tRNALeu Gene Research Articles

Disruption of de Novo Mitochondrial Thymidylate Synthesis Increases mtDNA Content

Mitochondrial DNA (mtDNA) synthesis is necessary for the replication and repair of damaged DNA; maintaining the proper balance of nucleotides is required to avoid mtDNA depletion and mutation. Mitochondrial deoxynucleotide triphosphates (dNTPs) are obtained from either salvage pathways or de novo biosynthesis within the mitochondria. In the mitochondria, de novo dTMP biosynthesis requires the enzymes serine hydroxymethyltransferase 2 (SHMT2), thymidylate synthase (TYMS), and dihydrofolate reductase‐like 1 (DHFRL1). In the nucleus, depletion of dTMP synthesis leads to misincorporation of deoxyuridine into nuclear DNA (nuDNA) resulting in genomic instability. SHMT2 in the mitochondria is responsible for transferring a one‐carbon unit from serine to tetrahydrofolate (THF), producing 5,10‐methyleneTHF and glycine in a reversible reaction; 5–10‐methyleneTHF is utilized by TYMS to create dTMP from dUMP.Our goal is to determine how mtDNA content is affected by disruptions of de novo mitochondrial dTMP synthesis, using two different approaches to decrease SHMT2 activity. First, SHMT2 expression in mammalian HeLa cells was decreased using siRNA technology. Second, HeLa cells were cultured in glycine concentrations ranging from 0mM to 10mM, which inhibits the SHMT2‐catalyzed synthesis of 5,10‐methyleneTHF. The mtDNA and nuDNA copy numbers were determined by qPCR using primers specific for the mitochondrial tRNALeu(UUR) gene and a single copy nuclear gene, β2‐microglobulin. Effects of SHMT2 inhibition on deoxyuridine misincorporation into mtDNA are being assessed.Our preliminary results suggest that mitochondrial de novo dTMP synthesis is affected by SHMT2 expression and glycine concentration. Knockdown of SHMT2 results in an increase (2.8 fold) in mtDNA copy number. Similarly, mtDNA copy number increases with increasing glycine concentration in culture media (4 fold). Supported by NIH R37DK58144.

A cryptic taxon rather than a hybrid species of Tragopogon (Asteraceae) from the Czech Republic

Tragopogon ×mirabilis Rouy is described as a diploid hybrid between T. porrifolius and T. pratensis. A population of T. ×mirabilis from Central Bohemia, Czech Republic, was recently investigated and, unlike previous reports of T. ×mirabilis, was found to be highly fertile. This fertile diploid hybrid population was considered to represent an alternative evolutionary pathway to polyploidy in Tragopogon. To determine the parentage of the plants from Bohemia, we investigated 12 samples of T. ×mirabilis with ITS, ETS, LFY and plastid (rpL16 gene, intron 1, tRNA-Leu (trnL) gene, intron, trnL-trnF intergenic spacer, psbA-trnH intergenic spacer, and trnG-trnT intergenic spacer) sequence data. None of the Bohemian plants have sequences that are consistent with a hybrid origin between T. porrifolius (incl. T. australis) and T. pratensis. Our data suggest that this fertile population of “T. ×mirabilis” may represent an unrecognised diploid species from the Angustissimi clade sensu Mavrodiev et al. (Int. J. Pl. Sci. 164: 1 – 19, 2005), a clade with a centre of distribution in the Caucasus, and hybrids of this unknown species with T. orientalis or T. hayekii (= T. orientalis L. var. hayekii Soo), a species closely related to T. pratensis and native to Bohemia. The Bohemian population of “Tragopogon ×mirabilis” clearly requires more investigation, but based on our data it does not appear to represent T. porrifolius × T. pratensis.

A maternally inherited diabetes and deafness patient with the 12S rRNA m.1555A>G and the ND1 m.3308T>C mutations associated with multiple mitochondrial deletions

