J protein cochaperone of the mitochondrial inner membrane required for protein import into the mitochondrial matrix

The major Hsp70 of the mitochondrial matrix (Ssc1 in yeast) is critically important for the translocation of proteins from the cytosol, across the mitochondrial inner membrane, and into the matrix. Tim44, a peripheral inner membrane protein with limited sequence similarity to the J domain of J-type cochaperones, tethers Ssc1 to the import channel. Here we report that, unlike a J protein, Tim44 does not stimulate the ATPase activity of Ssc1, nor does it affect the stimulation by either a known mitochondrial J protein or a peptide substrate. Thus, we conclude that Tim44 does not function as a J protein cochaperone of Ssc1; rather, it tethers Ssc1 to the import channel through interactions independent of those critical for J protein function. However, a previously unstudied essential gene, PAM18, encodes an 18-kDa protein that contains a J domain and is localized to the mitochondrial inner membrane. Pam18 stimulates the ATPase activity of Ssc1; depletion of Pam18 in vivo disrupts import of proteins into the mitochondrial matrix. We propose that Pam18 is the J protein partner for Ssc1 at the import channel and is critical for Ssc1's function in protein import.     

Biochemical indicators of vitamin A depletion in children with cholestasis

Biochemical indicators of vitamin A status were measured in 24 children (1 month to 6 years old) with severe cholestasis starting early in life and in 21 children (3 months to 13 years old) with liver disease but without cholestasis. Liver vitamin A concentrations, expressed as micrograms of retinol per gram of liver (mean +/- S.D.), were 6.3 +/- 7.1 (range: 0.14 to 28) and 143 +/- 108 (range: 18 to 424), respectively, in cholestatic and non-cholestatic children. In infants less than 6 months of age, liver vitamin A values less than 10 micrograms per gm were found in 14 of 17 cholestatic children but in none of 3 non-cholestatic subjects. Plasma vitamin A values, expressed as micrograms of retinol per deciliter (mean +/- S.D.), were 23 +/- 18 (range: 3 to 62) and 46 +/- 33 (range: 14 to 125), respectively, for the two groups. Plasma retinol values less than 10 micrograms per dl were always associated with liver concentrations less than 10 micrograms per gm. Plasma retinol-binding protein was only reduced to 71% of control values in cholestatic children. The fatty acid composition of liver retinyl esters was unaffected by any condition studied. Infants with chronic cholestasis are in a precarious nutritional status very early in life relative to liver reserves of vitamin A. Plasma vitamin A values, unless less than 10 micrograms retinol per dl, are poor indicators of inadequate vitamin A status.     

Content mixing and membrane integrity during membrane fusion driven by pairing of isolated v-SNAREs and t-SNAREs

Membrane bilayer fusion has been shown to be mediated by v- and t-SNAREs initially present in separate populations of liposomes and to occur with high efficiency at a physiologically meaningful rate. Lipid mixing was demonstrated to involve both the inner and the outer leaflets of the membrane bilayer. Here, we use a fusion assay that relies on duplex formation of oligonucleotides introduced in separate liposome populations and report that SNARE proteins suffice to mediate complete membrane fusion accompanied by mixing of luminal content. We also find that SNARE-mediated membrane fusion does not compromise the integrity of liposomes.     

Energy depletion retards the loss of membrane transport during reticulocyte maturation.

The effect of metabolic depletion on the maturation-associated loss of membrane functions has been studied by using sheep reticulocytes incubated in vitro at 37 degrees C for periods up to 41 hr. ATP was either maintained with glucose, adenosine plus inosine, or depleted with 2-deoxyglucose plus arsenate. Two membrane transport systems were studied: Na+-dependent glycine transport activity and the sodium pump, estimated from measurements of the number of [3H]ouabain binding sites per cell. Both transport systems were decreased during maturation. However, the decrease was much less in ATP-depleted cells compared to ATP-replete cells. It is concluded that the loss of certain functions during reticulocyte maturation is retarded by metabolic depletion.     

Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase

Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants.Although proteins localized to biological membranes constitute about a quarter of the products of an organism’s ORFs, only 0.1% of known protein structures are membrane proteins (1, 2). The lag in our knowledge of the physiology and structural biology of membrane proteins is due to many factors including the low expression levels of membrane proteins and the complex biophysical nature of their interactions at the water/lipid interface. Membrane proteins, which have one or more segments that traverse the lipid bilayer, require dedicated cellular machineries for their targeting to and integration into the membranes (3). Even though these cellular machineries have maintained conserved features in all domains of life, efforts to express membrane proteins in foreign organisms have often been unsuccessful. In many cases, the overexpression of indigenous membrane proteins leads to mis-targeting and inclusion body formation, protein degradation, or cell death (4).Promoting the correct orientation of proteins within the membrane provides an additional challenge to the insertion machinery. Proper assembly is a crucial step in the folding pathway of a membrane protein because a wrong orientation could be detrimental to functioning of the protein and to the viability of the cell. The overall architecture of a membrane protein, including its final membrane topology, is achieved by topogenic signals embedded in the amino acid sequence. Such signals include long stretches of mostly hydrophobic amino acids (around 20) that determine the portion of the protein to be embedded in the membrane. Furthermore, the abundance of positively charged amino acids in cytoplasmic domains of membrane proteins, generally referred to as the positive-inside rule, is a major contributor to membrane protein topology (5, 6).In Escherichia coli, the direct insertion of proteins into the inner membrane is mediated by the translocon protein complex SecYEG and/or the insertase YidC (7). Although the majority of inner membrane proteins tested require SecYEG for assembly, fewer are dependent on YidC for integration into the membrane (8). Nevertheless, both integration machineries are essential for E. coli viability. A previous study presented evidence that some proteins dependent on the YidC protein for proper assembly are ones that exhibit violations of the positive-inside rule (9). More recent work has also suggested that both decreasing transmembrane helix (TMH) hydrophobicity and increasing polarity, particularly the positive charge of the periplasmic loop of a model protein, increases dependence on YidC (10).In this paper, we describe a genetic selection for mutants of E. coli that promote the efficient membrane integration and expression of a foreign membrane protein, one that is ordinarily barely expressed in a wild-type E. coli. These studies arose out of our interest in protein disulfide bond formation in bacteria. The formation of disulfide bonds in E. coli depends on two enzymes: DsbA that directly catalyzes disulfide bond formation in protein substrates in the bacterial cell envelope and DsbB that maintains DsbA in the oxidized state (11). In contrast to E. coli, many bacteria (e.g., mycobacteria) use the membrane protein VKOR for oxidation of DsbA instead of DsbB (Fig. 1) (12). Despite the lack of homology of Mycobacterium tuberculosis VKOR (MtbVKOR) with DsbB, MtbVKOR, when cloned into E. coli, can substitute for the enzyme EcDsbB (12). Bacterial VKORs are so named because they were first identified as homologs of the mammalian integral membrane protein vitamin K epoxide reductase (VKORc1) involved in blood coagulation (12). VKORc1 is also the target of the widely used anticoagulant warfarin (13). Because we are carrying out high-throughput screening to identify compounds that might serve as leads for antibiotics against M. tuberculosis (14) we sought to express mammalian VKORc1 in E. coli as a simple counter-screen to exclude compounds that might be toxic to humans due to anticoagulant activity. However, when the mammalian VKORc1 was expressed from efficient E. coli promoters, it failed to substitute for EcDsbB. Therefore, we devised for this work a genetic selection for mutants that allowed functioning of VKORc1 in the E. coli disulfide bond-forming pathway and enhanced the expression of the protein VKORc1. The first mutations obtained resulted in amino acid substitutions that reduced the positive charge in a hydrophilic loop of VKORc1 (SI Appendix, Fig. S1) and gave measurable although weak restoration of disulfide bond formation. Then, after mutagenesis of an E. coli dsbB⁻ strain expressing one of these VKORc1 mutations, we identified and characterized chromosomal mutations that further enhanced the expression of VKORc1 function in disulfide bond formation. Mutations in the genes for the YidC protein and for the protease HslV were repeatedly isolated in independent selections. The yidC mutations mapped to a putative substrate-binding site in the insertase YidC, and the hslV mutations mapped to essential catalytic residues in HslV. The results reported here suggest that YidC may play a role in quality control of membrane proteins at the level of insertion. Alteration of this function can greatly enhance functional overexpression of other membrane proteins. Our findings shed light on the problems leading to the dysfunctional expression of at least some foreign membrane proteins in E. coli and on the mechanism of YidC-mediated membrane protein insertion and quality control. Our results also allow new potential approaches to studying the mechanism of action of VKORc1 and to obtaining inhibitors of this blood coagulation protein.Open in a separate windowFig. 1.Disulfide bond formation in E. coli is promoted by EcDsbB and MtbVKOR but not by vertebrate VKORc1.     

