Search

Return to work may be easily monitored as a surrogate of long-term functional outcome for benchmarking and performance improvement of trauma systems. We hypothesized that employment rates among survivors of traumatic brain injury (TBI) decrease following injury and remain depressed for an extended period of time. Data were obtained from a statewide surveillance system of 3522 TBI patients (aged >15 years) who were discharged alive from acute care hospitals and followed yearly using telephone interviews (1996-1999). The study population consisted of patients with severe TBI (head abbreviated injury score 3, 4, or 5) and complete follow-up for 3 years postinjury (n = 572). Patients were mostly young males (43 ± 19 years, 65% male) with blunt TBI (92%). The preinjury employment rate was 67%, which declined to 52% (P < 0.001) in the first year and slowly rose in subsequent years but never reached the preinjury level (54% in year 2, P < 0.001; 57% in year 3, P = 0.001). Increasing severity of TBI was associated with a lower employment rate. Patients who remained employed worked the same number of hours as they did before the injury (47.8 ± 10.5 hours). Female employment rates rose similar to rates for males. However, women who were employed full-time before TBI were more likely to work part-time after TBI than men (50% vs 24%, P < 0.001). In conclusion, survivors of severe injury do not attain preinjury employment levels for several years. Once validated in other studies, postinjury employment may be used as an indicator to monitor functional outcomes in trauma registries. [ABSTRACT FROM AUTHOR]

Purpose: Exercise like any medication requires the correct dose; to be effective the appropriate frequency, duration, and intensity are necessary. This study aimed to assess if a semi-supervised exercise training (ET) program would be more effective at improving aerobic fitness (VO 2PEAK ), exercise tolerance, and symptoms in individuals with postural orthostatic tachycardia syndrome (POTS) compared to the standard of care (SOC)., Methods: Subjects were randomized to either the ET or SOC groups (n 26 vs. 23; age 33 ± 11 vs. 37 ± 10 years; VO 2PEAK 66 ± 15 vs. 62 ± 15% predicted, ET vs. SOC respectively, p > 0.05). Composite Autonomic Symptom Score (COMPASS 31), 10 min stand test, and cardiopulmonary exercise test were performed at baseline and following 12 weeks. The ET group received an exercise consultation and eight semi-supervised in-person or virtual exercise sessions., Results: The ET group demonstrated a greater improvement in VO 2PEAK , higher or longer tolerance for baseline peak workload, and more often had a delayed symptom onset with exercise than the SOC group (ΔVO 2PEAK 3.4 vs. - 0.2 mL/min/kg, p < 0.0001, ΔWorkload 19 ± 17 vs. 0 ± 10 W; Workload time 63 ± 29 vs. 22 ± 30 s; onset-delay 80% vs. 30%, p < 0.05). Individuals in the ET group reported a significant improvement in orthostatic intolerance domain score (p = 0.02), but there was not a significant difference in the improvement in total COMPASS score (- 11.38 vs. - 6.49, p = 0.09)., Conclusion: Exercise training was more effective with greater improvements in aerobic fitness, orthostatic symptoms, and exercise tolerance for individuals with POTS when intensity and progression were personalized and delivered with minimal supervision compared to the SOC., (© 2023. The Author(s).)

Parks, Jordan K, Courtney M. Wheatley-Guy, Glenn M. Stewart, Caitlin C. Fermoyle, Bryan J. Taylor, Jesse Schwartz, Briana Ziegler, Kay Johnson, Alice Gavet, Loïc Chabridon, Paul Robach, and Bruce D. Johnson. Lung "Comet Tails" in healthy individuals: accumulation or clearance of extravascular lung water? High Alt Med Biol. 24:230-233, 2023-Ultrasound lung comet tails (or B-lines) tend to be limited in number (<5) or absent under ultrasound examination, and the appearance of diffuse B-lines with lung sliding has been suggested to identify pulmonary edema. Clinical evaluation of B-lines has been utilized as a bedside test to assess pulmonary congestion in patients with heart failure. Exposure to altitude or prolonged exercise can alter fluid regulation and can lead to pulmonary congestion or edema. As such, B-lines have been utilized in the field to monitor for pathological lung fluid accumulation. However, ultrasound lung comet lines might not be as reliable for identifying extravascular lung water (EVLW) as previously thought in healthy individuals exercising at altitude where an increase in the number of ultrasound lung comets would reflect fluid buildup in the interstitial space of the alveoli and pulmonary capillaries. This report will focus on reviewing the literature and our data from a group of ultraendurance runners that completed the Ultra Trail Mont Blanc race that demonstrates that lung comet tails may not always be evidence of pathological fluid accumulation in healthy individuals and as such should be used to assess EVLW in concert with other diagnostic testing.

Trineopentylphosphine (TNpP) in combination with palladium provides a highly effective catalyst for the Buchwald-Hartwig coupling of sterically demanding aryl bromides and chlorides with sterically hindered aniline derivatives. Excellent yields are obtained even when both substrates include 2,6-diisopropyl substituents. Notably, the reaction rate is inversely related to the steric demand of the substrates. X-ray crystallographic structures of Pd(TNpP)2, [Pd(4-t-Bu-C6H4)(TNpP)(μ-Br)]2, and [Pd(2-Me-C6H4)(TNpP)(μ-Br)]2 are reported. These structures suggest that the conformational flexibility of the TNpP ligand plays a key role in allowing the catalyst to couple hindered substrates. The Pd/TNpP system also shows good activity for the Suzuki coupling of hindered aryl bromides.

