Removal from the Membrane Affects the Interaction of Rat Osseous Plate Ecto-Nucleosidetriphosphate Diphosphohydrolase-1 with Substrates and Ions

We have characterized the kinetic properties of ectonucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1) from rat osseous plate membranes. A novel finding of the present study is that the solubilized enzyme shows high- and low-affinity sites for the substrate in contrast with a single substrate site for the membrane-bound enzyme. In addition, contrary to the Michaelian chraracteristics of the membrane-bound enzyme, the site-site interactions after solubilization with 0.5% digitonin plus 0.1% lysolecithin resulted in a less active ectonucleoside triphosphate diphosphohydrolase, showing activity of about 398.3 nmol Pi min(-1) mg(-1). The solubilized enzyme has M(r) of 66-72 kDa, and its catalytic efficiency was significantly increased by magnesium and calcium ions; but the ATP/ADP activity ratio was always < 2.0. Partial purification and kinetic characterization of the rat osseous plate E-NTPDase1 in a solubilized form may lead to a better understanding of a possible function of the enzyme as a modulator of nucleotidase activity or purinergic signaling in matrix vesicle membranes. The simple procedure to obtain the enzyme in a solubilized form may also be attractive for comparative studies of particular features of the active sites from this and other ATPases.

The crystal structure of free human hypoxanthineguanine phosphoribosyltransferase reveals extensive conformational plasticity throughout the catalytic cycle

Human hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyses the synthesis of the purine nucleoside monophosphates, IMP and GMP, by the addition of a 6-oxopurine base, either hypoxanthine or guanine, to the 1-beta-position of 5-phospho-U-D-ribosyl-1-pyrophosphate (PRib-PP). The mechanism is sequential, with PRib-PP binding to the free enzyme prior to the base. After the covalent reaction, pyrophosphate is released followed by the nucleoside monophosphate. A number of snapshots of the structure of this enzyme along the reaction pathway have been captured. These include the structure in the presence of the inactive purine base analogue, 7-hydroxy [4,3-d] pyrazolo pyrimidine (HPP) and PRib-PP. Mg2+, and in complex with IMP or GMP. The third structure is that of the immucillinHP.Mg2+.PPi complex, a transition-state analogue. Here, the first crystal structure of free human HGPRT is reported to 1.9 angstrom resolution, showing that significant conformational changes have to occur for the substrate(s) to bind and for catalysis to proceed. Included in these changes are relative movement of subunits within the tetramer, rotation and extension of an active-site alpha-helix (D137-D153), reorientation of key active-site residues K68, D137 and K165, and the rearrangement of three active-site loops (100-128, 165-173 and 186-196). Toxoplasina gondii HGXPRT is the only other 6-oxopurine phosphoribosyltransferase structure solved in the absence of ligands. Comparison of this structure with human HGPRT reveals significant differences in the two active sites, including the structure of the flexible loop containing K68 (human) or K79 (T gondii). (c) 2005 Elsevier Ltd. All rights reserved.

Letters that construct limits: intellectual and political projects in the letters of Capistrano de Abreu to Barao do Rio Branco (1886-1903)

The intention of this paper is to analyze the letters from Capistrano de Abreu to Barao do Rio Branco in the years between 1886 and 1903. The focus will be given to the divergences around the notion of territorial formation, a basic concept for these authors who were thinking about the construction of a historical narrative at the end of the 19(th) and beginning of the 20(th) century. Later, the question is the construction of the craft of the historian in the letters of Capistrano de Abreu and his distinction and proximity to the ideas of the Barao do Rio Branco.

