Mitochondrial DNA mutations and three major forms of mitochondrial myopathies: CPEO, MELAS and MERRF

Mitochondrial DNA (mtDNA) mutations were discovered during a few years around 1990 and thought to be assigned to the three major clinical forms of mitochondrial myopathies. Then, several mtDNA mutations were detected in each disease and the common mutations were found in more than one disease, suggesting genotype and phenotype heterogeneity. Heteroplasmy, tissue/cellular specificity and threshold effect have been extensively studied using skeletal muscle and culture cells from the patients, but the major part of the relations up between mtDNA genotype and clinical phenotype remains unknown. Because of the facts that mtDNA mutations have been found in other clinical forms (not myopathies), and frataxin, the product of the responsible gene for Freidreich ataxia, may be associated with mtDNA biogenesis, disease entity accompanied by mtDNA abnormalities could expand more in the future.     

Mitochondrial impairment as an early event in the process of apoptosis induced by glutathione depletion in neuronal cells: relevance to Parkinson''s disease

OBJECTIVE: To examine the effect of ZD7155, an angiotensin II receptor antagonist, on blood pressure, heart rate and occupancy of tissue angiotensin II receptor in two-kidney, one-clip Goldblatt hypertensive rats. METHODS: Goldblatt hypertension was produced in Sprague-Dawley rats. ZD7155 was administered orally at 3 mg/kg and blood pressure and heart rate were monitored for up to 48 h. In a second series of experiments, the rats were administered increasing doses of ZD7155 and monitored for 1 h. For each time and dose, rats were killed and their blood and tissues were collected. Tissue angiotensin II receptor binding was assessed by quantitative autoradiography in vitro. For a separate group of rats, the pressor response to 0.1 microg/kg angiotensin II was monitored before and after the administration of 3 mg/kg ZD7155. RESULTS: After oral administration of ZD7155, blood pressure was rapidly lowered and this lowering was sustained for up to 48 h. This effect was accompanied by sustained inhibition of angiotensin II receptor binding in the aorta, kidney and adrenal gland, together with an increase in plasma renin activity. Increasing doses of ZD7155 dose-dependently reduced blood pressure and inhibited angiotensin II receptor binding in the tissues. Degrees of inhibition varied among the different tissues and had different time courses. ZD7155 inhibited the pressor response to angiotensin II remarkably for 24 h. CONCLUSIONS: ZD7155 is a potent antihypertensive agent in two-kidney, one-clip Goldblatt hypertensive rats and this effect is accompanied by sustained inhibition of tissue angiotensin II binding.     

Mitochondrial mutations, cellular instability and ageing: modelling the population dynamics of mitochondria

All eukaryotic cells rely on mitochondrial respiration as their major source of metabolic energy (ATP). However, the mitochondria are also the main cellular source of oxygen radicals and the mutation rate of mtDNA is much higher than for chromosomal DNA. Damage to mtDNA is of great importance because it will often impair cellular energy production. However, damaged mitochondria can still replicate because the enzymes for mitochondrial replication are encoded entirely in the cell nucleus. For these reasons, it has been suggested that accumulation of defective mitochondria may be an important contributor to loss of cellular homoeostasis underlying the ageing process. We describe a mathematical model which treats the dynamics of a population of mitochondria subject to radical-induced DNA mutations. The model confirms the existence of an upper threshold level for mutations beyond which the mitochondrial population collapses. This threshold depends strongly on the division rate of the mitochondria. The model also reproduces and explains (i) the decrease in mitochondrial population with age, (ii) the increase in the fraction of damaged mitochondria in old cells, (iii) the increase in radical production per mitochondrion, and (iv) the decrease in ATP production per mitochondrion.     

Crystal structure and mechanism of human L-arginine:glycine amidinotransferase: a mitochondrial enzyme involved in creatine biosynthesis

L-arginine:glycine amidinotransferase (AT) catalyses the committed step in creatine biosynthesis by formation of guanidinoacetic acid, the immediate precursor of creatine. We have determined the crystal structure of the recombinant human enzyme by multiple isomorphous replacement at 1.9 A resolution. A telluromethionine derivative was used in sequence assignment. The structure of AT reveals a new fold with 5-fold pseudosymmetry of circularly arranged betabeta alphabeta-modules. These enclose the active site compartment, which is accessible only through a narrow channel. The overall structure resembles a basket with handles that are formed from insertions into the betabeta alphabeta-modules. Binding of L-ornithine, a product inhibitor, reveals a marked induced-fit mechanism, with a loop at the active site entrance changing its conformation accompanied by a shift of an alpha-helix by -4 A. Binding of the arginine educt to the inactive mutant C407A shows a similar mode of binding. A reaction mechanism with a catalytic triad Cys-His-Asp is proposed on the basis of substrate and product bound states.     

