Antioxidant effect of functionalized alkyl-organotellurides: a study in vitro

We evaluated the in vitro antioxidant effect of alkyl-organotellurides A-D on lipid peroxidation and protein carbonylation in rat liver homogenates. The thiol oxidase and thiol peroxidase-like activities of compounds were investigated. delta-Aminolevulinic acid dehydratase (delta-ALA-D) activity was determined in rat liver homogenates. Compounds A-D protected against lipid peroxidation induced by Fe(2+)/EDTA and sodium nitroprusside (SNP). According to the confidence limits of the IC(50) values of compounds A-D, the IC(50) values for organotellurides followed the order: C (0.30 mu M) <= B (0.40 mu M) < D (0.68 mu M) < A (2.90 mu M), for Fe(2+)/EDTA, and B (0.21 mu M) <= C (0.33 mu M) < D (0.43 mu M) < A (1.21 mu M) for SNP-induced lipid peroxidation. Compounds A-D reduced protein carbonyl content to control levels. The results demonstrated an inverse correlation between thiol oxidase and delta-ALA-D activities. This study supports an antioxidant effect of organotellurides A-D on rat liver.

High Matrix Metalloproteinase Activity Is a Hallmark of Periapical Granulomas

Introduction: The inability to distinguish periapical cysts from granulomas before performing root canal treatment leads to uncertainty in treatment outcomes because cysts have lower healing rates. Searching for differential expression of molecules within cysts or granulomas could provide information with regard to the identity of the lesion or suggest mechanistic differences that may form the basis for future therapeutic intervention. Thus, we investigated whether granulomas and cysts exhibit differential expression of extracellular matrix (ECM) molecules. Methods: Human periapical granulomas, periapical cysts, and healthy periodontal ligament tissues were used to investigate the differential expression of ECM molecules by microarray analysis. Because matrix metalloproteinases (MMP) showed the highest differential expression in the microarray analysis, MMPs were further examined by in situ zymography and immunohistochemistry. Data were analyzed by using one-way analysis of variance followed by the Tu-key test. Results: We observed that cysts and granulomas differentially expressed several ECM molecules, especially those from the MMP family. Compared with cysts, granulomas exhibited higher MMP enzymatic activity in areas stained for MMP-9. These areas were composed of polymorphonuclear cells (PMNs) in contrast to cysts. Similarly, MMP-13 was expressed by a greater number of cells in granulomas compared with cysts. Conclusion: Our findings indicate that high enzymatic MIMP activity in PMNs together with MMP-9 and MMP-13 stained cells could be a molecular signature of granulomas unlike periapical cysts. (J Endod 2009;35:1234-1242)

Prevalence of Vitamin D Insufficiency in Brazilian Adolescents

Background/Aims: Cutaneous sun exposure and dietary vitamin D intake are important determinants of vitamin D status. The objective of the present study was to evaluate the vitamin D status of a group of healthy adolescent students living in Brazil. Methods: One hundred and thirty-six adolescents, 64 boys and 72 girls, aged 16-20 years old, living in a rural town in the state of Sao Paulo, Brazil, participated in this study. Results: The mean dietary vitamin D intake was 140 (120-156) IU/day [3.5 (3.0-3.9) mu g/day]. Only 14.9% of the students met the daily adequate intake recommendation of vitamin D. Only 27.9% practice physical activity outdoors and 17.6% of the adolescents apply sunscreen daily. The mean 25(OH)D concentration was 73.0 (22.0) nmol/l [29.2 (8.8) ng/ml]. Vitamin D insufficiency was observed in 60% of adolescents. Conclusions: The present study suggests that even in a sunny climate like Brazil the prevalence of vitamin D insufficiency in adolescents is high. Most likely this is due to low intakes of vitamin D in this group. Due to the limited extent of natural dietary sources of vitamin D, a policy of vitamin D food fortification should be considered in the future, and in the meantime greater use of vitamin D supplements in this population group should be encouraged to provide the increased amounts of this essential nutrient for optimal health. Copyright (C) 2009 S. Karger AG, Basel

Chemical Composition, Seasonal Variation and Evaluation of Antimicrobial Activity of Essential Oils of Talauma ovata A. St. Hil. (Magnoliaceae)

The essential oils isolated by hydrodistillation from trunk bark and leaves of Talauma ovata A. St. Hil. (Magnoliaceae), collected in four seasons, were analyzed by capillary GC and GC/MS. Altogether 52 components were identified, The oils were characterized by predominance of cyclic sesquiterpenes. The main components were linalool, trans-beta-guaiene, germaerene D, germacrene B, spathulenol, caryophyllene oxide, viridiflorol and alpha-endesmol. The content of individual components was variable during the year. All oils were screened against several strains of bacteria and yeasts, using the agar well-diffusion technique. The antimicrobial activity of oils showed strong dependence with the season. Significant activity was found for oils obtained in the spring and summer.