Crystal structure of Escherichia coli xanthine phosphoribosyltransferase

Xanthine phosphoribosyltransferase (XPRT; EC 2.4.2.22) from Escherichia coil is a tetrameric enzyme having 152 residues per subunit. XPRT catalyzes the transfer of the phosphoribosyl group from 5-phospho-alpha-D-ribosyl l-pyrophosphate (PRib-PP) to the 6-oxopurine bases guanine, xanthine, and hypoxanthine to form GMP, XMP, and IMP, respectively. Crystals grown in the absence of substrate or product were used to determine the structure of XPRT at a resolution of 1.8 Angstrom by multiple isomorphous replacement. The core structure of XPRT includes a five-stranded parallel B-sheet surrounded by three or-helices, which is similar to that observed in other known phosphoribosyltransferase (PRTase) structures. The XPRT structure also has several interesting features. A glutamine residue in the purine binding site may be responsible for the altered 6-oxopurine base specificity seen in this enzyme compared to other 6-oxopurine PRTases. Also, we observe both a magnesium ion and a sulfate ion bound at the PRib-PP binding site of XPRT. The sulfate ion interacts with Arg-37 which has a cis-peptide conformation, and the magnesium ion interacts with Asp-89, a highly conserved acidic residue in the PRib-PP binding site motif. The XPRT structure also incorporates a feature which has not been observed in other PRTase structures. The C-terminal 12 residues of XPRT adopt an unusual extended conformation and make interactions with a neighboring subunit. The very last residue, Arg-152, could form part of the active site of a symmetry-related subunit in the XPRT tetramer.

Antileishmanial and antitrypanosomal activity of bufadienolides isolated from the toad Rhinella jimi parotoid macrogland secretion

Amphibian skin secretions are considered a rich source of biologically active compounds and are known to be rich in peptides, bufadienolides and alkaloids. Bufadienolides are cardioactive steroids from animals and plants that have also been reported to possess antimicrobial activities. Leishmaniasis and American Trypanosomiasis are parasitic diseases found in tropical and subtropical regions. The efforts toward the discovery of new treatments for these diseases have been largely neglected, despite the fact that the only available treatments are highly toxic drugs. In this work, we have isolated, through bioguided assays, the major antileishmanial compounds of the toad Rhinella jimi parotoid macrogland secretion. Mass spectrometry and (1)H and (13)C NMR spectroscopic analyses were able to demonstrate that the active molecules are telocinobufagin and hellebrigenin. Both steroids demonstrated activity against Leishmania (L.) chagasi promastigotes, but only hellebrigenin was active against Trypanosoma cruzi trypomastigotes. These steroids were active against the intracellular amastigotes of Leishmania, with no activation of nitric oxide production by macrophages. Neither cytotoxicity against mouse macrophages nor hemolytic activities were observed. The ultrastructural studies with promastigotes revealed the induction of mitochondrial damage and plasma membrane disturbances by telocinobufagin, resulting in cellular death. This novel biological effect of R. jimi steroids could be used as a template for the design of new therapeutics against Leishmaniasis and American Trypanosomiasis. (C) 2008 Elsevier Ltd. All rights reserved.

Diffuse analgesic effects of unilateral repetitive transcranial magnetic stimulation (rTMS) in healthy volunteers

We investigated the analgesic effects of unilateral repetitive transcranial magnetic stimulation (rTMS) of the motor cortex (M1) or dorsolateral prefrontal cortex (DLPFC) in two models of experimental pain in healthy volunteers. Two studies were carried out in parallel in two groups of 26 paid healthy volunteers. The effects of active or sham rTMS (frequency, 10 Hz; intensity, 80% resting motor threshold) applied to the right M1 or DLPFC were compared in a double-blind randomized cross-over design. In the first series of experiments, we analyzed the effects of rTMS on thermal (heat and cold) detection and pain thresholds measured on both hands and the left foot, by standardized quantitative sensory testing methods. In the second series of experiments, we measured the effects of M1 or DLPFC rTMS on the threshold and recruitment curves of the RIII nociceptive reflex evoked by ipsilateral electrical stimulation of the sural nerve and recorded on the biceps femoris of both lower limbs. In both studies, measurements were taken before and up to 60 min after the end of rTMS. Active rTMS of both M1 and DLPFC significantly increased the thermal pain thresholds, measured for both hands and the left foot, this effect being most marked for cold pain. These effects, which lasted at least 1 h after rTMS, were selective because they were not associated with changes in non-painful thermal sensations. By contrast, the second study showed that rTMS of M1 or DLPFC had no significant effect on the threshold or recruitment curve of the nociceptive flexion RIII reflex. Our findings demonstrate that unilateral rTMS of M1 or DLPFC induces diffuse and selective analgesic effects in healthy volunteers. The lack of effect on the RIII reflex suggests that such analgesic effects may not depend on the activation of descending inhibitory systems. (C) 2009 International Association for the Study of Pain. Published by Elsevier B. V. All rights reserved.