Discrete-event simulation can be used as an effective tool for healthcare administrators to "test" various operational decisions. The recent growth in hospital outpatient volumes and a constrained financial environment make discrete-event simulation a cost-effective way to diagnose inefficiency and create and test strategies for improvement. This study shows how discrete-event simulation was used in an adult medicine clinic within a large, tertiary care, academic medical center. Simulation creation steps are discussed, including information gathering, process mapping, data collection, model creation, and results. Results of the simulation indicated that system bottle-necks were present in the medication administration and check-out steps of the clinic process. The simulation predicted that matching resources to excessive demand at appropriate times for these bottleneck steps would reduce patients' mean time in the system (i.e., visit time) from 124.3 (s.d. +/- 65.7) minutes to 87.0 (s.d. +/- 36.4) minutes. Although other factors may affect real-world operations of a clinic, discrete-event simulation allows healthcare administrators and clinic operational decision makers to observe the effects of changing staffing and resource allocations on patient wait and throughput time. Discrete-event simulation is not a cure-all for clinic throughput problems, but can be a strong tool to provide evidentiary guidance for clinic operational redesign.

Objective: The Institute of Medicine has identified trauma center overcrowding as a crisis. We applied corporate Lean Six Sigma methodology to reduce overcrowding by quantifying patient dwell times in trauma resuscitation units (TRU) and to identify opportunities for reducing them., Methods: TRU dwell time of all patients treated at a Level I trauma center were measured prospectively during a 3-month period (n = 1,184). Delays were defined as TRU dwell time >6 hours. Using personnel trained in corporate Lean Six Sigma methodology, we created a detailed process map of patient flow through our TRU and measured time spent at each step prospectively during a 24/7 week-long time study (n = 43). Patients with TRU dwell time below the median (3 hours) were compared with those with longer dwell times to identify opportunities for improvement., Results: TRU delays occurred in 183 of 1,184 trauma patients (15%), and peaked on days with >15 patients or with presence of five simultaneous patients. However, 135 delays (74%) occurred on days when

A study of complex I (NADH:ubiquinone oxidoreductase) activity in Parkinson's disease (PD) brain has identified loss of activity only in substantia nigra although loss of activity of this enzyme has been identified in a number of non-brain tissues. We investigated this paradox by studying complex I and other complexes of the mitochondrial electron transport chain in frontal cortex from PD and aged control brain using a variety of assay conditions and tissue preparations. We found increasingly significant losses of complex I activity in PD frontal cortex as increasingly pure mitochondria were studied. Complexes II, III, and IV were comparable in PD and controls. Inclusion of bovine serum albumin in the assay increased enzyme activity but lessened discrimination between PD and controls. Complex I deficiency in PD brain is not confined to substantia nigra. Methodological issues are critical in demonstrating this loss of activity.

Background: Trauma has become a major cause of death and disability in developing countries. In India, most trauma patients receive initial care at general practitioner-staffed hospitals. We hypothesize that general practitioners (GPs) could improve their knowledge of trauma care after attending an educational course., Methods: A 2-day trauma course was conducted at a teaching hospital (170 bed) in Bangalore, India. Referral GPs, local surgeons and residents in training attended. A pre-course test was given to assess baseline trauma knowledge. The core didactic sessions included: resuscitation/recognition of shock states, airway prioritization, and evaluation/initial management of head, cardiothoracic, abdominal, pelvic/genitourinary, and thermal injuries. A post-course test was used to assess trauma knowledge obtained from the course. Paired t tests were performed on the test scores and demographic data were stratified by specialty and training status., Results: Of the 44 participants, 32 (72%) met study inclusion criteria: MBBS degree and course completion. The study population was 62.5% male with 47% surgeons and 53% GPs. Residents were 71.8% of the entire group. Overall, the pre- and post- course scores improved from 70.7% +/-11.2 to 87.5% +/-8.9, P = 0.000 (95%CI 12.1, 21.2). There was an increase of mean scores: 21.4% (SD +/-13.7) for GPs and 11.3% (SD +/-8.5) for surgeons (P = 0.02)., Conclusion: Although GPs had significantly lower pre-course scores than surgeons, at the end of the course, GPs performed as well as surgeons. These findings suggest allocation of limited educational resources for trauma care in India may be best used by GPs.

Background: Systolic blood pressure is used extensively to triage trauma patients as stable or unstable, contrary to Advanced Trauma Life Support recommendations. We hypothesized that systemic hypotension is a late marker of shock., Methods: The National Trauma Data Bank was queried (n = 115,830). Base deficit was used as a measure of circulatory shock. Systolic blood pressure was correlated with the presence and the severity of base-deficit derangement., Results: Systolic blood pressure correlated poorly with base deficit (r = .28). There was wide variation in systolic blood pressure within each base-deficit group. The mean and median systolic blood pressure did not decrease to less than 90 mm Hg until the base deficit was worse than -20, with mortality reaching 65%., Conclusions: We validated the Advanced Trauma Life Support principle that systemic hypotension is a late marker of shock. A normal blood pressure should not deter aggressive evaluation and resuscitation of trauma patients.

Idiopathic Parkinson's disease (PD) is characterized by a systemic loss of activity of complex I (NADH:ubiquinone oxidoreductase), the target enzyme of the parkinsonism producing neurotoxin, MPTP. Cybrid experiments strongly suggest that the loss of complex I activity arises from mitochondrial DNA. We prospectively evaluated low frequency, amino acid changing, heteroplasmic mutations in a narrow region of ND5, a mitochondrial gene encoding a complex I subunit, in brain tissue from PD and controls. The presence or absence of amino acid changing mutations correctly classified 15 of 16 samples. Heteroplasmic mutations in a specific region of ND5 largely segregate PD from controls and may be of major pathogenic importance in idiopathic PD.

