RESUMEN
In a study performed to determine which regions of the human T-cell lymphotrophic virus type III (HTLV-III) may represent vaccine candidates to prevent the acquired immune deficiency syndrome (AIDS), a synthetic peptide corresponding to amino acid sequence 735 to 752 of the precursor envelope glycoprotein of HTLV-III was used to immunize rabbits. The resulting rabbit antiserum to the synthetic peptide specifically recognized the precursor envelope glycoprotein (gp160) of HTLV-III. Human sera positive for antibody to HTLV-III reacted with this peptide. These findings indicate that synthetic peptides can be used to induce an immune response directed against a native envelope glycoprotein epitope of HTLV-III. The data are discussed in terms of using synthetic peptides to identify antigenic determinants involved in the induction of protective immunity and possibly as vaccine candidates against the etiologic agent of AIDS.
Asunto(s)
Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Deltaretrovirus/inmunología , Péptidos/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Especificidad de Anticuerpos , Humanos , Peso Molecular , Péptidos/síntesis química , Conejos , SolubilidadRESUMEN
Anti-idiotype reagents that recognize a common idiotype associated with the combining site of antibodies to hepatitis B surface antigen (anti-HBs) were used to manipulate the immune response to hepatitis B surface antigen in BALB/c mice. The injection of antibodies to the idiotype before antigenic stimulation resulted in an increase in the number of cells secreting immunoglobulin M antibodies to hepatitis B surface antigen. Anti-HBs-secreting cells were also induced by administration of antibodies to the idiotype without subsequent antigen exposure. These findings indicate that the immune response to hepatitis B surface antigen in mice is regulated through an idiotype-anti-idiotype network.
Asunto(s)
Anticuerpos Antivirales/biosíntesis , Anticuerpos contra la Hepatitis B/biosíntesis , Antígenos de Superficie de la Hepatitis B/inmunología , Animales , Anticuerpos Antiidiotipos/inmunología , Inmunoglobulina G/biosíntesis , Idiotipos de Inmunoglobulinas/inmunología , Inmunoglobulina M/biosíntesis , Ratones , Bazo/inmunología , Factores de TiempoRESUMEN
We have identified two distinct beta-myosin heavy chains (MHCs) present in baboon myocardium by electrophoresis in gradient pore gels and by Western blots with anti-MHC MAb. The two beta-MHCs have molecular masses of 210 and 200 kD and share several antigenic determinants including an epitope recognized by a beta-MHC-specific MAb. A fivefold increase in the level of the 200-kD beta-MHC was observed in the hypertrophied left ventricles of baboons with chronic (5.3 +/- 0.7 yr) renal hypertension. A 60% increase (P less than 0.01) in BP and a 100% increase (P less than 0.001) in left ventricular mass to body weight ratio occurred in hypertensive baboons compared with normotensive animals. The Ca2+-activated myosin ATPase activity in hypertrophied left ventricles was decreased by 35% (P less than 0.05) compared with controls. Normal levels of the 200-kD MHC were detected in the right ventricles and intraventricular septa of the hypertensive animals. These data suggest that cardiac MHCs of primates may exist in alternative molecular forms that are indistinguishable by nondenaturing gel electrophoresis and that increased concentration of a second beta-MHC is associated with ventricular hypertrophy (r = 0.55). The functional significance and mechanisms that control the concentration of beta-MHC subspecies remain to be determined.
Asunto(s)
Hipertensión Renal/metabolismo , Miocardio/análisis , Miosinas/aislamiento & purificación , Adenosina Trifosfatasas/metabolismo , Animales , Cardiomegalia/enzimología , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatología , Enfermedad Crónica , Modelos Animales de Enfermedad , Electroforesis en Gel de Poliacrilamida , Hipertensión Renal/enzimología , Peso Molecular , Contracción Miocárdica , Miocardio/enzimología , Miosinas/clasificación , Miosinas/fisiología , PapioRESUMEN
The molecular basis for charge heterogeneity in human hepatic alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) was determined by analysis of the carbohydrate and polypeptide components of the enzyme. Only small remnants of high mannose chains that contained neither sialic acid nor mannose 6-phosphate were detected in the carbohydrate structure. Four enzymatically active forms of alpha-glucosidase separated by chromatofocusing were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were found to contain different polypeptides. The absence of charged residues in oligosaccharide chains and variability in the polypeptide subunits of the charge forms of hepatic alpha-glucosidase suggest that charge heterogeneity results from differences in the protein structure of the charge forms. The pattern of differences in the polypeptide subunits suggests that the charge forms for hepatic alpha-glucosidase may be the product of proteolysis.
