RESUMEN
We describe here a clonal approach for efficient and robust construction of recombinant adenoviral genomes that holds certain advantages over existing approaches. Transgenes of interest are cloned into a small, conditionally replicating plasmid containing the left end of a recombinant adenoviral genome, encompassing pIX coding regions. Transformation of this plasmid into recombination-competent Escherichia coli bearing a plasmid containing the right end of a recombinant adenoviral genome, commencing from pIX coding regions, yields a stable co-integrated plasmid encoding a full adenoviral genome, by virtue of shared homology in pIX coding regions contained in both plasmids. The recombination process yielding the full adenoviral plasmid requires only one step, and always results in the formation of only the desired recombinant adenoviral genome. Thus, no screening is required to identify the correct plasmid encoding the desired recombinant adenoviral genome. In addition, the plasmid encoding the right-hand side of the adenoviral genome is itself incapable of producing contaminating adenovirus. We have successfully employed this approach to generate over 200 recombinant adenoviruses, obtaining only the desired recombinant adenoviral species each time. The process is amenable to medium-to-high-throughput parallel construction of adenoviral genomes, and as such should aid efforts aimed towards high-throughput functional annotation of therapeutic gene targets, which aim to leverage the benefits of adenoviruses as gene delivery and expression vectors.
Asunto(s)
Adenoviridae/genética , Ingeniería Genética , Vectores Genéticos/genética , Genoma Viral , Recombinación Genética , Animales , Clonación Molecular , Escherichia coli/genética , Luciferasas de Luciérnaga/genética , Plásmidos , TransgenesRESUMEN
The ABCA subfamily of ATP-binding cassette (ABC) transporters includes eleven members to date. In this study, we describe a new, unusually large gene on chromosome 7p12.3, ABCA13. This gene spans over 450 kb and is split into 62 exons. The predicted ABCA13 protein consists of 5,058 ami- no acid residues making it the largest ABC protein described to date. Like the other ABCA subfamily members, ABCA13 contains a hydrophobic, predicted transmembrane segment at the N-terminus, followed by a large hydrophilic region. In the case of ABCA13, the hydrophilic region is unexpectedly large, more than 3,500 amino acids, encoded by 30 exons, two of which are 4.8 and 1.7 kb in length. These two large exons are adjacent to each other and are conserved in the mouse Abca13 gene. Tissue profiling of the major transcript reveals the highest expression in human trachea, testis, and bone marrow. The expression of the gene was also determined in 60 tumor cell lines and the highest expression was detected in the SR leukemia, SNB-19 CNS tumor and DU-145 prostate tumor cell lines. ABCA13 has high similarity with other ABCA subfamily genes which are associated with human inherited diseases: ABCA1 with the cholesterol transport disorders Tangier disease and familial hypoalphalipoproteinemia, and ABCA4 with several retinal degeneration disorders. The ABCA13 gene maps to chromosome 7p12.3, a region that contains an inherited disorder affecting the pancreas (Shwachman-Diamond syndrome) as well as a locus involved in T-cell tumor invasion and metastasis (INM7), and therefore is a positional candidate for these pathologies.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Cromosomas Humanos Par 7 , Transportadoras de Casetes de Unión a ATP/biosíntesis , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Secuencia Conservada , ADN Complementario/aislamiento & purificación , Exones , Humanos , Ratones , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , ARN Mensajero/biosíntesis , Homología de Secuencia de Aminoácido , Distribución Tisular , Células Tumorales CultivadasRESUMEN
