60 research outputs found
Reinigung, Kristallisation und Röntgenstrukturanalyse der Proteine des Lysinbiosyntheseweges von Mycobacterium Tuberculosis und Strukturanalyse von Membranproteinen von Deinococcus radiodurans R1 und Escherichia coli K12
No full textSince the lysine biosynthetic pathway is present in bacteria, fungi and plants but absent from mammals its 9 annotated enzymes have drawn considerable attention as potential candidates for new drugs against tuberculosis. Within this work the 3 proteins Rv0858c, Rv1201c and Rv1293 have been selected for structural analysis. Rv0858c protein has been purified, crystallised and the structure determined to a resolution of 2.0 Å. In addition, pure protein of Rv1201c has been crystallised successfully and first attempts towards structure determination were carried out. For the third target, Rv1293, an existing dataset was used to re-build the model and to refine the structure. In addition this structure was compared in detail to the same target solved earlier in a different space group.A detailed bioinformatical analysis was carried out for all 9 annotated proteins of the lysine biosynthetic pathway. The entire analysis was based on the questions: are too many or too few proteins annotated within the pathway?, is the order of proteins correctly annotated and is there any hint towards complex formation?A second project dealt with the analysis of structures of several outer and inner membrane proteins. A new bacterial strain was therefore introduced to the lab and basic membrane protein laboratory procedures were established. Different well established protocols were applied in order to extract all the present membrane proteins from the outer membranes. Different candidate proteins were analysed by Maldi-tof mass spectroscopy and sequence comparisons.Additionally the inner membrane protein Nramp of Deinococcus radiodurans R1 was selected for structural analysis. The target sequence of this protein was amplified by PCR, cloned into different vectors and the gene product overexpressed in different host organisms. The overexpression procedure was optimised and a purification protocol established. The protein was crystallised in lipidic cubic phases as well as by counter diffusion. These crystals were optimised and diffraction was obtained to a resolution of approximately 30 Å.In order to set up a SAXS approach for membrane proteins, the model protein OmpF of E.coli was selected. The experimental set up was optimised and the shape of the obtained model reflected the high resolution structure which was clearly reproduced. Also, the Nramp transporter of the inner membrane was measured using this technique, but could not structurally characterised so far.publishe
Adenosiinireseptorien ja niiden interaktiokumppaneiden biokemiallinen ja biofysikaalinen tutkimus
No full textAbstractAdenosine receptors are heterotrimeric guanine nucleotide-binding (G protein)-coupled receptors (GPCRs) that mediate the effects of the endogenous agonist adenosine. The adenosine A₃ receptor (A₃R) is the least explored among the four human adenosine receptor subtype members (A₁, A2A, A2B and A₃) and it is implicated in both neuroprotective and neurodegenerative effects.During the course of this work, the production of the recombinant human A₃R in yeast and insect cells was evaluated and heteromerization between the human adenosine A2A receptor (A2AR) and the dopamine D₂ receptor (D₂R) was studied. A₃R with carboxyl-terminal GFP tag was expressed in the yeast Saccharomyces cerevisiae upto 15 mg per litre of culture. Another yeast Pichia pastoris increased the expression up to 108 mg/L of the same receptor when grown in bioreactors. Despite the very high expression levels, purification of A₃R from both yeasts was a daunting task, as the aggregation of the receptor could not be averted. In this study, insect cells have been found out to be more suitable host for A₃R expression: 10µg of the monomeric A₃R could be purified from one liter of insect cell culture.For successful crystallization thermostability of the A₃R was to be improved. This work has demonstrated that insertion of T4L, a fusion protein, in the third intracellular loop of A₃R increased the thermostability of the receptor by 10°C. As a next step, the combination