Effect of age on in vivo rates of mitochondrial protein synthesis in human skeletal muscle

A progressive decline in muscle performance in the rapidly expanding aging population is causing a dramatic increase in disability and health care costs. A decrease in muscle endurance capacity due to mitochondrial decay likely contributes to this decline in muscle performance. We developed a novel stable isotope technique to measure in vivo rates of mitochondrial protein synthesis in human skeletal muscle using needle biopsy samples and applied this technique to elucidate a potential mechanism for the age-related decline in the mitochondrial content and function of skeletal muscle. The fractional rate of muscle mitochondrial protein synthesis in young humans (24 ± 1 year) was 0.081 ± 0.004%·h−1, and this rate declined to 0.047 ± 0.005%·h−1 by middle age (54 ± 1 year; P < 0.01). No further decline in the rate of mitochondrial protein synthesis (0.051 ± 0.004%·h−1) occurred with advancing age (73 ± 2 years). The mitochondrial synthesis rate was about 95% higher than that of mixed protein in the young, whereas it was approximately 35% higher in the middle-aged and elderly subjects. In addition, decreasing activities of mitochondrial enzymes were observed in muscle homogenates (cytochrome c oxidase and citrate synthase) and in isolated mitochondria (citrate synthase) with increasing age, indicating declines in muscle oxidative capacity and mitochondrial function, respectively. The decrease in the rates of mitochondrial protein synthesis is likely to be responsible for this decline in muscle oxidative capacity and mitochondrial function. These changes in muscle mitochondrial protein metabolism may contribute to the age-related decline in aerobic capacity and muscle performance.

Cardiolipin is a normal component of human plasma lipoproteins

Anticardiolipin (anti-CL) antibodies, diagnostic for antiphospholipid antibody syndrome, are associated with increased risks of venous and arterial thrombosis. Because CL selectively enhances activated protein C/protein S-dependent anticoagulant activities in purified systems and because CL is not known to be a normal plasma component, we searched for CL in plasma. Plasma lipid extracts [chloroform/methanol (2:1, vol/vol)] were subjected to analyses by using TLC, analytical HPLC, and MS. A plasma lipid component was purified that was indistinguishable from reference CL (M:1448). When CL in 40 fasting plasma lipid extracts (20 males, 20 females) was quantitated by using HPLC, CL (mean ± SD) was 14.9 ± 3.7 μg/ml (range 9.1 to 24.2) and CL was not correlated with phosphatidylserine (3.8 ± 1.7 μg/ml), phosphatidylethanolamine (64 ± 20 μg/ml), or choline-containing phospholipid (1,580 ± 280 μg/ml). Based on studies of fasting blood donors, CL (≥94%) was recovered in very low density, low density, and high density lipoproteins (11 ± 5.3%, 67 ± 11.0%, and 17 ± 10%, respectively), showing that the majority of plasma CL (67%) is in low density lipoprotein. Analysis of relative phospholipid contents of lipoproteins indicated that high density lipoprotein is selectively enriched in CL and phosphatidylethanolamine. These results shows that CL is a normal plasma component and suggest that the epitopes of antiphospholipid antibodies could include CL or oxidized CL in lipoproteins or in complexes with plasma proteins (e.g., β2-glycoprotein I, prothrombin, protein C, or protein S) or with platelet or endothelial surface proteins.

Circadian rhythms in Neurospora crassa: Lipid deficiencies restore robust rhythmicity to null frequency and white-collar mutants

The conidiation rhythm in the fungus Neurospora crassa is a model system for investigating the genetics of circadian clocks. Null mutants at the frq (frequency) locus (frq9 and frq10) make no functional frq gene products and are arrhythmic under standard conditions. The white-collar strains (wc-1 and wc-2) are insensitive to most effects of light, and are also arrhythmic. All three genes are proposed to be central components of the circadian oscillator. We have been investigating two mutants, cel (chain-elongation) and chol-1 (choline-requirer), which are defective in lipid synthesis and affect the period and temperature compensation of the rhythm. We have constructed the double mutant strains chol-1 frq9, chol-1 frq10, chol-1 wc-1, chol-1 wc-2, cel frq9, cel frq10, and cel wc-2. We find that these double mutant strains are robustly rhythmic when assayed under lipid-deficient conditions, indicating that free-running rhythmicity does not require the frq, wc-1, or wc-2 gene products. The rhythms in the double mutant strains are similar to the cel and chol-1 parents, except that they are less sensitive to light. This suggests that the frq, wc-1, and wc-2 gene products may be components of a pathway that normally supplies input to a core oscillator to transduce light signals and sustain rhythmicity. This pathway can be bypassed when lipid metabolism is altered.

