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M Pineda

Publications and source records attributed to M Pineda.

At least 55 records · Page 3Linked to original sources
PubMed2000

Personal experience with the application of carbohydrate-deficient transferrin (CDT) assays to the detection of congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDG) are genetic multisystemic diseases due to various defects in the biosynthesis or processing of glycoproteins. Our aim is to present our experience in the selective screening of CDG syndrome in a paediatric population (421 patients) with clinical suspicion of the disease, analysing serum carbohydrate-deficient transferrin (CDT) by radioimmunoassay and/or immunoturbidimetry. We established the normal values for our paediatric population. The abnormal results were confirmed and classified by isoelectric focusing of serum sialotransferrins, and by enzymatic and molecular studies. We found 14 patients (3.3%) with abnormal serum CDT; 11 of them were classified as CDG type Ia (CDG-Ia) and the other three showed altered isoelectrofocusing patterns but remain untyped and are under investigation. In conclusion, both CDT assays proved to be useful tools for CDG screening. Isoelectric focusing is a simple procedure but it requires specific instruments that are not always available. Since the immunoturbidimetric procedure is commonly used to monitor for recent excessive alcohol consumption in clinical laboratories and does not require special equipment, it may also be reliably used to screen for CDG in children under clinical suspicion.

Adolescent↗
PubMed1999

Role of two upstream open reading frames in the translational control of oncogene mdm2.

Overexpression of oncoprotein MDM2 has been found in a significant number of human soft tissue tumors. In a subset of these tumors, overexpression is a result of enhanced translation of mdm2 mRNA. There are two transcripts from the mdm2 gene that differ only in their 5' leaders: a long form (L-mdm2) and a short form (S-mdm2) that arise from the use of different promoters. L-mdm2 mRNA contains two upstream open reading frames (uORFs) and this mRNA was loaded with ribosomes inefficiently in comparison with S-mdm2. The 5' leader of L-mdm2 was sufficient to transfer translational repression to a reporter gene and the two uORFs acted synergistically to achieve full suppression. In contrast, the 5' leader of S-mdm2 allowed efficient translation of an attached reporter gene in the tumor cells. These results are consistent with a model in which overexpression of MDM2 in certain tumors results from a change in mRNA structure due to a switch in promoter usage.

5' Untranslated Regions↗
PubMed1999

Identification of a membrane protein, LAT-2, that Co-expresses with 4F2 heavy chain, an L-type amino acid transport activity with broad specificity for small and large zwitterionic amino acids.

We have identified a new human cDNA, L-amino acid transporter-2 (LAT-2), that induces a system L transport activity with 4F2hc (the heavy chain of the surface antigen 4F2, also named CD98) in oocytes. Human LAT-2 is the fourth member of the family of amino acid transporters that are subunits of 4F2hc. The amino acid transport activity induced by the co-expression of 4F2hc and LAT-2 was sodium-independent and showed broad specificity for small and large zwitterionic amino acids, as well as bulky analogs (e.g. BCH (2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid)). This transport activity was highly trans-stimulated, suggesting an exchanger mechanism of transport. Expression of tagged N-myc-LAT-2 alone in oocytes did not induce amino acid transport, and the protein had an intracellular location. Co-expression of N-myc-LAT-2 and 4F2hc gave amino acid transport induction and expression of N-myc-LAT-2 at the plasma membrane of the oocytes. These data suggest that LAT-2 is an additional member of the family of 4F2 light chain subunits, which associates with 4F2hc to express a system L transport activity with broad specificity for zwitterionic amino acids. Human LAT-2 mRNA is expressed in kidney >>> placenta >> brain, liver > spleen, skeletal muscle, heart, small intestine, and lung. Human LAT-2 gene localizes at chromosome 14q11.2-13 (13 cR or approximately 286 kb from marker D14S1349). The high expression of LAT-2 mRNA in epithelial cells of proximal tubules, the basolateral location of 4F2hc in these cells, and the amino acid transport activity of LAT-2 suggest that this transporter contributes to the renal reabsorption of neutral amino acids in the basolateral domain of epithelial proximal tubule cells.

Amino Acid Sequence↗
PubMed1999

Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E.

