Proteomics analysis of tissue samples from patients with squamous cell carcinoma of the penis and positive to human papillomavirus

ABSTRACTPurpose:The aim of this study was to identify possible protein biomarkers and/or candidates for therapeutic targets in tissues of patients with SCCP, infected by HPV, applying one dimensional electrophoresis (1DE), followed by direct mass spectrometry (MS) analysis.Materials and Methods:Tissues from 10 HPV positive patients with SCCP and from 10 patients with HPV negative non-tumorous penile foreskins were analyzed applying 1D electrophoresis, followed by analysis with direct mass spectrometry (MS).Results:Sixty-three different proteins were identified in the first group and 50 in the second group. Recognition was possible for 28 proteins exclusively detected in Group 1 and 21 proteins presented only in Group 2.Conclusion:Some proteins in the first group are directly involved in the development of other types of cancer, and therefore, suitable for analysis. Complement C3 protein is a strong candidate for evaluating SCCP patients.

Estudo do mecanismo cinético de ação da álcool desidrogenase de Thermoanaerobium brockii sobre o sistema isopropanol/acetona

Neste trabalho, o mecanismo cinético de ação da álcool desidrogenase de Thermoanaerobium brockii (TBADH) atuando sobre o sistema isopropanol/acetona foi estudado, visando tanto a elucidação do mecanismo cinético da TBADH quanto explicar o sistema acoplado de reciclagem da coenzima utilizando altas concentrações de isopropanol, 30 (por cento v/v), como co-solvente e co-substrato no meio reacional e com somente uma enzima atuando nas duas reações. Para tanto, realizamos estudos de velocidade inicial de produtos, estudos de inibição por produto e estudos de inibição por formação de complexo "dead-end", nos quais pirazol foi utilizado como inibidor. Os estudos de velocidade inicial na ausência de produtos, forneceram gráficos de Lineweaver-Burk tanto com efeito de inclinação, indicando que o mecanismo cinético era do tipo sequencial. A análise do significado cinético do complexo ternário central, E-coenzima-substrato, sugeriu que o mecanismo cinético era mais provavelmente, do tipo Theorell_Chance Bi-Bi. Nos estudos de inibição por produto objetivemos, gráficos de duplos recíprocos que apresentaram quatro perfis de inibição do tipo competitiva linear (entre NADP e NADPH e entre isopropanol e acetona) e quatro perfis de inibição do tipo mista linear (entre NADP e acetona e entre NADPH e isopropanol), desta maneira os mecanismos cinéticos dos tipos sequencial ordenado Bi-Bi, em estado estacionário, e sequencial ao acaso Bi-Bi, em estado estacionário foram totalmente descartado. Através do estudo de inibição por formação de complexo "dead-end, o mecanismo cinético do tipo sequencial ao acso Bi-Bi, em rápido equilíbrio, foi descartado, ficando caracterizado que no mecanismo cinético proposto a coenzima atua como o primeiro substrato a se ligar à forma livre de enzima. Ambos os parâmetros cinéticos e termodinâmicos da reação foram estimados, pelos ajustes das respectivas equações de velocidade aos dados experimentais obtidos, utilizando-se um programa de computador de regressão não linear e um prorama de computador baseado no método de Hooke-Jeeves, método de procura direta comaceleração em distância, ambos desenvolvidos em nosso laboratório. O ajuste da equação de velocidade inicial para a inibição por formação de complexo "dead-end", forneceu uma estimativa da constante de dissociação...

The kinetic mechanism of the redox reaction isopropanoVacetone catalyzed by Thermoanaerobium broe/ai alcohol dehydrogenase (TBADH) was studied aiming both elucidation of the kinetic mechanism and gaining insight into the coupled-substrate approach that has been extensively used for NADPH regeneration, by using high concentrations of isopropanol, 30% (v/v), as a co-solvent and co-substrate in preparative-scale reductions of carbonyl compounds. For this purpose, initial velocity studies in the absence of products, product inhibition studies and dead-end inhibition studies, using pyrazole , were performed. Initial velocity studies in the absence of products in the forward (isopropanol oxidation) and reverse (acetone reduction) reactions showed that all the double reciprocal plots were of the intersecting type, indicating that the kinetic mechanism of TBADH is of the sequential type, involving ternary or central complexes not necessarily significant kinetically. Kinetic evaluation of the significance of the central complexes, E-coenzymes-substrates, suggested that the mechanism is most probably of the Theorell-Chance Bi-Bi type The patterns obtained in product inhibition studies consisted in four linear competitive inhibitions (between NADP and NADPH, as well as, between isopropanol and acetone) and four linear mixed type inhibitions (between NADP and acetone, as well as, between NADPH and isopropanol). With these studies both the steady-state sequential ordered Bi-Bi mechanism and the steady-state random sequential Hi-Hi mechanism could be ruled out. Dead-end inhibition studies, using pyrazole as inhibitor, showed that NADP is the first substrate to bind to the enzyme followed by the binding of isopropanol, then the rapid equilibrium random sequential Bi-Bi mechanism was also ruled out. Both the kinetic and thermodynamic parameters of the reaction were estimated with a non-linear regression analysis computer program and a computer program based on the Hooke-Jeeves direct search method, both developed in our laboratory. The fitting of the dead-end inhibition equation, with formation of a TBADHNADP-pyrazole complex to the experimental data obtained in the presence of the deadend inhibitor gave estimates of the pyrazole dissociation constant of the E-NADPpyrazole complex, as well as, of the NADP dissociation constant of the same complex. The values obtained for these dissociation constants, when compared to those obtained with horse liver alcohol dehydrogenase, enable us to suggest a model for the structure of the ternary complex TBADH-NADP-pyrazole, in which the interaction of pyrazole occurs only by a coordinate bond formed between one of the heterocyclic nitrogen atoms of the inhibitor with a zinc atom, probably present at the active site of the enzyme. The Theorell-Chance Bi-Bi kinetic mechanism, proposed in this study, satisfactorily explains the recycling process of the coenzyme NADPH, using high concentrations of isopropanol 30 % (v/v), as a co-solvent and co-substrate in preparative-scale enantioselective reactions reduction. Since the coenzyme is the first substrate to bind the free form of the enzyme in a sequential and compulsory order, the interaction of isopropanol with the free form of the enzyme is not possible. Thus preventing TBADH to be saturated with isopropanol. In this way, free TBADH will always be available to catalyze the reduction of the oxidized substrate and the NADP formed in this reaction, continuously reduced to NADPH by isopropanol oxidation concomitantly catalyzed by the same enzyme.