Application of the fluorometric DNA quantitative assay using 4'6-diamidine-2-phenylindole for the evaluation of lymphocyte blastogenic response

A fluorometric DNA microassay using the fluorescent reagent 4'6-diamidine-2-phenylindole dihydrochloride (DAPI) was developed for evaluating a lymphocyte DNA synthesis assay. Triton X-100 (0.05 per cent) was added to the cultured lymphocytes, then DAPI (0.8 micrograms ml-1) was added as a DNA-specific fluorescent intercalating agent. Results are expressed as fluorescence intensity (FI) and stimulation index. FIs measured by DAPI fluorometric assay were highly correlated with [3H]-thymidine incorporation (r = 0.868, P less than 0.01, n = 28). The method is simple, reliable and widely applicable for evaluating blastogenic responses.     

Efficacy of ivermectin against live mites and eggs of Sarcoptes scabiei in pigs

Sows infested with Sarcoptes scabiei var. suis (ssvs) were treated with 75, 150 and 300 micrograms/kg of ivermectin by a single subcutaneous injection at the neck region. Compared to the numbers of mites and eggs just before injection, those on post treatment weeks (PTW) 1, 2 and 4 showed significant decreases. Especially at 300 micrograms/kg, the counts showed almost all mites and eggs were eradicated on PTW 1, manifesting ivermectin to possess potential effect on ssve without apparent abnormal side effect. Potential mitocide effect of ivermectin on ssvs was revealed.     

Isolation of Fukuoka virus, a member of the Kern Canyon serogroup viruses of the family Rhabdoviridae, from cattle.

Four virus strains with identical antigenic properties were isolated from blood samples of 4 sentinel calves having a fever and leukopenea in cultures of HmLu-1 cells derived from baby hamster lung. The virus was identified as Fukuoka virus, classified as a member of the Kern Canyon serogroup viruses of the family Rhabdoviridae, on the basis of its antigenic properties.     

Nucleocapsid-like structures of Ebola virus reconstructed using electron tomography

Electron tomography (ET) is a new technique for high resolution, three-dimensional (3D) reconstruction of pleiomorphic macromolecular complexes, such as virus components. By employing this technique, we resolved the 3D structure of Ebola virus nucleocapsid-like (NC-like) structures in the cytoplasm of cells expressing NP, VP24, and VP35: the minimum components required to form these NC-like structures. Reconstruction of these tubular NC-like structures of Ebola virus showed them to be composed of left-handed helices spaced at short intervals, which is structurally consistent with other non-segmented negative-strand RNA viruses.     

Effects of heat stress on the redox status in the oviduct and early embryonic development in mice

This study examined the association between redox status in the oviduct and early embryonic death in heat-stressed mice. In Experiment 1, non-pregnant mice were heat-stressed at 35 C with 60% relative humidity for 12, 24, or 36 h, and the maternal redox status was verified by measuring the levels of reactive oxygen species (ROS) and free radical scavenging activity (FRSA) in the oviduct, and thiobarbituric acid reactive substances (TBARS) and glutathione peroxidase (GSH-Px) activity in the liver. In Experiment 2, zygotes were collected from mice heat-stressed for 12 h on the day of pregnancy, and their developmental abilities were assessed in vitro, along with the intensity of DNA damage at the 2-cell stage. The TBARS value and GSH-Px activity in the liver, and ROS level in the oviduct were significantly higher in heat-stressed mice, and this increase appeared to depend on the duration of the heat stress. Maternal heat stress significantly reduced the percentage of zygotes that developed to the morula and blastocyst and the total cell number in the blastocyst. In addition, DNA damage at the 2-cell stage was significantly higher in maternally heat-stressed embryos. These results suggest that heat stress induces systemic changes in redox status in the maternal body, and the resultant increase in oxidative stress in the oviduct is possibly involved in heat stress-induced early embryonic death .     

Insulin-like growth factor-I stimulated DNA replication in mouse endometrial stromal cells

