The beta1 adrenergic effects of antibodies against the C-terminal end of the ribosomal P2beta protein of Trypanosoma cruzi associate with a specific pattern of epitope recognition.

BALB/c mice immunized with recombinant Trypanosoma cruzi ribosomal P2beta protein (TcP2beta) develop a strong and specific antibody response against its 13 residue-long C-terminal epitope (peptide R13: EEEDDDMGFGLFD) that has a concomitant beta1-adrenergic stimulating activity. However, other animals that undergo similar immunizations seem tolerant to this epitope. To evaluate further the antibody response against the ribosomal P proteins, 25 BALB/c and 25 Swiss mice were immunized with TcP2beta. From the 50 animals, 31 developed a positive anti-R13 response, whereas 19 were non-responsive. From the 31 anti-R13 positive mice, 25 had anti-R13 antibodies that recognized the discontinuous motif ExDDxGF, and their presence correlated with the recording of supraventricular tachycardia. The other six had anti-R13 antibodies but with a normal electrocardiographic recording. These anti-R13 antibodies recognized the motif DDxGF shared by mammals and T. cruzi and proved to be a true anti-P autoantibody because they were similar to those elicited in Swiss, but not in BALB/c mice, by immunization with the C-terminal portion of the mouse ribosomal P protein. Our results show that the recognition of the glutamic acid in position 3 of peptide R13 defines the ability of anti-R13 antibodies to react with the motif AESDE of the second extracellular loop of the beta1-adrenergic receptor, setting the molecular basis for their pathogenic beta1 adrenoceptor stimulating activity.

Increased pituitary vascular endothelial growth factor-a in dopaminergic D2 receptor knockout female mice.

Vascular endothelial growth factor (VEGF)-A is an important angiogenic cytokine in cancer and pathological angiogenesis and has been related to the antiangiogenic activity of dopamine in endothelial cells. We investigated VEGF expression, localization, and function in pituitary hyperplasia of dopamine D2 receptor (D2R)-knockout female mice. Pituitaries from knockout mice showed increased protein and mRNA VEGF-A expression when compared with wild-type mice. In wild-type mice, prolonged treatment with the D2R antagonist, haloperidol, enhanced pituitary VEGF expression and prolactin release, suggesting that dopamine inhibits pituitary VEGF expression. VEGF expression was also increased in pituitary cells from knockout mice, even though these cells proliferated less in vitro when compared with wild-type cells, as determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium proliferation assay, proliferating cell nuclear antigen expression, and [(3)H]thymidine incorporation. In contrast to other animal models, estrogen did not increase pituitary VEGF protein and mRNA expression and lowered serum prolactin secretion in vivo and in vitro in both genotypes. VEGF (10 and 30 ng/ml) did not modify pituitary cell proliferation in either genotype and increased prolactin secretion in vitro in estrogen-pretreated cells of both genotypes. But conditioned media from D2R(-/-) cells enhanced human umbilical vein cell proliferation, and this effect could be partially inhibited by an anti-VEGF antiserum. Finally, using dual-labeling immunofluorescence and confocal laser microscopy, we found that in the hyperplastic pituitaries, VEGF-A was mostly present in follicle-stellate cells. In conclusion, pituitary VEGF expression is under dopaminergic control, and even though VEGF does not promote pituitary cellular proliferation in vitro, it may be critical for pituitary angiogenesis through paracrine actions in the D2R knockout female mice.

H1 and H2 histamine receptors mediate the production of inositol phosphates but not cAMP in human breast epithelial cells.

