RESUMEN
Prostate cancer cells are characterized by a remarkably low proliferative rate and the production of high levels of prostate-specific proteases. Protein-based toxins are attractive candidates for prostate cancer therapy because they kill cells via proliferation-independent mechanisms. However, the non-specific cytotoxicity of these potent cytotoxins must be redirected to avoid toxicity to normal tissues. Prostate-Specific Membrane Antigen (PSMA) is membrane-bound carboxypeptidase that is highly expressed by prostate cancer cells. Potent dipeptide PSMA inhibitors have been developed that can selectively deliver and concentrate imaging agents within prostate cancer cells based on continuous PSMA internalization and endosomal cycling. On this basis, we conjugated a PSMA inhibitor to the apoptosis-inducing human protease Granzyme B and the potent Pseudomonas exotoxin protein toxin fragment, PE35. We assessed selective PSMA binding and entrance into tumor cell to induce cell death. We demonstrated these agents selectively bound to PSMA and became internalized. PSMA-targeted PE35 toxin was selectively toxic to PSMA producing cells in vitro. Intratumoral and intravenous administration of this toxin produced marked tumor killing of PSMA-producing xenografts with minimal host toxicity. These studies demonstrate that urea-based PSMA inhibitors represent a simpler, less expensive alternative to antibodies as a means to deliver cytotoxic proteins to prostate cancer cells.
Asunto(s)
Sistemas de Liberación de Medicamentos , Inmunotoxinas/administración & dosificación , Calicreínas , Antígeno Prostático Específico , Neoplasias de la Próstata/tratamiento farmacológico , Urea , Carboxipeptidasas/metabolismo , Línea Celular Tumoral , Humanos , Calicreínas/antagonistas & inhibidores , Calicreínas/metabolismo , Masculino , Antígeno Prostático Específico/antagonistas & inhibidores , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/metabolismoAsunto(s)
Janus Quinasa 2/genética , Neutrófilos/metabolismo , Trombocitosis/diagnóstico , Trombocitosis/genética , Trombosis/etiología , Trombosis/genética , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Plaquetas/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factores de Tiempo , Resultado del TratamientoRESUMEN
The human platelet alloantigen 1 system (HPA-1) is determined by a polymorphism at position 33 in the N-terminus of human glycoprotein IIIa (GPIIIa). This naturally occurring substitution creates a conformation in the HPA-1a allelic form that can be antigenic when presented to an individual expressing the HPA-1b form. Anti-HPA-1a antibodies generated by this immune response can lead to the destruction of platelets, as seen in the clinical disorders, neonatal alloimmune thrombocytopenia (NAIT) and posttransfusion purpura (PTP). To understand better the structural requirements for recognition by these pathogenic antibodies, we investigated the N-terminal 66 amino acids from the HPA-1a form of human GPIIIa and the analogous amino acids from the nonimmunogenic murine homolog. Our objectives were to define further the boundaries of the HPA-1a epitope(s) in the N-terminus of human GPIIIa, to isolate the murine 5' nucleotide sequence and compare the deduced murine N-terminal sequence to that of human, and to mutate the murine sequence systematically to include an HPA-1a epitope(s). Murine amino acids that differed from human were changed by site-directed mutagenesis to the analogous residues in the HPA-1a form of human GPIIIa, starting and radiating from murine position 33 (site of human polymorphism). This systematic approach allowed us to pinpoint amino acids critical to a conformation recognized by anti-HPA-1a antibodies. Our results show that an HPA-1a epitope can be created within the N-terminus of murine GPIIIa and raise the possibility that murine models of HPA-1a sensitization can be developed.
Asunto(s)
Antígenos de Plaqueta Humana/química , Antígenos de Plaqueta Humana/inmunología , Epítopos/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Anticuerpos Monoclonales , Sitios de Unión de Anticuerpos , Unión Competitiva , Humanos , Integrina beta3 , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N-terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.
