Artificial incubation of Japanese crayfish (Cambaroides japonicus) eggs by using a simple, easy method with a microplate

We developed a simple, easy method with a microplate to artificially incubate Japanese crayfish (Cambaroides japonicus) eggs for their cultivation. We prepared 6-, 12-, 24-, 48-, and 96-well microplates containing sterile water heated to 5, 10, 15, and 20 °C. Fourteen experimental groups for each water temperature were prepared for each of different water volumes (0.125–10 ml) in each well. One embryonic egg was placed in each well. Experiments were also conducted with water collected from the lake where the eggs were harvested from and held at 15 °C. In the microplates with sterile water, high proportions of eggs hatched (60–100%) at 15 °C in all volumes of water, although the proportions of hatching were low (0–20%) at 5, 10, and 20 °C. All eggs died in the experiments that used lake water. We conclude that the 96-well would be the most effective size to hatch crayfish eggs in, because of its convenience. This method using a microplate is simpler and easier compared with methods of previous studies to artificially incubate crayfish eggs, and therefore it might be useful to incubate eggs of other freshwater crayfish species.     

Multiplex PCR and Genescan analysis to detect immunoglobulin heavy chain gene rearrangement in feline B-cell neoplasms

Lymphoid neoplasms are usually diagnosed on the basis of cytological and histopathological findings. However, in some cases, discrimination of lymphoid neoplasms from reactive lymphoid proliferation is difficult. PCR amplification of complementarity-determining region 3 (CDR3) of the immunoglobulin heavy-chain variable region (IGHV) gene can be used to assess clonality of B-cell populations as a supportive diagnostic tool for B-cell neoplasms. Because of the sequence variation and possible somatic hypermutation of the IGHV gene, sensitivity of the PCR-based assay to detect clonal IGHV gene rearrangement largely depends on the sequences and numbers of primer sets. Prior to the development of an efficient assay, we cloned and sequenced 97 IGHV complementary DNAs (48 IGHV-1 and 49 IGHV-3 clones) from normal cat spleens. On the basis of these sequences, we designed 6 forward primers at the variable region and 5 reverse primers at the joining region. Using each of 6 forward primers and a mixture of 5 reverse primers, we amplified CDR3 of IGHV genes and analyzed the PCR products by conventional PAGE and Genescan analyses using fluorescence-labeled primers. Twenty-six feline B-cell neoplasms diagnosed by histopathological and immunohistochemical examinations were subjected to the newly developed analysis of IGHV gene rearrangement. Clonal IGHV gene rearrangement was detected in 22 of 26 (84%) samples by both PAGE and Genescan analyses. To reduce the number of PCR reactions, we constructed a multiplex PCR analysis system using a mixture of IGHV-1- and IGHV-3-specific primers as forward primers and a mixture of 5 joining region reverse primers. Results of the multiplex PCR were 100% concordant with those obtained by each of the singleplex PCRs. The multiplex PCR-based assay and Genescan analysis developed in the present study would be useful and practical tools to detect clonal IGHV gene rearrangement in feline B-cell neoplasms.     

Evaluating antigen-specific cytotoxicity of CD8+ T cells in fish by granzyme B-like activity

Granzyme B plays an important role in granule-mediated apoptosis by CTL. It is a well characterized component of the cytolytic machinery in mammals and a candidate for the evaluation of cytotoxic activity of CTL as an alternative to conventional cytotoxicity assay. In this study, we examined the effects of granzyme inhibitors to assess the characteristics of fish granzymes in terms of substrate specificity and the involvement of granzyme B-like in the cytotoxic response. 3,4-dichloroisocoumarin (DCI), which inhibit the activity of serine protease including all members of the granzyme family, markedly suppressed the cytotoxic activity of CTL. However, CTL-mediated cytotoxicity was significantly but not completely suppressed by the addition of carbobenzyloxy-Ile-Glu-Thr-Asp-fluoromethyl ketone (Z-IETD-FMK) that specifically blocks granzyme B activity. These results suggest that additional serine proteases as well as granzyme B-like are involved in cytotoxicity of CTL in fish. We further compared cytotoxicity with the granzyme B-like hydrolytic activity against fluorogenic substrate acetyl-Ile-Glu-Thr-Asp-4-methylcoumaryl-7-amide (Ac-IETD-MCA) and found that granzyme B-like activity correlated well with the cytotoxicity of CTL in ginbuna crucian carp. Present results suggest that the granzyme activity assays is useful to assess cytotoxic activity of CTL in fish in which genetic information on granzymes and specific tools for cytotoxicity assay are not available because of well conserved catalytic triad residues and substrate binding sites in granzyme B throughout vertebrates.     

