RESUMEN
X-linked cerebral creatine deficiency (MIM 300036) is caused by deficiency of the creatine transporter encoded by the SLC6A8 gene. Here we report three patients with this condition from Israel. These unrelated patients were evaluated for global developmental delays and language apraxia. Borderline microcephaly was noted in one of them. Diagnosis was prompted by brain magnetic resonance imaging and spectroscopy which revealed normal white matter distribution, but absence of the creatine peak in all three patients. Biochemical testing indicated normal plasma levels of creatine and guanidinoacetate, but an increased urine creatine/creatinine ratio. The diagnosis was confirmed by demonstrating absent ([14])C-creatine transport in fibroblasts. Molecular studies indicated that the first patient is hemizygous for a single nucleotide change substituting a single amino acid (c.619 C > T, p.R207W). Expression studies in HeLa cells confirmed the causative role of the R207W substitution. The second patient had a three base pair deletion in the SLC6A8 gene (c.1222_1224delTTC, p.F408del) as well as a single base change (c.1254 + 1G > A) at a splicing site in the intron-exon junction of exon 8, the latter occurring de novo. The third patient, had a three base pair deletion (c.1006_1008delAAC, p.N336del) previously reported in other patients with creatine transporter deficiency. These three patients are the first reported cases of creatine transporter deficiency in Israel.
RESUMEN
Mutations in the insulin receptor gene cause the inherited insulin resistant syndromes Leprechaunism and Rabson-Mendenhall syndrome. These recessive conditions are characterized by intrauterine and post-natal growth restrictions, dysmorphic features, altered glucose homeostasis, and early demise. The insulin receptor gene (INSR) maps to the short arm of chromosome 19 and is composed of 22 exons. Here we optimize the conditions for sequencing this gene and report novel mutations in patients with severe insulin resistance. METHODS: PCR amplification of the 22 coding exons of the INSR gene was performed using M13-tailed primers. Bidirectional DNA sequencing was performed with BigDye Terminator chemistry and M13 primers and the product was analyzed on the ABI 3100 genetic analyzer. Data analysis was performed using Mutation Surveyor software comparing the sequence to a reference INSR sequence (Genbank NC_000019). RESULTS: We sequenced four patients with Leprechaunism or Rabson-Mendenhall syndromes as well as seven samples from normal individuals and confirmed previously identified mutations in the affected patients. Three of the four mutations identified in this group caused premature insertion of a stop codon. In addition, the INSR gene was sequenced in 14 clinical samples from patients with suspected insulin resistance and one novel mutation was found in an infant with a suspected diagnosis of Leprechaunism. DISCUSSION: Leprechaunism and Rabson-Mendenhall syndrome are very rare and difficult to diagnose. Diagnosis is currently based mostly on clinical criteria. Clinical availability of DNA sequencing can provide an objective way of confirming or excluding the diagnosis.
RESUMEN
The human immunodeficiency virus type-1 (HIV-1) accessory gene vpr encodes a conserved 96-amino-acid protein that is necessary and sufficient for the HIV-1-induced block of cellular proliferation. Expression of vpr in CD4+ lymphocytes results in G2 arrest, followed by apoptosis. In a previous study, we identified the ataxia telangiectasia-mutated (ATM) and Rad3-related protein (ATR) as a cellular factor that mediates Vpr-induced cell cycle arrest. In the present study, we report that the breast cancer-associated protein-1 (BRCA1), a known target of ATR, is activated in the presence of Vpr. In addition, the gene encoding the growth arrest and DNA damage-45 protein alpha (GADD45alpha), a known transcriptional target of BRCA1, is upregulated by Vpr in an ATR-dependent manner. We demonstrate that RNAi-mediated silencing of either ATR or GADD45alpha leads to nearly complete suppression of the proapoptotic effect of Vpr. Our results support a model in which Vpr-induced apoptosis is mediated via ATR phosphorylation of BRCA1, and consequent upregulation of GADD45alpha.
Asunto(s)
Apoptosis/fisiología , Proteínas de Ciclo Celular/metabolismo , Productos del Gen vpr/metabolismo , VIH-1/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Proteína BRCA1/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Inhibidores de Caspasas , Fase G2/fisiología , Células HeLa , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Serina/metabolismo , Subgrupos de Linfocitos T/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
Saccharomyces cerevisiae can accumulate iron through the uptake of siderophore-iron. Siderophore-iron uptake can occur through the reduction of the complex and the subsequent uptake of iron by the high affinity iron transporter Fet3p/Ftr1p. Alternatively, specific siderophore transporters can take up the siderophore-iron complex. The pathogenic fungus Candida albicans can also take up siderophore-iron. Here we identify a C. albicans siderophore transporter, CaArn1p, and characterize its activity. CaARN1 is transcriptionally regulated in response to iron. Through expression studies in S. cerevisiae strains lacking endogenous siderophore transporters, we demonstrate that CaArn1p specifically mediates the uptake of ferrichrome-iron. Iron-ferrichrome and gallium-ferrichrome, but not desferri-ferrichrome, could competitively inhibit the uptake of iron from ferrichrome. Uptake of siderophore-iron resulting from expression of CaARN1 under the control of the MET25-promoter in S. cerevisiae was independent of the iron status of the cells and of Aft1p, the iron-sensing transcription factor. These studies demonstrate that the expression of CaArn1p is both necessary and sufficient for the nonreductive uptake of ferrichrome-iron and suggests that the transporter may be the only required component of the siderophore uptake system that is regulated by iron and Aft1p.
