RESUMEN
Type 1 plasminogen deficiency is a rare genetic disorder. Type 1 plasminogen deficiency is characterized by fibrin-rich pseudomembrane formation on mucosal surfaces, particularly the conjunctiva. Tracheobronchial tree involvement is a less common reported manifestation of type 1 plasminogen deficiency. Pseudomembranes in the tracheobronchial tree may result in respiratory compromise and ultimately fail if not recognized and treated. Currently, there is no specific replacement therapy approved for the treatment of congenital plasminogen deficiency. In the present paper, we report that type 1 plasminogen deficiency with novel frameshift mutation and pulmonary involvement was treated initially with systemic fresh frozen plasma followed by pulmonary lavage with fresh frozen plasma and tissue plasminogen activator.
Asunto(s)
Conjuntivitis/genética , Mutación del Sistema de Lectura , Plasminógeno/deficiencia , Plasminógeno/genética , Enfermedades Cutáneas Genéticas/genética , Transfusión de Componentes Sanguíneos , Conjuntivitis/patología , Conjuntivitis/terapia , Humanos , Lactante , Pulmón/patología , Masculino , Enfermedades Cutáneas Genéticas/patología , Enfermedades Cutáneas Genéticas/terapia , Activador de Tejido Plasminógeno/uso terapéuticoAsunto(s)
Antibacterianos/uso terapéutico , Azitromicina/uso terapéutico , Conjuntivitis/diagnóstico , Plasminógeno/deficiencia , Rifamicinas/uso terapéutico , Enfermedades Cutáneas Genéticas/diagnóstico , Administración Oftálmica , Antibacterianos/administración & dosificación , Azitromicina/administración & dosificación , Preescolar , Terapia Combinada , Conjuntivitis/tratamiento farmacológico , Conjuntivitis/genética , Conjuntivitis/cirugía , Quimioterapia Combinada , Femenino , Humanos , Soluciones Oftálmicas , Plasma , Plasminógeno/análisis , Plasminógeno/genética , Recurrencia , Rifamicinas/administración & dosificación , Enfermedades Cutáneas Genéticas/tratamiento farmacológico , Enfermedades Cutáneas Genéticas/genética , Enfermedades Cutáneas Genéticas/cirugía , Irrigación TerapéuticaAsunto(s)
Antibacterianos/uso terapéutico , Conjuntivitis/diagnóstico , Conjuntivitis/terapia , Inmunosupresores/uso terapéutico , Plasminógeno/deficiencia , Plasminógeno/genética , Enfermedades Cutáneas Genéticas/diagnóstico , Enfermedades Cutáneas Genéticas/terapia , Preescolar , Terapia Combinada , Conjuntivitis/genética , Femenino , Predisposición Genética a la Enfermedad/genética , Humanos , Mutación/genética , Enfermedades Cutáneas Genéticas/genética , Resultado del TratamientoRESUMEN
The genome-wide mapping of the major gene expression regulators, the transcription factors (TFs) and their DNA binding sites, is of great importance for describing cellular behavior and phenotypic diversity. Presently, the methods for prediction of genomic TF binding produce a large number of false positives, most likely due to insufficient description of the physiochemical mechanisms of protein-DNA binding. Growing evidence suggests that, in the cell, the double-stranded DNA (dsDNA) is subject to local transient strands separations (breathing) that contribute to genomic functions. By using site-specific chromatin immunopecipitations, gel shifts, BIOBASE data, and our model that accurately describes the melting behavior and breathing dynamics of dsDNA we report a specific DNA breathing profile found at YY1 binding sites in cells. We find that the genomic flanking sequence variations and SNPs, may exert long-range effects on DNA dynamics and predetermine YY1 binding. The ubiquitous TF YY1 has a fundamental role in essential biological processes by activating, initiating or repressing transcription depending upon the sequence context it binds. We anticipate that consensus binding sequences together with the related DNA dynamics profile may significantly improve the accuracy of genomic TF binding sites and TF binding-related functional SNPs.