A spectrophotometric method has been developed for the quantitative determination of antioxidant capacity. The assay is based on the reduction of Mo(VI) to Mo(V) by the sample analyte and the subsequent formation of a green phosphate/Mo(V) complex at acidic pH. The method has been optimized and characterized with respect to linearity interval, repetitivity and reproducibility, and molar absorption coefficients for the quantitation of several antioxidants, including vitamin E. The phosphomolybdenum method, in combination with hexane monophasic extraction, has also been adapted for the specific determination of vitamin E in seeds. The results obtained with the proposed method were validated by comparison with a standard HPLC method. The phosphomolybdenum method is routinely applied in our laboratory to evaluate the total antioxidant capacity of plant extracts and to determine vitamin E in a variety of grains and seeds, including corn and soybean.

Antioxidants↗
PubMed1999

RT-PCR cloning, characterization and mRNA expression analysis of a cDNA encoding a type II asparagine synthetase in common bean.

Following a RT-PCR strategy based on the design of degenerate oligonucleotides resembling conserved domains of asparagine synthetase (AS; EC 6.3.5.4), we isolated a 2 kb cDNA clone (PVAS2) from root tissue of the common bean (Phaseolus vulgaris). PVAS2 encodes a protein of 584 amino acids with a predicted relative molecular mass of 65810 Da, an isoelectric point of 6.4, and a net charge of -7.2 at pH 7.0. The amino acid sequence of the protein encoded by PVAS2 is very similar to that encoded by the soybean SAS2 asparagine synthetase gene. The amino-terminal residues of the predicted PVAS2 protein are identical to the amino acids that constitute the glutamine-binding (GAT) domain of AS from other plant species, which suggests that the PVAS2 cDNA encodes a type II glutamine-dependent form of asparagine synthetase. Southern blot analysis indicates that the common bean AS is part of a small family composed of at least two genes. Expression analysis by Northern blot revealed that the PVAS2 transcript accumulates to a high level in roots and, to a lesser extent, in nodules and developing pods. Accumulation of the PVAS2 transcript in the root seems to be negatively regulated by light and sucrose, and positively regulated by nitrate.

Amino Acid Sequence↗
PubMed1999

Identification of a cytogenetic deletion and of four novel mutations (Q69X, I172F, G188V, G197R) affecting the gene for ornithine transcarbamylase (OTC) in Spanish patients with OTC deficiency.

A deletion of at least 11.5 cM in the paternal X chromosome mapping between microsatellites DXS989 and DXS1003 and encompassing the genes for ornithine transcarbamylase (OTC), retinitis pigmentosa GTPase regulator (RPGR) and dystrophin, was associated with the loss of band Xp21 in a female patient with OTC deficiency. Another four female patients were heterozygous for point mutations in the OTC gene: the nonsense mutation Q69X or the missense mutations I172F, G188V and G197R. In the OTC amino acid sequence, I172 and G197 are proximate to residues involved in catalysis, and G188 is within a loop joining helix 5 and strand 6 in the core of the ornithine-bindingdomain. Therefore, the mutations of these residues may cause structural changes affecting catalysis and/or the architecture of the ornithine domain. The mutation appeared "de novo" in the patients or, in one case, in the mother of the patient, in agreement with the predominance of "de novo" mutations in female patients of OTC deficiency. There was full agreement between the results of mutational analysis and of allopurinol testing in the patients and their female relatives, supporting the value of the allopurinol test in the detection of carriers of OTC deficiency. This deficiency is a genetically heterogeneous X-linked condition.

Allopurinol↗
PubMed1999

Neuronopathic juvenile glucosylceramidosis due to sap-C deficiency: clinical course, neuropathology and brain lipid composition in this Gaucher disease variant.

Glucosylceramide lipidosis results from a defective lysosomal degradation of this glycolipid. Lipid degradation is controlled by two components, the enzyme beta-glucocerebrosidase and a sphingolipid activator protein. While most Gaucher cases are due to mutations within the gene that codes for the lysosomal enzyme, only two patients have been described with normal enzyme levels and mutations in the gene for the sphingolipid activator protein C (sap-C). Here we present the detailed neurological manifestations, neuropathological findings and brain lipid composition in one sap-C-deficient patient. The patient was an 8-year-old boy who presented with transient losses of consciousness, myoclonic jerks and generalized seizures resistant to all antiepileptic drugs. He developed progressive horizontal ophthalmoplegia, pyramidal and cerebellar signs, and died at the age of 15.5 years. Neuropathological studies demonstrated neuronal cell loss and neuronophagia, massive intraneuronal lipid storage and lack of perivascular Gaucher cells. Electron microscopy examination showed different types of storage including lipofuscin granules as well as the cytosomes with parallel arrays of bilayers that are assumed to be formed by stored lipids. General brain lipid composition did not show a remarkable increase or loss of any of the major lipid fractions but the glucosylceramide concentration in the cortex of several anatomical regions showed a striking increase. Fatty acid composition of the ceramide moiety clearly suggests that gangliosides are the main precursors in the cerebral cortex, while it implies an additional and distinct source in the cerebellum. Studying the phenotypic consequences of mutant sphingolipid activator proteins is critical to a better understanding of the physiological significance of these proteins.

