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Biomedical subjects

R R Brentani

Publications and source records attributed to R R Brentani.

At least 19 recordsLinked to original sources
PubMed1997

Post-translational modifications of alpha5beta1 integrin by glycosaminoglycan chains. The alpha5beta1 integrin is a facultative proteoglycan.

Cell-fibronectin interactions, mediated through several different receptors, have been implicated in a wide variety of cellular properties. Among the cell surface receptors for fibronectin, integrins are the best characterized, particularly the prototype alpha5beta1 integrin. Using [125I]iodine cell surface labeling or metabolic radiolabeling with sodium [35S]sulfate, we identified alpha5beta1 integrin as the only sulfated integrin among beta1 integrin heterodimers expressed by the human melanoma cell line Mel-85. This facultative sulfation was confirmed not only by immunoprecipitation reactions using specific monoclonal antibodies but also by fibronectin affinity chromatography, two-dimensional electrophoresis, and chemical reduction. The covalent nature of alpha5beta1 integrin sulfation was evidenced by its resistance to treatments with high ionic, chaotrophic, and denaturing agents such as 4 M NaCl, 4 M MgCl2, 8 M urea, and 6 M guanidine HCl. Based on deglycosylation procedures as chemical beta-elimination, proteinase K digestion, and susceptibility to glycosaminoglycan lyases (chondroitinase ABC and heparitinases I and II), it was demonstrated that the alpha5beta1 heterodimer and alpha5 and beta1 integrin subunits were proteoglycans. The importance of alpha5beta1 sulfation was strengthened by the finding that this molecule is also sulfated in MG-63 (human osteosarcoma) and HCT-8 (human colon adenocarcinoma) cells.

Cell Adhesion↗
PubMed1996

Polymorphonuclear leukocytes present laminin peptides in endocytic compartments.

Rat and Human neutrophils presented cytoplasmic vacuoles immunoreactive for laminin at the electron microscopy level. Colocalization of the anti-laminin labeling with albumin-gold complexes and alkaline phosphatase activity in rat PMN suggest an endocytic nature for this compartment. Immunoblot analysis of human normal peripheral blood neutrophils revealed the presence of two laminin peptides around 100 kDa.

Animals↗
PubMed1996

alpha6beta1-Integrin, a major cell surface carrier of beta1-6-branched oligosaccharides, mediates migration of EJ-ras-transformed fibroblasts on laminin-1 independently of its glycosylation state.

EJ-ras oncogene-induced malignant transformation is characterized by a series of changes in cell surface carbohydrates and cell-cell and cell-matrix interactions. Here, we show that EJ-ras-transformed NIH-3T3 fibroblasts acquired a migratory phenotype on laminin-1 surfaces. Such a phenotype was accompanied by overexpression of: (a) functional alpha6beta1, but not other laminin binding beta1-integrins; and (b) glycoconjugates on the cell surface bearing large oligosaccharides recognized by leukoagglutinin from Phaseolus vulgaris (L-PHA). The internal pool of pre-beta1-integrins was differently regulated in EJ-ras-transformed cells compared with nontransfected fibroblasts. Conversion of pre-beta1- into mature beta1-integrins was faster in EJ-ras-transformed cells, a process associated with the overexpression of the alpha6-chain. Overexpression of L-PHA-reactive oligosaccharides is dependent on the activity of N-acetylglucosaminyltransferase V, which is increased in transformed cells [J. W. Dennis et al., Science (Washington DC), 236: 582-585, 1987]. We show that beta1-integrins were the major carriers of L-PHA-reactive oligosaccharides on the cell surface. This glycosylation pattern, however, was not necessary for either the cell surface expression of beta1-integrins or their functional activity in the migratory response to laminin-1. Moreover, EJ-ras-transformed fibroblasts aggregated spontaneously. These effects were not observed in c-jun-transfected fibroblasts, which were unable to migrate on laminin, did not overexpress either beta1-integrins or L-PHA-reactive oligosaccharides, and did not self-aggregate.

3T3 Cells↗
PubMed1996

Collagen/collagenase interaction: does the enzyme mimic the conformation of its own substrate?

In this report, we present a hypothesis on the mechanism used by interstitial collagenases to cleave their natural substrate, interstitial collagens. The hypothesis is based on the assumption that the proline hinge domain of interstitial collagenase adopts a collagen-like conformation. With a collagen-like domain, the enzyme is able to disturb the quaternary organization of the triple helix in the collagenase-susceptible site. A modeling analysis suggests that interaction between prolines of both collagen and collagenase forming a kind of "proline zipper" is involved in the destabilization step. This destabilization makes the three-collagen helix susceptible to the catalytic cleft of the catalytic core.

