RESUMO
Intrauterine growth restriction (IUGR) is one of the key features of fetal alcohol syndrome (FAS), and IUGR can be mediated by impaired placentation. Insulin-like growth factors (IGF) regulate placentation due to stimulatory effects on extravillous trophoblasts, which are highly motile and invasive. Previous studies demonstrated that extravillous trophoblasts express high levels of aspartyl-(asparaginyl) beta-hydroxylase (AAH), a gene that is regulated by IGF and has a critical role in cell motility and invasion. The present study examines the hypothesis that ethanol impaired placentation is associated with inhibition of AAH expression in trophoblasts. Pregnant Long Evans rats were fed isocaloric liquid diets containing 0% or 37% ethanol by caloric content. Placentas harvested on gestation day 16 were used for histopathological, mRNA, and protein studies to examine AAH expression in relation to the integrity of placentation and ethanol exposure. Chronic ethanol feeding prevented or impaired the physiological conversion of uterine vessels required for expansion of maternal circulation into placenta, a crucial process for adequate placentation. Real-time quantitative RT-PCR analysis demonstrated significant reductions in IRS-1, IRS-2, and significant increases in IGF-II and IGF-II receptor mRNA levels in ethanol-exposed placentas. These abnormalities were associated with significantly reduced levels of AAH expression in trophoblastic cells, particularly within the mesometrial triangle (deep placental bed) as demonstrated by real time quantitative RT-PCR, Western blot analysis, ELISA, and immunohistochemical staining. Ethanol-impaired placentation is associated with inhibition of AAH expression in trophoblasts. This effect of chronic gestational exposure to ethanol may contribute to IUGR in FAS.
Assuntos
Etanol/efeitos adversos , Transtornos do Espectro Alcoólico Fetal/fisiopatologia , Doenças Placentárias/etiologia , Placentação/fisiologia , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Idade Gestacional , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like II/genética , Oxigenases de Função Mista/genética , Placenta/efeitos dos fármacos , Placenta/metabolismo , Doenças Placentárias/genética , Placentação/efeitos dos fármacos , Placentação/genética , Gravidez , Ratos , Ratos Long-Evans , Receptor IGF Tipo 1/genética , Receptor de Insulina/genética , Fatores de TempoRESUMO
Hepadnavirus polymerases are multifunctional enzymes that play critical roles during the viral life cycle but have been difficult to study due to a lack of a well-defined panel of monoclonal antibodies (MAbs). We have used recombinant human hepatitis B virus (HBV) polymerase (Pol) expressed in and purified from baculovirus-infected insect cells to generate a panel of six MAbs directed against HBV Pol protein. Such MAbs were subsequently characterized with respect to their isotypes and functions in analytical and preparative assays. Using these MAbs as probes together with various deletion mutants of Pol expressed in insect cells, we mapped the B-cell epitopes of Pol recognized by these MAbs to amino acids (aa) 8 to 20 and 20 to 30 in the terminal protein (TP) region of Pol, to aa 225 to 250 in the spacer region, and to aa 800 to 832 in the RNase H domain. Confocal microscopy and immunocytochemical studies using various Pol-specific MAbs revealed that the protein itself appears to be exclusively localized to the cytoplasm. Finally, MAbs specific for the TP domain, but not MAbs specific for the spacer or RNase H regions of Pol, appeared to inhibit Pol function in the in vitro priming assay, suggesting that antibody-mediated interference with TP may now be assessed in the context of HBV replication.
