The Yeast Complex I Equivalent NADH Dehydrogenase Rescues pink1 Mutants

Pink1 is a mitochondrial kinase involved in Parkinson's disease, and loss of Pink1 function affects mitochondrial morphology via a pathway involving Parkin and components of the mitochondrial remodeling machinery. Pink1 loss also affects the enzymatic activity of isolated Complex I of the electron transport chain (ETC); however, the primary defect in pink1 mutants is unclear. We tested the hypothesis that ETC deficiency is upstream of other pink1-associated phenotypes. We expressed Saccaromyces cerevisiae Ndi1p, an enzyme that bypasses ETC Complex I, or sea squirt Ciona intestinalis AOX, an enzyme that bypasses ETC Complex III and IV, in pink1 mutant Drosophila and find that expression of Ndi1p, but not of AOX, rescues pink1-associated defects. Likewise, loss of function of subunits that encode for Complex I–associated proteins displays many of the pink1-associated phenotypes, and these defects are rescued by Ndi1p expression. Conversely, expression of Ndi1p fails to rescue any of the parkin mutant phenotypes. Additionally, unlike pink1 mutants, fly parkin mutants do not show reduced enzymatic activity of Complex I, indicating that Ndi1p acts downstream or parallel to Pink1, but upstream or independent of Parkin. Furthermore, while increasing mitochondrial fission or decreasing mitochondrial fusion rescues mitochondrial morphological defects in pink1 mutants, these manipulations fail to significantly rescue the reduced enzymatic activity of Complex I, indicating that functional defects observed at the level of Complex I enzymatic activity in pink1 mutant mitochondria do not arise from morphological defects. Our data indicate a central role for Complex I dysfunction in pink1-associated defects, and our genetic analyses with heterologous ETC enzymes suggest that Ndi1p-dependent NADH dehydrogenase activity largely acts downstream of, or in parallel to, Pink1 but upstream of Parkin and mitochondrial remodeling

Aconitase Causes Iron Toxicity in Drosophila pink1 Mutants

The PTEN-induced kinase 1 (PINK1) is a mitochondrial kinase, and pink1 mutations cause early onset Parkinson's disease (PD) in humans. Loss of pink1 in Drosophila leads to defects in mitochondrial function, and genetic data suggest that another PD-related gene product, Parkin, acts with pink1 to regulate the clearance of dysfunctional mitochondria (mitophagy). Consequently, pink1 mutants show an accumulation of morphologically abnormal mitochondria, but it is unclear if other factors are involved in pink1 function in vivo and contribute to the mitochondrial morphological defects seen in specific cell types in pink1 mutants. To explore the molecular mechanisms of pink1 function, we performed a genetic modifier screen in Drosophila and identified aconitase (acon) as a dominant suppressor of pink1. Acon localizes to mitochondria and harbors a labile iron-sulfur [4Fe-4S] cluster that can scavenge superoxide to release hydrogen peroxide and iron that combine to produce hydroxyl radicals. Using Acon enzymatic mutants, and expression of mitoferritin that scavenges free iron, we show that [4Fe-4S] cluster inactivation, as a result of increased superoxide in pink1 mutants, results in oxidative stress and mitochondrial swelling. We show that [4Fe-4S] inactivation acts downstream of pink1 in a pathway that affects mitochondrial morphology, but acts independently of parkin. Thus our data indicate that superoxide-dependent [4Fe-4S] inactivation defines a potential pathogenic cascade that acts independent of mitophagy and links iron toxicity to mitochondrial failure in a PD–relevant model.status: publishe

Serum (1-3)-β-d-Glucan as a Tool for Diagnosis of Pneumocystis jirovecii Pneumonia in Patients with Human Immunodeficiency Virus Infection or Hematological Malignancy ▿

(1-3)-β-d-Glucan (BG) reactivity was tested in serum samples from 28 patients with human immunodeficiency virus infection or a hematological malignancy and Pneumocystis jirovecii pneumonia (PCP) and 28 control patients. The sensitivity and specificity of BG detection with the Fungitell assay for PCP were 100 and 96.4%, respectively, using a cutoff value of 100 pg/ml. Serum BG testing looks promising for the noninvasive diagnosis of PCP. Our data suggest that a higher cutoff value for the diagnosis of PCP than for the diagnosis of invasive aspergillosis or candidiasis could be used safely and will improve the specificity of the test

KEEFEKTIFAN STRATEGI PEMBELAJARAN BERSAFARI BERMINAT, SANGAT MENGUASAI, FAKTA, RABUK PANCAINDRA, DAN DIKSI DALAM PEMBELAJARAN MENULIS CERPEN

