Carabid beetle (Coleoptera: Carabidae) assemblages in the intermediate successional stage after Mt. Bandai eruption of 1888: Effects of environmental variables on carabid beetles in the Urabandai area

We studied carabid beetle assemblage structure and species diversity in an intermediate successional stage (seral) forest established in areas affected by the 1888 eruption of Mt. Bandai and a climax forest that had not been affected by the eruption at the Urabandai area, Fukushima Prefecture, Japan. In total, 2,131 carabid beetles representing 31 species were collected using pitfall traps without bait. A comparison of carabid beetle assemblages between the two forest types revealed that the number of species observed was comparable, but their abundance was greater in the seral forest. The assemblage structure clearly differed between the two forest types. In the seral forest, forest generalists, such as Synuchus arcuaticollis and Pterostichus prolongatus, along with forest specialists including Carabus vanvolxemi and Pterostichus asymmetricus, were collected. In the climax forest, forest generalists, such as Synuchus cycloderus and Carabus albrechti tsukubanus, were more abundant than forest specialists. This suggests that the current seral forest in the Urabandai area possesses environmental variables that enable the coexistence of both forest specialist and generalist beetle species. A redundancy analysis showed that six species from the genera Synuchus and Pterostichus were associated with high canopy openness and high understory vegetation cover, whereas species recorded only in the climax forest were associated with deep litter. Therefore, it seems likely that carabid beetles in the Urabandai area were affected by these three environmental variables.     

Evolution of intrahepatic carbon, nitrogen, and energy metabolism in a D-galactosamine-induced rat liver failure model

A clearer picture of the hepatic metabolic pathways affected by fulminant hepatic failure (FHF) would help develop nutritional support and nonsurgical therapies for FHF. We characterized the evolution of hepatic metabolism in a rat model of FHF using an isolated perfused liver system together with a mass-balance model of intermediary metabolism. Principal component analysis (PCA) was used to identify potential new sensitive markers for FHF. To induce FHF, rats were given two D-galactosamine injections under fasting conditions. Controls were fasted only. Livers were harvested 1, 4, 8, and 12 h later and perfused with Eagle minimal essential medium supplemented with amino acids and bovine serum albumin, and equilibrated with 95% O2/5% CO2. At the 1 h time point, lactate release increased concomitant with a decrease in gluconeogenesis, TCA cycle and mitochondrial electron transport fluxes. At 4 h, amino acid metabolism and urea cycle fluxes were significantly depressed. By 8 h, gluconeogenesis had switched to glycolysis. By 12 h, amino acid metabolism was broadly inhibited, and there was a net release of many amino acids. Mass-balance analysis shows that the main source of ATP production in the FHF liver gradually changed from mitochondrial oxidative phosphorylation to glycolysis. PCA suggests that a linear combination of glucose, lactate, and glutamine concentrations in arterial plasma is a sensitive marker for FHF. We conclude that D-galactosamine causes early mitochondrial dysfunction while glycolytic ATP synthesis remains functional. Markers that are indirectly linked to these pathways may be used to evaluate the progression of FHF.     

Enzymatic profiling of human kallikrein 14 using phage-display substrate technology

The human KLK14 gene is one of the newly identified serine protease genes belonging to the human kallikrein family, which contains 15 members. KLK14 , like all other members of the human kallikrein family, is predicted to encode for a secreted serine protease already found in various biological fluids. This new kallikrein is mainly expressed in prostate and endocrine tissues, but its function is still unknown. Recent studies have demonstrated that KLK14 gene expression is up-regulated in prostate and breast cancer tissues, and that higher expression levels correlate with more aggressive tumors. In this work, we used phage-display substrate technology to study the substrate specificity of hK14. A phage-displayed random pentapeptide library with exhaustive diversity was screened with purified recombinant hK14. Highly specific and sensitive substrates were selected from the library. We show that hK14 has dual activity, trypsin- and chymotrypsin-like, with a preference for cleavage after arginine residues. A SwissProt database search with selected sequences identified six potential human protein substrates for hK14. Two of them, laminin alpha-5 and collagen IV, which are major components of the extracellular matrix, have been demonstrated to be hydrolyzed efficiently by hK14.     

Quantitative effects of thermal injury and insulin on the metabolism of the skeletal muscle using the perfused rat hindquarter preparation

Injury from a severe burn or trauma can propel the body into a hypermetabolic state that can lead to the significant erosion of lean muscle mass. Investigations describing this process have been somewhat limited due to the lack of adequate experimental models. Here we report the use of a perfused rat hindquarter preparation to study the consequences of a moderate burn injury (approximately 20% total body surface area), with or without the addition of exogenous insulin (12.5 mU/mL), on the fluxes of major metabolites across the isolated skeletal muscle. The metabolic flux data was further analyzed using metabolic flux analysis (MFA), which allows for the estimation of the impact of these conditions on the intracellular muscle metabolism. Results indicate that this model is able to capture the increased rate of proteolysis, glutamine formation, and the negative nitrogen balance associated with the burn-induced hypermetabolic state. The inclusion of exogenous insulin resulted in significant changes in several fluxes, including an increase in the metabolism of glucose and the flux through the pentose phosphate pathway, as well as a reduction in the metabolism of glutamine, alanine, and leucine. However, insulin administration did not affect the nitrogen balance or the rate of proteolysis in the muscle, as has been suggested using other techniques. The use of the perfused hindquarter model coupled with MFA is a physiologically relevant and experimentally flexible platform for the exploration of skeletal muscle metabolism under catabolic conditions, and it will be useful in quantifying the specific metabolic consequences of other therapeutic advances.     