Confronting professional impairment during the internship: Identification, due process, and remediation.

Although increasing efforts have been made in recent years to address impairment in practicing psychologists, little regarding how to deal with impairment early in the professional career has been reported. The predoctoral internship is a pivotal training experience, which makes it a critical stage at which to assess and deal with impairment. We identify criteria of impairment, suggest components to be included in any evaluation model, identify guidelines and procedures for ensuring due process, and recommend strategies for prevention and remediation. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Mitochondrial contact sites detected by creatine phosphokinase activity in the hearts of normal and diabetic rats: is mitochondrial contact sites formation a calcium-dependent process?

Reovirus type 2 (Reo-2) infection in DBA/1 sucking mice causes pancreatic islet-cell destruction, which results in a diabetes-like syndrome. To investigate the role of reactive oxygen species (ROS), the protective effect of dimethylthiourea (DMTU) was examined, this substance being an effective scavenger of hydroxyl radicals. The degree of cellular infiltration in and around pancreatic islets was the same in mice receiving either virus only or virus and DMTU. The latter had no effect on (1) the number or type of white blood cells, (2) lymphocyte function-associated antigen 1-alpha-positive splenocytes, or (3) viral multiplication in the pancreas. However, treatment with DMTU inhibited the elevation of blood glucose concentrations and reduced pancreatic islet-cell damage (beta-cell degranulation and necrosis). These results suggest that ROS play a role in the pathogenesis of Reo-2-induced diabetes-like syndrome.     

Import of cytochrome b2 to the mitochondrial intermembrane space: the tightly folded heme-binding domain makes import dependent upon matrix ATP

Cytochrome b2 is synthesized as a precursor in the cytoplasm and imported to the intermembrane space of yeast mitochondria. We show here that the precursor contains a tightly folded heme-binding domain and that translocation of this domain across the outer membrane requires ATP. Surprisingly, it is ATP in the mitochondrial matrix rather than external ATP that drives import of the heme-binding domain. When the folded structure of the heme-binding domain is disrupted by mutation or by urea denaturation, import and correct processing take place in ATP-depleted mitochondria. These results indicate that (1) cytochrome b2 reaches the intermembrane space without completely crossing the inner membrane, and (2) some precursors fold outside the mitochondria but remain translocation-competent, and import of these precursors in vitro does not require ATP-dependent cytosolic chaperone proteins.     

Cytoplasmic inclusion bodies: a study in several diseases and a review of the literature

Among 1400 muscle biopsies, we studied 16 cases with rimmed vacuoles, whose histology suggests cytoplasm inclusion bodies. We tried to correlate the clinical, laboratory and histopatological data in order to verify the specificity of cytoplasm inclusion bodies to certain diseases. The creatinekinase was increased in 10 cases. In all cases electromyography was abnormal. Muscle histochemistry revealed myopathy in 5 cases, mixed pattern in 7, denervation in 2 and in 2 cases, inflammatory myopathy. Electron microscopy showed the presence of filaments in 8 cases (nuclear, disseminated in cytoplasm or in the subsarcolemmal region). The patients were classified according to history, heredity, laboratory, electrophysiologic, histochemistry data and electron microscopy: in myositis with inclusion cytoplasmic bodies (4 cases), juvenile spinal muscular atrophy (6 cases), distal myopathies (3 cases), limb-girdle dystrophy (2 cases) and peripheral neuropathy (1 case). We present a revision on the pathogenesis and possible etiology of rimmed vacuoles and their relationship with several diseases.     

