Expression of c-erbB-2, p53 and c-myc proteins in male breast carcinoma: Comparison with traditional prognostic factors and survival

There are few data evaluating biological markers for men with breast cancer. The purpose of the present study was to analyze the expression of the oncogenes c-erbB-2 and c-myc and of the suppressor gene p53 by immunohistochemical techniques in archival paraffin-embedded tissue blocks of 48 male breast cancer patients, treated at the A.C. Camargo Cancer Hospital, Säo Paulo, SP, Brazil. The results were compared with clinicopathological prognostic features. Immunopositivity of c-erbB-2, p53 and c-myc was detected in 62.5, 16.7 and 20.8 percent of the cases analyzed, respectively. Estrogen and progesterone receptors were positive in 75 and 69 percent of the cases, respectively. Increasing staging was statistically associated with c-erbB-2 (P = 0.04) and weakly related to p53 positivity (P = 0.06). No significant correlation between specific survival rate (determined by the log rank test) and the molecular markers analyzed was found, whereas the number of compromised lymph nodes and advanced TNM (tumor, node, metastasis) staging were associated with diminished survival

Laminin-binding proteins in EJ-ras-transformed fibroblasts

Malignant transformation is accompanied by changes in cell-matrix interations. Upon transfection with EJ-ras oncogene, transformed fibroblasts, acquired a migratory phenotype towards laminin-1. The increase in integrin expression was responsible for the migratory activity of transformed fibroblasts. In addition alpha(6)beta(1) integrins, both galectin-3 and an unidentified laminin-binding polypeptide had their expression pattern altered upon transformation. Here, we review these two classes of laminin-binding proteins and their possible roles in cell-laminin interactions.

Detection of post-translational sulfation of alpha-5 beta-1 integrin and its role in integrin-fibronectin binding

Fibronectins are glycoproteins of the extracellular matrix composed of two 220-kDa polypeptide chains named A and B bound by two disulfide bridges. Both chains when digested with proteolytic enzymes give rise to six different domains named I to VI that are involved in the ligand properties of this molecule. Fibronectins bind fibrin, collagen, glycosaminoglycan residues and several integrins. In this study, using metabolic radiolabeling of alpha(5)beta(1) integrin with sodium sulfate, an immunoprecipitation reaction, inhibition of sulfate incorporation an a fibronectin-binding assay, we were able to detect this integrin as a sulfated molecule and this sulfation appears to regulate the integrin-fibronectin binding.

Cellular adhesion to laminin involves a chondroitin sulfate proteoglycan

Cell-extracellular matrix interactions are intimately involved in the regulation of many cellular processes such as embryonic development or tumor cell growth and metastasis. In our previous work we were able to detect a 90/100-kDa laminin binding chondroitin sulfate proteoglycan. A search for this molecule in different cell lines showed that it is only found in cells that adhere to laminin.

Adhesion to laminin is down-regulated upon retinoic acid-induced F9 cell differentiation: a role for alpha6/beta1 integrin

F9 mouse teratocarcinoma cells have a high capacity to adhere to laminin and we identified alpha6/beta1 integrin as the principal laminin-binding protein present in these cells. F9 cells differentiated into parietal endoderm when monolayer cultures were treated with retinoic acid and dibutyryl cyclic AMP. In this process a decreased adherence to laminin was observed due to a lower expression of alpha6/beta1 integrin on the cell surface

Regulation of extracellular matrix-degrading proteases

Extracellular matrix-degrading enzymes have a very important role in many normal and pathological processes. Members of the matrix metalloproteinase and plasminogen activator families are the major modulators of extracellular matrix degradation. Here, we discuss some topics about these enzymes giving special attention to the transcriptional and extracellular regulation of their expression.