Sequence and function of lysosomal and digestive cathepsin D-like proteinases of Musca domestica midgut

Musca domestica larvae display in anterior and middle midgut contents, a proteolytic activity with pH optimum of 3.0-3.5 and kinetic properties like cathepsin D. Three cDNAs coding for preprocathepsin D-like proteinases (ppCAD 1, ppCAD 2, ppCAD 3) were cloned from a M. domestica midgut cDNA library. The coded protein sequences included the signal peptide, propeptide and mature enzyme that has all conserved catalytic and substrate binding residues found in bovine lysosomal cathepsin D. Nevertheless, ppCAD 2 and ppCAD 3 lack the characteristic proline loop and glycosylation sites. A comparison among the sequences of cathepsin D-like enzymes from some vertebrates and those found in M. domestica and in the genomes of Aedes aegypti, Drosophila melanogaster, Tribolium castaneum, and Bombyx mori showed that only flies have enzymes lacking the proline loop (as defined by the motif: DxPxPx(G/A)P), thus resembling vertebrate pepsin. ppCAD 3 should correspond to the digestive cathepsin D-like proteinase (CAD) found in enzyme assays because: (1) it seems to be the most expressed CAD, based on the frequency of ESTs found. (2) The mRNA for CAD 3 is expressed only in the anterior and proximal middle midgut. (3) Recombinant procathepsin D-like proteinase (pCAD 3), after auto-activation has a pH optimum of 2.5-3.0 that is close to the luminal pH of M. domestica midgut. (4) Immunoblots of proteins from different tissues revealed with anti-pCAD 3 serum were positive only in samples of anterior and middle midgut tissue and contents. (5) CAD 3 is localized with immunogold inside secretory vesicles and around microvilli in anterior and middle midguit cells. The data support the view that on adapting to deal with a bacteria-rich food in an acid midgut region, M. domestica digestive CAD resulted from the same archetypical gene as the intracellular cathepsin D, paralleling what happened with vertebrates. The lack of the proline loop may be somehow associated with the extracellular role of both pepsin and digestive CAD 3. (C) 2009 Elsevier Ltd. All rights reserved.

Crotoxin alters lymphocyte distribution in rats: Involvement of adhesion molecules and lipoxygenase-derived mediators

Crotoxin is the main neurotoxic component of Crotalus durissus terrificus snake venom and modulates immune and inflammatory responses, interfering with the activity of leukocytes. In the present work, the effects of crotoxin on the number of blood and lymphatic leukocytes and on lymph nodes and spleen lymphocytes population were investigated. The toxin s.c. administered to male Wistar rats, decreases the number of lymphocytes in blood and lymph circulation and increases the content of B and T-lymphocytes in lymph nodes. These effects were detected 1-2 h after treatment. The crotoxin molecule is composed of two subunits, an acidic non-toxic polypeptide, named crotapotin and a toxic basic phospholipase A(2) (PLA(2)). PLA(2), but not crotapotin, decreased the number of circulating blood and lymph lymphocytes. Crotoxin promotes leukocyte adherence to endothelial cells of blood microcirculation and to lymph node high endothelial venules, which might contribute to the drop in the number of circulating lymphocytes. Crotoxin increases expression of the adhesion molecule LFA-1 in lymphocytes. The changes in the expression of the adhesion molecule might contribute, at least in part, for the increased leukocyte adhesion to endothelium. Zileuton, a 5-lipoxygenase inhibitor, blocked the decrease in the number of circulating leukocytes induced by crotoxin and also abolished the changes observed in leukocyte-endothelial interactions, suggesting the involvement of lipoxygenase-derived mediators in the effects of the toxin. (c) 2008 Elsevier Ltd. All rights reserved.

