Modulation of gastric acid secretion by cannabinoids in rats

The current study aimed to evaluate the role of cannabinoid receptors in the regulation of gastric acid secretion and oxidative stress in gastric mucosa. To fulfill this aim, gastric acid secretion stimulated with histamine (5 mg/kg, subcutaneous [SC]), 2‐deoxy‐ d ‐glucose (D‐G) (200 mg/kg, intravenous) or ‐carbachol (4 μg/kg, SC) in the 4‐hour pylorus‐ligated rats. The CB1R agonist ( N‐arachidonoyl dopamine, 1 mg/kg, SC) inhibited gastric acid secretion stimulated by D‐G and carbachol but not in histamine, reduced pepsin content, and increased mucin secretion. Furthermore, it decreased malondialdehyde (MDA) and nitric oxide (NO) contents with an increase in glutathione (GSH) and paraoxonase 1 (PON‐1). Meanwhile, CB2R antagonist (AM630, 1 mg/kg, SC) inhibited gastric acid secretion stimulated by D‐G and reduced MDA and NO contents with an increase in GSH and PON‐1. Meanwhile, CB1R antagonist rimonabant or CB2R agonist GW 405833 had no effect on stimulated gastric acid secretion. Therefore, both CB1R agonist and CB2R antagonist may exert antisecretory and antioxidant potential in the stomach.     

Combination of arsenic trioxide and cisplatin synergistically inhibits both hexokinase activity and viability of Ehrlich ascites carcinoma cells

Hexokinase‐2 is overexpressed in several carcinomas including breast cancer to sustain energy for rapidly dividing cells and associates with chemoresistance. However, the impact of chemo drugs (alone or in combination) on hexokinase activity and autophagic cell death is unclear. In this report, we used an in vivo murine adenocarcinoma model to validate the effects of As₂O₃ and cisplatin on hexokinase activity and autophagic cancer cell death. We found that the two drugs inhibit hexokinase activity and induce autophagic marker, beclin 1 expression. Interestingly, combining As₂O₃ with cisplatin synergistically enhanced these effects and alleviated oxidative stress often encountered in As₂O₃ treatment. Altogether, our data provide direct evidence that inhibition of hexokinase activity and induction of autophagic cell death are mediating the antineoplastic effects of As₂O₃ and cisplatin. Our findings raise the potential of combining As₂O₃ with cisplatin as an approach to augment cisplatin‐induced cell death and combat cisplatin chemoresistance in cancer.     

The abp gene in Geobacillus stearothermophilus T-6 encodes a GH27 β-L-arabinopyranosidase

In this study we demonstrate that the abp gene in Geobacillus stearothermophilus T-6 encodes a family 27 glycoside hydrolase β-L-arabinopyranosidase. The catalytic constants towards the chromogenic substrate pNP-β-L-arabinopyranoside were 0.8±0.1 mM, 6.6±0.3 s(-1), and 8.2±0.3 s(-1) mM(-1) for K(m), k(cat) and k(cat)/K(m), respectively. (13)C NMR spectroscopy unequivocally showed that Abp is capable of removing β-L-arabinopyranose residues from the natural arabino-polysaccharide, larch arabinogalactan. Most family 27 enzymes are active on galactose and contain a conserved Asp residue, whereas in Abp this residue is Ile67, which shifts the specificity of the enzyme towards arabinopyranoside.     

Dual Nuclease and Helicase Activities of Helicobacter pylori AddAB Are Required for DNA Repair, Recombination, and Mouse Infectivity

