Analysis of muscle mitochondrial function with techniques applicable to needle biopsy samples

Summary. Methods are described for the assessment of mitochondrial function in small (less than 50 mg) samples of human skeletal muscle using spectrophotometric assays to follow the reduction or oxidation of added cytochrome c. Activities of pyruvate plus malate cytochrome c reductase [(P + M)CR], α-oxo-glutarate cytochrome c reductase (OgCR) glutamate cytochrome c reductase (GCR), succinate cytochrome c reductase (SCR) and cytochrome c oxidase (COx) were measured in various rat tissues and samples of human skeletal muscle. In all tissues the activities were in the order (P + M) CR<SCR<COx, although there were wide variations in the absolute levels of activity. Rates of (P + M)CR and SCR activity measured spectrophotometrically in hypotonic medium were similar to the equivalent rates of oxygen uptake measured in isotonic medium with an oxygen electrode. Because the spectrophotometric assays have to be carried out in hypotonic medium, mitochondria are uncoupled and the method cannot be used to examine this aspect of mitochondrial function. Measurements can be reliably made on a single needle biopsy specimen of human muscle and by measuring a number of different activities the function of the whole electron transport chain and a number of tricarboxylic acid cycle enzymes can be determined. The clinical use of the methods is illustrated with results from selected patients with specific defects of mitochondrial metabolism. The methods offer a convenient way both for screening and the detailed and repeated study of patients.     

The transformer genes in the fig wasp Ceratosolen solmsi provide new evidence for duplications independent of complementary sex determination

Transformer (tra) is the key gene that turns on the sex‐determination cascade in Drosophila melanogaster and in some other insects. The honeybee Apis mellifera has two duplicates of tra, one of which (complementary sex determiner, csd) is the primary signal for complementary sex‐determination (CSD), regulating the other duplicate (feminizer). Two tra duplicates have been found in some other hymenopteran species, resulting in the assumption that a single ancestral duplication of tra took place in the Hymenoptera. Here, we searched for tra homologues and pseudogenes in the Hymenoptera, focusing on five newly published hymenopteran genomes. We found three tra copies in the fig wasp Ceratosolen solmsi. Further evolutionary and expression analyses also showed that the two duplicates (Csoltra‐B and Csoltra‐C) are under positive selection, and have female‐specific expression, suggesting possible sex‐related functions. Moreover, Aculeata species exhibit many pseudogenes generated by lineage‐specific duplications. We conclude that phylogenetic reconstruction and pseudogene screening provide novel evidence supporting the hypothesis of independent duplications rather an ancestral origin of multiple tra paralogues in the Hymenoptera. The case of C. solmsi is the first example of a non‐CSD species with duplicated tra, contrary to the previous assumption that derived tra paralogues function as the CSD locus.     

Molecular basis for odorant receptor tuning: a short C‐terminal sequence is necessary and sufficient for selectivity of mosquito Or8

A birth‐and‐death evolutionary model for odorant receptor gene repertoires presumes the creation of repertoires with the capacity for high‐level diversity and rapid ligand specificity change. This changes the recognised odour space, directly affecting fitness‐related behaviours and ultimately affecting adaptation to new environments and resources. The proximate molecular mechanisms underlying the tuning of odorant receptor repertoires, and thus peripheral olfaction, are unclear. In the present study, we report a concrete example of this model of odorant receptor evolution leading to rapid changes in receptor tuning that leave the peripheral neuronal circuitry intact. We identified a conserved odorant receptor gene in mosquitoes, Or8, which in Culex quinquefasciatus underwent a duplication and inversion event. The paralogues differ in only minor structural changes manifesting at the C‐terminus. We assessed the specificity of the paralogous odorant receptors and receptor neurones. We found that the functional tuning of the receptor was indeed reflected in minor differences in amino acid structure. Specifically, we found that enantiomeric specificity of these mosquito Or8 paralogues relies on eight C‐terminal amino acids encoded in the final exon of the gene; thus, the birth of a paralogous odorant receptor can change the tuning of the peripheral olfactory system.     

