Propionibacterium acnes induces acute TNFα-mediated apoptosis of hepatocytes followed by inflammatory T-cell-mediated granulomatous hepatitis in mice

The CD3+/TCR+ T-cell-mediated hepatic inflammation induced byPropionibacterium acnes could be divided into an acute and a chronic phase. The acute phase occurred within 72 h after injection and displayed hepatic apoptosis. Anti-TNF antibody inhibited both theP. acnes-induced hepatic apoptosis and lymphocyte infiltration seen in this phase, indicating the involvement of this cytokine. Thereafter, a chronic phase was manifested from days 7 to 14 after injection. It was characterized as granulomatous inflammation admixed with apoptosis of infiltrating lymphocytes and some hepatocytes. Immunohistochemical staining showed that the infiltrating lymphocytes displayed TNF, TNF type I receptor and a variety of cytokines including IL-1, IL-4, IL-6, IL-10, IFN or IL-12. Interestingly, in naive mice, the arteries in the liver constitutively expressed IFN. Its expression appeared to be substantially increased at 48 h, decreased at 72 h, and increased again on day 14 afterP. acnes injection. Furthermore, Fas or FasL was only detected on the lymphocytes within the granuloma. We conclude thatP. acnes can induce a TNF-mediated acute hepatic apoptosis which subsequently progress to a T-cell-mediated granulomatous hepatitis with increased expression of multiple cytokines and Fas/FasL.     

Synergistic effect of PEG-400 and cyclodextrin to enhance solubility of progesterone

Conclusion  PEG-400, polysorbate 80, and 2 CDs (Trappsol HPB and Captisol) were used in an attempt to improve the aqueous solubility of a model hydrophobic drug, progesterone. The aqueous solubility of progesterone improved significantly from 0.007 mg/mL by the addition of PEG-400, CDs, and polysorbate 80. In systems containing various amounts of PEG-400 and 3% Trappsol HPB in water (% wt/wt), the theoretical solubility was calculated by adding the solubilities in the individual systems. The observed solubility values were up to 96% higher than the theoretical values. The effect of synergism was significant in 5% to 50% PEG-400/water systems containing Trappsol HPB. Systems containing Captisol did not show such synergistic effects. In general, the addition of polysorbate 80 to the PEG-400/water systems containing CDs affected synergism negatively.     

Trypsin induces tumour necrosis factor-alpha secretion from a human leukemic mast cell line

Trypsin activating both proteinase-activated receptor (PAR) 2 and PAR4 plays an important role in inflammation. We have investigated the potential of trypsin to induce TNF-alpha secretion from the human leukemic mast cell line (HMC-1). HMC-1 cells co-express both PAR2 and PAR4, and their agonist trypsin signals to HMC-1 cells. Trypsin (100 nm), SLIGKV-NH(2) (100 microm, corresponding to the PAR2 tethered ligand), or GYPGQV-NH(2) (100 microm, corresponding to the PAR4 tethered ligand) induced tumour necrosis factor (TNF)-alpha secretion from HMC-1 cells. TNF-alpha secretion by trypsin was significantly blocked by pretreatment with 50 microm PD098059, MEK-1 inhibitor. Furthermore, trypsin stimulated the activation of extracellular signal-regulated kinase (ERK) in HMC-1 cells without any detectable activation of c-Jun N-terminal kinase (JNK) and p38 MAP kinase homologue. These results show that trypsin may induce TNF-alpha secretion following activation of ERK via both PAR2 and PAR4 on HMC-1 cells.     

Novel maltotriose esters enhance biodegradation of Aroclor 1242 by Burkholderia cepacia LB400

The objective of this research was to evaluate the effect of enzymatically synthesized maltotriose fatty acid monoesters (Ferrer, M., et al. 2000 Tetrahedron 56, 4053–4061) on Aroclor 1242 solubilization and biodegradation. Three forms of the surfactant, laurate, palmitate and stearate monoester, were tested. Potential enhancement of solubilization of hydrophobic substances mediated by these non-ionic surfactants was exploited in this study. A polychlorinated biphenyl (PCB) degrading organism, Burkholderia cepacia LB400, was also selected. It was found that all surfactants were effective in solubilizing Aroclor 1242 but the rate of Aroclor 1242 biodegradation proceeded rapidly only in the presence of 6-O-palmitoylmaltotriose. For example, the addition of 48 mg 6-O-palmitoylmaltotriose/l increased the apparent solubility from 140 to 305 g/l. As a result, only 8% of the Aroclor remained at the end of 24 h incubation. In contrast, 49.2% of the Aroclor 1242 remained in the absence of surfactant. It appears that maltotriose fatty acid monoesters can significantly increase the bioavailability, and thereby accelerate the biodegradation of highly chlorinated PCBs, particularly Aroclor 1242, by Burkholderia cepacia LB400. The possibility of obtaining these biodegradable surfactants with high yield, easy recovery and high purity by using a new enzymatic methodology, makes maltotriose esters available for bioremediation purposes.     