Amino Acid Sequence↗
PubMed1996

Laminin-binding proteins in EJ-ras-transformed fibroblasts.

Malignant transformation is accompanied by changes in cell-matrix interactions. 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 addtion 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.

Animals↗
PubMed1996

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 and 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.

Collagen↗
PubMed1996

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.

Cell Adhesion↗
PubMed1995

Glycosylation of beta-1 integrins in B16-F10 mouse melanoma cells as determinant of differential binding and acquisition of biological activity.

Studying B16-F10 cells we could identify beta-1 integrins as laminin, fibronectin and collagen receptors. Gradient ionic strength elution analysis of affinity chromatography showed differential interactions between laminin-binding beta-1 integrins (two beta-1 polypeptides of 105 and 120 kDa) and fibronectin and collagen-binding beta-1 integrins (elution of one major beta-1 polypeptide of 120 kDa) and their respective ligands. To evaluate this diversity we submitted B16-F10 extracts to IEF and SDS-PAGE and found that one beta-1 integrin formed acidic and larger isoforms, while another formed basic and smaller isoforms. To study this difference we also submitted material eluted from WGA-Sepharose columns to IEF but now only the acidic beta-1 isoform was found. Extracts of B16-F10 treated with neuraminidase showed only the basic beta-1 isoform, suggesting that terminal sialic acid residues may be responsible for this acidic pattern, an interpretation supported by the fact that MAA (Maackia ammurensis agglutinin) reacts only with the acidic isoform. Differential glycosylation of beta-1 integrin isoforms in B16-F10 was also demonstrated since the smaller laminin-binding beta-1 integrin isoform reacted only with GNA (Galanthus nivalis agglutinin), whereas the mature larger form reacted with DSA (Datura stramonium agglutinin) and MAA; thus this heterogeneity of beta-1 chains is essentially due to variable glycosylation. Autoradiography and immunoblotting analysis of material separated by 2-dimensional electrophoresis show that only the processed forms of beta-1 integrins are expressed at the cell surface.

Animals↗
PubMed1995

TP53 mutations in upper aerodigestive squamous cell carcinomas from a group of Brazilian patients.

BACKGROUND: Loss of function of the tumor suppressor gene TP53 contributes to the development of several tumors. PATIENTS AND METHODS: We screened DNA samples from 47 patients with upper respiratory system squamous cell carcinomas for the presence of TP53 mutations. Exons 4 to 8 of the TP53 gene were amplified by the polymerase chain reaction, and mutations were identified by single-strand conformation polymorphism analysis. RESULTS: The TP53 mutations were demonstrated in 23 cases (49%). Mutations were distributed as follows: exon 4, 5 cases; exon 5, 4 cases; exon 6, 6 cases; exon 7, 4 cases; and exon 8, 4 cases. Demographic variables, tumor site, stage, family history of cancer, and tobacco smoking were not predictors of TP53 mutations. There was an increasing number of mutations in the more undifferentiated tumors (P = 0.0594). CONCLUSIONS: These findings suggest that TP53 mutations are associated with tumor differentiation, but not with the risk of lymph node metastasis in the group of patients analyzed.

Adult↗
PubMed1995

Absence of p53 autoantibodies in a significant proportion of breast cancer patients.

We analysed antibodies specific for human p53 in sera from primary breast cancer patients using three different immunoassays and we related these results to the p53 level in tumour tissue detected by immunohistochemistry. Only 44% (11/25) of apparently enzyme-linked immunosorbent assay (ELISA)-positive sera were from patients with a high level of p53 protein in more than 50% of their tumour cells. Surprisingly, 36% (9/25) of the sera originated from patients with no detectable p53 protein at all. Immunoprecipitation data suggested that the reason for this discrepancy is that at least some of the antibodies detected as positive in the ELISA in these sera from patients with clinical stage I and stage II breast cancers may be induced by immunogens other than p53 protein. Many of these reactions give apparently positive signals in a variety of p53 assays, and very stringent analysis is required to avoid possible misinterpretation of these responses as a p53-specific B-cell response in human cancer patients.

Autoantibodies↗
PubMed1994

Extracellular matrix components of the mouse thymus microenvironment. IV. Modulation of thymic nurse cells by extracellular matrix ligands and receptors.

Extracellular matrix (ECM) proteins can influence cell migration and differentiation in a variety of cell systems. Within the thymus, these molecules are heterogeneously distributed, and their physiological role is poorly understood. This prompted us to carry out in vitro studies using the thymic nurse cell (TNC) model. We observed that fibronectin and laminin accelerate spontaneous in vitro release of thymocytes from TNC, whereas anti-ECM antibodies exhibited a blocking effect. Similar results were obtained with anti-ECM receptor reagents. Moreover, these antibodies abrogated in vitro reconstitution of TNC complexes and thymocyte adhesion to TNC-derived epithelial cultures. Our results indicate that lymphocyte traffic in TNC (comprising both entrance into and exit from the epithelial structure) is affected by interactions involving extracellular matrix ligands and receptors. In this respect, the dynamic analysis of thymic nurse cell complexes should be regarded as a relevant in vitro tool for functional studies of distinct adhesion molecules in intrathymic lymphocyte traffic.

