Menkes disease is a severe X-linked recessive disorder caused by a defect in the ATP7A gene, which encodes a membrane copper-transporting ATPase. Deficient activity of the ATP7A protein results in decreased intestinal absorption of copper, low copper level in serum and defective distribution of copper in tissues. The clinical symptoms are caused by decreased activities of copper-dependent enzymes and include neurodegeneration, connective tissue disorders, arterial changes and hair abnormalities. Without therapy, the disease is fatal in early infancy. Rapid diagnosis of Menkes disease and early start of copper therapy is critical for the effectiveness of treatment. We report a molecular biology-based strategy that allows early diagnosis of copper transport defects and implementation of individual therapies before the full development of pathological symptoms. Low serum copper and decreased activity of copperdependent mitochondrial cytochrome c oxidase in isolated platelets found in three patients indicated a possibility of functional defects in copper-transporting proteins, especially in the ATPA7 protein, a copper- transporting P-type ATPase. Rapid mutational screening of the ATP7A gene using high-resolution melting analysis of DNA indicated presence of mutations in the patients. Molecular investigation for mutations in the ATP7A gene revealed three nonsense mutations: c.2170C>T (p.Gln724Ter); c.3745G>T (p.Glu1249Ter); and c.3862C>T (p.Gln1288Ter). The mutation c.3745G>T (p.Glu1249Ter) has not been identified previously. Molecular analysis of the ATOX1 gene as a possible modulating factor of Menkes disease did not reveal presence of pathogenic mutations. Molecular diagnostics allowed early onset of individual therapies, adequate genetic counselling and prenatal diagnosis in the affected families.

• MeSH
• ATPasy transportující měď genetika   metabolismus   MeSH
• biologické modely MeSH
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• měď krev   metabolismus   MeSH
• Menkesova choroba genetika   metabolismus   MeSH
• metalochaperony genetika   metabolismus   MeSH
• mutace genetika   MeSH
• transportní proteiny genetika   metabolismus   MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Production of thyroid hormone is precisely regulated in a negative feed-back mechanism that depends critically on thyroid hormone receptor β (TRβ). This mechanism decreases production of thyrotropin- releasing hormone (TRH) and thyrotropin (TSH) in the hypothalamus and pituitary gland in response to high levels of circulating thyroid hormones (TH). Despite the wealth of accumulated knowledge, it is still not clear how exactly this negative regulation is executed. The syndrome of resistance to thyroid hormone (RTH), in which the levels of TH are not properly sensed, represents naturally occurring situations in which molecular components of this regulation are displayed and may be uncovered. TRβ, which is central to this regulation, is in the majority of RTH cases mutated in a way that preserves some functions of the receptor. Approximately 150 different mutations in TRβ have been identified to date. Here, we hypothesized that additional pathogenic mutations in TRβ are likely to exist in human population and analysed clinical cases with suspected RTH. In keeping with our prediction, analysis of 17 patients from nine families led to identification of four presumed pathogenic mutations of TRβ, including a previously unknown mutation, T273R. This suggests that threonine 273 is likely to be critical for the normal function of TRβ, possibly due to its role in helix 12 mobility and interaction with coactivators, and thus supports the concept that TRβ-dependent trans-activating function is necessary for the inhibition of TRH and TSH expression in response to elevated levels of TH.

• MeSH
• hormon uvolňující thyreotropin metabolismus   MeSH
• hormony štítné žlázy metabolismus   MeSH
• hypothalamus metabolismus   MeSH
• lidé MeSH
• mutace MeSH
• thyreotropin metabolismus   MeSH
• tyreoidální hormony, receptory beta genetika   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Porphyrias are metabolic disorders resulting from mutations in haem biosynthetic pathway genes. Hepatoerythropoietic porphyria (HEP) is a rare type of porphyria caused by the deficiency of the fifth enzyme (uroporphyrinogen decarboxylase, UROD) in this pathway. The defect in the enzymatic activity is due to biallelic mutations in the UROD gene. Currently, 109 UROD mutations are known. The human disease has an early onset, manifesting in infancy or early childhood with red urine, skin photosensitivity in sun-exposed areas, and hypertrichosis. Similar defects and links to photosensitivity and hepatopathy exist in several animal models, including zebrafish and mice. In the present study, we report a new mutation in the UROD gene in Egyptian patients with HEP. We show that the homozygous c.T163A missense mutation leads to a substitution of a conserved phenylalanine (amino acid 55) for isoleucine in the enzyme active site, causing a dramatic decrease in the enzyme activity (19 % of activity of wild-type enzyme). Inspection of the UROD crystal structure shows that Phe-55 contacts the substrate and is located in the loop that connects helices 2 and 3. Phe-55 is strictly conserved in both prokaryotic and eukaryotic UROD. The F55I substitution likely interferes with the enzyme-substrate interaction.