Objective: The pressure-relieving ability of three specialized support surface mattresses was investigated and compared to a standard hospital mattress by measuring total body tissue interface pressure in a laboratory experiment designed to simulate clinical conditions predisposing to formation of pressure ulcers., Summary Background Data: Pressure ulcers are commonly seen in the elderly; in patients with trauma, burns, spinal cord injury, neurological deficits, peripheral vascular disease, and stroke; and in other patient groups with debilitating or emaciating illness. They represent from 4 to 29% of all hospital admissions. This study was undertaken to compare three different types of specialty beds to a standard hospital mattress in order to find a mattress that prevents the occurrence of pressure ulcers., Methods: The tested beds and mattresses were the Orthoderm Convertible II (Bio-Clinic Inc., Ontario, Canada) low air-loss mattress on a Stryker PMS (Stryker Inc., Kalamazoo, Michigan, USA) bed; the FluidAir Elite (KCI Inc., San Antonio, Texas, USA) air-fluidized bed; the Pegasus Airwave mattress (Pegasus Airwave Inc., Boca Raton, Florida, USA) on a Stryker PMS bed; and a standard hospital mattress on a Stryker PMS bed. The nineteen test subjects were all healthy with exclusion criteria of spinal cord injury, immobility, or other neurological or physical impairment. The Force Management System (Numotech Inc., Northridge, California, USA) was used to measure tissue interface pressure., Results: The study data show that the Pegasus Airwave mattress total surface pressure values of the anatomical regions (total body, torso, hips) of the body are lower than those of the other three tested mattresses. The total body and hip pressure resulting from pressure maturation is significantly less on the Pegasus Airwave mattress than on to the hospital bed, the air-fluidized bed, and the low air-loss bed., Conclusions: The Pegasus Airwave mattress outperforms the other tested support surfaces by decreasing overall pressure. Therefore, the Pegasus Airwave mattress is an excellent addition to augment nursing care in the prevention and treatment of pressure sores.

Complex I activity is reduced in cytoplasmic hybrid (cybrid) cell lines that contain mitochondrial DNA (mtDNA) from sporadic Parkinson's disease (PD) patients. This implies that mtDNA aberration occurs in sporadic PD. To assess the integrity of mtDNA in autosomal dominant PD arising from mutation of the alpha-synuclein gene, we transferred mitochondrial genes from PD-affected members of the Italian-American Contursi kindred to cells previously depleted of their endogenous mtDNA. Unlike cybrid cell lines expressing mtDNA from persons with sporadic or maternally inherited PD, the resultant Contursi cybrid lines did not manifest complex I deficiency, indicating that in Contursi PD mtDNA integrity is relatively preserved. Compared to control cybrids, however, Contursi cybrid lines did show some evidence of oxidative stress. For reasons that are unclear, at least a limited amount of mtDNA damage may nevertheless develop in PD patients with alpha-synuclein mutation., (Copyright 2001 Academic Press.)

Sporadic, non-familial Parkinson's disease is characterized by a 15-30% reduction in complex I activity of the electron transport chain. A pharmacological model of reduced complex I activity was created by prolonged treatment of SH-SY5Y cells with low doses (5-20 nM) of rotenone, a selective inhibitor of complex I. Short-term (less than 2 week) exposure to rotenone did not influence calcium signaling, production of reactive oxygen species, or mitochondrial morphology. However, following 2 weeks of rotenone exposure, SH-SY5Y cells showed unusual calcium dynamics, specifically multiple calcium responses to carbachol, a muscarinic agonist. These secondary calcium responses were not seen in control SH-SY5Y cells and were dependent upon calcium influx. Mitochondrial membrane potential was also reduced in low dose rotenone-treated cells. These results demonstrate that a chronic, partial reduction in complex I activity, such as that seen in Parkinson's disease, can alter cell signaling events and perhaps increase the susceptibility of cells to calcium overload and subsequent cell death.

Beta amyloid (Abeta) peptides accumulate in Alzheimer's disease and are neurotoxic possibly through the production of oxygen free radicals. Using brain microdialysis we characterized the ability of Abeta to increase oxygen radical production in vivo. The 1-40 Abeta fragment increased 2,3-dehydroxybenzoic acid efflux more than the 1-28 fragment, in a manner dependent on nitric oxide synthase and NMDA receptor channels. We then examined the effects of Abeta peptides on mitochondrial function in vitro. Induction of the mitochondrial permeability transition in isolated rat liver mitochondria by Abeta(25-35) and Abeta(35-25) exhibited dose dependency and required calcium and phosphate. Cyclosporin A prevented the transition as did ruthenium red, chlorpromazine, or N-ethylmaleimide. ADP and magnesium delayed the onset of mitochondrial permeability transition. Electron microscopy confirmed the presence of Abeta aggregates and swollen mitochondria and preservation of mitochondrial structure by inhibitors of mitochondrial permeability transition. Cytochrome c oxidase (COX) activity was selectively inhibited by Abeta(25-35) but not by Abeta(35-25). Neurotoxic Abeta peptide can increase oxidative stress in vivo through mechanisms involving NMDA receptors and nitric oxide sythase. Increased intracellular Abeta levels can further exacerbate the genetically driven complex IV defect in sporadic Alzheimer's disease and may precipitate mitochondrial permeability transition opening. In combination, our results provide potential mechanisms to support the feed-forward hypothesis of Abeta neurotoxicity.

Progressive supranuclear palsy (PSP) is a neurodegenerative movement disorder of unknown etiology. We hypothesized that mitochondrial DNA (mtDNA) aberration could occur in this disease and contribute to its pathogenesis. To address this we created transmitochondrial cytoplasmic hybrid (cybrid) cell lines expressing mitochondrial genes from persons with PSP. The presence of cybrid mtDNA aberration was screened for by biochemical assay of mitochondrial gene products. Relative to a control cybrid set, complex I activity was reduced in PSP cybrid lines (p<0.005). Antioxidant enzyme activities were elevated in PSP cybrid lines. These data suggest that mtDNA aberration occurs in PSP, causes electron transport chain pathology, and can produce oxidative stress. Further study of mitochondrial dysfunction in PSP may yield insights into why neurodegeneration occurs in this disease.

