Multimeric HCV E2 protein obtained from <Emphasis Type="Italic">pichia pastoris</Emphasis> cells induces a strong immune response in mice

Production of immunogenic hepatitis C virus (HCV) envelope proteins will assist in the future development of preventive or therapeutics applications. Only properly folded monomeric E2 protein is able to bind a putative cellular co-receptor CD81, but this interaction may modulate cell immune function. Recombinant E2 proteins, similar to the native form, but lacking undesirable immunoregulatory features, might be promising components of vaccine candidates against HCV. To obtain E2 suitable for structural as well as functional studies, a recombinant E2 variant (E2₆₈₀) was produced in Pichia pastoris cells. E2₆₈₀, comprising amino acids 384 to 680 of the HCV polyprotein, was secreted into the culture supernatant in the N-glycosilated form and was mainly composed of disulide-linked multimers. Both monomeric and oligomeric forms of E2₆₈₀ were recognized by conformational-sensitive MAb H53. In addition, antibodies in sera from 70% of HCV-positive patients were reactive against E2₆₈₀. By immunizing E2₆₈₀ in BALB/c mice, both a specific cellular immune response and anti-E2₆₈₀ IgG antibody titers of 1:200,000 were induced. Our data suggest that recombinant E2₆₈₀ could be useful to successfully induce strong anti-HCV immunity.     

Membrane-associated phosphoinositides-specific phospholipase C forms from <Emphasis Type="Italic">Catharanthus roseus</Emphasis> transformed roots

We have previously reported that Catharanthus roseus transformed roots contain at least two phosphatidylinositol 4,5-bisphosphate-phospholipase C (PLC) activities, one soluble and the other membrane associated. Detergent, divalent cations, and neomycin differentially regulate these activities and pure protein is required for a greater understanding of the function and regulation of this enzyme. In this article we report a partia purification of membrane-associated PLC. We found that there are at least two forms of membrane-associated PLC in transformed roots of C. roseus. These forms were separated on the basis of their affinity for heparin. One form shows an affinity for heparin and elutes at approx 600 mM KCl. This form has a molecular mass of 67 kDa by size exclusion chromatography and Western blot analysis, whereas the other form does not bind to heparin and has a molecular mass of 57 kDa. Possible differential regulation of these forms during transformed root growth is discussed.     

Toxic effects of zinc on anaerobic microbiota from Zimapán Reservoir (Mexico)

The toxic effects of heavy metals have been extensively documented in different organisms. Nevertheless, a lack of information exists with regard to this topic in the case of autochthonous microorganism communities. The aim of this study was to evaluate the toxic effects of zinc on the anaerobic microorganisms present in the sediment and anoxic water of Zimapán Reservoir (Mexico), with particular focus on dissimilatory sulphate reducing bacteria. In the laboratory, a system of enrichment microcosms was set up with sediment and water from the reservoir. ATP, protein, carbohydrates and lactate and alcohol dehydrogenase activity were determined. The physicochemical parameters of the reservoir were evaluated over the course of one year. Sulphate reduction occurred in the reservoir throughout the year, but was most pronounced at the end of the wet season and during winter. In the field, increases in the rate of sulphate reduction coincided with the lowest levels of total phosphorus and hydrosoluble organic carbon. Zinc enrichment was observed to modify protein and carbohydrate content as well as to affect lactate and alcohol dehydrogenase activity. All responses followed a zinc concentration-response relationship and were dependent on reservoir physicochemical parameters. ATP content was used as a biomarker to evaluate the sublethal toxic effects of zinc. The acceptable threshold concentration of zinc in the aquatic and sediment enrichment microcosms was determined to be 0.06mgZn/L and 711.1mgZn/kg, respectively.     

Shifts in the psychophysical function in rats

The primary goal was to compare results from a free-operant procedure with pigeons [Machado, A., Guilhardi, P., 2000. Shifts in the psychometric function and their implications for models of timing. J. Exp. Anal. Behav. 74, 25-54, Experiment 2] with new results obtained with rats. The secondary goal was to compare the results of both experiments with dependent variables that were not used in the original publication. As in the original study with pigeons, rats were trained on a two-alternative free-operant psychophysical procedure in which left lever press responses were reinforced during the first and second quarters of a 60-s trial, and right lever press responses were reinforced during the third and fourth quarters of the trial. The quarters were reinforced according to four independent variable interval (VI) schedules of reinforcement. The VI duration was manipulated in each quarter, and shifts in the psychophysical functions that relate response rate with time since trial onset were measured. The results obtained with rats were consistent with those previously obtained with pigeons. In addition, results not originally reported were also consistent between rats and pigeons, and provided insights into the perception, memory, and decision processes in Scalar Expectancy Theory and Learning-to-Time Theory.     

