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The "epithelial barrier hypothesis" states that a barrier dysfunction can result in allergy development due to tolerance breakdown. This barrier alteration may come from the direct contact of epithelial and immune cells with the allergens, and indirectly, through deleterious effects caused by environmental changes triggered by industrialization, pollution, and changes in the lifestyle. Apart from their protective role, epithelial cells can respond to external factors secreting IL-25 IL-33, and TSLP, provoking the activation of ILC2 cells and a Th2-biased response. Several environmental agents that influence epithelial barrier function, such as allergenic proteases, food additives or certain xenobiotics are reviewed in this paper. In addition, dietary factors that influence the allergenic response in a positive or negative way will be also described here. Finally, we discuss how the gut microbiota, its composition, and microbe-derived metabolites, such as short-chain fatty acids, alter not only the gut but also the integrity of distant epithelial barriers, focusing this review on the gut-lung axis.
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OBJECTIVES: To evaluate whether the cutting-off and rescanning procedures have an impact on the accuracy (trueness and precision) of the intraoral digital scan. METHODS: A right quadrant digital scan (reference scan) of a participant was obtained using an intraoral scanner (IOS) (TRIOS 4; 3Shape A/S, Copenhagen, Denmark). The reference scan was duplicated 135 times and divided into 3 groups based on the number of rescanned mesh areas: 1 (G1 group), 2 (G2 group), and 3 (G3 group) mesh holes. Each group was subdivided into 3 subgroups depending on the mesh hole diameter: 2 mm- (G1-2, G2-2, and G3-2), 4 mm- (G1-4, G2-4, and G3-4), and 6 mm- (G1-6, G2-6, and G3-6) (n = 15). A software program (Geomagic; 3D Systems, Rock Hill, SC, USA) was used to assess the discrepancy between the reference and the experimental scans using the root mean square (RMS). Kruskal-Wallis and post hoc multiple comparison Dunn's tests were used to analyze the data (α=0.05). RESULTS: Trueness ranged from 5 to 20 µm and precision ranged from 2 to 10 µm. For trueness assessment, Kruskal-Wallis test revealed significant differences on the RMS error values among the groups tested (P<.05). The G3-6 group obtained the lowest trueness and lowest precision values, while the G1-2, G1-4, G2-2, G2-4, and G3-2 groups computed the highest trueness and precision values. When comparing groups with the same number of rescanned mesh holes but with different diameter, the higher the diameter of the rescanned mesh hole, the lower the trueness values computed; however, when comparing groups with the same diameter of the rescanned mesh hole but with differing number of rescanned mesh holes, no significant differences were found in the RMS values among the groups. For the precision evaluation, Levene's test showed a lack of equality of the variances, and therefore of the standard deviations. The F-test with Bonferroni correction identified significant differences between the SDs between group G3-6 and all the other groups. When comparing instead the interquartile range (IQRs) due to the non-normality of the data, groups G1 and G2 also showed lower IQR values or higher precision than groups G3. CONCLUSIONS: Cutting-off and rescanning procedures decreased the accuracy of the IOS tested. The higher the number and diameter of the rescanned areas, the lower the accuracy. CLINICAL SIGNIFICANCE: Cutting-off and rescanning procedures should be minimized in order to increase the accuracy of the IOS evaluated. The intended clinical use of the intraoral digital scan is a critical factor that might determine the scanning workflow procedures.
