Poly-(ADP-ribose) polymerase partially contributes to target cell death triggered by cytolytic T lymphocytes.

We hypothesized that CTL-induced target cell (TC) death is partially due to processes that follow the DNA damage in target cells and include the activation of poly-ADP-ribose transferase (PADPRT) by DNA strand breaks. According to this model, the activated PADPRT is expected to deplete NAD, ATP, and to contribute to the TC death. We used inhibitors of PADPRT and a PADPRT-deficient cell mutant, as well as other nucleated TC and SRBC to test the role of PADPRT in CTL-induced cytotoxicity. It is found that inhibitors of PADPRT (3-aminobenzamide, benzamide (aromatic amides)) and nicotinamide all inhibit the CTL-mediated lysis of both Ag-specific TC and of Ag-nonbearing TC. The effect of PADPRT inhibitors was not due to inhibition of the lethal hit delivery by CTL, because in parallel control experiments, the same inhibitors did not interfere with CTL-induced lysis of SRBC, cells that are devoid of nuclei and PADPRT. Moreover, the effect of inhibitors of PADPRT did not affect earlier stages of lethal hit delivery because 3-aminobenzamide and benzamide did not interfere with CTL-induced DNA fragmentation in TC at concentration which protected TC lysis. Importantly, a PADPRT-deficient cell line was also much more resistant to CTL-induced lysis as tested in retargeting (4 and 8 h) assays; this was expected if activation of PADPRT is indeed involved in TC death. Control experiments reveal that the relative resistance of the PADPRT-deficient cell mutant to CTL-induced lysis was not related to its impaired ability to form conjugates and to trigger CTL (as tested in granule exocytosis assay). In addition, PADPRT-deficient cells were as susceptible to CTL-induced DNA fragmentation as were the control cells; yet, they were resistant to CTL-induced 51Cr-release. Control cells and PADPRT-deficient mutant were equally susceptible to antibody+C'-mediated lysis. Our data support the view that the activation of PADPRT can contribute to the CTL-induced cytolysis of some TC, but is not involved in lysis of other TC, as evidenced by the ability of CTL to efficiently lyse SRBC. These data suggest that there could be multiple molecular pathways of TC death in CTL-mediated cytotoxicity and the relative contribution of PADPRT and/or other enzymes will reflect the individual make-up of a particular TC.     

Surface electrokinetic properties of two strains of Staphylococcus aureus during their cell growth cycle

Two strains of Staphylococcus aureus were investigated: S. aureus H, a normal wild-type strain, and 52A5, a mutant strain whose cell wall contains no teichoic acid but is made up entirely of mucopeptide. S. aureus H cells in the lag or stationary phase of growth had an electrophoretic mobility of ?1.10 μm/s/V/cm while those in the logarithmic phase had a mobility of ?0.80 μm/s/V/cm in saline at pH 7.2, 0.6 mM NaHCO₃, 25°C (I = 0.145 g-ions/l). S. aureus 52A5 cells in the same solution had a mobility of ?0.87 μm/s/V/cm in lag and stationary growth phases but a mobility of ?1.30 μm/s/V/cm in the logarithmic growth phase. The S. aureus H cell surfaces at lag phase had pKs of 3.2 and 9.5; at logarithmic phase, 4.2 and 9.0; and at stationary phase, 3.0 and 9.5. The 52A5 cell surfaces at lag phase had pKs of 2.3 and 10.3; at logarithmic phase, 1.7 and 8.5; at stationary phase, 2.6 and 10.2.     

Ultrasonic radiation induced lipid peroxidation in liposomal membrane

Summary Ultrasonic radiation produced a dose dependent linear increase in lipid peroxidation (MDA formation) in the liposomal membrane. The yield of MDA was significantly inhibited by butylated hydroxytoluene (BHT), the antioxidant, sodium formate, the OH radical scavenger, and EDTA, the metal ion chelator. Ascorbic acid at low concentration increased the ultrasonic induced MDA formation while high concentrations inhibited lipid peroxidation. A mechanism of ultrasound induced lipid peroxidation is suggested.     

