Cysteine protease activity of feline Tritrichomonas foetus promotes adhesion-dependent cytotoxicity to intestinal epithelial cells.

Trichomonads are obligate protozoan parasites most renowned as venereal pathogens of the reproductive tract of humans and cattle. Recently, a trichomonad highly similar to bovine venereal Tritrichomonas foetus but having a unique tropism for the intestinal tract was recognized as a significant cause of colitis in domestic cats. Despite a high prevalence, worldwide distribution, and lack of consistently effective drugs for treatment of the infection, the cellular mechanisms of T. foetus pathogenicity in the intestinal tract have not been examined. The aims of this study were to determine the pathogenic effect of feline T. foetus on porcine intestinal epithelial cells, the dependence of T. foetus pathogenicity on adhesion of T. foetus to the intestinal epithelium, and the identity of mediators responsible for these effects. Using an in vitro coculture approach to model feline T. foetus infection of the intestinal epithelium, these studies demonstrate that T. foetus promotes a direct contact-dependent activation of intestinal epithelial cell apoptosis signaling and progressive monolayer destruction. Moreover, these pathological effects were demonstrated to be largely dependent on T. foetus cell-associated cysteine protease activity. Finally, T. foetus cysteine proteases were identified as enabling cytopathic effects by promoting adhesion of T. foetus to the intestinal epithelium. The present studies are the first to examine the cellular mechanisms of pathogenicity of T. foetus toward the intestinal epithelium and support further investigation of the cysteine proteases as virulence factors in vivo and as potential therapeutic targets for ameliorating the pathological effects of intestinal trichomonosis.

The role of mitochondrial function and cellular bioenergetics in ageing and disease.

Mitochondria constitute an important topic of biomedical enquiry (one paper in every 154 indexed in PubMed since 1998 is retrieved by the keyword 'mitochondria') because of widespread recognition of their importance in cell physiology and pathology. Mitochondrial dysfunction is widely implicated in ageing and in the diseases of ageing, through dysfunction in adenosine triphosphate (ATP) synthesis, Ca(2+) homeostasis, central metabolic pathways or radical production. Nonetheless, the mechanisms and regulation of superoxide and hydrogen peroxide formation by mitochondria remain poorly described. Measurement of the capacities of different sites of superoxide and hydrogen peroxide production in isolated skeletal muscle mitochondria show that the maximum capacities of sites in complexes I, II and III and in several associated redox enzymes greatly exceed the native rates observed in the absence of respiratory chain inhibitors. In vitro, the native rates and the relative importance of different sites both depend on the substrate being oxidized, with sites IQ, IIF, GPDH, IF and IIIQo each being important with particular substrates. The techniques involved in measuring rates from each site should become applicable to cell cultures and in vivo in the future.

Mitochondrial uncoupling protein 2 in pancreatic ß-cells.

Pancreatic ß-cells have remarkable bioenergetics in which increased glucose supply upregulates the cytosolic ATP/ADP ratio and increases insulin secretion. This arrangement allows glucose-stimulated insulin secretion (GSIS) to be regulated by the coupling efficiency of oxidative phosphorylation. Uncoupling protein 2 (UCP2) modulates coupling efficiency and may regulate GSIS. Initial measurements of GSIS and glucose tolerance in Ucp2(-/-) mice supported this model, but recent studies show confounding effects of genetic background. Importantly, however, the enhancement of GSIS is robustly recapitulated with acute UCP2 knockdown in INS-1E insulinoma cells. UCP2 protein level in these cells is dynamically regulated, over at least a fourfold concentration range, by rapid proteolysis (half-life less than 1 h) opposing regulated gene transcription and mRNA translation. Degradation is catalysed by the cytosolic proteasome in an unprecedented pathway that is currently known to act only on UCP2 and UCP3. Evidence for proteasomal turnover of UCP2 includes sensitivity of degradation to classic proteasome inhibitors in cells, and reconstitution of degradation in vitro in mitochondria incubated with ubiquitin and the cytosolic 26S proteasome. These dynamic changes in UCP2 content may provide a fine level of control over GSIS in ß-cells.

Mitochondrial proton leak kinetics and relationship with feed efficiency within a single genetic line of male broilers.

