Mitochondrial DNA variations in ova and blastocyst: Implications in assisted reproduction

Mitochondrial DNA (mtDNA) of oocyte is critical for its function, embryo quality and development. Analysis of complete mtDNA of 49 oocytes and 18 blastocysts from 67 females opting for IVF revealed 437 nucleotide variations. 40.29% samples had either disease associated or non-synonymous novel or pathogenic mutation in evolutionarily conserved regions. Samples with disease associated mtDNA mutations had low fertilization rate and poor embryo quality, however no difference in implantation or clinical pregnancy rate was observed. Screening mtDNA from oocyte/blastocyst is a simple, clinically reliable method for diagnostic evaluation of female infertility and may reduce risk of mtDNA disease transmission.     

Ligand‐based pharmacophore modelling and screening of DNA minor groove binders targeting Staphylococcus aureus

The recognition of DNA by small molecules is of special importance in the design of new drugs. Many natural and synthetic compounds have the ability to interact with the minor groove of DNA. In the present study, identification of minor groove binding compounds was attained by the combined approach of pharmacophore modelling, virtual screening and molecular dynamics approach. Experimentally reported 32 minor groove binding compounds were used to develop the pharmacophore model. Based on the fitness score, best three pharmacophore hypotheses were selected and used as template for screening the compounds from drug bank database. This pharmacophore‐based screening provides many compounds with the same pharmacological properties. All these compounds were subjected to four phases of docking protocols with combined Glide‐quantum‐polarized ligand docking approach. Molecular dynamics results indicated that selected compounds are more active and showed good interaction in the binding site of DNA. Based on the scoring parameters and energy values, the best compounds were selected, and antibacterial activity of these compounds was identified using in vitro antimicrobial techniques. Copyright © 2014 John Wiley & Sons, Ltd.     

Reconstitution of TGF-β sensitivity in the VACO-411 human colon carcinoma line by somatic cell fusion with MCF-7

We characterized the mechanism of transforming growth factor beta (TGF-beta) resistance in the VACO-411 human colon carcinoma line. VACO-411 is unique for several reasons, including having a novel mutator phenotype and wild-type p53. Like many colon tumors, VACO-411 is not growth inhibited by TGF-beta. However, VACO-411 represents a subset of colon tumors that are resistant to TGF-beta-mediated growth inhibition, despite the expression of functional TGF-beta receptors. VACO-411 expresses cell surface TGF-beta receptor types I and II, and the coding regions of these receptors are wild type. To further characterize the nature of the VACO-411 defect, we fused VACO-411 with the human breast carcinoma line MCF-7. MCF-7 is also resistant to TGF-beta-mediated growth inhibition. However, unlike VACO-411, MCF-7 lacks cell surface expression of TGF-beta receptor type II, but does contain an intact postreceptor signaling pathway, as shown by regeneration of TGF-beta sensitivity following wild-type TGF-beta receptor type II transfection. In contrast to parental VACO-411 and MCF-7, the morphologically distinct cell hybrids were growth inhibited by TGF-beta. Therefore, the TGF-beta defect in VACO-411 is a postreceptor, loss-of-function mutation which can be genetically complemented. The data suggest that the VACO-411 defect in TGF-beta signaling will be able to be further complemented by microcell-mediated chromosome transfer.     

Molecular docking analysis of glycogen phosphorylase with inhibitors from Cissampelos pareira Linn.

Cissampelos pareira Linn. is a climbing herb known in Indian traditional medicine as laghupatha. It belongs to the Menispermaceae family. The enzyme glycogen phosphorylase (GP) is a promising target for the treatment of type-2 diabetes (T2DM). A variety of natural product inhibitors with both pharmaceutical and nutraceutical potential have been reported in the search for powerful, selective and drug-like GP inhibitors that could lead to hypoglycemic medicines. Therefore, it is of interest to document the molecular docking analysis data of glycogen phosphorylase with compounds from Cissampelos pareira Linn. We report the optimal binding features of 4 compounds namely Trans-N-feruloyltyramine, Coclaurine, Magnoflorine, and Curine with the target protein for further consideration in the context of T2DM.     

