Primary structure and thermostability of bigeye tuna myoglobin in relation to those of other scombridae fish

ABSTRACT:   In the present study, the cDNA encoding myoglobin (Mb) of bigeye tuna Thunnus obesus was cloned and its amino acid sequence deduced in order to investigate the relationship between the primary structure and thermostability of scombridae fish Mb. An open reading frame of bigeye tuna Mb cDNA contained 444 nucleotides encoding 147 amino acids. The primary structure of bigeye tuna Mb was highly conserved when compared with those of bluefin tuna and yellowfin tuna Mb, the sequence identity being 95.2–100.0%. It also showed relatively high identity (82.3–89.1%) with the counterparts of scombridae fish. Myoglobin was then isolated from the dark muscle of four scombridae fish including bigeye tuna. Differential scanning calorimetry and circular dichroism measurements on these Mb revealed that the thermostability of bigeye tuna Mb was lowest and that of skipjack Katsuwonus pelamis Mb highest among the scombridae fish Mb examined. The α-helical contents of scombridae fish Mb at 10°C were in the range of 39.8–44.8%, clearly lower than that of horse Mb (55.3%), suggesting instability of fish Mb. The melting temperatures of these Mb fell in the range of 75.7–79.9°C, lower than that of horse Mb (84.2°C). These results strongly suggest the instability of fish Mb.     

Type‐I interferon regulates matrix metalloproteinases clearance of the bovine endometrial spheroid

This study investigated the effect of type‐I interferon (IFN) on the expression of matrix metalloproteinases (MMPs) of the bovine endometrial stromal cells (BES) and epithelial cells (BEE). The cells were separated and purified from the caruncles and cultured in DMEM/F‐12 containing 10% fetal bovine serum. Spheroids were generated by using ascorbate. Zymograms of the supernatant showed that BEE predominantly expressed MMP‐9, whereas MMP‐2 was expressed in BES and homo‐spheroids. While MMPs expression was not detected in hetero‐spheroids. Real‐time quantitative PCR revealed that type‐I IFN and P4 suppressed the gene expression of MMP‐2 and MMP‐9 in hetero‐spheroids, respectively. On the other hand, gelatin zymography analysis of the supernatant showed that type‐I IFN strongly promote the clearance of MMPs. While zymograms of the MMPs stocked in the hetero‐spheroids were significantly reduced by type‐I IFN. Phenylmethanesulfonyl fluoride and leupeptin (both are serine proteinase inhibitors) significantly repressed the clearance of MMP‐2 and MMP‐9 induced by type‐I IFN. Moreover, collagen fibers in hetero‐spheroids significantly decreased after the treatment with type‐I IFN. In conclusion, it was suggested that type‐I IFN participate in the tissue remodeling by regulation the clearance of MMPs.     

Contribution of increasing CO2 and climate to carbon storage by ecosystems in the United States

The effects of increasing carbon dioxide (CO2) and climate on net carbon storage in terrestrial ecosystems of the conterminous United States for the period 1895-1993 were modeled with new, detailed historical climate information. For the period 1980-1993, results from an ensemble of three models agree within 25%, simulating a land carbon sink from CO2 and climate effects of 0.08 gigaton of carbon per year. The best estimates of the total sink from inventory data are about three times larger, suggesting that processes such as regrowth on abandoned agricultural land or in forests harvested before 1980 have effects as large as or larger than the direct effects of CO2 and climate. The modeled sink varies by about 100% from year to year as a result of climate variability.     

