Availability of culture filtrate protein-10 (CFP-10) secreted from Mycobacterium pseudoshottsii for mycobacteriosis diagnosis in ginbuna crucian carp Carrasius auratus langsdorfii

Mycobacteriosis in cultured fish is a challenge for the aquaculture industry worldwide. Treatment by chemical administration is difficult and no effective vaccine has been developed. Therefore, detection and isolation by early diagnosis are important for prevention of the spread of the disease. In mammals, interferon gamma release assays have been established for detection of tuberculosis; these tests are based on the delayed-type hypersensitivity (DTH) response against culture filtrate protein-10 (CFP-10) and the 6-kDa early secreted antigen target (ESAT-6) of Mycobacterium tuberculosis. On the other hand, little is known about the fish immune response against the ESAT-6 and CFP-10 proteins of mycobacteria, although these responses should find application in the diagnosis of mycobacteriosis in fish. In the present study, we identified ESAT-6 and CFP-10 from Mycobacterium pseudoshottsii and cloned the corresponding genes. Intraperitoneal injection of the corresponding DNA plasmid constructs in ginbuna crucian carp yielded increased expression of the fish interferon-γ1-1-encoding gene (IFN-γ1-1). In contrast, IFN-γ1-1 expression accompanied by DTH response was observed only in the CFP-10-DNA plasmid-injected fish. Furthermore, fish that had been prophylactically injected with CFP-10-DNA plasmid exhibited increased survival of M. pseudoshottsii infection. Taken together, these results suggested that CFP-10 may facilitate diagnosis of mycobacteriosis.     

Improving the accuracy of estimating blood calcium concentration in Holstein cows using electrocardiographic variables

We previously reported the possibility of using the electrocardiogram variable to estimate blood calcium (Ca) concentration in dairy cows based on the strong positive correlation between the blood Ca concentration and the inverse of the corrected ST peak interval (STc⁻¹). To improve the accuracy of the estimation of blood Ca concentration, we investigated the relationship between blood Ca concentration and STc⁻¹ for each postpartum day and available variables other than STc⁻¹. We measured multiple variables (milk yield, calving number, age, body temperature, etc.), including serum total Ca concentration (tCa), blood ionized Ca concentration (iCa) and STc⁻¹ in 462 Holstein cows on days 0, 1, 2, 3, 5, and 7 postpartum. A very high correlation was observed between iCa and tCa. The association between tCa and STc⁻¹ for each postpartum day had a high coefficient of determination of 0.61–0.79 postpartum 0–2 days but decreased after the third day. In the investigation using the data from postpartum days 0–2, STc⁻¹, heart rate interval, calving number, and age were highly correlated with tCa. In addition, a multiple regression equation was obtained with tCa as the objective variable and STc⁻¹ and calving number as explanatory variables. The estimation accuracy was improved as compared with the simple regression equation using only STc⁻¹ as the explanatory variable. This multiple regression equation was used for 11 cows suspected of having hypocalcemia, and it was able to correctly detect cows requiring early treatment, except for one cow.     

Effect of lodging on the level of mycotoxins in wheat,barley, and rice infected with the <Emphasis Type="Italic">Fusarium graminearum</Emphasis> species complex