Alzheimer's disease (AD) is characterized by the deposition in brain of beta-amyloid (Abeta) peptides, elevated brain caspase-3, and systemic deficiency of cytochrome c oxidase. Although increased Abeta deposition can result from mutations in amyloid precursor protein or presenilin genes, the cause of increased Abeta deposition in sporadic AD is unknown. Cytoplasmic hybrid ("cybrid") cells made from mitochondrial DNA of nonfamilial AD subjects show antioxidant-reversible lowering of mitochondrial membrane potential (delta(gYm), secrete twice as much Abeta(1-40) and Abeta(1-42), have increased intracellular Abeta(1-40) (1.7-fold), and develop Congo red-positive Abeta deposits. Also elevated are cytoplasmic cytochrome c (threefold) and caspase-3 activity (twofold). Increased AD cybrid Abeta(1-40) secretion was normalized by inhibition of caspase-3 or secretase and reduced by treatment with the antioxidant S(-)pramipexole. Expression of AD mitochondrial genes in cybrid cells depresses cytochrome c oxidase activity and increases oxidative stress, which, in turn, lowers delta(psi)m. Under stress, cells with AD mitochondrial genes are more likely to activate cell death pathways, which drive caspase 3-mediated Abeta peptide secretion and may account for increased Abeta deposition in the AD brain. Therapeutic strategies for reducing neurodegeneration in sporadic AD can address restoration of delta(psi)m and reduction of elevated Abeta secretion.

Neurodegeneration in amyotrophic lateral sclerosis (ALS) is characterized by the specific loss of central and peripheral motor neurons. While this pattern of neuronal demise gives rise to a distinct clinical syndrome, at the cellular and molecular level ALS pathology is similar to that seen in other neurodegenerative diseases. In particular, mitochondrial dysfunction in ALS is reminiscent of that observed in Alzheimer's and Parkinson's diseases. Mitochondria in persons with ALS demonstrate impaired electron transport, increased free radical generation, and an inability to adequately buffer cytosolic calcium shifts. These abnormalities are probably systemic and potentially due to mutation of mitochondrial DNA.

To investigate the role of chronic mitochondrial dysfunction on intracellular calcium signaling, we studied basal and stimulated cytosolic calcium levels in SH-SY5Y cells and a derived cell line devoid of mitochondrial DNA (Rho degrees ). Basal cytosolic calcium levels were slightly but significantly reduced in Rho degrees cells. The impact of chronic depletion of mitochondrial DNA was more evident following exposure of cells to carbachol, a calcium mobilizing agent. Calcium transients generated in Rho degrees cells following application of carbachol were more rapid than those in SH-SY5Y cells. A plateau phase of calcium recovery during calcium transients was present in SH-SY5Y cells but absent in Rho degrees cells. The rapid calcium transients in Rho degrees cells were due, in part, to increased reliance on Na(+)/Ca(2+) exchange activity at the plasma membrane and the plateau phase in calcium recovery in SH-SY5Y cells was dependent on the presence of extracellular calcium. We also examined whether mitochondrial DNA depletion influenced calcium responses to release of intracellular calcium stores. Rho degrees cells showed reduced responses to the uncoupler, FCCP, and the sarcoplasmic reticulum calcium ATPase inhibitor, thapsigargin. Acute exposure of SH-SY5Y cells to mitochondrial inhibitors did not mimic the results seen in Rho degrees cells. These results suggest that cytosolic calcium homeostasis in this neuron-like cell line is significantly altered as a consequence of chronic depletion of mitochondrial DNA.

Diseases linked to defective mitochondrial function are characterized by morphologically abnormal, swollen mitochondria with distorted cristae. Several lines of evidence now suggest that sporadic forms of Parkinson's disease (PD) and Alzheimer's disease (AD) are linked to mitochondrial dysfunction arising from defects in mitochondrial DNA (mtDNA). Human neuroblastoma (SH-SY5Y) cells that are deficient in mtDNA (Rho(0)) were repopulated with mitochondria from AD or PD patients or age-matched controls. These cytoplasmic hybrid (cybrid) cell lines differ only in the source of their mtDNA. Differences between cybrid cell lines therefore arise from differences in mtDNA and provide a model for the study of how impaired mitochondrial function alters the mitochondria themselves and how these changes adversely affect the neuronal cells they occupy. Cybrid cell mitochondria were labeled with the mitochondrial membrane potential-sensitive dye, JC-1. Analysis of these JC-1 labeled mitochondria by confocal microscopy revealed that mitochondrial membrane potential was significantly reduced in both PD and AD cybrid cells when compared with controls. Ultrastructural examination showed that control cybrid cells contained small, morphologically normal, round or oval mitochondria with a dark matrix and regular distribution of cristae. PD cybrid cells contained a significant and increased percentage of mitochondria that were enlarged or swollen and had a pale matrix with few remaining cristae (0.26-0.65 microm(2)). AD cybrid cells also contained a significantly increased percentage of enlarged or swollen mitochondria (0.25-5.0 microm(2)) that had a pale matrix and few remaining cristae. Other pathological features such as crystal-like intramitochondrial inclusions and cytoplasmic inclusion bodies were also found in PD and AD cybrids. These observations suggest that transfer of PD or AD mtDNA into Rho(0) cells was sufficient to produce pathological changes in mitochondrial ultrastructure that are similar to those seen in other mitochondrial disorders. These data were reported in abstract form (Trimmer et al., 1998, Soc. Neurosci. Abstr. 24: 476)., (Copyright 2000 Academic Press.)

Ht30/=Ht5). Cells with reduced mitochondrial activity also showed abnormal responses to the stimulation of NGF output. Thrombin and phorbol ester elevated NGF production from Ht100, Ht30 and Ht10 cells, but not from Ht5 cells. Ht30 cells, despite secreting less NGF basally than Ht100 cells, reached a similar or greater NGF output upon stimulation. Mitogens increased NGF output and NGF mRNA levels with the largest effect on NGF protein in Ht30 cells. Free radical production and the ability of cells to respond to NGF-inducing agents were related. These data suggest that chronic impairment of mitochondrial function associates with disturbances in cellular production of a signaling protein.

