Expression of Glycolysis-Related Proteins in Solid Papillary Carcinoma of the Breast According to Basement Membrane Status

PURPOSE: The aim of this study was to investigate the differences of expression in glycolysis-related proteins such as Glut-1, carbonic anhydrase (CA) IX, and monocarboxylate transporter (MCT) 4 according to the myoepithelial cell (MEC) and basement membrane (BM) status in solid papillary carcinoma (SPC) of the breast. MATERIALS AND METHODS: Immunohistochemical evaluation of Glut-1, CAIX, and MCT4, as well as p63 and type IV collagen, were performed on 23 SPC cases. RESULTS: Six and nine cases of SPC showed the presence and absence of myoepithelial cells, respectively, and eight cases belonged to the borderline status (p63-positive MEC on some areas of the outer tumor surface but not in others). BM was partially or completely absent in 14 cases and present in nine cases. SPC lacking BM more frequently showed high expression of CAIX than SPC with BM (p=0.037). CONCLUSION: In SPC of the breast, a strong expression of CAIX seems to be associated with an increasing degree of loss of BM, which can be interpreted as BM degradation due to the induction of extracellular acidity with increasing expression of CAIX.

Análisis de la distribución de los componentes de membrana basal laminina, fibronectina y colágeno IV en vasos sanguíneos de patología mamaria benigna y maligna / Analysis of the distribution of components in basal membrane laminin, fibronectin and type IV collagen in blood vessels of benign and malignant mammary pathology

Dentro de la compleja y aún poco comprendida biología de la formación de neovasos en tumores malignos se ha establecido que uno de los puntos restrictivos del proceso sería la precoz destrucción y remodelación que sufre la membrana basal perivascular, la cual estaría mediada por la activación de metalloproteinasas de matriz extracelular. Las diferencias en la cantidad y potencia de los distintos mediadores podrían traer como resultado un cambio en los patrones normales de distribución, cantidad y calidad de los componentes de membranas basales perivasculares en tejidos afectados por neoplasias respecto de lo que existe en la mama normal o en patologías benignas. Se estudiaron 118 piezas histológicas de patología mamaria entre 1990-2000. Se realizaron inmunomarcaciones con anticuerpos primarios antilaminina, fibronectina y colágeno de tipo IV para estudiar membrana basal perivascular. La cantidad de componentes de membrana basal de los vasos sanguíneos aumentan cuando una patología mamaria pasa de ser localizada a invasora y también a medida que se va perdiendo el grado de diferenciación histológica de las mismas. Las características moleculares de las membranas basales vasculares son diferentes en el tejido correspondiente a carcinomas mamarios invasores respecto de lo observado en tejido normal o en patologías sin disrupción de la membrana basal.

Life-long food restriction prevents renal membrane lipid deposition and lowers renal work in rats.

Renal cortical brush-border (BBM), basolateral membrane (BLM), and medullary plasma membrane (mPM) preparations were analyzed to assess the effects of life-long food restriction in aged rats on membrane lipid content. Young male Fischer 344 x Brown-Norway F1 rats consumed food ad libitum (young AL) or were food-restricted (FR, 60% of AL consumption) for either 6 weeks (young FR) or until the age of 30 months old (old FR). Senescent FR rats had 50 per cent decreases in fractional excretion of Na and K (p < 0.001) as compared with the young AL rats. Long-term FR reduced phosphate and titratable acid excretion by 80 per cent (p < 0.001). These values were not significantly different from those observed in young rats during 6 weeks of FR. Food restriction decreased renal Na, K-ATPase activity by 50 per cent (p < 0.001) in both old and young FR animals. Reduction of food intake, in old and young rats, decreased all BBM phospholipid concentrations (phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin) by 50 per cent than in the AL rats (p < 0.001). In BLM, chronic FR resulted only in lower phosphatidylcholine concentration (by 21%, p < 0.05) while phosphatidylethanolamine was increased approximately 80 per cent (p < 0.001). Total phospholipid content in mPM was progressively decreased by 23 per cent (p < 0.05) in the young FR group to be 55 per cent (p < 0.001) in the old FR rats. Cholesterol content was reduced in BBM and mPM by 38 per cent and 25 per cent (p < 0.05), respectively, during long-term FR. Both total phospholipid and cholesterol contents detected in mPM of the old FR rats were significantly lower than those obtained from the young FR animals (by 42%, p < 0.001 and 12%, p < 0.05, respectively). Plasma glucose, blood urea nitrogen, and body weight maintained at significantly lower levels during chronic FR. That life-long FR could prevent renal membrane lipid deposition and could lower renal work may explain the mechanisms that FR can delay the onset and diminish the severity of age-associated renal diseases.

