14 research outputs found
The noninferiority of the nonparticulate steroid dexamethasone vs the particulate steroids betamethasone and triamcinolone in lumbar transforaminal epidural steroid injections
No full textObjective: To assess whether a nonparticulate steroid (dexamethasone, 10mg) is less clinically effective than the particulate steroids (triamcinolone, 80mg; betamethasone, 12mg) in lumbar transforaminal epidural steroid injections (TFESIs) in subjects with radicular pain with or without radiculopathy. Design: Retrospective observational study with noninferiority analysis of dexamethasone relative to particulate steroids. Setting: Single academic radiology pain management practice. Subjects: Three thousand six hundred forty-five lumbar TFESIs at the L4-5, L5-S1, or S1 neural foramina, performed on 2,634 subjects. Methods-Outcome Measures: Subjects were assessed with a pain numerical rating scale (NRS, 0-10) and Roland-Morris disability questionnaire (R-M) prior to TFESI, and at 2 weeks and 2 months follow-up. For categorical outcomes, successful pain relief was defined as either ≥50percent reduction in NRS or pain 0-10; functional success was defined as ≥40percent reduction in R-M score. Noninferiority analysis was performed with δ=-10percent as the limit of noninferiority. Continuous outcomes (mean NRS, R-M scores) were analyzed for noninferiority with difference bounds of 0.3 for NRS scores and 1.0 for R-M scores. Results: With categorical outcomes, dexamethasone was demonstrated to be noninferior to the particulate steroids in pain relief and functional improvement at 2 months. Using continuous outcomes, dexamethasone was demonstrated to be superior to the particulate steroids in both pain relief and functional improvement at 2 months. Conclusion: This retrospective observational study reveals no evidence that dexamethasone is less effective than particulate steroids in lumbar TFESIs performed for radicular pain with or without radiculopathy. © 2013 Wiley Periodicals, Inc.Abdi Salahadin, 2005, Pain Physician, V8, P127; Abdi Salahadin, 2007, Pain Physician, V10, P185; Ahadian FM, 2011, REGION ANESTH PAIN M, V36, P572, DOI 10.1097-AAP.0b013e318232e843; ALTMAN DG, 1995, BRIT MED J, V311, P485; Benzon HT, 2007, ANESTHESIOLOGY, V106, P331, DOI 10.1097-00000542-200702000-00022; Bogduk N, 2004, PRACTICE GUIDELINES; Boswell Mark V, 2003, Pain Physician, V6, P319; Buenaventura RM, 2009, PAIN PHYSICIAN, V12, P233; Chang Chien GC, 2012, PAIN PHYSICIAN, V15, P515; Committee for Proprietary Medicinal Products, 2001, BR J CLIN PHARM, V52, P223; Derby R, 2008, PAIN MED, V9, P227, DOI 10.1111-j.1526-4637.2007.00341.x; Dreyfuss P, 2006, PAIN MED, V7, P237, DOI 10.1111-j.1526-4637.2006.00162.x; Ghahreman A, 2010, PAIN MED, V11, P1149, DOI 10.1111-j.1526-4637.2010.00908.x; Glaser Scott E, 2005, Pain Physician, V8, P309; Houten John K, 2002, Spine J, V2, P70, DOI 10.1016-S1529-9430(01)00159-0; Huntoon MA, 2004, REGION ANESTH PAIN M, V29, P494, DOI 10.1016-j.rapm.2004.05.002; Karppinen J, 2001, SPINE, V26, P1059, DOI 10.1097-00007632-200105010-00015; Karppinen J, 2001, SPINE, V26, P2587, DOI 10.1097-00007632-200112010-00013; Kaufmann TJ, 2013, PAIN MED, V14, P1126, DOI 10.1111-pme.12122; Kennedy DJ, 2009, PAIN MED, V10, P1389, DOI 10.1111-j.1526-4637.2009.00728.x; Lauridsen HH, 2006, BMC MUSCULOSKEL DIS, V7, DOI 10.1186-1471-2474-7-82; Lee JW, 2009, SKELETAL RADIOL, V38, P1077, DOI 10.1007-s00256-009-0735-5; Lennard TA, 2011, PAIN PROCEDURES CLIN, P7; Lyders EM, 2009, AM J NEURORADIOL, V30, P1691, DOI 10.3174-ajnr.A1567; MacVicar J, 2013, PAIN MED, V14, P14, DOI 10.1111-j.1526-4637.2012.01508.x; Murthy NS, 2010, PAIN MED, V11, P1756, DOI 10.1111-j.1526-4637.2010.00948.x; Park CH, 2010, PAIN MED, V11, P1654, DOI 10.1111-j.1526-4637.2010.00941.x; PATRICK DL, 1995, SPINE, V20, P1899, DOI 10.1097-00007632-199509000-00011; Reiw KD, 2006, J BONE JOINT SURG AM, V88, P1722; Riew KD, 2000, J BONE JOINT SURG AM, V82A, P1589; Roberts ST, 2009, PMandR, V1, P657, DOI 10.1016-j.pmrj.2009.04.008; Somayaji HS, 2005, SPINE, V30, pE106, DOI 10.1097-01.brs.0000153400.67526.07; Thomas E, 2003, CLIN RHEUMATOL, V22, P299, DOI 10.1007-s10067-003-0736-z; Vad VB, 2002, SPINE, V27, P11, DOI 10.1097-00007632-200201010-00005; Wybier M, 2008, JOINT BONE SPINE, V75, P523, DOI 10.1016-j.jbspin.2008.07.001; Wybier M, 2010, EUR RADIOL, V20, P181, DOI 10.1007-s00330-009-1539-787
