The highly reduced mitochondria (mitosomes) of Giardia intestinalis are recently discovered organelles for which, it was suggested, iron-sulfur cluster assembly was their only conserved function. However, only an incomplete set of the components required for FeS cluster biogenesis was localized to the mitosomes. Via proteomic analysis of a mitosome-rich cellular fraction together with immunofluorescence microscopy, we identified a novel mitosomal protein homologous to monothiol glutaredoxins containing a CGFS motif at the active site. Sequence analysis revealed the presence of long nonconserved N-terminal extension of 77 amino acids, which was absent in the mature protein. Expression of the complete and N-terminally truncated forms of the glutaredoxin indicated that the extension is involved in glutaredoxin import into mitosomes. However, the mechanism of preprotein processing is unclear, as the mitosomal processing peptidase is unable to cleave this type of extension. The recombinant mature protein was shown to form a homodimeric structure, which binds a labile FeS cluster. The cluster is stabilized by glutathione and dithiothreitol. Phylogenetic analysis showed that giardial glutaredoxin is related to the mitochondrial monothiol glutaredoxins involved in FeS cluster assembly. The identification of a mitochondrial-type monothiol glutaredoxin in the mitosomes of G. intestinalis thus completes the mitosomal FeS cluster biosynthetic pathway and provides further evidence for the mitochondrial origin of these organelles.

• MeSH
• Amino Acid Motifs MeSH
• Microscopy, Fluorescence MeSH
• Phylogeny MeSH
• Giardia lamblia metabolism   MeSH
• Glutaredoxins chemistry   MeSH
• Mitochondria metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Alignment MeSH
• Binding Sites MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

OBJECTIVES: Non-syndromic hearing loss (NSHL) is a genetically heterogeneous disorder with mostly autosomal recessive inheritance. So far 40 genes and the same amount of loci with as yet unknown genes were described with autosomal recessive NSHL. PATIENTS AND METHODS: A consanguineous Czech family with a child with NSHL was genotyped using SNP array and homozygous regions were compared with previously reported DFNB loci. RESULTS: GRXCR1 and ESRRB genes associated with autosomal recessive NSHL were located in two of the eight homozygous regions detected by SNP array genotyping. Mutation p.R291L in a homozygous state was found in the deaf child, the parents were heterozygous. The entire coding region of the ESRRB gene was sequenced in additional 39 patients of Czech origin with early NSHL and only two variants, p.V413I and p.P386S, were found in homozygous state, but are considered to be polymorphisms. CONCLUSION: Homozygosity mapping is a powerful method for identification of genes in heterogeneous recessive diseases. This is the first report of DFNB35 mutations in the Czech Republic and it seems to be a rare cause of NSHL. Additional mutations in ESRRB gene were reported in Pakistan, Tunisia and Turkey.

• MeSH
• Genetic Carrier Screening MeSH
• Exons MeSH
• Genotype MeSH
• Glutaredoxins genetics   MeSH
• Deafness genetics   MeSH
• Homozygote MeSH
• Polymorphism, Single Nucleotide MeSH
• Humans MeSH
• Mutation * MeSH
• Consanguinity MeSH
• Receptors, Estrogen genetics   MeSH
• Pedigree MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic MeSH

An arsenic (ars) four-gene operon, containing genes encoding a putative membrane permease (ArsP), a transcriptional repressor (ArsR), an arsenate reductase (ArsC) and an arsenical-resistance membrane transporter (Acr3) was first identified in urease-positive thermophilic Campylobacter (UPTC) isolate, CF89-12. UPTC CF89-12 and some other Campylobacter lari isolates contained their ars four-genes, similarly, differing from that in the reference C. lari RM2100 strain. Two putative promoters and a putative terminator were identified for the operon in UPTC CF89-12. In vivo transcription of the operon was confirmed in the UPTC cells. PCR experiments using two primer pairs designed in silico to amplify two arsR and arsC-acr3 segments, respectively, generated two amplicons, approximately 200 and 350 base pairs, with all 31 of 31 and 19 of 31 C. lari isolates (n = 17 for UPTC; n = 14 for UN C. lari), respectively. An inverted repeat forming a dyad structure, a potential binding site for a transcriptional repressor, was identified in the promoter region. Within the deduced 61 amino acids sequence of the putative arsR open reading frame from the UPTC CF89-12, a metal binding box and a DNA-binding helix-turn-helix motif were identified. The UPTC CF89-12 and some other UPTC isolates isolated from natural environment were resistant to arsenate.

