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Giraldo P.,Hospital Universitario Miguel Servet | Giraldo P.,Research Center Biomedica En Red Of Enfermedades Raras Ciberer Isciii | Giraldo P.,Instituto Aragones Of Ciencias Of La Salud Ics | Irun P.,Research Center Biomedica En Red Of Enfermedades Raras Ciberer Isciii | And 8 more authors.
Blood Cells, Molecules, and Diseases | Year: 2011

Recently, an acute restriction of imiglucerase has occurred as a result of viral contamination and manufacturing problems. A position statement from the European Working Group for Gaucher Disease and European Gaucher Alliance established a set of key recommendations for identifying and monitoring at-risk patients. In Spain, a profile of the shortage situation was obtained through follow-up of patients with Gaucher disease (GD) and compliance with the therapy recommendations. Here we describe a group of patients, with modified doses of imiglucerase, during the shortage. Fifty adult GD1 patients (25 males/25 females), previously on ERT, were analysed before and after the 6-month shortage. The mean age was 45.3 ± 15.3 years (range: 18-84). The mean Severity Score Index at diagnosis was 8.7 ± 3.8 (range: 3-19); 20% of patients were splenectomized; and 78% had bone disease. During the shortage, 23 patients (46%) discontinued therapy; as complications in this group only one patient suffered a bone crisis and another anaemia (Hb < 10.0 g/dL). The mean reduction of haemoglobin level (-2.7%) and platelet counts (-5.4%) were non-significant. Chitotriosidase (CT) activity was increased 135% (p<0.03) and CCL18/PARC 8.2% (p<0.08) in this group. Imiglucerase was reduced by 50% in 17 patients (34%) in this group, seven patients (41.0%) suffered bone pain, three of them true bone crisis and four (23.5%) required support therapy. The mean reduction of haemoglobin (-2.8%) and platelet counts (-10.7%), CT activity was increased 48.2% (p<0.03) and no changes were observed in CCL18/PARC concentration. In both groups no significant changes in visceral size were observed. In 3 patients (6%), imiglucerase was reduced 75% and 7 patients (14%) needed to switch to another ERT (4 patients) or miglustat (3 patients) due to a restart of symptomatic disease. In Spain the 6 first months shortage of imiglucerase have produced a 20% incidence of bone pain, one case of anaemia, and a significant increase in CT activity. Fourteen percent of patients had to switch to another therapy. No significant changes in blood counts, visceral volumes and CCL18/PARC concentration were observed. © 2010 Elsevier Inc.


Perez-Arellano I.,Research Center Principe Felipe | Perez-Arellano I.,Research Center Biomedica En Red Of Enfermedades Raras Ciberer Isciii | Carmona-Alvarez F.,Research Center Principe Felipe | Martinez A.I.,Research Center Principe Felipe | And 3 more authors.
Protein Science | Year: 2010

Pyrroline-5-carboxylate synthase (P5CS) is a bifunctional enzyme that exhibits glutamate kinase (GK) and γ-glutamyl phosphate reductase (GPR) activities. The enzyme is highly relevant in humans because it belongs to a combined route for the interconversion of glutamate, ornithine and proline. The deficiency of P5CS activity in humans is associated with a rare, inherited metabolic disease. It is well established that some bacteria and plants accumulate proline in response to osmotic stress. The alignment of P5CSs from different species and analysis of the solved structures of GK and GPR reveal high sequence and structural conservation. The information acquired from different mutant enzymes with increased osmotolerant properties, together with the position of the insertion found in the longer human isoform, permit the delimitation of the regulatory site of GK and P5CS and the proposal of a model of P5CS architecture. Additionally, the GK moiety of the human enzyme has been modeled and the known clinical mutations and polymorphisms have been mapped. Published by Wiley-Blackwell. © 2010 The Protein Society.


Martinez A.I.,Research Center Principe Felipe | Perez-Arellano I.,Research Center Principe Felipe | Perez-Arellano I.,Research Center Biomedica En Red Of Enfermedades Raras Ciberer Isciii | Pekkala S.,Research Center Principe Felipe | And 3 more authors.
Molecular Genetics and Metabolism | Year: 2010

Carbamoyl phosphate synthetase 1 (CPS1) plays a paramount role in liver ureagenesis since it catalyzes the first and rate-limiting step of the urea cycle, the major pathway for nitrogen disposal in humans. CPS1 deficiency (CPS1D) is an autosomal recessive inborn error which leads to hyperammonemia due to mutations in the CPS1 gene, or is caused secondarily by lack of its allosteric activator NAG.Proteolytic, immunological and structural data indicate that human CPS1 resembles Escherichia coli CPS in structure, and a 3D model of CPS1 has been presented for elucidating the pathogenic role of missense mutations. Recent availability of CPS1 expression systems also can provide valuable tools for structure-function analysis and pathogenicity-testing of mutations in CPS1. In this paper, we provide a comprehensive compilation of clinical CPS1 mutations, and discuss how structural knowledge of CPS enzymes in combination with in vitro analyses can be a useful tool for diagnosis of CPS1D. © 2010 Elsevier Inc.


Perez-Arellano I.,Research Center Principe Felipe | Perez-Arellano I.,Research Center Biomedica En Red Of Enfermedades Raras Ciberer Isciii | Gozalbo-Rovira R.,Research Center Principe Felipe | Martinez A.I.,Research Center Principe Felipe | And 2 more authors.
Protein Journal | Year: 2010

MCT-1 protein is encoded by an oncogene highly expressed in lymphomas. It is implicated in the interaction with the cap complex of the mRNA, through an RNA binding domain, named PUA. Targeted suppression of this domain attenuates the malignant phenotype and hence MCT-1 is a potential target for therapeutic intervention. In the present study 6×His-tagged MCT-1 expression and purification was assessed in insect cells using a baculovirus expression system. The gene was amplified by PCR from a human cDNA library, encoding an open reading frame of 181 amino acid residues. High MCT-1 production level (6 mg/L) was achieved in a two-step purification procedure. The protein was partially characterized by gel filtration chromatography, peptide mass fingerprinting and circular dichroism. A cap-binding assay confirmed its appropriate folding and functionality. Furthermore, a three dimensional model was built based on another known PUA domain structure. The abundant, pure and properly folded source of MCT-1 protein generated lays a foundation for future structure-function studies. © Springer Science+Business Media, LLC 2010.

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