Expression of brain-type creatine kinase and ubiquitous mitochondrial creatine kinase in the fetal rat brain: evidence for a nuclear energy shuttle

To test the hypothesis that embryonic brain cells utilize a creatine phosphate energy shuttle, we examined the pattern of creatine kinase (CK) isoform expression and localization in the fetal rat brain. Moderate levels of CK activity are present at embryonic day 14 (7 U/mg protein) and decrease slightly until 3 days postpartum followed by a rapid, fourfold up-regulation to adult levels by 1 month (18 U/mg protein). In parallel with changes in enzyme activity, there is a biphasic and coordinate pattern of expression of brain-type CK (BCK) and ubiquitous mitochondrial CK (uMtCK) determined by nondenaturing electrophoresis and immunoblot analysis. The localization of CK isoforms was examined by immunocytochemistry, and, during the fetal period, BCK and uMtCK immunoreactivity was detected throughout the central and peripheral nervous system, especially in neuroepithelial regions of the cerebral vesicles and spinal cord. In large cells within the olfactory neuroepithelium and ventral spinal cord, differential compartmentation of CK isoforms was evident, with BCK localized primarily in cell nuclei, whereas uMtCK immunoreactivity was present in the cell body (but not within nuclei). In olfactory bulb neuroepithelium, both isoforms were expressed in the middle zone of the germinal layer associated with DNA synthesis. In embryonic skeletal and cardiac muscle, which also express BCK, the same compartmentation of BCK was seen, with BCK localized primarily in the cell nucleus of cardiac and skeletal myoblasts. These results demonstrate a coordinate pattern of expression and compartmentation of BCK and uMtCK isoforms in the fetal brain that, in some cells, provides the anatomic basis for a nuclear energy shuttle.     

Impaired innervation of cultured human muscle overexpressing betaAPP experimentally and genetically: relevance to inclusion-body myopathies

To investigate whether abnormally accumulated betaAPP may be responsible for denervation of muscle fibers that are present in hereditary inclusion-body myopathy (h-IBM) and sporadic inclusion-body myositis (s-IBM), we cultured five h-IBM and eight normal muscle biopsies. In eight other experiments, a 3 kb human 751-betaAPP-cDNA was transferred, using adenovirus vector, into cultured normal myotubes immediately after myoblast fusion. In all experiments, cultured muscle fibers were co-cultured with fetal rat spinal cord. Controls had no detectable betaAPP epitopes, whereas betaAPP epitopes were greatly increased in cultured h-IBM muscle and in cultured normal muscle after betaAPP-gene transfer. Innervated normal cultured muscle fibers were continuously contracting and fully cross-striated, and they had acetylcholine receptors (AChRs) and acetylcholinesterase (AChE) accumulated only at the neuromuscular junctions (NMJs). By contrast, both groups of betaAPP-overexpressing cultured muscle fibers were not contracting and not cross-striated; and did not have NJMs containing AChRs and AChE. Our results suggest that over-expression of betaAPP in cultured muscle fibers inhibits their innervation, and that the accumulation of betaAPP in muscle fibers of both h- and s-IBM patients may be responsible for their not becoming or remaining properly innervated or reinnervated, i.e. a 'myogenous-dysinnervation' mechanism.     

Biliary stricture due to mucosal hyperplasia of the common bile duct: a case report

A 71-year-old man with a tumorous lesion of the common bile duct is presented. Although histological examination of tumor biopsy specimens taken at the time of duodenoscopy or percutaneous transhepatic choledochoscopy showed mucosal hyperplasia, cancer could not be ruled out; therefore, the patient underwent pancreatoduodenectomy. However, histology showed nodular mucosal hyperplasia and an ectopic pancreas adjacent to the papilla of Vater, which was suspected to have contributed to the development of the lesion by stimulating the mucosa of the papilla and the distal common bile duct.     

Early ischemia-induced alterations of the outer mitochondrial membrane and the intermembrane space: a potential cause for altered energy transfer in cardiac muscle?

