Geometrical constraints limiting the poly(ADP‐ribose) conformation investigated by molecular dynamics simulation

Poly(ADP‐ribosylation) is a post‐transductional modification that regulates protein's function. Most of the proteins subjected to this control mechanism belong to machineries involved in DNA damage repair, or DNA interacting proteins. Poly(ADP‐ribose) polymers are long chains of even 100 monomer length that can be branched at several positions but, not withstanding its importance, nothing is known concerning its structure. To understand, which are the geometrical parameters that confer to the polymer the structural constraints that determine its interaction with the target proteins, we have performed molecular dynamics of three chains of different length, made by 5, 25, and 30 units, the last one being branched. Analysis of the simulations allowed us to identify the main intra‐ and inter‐monomer dihedral angles that govern the structure of the polymer that however, does not reach a unique definite conformation. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 78–86, 2014.     

Protective effects of polyphenolics in red wine on diabetes associated oxidative/nitrative stress in streptozotocin‐diabetic rats

Increased accumulation of NT (3‐nitrotyrosine) and PARylated [poly(ADP‐ribosyl)ated] proteins in the tissues of diabetics are associated with diabetes complications (diabetes neuropathy, nephropathy and retinopathy). Red wine (its polyphenols are considered to be the main active components) can act as ROS (reactive oxygen species) scavengers, iron chelators and enzyme modulators. This study is novel in investigating the effect of red wine in preventing the accumulation of NT and PARylated proteins in the sciatic nerve, DRG (dorsal root ganglia), spinal cord, kidney and retina of diabetic animals. We have shown that during the experiment the body weight of control and diabetic groups of rats with consumption of red wine was significantly increased, by 52% and 19% accordingly. The significant increase in the content of NT in the sciatic nerve, DRG, spinal cord, kidney and retina, and PARylated proteins in the sciatic nerve, renal glomeruli and retinae of diabetic rats was partly or completely prevented by treatment with red wine. Red wine and its polyphenol preparations might be a promising option in the prevention and treatment of diabetic complications.     

Binding kinetics and activity of human poly(ADP‐ribose) polymerase‐1 on oligo‐deoxyribonucleotide substrates

Poly(ADP‐ribose) polymerase‐1 (PARP‐1) is a mammalian enzyme that attaches long branching chains of ADP‐ribose to specific nuclear proteins, including itself. Because its activity in vitro is dependent upon interaction with broken DNA, it has been postulated that PARP‐1 plays an important role in DNA strand‐break repair in vivo. The exact mechanism of binding to DNA and the structural determinants of binding remain to be defined, but regions of transition from single‐stranded to double‐strandedness may be important recognition sites. Here we employ surface plasmon resonance (SPR) to investigate this hypothesis. Oligodeoxynucleotide (ODN) substrates that mimic DNA with different degrees of single‐strandedness were used for measurements of both PARP‐1/DNA binding kinetics and PARP‐1's enzyme activities. We found that binding correlated with activity, but was unrelated to single‐strandedness of the ODN. Instead, PARP‐1 binding and activity were highest on ODNs that modeled a DNA double‐strand break (DSB). These results provide support for PARP‐1 recognizing and binding DSBs in a manner that is independent of single‐stranded features, and demonstrate the usefulness of SPR for simultaneously investigating both PARP‐1 binding and PARP‐1 auto‐poly(ADP‐ribosyl)ation activities within the same in vitro system. Copyright © 2009 John Wiley & Sons, Ltd.     

Crystal structures of the X‐domains of a Group‐1 and a Group‐3 coronavirus reveal that ADP‐ribose‐binding may not be a conserved property

The polyproteins of coronaviruses are cleaved by viral proteases into at least 15 nonstructural proteins (Nsps). Consisting of five domains, Nsp3 is the largest of these (180–210 kDa). Among these domains, the so‐called X‐domain is believed to act as ADP‐ribose‐1″‐phosphate phosphatase or to bind poly(ADP‐ribose). However, here we show that the X‐domain of Infectious Bronchitis Virus (strain Beaudette), a Group‐3 coronavirus, fails to bind ADP‐ribose. This is explained on the basis of the crystal structure of the protein, determined at two different pH values. For comparison, we also describe the crystal structure of the homologous X‐domain from Human Coronavirus 229E, a Group‐1 coronavirus, which does bind ADP‐ribose.     

