Cyclooxygenase-2 169C > G and 8473T > C gene polymorphisms and prostaglandin E2 level in breast cancer: A case–control study

Aims

Cyclooxygenase 2 (COX-2) with the resulting prostaglandin E2 (PGE2) is linked to increased risk of human breast cancer (BC). The aim of this study was to determine COX-2 169C > G and 8473T > C gene polymorphisms and PGE2 level at various stages of BC clarifying the role of COX-2 gene polymorphism and PGE2 in relation to BC.

Methods

The study population comprised 160 women at different stages of BC and 150 gender- and age-matched healthy control subjects. Plasma PGE2 was measured by ELISA, the COX-2 gene polymorphisms were determined using PCR-RFLP.

Results

The variant alleles COX-2 169G and 8473C were significantly associated with BC susceptibility [OR = 3.1, 95% CI (2.2–4.4), P < 0.001 for 169C>G and OR = 1.74, 95%CI (1.3–2.4), P = 0.005 for 8473C]. However, both COX-2 gene polymorphisms were not associated with breast cancer stage. Plasma PGE2 levels were significantly increased in patients compared to the controls. In early and late stages of BC, there was a significant increase in the plasma PGE2 levels towards the presence of homozygous GG compared with homozygous CC (P < 0.001) for 169 C>G, also towards the presence of CC than TT (P < 0.001) for 8473T>C SNP.

Conclusion

The 169C>G and 8473T>C polymorphisms of the COX-2 gene were associated with the BC in Egyptian women. Furthermore, individuals with COX-2 169GG and 8473CC genotypes showed significant increase in plasma PGE2 levels. PGE2 levels may serve as a predictor of poor prognosis in patients with BC.     

Functional properties of the newly observed γ-chain fetal hemoglobin variant Hb F-Monserrato-Sassari (HBG2:c.280T > C) or [γ93 (F9) Cys → Arg]

Background

HbF-Monserrato-Sassari is a newly discovered abnormal fetal hemoglobin observed in an apparently normal newborn baby during a hemoglobinopathies survey at birth in North Sardinian population.

Methods

Electrophoretic analysis of the cord blood lysate evidenced for an abnormal tetramer due to a mutated fetal globin chain. Electrospray ionisation-mass spectrometry and gene sequencing were used to identify the mutation. Oxygen binding ability of the variant Hb was determined.

Results

Sequencing of the γ globin genes revealed the TGT → CGT transition at codon 93 in one of the two ^Gγ genes, which leads to the Arg for Cys amino acid replacement at position 9 of the F α-helix. The amino acid substitution was confirmed by mass spectrometric analysis of the globin chains. Since modifications or substitutions at position β93 are known to affect the arrangement of a salt bridge at the α1β2 sliding contacts that are crucial for subunit cooperativity, the functional properties of the variant were studied to evaluate the effect of the replacement at the same position in the γ globin chain. With respect to normal HbF, the variant showed a significant increase in oxygen affinity and a slight decrease of both Bohr effect and cooperativity.

General significance

Result indicates a key role of the Cys γ93 residue for subunit cooperativity in the T → R transition of the HbF tetramer. Substitutions at the F9 position of the ^Gγ globin may result in stabilization of the high affinity R-state of the Hb tetramer. Because of the loss of Cys γ93 residue, this variant is considered to be potentially compromised in nitric oxide transport.     

Body temperatures during three long-distance polar swims in water of 0–3 °C

1.

We report body temperature responses in a single individual to 3 swims of 1000 m or longer in ice-cold water (0–3 °C) during which he swam the normal crawl stroke with his face in the water whilst wearing only a swimming costume, swimming cap and goggles.     

