The cell wall regulates dynamics and size of plasma-membrane nanodomains in Arabidopsis.

Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport, and cellular signaling. For these proteins to function effectively there needs to be structure within the PM allowing, for example, proteins in the same signaling cascade to be spatially organized. Here we demonstrate that several proteins with divergent functions are located in clusters of differing size in the membrane using subdiffraction-limited Airyscan confocal microscopy. Single particle tracking reveals that these proteins move at different rates within the membrane. Actin and microtubule cytoskeletons appear to significantly regulate the mobility of one of these proteins (the pathogen receptor FLS2) and we further demonstrate that the cell wall is critical for the regulation of cluster size by quantifying single particle dynamics of proteins with key roles in morphogenesis (PIN3) and pathogen perception (FLS2). We propose a model in which the cell wall and cytoskeleton are pivotal for regulation of protein cluster size and dynamics, thereby contributing to the formation and functionality of membrane nanodomains.

Simultaneous widefield single molecule orientation and FRET microscopy in cells.

We combine single molecule fluorescence orientation imaging with single-pair fluorescence resonance energy transfer microscopy, using a total internal reflection microscope. We show how angles and FRET efficiencies can be determined for membrane proteins at the single molecule level and provide data from the epidermal growth factor receptor system in cells.

Use of physiological doses of human growth hormone in haematological patients receiving intensive chemotherapy promotes haematopoietic recovery: a double-blind randomized, placebo-controlled study.

In vivo and in vitro studies suggest human growth hormone (hGH) receptors on bone marrow stem cells may be biologically active and could be exploited to promote haemopoetic recovery after intensive chemotherapy. Patients with haematological malignancies receiving intensive chemotherapy and requiring hospitalization were randomized in a double-blind, placebo-controlled single-centre trial. Patients were randomly assigned to receive either hGH 500 microg/day or placebo, for 6 weeks. There was no significant difference in patient characteristics at baseline between the placebo and treatment arms. Patients treated with hGH showed significantly faster recovery of platelets to 25 x 10(9)/l (median of 16 versus 19 days; P = 0.03) compared to the placebo-controlled arm (hazard ratio 1.47 favouring hGH, 95% confidence interval (CI), 1.03-2.08). Time to relapse did not differ significantly between arms. There was no change in the anthropometric parameters at the start and end of hGH/placebo therapy. The study drug was well tolerated. Treatment with hGH in physiological doses improves platelet recovery, but is not associated with a lower relapse rate or improved anthropometric parameters in patients receiving intensive chemotherapy.

Spatial analysis of Yersinia pestis and Bartonella vinsonii subsp. berkhoffii seroprevalence in California coyotes (Canis latrans).

Zoonotic transmission of sylvatic plague caused by Yersinia pestis occurs in California, USA. Human infections with various Bartonella species have been reported recently. Coyotes (Canis latrans) are ubiquitous throughout California and can become infected with both bacterial agents, making the species useful for surveillance purposes. This study examined the geographic distribution of 863 coyotes tested for Y. pestis and Bartonella vinsonii subsp. berkhoffii serologic status to gain insight into the natural history of B. vinsonii subsp. berkhoffii and to characterize the spatial distribution of the two agents. We found 11.7% of specimens positive to Y. pestis and 35.5% positive to B. vinsonii subsp. berkhoffii. The two pathogens had distinct spatial clusters: Y. pestis was more prevalent in eastern portions of the state and B. vinsonii subsp. berkhoffii in coastal regions. Prevalence of Y. pestis increased with increasing elevation, whereas prevalence of B. vinsonii subsp. berkhoffii decreased with increasing elevation. There were differences in the proportions of positive animals on a yearly basis to both pathogens.

The public awareness of aphasia: an international survey.

We surveyed 929 shoppers in Exeter (England), Louisiana (USA) and Sydney (Australia) to determine what they knew of aphasia. Between 10% and 18% said they had heard of aphasia but only between 1.5% and 7.6% had even some basic knowledge of aphasia. We found that more females knew something about aphasia than males and that older people were more likely to have heard of it, although those with some knowledge were significantly younger. Informants had heard of aphasia mainly through their work or the media and were mainly professionals like teachers, nurses, therapists, managers and administrators, followed by a retired/student group. We found some differences in awareness levels in the different locations we sampled. Results have implications for targeting awareness raising and campaigning.

Drag swab efficiency factors when sampling chicken manure.