Asunto(s)
Glicoproteínas de Membrana Plaquetaria/biosíntesis , Proteínas Recombinantes/biosíntesis , Alelos , Anticuerpos/aislamiento & purificación , Enfermedades Autoinmunes/sangre , Secuencia de Bases , Cromatografía de Afinidad , Clonación Molecular , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Epítopos/biosíntesis , Epítopos/aislamiento & purificación , Glutatión Transferasa/biosíntesis , Humanos , Recién Nacido , Datos de Secuencia Molecular , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Señales de Clasificación de Proteína/biosíntesis , Señales de Clasificación de Proteína/genética , Púrpura/sangre , Púrpura/etiología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Trombocitopenia/sangre , Trombocitopenia/inmunología , Reacción a la TransfusiónRESUMEN
We have used 75% to 90% pure murine erythroid colony-forming units (CFU-E) to delineate the processes and factors underlying their maturation. These CFU-E form 32 cell colonies and are drawn from what we term generation I of a six-generation long maturation sequence (Landschulz et al, Blood 79:2749, 1992). Applying assays of 59Fe-heme biosynthesis and colony numbers as measures of maturation and analyses of DNA degradation as an index of programmed cell death, we find that (1) erythropoietin (Epo) enhances maturation throughout most of its course; (2) Epo first seems able to forestall DNA degradation when CFU-E reach generation II; (3) the processes that Epo elicits thereafter start to persist when Epo is withdrawn; (4) insulin-like growth factor I (IGF-I) also forestalls DNA breakdown, but later loses effectiveness; (5) IGF-I adds little to maturation when Epo levels are high, but when Epo levels are low, enhances it substantially; and (6) for maturation to be entirely optimal, an unidentified serum factor(s) is probably required when Epo levels are high and is certainly needed when Epo levels are like those in normal animals. Quantitatively, about 40% of optimal in vitro erythropoiesis at normal Epo levels depends on Epo alone, another 30% or less on the addition of IGF-I, and the remaining 30% or more on the addition of unidentified serum factor(s). Applied together, these three or more factors lead to two-thirds of the maximum maturation realized with saturating Epo levels. Because we also find that heme accumulated in CFU-E culture can closely approach levels in red blood cells, we suppose that our conclusions apply as well to CFU-E maturation in vivo.
Asunto(s)
Células Precursoras Eritroides/citología , Eritropoyetina/farmacología , Sustancias de Crecimiento/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Animales , Sangre , Recuento de Células , División Celular , ADN/metabolismo , Células Precursoras Eritroides/metabolismo , Hemo/biosíntesis , Humanos , Ratones , Proteínas Recombinantes/farmacologíaRESUMEN
Murine erythroid colony-forming units (CFU-E) representing successive cell generations in a six-generation long in vitro maturation sequence were tested for their response to erythropoietin (Epo) by measurement of Epo-exposure times necessary to stimulate heme biosynthesis. Generation I CFU-E, which produce mainly 32-cell erythroid colonies, were isolated in 82% average purity from spleens of thiamphenicol-treated anemic animals via differential centrifugation. Generation II CFU-E, which produce mainly 16-cell colonies, were similarly isolated in 51% average purity. Although both types of CFU-E had equivalent dose sensitivity to and affinity for Epo, generation II CFU-E responded to shorter pulses of Epo than did generation I. Correlations between DNA cell-cycle profiles and 59Fe-heme biosynthesis resulting from pulsed exposures established that appreciable Epo response only begins when CFU-E attain early S-phase of generation II. Because CFU-E did not require Epo or other serum factors to pass from generation I to II and because the onset of Epo responsiveness coincided with the beginning of DNA replication in generation II, we suppose that differentiation has reprogrammed one or more of the events associated with generation II S-phase in CFU-E and that these alterations allow Epo to act. Further comparisons between CFU-E from generation I and II may allow us to identify the alterations in question and the nature of their interaction with Epo.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Células Precursoras Eritroides/citología , Eritropoyetina/farmacología , Animales , Separación Celular , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Células Precursoras Eritroides/efectos de los fármacos , Eritropoyetina/metabolismo , Cinética , Ratones , Receptores de Superficie Celular/metabolismo , Receptores de Eritropoyetina , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Fase S/efectos de los fármacos , Factores de TiempoRESUMEN