The ABCA subfamily of ABC transporters includes ten members to date. In this study, we describe an additional gene, ABCA12. Four full-length cDNA sequences have been obtained from human placenta that contain two different polyadenylation sites and two splicing forms, coding for ABCA12 isoforms of 2,595 and 2,516 amino acid residues. Both isoforms are predicted to have two ATP-binding domains (nucleotide binding domain, NBD) and two transmembrane (TM) domains, features shared by all other ABCA subfamily proteins. ABCA12 is most closely related to ABCA1, with an amino acid similarity of 47%. Northern blot analysis demonstrates that a 9.5-kb transcript is mainly expressed in the stom- ach. ABCA12 was mapped to human chromosome 2q34. Two other genes from ABCA subfamily are associated with human inherited diseases, ABCA1 with the cholesterol transport disorders Tangier disease and familial hypoalphalipoproteinemia, and ABCA4 with several retinal degeneration disorders. The ABCA12 gene is located in a region of chromosome 2q34 that harbors the genes for lamellar ichthyosis, polymorphic congenital cataract, and insulin-dependent diabetes mellitus (IDDM13), and therefore is a positional candidate for these pathologies.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Cromosomas Humanos Par 2 , Transportadoras de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/clasificación , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 2/química , Clonación Molecular , ADN Complementario/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Filogenia , Sitios de Empalme de ARN , ARN Mensajero/biosíntesis , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
We report here the genomic and transcriptional characterization in mouse and man of a novel transporter of the ABCA subclass, named ABCA7. As it is the case for other ABCA genes, the predicted protein encoded by ABCA7 is a full symmetric transporter, highly conserved across species. The ABCA7 gene maps to human chromosome 19 and to the homologous region at band B4-C1 on mouse chromosome 10. The preferential expression of ABCA7 in the spleen, thymus, and fetal liver is consistent with the finding, in both human and mouse promoter, of sites targeted by lymphomyeloid-specific transcription factors. This suggests that ABCA7 may play a pivotal role in the developmental specification of hematopoietic cell lineages.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Exones/genética , Intrones/genética , Regiones Promotoras Genéticas/genética , Transportadoras de Casetes de Unión a ATP/química , Secuencia de Aminoácidos , Animales , Línea Celular , Cromosomas Humanos Par 19/genética , Secuencia Conservada/genética , ADN Complementario/genética , Humanos , Hibridación Fluorescente in Situ , Hígado/embriología , Hígado/metabolismo , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Mensajero/análisis , ARN Mensajero/genética , Mapeo de Híbrido por Radiación , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Bazo/metabolismo , Timo/metabolismoRESUMEN
A wide variety of mutations in the parkin gene, including exon deletions and duplications, as well as point mutations, result in autosomal recessive early-onset parkinsonism. Interestingly, several of these anomalies were found repeatedly in unrelated patients and may therefore result from recurrent, de novo mutational events or from founder effects. In the present study, haplotype analysis, using 10 microsatellite markers covering a 4.7-cM region known to contain the parkin gene, was performed in 48 families, mostly from European countries, with early-onset autosomal recessive parkinsonism. The patients carried 14 distinct mutations in the parkin gene, and each mutation was detected in more than one family. Our results support the hypothesis that exon rearrangements occurred independently, whereas some point mutations, found in families from different geographic origins, may have been transmitted by a common founder.
Asunto(s)
Cromosomas Humanos Par 6 , Exones , Efecto Fundador , Reordenamiento Génico , Ligasas/genética , Mutación , Enfermedad de Parkinson/genética , Trastornos Parkinsonianos/genética , Población Blanca/genética , Edad de Inicio , Mapeo Cromosómico , Europa (Continente) , Familia , Genes Recesivos , Marcadores Genéticos , Humanos , Desequilibrio de Ligamiento , Repeticiones de Microsatélite/genética , Núcleo Familiar , Mutación Puntual , Eliminación de Secuencia , Ubiquitina-Proteína LigasasRESUMEN
A Dutch family with autosomal recessive early-onset parkinsonism showed a heterozygous missense mutation in combination with a heterozygous exon deletion in the parkin gene. Although the main clinical syndrome consisted of parkinsonism, the proband clinically had additional mild gait ataxia and pathologically showed neuronal loss in parts of the spinocerebellar system, in addition to selective loss of dopaminergic neurons in the substantia nigra pars compacta. Lewy bodies and neurofibrillary tangles were absent, but tau pathology was found.
