RESUMEN
BACKGROUND: Despite recent progress in the field of genetics, sporadic late-onset (> 40 years) cerebellar ataxia (SLOCA) etiology remains frequently elusive, while the optimal diagnostic workup still needs to be determined. We aimed to comprehensively describe the causes of SLOCA and to discuss the relevance of the investigations. METHODS: We included 205 consecutive patients with SLOCA seen in our referral center. Patients were prospectively investigated using exhaustive clinical assessment, biochemical, genetic, electrophysiological, and imaging explorations. RESULTS: We established a diagnosis in 135 (66%) patients and reported 26 different causes for SLOCA, the most frequent being multiple system atrophy cerebellar type (MSA-C) (41%). Fifty-one patients (25%) had various causes of SLOCA including immune-mediated diseases such as multiple sclerosis or anti-GAD antibody-mediated ataxia; and other causes, such as alcoholic cerebellar degeneration, superficial siderosis, or Creutzfeldt-Jakob disease. We also identified 11 genetic causes in 20 patients, including SPG7 (n = 4), RFC1-associated CANVAS (n = 3), SLC20A2 (n = 3), very-late-onset Friedreich's ataxia (n = 2), FXTAS (n = 2), SCA3 (n = 1), SCA17 (n = 1), DRPLA (n = 1), MYORG (n = 1), MELAS (n = 1), and a mitochondriopathy (n = 1) that were less severe than MSA-C (p < 0.001). Remaining patients (34%) had idiopathic late-onset cerebellar ataxia which was less severe than MSA-C (p < 0.01). CONCLUSION: Our prospective study provides an exhaustive picture of the etiology of SLOCA and clues regarding yield of investigations and diagnostic workup. Based on our observations, we established a diagnostic algorithm for SLOCA.
Asunto(s)
Ataxia Cerebelosa , Atrofia de Múltiples Sistemas , Ataxias Espinocerebelosas , Degeneraciones Espinocerebelosas , Humanos , Estudios Prospectivos , Ataxia Cerebelosa/epidemiología , Ataxia Cerebelosa/etiología , Ataxia Cerebelosa/diagnóstico , Degeneraciones Espinocerebelosas/complicaciones , Ataxias Espinocerebelosas/complicaciones , Atrofia de Múltiples Sistemas/complicaciones , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIIRESUMEN
The Brangus breed was developed to combine the superior characteristics of both of its founder breeds, Angus and Brahman. It combines the high adaptability to tropical and subtropical environments, disease resistance, and overall hardiness of Zebu cattle with the reproductive potential and carcass quality of Angus. It is known that the major histocompatibility complex (MHC, also known as bovine leucocyte antigen: BoLA), located on chromosome 23, encodes several genes involved in the adaptive immune response and may be responsible for adaptation to harsh environments. The objective of this work was to evaluate whether the local breed ancestry percentages in the BoLA locus of a Brangus population diverged from the estimated genome-wide proportions and to identify signatures of positive selection in this genomic region. For this, 167 animals (100 Brangus, 45 Angus and 22 Brahman) were genotyped using a high-density single nucleotide polymorphism array. The local ancestry analysis showed that more than half of the haplotypes (55.0%) shared a Brahman origin. This value was significantly different from the global genome-wide proportion estimated by cluster analysis (34.7% Brahman), and the proportion expected by pedigree (37.5% Brahman). The analysis of selection signatures by genetic differentiation (F st ) and extended haplotype homozygosity-based methods (iHS and Rsb) revealed 10 and seven candidate regions, respectively. The analysis of the genes located within these candidate regions showed mainly genes involved in immune response-related pathway, while other genes and pathways were also observed (cell surface signalling pathways, membrane proteins and ion-binding proteins). Our results suggest that the BoLA region of Brangus cattle may have been enriched with Brahman haplotypes as a consequence of selection processes to promote adaptation to subtropical environments.
