Under-representation of intrinsic terminators across bacterial genomic islands: Rho as a principal regulator of xenogenic DNA expression

Two transcription termination mechanisms - intrinsic and Rho-dependent - have evolved in bacteria. The Rho factor occurs in most bacterial lineages, and has been hypothesized to play a global regulatory role. Genome-wide studies using microarray, 2D-gel electrophoresis and ChIP-chip provided evidence that Rho serves to silence transcription from horizontally acquired genes and prophages in Escherichia coli K-12, implicating the factor to be a part of the ``cellular immune mechanism'' protecting against deleterious phages and aberrant gene expression from acquired xenogenic DNA. We have investigated this model by adopting an alternate in silico approach and have extended the study to other species. Our analysis shows that several genomic islands across diverse phyla have under-representation of intrinsic terminators, similar to that experimentally observed in E. coli K-12. This implies that Rho-dependent termination is the predominant process operational in these islands and that silencing of foreign DNA is a conserved function of Rho. From the present analysis, it is evident that horizontally acquired islands have lost intrinsic terminators to facilitate Rho-dependent termination. These results underscore the importance of Rho as a conserved, genome-wide sentinel that regulates potentially toxic xenogenic DNA. (C) 2012 Elsevier B.V. All rights reserved.

How Does the Spacer Length of Cationic Gemini Lipids Influence the Lipoplex Formation with Plasmid DNA? Physicochemical and Biochemical Characterizations and their Relevance in Gene Therapy

Lipoplexes formed by the pEGFP-C3 plasmid DNA (pDNA) and lipid mixtures containing cationic gemini surfactant of the 1,2-bis(hexadecyl dimethyl ammonium) Acmes family referred to as C16CnC16, where n = 2 3, 5, or 12, and the zwitterionic helper lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) have been studied from a wide variety of physical, chemical, and biological standpoints. The study has been carried out using several experimental methods, such as zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), cryo-TEM, gene transfection, cell viability/cytotoxicity, and confocal fluorescence microscopy. As reported recently in a communication (J. Am. Chem. Soc. 2011, 133, 18014), the detailed physicochemical and biological studies confirm that, in the presence of the studied series lipid mixtures, plasmid DNA is compacted with a large number of its associated Na+ counterions. This in turn yields a much lower effective negative charge, q(pDNA)(-), a value that has been experimentally obtained for each mixed lipid mixture. Consequently, the cationic lipid (CL) complexes prepared with pDNA and CL/DOPE mixtures to be used in gene transfection require significantly less amount of CL than the one estimated assuming a value of q(DNA)(-) = -2. This drives to a considerably lower cytotoxicity of the gene vector. Depending on the CL molar composition, alpha, of the lipid mixture, and the effective charge ratio of the lipoplex, rho(eff), the reported SAXS data indicate the presence of two or three structures in the same lipoplex, one in the DOPE-rich region, other in the CL-rich region, and another one present at any CL composition. Cryo-TEM and SAXS studies with C16CnC16/DOPE-pDNA lipoplexes indicate that pDNA is localized between the mixed lipid bilayers of lamellar structures within a monolayer of similar to 2 nm. This is consistent with a highly compacted supercoiled pDNA conformation compared with that of linear DNA. Transfection studies were carried out with HEK293T, HeLa, CHO, U343, and H460 cells. The alpha and rho(eff) values for each lipid mixture were optimized on HEK293T cells for transfection, and using these values, the remaining cells were also transfected in absence (-FBS-FBS) and presence (-FBS+FBS) of serum. The transfection efficiency was higher with the CLs of shorter gemini spacers (n = 2 or 3). Each formulation expressed GFP on pDNA transfection and confocal fluorescence microscopy corroborated the results. C16C2C16/DOPE mixtures were the most efficient toward transfection among all the lipid mixtures and, in presence of serum, even better than the Lipofectamine2000, a commercial transfecting agent Each lipid combination was safe and did not show any significant levels of toxicity. Probably, the presence of two coexisting lamellar structures in lipoplexes synergizes the transfection efficiency of the lipid mixtures which are plentiful in the lipoplexes formed by CLs with short spacer (n = 2, 3) than those with the long spacer (n = 5, 12).