A direct role for hepatitis B virus X protein in inducing mitochondrial membrane permeabilization

Hepatitis B virus X protein (HBx) acts as a multifunctional protein that regulates intracellular signalling pathways during HBV infection. It has mainly been studied in terms of its interaction with cellular proteins. Here, we show that HBx induces membrane permeabilization independently of the mitochondrial permeability transition pore complex. We generated mitochondrial outer membrane‐mimic liposomes to observe the direct effects of HBx on membranes. We found that HBx induced membrane permeabilization, and the region comprising the transmembrane domain and the mitochondrial‐targeting sequence was sufficient for this process. Membrane permeabilization was inhibited by nonselective channel blockers or by N‐(n‐nonyl)deoxynojirimycin (NN‐DNJ), a viroporin inhibitor. Moreover, NN‐DNJ inhibited HBx‐induced mitochondrial depolarization in Huh‐7 cells. Based on the results of this study, we can postulate that the HBx protein itself is sufficient to induce mitochondrial membrane permeabilization. Our finding provides important information for a strategy of HBx targeting during HBV treatment.     

Glycolysis protects sarcolemmal membrane integrity during total ischemia in the rat heart

Experimental evidence indicates that ischemic glycolysis improves myocardial tolerance to low flow ischemia and anoxia, and cellular membrane disruption signals and/or causes transition to irreversible ischemic injury. The objective of this study was to determine the impact of ischemic glycolysis on membrane integrity and myocardial viability during total ischemia. Phosphorus metabolites were measured by ³¹P NMR spectroscopy and cellular volumes were determined by ¹H and ⁵⁹Co NMR in conjunction with the extracellular marker cobalticyanide. Isolated rat hearts were submitted to 30 min of total ischemia, followed by 30 min of reperfusion. Glycogen contents were modulated by pre-ischemic perfusion with various substrates. Increased glycolytic activities, as determined from lactate production, delayed onset of ischemic contracture (p < 0.05), induced cytosolic acidification (p < 0.005) and cellular swelling during ischemia (p < 0.05), reduced post-ischemic diastolic tone (p < 0.05), improved recovery of high energy phosphates and contraction force (p < 0.005). Inhibition of glycolysis with iodoacetate and glycogen depletion with 2-deoxyglycose resulted in early onset of ischemic contracture (p < 0.005), elevated post-ischemic diastolic pressures (p < 0.05), reduced coronary flow rates and mechanical activities (p < 0.05). Cellular viability was evaluated by creatine kinase efflux, and membrane integrity was determined from cellular swelling during perfusion with hypoosmotic medium. High activities of ischemic glycolysis correlated with improved cellular viability and preserved membrane integrity, while low glycolytic fluxes were associated with membrane permeabilization (p < 0.05). The protective effect of ischemic glycolysis over sarcolemmal integrity was attributed to continuous provision of energy, undetected by ³¹P NMR spectroscopy. There was no evidence that ischemic swelling caused by glycolytic end-metabolites accumulation had detrimental consequences, and of excessive swelling during reperfusion. It is concluded that one of the cardio-protective mechanisms of ischemic glycolysis is energy-dependent preservation of sarcolemmal integrity and cellular viability. Received: 3 January 2001, Returned for revision: 25 January 2001, Revision received: 14 February 2001, Accepted: 15 March 2001     