Asunto(s)
ADN/química , Factor de Transcripción YY1/metabolismo , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , Células HeLa , Humanos , Simulación de Dinámica Molecular , Plasminógeno/genética , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
The widespread occurrence of antibiotic resistance among human pathogens is a major public health problem. Conventional antibiotics typically target bacterial killing or growth inhibition, resulting in strong selection for the development of antibiotic resistance. Alternative therapeutic approaches targeting microbial pathogenicity without inhibiting growth might minimize selection for resistant organisms. Compounds inhibiting gene expression of streptokinase (SK), a critical group A streptococcal (GAS) virulence factor, were identified through a high-throughput, growth-based screen on a library of 55,000 small molecules. The lead compound [Center for Chemical Genomics 2979 (CCG-2979)] and an analog (CCG-102487) were confirmed to also inhibit the production of active SK protein. Microarray analysis of GAS grown in the presence of CCG-102487 showed down-regulation of a number of important virulence factors in addition to SK, suggesting disruption of a general virulence gene regulatory network. CCG-2979 and CCG-102487 both enhanced granulocyte phagocytosis and killing of GAS in an in vitro assay, and CCG-2979 also protected mice from GAS-induced mortality in vivo. These data suggest that the class of compounds represented by CCG-2979 may be of therapeutic value for the treatment of GAS and potentially other gram-positive infections in humans.
Asunto(s)
Antibacterianos/uso terapéutico , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Quinazolinas/uso terapéutico , Infecciones Estreptocócicas/tratamiento farmacológico , Streptococcus pyogenes/efectos de los fármacos , Estreptoquinasa/antagonistas & inhibidores , Animales , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Depresión Química , Evaluación Preclínica de Medicamentos , Inducción Enzimática/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Especificidad del Huésped/genética , Humanos , Resistencia a la Kanamicina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Estructura Molecular , Fagocitosis/efectos de los fármacos , Plasminógeno/genética , Regiones Promotoras Genéticas/genética , Quinazolinas/aislamiento & purificación , Quinazolinas/farmacología , Bibliotecas de Moléculas Pequeñas , Streptococcus pyogenes/enzimología , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidad , Estreptoquinasa/biosíntesis , Estreptoquinasa/genética , Virulencia/efectos de los fármacos , Virulencia/genéticaRESUMEN
Recent data has shown that hormone therapy (HT) increases the risk of cardiovascular and thromboembolic disease, particularly in users of oral HT. Phytoestrogens are popular alternatives to oestrogen therapy; however, their effects on cardiovascular risk are unknown. We investigated the effect of the phytoestrogen, genistein on the expression of genes and proteins from the haemostatic system in the liver in an ovariectomised rat model. Fifty-nine virgin female Sprague-Dawley rats were fed with soy-free chow supplemented with 17ß estradiol (E2) (daily uptake 0.19 or 0.75 mg/kg body weight), or genistein (daily uptake 6 or 60 mg/kg body weight), for three months and compared to soy-free control rats. Gene expression of prothrombin, factor VII, fibrinogen alpha and fibrinogen beta was increased with E2 and genistein compared to the soy-free control group (p<0.001). Genistein increased factor VII significantly more than E2 (p<0.005). Plasminogen mRNA was increased in both treatment groups compared to the soy-free control, with genistein expression significantly higher than E2 (p<0.001). Tissue plasminogen inhibitor (tPA), plasminogen activator inhibitor-1 (PAI-1) and C-reactive protein (CRP) expression were also increased in both groups relative the soy-free control. Results of protein analysis largely concurred with those of the mRNA. Oestrogen receptor ß (ERß) was undetected while oestrogen receptor α (ERα) was detected in each sample group. Genistein can increase the expression of coagulation and fibrinolytic genes. This effect was similar and in some cases higher than 17ß estradiol. These results suggest that genistein may not be neutral with respect to the haemostatic system.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Terapia de Reemplazo de Estrógeno , Genisteína/farmacología , Hígado/efectos de los fármacos , Ovariectomía , Fitoestrógenos/farmacología , Animales , Coagulación Sanguínea/genética , Western Blotting , Proteína C-Reactiva/genética , Proteína C-Reactiva/metabolismo , Ensayo de Inmunoadsorción Enzimática , Factor VII/genética , Factor VII/metabolismo , Femenino , Fibrinógeno/genética , Fibrinógeno/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Plasminógeno/genética , Plasminógeno/metabolismo , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , Protrombina/genética , Protrombina/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Activador de Tejido Plasminógeno/genética , Activador de Tejido Plasminógeno/metabolismoRESUMEN
A 71-year-old woman and her elder sister developed ligneous conjunctivitis after ocular surgery. Laboratory tests demonstrated that the proband and her sister had 6.6% and 8.1% of plasminogen activity, and 1.2 and 1.4 mg/dl of antigen, respectively. Thus, they were diagnosed as having severe type I plasminogen deficiency, for the first time, in Japan. DNA sequencing and PCR-RFLP analyses revealed that these two cases are homozygotes of a novel A-to-G mutation at the obligatory splicing acceptor site in intron-C. Both cases were satisfactorily treated with a direct thrombin inhibitor, topical Argatroban, and topical plasma obtained from their healthy family members.