This study examines drag swabbing distance, media for moistening the drag swabs, and site selection when sampling a laying facility by drag swabbing manure piles. Manure piles at a laying facility in California's San Joaquin Valley were sampled with drag swabs over various distances. Samples were cultured for Salmonella spp. with standard laboratory methods, and most probable number calculations. Salmonella spp. counts were expected to be highly variable because of reported clustering. Therefore, total bacteria and Escherichia coli, which were assumed to have a more uniform distribution on the surface of the manure, were additionally used as proxies for Salmonella. Media for moistening the swabs were compared by seeding postswabbing samples with Salmonella typhimurium, and culturing at different delay times. Total bacterial counts were compared between samples that were obtained from either wet or dry surfaces. Numbers of Salmonella spp. and total bacteria peaked within 120 feet of swabbing distance. Higher total bacteria counts were obtained by swabbing wet areas rather than dry areas, but the distance that could be swabbed effectively was shorter in wet areas. Moistening media selected for the swab resulted in statistically different culture counts, but did not show any important difference in maintaining Salmonella viability over a 48-hr period when the samples were kept at refrigerated temperatures. Once swabs became fully loaded with fecal material, bacterial numbers failed to increase with further use. Overuse of a swab may result in failure to detect Salmonella enteritidis on chicken manure if the distribution of this organism is clustered.

The significance and mechanism of mitochondrial proton conductance.

There is a futile cycle of pump and leak of protons across the mitochondrial inner membrane. The contribution of the proton cycle to standard metabolic rate is significant, particularly in skeletal muscle, and it accounts for 20% or more of the resting respiration of a rat. The mechanism of the proton leak is uncertain: basal proton conductance is not a simple biophysical leak across the unmodified phospholipid bilayer. Equally, the evidence that it is catalysed by homologues of the brown adipose uncoupling protein, UCP1, is weak. The yeast genome contains no clear UCP homologue but yeast mitochondria have normal basal proton conductance. UCP1 catalyses a regulated inducible proton conductance in brown adipose tissue and the possibility remains open that UCP2 and UCP3 have a similar role in other tissues, although this has yet to be demonstrated.

Contribution of mitochondrial proton leak to respiration rate in working skeletal muscle and liver and to SMR.

Proton pumping across the mitochondrial inner membrane and proton leak back through the natural proton conductance pathway make up a futile cycle that dissipates redox energy. We measured respiration and average mitochondrial membrane potential in perfused rat hindquarter with maximal tetanic contraction of the left gastrocnemius-soleus-plantaris muscle group, and we estimate that the mitochondrial proton cycle accounted for 34% of the respiration rate of the preparation. Similar measurements in rat hepatocytes given substrates to cause a high rate of gluconeogenesis and ureagenesis showed that the proton cycle accounted for 22% of the respiration rate of these cells. Combining these in vitro values with literature values for the contribution of skeletal muscle and liver to standard metabolic rate (SMR), we calculate that the proton cycle in working muscle and liver may account for 15% of SMR in vivo. Although this value is less than the 20% of SMR we calculated previously using data from resting skeletal muscle and hepatocytes, it is still large, and we conclude that the futile proton cycle is a major contributor to SMR.

Cellular energy utilization and molecular origin of standard metabolic rate in mammals.

The molecular origin of standard metabolic rate and thermogenesis in mammals is examined. It is pointed out that there are important differences and distinctions between the cellular reactions that 1) couple to oxygen consumption, 2) uncouple metabolism, 3) hydrolyze ATP, 4) control metabolic rate, 5) regulate metabolic rate, 6) produce heat, and 7) dissipate free energy. The quantitative contribution of different cellular reactions to these processes is assessed in mammals. We estimate that approximately 90% of mammalian oxygen consumption in the standard state is mitochondrial, of which approximately 20% is uncoupled by the mitochondrial proton leak and 80% is coupled to ATP synthesis. The consequences of the significant contribution of proton leak to standard metabolic rate for tissue P-to-O ratio, heat production, and free energy dissipation by oxidative phosphorylation and the estimated contribution of ATP-consuming processes to tissue oxygen consumption rate are discussed. Of the 80% of oxygen consumption coupled to ATP synthesis, approximately 25-30% is used by protein synthesis, 19-28% by the Na(+)-K(+)-ATPase, 4-8% by the Ca2(+)-ATPase, 2-8% by the actinomyosin ATPase, 7-10% by gluconeogenesis, and 3% by ureagenesis, with mRNA synthesis and substrate cycling also making significant contributions. The main cellular reactions that uncouple standard energy metabolism are the Na+, K+, H+, and Ca2+ channels and leaks of cell membranes and protein breakdown. Cellular metabolic rate is controlled by a number of processes including metabolic demand and substrate supply. The differences in standard metabolic rate between animals of different body mass and phylogeny appear to be due to proportionate changes in the whole of energy metabolism. Heat is produced by some reactions and taken up by others but is mainly produced by the reactions of mitochondrial respiration, oxidative phosphorylation, and proton leak on the inner mitochondrial membrane. Free energy is dissipated by all cellular reactions, but the major contributions are by the ATP-utilizing reactions and the uncoupling reactions. The functions and evolutionary significance of standard metabolic rate are discussed.