• MeSH
• Arsenic * MeSH
• Arsenate Reductases genetics   MeSH
• Genes, Bacterial * MeSH
• RNA, Bacterial genetics   MeSH
• Campylobacter lari genetics   isolation & purification   MeSH
• DNA, Bacterial genetics   MeSH
• DNA Primers MeSH
• Genetic Loci MeSH
• Nucleic Acid Conformation MeSH
• Molecular Sequence Data MeSH
• Helix-Turn-Helix Motifs genetics   MeSH
• Operon genetics   MeSH
• Open Reading Frames MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Amino Acid Sequence MeSH
• Sequence Analysis, DNA MeSH
• Sequence Alignment MeSH
• Urease genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND/AIMS: Congenital sideroblastic anemias (SA) are characterized by the presence of ringed sideroblasts in the bone marrow. The most common form is X-linked SA, which results from mutations in erythroid-specific δ-aminolevulinate synthase (ALAS2), the first enzyme in heme biosynthesis. In addition, autosomal recessive mutations in the erythroid-specific mitochondrial transporter SLC25A38 and glutaredoxin 5 (GLRX5) have recently been identified in SA patients with isolated erythroid phenotype. MATERIALS AND METHODS: We studied 5 young males with congenital SA from the Czech Republic. Mutation analysis was performed on the complete coding regions of 3 candidate genes (ALAS2, SLC25A38 and GLRX5), and the enzyme activity of ALAS2 was measured by a continuous spectrophotometric assay. RESULTS: We found the previously published R452H and R452C ALAS2 mutations in 3 patients. A novel K156E substitution in ALAS2 was discovered in 1 pyridoxine-responsive patient. The functional study showed that this substitution severely decreases ALAS2 enzyme activity. In 1 pyridoxine-refractory patient, no mutations were detected in ALAS2, SLC25A38 or GLRX5. CONCLUSION: Our report extends the list of known ALAS2 mutations, with the addition of a novel K156E substitution that is responsive to pyridoxine treatment and contributes to the general knowledge of congenital SA cases characterized worldwide.

• MeSH
• 5-Aminolevulinate Synthetase genetics   metabolism   MeSH
• Adult MeSH
• Humans MeSH
• Mutation MeSH
• Pyridoxine therapeutic use   MeSH
• Base Sequence MeSH
• Sequence Alignment MeSH
• Anemia, Sideroblastic drug therapy   enzymology   genetics   MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Isocitrate dehydrogenase 2 (IDH2) is located in the mitochondrial matrix. IDH2 acts in the forward Krebs cycle as an NADP(+)-consuming enzyme, providing NADPH for maintenance of the reduced glutathione and peroxiredoxin systems and for self-maintenance by reactivation of cystine-inactivated IDH2 by glutaredoxin 2. In highly respiring cells, the resulting NAD(+) accumulation then induces sirtuin-3-mediated activating IDH2 deacetylation, thus increasing its protective function. Reductive carboxylation of 2-oxoglutarate by IDH2 (in the reverse Krebs cycle direction), which consumes NADPH, may follow glutaminolysis of glutamine to 2-oxoglutarate in cancer cells. When the reverse aconitase reaction and citrate efflux are added, this overall "anoxic" glutaminolysis mode may help highly malignant tumors survive aglycemia during hypoxia. Intermittent glycolysis would hypothetically be required to provide ATP. When oxidative phosphorylation is dormant, this mode causes substantial oxidative stress. Arg172 mutants of human IDH2-frequently found with similar mutants of cytosolic IDH1 in grade 2 and 3 gliomas, secondary glioblastomas, and acute myeloid leukemia-catalyze reductive carboxylation of 2-oxoglutarate and reduction to D-2-hydroxyglutarate, which strengthens the neoplastic phenotype by competitive inhibition of histone demethylation and 5-methylcytosine hydroxylation, leading to genome-wide histone and DNA methylation alternations. D-2-hydroxyglutarate also interferes with proline hydroxylation and thus may stabilize hypoxia-induced factor α.

• Publication type
• Journal Article MeSH

Diapause is a state of arrested development during which insects cope with many external and internal stressful factors. European corn borer, Ostrinia nubilalis, overwinters as a fifth instar freeze-tolerant diapausing larva. In order to explore diapause-linked stress tolerance processes, the expression of selected genes coding for stress-related proteins-glutathione S-transferase (Gst), thioredoxin (Trx), glutaredoxin (Grx), ferritin (Fer), metallothionein (Mtn), and heat shock proteins Hsp90, Hsc70, Hsp20.4, and Hsp20.1-was assessed in the fat body of diapause-destined, warm (22 °C) and cold (5 °C) acclimated diapausing larvae using the quantitative real-time PCR. Gene expression was normalised to mRNA transcripts for Actin and Rps03, and relative expression was calculated using non-diapausing larvae as a control group. During the initiation phase of diapause, the abundance of mRNA transcripts of Grx, Hsp90, Hsc70, and Hsp20.1 was significantly upregulated, Trx, Fer, Mtn, and Hsp20.1 were unchanged, while only Gst was clearly downregulated in comparison to non-diapause control. Later, in the early phase of diapause, the expression of most genes (except Trx and Hsp20.1) was upregulated in warm-acclimated larvae, while only Trx and Hsp90 were upregulated in cold-acclimated larvae. Furthermore, the relative expression of all genes (except Trx) increased gradually throughout the diapause in cold-acclimated larvae. This result indicates that the half-life of mRNAs is prolonged during diapause at low temperature, which may lead to a gradual accumulation of mRNA transcripts. Our results show that both diapause programming and temperatures affect the expression of stress-related genes in Ostrinia nubilalis.

• MeSH
• Diapause, Insect * MeSH
• Gene Expression MeSH
• Stress, Physiological MeSH
• Insect Proteins genetics   MeSH
• Larva genetics   metabolism   MeSH
• Lepidoptera classification   genetics   growth & development   metabolism   MeSH
• RNA, Messenger metabolism   MeSH
• Cold Shock Proteins and Peptides genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH