Expression,purification and characterization of the interferon-inducible,antiviral and tumour-suppressor protein,human RNase L

The interferon (IFN)-inducible, 2′,5′-oligoadenylate (2-5A)-dependent ribonuclease L (RNase L) plays key role in antiviral defense of mammalian cells. Induction by IFN and activation by double-stranded RNA lead to 2-5A cofactor synthesis, which activates RNase L by causing its dimerization. Active RNase L degrades single-stranded viral as well as cellular RNAs causing apoptosis of virus-infected cells. Earlier, we had reported that expression of recombinant human RNase L caused RNA-degradation and cell-growth inhibition in E. coli without the need for exogenous 2-5A. Expression of human RNase L in E. coli usually leads to problems of leaky expression, low yield and degradation of the recombinant protein, which demands number of chromatographic steps for its subsequent purification thereby, compromising its biochemical activity. Here, we report a convenient protocol for expression of full-length, soluble and biochemically active recombinant human RNase L as GST-RNase L fusion protein from E. coli utilizing a single-step affinity purification with an appreciable yield of the highly purified protein. Recombinant RNase L was characterized by SDS-PAGE, immunoblotting and MALDI-TOF analysis. A semi-quantitative agarose-gel-based ribonuclease assay was developed for measuring its 2-5A-dependent RNase L activity against cellular large rRNAs as substrates. The optimized expression conditions minimized degradation of the protein, making it a convenient method for purification of RNase L, which can be utilized to study effects of various agents on the RNase L activity and its protein–protein interactions.     

Cross‐Species Alleviation of Biotic and Abiotic Stresses by the Endophyte Pseudomonas aeruginosa PW09

A wheat endophytic bacterium (Pseudomonas aeruginosa PW09) was evaluated for its ability to trigger an induced systemic resistance response in cucumber against biotic and abiotic stresses. PW09 was applied to cucumber seeds, and the seedlings were subjected to Sclerotium rolfsii infection and NaCl (150 mm ). The role of PW09 was evaluated in alleviating the stresses by assessing plant mortality due to S. rolfsii infection and biomass accumulation under NaCl stress as well as at the physiological level through phenylpropanoid metabolism, antioxidant activities and proline accumulation. The endophyte reduced seedling mortality by 60% and increased biomass accumulation significantly under S. rolfsii (7%) and NaCl (18%) stresses, respectively, compared with endophyte‐untreated seedlings. Application of PW09 also induced higher accumulation of proline (1.3‐ and 1.4‐fold) and total phenolics (1.2‐ and 1.1‐fold) and activities of polyphenol oxidase (4.3‐ and 1.5‐fold), phenylalanine ammonia lyase (1.29‐ and 1.27‐fold) and superoxide dismutase (2.5‐ and 1.39‐fold) under S. rolfsii and NaCl stresses, indicating the ability of the wheat endophyte PW09 in alleviating both biotic and abiotic stresses in cucumber.     

Computational design of proteins stereochemically optimized in size, stability, and folding speed

Artificial proteins potentially barrier-free in the folding kinetics are approached computationally under the guidance of protein-folding theories. The smallest and fastest folding globular protein triple-helix-bundle (THB) is so modified as to minimize or eliminate its presumed barriers in folding speed. As the barriers may reside in the ordering of either secondary or tertiary structure, the elements of both secondary and tertiary structure in the protein are targeted for prenucleation with suitable stereochemically constrained amino acid residues. The required elements of topology and sequence for the THB are optimized independently; first the topology is optimized with simulated annealing in polypeptides of highly simplified alphabet; next, the sequence in side chains is optimized using the standard inverse design methods. The resultant three best-adapted THBs, variable in topology and distinctive in sequences, are assessed by comparing them with a few benchmark proteins. The results of mainly molecular dynamics (MD) comparisons, undertaken in explicit water at different temperatures, show that the designed sequences are favorably placed against the chosen benchmarks as THB proteins potentially thermostable in the native folds. Folding simulation experiments with MD establish that the designed sequences are rapid in the folding of individual helices, but not in the evolution of tertiary structure; energetic cum topological frustrations remain but could be the artifacts of the starting conformations that were chosen in the THBs in the folding simulations. Overall, a practical high-throughput approach for de novo protein design has been developed that may have fruitful application for any type of tertiary structure.     

Anti-apoptotic activity of caffeic acid, ellagic acid and ferulic acid in normal human peripheral blood mononuclear cells: a Bcl-2 independent mechanism

Polyphenols have been shown to induce apoptosis in a variety of tumor cells including leukemia both in vitro and in vivo. However, their action on normal human peripheral blood mononuclear cells (PBMCs) during oxidative stress remains to be explored. In this study, we have evaluated the anti-apoptotic and radical scavenging activities of dietary phenolics, namely caffeic acid (CA), ellagic acid (EA) and ferulic acid (FA). H2O2-induced apoptosis in normal human PBMCs was assayed by phosphotidylserine externalization, nucleosomal damage and DNA fragmentation. Incubation of PBMCs with 5 mM H2O2 led to increased Annexin-V binding to externalized phosphatidyl serine (PS), an event of pre-apoptotic stage of the cell. Peripheral blood mononuclear cells pretreated with phenolics could resist H2O2-induced apoptotic damage. Caffeic acid (60 and 120 microM) and EA (100 and 200 microM) caused no change in externalization of PS, whereas FA (100 and 200 microM) increased externalization of PS in PBMCs treated with H2O2. The effects of phenolics were abolished to a large extent by culturing the PBMCs for 24 h after washing the phenolics from the medium. Inhibitory activities of these phenolics on lipid peroxidation were in the order of EA相似文献   