Cerebellum↗
PubMed1999

Evaluation of hyperhomocysteinaemia in children with stroke.

Hyperhomocysteinaemia is associated with an increased risk of arterial vascular disease and thrombosis in adults. Our aim was to study the association of hyperhomocysteinaemia and stroke in children. Since some patients who had suffered a stroke developed seizures and received treatment with anti-epileptic (antifolate) drugs, we also examined the possible interaction between anti-epileptic drugs and hyperhomocysteinaemia. Plasma total homocysteine was measured in 68 children with stroke (23 of the 68 were taking anti-epileptic drugs) and 100 children undergoing anti-epileptic treatment but without history of stroke, and we compared the values with our reference values for similar ages (n = 195). Total homocysteine was determined by high profile liquid chromatography with fluorescence detection. Hyperhomocysteinaemia was defined as a homocysteine concentration above the 95th percentile for the reference values. Significant differences were found in total homocysteine values of children with stroke and those taking anti-epileptic drugs compared with our reference values for similar ages, except for the adolescent group. Total homocysteine values above the 95th percentile for the reference values were found in 36% of patients with stroke and 28% of children on anti-epileptic treatment. Total homocysteine concentrations in the 23 patients with both stroke and anti-epileptic drug treatment were similar to those of untreated patients with stroke in all age groups. In summary, systematic screening for hyperhomocysteinaemia should be included in the protocol to investigate the aetiology of stroke, even in paediatrics. Anti-epileptic treatment in children with stroke may be responsible for the mild hyperhomocysteinaemia observed in some of them. A dietary supplement of folate may be of benefit in children with stroke and in patients taking anti-epileptic drugs.

Adolescent↗
PubMed1999

Non-type I cystinuria caused by mutations in SLC7A9, encoding a subunit (bo,+AT) of rBAT.

Cystinuria (MIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids. Mutations in SLC3A1, encoding rBAT, cause cystinuria type I (ref. 1), but not other types of cystinuria (ref. 2). A gene whose mutation causes non-type I cystinuria has been mapped by linkage analysis to 19q12-13.1 (Refs 3,4). We have identified a new transcript, encoding a protein (bo, +AT, for bo,+ amino acid transporter) belonging to a family of light subunits of amino acid transporters, expressed in kidney, liver, small intestine and placenta, and localized its gene (SLC7A9) to the non-type I cystinuria 19q locus. Co-transfection of bo,+AT and rBAT brings the latter to the plasma membrane, and results in the uptake of L-arginine in COS cells. We have found SLC7A9 mutations in Libyan-Jews, North American, Italian and Spanish non-type I cystinuria patients. The Libyan Jewish patients are homozygous for a founder missense mutation (V170M) that abolishes b o,+AT amino-acid uptake activity when co-transfected with rBAT in COS cells. We identified four missense mutations (G105R, A182T, G195R and G295R) and two frameshift (520insT and 596delTG) mutations in other patients. Our data establish that mutations in SLC7A9 cause non-type I cystinuria, and suggest that bo,+AT is the light subunit of rBAT.

Amino Acid Sequence↗
PubMed1999

Identification of SLC7A7, encoding y+LAT-1, as the lysinuric protein intolerance gene.