Poly (ADP‐ribose) polymerase‐1 binds to BCL2 major breakpoint region and regulates BCL2 expression

BCL2, originally identified as a proto‐oncogene in B‐cell lymphoma, is a key regulator of apoptosis. Although it is more than 200 kb in length, at least 70% of the t(14;18) translocation in follicular lymphomas occurs at the BCL2 major breakpoint region (mbr), located in the 3′‐untranslated region (3'‐UTR). We have previously found that the mbr is a regulatory element which positively regulates BCL2 expression and this regulatory function was closely associated with SATB1, which binds to a 37 bp mbr (37 mbr) in the 3′‐end of the mbr directly. However, the precise molecular mechanisms by which the mbr regulates gene expression are not fully understood. In this study, we purified Poly(ADP‐ribose) polymerase‐1 (PARP‐1) from the DNA–protein complexes formed by 37 mbr in Jurkat cells and demonstrated that PARP‐1 participates in the 37 mbr–protein complex's formation in vitro and in vivo. Functional analysis showed that overexpression of PARP‐1 decreases 37 mbr regulatory function and BCL2 expression. Conversely, knockdown of PARP‐1 with RNAi increases BCL2 expression. Taken together, the present findings indicate that PARP‐1 is a component of BCL2 37 mbr–protein complexes, and PARP‐1 is involved in the regulation of BCL2 expression. These findings are helpful in understanding the regulatory mechanisms of BCL2 expression. J. Cell. Biochem. 110: 1208–1218, 2010. Published 2010 Wiley‐Liss, Inc.     

Poly(ADP‐ribose) polymerase inhibition with HYDAMTIQ reduces allergen‐induced asthma‐like reaction,bronchial hyper‐reactivity and airway remodelling

Activation of poly(ADP‐ribose) polymerases (PARPs) is considered a key event in the molecular and cellular processes leading from acute asthma attacks to bronchial hyper‐reactivity, leucocyte recruitment, chronic inflammation, airway remodelling and lung damage. The present investigation has been carried out to investigate the action of hydroxyl‐dimethylaminomethyl‐thieno[2,3‐c]isoquinolin‐5(4H)‐one (HYDAMTIQ), a new potent PARP inhibitor, in the process leading from asthma‐like events to airway damage. Ovalbumin‐sensitized guinea pigs exposed two times to allergen inhalation were treated for 8 days with vehicle or HYDAMTIQ. Asthma‐like signs, bronchial hyper‐reactivity to methacholine, cytokine production, histamine release from mast cells, airway remodelling, collagen deposition and lung damage were evaluated. Repeated HYDAMTIQ administration (1‐10 mg/kg/day i.p.) reduced lung PARP activity, delayed the appearance and reduced the severity of allergen‐induced cough and dyspnoea and dampened the increased bronchial responses to methacholine. HYDAMTIQ‐treated animals presented reduced bronchial or alveolar abnormalities, lower number of eosinophils and other leucocytes in the lung and decreased smooth muscle or goblet cell hyperplasia. The treatment also reduced lung oxidative stress markers, such as malondialdehyde or 8‐hydroxy‐2′‐deoxyguanosine and the lung content of pro‐inflammatory cytokines (TNF‐α, interleukin (IL)‐1β, IL‐5, IL‐6 and IL‐18). Finally, mast cells isolated from the peritoneal or pleural cavities of sensitized, HYDAMTIQ‐treated animals had a reduced ability to release histamine when exposed to ovalbumin in vitro. Our findings support the proposal that PARP inhibitors could have a therapeutic potential to reduce chronic lung inflammation, airway damage and remodelling in severe unresponsive asthmatic patients.     

Spectrofluorimetric determination of aluminium using 2‐hydroxy‐1‐naphthylidene‐(8‐aminoquinoline)

A sensitive and selective spectrofluorimetric method has been developed for the rapid determination of aluminium. This method is based on the complex formation between aluminium and 2‐hydroxy‐1‐naphthylidene‐(8‐aminoquinoline) (HNAQ). The optimum conditions for the complex formation were a metal‐to‐ligand (M : L) stoichiometric ratio of 1:1, a pH of 5.5 and a 0.20 m acetate buffer. The fluorescence of the complex was monitored at an emission wavelength of 502 nm with excitation at 438 nm. Under these conditions, linear calibration curves were obtained in the ranges 0.05–1 and 1–5 ppm. The detection limit was 3.4 ppb for the former and 13.5 ppb for the latter. The maximum relative standard deviation of the method for an aluminium standard of 200 ppb was 1.5% (n = 5). This method was successfully applied for the determination of aluminium in drinking water, pharmaceutical antacid tablets and suspension samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Interleukin‐8 induction due to diffusely adherent Escherichia coli possessing Afa/Dr genes depends on flagella and epithelial Toll‐like receptor 5

