RESUMEN
Actin is a highly conserved protein that is found in all eukaryotic cells, and has been widely used as an internal control gene in gene expression studies. In this study, we cloned an actin gene (named Ecß-actin) from Exopalaemon carinicauda and determined its expression levels. The full-length cDNA of Ecß-actin was 1335 bp long, comprising a 1131-bp ORF encoding 376 amino acids, a 65-bp 5'-UTR, and a 139-bp 3'-UTR with a poly(A) tail. The A + T content was approximately 79% in the 3'-UTR of the Ecß-actin mRNA. The 3'-UTR contained two repeats of the AUUUA motif. The putative protein Ecß-actin showed high identity (97-99%) with other actins from various species. Phylogenetic analysis revealed that Ecß-actin belongs to Crustacea, although it formed a singleton sub-branch that was located a short distance from crabs and other shrimp species. Ecß- actin expression was detected in the hepatopancreas, ovary, muscle, gill, stomach, and hemocytes, and was strongly expressed in the hemocytes and ovary of E. carinicauda. Ecß-actin mRNA expression varied during ovarian development, with high levels observed at stages I and V. Therefore, caution should be taken when using the Ecß-actin gene as an endogenous control gene.
Asunto(s)
Actinas/biosíntesis , Palaemonidae/genética , Filogenia , Actinas/genética , Animales , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Hepatopáncreas/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
The effect of all-trans retinoic acid (ATRA) on the expression of α-smooth muscle actin (α-SMA) in rats with pulmonary arterial hypertension (PAH) was studied, and the mechanism of the effect of ATRA on PAH was proposed. Thirty male SD rats were randomly divided into normal control, monocrotaline (MCT) model, and ATRA [30 mg/(kg.day)]intervention groups (N = 10 each). The mean pulmonary arterial pressure was recorded. Right ventricular hypertrophy index (RVHI) was calculated (weight of right ventricle: total weight of left ventricle and interventricular septum). The percentages of wall thickness of pulmonary arteriole (WT) to external diameter of artery (WT%) and vascular wall area (WA) to total vascular area (WA%) were determined. Real-time fluorescence-based quantitative PCR and western blot analyses were employed to detect the α-SMA mRNA and protein expressions. The mean pulmonary arterial pressure, RVHI, WT%, and WA% were all obviously higher in the model group than in the control and intervention groups. The values of these indicators in the intervention group were also higher than those in the control group (P < 0.01). The mRNA and protein expression levels of α-SMA were significantly higher in the lung tissue of model rats than those in the control and intervention groups. However, the intervention group showed no statistically significant differences in α-SMA mRNA and protein expression levels compared to the control (P < 0.05). ATRA inhibited the α-SMA mRNA and protein expressionin the lung tissues of rats with MCT-induced PAH, and could be used to treat PAH.
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Actinas/biosíntesis , Hipertensión Pulmonar/tratamiento farmacológico , Hipertensión Pulmonar/metabolismo , Tretinoina/farmacología , Actinas/genética , Animales , Modelos Animales de Enfermedad , Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/genética , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Masculino , Monocrotalina , Arteria Pulmonar/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
The present study evaluated whether the changes in the labeling pattern of cytoskeletal proteins in osteogenic cells cultured on bioactive glass-based materials are due to altered mRNA and protein levels. Primary rat-derived osteogenic cells were plated on Bioglass® 45S5, Biosilicate®, and borosilicate (bioinert control). The following parameters were assayed: (i) qualitative epifluorescence analysis of actin and tubulin; (ii) quantitative mRNA and protein expression for actin and tubulin by real-time PCR and ELISA, respectively, and (iii) qualitative analysis of cell morphology by scanning electron microscopy (SEM). At days 3 and 7, the cells grown on borosilicate showed typical actin and tubulin labeling patterns, whereas those on the bioactive materials showed roundish areas devoid of fluorescence signals. The cultures grown on bioactive materials showed significant changes in actin and tubulin mRNA expression that were not reflected in the corresponding protein levels. A positive correlation between the mRNA and protein as well as an association between epifluorescence imaging and quantitative data were only detected for the borosilicate. SEM imaging of the cultures on the bioactive surfaces revealed cells partly or totally coated with material aggregates, whose characteristics resembled the substrate topography. The culturing of osteogenic cells on Bioglass® 45S5 and Biosilicate® affect actin and tubulin mRNA expression but not the corresponding protein levels. Changes in the labeling pattern of these proteins should then be attributed, at least in part, to the presence of a physical barrier on the cell surface as a result of the material surface reactions, thus limiting fluorescence signals.
