Nitric oxide regulates adhesiveness, invasiveness, and migration of anoikis-resistant endothelial cells.

Anoikis is a type of apoptosis that occurs in response to the loss of adhesion to the extracellular matrix (ECM). Anoikis resistance is a critical mechanism in cancer and contributes to tumor metastasis. Nitric oxide (NO) is frequently upregulated in the tumor area and is considered an important player in cancer metastasis. The aim of this study was to evaluate the effect of NO on adhesiveness, invasiveness, and migration of anoikis-resistant endothelial cells. Here, we report that anoikis-resistant endothelial cells overexpress endothelial nitric oxide synthase. The inhibition of NO release in anoikis-resistant endothelial cells was able to decrease adhesiveness to fibronectin, laminin, and collagen IV. This was accompanied by an increase in cell invasiveness and migration. Furthermore, anoikis-resistant cell lines displayed a decrease in fibronectin and collagen IV protein expression after L-NAME treatment. These alterations in adhesiveness and invasiveness were the consequence of MMP-2 up-regulation observed after NO release inhibition. The decrease in NO levels was able to down-regulate the activating transcription factor 3 (ATF3) protein expression. ATF3 represses MMP-2 gene expression by antagonizing p53-dependent trans-activation of the MMP-2 promoter. We speculate that the increased release of NO by anoikis-resistant endothelial cells acted as a response to restrict the MMP-2 action, interfering in MMP-2 gene expression via ATF3 regulation. The up-regulation of nitric oxide by anoikis-resistant endothelial cells is an important response to restrict tumorigenic behavior. Without this mechanism, invasiveness and migration potential would be even higher, as shown after L-NAME treatment.

Nitric oxide regulates adhesiveness, invasiveness, and migration of anoikis-resistant endothelial cells

Anoikis is a type of apoptosis that occurs in response to the loss of adhesion to the extracellular matrix (ECM). Anoikis resistance is a critical mechanism in cancer and contributes to tumor metastasis. Nitric oxide (NO) is frequently upregulated in the tumor area and is considered an important player in cancer metastasis. The aim of this study was to evaluate the effect of NO on adhesiveness, invasiveness, and migration of anoikis-resistant endothelial cells. Here, we report that anoikis-resistant endothelial cells overexpress endothelial nitric oxide synthase. The inhibition of NO release in anoikis-resistant endothelial cells was able to decrease adhesiveness to fibronectin, laminin, and collagen IV. This was accompanied by an increase in cell invasiveness and migration. Furthermore, anoikis-resistant cell lines displayed a decrease in fibronectin and collagen IV protein expression after L-NAME treatment. These alterations in adhesiveness and invasiveness were the consequence of MMP-2 up-regulation observed after NO release inhibition. The decrease in NO levels was able to down-regulate the activating transcription factor 3 (ATF3) protein expression. ATF3 represses MMP-2 gene expression by antagonizing p53-dependent trans-activation of the MMP-2 promoter. We speculate that the increased release of NO by anoikis-resistant endothelial cells acted as a response to restrict the MMP-2 action, interfering in MMP-2 gene expression via ATF3 regulation. The up-regulation of nitric oxide by anoikis-resistant endothelial cells is an important response to restrict tumorigenic behavior. Without this mechanism, invasiveness and migration potential would be even higher, as shown after L-NAME treatment.

Caracterização nutricional das folhas de Moringa oleifera (MOL) para frangos de corte / Chemical and nutritional characterization of moringa oleifera leaves for broilers