Do Patients With Cardiac Problems Develop Disease-Specific Threat Schemas?: Evaluation of Conceptual Implicit Memory

Researchers have used cognitive psychology paradigms to evaluate emotional sequela of cardiovascular disease, although there have been no prior studies assessing implicit memory bias in a population of patients with cardiac problems. The purpose of the present investigation was to assess whether cardiac events precipitate disease-specific information processing changes in implicit memory. Results provided very limited support for a cardiac-specific memory bias, using a measure of conceptual implicit memory. A trend for patients with cardiac problems to rate white noise surrounding cardiac words as less loud, indication of an implicit memory bias, was present only at one white noise level evaluated, t(15) = –1.91, p = .08. Despite limited evidence for a cardiac-specific threat schema, further investigation appears warranted. Information processing paradigms adapted from cognitive psychology offer an alternative to experimental approaches entirely dependent on participant self-report.     

Influence of soil pH on the germination and survival of Eucalyptus regnans F.Muell. in southern Tasmania

Alkaline soil conditions present in localised areas shortly after slash burning of a logging coupe in southern Tasmania were found to reduce both germination of Eucalyptus regnans seed and survival of young seedlings. In vitro studies showed that high pH conditions could interfere with the germination of E. regnans seed at otherwise conducive temperatures although the influence of elevated pH on seed viability was reduced at temperatures sufficiently low to maintain seed dormancy. The transition to a detrimental effect appeared to be in the pH range 8.4 to 9.0.

Field and glasshouse trials showed that some of the surface soils could be sufficiently alkaline to have a detrimental effect on seedling establishment, especially where seed was sown very soon after burning although observed effects may not have been due to pH alone. Such soils would nevertheless represent only a small proportion of the total sowing area, with toxicity being limited to a period of a few months following burning.

Although elevation of soil pH by fire is well known, it appears that this as a factor contributing to seedling mortality has been overlooked.     

Experimental evidence for in vitro fluid transport in the presence of a traditional medicinal fruit extract across rat everted intestinal sacs

The present study was designed to investigate the effects of aqueous fruit extract of Momordica charantia (MC), a traditional medicinal plant, on the transport of fluid in vitro. Everted intestinal sacs from rats were mounted in an organ bath containing Krebs solution. We compared the effect of MC extract on water transport with increasing inorganic phosphate concentration with or without D-glucose in the buffer. In the control experiments, fluid uptake was enhanced significantly (P < 0.05) at high inorganic phosphate concentration (8-10 mM) in the presence of 5.5 mM D-glucose. Addition of 3.0 mg/mL MC extract to the serosal side inhibits the uptake of fluid significantly (P < 0.05). At high inorganic phosphate concentration (8-10 mM), fluid uptake was not inhibited (P > 0.05) when incubated with 3.0 mg/mL MC fruit extract. It is hypothesized that an increase in inorganic phosphate enhances oxidative phosphorylation thereby increasing the fluid uptake across everted intestinal sacs of rat. These findings seem to indicate that the MC-induced reduction on intestinal fluid absorption capacity could be mainly the result of an interference with the carrier-mediated coupled entrance of glucose and Na(+) across the brush-border membrane.     

Thrombin induces activation and translocation of Bid,Bax and Bak to the mitochondria in human platelets

Summary. Background: Thrombin is a physiological platelet agonist that activates apoptotic events, including cytochrome c release and phosphatidylserine exposure; however, the mechanisms underlying these events remain unclear. Objectives: The present study is aimed to investigate whether thrombin induces activation and mitochondrial translocation of Bid, Bax and Bak. Methods: Changes in the mitochondrial membrane potential were registered using the dye JC‐1; Bid, Bax and Bak translocation to the mitochondria was detected by immunoprecipitation and Western blotting in samples from mitochondrial and cytosolic fractions. Results: Treatment of platelets with thrombin or ADP induces activation and mitochondrial association of active Bid, Bax and Bak. Translocation of Bid and Bax to the mitochondria was reduced by cytochalasin D, latrunculin A or jasplakinolide. Platelet exposure to exogenous H₂O₂ (10 μm ) results in activation of Bid and Bax, which was found to be similar to the effect of thrombin. Thrombin evokes mitochondrial membrane depolarization, which is attenuated by catalase. Conclusion: Our results indicate that thrombin induces activation and mitochondrial translocation of Bid, Bax and Bak, which is likely to be one of the apoptotic events in human platelets.     

