Quercetin repressed the stress response factor (sigB) and virulence genes (prfA, actA, inlA, and inlC), lower the adhesion, and biofilm development of L. monocytogenes.

The present study explored the effect of quercetin on the expression of virulence genes actA, inlA, inlC, and their regulatory components, sigB and prfA, in L. monocytogenes. Furthermore, the physicochemical changes on the surface, membrane permeability, and biofilm formation of quercetin-treated bacteria were evaluated. An inhibitory dose-dependent effect of quercetin (0.1-0.8 mM) was observed on the cell attachment on stainless steel at 2 and 6 h at 37 °C. Quercetin at 0.8 mM prevented the biofilm formation on stainless steel surfaces after 6 h of incubation at 37 °C, while the untreated bacteria formed biofilms with a cell density of 5.1 Log CFU/cm2. The microscopic analysis evidenced that quercetin at 0.2 mM decreased the biovolume and covered area of the attached micro-colonies. Also, sigB, prfA, inlA, inlC, and actA genes were downregulated by 7-29 times lower compared to untreated bacteria. In addition, quercetin decreased the superficial cell charge, increased the membrane permeability, and its surface hydrophobicity. These results demonstrated that quercetin prevented biofilm formation, repressed the genes of stress and virulence of L. monocytogenes and also altered the physicochemical cell properties.

Selection and molecular identification of fungal isolates that produce xylanolytic enzymes.

Xylan is a heteropolysaccharide and its complete hydrolysis involves a complex set of xylanolytic enzymes. Fungal xylanases have been widely used in the holocellulose industry to obtain by-products or for its elimination. The aim of this study was to select and identify filamentous fungi from different ecosystems that produce extracellular xylanases showing biotechnological potential. One hundred three fungal isolates were obtained from orchard, horticultural, and forest ecosystems. The ability of fungi to degrade xylan was measured by quantifying their xylanolytic indices after growth on solid culture media and their extracellular xylanolytic and cellulolytic activities after submerged fermentation. All fungal isolates grew on solid medium supplemented with xylan as the sole carbon source, but only 44% of isolates showed xylanolytic indices greater than 1.0. In submerged fermentation, 39% of the fungi tested showed no cellulolytic activity. Filamentous fungi were chosen from correspondence analysis and were identified by molecular tools using internal transcribed spacers. One of the 9 isolates selected belonged to the Phoma genus and the remaining were from the Fusarium genus. Fusarium solani (isolate 59) showed the highest xylanolytic index (0.964 ± 0.042), rapid growth on solid medium (1.233 ± 0.050 cm/day), significant xylanolytic activity (3.823 ± 0.210 U/mg), and a total deficiency of cellulolytic activity compared to other fungal isolates. In the zymogram, a clear zone was observed, indicating that F. solani possesses at least 1 xylanase. Fusarium solani was selected for its ability to produce extracellular xylanases with biotechnological potential.

Isolation and molecular characterization of a novel strain of Bacillus with antifungal activity from the sorghum rhizosphere.

We looked for bacterial strains with antifungal activity in the sorghum rhizosphere. A prescreening procedure to search for hemolytic activity among the isolated strains allowed us to detect good fungitoxic activity in a bacterial isolate that we named UM96. This bacterial isolate showed strong growth inhibition in bioassays against the pathogens Diaporthe phaseolorum, Colletotrichum acutatum, Rhizoctonia solani, and Fusarium oxysporum. The supernatant of isolate UM96 also showed strong hemolytic activity, which was not observed in the protease-treated supernatant. However, the supernatant that was treated with protease had similar antagonistic effects to those exhibited by the supernatant that was not treated with this enzyme. These results suggest that a bacteriocin-like compound is responsible for the hemolytic activity; whereas, as far as antifungal effect is concerned, an antibiotic of nonribosomal origin, such as a lipopeptide, might be acting. Further molecular characterization by partial 16S rDNA sequencing placed isolate UM96 in a cluster with Bacillus amyloliquefaciens; however, the highest identity match found in databases of Bacillus species was 91% identity. This suggests that Bacillus sp UM96 might be a novel species.