OBJECTIVE: In the present work we studied the H1 and H2 histamine receptor expression and function in HBL-100 and MCF-10A cells, derived from non-tumorigenic human breast epithelia, and in MCF-10T, the H-ras-transfected MCF-10A counterpart. The signal transduction pathways associated with these receptors, and the expression of proto-oncogenes c-fos, c-myc and c-jun at the mRNA and protein levels, were examined. RESULTS: Saturation analysis using intact cells, showed two binding sites for [3H]tiotidine and [3H]mepyramine. Pretreatment of purified membrane with guanosine 5'-ythiotriphosphate resulted in the loss of the low affinity component for [3H]tiotidine binding, and of the high affinity component for [3H]mepyramine. In both cases, there was no modification in the total number of sites for both ligands. Neither H1 nor H2 agonists stimulated cyclic AMP production, though this pathway is functional in these cells. On the other hand, both H1 and H2 agonists enhanced phosphoinositide turnover in a dose-dependent fashion, and this induction is pertussis toxin-insensitive. H1 and H2 agonists did not influence the expression of c-myc or c-fos mRNA, nor their encoded proteins. CONCLUSIONS: These results indicate that the three cell lines examined showed functional H1 and H2 histamine receptors, which are involved in the metabolic turnover of inositol phosphates but are ineffective in the modulation of the cyclic AMP response. The fact that H2 receptors have lost their ability to stimulate cyclic AMP production would imply the loss of a regulatory mechanism of cell growth.

Histamine H2 receptor desensitization: involvement of a select array of G protein-coupled receptor kinases.

The histamine H2 receptor (H2r) belongs to the heptahelical receptor family; upon agonist binding, members of this family activate a G protein and the downstream effector adenylyl cyclase. Like other G protein-coupled receptors, exposure of H2r to agonists produces a desensitization of the response. The present study focused on the desensitization mechanism of this receptor. Using transiently transfected COS-7 cells expressing tagged-H2r, the desensitization induced by amthamine, characterized by decreased cAMP production, was studied. Results show that the receptor was rapidly desensitized with a t(1/2) = 0.49 +/- 0.01 min. Because of the rapid nature of H2r desensitization, receptor phosphorylation was examined as a likely mechanism for signal attenuation. H2r desensitization was not affected by protein kinases A and C (PKA and PKC) inhibitors but was remarkably reduced by Zn(2+), an inhibitor of G protein-coupled receptor kinases (GRKs). Cotransfection experiments using tagged H2r and different GRKs (2, 3, 5, or 6), demonstrated that GRK2 and GRK3 were the most potent in augmenting desensitization, causing a reduction in the maximal response to amthamine and a decrease of the t(1/2) for desensitization, whereas GRK5 and GRK6 did not affect the signaling. Receptor phosphorylation correlates with desensitization for each GRK studied, whereas phosphorylation that is dependent on protein kinases A and C seemed irrelevant in receptor signal termination. These results indicate that in H2r-transfected COS-7 cells, exposure to an agonist caused desensitization controlled by H2r phosphorylation via GRK2 and GRK3.

Receptores histamínicos H2, AMPc y diferenciación celular leucémica / H2 histaminics receptora, AMPc and leukemia celular diferenciation

Con el objeto de explorar su potencial empleo en terapéutica no citotóxicas, se estudió, en células leucémicas humanas, la vía de señalización y probable rol regulatorio del receptor a histamina H2. Mediante ensayos de binding, se detectaron sitios de unión específica de tipo H2, en casi todas las muestras de M.O.y S.P. de pacientes con L.A., con diferentes grados de infiltración. esto sugiere la presencia del receptor H2, en células hemopoyéticas normales y transformadas. La líneas U-937, modelo de célula monoblástica, presenta receptores H2, acoplados a AMPc. Su estímulo no produjo cambios proliferativos, ni deferenciación celular, pero sí un aumento transitorio, vía proteín kinasa A (PKA), en la expresión de Fos y Hun, sin reducción de Myc. Se hipotizó que el fracaso del estímulo H2, para diferenciar las células U-937 podría deberse a que su activación de la PKA es breve. Concordante con lo anterior, los receptores H2, mostraron una veloz de desensibilización homóloga (T. 1/2 = 20ï). En cambio la forskolina, un activador directo de la adenil ciclasa, no desensibilizó su estímulo ni aún después de 24 horas de incubación. La forskolina también inhibió la proliferación U-937 a las mismas concentraciones en que estimuló la síntesisde AMPc e indujo su diferenciación fagocitaria, con reducción del NBT y respuesta quimiotáctica al C5a. Conclusiones: 1) La desensibilización veloz de un receptor que transduce una señal diferenciadora, como el H2, en las células U-937, podría ser un mecanismo fisiopatogénico de la malignificación, al bloquear la recepción de estímulos madurativos por la célula neoplásica. 2) Dados estos resultados, y los efectos diferenciadores del dibutril AMPc (DBAMPc) en líneas celulares mieloides, los agentes que elevan el AMPc merecen ser valorados en la terapia de las LMA