The physiological significance of mitochondrial proton leak in animal cells and tissues.

Mitochondrial proton leak is an important component of cellular metabolism in animals and may account for as much as one quarter to one third of the Standard Metabolic Rate of the rat. The activity of the proton leak pathway is different in a wide range of animal species and in different thyroid states. Such differences imply some function for proton leak and candidates for this function include thermogenesis, protection against reactive oxygen species, endowment of metabolic sensitivity and maintenance of carbon fluxes.

The proton permeability of liposomes made from mitochondrial inner membrane phospholipids: comparison with isolated mitochondria.

Unilamellar liposomes with native phospholipid fatty acid composition were prepared from rat liver mitochondrial inner membrane phospholipids by extrusion in medium containing 50 mm potassium. They were diluted into low potassium medium to establish a transmembrane potassium gradient. A known membrane potential was imposed by addition of valinomycin, and proton flux into liposomes was measured. Valinomycin in the range 10 pm-1nm was sufficient to fully establish membrane potential. Valinomycin concentrations above 3 nm catalyzed additional proton flux and were avoided. At 300 pm valinomycin, proton flux depended nonlinearly on membrane potential. At 160 mV membrane potential the flux was 30 nmol H+/min/mg phospholipid-approximately 5% of the proton leak flux under comparable conditions in isolated mitochondria, indicating that leak pathways through bulk phospholipid bilayer account for only a small proportion of total mitochondrial proton leak.

Contribution of mitochondrial proton leak to skeletal muscle respiration and to standard metabolic rate.

We have tested the hypothesis that the leak of protons across the mitochondrial inner membrane (proton leak) is a significant contributor to standard metabolic rate (SMR). We found that proton leak accounts for around one-half of the resting respiration rate of perfused rat skeletal muscle. Proton leak is known to make a significant (26%) contribution to the resting respiration rate of isolated rat hepatocytes (M. D. Brand, L.-F. Chien, E. K. Ainscow, D. F. S. Rolfe, and R. K. Porter. Biochim. Biophys. Acta 1187: 132-139, 1994). If the importance of proton leak in these isolated and perfused systems is similar to its importance in vivo, then using literature values for the contribution of liver and skeletal muscle to SMR, we can calculate that proton leak in liver and skeletal muscle alone accounts for 11-26% (mean 20%) of the SMR of the rat. If proton leak activity in the other tissues of the rat is similar to that in liver cells, then the contribution of proton leak to rat SMR would be 16-31% (mean 25%).

Proton leak and control of oxidative phosphorylation in perfused, resting rat skeletal muscle.

The distribution of control over the resting respiration rate (and other variables) was assessed in perfused rat skeletal muscle. The results indicate that the general pattern of control in resting muscle is similar to that seen in isolated rat liver cells (Brown et al. (1990) Biochem. J. 192, 355-362). Control over resting mitochondrial oxygen consumption was distributed between reactions involved in substrate oxidation (flux control coefficient = 0.44 +/- 0.28) and those involved in ATP turnover (0.21 +/- 0.09). The mitochondrial proton leak also had an important share of the control (0.38 +/- 0.21). Since proton leak also has significant control over the resting respiration rate of isolated liver cells and liver and skeletal muscle account for around 60% of the standard metabolic rate of a rat, then these results indicate the potential importance of proton leak as a mechanism of regulating metabolic rate.

Copper-associated hepatopathies in dogs.

Copper-associated hepatotoxicity in certain breeds has been identified for almost 20 years, and the list of affected breeds is growing. The importance for copper analysis of liver specimens is emphasized by the realization that copper accumulation occurs in many hepatic disease states, and it may become pathologic. Many treatment possibilities exist. It is important that therapy be tailored to the needs of the animal and the severity of accumulation. To determine efficacy of treatment and when an endpoint has been reached, follow-up liver biopsies are essential.