Prognostic significance of the combined expression of matrix metalloproteinase-9, urokinase type plasminogen activator and its receptor in breast cancer as measured by Northern blot analysis.

Using Northern blot analysis we have measured the co-expression of the matrix metalloprotease MMP-9, plasminogen activator urokinase type (uPA) and its receptor (uPAR) mRNAs in 81 biopsies of breast carcinomas with the objective of analyzing the impact of these factors on the overall survival probability of the patients (median follow-up time: 4 years). Individual mRNA levels of either uPA or uPAR showed parallel variations with MMP-9 mRNA, suggesting a coordinate transcription of these markers. When median values were used as cutoff points to discriminate between high and low factor expression, no association was found with patient outcome and MMP-9 or uPA mRNA distribution. However, increased uPAR mRNA levels were associated with poor prognosis (p = 0.01). The combination of MMP-9 and uPAR mRNA measurements has not enhanced prognostic information compared to information supplied by the receptor alone (p = 0.01). The combination of MMP-9 and high levels of uPA mRNA led to a significant association with poor outcome (p = 0.03). Multivariate analysis supported the notion that increased uPAR mRNA production in primary breast cancer may be a predictor of overall survival.

Expression of a novel factor, com1, in early tumor progression of breast cancer.

Tumor cells and their surrounding microenvironment produce a variety of growth factors and proteolytic enzymes to promote tumor growth and metastasis. We have recently identified a novel factor, termed com1, which is up-regulated in human breast carcinoma cells upon formation of experimental metastatic tumors and assumed to act as a growth-promoting factor in breast cancer. In attempts to explore the biological role of com1 in clinical tumor growth and metastasis, expression of com1 mRNA in primary carcinomas from 81 breast cancer patients and 27 samples of uninvolved adjacent breast tissue from these patients was compared and related to known prognostic parameters and outcome. The levels of com1 mRNA were significantly up-regulated (P < 0.0001) in the tumors compared to the normal breast tissues. Tumor expression of com1 mRNA, however, did not correlate with the mRNA levels for urokinase-type plasminogen activator (uPA), its receptor, or the type 1 inhibitor, which are factors that define a phenotype of tumor aggressiveness when elevated. And whereas the mRNA levels of uPA and the uPA receptor were elevated in tumors from the patients who subsequently had poor outcome, no correlations were observed between tumor com1 mRNA expression and prognosis or histological and biochemical characteristics of the tumors. We therefore assume that com1 may mediate some growth-promoting function early in development of the primary breast carcinoma, but not in later stages of tumorigenesis or metastasis.

Expression of gelatinases A and B, stromelysin-3 and matrilysin genes in breast carcinomas: clinico-pathological correlations.

The purpose of this study was to investigate the association among matrix metalloproteinases (gelatinases A and B, stromelysin-3 (ST3) and matrilysin) mRNAs expressed in primary breast carcinomas and standard prognostic parameters and clinical outcome. mRNA levels were determined by Northern analysis in samples of 81 breast cancer patients (median follow-up, 40 months) and 27 samples of uninvolved adjacent breast tissue. Proteases were expressed by the majority of the tumors and normal breast tissues examined. ST3, gelatinase A and matrilysin mRNAs were more often expressed at high levels in carcinomatous than in normal breast tissues. Differences in the distribution of gelatinase B mRNA were not found. However, paired normal tissues generally produced weaker signals when compared to matched tumor samples. Univariate analysis showed no significant association of gelatinase A and matrilysin mRNAs with the classical prognostic markers (age, menopausal status, stage, size, nodal status, vascular infiltrate, necrosis, steroid receptors, metastasis and survival). Overexpression of ST3 was more frequently found in tumors of post-menopausal women (P < 0.022). Elevated expression of gel B mRNA was associated with the presence of vascular infiltrate (P < 0.026), necrosis (P < 0.039), PR negative tumors (P < 0.014) and inversely correlated to the number of survivors (P < 0.021). Multivariate analysis including 68 patients for whom all information was available indicated that neither stromelysin correlated significantly with pathological, clinical or biochemical features. High levels of gelatinase A and B mRNAs were inversely associated with the number of survivors. Our findings suggest that measurements of gelatinase A and B mRNAs expression in breast carcinoma may help to identify patients with an aggressive form of the disease.

