No detectable misrejoining in double-minute chromosomes.

Pulsed-field gel electrophoresis combined with Southern hybridization and rare-cutting restriction endonuclease digestion has been used recently to quantify misrejoining of DNA double-strand breaks (DSBs) resulting from exposure to ionizing radiation. Measurements are made 24 h after a high dose of radiation. These studies have suggested that a large fraction of DSBs are misrejoined to result in gross rearrangements. In the experiments described here, we show that elimination of broken DNA also eliminates "misrejoined" DNA. Mouse cells resistant to high levels of methotrexate by virtue of 100-fold amplification of the dyhydrofolate reductase (Dhfr) gene were treated with 50 and 100 Gy of ionizing radiation. The cells were allowed to repair the damage for 24 h. After the repair period, the cells were immobilized in agarose. Aliquots of each sample were pre-electrophoresed to remove linear DNA molecules smaller than 6 Mbp resulting from apoptosis or necrosis. The samples repairing damage from 50 or 100 Gy that did not receive the pre-electrophoresis showed high levels of label in a region of the lane that could be due to misrejoining DNA molecules. However, when the DNA from cells undergoing apoptosis or necrosis was removed from these samples, the levels of "misrejoined" DNA were reduced to levels far below those of unirradiated controls. These results suggest that other radiation-induced effects present 24 h after irradiation with 50 or 100 Gy are more significant than misrejoining for altering hybridization to regions of the lane outside the specific bands. Measurements of misrejoining using PFGE, rare-cutting restriction endonucleases, and Southern hybridization are likely to be compromised by nonspecific hybridization to broken and difficult-to-digest DNA resulting from apoptosis or necrosis.     

Chemopreventive action of Phyllanthus urinaria Linn on DMBA-induced skin carcinogenesis in mice

The inhibition of tumor incidence by hydro-alcoholic extract of the whole plant of P. urinaria was evaluated in 6-7 weeks old female albino mice on two-stage process of skin carcinogenesis induced by a single application of 7,12-dimethylbenz(a)anthracene (50 microg/50 microl of acetone), and 2 weeks later, promoted by repeated application of croton oil (1% in acetone/three times a week) till the end of the experiment (15 weeks). Topical application of the extract at a dose of 5 mg/kg body weight/day for 15 weeks at the peri-initiational stage (i.e., 7 days before and 7 days after DMBA application), promotional stage (i.e., from the time of croton oil application) and both peri and post-initiational stages (i.e., 7 days prior to DMBA application and continued till the end of the experiment) on the shaven backs of the mice recorded a significant reduction in tumor incidence to 50, 33.3 and 16.7% respectively in comparison to the control (i.e., the mice treated with DMBA and croton oil only) where tumor incidence was found to be 81.8%. The average number of papillomas per mouse was also significantly reduced. The results suggest a possible chemopreventive property of P. urinaria against DMBA-induced skin papillomagenesis in mice.     

Ethnopharmacological Survey of Plants Used for the Treatment of Stomach,Diabetes, and Ophthalmic Diseases in Sudhan Gali,Kashmir , Pakistan

The present paper represents the ethnopharmacological survey of Sudhan Gali, Kashmir, Pakistan. The study revealed that 12 plant species belonging to 11 families were used for the treatment of stomach, diabetes and ophthalmic diseases by the local people in Sudhan Gali. Achillea millefolium , Aconitum heterophyllum, Berberis lycium, Polygonum amplexicaule, Mentha longifolia, Paeonia emodi, Plantago lanceolata were locally used for stomach related problems treatment; Berberis lycium, Skimmia lareola, Solanum dulcamara for diabetes and Geranium wallichianum, Artemisia vulgaris, Solanum dulcamara, and Corydalis crassifolia used for the treatment of ophthalmic diseases. Two species Berberis lycium and Solanum dulcamara have multipurpose value. Former is used to treat stomach as well as diabetes while latter is used to treat not only to diabetes but also ophthalmic diseases. According to IUCN categories , out of these 12 plant species collected and marketed, Polygonum amplexicaule and Paeonia emodi are endangered, Aconitum heterophyllum; Berberis lycium species are vulnerable while Plantago lanceolata and Skimmia lareola species are rare.The availability of these medicinal plants has decreased during the past 20 years and these are facing a drastic biotic pressure due to their extensive usage and non-scientific methods of collection. It is quite evident that these valuable native medicinal plants species are going to decline in number and ultimately will become extinct if no timely proper conservation strategies are adopted.     