Asunto(s)
Adaptación Fisiológica/genética , Bovinos/genética , Genoma/genética , Haplotipos , Complejo Mayor de Histocompatibilidad/genética , Reproducción/genética , Animales , Cruzamiento , Bovinos/clasificación , Bovinos/fisiología , Sitios Genéticos/genética , Genotipo , Complejo Mayor de Histocompatibilidad/inmunología , Masculino , Linaje , Polimorfismo de Nucleótido Simple/genética , Selección GenéticaRESUMEN
Methods for individual identification are usually employed for traceability, whereas breed identification is useful to detect commercial frauds. In this study, Chinese Yellow Cattle (CYC) samples plus data from six Bos taurus breeds, two Bos indicus breeds, and one composite breed were used to develop an allocation test based on 22 microsatellites. The test allowed discriminating all foreign breeds from the CYC, although some CYC individuals were wrongly allocated as Limousin or Holstein, probably due to the recent introduction of these breeds into China. In addition, CYC evidenced a previously reported Zebu cline (south-north) and a possible structure within the B. taurus component that should be confirmed. An independent test performed with meat samples of unknown breed origin from Argentina allocated 92% of them to either Angus, Hereford, or their crossbreed, but none was identified as CYC. We conclude that the test is a suitable tool to certify meat of foreign breed origin and to detect adulterations of CYC beef labeled as imported meat.
Asunto(s)
Bovinos/genética , ADN/genética , Animales , Argentina , Cruzamiento , China , Variación Genética/genética , Técnicas de Genotipaje/métodos , Técnicas de Genotipaje/estadística & datos numéricosRESUMEN
The use of probiotics for farm animals has increased considerably over the last 15 years. Probiotics are defined as live microorganisms which can confer a health benefit for the host when administered in appropriate and regular quantities. Once ingested, the probiotic microorganisms can modulate the balance and activities of the gastrointestinal microbiota, whose role is fundamental to gut homeostasis. It has been demonstrated that numerous factors, such as dietary and management constraints, can strongly affect the structure and activities of the gut microbial communities, leading to impaired health and performance in livestock animals. In this review, the most important benefits of yeast and bacterial probiotics upon the gastrointestinal microbial ecosystem in ruminants and monogastric animals (equines, pigs, poultry, fish) reported in the recent scientific literature are described, as well as their implications in terms of animal nutrition and health. Additional knowledge on the possible mechanisms of action is also provided.
Asunto(s)
Fenómenos Fisiológicos Bacterianos , Tracto Gastrointestinal/microbiología , Ganado/fisiología , Probióticos/metabolismo , Levaduras/fisiología , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Tracto Gastrointestinal/fisiología , Salud , Ganado/microbiologíaRESUMEN
Lipoprotein associated phospholipase A2 (Lp-PLA2) modulates low-density lipoprotein (LDL) oxidation by hydrolysing oxidised phospholipids present on particle surfaces. We investigated whether Lp-PLA2 activity and PLA2G7 A379V genotype were related to mediators of atherosclerosis in a diabetic study. Plasma Lp-PLA2 activity (taken in men only) and A379V genotype were investigated with regards to metabolic syndrome (MS), UKPDS risk score, and oxidised LDL (oxLDL/LDL), in a cohort of Caucasian men and women (n=783, age 62.5+/-13.7 years). After adjustment for type of diabetes, CHD status, and statin use, those individuals with features defining the MS (WHO guidelines) had higher Lp-PLA2 activity (35.6+/-11.9 nmol/min/ml) compared to those without (33.0+/-10.8 nmol/min/ml) (p=0.02). Quartiles of UKPDS coronary heart disease (CHD) risk score were also positively associated with Lp-PLA2 activity (p=0.006, p=0.004 linear trend). Those men in the highest quartile of oxLDL/LDL level had the lowest Lp-PLA2 activity (31.3+/-10.5 nmol/min/ml) when compared to the middle two (32.3+/-9.8 and 35.9+/-10.9 nmol/min/ml, respectively) and lowest quartile (35.6 +/-12.5 nmol/min/ml; p=0.03, p=0.004 linear trend). There was no significant association between A379V genotype and Lp-PLA2 enzyme activity (p=0.34) or oxLDL/LDL (p=0.32). Lp-PLA2 activity is an independent predictor of CHD risk and MS in a sample of subjects with diabetes mellitus. The association of Lp-PLA2 activity with oxLDL/LDL suggests that Lp-PLA2 may be a modulating factor in the process of atherosclerosis.
