RESUMEN
Objetivo: Describir las características craneofaciales, dentoalveolares, de tejido blando, vía aérea y el patrón de actividad muscular determinadas a través de los estudios cefalométricos y electromiográficos de individuos incompetentes labiales y con presencia de anomalías dentomaxilares de 7 a 12 años de edad. Materiales y método: Cuarenta y seis participantes con incompetencia labial fueron sometidos a una toma de radiografía lateral de perfil para el análisis cefalométrico. Para el estudio electromiográfico se consideró el patrón de actividad de los músculos Orbicular superior de los labios, orbicular inferior de los labios y temporal anterior en funciones: reposo, fonoarticulación, deglución, máximo apriete labial. Resultados: Se observó clase II esqueletal y molar, retrusión mandibular, biprotrusión incisal, biprotrusión labial, disminución de vía aérea superior. La mayor actividad muscular fue observada en máximo apriete labial. Conclusión: Los niños y niñas con incompetencia labial y anomalías dentomaxilares presentan alteraciones en las características craneofaciales, dentoalveolares, de tejido blando, vía aérea y actividad muscular determinadas a través de los estudios cefalométricos y electromiográficos.
Objective: To describe craniofacial, dentoalveolar, soft issue and airway features, and the muscular activity, determined through a cephalometric and electromyographic study in individuals with lip incompetence and dentomaxillary anomalies aged 7 to 12 years. Methods: Forty-six participants with lip incompetence underwent lateral profile radiography for cephalometric analysis. For the electromyographic study, the activity of the superior orbicularis oris, inferior orbicularis oris and anterior temporalis muscles was considered in the following functions: rest, speaking, swallowing, and reciprocal compression of the lips. Results: Skeletal and molar class II, mandibular retrusion, labial biprotrusion, incisal biprotrusion, and upper airway dysfunction were found. The highest muscular activity was observed in reciprocal compression of the lips. Conclusion: Children with lip incompetence and dentomaxillary anomalies have alterations in the craniofacial, dentoalveolar, soft issue, and airway features, and in the muscular activity , determined through a cephalometric and electromyographic study.
Asunto(s)
Humanos , Masculino , Femenino , Niño , Anomalías Dentarias , Cefalometría , Electromiografía , Labio , Epidemiología Descriptiva , Estudio ObservacionalRESUMEN
Proprotein convertase subtilisin/kexin 9 (PCSK9) is a key regulator of lipid metabolism by degrading liver LDL receptors. Structural studies have provided molecular details of PCSK9 function. However, the N-terminal acidic stretch of the PCSK9 prodomain (Q31-T60) has eluded structural investigation, since it is in a disordered state. The interest in this region is intensified by the presence of human missense mutations associated with low and high LDL-c levels (E32K, D35Y, and R46L, respectively), as well as two posttranslationally modified sites, sulfated Y38 and phosphorylated S47. Herein we show that a segment within this region undergoes disorder-to-order transition. Experiments with acidic stretch-derived peptides demonstrated that the folding is centered at the segment Y38-L45, which adopts an α-helix as determined by NMR analysis of free peptides and by X-ray crystallography of peptides in complex with antibody 6E2 (Ab6E2). In the Fab6E2-peptide complexes, the structured region features a central 2 1/4-turn α-helix and encompasses up to 2/3 of the length of the acidic stretch, including the missense mutations and posttranslationally modified sites. Experiments with helix-breaking proline substitutions in peptides and in PCSK9 protein indicated that Ab6E2 specifically recognizes the helical conformation of the acidic stretch. Therefore, the observed quantitative binding of Ab6E2 to native PCSK9 from various cell lines suggests that the disorder-to-order transition is a true feature of PCSK9 and not limited to peptides. Because the helix provides a constrained spatial orientation of the missense mutations and the posttranslationally modified residues, it is probable that their biological functions take place in the context of an ordered conformational state.