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Técnica de Moldagem Odontológica , Modelos Dentários , Desenho Assistido por Computador , Humanos , Imageamento Tridimensional , Telas CirúrgicasRESUMO
PURPOSE: To measure the impact of different scanning patches on the accuracy (trueness and precision) of an intraoral scanner (IOS). MATERIAL AND METHODS: A typodont was digitized using an industrial optical scanner (GOM Atos Q 3D 12 M) to obtain a reference mesh. The typodont was scanned using an IOS (TRIOS 3). Three groups were generated based on the rescan areas created: no mesh holes (G0 group), 3 mesh holes distributed on the digital scan (G1 group), and 3 mesh holes located on the left quadrant of the digital scan (G2 group). In the G0 group, a digital scan was completed following the manufacturer's scanning protocol. In the G1 group, a digital scan was obtained following the same protocol as G0 group. Three 12-mm diameter holes were created in the occlusal surfaces of the left second first molar, incisal edges of the central incisors, and right first molar of the digital scan using the IOS software. In the G2 group, a digital scan was obtained following the same protocol as G0 group. Three 12-mm diameter holes in the digital scan were created in the occlusal surface of the left first molar and left second and first premolars using the IOS software program. The discrepancy between the control and the experimental digital scans was measured using the root mean square calculation. The Kolmogorov-Smirnov test demonstrated that data were normally distributed. One-way ANOVA followed by post hoc multiple comparison Bonferroni test were used to analyze the data (α = .05). RESULTS: Trueness values ranged from 15 to 26 µm and the precision ranged from 21 to 150 µm. Significant differences in trueness mean values were found among the groups tested (F(2, 42) = 6.622, P = .003); the Bonferroni test indicated significant mean differences between the G0 and G2 groups (mean difference=0.11, SE=0.003, and P = .002). For precision evaluation, significant precision differences were found between the groups tested (F(2, 39)=9.479, P < .001); the Bonferroni test revealed significant precision differences between G0 and G2 groups (mean difference=-0.12, SE=0.030, and P = .001). CONCLUSIONS: Rescanning mesh holes and stitching procedures decreased the trueness and precision of the IOS tested; furthermore, the number and dimensions of mesh holes rescanned represented an important factor that influenced the scanning accuracy of IOS tested. CLINICAL SIGNIFICANCE: It is a fundamental procedure obtaining intraoral digital scans without leaving mesh holes, so the rescanning techniques are minimized and, therefore, the scanning accuracy of the intraoral scanner tested is maximized.
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Técnica de Moldagem Odontológica , Modelos Dentários , Desenho Assistido por Computador , Imageamento Tridimensional , Telas CirúrgicasRESUMO
STATEMENT OF PROBLEM: Diagnostic stone casts can be digitized by using dental optical scanners based on structured light scanning technology. Nondental structured light scanning scanners could also be used; however, the accuracy of these nondental scanners remains unclear. PURPOSE: The purpose of this in vitro study was to measure the scanning accuracy (trueness and precision) of 3 nondental extraoral structured light scanners. MATERIAL AND METHODS: A representative maxillary diagnostic cast was obtained and digitized by using an extraoral dental scanner (Advaa Lab Scan; GC Europe), and a reference or control standard tessellation language file was obtained. Three nondental extraoral scanners were evaluated: groups ND-1 (Space Spider; Artec), ND-2 (Capture Mini; Geomagic), and ND-3 (DAVID SLS3; David). Ten digital scans per group were recorded at a constant room temperature (23 °C) by an experienced geodetic engineer following the manufacturer's recommendations. The control or reference file was used as a reference to measure the discrepancy between the digitized diagnostic cast and 3 different nondental scans by using an open-source software (CloudCompare v.2.6.1; CloudCompare) and the iterative closest point technique. The Shapiro-Wilk test revealed that the data were normally distributed. The data were analyzed by using 1-way ANOVA, followed by post hoc Bonferroni tests (α=.05). RESULTS: Significant differences between the 3 experimental nondental scanners and the control or reference scan (P<.001) were found. The ND-2 group had the lowest absolute mean error (trueness) and standard deviation (precision) (39 ±139 µm), followed by the ND-3 group (125 ±113 µm) and the ND1 group (-397 ±25 µm). No statistically significant differences were found in the mean error between the ND-2 and ND-3 groups (P=.228). CONCLUSIONS: Only 1 nondental extraoral scanner tested obtained trueness mean values similar to those of the reference dental scanner. In all groups, the precision mean values were higher than their trueness values, indicating low relative precision.
