Localization and responsiveness of a cowpea apyrase VsNTPase1 to phytopathogenic microorganisms

Apyrases (NTPases) are associated with both compatible and incompatible interactions between plants and microorganisms. Previously we reported that the ATPase activities of cell-wall-bound apyrases of several leguminous plants, such as pea, cowpea, soybean, and kidney bean, were enhanced by a glycoprotein elicitor and were inhibited in a species-specific manner by mucin-type glycopeptide suppressors secreted from a pea pathogenic fungus, Mycosphaerella pinodes. In this study, we isolated two apyrase genes, VsNTPase1 and VsNTPase2, from a cDNA library of Vigna sinensis Endl. cv. Sanjakusasage. Based on phylogenetic analysis, VsNTPase1 may belong to a group that responds to environmental stimuli. In a transient assay using DNA bombardment, a fusion protein of green fluorescent protein (GFP) and the N-terminal putative signal sequence of VsNTPase1 was distributed in the nucleus, cytoplasm (cytoskeletal structure), and cell wall. On the other hand, a fusion protein of GFP and the N-terminal putative VsNTPase2-signal sequence was localized in the cytoplasm, especially in small particles (perhaps mitochondria). A recombinant VsNTPase1 expressed in Spodoptera frugiperda 21 cells responded directly to signal molecules from several phytopathogenic microorganisms. Here, we discuss the role of apyrases in recognizing and responding to exogenous signals. The nucleotide sequences of VsNTPase1 and VsNTPase2 in this article have been submitted to DDBJ as accession numbers AB196769 and AB196770, respectively.     

Immunohistological study on bovine, swine and ovine skeletal muscle fibers for the localization of fatty acid translocase FAT/CD36

The present immunohistological study was conducted to investigate the localization of fatty acid translocase CD36 (FAT/CD36) in the skeletal muscle (Biceps femoris) fibers of bovine, swine and ovine. The results showed that CD36 was mainly localized in type I muscle fiber of these animals. In contrast, FAT/CD36 localization in type II fiber was insignificant in the types of muscle in the present experiments, suggesting that type II fiber of bovine, swine and ovine might lack fatty acid translocase FAT/CD36.     

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.     

Efficacy of bovine viral diarrhea vaccine used in Japan against bovine viral diarrhea virus type 2 strain 890

Bovine viral diarrhea virus (BVDV) has been segregated into two genotypes, type 1 and type 2. To determine the efficacy of the commercially available bovine viral diarrhea type 1 vaccine used in Japan against BVDV type 2, calves were infected with BVDV type 2 strain 890 4 weeks after administration of the vaccine. The vaccinated calves did not develop any clinical signs and hematological changes such as observed in unvaccinated calves after the challenge. Furthermore, the challenge virus was not recovered from the vaccinated calves throughout the duration of the experiment, whereas it was recovered from all unvaccinated calves. The bovine viral diarrhea vaccine used in Japan is efficacious against infection with BVDV type 2 strain 890.     

Estimation of beef marbling in the Longissimus muscle with computer image analysis of ultrasonic pictures of the Iliocostalis muscle area

This study investigated an objective method for estimating beef marbling using ultrasonic images of the Iliocostalis muscle and the Lomgissimus muscle area sections. Thirty‐one Japanese Black cattle steers were used in this study. The end of the left side shoulder blade bone was scanned using an ultrasonic device. Ultrasonic images were captured of the Longissimus muscle area and that around the Iliocostalis muscle area. Twenty items were measured in the two images using computer image analysis software. The level of beef marbling was measured according to the Beef Marbling Standard (BMS) for carcass grading, and the percentage of ether‐extractable fat content in the Longissimus muscle (EE). The difference in the gray level between the Iliocostalis muscle and intermuscular fat (X10) was used to estimate the BMS and the EE, which were highly correlated (r² = 67.72% and 61.30%). An equation was developed using four parameters from the two ultrasonic images, which could estimate the BMS (r² = 85.88%). This equation could also estimate the EE (r² = 68.98%). The equations used to estimate beef marbling were based on one to four parameters that included X10. Thus, ultrasonic images of the Iliocostalis muscle area section are important for estimating beef marbling.     

Use of Mg-L-Ascorbyl-2-Phosphate as a Vitamin C Source in Shrimp Diets

To overcome the lability of vitamin C (L-ascorbic acid or L-AsA), an essential nutrient of shrimp diets, the efficacy of Mg-L-ascorbyl-2-phosphate (MAP), a relatively stable derivative of L-AsA, was examined.
Groups of juvenile Penaeus japonicus were fed five different test diets containing different amounts of MAP for 100 days, to determine if MAP could be utilized by shrimp. Normal growth was observed on test groups fed diets containing 1,078, 430 and 215 mg MAP per kg, whereas mass mortality occurred in populations fed diets containing 43 and 0 mg MAP per kg. The dead shrimp found in the latter two treatments exhibited blackened lesions underneath the exoskeleton. The symptom looked similar to what was reported in previous papers as L-AsA deficiency syndrome. The feeding experiment suggests that MAP can be utilized by P.japonicus as a suitable replacement for L-AsA, and a supplement of 215 to 430 mg in 1 kg is sufficient to prevent clinical signs of vitamin C deficiency.
Repeated measurements of remaining L-AsA and MAP, made after diet processing and storage, demonstrated that MAP was significantly more stable than L-AsA and was less prone to leaching in sea water.     

Development of myofibrillar ATPase assay system on pH stat

The ATPase assay system on pH stat apparatus was developed. For the ATPase activity measurement, hydrogen ion (H⁺) concentration delivered from inorganic phosphate (Pi) as a hydrolysis production of ATP was estimated by titrating with 20 mM NaOH solution instead of colorimetric measurement of Pi. Inclusion of 0.5 M KCl in the ATP stock solution and 2 mM Tris-maleate (pH 7.0) buffer in the reaction medium reduced the extent of pH change upon addition of ATP to initiate the ATPase reaction. The amount of H⁺ liberated from Pi was strongly affected by the set pH for the ATPase assay with a promotion at alkaline pH. Thus, it was required to estimate the coefficient to convert H⁺ to a Pi concentration at a specific pH. The specified coefficient at pH 7.0 was 1.248. ATPase assay on pH stat allowed us to follow the ATP hydrolysis by myofibrils continuously showing a curvature profile at low salt medium (≤0.2 M KCl) or a linear profile at high salt (≥0.3 M KCl).