RESUMEN
BACKGROUND: Merkel cell polyomavirus (MCPyV), a human polyomavirus that is unequivocally linked to merkel cell carcinoma (MCC), has been found in association with keratinocytes carcinomas (KC), especially basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC). Nevertheless, there is scarce information about the possible involvement of MCPyV in the development of KC. OBJECTIVES: To assess the presence of MCPyV DNA and Large-T Antigen (LT-Ag) via Polymerase Chain Reaction (PCR) and Immunohistochemistry (IHC) in cases of KC, and to correlate its presence with immunohistochemical markers p16, p53, and ki67, tumor type and subtype, sun-exposed location, and epidemiological data. METHODS: The prevalence of MCPyV DNA, LT-Ag, and immunohistochemical markers p16, p53, and ki67 was assessed by PCR and Immunohistochemistry (IHC) in 127 cases of KC, these results were correlated with tumor type and subtype, sun-exposed location, and epidemiological data. RESULTS: The MCPyV DNA was detected in 42.57% (43 of 101) cases by PCR, the LT-Ag was detected in 16.4% (20 of 122) of cases, p16 in 81.5% (97 of 119), p53 in 66.4% (83 of 125), ki67 in 89% (73 of 82). No correlation between MCPyV LT-Ag and DNA confronted with tumor type, subtype, location site, and immunohistochemical markers was found. A single correlation between the MCPyV LT-Ag and cSCC tumors and peri-tumoral lymphocyte cells was noted. STUDY LIMITATIONS: Further steps need to be taken to better evaluate the MCPyV influence and its possible role in KC carcinogenesis, as the evaluation of the virus genome state, the gene sequence that encodes LT-Ag in the KC tumor cells, and in situ hybridization for viral DNA or RNA in these cells. CONCLUSIONS: Despite the frequent detection of MCPyV in KC, the data available so far does not support the hypothesis of a causal relationship between them.
Asunto(s)
Antígenos Virales de Tumores , Biomarcadores de Tumor , Carcinoma de Células de Merkel , Carcinoma de Células Escamosas , Antígeno Ki-67 , Poliomavirus de Células de Merkel , Neoplasias Cutáneas , Proteína p53 Supresora de Tumor , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Antígenos Virales de Tumores/análisis , Biomarcadores de Tumor/análisis , Carcinoma Basocelular/virología , Carcinoma Basocelular/patología , Carcinoma de Células de Merkel/virología , Carcinoma de Células de Merkel/patología , Carcinoma de Células Escamosas/virología , Carcinoma de Células Escamosas/patología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/análisis , ADN Viral/análisis , Inmunohistoquímica , Queratinocitos/virología , Queratinocitos/patología , Antígeno Ki-67/análisis , Poliomavirus de Células de Merkel/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Infecciones por Polyomavirus/virología , Neoplasias Cutáneas/virología , Neoplasias Cutáneas/patología , Proteína p53 Supresora de Tumor/análisis , Infecciones Tumorales por Virus/virologíaRESUMEN
Bovine polyomavirus-1 (BoPyV-1, Epsilonpolyomavirus bovis) is widespread in cattle and has been detected in commercialized beef at supermarkets in the USA and Germany. BoPyV-1 has been questioned as a probable zoonotic agent with documented increase in seropositivity in people exposed to cattle. However, to date, BoPyV-1 has not been causally associated with pathology or disease in any animal species, including humans. Here we describe and illustrate pathological findings in an aborted bovine fetus naturally infected with BoPyV-1, providing evidence of its pathogenicity and probable abortigenic potential. Our results indicate that: (i) BoPyV-1 can cause severe kidney lesions in cattle, including tubulointerstitial nephritis with cytopathic changes and necrosis in tubular epithelial cells, tubular and interstitial inflammation, and interstitial fibroplasia; (ii) lesions are at least partly attributable to active viral replication in renal tubular epithelial cells, which have abundant intranuclear viral inclusions; (iii) BoPyV-1 large T (LT) antigen, resulting from early viral gene expression, can be detected in infected renal tubular epithelial cells using a monoclonal antibody raised against Simian Virus-40 polyomavirus LT antigen; and (iv) there is productive BoPyV-1 replication and virion assembly in the nuclei of renal tubular epithelial cells, as demonstrated by the ultrastructural observation of abundant arrays of viral particles with typical polyomavirus morphology. Altogether, these lesions resemble the "cytopathic-inflammatory pathology pattern" proposed in the pathogenesis of Human polyomavirus-1-associated nephropathy in immunocompromised people and kidney allograft recipients. Additionally, we sequenced the complete genome of the BoPyV-1 infecting the fetus, which represents the first whole genome of a BoPyV-1 from the Southern Hemisphere. Lastly, the BoPyV-1 strain infecting this fetus was isolated, causing a cytopathic effect in Madin-Darby bovine kidney cells. We conclude that BoPyV-1 is pathogenic to the bovine fetus under natural circumstances. Further insights into the epidemiology, biology, clinical relevance, and zoonotic potential of BoPyV-1 are needed.