Asunto(s)
Actinas/biosíntesis , Cerámica , Perfilación de la Expresión Génica , Vidrio , Osteoblastos/metabolismo , Tubulina (Proteína)/biosíntesis , Actinas/análisis , Actinas/genética , Animales , Células Cultivadas , Dobutamina , Microscopía Electrónica de Rastreo , Osteoblastos/citología , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Tiempo , Tubulina (Proteína)/análisis , Tubulina (Proteína)/genéticaRESUMEN
We examined the influence of cyclophilin-D (CypD) protein expression level on endothelial cell oxidative damage resistance. A model of CypD protein expression or high expression in endothelial cells was established through gene silencing or cloning. The comparable groups were normal endothelial cells cultured in phosphate-buffered solution in liquid handling cells containing 500 mM H2O2 for 90 or 120 min, and then the medium was replaced with common nutrient solution and cultured again for 24 h. The apoptosis rate and nitric oxide (NO) levels of each group were tested. The cell apoptosis rate of the CyPD low expression group (32.51 ± 6.6 %) was significantly lower than that of the control group (52.57 ± 5.84%, P = 0.001), and total NO production was 24.06 ± 3 and 13.03 ± 3.55 µM. The apoptosis rate of the CyPD high expression group (24.24 + 3.08%) was significantly higher than that of the control group (7.7 + 0.68%, P < 0.001); total NO production was 3.55 ± 1.53 and 8.46 ± 0.77 µM, which was significantly different (P = 0.008). CypD protein could increase oxidative stress and cause endothelial cell injury and apoptosis.
Asunto(s)
Apoptosis/fisiología , Ciclofilinas/genética , Células Endoteliales de la Vena Umbilical Humana/patología , Óxido Nítrico/metabolismo , Estrés Oxidativo , Actinas/biosíntesis , Apoptosis/efectos de los fármacos , Secuencia de Bases , Línea Celular , Supervivencia Celular , Ciclofilinas/biosíntesis , Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Oxidación-Reducción , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Interferente PequeñoRESUMEN
Infantile myofibroma is a rare mesenchymal benign tumor mostly found in the head and neck region. The aim of this study was to describe a small case series of head and neck solitary infantile myofibroma, emphasizing the importance of the histopathological and immunohistochemical features, and the potential diagnostic challenges. For the study, clinical and imaging data were obtained from the medical records. All cases were histologically reviewed, and immunohistochemical analyses were performed to confirm the diagnosis. Four cases of head and neck solitary infantile myofibroma were identified. All patients were females and presented a mean age of 3 years old (ranging from 2 to 6 years). The site of the tumors were the mandible, right cheek, subcutaneous tissue adjacent to basal cortical of the mandible and upper anterior gingiva. No symptoms, such as pain or paresthesia, were reported. Computerized tomography revealed well-delimited tumors. All tumors were positive for vimentin and alpha-smooth muscle actin. All patients underwent surgical excision and no signs of recurrence were observed after long-term follow-up. In summary, head and neck solitary infantile myofibromas are rare and present excellent prognosis. The correlation between clinical, histopathological and immunohistochemical features are essential for a correct diagnosis.
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Actinas/biosíntesis , Neoplasias de Cabeza y Cuello , Miofibroma , Proteínas de Neoplasias/biosíntesis , Tomografía Computarizada por Rayos X , Vimentina/biosíntesis , Niño , Preescolar , Femenino , Neoplasias de Cabeza y Cuello/diagnóstico por imagen , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Inmunohistoquímica , Miofibroma/diagnóstico por imagen , Miofibroma/metabolismoRESUMEN
The therapeutic potential of mesenchymal stem cells (MSCs) and their conditioned medium (MSC-CM) has been extensively studied. MSCs can repair tissue, reduce local inflammation, and modulate the immune response. Persistent renal tubular interstitial inflammation results in fibrosis and leads to chronic kidney disease (CKD). Unilateral ureteral obstruction (UUO) is a very well-accepted renal fibrosis model. In this study, we evaluated factors influenced by the administration of MSCs or MSC-CM in the UUO model. MSCs extracted from rat bone marrow were cultivated in vitro and characterized by flow cytometry and cellular differentiation. Eight groups of female rats were used in experiments (n = 7, each), including Sham, UUO, UUO + MSC (obstruction + MSC), and UUO + CM (obstruction + MSC-CM) for 7 days of obstruction and Sham, UUO, UUO + MSC, and UUO + CM for 14 days of obstruction. The MSCs or MSC-CM was administered via the abdominal vena cava after total ligation of the left ureter. After 7 or 14 days, rats were euthanized, and serum and obstructed kidney samples were collected. MSCs or MSC-CM decreased the expression of molecules, such as Col1a1, α-SMA, and TNF-α. We also observed reductions in the levels of caspase 3, α-SMA, and PCNA in treated animals by immunohistochemistry. Our results suggest that the intravenous administration of MSCs or MSC-CM improves fibrosis progression and factors involved in apoptosis, inflammation, cell proliferation, and epithelial-mesenchymal transition in Wistar rats subjected to UUO, indicating a potential tool for preventing CKD.