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Técnica de Moldagem Odontológica , Modelos Dentários , Desenho Assistido por Computador , Imageamento Tridimensional , MaxilaRESUMO
Colorectal cancer is the third most common form of cancer in developed countries and, despite the improvements achieved in its treatment options, remains as one of the main causes of cancer-related death. In this review, we first focus on colorectal carcinogenesis and on the genetic and epigenetic alterations involved. In addition, noncoding RNAs have been shown to be important regulators of gene expression. We present a general overview of what is known about these molecules and their role and dysregulation in cancer, with a special focus on the biogenesis, characteristics, and function of microRNAs. These molecules are important regulators of carcinogenesis, progression, invasion, angiogenesis, and metastases in cancer, including colorectal cancer. For this reason, miRNAs can be used as potential biomarkers for diagnosis, prognosis, and efficacy of chemotherapeutic treatments, or even as therapeutic agents, or as targets by themselves. Thus, this review highlights the importance of miRNAs in the development, progression, diagnosis, and therapy of colorectal cancer and summarizes current therapeutic approaches for the treatment of colorectal cancer.
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Carcinogênese/genética , Neoplasias Colorretais/genética , Epigênese Genética/genética , RNA não Traduzido/genética , Biomarcadores Tumorais , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Modelos Genéticos , Neovascularização Patológica/genética , PrognósticoRESUMO
Annexin A13 is the founder member of the vertebrate family of annexins, which are comprised of a tetrad of unique conserved domains responsible for calcium-dependent binding to membranes. Its expression is restricted to epithelial intestinal and kidney cells. Alternative splicing in the N-terminal region generates two isoforms, A13a and A13b, differing in a deletion of 41 residues in the former. We have confirmed the expression of both isoforms in human colon adenocarcinoma cells at the mRNA and protein levels. We have cloned, expressed, and purified human annexin A13a for the first time to analyze its structural characteristics. Its secondary structure and thermal stability differs greatly from the A13b isoform. The only tryptophan residue (Trp186) is buried in the protein core in the absence of calcium but is exposed to the solvent after calcium binding even though circular dichroism spectra are quite similar. Non-myristoylated annexin A13a binds in a calcium-dependent manner to acidic phospholipids but not to neutral or raft-like liposomes. Calcium requirements for binding to phosphatidylserine are around 6-fold lower than those required by the A13b isoform. This fact could account for the different subcellular localization of both annexins as binding to basolateral membranes seems to be calcium-dependent and myristoylation-independent.
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4F2hc is a type-II glycoprotein whose covalent-bound association with one of several described light chains yields a heterodimer mainly involved in large neutral amino acid transport. Likewise, it is well known that the heavy chain interacts with ß-integrins mediating integrin-dependent events such as survival, proliferation, migration and even transformation. 4F2hc is a ubiquitous protein whose overexpression has been related to tumor development and progression. Stable silencing of 4F2hc in HeLa cells using an artificial miRNA impairs in vivo tumorigenicity and leads to an ineffective proliferation response to mitogens. 4F2hc colocalizes with ß1-integrins and CD147, but this interaction does not occur in lipid rafts in HeLa cells. Moreover, silenced cells present defects in integrin- (FAK, Akt and ERK1/2) and hypoxia-dependent signaling, and reduced expression/activity of MMP-2. These alterations seem to be dependent on the inappropriate formation of CD147/4F2hc/ß1-integrin heterocomplexes on the cell surface, arising when CD147 cannot interact with 4F2hc. Although extracellular galectin-3 accumulates due to the decrease in MMP-2 activity, galectin-3 signaling events are blocked due to an impaired interaction with 4F2hc, inducing an increased degradation of ß-catenin. Furthermore, cell motility is compromised after protein silencing, suggesting that 4F2hc is related to tumor invasion by facilitating cell motility. Therefore, here we propose a molecular mechanism by which 4F2hc participates in tumor progression, favoring first steps of epithelial-mesenchymal transition by inhibition of ß-catenin proteasomal degradation through Akt/GSK-3ß signaling and enabling cell motility.