Participation of glycosylation sites in the binding of Staphylococcus aureus to laminin

1. Microbial pathogenicity is in many instances associated with the ability to adhere to host surfaces or to extracellular matrix components. 2. Laminin is a major glycoprotein of basement membranes which can promote specific bacterial adhesion. Staphylococcus aureus is a pathogenic bacterium which presents a laminin receptor of about 50-kDa molecular mass (Lopes JD, Reis M per cent Brentani RR (1985). Science, 229: 275-277). 3. Adhesion inhibition assays of [125iodine]-labeled bacteria to laminin demonstrate that the receptor binding site is contained in the pepsin-derived (P1) laminin fragment. 4. Cell adhesion to laminin is unaffected by periodate oxidation of sugars on the surface of bacteria or by removal of divalent cations by ethylenediaminetetraacetic acid (EDTA). In contrast, bacterial adhesion is reduced when laminin is deglycosylated with N-glycosidase F or when bacteria are submitted to controlled trypsin digestion. 5. Laminin binding to the S. aureus 50-kDa band in immunoblotting assays has confirmed all of these results obtained in cell adhesion experiments

Identification and characterization of highly conserved antigenic determinants in the laminin molecule

1. Fragments P1 and E8, the results of two different enzymatic digestions of the laminin molecule, represent interaction sites of laminin with specific. By using negative and positive affinity purification of a rabbit antiserum against mouse laminin we have generated antibodies to these two fragments. 2. Antibodies against P1 were able to immunoprecipitate fragment E8 from elastase-digested laminin. By liquid phase competition experiments we demonstrated that the epitopes shared by P1 and E8 are a minor portion of the antigenic determinants of P1. When we checked for the presence of these shared epitopes in the human laminin molecule, they were the major fraction of the interspecies antigenic conservation. 3. A similar approach usisng polyclonal antibodies against human laminin has confirmed these reults. 4. The shared epitopes present in both mouse and human laminin molecules seem to be spatially determined, because antibodies against these sites did not bind to fully denatured laminin. 5. Since human and mouse laminin bind to cell receptors and to other extracellular matrix proteins from both species, we conclude that these antigenic determinants may represent the actual sites for at least some of these interactions

Hormonal regulation of ribosomal RNA synthesis in immature rat uterus

Fraçöes nucleares e nucleolares de útero de ratas jovens injetadas com 17-ß-estadiol näo radioativo foram isoladas e caracterizadas morfológica e bioquimicamente. A análise destas preparaçöes revelou que estas encontravam-se substancialmente puras, com a alfa-amanitina inibindo apenas apenas 20%% da atividade transcricional em nucléolos isolados. O emprego destas fraçöes nos experimentos utilizando a técnica de troca do 3H-estradiol pelo 17-ß-estradiol näo radioativo injetado préviamente nos animais, levou à determinaçäo de curvas de cinética, saturaçäo e de Scatchard para núcleos e nucléolos. Determinou-se alguns parâmetros físico-químicos que definem a ligaçäo do complexo receptor 17-ß-estradiol em núcleos e nucléolos. Os resultados obtidos mostram que tanto núcleos quanto nucléolos apresentam sítios aceptores para o complexo receptor-17-ß-estradiol, sendo esta ligaçäo saturável e específica devido à inibiçäo por um inibidor competitivo,m o diestilstilbestrol, com valores de Kd = 1,06 x 10**-9 M e 20,41 x10**-8 M; Ka = 0,94 x 10**10 M e 20,05 x 10 M; Bo = 0,49 x 10**9 M e 0,35 x 10**-9 M para nucléolos, respectivamente. Em nucléolos a curva de cinética sugere uma acumulaçäo tardia do complexo receptor-hormônio de características diferentes daquelas apresentadas em núcleos, onde existe uma acumulaçäo precoce deste complexo. A partir dos resultados apresentados neste estudo, conclue-se que em útero de ratas jovens pode haver uma açäo direta do hormônio sobre os genes ribossomais. Tal mecanismo caracteriza um sistema de regulaçäo extremamente preciso e complexo, com a presença de sítios aceptores específicos para a açäo do complexo receptor-hormônio sobre o genoma ribossomal. Tendo estabelecido este ponto, estudos adicionais säo necessários para determinar os mecanismos pelos quais os receptores estimulam as velocidades de transcriçäo e suas possíveis implicaçöes no controle gênico

