A new method for counting of quail leukocytes by flow cytometry

An automatic counting method was developed for fish blood cells using a fluorescent dye, 3, 3-dihexyloxacarbocyanine (DiOC (6)(3)), that selectively stain lipid bilayers in living cells. In the present study, the DiOC(6)(3) method was applied to quail (Cotumix cotumix japonica) blood cells. After quail blood cells were stained with DiOC(6)(3), absolute counts and relative proportions of erythrocytes, granulocytes, monocytes, and lymphocytes plus thrombocytes in whole blood were obtained by means of flow cytometry (FC). The number of each cell types by the FC was in good agreement with those counted microscopically. This method will offer new possibilities for routine blood cell counting for avian medicine.     

Precipitation chemistry in Japan 1989–1993

Precipitation chemistry in Japan was discussed on a wet-only sample database obtained in a nationwide survey from April 1989 to March 1993. Wet-only samples were collected at 29 stations over Japan on a biweekly basis. Commonly determined chemical parameters were measured in laboratories. The volume-weighted annual mean pH at each site ranged from 4.50 to 5.83 with a mean of 4.76. Concentration ranges and means (parenthesized) on an equivalent basis for major ions were as follows: nss-SO₄ ^2–; 5.2–58.9 (38.6), NO₃ ^–; 1.8–25.0 (14.1), NH₄ ⁺; 0.55–29.8 (18.3), nss-Ca²⁺; 2.0–34.5(14.2), Na⁺; 6.4–275.3 (49.1), Cl^–; 13.7–322.4 (63.5) eq L^–1. Acid-base relationships for Phase-II records were quantitatively discussed in terms of three measures: pH, fractional acidity, and our proposed pA_i.     

Use of soil color meter for aqueous iron and ammonium measurements

Abstract

In this paper, we proposed a new approach for on-site colorimetric analysis of ferrous ions (Fe²⁺) and ammonium-nitrogen (NH₄ ⁺-N) using a soil color meter as an alternative method to conventional spectrophotometry. The soil color meter we used can express solution color numerically on the basis of L*a*b* color space. After coloring of water by the 1, 10 phenanthroline method and the Indophenol blue method, the color of solution was measured by the soil color meter. A linear relationship between Fe²⁺ and a* or b* values, and systematic change of NH₄ ⁺-N with L* value, enable us to make a calibration curve. The Fe²⁺ and NH₄ ⁺-N concentrations in groundwater samples (Fe²⁺: 0.3–1.3 mg L^?1; NH₄ ⁺-N: 0.02–0.62 mg L^?1) determined by the proposed method agreed well with those determined by conventional spectrophotometry with the difference being ± 0.05 mg L^?1 and ± 0.02 mg L^?1, respectively. Since a similar apparatus is widely used in the soil science field, this technique would facilitate field surveys.     

CD38 gene disruption inhibits the contraction induced by alpha-adrenoceptor stimulation in mouse aorta

CD38 is an ectoenzyme with ADP-ribosyl cyclase and hydrolase activities, which synthesizes cyclic ADP-ribose from NAD and hydrolyzes cyclic ADP-ribose to ADP-ribose. It has been shown that cyclic ADP-ribose is a potent Ca(2+) mobilizing messenger in many cells. To know the physiological role of cyclic ADP-ribose in vascular smooth muscle, we examined the effects of various agonists in the aorta isolated from CD38 knockout (CD38(-/-)) mouse. Western blot analysis showed that CD38 protein was detected in the aorta isolated from wild-type (CD38(+/+)) mouse, but not from CD38(-/-) mouse. In the aortae isolated from both CD38(+/+) and CD38(-/-) mice, KCl, phenylephrine and norepinephrine induced concentration-dependent contraction. KCl produced similar concentration-dependent responses in the aortae from both CD38(+/+) and CD38(-/-) mice. Maximum force of contraction induced by KCl (65 mM) was same in the size. Phenylephrine- and norepinephrine-induced contractions were, however, significantly smaller in the aortae from CD38(-/-) mice than in those from CD38(+/+) mice. 5-Hydroxytryptamine, endothelin-1, caffeine and thapsigargin-induced contractions were not significantly different in these two aortae. These results suggest that CD38 gene disruption inhibits alpha-adrenoceptor-induced vascular contractions and cyclic ADP-ribose-mediated signal transduction system is committed in these responses.     