Asunto(s)
Medios de Cultivo Condicionados/farmacología , Fibrosis/terapia , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Insuficiencia Renal Crónica/prevención & control , Obstrucción Ureteral/terapia , Actinas/biosíntesis , Animales , Células de la Médula Ósea/citología , Caspasa 3/metabolismo , Diferenciación Celular , Proliferación Celular , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Células Cultivadas , Colágeno Tipo I/biosíntesis , Cadena alfa 1 del Colágeno Tipo I , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal/inmunología , Femenino , Inflamación/inmunología , Inflamación/terapia , Túbulos Renales/patología , Masculino , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas , Ratas Wistar , Insuficiencia Renal Crónica/patología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
BACKGROUND: The therapeutic potential of adult stem cells in the treatment of chronic diseases is becoming increasingly evident. In the present study, we sought to assess whether treatment with mesenchymal stem cells (MSCs) efficiently retards progression of chronic renal failure (CRF) when administered to experimental models of less severe CRF. METHODS: We used two renal mass reduction models to simulate different stages of CRF (5/6 or 2/3 mass renal reduction). Renal functional parameters measured were serum creatinine (SCr), creatinine clearance (CCr), rate of decline in CCr (RCCr), and 24-h proteinuria (PT24h). We also evaluated renal morphology by histology and immunohistochemistry. MSCs were obtained from bone marrow aspirates and injected into the renal parenchyma of the remnant kidneys of both groups of rats with CRF (MSC5/6 or MSC2/3). RESULTS: Animals from groups MSC5/6 and CRF2/3 seemed to benefit from MSC therapy because they showed significantly reduction in SCr and PT24h, increase in CCr and slowed the RCCr after 90 days. Treatment reduced glomerulosclerosis but significant improvement did occur in the tubulointerstitial compartment with much less fibrosis and atrophy. MSC therapy reduced inflammation by decreasing macrophage accumulation proliferative activity (PCNA-positive cells) and fibrosis (α-SM-actin). Comparisons of renal functional and morphological parameters responses between the two groups showed that rats MSC2/3 were more responsive to MSC therapy than MSC5/6. CONCLUSION: This study showed that MSC therapy is efficient to retard CRF progression and might be more effective when administered during less severe stages of CRF.
Asunto(s)
Fallo Renal Crónico/terapia , Trasplante de Células Madre Mesenquimatosas/métodos , Actinas/biosíntesis , Actinas/genética , Animales , Proliferación Celular , Creatinina/metabolismo , Progresión de la Enfermedad , Femenino , Fibrosis/patología , Glomeruloesclerosis Focal y Segmentaria/patología , Glomeruloesclerosis Focal y Segmentaria/terapia , Riñón/patología , Fallo Renal Crónico/patología , Pruebas de Función Renal , Macrófagos/patología , Células Madre Mesenquimatosas , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Antígeno Nuclear de Célula en Proliferación/genética , Proteinuria/metabolismo , Ratas , Ratas WistarRESUMEN
Rapid and efficient growth is a major consideration and challenge for global mariculture. The differential growth rate of the sea cucumber, Apostichopus japonicus, has significantly hampered the total production of the industry. In the present study, forward and reverse suppression subtractive hybridization libraries were constructed and sequenced from a fast-growth group and a slow-growth group of the sea cucumber. A total of 142 differentially expressed sequence tags (ESTs) with insertions longer than 150 bp were identified and further analyzed. Fifty-seven of these ESTs (approximately 40%) were functionally annotated for cell structure, energy metabolism, immunity response, and growth factor categories. Six candidate genes, arginine kinase, cytochrome c oxidase subunit I, HSP70, ß-actin, ferritin, and the ADP-ribosylation factor, were further validated by quantitative PCR. Significant differences were found between the fast- and slow-growth groups (P < 0.05) for the expression levels of arginine kinase, cytochrome c oxidase, HSP70, the ADP-ribosylation factor, and ß-actin. However, no significant difference was observed for ferritin. Our results provide promising candidate gene markers for practical size screening, and also further promote marker-assisted selective breeding of this species.