of point mutations based on alanine-scanning mutagenesis and a fusion protein approach could be useful to stabilize and further crystallize the A3R. This work has demonstrated that the amounts of A3R expressed in insect cells and the final yield of the receptor isolated by affinity purifications, forms a good basis for the beginning of biochemical characterization.Receptor heteromerization is a mechanism used by GPCRs to diversify their signaling properties and functions. The human A2AR and D₂R heteromers exist in the GABAergic enkephalinergic neurons. The domains responsible for forming intermolecular contacts were purified from Escherichia coli (E. coli). Using biochemical/biophysical techniques such as native-PAGE and mass spectrometry, It was validated that purified carboxyl-terminus of the A2AR and the 3rd intracellular loop of D₂R form heterodimers. The investigation of purified calmodulin protein binding to the 3rd intracellular loop of D₂R showed that the protein-protein interactions are calcium dependent.TiivistelmäAdenosiinireseptorit kuuluvat G-proteiinikytkeiset reseptorit (GPCR:t) proteiiniperheeseen. Adenosiinireseptorit välittävät endogeenisen ligandinsa adenosiinin vaikutuksia solukalvolta solunsisäisiin signaalijärjestelmiin. Adenosiini A₃ reseptori (A₃R) on adenosiinireseptorien neljästä alatyypistä (A₁, A2A, A2B ja A₃) vähiten tutkittu. Aikaisempien tutkimusten perusteella A3 reseptori yhdistetään sekä hermosoluja suojaaviin että rappeuttaviin tapahtumiin.Tässä työssä arvioitiin sekä ihmisen rekombinantti-A₃R:n tuottumista hiiva- ja hyönteissoluissa että tutkittiin ihmisen adenosiini A2A reseptorin (A2AR) ja dopamiini D₂ reseptorin (D₂R) heteromerisoitumista. Rekombinantti A₃ reseptori- vihreä fluoresoiva proteiini (GFP) fuusioproteiinia tuotettiin Saccharomyces cerevisiae -hiivassa 15 mg litrassa kasvatusliuosta. Pichia pastoris -hiivakanta taas kasvatti saman reseptorin tuottumista aina 108 mg/l saakka, kun tuotto tehtiin bioreaktorissa. Hyvin korkeasta tuottotasosta huolimattaA₃R:n puhdistus hiivasta oli ylitsepääsemätön tehtävä, sillä reseptorin saostumista ei voinut välttää. Työssä havaittiin, että hyönteissolut sopivat paremmin A₃R:n tuottoon: noin 10 µg monomeerista A₃R:a voitiin puhdistaa litran hyönteissoluviljelmästä.Reseptorin stabiilisuuden lisääminen helpottaa reseptorin biokemiallista ja biofysikaalista karakterisointia. Tässä työssä osoitettiin, että T4L-proteiinin lisääminen A₃R:n kolmannen solunsisäisen silmukan paikalle lisää reseptorin lämpöstabiilisuutta 10 °C. Jatkotutkimuksissa voitaisiin käyttää alaniiniskannausmutageneesiin perustuvien pistemutaatioiden ja fuusioproteiinin yhdistelmää A₃R:n lisästabilointiin ja kiteytykseen. Tämän työn perusteella määrät, joilla A₃R tuottuu hyönteissoluissa ja jotka saadaan eristettyä affiniteettipuhdistuksilla, muodostavat hyvän perustan proteiinin biokemialliselle karakterisoinnille.Reseptorin heteromerisoituminen on GPCR:en käyttämä mekanismi signalointiominaisuuksien ja toimintojen monipuolistamiseksi. Ihmisessä A2AR ja D₂R heteromeereja on GABAergisissä enkefalinergisissä hermosoluissa. Molekyylien välisiin kontakteihin osallistuvat domeenit puhdistettiin Escherichia coli (E. coli) -bakteerista. Biokemiallisia ja biofysikaalisia tekniikoita kuten natiivi-PAGE:a ja massaspektrometriaa käyttäen vahvistettiin, että puhdistettu A2AR:n karboksiterminaalinen osa ja D₂R:n kolmas solunsisäinen silmukka muodostavat heterodimeereja. Myös tutkittaessa puhdistetun kalmoduliini-proteiinin sitoutumista D₂R:n kolmanteen solunsisäiseen silmukkaan osoitettiin proteiini-proteiini -vuorovaikutuksen olevan kalsiumista riippuvainen.Academic dissertation to be presented with the assent of the Doctoral Training Committee of Health and Biosciences of the University of Oulu for public defence in the Leena Palotie auditorium (101A) of the Faculty of Medicine (Aapistie 5 A), on 26 February 2016, at 12 noonAbstract
Adenosine receptors are heterotrimeric guanine nucleotide-binding (G protein)-coupled receptors (GPCRs) that mediate the effects of the endogenous agonist adenosine. The adenosine A₃ receptor (A₃R) is the least explored among the four human adenosine receptor subtype members (A₁, A2A, A2B and A₃) and it is implicated in both neuroprotective and neurodegenerative effects.
