Role of P2z purinergic receptors in ATP-mediated killing of tumor necrosis factor (TNF)-sensitive and TNF-resistant L929 fibroblasts.

Two closely related cell lines were characterized in their responses to extracellular ATP (ATPo): the fibroblast cell line L929 and a TNF-resistant variant L929/R. Both lines showed ATPo-activated increases in intracellular Ca2+, inward current, and sustained depolarization of the plasma membrane, cell responses compatible with activation of purinergic receptors of the P2y, P2x, or P2z subtype; however, only the L929/R variant was susceptible to ATPo-dependent early permeabilization of the plasma membrane to hydrophilic solutes of M(r) below 900, a response uniquely caused by the activation of P2z receptors. Both cell types were susceptible to the cytotoxic effect of ATPo, but killing of the L929/R variant required much shorter incubations in the presence of this nucleotide. Morphologic examination of ATPo-challenged L929 and L929/R cells showed that cell death occurred by two alternative mechanisms: colloido-osmotic lysis or apoptosis. Occurrence of apoptosis was confirmed by agarose gel analysis of cellular DNA. Although ATPo caused a fast mobilization of intracellular Ca2+, neither colloido-osmotic lysis nor apoptosis were Ca2+ dependent. Our results show that the L929/R variant, but not the L929 parental fibroblast cell line, expresses functional purinergic receptors of the P2z subtype. The presence of P2z receptors confers to L929/R cells enhanced susceptibility to ATPo-mediated cytotoxicity.     

Large scale purification of factor X by hydrophobic chromatography

Factor X is a critical enzyme in the blood coagulation cascade, however, in recent years the coagulation zymogen factor X has received additional interest as a selective proteinase to allow production of functional eukaryotic proteins in a prokaryotic expression system. Traditional factor X purification schemes suffer from low yields, low capacity, lengthy dialysis steps, and contamination by the autoproteolytic activated enzyme factor Xa. By incorporating a reversible inhibitor of factor X activation, we were able to recover 67% of the factor X present without any detectable activated enzyme. Six liters of plasma could be processed onto a 50 mL phenylalanine-Sepharose hydrophobic chromatography column without saturating the matrix. The final product is devoid of detectable proteolytic activity. At time of use, the zymogen is specifically activated with a Sepharose-bound activating enzyme isolated from Russell's Viper Venom, resulting in factor Xa free of other detectable proteinases.     

Responses of mouse lymphocytes to extracellular ATP. II. Extracellular ATP causes cell type-dependent lysis and DNA fragmentation

Extracellular ATP (ATPo) caused dose-dependent lysis of YAC-1 and P-815 mouse tumor cells. This event, assessed by 51Cr release, was accompanied by sustained depolarization of the plasma membrane potential and Ca2+ influx. Plasma membrane depolarization and Ca2+ influx occurred within a few seconds of ATPo addition to both cell types, whereas 51Cr was released without apparent lag in YAC-1 cells and after 2 h in P-815 cells. Furthermore, a rise in [Ca2+]i was required for ATPo-dependent lysis of YAC-1 but not P-815 cells. In P-815 cells, ATPo caused an early and [Ca2+]i-independent DNA fragmentation that occurred at lower nucleotide concentrations than those required to trigger 51Cr release. Instead in YAC-1 cells very low concentrations of ATPo caused early lysis (ED50 for lysis about 200 microM) accompanied by only barely detectable DNA fragmentation. Previous studies disclosed that lymphokine-activated killer cells are fully resistant to the membrane-perturbing effects of ATPo. We show that lymphokine-activated killer cells also do not undergo DNA fragmentation even in the presence of high ATPo concentrations. This study complements previous observations on the lytic effects of ATPo and shows that this nucleotide can also cause DNA fragmentation, one of the earliest target cell alterations observed during CTL-mediated lysis.     

The Toluene o-Xylene Monooxygenase Enzymatic Activity for the Biosynthesis of Aromatic Antioxidants

Monocyclic phenols and catechols are important antioxidant compounds for the food and pharmaceutic industries; their production through biotransformation of low-added value starting compounds is of major biotechnological interest. The toluene o-xylene monooxygenase (ToMO) from Pseudomonas sp. OX1 is a bacterial multicomponent monooxygenase (BMM) that is able to hydroxylate a wide array of aromatic compounds and has already proven to be a versatile biochemical tool to produce mono- and dihydroxylated derivatives of aromatic compounds. The molecular determinants of its regioselectivity and substrate specificity have been thoroughly investigated, and a computational strategy has been developed which allows designing mutants able to hydroxylate non-natural substrates of this enzyme to obtain high-added value compounds of commercial interest. In this work, we have investigated the use of recombinant ToMO, expressed in cells of Escherichia coli strain JM109, for the biotransformation of non-natural substrates of this enzyme such as 2-phenoxyethanol, phthalan and 2-indanol to produce six hydroxylated derivatives. The hydroxylated products obtained were identified, isolated and their antioxidant potential was assessed both in vitro, using the DPPH assay, and on the rat cardiomyoblast cell line H9c2. Incubation of H9c2 cells with the hydroxylated compounds obtained from ToMO-catalyzed biotransformation induced a differential protective effect towards a mild oxidative stress induced by the presence of sodium arsenite. The results obtained confirm once again the versatility of the ToMO system for oxyfunctionalization reactions of biotechnological importance. Moreover, the hydroxylated derivatives obtained possess an interesting antioxidant potential that encourages the use of the enzyme for further functionalization reactions and their possible use as scaffolds to design novel bioactive molecules.     