Synthesis, crystal structures, and spectroscopic characterization of the neutral monomeric tetrahedral [M(Diap)2(OAc)2] · H2O complexes (M = Zn,Cd; Diap = 1,3-diazepane-2-thione; OAc = acetate) with N-H?O and O-H?O intra- and intermolecular hydrogen bonding interactions

The title complexes, [M(Diap)₂(OAc)₂] · H₂O (M = Zn,Cd; Diap = 1,3-diazepane-2-thione; OAc = acetate) with an MO₂S₂ configuration, have been characterized by X-ray crystallography as well as FT-IR, ¹H and ¹³C NMR spectroscopy. In these complexes, the metal atoms lie in a pseudo-tetrahedral environment and are coordinated by the thione sulfur atoms of two neutral 1,3-diazepane-2-thione ligands and one oxygen atom from each of two monodentate acetate anions. In both complexes, there are two intramolecular N-H?O hydrogen bonds, each being between one NH group of a Diap ligand and the uncoordinated O atom of an OAc ligand. The water molecule is also involved in hydrogen bonds, as an acceptor and as a donor twice, linking together three symmetry-related complexes. The Cd complex undergoes a structural phase transition from a monoclinic form at 150 K with Z′ = 2 to a smaller monoclinic cell at room temperature with Z′ = 1 without loss of crystallinity. The Zn complex does not exhibit an equivalent phase transition, and at 150 K is isostructural with the room-temperature form of the Cd complex. All three crystallographically independent molecules found for the Cd complex (two at low temperature and one at room temperature) have essentially the same structure except for small changes in the conformations of the ligands. Tetrahedral coordination with monodentate carboxylate ligands is common for Zn complexes of this kind, but is unusual for Cd complexes, and is the result of the bulky Diap ligands.     

A family of lanthanide-containing molecular open frameworks with high porosity: [Ln(abdc)(Habdc), nH2O]∞ with Ln = La-Eu and 8 ? n ? 11

Reactions in water between the di-sodium salt of amino terepthalic acid (C₈H₃NO₄Na₂) and a lanthanide chloride lead to a family of 3D-coordination polymers with general chemical formula [Ln(C₈H₃NO₄)(C₈H₄NO₄), O]_∞ where Ln = La-Eu (except Pm) and 8 ? n ? 11. All these compounds are isostructural. High quality single crystals of [Ln(C₈H₃NO₄)(C₈H₄NO₄), nH₂O]_∞ with Ln = La-Sm (except Pm) and 8 ? n ? 11 have been obtained by slow diffusion in agar-agar gels. The crystal structure has been solved for the Nd-containing compound. This compound crystallizes in the cubic system, space group Ia-3 (no. 206) with a = 26.8056(5) Å. The crystal structure can be described as the juxtaposition of large channels with square cross-section.The channels are filled by highly disordered crystallization water molecules. The dehydration of the compounds by freeze-drying is possible and most of the crystallization water molecules can be removed without destruction of the molecular skeleton. The partially dehydrated compounds have general chemical formula [Ln(abdc)(Habdc), 2H₂O]_∞ with Ln = La-Eu except Pm. The porosity of the Nd-containing compound has been estimated by computational methods to 2170 m² g⁻¹. This dehydrated compound reversibly binds water when exposed to wet atmosphere restoring the initial hydrated phase.     

PPARβ mRNA expression,reduced by n − 3 PUFA diet in mammary tumor,controls breast cancer cell growth