Enhanced immunoprotective potential of Mycobacterium tuberculosis Ag85 complex protein based vaccine against airway Mycobacterium tuberculosis challenge following intranasal administration

This study examined the role of intranasal vaccination with Mycobacterium tuberculosis antigen85 complex proteins formulated in dimethyldioctadecylammonium bromide against airway Mycobacterium tuberculosis challenge in mice. Intranasal vaccination with antigen85A and antigen85B induced a significantly higher level of interferon-gamma, interleukin-12 and interleukin-4 in cervical lymph nodes together with IgA and IgG, predominantly IgG2a isotype in nasal secretion over subcutaneous vaccination. Further, intranasal vaccination with antigen85A and antigen85B imparted protection comparable with that obtained from intranasal or subcutaneous Mycobacterium bovis bacillus Calmette-Guerin immunization. These results suggest that mucosal vaccination via the intranasal route is of importance in the development of vaccine for tuberculosis.     

Synthesis, SAR exploration, and X-ray crystal structures of factor XIa inhibitors containing an alpha-ketothiazole arginine

Using an alpha-ketothiazole arginine moiety as a key recognition element, a series of small peptidomimetic molecules was designed and synthesized, and their co-crystal structures with factor XIa were studied in an effort to develop smaller, less peptidic inhibitors as antithrombotic agents.     

Biosorption of Cd(II) and Pb(II) onto brown seaweed, Lobophora variegata (Lamouroux): kinetic and equilibrium studies

The present work deals with the biosorption performance of raw and chemically modified biomass of the brown seaweed Lobophora variegata for removal of Cd(II) and Pb(II) from aqueous solution. The biosorption capacity was significantly altered by pH of the solution delineating that the higher the pH, the higher the Cd(II) and Pb(II) removal. Kinetic and isotherm experiments were carried out at the optimal pH 5.0. The metal removal rates were conspicuously rapid wherein 90% of the total sorption occurred within 90 min. Biomass treated with CaCl₂ demonstrated the highest potential for the sorption of the metal ions with the maximum uptake capacities i.e. 1.71 and 1.79 mmol g⁻¹ for Cd(II) and Pb(II), respectively. Kinetic data were satisfactorily manifested by a pseudo-second order chemical sorption process. The process mechanism consisting of both surface adsorption and pore diffusion was found to be complex. The sorption data have been analyzed and fitted to sorption isotherm of the Freundlich, Langmuir, and Redlich–Peterson models. The regression coefficient for both Langmuir and Redlich–Peterson isotherms were higher than those secured for Freundlich isotherm implying that the biosorption system is possibly monolayer coverage of the L. variegata surface by the cadmium and lead ions. FT-IR studies revealed that Cd(II) and Pb(II) binding to L. variegata occurred primarily through biomass carboxyl groups accompanied by momentous interactions of the biomass amino and amide groups. In this study, we have observed that L. variegata had maximum biosorption capacity for Cd(II) and Pb(II) reported so far for any marine algae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Acute Vascular Disruption by 5,6-Dimethylxanthenone-4-Acetic Acid in an Orthotopic Model of Human Head and Neck Cancer

The sustenance of most solid tumors including head and neck cancers (HNCs) is strongly dependent on the presence of a functioning vascular network. In this study, we examined the acute effects of a tumor vascular disrupting agent (VDA), 5,6-dimethylxanthenone-4-acetic acid (DMXAA; ASA404), in an orthotopic model of human HNC. Noninvasive magnetic resonance imaging (MRI) was used to monitor the vascular response of orthotopic FaDu xenografts to VDA therapy. Untreated tumors showed a marked but heterogeneous pattern of enhancement after contrast agent injection on serial T1-weighted (T1W) MR images. After VDA treatment, T2W and T1W MRI revealed evidence of hemorrhaging and lack of functioning vessels (enhancement) within the tumor. Quantitative estimates of relative vascular volume also showed a significant (P < .01) reduction in DMXAA-treated tumors 24 hours after therapy compared with untreated controls. Histology and immunostaining of untreated orthotopic FaDu tumors revealed poorly differentiated squamous cell carcinoma histology with distinctly visible CD31⁺ endothelial cells. In sharp contrast, minimal CD31 staining with irregular endothelial fragments and faint outlines of blood vessels were seen in DMXAA-treated tumor sections. CD31 immunostaining and histology also highlighted the selectivity of vascular damage and tissue necrosis after VDA therapy with no evidence of toxicity observed in normal salivary gland, heart, liver, and skeletal muscle tissues. Together, our results demonstrate a potent and selective vascular disruptive activity of DMXAA in an orthotopic HNC model. Further evaluation into its antitumor effects alone and in combination with other agents is warranted.