Our aim was to carefully analyse the time-dependent changes that affect the mitochondrial function of myocardial cells during and after an ischemic episode. To this end, variables characterizing mitochondrial function have been evaluated on myocardial samples from isolated rat hearts subjected to different conditions of ischemia. The technique of permeabilized fibers was used in order to evaluate the mitochondrial function whilst retaining intracellular structure. The earliest alteration that could be detected was a decrease in the stimulatory effect of creatine on mitochondrial respiration. This alteration became more pronounced as the severity (or duration) of the ischemia increased. Afterwards, a significant decrease in the apparent Km of mitochondrial respiration for ADP also appeared, followed by a diminution of the maximal respiration rate which was partly restored by adding cytochrome c. Finally, for the most severe conditions of ischemia, the basal respiratory rate also increased. These observations are indicative of a sequence of alterations affecting first the intermembrane space, then the outer mitochondrial membrane, and finally the inner membrane. The discussion is focused on the very early alterations, that could not be detected using the conventional techniques of isolated mitochondria. We postulate that these alterations to the intermembrane space and outer mitochondrial membrane can induce disturbances both in the channelling of energy from the mitochondria, and on the signalling towards the mitochondria. The potential consequences on the regulation of the production of energy (ATP, PC) by the mitochondria are evoked.     

The tryptophan residues of mitochondrial creatine kinase: roles of Trp-223, Trp-206, and Trp-264 in active-site and quaternary structure formation

The 5 tryptophan residues of chicken sarcomeric mitochondrial creatine kinase (Mib-CK) were individually replaced by phenylalanine or cysteine using site-directed mutagenesis. The mutant proteins were analyzed by enzyme kinetics, fluorescence spectroscopy, circular dichroism, and conformational stability studies. In the present work, Trp-223 is identified as an active-site residue whose replacement even by phenylalanine resulted in > or = 96% inactivation of the enzyme. Trp-223 is responsible for a strong (18-21%) fluorescence quenching effect occurring upon formation of a transition state-analogue complex (TSAC;Mib-CK.creatine.MgADP.NO3-), and Trp-223 is probably required for the conformational change leading to the TSAC-induced octamer dissociation of Mib-CK. Replacement of Trp-206 by cysteine led to a destabilization of the active-site structure, solvent exposure of Trp-223, and to the dissociation of the Mib-CK dimers into monomers. However, this dimer dissociation was counteracted by TSAC formation or the presence of ADP alone. Trp-264 is shown to be located at the dimer-dimer interfaces within the Mib-CK octamer, being the origin of another strong (25%) fluorescence quenching effect, which was observed upon the TSAC-induced octamer dissociation. Substitution of Trp-264 by cysteine drastically accelerated the TSAC-induced dissociation and destabilized the octameric structure by one-fourth of the total free interaction energy, probably by weakening hydrophobic contacts. The roles of the other 2 tryptophan residues, Trp-213 and Trp-268, could be less well assigned.     

Factors affecting the susceptibility of staphylococci to killing by the cationic proteins from rabbit polymorphonuclear leucocytes: the effects of alteration of cellular energetics and of various iron compounds

Anaerobiosis, various respiratory inhibitors and certain agents altering cellular energetics profoundly affect the staphylocidal action of the cationic proteins from rabbit polymorphonuclear leucocytes. It is suggested that sensitivity to these proteins depends on the structure of the cell membrane as influenced by (1) the oxidation level of the cytochrome chain and (2) its energized state. Agents such as amytal and rotenone, which cause a block at the beginning of the chain and would increase its oxidation level, enhance killing, whereas those causing a block in or at the end of the chain, such a 2-n-heptyl-4-hydroxyquinoline-N-oxide, cyanide and anaerobiosis, which would cause reduction of a part or whole of the chain, prevent killing. Among agents altering the energized state of the membrane, dicyclohexyl-carbodi-imide, an ATPase inhibitor, does not prevent killing, whereas 2,4-dinitrophenol, carbonylcyanide-trifluoromethoxy-phenylhydrazone and 5-Cl, 3-t-butyl, 2'-Cl, 4'-NO2-salicylanilide, all uncouplers and ionophores for a specific ion, do prevent killing, although gramicidin, a relatively nonspecific ionophore, does not. The paper also contains an extension of previous work on the effect of iron and haematin, to include various other iron compounds and haematin derivatives.     