Asunto(s)
Etiquetas de Secuencia Expresada , Regulación del Desarrollo de la Expresión Génica , Stichopus/genética , Factores de Ribosilacion-ADP/biosíntesis , Actinas/biosíntesis , Animales , Arginina Quinasa/biosíntesis , Complejo IV de Transporte de Electrones/biosíntesis , Proteínas HSP70 de Choque Térmico/biosíntesis , Stichopus/crecimiento & desarrolloRESUMEN
Seizure susceptibility appears to be greater in males than females during the early developmental stages of the brain when the gamma-aminobutyric acid (GABA), acting through its GABA-A receptor, predominantly produces neuronal depolarization. GABA-mediated excitation has been observed when the NKCC1 (chloride importer) expression level is higher than KCC2 (chloride exporter). In this study, the relative protein expression of NKCC1 and KCC2 over ß-actin was evaluated in the hippocampus and entorhinal cortex of male and female rats during postnatal days (PND) 1, 3, 5, 7, 9, 11, 13 and 15 using Western blotting assays. For both cerebral regions in the females, the NKCC1/ß-actin expression ratio was constant during all evaluated ages, whereas the KCC2/ß-actin expression ratio increased gradually until reaching a maximal level at PND9 that was nearly three- and ten-fold higher in the hippocampus and entorhinal cortex, respectively, compared with the initial level. In males, the NKCC1/ß-actin expression ratio was constant during the first week, peaking almost three-fold higher than the initial level at PND9 in the hippocampus and at PND11 in the entorhinal cortex and then returning to the initial values at PND13, whereas the KCC2/ß-actin expression ratio increased gradually to reach a maximal and steady level at PND5, which were nearly two- and four-fold higher in the hippocampus and entorhinal cortex, respectively, compared with the intial level. In conclusion, the NKCC1/ß-actin and KCC2/ß-actin expression ratios displayed a specific expression profile for each gender and cerebral region, which could be related with the differences in seizure susceptibility observed between genders.
Asunto(s)
Corteza Entorrinal/metabolismo , Hipocampo/metabolismo , Caracteres Sexuales , Miembro 2 de la Familia de Transportadores de Soluto 12/biosíntesis , Simportadores/biosíntesis , Actinas/biosíntesis , Animales , Animales Recién Nacidos/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratas , Factores de Tiempo , Cotransportadores de K ClRESUMEN
Helminth ß-tubulins are the targets of benzimidazole (BZM) carbamate compounds. The specificity of the interactions between such compounds and their in vivo targets depends on the presence of specific amino acid residues in the target molecules. To discover new and effective anthelmintic drugs, we used a medicinal chemistry approach to synthesize a series of BZM derivatives that exploited the BZM moiety as a template. We have previously found that one compound, 2-(trifluoromethyl)-1H-benzimidazole (RCB20), has better in vitro and in vivo activity than albendazole sulfoxide (ABZSO). In the present study, the effect of RCB20 and ABZSO treatment on expression of Taenia crassiceps cysticerci cytoskeletal proteins such as actin, myosin II, and tubulin isoforms was examined. The effects of RCB20 and ABZSO after 11 days treatment of the parasites was evaluated by light, confocal, and electron microscopy, and by immunochemistry and immunohistochemistry. The RCB20-induced effects were more rapid than the ABZSO-induced effects on the parasites. In the RCB20-treated parasites, we observed gross-structural damage at the whole parasite level, particularly in the inner tissues and flame cells. Changes in the expression patterns of the cytoskeletal proteins, as assessed by immunohistochemistry and immunoblotting, revealed that the most important drug-induced effect on the parasites was a reduction in the expression level of tyrosinated α-tubulins. Our research findings suggest that RCB20 treatment affected posttranslational modification of parasite α-tubulin molecules, which involved removal of the α-tubulin carboxy-terminal tyrosine.