Immunohistochemical and histoplanimetrical study on the spatial relationship between the settlement of indigenous bacteria and the secretion of bactericidal peptides in rat alimentary tract

To clarify the regulatory mechanism by bactericidal peptides secretion, the secretion of bactericidal peptides was immunohistochemically and histoplanimetrically compared with the degree of Gram-positive/negative bacterial colonization throughout the rat alimentary tract. In the associated exocrine glands from the oral cavity to the stomach, no comparable differences were observed under the changes of development of indigenous bacterial colonies. In the small intestine, immunopositive granules for lysozyme and secretory phospholipase A2 (sPLA2) were markedly decreased, whereas immunopositive vacuoles in the Paneth cells were more increased at sites with hyper-development of indigenous bacterial colonies in the intervillous spaces than at sites with no or less development. No changes in exocrine glands were observed in the large intestine because of the constant existence of large quantities of bacteria. Gram-positive bacterial colonies on the mucosal surfaces were dominant from the oral cavity to the stomach. Gram-negative bacteria were dominant in the large intestine, and the distributions of both Gram-positive and negative bacteria were intermediate in the small intestine. These findings suggest that lysozyme and sPLA2 secreted from the Paneth cells contribute to the regulation of the proliferation of indigenous bacteria in the intervillous spaces of the small intestine, and that the inversion of distributions of Gram-positive and -negative bacteria in the alimentary tract might be caused by the secretion of lysozyme and sPLA2 in the small intestine.     

The effects of acute exposure to deoxynivalenol on some inflammatory parameters in miniature pigs

Seven miniature pigs were injected intravenously with deoxynivalenol (DON) at 1 mg/kg body weight; afterward, the number of leukocytes in peripheral blood, the luminol-dependent chemiluminescence of neutrophils, the serum or plasma concentration of cytokines and acute-phase proteins were evaluated to determine the effects of acute exposure to DON on inflammatory responses. White blood cell counts were transiently increased at 3, 6, and 12 hr post-injection (PI) due to the increased number of neutrophils. The luminol-dependent chemiluminescence value of neutrophils was significantly elevated at 24 hr PI, indicating the activation of the bactericidal function of neutrophils. Significant increases of interleukin (IL)-8 and tumor necrosis factor-α at 3 hr PI and IL-6 at 6 hr PI were detected in the serum. The concentration of haptoglobin and serum amyloid A was significantly increased at 24 hr PI. These results suggest that acute exposure to DON induced a temporary recruitment of neutrophils in the peripheral blood by IL-8 and subsequent activation of the bactericidal function, and a transient increase of proinflammatory cytokines and acute-phase proteins, indicating the immunomodulatory effects of DON in pigs.     

A simplified PCR assay for fast and easy mycoplasma mastitis screening in dairy cattle

A simplified polymerase chain reaction (PCR) assay was developed for fast and easy screening of mycoplasma mastitis in dairy cattle. Species of major mycoplasma strains [Mycoplasma (M.) bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens and M. canadense] in cultured milk samples were detected by this simplified PCR-based method as well as a standard PCR technique. The minimum concentration limit for detecting mycoplasma by the simplified PCR was estimated to be about 2.5 × 10³ cfu/mL and was similar to that of the standard PCR. We compared the specificity and sensitivity of the simplified PCR to those of a culture method. Out of 1,685 milk samples cultured in mycoplasma broth, the simplified PCR detected Mycoplasma DNA in 152 that were also positive according to the culture assay. The sensitivity and specificity of the simplified PCR were 98.7% and 99.7%, respectively, for detecting mycoplasma in those cultures. The results obtained by the simplified PCR were consistent with ones from standard PCR. This newly developed simplified PCR, which does not require DNA purification, can analyze about 300 cultured samples within 3 h. The results from our study suggest that the simplified PCR can be used for mycoplasma mastitis screening in large-scale dairy farms.     

