1,279 research outputs found
Cellular localization and associations of the major lipolytic proteins in human skeletal muscle at rest and during exercise
Lipolysis involves the sequential breakdown of fatty acids from triacylglycerol and is increased during energy stress such as exercise. Adipose triglyceride lipase (ATGL) is a key regulator of skeletal muscle lipolysis and perilipin (PLIN) 5 is postulated to be an important regulator of ATGL action of muscle lipolysis. Hence, we hypothesized that non-genomic regulation such as cellular localization and the interaction of these key proteins modulate muscle lipolysis during exercise. PLIN5, ATGL and CGI-58 were highly (>60%) colocated with Oil Red O (ORO) stained lipid droplets. PLIN5 was significantly colocated with ATGL, mitochondria and CGI-58, indicating a close association between the key lipolytic effectors in resting skeletal muscle. The colocation of the lipolytic proteins, their independent association with ORO and the PLIN5/ORO colocation were not altered after 60 min of moderate intensity exercise. Further experiments in cultured human myocytes showed that PLIN5 colocation with ORO or mitochondria is unaffected by pharmacological activation of lipolytic pathways. Together, these data suggest that the major lipolytic proteins are highly expressed at the lipid droplet and colocate in resting skeletal muscle, that their localization and interactions appear to remain unchanged during prolonged exercise, and, accordingly, that other post-translational mechanisms are likely regulators of skeletal muscle lipolysis
BAFF and MyD88 signals promote a lupuslike disease independent of T cells
Systemic lupus erythernatosus (SLE) is a systemic autoimmune disease characterized by the production of autoantibodies. However, the underlying cause of disease appears to relate to defects in T cell tolerance or T cell help to 13 cells. Transgenic (Tg) mice over-expressing the cytokine 13 cell-activating factor of the tumor necrosis factor family (BAFF) develop an autoimmune disorder similar to SLE and show impaired B cell tolerance and altered T cell differentiation. We generated BAFF Tg mice that were completely deficient in T cells, and, surprisingly, these mice developed an SLE-like disease indistinguishable from that of BAFF Tg mice. Autoimmunity in BAFF Tg mice did, however, require 13 cell-intrinsic signals through the Toll-like receptor (TLR)-associated signaling adaptor MyD88, which controlled the production of proinflammatory autoantibody isotypes. TLR7/9 activation strongly up-regulated expression of transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), which is a receptor for BAFF involved in 13 cell responses to T cell-independent antigens. Moreover, BAFF enhanced TLR7/9 expression on 13 cells and TLR-mediated production of autoantibodies. Therefore, autoirnmunity in BAFF Tg mice results from altered 13 cell tolerance, but requires TLR signaling and is independent of T cell help. It is possible that SLE patients with elevated levels of BAFF show a similar basis for disease
PLIN5 deletion remodels intracellular lipid composition and causes insulin resistance in muscle
Defective control of lipid metabolism leading to lipotoxicity causes insulin resistance in skeletal muscle, a major factor leading to diabetes. Here, we demonstrate that perilipin (PLIN) 5 is required to couple intramyocellular triacylglycerol lipolysis with the metabolic demand for fatty acids. PLIN5 ablation depleted triacylglycerol stores but increased sphingolipids including ceramide, hydroxylceramides and sphingomyelin. We generated perilipin 5 (Plin5)-/- mice to determine the functional significance of PLIN5 in metabolic control and insulin action. Loss of PLIN5 had no effect on body weight, feeding or adiposity but increased whole-body carbohydrate oxidation. Plin5-/- mice developed skeletal muscle insulin resistance, which was associated with ceramide accumulation. Liver insulin sensitivity was improved in Plin5-/- mice, indicating tissue-specific effects of PLIN5 on insulin action. We conclude that PLIN5 plays a critical role in coordinating skeletal muscle triacylglycerol metabolism, which impacts sphingolipid metabolism, and is requisite for the maintenance of skeletal muscle insulin action. © 2014 The Authors
Pigmentsynthese bei Exophiala dermatitidis : Einfluss auf Phagozytose und Oxidative Burst
Indolderivate wurden von der Arbeitsgruppe um Prof. Mayser 1998 erstmals bei der Hefe
Malassezia furfur beschrieben, bei der sie vermutlich in einem klinischen Zusammenhang mit
der Hautkrankheit Pityriasis versicolor stehen. Im Jahr 2006 konnten Nies et al. solche
Indolderivate erstmals bei Exophiala dermatitidis nachweisen. Ziel der vorliegenden Arbeit
war die vergleichende Untersuchung der Pigmentspektren verschiedener Stämme von
Exophiala dermatitidis und ihre Überprüfung auf eine mögliche Rolle in der Pathogenese von
Exophiala-dermatitidis-Infektionen.