During the course of this work, the production of the recombinant human A₃R in yeast and insect cells was evaluated and heteromerization between the human adenosine A2A receptor (A2AR) and the dopamine D₂ receptor (D₂R) was studied. A₃R with carboxyl-terminal GFP tag was expressed in the yeast Saccharomyces cerevisiae upto 15 mg per litre of culture. Another yeast Pichia pastoris increased the expression up to 108 mg/L of the same receptor when grown in bioreactors. Despite the very high expression levels, purification of A₃R from both yeasts was a daunting task, as the aggregation of the receptor could not be averted. In this study, insect cells have been found out to be more suitable host for A₃R expression: 10µg of the monomeric A₃R could be purified from one liter of insect cell culture.
For successful crystallization thermostability of the A₃R was to be improved. This work has demonstrated that insertion of T4L, a fusion protein, in the third intracellular loop of A₃R increased the thermostability of the receptor by 10°C. As a next step, the combination of point mutations based on alanine-scanning mutagenesis and a fusion protein approach could be useful to stabilize and further crystallize the A3R. This work has demonstrated that the amounts of A3R expressed in insect cells and the final yield of the receptor isolated by affinity purifications, forms a good basis for the beginning of biochemical characterization.
Receptor heteromerization is a mechanism used by GPCRs to diversify their signaling properties and functions. The human A2AR and D₂R heteromers exist in the GABAergic enkephalinergic neurons. The domains responsible for forming intermolecular contacts were purified from Escherichia coli (E. coli). Using biochemical/biophysical techniques such as native-PAGE and mass spectrometry, It was validated that purified carboxyl-terminus of the A2AR and the 3rd intracellular loop of D₂R form heterodimers. The investigation of purified calmodulin protein binding to the 3rd intracellular loop of D₂R showed that the protein-protein interactions are calcium dependent.Tiivistelmä
Adenosiinireseptorit kuuluvat G-proteiinikytkeiset reseptorit (GPCR:t) proteiiniperheeseen. Adenosiinireseptorit välittävät endogeenisen ligandinsa adenosiinin vaikutuksia solukalvolta solunsisäisiin signaalijärjestelmiin. Adenosiini A₃ reseptori (A₃R) on adenosiinireseptorien neljästä alatyypistä (A₁, A2A, A2B ja A₃) vähiten tutkittu. Aikaisempien tutkimusten perusteella A3 reseptori yhdistetään sekä hermosoluja suojaaviin että rappeuttaviin tapahtumiin.
Tässä työssä arvioitiin sekä ihmisen rekombinantti-A₃R:n tuottumista hiiva- ja hyönteissoluissa että tutkittiin ihmisen adenosiini A2A reseptorin (A2AR) ja dopamiini D₂ reseptorin (D₂R) heteromerisoitumista. Rekombinantti A₃ reseptori- vihreä fluoresoiva proteiini (GFP) fuusioproteiinia tuotettiin Saccharomyces cerevisiae -hiivassa 15 mg litrassa kasvatusliuosta. Pichia pastoris -hiivakanta taas kasvatti saman reseptorin tuottumista aina 108 mg/l saakka, kun tuotto tehtiin bioreaktorissa. Hyvin korkeasta tuottotasosta huolimattaA₃R:n puhdistus hiivasta oli ylitsepääsemätön tehtävä, sillä reseptorin saostumista ei voinut välttää. Työssä havaittiin, että hyönteissolut sopivat paremmin A₃R:n tuottoon: noin 10 µg monomeerista A₃R:a voitiin puhdistaa litran hyönteissoluviljelmästä.