G protein betagamma subunits interact with alphabeta- and gamma-tubulin and play a role in microtubule assembly in PC12 cells

The betagamma subunit of G proteins (Gbetagamma) is known to transfer signals from cell surface receptors to intracellular effector molecules. Recent results suggest that Gbetagamma also interacts with microtubules and is involved in the regulation of the mitotic spindle. In the current study, the anti-microtubular drug nocodazole was employed to investigate the mechanism by which Gbetagamma interacts with tubulin and its possible implications in microtubule assembly in cultured PC12 cells. Nocodazole-induced depolymerization of microtubules drastically inhibited the interaction between Gbetagamma and tubulin. Gbetagamma was preferentially bound to microtubules and treatment with nocodazole suggested that the dissociation of Gbetagamma from microtubules is an early step in the depolymerization process. When microtubules were allowed to recover after removal of nocodazole, the tubulin-Gbetagamma interaction was restored. Unlike Gbetagamma, however, the interaction between tubulin and the alpha subunit of the Gs protein (Gsalpha) was not inhibited by nocodazole, indicating that the inhibition of tubulin-Gbetagamma interactions during microtubule depolymerization is selective. We found that Gbetagamma also interacts with gamma-tubulin, colocalizes with gamma-tubulin in centrosomes, and co-sediments in centrosomal fractions. The interaction between Gbetagamma and gamma-tubulin was unaffected by nocodazole, suggesting that the Gbetagamma-gamma-tubulin interaction is not dependent on assembled microtubules. Taken together, our results suggest that Gbetagamma may play an important and definitive role in microtubule assembly and/or stability. We propose that betagamma-microtubule interaction is an important step for G protein-mediated cell activation. These results may also provide new insights into the mechanism of action of anti-microtubule drugs.     

A novel method to screen genomic libraries that combines genomic immunization with the prime-boost strategy

We employed a prime-boost regimen in combination with the expression library immunization protocol to improve the protective effectiveness of a genomic library used as immunogen. To demonstrate the feasibility of this novel strategy, we used as a prime a serogroup B Neisseria meningitidis random genomic library constructed in a eukaryotic expression vector. Mice immunized with different fractions of this library and boosted with a single dose of meningococcal outer membrane vesicles elicited higher bactericidal antibody titers compared with mice primed with the empty vector. After the boost, passive administration of sera from mice primed with two of these fractions significantly reduced the number of viable bacteria in the blood of infant rats challenged with live N. meningitidis. The method proposed could be applied to the identification of subimmunogenic antigens during vaccine candidate screening by employing expression library immunization.     

Diminished redundancy of outer membrane factor proteins in rhizobiales: a nodT homolog is essential for free-living Rhizobium etli

Rhizobium etli is a gram-negative soil bacterium that induces nitrogen-fixing nodules on common bean roots (Phaseolus vulgaris). R. etli encodes two genes homologous to nodT of Rhizobium leguminosarum. nodTch is chromosomal and forms an operon with new genes resembling a multi-drug efflux pump of the resistance-nodulation-cell division (RND) family. nodTch is the last gene of this operon and can also be independently transcribed; the gene product is located in the bacterial outer membrane. Cell survival requires nodTch under all conditions tested. A second nodT gene, nodTpc, is encoded by plasmid c; it is constitutively transcribed but does not complement the essential function encoded by nodTch. NodT proteins belong to the outer membrane efflux proteins of the TolC superfamily. The number of duplications in the tolC gene family positively correlates with genome size in gram-negative bacteria. Nonetheless, some alpha-proteobacteria, including R. etli, encode fewer outer membrane factor exporters than expected suggesting further roles in addition to detoxification.     

Forest cover loss in the Nevado de Toluca volcano protected area (Mexico) after the change to a less restrictive category in 2013

Biodiversity and Conservation - In 2013, the protection status of the Nevado de Toluca Volcano was changed from National Park to a less restrictive category. Much controversy has arisen surrounding...     

A family of amphiphilic dioxidovanadium(V) hydrazone complexes as potent carbonic anhydrase inhibitors along with anti-diabetic and cytotoxic activities

BioMetals - A family of dioxidovanadium(V) complexes (1–4) of the type [Na(H2O)x]+[VVO2(HL1?4)]? (x?=?4, 4.5 and 7) where HL2? represents the dianionic form of...