Asunto(s)
Conjuntivitis/terapia , Plasminógeno/deficiencia , Plasminógeno/genética , Anciano , Transfusión de Componentes Sanguíneos , Conjuntivitis/tratamiento farmacológico , Conjuntivitis/genética , Femenino , Humanos , Japón , Plasma , Trombina/antagonistas & inhibidoresRESUMEN
Ligneous conjunctivitis (LC) is a rare disease characterized by wood-like pseudomembranes developing on the ocular and extraocular mucosae secondary to plasminogen (PLG) deficiency. In this paper, we report two cases of LC in two sisters of 57 and 62 years of age that presented with recurrent, bilateral pseudomembranes on conjunctiva and a history of consanguinity and deafness. Pseudomembranes showed superficial and/or subepithelial deposits of eosinophilic amorphous hyaline, amyloid-like material with a variable proportion of granulation tissue, and inflammatory cells. The eosinophilic deposits were negative for Congo red stain, immunoreactive for fibrinogen, and consistently negative for amyloid A component, transthyretin, beta(2)-microglobulin, albumin, fibronectin, collagen type IV, vimentin, and cytokeratins. Among inflammatory cells, a percentage of positivity of roughly 60% for lymphocytes T (CD3+) and 40% for lymphocytes B (CD8+), with a relation of cytotoxic/helper (CD8/4) T cells of 3:2, was found. In one case, nasal polyps and recurrent gastric peptic ulcer were also characterized by the same subepithelial hyaline deposits. A novel homozygous point mutation c.1856 C>T was found in exon 15 of the PLG gene in both patients. Amniotic membrane transplantation was done in one case with promising results.
Asunto(s)
Conjuntivitis/genética , Conjuntivitis/patología , Pólipos Nasales/genética , Pólipos Nasales/patología , Úlcera Gástrica/genética , Úlcera Gástrica/patología , Amnios/trasplante , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/patología , Conjuntivitis/diagnóstico , Exones/genética , Ojo/metabolismo , Ojo/patología , Femenino , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patología , Humanos , Hialina/metabolismo , Persona de Mediana Edad , Membrana Mucosa/metabolismo , Membrana Mucosa/patología , Pólipos Nasales/diagnóstico , Plasminógeno/deficiencia , Plasminógeno/genética , Plasminógeno/metabolismo , Mutación Puntual/genética , Hermanos , Úlcera Gástrica/diagnósticoRESUMEN
Plasmin(ogen) kringles 1 and 4 are involved in anchorage of plasmin(ogen) to fibrin and cells, an essential step in fibrinolysis and pericellular proteolysis. Their contribution to these processes was investigated by selective neutralization of their lysine-binding function. Blocking the kringle 1 lysine-binding site with monoclonal antibody 34D3 fully abolished binding and activation of Glu-plasminogen and prevented both fibrinolysis and plasmin-induced cell detachment-induced apoptosis. In contrast, blocking the kringle 4 lysine-binding site with monoclonal antibody A10.2 did not impair its activation although it partially inhibited plasmin(ogen) binding, fibrinolysis and cell detachment. This remarkable, biologically relevant, distinctive response was not observed for plasmin or Lys-plasminogen; each antibody inhibited their binding and activation of Lys-plasminogen to a limited extent, and full inhibition of fibrinolysis required simultaneous neutralization of both kringles. Thus, in Lys-plasminogen and plasmin, kringles 1 and 4 act as independent and complementary domains, both able to support binding and activation. We conclude that Glu-/Lys-plasminogen and plasmin conformations are associated with transitions in the lysine-binding function of kringles 1 and 4 that modulate fibrinolysis and pericellular proteolysis and may be of biological relevance during athero-thrombosis and inflammatory states. These findings constitute the first biological link between plasmin(ogen) transitions and functions.