Receptores histamínicos H2, AMPc y diferenciación celular leucémica / H2 histaminics receptora, AMPc and leukemia celular diferenciation

Con el objeto de explorar su potencial empleo en terapéutica no citotóxicas, se estudió, en células leucémicas humanas, la vía de señalización y probable rol regulatorio del receptor a histamina H2. Mediante ensayos de binding, se detectaron sitios de unión específica de tipo H2, en casi todas las muestras de M.O.y S.P. de pacientes con L.A., con diferentes grados de infiltración. esto sugiere la presencia del receptor H2, en células hemopoyéticas normales y transformadas. La líneas U-937, modelo de célula monoblástica, presenta receptores H2, acoplados a AMPc. Su estímulo no produjo cambios proliferativos, ni deferenciación celular, pero sí un aumento transitorio, vía proteín kinasa A (PKA), en la expresión de Fos y Hun, sin reducción de Myc. Se hipotizó que el fracaso del estímulo H2, para diferenciar las células U-937 podría deberse a que su activación de la PKA es breve. Concordante con lo anterior, los receptores H2, mostraron una veloz de desensibilización homóloga (T. 1/2 = 20´). En cambio la forskolina, un activador directo de la adenil ciclasa, no desensibilizó su estímulo ni aún después de 24 horas de incubación. La forskolina también inhibió la proliferación U-937 a las mismas concentraciones en que estimuló la síntesisde AMPc e indujo su diferenciación fagocitaria, con reducción del NBT y respuesta quimiotáctica al C5a. Conclusiones: 1) La desensibilización veloz de un receptor que transduce una señal diferenciadora, como el H2, en las células U-937, podría ser un mecanismo fisiopatogénico de la malignificación, al bloquear la recepción de estímulos madurativos por la célula neoplásica. 2) Dados estos resultados, y los efectos diferenciadores del dibutril AMPc (DBAMPc) en líneas celulares mieloides, los agentes que elevan el AMPc merecen ser valorados en la terapia de las LMA (AU)

Rapid desensitization and slow recovery of the cyclic AMP response mediated by histamine H(2) receptors in the U937 cell line.

The present study focused on the desensitization process of the H(2) receptor in U937 cells and the recovery of the cyclic AMP (cAMP) response. Treatment of U937 leukemic cells with the H(2) histamine receptor agonists (+/-)-N(1)-[3-(3, 4-difluorophenyl)-3-(pyridin-2-yl)propyl]-N(2)-[3-(1H-imidazol-4-yl)p ropyl]guanidine (BU-E-75) and amthamine produced a rapid desensitization characterized by decreased cAMP production (T(1/2) = 20 min). Pretreatment with 10 microM BU-E-75 did not induce modifications in the responses to prostaglandin E(2), isoproterenol, or forskolin. H(2) receptor desensitization was not affected by protein kinase A and C inhibitors, but was reduced drastically by Zn(2+) and heparin, known to act as inhibitors of G protein-coupled receptor kinases. Recovery studies of the cAMP response showed that cAMP levels reached 50% of the initial values within 5 hr. Furthermore, desensitization produced an important decrease in the basal level of this cyclic nucleotide. The minimal value was observed 12 hr later, and corresponded to approximately 1.3% of the initial basal level (7.5 vs 0.1 pmol/10(6) cells). This result could be explained by an increase in phosphodiesterase activity following 10 microM BU-E-75 treatment. When cells were exposed for 2 hr to an H(2) agonist, binding assays showed no modification in the number of H(2) receptors; internalization began just after 8 hr. Although the initial desensitization seems to involve G protein-coupled receptor kinases, results indicate that additional mechanisms of regulation were triggered by the H(2) agonists.