Asunto(s)
Ligasas/genética , Trastornos Parkinsonianos/genética , Trastornos Parkinsonianos/patología , Ubiquitina-Proteína Ligasas , Anciano , Astrocitos/patología , Encéfalo/patología , Femenino , Humanos , Masculino , Países Bajos , Mutación Puntual/genética , Reacción en Cadena de la Polimerasa , Proteínas tau/análisisRESUMEN
To gain insight into the evolution and function of apolipoprotein A-IV (apoA-IV) we compared structural and interfacial properties of chicken apoA-IV, human apoA-IV, and a recombinant human apoA-IV truncation mutant lacking the carboxyl terminus. Circular dichroism thermal denaturation studies revealed that the thermodynamic stability of the alpha-helical structure in chicken apoA-IV (DeltaH = 71.0 kcal/mol) was greater than that of human apoA-IV (63.6 kcal/mol), but similar to that of human apoA-I (73.1 kcal/mol). Fluorescence chemical denaturation studies revealed a multiphasic red shift with a 65% increase in relative quantum yield that preceded loss of alpha-helical structure, a phenomenon previously noted for human apoA-IV. The elastic modulus of chicken apoA-IV at the air/water interface was 13.7 mN/m, versus 21.7 mN/m for human apoA-IV and 7.6 mN/m for apoA-I. The interfacial exclusion pressure of chicken apoA-IV for phospholipid monolayers was 31.1 mN/m, versus 33.0 mN/m for human A-I and 28.5 mN/m for apoA-IV. We conclude that the secondary structural features of chicken apoA-IV more closely resemble those of human apoA-I, which may reflect the evolution of apoA-IV by intraexonic duplication of the apoA-I gene. However, the interfacial properties of chicken apoA-IV are intermediate between those of human apoA-I and apoA-IV, which suggests that chicken apoA-IV may represent an ancestral prototype of mammalian apoA-IV, which subsequently underwent further structural change as an evolutionary response to the requisites of mammalian lipoprotein metabolism.
Asunto(s)
Apolipoproteínas A/química , Aire , Animales , Pollos , Dicroismo Circular , Humanos , Desnaturalización Proteica , Proteínas Recombinantes/química , Propiedades de Superficie , Termodinámica , AguaRESUMEN
The ABCA1 gene, a member of the ATP-binding cassette A (ABCA1) transporter superfamily, encodes a membrane protein that facilitates the cellular efflux of cholesterol and phospholipids. Mutations in ABCA1 lead to familial high density lipoprotein deficiency and Tangier disease. We report the complete human ABCA1 gene sequence, including 1,453 bp of the promoter, 146,581 bp of introns and exons, and 1 kb of the 3' flanking region. The ABCA1 gene spans 149 kb and comprises 50 exons. Sixty-two repetitive Alu sequences were identified in introns 1-49. The transcription start site is 315 bp upstream of a newly identified initiation methionine codon and encodes an ORF of 6,783 bp. Thus, the ABCA1 protein is comprised of 2,261 aa. Analysis of the 1,453 bp 5' upstream of the transcriptional start site reveals multiple binding sites for transcription factors with roles in lipid metabolism. Comparative analysis of the mouse and human ABCA1 promoter sequences identified specific regulatory elements, which are evolutionarily conserved. The human ABCA1 promoter fragment -200 to -80 bp that contains binding motifs for SP1, SP3, E-box, and AP1 modulates cellular cholesterol and cAMP regulation of ABCA1 gene expression. These combined findings provide insights into ABCA1-mediated regulation of cellular cholesterol metabolism and will facilitate the identification of new pharmacologic agents for the treatment of atherosclerosis in humans.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Regiones Promotoras Genéticas , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Elementos Alu , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Transporte Biológico , Colesterol/metabolismo , Clonación Molecular , Humanos , Hipolipoproteinemias/genética , Ratones , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Enfermedad de Tangier/genética , Factores de TranscripciónRESUMEN
BACKGROUND: Mutations in the parkin gene have recently been identified in patients with early-onset Parkinson's disease, but the frequency of the mutations and the associated phenotype have not been assessed in a large series of patients. METHODS: We studied 73 families in which at least one of the affected family members was affected at or before the age of 45 years and had parents who were not affected, as well as 100 patients with isolated Parkinson's disease that began at or before the age of 45 years. All subjects were screened for mutations in the parkin gene with use of a semiquantitative polymerase-chain-reaction assay that simultaneously amplified several exons. We sequenced the coding exons in a subgroup of patients. We also compared the clinical features of patients with parkin mutations and those without mutations. RESULTS: Among the families with early-onset Parkinson's disease, 36 (49 percent) had parkin mutations. The age at onset ranged from 7 to 58 years. Among the patients with isolated Parkinson's disease, mutations were detected in 10 of 13 patients (77 percent) with an age at onset of 20 years or younger, but in only 2 of 64 patients (3 percent) with an age at onset of more than 30 years. The mean (+/-SD) age at onset in the patients with parkin mutations was younger than that in those without mutations (32+/-11 vs. 42+/-11 years, P<0.001), and they were more likely to have symmetric involvement and dystonia at onset, to have hyperreflexia at onset or later, to have a good response to levodopa therapy, and to have levodopa-induced dyskinesias during treatment. Nineteen different rearrangements of exons (deletions and multiplications) and 16 different point mutations were detected. CONCLUSIONS: Mutations in the parkin gene are a major cause of early-onset autosomal recessive familial Parkinson's disease and isolated juvenile-onset Parkinson's disease (at or before the age of 20 years). Accurate diagnosis of these cases cannot be based only on the clinical manifestations of the disease.