Asunto(s)
Apoptosis , Fibrinolisina/farmacología , Fibrinólisis/efectos de los fármacos , Kringles/fisiología , Fragmentos de Péptidos/metabolismo , Plasminógeno/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Antifibrinolíticos/química , Sitios de Unión , Células CHO , Adhesión Celular/efectos de los fármacos , Cricetinae , Fibrina/metabolismo , Ácido Glutámico/química , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Humanos , Lisina/química , Lisina/genética , Lisina/metabolismo , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/genética , Plasminógeno/antagonistas & inhibidores , Plasminógeno/genética , Unión Proteica/efectos de los fármacos , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
OBJECTIVE: To observe the effect of rhubarb in treating secondary damage of central nerve system (CNS) in rats with acute hemorrhagic stroke (AHS) and to explore the possible mechanism. METHODS: The rat's AHS model was established by autologous blood injection, the effect of rhubarb on the secondary damage of CNS, plasminogen (PLG) in brain and tissue type plasminogen activator (t-PA) were observed. RESULTS: (1) The nerve function deficit signs reappeared in about 70% model rats 4 - 6 days after modeling and reached the peak at day 6 - 8, scored as 1.63 +/- 0.72 on day 4 and as 2.32 +/- 1.12 on day 7; (2) Rhubarb could effectively improve the secondary nerve function damage, with the nerve deficit scores kept to 1.24 +/- 0.19 from day 4 on, and no sign of secondary CNS damage was shown. The nerve deficit score was 1.22 +/- 0.15 on day 7 in the rhubarb treated group, showing significant difference as compared with that in the model group (P<0.05); (3) The specific amplified products of t-PA mRNA on day 3 and that of PLG mRNA on day 7 in CNS of model group were significantly higher than those in the sham operated group and the rhubarb treated group. CONCLUSION: Rhubarb could effectively reduce the secondary CNS damage in rats with AHS, it might be related with the suppressive effect of rhubarb on tPA mRNA and PLG mRNA expression in CNS.
Asunto(s)
Medicamentos Herbarios Chinos/farmacología , Hemorragias Intracraneales , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Plasminógeno/biosíntesis , Rheum , Animales , Hemorragias Intracraneales/sangre , Hemorragias Intracraneales/patología , Hemorragias Intracraneales/fisiopatología , Masculino , Plasminógeno/genética , Inhibidor 1 de Activador Plasminogénico/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Rheum/química , Accidente Cerebrovascular/sangre , Accidente Cerebrovascular/patología , Accidente Cerebrovascular/fisiopatologíaRESUMEN
PURPOSE: To investigate the effect of different doses of adjuvant angiostatin affecting hepatic micrometastasis in a murine model of metastatic ocular melanoma. METHODS: Angiostatin and plasminogen expression was detected in three murine melanoma cell lines (Queens, B16F10, and B16LS9). The three cell lines were heterotopically inoculated into the posterior compartment (PC) of the right eyes of C57BL/6 mice. After enucleation, the mice were given injections of 100 microl PBS and low dose (0.1 microg/microl) or high dose (0.3 microg/microl) murine recombinant angiostatin every day for 14 days after enucleation. The mice were sacrificed at 21 days post-enucleation and hepatic micrometastases were counted. In vitro migration/invasion assays were performed with low (0.1 microg/microl) and high (50 microg/microl) concentration angiostatin supplementation. Quantitative RT-PCR detected mRNA and Western analysis determined protein expression of VEGF for all cell lines. Evaluation of TdT mediated dUTP nick end labeling (TUNEL) and MIB1 immunostaining of the micrometastases determined apoptosis and proliferation ratios. RESULTS: There was a decrease in micrometastasis in the low dose group for Queens (p<0.05), B16F10 (p<0.05), and B16LS9 melanoma (p<0.01) cell lines. Two of the cell lines (B16F10 and B16LS9) elaborated plasminogen and were able to cleave plasminogen into K1-K4 (angiostatin). There was a decrease in the in vitro migration and invasion after supplementation with low concentration compared to high concentration angiostatin (p<0.01). VEGF mRNA and protein expression decreased in all cells lines in low concentration angiostatin, with the greatest decrease in B16LS9 cells (p<0.05). Apoptosis ratios were increased (p<0.01) and proliferation ratios were decreased (p<0.01) in hepatic micrometastases after treatment with low dose angiostatin. CONCLUSIONS: There were significantly fewer micrometastases in treated compared to controls with low dose compared to high dose angiostatin. This treatment results in apoptosis in the micrometastases. The mechanism appears to be related an anti-migratory effect and altered VEGF expression by melanoma cells.
