Caffeine abolishes the mammalian G(2)/M DNA damage checkpoint by inhibiting ataxia-telangiectasia-mutated kinase activity

Recent evidence indicates that arrest of mammalian cells at the G(2)/M checkpoint involves inactivation and translocation of Cdc25C, which is mediated by phosphorylation of Cdc25C on serine 216. Data obtained with a phospho-specific antibody against serine 216 suggest that activation of the DNA damage checkpoint is accompanied by an increase in serine 216 phosphorylated Cdc25C in the nucleus after exposure of cells to gamma-radiation. Prior treatment of cells with 2 mM caffeine inhibits such a change and markedly reduces radiation-induced ataxia-telangiectasia-mutated (ATM)-dependent Chk2/Cds1 activation and phosphorylation. Chk2/Cds1 is known to localize in the nucleus and to phosphorylate Cdc25C at serine 216 in vitro. Caffeine does not inhibit Chk2/Cds1 activity directly, but rather, blocks the activation of Chk2/Cds1 by inhibiting ATM kinase activity. In vitro, ATM phosphorylates Chk2/Cds1 at threonine 68 close to the N terminus, and caffeine inhibits this phosphorylation with an IC(50) of approximately 200 microM. Using a phospho-specific antibody against threonine 68, we demonstrate that radiation-induced, ATM-dependent phosphorylation of Chk2/Cds1 at this site is caffeine-sensitive. From these results, we propose a model wherein caffeine abrogates the G(2)/M checkpoint by targeting the ATM-Chk2/Cds1 pathway; by inhibiting ATM, it prevents the serine 216 phosphorylation of Cdc25C in the nucleus. Inhibition of ATM provides a molecular explanation for the increased radiosensitivity of caffeine-treated cells.     

Preparation and Evaluation of Differently Sulfonated Styrene–Divinylbenzene Cross-linked Copolymer Cationic Exchange Resins as Novel Carriers for Drug Delivery

The differently sulfonated styrene–divinylbenzene cross-linked copolymer cationic exchange resins were prepared by oil-in-water polymerization and varied degrees of sulfonation. Several characteristics of the obtained resins were evaluated, i.e., Fourier transform infrared spectra, the ion-exchange capacity, microscopic morphology, size, and swelling. The resin characteristics were altered in relation to the degree of sulfonation, proving that differently sulfonated resins could be prepared. The behavior of chlorpheniramine (CPM) loading and in vitro release in the USP simulated gastric (SGF) and intestinal fluids (SIF) of the obtained resins were also evaluated. The CPM loaded in the resinates (drug-loaded resins) increased with the increasing degree of sulfonic group and hence the drug binding site in the employed resins. The CPM release was lower from the resins with the higher degree of sulfonic group due to the increase in the diffusive path depth. The CPM release was obviously lower in SGF than SIF because CPM, a weak base drug, ionized to a greater extent in SGF and then preferred binding with rather than releasing from the resins. In conclusion, the differently sulfonated resins could be utilized as novel carriers for drug delivery.     

Development and in vitro characterization of a bivalent DNA containing HN and F genes of velogenic Newcastle disease virus

Newcastle disease (ND) is highly contagious, economically important viral disease affecting most of avian species worldwide. Newcastle disease virus (NDV) has single stranded negative sense RNA genome which encodes for six structural and two non-structural proteins. Envelope glycoproteins i.e. hemagglutinin-neuraminidase (HN) and the fusion (F), elicit protective immune response. In this study, HN and F genes of velogenic (virulent) strain were amplified and cloned at multiple cloning sites A and B, respectively into pIRES bicistronic vector for use as bivalent DNA vaccine against ND. The recombinant plasmid was characterized for its orientation by restriction enzyme digestion and PCR. Expression of HN and F genes was assessed in transfected Vero cells at RNA level using RT-PCR in total RNA as well as protein level using IFAT, IPT and western blot using NDV specific antiserum. All these experiments confirmed that HN and F genes cloned in recombinant pIRES.nd.hn.f are functionally active. The recombinant construct is being evaluated as DNA vaccine against ND.     

Influence of different photoperiods on development of gonad, cloacal gland and circulating thyroid hormones in male Japanese quail Coturnix coturnix japonica.