Asunto(s)
Ligasas , Mutación , Trastornos Parkinsonianos/genética , Proteínas/genética , Ubiquitina-Proteína Ligasas , Adolescente , Adulto , Edad de Inicio , Secuencia de Bases , Niño , Exones/genética , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Mutación Puntual , Reacción en Cadena de la Polimerasa , Eliminación de SecuenciaRESUMEN
ApoA-I(R151)Paris is a natural apolipoprotein (apo) A-I variant that is associated with low levels of high-density lipoprotein cholesterol (HDL-cholesterol) and the partial deficiency of lecithin:cholesterol acyl-transferase (LCAT) in the plasma of heterozygous carriers. We compared the abilities of recombinant normal apoA-I and recombinant apoA-I(R151C)Paris to clear an emulsion of dimyristoylphosphatidylcholine (DMPC), to form reconstituted lipoproteins with dipalmitoylphosphatidylcholine (DPPC), to activate LCAT, and to promote efflux of biosynthetic cholesterol from porcine aortic smooth muscle cells (SMCs) or of exogenous cholesterol from lipid-loaded mouse peritoneal macrophages. Recombinant apoA-I(R151C)Paris occurred in monomeric and dimeric forms at a ratio of 60:40. Normal apoA-I and apoA-I(R151C)Paris cleared DMPC emulsions at equal rates. Both isoforms associated completely with DPPC during cholate dialysis. Normal apoA-I formed one single particle with a mean diameter of 9.3 nm, whereas apoA-I(R151)Paris gave rise to three particles with mean diameters of 9.3 nm (containing 74% of apoA-I), 10.6 nm, and 12.1 nm, respectively. Compared to normal apoA-I, apoA-I(R151C)Paris had a reduced LCAT-cofactor activity with a 60% lower Vmax/Km ratio due to a 50% higher affinity constant, Km. During incubations for 10 min and 360 min, normal apoA-I/DPPC complexes and apoA-I(R151C)Paris/DPPC complexes were equally efficient in releasing biosynthetic cholesterol from SMCs. In the lipid-free form, apoA-I(R151C)Paris induced normal hydrolysis of cholesteryl esters and normal cholesterol efflux from lipid-loaded mouse-peritoneal macrophages. In conclusion, in addition to its ability to form homo- and heterodimers, apoA-I(R151C)Paris is characterized by defective LCAT-cofactor activity but by normal lipid binding and cholesterol-efflux-promoting abilities.
Asunto(s)
Apolipoproteína A-I/metabolismo , Colesterol/metabolismo , Metabolismo de los Lípidos , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/química , Sustitución de Aminoácidos , Animales , Apolipoproteína A-I/química , Dimiristoilfosfatidilcolina/química , Electroforesis en Gel de Poliacrilamida , Emulsiones , Activación Enzimática , Técnicas In Vitro , Lípidos/química , Macrófagos Peritoneales/enzimología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/citología , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/metabolismo , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , PorcinosRESUMEN
Tangier disease is characterized by low serum high density lipoproteins and a biochemical defect in the cellular efflux of lipids to high density lipoproteins. ABC1, a member of the ATP-binding cassette family, recently has been identified as the defective gene in Tangier disease. We report here the organization of the human ABC1 gene and the identification of a mutation in the ABC1 gene from the original Tangier disease kindred. The organization of the human ABC1 gene is similar to that of the mouse ABC1 gene and other related ABC genes. The ABC1 gene contains 49 exons that range in size from 33 to 249 bp and is over 70 kb in length. Sequence analysis of the ABC1 gene revealed that the proband for Tangier disease was homozygous for a deletion of nucleotides 3283 and 3284 (TC) in exon 22. The deletion results in a frameshift mutation and a premature stop codon starting at nucleotide 3375. The product is predicted to encode a nonfunctional protein of 1,084 aa, which is approximately half the size of the full-length ABC1 protein. The loss of a Mnl1 restriction site, which results from the deletion, was used to establish the genotype of the rest of the kindred. In summary, we report on the genomic organization of the human ABC1 gene and identify a frameshift mutation in the ABC1 gene of the index case of Tangier disease. These results will be useful in the future characterization of the structure and function of the ABC1 gene and the analysis of additional ABC1 mutations in patients with Tangier disease.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Glicoproteínas/genética , Enfermedad de Tangier/genética , Transportador 1 de Casete de Unión a ATP , Animales , Secuencia de Bases , ADN , Exones , Femenino , Humanos , Intrones , Masculino , Ratones , LinajeRESUMEN