Hierfür wurden der Wildtyp und seine Mutanten Mel-1, Mel-3 und Mel-4 zur
Pigmentsynthese auf 1b-Minimalagar angezüchtet. Nach vierwöchiger Wachstumsphase
wurde das dabei entstandene Pigment als Rohextrakt gewonnen, durch chromatographische
Methoden vorgereinigt, sowie einzelne farbige Bestandteile weiter aufgereinigt. Die
Stukturaufklärung isolierter Reinsubstanzen erfolgte an der TU München. Grundlage zur
Untersuchung des Oxidative Burst stellte der von Schnitzler et al. (1999) etablierte
Vollblutansatz mit Dihydrorhodamine 123 als Indikator für die Sauerstoffradikalsynthese von
neutrophilen Granulozyten dar. Die anschließende Messung erfolgte am Durchflußzytometer.
Im Rahmen der Untersuchung der Pigmentspektren konnte beim Wildtyp von Exophiala
dermatitidis erstmals die Synthese von Pityriacitrin, Pityriarubin C, Exophialin und
Desoxyexophialin nachgewiesen werden.
Für die Mutante Mel-1 konnte erstmals das bislang nur durch vergleichende DC
nachgewiesene Pityriacitrin in der HPLC, sowie durch Analyse der TU München bestätigt
werden. Desweiteren konnte Exophialin aus zwei violetten Banden mit einem Rf-Wert von
0,38 und 0,56 isoliert werden.
Unter den Syntheseprodukten der Mutante Mel-3 wurde erstmals Exophialin, Pityriarubin C
und Pityriacitrin nachgewiesen.
Beim Vergleich des Wildtyps von Exophiala dermatitidis mit seiner Melaninmangel-Mutante
Mel-3 hinsichtlich ihrer Auswirkung auf den Oxidative Burst in Vollblutproben konnte eine
deutliche Hemmung des Oxidative Burst durch den Wildtyp gegenüber Mel-3 aufgezeigt
werden. Dies läßt den Rückschluß auf eine deutliche Schutzwirkung des vom Wildtyp
synthetisierten Melanins vor der ROS-Synthese zu.
Auch die untersuchten Pigment-Rohextrakte von Wildtyp, Mel-1, Mel-3 und Mel-4 hatten alle
eine hemmende Wirkung auf die ROS-Synthese, wobei der Rohextrakt von Mel-3 den
stärksten Einfluß hatte.
Bei den Sephadex-Fraktionen der Mutante Mel-3 ergaben unterschiedliche Einflüsse auf die
ROS-Synthese. Die Fraktionen zwei und sechs zeigten in allen Zeitstufen zwischen 0 und 60
Minuten die stärkste Unterdrückung, wohingegen bei den Fraktionen drei und acht zu keinem
Zeitpunkt Auswirkungen auf die ROS-Synthese beobachtet wurden.