Role of CFTR and ClC-5 in Modulating Vacuolar H(+)-ATPase Activity in Kidney Proximal Tubule

Background/Aims: It has been widely accepted that chloride ions moving along chloride channels act to dissipate the electrical gradient established by the electrogenic transport of H(+) ions performed by H(+)-ATPase into subcellular vesicles. Largely known in intracellular compartments, this mechanism is also important at the plasma membrane of cells from various tissues, including kidney. The present work was performed to study the modulation of plasma membrane H(+)-ATPase by chloride channels, in particular, CFTR and ClC-5 in kidney proximal tubule. Methods and Results: Using in vivo stationary microperfusion, it was observed that ATPase-mediated HCO(3)(-) reabsorption was significantly reduced in the presence of the Cl(-) channels inhibitor NPPB. This effect was confirmed in vitro by measuring the cell pH recovery rates after a NH(4)Cl pulse in immortalized rat renal proximal tubule cells, IRPTC. In these cells, even after abolishing the membrane potential with valinomycin, ATPase activity was seen to be still dependent on Cl(-). siRNA-mediated CFTR channels and ClC-5 chloride-proton exchanger knockdown significantly reduced H(+)-ATPase activity and V-ATPase B2 subunit expression. Conclusion: These results indicate a role of chloride in modulating plasma membrane H(+)-ATPase activity in proximal tubule and suggest that both CFTR and ClC-5 modulate ATPase activity. Copyright (C) 2010 S. Karger AG, Basel

Localization of Cathepsins D and B at the Maternal-Fetal Interface and the Invasiveness of the Trophoblast during the Postimplantation Period in the Mouse

A survey of existing data suggests that trophoblast cells produce factors involved in extracellular matrix degradation. In this study, we correlated the expression of cathepsins D and B in the murine ectoplacental cone with the ultrastructural progress of decidual invasion by trophoblast cells. Both proteases were immunolocalized at implantation sites in lysosome-endosome-like compartments of trophoblast giant cells. Cathepsin D, but not cathepsin B, was also detected ultrastructurally in extracellular compartments surrounded by processes of the invading trophoblast containing extracellular matrix components and endometrial cell debris. The expression of cathepsins D and B by trophoblast cells was confirmed by RT-PCR in ectoplacental cones isolated from implantation chambers at gestation day 7.5. Our data addressed a positive relationship between the expression and presence of cathepsin D at the extracellular compartment of the maternal-fetal interface and the invasiveness of the trophoblast during the postimplantation period, suggesting a participation of invading trophoblast cells in the cathepsin D release. Such findings indicate that mouse trophoblast cells might exhibit a proteolytic ability to partake in the decidual invasion process at the maternal-fetal interface. Copyright (C) 2010 S. Karger AG, Basel

Ethanol-induced sensitization depends preferentially on D(1) rather than D(2) dopamine receptors

Behavioral sensitization, defined as a progressive increase in the locomotor stimulant effects elicited by repeated exposure to drugs of abuse, has been used as an animal model for drug craving in humans. The mesoaccumbens dopaminergic system has been proposed to be critically involved in this phenomenon; however, few studies have been designed to systematically investigate the effects of dopaminergic antagonists on development and expression of behavioral sensitization to ethanol in Swiss mice. We first tested the effects of D(1) antagonist SCH-23390 (0-0.03 mg/kg) or D(2) antagonist Sulpiride (0-30 mg/kg) on the locomotor responses to an acute injection of ethanol (2.0 g/kg). Results showed that all tested doses of the antagonists were effective in blocking ethanol`s stimulant effects. In another set of experiments, mice were pretreated intraperitoneally with SCH-23390 (0.01 mg/kg) or Sulpiride (10 mg/kg) 30 min before saline or ethanol injection, for 21 days. Locomotor activity was measured weekly for 20 min. Four days following this pretreatment, all mice were challenged with ethanol. Both antagonists attenuated the development of ethanol sensitization, but only SCH-23390 blocked the expression of ethanol sensitization according to this protocol. When we tested a single dose (30 min before tests) of either antagonist in mice treated chronically with ethanol, both antagonists attenuated ethanol-induced effects. The present findings demonstrate that the concomitant administration of ethanol with D(1) but not D(2) antagonist prevented the expression of ethanol sensitization, suggesting that the neuroadaptations underlying ethanol behavioral sensitization depend preferentially on D(1) receptor actions. (C) 2010 Elsevier Inc. All rights reserved.