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Antihelmínticos/farmacología , Bencimidazoles/farmacología , Expresión Génica/efectos de los fármacos , Taenia/efectos de los fármacos , Tubulina (Proteína)/biosíntesis , Actinas/biosíntesis , Albendazol/análogos & derivados , Albendazol/farmacología , Animales , Cysticercus/anatomía & histología , Cysticercus/efectos de los fármacos , Inmunoquímica , Microscopía , Miosina Tipo II/biosíntesis , Taenia/anatomía & histologíaRESUMEN
BACKGROUND: Fibroblasts play a critical role during wound healing and chronic inflammation through the synthesis and assembly of extracellular matrix (ECM) molecules. These responses may be modulated by soluble cytokines and growth factors present in tissues. In the present study, we evaluate whether transforming growth factor-ß1 (TGF-ß1) and tumor necrosis factor-α (TNF-α) modulate myofibroblastic differentiation and the production of ECM components. METHODS: Primary cultures of human gingival fibroblasts (HGFs) were stimulated with recombinant TGF-ß1 and TNF-α. Protein levels of α-smooth muscle actin (α-SMA), type I collagen, heat shock protein-47 (HSP-47), fibronectin (FN), ED-A-FN, and periostin and activation of the Smad pathway were evaluated through Western blot analysis. α-SMA and actin fibers were identified by immunofluorescence. TGF-ß1, TNF-α, and α-SMA were identified by immunohistochemistry in biopsies of inflamed human gingival tissues. TGF-ß1 activity was evaluated using a plasminogen activator inhibitor-1 (PAI-1) reporter transfected in HGFs. RESULTS: TGF-ß1 stimulated the differentiation of myofibroblasts as evidenced by strong expression of α-SMA and ED-A-FN. Moreover, TGF-ß1 induced the production of type I collagen, HSP-47, FN, and periostin. Costimulation with TNF-α and TGF-ß1 significantly reduced the expression of all the above-mentioned proteins. TNF-α also inhibited the activation of the Smad2/3 pathway and the activity of the PAI-1 reporter. CONCLUSIONS: TNF-α inhibits several cell responses induced by TGF-ß1, including the differentiation of myofibroblasts, the activation of the Smad signaling pathway, and the production of key molecules involved in tissue repair, such as type I collagen, FN, and periostin. The interaction between cytokines may explain the delayed tissue repair observed in chronic inflammation of gingival tissues.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Matriz Extracelular/efectos de los fármacos , Encía/metabolismo , Miofibroblastos/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Actinas/biosíntesis , Adulto , Moléculas de Adhesión Celular/biosíntesis , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Periodontitis Crónica/metabolismo , Colágeno Tipo I/biosíntesis , Femenino , Fibronectinas/biosíntesis , Encía/citología , Humanos , Masculino , Persona de Mediana Edad , Inhibidor 1 de Activador Plasminogénico/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Smad/metabolismoRESUMEN
A novel application of diffuse reflectance and fluorescence spectroscopy in the assessment of liver fibrosis is here reported. To induce different stages of liver fibrosis, a sufficient number of male Wistar rats were differentially exposed to chronic administration with carbon tetrachloride. Then, diffuse reflectance and fluorescence spectra were in vivo measured from the liver surface of each animal by a minimal invasive laparoscopic procedure. The liver fibrosis degree was conventionally determined by means of histological examination using the Mason's Trichrome stain, accompanied by hepatic expression of α-sma, and evaluation of the ALT/AST serum levels. The liver from rats exhibiting higher grades of fibrosis showed a significant increase in diffuse reflectance and fluorescence intensity when compared with control animals. At 365 nm, the diffuse reflectance spectrum exhibited an increase of 4 and 3-fold in mild and advanced fibrotic rats, respectively, when compared to the control group. Similarly, the fluorescence emission at 493 nm was 2-fold higher in fibrotic animals than in controls. By using fluorescence intensity, discrimination algorithms indicated 73% sensitivity and 94% specificity for recognition of hepatic fibrosis, while for diffuse reflectance, these values increased up to 85% and 100%, respectively. Taking into consideration there is a special need for developing new diagnostic approaches focused on detecting different stages of liver fibrosis with minimal invasiveness, these results suggest that diffuse reflectance and fluorescence spectroscopy could be worthy of further exploration in patients with liver disease.
Asunto(s)
Cirrosis Hepática/patología , Hígado/patología , Espectrometría de Fluorescencia/métodos , Actinas/biosíntesis , Animales , Tetracloruro de Carbono/toxicidad , Laparoscopía/métodos , Cirrosis Hepática/inducido químicamente , Pruebas de Función Hepática , Masculino , Ratas , Ratas WistarRESUMEN
BACKGROUND: The aim of this study was to investigate whether mutations in the genes H-ras and K-ras were related to the mechanism of invasion as a result of the immunoexpression of H-Ras, Ki-67, alpha-smooth muscle actin (SMA) and vascular endothelial growth factor (VEGF) during 4-nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinogenesis. METHODS: Male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution through their drinking water for 4, 12 and 20 weeks. Ten animals were used as negative control. RESULTS: Although no histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure, Ki-67 was overexpresssed in the 'normal' oral epithelium. In pre-neoplastic lesions at 12 weeks following carcinogen exposure, the levels of Ki-67 were increased (P < 0.05) when compared to negative control. Ki-67, alpha-SMA and VEGF were also overexpressed in squamous cell carcinomas induced after 20 weeks of treatment with 4NQO. No significant statistical differences (P > 0.05) were found in H-ras protein expression for all experimental periods evaluated that corresponded to normal oral mucosa, hyperplasia, dysplasia and squamous cell carcinomas. In the same way, no mutations in H-ras or K-ras genes were found. CONCLUSIONS: Our results support the idea that expression of Ki-67 plays a crucial role during malignant transformation being closely related to neoplastic conversion of the oral mucosa cells. However, it seems that mutations in the ras genes are not involved to experimental tongue carcinogenesis induced by 4NQO.
