Glucose uptake activity in murine red blood cells infected with Babesia microti and Babesia rodhaini

The glucose uptake activity in Babesia rodhaini and B. microti - infected red blood cell (IRBC) was investigated in mice using 2-deoxy-D-glucose (2DOG) and L-glucose (L-Glc), a non-metabolizable analogue of D-glucose and non-incorporative glucose to non-infected RBC (NRBC), respectively. The uptake activities of both DOG and L-Glc were higher in IRBCs than those in NRBC. The concentration dependent uptake of 2DOG and L-Glc in both IRBC revealed a linear curve, indicating non-transporter mediated uptake. In addition, B. microti IRBC showed higher 2DOG uptake than B. rodhaini IRBC, whereas no difference was observed in L-Glc uptake. These results indicated that some new glucose uptake system, at least two systems, developed in both IRBC. The new systems were sodium independent, non-competitive to L-Glc, and sensitive to temperature. One of two systems had no kinetical difference between B. rodhaini and B. microti IRBC, however another one might have higher uptake activity in B. microti IRBC compared to that in B. rodhaini IRBC.     

A comparative study of the effects of glycerol and hydroxyethyl starch in canine red blood cell cryopreservation

Hydroxyethyl starch (HES) is a nonpenetrating extracellular cryoprotectant. In contrast to glycerol, it does not require labor-intensive removal from thawed red blood cells (RBCs) prior to transfusion. In this study, we compared glycerol and HES, and assessed HES as a substitute for glycerol in cryopreserved canine RBCs. The RBCs were preserved for 2 months in liquid nitrogen using a 20% (w/v) glycerol solution, and variable concentrations of HES solution. We evaluated the two cryoprotectants by the percentage of post-thaw hemolysis from the total free hemoglobin, saline stability, osmotic fragility, and by observing the erythrocyte morphology using a scanning electron microscope after thawing. The optimal concentration of HES was 12.5% (w/v) for the cryopreservation of canine RBCs. The thaw hemolysis, saline stability, and osmotic fragility index were 25.6 +/- 4.7%, 87.8 +/- 6.9%, and 0.445 +/- 0.024% NaCl respectively. These parameters resemble the results of RBCs frozen in a 20% (w/v) glycerol solution, which are 24.7 +/- 5.2%, 99.2 +/- 0.1%, and 0.485 +/- 0.023% NaCl respectively. From a morphological point of view, 12.5% (w/v) HES showed the best cryoprotection of RBCs compared to the other concentrations of HES. These results suggest that HES could be a possible substitute for glycerol for the cryopreservation of canine RBCs.     

Cathepsin gene expression in mouse placenta during the latter half of pregnancy

Gene expressions and their interaction are complex and have not been definitely clarified in the placenta. To identify interactions of gene products previously not studied, we applied cDNA subtraction analyses to the placenta between days 12 and 16, days 12 and 14, days 14 and 16 of pregnancy. Among subtracted cDNAs cathepsin M, Q and R in PECs were specifically identified on days 14 and 16 pregnancy. All of these gene expressions exhibited a similar pattern to the mPL-II gene expression determined by northern blot and RT-PCR analyses. By means of in situ hybridization, these mRNAs were localized in the basal and labyrinth zones of the placenta on day 16 of pregnancy. Double staining studies of cathepsin Q or cathepsin R mRNA by in situ hybridization followed by immunohistochemical staining of mPL-II in the same section revealed that signals for cathepsin Q and cathepsin R mRNAs were colocalized in mPL-II immunopositive trophoblast cells in the basal and labyrinth zones of the placenta on day 16 of pregnancy. Possible association of cathepsins with mPL-II may play important roles in placental functions during the latter half of pregnancy in mice.     

Simultaneous observation of the GnRH pulse generator activity and plasma concentrations of metabolites and insulin during fasting and subsequent refeeding periods in Shiba goats

The time course of GnRH pulse generator activity and plasma concentrations of energy substrates and insulin were simultaneously observed in female goats during 4-day fasting and subsequent refeeding in the presence or absence of estrogen for a better understanding of the mechanism of energetic control of gonadotropin secretion in ruminants. The GnRH pulse generator activity was electrophysiologically assessed with the intervals of characteristic increases in multiple-unit activity (MUA volleys) in the mediobasal hypothalamus. In estradiol-treated ovariectomized (OVX+E2) goats, the MUA volley intervals increased as fasting progressed. Plasma concentrations of non-esterified fatty acid and ketone body increased, while those of acetic acid and insulin decreased during fasting. The MUA volley intervals and plasma concentrations of those metabolites and insulin were restored to pre-fasting levels after subsequent refeeding. In ovariectomized (OVX) goats, changes in plasma metabolites and insulin concentrations were similar to those in OVX+E2 goats, but the MUA volley intervals were not altered. The present results demonstrated that fasting suppressed GnRH pulse generator activity in an estrogen-dependent manner. Changes in plasma concentrations of energy substrates and insulin during fasting were associated with the GnRH pulse generator activity in the presence of estrogen, but not in the absence of the steroid in female goats.     