Maternally inherited diabetes and deafness (MIDD) is a mitochondrial syndrome characterized by the onset of sensorineural hearing loss and diabetes in adults. Some patients may have other additional clinical features common in mitochondrial disorders such as pigmentary retinopathy, ptosis, cardiomyopathy, myopathy and renal affections. We report a 40-year-old Tunisian patient presenting maternally inherited type 2 diabetes and deafness (MIDD). A molecular genetic analysis was conducted in the patient and his twin sister, but no reported mutations in the tRNALeu(UUR) and tRNAGlu genes were found, especially the two mitochondrial m.3243A>G and the m.14709T>C mutations in muscle and blood leukocytes. The results showed the presence of the mitochondrial NADH deshydrogenase 1 (ND1) homoplasmic m.3308T>C mutation the 2 tested tissues (blood leukocytes and skeletal muscle) of the proband and in the patient’s sister blood leukocytes. In addition, we identified the mitochondrial 12S rRNA m.1555A>G mutation in muscle and blood leukocytes. The Long-range PCR amplification revealed the presence of multiple deletions of the mitochondrial DNA extracted from the patient’s skeletal muscle removing several tRNA and protein-coding genes. Our study reported a Tunisian patient with clinical features of MIDD in whom we detected the 12S rRNA m.1555A>G and the ND1 m.3308T>C mutations with mitochondrial multiple deletions.

Chloroplast genome of one brown seaweed, Saccharina japonica (Laminariales, Phaeophyta): Its structural features and phylogenetic analyses with other photosynthetic plastids

The chloroplast genome sequence of one brown seaweed, Saccharina japonica, was fully determined. It is characterized by 130,584base pairs (bp) with a large and a small single-copy region (LSC and SSC), separated by two copies of inverted repeats (IR1 and IR2). The inverted repeat is 5015bp long, and the sizes of SSC and LSC are 43,174bp and 77,378bp, respectively. The chloroplast genome of S. japonica consists of 139 protein-coding genes, 29 tRNA genes, and 3 ribosomal RNA genes. One intron was found in one tRNA-Leu gene in the chloroplast genome of S. japonica. Four types of overlapping genes were identified, ycf24 overlapped with ycf16 by 4 nucleotides (nt), ftrB overlapped with ycf12 by 6 nt, rpl4 and rpl23 overlapped by 8 nt, finally, psbC overlapped with psbD by 53 nt. With two sets of concatenated plastid protein data, 40-protein dataset and 26-protein dataset, the chloroplast phylogenetic relationship among S. japonica and the other photosynthetic species was evaluated. We found that the chloroplast genomes of haptophyte, cryptophyte and heterokont were not resolved into one cluster by the 40-protein dataset with amino acid composition bias, although it was recovered with strong support by the 26-protein dataset.

Discrimination of Lonicera japonica and Lonicera confusa using chemical analysis and genetic marker

Objective : Lonicera japonica THUNB. a traditional herbal medicine, has been commonly used anti-inflammatory disease. It has been very complicated with respect to its sources on the market. The significant selection of medicine depends on its origin. However, it is difficult to discrimination criteria for confirming L. japonica authenticity using the senses. This study was performed to determine the discriminant analysis of L. japonica and L. confusa. Methods : The identification of L. japonica and L. confusa were performed by the classification and identification committee of the national center for standardization of herbal medicines. And we examined its differences using HPLC and genetic marker analysis. Results : The analytical pattern of High Performance Liquid Chromatography was determined from the corresponding peak curves ((E)-aldosecologanin, chlorogenic acid, luteolin 7-O-glucoside, sweroside). For L. japonica, additional unknown peaks were detected at 13.8 min, 20.6 min, and 36.9 min. And, we developed genetic marker using the the tRNA-Leu gene, trnL-trnF intergenic spacer and tRNA-Phe region of chloroplast DNA. By the method, 164 bp PCR product amplified from L. confusa was distinguished into L. japonica and L. confusa efficiently. Conclusion : Base on these results, two techniques provide effective approaches to distinguish L. japonica from L. confusa.

Screening of effective pharmacological treatments for MELAS syndrome using yeasts, fibroblasts and cybrid models of the disease

MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is a mitochondrial disease most usually caused by point mutations in tRNA genes encoded by mitochondrial DNA (mtDNA). Approximately 80% of cases of MELAS syndrome are associated with a m.3243A > G mutation in the MT-TL1 gene, which encodes the mitochondrial tRNALeu (UUR). Currently, no effective treatments are available for this chronic progressive disorder. Treatment strategies in MELAS and other mitochondrial diseases consist of several drugs that diminish the deleterious effects of the abnormal respiratory chain function, reduce the presence of toxic agents or correct deficiencies in essential cofactors. We evaluated the effectiveness of some common pharmacological agents that have been utilized in the treatment of MELAS, in yeast, fibroblast and cybrid models of the disease. The yeast model harbouring the A14G mutation in the mitochondrial tRNALeu(UUR) gene, which is equivalent to the A3243G mutation in humans, was used in the initial screening. Next, the most effective drugs that were able to rescue the respiratory deficiency in MELAS yeast mutants were tested in fibroblasts and cybrid models of MELAS disease. According to our results, supplementation with riboflavin or coenzyme Q(10) effectively reversed the respiratory defect in MELAS yeast and improved the pathologic alterations in MELAS fibroblast and cybrid cell models. Our results indicate that cell models have great potential for screening and validating the effects of novel drug candidates for MELAS treatment and presumably also for other diseases with mitochondrial impairment.

