Glycophorins are the major sialoglycoproteins in red blood cell membranes, possessing various physiological and pathological roles. We examined membrane glycoproteins in canine red cells and cloned cDNAs for two major glycophorins, glycophorins A (GPA) and C (GPC) from bone marrow cells. Periodic acid-Schiff staining and immunoblotting analyses showed that canine red cell membranes contained several glycoproteins immunoreactive to an anti-bovine GPC antibody, whereas the most abundant sialoglycoproteins, the candidates for GPA, did not react with an anti-human GPA antibody. The amino acid sequences of the extracellular domains of GPA and GPC had no significant homology to those from other mammalian species, including humans, and had O-linked and/or N-linked glycosylation sites. On the other hand, the C-terminal cytoplasmic domain and/or the transmembrane helices of GPA and GPC were conserved among species, indicating some functional significance of those regions in red cell membranes that include dimerization of GPA in the membrane-spanning region, and association of GPC with membrane skeletal proteins through binding with protein 4.1 and p55 in the cytoplasmic domain. These findings provide insights for clinical studies to evaluate the involvement of GPA and GPC in the pathogenesis of red cell diseases.

alpha-Hemoglobin stabilizing protein (AHSP) functions as the erythroid-specific molecular chaperon for alpha-globin. AHSP gene expression has been reported to be downregulated in hematopoietic tissues of animals suffering from prion diseases though the mechanism remains to be clarified. Herein, we demonstrate that MELhipod8 cells, a subclone of murine erythroleukemia (MEL) cells, have prion protein (PrPsup(C)) on the cell surface and have highly inducible expression of the AHSP and alpha- and beta-globin genes, resembling the expression pattern of the PrP and AHSP genes in bipotential erythroid- and megakaryocyte-lineage cells followed by erythroid differentiation in normal erythropoiesis. Moreover, MELhipod8 cells exhibit greater effective erythroid differentiation with a population of hemoglobinized normoblast-like cells than that observed for the parental MEL cells. These findings suggest that MELhipod8 cells could provide a mechanism for downregulation of the AHSP gene in prion diseases.

The health hazards of individual organophosphorus insecticides have been characterized by their acute toxicity, mainly by investigating their cholinesterase inhibition. However, the chronic effects of most of these toxicants on the drug-metabolizing enzymes have not been investigated. Profenofos (O-4-bromo-2-chlorophenyl O-ethyl S-propyl phosphorothioate) is an organophosphorus pesticide widely used in cotton cultivation. In the present study, we investigated the effect of profenofos on male-specific cytochrome P450 (CYP) enzymes in adult Wistar rats. We orally administered 17.8 mg/kg body weight, twice weekly for 65 days. Profenofos downregulated levels of hepatic and testicular CYP2C11 and CYP3A2 mRNA and protein expression. Testicular aromatase (CYP19A) mRNA was decreased in the profenofos-treated rats compared to controls. Overall, the present study suggests that profenofos acts as an endocrine disruptor of male-specific CYP enzymes and affects testosterone concentration, which implicates its deleterious effects on animal or human males chronically exposed to organophosphorus pesticide.

Nigella sativa (family Ranunculaceae ) is an annual plant that has been traditionally used on the Indian subcontinent and in Middle Eastern countries. In this study, we investigated the effect of N. sativa oil on the drug-metabolizing cytochrome P450 (CYP) enzymes and whether it has a protective effect against the acute hepatotoxicity of CCl4. Intraperitoneal injection of rats with CCl4 drastically decreased CYP2E1, CYP2B, CYP3A2, CYP2C11, and CYP1A2 mRNA and protein expressions. Oral administration of 1 ml/kg N. sativa oil every day for one week prior to CCl4 injection alleviated CCl4-induced suppression of CYP2B, CYP3A2, CYP2C11, and CYP1A2. Moreover, CCl4 increased iNOS and TNFalpha mRNA, while N. sativa oil administration for one week prior to CCl4 injection downregulated the CCl4-induced iNOS mRNA and up-regulated IL-10 mRNA. These results indicate that N. sativa oil administration has a protective effect against the CCl4-mediated suppression of hepatic CYPs and that this protective effect is partly due to the downregulation of NO production and up-regulation of the anti-inflamnatory IL-10.

To establish vaccine strains of H5 and H7 influenza viruses, A/duck/Hokkaido/Vac-1/04 (H5N1) [Vac-1/04 (H5N1)], A/duck/Hokhaido/Vac-3/07 (H5N1) [Vac-3/07 (H5N1)], and A/duck/Hokkaido/Vac-2/04 (H7N7) [Vac-2/04 (H7N7)] were generated from non-pathogenic avian influenza viruses isolated from migratory ducks. Vac-1/04 (H5N1) and Vac-3/07 (H5N1) were generated by genetic reassortment between H5N2 or H5N3 virus as an HA gene provider and H7N1 or H6N1 virus as an NA gene provider. Vac-2/04 (H7N7) was a genetic reassortant obtained using H7N7 and H9N2 viruses to give high growth character of the H9N2 virus in chicken embryonated eggs. The results of sequence analyses and experimental infections revealed that these H5N1 and H7N7 reassortant viruses were non-pathogenic in chickens and embryos, and had good growth potential in embryonated eggs. These viruses should be useful to develop vaccines against H5 and H7 highly pathogenic avian influenza viruses.