One day old chicks of Japanese quail were exposed to different photoperiods (LD, 8:16, 13.5:10.5, 16:8 and LL) and observations (testes weight, cloacal gland size, body weight and circulating thyroxine and triiodothyronine) were taken at the age of 3, 5, 7, 9 and 16 weeks. Results indicate that immediate reproductive development occurred in birds exposed to long photoperiods (greater than 12 hr). Growth under LD 8:16, was not apparent till 7th week and by 16 weeks, degree of gonadal development was similar in all the birds, irrespective of photoperiodic treatment. Whereas body weight of the intermediate and long day (LD 13.5:10.5, 16:8 and LL) treated birds increased upto 5th week and remained constant thereafter. But the chicks maintained under short day length (LD 8:16), showed spontaneous increase till the end of the study and birds were much heavier compared to all other groups. Plasma T4 concentration increased with increasing age till 9th week and remained unaltered thereafter. On the other hand T3 level did not change till 7th week followed by a decline. It is suggested that the initiation and degree of gonadal growth in quail depends on the availability of daily photoperiod, until the achievement of full breeding condition. Peak level of T4 observed in 9 week old birds may be involved in the development of photorefractoriness at that age.     

Meloxicam Taste-Masked Oral Disintegrating Tablet with Dissolution Enhanced by Ion Exchange Resins and Cyclodextrin

The purpose of this study was to develop taste-masked oral disintegrating tablets (ODTs) using the combination of ion exchange resin and cyclodextrin, to mask the bitter taste and enhance drug dissolution. Meloxicam (MX) was selected as a model drug with poor water solubility and a bitter taste. Formulations containing various forms of MX (free drug, MX-loaded resin or resinate, complexes of MX and 2-hydroxypropyl-β-cyclodextrin (HPβCD) or MX/HPβCD complexes, and a mixture of resinate and MX/HPβCD complexes) were made and tablets were prepared by direct compression. The ODTs were evaluated for weight variation, thickness, diameter, hardness, friability, disintegration time, wetting time, MX content, MX release, degree of bitter taste, and stability. The results showed that thickness, diameter, weight, and friability did not differ significantly for all of these formulations. The tablet hardness was approximately 3 kg/in.², and the friability was less than 1%. Tablets formulated with resinate and the mixture of resinate and MX/HPβCD complexes disintegrated rapidly within 60 s, which is the acceptable limit for ODTs. These results corresponded to the in vivo disintegration and wetting times. However, only tablets containing the mixture of resinate and MX/HPβCD complexes provided complete MX dissolution and successfully masked the bitter taste of MX. In addition, this tablet was stable at least 6 months. The results from this study suggest that the appropriate combination of ion exchange resin and cyclodextrin could be used in ODTs to mask the bitter taste of drug and enhance the dissolution of drugs that are weakly soluble in water.     

Identification and characterization of Cryptococcus neoformans protein fractions that induce protective immune responses

Cryptococcus neoformans, the main causative agent of cryptococcosis, is a fungal pathogen that causes life‐threatening meningoencephalitis in immunocompromised patients. To date, there is no vaccine or immunotherapy approved to treat cryptococcosis. Cell‐ and antibody‐mediated immune responses collaborate to mediate optimal protection against C. neoformans infections. Accordingly, we identified cryptococcal protein fractions capable of stimulating cell‐ and antibody‐mediated immune responses and determined their efficacy to elicit protection against cryptococcosis. Proteins were extracted from C. neoformans and fractionated based on molecular mass. The fractions were then evaluated by immunoblot analysis for reactivity to serum extracted from protectively immunized mice and in cytokine recall assays for their efficacy to induce pro‐inflammatory and Th1‐type cytokine responses associated with protection. MS analysis revealed a number of proteins with roles in stress response, signal transduction, carbohydrate metabolism, amino acid synthesis, and protein synthesis. Immunization with select protein fractions containing immunodominant antigens induced significantly prolonged survival against experimental pulmonary cryptococcosis. Our studies support using the combination of immunological and proteomic approaches to identify proteins that elicit antigen‐specific antibody and Th1‐type cytokine responses. The immunodominant antigens that were discovered represent attractive candidates for the development of novel subunit vaccines for treatment and/or prevention of cryptococcosis.     

