8 research outputs found

    Table_1_Clinical, neurophysiological evaluation and genetic features of axonal Charcot–Marie–Tooth disease in a Chinese family.doc

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    Charcot–Marie–Tooth disease (CMT) is a group of inherited peripheral neuropathies related to variants in the mitochondrial transfer RNA (mt-tRNAval) gene. Here, we report a Chinese family harboring the m.1661A>G variant in the mt-tRNAval gene. Clinical evaluation, neuroelectrodiagnostic testing, and nerve biopsy were performed on four affected family members. Weakness, spasms, and pain in the limbs (especially in the lower limbs) were the main complaints of the proband. Physical examination revealed atrophy and weakness in the distal limbs, increased muscle tone, and hyperreflexia in four limbs. Neuroelectrodiagnostic tests and nerve biopsy supported an axonal polyneuropathy. This study furthers the understanding of phenotype diversity caused by variants in the mt-tRNAval gene in CMT.</p

    The flow chart to illustrate the strategy for this study.

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    <p>The sketch depicted the overall framework for this study. Genetic engineering of native NHase was carried out in two tiers. Firstly, the codons were optimized. Secondly, the genetic elements, RBS and spacer, were modified and optimized. Resorting to these modifications, the optimized NHases were over-produced in recombinant E. coli. Accordingly, the enzymatic properties were characterized and a transformation pipeline by whole-cell catalysis was primarily established.</p

    Determination of the molecular masses of purified proteins.

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    <p>Determination of the molecular masses and structures of NhlBA and NhhBA by Superdex 200 10/300 GL pg column. The marker proteins were used for gel filtration: (i) glutamate dehydrogenase (yeast) (290 kDa), (ii) lactate dehydrogenase (pig heart) (142 kDa), (iii) enolase (yeast) (67 kDa), (iv) myokinase (yeast) (32 kDa), and (v) cytochrome c (horse heart) (12.4 kDa).</p

    Gene organization for the construction of a set of plasmids.

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    <p>(A) Organization of the expression cassette for L-NHase. <i>nhlBAE</i>, wild-type L-NHase gene; <i>nhlBrbsAE</i>, the intervening sequences shortened gene with one enhanced RBS ahead of <i>nhlA; nhlBrbsArbsE</i>, the intervening sequences shortened gene with two enhanced RBSes ahead of <i>nhlA</i> and <i>nhlE</i>. (B) Gene organization of the constructed genes used for H-NHase expression.</p

    Production of NHase in recombinant <i>E</i>. <i>coli</i> and in native <i>R</i>. <i>rhodochrous</i> J1.

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    <p>Cell growth and NHase activity of (A) the genetically engineered <i>E</i>. <i>coli</i> for H-NHase, (B) L-NHase, and (C) <i>R</i>. <i>rhodochrous</i> J1. (D) Comparison of nicotinamide-tolerance of NHases from the cells of <i>R</i>. <i>rhodochrous</i> J1 (column 1) and the H-NHase, and L-NHase from the genetically engineered <i>E</i>. <i>coli</i> (column 2 and 3). The reduction ratio (the proportion of the reduced 3-cyanopyridine amount in the reaction with and without 0.5 M nicotinamide) was calculated.</p

    SDS-PAGE analysis of the expression of L-NHases and H-NHase.

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    <p>(A) The expression of L-NHases. M: Marker; 1: control, BL21(DE3)/pET24a; 2: BL21(DE3)/pET24a-<i>nhlBAE</i>; 3: BL21(DE3)/pET24a-<i>nhlBrbsAE</i>; 4: BL21(DE3)/pET24a-<i>nhlBrbsArbsE</i>. (B) The expression of H-NHase. M: Marker; 1: control, BL21(DE3)/pET24a; 2: BL21(DE3)/pET24a-<i>nhhBAG</i>; 3: BL21(DE3)/pET24a-<i>nhhBrbsArbsG</i>. (C) The purified L-NHases and H-NHase. M: Marker; 1, L-NHase encoded by <i>nhlBrbsAE</i>; 2, L-NHase encoded by <i>nhlBrbsArbsE</i>; 3, H-NHase encoded by <i>nhhBrbsArbsG</i>.</p

    Thermo-stability, substrate and product tolerance of L-NHase and H-NHase.

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    <p>(A) Thermo-stability of NHases; (B) Substrate tolerance of enzymes; (C) Product tolerance of enzymes.</p
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