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Cadeia Pesada da Proteína-1 Reguladora de Fusão/biossíntese , Galectina 3/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Sistema de Sinalização das MAP Quinases , Metaloproteinase 2 da Matriz/biossíntese , Neoplasias Experimentais/metabolismo , beta Catenina/metabolismo , Animais , Basigina/genética , Basigina/metabolismo , Movimento Celular/genética , Transição Epitelial-Mesenquimal/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Cadeia Pesada da Proteína-1 Reguladora de Fusão/genética , Galectina 3/genética , Células HeLa , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Metaloproteinase 2 da Matriz/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transplante Heterólogo , beta Catenina/genéticaRESUMO
Annexins constitute an evolutionary conserved multigene protein superfamily characterized by their ability to interact with biological membranes in a calcium dependent manner. They are expressed by all living organisms with the exception of certain unicellular organisms. The vertebrate annexin core is composed of four (eight in annexin A6) homologous domains of around 70 amino acids, with the overall shape of a slightly bent ring surrounding a central hydrophilic pore. Calcium- and phospholipid-binding sites are located on the convex side while the N-terminus links domains I and IV on the concave side. The N-terminus region shows great variability in length and amino acid sequence and it greatly influences protein stability and specific functions of annexins. These proteins interact mainly with acidic phospholipids, such as phosphatidylserine, but differences are found regarding their affinity for lipids and calcium requirements for the interaction. Annexins are involved in a wide range of intra- and extracellular biological processes in vitro, most of them directly related with the conserved ability to bind to phospholipid bilayers: membrane trafficking, membrane-cytoskeleton anchorage, ion channel activity and regulation, as well as antiinflammatory and anticoagulant activities. However, the in vivo physiological functions of annexins are just beginning to be established.
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Bile acids are natural detergents mainly involved in facilitating the absorption of dietary fat in the intestine. In addition to this absorptive function, bile acids are also essential in the maintenance of the intestinal epithelium homeostasis. To accomplish this regulatory function, bile acids may induce programmed cell death fostering the renewal of the epithelium. Here we first discuss on the different molecular pathways of cell death focusing on apoptosis in colon epithelial cells. Bile acids may induce apoptosis in colonocytes through different mechanisms. In contrast to hepatocytes, the extrinsic apoptotic pathway seems to have a low relevance regarding bile acid cytotoxicity in the colon. On the contrary, these molecules mainly trigger apoptosis through direct or indirect mitochondrial perturbations, where oxidative stress plays a key role. In addition, bile acids may also act as regulatory molecules involved in different cell signaling pathways in colon cells. On the other hand, there is increasing evidence that the continuous exposure to certain hydrophobic bile acids, due to a fat-rich diet or pathological conditions, may induce oxidative DNA damage that, in turn, may lead to colorectal carcinogenesis as a consequence of the appearance of cell populations resistant to bile acid-induced apoptosis. Finally, some bile acids, such as UDCA, or low concentrations of hydrophobic bile acids, can protect colon cells against apoptosis induced by high concentrations of cytotoxic bile acids, suggesting a dual behavior of these agents as pro-death or pro-survival molecules.
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Ácidos e Sais Biliares/metabolismo , Colo/metabolismo , Animais , Apoptose , Morte Celular , Neoplasias do Colo/metabolismo , Humanos , Estresse OxidativoRESUMO
A critical risk factor in colorectal carcinogenesis and tumor therapy is the resistance to the apoptotic effects of different compounds from the intestinal lumen, among them butyrate (main regulator of colonic epithelium homeostasis). Insensitivity to butyrate-induced apoptosis yields resistance to other agents, as bile acids or chemotherapy drugs, allowing the selective growth of malignant cell subpopulations. Here we analyze bile acid-induced apoptosis in a butyrate-resistant human colon adenocarcinoma cell line (BCS-TC2.BR2) to determine the mechanisms that underlay the resistance to these agents in comparison with their parental butyrate-sensitive BCS-TC2 cells. This study demonstrates that DCA and CDCA still induce apoptosis in butyrate-resistant cells through increased ROS production by activation of membrane-associated enzymes and subsequent triggering of the intrinsic mitochondrial apoptotic pathway. Although this mechanism is similar to that described in butyrate-sensitive cells, cell viability is significantly higher in resistant cells. Moreover, butyrate-resistant cells show higher Bcl-2 levels that confer resistance to bile acid-induced apoptosis sequestering Bax and avoiding Bax-dependent pore formation in the mitochondria. We have confirmed that this resistance is reverted using the Bcl-2 inhibitor ABT-263, thus demonstrating that the lower sensitivity of butyrate-resistant cells to the apoptotic effects of bile acids is mainly due to increased Bcl-2 levels.
