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Y Indo

Publications and source records attributed to Y Indo.

50 records · Page 3Linked to original sources
PubMed1990

Molecular basis of medium chain acyl-coenzyme A dehydrogenase deficiency. An A to G transition at position 985 that causes a lysine-304 to glutamate substitution in the mature protein is the single prevalent mutation.

We sequenced polymerase chain reaction (PCR)-amplified variant medium chain acyl-CoA dehydrogenase (MCAD) cDNAs in cultured fibroblasts from three MCAD-deficient patients. In all three patients, an A to G transition was identified at position 985 of the coding region. Since no appropriate restriction sites for detecting this point mutation were found, we devised a PCR method that amplifies an 87-bp fragment from position 955. In the 5' primer encompassing positions 955 to 984, A-981 was artificially substituted with C. With the presence of C-981 and G-985, an Nco I restriction site is introduced in the mutant copies. When cDNA or genomic DNA from fibroblasts of nine MCAD-deficient patients were tested with this method, the copies from all of them completely cleaved into two shorter fragments by Nco I, indicating their homozygosity for the A----G-985 transition. In contrast, the copies from all eight controls remained intact. Thus, this A----G-985 transition is the single prevalent mutation causing MCAD deficiency, a highly unusual feature for any genetic disorder. The PCR/Nco I digestion method is suitable for the diagnosis of MCAD deficiency.

Acyl-CoA Dehydrogenase↗
PubMed1989

Molecular cloning and nucleotide sequence of cDNAs encoding the precursors of rat long chain acyl-coenzyme A, short chain acyl-coenzyme A, and isovaleryl-coenzyme A dehydrogenases. Sequence homology of four enzymes of the acyl-CoA dehydrogenase family.

cDNAs encoding the entire coding regions of the precursors (p) of rat long chain acyl-CoA (LCAD), short chain acyl-CoA (SCAD) and isovaleryl-CoA dehydrogenase (IVD) have been cloned and sequenced. Three cDNAs for rat liver LCAD together cover a 1440-base pair region. These cDNAs encode the entire 430-amino acid sequence of pLCAD, including the 30-amino acid leader peptide and the 400-amino acid mature LCAD. A single 1773 base pair cDNA for rat SCAD covers the entire coding region (414 amino acids), including the 26-amino acid leader peptide and the 388-amino acid mature peptide. Four identified IVD cDNAs, when combined, encompass a 2104 base region, and encode 424 amino acids including a 30-amino acid leader peptide and the 394-amino acid mature peptide. The identities of all cDNA clones have been confirmed by matching the amino acid sequences predicted from the respective cDNAs to the amino-terminal and tryptic peptide sequences derived from the corresponding purified rat enzyme. Comparison of the sequences of four rat acyl-CoA dehydrogenases, including LCAD, MCAD, SCAD, and IVD, and two of their human counterparts (MCAD and SCAD) reveals a high degree of homology (57 invariant and 92 near invariant residues: 30.6-35.4% of identical residues in pairwise comparisons), suggesting that these enzymes belong to a gene family and have evolved from a common ancestral gene.

Acyl-CoA Dehydrogenase↗
PubMed1989

Primary structure and gene localization of human prolidase.

Complementary DNA clones of prolidase (imidodipeptidase, EC 3.4.13.9) were isolated from human liver and placental cDNA libraries. Two clones named lambda PL21 and lambda PP6 from the liver and placental cDNA libraries, respectively, were analyzed in detail. The first clone, lambda PL21, carried a cDNA insert of 1.7 kilobase pairs and covered all the coding region of human prolidase mRNA. The second clone, lambda PP6, contained a 1.8-kilobase insert with a full-length 3'-untranslated region. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the two clones with the partial amino acid sequence determined by Edman degradation of peptides derived from human erythrocyte prolidase established that both clones code for human prolidase. The amino terminus of the human mature enzyme is blocked and seems to begin with the sequence X-Ala-Ala-Ala. Presumably no processing occurs at the carboxyl terminus. The mature enzyme is composed of 492 residues, corresponding to Mr 54,305. The sequence of prolidase is unique and not similar to any known protein, except for a significant similarity to regions of F1-ATPase alpha and beta subunits from various sources. The gene has been mapped to the short arm of chromosome 19 (19p13.2). Elucidation of the complete amino acid sequence and the gene location of prolidase should provide the basis for understanding structure-function relationships and also inherited disorders caused by deficiency of this metabolically important enzyme.

Amino Acid Sequence↗
PubMed1989

Short chain acyl-coenzyme A dehydrogenase (SCAD) deficiency. Immunochemical demonstration of molecular heterogeneity due to variant SCAD with differing stability.