Lysinuric protein intolerance (LPI; OMIM 222700) is a rare, recessive disorder with a worldwide distribution, but with a high prevalence in the Finnish population; symptoms include failure to thrive, growth retardation, muscle hypotonia and hepatosplenomegaly. A defect in the plasma membrane transport of dibasic amino acids has been demonstrated at the baso-lateral membrane of epithelial cells in small intestine and in renal tubules and in plasma membrane of cultured skin fibroblasts from LPI patients. The gene causing LPI has been assigned by linkage analysis to 14q11-13. Here we report mutations in SLC7A7 cDNA (encoding y+L amino acid transporter-1, y+LAT-1), which expresses dibasic amino-acid transport activity and is located in the LPI region, in 31 Finnish LPI patients and 1 Spanish patient. The Finnish patients are homozygous for a founder missense mutation leading to a premature stop codon. The Spanish patient is a compound heterozygote with a missense mutation in one allele and a frameshift mutation in the other. The frameshift mutation generates a premature stop codon, eliminating the last one-third of the protein. The missense mutation abolishes y+LAT-1 amino-acid transport activity when co-expressed with the heavy chain of the cell-surface antigen 4F2 (4F2hc, also known as CD98) in Xenopus laevis oocytes. Our data establish that mutations in SLC7A7 cause LPI.

Adolescent↗
PubMed1999

Diagnosis and follow-up of a case of peroxisomal disorder with peroxisomal mosaicism.

Peroxisomal disorder phenotypes are the result of mutations that cause defective peroxisomal assembly or alterations in the import mechanism of peroxisomal proteins that lead to multiple peroxisomal dysfunctions, or the result of a peroxisomal enzymatic deficiency with a single peroxisomal dysfunction. With complementation analysis, 16 groups have been found. Assignment of the genetic defect has been described for some of the complementation groups. We describe the clinical evolution and follow-up over 10 years of a patient who belongs to complementation group 4, although he showed a milder clinical course. It has been found in fibroblasts different peroxisome populations, normal processing and expression of beta-oxidation PTS1 and PTS2 proteins, abnormal ALD protein distribution and normal plasmalogen biosynthesis; abnormal beta-oxidation metabolites have also been detected in serum. Ultrastructural studies in liver showed peroxisomal mosaicism as in fibroblasts. It has been taken into account that peroxisomal mosaicism may lead to variability in peroxisomal diagnostic parameters, making difficult the final diagnosis in these patients.

Adolescent↗
PubMed1999

Early onset cerebellar ataxia and preservation of tendon reflexes: clinical phenotypes associated with GAA trinucleotide repeat expanded and non-expanded genotypes.

The aim of this study was to determine phenotypie characteristics of patients with early onset cerebellar ataxia (EOCA) with preserved tendon reflexes. The series comprises 25 patients, representing 10% of all ataxic patients who have been genetically studied in our laboratory since 1990. There were 11 males and 14 females. Fourteen patients were homozygous for the GAA expansion on chromosome 9q13 (group 1) and therefore a diagnosis of Friedreich's ataxia with retained reflexes (FARR) was given. The remaining 11 patients had two normal non-expanded alleles (group 2) and a working diagnosis of EOCA with retained reflexes (EOCARR) was established. Mean ages of onset were 13.7 +/- 5.9 years (3-25) for group 1 and 10.3 +/- 7.3 for group 2; the difference was not significant. Frequencies of symptoms and signs were also comparable for both groups the only significant differences being the higher frequency of nystagmus, cardiomyopathy and sensory neuropathy in group 1 patients. There was a tendency for FARR patients to have higher frequencies of hypopallesthesia in the lower limbs and skeletal deformities. In none of the cases diabetes mellitus was observed. We conclude that differentiation of FARR and EOCARR may be suspected by classical clinical and electrophysiological data and confirmed by analysis of the GAA repeat.

Adolescent↗
PubMed1999

[Treatment of generalized peroxisomal disorders with docosahexaenoic acid ethyl ether].

INTRODUCTION: We found that patients with the Zellweger syndrome and other generalized peroxisomal disorders have a dramatic decrease of docosahexaenoic acid (DHA, 22:6n-3) in the blood, brain, retina and other tissues. DHA is believed to play an important role in the brain and retina. DEVELOPMENT: Patients with the Zellweger syndrome and its variants have severe cerebral and retinal defects that could be related to their DHA deficiency. With this rationale, we have been treating peroxisomal-disorder patients with a DHA derivative of a high degree of purity (DHA ethyl ester, > 90% pure) since 1991. So far, we have treated 13 DHA-deficient peroxisomal patients, one with the classic Zellweger syndrome and 12 with milder variants of the disease. This paper presents the follow-up of these DHA-treated patients. In summary, we have found important improvements in liver function, in the plasmalogen levels and in the two ratios 26:0/22:0 y 26:1/22:0, diagnostic of the disease. We have also found clear clinical improvements in most cases. Most significantly, magnetic resonance imaging has shown advances in brain myelination, so far in 6 of the treated patients. CONCLUSION: We strongly recommend treatment with DHA ethyl ester in all DHA-deficient patients with generalized peroxisomal disorders. Logically, treatment should be started as soon as possible, in the hope of preventing cerebral and visual damage.