Asunto(s)
Glucosidasas/aislamiento & purificación , alfa-Glucosidasas/aislamiento & purificación , Electroquímica , Humanos , Focalización Isoeléctrica , Hígado/enzimología , Lisosomas/enzimología , Peso Molecular , Oligosacáridos/aislamiento & purificación , Fragmentos de Péptidos/aislamiento & purificaciónRESUMEN
OBJECTIVE: The aim was to determine the extent to which myosin heavy chain and light chain isoform transitions in atrial myocardium are coordinately regulated under pathological conditions in tissue from normal baboons, hypertensive baboons with myocardial hypertrophy, and baboons in which hypertrophy had regressed. METHODS: Quantitative distributions of myosin heavy chain (MHC) and regulatory myosin light chain (MLC2) isoforms in atrial myocardium from 35 adult baboons were determined by electrophoresis under denaturing conditions and laser densitometry. RESULTS: A significant association was observed between the ratios of MHC and MLC2 isoforms in atrial myocardium (r = 0.73, p < 0.001, n = 69). Expressions of alpha MHC and atrial MLC2 (ALC2) isoforms were correlated in atrial myocardium, as were those of beta MHC and ventricular MLC2 (VLC2) isoforms. In a subset of baboons with experimentally induced renal hypertension (n = 12) both beta MHC and VLC2 isoforms were found at higher levels in left atria than were present in normotensive baboons (p = 0.006, n = 15). Left atria from hypertensive baboons with regressed LVH contained intermediate levels of both beta MHC and VLC2 isoforms. CONCLUSIONS: There is tight coupling between the expression of myosin subunit isoforms under pathological conditions from a primate species closely related to humans. The data suggest that the synthesis of these subunits of myosin may be coordinated at the molecular level.
Asunto(s)
Cardiomegalia/metabolismo , Hipertensión Renal/metabolismo , Miocardio/metabolismo , Miosinas/metabolismo , Animales , Western Blotting , Densitometría , Electroforesis , Atrios Cardíacos/metabolismoRESUMEN
A variety of methods exist for the immortalization of B lymphocytes by Epstein-Barr virus due to the simplicity of such techniques to establish cell lines with stable genomic DNA. Two different methods for immortalizing lymphoblastoid cell lines were compared for differences in techniques and materials, time between initiation and immortalization, and success rate of immortalization. An incubation period in Epstein-Barr virus and the use of conditioned media improved immortalization efficiency from 86 to 98% and decreased the time (usually weeks) from culture initiation to cryopreservation. The resulting cell bank was used to produce DNA for genetic studies focusing on the genes involved in non-insulin-dependent diabetes mellitus.
Asunto(s)
Linfocitos B/citología , Transformación Celular Viral , Herpesvirus Humano 4 , Criopreservación , Medios de Cultivo Condicionados , HumanosRESUMEN
Corneal tumours were induced in almost 100% of grey, short-tailed South American opossums (Monodelphis domestica) exposed three times weekly to ultraviolet radiation (UVR) for periods of a year or more. Five tumours, representing the morphological spectrum of UVR-induced corneal tumours (two fibrosarcomas, one malignant fibrous histiocytoma, one putative haemangiosarcoma, and one squamous cell carcinoma overlying a sarcoma), were assayed immunohistochemically for reactivity with antibodies against the intermediate filaments vimentin, smooth muscle actin (alpha isoform), muscle-specific actins (alpha and gamma isoforms), desmin and cytokeratin, and with antibodies against the vascular endothelial marker von Willebrand factor. The squamous cell carcinoma was cytokeratin-positive. Other tumours were cytokeratin-negative and vimentin-positive. Three tumours had scattered individual cells and groups of cells immunoreactive with antibodies against smooth muscle actin and muscle-specific actins; two tumours (a fibrosarcoma and the malignant fibrous histiocytoma) had small numbers of desmin-positive cells. The putative haemangiosarcoma contained two populations of neoplastic cells, von Willebrand factor-positive vascular endothelial cells and smooth muscle actin-positive spindle cells. It was concluded (1) that UVR-induced corneal tumours may be composed of cells derived from resident epithelial cells, immigrant vascular endothelial cells, or fibroblast-like cells of unknown origin, and (2) that such tumours may contain more than one neoplastic cell type.