Bone Marrow Cells in Murine Colitis: Multi-Signal Analysis Confirms Pericryptal Myofibroblast Engraftment without Epithelial Involvement
No full textThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credite
Implications of differences between temperate and tropical freshwater ecosystems for the ecological risk assessment of pesticides
No full textDespite considerable increased pesticide use over the past decades, little research has been done into their fate and effects in surface waters in tropical regions. In the present review, possible differences in response between temperate and tropical freshwaters to pesticide stress are discussed. Three underlying mechanisms for these differences are distinguished: (1) climate related parameters, (2) ecosystem sensitivity, and (3) agricultural practices. Pesticide dissipation rates and vulnerability of freshwaters appear not to be consistently higher or lower in tropical regions compared to their temperate counterparts. However, differences in fate and effects may occur for individual pesticides and taxa. Furthermore, intensive agricultural practices in tropical countries lead to a higher input of pesticides and spread of contamination over watersheds. Field studies in tropical farms on pesticide fate in the enclosed and surrounding waterways are recommended, which should ultimately lead to the development of surface water scenarios for tropical countries like developed by the Forum for the co-ordination of pesticide fate models and their use for temperate regions. Future tropical effect assessment studies should evaluate whether specific tropical taxa, not represented by the current standard test species in use, are at risk. If so, tropical model ecosystem studies evaluating pesticide concentration ranges need to be conducted to validate whether selected surrogate indigenous test species are representative for local tropical freshwater ecosystems
Comparative analysis of Mycobacterium tuberculosis pe and ppe genes reveals high sequence variation and an apparent absence of selective constraints.
No full textContains fulltext :
110619.pdf (Publisher’s version ) (Open Access)Mycobacterium tuberculosis complex (MTBC) genomes contain 2 large gene families termed pe and ppe. The function of pe/ppe proteins remains enigmatic but studies suggest that they are secreted or cell surface associated and are involved in bacterial virulence. Previous studies have also shown that some pe/ppe genes are polymorphic, a finding that suggests involvement in antigenic variation. Using comparative sequence analysis of 18 publicly available MTBC whole genome sequences, we have performed alignments of 33 pe (excluding pe_pgrs) and 66 ppe genes in order to detect the frequency and nature of genetic variation. This work has been supplemented by whole gene sequencing of 14 pe/ppe (including 5 pe_pgrs) genes in a cohort of 40 diverse and well defined clinical isolates covering all the main lineages of the M. tuberculosis phylogenetic tree. We show that nsSNP's in pe (excluding pgrs) and ppe genes are 3.0 and 3.3 times higher than in non-pe/ppe genes respectively and that numerous other mutation types are also present at a high frequency. It has previously been shown that non-pe/ppe M. tuberculosis genes display a remarkably low level of purifying selection. Here, we also show that compared to these genes those of the pe/ppe families show a further reduction of selection pressure that suggests neutral evolution. This is inconsistent with the positive selection pressure of "classical" antigenic variation. Finally, by analyzing such a large number of genes we were able to detect large differences in mutation type and frequency between both individual genes and gene sub-families. The high variation rates and absence of selective constraints provides valuable insights into potential pe/ppe function. Since pe/ppe proteins are highly antigenic and have been studied as potential vaccine components these results should also prove informative for aspects of M. tuberculosis vaccine design
Role of the soluble epoxide hydrolase and cytochrome P450-derived epoxyeicosatrienoic acids in hypoxic pulmonary vasoconstriction and hypoxia-induced pulmonary vascular remodelling