Effect of vitamin D status on chick kidney proteins: detection of a 45-kilodalton mitochondrial protein suppressed by vitamin D

Two-dimensional polyacrylamide gel electrophoresis along with L-[35S]methionine radiolabeling studies were used to examine the effect of chronic vitamin D status on the composition and relative abundance of chick kidney proteins. Comparison of silver-stained gels revealed no extensive differences in either the electrophoretic mobility or the amounts of kidney proteins present in the mitochondrial fraction from vitamin D-replete and vitamin D-deficient chicks. A similar result was obtained in studies with L-[35S]methionine-labeled proteins. Vitamin D deficiency specifically elevated levels of a 45-kilodalton mitochondrial protein (pI 5.0 to 5.5) by approximately 5- to 12-fold relative to amounts present in vitamin D-replete tissue. This protein could not be detected in postmitochondrial supernatant fractions and was only faintly visible in crude kidney homogenates. The specificity of the observed suppression of this 45-kilodalton protein by vitamin D suggests that it may play an important role in renal functions influenced by the vitamin D endocrine system.     

Mitigation of age-dependent accumulation of defective mitochondrial genomes

Unknown processes promote the accumulation of mitochondrial DNA (mtDNA) mutations during aging. Accumulation of defective mitochondrial genomes is thought to promote the progression of heteroplasmic mitochondrial diseases and degenerative changes with natural aging. We used a heteroplasmic Drosophila model to test 1) whether purifying selection acts to limit the abundance of deleterious mutations during development and aging, 2) whether quality control pathways contribute to purifying selection, 3) whether activation of quality control can mitigate accumulation of deleterious mutations, and 4) whether improved quality control improves health span. We show that purifying selection operates during development and growth but is ineffective during aging. Genetic manipulations suggest that a quality control process known to enforce purifying selection during oogenesis also suppresses accumulation of a deleterious mutation during growth and development. Flies with nuclear genotypes that enhance purifying selection sustained higher genome quality, retained more vigorous climbing activity, and lost fewer dopaminergic neurons. A pharmacological agent thought to enhance quality control produced similar benefits. Importantly, similar pharmacological treatment of aged mice reversed age-associated accumulation of a deleterious mtDNA mutation. Our findings reveal dynamic maintenance of mitochondrial genome fitness and reduction in the effectiveness of purifying selection during life. Importantly, we describe interventions that mitigate and even reverse age-associated genome degeneration in flies and in mice. Furthermore, mitigation of genome degeneration improved well-being in a Drosophila model of heteroplasmic mitochondrial disease.

