Characterization of 38 kDa and 42 kDa chitinase isozymes from the liver of Japanese common squid Todarodes pacificus

ABSTRACT: Characterization was investigated on the 38 kDa and 42 kDa chitinase (EC3.2.1.14) isozymes from the liver of Japanese common squid Todarodes pacificus . Optimum pH toward colloidal chitin was observed at pH 3.0 for the 38 kDa chitinase, and pH 3.0 and 9.0 for the 42 kDa chitinase. K _m and k _cat of the 38 kDa and 42 kDa chitinases toward a longer substrate, glycol chitin, were 0.071 mg/mL and 1.22/s, and 0.074 mg/mL and 0.196/s, respectively. Alternatively, strong substrate inhibition of both chitinases were observed toward a short substrate, N -acetylchitopentaose (GlcNAc₅). Both chitinases decomposed not only chitin but also chitosan (D. A. 95%). The cleavage pattern and reaction rate were investigated using N -acetylchitooligosaccharides (GlcNAc_n, n  = 2–6). Both chitinases hydrolyzed GlcNAc_n ( n  = 4,5, and 6). The release of GlcNAc was not observed. The speed of the reaction was observed to be in the following order: GlcNAc₄ > GlcNAc₅ > GlcNAc₆ for the 38 kDa chitinase, and GlcNAc₆ > GlcNAc₅ > GlcNAc₄ for the 42 kDa chitinase. Both the chitinases released p -nitrophenol from p -nitrophenyl GlcNAc_n ( n  = 2, 3, and 4). N-terminal amino acid sequences of the 38 kDa and 42 kDa chitinases were YLLSXYFTNWSQYRPGAGKYFPQNI and EYRKVXYYTNWSQYREVPAKFFPEN, respectively.     

Isolation and characterization of an attenuated strain of an Orthotospovirus,melon yellow spot virus

Journal of General Plant Pathology - An attenuated mutant strain of melon yellow spot virus (MYSV), an Orthotospovirus, designated as SA08-8, was obtained from a pathogenic isolate, C95S, via high...     

Pseudorabies virus infection in wild boars in Japan

In Japan, most pig populations are now free from pseudorabies virus (PRV) due to the recent success of an extensive eradication program. However, PRV infection persists in Japanese wild boars (Sus scrofa leucomystax), representing another potential reservoir for the virus in Japan. In this study, the seroprevalence of PRV in wild boars captured in three different prefectures was ascertained. A virus neutralization (VN) test showed that 6 of 173 serum samples (3%) were positive for VN antibody; glycoprotein E-ELISA revealed infection with the wild-type, but not the available vaccine strain, PRV. These results indicate that PRV has continued to spread among wild boars in Japan.     

Genomic in situ hybridization (GISH) analysis of intergeneric hybrids in Colchicaceae

Some intergeneric hybrids produced by the crosses among several colchicaceous ornamentals, Gloriosa superba ??Lutea?? (2n = 2x = 22), G. ??Marron Gold?? (2n = 4x = 44), G. ??Verschild?? (2n = 7x = 77), Littonia modesta Hook. (2n = 2x = 22), and Sandersonia aurantiaca Hook. (2n = 2x = 24), were subjected to genomic in situ hybridization (GISH) analysis in order to clarify their genome constitutions. Chromosome preparation was made from root tip cells of L. modesta × G. superba ??Lutea??, L. modesta × S. aurantiaca, S. aurantiaca × G. superba ??Lutea??, L. modesta × G. ??Marron Gold??, S. aurantiaca × G. ??Marron Gold?? and G. ??Verschild?? × S. aurantiaca. Total DNA of one parent was labeled with digoxigenin or biotin and used as probe, and chromosomes were counterstained with 4??-6-diamidono-2-phenylindole (DAPI). For all the nine intergeneric hybrids, chromosomes from each parent could be clearly distinguished by GISH analysis. Thus GISH analysis is a powerful tool for identifying the genome constitution of intergeneric hybrids in colchicaceous ornamentals. The results obtained by GISH analysis study may be important for further progress in breeding of colchicaceous ornamentals.     

