Synthesis, characterization and DNA-binding properties of rac-[Ru(5,6-dmp)2(dppz)]2+--enantiopreferential DNA binding and co-ligand promoted exciton coupling

The new mixed ligand complex [Ru(5,6-dmp)2(dppz)]Cl2 [5,6-dmp = 5,6-dimethyl-1,10-phenanthroline, dppz = dipyrido[3,2-a:2',3'-c]phenazine] has been isolated and its DNA-binding properties studied by employing UV-visible (UV-Vis), steady-state and time-resolved emission and circular dichroism spectral methods, viscometry, thermal denaturation and cyclic/differential pulse voltammetric techniques. The complex acts as a 'molecular light-switch' on binding to DNA, but the enhancement in emission intensity is only 75% of that of the parent complex [Ru(phen)2(dppz)]2+ (phen = 1,10-phenanthroline). The emission decay curves and quenching studies suggest two different DNA-binding modes both involving intercalation of the dppz ligand of [Ru(5,6-dmp)2(dppz)]Cl2. The characteristic red-shift of the induced CD signal, which is not observed for the phen analogue, arises from exciton coupling. The hydrophobicity and polarizability of 5,6-dmp co-ligand strongly favour the formation of a stable structural and electronic scaffold on the DNA surface for the unbound molecules to couple with the DNA-bound complexes facilitating spontaneous assembly of novel extended molecular aggregates using DNA as a helical nanotemplate. This observation is consistent with the shift in Ru(II)/Ru(III) redox potential to more positive values with a dramatic drop in peak current on binding of the 5,6-dmp complex to calf thymus (CT) DNA. Equilibrium dialysis experiments monitored by CD spectroscopy unambiguously reveal the preferential binding of the delta-enantiomer to the right-handed calf thymus (CT) DNA. The 5,6-dmp complex exhibits preferential binding to [d(AT)6]2 over [d(GC)6]2 and the complex aggregates formed consist of six [Ru(5,6-dmp)2(dppz)]2+ cations per base pair of [d(AT)6]2; however, only one [Ru(phen)2(dppz)]2+ cation per base pair is involved in DNA binding.     

Structures, spectra, and DNA-binding properties of mixed ligand copper(II) complexes of iminodiacetic acid: the novel role of diimine co-ligands on DNA conformation and hydrolytic and oxidative double strand DNA cleavage

The coordination geometry around copper(II) in [Cu(imda)(phen)(H2O)] (1) (H2imda = iminodiacetic acid, phen = 1,10-phenanthroline) is described as distorted octahedral while those in [Cu(imda)(5,6-dmp)] (2) (5,6-dmp = 5,6-dimethyl-1,10-phenanthroline) and [Cu(imda)(dpq)] (3) (dpq = dipyrido-[3,2-d:2',3'-f]-quinoxaline) as trigonal bipyramidal distorted square-based pyramidal with the imda anion facially coordinated to copper(II). Absorption spectral (Kb: 1, 0.60+/-0.04x10(3); 2, 3.9+/-0.3x10(3); 3, 1.7+/-0.5x10(4) M(-1)) and thermal denaturation studies (deltaTm: 1, 5.70+/-0.05; 2, 5.5+/-10; 3, 10.6+/-10 degrees C) and viscosity measurements indicate that 3 interacts with calf thymus DNA more strongly than 1 and 2. The relative viscosities of DNA bound to 1 and 3 increase while that of DNA bound to 2 decreases indicating formation of kinks or bends and/or conversion of B to A conformation as revealed by the decrease in intensity of the helicity band in the circular dichroism spectrum of DNA. While 1 and 3 are bound to DNA through partial intercalation, respectively, of phen ring and the extended planar ring of dpq with DNA base stack, the complex 2 is involved in groove binding. All the complexes show cleavage of pBR322 supercoiled DNA in the presence of ascorbic acid with the cleavage efficiency varying in the order 3 > 1 > 2. The highest oxidative DNA cleavage of dpq complex is ascribed to its highest Cu(II)/Cu(I) redox potential. Oxidative cleavage studies using distamycin reveal minor groove binding for the dpq complex but a major groove binding for the phen and 5,6-dmp complexes. Also, all the complexes show hydrolytic DNA cleavage activity in the absence of light or a reducing agent with cleavage efficiency varying in the order 1 > 3 > 2.     