Altered leukocyte responsiveness in dairy cows with naturally occurring chronic Staphylococcus aureus mastitis

Changes in inflammatory parameters, leukocyte surface markers, functional responses and cytokine mRNA expression of leukocytes of dairy cows with naturally occurring chronic Staphylococcus aureus (S. aureus) mastitis and healthy cows were determined to elucidate the leukocyte responses to S. aureus infection of the mammary gland. Increased values in inflammatory parameters and matrix metalloproteinase activities in milk revealed the characteristics of cows with chronic mastitis. Expression of L-selectin and CD18 molecules on neutrophils and proportion of CD8 cells in milk from cows with S. aureus mastitis were significantly (P<0.05) increased compared with those found in healthy cows. The FcR-stimulated CL response of blood neutrophils was significantly (P<0.05) decreased in cows with S. aureus mastitis. Significantly (P<0.05) decreased mitogenic responses of lymphocytes were found in cows with S. aureus mastitis; however, the values were not restored to those of healthy cows when stimulated with both mitogens and the cytokine IL-1β. The mRNA expression of TNF-α, IL-1β and IL-8 on milk leukocytes from cows with S. aureus was found to be increased compared with that of healthy cows. The changes of immune responses found in cows with S. aureus mastitis appear to be influenced by the severity and duration of inflammation in infected quarters. The down-regulation of the leukocyte functions found in cows with S. aureus mastitis appears to be associated with the progress of the chronic stage of S. aureus mastitis.     

A rapid and simple method to obtain canine peripheral blood-derived macrophages

Macrophages play an important role in a variety of situations, including pathogen elimination, inflammation, and tissue repair. However, these cells are not fully studied in dogs, in part, due to the difficulty of efficiently isolating and culturing them in vitro. In this study, we cultured canine peripheral blood mononuclear cells (PBMCs) with 10 ng/ml of phorbol 12-myristate-13-acetate (PMA) for 5 days to obtain macrophages. A high number of round-adherent cells were obtained without the addition of any cytokine. These cells showed active phagocytic activity and a cell surface antigen profile different from dendritic cells. Our method facilitates a high yield of macrophages in a short cultivation period compared with previous studies. This method might be a powerful tool to study macrophage functions in dogs.     

Relationships of survival time, productivity and cause of death with telomere lengths of cows produced by somatic cell nuclear transfer

The reproductive ability, milk-producing capacity, survival time and relationships of these parameters with telomere length were investigated in 4 groups of cows produced by somatic cell nuclear transfer (SCNT). Each group was produced using the same donor cells (6 Holstein (1H), 3 Holstein (2H), 4 Jersey (1J) and 5 Japanese Black (1B) cows). As controls, 47 Holstein cows produced by artificial insemination were used. The SCNT cows were artificially inseminated, and multiple deliveries were performed after successive rounds of breeding and conception. No correlation was observed between the telomere length and survival time in the SCNT cows. Causes of death of SCNT cows included accidents, accident-associated infections, inappropriate management, acute mastitis and hypocalcemia. The lifetime productivity of SCNT cows was superior to those of the controls and cell donor cows. All SCNT beef cows with a relatively light burden of lactation remained alive and showed significantly prolonged survival time compared with the cows in the SCNT dairy breeds. These results suggest that the lifetime productivity of SCNT cows was favorable, and their survival time was more strongly influenced by environmental burdens, such as pregnancy, delivery, lactation and feeding management, than by the telomere length.     

Screening of therapeutic targets for canine mast cell tumors from a variety of kinase molecules

Options of systemic treatment for canine MCT have been still limited and most canine cases with MCTs eventually undergo relapses even after achievement of a remission. Thus additional therapies are required to establish for the tumor. To identify the novel candidate therapeutic targets for canine MCT, the mRNA expression and phosphorylation statuses of several receptor or non-receptor kinases as well as the inhibitory effect of 95 specific inhibitors on the growth were assessed in three canine MCT cell lines (HRMC, VIMC1 and CMMC1). Among the 14 targets, the mRNAs of 11, 7 and 7 kinases were amplified in HRMC, VIMC1 and CMMC1, respectively. The mRNAs of VEGFR3, PDGFRα, SRC, YES, LCK and FYN were detected in all cell lines. The phosphorylation of 12, 8 and 7 kinases was observed by using specific antibody arrays in HRMC, VIMC1 and CMMC1, respectively. DTK, EPHB6, AMPKα1, CREB, STAT5a and STAT5b were phosphorylated in all cell lines. The 10, 9 and 17 inhibitors exhibited the biological activity against the growth of HRMC, VIMC1 and CMMC1, respectively. Only three inhibitors such as SB218078 (for Chk1), PDGF RTK inhibitor IV (for PDGFR) and radicicol (for Hsp90) suppressed the growth of all three cell lines. The present study indicated that several kinases, such as Chk1, PDGFR and Hsp90, could be used as therapeutic targets in the treatment for canine MCT. Further studies and clinical trials are warranted to apply the inhibitors for the treatment of the tumor.     