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.

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.

Monoclonal antibody-based immunoassay for carcinoembryonic antigen (CEA) measurement in human serum

Clinical oncology requires methods to detect tumor markers in patients sera and tissues Presently, monoclonal antibodies (MAbs based enzyme immunoassays are amongst the most advantageous techniques. Here we present a sensitive and specific double-antibody enzyme immunoassay for serum me surement of carcinoembryonic antigen (CEA). It has been developed with MAbs which recognize nonoverlapping peptide epitopes on the antigen molecule. The capture MAb 6C7 is GOLD 1 highly specific antibody while the tracer MAb 5.D11, labeled with biotin, is GOLD 4 that cross-reacts with nonspecific cross-reacting antigen (NCA) expressed on granulocytes. In addition the biotinylate MAb is shown to be also useful to detect CEA by immunohistochemistry.

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.

c-erbB-2 DNA amplification and mRNA expression in human primary breast tumors and its relationship to other prognostic factors.

In order to evaluate the involvement of cerbB-2 oncogene in the etiology and progression of breast cancer, DNA samples from 157 primary human mammary carcinomas were subjected to Southern and dot blot analyses for the presence of c-erbB-2 protooncogene alterations. None of 157 carcinomas analyzed showed c-erbB-2 rearrangement. Amplification of the c-erbB-2 was found in 28.6% (45/157) of the samples. Gene expression could be analyzed in only 97 of these tumors. High levels of c-erbB-2 transcripts were detected in 25.7% (25/97) of the tumor RNA preparations. Although there was a good correlation between c-erbB-2 overexpression and gene amplification, a significant proportion of the tumors showed overexpression in the absence of gene amplification or gene amplification without overexpression. A significant correlation between c-erbB-2 proto-oncogene alterations with the number of positive lymph nodes and tumor necrosis was found, suggesting that c-erbB-2 genetic alterations have a biological importance in the etiology of breast cancer.

Hippocampal mossy fiber-CA3 synapses after chronic alcohol consumption and withdrawal.

A quantitative study of the CA3 pyramidal cells and of the mossy fiber-CA3 synapses (MF-CA3) of the rat hippocampal formation was performed in rats alcohol-fed for 6, 12 and 18 months and respective age-matched controls. Additional groups alcohol-fed for 6 and 12 months and withdrawn for 6 months were also studied. The numerical densities of the CA3 pyramids and of the synapses were calculated applying the disector method to adjacent sections of the CA3 pyramidal cell layer and the stratum lucidum respectively. The results showed a progressive loss of pyramidal cells in alcohol-treated and withdrawal groups and a significant decrease of MF-CA3 synapses after 18 months of alcohol feeding. Taking into account that both hippocampal granule and CA3 pyramidal cells are reduced, the maintenance of the relative number of MF-CA3 synapses in 6- and 12-month alcohol-fed rats suggests the formation of new contacts. The increased proportion of the MF plasmalemma occupied by synapses can also be interpreted as an additional compensation process. These data show that MF-CA3 synapses display plastic and degenerative changes after chronic alcohol consumption and withdrawal which presumably will lead to functional modifications of the hippocampal circuitry.

Plasminogen activator expression and steroid hormone receptors in female breast cancer: a multifactorial study.