Asunto(s)
Candida albicans/química , Candida albicans/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/fisiología , Proteínas de Transporte de Catión , Ferricromo/metabolismo , Proteínas Fúngicas , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Northern Blotting , Western Blotting , Proteínas Portadoras/biosíntesis , Relación Dosis-Respuesta a Droga , Hierro/metabolismo , Hierro/farmacocinética , Hierro/farmacología , Cinética , Metionina/metabolismo , Datos de Secuencia Molecular , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Homología de Secuencia de Aminoácido , Sideróforos/farmacocinéticaRESUMEN
In the yeast Saccharomyces cerevisiae, uptake of iron is largely regulated by the transcription factor Aft1. cDNA microarrays were used to identify new iron and AFT1-regulated genes. Four homologous genes regulated as part of the AFT1-regulon (ARN1-4) were predicted to encode members of a subfamily of the major facilitator superfamily of transporters. These genes were predicted to encode proteins with 14 membrane spanning domains and were from 26 to 53% identical at the amino acid level. ARN3 is identical to SIT1, which is reported to encode a ferrioxamine B permease. Deletion of ARN3 did not prevent yeast from using ferrioxamine B as an iron source; however, deletion of ARN3 and FET3, a component of the high affinity ferrous iron transport system, did prevent uptake of ferrioxamine-bound iron and growth on ferrioxamine as an iron source. The siderophore-mediated transport system and the high affinity ferrous iron transport system were localized to separate cellular compartments. Epitope-tagged Arn3p was expressed in intracellular vesicles that co-sediment with the endosomal protein Pep12. In contrast, Fet3p was expressed on the plasma membrane and was digested by extracellular proteases. These data indicate that S. cerevisiae has two pathways for ferrrioxamine-mediated iron uptake, one occurring at the plasma membrane and the other occurring in an intracellular compartment.
Asunto(s)
Proteínas Portadoras/genética , Deferoxamina/farmacocinética , Proteínas Fúngicas/metabolismo , Hierro/metabolismo , Proteínas de la Membrana/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Ceruloplasmina/genética , Ceruloplasmina/metabolismo , Deferoxamina/farmacología , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Eliminación de Gen , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Datos de Secuencia Molecular , Regulón , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción/genéticaRESUMEN
Biomimetic analogues 1 of the microbial siderophore (iron carrier) ferrichrome were labeled via piperazine with various fluorescent markers at a site not interfering with iron binding or receptor recognition (compounds 10-12). These iron carriers were built from a tetrahedral carbon symmetrically extended with three strands, each containing an amino acid (G = glycyl, A = alanyl, L = leucyl and P = phenylalanyl) and terminated by a hydroxamic acid, which together define an octahedral iron-binding domain. A fourth exogenous strand provided the site for connecting various fluorescent markers via a short bifunctional linker. Iron(III) titrations, along with fluorescence spectroscopy, generated quenching of fluorescence emission of some of the probes used. The quenching process fits the Perrin model which reinforces the intramolecular quenching process, postulated previously.1 All tested compounds, regardless of their probe size, polarity, or the linker binding them to the siderophore analogue, promote growth of Pseudomonas putida with the same efficacy as the nonlabeled analogues 1, with the added benefit of signaling microbial activity by fluorescence emission. All G derivatives of compounds 10-12 were found to parallel the behavior of natural ferrichrome, whereas A derivatives mediated only a modest iron(III) uptake by P. putida. Incubation of various Pseudomonas strains with iron(III)-loaded G derivatives resulted in the build-up of the labels' fluorescence in the culture medium to a much larger extent than from the corresponding A derivatives. The fluorescence buildup corresponds to iron utilization by the cells and the release of the fluorescent labeled desferrisiderophore from the cell to the media. The fact that the microbial activity of these compounds is not altered by attachment of various fluorescent markers via a bifunctional linker proposes their application as diagnostic tools for detecting and identifying pathogenic microorganisms.
Asunto(s)
Diseño de Fármacos , Ferricromo/química , Colorantes Fluorescentes/química , Quelantes del Hierro/química , Compuestos Férricos/química , Compuestos Férricos/metabolismo , Ferricromo/análogos & derivados , Ferricromo/síntesis química , Ferricromo/farmacología , Quelantes del Hierro/síntesis química , Quelantes del Hierro/farmacología , Imitación Molecular , Pseudomonas putida/efectos de los fármacos , Pseudomonas putida/crecimiento & desarrollo , Pseudomonas putida/metabolismoRESUMEN
In this study, we monitored and compared the uptake of iron in the fungus Ustilago maydis by using biomimetic siderophore analogs of ferrichrome, the fungal native siderophore, and ferrioxamine B (FOB), a xenosiderophore. Ferrichrome-iron was taken up at a higher rate than FOB-iron. Unlike ferrichrome-mediated uptake, FOB-mediated iron transport involved an extracellular reduction mechanism. By using fluorescently labeled siderophore analogs, we monitored the time course, as well as the localization, of iron uptake processes within the fungal cells. A fluorescently labeled ferrichrome analog, B9-lissamine rhodamine B, which does not exhibit fluorescence quenching upon iron binding, was used to monitor the entry of the compounds into the fungal cells. The fluorescence was found intracellularly 4 h after the application and later was found concentrated in two to three vesicles within each cell. The fluorescence of the fluorescently labeled FOB analog CAT18, which is quenched by iron, was visualized around the cell membrane after 4 h of incubation with the ferrated (nonfluorescent) compounds. This fluorescence intensity increased with time, demonstrating fungal iron uptake from the siderophores, which remained extracellular. We here introduce the use of fluorescent biomimetic siderophores as tools to directly track and discriminate between different pathways of iron uptake in cells.