The Association of Lipodystrophy and Oxidative Stress Biomarkers in HIV-Infected Men

The aim of this study was to describe the status of oxidative stress and antioxidant biomarkers and their association with metabolic and body composition components of HIV-lipodystrophy syndrome. In a cross-sectional study of blood samples from HIV-infected men with lipodystrophy syndrome (HIV+LIPO+ = 10), HIV-infected men without lipodystrophy syndrome (HIV+LIPO- = 22), and healthy subjects (control = 12), the following oxidative stress biomarkers were analyzed: total hydroperoxide, thiobarbituric acid reactive substances (TBARS), and advanced oxidation protein products (AOPP). In addition, antioxidant biomarkers, including total glutathione, uric acid, alpha-tocopherol, and metabolic components were tested. Dual-energy x-ray absorciometry (DXA) was used to measure the fat mass. The duration of HIV infection and the duration and type of highly active antiretroviral therapy were similar between the two HIV-infected groups. Higher levels of total hydroperoxide were observed in the HIV+LIPO+ (50 +/- 33 H(2)O(2)/L) group compared to the HIV+LIPO-(19 +/- 13 H(2)O(2)/L) and control (5 +/- 5 H(2)O(2)/L) groups (p < 0.05). Similarly, higher levels of AOPP were observed in the HIV+LIPO+ (326 +/- 173 mu mol/L) group compared to the HIV+LIPO- (105 +/- 92 mu mol/L) and control groups (80 +/- 20 mu mol/L) (p < 0.05). Total hydroperoxide significantly correlated with insulin serum levels in the HIV+LIPO+ (r = 0.47, p < 0.05) and HIV+LIPO- groups (r = 0.29, p < 0.05), while AOPP significantly correlated with insulin serum levels in the HIV+LIPO+ (r = 0.73, p < 0.05) and HIV+LIPO- (r = 0.54, p < 0.05) groups. Therefore, higher lipid and protein oxidation were found in HIV-infected patients with lipodystrophy syndrome, and both were associated with insulin levels.

Opioidergic and GABAergic mechanisms in the rostral ventromedial medulla modulate the nociceptive response of vocalization in guinea pigs

Vocalization generated by the application of a noxious stimulus is an integrative response related to the affective-motivational component of pain. The rostral ventromedial medulla (RVM) plays an important role in descending pain modulation, and opiates play a major role in modulation of the antinociception mediated by the RVM. Further, it has been suggested that morphine mediates antinociception indirectly, by inhibition of tonically active GABAergic neurons. The current study evaluated the effects of the opioids and GABA agonists and antagonists in the RVM on an affective-motivational pain model. Additionally, we investigated the opioidergic-GABAergic interaction in the RVM in the vocalization response to noxious stimulation. Microinjection of either morphine (4.4 nmo1/0.2 mu l) or bicuculline (0.4 nmo1/0.2 mu l) into the RVM decreased the vocalization index, whereas application of the GABA(A) receptor agonist, musci-mol (0.5 nmo1/0.2 mu l) increased the vocalization index during noxious stimulation. Furthermore, prior microinjection of either the opioid antagonist naloxone (2.7 nmo1/0.2 mu l) or muscimol (0.25 nmo1/0.2 mu l) into the RVM blocked the reduction in vocalization index induced by morphine. These observations suggest an antinociceptive and pro-nociceptive role of the opioidergic and GABAergic neurotransmitters in the RVM, respectively. Our data show that opioids have an antinociceptive effect in the RVM, while GABAergic neurotransmission is related to the facilitation of nociceptive responses. Additionally, our results indicate that the antinociceptive effect of the opioids in the RVM could be mediated by a disinhibition of tonically active GABAergic interneurons in the downstream projection neurons of the descending pain control system; indicating an interaction between the opioidergic and GABAergic pathways of pain modulation. (C) 2010 Elsevier Inc. All rights reserved.

