RESUMEN
Cancer cells are able to reach distant tissues by migration and invasion processes. Enhanced ability to cope with physical stresses leading to cell membrane damages may offer to cancer cells high survival rate during metastasis. Consequently, down-regulation of the membrane repair machinery may lead to metastasis inhibition. We show that migration of MDA-MB-231 cells on collagen I fibrils induces disruptions of plasma membrane and pullout of membrane fragments in the wake of cells. These cells are able to reseal membrane damages thanks to annexins (Anx) that are highly expressed in invasive cancer cells. In vitro membrane repair assays reveal that MDA-MB-231 cells respond heterogeneously to membrane injury and some of them possess a very efficient repair machinery. Finally, we show that silencing of AnxA5 and AnxA6 leads to the death of migrating MDA-MB-231 cells due to major defect of the membrane repair machinery. Disturbance of the membrane repair process may therefore provide a new avenue for inhibiting cancer metastasis.
Asunto(s)
Anexina A5/metabolismo , Membrana Celular/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Línea Celular Tumoral , Membrana Celular/patología , Supervivencia Celular , Humanos , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias/patologíaRESUMEN
An effective prophylactic hepatitis B virus (HBV) vaccine has long been available but is ineffective for chronic infection. The primary cause of chronic hepatitis B (CHB) and greatest impediment for a therapeutic vaccine is the direct and indirect effects of immune tolerance to HBV antigens. The resulting defective CD4+/CD8+ T cell response, poor cytokine production, insufficient neutralizing antibody (nAb) and poor response to HBsAg vaccination characterize CHB infection. The objective of this study was to develop virus-like-particles (VLPs) that elicit nAb to prevent viral spread and prime CD4+/CD8+ T cells to eradicate intracellular HBV. Eight neutralizing B cell epitopes from the envelope PreS1 region were consolidated onto a species-variant of the HBV core protein, the woodchuck hepatitis core antigen (WHcAg). PreS1-specific B cell epitopes were chosen because of preferential expression on HBV virions. Because WHcAg and HBcAg are not crossreactive at the B cell level and only partially cross-reactive at the CD4+/CD8+ T cell level, CD4+ T cells specific for WHcAg-unique T cell sites can provide cognate T-B cell help for anti-PreS1 Ab production that is not curtailed by immune tolerance. Immunization of immune tolerant HBV transgenic (Tg) mice with PreS1-WHc VLPs elicited levels of high titer anti-PreS1 nAbs equivalent to wildtype mice. Passive transfer of PreS1 nAbs into human-liver chimeric mice prevented acute infection and cleared serum HBV from mice previously infected with HBV in a model of CHB. At the T cell level, PreS1-WHc VLPs and hybrid WHcAg/HBcAg DNA immunogens elicited HBcAg-specific CD4+ Th and CD8+ CTL responses.
Asunto(s)
Hepatitis B Crónica , Hepatitis B , Animales , Linfocitos T CD8-positivos , Hepatitis B/prevención & control , Antígenos del Núcleo de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B , Vacunas contra Hepatitis B , Virus de la Hepatitis B , Hepatitis B Crónica/prevención & control , Tolerancia Inmunológica , RatonesRESUMEN
We have investigated the ability of hepatitis C virus non-structural (NS) 3/4A-DNA-based vaccines to activate long-term cell-mediated immune responses in mice. Wild-type and synthetic codon optimized (co) NS3/4A DNA vaccines have previously been shown to be immunogenic in mice, rabbits and humans, although we have very poor knowledge about the longevity of the immune responses primed. We therefore analyzed the functionality of primed NS3/4A-specific immune responses in BALB/c (H-2(d)) and/or C57BL/6J (H-2(b)) mice 1, 2, 3, 4, 6, 12 and 16 months after the last immunization. Mice were immunized one, two, three or four times using gene gun delivery to the skin or by intramuscular administration. Immunological responses after immunization were monitored by protection against in vivo challenge of NS3/4A-expressing syngeneic tumor cells. In addition, functionality of the NS3/4A-specific T cells was analyzed by a standard cytotoxicity assay. First, we identified a new unique murine H-2(d)-restricted NS3/4A cytotoxic T lymphocyte (CTL) epitope, which enabled us to study the epitope-specific immune responses. Our results show that the coNS3/4A vaccine was highly immunogenic by determination of interferon-γ/tumor necrosis factor-α production and lytic cytotoxic T cells, which could efficiently inhibit in vivo tumor growth. Importantly, we showed that one to four monthly immunizations protected mice from tumor development when challenged up to 16 months after the last immunization. When determining the functionality of NS3/4A-specific T cells in vitro, we showed detectable lytic activity up to 12 months after the last immunization. Thus, NS3/4A-based DNA vaccines activate potent cellular immune responses that are present and function in both BALB/c and C57BL/6J mice up to 12-16 months after the last immunization. The induction of long-term immunity after NS3/4A DNA immunization has not been shown previously and supports the use of NS3/4A in hepatitis C virus vaccine compositions.