No full textHypoxic pulmonary vasoconstriction (HPV) redistributes pulmonary blood flow from areas of low oxygen partial pressure to areas of normal or relativity high oxygen availability, thus optimising the matching of perfusion to ventilation and preventing arterial hypoxemia. Generalised alveolar hypoxia results in a sustained increase in pulmonary artery pressure which in turn leads to structural changes in the walls of the pulmonary vasculature (pulmonary vascular remodelling). Recent findings have indicated a role for cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) in hypoxia-induced pulmonary vasoconstriction. Given that the intracellular concentration of EETs is determined by the soluble epoxide hydrolase (sEH), which metabolises EETs to their less active dihydroxyeicosatrienoic acids (DHETs), we assessed the influence of the sEH and EETs on pulmonary artery pressure, acute and chronic HPV, and pulmonary vascular remodelling in the mouse lung. In isolated lungs from wild-type mice, acute HPV was significantly increased by sEH inhibition, an effect abolished by pre-treatment with CYP epoxygenase inhibitors and the EET antagonist 14,15-EEZE. The acute hypoxia-induced vasoconstriction and EET production were greater in lungs from sEH-/- mice than from wild-type mice and sEH inhibition had no further effect on HPV in lungs from the former animals, while MSPPOH (CYP epoxygenase inhibitor) and 14,15-EEZE decreased the response. Exogenous application of 11,12-EET increased pulmonary artery pressure in a concentration-dependent manner and enhanced acute HPV in wild-type lungs, while 14,15-EET and 11,12-DHET were without significant effect on pulmonary artery pressure. 5-HT2A receptor antagonism or Rho kinase inhibition shifted the EET concentration-response curve to the right and abrogated the EET- and sEH inhibition-induced potentiation of acute hypoxic vasoconstriction. In lungs from wild-type and sEH-/- mice, hypoxic preconditioning (hypoxic ventilation for 10 minutes) enhanced the 5-HT response. 1-Adamantyl-3-cyclohexylurea (ACU), a sEH inhibitor, further amplified the hypoxia-induced 5-HT-hypersensitivity in wild-type mice. However, after hypoxic preconditioning, the sEH-/- lungs displayed a striking leftward shift in the 5-HT response. 11,12-EET can activate TRPC6 channels in endothelial cells by eliciting its translocation to the plasma membrane, more specifically to membrane domains enriched with the caveolae marker caveolin-1. This effect was also observed in rat pulmonary artery smooth muscle cells overexpressing the channel. Exposure of the latter cells to acute hypoxia also stimulated the intracellular translocation of TRPC6 to caveolae, an effect that was sensitive to the EET antagonist. The EET-induced translocation of TRPC6 channels was prevented by a 5-HT2A receptor antagonist but not by a Rho kinase inhibitor. Moreover, while acute hypoxia and 11,12-EET increased pulmonary pressure in lungs from TRPC6+/- mice, lungs from TRPC6-/- mice did not respond to either stimuli. These results indicate that the sEH and CYP-derived EETs are involved in acute HPV and that EET-induced pulmonary contraction under normoxic and hypoxic conditions involves a TRPC6 channel, a 5-HT2A receptor-dependent pathway and Rho kinase activation. In the second part of the study the role of the sEH in the development of pulmonary hypertension and vascular remodelling induced in mice by exposure to hypoxia (10% O2) for 21 days was analysed. In wild-type mice, chronic hypoxia decreased the pulmonary expression/activity of the sEH, induced right heart hypertrophy and erythropoiesis, and increased the number of partially and fully muscularised pulmonary resistance arteries (by 3-fold). Moreover, in HEK 293 cells, hypoxia (1% O2 up to 24 h) decreased sEH promoter activity by 50%. In isolated lungs, pre-exposure to chronic hypoxia significantly increased baseline perfusion pressures and potentiated the acute HPV. While an sEH inhibitor, ACU, potentiated acute HPV in lungs from mice maintained in normoxic conditions, it had no effect on HPV in lungs from mice exposed to hypoxia. The EET antagonist, 14,15-EEZE, abolished the sEH inhibitor-dependent increase in acute HPV in normoxic lungs and decreased HPV in chronic hypoxic lungs. Hypoxia-induced right heart hypertrophy and erythropoiesis were more pronounced in sEH-/- than in wild-type mice. Under normoxic and hypoxic