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.

Mitochondrial DNA deletion mutations colocalize with segmental electron transport system abnormalities, muscle fiber atrophy, fiber splitting, and oxidative damage in sarcopenia.

The in vivo cellular impact of age-associated mitochondrial DNA mutations is unknown. We hypothesized that mitochondrial DNA deletion mutations contribute to the fiber atrophy and loss that cause sarcopenia, the age-related decline of muscle mass and function. We examined 82,713 rectus femoris muscle fibers from Fischer 344 x Brown Norway F1 hybrid rats of ages 5, 18, and 38 months through 1000 microns by serial cryosectioning and histochemical staining for cytochrome c oxidase and succinate dehydrogenase. Between 5 and 38 months of age, the rectus femoris muscle in the hybrid rat demonstrated a 33% decrease in mass concomitant with a 30% decrease in total fibers at the muscle mid-belly. We observed significant increases in the number of mitochondrial abnormalities with age from 289 +/- 8 ETS abnormal fibers in the entire 5-month-old rectus femoris to 1094 +/- 126 in the 38-month-old as calculated from the volume density of these abnormalities. Segmental mitochondrial abnormalities contained mitochondrial DNA deletion mutations as revealed by laser capture microdissection and whole mitochondrial genome amplification. Muscle fibers harboring mitochondrial deletions often displayed atrophy, splitting and increased steady-state levels of oxidative nucleic damage. These data suggest a causal role for age-associated mitochondrial DNA deletion mutations in sarcopenia.

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.

Adaptation and selection of prion protein strain conformations following interspecies transmission of transmissible mink encephalopathy.

Interspecies transmission of the transmissible spongiform encephalopathies (TSEs), or prion diseases, can result in the adaptation and selection of TSE strains with an expanded host range and increased virulence such as in the case of bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease. To investigate TSE strain adaptation, we serially passaged a biological clone of transmissible mink encephalopathy (TME) into Syrian golden hamsters and examined the selection of distinct strain phenotypes and conformations of the disease-specific isoform of the prion protein (PrP(Sc)). The long-incubation-period drowsy (DY) TME strain was the predominate strain, based on the presence of its strain-specific PrP(Sc) following interspecies passage. Additional serial passages in hamsters resulted in the selection of the hyper (HY) TME PrP(Sc) strain-dependent conformation and its short incubation period phenotype unless the passages were performed with a low-dose inoculum (e.g., 10(-5) dilution), in which case the DY TME clinical phenotype continued to predominate. For both TME strains, the PrP(Sc) strain pattern preceded stabilization of the TME strain phenotype. These findings demonstrate that interspecies transmission of a single cloned TSE strain resulted in adaptation of at least two strain-associated PrP(Sc) conformations that underwent selection until one type of PrP(Sc) conformation and strain phenotype became predominant. To examine TME strain selection in the absence of host adaptation, hamsters were coinfected with hamster-adapted HY and DY TME. DY TME was able to interfere with the selection of the short-incubation HY TME phenotype. Coinfection could result in the DY TME phenotype and PrP(Sc) conformation on first passage, but on subsequent passages, the disease pattern converted to HY TME. These findings indicate that during TSE strain adaptation, there is selection of a strain-specific PrP(Sc) conformation that can determine the TSE strain phenotype.

Programmable color liquid-crystal television spatial light modulator: transmittance properties and application to speckle-correlation displacement measurement.

Drive electronics developed for a color liquid-crystal television (LCTV) display enable data to be written onto individual pixels. Display transmittance characteristics obtained with the new and the original TV drive electronics are compared. The enhanced performance obtained through this development has some potential for spatial light modulator applications in color, optical information processing based on the low-cost LCTV. As an example, we describe a novel, to our knowledge, speckle metrology technique used to display fringes and to output correlation peaks resulting from in-plane object displacement. This requires only a single LC display to encode, simultaneously in three pixel colors, speckle and fringe patterns for real-time measurements. Relative merits of this technique, including displacement range and temporal resolution, are discussed.

Use of mediastinoscopy for foreign body removal.

Foreign body removal from the aerodigestive tract can be a challenging endeavor despite improvements in technology. Rigid bronchoscopy has been demonstrated to be a safe and effective means of airway foreign body removal with appropriate training and expertise. However, potential complications exist and include extraluminal impaction of a penetrating foreign body during removal. This report details such a complication and the first known use of mediastinoscopy to remove the impacted foreign body to avoid the need for thoracotomy.

In situ detection of the Clostridium botulinum type C1 toxin gene in wetland sediments with a nested PCR assay.

A nested PCR was developed for detection of the Clostridium botulinum type C1 toxin gene in sediments collected from wetlands where avian botulism outbreaks had or had not occurred. The C1 toxin gene was detected in 16 of 18 sites, demonstrating both the ubiquitous distribution of C. botulinum type C in wetland sediments and the sensitivity of the detection assay.

The host range of chronic wasting disease is altered on passage in ferrets.

Chronic wasting disease (CWD), a member of the transmissible spongiform encephalopathies (TSEs), was first identified in captive mule and black-tail deer in 1967. Due to the failure to transmit CWD to rodents, we investigated the use of ferrets (Mustela putorius furo) as a small animal model of CWD. The inoculation of CWD into ferrets resulted in an incubation period of 17-21 months on primary passage that shortened to 5 months by the third ferret passage. The brain tissue of animals inoculated with ferret-passaged CWD exhibited spongiform degeneration and reactive astrocytosis. Western blot analysis of ferret-passaged CWD demonstrated the presence of PrP-res. Unlike mule deer CWD, ferret-passaged CWD was transmissible to Syrian golden hamsters (Mesocricetus auratus). Increasing the passage number of CWD in ferrets increased the pathogenicity of the agent for hamsters. This increase in host range of a field isolate on interspecies transmission emphasizes the need for caution when assessing the potential risk of transmission of TSEs, such as bovine spongiform encephalopathy, to new host species.

