Mitochondrial DNA sequence and gene organization in the Australian Blacklip Abalone Haliotis rubra (Leach)

The complete mitochondrial DNA of the blacklip abalone Haliotis rubra (Gastropoda: Mollusca) was cloned and 16,907 base pairs were sequenced. The sequence represents an estimated 99.85% of the mitochondrial genome, and contains 2 ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes found in other metazoan mtDNA. An AT tandem repeat and a possible C-rich domain within the putative control region could not be fully sequenced. The H. rubra mtDNA gene order is novel for mollusks, separated from the black chiton Katharina tunicata by the individual translocations of 3 tRNAs. Compared with other mtDNA regions, sequences from the ATP8, NAD2, NAD4L, NAD6, and 12S rRNA genes, as well as the control region, are the most variable among representatives from Mollusca, Arthropoda, and Rhynchonelliformea, with similar mtDNA arrangements to H. rubra. These sequences are being evaluated as genetic markers within commercially important Haliotis species, and some applications and considerations for their use are discussed.

Recent advances in chloroplast and mitochondrial division

Stomatin, originally identified as a major protein of the human erythrocyte membrane, is widely expressed in various tissues. Orthologues are found in vertebrates, invertebrates, plants, and microorganisms. Related proteins exhibit a common core structure, termed the prohibitin (PHB) domain, with varying extensions. Stomatin has an unusual topology, similar to caveolin-1, with a hydrophobic domain embedded at the cytoplasmic side of the membrane. Additional anchoring is provided by palmitoylation and the membrane affinity of the PHB domain. Stomatin associates with cholesterol-rich microdomains (lipid rafts), forms oligomers, and thereby displays a scaffolding function by generating large protein-lipid complexes. It regulates the activity of various membrane proteins by reversibly recruiting them to lipid rafts. This mechanism of regulation has been shown for GLUT-1 and may also apply for ion channels. Stomatin is located at the plasma membrane, particularly in microvilli, in endocytic and exocytic vesicles, and cytoplasmic granules. Stomatin-carrying endosomes are highly dynamic and interact with lipid droplets suggesting a role in intracellular lipid transport. This subcellular distribution and the caveolin-like protein structure suggest important membrane organizing functions for stomatin. A general picture emerges now that cell membranes contain cholesterol-rich domains that are generated and regulated by scaffolding proteins like caveolins, stomatins, and flotillin/reggie proteins.

The Omp85 family of proteins is essential for outer membrane biogenesis in mitochondria and bacteria

Integral proteins in the outer membrane of mitochondria control all aspects of organelle biogenesis, being required for protein import, mitochondrial fission, and, in metazoans, mitochondrial aspects of programmed cell death. How these integral proteins are assembled in the outer membrane had been unclear. In bacteria, Omp85 is an essential component of the protein insertion machinery, and we show that members of the Omp85 protein family are also found in eukaryotes ranging from plants to humans. In eukaryotes, Omp85 is present in the mitochondrial outer membrane. The gene encoding Omp85 is essential for cell viability in yeast, and conditional omp85 mutants have defects that arise from compromised insertion of integral proteins like voltage-dependent anion channel (VDAC) and components of the translocase in the outer membrane of mitochondria (TOM) complex into the mitochondrial outer membrane.

Nitric oxide protects against mitochondrial permeabilizastion induced by gluthathione depletion: role of S-nitrosylation?

Nitric oxide (NO) is known to mediate a multitude of biological effects including inhibition of respiration at cytochrome c oxidase (COX), formation of peroxynitrite (ONOO⁻) by reaction with mitochondrial superoxide (O2^{• −}), and S-nitrosylation of proteins. In this study, we investigated pathways of NO metabolism in lymphoblastic leukemic CEM cells in response to glutathione (GSH) depletion. We found that NO blocked mitochondrial protein thiol oxidation, membrane permeabilization, and cell death. The effects of NO were: (1) independent of respiratory chain inhibition since protection was also observed in CEM cells lacking mitochondrial DNA (ρ⁰) which do not possess a functional respiratory chain and (2) independent of ONOO⁻ formation since nitrotyrosine (a marker for ONOO⁻ formation) was not detected in extracts from cells treated with NO after GSH depletion. However, NO increased the level of mitochondrial protein S-nitrosylation (SNO) determined by the Biotin Switch assay and by the release of NO from mitochondrial fractions treated with mercuric chloride (which cleaves SNO bonds to release NO). In conclusion, these results indicate that NO blocks cell death after GSH depletion by preserving the redox status of mitochondrial protein thiols probably by a mechanism that involves S-nitrosylation of mitochondrial protein thiols.