The effect of numerous anticancer drugs on breast cancer cell lines and rodent mammary tumors can be enhanced by a treatment with long-chain n − 3 polyunsaturated fatty acids (n − 3 PUFA) such as docosahexaenoic acid (DHA, 22:6n − 3) which is a natural ligand of peroxisome proliferator-activated receptors (PPAR). In order to identify the PPAR regulating breast cancer cell growth, we tested the impact of siRNA, selected to suppress PPARα, PPARβ or PPARγ mRNA in MDA-MB-231 and MCF-7 breast cancer cell lines. The siPPARβ was the most effective to inhibit breast cancer cell growth in both cell lines. Using PPARα, PPARβ and PPARγ pharmacological antagonists, we showed that PPARβ regulated DHA-induced inhibition of growth in MDA-MB-231 and MCF-7 cells. In addition, the expressions of all 3 PPAR mRNA were co-regulated in both cell lines, upon treatments with siRNA or PPAR antagonists. PPAR mRNA expression was also examined in the NitrosoMethylUrea (NMU)-induced rat mammary tumor model. The expressions of PPARα and PPARβ mRNAs were correlated in the control group but not in the n − 3 PUFA group in which the expression of PPARβ mRNA was reduced. Although PPARα expression was also increased in the n − 3 PUFA-enriched diet group under docetaxel treatment, it is only the expression of PPARβ mRNA that correlated with the regression of mammary tumors: those that most regressed displayed the lowest PPARβ mRNA expression. Altogether, these data identify PPARβ as an important player capable of modulating other PPAR mRNA expressions, under DHA diet, for inhibiting breast cancer cell growth and mammary tumor growth.     

Phase studies of model biomembranes: Macroscopic coexistence of Lα + Lβ, with light-induced coexistence of Lα + Lo Phases

Phase diagrams of 3-component lipid bilayer mixtures containing cholesterol reveal major differences among the different types of lipids. Here we report that mixtures of cholesterol together with POPC and a high-melting temperature PC or sphingomyelin show different phase behavior from similar mixtures that contain DOPC or di-phytanoyl-PC instead of POPC. In particular, only one region of macroscopic phase coexistence occurs with POPC, a region of coexisting liquid disordered and solid phases, {Lα + Lβ}. Fluorescence microscopy imaging is useful for these studies, but is subject to artifactual light-induced domain formation, as reported by Ayuyan and Cohen [A.G. Ayuyan, F.S. Cohen, Lipid peroxides promote large rafts: Effects of excitation of probes in fluorescence microscopy and electrochemical reactions during vesicle formation, Biophys. J. 91 (2006) 2172-2183.]. This artifact can be attenuated by decreased illumination and low dye concentration. The use of the free radical scavenger n-propyl gallate can reduce the artifact, but this molecule enters the bilayer and itself perturbs the phase behavior. We suggest that the light-induced domain separation artifact might actually arise from pre-existing lipid clusters that are induced to coalesce, and therefore indicates highly nonrandom mixing of the lipid components.     

Unraveling the mechanism of octenidine and chlorhexidine on membranes: Does electrostatics matter?

The increasing problem of antibiotic resistance in bacteria requires the development of new antimicrobial candidates. There are several well-known substances with commercial use, but their molecular mode of action is not fully understood. In this work, we focus on two commonly used antimicrobial agents from the detergent family—octenidine dichloride (OCT) and chlorhexidine digluconate (CHX). Both of them are reported to be agents selectively attacking the cell membrane through interaction inducing membrane disruption by emulsification. They are believed to present electrostatic selectivity toward charged lipids. In this study, we tested this hypothesis and revised previously proposed molecular mechanisms of action. Employing a variety of techniques such as molecular dynamics, ζ potential with dynamic light scattering, vesicle fluctuation spectroscopy, carboxyfluorescein leakage measurement, and fluorescence trimethylammonium-diphenylhexatriene- and diphenylhexatriene-based studies for determination of OCT and CHX membrane location, we performed experimental studies using two model membrane systems—zwitterionic PC and negatively charged PG (18:1/18:1):PC (16:0/18:1) 3:7, respectively. These studies were extended by molecular dynamics simulations performed on a three-component bacterial membrane model system to further test interactions with another negatively charged lipid, cardiolipin. In summary, our study demonstrated that detergent selectivity is far more complicated than supposed simple electrostatic interactions. Although OCT does disrupt the membrane, our results suggest that its primary selectivity was more linked to mechanical properties of the membrane. On the other hand, CHX did not disrupt membranes as a primary activity, nor did it show any sign of electrostatic selectivity toward negatively charged membranes at any stage of interactions, which suggests membrane disruption by influencing more discrete membrane properties.     