Assuntos
Anticorpos Monoclonais/imunologia , Produtos do Gene pol/imunologia , Anticorpos Anti-Hepatite B/imunologia , Vírus da Hepatite B/enzimologia , DNA Polimerase Dirigida por RNA/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Western Blotting , Linhagem Celular , Mapeamento de Epitopos , Técnica Indireta de Fluorescência para Anticorpo , Anticorpos Anti-Hepatite B/biossíntese , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Precipitina , Células Tumorais CultivadasRESUMO
Neuronal thread proteins (NTPs) comprise a family of molecules expressed in brain and primitive neuroectodermal tumor cell lines. In Alzheimer's disease (AD), increased CNS levels of the 21 kD NTP species are correlated with dementia. The present study characterizes the nature and distribution of NTP expression using recently generated brain-derived polyclonal and monoclonal antibodies (MoAbs) to recombinant AD7c-NTP protein. In AD, high levels of NTP immunoreactivity were detected in neuronal perikarya, neuropil fibers, and white matter fibers (axons). In addition, 4 of the 23 AD7c-NTP MoAbs labeled degenerating neurons (with or without neurofibrillary tangles), axonal spheroids, dystrophic neurites, or irregular, wavy threadlike neuropil fibers in AD. Increased neuronal AD7c-NTP immunoreactivity in AD colocalized with perikaryal accumulations of tau-1, phosphorylated neurofilament, and the ganglioside, A2B5. In addition, AD7c-NTP immunoreactivity was detected in early neuritic plaques along with beta-amyloid-containing fibrils, but not in mature plaques, nor was it colocalized in beta A4-immunoreactive fibrils. This study demonstrates the profiles of NTP overexpression in relation to paired helical filament-associated neurodegenerative lesions in AD.
Assuntos
Doença de Alzheimer/metabolismo , Proteínas de Ligação ao Cálcio/análise , Proteínas do Tecido Nervoso/análise , Idoso , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/imunologia , Anticorpos Monoclonais , Especificidade de Anticorpos , Biomarcadores , Western Blotting , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/imunologia , Artérias Cerebrais/química , Córtex Cerebral/citologia , Veias Cerebrais/química , Feminino , Gangliosídeos/análise , Gangliosídeos/imunologia , Humanos , Imuno-Histoquímica , Litostatina , Masculino , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/imunologia , Neuritos/química , Neurônios/química , Proteínas Recombinantes/imunologia , Proteínas tau/análise , Proteínas tau/imunologiaRESUMO
Three novel monoclonal antibodies (MAbs) were established against a recombinant hepatitis C virus (HCV) core protein derived from cloned genotype 1b HCV cDNA. MAbs C7-50 and C8-59 recognize a conserved linear epitope represented by amino acid residues 21 to 40 of the nucleocapsid protein. MAb C8-48 is directed against a strain-specific conformational epitope located within the first 82 amino acids. A sensitive two-site MAb-based immunoradiometric assay was established using antibodies directed against distinct epitopes on the nucleocapsid protein. Processed 21 kDa core protein was detected by immunoblotting in human hepatocellular carcinoma cell lines and primary adult rat hepatocytes transfected with a cytomegalovirus promoter-driven expression construct. Immunofluorescence microscopy studies revealed a granular and vesicular cytoplasmic staining pattern. MAb C7-50 was used successfully to detect HCV core antigen in chronically infected chimpanzee liver tissue. These MAbs represent important reagents for the study of HCV biology and for the development of immunodiagnostic assays.
Assuntos
Anticorpos Monoclonais/imunologia , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/imunologia , Proteínas do Core Viral/imunologia , Animais , Ligação Competitiva , Linhagem Celular , Mapeamento de Epitopos , Técnica Indireta de Fluorescência para Anticorpo , Hepacivirus/isolamento & purificação , Hepatite C/imunologia , Hepatite C/virologia , Humanos , Fígado/imunologia , Fígado/virologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Pan troglodytes , Ratos , Células Tumorais CultivadasRESUMO
p36 plays a direct role in DNA synthesis and is overexpressed in transformed cells. It is also an important component linking the cell membrane to intracellular cytoskeletal components. Experiments were performed in primary rat hepatocyte culture stimulated with epidermal growth factor (EGF) to determine if p36 expression was related to DNA synthesis or to the effect of the extracellular matrix on hepatocyte differentiation; ethanol was employed as an agent to inhibit hormone stimulated hepatocyte DNA synthesis. It was found that hepatocyte p36 expression was highly dependent on the type of extracellular matrix and the time in culture. There was no correlation of p36 expression with DNA synthesis and, therefore, p36 levels appeared more closely related to the differentiated phenotype, induced by the extracellular matrix interactions rather than cellular proliferation.
