An arachidonic acid generation/export system involved in the regulation of cholesterol transport in mitochondria of steroidogenic cells

Recent studies demonstrated the importance of the mitochondrial ATP in the regulation of a novel long-chain fatty acid generation/export system in mitochondria of diabetic rat heart. In steroidogenic systems, mitochondrial ATP and intramitochondrial arachidonic acid (AA) generation are important for steroidogenesis. Here, we report that mitochondrial ATP is necessary for the generation and export of AA, steroid production and steroidogenic acute regulatory protein induction supported by cyclic 3'-5'-adenosine monophosphate in steroidogenic cells. These results demonstrate that ATP depletion affects AA export and provide new evidence of the existence of the fatty acid generation and export system involved in mitochondrial cholesterol transport.     

<Emphasis Type="Italic">Drosophila</Emphasis> as a platform to predict the pathogenicity of novel aminoacyl-tRNA synthetase mutations in CMT

Charcot-Marie-Tooth disease (CMT) is the major form of inherited peripheral neuropathy in humans. CMT is clinically and genetically heterogeneous and four aminoacyl-tRNA synthetases have been implicated in disease etiology. Mutations in the YARS gene encoding a tyrosyl-tRNA synthetase (TyrRS) lead to Dominant Intermediate CMT type C (DI-CMTC). Three dominant YARS mutations were so far associated with DI-CMTC. To further expand the spectrum of CMT causing genetic defects in this tRNA synthetase, we performed DNA sequencing of YARS coding regions in a cohort of 181 patients with various types of peripheral neuropathy. We identified a novel K265N substitution that in contrast to all previously described mutations is located at the anticodon recognition domain of the enzyme. Further genetic analysis revealed that this variant represents a benign substitution. Using our recently developed DI-CMTC Drosophila model, we tested in vivo the pathogenicity of this new YARS variant. We demonstrated that the developmental and behavioral defects induced by all DI-CMTC causing mutations were not present upon ubiquitous or panneuronal TyrRS K265N expression. Thus, in line with our genetic studies, functional analysis confirmed that the K265N substitution does not induce toxicity signs in Drosophila. The consistency observed throughout this work underscores the robustness of our DI-CMTC animal model and identifies Drosophila as a valid read-out platform to ascertain the pathogenicity of novel mutations to be identified in the future.     

Molecular characterization of Corynebacterium pseudotuberculosis isolated from goats using ERIC-PCR

Corynebacterium pseudotuberculosis, the infectious agent of caseous lymphadenitis (CLA), is responsible for substantial economic losses in goat and sheep production. Molecular characterization of C. pseudotuberculosis isolates by enterobacterial repetitive intergenic consensus (ERIC)-PCR has shown promising results in genotyping strains isolated from sheep with CLA. We evaluated the genetic diversity of C. pseudotuberculosis isolates collected from the Sert?o region of the Pernambuco (PE) State, Brazil, and investigated the potential of ERIC-PCR as a tool for the molecular typing of strains of C. pseudotuberculosis isolated from goats. Thirty-two C. pseudotuberculosis strains isolated from goats in the municipalities of Floresta and Ibimirim, PE, C. pseudotuberculosis type strain ATCC 19410, the 1002 vaccine strain, and a field isolate of Rhodococcus equi were fingerprinted using the primers ERIC-1R and ERIC-2 and the primer pair ERIC- 1R+ERIC-2. Using 100% similarity as the cutoff, 8, 10, and 7 genotypes were obtained with ERIC-1-PCR, ERIC-2-PCR, and ERIC-1+2-PCR, respectively. The Hunter-Gaston discriminatory index calculated for the ERIC-1-PCR was 0.75. The index for the ERIC-2-PCR was 0.88, and the index for the ERIC-1+2-PCR was 0.79. Among goat isolates of C. pseudotuberculosis, three, two and four genotypes (found by ERIC-1-PCR, ERIC-2-PCR, and ERIC-1+2-PCR, respectively) had been previously described among sheep isolates from Minas Gerais State, Brazil. These results showed that ERIC-PCR has good discriminatory power and typeability, making it a useful tool for discrimination among C. pseudotuberculosis isolates from goats.     

Sequence verification of synthetic DNA by assembly of sequencing reads

Gene synthesis attempts to assemble user-defined DNA sequences with base-level precision. Verifying the sequences of construction intermediates and the final product of a gene synthesis project is a critical part of the workflow, yet one that has received the least attention. Sequence validation is equally important for other kinds of curated clone collections. Ensuring that the physical sequence of a clone matches its published sequence is a common quality control step performed at least once over the course of a research project. GenoREAD is a web-based application that breaks the sequence verification process into two steps: the assembly of sequencing reads and the alignment of the resulting contig with a reference sequence. GenoREAD can determine if a clone matches its reference sequence. Its sophisticated reporting features help identify and troubleshoot problems that arise during the sequence verification process. GenoREAD has been experimentally validated on thousands of gene-sized constructs from an ORFeome project, and on longer sequences including whole plasmids and synthetic chromosomes. Comparing GenoREAD results with those from manual analysis of the sequencing data demonstrates that GenoREAD tends to be conservative in its diagnostic. GenoREAD is available at www.genoread.org.     

