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.     

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.     

Arteriovenous shunts resembling patent ductus arteriosus in dogs: 3 cases

Three dogs presented for the evaluation of cardiac murmurs were diagnosed with aberrant arteriovenous shunts. All cases demonstrated the following findings: 1) relatively soft continuous murmur loudest at the left heart base resembling patent ductus arteriosus (PDA); 2) shunt flow signals in the pulmonary artery on echocardiography; and 3) no PDA on selective angiography, but evidence of anomalous shunting vessels from thoracic aorta to pulmonary vasculature. An aberrant arteriovenous shunt should be considered when a continuous murmur of relatively small intensity is heard.     

Effects of small intestinal ischemia and reperfusion on expression of tumor necrosis factor-alpha and interleukin-6 messenger RNAs in the jejunum, liver, and lungs of dogs

OBJECTIVE: To determine the effects of intestinal ischemia and reperfusion on the expression of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 mRNAs in the jejunum, liver, and lungs of dogs. ANIMALS: 8 healthy adult Beagles. PROCEDURES: In each dog, the cranial mesenteric artery was occluded for 0 (control group; n=4) or 60 (I-R group; 4) minutes, followed by reperfusion for 480 minutes; serum TNF-alpha and IL-6 activities and expression levels of TNF-alpha and IL-6 mRNAs in jejunal, hepatic, and lung tissues were measured before and at the end of the ischemic period and at intervals during reperfusion. For each variable, values were compared between the control and I-R groups at each time point. RESULTS: Compared with the control group, serum IL-6 activity increased significantly after 180 minutes of reperfusion in the I-R group; also, jejunal TNF-alpha mRNA expression increased significantly after 60 (peak) and 180 minutes of reperfusion. In the I-R group, expressions of IL-6 mRNA in the liver and TNF-alpha and IL-6 mRNAs in the lungs increased significantly at 480 minutes of reperfusion, compared with the control group. Serum TNF-alpha activity, expression of IL-6 mRNA in the jejunum, and expression of TNF-alpha mRNA in the liver in the control and I-R groups did not differ. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that the liver, lungs, and jejunum contributed to the production of TNF-alpha and IL-6 after intestinal ischemia and reperfusion in dogs, suggesting that intestinal ischemia and reperfusion induce a systemic proinflammatory cytokine response in dogs.     

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.     

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.     

Ecology,physiology and genetics of a phycodnavirus infecting the noxious bloom-forming raphidophyte <Emphasis Type="Italic">Heterosigma akashiwo</Emphasis>

Abstract:   Heterosigma akashiwo virus (HaV) is a large icosahedral virus (∼0.2 μm) harboring a double-stranded DNA (dsDNA) genome (∼294 kbp). The virus is the only member of the genus Raphidovirus in the family Phycodnaviridae. Since its first discovery, a number of ecologic, physiologic and genetic studies about HaV have been conducted; especially, the relationship between H. akashiwo and HaV in nature was studied and viral infection is now regarded as a significant factor influencing the dynamics and termination of H. akashiwo blooms. HaV infection has considerable impacts on H. akashiwo populations in both aspects of fluctuation in biomass (quantity) and changes in clonal composition (quality). Partial sequencing of the HaV genome revealed that a number of genes showed considerable similarity to those of other protist-infecting viruses; still, the phylogenetic position of HaV suggested a number of enigmas in host–virus coevolution. Here are summarized the ecology, physiology and genetics of HaV especially from the viewpoint of the host–virus relationship.     

Membrane proteins of the endoplasmic reticulum induce high-curvature tubules

The tubular structure of the endoplasmic reticulum (ER) appears to be generated by integral membrane proteins, the reticulons and a protein family consisting of DP1 in mammals and Yop1p in yeast. Here, individual members of these families were found to be sufficient to generate membrane tubules. When we purified yeast Yop1p and incorporated it into proteoliposomes, narrow tubules (approximately 15 to 17 nanometers in diameter) were generated. Tubule formation occurred with different lipids; required essentially only the central portion of the protein, including its two long hydrophobic segments; and was prevented by mutations that affected tubule formation in vivo. Tubules were also formed by reconstituted purified yeast Rtn1p. Tubules made in vitro were narrower than normal ER tubules, due to a higher concentration of tubule-inducing proteins. The shape and oligomerization of the "morphogenic" proteins could explain the formation of the tubular ER.     

Isolation and characterization of a novel immunomodulatory alpha-glucan-protein complex from the mycelium of Tricholoma matsutake in basidiomycetes

Tricholoma matsutake, a high-class edible mushroom in Japan, has been reported to have excellent biological activities, but difficulty in cultivating the fruit bodies and limited bulk availability have restricted detailed studies. We have developed a method of culturing in tanks, enabling the bulk supply of the mycelia. The preparation (CM6271) exerts modulative effects on the immune competence of mice and rats. In this study, a sodium hydroxide extract of CM6271 was defatted followed by fractionation with a combination of ion exchange chromatography and gel filtration in order to identify the components involved in the expression of the activity, and a single peak fraction (MPG-1) was obtained with reversed phase chromatography. MPG-1 was a glycoprotein (sugar:protein ratio, 94.3:5.7) with a relative molecular mass of 360 kDa, and the sugar moiety contained about 90% glucose. NMR spectra and methylation analysis revealed that the alpha-1,4-linkage was the predominant glucan linkage with alpha-1,6- and alpha-1,2-linkages in the minority. The amino acid composition in the protein moiety was rich in glutamine, alanine, asparagine, leucine, glycine, valine, serine, threonine, isoleucine, and proline. MPG-1 was resistant to degradation with amylase or protease. The oral administration of MPG-1 promoted, in a dose-dependent manner, the recovery of the mouse natural killer cell activity and serum IL-12 level that had been reduced by the loading of restraint stress. The dose of MPG-1 (25 mg/kg) required for the expression of the effect decreases to 1/12 of that of CM6271 (300 mg/kg). Furthermore, MPG-1 formed a complex with TGF-beta1 in vitro, modulating the biological activity of TGF-beta1 by binding to its active form. These results indicate that the mycelium of T. matsutake contains a novel alpha-glucan-protein complex with immunomodulatory activities.     

Conjugated linoleic acid-induced fatty liver can be attenuated by combination with docosahexaenoic acid in C57BL/6N mice

We investigated the effect of dietary combination of conjugated linoleic acid (CLA) and docosahexaenoic acid (DHA) to attenuate CLA-induced fatty liver in C57BL/6N mice. Mice were fed semisynthetic diets that contained either 6% high linoleic safflower oil (HL-SAF), 4% HL-SAF + 2% CLA, or 3.5% HL-SAF + 2% CLA + 0.5% DHA for 4 weeks. This 4 week feeding of CLA showed hepatic lipid accumulation concomitant with the decrease in adipose tissue weight in mice. However, 0.5% supplementation of DHA to the CLA diet could alleviate fatty liver without decreasing the antiobesity effect of CLA. The CLA diet promoted fatty acid synthesis in the liver, but DHA supplementation significantly attenuated the increase in enzyme activity induced by CLA. On the other hand, serum adipocytokines, leptin and adiponectin, were drastically decreased by CLA feeding, and DHA supplementation did not affect those levels. These results show that DHA supplementation to the CLA diet can attenuate CLA-induced fatty liver through the reduction of hepatic fatty acid synthesis without affecting adipocytokine production in C57BL/6N mice.