237 research outputs found
Electron transfer kinetics between soluble modules of Paracoccus denitrificans cytochrome c1 and its physiological redox partners
No full textThe transient electron transfer (ET) interactions between cytochrome c1 of the bc1-complex from Paracoccus denitrificans and its physiological redox partners cytochrome c552 and cytochrome c550 have been characterized functionally by stopped-flow spectroscopy. Two different soluble fragments of cytochrome c1 were generated and used together with a soluble cytochrome c552 module as a model system for interprotein ET reactions. Both c1 fragments lack the membrane anchor; the c1 core fragment (c1CF) consists of only the hydrophilic heme-carrying domain, whereas the c1 acidic fragment (c1AF) additionally contains the acidic domain unique to P. denitrificans. In order to determine the ionic strength dependencies of the ET rate constants, an optimized stopped-flow protocol was developed to overcome problems of spectral overlap, heme autoxidation and the prevalent non-pseudo first order conditions. Cytochrome c1 reveals fast bimolecular rate constants (10(7) to 10(8) M(-1) s(-1)) for the ET reaction with its physiological substrates c552 and c550, thus approaching the limit of a diffusion-controlled process, with 2 to 3 effective charges of opposite sign contributing to these interactions. No direct involvement of the N-terminal acidic c1-domain in electrostatically attracting its substrates could be detected. However, a slight preference for cytochrome c550 over c552 reacting with cyochrome c1 was found and attributed to the different functions of both cytochromes in the respiratory chain of P. denitrificans
Studies on bacterial transmission pathways in a high endemic area, with a focus on Helicobacter pylori
No full textEven though half of the world’s population is infected with Helicobacter pylori, which causes gastritis, peptic ulcer and gastric cancer, the transmission routes of these bacteria remain unknown despite extensive epidemiological studies. Enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae are two of the most common causes of acute watery diarrhea in developing countries. The main aim of this thesis was to study transmission pathways of these bacteria, with a focus on H. pylori, through analyses of clinical and water samples from Dhaka, Bangladesh, an area with high prevalence of gastrointestinal diseases.
To determine the bacterial numbers in clinical and water samples we developed highly sensitive quantitative real-time PCR assays targeting specific and conserved virulence genes of H. pylori (cagA, flaA, glmM, hpaA, ureA and vacA), ETEC (eltA and estB) and V. cholerae (ctxB and tcpA). The assays were used for quantification of bacterial DNA and reverse-transcribed gene transcripts.
Twenty-six of 39 (67 %) drinking and environmental water samples from a poor area in Dhaka were positive by real-time PCR for ETEC, whereas all 75 drinking and environmental water and 21 drinking water biofilms from the same location were negative for H. pylori, suggesting that ETEC may be waterborne while H. pylori is not.
H. pylori transmission during epidemics of gastroenteritis was then explored by analyzing vomitus and stool samples collected from diarrhea patients admitted to the ICDDR, B hospital in Dhaka. All samples were positive for V. cholerae, with higher numbers in stool (median 2.5 x 106 genomes) than vomitus (median 2.7 x 104 genomes) and a strong correlation between DNA real-time PCR and quantitative culture. Analyses for H. pylori showed that 23 of 26 (88 %, median genome number = 4.35 x 105 ml-1) vomitus and 17 of 23 (74 %, median genome number = 7.33 x 102 ml-1) stool samples were positive in real-time PCR, but H. pylori could not be isolated by culture. The results indicate that high numbers of H. pylori are shed in vomitus during acute gastroenteric disease and indicate that H. pylori may be transmitted by this route. To establish possible infectivity of these bacteria, the gene expression of H. pylori in vomitus, stool and biopsies from infected individuals and in in vitro cultures was analyzed. Vomitus, biopsies and in vitro cultures showed high expression of cagA, flaA and ureA and lower expression of hpaA and vacA, whereas no expression was detected in diarrheal stool. Expression analyses of the same genes in a C57Bl/6 H. pylori strain SS1 infection mouse model showed a similar relative transcription pattern as in biopsies, in vitro cultures and vomitus and that expression is up-regulated during exponential growth.
