Effects of Lonomia obliqua caterpillar venom upon the proliferation and viability of cell lines

Many active principles produced by animals, plants and microorganisms have been employed in the development of new drugs for the treatment of human diseases. Among animals known to produce pharmacologically active molecules that interfere in human cell physiology, the caterpillar Lonomia obliqua has become the focus of toxicological studies due to recent findings about its venom constituents. The objective of this study was to investigate the effects of L. obliqua venom upon the viability and the proliferation of different cell lineages and to propose mechanisms for the herein observed induction of cell proliferation in glioma cell lines. MTT analyses indicate that L. obliqua venom increases the viability of tumor cell lines U138-MG and HT-29; on the other hand, it inhibits the viability of V-79 nontumor cells. Cell count based on the trypan blue exclusion method suggests a proliferating activity of the venom upon U138-MG cells. Exposure of U138-MG to crude venom extract led to a decrease in the production of nitric oxide, and activation of the cAMP signaling pathway inhibited the effects of the venom, indicating that these mechanisms may influence cell proliferation triggered by the venom. Despite the proliferative effects of crude venom on U138-MG and HT-29 cell cultures, a protein purified from L. obliqua hemolymph previously shown to have cytoprotective activity had no effect on U138-MG and HT-29; however, this same protein increased the viability of V-79 cells that had previously been exposed to the cytotoxic activity of the crude venom extract. This study indicates that the venom and the antiapoptotic protein act differently and have different effects on cell cultures, depending on the cell line analyzed. Biomolecules displaying either mitogenic or cytotoxic activities are of great biotechnological interest. Further studies encompassing the purification of active principles from L. obliqua venom are necessary to further elucidate its effects on different cell types.     

Communities of archaea and bacteria in a subsurface radioactive thermal spring in the Austrian Central Alps, and evidence of ammonia-oxidizing Crenarchaeota

Scanning electron microscopy revealed great morphological diversity in biofilms from several largely unexplored subterranean thermal Alpine springs, which contain radium 226 and radon 222. A culture-independent molecular analysis of microbial communities on rocks and in the water of one spring, the "Franz-Josef-Quelle" in Bad Gastein, Austria, was performed. Four hundred fifteen clones were analyzed. One hundred thirty-two sequences were affiliated with 14 bacterial operational taxonomic units (OTUs) and 283 with four archaeal OTUs. Rarefaction analysis indicated a high diversity of bacterial sequences, while archaeal sequences were less diverse. The majority of the cloned archaeal 16S rRNA gene sequences belonged to the soil-freshwater-subsurface (1.1b) crenarchaeotic group; other representatives belonged to the freshwater-wastewater-soil (1.3b) group, except one clone, which was related to a group of uncultivated Euryarchaeota. These findings support recent reports that Crenarchaeota are not restricted to high-temperature environments. Most of the bacterial sequences were related to the Proteobacteria (alpha, beta, gamma, and delta), Bacteroidetes, and Planctomycetes. One OTU was allied with Nitrospina sp. (delta-Proteobacteria) and three others grouped with Nitrospira. Statistical analyses suggested high diversity based on 16S rRNA gene analyses; the rarefaction plot of archaeal clones showed a plateau. Since Crenarchaeota have been implicated recently in the nitrogen cycle, the spring environment was probed for the presence of the ammonia monooxygenase subunit A (amoA) gene. Sequences were obtained which were related to crenarchaeotic amoA genes from marine and soil habitats. The data suggested that nitrification processes are occurring in the subterranean environment and that ammonia may possibly be an energy source for the resident communities.     

Conservation of species interactions to achieve self-sustaining ecosystems

A desirable goal of nature management is to re-establish self-sustaining ecosystems that ensure the persistence of natural habitats and species without requiring active management. Such self-sustainability relies on functional species interactions; yet, species interactions are often overlooked in the conservation literature, and when designing species-specific management efforts. Some interactions may not be restored under general management (e.g. land protection), and may require additional specific management interventions. Interventions targeting these specific interactions fall in a gap between general and species-specific management, effectively bridging community- and population-level approaches to conservation management. We propose that managers should explicitly identify cases where active management of specific interaction partners is required to achieve population self-sustainability. In addition, they should ensure that general management interventions do not inadvertently conflict with naturally occurring interactions, potentially thwarting conservation targets. Interaction functionality may be restored by relying on native species and by identifying the spatial context in which interactions are most likely to re-establish, considering distributional range overlap of interaction partners, local variation in individual encounter rate or even spatial variation in the expected success rate (efficiency) of the focal interaction.     