Green synthesis,characterization, enhanced functionality and biological evaluation of silver nanoparticles based on Coriander sativum

The present study focused on the green synthesis of silver nanoparticles from Coriander sativum (CS) containing structural polymers, phenolic compounds and glycosidic bioactive macromolecules. Plant phenolic compounds can act as antioxidants, lignin, and attractants like flavonoids and carotenoids. Henceforth, silver nanoparticles (AgNPs) were prepared extracellularly by the combinatorial action of stabilizing and reduction of the CS leaf extract. The biologically synthesized CS-AgNPs were studied by UV-spectroscopy, zeta potential determination, scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis to characterize and confirm the formation of crystalline nanoparticles. The synthesized nanoparticles demonstrated strong antimicrobial activity against all microbial strains examined with varying degrees. The scavenging action on free radicals by CS-AgNPs showed strong antioxidant efficiency with superoxide and hydroxyl radicals at different concentrations as compared with standard ascorbic acid. The presence of in vitro anticancer effect was confirmed at different concentrations on the MCF-7 cell line as revealed with decrease in cell viability which was proportionately related to the concentration of CS-AgNPs illustrating the toxigenic nature of synthesized nanoparticles on cancerous cells.     

Attenuation of oxidative damage-associated cognitive decline by Withania somnifera in rat model of streptozotocin-induced cognitive impairment

Oxidative stress is a critical contributing factor to age-related neurodegenerative disorders. Therefore, the inhibition of oxidative damage, responsible for chronic detrimental neurodegeneration, is an important strategy for neuroprotective therapy. Withania somnifera (WS) extract has been reported to have potent antioxidant and free radical quenching properties in various disease conditions. The present study evaluated the hypothesis that WS extract would reduce oxidative stress-associated neurodegeneration after intracerebroventricular injection of streptozotocin (ICV-STZ) in rats. To test this hypothesis, male Wistar rats were pretreated with WS extract at doses of 100, 200, and 300 mg/kg body weight once daily for 3 weeks. On day 22nd, the rats were infused bilaterally with ICV-STZ injection (3 mg/kg body weight) in normal saline while sham group received only saline. Two weeks after the lesioning, STZ-infused rats showed cognitive impairment in the Morris water maze test. The rats were sacrificed after 3 weeks of the lesioning for the estimation of the contents of lipid peroxidation, reduced glutathione, and activities of glutathione reductase, glutathione peroxidase, and catalase. Pretreatment with WS extract attenuated behavioral, biochemical, and histological alterations significantly in dose-dependent manner in the hippocampus and cerebral cortex of ICV-STZ-infused rats. These results suggest that WS affords a beneficial effect on cognitive deficit by ameliorating oxidative damage induced by streptozotocin in a model of cognitive impairment.     

Synthesis and enantiopreferential DNA-binding profile of late 3d transition metal R- and S-enantiomeric complexes derived from N,N-bis-(1-benzyl-2-ethoxyethane): Validation of R-enantiomer of copper(II) complex as a human topoisomerase II inhibitor

To evaluate the biological preference of chiral drug candidates for molecular target DNA, new potential metal‐based chemotherapeutic agents 1 , 1a , 1b , 2 , 2a , 2b , 3 , 3a , 3b of late 3d transition metals Ni(II), Cu(II), and Zn(II), respectively, derived from (R)‐ and (S)‐2‐amino‐2‐phenylethanol with  CH₂ CH₂ linker were synthesized and thoroughly characterized. Interaction studies of 1 , 1a , 1b , 2 , 2a , 2b , 3 , 3a , 3b with calf thymus DNA in Tris buffer were studied by electronic absorption titrations, luminescence titrations, cyclic voltammetry, and circular dichroism. The results reveal that the extent of DNA binding of R‐enantiomer of copper 1a was highest in comparison to rest of the complexes via electrostatic interaction mode. The nuclease activity of 1a , 1b with supercoiled pBR322 DNA was further examined by gel electrophoresis, which reveals that complex 1a exhibits a remarkable DNA cleavage activity (concentration dependent) with pBR322DNA, and the cleavage activity of both enantiomers of complex 1 was significantly enhanced in the presence of activators. The activating efficiency follows the order Asc > H₂O₂ > MPA for 1a , and reverse order was observed for 1b , because of the differences in enantioselectivity and conformation. Further, it was observed that cleavage reaction involves singlet oxygen species and superoxide radicals via oxidative cleavage mechanism. In addition, complex 1a exhibits significant inhibitory effects on the topoisomerase II (topo II) activity at a very low concentration ∼24 μM, which suggest that complex 1a is indeed catalytic inhibitor or (poison) of human topo II. Chirality 2011 © 2011 Wiley‐Liss, Inc.     