Effect of administration of acid‐ and bile‐tolerant lactobacilli on pigs intestinal microbial population

The effects of the administration of four Lactobacillus strains as probiotics on the constitution of microbial populations in the intestine of pigs were investigated by using a litter of pigs. The experimental group was fed a diet blended with the freeze‐dried powder containing lactobacilli at a concentration of 0.2% (W/W). After 4 weeks of probiotics administration, the total viable count of anaerobes in the feces was not significantly different between the pigs fed a diet supplemented with probiotics and the pigs given the non‐supplemented diet. However, viable counts of eubacteria were significantly higher (P < 0.01) in the feces of pigs fed a diet supplemented with probiotics than in the pigs given the non‐supplemented diet. Conversely, viable counts of clostridia were significantly lower (P < 0.01) in the feces of pigs fed a diet supplemented with probiotics than the pigs given the non‐supplemented diet. The total volatile fatty acid concentrations in the feces of pigs that were given a diet supplemented with probiotics were significantly lower (P < 0.05) than those given the non‐supplemented diet. Further, the molar proportion of n‐butyric acids in the feces of pigs that were given a diet supplemented with probiotics, was significantly lower (P < 0.05) than those given the non‐supplemented diet.     

Expression in Escherichia coli and purification of the functional feline granulocyte colony-stimulating factor

Feline granulocyte colony-stimulating factor (G-CSF) with an N-terminal histidine hexamer tag was expressed as inclusion bodies in E. coli. The G-CSF solubilized in 6 M guanidine solution was absorbed onto a Ni-NTA column and, after washing with decreasing concentrations of guanidine, eluted with imidazole in a soluble and apparently pure form. The activity of the recombinant feline G-CSF was 3×10⁶ U/mg protein, as assayed by its stimulatory effect on NFS-60 cell proliferation. When a low level of purified feline G-CSF was administered once a day for two successive days to cats, the number of neutrophil increased 4-fold while the levels of other blood cell types remained virtually unchanged. Daily administration of G-CSF for a total of 11 days led to a more than 10-fold increase in neutrophils, an 8-fold increase in the number of monocytes and 2-fold increase in lymphocytes. No severe side effects or antibody production was observed in cats after administration of G-CSF.     

Comparison of estrogen responsive genes in the mouse uterus, vagina and mammary gland

Female reproductive organs are mainly regulated by estrogen and progesterone. Specifically, the uterus, vagina and mammary gland show organ-specific mitosis and morphological changes during proliferative events, such as estrous cycle, gestation and lactation. The mechanism underlying these organ-specific estrogen-dependent events is still unknown. We examined, therefore, global gene expression in the mature uterus, vagina and mammary gland of ovariectomized adult mice 6 hr after an injection of 5 microg/kg 17beta-estradiol (E2) using a microarray method in order to identify primary E2-responsive genes. Half of the E2 up-regulated genes in the uterus were similar to those in the vagina. E2 up-regulated the expression of Insulin-like growth factor 1 (Igf-1) genes in the uterus and vagina. In the vagina, E2 up-regulated the expression of IGF binding proteins (Igfbp2 and Igfbp5). In the mammary gland, unlike the uterus and vagina, no gene showed altered expression 6 hr after the E2 exposure. These results suggest that expression of Igf-1 and morphogenesis genes is regulated by E2 in an organ-specific manner, and it is supported by the results of BrdU labeling showing E2-induced mitosis in the uterus and vagina except the mammary gland. The differences in organ specificity in response to E2 may be attributed by differences in gene expression regulated by E2 in female reproductive organs. The candidate estrogen-responsive genes in the uterus and vagina identified by profiling provide an important foundation understanding functional mechanisms of estrogen regulating morphogenesis and maintenance of each reproductive organ.