Étude structuro-fonctionnelle de SecP43 et caractérisation de ses interactions avec SepSecS et l’ARNtSec in vitro

Selenoproteins are proteins containing selenium in the form of the 21st amino acid, selenocysteine. Selenocysteine (Sec) is directly synthesized onto its cognate tRNA (tRNA[Ser]Sec or tRNASec) and inserted into selenoproteins co-translationally with the help of various cis- and trans-acting factors. Among those factors, SecP43 has been reported to possibly play an essential role in the methylation at the 2’-hydroxylribosyl moiety in the wobble position (Um34) of Sec-tRNA[Ser]Sec and consequently reduce the expression of glutathione peroxidase 1. SecP43 also called tRNASec-associated protein has also been reported to interact in with SepSecS and tRNASec in vivo and the targeted removal of one of these proteins affected the binding of the other to the Sec-tRNASec. The initial aim of the project was to solve the structure of SecP43 by means of x-ray crystallography. Secondly, we were interested in characterizing the interaction of the latter with some of the components of the selenocysteine insertion machinery. These factors are SepSecS and tRNASec. We were able to optimize the expression and the purification of soluble form of the human homologue of SecP43 and of SepSecS by using an adapted auto-induction protocol. This was a major challenge considering that full length SecP43 has not been expressed and purify to date. We did not succeed in crystallizing SecP43. Our failure to crystallize SecP43 is probably due to the fact that it is a partially folded protein as we were able to demonstrate by SAXS (Small Angle X-ray Scattering). The SecP43 envelope calculated by SAXS displayed a rod-shape like structure. In order to enhance the stability of SecP43 required for crystallization, binding affinity studies were conducted to characterize the interaction between SecP43, tRNASec and SepSecS. We did not detect an interaction between SecP43 and tRNASec by using EMSA (Electrophoretic Mobility Shift Assay) and gel filtration. We also could not detect an interaction between SecP43 and SepSecS using a cross-linking assay. In contrast, the tRNASec/SepSecS interaction was demonstrated by EMSA and the addition of SecP43 seemed to reduce the binding affinity. Therefore, SecP43 might induce a conformational change in SepSecS in the presence of tRNASec.

Die Rolle des Zelladhäsionsmoleküls L1 in der Tumorprogression

Das neuronale Adhäsionsmolekül L1 wird neben den Zellen des Nervensystems auf vielen humanen Tumoren exprimiert und ist dort mit einer schlechten Prognose für die betroffenen Patienten assoziiert. Zusätzlich zu seiner Funktion als Oberflächenmolekül kann L1 durch membranproximale Spaltung in eine lösliche Form überführt werden. In der vorliegenden Arbeit wurde der Einfluss von L1 auf die Motilität von Tumorzellen untersucht. Lösliches L1 aus Asziten führte zu einer Integrin-vermittelten Zellmigration auf EZM-Substraten. Derselbe Effekt wurde durch Überexpression von L1 in Tumorlinien beobachtet. Weiterhin führt die L1-Expression zu einer erhöhten Invasion, einem verstärkten Tumorwachstum in NOD/SCID Mäusen und zur konstitutiven Aktivierung der MAPK ERK1/2. Eine Mutation in der zytoplasmatischen Domäne von hL1 (Thr1247Ala/Ser1248Ala)(hL1mut) führte hingegen zu einer Blockade dieser Funktionen. Dies weist daraufhin, dass nicht nur lösliches L1, sondern auch die zytoplasmatische Domäne von L1 funktionell aktiv ist. Im zweiten Teil der Arbeit wurde der Mechanismus, der L1-vermittelten Signaltransduktion untersucht. Die zytoplasmatische Domäne von L1 gelangt nach sequenzieller Proteolyse durch ADAM und Presenilin-abhängiger γ-Sekretase Spaltung in den Zellkern. Diese Translokation im Zusammenspiel mit der Aktivierung der MAPK ERK1/2 durch L1-Expression führt zu einer L1-abhängigen Genregulation. Die zytoplasmatische Domäne von hL1mut konnte ebenfalls im Zellkern detektiert werden, vermittelte jedoch keine Genregulation und unterdrückte die ERK1/2 Phosphorylierung. Die L1-abhängige Induktion von ERK1/2-abhängigen Genen wie Cathepsin B, β3 Integrin und IER 3 war in Zellen der L1-Mutante unterdrückt. Die Expression des Retinsäure-bindenden Proteins CRABP-II, welches in hL1 Zellen supprimiert wird, wurde in der L1-Mutante nicht verändert. Weitere biochemische Untersuchungen zeigen, dass die zytoplasmatische Domäne von L1 Komplexe mit Transkriptionsfaktoren bilden kann, die an Promoterregionen binden können. Die dargestellten Ergebnisse belegen, dass L1-Expression in Tumoren an drei Funktionen beteiligt ist; (i) L1 erhöht Zellmotilität, (ii) fördert Tumorprogression durch Hochregulation von pro-invasiven und proliferationsfördernden Genen nach Translokation in den Nukleus und (iii) schützt die Zellen mittels Regulation pro- bzw. anti-apoptotischer Gene vor Apoptose. Die mutierte Phosphorylierungsstelle im L1-Molekül ist essentiell für diese Prozesse. Die Anwendung neuer Therapien für Patienten mit L1-positiven Karzinomen kann mit Hinblick auf die guten Erfolge der Antikörper-basierenden Therapie mit dem mAk L1-11A diskutiert werden.