Studies were conducted to assess proton leak kinetics (proton conductance) in breast muscle mitochondria isolated from broiler breeder males within a single genetic line exhibiting either high (HFE) or low (LFE) feed efficiency. Proton leak kinetics were determined by simultaneously measuring mitochondrial membrane potential and state 2 (resting) respiration rate in breast muscle mitochondria as succinate oxidation was progressively decreased by malonate. Control proton conductance was similar in HFE and LFE mitochondria and decreased to a similar extent in both groups in response to BSA. Although treatment of mitochondria with Glu or guanosine diphosphate had no effect, retinal increased and carboxyatractylate alone or in combination with Glu decreased proton conductance relative to control proton conductance in both HFE and LFE mitochondria. After treatment with either guanosine diphosphate or carboxyatractylate alone, proton conductance was lower in HFE compared with LFE mitochondria. With the exception of BSA, proton conductance in HFE mitochondria after the various chemical treatments was either less than or equal to, and never greater than, proton conductance in the LFE mitochondria. The results suggest that there are subtle differences in membrane characteristics (e.g., lipids, integral membrane proteins) that affect proton conductance in broiler muscle mitochondria that may in turn play a role in the phenotypic expression of feed efficiency.

The efficiency and plasticity of mitochondrial energy transduction.

Since it was first realized that biological energy transduction involves oxygen and ATP, opinions about the amount of ATP made per oxygen consumed have continually evolved. The coupling efficiency is crucial because it constrains mechanistic models of the electron-transport chain and ATP synthase, and underpins the physiology and ecology of how organisms prosper in a thermodynamically hostile environment. Mechanistically, we have a good model of proton pumping by complex III of the electron-transport chain and a reasonable understanding of complex IV and the ATP synthase, but remain ignorant about complex I. Energy transduction is plastic: coupling efficiency can vary. Whether this occurs physiologically by molecular slipping in the proton pumps remains controversial. However, the membrane clearly leaks protons, decreasing the energy funnelled into ATP synthesis. Up to 20% of the basal metabolic rate may be used to drive this basal leak. In addition, UCP1 (uncoupling protein 1) is used in specialized tissues to uncouple oxidative phosphorylation, causing adaptive thermogenesis. Other UCPs can also uncouple, but are tightly regulated; they may function to decrease coupling efficiency and so attenuate mitochondrial radical production. UCPs may also integrate inputs from different fuels in pancreatic beta-cells and modulate insulin secretion. They are exciting potential targets for treatment of obesity, cachexia, aging and diabetes.

Respiration rate of hepatocytes varies with body mass in birds.

Hepatocytes were isolated from eight species of birds ranging from 13 g zebra finches to 35 kg emus. This represents a 2800-fold range of body mass (Mb). Liver mass (g) was allometrically related to species body mass by the equation: liver mass=19.6 x Mb(0.91). There was a significant allometric decline in hepatocyte respiration rate (HRR; nmol O2 mg(-1) dry mass min(-1)) with species body mass (kg) described by the relationship: HRR=5.27 x Mb(-0.10). The proportions of hepatocyte oxygen consumption devoted to (i) mitochondrial ATP production, (ii) mitochondrial proton leak and (iii) non-mitochondrial processes were estimated by using excess amounts of appropriate inhibitors. It was found that although hepatocyte respiration rate varied with body mass in birds, these processes constitute a relatively constant proportion of hepatocyte metabolic rate irrespective of the size of the bird species. The respective percentages were 54%, 21% and 25%. The portion of hepatocyte respiration devoted to ATP production for use by the sodium pump was estimated and found to be a relatively constant 24% of hepatocyte respiration and 45% of mitochondrial ATP production in different-sized bird species. These results are discussed in the context of competing theories to explain the metabolism-body size allometry, and are found to support the 'allometric cascade' model.

Mitochondrial matrix reactive oxygen species production is very sensitive to mild uncoupling.

Mitochondria produce ROS (reactive oxygen species) as a by-product of aerobic respiration. Several studies in mammals and birds suggest that the most physiologically relevant ROS production is from complex I following reverse electron flow, and is highly sensitive to membrane potential. A study of Drosophila mitochondria respiring glycerol 3-phosphate revealed that membrane potential-sensitive ROS production from complex I following reverse electron flow was on the matrix side of the inner membrane. A 10 mV decrease in membrane potential was enough to abolish around 70% of the ROS produced by complex I under these conditions. Another important ROS generator in this model, glycerol-3-phosphate dehydrogenase, produced ROS mostly to the cytosolic side; this ROS production was totally insensitive to a small decrease in membrane potential (10 mV). Thus mild uncoupling may be particularly significant for ROS production from complex I on the matrix side of the mitochondrial inner membrane.

