From bivalves to birds : oxidative stress and longevity

1. The oxidative stress theory of ageing predicts that animals living longer will have less cumulative oxidative damage together with structural characteristics that make them more resistant to oxidative damage itself.
2. Although a general relationship between body size, metabolism and longevity does not exist in marine invertebrates, they are generally characterized by low rates of metabolism and reactive oxygen species (ROS) formation associated with lower antioxidant enzyme activities compared to vertebrates.
3. Birds and mammals have very similar size-affected metabolic rates and their metabolic intensity explains only some of the variation in maximum lifespan potential (MLSP).Within each class, smaller animals have higher rates of metabolism and ROS production and membranes that are more susceptible to oxidative damage and autocatalytic propagation of free radicals than larger ones.
4. Although the high variation in life-history strategies is accompanied by substantial variation in MLSP, there is a consistent positive correlation between rates of ROS formation and antioxidant levels among most animals examined so far for these traits. The consensus of these studies is that ROS and antioxidant levels are inversely related to MLSP.
5. The lack of a clear stoichiometric relation between variables contributing to oxidative stress limits our capacity to infer longevity consequences from measures of pro-oxidant or antioxidant status among or within species

Functional analysis of yeast snoRNA and snRNA 3' end formation mediated by uncoupling of cleavage and polyadenylation

Many nuclear and nucleolar small RNAs are accumulated as nonpolyadenylated species and require 3′-end processing for maturation. Here, we show that several genes coding for box C/D and H/ACA snoRNAs and for the U5 and U2 snRNAs contain sequences in their 3′ portions which direct cleavage of primary transcripts without being polyadenylated. Genetic analysis of yeasts with mutations in different components of the pre-mRNA cleavage and polyadenylation machinery suggests that this mechanism of 3"-end formation requires cleavage factor IA (CF IA) but not cleavage and polyadenylation factor activity. However, in vitro results indicate that other factors participate in the reaction besides CF IA. Sequence analysis of snoRNA genes indicated that they contain conserved motifs in their 3" noncoding regions, and mutational studies demonstrated their essential role in 3"-end formation. We propose a model in which CF IA functions in cleavage and polyadenylation of pre-mRNAs and, in combination with a different set of factors, in 3"-end formation of nonpolyadenylated polymerase II transcripts.

Molecular basis of copper transport: cellular and physiological functions of Menkes and Wilson disease proteins (ATP7A and ATPB)

Eukaryotic cells prevent copper-induced, free radical damage to cell components by employing copper-binding proteins and transporters that minimize the likelihood of free copper ions existing in the cell. In the cell, copper is actively transported from the cytoplasm during the biosynthesis of secreted coppercontaining proteins and, as a protective measure, when there is an excess of copper. In humans, this is accomplished by two related copper-transporting ATPases (ATP7A and ATP7B), which are the affected genes in two distinct human genetic disorders of copper transport, Menkes disease (copper deficiency) and Wilson disease (copper toxicosis). The study of these ATPases has revealed their molecular mechanisms of copper transport and their roles in physiological copper homeostasis. Both ATP7A and ATP7B are expressed in specific brain regions and neurological abnormalities are important clinical features in Menkes and Wilson disease.

Immunolocalisation of sodium/proton exchanger-like proteins in the gills of elasmobranchs