In conclusion, our results suggest that H. pylori may be disseminated through vomitus during outbreaks of gastrointestinal infections in Bangladesh and that waterborne transmission is less likely whereas waterborne transmission of ETEC may occur. Furthermore, the studies indicate that experimental murine infection and vomitus from H. pylori infected subjects may be suitable models of H. pylori virulence gene expression in vivo
A clinician’s experience of using the Cardiac Reader NT-proBNP point-of-care assay in a clinical setting
No full textThe evaluation of natriuretic peptides has become increasingly valuable in a clinical setting, where information is often needed promptly. Objectives: To compare the usefulness of the recently released Roche Cardiac Reader ® NT-proBNP assay against the Roche Elecsys® NT-proBNP laboratory system in a clinical setting. Design and Results: Blood samples from 440 patients admitted for acute coronary syndromes, worsening of heart failure, or as policlinic heart failure patients were evaluated. The relation between the assays was analysed and the diagnostic concordance calculated. A good correlation was found between the assays (r=0.96, 95% CI: 0.94-0.97) with a diagnostic concordance of 0.93. A separate analysis was performed in the range where most clinical decisions are made (60-3000 ng/L), with a diagnostic concordance of 88%. The usefulness in a clinical setting where time is important was high. Conclusion: The Roche Cardiac Reader® NT-proBNP assay has been evaluated in a clinical setting. The point-of-care method shows good results, although with a restricted analytical range compared with the reference.Original publication: Alehagen U, and Janzon M, A clinician’s experience of using the Cardiac Reader NT-proBNP point-of-care assay in a clinical setting, 2008, European Journal of Heart Failure, (10), 3, 260-266. http://dx.doi.org/10.1016/j.ejheart.2008.01.005. Copyright: Elsevier B.V., http://www.elsevier.com
Gender difference in prevalence and prognostic impact of renal insufficiency in patients with ST-elevation myocardial infarction treated with primary percutaneous coronary intervention
No full textObjective To evaluate if female gender is associated with renal insufficiency in patients with ST-elevation myocardial infarction (STEMI) and if there is a gender difference in the prognostic importance of renal insufficiency in STEMI. Design Single-centre observational study. Setting One tertiary cardiac centre. Patients All consecutive patients with STEMI planned for primary percutaneous coronary intervention in one Swedish county in 2005 (98 women and 176 men). Main outcome measures Logistic regression analyses were conducted to evaluate the predictors of renal insufficiency, associations between estimated glomerular filtration rate (eGFR) and outcome in each gender and a possible interaction between gender and eGFR regarding outcome. Results Renal insufficiency was defined as eGFR less than 60 ml/min per 1.73 m(2). 67% of women had renal insufficiency compared with 26% of men, OR 5.06 (95% CI 2.66 to 9.59) after multivariable adjustment. In women each 10 ml/min per 1.73 m 2 increment of eGFR was associated with a 63% risk reduction for 1-year mortality, OR 0.37 (95% CI 0.15 to 0.89). No such association was found in men, OR 1.05 (95% CI 0.63 to 1.76). A trend towards a significant interaction between gender and eGFR regarding 1-year mortality was found, OR 2.05 (95% CI 0.93 to 4.50). Conclusions A considerable gender difference in the prevalence of renal insufficiency in STEMI was found and renal insufficiency seemed to be a more important prognostic marker in women. These results are important as previous STEMI studies have shown higher multivariable adjusted mortality in women than in men but renal function has seldom been taken into consideration.Original Publication:Sofia Lawesson, Tim Tödt, Joakim Alfredsson, Magnus Janzon, Ulf Stenestrand and Eva Swahn, Gender difference in prevalence and prognostic impact of renal insufficiency in patients with ST-elevation myocardial infarction treated with primary percutaneous coronary intervention, 2011, HEART, (97), 4, 308-314.http://dx.doi.org/10.1136/hrt.2010.194282Copyright: BMJ Publishing Group; 1999http://group.bmj.com
Estimating attendance for breast cancer screening in ethnic groups in London.
