Role of zinc in mitigating the toxic effects of chlorpyrifos on hematological alterations and electron microscopic observations in rat blood

The present study determined the protective potential of zinc in attenuating the toxicity induced by chlorpyrifos in rat blood. Male Sparque Dawley (SD) rats received either oral chlorpyrifos (13.5 mg/kg body weight) treatment every alternate day, zinc alone (227 mg/l in drinking water) or combined chlorpyrifos plus zinc treatment for a total duration of 8 weeks. The effects of different treatments were studied on various parameters in rat blood including haemoglobin (Hb) levels, total leukocyte count (TLC), differential leukocyte count (DLC), zinc protoporphyrins (ZPP), serum trace elemental concentrations and Scanning Electron Microscopic (SEM) observation of the blood cells. Chlorpyrifos treatment to normal control animals resulted in a significant decrease in TLC and ZPP concentration after 4 and 8 weeks. Chlorpyrifos treated animals also showed significant neutrophilia and lymphopenia after 8 weeks of toxicity. In addition, a significant decrease in serum zinc and iron concentrations were observed following chlorpyrifos intoxication, however, these animals responded with increased serum copper levels following the toxic treatment with this organophosphate. SEM studies of the red blood cells from chlorpyrifos treated animals indicated marked alterations in the topographical morphology of the various cell types, with the prominent feature being common aniscocytosis of the erythrocytes. Oral zinc treatment to the chlorpyrifos treated animals significantly improved the total leukocyte, neutrophil and lymphocyte counts, as well as the otherwise reduced concentrations of ZPP and the levels of various serum trace elements. Protective effects of zinc were also evident in the electron microscopic observations where most blood cell types depicted reverted to a close to the normal appearance. Based upon these data, the present study is first of its kind and suggests that zinc treatment considerably attenuates chlorpyrifos induced toxicity induced in restoring the altered hematological indices and morphological changes.     

Certolizumab pegol

Certolizumab pegol (Cimzia^®) is currently the only PEGylated anti-TNFα biologic approved for the treatment of rheumatoid arthritis and Crohn disease. The product, developed by UCB, is a humanized antigen-binding fragment (Fab’) of a monoclonal antibody that has been conjugated to polyethylene glycol. Certolizumab pegol was approved as a treatment for rheumatoid arthritis in the EU, US and Canada in 2009, and as a treatment for Crohn disease in Switzerland in 2007 and the US in 2008. Certolizumab pegol is entering into an increasingly competitive marketplace, especially in rheumatoid arthritis, but clinical data demonstrate benefits across a range of clinical, radiographic and patient reported outcomes.Key words: certolizumab pegol, rheumatoid arthritis, Crohn disease, TNFα, PEGylated, methotrexate     

An Optimized Pentaplex PCR for Detecting DNA Mismatch Repair-Deficient Colorectal Cancers

Purpose

Microsatellite instability (MSI) is used to screen colorectal cancers (CRC) for Lynch Syndrome, and to predict outcome and response to treatment. The current technique for measuring MSI requires DNA from normal and neoplastic tissues, and fails to identify tumors with specific DNA mismatch repair (MMR) defects. We tested a panel of five quasi-monomorphic mononucleotide repeat markers amplified in a single multiplex PCR reaction (pentaplex PCR) to detect MSI.

Experimental Design

We investigated a cohort of 213 CRC patients, comprised of 114 MMR-deficient and 99 MMR-proficient tumors. Immunohistochemical (IHC) analysis evaluated the expression of MLH1, MSH2, PMS2 and MSH6. MSI status was defined by differences in the quasi-monomorphic variation range (QMVR) from a pool of normal DNA samples, and measuring differences in allele lengths in tumor DNA.