Asunto(s)
Inhibidores de la Angiogénesis/administración & dosificación , Angiostatinas/administración & dosificación , Antineoplásicos/administración & dosificación , Neoplasias del Ojo/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Melanoma Experimental/tratamiento farmacológico , Animales , Apoptosis , Western Blotting , Proliferación Celular , Modelos Animales de Enfermedad , Enucleación del Ojo , Neoplasias del Ojo/genética , Neoplasias del Ojo/patología , Femenino , Etiquetado Corte-Fin in Situ , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundario , Melanoma Experimental/genética , Melanoma Experimental/secundario , Ratones , Ratones Endogámicos C57BL , Invasividad Neoplásica , Plasminógeno/genética , Plasminógeno/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes/administración & dosificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Severe type I plasminogen deficiency is the underlying cause of ligneous conjunctivitis, a rare disease characterized by wood-like pseudomembranes developing on the ocular and extraocular mucosa. Two unrelated female patients with ligneous conjunctivitis and moderate hypoplasminogenemia are described. Being of fertile age, they were treated with oral contraceptives, which determined a marked increase in plasminogen levels. Moreover, a palpebral pseudomembrane stopped growing in one patient and disappeared completely in the other while on the estroprogestinic treatment. In patient n. 2, who also suffered from von Willebrand's disease, prior Cushing's disease induced an increase in both von Willebrand factor and plasminogen levels, which dropped after curative hypophysectomy. Genetic plasminogen study showed a 19Lys>Glu mutation in a heterozygous state in the first proposita and in a homozygous state in the second proband. In addition, both index patients were homozygous for a new intron F-14T>G mutation, which was found to reduce the acceptor splicing site prediction score. In conclusion, oral contraceptive therapy may improve plasminogen deficiency and deserves attention as an alternative therapeutic approach in selected cases of ligneous conjunctivitis with low, but not absent, plasminogen synthesis.