Temperature-dependent lipid content and fatty acid composition of three thermophilic bacteria

The lipid content and fatty acid composition of a strain ofBacillus caldolyticus and of two facultative thermophiles (B. flavothermus and strain NZ-2) were analysed after growth at different temperatures. In all three strains the amount of membrane, as a fraction of total cellular dry mass, was found to increase with temperature, however, in varying degrees. Changes of lipid content and protein/lipid ratio inB. caldolyticus between 60°C and 100°C and in strain NZ-2 between 45°C and 70°C were minor; inB. f avothermus the alterations in the 50°C–70°C range were more pronounced. The same was found for changes observed in the phospholipid/total lipid and phospholipid/membrane ratios, and also in the amounts of individual phospholipids. The alterations of the fatty acid composition were most significant inB. caldolyticus, especially between 80°C and 95°C. In contrast, the main changes inB. flavothermus and NZ-2 were found to occur between 30°C and 50°C, and between 45°C and 60°C, respectively. Based on these data, strain NZ-2 could be characterized as the least andB. flavothermus as the most versatile of the three organisms.     

Morphine-Induced Preconditioning: Involvement of Protein Kinase A and Mitochondrial Permeability Transition Pore

Background

Morphine induces myocardial preconditioning (M-PC) via activation of mitochondrial large conductance Ca²⁺-sensitive potassium (mK_Ca) channels. An upstream regulator of mK_Ca channels is protein kinase A (PKA). Furthermore, mK_Ca channel activation regulates mitochondrial bioenergetics and thereby prevents opening of the mitochondrial permeability transition pore (mPTP). Here, we investigated in the rat heart in vivo whether 1) M-PC is mediated by activation of PKA, and 2) pharmacological opening of the mPTP abolishes the cardioprotective effect of M-PC and 3) M-PC is critically dependent on STAT3 activation, which is located upstream of mPTP within the signalling pathway.

Methods

Male Wistar rats were randomised to six groups (each n = 6). All animals underwent 25 minutes of regional myocardial ischemia and 120 minutes of reperfusion. Control animals (Con) were not further treated. Morphine preconditioning was initiated by intravenous administration of 0.3 mg/kg morphine (M-PC). The PKA blocker H-89 (10 μg/kg) was investigated with and without morphine (H-89+M-PC, H-89). We determined the effect of mPTP opening with atractyloside (5 mg/kg) with and without morphine (Atr+M-PC, Atr). Furthermore, the effect of morphine on PKA activity was tested in isolated adult rat cardiomyocytes. In further experiments in isolated hearts we tested the protective properties of morphine in the presence of STAT3 inhibition, and whether pharmacological prevention of the mPTP-opening by cyclosporine A (CsA) is cardioprotective in the presence of STAT3 inhibition.

Results

Morphine reduced infarct size from 64±5% to 39±9% (P<0.05 vs. Con). H-89 completely blocked preconditioning by morphine (64±9%; P<0.05 vs. M-PC), but H-89 itself had not effect on infarct size (61±10%; P>0.05 vs. Con). Also, atractyloside abolished infarct size reduction of morphine completely (65±9%; P<0.05 vs. M-PC) but had no influence on infarct size itself (64±5%; P>0.05 vs. Con). In isolated hearts STAT3 inhibitor Stattic completely abolished morphine-induced preconditioning. Administration of Stattic and mPTP inhibitor cyclosporine A reduced infarct size to 31±6% (Stat+CsA, P<0.05 vs. Con). Cyclosporine A alone reduced infarct size to 26±7% (CsA P<0.05 vs. Con). In cardiomyocytes, PKA activity was increased by morphine.

Conclusion

Our data suggest that morphine-induced cardioprotection is mediated by STAT3-activation and inhibition of mPTP, with STA3 located upstream of mPTP. There is some evidence that protein kinase A is involved within the signalling pathway.     