Objetivou-se determinar os valores energéticos e nutricionais das folhas de Moringa oleifera (MOL) para frangos de corte. Utilizaram-se 90 pintos machos, Cobb-500, com 14 dias de idade, distribuídos em delineamento inteiramente ao acaso, com cinco tratamentos e seis repetições de três aves. Os tratamentos consistiram de: uma dieta referência e quatro dietas com substituição de 10%, 20%, 30% e 40% da dieta referência pelas folhas de MOL. O período experimental teve duração de oito dias, utilizando-se a metodologia de coleta total de excretas. Foram determinados os valores de energia metabolizável aparente (EMA), aparente corrigida para o nitrogênio (EMAn), coeficiente de metabolizabilidade aparente da matéria seca (CMAMS), da proteína bruta (CMAPB) e da energia bruta (CMAEB). Os resultados obtidos foram submetidos à análise de variância e à análise de regressão a 5% de probabilidade. Houve efeito quadrático das variáveis à medida que a moringa era adicionada à ração referência. Na derivação das equações de regressão, o nível que proporcionou os melhores valores de EMA, EMAn e CMEB foi de 37,7% de substituição. O farelo de folhas MOL apresentou médias de 3140kcal/kg de EMA, 2845kcal/kg de EMAn, 76,92% de CMAEB, 76,63% de CMAMS e 73,42% de CMAPB.(AU)

This study aimed to determine the energy and nutritional value of the leaves of Moringa oleifera (MOL) for broilers. We used 90 male chicks, Cobb-500, with 14 days of age in a completely randomized design with five treatments and six repetitions of three birds. The treatments were: reference diet and 4 diets with substitution of 10%, 20%, 30%, and 40% of the diet by reference sheet MOL. The trial lasted eight days, using the method of total excreta collection. The apparent metabolizable energy (AME), apparent corrected for nitrogen (AMEn), apparent metabolizable coefficient of dry matter (AMCDM), crude protein (AMCCP) and gross energy (AMCGE). The results were submitted to analysis of variance and regression analysis at 5% probability. There was a quadratic effect of the variables as the moringa was added to the reference diet. In the derivation of the regression equations the level that provided the best values of AME, AMEn, AMCGE was 37.7% substitution. The leaves meal MOL presented average 3140kcal / kg of AME, 2845kcal / kg AMEn, 76.92% of AMCGE, 76.63% of AMCDM and 73.42% of AMCCP.(AU)

Coupling of vinculin to F-actin demands Syndecan-4 proteoglycan.

Syndecans are heparan sulfate proteoglycans characterized as transmembrane receptors that act cooperatively with the cell surface and extracellular matrix proteins. Syn4 knockdown was performed in order to address its role in endothelial cells (EC) behavior. Normal EC and shRNA-Syn4-EC cells were studied comparatively using complementary confocal, super-resolution and non-linear microscopic techniques. Confocal and super-resolution microscopy revealed that Syn4 knockdown alters the level and arrangement of essential proteins for focal adhesion, evidenced by the decoupling of vinculin from F-actin filaments. Furthermore, Syn4 knockdown alters the actin network leading to filopodial protrusions connected by VE-cadherin-rich junction. shRNA-Syn4-EC showed reduced adhesion and increased migration. Also, Syn4 silencing alters cell cycle as well as cell proliferation. Moreover, the ability of EC to form tube-like structures in matrigel is reduced when Syn4 is silenced. Together, the results suggest a mechanism in which Syndecan-4 acts as a central mediator that bridges fibronectin, integrin and intracellular components (actin and vinculin) and once silenced, the cytoskeleton protein network is disrupted. Ultimately, the results highlight Syn4 relevance for balanced cell behavior.

Prediction of the metabolizable energy requirements of free-range laying hens.

This experiment was conducted with the aim of estimating the ME requirements of free-range laying hens for maintenance, weight gain, and egg production. These experiments were performed to develop an energy requirement prediction equation by using the comparative slaughter technique and the total excreta collection method. Regression equations were used to relate the energy intake, the energy retained in the body and eggs, and the heat production of the hens. These relationships were used to determine the daily ME requirement for maintenance, the efficiency energy utilization above the requirements for maintenance, and the NE requirement for maintenance. The requirement for weight gain was estimated from the energy content of the carcass, and the diet's efficiency energy utilization was determined from the weight gain, which was measured during weekly slaughter. The requirement for egg production was estimated by considering the energy content of the eggs and the efficiency of energy deposition in the eggs. The requirement and efficiency energy utilization for maintenance were 121.8 kcal ME/(kg∙d)and 0.68, respectively. Similarly, the NE requirement for maintenance was 82.4 kcal ME/(kg∙d), and the efficiency energy utilization above maintenance was 0.61. Because the carcass body weight and energy did not increase during the trial, the weight gain could not be estimated. The requirements for egg production requirement and efficiency energy utilization for egg production were 2.48 kcal/g and 0.61, respectively. The following energy prediction equation for free-range laying hens (without weight gain) was developed: ME /(hen ∙ d) = 121.8 × W + 2.48 × EM, in which W = body weight (kg) and EM = egg mass (g/[hen ∙ d]).