Background/aims: We assessed mitochondrial morphology by electron microscopy and the prevalence of a mitochondrial gene deletion in patients with non-alcoholic steatohepatitis (NASH), alcohol-related liver disease and non-fatty liver diseases. Respiratory chain function using a cytoplasmic hybrid (cybrid) assay was further studied in NASH patients and healthy controls., Methods: Electron microscopy was performed in 26 specimens. Fifteen patients were studied by polymerase chain reaction to detect a 520-bp deletion product of the mitochondrial genome (dmtDNA). Cybrids were created by fusion of platelets with anaerobic neuroblastoma cells in six NASH patients and 12 controls., Results: Eight of ten NASH, one of seven alcoholics and two of nine other patients had linear crystalline inclusions in megamitochondria (p<0.05). Three of five patients with alcohol-related liver disease had dmtDNA compared to one of five NASH patients and one of five non-steatohepatitis controls. Cybrid respiratory chain function in platelets was not different from that of controls., Conclusions: Respiratory chain dysfunction, if present in NASH, is not expressed in platelet-derived mitochondria. In contrast to alcohol-related liver disease with active drinking, NASH patients do not commonly express the 5-kb mitochondrial DNA gene deletion in liver tissue. As previously described in early alcohol-related liver disease, crystalline inclusions of unknown composition are seen in hepatic mitochondria in NASH. Their presence suggests either an adaptive process or mitochondrial injury.

Electron transport chain (ETC) dysfunction may arise from mitochondrial genetic, nuclear genetic, or toxic etiologies. Cytoplasmic hybrid (cybrid) systems can help distinguish between these possibilities by facilitating expression of suspect mitochondrial DNA (mtDNA) within a nuclear and environmentally controlled context. Perpetuation of ETC dysfunction in cybrids is consistent with an mtDNA pathogenesis while defect correction is not. We previously used cybrids to screen sporadic Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis patients for mtDNA mutation with positive results. To further address the fidelity of these experiments, we created and characterized cybrids expressing mtDNA from persons with Huntington's disease (HD), an autosomal dominant, nuclear DNA-determined disorder in which mitochondrial ETC functioning is abnormal. On ETC, oxidative stress, and calcium homeostasis assays HD cybrid lines were indistinguishable from control cybrid lines. These data support the use of the cybrid technique for mtDNA mutation screening in candidate diseases., (Copyright 1999 Academic Press.)

The mitochondrial transition pore (MTP) is implicated as a mediator of cell injury and death in many situations. The MTP opens in response to stimuli including reactive oxygen species and inhibition of the electron transport chain. Sporadic Parkinson's disease (PD) is characterized by oxidative stress and specifically involves a defect in complex I of the electron transport chain. To explore the possible involvement of the MTP in PD models, we tested the effects of the complex I inhibitor and apoptosis-inducing toxin N-methyl-4-phenylpyridinium (MPP+) on cyclosporin A (CsA)-sensitive mitochondrial swelling and release of cytochrome c. In the presence of Ca2+ and Pi, MPP+ induced a permeability transition in both liver and brain mitochondria. MPP+ also caused release of cytochrome c from liver mitochondria. Rotenone, a classic non-competitive complex I inhibitor, completely inhibited MPP(+)-induced swelling and release of cytochrome c. The MPP(+)-induced permeability transition was synergistic with nitric oxide and the adenine nucleotide translocator inhibitor atractyloside, and additive with phenyl arsine oxide cross-linking of dithiol residues. MPP(+)-induced pore opening and cytochrome c release were blocked by CsA, the Ca2+ uniporter inhibitor ruthenium red, the hydrophobic disulfide reagent N-ethylmaleimide, butacaine, and the free radical scavenging enzymes catalase and superoxide dismutase. MPP+ neurotoxicity may derive from not only its inhibition of complex I and consequent ATP depletion, but also from its ability to open the MTP and to release mitochondrial factors including Ca2+ and cytochrome c known to be involved in apoptosis.

Recent data suggesting complex I dysfunction in Parkinson's disease (PD) arises from mitochondrial DNA (mtDNA) mutation does not conclusively answer whether the responsible genetic lesion is inherited (primary) or somatic (secondary). To address this question, we identified a family in which multiple members over three generations are affected with PD through exclusively maternal lines. Cytoplasmic hybrids (cybrids) were created for 15 family members over two generations by transferring each individual's mtDNA to mtDNA-depleted human neuroblastoma cells. Eight of the 15 cybrid lines contained mtDNA obtained from maternally descended family members and seven contained mtDNA from paternally descended family members. After 6 weeks of culture, cybrid cell lines were assayed for complex I activity and oxidative stress, and mitochondrial morphology was analyzed by electron microscopy. Compared with the cybrid lines containing mtDNA from paternal descendants, cybrid lines containing mtDNA from maternal descendants had lower complex I activity, increased reactive oxygen species production, increased radical scavenging enzyme activities, and more abnormal mitochondrial morphologic features. These findings were present in cybrid lines containing mtDNA from maternal descendants with PD as well as in currently asymptomatic young maternal descendants, and support a precedent for inherited mtDNA mutation in some persons with PD.

Mitochondria are abnormal in persons with amyotrophic lateral sclerosis (ALS) for unknown reasons. We explored whether aberration of mitochondrial DNA (mtDNA) could play a role in this by transferring mitochondrial DNA (mtDNA) from ALS subjects to mtDNA-depleted human neuroblastoma cells. Resulting ALS cytoplasmic hybrids (cybrids) exhibited abnormal electron transport chain functioning, increases in free radical scavenging enzyme activities, perturbed calcium homeostasis, and altered mitochondrial ultrastructure. Recapitulation of defects previously observed in ALS subjects and ALS transgenic mice by expression of ALS mtDNA support a pathophysiologic role for mtDNA mutation in some persons with this disease., (Copyright 1998 Academic Press.)

The parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridine (MPP+) acts through inhibition of complex I of the electron transport chain. Recent evidence suggests that it may also act through inhibition of 2-ketoglutarate dehydrogenase complex (KDHC). We confirmed this observation in isolated rat liver mitochondria but found that this inhibition is prevented by preincubation with the radical quencher, cysteine (Cys). KDHC is also inhibited by the NO generator S-nitroso-N-acetyl-penicillamine (SNAP) and this inhibition is similarly blocked by cysteine. MPP+ may inhibit KDHC secondary through a radical-mediated event rather than through direct interaction with KDHC.

