The isatin-Schiff base copper(II) complex Cu(isaepy)(2) acts as delocalized lipophilic cation, yields widespread mitochondrial oxidative damage and induces AMP-activated protein kinase-dependent apoptosis

We previously demonstrated that Bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl) pyridine-N, N`] copper(II) [Cu(isaepy)(2)] was an efficient inducer of the apoptotic mitochondrial pathway. Here, we deeply dissect the mechanisms underlying the ability of Cu(isaepy)(2) to cause mitochondriotoxicity. In particular, we demonstrate that Cu(isaepy)(2) increases NADH-dependent oxygen consumption of isolated mitochondria and that this phenomenon is associated with oxy-radical production and insensitive to adenosine diphosphate. These data indicate that Cu(isaepy)(2) behaves as an uncoupler and this property is also confirmed in cell systems. Particularly, SH-SY5Y cells show: (i) an early loss of mitochondrial transmembrane potential; (ii) a decrease in the expression levels of respiratory complex components and (iii) a significant adenosine triphosphate (ATP) decrement. The causative energetic impairment mediated by Cu(isaepy)(2) in apoptosis is confirmed by experiments carried out with rho(0) cells, or by glucose supplementation, where cell death is significantly inhibited. Moreover, gastric and cervix carcinoma AGS and HeLa cells, which rely most of their ATP production on oxidative phosphorylation, show a marked sensitivity toward Cu(isaepy)(2). Adenosine monophosphate-activated protein kinase (AMPK), which is activated by events increasing the adenosine monophosphate: ATP ratio, is deeply involved in the apoptotic process because the overexpression of its dominant/negative form completely abolishes cell death. Upon glucose supplementation, AMPK is not activated, confirming its role as fuel-sensing enzyme that positively responds to Cu(isaepy)(2)-mediated energetic impairment by committing cells to apoptosis. Overall, data obtained indicate that Cu(isaepy)(2) behaves as delocalized lipophilic cation and induces mitochondrial-sited reactive oxygen species production. This event results in mitochondrial dysfunction and ATP decrease, which in turn triggers AMPK-dependent apoptosis.

Ensaio clínico duplo-cego controlado e randômico, comparando a eficácia da Clofazimina com a Cloroquina, no tratamento das lesões cutâneas do Lúpus Eritematoso Sistêmico

PURPOSE: To evaluate the capacity of clofazimine (CFZ) to control cutaneous activity of systemic lupus erythematosus (SLE), compared with chloroquine diphosphate (CDP). METHODS: A prospective, randomized, controlled, double blind clinical trial was carried out in thirty-three patients with SLE and cutaneous lesions (malar rash and/or discoid lupus and/or subacute cutaneous lupus), after approval by the respective Ethics Committee. Sixteen patients received clofazimine - 100mg/day (CFZ group) and 17 received chloroquine diphosphate - 250mg/day (CDP group), during six months. Both groups applied broad-spectrum sunscreens twice a day. The dose of prednisone was kept stable during the study. Cutaneous lesions were evaluated by 2 blinded observers and photographed at baseline and at months 1, 2, 4 and 6. RESULTS: Thirty-three patients began and 27 completed the 6 months of treatment. The groups were homogeneous and comparable in terms of demographic and clinical characteristics. Five CFZ-patients and one CDP-patients dropped out due to severe flare of disease requiring other treatment. At the end of the study, 12 CFZ-patients (75%) and 14 CDP-patients (82,4%) presented complete or near-complete remission of skin lesions; intention-to-treat analysis showed no significant difference in the response rates between groups. Side effects in both groups were frequent, but patients didn t have to discontinue the drugs. CONCLUSIONS: Clofazimine and chloroquine were effective in controlling cutaneous lesions in SLE patients

In vitro and in vivo evaluation of a primaquine prodrug without red blood cell membrane destabilization property

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Platelet aggregation and TGF-beta(1) plasma levels in pregnant women with preeclampsia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Geranylation of benzoic acid derivatives by enzymatic extracts from Piper crassinervium (Piperaceae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Biosynthetic origins of the isoprene units of gaudichaudianic acid in Piper gaudichaudianum (Piperaceae)

The biosynthesis of (2S)-2-methyl-2-(4'-methyl-3-pentenyl)-8-(3-methyl-2-butenyl)-2H-1-benzopyran-6-carboxylic acid (gaudichaudianic acid), the major metabolite in leaves and roots of Piper gaudichaudianum Kunth (Piperaceae), has been investigated employing [1(-13) C]-D-glucose as precursor. The labelling pattern in the isolated gaudichaudianic acid was determined by quantitative 13 C NMR spectroscopy analysis and was consistent with involvement of both mevalonic acid and 2-C-methyl-D-erythritol-4-phosphate pathways in the formation of the dimethylallyl- and geranyl-derived moieties. The results confirmed that both plastidic and cytoplasmic pathways are able to provide isopentenyl diphosphate units for prenylation of p-hydroxybenzoic acid. (c) 2007 Elsevier Ltd. All rights reserved.