Using a [35S]methionine labeling/immunoprecipitation technique, we have previously shown that cultured skin fibroblast from three patients with short chain acyl-CoA dehydrogenase (SCAD) deficiency each synthesize a normal-sized (41 kD) variant SCAD in an amount comparable to that of normal cells. In the current study, these same cell lines were reexamined with immunoblot analysis. In one cell line (YH2065) no SCAD protein was detectable. In the other two deficient cell lines, the amount of variant SCAD was similar to, or only slightly less than, normal. These results suggested that SCAD-YH2065 is labile. In the pulse-labeling experiments, labeled SCAD was readily detectable for at least 30 h in a normal control and two other SCAD-deficient cell lines. In contrast, the labeled SCAD band in YH2065 cells was barely detectable at 6 h and undetectable at 20 h. [35S]Methionine-labeling in the presence of rhodamine 6G demonstrated that SCAD-YH2065 was synthesized as a 44-kD precursor and imported normally into mitochondria, as were the normal SCAD and two other variant SCADs, excluding the possibility that SCAD-YH2065 is a truncated precursor that cannot be imported into mitochondria. These results indicate that the mutations responsible for SCAD deficiency are heterogeneous, and emphasize the importance of using both radiolabeling and immunoblotting when evaluating such genetic defects at the protein level.

Acyl-CoA Dehydrogenases↗
PubMed1988

Maple syrup urine disease: a possible biochemical basis for the clinical heterogeneity.

Nine patients with maple syrup urine disease (MSUD), of whom eight were detected by mass-screening of neonates for inherited metabolic disease, were studied to determine possible relationships between clinical features and properties of the branched-chain alpha-keto acid dehydrogenase complex (BCKDH) in cultured lymphoblastoid cells. Based on their tolerance for leucine and on the clinical manifestations observed after 2 years of age, most could be classified into three types; classical (tolerate less than 600 mg of leucine per day, N = 2), intermediate (N = 3) and intermittent (N = 3) types. In the other patient two of these three phenotypes were present. The BCKDH activities measured at a lower alpha-ketoisovaleric acid concentration (0.054 mM) were 0.026 +/- 0.015 in classical, 0.118 +/- 0.016 in intermediate and 0.625 +/- 0.139 in intermittent types and 7.052 +/- 0.779 (nmol/h per milligram of protein) in two controls, respectively; the differences being statistically significant (P less than 0.01, classical vs intermediate types; P less than 0.01, intermediate vs intermittent types; P less than 0.01, intermittent vs control). Kinetic and immunochemical analyses of the BCKDH revealed that, although there are a few exceptions, classical, intermediate and intermittent types correspond to the enzyme properties of sigmoidal kinetics with E1 beta subunit deficiency, near-sigmoidal kinetics with E1 beta subunit deficiency and hyperbolic kinetics with E2 subunit deficiency of the BCKDH, respectively.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1987

Altered kinetic properties of the branched-chain alpha-keto acid dehydrogenase complex due to mutation of the beta-subunit of the branched-chain alpha-keto acid decarboxylase (E1) component in lymphoblastoid cells derived from patients with maple syrup urine disease.

Branched-chain alpha-keto acid dehydrogenase (BCKDH) complexes of lymphoblastoid cell lines derived from patients with classical maple syrup urine disease (MSUD) phenotypes were studied in terms of their catalytic functions and analyzed by immunoblotting, using affinity purified anti-bovine BCKDH antibody. Kinetic studies on three cell lines derived from patients with the classical phenotype showed sigmoidal or near sigmoidal kinetics for overall BCKDH activity and a deficiency of the E1 component activity. An immunoblot study revealed a markedly decreased amount of the E1 beta subunit accompanied by weak staining of the E1 alpha subunit. The E2 and E3 component exhibited a cross-reactive peptide. Thus, in at least some patients with MSUD, mutations of the E1 beta subunit might provide an explanation for the altered kinetic properties of the BCKDH complex.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1987

Immunochemical studies of human prolidase with monoclonal and polyclonal antibodies: absence of the subunit of prolidase in erythrocytes from a patient with prolidase deficiency.

Prolidase was highly purified from human liver and erythrocytes. NaDodSO4/acrylamide gel electrophoresis revealed that these preparations contained a major protein with MW = 56,000. The mass of prolidase was estimated on gel filtration to be MW = 97,000, for both enzyme preparations. A monoclonal antibody was raised against the liver enzyme and a specific antiserum against the erythrocyte enzyme. The monoclonal antibody (EP-2) recognized prolidase from erythrocytes and liver, in equal proportions. The antiserum also recognized the enzyme from erythrocytes and liver. Immunoprecipitation studies with these antibodies suggested only a single species of prolidase in erythrocytes and liver. Using an immobilized monoclonal antibody (EP-2) as an immunoadsorbent, prolidase was partially purified from crude extracts, and the protein of the partially purified enzyme was identified by immunoblotting using antiserum. A protein band with a MW = 56,000 was demonstrated specifically when crude extracts from the liver and erythrocytes were examined using NaDodSO4/acrylamide gel electrophoresis. The subunit protein was absent in erythrocytes from a patient with prolidase deficiency. We propose that the absence of the subunit is one cause of the prolidase deficiency.