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Apoptose/efeitos dos fármacos , Ácidos e Sais Biliares/farmacologia , Butiratos/farmacologia , Neoplasias do Colo/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Western Blotting , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Citometria de Fluxo , Fármacos Gastrointestinais/farmacologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais Cultivadas , Proteína X Associada a bcl-2/antagonistas & inibidoresRESUMO
Ribotoxins are a family of toxic proteins that exert a highly specific cleavage at the universally conserved sarcin/ricin loop (SRL) of the larger rRNA molecule. Before this ribonucleolytic action, passage through the cell membrane is a necessary step for ribotoxin internalization and the limiting factor for cytotoxicity. Although extensive knowledge of their ribonucleolytic activity and substrate recognition has been accumulated, little is known about the mechanisms of cell entry of ribotoxins. Hirsutellin A (HtA) is a recently described member of this family, which accommodates the main abilities of previously characterized ribotoxins into a shorter sequence, but exhibits some differences regarding membrane interaction properties. This work investigates the contribution of tryptophan (Trp) residues 71 and 78 to both endoribonucleolytic activity and cellular toxicity of this ribotoxin. Substitution mutants W71F and W78F, as well as the double mutant W71/78F, were obtained and assayed against isolated ribosomes, synthetic SRL, and human tumor cells. The results provide evidence that cell membrane passage and internalization, as well as substrate-specific recognition, require the participation of the region involving both Trp 71 and Trp 78. Additionally, the mutant W71/78F is the first non-cytotoxic but specific ribosome-cleaving ribotoxin mutant obtained to date.
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Citotoxinas/química , Citotoxinas/toxicidade , Proteínas Fúngicas/química , Proteínas Fúngicas/toxicidade , RNA Ribossômico/metabolismo , Ribonucleases/química , Ribonucleases/metabolismo , Triptofano/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Sequência Conservada , Citotoxinas/genética , Citotoxinas/metabolismo , Endorribonucleases/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Inibidores da Síntese de Proteínas/química , Inibidores da Síntese de Proteínas/metabolismo , Inibidores da Síntese de Proteínas/toxicidade , Transporte Proteico , Ribonucleases/genética , Ribonucleases/toxicidade , Ricina/química , Especificidade por Substrato , Triptofano/genéticaRESUMO
INTRODUCTION: According to Spanish health regulations, primary care professionals have the responsibility to carry out health-promoting community activities (CAs). However, in practice, their implementation is not as widespread as it should be. The aims of this study were to identify factors within the team, the community and the professionals that influence the development of these activities and to describe the community interventions in progress. METHODS AND ANALYSIS: This study is an observational analytical retrospective study. The information will be collected from five Spanish regions: Catalonia, Madrid, the Balearic Islands, Navarra and Aragón. The authors will contact primary care teams (PCTs) and identify the CAs from the previous year. The research team will conduct a peer review whether the inclusion criteria are met. In the health centres where CAs are implemented, the authors will select professionals carrying them out and randomly select an identical number of professionals not doing these activities. In the centres where no CA is implemented, three professionals will be randomly selected. The selected professionals will complete the questionnaires for individual-level variables. Information about the registered population and the PCTs will be collected through questionnaires and secondary sources. OUTCOMES: Variables will be collected from the community, the PCTs, the individual professionals and CAs. ANALYSIS: A descriptive analysis of all the variables will be carried out, along with a bivariate and a logistic regression analysis, with CAs being the primary outcome. ETHICS AND DISSEMINATION: This study has been approved by the Research Ethics Committee of the Jordi Gol y Gurina Foundation in Barcelona and area 11 in Madrid. The questionnaire distributed to the professionals will be anonymous.
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MMP-11 (stromelysin-3) is a matrix metalloproteinase associated with tumor progression and poor prognosis. Its expression was initially described exclusively in stromal cells surrounding tumors, but more recently it has also been detected in macrophages and hepatocarcinoma cells. Here we show MMP-11 expression in human epithelial colon adenocarcinoma cell lines (Caco-2, HT-29 and BCS-TC2). Treatment of BCS-TC2 cells with butyrate and trichostatin A (TSA) (histone deacetylase inhibitors) increases MMP11 promoter activity and protein expression. Using electrophoretic mobility shift assay (EMSA) and supershift assays, we demonstrate for the first time that Sp1 is able to bind to the GC-boxes within the MMP11 proximal promoter region; this binding has been confirmed by chromatin immunoprecipitation. Sp1 is involved in MMP11 basal expression and it is essential for the upregulation of transcription by histone deacetylase inhibitors as deduced from mutant constructs lacking the Sp1 sites and by inhibition of its binding to the promoter with mithramycin. This regulation requires the formation of Sp1/Smad2 heterocomplexes, which is stimulated by an increase in the acetylation status of Smad after butyrate or TSA treatments. We have also found that ERK1/2-mitogen-activated protein kinase (MAPK), but not p38-MAPK or JNK, is involved in the upregulation of MMP11 by HDAC inhibitors.