Asunto(s)
Inmunidad Adaptativa , Hepacivirus/inmunología , Vacunas de ADN/inmunología , Animales , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Factores de Tiempo , Vacunación/métodos , Vacunas de ADN/administración & dosificaciónRESUMEN
BACKGROUND: Hepatitis C virus (HCV) effectively establishes persistent infection in human livers. The non-structural (NS) 3/4A complex participates in this process by cleavage of interferon beta (IFN beta) promoter stimulator-1 (IPS-1; also termed Cardif/MAVS/VISA), which inhibits responses to double stranded (ds) RNA. However, it is not known whether this effect extends beyond innate responses. AIMS: To test if HCV NS3/4A affects innate and adaptive immune responses in vivo. METHODS: NS3 levels were semi-quantified in human liver biopsies, transfected cells, and in transgenic (Tg) mouse livers by western blot. The effect of NS3/4A on dsRNA-mediated signalling and on the integrity of IPS-1 was analysed using in vitro translation, transfected cells and Tg mice. Cytotoxic T cell (CTL)-mediated clearance of transient firefly luciferase (FLuc)- and/or NS3/4A-Tg hepatocytes was determined using in vivo imaging and western blot. RESULTS: NS3 protein levels were in a comparable range (0.1-49 microg/g tissue) in infected human livers and Tg mouse livers. Importantly, these levels of NS3/4A reduced murine innate responses to synthetic dsRNA in vivo, supporting the possibility that this occurs also in infected humans. The likely explanation for this was the NS3/4A-mediated cleavage of mouse IPS-1, albeit less efficiently than human IPS-1. Despite this, FLuc- and/or NS3/4A-expressing murine hepatocytes were effectively eliminated by hepatic CTLs, utilising the classical molecules for virus-infected cell lysis, including CD8, IFN gamma, perforin and FasL. CONCLUSIONS: Although HCV NS3/4A inhibits the innate immunity, this does not prevent CTL-mediated clearance of NS3/4A-expressing hepatocytes in vivo. Thus, other HCV proteins are most likely responsible for interfering with the adaptive immunity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Hepacivirus , Hepatitis C Crónica/inmunología , Hepatocitos/virología , Linfocitos T/inmunología , Proteínas no Estructurales Virales/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Hepacivirus/inmunología , Hepacivirus/metabolismo , Hepatitis C Crónica/virología , Hepatocitos/inmunología , Humanos , Inmunidad Innata , Interferón beta/inmunología , Hígado/metabolismo , Neoplasias Hepáticas/inmunología , Masculino , Ratones , Ratones Endogámicos , Ratones Transgénicos , FN-kappa B/metabolismo , ARN Bicatenario/inmunología , Especificidad de la Especie , Células Tumorales CultivadasRESUMEN
BACKGROUND: The hepatitis C virus (HCV) establishes chronic infection by incompletely understood mechanisms. The non-structural (NS) 3/4A protease/helicase has been proposed as a key complex in modulating the infected hepatocyte, although nothing is known about the effects this complex exerts in vivo. AIM: To generate mice with stable and transient hepatocyte expression of the HCV NS3/4A proteins to study its effects in vivo. METHODS: NS3/4A expression was determined by western blot and immunohistochemistry. Two independent pathologists determined the liver histology. Hepatic