Asunto(s)
Conjuntivitis/tratamiento farmacológico , Anticonceptivos Orales Combinados/uso terapéutico , Anticonceptivos Hormonales Orales/uso terapéutico , Etinilestradiol/uso terapéutico , Levonorgestrel/uso terapéutico , Plasminógeno/deficiencia , Adolescente , Adulto , Sustitución de Aminoácidos , Terapia Combinada , Conjuntivitis/genética , Conjuntivitis/cirugía , Síndrome de Cushing/complicaciones , Síndrome de Cushing/cirugía , Análisis Mutacional de ADN , Etinilestradiol/administración & dosificación , Exones/genética , Femenino , Genes Recesivos , Genotipo , Humanos , Hipofisectomía , Intrones/genética , Levonorgestrel/administración & dosificación , Mutación Missense , Linaje , Plasminógeno/genética , Mutación Puntual , Sitios de Empalme de ARN , Enfermedades de von Willebrand/complicacionesRESUMEN
PURPOSE: The etiology of ligneous conjunctivitis is now known to be due to an underlying type 1 plasminogen deficiency. We hereby report the clinical features of three cases and their response to topically administered plasminogen. DESIGN: Observational case series. METHODS: Two Caucasian females aged 5 years and an 18-month male of north African descent presented with a membranous conjunctivitis, which recurred after surgical excision. Case 1 presented before the association with plasminogen deficiency was known with a bilateral chronic membranous mucopurulent conjunctivitis from the age of 14 months associated with bronchiolitis and gingival hyperplasia. A diagnosis of ligneous conjunctivitis was entertained and a number of drops were instituted. At the age of 4 years plasminogen levels were ordered. Case 2 presented at the age of 4 years with a unilateral chronic membranous conjunctivitis. Plasminogen levels were requested as soon as a diagnosis of ligneous conjunctivitis was suspected. Case 3 was born with congenital hydrocephalus. Conjunctivitis was treated with antibiotics from the age of 1 month. He presented to the eye clinic at the age of 5 months when a clinical diagnosis of ligneous conjunctivitis was entertained and treated with a number of medications. Plasminogen levels were available at 9 months of age. RESULTS: The two female patients returned plasminogen levels of 0.25 U/ml and 0.3 U/ml, well below the normal level of 0.7-1.0 U/ml. Functional plasminogen levels in the male infant were not recordable with plasminogen antigen levels of 0.125 U/ml (normal range, 0.52-1.82). All cases have responded well to excision of the membranes and institution of topical plasminogen drops. There has been no recurrence with more than 12 months' follow-up. CONCLUSIONS: With the knowledge of the etiology of ligneous conjunctivitis, efforts are underway to identify the best method of delivery of plasminogen. Topical plasminogen concentrate from fresh frozen plasma holds promise as the definitive treatment for this chronic membranous conjunctivitis
Asunto(s)
Conjuntivitis/tratamiento farmacológico , Plasminógeno/uso terapéutico , Administración Tópica , Preescolar , Conjuntivitis/enzimología , Conjuntivitis/genética , Femenino , Humanos , Lactante , Masculino , Soluciones Oftálmicas , Plasminógeno/deficiencia , Plasminógeno/genéticaRESUMEN
BACKGROUND: The role of plasminogen system components in focal cerebral ischemic infarction (FCI) was studied in mice deficient in plasminogen (Plg-/-), in tissue or urokinase plasminogen activator (tPA-/- or uPA-/-), or in plasminogen activator inhibitor-1 or alpha2-antiplasmin (PAI-1(-/-) or alpha2-AP-/-). METHODS AND RESULTS: FCI was produced by ligation of the left middle cerebral artery and measured after 24 hours by planimetry of stained brain slices. In control (wild-type) mice, infarct size was 7.6+/-1.1 mm3 (mean+/-SEM), uPA-/- mice had similar infarcts (7.8+/-1.0 mm3, P=NS), tPA-/- mice smaller (2.6+/-0.80 mm3, P<0.0001), PAI-1(-/-) mice larger (16+/-0.52 mm3, P<0.0001), and Plg-/- mice larger (12+/-1.2 mm3, P=0.037) infarcts. alpha2-AP-/- mice had smaller infarcts (2. 2+/-1.1 mm3, P<0.0001 versus wild-type), which increased to 13+/-2.5 mm3 (P<0.005 versus alpha2-AP-/-) after intravenous injection of human alpha2-AP. Injection into alpha2-AP-/- mice of Fab fragments of affinospecific rabbit IgG against human alpha2-AP, after injection of 200 microg human alpha2-AP, reduced FCI from 11+/-1.5 to 5.1+/-1.1 mm3 (P=0.004). CONCLUSIONS: Plg system components affect FCI at 2 different levels: (1) reduction of tPA activity (tPA gene inactivation) reduces whereas its augmentation (PAI-1 gene inactivation) increases infarct size, and (2) reduction of Plg activity (Plg gene inactivation or alpha2-AP injection) increases whereas its augmentation (alpha2-AP gene inactivation or alpha2-AP neutralization) reduces infarct size. Inhibition of alpha2-AP may constitute a potential avenue to treatment of FCI.