Tissue diffusion and retention of metalloproteinases in ascending aortic aneurysms and dissections

Histopathological alterations in human aneurysms and dissections of the thoracic ascending aorta include areas of mucoid degeneration within the medial layer, colocalized with areas of cell disappearance and disruption of extracellular matrix elastic and collagen fibers. We studied the presence of matrix metalloproteinases in relation to their capacity to diffuse through the tissue or to be retained in areas of mucoid degeneration in aneurysms and dissections of the ascending aorta. Ascending aortas from 9 controls, 33 patients with aneurysms, and 14 with acute dissections, all collected at surgery, were analyzed. The morphological aspect was similar whatever the etiology or phenotypic expression of the pathological aortas, involving areas of extracellular matrix breakdown and cell rarefaction associated with mucoid degeneration. Release of proMMP-2, constitutively expressed by smooth muscle cells, was not different between controls and aneurysmal aortas, whereas the aneurysmal aortas released more of the active form. Release of pro and active MMP-9 was also similar between controls and aneurysmal aortas. Immunohistochemical staining of MMP-2 and MMP-9 was weak in both control and pathological aortas. In contrast, released MMP-7 (matrilysin) and MMP-3 (stromelysin-1) could not be detected in conditioned media but were present in tissue extracts with no detectable quantitative difference between controls and pathological aortas. Immunohistochemical staining of MMP-7 and MMP-3 revealed their retention in areas of mucoid degeneration, and semiquantitative evaluation of immunostaining showed more MMP-7 in pathological aortas than in controls. In conclusion, areas of mucoid degeneration, the hallmark of aneurysms, and dissections of thoracic ascending aortas, whatever their etiology, are not inert and can retain specific proteases. (c) 2009 Elsevier Inc. All rights reserved.

Expressed sequence tags (ESTs) from the salivary glands of the tick Amblyomma cajennense (Acari : Ixodidae)

The neotropical tick Amblyomma cajennense is a significant pest to domestic animals, the most frequently human-biting tick in South America and the main vector of Brazilian spotted fever (caused by Rickettsia rickettsii), a deadly human disease. The purpose of this study is to characterize the adult A. cajennense salivary gland transcriptome by expressed sequence tags (ESTs). We report the analysis of 1754 clones obtained from a cDNA library, which reveal mainly transcripts related to proteins involved in the hemostatic processes, especially proteases and their inhibitors. Remarkably, five types of possible serine protease inhibitors were found, including a molecule with a distinguished structure that contains repeats of the active motif of hirudin inhibitors. Besides, other components that may be active over the host immune system or acting as defensins against infecting microorganisms were also described, including a molecule similar to insect venom allergens. The conjunction of components from this transcriptome suggests a diverse strategy of A. cajennense tick during feeding, but emphasized in the coagulation system. (c) 2008 Published by Elsevier Ltd.

Protective action of indole-3-acetic acid on induced hepatocarcinoma in mice

In this study, we report the protective effects of IAA on diethylnitrosamine (DEN)-induced hepatocarcinogenesis. BALB/c mice received daily IAA at 50 (T(50)), 250 (T(250)), and 500 (T(500)) mg K(-1) per body mass by gavage for 15 days. At day 15, animals were administered DEN and sacrificed 4 h later. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed in sera. In addition., hepatomorphologic alterations, activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR), gene expression of antioxidant enzymes and DNA integrity were evaluated in the liver. IAA administration did not show any alterations in any of the parameters available, except for a reduction of the gene expression for antioxidant enzyrries by 55, 56, 27, and 28% for SOD, CAT, GPx, and GR upon T(500). respectively compared with the control. Several hepatic alterations were observed by DEN exposure. Moreover, IAA administration at 3 doses was shown to provide a total prevention of the active reduction of CAT and GR induced by DEN exposure compared with the control. IAA at T5(00) was shown to give partial protection (87, 71, 57, and 90% for respectively SOD, CAT. GPx. and GR) on the down-regulation of the enzymes induced by DEN and this auxin showed a partial protection (50%) on DEN-induced DNA fragmentation for both parameters when compared to DEN alone. This work showed IAA hepatocarcinogenesis protection for the first time by means of a DEN-protective effect on CAT and GR activity. and by affecting antioxidant gene expression and DNA fragmentation. Copyright (C) 2008 John Wiley & Sons, Ltd.