DAEC is considered potentially diarrheagenic. For diffuse adhesion, the role of the Afa, which was originally identified as a uropathogenic factor, is now understood. However, the role of DAEC in diarrheal disease remains controversial because DAEC is often isolated not only from patients but also from healthy individuals. Previously, we suggested that Afa/Dr DAEC, which can induce high levels of IL‐8 secretion in cultures of human carcinoma epithelial cells (HEp‐2, Caco‐2), is enterovirulent. In the present study, we examined whether IL‐8 secretion induced by certain Afa/Dr DAEC strains was primarily due to flagella via TLR5. All IL‐8 high‐inducing strains were highly motile in swarming tests. Partially purified flagella induced IL‐8 in a dose‐dependent manner. However, IL‐8 induction was inhibited by small‐interfering RNA against TLR5 or by treating flagella with disialoganglioside‐GD1a, a TLR5 blocker. TLR5 is reportedly located on the basolateral side of intestinal epithelia; flagella should not have reached TLR5 from the apical side beyond tight junctions. Reduction in the number of intracellular organisms by wortmannin, a PI3K inhibitor, did not reduce IL‐8 secretion. Afa/Dr DAEC seemed to loosen the tight junctions because it quickly reduced transepithelial electrical resistance after infection. Decreased resistance led to increased IL‐8 production. In conclusion, diffuse adhesion itself is insufficient to induce high levels of IL‐8, and simultaneous stimulation by flagella via TLR5 is likely required for additional induction. Clinically, high motility may be a candidate criterion for predicting the ability of Afa/Dr DAEC strains to induce higher levels of IL‐8 secretion.     

Down‐regulation of c‐fos by shRNA sensitizes adriamycin‐resistant MCF‐7/ADR cells to chemotherapeutic agents via P‐glycoprotein inhibition and apoptosis augmentation

Multidrug resistance (MDR) is a major hurdle in the treatment of cancer. Research indicated that the main mechanisms of most cancers included so‐called “pump” (P‐glycoprotein, P‐gp) and “non‐pump” (apoptosis) resistance. Identification of novel signaling molecules associated with both P‐gp and apoptosis will facilitate the development of more effective strategies to overcome MDR in tumor cells. Since the proto‐oncogene c‐fos has been implicated in cell adaptation to environmental changes, we analyzed its role in mediating “pump” and “non‐pump” resistance in MCF‐7/ADR, an adriamycin (ADR)‐selected human breast cancer cell line with the MDR phenotype. Elevated expression of c‐fos in MCF‐7/ADR cells and induction of c‐fos by ADR in the parental drug‐sensitive MCF‐7 cells suggested a link between c‐fos and MDR phenotype. Down‐regulation of c‐fos expression via shRNA resulted in sensitization of MCF‐7/ADR cells to chemotherapeutic agents, including both P‐gp and non‐P‐gp substrates. Further results proved that c‐fos down‐regulation in MCF‐7/ADR cells resulted in decreased P‐gp expression and activity, enhanced apoptosis, and altered expression of apoptosis‐associated proteins (i.e., Bax, Bcl‐2, p53, and PUMA). All above facts indicate that c‐fos is involved in both P‐gp‐ and anti‐apoptosis‐mediated MDR of MCF‐7/ADR cells. Based on these results, we propose that c‐fos may represent a potential molecular target for resistant cancer therapy, and suppressing c‐fos gene expression may therefore be an effective means to temper breast cancer cell's MDR to cytotoxic chemotherapy. J. Cell. Biochem. 114: 1890–1900, 2013. © 2013 Wiley Periodicals, Inc.     

Microparticle‐mediated gene delivery for the enhanced expression of a 19‐kDa fragment of merozoite surface protein 1 of Plasmodium falciparum

The 19 kDa carboxyl‐terminal fragment of merozoite surface protein 1 (MSP1₁₉) is a major component of the invasion‐inhibitory response in individual immunity to malaria. A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic‐co‐glycolic acid) (PLGA) microparticles of malaria DNA vaccines encoding MSP1₁₉ is presented here. After condensing the plasmid DNA (pDNA) molecules with a cationic polymer polyethylenimine (PEI), a 40 kHz ultrasonic atomization frequency was used to formulate PLGA microparticles at a flow rate of 18 mL h^?1. High levels of gene expression and moderate cytotoxicity in COS‐7 cells were achieved with the condensed pDNA at a nitrogen to phosphate (N/P) ratio of 20, thus demonstrating enhanced cellular uptake and expression of the transgene. The ability of the microparticles to convey pDNA was examined by characterizing the formulated microparticles. The microparticles displayed Z‐average hydrodynamic diameters of 1.50–2.10 μm and zeta potentials of 17.8–23.2 mV. The encapsulation efficiencies were between 78 and 83%, and 76 and 85% of the embedded malaria pDNA molecules were released under physiological conditions in vitro. These results indicate that PLGA‐mediated microparticles can be employed as potential gene delivery systems to antigen‐presenting cells in the prevention of malaria. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