• MeSH
• alely * MeSH
• dítě MeSH
• genetická predispozice k nemoci * MeSH
• hypertrichóza komplikace   MeSH
• jizva komplikace   MeSH
• lidé MeSH
• mladiství MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutace genetika   MeSH
• mutační analýza DNA MeSH
• mutační rychlost MeSH
• porfyrie hepatoerytropoetická komplikace   enzymologie   genetika   MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• rodina MeSH
• rodokmen MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• uroporfyrinogendekarboxylasa chemie   genetika   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Egypt MeSH

The prorenin receptor (ATP6AP2) is a multifunctional transmembrane protein; it is a constituent of proton-translocating V-ATPase, a non-proteolytic activator of renin and an adaptor in the Wnt/β-catenin pathway. Here, we studied vha-20, one of the two prorenin receptor homologues that are identified by sequence similarity in the C. elegans genome. We show that vha-20 (R03E1.2) is prominently expressed in the intestine, in the excretory cell and in amphid neurons, tissues critical for regulation of ion and water management. The expression of vha-20 in the intestine is dependent on NHR-31, a nuclear receptor related to HNF4. VHA-20 is indispensable for normal larval development, acidification of the intestine, and is required for nutrient uptake. Inhibition of vha-20 by RNAi leads to complex deterioration of water and pH gradients at the level of the whole organism including distention of pseudocoelome cavity. This suggests new roles of prorenin receptor in the regulation of body ion and water management and in acidification of intestinal lumen in nematodes.

• MeSH
• Caenorhabditis elegans fyziologie   MeSH
• homeostáza fyziologie   MeSH
• koncentrace vodíkových iontů MeSH
• larva fyziologie   MeSH
• polymerázová řetězová reakce MeSH
• proteiny Caenorhabditis elegans genetika   metabolismus   MeSH
• stanovení celkové genové exprese MeSH
• transkriptom MeSH
• vakuolární protonové ATPasy genetika   metabolismus   MeSH
• voda metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Perilipins are lipid droplet surface proteins that contribute to fat metabolism by controlling the access of lipids to lipolytic enzymes. Perilipins have been identified in organisms as diverse as metazoa, fungi, and amoebas but strikingly not in nematodes. Here we identify the protein encoded by the W01A8.1 gene in Caenorhabditis elegans as the closest homologue and likely orthologue of metazoan perilipin. We demonstrate that nematode W01A8.1 is a cytoplasmic protein residing on lipid droplets similarly as human perilipins 1 and 2. Downregulation or elimination of W01A8.1 affects the appearance of lipid droplets resulting in the formation of large lipid droplets localized around the dividing nucleus during the early zygotic divisions. Visualization of lipid containing structures by CARS microscopy in vivo showed that lipid-containing structures become gradually enlarged during oogenesis and relocate during the first zygotic division around the dividing nucleus. In mutant embryos, the lipid containing structures show defective intracellular distribution in subsequent embryonic divisions and become gradually smaller during further development. In contrast to embryos, lipid-containing structures in enterocytes and in epidermal cells of adult animals are smaller in mutants than in wild type animals. Our results demonstrate the existence of a perilipin-related regulation of fat metabolism in nematodes and provide new possibilities for functional studies of lipid metabolism.

• Publikační typ
• časopisecké články MeSH

NCoR and SMRT are two paralogous vertebrate proteins that function as corepressors with unliganded nuclear receptors. Although C. elegans has a large number of nuclear receptors, orthologues of the corepressors NCoR and SMRT have not unambiguously been identified in Drosophila or C. elegans. Here, we identify GEI-8 as the closest homologue of NCoR and SMRT in C. elegans and demonstrate that GEI-8 is expressed as at least two isoforms throughout development in multiple tissues, including neurons, muscle and intestinal cells. We demonstrate that a homozygous deletion within the gei-8 coding region, which is predicted to encode a truncated protein lacking the predicted NR domain, results in severe mutant phenotypes with developmental defects, slow movement and growth, arrested gonadogenesis and defects in cholinergic neurotransmission. Whole genome expression analysis by microarrays identified sets of de-regulated genes consistent with both the observed mutant phenotypes and a role of GEI-8 in regulating transcription. Interestingly, the upregulated transcripts included a predicted mitochondrial sulfide:quinine reductase encoded by Y9C9A.16. This locus also contains non-coding, 21-U RNAs of the piRNA class. Inhibition of the expression of the region coding for 21-U RNAs leads to irregular gonadogenesis in the homozygous gei-8 mutants, but not in an otherwise wild-type background, suggesting that GEI-8 may function in concert with the 21-U RNAs to regulate gonadogenesis. Our results confirm that GEI-8 is the orthologue of the vertebrate NCoR/SMRT corepressors and demonstrate important roles for this putative transcriptional corepressor in development and neuronal function.