Unlike nuclear genotype, which is largely stable during one’s lifetime, when genetically distinct mitochondrial genomes co-reside (heteroplasmy), their relative proportions shift during growth, development, and aging. This shift is not random (1–7). Mutant mitochondrial DNA (mtDNA) variants accumulate during aging and in the progression of some mitochondrial diseases (1–5). Stereotyped changes in abundance of particular alleles in different tissues in human and mouse indicate that selective forces favor different mitochondrial genomes (3, 6, 7). However, despite the importance of mitochondrial function to health and well-being, we have limited understanding of the processes underlying the accumulation of mitochondrial mutations with age.The nuclear genome encodes mechanisms of quality control that survey the function of mitochondria and eliminate or compromise the proliferation of defective mitochondria (8, 9). Two described mechanisms use PINK1, the product of a gene discovered as one of the causes of early-onset familial Parkinson’s disease, as a sensor of mitochondrial function. PINK1 accumulates on the surface of mitochondria having a reduced membrane potential, and its kinase activity in this location signals several downstream events (10–15). In one pathway defined largely in a cell culture model, PINK1 activates PARKIN, the product of another Parkinson’s disease gene, which then triggers elimination of compromised mitochondria by mitophagy (16). In a second pathway, acting in the Drosophila female germ line (17, 18), PINK1 acts in a PARKIN-independent pathway that targets a protein called Larp to inhibit its role in promoting biogenesis of the mitochondria (18) (SI Appendix, Fig. S2).Since deleterious mtDNA mutations compromise electron transport of mitochondria, it seems that quality control would put genomes carrying such mutations at a disadvantage, creating a purifying selection that leads to their elimination. However, this outcome is far from certain. Various factors such as the sharing of gene products among mitochondria as a result of dynamic fission and fusion could mask the consequences of heteroplasmic mutations, shielding them from quality control. Indeed, a number of studies suggest a contrast between the germ line, which exhibits purifying selection, and adult somatic tissues, which often show accumulation of mutations. Studies in Drosophila show that purifying selection acting in the female germ line eliminates deleterious mutations in a few generations (18–21). Genetic dissection revealed that this purifying selection depends on the PINK1/LARP pathway of quality control (18). On the other hand, an elegant study that induced heteroplasmic deletions in the flight muscle of the adult fly detected no substantial indications of purifying selection unless additional stressors were introduced (22). Furthermore, a detailed study in Drosophila carrying a proofreading defective mitochondrial DNA polymerase (mutator line) showed that adult flies accumulate a spectrum of mutations biased toward deleterious mutations (23). Since deleterious mutations would potentially be removed by purifying selection, this finding argues either that it was not operating or that it was opposed by a stronger selection favoring deleterious mutations (23). Similarly, studies in mouse suggest a discordance between germ line and soma. When mutations in the mitochondrial genome that were introduced in a mutator line were passed through subsequent generations in a wild-type background, there was selective elimination of deleterious mutations, a strong signal of purifying selection (7). In contrast, zygotically accumulated mutations in the mutator mouse exhibited high levels of deleterious mutations, suggesting a lack of purifying selection in the soma. Furthermore, Parkin mutant mice did not show a significant increase in mutations in adult wild-type or mutator mice, suggesting that Parkin-dependent quality control does not contribute to purifying selection (16). While these studies suggest major changes in the efficiency of purifying selection, other studies have suggested continued purifying selection in some circumstances. For example, adult human T cells of mitochondrial disease patients show exceptionally low heteroplasmy levels, suggesting cell type-specific action of purifying selection (24). We sought to measure purifying selection and to understand the nature of