Asunto(s)
ADN/genética , Diabetes Mellitus/enzimología , Fosfolipasas A/genética , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Anciano , LDL-Colesterol/sangre , Enfermedad Coronaria/sangre , Enfermedad Coronaria/etiología , Diabetes Mellitus/genética , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Oxidación-Reducción , Fosfolipasas A/sangre , Fosfolipasas A2 , Reacción en Cadena de la Polimerasa , Pronóstico , Factores de RiesgoRESUMEN
Churg-Strauss syndrome (CSS) is a disorder characterised by hypereosinophilia and systemic vasculitis complicating a preexisting asthma. Twenty cases have been studied. Mean duration of asthma before CSS was 8 years, the peripheral-blood eosinophilia, always > 1,700/microL, went above 5,000/microL in 17 cases. The clinical manifestations were the following: 20 impairements of general state with fever, 13 peripheral neuropathies, 15 cutaneous injuries, 10 pericardial or myocardial attacks, 10 digestive impairements, 9 muscular and articular diseases, 7 renal diseases--all of them linked with the vasculitis--and 9 upper respiratory tract involvements. Pleuropulmonary or cardiac anomalies have been discovered at the chest X-ray in 14 cases. The diagnosis has been histologicaly confirmed in 15 cases. No clinical or biological or evolutive distinction was noticed between the 15 positive biopsy patients and the 5 negative ones. During 8.4 (+/- 7.9 years) the evolution was caracterised by relapses which have always been announced by increasing eosinophilia. Eighteen patients have been successfully treated with corticoids alone or associated with cyclophosphamid in 9. Survival at 5 years was 85%. Five deaths occured because of CSS (2 because of an unadapted treatment). We have to focus the need for an emergency treatment of CSS. The diagnosis can be done by clinical investigation only, before any anatomopathologic results.
Asunto(s)
Síndrome de Churg-Strauss , Adolescente , Adulto , Anciano , Síndrome de Churg-Strauss/diagnóstico , Síndrome de Churg-Strauss/terapia , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
The ubiquitin-proteasome pathway regulates gene expression through protein degradation. Here we show that the F-box protein betaTrCP, the receptor component of the SCF E3 ubiquitin ligase responsible for IkappaBalpha and beta-catenin degradation, is colocalized in the nucleus with ATF4, a member of the ATF-CREB bZIP family of transcription factors, and controls its stability. Association between the two proteins depends on ATF4 phosphorylation and on ATF4 serine residue 219 present in the context of DSGXXXS, which is similar but not identical to the motif found in other substrates of betaTrCP. ATF4 ubiquitination in HeLa cells is enhanced in the presence of betaTrCP. The F-box-deleted betaTrCP protein behaves as a negative transdominant mutant that inhibits ATF4 ubiquitination and degradation and, subsequently, enhances its activity in cyclic AMP-mediated transcription. ATF4 represents a novel substrate for the SCF(betaTrCP) complex, which is the first mammalian E3 ubiquitin ligase identified so far for the control of the degradation of a bZIP transcription factor.