Many quantitative trait loci (QTLs) contributing to genetically complex conditions have been discovered, but few causative genes have been identified. This is mainly due to the large size of QTLs and the subtle connection between genotype and quantitative phenotype associated with these conditions. Transgenic mice have been successfully used to analyse well-characterized genes suspected of contributing to quantitative traits. Although this approach is powerful for examining one gene at a time, it can be impractical for surveying the large genomic intervals containing many genes that are typically associated with QTLs. To screen for genes contributing to an asthma QTL mapped to human chromosome 5q3 (refs 6,7), we characterized a panel of large-insert 5q31 transgenics based on studies demonstrating that altering gene dosage frequently affects quantitative phenotypes normally influenced by that gene. This panel of human YAC transgenics, propagating a 1-Mb interval of chromosome 5q31 containing 6 cytokine genes and 17 partially characterized genes, was screened for quantitative changes in several asthma-associated phenotypes. Multiple independent transgenic lines with altered IgE response to antigen treatment shared a 180-kb region containing 5 genes, including those encoding human interleukin 4 (IL4) and interleukin 13 (IL13 ), which induce IgE class switching in B cells. Further analysis of these mice and mice transgenic for mouse Il4 and Il13 demonstrated that moderate changes in Il4 and Il13 expression affect asthma-associated phenotypes in vivo. This functional screen of large-insert transgenics enabled us to identify genes that influence the QTL phenotype in vivo.
Asunto(s)
Asma/genética , Carácter Cuantitativo Heredable , Animales , Asma/fisiopatología , Pruebas de Provocación Bronquial , Broncoconstricción/efectos de los fármacos , Cromosomas Artificiales de Levadura , Citometría de Flujo , Expresión Génica , Pruebas Genéticas , Humanos , Inmunoglobulina E/sangre , Interleucina-13/genética , Interleucina-4/genética , Cloruro de Metacolina/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , ARN/genética , ARN/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismo , Células Th2/citología , Células Th2/efectos de los fármacos , Células Th2/metabolismoRESUMEN
Tangier disease (TD) was first discovered nearly 40 years ago in two siblings living on Tangier Island. This autosomal co-dominant condition is characterized in the homozygous state by the absence of HDL-cholesterol (HDL-C) from plasma, hepatosplenomegaly, peripheral neuropathy and frequently premature coronary artery disease (CAD). In heterozygotes, HDL-C levels are about one-half those of normal individuals. Impaired cholesterol efflux from macrophages leads to the presence of foam cells throughout the body, which may explain the increased risk of coronary heart disease in some TD families. We report here refining of our previous linkage of the TD gene to a 1-cM region between markers D9S271 and D9S1866 on chromosome 9q31, in which we found the gene encoding human ATP cassette-binding transporter 1 (ABC1). We also found a change in ABC1 expression level on cholesterol loading of phorbol ester-treated THP1 macrophages, substantiating the role of ABC1 in cholesterol efflux. We cloned the full-length cDNA and sequenced the gene in two unrelated families with four TD homozygotes. In the first pedigree, a 1-bp deletion in exon 13, resulting in truncation of the predicted protein to approximately one-fourth of its normal size, co-segregated with the disease phenotype. An in-frame insertion-deletion in exon 12 was found in the second family. Our findings indicate that defects in ABC1, encoding a member of the ABC transporter superfamily, are the cause of TD.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Glicoproteínas/genética , Mutación , Enfermedad de Tangier/genética , Transportador 1 de Casete de Unión a ATP , Secuencia de Aminoácidos , Apolipoproteínas E/sangre , Secuencia de Bases , Cromosomas Humanos Par 9 , Exones , Femenino , Biblioteca de Genes , Marcadores Genéticos , Humanos , Lipoproteínas HDL , Masculino , Modelos Biológicos , Modelos Genéticos , Datos de Secuencia Molecular , LinajeRESUMEN
Autosomal recessive juvenile parkinsonism (AR-JP, PARK2; OMIM 602544), one of the monogenic forms of Parkinson's disease (PD), was initially described in Japan. It is characterized by early onset (before age 40), marked response to levodopa treatment and levodopa-induced dyskinesias. The gene responsible for AR-JP was recently identified and designated parkin. We have analysed the 12 coding exons of the parkin gene in 35 mostly European families with early onset autosomal recessive parkinsonism. In one family, a homozygous deletion of exon 4 could be demonstrated. By direct sequencing of the exons in the index patients of the remaining 34 families, eight previously undescribed point mutations (homozygous or heterozygous) were detected in eight families that included 20 patients. The mutations segregated with