Recovery of norbixin from a raw extraction solution of annatto pigments using colloidal gas aphrons (CGAs)

Annatto dyes are widely used in food and are finding increasing interest also for their application in the pharmaceutical and cosmetics industry. Bixin is the main pigment extracted from annatto seeds and accounts for 80% of the carotenoids in the outer coat of the seeds; norbixin being the water-soluble form of the bixin. Typically annatto dyes are extracted from the seeds by mechanical means or solutions of alkali, edible oil or organic solvents, or a combination of the two depending on the desired final product. In this work CGAs are investigated as an alternative separation method for the recovery of norbixin from a raw extraction solution of annatto pigments in KOH. A volume of CGAs generated from a cationic surfactant (CTAB) solution is mixed with a volume of annatto solution and when the mixture is allowed to settle it separates into the top aphron phase and the bottom liquid phase. Potassium norbixinate presented in the annatto solution will interact with the surfactant in the aphron phase, which results in the effective separation of norbixin. Recovery= 94% was achieved at a CTAB to norbixin molar ratio of 3.3. In addition a mechanism of separation is proposed here based on the separation results with the cationic surfactant and an anionic surfactant (bis-2-ethyl hexyl sulfosuccinate, AOT) and measurements of surfactant to norbixin ratio in the aphron phase; electrostatic interactions between the surfactant and norbixin molecules result in the fort-nation of a coloured complex and effective separation of norbixin. (c) 2005 Elsevier B.V. All rights reserved.

Basic aminopeptidase activity is an emerging biomarker in collagen-induced rheumatoid arthritis

The objective of this study was to investigate the catalytic activity of basic aminopeptidase (APB) and its association with periarticular edema and circulating tumor necrosis factor (TNF)-alpha and type II collagen (CII) antibodies (AACII) in a rat model of rheumatoid arthritis (RA) induced by CII (CIA). Edema does not occur in part of CH-treated, even when AACII is higher than in control. TNF-alpha is detectable only in edematous CII-treated. APB in synovial membrane is predominantly a membrane-bound activity also present in soluble form and with higher activity in edematous than in non-edematous CH-treated or control. Synovial fluid and blood plasma have lower APB in non-edematous than in edematous CII-treated or control. In peripheral blood mononuclear cells (PBMCs) the highest levels of APB are found in soluble form in control and in membrane-bound form in non-edematous CII-treated. CII treatment distinguishes two categories of rats: one with arthritic edema, high AACII, detectable TNF-alpha, high soluble and membrane-bound APB in synovial membrane and low APB in the soluble fraction of PBMCs, and another without edema and with high AACII, undetectable TNF-alpha, low APB in the synovial fluid and blood plasma and high APB in the membrane-bound fraction of PBMCs. Data suggest that APB and CIA are strongly related. (C) 2011 Elsevier B.V. All rights reserved.