Evolutionary conservation of laminin-binding proteins

The virulence of pathogens and metastatic capacity of cancer cells seems to correlate with the ability to adhere to cells and/or to basement components. A key feature of this mechanism in the expression of specific receptors for the basement membrane protein laminin. There different receptors have been already described in cells phylogenetically very distant, such as human white blood cells, Trichomonas vaginalis and Stapgylococcus aureus, all recognizing laminin with the same range of affinity. We have shown that laminin, which is also found in the circulation, enchances phagocytosis of S. aureus by macrophages in a species-specific fashion. Also, monoclonal antibodies (MAb) raised against the bacterial receptor inhibit the phagocytic enhancement mediated by laminin and recognize laminin-binding proteins in unicellular parasites and mammalian cells. The same Mab 1.H12 elutes a 52-kDa protein from bacterial extracts and a 67-kDa band from cancer cells extracts. Since the MAb is a monospecific reagent, results with 1.H12 strongly suggest and evolutionary conservation of the biding site of phylogenetically different laminin receptors

A direct method for cDNA cloning employing translation released mRNA

1. A new cloning procedure is described for cDNA synthesis from mRNA released by in vitro translation of polysomes in a cell-free amino acid incorporating system. The usefulness of the method lies in the feasibility of employing nanogram amounts of mRNA. 2. Complementary DNA is synthesized derectly in the translation mixture simply by adjusting the concentration of some components and removing ribosomes by boiling and centrifugation. 3. As an example, we report here the construction and characterization of cDNA clone corresponding to chick alfa (1) procollagen starting from a collagen-synthesizing polysome fraction obtained from chick embryos

As tecnicas envolvidas em engenharia genetica: suas perspectivas e dificuldades. / The involved technics in genetic engineering: its perspectives and difficulties

A essencia da Engenharia Genetica esta na juncao de fragmentos de DNA ou genes de diferentes organismos com o DNA de vetores plasmidiais ou virais, a fim de serem introduzidos e propagados em bacterias ou outras celulas hospedeiras. Com o objetivo de analisar a estrutura genica, os processos envolvidos em sua regulacao e expressao ou, ainda, para produzir proteinas especificas de interesse medico ou comercial, uma serie de tecnicas vem sendo descritas. Este artigo resume a maioria dos metodos empregados, conhecidos em conjunto como a tecnologia do DNA recombinante

Insulina humana: obtencao de uma bacteria contendo a informacao para a sua sintese. / Human insulin: construction of a bacteria containing the information for its synthesis

Empregando a tecnologia de DNA recombinante (clonagem molecular), um clone bacteriano contendo sequencias de DNA complementares a insulina humana foi construido. O RNA mensageiro utilizado para a sintese do DNA complementar foi isolado a partir de um insulinoma de pancreas humano. O plasmideo recombinante pl2, que abriga uma insercao de 400 pares de bases, contem toda a informacao para proinsulina, alem de se estender por 5 a 10 pares de bases na regiao do pre-peptideo. Essas investigacoes deverao beneficiar o estudo da regulacao genica de insulina, seja no individuo normal, seja no estado diabetico. Outrossim, poder-se-a desenvolver um sistema biologico alternativo para a producao deste hormonio em larga escala

Traducao "in vitro" e transcricao reversa do mRNA de procolageno. / Translation \"in vitro\" and reverse transcription of procollagen mRNA

Uma fracao de RNA mensageiro (mRNA) foi isolada a partir de RNA total extraido de polissomos de embrioes de galinha e purificada por dois ciclos de cromatografia em oligo (dt) - celulose. A analise deste material por eletroforese em condicoes denaturantes revelou a presenca de um pico homogeneo de moleculas, com um coeficiente de sedimentacao em torno de 28S. A adicao deste mRNA a um sistema de sintese proteica derivado de reticulocitos de coelho induziu a formacao de polipeptideos sensiveis a colagenase bacteriana purificada, com mobilidade eletroforetica igual a das cadeias do procolageno padrao. DNA complementar (cDNA) a mRNA de procolageno foi obtido pelo uso de transcriptase reserva. A analise deste cDNA em gel de agarose alcalino, revelou um tamanho medio correspondente a 565000 daltons. Atraves de experimentos de hibridizacao dos cDNAs de procolageno e globina com seus respectivos mRNAs, em que se assumiu a pureza da preparacao do mRNA de globina como 100%, pode-se estimar, por comparacao, que o mRNA de procolageno apresenta-se 78% puro