of ras proteins on ligand substitution of GDP with GTP.     

Identification and partial purification of a novel tumor-derived protein that induces tissue factor on cultured human endothelial cells

Conditioned medium of a human bladder carcinoma cell line (J82) was found to induce tissue factor synthesis in cultured human umbilical vein endothelial cells (HUVEC). A protein present in the J82 conditioned medium was partially purified by FPLC using a combination of MONO Q and Superose 6 columns. The bladder carcinoma-derived cytokine (BCDC) exhibited a Mr of 22 kDa by gel permeation HPLC. Polyclonal antibody against either interleukin-1, tumor necrosis factor, or transforming growth factor-beta failed to inhibit the ability of the conditioned medium to induce HUVEC tissue factor activity, suggesting that this tumor cell line secretes a novel cytokine responsible for HUVEC tissue factor induction.     

Selection of High Ubiquinone 10-Producing Strain of Tobacco Cultured Cells by Cell Cloning Technique

A novel enzyme, which was named N^α-benzyloxycarbonyl amino acid urethane hydrolase, was purified from a cell-free extract of Streptococcus faecalis R ATCC 8043, using N^α-benzyloxycarbonyl glycine as substrate. The enzyme was purified 1300-fold with an activity yield of 8%. The purified enzyme was homogeneous by disc electrophoresis. The molecular weight of the native enzyme is about 220,000 by gel filtration, and a molecular weight of 32,000 was determined for the reduced and denatured enzyme by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point was 4.48. The enzyme was inhibited by p-chloromercuribenzoate. The presence of divalent cations (i.e., Co²⁺ or Zn²⁺) is essential for its activity.     

Skin temperature rise induced by calcitonin gene-related peptide in gonadotropin-releasing hormone analogue-treated female rats and alleviation by Keishi-bukuryo-gan, a Japanese herbal medicine

The effects of Keishi-bukuryo-gan on calcitonin gene-related peptide (CGRP)-induced elevation of skin temperature were investigated in gonadotropin-releasing hormone (GnRH) analogue-treated female rats. Leupline (1.0 mg/kg) as the GnRH analogue was subcutaneously (s.c.) injected into female rats. After Keishi-bukuryo-gan (100-1,000 mg/kg, p.o.) or 17beta-estradiol (0.010 mg/kg, s.c.) was administered to GnRH analogue-treated rats for 14 days, CGRP-induced skin temperature elevation, concentration of plasma 17beta-estradiol and pituitary gonadotropin (luteinizing hormone; LH, and follicle stimulating hormone; FSH) were measured. In addition, effects of 17beta-estradiol and Keishi-bukuryo-gan on the proliferation of estrogen-dependent human breast cancer (MCF-7) cells were investigated under in vitro conditions. GnRH analogue significantly lowered the concentrations of plasma 17beta-estradiol and pituitary gonadotropins. Tissue weights of the ovaries and uterus were also decreased by the analogue. Under the condition of estrogen deficiency, intravenous (i.v.) injection of exogenous CGRP (10 microg/kg) elevated the skin temperature of the hind paws more significantly than it did in sham-treated control rats. Estrogen supplementation inhibited this elevation of skin temperature with restoration of both the lowered plasma estrogen level and the decreased uterine weight in GnRH analogue-treated rats. On the other hand, Keishi-bukuryo-gan inhibited the elevation of skin temperature in a dose-dependent manner without restoring the plasma estrogen level and uterine weight. In addition, in an in vitro study, MCF-7 cells proliferated in a dose-dependent manner by the addition of 17beta-estradiol (10(-13)-10(-8) M) to the medium. However, Keishi-bukuryo-gan (10(-6)-10(-4) mg/ml) did not activate the MCF-7 cell proliferation. These results suggest that Keishi-bukuryo-gan, which does not exhibit estrogen activity, may be useful for the treatment of hot flashes in women who are undergoing medical ovariectomy with a GnRH analogue.     

Segmental body composition assessment for obese Japanese adults by single-frequency bioelectrical impedance analysis with 8-point contact electrodes

This study aimed to determine the accuracy of segmental body composition variables estimated by single-frequency BIA with 8-point contact electrodes (SF-BIA8), compared with dual-energy X-ray absorptiometry (DXA). Subjects were 72 obese Japanese adults (43 males and 29 females) aged 30 to 66 years. Segmental body composition variables (fat free mass: FFM, fat mass: FM, and percent fat mass: %FAT) were measured by these techniques. The correlations between impedance values and FFM measured by DXA were calculated. To examine the consistency in predicted values (SF-BIA8) with the reference (DXA), significant mean differences were tested by t-test and the degree of the difference was assessed by effect size. Correlations between the reference and predicted values were calculated. Additionally, the standard error of estimation (SEE) when estimating the reference from the predictor and the relative value of the SEE to the mean value of the DXA measurement (%SEE) were calculated. Systematic error was examined by Bland-Altman plots. High correlations were found between impedance and FFM measured by SF-BIA8. FFM in the extremities showed high correlations with the reference values, but systematic error was found. SF-BIA8 tended to overestimate FFM in the trunk. The consistencies in %FAT and FM with the reference value are inferior to those for FFM, and SEE values in %FAT and FM were greater than those for FFM. The accuracy of the estimated values in the trunk (FFM, %FAT, and FM) are inferior to those of the total body and extremities.     

Cloning of human muscle phosphofructokinase cDNA

Three overlapping cDNA clones for human muscle phosphofructokinase (HMPFK) covering the complete coding sequence were isolated. The sequence included a poly(A) tail, a 399 bp 3'-untranslated region, a 2337 bp coding region for 779 amino acid residues and a part of the 5'-untranslated region. Homologies between HMPFK and rabbit muscle phosphofructokinase (RMPFK) were 96% of the amino acids and 89% of the nucleotides in the coding region. Like RMPFK, HMPFK also possessed the internal homology between C- and N-halves in its primary structure. Cloning of HMPFK cDNA will help to identify the molecular defect in patients with glycogenosis type VII (HMPFK deficiency).