Oxidative Stress Produced by Xanthine Oxidase Induces Apoptosis in Human Extravillous Trophoblast Cells

Oxidative stress has been recognized as an important factor in the pathophysiology of preeclampsia. It has been reported that the expression of xanthine oxidase (XO) in the cytotrophoblast and plasma hydrogen peroxide (H₂O₂) level are significantly higher in preeclamptics than in control women. The aim of this study was to clarify the biological influence of reactive oxygen species (ROS) produced by XO on extravillous trophoblast (EVT) cells. TCL1 cells, a human immortalized EVT cell line, were incubated with xanthine and XO (X/XO). We then measured the cell number, urate level of the culture media and the apoptotic cell ratio. Similar experiments were performed with additional administration of allopurinol, catalase, L-NAME or D-NAME, and with administration of H₂O₂ in substitution for X/XO. We assessed the effects of H₂O₂ on invasion ability, tube-like formation and protein expression of HIF1A and ITGAV of TCL1. Finally, the apoptotic cell ratio using primary cultured trophoblasts was measured following exposure to H₂O₂. X/XO decreased the relative cell number and increased the urate level and apoptotic cell ratio significantly. Elevation of the urate level and apoptotic cell ratio was attenuated by allopurinol and catalase, respectively. L-NAME and D-NAME had no influence on these effects. H₂O₂ also decreased the relative cell number. Pretreatment with H₂O₂ significantly inhibited the invasion ability, tube-like formation and HIF1A and ITGAV of TCL1. H₂O₂ also induced apoptosis in primary cultured trophoblasts. In conclusion, ROS produced by XO induced apoptosis and affected EVT function including invasion and differentiation.     

Differential safening activity of dymron and fenclorim on pretilachlor injury in rice seedlings in soil

The safening activity of dymron [1-(α,α-dimethylbenzyl)-3-( p -tolyl)urea] and fenclorim [4,6-dichloro-2-phenylpyrimidine] on the phytotoxic activity of pretilachlor [2-chloro-2',6'-diethyl- N- (2-propoxyethyl)acetanilide] on rice seedlings was examined in both water and soil culture. The safening activity of fenclorim in water culture was greater than that of dymron, whereas the activity of fenclorim in soil was lower than that of dymron. The fenclorim concentration in soil water was lower than that of dymron at all times when determined after the application at the same concentrations. The phytotoxic activity of pretilachlor and the safening activities of dymron and fenclorim were well correlated with the concentration of each in soil water but not with the amount in total soil. The adsorption of fenclorim on soil solids was greater than those of dymron and pretilachlor. It was suggested that both the phytotoxic activity of pretilachlor and the safening activities of dymron and fenclorim were dependent on their concentrations in soil water, which were primarily dominated by the adsorption on soil.     

Gradients in N‐cycling attributes along forestry and agricultural land‐use systems are indicative of soil capacity for N supply

Indicators of soil quality associated with N‐cycling were assessed under different land‐use systems (native forest – NAT, reforestation with Araucaria angustifolia or Pinus taeda and agricultural use – AGR) to appraise the effects on the soil potential for N supply. The soil total N ranged from 2 to 4 g/kg (AGR and NAT, respectively), and the microbial biomass N ranged from 80 to 250 mg/kg, being higher in NAT and A. angustifolia, and lower in P. taeda and AGR sites. Activities of asparaginase (ca. 50–200 mg NH₄⁺‐N/kg per h), glutaminase (ca. 200–800 mg NH₄⁺‐N/kg per h) and urease (ca. 80–200 mg NH₄⁺‐N/kg/h) were also more intense in the NAT and A. angustifolia‐reforested soils, indicating greater capacity for N mineralization. The NAT and AGR soils showed the highest and the lowest ammonification rate, respectively (ca. 1 and 0.4 mg NH₄⁺‐N/kg per day), but the inverse for nitrification rate (ca. 12 and 26%), indicating a low capacity for N supply, in addition to higher risks of N losses in the AGR soil. A multivariate analysis indicated more similarity between NAT and A. angustifolia‐reforested sites, whilst the AGR soil was different and associated with a higher nitrification rate. In general, reforestation with the native species A. angustifolia had less impact than reforestation with the exogenous species P. taeda, considering the soil capacity for N supply. However, AGR use caused more changes, generally decrease in indicators of N‐cycling, showing a negative soil management effect on the sustainability of this agroecosystem.     

Analysis of blood clotting with the total thrombus analysis system in healthy dogs

To date, coagulation tests are unable to reflect in vivo coagulation status in the same system, including platelet function, fibrin clot formation, and whole blood flow. The Total Thrombus Analysis System (T-TAS), which is a microfluidic assay that simulates conditions in vivo, measures whole blood flow at defined shear rates under conditions designed to assess platelet function (PL-chip) or coagulation and fibrin clot formation (AR-chip). The T-TAS records occlusion start time, occlusion time, and area under the curve. We evaluated this test in healthy control dogs. We also investigated the effect in vivo of acetylsalicylic acid (ASA), and the effect in vitro of an anticoagulation drug (dalteparin; low-molecular-weight heparin; LMWH). The CV of the AUC of both chips was good (CVs of 6.45% [PL] and 1.57% [AR]). The inhibition of platelet function by ASA was evident in the right-shift in the PL test pressure curve. The right-shift in the AR test pressure curves showed that the administration of LMWH inhibited both platelets and the coagulation cascade. The T-TAS may be useful in the evaluation of canine blood coagulation.