Outcome of unrelated bone marrow donor searches in 174 children resulting in 45 patients transplanted in the HLA-matched and -mismatched situation. (25/628)

The aim of the study was to evaluate the outcome of unrelated bone marrow donor (UBMD) searches initiated for 174 children between 1986 and 1997. Seven patients were registered twice so that a total of 181 UBMD searches took place. At the time of registration, patients suffered from hematological malignancies (n = 121), non-malignant hemopathies (n = 26) and inborn errors (n = 34). Forty-five of the patients (26%) were given transplants from unrelated donors of whom 26 (58%) were HLA-mismatched transplants. Our strategy accepted HLA mismatches at the time of donor selection, using Thymoglobuline as part of the conditioning regimen. Of the 45 patients given unrelated donor transplants, overall survival was 60% at 3 years and concerned 27 patients of whom 14 were from HLA-mismatched donors. Disease-free survival for hematological malignancies was 65% in HLA-matched transplants and 50% in HLA-mismatched transplants. For some patients (16%) urgency led us to use alternative options: non-identical related donor (n = 14), autograft (n = 10), related cord blood transplant (n = 4). For others, UBMD searches were stopped because of favorable evolution (n = 29), death (n = 24), disease progression (n = 22) or other reasons (n = 21). By the end of the follow-up period, 88 patients had died (50%), 75 (43%) are currently alive with or without being transplanted of whom eight are still having active searches and 11 are no longer contactable. In conclusion, in severe disease in children, an immediate transplant from a partially matched donor might be preferable to a prolonged search for a full match. Consequently, this strategy increases the number of patients for whom a suitable donor can be found. We have chosen this option in order not to delay BMT; in so doing we have obtained encouraging results which include high overall survival, low incidence of acute GVHD grade III-IV and low percentage of relapse even in mismatched pairs.  (+info)

Structural requirements for the stability and microsomal transport activity of the human glucose 6-phosphate transporter. (26/628)

Deficiencies in glucose 6-phosphate (G6P) transporter (G6PT), a 10-helical endoplasmic reticulum transmembrane protein of 429 amino acids, cause glycogen storage disease type 1b. To date, only three missense mutations in G6PT have been shown to abolish microsomal G6P transport activity. Here, we report the results of structure-function studies on human G6PT and demonstrate that 15 missense mutations and a codon deletion (delta F93) mutation abolish microsomal G6P uptake activity and that two splicing mutations cause exon skipping. While most missense mutants support the synthesis of G6PT protein similar to that of the wild-type transporter, immunoblot analysis shows that G20D, delta F93, and I278N mutations, located in helix 1, 2, and 6, respectively, destabilize the G6PT. Further, we demonstrate that G6PT mutants lacking an intact helix 10 are misfolded and undergo degradation within cells. Moreover, amino acids 415-417 in the cytoplasmic tail of the carboxyl-domain, extending from helix 10, also play a critical role in the correct folding of the transporter. However, the last 12 amino acids of the cytoplasmic tail play no essential role(s) in functional integrity of the G6PT. Our results, for the first time, elucidate the structural requirements for the stability and transport activity of the G6PT protein.  (+info)

Clinical, biochemical and molecular genetic correlations in adenylosuccinate lyase deficiency. (27/628)

