RESUMEN
OBJECTIVES: The aim of the study was to assess the interest to combine cytological examination and human papillomavirus (HPV) typing of anal and cervical Papanicolaou (Pap) smears of HIV-infected patients on combination antiretroviral therapy (cART), to evaluate whether differences in prevalence exist between anal and cervical squamous intraepithelial lesions in patients with high-risk oncogenic HPV infection. METHODS: Anal and/or cervical Pap smears were obtained by anoscopy and/or colposcopy in 238 subjects recruited consecutively in 2015: anal smears were obtained from 48 male and female patients [42 men; 35 men who have sex with men (MSM)] and cervical smears from 190 female patients. Cytological Bethesda classification was coupled with HPV typing. HPV typing was performed, on the same smears, using the Xpert® HPV Assay, which detects only high-risk HPV (hrHPV), and the Anyplex® II HPV28 Detection assay, which detects hrHPV and low-risk (lr) HPV. RESULTS: Our data showed clear-cut differences between the anal and cervical samples. Compared with the cervical samples, the anal samples exhibited (1) more numerous cytological lesions, which were histologically proven; (2) a higher hrHPV infection prevalence; (3) a higher prevalence of multiple hrHPV coinfections whatever HPV typing kit was used; (4) a predominance of HPV16 and HPV18/45 types. Overall, there was an almost perfect agreement between the two HPV typing assays (absolute agreement = 90.3%). CONCLUSIONS: Co-testing consisting of cytology and HPV typing is a useful screening tool in the HIV-infected population on cART. It allows detection of prevalence differences between anal and cervical HPV-related lesions. As recently recommended, anal examination should be regularly performed especially in HIV-infected MSM but also in HIV-infected women with genital hrHPV lesions.
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Antirretrovirales/uso terapéutico , Coinfección/diagnóstico , Técnicas Citológicas/métodos , Infecciones por VIH/complicaciones , Técnicas de Diagnóstico Molecular/métodos , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/diagnóstico , Adulto , Anciano , Canal Anal/patología , Canal Anal/virología , Cuello del Útero/patología , Cuello del Útero/virología , Coinfección/virología , Femenino , Genotipo , Infecciones por VIH/tratamiento farmacológico , Humanos , Masculino , Tamizaje Masivo/métodos , Persona de Mediana Edad , Papillomaviridae/clasificación , Papillomaviridae/genética , Infecciones por Papillomavirus/virología , Prevalencia , Estudios Prospectivos , Adulto JovenRESUMEN
BACKGROUND AND PURPOSE: Human internal mammary arteries (IMA) and saphenous veins (SV) are frequently used for coronary artery bypass graft surgery. Intra- and postoperatively, the bypass grafts are exposed to inflammatory conditions, under which there is a striking increase in the synthesis of prostaglandin E(2) (PGE(2) ). In this context, the physiological response of these vascular grafts to PGE(2) is highly relevant. The aim of this study was thus to characterize the PGE(2) receptor subtypes (EP(1) , EP(2) , EP(3) or EP(4) ) involved in modulation of the vascular tone in these two vessels. EXPERIMENTAL APPROACH: Rings of IMA and SV were prepared from 48 patients. The rings were mounted in organ baths for isometric recording of tension, and a pharmacological study was performed, together with associated reverse transcriptase PCR and immunohistochemistry experiments. KEY RESULTS: PGE(2) induced contractions of IMA (E(max) = 1.43 ± 0.20 g; pEC(50) = 7.50 ± 0.10); contractions were also observed with the EP(3) receptor agonists, sulprostone, 17-phenyl-PGE(2) , misoprostol or ONO-AE-248. In contrast, PGE(2) induced relaxation of the precontracted SV (E(max) =-0.22 ± 0.02 g; pEC(50) = 7.14 ± 0.09), as did the EP(4) receptor agonist, ONO-AE1-329. These results were confirmed by the use of selective EP receptor antagonists (GW627368X, L-826266, ONO-8713, SC-51322) and by molecular biology and immunostaining. CONCLUSIONS AND IMPLICATIONS: PGE(2) induced potent and opposite effects on the human vascular segments used for grafting, namely vasoconstriction of the IMA and vasodilatation of the SV via EP(3) and EP(4) receptors respectively. These observations suggest that EP(3) and EP(4) receptors could constitute therapeutic targets to increase vascular graft patency.