Asunto(s)
Isquemia Encefálica/metabolismo , Infarto Cerebral/metabolismo , Inhibidor 1 de Activador Plasminogénico/fisiología , Plasminógeno/fisiología , Activador de Tejido Plasminógeno/fisiología , Activador de Plasminógeno de Tipo Uroquinasa/fisiología , alfa 2-Antiplasmina/fisiología , Adenoviridae/genética , Animales , Encéfalo/patología , Isquemia Encefálica/patología , Infarto Cerebral/patología , Evaluación Preclínica de Medicamentos , Femenino , Fibrinólisis , Marcación de Gen , Terapia Genética , Vectores Genéticos/genética , Humanos , Inmunoglobulina G/farmacología , Inmunoglobulina G/uso terapéutico , Masculino , Ratones , Ratones Noqueados , Plasminógeno/deficiencia , Plasminógeno/genética , Inhibidor 1 de Activador Plasminogénico/deficiencia , Inhibidor 1 de Activador Plasminogénico/genética , Conejos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/fisiología , Proteínas Recombinantes de Fusión/uso terapéutico , Activador de Tejido Plasminógeno/deficiencia , Activador de Tejido Plasminógeno/genética , Transfección , Activador de Plasminógeno de Tipo Uroquinasa/deficiencia , Activador de Plasminógeno de Tipo Uroquinasa/genética , alfa 2-Antiplasmina/deficiencia , alfa 2-Antiplasmina/genética , alfa 2-Antiplasmina/inmunología , alfa 2-Antiplasmina/uso terapéuticoAsunto(s)
Conjuntivitis/genética , Fragmentos de Péptidos/uso terapéutico , Plasminógeno/deficiencia , Plasminógeno/uso terapéutico , Factores de Coagulación Sanguínea/análisis , Conjuntivitis/tratamiento farmacológico , Semivida , Homocigoto , Humanos , Hidrocefalia/genética , Recién Nacido , Masculino , Fragmentos de Péptidos/farmacocinética , Plasminógeno/análisis , Plasminógeno/genética , Plasminógeno/farmacocinética , Mutación Puntual , Sistema Respiratorio/metabolismo , Mapeo Restrictivo , Análisis de Secuencia de ADN , Viscosidad , Cicatrización de Heridas/genéticaRESUMEN
Despite recent advances in neurosurgery, radiation, and chemotherapy, the prognosis of patients with malignant gliomas remains dismal. Based on the observation that solid tumor growth is angiogenic dependent, and gliomas are among the most angiogenic of all tumors, therapeutic strategies aimed at inhibiting angiogenesis are theoretically attractive. Angiostatin, an internal peptide fragment of plasminogen, has recently been shown to potently inhibit endothelial proliferation in vitro and tumor growth in vivo. Long-term systemic delivery of proteins, however, poses a number of difficult logistic and pharmacological problems and may not be necessary or optimal for treating locally aggressive tumors such as gliomas. We now demonstrate that retroviral and adenoviral vectors that transduce the angiostatin cDNA can be used to inhibit endothelial cell growth in vitro and angiogenesis in vivo. Vector-mediated inhibition of tumor-associated angiogenesis results in increased apoptotic tumor cell death, leading to inhibition of tumor growth. These studies support a potential role of vector-mediated transduction of the cDNA encoding angiostatin as a potential novel therapeutic strategy for the treatment of malignant brain tumors and confirm the antitumor activity of angiostatin and the concept of dormancy therapy.