Asunto(s)
Genes ras/genética , Mutación , Invasividad Neoplásica/genética , Neoplasias Experimentales/genética , Neoplasias de la Lengua/genética , 4-Nitroquinolina-1-Óxido , Actinas/biosíntesis , Animales , Transformación Celular Neoplásica/metabolismo , Antígeno Ki-67/biosíntesis , Masculino , Neoplasias Experimentales/metabolismo , Ratas , Ratas Wistar , Neoplasias de la Lengua/inducido químicamente , Neoplasias de la Lengua/metabolismo , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Proteínas ras/biosíntesisRESUMEN
Constitutive overexpression of Cyp6g1 and Cyp6a2 genes in DDT-resistant line Oregon-flare of the Drosophila melanogaster wing spot test (SMART) has been reported. Cyp6g1 and Cyp6a2 expression levels were compared against the ß-actin gene in the standard (ST) and high bioactivation (HB) crosses of the Somatic Mutation and Recombination test (SMART) treated with sulforaphane or phenobarbital as the control inductor. The CYP450s' enzymatic activity was determined by overall NADH consumption. The expression levels of both genes and the CYP450s activity was higher in the HB cross. The Cyp6g1 levels were higher than those of Cyp6a2 in both crosses, but lower than the expression of ß-actin. Sulforaphane decreased Cyp6g1 in the HB cross and increased it in the ST cross; Cyp6a2 expression was inhibited in the ST cross. Sulforaphane resulted mutagenic in the ST cross, which could be related to the inhibition of Cyp6a2. Phenobarbital did not modify the Cyp6g1 levels but increased the Cyp6a2 and CYP450s basal activity. Although the transcript levels were always higher in the HB cross than in the ST, the expression of Cyp6a2 and Cyp6g1 was not constitutive and was independent one from the other. Sulforaphane modulated both genes in a differential way in each cross and, in contrast to its putative protective effect, it resulted to be mutagenic.
Asunto(s)
Anticarcinógenos/farmacología , Sistema Enzimático del Citocromo P-450/biosíntesis , Proteínas de Drosophila/biosíntesis , Mutágenos , Tiocianatos/farmacología , Alas de Animales/anatomía & histología , Actinas/biosíntesis , Actinas/genética , Animales , Anticarcinógenos/toxicidad , Cruzamientos Genéticos , Sistema Enzimático del Citocromo P-450/genética , Familia 6 del Citocromo P450 , Proteínas de Drosophila/genética , Drosophila melanogaster , Vectores Genéticos , Hipnóticos y Sedantes/toxicidad , Isotiocianatos , Larva/metabolismo , Pruebas de Mutagenicidad , NAD/metabolismo , Oligonucleótidos/síntesis química , Oligonucleótidos/genética , Fenobarbital/toxicidad , Recombinación Genética/genética , Sulfóxidos , Tiocianatos/toxicidadRESUMEN
This study describes increased sarcolemmal permeability and myofilamentar damage that occur together with lipid peroxidation and protein nitration in the myocardium in severe sepsis induced by cecal ligation and puncture. Male C57BL/6 mice were submitted to moderate and severe septic injury and sham operation. Using light and laser confocal microscopy, diffuse foci of myocytolysis associated with focal disruption of the actin/myosin contractile apparatus could be seen in hearts with severe septic injury. The myocardial expressions of the sarcomeric proteins myosin and actin were downregulated by both severe and moderate injuries. The detection of albumin staining in the cytoplasm of myocytes to evaluate sarcolemmal permeability provided evidence of severe and mild injury of the plasma membrane in hearts with severe and moderate septic injury, respectively. The administration of a superoxide scavenger caused marked reduction of sarcolemmal permeability, indicating the involvement of free radicals in its genesis. On electron microscopy, these changes were seen to correspond to spread blocks of a few myocytes with fragmentation and dissolution of myofibrils, intracellular edema, and, occasionally, rupture of the sarcolemma. In addition, oxidative damage to lipids, using anti-4-hydroxynonenal, an indicator of oxidative stress and disruption of plasma membrane lipids, and to proteins, using antinitrotyrosine, a stable biomarker of peroxynitrite-mediated protein nitration, was demonstrated. These findings make plausible the hypothesis that increased sarcolemmal permeability might be a primary event in myocardial injury in severe sepsis possibly due to oxidative damage to lipids and proteins that could precede phenotypic changes that characterize a septic cardiomyopathy.