Na⁺/H⁺ exchangers are integral membrane proteins that exchange Na⁺ and H⁺ across cell membranes. The Na⁺/H⁺ exchangers 2 and 3 are epithelial isoforms in mammals and contribute to acid–base homeostasis. The gills of fishes, including elasmobranchs, are also associated with acid/base balance, and are probably the primary acid/base regulatory organ. This study examines the presence of Na⁺/H⁺ exchangers 2 and 3 using immunohistochemistry and immunoblotting in the gills of four species of elasmobranchs, the banjo ray (Trygonorrhina fasciata), southern eagle ray (Myliobatis australis), the gummy shark (Mustelus antarcticus) and the Australian angel shark (Squatina australis) using heterologous antibodies. Na⁺/H⁺ exchanger 2-like immunoreactivity was observed in the gills of the banjo ray, eagle ray and angel shark. In the banjo and eagle rays, this Na⁺/H⁺ exchanger-like immunoreactivity co-localised with immunoreactivity to Na⁺/K⁺-ATPase, a marker for the mitochondrial-rich cells of fishes. Na⁺/H⁺ exchanger 3-like immunoreactivity was only observed in the gills of the angel and gummy sharks, some Na⁺/H⁺ exchanger 3-like cells also showed Na⁺/K⁺-ATPase immunoreactivity. However, immunoblotting of banjo and eagle ray gill membranes demonstrated Na⁺/H⁺ exchanger 3-like immunoreactivity, which was not consistent with the immunohistochemical results. These data demonstrate the presence of epithelial Na⁺/H⁺ exchangers 2 and 3 in the gills of elasmobranchs and a link with acid/base regulation is suggested.

A short-term, high-fat diet up-regulates lipid metabolism and gene expression in human skeletal muscle

Background: Dietary fatty acids may be important in regulating gene expression. However, little is known about the effect of changes in dietary fatty acids on gene regulation in human skeletal muscle.
Objective: The objective was to determine the effect of altered dietary fat intake on the expression of genes encoding proteins necessary for fatty acid transport and ß-oxidation in skeletal muscle.
Design: Fourteen well-trained male cyclists and triathletes with a mean (± SE) age of 26.9 ± 1.7 y, weight of 73.7 ± 1.7 kg, and peak oxygen uptake of 67.0 ± 1.3 mL ˙ kg-1 ˙ min-1 consumed either a high-fat diet (HFat: > 65% of energy as lipids) or an isoenergetic high-carbohydrate diet (HCho: 70–75% of energy as carbohydrate) for 5 d in a crossover design. On day 1 (baseline) and again after 5 d of dietary intervention, resting muscle and blood samples were taken. Muscle samples were analyzed for gene expression [fatty acid translocase (FAT/CD36), plasma membrane fatty acid binding protein (FABPpm), carnitine palmitoyltransferase I (CPT I), ß-hydroxyacyl-CoA dehydrogenase (ß-HAD), and uncoupling protein 3 (UCP3)] and concentrations of the proteins FAT/CD36 and FABPpm.
Results: The gene expression of FAT/CD36 and &szlig; -HAD and the gene abundance of FAT/CD36 were greater after the HFat than after the HCho diet (P < 0.05). Messenger RNA expression of FABPpm, CPT I, and UCP-3 did not change significantly with either diet.
Conclusions: A rapid and marked capacity for changes in dietary fatty acid availability to modulate the expression of mRNA-encoding proteins is necessary for fatty acid transport and oxidative metabolism. This finding is evidence of nutrient-gene interactions in human skeletal muscle.

Effect of elevated lipid concentrations on human skeletal muscle gene expression

Dietary fatty acids regulate the abundance and activity of various proteins involved in the regulation of fat oxidation by functioning as regulators of gene transcription. To determine whether the transcription of key lipid metabolic proteins necessary for fat metabolism within human skeletal muscle are regulated by acute elevations in circulating free fatty acid (FFA) concentrations, 7 healthy men underwent 3 randomized resting infusions of Intralipid (20%) with heparin sodium, saline and heparin sodium, or saline only for 5 hours. These infusions significantly elevated plasma FFA concentrations by 15-fold (to 1.67 ± 0.13 mmol/L) in the Intralipid infusion trial, with modest elevations observed in the saline and heparin sodium and saline alone infusion groups (0.67 ± 0.09 and 0.49 ± 0.087 mmol/L, P < .01 both vs Intralipid infusion). Analysis of messenger RNA (mRNA) concentration demonstrated that pyruvate dehydrogenase kinase isoform 4 (PDK4) mRNA, a key negative regulator of glucose oxidation, was increased in all trials with a 24-fold response after Intralipid infusion, 15-fold after saline and heparin infusion, and 9-fold after saline alone. The PDK4 increases were not significantly different between the 3 trials. The mRNA concentration of the major uncoupling protein within skeletal muscle, uncoupling protein 3, was not elevated in parallel to the increased plasma FFA as similar (not, vert, similar2-fold) increases were evident in all trials. Additional genes involved in lipid transport (fatty acid translocase/CD36), oxidation (carnitine palmitoyltransferase I), and metabolism (1-acylglycerol-3-phosphate O-acyltransferase 1, hormone-sensitive lipase, and peroxisomal proliferator-activated receptor-γ coactivator-1α) were not altered by increased circulating FFA concentrations. The present data demonstrate that of the genes analyzed that encode proteins that are key regulators of lipid homeostasis within skeletal muscle, only the PDK4 gene is uniquely sensitive to increasing FFA concentrations after increased plasma FFA achieved by intravenous lipid infusion.