In order to evaluate the relationship of plasminogen activator (PA) activity to the expression of estrogen (ER) and progesterone (PR) receptors, we assayed primary tumor specimens from 121 cases of female breast cancer. Other clinical and histopathological variables were also investigated with respect to their possible association with PA activity in the samples. Statistical correlations were examined by stratified analysis techniques and by multiple regression methods. PA activity was higher in tumors which were ER+PR+ than in those exhibiting other subsets of joint receptor expression, a finding which was particularly predominant in tumors from post-menopausal women. Of the other variables examined, only disease stage, nodal status and vascular infiltration presented marginal positive statistical associations with PA activity, although this was not confirmed by multivariate analysis. The latter technique showed that the presence of PR was sufficient to explain the variation in PA levels. However, ER emerged as the sole significantly explanatory factor when ER+PR- patients were removed from analysis. Both PR and age (negatively) were independent contributory factors for PA activity in the analysis of post-menopausal women. None of the variables examined emerged as being significantly associated with PA when data from pre-menopausal patients were used. These findings indicate that PA activity in breast tumor samples is statistically associated with the expression of functional estradiol receptors, although to a lesser extent than PR.

Androgen, estrogen, and progesterone receptor levels in malignant and benign breast tumors: a multivariate analysis approach.

Androgens have been frequently used in the treatment of breast cancer. However, objective responses seem to vary according to the steroid hormone receptor expression of the tumor. We have studied the relationship between concentrations of androgen receptors (AR) and those of estrogen (ER) and progesterone (PR) receptors by multiple regression and stratified analysis techniques in 154 cases of primary breast carcinomas and 39 cases of benign tumors. Both the proportion of AR-positive tumors and the concentration of AR were dependent upon the coexpression of ER and PR by the specimens. This association was evident for malignant tumors with predominance of the positive correlation between AR and ER over that of AR and PR in post-menopausal patients. In premenopausal women, ER and PR concentrations were similarly correlated with AR levels. The PR, but not the ER concentration, was positively correlated to AR levels in benign breast tumors. These findings were confirmed by multiple regression, taking into consideration additional information about the patients to build statistical models allowing prediction of AR. Among the other variables considered in building these models--age, menopausal status, weight, height, and clinical stage--only height (using data from all patients) and age (data from post-menopausal women) emerged in addition to ER and PR as significantly explaining the variability of AR as the dependent variable.

Steroid hormone receptors in male breast diseases.

Estrogen (ER), progesterone (PR), glucocorticoid (GR) and androgen (AR) receptors were assayed in tumor samples from 8 cases of male breast cancer (MBC) and 20 cases of male gynecomastia. Seven out of eight (87.5%) male tumor samples had positive ER assays with values ranging from 12 to 180 fmol/mg protein. Of the seven ER positive cases of MBC, six, had positive PR activity with high titers. Positive GR and AR values were also detected in 75% of MBC cases. Concentrations of all four receptors were significantly correlated with each other. With gynecomastic tissue, the proportion of receptor-positive patients was 20% ER, 20% PR, 20% AR, and 45% GR. Except for GR, steroid receptor values for MBC individuals were significantly higher than those of gynecomastia patients.

Changes in the pulmonary glucocorticoid receptor content in the course of interstitial disease.

The level of cytosolic glucocorticoid receptors (GR) was measured in 12 open-chest lung biopsies of interstitial pulmonary diseases. The results showed an increase in the GR content in the diseased lungs correlated to the degree of septal cellularity in nine cases. Two pulmonary sarcoidosis and one end-stage idiopathic pulmonary fibrosis patients presented higher levels of GR than those predicted by the septal cellularity. It was concluded that the GR content of the lungs increases in the course of interstitial diseases, reflecting the number of cells that express cytosolic GR in pulmonary parenchyma.