Avaliação da toxicidade aguda do inseticida metomil e o seu efeito sobre a atividade da acetilcolinesterase do peixe Danio rerio

Methomyl (Lannate®) is an insecticide from the carbamate group, frequently used in pest control in various types of crops. This compound works inhibiting the activity of the enzyme acetylcholinesterase. The use of physicochemical and ecotoxicological analysis is the most efficient strategy for the correct characterization and control of residues of metomil. The main objectives of this study were to evaluate the acute toxicity of methomyl in 96 hours of exposure and, through a sublethal assay of 5 hours, to assess its effect on the activity of acetylcholinesterase present in brain and squeletic dorsal muscle of the Danio rerio fish. The results showed that the LC50-96 found to D. rerio was 3.4 mg/L and it was found through the average of four definitive tests. In vitro assays were used to test the inhibitory action of methomyl directly over soluble AChE, extracted from the squeletic dorsal muscle, with maximum inhibition of 68.57% to the insecticide concentrations of 0.2 mg/L. In sublethal tests with D. rerio, inhibitory effect of methomyl was found over the soluble form of AChE in the squeletic dorsal muscle, both in one and five hours of fish exposure to the insecticide. In both period, the average values of inhibition were around 61%. In the same condition, no significant inhibitory effect of methomyl soluble and membrane AChE of the D. rerio was observed in the 0.42, 0.85, 1.70 and 2.50 mg/L concentrations and in both times of fish exposure

Functional shikimate dehydrogenase from Mycobacterium tuberculosis H37Rv: Purification and characterization

Tuberculosis (TB) poses a major worldwide public health problem. The increasing prevalence of TB, the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, the causative agent of TB, and the devastating effect of co-infection with HIV have highlighted the urgent need for the development of new antimycobacterial agents. Analysis of the complete genome sequence of M. tuberculosis shows the presence of genes involved in the aromatic amino acid biosynthetic pathway. Experimental evidence that this pathway is essential for M. tuberculosis has been reported. The genes and pathways that are essential for the growth of the microorganisms make them attractive drug targets since inhibiting their function may kill the bacilli. We have previously cloned and expressed in the soluble form the fourth shikimate pathway enzyme of the M. tuberculosis, the aroE-encoded shikimate dehydrogenase (mtSD). Here, we present the purification of active recombinant aroE-encoded M. tuberculosis shikimate dehydrogenase (mtSD) to homogeneity, N-terminal sequencing, mass spectrometry, assessment of the oligomeric state by gel filtration chromatography, determination of apparent steady-state kinetic parameters for both the forward and reverse directions, apparent equilibrium constant, thermal stability, and energy of activation for the enzyme-catalyzed chemical reaction. These results pave the way for structural and kinetic studies, which should aid in the rational design of mtSD inhibitors to be tested as antimycobacterial agents. (c) 2005 Elsevier B.V. All rights reserved.

Use of vitamin D to reduce lameness in broilers reared in harsh environments

In tropical poultry-producing countries, poultry houses usually have little environmental control. This study investigated the effect of dietary vitamin D on the incidence of leg abnormalities of a fast-growing broiler strain reared under harsh conditions. In this study, 300 one-day-old male broilers were distributed in two treatments with three replicates of 50 birds each. One group was fed a placebo and the other group was fed 25-hydroxycholecalciferol (25-OH-D3) in a soluble form. The environmental variables were weekly recorded during until birds were 49 days old. Birds were weekly gait scored, and their walking speed and vertical force while walking were assessed. Post-mortem examination was performed and skeletal lesions were assessed. Control birds presented more leg problems than those that ingested 25-OH-D3. These results suggest that dietary vitamin D is effective in decreasing the severity of lameness by reducing tibial dyschondroplasia and other leg abnormalities.