Asunto(s)
Córnea/efectos de la radiación , Neoplasias del Ojo/patología , Rayos Ultravioleta/efectos adversos , Actinas/análisis , Animales , Carcinoma de Células Escamosas/etiología , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Córnea/química , Córnea/patología , Desmina/análisis , Neoplasias del Ojo/etiología , Neoplasias del Ojo/metabolismo , Femenino , Fibrosarcoma/etiología , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Hemangiosarcoma/etiología , Hemangiosarcoma/metabolismo , Hemangiosarcoma/patología , Histiocitoma Fibroso Benigno/etiología , Histiocitoma Fibroso Benigno/metabolismo , Histiocitoma Fibroso Benigno/patología , Inmunohistoquímica , Queratinas/análisis , Masculino , Músculo Liso/química , Zarigüeyas , Traumatismos Experimentales por Radiación/etiología , Traumatismos Experimentales por Radiación/metabolismo , Traumatismos Experimentales por Radiación/patología , Vimentina/análisis , Factor de von Willebrand/análisisRESUMEN
A sensitive and specific immunological assay for detection of human lysosomal alpha-glucosidase was developed using a mouse monoclonal antibody incorporated into a biotin-avidin amplified ELISA. The immunoassay was more than 60 times more sensitive than the currently used enzymatic assay for alpha-glucosidase activity using a fluorimetric substrate. This methodology provides an alternative approach with increased sensitivity for screening individuals for alpha-glucosidase deficiency.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Glucosidasas/inmunología , alfa-Glucosidasas/inmunología , Avidina , Biotina , Ensayo de Inmunoadsorción Enzimática/métodos , Fibroblastos/enzimología , Humanos , Lisosomas/enzimologíaRESUMEN
Two transient, high molecular weight precursors of human acid alpha-glucosidase were detected by immune precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The high molecular weight precursors were rapidly converted into lower molecular weight forms corresponding to previously identified intermediates of acid alpha-glucosidase. An accumulation of these precursors was observed in fibroblasts treated with monensin and nigericin, suggesting that these precursors are intermediates of acid alpha-glucosidase undergoing transport through the Golgi complex.
Asunto(s)
Líquidos Corporales/metabolismo , Fibroblastos/enzimología , Glucosidasas/metabolismo , Líquido Intracelular/metabolismo , alfa-Glucosidasas/metabolismo , Animales , Transporte Biológico Activo , Electroforesis en Gel de Poliacrilamida , Humanos , Peso Molecular , Monensina/farmacología , Nigericina/farmacología , ConejosRESUMEN
A newly developed microtechnique for quantitating activity of myosin ATPase (EC 3.6.1.32) is more sensitive and less time-consuming than existing spectrophotometric methods. Measurement of ATPase activity using the new method can be accomplished in a final volume of 0.25 ml, allowing the assay to be conducted in individual wells of 96-well microplates commonly used for the enzyme-linked immunosorbent assay (ELISA). The microassay is performed by adding purified myosin to microplate wells followed by addition of ATP to initiate the enzymatic reaction. The reaction is subsequently terminated by addition of an acidic solution containing malachite green and ammonium molybdate. The level of inorganic phosphate produced by enzymatic hydrolysis of ATP is measured by scanning the microplates using a microELISA plate reader. An entire 96-well microplate can be scanned in less than 2 min, and data from the microassay can be transferred directly to a microprocessor for statistical analysis. The microassay is capable of detecting between 0.2 and 3 nmol of inorganic phosphate in a reaction volume of 50 microliter, and the ATPase activity of as little as 10 ng of rat cardiac myosin can be measured. The increased sensitivity compared with that of other spectrophotometric assays and ease of performing the microassay enable a detailed analysis of the enzymatic properties of cardiac myosin to be conducted on large numbers of small tissue specimens. Several kinetic properties of rat cardiac myosin were determined using this technique.
Asunto(s)
Adenosina Trifosfatasas/aislamiento & purificación , Animales , Humanos , Masculino , Microquímica , Miocardio/enzimología , Fosfatos/aislamiento & purificación , RatasRESUMEN
The further characterization of internal image anti-idiotypic antibodies (anti-Id) that represent a potential alternative vaccine candidate for type B viral hepatitis is described. The anti-Id preparation contains an internal image component or related epitope that mimics hepatitis B surface antigen (HBsAg) and binds to murine hybridoma cells that secrete antibodies to HBsAg (anti-HBs). This binding to anti-HBs-secreting hybridomas was partially inhibited by intact HBsAg particles and was associated with the expression of an interspecies idiotype. Immunoprecipitation studies demonstrated that the anti-Id bound to immunoglobulin molecules expressed on the surface of the hybridoma cells. These data suggest that internal image anti-Id, which induces an in vivo antibody response by antigenic mimicry in the absence of HBsAg, binds to anti-HBs molecules on the surface of cells actively secreting anti-HBs. The possible mechanism for internal image anti-Id-based antibody vaccines that mimic the overall conformation of antigens associated with infectious agents is discussed.