Exercise does not alter subcellular localization, but increases phosphorylation of insulin-signaling proteins in human skeletal muscle

The subcellular localization of insulin signaling proteins is altered by various stimuli such as insulin, insulin-like growth factor I, and oxidative stress and is thought to be an important mechanism that can influence intracellular signal transduction and cellular function. This study examined the possibility that exercise may also alter the subcellular localization of insulin signaling proteins in human skeletal muscle. Nine untrained males performed 60 min of cycling exercise (~67% peak pulmonary O₂ uptake). Muscle biopsies were sampled at rest, immediately after exercise, and 3 h postexercise. Muscle was fractionated by centrifugation into the following crude fractions: cytosolic, nuclear, and a high-speed pellet containing membrane and cytoskeletal components. Fractions were analyzed for protein content of insulin receptor, insulin receptor substrate (IRS)-1 and -2, p85 subunit of phosphatidylinositol 3-kinase, Akt, and glycogen synthase kinase-3 (GSK-3). There was no significant change in the protein content of the insulin signaling proteins in any of the crude fractions after exercise or 3 h postexercise. Exercise had no significant effect on the phosphorylation of IRS-1 Tyr612 in any of the fractions. In contrast, exercise increased (P < 0.05) the phosphorylation of Akt Ser⁴⁷³ and GSK-3α/ß Ser^9/21 in the cytosolic fraction only. In conclusion, exercise can increase phosphorylation of downstream insulin signaling proteins specifically in the cytosolic fraction but does not result in changes in the subcellular localization of insulin signaling proteins in human skeletal muscle. Change in the subcellular protein localization is therefore an unlikely mechanism to influence signal transduction pathways and cellular function in skeletal muscle after exercise.

Decreased fatty acit Â-oxidation in riboflavin-responsive, multiple acylocoenzyme a dehydrogenase-deficient patients is associated with an increase in uncoupling protein-3

Riboflavin-responsive, multiple acylcoenzyme A dehydrogenase deficiency (RR-MAD), a lipid storage myopathy, is characterized by, among others, a decrease in fatty acid (FA) ß-oxidation capacity. Muscle uncoupling protein 3 (UCP3) is up-regulated under conditions that either increase the levels of circulating free FA and/or decrease FA ß-oxidation. Using a relatively large cohort of seven RR-MAD patients, we aimed to better characterize the metabolic disturbances of this disease and to explore the possibility that it might increase UCP3 expression. A battery of biochemical and molecular tests were performed, which demonstrated decreases in FA ß-oxidation and in the activities of respiratory chain complexes I and II. These metabolic alterations were associated with increases of 3.1- and 1.7-fold in UCP3 mRNA and protein expression, respectively. All parameters were restored to control values after riboflavin treatment. We postulate that the up-regulation of UCP3 in RR-MAD is due to the accumulation of muscle FA/acylCoA. RR-MAD is an optimal model to support the hypothesis that UCP3 is involved in the outward translocation of an excess of FA from the mitochondria and to show that, in humans, the effects of FA on UCP3 expression are direct and independent of fatty acid ß-oxidation.