immunity was characterised by quantifying intrahepatic immune cell subsets. Liver damage was induced using carbon tetrachloride (CCl(4)), lipopolysaccaride (LPS), tumour necrosis factor alpha (TNFalpha), and anti-Fas antibody. RESULTS: Expression of NS3/4A was restricted to the cytoplasm of hepatocytes, and did not cause liver cancer or any spontaneous liver pathology. However, the presence of NS3/4A modulated the intrahepatic immunity, as follows: first, the CD4+ T cell and type I/II dendritic cell subsets were reduced in transgenic livers; second, NS3/4A protected hepatocytes from liver damage mediated in vivo by CCl(4), LPS, TNFalpha, but not FAS; and third, both stable and transiently NS3/4A transgenic mice were resistant to lethal doses of liver targeted TNFalpha, and the resistance could be reverted by treatment with a p38 mitogen activated protein kinase inhibitor (MAPK). CONCLUSIONS: Hepatic expression of NS3/4A does not induce spontaneous liver disease. NS3/4A does, however, alter the intrahepatic immune cell subsets and protects hepatocytes against TNFalpha induced liver damage in vivo. The TNFalpha resistance can be reverted by treatment with a p38 MAPK inhibitor. This represents a new immune evasion strategy conferred by NS3/4A.
Asunto(s)
Hepacivirus/inmunología , Hepatopatías/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Hepatocitos/metabolismo , Inmunohistoquímica , Subgrupos Linfocitarios/inmunología , Ratones , Ratones Transgénicos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
BACKGROUND: The hepatitis C virus (HCV) mutates within human leucocyte antigen (HLA) class I restricted immunodominant epitopes of the non-structural (NS) 3/4A protease to escape cytotoxic T lymphocyte (CTL) recognition and promote viral persistence. However, variability is not unlimited, and sometimes almost absent, and factors that restrict viral variability have not been defined experimentally. AIMS: We wished to explore whether the variability of the immunodominant CTL epitope at residues 1073-1081 of the NS3 protease was limited by viral fitness. PATIENTS: Venous blood was obtained from six patients (four HLA-A2+) with chronic HCV infection and from one HLA-A2+ patient with acute HCV infection. METHODS: NS3/4A genes were amplified from serum, cloned in a eukaryotic expression plasmid, sequenced, and expressed. CTL recognition of naturally occurring and artificially introduced escape mutations in HLA-A2-restricted NS3 epitopes were determined using CTLs from human blood and genetically immunised HLA-A2-transgenic mice. HCV replicons were used to test the effect of escape mutations on HCV protease activity and RNA replication. RESULTS: Sequence analysis of NS3/4A confirmed low genetic variability. The major viral species had functional proteases with 1073-1081 epitopes that were generally recognised by cross reactive human and murine HLA-A2 restricted CTLs. Introduction of mutations at five positions of the 1073-1081 epitope prevented CTL recognition but three of these reduced protease activity and RNA replication. CONCLUSIONS: Viral fitness can indeed limit the variability of HCV within immunological epitopes. This helps to explain why certain immunological escape variants never appear as a major viral species in infected humans.