Determination of the affinity and kinetic constants for the interaction between the human virus echovirus 11 and its cellular receptor, CD55

The biochemical properties of the molecular interactions mediating viral-cell recognition are poorly characterized. In this study, we use surface plasmon resonance to study the affinity and kinetics of the interaction of echovirus 11 with its cellular receptor decay-accelerating factor (CD55). As reported for interactions between cell-cell recognition molecules, the interaction has a low affinity (KD approximately 3.0 microM) as a result of a very fast dissociation rate constant (kon approximately 10(5) M-1.s-1, koff approximately 0.3 s-1). This contrasts with the interaction of soluble ICAM-1 (sICAM-1, CD54) with human rhinovirus 3 which has been reported to have a similar affinity but 10(2)-10(3)-fold slower kinetics (Casasnovas, J. M., and Springer, T. A. (1995) J. Biol. Chem. 270, 13216-13224). The extracellular portion of decay-accelerating factor comprises four short consensus repeat domains (domains 1-4) and a mucin-like stalk. By comparison of the binding affinity for echovirus 11 of various fragments of decay-accelerating factor, we are able to conclude that short consensus repeat domain 3 contributes approximately 80% of the binding energy.     

Role of oxidative stress and the glutathione system in loss of dopamine neurons due to impairment of energy metabolism

Alterations in the glutathione system and impairment in energy metabolism have both been implicated in the loss of dopamine neurons in Parkinson's disease. This study examined the importance of cellular glutathione and the involvement of oxidative stress in the loss of mesencephalic dopamine and GABA neurons due to inhibition of energy metabolism with malonate, the reversible, competitive inhibitor of succinate dehydrogenase. Consistent with previous findings, exposure to malonate for 24 h followed by 48 h of recovery caused a dose-dependent loss of the dopamine population with little effect on the GABA population. Toxicity was assessed by simultaneous measurement of the high-affinity uptake of [3H]dopamine and [14C]GABA. Total glutathione content in rat mesencephalic cultures was decreased by 65% with a 24-h pretreatment with 10 microM buthionine sulfoxamine. This reduction in glutathione level greatly potentiated damage to both the dopamine and GABA populations and removed the differential susceptibility between the two populations in response to malonate. These findings point to a role for oxidative stress occurring during energy impairment by malonate. Consistent with this, several spin-trapping agents, alpha-phenyl-tert-butyl nitrone and two cyclic nitrones, MDL 101,002 and MDL 102,832, completely prevented malonate-induced damage to the dopamine neurons in the absence of buthionine sulfoxamine. The spin-trapping agents also completely prevented toxicity to both the dopamine and GABA populations when cultures were exposed to malonate after pretreatment with buthionine sulfoxamine to reduce glutathione levels. Counts of tyrosine hydroxylase-positive neurons verified enhancement of cell loss by buthionine sulfoxamine plus malonate and protection against cell loss by the spin-trapping agents. NMDA receptors have also been shown to play a role in malonate-induced dopamine cell loss and are associated with the generation of free radicals. Consistent with this, toxicity to the dopamine neurons due to a 1-h exposure to 50 microM glutamate was attenuated by the nitrone spin traps. These findings provide evidence for an oxidative challenge occurring during inhibition of energy metabolism by malonate and show that glutathione is an important neuroprotectant for midbrain neurons during situations when energy metabolism is impaired.     

The sorting of mitochondrial DNA and mitochondrial proteins in zygotes: preferential transmission of mitochondrial DNA to the medial bud

Green fluorescent protein (GFP) was used to tag proteins of the mitochondrial matrix, inner, and outer membranes to examine their sorting patterns relative to mtDNA in zygotes of synchronously mated yeast cells in rho+ x rho0 crosses. When transiently expressed in one of the haploid parents, each of the marker proteins distributes throughout the fused mitochondrial reticulum of the zygote before equilibration of mtDNA, although the membrane markers equilibrate slower than the matrix marker. A GFP-tagged form of Abf2p, a mtDNA binding protein required for faithful transmission of rho+ mtDNA in vegetatively growing cells, colocalizes with mtDNA in situ. In zygotes of a rho+ x rho+ cross, in which there is little mixing of parental mtDNAs, Abf2p-GFP prelabeled in one parent rapidly equilibrates to most or all of the mtDNA, showing that the mtDNA compartment is accessible to exchange of proteins. In rho+ x rho0 crosses, mtDNA is preferentially transmitted to the medial diploid bud, whereas mitochondrial GFP marker proteins distribute throughout the zygote and the bud. In zygotes lacking Abf2p, mtDNA sorting is delayed and preferential sorting is reduced. These findings argue for the existence of a segregation apparatus that directs mtDNA to the emerging bud.