conditions the muscularisation of resistance pulmonary arteries was greater in lungs from sEH-/- mice than in lungs from wild-type mice. sEH-/- mice also displayed an enhanced acute HPV, compared to that observed in wild-type mice and chronic exposure to hypoxia did not further potentiate acute HPV. However, in the presence of 14,15-EEZE responses returned to levels observed in normoxic lungs from wild-type animals. Furthermore, immunohistochemistry demonstrated an extensive expression of the sEH in the medial wall of pulmonary arteries from human donor lungs. Whereas sEH expression was not detectable in samples from pulmonary hypertension patients, indicating that the sEH is involved in hypoxia-induced pulmonary vascular remodelling and hypoxic pulmonary vasoconstriction. Taken together, the results presented in this thesis indicate that the expression/activity of the sEH is an important determinant of the magnitude of acute and chronic hypoxia-induced pulmonary vasoconstriction and pulmonary vascular remodelling by inactivating vasoconstrictor CYP-derived EETs. As sEH inhibitors are currently being developed for the treatment of human systemic hypertension, it should be noted that these compounds may even promote the development of pulmonary hypertension.Die akute hypoxische pulmonale Vasokonstriktion (HPV) ist ein physiologischer Mechanismus, der es erlaubt, den lokalen Blutfluss der Lunge an alveolaren O2- Mangel anzupassen. Durch die Hypoxie-bedingte Widerstandserhöhung besteht die Möglichkeit, die Durchblutung schlecht ventilierter Lungenbezirke einzuschränken und den Blutstrom in gut ventilierte Gebiete zu leiten. Die HPV wurde erstmals im Jahr 1946 durch von Euler und Liljestrand beschrieben und ist bis heute Gegenstand intensiver Forschung. Dennoch sind die Signaltransduktionswege und die Sauerstoff-Sensoren, die in die HPV involviert sind, weitgehend unbekannt. Als Sensoren werden unter anderem die Mitochondrien und die NADPH-Oxidasen diskutiert. Allerdings konnten neuere Studien zeigen, dass die glatten Muskelzellen der pulmonalen Widerstandsgefäße Effektorzellen darstellen. Des Weiteren konnte nachgewiesen werden, dass Mediatoren - wie 5-Hydroxytryptamin (5-HT) und Endothelin-1 - die akute HPV modulieren und ein nicht selektiver Kationen-Kanal (TRPC6-Kanal) sowie die Aktivierung der Rho-Kinase wesentlich an der akuten HPV beteiligt sind. Bei chronischer Hypoxie, wie sie z. B. bei dauerhaftem Aufenthalt in großen Höhen entsteht, kommt es zu einer globalen Vasokonstriktion der Lunge. Die Folge ist ein pulmonaler Bluthochdruck mit einer Hypertrophie der glatten Muskulatur der Pulmonalgefäße („pulmonales vaskuläres Remodelling“). Hinzu kommt, dass eine dauerhafte Hypoxie zur Produktion von endothelialen Substanzen führt, die sowohl die Kontraktilität als auch die Proliferation der glatten Muskelzellen beeinflussen. Es konnte in mehreren Studien nachgewiesen werden, dass eine chronische Hypoxie zu einer verminderten Bildung von Stickstoffmonoxid und Prostazyklinen sowie zu einer vermehrten Produktion von 5-HT, Endothelin-1 und verschiedenen Wachstumsfaktoren führt. Über eine Beteiligung des 5-HT-Transporters, der TRPC-Kanäle und der Rho-Kinase in der chronischen Hypoxie-bedingten pulmonalen Hypertension wird ebenfalls diskutiert. Enzyme der Cytochrom P450 (CYP)-Epoxygenase-Familie werden extrahepatisch vor allem im Herz, dem Gefäßsystem, dem Gastrointestinaltrakt, der Niere und der Lunge exprimiert. Diese liefern durch Metabolisierung von Arachidonsäure vier verschiedene Isomere der Epoxyeicosatriensäuren (5,6-, 8,9-, 11,12-, und 14,15-EETs), die sowohl den Gefäßtonus modulieren als auch antiinflammatorische und angiogenetische Eigenschaften haben. Nach ihrer Synthese stehen den EETs zwei Wege offen: Zum einen können sie in Phospholipide inkorporiert, zum anderen durch die Cyclooxygenase (5,6-EET) und die lösliche Epoxidhydrolase (sEH; 8,9-, 11,12- und 14,15-EET) zu ihren weniger aktiven Dihydroxyeicosatriensäuren (DHETs) metabolisiert werden. In Koronar- und Nierenarterien unterschiedlicher Spezies stellen EETs den sogenannten endothelialen hyperpolarisierenden Faktor dar, der zur Hyperpolarisation und Relaxation der glatten Gefäßmuskulatur führt. Kürzlich konnte unsere Arbeitsgruppe nachweisen, dass 11,12-EET die Agonisten-induzierte endotheliale Hyperpolarisation durch Translokation