Oral bisphosphonate use in the elderly is not associated with acute kidney injury

Intravenous bisphosphonates can cause acute kidney injury; however, this risk was not found with oral bisphosphonates in randomized clinical trials with restrictive eligibility criteria. In order to provide complementary safety data, we studied the risk of acute kidney injury in a population-based cohort of 122,727 patients aged 66 years and older discharged from hospital following a new fragility fracture and no history of bisphosphonate use in the prior year. Bisphosphonate treatment was identified within 120 days after discharge and event rates were measured from 90 days of therapy initiation. The primary outcome was hospitalization with acute kidney injury with secondary outcomes of new nephrology consultation and, in a subset of patients with laboratory values, acute kidney injury was defined as an increase in serum creatinine. We identified 18,286 bisphosphonate users and 104,441 non-users with a mean age of 81 years. Of 5772 patients with laboratory values, 40% had chronic kidney disease (eGFR <60 ml/min per 1.73 m(2)). Overall, there was no statistically significant difference in the risk of acute kidney injury among bisphosphonate users compared to non-users (adjusted odds ratio 1.03), and no significant differences in other outcomes or in subgroups of patients with baseline chronic kidney disease. Thus, in this older population-based cohort, oral bisphosphonate use was not associated with acute kidney injury.

Screening of the most common mitochondrial DNA mutations among Egyptian pediatric patients with mitochondrial disorders: A one year study

The mitochondrion is the only extranuclear organelle containing DNA (mtDNA). As such, genetically determined mitochondrial diseases may result from a molecular defect involving the mitochondrial or the nuclear genome. The first is characterized by maternal inheritance and the second by Mendelian inheritance. Ragged-red fibers (RRF) are commonly seen with primary lesions of mtDNA, but this association is not invariant. Conversely, RRF are seldom associated with primary lesions of nuclear DNA. Large-scale rearrangements (deletions and insertions) and point mutations of mtDNA are commonly associated with RRF and lactic acidosis, e.g. Kearns-Sayre syndrome (KSS) (major large-scale rearrangements), Pearson syndrome (large-scale rearrangements), myoclonus epilepsy with RRF (MERRF) (point mutation affecting tRNAlys gene), mitochondrial myopathy, lactic acidosis, and stroke-like episodes (MELAS) (two point mutations affecting tRNAleu(UUR) gene) and a maternally-inherited myopathy with cardiac involvement (MIMyCa) (point mutation affecting tRNAleu(UUR) gene). However, RRF and lactic acidosis are absent in Leber hereditary optic neuropathy (LHON) (one point mutation affecting ND4 gene, two point mutations affecting ND1 gene, and one point mutation affecting the apocytochromeb subunit of complex III), and the condition associated with maternally inherited sensory neuropathy (N), ataxia (A), retinitis pigmentosa (RP), developmental delay, dementia, seizures, and limb weakness (NARP) (point mutation affecting ATPase subunit 6 gene). The point mutations in MELAS, MIMyCa, and MERRF, and the large-scale mtDNA rearrangements in KSS and Pearson syndrome have a broader biochemical impact since these molecular defects involve the translational sequence of mitochondrial protein synthesis. The nuclear defects involving mitochondrial function generally are not associated with RRF. The biochemical classification of mitochondrial diseases principally catalogues these nuclear defects. This classification divides mitochondrial diseases into five categories. Primary and secondary deficiencies of carnitine are examples of a substrate transport defect. A lipid storage myopathy is often present. Disturbances of pyruvate or fatty acid metabolism are examples of substrate utilization defects. Only four defects of the Krebs cycle are known: fumarase deficiency, dihydrolipoyl dehydrogenase deficiency, α-ketoglutarate dehydrogenase deficiency, and combined defects of muscle succinate dehydrogenase and aconitase. Luft disease is the singular example of a defect in oxidation-phosphorylation coupling. Defects of respiratory chain function are manifold. Two clinical syndromes predominate, one involving limb weakness, and the other primarily affecting brain function. Leigh syndrome may result from different enzyme defects, most notably pyruvate dehydrogenase complex deficiency, cytochromec oxidase deficiency, complex I deficiency, and complex V deficiency associated with the recently described NARP point mutation. A new group of mitochondrial diseases has emerged. These conditions result from defective interaction between the nuclear and mitochondrial genomes. The first example is an autosomal dominant condition associated with multiple mtDNA deletions. The second example is a fatal infantile disorder associated with reduced abundance of mtDNA, termed the mitochondrial DNA depletion syndrome.