Cholecystokinin-8 protects central cholinergic neurons against fimbria–fornix lesion through the up-regulation of nerve growth factor synthesis

In this study, we demonstrate that cholecystokinin-8 (CCK-8) induces an increase in both nerve growth factor (NGF) protein and NGF mRNA in mouse cortex and hippocampus when i.p. injected at physiological doses. By using fimbria–fornix-lesioned mice, we have also demonstrated that repeated CCK-8 i.p. injections result in recovery of lesion-induced NGF deficit in septum and restore the baseline NGF levels in hippocampus and cortex. Parallel to the effects on NGF, CCK-8 increases choline acetyltransferase (Chat) activity in forebrain when injected in unlesioned mice and counteract the septo-hippocampal Chat alterations in fimbria–fornix-lesioned mice. To assess the NGF involvement in the mechanism by which CCK-8 induces brain Chat, NGF antibody was administrated intracerebrally to saline- and CCK-8-injected mice. We observe that pretreatment with NGF antibody causes a marked reduction of NGF and Chat activity in septum and hippocampus of both saline- and CCK-8-injected mice. This evidence indicates that the CCK-8 effects on cholinergic cells are mediated through the synthesis and release of NGF. Taken together, our results suggest that peripheral administration of CCK-8 may represent a potential experimental model for investigating the effects of endogenous NGF up-regulation on diseases associated with altered brain cholinergic functions.

Region-specific differentiation of neural tube-derived neuronal restricted progenitor cells after heterotopic transplantation

Spinal cord neuronal restricted progenitor (NRP) cells, when transplanted into the neonatal anterior forebrain subventricular zone, migrate to distinct regions throughout the forebrain including the olfactory bulb, frontal cortex, and occipital cortex but not to the hippocampus. Their migration pattern and differentiation potential is distinct from anterior forebrain subventricular zone NRPs. Irrespective of their final destination, NRP cells do not differentiate into glia. Rather they synthesize neurotransmitters, acquire region-specific phenotypes, and receive synapses from host neurons after transplantation. Spinal cord NRPs express choline acetyl transferase even in regions where host neurons do not express this marker. The restricted distribution of transplanted spinal cord NRP cells and their acquisition of varied region-specific phenotypes suggest that their ultimate fate and phenotype is dictated by a combination of intrinsic properties and extrinsic cues from the host.

Lamellar lipoproteins uniquely contribute to hyperlipidemia in mice doubly deficient in apolipoprotein E and hepatic lipase

Remnants of triglyceride-rich lipoproteins containing apolipoprotein (apo) B-48 accumulate in apo E-deficient mice, causing pronounced hypercholesterolemia. Mice doubly deficient in apo E and hepatic lipase have more pronounced hypercholesterolemia, even though remnants do not accumulate appreciably in mice deficient in hepatic lipase alone. Here we show that the doubly deficient mice manifest a unique lamellar hyperlipoproteinemia, characterized by vesicular particles 600 Å–1,300 Å in diameter. As seen by negative-staining electron microscopy, these lipoproteins also contain an electron-lucent region adjacent to the vesicle wall, similar to the core of typical lipoproteins. Correlative chemical analysis indicates that the vesicle wall is composed of a 1:1 molar mixture of cholesterol and phospholipids, whereas the electron-lucent region appears to be composed of cholesteryl esters (about 12% of the particle mass). Like the spherical lipoproteins of doubly deficient mice, the vesicular particles contain apo B-48, but they are particularly rich in apo A-IV. We propose that cholesteryl esters are removed from spherical lipoproteins of these mice by scavenger receptor B1, leaving behind polar lipid-rich particles that fuse to form vesicular lipoproteins. Hepatic lipase may prevent such vesicular lipoproteins from accumulating in apo E-deficient mice by hydrolyzing phosphatidyl choline as scavenger receptor B1 removes the cholesteryl esters and by gradual endocytosis of lipoproteins bound to hepatic lipase on the surface of hepatocytes.

Point mutant mice with hypersensitive α4 nicotinic receptors show dopaminergic deficits and increased anxiety

Knock-in mice were generated that harbored a leucine-to-serine mutation in the α4 nicotinic receptor near the gate in the channel pore. Mice with intact expression of this hypersensitive receptor display dominant neonatal lethality. These mice have a severe deficit of dopaminergic neurons in the substantia nigra, possibly because the hypersensitive receptors are continuously activated by normal extracellular choline concentrations. A strain that retains the neo selection cassette in an intron has reduced expression of the hypersensitive receptor and is viable and fertile. The viable mice display increased anxiety, poor motor learning, excessive ambulation that is eliminated by very low levels of nicotine, and a reduction of nigrostriatal dopaminergic function upon aging. These knock-in mice provide useful insights into the pathophysiology of sustained nicotinic receptor activation and may provide a model for Parkinson's disease.

