Identification of osteopontin phosphorylation sites involved in bone remodeling and inhibition of pathological calcification

Osteopontin is a noncollagenous, phosphorylated extracellular glycoprotein, expressed in mineralized and nonmineralized tissues, organs and body fluids. The protein contains an RGD tripeptide cell-binding motif, and is subjected to a variety of posttranslational modifications that play important roles in its multiple biological functions, such as bone remodeling and inhibition of pathological calcification. In this study, we have expressed bovine osteopontin in a prokaryotic system and identified the seven amino acid residues phosphorylated in vitro by CKII.     

Sequence structure of hidden 10.4-base repeat in the nucleosomes of C. elegans

By measuring prevailing distances between YY, YR, RR, and RY dinucleotides in the large database of the nucleosome DNA fragments from C. elegans, the consensus sequence structure of the nucleosome DNA repeat of C. elegans was reconstructed: (YYYYYRRRRR)n. An actual period was estimated to be 10.4 bases. The pattern is fully consistent with the nucleosome DNA patterns of other eukaryotes, as established earlier, and, thus, the YYYYYRRRRR repeat can be considered as consensus nucleosome DNA sequence repeat across eukaryotic species. Similar distance analysis for [A, T] dinucleotides suggested the related pattern (TTTYTARAAA)n where the TT and AA dinucleotides display rather out of phase behavior, contrary to the "AA or TT" in-phase periodicity, considered in some publications. A weak 5-base periodicity in the distribution of TA dinucleotides was detected.     

Glutathione modulates Ca(2+) influx and oxidative toxicity through TRPM2 channel in rat dorsal root ganglion neurons

Glutathione (GSH) is the most abundant thiol antioxidant in mammalian cells and maintains thiol redox in the cells. GSH depletion has been implicated in the neurobiology of sensory neurons. Because the mechanisms that lead to melastatin-like transient receptor potential 2 (TRPM2) channel activation/inhibition in response to glutathione depletion and 2-aminoethyldiphenyl borinate (2-APB) administration are not understood, we tested the effects of 2-APB and GSH on oxidative stress and buthionine sulfoximine (BSO)-induced TRPM2 cation channel currents in dorsal root ganglion (DRG) neurons of rats. DRG neurons were freshly isolated from rats and the neurons were incubated for 24 h with BSO. In whole-cell patch clamp experiments, TRPM2 currents in the rat were consistently induced by H₂O₂ or BSO. TRPM2 channels current densities and cytosolic free Ca²⁺ content of the neurons were higher in BSO and H₂O₂ groups than in control. However, the current densities and cytosolic Ca²⁺ release were also higher in the BSO + H₂O₂ group than in the H₂O₂ alone. When intracellular GSH is introduced by pipette TRPM2 channel currents were not activated by BSO, H₂O₂ or rotenone. BSO and H₂O₂-induced Ca²⁺ gates were blocked by the 2-APB. Glutathione peroxidase activity, lipid peroxidation and GSH levels in the DRG neurons were also modulated by GSH and 2-APB inhibition. In conclusion, we observed the protective role of 2-APB and GSH on Ca²⁺ influx through a TRPM2 channel in intracellular GSH depleted DRG neurons. Since cytosolic glutathione depletion is a common feature of neuropathic pain and diseases of sensory neuron, our findings are relevant to the etiology of neuropathology in DRG neurons.     

Practice and Attitude of Cigarette Smoking: A Community-Based Study

Background

In Saudi Arabia many studies have addressed cigarette smoking from various perspectives. Most of these studies, however, were conducted among males and confined to Riyadh, the capital city. Such limitations have enhanced the need for community-based epidemiological studies that include both genders and various age groups and socio-demographic features, as well as different regions.

Objective

This cross-sectional study aims to assess the prevalence of cigarette smoking and to discuss the association between cigarette smoking habits and socio-demographic factors among community members of the Jazan area in southwest Saudi Arabia.

Methods

A pre-coded questionnaire was designed and tested for data consistency. A well-trained health team was assigned to gather the data from the 30 primary healthcare centers distributed across eight provinces. The response rate was 92.8% (4,326 respondents ≥13 years old). The associations among the subjects'' socio-demographic characteristics were examined by the chi-square test. A multiple logistic regression and odds ratios were calculated as well.

Results

A total of 1,017 (23.5%), 1,042 (24.1%), and 3,284 (75.9%) respondents were, respectively, current smokers (TCS), ever-smokers (TES), and non-smokers (TNS). Though current smokers seem to be more prevalent in urban populations (13.8%) than in rural populations (9.7%), the association of urbanization with a current smoking habit is insignificant.

Conclusion

Having fun, relieving stress, and the influence of parents, particularly of mothers, were the main motives that encouraged participants'' cigarette-smoking habits. This situation was worsened by the fact that accessing cigarettes was either very easy or easy for over 90% of the respondents.     