Asunto(s)
Antígenos de Superficie de la Hepatitis B/inmunología , Idiotipos de Inmunoglobulinas/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Animales , Unión Competitiva , Epítopos , Hibridomas/inmunología , Ratones , Conformación Proteica , Vacunas Virales/inmunologíaRESUMEN
The gene encoding human phenol-preferring phenol sulfotransferase (STP) has been cloned and mapped to chromosome 16p. A HindIII RFLP in this gene is described.
Asunto(s)
Arilsulfotransferasa/genética , Polimorfismo de Longitud del Fragmento de Restricción , Cromosomas Humanos Par 16 , Desoxirribonucleasa HindIII , Femenino , Humanos , Masculino , LinajeRESUMEN
A biotin-avidin-amplified enzyme-linked immunosorbent assay (B-A ELISA) has been developed to detect herpes simplex virus type 1 (HSV-1) and HSV-2 antigens in clinical specimens. The test was designed as a solid-phase, double-antibody, sandwich assay in which plates were coated with a polyclonal rabbit immunoglobulin G anti-HSV reagent, and the sandwich antibody was a biotin-labeled mouse immunoglobulin M monoclonal antibody that reacts with a common antigen associated with HSV-1 and HSV-2. The test can be completed in 4 h if antibody-coated plates are available. The detection limit of the B-A ELISA, determined by titration of virus stocks, was found to be approximately 90 PFU or 6 X 10(3) physical particles of either HSV-1 or HSV-2 per 50 microliter of virus stock. The following results were obtained in a study in which swabs were taken from a variety of lesions and assayed for infectivity in tissue culture and by B-A ELISA. Of 421 suspected HSV lesions tested, 69 were positive by both tests and 159 were negative by both tests. A total of 122 were positive by B-A ELISA but negative for infectivity. Seventy-one were negative by B-A ELISA but contained infectious virus. The HSV specificity of the assay was substantiated by partial blocking of reactivity with rabbit immunoglobulin G anti-HSV and by the absence of reactivity with a nonspecific biotin-labeled mouse immunoglobulin M monoclonal antibody.
Asunto(s)
Antígenos Virales/análisis , Ensayo de Inmunoadsorción Enzimática , Herpes Simple/diagnóstico , Técnicas para Inmunoenzimas , Simplexvirus/inmunología , Avidina , Biotina , HumanosRESUMEN
Hepatitis B surface antigen (HBsAg), produced by a human hepatoma which had been transplanted into athymic nude mice, was specifically detected in vivo by 131I-labeled monoclonal antibodies (McAb) directed against distinct epitopes of HBsAg (anti-HBs). Significantly higher levels of radioactivity were present in the hepatoma secreting HBsAg when compared to either a non-HBsAg producing epidermoid tumor or most other tissues obtained from nude mice treated with the 131I-labeled anti-Hbs McAb. A radiolabeled control McAb that did not recognize HBsAg failed to discriminate between either the HBsAg positive and negative tumors or other tissues from nude mice. These data demonstrate the in vivo immunological specificity of anti-HBs McAb for HBsAg associated with a hepatoma tumor.
Asunto(s)
Carcinoma Hepatocelular/inmunología , Antígenos de Superficie de la Hepatitis B/análisis , Neoplasias Hepáticas/inmunología , Animales , Anticuerpos Monoclonales , Línea Celular , Epítopos , Ratones , Ratones DesnudosRESUMEN
Two IgM monoclonal antibodies (McAb) that recognized distinct antigenic determinants of herpes simplex virus type 2 (HSV2) were shown to share idiotypes. Seven other IgM McAb generated to either HSV2 or the major envelope glycoprotein of HSV2, VP119, did not express this shared idiotype. The idiotypic determinants were associated with the antibody-combining site, because HSV2 inhibited the idiotype-anti-idiotype reactions. Anti-HSV2 sera raised in BALB/c mice also expressed the idiotype shared by the two anti-HSV2 McAb.