Penelitian ini dilatarbelakangi oleh kelemahan siswa dalam menulis cerpen, sehingga diperlukan strategi pembelajaran yang tepat untuk diterapkan dalam pembelajaran menulis cerpen. Berdasarkan latar belakang tersebut, penulis menyusun rumusan masalah: 1) Bagaimana kemampuan siswa dalam menulis cerpen sebelum dan sesudah menggunakan Strategi Bersafari pada kelas eksperimen; 2) Bagaimana kemampuan siswa dalam menulis cerpen sebelum dan setelah tes tanpa menggunakan Strategi Bersafari pada kelas kontrol; 3)Adakah perbedaan yang signifikan antara kemampuan siswa dalam menulis cerpen dengan menggunakan Strategi Bersafari di kelas eksperimen dan tanpa menggunakan Strategi Bersafari di kelas kontrol. Adapun tujuan dari penelitian ini adalah untuk mendeskripsikan hal-hal yang tercantum dalam rumusan masalah tersebut. Strategi pembelajaran yang digunakan dalam penelitian ini adalah Strategi Bersafari. Metode yang digunakan dalam penelitian ini adalah metode eksperimen semu dengan desain pretest-posttest control group. Teknik pengolahan data dalam penelitian ini berupa pengolahan data kuantitatif yang meliputi uji reliabilitas, normalitas, homogenitas, dan uji hipotesis. Terdapat perbedaan yang signifikan antara kemampuan siswa dalam menulis cerpen di kelas eksperimen dan di kelas kontrol. Hal ini bisa dlihat dari pemerolehan hasil posttest antara kedua kelas. Hasil posttest kelas eskperimen sebesar 73, sementara hasil posttest kelas kontrol sebesar 67. Berdasarkan hasil uji hipotesis dengan menentukan thitung, diperoleh nilai thitung = 5,81 dan ttabel = 2,36, sehingga thitung = 5,81 > ttabel = 2,36. Maka hipotesis H1 diterima, yaitu terdapat perbedaan yang signifikan antara kemampuan siswa dalam menulis cerpen di kelas eksperimen dan di kelas kontrol

Vitamin K2 is a mitochondrial electron carrier that rescues pink1 deficiency

Human UBIAD1 localizes to mitochondria and converts vitamin K(1) to vitamin K(2). Vitamin K(2) is best known as a cofactor in blood coagulation, but in bacteria it is a membrane-bound electron carrier. Whether vitamin K(2) exerts a similar carrier function in eukaryotic cells is unknown. We identified Drosophila UBIAD1/Heix as a modifier of pink1, a gene mutated in Parkinson's disease that affects mitochondrial function. We found that vitamin K(2) was necessary and sufficient to transfer electrons in Drosophila mitochondria. Heix mutants showed severe mitochondrial defects that were rescued by vitamin K(2), and, similar to ubiquinone, vitamin K(2) transferred electrons in Drosophila mitochondria, resulting in more efficient adenosine triphosphate (ATP) production. Thus, mitochondrial dysfunction was rescued by vitamin K(2) that serves as a mitochondrial electron carrier, helping to maintain normal ATP production.status: publishe

Acon inactivation and Parkin-mediated mitophagy act in parallel in <i>pink1</i> mutants.

<p>Analysis of <i>parkin</i> mutants (<i>pakin^1/Δ21</i>) and parkin flies heterozygous for <i>acon^1or2</i> (A) Flight ability of 5-day-old adult flies and (B) ATP content in the head-thorax of 5-day-old flies. Data collected from at least 5 independent experiments. (C) GFP-labeled mitochondria in flight muscles (<i>daGal4 UAS-mitoGFP</i>). Scale bar: muscle 10 µm. (D) Quantification of average mitochondrial aggregate size. 5 images from n≥6 thoraxes. (E) Flight ability of control flies, <i>pink1^B9</i>, <i>pink1^B9</i> heterozygous for <i>acon^1or2</i> heterozygous for <i>acon^1or2</i> or overexpressing <i>drp1</i> and <i>pink1</i> mutants with a combination of <i>acon^1or2</i> heterozygousity and <i>drp1</i> overexpression. * Significantly different from <i>pink1^B9</i>, One-way ANOVA, post hoc Dunnett p<0.01, ns: not significantly different.</p

Acon[4Fe-4S] cluster induces mitochondrial defect in DA neurons that is not rescued by increased mitophagy.