Curative effect of Semecarpus anacardium Linn. nut milk extract against adjuvant arthritis -- with special reference to bone metabolism

Localised bone loss in the form of bone erosions and peri-articular osteopenia constitutes an important criteria for the diagnosis of rheumatoid arthritis. In the present study, the effect of Semecarpus anacardium Linn. nut milk extract (SA) on the metabolism of bone turn over has been studied by analyzing various markers of bone turnover and by histological and radiological analysis of the joints in adjuvant arthritis in rats. Arthritis was induced in rats by injecting Freund's complete adjuvant containing 10mg of heat killed mycobacterium tuberculosis in 1 ml paraffin oil (0.1 ml) into the left hind paw of the rat intradermally. After 14 days of induction, SA (150 mg/kg body weight/day) was administered orally by gastric intubations for 14 days. SA significantly reverted the alterations in the bone turnover observed in arthritic animals by modulating the levels of calcium, phosphorus and the activities of the enzymes names tartrate resistant acid phosphatase, acid phosphatase and alkaline phosphatase. The drug increased the bone weights that were found to be decreased during arthritis. Protective effect of SA was also observed by the decrease in the levels and expression of tumour necrosis factor alpha (TNF-alpha) as well as the histopathological and radiological observations. From all these observations it can be concluded that SA possesses strong anti-arthritic property by regulating bone turnover.     

A novel pathway involving progesterone receptor, endothelin-2, and endothelin receptor B controls ovulation in mice

The steroid hormone progesterone (P) plays a pivotal role during ovulation. Mice lacking P receptor (Pgr) gene fail to ovulate due to a defect in follicular rupture. The P receptor (PGR)-regulated pathways that modulate ovulation, however, remain poorly understood. To identify these pathways, we performed gene expression profiling using ovaries from mice subjected to gonadotropin-induced superovulation in the presence and in the absence of CDB-2914, a synthetic PGR antagonist. Prominent among the genes that were down-regulated in response to CDB-2914 was endothelin (ET)-2, a potent vasoactive molecule. ET-2 mRNA was transiently induced in mural granulosa cells of the preovulatory follicles immediately preceding ovulation. This induction was absent in the ovaries of PGR null mice, indicating a critical role of this receptor in ET-2 expression. To investigate the functional role of ET-2 during ovulation, we employed selective antagonists of endothelin receptors, ETR-A and ETR-B. Mice treated with an ETR-B antagonist exhibited a dramatic (>85%) decline in the number of released oocytes. Strong expression of ETR-B was observed in the mural and cumulus granulosa cells of the preovulatory follicles as well as in the capillaries lining the inner border of the theca interna. We also identified cGMP-dependent protein kinase II, a previously reported PGR-regulated gene, as a downstream target of ET-2 during ovulation. Collectively, our studies uncovered a unique pathway in which ET-2, produced by PGR in mural granulosa cells, acts in a paracrine or autocrine manner on multiple cell types within the preovulatory follicle to control the final events leading to its rupture.     

Pin1 Promotes Transforming Growth Factor-��-induced Migration and Invasion

Transforming growth factor-β (TGF-β) regulates a wide variety of biological activities. It induces potent growth-inhibitory responses in normal cells but promotes migration and invasion of cancer cells. Smads mediate the TGF-β responses. TGF-β binding to the cell surface receptors leads to the phosphorylation of Smad2/3 in their C terminus as well as in the proline-rich linker region. The serine/threonine phosphorylation sites in the linker region are followed by the proline residue. Pin1, a peptidyl-prolyl cis/trans isomerase, recognizes phosphorylated serine/threonine-proline motifs. Here we show that Smad2/3 interacts with Pin1 in a TGF-β-dependent manner. We further show that the phosphorylated threonine 179-proline motif in the Smad3 linker region is the major binding site for Pin1. Although epidermal growth factor also induces phosphorylation of threonine 179 and other residues in the Smad3 linker region the same as TGF-β, Pin1 is unable to bind to the epidermal growth factor-stimulated Smad3. Further analysis suggests that phosphorylation of Smad3 in the C terminus is necessary for the interaction with Pin1. Depletion of Pin1 by small hairpin RNA does not significantly affect TGF-β-induced growth-inhibitory responses and a number of TGF-β/Smad target genes analyzed. In contrast, knockdown of Pin1 in human PC3 prostate cancer cells strongly inhibited TGF-β-mediated migration and invasion. Accordingly, TGF-β induction of N-cadherin, which plays an important role in migration and invasion, is markedly reduced when Pin1 is depleted in PC3 cells. Because Pin1 is overexpressed in many cancers, our findings highlight the importance of Pin1 in TGF-β-induced migration and invasion of cancer cells.     

Sharpening gene editing toolbox in Arabidopsis for plants

Journal of Plant Biochemistry and Biotechnology - Arabidopsis thaliana is considered as an indispensable model system across various disciplines of modern plant biology. The short life cycle, well...     