Patient-derived fibroblasts indicate oxidative stress status and may justify antioxidant therapy in OXPHOS disorders

Oxidative phosphorylation disorders are often associated with increased oxidative stress and antioxidant therapy is frequently given as treatment. However, the role of oxidative stress in oxidative phosphorylation disorders or patients is far from clear and consequently the preventive or therapeutic effect of antioxidants is highly anecdotic. Therefore, we performed a systematic study of a panel of oxidative stress parameters (reactive oxygen species levels, damage and defense) in fibroblasts of twelve well-characterized oxidative phosphorylation patients with a defect in the POLG1 gene, in the mitochondrial DNA-encoded tRNA-Leu gene (m.3243A>G or m.3302A>G) and in one of the mitochondrial DNA-encoded NADH dehydrogenase complex I (CI) subunits. All except two cell lines (one POLG1 and one tRNA-Leu) showed increased reactive oxygen species levels compared with controls, but only four (two CI and two tRNA-Leu) cell lines provided evidence for increased oxidative protein damage. The absence of a correlation between reactive oxygen species levels and oxidative protein damage implies differences in damage prevention or correction. This was investigated by gene expression studies, which showed adaptive and compensating changes involving antioxidants and the unfolded protein response, especially in the POLG1 group. This study indicated that patients display individual responses and that detailed analysis of fibroblasts enables the identification of patients that potentially benefit from antioxidant therapy. Furthermore, the fibroblast model can also be used to search for and test novel, more specific antioxidants or explore ways to stimulate compensatory mechanisms.

The complete mitochondrial genome of Hoplobatrachus rugulosus (Anura: Dicroglossidae)

The mitochondrial genome of Hoplobatrachus rugulosus (Anura: Dicroglossidae) is a circular molecule of 20,309 bp in length, containing 39 genes (including the extra copy of ND5 and tRNAMet genes). The following four distinctive features are observed: the cluster of rearranged tRNA genes (TPF tRNA gene cluster), the translocation of tRNALeu(CUN) and ND5 genes, the tandem duplication of tRNAMet genes (Met1 and Met2), and the duplicated d-loop–ND5 regions.

Accurate Localization of the Mobile Genomic Islands in &lt;i&gt;Pseudomonas putida&lt;/i&gt;

Pseudomonas putida is a safety gammaproteobacterium that plays an important role in bioremediation. Twenty nine mobile genomic islands were accurately localized in four strains of P. putida, six in P. putida F1, six in P. putida GB-1, nine in P. putida KT2440, and eight in P. putida W619, respectively. The integration sites include the tRNA gene, such as tRNAMet gene, tRNASer gene, tRNALeu gene, tRNAGly gene, tRNAThr gene, tRNACys gene, tRNAPro gene, and some structural genes, such as arsenate reductase gene, DNA mismatch repair protein MutS gene, thymidylate synthase gene, and 6-pyruvoyl tetrahydropterin synthase gene. 6-pyruvoyl tetrahydropterin synthase gene was firstly determined as the integration site of the genomic islands. The action sites of the lambda integrases are the stem-loop sequence, and the action sites of the P4 integrase are the asymmetric sequence. KT2440GI-5 can produce R2-type pyocin particle that is a bacteriocin and can kill sensitive bacterium. KT2440GI-9 can code ectoine-induced proteins that cause the cells to survive in high salt concentration.