Forskolin induces U937 cell line differentiation as a result of a sustained cAMP elevation.

The present study examines the effects of forskolin on U937 cell differentiation. We recently reported that dibutyryl cAMP (dbcAMP), but not cAMP-elevating agents such as histamine, promotes U937 cell differentiation. cAMP production elicited by stimulation of histamine H2 receptors showed a rapid, homologous desensitization, which might explain the dissimilar responses to histamine and dbcAMP. Forskolin induced an increase in cAMP levels in a concentration-dependent manner (EC50=30 microM) for an extended period of at least 24 h. Forskolin but not histamine (up to 100 microM), also inhibited cell growth in a dose-dependent fashion (EC50=22 microM). After 3 days of incubation, 75 microM forskolin induced U937 cell differentiation as judged by an increased rate of reduction of nitrobluetetrazolium (mean+/-S.E.M.: 21.3+/-6.6% in treated cells vs. 3.2+/-1.9% in the control group, P < 0.001) and an augmented chemotactic response to complement 5a (C5a) (33.2+/-5.9% in forskolin-treated vs. 0.34+/-0.12% in control cells, P < 0.01). Furthermore, c-Myc levels decreased following forskolin treatment, while the histamine H2 receptor agonist dimaprit had no effect. We conclude that forskolin induces U937 cell differentiation through a sustained rise in cAMP levels.

Expression of a recombinant Fab antibody fragment against cruzipain, the major cysteine proteinase of Trypanosoma cruzi.

Cruzipain, the major proteinase of Trypanosoma cruzi, plays an important role in the biology of this parasite. This study reports the development of a recombinant Fab antibody, using RNA isolated from the anti-Ag163B6 hybridoma against cruzipain. This procedure involves the use of cDNAs obtained with the aid of a specific set of primers complementary to the complete light kappa chain (L kappa) and the first two domains of the IgG1 heavy chain (VH/CH1). These products were subsequently cloned in the pComb3 system, from which the gIII gene had been removed, and expressed in Escherichia coli cells. The recombinant Fab molecule recognized cruzipain by ELISA, in a fashion similar to the original mAb anti-Ag163B6. Nucleotide sequence analysis of the recombinant molecule, together with its immunological recognition by specific anti-mouse IgG (Fab)2, indicated the immunoglobulin nature of the recombinant product. Moreover, both the mAb anti-Ag163B6 and the soluble Fab fragment described here react similarly with the intact parasite surface, as observed in an indirect immunofluorescence assay. In conclusion, our recombinant Fab anti-Ag 163B6 allows the possible use of this molecule for diagnosis, antigen purification, and eventually treatment of Chagas-afflicted individuals.

Differentiating agents modulate topoisomerase I activity in U-937 promonocytic cells.

Phorbol 12-myristate 13-acetate (PMA), N,N'-hexamethylenebisacetamide (HMBA) and retinoic acid induce cell differentiation in U-937 promonocytic cells. This report examines the effects of these agents on DNA topoisomerase I activity. A decrease in enzyme activity could be detected as early as 30 min after treatment with all three differentiating compounds and lasted at least 48 h. No alteration in the levels of DNA topoisomerase I transcript or protein was observed during these treatments. The results might be explained by post-translational events that render DNA topoisomerase type I less active.