the disease in the families and were not detected on 110-166 control chromosomes. Four mutations caused truncation of the parkin protein. Three were frameshifts (202-203delAG, 255delA and 321-322insGT) and one a nonsense mutation (Trp453Stop). The other four were missense mutations (Lys161Asn, Arg256Cys, Arg275Trp and Thr415Asn) that probably affect amino acids that are important for the function of the parkin protein, since they result in the same phenotype as truncating mutations or homozygous exon deletions. Mean age at onset was 38 +/- 12 years, but onset up to age 58 was observed. Mutations in the parkin gene are therefore not invariably associated with early onset parkinsonism. In many patients, the phenotype is indistinguishable from that of idiopathic PD. This study has shown that a wide variety of different mutations in the parkin gene are a common cause of autosomal recessive parkinsonism in Europe and that different types of point mutations seem to be more frequently responsible for the disease phenotype than are deletions.
Asunto(s)
Genes Recesivos/genética , Ligasas , Enfermedad de Parkinson/genética , Proteínas/genética , Ubiquitina-Proteína Ligasas , Sitios de Unión , Europa (Continente) , Exones/genética , Salud de la Familia , Femenino , Eliminación de Gen , Genotipo , Humanos , Masculino , Mutación , Linaje , Fenotipo , Mutación Puntual , Polimorfismo GenéticoRESUMEN
BACKGROUND: Apolipoprotein (apo) A-I is the major component of HDL, and it displays antiatherogenic properties. METHODS AND RESULTS: The human apoA-I gene has been transferred into different mouse models by use of a recombinant adenovirus under the control of an RSV-LTR promoter (AV RSV apoA-I). Administration of AV RSV apoA-I to C57BL/6 mice resulted in moderate expression of human apoA-I for 3 weeks, leading to a transient elevation (40% at day 11 after injection) of HDL cholesterol concentration. In contrast, administration of AV RSV apoA-I to human apoA-I-transgenic mice induced a large increase of human apoA-I and HDL cholesterol concentrations (300% and 360%, respectively, at day 14 after injection) for 10 weeks, indicating that an immune response to the transgene was one major hurdle for long-term duration of expression. Recombinant adenovirus expressing human apolipoprotein A-I (AV RSV apoA-I) was also injected into human apoA-I-transgenic/apoE-deficient mice, which are prone to develop atherosclerosis. Over a 6-week period, overexpression of human apoA-I inhibited fatty streak lesion formation by 56% in comparison with control. CONCLUSIONS: Somatic gene transfer of human apoA-I prevents the development of atherosclerosis in the mouse model.
Asunto(s)
Apolipoproteína A-I/genética , Arteriosclerosis/terapia , Técnicas de Transferencia de Gen , Adenoviridae/genética , Animales , Apolipoproteínas E/deficiencia , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Vectores Genéticos , Humanos , Ratones , Ratones Endogámicos C57BL , Valores de ReferenciaRESUMEN
The authors previously demonstrated that the gene for human lipoprotein lipase (hLPL), an enzyme crucial to the breakdown of triglyceride (TG)-rich dietary fats, corrects the hypertriglyceridemia in lipoprotein lipase (LPL)-deficient knockout mice after adenoviral (Ad)-mediated LPL gene transfer. They have now extended their observations to primary cultured mouse hepatocytes and intact animals of normal LPL genotype, and confirm effective overexpression of hLPL from the liver and a sustained TG-lowering effect in plasma over 60 days. A typical first-generation Ad-vector containing the hLPL cDNA (Ad-LPL) resulted in efficient gene transfer into isolated mouse hepatocytes and significant de novo synthesis of active hLPL protein. In this experiment, 5 x 10(9) viral particles (5 x 10(7) pfu) of either Ad-LPL or an Ad-LacZ control vector were injected into CD1 mice of normal LPL genotype. Hepatic expression of hLPL was confirmed at Day 7 postinjection by in situ hybridization and direct measurement of LPL in the liver. This correlated with a total LPL activity (human + mouse) in postheparin plasma (PHP) of 1020.5 standard deviation [SD] 93.6 mU/mL, versus 479.5 SD 129.7 mU/mL (p < 0.001) in Ad-LacZ controls at Day 7. Respective hLPL activity comprised 49% of the total. Significantly raised levels of hLPL protein mass persisted until Day 60. Corresponding plasma TGs decreased to 39% of Ad-LacZ controls at Day 7, and, despite absent hLPL activity from Day 28 on, serum TGs remained significantly lower in Ad-LPL mice up to Day 42. Fast phase liquid chromatography analysis showed a dramatic depletion in TG-rich lipoproteins, mainly very low density lipoproteins (VLDL) and chylomicron fractions. Therefore, Ad-mediated overexpression of hepatic LPL was found to significantly decrease plasma TG levels unrelated to primary LPL deficiency.
