Antioxidant and oxidative stress parameters in brain of Heteropneustes fossilis under air exposure condition; role of mitochondrial electron transport chain

Many fishes are exposed to air in their natural habitat or during their commercial handling. In natural habitat or during commercial handling, the cat fish Heteropneustes fossilis is exposed to air for > 24 h. Data on its oxidative metabolism in the above condition are not available. Oxidative stress (OS) indices (lipid and protein oxidation), toxic reactive oxygen species (ROS: H2O2) generation, antioxidative status (levels of superoxide dismutase, catalase, glutathione peroxidase and reductase, ascorbic acid and nonprotein sulfhydryl) and activities of electron transport chain (ETC) enzymes (complex I-IV) were investigated in brain tissue of H. fossilis under air exposure condition (0, 3, 6, 12 and 18 h at 25 degrees C). Decreased activities of antioxidant (except catalase) and ETC enzymes (except complex II) with increased H2O2 and OS levels were observed in the tissue under water deprivation condition. Positive correlation was observed for complex II activity and non-protein thiol groups with time period of air exposure. The critical time period to induce OS and to reduce most of the studied antioxidant level in brain was found to be 3-6 h air exposure. The data can be useful to minimize the stress generated during commercial handling of the live fishes those exposed to air in general and H. fossilis in particular. (C) 2013 Elsevier Inc. All rights reserved.

Aza-induced cardiomyocyte differentiation of P19 EC-cells by epigenetic co-regulation and ERK signaling

Stem cells in cell based therapy for cardiac injury is being potentially considered. However, genetic regulatory networks involved in cardiac differentiation are not clearly understood. Among stem cell differentiation models, mouse P19 embryonic carcinoma (EC) cells, are employed for studying (epi)genetic regulation of cardiomyocyte differentiation. Here, we comprehensively assessed cardiogenic differentiation potential of 5-azacytidine (Aza) on P19 EC-cells, associated gene expression profiles and the changes in DNA methylation, histone acetylation and activated-ERK signaling status during differentiation. Initial exposure of Aza to cultured EC-cells leads to an efficient (55%) differentiation to cardiomyocyte-rich embryoid bodies with a threefold (16.8%) increase in the cTnI(+) cardiomyocytes. Expression levels of cardiac-specific gene markers i.e., Isl-1, BMP-2, GATA-4, and alpha-MHC were up-regulated following Aza induction, accompanied by differential changes in their methylation status particularly that of BMP-2 and alpha-MHC. Additionally, increases in the levels of acetylated-H3 and pERK were observed during Aza-induced cardiac differentiation. These studies demonstrate that Aza is a potent cardiac inducer when treated during the initial phase of differentiation of mouse P19 EC-cells and its effect is brought about epigenetically and co-ordinatedly by hypo-methylation and histone acetylation-mediated hyper-expression of cardiogenesis-associated genes and involving activation of ERK signaling.

Overexpression of the Rv0805 phosphodiesterase elicits a cAMP-independent transcriptional response

The Rv0805 gene in Mycobacterium tuberculosis encodes a metallophosphoesterase which shows cAMP-hydrolytic activity. Overexpression of Rv0805 has been used as a tool to lower intracellular cAMP levels and thereby elucidate the roles of cAMP in mycobacteria. Here we show that levels of cAMP in M. tuberculosis were lowered by only similar to 30% following overexpression of Rv0805, and transcript levels of a number of genes, which include those associated with virulence and the methyl citrate cycle, were altered. The genes that showed altered expression were distinct from those differentially regulated in a strain deleted for the cAMP-receptor protein (CRPMt), consistent with the relatively low dependence on cAMP of CRPMt binding to DNA. Using mutants of Rv0805 we show that the transcriptional signature of Rv0805 overexpression is a combination of catalysis-dependent and independent effects, and that the structurally flexible C-terminus of Rv0805 is crucial for the catalysis-independent effects of the protein. Our study demonstrates the dissociation of Rv0805 and cAMP-regulated gene expression, and reveals alternate functions for this phosphodiesterase from M. tuberculosis.

MPK-09, a small molecule inspired from bioactive styryllactone restores the wild-type function of mutant p53

In the search for more efficacious and less toxic cancer drugs, the tumor suppressor p53 protein has long been a desirable therapeutic target. In the recent past, few independent studies have demonstrated that the antitumor activity of wild-type p53 can be restored in cancer cells harboring mutant form of p53 using small molecule activators. In this study, we describe a novel small molecule MPK-09, which is selective and highly potent against allele specific p53 mutations mainly, R175H, R249S, R273H, R273C, and E285K. Except E285K, all other mutations tested are among the six ``hot spot'' p53 mutations reported in majority of human cancer. Furthermore, our study conclusively demonstrates that the apoptotic activity of the small molecule MPK-09 against cancer cells harboring R273C and E285K mutations is due to restoration of the wild-type conformation to the corresponding mutant form of p53.