quality control in the soma during growth, development, and aging using an experimental model developed in Drosophila.A previously described heteroplasmic line of Drosophila melanogaster carries a wild-type mitochondrial genome (Yak-mt) from another species, Drosophila yakuba, and a D. melanogaster genome (Mel-mt^ts) crippled by a temperature-sensitive mutation in cytochrome oxidase subunit 1 (mt:CoI^T300I) [Fig. 1A and (25)]. The Mel-mt^ts genome has an intrinsic advantage in replication. At a permissive temperature, it gradually displaces the Yak-mt genome, resulting in the loss of the D. yakuba genome in a few generations (25). At 29 °C, a temperature at which the CoI^T3000I mutant cannot support viability (26), purifying selection counters the replicative advantage of the mutant Mel-mt^ts genome, preventing it from taking over (25). qPCR gives a measure of the ratio Yak-mt/total-mt. The difference in this ratio between permissive and restrictive temperatures allows us to assess the impact of a functional disparity on competition between the two genomes and provides a measure of purifying selection.Open in a separate windowFig. 1.Quality control modulates the ratio of heteroplasmic mitochondrial genomes during development. (A) Heteroplasmy for schematized Yak-mt and Mel-mt^ts genomes was established by transferring cytoplasm of D. yakuba embryos into D. melanogaster embryos carrying the doubly mutant genome mt:ND2^del + mt:Col^T300I (Mel-mt^ts). (B) The proportion of Yak-mt (Yak-mt/total-mt) following development from egg to adult shows action of quality control. Eggs (2-h collection at 29 °C) were assayed (gray bar) or allowed to develop to 5 d after eclosion at 22 °C (blue) or 29 °C (amber). Adult females have a large contribution from oocyte mtDNA. (C) Quality control operates in multiple tissues with different effectiveness. The blue and amber bars (22 °C or 29 °C, respectively) show the proportion of Yak-mt in different tissues of late third instar larvae. (D) The impact of purifying selection declined with age in the CNS. (E) The decline in relative abundance of wild-type, indicated by the dashed regression lines (orange for 29°C and blue for 22°C), Yak-mt following eclosion was fast. (F) The signature of quality control is absent during maturation of adults. Yak-mt/total-mt ratios from gut and brain taken from 5-d (gray bars) or 20-d adults aged at 22 °C or 29 °C (blue and amber bars, respectively). Here and below, *P < 0.05; **P < 0.01; and ***P < 0.001 by one-way ANOVA/Tukey’s multiple comparison test. Data represent eight independent biological repeats, with each repeat being an average of ratios assessed in three samples of eggs or adults. For tissues, data represent tissues dissected from eight individuals. Error bars represent standard error. In E, slopes differ (*P < 0.05) by linear regression. Cyto, cytoplasm; AED, after egg deposition; ns, not significant.Past work using this heteroplasmic line focused on changes in the relative abundance of the two genomes from one generation to the next and uncovered the action of purifying selection during oogenesis (18–21, 25, 27). It is notable that this selection depends on quality control, occurs by competition between mitochondria within the oocyte, and does not involve selection for organismal fitness (18, 21). Changes in the ratio of Yak-mt/total-mt during the lifetime of the fly suggested that maintenance of this ratio is dynamic (25). Here, we examined changes in the relative abundance of the two genomes in the soma. Shifts in the ratio of Yak-mt/total-mt provide a measure of the effectiveness of purifying selection during the life of the fly and a means of assessing whether purifying selection can be genetically or pharmacologically modified. We found evidence that purifying selection is active in the soma during growth and development. The influence of mutations in quality control genes suggests that somatic mechanisms of quality control overlap those operating in the germ line. However, the effectiveness of purifying selection declines with age and has no obvious impact in tested tissues beyond 5 d after eclosion of adult flies. Importantly, we found that quality control can be stimulated in the adult by genetic alterations or by feeding kinetin and that such measures forestall mutational accumulation and aging phenotypes in Drosophila and can reverse mutational accumulation in aged mice.     