Endosomal trafficking of the Menkes copper ATPase ATP7A is mediated by vesicles containing the Rab7 and Rab5 GTPase proteins

The Cu-ATPase ATP7A (MNK) is localized in the trans-Golgi network (TGN) and relocalizes in the plasma membrane via vesicle-mediated traffic following exposure of the cells to high concentrations of copper. Rab proteins are organelle-specific GTPases, markers of different endosomal compartments; their role has been recently reviewed (Trends Cell Biol. 11(2001) 487). In this article we analyze the endosomal pathway of trafficking of the MNK protein in stably transfected clones of CHO cells, expressing chimeric Rab5-myc or Rab7-myc proteins, markers of early or late endosome compartments, respectively. We demonstrate by immunofluorescence and confocal and electron microscopy techniques that the increase in the concentration of copper in the medium (189 μM) rapidly induces a redistribution of the MNK protein from early sorting endosomes, positive for Rab5-myc protein, to late endosomes, containing the Rab7-myc protein. Cell fractionation experiments confirm these results; i.e., the MNK protein is recruited to the endosomal fraction on copper stimulation and colocalizes with Rab5 and Rab7 proteins. These findings allow the first characterization of the vesicles involved in the intracellular routing of the MNK protein from the TGN to the plasma membrane, a key mechanism allowing appropriate efflux of copper in cells grown in high concentrations of the metal.

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.

Characterisation of proteins in the milk of fur seals

Milk protein composition was investigated throughout the lactation periods of the Australian fur seal (Arctocephalus pusillus doriferus) and Antarctic fur seal (Arctocephalus gazella). The mean protein content of the milk was found to be 10.9% and 10.6% respectively. The concentration of total protein did not change during lactation, although a decline in casein content of the milk in late lactation was apparent. Milk protein concentration during a foraging/suckling cycle of the Antarctic fur seal analysed at the time of arrival on shore, and 24 h and 72 h after arrival was 12.8%, 11.4% and 12.5% respectively. Re-feeding animals at 72 h resulted in a significant increase in milk protein content to 14.9%. Characterisation of milk protein by SDS-PAGE analysis revealed 5 casein and 10 major whey protein bands. Amino-terminal sequencing indicated that the majority of the whey fraction of the milk is β-lactoglobulin (β-LG). The limited amino acid sequence indicated 3 different β-LGs were secreted in the milk. Subsequently, RT-PCR was used to extend the sequence of one of the β-LGs and translation of the 464 bp fragment indicated that it shared 79% sequence identity with feline β-LG II.

Alterations in the proportions of skeletal muscle proteins following a unilateral lesion to the substantia nigra pars compacta of rats

It is well established that mammalian skeletal muscles exhibit a considerable degree of plasticity and one of the main determining factors of this plasticity is the activity pattern and duration of motoneurone discharge. Lesions to the right substantia nigra pars compacta (SNpc) of six adult rats were made to determine whether altered output from the SNpc ultimately leads to a change in the expression of proteins in contralateral skeletal muscles. After 4 months, altered motor performance was identified by the administration of amphetamine. After 7 months, 30–70% of dopaminergic cells in the SNpc had been destroyed. The protein content of muscles was then quantified from densitometric scans of gels, and expressed as a % of the amount of actin (the protein used as a reference in this study). The lesion affected the expression of different protein isoforms in the fast- and slow-twitch muscles. In slow-twitch soleus muscles, the lesion decreased the proportion of α-tropomyosin and increased the proportion of β-tropomyosin. In the fast-twitch extensor digitorum longus muscles, the lesion increased the proportion of the fast isoform of troponin-T_1f, and decreased the proportions of the two isoforms of myosin light chain. This study establishes a connection between the chronic effects of a lesion to the SNpc, with a loss of dopaminergic neurones, impaired motor performance, and altered expression of proteins in skeletal muscle. The implication of these results is that the altered motor function observed in Parkinson’s disease may be associated with alterations to the expression of skeletal muscle proteins.