Decline of neural tube defects cases after a folic acid campaign in Nuevo León,México

BACKGROUND: Nuevo León is a state in northeastern Mexico, near the border of Texas. Mean mortality rate from 1996-98 due to anencephaly cases was 0.6/1,000. In 1999 a surveillance program for the registry and prevention of neural tube defects (NTD) cases was initiated. METHODS: Cases were obtained from hospitals and OB-GYN clinics by immediate notification, death certificates, or fetal death registries. Only isolated cases of NTD were included. In August 1999 a folic acid campaign was initiated with the free distribution of the vitamin to low-income women with a recommendation to take a 5.0-mg pill once a week. Number of cases and rates from 1999 to 2001 were compared (chi(2) test). RESULTS: After 2 years there has been a significant reduction in the number of cases and rates. In 1999 there were 95 NTD cases and in the years 2000 and 2001 there were only 59 and 55 respectively (P < 0.001). NTD rate decreased from 1.04/1,000 in 1999 to 0.58/1,000 in 2001. Anencephaly and spina bifida rates decreased from 0.55/1,000 to 0.29/1,000 and from 0.47/1,000 to 0.22/1,000 respectively, from 1999-2001. Decrease of female cases was higher than male cases for both phenotypes. CONCLUSION: After 2 years there was a 50% decrease in the incidence of anencephaly and spina bifida cases with a significant reduction of infant mortality and disability. These results encourage us to propose the use of a single tablet of 5.0-mg of folic acid per week as an alternative to supplementation on a daily basis.     

3-Hydroxy-3-methylglutaric and 3-methylglutaric acids impair redox status and energy production and transfer in rat heart: relevance for the pathophysiology of cardiac dysfunction in 3-hydroxy-3-methylglutaryl-coenzyme A lyase deficiency

3-Hydroxy-3-methylglutaryl-coenzyme A lyase (HL) deficiency is characterized by tissue accumulation of 3-hydroxy-3-methylglutaric (HMG), and 3-methylglutaric (MGA) acids. Affected patients present cardiomyopathy, whose pathomechanisms are not yet established. We investigated the effects of HMG and MGA on energy and redox homeostasis in rat heart using in vivo and in vitro models. In vivo experiments showed that intraperitoneal administration of HMG and MGA decreased the activities of the respiratory chain complex II and creatine kinase (CK), whereas HMG also decreased the activity of complex II–III. Furthermore, HMG and MGA injection increased reactive species production and carbonyl formation, and decreased glutathione concentrations. Regarding the enzymatic antioxidant defenses, HMG and MGA increased glutathione peroxidase (GPx) and glutathione reductase (GR) activities, while only MGA diminished the activities of superoxide dismutase (SOD) and catalase, as well as the protein content of SOD1. Pre-treatment with melatonin (MEL) prevented MGA-induced decrease of CK activity and SOD1 levels. In vitro results demonstrated that HMG and MGA increased reactive species formation, induced lipid peroxidation and decreased glutathione. We also verified that reactive species overproduction and glutathione decrease provoked by HMG and MGA were abrogated by MEL and lipoic acid (LA), while only MEL prevented HMG- and MGA-induced lipoperoxidation. Allopurinol (ALP) also prevented reactive species overproduction caused by both metabolites. Our data provide solid evidence that bioenergetics dysfunction and oxidative stress are induced by HMG and MGA in heart, which may explain the cardiac dysfunction observed in HL deficiency, and also suggest that antioxidant supplementation could be considered as adjuvant therapy for affected patients.     

The plant glycosyltransferase clone collection for functional genomics

The glycosyltransferases (GTs) are an important and functionally diverse family of enzymes involved in glycan and glycoside biosynthesis. Plants have evolved large families of GTs which undertake the array of glycosylation reactions that occur during plant development and growth. Based on the Carbohydrate‐Active enZymes (CAZy) database, the genome of the reference plant Arabidopsis thaliana codes for over 450 GTs, while the rice genome (Oryza sativa) contains over 600 members. Collectively, GTs from these reference plants can be classified into over 40 distinct GT families. Although these enzymes are involved in many important plant specific processes such as cell‐wall and secondary metabolite biosynthesis, few have been functionally characterized. We have sought to develop a plant GTs clone resource that will enable functional genomic approaches to be undertaken by the plant research community. In total, 403 (88%) of CAZy defined Arabidopsis GTs have been cloned, while 96 (15%) of the GTs coded by rice have been cloned. The collection resulted in the update of a number of Arabidopsis GT gene models. The clones represent full‐length coding sequences without termination codons and are Gateway^® compatible. To demonstrate the utility of this JBEI GT Collection, a set of efficient particle bombardment plasmids (pBullet) was also constructed with markers for the endomembrane. The utility of the pBullet collection was demonstrated by localizing all members of the Arabidopsis GT14 family to the Golgi apparatus or the endoplasmic reticulum (ER). Updates to these resources are available at the JBEI GT Collection website http://www.addgene.org/ .     

