EFFECTS OF SARAFOTOXIN S6c ON RENAL HAEMODYNAMICS AND URINE FORMATION IN ANAESTHETIZED DOGS

1. The effects of sarafotoxin S6c (S6c), a selective endothelin ETB receptor agonist, on renal haemodynamics and urine formation were examined in anaesthetized dogs. 2. Intrarenal arterial infusion of S6c at a rate of 1 or 5 ng/kg per min produced a transient increase in renal blood flow (RBF), with no change in systemic blood pressure and heart rate; RBF then decreased gradually to below the basal value. There were significant and dose-dependent increases in urine flow and free water clearance and decreases in urine osmolality during S6c infusion, whereas urinary excretion of sodium and glomerular filtration rate (GFR) remained unchanged. Simultaneously, S6c administration elicited a marked increase in urinary excretion of nitric oxide (NO) metabolites, N0₂^? and N0₃^? (UNO*V). 3. In dogs simultaneously administered S6c (5 ng/kg per min) and iV^G-nitro-L-arginine (NOARG; 40 (jig/kg per min), a NO synthase inhibitor, the renal vasodilator effect of S6c was abolished and marked reductions in RBF and GFR were observed. The S6c-induced diuretic action was not affected by NOARG. In the presence of NOARG, there was a small amount of UNO_xV at the basal level and the administration of S6c did not increase UNOxV. 4. These results suggest that an intrarenal arterial infusion of S6c enhances the production of NO in the kidney and that this enhancement contributes to the peptide-induced renal vasodilation. In contrast, it is unlikely that S6c-induced water diuresis is related to NO production stimulated by this peptide.     

Locus of a virus neutralization epitope on the Japanese encephalitis virus envelope protein determined by use of long PCR-based region-specific random mutagenesis

We prepared recombinant Japanese encephalitis (JE) virus populations possessing random mutations at the envelope (E) protein region by a long PCR-based method. Neutralization-resistant mutants were selected from these populations by application of JE-specific virus neutralizing monoclonal antibody (mAb) 503, which possessed a 51,200-fold neutralization titer. We classified the mutants into three groups, each bearing two amino acid alterations at the E protein region: 52, Gln-Arg, and 136, Lys-Glu; 136, Lys-Glu, and 275, Ser-Pro; and 126, Ile-Thr, and 136, Lys-Glu, respectively. Three different genetically engineered variants, each bearing a single mutation, 126, Ile-Thr; 136, Lys-Glu; and 275, Ser-Pro, respectively, showed partial but not complete recovery of reactivity to mAb 503. Our results indicate that the amino acid substitutions at amino acid positions 52, 126, 136, and 275 altered the structure of the neutralization epitope for mAb 503 on the E protein. All these mutations were clustered at the junction of domains I and II of the E protein and it is likely that the epitope for mAb 503 is composed of at least E(0)-e, D(0)-a, and k strands of the E protein. We also demonstrated the efficacy of the long PCR-based recombinant virus technique as a useful tool for the creation of a variety of mutants bearing random mutations at targeted areas of the virus genome.     

Supplementation Effect of Early Pregnancy Factor-Positive Serum into Bovine In Vitro Fertilization Culture Medium

PROBLEM: Early pregnancy factor (EPF) has been detected in pregnant bovine serum, and its activity appeared from 24 to 48 hr after insemination. However, in bovine in vitro fertilization (IVF), an EPF-like substance(s) had been detected in the culture medium of fertilized ovum. Therefore, we think that EPF and EPF-like substance(s) are very important materials for the development of the embryo. In this study, we examined the development of the embryo when fertilized bovine ova were cultured with IVF culture medium supplemented with EPF-positive or -negative serum. METHOD OF STUDY: EPF activity of each serum (fetal calf serum [FCS], calf serum [CS], estrus bovine serum, and pregnant bovine serum) was assessed by the bovine-rosette inhibition test. The sera were supplemented in TCM-199 culture medium, and IVF bovine ova were cultured with the media for 6 or 7 days, respectively. The culture media of each group were evaluated for EPF activity by the bovine-rosette inhibition test 48 hr after IVF. The cleavage rate of each group was calculated at 48 hr, and 6 or 7 days after IVF. The culture medium of cumulus cells was also tested for EPF activity. RESULTS: Only the pregnant bovine sera were EPF positive. All the culture media 48 hr after IVF became EPF positive. Additionally, the culture medium of cumulus cells did not have EPF activity. There was no significant difference in the cleavage rate of the EPF-positive and - negative sera 48 hr after IVF. However, the cleavage rate of EPF-positive sera tended to be higher than the negative sera. In contrast, the blastocyst development rates of EPF-positive sera were significantly higher than those of the negative sera 6 to 7 days after IVF (P < 0.05). CONCLUSIONS: The data suggest that an EPF-like substance(s) may be secreted from the in vitro fertilized bovine ovum but not from the cumulus cell, and that the EPF in the pregnant serum may accelerate the development of the bovine embryo in IVF.     