Effect of accurate exercise on uncoupling protein3 is a fat metabolism-mediated effect

Human and rodent uncoupling protein (UCP)3 mRNA is upregulated after acute exercise. Moreover, exercise increases plasma levels of free fatty acid (FFA), which are also known to upregulate UCP3. We investigated whether the upregulation of UCP3 after exercise is an effect of exercise per se or an effect of FFA levels or substrate oxidation. Seven healthy untrained men [age: 22.7 ± 0.6 yr; body mass index: 23.8 ± 1.0 kg/m²; maximal O₂ uptake (VO_{2 max}): 3,852 ± 211 ml/min] exercised at 50% VO_{2 max} for 2 h and then rested for 4 h. Muscle biopsies and blood samples were taken before and immediately after 2 h of exercise and 1 and 4 h in the postexercise period. To modulate plasma FFA levels and fat/glucose oxidation, the experiment was performed two times, one time with glucose ingestion and one time while fasting. UCP3 mRNA and UCP3 protein were determined by RT-competitive PCR and Western blot. In the fasted state, plasma FFA levels significantly increased (P < 0.0001) during exercise (293 ± 25 vs. 1,050 ± 127 μmol/l), whereas they were unchanged after glucose ingestion (335 ± 54 vs. 392 ± 74 µmol/l). Also, fat oxidation was higher after fasting (P < 0.05), whereas glucose oxidation was higher after glucose ingestion (P < 0.05). In the fasted state, UCP3L mRNA expression was increased significantly (P < 0.05) 4 h after exercise (4.6 ± 1.2 vs. 9.6 ± 3.3 amol/µg RNA). This increase in UCP3L mRNA expression was prevented by glucose ingestion. Acute exercise had no effect on UCP3 protein levels. In conclusion, we found that acute exercise had no direct effect on UCP3 mRNA expression. Abolishing the commonly observed increase in plasma FFA levels and/or fatty acid oxidation during and after exercise prevents the upregulation of UCP3 after acute exercise. Therefore, the previously observed increase in UCP3 expression appears to be an effect of prolonged elevation of plasma FFA levels and/or increased fatty acid oxidation.

Expression of uncoupling protein-3 subsarcolemmal and intermyofibrillar mitochondria of various mouse muscle types and its moduclation by fasting

Uncoupling protein-3 (UCP3) is a mitochondrial inner-membrane protein abundantly expressed in rodent and human skeletal muscle which may be involved in energy dissipation. Many studies have been performed on the metabolic regulation of UCP3 mRNA level, but little is known about UCP3 expression at the protein level. Two populations of mitochondria have been described in skeletal muscle, subsarcolemmal (SS) and intermyofibrillar (IMF), which differ in their intracellular localization and possibly also their metabolic role. To examine if UCP3 is differentially expressed in these two populations and in different mouse muscle types, we developed a new protocol for isolation of SS and IMF mitochondria and carefully validated a new UCP3 antibody. The data show that the density of UCP3 is higher in the mitochondria of glycolytic muscles (tibialis anterior and gastrocnemius) than in those of oxidative muscle (soleus). They also show that SS mitochondria contain more UCP3 per mg of protein than IMF mitochondria. Taken together, these results suggest that oxidative muscle and the mitochondria most closely associated with myofibrils are most efficient at producing ATP. We then determined the effect of a 24-h fast, which greatly increases UCP3 mRNA (16.4-fold) in muscle, on UCP3 protein expression in gastrocnemius mitochondria. We found that fasting moderately increases (1.5-fold) or does not change UCP3 protein in gastrocnemius SS or IMF mitochondria, respectively. These results show that modulation of UCP3 expression at the mRNA level does not necessarily result in similar changes at the protein level and indicate that UCP3 density in SS and IMF mitochondria can be differently affected by metabolic changes.