Extracting meaning from microarray data.

Gene expression is complex: many mRNAs change in abundance in response to a new condition. But while some of these expression changes may be direct, many may be downstream, indirect effects. One of the major problems of microarray data analysis is distinguishing between these changes. Some of the most common methods of analysis are discussed, in the context of their ability to distinguish between direct and indirect expression changes. The application of modular control analysis to microarray data in order to partition and quantify the importance of mRNA clusters in mediating responses is described.

Proton leak in hepatocytes and liver mitochondria from archosaurs (crocodiles) and allometric relationships for ectotherms.

It has previously been shown that mitochondrial proton conductance decreases with increasing body mass in mammals and is lower in a 250-g lizard than the laboratory rat. To examine whether mitochondrial proton conductance is extremely low in very large reptiles, hepatocytes and mitochondria were prepared from saltwater crocodiles ( Crocodylus porosus) and freshwater crocodiles ( Crocodylus johnstoni). Respiration rates of hepatocytes and liver mitochondria were measured at 37 degrees C and compared with values obtained for rat or previously measured for other species. Respiration rates of hepatocytes from either species of crocodile were similar to those reported for lizards and approximately one fifth of the rates measured using cells from mammals (rat and sheep). Ten-to-thirty percent of crocodile hepatocyte respiration was used to drive mitochondrial proton leak, similar to the proportion in other species. Respiration rates of crocodile liver mitochondria were similar to those of mammalian species. Proton leak rate in isolated liver mitochondria was measured as a function of membrane potential. Contrary to our prediction, the mitochondrial proton conductance of liver mitochondria from crocodiles was greater than that of liver mitochondria from lizards and was similar to that of rats. The acyl composition of liver mitochondrial phospholipids from the crocodiles was more similar to that in mitochondria from rats than in mitochondria from lizards. The relatively high mitochondrial proton conductance was associated with a relatively small liver, which seems to be characteristic of crocodilians. Comparison of data from a number of diverse ectothermic species suggested that hepatocyte respiration rate may decrease with body mass, with an allometric exponent of about -0.2, similar to the exponent in mammalian hepatocytes. However, unlike mammals, liver mitochondrial proton conductance in ectotherms showed no allometric relationship with body size.

Structure and function of mitochondria in hepatopancreas cells from metabolically depressed snails.

Mitochondria in cells isolated from the hepatopancreas of aestivating land snails (Helix aspersa) consume oxygen at 30% of the active control rate. The aim of this study was to investigate whether the lower respiration rate is caused by a decrease in the density of mitochondria or by intrinsic changes in the mitochondria. Mitochondria occupied 2% of cellular volume, and the mitochondrial inner membrane surface density was 17 microm(-1), in cells from active snails. These values were not different in cells from aestivating snails. The mitochondrial protein and mitochondrial phospholipid contents of cells were also similar. There was little difference in the phospholipid fatty acyl composition of mitochondria isolated from metabolically depressed or active snails, except for arachidonic acid, which was 18% higher in mitochondria from aestivating snails. However, the activities of citrate synthase and cytochrome c oxidase in mitochondria isolated from aestivating snails were 68% and 63% of control, respectively. Thus the lower mitochondrial respiration rate in hepatopancreas cells from aestivating snails was not caused by differences in mitochondrial volume or surface density but was associated with intrinsic changes in the mitochondria.

Coenzyme Q induces GDP-sensitive proton conductance in kidney mitochondria.

Addition of coenzyme Q(10) (CoQ) at low concentration (29 nmol/mg of protein) to kidney but not liver mitochondria resulted in an increase in proton conductance. This uncoupling activity required fatty acid and was completely inhibited by GDP. CoQ activated when it was likely to be reduced but not when it was likely to become oxidized. However, the redox state of endogenous CoQ did not affect mitochondrial proton conductance. Stimulation by CoQ was not inhibited by cyclosporin A, carboxyatractylate, bongkrekate and catalase but could be reversed by superoxide dismutase. We conclude that CoQ acted in mitochondria through production of superoxide, which mediated uncoupling, probably by acting through uncoupling protein 2.

Signaling takes a breath--new quantitative perspectives on bioenergetics and signal transduction.

Immune cells are in constant need of energy for both basic housekeeping and specific immune functions. Increased energy demand during lymphocyte stimulation is coordinated by signal transduction pathways. This review explores the interface between lymphocyte signaling and energy metabolism. In particular, it discusses recent work that allows weighing signaling routes with respect to their role in the regulation of energy metabolism during lymphocyte activation.