Asunto(s)
Proproteína Convertasa 9/genética , Secuencia de Aminoácidos , Línea Celular Tumoral , Cristalografía por Rayos X/métodos , Células Hep G2 , Humanos , Mutación Missense/genética , Péptidos/genética , Procesamiento Proteico-Postraduccional/genética , Receptores de LDL/genética , Serina Endopeptidasas/genéticaRESUMEN
Tissue factor is the cell membrane-anchored cofactor for factor VIIa and triggers the coagulation reactions. The initial step is the conversion of factor VII to factor VIIa which, in vitro, is efficiently catalyzed by low concentrations of factor Xa. To identify the tissue factor region that interacts with the activator factor Xa during this process, we evaluated a panel of soluble tissue factor (1-219) mutants for their ability to support factor Xa-mediated activation of factor VII. The tissue factor residues identified as most important for this interaction (Tyr157, Lys159, Ser163, Gly164, Lys165, Lys166, and Tyr185) were identical to those found to be important for the interaction of substrate factor X with the tissue factor.factor VIIa complex. The residues form a continuous surface-exposed patch with an area of about 500 A(2), which appears to be located outside the tissue factor-factor VII contact zone. In agreement, the two monoclonal antibodies 5G6 and D3H44-F(ab')(2), whose epitopes overlap with this identified region, inhibited the rates of factor VII activation by 86% and 95%, respectively. These antibodies also strongly inhibited the conversion of (125)I-labeled factor VII when cell membrane-expressed, full-length tissue factor (1-263) was employed. Together the results suggest the usage of a common surface region of tissue factor in its dual role-as a cofactor for factor Xa-mediated factor VII activation and as a cofactor for factor VIIa-mediated factor X activation. The finding that factor Xa and factor X may engage in similar, if not identical, molecular interactions with tissue factor further indicates that factor Xa and factor X are similarly oriented toward their respective interaction partners in the ternary catalytic complexes.
Asunto(s)
Factor VII/metabolismo , Factor Xa/metabolismo , Tromboplastina/metabolismo , Ácido 1-Carboxiglutámico/metabolismo , Línea Celular , Membrana Celular/enzimología , Membrana Celular/genética , Membrana Celular/metabolismo , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Precursores Enzimáticos/metabolismo , Factor VIIa/metabolismo , Humanos , Radioisótopos de Yodo , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Mapeo Peptídico , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Solubilidad , Resonancia por Plasmón de Superficie , Tromboplastina/genéticaRESUMEN
BACKGROUND: Thrombosis and neointima formation limit the efficacy of coronary angioplasty (PTCA). Clinical trials have implicated the adhesion molecules integrin alpha(IIb)beta(3) and integrin alpha(v)beta(3) in these processes. The roles of these molecules in vascular smooth muscle cell adhesion, platelet aggregation, and the thrombotic and neointimal response to oversize porcine PTCA was investigated by use of a selective alpha(IIb)beta(3) antagonist (lamifiban), a selective alpha(v)beta(3) antagonist (VO514), and a combined alpha(IIb)beta(3)/alpha(v)beta(3) antagonist (G3580). METHODS AND RESULTS: In vitro, both alpha(v)beta(3) inhibitors caused dose-dependent inhibition of porcine vascular smooth muscle cell adhesion to vitronectin but not to collagen type IV, fibronectin, or laminin, whereas selective alpha(IIb)beta(3) inhibition had no effect. Intravenous infusions of either alpha(IIb)beta(3) inhibitor in swine profoundly inhibited ex vivo platelet aggregation to ADP, whereas selective alpha(v)beta(3) inhibition had no effect. In a porcine PTCA model, intravenous infusions of the integrin antagonists were administered for 14 days after oversized balloon angioplasty injury. After PTCA, there was regional upregulation of integrin alpha(v)beta(3) in the developing neointima, as assessed by immunohistochemistry. Six hours after PTCA, obstruction of lumen by thrombus was reduced significantly by alpha(IIb)beta(3) inhibition compared with either control or alpha(v)beta(3) inhibition (mean control, 18.7%; VO514, 18.5%; lamifiban, 6.4%; G3580, 7.9%). Twenty-eight days after PTCA, there was a significant reduction of neointima with inhibitors of either integrin (mean intima/media ratio: control, 3.08; VO514, 1.33; lamifiban, 0.97; G3580, 1.32). CONCLUSIONS: We conclude that both integrin alpha(IIb)beta(3) and integrin alpha(v)beta(3) participate in neointima development after experimental angioplasty.