Asunto(s)
Dinoprostona/metabolismo , Arterias Mamarias/efectos de los fármacos , Arterias Mamarias/trasplante , Vena Safena/efectos de los fármacos , Vena Safena/trasplante , Acrilamidas/farmacología , Anciano , Puente de Arteria Coronaria/métodos , Dinoprostona/análogos & derivados , Dinoprostona/farmacología , Femenino , Humanos , Isoindoles/farmacología , Masculino , Arterias Mamarias/metabolismo , Éteres Metílicos/farmacología , Misoprostol/farmacología , Naftalenos/farmacología , Receptores de Prostaglandina E/agonistas , Receptores de Prostaglandina E/antagonistas & inhibidores , Receptores de Prostaglandina E/metabolismo , Vena Safena/metabolismo , Sulfonamidas/farmacología , Injerto Vascular/métodosRESUMEN
BACKGROUND AND PURPOSE: PGE2 has been shown to induce relaxations in precontracted human pulmonary venous preparations, while in pulmonary arteries this response was not observed. We investigated and characterized the prostanoid receptors which are activated by PGE2 in the human pulmonary veins. EXPERIMENTAL APPROACH: Human pulmonary arteries and veins were cut as rings and set up in organ baths in presence of a TP antagonist. A pharmacological study was performed using selective EP1-4 ligands. The cellular localization of the EP4 receptors by immunohistochemistry and their corresponding transcripts were also investigated in these vessels. KEY RESULTS: PGE2 and the EP4 agonists (L-902688, ONO-AE1-329) induced potent vasodilatation of the human pulmonary vein, pEC50 values: <7.22+/-0.20, 8.06+/-0.12 and 7.80+/-0.09, respectively. These relaxations were inhibited by the EP(4) antagonist GW627368X and not modified in presence of the DP antagonist L-877499. Higher concentrations (>or=1 microM) of the EP2 agonist ONO-AE1-259 induced relaxations of the veins. The EP4 agonists had no effect on the precontracted arteries. Finally, the EP(1) antagonists ONO-8713 and SC-51322 potentiated the relaxation of the veins induced by PGE2. EP4 and EP1 receptors were detected by immunohistochemistry in the veins but not in the arteries. EP4 mRNA accumulation was also greater in the veins when compared with the arterial preparations. CONCLUSIONS AND IMPLICATIONS: Of the 4 EP receptor subtypes, smooth muscle cells in the human pulmonary vein express the EP4 and EP1 receptor subtypes. The relaxations induced by PGE2 in this vessel result from the activation of the EP4 receptor.
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Dinoprostona/farmacología , Venas Pulmonares/metabolismo , Receptores de Prostaglandina E/efectos de los fármacos , Receptores de Prostaglandina E/metabolismo , Anciano , Femenino , Humanos , Inmunohistoquímica , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/metabolismo , Venas Pulmonares/efectos de los fármacos , Subtipo EP1 de Receptores de Prostaglandina E , Subtipo EP4 de Receptores de Prostaglandina E , Vasodilatación/efectos de los fármacosRESUMEN
The cellular localization of prostaglandin E2 receptors (EP) and their corresponding transcripts were investigated in human gastric and vascular tissues. A strong staining of the EP3 receptor on the gastric glands, mucous cells, media of the mammary and pulmonary arteries was observed by immunohistochemistry. We identified a new mRNA splice variant of the EP3 gene in human gastric fundic mucosa, mammary artery and pulmonary vessels. This EP3-Ic transcript contains exons 1, 2, 3, 5 and 6 of the EP3 gene and should be translated in the EP3-I isoform. In addition, the EP3-Ib, EP3-II, EP3-III, EP3-IV and EP3-e mRNAs were detected in these tissues.
