Mitochondrial DNA deletion mutations are concomitant with ragged red regions of individual, aged muscle fibers: analysis by laser-capture microdissection.

Laser-capture microdissection was coupled with PCR to define the mitochondrial genotype of aged muscle fibers exhibiting mitochondrial enzymatic abnormalities. These electron transport system (ETS) abnormalities accumulate with age, are localized segmentally along muscle fibers, are associated with fiber atrophy and may contribute to age-related fiber loss. DNA extracted from single, 10 microm thick, ETS abnormal muscle fibers, as well as sections from normal fibers, served as templates for PCR-based deletion analysis. Large mitochondrial (mt) DNA deletion mutations (4.4-9.7 kb) were detected in all 29 ETS abnormal fibers analyzed. Deleted mtDNA genomes were detected only in the regions of the fibers with ETS abnormalities; adjacent phenotypically normal portions of the same fiber contained wild-type mtDNA. In addition, identical mtDNA deletion mutations were found within different sections of the same abnormal region. These findings demonstrate that large deletion mutations are associated with ETS abnormalities in aged rat muscle and that, within a fiber, deletion mutations are clonal. The displacement of wild-type mtDNAs with mutant mtDNAs results in concomitant mitochondrial enzymatic abnormalities, fiber atrophy and fiber breakage.

Distribution of Lyb-4.1 and other membrane antigens on murine lymphoid tumors.

The distribution of membrane antigens on 6 DBA/2-derived tumors (L1210, L5178Y, P815, ABLS 11, ABLS 12, and ABLS 13) was studied by direct cytotoxicity and quantitative absorption assays. Lyb-4.1 antigen was found solely on the L1210 tumor. Iad antigens were absent from all tumors, and H-2Kd and H-2Dd antigens were present on all tumors. Immunoglobulin was adsorbed to the ascites tumors and lost after 3 days or more in tissue culture. These studies were performed to characterize the distribution of DBA/2 membrane antigens on DBA/2-derived tumors as a base line for functional and chemical studies with these tumors and with their solubilized proteins.

Heterogeneity in the membrane proteins of human lymphoid cell lines as seen in sodium dodecyl sulfate-polyacrylamide electrophoresis slab gels.

The proteins of [35S]methionine-labeled membranes of six human lymphoid cell lines were examined by electrophoresis in sodium dodecyl sulfate-polyacrylamide gradient slab gels in order to identify molecular differences among these tumors. The lymphoid cells were internally labeled with [35S]methionine, their membranes were isolated, and the reduced and alkylated membrane proteins were treated electrophoretically in sodium dodecyl sulfate-polyacrylamide gradient slab gels. The gel patterns of over 100 membrane proteins per cell were highly complex but reproducible and, in that sense, constituted fingerprints of the individual tumors. Several proteins occurred uniquely on one or a few tumors. Some protein bands were identified to be serologically recognized membrane antigens by electrophoresis of immunopurified antigen in parallel to membrane samples. p44,12, a complex of proteins with molecular weights of 44,000 and 12,000 (HLA-A and -B antigens and beta2-microglobulin), and p29,34, (HLA-D antigen) were identified in this manner. High-resolution sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis can be used to catalog and describe lymphocyte membrane proteins and perhaps to identify subsets of lymphoid cancers.

Nucleotide sequence of a middle repetitive DNA from honey bees.

The nucleotide sequence of a 264-bp clone, pAfr3.4, which represents a related, tandemly arrayed, middle repetitive DNA family within various subspecies of the honey bee (Apis mellifera L.), has been determined.

Age-associated alterations of the mitochondrial genome.

Age-associated alterations of the mitochondrial genome occur in several different species; however, their physiological relevance remains unclear. The age-associated changes of mitochondrial DNA (mtDNA) include nucleotide point mutations and modifications, as well as deletions. In this review, we summarize the current literature on age-associated mtDNA mutations and deletions and comment on their abundance. A clear need exists for a more thorough evaluation of the total damage to the mitochondrial genome that accumulates in aged tissues.

Oxidative stress and aging reduce COX I RNA and cytochrome oxidase activity in Drosophila.