Structural investigation of CDCA3‐Cdh1 protein–protein interactions using in vitro studies and molecular dynamics simulation

The anaphase‐promoting complex/cyclosome (APC/C) ubiquitin ligase and its cofactor, Cdh1, regulate the expression of several cell‐cycle proteins and their functions during mitosis. Levels of the protein cell division cycle‐associated protein 3 (CDCA3), which is functionally required for mitotic entry, are regulated by APC/C^Cdh1. CDCA3 is an intrinsically disordered protein and contains both C‐terminal KEN box and D‐box recognition motifs, enabling binding to Cdh1. Our previous findings demonstrate that CDCA3 has a phosphorylation‐dependent non‐canonical ABBA‐like motif within the linker region bridging these two recognition motifs and is required for efficient binding to Cdh1. Here, we sought to identify and further characterize additional residues that participate within this ABBA‐like motif using detailed in vitro experiments and in silico modeling studies. We identified the role of H‐bonds, hydrophobic and ionic interactions across the CDCA3 ABBA‐like motif in the linker region between KEN and D‐box motifs. This linker region adopts a well‐defined structure when bound to Cdh1 in the presence of phosphorylation. Upon alanine mutation, the structure of this region is lost, leading to higher flexibility, and alteration in affinities due to binding to alternate sites on Cdh1. Our findings identify roles for the anchoring residues in the non‐canonical ABBA‐like motif to promote binding to the APC/C^Cdh1 and regulation of CDCA3 protein levels.     

Genetic Variation within the Mx Gene of Commercially Selected Chicken Lines Reveals Multiple Haplotypes,Recombination and a Protein under Selection Pressure

The Mx protein is one of the best-characterized interferon-stimulated antiviral mediators. Mx homologs have been identified in most vertebrates examined; however, their location within the cell, their level of activity, and the viruses they inhibit vary widely. Recent studies have demonstrated multiple Mx alleles in chickens and some reports have suggested a specific variant (S631N) within exon 14 confers antiviral activity. In the current study, the complete genome of nine elite egg-layer type lines were sequenced and multiple variants of the Mx gene identified. Within the coding region and upstream putative promoter region 36 SNP variants were identified, producing a total of 12 unique haplotypes. Each elite line contained from one to four haplotypes, with many of these haplotypes being found in only one line. Observation of changes in haplotype frequency over generations, as well as recombination, suggested some unknown selection pressure on the Mx gene. Trait association analysis with either individual SNP or haplotypes showed a significant effect of Mx haplotype on several egg production related traits, and on mortality following Marek''s disease virus challenge in some lines. Examination of the location of the various SNP within the protein suggests synonymous SNP tend to be found within structural or enzymatic regions of the protein, while non-synonymous SNP are located in less well defined regions. The putative resistance variant N631 was found in five of the 12 haplotypes with an overall frequency of 47% across the nine lines. Two Mx recombinants were identified within the elite populations, indicating that novel variation can arise and be maintained within intensively selected lines. Collectively, these results suggest the conflicting reports in the literature describing the impact of the different SNP on chicken Mx function may be due to the varying context of haplotypes present in the populations studied.     

Template synthesis of novel carboxamide dinuclear copper (II) complex: spectral characterization and reactivity towards calf-thymus DNA

Dinuclear complexes Bis [aqua 1,8-(1,2-dicarboxamido benzene) 3,6-diazaoctane copper (II)/nickel (II)] tetrachloride (1 and 2) were synthesized by a two component one-pot metal template condensation between phthalic anhydride and 1,8-diamino 3,6-diazaoctane. Elemental analysis, molar conductance measurements, electronic absorption, infra-red, electron paramagnetic resonance, nuclear magnetic resonance, atomic absorption, and electron spray mass spectral studies have been performed to probe the nature and structure of the complexes. The interaction of copper (II) complex with calf thymus (CT-DNA) has been studied by using absorption, emission and circular dichoric spectral methods, viscometry, and cyclic voltammetry. A strong hyperchromism along with a red shift in UV bands and hypochromism in the ligand field band of the complex 1 on interaction with CT-DNA imply a covalent mode of DNA binding. This is further confirmed by studying the reactivity of complex 1 using circular dichroism and viscosity measurements. The variation in relative emission intensity of DNA-bound ethidium bromide observed upon treatment with the complex 1 parallel the trend of DNA binding studies. Cyclic voltammetry studies reveal that the complex 1 prefers to bind to DNA in Cu(II) rather than Cu(I) oxidation state.