Erythropoietin (Epo) response and binding was assessed in purified murine CFU-E and their descendants. Several features emerged. First, Epo on CFU-E is in rapid flux: Half-time for 125I-Epo internalization is approximately four to five minutes. Second, computer-aided Scatchard analyses indicate that greater than 70 high-affinity Epo-receptor sites on anemic animal CFU-E are sometimes already occupied by Epo acquired in vivo. When this is removed, 40% of greater than or equal to 370 sites per CFU-E belong to a high-affinity class (dissociation constant, kd: 73 pmol/L +/- 15 [SE]) and 60% belong to a low-affinity class (kd: 813 pmol/L +/- 246). Third, the few small colonies that develop from CFU-E in the absence of Epo are shown, by serial assay of 59Fe-heme biosynthesis, to stem from contaminating erythroblasts: a result consistent with our finding that, after eight-hour CFU-E culture, most erythroblasts no longer require appreciable Epo for growth. Thus, although the early need for Epo by CFU-E is nearly absolute, this need is not met by the often substantial Epo already on board. The inference is that repeated occupancy of the rapidly turning over Epo receptors is required. Fourth, Epo bound and/or internalized by CFU-E descendants decreases to 40% of zero-time levels after 14 hours in Epo-supplemented culture and disappears after 28 hours. Scatchard analyses indicate that 73 pmol/L kd receptor sites become undetectable at seven to eight hours, whereas 813 pmol/L kd sites are undiminished and only one-third less by 16 hours. This apparent disappearance of high-affinity sites and persistence of low-affinity sites suggests that (a) at least two gene products mediate Epo binding, eg, two different receptor polypeptides or one receptor and one cofactor which modulates affinity; (b) high-affinity sites mediate the growth function of Epo during the first eight hours of culture; and (c) lingering low-affinity receptors may mediate some unrecognized Epo function. Fifth, the efficiency with which 106- and 91-Kd CFU-E membrane polypeptides can be cross-linked to 125I-Epo is two- to threefold higher for cells labeled at high Epo concentrations than at low ones, which suggests that these polypeptides largely reflect low-affinity site reactions.
Asunto(s)
Eritropoyesis , Eritropoyetina/fisiología , Células Madre Hematopoyéticas/fisiología , Receptores de Superficie Celular/fisiología , Animales , Células Cultivadas , Endocitosis , Técnicas In Vitro , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Peso Molecular , Receptores de Eritropoyetina , TemperaturaRESUMEN
Patients with unilateral cerebral stroke and healthy controls were studied for limb and hemispatial hypometria. Subjects were required to reproduce lateral and medial horizontal displacements of each arm, with their eyes closed. Patients exhibited significant hypometria when moving toward hemispace contralateral to the lesion, and also when moving the limb contralateral to the lesion. Possible mechanisms for the hemispatial-limb hypometria are discussed.
Asunto(s)
Brazo/fisiopatología , Trastornos Cerebrovasculares/complicaciones , Trastornos del Movimiento/etiología , Adulto , Anciano , Trastornos Cerebrovasculares/fisiopatología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Trastornos del Movimiento/fisiopatologíaRESUMEN
Limbic evoked potentials were recorded from intracerebral electrodes bilaterally implanted in the hippocampi of 16 epileptic patients undergoing evaluation for surgical resection of a seizure focus. Spectral analysis of the limbic evoked potentials revealed consistently lower spectral power of both low-band (1-12.6 Hz) and high-band (13-20 Hz) components recorded form the hippocampus that was associated with the electrographically confirmed seizure focus as compared to the contralateral side. Limbic evoked potential recordings appear to be useful in the determination of a focal functional deficit and thus in identifying the site of epileptogenic pathology. Further, the results support the hypothesis of a local generator for limbic evoked potentials and demonstrate that limbic evoked potentials in humans include not only low-frequency, high-amplitude electric field changes, but also a high-frequency component.
