Contrasting roles for Myc and Mad proteins in cellular growth and differentiation.

The positive effects of Myc on cellular growth and gene expression are antagonized by activities of another member of the Myc superfamily, Mad. Characterization of the mouse homolog of human mad on the structural level revealed that domains shown previously to be required in the human protein for anti-Myc repression, sequence-specific DNA-binding activity, and dimerization with its partner Max are highly conserved. Conservation is also evident on the biological level in that both human and mouse mad can antagonize the ability of c-myc to cooperate with ras in the malignant transformation of cultured cells. An analysis of c-myc and mad gene expression in the developing mouse showed contrasting patterns with respect to tissue distribution and developmental stage. Regional differences in expression were more striking on the cellular level, particularly in the mouse and human gastrointestinal system, wherein c-Myc protein was readily detected in immature proliferating cells at the base of the colonic crypts, while Mad protein distribution was restricted to the postmitotic differentiated cells in the apex of the crypts. An increasing gradient of Mad was also evident in the more differentiated subcorneal layers of the stratified squamous epithelium of the skin. Together, these observations support the view that both downregulation of Myc and accumulation of Mad may be necessary for progression of precursor cells to a growth-arrested, terminally differentiated state.

Tumorigenic activity of the BCR-ABL oncogenes is mediated by BCL2.

BCR-ABL is a chimeric oncogene generated by translocation of sequences from the c-abl protein-tyrosine kinase gene on chromosome 9 into the BCR gene on chromosome 22. Alternative chimeric proteins, p210BCR-ABL and p190BCR-ABL, are produced that are characteristic of chronic myelogenous leukemia and acute lymphoblastic leukemia, respectively. Their role in the etiology of human leukemia remains to be defined. Transformed murine hematopoietic cells can be used as a model of BCR-ABL function since these cells can be made growth factor independent and tumorigenic by the action of the BCR-ABL oncogene. We show that the BCR-ABL oncogenes prevent apoptotic death in these cells by inducing a Bcl-2 expression pathway. Furthermore, BCR-ABL-expressing cells revert to factor dependence and nontumorigenicity after Bcl-2 expression is suppressed. These results help to explain the ability of BCR-ABL oncogenes to synergize with c-myc in cell transformation.

Clonagem molecular, expressão, purificação e caracterização estrutural da endoglucanase de Trichoderma harzianum visando o desenvolvimento de coquetéis enzimáticos para a produção de etanol lignocelulósico

O aumento na demanda mundial por energia, a perspectiva de encolhimento dos recursos energéticos e a preocupação global com a questão ambiental, despertaram o interesse por fontes alternativas de energia. A biomassa lignocelulósica é abundante e de baixo custo, com potencial para complementar a produção em larga escala de combustíveis. A degradação das moléculas constituintes da parede celular à açúcares fermentescíveis e então à etanol, ocorre através da hidrólise enzimática da biomassa. Contudo, a utilização de enzimas para esse fim encontra-se em estágio exploratório e representa um gargalo na implementação de tecnologias de etanol 2G em escala industrial, desencadeando a busca de celulases bioquimicamente mais ativas, estáveis e economicamente viáveis. O presente trabalho visou a caracterização da endoglucanase I do fungo Trichoderma harzianum, e para isso foi realizada expressão, ensaios bioquímicos e biofísicos do domínio catalítico (ThCel7B-CCD) e da proteína inteira (ThCel7B-full). A enzima exibiu um perfil acidofílico, com atividade ótima em pH 3,0 a 55°C. A proteína também se mostrou capaz de hidrolisar uma variedade de substratos, sendo a maior atividade hidrolítica em β-glucano (75 U mg-1). Ao analisar a estabilidade térmica medida a 55°C em pH 5, a atividade residual manteve-se intacta por mais de 2 meses. Outra característica relevante foi o elevado grau de sinergismo entre ThCel7B e ThCel7A. Análises de microscopia eletrônica de flocos de aveia submetidas à hidrólise com ThCel7B evidenciaram os efeitos de degradação do substrato em relação às amostras controle. O conjunto desses resultados, além de importante para a compreensão do mecanismo molecular de ThCel7B e de outras endoglucanases da família GH7, também revelou uma enzima de interesse biotecnológicos uma vez que o comportamento ácido e sua estabilidade térmica são características relevantes para aplicações industriais sob condições extremamente ácidas.