Ultrashort nanosecond electric pulses evoke heterogeneous patterns of Ca2+ release from the endoplasmic reticulum of adrenal chromaffin cells

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Extracting proteins involved in disease progression using temporally connected networks

Background

Metabolic disorders such as obesity and diabetes are diseases which develop gradually over time in an individual and through the perturbations of genes. Systematic experiments tracking disease progression at gene level are usually conducted giving a temporal microarray data. There is a need for developing methods to analyze such complex data and extract important proteins which could be involved in temporal progression of the data and hence progression of the disease.

Results

In the present study, we have considered a temporal microarray data from an experiment conducted to study development of obesity and diabetes in mice. We have used this data along with an available Protein-Protein Interaction network to find a network of interactions between proteins which reproduces the next time point data from previous time point data. We show that the resulting network can be mined to identify critical nodes involved in the temporal progression of perturbations. We further show that published algorithms can be applied on such connected network to mine important proteins and show an overlap between outputs from published and our algorithms. The importance of set of proteins identified was supported by literature as well as was further validated by comparing them with the positive genes dataset from OMIM database which shows significant overlap.

Conclusions

The critical proteins identified from algorithms can be hypothesized to play important role in temporal progression of the data.

     

Scavenging of superoxide radical by ascorbic acid

Using acetaldehyde and xanthine oxidase as the source of suPeroxide radical, the second order rate constant for the reaction between ascorbic acid and superoxide radical was estimated to be 8.2 X 10⁷ M^-1 s^-1. In rats, the average tissue concentration of ascorbic acid was of the order of 10^-3 M and that of superoxide dismutase was of the order of 10^-6 M. So, taking together both the rate constants and the tissue concentrations, the efficacy of ascorbic acid for scavenging superoxide radical in animal tissues appears to be better than that of suPeroxide dismutase. The significance of ascorbic acid as a scavenger of superoxide radical has been discussed from the point of view of the evolution of ascorbic acid synthesizing capacity of terrestrial vertebrates.     

A novel mechanism for protein-assisted group I intron splicing

Previously it was shown that the Aspergillus nidulans (A.n.) mitochondrial COB intron maturase, I-AniI, facilitates splicing of the COB intron in vitro. In this study, we apply kinetic analysis of binding and splicing along with RNA deletion analysis to gain insight into the mechanism of I-AniI facilitated splicing. Our results are consistent with I-AniI and A.n. COB pre-RNA forming a specific but labile encounter complex that is resolved into the native, splicing-competent complex. Significantly, kinetic analysis of splicing shows that the resolution step is rate limiting for splicing. RNA deletion studies show that I-AniI requires most of the A.n. COB intron for binding suggesting that the integrity of the I-AniI-binding site depends on overall RNA tertiary structure. These results, taken together with the observation that A.n. COB intron lacks significant stable tertiary structure in the absence of protein, support a model in which I-AniI preassociates with an unfolded COB intron via a "labile" interaction that facilitates correct folding of the intron catalytic core, perhaps by resolving misfolded RNAs or narrowing the number of conformations sampled by the intron during its search for native structure. The active intron conformation is then "locked in" by specific binding of I-Anil to its intron interaction site.     