No full textBACKGROUND: Breast screening uptake in London is below the Government's target of 70% and we investigate whether ethnicity affects this. Information on the ethnicity for the individual women invited is unavailable, so we use an area-based method similar to that routinely used to derive a geographical measure for socioeconomic deprivation. METHODS: We extracted 742,786 observations on attendance for routine appointments between 2004 and 2007 collected by the London Quality Assurance Reference Centre. Each woman was assigned to a lower super output (LSOA) based on her postcode of residence. The proportions of the ethnic groups within each LSOA are known, so that the likelihood of a woman belonging to White, Black and Asian groups can be assigned. We investigated screening attendance by age group, socioeconomic deprivation using the Index of Deprivation 2004 income quintile, invitation type and breast screening service. Using logistic regression analysis we calculated odds ratios for attendance based on ethnic composition of the population, adjusting for age, socioeconomic status, the invitation type and screening service. RESULTS: The unadjusted attendance odds ratios were high for the White population (OR: 3.34 95% CI [3.26-3.42]) and low for the Black population (0.13 [0.12-0.13]) and the Asian population (0.55 [0.53-0.56]). Multivariate adjustment reduced the differences, but the Black population remained below unity (0.47 [0.44-0.50]); while the White (1.30 [1.26-1.35]) and Asian populations (1.10 [1.05-1.15]) were higher. There was little difference in the attendance between age groups. Attendance was highest for the most affluent group and fell sharply with increasing deprivation. For invitation type, the routine recall was higher than the first call. There were wide variations in the attendance for different ethnic groups between the individual screening services. CONCLUSIONS: Overall breast screening attendance is low in communities with large Black populations, suggesting the need to improve participation of Black women. Variations in attendance for the Asian population require further investigation at an individual screening service level
Dissertatio academica de gente antiqua Troll. Cujus sectionem quartam approb. ampliss. fac. phil. Ups. praeside mag. Jac. Fr. Neikter ... publico examini defert Laurentius Janzon, Ostrogothus. In audit. Gust. maj. d. XXVIII Maji MDCCXCVI. h. p. m. s., D. 4 [Elektronisk resurs]
No full textEvaluation metabolischer Sensor Chip Messungen mit MCF-7 Brustkrebs Zellen
No full textThe aim of this project was the analysis of metabolic changes of MCF-7 cells under limiting conditions. Concentrations of nutrients and growth factors as well as the extracellular pH were varied, and their influence on chemosensitivity was investigated. A prototype of an electronic sensor-chip system was evaluated by measuring the metabolic activity of tumor cells and comparing the results to biochemical-metabolic parameters.Änderungen im Tumorstoffwechsel und der Chemosensitivität wurden an MCF-7 Zellen unter variablen und limitierenden Wachstumsbedingungen untersucht, um diese mit Daten aus Messungen eines neuentwickelten Sensorchip-Testsystems vergleichen zu können. Die Reduktion von Glukose und Glutamin, wie auch die Änderungen im extrazellulären pH führten zu unterschiedlichen Ergebnissen je nach den biochemischen und elektrochemischen Messparametern
Electron transfer between complex III and IV of the respiratory chains of Paracoccus denitrificans and Thermus thermophilus : a functional and kinetic characterization of interactions using soluble fragments