Lodging is one possible risk factor that leads to increased cereal mycotoxin contamination, but few reports have been published on the subject. We examined the effects of lodging on the level of deoxynivalenol (DON) and nivalenol (NIV) contamination in wheat, barley, and rice infected with the Fusarium graminearum species complex. Case-control and intervention studies were applied to test the hypothesis that lodging increases the level of mycotoxin contamination. A total of 66 grain samples were collected from each field in 12 Japanese prefectures from 2002 to 2006. Each sample set consisted of grains from lodged and nonlodged plants. The concentration of DON + NIV in lodged plants was significantly higher than in nonlodged plants. All samples of wheat and barley were contaminated with DON and NIV; however, most of the lodged rice samples were contaminated only with NIV. In intervention trials to investigate the effects of lodging duration, a small area of wheat inoculated with the pathogen was completely lodged by trampling. Even with 5 days of lodging, the levels of DON + NIV in wheat grain at harvest increased by 27–51% compared to nonlodged control plots. For rice, half of each plot area was completely lodged by trampling 20 days before harvest. The level of NIV in lodged rice grain was significantly higher than that in nonlodged rice at optimum and delayed harvests, because lodging significantly increased the level of Fusarium mycotoxins in the three crops. Thus, practices (e.g., rational use of fertilizers) to avoid lodging should reduce the risk of mycotoxin contamination. This is the first epidemiological study on the effect of lodging on mycotoxin production by the F. graminearum species complex in wheat, barley, and rice.     

Effect of the liquid form of traditional Chinese medicine,Hozen-S,on gastric motility in dogs

Juzen-taiho-to, a traditional Chinese herbal medicine, is used for patients with anorexia and fatigue in human medicine. In our previous study, granulated Juzen-taiho-to improved vincristine-induced gastrointestinal adverse effects through increasing gastric motility in dogs. As the effect of Hozen-S, the sweet liquid form of Juzen-taiho-to, on dog gastric motility has not been investigated, we examined the effect of administration of Hozen-S on gastric motility. Furthermore, we assessed dog plasma ghrelin level to further elucidate the mechanism of the effect of Hozen-S on gastric contraction. Finally, we assessed the palatability of Hozen-S compared to granulated Juzen-taiho-to and its effect on body weight in dogs. Administration of Hozen-S significantly increased gastric motility, plasma ghrelin concentration, and body weight. A palatability evaluation revealed that the dogs preferred Hozen-S to granulated Juzen-taiho-to. In conclusion, Hozen-S administration to dogs promoted gastric motility by raising plasma ghrelin levels. Considering these functional and palatability data, Hozen-S may replace granulated type Juzen-taiho-to and become a prominent traditional Chinese veterinary medicament.     

Growth and spawning of hatchery-reared Chinese white prawn Penaeus (Fenneropenaeus) chinensis released in the Ariake Sea, Japan

SUMMARY: Growth and reproduction of hatchery-reared Chinese white prawn Penaeus chinensis released in the Ariake Sea, Japan, were examined. Chinese white prawn grew rapidly, reaching a body length of 154 mm in males and 198 mm in females by November (219–229 days after hatching). Maximum body length of sampled individuals was 164 mm in males and 223 mm in females. Growth curve of the Chinese white prawn was fitted to the Pitcher and MacDonald's formula, L_t = 155.0{1 − e ^{2.925sin[2π(t − 16.151)/365] − 0.0623( t − 10.712)}} for males and the logistic curve, L_t = 200.3/[1 + e ^{(1.985–0.034 t )}] for females (where L_t is the body length t days after release and t is the number of days after release). Females reached sexual maturity in late February and spawning occurred until April. Minimum size at ripe and spawned stages was 189 mm and 193 mm body length, respectively.     

Biotechnology and apple breeding in Japan

Apple is a fruit crop of significant economic importance, and breeders world wide continue to develop novel cultivars with improved characteristics. The lengthy juvenile period and the large field space required to grow apple populations have imposed major limitations on breeding. Various molecular biological techniques have been employed to make apple breeding easier. Transgenic technology has facilitated the development of apples with resistance to fungal or bacterial diseases, improved fruit quality, or root stocks with better rooting or dwarfing ability. DNA markers for disease resistance (scab, powdery mildew, fire-blight, Alternaria blotch) and fruit skin color have also been developed, and marker-assisted selection (MAS) has been employed in breeding programs. In the last decade, genomic sequences and chromosome maps of various cultivars have become available, allowing the development of large SNP arrays, enabling efficient QTL mapping and genomic selection (GS). In recent years, new technologies for genetic improvement, such as trans-grafting, virus vectors, and genome-editing, have emerged. Using these techniques, no foreign genes are present in the final product, and some of them show considerable promise for application to apple breeding.     