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Isoformas de Proteínas/genética , ARN Mensajero/genética , Receptores de Prostaglandina E/genética , Secuencia de Bases , Femenino , Fundus Gástrico/metabolismo , Mucosa Gástrica/metabolismo , Humanos , Inmunohistoquímica , Masculino , Arterias Mamarias/metabolismo , Persona de Mediana Edad , Datos de Secuencia Molecular , Arteria Pulmonar/metabolismo , Venas Pulmonares/metabolismo , Subtipo EP3 de Receptores de Prostaglandina E , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The recently identified subfamily of WNK protein kinases is characterized by a unique sequence variation in the catalytic domain and four related human WNK genes were identified. Here, we describe the cloning and functional analysis of the human family member WNK2. We show that the depletion of endogenous WNK2 expression by RNA interference in human cervical HeLa cancer cells led to the activation of the extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinases but, in contrast to the depletion of WNK1, had no effect on ERK5. Furthermore, expression of a kinase-dead WNK2-K207M mutant also activated ERK1/2 suggesting that WNK2 catalytic activity is required. Depletion of WNK2 expression increased G1/S progression and potentiated the cellular response to low epidermal growth factor concentrations. The molecular mechanism of ERK1/2 activation in WNK2-depleted cells lies downstream of the Raf kinases and involves MEK1 phosphorylation at serine 298 in both HeLa and HT29 colon cancer cells. This modification is linked to the upregulation of MEK1 activity toward ERK1/2. Together, these results provide evidence that WNK2 is involved in the modulation of growth factor-induced cancer cell proliferation through the MEK1/ERK1/2 pathway. The data identify WNK2 as a candidate tumor suppressor gene and suggest a coordinated activity of WNK kinases in the regulation of cell proliferation.
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División Celular/efectos de los fármacos , MAP Quinasa Quinasa 1/metabolismo , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Empalme Alternativo , Clonación Molecular , Replicación del ADN , Activación Enzimática , Células HeLa , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/deficiencia , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND AND AIMS: Leptin, the product of the ob gene, has been suggested to increase the risk of colon cancer. However, we have shown that although leptin stimulates epithelial cell proliferation it reduces the development of carcinogen induced preneoplastic lesions in the rat colon. Here, we explored the effect of leptin in vitro on proliferation of human colon cancer cells, and in vivo on the growth of HT-29 xenografts in nude mice and the development of intestinal tumours in Apc(Min/+) mice. METHODS: Proliferation of HT-29, LoVo, Caco2, and SW 480 cells was assessed in the absence or presence of leptin (20-500 ng/ml) by 3H-thymidine incorporation and cell count. Leptin (800 microg/kg/day) or its vehicle was delivered for four weeks to nude mice, inoculated with HT-29 cells on day 0, and for six weeks to Apc(Min/+) mice. RESULTS: Leptin dose dependently stimulated cell DNA synthesis and growth in all cell lines. In nude mice, leptin caused a 4.3-fold increase in plasma leptin levels compared with pair fed controls. This hyperleptinaemia, despite leptin receptor expression in tumours, did not induce significant variation in tumour volume or weight. Tumour Ki-67 index was even inhibited. In leptin treated Apc(Min/+) mice, a 2.4-fold increase in plasma leptin levels did not modify the number, size, or distribution of intestinal adenomas compared with pair fed controls. CONCLUSIONS: Leptin acts as a growth factor on colon cancer cells in vitro but does not promote tumour growth in vivo in the two models tested. These findings do not support a pivotal role for hyperleptinaemia in intestinal carcinogenesis.