Receptores de estrógeno e progesterona em cérvix uterina / Estrogen and progesterone receptors in uterine cervix

O objetivo deste trabalho foi a determinaçäo das concentraçöes dos receptores de estrógeno (ER) e progesterona (PR) em exocérvix humana. Ensaiamos ER e PR em vários "pools" de 25 fragmentos de exocérvix de mulheres normais obtidos por curetagem e em endométrios obtidos de pacientes submetidas à histerectomia por condiçöes näo malignas. Os "pools" de fragmentos de exocérvix foram separados em quatro tipos: 1) os referentes às mulheres na fase luteal; 2) na fase proliferativa do ciclo menstrual; 3) em uso de anticoncepcionais e 4) na menopausa. As dosagens dos receptores de estrógeno e progesterona foram feitas pelo método de carväo-dextrana. As concentraçöes de ER e PR encontradas na exocérvix foram respectivamente 15% e 3,6% das encontradas no endométrio, mas com constantes de afinidade semelhantes. Näo detectamos flutuaçöes na concentraçäo de ER e PR em exocérvix durante a fase proliferativa e luteal. Nas amostras de exocérvix de usuárias de anticoncepcionais, apenas o nível de PR foi estatisticamente menor quando comparado aos níveis obtidos na fase luteal e proliferativa. Os valores de ER e PR em exocérvix de mulheres pós-menopáusicas säo estatisticamente maiores do que os encontrados em mulheres pré-menopáusicas. Näo há diferenças em relaçäo ao tempo de menopausa. O efeito de estrógeno e progesterona na porçäo cervical é aparentemente limitado pela baixa concentraçäo dos seus respectivos receptores

[Estrogen and progesterone receptors in uterine cervix]. / Receptores de estrogeno e progesterona em cervix uterina.

PIP: This report describes the measurement of estrogen (ER) and progesterone receptors (PR) in cytosols of human exocervix and endometrium, using a charcoal dextran method. Endometrial tissues were obtained from patients undergoing hysterectomy. Curettage specimens of exocervix were obtained from healthy women throughout the menstrual cycle, from women taking combined oral contraceptives, and from postmenopausal women. The highest ER and PR concentrations and ER/PR ratio were detected in endometrium. Exocervix ER and PR levels were lower as compared to endometrium (14% and 3.6% respectively) and no cyclic variations were detectable. Specimens of exocervix from women taking oral contraceptives showed a significant PR decrease. In postmenopausal women, cervical ER and PR levels were significantly higher than in premenopausal women. No difference in binding specificity of estradiol and progesterone to their receptors could be found between endometrium and exocervix. The authors concluded that the effect of estrogen and progesterone on the cervix is limited by the low cytoplasmic receptor levels.^ieng

Steroid receptors in pulmonary lymphangiomyomatosis.

As an initial step in investigating the role of steroid hormones in lymphangiomyomatosis, the cytosolic receptors for steroid hormones were determined by a dextran charcoal method. Specific saturable receptors were found for estrogens (measured with [3H] estradiol +/- unlabeled diethylstilbestrol), progestins (measured with [3H] R5020 +/- unlabeled R5020), and glucocorticoids (measured with [3H] dexamethasone +/- unlabeled dexamethasone); they were absent for androgens (measured with [3H] R1881 +/- unlabeled R1881). Even though receptor levels were of low absolute value, they were significant because specimens of normal lung display no receptor at all. Steroid hormones may have direct effects on pulmonary lymphangiomyomatosis tissue mediated by specific receptors.

Steroid receptors in breast angiosarcoma.

The presence of receptors for estrogen, glucocorticoid and progesterone was determined in the cytosol of two breast angiosarcomas. Estrogen and glucocorticoid receptors were present in both of them. Progesterone receptors were present in one of the two tumors assayed. Occupied nuclear estrogen receptors have been found in the nuclear extracts of both tumors. Unoccupied nuclear receptors were found only in the progesterone-positive tumor. Density gradient analysis suggested that glucocorticoid and estrogen bindings were located predominantly in the 6S and 7 to 8S regions, whereas receptor for progesterone sedimented at 4S.