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Adenocarcinoma , Neoplasias do Colo , Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Metaloproteinase 11 da Matriz/metabolismo , Proteínas Smad/metabolismo , Fator de Transcrição Sp1/metabolismo , Butiratos/farmacologia , Linhagem Celular Tumoral , Humanos , Ácidos Hidroxâmicos/farmacologia , Metaloproteinase 11 da Matriz/genética , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Regiões Promotoras Genéticas , Proteínas Smad/genética , Fator de Transcrição Sp1/genéticaRESUMO
Ribotoxins are fungal extracellular ribonucleases that specifically cleave ribosomes leading to cell-death via apoptosis. α-Sarcin is the ribotoxin studied in deepest detail, and therefore constitutes the referential protein for the whole family. It has been demonstrated that ribotoxin activity depends on a very precise structural microenvironment in which electrostatic interactions among residues in the active site are of the highest importance. Hirsutellin A (HtA) has been recently described as the smallest ribotoxin known to date, encompassing all the abilities of previously characterized members of this family into a shorter sequence. Comparison of HtA and α-sarcin three-dimensional structures suggested that residues presumably forming the catalytic triad of HtA would be His 42, Glu 66, and His 113. Within this same idea, the presence of an Asp residue (Asp 40) in a position equivalent to α-sarcin Tyr 48 is highlighted as a novelty in this field. In this work, substitution mutants H42Q, E66Q and H113Q, as well as double and triple mutants in all possible combinations, are studied regarding their ribonucleolytic activity and cytotoxicity. Implication of these three residues in the ribotoxin activity of HtA is confirmed, though none of them is strictly essential for ribosomal cleavage. Studies with mutants D40N and D40N/E66Q demonstrate an important role for Asp 40 in the activity of HtA and establish a new set of electrostatic interactions different from the one described for already known ribotoxins.
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Ácido Aspártico/metabolismo , Proteínas Fúngicas/genética , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Ribossomos/metabolismo , Ácido Aspártico/genética , Domínio Catalítico , Clonagem Molecular , Endorribonucleases/química , Endorribonucleases/genética , Endorribonucleases/farmacologia , Proteínas Fúngicas/química , Proteínas Fúngicas/farmacologia , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Inibidores da Síntese de Proteínas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Eletricidade EstáticaRESUMO
The continuous exposure of the colonic epithelium to high concentrations of bile acids may exert cytotoxic effects and has been related to pathogenesis of colon cancer. A better knowledge of the mechanisms by which bile acids induce toxicity is still required and may be useful for the development of new therapeutic strategies. We have studied the effect of deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) treatments in BCS-TC2 human colon adenocarcinoma cells. Both bile acids promote cell death, being this effect higher for CDCA. Apoptosis is detected after 30 min-2 h of treatment, as observed by cell detachment, loss of membrane asymmetry, internucleosomal DNA degradation, appearance of mitochondrial transition permeability (MPT), and caspase and Bax activation. At longer treatment times, apoptosis is followed in vitro by secondary necrosis due to impaired mitochondrial activity and ATP depletion. Bile acid-induced apoptosis is a result of oxidative stress with increased ROS generation mainly by activation of plasma membrane enzymes, such as NAD(P)H oxidases and, to a lower extent, PLA2. These effects lead to a loss of mitochondrial potential and release of pro-apoptotic factors to the cytosol, which is confirmed by activation of caspase-9 and -3, but not caspase-8. This initial apoptotic steps promote cleavage of Bcl-2, allowing Bax activation and formation of additional pores in the mitochondrial membrane that amplify the apoptotic signal.
