Caloric restriction prevents age-associated accrual of oxidative damage to mouse skeletal muscle mitochondria

The purpose of this study was to understand the nature of the causes underlying the senescence-related decline in skeletal muscle mass and performance. Protein and lipid oxidative damage to upper hindlimb skeletal muscle mitochondria was compared between mice fed ad libitum and those restricted to 40% fewer calories—a regimen that increases life span by 30–40% and attenuates the senescence-associated decrement in skeletal muscle mass and function. Oxidative damage to mitochondrial proteins, measured as amounts of protein carbonyls and loss of protein sulfhydryl content, and to mitochondrial lipids, determined as concentration of thiobarbituric acid reactive substances, significantly increased with age in the ad libitum-fed (AL) C57BL/6 mice. The rate of superoxide anion radical generation by submitochondrial particles increased whereas the activities of antioxidative enzymes superoxide dismutase, catalase, and glutathione peroxidase in muscle homogenates remained unaltered with age in the AL group. In calorically-restricted (CR) mice there was no age-associated increase in mitochondrial protein or lipid oxidative damage, or in superoxide anion radical generation. Crossover studies, involving the transfer of 18- to 22-month-old mice fed on the AL regimen to the CR regimen, and vice versa, indicated that the mitochondrial oxidative damage could not be reversed by CR or induced by AL feeding within a time frame of 6 weeks. Results of this study indicate that mitochondria in skeletal muscles accumulate significant amounts of oxidative damage during aging. Although such damage is largely irreversible, it can be prevented by restriction of caloric intake.     

Developmental patterns in the antioxidant defenses of the housefly, Musca domestica

The activities of superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione S-transferases, GSSG reductase, thiol transferases, gamma glutamylcysteine synthetase, and glucose-6-phosphate dehydrogenase, and the concentrations of H2O2 and reduced and oxidized glutathione were determined in the various developmental stages of houseflies. Housefly development was correlated with a progressive increase of cellular oxidizing equivalents and a loss of cellular reducing capacity. The loss of reducing equivalents appeared to result from a decrease in the activity of enzymes involved in glutathione and NADPH synthesis and a concomitant increase in glutathione-oxidizing enzymes. Relatively little change was observed in SOD activity during housefly development; however, the electrophoretic pattern of MnSOD varied in a manner specific to developmental stage. A striking increase in H2O2 concentration occurred prior to pupation possibly due to changes in substrate catabolism. These results support the hypothesis that the cellular environment becomes progressively more oxidizing during development.     

Fine structure of the Malpighian tubules in the housefly, Musca domestica

The epithelium of the Malpighian tubules in the housefly is comprised of four distinct cellular types. Type I cells are characterized by the presence of intimate associations between infoldings of basal plasma membrane and mitochondria. On the luminal surface, cytoplasm is extended into microvilli which contain mitochondria. Membrane-bound vacuoles in the cytoplasm seem to progressively accumulate granular material. Type II cells have dilated canaliculi. Microvilli lack mitochondria. The Type III cell has not been reported previously in Malpighian tubules. It has very well-developed granular endoplasmic reticulum which contains intracisternal bundles of tubules. Cytoplasm contains numerous electron dense bodies. Type IV cells occur in the common duct region of the Malpighian tubules. Mitochondria do not extend into the microvilli.     

Alterations in superoxide dismutase, glutathione, and peroxides in the plasmodial slime mold Physarum polycephalum during differentiation

Changes in the level of antioxidant defenses and the concentration of free radical by-products were examined in differentiating (M3cVII and LU897 X LU863), non-differentiating (LU887 X LU897), and heterokaryon microplasmodia of the slime mold Physarum polycephalum during spherulation in salts-only medium. As differentiation proceeded, superoxide dismutase activity increased by as much as 46 fold; glutathione concentration and the rate of oxygen consumption decreased; cyanide-resistant respiration, hydrogen peroxide, and organic peroxide concentrations increased. The non-differentiating culture failed to exhibit any of these changes. A heterokaryon obtained by the fusion of differentiating and non-differentiating strains was observed to differentiate at a very retarded rate and to exhibit the changes observed in the spherulating strains at a correspondingly slower rate. These observations suggest that a free radical mechanism may be involved in the differentiation of Physarum microplasmodia into spherules.     