Functional consequences of sequence alterations in the ATM gene

The product of the gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) is a high molecular weight, protein (similar to350 kDa) containing a C-terminal protein kinase domain and a number of other putative domains not yet functionally defined. The majority of ATM gene mutations in A-T patients are truncating, resulting in prematurely terminated products that are highly unstable. Missense mutations within the kinase domain and elsewhere in the molecule alter the stability of the protein and lead to loss of protein kinase activity. Only rarely are patients observed with two missense mutations and this gives rise to a milder disease phenotype. Evidence for a dominant interfering effect on normal ATM kinase activity has been reported in cell lines transfected with missense mutant ATM and in cell lines from some A-T heterozygotes. The dominant negative effect of mutant ATM is manifested by an enhancement of cellular radiosensitivity and may be responsible for the cancer predisposition observed in carriers of ATM missense mutations. In this review, we explore the domain structure of the ATM molecule, sites of interaction with other proteins and the consequences of specific amino acid changes on function. (C) 2003 Elsevier B.V. All rights reserved.

Intracellar signalling in the HGT-1 gastric cell line and in rat isolated parietal cells

The work presented in this thesis was undertaken to increase understanding of the intracellular mechanisms regulating acid secretion by gastric parietal cells. Investigation of the effects of protein kinase C on secretory activity induced by a variety of agents was a major objective. A further aim was to establish the sites at which epidermal growth factor (EGF) acts to stimulate prostaglandin E2 (PGE2) production and to inhibit acid secretion. These investigations were carried out by using the HGT-1 human gastric cancer cell line and freshly isolated rat parietal cells. In HGT-1 cells, the cyclic AMP response to histamine and to truncated glucagon-like peptide 1 (TGLP-1) was reduced when protein kinase C was activated by 12-0-tetradecanoylphorbol 13-acetate (TPA). Receptor-binding studies and experiments in which cyclic AMP production in HGT-1 cells was stimulated by gastric inhibitory polypeptide, cholera toxin and forskolin suggested that the effect of TPA was mediated by uncoupling of the histamine H2 receptor from the guanine nucleotide regulatory protein Gs, possibly by phosphorylation of the receptor. An involvement of protein kinase C α in this effect was suggested because an antibody to this isoform specifically prevented the inhibitory effects of TPA on histamine-stimulated adenylate cyclase activity in a membrane fraction prepared from HGT-1 cells. Carbachol-stimulated secretory activity in parietal cells was specifically inhibited by Ro 31-8220, a bisindolylmaleimide inhibitor of protein kinase C. Thus protein kinase C may play a role in the activation of the secretory response to carbachol. In parietal cells prelabelled with [3H]-arachidonic acid or [3H]myristic acid, EGF did not affect [3H]-fatty acid or [3H] - diacylglycerol content. No evidence for effects of EGF on phosphatidylinositol glycan-specific phospholipase C, phospholipase A2 or on low Km cyclic AMP phosphodiesterase activities were found.

Catching transcriptional regulation by thermostatistical modeling

Gene expression is frequently regulated by multiple transcription factors (TFs). Thermostatistical methods allow for a quantitative description of interactions between TFs, RNA polymerase and DNA, and their impact on the transcription rates. We illustrate three different scales of the thermostatistical approach: the microscale of TF molecules, the mesoscale of promoter energy levels and the macroscale of transcriptionally active and inactive cells in a cell population. We demonstrate versatility of combinatorial transcriptional activation by exemplifying logic functions, such as AND and OR gates. We discuss a metric for cell-to-cell transcriptional activation variability known as Fermi entropy. Suitability of thermostatistical modeling is illustrated by describing the experimental data on transcriptional induction of NF?B and the c-Fos protein.