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Adenocarcinoma/patologia , Apoptose/efeitos dos fármacos , Ácido Quenodesoxicólico/toxicidade , Neoplasias do Colo/patologia , Ácido Desoxicólico/toxicidade , Estresse Oxidativo/fisiologia , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Ácido Quenodesoxicólico/farmacologia , Ácido Desoxicólico/farmacologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Necrose/induzido quimicamente , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Fungal ribotoxins are toxic secreted ribonucleases that cleave a conserved single phosphodiester bond located at the sarcin/ricin loop of the larger rRNA. This cleavage inactivates ribosomes leading to protein biosynthesis inhibition and cell death. It has been proposed that interactions other than those found at the active site of ribotoxins are needed to explain their exquisite specific activity. The study presented shows the ability of a catalytically inactive α-sarcin mutant (H137Q) to bind eukaryotic ribosomes and interfere with in vitro protein biosynthesis. The results obtained are compatible with previous observations that α-sarcin can promote cell death by a mechanism that is independent of rRNA cleavage, expanding the potential set of activities performed by this family of toxins.
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Endorribonucleases/farmacologia , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Animais , Endorribonucleases/genética , Endorribonucleases/metabolismo , Proteínas Fúngicas/genética , Modelos Biológicos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas Mutantes/farmacologia , Ligação Proteica , Inibidores da Síntese de Proteínas/metabolismo , Coelhos , Ribossomos/metabolismo , Saccharomyces cerevisiaeRESUMO
4F2hc (CD98hc) is a multifunctional type II membrane glycoprotein involved in several functions as amino acid transport, cell fusion, ß1-integrin-signaling and transformation. 4F2hc ectodomain has been crystallized and its three-dimensional structure determined. We have carried out a spectroscopical/structural characterization of the recombinant ectodomain in order to obtain information on its dynamic structure in solution and on its ability to form homodimers by itself in the absence of the transmembrane helix and of the potential interactions with the plasma membrane. Analytical ultracentrifugation and crosslinking experiments showed that the ectodomain is monomeric in solution. The secondary structure determined by far-UV circular dichroism (CD) spectroscopy (around 30% α-helix and 20% ß-sheets, 12% antiparallel and 8% parallel) reveals a compact and thermally stable structure with a high melting temperature (57-59°C). Tryptophan residues are mainly buried and immobilized in the hydrophobic core of the protein as suggested by near-UV CD spectrum, the position of the Trp maximum fluorescence emission (323nm) and from the acrylamide quenching constant (2.6M(-1)). Urea unfolding equilibrium has been studied by far-UV CD and fluorescence spectroscopy to gain information on the folding/unfolding process of the ectodomain. The analyses suggest the existence of two intermediate states as reported for other TIM barrel-containing proteins rather than an independent unfolding of each domain [A, (ßα)(8) barrel; C, antiparallel ß(8) sandwich]. Folding seems to be directed by the initial formation of hydrophobic clusters within the first strands of the ß-barrel of domain A followed by additional hydrophobic interactions in domain C.
Assuntos
Cadeia Pesada da Proteína-1 Reguladora de Fusão/química , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Cadeia Pesada da Proteína-1 Reguladora de Fusão/genética , Humanos , Dobramento de Proteína , Multimerização Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Espectrometria de FluorescênciaRESUMO
The use of biological materials in the construction of bioprostheses requires the application of different chemical procedures to improve the durability of the material without producing any undesirable effects. A number of crosslinking methods have been tested in biological tissues composed mainly of collagen. The aim of this study was to evaluate the in vitro biocompatibility, the mechanical properties, and in vivo calcification of chemically modified bovine pericardium using glutaraldehyde acetals (GAAs) in comparison with glutaraldehyde (GA) treatment. Homsy's tests showed that the most cytotoxic treatment is GA whereas GAA treatments showed lower cytotoxicity. Regarding the mechanical properties of the modified materials, no significant differences in stress at rupture were detected among the different treatments. Zeta-Potential showed higher negative values for GA treatment (-4.9 +/- 0.6 mV) compared with GAA-0.625% (-2.2 +/- 0.5 mV) and GAA-1% (-2.2 +/- 0.4 mV), which presented values similar to native tissue. Similar results were obtained for calcium permeability coefficients which showed the highest values for GA treatment (0.12 +/- 0.02 mm(2)/min), being significantly lower for GAA treatments or non-crosslinked pericardium. These results confirmed the higher propensity of the GA-treated tissues for attraction of calcium cations and were in good agreement with the calcification degree obtained after 60 days implantation into young rats, which was significantly higher for the GA group (22.70 +/- 20.80 mg/g dry tissue) compared with GAA-0.625% and GAA-1% groups (0.49 +/- 0.28 mg/g dry tissue and 3.51 +/- 3.27 mg/g dry tissue, respectively; P < 0.001). In conclusion, GAA treatments can be considered a promising alternative to GA treatment.