Seasonal patterns of microcystin-producing and non-producing Planktothrix rubescens genotypes in a deep pre-alpine lake

The filamentous cyanobacterium Planktothrix rubescens produces secondary metabolites called microcystins (MC) that are potent toxins for most eukaryotes, including zooplankton grazers, cattle and humans. P. rubescens occurs in many deep and thermally stratified lakes throughout Europe. In Lake Zurich (Switzerland), it re-appeared in the 1970s concomitant with decreasing eutrophication. Since then, P. rubescens has become the dominant species in this major drinking water reservoir, where it forms massive metalimnetic blooms during late summer. These cyanobacteria harbor subpopulations of non-MC producers, but little is known about the environmental factors affecting the success of such genotypes. The non-MC-producing subpopulation of P. rubescens was studied using a quantitative real-time PCR (qPCR) assay on the MC synthetase (mcy) gene cluster that targets a deletion on the mcyH and mcyA genes, which inactivates MC biosynthesis. Two complementary qPCR assays were used to assess the total population abundance (based on the 16S rDNA gene) and the mcy gene copy number (based on a conserved region in the adenylation domain of the mcyB gene). The objective was to evaluate the seasonal patterns of the share of non-MC-producing filaments in the total P. rubescens population. The mcyHA mutants were present in low proportions (up to 14%) throughout the year. Their highest relative abundances occurred during the winter mixis, when total concentrations of P. rubescens were minimal. The MC deficient mutants seemed to better survive in sparse populations, possibly because of lower grazing pressure and a consequently reduced need for MC-mediated protection. Alternatively, the mutants might cope better with the sub-optimal, stressful pressure and light conditions during the winter mixis. Altogether, our results suggest that subtle trade-offs might seasonally determine the proportions of non-MC producers within P. rubescens populations.     

Photosynthetic characteristics and productivity of spring rape plants as related to crop density

Optimal density of spring rape (Brassica napus L.) crop stand was determined by plant photosynthetic characteristics at the beginning of flowering. As crop density increased from 100 to 350 plants/m², leaf surface index (LSI) of the crop was found to increase by 18.2–80.2%, and LSI decreased by 38.8–67.3% as compared with the sparsest crop (50–100 plants/m²). LSI depended on the rate of incident PAR reaching 0.5 and 0.25 heights of the crop stand and to the soil surface. When crop density increased from 100 to 350 plants/m², the photosynthetic potential (PP) of the crop increased 1.8 times as compared with the sparsest crop. PP of the densest rape crop stand was 3 times lower than in the sparsest crop. When the crop density increased from 100 to 250 plants/m², the daily increment in biomass calculated per leaf surface unit increased by 27.0% as compared with the sparsest crop and depended on LSI. When leaf area decreased, the daily increment in biomass calculated per leaf surface unit declined; in the densest stand, this characteristic was by 58.3% lower than in the sparsest crop. Rape productivity at the flowering stage depended on the crop density, LSI of plants, rate of PAR reaching 0.5 and 0.25 heights of the crop stand and to the soil surface, PP, and the daily increment in biomass calculated per leaf surface unit. Crop productivity at the flowering stage and the rape seed yield were associated by a significant parabolic relationship. When crop density increased from 100 to 350 plants/m², seed yield per plant considerably decreased (by 33.1–78.5%) as compared with the sparsest crop. The greatest influence on seed yield per plant was exerted by LSI and the daily increment in biomass calculated per leaf surface unit. When crop density increased to 250–300 plants/m², the seed yield considerably rose (by 28.6–58.8%) as compared with the sparsest crop; when this index reached 300–350 plants/m², the seed yield decreased because plant growth was suppressed, with the productivity reduced. The results thus obtained suggest that the photometric characteristics of spring rape were at optimum at crop density of 100–250 plants/m². The agroclimatic conditions of Lithuania ensure potential for rapid accumulation of total biomass and high seed yield.     

Thrombin causes the Enrichment of Rat Brain Primary Cultures with Ependymal Cells Via Protease-Activated Receptor 1

Ependymal cell culture models from rat have been developed over the last 20 years to facilitate biochemical studies on this least-studied glial cell type. The cell culture protocol calls for the presence of thrombin, which is essential for obtaining a high proportion of multiciliated ependymal cells. The serine protease appears to act via protease-activated receptor 1 to prevent the apoptosis of ependymal precursors and enhance their proliferation without affecting contaminating cells. Unciliated precursors differentiate into polyciliated ependymocytes by passing through a stage of monociliation. The message for protease-activated receptor (PAR) 1 is initially abundant in the cultures, but its level declines as the cells differentiate. Besides PAR 1, signalling through PAR 2 also promotes ciliation in rat brain primary cultures, albeit to a lesser degree than the thrombin receptor. Thrombin and other proteases may be involved in the regulation of ventricular wall development. This action would be mediated mainly by PAR1.     

Giardia duodenalis cysteine proteases cleave proteinase-activated receptor-2 to regulate intestinal goblet cell mucin gene expression

Giardia duodenalis cysteine proteases have been identified as key virulence factors and have been implicated in alterations to intestinal goblet cell activity and mucus production during Giardia infection. The present findings demonstrate a novel mechanism by which Giardia cysteine proteases modulate goblet cell activity via cleavage and activation of protease-activated receptor 2. Giardia duodenalis (assemblage A) increased MUC2 mucin gene expression in human colonic epithelial cells in a manner dependent upon both protease-activated receptor 2 activation and Giardia cysteine protease activity. Protease-activated receptor 2 cleavage within the N-terminal activation domain by Giardia proteases was confirmed using a nano-luciferase tagged recombinant protease-activated receptor 2. In keeping with these observations, the synthetic protease-activated receptor 2-activating peptide 2fLIGRLO-amide increased Muc2 gene expression in a time-dependent manner. Calcium chelation and inhibition of the ERK1/2 mitogen activated protein kinase pathway inhibited Muc2 upregulation during Giardia infection, consistent with canonical protease-activated receptor 2 signaling pathways. Giardia cysteine proteases cleaved both recombinant protease-activated receptor 1 and protease-activated receptor 2 within their extracellular activation domains with isolate-dependent efficiency that correlated with the production of cysteine protease activity. Protease-activated receptors represent a novel target for Giardia cysteine proteases, and these findings demonstrate that protease-activated receptor 2 can regulate mucin gene expression in intestinal goblet cells.