Tumor Cell Death Mediated by Peptides That Recognize Branched Intermediates of DNA Replication and Repair

Effective treatments for cancer are still needed, both for cancers that do not respond well to current therapeutics and for cancers that become resistant to available treatments. Herein we investigated the effect of a structure-selective d-amino acid peptide wrwycr that binds replication fork mimics and Holliday Junction (HJs) intermediates of homologous recombination (HR) in vitro, and inhibits their resolution by HJ-processing enzymes. We predicted that treating cells with HJ-binding compounds would lead to accumulation of DNA damage. As cells repair endogenous or exogenous DNA damage, collapsed replication forks and HJ intermediates will accumulate and serve as targets for the HJ-binding peptides. Inhibiting junction resolution will lead to further accumulation of DNA breaks, eventually resulting in amplification of the damage and causing cell death. Both peptide wrwycr and the related wrwyrggrywrw entered cancer cells and reduced cell survival in a dose- and time-dependent manner. Early markers for DNA damage, γH2AX foci and 53BP1 foci, increased with dose and/or time exposure to the peptides. DNA breaks persisted at least 48 h, and both checkpoint proteins Chk1 and Chk2 were activated. The passage of the cells from S to G2/M was blocked even after 72 h. Apoptosis, however, was not induced in either HeLa or PC3 cells. Based on colony-forming assays, about 35% peptide-induced cytotoxicity was irreversible. Finally, sublethal doses of peptide wrwycr (50–100 µM) in conjunction with sublethal doses of several DNA damaging agents (etoposide, doxorubicin, and HU) reduced cell survival at least additively and sometimes synergistically. Taken together, the results suggest that the peptides merit further investigation as proof-of-principle molecules for a new class of anti-cancer therapeutics, in particular in combination with other DNA damaging therapies.     

The Werner and Bloom syndrome proteins help resolve replication blockage by converting (regressed) holliday junctions to functional replication forks

Cells cope with blockage of replication fork progression in a manner that allows DNA synthesis to be completed and genomic instability minimized. Models for resolution of blocked replication involve fork regression to form Holliday junction structures. The human RecQ helicases WRN and BLM (deficient in Werner and Bloom syndromes, respectively) are critical for maintaining genomic stability and thought to function in accurate resolution of replication blockage. Consistent with this notion, WRN and BLM localize to sites of blocked replication after certain DNA-damaging treatments and exhibit enhanced activity on replication and recombination intermediates. Here we examine the actions of WRN and BLM on a special Holliday junction substrate reflective of a regressed replication fork. Our results demonstrate that, in reactions requiring ATP hydrolysis, both WRN and BLM convert this Holliday junction substrate primarily to a four-stranded replication fork structure, suggesting they target the Holliday junction to initiate branch migration. In agreement, the Holliday junction binding protein RuvA inhibits the WRN- and BLM-mediated conversion reactions. Importantly, this conversion product is suitable for replication with its leading daughter strand readily extended by DNA polymerases. Furthermore, binding to and conversion of this Holliday junction are optimal at low MgCl(2) concentrations, suggesting that WRN and BLM preferentially act on the square planar (open) conformation of Holliday junctions. Our findings suggest that, subsequent to fork regression events, WRN and/or BLM could re-establish functional replication forks to help overcome fork blockage. Such a function is highly consistent with phenotypes associated with WRN- and BLM-deficient cells.     

A heteropolysaccharide from aqueous extract of an edible mushroom, Pleurotus ostreatus cultivar: structural and biological studies

A water soluble polysaccharide isolated from the hot aqueous extract of Pleurotusostreatus cultivar was found to contain d-glucose and d-galactose in a molar ratio of nearly 7:1. Structural investigation was carried out using acid hydrolysis, methylation analysis, periodate oxidation, Smith degradation, and NMR studies (¹H, ¹³C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC). On the basis of the above mentioned experiments the structure of the repeating unit of the polysaccharide was established as:This heteroglycan stimulates macrophages, splenocytes, and thymocytes.     

Hematological and plasma chemistry of Indian major carp,Labeo rohita (Hamilton, 1822)

Hematological and plasma chemistry indices are simple and essential diagnostic tools for monitoring the physiological and health status of fish. Aim of the present study was to obtain reference values for the hematological and plasma chemistry of wild populations of Labeo rohita captured in a freshwater pond between July 2008 and June 2010. These reference values and the mean were evaluated according to sex and season. In summer, the red blood cells (1.84 × 106 38 per cubic mm), haemoglobin (8.52 gm dl^?1) and haematocrit (31.49%) were highest in males, whereas the maximum values for white blood cells (5.635 × 103 40 per cubic mm) were found in females, however, no significant variation of mean corpuscular volume (MCV) or mean corpuscular haemoglobin (MCH) was observed between sexes. Various blood parameter levels between the sexes in summer were notably different from those measured in other seasons except for MCH and MCHC values (p < 0.05). Compared to most teleosts, the L. rohita has similar mean values for PCV and Hb. Throughout summer the glucose (76.0 mg dl^?1), lipid (3.41 gm dl^?1) and cholesterol (145.0 mg dl^?1) levels were highest. In spring the plasma protein levels were higher in males, but higher in winter for females. Consequently, the seasons are key factors when using blood parameters as biomarkers for environmental alterations.     

A Protein-Conjugate Approach to Develop a Monoclonal Antibody-Based Antigen Detection Test for the Diagnosis of Human Brucellosis

Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases.     

Application of Heavy Metal Rich Tannery Sludge on Sustainable Growth,Yield and Metal Accumulation by Clarysage (Salvia sclarea L.)

A field experiment was conducted to evaluate the effective utilization of tannery sludge for cultivation of clarysage (Salvia sclarea) at CIMAP research farm, Lucknow, India during the year 2012–2013. Six doses (0, 20, 40, 60, 80, 100 tha^?1) of processed tannery sludge were tested in randomised block design with four replications. Results revealed that maximum shoot, root, dry matter and oil yield were obtained with application of 80 tha^?1of tannery sludge and these were 94, 113 and 61% higher respectively, over control. Accumulation of heavy metals (Cr, Ni, Fe, Pb) were relatively high in shoot portion of the plant than root. Among heavy metals, magnitude of chromium accumulation was higher than nickel, iron and lead in shoot as well as in root. Linalool, linalyl acetate and sclareol content in oil increased by 13,8 and 27% respectively over control, with tannery sludge application at 80 tha^?1. Heavy metals such as chromium, cadmium and lead content reduced in postharvest soil when compared to initial status. Results indicated that clarysage (Salvia sclarea) can be grown in soil amended with 80 tha^?1sludge and this can be a suitable accumulator of heavy metals for phytoremediation of metal polluted soils.     

Biocomputational Analysis and In Silico Characterization of an Angiogenic Protein (RNase5) in Zebrafish (Danio rerio)

International Journal of Peptide Research and Therapeutics - Several types of RNase protein has been identified and characterized from different group of organism using advanced biocomputational...