Enhancement of glucocorticoid receptor-mediated gene expression by constitutively active heat shock factor 1

To further define the role of heat shock factor 1 (HSF1) in the stress potentiation of glucocorticoid receptor (GR) activity, we placed a constitutively active mutant of human HSF1 (hHSF1-E189) under the control of a doxycycline (DOX)-inducible vector. In mouse L929 cells, DOX-induced expression of hHSF1-E189 correlated with in vivo occupancy of the human heat shock protein 70 (hHsp70) promoter (chromatin-immunoprecipitation assay) and with increased activity under nonstress conditions at the hHsp70 promoter controlling expression of chloramphenicol acetyl transferase (CAT) (p2500-CAT). Comparison of hHSF1-E189 against stress-activated, endogenous HSF1 for DNA-binding, p2500-CAT, and Hsp70 protein expression activities showed the mutant factor to have lower, but clearly detectable, activities as compared with wild-type factor. Thus, the hHSF1-E189 mutant is capable of replicating these key functions of endogenous HSF1, albeit at reduced levels. To assess the involvement of hHSF1-E189 in GR activity, DOX-induced expression of hHSF1-E189 was performed in L929 cells expressing the minimal pGRE(2)E1B-CAT reporter. hHSF1-E189 protein expression in these cells was maximal at 24 h of DOX and remained constant up to 72 h. hHSF1-E189 expressed under these conditions was found both in the cytosolic and nuclear compartments, in a state capable of binding DNA. More importantly, GR activity at the pGRE(2)E1B-CAT promoter was found to increase after DOX-induced expression of hHSF1-E189. The potentiation of GR by hHSF1-E189 occurred at saturating concentrations of hormone and was dependent on at least 48 h of hHSF1-E189 up-regulation, suggesting that time was needed for an HSF1-induced factor to accumulate to a threshold level. Initial efforts to characterize how hHSF1-E189 controls GR signaling showed that it does not occur through alterations of GR protein levels or changes in GR hormone binding capacity. In summary, our observations provide the first molecular evidence for the existence of HSF1-regulated genes that serve to elevate the response of steroid receptors under stress conditions.     

Mitochondrial DNA variations in Madras motor neuron disease

Although the Madras motor neuron disease (MMND) was found three decades ago, its genetic basis has not been elucidated, so far. The symptom at onset was impaired hearing, upper limb weakness and atrophy. Since some clinical features of MMND overlap with mitochondrial disorders, we analyzed the complete mitochondrial genome of 45 MMND patients and found 396 variations, including 13 disease-associated, 2 mt-tRNA and 33 non-synonymous (16 MT-ND, 10 MT-CO, 3 MT-CYB and 4 MT-ATPase). A rare variant (m.8302A>G) in mt-tRNA^Leu was found in three patients. We predict that these variation(s) may influence the disease pathogenesis along with some unknown factor(s).     

A mitochondrial DNA variant 10398G>A in breast cancer among South Indians: An original study with meta-analysis

The m.10398G > A polymorphism in the MT-ND3 gene has been linked to the manifestation of several neurodegenerative disorders and cancers. Several research groups have analyzed the association between m.10398G > A polymorphism and breast cancer; however, the results do not follow a consensus. We have studied this polymorphism in three Dravidian populations from South India. Analysis on 716 cases and 724 controls found no association between m.10398G > A polymorphism and breast cancer [OR = 0.916 (0.743–1.128); P = 0.409]. Menopausal stratification also revealed no significant association in either pre-menopausal or post-menopausal breast cancer groups. In addition, we undertook a meta-analysis on 16 study groups, comprising a total of 7202 cases and 7490 controls. The pooled odds ratio suggested no significant association of m.10398G > A substitution with breast cancer [OR = 1.016 (0.85–1.22); P = 0.86]. In conclusion, there is no evidence of association between m.10398G > A polymorphism and breast cancer risk among South Indian women. Meta-analysis suggested no overall correlation between this polymorphism and breast cancer risk.