Spindle formation and microtubule organization during first division in reconstructed rat embryos produced by somatic cell nuclear transfer

The present study was conducted to demonstrate the spindle formation and behavior of chromosomes and microtubules during first division in reconstructed rat embryos produced by somatic cell nuclear transfer (SCNT) with cumulus cell nuclei. To demonstrate the effect of oocyte aging after ovulation on the cleavage of SCNT embryos, micromanipulation was carried out 11, 15 and 18 h after injection of hCG. SCNT oocytes were activated by incubation in culture medium supplemented with 5 microM ionomycin for 5 min followed by treatment with 2 mM 6-dimethylaminopurine (6-DMAP) in mR1ECM for 2-3 h. For immunocytochemical observation, the SCNT embryos were incubated with monoclonal anti-alpha-tubulin antibody and then fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Cleavage rates were significantly higher for oocytes collected after 15 and 18 h rather than for those collected 11 h after injection of hCG (56 and 53%, respectively vs. 28%; P<0.05). Premature chromosome condensation occurred before activation of the SCNT oocytes, but adequate spindle formation was only rarely observed. The distribution of microtubules in SCNT embryos after activation was different from those of fertilized and parthenogenic oocytes, i.e., a dense microtubule organization shaped like a ring was observed. Eighteen to 20 h post-activation, most SCNT embryos were in the 2-cell stage, but no nucleoli were clearly visible, which was quite different from the fertilized oocytes. In addition, first division with and without small cellular bodies containing DNA was observed in the rat SCNT embryos in some cases. The present study suggests that reorganization of transferred nuclei in rat SCNT embryos may be inadequate in terms of formation of the mitotic assembly and nucleolar reorganization.     

Effect of Extraction Variables on the Biodegradable Chelant-Assisted Removal of Toxic Metals from Artificially Contaminated European Reference Soils

Development of aminopolycarboxylate chelants (APCs) having enhanced biodegradability is gaining increasing focus to replace the EDTA and its homologs with those used widely for the ex situ treatment of contaminated soils and are potential eco-threats. The paper reports the chelant-assisted extraction of the toxic metals (Cd, Cu, Pb, and Zn) from the metal-spiked European reference soils (Eurosoil 1 and Eurosoil 4) using biodegradable APCs, namely EDDS, GLDA, and HIDS. The effects of chelant-to-metal molar ratio, solution pH, and metal/chelant stability constants were evaluated, and compared with that of EDTA. The selectivity aptitude of the biodegradable chelants towards the toxic metals was assumed from the speciation calculations, and a proportionate correlation was observed at neutral pH. Pre- and post-extractive solid phase distributions of the target metals were defined using the sequential extraction procedure and dissolution of metals from the theoretically immobilized fraction was witnessed. The effect of competing species (Al, Ca, Fe, Mg, and Mn) concentrations was proven to be minimized with an excess of chelant in solution. The highlight of the outcomes is the superior decontamination ability of GLDA, a biodegradable APC, at minimum chelant concentration in solution and applicability at a wide range of pH environments.     

A serosurvey of Borna disease virus infection in wild rats by a capture ELISA

For a serological diagnostic test for Borna disease (BD), we developed a capture ELISA with specificity and sensitivity based on detection of antibodies against BD virus (BDV) p40 protein. Using our capture ELISA system, the antibody response of rats inoculated intracerebrally with BDV at 4 weeks after birth showed a sharp increase from 1 to 4 weeks postinoculation (p.i.) and a steady level after 5 weeks p.i. To investigate prevalence of BDV infection among wild rats, we examined sera of Rattus norvegicus in Kami-iso town, Oshima district, Hokkaido, suggesting that rats in this area had not been infected by BDV.     