MERRF and Kearns-Sayre Overlap Syndrome Due to the Mitochondrial DNA M.3291T&gt;C Mutation (IN7-1.008)

Objective: To report a patient with a complex phenotype of myoclonus epilepsy with ragged-red fibers (MERRF) syndrome and Kearns–Sayre syndrome (KSS), who harbored a m.3291T>C mutation in the tRNALeu(UUR) gene. This mutation has never been reported in association with a MERRF/KSS syndrome. Background Individuals who harbor a pathogenic mtDNA mutation can manifest overlapping features of typical mitochondrial syndromes. MERRF is a multisystem disorder characterized by myoclonic seizures, cerebellar ataxia, mitochondrial myopathy, and ragged-red fibers (RRF) on muscle biopsy, commonly caused by the m.8344A>G mutation in the tRNALys gene. KSS is a mitochondrial disorder characterized by onset before 20 years of age of progressive external ophthalmoplegia or pigmentary retinopathy, together with at least one of a triad of cerebellar ataxia, heart block, and elevated cerebrospinal fluid protein. Typically, KSS is due to single, large-scale deletions of mtDNA. Design/Methods: Clinical data were collected. Muscle histochemical and biochemical analysis was performed. Muscle DNA was screened for mtDNA large-scale rearrangements and point mutations. Mutation level was quantified by restriction fragment length polymorphism (RFLP) analysis. Results: A 48-year-old man presented with progressive myoclonus epilepsy, cerebellar ataxia, hearing loss, myopathic weakness, ophthalmoparesis, pigmentary retinopathy, and bifascicular heart block. Muscle biopsy demonstrated multiple RRF. Biochemical analysis of respiratory chain enzymes was normal. Genetic tests were negative for the common MERRF mtDNA mutations, and no deletions in mitochondrial DNA were detected. Direct sequencing mtDNA revealed a T-to-C transition at nucleotide position 3291 in the tRNALeu(UUR) gene. PCR RFLP showed 92% mutant genome in muscle. Conclusions: This is the first report associating a m.3291T>C mutation in the tRNALeu(UUR) gene with an adult-onset MERRF/KSS overlap. Our data reinforce the well-established concept that, in mtDNA-related disorders, the same genetic abnormality can be associated with a wide spectrum of phenotypes, and the observation that tRNALeu(UUR) is a hotspot for mtDNA mutations. Supported by: The National Institutes of Health (NIH HD32062) and by the Marriott Mitochondrial Disorders Clinical Research Fund (MMDCRF). M.H. was supported by NIH grants (R01HD57543, R01HD056103, and RCNS070232) and by the Muscular Dystrophy Association. Disclosure: Dr. Emmanuele has nothing to disclose. Dr. Silvers has nothing to disclose. Dr. Sotiriou has nothing to disclose. Dr. Tanji has nothing to disclose. Dr. DiMauro has nothing to disclose. Dr. Hirano has received personal compensation for activities with Athena Diagnostics as a speaker.Dr. Hirano has received personal compensation in an editorial capacity for Current Neurology and Neuroscience Reports.Dr. Hirano has received research support from Santhera Pharmaceutical and Edison Pharmaceuticals.

Is mitochondrial tRNALeu(UUR) 3291T>C mutation pathogenic?

According to a recent report by Sunami et al., a maternally inherited Japanese family with variable phenotypes including mitochondrial myopathy, recurrent headache, and myoclonus and epilepsy had been described to be associated with mitochondrial tRNALeu(UUR) 3291T>C mutation. In order to verify this association, we reanalyzed the clinical and molecular datasets obtained from Sunami's work; in addition, a phylogenetic approach was employed to evaluate the conservation index of this mutation among different species. We further utilized RNA Fold Web Server to predict the minimum free energy (MFE) of tRNALeu(UUR) gene with and without this mutation. Most strikingly, a low level of conservation was found regarding 3291T>C mutation and a slight change in MFE had been observed between the wild type and the mutant. Our negative results gave no support for an active role for this mutation on the clinical expression of mitochondrial disorders.