Nach weiterer Unterteilung der hemmenden Fraktion sechs der Mutante Mel-3 mittels
präparativer Dünnschichtchromatographie in acht Banden konnte eine deutliche Hemmung
des Oxidative Burst durch die Banden 5-8 verzeichnet werden. In diesem Bereich kam neben
zahlreichen Fluorochromen und Pityriarubin C eine prominente Bande von grauer Farbe vor.
Diese Substanz hemmte die ROS-Synthese deutlich, wohingegen Pityriarubin C keinen
Einfluß darauf hatte. Auch die acht Banden der Fraktion zwei der Mutante Mel-3
unterschieden sich in ihrer Auswirkung auf den Oxidative Burst. Hier wirkten sich nur die
Fraktionen 1,4,5 hemmend auf die ROS-Synthese aus.
Die weiterhin untersuchten Reinsubstanzen Exophialin und Malassezin hatten selbst in hohen
Konzentrationen keinen signifikanten Einfluß auf die ROS-Synthese.
Insgesamt konnte gezeigt werden, daß Exophiala dermatitidis Wildtyp und seine Mutanten
unter den eingesetzten Anzuchtbedingungen ein sehr unterschiedliches Spektrum an
pigmentierten Indolderivaten bilden, das sich stets reproduzieren ließ. Es wurde eine
Hemmung der ROS-Synthese durch diese Pigmente in Form von Rohextrakten einzelner
Stämme, in Form weiter aufgereinigten Säulenchromatographie-Fraktionen, sowie in Form
von Einzelbanden beobachtet. Die läßt Rückschlüsse auf deren wichtige Rolle in der
Pathogenese von Exophiala dermatitidis-Infektionen bei Mensch und Tier zu. Die aus diesen
Resultaten hervorgehenden neuen Fragestellungen sind weiteren Untersuchungen
vorbehalten.In 1998, the working group of Prof. Mayser first described some particular indole derivatives
in the yeast Malassezia furfur, which might be clinically related to the skin disease pityriasis
versicolor. In 2006, Nies et al. were the first ones able to show the production of these indole
derivatives in Exophiala dermatitidis. The aim of this study was to compare the pigment
spectrum of different species of Exophiala dermatitidis and to clarify whether they are part of
the pathogenese of infections with Exophiala dermatitidis.
The wildtype and its mutants Mel-1, Mel-3 and Mel-4 were cultivated on minimal agar as a
base for pigment production. The pigment was extracted after a period of growth of 4 weeks
and chromatographically purified to obtain pure substances. Structural analyses of the isolated
substances were performed at Munich Technological University. Following the studies of
Schnitzler et al. (1999), unlabeled yeast cells were incubated in heparinized whole blood and
dihydrorhodamine 123 was added as an indicator for the ROS-synthesis in the granulocytes.
Flow cytometric analysis was performed in a FACScan flow cytometer.
By investigating the pigment spectrum of Exophiala dermatitidis, the synthesis of
Pityriacitrin, Pityriarubin C, Exophialin and Desoxyexophialin in the wildtype could be
shown.
In former studies, the pigments of the mutant Mel-1 were already investigated by comparative
DC and the indole pyruvate Pityriacitrin was found to be produced by this yeast. In the
present study, this achievement could be approved by HPLC and the analysis of Munich
Technological University. Exophialin, an almost ankwown indole derivate which was isolated
from Mel-3 for the first time by Nies et al. (2006) was found in two violet DC-bands of Mel-1
(Rf=0,38; Rf=0,56).
The indole derivates Exophialin, Pityriarubin C and Pityriacitrin were detected in the mutant
Mel-3 in this present study.
The degree of the oxidative burst evoked by Exophiala dermatitidis wildtype was found to be
lower when it was compared with its mutant Mel-3. The wildtype is probably protected from
the oxidative burst by its synthesised Melanin.