Mecanismos de absorçäo de aminoácidos e oligopeptídios: controle e implicaçöes na dietoterapia humana / Mechanisms of amino acids and oligopeptides absorption in humans

Os mecanismos envolvidos na absorção de aminoácidos e di-tripeptídeos são revistos direcionadamente às implicações na nutrição humana. As membranas borda em escova e basolateral são atravessadas por aminoácidos e ditripeptídeos mediante mecanismos passivos (difusão simples ou facilitada) ou ativos (c0-transportadores de Na+ ou H+). O sistema ativo dependente de Na+ ocorre principalmente na borda em escova e a difusão simples na basolateral. Ambas as membranas apresentam o transporte passivo facilitado. Os aminoácidos livres usam tanto o sistema passivo, como o ativo, enquanto os di-tripeptídeos utilizam mais o sistema ativo (H+). A borda em escova possui sistemas distintos para aminoácidos livres e di-tripeptídios, com os primeiros utilizando preferentemente o Na+ dependente e os demais o H+ - dependente. Os aminoácidos livres são transportados em diferentes velocidades por carreadores, em mecanismos saturáveis e competitivos, com especificidade para aminoácidos básicos, ácidos e neutros. Os di e tripeptídeos têm pelo menos dois carreadores, ambos H+ dependentes. Na membrana basolateral os aminoácidos livres são transportados por difusão simples enquanto os di-tripeptídios o são ativamente por processo de troca aniônica. A principal regulação do transporte transmembrana de aminoácidos e di-tripepídios é a concentração desses substratos ao nível da membrana. Afora isso, di e tripeptídios são mais eficientemente absorvidos que os aminoácidos livres os quais por sua vez o são melhor que os tetra ou mais peptídios. Desta forma, o di-tripeptídios resultam em maior retenção nitrogenada que os demais e são particularmente úteis nos casos de redução da capacidade absortiva intestinal. Os peptídios não absorvidos são fermentados pelas bactérias colônicas resultando ácidos graxos de cadeia curta, ácidos dicarboxílicos, compostos fenólicos e amônia. Os ácidos graxos e a amônia são fixados pelas bactérias para produção de energia e aminoácidos, respectivamente.

Detection of the basement membrane-degrading proteolytic activity of Paracoccidioides brasiliensis after SDS-PAGE using agarose overlays containing Abz-MKALTLQ-EDDnp

We have characterized, in the Paracoccidioides brasiliensis yeast phase, an exocellular SH-dependent serine proteinase activity against Abz-MKRLTL-EDDnp and analogous fluorescent-quenched peptides, and showed that it is also active against constituents of the basement membrane in vitro. In the present study, we separated the components of P. brasiliensis culture filtrates by electrophoresis and demonstrated that the serine-thiol exocellular proteinase has a diffuse and heterogeneous migration by SDS-PAGE, localizing in a region between 69 and 43 kDa. The hydrolytic activity was demonstrable after SDS-PAGE using buffered agarose overlays of Abz-MKALTLQ-EDDnp, following incubation at 37oC, and detection of fluorescent bands with a UV transilluminator. Hydrolysis was more intense when incubation was carried out at basic pH, and was completely inhibited with 2.5 mM PMSF and partially with sodium 7-hydroxymercuribenzoate (2.5 mM p-HMB), suggesting its serine-thiol nature. A proteolytic band with similar characteristics was observed in conventional gelatin zymograms, but could not be correlated with a silver-stained component. Detection of the serine-thiol proteinase in substrate gels after SDS-PAGE provides a useful way of monitoring purification of the basement membrane degrading enzyme

Changes of glomerular basement membrane components in Vacor-induced diabetic nephropathy

OBJECTIVES: The thickening of the glomerular basement membrane in rats after Vacor ingestion was examined by electron microscopy. This study was performed to elucidate which biochemical components changed in the glomerular basement membrane after Vacor-induced diabetic glomerulopathy. METHODS: Immunohistochemical analyses of type IV collagen, laminin, fibronectin and chondroitin sulfate proteoglycan were performed. A single dose of Vacor (molecular weight 272), 80 mg/kg, was administered to adult male Wistar rats by orogastric canule, and the animals were sacrificed at 0.5, 1, 3, 7, 14, 28 and 56 days after administration. RESULTS: Mild thickening of the glomerular basement membrane was evident 7 days after Vacor administration, and the width of the glomerular basement membrane was more than twice that of normal controls at 28 and 56 days. Significantly increased expressions of type IV collagen, laminin, fibronectin and neutral polysaccharide in the thickened glomerular basement membrane were noted 14 to 56 days after administration, and a mildly increased expression of chondroitin sulfate proteoglycan appeared between 3 to 7 days. CONCLUSION: These abnormally increased glomerular basement membrane components might be part of what causes diabetic nephropathy after Vacor administration.