Quality improvement of the thermal gel of horse mackerel by sodium citrate and its inhibition of the insoluble metalloproteinase

Modori-inducing proteinases (MIPs) in horse mackerel and the effect of sodium citrate on the modori phenomenon were investigated. Cysteine protease caused the modori phenomenon only at 60 ℃, while metalloproteinase caused it at both 60 ℃ and 70 ℃. Moreover, the autolysis of myofibrillar proteins was mainly inhibited by ethylene diamine tetra-acetic acid (EDTA), verifying the existence of insoluble metalloproteinase. From the results comparing thermal gel properties and protease activities, we confirmed that increased activity of insoluble metalloproteinase corresponded to the weakening of the thermal gel. Thus, insoluble metalloproteinase was the major MIP in horse mackerel. Moreover, sodium citrate could markedly inhibit insoluble metalloproteinase activity at 60 ℃. The addition of sodium citrate significantly improved the breaking force of the thermal gel at 60 ℃ and 70 ℃, suggesting that sodium citrate could suppress the modori phenomenon by inhibiting insoluble metalloproteinase. Therefore, it is desirable to use sodium citrate in horse mackerel surimi-based products to prevent the modori phenomenon.

     

Identification of novel haze-active beer proteins by proteome analysis

Colloidal haze reduces beer quality considerably. Four haze samples were analyzed by two-dimensional gel electrophoresis (2DE) in order to identify haze-active proteins. Several protein spots were observed in all of the four haze samples. Using mass spectrometry analysis followed by a database search identified these spots as barley dimeric alpha-amylase inhibitor (BDAI-1), CMb component of tetrameric alpha-amylase inhibitor (CMb) and trypsin inhibitor CMe precursor (CMe). These proteins were considered to be haze-active. Since haze-active proteins are adsorbed by silica gel in the beer filtration process, we eluted proteins adsorbed onto silica gel (PAS) and identified their species. These major PAS were identified as protein Z4, protein Z7 and trypsin/amylase inhibitor pUP13 (TAI), rather than BDAI-1, CMb and CMe. Furthermore, we analyzed proline compositions in the beer proteins, PAS and the haze proteins. Consequently, we found that the proline compositions of PAS were higher (ca. 20 mol%) than those in the beer proteins (ca. 10 mol%), although those of the haze-active proteins such as BDAI-1, CMb and CMe were 6.6–8.7 mol%. Our results suggest that BDAI-1, CMb and CMe are not predominant haze-active proteins, but growth factors of beer colloidal haze.     

Inhibitory function of whey acidic protein in the cell-cycle progression of mouse mammary epithelial cells (EpH4/K6 cells)

Although the biological role for whey acidic protein (WAP) in milk has been suggested, its true function is not known. This paper describes evidence for WAP function in the cell-cycle progression of EpH4/K6 (EpH4), mammary epithelial cells in vitro. The forced expression of exogenous WAP significantly impaired the proliferation of EpH4 cells, whereas it did not affect that of NIH3T3 cells. Apoptosis was not enhanced in the EpH4 cells with stable expression of WAP (WAP-clonal EpH4 cells). The analyses of BrdU incorporation revealed that forced WAP expression significantly reduced incorporation of BrdU in WAP-clonal EpH4 cells compared with control cells transfected with empty plasmid. Among G1 cyclins, the level expression of cyclins D1 was significantly lower in the WAP-clonal EpH4 cells than in control cells. The inhibitory action of WAP on the proliferation of EpH4 cells was enhanced by the presence of extracellular matrix (ECM), but not by the presence of a single component comprising ECM. The cultured medium of WAP-clonal EpH4 cells inhibited the proliferation of control cells without WAP expression. The present results indicate that WAP plays a negative regulatory role in the cell-cycle progression of mammary epithelial cells through an autocrine/paracrine mechanism.