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Cardiomiopatías/fisiopatología , Sarcolema/fisiología , Sepsis/fisiopatología , Actinas/biosíntesis , Aldehídos/metabolismo , Animales , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Ciego/lesiones , Regulación hacia Abajo , Ligadura , Peroxidación de Lípido , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Miocardio/metabolismo , Miocardio/patología , Miosinas/biosíntesis , Permeabilidad/efectos de los fármacos , Proteínas/metabolismo , Punciones , Sepsis/metabolismoRESUMEN
The consumption of drinking water rich in fluoride has toxic effects on the central nervous system. In cell biology research, fluoride is currently used as a phosphatase inhibitor. The aim of the present study was to evaluate the effect of fluoride on different physiological processes in GH4C1 pituitary tumour cells. We used a range of different fluoride concentrations, from levels below normal human serum concentrations (0.23 and 1.2 micromol/L) to those observed in chronically exposed persons (10.7 micromol/L) and above (107 and 1072 micromol/L). Treatment of 10.7 micromol/L fluoride resulted in a discrete induction of DNA synthesis, without a change in cell number. Cell migration, a behaviour stimulated by growth factors, was increased in cells treated with 2.4 micromol/L. At this fluoride concentration, changes in phosphorylation status of both cytoskeletal and cytosolic protein fractions, as well as in actin cytoskeletal arrangements were observed. The GH4C1 fluoride treated cells had significantly less cellular protein than control cells, suggesting an effect of fluoride on hormone secretion and protein synthesis in this endocrine cell. The bioreduction of MTT was significantly increased with a wide range of fluoride concentrations. With the highest fluoride concentration, 1072 micromol/L, all of the analysed parameters were significantly reduced, suggesting that this dose is highly toxic in GH4C1 cells. Our results show that biologically relevant concentrations of fluoride are capable of increasing cell migration in tumour cells, suggesting that exposure to fluoride could stimulate tumour invasion.
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Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Fluoruros/farmacología , Actinas/biosíntesis , Actinas/genética , Animales , Western Blotting , Adhesión Celular/efectos de los fármacos , Recuento de Células , Línea Celular Tumoral , Colorantes , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , ADN de Neoplasias/biosíntesis , Relación Dosis-Respuesta a Droga , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/metabolismo , Fosforilación , Prolactina/metabolismo , Ratas , Sales de Tetrazolio , TiazolesRESUMEN
BACKGROUND/AIMS: Podocytes are critical in maintaining the filtration barrier of the glomerulus and are dependent on the slit diaphragm. We hypothesized that disturbances of podocyte biology contribute to proteinuria in women with preeclampsia (PE). METHODS: A human podocyte cell line was stimulated with serum from women with PE (patients) and healthy pregnant women (controls); the main changes in 3 important podocyte proteins: podocin, CD2AP and actin were established by immunofluorescence and Western blot; we also searched for changes in cell plasticity by measuring the resistance of cultured podocytes. RESULTS: Different distributions of CD2AP, podocin and actin were observed in the podocytes stimulated with patient sera compared to podocytes stimulated with control sera. We also found that the mean resistance value of podocytes cultured with serum from women with PE was significantly lower than podocytes cultured with serum from controls. There was no difference in the protein expression level of podocin and CD2AP between patients and controls. CONCLUSIONS: We present evidence that there are differences in podocytes when stimulated with sera from women with PE compared to those stimulated with healthy pregnancy sera. This is the first time that podocyte alterations have been directly related to PE; these descriptive findings could be considered as an interesting beginning for further studies relating podocytes and PE.
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Fenómenos Fisiológicos Sanguíneos , Podocitos/fisiología , Preeclampsia , Actinas/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Células Cultivadas , Proteínas del Citoesqueleto/biosíntesis , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/biosíntesis , Podocitos/metabolismo , EmbarazoRESUMEN
Transforming growth factor-beta 1 (TGF-beta1) has been related to induce the expression of alpha-smooth muscle actin (alpha-SMA) in fibroblasts during repair. Because pulpal fibroblasts seem to be somewhat different from other fibroblasts, the present study investigated in vitro whether TGF-beta1 enhances the expression of alpha-SMA in human pulpal fibroblasts. TGF-beta1 was added in doses between 5-10 ng/mL to cultures of both dental pulp and gingival human fibroblasts. The expression of alpha-SMA was analyzed by immunofluorescence and Western blotting, whereas the ultrastructure was evaluated by electron microscopy. In addition, the expression of tenascin, osteonectin, and vimentin was also investigated. Both cell types were immunoreactive for alpha-SMA even without TGF-beta1. When TGF-beta1 was added to cell cultures, the expression of alpha-SMA increased dramatically in pulpal fibroblasts, independent of the concentration used. It was confirmed by the Western blotting analysis. Ultrastructure revealed myofilaments and indented nuclei in both fibroblasts treated with TGF-beta1. Tenascin and osteonectin were only immunolabeled in pulpal fibroblasts treated or not with TGF-beta1. Both fibroblast types were positive for vimentin. The present findings showed that TGF-beta1 up-regulated the expression of alpha-SMA, thus inducing pulpal fibroblasts to acquire the myofibroblast phenotype.