Oxygen at physiological concentrations. A potential, paradoxical mediator of reperfusion injury to mitochondria induced by phosphate

Cellular injury induced by reperfusion after myocardial ischemia is manifested by striking mitochondrial damage as well as other hallmarks such as contraction band necrosis. Calcium has been implicated as a mediator of irreversible cellular injury in several systems. To identify other potential mediators of the mitochondrial injury associated with reperfusion, interactions between inorganic phosphate, oxygen, and mitochondria harvested from rabbit hearts were evaluated in vitro. Mitochondria exhibited rapid inactivation of oxidative phosphorylation after preincubation at 25 degrees C when phosphate and oxygen were present. Inactivation was partially but not completely precluded by EDTA, EGTA, magnesium, diltiazem, or ruthenium red, results in concert with findings of others suggesting involvement of a deleterious influx of calcium into mitochondria; exogenous calcium enhanced inactivation. However, the present data indicate that inactivation is prevented by incubation of mitochondria in the absence of oxygen, and demonstrate for the first time that injury elicited by phosphate is dependent on oxygen at physiological concentrations either because calcium and/or phosphate influx is linked to aerobic metabolism or because oxygen exerts deleterious effects on mitochondria, which may render them particularly susceptible to calcium influx. Since intracellular inorganic phosphate concentration increases markedly with ischemia, reperfusion with oxygenated medium may paradoxically augment mitochondrial injury in this setting. Thus, in the presence of increased intracellular concentrations of calcium and phosphate induced by ischemia, subsequent reestablishment of physiological levels of intracellular oxygen tension may promote mitochondrial damage, which is known to increase with reperfusion.     

Characterization of an atypical creatine kinase from human heart tissue,with properties similar to those of mitochondrial creatine kinase

The 600 × g paniculate fraction, obtained from the homogenates of human heart muscle, contained large quantities of an atypical creatine kinase (CK-Z). Creatine kinase Z migrated cathodically relative to CK-MM on agarose gel electrophoresis, and was not inhibited by antibodies directed against human CK-MM and CK-BB. Creatine kinase Z had an apparent K_m for Mg-ADP and creatine phosphate of 0.04 mmol/1 and 1.3 mmol/1, respectively. This enzyme existed in two molecular forms; one form of molecular weight 33000–38000 in the presence of a buffer containing Tris-HCl (0.05 mol/1), EDTA (0.001 mol/1), and 2-mercaptoethanol (0.010 mol/1), pH 8.0; and another form having a molecular weight of 62000–68000 in the presence of a buffer containing sodium phosphate (0.020 mol/1) and EDTA (0.001 mol/1), pH 8.0.Creatine kinase Z had biochemical properties which were different from those of the other soluble creatine kinase isoenzymes (MM, MB and BB), but similar to those reported for mitochondrial creatine kinases isolated from other animal tissues.     

Complicated migraine studied by phosphorus magnetic resonance spectroscopy

The brain and skeletal muscle of eight adult patients with migraine with prolonged auras or migraine strokes leaving a permanent hemianopic defect were studied by phosphorus magnetic resonance spectroscopy. Biochemical assays performed on muscle biopsy and platelets had revealed abnormal mitochondrial enzyme activities. Brain magnetic resonance spectroscopy showed an abnormally low phosphocreatine to inorganic phosphate ratio in all patients, apparently due to decreased phosphocreatine and increased inorganic phosphate contents. Muscle phosphorus magnetic resonance spectroscopy showed low recovery from exercise in seven patients. Three patients had an increased phosphocreatine/inorganic phosphate ratio at rest, and the exercise transfer characteristics were abnormal in four patients for relatively low levels of exercise. The mitochondrial metabolic defects present in platelets and muscle of complicated migraine patients are therefore also expressed in the brain.     

The structure, sequence and developmental pattern of expression of the white gene in the blowfly Lucilia cuprina

We have sequenced the complete coding region of the white gene of Lucilia cuprina. Strong sequence identity exists between this gene and its homologue from Drosophila melanogaster at both nucleotide and derived amino acid levels (68% and 78% respectively). The exon/intron structure of the two genes is also largely conserved, although the Lucilia gene contains one extra Won. Expression of the gene peaks during mid-pupal stage, with secondary peaks in late larval and early adult stages. Comparisons between this and other white genes will contribute to a better understanding of ATP-binding transmembrane transport proteins. The white gene should also serve as a useful marker gene in the development of a gene transformation system for the sheep blowfly.     