Circadian rhythm of anti-fungal prenylated chromene in leaves of Piper aduncum

Leaves of Piper aduncum accumulate the anti-fungal chromenes methyl 2,2-dimethyl-2H-1-chromene-6-carboxylate (1) and methyl 2,2-dimethyl-8-(3'-methyl-2'-butenyl)-2H-1-chromene-6-carboxylate (2). The enzymatic formation of 2 from dimethylallyl diphosphate and I was investigated using cell-free extracts of the title plant. An HPLC assay for the prenylation reaction was developed and the enzyme activity measured in the protein extracts. The prenyltransferase that catalyses the transfer of the dimethylallyl group to C-2' of 1 was soluble and required dimethylallyl diphosphate as the prenyl donor. In the leaves, the biosynthesis of the prenylated chromene 2 was time-regulated and prenyltransferase activity depended upon circadian variation. Preliminary characterisation and purification experiments on the prenyltransferase from P. aduncum have been performed. Copyright (C) 2005 John Wiley & Sons, Ltd.

Phosphate functionalization of spongiolite surface

Spongiolite from Mato Grosso do Sul (Brazil), natural inorganic composite constituted of silica needles, was treated with concentrated phosphoric acid at high temperatures. Superficial coating of the needles was proved to be constituted of silicon diphosphate, a compound offering six-coordinated silicon sites. Owing to the affinity of three charged ions to phosphate groups, this coating acts as specific adsorbent for the rare earth elements which prefer octahedral coordination (starting from samarium, yttrium included). The uptake of lanthanum and neodymium are significantly lower due to different coordination tendencies. Lanthanide fixation upon silica with PO4 groups anchored on its surface may be useful in the manufacturing of special phosphate-silicate glasses. (C) 2003 Elsevier B.V. All rights reserved.

Evaluation of platelet number and function and fibrinogen level in patients bitten by snakes of the Bothrops genus.

Platelet function and plasma fibrinogen levels were evaluated in 14 patients, 10 males and 4 females, aged 13-59 years bitten by Bothrops genus snakes. There was a statistical difference (p < 0.05) among plasma fibrinogen levels evaluated 24 and 48 hours after envenomation. There was a tendency towards normalization after 48 hours of treatment. The low platelet number was clear in 24-48 hour evaluations with a tendency towards normalization after 48 hours of treatment (p < 0.05). When platelet function was stimulated by collagen and epinephrine, it appeared to be within normal values. On the other hand, when it was stimulated by adenosine diphosphate (ADP), platelet function was hypoaggregated by a single micromol concentration until 48 hours after treatment. At a 3 micromol concentration, there were alterations only before specific treatment (p < 0.05). Fibrinogen levels and fibrin degradation product (FDP) levels appeared to be altered in 83.33% of patients evaluated. The authors suggest that platelet hypoaggregation is related to decreased fibrinogen and increased FDP levels.

Pharmacological antagonism of Anchietia salutaris extracts on the contraction induced by prostaglandin D2 and U46619 in guinea-pig lung parenchymal strips

Anchietia salutaris tea is traditionally used in Brazil to treat allergies, suggesting it contains compounds with antagonistic activity on the allergic mediators. We have evaluated extracts and semi-purified fractions of Anchietia salutaris as a source of compounds having this type of antagonism on the contraction induced in guinea-pig lung parenchymal strips and on platelet aggregation and shape change. After 10 min pre-incubation dichloromethane extracts containing 30 or 100 μg mL-1 inhibited the contraction induced by prostaglandin D2 (PGD2) in guinea-pig lung parenchymal strips with dose ratios (DR) of 0.76 ± 0.14 and 0.93 ± 0.19, respectively; the amount of inhibition depended both on the concentration and on the time of preincubation (DR after 30 min pre-incubation was 1.21 ± 0.51). The dichloromethane extract and its semi-purified fractions also inhibited the contractions induced by U46619, a more potent, stable, synthetic agonist of thromboxane A2 (TxA2) prostanoid (TP) receptors, the receptors acted upon by PGD2 to produce lung contractions. The dichloromethane extract did not inhibit the lung parenchymal contractions induced by histamine, leukotriene D4 (LTD4) or platelet-activating factor (PAF). Platelet aggregation induced by U46619, adenosine 5'-diphosphate (ADP) or PAF was not inhibited by the dichloromethane extract. Indeed, the extract potentiated platelet aggregation induced by low concentrations of these agonists and also potentiated the shape change induced by U46619. These results imply that the dichloromethane extract of Anchietia salutaris and its semipurified fractions contain an active principle that competitively inhibits TxA2 TP receptors, the stimulation of which causes lung parenchymal contraction. The inhibition seems to be selective for this receptor subtype, because the extract fails to inhibit platelet aggregation or shape change. This provides additional support of earlier reports suggesting the occurrence of TP receptor subtypes.