des TRRPC6-Kanals in mit Caveolin-1 angereicherte Membrangebiete, erhöht. Des Weiteren legen verschiedene Untersuchungen eine Verbindung zwischen der sEH und kardiovaskulären Erkrankungen nahe, da eine Hemmung dieses Enzyms der Entstehung einer Hypertonie entgegenwirkt. Dagegen ist die Rolle von CYP-Epoxygenase-generierten EETs und der sEH in der pulmonalen Zirkulation weitgehend unbekannt. Hierzu wurden in den letzten Jahren widersprüchliche Ergebnisse publiziert. Jedoch konnte unsere Arbeitsgruppe vor kurzem nachweisen, dass eine CYP-Epoxygenase in die HPV und das chronische Hypoxie-induzierte pulmonale vaskuläre Remodelling involviert ist. Ziel der vorliegenden Arbeit war es daher, ausführlich die Rolle der sEH und der CYP-Epoxygenase-generierten EETs in der akuten und chronischen HPV sowie im Hypoxie-bedingten pulmonalen vaskulären Remodelling zu analysieren. Dazu wurden CYP-Epoxygenase-Inhibitoren, ein EET-Antagonist und sEH-Inhibitoren verwendet. Um den molekularen Mechanismus der Hypoxie- und EET-induzierten pulmonalen Vasokonstriktion und des pulmonalen vaskulären Remodelling zu untersuchen, wurden kultivierte pulmonale glatte Muskelzellen, Promotor-Aktivitäts-Assays und genetisch veränderte Tiere (sEH-/- und TRPC6-/- Mäusen) verwendet. Zunächst wurde die Rolle der sEH und EETs in der akuten HPV anhand der isolierten perfundierten Mauslunge untersucht. In Lungen von Wildtyp-Mäusen führte eine Hemmung der sEH zu einer deutlichen Steigerung der Amplitude der akuten HPV. Darüber hinaus konnte nachgewiesen werden, dass eine vorherige Inkubation mit CYP-Epoxygenase-Inhibitoren oder dem EET-Antagonisten 14,15-EEZE den Effekt der sEH-Inhibitoren auf die akute HPV signifikant reduzierte. Im Vergleich zu Lungen von Wildtyp-Mäusen zeigten sEH-/--Lungen eine deutlich erhöhte akute Hypoxie-bedingte Vasokonstriktion. Erwartungsgemäß hatte eine Hemmung der sEH in Lungen von sEH-/--Mäusen keine Wirkung, während MSPPOH, ein CYP-Epoxygenase-Inhibitor, und 14,15-EEZE die gesteigerte HPV signifikant reduzierten. Weiterhin konnte in der vorliegenden Arbeit bewiesen werden, dass eine hypoxische Inkubation (1% O2, 10 Minuten) von Maus-Lungenmikrosomen die EET-Produktion erheblich steigerte. Dieser Effekt war deutlich potenziert in Lungenmikrosomen von sEH-/--Mäusen. Eine exogene Applikation von 11,12-EET in der isolierten perfundierten Mauslunge steigerte signifikant den basalen pulmonalarteriellen Druck und die akute HPV, während 14,15-EET und 11,12-DHET keine Effekte auf den pulmonalarteriellen Druck ausübten. Eine Inhibition des 5-HT2A-Rezeptors oder der Rho-Kinase führte sowohl zu einer deutlichen Reduktion der 11,12-EET-induzierten Vasokonstriktion, als auch zu einer signifikanten Hemmung der durch die EET und sEH-Inhibition gesteigerten akuten HPV. In isolierten Lungen von Wildtyp- und sEH-/--Mäusen führte eine hypoxische Präkonditionierung (hypoxische Ventilation der isolierten Lunge für 10 Minuten) zu einer Verstärkung der 5-HT-induzierten Vasokonstriktion. Der Verlust der sEH-Aktivität, entweder durch Einsatz spezifische Hemmstoffe oder durch genetische Deletion, führte zu einer weiteren deutlichen Verstärkung der 5-HT Dosiswirkungskurve nach hypoxischer Vorbehandlung. In pulmonalarteriellen glatten Muskelzellen, die TRPC6 überexprimieren, konnte nachgewiesen werden, dass 11,12-EET diesen Kanal durch Translokation in mit Caveolin-1 angereicherte Membrangebiete, aktiviert. Darüber hinaus führte eine akute Hypoxie in TRPC6-überexprimierenden glatten Muskelzellen ebenfalls zu einer TRPC6-Kanal-Translokation. Dieser Effekt wurde durch den EET-Antagonisten deutlich gehemmt. Ferner konnte gezeigt werden, dass die EET-induzierte TRPC6-Translokation durch einen 5-HT2A-Rezeptor-Antagonisten reduziert wurde, wogegen ein Rho-Kinase-Inhibitor ohne Effekt war. Außerdem bewirkte eine akute Hypoxie und die Zugabe von 11,12-EET einen Anstieg des pulmonalarteriellen Drucks in isolierten perfundierten Lungen von TRPC6+/--Mäusen. TRPC6-/--Mäuse reagierten auf keinen der beiden Stimuli. Diese Befunde zeigen, dass die Aktivität der sEH und CYP-Epoxygenase-generierte EETs die akute HPV modulieren und die EET-induzierte Kontraktion unter normoxischen und hypoxischen Bedingungen über einen TRPC6-Kanal-, einen 5-HT2A-Rezeptor-abhängigen Signaltransduktionsweg und die Aktivierung der Rho-Kinase verläuft. Ein weiteres