Assuntos
Bioprótese , Calcificação Fisiológica , Glutaral/química , Coração Artificial , Pericárdio/química , Animais , Bioprótese/efeitos adversos , Cálcio/metabolismo , Bovinos , Reagentes de Ligações Cruzadas/química , Coração Artificial/efeitos adversos , Teste de Materiais , Pericárdio/metabolismo , RatosRESUMO
Annexins are calcium-dependent phospholipid-binding proteins involved in calcium signaling and intracellular membrane trafficking among other functions. Vesicle aggregation is a crucial event to make possible the membrane remodeling but this process is energetically unfavorable, and phospholipid membranes do not aggregate and fuse spontaneously. This issue can be circumvented by the presence of different agents such as divalent cations and/or proteins, among them some annexins. Although human annexin A5 lacks the ability to aggregate vesicles, here we demonstrate that its highly similar chicken ortholog induces aggregation of vesicles containing acidic phospholipids even at low protein and/or calcium concentration by establishment of protein dimers. Our experiments show that the ability to aggregate vesicles mainly resides in the N-terminus as truncation of the N-terminus of chicken annexin A5 significantly decreases this process and replacement of the N-terminus of human annexin A5 by that of chicken switches on aggregation; in both cases, there are no changes in the overall protein structure and only minor changes in phospholipid binding. Electrostatic repulsions between negatively charged residues in the concave face of the molecule, mainly in the N-terminus, seem to be responsible for the impairment of dimer formation in human annexin A5. Taking into account that chicken annexin A5 presents a high sequence and structural similarity with mammalian annexins absent in birds, as annexins A3 and A4, some of the physiological functions exerted by these proteins may be carried out by chicken annexin A5, even those that could require calcium-dependent membrane aggregation.
Assuntos
Anexina A5/química , Anexina A5/fisiologia , Vesículas Transportadoras/fisiologia , Animais , Anexina A5/genética , Anexina A5/metabolismo , Cálcio/metabolismo , Galinhas , Dicroísmo Circular , Clonagem Molecular , Escherichia coli/genética , Humanos , Concentração de Íons de Hidrogênio , Lipossomos/metabolismo , Microscopia de Fluorescência , Modelos Moleculares , Fosfolipídeos , Multimerização Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vesículas Transportadoras/metabolismoRESUMO
The mite fungal pathogen Hirsutella thompsonii produces a single polypeptide chain, insecticidal protein named hirsutellin A (HtA) that is composed of 130 amino acid residues. This protein has been purified from its natural source and produced as a recombinant protein in Escherichia coli. Spectroscopic analysis has determined that the two protein forms are indistinguishable. HtA specifically inactivates ribosomes and produces the alpha-fragment characteristic of ribotoxin activity on rRNA. Behaving as a cyclizing ribonuclease, HtA specifically cleaves oligonucleotides that mimick the sarcin/ricin loop of the ribosome, as well as selected polynucleotides and dinucleosides. HtA interacts with phospholipid membranes as do other ribotoxins. As a consequence of its ribonuclease activity and its ability to interact with cell membranes, HtA exhibits cytotoxic activity on human tumor cells. On the basis of these results, HtA is considered to be a member of the ribotoxin group of proteins, although it is significantly smaller (130 aa) than all known ribotoxins that are composed of 149/150 amino acids. Ribotoxins are members of a larger family of fungal ribonucleases whose members of smaller size (100/110 aa) are not cytotoxic. Thus, the characterization of the fungal ribotoxin HtA represents an important milestone in the study of the diversity and the function of fungal ribonucleases.