Expression of nonstructural rotavirus protein NSP4 mimics Ca2+ homeostasis changes induced by rotavirus infection in cultured cells

Rotavirus infection modifies Ca²⁺ homeostasis, provoking an increase in Ca²⁺ permeation, the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyto), and total Ca²⁺ pools and a decrease in Ca²⁺ response to agonists. A glycosylated viral protein(s), NSP4 and/or VP7, may be responsible for these effects. HT29 or Cos-7 cells were infected by the SA11 clone 28 strain, in which VP7 is not glycosylated, or transiently transfected with plasmids coding for NSP4-enhanced green fluorescent protein (EGFP) or NSP4. The permeability of the plasma membrane to Ca²⁺ and the amount of Ca²⁺ sequestered in the endoplasmic reticulum released by carbachol or ATP were measured in fura-2-loaded cells at the single-cell level under a fluorescence microscope or in cell suspensions in a fluorimeter. Total cell Ca²⁺ pools were evaluated as ⁴⁵Ca²⁺ uptake. Infection with SA11 clone 28 induced an increase in Ca²⁺ permeability and ⁴⁵Ca²⁺ uptake similar to that found with the normally glycosylated SA11 strain. These effects were inhibited by tunicamycin, indicating that inhibition of glycosylation of a viral protein other than VP7 affects the changes of Ca²⁺ homeostasis induced by infection. Expression of NSP4-EGFP or NSP4 in transfected cells induced the same changes observed with rotavirus infection, whereas the expression of EGFP or EGFP-VP4 showed the behavior of uninfected and untransfected cells. Increased ⁴⁵Ca²⁺ uptake was also observed in cells expressing NSP4-EGFP or NSP4, as evidenced in rotavirus infection. These results indicate that glycosylated NSP4 is primarily responsible for altering the Ca²⁺ homeostasis of infected cells through an initial increase of cell membrane permeability to Ca²⁺.     

The solution structure of a tetraheme cytochrome from Shewanella frigidimarina reveals a novel family structural motif

The bacteria belonging to the genus Shewanella are facultative anaerobes that utilize a variety of terminal electron acceptors which includes soluble and insoluble metal oxides. The tetraheme c-type cytochrome isolated during anaerobic growth of Shewanella frigidimarina NCIMB400 ( Sfc) contains 86 residues and is involved in the Fe(III) reduction pathways. Although the functional properties of Sfc redox centers are quite well described, no structures are available for this protein. In this work, we report the solution structure of the reduced form of Sfc. The overall fold is completely different from those of the tetraheme cytochromes c 3 and instead has similarities with the tetraheme cytochrome recently isolated from Shewanella oneidensis ( Soc). Comparison of the tetraheme cytochromes from Shewanella shows a considerable diversity in their primary structure and heme reduction potentials, yet they have highly conserved heme geometry, as is the case for the family of tetraheme cytochromes isolated from Desulfovibrio spp.     

Plant gamma-glutamyl hydrolases and folate polyglutamates: characterization, compartmentation, and co-occurrence in vacuoles

gamma-Glutamyl hydrolase (GGH, EC 3.4.19.9) catalyzes removal of the polyglutamyl tail from folyl and p-aminobenzoyl polyglutamates. Plants typically have one or a few GGH genes; Arabidopsis has three, tandemly arranged on chromosome 1, which encode proteins with predicted secretory pathway signal peptides. Two representative Arabidopsis GGH proteins, AtGGH1 and AtGGH2 (the At1g78660 and At1g78680 gene products, respectively) were expressed in truncated form in Escherichia coli and purified. Both enzymes were active as dimers, had low K(m) values (0.5-2 microm) for folyl and p-aminobenzoyl pentaglutamates, and acted as endopeptidases. However, despite 80% sequence identity, they differed in that AtGGH1 cleaved pentaglutamates, mainly to di- and triglutamates, whereas AtGGH2 yielded mainly monoglutamates. Analysis of subcellular fractions of pea leaves and red beet roots established that GGH activity is confined to the vacuole and that this activity, if not so sequestered, would deglutamylate all cellular folylpolyglutamates within minutes. Purified pea leaf vacuoles contained an average of 20% of the total cellular folate compared with approximately 50 and approximately 10%, respectively, in mitochondria and chloroplasts. The main vacuolar folate was 5-methyltetrahydrofolate, of which 51% was polyglutamylated. In contrast, the principal mitochondrial and chloroplastic forms were 5-formyl- and 5,10-methenyltetrahydrofolate polyglutamates, respectively. In beet roots, 16-60% of the folate was vacuolar and was again mainly 5-methyltetrahydrofolate, of which 76% was polyglutamylated. These data point to a hitherto unsuspected role for vacuoles in folate storage. Furthermore, the paradoxical co-occurrence of GGH and folylpolyglutamates in vacuoles implies that the polyglutamates are somehow protected from GGH attack.