Development and characterization of monoclonal antibodies against canine trypsin

Canine cationic trypsin was purified by salting-out, gel filtration and affinity chromatography. Purity was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was ca. 28 kDa by SDS-PAGE.

Thirty hybridomas were obtained which produced mAb to canine cationic trypsin by the cell fusion technique. Twenty-two of these recognized cationic trypsin only, while eight hybridomas recognized both cationic and anionic trypsin. Several of the anti-canine cationic trypsin mAb were purified by salting-out and DEAE ion-change chromatography using ascites fluid of immunized BALB/c mice. The mAb proved to have very high specificity to canine cationic trypsin as shown by immunoblotting and it may be possible to use them to develop clinical assays.     

Transgenic cucumber expressing an endogenous class III chitinase gene has reduced symptoms from Botrytis cinerea

A class III chitinase gene (CHI2) is induced in cucumber plants (Cucumis sativa L.) in response to infection by pathogenic microorganisms. Infection of Botrytis cinerea, causal agent of gray mold disease on cucumber, also induces CHI2 expression. To investigate whether CHI2 is involved in resistance to gray mold disease, transgenic cucumber plants were produced to overexpress the CHI2 gene. One line was analyzed in detail in terms of disease resistance. The transgenic cucumber plant (CC2) constitutively expressed CHI2 and reduced the symptoms of B. cinerea for 4 days after inoculation compared with nontransgenic plants. However, this inhibitory effect was not absolute, and CC2 eventually developed serious disease symptoms. Chitinase activity of the crude extract from CC2 leaves was higher than that from nontransgenic plants. A high-molecular-weight fraction containing CHI2 from CC2 leaves had fungistatic activity against B. cinerea. Interestingly, the low-molecular-weight fraction from CC2 leaves with CHI2 removed also had fungistatic activity against B. cinerea. Not only the introduced chitinase activity but also the endogenous defense reactions activated by overexpression of CHI2 may be involved in the enhanced gray mold disease resistance in CC2.     

Assessment of water quality in small agricultural ponds in Nara,Japan

Paddy and Water Environment - In Japan, since ancient times, numerous small- to medium-size agricultural ponds have been constructed to irrigate paddy rice fields. Economic growth and urbanization...     

Effect of the timing of first cleavage on in vitro developmental potential of nuclear-transferred bovine oocytes receiving cumulus and fibroblast cells

The aim of the present study was to examine whether cumulus and fibroblast cell nuclear-transferred oocytes, which have high and low potential to develop into normal calves, respectively, are different in terms of in their patterns of timing of first cleavage and in their relationships between timing of first cleavage and in vitro developmental potential. The timing of first cleavage was similar in both types of nuclear-transferred and in vitro fertilized oocytes. More than 86% of the oocytes cleaved within 24 h after activation or in vitro fertilization; these oocytes contributed to more than 98% of the total number of blastocysts in all three groups. The potential of oocytes that cleaved at different intervals to develop into blastocysts differed among the groups. The developmental potential of the cumulus cell nuclear-transferred oocytes and in vitro fertilized oocytes decreased with the increase in time required for cleavage. Fibroblast cell nuclear-transferred oocytes that cleaved at 20 h, an intermediate cleaving time, had higher potential to develop into blastocysts. The results of the present study suggest that the type of donor nucleus used for nuclear transfer affects the timing of first cleavage.     

Ovarian activity and pregnancy in the Siberian tiger, Panthera tigris altaica, assessed by fecal gonadal steroid hormones analyses

Feces were collected from two female and one male Siberian tigers, Panthera tigris altaica. Steroid hormones were extracted from lyophilized feces and quantified by enzyme immunoassay. The fecal contents of estradiol-17beta (E(2)) and testosterone in the females and male, respectively, changed markedly throughout the year. The fecal E(2) contents of females Nos. 179 and 238 increased at 26.4 +/- 8.0 and 28.0 +/- 14.2 day intervals, respectively. However, the fecal contents of progesterone (P(4)) in the female kept alone did not change. In contrast, the other female, which was kept with a male, had increased fecal P(4) contents after copulation. The fecal progesterone levels of the pregnant female remained high during her 106-day pregnancy.     