Adenylosuccinate lyase (ADSL) deficiency (MIM 103050) is an autosomal recessive inborn error of purine synthesis characterized by the accumulation in body fluids of succinylaminoimidazolecarboxamide (SAICA) riboside and succinyladenosine (S-Ado), the dephosphorylated derivatives of the two substrates of the enzyme. Because ADSL-deficient patients display widely variable degrees of psychomotor retardation, we have expressed eight mutated ADSL enzymes as thioredoxin fusions and compared their properties with the clinical and biochemical characteristics of 10 patients. Three expressed mutated ADSL enzymes (M26L, R426H and T450S) were thermolabile, four (A2V, R141W, R303C and S395R) were thermostable and one (del206-218), was inactive. Thermolabile mutations decreased activities with SAICA ribotide (SAICAR) and adenylosuccinate (S-AMP) in parallel, or more with SAICAR than with S-AMP. Patients homozygous for one of these mutations, R426H, displayed similarly decreased ADSL activities in their fibroblasts, S-Ado:SAICA riboside ratios of approximately 1 in their cerebrospinal fluid and were profoundly retarded. With the exception of A2V, thermostable mutations decreased activity with S-AMP to a much more marked extent than with SAICAR. Two unrelated patients homozygous for one of the thermostable mutations, R303C, also displayed a much more marked decrease in the activity of fibroblast ADSL with S-AMP than with SAICAR, had S-Ado:SAICA riboside ratios between 3 and 4 in their cerebrospinal fluid and were mildly retarded. These results suggest that, in some cases, the genetic lesion of ADSL determines the ratio of its activities with S-AMP versus SAICAR, which in turn defines the S-Ado:SAICA riboside ratio and the patients' mental status.  (+info)

Use of the NADH-quinone oxidoreductase (NDI1) gene of Saccharomyces cerevisiae as a possible cure for complex I defects in human cells. (28/628)

The Ndi1 enzyme of Saccharomyces cerevisiae is a single subunit rotenone-insensitive NADH-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane. We have shown previously that the NDI1 gene can be functionally expressed in Chinese hamster cells (Seo, B. B., Kitajima-Ihara, T., Chan, E. K., Scheffler, I. E., Matsuno-Yagi, A., and Yagi, T. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9167-9171) and human embryonal kidney 293 (HEK 293) cells (Seo, B. B., Matsuno-Yagi, A., and Yagi, T. (1999) Biochim. Biochem. Acta 1412, 56-65) and that the Ndi1 protein is capable of compensating respiratory deficiencies caused by defects in the host NADH-quinone oxidoreductase (complex I). To extend the potential use of this enzyme to repair complex I deficiencies in vivo, we constructed a recombinant adeno-associated virus vector carrying the NDI1 gene (rAAV-NDI1). With rAAV-NDI1 as the gene delivery method, we were able to achieve high transduction efficiencies (nearly 100%) even in 143B cells that are difficult to transfect by lipofection or calcium phosphate precipitation methods. The NDI1 gene was successfully introduced into non-proliferating human cells using rAAV-NDI1. The expressed Ndi1 protein was shown to be functionally active just as seen for proliferating cells. Furthermore, when cells were cultured under the conditions where energy has to be provided by respiration, the NDI1-transduced cells were able to grow even in the presence of added complex I inhibitor such as rotenone and 1-methyl-4-phenylpyridinium ion. In contrast, control cells that did not receive the NDI1 gene failed to survive as anticipated. The Ndi1 protein has a great potential as a molecular remedy for complex I defects, and it is highly likely that the same strategy can be extended to correction of other mitochondrial disorders.  (+info)

Gating of inward-rectifier K+ channels by intracellular pH. (29/628)

Inward rectifier K+ channels of the Kir1.1 (ROMK) and Kir4.1 subtype are predominantly expressed in epithelial cells where they are responsible for K+ transport across the plasma membrane. Uniquely among the members of the Kir family, these channels are gated by intracellular pH in the physiological range. pH-gating involves structural rearrangements in cytoplasmic domains and the P-loop of the Kir protein. The energy for the gating transition is delivered by protonation of a lysine residue that is located prior to the first transmembrane segment and serves as a 'pH sensor'. The anomalous titration required for lysine operating in the neutral pH range results from its close interaction with two positively charged arginines from the distant N- and C-termini termed the R/K/R triad. Disturbance of this triad as results from a number of point mutations found in patients with hyperprostaglandin E syndrome (HPS) increases the pKa of the pH sensor and results in channels being permanently inactivated under physiological conditions. This article will focus on the mechanism of pH-gating, its implications for the tertiary structure of Kir proteins and on its significance for the pathogenesis of HPS.  (+info)

Inherited disorders of renal magnesium handling. (30/628)