Results

Amplification of 426 normal alleles allowed optimization of the QMVR at each marker, and eliminated the requirement for matched reference DNA to define MSI in each sample. Using ≥2/5 unstable markers as the criteria for MSI resulted in a sensitivity of 95.6% (95% CI = 90.1–98.1%) and a positive predictive value of 100% (95% CI = 96.6%–100%). Detection of MSH6-deficiency was limited using all techniques. Data analysis with a three-marker panel (BAT26, NR21 and NR27) was comparable in sensitivity (97.4%) and positive predictive value (96.5%) to the five marker panel. Both approaches were superior to the standard approach to measuring MSI.

Conclusions

An optimized pentaplex (or triplex) PCR offers a facile, robust, very inexpensive, highly sensitive, and specific assay for the identification of MSI in CRC.     

Mechanism of biochemical action of substituted 4-methylbenzopyran-2-ones. Part 4: hyperbolic activation of rat liver microsomal NADPH-cytochrome C reductase by the novel acetylator 7,8-diacetoxy-4-methylcoumarin

The effect of 7,8-diacetoxy-4-methylcoumarin (DAMC) has been studied on hepatic NADPH cytochrome C reductase-- an enzyme participating in the microsomal electron transport. The preincubation of liver microsomes with DAMC resulted in a time-dependent activation of NADPH cytochrome C reductase. The catalytic activity of the enzyme enhanced nearly 600% by 25 microM concentration of DAMC after 10 min of preincubation. The action of DAMC on the reductase resulted in enhanced v(max) while Km remained constant. A plot of 1/v(max) as a function of DAMC concentration resulted in a non-linear, but rectangular hyperbola indicative of hyperbolic activation. DAMC was also proved to be effective in significantly enhancing the activity of NADPH cytochrome C reductase in vivo. 7,8-Dihydroxy-4-methylcoumarin (DHMC), the deacetylated product of DAMC failed to irreversibly activate the enzyme. The activation effect of DAMC upon the enzyme was abolished by p-hydroxymercury benzoate. The role of a transacetylase in transferring the acetyl group of DAMC to the amino acid(s) of the active site of NADPH cytochrome C reductase causing irreversible enzyme activation is enunciated.     

Transformation of tomato with a bacterial codA gene enhances tolerance to salt and water stresses

Genetically engineered tomato (Lycopersicon esculentum) with the ability to synthesize glycinebetaine was generated by introducing the codA gene encoding choline oxidase from Arthrobacter globiformis. Integration of the codA gene in transgenic tomato plants was verified by PCR analysis and DNA blot hybridization. Transgenic expression of gene was verified by RT-PCR analysis and RNA blot hybridization. The codA-transgenic plants showed higher tolerance to salt stress during seed germination, and subsequent growth of young seedlings than wild-type plants. The codA transgene enhanced the salt tolerance of whole plants and leaves. Mature leaves of codA-transgenic plants revealed higher levels of relative water content, chlorophyll content, and proline content than those of wild-type plants under salt and water stresses. Results from the current study suggest that the expression of the codA gene in transgenic tomato plants induces the synthesis of glycinebetaine and improves the tolerance of plants to salt and water stresses.     

Unravelling the antimetastatic potential of pentoxifylline, a methylxanthine derivative in human MDA-MB-231 breast cancer cells

Pentoxifylline (PTX), a methylxanthine derivative is a non-steroidal immunomodulating agent with unique hemorheologic properties. It is used in the treatment of intermittent claudication as it increases the amount of oxygen reaching tissues by increasing the flexibility of red blood cells. Recently, it has also shown to exhibit anti-metastatic and anti-angiogenic activities in B16F10 melanoma cells both in vitro as well as in vivo. As per the reports, the choice of drug in the treatment of breast cancer is paclitaxel, but the major limitation is its toxicity. However, the effects of PTX on metastatic processes in breast cancer are not currently known. Therefore, in this study, we have examined the effect of PTX in MDA-MB-231 human breast cancer cells. The MTT assay showed dose- and time-dependent decreases in cellular proliferation. The non-toxic concentration of PTX selected were 1, 2.5 and 5 mM for 24 h. PTX induced a G0-G1 cell-cycle arrest leading to apoptosis. Further, it affected adhesion to both the matrigel and collagen type-IV in a time- and dose-dependent manner. The PTX impeded the migration of MDA-MB-231 cells and also decreased the activities of both MMP-2 and MMP-9. Thus, PTX at non-toxic doses affected cellular proliferation, adhesion, migration and invasion. These results demonstrate its anti-metastatic effect on MDA-MB-231 cells, and further studies need to be carried out to understand the mechanism of action.     