Asunto(s)
Adenoviridae/genética , Técnicas de Transferencia de Gen , Lipólisis/genética , Lipoproteína Lipasa/genética , Hígado/enzimología , Animales , Células Cultivadas , Quilomicrones/sangre , Regulación Enzimológica de la Expresión Génica/genética , Humanos , Hibridación in Situ , Lipoproteína Lipasa/metabolismo , Lipoproteínas/sangre , Hígado/virología , Masculino , Ratones , Ratones Endogámicos , Triglicéridos/sangreRESUMEN
Rat hepatocytes cocultured with rat liver epithelial cells (RLEC) were used to investigate the influence of all-trans retinoic acid (RA) on the regulation of apolipoproteins (Apo) A-I and A-II gene expression, the major protein constituent of high-density lipoproteins. In contrast to rat hepatocytes in conventional primary culture, Apo A-I and Apo A-II gene expression remained high and stable for several days in parenchymal cells in coculture. Treatment of cocultured rat hepatocytes with RA resulted in a specific decrease in Apo A-I mRNA levels whereas no marked difference in Apo A-II mRNA levels was observed. Such a negative effect of RA was already detected as early as 2 days of treatment and was effective for the entire experimental period (6 days). As controls, RARbeta mRNA levels increased whereas those of GAPDH mRNA were not affected by the RA treatment. The decrease in Apo A-I mRNA levels was associated with lower amounts of Apo A-I secreted in the culture medium within day 1 of treatment. This effect required active transcription and protein synthesis. These results show that, contrary to primary pure hepatocyte cultures and hepatoma cell lines, cocultures of rat hepatocytes reproduce the in vivo results suggesting that only well differentiated hepatocytes may correctly respond to RA. Furthermore, they demonstrate that RA can directly act on hepatocytes and differently affect Apo A-I and Apo A-II gene expression.
Asunto(s)
Apolipoproteína A-I/genética , Regulación hacia Abajo/genética , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/metabolismo , Tretinoina/farmacología , Albúminas/efectos de los fármacos , Albúminas/metabolismo , Animales , Apolipoproteína A-I/biosíntesis , Apolipoproteína A-I/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Regulación hacia Abajo/efectos de los fármacos , Células Epiteliales/metabolismo , Hígado/citología , Hígado/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The in vivo analysis of lipoprotein(a) (Lp(a)), an independent atherosclerosis risk factor in humans, has been limited in part by its restricted distribution among mammals. Although transgenic mice have been created containing Lp(a), the relatively small size of the mouse has precluded some studies. To examine the properties of this molecule in a significantly larger mammal, we have used a 270-kilobase yeast artificial chromosome clone containing the human apolipoprotein(a) (apo(a)) gene and a 90-kilobase P1 phagemid clone containing the human apolipoprotein B (apoB) gene to create transgenic rabbits that express either or both transgenes. Expression of both transgenes was tissue specific and localized predominantly to the liver. Average apolipoprotein plasma levels in the rabbits were 2.5 mg/dl for apo(a) and 17.6 mg/dl for human apoB. In contrast to observations in apo(a) transgenic mice, we found that apo(a) plasma levels in the rabbits were stable throughout sexual maturity. Also, apo(a) formed a covalent association with the endogenous rabbit apoB albeit with a lower efficiency than its association with human apoB. The analysis of Lp(a) transgenic rabbits has provided new insights into apo(a) expression and Lp(a) assembly. In addition, these transgenic rabbits potentially will provide an improved experimental model for the in vivo analysis of Lp(a) and its role in promoting atherosclerosis and restenosis.