Much evidence has suggested that sex steroid hormone-induced growth of uterine cells is mediated by polypeptide growth factors synthesized in uterine tissues. The present study aimed to clarify the effect of insulin-like growth factor-I (IGF-I) on the proliferation of mouse endometrial stromal cells obtained from immature mice. IGF-I and IGF-I receptor (type I) mRNAs were detected in the endometrial stromal cells. IGF-I increased bromodeoxyuridine (BrdU) uptake in the endometrial stromal cells, indicating an increase in DNA replication. E2 increased IGF-I mRNA levels in the endometrial stromal cells. IGF-I receptor is a tyrosine kinase receptor, and treatment with genistein, a tyrosine kinase inhibitor, reduced IGF-I-induced BrdU-uptake in the endometrial stromal cells. IGF-I signaling pathways involve mitogen-activated protein (MAP) kinase and phosphatidylinositol-3 kinase (PI-3 kinase). Treatment with 10(-7) M of the MAP kinase inhibitor PD098059 and 10(-5) M of the PI-3 kinase inhibitor LY294002 decreased IGF-I-induced BrdU-uptake in the endometrial stromal cells. However, LY294002 (10(-5) M) also decreased the BrdU-uptake in the absence of IGF-I treatment. These results suggest that endometrial IGF-I is involved in the proliferation of endometrial stromal cells in a paracrine or autocrine manner, and that the MAP kinase pathway is involved in DNA replication of endometrial stromal cells.     

Enzyme-linked immunosorbent assay for detection of canine chromogranin A by use of immunological cross-reactivity of rabbit anti-bovine chromogranin A antibody

Bovine and canine chromogranin A were extracted and purified from each specie's adrenal glands. Isolated bovine 70 kDa protein showed 100% identity to bovine CgA reported previously, whereas isolated canine 68 kDa protein showed 83.3% identity to bovine CgA by the NH(2)-terminal amino acid sequence analysis. Rabbit antibody to purified bovine protein (CgA) was found to immunologically cross-reacted with purified canine protein (CgA). In sandwich ELISA with anti-bovine CgA, concentration-dependent curves were obtained ranging from 0.3 to 20 mug/ml for canine CgA. From these findings, sandwich ELISA with anti-bovine CgA is found to be useful to determine the concentration of canine CgA.     

Intracranial elastance in isoflurane-anesthetized horses

OBJECTIVE: To determine whether high intracranial pressure (ICP) during spontaneous ventilation (SV) in anesthetized horses coincides with an increase in intracranial elastance (ie, change in ICP per unit change of intracranial volume). ANIMALS: 6 adult horses. PROCEDURE: Anesthesia was induced and maintained in each horse for 5 hours with isoflurane at a constant dose equal to 1.2 times the minimum alveolar concentration. Direct ICP measurements were obtained by use of a strain gauge transducer inserted in the subarachnoid space, and arterial blood pressure was measured from a carotid artery. Physiologic responses were recorded after 15 minutes of normocapnic controlled ventilation (CV) and then after 10 minutes of SV. Aliquots (3 mL) of CSF were removed from each horse during SV until ICP returned to CV values. Slopes of pressure-volume curves yielded intracranial elastance. RESULTS: Intracranial elastance ranged from 0.2 to 3.7 mm Hg/mL after removal of the first aliquot of CSF Slopes of pressure-volume curves were largest following removal of the initial CSF aliquot, but shallow portions of curves were detected at relatively high ICPs (25 to 35 mm Hg). A second-order relationship between SV ICP and initial intracranial elastance was found. CONCLUSIONS AND CLINICAL RELEVANCE: In horses anesthetized with isoflurane, small changes in intracranial volume can cause large changes in ICP Increased intracranial elastance could further exacerbate preexisting intracranial hypertension. However, removal of small volumes of CSF may cause rapid compensatory replacement from other intracranial compartments, which suggests steady-state maintenance of an increase in intracranial volume during isoflurane anesthesia in horses.     

The influence of age and body mass index on relative accuracy of energy intake among Japanese adults

OBJECTIVE: To examine relationships between the ratio of energy intake to basal metabolic rate (EI/BMR) and age and body mass index (BMI) among Japanese adults. DESIGN: Energy intake was assessed by 4-day semi-weighed diet records in each of four seasons (16 days in total). The EI/BMR ratio was calculated from reported energy intake and estimated basal metabolic rate as an indicator of reporting accuracy. SETTING: Residents in three areas in Japan, namely Osaka (urban), Nagano (rural inland) and Tottori (rural coastal). SUBJECTS: One hundred and eighty-three healthy Japanese men and women aged > or =30 years. RESULTS: The oldest age group (> or =60 years) had higher EI/BMR values than the youngest age group (30-39 years) in both sexes (1.74 vs. 1.37 for men; 1.65 vs. 1.43 for women). In multiple regression analyses, age correlated positively (partial correlation coefficient, beta = 0.012, P < 0.001 for men; beta = 0.011, P < 0.001 for women) and BMI correlated negatively (beta = -0.031, P < 0.001 for men; beta = -0.025, P < 0.01 for women) with EI/BMR. CONCLUSION: Age and BMI may influence the relative accuracy of energy intake among Japanese adults.