66 research outputs found
Using enhanced number and brightness to measure protein oligomerization dynamics in live cells
Protein dimerization and oligomerization are essential to most cellular functions, yet measurement of the size of these oligomers in live cells, especially when their size changes over time and space, remains a challenge. A commonly used approach for studying protein aggregates in cells is number and brightness (N&B), a fluorescence microscopy method that is capable of measuring the apparent average number of molecules and their oligomerization (brightness) in each pixel from a series of fluorescence microscopy images. We have recently expanded this approach in order to allow resampling of the raw data to resolve the statistical weighting of coexisting species within each pixel. This feature makes enhanced N&B (eN&B) optimal for capturing the temporal aspects of protein oligomerization when a distribution of oligomers shifts toward a larger central size over time. In this protocol, we demonstrate the application of eN&B by quantifying receptor clustering dynamics using electron-multiplying charge-coupled device (EMCCD)-based total internal reflection microscopy (TIRF) imaging. TIRF provides a superior signal-to-noise ratio, but we also provide guidelines for implementing eN&B in confocal microscopes. For each time point, eN&B requires the acquisition of 200 frames, and it takes a few seconds up to 2 min to complete a single time point. We provide an eN&B (and standard N&B) MATLAB software package amenable to any standard confocal or TIRF microscope. The software requires a high-RAM computer (64 Gb) to run and includes a photobleaching detrending algorithm, which allows extension of the live imaging for more than an hour
The Essentials of Protein Import in the Degenerate Mitochondrion of Entamoeba histolytica
Several essential biochemical processes are situated in mitochondria. The metabolic transformation of mitochondria in distinct lineages of eukaryotes created proteomes ranging from thousands of proteins to what appear to be a much simpler scenario. In the case of Entamoeba histolytica, tiny mitochondria known as mitosomes have undergone extreme reduction. Only recently a single complete metabolic pathway of sulfate activation has been identified in these organelles. The E. histolytica mitosomes do not produce ATP needed for the sulfate activation pathway and for three molecular chaperones, Cpn60, Cpn10 and mtHsp70. The already characterized ADP/ATP carrier would thus be essential to provide cytosolic ATP for these processes, but how the equilibrium of inorganic phosphate could be maintained was unknown. Finally, how the mitosomal proteins are translocated to the mitosomes had remained unclear. We used a hidden Markov model (HMM) based search of the E. histolytica genome sequence to discover candidate (i) mitosomal phosphate carrier complementing the activity of the ADP/ATP carrier and (ii) membrane-located components of the protein import machinery that includes the outer membrane translocation channel Tom40 and membrane assembly protein Sam50. Using in vitro and in vivo systems we show that E. histolytica contains a minimalist set up of the core import components in order to accommodate a handful of mitosomal proteins. The anaerobic and parasitic lifestyle of E. histolytica has produced one of the simplest known mitochondrial compartments of all eukaryotes. Comparisons with mitochondria of another amoeba, Dictystelium discoideum, emphasize just how dramatic the reduction of the protein import apparatus was after the loss of archetypal mitochondrial functions in the mitosomes of E. histolytica
A Chaperone Trap Contributes to the Onset of Cystic Fibrosis
Protein folding is the primary role of proteostasis network (PN) where chaperone interactions with client proteins determine the success or failure of the folding reaction in the cell. We now address how the Phe508 deletion in the NBD1 domain of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein responsible for cystic fibrosis (CF) impacts the binding of CFTR with cellular chaperones. We applied single ion reaction monitoring mass spectrometry (SRM-MS) to quantitatively characterize the stoichiometry of the heat shock proteins (Hsps) in CFTR folding intermediates in vivo and mapped the sites of interaction of the NBD1 domain of CFTR with Hsp90 in vitro. Unlike folding of WT-CFTR, we now demonstrate the presence of ΔF508-CFTR in a stalled folding intermediate in stoichiometric association with the core Hsps 40, 70 and 90, referred to as a ‘chaperone trap’. Culturing cells at 30 C resulted in correction of ΔF508-CFTR trafficking and function, restoring the sub-stoichiometric association of core Hsps observed for WT-CFTR. These results support the interpretation that ΔF508-CFTR is restricted to a chaperone-bound folding intermediate, a state that may contribute to its loss of trafficking and increased targeting for degradation. We propose that stalled folding intermediates could define a critical proteostasis pathway branch-point(s) responsible for the loss of function in misfolding diseases as observed in CF