Drosophila melanogaster displays an age-associated increase in oxidative damage and a decrease in mitochondrial transcripts. To determine if these changes result in energy production deficiencies, we measured the electron transport system (ETS) enzyme activity, and ATP levels with age. No statistically significant influences of age on activities of complexes I and II or citrate synthase were observed. In contrast, from 2 to 45 days post-eclosion, declines were found in complex IV cytochrome c oxidase activity (COX, 40% decline) and ATP abundance (15%), while lipid peroxidation increased 71%. We next examined flies that were either genetically or chemically oxidatively stressed to determine the effect on levels of mitochondrial-encoded cytochrome oxidase I RNA (coxI) and COX activity. A catalase null mutant line had 48% of coxI RNA compared to the wild type. In Cu/Zn superoxide dismutase (cSOD) null flies, the rate of coxI RNA decline was greater than in controls. CoxI RNA also declined with increasing hydrogen peroxide (H2O2) treatment, which was reflected in reduced cytochrome c oxidase (COX) activity. These results show that oxidative stress is closely associated with reductions in mitochondrial transcript levels and support the hypothesis that oxidative stress may contribute to mitochondrial dysfunction and aging in D. melanogaster.

Association of age-related mitochondrial abnormalities with skeletal muscle fiber atrophy.

The hypothesis that mitochondrial dysfunction contributes to the senescent loss of skeletal muscle was investigated in quadriceps from 2- to 39-year old rhesus monkeys. Histological approaches, both cross-sectional (a single cross-section of the muscle) and longitudinal (multiple cross-sections of individual fibers spanning a 350-1600 microm region), were used to identify muscle fibers with abnormal mitochondrial electron transport system (ETS) enzyme activities and mitochondrial DNA deletions. Fibers were examined for two ETS activities, succinate dehydrogenase (SDH, ETS complex II) and cytochrome c oxidase (COX, ETS complex IV). The number of individual fibers containing ETS abnormalities (predominately negative for cytochrome c oxidase activity and/or hyperreactive for succinate dehydrogenase) increased with age. Deletions of the mitochondrial genome were observed in 89% of these ETS abnormal fibers. Longitudinal analysis allowed characterization of the ETS abnormal phenotype along their length. A decrease in cross-sectional area in 14% of the ETS abnormal fibers supports the hypothesis that deleted mitochondrial genomes may contribute to age-related fiber atrophy.

Direct repeat sequences are not required at the breakpoints of age-associated mitochondrial DNA deletions in rhesus monkeys.

The large majority of mitochondrial DNA (mtDNA) deletions analyzed from mitochondrial myopathies and aging humans have been found to be flanked by direct repeats, a finding which has led to the slip-replication hypothesis of deletion formation. In this study, we have characterized 13 mtDNA deletion breakpoints from skeletal muscle harvested from 9- to 27-year-old rhesus monkeys. Seven of the deletions, five of which were unique to a particular animal, did not have direct repeats at the deletion breakpoints. In contrast, two of the three deletions common to several animals had direct repeats flanking the breakpoints. It appears, therefore, that at least two different mechanisms exist by which mtDNA deletions are formed during aging, one requiring and one independent of flanking direct repeats. Furthermore, the species in which mtDNA deletions are detected may determine which mechanism predominates.

High levels of mitochondrial DNA deletions in skeletal muscle of old rhesus monkeys.

Mitochondrial DNA (mtDNA) deletions increase in abundance with age in many tissues, however, their calculated low levels (usually < 0.1%) in samples from tissue homogenates containing thousands of cells argue against physiologic significance. Through the analysis of defined numbers of cells (skeletal muscle fibers) from rhesus monkeys, we report that the calculated abundance of specific mtDNA deletions is dependent upon the number of fibers analyzed: as the number of fibers decreases, the calculated deletion abundance increases. Also, most mtDNA deletions appear to occur in a mosaic pattern, varying from cell to cell in size, number and abundance. These data support the hypothesis that mtDNA deletions can focally accumulate to high levels contributing to declines in mass and function of aging skeletal muscle.

Tröger's base. An alternate synthesis and a structural analog with thromboxane A2 synthetase inhibitory activity.