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Neoplasias del Colon/patología , Genes APC , Leptina/fisiología , Adenoma/genética , Adenoma/patología , Adenoma/fisiopatología , Animales , Apoptosis/fisiología , División Celular/fisiología , Línea Celular Tumoral , Colon/patología , Colon/fisiopatología , Neoplasias del Colon/genética , Neoplasias del Colon/fisiopatología , ADN de Neoplasias/biosíntesis , Humanos , Mucosa Intestinal/patología , Mucosa Intestinal/fisiopatología , Leptina/sangre , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones DesnudosRESUMEN
To analyze the implication of PTEN in the control of tumor cell invasiveness, the canine kidney epithelial cell lines MDCKras-f and MDCKts-src, expressing activated Ras and a temperature-sensitive v-Src tyrosine kinase, respectively, were transfected with PTEN expression vectors. Likewise, the human PTEN-defective glioblastoma cell lines U87MG and U373MG, the melanoma cell line FM-45, and the prostate carcinoma cell line PC-3 were transfected. We demonstrate that ectopic expression of wild-type PTEN in MDCKts-src cells, but not expression of PTEN mutants deficient in either the lipid or both the lipid and protein phosphatase activities, reverted the morphological transformation, induced cell-cell aggregation, and suppressed the invasive phenotype in an E-cadherin-dependent manner. In contrast, overexpression of wild-type PTEN did not counteract Ras-induced invasiveness of MDCKras-f cells expressing low levels of E-cadherin. PTEN effects were not associated with marked changes in accumulation or phosphorylation levels of E-cadherin and associated catenins. Wild-type, but not mutant, PTEN also reverted the invasive phenotype of U87MG, U373MG, PC-3, and FM-45 cells. Interestingly, PTEN effects were mimicked by N-cadherin-neutralizing antibody in the glioblastoma cell lines. Our data confirm the differential activities of E- and N-cadherin on invasiveness and suggest that the lipid phosphatase activity of PTEN exerts a critical role in stabilizing junctional complexes and restraining invasiveness.
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Glioblastoma/fisiopatología , Uniones Intercelulares/fisiología , Melanoma/fisiopatología , Invasividad Neoplásica , Monoéster Fosfórico Hidrolasas/genética , Neoplasias de la Próstata/fisiopatología , Proteínas Supresoras de Tumor/genética , Animales , Línea Celular , Glioblastoma/patología , Humanos , Masculino , Melanoma/patología , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/fisiología , Neoplasias de la Próstata/patología , Proteínas Recombinantes/metabolismo , Transfección , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
Because the crucial role of angiogenesis has been demonstrated in tumor growth and metastasis, the present study was undertaken to characterize the relative expression of vascular endothelial growth factors VEGF (vascular endothelial growth factor), VEGF-B, VEGF-C, and their receptors KDR (kinase insert domain-containing receptor), FLT-1 (fms-like tyrosine kinase), and FLT-4 in human colonic cancers, in relation to the Astler-Coller pathological classification, and to prognosis. VEGF and VEGF-B gene expression was quantified by Northern blot in 72 tumor samples matched with control tissues. VEGF gene expression was 1.4 times higher in adenocarcinomas than in control tissues (p = 0.02), but did not increase further between Astler-Coller tumor stages A and D, and did not correlate with disease recurrence for patients at stages B2 or C. In adenomas, VEGF mRNA levels were not significantly different from those in the paired control colonic mucosa. The expression pattern of VEGF isoforms, mainly identified by RT-PCR (reverse-transcriptase-coupled polymerase chain reaction) as VEGF121 and VEGF165 and to a lesser extent VEGF189, was comparable in tumor and control tissues. VEGF-B mRNA levels were unchanged during the neoplastic progression of colonic mucosa. In contrast to KDR and FLT-4, the expression of VEGF-C and FLT-1 genes increased in some pathological tissues. These results provide evidence that the early and sustained increase in VEGF transcripts and the expression of multiple angiogenic factors and receptors contribute to the development of colon cancer, and thus constitute a putative target for anti-angiogenic drug therapy.
