Administration of glucocorticoids markedly increases the numbers of granulocytes and extrathymic T cells in the bone marrow.

Glucocorticoids, steroid hormones, are widely used as an anti-inflammatory drug. However, clinicians have sometimes encountered adverse drug reactions such as ulcers and tissue damage. In this study, we investigated how such adverse reactions of glucocorticoids are evoked, using an experimental mice model. When hydrocortisone (0.5 or 1.0 mg/day/mouse) was administered daily for 2 weeks, severe leukocytopenia was induced in all immune system organs. However, granulocytes (Gr-1(+)Mac-1(+)) were increased in number in the bone marrow and peripheral blood. This seemed to be due to an elevated level of myelopoiesis in the bone marrow. As well as increasing in number, granulocytes were functionally activated as estimated by the Ca2+ influx and superoxide production. The proportion of primordial T cells (CD3(int)IL-2Rbeta+) in the thymus and the number of primordial T cells in the bone marrow also increased. Mice administered hydrocortisone became susceptible to stress. Thus, these mice showed gastric ulcers when they were exposed to restraint stress for 12 h. These results suggest that activated granulocytes and primordial T cells might provide a mechanism involved in steroid ulcers and tissue damage, possibly through the superoxide production of granulocytes and the autoreactivity of primordial T cells.     

Predator-avoidance behavior in anuran tadpoles: a new bioassay for characterization of water-soluble cues

Most surveys of large wood in streams are conducted by counting and measuring every piece of large wood within a reach, a technique that is effective but time-consuming. In this study we evaluated an alternative method that takes less time and can be employed in studies in which an estimate of total large wood volume along a stream reach is the primary metric of interest. In first- through third-order streams we estimated in-stream large wood volume and large wood frequency, comparing large wood census estimates to those from a modified a line-intercept technique that has been commonly used in terrestrial forest surveys. Estimates of large wood volume from line transects located in the geographic center of the stream (parallel to stream axis and equidistant from bankfull margins) were highly correlated with those from the wood census (P < 0.001, r ² = 0.88, Pearson’s r = 0.935), but produced slightly greater estimates of large wood volume (regression slope = 1.28, SE = 0.16). Line-intercept estimates of large wood frequency (number per 100 m of stream) were significantly correlated to the wood census counts, but the line-intercept method underestimated frequency by about 50% (P = 0.016). Differences in the estimated large wood volume between line-intercept and wood census surveys were associated with variability in the diameter of the large wood, but unrelated to stream bankfull width, for the range of stream sizes evaluated in this study (≈ 2 to 11 m). Our results suggest that in small constrained streams, line-intercept surveys are an effective method for estimating in-stream large wood volume and that these estimates better approximate results from whole-stream census techniques where the diameter of in-stream wood is relatively consistent. Handling editor: K. Martens     

Paradoxical effect of sudan III on the in vivo and in vitro genotoxicity elicited by 7,12-dimethylbenz(a)anthracene

Effect of the induction of drug metabolizing enzymes by Sudan III on the in vivo and in vitro genotoxicity elicited by 7,12-dimethyl-benz(a)anthracene (DMBA) was investigated. A significant suppression of DMBA-induced micronucleated reticulocytes was observed in C57BL/6 mice treated with Sudan III intraperitoneally for 3 or 5 days before injection of the DMBA. However, the preincubation of DMBA with hepatic microsomes from Sudan III-treated rats caused a marked increase in the in vitro mutagenicity in the Ames assay, paradoxically. Sudan III was found to induce CYP 1A1, 7-ethoxycoumarin O-deethylase activity as well as both UDP-glucuronyl transferase and glutathione S-transferase activities. The increase of mutagenicity of DMBA observed in the Ames assay using hepatic microsomes from Sudan III-treated rats was inhibited by the addition of uridine 5′-diphosphoglucuronic add or reduced glutathione with cytosol. Mutagenic metabolites of DMBA formed by CYP1A1 appeared to be effectively detoxified by these phase II enzymes. The results of this study suggest that Sudan III-induced prevention of in vivo mutagenesis is due to the induction of both CYP 1A1 and detoxifying phase II enzymes. The induced CYP1A1 may accelerate formation of active metabolic intermediates, but phase II enzymes are also induced and detoxify these intermediates to inactive metabolites. This would reduce residence time of the carcinogen in the body and the time of exposure to active metabolites for target organs.     