Asunto(s)
Núcleo Celular/metabolismo , Péptido Sintasas/metabolismo , Factores de Transcripción/metabolismo , Factor de Transcripción Activador 4 , Secuencias de Aminoácidos , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Humanos , Mutación , Fosforilación , Pruebas de Precipitina , Proteínas Ligasas SKP Cullina F-box , Serina , Factores de Transcripción/genética , Transcripción Genética , Proteínas con Repetición de beta-TransducinaRESUMEN
The Rp1-D gene, which conveys a chlorotic-fleck resistant reaction to Puccinia sorghi, effectively controlled common rust on sweet corn in North America for nearly 15 years. Biotypes of P. sorghi virulent on plants with the Rp1-D gene were widespread in North America for the first time in 1999 and again in 2000 (1,2). Many Rp-resistant sweet corn hybrids that are developed and grown in North America also are grown in Europe, including France where virulence against the Rp1-D gene has not been reported previously. In September 2000, uredinia of common rust were observed on and collected from sweet corn hybrids with the Rp1-D gene in commercial fields and hybrid trials in the Landes and Pyrénées Atlantiques departments of the Aquitaine region of southwestern France. Severity of rust generally was below 5% on these plants except for a few hybrids for which severity was about 20 to 30%. Common rust was not observed on hybrids with the Rp-G gene. Urediniospores were increased as a bulk population on the susceptible sweet corn hybrid Sterling in a greenhouse. Plants with each of 10 single Rp genes (Rp1-A, Rp1-C, Rp1-D, Rp1-E, Rp1-F, Rp1-I, Rp1-K, Rp1-L, Rp1-N, and Rp-G) or each of six compound rust resistance genes (Rp1-D5, Rp1-JC, Rp1-JFC, Rp-GDJ, Rp-GFJ, and Rp-G5JC) were assayed for reactions to this population of P. sorghi. Two to six different sources of seed of each single Rp gene and two different sources of seed of each compound rust resistance gene were replicates with a single pot of 6 to 18 plants grown from a specific seed source. Plants were inoculated three times on successive days by placing 2 or 3 ml of a urediniospore suspension in the whorl of two- to four-leaved seedlings. Reactions were rated 10 days after the last inoculation. Plants without symptoms or with chlorotic-fleck resistant reactions were inoculated again and rated 10 days later. Uredinia did not form on plants with compound rust resistance genes. Plants with the genes Rp1-E, Rp1-I, Rp1-K, and Rp-G also were resistant although a few, very small uredinia (i.e., type-1 uredinia) were observed on a few plants. Plants with the genes Rp1-A, Rp1-C, Rp1-D, Rp1-F, Rp1-L, and Rp1-N were fully susceptible. This pattern of virulence is the same as that observed during the past two years in North American populations of P. sorghi virulent against Rp1-D. Rp-resistance currently available in most sweet corn hybrids will not be effective in France if these virulent biotypes become prevalent. References: (1) J. K. Pataky et al. Plant Dis. 85:165, 2001. (2) M. C. Pate et al. Plant Dis. 84:1154, 2000.
RESUMEN
We report three personal cases of hydrocarbide aspiration pneumonia. High-viscosity non-volatile hydrocarbides (paraffin oil, for instance) cause often pseudotumoral exogenous fat-aspiration lung disease. Low-viscosity volatile hydrocarbides (petroleum, gasoline, white spirit, for instance) cause acute pseudo-infectious lung disease with dyspnea and fever which usually resolves within a few weeks but which may also be life-threatening. Purely symptomatic treatment has greatly progressed with advances in intensive ventilatory assistance. Gastric emptying with emetic agents or lavage procedures is dangerous and must be avoided except for exceptional cases. When required, the airways must be protected with tracheal intubation. Volatile hydrocarbides should be stored in protected areas in containers with safety stoppers which children cannot open.
