Immunohistochemical detection of phosphatidylserine and thrombospondin on denucleating erythroblasts in rat bone marrow

The hypothesis that apoptotic factors play some roles in the denucleation of erythroblasts has been confirmed by the immunohistological detection of both phosphatidylserine and thrombospondin as phagocytosis-inducing factors in general apoptotic events. Both phosphatidylserine and thrombospondin were detected on the surface of cell membrane of mature erythroblasts, while thrombospondin was also detected in more immature erythroblasts. The intensities of their immune reactions increased as the erythroids matured. During denucleation, the positivities of both phosphatidylserine and thrombospondin were restricted on the surface of the cell membrane surrounding the protruding nuclei. Thus, the apoptotic process involves denucleation of erythroblasts and phosphatidylserine, and thrombospondin acts as phagocytosis-inducing factors in the denucleation event.     

Evaluation of a PCR-restriction fragment length polymorphism (PCR-RFLP) assay for molecular epidemiological study of Shiga toxin-producing Escherichia coli

In this study, we have evaluated our recently developed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for the molecular subtyping of Shiga toxin-producing Escherichia coli (STEC). A total of 200 STEC strains including O157 (n=100), O26 (n=50), O111 (n=10), and non-O26/O111/O157 (n=40) serogroups isolated during 2005-2006 in Japan, which were identified to be clonally different by pulsed-field gel electrophoresis (PFGE) were further analyzed by the PCR-RFLP assay in comparison to PFGE. Ninety-five of O157, 48 of O26, five of O111 and 19 of non-O26/O111/O157 STEC strains yielded one to three amplicons ranging from 6.0 to 15.5 kb in size by the specific primer set targeting region V which is located in the upstream of stx genes. These strains were classified into 41 (O157), 8 (O26), 4 (O111) and 17 (non-O26/O111/O157) groups based on the RFLP patterns obtained by subsequent restriction digestion, respectively. Although the discriminatory power of PCR-RFLP assay was somewhat less than that of PFGE, it is more convenient for molecular subtyping of STEC strains especially for O157, the most important serogroup implicated in human diseases, as well as to identify the outbreak-associated isolates because of its simplicity, rapidity, ease and good reproducibility.     

Molecular typing and antimicrobial resistance of Salmonella enterica subspecies enterica serovar Choleraesuis isolates from diseased pigs in Japan

Salmonella enterica serovar Choleraesuis (S. Choleraesuis) isolates derived from diseased pigs in Japan during 2001 and 2005 were analyzed for biotype, based on H(2)S production and dulcitol fermentation, pulsed-field gel electrophoresis (PFGE) profile, and antimicrobial resistance profile. S. Choleraesuis biotype Choleraesuis (biotype Choleraesuis) was classified into one genotype, while varietas Kunzendorf (var. Kunzendorf) was classified into two genotypes. The isolates of var. Kunzendorf belonging to one genotype were isolated in a limited area of Japan. Variation in the antimicrobial resistance pattern was observed in isolates of both biotypes Choleraesuis and var. Kunzendorf. We have also shown that the PFGE profile was associated with the biotype and isolation region of each isolate.     

Effect of CD4+CD25+ T cell-depletion on acute lethal infection of mice with Trypanosoma congolense

Despite the immense socio-economic repercussions of African trypanosomosis (AT), there is currently no effective control measure against the disease. Characterization of mechanisms governing resistance and/or susceptibility to AT could suggest interventions that might lead to more effective disease control. The present study was designed in an attempt to address the possible role of CD4+CD25+ T cells during an acute lethal infection of mice with Trypanosoma congolense, the causative agent of AT in domestic animals, through selective depletion using anti-CD25 monoclonal antibody. Accordingly, CD4+CD25+ T-cell-depletion resulted in a significant reduction or delay in parasitemia, pathology, and mortality, as compared to controls. The apparent resistance in CD4+CD25+-T-cell-depleted mice correlated with a profound suppression of Th2 cytokines in vitro and in vivo, culminating in a net Th1 cytokine environment. Cumulatively, these findings suggest that CD4+CD25+ T-cell- depletion improves the trypanotolerance of highly susceptible BALB/c mice acutely infected with the lethal T. congolense.     