Environmental modulation of ethanol-induced locomotor activity: Correlation with neuronal activity in distinct brain regions of adolescent and adult Swiss mice

Drug abuse is a concerning health problem in adults and has been recognized as a major problem in adolescents. induction of immediate-early genes (IEG), such as c-Fos or Egr-1, is used to identify brain areas that become activated in response to various stimuli, including addictive drugs. It is known that the environment can alter the response to drugs of abuse. Accordingly, environmental cues may trigger drug-seeking behavior when the drug is repeatedly administered in a given environment. The goal of this study was first to examine for age differences in context-dependent sensitization and then evaluate IEG expression in different brain regions. For this, groups of mice received i.p. ethanol (2.0 g/kg) or saline in the test apparatus, while other groups received the solutions in the home cage, for 15 days. One week after this treatment phase, mice were challenged with ethanol injection. Acutely, ethanol increased both locomotor activity and IEG expression in different brain regions, indistinctly, in adolescent and adult mice. However, adults exhibited a typical context-dependent behavioral sensitization following repeated ethanol treatment, while adolescent mice presented gradually smaller locomotion across treatment, when ethanol was administered in a paired regimen with environment. Conversely, ethanol-treated adolescents expressed context-independent behavioral sensitization. Overall, repeated ethanol administration desensitized IEG expression in both adolescent and adult mice, but this effect was greatest in the nucleus accumbens and prefrontal cortex of adolescents treated in the context-dependent paradigm. These results suggest developmental differences in the sensitivity to the conditioned and unconditioned locomotor effects of ethanol. (C) 2008 Elsevier B.V. All rights reserved.

Effects of adolescent exposure to cocaine on locomotor activity and extracellular dopamine and glutamate levels in nucleus accumbens of DBA/2J mice

Adolescents differ from adults in their acute sensitivity to several drugs of abuse, but little is known about the long-term neurobehavioral effects of adolescent drug exposure. To explore this further, we evaluated the locomotor responses to repeated cocaine administration in adolescent and adult male DBA/2J mice and alterations in extracellular levels of dopamine (DA) and glutamate (GLU) in the nucleus accumbens (NAc) in response to a subsequent cocaine challenge. Adolescent and adult mice were treated daily with saline or cocaine (10 mg/kg, i.p) for 9 consecutive days. Ten days following the last injection, animals were implanted with microdialysis probes and 24 h later microdialysis samples were collected before and after an acute cocaine challenge. Adolescents but not adults demonstrated development of behavioral sensitization to cocaine. Microdialysis procedures revealed that cocaine-treated mice displayed greater peak increases in extracellular DA in response to a subsequent cocaine challenge as compared to saline-treated mice, in contrast with lower peak increases in extracellular GLU. While adults exhibited greater peaks in extracellular DA in response to cocaine than adolescents did, adolescent mice presented a more rapid onset of peak extracellular DA levels than adults. Our results indicate differences in the behavioral and neurochemical responses to cocaine in adolescent versus adult mice, which may be relevant to the increased risk of developing addiction in humans who are exposed to drugs of abuse during adolescence. (C) 2007 Elsevier B.V. All rights reserved.

Alboserpin, a Factor Xa Inhibitor from the Mosquito Vector of Yellow Fever, Binds Heparin and Membrane Phospholipids and Exhibits Antithrombotic Activity

The molecular mechanism of factor Xa (FXa) inhibition by Alboserpin, the major salivary gland anticoagulant from the mosquito and yellow fever vector Aedes albopictus, has been characterized. cDNA of Alboserpin predicts a 45-kDa protein that belongs to the serpin family of protease inhibitors. Recombinant Alboserpin displays stoichiometric, competitive, reversible and tight binding to FXa (picomolar range). Binding is highly specific and is not detectable for FX, catalytic site-blocked FXa, thrombin, and 12 other enzymes. Alboserpin displays high affinity binding to heparin (K(D) similar to 20 nM), but no change in FXa inhibition was observed in the presence of the cofactor, implying that bridging mechanisms did not take place. Notably, Alboserpin was also found to interact with phosphatidylcholine and phosphatidylethanolamine but not with phosphatidylserine. Further, annexin V (in the absence of Ca(2+)) or heparin outcompetes Alboserpin for binding to phospholipid vesicles, suggesting a common binding site. Consistent with its activity, Alboserpin blocks prothrombinase activity and increases both prothrombin time and activated partial thromboplastin time in vitro or ex vivo. Furthermore, Alboserpin prevents thrombus formation provoked by ferric chloride injury of the carotid artery and increases bleeding in a dose-dependent manner. Alboserpin emerges as an atypical serpin that targets FXa and displays unique phospholipid specificity. It conceivably uses heparin and phosphatidylcholine/phosphatidylethanolamine as anchors to increase protein localization and effective concentration at sites of injury, cell activation, or inflammation.