DNA aptamers bind specifically and selectively to (1 → 3)-β-d-glucans

(1 → 3)-β-d-Glucans are structural cell wall components of fungi, plants, and some bacteria and have been linked with human respiratory symptoms following aerosol exposure. A clear interpretation of the health impact of (1 → 3)-β-d-glucans is limited by the high cost and uncertainties associated with current glucan quantitation methods. The objective of this research is to develop DNA aptamers for the measurement of (1 → 3)-β-d-glucans. Aptamers are synthetic DNA functional binding molecules that fold into unique conformations, allowing them to bind specifically to their target. Through the in vitro selection process SELEX, we have produced aptamers that are able to bind with sub-micromolar affinity to curdlan, a linear unbranched form of (1 → 3)-β-d-glucans. These aptamers display high selectivity to curdlan and do not bind to non-(1 → 3)-β-d-polysaccharides, suggesting specificity for the β-(1 → 3)-glycosidic linkage. The aptamers produced here will enable the production of more cost-effective, less ambiguous assays for the environmental measurement of (1 → 3)-β-d-glucans.     

A new β-chain haemoglobin variant with increased oxygen affinity: Hb Roma [β115(g17)Ala → Val]

Background

Haemoglobin Roma [β115(G17)Ala → Val] is a new adult haemoglobin variant found in a patient presenting a mild hypochromia and microcytosis. We studied this previously uncharacterised variant in order to evaluate the effect on the structural and funcional properties of the Ala → Val substitution at the α1β1 interface.

Methods and results

The variant chain was identified by direct DNA sequencing of the β-globin gene, which revealed a GCC → GTC mutation in codon 115. This mutation was confirmed by mass spectrometric analysis of the tetramers and peptides. The oxygen-binding properties of the haemoglobin A/haemoglobin Roma mixture, in which the variant makes up 25% of the haemoglobins, showed a significant increase in oxygen affinity with respect to normal haemoglobin A, both in the absence and presence of 2,3-bisphosphoglycerate. The role of the βG17 position, situated at the α₁β₁ interface, has been examined using computational models of haemoglobin Roma and other known βG17 variants, in comparison with normal haemoglobin A.

Conclusions

This study suggests that the β115(G17)Ala → Val substitution at the α1β1 interface is responsible for increased oxygen affinity and mild destabilisation of the haemoglobin Roma.

General significance

An amino acid substitution at the G17 position of the α1β1 interface may result in stabilisation of the high affinity R-state of the haemoglobin molecule.     

Succinate bridged dimeric Cu(II) system containing sandwiched non-coordinating succinate dianion: Crystal structure, spectroscopic and thermal studies of [(phen)2Cu(μ-L)Cu(phen)2]L · 12.5H2O (H2L = succinic acid; phen = 1,10-phenanthroline)

A mixed ligand and dimeric Cu^II complex [(phen)₂Cu(μ-L)Cu(phen)₂]L · 12.5H₂O (H₂L = succinic acid) containing bridging succinate moiety and also non-coordinated succinate dianion was prepared from polymeric Cu(II) succinate by nucleophilic reaction with o-phenanthroline (phen) followed by depolymerization. The dimeric product was characterized by crystallographic, spectroscopic and thermoanalytical studies. The complex crystallizes in triclinic crystal system and is composed of succinate bridged [(phen)₂Cu(μ-L)Cu(phen)₂]²⁺ complex cations, non-coordinated succinate anions and hydrogen bonded water molecules. Within the dimeric cationic unit, each of the Cu atoms is octahedrally coordinated by four N atoms of both phen ligands and both O atoms of a carboxylate moiety of the bridging succinate group in chelating form. Through intermolecular π-π stacking interactions, the complex cations form positively charged 2-D layers, between which the non-coordinating succinate anions and water molecules are sandwiched. Both the electronic and EPR studies indicate that the dimeric complex undergoes partial dissociation in solution state to exist in two structural forms. The kinetic and thermodynamic parameters involved in three stage thermal decompositions of the dimeric complex could also be evaluated using Coats-Redfern method.     