Asunto(s)
Epilepsia/fisiopatología , Potenciales Evocados Auditivos , Sistema Límbico/fisiopatología , Adolescente , Adulto , Amígdala del Cerebelo/fisiopatología , Electroencefalografía , Epilepsia/psicología , Femenino , Hipocampo/fisiopatología , Humanos , Pruebas de Inteligencia , Masculino , Persona de Mediana Edad , Pruebas NeuropsicológicasRESUMEN
Hypophysectomized rats showed aberrant and retarded rates of kindled epilepsy. In hypophysectomized rats administered adrenocorticotropic hormone (ACTH) subunits ACTH4-10 and ACTH1-24, the normal kindling pattern was restored. However, in hypophysectomized animals which also had lesions of the thalamus (nucleus parafascicularis), ACTH4-10 did not restore the normal pattern of kindling. There have been many conjectures that kindling may be a subcase of learning. These results are compatible with this hypothesis, since the same procedures act in an analogous fashion within avoidance conditioning paradigms.
Asunto(s)
Hormona Adrenocorticotrópica/análogos & derivados , Hormona Adrenocorticotrópica/farmacología , Cosintropina/farmacología , Epilepsia/fisiopatología , Hipofisectomía , Excitación Neurológica/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Tálamo/fisiopatología , Glándulas Suprarrenales/patología , Glándulas Suprarrenales/fisiopatología , Animales , Conducta Animal/fisiología , Electroencefalografía , Masculino , Tamaño de los Órganos , Ratas , Ratas Endogámicas , Tálamo/patologíaRESUMEN
The recycling of cellular glycoproteins to the site of Golgi mannosidase I, an enzyme of asparagine-linked oligosaccharide synthesis, was studied in K562 human erythroleukemia cells. Cells were metabolically labeled in the presence of deoxymannojirimycin, a reversible inhibitor of Golgi mannosidase I. This generates glycoproteins with immature oligosaccharides in their normal locations. Transport to the mannosidase I compartment was then assessed by testing for the conversion of oligosaccharides into mature forms during reculture without deoxymannojirimycin. Transferrin receptor (TfR) was acted on by mannosidase I during reculture, suggesting that it returned to the region of the Golgi complex where this enzyme resides. The slow rate of this transport (t1/2 greater than 6 h) implies that it is probably different than TfR movement during transferrin internalization (t1/2 = 10-20 min) and TfR transport to the sialyltransferase compartment in the Golgi complex (t1/2 = 2-3 h) (Snider, M. D., and O. C. Rogers, 1985, J. Cell Biol., 100:826-834). The total cell glycoprotein pool was also transported to the mannosidase I compartment with a half-time of 4 h. Because this transport is 5-10 times faster than the rate of de novo glycoprotein synthesis in these cells, it is likely that most of the glycoprotein traffic through the Golgi complex is composed of recycling molecules.
Asunto(s)
Glicoproteínas/metabolismo , Aparato de Golgi/metabolismo , Manosidasas/metabolismo , Glicoproteínas de Membrana , Proteínas de la Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , 1-Desoxinojirimicina , Alcaloides/farmacología , Transporte Biológico/efectos de los fármacos , Compartimento Celular , Línea Celular , Glucosamina/análogos & derivados , Glucosamina/farmacología , Humanos , Membranas Intracelulares/metabolismo , Manosidasas/antagonistas & inhibidores , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Receptores de Transferrina , SwainsoninaRESUMEN
The intracellular movement of cell surface transferrin receptor (TfR) after internalization was studied in K562 cultured human erythroleukemia cells. The sialic acid residues of the TfR glycoprotein were used to monitor transport to the Golgi complex, the site of sialyltransferases. Surface-labeled cells were treated with neuraminidase, and readdition of sialic acid residues, monitored by isoelectric focusing of immunoprecipitated TfR, was used to assess the movement of receptor to sialyltransferase-containing compartments. Asialo-TfR was resialylated by the cells with a half-time of 2-3 h. Resialylation occurred in an intracellular organelle, since it was inhibited by treatments that allow internalization of surface components but block transfer out of the endosomal compartment. Moreover, roughly half of the resialylated molecules were cleaved when cells were retreated with neuraminidase after culturing, indicating that this fraction of the molecules had returned to the cell surface. These results suggest that TfR is transported from the cell surface to the Golgi complex, the intracellular site of sialyltransferases, and then returns to the cell surface. This pathway, which has not been previously described for a cell surface receptor, may be different from the route followed by TfR in iron uptake, since reported rates of transferrin uptake and release are significantly more rapid than the resialylation of asialo-TfR.