<p>(A) Quantification of Western blots of fly heads expressing <i>UAS-acon</i> (see text) in DA neurons probed with anti-Acon normalized to tubulin, relative to control. Data from 3 independent experiments. (B) Mitochondrial Aconitase activity relative to the control. Data collected from 5 independent mitochondrial preparations. (C,E) GFP-labeled mitochondria in dopaminergic (DA) neurons DA neurons labeled with anti-tyrosine hydroxylase (magenta). White arrows indicate mitochondrial aggregates. Scale bar: 2.5 µm. (D,F) Quantification of average mitochondrial aggregate size. 5 neurons per brain from n≥10 brains were analyzed. The genotype of control is: <i>w¹¹¹⁸</i>; <i>pleGal4 UAS-mitoGFP</i>/+ ; and of mutants that express wild type or mutant Acon is: <i>w¹¹¹⁸</i>; <i>UAS-acon^*/+</i>; <i>pleGal4 UAS-mitoGFP</i>/+. Significantly different <b>*</b> from control, ° from ple>acon^wt, °° from ple>acon^S677A One-way ANOVA, post hoc Dunnett p<0.01, ns: not significantly different. Data are shown as Mean ± SEM.</p

Partial loss of Acon suppresses <i>pink1^B9</i> phenotypes.

<p>(A–D) Flight ability of 5-day-old adult flies (A, B) and ATP content in the head-thorax of 5-day-old flies (C, D). Data collected from at least 5 independent experiments. * Significantly different from <i>pink1^B9</i>, One-way ANOVA, post hoc Dunnett p<0.01, ns: not significantly different. (E) TEM analysis of thorax. Black arrows indicate swollen mitochondria. Scale bar: ×5000 5 µm; ×2000 2 µm. (F, H) GFP-labeled mitochondria in flight muscles (F; <i>daGal4 UAS-mitoGFP</i>) and in dopaminergic (H; <i>pleGal4 UAS-mitoGFP</i>) neurons that are double-labeled with anti-tyrosine hydroxylase (magenta). White arrows indicate mitochondrial aggregates. Scale bar: muscle 10 µm; DA neurons 2.5 µm. (G, I) Quantification of average mitochondrial aggregate size. 5 images from n≥6 thoraxes and about 5 neurons per brain from n≥10 brains were analyzed. * Significantly different from <i>pink1^B9</i>, One-way ANOVA, post hoc Dunnett p<0.01, ns not significantly different. In all panel “control” is <i>pink1^RV</i>, a precise <i>P element</i> excision; “genomic” indicates the insertion of a construct on the third chromosome that encompasses the wild type <i>acon</i> gene, data are shown as Mean ± SEM.</p

Model of oxidative Acon inactivation in <i>pink1</i> mutants.

<p>Pink1 loss induces increased superoxide production that inactivates the Acon[4Fe-4S] resulting in the generation of Fe²⁺ and H₂O₂. These combine to form hydroxyl radicals that lead to mitochondrial failure. Mitoferritin chelates Fe²⁺ and is thus able to rescue mitochondrial failure in <i>pink1</i> mutants. Acon inactivation act in parallel to Parkin-mediated mitophagy in controlling mitochondrial integrity.</p

Oxidative inactivation of Acon in <i>pink1^B9</i> mutants.

<p>(A) Superoxide production measured as fluorescence change of DHE, a superoxide sensitive dye, in isolated mitochondria from control (<i>pink1^RV</i>), <i>pink1^B9</i>, <i>pink1^B9</i> mutant flies heterozygous for <i>acon^{1 or 2}</i> flies as well as in <i>pink1^RV</i> isolated mitochondria incubated with antimycin. Changes normalized to control. Data collected from 5 independent mitochondrial preparations. * Significantly different from control, One-way ANOVA, post hoc Dunnett p<0.01. (B) Relative mitochondrial Aconitase activity in <i>pink1^B9</i> normalized to Acon protein levels. Data collected from 5 independent mitochondrial preparations. * Significantly different from control, Student's t test p<0.01. (C) H₂O₂ and derivates content measured as H₂O₂-sensitive DCHF fluorescence in fly lysates of indicated genotypes, relative to total protein content. Data collected from 5 independent mitochondrial preparations. * Significantly different from control and ° significantly different from <i>pink1^B9</i>, One-way ANOVA, post hoc Dunnett p<0.01 (D) Fe²⁺ levels in isolated mitochondria from flies with indicated genotype. Quencing of Fe²⁺-sensitive RPA and Fe²⁺-insensitive RPAC dye are represented as percent of initial fluorescence. Data collected from 5 independent mitochondrial preparations. * Significantly different from control and ° significantly different from <i>pink1^B9</i>, One-way ANOVA, post hoc Dunnett p<0.01. Data are shown as Mean ± SEM.</p