Asunto(s)
Acetatos/uso terapéutico , Trombosis/prevención & control , Tirosina/análogos & derivados , Tirosina/uso terapéutico , Acetatos/farmacología , Angioplastia Coronaria con Balón/efectos adversos , Animales , Adhesión Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Inmunohistoquímica , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Inhibidores de Agregación Plaquetaria/uso terapéutico , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/biosíntesis , Receptores de Vitronectina/antagonistas & inhibidores , Receptores de Vitronectina/biosíntesis , Porcinos , Trombosis/etiología , Túnica Íntima/efectos de los fármacos , Tirosina/farmacologíaRESUMEN
Many reports indicate that nitric oxide (NO) could be involved in migraine without aura (MWA), an extremely diffuse clinical event. Since monocyte may be a relevant source of NO, we analysed monocyte activation in MWA patients, in a period in which they were free of symptoms. NO basal production by MWA peripheral monocytes was significantly higher than in healthy subjects (91.25+/-8.6 microM/10(6) cells vs. 22.6+/-3.2 microM/106 cells). Interestingly, even the release of prostaglandin E2 (PGE2), was higher in MWA patients than in healthy subjects (3137+/-320 pg/10(6) cells vs. 1531+/-220 pg/10(6) cells). The incubation of monocytes from healthy subjects and MWA patients with N-nitro-L-arginine methyl ester caused a marked decrease of both NO and PGE2 release. We hypothesise that NOS and cyclooxygenase pathways in monocytes are linked and are, in MWA patients, up-regulated, even in a symptoms-free period. NO and PGE2 hyperproduction could therefore be involved in the neurovascular modifications leading to migraine attacks.
Asunto(s)
Dinoprostona/metabolismo , Migraña sin Aura/metabolismo , Monocitos/metabolismo , Óxido Nítrico Sintasa/metabolismo , Adulto , Intervalos de Confianza , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Migraña sin Aura/sangre , Monocitos/efectos de los fármacos , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/efectos de los fármacos , Nitritos/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismoRESUMEN
The enzymatic activity of coagulation factor VIIa is controlled by its cellular cofactor tissue factor (TF). TF binds factor VIIa with high affinity and, in addition, participates in substrate interaction through its C-terminal fibronectin type III domain. We analyzed surface-exposed residues in the C-terminal TF domain to more fully determine the area on TF important for substrate activation. Soluble TF (sTF) mutants were expressed in E. coli, and their ability to support factor VIIa-dependent substrate activation was measured in the presence of phospholipid vesicles or SW-13 cell membranes. The results showed that factor IX and factor X interacted with the same TF region located proximal to the putative phospholipid surface. According to the degree of activity loss of the sTF mutants, this TF region can be divided into a main region (residues Tyr157, Lys159, Ser163, Gly164, Lys165, Lys166, Tyr185) forming a solvent-exposed patch of 488 A(2) and an extended region which comprises an additional 7-8 residues, including the distally positioned Asn199, Arg200, and Asp204. Some of the identified TF residues, such as Trp158 and those within the loop Lys159-Lys165, are near the factor VIIa gamma-carboxyglutamic acid (Gla) domain, suggesting that the factor VIIa Gla-domain may also participate in substrate interaction. Moreover, the surface identified as important for substrate interaction carries a net positive charge, suggesting that charge interactions may significantly contribute to TF-substrate binding. The calculated surface-exposed area of this substrate interaction region is about 1100 A(2), which is approximately half the size of the TF area that is in contact with factor VIIa. Therefore, a substantial portion of the TF surface (3000 A(2)) is engaged in protein-protein interactions during substrate catalysis.
Asunto(s)
Factor IX/metabolismo , Factor X/metabolismo , Tromboplastina/metabolismo , Amidas/metabolismo , Anticuerpos/inmunología , Activación Enzimática , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fosfolípidos/metabolismo , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Tromboplastina/química , Tromboplastina/genética , Tromboplastina/inmunologíaRESUMEN
C3 production, release and CRs expression during the neutrophilic differentiation of a murine non tumorigenic cell line is investigated. The murine non tumorigenic cell line 32DCl3(G) which undergoes terminal differentiation into polymorphonuclear granulocytes when cultured in presence of G-CSF was selected as a suitable in vitro model for this study. The results show that as the cells progress into the differentiation program, levels of C3 mRNA increase, accompanied by increased C3 production. As differentiation progresses the cells gradually express CRs on their surface; these are undetectable on the surface of undifferentiated cells. As a consequence of CRs appearance, cells become able to bind C3 through receptorial binding. Differences were found in the modality of C3 secretion: differentiated cells tend to store C3 in their intracellular compartments rather than secrete it continuously into the medium and they respond to membrane stimulation with increased secretion of C3. Treatment of 32DCl3(G) with TNF-alpha increased C3 production in a time- and dose-dependent fashion. Cell response to this stimulus progressively increases during the differentiation process suggesting that they acquire functionality in the signal transduction mechanisms.