Suppression of the Insulin Receptors in Adult Schistosoma japonicum Impacts on Parasite Growth and Development: Further Evidence of Vaccine Potential

To further investigate the importance of insulin signaling in the growth, development, sexual maturation and egg production of adult schistosomes, we have focused attention on the insulin receptors (SjIRs) of Schistosoma japonicum, which we have previously cloned and partially characterised. We now show, by Biolayer Interferometry, that human insulin can bind the L1 subdomain (insulin binding domain) of recombinant (r)SjIR1 and rSjIR2 (designated SjLD1 and SjLD2) produced using the Drosophila S2 protein expression system. We have then used RNA interference (RNAi) to knock down the expression of the SjIRs in adult S. japonicum in vitro and show that, in addition to their reduced transcription, the transcript levels of other important downstream genes within the insulin pathway, associated with glucose metabolism and schistosome fecundity, were also impacted substantially. Further, a significant decrease in glucose uptake was observed in the SjIR-knockdown worms compared with luciferase controls. In vaccine/challenge experiments, we found that rSjLD1 and rSjLD2 depressed female growth, intestinal granuloma density and faecal egg production in S. japonicum in mice presented with a low dose challenge infection. These data re-emphasize the potential of the SjIRs as veterinary transmission blocking vaccine candidates against zoonotic schistosomiasis japonica in China and the Philippines.

Caractérisation de l'impact des conditions opératoires sur l'efficience d'un procédé de concentration du lait par ultrafiltration

Les concentrés de protéines de lait sont couramment utilisés comme ingrédients lors de la standardisation du lait de fromagerie. La concentration des protéines est généralement réalisée par ultrafiltration (UF) à l’aide de membranes polymériques ayant un seuil de coupure de 10 kDa, et ce, jusqu’à un facteur de concentration volumique de 3.5X. Dans l’optique d’améliorer l’efficience du procédé d’UF, l’étude avait pour but de caractériser l’impact du mode opératoire (pression transmembranaire constante (465 et 672 kPa) et flux constant) ainsi que la température (10°C et 50°C) sur la performance du système jusqu’à un facteur de concentration volumique de 3.6X. Le module de filtration à l’échelle pilote comprenait une membrane d’UF en polyéthersulfone de 10 kDa d’une surface de 2,04 m2. La performance du système a été caractérisée sur le flux de perméation, la sélectivité et la consommation énergétique totale. L’étude a montré que le flux de perméation était 1,9 fois plus élevé à une température de 50°C comparativement à 10°C lors de l’UF du lait. Le coefficient de rejet des protéines n’a pas été affecté significativement par la pression transmembranaire et la température (P< 0,05). L’effet de la température a été observé au niveau de la teneur en calcium, laquelle était plus élevée de 12% dans les rétentats générés à 50C. La consommation énergétique totale du système d’UF était plus élevée à 10C comparativement à 50C, représentant 0,32±0,02 et 0,26±0,04 kWh/kg rétentat respectivement. Les résultats montrent que le ratio d’efficience énergétique (rapport entre le flux de perméation et la consommation énergétique) optimal a été obtenu à faible pression transmembranaire constante et à 50C. L’approche développée dans le cadre de ce projet fournira des outils aux industriels laitiers pour améliorer l’éco-efficience de leurs procédés de séparation baromembranaire.

An immunoglobulin C kappa-reactive single chain antibody fusion protein induces tolerance through receptor editing in a normal polyclonal immune system.

Understanding immune tolerance mechanisms is a major goal of immunology research, but mechanistic studies have generally required the use of mouse models carrying untargeted or targeted antigen receptor transgenes, which distort lymphocyte development and therefore preclude analysis of a truly normal immune system. Here we demonstrate an advance in in vivo analysis of immune tolerance that overcomes these shortcomings. We show that custom superantigens generated by single chain antibody technology permit the study of tolerance in a normal, polyclonal immune system. In the present study we generated a membrane-tethered anti-Igkappa-reactive single chain antibody chimeric gene and expressed it as a transgene in mice. B cell tolerance was directly characterized in the transgenic mice and in radiation bone marrow chimeras in which ligand-bearing mice served as recipients of nontransgenic cells. We find that the ubiquitously expressed, Igkappa-reactive ligand induces efficient B cell tolerance primarily or exclusively by receptor editing. We also demonstrate the unique advantages of our model in the genetic and cellular analysis of immune tolerance.