Evaluation of the Accuracy of Fiberglas‐Gypsum Blocks for Measuring Soil Moisture Changes 1
EFFECT OF CROPPING SYSTEMS AND FERTILIZERS ON MEAN WEIGHT-DIAMETER OF AGGREGATES OF BRETON PLOT SOILS
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Hirntodinduzierte Aktivierung proinflammatorischer Reaktionen in potentiellen Spenderorganen
Eine Serie unspezifischer inflammatorischer Ereignisse,
ausgelöst durch explosiven Hirntod, scheint in potentiellen
Spenderorganen eine Reihe von funktionellen und immunologischen
Veränderungen zu verursachen. Ziel dieser Studie ist es, den
Verlauf pro-inflammatorischer Zytokine in Herz, Lunge und Niere
hirntoter Tiere aufzuzeigen.Bei 4 Hausschweinen wurde Hirntod durch akuten
Druckanstieg über einen intrakraniell eingebrachten Foley-
Katheter herbeigeführt. Weitere 4 Schweine wurden scheinoperiert
und dienten somit als Kontrolltiere. Jedes Experiment wurde 6
Stunden nach Hirntodinduktion beendet und die Organe wurden
entnommen. Wir bestimmten die mRNA-Expression für TNF-α, IL-1β
und IL-6 mittels rt-PCR in Herz, Lunge und Niere. Die Ergebnisse
werden als Relation der Dichtewerte der Zytokine zum
Housekeeping-Gen β-actin in der Gelelektrophorese
beschrieben.Die 6h-Werte für IL-1β- und IL-6-mRNA waren in allen
untersuchten Organen von hirntoten Tieren signifikant höher als
in den von scheinoperierten. TNF-α mRNA-Expression war in
Lungegewebe hirntoter Tiere ebenfalls signifikant höher,
wohingegen in Herz- und Nierengewebe die Werte für TNF-α mRNA in
der Hirntod-Gruppe eher niedriger waren als in der
Kontrollgruppe (statistisch nicht signifikant).Explosiver Hirntod führt zu Zytokininduktion in den
peripheren Organen. Aufgrund unserer Ergebnisse schlussfolgern
wir, dass es sich hierbei um eine organ-spezifische Regulation
handelt.Organ dysfunction after explosive brain death has been
well documented. In addition, a series of nonspecific
inflammatory events may increase the intensity of the
immunological host response. The aim of the present study was to
monitor the course of proinflammatory cytokines in heart, lung
and kidney after brain death induction.Brain death (BD) was induced in 4 pigs by inflation of an
intracranial Foley catheter and separately 4 pigs were
sham-operated. Each experiment was terminated 6 hours after
brain death/sham operation and the organs were harvested. We
determined the mRNA-expression for TNF-α, IL-1β and IL-6 in the
heart, lung and kidney using rt-PCR techniques. The results are
presented as absorbance intensity (Mean±SEM). β-actin was used
for standardization.After 6 hours, IL-1β and IL-6 mRNA expression increased
significantly in all investigated organs in the brain dead
animals compared to sham-operated. TNF-α transcripts increased
significantly only in lung, while in heart and kidney TNF-α mRNA
tended even to lower values in brain dead animals.Brain death was associated with an increase in mRNA
concentration of IL-1β and IL-6 in lung, heart and kidney, while
TNF-α mRNA expression was only up-regulated in lung. We suggest
that brain death initiates a cascade of organ-specific
inflammatory events that may play a crucial role in early graft
dysfunction and rejection via a priming effect on the
transplanted organ
Echocardiographic Assessment of Cardiac Chamber Size and Left Ventricular Function in Aerobically Trained Athletes
Summary: We compared the echocardiographic assessment of left ventricular function in 20 aerobically trained male athletes (VO2 max 61 ml/kg/min) with 20 control subjects of similar age and size (VO2 max 37 ml/kg/min). On average, the left ventricle was larger in athletes and the extent of myocardial contraction (fractional shortening) was reduced. In nine athletes, fractional shortening was less than our laboratory lower limit of normal (26%) and in four athletes the left ventricle was enlarged and fractional shortening was reduced. However, the peak rate of circumferential shortening was not reduced in athletes. We concluded that athletes have normal myocardial contractility but some have a reduced extent of myocardial contraction which could be mistaken for the effects of myocardial disease.</p
NOTE ON EFFECTS OF WETTING AND DRYING ON READINGS OF ELECTRICAL RESISTANCE BLOCKS IN SOIL
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DECORIN UPREGULATION FOLLOWING LVAD SUPPORT SUGGESTS A ROLE IN REVERSE REMODELING
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TRYPTASE mRNA EXPRESSION INCREASES IN MYOCARDIUM FROM PATIENTS WITH DILATED CARDIOMYOPATHY AFTER VENTRICULAR ASSIST DEVICE SUPPORT
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