DAHP synthase from Mycobacterium tuberculosis H37Rv: Cloning, expression, and purification of functional enzyme

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains the leading cause of mortality due to a bacterial pathogen. According to the 2004 Global TB Control Report of the World Health Organization, there are 300,000 new cases per year of multi-drug resistant strains (MDR-TB), defined as resistant to isoniazid and rifampicin, and 79% of MDR-TB cases are now super strains, resistant to at least three of the four main drugs used to treat TB. Thus there is a need for the development of effective new agents to treat TB. The shikimate pathway is an attractive target for the development of antimycobacterial agents because it has been shown to be essential for the viability of M. tuberculosis, but absent from mammals. The M. tuberculosis aroG-encoded 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (mtDAHPS) catalyzes the first committed step in this pathway. Here we describe the PCR amplification, cloning, and sequencing of aroG structural gene from M. tuberculosis H37Rv. The expression of recombinant mtDAHPS protein in the soluble form was obtained in Escherichia coli Rosetta-gami (DE3) host cells without IPTG induction. An approximately threefold purification protocol yielded homogeneous enzyme with a specific activity value of 0.47 U mg-1 under the experimental conditions used. Gel filtration chromatography results demonstrate that recombinant mtDAHPS is a pentamer in solution. The availability of homogeneous mtDAHPS will allow structural and kinetics studies to be performed aiming at antitubercular agents development. © 2004 Elsevier Inc. All rights reserved.

Expressão e análise de proteínas recombinantes de Xanthomonas axonopodis pv. citri em E. coli, visando análise estrutural por cristalografia

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Análise imunológica comparada da eficiência e especificidade da hialuronidase recombinante de Polybia paulista (Hymenoptera, Vespidae) expressa em bactéria e levedura

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

Estudo das interações entre a proteína TRF de Leishmania amazonensis (LaTRF) e suas possíveis parcerias

Pós-graduação em Ciências Biológicas (Genética) - IBB

Protonation Pattern, Tautomerism, Conformerism, and Physicochemical Analysis in New Crystal Forms of the Antibiotic Doxycycline

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Determinação de fatores angiogênicos e anti-angiogênicos em plasma de gestantes portadoras de pré-eclâmpsia precoce e tardia

Changes in circulating angiogenic factors have been implicated in the pathogenesis of preeclampsia. Thus, evaluation of angiogenesis agonist and antagonist factors is of greater importance to understand the mechanisms responsible for this disorder. The objective of the present study was to evaluate whether circulating angiogenic and anti-angiogenic factors may differentiate early-onset from late-onset preeclampsia. The study was conducted in 86 women with preeclampsia diagnosed in the third trimester of pregnancy. Preeclampsia was classified according to the onset of clinical manifestation in early-onset (before 34 weeks of gestation; n=31) or in late-onset (from 34 weeks of gestation on; n=55) preeclampsia. Serum was obtained from the patients in the moment of the diagnosis and assayed for placental growth factor (PlGF), vascular endothelial growth factor (VEGF), soluble Endoglin (sEng) and soluble form of vascular endothelial growth factor receptor (sVEGFR-1) determination by enzyme-linked immunosorbent assay. The results showed that early-onset preeclampsia was characterized by significant lower levels of PlGF (median 38.3 vs 123.5 pg/mL) and VEGF (median 23.1 vs 35.3 pg/mL) in serum as well as by higher serum levels of sEng (median 54.7 vs 42.1 pg/mL) and sVEGFR-1 (median 5211.0 vs 4657.6 pg/mL) compared with late-onset preeclampsia. In this study serum levels of angiogenic and anti-angiogenic factors prove useful in differentiating early-onset from late-onset preeclampsia in the third trimester of pregnancy. Therefore, these findings suggest that angiogenic factors determination may indicate that early- and late-onset preeclampsia have different pathophysiology

Caracterização funcional da proteína XAC1201 envolvida na patogenicidade de Xanthomonas citri subsp. citri

Pós-graduação em Agronomia (Genética e Melhoramento de Plantas) - FCAV

Marcadores de ativação imune, inflamação crônica e estresse oxidativo em pessoas que vivem com o HIV/Aids: a busca do melhor prognóstico

Pós-graduação em Doenças Tropicais - FMB