Alzheimer's disease (AD) brains exhibit oxidative stress and a biochemical defect of complex IV (cytochrome oxidase, COX) of the mitochondrial electron transport chain (ETC). This defect can be transferred through mitochondrial DNA (mtDNA) into clonal SY5Y cells depleted of their mtDNA. The resulting cytoplasmic hybrids or "cybrids" retain the complex IV defect and exhibit oxidative stress. We measured the mitochondrial membrane potential (delta psi m) in AD and control cybrids via H3-tetraphenylphosphonium ion (H3-TPP+) accumulation. AD cybrids exhibited a significant (about 30%) decrease in H3-TPP+ accumulation relative to controls. Acute treatment of normal SY5Ys with azide, a COX inhibitor, moderately decreased H3-TPP+ retention and strongly inhibited COX activity in a dose-dependent manner. As the mitochondrial transition pore (MTP) can be activated by reactive oxygen species and ETC inhibitors, and its opening causes delta psi m dissipation, we tested the effects of the MTP inhibitor cyclosporin A (CsA) on TPP+ accumulation. 5mM CsA increased basal H3-TPP+ accumulation in SY5Y cells about 10-fold, corresponding to about a 2-fold increase in delta psi m. In the AD cybrids, CsA increased the apparent delta psi m to the same final levels as it did in controls. These results indicate that low-conductance MTP activity contributes significantly to resting delta psi m in SY5Y cells. We propose the novel hypothesis that the COX defect and resulting oxidative stress in AD may pathologically activate the MTP, resulting in lower delta psi m and the release of mitochondrial factors involved in apoptosis.

The mitochondrial electron transport chain enzyme cytochrome c oxidase (COX) is defective in patients with sporadic Alzheimer's disease (AD). This defect arises from the mutation of mitochondrial DNA (mtDNA). To develop a tissue culture system that would express this genetically derived bioenergetic lesion and permit characterization of its functional consequences, we depleted Ntera2/D1 (NT2) teratocarcinoma cells of endogenous mtDNA and repopulated them with platelet mtDNA from AD patients. Cytochrome c oxidase activity was depressed in the resulting AD cytoplasmic hybrids (cybrids) compared with cybrids prepared with mtDNA from non-AD controls. Reactive oxygen species (ROS) production and free radical scavenging enzyme activities were significantly elevated in AD cybrids. A COX defect in NT2 AD cybrid lines indicates that AD patients possess mtDNA COX gene mutations that are sufficient for determining this biochemical lesion. Expression of unique functional characteristics (increased ROS production and free radical scavenging enzyme activities) relevant to neurodegeneration demonstrates the utility of these cells in defining AD pathophysiology at a cellular level. This in vitro tissue culture model of AD may prove useful in drug screening.

Alzheimer's disease (AD) is associated with defects in mitochondrial function. Mitochondrial-based disturbances in calcium homeostasis, reactive oxygen species (ROS) generation, and amyloid metabolism have been implicated in the pathophysiology of sporadic AD. The cellular consequences of mitochondrial dysfunction, however, are not known. To examine these consequences, mitochondrially transformed cells (cybrids) were created from AD patients or disease-free controls. Mitochondria from platelets were fused to rho0 cells created by depleting the human neuroblastoma line SH-SY5Y of its mitochondrial DNA (mtDNA). AD cybrids demonstrated a 52% decrease in electron transport chain (ETC) complex IV activity but no difference in complex I activity compared with control cybrids or SH-SY5Y cells. This mitochondrial dysfunction suggests a transferable mtDNA defect associated with AD. ROS generation was elevated in the AD cybrids. AD cybrids also displayed an increased basal cytosolic calcium concentration and enhanced sensitivity to inositol-1,4, 5-triphosphate (InsP3)-mediated release. Furthermore, they recovered more slowly from an elevation in cytosolic calcium induced by the InsP3 agonist carbachol. Mitochondrial calcium buffering plays a major role after this type of perturbation. beta-amyloid (25-35) peptide delayed the initiation of calcium recovery to a carbachol challenge and slowed the recovery rate. Nerve growth factor reduced the carbachol-induced maximum and moderated the recovery kinetics. Succinate increased ETC activity and partially restored the AD cybrid recovery rate. These subtle alterations in calcium homeostasis and ROS generation might lead to increased susceptibility to cell death under circumstances not ordinarily toxic.

The mitochondrial electron transport enzyme NADH:ubiquinone oxidoreductase (complex I), which is encoded by both mitochondrial DNA and nuclear DNA, is defective in multiple tissues in persons with Parkinson's disease (PD). The origin of this lesion and its role in the neurodegeneration of PD are unknown. To address these questions, we created an in vitro system in which the potential contributions of environmental toxins, complex I nuclear DNA mutations, and mitochondrial DNA mutations could be systematically analyzed. A clonal line of human neuroblastoma cells containing no mitochondrial DNA was repopulated with mitochondria derived from the platelets of PD or control subjects. After 5 to 6 weeks in culture, these cytoplasmic hybrid (cybrid) cell lines were assayed for electron transport chain activities, production of reactive oxygen species, and sensitivity to induction of apoptotic cell death by 1-methyl-4-phenyl pyridinium (MPP+). In PD cybrids we found a stable 20% decrement in complex I activity, increased oxygen radical production, and increased susceptibility to 1-methyl-4-phenyl pyridinium-induced programmed cell death. The complex I defect in PD appears to be genetic, arising from mitochondrial DNA, and may play an important role in the neurodegeneration of PD by fostering reactive oxygen species production and conferring increased neuronal susceptibility to mitochondrial toxins.

We report previously undescribed or atypical clinical and biochemical manifestations of the mitochondrial DNA MERRF mutation at nucleotide 8344 in members of a multigenerational family with maternally inherited, highly variable neurodegenerative disorder. The more profound neurologic abnormalities include Leigh disease, spinocerebellar degeneration, and atypical Charcot-Marie-Tooth disease.