Purification and characterization of a bacteriocin from an oenological strain of Leuconostoc mesenteroides subsp. cremoris

Malolactic fermentation (MLF), which improves organoleptic properties and biologic stability of some wines, may cause wine spoilage if uncontrolled. Bacteriocins were reported as efficient preservatives to control MLF through their bactericidal effect on malolactic bacteria. Leuconostoc mesenteroides subsp. cremoris W3 isolated from wine produces an inhibitory substance that is bactericidal against malolactic bacteria in model wine medium. Treatment of the culture supernatant of strain W3 with proteases eliminated the inhibitory activity, which proved that it is a true bacteriocin and we tentatively termed it mesentericin W3. The bacteriocin inhibited the growth of food-borne pathogenic bacteria such as Enterococcus faecalis, Listeria monocytogenes, and malolactic bacteria. It was active over a wide pH range and stable to organic solvents and heat. Mesentericin W3 was purified to homogeneity by a pH-mediated cell adsorption–desorption method, cation exchange, hydrophobic interaction, and reverse-phase chromatography. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectroscopy (MS) and partial amino acid sequence analysis revealed that mesentericin W3 was identical to mesentericin Y105.     

Formation of Adeno-Associated Virus Circular Genomes Is Differentially Regulated by Adenovirus E4 ORF6 and E2a Gene Expression

A central feature of the adeno-associated virus (AAV) latent life cycle is persistence in the form of both integrated and episomal genomes. However, the molecular processes associated with episomal long-term persistence of AAV genomes are only poorly understood. To investigate these mechanisms, we have utilized a recombinant AAV (rAAV) shuttle vector to identify circular AAV intermediates from transduced HeLa cells and primary fibroblasts. The unique structural features exhibited by these transduction intermediates included circularized monomer and dimer virus genomes in a head-to-tail array, with associated specific base pair alterations in the 5′ viral D sequence. In HeLa cells, the abundance and stability of AAV circular intermediates were augmented by adenovirus expressing the E2a gene product. In the absence of E2a, adenovirus expressing the E4 open reading frame 6 gene product decreased the abundance of AAV circular intermediates, favoring instead the linear replication form monomer (Rf_m) and dimer (Rf_d) structures. In summary, the formation of AAV circular intermediates appears to represent a new pathway for AAV genome conversion, which is consistent with the head-to-tail concatemerization associated with latent-phase persistence of rAAV. A better understanding of this pathway may increase the utility of rAAV vectors for gene therapy.     

Photosynthetic formation of inorganic pyrophosphate in phototrophic bacteria

In this paper we report studies on photosynthetic formation of inorganic pyrophosphate (PP_i) in three phototrophic bacteria. Formation of PP_i was found in chromatophores from Rhodopseudomonas viridis but not in chromatophores from Rhodopseudomonas blastica and Rhodobacter capsulatus. The maximal rate of PP_i synthesis in Rps. viridis was 0.15 mol PP_i formed/(min*mol Bacteriochlorophyll) at 23°C. The synthesis of PP_i was inhibited by electron transport inhibitors, uncouplers and fluoride, but was insensitive to oligomycin and venturicidin. The steady state rate of PP_i synthesis under continuous illumination was about 15% of the steady-state rate of ATP synthesis. The synthesis of PP_i after short light flashes was also studied. The yield of PP_i after a single 1 ms flash was equivalent to approximately 1 mol PP_i/500 mol Bacteriochlorophyll. In Rps. viridis chromatophores, PP_i was also found to induce a membrane potential, which was sensitive to carbonyl cyanide p-trifluoromethoxyphenylhydrazone and NaF.Abbreviations BChl Bacteriochlorophyll - F₀F₁-ATPase Membrane bound proton translocating ATP synthase - FCCP Carbonyl cyanide p-trifluoromethoxyphenylhydrazone - H⁺-PPase Membrane bound proton translocating PP_i synthase - TPP⁺ Tetraphenyl phosphonium ion - TPB^- Tetraphenyl boron ion - Transmembrane electrical potential difference     

Two-hydronic-reactive states of acetylcholinesterase, mechanistically relevant acid-base catalyst of pKa 6.5 and a modulatory group of pKa 5.5

Variation of experimentally observed pKa values in pH-dependent kinetic studies using acetylcholinesterase (AcChE) is rationalized by proposal of two-hydronic-reactive states, EH and EH2, of the free AcChE molecule. Two kinetically influential ionizations with pKa 6.5 for the general acid-base catalyst, possibly the imidazole group of histidine, and a modulatory group with pKa 5.5 residing at the juxtaposal modulatory site, provided fundamental bases for the observed variation in pK(app) values. Appropriate equations applicable to the proposed kinetic model in conjunction with pKa values (pKI 5.5, pKII 6.5) and relative varied values of the pH-independent rate constants, k'cat/K'm and kcat/Km, of the reactive states were used to generate computer simulation error-free pH-rate profiles. A series of theoretical apparently simple sigmoidal pH-rate profiles with characterizing parameters pK(app) varying between 5.5-6.5 were obtained. Ionization of a modulatory group with pKa 5.5 alone modifies the reaction mechanism of AcChE, and binding of substrates and inhibitors at this site provides modulation of catalysis/binding at the active center. Analysis of the relative magnitudes of pH-independent rate constants for the two reactive states revealed that in terms of the overall catalysis, the EH state shows favorable reactivity towards the cationic reagents with reactivity 1.0, as compared to the EH2 state with reactivities 0.25-0.55. Neutral reagents, in general, make use of the EH2 state more than cationic reagents, with reactivities 1.0 for the EH state and 0.3-1.0 for the EH2 state. Further analysis showed that this discrimination between the two reactive states, by both types of reagents, occurs predominantly through the difference in binding constants K'm and Km. Relative binding of a given cationic reagent to the respective reactive states ranges from K'm = 1.8 X Km to 4.0 X Km, and from K'm = 1.0 X Km to 2.0 X Km for the neutral reagents.