Blood preservation. XXXIV. DHA maintains 2,3-DPG and its adverse effect on ATP is reversed with extra phosphate

We have found that the addition of 10 mM inorganic phosphate to DHA in CPD-adenine maintains ATP levels at normal or higher than normal values for six weeks of storage. 2,3-DPG values are slightly lowered by the extra phosphate, but are still maintained at approximately half normal for four weeks by the DHA. The addition of a higher phosphate concentration, 20 mM, to DHA produced lower levels of ATP and 2,3-DPG than those observed with 10 mM phosphate, although both levels were better than in the CPD-adenine control. pH values in this experiment were lowest in the three preservatives containing DHA, probably indicating increased lactate production due to metabolism of this triose sugar, in addition to dextrose present in CPD.     

Erythrocyte Energy Metabolism in Hereditary Spherocytosis

The incorporation of extracellular orthophosphate-³²P into cellular ATP, 2,3-diphosphoglyceric acid, and inorganic phosphate has been measured over a period of 6 hours in vitro in red blood cells from normal subjects and from patients with hereditary spherocytosis who had undergone splenectomy. The pattern of labeling of the intracellular compounds was found to be the same in both types of red blood cells, as reported by other workers using much shorter periods of incubation. In addition, in the present study it was possible to compare the net flux of extracellular phosphate into ATP between the two groups of erythrocytes. These latter results suggest that the actual turnover rate of ATP was not abnormal in these patients with hereditary spherocytosis.     

Parkin expression reverses mitochondrial dysfunction in fused in sarcoma‐induced amyotrophic lateral sclerosis

Fused in sarcoma (FUS) is a DNA/RNA‐binding protein associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. The exact molecular mechanisms by which FUS results in neurotoxicity have not yet been fully elucidated. Here, we found that parkin is a genetic suppressor of defective phenotypes induced by exogenous human wild type FUS in Drosophila. Although parkin overexpression did not modulate the FUS protein expression level, the locomotive defects in FUS‐expressing larvae and adult flies were rescued by parkin expression. We found that FUS expression in muscle tissues resulted in a reduction of the levels and assembly of mitochondrial complex I and III subunits, as well as decreased ATP. Remarkably, expression of parkin suppressed these mitochondrial dysfunctions. Our results indicate parkin as a neuroprotective regulator of FUS‐induced proteinopathy by recovering the protein levels of mitochondrial complexes I and III. Our findings on parkin‐mediated neuroprotection may expand our understanding of FUS‐induced ALS pathogenesis.     

Biochemical analysis of a lithopedion

Lithopedions are very rare; the medical literature mentions 330 cases. In the present case, a calcified lithopedion was found in the abdominal cavity of a 69-year-old woman who had died of unrelated causes.In general, the different organs of the lithopedion were easily distinguishable macroscopically. Microscopic examination detected reasonably well preserved structure only in skeletal muscle.Dry weight studies showed a definite dehydration (mummification) had taken place.Chemical analysis revealed a shell consisting mostly of inorganic constituents.Quantitative inorganic analysis of tissues was done and results compared to normal tissues. These results showed significant decrease of K and Ci, significant increase in Mg, P, Na and enormous increase in Ca. These data are compatible with the histologically observed calcification of the tissues.Under proper conditions, presumably dormant enzymes were reactivated in brain, liver, and muscle, although the total activity was low. Discernible LDH isoenzymes were found in liver and muscle. Discernible CK isoenzymes were found in brain and muscle.     