Asunto(s)
Trasplante de Riñón , Nefritis Intersticial , Infecciones por Polyomavirus , Poliomavirus , Infecciones Tumorales por Virus , Animales , Anticuerpos Monoclonales , Antígenos Virales de Tumores , Bovinos , Feto/patología , Humanos , Riñón , Trasplante de Riñón/efectos adversos , Nefritis Intersticial/complicaciones , Nefritis Intersticial/patología , Infecciones por Polyomavirus/complicaciones , Virus 40 de los Simios , Infecciones Tumorales por Virus/complicacionesRESUMEN
Diagnosis and prognosis of breast cancer is based on disease staging identified through histopathological and molecular biology techniques. Animal models are used to gain mechanistic insights into the development of breast cancer. C(3)1-TAg is a genetically engineered mouse model that develops mammary cancer. However, carcinogenesis caused by this transgene was characterized in the Friend Virus B (FVB) background. As most genetic studies are done in mice with C57BL/6 J background, we aimed to define the histological alterations in C3(1)-TAg C57BL/6 J animals. Our results showed that C3(1)-TAg animals with C57BL/6 J background develop solid-basaloid adenoid cystic carcinomas with increased fibrosis, decreased area of adipocytes, and a high proliferative index, which are triple-negative for progesterone, estrogen, and human epidermal growth factor receptor 2 (HER2) receptors. Our results also revealed that tumor development is slower in the C57BL/6 J background when compared with the FVB strain, providing a better model to study the different stages in breast cancer progression.
Asunto(s)
Antígenos Virales de Tumores/genética , Neoplasias de la Mama/genética , Carcinoma Adenoide Quístico/genética , Modelos Genéticos , Animales , Antígenos Virales de Tumores/inmunología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Carcinoma Adenoide Quístico/inmunología , Carcinoma Adenoide Quístico/patología , Femenino , Virus de la Leucemia Murina de Friend/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
An impedimetric biosensor was developed for the selective detection of the cancer-associated T antigen, using the lectin from Arachis hypogaea (peanut agglutinin, PNA) as the recognition element. The increase in the biosensor's impedance after sample incubation was indicative of lectin recognition and complex formation between PNA and glycoproteins containing T antigen. When using asialofetuin as model glycoprotein, a minimum amount of 100â¯ng of glycoprotein could be detected, generating an increase in impedance of 7.2%. Albumin did not cause interference in the detection of T-carrying glycoproteins up to a concentration of 0.01â¯mgâ¯ml-1. The biosensor was used to evaluate the T-antigen expression in serum samples and was able to discriminate between control samples (of individuals without cancer) and case samples from patients with diverse types of carcinomas (skin, colon, breast, prostate, stomach, kidney, lung, liver and rectum) in which an increase in the expression of T antigen is well-known. The same samples were analyzed with a Vicia villosa agglutinin biosensor that has specificity for the cancer-associated Tn antigen, to compare the expression of both antigens in the diverse carcinomas. The results were different for both biosensors, confirming that the use of different lectins allows to monitor different antigen expression. Furthermore, combining different lectins, glycosylation profiles for each carcinoma type can be obtained. This work demonstrates the feasibility of employing PNA to selectively recognize the T epitope in glycoproteins and the proposed biosensor could be used for high-throughput, label-free profiling of the cancer-associated T antigen in serum samples.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/sangre , Antígenos Virales de Tumores/sangre , Técnicas Biosensibles/instrumentación , Neoplasias/sangre , Aglutinina de Mani/química , Arachis/química , Diseño de Equipo , Humanos , Lectinas de Plantas/química , Vicia/químicaRESUMEN
A novel polyomavirus (PyVs) comprising 5,422 bp was identified by high-throughput sequencing (HTS) in pooled organs of nutria (Myocastor coypus). The new genome displays the archetypal organization of PyVs, which includes open reading frames for the regulatory proteins small T antigen (sTAg) and large T antigen (LTAg), as well as for the capsid proteins VP1, VP2 and VP3. Based on the International Committee on Taxonomy of Viruses (ICTV) Polyomaviridae Study Group criteria, this genome comprises a new PyVs species for the Alphapolyomavirus genus and is putatively named "Myocastor coypus Polyomavirus 1" . The complete genome sequence of this Myocastor coypus Polyomavirus 1 (McPyV1) isolate is publically available under the GenBank accession no. MH182627.
Asunto(s)
Infecciones por Polyomavirus/veterinaria , Poliomavirus/aislamiento & purificación , Enfermedades de los Roedores/virología , Roedores/virología , Animales , Antígenos Virales de Tumores/genética , Proteínas de la Cápside/genética , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Sistemas de Lectura Abierta , Filogenia , Poliomavirus/clasificación , Poliomavirus/genética , Infecciones por Polyomavirus/virología , RatasRESUMEN
The nearly complete genome sequence of a novel polyomavirus from blood samples of Akodon montensis and Calomys tener collected in Brazil was determined by high-throughput sequencing. This virus showed a typical polyomaviruses genome organization, and it was classified as a member of the genus Betapolyomavirus. Our results expand the host range and viral diversity of the family Polyomaviridae.
Asunto(s)
Antígenos Virales de Tumores/genética , Genoma Viral/genética , Polyomaviridae , Sigmodontinae/virología , Secuencia de Aminoácidos/genética , Animales , Brasil , Especificidad del Huésped , Filogenia , Polyomaviridae/clasificación , Polyomaviridae/genética , Polyomaviridae/aislamiento & purificaciónRESUMEN
BACKGROUND: Mammary cell cultures are convenient tools for in vitro studies of mammary gland biology. However, the heterogeneity of mammary cell types, e.g., glandular milk secretory epithelial or myoepithelial cells, often complicates the interpretation of cell-based data. The present study was undertaken to determine the relevance of bovine primary mammary epithelial cells isolated from American Holstein (bMECUS) or Swiss Holstein-Friesian (bMECCH) cows, and of primary bovine mammary alveolar epithelial cells stably transfected with simian virus-40 (SV-40) large T-antigen (MAC-T) for in vitro analyses. This was evaluated by testing their expression pattern of cytokeratin (CK) 7, 18, 19, vimentin, and α-smooth muscle actin (α-SMA). RESULTS: The expression of the listed markers was assessed using real-time quantitative PCR, flow cytometry and immunofluorescence microscopy. Characteristic markers of the mesenchymal (vimentin), myoepithelial (α-SMA) and glandular secretory cells (CKs) showed differential expression among the studied cell cultures, partly depending on the analytical method used. The relative mRNA expression of vimentin, CK7 and CK19, respectively, was lower (P < 0.05) in immortalized than in primary mammary cell cultures. The stain index (based on flow cytometry) of CK7 and CK19 protein was lower (P < 0.05) in MAC-T than in bMECs, while the expression of α-SMA and CK18 showed an inverse pattern. Immunofluorescence microscopy analysis mostly confirmed the mRNA data, while partly disagreed with flow cytometry data (e.g., vimentin level in MAC-T). The differential expression of CK7 and CK19 allowed discriminating between immortal and primary mammary cultures. CONCLUSIONS: The expression of the selected widely used cell type markers in primary and immortalized MEC cells did not allow a clear preference between these two cell models for in vitro analyses studying aspects of milk composition. All tested cell models exhibited to a variable degree epithelial and mesenchymal features. Thus, based on their characterization with widely used cell markers, none of these cultures represent an unequivocal alveolar mammary epithelial cell model. For choosing the appropriate in vitro model additional properties such as the expression profile of specific proteins of interest (e.g., transporter proteins) should equally be taken into account.