Metal(II)-organic coordination polymers with two distinct atropisomeric building units from an axially prochiral inner salt through interchain C-H?X hydrogen bonds (X = O, Cl; metal = cobalt, nickel)

Two isostructural photoluminescent metal-organic coordination polymers, namely, [M(H-mpypdc)(Cl)(H₂O)₃]_n (mpypdc = 2,6-dimethyl-4-(pyridin-4-yl)pyridine-3,5-dicarboxylate; M = Co (2) and Ni (3)), were synthesized from H₂mpypdc (1), and characterized by single crystal X-ray analyses. New ligand 1 is an inner salt. In the metal-organic coordination polymeric chains, two distinct S- and R-atropisomeric units are generated from the axially prochiral 1 through C-H?X (X = O, Cl) hydrogen bonds. There have interesting 2D supramolecular networks [M(Cl)(H₂O)₂]_n in the crystal structures of title compounds. The photoluminescence of new compounds are also investigated in solid state at ambient temperature.     

δ-Opioid receptor-stimulated Akt signaling in neuroblastoma × glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

δ-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the “pro-survival” kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma × glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen^2,5]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G_i/o proteins, because pre-treatment with pertussis toxin, but not over-expression of the G_q/11 scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the Gβγ scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.     

Interleukin-1α − 899 (+ 4845) C→T polymorphism increases the risk of chronic periodontitis: Evidence from a meta-analysis of 23 case–control studies

Many epidemiological studies have indicated that interleukin-1α (IL-1α) − 899 (+ 4845) C→T polymorphism increases the risk of chronic periodontitis (CP), whereas some studies have reported opposite results. Accordingly, the aim of this meta-analysis is to investigate the association of the IL-1α − 899 (+ 4845) C→T polymorphism with CP. We searched the PubMed database up to May 1, 2013 and finally obtained 23 case–control studies. After data extraction, we performed meta-analysis using Comprehensive Meta-Analysis v2.2 software. The overall result based on the fixed-effect model showed that IL-1α − 899 (+ 4845) C→T polymorphism was significantly associated with increased risk of CP: [odds ratio (OR) = 1.29, 95% confidence interval (CI) = 1.15–1.44, p < 0.001] for T vs. C; (OR = 1.59, 95%CI = 1.22–2.07, p = 0.0005) for TT vs. CC; (OR = 1.30, 95% CI = 1.12–1.51, p = 0.0004) for CT vs. CC; and (OR = 1.40, 95% CI = 1.21–1.61, p < 0.001) for (CT+TT) vs. CC; (OR = 1.47, 95% CI = 1.16–1.87, p = 0.002) for TT vs. (CT+CC). Stratified analyses revealed that there was a significantly increased risk for Caucasians and Asians. In conclusion, current evidence showed that IL-1α − 899 (+ 4845) C→T polymorphism probably increased the risk of CP.     

IF<Subscript>1</Subscript> distribution in HepG2 cells in relation to ecto–F<Subscript>0</Subscript>F<Subscript>1</Subscript>ATPsynthase and calmodulin

F₀F₁ATPsynthase is now known to be expressed as a plasma membrane receptor for several extracellular ligands. On hepatocytes, ecto–F₀F₁ATPsynthase binds apoA–I and triggers HDL endocytosis concomitant with ATP hydrolysis. Considering that inhibitor protein IF₁ was shown to regulate the hydrolytic activity of ecto–F₀F₁ATPsynthase and to interact with calmodulin (CaM) in vitro, we investigated the subcellular distributions of IF₁, calmodulin (CaM), OSCP and β subunits of F₀F₁ATPsynthase in HepG2 cells. Using immunofluorescence and Western blotting, we found that around 50% of total cellular IF₁ is localized outside mitochondria, a relevant amount of which is associated to the plasma membrane where we also found Ca²⁺–CaM, OSCP and β. Confocal microscopy showed that IF₁ colocalized with Ca²⁺–CaM on plasma membrane but not in mitochondria, suggesting that Ca²⁺–CaM may modulate the cell surface availability of IF₁ and thus its ability to inhibit ATP hydrolysis by ecto–F₀F₁ATPsynthase. These observations support a hypothesis that the IF₁–Ca²⁺–CaM complex, forming on plasma membrane, functions in the cellular regulation of HDL endocytosis by hepatocytes.     