Asunto(s)
Complemento C3/biosíntesis , Receptores de Complemento/biosíntesis , Células Madre/inmunología , Animales , Calcimicina/farmacología , Diferenciación Celular , Línea Celular , Complemento C3/genética , Citocalasina D/farmacología , Relación Dosis-Respuesta a Droga , Ratones , Ratones Endogámicos C57BL , Células Madre/citología , Células Madre/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Factores de Tiempo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
C3 molecules from normal murine serum are mainly bound to Lewis lung carcinoma cells (3LL) that do not express CRs, mainly through covalent binding as determined by the appearance of bands stained with anti-C3 and larger than 190 kD in immunoblots of proteins in whole cell extracts. Methylamine-treated, or zymosan-treated normal mouse serum, heat inactivated, or EDTA-treated murine serum resulted in low C3 deposition on 3LL cells, as indicated by fluorescence tests and immunoblotting. Cytofluorimetric studies showed that C3 molecules bound to 3LL cells were internalized in a time- and temperature-dependent process. This was confirmed by electronmicroscopic studies. The conditions allowing C3 fixation to acceptor sites and subsequent internalization increased cell proliferation. This was also true, when serum from mice genetically deficient in C5 was used which stresses the role of C3 in contrast to effects of membrane attack complex formation.
Asunto(s)
Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patología , Complemento C3/metabolismo , Endocitosis , Receptores de Superficie Celular/metabolismo , Animales , Transporte Biológico , División Celular , Ratones , Unión ProteicaRESUMEN
Recent studies have pointed to the relationship between iron deficiency anemia and celiac disease, although data on the prevalence of celiac disease in anemic patients have been conflicting, and there is no agreement on the best screening procedure for CD in these patients. Our aims were to evaluate the relationship between anemia and celiac disease (CD) from two different points of view--the hematology clinic and the pediatric gastroenterology department--and to evaluate the utility of anti-endomysial antibody determination in screening anemic patients for CD using human umbilical cord as substrate. We studied 130 patients with CD (58 males, 72 females; median age 18 months) diagnosed at a department of Pediatric Gastroenterology, and 85 patients with iron deficiency anemia (38 males, 47 females; median age 48 years) observed at a hematology outpatient clinic. From the 85 adult patients with iron deficiency anemia, we selected a subgroup of 25 subjects with no improvement in Hb after two months of iron therapy (80 mg/day orally). Routine hematochemical tests were performed in all 215 patients. All pediatric and adult subjects underwent immunological screening for celiac disease (AGA and EmA assay); intestinal biopsy was also performed on patients testing positive. In the adult anemic patients a serum sample was stored at -20 degrees C on first observation, and after 6-18 months EmA on human umbilical cord were assayed. In the pediatric patients with CD, anemia was observed in 91/130 patients (70% of cases, the most frequent symptom after poor growth); however, this was the only presenting symptom of CD in 2/130 patients (1.5% of cases). Anemia was sideropenic in 41/91 patients (iron <45 microg/dl, ferritin <15 microg/liter). In the adult patients with iron deficiency anemia, immunological screening (AGA and EmA) showed suspected CD in 5/85 cases (5.8%), with diagnosis confirmed on intestinal biopsy. These five patients were in the subgroup of iron supplementation therapy nonresponders. CD prevalence in the refractory anemia subgroup was, therefore, 5/25 (20%). On diagnosis the hematological indices of the anemia + CD patients were not different than those of the refractory anemia patients without CD. The median age of the CD + anemia patients was significantly lower than that of the whole group of anemic subjects, and there was also a prevalence of females (4/5 cases). The results of the EmA determination on human umbilical cord in the adult anemic patients showed a perfect concordance with those using a traditional kit that uses monkey esophagus as substrate. In the pediatric age group many cases of CD with anemia as the only sign of the disease are probably not diagnosed. In our adult patients with sideropenic anemia, CD prevalence was 5-6%; however, the observation of anemic patients not responding to oral iron therapy makes a diagnosis of CD much more probable. EmA determination on human umbilical cord is the most logical approach to screen anemic patients for suspected CD.