Comparative Analysis of DNA Word Abundances in Four Yeast Genomes Using a Novel Statistical Background Model

Previous studies have shown that the identification and analysis of both abundant and rare k-mers or “DNA words of length k” in genomic sequences using suitable statistical background models can reveal biologically significant sequence elements. Other studies have investigated the uni/multimodal distribution of k-mer abundances or “k-mer spectra” in different DNA sequences. However, the existing background models are affected to varying extents by compositional bias. Moreover, the distribution of k-mer abundances in the context of related genomes has not been studied previously. Here, we present a novel statistical background model for calculating k-mer enrichment in DNA sequences based on the average of the frequencies of the two (k-1) mers for each k-mer. Comparison of our null model with the commonly used ones, including Markov models of different orders and the single mismatch model, shows that our method is more robust to compositional AT-rich bias and detects many additional, repeat-poor over-abundant k-mers that are biologically meaningful. Analysis of overrepresented genomic k-mers (4≤k≤16) from four yeast species using this model showed that the fraction of overrepresented DNA words falls linearly as k increases; however, a significant number of overabundant k-mers exists at higher values of k. Finally, comparative analysis of k-mer abundance scores across four yeast species revealed a mixture of unimodal and multimodal spectra for the various genomic sub-regions analyzed.     

Senescence of Primary Amniotic Cells via Oxidative DNA Damage

Objective

Oxidative stress is a postulated etiology of spontaneous preterm birth (PTB) and preterm prelabor rupture of the membranes (pPROM); however, the precise mechanistic role of reactive oxygen species (ROS) in these complications is unclear. The objective of this study is to examine impact of a water soluble cigarette smoke extract (wsCSE), a predicted cause of pregnancy complications, on human amnion epithelial cells.

Methods

Amnion cells isolated from fetal membranes were exposed to wsCSE prepared in cell culture medium and changes in ROS levels, DNA base and strand damage was determined by using 2′7′-dichlorodihydro-fluorescein and comet assays as well as Fragment Length Analysis using Repair Enzymes (FLARE) assays, respectively. Western blot analyses were used to determine the changes in mass and post-translational modification of apoptosis signal-regulating kinase (ASK1), phospho-p38 (P-p38 MAPK), and p19^arf. Expression of senescence-associated β-galectosidase (SAβ-gal) was used to confirm cell ageing in situ.

Results

ROS levels in wsCSE-exposed amnion cells increased rapidly (within 2 min) and significantly (p<0.01) at all-time points, and DNA strand and base damage was evidenced by comet and FLARE assays. Activation of ASK1, P-p38 MAPK and p19^Arf correlated with percentage of SAβ-gal expressing cells after wsCSE treatment. The antioxidant N-acetyl-L-cysteine (NAC) prevented ROS-induced DNA damage and phosphorylation of p38 MAPK, whereas activation of ASK1 and increased expression of p19^Arf were not significantly affected by NAC.

Conclusions

The findings support the hypothesis that compounds in wsCSE induces amnion cell senescence via a mechanism involving ROS and DNA damage. Both pathways may contribute to PTB and pPROM. Our results imply that antioxidant interventions that control ROS may interrupt pathways leading to pPROM and other causes of PTB.     

Investigation of genetic risk factors for chronic adult diseases for association with preterm birth

Preterm birth (PTB) is the leading cause of infant mortality. PTB pathophysiology overlaps with those of adult cardiovascular, immune and metabolic disorders (CIMD), with mechanisms including inflammation, immunotolerance, thrombosis, and nutrient metabolism. Whereas many genetic factors for CIMD have been identified, progress in PTB has lagged. We hypothesized that highly validated genetic risk factors for CIMD may also be associated with PTB. We conducted case–control study of four female cohorts with spontaneous PTB (n = 673) versus term (n = 1119). Of 35 SNPs genotyped, there were 13 statistically significant associations (P < 0.05), which were more than expected (binomial test; P = 0.02). In US White (307 cases/342 controls), the G allele of HLA-DQA1 (A/G) rs9272346 was protective for PTB in the initial discovery cohort (P = 0.02; OR = 0.65; 95 % CI 0.46, 0.94). This protective association replicated (P = 0.02; OR = 0.85; 95 % CI 0.75, 0.97) nominally in the Danish Cohort (883 cases, 959 controls), but lost significance upon multiple testing correction. We observed more statistically significant associations than expected, suggesting that chance is an unlikely explanation for one or more of the associations. Particularly, a protective association of the G allele of HLA-DQA1 was found in two independent cohorts, and in previous studies, this same allele was found to protect against type-1-diabetes (meta-analysis P value 5.52 × 10^–219). Previous investigations have implicated HLA phenotypic variation in recurrent fetal loss and in chronic chorioamnionitis. Given the limited sample size in his study, we suggest larger studies to further investigate possible HLA genetic involvement in PTB.     

Phosphoroamidate compounds of 1,1′-Bi-2-napthol: Synthesis, structural characterization and solvent-free ring-opening polymerization of ε-caprolactone and l-lactide

New phosphoroamidate compounds with 1,1′-Bi-2-napthol (binol) ligand were synthesized from the corresponding phosphorochloridate intermediates and benzyl amine or benzyl amine derivatives. They were completely characterized using different spectroscopic methods and single crystal X-ray diffraction studies. These compounds effectively catalyze the ring-opening polymerization of ε-caprolactone (CL) and l-lactide (LA). This methodology of polymer synthesis is green and environmental benign since phosphorus is a natural constituent of human anatomy and these polymers being completely biodegradable.