• MeSH
• Caenorhabditis elegans genetika   fyziologie   MeSH
• delece genu MeSH
• gonády růst a vývoj   MeSH
• korepresor 1 jaderného receptoru genetika   MeSH
• korepresor 2 jaderného receptoru genetika   MeSH
• korepresorové proteiny genetika   metabolismus   MeSH
• mikročipová analýza MeSH
• molekulární sekvence - údaje MeSH
• neurony fyziologie   MeSH
• proteiny Caenorhabditis elegans genetika   metabolismus   MeSH
• receptory cytoplazmatické a nukleární metabolismus   MeSH
• regulace genové exprese genetika   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• sekvenční seřazení MeSH
• stanovení celkové genové exprese MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Nuclear receptors (NRs), or nuclear hormone receptors (NHRs), are transcription factors that regulate development and metabolism of most if not all animal species. Their regulatory networks include conserved mechanisms that are shared in-between species as well as mechanisms that are restricted to certain phyla or even species. In search for conserved members of the NHR family in Schmidtea mediterranea, we identified a molecular signature of a class of NRs, NR2E1, in the S. mediterranea genome and cloned its complete cDNA coding sequence. The derived amino acid sequence shows a high degree of conservation of both DNA-binding domain and ligand- binding domain and a remarkably high homology to vertebrate NR2E1 and C. elegans NHR-67. Quantitative PCR detected approximately ten-fold higher expression of Smed-tlx-1 in the proximal part of the head compared to the tail region. The expression of Smed-tlx-1 is higher during fed state than during fasting. Smed-tlx-1 down-regulation by RNA interference affects the ability of the animals to maintain body plan and induces defects of brain, eyes and body shape during fasting and re-growing cycles. These results suggest that SMED-TLX-1 is critical for tissue and body plan maintenance in planaria.

• MeSH
• fylogeneze MeSH
• klonování DNA MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• omezení příjmu potravy fyziologie   MeSH
• orgánová specificita genetika   MeSH
• proteiny červů genetika   chemie   metabolismus   MeSH
• receptory cytoplazmatické a nukleární genetika   chemie   metabolismus   MeSH
• regulace genové exprese MeSH
• RNA interference MeSH
• rozvržení tělního plánu genetika   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• stravovací zvyklosti fyziologie   MeSH
• Turbellaria embryologie   fyziologie   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

Mutations in the hydroxymethylbilane synthase (HMBS) gene are responsible for the inherited disorder of acute intermittent porphyria (AIP). AIP is diagnosed on the basis of characteristic clinical symptoms, elevated levels of urinary porphyrin precursors aminolevulinic acid (ALA) and porphobilinogen (PBG) and a decreased erythrocytic HMBS activity, although an identifiable HMBS mutation provides the ultimate proof for AIP. Six Israeli AIP families underwent biochemical and mutation analysis in order to establish an AIP diagnosis. Variability with respect to the ALA/PBG levels and HBMS activity was found among the index patients. Indeed, each family carried a unique mutation in the HMBS gene. A novel missense c.95G>C (p.R32P) was shown to be a de novo mutation in one family, along with five known mutations p.T59I, p.D178N, p.V215M, c.730_731delCT and c.982_983delCA identified in the rest of the families. Both R32P and D178N were expressed in a prokaryotic system. Recombinant p.R32P was enzymatically inactive as demonstrated by a <1% residual activity, whereas p.D178N possessed 81% of the activity of the wild type enzyme. However, the p.D178N mutant did display a shift in optimal pH and was thermo labile compared to the wild type. Among the four missense mutations, p.R32P and p.V215M had not only harmful effects on the enzyme in vitro but also were associated with high levels of ALA/PBG in patients. On the other hand, the in vitro effect of both p.T59I and p.D178N, and the impact of these mutations on the enzyme structure and function as interpreted by the 3-D structure of the Escherichia coli enzyme, were weaker than that of p.R32P and p.V215M. Concomitantly, patients carrying the p.T59I or p.D178N had normal or borderline increases in ALA/PBG concentrations although they presented characteristic clinical symptoms. These findings provided further insights into the causal relationship between HMBS mutations and AIP.