Asunto(s)
Actinas/análisis , Células Epiteliales/citología , Queratinas/análisis , Glándulas Mamarias Animales/citología , Vimentina/análisis , Análisis de Varianza , Animales , Antígenos Virales de Tumores , Bovinos , Línea Celular , Células Cultivadas , Células Epiteliales/química , Femenino , Citometría de Flujo/métodos , Glándulas Mamarias Animales/química , Microscopía Fluorescente/métodos , Cultivo Primario de Células , Reacción en Cadena en Tiempo Real de la Polimerasa , Virus 40 de los SimiosRESUMEN
BACKGROUND: Mammary cell cultures are convenient tools for in vitro studies of mammary gland biology. However, the heterogeneity of mammary cell types, e.g., glandular milk secretory epithelial or myoepithelial cells, often complicates the interpretation of cell-based data. The present study was undertaken to determine the relevance of bovine primary mammary epithelial cells isolated from American Holstein (bMEC US) or Swiss Holstein-Friesian (bMEC CH) cows, and of primary bovine mammary alveolar epithelial cells stably transfected with simian virus-40 (SV-40) large T-antigen (MAC-T) for in vitro analyses. This was evaluated by testing their expression pattern of cytokeratin (CK) 7, 18, 19, vimentin, and α-smooth muscle actin (α-SMA. RESULTS: The expression of the listed markers was assessed using real-time quantitative PCR, flow cytometry and immunofluorescence microscopy. Characteristic markers of the mesenchymal (vimentin), myoepithelial (α-SMA) and glandular secretory cells (CKs) showed differential expression among the studied cell cultures, partly depending on the analytical method used. The relative mRNA expression of vimentin, CK7 and CK19, respectively, was lower (P < 0.05) in immortalized than in primary mammary cell cultures. The stain index (based on flow cytometry) of CK7 and CK19 protein was lower (P < 0.05) in MAC-T than in bMECs, while the expression of α-SMA and CK18 showed an inverse pattern. Immunofluorescence microscopy analysis mostly confirmed the mRNA data, while partly disagreed with flow cytometry data (e.g., vimentin level in MAC-T). The differential expression of CK7 and CK19 allowed discriminating between immortal and primary mammary cultures. CONCLUSIONS: The expression of the selected widely used cell type markers in primary and immortalized MEC cells did not allow a clear preference between these two cell models for in vitro analyses studying aspects of milk composition. All tested cell models exhibited to a variable degree epithelial and mesenchymal features. Thus, based on their characterization with widely used cell markers, none of these cultures represent an unequivocal alveolar mammary epithelial cell model. For choosing the appropriate in vitro model additional properties such as the expression profile of specific proteins of interest (e.g., transporter proteins) should equally be taken into account.
Asunto(s)
Animales , Bovinos , Femenino , Actinas/análisis , Células Epiteliales/citología , Queratinas/análisis , Glándulas Mamarias Animales/citología , Vimentina/análisis , Análisis de Varianza , Antígenos Virales de Tumores , Línea Celular , Células Cultivadas , Células Epiteliales/química , Citometría de Flujo/métodos , Glándulas Mamarias Animales/química , Microscopía Fluorescente/métodos , Cultivo Primario de Células , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Polyomavirus BK (BKPyV) T-antigens (large and small tumor antigens, or Lt-ag and st-ag, respectively), control key aspects of viral replication and are able to regulate cell cycle, promoting cell proliferation. However, the structural effects of genetic mutations on T-antigens are poorly investigated. In this study, 214 sequences of T-antigens from individuals with different BKPyV infections (16 renal transplant with nephropathy; 78 asymptomatic renal transplant; 24 hematopoietic stem cell transplant with hemorrhagic cystitis; 96 healthy non-transplant), were analyzed from the genetic and structural standpoints. We found a high concentration of non-synonymous mutations at inter-domains and hexamerization regions of both proteins, being five of them under positive selection in the Lt-ag but none in the st-ag. The in silico analysis indicated that two mutations, located at positions 164 in the st-ag and 592 in the Lt-ag, would significantly affect the interaction with PP2A and p53 cell targets, respectively, although they were not associated to a specific clinical status. No mutations were detected on the J-domains or at the ATPase motif. In sum, the profile of the mutations found seem not to be associated to increased morbidity. This is the first work to analyze structural modifications on T-antigens in different BKPyV infections, and managed to map conserved and variable regions of the T-antigens, which will be helpful for the study of new antiviral drugs.