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Colon/metabolismo , Neoplasias del Colon/metabolismo , Factores de Crecimiento Endotelial/genética , Expresión Génica , Mucosa Intestinal/metabolismo , Linfocinas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factores de Crecimiento/genética , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD34/genética , Southern Blotting , Western Blotting , Femenino , Humanos , Masculino , Persona de Mediana Edad , ARN Mensajero/análisis , Receptores de Factores de Crecimiento Endotelial Vascular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
This study was designed to characterize platelet-activating factor receptor (PAF-R) expression and function in normal and cancerous human colonic epithelial cells. PAF-R gene transcripts were analyzed by reverse transcription-polymerase chain reaction and Southern blot, using three sets of primers corresponding either to the coding region of the human PAF-R sequence (polymerase chain reaction product: 682 base pairs (bp)) or to the leukocyte- and tissue-type transcripts of 166 and 252 bp, respectively. An elongated splice variant was identified in the 5'-untranslated region of the tissue-type PAF-R transcript (334 bp) in colonic epithelial crypts and tumors. In human colonic PCmsrc cells transformed by c-src oncogene, the hepatocyte growth factor (HGF)-dependent invasiveness of collagen gels was abolished by 0.1 microM PAF and restored by the PAF-R antagonists WEB2086 and SR27417. PAF blocked HGF-induced tyrosine phosphorylation of p125 focal adhesion kinase. The phosphatidylinositol 3'-kinase (PI3'-K) inhibitors wortmannin and LY294002 totally blocked the HGF-induced invasion. Similar effects were observed in ts-srcMDCK kidney epithelial cells transformed by a v-Src temperature-sensitive mutant: (i) PAF and wortmannin exerted additive inhibitory effects on Src-induced invasion and (ii) activated and dominant negative forms of p110alpha PI3'-K, respectively, amplified and abrogated the Src- and HGF-dependent invasiveness of parental and ts-srcMDCK cells. We also provided the first evidence for the contribution of rapamycin-insensitive, pertussis toxin-dependent G-protein pathways to the integration of the signals emerging from activated Met and PAF receptors. These results indicate that PI3'-K is a critical transducer of invasiveness and strongly suggest that PAF exerts a negative control on invasion by inhibiting this signaling pathway. A possible beneficial role of PAF analogs on tumor invasion is therefore proposed.
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Movimiento Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Genes src/genética , Factor de Crecimiento de Hepatocito/farmacología , Factor de Activación Plaquetaria/farmacología , Glicoproteínas de Membrana Plaquetaria/genética , Receptores de Superficie Celular , Receptores Acoplados a Proteínas G , Androstadienos/farmacología , Animales , Azepinas/farmacología , Moléculas de Adhesión Celular/metabolismo , Cromonas/farmacología , Neoplasias del Colon/metabolismo , Perros , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Proteínas de Unión al GTP/fisiología , Humanos , Mucosa Intestinal/metabolismo , Riñón/metabolismo , Morfolinas/farmacología , Toxina del Pertussis , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/fisiología , Tiazoles/farmacología , Transformación Genética/genética , Triazoles/farmacología , Células Tumorales Cultivadas , Factores de Virulencia de Bordetella/farmacología , WortmaninaRESUMEN
Molecular markers can improve staging and predict aggressive clinical behavior in esophageal cancer, thus helping to define appropriate therapeutic protocols and to identify patients who will benefit from surgery. We therefore characterized, by Northern blot and/or immunohistochemistry, the relative expression of three effectors involved in the invasion, angiogenesis, and dissemination of tumor cells in esophageal cancer versus nontumoral mucosae: (a) stromelysin-3 (ST3), a member of the metalloproteinase family; (b) basement membrane 40/secreted protein acidic and rich in cysteine (BM-40/SPARC), an extracellular matrix-associated protein involved in angiogenesis; and (c) the hepatocyte growth factor receptor MET, which triggers the scattering of epithelial cells. Results were analyzed in relation to clinicopathological parameters (cpTNE) including tumor size (T), lymph node status (N), periesophageal tissue invasion (E), disease recurrence, and overall survival. The ST3, BM-40/SPARC, and MET genes were found to be overexpressed in tumor samples compared to control mucosa. BM-40/SPARC and MET mRNA levels were not linked to any one of the cpTNE, indicating that this overexpression occurs at an early stage of neoplastic progression. In contrast, ST3 expression, identified by immunohistochemistry in fibroblastic cells surrounding neoplastic islets, correlated with tumor size and periesophageal tissue invasion. Of the 36 patients studied, those with high ST3 levels had shorter disease-free survival than those with low levels, but there was no relationship between the cpTNE and disease recurrence or survival. Our study demonstrates that ST3, BM-40/SPARC, and MET are involved in different steps of esophageal carcinogenesis and that ST3 overexpression is a marker of aggressive clinical behavior. We conclude that in esophageal cancer, ST3 might help to assess survival and the risk of recurrence after surgical resection.