Asunto(s)
Anemia Ferropénica/etiología , Enfermedad Celíaca/complicaciones , Anemia Ferropénica/diagnóstico , Enfermedad Celíaca/diagnóstico , Enfermedad Celíaca/epidemiología , Femenino , Humanos , Inmunoglobulina A/análisis , Inmunoglobulina A/inmunología , Lactante , Masculino , Persona de Mediana Edad , Miofibrillas/inmunología , Estudios ProspectivosRESUMEN
Ro 44-3888 is a potent and selective antagonist of GP IIb/IIIa. Following I.V. administration to rhesus monkeys, the (mean +/- SD.) clearance, volume of distribution and terminal half-life of Ro 44-3888 were 4.4 +/- 1.8 ml/min/kg, 0.8 +/- 0.4 l/kg and 2.5 +/- 0.8 h respectively. Oral administration of Ro 48-3657 (1 mg/kg), a doubly protected prodrug form, produced peak concentrations of Ro 44-3888 (152 +/- 51 ng/ml), 4.2 +/- 2.2 h after dosing. Terminal half-life and estimated bioavailability were 5.1 +/- 1.6 h and 33 +/- 6% respectively. No effect on blood pressure, heart rate or platelet counts were seen. Adenosine diphosohate (ADP) induced platelet aggregation (PA) and cutaneous bleeding times (CBT) were determined prior to and after the last of 8 daily oral administrations of Ro 48-3657 (0.25 or 0.5 mg/kg) to eight rhesus monkeys. Peak and trough plasma concentrations were proportional to dose and steady state was achieved after the second administration. Inhibition of PA and prolongation of CBT were concentration dependent. The ex vivo IC50 (82 nM) for ADP-mediated PA correlated with a value (58 nM) determined in vitro. The CBT response curve was displaced to the right of the PA curve. CBT was prolonged to > or = 25 min when levels of Ro 44-3888 exceeded 190 nM and PA was > 90% inhibited. Therefore, in rhesus monkeys, Ro 48-3657 is reproducibly absorbed and converted to its active form, is well tolerated, and has a concentration-dependent effect on PA and CBT. These properties make Ro 48-3657 an attractive candidate for evaluation in patients at high risk for arterial thrombosis.
Asunto(s)
Amidinas/farmacología , Piperidinas/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Profármacos/farmacocinética , Administración Oral , Amidinas/efectos adversos , Amidinas/farmacocinética , Animales , Esquema de Medicación , Pruebas Hematológicas , Macaca mulatta , Oximas/farmacología , Piperidinas/efectos adversos , Piperidinas/farmacocinética , Profármacos/efectos adversos , Profármacos/farmacologíaRESUMEN
The potent and selective GP IIb-IIIa antagonist lamifiban (1, Ro 44-9883) is currently in clinical development as an injectable antithrombotic agent for treating and preventing acute coronary syndromes. However, for secondary prevention of thrombotic occlusions, orally active inhibitors are needed. By means of a prodrug strategy, the modest oral absorption of 1 in mice was improved by a factor of 9. In addition, these studies demonstrated that an amidoxime group can serve as a prodrug functionality for an amidino group. Application of this principle to the structurally related amidino carboxylate 13 led to the amidoxime ester 18 which was absorbed approximately 20 times better, after oral administration to mice, than 13. Due to the modification of the amidino group as well as of the carboxylate group, 18 completely lost its ability to interact with purified platelet GP IIb-IIIa. After oral administration of 18 to rats, dogs, and rhesus monkeys, the bioavailability of the active derivative 13 was 26 +/- 5, 25 +/- 6, and 33 +/- 6%, respectively, and the elimination half-life was 4.1 +/- 1.7, 11.4 +/- 1.1, and 5.1 +/- 1.4 h, respectively. On the basis of these properties, the orally active 18 (Ro 48-3657), a double prodrug of the potent and selective non-peptide GP IIb-IIIa antagonist 13 (Ro 44-3888), was selected as clinical candidate for evaluation as a prophylactic agent in patients at high risk for arterial thrombosis.