No full textAdenosintriphosphat (ATP) als universelles Energieäquivalent der Zelle wird durch die oxidative Phosphorylierung synthetisiert, bei der Elektronen entlang des elektrochemischen Gefälles der Atmungskette über verschiedene Redoxkomplexe transferiert und durch die chemiosmotische Kopplung Protonen über die Membran gepumpt werden. Der Protonengradient wird dann von der ATP-Synthase genutzt, um ADP zu ATP zu phosphorylieren. Zentraler Redoxkomplex der Atmungskette vieler Pro- und Eukaryonten ist der bc1-Komplex, der Elektronen von Ubichinol auf Cytochrom c überträgt, von wo sie nachfolgend auf die Cytochrom c-Oxidase transferiert werden. Das mesophile Bodenbakterium Paracoccus denitrificans wird als Modellsystem für den mitochondrialen Elektronentransfer (ET) verwendet, da es eine aerobe Atmungskette exprimiert, die der mitochondrialen homolog ist, allerdings einen wesentlich einfacheren Aufbau der einzelnen Redoxkomplexe aufweist. Auch Thermus thermophilus als extrem thermophiles Bakterium bildet eine vergleichbare aerobe Atmungskette aus, wobei deren Proteine allerdings eine hohe Thermostabilität aufweisen, wodurch sie in das Interesse der Forschung gerückt sind. Ziel dieser Arbeit war die funktionelle Charakterisierung des ET zwischen Komplex III und IV der Atmungsketten von P. denitrificans und T. thermophilus, die aufgrund ihrer mesophilen und thermophilen Eigenschaften unterschiedliche Mechanismen in der Wechselwirkung ihrer Redoxproteine aufweisen. Es wurden lösliche Redoxfragmente verwendet, um anhand von Mutagenesestudien, Stopped-Flow (SF)- und Laserflash-Kinetiken (LF) unter pre-steady state-Bedingungen sowie FRET-Experimenten den ET zu untersuchen. Für die LF-Experimente wurde eine photoaktive Rutheniumverbindung kovalent an Cyt c552 gekoppelt, durch die bei Laseranregung das Häm photooxidiert und dadurch der ET von einem reduzierten Redoxpartner induziert werden kann. Die Strukturdaten des bc1/Cyt c-Cokomplexes aus Hefe zeigen, dass direkte Kontakte zwischen Cyt c1 und Cyt c durch unpolare Wechselwirkungen und eine zentrale Kation-p-Interaktion vermittelt werden. Daher wurden anhand von Sequenz- und Struktur-alignments äquivalente Aminosäurepositionen im Paracoccus Cyt c1 identifiziert, die an der Wechselwirkung zu Cyt c552 beteiligt sein könnten. Diese wurden durch gerichtete Mutagenese in ihrem Raumvolumen, ihrer Polarität oder Ladung variiert und in kinetischen Studien untersucht. Für die LF-Kinetiken sowie für die FRET-Experimente wurden Oberflächen-Cysteinmutanten des Cyt c1 bzw. c552 generiert, über deren SH-Gruppen kovalent der Rutheniumkomplex bzw. Fluorophore gekoppelt werden konnten. Die ET-Reaktion zeigt zwei Phasen in Abhängigkeit von der Ionenstärke. Bei niedrigen Ionenstärken ergeben sich Geschwindigkeitskonstanten von 109 M -1s-1 und eine geringe Ionenstärkeabhängigkeit (etwa eine effektive Ladung), wohingegen bei Ionenstärken ab 35 mM 2-3 effektive Ladungen pro Redoxpartner beteiligt sind und bimolekulare Geschwindigkeitskonstanten von 107 bis 109 M-1s-1 erhalten werden. Diese Ergebnisse deuten auf die Bildung eines Encounter-Komplexes bei niedrigen Ionenstärken hin, der ein Ensemble verschiedener Distanzen und relativer Orientierungen der Redoxpartner zueinander darstellt. Diese Annahme wurde ebenfalls durch FRET-Experimente bestätigt, durch die selbst bei niedrigen Ionenstärken keine definierten Abstände erhalten werden konnten. Die Geschwindigkeitskonstanten der bimolekularen Reaktion weisen auf einen diffusionskontrollierten Prozess hin, an dem 2 bis 3 effektive Ladungen an der elektrostatischen Annäherung der beiden Redoxpartner beteiligt sind. Diese Ergebnisse werden durch vorherige kinetische Untersuchungen und EPR-Studien des ET von Cyt c552 zum CuA-Fragment der aa3-Oxidase bekräftigt, für den die gleiche Anzahl effektiver Ladungen und die Bildung eines Encounter-Komplex gefunden wurden. Lediglich c1-Mutanten, deren variierte Aminosäuren sich direkt oberhalb der Hämspalte und in der Sequenz um bzw. innerhalb des Hämbindemotivs befinden, bewirken eine Verlangsamung der Gesamtreaktion und ein leicht verändertes Ionenstärkeverhalten, das auf eine leicht verschobene Interaktionsfläche hindeutet. Es wurde durch Sequenz- und Strukturalignments kein aromatischer Rest in direkter Nähe der entsprechenden Position im Paracoccus Cyt c1 gefunden, der wie im Hefekomplex eine stabilisierende Kation-p-Interaktion zwischen den Redoxpartnern vermittelt. Zur Untersuchung des ET zwischen Komplex III und Komplex IV aus T. thermophilus wurde die hydrophile Cytochrom c-Domäne der caa3-Oxidase in Analogie zum Paracoccus-System als lösliches Redoxfragment kloniert, heterolog