DNA-Launched Alphavirus Replicons Encoding a Fusion of Mycobacterial Antigens Acr and Ag85B Are Immunogenic and Protective in a Murine Model of TB Infection

There is an urgent need for effective prophylactic measures against Mycobacterium tuberculosis (Mtb) infection, particularly given the highly variable efficacy of Bacille Calmette-Guerin (BCG), the only licensed vaccine against tuberculosis (TB). Most studies indicate that cell-mediated immune responses involving both CD4+ and CD8+ T cells are necessary for effective immunity against Mtb. Genetic vaccination induces humoral and cellular immune responses, including CD4+ and CD8+ T-cell responses, against a variety of bacterial, viral, parasitic and tumor antigens, and this strategy may therefore hold promise for the development of more effective TB vaccines. Novel formulations and delivery strategies to improve the immunogenicity of DNA-based vaccines have recently been evaluated, and have shown varying degrees of success. In the present study, we evaluated DNA-launched Venezuelan equine encephalitis replicons (Vrep) encoding a novel fusion of the mycobacterial antigens α-crystallin (Acr) and antigen 85B (Ag85B), termed Vrep-Acr/Ag85B, for their immunogenicity and protective efficacy in a murine model of pulmonary TB. Vrep-Acr/Ag85B generated antigen-specific CD4+ and CD8+ T cell responses that persisted for at least 10 wk post-immunization. Interestingly, parenterally administered Vrep-Acr/Ag85B also induced T cell responses in the lung tissues, the primary site of infection, and inhibited bacterial growth in both the lungs and spleens following aerosol challenge with Mtb. DNA-launched Vrep may, therefore, represent an effective approach to the development of gene-based vaccines against TB, particularly as components of heterologous prime-boost strategies or as BCG boosters.     

Evaluation of keratinolytic potential of some fungal isolates from gelatin factory campus.

During hair degradation, majority of organic sulphur was oxidized to inorganic sulphate and thiosulphate by four fungal isolates (Cylindrocarpon lichenicola, Graphium cuneiferum, Microsporum gypseum, and M. fulvum) from gelatin factory soil. Inorganic thiosulphate, an unusual metabolite, was regularly detected in the culture filtrates of all fungi, although in less amounts. Maximum quantity (44 micrograms/ml) was released by G. cuneiferum on 50th day of incubation. All four fungi showed significant extracellular keratinase activity on human hair. Sulphydryl compounds were present in low amounts throughout the experiment. Detection of inorganic sulphate and thiosulphate with significant release of total protein and keratinase and changes in alkalinity, established the role of sulphitolysis and peptidolysis during keratin biodegradation by fungi which ultimately results in complete keratin degradation.     

Keratinolysis by Absidia cylindrospora and Rhizomucor pusillus: biochemical proof

Absidia cylindrospora and Rhizomucor pusillus, causal agents of phycomycoses, were cultured on sterile natural keratins in a mineral solution and the keratin degradation products analyzed. The excess of sulphur was removed by oxidation to inorganic sulphate and thiosulphate, which were the main products of sulphitolysis of keratin. The proteolytic activity of the two fungi depended on the nature of the keratin substrate. Human scalp hair was the most favoured keratin substrate by both the fungi.     

Phosphorylation by protein kinase C modulates agonist binding to striatal dopamine D2 receptors

The effect of purified protein kinase C (PKC) on dopamine D2 receptor binding was studied. Saturation binding with [3H]spiperone was not affected. In competition experiments using agonists PKC-treated membranes showed a significant reduction in the proportion of high affinity sites, and the influence of GTP gamma S was abolished. These results suggest that PKC-dependent mechanisms can regulate the coupling between the dopamine D2 receptor and its G-protein.     