Asunto(s)
Anticuerpos Monoclonales/análisis , Anticuerpos Antivirales/análisis , Herpes Simple/inmunología , Idiotipos de Inmunoglobulinas/análisis , Animales , Anticuerpos Monoclonales/biosíntesis , Sitios de Unión de Anticuerpos , Unión Competitiva , Chlorocebus aethiops , Epítopos/análisis , Humanos , Idiotipos de Inmunoglobulinas/inmunología , Inmunoglobulina M , Ratones , Ratones Endogámicos BALB C , ConejosRESUMEN
Contact between sooty mangabeys (SMs) and a pigtailed macaque prompted the serological screening of SMs for evidence of infection with B virus. Serological tests detected SM antibodies that reacted with B virus polypeptides. Additional testing was performed with sera from SMs with no previous contact with macaques. Results from these tests indicated that 56% (33/59) of the SMs had antibodies that reacted with B virus and SA8. SM antibodies also reacted with herpesvirus papio 2 and to a lesser extent with human alpha herpesviruses (HSV-1 and HSV-2). There was an age-related increase in the presence of these antibodies in SMs that was consistent with the serological pattern of reactivity observed in other nonhuman primate species infected with alpha herpesviruses. These data suggest that SMs may be a host for a herpesvirus that is antigenically similar to those viruses present in other Old World nonhuman primates.
Asunto(s)
Cercocebus atys/virología , Infecciones por Herpesviridae/diagnóstico , Infecciones por Herpesviridae/veterinaria , Herpesviridae/aislamiento & purificación , Enfermedades de los Monos/diagnóstico , Enfermedades de los Monos/virología , Factores de Edad , Animales , Anticuerpos Antivirales , Especificidad de Anticuerpos , Antígenos Virales/inmunología , Western Blotting , Cercocebus atys/inmunología , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Herpesviridae/inmunología , Infecciones por Herpesviridae/inmunología , Humanos , Inmunohistoquímica , Masculino , Enfermedades de los Monos/inmunología , Pruebas SerológicasRESUMEN
Antiidiotype reagents that recognize idiotypic determinants associated with the combining site of monoclonal antibodies to herpes simplex virus type 2 ( HSV2 ) were used to manipulate the immune response to HSV2 in BALB/c mice. The injection of antiidiotype antibodies into mice before challenge with a 50% lethal dose of HSV2 resulted in a shorter survival time than that of mice receiving either preimmune rabbit immunoglobulin G or antiidiotype reagents against hepatitis B surface antigen before HSV2 challenge. These findings indicate that the immune response to HSV2 in mice can be modulated through idiotype- antiidiotype networks, thereby increasing the pathogenicity of HSV2 infections.
Asunto(s)
Herpes Simple/inmunología , Inmunidad Activa , Idiotipos de Inmunoglobulinas/inmunología , Simplexvirus/inmunología , Animales , Anticuerpos Monoclonales , Epítopos/análisis , Ratones , Ratones Endogámicos BALB C , Simplexvirus/patogenicidadRESUMEN
17 monoclonal antibodies generated against purified hepatitis B surface antigen (HBsAg), subtype ayw, were characterized by solid-phase radioimmunoassays. Eleven of these antibodies had specificity against the group-specific alpha determinant of HBsAg, two demonstrated antibody activity against the w HBsAg subtype, one against human serum albumin, and three against human IgG. All monoclonal antibodies were of the IgG class.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos contra la Hepatitis B/análisis , Antígenos de Superficie de la Hepatitis B/inmunología , Especificidad de Anticuerpos , Reacciones Cruzadas , Epítopos , Inmunoglobulina G/inmunología , Albúmina Sérica/inmunologíaRESUMEN
To examine cardiac myosin gene structure and expression in a non-human primate model for human heart development and disease, we have constructed a cDNA library from baboon atrium and used baboon beta-myosin heavy chain (beta-MHC)* cDNA probes to isolate atrial MHC clones. The nucleotide sequence of one such clone, lambda BMHC alpha 3, contains sequences that encode part of the light meromyosin region (LMM) and the 3' untranslated region of the baboon alpha-MHC. To study cardiac MHC gene transcription, we constructed probes from the baboon alpha-MHC cDNA for S1 nuclease analyses of RNA from atria and ventricles. To examine translational regulation of cardiac MHC gene expression, we used monoclonal antibodies (MAb) against specific alpha- and beta-MHC epitopes for Western blot analyses. In atria and ventricles from adult baboons, we detected predominantly alpha- and beta-MHC gene transcripts, respectively. In ventricles from fetal baboons at two stages of development (140 and 160 days gestation), we also detected predominantly beta-MHC gene transcripts and isoforms. To investigate changes induced by parturition, we obtained ventricles from baboons that were prematurely delivered at 140 days gestation and supported for 10 days in an extrauterine environment. In contrast to adult and fetal patterns, we observed an increase in alpha-MHC transcripts and isoforms in ventricles of premature baboons. Because alpha-MHC gene expression is increased in premature baboons (total age of 150 days) compared to their older 160 day fetal counterparts, the induction of ventricular alpha-MHC synthesis must have resulted from factor(s) associated with parturition or prolonged mechanical ventilation rather than at predetermined stages of gestational development.