Reseptorin stabiilisuuden lisääminen helpottaa reseptorin biokemiallista ja biofysikaalista karakterisointia. Tässä työssä osoitettiin, että T4L-proteiinin lisääminen A₃R:n kolmannen solunsisäisen silmukan paikalle lisää reseptorin lämpöstabiilisuutta 10 °C. Jatkotutkimuksissa voitaisiin käyttää alaniiniskannausmutageneesiin perustuvien pistemutaatioiden ja fuusioproteiinin yhdistelmää A₃R:n lisästabilointiin ja kiteytykseen. Tämän työn perusteella määrät, joilla A₃R tuottuu hyönteissoluissa ja jotka saadaan eristettyä affiniteettipuhdistuksilla, muodostavat hyvän perustan proteiinin biokemialliselle karakterisoinnille.
Reseptorin heteromerisoituminen on GPCR:en käyttämä mekanismi signalointiominaisuuksien ja toimintojen monipuolistamiseksi. Ihmisessä A2AR ja D₂R heteromeereja on GABAergisissä enkefalinergisissä hermosoluissa. Molekyylien välisiin kontakteihin osallistuvat domeenit puhdistettiin Escherichia coli (E. coli) -bakteerista. Biokemiallisia ja biofysikaalisia tekniikoita kuten natiivi-PAGE:a ja massaspektrometriaa käyttäen vahvistettiin, että puhdistettu A2AR:n karboksiterminaalinen osa ja D₂R:n kolmas solunsisäinen silmukka muodostavat heterodimeereja. Myös tutkittaessa puhdistetun kalmoduliini-proteiinin sitoutumista D₂R:n kolmanteen solunsisäiseen silmukkaan osoitettiin proteiini-proteiini -vuorovaikutuksen olevan kalsiumista riippuvainen
Old Gate Gets a New Look
No full textA proposed structure for a protein that enables potassium ions to move in and out of cells raises new questions about how its “gate” works.</jats:p
Do Horizontal Forces Matter For Horizontal Running?
No full textDO HORIZONTAL FORCES MATTER FOR HORIZONTAL RUNNING?
Kenneth P. Clark, Laurence J. Ryan, and Peter G. Weyand
Southern Methodist University, Locomotor Performance Laboratory, Department of Applied Physiology and Wellness, Dallas, TX 75206
Classification of First Author: Doctoral Student
Introduction: The application of ground force is widely recognized as the critical determinant of running speed. At maximal speeds, 90-98% of the total force applied is directed vertically into the running surface while horizontal (fore-aft) contributions are relatively small. Despite their small magnitude, horizontal forces are clearly essential for balance and may be important for other reasons. However, the pattern of horizontal force application across faster speeds is not well understood. Objective: For moderate to top speeds, we aimed to determine whether: 1) the horizontal forces required increase substantially, and 2) horizontal forces become larger relative to vertical forces. Participants: Two male and three female athletes volunteered for the study (age: 19.0 ± 0.6 years, height: 1.75 ± 0.06 m, mass: 71.0 ± 8.2 kg). Data Collection: Trials were completed on a high-speed, three-axis force treadmill (AMTI, Watertown, MA), with ground force data acquired at 1,000 Hz. Data was analyzed from each individual’s top speed and submaximal trials at 5.0 and 7.0 m/s. Top speed was determined by the fastest speed where the participant could complete eight steps without drifting backward 0.2 m. Outcome Measures: Because center of mass motion is determined by the mass-specific force applied and the time of force application, (i.e. impulse, or product of average force and time of application, or area under the force-time curve), we analyzed both average vertical and horizontal forces and impulses for every step. Average horizontal forces and impulses were calculated as the absolute value for the braking and propulsive phases of the horizontal force-time curve. Forces were standardized to body weight (Wb) and impulses calculated in body weight • seconds (Wb•s). The ratio of average vertical impulse to average horizontal impulse was calculated for each runner across speeds. Results: From 5.0 m/s to top speed, mean vertical and horizontal forces increased from 1.70 to 1.99 Wb and 0.29 to 0.34 Wb, respectively, and mean vertical and horizontal impulses decreased from 0.30 to 0.24 Wb•s and 0.05 to 0.04 Wb•s, respectively. From 5.0 m/s to top speed, the ratio of vertical to horizontal impulses varied by only 5.2% on average over a 1.5 to 2.0-fold range of speeds for the individuals tested and did so without consistent direction. Conclusions: The average horizontal forces and the ratio of vertical to horizontal impulses did not vary appreciably across a range of faster running speeds in a small sample of athletic subjects