Occurrence of swine salmonellosis in postweaning multisystemic wasting syndrome (PMWS) affected pigs concurrently infected with porcine reproduction and respiratory syndrome virus (PRRSV)

Fourteen diseased pigs from four farms in which there had been an outbreak of salmonellosis were investigated. Granulomatous inflammation with depletion of lymphocytes was observed in the swollen lymph nodes in these pigs. Antigens to porcine circovirus type 2 (PCV2) were immunolabeled in the lesions along with detection of viral DNA as PCV2 by polymerase chain reaction (PCR). In addition, antigens to porcine reproductive respiratory syndrome virus (PRRSV) were immunodetected in the lungs and Salmonella Choleraesuis was isolated from the affected pigs. The nine salmonellosis affected pigs, five (55.6%) with salmonellosis and PMWS concurrently infected with PRRSV were much higher than those infected with salmonellosis and PMWS (22.2%) or with salmonellosis and PPPRV (22.2%).     

PCR-based discrimination of Toxoplasma gondii from pigs at an abattoir in Okinawa, Japan

To determine the prevalence of the 3 primary clonal lineages of Toxoplasma gondii (strain types I, II, and III) in pigs in Okinawa Prefecture, we analyzed lymph node samples that had been collected at an abattoir by PCR analysis using primers specific for the Toxoplasma gondii SAG2 locus. This study revealed the presence of this parasite in 57 out of 101 samples examined. Restriction fragment length polymorphism (RFLP) in PCR-amplified SAG2 products was used to group strains into one of the three genotypes of T. gondii. Genotypes I and II were equally predominant, accounting for 22 (44.9%) and 23 (46.9%) of 49 SAG2-positive samples, respectively, while the type III strain was found in only 4 (8.2%) of the 49 samples. The other 8 samples were indistinguishable by PCR-RFLP analysis. Polymorphisms for the 3 genotypes were confirmed at the sequence level for several samples using the sequences from the RH strain, the Beverley strain, and the C56 strain as references. On the other hand, the dihydropteroate synthase gene, which is responsible for sulfonamide resistance, was amplified in 40 of 54 SAG2-positive samples by PCR with the specific primers, and further RFLP and sequence analysis revealed that none of them carried the drug-resistant form of the dhps gene. This is the first report of genotyping of T. gondii distributed in Japan.     

Establishment and biological characterization of canine histiocytic sarcoma cell lines

Seven novel cell lines from canine histiocytic sarcoma (HS), three of which were disseminated cutaneous HS and four of which were synovial HS, were established. All of the established cell lines had the same morphological (by light and electron microscopic findings), cytochemical (alpha-naphthyl butyrate esterase-positive), and immunohistochemical (vimentin- and lysozyme-positive, and cyto-keratin-negative) characteristics as the original HS tumor cells. All of the established cell lines injected into nude mice subcutaneously produced solid tumors. Because the established cell lines also showed phagocytic and processing activities, the HS tumor cells appear to originate from the mononuclear phagocytic system cells, despite their differences in locations or organs.     

Expression and subcellular localization of GSE protein in germ cells and preimplantation embryos

We previously identified a novel gonad-specific expression gene (Gse) and investigated its expression during gametogenesis in the mouse testis and ovary. In this study, we generated a polyclonal antibody to GSE protein and determined the profiles of the protein's expression in germ cells and preimplantation embryos in detail using immunocytochemical and immunofluorescence staining. In a Western blot analysis, the anti-GSE antibody recognized long and short isoforms (approximately 27.6 kDa and 23.1 kDa) of the protein in the mouse testis and the long isoform in the ovary. In the mouse testis, GSE protein was expressed in spermatocytes I in the pachytene stage, round spermatids, and elongated spermatids. In the mouse ovary, the protein was located in the cytoplasm and nucleus of all oocytes regardless of the stage of the ovarian follicles. In preimplantation embryos from the pronuclear to blastocyst stage, however, GSE protein was mainly detected in the nuclei of cells. At the blastocyst stage, the protein was confirmed to have accumulated in the inner cell mass (ICM), whereas it had mostly disappeared from the trophectoderm (TE). These findings suggest that GSE protein may play a role in the establishment of nuclear totipotency and may be associated with early lineage specification.     

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.