The genetic basis and cellular defects of a number of primary magnesium wasting diseases have been elucidated over the past decade. This review correlates the clinical pathophysiology with the primary defect and secondary changes in cellular electrolyte transport. The described disorders include (1) hypomagnesemia with secondary hypocalcemia, an earlyonset, autosomal-recessive disease segregating with chromosome 9q12-22.2; (2) autosomal-dominant hypomagnesemia caused by isolated renal magnesium wasting, mapped to chromosome 11q23; (3) hypomagnesemia with hypercalciuria and nephrocalcinosis, a recessive condition caused by a mutation of the claudin 16 gene (3q27) coding for a tight junctional protein that regulates paracellular Mg(2+) transport in the loop of Henle; (4) autosomal-dominant hypoparathyroidism, a variably hypomagnesemic disorder caused by inactivating mutations of the extracellular Ca(2+)/Mg(2+)-sensing receptor, CASR: gene, at 3q13.3-21 (a significant association between common polymorphisms of the CASR: and extracellular Mg(2+) concentration has been demonstrated in a healthy adult population); and (5) Gitelman syndrome, a recessive form of hypomagnesemia caused by mutations in the distal tubular NaCl cotransporter gene, SLC12A3, at 16q13. The basis for renal magnesium wasting in this disease is not known. These inherited conditions affect different nephron segments and different cell types and lead to variable but increasingly distinguishable phenotypic presentations. No doubt, there are in the general population other disorders that have not yet been identified or characterized. The continued use of molecular techniques to probe the constitutive and congenital disturbances of magnesium metabolism will increase the understanding of cellular magnesium transport and provide new insights into the way these diseases are diagnosed and managed.  (+info)

Mutations of the SCO1 gene in mitochondrial cytochrome c oxidase deficiency with neonatal-onset hepatic failure and encephalopathy. (31/628)

Cytochrome c oxidase (COX) catalyzes both electron transfer from cytochrome c to molecular oxygen and the concomitant vectorial proton pumping across the inner mitochondrial membrane. Studying a large family with multiple cases of neonatal ketoacidotic comas and isolated COX deficiency, we have mapped the disease locus to chromosome 17p13.1, in a region encompassing two candidate genes involved in COX assembly-namely, SCO1 and COX10. Mutation screening revealed compound heterozygosity for SCO1 gene mutations in the patients. The mutated allele, inherited from the father, harbored a 2-bp frameshift deletion (DeltaGA; nt 363-364) resulting in both a premature stop codon and a highly unstable mRNA. The maternally inherited mutation (C520T) changed a highly conserved proline into a leucine in the protein (P174L). This proline, adjacent to the CxxxC copper-binding domain of SCO1, is likely to play a crucial role in the tridimentional structure of the domain. Interestingly, the clinical presentation of SCO1-deficient patients markedly differs from that of patients harboring mutations in other COX assembly and/or maturation genes.  (+info)

Inherited depression of arterial lipoamide dehydrogenase activity associated with susceptibility to atherosclerosis in pigeons. (32/628)

The activity of lipoamide dehydorgenase (E.C.1.6.4.3) was measured in arterial homogenates from very young pigeons (5-8 weeks old) known to differ in their susceptibility to atherosclerosis. The activity of the arterial enzyme was significantly lower in the atherosclerosis-susceptible White Carneau pigeons than it was in the atherosclerosis-resistant Show Racer pigeons. Lipoamide dehydrogenase is a component of the pyruvate dehydrogenase and alpha-ketoglutarate multienzyme complexes. The first complex catalyzes the conversion of pyruvate to oxaloacetate via acetyl-CoA, and this reaction represents a crucial link between glycolysis and the Krebs cycle. The second complex is essential for the oxidative breakdown of carbohydrates, fats, and amino acids via the Krebs cycle. Reduced activity of these complexes, resulting from low activity of lipoamide dehydrogenase, favors reduction of pyruvate to lactate and a shift to glycolysis. This situation is in accord with other results obtained in avian and human arteries which appear to indicate a higher rate of glycolysis in atherosclerosis-susceptible and atherosclerotic arteries. It appears that the increased dependence of the White Carneau arteries on glycolysis, suggested by the reduced lipoamide dehydrogenase activity, facilitates the development of atherosclerosis in this pigeon strain.  (+info)