Asunto(s)
Hidrocarburos/efectos adversos , Neumonía por Aspiración/inducido químicamente , Neumonía Lipoidea/inducido químicamente , Adulto , Anciano , Animales , Biopsia , Niño , Perros , Femenino , Estudios de Seguimiento , Humanos , Lactante , Pulmón/patología , Masculino , Parafina/efectos adversos , Petróleo/efectos adversos , Neumonía por Aspiración/diagnóstico por imagen , Neumonía por Aspiración/patología , Neumonía por Aspiración/terapia , Neumonía Lipoidea/diagnóstico por imagen , Neumonía Lipoidea/patología , Neumonía Lipoidea/terapia , Radiografía Torácica , Factores de Tiempo , Tomografía Computarizada por Rayos XAsunto(s)
Enfermedades Pleurales , Sarcoidosis , Adulto , Antiinflamatorios/administración & dosificación , Antiinflamatorios/uso terapéutico , Líquido del Lavado Bronquioalveolar , Estudios de Seguimiento , Humanos , Masculino , Enfermedades Pleurales/diagnóstico , Prednisona/administración & dosificación , Prednisona/uso terapéutico , Radiografía Torácica , Sarcoidosis/diagnóstico , Factores de Tiempo , Tomografía Computarizada por Rayos XAsunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/tratamiento farmacológico , Enfermedad Celíaca/terapia , Infecciones por Citomegalovirus/tratamiento farmacológico , Enteropatías Perdedoras de Proteínas/terapia , Adulto , Fármacos Anti-VIH/administración & dosificación , Foscarnet/administración & dosificación , Foscarnet/uso terapéutico , Ganciclovir/administración & dosificación , Ganciclovir/uso terapéutico , Inhibidores de la Proteasa del VIH/administración & dosificación , Humanos , Lamivudine/administración & dosificación , Masculino , Nelfinavir/administración & dosificación , Nutrición Parenteral , Pentamidina , Inhibidores de la Transcriptasa Inversa/administración & dosificación , Estavudina/administración & dosificación , Factores de Tiempo , Resultado del TratamientoRESUMEN
Abstract This study evaluated the concordance of concomitant urinalysis and clinical assessments of drug abusers included in a methadone maintenance programme. The agreement between a clinical subjective score and an objective biological score, both measuring the evolution of illicit substance consumption over 12 months, was analysed. The clinical score, established by physicians and applied during patient interviews, was determined at entry into the programme and re-evaluated after 6 and 12 months. Forty-one patients were evaluated. The urinalysis score was based on regular screening of urine samples with the EMIT method. Agreement between the two scores was determined by using the kappa coefficient for each substance (opiates, benzodiazepines and cocaine) for each time-point. The calculated kappa coefficients showed poor agreement between the two scores, but could indicate the complementarity of these clinical and biological appraisals. Indeed, the urinalysis objectively detected change in drug use before the clinician. Thus, urinalysis monitoring should be considered as an additional and complementary biological procedure for patient follow-up by physicians.
RESUMEN
Defects in beta-catenin regulation contribute to the neoplastic transformation of mammalian cells. Dysregulation of beta-catenin can result from missense mutations that affect critical sites of phosphorylation by glycogen synthase kinase 3beta (GSK3beta). Given that phosphorylation can regulate targeted degradation of beta-catenin by the proteasome, beta-catenin might interact with an E3 ubiquitin ligase complex containing an F-box protein, as is the case for certain cell cycle regulators. Accordingly, disruption of the Drosophila F-box protein Slimb upregulates the beta-catenin homolog Armadillo. We reasoned that the human homologs of Slimb - beta-TrCP and its isoform beta-TrCP2 (KIAA0696) - might interact with beta-catenin. We found that the binding of beta-TrCP to beta-catenin was direct and dependent upon the WD40 repeat sequences in beta-TrCP and on phosphorylation of the GSK3beta sites in beta-catenin. Endogenous beta-catenin and beta-TrCP could be coimmunoprecipitated from mammalian cells. Overexpression of wild-type beta-TrCP in mammalian cells promoted the downregulation of beta-catenin, whereas overexpression of a dominant-negative deletion mutant upregulated beta-catenin protein levels and activated signaling dependent on the transcription factor Tcf. In contrast, beta-TrCP2 did not associate with beta-catenin. We conclude that beta-TrCP is a component of an E3 ubiquitin ligase that is responsible for the targeted degradation of phosphorylated beta-catenin.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al GTP/metabolismo , Transactivadores , Animales , Cadherinas/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Drosophila , Proteínas de Unión al GTP/química , Genes Reporteros , Células HeLa , Humanos , Fosforilación , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Secuencias Repetitivas de Aminoácido , Transfección , Ubiquitina-Proteína Ligasas , beta Catenina , Proteínas con Repetición de beta-TransducinaRESUMEN