When the raw extracts of the wildtype, Mel-1, Mel-3 and Mel-4 were added in the
experimental series of oxidative burst, evoked oxidative burst levels were also found to be
lower compared with the contol without any indole derivates. The raw extract of Mel-3
inhibited the oxidative burst most. The sephadex-fractions of the mutant Mel-3 had different
influences on the synthesis of ROS. Fractions two and six suppressed the oxidative burst in all
time levels between 0 and 60 minutes strongly, while fractions three and eight had no
influence on the synthesis of ROS at any time.
Fraction six was subdivided by preparative thin layer chromatography into eight bands and
the bands 5-8 were able to inhibit evoked oxidative burst levels. Beside the already known
Pityriarubin C and other fluorochromes these three bands 5-8 include an unknown greycoloured
pigment. This grey pigment was able to inhibit the synthesis of ROS strongly, while
Pityriarubin C had no influence on the evoked oxidative burst. The eight bands of fraction two
of the mutant Mel-3 differed in the way tey influenzed the oxidative burst. Only fractions
1,4,5 led to a decreased oxidative burst level. Although added in high concentrations, the pure
substances Exophialin and Malassezin did not have any influence on the synthesis of ROS.
I summary can be said that the wildtype of Exophiala dermatitidis an its mutants are able to
produce a very bright and different spectrum of pigmented indole derivates when cultivated
on minimal agar. This pigment spectrum is reproducable at any time. An inhibition of the
ROS synthesis was shown by these indole derivates when they were added as raw extracts,
fractions of thin layer chromatographie and as single bunches. The indole derivates seem to
play an important role in the pathogenesis of infections in human beings and animals caused
by Exophiala dermatitidis
Absence of renal hypoxia in the subacute phase of severe renal ischemia reperfusion injury
 This is the author accepted manuscript. The final version is available from American Physiological Society via the DOI in this recordTissue hypoxia has been proposed as an important event in renal ischemia reperfusion injury (IRI) particularly during the period of ischemia and in the immediate hours following reperfusion. However, little is known about renal oxygenation during the subacute phase of IRI. We employed four different methods to assess the temporal and spatial changes in tissue oxygenation during the subacute phase (24 h and 5 days after reperfusion) of a severe form of renal IRI in rats. We hypothesized that the kidney is hypoxic 24 h and 5 days after an hour of bilateral renal ischemia, driven by a disturbed balance between renal oxygen delivery (DO2) and oxygen consumption (VO2). Renal DO2 was not significantly reduced in the subacute phase of IRI. In contrast, renal VO2 was 55% less 24 h, and 49% less 5 days after reperfusion than after sham-ischemia. Inner medullary tissue PO2, measured by radiotelemetry was 25 {plus minus} 12% greater 24 h after ischemia than after sham-ischemia. By 5 days after reperfusion, tissue PO2 was similar to that in rats subjected to sham-ischemia. Tissue PO2 measured by Clark electrode was consistently greater 24 h, but not 5 days, after ischemia than after sham-ischemia. Cellular hypoxia, assessed by pimonidazole adduct immunohistochemistry, was largely absent at both time-points and tissue levels of hypoxia inducible factors were downregulated following renal ischemia. Thus, in this model of severe IRI, tissue hypoxia does not appear to be an obligatory event during the subacute phase, likely due to the markedly reduced oxygen consumption.British Heart FoundationBritish Heart FoundationNational Health and Medical Research Council of AustraliaEuropean Union, Seventh Framework Programm
Severe COVID anxiety among adults in the United Kingdom: cohort study and nested feasibility trial
BACKGROUND: People with severe COVID anxiety have poor mental health and impaired functioning, but the course of severe COVID anxiety is unknown and the quality of evidence on the acceptability and impact of psychological interventions is low. METHODS: People with severe COVID anxiety have poor mental health and impaired functioning, but the course of severe COVID anxiety is unknown and the quality of evidence on the acceptability and impact of psychological interventions is low. RESULTS: 204 (70.2%) of 285 people who took part in the cohort study completed the six month follow-up, for whom levels of COVID anxiety fell from 12.4 at baseline to 6.8 at six months (difference = -5.5, 95% CI = -6.0 to -4.9). Reductions in COVID anxiety were lower among older people, those living with a vulnerable person, those with lower baseline COVID anxiety, and those with higher levels of generalised anxiety and health anxiety at baseline. 36 (90%) of 40 participants enrolled in the nested feasibility trial were followed up at six months. 17 (80.9%) of 21 people in the active arm of the trial received four or more sessions of CBT-HA. We found improved mental health and social functioning among those in the active, but not the control arm of the trial (Mean difference in total score on the Work and Social Adjustment Scale between baseline and follow up, was 9.7 (95% CI = 5.8–13.6) among those in the active, and 1.0 (95% C.I. = -4.6 to 6.6) among those in the control arm of the trial. CONCLUSIONS: While the mental health of people with severe COVID anxiety appears to improve over time, many continue to experience high levels of anxiety and poor social functioning. Health anxiety is highly prevalent among people with severe COVID anxiety and may provide a target for psychological treatment. TRIAL REGISTRATION: Retrospectively registered at ISRCTN14973494 on 09/09/2021
The use of rapid review methods in health technology assessments: 3 case studies.