exprimiert und charakterisiert. Dieses Fragment wurde in SF-Studien eingesetzt, um den ET zu seinen potentiellen Redoxpartnern Cyt cbc des bc-Komplexes und Cyt c552 zu charakterisieren. Es konnte gezeigt werden, dass das ccaa3-Fragment Elektronen von beiden Redoxpartnern empfängt, wobei die ET-Reaktion mit Cyt c552 so schnell verläuft, dass sie durch SF-Techniken nur schlecht aufgelöst und lediglich eine Größenordnung der Geschwindigkeitskonstanten abgeschätzt werden kann (kon ~ 1010 M-1s-1). Die Reaktion mit Cyt cbc verläuft auch noch schnell (kon = 108-109 M-1s-1) und ist nahezu unabhängig von der Ionenstärke, was eine hydrophobe Wechselwirkung zwischen beiden Redoxpartnern bestätigt. Die Ergebnisse deuten erstmals darauf hin, dass ein ET direkt zwischen zwei Redoxproteinkomplexen (bc-Komplex und caa3-Oxidase) stattfindet, ohne dass ein Elektronenüberträger (wie z. B. Cyt c552) zwischengeschaltet ist. Um die Übertragbarkeit der Erkenntnisse, die hier über den ET an löslichen Redoxfragmenten gewonnen wurden, in einem Proteinkomplex zu verifizieren, wurde ein bc1-Komplex verwendet, in dem die für P. denitrificans einzigartige saure Cyt c1-Domäne deletiert ist. Der Komplex wurde für eine erleichterte Aufreinigung mit einem His-tag versehen, homolog exprimiert und erste Aufreinigungs- und Charakterisierungsschritte unternommen. Er soll zukünftig eingesetzt werden, um die Mutationen des löslichen c1-Fragmentes in den Komplex zu überführen und die Mutanten kinetisch mittels pre-steady state- und steady state-Techniken im Vergleich zum bc1WT-Komplex zu untersuchen, ohne dass die Effekte der Punktmutationen durch die hohe negative Ladungsdichte der sauren Domäne überlagert werden.Adenosine triphosphate as the universal energy source of cells is produced by oxidative phosphorylation, where electrons are passed through different transmembrane redox complexes along the electrochemical gradient of the respiratory chain. This electron transfer (ET) is coupled to proton pumping across the membrane to generate a gradient which is then used by the ATP-synthase to drive phosphorylation of ADP to ATP. The cytochrome bc1-complex represents the central part of this aerobic respiratory chain of many pro- and eukaryotic organisms and transfers electrons from ubiquinol to cytochrome c, from where they are passed subsequently to cytochrome c-oxidase. The mesophilic soil bacterium Paracoccus denitrificans is used as a model system for mitochondrial electron transfer processes because it has an aerobic respiratory chain which is homologous to the mitochondrial one, but with a much simpler subunit composition of its redox complexes; this bacterium is a member of the a-subdivision of the proteobacteria, presumably harbouring the mitochondrial precursor. Also the extremely thermophilic genus Thermus thermophilus provides a similar aerobic respiratory chain. Proteins of T. thermophilus have gained interest because of their thermostability and their decreased sensitivity against extreme pH-values and proteolytic degradation. The aim of this work was the characterization of the electron transfer between complex III and complex IV of the aerobic respiratory chains of P. denitrificans and T. thermophilus. Because of their mesophilic or thermophilic properties both organisms show different mechanisms of interaction between their redox components.Soluble redox fragments have been successfully used before to study the electron transfer reactions of P. denitrificans and T. thermophilus (Lappalainen, Watmough et al. 1995; Reincke, Thony-Meyer et al. 1999; Maneg, Ludwig et al. 2003; Maneg, Malatesta et al. 2004; Mooser, Maneg et al. 2005). In this work soluble fragments of cytochrome c1 (c1CF) (Eichhorn, Dissertation 2003) and cytochrome c552 (c552F) (Reincke, Thony-Meyer et al. 1999) were used to determine the interaction of both redox partners in a dynamic and functional approach by mutagenesis studies, pre-steady state kinetics and fluorescence resonance energy transfer (FRET) experiments
Some records of Pteromalus leucanthemi Janzon (Hymenoptera: Pteromalidae) from Kent
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