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Adenocarcinoma/patología , Carcinoma de Células Escamosas/patología , Neoplasias Esofágicas/patología , Metaloendopeptidasas/biosíntesis , Osteonectina/biosíntesis , Proteínas Proto-Oncogénicas c-met/biosíntesis , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidad , Adenocarcinoma/cirugía , Adulto , Anciano , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/cirugía , Supervivencia sin Enfermedad , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/mortalidad , Neoplasias Esofágicas/cirugía , Esofagectomía , Regulación Neoplásica de la Expresión Génica , Humanos , Metástasis Linfática , Metaloproteinasa 11 de la Matriz , Metaloendopeptidasas/análisis , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Estadificación de Neoplasias , Osteonectina/análisis , Reacción en Cadena de la Polimerasa , Pronóstico , Proteínas Proto-Oncogénicas c-met/análisis , Tasa de Supervivencia , Factores de TiempoRESUMEN
INTRODUCTION: Angiogenesis activation plays a crucial role in tumoral growth and metastases dissemination. This review summarizes and analyzes current knowledge on molecular mechanisms related to angiogenesis and the prognostic value of its effectors. It also focuses on the therapeutical relevance of various drugs that might inhibit angiogenesic processes. CURRENT KNOWLEDGE AND KEY POINTS: Tumor angiogenesis involves complex interactions between tumoral, stromal, endothelial cells, fibroblasts and the extracellular matrix. Normal and malignant angiogenesis depends on the balance of proangiogenic and antiangiogenic factors. Endothelial cells are activated by growth factors, such as Vascular Endothelial Growth Factor (VEGF), and proliferate; they release proteases able to induce degradation of the basement membrane and extracellular matrix, and undergo migration and tubulogenesis. Angiostatin and endostatin are two powerful inhibitors of angiogenesis in experimental models. Assessment of intratumoral microvessel density and quantification of angiogenic factors, including VEGF, are of prognostic value in most cancers, particularly in breast cancer. However, the use of these prognosis markers in clinical practice is still controversial due to the lack of prospective studies and to technical limits inherent to the scoring and standardization of immunohistochemical methods. FUTURE PROSPECTS AND PROJECTS: Better understanding of the molecular basis of angiogenesis allows the development of new therapeutical strategies. Biochemical targets of antiangiogenic therapy are: the interaction between angiogenic factors and their receptors; the interaction of endothelial cells with the extracellular matrix; and intracellular signaling pathways. Angiogenesis inhibitors may not cause tumor regression, but inhibit cellular growth and produce "disease dormancy". Extensive phase I to III clinical trials involving antiangiogenesis therapy are in progress.