Asunto(s)
Hepacivirus/genética , Hepatitis C/inmunología , Tolerancia Inmunológica , Proteínas no Estructurales Virales/genética , Enfermedad Aguda , Adulto , Animales , Citotoxicidad Inmunológica , Epítopos de Linfocito T/inmunología , Femenino , Genes Virales , Variación Genética/inmunología , Antígeno HLA-A2/metabolismo , Hepacivirus/inmunología , Hepatitis C/virología , Hepatitis C Crónica/inmunología , Hepatitis C Crónica/virología , Humanos , Epítopos Inmunodominantes/genética , Epítopos Inmunodominantes/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Mutación , Fragmentos de Péptidos/inmunología , ARN Viral/genética , Linfocitos T Citotóxicos/inmunología , Proteínas no Estructurales Virales/inmunología , Replicación Viral/genética , Replicación Viral/inmunologíaRESUMEN
We have recently shown that the NS3-based genetic immunogens should contain also hepatitis C virus (HCV) nonstructural (NS) 4A to utilize fully the immunogenicity of NS3. The next step was to try to enhance immunogenicity by modifying translation or mRNA synthesis. To enhance translation efficiency, a synthetic NS3/4A-based DNA (coNS3/4A-DNA) vaccine was generated in which the codon usage was optimized (co) for human cells. In a second approach, expression of the wild-type (wt) NS3/4A gene was enhanced by mRNA amplification using the Semliki forest virus (SFV) replicon (wtNS3/4A-SFV). Transient tranfections of human HepG2 cells showed that the coNS3/4A gene gave 11-fold higher levels of NS3 as compared to the wtNS3/4A gene when using the CMV promoter. We have previously shown that the presence of NS4A enhances the expression by SFV. Both codon optimization and mRNA amplification resulted in an improved immunogenicity as evidenced by higher levels of NS3-specific antibodies. This improved immunogenicity also resulted in a more rapid priming of cytotoxic T lymphocytes (CTLs). Since HCV is a noncytolytic virus, the functionality of the primed CTL responses was evaluated by an in vivo challenge with NS3/4A-expressing syngeneic tumor cells. The priming of a tumor protective immunity required an endogenous production of the immunogen and CD8+ CTLs, but was independent of B and CD4+ T cells. This model confirmed the more rapid in vivo activation of an NS3/4A-specific tumor-inhibiting immunity by codon optimization and mRNA amplification. Finally, therapeutic vaccination with the coNS3/4A gene using gene gun 6-12 days after injection of tumors significantly reduced the tumor growth in vivo. Codon optimization and mRNA amplification effectively enhances the overall immunogenicity of NS3/4A. Thus, either, or both, of these approaches should be utilized in an NS3/4A-based HCV genetic vaccine.
Asunto(s)
Codón , Hepacivirus/genética , Hepatitis C/prevención & control , ARN Mensajero/genética , Vacunas de ADN/genética , Vacunas contra Hepatitis Viral/genética , Animales , Anticuerpos Antivirales/sangre , Cricetinae , Femenino , Citometría de Flujo , Amplificación de Genes , Ingeniería Genética/métodos , Hepacivirus/inmunología , Hepatitis C/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos Animales , Linfocitos T Citotóxicos/inmunología , Vacunas de ADN/inmunología , Vacunas contra Hepatitis Viral/inmunologíaRESUMEN
The hepatitis C virus (HCV) protease and helicase encompasses the nonstructural (NS) 3 protein and the cofactor NS4A, which targets the NS3/4A-complex to intracellular membranes. We here evaluate the importance of NS4A in NS3-based genetic immunogens. A full-length genotype 1 NS3/4A gene was cloned into a eucaryotic expression vector in the form of NS3/4A and NS3 alone. Transient transfections revealed that the inclusion of NS4A increased the expression levels of NS3. Subsequently, immunization with the NS3/4A gene primed 10- to 100-fold higher levels of NS3-specific antibodies as compared to immunization with the NS3 gene. Humoral responses primed by the NS3/4A gene had a higher IgG2a/IgG1 ratio (>20) as compared to the NS3 gene (3.0), suggesting a T helper 1-skewed response. Low dose i.m. (10 microg) immunization with the NS3/4A gene inhibited the growth of NS3/4A-expressing tumor cells in vivo, whereas the NS3 gene alone or NS3 protein did not. We then evaluated the efficiency of the NS3/4A gene administered by the gene gun, at the same doses used for humans, in priming cytotoxic T lymphocyte (CTL) responses. Three to four 4 microg doses of the NS3/4A gene primed CTL at a precursor frequency of 2-4%, which inhibited the growth of NS3/4A-expressing tumor cells in vivo. Thus, NS4A enhances the expression levels and immunogenicity of NS3, and an NS3/4A gene delivered transdermally could be a therapeutic vaccine candidate.