The structure of postsynaptic densities isolated from dog cerebral cortex: I. overall morphology and protein composition

A postsynaptic density (PSD) fraction, including some adherent subsynaptic web material, has been isolated from dog cerebral cortex by a short-procedure modification of methods of Davis and Bloom (21, 22) and Cotman and Taylor (20), using Triton X-100. The fraction has been visualized by thin-section, replica, and negative (phosphotungstic acid) staining electron microscopy and its proteins separated by high-resoltuion SDS gel electrophoresis. Morphologically, the preparation seems to be quite pure, with very little membrane contamination. The density is composed of protein, no nuclei acids, and very little phospholipids being detectable. The fraction had no ATPase or GTPase activity, but it did have a very small amount of cytochrome c oxidase activity (of a specific activity less than 0.5 percent that of a mitochondrial fraction) and a small amount of 5'- nucleotidase activity (of a specific activity between 6 and 7 percent that of a synaptic membrane fraction). Electron micrographs reveal cup-shaped structures approximately 400nm long and approximately 40nm wide, made up of apparent particles 13-28nm in diameter. However, en face views, and particularly micrographs of replicas and PTA-stained preparations, reveal a disk-shaped structure, outside diameter approximately 400 nm, in which filaments are seen to extend from the central part of the density. High resolution gel electrophoresis studies indicated some 15 major proteins and perhaps 10 or more minor ones; the predominant protein had a mol wt of 51,000, followed by ones at 45,000, 40,000, 31,000, 26,000, and several at 100,000. A comparison by gel electrophoresis of density fraction proteins with those of a lysed synaptosomal membrane fraction containing some adherent densities indicated some comigrating proteins, but the major membrane fraction protein, mol wt 52,000, was not found in the density fraction. Antibodies raised against the density fraction reacted with a preparation of solubilized synaptic membrane proteins. By both these criteria, it was considered that the density and the synaptic membrane have some proteins in common. By separately mixing (125)I-labeled myelin, synaptic vesicle, and mitochondrial fraction proteins with synaptosomes, and then isolating the density fraction from the mixture, it was concluded that a major 26,000 mol wt density fraction protein was common to both mitochondria and density, that none of the proteins of the density were contaminants from the mitochondrial fraction, that a minor approximately 150,000 band was a contaminant from the synaptic vesicle fraction, and that the moderately staining PSD fraction protein of 17,000 mol wt band was the result of contamination by the major basic protein of myelin. On the basis of the marker enzymatic assays and the mixing experiments, it is considered that the density fraction is moderately pure biochemically, and that its protein composition, aside from a few exceptions noted above, reflects its in situ character.     

The structure of postsynaptic densities isolated from dog cerebral cortex: II. characterization and arrangement of some of the major proteins within the structure

An attempt was made to identify some of the proteins of the postsynaptic density (PSD) fraction isolated from dog cerebral cortex. The major protein has been tentatively labeled "neurofilament" protein, on the basis of its 51,000 mol wt correspondence to a protein found in neurofilament preparations. Other proteins are akin to some dog myofibrillar proteins, on the basis if immunological crossreaction and equal sodium dodecyl sulfate (SDS)-gel electrophoretic mobilities. While a protein similar to dog muscle myosin is not present in the PSD fraction, a major protein present is actin, as evident from reactivity with antiactin serum, from SDS-gel mobility, and from amino acid composition. Only very little tubulin may be present in the PSD fraction, as determined by gel electrophoresis. Various treatments of the PSD fraction were attempted in order to extract some proteins, as revealed by gel electrophoresis, and to observe the structural changes of the PSD fraction residue after extraction of these proteins. The PSD is remarkably resistant to various extraction conditions, with only 4 M guanidine being found to extract most of the proteins, except the 51,000 mol wt protein. Disulfide reducing agents such as dithiothreitol (DTT), blocking agents such as p-chloromercuribenzoate (PCMB) (both in the presence of deoxycholate [DOC]), a Ca++ extractor, ethylene glycol-bis (beta- aminoethyl ether) N,N,N',N'-tetraacetate (EGTA), and guanidine caused an opening up of the native dense PSD structure, revealing approximately 10-nm filaments, presumably consisting of "neurofilament" protein. Both DTT-DOC and PCMB-DOC removed chiefly actin but also some other proteins. EGTA, in greatly opening up the structure, as observed in the electron microscope, revealed both 10-nm and 3- to 5-nm filaments; the later could be composed of actin, since actin was still in the residue after the treatment. EGTA removed a major 18,000 mol wt component and two minor proteins of 68,000 and 73,000 mol wt. Based on the morphological and biochemical evidence, a picture is presented of the PSD as a structure partly made up of 10-nm and 3- to 5-nm filaments, held together through Ca++ interaction and by bonds amendable to breakage by sulfhydrylblocking and disulfide-reducing reagents; either removal of Ca++ and/or rupture of these disulfide bonds opens up the structure. On the basis of the existence of filamentous proteins and the appearance of the PSD after certain treatments as a closed or open structure, a theory is presented with envisages the PSD to function as a modulator in the conduction of the nerve impulse, by movements of its protein relative.     