[Rupture of sinus of Valsalva aneurysm]. / Rotura de aneurisma do seio de Valsalva.

A 35 year-old man with aneurysm of the noncoronary sinus of Valsalva ruptured into the right atrium, detected by echodopplercardiogram and submitted to surgical correction is reported. The authors discuss peculiar aspects of this disease, with emphasis to the echodopplercardiographic diagnosis.

Monoclonal antibodies against human breast tumor-associated antigens: characterization of B21 antigen.

BACKGROUND: Monoclonal antibodies (MAbs) show promise in the early detection and monitoring of cancer and may have therapeutic applications as well. PURPOSE: The purpose of this study was to characterize MAb B21, a novel murine-derived antibody that has strong reactivity with MCF-7 and T47D cell lines from human breast cancer. METHODS: A number of MAbs that react with breast cancer cell lines were obtained from cultured mouse spleen cells, and one, MAb B21, was selected for detailed analysis. MAb B21 was characterized by immunocytochemical, immunofluorescence, immunoprecipitation, and Western blotting procedures. RESULTS: We found a strong reactivity of MAb B21 with cultured breast cancer cells and cells from human breast tumors, although some reactivity was seen sporadically in non-breast or normal tissue. Negligible reactivity was detected in a series of non-breast cell lines and with normal breast epithelial cell line MCF-10A. However, when MCF-10A cells were permeabilized, allowing the antibody to penetrate within the cells, reaction became apparent. MCF-10A cells, when transfected with the oncogene c-Ha-ras (MCF-10T), gave a positive immunostaining similar to that observed with MCF-7 and T47D cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of L-[35S]methionine-labeled MCF-7 and T47D cell extracts showed distinct immunoprecipitated components, with molecular weight values ranging from 150,000 to 20,000 with the addition of MAb B21. Western blot assays using MAb B21 of SDS-PAGE fractionated/electroblotted proteins from breast cancer cell lines and MCF-10A cells showed specific reaction with a 95,000 component. CONCLUSIONS: Our results indicate that B21 antigen is expressed in neoplastic cells of epithelial origin, mainly breast cancer, and to a minor extent in other cell lines. In addition, MAb B21 recognizes an antigen that is differentially localized during cell transformation. IMPLICATIONS: Our future studies will address the full characterization of MAb B21 and explore its capacity as a tool for therapeutic manipulation.

Vacuolar apical compartment (VAC) in breast carcinoma cell lines (MCF-7 and T47D): failure of the cell-cell regulated exocytosis mechanism of apical membrane.

We have previously shown that an integral plasma membrane glycoprotein (AP2) is highly polarized to the apical domain in confluent Madin-Darby canine kidney (MDCK) epithelial cells. However, when the monolayers are prevented from forming intercellular contacts, approximately 60% of the AP2 cellular content is stored in the intracellular vacuolar apical compartment (VAC). In the current work we found that AP2 was present in the non-tumorigenic human mammary epithelial cell line MCF-10A, in the breast carcinoma cell lines MCF-7 and T47D, and in breast ductal carcinomas in vivo. By radioimmunoassay, an intracellular compartment of AP2 was identified in the mammary cell lines in culture. In MCF-10A, this compartment behaved as in MDCK cells; namely it was observed only when the cells cannot form cell-cell contacts. However, in the carcinoma cell lines MCF-7 and T47D, a significant AP2 intracellular compartment was observed also under conditions permissive for the formation of intercellular contacts. These results were confirmed by immunofluorescence and immunoelectron microscopy experiments that showed VACs in MCF-7 and T47D, even in cells with extensive intercellular contacts. In MCF-7 cells, the addition of serum caused a partial decrease of the AP2 intracellular compartment. The exocytosis of VACs occurred towards the center of multi-cellular groups, forming intercellular lumens, similar to those transiently observed in MDCK cells and to structures described by others during embryo development. Altogether, these results suggest that VAC exocytosis is controlled by cell-cell contact signalling, which may be defective in carcinoma cells.

