Dietary effect on immunological energetics in mice

Defense against natural aggressors, such as bacterial infections, requires both energy and an immune-cellular response. However, the question as to how these two components are interconnected in small endotherms by means of the host diet remains only poorly understood. Here, we tested in laboratory mice whether dietary proteins and carbohydrates can modulate the interplay between energy expenditure, food intake and the innate and adaptive immune response when confronting a bacterial challenge (Bacillus Calmette-Guérin, BCG). We observed that mice fed with a high protein diet (HP) developed a better immune response associated to increased numbers of circulating monocytes. In addition, HP diet directly influenced the peripheral blood proportions of both T and B lymphocytes even before the BCG challenge. Interestingly, animals that developed this type of immune response after BCG challenge showed an increased rate of metabolism and food consumption before being challenged. Thus, HP diet induced in non-challenged animals a similar energy expenditure and food intake described by BCG-treated mice. These data suggest that a high amount of proteins in diet can modify the energetic and nutrient dynamic in the host causing a better immune reaction against a microbial challenge.     

Detection and Quantification of Functional Genes of Cellulose- Degrading,Fermentative, and Sulfate-Reducing Bacteria and Methanogenic Archaea

Cellulose degradation, fermentation, sulfate reduction, and methanogenesis are microbial processes that coexist in a variety of natural and engineered anaerobic environments. Compared to the study of 16S rRNA genes, the study of the genes encoding the enzymes responsible for these phylogenetically diverse functions is advantageous because it provides direct functional information. However, no methods are available for the broad quantification of these genes from uncultured microbes characteristic of complex environments. In this study, consensus degenerate hybrid oligonucleotide primers were designed and validated to amplify both sequenced and unsequenced glycoside hydrolase genes of cellulose-degrading bacteria, hydA genes of fermentative bacteria, dsrA genes of sulfate-reducing bacteria, and mcrA genes of methanogenic archaea. Specificity was verified in silico and by cloning and sequencing of PCR products obtained from an environmental sample characterized by the target functions. The primer pairs were further adapted to quantitative PCR (Q-PCR), and the method was demonstrated on samples obtained from two sulfate-reducing bioreactors treating mine drainage, one lignocellulose based and the other ethanol fed. As expected, the Q-PCR analysis revealed that the lignocellulose-based bioreactor contained higher numbers of cellulose degraders, fermenters, and methanogens, while the ethanol-fed bioreactor was enriched in sulfate reducers. The suite of primers developed represents a significant advance over prior work, which, for the most part, has targeted only pure cultures or has suffered from low specificity. Furthermore, ensuring the suitability of the primers for Q-PCR provided broad quantitative access to genes that drive critical anaerobic catalytic processes.The gene encoding the 16S small ribosomal subunit has served as a highly suitable target for studying bacterial species. When one obtains 16S rRNA gene sequence information, it is sometimes possible to infer function from an identical match to a well-characterized pure culture. More commonly, however, the similarity to pure cultures is low, and/or the highest similarities correspond to 16S rRNA gene sequences identified without isolation or phenotypic characterization. In either case, care must be taken, because distinct phenotypes [e.g., dissimilatory Fe(III) reduction, chlorate reduction] are found in microorganisms with highly similar (e.g., 99.5%) 16S rRNA gene sequences (1). In addition, 16S rRNA gene surveys of broad phylogenetic groups can be time-, labor-, and cost-intensive. For example, it is estimated that the 16S rRNA gene-based detection of all recognized lineages of sulfate-reducing bacteria (SRB) would require approximately 132 16S rRNA gene-targeted microarray probes (32).A more-direct approach for the study of microbes that span phylogenetic groups is to target them as a physiologically coherent guild by using specific genetic markers (functional genes) for the functions of interest. Functional genes have been successfully targeted in bioremediation studies to investigate microbial populations responsible for the degradation of various contaminants. Some examples include the use of the large alpha subunit of benzylsuccinate synthase to monitor anaerobic hydrocarbon-degrading bacteria (5), the monitoring of ars genes for the identification and quantification of arsenic-metabolizing bacteria (45), and the detection of catechol 1,2-dioxygenase in aromatic-hydrocarbon-degrading Rhodococcus spp. (48). In the field of mine drainage/metal remediation, functional genes have been used to target SRB (17, 26), but the methods have suffered both from a lack of broad specificity for SRB and from the inability to distinguish SRB from sulfur-oxidizing bacteria (SOB). A general challenge to the functional-gene approach has been the relative lack of characterization and unavailability of target sequences. As a consequence, the primer sets that are available tend to be more relevant to pure cultures than to complex environmental samples.Microbial communities in natural and engineered anaerobic environments that utilize cellulose as the primary carbon source, such as those in rumina (56), termite guts (54), decomposing wood (7), sulfate-reducing and methanogenic sediments (9, 22), wetlands (28), and sulfate-reducing bioreactors (26), are particularly challenging to characterize. 16S rRNA gene-based studies have revealed the complexity of these microbial communities and their high levels of phylogenetic and functional diversity. In such anaerobic environments, mineralization of complex organic matter occurs through the concerted action of a variety of microorganisms. Primary fermenters, such as cellulose degraders, break down the complex molecules and ferment the hydrolysis products. Secondary fermenters also ferment the hydrolysis products. When sulfate is available, SRB utilize the fermentation products as carbon and energy sources. In addition, methanogens can also utilize some of the fermentation products. In many cases, functionally important members, such as SRB, are present only as a small fraction of the community (36, 38), making them difficult to detect by use of 16S rRNA gene-targeted fingerprinting methods. Furthermore, the phylogenetic diversity of cellulose degraders, fermenters, and SRB prevents their quantification using a small number of 16S rRNA gene-targeted probes.In this study, degenerate PCR primers were developed, validated, and demonstrated for the amplification of key functional groups in anaerobic environments possessing genes encoding glycoside hydrolases of families 5 (collectively designated cel5 in this study) and 48 (collectively designated cel48 in this study) (cellulose degradation), the alpha subunit of iron hydrogenase (hydA) (fermentation), dissimilatory sulfite reductase (dsrA) (sulfate reduction), and methyl coenzyme M reductase (mcrA) (methanogenesis). This work is particularly novel considering that the vast majority of existing methods are suitable only for pure cultures, especially in the cases of cel5, cel48, and hydA (21, 44, 47). Thus, the approach provides access to uncultured and unsequenced markers, a critical feature for the study of key anaerobic processes in complex environments. Specificity was also enhanced where possible, notably in the case of dsrA, for which existing primers either do not distinguish SRB from SOB (14, 17) or have good alignment with only a narrow range of SRB (31, 52). Finally, all primers in this study were designed and validated for quantitative PCR (Q-PCR), in order to provide valuable quantitative functional information about complex anaerobic communities. The approach is demonstrated on mine drainage remediation systems and is expected to be of broad value to a variety of fields, including advancing the understanding of biohydrogen production, global carbon cycling, and other important biogeochemical processes.     