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.

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.

Reversibility of scrapie inactivation is enhanced by copper.

The only known difference between the cellular (PrPC) and scrapie-specific (PrPSc) isoforms of the prion protein is conformational. Because disruption of PrPSc structure decreases scrapie infectivity, restoration of the disease-specific conformation should restore infectivity. In this study, disruption of PrPSc (as monitored by the loss of proteinase K resistance) by guanidine hydrochloride (GdnHCl) resulted in decreased infectivity. Upon dilution of the GdnHCl, protease resistance of PrP was restored and infectivity was regained. The addition of copper facilitated restoration of both infectivity and protease resistance of PrP in a subset of samples that did not renature by the simple dilution of the GdnHCl. These data demonstrate that loss of scrapie infectivity can be a reversible process and that copper can enhance this restoration of proteinase K resistance and infectivity.

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.

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.

Developing genetic privacy legislation: the South Carolina experience.

The availability of presymptomatic and predisposition genetic testing has spawned the need for legislation prohibiting health insurance discrimination on the basis of genetic information. The federal effort, the Health Insurance Portability and Accountability Act (HIPAA) of 1996, falls short by protecting only those who access insurance through group plans. A committee of University of South Carolina professionals convened in 1996 to develop legislation in support of genetic privacy for the state of South Carolina. The legislation prevents health insurance companies from denying coverage or setting insurance rates on the basis of genetic information. It also protects the privacy of genetic information and prohibits performance of genetic tests without specific informed consent. In preparing the bill, genetic privacy laws from other states were reviewed, and a modified version of the Virginia law adopted. The South Carolina Committee for the Protection of Genetic Privacy version went a step further by including enforcement language and excluding Virginia's sunset clause. The definition of genetic information encompassed genetic test results, and importantly, includes family history of genetic disease. Our experience in navigating through the state legislature and working through opposition from the health insurance lobby is detailed herein.

Caloric restriction reduces fiber loss and mitochondrial abnormalities in aged rat muscle.

The influence of caloric restriction (CR) initiated at 17 months of age was investigated on selected age-associated measures in skeletal muscle. Tissue 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 vastus lateralis muscle of the CR50 rats. In the old rats from all groups, individual fibers were found with either no detectable cytochrome c oxidase activity (COX-), hyperreactivity for succinate dehydrogenase activity (SDH++; also known as ragged red fibers [RRF]), or both COX- and SDH++. Muscle from the CR50 rats contained significantly fewer COX- and SDH++ fibers than did the muscle from 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 increases in the number of skeletal muscle fibers showing mitochondrial enzyme abnormalities. CR also decreased the accumulation of mtDNA deletions.

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.

A citizens' AIDS Task Force: overcoming obstacles.

This article analyzes the experience of a state-wide Task Force on AIDS using grassroots techniques to construct policy responsive to the needs of people living with HIV. The four primary obstacles to effective policy making were the need (1) to include disenfranchised groups; (2) to avoid the domination of governmental bureaucracy so that community-based organizations could offer solutions, as well as services; (3) to overcome resistance to anonymous testing; and (4) to reach "hard to reach" populations. Task Force members' perspectives colored what were deemed appropriate policies. Members tended to polarize into two groups: those community-based groups and individuals who focused on the needs of people with HIV on one side, and on the other side, more institutional players who wanted to identify and isolate "HIV carriers." The article closes by analyzing the resulting HIV Omnibus Bill. Those who focused on the needs of people with HIV seized the opportunity to draft and successfully pass an omnibus bill through the Arizona Legislature. This success demonstrates that highly organized communities can affect policy making, even to the extent that it offsets more institutionalized power.

Adult-onset energy restriction of rhesus monkeys attenuates oxidative stress-induced cytokine expression by peripheral blood mononuclear cells.

We previously reported that energy restriction (ER) of mice attenuated age-associated increases in serum levels of interleukin-6 (IL-6). Here, we studied peripheral blood mononuclear cells (PBMC) from male rhesus monkeys to investigate the following: 1) the production of IL-6 and other cytokines become dysregulated with aging; 2) ER influences cytokine production and mRNA expression; and, 3) oxidative stress, as induced in vitro by xanthine and xanthine oxidase (X/XOD), influences cytokine mRNA and protein levels. Two types of comparisons were made as follows: 1) between normally fed young (6-9 y) and old monkeys (22-33 y); and 2) between middle-aged monkeys (15-21 y) fed either a normal energy intake or subjected to ER (for 5.5 y at 30% less than base-line intake). IL-6 protein levels and X/XOD-induced IL-6 mRNA levels in PBMC from old monkeys were significantly greater than those in PBMC from young animals. In contrast, interleukin-1beta (IL-1beta) and interleukin-8 mRNA levels were not strongly influenced by advancing age. X/XOD, which increased levels of protein carbonyls (indicative of oxidative damage) in PBMC, induced the expression of all three cytokines. ER reduced IL-6 protein and mRNA levels induced by X/XOD and the unstimulated mRNA levels of IL-1beta. These results indicate that, in a nonhuman primate model, oxidative stress may contribute to age-associated increases in the levels of certain cytokines and that adult-onset ER partially ameliorates this alteration.