Energia metabolizável da levedura de cana-de-açúcar submetida a diferentes temperaturas e tempos de secagem para frangos de corte e poedeiras / Metabolizable energy of sugar cane yeast submitted to different temperatures and drying times for broilers and layers hens

Objetivou-se determinar a temperatura e o tempo de secagem por rolos rotativos, aos quais a, levedura de cana-de-açúcar é submetida que permitam seu melhor aproveitamento energético por galinhas poedeiras e frangos de corte. Para isso foram realizados três ensaios de metabolismo para determinar os valores de energia metabolizável aparente (EMA), aparente corrigida para nitrogênio (EMAn) e os coeficientes de metabolizabilidade aparente da matéria seca (CMMS) e da energia bruta (CMEB). O primeiro ensaio foi conduzido com galinhas poedeiras (E1), o segundo com frangos de corte (E2) em crescimento e o terceiro com frangos de corte em diferentes idades (E3)...

This study aimed to determine the temperature and drying time through rotative rolls, that sugar cane yeast is subjected to in order to allow best energy utilization by laying hens and broilers. Three metabolism trials were conducted to determine the values of apparent metabolizable energy (AME) and apparent corrected for nitrogen balance (AMEn), coefficient of apparent metabolizable dry matter (CAMDM) and gross energy (CAMGE). The first experiment was conducted with laying hens (E1), the second with broilers (E2) in growth and the third with broilers at different ages (E3)...

Immunohistochemical expression of vascular endothelial growth factor in canine mammary tumours.

The histopathological and clinical aspects of canine mammary tumours (CMTs) have been widely studied, but the variation in the biological behaviour of these neoplasms hampers the identification of prognostic factors. Sustained angiogenesis has been suggested to be one of the most important factors underlying tumour growth and invasion. This process involves the action of several growth factors including vascular endothelial growth factor (VEGF). The present study characterizes the relationship between immunohistochemical expression of VEGF and gross (e.g. size and tissue fixation) and microscopical (e.g. type, growth, necrosis, lymphoid infiltration, lymph node metastasis, histological grade and proliferation index) features of CMTs. Forty-eight benign and 64 malignant CMTs were evaluated. Statistical analysis failed to show a significant relationship between VEGF expression and the pathological features, suggesting that VEGF expression occurs in both benign and malignant tumours and is independent of histological type, proliferation, tissue invasion or local metastatic capacity.

Immunohistochemical expression of caveolin-1 in normal and neoplastic canine mammary tissue.

Caveolins are the major structural proteins of caveolae and play a role in tumour surveillance. The expression of caveolin-1 (Cav-1) was investigated immunohistochemically in samples of normal canine mammary tissue (n=5), benign (n=23) and malignant (n=45) mammary tumours, tumour emboli (n=10) and metastatic lesions (n=10). Cav-1 was not expressed by normal luminal epithelium, but was consistently expressed by normal myoepithelial cells. There was no epithelial expression of Cav-1 by the benign mammary lesions, but the molecule was detected in 35.6% of the malignant lesions, 80% of the tumour emboli and in 40% of the metastatic lesions. These findings link increased expression of Cav-1 to neoplastic transformation and suggest that Cav-1 expression is associated with more malignant canine mammary tumours and their vascular invasion and regional lymph node metastasis.

Caveolin-1 in diagnosis and prognosis of canine mammary tumours: comparison of evaluation systems.