Assuntos
Anexina A2/biossíntese , Etanol/farmacologia , Matriz Extracelular/fisiologia , Fígado/metabolismo , Proteínas Quinases/metabolismo , Animais , Northern Blotting , Células Cultivadas , Colágeno/farmacologia , DNA/biossíntese , Combinação de Medicamentos , Fator de Crescimento Epidérmico/farmacologia , Feminino , Cinética , Laminina/farmacologia , Fígado/efeitos dos fármacos , Proteoglicanas/farmacologia , RNA/análise , Ratos , Especificidade por Substrato , Timidina/metabolismo , Fatores de TempoRESUMO
An unusual protein was isolated from acid extracts of normal human pancreas and pancreatic secretion in the form of uniform 7-10-nm long single threads without visible axial periodicity or other structure, as seen in the electron microscope. It accounts for as much as 300 micrograms/ml in some pancreatic secretions as measured by specific radioimmunoassay. The protein undergoes a freely reversible, pH dependent, globule-fibril transformation, being stable in the fibril form between pH 5.4 and 9.2. The monomer at acid pH has an apparent molecular weight of approximately 14,000 and consists of a single polypeptide chain, the amino acid composition of which is rich in aromatic amino acids and lacks carbohydrate, fatty acid, and phosphate. The amino acid sequence of 45 residues from the amino terminus shows no homology with any other reported protein sequences other than that of the A chain of the bovine pancreas thread protein (reported elsewhere). A sensitive radioimmunoassay employing monoclonal antibodies against human pancreatic thread protein failed to detect the antigen in a wide range of human tissues other than pancreas, nor was the antigen measurable in normal human sera. Immunohistochemistry utilizing these antibodies revealed the antigen as a component of the cytoplasm of some but not all the pancreatic acinar cells. A physiologic function has not yet been determined for this protein.
Assuntos
Proteínas do Tecido Nervoso , Pâncreas/análise , Suco Pancreático/análise , Sequência de Aminoácidos , Aminoácidos/análise , Anticorpos Monoclonais/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Humanos , Concentração de Íons de Hidrogênio , Técnicas Imunoenzimáticas , Litostatina , Peso Molecular , Conformação Proteica , Radioimunoensaio , SolubilidadeRESUMO
Several libraries of monoclonal antibodies have been produced by immunization of Balb/c mice with single cell suspensions of nontrypsin-treated human hepatocellular carcinoma cell (HCC) lines in order to study the antigenic properties of transformed hepatocytes. The antibodies were characterized with regards to specificity for hepatoma-associated antigens and their capability for use as reagents in radioimmunoassays (RIAs) and tumor localization in vivo. Three such antibodies namely, P215457, PM4E9917, P232524 of the IgG2a, IgG2a, and IgG1 isotypes, respectively, not only recognized separate and distinct antigenic determinants on four human hepatoma cell lines but also reacted with epitopes present on chemically induced rat hepatoma cell lines. In contrast, only 1 of 38 other human malignant and transformed cell lines demonstrated reactivity with the three antibodies; normal human tissues were also found to be unreactive. Monoclonal antibody P215457 densely stained the plasma membrane by indirect immunofluorescence, showed rapid binding activity to HCC cells in suspension, and precipitated a 50,000-mol wt cell surface protein; antibody PM4E9917 also stained the plasma membrane and precipitated a 65,000-mol wt protein, whereas P232534 recognized cytoplasmic antigenic determinants. With these antibodies "simultaneous sandwich" RIAs were established that detect soluble hepatoma-associated antigens in culture supernatants. Finally, the Fab fragment of P215457 was found to be useful in tumor localization in vivo. This antibody fragment when labeled with 131I was shown to localize by radionuclide-imaging studies in human hepatoma grown in nude mice. Thus, these investigations demonstrate that monoclonal antibodies may be produced against epitopes that reside almost exclusively on transformed hepatocytes and such antibodies may be successfully employed in the development of in vitro and in vivo immunoassays.