The sudden emergence of severe acute respiratory syndrome (SARS) at the end of 2002 resulted in 774 reported deaths from more than 8000 cases worldwide. As no effective vaccines or antiviral agents are available, the most effective measure to prevent the expansion of a SARS epidemic is the rapid diagnosis and isolation of SARS patients. To establish specific diagnostic methods, we generated nine clones of monoclonal antibodies to nucleocapsid protein (NP) of SARS-coronavirus (SARS-CoV). On immunofluorescent antibody assay and Western blotting analysis, none of the monoclonal antibodies showed cross-reactivity to authentic and recombinant NPs of human coronavirus (HCoV) 229E strain. To determine the region on the NP molecule where the monoclonal antibodies bind, we generated four truncated recombinant NPs and analyzed the reactivity between monoclonal antibodies and truncated NPs. Two monoclonal antibodies reacted with a truncated NP covering from amino acid residues 111 to 230, and seven reacted with another truncated NP covering from amino acid residues 221 to 340. Epitope mapping analysis indicated that monoclonal antibody SN5-25 recognized the amino acid sequence Qsup(245)TVTKKsup(250) on SARS-NP. Within the epitope, Q245, T246, V247, K249, and K250 appeared to form an essential motif for monoclonal antibody SN5-25 to bind. The information about binding sites and epitopes of monoclonal antibodies may be useful for the development of new diagnostic methods for SARS and for analyzing the function of N protein of SARS-CoV.

In the present study, we employed flow cytometry to evaluate the level of parasitemia of Babesia gibsoni infecting canine erythrocytes in vivo and in vitro by using fluorescent nucleic acid staining. Peripheral blood samples from a B. gibsoni-infected dog and cultured B. gibsoni parasitizing in canine erythrocytes were stained with a membrane-permeable fluorescent nucleic acid stain, SYTO16. In this study, we utilized normal canine erythrocytes (LK erythrocytes) and canine erythrocytes containing high concentrations of potassium, reduced glutathione, and some free amino acids (HK erythrocytes) as host cells for culture. In vivo parasitized cells were discriminated completely from unparasitized cells and a correlation (r=0.998) between the percentage of SYTO16-positive cells and parasitemia in vivo was observed. On the other hand, in vitro erythrocytes could not be divided clearly into parasitized and unparasitized cells. However, when LK erythrocytes were used as host cells, the percentage of SYTO16-positive cells was the almost same as, and was well correlated (r=0.932) with the level of parasitemia. When HK erythrocytes were used as host cells, the percentage of SYTO16-positive cells was almost half of, but was correlated (r=0.982) with the level of parasitemia. Therefore, we attempted to observe the changes in the percentage of parasitized cells after treatment with antiprotozoal drug or mitochondria inhibitors by using flow cytometry. The changes in the percentage of SYTO16-positive cells corresponded well with the changes of the level of parasitemia when the parasites in HK erythrocytes were cultured with each compound. The present results suggest that flow cytometric detection using SYTO16 is a rapid and reliable method for monitoring parasitemia both in vivo and in vitro.

Bovine nasal and oral discharges were used as samples for bovine viral diarrhea virus (BVDV) gene detection. Viral genes in serum (S), nasal discharge (N) and oral discharge (O) were quantified with real-time polymerase chain reaction using SYBR Green by the relative quantification method, and findings were compared among samples. Although the quantity of the BVDV gene in S was greater than those in N and O, all samples were available to identify persistently infected (PI) cattle with BVDV by reverse transcription polymerase chain reaction (RT-PCR). The swab samples were able to be stored for a few days at 4degC with a little decrease of amplification signal in RT-PCR. Oral swab sampling was easier than nasal swab sampling, and was also less uncomfortable for the cattle than other sampling methods without pain or unnecessary retention. This sampling method can be performed without any special technique and equipment. Therefore, the oral swab sampling method is useful for screening to detect BVDV PI cattle by RT-PCR.

Although mammals produce either sperm or eggs depending on their sex, newborn MRL/MpJ male mice contain oocytes within their testes. In our previous study, the testicular oocyte appears as early as day 0 afterbirth and has morphological characteristics as an oocyte such as zona pellucida and follicular epithelial cells. Based on the observation of F1 between MRL/MpJ and C57BL/6, one of the genes causing the appearance of testicular oocyte exists on the Y chromosome. In the present study, we found testicular oocytes within newborn AKR mice. We have also analyzed the Sry genes from several inbred mouse strains and identified a shortened glutamine repeat near the C-terminal region that is unique to MRL and AKR. These results suggest that polymorphism of glutamine repeat within SRY correlates with the appearance of testicular oocyte and this phenotype is derived from AKR, one of the original strains of MRL mice.

Information on steroid hormone receptor distribution in the uterus is essential to understand the roles of their ligands in pregnancy. This study examined the spatio-temporal localization of estrogen receptor alpha (ERalpha) and progesterone receptor (PR) in the uterus of sika deer (Cervus nippon) to determine the estrogen and progesterone action site during pregnancy. Ovaries and uteri were collected from 21 pregnant sika deer with single fetus and two corpora lutea, ranging from Day 20 to Day 207 of pregnancy. In addition, genital organs were also collected from three sika deer whose gestational status was unknown: one female had only one developing corpus luteum: =Day 4 (metestrus) and two females had two corpora lutea, one of which was at the developing stage equivalent to diestrus or early pregnancy: Day 7 (diestrus). Staining of ERalpha and PR was clear in all cell types during metestrus. During diestrus, the presence of ERalpha was also clear in deep glandular epithelium, stroma and myometrium, whereas it was suppressed in luminal epithelium and shallow glandular epithelium. Staining of PR was suppressed in luminal epithelium but was detectable in other cell types. Staining of ERalpha in all cell types and PR in luminal epithelium and glandular epithelium became undetectable by Day 28. PR was presented in stroma and myometrium throughout pregnancy. The distribution pattern of ERalpha and PR was different during diestrus from that in a ruminant. This could be attributed to estrogen secretion from the maturing and ovulating follicles in the presence of developed corpus luteum.