Asunto(s)
Terapia Genética/métodos , Hepacivirus/genética , Mieloma Múltiple/terapia , Vacunas de ADN/administración & dosificación , Animales , Biolística , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mieloma Múltiple/inmunología , Mieloma Múltiple/virología , Estadísticas no Paramétricas , Linfocitos T Citotóxicos/inmunología , Células TH1/inmunología , Proteínas no Estructurales Virales/genéticaRESUMEN
The toxic metal lead is a widespread environmental health hazard that can adversely affect human health. In an effort to better understand the cellular and molecular consequences of lead exposure, we have employed cDNA microarrays to analyze the effects of acute lead exposure on large-scale gene expression patterns in immortalized rat astrocytes. Our studies identified many genes previously reported to be differentially regulated by lead exposure. Additionally, we have identified novel putative targets of lead-mediated toxicity, including members of the family of calcium/phospholipid binding annexins, the angiogenesis-inducing thrombospondins, collagens, and tRNA synthetases. We demonstrate the ability to distinguish lead-exposed samples from control or sodium samples solely on the basis of large-scale gene expression patterns using two complementary clustering methods. We have confirmed the altered expression of candidate genes and their encoded proteins by RT-PCR and Western blotting, respectively. Finally, we show that the calcium-dependent phospholipid binding protein annexin A5, initially identified as a differentially regulated gene by our microarray analysis, is directly bound and activated by nanomolar concentrations of lead. We conclude that microarray technology is an effective tool for the identification of lead-induced patterns of gene expression and molecular targets of lead.
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Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Plomo/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Aminoacil-ARNt Sintetasas/genética , Animales , Anexinas/genética , Anexinas/metabolismo , Western Blotting , Línea Celular Transformada , Colágeno/genética , ADN Complementario/análisis , Plomo/metabolismo , Plomo/toxicidad , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trombospondinas/genéticaRESUMEN
Lead poisoning can cause a wide range of symptoms with particularly severe clinical effects on the CNS. Lead can increase spontaneous neurotransmitter release but decrease evoked neurotransmitter release. These effects may be caused by an interaction of lead with specific molecular targets involved in neurotransmitter release. We demonstrate here that the normally calcium-dependent binding characteristics of the synaptic vesicle protein synaptotagmin I are altered by lead. Nanomolar concentrations of lead induce the interaction of synaptotagmin I with phospholipid liposomes. The C2A domain of synaptotagmin I is required for lead-mediated phospholipid binding. Lead protects both recombinant and endogenous rat brain synaptotagmin I from proteolytic cleavage in a manner similar to calcium. However, lead is unable to promote the interaction of either recombinant or endogenous synaptotagmin I and syntaxin. Finally, nanomolar concentrations of lead are able to directly compete with and inhibit the ability of micromolar concentrations of calcium to induce the interaction of synaptotagmin I and syntaxin. Based on these findings, we conclude that synaptotagmin I may be an important, physiologically relevant target of lead.