Asunto(s)
Antígenos Virales de Tumores/genética , Virus BK/clasificación , Virus BK/genética , Variación Genética , Mutación Missense , Infecciones por Polyomavirus/virología , Infecciones Tumorales por Virus/virología , Virus BK/aislamiento & purificación , ADN Viral/química , ADN Viral/genética , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Análisis de Secuencia de ADNRESUMEN
Lung cancer is a leading pathology strongly associated with the smoking habit. However, a viral etiology for a subset of patients developing lung cancer has been suggested. Polyomaviruses (PyVs) are small double stranded DNA viruses associated with the development of some human diseases. However, a causal role of these viruses in human cancer has been difficult to demonstrate. In this study, eighty-six non-small cell lung carcinomas (NSCLCs), including adenocarcinomas (AdCs) and squamous cell lung carcinomas (SQCs) from Chile were analyzed for the presence of PyVs using polymerase chain reaction (PCR). All of the specimens were positive for a fragment of the betaglobin gene. We found that 4/86 (4.7%) of lung carcinomas were positive for PyVs. After sequencing and BlastN alignment, all four cases were identified as Merkel cell polyomaviruses (MCV) that corresponded to two AdCs and two SQCs. A non-significant statistical association was found between the presence of MCV and clinic-pathological features of the patients and tumors. In addition, 1/4 (25%) of the carcinomas were actively expressing large T antigen (LT) transcripts, as demonstrated by reverse-transcriptase PCR (RT-PCR). Thus a possible role of MCV in a very small subset of patients with lung cancer cannot be ruled out and warrants more investigation.
Asunto(s)
Carcinoma de Células de Merkel/virología , Carcinoma de Pulmón de Células no Pequeñas/virología , Neoplasias Pulmonares/virología , Poliomavirus de Células de Merkel/aislamiento & purificación , Infecciones por Polyomavirus/virología , Infecciones Tumorales por Virus/virología , Anciano , Antígenos Virales de Tumores/biosíntesis , Secuencia de Bases , Chile/epidemiología , ADN Viral/genética , ADN Viral/aislamiento & purificación , Femenino , Humanos , Masculino , Poliomavirus de Células de Merkel/genética , Persona de Mediana Edad , Datos de Secuencia MolecularRESUMEN
In this work, quaternary conformational studies of peanut agglutinin (PNA) have been carried out using small-angle X-ray scattering (SAXS). PNA was submitted to three different conditions: pH variation (2.5, 4.0, 7.4 and 9.0), guanidine hydrochloride presence (0.5-2M) at each pH value, and temperature ranging from 25 to 60°C. All experiments were performed in the absence and presence of T-antigen to evaluate its influence on the lectin stability. At room temperature and pH 4.0, 7.4 and 9.0, the SAXS curves are consistent with the PNA scattering in its crystallographic native homotetrameric structure, with monomers in a jelly roll fold, associated by non-covalent bonds resulting in an open structure. At pH 2.5, the results indicate that PNA tends to dissociate into smaller sub-units, as dimers and monomers, followed by a self-assembling into larger aggregates. Furthermore, the conformational stability under thermal denaturation follows the pH sequence 7.4>9.0>4.0>2.5. Such results are consistent with the conformational behavior found upon GndHCl influence. The presence of T-antigen does not affect the protein quaternary structure in all studied systems within the SAXS resolution.