• MeSH
• akutní intermitentní porfyrie etnologie   genetika   MeSH
• bodová mutace MeSH
• dospělí MeSH
• exony genetika   MeSH
• financování organizované MeSH
• hydroxymethylbilansynthasa genetika   chemie   MeSH
• konformace proteinů MeSH
• kyselina aminolevulová moč   MeSH
• lidé středního věku MeSH
• lidé MeSH
• missense mutace MeSH
• mladiství MeSH
• mladý dospělý MeSH
• molekulární modely MeSH
• mutační analýza DNA MeSH
• porfobilinogen metabolismus   MeSH
• proteiny z Escherichia coli chemie   MeSH
• sekvenční delece MeSH
• senioři nad 80 let MeSH
• stabilita proteinů MeSH
• substituce aminokyselin MeSH
• vztahy mezi strukturou a aktivitou MeSH
• židé genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• ženské pohlaví MeSH
• Geografické názvy
• Evropa MeSH
• Indie MeSH
• Izrael MeSH

Acute intermittent porphyria is an autosomal dominantly inherited disorder, classified as acute hepatic porphyria, caused by a deficiency of hydroxymethylbilane synthase (EC 2.5.1.61, EC 4.3.1.8, also known as porphobilinogen deaminase, uroporphyrinogen I synthase), the third enzyme in heme biosynthesis. Clinical features include autonomous, central, motor or sensory symptoms, but the most common clinical presentation is abdominal pain caused by neurovisceral crises. A diagnosis of acute intermittent porphyria is crucial to prevent life-threatening acute attacks. Detection of DNA variations by molecular techniques allows a diagnosis of acute intermittent porphyria in situations where the measurement of porphyrins and precursors in urine and faeces and erythrocyte hydroxymethylbilane synthase activity is inconclusive. In the present study, we identified gene defects in six Czech patients with acute intermittent porphyria, as diagnosed based on biochemical findings, and members of their families to confirm the diagnosis at the molecular level and/or to provide genetic counselling. Molecular analyses of the hydroxymethylbilane synthase gene revealed seven mutations. Four were previously reported: c.76C>T, c.77G>A, c.518G>A, c.771 + 1G>T (p.Arg26Cys, p.Arg26His, p.Arg173Gln). Three were novel mutations: c.610C>A, c.675delA, c.750A>T (p.Gln204Lys, p.Ala226ProfsX28, p.Glu250Asp). Of particular interest, one patient had two mutations (c.518G>A; c.610C>A), both located in exon 10 of the same allele. To establish the effects of the mutations on enzyme function, biochemical characterization of the expressed normal recombinant and mutated proteins was performed. Prokaryotic expression of the mutant alleles of the hydroxymethylbilane synthase gene revealed that, with the exception of the p.Gln204Lys mutation, all mutations resulted in little, if any, enzymatic activity. Moreover, the 3D structure of the Escherichia coli and human protein was used to interpret structure-function relationships for the mutations in the human isoform.

• MeSH
• akutní intermitentní porfyrie diagnóza   enzymologie   genetika   MeSH
• Escherichia coli enzymologie   genetika   metabolismus   MeSH
• financování organizované MeSH
• hydroxymethylbilansynthasa genetika   metabolismus   MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• mutační analýza DNA MeSH
• sekvence nukleotidů MeSH
• substituce aminokyselin MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH

Based on Internet search, we were contacted by a 50-year-old man suffering from severe abdominal pain. Acute hepatic porphyria was considered from positive Watson-Schwartz test. He, not being a health professional, searched for centres with ability to do molecular diagnosis and for information about therapeutic possibilities. He asked his physician for haem-arginate (Normosang, Orphan Europe, Paris) treatment, arranged sending his blood to our laboratory and mediated genetic counselling for him and his family. Molecular analyses of the PBGD gene revealed a novel mutation in exon 15, the 973insG. Subsequently, genetic analysis was performed in 18 members of the proband's extensive family. In 12 members of the family, the same mutation was found. The mutation, which consisted of one nucleotide insertion, resulted in addition of four different amino acids leading to a protein that is prematurely truncated by the stop codon. The effect of this mutation was investigated by expression of the wildtype and mutated PBGD in a prokaryotic expression system. The mutation resulted in instability of the protein and loss of enzymatic function. The increasing access to a number of disease- and symptom-oriented web pages presents a new and unusual venue for gaining knowledge and enabling self-diagnosis and self-help. It is, therefore, important that diseaseoriented Internet pages for public use should be designed with clarity and accurate current knowledge based background.

• MeSH
• akutní intermitentní porfyrie diagnóza   genetika   krev   MeSH
• diagnostické techniky molekulární metody   využití   MeSH
• dospělí MeSH
• elektroforéza metody   využití   MeSH
• financování organizované využití   MeSH
• mutace genetika   MeSH
• mutační analýza DNA metody   využití   MeSH
• muži MeSH
• polymerázová řetězová reakce metody   využití   MeSH
• porfobilinogen izolace a purifikace   krev   MeSH
• Check Tag
• dospělí MeSH
• Publikační typ
• kazuistiky MeSH