Apurinic/apyrimidinic endonuclease1/redox factor-1 (Ape1/Ref-1) is essential for IL-21-induced signal transduction through ERK1/2 pathway

IL-21 is a pleiotropic cytokine that regulates T-cell and B-cell differentiation, NK-cell activation, and dendritic cell functions. IL-21 activates the JAK-STAT, ERK, and PI3K pathways. We report here that Ape1/Ref-1 has an essential role in IL-21-induced cell growth signal transduction. Overexpression of Ape1/Ref-1 enhances IL-21-induced cell proliferation, but it is suppressed by overexpressing an N-terminal deletion mutant of Ape1/Ref-1 that lacks the redox domain. Furthermore, knockdown of the Ape1/Ref-1 mRNA dramatically compromises IL-21-induced ERK1/2 activation and cell proliferation with increasing cell death. These impaired activities are recovered by the re-expression of Ape1/Ref-1 in the knockdown cells. Our findings are the first demonstration that Ape1/Ref-1 is an indispensable molecule for the IL-21-mediated signal transduction through ERK1/2 activation.     

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g., other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO₂ drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.     

Role of fusogenic non-PC liposomes in elicitation of protective immune response against experimental murine salmonellosis

Earlier we have demonstrated that novel fusogenic liposomes made up of lipid from Escherichia coli (escheriosomes) have strong tendency to fuse with the plasma membrane of target cells and thereby delivering the entrapped contents into their cytosol. The delivery of entrapped antigen in cytosol of the target cells ensues its processing and presentation along with MHC class I pathway that eventually elicit antigen specific cytotoxic T cells. The result of the present study revealed that immunization of BALB/c mice with escheriosome-encapsulated Salmonella typhimurium (S. typhimurium) cytosolic antigens resulted in the augmentation of antigen specific cytotoxic T cell lymphocyte as well as IgG responses. In contrast, free or conventional liposome (PC liposome) encapsulated antigen failed to induce CD8+ CTLs in the immunized animals. Further, immunization with escheriosome-encapsulated antigen resulted in significant enhancement in the release of IFN-gamma and IgG2a in the experimental animals. Interestingly, the immunization with escheriosome-encapsulated antigen resulted in upregulation of CD80 and CD86 on the surface of antigen presenting cells (APCs) as well. Finally, the results of the present study reveal that immunization of animals with escheriosomes encapsulated antigen protected them against virulent S. typhimurium infection. This was evident by increased survival, and reduced bacterial burden in vital organs of the immunized animals. The data of the present study suggest that escheriosomes can emerge as an effective vehicle for intracellular delivery of antigen and thus hold promise in development of liposome based vaccine against Salmonella and other intracellular pathogens.     

Impaired IL-7 signaling may explain a case of atypical JAK3-SCID

Janus kinase 3-severe combined immunodeficiency (JAK3-SCID) is an autosomal recessive immunodeficiency disease caused by various mutations in the JAK3 gene. Typical JAK3-SCID is characterized by a phenotype in which B cells are present but T and NK cells are not, the T^?B⁺NK^? phenotype, and by impaired signaling through cytokine receptors that use the common gamma chain (γc) subunit. An atypical JAK3-SCID case carrying a single glutamate to glycine substitution mutation (E481G) in the JH3 domain of one JAK3 allele, and a deletion mutation (del482-596) in the JH3 and JH2 domains of the other allele was reported previously. Although this patient had CD4⁺ T cells and NK cells unlike typical cases, the CD4⁺ T cells were functionally impaired. We report here that the JAK3-E481G mutant transduced IL-2-, IL-4-, IL-15-, and IL-21-induced signals as efficiently as wild-type JAK3. However, this mutant failed to respond to IL-7 by phosphorylating JAK1, JAK3, or STAT5. The other mutant JAK3, JAK3-del482-596, was non-functional. Thus, an impaired IL-7 signal may cause SCID and compromise T-cell differentiation, even if the IL-15 signal is preserved and supports NK-cell development, as in this patient.     