Metabolically induced heteroplasmy shifting and l-arginine treatment reduce the energetic defect in a neuronal-like model of MELAS

The m.3243A>G variant in the mitochondrial tRNALeu(UUR) gene is a common mitochondrial DNA (mtDNA) mutation. Phenotypic manifestations depend mainly on the heteroplasmy, i.e. the ratio of mutant to normal mtDNA copies. A high percentage of mutant mtDNA is associated with a severe, life-threatening neurological syndrome known as MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). MELAS is described as a neurovascular disorder primarily affecting the brain and blood vessels, but the pathophysiology of the disease is poorly understood.We developed a series of cybrid cell lines at two different mutant loads: 70% and 100% in the nuclear background of a neuroblastoma cell line (SH-SY5Y). We investigated the impact of the mutation on the metabolism and mitochondrial respiratory chain activity of the cybrids. The m.3243A>G mitochondrial mutation induced a metabolic switch towards glycolysis in the neuronal cells and produced severe defects in respiratory chain assembly and activity. We used two strategies to compensate for the biochemical defects in the mutant cells: one consisted of lowering the glucose content in the culture medium, and the other involved the addition of l-arginine. The reduction of glucose significantly shifted the 100% mutant cells towards the wild-type, reaching a 90% mutant level and restoring respiratory chain complex assembly. The addition of l-arginine, a nitric oxide (NO) donor, improved complex I activity in the mutant cells in which the defective NO metabolism had led to a relative shortage of NO. Thus, metabolically induced heteroplasmy shifting and l-arginine therapy may constitute promising therapeutic strategies against MELAS.

Increased Prevalence 12308 A > G mutation in Mitochondrial tRNALeu (CUN) Gene Associated with earlier Age of Onset in Friedreich Ataxia

Increased Prevalence 12308 A > G mutation in Mitochondrial tRNALeu (CUN) Gene Associated with earlier Age of Onset in Friedreich Ataxia

Clinical and cellular consequences of the mutation m.12300G>A in the mitochondrial tRNALeu(CUN) gene

We report, for the first time, a patient with an overlap MERRF–NARP syndrome who carries the mutation m.12300G>A in the mitochondrial tRNALeu(CUN) gene. The mutation was heteroplamic and more abundant in her muscle and fibroblast than in blood from her oligosymptomatic mother. Single muscle fiber analysis revealed that the proportion of mutant mtDNA in ragged red fibers was higher than that in normal fibers. Combined defects of mitochondrial respiratory chain complexes were detected in muscle, fibroblasts and transmitochondrial hybrid cells. Significant reduction of total ATP and mitochondrial membrane potential and an increased production of reactive oxygen species were observed.

The search for mitochondrial tRNALeu(UUR) A3243G mutation among type 2 diabetes mellitus patients in the Nigerian population

The study aimed to compare the incidence of the pathogenic point mutation A3243G in the gene tRNA Leu(UUR) indicating sub-type 2 diabetes mellitus conducted within the Nigerian population with that reported in other populations. 112 patients diagnosed with type 2 diabetes (T2D) mellitus according to the World Health Organization criteria were selected based on family history and re-evaluated for associated disorders from the diabetic clinics in the Northern part of Nigeria. The mtDNA of these patients was extracted and the tRNA Leu(UUR) gene screened for A3243G by PCR-RFLP method. Probands with maternal history were further investigated for other mutations using PCR-sequencing methods. None of the 112 patients were found to carry the A3243G mutation in the mitochondrial tRNA Leu(UUR) gene in the homoplasmic or in the heteroplasmic form. However, C3254T was identified in two of our patients. This mutation was reported to be associated with gestational diabetes and linked with population from sub-Saharan Africa. The A3243G mutation in mitochondrial tRNA Leu(UUR) is not a frequent cause of maternal diabetes in the Nigerian population contrary to other reported populations. However, further screening of an enlarged selected study group is necessary to fully determine the prevalence of this mutation in this population. This further search will help to fully appreciate the prevalence of maternal inheritance and diabetic deafness (MIDD) as extensively reported in other populations. Key words: Maternal diabetes, mitochondrial gene, maternal Inheritance and diabetic deafness, Nigeria, sub-Saharan Africa.

An Autopsy Case of Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis, and Stroke-Like Episodes (MELAS) with Intestinal Bleeding in Chronic Renal Failure

A 50-year-old man who underwent hemodialysis (HD) at local outpatient HD center due to end-stage renal disease (ESRD) was transferred to our hospital because of pneumonia. He had severe emaciation and past history of congestive heart failure. Presenting symptoms almost consistently involved difficulty in hearing and recurrent attacks of migraine-like headaches. He was diagnosed with dilated cardiomyopathy, showing diastolic mechanical dyssynchrony by tissue Doppler echocardiography. On the day of death, he had hematemesis and hemorrhagic shock. Autopsy revealed perforation of duodenum, and genetic analysis using mitochondrial DNA from cardiac muscle and iliopsoas muscle revealed a 3243A > G mutation in the mitochondrial tRNALeu(UUR) gene, which is related to mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Multiple organ failure due to the mutation of mitochondrial DNA with gastrointestinal bleeding is not a common.