Activation of NF-kappaB transcription factors requires phosphorylation and ubiquitin-proteasome-dependent degradation of IkappaB proteins. We provide evidence that a human F-box protein, h-betaTrCP, a component of Skp1-Cullin-F-box protein (SCF) complexes, a new class of E3 ubiquitin ligases, is essential for inducible degradation of IkappaBalpha. betaTrCP associates with Ser32-Ser36 phosphorylated, but not with unmodified IkappaBalpha or Ser32-Ser36 phosphorylation-deficient mutants. Expression of a F-box-deleted betaTrCP inhibits IkappaBalpha degradation, promotes accumulation of phosphorylated Ser32-Ser36 IkappaBalpha, and prevents NF-kappaB-dependent transcription. Our findings indicate that betaTrCP is the adaptor protein required for IkappaBalpha recognition by the SCFbetaTrCP E3 complex that ubiquitinates IkappaBalpha and makes it a substrate for the proteasome.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cisteína Endopeptidasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas I-kappa B , Complejos Multienzimáticos/metabolismo , Péptido Sintasas/metabolismo , Células HeLa , Humanos , Modelos Químicos , Inhibidor NF-kappaB alfa , FN-kappa B/biosíntesis , FN-kappa B/genética , Fosforilación , Complejo de la Endopetidasa Proteasomal , Proteínas Quinasas Asociadas a Fase-S , Proteínas Ligasas SKP Cullina F-box , Serina/metabolismo , Transcripción Genética , Proteínas con Repetición de beta-TransducinaRESUMEN
HIV-1 Vpu interacts with CD4 in the endoplasmic reticulum and triggers CD4 degradation, presumably by proteasomes. Human beta TrCP identified by interaction with Vpu connects CD4 to this proteolytic machinery, and CD4-Vpu-beta TrCP ternary complexes have been detected by coimmunoprecipitation. beta TrCP binding to Vpu and its recruitment to membranes require two phosphoserine residues in Vpu essential for CD4 degradation. In beta TrCP, WD repeats at the C terminus mediate binding to Vpu, and an F box near the N terminus is involved in interaction with Skp1p, a targeting factor for ubiquitin-mediated proteolysis. An F-box deletion mutant of beta TrCP had a dominant-negative effect on Vpu-mediated CD4 degradation. These data suggest that beta TrCP and Skp1p represent components of a novel ER-associated protein degradation pathway that mediates CD4 proteolysis.
Asunto(s)
Antígenos CD4/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Unión al GTP/genética , VIH-1 , Proteínas Reguladoras y Accesorias Virales/metabolismo , Sitios de Unión/inmunología , Proteínas de Ciclo Celular/metabolismo , Retículo Endoplásmico/química , Retículo Endoplásmico/virología , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana , Humanos , Células Jurkat , Datos de Secuencia Molecular , Mutagénesis/fisiología , Secuencias Repetitivas de Ácidos Nucleicos , Proteínas Quinasas Asociadas a Fase-S , Homología de Secuencia de Aminoácido , Serina/metabolismo , Ubiquitinas/metabolismo , Proteínas con Repetición de beta-TransducinaRESUMEN
Dictyostelium discoideum cells are highly resistant to xenobiotics. We previously observed that these primitive eukaryotic cells contain a 170-kDa P-glycoprotein, mediating multidrug resistance in mammalian cells, but nonfunctional in Dictyostelium cells. We show here that D. discoideum cells vitally stained with the DNA-specific dye, Hoechst 33342, release fluorescent material in their culture medium. Electron microscopy and lipid analysis demonstrate the vesicular nature of this material. Moreover, nucleic acids associate with these extracellular vesicles independently of Hoechst vital staining. The main vesicular DNA component exhibits a size > 21 kb. Shedding of microvesicles during cell growth is not concomitant with programmed cell death. We propose that these extracellular vesicles are involved in a new cellular resistance mechanism against xenobiotics. Furthermore, since the association of DNA with vesicles occurs in physiological growth conditions and independently of vital staining, the new shedding process might be involved in a more general intercellular mechanism.