Metal–organic chemical vapor deposition of Bi2Mn4O10 films on SrTiO3 〈1 0 0〉

Bi₂Mn₄O₁₀ films were deposited on SrTiO₃ (1 0 0) substrates via metal–organic chemical vapor deposition (MOCVD) from the Bi(phenyl)₃ and Mn(tmhd)₃ (Htmhd = 2,2,6,6-tetramethyl-3,5-heptanedione) precursors. The films were deposited in the temperature range of 600–800 °C. The X-ray diffraction (XRD) characterization indicates that the Bi₂Mn₄O₁₀ phase is stable within the investigated range, but the temperature plays a crucial role in determining the out-of-plane orientation of the films. The SEM shows very homogeneous surfaces with a fiber texture morphology at the highest deposition temperature. The AFM data indicate a textured surface with a root mean square roughness of 77.67 nm for films deposited at 800 °C.     

Comparative genomic study of <Emphasis Type="Italic">spo0E</Emphasis> family genes and elucidation of the role of Spo0E in <Emphasis Type="Italic">Bacillus anthracis</Emphasis>

The propensity of bacterium to sporulate or retain the vegetative form depends on the amount of phosphorylated Spo0A (Spo0A^-P), regulated by Spo0E multigene family of phosphatases (Spo0E, YisI and YnzD). Phylogenetic analysis revealed that Spo0E multigene family of phosphatases (SMFP) descends in two distinct clades of aerobic (Bacillus cluster) and anaerobic (Clostridia cluster) sporulating bacteria. High sequence conservation within species gives a notion that these members could have evolved through lineage and species-specific duplication event. Of the five genes in Bacillus cereus group, three are pathogen specific, and their synteny suggests that these paralogs could be involved in the regulation of amino acid metabolism and its transport. Overexpression of B. subtilis Spo0E, an ortholog of SMFP members in B. anthracis (BAS1251), resulted in sporulation deficient phenotype in B. anthracis. B. anthracis Spo0A^-P binds to a consensus DNA sequence 5′-TGNCGAA-3′ (‘0A-like box’) and loses its DNA binding ability following treatment with B. subtilis Spo0E. Thus, B. subtilis Spo0E acts on B. anthracis Spo0A^-P and, therefore could complement the function of BAS1251. Further, since ‘0A-like box’ are present in the promoter region of abrB gene, a known regulator of anthrax toxin gene expression, cross talk among SMFP members and Spo0A^-P–AbrB could regulate the expression of anthrax toxin genes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Preparation of (1 → 3)-β-d-glucan oligosaccharides by hydrolysis of curdlan with commercial α-amylase

Water soluble (1 → 3)-β-d-glucan oligosaccharides were prepared by hydrolyzing curdlan with α-amylase. The hydrolysis process was monitored by the DE values of the hydrolysates. Under the optimized conditions (pH, 5.98; temperature, 55.92 °C; α-amylase amount, 31.94 mg α-amylase/500 mL of reaction mixture containing 5 g curdlan; reaction time, 30 min), maximum DE value (15.62%) was obtained. The resulting products were composed of (1 → 3)-β-d-glucan oligosaccharides of DP 2-9. The hydrolysates were filtered, concentrated to ∼20% (w/v), and precipitated with 5 volumes of ethanol, which were then freeze dried to yield a water soluble powder. The (1 → 3)-β-d-glucan oligosaccharides content of the product and the yield were 97.7% and 97.6% (w/w), respectively.