Cloning and characterization of the carbapenem biosynthetic genes from Streptomyces fulvoviridis

Carbapenem non-producing mutants were isolated from Streptomyces fulvoviridis and divided into six cosynthesis groups. By using one of the mutants as the host and plasmid pIJ385 as the vector, we cloned carbapenem biosynthetic genes from the parental S. fulvoviridis strain. A cloned 6-kb DNA fragment complemented the defects of three mutants each of which had a mutation in different genes. Southern blot hybridization using the cloned 6-kb fragment as probe showed the presence of the nucleotide sequences homologous to the probe in other carbapenem-producing Streptomyces spp. In addition, Streptomyces griseus, a carbapenem non-producer, possessed the sequence homologous to the probe and showed co-synthesis phenomena with some of the carbapenem non-producing mutants of S. fulvoviridis.     

Characterization of an alkaline protease from Bacillus sp. no. AH-101

Summary The Bacillus sp. no. AH-101 alkaline protease showed higher hydrolysing activity against insoluble fibrous natural proteins such as elastin and keratin in comparison with subtilisins and Proteinase K. The optimum pH of the enzyme toward elastin and keratin was pH 10.5 and pH 11.0–12.0 respectively. The specific activity toward elastin and keratin was 10 600 units/mg protein and 3970 units/mg protein, respectively. The enzymatic activity was not inhibited by p-chloromercuribenzoic acid and iodoacetic acid. Carbobenzoxy-glycyl-glycyl-L-phenylalanyl chloromethyl ketone completely inhibited the caseinolytic activity, but 36% elastolytic activity remained. No inhibitory effect on caseinolytic and elastolytic activity was shown by tosyl-L-phenylalanyl-chloromethyl ketone, tosyl-L-lysine chloromethyl ketone, carbobenzoxy-L-phenylalanyl chloromethyl ketone, and elastatinal. The amino acid composition and amino terminal sequence of the enzyme were determined. The no. AH-101 alkaline protease was compared with subtilisin BPN', subtilisin Carlsberg, no. 221, and Ya-B alkaline proteases. Extensive sequence homology existed among these enzymes. Offprint requests to: H. Takami     

Alleviation of High-Fat Diet-Induced Fatty Liver Damage in Group IVA Phospholipase A2-Knockout Mice

Hepatic fat deposition with hepatocellular damage, a feature of non-alcoholic fatty liver disease, is mediated by several putative factors including prostaglandins. In the present study, we examined whether group IVA phospholipase A₂ (IVA-PLA₂), which catalyzes the first step in prostanoid biosynthesis, is involved in the development of fatty liver, using IVA-PLA₂-knockout mice. Male wild-type mice on high-fat diets (20% fat and 1.25% cholesterol) developed hepatocellular vacuolation and liver hypertrophy with an increase in the serum levels of liver damage marker aminotransferases when compared with wild-type mice fed normal diets. These high-fat diet-induced alterations were markedly decreased in IVA-PLA₂-knockout mice. Hepatic triacylglycerol content was lower in IVA-PLA₂-knockout mice than in wild-type mice under normal dietary conditions. Although high-fat diets increased hepatic triacylglycerol content in both genotypes, the degree was lower in IVA-PLA₂-knockout mice than in wild-type mice. Under the high-fat dietary conditions, IVA-PLA₂-knockout mice had lower epididymal fat pad weight and smaller adipocytes than wild-type mice. The serum level of prostaglandin E₂, which has a fat storage effect, was lower in IVA-PLA₂-knockout mice than in wild-type mice, irrespective of the kind of diet. In both genotypes, high-fat diets increased serum leptin levels equally between the two groups, but did not affect the serum levels of adiponectin, resistin, free fatty acid, triacylglycerol, glucose, or insulin. Our findings suggest that a deficiency of IVA-PLA₂ alleviates fatty liver damage caused by high-fat diets, probably because of the lower generation of IVA-PLA₂ metabolites, such as prostaglandin E₂. IVA-PLA₂ could be a promising therapeutic target for obesity-related diseases including non-alcoholic fatty liver disease.