Asunto(s)
Bencimidazoles/metabolismo , ADN/metabolismo , Dictyostelium/metabolismo , Colorantes Fluorescentes/metabolismo , Animales , Resistencia a Múltiples Medicamentos , Espacio Extracelular/metabolismo , Peso Molecular , Xenobióticos/farmacologíaRESUMEN
We have cloned the nef, vif, vpr and vpu genes of HIV1 in the pGEX system to produce auxiliary proteins of HIV1 as N-terminal fusions with glutathione S-transferase (GST). Some GST proteins are difficult to obtain under standard conditions. The synthesis and solubility varied considerably from one protein to another. We investigated the reasons for the poor production of GST-Vpr, GST-Vpu and GST-Vif. Interestingly, using this GST prokaryotic model, we demonstrated that Vpr, which is known to block the cell cycle of mammalian and yeast cells at the G2 phase, is also bacteriostatic for Escherichia coli. The effect on E. coli was specific to Vpr, and was not linked to the expression of the other HIV1 proteins. This suggests that Vpr interferes with components of cell replication that are conserved from prokaryotes to eukaryotes. Thus, E. coli appears to be a convenient model system for studies on the function of Vpr.
Asunto(s)
Escherichia coli/crecimiento & desarrollo , Productos del Gen vpr/fisiología , VIH-1 , División Celular , Escherichia coli/metabolismo , Expresión Génica , Productos del Gen nef/genética , Productos del Gen nef/fisiología , Productos del Gen vif/genética , Productos del Gen vif/fisiología , Productos del Gen vpr/genética , Glutatión Transferasa/genética , Proteínas del Virus de la Inmunodeficiencia Humana , Humanos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/fisiología , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/fisiología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Productos del Gen vif del Virus de la Inmunodeficiencia Humana , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
A serious limitation in the use of the DNA-cleaving, antitumoral-antibiotic, bleomycin during chemotherapy is pulmonary toxicity. Lung injury induced by bleomycin is characterized by an increased deposition of interstitial extracellular matrix proteins in the alveolar wall that compromises respiratory function. Several drugs have been tested in animal models to prevent the pulmonary toxicity of bleomycin, but have not led to a useful clinical treatment because of their adverse effects on other tissues. We have shown that transgenic mice expressing Streptoalloteichus hindustanus (Sh) ble bleomycin resistance protein in pulmonary epithelial cells in the lungs are protected against bleomycin-induced toxicity in lungs. In the present study, we used intranasal administration by adenovirus-mediated gene transfer of the bleomycin resistance Sh ble gene to mouse lung for prevention of bleomycin-induced pulmonary fibrosis. We constructed recombinant adenoviruses Ad.CMVble and Ad.RSVble harboring the bleomycin resistance Sh ble gene under the control of the cytomegalovirus early promoter and the Rous sarcoma virus early promoter, respectively. Transgene expression was detected in epithelia of conducting airways and alveolar septa by immunostaining with a rabbit polyclonal antibody directed against the bleomycin resistance protein and persisted for the duration of drug treatment; i.e., up to 17 d. No toxic effect was seen in adenovirus-treated mice. Pretreatment of mice with Ad.CMVble or Ad.RSVble completely prevented collagen deposition 42-133 d after bleomycin treatment, as measured by lung OH-proline content. Histologic studies indicated that there was little or no lung injury in the adenovirus/bleomycin-treated mice compared with the bleomycin-treated mice. These observations may lead to new approaches for the prevention of bleomycin-induced pulmonary fibrosis.