Asunto(s)
Neoplasias Encefálicas/irrigación sanguínea , ADN Complementario/genética , Terapia Genética/métodos , Glioblastoma/irrigación sanguínea , Glioma/irrigación sanguínea , Neovascularización Patológica/terapia , Fragmentos de Péptidos/genética , Plasminógeno/genética , Adenoviridae/genética , Adenoviridae/metabolismo , Angiostatinas , Animales , Northern Blotting , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/terapia , División Celular/fisiología , ADN Complementario/metabolismo , Vectores Genéticos , Glioblastoma/patología , Glioblastoma/terapia , Glioma/patología , Glioma/terapia , Humanos , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Fragmentos de Péptidos/biosíntesis , Plasminógeno/biosíntesis , Ratas , Ratas Endogámicas F344 , TransfecciónRESUMEN
Insight into the structural features of human lipoprotein(a) [Lp(a)] which underlie its functional implication in fibrinolysis may be gained from comparative studies of apo(a). Indeed, cloning of rhesus monkey apo(a) has shown that a Trp72 --> Arg mutation in the lysine-binding site (LBS) of KIV-10 leads to loss of lysine-binding properties of the rhesus Lp(a) particle. Consequently, comparative studies of apo(a) sequences in different Old World monkey species should further our understanding of the molecular role of Lp(a) in the fibrinolytic process. In contrast to other Old World monkeys, including rhesus monkey, cynomolgus, and baboon, the chimpanzee exhibits an elevated level of Lp(a) and a distinct isoform distribution as compared to humans [Doucet et al. J. Lipid Res. (1994) 35, 263-270]. Clearly then, the chimpanzee is an interesting animal model for study of the structure, function, and potential pathophysiological roles of Lp(a). We have cloned and sequenced the region of chimpanzee apo(a) cDNA spanning KIV-3 to the stop codon. The global organization of this region is similar to that of human apo(a) with the presence of KV, which is absent in rhesus monkey apo(a). Nucleotide sequence comparison indicates a variation of 1.4% between chimpanzee and man and 5.1% between chimpanzee and rhesus monkey. The differences concerned single base changes. An Asp57 --> Asn mutation was detected in KIV-10; this residue is critical to the LBS of KIV-10 in human apo(a). To verify that the Asp57 --> Asn substitution was specific to apo(a), we have also cloned the cDNA-encoding plasminogen, which exhibited an Asp at the corresponding position in kringle IV. Using an in vitro binding assay, we have demonstrated that chimpanzee Lp(a) exhibits poor lysine-specific interaction with both intact and plasmin-degraded fibrin as compared to its human counterpart. We propose that the Asn57 substitution in KIV-10 of chimpanzee apo(a) is responsible for this property. Chimpanzee Lp(a) therefore represents an appropriate particle with which to explore the potential effects of Lp(a) on the fibrinolytic system, such as the inhibition of plasminogen activation or inhibition of t-PA activity.
Asunto(s)
Apolipoproteínas/química , Apolipoproteínas/genética , ADN Complementario/aislamiento & purificación , Fibrina/metabolismo , Kringles/genética , Lipoproteína(a) , Mutación , Sustitución de Aminoácidos/genética , Animales , Apolipoproteínas/aislamiento & purificación , Apoproteína(a) , Asparagina/genética , Ácido Aspártico/genética , Clonación Molecular , ADN Complementario/química , Femenino , Humanos , Macaca mulatta , Pan troglodytes , Plasminógeno/química , Plasminógeno/genética , Unión Proteica/genética , Estructura Terciaria de ProteínaRESUMEN
Regions of the human plasminogen (Pg) cDNA containing its kringle 4 (K4) and K5 domains have been expressed in Escherichia coli, and binding constants of omega-amino acid ligands for recombinant (r)-[K4Pg] and r-[K5Pg] have been obtained. In each case, the results showed that of a series of aliphatic alpha, omega-amino acid analogues, 6-aminohexanoic acid showed maximal affinity for these modules, and all ligands interacted more strongly with r-[K4Pg] than with r-[K5Pg]. Site-directed mutagenesis investigations demonstrated that the major amino acid side chain contributors to ligand binding were similar for each of these kringles. Ligand binding was stabilized by charged groups at Asp56 of r-[K4Pg] and Asp57 of r-[K5Pg] as well as by Arg69 of both r-[K4Pg] and r-[K5Pg]. Some hydrophobic amino acids that contributed significantly to the binding strength of the ligands were identified. These were provided by homologous residues in each of the domains, viz. Trp60 and Trp70 of r-[K4Pg] and Trp62 and Tyr72 of r-[K5Pg]. Tyr74 of r-[K5Pg] also substantially contributed to its ligand binding energy.