Molecular model of shikimate kinase from Mycobacterium tuberculosis

Tuberculosis (TB) resurged in the late 1980s and now kills approximately 3 million people a year. The reemergence of tuberculosis as a public health threat has created a need to develop new anti-mycobacterial agents. The shikimate pathway is an attractive target for herbicides and anti-microbial agents development because it is essential in algae, higher plants, bacteria, and fungi, but absent from mammals. Homologs to enzymes in the shikimate pathway have been identified in the genome sequence of Mycobacterium tuberculosis. Among them, the shikimate kinase I encoding gene (aroK) was proposed to be present by sequence homology. Accordingly, to pave the way for structural and functional efforts towards anti-mycobacterial agents development, here we describe the molecular modeling of M. tuberculosis shikimate kinase that should provide a structural framework on which the design of specific inhibitors may be based. © 2002 Elsevier Science (USA). All rights reserved.

Biosynthetic origins of the isoprene units of 4-nerolidylcatechol in Potomorphe umbellata

The biosynthetic origins of the isoprene units of 4-nerolidylcatechol (1), the major constituent of Potomorphe umbellata, have been studied through feeding experiments with [14C]- and [13C]-glucose, and with precursors of the mevalonic acid and triose/pyruvate pathways, namely, [2- 14C]-mevalonolactone and [U-14C]-glyceraldehyde-3- phosphate, respectively. The pattern of incorporation of label from [1- 13C]-glucose into 1 was determined by quantitative 13C NMR spectroscopy. The labelling pattern revealed that the additive was specifically incorporated, and that the isoprene units of the sesquiterpenoid moiety of 4-nerolidylcatechol were derived from both the mevalonic acid and the triose/pyruvate pathways. The results indicate that both plastidic and cytoplasmic pathways are able to provide isopentenyl diphosphate units for the biosynthesis of 1. ©2005 Sociedade Brasileira de Química.

UMP kinase from Mycobacterium tuberculosis: Mode of action and allosteric interactions, and their likely role in pyrimidine metabolism regulation

The pyrH-encoded uridine 5′-monophosphate kinase (UMPK) is involved in both de novo and salvage synthesis of DNA and RNA precursors. Here we describe Mycobacterium tuberculosis UMPK (MtUMPK) cloning and expression in Escherichia coli. N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtUMPK. MtUMPK catalyzed the phosphorylation of UMP to UDP, using ATP-Mg 2+ as phosphate donor. Size exclusion chromatography showed that the protein is a homotetramer. Kinetic studies revealed that MtUMPK exhibits cooperative kinetics towards ATP and undergoes allosteric regulation. GTP and UTP are, respectively, positive and negative effectors, maintaining the balance of purine versus pyrimidine synthesis. Initial velocity studies and substrate(s) binding measured by isothermal titration calorimetry suggested that catalysis proceeds by a sequential ordered mechanism, in which ATP binds first followed by UMP binding, and release of products is random. As MtUMPK does not resemble its eukaryotic counterparts, specific inhibitors could be designed to be tested as antitubercular agents. © 2010 Elsevier Inc. All rights reserved.

Influence of experimental canine ehrlichiosis on the E-ADA activity and purine levels in serum and possible functional correlations with pathogenesis

The aim of this study was to evaluate adenosine deaminase activity and purines levels in serum of dogs experimentally infected by Ehrlichia canis. Banked serum samples of dogs divided into two groups with five animals each: healthy animals and animals infected by E. canis. The concentration of purines (adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine, inosine, hypoxanthine, xanthine and uric acid), and adenosine deaminase (E-ADA) activity in sera were evaluated. Samples were collected on days 12 and 30 post-infection (PI). The E-ADA activity showed a significant reduction on day 12 PI, and increased on day 30 PI in dogs infected with E. canis. On day 12, an increase in seric concentration of ATP, ADP and adenosine was verified, and different levels of hypoxanthine, xanthine and uric acid had a drastic reduction in infected compared healthy dogs (P< 0.05). However, on day 30 PI, the levels of seric ADP and AMP decreased, unlike the concentration of xanthine and uric acid that increased significantly in infected dogs (P< 0.05). Therefore, the activity of E-ADA and purine levels are altered in experimental canine ehrlichiosis, probably with the purpose of modulating the pathogenesis of the disease related to immune response, oxidative stress and coagulation disorders in acute phase. © 2013 Elsevier B.V.