BACKGROUND: Rapid reviews are of increasing importance within health technology assessment due to time and resource constraints. There are many rapid review methods available although there is little guidance as to the most suitable methods. We present three case studies employing differing methods to suit the evidence base for each review and outline some issues to consider when selecting an appropriate method. METHODS: Three recently completed systematic review short reports produced for the UK National Institute for Health Research were examined. Different approaches to rapid review methods were used in the three reports which were undertaken to inform the commissioning of services within the NHS and to inform future trial design. We describe the methods used, the reasoning behind the choice of methods and explore the strengths and weaknesses of each method. RESULTS: Rapid review methods were chosen to meet the needs of the review and each review had distinctly different challenges such as heterogeneity in terms of populations, interventions, comparators and outcome measures (PICO) and/or large numbers of relevant trials. All reviews included at least 10 randomised controlled trials (RCTs), each with numerous included outcomes. For the first case study (sexual health interventions), very diverse studies in terms of PICO were included. P-values and summary information only were presented due to substantial heterogeneity between studies and outcomes measured. For the second case study (premature ejaculation treatments), there were over 100 RCTs but also several existing systematic reviews. Data for meta-analyses were extracted directly from existing systematic reviews with new RCT data added where available. For the final case study (cannabis cessation therapies), studies included a wide range of interventions and considerable variation in study populations and outcomes. A brief summary of the key findings for each study was presented and narrative synthesis used to summarise results for each pair of interventions compared. CONCLUSIONS: Rapid review methods need to be chosen to meet both the nature of the evidence base of a review and the challenges presented by the included studies. Appropriate methods should be chosen after an assessment of the evidence base
Use of AUDIT-C alcohol screening tool in NHS general dental practices in North London
Background: The numerous health risks of excessive alcohol consumption are well documented. Individuals at risk of harm from alcohol consumption can be identified through alcohol screening tools; however, there is limited research regarding their use in general dental practices. Methods: Data were collected as part of a feasibility trial evaluating delivery of brief alcohol advice in general dental practices in North London. Patient demographics and health-related behaviours were collected, and the Alcohol Use Disorders Identification Test-Consumption (AUDIT-C) tool was used to assess alcohol consumption patterns. Results: The analytical sample comprised 552 dental patients, of whom approximately half (46%) were drinking alcohol at hazardous levels. Males, younger adults, those who consumed red meat weekly and smokers all had significantly increased risks of excessive alcohol consumption. Smokers were more likely to consume excessive levels of alcohol irrespective of smoking frequency. Notable sex differences in alcohol consumption were identified, with males being more likely to consume alcohol frequently and in larger quantities than females. Conclusion: The AUDIT-C tool can be used in general dental practice to screen for harmful levels of alcohol consumption. Clear associations exist between patient demographics, health behaviours and excessive alcohol consumption