Asunto(s)
Aglutinina de Mani/química , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Antígenos Virales de Tumores/metabolismo , Guanidina/metabolismo , Concentración de Iones de Hidrógeno , Conformación Proteica , TemperaturaRESUMEN
Los marcadores tumorales, también denominados marcadores biológicos o biomarcadores, se definen como moléculas, sustancias o procesos que se alteran cualitativa o cuantitativamente como resultado de una condición precancerosa o un cáncer, detectables mediante una prueba de laboratorio en sangre, en líquidos orgánicos o en tejidos. La naturaleza de los marcadores tumorales es muy variable: va desde ácido nucleico, ADN o ARN, una proteína o un péptido, hasta procesos complejos como un anticuerpo, la apoptosis, la amilogénesis y la proliferación. Desde el punto de vista de su origen, los marcadores tumorales se producen por el tumor mismo, como la gonadotropina coriónica en el coriocarcinoma, o como respuesta a la lesión tumoral en el tejido circundante, como el antígeno carcinoembrionario en el cáncer de mama. No hay un marcador tumoral ideal, definido como aquel con una sensibilidad y especificidad del 100. Los marcadores tumorales pueden ser utilizados para el cribado en población con riesgo de presentar un cáncer para su detección precoz con enfermedad confinada y potencialmente curable, como parte del diagnóstico, en el diagnóstico diferencial, como prueba de valor pronóstico y predictivo, como herramienta para evaluar el tratamiento administrado, y para la detección de las recaídas cuando éstas se presentan y el paciente tiene una nueva oportunidad de tratamiento, antes de que las manifestaciones clínicas reaparezcan. En este módulo se analizan los principales marcadores tumorales disponibles en el medio, como el antígeno carcinoembrionario, la alfafetoproteína, el antígeno específico de próstata, el CA 15-3, el CA 125, el CA 19-9, el Cyfra 21-1, la gonadotropina coriónica, la calcitonina, la ferritina, la beta 2 microglobulina, entre otros marcadores. Además, se hará referencia a marcadores subrogados de cáncer, como la presencia de la infección por Helicobacter pylori y el virus del papiloma humano.
Asunto(s)
Humanos , Antígenos de Superficie , Antígenos Virales de Tumores , Antígeno Carcinoembrionario , Papiloma , Infecciones Tumorales por VirusRESUMEN
Cancer-associated mucins show frequent alterations of oligosaccharide chain profile. Terminal structures may be deleted, thereby exposing normally 'cryptic' structures such as Tn (GalNAcα-O-Ser/Thr) and T antigen (Galß1-3GalNAcα-O-Ser/Thr). Overexpression of these commonly hidden glycoforms, and reduced level of naturally occurring anti-T or anti-Tn antibodies, is associated with epithelial tumor progression and aggressiveness. The lectin from the common edible mushroom Agaricus bisporus (ABL) shows high affinity binding to T antigen, and reversible noncytotoxic inhibitory effect on epithelial tumor cell proliferation. The aim of this study was to induce immune response with tumor-associated glycan specificity and biological activity similar to those of ABL. An anti-idiotypic (Id) antibody strategy was developed using ABL as first template. ABL was purified by affinity chromatography and assayed as immunogen in rabbit. Rabbit IgG was purified from anti-ABL serum using a protein G column, and specific anti-ABL IgG was obtained by affinity chromatography using immobilized ABL. Affinity-purified anti-ABL IgG contained an antibody fraction that recognizes the carbohydrate-binding site of ABL. This IgG was used as immunogen in mouse to yield anti-Id antibody recognizing tumor-associated glycans such as Tn and T antigen. Competitive assays showed that α-anomeric GalNAc is the main binding subsite of anti-Id antibody in glycan recognition. Anti-Id antibody bound human epithelial tumor cells, as shown by cell enzyme-linked immunosorbent assay and immunofluorescence. Anti-Id antibody raised by immunization with affinity-purified anti-ABL IgG had antiproliferative effect on human epithelial tumor cells through apoptosis induction similar to that of ABL. The anti-Id immune response developed here has potential application in cancer therapy.
Asunto(s)
Anticuerpos Antiidiotipos/farmacología , Carcinoma/inmunología , Lectinas/inmunología , Agaricus/inmunología , Animales , Antígenos Virales de Tumores/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Carcinoma/tratamiento farmacológico , Carcinoma/patología , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/inmunología , Células Cultivadas , Cromatografía de Afinidad , Epítopos/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunización , Inmunoglobulina G/inmunología , Lectinas/aislamiento & purificación , Lectinas/metabolismo , Ratones , Unión Proteica/inmunología , ConejosRESUMEN
Alternative splicing plays a key role in generating protein diversity. Transfections with minigenes revealed coordination between two distant, alternatively spliced exons in the same gene. Mutations that either inhibit or stimulate inclusion of the upstream alternative exon deeply affect inclusion of the downstream one. However, similar mutations at the downstream alternative exon have little effect on the upstream one. This polar effect is promoter specific and is enhanced by inhibition of transcriptional elongation. Consistently, cells from mutant mice with either constitutive or null inclusion of a fibronectin alternative exon revealed coordination with a second alternative splicing region, located far downstream. Using allele-specific RT-PCR, we demonstrate that this coordination occurs in cis and is also affected by transcriptional elongation rates. Bioinformatics supports the generality of these findings, indicating that 25% of human genes contain multiple alternative splicing regions and identifying several genes with nonrandom distribution of mRNA isoforms at two alternative regions.