Caveolin-1 (Cav-1) is a major structural protein of caveolae, plasma membrane invaginations related to several cellular processes including regulation of signal transduction. In recent years there has been some controversy regarding the distribution of Cav-1 in normal and neoplastic mammary cell types, which may be attributed to different scoring systems adopted in different studies. The present study compares Cav-1 immunoexpression in normal (n=17) and neoplastic (n=79) canine mammary tissues assessed by two different scoring methods (previously reported by others with conflicting results) and associates Cav-1 expression with metastasis and overall survival (OS). Results obtained with both scoring methods were similar, revealing absence of immunoreactivity in normal luminal epithelium and in benign neoplasms and clearly associating Cav-1 expression with malignant transformation. The data suggest that Cav-1 expression is associated with highly malignant subtypes of mammary tumours (i.e. basal-like carcinoma), invasion and metastasis, thus supporting the hypothesis that it may play a major role in the epithelial-mesenchymal transition process. Furthermore, one of the scoring systems employed associated Cav-1 expression with unfavourable prognosis in canine mammary carcinomas, showing a strong correlation between Cav-1-positive carcinomas and shorter OS.

Influence of catechol-O-methyltransferase (COMT) genotypes on the prognosis of canine mammary tumors.

Catechol-O-methyltransferase (COMT) is an important enzyme involved in inactivation of catechol estrogens, which are metabolites with carcinogenic properties. Some investigations in human breast cancer associate a genetic polymorphism in the COMT gene (COMT val158met) with an increased risk and poor clinical progression of the disease. In dogs, there are 2 recognized single nucleotide polymorphisms in the COMT gene (COMTG216A and COMTG482A); however, their influence on the outcome of mammary neoplasms has never been investigated. The purpose of this study is to investigate the influence of COMT in the clinical progression of canine mammary tumors, namely in recurrence, metastasis and survival by testing 2 SNPs (G216A and G482A), and 2 genotypes of the COMT gene. A case series was conducted analyzing genomic DNA samples by polymerase chain reaction-restriction fragment length polymorphism from 80 bitches with mammary tumors. Animals were submitted to an active follow-up study for a period of 24 months after surgery. We observed that bitches carrying both genetic variations simultaneously are more likely to develop recurrence of mammary lesions. Our results demonstrate a possible role for COMT genotypes in the outcome of mammary neoplasms in the dog. Identifying a genetic factor predictive of recurrence may be useful in selecting the most effective surgical approach for canine mammary neoplasms.

COX-2 expression in canine normal and neoplastic mammary gland.

COX-2 expression was examined immunohistochemically in samples of normal canine mammary tissue (n=22) and benign (n=36) and malignant (n=45) mammary tumours including metastases (n=12). COX-2 was constitutively expressed in normal mammary tissue with membranous apical labelling of glandular epithelium, suggesting a role for this molecule in normal mammary physiology. By contrast, in neoplastic lesions and in adjacent non-neoplastic mammary tissue COX-2 was expressed in the cytoplasm of epithelial cells, suggesting that internalization of the molecule is associated with oncogenesis. Marked expression of COX-2 was observed in 8.3% of benign neoplasms and in 42.2% of malignant neoplasms, mainly in poorly differentiated areas. The majority of metastatic lesions (58.3%) exhibited strong COX-2 labelling and in almost all cases (83.3%) the labelling intensity was similar or stronger to that of the primary neoplasm. This finding is consistent with the hypothesis that COX-2 metabolites are important promoters of angiogenesis and invasiveness and therefore contribute to metastatic spread.

Estrogens metabolism associated with polymorphisms: influence of COMT G482a genotype on age at onset of canine mammary tumors.