Genetic and biological comparison of tick-borne encephalitis viruses from Hokkaido and far-eastern Russia

We compared the biological properties of Oshima 5-10 (tick-borne encephalitis [TBE] virus isolated in Hokkaido, Japan) and Sofjin-HO (Far-Eastern subtype TBE virus) including plaque formation, virus replication and virus protein synthesis in BHK-21 cell cultures to reveal strain differences. We also determined the complete nucleotide sequences of both strains and compared the deduced amino acid sequences. Plaques of Oshima 5-10 were smaller than those of Sofjin-HO. Virus titers in culture fluid of Oshima 5-10 were 1/100 of those of Sofjin-HO at 9 and 12 hr after infection. Less viral protein and RNA syntheses of strain Oshima 5-10 was observed than with Sofjin-HO. Genetic analysis revealed 1.4% of amino acids to differ with Sofjin-HO. No difference between the two strains was detected in the motif sequence of the viral enzyme, cleavage sites of viral protein or glycosylation sites of NS1.     

Defense Mechanism of Rice Plant to Respiratory Inhibition by a Promising Candidate Blasticide, SSF126

A new candidate systemic rice blasticide, SSF126, dose-dependently inhibited NADH oxidation by submitochondrial particles from rice roots. However, oxidation by the root submitochondrial particles was much less susceptible to SSF126 compared to that by submitochondrial particles from mycelial cells ofPyricularia grisea, a pathogen causing rice blast. Interestingly, SSF126 did not completely suppress the respiration by intact rice roots, and the respiratory activity of the roots recovered from inhibition time-dependently even in the presence of SSF126 at concentrations sufficient to fully block oxidation by the submitochondrial particles. This recovery was not due to selective extrusion of SSF126 from the roots, but to switching from the cytochrome pathway to the alternative cyanide-resistant respiratory pathway. In immunoblots of the alternative oxidase, high molecular mass species were detected in the mitochondria from rice roots in addition to low molecular mass species. Quantification of high and low molecular mass species revealed an increase in the amount of a protein corresponding to a 36-kDa species equivalent to a decrease in the amount of a protein corresponding to a 72-kDa species following a 5-h incubation with SSF126. This conversion of the alternative oxidase to the low molecular mass species in the mitochondria was correlated with the respiratory recovery found in intact rice roots, suggesting that the low molecular mass species is the active form of the alternative oxidase and the high molecular mass species is the inactive form. These results suggest that rice plants can block the severe injury caused by limiting the cytochrome pathway by SSF126 through utilization of the alternative pathway promoted by the interconversion of the alternative oxidase protein.     

Effects of heating on the interaction of lipid and zein in a dry powder system

The effects of heat treatment on the interaction of lipid and zein in a dry powder system were investigated. Linolenic acid ethyl ester (LAE) was mixed with the zein powder. The glass transition temperature (T(g)) for the dry powder zein was shown to be approximately 107 degrees C by differential scanning calorimetry. The thermogram of the zein-LAE mixed powder showed an exothermic transition near the T(g) of zein. Fourier transform infrared (FT-IR) spectroscopy was used for detecting the structural changes in zein by heat treatment, that is, elevating the temperature from 25 to 160 degrees C. The heat treatment of the powdery zein with and without LAE caused increases in the alpha-helix, beta-turn, and beta-sheet, concomitant with decreases in the intermolecular hydrogen-bonded beta-sheet and random coil. Such changes in the secondary structure were more drastic for the powder with LAE. The heating of the zein-LAE mixed powder also caused decreases in the peaks originating from LAE in the FT-IR spectra. These results suggest that the heat treatment induced the interaction of the zein and LAE in the powdery system. The influence of heating on the antioxidative activity of dry powder zein was studied by measurements of the peroxide value. When zein-LAE mixed powder was heated before storage, the oxidation of LAE was inhibited for 7 days, whereas LAE was oxidized within 1 day in the absence of heat treatment.     

Inhibition of mouse hepatitis virus multiplication by an oligonucleotide complementary to the leader RNA.

An oligonucleotide complementary to a leader RNA of positive-stranded mouse hepatitis virus (MHV) was tested for the effect on the viral multiplication in mouse DBT cells. A 14-mer antisense oligonucleotide contained a sequence complementary to the conserved pentanucleotide sequence, UCUAA, of the leader RNA. A treatment of MHV-infected cells with the antisense oligonucleotide at concentrations from 5 to 25 microM had an inhibitory effect on the viral multiplication and reduced the synthesis of viral specific mRNA and proteins. No inhibitory effect was observed when the cells were treated with sense oligonucleotide and oligonucleotide which contained unrelated sequences at concentrations from 1 to 10 microM. These results showed that antisense oligonucleotide against the leader RNA reduced the multiplication of positive-stranded RNA virus, MHV.     