Ribosome inactivating proteins (RIPs) from momordica charantia for anti viral therapy

This review describes the nature and applications of ribosome inactivating proteins (RIPs) from Momordica charantia (bitter melon). RIPs from the plant kingdom have received much attention in biomedical research because they target conserved host protein synthesis machinery and show specificity towards human and animal cell targets. Recent studies aimed at unravelling the enzymatic activities of the M charantia RIPs provide a structural basis for their activities. It has been reported that RIPs are member of the single chain ribosome inactivating protein (SCRIP) family which act irreversibly on ribosome by removing adenine residue from eukaryotic ribosomal RNA. Various activities of RIPs include anti-tumor, broad anti-viral, ribonuclease and deoxyribonuclease. MAP30 (Momordica Anti-HIV Protein), alpha- and beta-momorcharins inhibit HIV replication in acutely and chronically infected cells and thus are considered potential therapeutic agent in HIV infection and AIDS. Further, MAP30 improved the efficacy of anti-HIV therapy when used in combination with other anti-viral drugs. MAP30 holds therapeutic promise over other RIPs because not only it is active against infection and replication of both HSV and HIV but is non toxic to normal cells. Here we review the nature, action, structure function relationship and applications of RIPs from Momordica charantia and evaluate their potential for anti-cancer and anti-viral therapy.

Mitochondrial division in dictyostellium discoideum

Mitochondria, the cellular powerplants, are essential to eukaryotic life and have evolved from free-living bacteria. Using molecular biology, this thesis has deepened our understanding of the evolution of mitochondrial division through the study of two, key bacterially-derived proteins in the slime mold, dictyostelium.

Ribosome inactivating proteins from plants inhibiting viruses

Many plants contain ribosome inactivating proteins (RIPs) with N-glycosidase activity, which depurinate large ribosomal RNA and arrest protein synthesis. RIPs so far tested inhibit replication of mRNA as well as DNA viruses and these proteins, isolated from plants, are found to be effective against a broad range of viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes simplex virus (HSV). Most of the research work related to RIPs has been focused on antiviral activity against HIV; however, the exact mechanism of antiviral activity is still not clear. The mechanism of antiviral activity was thought to follow inactivation of the host cell ribosome, leading to inhibition of viral protein translation and host cell death. Enzymatic activity of RIPs is not limited to depurination of the large rRNA, in addition they can depurinate viral DNA as well as RNA. Recently, Phase I/II clinical trials have demonstrated the potential use of RIPs for treating patients with HIV disease. The aim of this review is to focus on various RIPs from plants associated with anti-HIV activity.

New drug targets in depression : inflammatory, cell-mediated immune, oxidative and nitrosative stress, mitochondrial, antioxidant, and neuroprogressive pathways. And new drug candidates—Nrf2 activators and GSK-3 inhibitors

This paper reviews new drug targets in the treatment of depression and new drug candidates to treat depression. Depression is characterized by aberrations in six intertwined pathways: (1) inflammatory pathways as indicated by increased levels of proinflammatory cytokines, e.g. interleukin-1 (IL-1), IL-6, and tumour necrosis factor α. (2) Activation of cell-mediated immune pathways as indicated by an increased production of interferon γ and neopterin. (3) Increased reactive oxygen and nitrogen species and damage by oxidative and nitrosative stress (O&NS), including lipid peroxidation, damage to DNA, proteins and mitochondria. (4) Lowered levels of key antioxidants, such as coenzyme Q10, zinc, vitamin E, glutathione, and glutathione peroxidase. (5) Damage to mitochondria and mitochondrial DNA and reduced activity of respiratory chain enzymes and adenosine triphosphate production. (6) Neuroprogression, which is the progressive process of neurodegeneration, apoptosis, and reduced neurogenesis and neuronal plasticity, phenomena that are probably caused by inflammation and O&NS. Antidepressants tend to normalize the above six pathways. Targeting these pathways has the potential to yield antidepressant effects, e.g. using cytokine antagonists, minocycline, Cox-2 inhibitors, statins, acetylsalicylic acid, ketamine, ω3 poly-unsaturated fatty acids, antioxidants, and neurotrophic factors. These six pathways offer new, pathophysiologically guided drug targets suggesting that novel therapies could be developed that target these six pathways simultaneously. Both nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activators and glycogen synthase kinase-3 (GSK-3) inhibitors target the six above-mentioned pathways. GSK-3 inhibitors have antidepressant effects in animal models of depression. Nrf2 activators and GSK-3 inhibitors have the potential to be advanced to phase-2 clinical trials to examine whether they augment the efficacy of antidepressants or are useful as monotherapy.

Ribosome-inactivating proteins : current status and biomedical applications

Ribosome-inactivating proteins (RIPs) are mainly present in plants and function to inhibit protein synthesis through the removal of adenine residues from eukaryotic ribosomal RNA (rRNA). They are broadly classified into two groups: type I and type II. Type I RIPs are a diverse family of proteins comprising a single polypeptide chain, whereas type II RIPs are heterodimeric glycoproteins comprising an A-chain (functionally equivalent to a type I RIP) linked via a disulphide bond to a B chain, mediating cell entry. In this review, we describe common type I and type II RIPs, their diverse biological functions, mechanism of cell entry, stability in plasma and antigenicity. We end with a discussion of promising applications for RIPs in biomedicine.

Xanthine oxidase inhibition attenuates skeletal muscle signaling following acute exercise but does not impair mitochondrial adaptations to endurance training.