Asunto(s)
Empalme Alternativo , Genes/genética , Alelos , alfa-Globulinas/genética , Animales , Antígenos Virales de Tumores/genética , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Biología Computacional , Proteínas de Unión al ADN/genética , Diclororribofuranosil Benzoimidazol/farmacología , Exones/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibronectinas/genética , Humanos , Ratones , Ratones Noqueados , Modelos Genéticos , Proteínas Nucleares/genética , Regiones Promotoras Genéticas/genética , Isoformas de Proteínas/genética , ARN Polimerasa II/antagonistas & inhibidores , ARN Polimerasa II/metabolismo , Empalme del ARN , Proteínas de Unión al ARN/genética , Factores de Empalme Serina-Arginina , Factores de Transcripción/genética , TransfecciónRESUMEN
In this work we identified in adult and juvenile freshwater prawn, Macrobrachium rosenbergii, three major type of circulating hemocytes: fusiform; rounded; and large ovoid hemocytes. Rounded and large hemocytes represent the first defense line, since this type of cells exerts phagocytic activity as well as lectin synthesis. Considering that glycosylation plays important roles in cell communication and as a target for pathogenic microorganisms, in this report was also described the main glycosidic modifications that occur in the large and rounded hemocytes from the freshwater prawn during maturation as determined with lectins. Neu5Acalpha2,6Gal, was identified homogeneously distributed in the membrane in 90% of hemocytes from juvenile organisms. Maturation of the freshwater prawn induced a decrease or complete loss of Neu5Acalpha2,6Gal residues that were replaced with Neu5Acalpha2,3 molecules in practically all hemocytes from adult organisms. This change was paralleled by a diminution in 9-O-acetyl-neuraminic acid (Neu5,9Ac(2)) expression. T and Tn antigens (Galbetal,3 GalNAcalpha1-0-Ser/Thr or GalNAcalpha1-0-Ser/Thr, respectively), as well as N-glycosidically linked glycans, seem to be highly conserved throughout maturation. Our results show that sialylation of freshwater prawn hemocytes is modulated throughout the maturation process.
Asunto(s)
Glicósidos/metabolismo , Hemocitos/metabolismo , Lectinas/metabolismo , Ácido N-Acetilneuramínico/sangre , Palaemonidae/metabolismo , Animales , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Antígenos Virales de Tumores/metabolismo , Glicosilación , Hemocitos/ultraestructura , Microscopía Electrónica de Rastreo , Palaemonidae/clasificación , Fagocitosis/fisiología , Polisacáridos/metabolismo , Coloración y EtiquetadoRESUMEN
Amaranthus leucocarpus lectin is a homodimeric glycoprotein of 35 kDa per sub-unit, which interacts specifically with N-acetyl-galactosamine. In this work, we compared different glycoproteins that contain Galbeta1-3 GalNAcalpha1-3 Ser/Thr or GalNAcalpha1-3 Ser/Thr in their structure as ligands to purify the A. leucocarpus lectin. From the glycoproteins tested, fetuin was the most potent inhibitor of the hemagglutinating activity and the better ligand for lectin purification; however, the use of desialylated stroma from erythrocytes represented the cheapest method to purify this lectin. O-linked glycans released from the glycoproteins used as affinity matrix and those from different erythrocytes were less inhibitory than parental glycoproteins. The NH2-terminal of the lectin is blocked; moreover, this is the only example of a lectin isolated from this genus to be a glycoprotein. Analysis of the glycoprotein sequences with inhibitory activity for the lectin, showed a different pattern in the O-glycosylation, which confirms that A. leucocarpus lectin recognizes conformation and, probably, distances among O-linked glycans moieties.