Asunto(s)
Amidinas/farmacología , Oximas/farmacología , Piperidinas/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Profármacos/farmacología , Acetatos/farmacología , Administración Oral , Amidinas/química , Amidinas/farmacocinética , Animales , Anticoagulantes/administración & dosificación , Anticoagulantes/síntesis química , Anticoagulantes/farmacocinética , Anticoagulantes/farmacología , Perros , Macaca mulatta , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ratones , Estructura Molecular , Oximas/química , Oximas/farmacocinética , Piperidinas/química , Piperidinas/farmacocinética , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/farmacocinética , Profármacos/administración & dosificación , Profármacos/síntesis química , Profármacos/farmacocinética , Ratas , Tirosina/análogos & derivados , Tirosina/farmacologíaRESUMEN
PGE2 has been shown to be able to interfere with various lymphocyte and macrophage functions, but its effects on macrophage activation are still unclear. In this study, carried out on peritoneal macrophages obtained from healthy, tumour-bearing and Corynebacterium parvum-treated mice, we demonstrated that PGE2 is involved in the down-regulation of macrophage activation, but it cannot exert its inhibiting effect when macrophages are further stimulated with activating cytokines, such as IFN gamma and TNF alpha. Our findings provide new insight into how macrophage tumoricidal activity may be induced and maintained even in presence of significant levels of PGE2.
Asunto(s)
Antineoplásicos/farmacología , Dinoprostona/antagonistas & inhibidores , Interferón gamma/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Animales , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Regulación hacia Abajo/efectos de los fármacos , Femenino , Infecciones por Bacterias Grampositivas/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Propionibacterium acnes/efectos de los fármacosRESUMEN
Macrophages from mice bearing Lewis lung carcinoma release higher amounts of C3 molecules than macrophages from healthy mice. The C3 pro-releasing activity operating in vivo was suspected to be due to an immunological network. Indeed, the supernatants of splenocytes from tumor bearing mice, but not from normal mice, induced in vitro an increased release of C3 molecules by macrophages. Recombinant IFN gamma and TNF alpha were strong inducers of C3 release, while IL-2 acted poorly. The C3 pro-releasing activity of splenocyte supernatants was largely prevented by their pretreatment with specific mAb anti IFN gamma or anti TNF alpha, but not completely prevented by the simultaneous neutralization of the two cytokines. Taken together, these results show that murine macrophages increase the release of C3 molecules upon treatment with IFN gamma or TNF alpha and that these cytokines released in vivo by splenocytes from tumor bearing mice may account, together with a yet unknown factor, for a humoral network causing the increased release of C3 by peritoneal macrophages.
Asunto(s)
Complemento C3/metabolismo , Interferón gamma/fisiología , Macrófagos Peritoneales/inmunología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Células Cultivadas , Medios de Cultivo Condicionados , Ensayo de Inmunoadsorción Enzimática , Femenino , Interleucina-2/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/farmacología , Bazo/inmunologíaRESUMEN
32DCl3(G) cells are a diploid, nontumorigenic, IL-3-dependent hemopoietic progenitor cell line, which undergoes terminal differentiation into neutrophilic granulocytes when cultured in presence of G-CSF. The infection with BALB-Moloney murine sarcoma virus, containing a v-HA-ras oncogene, renders this cell line IL-3 independent and continuously growing in the presence of G-CSF, nontumorigenic and with an apparent block at the level of promyelocyte/myelocyte (32D-Ha-ras). After infection with Abelson murine leukemia virus containing a v-abl oncogene, the cell line originates (32D-abl) that is also IL-3 independent but is tumorigenic and unable to differentiate in the presence of G-CSF. This cellular model allowed us to study the relationship between distinct steps of cell differentiation, neoplastic transformation, and C3 synthesis, activation, and characteristics of binding. We demonstrated that C3 synthesis, release, and cleavage are properties already present in the progenitor 32DCl3(G) cells. The more differentiated 32D-Ha-ras cells acquired C3 acceptor sites, apparently completely saturated by the constitutively released molecules. The transformation with Abelson murine leukemia virus, although it did not affect all these properties, let the cells bind considerable amounts of C3-related fragments activated in normal murine serum through the alternative pathway. This last event was a result of the acquisition of PMSF-sensitive serine proteases associated with the plasma membrane.