Increment of DNA topoisomerases in chemically and virally transformed cells.

The activities of topoisomerases I and II were assayed in subcellular extracts obtained from nontumorigenic BALB/c 3T3 A31 and normal rat kidney (NRK) cell lines and from the same cells transformed by benzo[a]pyrene (BP-A31), Moloney (M-MSV-A31) and Kirsten (K-A31) sarcoma viruses, and simian virus 40 (SV-NRK). The enzymatic activity of topoisomerase I was monitored by the relaxation of negatively supercoiled pBR322 DNA and by the formation of covalent complexes between 32P-labeled DNA and topoisomerase I. Topoisomerase II activity was determined by decatenation of kinetoplast DNA (k-DNA). It was found that nuclear and cytoplasmic type I topoisomerase specific activities were higher in every transformed cell line than in the normal counterparts. These differences cannot be attributed to an inhibitory factor present in A31 cells. When chromatin was treated at increasing ionic strengths, the 0.4 M NaCl extract showed the highest topoisomerase I specific activity. Moreover, in this fraction the transformed cells exhibited the most significant increment in the enzymatic activity as compared with nontransformed cultures. Spontaneously transformed A31 cells showed topoisomerase I activity similar to that of extracts of cells transformed by benzo[a]pyrene. Topoisomerase II specific activity was also increased in SV-NRK cells, as judged by the assay for decatenation of k-DNA to yield minicircle DNA.

Informe especial: estudios tendientes a determinar las posibles propiedades antitumorales del veneno de cobra y del complejo crotoxina A y B / Special report: studies to determine the possible antitumoral properities of Cobra venom and of crotoxin complex A and B

La presente investigación fue encomendada por el CONICET (Consejo Nacional de Investigaciones Cientificas y Técnicas) para determinar las posibles propriedades antineoplásicas del veneno de Cobra (Naja Naja Siamensis) VC) y del Complejo Crotoxina a y B (CCAB), purificado a partir del veneno de Crotalus durissus terificus (cascavel sudamericana). La coordinación de las investigaciones estuvo a cargo de los Dres. Baldi y Mordoh, y la ejecución de la parte experimental fue llevada a cabo por los restantes autores. Se utilizaron diversos sistemas experimentales: 1) lúneas celulares in vitro: de origen murino, normales (3T3 A31), o transformadas (M-A31, Ki-A31 y BP-A31) o de origen humano (adenocarcinoma mamario: MCF-7 y T47D). el efecto de las drogas fue determinado a 1, 10 y 100 ng/ml de VC y CCAB, solas o en combinación. En ninguna de las líneas celulares mencionadas se observaron cambios significativos en la velocidad de crecimiento o en la morfología celular; 2) desarrollo in vivo del Sarcoma 180 (S180) en ratones Balb/c: el VC a dosis de 21 ó 26 ng/g (inyecciones i.p. a los días 10, 11, 20, 21, 30 y 31 post-inoculo tumoral) no afectaron el crecimiento ni la sobrevida de los animales. Dosis de CCAB de 6 ng/g o 9 ng/g ]con el mismo esquema de inoculación anterior) no afectaron el crecimiento de s180 hasta los 25 días de desarrollo tumoral. Entre los días 25 y 30 la dosis de 9 ng/g determinó una evolución tumoral más rápida. La combinación de 13 ng/g VC y 4,5 ng/g CCAB con iguales tiempos de inyección no afectaron el desarrollo tumoral. La histología de los tumores de los animales tratados y controles no evidenció cambios significativos; 3) desarrollo in vivo del carcinoma de Ehrlich en ratones Swiss: se probó el efecto de CCAB (9 ng/g inyectados i.p. a los días 8, 9, 16, y 17) sin obtenerse modificación del desarrollo tumoral;...