The mechanism of folate transport in rabbit reticulocytes

Folate transport in phenylhydrazine-induced rabbit reticulocytes was studied with the non-metabolized folate-analog, methotrexate. The time-course of methotrexate uptake into a mixed population of reticulocytes and mature erythrocytes is a two-component process consisting of a small, but rapid, initial uptake phase followed by a much slower uptake component which remains essentially constant over the period of observation. The velocity of the latter uptake component is directly proportional to the per cent reticulocytes and appears to represent a unidirectional influx of methotrexate into these cells. Uptake of methotrexate into reticulocytes was found to have the following characteristics: (a) temperature sensitivity, Q(10) of 4; (b) uptake velocity as a function of the extracellular methotrexate concentration approximated Michaelis-Menten kinetics with a maximum transport velocity of 48 pmoles/min per g dry wt; the extracellular methotrexate level at which the uptake velocity was one-half maximum was 1.4 muM; (c) 5-formyltetrahydrofolate markedly inhibited methotrexate uptake but pteroylglutamic acid inhibition was weak; (d) uptake was stimulated in cells preincubated with 5-formyltetrahydrofolate, indicative of hetero-exchange diffusion; (e) uptake was independent of extracellular sodium but was inhibited by anions including nitrate, phosphate, and glucose-6-phosphate; (f) uptake was enhanced by azide plus iodoacetate.These data indicate that folate transport in rabbit reticulocytes is mediated by a carrier mechanism which disappears with reticulocyte maturation. The mechanism of folate transport in rabbit reticulocytes is qualitatively similar to tumor cells previously studied; both appear to have an energy-dependent mechanism limiting folate uptake, and influx in both is inhibited by structurally unrelated inorganic and organic anions. These studies suggest that circulating pteroylglutamic acid is of little importance in meeting the folate requirements of folate-dependent tissues and raise the possibility that clinical conditions associated with alterations in the anionic composition of the blood may be accompanied by impaired utilization of the folates.     

Relationship of red-cell 2,3-diphosphoglycerate with anaemia,hypoxaemia and acid—base status in patients with cirrhosis of the liver

The red-cell 2,3-diphosphoglycerate (DPG) concentration is determined in 60 patients with hepatic cirrhosis, in 33 with ferropenic anaemia and in 86 healthy subjects. In all cases, the erythrocyte volume fraction and the haemoglobin concentration are simultaneously measured, while the cirrhotic patients undergo, at the same time, analyses of the arterial pH, po₂ and pco₂ and of the levels of inorganic phosphate, bicarbonate and lactate in their venous blood. In the 60 cirrhotic patients the red-cell DPG concentration (7.40 ± 1.23 mmol/1) is significantly higher (P<0.001) than in the 86 control subjects (4.58 ± 0.59 mmol/1) and the 33 patients with ferropenic anaemia (5.86 ± 1.06 mmol/1), although the level of anaemia in the latter is greater (P<0.001) than in the patients with liver cirrhosis. The DPG concentration found in the cirrhotic patients was far higher (P<0.001) than the theoretical value attributable to them by virtue of their grade of anaemia (5.21 ± 0.95 mmol/1), which value is deduced mathematically from the equation of the regression line between haemoglobin and DPG normal in patients with ferropenic anaemia. Anaemia, hypoxaemia and acid-base disturbances are disorders frequently associated with cirrhosis of the liver. In the present study we deduce that alkalosis, and therefore the plasma pH level, is the most important factor causing the increased DPG concentration in patients with liver cirrhosis for any level of haemoglobin, with respect to other subjects with anaemia.     

Bench-to-bedside review: Cytopathic hypoxia

The rate of oxygen consumption by certain tissues is impaired when mice or rats are injected with lipopolysaccharide. A similar change in the rate of oxygen consumption is observed when Caco-2 human enterocyte-like cells are incubated in vitro with cytomix, a cocktail of cytokines containing tumor necrosis factor, IL-1β, and IFN-γ. The decrease in the rate of oxygen consumption is not due to a change in oxygen delivery (e.g. on the basis of diminished microvascular perfusion), but rather to an acquired intrinsic defect in cellular respiration, a phenomenon that we have termed 'cytopathic hypoxia'. A number of different biochemical mechanisms have been postulated to account for cytopathic hypoxia in sepsis, including reversible inhibition of cytochrome a,a ₃ by nitric oxide, and irreversible inhibition of one or more mitochondrial respiratory complexes by peroxynitrite. Recently, however, our laboratory has obtained data to suggest that the most important mechanism underlying the development of cytopathic hypoxia is depletion of cellular stores of nicotinamide adenine dinucleotide (NAD⁺/NADH) as a result of activation of the enzyme, poly(ADP-ribose) polymerase-1. If cytopathic hypoxia is important in the pathophysiology of established sepsis and multiorgan dysfunction syndrome, then efforts in the future will need to focus on pharmacological interventions designed to preserve normal mitochondrial function and energy production in sepsis.