Infliximab as monotherapy in giant cell arteritis
No full text[No abstract available]Andonopoulos AP, 2003, ANN RHEUM DIS, V62, P1116, DOI 10.1136-ard.62.11.1116; Cantini F, 2001, ARTHRITIS RHEUM, V44, P2933, DOI 10.1002-1529-0131(200112)44:122933::AID-ART4823.0.CO;2-Y; Field M, 1997, RHEUMATOL INT, V17, P113, DOI 10.1007-s002960050019; Hernandez-Rodriguez J, 2004, RHEUMATOLOGY, V43, P294, DOI 10.1093-rheumatology-keh058; Hernandez-Rodriguez J, 2002, ARTHRIT RHEUM-ARTHR, V47, P29, DOI 10.1002-art1.10161; NESHER G, 1994, J RHEUMATOL, V21, P1283; Rozin AP, 2004, ANN RHEUM DIS, V63, P751; Uthman I, 2004, SEMIN ARTHRITIS RHEU, V33, P422, DOI 10.1016-j.semarthrit.2003.12.005; Weyand CM, 2000, ARTHRITIS RHEUM, V43, P1041, DOI 10.1002-1529-0131(200005)43:51041::AID-ANR123.0.CO;2-7; Weyand CM, 2003, ANN INTERN MED, V139, P50529161
Structure-function relationships of olfactory and taste receptors
No full textThe field of chemical senses has made major progress in understanding the cellular mechanisms of olfaction and taste in the past two decades. However, the molecular understanding of odour and taste recognition is still lagging far behind and will require solving multiple structures of the relevant full-length receptors in complex with native ligands to achieve this goal. However, the development of multiple complimentary strategies for the structure determination of G protein-coupled receptors (GPCRs) makes this goal realistic. The common conundrum of how multi-specific receptors that recognize a large number of different ligands results in a sensory perception in the brain will only be fully understood by a combination of high-resolution receptor structures and functional studies. This review discusses the first steps on this pathway, including biochemical and physiological assays, forward genetics approaches, molecular modelling and the first steps towards the structural biology of olfactory and taste receptors
CRIS—A Novel cAMP-Binding Protein Controlling Spermiogenesis and the Development of Flagellar Bending
No full textThe second messengers cAMP and cGMP activate their target proteins by binding to a conserved cyclic nucleotide-binding domain (CNBD). Here, we identify and characterize an entirely novel CNBD-containing protein called CRIS (cyclic nucleotide receptor involved in sperm function) that is unrelated to any of the other members of this protein family. CRIS is exclusively expressed in sperm precursor cells. Cris-deficient male mice are either infertile due to a lack of sperm resulting from spermatogenic arrest, or subfertile due to impaired sperm motility. The motility defect is caused by altered Ca(2+) regulation of flagellar beat asymmetry, leading to a beating pattern that is reminiscent of sperm hyperactivation. Our results suggest that CRIS interacts during spermiogenesis with Ca(2+)-regulated proteins that--in mature sperm--are involved in flagellar bending
Reproducibility and accuracy of microscale thermophoresis in the NanoTemper Monolith: a multi laboratory benchmark study (European Biophysics Journal, (2021), 50, 3-4, (411-427), 10.1007/s00249-021-01532-6)
No full textThe article “Reproducibility and accuracy of microscale thermophoresis in the NanoTemper Monolith: a multi laboratory benchmark study” written by López-Méndez, B., Baron, B., Brautigam, C. A., Jowitt, T. A., Knauer, S. H., Uebel, S., Williams, M. A., Sedivy, A., Abian, O., Abreu, C., Adamczyk, M., Bal, W., Berger, S., Buell, A. K., Carolis, C., Daviter, T., Fish, A., Garcia-Alai, M., Guenther, C., Hamacek, J., Holková, J., Houser, J., Johnson, C., Kelly, S., Leech, A., Mas, C., Matulis, D., McLaughlin, S. H., Montserret, R., Nasreddine, R., Nehmé, R., Nguyen, Q., Ortega-Alarcón, D., Perez, K., Pirc, K., Piszczek, G., Podobnik, M., Rodrigo, N., Rokov-Plavec, J., Schaefer, S., Sharpe, T., Southall, J., Staunton, D., Tavares, P., Vanek, O., Weyand, M., Wu, D. was originally published Online First without Open Access. After publication in volume 50, issue 3–4, pages 411–427 the author decided to opt for Open Choice and to make the article an Open Access publication. Therefore, the copyright of the article has been changed topublishersversionpublishe