Manufacturing DNA microarrays of high spot homogeneity and reduced background signal

Analyses on DNA microarrays depend considerably on spot quality and a low background signal of the glass support. By using betaine as an additive to a spotting solution made of saline sodium citrate, both the binding efficiency of spotted PCR products and the homogeneity of the DNA spots is improved significantly on aminated surfaces such as glass slides coated with the widely used poly-l-lysine or aminosilane. In addition, non-specific background signal is markedly diminished. Concomitantly, during the arraying procedure, the betaine reduces evaporation from the microtitre dish wells, which hold the PCR products. Subsequent blocking of the chip surface with succinic anhydride was improved considerably in the presence of the non-polar, non-aqueous solvent 1,2-dichloroethane and the acylating catalyst N-methylimidazole. This procedure prevents the overall background signal that occurs with the frequently applied aqueous solvent 1-methyl-2-pyrrolidone in borate buffer because of DNA that re-dissolves from spots during the blocking process, only to bind again across the entire glass surface.

Oxaloacetate Transport into Plant Mitochondria1

The properties of oxaloacetate (OA) transport into mitochondria from potato (Solanum tuberosum) tuber and pea (Pisum sativum) leaves were studied by measuring the uptake of 14C-labeled OA into liposomes with incorporated mitochondrial membrane proteins preloaded with various dicarboxylates or citrate. OA was found to be transported in an obligatory counterexchange with malate, 2-oxoglutarate, succinate, citrate, or aspartate. Phtalonate inhibited all of these countertransports. OA-malate countertransport was inhibited by 4,4′-dithiocyanostilbene-2,2′-disulfonate and pyridoxal phosphate, and also by p-chloromercuribenzene sulfonate and mersalyl, indicating that a lysine and a cysteine residue of the translocator protein are involved in the transport. From these and other inhibition studies, we concluded that plant mitochondria contain an OA translocator that differs from all other known mitochondrial translocators. Major functions of this translocator are the export of reducing equivalents from the mitochondria via the malate-OA shuttle and the export of citrate via the citrate-OA shuttle. In the cytosol, citrate can then be converted either into 2-oxoglutarate for use as a carbon skeleton for nitrate assimilation or into acetyl-coenzyme A for use as a precursor for fatty acid elongation or isoprenoid biosynthesis.

Evidence for an Intrinsic Toxicity of Phosphatidylcholine to Sec14p-dependent Protein Transport from the Yeast Golgi Complex

Yeast phosphatidylinositol-transfer protein (Sec14p) is essential for Golgi secretory function and cell viability. This requirement of Sec14p is relieved by genetic inactivation of the cytidine diphosphate-choline pathway for phosphatidycholine (PtdCho) biosynthesis. Standard phenotypic analyses indicate that inactivation of the phosphatidylethanolamine (PtdEtn) pathway for PtdCho biosynthesis, however, does not rescue the growth and secretory defects associated with Sec14p deficiency. We now report inhibition of choline uptake from the media reveals an efficient “bypass Sec14p” phenotype associated with PtdEtn-methylation pathway defects. We further show that the bypass Sec14p phenotype associated with PtdEtn-methylation pathway defects resembles other bypass Sec14p mutations in its dependence on phospholipase D activity. Finally, we find that increased dosage of enzymes that catalyze phospholipase D-independent turnover of PtdCho, via mechanisms that do not result in a direct production of phosphatidic acid or diacylglycerol, effect a partial rescue of sec14-1ts-associated growth defects. Taken together, these data support the idea that PtdCho is intrinsically toxic to yeast Golgi secretory function.

Methionine adenosyltransferase 1A knockout mice are predisposed to liver injury and exhibit increased expression of genes involved in proliferation

Liver-specific and nonliver-specific methionine adenosyltransferases (MATs) are products of two genes, MAT1A and MAT2A, respectively, that catalyze the formation of S-adenosylmethionine (AdoMet), the principal biological methyl donor. Mature liver expresses MAT1A, whereas MAT2A is expressed in extrahepatic tissues and is induced during liver growth and dedifferentiation. To examine the influence of MAT1A on hepatic growth, we studied the effects of a targeted disruption of the murine MAT1A gene. MAT1A mRNA and protein levels were absent in homozygous knockout mice. At 3 months, plasma methionine level increased 776% in knockouts. Hepatic AdoMet and glutathione levels were reduced by 74 and 40%, respectively, whereas S-adenosylhomocysteine, methylthioadenosine, and global DNA methylation were unchanged. The body weight of 3-month-old knockout mice was unchanged from wild-type littermates, but the liver weight was increased 40%. The Affymetrix genechip system and Northern and Western blot analyses were used to analyze differential expression of genes. The expression of many acute phase-response and inflammatory markers, including orosomucoid, amyloid, metallothionein, Fas antigen, and growth-related genes, including early growth response 1 and proliferating cell nuclear antigen, is increased in the knockout animal. At 3 months, knockout mice are more susceptible to choline-deficient diet-induced fatty liver. At 8 months, knockout mice developed spontaneous macrovesicular steatosis and predominantly periportal mononuclear cell infiltration. Thus, absence of MAT1A resulted in a liver that is more susceptible to injury, expresses markers of an acute phase response, and displays increased proliferation.