Previously, our laboratory has demonstrated inhibition of mitochondrial state 3 (ADP-dependent) respiration 5 min after resuscitation from an asphyxial insult in lambs less than 3 days of age. Older lambs were resistant to this transient mitochondrial dysfunction. This study was designed to examine if age-related differences in baseline state 3 mitochondrial respiration, electron transport chain activity, or susceptibility to oxygen free radical-mediated lipid peroxidation were related to the previously observed differences in postasphyxial mitochondrial respiration. Mitochondrial respiration was measured in 24 nonasphyxiated control lambs aged 1-10 days using four different substrates. Electron transport chain activity was assessed in 15 of these lambs, and lipid peroxidation measured as conjugated diene production was measured in 11 of these lambs. These lambs were all ventilated to maintain normal blood gases for a time period equal to the length of the hypoxic insult in asphyxiated lambs (see below), after which samples of brain were removed for isolation of mitochondria. A second group of 11 lambs (seven < or = 3 days of age and four > 3 days of age) were asphyxiated. The insult was a 75-to-90-min episode of hypoxia and hypercarbia that resulted in bradycardia and systemic hypotension over the final 15 min of the insult. At the end of asphyxia, the lambs were resuscitated and returned to control ventilator settings. Samples of brain were removed 5 min after resuscitation. Postasphyxia electron transport chain activity and lipid peroxidation were measured. All measurements described above were done in both nonsynaptic (primarily glial in origin) and synaptic mitochondria. State 3 mitochondrial respiration varied significantly with age, decreasing by an average of 41.2% +/- 11.1% (mean +/- SEM) from Day 2 to Day 5-6 and then increasing back to levels similar to Day 2 by Day 8-10 in nonsynaptic mitochondria. State 3 respiration in synaptic mitochondria decreased 60.6% +/- 5.2% from Day 2 to Day 5-6 before returning to levels similar to Day 2 by Day 8-10. Resting (nonADP-dependent) state 4 respiration demonstrated similar developmental patterns. Electron transport chain activities did not vary with age in the nonasphyxiated control animals. In addition, an asphyxial insult did not diminish electron transport chain activities in either lambs < or = 3 days old or those > 3 days of age.(ABSTRACT TRUNCATED AT 400 WORDS)

We evaluated a simplified method for preparation and analysis of platelet cytochrome c oxidase activity in Alzheimer's disease (AD) and control patients. Mean cytochrome c oxidase activity in controls (n = 17) was 0.233 sec-1/mg whereas mean cytochrome c oxidase activity in Alzheimer patients (n = 19) was 0.193 sec-1/mg, p = 0.033. Complex III (ubiquinol:cytochrome c oxidoreductase), complex II (succinic dehydrogenase), and citrate synthase were all assayed as internal controls and were not significantly different in controls and Alzheimer patients. There is a relatively specific loss of platelet cytochrome c oxidase activity in Alzheimer disease patients.

Succinate-driven respiration in azide-inhibited rat brain submitochondrial particles (smps) produces azidyl and hydroxyl radicals that were detected by spin trapping with 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO). Production of radicals required succinate and oxygen and was eliminated by heat denaturation, which indicates that radical production is a result of respiration. The concentrations of both DMPO/.OH and DMPO/.N3 were decreased by addition of catalase to the smps, which indicates that H2O2 is involved in radical production. In the absence of azide anion, DMPO/.OH was not detected in the same system, even after five additions of succinate over a period of 24 h. It is proposed that azide inhibition of cytochrome c oxidase results in increased production of superoxide, which is efficiently converted to hydrogen peroxide by membrane-bound superoxide dismutase. Hydrogen peroxide activates endogenous peroxidase to react with azide anion forming azidyl radical, which damages the peroxidase, resulting in decreased production of azidyl radical with successive additions of succinate. Hydroxyl radical is produced from the hydrogen peroxide that is not removed by peroxidase. The increased production of superoxide in the azide-inhibited system suggests that loss of cytochrome c oxidase activity can lead to increased radical production if other proteins in the respiratory chain remain active. In the azide-inhibited system, reaction of azide anion with H2O2-activated endogenous peroxidase and spin-trapping of the resulting azidyl radical is a convenient monitor of H2O2 production.

The anticonvulsant valproic acid (VPA, 2-n-propylpentanoic acid) causes inhibition of the citric acid cycle and elevations of central nervous system (CNS) gamma-aminobutyric acid (GABA) levels, which correlates with anticonvulsant action. No unifying mechanism for these actions of VPA has won general acceptance. alpha-Ketoglutarate dehydrogenase complex (KDHC) is a critical control enzyme in the CNS. We hypothesized that VPA may be an inhibitor of this enzyme since decreased KDHC activity would reduce substrate flux through the citric acid cycle and may increase flux into GABA synthesis. To test this hypothesis, inhibition of purified beef brain KDHC by VPA and its metabolites 2-n-propylpent-2-enoic acid (delta 2,3 VPE) and their coenzyme A (CoA) derivatives were studied. Preincubation of the NADH-reduced enzyme with delta 2,3 VPE, VPA-CoA, and delta 2,3 VPE-CoA caused time-dependent inactivation, reversible by addition of CoA. Under steady-state conditions, delta 2,3 VPE and VPA-CoA were competitive inhibitors of KDHC and delta 2,3 VPE-CoA was a mixed inhibitor. These observations have implications for the molecular mechanisms of VPA action. VPA derivatives cause inactivation and inhibition of KDHC, which may explain the anticonvulsant and some toxic actions of VPA.

We assayed cytochrome oxidase and other electron transport chain activities in platelet mitochondria isolated from patients with Alzheimer's disease (AD). Five of 6 patients had striking reductions of platelet cytochrome oxidase activity (patient mean, 83.72 +/- 82.99 nmol/min/mg; control mean, 167.14 +/- 36.21 nmol/min/mg; n = 8). Other electron transport chain catalytic activities were not significantly different than control values. AD may be a systemic illness, a primary defect in cytochrome oxidase may be pathogenically important in its production, and the mitochondrial genes encoding cytochrome oxidase subunits may be important in producing the defect.