Helicobacter pylori infection of the human stomach is associated with disease-causing inflammation that elicits DNA damage in both bacterial and host cells. Bacteria must repair their DNA to persist. The H. pylori AddAB helicase-exonuclease is required for DNA repair and efficient stomach colonization. To dissect the role of each activity in DNA repair and infectivity, we altered the AddA and AddB nuclease (NUC) domains and the AddA helicase (HEL) domain by site-directed mutagenesis. Extracts of Escherichia coli expressing H. pylori addA^NUCB or addAB^NUC mutants unwound DNA but had approximately half of the exonuclease activity of wild-type AddAB; the addA^NUCB^NUC double mutant lacked detectable nuclease activity but retained helicase activity. Extracts with AddA^HELB lacked detectable helicase and nuclease activity. H. pylori with the single nuclease domain mutations were somewhat less sensitive to the DNA-damaging agent ciprofloxacin than the corresponding deletion mutant, suggesting that residual nuclease activity promotes limited DNA repair. The addA^NUC and addA^HEL mutants colonized the stomach less efficiently than the wild type; addB^NUC showed partial attenuation. E. coli ΔrecBCD expressing H. pylori addAB was recombination-deficient unless H. pylori recA was also expressed, suggesting a species-specific interaction between AddAB and RecA and also that H. pylori AddAB participates in both DNA repair and recombination. These results support a role for both the AddAB nuclease and helicase in DNA repair and promoting infectivity.Infection of the stomach with Helicobacter pylori causes a variety of diseases including gastritis, peptic ulcers, and gastric cancer (1). A central feature of the pathology of these conditions is the establishment of a chronic inflammatory response that acts both on the host and the infecting bacteria (2). Both epithelial (3, 4) and lymphoid (5, 6) cells in the gastric mucosa of infected individuals release DNA-damaging agents that can introduce double-stranded (ds)² breaks into the bacterial chromosome (7). The ds breaks must be repaired for the bacteria to survive and establish chronic colonization of the stomach. Homologous recombination is required for the faithful repair of DNA damage and bacterial survival. Alteration of the expression of one of a series of cell surface proteins on H. pylori occurs by an apparent gene conversion of babA, the frequency of which is reduced in repair-deficient strains (8, 9). This change in the cell surface, which may allow H. pylori to evade the host immune response, is a second means by which recombination can promote efficient colonization of the stomach by H. pylori.The initiation or presynaptic steps of recombination at dsDNA breaks in most bacteria involves the coordinated action of nuclease and helicase activities provided by one of two multisubunit enzymes, the AddAB and RecBCD enzymes (10). Escherichia coli recBCD null mutants have reduced cell viability, are hypersensitive to DNA-damaging agents, and are homologous recombination-deficient (11–14). Similarly, H. pylori addA and addB null mutants are hypersensitive to DNA-damaging agents, have reduced frequencies of babA gene conversion, and colonize the stomach of mice less efficiently than wild-type strains (8).The activities of RecBCD enzyme from E. coli (15–19) and AddAB from H. pylori (8) or Bacillus subtilis (20–23) indicate some common general features of the presynaptic steps of DNA repair. In the case of E. coli, repair begins when the RecBCD enzyme binds to a dsDNA end and unwinds the DNA using its ATP-dependent helicase activities (17, 24). Single-stranded (ss) DNA produced during unwinding, with or without accompanying nuclease, is coated with RecA protein (16, 25). This recombinogenic substrate engages in strand exchange with a homologous intact duplex to form a joint molecule. Joint molecules are thought to be converted into intact, recombinant DNA either by replication or by cutting and ligation of exchanged strands (26).Although the AddAB and RecBCD enzymes appear to play similar roles in promoting recombination and DNA repair, they differ in several ways. RecBCD is a heterotrimer, composed of one copy of the RecB, RecC, and RecD gene products (27), whereas AddAB has two subunits, encoded by the addA and addB genes (21, 28). The enzyme subunit(s) responsible for helicase activity can be inferred from the presence of conserved protein domains or the activity of purified proteins. AddA, RecB, and RecD are superfamily I helicases with six highly conserved helicase motifs, including the conserved Walker A box found in many enzymes that bind ATP (29–32). A Walker A box is defined by the consensus sequence (G/A)XXGXGKT (X is any amino acid (29). RecBCD enzymes in which the conserved Lys in this motif is changed to Gln have a reduced affinity for ATP binding (33, 34) and altered helicase activity (17, 35–37).A nuclease domain with the conserved amino acid sequence LDYK is found in RecB, AddA, AddB, and many other nucleases (38). The conserved Asp plays a role in Mg²⁺ binding at the active site; Mg²⁺ is required for nuclease activity (39). The recB1080 mutation, which changes codon 1080 from the conserved Asp in this motif to Ala, eliminates nuclease activity (39).We have recently shown that addA and addB deletion mutants are hypersensitive to DNA-damaging agents and impaired in colonization of the mouse stomach compared with wild-type strains (8). To determine the roles of the individual helicase and nuclease activities of H. pylori AddAB in DNA repair and infectivity, we used site-directed mutagenesis to inactivate the conserved nuclease domains of addA and addB and the conserved ATPase (helicase) domain of AddA. Here, we report that loss of the AddAB helicase is sufficient to impair H. pylori DNA repair and infectivity and, when the genes are expressed in E. coli, homologous recombination. AddAB retains partial activity in biochemical and genetic assays when either of the two nuclease domains is inactivated but loses all detectable nuclease activity when both domains are inactivated. Remarkably, H. pylori AddAB can produce recombinants in E. coli only in the presence of H. pylori RecA, suggesting a species-specific interaction in which AddAB facilitates the production of ssDNA-coated with RecA protein. Our results show that both the helicase and nuclease activities are required for the biological roles of H. pylori AddAB.     