Mesothelial/monocytic incidental cardiac excrescences (cardiac MICE) associated with acute aortic dissection: a study of two cases
No full textAcute aortic dissection is a life-threatening condition mainly caused by hypertension, atherosclerotic disease and other degenerative diseases of the connective tissue of the aortic wall. Mesothelial/monocytic incidental cardiac excrescences (cardiac MICE) is a rare benign reactive tumor-like lesion composed of admixture of histiocytes, mesothelial cells, and inflammatory cells set within a fibrinous meshwork without a vascular network or supporting stroma. Cardiac MICE occurring in association with aortic dissection is exceptionally rare (only one such case reported to date). We herein report on the surgical repair of two Stanford type A aortic dissections caused by idiopathic giant cell aortitis in a 66-year-old-woman and by atherosclerotic disease in a 58-year-old-man, respectively. In both cases, the dissections could be visualized via computed tomography. Histopathology showed cardiac incidental MICE within the external aortic wall near the pericardial surface which was confirmed by immunohistochemistry
Accuracy of walking metabolic prediction equations using a large diverse data set
No full text2014 Summer.Includes bibliographical references.Walking metabolic rate prediction equations are commonly used to estimate oxygen consumption, exercise intensity and energy expenditure across a wide range of ages and anthropometrics. Despite their widespread use, independent validations of these equations using metabolic data from a large number of individuals are uncommon. PURPOSE: To assess the accuracy of the commonly used ACSM and Pandolf walking metabolic rate prediction equations, along with two new walking metabolic rate predictions equations developed by Weyand et al. and Browning et al., using data from a large number of adults. METHODS: We used demographic, anthropometric, walking speed, and oxygen consumption data from several laboratories (N = 450 (164 Males, 286 females), 18-85 years old, 16.5-44 kg/m2). We estimated oxygen consumption using each prediction equation in 1,078 walking trials ranging from 0.55-2.18 m/s, and 0.5-12% grade. Comparisons between predictive methods were made for all walking trials, as well as among normal weight participants during level and gradient walking, and overweight and obese participants during level and gradient walking. We computed the mean prediction difference (MPD) as the difference between predicted vs. measured rates of oxygen consumption (ml/kg/min) for each trial, and examined the relationship between the MPD and measured oxygen consumption (ml/kg/min) using modified Bland-Altman plots. Linear regression was used to determine the intercept (fixed bias) and slope (proportional bias) for each equation. The absolute value of the mean prediction difference, and Root Mean Square Error (RMSE) values were also calculated for each equation and population. RESULTS: For level walking, all prediction equations had mean prediction differences that were statistically different from zero (P ≤ 0.05) except for the Browning et al., equation when applied to normal weight individuals and the Pandolf equation when applied to overweight and obese individuals. Most importantly, all prediction equations had significant (P ≤ 0.05) fixed and proportional bias, and demonstrated large RMSE (7.8-23.5% of mean measured metabolic rate) that were similar across equations and population. In addition, prediction error increased as measured metabolic rate increased for all equations. CONCLUSION: The metabolic prediction equations evaluated here each had considerable error when compared to measured values, regardless of the population in which the equation was created and/or validated. Improvements in prediction equations may require using approaches that aim to minimize RMSE and/or developing population/intensity specific equations
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