The ‘Coptic question’ in post-revolutionary Egypt: citizenship,democracy, religion

Relations between the Coptic minority and the Egyptian state have gone through different stages, with Copts experiencing greater or lesser degrees of integration into, or alienation from, the social and civil fabric of Egypt. This paper traces the long and ongoing path of the Coptic community from dhimmah to citizenship, a path that is not yet concluded, with a particular focus on the relations between the Copts and Al-Azhar, especially in the transition period opened by the 25 January Revolution. New discourses among both Islamic and Christian intellectuals are examined in order to search out the conceptions of citizenship emerging both in Al-Azhar and Islamist scholars’ thought and in Coptic circles.     

Cross-generational impact of a male murine pheromone 2-sec-butyl-4,5-dihydrothiazole in female mice

The current understanding of the activity of mammalian pheromones is that endocrine and behavioural effects are limited to the exposed individuals. Here, we demonstrate that the nasal exposure of female mice to a male murine pheromone stimulates expansion of mammary glands, leading to prolonged nursing of pups. Subsequent behavioural testing of the pups from pheromone-exposed dams exhibited enhanced learning. Sialic acid components in the milk are known to be involved in brain development. We hypothesized that the offspring might have received more of this key nutrient that promotes brain development. The mRNA for polysialyltransferase, which produces polysialylated neural cell adhesion molecules related to brain development, was increased in the brain of offspring of pheromone-exposed dams at post-natal day 10, while it was not different at embryonic stages, indicating possible differential brain development during early post-natal life.     

An alternative gas-phase in vitro exposure system for toxicity testing: the interaction between nitrous oxide and A549 cells

An original in vitro approach was adopted to expose cells to volatile agents. The anaesthetic nitrous oxide (N(2)O) was chosen as the model agent, and type II pneumocyte-like cells (A549 cells) were used as the target to represent the lungs. A time-lapse microscopy station was equipped with a manual gas mixer that allowed the generation of a mixture of N(2)O/air/CO(2) in the gas phase, to provide a uniform distribution of the volatile agent. The dissolution of N(2)O in the culture medium was monitored by gas chromatography-electron capture detection. Biochemical alterations, in terms of homocysteine accumulation, demonstrated that intracellular methionine synthase had been inactivated by N(2)O absorbed by the cells, a process that also occurs in vivo. Toll-like receptors, which are key molecules in inflammatory lung diseases, were also investigated at the molecular level. Our experiments indicated that biochemical and molecular alterations occurred in the cells, even under conditions where neither morphologic changes nor consistent alterations in cell proliferation were evident. This in vitro exposure system can be efficiently adopted for looking at the repeat-dose effects of volatile agents on respiratory tissues. Moreover, it could be of further benefit for identifying the wide range of specific cell targets, and for monitoring relevant endpoints in the cellular and molecular processes that occur during exposure to volatile compounds.     

Structure-cytotoxicity relationships in bovine seminal ribonuclease: new insights from heat and chemical denaturation studies on variants

Bovine seminal ribonuclease (BS-RNase), a homodimeric protein displaying selective cytotoxicity towards tumor cells, is isolated as a mixture of two isoforms, a dimeric form in which the chains swap their N-termini, and an unswapped dimer. In the cytosolic reducing environment, the dimeric form in which the chains swap their N-termini is converted into a noncovalent dimer (termed NCD), in which the monomers remain intertwined through their N-terminal ends. The quaternary structure renders the reduced protein resistant to the ribonuclease inhibitor, a protein that binds most ribonucleases with very high affinity. On the other hand, upon selective reduction, the unswapped dimer is converted in two monomers, which are readily bound and inactivated by the ribonuclease inhibitor. On the basis of these considerations, it has been proposed that the cytotoxic activity of BS-RNase relies on the 3D structure and stability of its NCD derivative. Here, we report a comparison of the thermodynamic and chemical stability of the NCD form of BS-RNase with that of the monomeric derivative, together with an investigation of the thermal dissociation mechanism revealing the presence of a dimeric intermediate. In addition, we report that the replacement of of Arg80 by Ser significantly decreases the cytotoxic activity of BS-RNase and the stability of the NCD form with respect to the parent protein, but does not affect the ribonucleolytic activity or the dissociation mechanism. The data show the importance of Arg80 for the cytotoxicity of BS-RNase, and also support the hypothesis that the reduced derivative of BS-RNase is responsible for its cytotoxic activity.     

Shape--but not size--codivergence between male and female copulatory structures in Onthophagus beetles

Genitalia are among the fastest evolving morphological traits in arthropods. Among the many hypotheses aimed at explaining this observation, some explicitly or implicitly predict concomitant male and female changes of genital traits that interact during copulation (i.e., lock and key, sexual conflict, cryptic female choice and pleiotropy). Testing these hypotheses requires insights into whether male and female copulatory structures that physically interact during mating also affect each other's evolution and patterns of diversification. Here we compare and contrast size and shape evolution of male and female structures that are known to interact tightly during copulation using two model systems: (a) the sister species O. taurus (1 native, 3 recently established populations) and O. illyricus, and (b) the species-complex O. fracticornis-similis-opacicollis. Partial Least Squares analyses indicated very little to no correlation between size and shape of copulatory structures, both in males and females. Accordingly, comparing shape and size diversification patterns of genitalia within each sex showed that the two components diversify readily--though largely independently of each other--within and between species. Similarly, comparing patterns of divergence across sexes showed that relative sizes of male and female copulatory organs diversify largely independent of each other. However, performing this analysis for genital shape revealed a signature of parallel divergence. Our results therefore suggest that male and female copulatory structures that are linked mechanically during copulation may diverge in concert with respect to their shapes. Furthermore, our results suggest that genital divergence in general, and co-divergence of male and female genital shape in particular, can evolve over an extraordinarily short time frame. Results are discussed in the framework of the hypotheses that assume or predict concomitant evolutionary changes in male and female copulatory organs.