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Actinas/biosíntesis , Pulpa Dental/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Western Blotting , Células Cultivadas , Pulpa Dental/citología , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Encía/citología , Humanos , Microscopía Electrónica de Transmisión , Mioblastos del Músculo Liso/metabolismo , Osteonectina/biosíntesis , Fenotipo , Tenascina/biosíntesis , Factor de Crecimiento Transformador beta1/fisiología , Vimentina/biosíntesisRESUMEN
Ether à go-go (EAG) potassium channels possess oncogenic properties and have gained great interest as research tools for cancer detection and therapy. Besides, EAG electrophysiological properties are regulated through the cell cycle and determined by cytoskeletal interactions. Thus, because of the pivotal role of extracellular matrix (ECM) and cytoskeleton in cancer progression, we studied the effect of ECM components on adhesion, viability, actin organization and EAG currents in wild-type CHO cells (CHO-wt) and cells expressing human EAG channels (CHO-hEAG). At short incubation times, adhesion and viability of CHO-hEAG cells grown on collagen, heparin or poly-lysine were lower than CHO-wt cells, however, only CHO-hEAG sustained growing under total serum starvation. CHO-hEAG cells grown on poly-lysine did not organize their cytoskeleton but when grown on collagen or fibronectin displayed lamellipodia and stress fibers, respectively. Interestingly, EAG expressing cells displayed special actin structures suggesting a dynamic actin cytoskeleton, such structures were not exhibited by wild-type cells. EAG current density was significantly lower in cells grown on collagen at short incubation times. Finally, we studied potential associations between hEAG channels and integrins or actin filaments by confocal microscopy. No association between beta1-integrins and hEAG channels was found, however, a very strong co-localization was observed between hEAG channels and actin filaments, supported by immunoblot experiments in which hEAG channels were found in the insoluble fraction (associated to cytoskeleton). Our results suggest ECM components as potential modulators of oncogenic human-EAG expressing cells and emphasize the relationship between potassium channels, cytoskeleton, ECM and cancer.
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Actinas/biosíntesis , Adhesión Celular/fisiología , Citoesqueleto/metabolismo , Canales de Potasio Éter-A-Go-Go/biosíntesis , Matriz Extracelular/fisiología , Animales , Células CHO , Proliferación Celular , Supervivencia Celular/fisiología , Cricetinae , Cricetulus , Electrofisiología , Canales de Potasio Éter-A-Go-Go/genética , Técnica del Anticuerpo Fluorescente , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Humanos , Inmunohistoquímica , Cadenas beta de Integrinas/biosíntesis , Microscopía Confocal , Técnicas de Placa-Clamp , TransfecciónRESUMEN
BACKGROUND/AIM: Previously, we have shown that myofibroblasts, the main cell type associated with interstitial fibrosis, may be implicated with the gingival overgrowth observed in hereditary gingival fibromatosis (HGF) patients. The goal of this study was to determine whether transforming growth factor-beta1 (TGF-beta1) stimulates myofibroblast generation in gingival fibroblast cultures. Moreover, we analysed how interferon-gamma (IFN-gamma) interferes in this process. MATERIAL AND METHODS: Fibroblast cultures from normal gingiva and myofibroblast cells from HGF were included in this study. To determine the effects of TGF-beta1 and IFN-gamma stimulation in these cells, the expression of the specific myofibroblast marker smooth muscle isoform of alpha-actin (alpha-SMA) was examined by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot and immunofluorescence. Enzyme-linked immunosorbent assay (ELISA) for type I collagen was performed to measure the myofibroblast activity. RESULTS: Our results demonstrated that TGF-beta1 promotes a dose- and time-dependent increase in the expression of alpha-SMA, whereas IFN-gamma blocks it and markedly prevents the fibroblast-myofibroblast switch induced by TGF-beta1 on normal gingiva cultures. IFN-gamma altered HGF myofibroblasts metabolism with a decrease of both alpha-SMA and type I collagen expression. Additionally, IFN-gamma treatment stimulated SMAD7 expression and inhibited connective tissue growth factor, which has been considered a key molecule to promote the transdifferentiation of myofibroblasts via TGF-beta1 activation. CONCLUSIONS: These findings demonstrate that TGF-beta1 induces gingival fibroblast-myofibroblast transdifferentiation, whereas IFN-gamma blocks this process. More importantly, this study suggests that IFN-gamma may be clinically effective in attenuating excessive accumulation of extracellular matrix produced by myofibroblasts in HGF.