Asunto(s)
Transformación Celular Neoplásica/inmunología , Complemento C3/biosíntesis , Complemento C3b/metabolismo , Células Madre Hematopoyéticas/metabolismo , Animales , Sitios de Unión , Diferenciación Celular , Línea Celular , Transformación Celular Viral , Células Cultivadas , Vía Alternativa del Complemento , Genes ras , Hematopoyesis , Células Madre Hematopoyéticas/citología , Ratones , Ratones Endogámicos C3HRESUMEN
The present investigation shows that C3 (hemolytic inactive) as well as C3b and C3d bind Raji, a CR2-positive Burkitt lymphoma-derived cell line. Pretreatment of the cells with OKB-7 inhibited the binding of C3, whereas pretreatment with HB-5 inhibited the binding of C3b. Furthermore, the cells coated either with OKB-7 or HB-5 bound high amounts of C3d. TPA-treated cells showed binding for C3b and weak binding for C3 and C3d. Taken together, the data suggest that Raji cells may express three binding sites for C3, C3b and C3d which can be differently modulated by anti-CR2 MoAbs and TPA.
Asunto(s)
Linfoma de Burkitt/metabolismo , Complemento C3/metabolismo , Complemento C3b/metabolismo , Complemento C3d/metabolismo , Receptores de Complemento 3d/metabolismo , Anticuerpos Monoclonales/inmunología , Hemólisis , Humanos , Técnicas In Vitro , Antígeno de Macrófago-1/metabolismo , Receptores de Complemento 3b/metabolismo , Temperatura , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
The natriuretic peptide receptor type A (NPR-A) is a receptor-guanylyl cyclase whose cytoplasmic enzymatic activity is stimulated by atrial natriuretic peptide binding to the extracellular domain. NPR-A expressed in COS cells is heterogeneously glycosylated, and the more highly glycosylated protein is also phosphorylated. Upon hormone binding, dephosphorylation occurs from both serine and threonine residues, probably within the kinase homology domain of NPR-A, and may be involved with receptor desensitization. Using site-specific mutations in the kinase homology domain of NPR-A, we have identified several residues that are important for regulating the guanylyl cyclase activity of NPR-A. Some of these amino acids are probably essential for maintaining the proper tertiary structure of the intracellular domain, and others may form loops that allow for binding of ATP, which is required for proper enzymatic activity. The site-specific mutants which have greatly reduced enzymatic activity are not phosphorylated and are incompletely glycosylated. These results suggest a correlation between phosphorylation and complete glycosylation of NPR-A and that both are required for hormone-induced enzymatic activity.
Asunto(s)
Factor Natriurético Atrial/metabolismo , Guanilato Ciclasa/metabolismo , Receptores del Factor Natriurético Atrial/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Clonación Molecular , ADN de Cadena Simple , Activación Enzimática , Glicosilación , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Receptores del Factor Natriurético Atrial/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
An expression plasmid encoding the human 75-kDa tumor necrosis factor (TNF) type 2 receptor (TNF-R2) was constructed and used to generate a stable human cell line (293/TNF-R2) overexpressing TNF-R2. Ligand binding analysis revealed high affinity binding (Kd = 0.2 nM) with approximately 94,000 +/- 7,500 sites/cell for 125I-TNF-alpha and approximately 5-fold lower affinity for TNF-beta (Kd = 1.1 nM) with 264,000 +/- 2,000 sites/cell. Cross-linking of 125I-TNF-alpha and 125I-TNF-beta to 293/TNF-R2 cells yielded predominant complexes with apparent molecular weights of 211,000 for TNF-alpha and 205,000 and 244,000 for TNF-beta, suggesting these complexes contain two or three TNF-R2 molecules. Immunoprecipitation of TNF-R2 from 32P-labeled 293/TNF-R2 cells demonstrated that the receptor is phosphorylated. The majority (97%) of 32Pi incorporation was found in serine residues with a very low level of incorporation (3%) in threonine residues. TNF-alpha treatment of 293/TNF-R2 cells did not significantly affect the degree or pattern of phosphorylation. Cell surface-bound 125I-TNF-alpha was slowly internalized by the 293/TNF-R2 cell line with a t1/2 = 25 min. Shedding of the extracellular domain of TNF-R2 was induced by 4 beta-phorbol 12-myristate 13-acetate but not by TNF-alpha or TNF-beta.