Catechol-O-methyltransferase (COMT) is an important enzyme participating in inactivation of carcinogenic oestrogen metabolites. In humans there is a single nucleotide polymorphism in COMT gene (COMT val158met) that has been associated with an increased risk for developing breast cancer. In dogs, there is a single nucleotide polymorphism in COMT gene (G482A), but its relation with mammary carcinogenesis has never been investigated. The aim of this study was to focus on the evaluation of such polymorphism as a risk factor for the development of mammary tumors in bitches and on the analysis of its relationship with some clinicopathologic features (dog's age and weight, number and histologic type of the lesions, lymph node metastasis) of canine mammary neoplasms. A case-control study was conducted analyzing 90 bitches with mammary tumors and 84 bitches without evidence of neoplastic disease. The COMT G482A polymorphism was analyzed by PCR-RFLP. We found a protective effect of the polymorphism in age of onset of mammary tumors, although we could not establish a significant association between COMT genotype and other clinicopathologic parameters nor with mammary tumor risk overall. Animals carrying the variant allele have a threefold likelihood of developing mammary tumors after 9 years of age in comparison with noncarriers. The Kaplan-Meier method revealed significant differences in the waiting time for onset of malignant disease for A allele carrier (12.46 years) and noncarrier (11.13 years) animals. This investigation constitutes the first case-control study designed to assess the relationship between polymorphic genes and mammary tumor risk in dogs. Our results point to the combined effect of COMT genotype with other genetic and/or environmental risk factors as important key factors for mammary tumor etiopathogenesis.

EJ-ras oncogene transfection of endothelial cells upregulates the expression of syndecan-4 and downregulates heparan sulfate sulfotransferases and epimerase.

The EC rabbit endothelial cell line was transfected with the EJ-ras oncogene (EJ-ras EC). EJ-ras EC cells display over expression of the Ras oncogene, morphological changes and deregulation of the cell cycle, becoming more densely populated and serum-independent. In addition, EJ-ras-transfectant cells show higher levels of the syndecan-4 mRNA. In addition to the increase in the core protein, a parallel increase in the glycosylation of the syndecan-4 protein, a proteoglycan that bears heparan sulfate chains, also occurs. This increase is observed both for the heparan sulfate proteoglycan synthesized by the cells and for that secreted to the culture medium. This enhancement in heparan sulfate synthesis was observed through metabolic labeling of the cells, immunoprecipitation of syndecan-4 and heparitinases treatment. Furthermore, the EJ-ras-transfectant cells do not exhibit decreased synthesis of heparan sulfate during the G(1)-S phase transition, as observed for the parental cell line. Also, heparan sulfate synthesis is not stimulated by PMA as displayed by parental endothelial cells. Significant structural changes of heparan sulfate, such as decreased O-sulfation, were observed in the EJ-ras-transfected cells. Decreases in the mRNA levels of some enzymes (glucuronosyl C-5 epimerase, iduronosyl-2-O-sulfotransferase, glucosaminyl-6-O-sulfotransferase-1 and N-deacetylase/N-sulfotransferase-1), involved in the biosynthetic pathway of heparan sulfate, were also observed. The results suggest that overexpression of the EJ-ras oncogene alters the cell cycle, through signal transduction cascades, upregulates the expression of syndecan-4, and downregulates enzymes involved in the heparan sulfate biosynthesis related to chain modification, leading to the structural changes of the heparan sulfate syndecan-4 proteoglycan in endothelial cells.

Specific structural features of syndecans and heparan sulfate chains are needed for cell signaling.

The syndecans, heparan sulfate proteoglycans, are abundant molecules associated with the cell surface and extracellular matrix and consist of a protein core to which heparan sulfate chains are covalently attached. Each of the syndecan core proteins has a short cytoplasmic domain that binds cytosolic regulatory factors. The syndecans also contain highly conserved transmembrane domains and extracellular domains for which important activities are becoming known. These protein domains locate the syndecan on cell surface sites during development and tumor formation where they interact with other receptors to regulate signaling and cytoskeletal organization. The functions of cell surface heparan sulfate proteoglycan have been centered on the role of heparan sulfate chains, located on the outer side of the cell surface, in the binding of a wide array of ligands, including extracellular matrix proteins and soluble growth factors. More recently, the core proteins of the syndecan family transmembrane proteoglycans have also been shown to be involved in cell signaling through interaction with integrins and tyrosine kinase receptors.