Need for flagella for complete virulence of Pseudomonas syringae pv. tabaci: genetic analysis with flagella-defective mutants ?fliC and ?fliD in host tobacco plants

The flagellins purified from Pseudomonas syringae pv. tabaci induce a hypersensitive reaction in nonhost tomato cells. To investigate the role of flagella and flagellin in the compatible interaction, we generated two types of flagella-defective mutant. The fliC mutant lost the fliC gene that encodes flagellin protein, whereas the fliD mutant lost the fliD gene that encodes HAP2-capping protein. The two mutants had markedly reduced ability to cause disease symptoms in tobacco leaves. Furthermore, propagation of the mutants in tobacco leaves was less than that in wild-type pv. tabaci. Compared to the inoculation with wild-type pv. tabaci, inoculation with the two mutants did not markedly induce the expression of typical defense response-related genes such as PAL and hsr203J. Complementation of each fliC and fliD gene to the corresponding deficient mutant restored motility and virulence. These results indicate that flagella of P. syringae pv. tabaci are indispensable organelles for complete virulence on host tobacco plants.     

Spectral analysis of heart rate variability in dogs with mild mitral regurgitation

OBJECTIVE: To assess autonomic function in dogs with mild mitral regurgitation (MR) that did not have clinical signs of the condition. ANIMALS: 6 healthy adult Beagles. PROCEDURE: Mild MR was experimentally induced. A 24-hour ambulatory ECG was recorded before and after induction of MR. Heart rate variability was analyzed in frequency domains by use of the ambulatory ECG. Low-frequency (LF) and high-frequency (HF) power were calculated by integrating over their frequency intervals, and the ratio of LF to HF was also calculated. Measurements of frequency domains were analyzed for 4 time periods (midnight to 6 AM, 6 AM to noon, noon to 6 PM, and 6 PM to midnight). RESULTS: Dogs with experimentally induced MR were classified as International Small Animal Cardiac Health Council class Ia. The HF power of dogs with MR was significantly decreased between 6 AM and noon. The ratio of LF to HF in dogs with MR was significantly increased for the periods between midnight and 6 AM, 6 AM and noon, and noon and 6 PM. CONCLUSIONS AND CLINICAL RELEVANCE: Compensatory response through autonomic modulation was observed in dogs with mild MR that did not have abnormalities, except for cardiac murmur, during clinical examination. This result suggests that treatment during the early stages of mild MR may be beneficial. Additional studies are necessary to determine whether such treatment will delay the onset of congestive heart failure and prolong survival in dogs affected with mild MR.     

Evaluation of intestinal intramucosal pH,arterial and portal venous blood gas values,and intestinal blood flow during small intestinal ischemia and reperfusion in dogs

OBJECTIVES: To determine whether small intestinal ischemia and reperfusion affects intestinal intramucosal pH (pHi), arterial and portal venous blood gas values, and intestinal blood flow (IBF) and to investigate relationships between regional intestinal tissue oxygenation and systemic variables in dogs. ANIMALS: 15 healthy adult Beagles. PROCEDURE: Occlusion of superior mesenteric artery (SMA) for 0, 30, or 60 minutes, followed by reperfusion for 180 minutes, was performed; IBF, pHi, arterial and portal venous blood gas values, arterial pressure, and heart rate were measured at various time points; and intestinal mucosal injury was histologically graded. RESULTS: Occlusion of the SMA induced significant decreases in pHi and IBF. After the release of the occlusion, IBF returned rapidly to baseline values, but improvement in pHi was slow. Arterial and portal venous blood gas analyses were less sensitive than tonometric measurements of pHi, and there was no correlation between results of blood gas analyses and tonometric measurements. Histologic score for intestinal mucosal injury increased significantly, depending on duration of ischemia, and there was a correlation between tonometric results and the histologic score. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that it is difficult to accurately evaluate local oxygenation disorders by monitoring at the systemic level, whereas clinically pHi is the only reliable indicator of inadequate regional intestinal tissue oxygenation in dogs.     

Transformation of trifoliate orange with rice chitinase gene and the use of the transformed plant as a rootstock

RCC2, cDNA clone encoding rice class-I chitinase, was introduced into trifoliate orange (Poncirus trifoliata Raf.). Chitinase activity of the transformed plants was higher than that of the non-transformed plants. The introduced gene was not detected in the scions grafted onto the transformed plants. The effect of the introduction of foreign gene into rootstocks on the scions was discussed.     

Root and stem rot of chrysanthemum caused by five <Emphasis Type="Italic">Pythium</Emphasis> species in Japan

Root and stem rot with wilt of above ground parts of cultivated chrysanthemums was first found in Ibaraki, Toyama and Kagawa prefectures, Japan in 2002 and 2003. Pythium species were isolated from the diseased tissues and identified as P. dissotocum, P. oedochilum, P. sylvaticum, P. ultimum var. ultimum and asexual strains of P. helicoides based on their morphologies and sequences of rDNA-ITS region. All the Pythium species were strongly pathogenic to chrysanthemums in pot conditions and were reisolated from the inoculated plants. Because Pythium root and stem rot of chrysanthemum has never been reported in Japan, we propose that this is a new disease that can be caused by the five Pythium species.