Macroscopic study of the functional significance of the forearm muscles in the giant panda

The extensor and flexor group muscles and their related muscles were functional-morphologically observed in the dead body of the giant panda to clarify the action of the forearm and the palm in the manipulation of the species. The Musculus flexor carpi ulnaris had two developed heads, however, we can conclude that the contraction of this muscle slightly changes the angle of the accessory carpal bone to the ulna. The data pointed out that the accessory carpal bone acts as a supporting post, when the giant panda seizes the object. The M. abductor digiti I longus possessed the well-developed origin in both ulna and radius. These findings suggest that this muscle may function as a supinator of the forearm. We also suggest that the well-developed M. pronator quadratus and M. pronator teres, and the proximal part of the M. abductor digiti I longus and the M. supinator may efficiently contribute to the pronator-spinator action of the forearm, when the giant panda brings the food to its mouth using the manipulation system equipped in the palm region.     

In vivo characterization of primitive hematopoietic cells in clonal ginbuna crucian carp (Carassius auratus langsdorfii)

Primitive hematopoietic cells in mammalian bone marrow are purified by flow cytometry using Hoechst 33342 (Hoechst) and rhodamine-123 (Rho), because these dyes efflux activities of hematopoietic cells widely conserved in mammals. Hematopoietic stem cells (HSCs) are identified as side population (SP) cells, characterized by specific Hoechst efflux pattern in flow cytometric analysis. We previously demonstrated that SP cells from teleost body kidney (BK) had the HSC activity by a transplantation experiment using clonal ginbuna crucian carp (Carassius auratus langsdorfii). In the present study, to isolate HSCs and hematopoietic progenitor cells (HPCs) from teleosts using Hoechst and Rho, we compared the hematopoietic activity of Rho-negative (Rho(-)) cells with that of SP cells by ginbuna transplantation experiments. Rho(-) cells were clearly identified from ginbuna BK, and the majority of these cells (85%) showed a non-SP phenotype. Transplantation experiments showed that long-term repopulating activity (HSC activity) of Rho(-) cells was lower than that of SP cells, while Rho(-) cells had higher short-term repopulating activity (HPC activity) than SP cells. These results suggest that Rho(-) cells in ginbuna BK contain various stages of hematopoietic cells, while SP cells are highly enriched for HSCs, and that these dyes are useful for purification of HSCs and HPCs in teleosts.     

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.     

Expression profiles of interferon gamma genes in response to immunostimulants and alloantigen in ginbuna crucian carp Carassius auratus langsdorfii

Interferon gamma (IFNγ), a Th1-derived cytokine is one of the key molecules inducing cell-mediated immune responses in mammals. The expression of 2 distinct IFNγ (ifng1 and ifng2) and IFNγrel (ifngrel) genes was examined in kidney leukocytes from clonal ginbuna crucian carp Carassius auratus langsdorfii. The expression of IFNγ and IFNγrel genes was induced in kidney leukocytes by stimulation with lipopolysaccharide or phytohemagglutinin in a dose-dependent manner. The expression levels of IFNγ and IFNγrel genes in kidney leukocytes from allo-sensitized fish cocultured with allogeneic cells were higher than those from fish cocultured with isogeneic cells. The highest expression of ifng1 was observed at day 1, whereas that of ifng2 and ifngrel was detected at day 2 after cocultivation with allogeneic cells. However, the expression pattern of ifng2 and ifngrel in kidney leukocytes from allo-sensitized fish by scale-grafting was similar to those from non-sensitized fish. These results indicate that ifng1 is a major isoform of IFNγ related to antigen-specific cell-mediated immunity (CMI), and will be a useful marker to evaluate induction of CMI in ginbuna crucian carp. In addition, IFNγ would be a crucial type-II interferon compared to IFNγrel, relevant to cell-mediated immunity in fish as in mammals.