Ziel der vorliegenden Arbeit war, die Rolle der sEH in der Entwicklung einer Hypoxie-bedingten pulmonalen Hypertension und des pulmonalen vaskulären Remodelling aufzuklären. Dafür wurden Mäuse 21 Tage in Hypoxie-Kammern hypoxischen Bedingungen (10% O2) ausgesetzt oder unter normoxischen Bedingungen gehalten. Eine chronische Hypoxie führte in Wildtyp-Mäusen zu einer verminderten pulmonalen Expression und folglich auch Aktivität der sEH. Zusätzlich induzierte eine dauerhafte Hypoxie eine Rechtsherzhypertrophie und Hämatopoese in Wildtyp-Mäusen und erhöhte signifikant die Anzahl der teil- und vollmuskularisierten pulmonalen Widerstandsgefäße. In isolierten perfundierten Lungen aus chronisch hypoxischen Mäusen war der Basisdruck signifikant erhöht und die akute HPV deutlich potenziert. Während der sEH-Inhibitor 1-Adamantyl-3-Cyclohexylurea die akute HPV in Lungen von Tieren, die unter normoxischen Bedingungen gehalten wurden, steigerte, hatte er keinen Effekt auf die Antwort in Lungen von chronisch hypoxischen Mäusen. Der EET-Antagonist 14,15-EEZE hemmte signifikant den sEH-Inhibitor-induzierten Anstieg der akuten HPV in normoxischen Lungen und reduzierte die potenzierte akute HPV in Lungen von chronisch hypoxischen Tieren. sEH-/--Mäuse zeigten im Vergleich zu Wildtyp-Mäusen eine verstärkte Hypoxie-induzierte Rechtsherzhypertrophie und Hämatopoese. Darüber hinaus konnte in Lungen von hypoxischen und normoxischen sEH-/--Mäusen eine erhöhte Muskularisierung der pulmonalen Widerstandsgefäße nachgewiesen werden. Außerdem zeigten sEH-/--Lungen eine verstärkte akute HPV. Eine vorherige hypoxische Exposition hatte keinen potenzierenden Effekt auf die akute HPV. Allerdings reduzierte 14,15-EEZE die akute HPV in sEH-/--Lungen auf das Niveau der HPV von normoxischen Wildtyp-Lungen. Überdies führte Hypoxie (1% O2, 24 Stunden) in humanen embryonalen Nierenzellen (HEK 293), die mit sEH Promotor-Konstrukten transfiziert wurden, zu einer verminderten sEH-Promotor-Aktivität. Über die Expression der sEH in Lungen von Patienten mit pulmonaler Hypertonie ist bisher wenig bekannt. In pulmonalen Gewebeschnitten des lungengesunden Kontrollkollektivs konnte eine starke sEH-Expression in der Media der Pulmonalarterien nachgewiesen werden, während in Gewebeschnitten von Patienten mit pulmonaler Hypertonie keine sEH-Expression detektiert werden konnte. Zusammenfassend legen die Resultate des zweiten Teils dieser Arbeit nahe, dass die sEH in die Entstehung der Hypoxie-bedingten pulmonalen Hypertension und des daraus folgenden pulmonalen vaskulären Remodellings involviert ist. Die Ergebnisse der vorliegenden Arbeit zeigen, dass die Aktivität/Expression der sEH die akute hypoxische Vasokonstriktion sowie die chronische HPV und die damit einhergehenden Veränderungen der pulmonalen Gefäße beeinflusst. Ihren Effekt vermittelt die sEH über den Abbau der vasokonstringierenden EETs - vor allem ist hier 11,12-EET relevant - zu weniger aktiven Diolen. Außerdem konnte hier nachgewiesen werden, dass an der EET-induzierten Vasokonstriktion unter normoxischen und unter akut hypoxischen Bedingungen der TRPC6-Kanal, der 5-HT2A-Rezeptor und die Rho-Kinase an der Signaltransduktion beteiligt sind. Wie chronische Hypoxie die Aktivität/Expression der sEH und folglich auch die Entwicklung einer pulmonalen Hypertension und des pulmonalen vaskulären Remodelling beeinflusst, bleibt offen. Gleiches gilt für eine Involvierung des für die akute HPV beschriebenen Signaltransduktionsweges (5-HT2A-Rezeptor, TRPC6-Kanal) in diese Prozesse. In verschiedenen Studien wurde nachgewiesen, dass TRPC6-Kanäle in pulmonaler Hypertension verstärkt exprimiert werden. TRPC6-/--Mäuse zeigten im Vergleich zu Wildtyp-Mäusen jedoch keine Unterschiede in der Entwicklung einer chronisch Hypoxie-induzierten pulmonalen Hypertension. sEH-Inhibitoren werden momentan für den Einsatz in der Therapie der Hypertonie entwickelt. Angesichts der Tatsache, dass eine CYP-Epoxygenase, EETs und die Aktivität/Expression der sEH in die HPV und in das chronische Hypoxie-induzierte pulmonale vaskuläre Remodelling involviert sind, besteht die Möglichkeit, dass diese Substanzen die Entwicklung einer pulmonalen Hypertension fördern
The key role of nitric oxide in hypoxia: hypoxic vasodilation and energy supply-demand matching