Antibodies, Monoclonal↗
PubMed1985

Effects of dietary zinc deficiency on hepatic ornithine carbamoyltransferase and alcohol dehydrogenase activities in rats.

Hepatic ornithine carbamoyltransferase (OCT) and alcohol dehydrogenase (ADH) activities were measured in six groups of rats: (A) fed a severe zinc-(Zn-) deficient diet (1.98 ppm) for 5 weeks; (B) pair-fed control for group (A); (C) fed a less severe Zn-deficient diet (6.10 ppm) for 5 weeks; (D) pair-fed control for group (C); (E) fed a Zn-supplemented control diet (90.4 ppm) for 5 weeks; and (F) first fed the severe Zn-deficient diet for 5 weeks and then replaced on the Zn-supplemented control diet until a body weight corresponding to the final weight of group (E) was obtained. Hepatic OCT was similar in all these six groups. On the contrary, hepatic ADH was significantly reduced in groups (A) and (C) and in each of the corresponding pair-fed groups, (B) and (D). No differences were found between groups (A) and (B) or between groups (C) and (D). In group (F), ADH activity improved to a level equivalent to that in group (E). The changes in ADH activities were accompanied by changes in the hepatic Zn content. Thus, it is clear that: (1) the hepatic Zn content may not be affected by the amount of Zn intake alone, but by the combination of Zn and food intake; and (2) ADH, and not OCT, reflected the hepatic Zn content.

Alcohol Dehydrogenase↗
PubMedDate not supplied

Conversion of thyroxine into tri-iodothyronine in zinc deficient rat liver.

Hepatic thyroxine (T4) to tri-iodothyronine (T3) conversion was measured in six animal groups: Group A was fed a severe zinc-deficient diet (1.98 ppm) for 5 weeks; group B was a pair-fed control group for group A; group C was fed a less severe zinc-deficient diet (6.10 ppm) for 5 weeks; group D was a pair-fed control group for group C; group E was fed a zinc-supplemented control diet (90.4 ppm) for 5 weeks; and group F was first fed the severe zinc-deficient diet for 5 weeks and then placed on the zinc-supplemented control diet until a body weight corresponding to the final weight of group E was obtained. Serum T3 and T4 levels and T4 to T3 conversion were significantly reduced in group A. A significant positive correlation was observed between T4 to T3 conversion and alcohol dehydrogenase (zinc-metalloenzyme) activity in liver tissue from the six groups. Thus, it appeared that an appropriate amount of zinc might be one of the factors in thyroxine to tri-iodothyronine conversion in liver tissue normally.

Alcohol Dehydrogenase↗
PubMedDate not supplied

[Congenital insensitivity to pain with anhidrosis associated with congenital myasthenic syndrome].

INTRODUCTION: Congenital insensitivity to pain with anhidrosis (CIPA) or hereditary sensory and autonomic neuropathy type IV (HSAN IV) is a rare autosomal recessive disorder featuring recurrent fever episodes, inability to sweat, absent response to noxious stimuli, self mutilating behavior and mental retardation. It has been associated with mutations in the NTRK1 gene, located in 1q21-22 and encoding a high-affinity NGF receptor. CASE REPORT: An 8-year-old boy, the first son of consanguineous parents, presented with hypotonia, episodic hyperpyrexia and global developmental delay since the neonatal period. In addition to these signs, typical of CIPA, he displayed some other not previously described in this disease, such as facial dysmorphism, a severe swallowing disorder and a myogenic EMG pattern, that led to the initial suspicion of a muscle disorder. Molecular genetics studies uncovered a mutation c.C2011T in exon 15 of the NTRK1 gene. Genetic counselling was possible in the following pregnancy of the couple, where the female fetus was found to harbour the mutation in heterozygosity. The subsequent diagnosis of a congenital myasthenic syndrome in this sister led to neurophysiological re-evaluation of the probandus, in whom a myasthenic pattern of muscle activation was also found. CONCLUSIONS: A patient with CIPA and congenital myasthenic syndrome is described. CIPA must be the first diagnostic hypothesis when assessing a patient with insensitivity to pain, anhidrosis and self-mutilation. Given the rather homogeneous presentation of CIPA, the occurrence of atypical myopathic manifestations should raise the suspicion of a concurrent disorder. The present consanguineous kindred illustrates a rare instance of transmission of two mutated alleles giving rise to two unrelated, infrequent neurological syndromes.

Alleles↗