Widespread formation of intracellular calcium carbonates by the bloom-forming cyanobacterium Microcystis

The formation of intracellular amorphous calcium carbonates (iACC) has been recently observed in a few cultured strains of Microcystis, a potentially toxic bloom-forming cyanobacterium found worldwide in freshwater ecosystems. If iACC-forming Microcystis are abundant within blooms, they may represent a significant amount of particulate Ca. Here, we investigate the significance of iACC biomineralization by Microcystis. First, the presence of iACC-forming Microcystis cells has been detected in several eutrophic lakes, indicating that this phenomenon occurs under environmental conditions. Second, some genotypic (presence/absence of ccyA, a marker gene of iACC biomineralization) and phenotypic (presence/absence of iACC) diversity have been detected within a collection of strains isolated from one single lake. This illustrates that this trait is frequent but also variable within Microcystis even at a single locality. Finally, one-third of publicly available genomes of Microcystis were shown to contain the ccyA gene, revealing a wide geographic and phylogenetic distribution within the genus. Overall, the present work shows that the formation of iACC by Microcystis is common under environmental conditions. While its biological function remains undetermined, this process should be further considered regarding the biology of Microcystis and implications on the Ca geochemical cycle in freshwater environments.     

Role of cadmium in activating nuclear protein kinase C and the enzyme binding to nuclear protein.

Specific effects of cadmium on nuclear protein kinase C activity were found with 3T3/10T1/2 mouse fibroblast and rat liver nuclei. Treatment of the mouse fibroblasts in culture with 12-O-tetradecanoylphorbol-13-acetate resulted in the stimulation of nuclear protein kinase C activity in a "fixed" pool which is defined by its resistance to chelator extraction, whereas the chelator extractable enzyme activity, defined as the "labile" pool was unaffected. Cadmium was found to potentiate the effect of the phorbol ester, directed specifically to nuclei, since the particulate protein kinase C activity was not changed under similar treatment. In a reconstituted system consisting of rat liver nuclei and rat brain protein kinase C, cadmium stimulated the binding of the enzyme to a 105-kDa nuclear protein. The binding of a 105-kDa protein to protein kinase C is attributed strictly due to the cadmium effect, whereas a 50-kDa protein binding to protein kinase C was only enhanced by cadmium. We propose a mechanistic model, where cadmium substitutes zinc in the regulatory domain of protein kinase C rendering the putative protein-protein binding site exposed.     

Aggregation properties of cold-active lipase produced by a psychrotolerant strain of Pseudomonas palleroniana (GBPI_508)

Abstract

The present study aims to exploit microbial potential from colder region to produce lipase enzyme stable at low temperatures. A newly isolated bacterium GBPI_508 from Himalayan environment, was investigated for the production of cold-active lipase emphasizing on its aggregation properties. Plate based assays followed by quantitative production of enzyme was estimated under different culture conditions. Further characterization of partially purified enzyme was done for molecular weight determination and activity and stability under varying conditions of pH, temperature, and in presence of organic solvents, inhibitors, and metal ions. The psychrotolerant bacterium was identified as Pseudomonas palleroniana following 16S rRNA gene sequencing. Maximum lipase production by GBPI_508 was recorded in 7?days at 25?°C utilizing yeast extract as nitrogen source and olive oil as substrate in the lipase production medium. Triton X-100 (1%) in the medium as emulsifier significantly enhanced the lipase production. Lipase produced by bacterium showed aggregation which was confirmed by dynamic light scattering and native PAGE. SDS-PAGE followed by zymogram analysis of partially purified enzyme showed two active bands of ～50?kDa and ～54?kDa. Optimum activity of partially purified enzymatic preparation was recorded at 40?°C while the activity remained nearly consistent from pH 7.0 to 12.0, whereas, maximum stability was recorded at pH values 7.0 and 11.0 at 25?°C. Interestingly, lipase in the partially purified fraction retained 60% enzyme activity at 10?°C. Medium chain pNP ester (C10) was the most preferred substrate for the lipase of GBPI_508. The lipase possessed >50% residual activity when incubated with different organic solvents (25% v/v) except toluene and dichloromethane which inhibited the activity below 50%. Partially purified enzyme was also stable in the presence of metal ions and inhibitors. The study suggests applicability of GBPI_508 lipase in low temperature conditions such as cold-active detergent formulations and cold bioremediation.