Stimulation of "stress-regulated" mitogen-activated protein kinases (stress-activated protein kinases/c-Jun N-terminal kinases and p38-mitogen-activated protein kinases) in perfused rat hearts by oxidative and other stresses.

"Stress-regulated" mitogen-activated protein kinases (SR-MAPKs) comprise the stress-activated protein kinases (SAPKs)/c-Jun N-terminal kinases (JNKs) and the p38-MAPKs. In the perfused heart, ischemia/reperfusion activates SR-MAPKs. Although the agent(s) directly responsible is unclear, reactive oxygen species are generated during ischemia/reperfusion. We have assessed the ability of oxidative stress (as exemplified by H2O2) to activate SR-MAPKs in the perfused heart and compared it with the effect of ischemia/reperfusion. H2O2 activated both SAPKs/JNKs and p38-MAPK. Maximal activation by H2O2 in both cases was observed at 0.5 mM. Whereas activation of p38-MAPK by H2O2 was comparable to that of ischemia and ischemia/reperfusion, activation of the SAPKs/JNKs was less than that of ischemia/reperfusion. As with ischemia/reperfusion, there was minimal activation of the ERK MAPK subfamily by H2O2. MAPK-activated protein kinase 2 (MAPKAPK2), a downstream substrate of p38-MAPKs, was activated by H2O2 to a similar extent as with ischemia or ischemia/reperfusion. In all instances, activation of MAPKAPK2 in perfused hearts was inhibited by SB203580, an inhibitor of p38-MAPKs. Perfusion of hearts at high aortic pressure (20 kilopascals) also activated the SR-MAPKs and MAPKAPK2. Free radical trapping agents (dimethyl sulfoxide and N-t-butyl-alpha-phenyl nitrone) inhibited the activation of SR-MAPKs and MAPKAPK2 by ischemia/reperfusion. These data are consistent with a role for reactive oxygen species in the activation of SR-MAPKs during ischemia/reperfusion.

Deletion of Epstein-Barr virus latent membrane protein 1 gene in United States and Brazilian Hodgkin's disease and reactive lymphoid tissue: High frequency of a 30-bp deletion

A 30-basepair (bp) deletion in the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) gene has been reported in nasopharyngeal carcinoma and EBV-associated malignant lymphomas. Prior studies have found the deletion in about 10% to 28% of cases of Hodgkin's disease (HD), particularly in cases with aggressive histology. We studied the prevalence of 30-bp LMP1 gene deletion in EBV-positive HD in the United States (US) (12 cases) and Brazil (26 cases) with comparison to reactive lymphoid tissues (21 cases) and HD without EBV-positive Reed-Sternberg cells (15 cases). We studied the status of the LMP1 gene by Southern blot hybridization of polymerase chain reaction (PCR) products obtained after amplification with primers spanning the site of the deletion. We also performed EBV typing, EBER1 in situ hybridization, and LMP1 protein immunohistochemistry. EBV was detected in 12/26 (46%) cases of HD from the US and 26/27 (96%) cases of Brazilian HD. The 30-bp LMP1 gene deletion was observed in 4/12 (33%) cases of EBV-positive HD from US, and 12/26 (46%) cases of Brazilian EBV-positive HD, including 3 cases of type B EBV, as compared with 12/21 (57%) reactive lymphoid tissues and 9/15 (60%) cases of EBV-negative HD. US and Brazilian HD showed a higher prevalence of the 30-bp LMP1 gene deletion, compared with studies of others. The unexpected finding of high incidence of 30-bp deletion in LMP1 gene in reactive lymphoid tissue and HD without EBV-positive Reed-Sternberg cells suggests that this deletion may not be relevant to HD pathogenesis in most cases. Copyright (C) 1997 by W.B. Saunders Company.