Effect of ovary lipid of skipjack tuna on rat serum components after stress application

Lipid extracted from the ovary of skipjack tuna by the method that we developed is rich in phospholipid-type docosahexaenoic acid. The ovary lipid of skipjack tuna (OLS) was studied for its anti-stress activity in male Wistar rats, focusing on stress-related blood components: recovery from stress was examined after application of water immersion restraint stress. As a result, serum corticosterone (CORT) secretion was inhibited and decreased rapidly after stress application in rats given OLS compared with control rats. As CORT acts as a glucocorticoid, non-esterified fatty acid (NEFA) is expected to increase by stress application. However, the concentration tended to be lower in rats given OLS than in control rats. With respect to OLS concentration, OLS increased serum dehydroepiandrosterone, secretion concentration-dependently. In addition, as with the recovery study, it tended to inhibit the increase in NEFA. These results indicate that OLS may have an anti-stress activity against acute stress.     

Purification and characterization of feline ghrelin and its possible role

Ghrelin, a novel 28-amino acid peptide with an n-octanoyl modification at Ser3, has been isolated from rat and human stomach as an endogenous ligand for the growth hormone secretagogue receptor. Here, we purified feline ghrelin and examined its possible physiological role in cats. The major active form of feline ghrelin is a 28-amino acid peptide octanoylated (C8:0) at Ser3; except for one amino acid residue replacement, this structure is identical to those of rat and human ghrelins. However, much structural divergence in peptide length and fatty acid modification was observed in feline ghrelin: peptides consisting of 27 or 26 amino acids lacking Gln14 and/or Arg28 were found, and the third serine residue was modified by octanoic acid (C8:0), decanoic acid (10:0), or unsaturated fatty acids (C8:1, C10:1 and C10:2). In agreement with the structural divergence, two kinds of cDNA with different lengths were isolated. Administration of synthetic rat ghrelin increased plasma growth hormone levels in cats, with a potency similar to that in rat or human. Plasma levels of ghrelin in cats increased approximately 2.5-fold after fasting. The present study indicates the existence of structural divergence in feline ghrelin and suggests that, as in other animals, ghrelin may play important roles in GH release and feeding in cats.     

Comparison and phylogenetic analysis of the heat shock protein 70 gene of Babesia parasites from dogs

The heat shock protein 70 (hsp70) genes of Babesia gibsoni, B. canis canis, B. canis vogeli, and B. canis rossi isolated from infected dogs were cloned by polymerase chain reaction (PCR) and sequenced. In the nucleotide sequence and the predicted amino acid sequence of the gene, the parasites were very similar to each other. The nucleotide sequences of the hsp70 gene had more variety than those of 18S nuclear subunit ribosomal DNA (18S rDNA). A phylogenetic analysis of these sequences and comparisons with sequences from other Babesia and Theileria species revealed that all canine babesial isolates analyzed in the present study were closely related to each other and formed one cluster. Additionally, a phylogenetic analysis of Babesia and Theileria species showed that these parasites could be divided into three groups: group A including canine babesial isolates, B. divergens, B. odocoilei, B. bovis, B. caballi, and B. ovis; group B including Theileria annulata, T. orientalis, and T. cervi; and group C including B. microti and B. rodhaini. These results suggested that a phylogenetic analysis of the hsp70 gene sequence might be helpful in classifying Babesia and Theileria species, and that canine babesial isolates might be closely related to each other, indicating their evolution from the same ancestry.     

Effects of low root temperature on sap flow rate,soluble carbohydrates,nitrate contents and on cytokinin and gibberellin levels in root xylem exudate of sand‐grown tomato

Tomato (Lycopersicon esculuntum Mill.) grown in open fields in dry land areas or in non‐controlled greenhouses are subjected to substantial daily changes in root temperature. In the field, root‐zone temperatures fluctuate both diurnally and during the growing season. The purpose of this study was to monitor root‐zone temperature effects on tomato initial growth, transpiration, sap flow rate, leaf and air temperatures differences, nitrate accumulation, total nitrogen, and soluble carbohydrates in the shoot and roots as well as levels of endogenous cytokinins and gibberellins in xylem exudate. Tomato seedlings were grown in three growth cabinets with variable control of root temperatures. Three day/night root temperature regimes (12/12, 16/8 and 20/20°C) were employed. Low day root temperatures of 12 and 16°C reduced shoot dry weight by 47 and 26%, root dry weight by 36 and 14%, shoot nitrate by 79 and 50%, root nitrate by 49 and 16%, levels of cytokinins in root xylem exudate by 27 and 13% and gibberellins by 65 and 23%, in relation to the respective values of 20°C day root temperature. Soluble carbohydrates in the shoot and roots were increased significantly (18 and 111%) by 12°C root temperature. The main effects of low root temperatures on shoot growth stem from slow upward transport of plant hormones and nitrate rather than reduction in their rate of biosynthesis or entry to the root, respectively.