Use of Cellulolytic Marine Bacteria for Enzymatic Pretreatment in Microalgal Biogas Production

In this study, we designed and evaluated a microalgal pretreatment method using cellulolytic bacteria that naturally degrades microalgae in their native habitat. Bacterial strains were isolated from each of two mollusk species in a medium containing 1% carboxymethyl cellulose agar. We selected nine bacterial strains that had endoglucanase activity: five strains from Mytilus chilensis, a Chilean mussel, and four strains from Mesodesma donacium, a clam found in the Southern Pacific. These strains were identified phylogenetically as belonging to the genera Aeromonas, Pseudomonas, Chryseobacterium, and Raoultella. The cellulase-producing capacities of these strains were characterized, and the degradation of cell walls in Botryococcus braunii and Nannochloropsis gaditana was tested with “whole-cell” cellulolytic experiments. Aeromonas bivalvium MA2, Raoultella ornithinolytica MA5, and Aeromonas salmonicida MC25 degraded B. braunii, and R. ornithinolytica MC3 and MA5 degraded N. gaditana. In addition, N. gaditana was pretreated with R. ornithinolytica strains MC3 and MA5 and was then subjected to an anaerobic digestion process, which increased the yield of methane by 140.32% and 158.68%, respectively, over that from nonpretreated microalgae. Therefore, a “whole-cell” cellulolytic pretreatment can increase the performance and efficiency of biogas production.     

Selectivity in subunit composition of Ena/VASP tetramers

The members of the actin regulatory family of Ena/VASP proteins form stable tetramers. The vertebrate members of the Ena/VASP family, VASP, Mena and EVL, have many overlapping properties and expression patterns, but functional and regulatory differences between paralogues have been observed. The formation of mixed oligomers may serve a regulatory role to refine Ena/VASP activity. While it has been assumed that family members can form mixed oligomers, this possibility has not been investigated systematically. Using cells expressing controlled combinations of VASP, Mena and EVL, we evaluated the composition of Ena/VASP oligomers and found that VASP forms oligomers without apparent bias with itself, Mena or EVL. However, Mena and EVL showed only weak hetero-oligomerization, suggesting specificity in the association of Ena/VASP family members. Co-expression of VASP increased the ability of Mena and EVL to form mixed oligomers. Additionally, we found that the tetramerization domain (TD) at the C-termini of Ena/VASP proteins conferred the observed selectivity. Finally, we demonstrate that replacement of the TD with a synthetic tetramerizing coiled coil sequence supports homo-oligomerization and normal VASP subcellular localization.     