Variation of parameters affecting the start-up of methanogenic fluidized bed reactors

Summary The influence of the medium composition, the inoculum and the inoculation procedure on initial biofilm development in methanogenic fluidized bed reactors was studied on laboratory scale. Trace minerals but not vitamins were found to be essential for biofilm development. Inoculation with heterogeneous bacterial cultures of potentially sand-colonizing microorganisms and/or with pure cultures ofMethanothrix soehngenii did not accelerate biofilm development significantly as compared to inoculation with effluent from a fully operative fluidized bed reactor.     

Precise localization of antigens on follicular dendritic cells

Summary Horse-spleen ferritin or bovine serum albumin conjugated to colloidal gold (BSA-gold) were injected subcutaneously in preimmunized mice. In draining lymph nodes both antigens were located in macrophages or between the cytoplasmic processes of follicular dendritic cells (FDC). Some of the antigens remained trapped on FDC until day 31 after injection. Simultaneous injection of both antigens showed that they were located between the infoldings of the same FDC. These cells are thus able to retain at least two different antigens on their surface. The peculiar arrangement of ferritin between the cytoplasmic infoldings suggests that this antigen is fixed on both cell membranes by specific antibodies. The trapped immune complexes could thus stabilize the FDC membrane system.The antigen retention requires the presence of specific antibodies since BSA-gold or ferritin injected without preimmunization were not found between FDC processes. Nonantigenic materials, such as colloidal gold or carbon particles, are not trapped by FDC, except when injected in large amounts.The antigens were trapped on the surface of FDC, however unfrequently in close contact with lymphocytes. FDC might protect lymphocytes against an excess of immune complexes and act as regulators of contacts between lymphocytes and immune complexes.Abbreviations BSA bovine serum albumin - BSA-gold BSA conjugated to colloidal gold particles - FDC follicular dendritic cells     

A decade of improvements in quantification of gene expression and internal standard selection

Major improvements have been made in mRNA quantification and internal standard selection over the last decade. Our aim in this paper is to present the main developments that are of interest for practical laboratory work, contrasting the situation as it is now with the one of ten years ago, and presenting some excellent examples of what can be done today. Specifically, we will mainly discuss Real-Time RT-PCR major improvements that have been performed in the following areas: the most commonly used quantification techniques, the mathematical and software tools created to help researchers in their work on internal standard selection, the availability of detection chemistries and technical information and of commercial tools and services. In addition to mRNA quantification, we will also discuss some aspects of non-coding RNA and protein quantification. In addition to technical improvements, the development of international cooperation and the creation of technical databases are likely to represent a major tool for the future in the standardization of gene expression quantification.     

Rooting characteristics of lettuce grown in irrigated sand beds

To avoid the current water pollution from intensive glasshouse horticulture, closed systems have to be developed with recirculating drainage water. For crops with a high planting density, such as letuuce, shallow beds of coarse sand may be used if water and nutrient supply can be regulated adequately. The aim of the present study was to determine the rooting characteristics and root distribution of lettuce in sand beds, as affected by substrate depth, the distance to a drain, drip lines and drip points, and the excess of nutrient solution applied. The hypothesis was tested that a small excess and a large distance between drip points leads to local salt accumulations in the root environment and thus to a less homogeneous root distribution.The data confirmed both parts of the hypothesis: spatial patterns in salt distribution were found. Detailed measurements in a sand bed with only one drip line per two crop rows and an amount of fertigation solution added of 2 times the estimated evapotranspiration, showed that root length density was negatively correlated with salt content when comparisons were made within the same layer. Crop yield per row was influenced in the extreme treatment, i.e. one drip line per two crop rows and an amount of fertigation solution added of 1.3 times the estimated evapotranspiration, but yield per bed was still unaffected. The increased heterogeneity of the crop will cause problems at harvest and indicates that the most extreme treatment included in the comparison is just beyond the limit of acceptable heterogeneity in the root medium. Lettuce can be grown on sand beds with a recirculating nutrient solution provided that drip lines are well distributed in the bed and the daily nutrient solution excess is more than 30% of demand.