Lacrimal Proline Rich 4 (LPRR4) Protein in the Tear Fluid Is a Potential Biomarker of Dry Eye Syndrome

Dry eye syndrome (DES) is a complex, multifactorial, immune-associated disorder of the tear and ocular surface. DES with a high prevalence world over needs identification of potential biomarkers so as to understand not only the disease mechanism but also to identify drug targets. In this study we looked for differentially expressed proteins in tear samples of DES to arrive at characteristic biomarkers. As part of a prospective case-control study, tear specimen were collected using Schirmer strips from 129 dry eye cases and 73 age matched controls. 2D electrophoresis (2DE) and Differential gel electrophoresis (DIGE) was done to identify differentially expressed proteins. One of the differentially expressed protein in DES is lacrimal proline rich 4 protein (LPRR4). LPRR4 protein expression was quantified by enzyme immune sorbent assay (ELISA). LPRR4 was down regulated significantly in all types of dry eye cases, correlating with the disease severity as measured by clinical investigations. Further characterization of the protein is required to assess its therapeutic potential in DES.

Active-contour-based automated image quantitation techniques for Western Blot analysis

Western Blot analysis is an analytical technique used in Molecular Biology, Biochemistry, Immunogenetics and other Molecular Biology studies to separate proteins by electrophoresis. The procedure results in images containing nearly rectangular-shaped blots. In this paper, we address the problem of quantitation of the blots using automated image processing techniques. We formulate a special active contour (or snake) called Oblong, which locks on to rectangular shaped objects. Oblongs depend on five free parameters, which is also the minimum number of parameters required for a unique characterization. Unlike many snake formulations, Oblongs do not require explicit gradient computations and therefore the optimization is carried out fast. The performance of Oblongs is assessed on synthesized data and Western Blot Analysis images.

Synthesis, crystal structure, DNA binding and cleavage activities of oximato bridged cationic dinuclear copper(II) complex having labile ligands

Oximato bridged dinuclear copper(II) complex Cu(L)(CH3OH)](2)(ClO4)(2) with an oxime-Schiff base ligand, viz. 3-2-(dimethylamino)ethyl]imino]-2-butanoneoxime (HL), has been synthesized and structurally characterized. The dinuclear copper(II) complex crystallizes in monoclinic space group P2(1)/n with the unit cell parameters, a = 13.3564(9) angstrom, b = 12.0821(8) angstrom, c = 17.5045(11) angstrom, beta = 90.097, V = 2824.8(3) angstrom(3), Z = 4, R = 0.0769. The complex shows quasi-reversible cyclic voltammetric response at 0.844V (Delta E-p = 276 mV) at 100 mVs(-1). The binding studies of the complex with calf thymus DNA has been investigated using absorption spectrophotometry. Cleavage activity of the complex has been carried out on double stranded pBR 322 plasmid DNA by using gel electrophoresis experiments in the absence and in the presence of the oxidant, viz., H2O2.

Palladium(II) complexes as biologically potent metallo-drugs: Synthesis, spectral characterization, DNA interaction studies and antibacterial activity

Four novel mononuclear Pd(II) complexes have been synthesized with the biologically active Schiff base ligands (L-1-L-4) derived from 3-amino-2-methyl-4(3H)-quinazolinone. The structure of the complexes has been proposed by elemental analysis, molar conductance, IR, H-1 NMR, mass, UV-Vis spectrometric and thermal studies. The investigation of interaction of the complexes with calf thymus DNA (CT-DNA) has been performed with absorption and fluorescence spectroscopic studies. The nuclease activity was done using pUC19 supercoiled DNA by gel-electrophoresis. All the ligands and their Pd(II) complexes have also been screened for their antibacterial activity by discolor diffusion technique. (C) 2013 Elsevier B.V. All rights reserved.

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.

Synthesis, crystal structure, DNA interaction and cleavage activities of mononuclear and trinuclear copper(II) complexes

Mono- and trinuclear copper(II) complexes with 2-1-(2-dimethylamino-ethylamino)-ethyl]-phenol (HL) have been synthesized and structurally characterized. The mononuclear complex Cu(L)(H2O)(ONO2)] (1) crystallizes in monoclinic space group P2(1) /n with a square pyramidal Cu(II) center coordinated by the tridentate Schiff base (L) and a water ligand in the equatorial plane and an oxygen atom from nitrate in the axial position. The trinuclear complex (CuL)(3)(mu(3)-OH)](ClO4)(2)center dot H2O (2) crystallizes in hexagonal space group P6(3); all three copper atoms are five-coordinate with square pyramidal geometries. The interactions of these complexes with calf-thymus DNA have been investigated using absorption spectrophotometry. The mononuclear complex binds more strongly than the trinuclear complex. The DNA cleavage activity of these complexes has been studied on double-stranded pBR 322 plasmid DNA by gel electrophoresis experiments in the absence and in the presence of added oxidant (H2O2). The trinuclear complex cleaves DNA more efficiently than the mononuclear complex in the presence of H2O2.