MicroRNA‐146b‐5p overexpression attenuates premature ovarian failure in mice by inhibiting the Dab2ip/Ask1/p38‐Mapk pathway and γH2A.X phosphorylation

ObjectiveTo examine the role of high‐fat and high‐sugar (HFHS) diet‐induced oxidative stress, which is a risk factor for various diseases, in premature ovarian failure (POF).Materials and methodsOvarian granulosa cells (OGCs) were isolated from mice and cultured in medium supplemented with HFHS and poly (lactic‐co‐glycolic acid) (PLGA)‐cross‐linked miR‐146b‐5p nanoparticles (miR‐146@PLGA). RNA and protein expression levels were examined using quantitative real‐time polymerase chain reaction and Western blotting, respectively. HFHS diet‐induced POF model mice were administered miR‐146@PLGA.ResultsThe ovarian tissue of mice fed a HFHS diet exhibited the typical pathological characteristics of POF. HFHS supplementation induced oxidative stress injury in the mouse OGCs, activation of the Dab2ip/Ask1/p38‐Mapk signalling pathway and phosphorylation of γH2A.X in vitro and in vivo. The results of the luciferase reporter assay revealed that miR‐146 specifically downregulated p38‐Mapk14 expression. Meanwhile, co‐immunoprecipitation and Western blot analyses revealed that HFHS supplementation upregulated nuclear p38‐Mapk14 expression and consequently enhanced γH2A.X (Ser139) phosphorylation. The HFHS diet‐induced POF mouse model treated with miR‐146@PLGA exhibited downregulated p38‐Mapk14 expression in the OGCs, mitigated OGC ageing and alleviated the symptoms of POF.ConclusionsThis study demonstrated that HFHS supplementation activates the Dab2ip/Ask1/p38‐Mapk signalling pathway and promotes γH2A.X phosphorylation by inhibiting the expression of endogenous miR‐146b‐5p, which results in OGC ageing and POF development.     

A quantitative anharmonic analysis of the amide A band in α‐helical poly(L‐alanine)

Polarized ir spectra of oriented films of α‐helical poly(l ‐alanine) (α‐PLA) have been obtained as a function of residual solvent dichloroacetic acid (DCA). The amide A, B, II, and V regions exhibit multiple bands whose structure depends on the residual DCA content, and those associated with the α_I‐PLA structure have been identified. A calculation of the relevant cubic anharmonic force constants indicates that, contrary to previous assignments, the overtone of amide II(A) is in Fermi resonance with the NH stretch fundamental, whose unperturbed frequency we now find to be at 3314 cm⁻¹, significantly higher than the previously suggested 3279 cm⁻¹. The presence of a structure in addition to the standard α_I‐PLA is indicated by our analysis. © 1999 John Wiley & Sons, Inc. Biopoly 49: 195–207, 1999     

Morphology‐Limited Free Carrier Generation in Donor/Acceptor Polymer Blend Solar Cells Composed of Poly(3‐hexylthiophene) and Fluorene‐Based Copolymer

The charge generation and recombination dynamics in polymer/polymer blend solar cells composed of poly(3‐hexylthiophene) (P3HT, electron donor) and poly[2,7‐(9,9‐didodecylfluorene)‐alt‐5,5‐(4′,7′‐bis(2‐thienyl)‐2′,1′,3′‐benzothiadiazole)] (PF12TBT, electron acceptor) are studied by transient absorption measurements. In the unannealed blend film, charge carriers are efficiently generated from polymer excitons, but some of them recombine geminately. In the blend film annealed at 160 °C, on the other hand, the geminate recombination loss is suppressed and hence free carrier generation efficiency increases up to 74%. These findings suggest that P3HT and PF12TBT are intermixed within a few nanometers, resulting in impure PF12TBT and disordered P3HT domains. The geminate recombination is likely due to charge carriers generated on isolated polymer chains in the matrix of the other polymer and at the domain interface with disordered P3HT. The undesired charge loss by geminate recombination is reduced by both the purification of the PF12TBT‐rich domain and crystallization of the P3HT chains. These results show that efficient free carrier generation is not inherent to the polymer/fullerene domain interface, but is possible with polymer/polymer systems composed of crystalline donor and amorphous acceptor polymers, opening up a new potential method for the improvement of solar cell materials.