Genotyping of Epstein-Barr virus in Brazilian Burkitt's lymphoma and reactive lymphoid tissue - Type A with a high prevalence of deletions within the latent membrane protein gene

Both Epstein-Barr virus (EBV) types A and B are found in endemic Burkitt's lymphoma (BL) occurring in equatorial Africa. We studied 17 cases of Brazilian BL previously demonstrated to be EBV-positive to determine the EBV type as well as the presence of a characteristic 30 bp deletion within the 3' end of the latent membrane protein-1 (LMP-1) gene that may be important to the pathogenesis of several EBV-associated neoplasms. All case in which the age was known were children. We found type A EBV in 13 of 14 (93%) evaluable cases, and type B in one case. The LMP-1 deletion was found in 12 of 15 (80%) evaluable cases, including the one case of type B EBV, and a similar high prevalence (59%) of the deletion was detected in EBV-positive normal and reactive lymphoid tissues from individuals from the same geographic region. The high proportion of cases associated with type A EBV suggests that immunodeficiency is not an important factor in the pathogenesis of Brazilian BL, in contrast to endemic African BL. The presence of the LMP-1 deletion in a high prevalence in the normal population in this region is unexplained.

Inflammatory response of endothelial cells to hepatitis C virus recombinant envelope glycoprotein 2 protein exposure

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

Gestational protein malnutrition impairs c-myc and p63 protein expression, increases prostatic intraepithelial neoplasia incidence and prostatitis aggressiveness in adult male offspring subjected to hormonal handling

Background: Prostate cancer is the second most common cancer diagnosed in men; however its etiology remains unknown. Previous studies have shown that environmental adverse factors, such as maternal nutritional status during pregnancy, can influence fetal development and predispose people to diseases in adult life. The feeding of low-protein diets to pregnant rats result in fetal growth disturbance, androgen/estrogen unbalance and changes in the expression and sensibility of hormone receptors in male offspring. These alterations can promote permanent changes in androgen dependent organs, such as in the prostate. In this sense, we hypothesized that the hormonal unbalance that occurs during aging can lead to an increase in the susceptibility to prostatic disorders. Aim: To evaluate our hypothesis, malnourished male rat offspring were submitted to simultaneous estrogen and testosterone exposure in adulthood, to drive lesions in the rat ventral prostate gland (VP). Methods: 17 week-old Wistar rats (n=48) that received in utero normal protein diet (NP group, AIN93G=17% protein) or low protein diet (RP group, AIN93G modified=6% protein) were given implants with 17β-estradiol plus testosterone administration (NPH and RPH groups) for 17 weeks. The animals were killed at the age of 34 weeks and the VP were excised, weighted and processed for histopathological, immunohistochemical (Ki67, AR, p63, e-caderin, laminin, c-myc and GSTP), biochemical and ultrastructural analysis. Results: Both absolute and relative VP weight from NPH animals were about 30% higher than RPH. Serological data showed that estradiol levels were similar in both groups, but testosterone levels were lower in the RPH male offspring. The steroid hormone exposure in adult life promoted prostate lesions in both RPH and NPH offspring associated with reactive stroma. VP from RPH group exhibited heightened susceptibility to prostatic intraepithelial neoplasia (mainly cribriform and signet ring-cell patterns) and increased the incidence and aggressiveness of prostatitis. In this group, a higher proportion of basal cells, increased proliferation index, lower expression ofthe androgen receptor and increased focus of collagenous micronodules closely associated to epithelial neoplasias were also observed. Conclusion:These observations suggest that maternal protein restriction alters adult prostate response to androgen/estrogen handling and increases susceptibility to prostate diseases. Ethical protocol:CEEA,476/2013 IBB-UNESP; Funding Support: 2009/50204-6 and 2013/09649-0.