Specific structural features of syndecans and heparan sulfate chains are needed for cell signaling

The syndecans, heparan sulfate proteoglycans, are abundant molecules associated with the cell surface and extracellular matrix and consist of a protein core to which heparan sulfate chains are covalently attached. Each of the syndecan core proteins has a short cytoplasmic domain that binds cytosolic regulatory factors. The syndecans also contain highly conserved transmembrane domains and extracellular domains for which important activities are becoming known. These protein domains locate the syndecan on cell surface sites during development and tumor formation where they interact with other receptors to regulate signaling and cytoskeletal organization. The functions of cell surface heparan sulfate proteoglycan have been centered on the role of heparan sulfate chains, located on the outer side of the cell surface, in the binding of a wide array of ligands, including extracellular matrix proteins and soluble growth factors. More recently, the core proteins of the syndecan family transmembrane proteoglycans have also been shown to be involved in cell signaling through interaction with integrins and tyrosine kinase receptors.

Microbiological assay for gatifloxacin in pharmaceutical formulations.

A simple, sensitive and specific agar diffusion bioassay for the antibacterial gatifloxacin was developed using a strain of Bacillus subtilis ATCC 9372 as the test organism. Gatifloxacin could be measured in tablets and raw material at concentration ranging 4-16 microgml(-1). The calibration graph for gatifloxacin was linear from 4.0 to 16.0 microgml(-1). A prospective validation of the method demonstrated that the method was linear (r2=0.9993), precise (R.S.D.=1.14%) and accurate. The results confirmed its precision and did not differ significantly from others methods described in the literature. The validated method yielded good results in terms of the range, linearity, precision, accuracy, specificity and recovery. We concluded that the microbiological assay is satisfactory for in vitro quantification of the antibacterial activity of gatifloxacin.

Determination of beta2-agonists in bovine urine: comparison of two extraction/clean-up procedures for high-resolution gas chromatography-mass spectrometry analysis.

Two extraction/clean-up analytical procedures were investigated and compared regarding their recovery and matrix-purification efficiency for screening beta2-agonist residues in fortified bovine urine by high-resolution gas chromatography-mass spectrometry (GC-MS). The first procedure, based on an analytical method originally developed for detecting anabolic steroids, consists of the employment of the nonionic resin, Amberlite XAD-2, a styrene-divinylbenzene copolymer for solid-phase extraction (SPE), followed by liquid-liquid extraction with diethyl ether. The second focuses on the use of a mixed SPE cartridge (reversed-phase and ion-exchange sorbent, Bond Elut Certify). In both cases, the trimethylsilylated derivatives were analyzed by GC-MS with an ion-trap detector. Clenbuterol, salbutamol, and terbutaline were used to spike urine samples during the comparison experimental phase. Afterwards, tulobuterol, mabuterol, mapenterol, cimbuterol, and brombuterol were included in the evaluation of the second procedure (the Bond Elut Certify procedure). At this stage, the detection was accomplished by GC-MS (quadrupole mass analyzer) with selective ion monitoring acquisition. The isotopic dilution method with the hexadeuterated analogues of clenbuterol and salbutamol was applied to prepare calibration curves and calculate recovery percentages. With XAD-2 resin, terbutaline and salbutamol (resorcinol and phenol-type beta2-agonists, respectively) could not be detected at 20 ng/mL or at 40 ng/mL. In spite of clenbuterol having been detected at 20 ng/mL, the results obtained were not reproducible. The use of the reversed-phase and ion-exchange sorbent Bond Elut Certify allowed multiresidue detection and showed several advantages for the screening of clenbuterol such as higher recoveries, cleaner final extracts, reduced sample preparation time, less labor intensive, and easier solvent consumption and disposal. Recoveries over 88% (concentrations ranging from 0.5 to 10 ppb) and limits of detection equal to 0.5 ppb were met for all the beta2-agonists studied with the last method.

Controlled introduction of selenium into Chlorella cells.

Selenium (Se) is an important element in the antioxidant system of the human body, and Chlorella, well-known for its therapeutic effects, is the ideal carrier to offer it in the wanted organic form. The kinetics of Se absorption by growing algal cells and its distribution in the cells are studied using radioactive 75Se labelled solutions. There is a rapid Se absorption within the first few minutes at the cell surfaces where it is irreversibly fixed and cannot be absorbed by the human body. In the final state, reached after 24-48 hr, about 40% of the total fixed Se is inside the cells in the wanted organic-bound form.