Role of CTLA4 in the Proliferation and Survival of Chronic Lymphocytic Leukemia

Earlier, we reported that CTLA4 expression is inversely correlated with CD38 expression in chronic lymphocytic leukemia (CLL) cells. However, the specific role of CTLA4 in CLL pathogenesis remains unknown. Therefore, to elucidate the possible role of CTLA4 in CLL pathogenesis, CTLA4 was down-regulated in primary CLL cells. We then evaluated proliferation/survival in these cells using MTT, ³H-thymidine uptake and Annexin-V apoptosis assays. We also measured expression levels of downstream molecules involved in B-cell proliferation/survival signaling including STAT1, NFATC2, c-Fos, c-Myc, and Bcl-2 using microarray, PCR, western blotting analyses, and a stromal cell culture system. CLL cells with CTLA4 down-regulation demonstrated a significant increase in proliferation and survival along with an increased expression of STAT1, STAT1 phosphorylation, NFATC2, c-Fos phosphorylation, c-Myc, Ki-67 and Bcl-2 molecules. In addition, compared to controls, the CTLA4-downregulated CLL cells showed a decreased frequency of apoptosis, which also correlated with increased expression of Bcl-2. Interestingly, CLL cells from lymph node and CLL cells co-cultured on stroma expressed lower levels of CTLA4 and higher levels of c-Fos, c-Myc, and Bcl-2 compared to CLL control cells. These results indicate that microenvironment-controlled-CTLA4 expression mediates proliferation/survival of CLL cells by regulating the expression/activation of STAT1, NFATC2, c-Fos, c-Myc, and/or Bcl-2.     

Expression of CspE by a Psychrotrophic Bacterium Enterobacter ludwigii PAS1, Isolated from Indian Himalayan Soil and In silico Protein Modelling, Prediction of Conserved Residues and Active Sites

Proteome analysis of Enterobacter ludwigii PAS1 provide a powerful set of tool to study the cold shock proteins along with that combination of bioinformatics is useful for interpretation of comparative results from many species. There is a considerable interest in the use of psychrotrophic bacteria for nitrogen fixation, especially at hilly regions, thus better understanding of cold adaptation mechanisms too. The psychrotrophic E. ludwigii PAS1 grown at 30 and 4 °C, isolated from Himalaya soil was undertaken for proteomic responses during optimal and cold shock conditions. Comparative proteomic analyses using two-dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF MS revealed the presence of Cold shock protein E (CspE). Three-dimensional structure of CspE of E. ludwigii PAS1 divulge the presence of five antiparallel β-sheets forming a β-barrel structure with surface exposed aromatic and basic residues that were responsible for nucleic acid binding and also reveals the presence of highly conserved nucleic acid-binding motifs RNP1 and RNP2 in Csp family.     

Overexpression of WsSGTL1 Gene of Withania somnifera Enhances Salt Tolerance,Heat Tolerance and Cold Acclimation Ability in Transgenic Arabidopsis Plants

Background

Sterol glycosyltrnasferases (SGT) are enzymes that glycosylate sterols which play important role in plant adaptation to stress and are medicinally important in plants like Withania somnifera. The present study aims to find the role of WsSGTL1 which is a sterol glycosyltransferase from W. somnifera, in plant’s adaptation to abiotic stress.

Methodology

The WsSGTL1 gene was transformed in Arabidopsis thaliana through Agrobacterium mediated transformation, using the binary vector pBI121, by floral dip method. The phenotypic and physiological parameters like germination, root length, shoot weight, relative electrolyte conductivity, MDA content, SOD levels, relative electrolyte leakage and chlorophyll measurements were compared between transgenic and wild type Arabidopsis plants under different abiotic stresses - salt, heat and cold. Biochemical analysis was done by HPLC-TLC and radiolabelled enzyme assay. The promoter of the WsSGTL1 gene was cloned by using Genome Walker kit (Clontech, USA) and the 3D structures were predicted by using Discovery Studio Ver. 2.5.

Results

The WsSGTL1 transgenic plants were confirmed to be single copy by Southern and homozygous by segregation analysis. As compared to WT, the transgenic plants showed better germination, salt tolerance, heat and cold tolerance. The level of the transgene WsSGTL1 was elevated in heat, cold and salt stress along with other marker genes such as HSP70, HSP90, RD29, SOS3 and LEA4-5. Biochemical analysis showed the formation of sterol glycosides and increase in enzyme activity. When the promoter of WsSGTL1 gene was cloned from W. somnifera and sequenced, it contained stress responsive elements. Bioinformatics analysis of the 3D structure of the WsSGTL1 protein showed functional similarity with sterol glycosyltransferase AtSGT of A. thaliana.

Conclusions

Transformation of WsSGTL1 gene in A. thaliana conferred abiotic stress tolerance. The promoter of the gene in W.somnifera was found to have stress responsive elements. The 3D structure showed functional similarity with sterol glycosyltransferases.