Merocyanine 540-sensitized photoinactivation of leukemia cells: role of oxygen and effects on plasma membrane integrity and mitochondrial respiration

Merocyanine 540 (MC 540) is a photosensitizing dye that is used clinically for the purging of autologous bone marrow grafts and preclinically for the inactivation of enveloped viruses in blood products. In this paper we present evidence that the MC 540-sensitized photoinactivation of leukemia cells is an oxygen-dependent process and that unsaturated plasma membrane lipids are substrates for singlet oxygen and/or other activated oxygen species generated by photoirradiated MC 540. A comparison of the inhibition of clonal growth, the inhibition of mitochondrial respiration, and the exclusion of trypan blue by the plasma membrane after exposure to MC 540 and graded doses of light showed that mitochondrial respiration is compromised relatively early in the course of the dye-mediated photoinactivation of cells, well before the plasma membrane loses its capacity to exclude trypan blue. It also showed that trypan blue exclusion assays can greatly underestimate the cytotoxic effects of MC 540-sensitized photoirradiation.     

Effects of vitamin A and ethanol on liver plasma membrane fluidity

To study possible mechanisms whereby vitamin A and ethanol may affect liver plasma membranes, rats were fed liquid diets containing either 6 international units of vitamin A per kcal or 5 times more, with or without ethanol (36% of total energy as isocaloric substitution for carbohydrate). Vitamin A supplementation resulted in 2- to 3-fold increases of liver plasma membrane free retinol (p less than 0.005) and retinyl esters (p less than 0.001), particularly esters of palmitate and oleate, whereas cholesterol esters did not change. The fluorescent probe 1,6-diphenyl-1,3,5-hexatriene revealed decreased fluidity as measured by an increase in fluorescence polarization which correlated significantly with retinyl palmitate plus oleate content in membranes. In rats fed ethanol chronically, we first verified our previous observation of a decrease in liver plasma membrane fluorescence polarization. We now find this effect to be associated with (and possibly due to) an increase of cholesterol ester content. In linear regression analysis, the change in fluorescence polarization correlated positively with vitamin A (p less than 0.02) and negatively with cholesterol ester contents (p less than 0.001). Ethanol feeding partially offset the effect of vitamin A supplementation on fluorescence polarization. We conclude from these observations that liver plasma membranes contain a significant amount of vitamin A, that vitamin A supplementation increases membrane fluorescence polarization and that chronic ethanol administration can interfere with this effect.     

Cardiac features of a novel autosomal recessive dilated cardiomyopathic syndrome due to defective importation of mitochondrial protein

Dilated cardiomyopathy as seen in children is clinically and genetically heterogeneous, with an increasing proportion of cases known to be caused by disorders of single genes. An autosomal recessive syndrome with a high incidence of dilated cardiomyopathy was recently described in the Canadian Dariusleut Hutterite population. It is caused by homozygous mutations in a novel gene, DNAJC19, presumed to play a role in importation of mitochondrial proteins. We discuss the cardiac features of this syndrome, and its relationship to cardiac mitochondrial function.     

Relationship of mitochondrial membrane potential to hemoglobin synthesis during Friend cell maturation

Previous studies have shown that exposure to imidazole dissociates hemoglobin synthesis from other aspects of cell maturation in dimethylsulfoxide (DMSO)-treated mouse erythroleukemia (MEL) cells. In the present study, we have found that imidazole causes hyperpolarization of the mitochondrial membrane in MEL cells exposed to DMSO, in contrast to the depolarization observed with DMSO alone. Like the defect in hemoglobin synthesis, membrane hyperpolarization is reversible upon removal of imidazole and incubation of cells with DMSO alone. These correlations suggest that alterations in the electrostatic properties of the mitochondrial membrane, due directly or indirectly to the effects of imidazole, interfere with heme synthesis but not with other aspects of the maturation process in these developing erythroid cells.     

MitoNEET is an iron-containing outer mitochondrial membrane protein that regulates oxidative capacity

Members of the thiazolidinedione (TZD) class of insulin-sensitizing drugs are extensively used in the treatment of type 2 diabetes. Pioglitazone, a member of the TZD family, has been shown to bind specifically to a protein named mitoNEET [Colca JR, McDonald WG, Waldon DJ, Leone JW, Lull JM, Bannow CA, Lund ET, Mathews WR (2004) Am J Physiol 286:E252-E260]. Bioinformatic analysis reveals that mitoNEET is a member of a small family of proteins containing a domain annotated as a CDGSH-type zinc finger. Although annotated as a zinc finger protein, mitoNEET contains no zinc, but instead contains 1.6 mol of Fe per mole of protein. The conserved sequence C-X-C-X(2)-(S/T)-X(3)-P-X-C-D-G-(S/A/T)-H is a defining feature of this unique family of proteins and is likely involved in iron binding. Localization studies demonstrate that mitoNEET is an integral protein present in the outer mitochondrial membrane. An amino-terminal anchor sequence tethers the protein to the outer membrane with the CDGSH domain oriented toward the cytoplasm. Cardiac mitochondria isolated from mitoNEET-null mice demonstrate a reduced oxidative capacity, suggesting that mito- NEET is an important iron-containing protein involved in the control of maximal mitochondrial respiratory rates.     