We evaluated electron transport chain activity in platelet mitochondria taken from HD patients. All 5 patients studied had striking depressions of NADH:ubiquinone oxidoreductase activity (complex I) (5.36 +/- 2.91 nmol/min/mg; control mean, 19.12 +/- 5.64 nmol/min/mg). Other electron transport chain activities were not significantly different from control values. HD may be caused by a mutation in 1 of the nuclear coded subunits of NADH:ubiquinone oxidoreductase.

Polarographic assays of oxidative phosphorylation in muscle mitochondria indicated abnormal pyruvate-malate metabolism in Friedreich ataxia (FA). Pursuing this clue, more specific assays were performed. Mitochondrial malic enzyme (MEm; malate: NADP+ oxidoreductase) specific activity was 10% of controls in fibroblasts from eight FA patients (p less than 0.0001). Cytosolic malic enzyme was modestly increased in FA fibroblasts. Mitochondrial and cytosolic malate dehydrogenase and aspartate aminotransferase, and malate transport on the dicarboxylate and alpha-ketoglutarate carriers were normal in fibroblasts or leukocytes. MEm activity is normally highest in the nervous system and heart is important in regulating carbohydrate metabolism. MEm deficiency could cause FA; further studies are required to substantiate this hypothesis.

Friedreich's disease (FD) obligate heterozygotes have reduced mitochondrial malic enzyme (MEm) activity in cultured fibroblasts. This indicates that the MEm deficiency in homozygous affected patients is genetically determined. Heterozygote MEm activity was only 20% of the control mean activity, lower than the 50% expected in an autosomal-recessive disorder. This may result from negative interactions between mutant and normal subunits in the tetrameric enzyme. These data support the idea that MEm deficiency causes FD, but further studies are required to prove this hypothesis.

We examined mitochondrial oxidative function 5 minutes and 2 hours after a gradual asphyxial insult in newborn lambs. We subjected 16 ventilated newborn lambs to 75-90 minutes of hypoxia and hypercarbia that resulted in bradycardia and systemic hypotension over the final 15 minutes of the insult. At the end of asphyxia, the lambs were resuscitated and returned to control ventilator settings. Samples of brain were removed 5 minutes (n = 8) and 2 hours (n = 8) after asphyxia. Each group of eight lambs was subdivided into those less than 3 or greater than 3 days old to evaluate the effect of age on postasphyxia mitochondrial function. After classification into nonsynaptic and synaptic mitochondria, mitochondrial respiration (oxygen consumption) was measured using five different substrates. Data from asphyxiated lambs were compared with that from a control group of ventilated nonasphyxiated lambs (n = 8). In the lambs less than 3 days old, there was significant depression of mean +/- SEM nonsynaptic mitochondrial state 3 (adenosine diphosphate-dependent) respiration to 29.5 +/- 5.2% of control with four of the five substrates and of state 4 respiration to 33.7 +/- 0.9% of control with three of the five substrates 5 minutes after asphyxia. By 2 hours after asphyxia, mean +/- SEM nonsynaptic mitochondria state 3 respiration increased to 70.4 +/- 6.4% of control while state 4 respiration increased to 58.2 +/- 4.5% of control. In contrast, lambs greater than 3 days old exhibited no inhibition of nonsynaptic mitochondrial function after asphyxia.(ABSTRACT TRUNCATED AT 250 WORDS)

The protonmotive force (delta p) of muscle mitochondria was measured by estimating the distribution of 14C-labeled TPMP (trimethylphenylphosphonium iodide) and 14C-labeled acetate across the inner membrane of muscle mitochondria. The matrix volume was simultaneously determined using 3H-labeled H2O and 3H-labeled mannitol and repeated drying to distinguish the label in these 2 compounds. Rapid separation of mitochondria from the incubation medium by centrifugation through silicone oil avoids the problems of potential anaerobic conditions associated with conventional centrifugation and large volumes of trapped media associated with filtration. The value for delta p (mean +/- SD) was 192+/- 26 mV in 30 determinations with rat muscle mitochondria during state 4. Measurement of oxygen consumption allowed calculation of membrane conductance (Cm,H+) which was 0.49 +/- 0.18 nmol of H+/min/mg protein/mV. The values for delta p and Cm,H+ are reported for a variety of experimental conditions and are consistent with Mitchell's chemiosmotic theory. Biopsy specimens obtained from human muscle gave state-4 delta p values of 197+/- 30 mV (n =5) and Cm,H+ values of 0.52 +/- 0.12 nmol of H+/min/mg/mV (n = 4). This delta p assay is the first described for coupled mammalian muscle mitochondria and will be useful in assessing membrane function.

Sodium valproate (VP) inhibited oxidative phosphorylation in isolated rat liver mitochondria. State 3 rates of oxygen consumption with glutamate as substrate were 80% of control values at a low VP concentration (24 microM). At 240 microM, there was more than 50% inhibition of glutamate and alpha-ketoglutarate state 3 rates. Succinate state 3 rates were 80% of control values, and uncoupling was noted at 2400 microM VP. These VP effects were similar to those of propionate and isovalerate, suggesting a common mechanism of toxicity. Inhibition of mitochondrial oxidative phosphorylation may explain why VP intoxication causes a hepatocerebral disorder that resembles Reye syndrome.

Lipoamide dehydrogenase (LAD) kinetic values, Km and Vmax, were normal in 11 patients with Friedreich ataxia. Fibroblast activities of the pyruvate and alpha-ketoglutarate dehydrogenase complex, and LAD activities, were also normal. There was no reduction in oxidative decarboxylation of pyruvate, alpha-ketoglutarate, or several other substrates in intact fibroblasts. Methodologic differences may account for differences of opinion about putative abnormalities of the alpha-ketoacid dehydrogenase complexes.