Heteropentameric cholera toxin B subunit chimeric molecules genetically fused to a vaccine antigen induce systemic and mucosal immune responses: a potential new strategy to target recombinant vaccine antigens to mucosal immune systems

Noninvasive mucosal vaccines are attractive alternatives to parenteral vaccines. Although the conjugation of vaccine antigens with the B subunit of cholera toxin (CTB) is one of the most promising strategies for vaccine delivery to mucosal immune systems, the molecule cannot tolerate large-protein fusion, as it severely impairs pentamerization and loses affinity for GM1-ganglioside. Here we report a new strategy, in which steric hindrance between CTB-antigen fusion subunits is significantly reduced through the integration of unfused CTB "molecular buffers" into the pentamer unit, making them more efficiently self-assemble into biologically active pentamers. In addition, the chimeric protein took a compact configuration, becoming small enough to be secreted, and one-step affinity-purified proteins, when administered through a mucosal route, induced specific immune responses in mice. Since our results are not dependent on the use of a particular expression system or vaccine antigen, this strategy could be broadly applicable to bacterial enterotoxin-based vaccine design.     

The ex vivo effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on rat intra- and extraneuronal monoamine oxidase activity

After i.p. injection of 30 mg/kg 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) daily for 4 days and sacrificing the rats 4 h after the last injection, striatal monoamine oxidase (MAO)-A and -B activities, assayed by conventional method with 5-hydroxytryptamine (5-HT) and benzylamine, were not changed. By an uptake technique, with dopamine as the substrate for both uptake and MAO, intrasynaptosomal MAO-A and -B activities were found to be greatly reduced with a greater MAO-A reduction. Intrasynaptosomal 5-HT oxidation by MAO-A was not changed in other forebrain regions treated with these MPTP doses. Similar results were also found with two brain preparations treated with single MPTP doses (30 mg/kg). This reduction in intrasynaptosomal MAO activity was completely absent after treatment with lower MPTP doses (15 mg/kg, daily for 5 days) and 5 days of a withdrawal period. The decrease in MAO activity might have been due to the decrease in DA transport into striatal synaptosomes during the enzyme assay and/or to reversible inhibition of intrasynaptosomal MAO by MPP+.     

Analgesic action of loperamide,an opioid agonist,and its blocking action on voltage-dependent Ca2+ channels

We investigated the relationship between the antinociceptive effect of the opiate agonist loperamide at the spinal level and its inhibitory effect on calcium influx. Intrathecal administration of loperamide showed a significant antinociceptive effect in the formalin test, which was not prevented by naloxone. On the other hand, no significant effects were observed by nicardipine, an L-type specific blocker, or by BAY K8644, an L-type specific agonist, suggesting no significant role of L-type calcium channels in nociceptive signal transduction. Loperamide suppressed the calcium influx in dorsal root ganglion neurons. As the antinociceptive effect of loperamide was not affected by naloxone or other calcium channel blocking toxins, and loperamide showed a direct inhibitory effect on calcium-influx, the analgesic effect of intrathecally injected loperamide might be due to its blockade of the voltage-dependent calcium channels at the terminals of the primary afferent fibers.     

Histopathologic changes in serum bile acid fractions in pressure ulcer patients

BACKGROUND/AIMS: Bile acids are synthesized in the liver and released into the intestinal tract to aid in digestion and absorption by increasing permeability via alteration of the cell membrane. Bedridden elderly patients typically have pressure ulcers that may be due to both physical local pressure as well as skin cell changes induced by the physiologic effects of bile acids. METHODOLOGY: This study investigated 31 elderly bedridden patients with pressure ulcers (mean age, 81.7 years) and 19 healthy elderly (mean age, 79.7 years). Five serum bile acid fractions were summed to determine total bile acid, and transaminase and cholesterol levels were also measured. RESULTS: Total cholesterol levels were significantly lower (p<0.05) in pressure ulcer patients and transaminase levels were not significantly different between the two groups. The primary bile acids were generally higher and the secondary and tertiary bile acids lower in pressure ulcer patients. In particular, the secondary bile acid deoxycholic acid was significantly higher in all pressure ulcer patients. When analyzed by grade of pressure ulcer, the primary bile acids were significantly lower in pressure ulcer patients. CONCLUSIONS: Secondary bile acid fraction deoxycholic acid measurements may indicate bedridden patients at higher risk for pressure ulcers.     

A trial of infection control measure by clinical microbiology laboratory

In 1998, we developed a Total Infection Control System in Saga Medical School Hospital, and would like to introduce it for the practical use. This system was named "Dr. FLEMING" (Flexible Microbiological Test & Information System for the New Generation) and is expected to help physicians by providing highly valuable test results and useful information. For example, bacterial identification and drug susceptibility test can be completed within 4-6 hrs after bacterial colony is isolated, and the test report the contains full-colored pictures to enhance understanding. In addition, we have made an information center for infectious disease, where physicians can have access to various data bases outside our hospital. Furthermore, we offer many kinds of useful information to physicians working at other medical facilities to assist their clinical practice of infectious diseases.