Exploration of deleterious single nucleotide polymorphisms in the components of human P bodies: An in silico approach

P bodies are 100-300 nm sized organelles involved in mRNA silencing and degradation. A total of 60 human proteins have been reported to localize to P bodies. Several human SNPs contribute to complex diseases by altering the structure and function of the proteins. Also, SNPs alter various transcription factors binding, splicing and miRNA regulatory sites. Owing to the essential functions of P bodies in mRNA regulation, we explored computationally the functional significance of SNPs in 7 P body components such as XRN1, DCP2, EDC3, CPEB1, GEMIN5, STAU1 and TRIM71. Computational analyses of non-synonymous SNPs of these components was initiated using well utilized publicly available software programs such as the SIFT, followed by PolyPhen, PANTHER, MutPred, I-Mutant-2.0 and PhosSNP 1.0. Functional significance of noncoding SNPs in the regulatory regions were analysed using FastSNP. Utilizing miRSNP database, we explored the role of SNPs in the context that alters the miRNA binding sites in the above mentioned genes. Our in silico studies have identified various deleterious SNPs and this cataloguing is essential and gives first hand information for further analysis by in vitro and in vivo methods for a better understanding of maintenance, assembly and functional aspects of P bodies in both health and disease. (C) 2013 Elsevier B.V. All rights reserved.

Gene Transfection in High Serum Levels: Case Studies with New Cholesterol Based Cationic Gemini Lipids

Background: Six new cationic gemini lipids based on cholesterol possessing different positional combinations of hydroxyethyl (-CH2CH2OH) and oligo-oxyethylene -(CH2CH2O)(n)- moieties were synthesized. For comparison the corresponding monomeric lipid was also prepared. Each new cationic lipid was found to form stable, clear suspensions in aqueous media. Methodology/Principal Findings: To understand the nature of the individual lipid aggregates, we have studied the aggregation properties using transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurements and X-ray diffraction (XRD). We studied the lipid/DNA complex (lipoplex) formation and the release of the DNA from such lipoplexes using ethidium bromide. These gemini lipids in presence of a helper lipid, 1, 2-dioleoyl phophatidyl ethanol amine (DOPE) showed significant enhancements in the gene transfection compared to several commercially available transfection agents. Cholesterol based gemini having -CH2-CH2-OH groups at the head and one oxyethylene spacer was found to be the most effective lipid, which showed transfection activity even in presence of high serum levels (50%) greater than Effectene, one of the potent commercially available transfecting agents. Most of these geminis protected plasmid DNA remarkably against DNase I in serum, although the degree of stability was found to vary with their structural features. Conclusions/Significance: -OH groups present on the cationic headgroups in combination with oxyethylene linkers on cholesterol based geminis, gave an optimized combination of new genera of gemini lipids possessing high transfection efficiency even in presence of very high percentage of serum. This property makes them preferential transfection reagents for possible in vivo studies.

Classification of Myopathies on Molecular basis in Drosophila using Raman spectroscopy

Myopathies are muscular diseases in which muscle fibers degenerate due to many factors such as nutrient deficiency, infection and mutations in myofibrillar etc. The objective of this study is to identify the bio-markers to distinguish various muscle mutants in Drosophila (fruit fly) using Raman Spectroscopy. Principal Components based Linear Discriminant Analysis (PC-LDA) classification model yielding >95% accuracy was developed to classify such different mutants representing various myopathies according to their physiopathology.

Effect of myonuclear number and mitochondrial fusion on Drosophila indirect flight muscle organization and size

Mechanisms involved in establishing the organization and numbers of fibres in a muscle are not completely understood. During Drosophila indirect flight muscle (IFM) formation, muscle growth is achieved by both incorporating hundreds of nuclei, and hypertrophy. As a result, IFMs provide a good model with which to understand the mechanisms that govern overall muscle organization and growth. We present a detailed analysis of the organization of dorsal longitudinal muscles (DLMs), a subset of the IFMs. We show that each DLM is similar to a vertebrate fascicle and consists of multiple muscle fibres. However, increased fascicle size does not necessarily change the number of constituent fibres, but does increase the number of myofibrils packed within the fibres. We also find that altering the number of myoblasts available for fusion changes DLM fascicle size and fibres are loosely packed with myofibrils. Additionally, we show that knock down of genes required for mitochondrial fusion causes a severe reduction in the size of DLM fascicles and fibres. Our results establish the organization levels of DLMs and highlight the importance of the appropriate number of nuclei and mitochondrial fusion in determining the overall organization, growth and size of DLMs. (C) 2013 Elsevier Inc. All rights reserved.