Asunto(s)
Plomo/farmacología , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Vesículas Sinápticas/metabolismo , Sustitución de Aminoácidos , Animales , Ácido Aspártico , Sitios de Unión , Proteínas de Unión al Calcio/metabolismo , Glutatión Transferasa/metabolismo , Liposomas , Glicoproteínas de Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/efectos de los fármacos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fosfolípidos/metabolismo , Proteínas Qa-SNARE , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Sinaptotagmina I , Sinaptotagminas , Terbio/farmacocinéticaRESUMEN
The movement of hydrolases and other proteins to lysosomes is accomplished by vesicle trafficking. Specific vesicles are targeted from the trans-Golgi network via a prelysosomal compartment to lysosomes. The specificity of vesicle transport is thought to occur through the interaction of vesicle proteins with receptors on a particular target membrane. The syntaxins are a family of transmembrane proteins that have been implicated as vesicle receptors involved in vesicle docking and fusion. Syntaxins 1-4 are localized to the plasma membrane, and in particular, syntaxin 1a mediates synaptic vesicle docking in the nerve terminal. Syntaxins 5 and 6 have been localized to cis-Golgi and trans-Golgi network compartments, respectively. We now report the identification of syntaxin 7 from a human brain cDNA library. The syntaxin 7 gene is localized to human chromosome 6. By Northern analysis, the syntaxin RNA was found to be broadly distributed. Based on its homology to yeast and plant vacuolar syntaxins, we propose that syntaxin 7 has a role in vesicle trafficking between the Golgi complex and lysosomes. In vitro binding studies reveal that syntaxin 7 binds alphaSNAP, a key regulator of transport vesicle fusion at multiple stages of the secretory pathway.
Asunto(s)
Lisosomas/metabolismo , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Homología de Secuencia de Aminoácido , Proteínas de Transporte Vesicular , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico , Química Encefálica , Proteínas Portadoras/metabolismo , Mapeo Cromosómico , Clonación Molecular , Feto , Aparato de Golgi/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Especificidad de Órganos , Filogenia , Unión Proteica , Proteínas Qa-SNARE , ARN Mensajero/análisis , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida , Sintaxina 1RESUMEN
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.
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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
Southern blot analysis of genomic DNA from different strains of rat indicated that there were multiple copies of the gene encoding the second enzyme of the heme biosynthetic pathway, delta-aminolevulinate dehydratase (ALA-D). Two types of genomic clones were isolated from a Sprague-Dawley rat library. One appears to be the expressed gene, whereas the nucleotide sequence of the other suggests that it contains an ALA-D processed pseudogene because (1) there are no introns, (2) there are multiple mutations that alter the predicted amino acid sequence of ALA-D and cause premature termination, (3) there is a 3' polyadenylated tract, and (4) there is an 8-bp direct repeat flanking the gene. The rat genome is unusual in this respect since ALA-D pseudogenes have not been detected in Southern blot analyses of other mammals, including human, gorilla, chimpanzee, orangutan, rabbit, mouse, and Chinese hamster.
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Porfobilinógeno Sintasa/genética , Seudogenes , Animales , Secuencia de Bases , Southern Blotting , Intrones , Datos de Secuencia Molecular , Mutación , Poli A , Ratas , Ratas Endogámicas , Mapeo RestrictivoRESUMEN
We have isolated several cDNA clones encoding delta-aminolevulinate dehydrase [ALAD; porphobilinogen synthase; 5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing), EC 4.2.1.24], the second enzyme in the heme biosynthetic pathway. We used a rabbit polyclonal antibody developed against the purified 35-kDa subunit of rat liver ALAD to screen a lambda gt11 cDNA expression library constructed from rat liver mRNA. A prototype clone (ALAD-1) contained a 680-base-pair insert and expressed a 140-kDa beta-galactosidase gene cDNA insert fusion protein immunoreactive with both polyclonal and monoclonal anti-ALAD. Identity of ALAD-1 was verified by hydridization to ALAD mRNA that had been enriched via immunopurification of rat liver polysomes with anti-ALAD. Intensity of such hybridization to a 1500-nucleotide-long mRNA was approximately equal to 200-fold greater than that realized with whole liver mRNA, a result consistent with the degree of immunoenrichment of ALAD mRNA found independently by analysis of cell-free translation products. A second ALAD cDNA clone (ALAD-3) was isolated when the rat liver cDNA expression library was rescreened with ALAD-1. The identity of both ALAD cDNA clones was established by correspondence between their nucleotide sequence and the reported amino-terminal protein sequence of bovine ALAD. Hybridization of ALAD cDNA to mouse genomic DNA indicates that the previously unexplained incremental differences in ALAD enzymatic activity among inbred mouse strains has arisen through alterations in ALAD gene dose.