Superoxide dismutase induces differentiation in microplasmodia of the slime mold Physarum polycephalum

Evidence is presented that supports a role for the enzyme superoxide dismutase (SOD) in the differentiation of the slime mold, Physarum polycephalum. SOD activity increases 46-fold during differentiation. A strain of Physarum that does not differentiate exhibits no change in SOD activity. Addition of SOD, via liposomes, to the nondifferentiating strain induces differentiation; this effect is enhanced by an inhibitor of glutathione synthesis. Other antioxidants selected for study failed to induce differentiation. Conversely, oxidative treatments including introduction of D-amino acid oxidase, via liposomes, induced differentiation. Cellular oxidation is the probable cause of the SOD effect.     

The role of target size in neuronal survival

A loss of about half of the trochlear motor neurons occurs during the course of normal development in duck and quail embryos. The role of the size of the target muscle in controlling the number of surviving motor neurons was examined by making motor neurons innervate targets either larger or smaller in size than their normal target. In one experiment the smaller trochlear motor neuron pool of the quail embryo was forced to innervate the larger superior oblique muscle of the duck embryo. This was accomplished by grafting the midbrain of a quail embryo in the place of the midbrain of a duck embyro. Results indicated that no additional quail trochlear motor neurons were rescued in spite of a considerable increase in target size. In another experiment the larger trochlear motor neuron pool of the duck embryo was made to innervate the smaller superior oblique muscle of the quail embryo. This resulted in loss of some additional neurons; however, the number of surviving motor neurons was not proportionate to the reduction in target size. These experiments failed to provide support for the hypothesis that the size of the target muscle controls the number of surviving motor neurons. Although contact with target is necessary for survival of neurons, factors other than the number or size of target cells are involved in the control of motor neuron numbers during development. © 1992 John Wiley & Sons, Inc.     

Mitochondrial changes in flight muscles of normal and flightless Drosophila melanogaster with age

Fine structural changes in mitochondrial morphology pertaining to size, number and growth were examined in flight muscles of normal and experimentally dewinged male Drosphila melanogaster ranging up to 26 days of age. In the normal winged flies, the number of mitochondria decreases during the first week of adult life whereas the size of individual mitochondrial profile increases significantly. Changes in mitochondrial size and number are due to the fusion of mitochondria. Fused mitochondria are extremely large in size and irregular in shape. In 26-day old normal flies, the number of mitochondria increases while the mitochondrial size is reduced indicating mitochondrial division. In comparison to the normal flies, dewinged flies exhibit a similar degree of mitochondrial fusion and growth during the first week of life. However, the extent of mitochondrial fission in 26-day old dewinged flies is greater than in the normal flies of this age. Structural mechanisms of mitochondrial fusion and fission are described. The objective of this study was to examine the relative effects of age and flight activity on the mitochondria.     

Synthesis and in vitro evaluation of a series of diketopiperazine inhibitors of plasminogen activator inhibitor-1

We have synthesized and evaluated a series of diketopiperazine-based inhibitors of PAI-1. These studies resulted in the identification of 34 which inhibited PAI-1 in vitro with an IC(50)=0.2 microM. The synthesis and SAR of these compounds are described.