Assuntos
Antígenos de Neoplasias/imunologia , Carcinoma Hepatocelular/imunologia , Neoplasias Hepáticas/imunologia , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Antígenos de Superfície/imunologia , Linhagem Celular , Imunofluorescência , Humanos , Neoplasias Hepáticas Experimentais/diagnóstico por imagem , Camundongos , Peso Molecular , Radioimunoensaio , CintilografiaRESUMO
A library of murine monoclonal antibodies reactive with human hepatoma cells was generated following immunization of Balb/c mice with an intact cloned human hepatoma cell line, designated PLC/PRF/5-NR. We report the characterization of one such IgG2a antibody, designated anti-PLC1. This antibody specifically stains parental PLC/PRF/5 cell membranes and membranes of SK-Hep 1 and Mahlavu human hepatoma cells grown in culture, using indirect immunofluorescence and horseradish immunoperoxidase techniques. A similar pattern of membranous staining was observed in solid tumors derived from the three hepatoma cell lines which were injected subcutaneously into athymic nude rats and mice. Spontaneous capping on the cell surface was observed in 7 to 30% of the three human hepatocellular carcinoma cell types when incubated in suspension with monoclonal anti-PLC1 at 37 degrees C. Treatment of cells with trypsin or sustained growth in culture did not affect the intensity of membranous staining. Monoclonal anti-PLC1 appeared specific, and antibodies did not stain a variety of human carcinoma cell lines and primary tumors of nonhepatic origin, or several normal human and murine tissues. Purified 125I-labeled monoclonal anti-PLC1 bound specifically to the three hepatoma cell lines in culture. Specificity of the antigen-antibody reaction was demonstrated by competitive binding inhibition in experiments using unlabeled homologous antibody. Binding of 125I-anti-PLC1 was not inhibited by unlabeled monoclonal antibodies to HBsAg or to alpha-fetoprotein. Two hepatoma cell lines secrete a protein that specifically blocks binding of 125I-anti-PLC1 antibodies to cell surface antigenic determinants. This "hepatoma-associated" protein was subsequently purified by affinity chromatography from supernates derived from the three hepatoma cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Neoplasias/análise , Antígenos de Superfície/análise , Carcinoma Hepatocelular/imunologia , Sítios de Ligação de Anticorpos , Linhagem Celular , Cromatografia de Afinidade , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Neoplasias Hepáticas , Proteínas de Neoplasias/isolamento & purificação , Células-Tronco Neoplásicas/imunologiaRESUMO
Using a monoclonal IgM antibody (anti-HBs) to hepatitis B surface antigen (HBsAg) in a radioimmunoassay for hepatitis B, we have detected high binding activity in human serum that was unreactive in assays employing conventional anti-HBs reagents. The binding material was isolated from serum by affinity chromatography on monoclonal IgM anti-HBs, and comparison of the material with HBsAg (by sodium dodecyl sulfate/polyacrylamide gel electrophoresis) demonstrated that the two shared several similar polypeptides. Furthermore, comparison of the binding properties of HBsAg and concentrated monoclonal immunoreactive material with conventional and monoclonal anti-HBs reagents demonstrated some antigenic crossreactivity. The molecular weight of the monoclonal immunoreactive material was approximately 2 X 10(6). Immunoprecipitation of the material with monoclonal IgM antibodies and examination by electron microscopy revealed clumped and "spiculated" particles that resembled 22-nm hepatitis B particles coated with the same antibody. Thus, this study suggests that the high-binding-activity material, detected in serum only by the monoclonal radioimmunoassay, is not identical with HBsAg, but it shares some common properties.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Anti-Hepatite B/imunologia , Antígenos da Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Adulto , Reações Antígeno-Anticorpo , Antígenos da Hepatite B/análise , Antígenos de Superfície da Hepatite B/análise , Humanos , Imunoglobulina M , Masculino , Peso Molecular , Peptídeos/análise , RadioimunoensaioRESUMO
High-affinity monoclonal IgG and IgM antibodies to hepatitis B surface antigen (HBsAg) have been prepared and their functional capabilities explored by means of solid-phase radioimmunoassays. 125I-labeled HBsAg binding studies indicated that monoclonal IgM antibodies against HBsAg (anti-HBs) coupled to a solid-phase support quantitatively bound more HbsAg at a faster rate than conventionally prepared anti-HBs reagents or other high-affinity IgG monoclonal anti-HBs antibodies. Consequently, IgM anti-HBs was also radiolabeled, and an IgM-IgM radioimmunoassay was developed for the immunodiagnosis of hepatitis B. The lower limit of this assay was approximately 100 pg +/- 30 (SEM) of HBsAg per ml of serum. Compared to available commercial radioassays, preliminary studies have shown the IgM-IgM assay to have increased sensitivity, which improved the detection of a HBsAg-associated determinant in acute hepatitis and post transfusion hepatitis. It is probable that the multivalent interaction between monoclonal IgM anti-HBs and the polydeterminant HBsAg is important in augmenting the performance of this monoclonal assay.