Cyclic AMP in Mycobacteria: the second messenger comes first

Cyclic AMP (cAMP) has emerged as a pivotal molecule for signalling in all life forms. Mycobacterial genomes have been found to encode for numerous proteins that are involved in cAMP generation, degradation and utilization. Many of these proteins have domain organizations unique to mycobacteria. This review summarizes recent advances in mechanisms of cAMP synthesis and degradation, focusing on the processes by which cAMP modulates mycobacterial signalling. We explore its impact on the physiology of the organism and on the discourse between M. tuberculosis and its host.

Effects of a Delocalizable Cation on the Headgroup of Gemini Lipids on the Lipoplex-Type Nanoaggregates Directly Formed from Plasmid DNA

Lipoplex-type nanoaggregates prepared from pEGFP-C3 plasmid DNA (pDNA) and mixed liposomes, with a gemini cationic lipid (CL) 1,2-bis(hexadecyl imidazolium) alkanes], referred as (C(16)Im)(2)C-n (where C-n is the alkane spacer length, n = 2, 3, 5, or 12, between the imidazolium heads) and DOPE zwitterionic lipid, have been analyzed by zeta potential, gel electrophoresis, SAXS, cryo-TEM, fluorescence anisotropy, transfection efficiency, fluorescence confocal microscopy, and cell viability/cytotoxicity experiments to establish a structure-biological activity relationship. The study, carried out at several mixed liposome compositions, alpha, and effective charge ratios, rho(eff), of the lipoplex, demonstrates that the transfection of pDNA using CLs initially requires the determination of the effective charge of both. The electrochemical study confirms that CLs with a delocalizable positive charge in their headgroups yield an effective positive charge that is 90% of their expected nominal one, while pDNA is compacted yielding an effective negative charge which is only 10-25% than that of the linear DNA. SAXS diffractograms show that lipoplexes formed by CLs with shorter spacer (n = 2, 3, or 5) present three lamellar structures, two of them in coexistence, while those formed by CL with longest spacer (n = 12) present two additional inverted hexagonal structures. Cryo-TEM micrographs show nanoaggregates with two multilamellar structures, a cluster-type (at low alpha value) and a fingerprint-type, that coexist with the cluster-type at moderate alpha composition. The optimized transfection efficiency (TE) of pDNA, in HEK293T, HeLa, and H1299 cells was higher using lipoplexes containing gemini CLs with shorter spacers at low a value. Each lipid formulation did not show any significant levels of toxicity, the reported lipoplexes being adequate DNA vectors for gene therapy and considerably better than both Lipofectamine 2000 and CLs of the 1,2-bis(hexadecyl ammnoniun) alkane series, recently reported.

Classification of Myopathies on Molecular basis in Drosophila using Raman spectroscopy

Myopathies are muscular diseases in which muscle fibers degenerate due to many factors such as nutrient deficiency, infection and mutations in myofibrillar etc. The objective of this study is to identify the bio-markers to distinguish various muscle mutants in Drosophila (fruit fly) using Raman Spectroscopy. Principal Components based Linear Discriminant Analysis (PC-LDA) classification model yielding >95% accuracy was developed to classify such different mutants representing various myopathies according to their physiopathology.

Expression and function of cyclooxygenase-2 is necessary for hamster blastocyst hatching

Blastocyst hatching is critical for successful implantation leading to pregnancy. Its failure causes infertility. The phenomenon of blastocyst hatching in humans is poorly understood and the available information on this stems from studies of rodents such as mice and hamsters. We and others showed that hamster blastocyst hatching is characterized by firstly blastocyst deflation followed by a dissolution of the zona pellucida (zona) and accompanied by trophectodermal projections (TEPs). We also showed that embryo-derived cathepsins (Cat) proteases, specifically Cat-L, -B and -P act as zonalysins and are responsible for hatching. In this study, we show the expression and function of one of the potential regulators of embryogenesis, cyclooxygenase (COX)-2 during blastocyst development and hatching. The expression of COX-2 mRNA and protein was observed in 8-cell through hatched blastocyst stages and it was also localized to blastocysts TEPs. Specific COX-2 inhibitors, NS-398 and CAY-10404, inhibited blastocyst hatching; percentages achieved were only 28.4 5.3 and 32.3 5.4, respectively, compared with 90 with untreated embryos. Interestingly, inhibitor-treated blastocysts failed to deflate, normally observed during hatching. Supplementation of prostaglandins (PGs)-E-2 or -I-2 to cultured embryos reversed the inhibitors effect on hatching and also the deflation behavior. Importantly, the levels of mRNA and protein of Cat-L, -B and -P showed a significant reduction in the inhibitor-treated embryos compared with untreated embryos, although its mechanism remains to be examined. These data provide the first evidence that COX-2 is critical for blastocyst hatching in the golden hamster.