Animals↗
PubMed1994

Detailed deletion mapping of chromosome segment 17q12-21 in sporadic breast tumours.

Linkage studies have indicated that a gene on chromosome arm 17q, designated BRCA1, confers susceptibility to familial breast and ovarian cancer. To investigate the possible involvement of the BRCA1 gene in sporadic breast cancer we have analysed loss of heterozygosity (LOH) in a panel of 100 sporadic primary breast tumours using 10 PCR-based polymorphic markers from 17q12-21. Allele losses were detected in 40 of 100 tumours informative for at least one of the markers analysed. Of these 40 deleted tumours, 27 showed partial or interstitial loss on 17q. The pattern of LOH in the tumours with partial or interstitial LOH revealed three putative distinct deleted regions on 17q12-21. The first lies on the proximal long arm between D17S250 and THRA1; the second one lies between D17S776 and D17S579, the region containing the BRCA1 gene; and the third is telomeric to D17S733. The most frequently deleted region overlaps with the minimal region containing the BRCA1 gene, suggesting that this gene might also be associated with the development or progression of a proportion of sporadic breast tumours.

BRCA1 Protein↗
PubMed1994

A monoclonal autoantibody against a complementary peptide recognizes interstitial collagenase.

A monoclonal autoantibody (mab 16) against the complementary peptide TKKTLRT, which was deduced from the collagenase-sensitive site in collagen, is described. Mab 16 recognized interstitial collagenase as visualized by ELISA and immunoprecipitation assays. Moreover, mab 16 was able to inhibit partially the collagenolytic activity of keratinocyte supernantant. Finally, it was possible to immunopurify collagenase using a mab 16/Sepharose column.

Amino Acid Sequence↗
PubMed1994

Regulation of vitronectin receptor expression by retinoic acid on human melanoma cells.

The integrin family of adhesion receptors is likely to be important for tumor cell invasion and dissemination. We have studied the effects of the differentiating agents retinoic acid on integrin expression by the human melanoma cell line MeWo. Our results show that this agent inhibits cellular proliferation, increases melanin content and induces morphological changes in MeWo cells. Functionally, these alterations are associated with an enhanced adhesion to matrix protein vitronectin and higher levels of expression of vitronectin receptor on the cell surface. This is accompanied by increased levels of alpha v integrin mRNA. Although the mechanism by which retinoic acid regulates the expression of vitronectin receptor in MeWo cells needs further examination, this system may represent a good model for understanding the role of this receptor in melanoma progression, as well the molecular basis for retinoic acid therapy in these tumors.

Blotting, Northern↗
PubMed1994

Allelic loss on distal chromosome 17p is associated with poor prognosis in a group of Brazilian breast cancer patients.

We examined loss of heterozygosity (LOH) for two loci on chromosome 17p (D17S5 and TP53), and erbB-2 gene amplification, in primary breast cancers from 67 Brazilian patients. We identified two distinct regions of LOH on chromosome 17p, one spanning TP53 and the other a more telomeric region (D17S5). Based on a short-term follow-up, Kaplan-Meier analyses of patients' disease-free survival showed that patients with LOH for D17S5, but retaining heterozygosity for TP53, were at higher risk of recurrence (P = 0.007) than those who retained heterozygosity for D17S5. Bivariate analyses indicated that patients with LOH for D17S5 alone had an increased risk of recurrence (hazard ratio = 7.2) over patients with erbB-2 amplification (hazard ratio = 3.7), when compared with patients with neither alteration (hazard ratio = 1.0). Further, lymph node-positive patients whose tumours had both LOH for D17S5 and erbB-2 gene amplification had a higher risk of recurrence than patients whose tumours had neither of these genetic alterations. Our data confirm previous reports of a putative tumour-suppressor gene, distinct from TP53, on distal chromosome 17p which is associated with breast cancer. They further suggest that LOH for loci in this region may provide an independent indicator to identify patients with poor prognosis.

Adult↗
PubMed1994

Adhesion to laminin is down-regulated upon retinoic acid-induced F9 cell differentiation: a role for alpha 6/beta 1 integrin.

F9 mouse teratocarcinoma cells have a high capacity to adhere to laminin and we identified alpha 6/beta 1 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 alpha 6/beta 1 integrin on the cell surface.

Animals↗