Abstract 3685: C-Jun modulates alcohol-induced RNA Pol III-dependent transcription through Brf1 and TBP pathway

Abstract Cancer cells have a consistent cytological feature of nucleolar hypertrophy, enlarged nucleoli, where RNA polymerase III genes (Pol III genes) are transcripted. This implies that transformation in situ is closely linked to the deregulation of Pol III gene transcription, because the size of the nucleolus reflects the levels of rRNA synthesis. Deregulation of Pol III genes is tightly associated cell transformation and tumorgenesis. However, very little is known about whether alcohol consumption affects Pol III gene transcription. By using cell culture model and animal model, we have found, for the first time that alcohol induces Pol III gene transcription in JNK1 and ADH dependent manner. Alcohol increases c-Jun expression in HepG2-ADH cells. To determine whether the alcohol-mediated increases c-jun required to stimulate TBP and Brf1 expression are due to enhance occupancy of c-jun at their promoters, chromatin immunoprecipitation (ChIP) assays were performed. Ethanol induces a marked increase in the recruitment of c-jun to the TBP promoter, while a modest increase in the occupancy of both c-fos and Elk-1 was observed. These results support the idea that alcohol mediates an increase in TBP expression primarily through enhanced expression of c-jun and its recruitment to the TBP promoter. Sequence analysis reveals both putative AP-1 and Elk-1 binding sites in regions 5’ of the transcription start site of the Brf1 gene. ChIP analysis reveals that ethanol induces an increase in the occupancy of c-jun to the region containing an AP-1 binding site, while no significant increase in binding of Elk-1 to the Elk-1 binding site is observed. Interestingly, c-jun, c-fos and Elk-1 are also found to occupy the tRNALeu gene. Upon ethanol treatment, an increase in TBP recruitment to the tRNALeu gene is observed, as well as a marked increase in the occupancy of c-jun. However, alcohol does not induce any apparent change in recruitment of either c-fos or Elk-1 to the tRNA gene. These results suggest that c-jun may function directly at sequences near tRNA genes to induce transcription. Together, these results support the idea that alcohol enhances expression of TBP, Brf1, and tRNA gene by facilitating c-jun recruitment to these promoters. *: This project was supported by NIH grant AA017288 to S.Z. **: Corresponding author: szhong@usc.edu. Tel: 626-628-7693 or 323-442-114 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3685. doi:10.1158/1538-7445.AM2011-3685

Accurate localization and excision of genomic islands in four strains of Pseudomonas aeruginosa and Pseudomonas fluorescens

Mobile genomic islands (GIs) can be excised from the chromosome, then form a circular intermediate and be reintegrated into the chromosome by the GI internal integrase. Some mobile GIs can also be transferred into a new receptor cell by transformation, conjugation, or transduction. The action sites of the integrase are usually flanked direct repeats (DRs) of the GIs. Accurate localization of the flanking sequences is a precondition for determining the mobility of the GI. Mobile GIs are generally associated with transfer RNAs (tRNAs). Based on the correlation between flanking sequences and tRNA sequences, the flanking sequences of 11 putative mobile GIs in Pseudomonas aeruginosa PAO1, P. aeruginosa PA14, P. fluorescens Pf-5 and P. fluorescens Pf0-1 were identified. Among the 11 GIs, Pf0-1GI-1 is responsible for benzoate degradation. PAO1GI-1, Pf5GI-2, Pf5GI-3, and Pf5GI-4 were confirmed experimentally to be excised from a chromosome to form a circular intermediate. The action sites of the integrases are these GIs direct repeats. Due to distinct DRs, cutting sites for the internal integrase of PAO1GI-1, Pf5GI-2, Pf5GI-3 and Pf5GI-4 were determined outside the T-loop of the tRNAGly gene, outside the anticodon loop of the tRNASer gene and tRNALys gene, and at the asymmetric 3′-end of the tRNALeu gene, respectively. PAO1GI-1 and other mobile GIs may be transferred into many different strains that belong to different phyla because of the clear flanking sequences. This study describes basic information about the action sites of the integrases, assesses the mobility of GIs, and can help design and transfer mobile GIs to candidate strains.