The synthesis of 2,8-dimethyl-6H,12H-5,11-methanodibenzo[b,f][1,5]diazocine (Tröger's base) from p-toluidine and of two Tröger's base analogs from other anilines by reaction with hexamethylenetetramine in trifluoroacetic acid is described. 2,3,6,7-Tetrahydro-9-methyl-2,6-di-p-tolyl-1H,5H-pyrimido[5,6,1-ij] quinazoline is formed as a secondary product in the reaction of p-toluidine and hexamethylenetetramine. One of the Tröger's base analogs, 2,8-bis(3'-pyridylmethyl)-6H,12H-5,11-methanodibenzo[b,f][1,5]d iazocine (5), is an effective inhibitor of the enzyme, thromboxane A2 (TxA2) synthase, with an ED50 of 30 ng/mL in a specified in vitro assay. Three analogs having substituents on the bridging methylene group of the bicyclic nucleus of the Tröger's base structure were prepared, but all were considerably less active than the aforementioned compound in the inhibition assay. The structures of these inhibitors of TxA2 synthase fall outside the classical structure-activity relationship that has been established for this class of enzyme inhibitors.

Thromboxane A2 synthase inhibitors. 5-(3-Pyridylmethyl)benzofuran-2-carboxylic acids.

The synthesis and screening of a series of 5-(3-pyridylmethyl)benzofuran-2-carboxylic acids as selective thromboxane A2 (TxA2) synthase inhibitors is outlined. The ability of these compounds to inhibit TxA2 biosynthesis was assayed using microsomal enzyme from human platelets. Substitution of the benzofuran ring caused small changes in potency; modification of the carboxylic acid group caused modest reductions in potency, and substitution of the pyridine ring resulted in large reductions of potency. 5-(3-Pyridylmethyl)benzofuran-2-carboxylic acid sodium salt (9b, sodium furegrelate) was chosen for further evaluation as a TxA2 synthase inhibitor.

Pyrimidine and triazine 3-oxide sulfates: a new family of vasodilators.

Di- and triaminopyrimidine 3-oxides (e.g., 2,4-diamino-6-piperidinylpyrimidine 3-oxide and 2,4-diamino-6-(diallylamino)triazine 3-oxide) react with sources of sulfur trioxide, such as sulfur trioxide trimethylamine or chlorosulfuryl chloride, to yield the corresponding heterocyclic O-sulfates. These sulfates are inner salts with unusual physical properties. The structure of the O-sulfate of 2,4-diamino-6-piperidinylpyrimidine 3-oxide was confirmed by X-ray. These O-sulfates are hypotensives. They apparently act by direct vasodilation.

Triazolobenzodiazepines: a new class of stimulators of tissue-type plasminogen activator synthesis in human endothelial cells.

In our search for compounds that can stimulate endogenous fibrinolysis, we have found that certain triazolobenzodiazepines enhance the production of tissue-type plasminogen activator (t-PA) by vascular endothelial cells maintained in vitro, with no or even a lowering effect on plasminogen activator inhibitor type-1 (PAI-1) production. The most active compounds tested, U-34599, U-46195 and U-51477, were studied in more detail and showed a time- and dose-dependent increase in the production of t-PA by human umbilical vein endothelial cells. At optimal stimulatory concentrations (about 10 microM), the three compounds stimulated t-PA expression about 2-fold after 24 hr and maximally about 4-fold after 48 hr of incubation; this maximal increase in t-PA synthesis was sustained at prolonged incubations of 72 or 96 hr. The triazolobenzodiazepine effects on t-PA production were accompanied by parallel increases in t-PA mRNA levels, without marked changes in PAI-1 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA concentrations. Numerous analogues of the three lead compounds were then tested to determine the relationship between benzodiazepine structure and the ability to stimulate t-PA production. No positive correlation was found between the ability of the various triazolobenzodiazepines to stimulate t-PA production and their affinity for the benzodiazepine receptor. In agreement with this, no specific binding of [3H]flunitrazepam, a specific ligand for benzodiazepine receptors, to endothelial cell membrane preparations was observed. Thus, it is unlikely that the triazolobenzodiazepines act through central-type benzodiazepine receptors to stimulate t-PA production. Similarly, no evidence was found for the presence of peripheral-type benzodiazepine receptors on endothelial cell membranes. The ability of the benzodiazepines to stimulate t-PA production, however, appeared to be related to their platelet-activating factor (PAF) antagonist activity. Despite this finding, several non-benzodiazepine PAF antagonists did not stimulate t-PA production. While the precise mechanism of action is not yet clear, selected benzodiazepine analogues possessing PAF antagonist activity stimulate the production of t-PA by endothelial cells in vitro.