Remarkable anticancer activity of ferrocenylterpyridine platinum(II) complexes in visible light with low dark toxicity

Ferrocenyl platinum(II) complexes (1-3), viz. Pt(Fc-tpy)Cl]Cl (1), Pt(Fc-tpy)(NPC)]Cl (2, HNPC = N-propargyl carbazole) and Pt(Fc-bpa)Cl]Cl (3), were prepared, characterized and their anti-proliferative properties in visible light in human keratinocyte (HaCaT) cell lines have been studied. Pt(Ph-tpy)Cl]Cl (4) was prepared and used as a control. Complexes 1 and 3, structurally characterized by X-ray crystallography, show distorted square-planar geometry for the platinum(II) centre. Complexes 1 and 2 having the Fc-tpy ligand showed an intense absorption band at similar to 590 nm. The ferrocenyl complexes are redox active showing the Fc(+)-Fc couple near 0.6 V vs. SCE in DMF-0.1 M tetrabutylammonium perchlorate (TBAP). Complexes 1-3 showed external binding to calf thymus DNA. Both 1 and 2 showed remarkable photocytotoxicity in HaCaT cell lines giving respective IC50 values of 9.8 and 12.0 mu M in visible light of 400-700 nm with low dark toxicity (IC50 > 60 mu M). Fluorescent imaging studies showed the spread of the complexes throughout the cell localising both in cytoplasm and the nucleus. The ferrocenyl complexes triggered apoptosis on light exposure as evidenced from the Annexin V-FITC/PI and DNA ladder formation assays. Spectral studies revealed the formation of ferrocenium ions upon photo-irradiation generating cytotoxic hydroxyl radicals via a Fenton type mechanism. The results are rationalized from a TDDFT study that shows involvement of ferrocene and the platinum coordinated terpyridine moiety as respective HOMO and LUMO.

Getting a kick out of it: multimodal signalling during male–male encounters in the foot-flagging frog Micrixalus aff. saxicola from the Western Ghats of India

Several anuran species use multimodal signals to communicate in diverse social contexts. Our study describes acoustic and visual behaviours of the Small Torrent Frog (Micrixalus aff. saxicola), a diurnal frog endemic to the Western Ghats of India. During agonistic interactions males display advertisement calls, foot-flagging and tapping (foot lifting) behaviours to signal the readiness to defend perching sites in perennial streams. Results from a quantitative video analysis of male–male interactions indicate that footflagging displays were used as directional signals toward the opponent male, but were less abundant than calls. The acoustic and visual signals were not functionally linked. The call of Micrixalus aff. saxicola thereby did not act as an alert signal. Analysis of behavioural transitions revealed that kicking behaviours (physical attacks) significantly elicited kicks from interacting males. We suggest that foot-flagging displays ritualized from this frequently observed fighting technique to reduce physical attacks.

Mitochondria-Targeting Oxidovanadium(IV) Complex as a Near-IR Light Photocytotoxic Agent

Oxidovanadium(IV) complexes VO(L-1)(phen)]Cl (1) and VO(L-2)(L-3)]Cl (2), in which HL1 is 2-{(benzimidazol-2-yl)methylimino]-methyl}phenol (sal-ambmz), HL2 is 2-({1-(anthracen-9-yl)methyl]-benzimidazol-2-yl}methylimino)-met hyl]phenol (sal-an-ambmz), phen is 1,10-phenanthroline and L-3 is dipyrido3,2-a:2,3-c]phenazine (dppz) conjugated to a Gly-Gly-OMe dipeptide moiety, were prepared, characterized, and their DNA binding, photoinduced DNA-cleavage, and photocytotoxic properties were studied. Fluorescence microscopy studies were performed by using complex 2 in HeLa and HaCaT cells. Complex 1, structurally characterized by X-ray crystallography, has a vanadyl group in VO2N4 core with the VO2+ moiety bonded to N,N-donor phen and a N,N,O-donor Schiff base. Complex 2, having an anthracenyl fluorophore, showed fluorescence emission bands at 397, 419, and 443nm. The complexes are redox-active exhibiting the V(IV)/V(III) redox couple near -0.85V versus SCE in DMF 0.1M tetrabutylammonium perchlorate (TBAP). Complex 2, having a dipeptide moiety, showed specific binding towards poly(dAdT)(2) sequence. The dppz-Gly-Gly-OMe complex showed significant DNA photocleavage activity in red light of 705nm through a hydroxyl radical ((OH)-O-.) pathway. Complex 2 showed photocytotoxicity in HaCaT and HeLa cells in visible light (400-700nm) and red light (620-700nm), however, the complex was less toxic in the dark. Fluorescence microscopy revealed the localization of complex 2 primarily in mitochondria. Apoptosis was found to occur inside mitochondria (intrinsic pathway) caused by ROS generation.