Asunto(s)
Apolipoproteínas/genética , Lipoproteína(a) , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Apolipoproteínas/metabolismo , Apoproteína(a) , Sitios de Unión , Cromosomas Artificiales de Levadura , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Seudoembarazo , Conejos , Homología de Secuencia de AminoácidoRESUMEN
Humans homozygous or heterozygous for mutations in the lipoprotein lipase (LPL) gene demonstrate significant disturbances in plasma lipoproteins, including raised triglyceride (TG) and reduced HDL cholesterol levels. In this study we explored the feasibility of adenovirus-mediated gene replacement therapy for LPL deficiency. A total of 5 x 10(9) plaque-forming units (pfu) of an E1/E3-deleted adenovirus expressing either human LPL (Ad-LPL) or the bacterial beta-galactosidase gene (Ad-LacZ) as a control were administered to mice heterozygous for targeted disruption in the LPL gene (n = 57). Peak expression of total postheparin plasma LPL activity was observed at day 7 in Ad-LPL mice versus Ad-LacZ controls (834 +/- 133 vs 313 +/- 89 mU/mL, P < .01), and correlated with human-specific LPL activity (522 +/- 219 mU/mL) and mass (9214 +/- 782 ng/mL), a change that was significant to 14 and 42 days, respectively. At day 7, plasma TGs were significantly reduced relative to Ad-LacZ mice (0.17 +/- 0.07 vs 1.90 +/- 0.89 mmol/L, P < .01) but returned to endogenous levels by day 42. Ectopic liver expression of human LPL was confirmed by in situ hybridization analysis and from raised LPL activity and mass in liver homogenates. Analysis of plasma lipoprotein composition revealed a marked decrease in VLDL-derived TGs. Severely impaired oral and intravenous fat-load tolerance in LPL-deficient mice was subsequently corrected after Ad-LPL administration and closely paralleled that observed in wild-type mice. These findings suggest that liver-targeted adenovirus-mediated LPL gene transfer offers an effective means for transient correction of altered lipoprotein metabolism and impaired fat tolerance due to LPL deficiency.
Asunto(s)
Terapia Genética , Hiperlipoproteinemia Tipo I/terapia , Hipertrigliceridemia/terapia , Lipoproteína Lipasa/genética , Adenoviridae/genética , Animales , Grasas de la Dieta , Vectores Genéticos/genética , Heterocigoto , Humanos , Hipertrigliceridemia/etiología , Lipoproteína Lipasa/sangre , Lipoproteína Lipasa/fisiología , Hígado/enzimología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Recombinantes de Fusión/fisiologíaRESUMEN
Previous studies have indicated that in HepG2 cells HDL3-signalling involves glycosylphosphatidylinositol (GPI) anchored proteins. HDL3-binding to HepG2 cells was found to be enhanced by cellular preincubation with PI-PLC inhibitors and sensitive to a cellular preincubation with exogenous PI-PLC, suggesting that HDL3 binds directly on GPI-anchored proteins to initiate signaling. Moreover HDL3-binding was found to be partly inhibited by antibodies against the HDL-binding protein (AbHBP). HDL3, when binding to HepG2 cells, promoted the release in the culture medium of a 110 kDa protein that binds AbHBP, while a cellular preincubation with antibodies against the inositol-phosphoglycan (IPG) moiety of GPI-anchor (AbIPG), used to block lipolytic cleavage of the GPI-anchor, inhibits HDL3-induced release of the 110 kDa protein in the culture medium. In [3H]-PC prelabeled HepG2 cells, AbHBP were found to stimulate PC-hydrolysis and DAG generation within 5 min as did HDL3 stimulation. Cellular preincubation with AbIPG was found to inhibit only the HDL3-signal and not the AbHBP-signal, while a prior cellular pretreatment with PI-PLC from Bacillus cereus was found to inhibit the HDL3-and AbHBP-signal. Moreover cellular preincubation with AbHBP for 1 h at 37 degrees C was found to inhibit HDL3-signalling pathways. Our results suggest that in HepG2 cells a 110 kDa protein, which could be HBP, can be anchored to the membrane via GPI, and can function in HDL3-signalling pathways as binding sites.