Abstracts of nippon Dozyohiryogaku Zasshi

The present study was carried out to analyze the factors that affected the growth of sugar beet in four different soil types by using concrete-framed plots as follows: soil acidity (soil pH, exchange acidity y ₁) and nitrification of fertilizer introduced by row application. Comparison of the value of the exchange acidity y ₁ of the four soil types with the pH value adjusted to the same level (pH 5.1) revealed that the Humic Gray Upland soil displayed the highest y ₁ value (y ₁: 18.0), followed by the Humic Volcanogenous Regosol (y ₁: 6.9), Haplic Brown Lowland soil (y ₁: 5.3), and Low-humic Andosol (y ₁: 2.2). Al release to the soil solution was considered to occur at soil pH values of 4.8 and lower except in the Low-humic Andosol. Al concentration in the soil solution of the Low-humic Andosol was substantially lower than that of the other soils. On the other hand, the soil pH value decreased temporarily by nitrification of the fertilizer introduced by row application, especially in the Humic Gray Upland soil. In this case, the soil pH value became lower than 4.8 for a time. At this pH level, Al release to the soil solution was assumed to occur. As described above, the soils displayed different properties in terms of soil acidity. In the four soils, although the growth of sugar beet was significantly related to both soil pH and exchange acidity y ₁ values before sowing, these relations were not strictly valid. On the other hand, the linear correlation coefficients of the relationships between the growth of sugar beet (leaf length) and NO₃-N content in rows were higher than those of the soil pH and exchange acidity y ₁. No appreciable variation associated with the differences in the soil types was observed in this relation. Furthermore, the values of both soil pH and exchange acidity y ₁ were closely related to the NO₃-N content in rows and the relationship between the NO₃-N content and y ₁ value appeared to be somewhat closer than that with the soil pH. These closer relations had two important implications. Firstly, NO₃-N content reflected the nitrogen nutrient conditions. NO₃-N promoted the growth of sugar beet directly. Secondly, the NO₃-N content was affected by the soil acidity, which is expressed by the value of the exchange acidity y ₁. Low NO₃-N content indicated indirectly the toxicity of soil acidity to sugar beet growth. It was thus suggested that in the present study, nitrification of the fertilizer expressed by the NO₃-N content was a beneficial factor for the growth of sugar beet regardless of the soil types. Finally, to promote the nitrification of fertilizer and to minimize the Al toxicity enhanced by the decrease of the soil pH associated with nitrification, it is important to avoid low values for the soil pH.     

Prediction of nitrogen uptake by sugar beet (Beta vulgaris L.) by scoring organic matter and nitrogen management (N-score), in Hokkaido,Japan

Studies were carried out on the interaction between potato scab, soil pH and exchangeable calcium in two different fields, one consisting of volcanic ash soil and the other, of red-yellow soil. Severity of potato scab in the field with red-yellow soil was correlated with the soil pH, unlike in the field with volcanic ash soil. The amount of exchangeable calcium in both soils was positively correlated with the scab index of potato tuber, when the content of exchangeable calcium in soil exceeded 150 mg per 100 g soil. From these results, it was concluded that the content of exchangeable calcium is as a more reliable parameter than the soil pH to evaluate the severity of potato scab.     

Genetic and environmental variances of body size and morphological traits in communally reared clonal lines from gynogenetic diploid ayu, Plecoglossus altivelis

Four clonal lines of ayu, Plecoglossus altivelis, were produced from mitotic-gynogenetic diploids, mixed before hatching and reared communally. After 9 months, the clonal fish were sacrificed for measurement of body size, morphometric relationships, and meristic counts. DNA fingerprinting was used to confirm the clonal nature of the fish and to identify the clonal line of origin for each fish.

Significant differences were observed among clonal lines for almost all body size, morphometric and meristic measures. Such differences are suggested to represent genotypic difference among clonal lines given the common environmental conditions provided to all the experimental groups, assuming that the genotype-environment interaction was negligible.

By applying the human twin model, genetic and environmental variances in the clonal population was estimated after the clonal lines were separated by DNA fingerprinting. Heritability estimates for data collected at 9 months were relatively high for body size and varied from low to high in meristic and morphometric traits. These results suggest the possible usage of clonal lines as a control fish for estimation of heritability of traits important to aquaculture.