Rad51-Rad52 Mediated Maintenance of Centromeric Chromatin in Candida albicans

Specification of the centromere location in most eukaryotes is not solely dependent on the DNA sequence. However, the non-genetic determinants of centromere identity are not clearly defined. While multiple mechanisms, individually or in concert, may specify centromeres epigenetically, most studies in this area are focused on a universal factor, a centromere-specific histone H3 variant CENP-A, often considered as the epigenetic determinant of centromere identity. In spite of variable timing of its loading at centromeres across species, a replication coupled early S phase deposition of CENP-A is found in most yeast centromeres. Centromeres are the earliest replicating chromosomal regions in a pathogenic budding yeast Candida albicans. Using a 2-dimensional agarose gel electrophoresis assay, we identify replication origins (ORI7-LI and ORI7-RI) proximal to an early replicating centromere (CEN7) in C. albicans. We show that the replication forks stall at CEN7 in a kinetochore dependent manner and fork stalling is reduced in the absence of the homologous recombination (HR) proteins Rad51 and Rad52. Deletion of ORI7-RI causes a significant reduction in the stalled fork signal and an increased loss rate of the altered chromosome 7. The HR proteins, Rad51 and Rad52, have been shown to play a role in fork restart. Confocal microscopy shows declustered kinetochores in rad51 and rad52 mutants, which are evidence of kinetochore disintegrity. CENP-A(CaCse4) levels at centromeres, as determined by chromatin immunoprecipitation (ChIP) experiments, are reduced in absence of Rad51/Rad52 resulting in disruption of the kinetochore structure. Moreover, western blot analysis reveals that delocalized CENP-A molecules in HR mutants degrade in a similar fashion as in other kinetochore mutants described before. Finally, co-immunoprecipitation assays indicate that Rad51 and Rad52 physically interact with CENP-A(CaCse4) in vivo. Thus, the HR proteins Rad51 and Rad52 epigenetically maintain centromere functioning by regulating CENP-A(CaCse4) levels at the programmed stall sites of early replicating centromeres.

Serum proteomics of hepatitis C virus infection reveals retinol-binding protein 4 as a novel regulator

Persistent infection of hepatitis C virus (HCV) can lead to liver cirrhosis and hepatocellular carcinoma, which are currently diagnosed by invasive liver biopsy. Approximately 15-20% of cases of chronic liver diseases in India are caused by HCV infection. In North India, genotype 3 is predominant, whereas genotype 1 is predominant in southern parts of India. The aim of this study was to identify differentially regulated serum proteins in HCV-infected Indian patients (genotypes 1 and 3) using a two-dimensional electrophoresis approach. We identified eight differentially expressed proteins by MS. Expression levels of one of the highly upregulated proteins, retinol-binding protein 4 (RBP4), was validated by ELISA and Western blotting in two independent cohorts. We also confirmed our observation in the JFH1 infectious cell culture system. Interestingly, the HCV core protein enhanced RBP4 levels and partial knockdown of RBP4 had a positive impact on HCV replication, suggesting a possible role for this cellular protein in regulating HCV infection. Analysis of RBP4-interacting partners using a bioinformatic approach revealed novel insights into the possible involvement of RBP4 in HCV-induced pathogenesis. Taken together, this study provided information on the proteome profile of the HCV-infected Indian population, and revealed a link between HCV infection, RBP4 and insulin resistance.

Characterization of Precursor PfHsp60 in Plasmodium falciparum Cytosol during Its Asexual Development in Human Erythrocytes

Mitochondrial heat shock protein 60 (Hsp60) is a nuclear encoded gene product that gets post-translationally translocated into the mitochondria. Using multiple approaches such as immunofluorescence experiments, isoelectric point analysis with two-dimensional gel electrophoresis, and mass spectrometric identification of the signal peptide, we show that Hsp60 from Plasmodium falciparum (PfHsp60) accumulates in the parasite cytoplasm during the ring, trophozoite, and schizont stages of parasite development before being imported into the parasite mitochondria. Using co-immunoprecipitation experiments with antibodies specific to cytoplasmic PfHsp90, PfHsp70-1, and PfHsp60, we show association of precursor PfHsp60 with cytoplasmic chaperone machinery. Metabolic labeling involving pulse and chase indicates translocation of the precursor pool into the parasite mitochondrion during chase. Analysis of results obtained with Geldanamycin treatment confirmed precursor PfHsp60 to be one of the clients for PfHsp90. Cytosolic chaperones bind precursor PfHsp60 prior to its import into the mitochondrion of the parasite. Our data suggests an inefficient co-ordination in the synthesis and translocation of mitochondrial PfHsp60 during asexual growth of malaria parasite in human erythrocytes.