Purification and Characterization of NAD-Isocitrate Dehydrogenase from Chlamydomonas reinhardtii1

NAD-isocitrate dehydrogenase (NAD-IDH) from the eukaryotic microalga Chlamydomonas reinhardtii was purified to electrophoretic homogeneity by successive chromatography steps on Phenyl-Sepharose, Blue-Sepharose, diethylaminoethyl-Sephacel, and Sephacryl S-300 (all Pharmacia Biotech). The 320-kD enzyme was found to be an octamer composed of 45-kD subunits. The presence of isocitrate plus Mn2+ protected the enzyme against thermal inactivation or inhibition by specific reagents for arginine or lysine. NADH was a competitive inhibitor (Ki, 0.14 mm) and NADPH was a noncompetitive inhibitor (Ki, 0.42 mm) with respect to NAD+. Citrate and adenine nucleotides at concentrations less than 1 mm had no effect on the activity, but 10 mm citrate, ATP, or ADP had an inhibitory effect. In addition, NAD-IDH was inhibited by inorganic monovalent anions, but l-amino acids and intermediates of glycolysis and the tricarboxylic acid cycle had no significant effect. These data support the idea that NAD-IDH from photosynthetic organisms may be a key regulatory enzyme within the tricarboxylic acid cycle.

Aluminum-Resistant Arabidopsis Mutants That Exhibit Altered Patterns of Aluminum Accumulation and Organic Acid Release from Roots1

Al-resistant (alr) mutants of Arabidopsis thaliana were isolated and characterized to gain a better understanding of the genetic and physiological mechanisms of Al resistance. alr mutants were identified on the basis of enhanced root growth in the presence of levels of Al that strongly inhibited root growth in wild-type seedlings. Genetic analysis of the alr mutants showed that Al resistance was semidominant, and chromosome mapping of the mutants with microsatellite and random amplified polymorphic DNA markers indicated that the mutants mapped to two sites in the Arabidopsis genome: one locus on chromosome 1 (alr-108, alr-128, alr-131, and alr-139) and another on chromosome 4 (alr-104). Al accumulation in roots of mutant seedlings was studied by staining with the fluorescent Al-indicator dye morin and quantified via inductively coupled argon plasma mass spectrometry. It was found that the alr mutants accumulated lower levels of Al in the root tips compared with wild type. The possibility that the mutants released Al-chelating organic acids was examined. The mutants that mapped together on chromosome 1 released greater amounts of citrate or malate (as well as pyruvate) compared with wild type, suggesting that Al exclusion from roots of these alr mutants results from enhanced organic acid exudation. Roots of alr-104, on the other hand, did not exhibit increased release of malate or citrate, but did alkalinize the rhizosphere to a greater extent than wild-type roots. A detailed examination of Al resistance in this mutant is described in an accompanying paper (J. Degenhardt, P.B. Larsen, S.H. Howell, L.V. Kochian [1998] Plant Physiol 117: 19–27).

A unique phospholipid organization in bovine erythrocyte membranes

Ruminant erythrocytes are remarkable for their choline-phospholipid anomalies; namely, low or absent phosphatidylcholine (PC) along with high sphingomyelin levels. Here, we report another anomaly in bovine erythrocytes that affects aminophospholipids: phosphatidylethanolamine (PE) shows an extreme asymmetry, with only 2% of the total present in the outer leaflet. Furthermore, we found that phospholipase A2, an enzyme located on the external surface of the erythrocytes, shows higher activity against PC than against PE. In addition, we observed that acylation of PE is by far the most important biosynthetic event in this system. We propose that deacylation of PE and PC by phospholipase A2 to generate lysocompounds, followed by selective reacylation of lyso-PE in the inner leaflet, can account for the compositional and architectural peculiarities of bovine erythrocyte membranes.

Energy metabolism, enzymatic flux capacities, and metabolic flux rates in flying honeybees.

Honeybees rely primarily on the oxidation of hexose sugars to provide the energy required for flight. Measurement of VCO2 (equal to VO2, because VCO2/VO2 = 1.0 during carbohydrate oxidation) during flight allowed estimation of steady-state flux rates through pathways of flight muscle energy metabolism. Comparison of Vmax values for flight muscle hexokinase, phosphofructokinase, citrate synthase, and cytochrome c oxidase with rates of carbon and O2 flux during flight reveal that these enzymes operate closer to Vmax in the flight muscles of flying honeybees than in other muscles previously studied. Possible mechanistic and evolutionary implications of these findings are discussed.