线粒体TOM系统在心血管疾病中的研究现状

线粒体内稳态的维持与其内部蛋白密切相关,而绝大多数蛋白进入线粒体内部发挥作用均需通过线粒体的线粒体外膜转位酶(translocase of the outer mitochondrial membrane,TOM)系统的转运。研究表明,线粒体TOM系统相关组成亚基Tom70、Tom20和Tom40等参与了心血管疾病的发生发展,这为我们从线粒体蛋白水平研究心血管疾病的机制及开发新的治疗措施提供了思路。本文针对线粒体TOM系统在心血管相关疾病(如心肌缺血/再灌注、高血压及心功能衰竭)方面的研究现状进行了综述。     

Clinical and molecular features of mitochondrial DNA depletion syndromes

Summary  Mitochondrial DNA depletion syndromes (MDSs) form a group of autosomal recessive disorders characterized by profoundly decreased mitochondrial DNA copy numbers in affected tissues. Three main clinical presentations are known: myopathic, encephalomyopathic and hepatocerebral. The first is associated with mutations in thymidine kinase 2 (TK2) and p53-induced ribonucleotide reductase B subunit (RRM2B); the second with mutations in succinate synthase A (SUCLA2) and B (SUCLG1); the third with mutations in Twinkle (PEO1), pol-γA (POLG1), deoxyguanosine kinase (DGUOK) and MPV17 (MPV17). In this work, we review the MDS-associated phenotypes and present our own experience of 32 MDS patients, with the aim of defining the mutation frequency of the known genes, the clinical spectrum of the diseases, and the genotype–phenotype correlations. Five of our patients carried previously unreported mutations in one of the eight MDS genes. Competing interests: None declared References to electronic databases: MDS myopathic form: OMIM #609560. MDS encephalomyopathic form: OMIM #612073. MDS, encephalomyopathic form, with renal tubulopathy: OMIM #612075. MDS hepatocerebral form: OMIM #251880. Presented at the Annual Symposium of the SSIEM, Lisbon, Portugal, 2–5 September 2008     

Retinol-binding protein: the serum transport protein for vitamin A

The information available regarding the chemical structure of RBP, the structure of the RBP gene, and the expression of the RBP gene has expanded dramatically in recent years. Still many questions concerning RBP remain to be answered. The longstanding and important questions concerning the possible existence and the biochemical characteristics of the RBP cell surface receptor are in need of resolution. The factors that regulate RBP secretion from the liver still remain to be fully elucidated. Additional information concerning the physiological role of RBP synthesis in extrahepatic tissues is needed. Considering what is now known, it is clear that in the future much intense research will be required before the many important questions regarding the structure, synthesis, secretion, and physiological roles of RBP can be answered.     

Free radical theory of aging: Consequences of mitochondrial aging

Mitochondria may serve as biologic clocks. This paper provides plausible explanations, based on mitochondrial aging, of some aging phenomenon: a) the inverse relationship between basal metabolic rate and life span; b) the antioxidants studies thus far which increase the average life span of mice, depress body weight and fail to lengthen maximum life span; c) the association of degenerative diseases with the terminal part of the life span; d) the exponential nature of the mortality curve, and e) the beneficial effect of caloric restriction on degenerative diseases and life span. A short discussion is also presented of the effect of exercise on life span and aging of muscle mitochondria.     

Innate immunity, gut integrity, and vitamin A in Gambian and Indian infants

Gut integrity, which can be measured by the urinary lactulose:mannitol excretion test, deteriorates with the introduction of weaning foods. In The Gambia, gut integrity measured monthly over 15 months in 119 infants (aged 2-15 months) was least impaired from April to June. This coincides with the time of year of maximum vitamin A (VA) intake-the mango season. Subsequently, two VA intervention studies were done in infants in India. Eighty infants attending a community health center received 16,700 IU weekly or placebo. In another study, 94 hospitalized infants were given 200, 000 IU VA or placebo: 31 received VA on admission, while the rest (32 VA, 31 placebo) received treatment on discharge. All VA-treated groups had more rapid improvement in gut integrity than the placebo groups, but no group had gut integrity normalized by Western standards. The data suggest that VA status may influence gut integrity.