Antimicrobial activity and phytochemical analyses of Polygonum aviculare L. (Polygonaceae), naturally growing in Egypt

Polygonum aviculare (Polygonaceae) is an herb commonly distributed in Mediterranean coastal regions in Egypt and used in folkloric medicine. Organic and aqueous solvent extracts and fractions of P. aviculare were investigated for antimicrobial activities on several microorganisms including bacteria and fungi. Phytochemical constituents of air-dried powered plant parts were extracted using aqueous and organic solvents (acetone, ethanol, chloroform and water). Antimicrobial activity of the concentrated extracts was evaluated by determination of the diameter of inhibition zone against both Gram-negative and Gram-positive bacteria and fungi using paper disc diffusion method.Results of the phytochemical studies revealed the presence of tannins, saponins, flavonoids, alkaloids and sesquiterpenes and the extracts were active against both Gram-negative and Gram-positive bacteria. Chloroform extract gave very good and excellent antimicrobial activity against all tested bacteria and good activity against all tested fungi except Candida albicans. Structural spectroscopic analysis that was carried out on the active substances in the chloroform extract led to the identification of panicudine (6-hydroxy-11-deoxy-13 dehydrohetisane).Evaluation of the antimicrobial activity of panicudine indicated significant activity against all tested Gram-negative and Gram-positive organisms. Panicudine displayed considerable activity against the tested fungi with the exception of C. albicans. Antimicrobial activity of the extracts was unaffected after exposure to different heat treatments, but was reduced at alkaline pH. Studies of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of panicudine on the tested organisms showed that the lowest MIC and the MBC were demonstrated against Salmonella paratyphi, Bacillus subtilis and Salmonella typhi and the highest MIC and MBC were against Staphylococcus aureus.     

Preferential Cu2+ coordination by His96 and His111 induces beta-sheet formation in the unstructured amyloidogenic region of the prion protein

The prion protein (PrP) is a Cu(2+) binding cell surface glycoprotein that can misfold into a beta-sheet-rich conformation to cause prion diseases. The majority of copper binding studies have concentrated on the octarepeat region of PrP. However, using a range of spectroscopic techniques, we show that copper binds preferentially to an unstructured region of PrP between residues 90 and 115, outside of the octarepeat domain. Comparison of recombinant PrP with PrP-(91-115) indicates that this prion fragment is a good model for Cu(2+) binding to the full-length protein. In contrast to previous reports we show that Cu(2+) binds to this region of PrP with a nanomolar dissociation constant. NMR and EPR spectroscopy indicate a square-planar or square-pyramidal Cu(2+) coordination utilizing histidine residues. Studies with PrP analogues show that the high affinity site requires both His(96) and His(111) as Cu(2+) ligands, rather than a complex centered on His(96) as has been previously suggested. Our circular dichroism studies indicate a loss of irregular structure on copper coordination with an increase in beta-sheet conformation. It has been shown that this unstructured region, between residues 90 and 120, is vital for prion propagation and different strains of prion disease have been linked with copper binding. The role of Cu(2+) in prion misfolding and disease must now be re-evaluated in the light of these findings.