Repeatability of nutrient intakes estimated by a semiquantitative food frequency questionnaire in elderly subjects.

In order to evaluate the repeatability of nutrient values estimated from a semiquantitative food frequency questionnaire being used in a longitudinal study of the relationships between diet, hemostatic factors, and stroke risk in the elderly in Western Sydney, a subsample of 62 participants (24 men, 38 women) completed a repeat questionnaire approximately 1 month after baseline data were collected. The mean age was 78 years (range, 65 to 88; median, 78). Nutrient values calculated from the repeat questionnaire were not significantly different from the baseline results by paired t test. Intraclass correlation coefficients ranged from 0.63 for beta carotene to 0.82 for saturated fat. Quadratic weighted kappa values were calculated for quintile categories and these ranged from 0.50 for fiber to 0.86 for ethanol. These values are comparable to previously published results in elderly subjects and confirm that repeatability of nutrient intakes estimated using semiquantitative food frequency questionnaires is very high in the elderly. Older subjects may be more established in their dietary habits than younger subjects, so any tendency for repeatability to decrease due to impaired memory associated with advanced age is offset by a lower intraindividual variability in dietary habit.

Influences of caloric restriction on age-associated skeletal muscle fiber characteristics and mitochondrial changes in rats and mice.

The effect of caloric restriction (CR) initiated in adult rats (17 months of age) on the abundance of deleted mitochondrial genomes, mitochondrial enzymatic abnormalities, and fiber number was examined in rat skeletal muscle. Vastus lateralis muscle from young (3-4 months) ad libitum-fed, old (30-32 months) restricted (35% and 50% CR, designated CR35 and CR50, respectively), and old ad libitum-fed rats (29 months) was studied. CR preserved fiber number and fiber-type composition in the CR50 rats. In the old rats from all groups, individual fibers were found with either no detectable cytochrome-c oxidase activity (COX-), hyperactive for succinate dehydrogenase activity (SDH++), or both COX- and SDH++. Muscle from the CR50 rats contained significantly fewer COX- and SDH++ fibers than did the muscle from the CR35 rats. CR50 rats also had significantly lower numbers of mtDNA deletion products in two (adductor longus and soleus) of the four muscles examined compared to CR35 rats. These data indicate that CR begun in late middle age can retard age-associated fiber loss and fiber-type changes as well as lower the number of skeletal muscle fibers exhibiting mitochondrial enzyme abnormalities. CR can also decrease the accumulation of deleted mitochondrial genomes.

The mesentery as a laminated vascular bed for hepatocyte transplantation.

The small bowel mesentery provides a unique structure of a large vascularized surface area to support hepatocyte transplantation. Cell-seeded polymeric matrices can be juxtaposed in a relatively atraumatic manner between leaves of mesentery such that adequate exchange of nutrients and diffusion of gases can proceed in the interim while neovascularization occurs. Hepatocytes obtained from (RHA) Wistar rats by collagenase perfusion were seeded onto non-woven filamentous sheets of polyglycolic acid 1 x 3 cm in size and 2 mm thickness to a density of 500,000 cells/cm2. Twenty-six recipient Gunn rats (UDP-glucuronyl transferase deficient) underwent laparotomy. Hepatocyte-ladened polymer sheets were placed between leaves of mesentery. Eight sheets were placed per animal and the leaves were approximated, creating a functional implant 1 x 3 x 2 cm. Biopsies between 5-99 days after implantation revealed neovascularization, moderate inflammatory reaction and the presence of viable hepatocytes in 96% (25/26). Immunoperoxidase studies using anti-albumin antibody substantiated hepatocyte specific function in implants. HPLC profiles of bile from Gunn rats transplanted with hepatocytes from congeneic (RHA) rats demonstrated the presence of bilirubin conjugates. There were no conjugation fractions seen in control gunn rats without hepatocyte transplantation. Although total serum bilirubin did not significantly decrease, conjugated bilirubin was identified in 46% (12/26) animals after transplantation with congeneic hepatocytes. We conclude that the mesentery of the small bowel provides a large vascularized surface for cell transplantation. Large numbers of metabolically active hepatocytes can engraft, vascularize, and show function. The mesentery may be a potential bed for clinical hepatocyte transplantation.