Comparative proteomic analysis of primordial follicles from ovaries of immature and aged rats

Age related decline in reproductive performance in women is well documented and apoptosis has been considered as one of the reasons for the decline of primordial follicle reserve. Recently we observed a decline in the efficiency of DNA repair ability in aged rat primordial follicles as demonstrated by decreased mRNA levels of DNA repair genes BRCA1 and H2AX. In the present study, a two-dimensional electrophoresis (2DE) proteomic approach was employed to identify differentially expressed proteins in primordial follicles isolated from ovaries of immature (approximate to 20 days) and aged (approximate to 400-450 days) rats. Using MALDI-TOF/TOF MS, we identified 13 differentially expressed proteins (p<0.05) which included seven up-regulated and six down-regulated proteins in aged primordial follicles. These proteins are involved in a wide range of biological functions including apoptosis, DNA repair, and the immune system. Interestingly, the differentially expressed proteins such as FIGNL1 (DNA repair) and BOK (apoptotic protein) have not been previously reported in the rat primordial follicles and these proteins can be related to some common features of ovarian aging such as loss of follicle reserve and genome integrity. The quantitative differences of two important proteins BOK and FIGNL1 observed by the proteomic analysis were correlated with the transcript levels, as determined by semi-quantitative RT-PCR. Our results improve the current knowledge about protein factors associated with molecular changes in rat primordial follicles as a function of aging and our understanding of the proteomic processes involved in degenerative changes observed in aging primordial follicles.

Difference in Enzyme Activity and Conformation of Glucose Oxidase Before and After Purification

EEnzyme activity of commercial glucose oxidase was enhanced after purification through a strong anionic exchange resin. In order to get a better insight into this phenomenon, surface pressure–area ( –A) isotherms and surface pressure–time ( –t) isotherms was used to study the interaction and the absorption at different pH values of the subphases between octadecylamine and glucose oxidase purified by a styrene system quaternary ammonium type strongly basic anionic exchange resin. Circular dichroism (CD), electrophoresis and enzyme activity measurements were conducted to study these phenomena. A preliminary hypothesis has been suggested to explain why the enzyme activity of purified glucose oxidase was higher than that of the commercial one. © 2002 Elsevier Science B.V. All rights reserved.

Simultaneous Laser-Induced Fluorescence And Contactless-Conductivity Detection For Microfluidic Chip

A combined detection system involving simultaneous LIF and contactless-conductometric measurements at the same place of the microfluidic chip was described. The LIF measurement was designed according to the confocal principle and a moveable contactless-conductivity detector was used in (CD)-D-4. Both measurements were mutually independent and advantageous in analyses of mixtures. Various experimental parameters affecting the response were examined and optimized. The performances were demonstrated by simultaneous detection of Rhodamine B. And the results showed that the combined detection system could be used sensitively and reliably. (C) 2008 Yong Yu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Adaptibilidad y estabilidad de naranjilla (Solaum quitoense LAM var. Septentrionale) y cocona (Solanum sessiliflorum DUNAL var. Sessiliflorum) en fincas de pequeños agricultores de granos básicos en Jinotega

La naranjilla ( Solanum quitoense LAM), y la cocona ( Solanum sessiliflorum DUNAL), son cultivadas mayormente en Jinotega y Matagalpa. Ambos cultivos son poco conocidos, al igual su manejo agronómico. El objetivo de este ensayo fue estudiar la adaptabilidad y estabilidad de ambos cultivos. Para esto se establecieron parcelas en 23 fincas, sin repeticiones dentro de cada finca. Entre los resultados se obtuvieron los siguientes: Tanto el factor finca como la interacción finca*variedad resultaron significativos. En general la naranjilla mostró una mayor adaptabilidad a los ambientes más productivos, aunque resultó menos estable que la cocona (b = 1.5). La cocona mostró una estabilidad mayor (b = 0.49) y adaptada a los ambientes menos productivos. La grafica bidimensional del AMMI reflejó que ambos cultivos y las Fincas Siete y Ocho contribuyeron grandemente a la interacción detectada. En cuanto a la variación observada entre sitios las variables independientes profundidad de suelo, Siembra T y pendiente del terreno mostraron cierta asociación con la variación en el rendimiento observado entre fincas.