Expressäo de colágeno IV e laminina urinários no diabetes mellitus induzidopor drogas (aloxana e estreptozotocina) e avaliaçäo morfo-funcional renal

Resumo: A excreçäo urinária de componentes da mebrana basal, específicamente colágeno IV e laminina, foi determinada através de "immunoblotting", e estudada em ratos Wistar machos, diabéticos-induzido por drogas (aloxana e estreptozotacina)EV, e comparada a controles, em funçäo do tempo de doença. Estudamos, também, as funçöes renais: gloerular e tubular, avalinado, respectivamente, clearance de creatina e de lítio. Além disso, aspectos morfológicos renais foram analizados utilizando-se técnicas de PAS e de imuno-histoquímica, para colágeno IV e laminina, com o objetivo de verificar possíveis alteraçöes da membrana basal. Assim pudemos obeservar excreçöes de fraçöes urinárias de colágeno IV (88 e 75 kDa) e laminina (108 a 88 kDa), em ambos os modelos de DM, em todos os tempos estudados . Entretanto, a fraçäo de 57 kDa da laminina, nos animais diabéticos-induzido por estreptozotocina, somente foi expressa a partir da quinta semana da doença, enquanto nos diabéticos-induzidopor aloxana sua excreçäo foi verificada precocedente (segundo dia). As alteraçöes da funçäo renal foram semelhantes nos dois modelos, porém de maneira mais intensa no modelo aloxânico. Dentre elas destacaram-se: diminuiçäo progressiva da taxa de filtraçäo glomerular, aumento da fraçäo de escreçäo de sódio;e, dimuiçäo da reabsorçäo deste íon, näo compensada por aumento da reabsorçäo distal.

Increased glycosylation of human lens epithelial basement membrane in diabetes mellitus

We studied the nonenzymatic glycosylation of lens epithelial basement membranes (LEBM) of senile cataractous lenses of both diabetic and nondiabetic patients. The human LEBMs were isolated from surgically removed senile cataracts and purified by osmotic lysis and detergent treatments. Glycosylation assay of LEBMs was done using the colorimetric method of Fluckiger and Winterhalter. The glycosylation value ranged from 16.39 to 92.56 n mol/mg protein overall, with a mean of 63.54 +/- 24.56 n mol/mg protein for the diabetic specimens and a mean of 29.97 +/- 14.48 n mol/mg protein for the nondiabetic controls (P = 0.009). The study confirms our previous observation of in vivo glycosylation of the LEBM and further establishes that diabetic patients have a twofold increase in the amount of LEBM glycosylation when compared to their nondiabetic counterparts.

Increased glycosylation of human lens epithelial basement membrane in diabetes mellitus

We studied the nonenzymatic glycosylation of lens epithelial basement membranes (LEBM) of senile cataractous lenses of both diabetic and nondiabetic patients. The human LEBMs were isolated from surgically removed senile cataracts and purified by osmotic lysis and detergent treatments. Glycosylation assay of LEBMs was done using the colorimetric method of Fluckiger and Winterhalter. The glycosylation value ranged from 16.39 to 92.56 n mol/mg protein overall, with a mean of 63.54 +/- 24.56 n mol/mg protein for the diabetic specimens and a mean of 29.97 +/- 14.48 n mol/mg protein for the nondiabetic controls (P = 0.009). The study confirms our previous observation of in vivo glycosylation of the LEBM and further establishes that diabetic patients have a twofold increase in the amount of LEBM glycosylation when compared to their nondiabetic counterparts.

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

Two-site immunoassays for the measurement of serum laminin: correlation with breast cancer staging and presence of auto-antibodies

Binding to and destruction of basement membrane (BM) are necessary steps for cancer cells to extravasate and metastasize. Serum levels of released BM components may correlate with the staging of human cancers or with inflammatory disorders. Furthermore, released material may also induce autoantibodies. Since Iaminin, an 800-kDa glycoprotein, is present in the extracellular matrix, serum laminin levels may be markers of BM injury. A two-site enzyme immunoassay and a radioimmunoassay were developed to test sera from patients with breast cancer or systemic lupus erythematosus (SLE). A significant difference in laminin concentrations was demonstrated between early (T0-T2) and advanced (T3-T4) tumors (P = 0.001). However, specimens from SLE patients did not differ in laminin concentration from normal individuals and no correlation was observed between laminin levels and anti-laminin auto-antibody titers. These results suggest that serum laminin levels are useful markers of BM damage and could be of prognostic value in cancer