Comparison of virulence of various hantaviruses related to hemorrhagic fever with renal syndrome in newborn mouse model

The virulence of hantaviruses that are antigenically related but have different genetic characteristics from the prototype of hantavirus, Hantaan (HTN) virus, was examined in newborn mice. The H5 and B78 strains of the Amur (AMR) genotype, the Bao14 strain of the Far East (FE) genotype, and the 76-118 strain of HTN virus were inoculated subcutaneously (1focus-forming unit; FFU) into newborn mice. All of the AMR and FE genotype viruses inoculated mice were died by 16 days post-infection (dpi) and 21 dpi, respectively, while 50% of the HTN virus inoculated mice survived until 30 dpi. The AMR and FE genotype viruses inoculated mice had high viral titers in the lung (1.3x10(6) to 1.3x10(8) FFU/gram [g] tissue) , brain (2.1x10(7) to 1.2x10(9) FFU/g tissue), and kidney(2.5x10(5) to 1.6x10(7) FFU/g tissue), and showed a detectable level of antibodies (titers 1:16-1:32) at 14 dpi. In contrast, the HTN virus infected mice had viruses only in the lungs at low titers (1.1-5.3x10(5) FFU/g tissue). Observations of body-weight changes revealed that the AMR and FE genotype viruses inoculated mice had lower growth rates than the HTN virus inoculated mice. These data suggest that the AMR and FE genotype viruses are more virulent than the HTN virus in newborn mice.     

Gene expressions of canine angiopoietin-1 and -2 in normal tissues and spontaneous tumours

The angiopoietin (Ang) family of proteins are central to the regulation of angiogenesis. The purposes of this study were to determine cDNA sequences of canine Ang-1 and Ang-2 and investigate their expressions in normal tissues and spontaneous tumours. The cDNA sequences of canine Ang-1 and Ang-2 were 1,494 and 1,488 bp, and the deduced amino acid sequences were 497 and 495 residues, respectively. The cDNA sequences of canine Ang-1 and Ang-2 showed high homology with those of the other mammalian species. Canine Ang-1 and Ang-2 mRNA were detectable in all 22 normal tissues and spontaneous tumours. Higher mRNA expression level of canine Ang-2 was demonstrated in mammary simple carcinomas, haemangiosarcoma and hepatocellular carcinoma in comparison with normal tissues.     

Evaluation of the clinical efficacy of benazepril in the treatment of chronic renal insufficiency in cats

BACKGROUND: Chronic renal insufficiency (CRI) is a common disease in cats. Angiotensin-converting enzyme inhibitors (ACEI) have beneficial effects in humans with CRI by reducing the loss of protein in the urine and increasing life expectancy. HYPOTHESIS: The ACEI benazepril has beneficial effects on survival, clinical variables, or both as compared with placebo in cats with CRI. ANIMALS: 61 cats with naturally occurring CRI. METHODS: The cats were enrolled into a prospective, randomized, double-blind, placebo-controlled clinical trial. Cats received placebo or 0.5-1 mg/kg benazepril once daily for up to 6 months. RESULTS: Urine protein/urine creatinine ratios were significantly (P < .05) lower with benazepril as compared with placebo at days 120 and 180. Three cats with placebo and 1 cat with benazepril were removed prematurely from the study because of deterioration of CRI or death. Cats were classified into 4 stages of CRI according to the International Renal Interest Society (IRIS) classification scheme. Incidence rates of cats with IRIS classification stage 2 or stage 3 that remained in stage 2 or 3 without progressing to stage 4 were higher with benazepril (93 +/- 5%) as compared with placebo (73 +/- 13%). CLINICAL IMPORTANCE: These results suggest a potential for benazepril to delay the progression of disease, extend survival time, or both in cats with CRI.