Radiation-associated valvular heart disease in Hodgkin's disease is associated with characteristic thickening and fibrosis of the aortic-mitral curtain.

Radiation-associated valvular dysfunction is characterized by variable aortic and mitral valve thickening. A review of three patients assessed echocardiographically revealed that radiation-associated valvular dysfunction after radiation treatment for Hodgkin's disease may be characterized by a unique and consistent pattern of thickening of the aortic and mitral valves involving the aortic-mitral curtain.

A mitochondrial uncoupling artifact can be caused by expression of uncoupling protein 1 in yeast.

Uncoupling protein 1 (UCP1) from mouse was expressed in yeast and the specific (GDP-inhibitable) and artifactual (GDP-insensitive) effects on mitochondrial uncoupling were assessed. UCP1 provides a GDP-inhibitable model system to help interpret the uncoupling effects of high expression in yeast of other members of the mitochondrial carrier protein family, such as the UCP1 homologues UCP2 and UCP3. Yeast expressing UCP1 at modest levels (approx. 1 microg/mg of mitochondrial protein) showed no growth defect, normal rates of chemically uncoupled respiration and an increased non-phosphorylating proton conductance that was completely GDP-sensitive. The catalytic-centre activity of UCP1 in these yeast mitochondria was similar to that in mammalian brown-adipose-tissue mitochondria. However, yeast expressing UCP1 at higher levels (approx. 11 microg/mg of mitochondrial protein) showed a growth defect. Their mitochondria had depressed chemically uncoupled respiration rates and an increased proton conductance that was partly GDP-insensitive. Thus, although UCP1 shows native behaviour at modest levels of expression in yeast, higher levels (or rates) of expression can lead to an uncoupling that is not a physiological property of the native protein and is therefore artifactual. This observation might be important in the interpretation of results from experiments in which the functions of UCP1 homologues are verified by their ability to uncouple yeast mitochondria.

Mitochondrial proton leak and the uncoupling protein 1 homologues.

Mitochondrial proton leak is the largest single contributor to the standard metabolic rate (SMR) of a rat, accounting for about 20% of SMR. Yet the mechanisms by which proton leak occurs are incompletely understood. The available evidence suggests that both phospholipids and proteins in the mitochondrial inner membrane are important determinants of proton conductance. The uncoupling protein 1 homologues (e.g. UCP2, UCP3) may play a role in mediating proton leak, but it is unlikely they account for all of the observed proton conductance. Experimental data regarding the functions of these proteins include important ambiguities and contradictions which must be addressed before their function can be confirmed. The physiological role of the proton leak, and of the uncoupling protein 1 homologues, remains similarly unclear.

Physiological levels of mammalian uncoupling protein 2 do not uncouple yeast mitochondria.

We assessed the ability of human uncoupling protein 2 (UCP2) to uncouple mitochondrial oxidative phosphorylation when expressed in yeast at physiological and supraphysiological levels. We used three different inducible UCP2 expression constructs to achieve mitochondrial UCP2 expression levels in yeast of 33, 283, and 4100 ng of UCP2/mg of mitochondrial protein. Yeast mitochondria expressing UCP2 at 33 or 283 ng/mg showed no increase in proton conductance, even in the presence of various putative effectors, including palmitate and all-trans-retinoic acid. Only when UCP2 expression in yeast mitochondria was increased to 4 microg/mg, more than an order of magnitude greater than the highest known physiological concentration, was proton conductance increased. This increased proton conductance was not abolished by GDP. At this high level of UCP2 expression, an inhibition of substrate oxidation was observed, which cannot be readily explained by an uncoupling activity of UCP2. Quantitatively, even the uncoupling seen at 4 microgram/mg was insufficient to account for the basal proton conductance of mammalian mitochondria. These observations suggest that uncoupling of yeast mitochondria by UCP2 is an overexpression artifact leading to compromised mitochondrial integrity.

Mitochondrial uncoupling as a target for drug development for the treatment of obesity.