Transient expression in electroporated pea protoplasts: Elicitor responsiveness of a phenylalanine ammonia-lyase promoter

Summary High yields of viable pea protoplasts were produced from suspension cultured cells and the conditions for the optimum transient expression of the chloramphenicol acetyltransferase (CAT) gene fused to the CaMV 35S promoter after electroporation were investigated. Conditions for elicitor induction of a member of the phenylalanine ammonia-lyase (PAL) gene family in pea was also investigated using a chimeric gene carrying 480 bp of the putative promoter region of gPAL1 connected to bacterial cat gene and nos terminator. CAT activity was considerably induced by the treatment with fungal elicitor (>100 g/ml glucose equivalent) isolated from Mycosphaerella pinodes, a pea pathogen.Abbreviations CAT chloramphenicol acetyltransferase - PAL phenylalanine ammonia-lyase - CM acetylated chloramphenicol - GSH reduced glutathione - NOS nopaline synthase - ES electroporation solution - CaMV cauliflower mosaic virus - GUS -glucuronidase - CHS chalcone synthase - 2,4-D 2, 4-dichlorophenoxyacetic acid Present address: Research Institute, Takasago Perfumery Inc, 5-31-36, Kamata, Minato, Tokyo, 144, Japan Toso Inc, 4560 Oaza-Tomita, Shin-nanyo, Yamaguchi, 746, Japan Central Laboratory of Green Complex, Kasetsert University, Kamphaensaen, Nakohn Pathom, Thailand     

Lactobacillus gasseri SBT2055 Induces TGF-β Expression in Dendritic Cells and Activates TLR2 Signal to Produce IgA in the Small Intestine

Probiotic bacteria provide benefits in enhancing host immune responses and protecting against infection. Induction of IgA production by oral administration of probiotic bacteria in the intestine has been considered to be one reason for this beneficial effect, but the mechanisms of the effect are poorly understood. Lactobacillus gasseri SBT2055 (LG2055) is a probiotic bacterium with properties such as bile tolerance, ability to improve the intestinal environment, and it has preventive effects related to abdominal adiposity. In this study, we have found that oral administration of LG2055 induced IgA production and increased the rate of IgA⁺ cell population in Peyer''s patch and in the lamina propria of the mouse small intestine. The LG2055 markedly increased the amount of IgA in a co-culture of B cells and bone marrow derived dendritic cells (BMDC), and TLR2 signal is critical for it. In addition, it is demonstrated that LG2055 stimulates BMDC to promote the production of TGF-β, BAFF, IL-6, and IL-10, all critical for IgA production from B cells. Combined stimulation of B cells with BAFF and LG2055 enhanced the induction of IgA production. Further, TGF-β signal was shown to be critical for LG2055-induced IgA production in the B cell and BMDC co-culture system, but TGF-β did not induce IgA production in a culture of only B cells stimulated with LG2055. Furthermore, TGF-β was critical for the production of BAFF, IL-6, IL-10, and TGF-β itself from LG2055-stimulated BMDC. These results demonstrate that TGF-β was produced by BMDC stimulated with LG2055 and it has an autocrine/paracrine function essential for BMDC to induce the production of BAFF, IL-6, and IL-10.     

Paradoxical effects of chondroitin sulfate-E on Japanese encephalitis viral infection

Glycosaminoglycans (GAGs) have diverse functions in the body and are involved in viral infection. The purpose of this study was to evaluate the possible roles of the E-disaccharide units GlcAβ1–3GalNAc(4,6-O-disulfate) of chondroitin sulfate (CS), a GAG involved in neuritogenesis and neuronal migration, in Japanese encephalitis virus (JEV) infection. Soluble CS-E (sCS-E) derived from squid cartilage inhibited JEV infection in African green monkey kidney-derived Vero cells and baby hamster kidney-derived BHK cells by interfering with viral attachment. In contrast, sCS-E enhanced viral infection in the mouse neuroblastoma cell line Neuro-2a, despite the fact that viral attachment to Neuro-2a cells was inhibited by sCS-E. This enhancement effect in Neuro-2a cells seemed to be related to increased viral RNA replication and was also observed in a rat infection model in which intracerebral coadministration of sCS-E with JEV in 17-day-old rats resulted in higher brain viral loads than in rats infected without sCS-E administration. These results show the paradoxical effects of sCS-E on JEV infection in different cell types and indicate that potential use of sCS-E as an antiviral agent against JEV infection should be approached with caution considering its effects in the neuron, the major target of JEV.     

Studies on Proteinases from Gliocladium roseum

The alkaline proteinases of Gliocladium roseum (Link) Bainier were purified in crystalline forms by procedures of alcoholic precipitation, fuller’s earth- and acrinol-treatment, and isolated in two types. (Proteinases I and II). Both of these proteinases were homogeneous on zone electrophoresis with polyacrylamide gel (Gyanogum 41), and had the optimal pH values of 11 (Proteinase I) and 10 (Proteinase II), and the optimal temperature of 45°C.

The enzymatic reaction of proteinase I was remarkably promoted by Fe⁺⁺ and Co⁺⁺, and that of proteinase II was promoted by Fe⁺⁺, Go⁺⁺ and Ca⁺⁺, and both proteinases were protected from heat-inactivation by Ca⁺⁺ Proteinase II was activated remarkably by Cl^? under the existence of Fe⁺⁺, but proteinase I was unaffected by the anion.

The order of strength of proteolytic power of these proteinases and chymotrypsin on casein was as follows; proteinase I> proteinase II> chymotrypsin.