Characteristics of mitochondrial proton leak and control of oxidative phosphorylation in the major oxygen-consuming tissues of the rat.

Maintenance of an electrochemical proton gradient across the mitochondrial inner membrane against the significant proton permeability of the membrane accounts for 25-30% of resting oxygen consumption in hepatocytes. It has been proposed that proton leak could be a significant contributor to resting metabolic rate in mammals if it were present in other tissues. Mitochondria were isolated from the major oxygen-consuming tissues (liver, kidney, brain and skeletal muscle) of the rat. In each tissue, the mitochondria showed significant proton leak with the same characteristic non-linear dependence on membrane potential. Liver and kidney mitochondria showed similar membrane proton permeability per mg of mitochondrial protein; brain and muscle permeabilities were greater when expressed in this way. Differences in the kinetic response of the substrate oxidation and phosphorylating systems to membrane potential were observed. The substrate oxidation system was more active in kidney, brain and skeletal muscle mitochondria than in liver mitochondria per mg of mitochondrial protein. Liver and kidney phosphorylating systems were less active than brain and skeletal muscle per mg of mitochondrial protein. The control of oxidative phosphorylation was also assessed. The distribution of control in mitochondria isolated from the four tissue types was found to be similar.

The causes and functions of mitochondrial proton leak.

The non-linear relationship between respiration rate and protonmotive force in isolated mitochondria is explained entirely by delta p-dependent changes in the proton conductance of the mitochondrial inner membrane and is not caused by redox slip in the proton pumps. Mitochondrial proton leak occurs in intact cells and tissues: the futile cycle of proton pumping and proton leak accounts for 26% +/- 7% of the total oxygen consumption rate or 33% +/- 7% of the mitochondrial respiration rate of isolated hepatocytes (mean +/- S.D. for 43 rats); 52% of the oxygen consumption rate of resting perfused muscle and up to 38% of the basal metabolic rate of a rat, suggesting that heat production may be an important function in the proton leak in homeotherms. Together with non-mitochondrial oxygen consumption, it lowers the effective P/O ratio in cells from maximum possible values of 2.33 (palmitate oxidation) or 2.58 (glucose oxidation) to as low as 1.1 in liver or 0.8 in muscle. The effective P/O ratio increases in response to ATP demand; the ability to allow rapid switching of flux from leak to ATP turnover may be an even more important function of the leak reaction than heat production. The mitochondrial proton conductance in isolated mitochondria and in hepatocytes is greatly modulated by thyroid hormones, by phylogeny and by body mass. Usually the reactions of ATP turnover change in parallel so that the coupling ratio is not greatly affected. Changes in proton leak in tissues are brought about in the short term by changes in mitochondrial protonmotive force and in the longer term by changes in the surface area and proton permeability of the mitochondrial inner membrane. Permeability changes are probably caused by changes in the fatty acid composition of the membrane phospholipids.

Monitoring of dairy herds for Brucella abortus infection when prevalence is low.

A total of 2,698 dairy herds were surveyed in 1981-1982 in New South Wales and north eastern Victoria in a review of the methods used to monitor them for the presence of Brucella abortus. The methods used to monitor dairy herds were testing of all breeding cows over 1 year of age using the rose bengal test (RBT) and complement fixation test (CFT), the bulk milk ring test (BMRT), and testing of blood samples collected at abattoirs using the RBT and CFT. The surveyed herds had at least one whole herd test, and BMRT was done at regular intervals in the period of the survey. Of the 99 (3.7%) herds that reacted to the BMRT, 91 (3.4%) herds had false positive reactions and 8 (0.3%) herds were declared infected on follow-up herd testing. False-positive reactions were obtained in 22 herds on more than one occasion. Common causes of false positive reactions to the BMRT were thought to be previous vaccination with Strain 19 and sampling in very early or late lactation. Of the 98 (3.63%) herds that reacted to the whole herd serological tests, 80 (2.96%) herds had false-positive reactions and 18 (0.67%) herds were declared infected. Strain 19 vaccination was thought to be an important cause of false-positive reactions. Fifty-three (2.0%) herds showed suspicious reactions on abattoir monitoring but none was declared infected on follow-up testing. Of the 18 herds with infected or equivocal status, the BMRT identified?(ABSTRACT TRUNCATED AT 250 WORDS)