Use of fetal biometry to determine fetal age in late pregnancy in llamas

Sixty-three pregnant llamas of known breeding date were used in this study. Forty-six of them were submitted to surgery between 186 and 320 days of gestation (52-91% of average gestation period, respectively). Under general anesthesia their fetuses were exteriorized and fetal weight (W), biparietal diameter (BPD) and femoral (F), tarsus-hoof (T-H), tibial (T)) and fronto-occipital (F-O) length were determined. Additionally, the same variables were determined on 16 newborn llamas. The weight was measured in kg and the length in cm. All the collected data was entered into a spreadsheet and different regression analyses as a function of gestational age (GA) were assessed. The best fit equations and their correlation for linear regression were the following: GA=169.448+16.66(*)W, r=0.99; GA=-51.713+44.77(*)BPD, r=0.88; GA=-72.139+39.48(*)F-O, r=0.71; GA=39.304+8.35(*)T-H, r=0.97; GA=91.276+8.23(*)T, r=0.86; GA=102.029+9.94(*)F, r=0.91. For multiple regression, the dependent variable GA can be predicted by the following equation: GA=67.462+11.163(*)W+20.297(*)BPD. Results of the present study indicated measured variables to be highly correlated with GA. This could be useful on daily basis in clinical examination of the neonates, in assessment of fetal growth and well being with cesarean sections, in the determination of GA in late gestation abortions, and in perinatal and reproductive research in the llama.     

Drosophila melanogaster S2 cells for expression of heterologous genes: From gene cloning to bioprocess development

In the present review we discuss strategies that have been used for heterologous gene expression in Drosophila melanogaster Schneider 2 (S2) cells using plasmid vectors. Since the growth of S2 cells is not dependent on anchorage to solid substrates, these cells can be easily cultured in suspension in large volumes. The factors that most affect the growth and gene expression of S2 cells, namely cell line, cell passage, inoculum concentration, culture medium, temperature, dissolved oxygen concentration, pH, hydrodynamic forces and toxic metabolites, are discussed by comparison with other insect and mammalian cells. Gene expression, cell metabolism, culture medium formulation and parameters involved in cellular respiration are particularly emphasized. The experience of the authors with the successful expression of a biologically functional protein, the rabies virus glycoprotein (RVGP), by recombinant S2 cells is presented in the topics covered.     

Effect of clofibrate on brain mevalonate-5-pyrophosphate decarboxylase

The effect of clofibrate on the activity of the three mevalonate-activating enzymes has been studied for the first time in brain by reactions carried out using [2-¹⁴C] mevalonic acid as substrate and 105,000g supernatants from 14-day-old chick brain. Mevalonate-5-pyrophosphate decarboxylase was clearly inhibited, while mevalonate kinase and mevalonate-5-phosphate kinase were not significantly affected. The effect of clofibrate on decarboxylase activity was progressive with increasing concentrations (1.25–5.00 mM) of the inhibitor. A transient inhibition and a subsequent activation as a function of clofibrate concentration seemed to occur for mevalonate kinase. Direct measurements of decarboxylase activity utilizing [2-¹⁴C] pyrophosphomevalonate as the specific substrate of this enzyme corroborated these results. Kinetic studies showed that clofibrate competes with the substrate ATP.     

Biological role of connexin intercellular channels and hemichannels

Gap junctions (GJ) and hemichannels (HC) formed from the protein subunits called connexins are transmembrane conduits for the exchange of small molecules and ions. Connexins and another group of HC-forming proteins, pannexins comprise the two families of transmembrane proteins ubiquitously distributed in vertebrates. Most cell types express more than one connexin or pannexin. While connexin expression and channel activity may vary as a function of physiological and pathological states of the cell and tissue, only a few studies suggest the involvement of pannexin HC in acquired pathological conditions. Importantly, genetic mutations in connexin appear to interfere with GJ and HC function which results in several diseases. Thus connexins could serve as potential drug target for therapeutic intervention. Growing evidence suggests that diseases resulting from HC dysfunction might open a new direction for development of specific HC reagents. This review provides a comprehensive overview of the current studies of GJ and HC formed by connexins and pannexins in various tissue and organ systems including heart, central nervous system, kidney, mammary glands, ovary, testis, lens, retina, inner ear, bone, cartilage, lung and liver. In addition, present knowledge of the role of GJ and HC in cell cycle progression, carcinogenesis and stem cell development is also discussed.