De novo design and characterization of a helical hairpin eicosapeptide; emergence of an anion receptor in the linker region

De novo design of supersecondary structures is expected to provide useful molecular frameworks for the incorporation of functional sites as in proteins. A 21 residue long, dehydrophenylalanine-containing peptide has been de novo designed and its crystal structure determined. The apolar peptide folds into a helical hairpin supersecondary structure with two right-handed helices, connected by a tetraglycine linker. The helices of the hairpin interact with each other through a combination of C-H.O and N-H.O hydrogen bonds. The folding of the apolar peptide has been realized without the help of either metal ions or disulphide bonds. A remarkable feature of the peptide is the unanticipated occurrence of an anion binding motif in the linker region, strikingly similar in conformation and function to the "nest" motif seen in several proteins. The observation supports the view for the possible emergence of rudimentary functions over short sequence stretches in the early peptides under prebiotic conditions.     

A convenient synthesis and hepatoprotective activity of imidazo[1,2-c]pyrimido[5,4-e]pyrimidine, tetraazaacenaphthene and tetraazaphenalene from cyclic ketene aminals through tandem addition-cyclization reactions

A novel one-pot synthesis of imidazo[1,2-c]pyrimido[5,4-e]pyrimidinones (2), tetraazaacenaphthene-3,6-diones (4), tetarazaphenalene-1,7-dione (4d) is delineated from the reaction of cyclic ketene aminal (1) and alkyl or aryl isothiocyanate through tandem addition-cyclization reactions. However, reaction of ketene aminal (1a) with alkyl isothiocyanate only yielded angularly cyclized product 5 which did not react further to yield 6. The structure of 2c and 4d was ascertained by single crystal X-ray diffraction analysis which demonstrated a network of various inter- and intramolecular interactions, responsible for the stability and packing of the molecules in the crystalline state. Some of the compounds (2a--h) were screened for hepatoprotective activity but only 2a was found most effective.     

Differential Proteomics in Response to Low Temperature Diazotrophy of Himalayan Psychrophilic Nitrogen Fixing Pseudomonas migulae S10724 Strain

Himalayas are considered as a reservoir of diversified and dynamic gene pool. This study describes the response of a Himalayan psychrophilic diazotroph to low temperature diazotrophy. Seven cold adaptive N₂ fixing bacteria were isolated and identified as Bacillus sp., Arthrobacter sp., Rhodococcus sp., Pseudomonas sp., etc. In order to examine the physiological response to low temperature diazotrophy, differential proteomic analysis of Pseudomonas migulae S10724 strain was carried out using two dimensional electrophoresis and MALDI–TOF–MS. Functional assessment of 66 differentially expressed proteins revealed several mechanisms thought to be involved in low temperature adaptation and nitrogen fixation, including general stress adaptation, protein and nucleic acid synthesis, energy metabolism, cell growth/maintenance, etc. Major fraction of the upregulated proteins was stress proteins, while majority of the downregulated proteins were related to cell division. Furthermore, MALDI–TOF–MS-based identification of randomly selected peptides encountered two exclusively expressed proteins: NifU family SUF system FeS assembly protein and membrane protein, suppressor for copper-sensitivity B precursor which might have a crucial role at low temperature nitrogen fixation. To the best of our knowledge, this is the first report of the isolation and differential proteomic analysis of psychrophilic diazotroph from Himalayan high altitude rhizospheric soil.