Asunto(s)
Receptores de Superficie Celular/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Cromatografía de Afinidad , Biblioteca de Genes , Humanos , Cinética , Linfotoxina-alfa/farmacología , Peso Molecular , Fosforilación , Plásmidos , ARN Mensajero/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/aislamiento & purificación , Receptores del Factor de Necrosis Tumoral , Proteínas Recombinantes/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Transfección , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Stimulation of platelets activates GPIIbIIIa, the heterodimeric integrin receptor, to bind fibrinogen (Fg), which results in platelet aggregation. GPIIbIIIa/Fg binding inhibitors are potentially suitable for acute use during and after thrombolytic therapy as antithrombotic agents. Incorporation of the tripeptide sequence Arg-Gly-Asp (RGD), a common structural element of many integrin ligands, into cyclic peptides produced a series of peptides of the general structure BrAc-(AA1)-RGD-Cys-OH, which were prepared by solid-phase peptide synthesis. Cyclization was accomplished by reaction of the N-terminal bromoacetyl group with the cysteine sulfhydryl at pH 8 at high dilution, resulting in thioether-bridged cyclic peptides [cyclo-S-Ac-(AA1)-RGD-Cys-OH]. Use of alpha-substituted bromoacetyl groups gave rise to an analogous series of acetyl-substituted thioether-bridged cyclic peptides. Oxidation of the thioethers produced separable diastereomeric sulfoxide-bridged cyclic peptides. After thorough evaluation in a GPIIbIIIa ELISA assay and a platelet aggregation assay, G-4120 (70A; AA1 = D-Tyr; sulfoxide bridge) was selected for further investigation as an antithrombotic agent. G-4120 was equipotent in the platelet aggregation assay to kistrin, a highly potent inhibitor of fibrinogen-mediated platelet aggregation isolated from snake venom (IC50 = 0.15 microM).
Asunto(s)
Fibrinolíticos/síntesis química , Péptidos Cíclicos/síntesis química , Péptidos/síntesis química , Inhibidores de Agregación Plaquetaria/síntesis química , Receptores Inmunológicos/química , Receptores de Péptidos , Sulfóxidos/síntesis química , Secuencia de Aminoácidos , Ciclización , Ensayo de Inmunoadsorción Enzimática , Fibrinolíticos/química , Fibrinolíticos/farmacología , Humanos , Conformación Molecular , Datos de Secuencia Molecular , Estructura Molecular , Péptidos/química , Péptidos/farmacología , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/farmacología , Glicoproteínas de Membrana Plaquetaria/metabolismo , Receptores Inmunológicos/metabolismo , Relación Estructura-Actividad , Sulfóxidos/química , Sulfóxidos/farmacología , Difracción de Rayos XRESUMEN
Peritoneal macrophages obtained from Lewis Lung carcinoma (3LL) tumor bearing mice release high amounts of soluble factors such as C3,H2O2 and lysosomal enzymes but fail to exert cytotoxic activity on tumor cells. In the present work we show that they acquire this property and become fully activated after in vitro incubation with supernatants derived from cultures of splenocytes from tumor bearing syngeneic mice. The presence of IFN gamma in the above supernatants was detected by immunoblotting analysis and by bioassay. The role played by IFN gamma in macrophage activation was investigated.
Asunto(s)
Interferón gamma/metabolismo , Activación de Macrófagos/inmunología , Neoplasias Experimentales/inmunología , Animales , Complemento C3/metabolismo , Femenino , Técnicas In Vitro , Interferón gamma/inmunología , Interleucina-2/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Bazo/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We tested the capacity of Lewis Lung carcinoma cells (3LL) to activate the alternative pathway of complement and to bind the C3 fragments on the plasma membrane. C3 fragments were detected by cytofluorometry and by immunoblotting. In time, the fixed C3b molecules were further cleaved into iC3b. The presence of C3b/iC3b on the target enhanced the formation of conjugates with macrophages. In spite of increased contacts, macrophages from tumor bearing mice were not cytotoxic. Only preactivated macrophages, by in vivo treatment with Corynebacterium parvum, were shown to be cytotoxic; this function was potentiated when the target cells were opsonized with C3b/iC3b.