Mitochondrial proton cycling is responsible for a significant proportion of basal or standard metabolic rate, so further uncoupling of mitochondria may be a good way to increase energy expenditure and represents a good pharmacological target for the treatment of obesity. Uncoupling by 2,4-dinitrophenol has been used in this way in the past with notable success, and some of the effects of thyroid hormone treatment to induce weight loss may also be due to uncoupling. Diet can alter the pattern of phospholipid fatty acyl groups in the mitochondrial membrane, and this may be a route to uncoupling in vivo. Energy expenditure can be increased by stimulating the activity of uncoupling protein 1 (UCP1) in brown adipocytes either directly or through beta 3-adrenoceptor agonists. UCP2 in a number of tissues, UCP3 in skeletal muscle and the adenine nucleotide translocase have also been proposed as possible drug targets. Specific uncoupling of muscle or brown adipocyte mitochondria remains an attractive target for the development of antiobesity drugs.

Quantitation of signal transduction.

Conventional qualitative approaches to signal transduction provide powerful ways to explore the architecture and function of signaling pathways. However, at the level of the complete system, they do not fully depict the interactions between signaling and metabolic pathways and fail to give a manageable overview of the complexity that is often a feature of cellular signal transduction. Here, we introduce a quantitative experimental approach to signal transduction that helps to overcome these difficulties. We present a quantitative analysis of signal transduction during early mitogen stimulation of lymphocytes, with steady-state respiration rate as a convenient marker of metabolic stimulation. First, by inhibiting various key signaling pathways, we measure their relative importance in regulating respiration. About 80% of the input signal is conveyed via identifiable routes: 50% through pathways sensitive to inhibitors of protein kinase C and MAP kinase and 30% through pathways sensitive to an inhibitor of calcineurin. Second, we quantify how each of these pathways differentially stimulates functional units of reactions that produce and consume a key intermediate in respiration: the mitochondrial membrane potential. Both the PKC and calcineurin routes stimulate consumption more strongly than production, whereas the unidentified signaling routes stimulate production more than consumption, leading to no change in membrane potential despite increased respiration rate. The approach allows a quantitative description of the relative importance of signal transduction pathways and the routes by which they activate a specific cellular process. It should be widely applicable.

Processes contributing to metabolic depression in hepatopancreas cells from the snail Helix aspersa.

Cells isolated from the hepatopancreas of the land snail Helix aspersa strongly depress respiration both immediately in response to lowered P(O2) (oxygen conformation) and, in the longer term, during aestivation. These phenomena were analysed by dividing cellular respiration into non-mitochondrial and mitochondrial respiration using the mitochondrial poisons myxothiazol, antimycin and azide. Non-mitochondrial respiration accounted for a surprisingly large proportion, 65+/-5 %, of cellular respiration in control cells at 70 % air saturation. Non-mitochondrial respiration decreased substantially as oxygen tension was lowered, but mitochondrial respiration did not, and the oxygen-conforming behaviour of the cells was due entirely to the oxygen-dependence of non-mitochondrial oxygen consumption. Non-mitochondrial respiration was still responsible for 45+/-2 % of cellular respiration at physiological oxygen tension. Mitochondrial respiration was further subdivided into respiration used to drive ATP turnover and respiration used to drive futile proton cycling across the mitochondrial inner membrane using the ATP synthase inhibitor oligomycin. At physiological oxygen tensions, 34+/-5 % of cellular respiration was used to drive ATP turnover and 22+/-4 % was used to drive proton cycling, echoing the metabolic inefficiency previously observed in liver cells from mammals, reptiles and amphibians. The respiration rate of hepatopancreas cells from aestivating snails was only 37 % of the control value. This was caused by proportional decreases in non-mitochondrial and mitochondrial respiration and in respiration to drive ATP turnover and to drive proton cycling. Thus, the fraction of cellular respiration devoted to different processes remained constant and the cellular energy balance was preserved in the hypometabolic state.

AMP decreases the efficiency of skeletal-muscle mitochondria.

Mitochondrial proton leak in rat muscle is responsible for approx. 15% of the standard metabolic rate, so its modulation could be important in regulating metabolic efficiency. We report in the present paper that physiological concentrations of AMP (K(0.5)=80 microM) increase the resting respiration rate and double the proton conductance of rat skeletal-muscle mitochondria. This effect is specific for AMP. AMP also doubles proton conductance in skeletal-muscle mitochondria from an ectotherm (the frog Rana temporaria), suggesting that AMP activation is not primarily for thermogenesis. AMP activation in rat muscle mitochondria is unchanged when uncoupling protein-3 is doubled by starvation, indicating that this protein is not involved in the AMP effect. AMP activation is, however, abolished by inhibitors and substrates of the adenine nucleotide translocase (ANT), suggesting that this carrier (possibly the ANT1 isoform) mediates AMP activation. AMP activation of ANT could be important for physiological regulation of metabolic rate.