Identification and evaluation of glutathione conjugate gamma-l-glutamyl-l-cysteine for improved drug delivery to the brain

Abstract

Glutathione (GU), an endogenous antioxidant tripeptide, is frequently transferred in the human brain through N-methyl-d-aspartate receptor (NMDAR), profusely expressed at the blood–brain barrier (BBB) junction. GU, also modifies the characteristics of tight junction proteins (occludin and claudin) at the site of BBB by depolarizing the enzyme, protein tyrosine phosphatase that manifests its usefulness for passive delivery of nanocarriers to the brain. GU, thus, represents itself as an ideal ligand for the surface decoration of nanocarriers to successfully administer them across the brain via receptor-mediated drug delivery pathway. Hence, we have employed here, in-silico approaches to identify the potential GU-like molecules, as appropriate ligand(s) for surface engineering of nanoconstruct with the purpose of attaining targeted drug delivery to the brain. Structure-based virtual screening methods was used to filter PubChem database for the identification of bioactive compounds with >95% structure similarity with GU. We have further screened the compounds against NMDAR using molecular docking approach. Top hits were selected based on their high binding affinities and selectivity towards NMDAR, and their binding pattern was analysed in detail. Finally, all atom molecular dynamics simulation for 100?ns was carried out on free NMDAR and in-presence of the selected GU-like compound, gamma-l-glutamyl-l-cysteine to evaluate complex stability and structural dynamics. In conclusion, gamma-l-glutamyl-l-cysteine may act as potential binding partner of NMDAR which can further be evaluated in drug delivery system to brain across the BBB.

Communicated by Ramaswamy H. Sarma     

Phylogenetic diversity and biotechnological potentials of marine bacteria from continental slope of eastern Arabian Sea

Marine environments are substantially untapped source for the isolation of bacteria with the capacity to produce various extracellular hydrolytic enzymes, which have important ecological roles and promising biotechnological applications. Hydrolases constitute a class of enzymes widely distributed in nature from bacteria to higher eukaryotes. Marine microbial communities are highly diverse and have evolved during extended evolutionary processes of physiological adaptations under the influence of a variety of ecological conditions and selection pressures. A number of marine hydrolases have been described, including amylases, lipases and proteases, which are being used extensively for biotechnological applications. The present study was carried out to isolate marine bacteria from continental slope sediments of the eastern Arabian Sea and explore their biotechnological potential. Among the 119 isolates screened, producers of amylases (15%), caseinases (40%), cellulases (40%), gelatinases (60%), lipases (26%), ligninases (33%), phytase (11%) and Malachite Green dye degraders (16%) were detected. Phylogenetic analysis based on 16S rRNA gene sequencing showed that predominant marine sediment bacteria possessing more than four enzymatic activities belonged to the phyla Firmicutes and Proteobacteria, was assigned to the genera Bacillus, Planococcus, Staphylococcus, Chryseomicrobium, Exiguobacterium and Halomonas. Biodegradation of the dye Malachite Green using the liquid decolorization assay showed that both the individual cultures (Bacillus vietnamensis, Planococcus maritimus and Bacillus pumilus) and their consortium were able to decolorize more than 70% of dye within 24?h of incubation. This is the first report on diversity and extracellular hydrolytic enzymatic activities and bioremediation properties of bacteria from continental slope sediment of eastern Arabian Sea.     

Anti-tumor activity of recombinant tumor necrosis factor on mouse fibrosarcoma in vivo and in vitro

The experiments presented were designed first to determine the effects of rTNF on the methylcholanthrene-induced fibrosarcoma (FSA-1) in C3H/JSed mice and second to determine whether the observed effects are the result of direct action by rTNF on the tumor or whether rTNF acts as a mediator of other effector mechanisms. Mice received syngeneic FSA-1 fibrosarcoma cells either s.c. or i.v. in order to evaluate growth of transplantable solid tumor or lung metastases, respectively. The range of dosages, from 10(2) to 2 x 10(5) U of rTNF, was administered i.v. at different intervals after the tumor cell injection. Early injection of 10(3) to 10(4) U of rTNF reduced the growth of s.c. injected tumor and the number of lung metastases in i.v. injected mice. In both cases, survival of mice was also prolonged. However, in vitro treatment of FSA-1 tumor cells with rTNF did not result in the reduction of their proliferating activity after injection into mice, although direct cytostatic and moderate cytotoxic activity of rTNF in vitro was demonstrated. To identify whether other cellular mechanisms are involved in the effects observed in vivo, the anti-tumor activity of rTNF-treated spleen cells was evaluated in vitro using a 75Se release assay. Whereas nontreated spleen cells demonstrated very low cytotoxic activity in this system, the cells from rTNF-treated mice showed marked increase in the cytotoxicity against syngeneic tumor cells. These results suggest that the anti-tumor activity of rTNF represents a combination of its direct effect on tumor cells and indirect effects involving host immune mechanisms.