PhyA, a secreted protein of Xanthomonas oryzae pv. oryzae, is required for optimum virulence and growth on phytic acid as a sole phosphate source

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. We have identified a novel virulence deficient mutant (BXO1691) of X. oryzae pv. oryzae that has a Tn5 insertion in an open reading frame (phyA; putative phytase A) encoding a 373-amino acid (aa) protein containing a 28-aa predicted signal peptide. Extracellular protein profiles revealed that a 38-kDa band is absent in phyA mutants as compared with phyA+ strains. A BLAST search with phyA and its deduced polypeptide sequence indicated significant similarity with conserved hypothetical proteins in Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris and limited homology to secreted phytases of Bacillus species. Homology modeling with a Bacillus phytase as the template suggests that the PhyA protein has a similar six-bladed beta-propeller architecture and exhibits conservation of certain critical active site residues. Phytases are enzymes that are involved in degradation of phytic acid (inositol hexaphosphate), a stored form of phosphate in plants. The phyA mutants exhibit a growth deficiency in media containing phytic acid as a sole phosphate source. Exogenous phosphate supplementation promotes migration of phyA X. oryzae pv. oryzae mutants in rice leaves. These results suggest that the virulence deficiency of phyA mutants is, at least in part, due to inability to use host phytic acid as a source of phosphate. phyA-like genes have not been previously reported to be involved in the virulence of any plant pathogenic bacterium.     

The Relative Expression of Mig6 and EGFR Is Associated with Resistance to EGFR Kinase Inhibitors

The sensitivity of only a few tumors to anti-epidermal growth factor receptor EGFR tyrosine kinase inhibitors (TKIs) can be explained by the presence of EGFR tyrosine kinase (TK) domain mutations. In addition, such mutations were rarely found in tumor types other than lung, such as pancreatic and head and neck cancer. In this study we sought to elucidate mechanisms of resistance to EGFR-targeted therapies in tumors that do not harbor TK sensitizing mutations in order to identify markers capable of guiding the decision to incorporate these drugs into chemotherapeutic regimens. Here we show that EGFR activity was markedly decreased during the evolution of resistance to the EGFR tyrosine kinase inhibitor (TKI) erlotinib, with a concomitant increase of mitogen-inducible gene 6 (Mig6), a negative regulator of EGFR through the upregulation of the PI3K-AKT pathway. EGFR activity, which was more accurately predicted by the ratio of Mig6/EGFR, highly correlated with erlotinib sensitivity in panels of cancer cell lines of different tissue origins. Blinded testing and analysis in a prospectively followed cohort of lung cancer patients treated with gefitinib alone demonstrated higher response rates and a marked increased in progression free survival for patients with a low Mig6/EGFR ratio (approximately 100 days, P = 0.01).     

EpCAM Knockdown Alters MicroRNA Expression in Retinoblastoma- Functional Implication of EpCAM Regulated MiRNA in Tumor Progression

The co-ordinated regulation of oncogenes along with miRNAs play crucial role in carcinogenesis. In retinoblastoma (RB), several miRNAs are known to be differentially expressed. Epithelial cell adhesion molecule (EpCAM) gene is involved in many epithelial cancers including, retinoblastoma (RB) tumorigenesis. EpCAM silencing effectively reduces the oncogenic miR-17-92 cluster. In order to investigate whether EpCAM has wider effect as an inducer or silencer of miRNAs, we performed a global microRNA expression profile in EpCAM siRNA knockdown Y79 cells. MicroRNA profiling in EpCAM silenced Y79 cells showed seventy-three significantly up regulated and thirty-six down regulated miRNAs. A subset of these miRNAs was also validated in tumors. Functional studies on Y79 and WERI-Rb-1 cells transfected with antagomirs against two miRNAs of miR-181c and miR-130b showed striking changes in tumor cell properties in RB cells. Treatment with anti-miR-181c and miR-130b showed significant decrease in cell viability and cell invasion. Increase in caspase-3 level was noticed in antagomir transfected cell lines indicating the induction of apoptosis. Possible genes altered by EpCAM influenced microRNAs were predicted by bioinformatic tools. Many of these belong to pathways implicated in cancer. The study shows significant influence of EpCAM on global microRNA expression. EpCAM regulated miR-181c and miR-130b may play significant roles in RB progression. EpCAM based targeted therapies may reduce carcinogenesis through several miRNAs and target genes.