The aim of this research was to examine the impact of the xanthine oxidase (XO) inhibitor allopurinol on the skeletal muscle activation of cell signaling kinases' and adaptations to mitochondrial proteins and antioxidant enzymes following acute endurance exercise and endurance training. Male Sprague-Dawley rats performed either acute exercise (60 min of treadmill running, 27 m/min, 5% incline) or 6 wk of endurance training (5 days/wk) while receiving allopurinol or vehicle. Allopurinol treatment reduced XO activity to 5% of the basal levels (P < 0.05), with skeletal muscle uric acid levels being almost undetectable. Following acute exercise, skeletal muscle oxidized glutathione (GSSG) significantly increased in allopurinol- and vehicle-treated groups despite XO activity and uric acid levels being unaltered by acute exercise (P < 0.05). This suggests that the source of ROS was not from XO. Surprisingly, muscle GSSG levels were significantly increased following allopurinol treatment. Following acute exercise, allopurinol treatment prevented the increase in p38 MAPK and ERK phosphorylation and attenuated the increase in mitochondrial transcription factor A (mtTFA) mRNA (P < 0.05) but had no effect on the increase in peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear respiratory factor-2, GLUT4, or superoxide dismutase mRNA. Allopurinol also had no impact on the endurance training-induced increases in PGC-1α, mtTFA, and mitochondrial proteins including cytochrome c, citrate synthase, and β-hydroxyacyl-CoA dehydrogenase. In conclusion, although allopurinol inhibits cell signaling pathways in response to acute exercise, the inhibitory effects of allopurinol appear unrelated to exercise-induced ROS production by XO. Allopurinol also has little effect on increases in mitochondrial proteins following endurance training.

Age-related proteostasis and metabolic alterations in Caspase-2-deficient mice.

Ageing is a complex biological process for which underlying biochemical changes are still largely unknown. We performed comparative profiling of the cellular proteome and metabolome to understand the molecular basis of ageing in Caspase-2-deficient (Casp2(-/-)) mice that are a model of premature ageing in the absence of overt disease. Age-related changes were determined in the liver and serum of young (6-9 week) and aged (18-24 month) wild-type and Casp2(-/-) mice. We identified perturbed metabolic pathways, decreased levels of ribosomal and respiratory complex proteins and altered mitochondrial function that contribute to premature ageing in the Casp2(-/-) mice. We show that the metabolic profile changes in the young Casp2(-/-) mice resemble those found in aged wild-type mice. Intriguingly, aged Casp2(-/-) mice were found to have reduced blood glucose and improved glucose tolerance. These results demonstrate an important role for caspase-2 in regulating proteome and metabolome remodelling during ageing.

Bid chimeras indicate that most BH3-only proteins can directly activate Bak and Bax, and show no preference for Bak versus Bax

The mitochondrial pathway of apoptosis is initiated by Bcl-2 homology region 3 (BH3)-only members of the Bcl-2 protein family. On upregulation or activation, certain BH3-only proteins can directly bind and activate Bak and Bax to induce conformation change, oligomerization and pore formation in mitochondria. BH3-only proteins, with the exception of Bid, are intrinsically disordered and therefore, functional studies often utilize peptides based on just their BH3 domains. However, these reagents do not possess the hydrophobic membrane targeting domains found on the native BH3-only molecule. To generate each BH3-only protein as a recombinant protein that could efficiently target mitochondria, we developed recombinant Bid chimeras in which the BH3 domain was replaced with that of other BH3-only proteins (Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk). The chimeras were stable following purification, and each immunoprecipitated with full-length Bcl-xL according to the specificity reported for the related BH3 peptide. When tested for activation of Bak and Bax in mitochondrial permeabilization assays, Bid chimeras were ~1000-fold more effective than the related BH3 peptides. BH3 sequences from Bid and Bim were the strongest activators, followed by Puma, Hrk, Bmf and Bik, while Bad and Noxa were not activators. Notably, chimeras and peptides showed no apparent preference for activating Bak or Bax. In addition, within the BH3 domain, the h0 position recently found to be important for Bax activation, was important also for Bak activation. Together, our data with full-length proteins indicate that most BH3-only proteins can directly activate both Bak and Bax.

Phylogeny of Triatoma sherlocki (Hemiptera: Reduviidae: Triatominae) Inferred from Two Mitochondrial Genes Suggests Its Location Within the Triatoma brasiliensis Complex

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Pancreatic islets from dexamethasone-treated rats show alterations in global gene expression and mitochondrial pathways

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Overexpression of plant uncoupling mitochondrial protein in transgenic tobacco increases tolerance to oxidative stress

An Arabidopsis thaliana cDNA clone encoding a plant uncoupling mitochondrial protein (AtPUMP1) was overexpressed in transgenic tobacco plants. Analysis of the AtPUMP1 mRNA content in the transgenic lines, determined by Northern blot, revealed variable levels of transgene expression. Antibody probing of Western blots of mitochondrial proteins from three independent transgenic lines showed significant accumulation of AtPUMP1 in this organelle. Overproduction of AtPUMP1 in transgenic tobacco plants led to a significant increase in tolerance to oxidative stress promoted by exogenous hydrogen peroxide as compared to wild-type control plants. These results provide the first biological evidence for a role of PUMP in protection of plant cells against oxidative stress damage.