No full textSignificance: a mismatch between energy supply and demand induces tissue hypoxia with the potential to cause cell death and organ failure. Whenever arterial oxygen concentration is reduced, increases in blood flow - 'hypoxic vasodilation' - occur in an attempt to restore oxygen supply. Nitric oxide is a major signalling and effector molecule mediating the body's response to hypoxia, given its unique characteristics of vasodilation (improving blood flow and oxygen supply) and modulation of energetic metabolism (reducing oxygen consumption and promoting utilization of alternative pathways). Recent advances: this review covers the role of oxygen in metabolism and responses to hypoxia, the hemodynamic and metabolic effects of nitric oxide, and mechanisms underlying the involvement of nitric oxide in hypoxic vasodilation. Recent insights into nitric oxide metabolism will be discussed, including the role for dietary intake of nitrate, endogenous nitrite reductases, and release of nitric oxide from storage pools. The processes through which nitric oxide levels are elevated during hypoxia are presented, namely (i) increased synthesis from nitric oxide synthases, increased reduction of nitrite to nitric oxide by heme- or pterin-based enzymes and increased release from nitric oxide stores, and (ii) reduced deactivation by mitochondrial cytochrome c oxidase. Critical issues: several reviews covered modulation of energetic metabolism by nitric oxide, while here we highlight the crucial role NO plays in achieving cardiocirculatory homeostasis during acute hypoxia through both vasodilation and metabolic suppression Future directions: we identify a key position for nitric oxide in the body's adaptation to an acute energy supply-demand mismatc
Innovative approaches to monitor mutant huntingtin and to facilitate its degradation in Huntington's disease models
No full textHuntington’s disease (HD) is a dominant genetic neurodegenerative disease caused by a mutation in the exon 1 of the huntingtin gene. The clinical symptoms, such as motor disturbances (chorea), cognitive decline and psychiatric impairments are usually developed by the patients in mid-life. Mutant huntingtin protein presents an amplification of a polyglutamine repeat at its N-terminus, which induces conformational changes and leads to neurotoxicity, impairment of cell homeostasis and neuronal cell death. The neuropathology of HD is characterized by a progressive degeneration of the brain starting from the striatum and spreading to other regions such as cortex, hypothalamus and cerebellum. In addition to the diffused brain atrophy, HD patients are also affected by multiple peripheral symptoms which contribute to worsening disease progression and eventually lead to death approximately two decades after onset.
The mechanisms leading to the toxicity induced by mutant huntingtin are not well understood. However the acquisition of a misfolded conformation and the formation of intracellular inclusions constituted by shorter fragments of the mutant protein are considered important in the neurodegenerative process.
In my thesis project I have investigated mechanisms to enhance the cellular degradation of mutant huntingtin. A second focus was on the development of an immunoassay to detect and quantify aggregates in HD models.
I analyzed the data obtained form a high through-put screen aimed to identify small molecular weight compounds decreasing mutant huntingtin levels in cells. Among all compounds screened, only inhibitors of heat shock protein 90 (Hsp90) showed a significant effect on mutant huntingtin clearance. I therefore investigated the mechanisms of Hsp90 chaperone inhibition and the reduction of soluble mutant huntigtin levels. Data from biochemical assays demonstrated that mutant huntingtin degradation is enhanced upon compound treatment and that the protein is cleared through the ubiquitin-proteasome system. This was independent from the heat shock response induced after pharmacological Hsp90 inhibition. Co-immunoprecipitation experiments suggested that mutant huntingtin is a client protein of Hsp90. The results were replicated in different cellular models including full length mutant huntingtin expressed from the endogenous locus, thus highlighting the importance of Hsp90 in stabilizing soluble mutant huntingtin and suggesting the possible application of Hsp90 inhibitors as therapies in HD.