Effects of prostaglandin D3 on nerve transmission in nictitating membrane of cats.

In anesthetized cats, intra-arterial injection of PGD3 toward the nictitating membrane caused long-lasting, dose-related decreases in the response of the nictitating membrane to sympathetic nerve stimulation. During peak depression of nerve transmission the response of the nictitating membrane to intra-arterial norepinephrine was not depressed suggesting that PGD3 suppressed the release of norepinephrine. PGD3 was as potent as PGD2 for modulating sympathetic nerve transmission but was less effective in activating a vagally mediated bradycardia. The results show that the PGD3 can modulate autonomic nerve transmission.

The effects of a sensory extinction procedure on stereotypic sounds of two autistic children.

A reversal design was used to investigate the effects of a sensory extinction procedure on stereotypic sounds produced by two autistic children. White noise programmed through earphones was used to mask auditory stimuli resulting from aberrant vocalizations (termed "slurring," "snorts," and "arias") and from clapping hands and dropping objects. This sensory extinction procedure substantially reduced the stereotypic vocalizations but had little practical effect on the clapping and object-dropping responses. The discussion addresses some of the limitations and potential uses of sensory extinction procedures.

Decreased mitochondrial RNA levels without accumulation of mitochondrial DNA deletions in aging Drosophila melanogaster.

Declines in electron transport system (ETS) activity have been reported to occur with advancing age in Drosophila melanogaster and many other animals. It has been proposed that these changes are importantly involved in the aging process. ETS decline has been attributed to mitochondrial nucleic acid damage. We analyzed various ages of D. melanogaster (embryos to 60-day-old adults) for the presence of mutated mitochondrial DNA (mtDNA) genomes. Although mtDNA genomes with large DNA deletions (up to 5 kb) were identified, abundance was low and remained constant throughout adult life. Therefore, these mtDNA deletions do not appear to be sufficiently abundant to cause large declines in ETS activity. Next, we analyzed various ages of D. melanogaster for the abundance of four mitochondrial-encoded and two nuclear-encoded ETS transcripts. The abundance of the mitochondrial transcripts declined 5-10-fold, while the nuclear-encoded transcripts declined 2-5-fold with advancing age. Separation of flies on the basis of flight loss was used to distinguish physiologic age from chronological age. Insects capable of flight at 30 days of age were found to have a 4-fold higher abundance of cox I mitochondrial-encoded RNA compared to flightless insects. No difference, however, was apparent in the nuclear-encoded beta-ATPase RNA level, suggesting only mitochondrial RNA (mtRNA) declines are associated with life expectancy.

Cellular phenotypes of age-associated skeletal muscle mitochondrial abnormalities in rhesus monkeys.

Rhesus monkey vastus lateralis muscle was examined histologically for age-associated electron transport system (ETS) abnormalities: fibers lacking cytochrome c oxidase activity (COX(-)) and/or exhibiting succinate dehydrogenase hyperreactivity (SDH(++)). Two hundred serial cross-sections (spanning 1600 microm) were obtained and analyzed for ETS abnormalities at regular intervals. The abundance and length of ETS abnormal regions increased with age. Extrapolating the data to the entire length of the fiber, up to 60% of the fibers were estimated to display ETS abnormalities in the oldest animal studied (34 years) compared to 4% in a young adult animal (11 years). ETS abnormal phenotypes varied with age and fiber type. Middle-aged animals primarily exhibited the COX(-) phenotype, while COX(-)/SDH(++) abnormalities were more common in old animals. Transition region phenotype was affected by fiber type with type 2 fibers first displaying COX(-) and then COX(-)/SDH(++) while type 1 fibers progressed from normal to SDH(++) and then to COX(-)/SDH(++). In situ hybridizations studies revealed an association of ETS abnormalities with deletions of the mitochondrial genome. By measuring cross-sectional area along the length of ETS abnormal fibers, we demonstrated that some of these fibers exhibit atrophy. Our data suggest mitochondrial (mtDNA) deletions and associated ETS abnormalities are contributors to age-associated fiber atrophy.