Asunto(s)
Endocitosis , Leucemia Eritroblástica Aguda/metabolismo , Receptores de Superficie Celular/metabolismo , Transporte Biológico Activo , Línea Celular , Membrana Celular/metabolismo , Aparato de Golgi/metabolismo , Humanos , Neuraminidasa/farmacología , Receptores de Transferrina , Ácidos Siálicos/metabolismo , Transferrina/farmacologíaRESUMEN
The transport of sugar residues into the endoplasmic reticulum (ER) during glycoprotein synthesis was studied by examining the transmembrane orientations of the oligosaccharide-lipid precursors of asparagine-linked oligosaccharides. Using the lectin concanavalin A, the lipid-linked oligosaccharides Man3-5GlcNAc2 were found on the cytoplasmic side of ER-derived vesicles in vitro while lipid-linked Man6-9GlcNAc2 and Glc1-3Man9GlcNAc2 were found facing the lumen. These results suggest that Man5GlcNAc2-lipid is synthesized on the cytoplasmic side of the ER membrane and then translocated to the luminal side. Glc3Man9GlcNAc2-lipid is then completed on the luminal side where it serves as the donor in peptide glycosylation. Translocation of Man5GlcNAc2-lipid offers a mechanism for the export of sugar residues from the cytoplasm during glycoprotein synthesis. This translocation may be the reason for the participation of lipid-linked mono- and oligosaccharides in glycoprotein synthesis.
Asunto(s)
Glucolípidos/metabolismo , Glicoproteínas/biosíntesis , Animales , Transporte Biológico , Compartimento Celular , Células Cultivadas , Concanavalina A , Cricetinae , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
The advantages offered by glass-ionomer cements would be greatly extended if they formed an attachment to ceramic materials.
Asunto(s)
Recubrimiento Dental Adhesivo/métodos , Cementos Dentales , Porcelana Dental , Cementos de Ionómero Vítreo , Adhesividad , Propiedades de SuperficieRESUMEN
A technique for electroforming pure gold margins on the abutment castings of bridge substructures made from gold alloys or base metals alloys has been described. The margin of gold can be burnished to compensate for errors inherent in the production of these castings and ceramic procedures. A modification of this procedure permits marginal adjustment to completed crowns and bridges with deficient margins.
Asunto(s)
Coronas , Porcelana Dental , Oro , Incrustaciones , Aleaciones Dentales , Preparación de la Cavidad Dental , Diseño de Dentadura , Reparación de la Dentadura , Dentadura Parcial , Electroquímica , Aleaciones de Oro , Propiedades de SuperficieRESUMEN
The use of gold electroforms provides (1) a porcelain jacket crown and adaptable gold margin; (2) a matrix for porcelain inlays; (3) an adaptable margin for cast ceramic bonded to metal crowns or abutments. Further, by the application of electrodeposited tin to the gold, excellent bonding of porcelain occurs. This system can be used advantageously as a substrate for bonding porcelain to metals.
Asunto(s)
Coronas , Recubrimiento Dental Adhesivo , Porcelana Dental , Oro , Cementación , Equipo Dental , Diseño de Dentadura , Electroquímica/instrumentación , Electrodos , Propiedades de Superficie , EstañoRESUMEN
A technique has been developed and described for the examination of the interface between dissimilar metals, utilizing electrolytic etching and gold electro-deposition procedures. This procedure permitted etching of both the constituents of the silver solder-stainless steel interface without differential leveling. The grain boundaries at the surface of the stainless steel interface were accentuated by the chemical action of the flux during the joining procedure and the notched grain boundaries influenced the nucleation of the silver solder. No evidence of alloying was found within the resolution of the instruments used.
Asunto(s)
Soldadura Dental , Plata , Acero Inoxidable , Cadmio/análisis , Cromo/análisis , Cobre/análisis , Microanálisis por Sonda Electrónica , Metalurgia , Níquel/análisis , Plata/análisis , Acero Inoxidable/análisis , Propiedades de Superficie , Zinc/análisisRESUMEN
The results obtained using single impulse and multiple impulse welds for joining orthodontic band and wire material showed that the single impulse produced an autogenous nugget of fused parent material and several nuggets were formed with the multiple impulse weld. Excessive current was deleterious.