Carbohydrate supplementation during prolonged cycling exercise spares muscle glycogen but does not affect intramyocellular lipid use
Using contemporary stable-isotope methodology and fluorescence microscopy, we assessed the impact of carbohydrate supplementation on whole-body and fiber-type-specific intramyocellular triacylglycerol (IMTG) and glycogen use during prolonged endurance exercise. Ten endurance-trained male subjects were studied twice during 3 h of cycling at 63 ± 4% of maximal O2 uptake with either glucose ingestion (CHO trial; 0.7 g CHO kg−1 h−1) or without (CON placebo trial; water only). Continuous infusions with [U-13C] palmitate and [6,6-2H2] glucose were applied to quantify plasma free fatty acids (FFA) and glucose oxidation rates and to estimate intramyocellular lipid and glycogen use. Before and after exercise, muscle biopsy samples were taken to quantify fiber-type-specific IMTG and glycogen content. Plasma glucose rate of appearance (Ra) and carbohydrate oxidation rates were substantially greater in the CHO vs CON trial. Carbohydrate supplementation resulted in a lower muscle glycogen use during the first hour of exercise in the CHO vs CON trial, resulting in a 38 ± 19 and 57 ± 22% decreased utilization in type I and II muscle-fiber glycogen content, respectively. In the CHO trial, both plasma FFA Ra and subsequent plasma FFA concentrations were lower, resulting in a 34 ± 12% reduction in plasma FFA oxidation rates during exercise (P < 0.05). Carbohydrate intake did not augment IMTG utilization, as fluorescence microscopy revealed a 76 ± 21 and 78 ± 22% reduction in type I muscle-fiber lipid content in the CHO and CON trial, respectively. We conclude that carbohydrate supplementation during prolonged cycling exercise does not modulate IMTG use but spares muscle glycogen use during the initial stages of exercise in endurance-trained men
Ser649 and Ser650 Are the Major Determinants of Protein Kinase A-Mediated Activation of Human Hormone-Sensitive Lipase against Lipid Substrates
BACKGROUND: Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. Its activity is regulated by reversible protein phosphorylation. In rat HSL Ser563, Ser659 and Ser660 have been shown to be phosphorylated by protein kinase A (PKA) in vitro as well as in vivo. METHODOLOGY/PRINCIPAL FINDINGS: In this study we employed site-directed mutagenesis, in vitro phosphorylation and mass spectrometry to show that in vitro phosphorylation of human HSL by PKA occurs primarily on Ser649 and Ser650 (Ser659 and Ser660 in rat HSL). The wild type enzyme and four mutants were expressed in C-terminally His-tagged form in Sf9 insect cells and purified to homogeneity. HSL variants in which Ser552 and/or Ser554 were mutated to Ala or Glu retained both lipolytic and non-lipolytic activity and were phosphorylated by PKA and activated to a similar extent as the wild type enzyme. (32)P-labeling studies revealed that the bulk of the phosphorylation was on the Ser649/Ser650 site, with only a minor phosphorylation of Ser552 and Ser554. MS/MS analysis demonstrated that the peptide containing Ser649 and Ser650 was primarily phosphorylated on Ser650. The mutant lacking all four serines had severely reduced lipolytic activity, but a lesser reduction in non-lipolytic activity, had S(0.5) values for p-nitrophenol butyrate and triolein comparable to those of wild type HSL and was not phosphorylated by PKA. PKA phosphorylation of the wild type enzyme resulted in an increase in both the maximum turnover and S(0,5) using the TO substrate. CONCLUSIONS: Our results demonstrate that PKA activates human HSL against lipid substrates in vitro primarily through phosphorylation of Ser649 and Ser650. In addition the results suggest that Ser649 and Ser650 are located in the vicinity of a lipid binding region and that PKA phosphorylation controls the accessibility of this region
- …