Brain↗
PubMed1998

Identification and characterization of a membrane protein (y+L amino acid transporter-1) that associates with 4F2hc to encode the amino acid transport activity y+L. A candidate gene for lysinuric protein intolerance.

We have identified a new human cDNA (y+L amino acid transporter-1 (y+LAT-1)) that induces system y+L transport activity with 4F2hc (the surface antigen 4F2 heavy chain) in oocytes. Human y+LAT-1 is a new member of a family of polytopic transmembrane proteins that are homologous to the yeast high affinity methionine permease MUP1. Other members of this family, the Xenopus laevis IU12 and the human KIAA0245 cDNAs, also co-express amino acid transport activity with 4F2hc in oocytes, with characteristics that are compatible with those of systems L and y+L, respectively. y+LAT-1 protein forms a approximately 135-kDa, disulfide bond-dependent heterodimer with 4F2hc in oocytes, which upon reduction results in two protein bands of approximately 85 kDa (i.e. 4F2hc) and approximately 40 kDa (y+LAT-1). Mutation of the human 4F2hc residue cysteine 109 (Cys-109) to serine abolishes the formation of this heterodimer and drastically reduces the co-expressed transport activity. These data suggest that y+LAT-1 and other members of this family are different 4F2 light chain subunits, which associated with 4F2hc, constitute different amino acid transporters. Human y+LAT-1 mRNA is expressed in kidney >> peripheral blood leukocytes >> lung > placenta = spleen > small intestine. The human y+LAT-1 gene localizes at chromosome 14q11.2 (17cR approximately 374 kb from D14S1350), within the lysinuric protein intolerance (LPI) locus (Lauteala, T., Sistonen, P. , Savontaus, M. L., Mykkanen, J., Simell, J., Lukkarinen, M., Simmell, O., and Aula, P. (1997) Am. J. Hum. Genet. 60, 1479-1486). LPI is an inherited autosomal disease characterized by a defective dibasic amino acid transport in kidney, intestine, and other tissues. The pattern of expression of human y+LAT-1, its co-expressed transport activity with 4F2hc, and its chromosomal location within the LPI locus, suggest y+LAT-1 as a candidate gene for LPI.

Amino Acid Metabolism, Inborn Errors↗
PubMed1998

Biochemical monitoring of the treatment in paediatric patients with mitochondrial disease.

Treatment strategies in mitochondrial diseases consist of several drugs that diminish the deleterious effects of the abnormal respiratory chain function, reduce the presence of toxic agents or correct deficiencies in essential cofactors. In this study we evaluated the monitoring of tocopherol, carnitine and ubiquinone concentrations in a group of paediatric patients during a follow-up period of 18 months and the response to treatment of these patients by means of the determination of blood lactate, plasma alanine and oxygen consumption by lymphocytes in relation to the clinical status of the patients. Tocopherol, carnitine and ubiquinone concentrations were easily corrected with therapy. Blood lactate proved the best biochemical tool to assess the response to treatment in paediatric patients. According to our results, improvement or stabilization of the clinical course seems to be more related to the biochemical or molecular defect than to the effectiveness of the treatment.

Adolescent↗
PubMed1998

Acute necrotizing encephalopathy of childhood (infantile bilateral thalamic necrosis): two non-Japanese cases.

This report describes two Spanish cases of acute necrotizing encephalopathy of childhood, a rare disease first described in Japan by Mizuguchi and colleagues. Similar cases have been reported from Taiwan and other countries of the Far East. Two cases have been reported from the UK and one from the USA. The disease affects young children and is characterized by acute encephalopathy after a viral illness, with seizures and decreased levels of consciousness. The hallmark of the disease is diffuse and symmetrical CNS lesions of both thalami, internal capsules, upper brainstem tegmentum, and cerebral white matter. The aetiology is unknown but an infectious or parainfectious process seems likely. Because of the predominant involvement of the thalami, we propose the term 'infantile bilateral thalamic necrosis', a more specific term and one which distinguishes the entity from other basal ganglia diseases in childhood.

Brain↗