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Neoplasias/complicaciones , Neovascularización Patológica , Inductores de la Angiogénesis/fisiología , Angiostatinas , Biomarcadores de Tumor , Colágeno/fisiología , Modelos Animales de Enfermedad , Endostatinas , Factores de Crecimiento Endotelial/fisiología , Humanos , Linfocinas/fisiología , Biología Molecular , Neovascularización Patológica/diagnóstico , Neovascularización Patológica/etiología , Neovascularización Patológica/fisiopatología , Neovascularización Patológica/terapia , Fragmentos de Péptidos/fisiología , Plasminógeno/fisiología , Pronóstico , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
We have carried out a genetic analysis of Escherichia coli HlyB using in vitro(hydroxylamine) mutagenesis and regionally directed mutagenesis. From random mutagenesis, three mutants, temperature sensitive (Ts) for secretion, were isolated and the DNA sequenced: Gly10Arg close to the N-terminus, Gly408Asp in a highly conserved small periplasmic loop region PIV, and Pro624Leu in another highly conserved region, within the ATP-binding region. Despite the Ts character of the Gly10 substitution, a derivative of HlyB, in which the first 25 amino acids were replaced by 21 amino acids of the lambda Cro protein, was still active in secretion of HlyA. This indicates that this region of HlyB is dispensable for function. Interestingly, the Gly408Asp substitution was toxic at high temperature and this is the first reported example of a conditional lethal mutation in HlyB. We have isolated 4 additional mutations in PIV by directed mutagenesis, giving a total of 5 out of 12 residues substituted in this region, with 4 mutations rendering HlyB defective in secretion. The Pro624 mutation, close to the Walker B-site for ATP binding in the cytoplasmic domain is identical to a mutation in HisP that leads to uncoupling of ATP hydrolysis from the transport of histidine. The expression of a fully functional haemolysin translocation system comprising HlyC,A,B and D increases the sensitivity of E. coli to vancomycin 2.5-fold, compared with cells expressing HlyB and HlyD alone. Thus, active translocation of HlyA renders the cells hyperpermeable to the drug. Mutations in hlyB affecting secretion could be assigned to two classes: those that restore the level of vancomycin resistance to that of E. coli not secreting HlyA and those that still confer hypersensitivity to the drug in the presence of HlyA. We propose that mutations that promote vancomycin resistance will include mutations affecting initial recognition of the secretion signal and therefore activation of a functional transport channel. Mutations that do not alter HlyA-dependent vancomycin sensitivity may, in contrast, affect later steps in the transport process.
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Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfatasas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos/genética , Proteínas Hemolisinas/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Ácido Aspártico/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Farmacorresistencia Microbiana/genética , Glicina/genética , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Leucina/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Prolina/genética , Proteínas Recombinantes de Fusión , Proteínas Represoras/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal/genética , Temperatura , Vancomicina , Proteínas Virales , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Using low temperature EPR spectroscopy, signals of iron-sulfur centers with g-factors of 2.02 and 1.94 were detected in the respiratory chain of St. aureus membranes. According to their relaxation parameters and redox properties, these iron-sulfur centers are similar to iron-sulfur centers S-1 and S-3 corresponding to succinate dehydrogenases of mitochondria and bacterial membranes.
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Proteínas Hierro-Azufre/metabolismo , Oxígeno/metabolismo , Staphylococcus aureus/metabolismo , Membrana Celular/enzimología , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Oxidación-Reducción , Succinato Deshidrogenasa/metabolismoRESUMEN
Two cytochrome b with peaks at 554 and 558 nm and cytochrome a with alpha-peak at 603 nm were found in intact cells and membranes of Staphylococcus aureus using low-temperature spectrophotometry and registration of second- and fourth-order finite difference spectra of cytochromes. Analysis of the cytochrome functioning in membranes isolated from the cells at the exponential and stationary growth phases revealed no difference in the set of these carriers. Analysis of cytochrome reduction with different substrates demonstrated identity of the cytochrome composition in the respiratory chain, reduced with NADH, lactate, alpha-glycerophosphate, malate and succinate. Cytochrome omicron with gamma-peak at 416 nm in the CO-spectra was found to be involved in oxidation of all the substrates tested both in intact cells and membranes of Staphylococcus aureus.