The success of endosseous implants in human immunodeficiency virus-positive patients receiving antiretroviral therapy A pilot study

Background. In a pilot study, the authors aimed to determine the success rate of dental implants placed in patients who were positive for human immunodeficiency virus (HIV) and were receiving different regimens of highly active anti-retroviral therapy (HAART). They considered patients` levels of cluster of differentiation (CD) 4(+) cells and viral load, and they attempted to verify whether patients with baseline biochemical signs of bone mineral density loss could experience osseointegration impairment. Materials and Methods. One of the authors, a dentist, placed dental implants in the posterior mandibles of 40 volunteers, divided into three groups: one composed of HIV-positive patients receiving protease inhibitor (PI)-based HAART; a second composed of HIV-positive patients receiving nonnucleoside reverse transcriptase inhibitor based HAART (without PI); and a control group composed of HIV-negative participants. The authors assessed pen-implant health six and 12 months after implant loading. They analyzed the success of the implants in relation to CD4(+) cell counts, viral load and baseline pyridinoline and deoxypyridinoline values. Results. The authors followed 59 implants for 12 months after loading. Higher baseline levels of pyridinoline and deoxypyridinoline found in HIV-positive participants did not interfere with osseointegration after 12 months of follow-up. Average pen-implant bone loss after 12 months was 0.49 millimeters in group 1, 0.47 mm in group 2, and 0.55 mm in the control group. Conclusions. The placement of dental implants in HIV-positive patients is a reasonable treatment option, regardless of CD4(+) cell count, viral load levels and type of antiretroviral therapy. Longer, follow-up periods are necessary to ascertain the predictability of the long-term success of dental implants in these patients. Clinical Implications. Limited published scientific evidence is available to guide clinicians in regard to possible increased risks associated with dental implant placement in HIV-positive patients.

In situ electron paramagnetic resonance (EPR) study of surface oxygen species on Au/ZnO catalyst for low-temperature carbon monoxide oxidation

Some paramagnetic superoxide ions detectable by electron paramagnetic resonance (EPR) can be generated on Au/ZnO catalyst by oxygen adsorption at room temperature as well as at 553 K. In both the cases, the O-2(-) ions are present on the catalyst surface. The disappearance of the O-2(-) signal by the introduction of carbon monoxide over the catalyst surface implies that the O-2(-) ions are either the active oxygen species or the precursors of the active oxygen species. The CO3- species produced are also detected by EPR. (C) 2001 Elsevier Science B.V. All rights reserved.

Endocytosis of uncleaved tumor necrosis factor-alpha in macrophages

Activated monocytes and macrophages secrete the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) TNF-alpha is produced as a 26 kd transmembrane protein that is cleaved to release a 17 kd soluble protein. TNF-alpha in both forms is biologically active. The intracellular trafficking of membrane-associated TNF-alpha in lipopolysaccharide-activated mouse macrophages was assessed after treatment with the metalloprotease inhibitor BB-3103, which prevents the cleavage of pro-TNF-alpha. Immunoprecipitation and immunofluorescence studies showed sustained expression of cell-associated TNF-alpha in the presence of the inhibitor. Cell immunoreactivity and surface biotinylation revealed that uncleaved TNF-alpha accumulated on the cell surface and was endocytosed, appearing in intracellular vesicles. Perturbation of post-Golgi traffic blocked the surface expression of 26 kd TNF-alpha. Tracking a bolus of TNF-alpha over time in cycloheximide-treated cells confirmed that uncleaved TNF-alpha is first transported to the cell surface and subsequently endocytosed. Vesicular structures immunoreactive for TNF-alpha were identified as endosomes by double labeling. The secretory and membrane-associated endocytic trafficking of TNF-alpha provides a mechanism for modulating the quantity of biologically active 26 kd TNF-alpha expressed on macrophages, allowing regulation of paracrine and autocrine responses.

Olfaction in the Queensland fruit fly, Bactrocera tryoni. I: Identification of olfactory receptor neuron types responding to environmental odors

The electroantennogram method was used to investigate the number of distinct olfactory receptor neuron types responding to a range of behaviorally active volatile chemicals in gravid Queensland fruit flies, Bactrocera tryoni. Three receptor neuron types were identified. One type responds to methyl butyrate, 2-butanone, farnesene, and carbon dioxide; a second to ethanol; and a third to n-butyric acid and ammonia. The receptor neuron type responding to methyl butyrate, 2-butanone, farnesene, and carbon dioxide consists of three subtypes. The presence of a limited number of receptor neuron types responding to a diverse set of chemicals and the reception of carbon dioxide by a receptor neuron type that responds to other odorants are novel aspects of the peripheral olfactory discrimination process.