In the second project I developed a sensitive method to detect mutant protein aggregates in HD models. To this purpose I implemented the already established time resolved fluorescence resonance energy transfer (TR-FRET) based immunoassay for the detection of soluble mutant and wild-type huntingtin. A mixture of either donor or acceptor fluorophore labeled single monoclonal antibody directed against an epitope exposed on the huntingtin aggregate surface was used. This strategy allowed for energy transfer and therefore a measurable TR-FRET signal, only in presence of mutant aggregated protein. I could demonstrate the sensitivity of the bioassay on a microtiter set up both as a single assay and in a duplex combination with the previously developed TR-FRET assay for soluble huntingtin.
I applied the TR-FRET for aggregated huntingtin to samples from R6/2 and HdhQ150 mice, expressing exon 1 and full length mutant huntingtin, respectively. In brain homogenates from both models there was an age-dependent, inverse correlation between soluble and aggregated mutant huntingtin. These findings supported the importance of the relation between aggregated and soluble protein in disease progression. Furthermore, I detected the inverse correlation also in peripheral tissues of R6/2 mice where the presence of aggregates was previously demonstrated with other methods. An in-depth analysis of R6/2 samples in a combination of TR-FRET and size exclusion chromatography suggested a differential specificity of the two antibody combinations used for different aggregate populations. The TR-FRET method provides a new means to characterize the aggregation process as well as to test the efficacy of possible disease modifying treatments for HD
The role of protein kinases in DNA replication in Saccharomyces cerevisiae
No full textThe initiation of DNA replication at the onset of S phase in eukaryotic cells is a critically important and tightly regulated process. Multiple origins of replication in the genome must be co-ordinately regulated such that duplication of the chromosomes is complete before cell division, whilst also ensuring that no sections of the DNA are over-replicated. In G1 phase of the cell cycle, a large ‘pre-replicative complex’ (pre-RC) forms at origins consisting of a hexameric Origin Recognition Complex (ORC) as well as Cdc6, Cdt1 and another hexameric complex known as the Minichromosome Maintenance (MCM) complex. At the onset of S phase, two cell cycle regulated protein kinases, the Cyclin Dependent Kinase (CDK) and Cdc7, are activated. Phosphorylation of various proteins by these two enzymes triggers formation of large ‘replisome’ complexes, initiation of DNA replication from each origin, and disassembly of the pre-RCs. Pre-RC re-assembly is subsequently inhibited until kinase activity falls again after cell division.
In this study, we have set about identifying substrates of both CDK and Cdc7 involved in DNA replication in the budding yeast Saccharomyces cerevisiae. Two techniques are employed, the in vitro phosphorylation of arrays of peptides and phosphorylation of pre-RCs assembled in cell-free yeast extracts. Peptide arrays provide a high throughput technique for screening large numbers of potential substrates in a single experiment, whilst pre-RC phosphorylation allows consideration of both tertiary and quaternary structures of the in vivo kinase substrate. Several potential novel substrates of both CDK and Cdc7 are revealed. Pre-RC phosphorylation also reveals a previously unreported phosphorylation of Orc1 by a third kinase which has been identified as Casein Kinase II (CKII)
Intrinsic properties of Hellenic “Marls”
No full textPost-alpine deposits cover substantial areas of Hellas. The fine grained facies of these deposits, comprise a wide range of over consolidated materials of varying grading and mineralogy. On average, however, these deposits cover different depositional environments and may be classed as silty clays and clayey silts of low to intermediate plasticity containing calcite in their mineralogical suite. Such deposits have been collectively known to practising engineers as marls. A concerted effort to collect field and laboratory data and interpret the behaviour of these materials was recently launched in view of the involvement of the aforesaid mentioned deposits in a series of geotechnical problems. This thesis aspires to contribute to this knowledge and provide a framework on which the field performance of such materials may be interpreted. To that end, disturbed and high quality undisturbed samples from the geologically dissimilar areas of Korinthos, Preveza-Igoumenitsa road axis and Amalias- Goumeron were obtained and tested in order to ascertain their physical characteristics and mineralogical composition. Further, engineering properties which are independent of stress history, i.e. intrinsic, of the materials sampled, were determined. These were the residual strength as determined by ring shearing and the compressibility of reconstituted samples. The performance of the samples was assessed in terms of grain size distribution and mineralogy. The results show that any attempt to explain or predict intrinsic properties of Hellenic fine grained calcareous sediments without taking simultaneous account of gradation and mineralogy is incomplete and therefore inaccurate
