Characterization of the inversion reaction of phiCh1 and the establishment of a transformation system for Natrialba magadii

Bis zum jetzigen Zeitpunkt wurden bakterielle und haloarchaeale Modellsysteme zur Untersuchung von phiCh1 und Nab. magadii verwendet. Durch ein Transformationsystem können Experimente auch im natürlichen System durchgeführt werden. Einige Shuttle-Vektoren zwischen Nab. magadii und E. coli wurden ausgehend vom Genom und im Besonderen vom mutmaßlichen Replikationsurspung des Phagen phiCh1 konstruiert. Anhand der Transformation dieser Vektoren in Nab. magadii konnte der Replikationsurspung genauer untersucht werden. Dabei wurde bestätigt, dass die offenen Leserahmen 49 und 55 des Ch1 Genoms als Repressoren fungieren und eine wichtige Rolle in der Regulation der Virusentwicklung spielen. Um eine mögliche Interaktion dieser Repressoren zu vermeiden, wurde der Vektor pRo-5, der diese Regionen nicht enthält, für weitere Experimente verwendet. Das Plasmid pRo-5 ist stabil in Nab. magadii und scheint nicht ins Genom zu integrieren. Das Wirtsspektrum dieses Systems ist allerdings nicht auf Nab. magadii begrenzt, da einige verschiedene halophile und haloalkaliphile Archaea transformiert werden konnten. Der zweite Teil der Arbeit beschäftigt sich mit dem Infektionsprozess des Phagen phiCh1. Ein möglicher Einfluss von Int1, einer Integrase der lambda-Familie, auf die Variation der „Tail-fibre“ Proteine, die von ORF34 und ORF36 kodiert werden, wurde untersucht. Das int1 Gen wird von diesen zwei offenen Leserahmen flankiert, die mehrere Wiederholungen von invertierten Sequenzen beinhalten. Es konnte gezeigt werden, dass Int1 für eine Inversion in dieser Region verantwortlich ist, die zum Austauch der C-terminalen Teile von ORF34 und ORF36 führen. Diese Inversionsreaktion ermöglicht ein Andocken des Phagen an spezielle Strukturen der Wirtszelle und ist daher essentiell für eine Infektion.Until now bacterial and haloarchaeal modell systesm were used to investigate phiCh1 and Nab. magadii. With the establishment of a transfomation system experiments can be performed in the natural environment. Several shuttle-vectors between Nab. magadii and E. coli were constructed, based on the genome and in particular on the putative origin of replication of the phage Ch1. The transformation of these vectors into Nab. magadii allowed a detailed investigation of the components of the replication origin. This leads to the confirmation that the open reading frames 49 and 55 of the phiCh1 genome act as repressors and carry out an important role in the regulation of virus development. The vector pRo-5, that lacks these regions, was used for further studies. This plasmid was quite stable in Nab. magadii and seems not to integrate into the genome. In addition the host range of this system is not limited to Nab. magadii as several different halophilic and haloalkaliphilic Archaea were successfully transformed. The second part of this thesis deals with the infection process of the phage phiCh1. A possible influence of the lambda-like integrase Int1 on the variation of the tail fibre proteins ORF34 and ORF36 was investigated. The int1 gene is flanked by these two open reading frames which contain multiple inverted repeat sequences. It could be shown that Int1 is responsible for an inversion in this region that leads to the exchange of the C-terminal parts of ORF34 and ORF36. This inversion reaction allows the docking of the phage on special structures of the host cell and is therefore essential for an infection

Modulation of the endothelial nitric oxide synthase system by natural compounds

Das Endothelium is ein wichtiger Regulator für das Aufrechterhalten der vaskulären Homeostase. Es erhält das Gleichgewicht zwischen Vasodilation und Vasokonstriktion, Hemmung und Stimulierung der Proliferation und Migration glatter Muskelzellen, sowie Thrombogenese und Fibrinolyse. Ist diese Balance gestört, spricht man von endothelialer Dysfunktion, welche ein Schlüsselereignis in der Entstehung von Atherosklerose, die den meisten kardiovaskulären Erkrankungen zu Grunde liegt, ist. Das Hauptcharakteristikum endothelialer Dysfunktion ist eine verminderte Endothelium-abhängige Vasodilation auf Grund einer reduzierten Aktivität der endothelialen Stickstoffmonoxid-Synthase (eNOS) oder ihres Syntheseproduktes, Stickstoffmonoxid (NO). Für einige Naturstoffe, die in der Nahrung oder Phytopharmaka enthalten sind, wurde eine modulierende Wirkung auf die eNOS gezeigt. Die Identifizierung von bisher unbekannten, eNOS aktivierenden Naturstoffen und die Aufklärung ihres Wirkungsmechanismus stellt eine wertvolle Information für zukünftige therapeutische Anwendungen dar. In dieser Arbeit wurden zwei ausgewählte Substanzen auf ihre Wirkungsweise im Bezug auf eNOS Aktivität untersucht. Frühere Studien zeigten, dass Ascorbate (Vitamin C) den essentiellen eNOS Kofaktor Tetrahydrobiopterin (BH4) nach Langzeitstimulierung stabilisiert und regeneriert. Zusätzlich waren Infusionen von Ascorbate in der Lage, sehr schnell die Endothelium bedingte Vasodilation bei Patienten mit verschiedenen kardiovaskulären Krankheiten zu erhöhen. Hier konnten wir zeigen, dass dieser schnelle Effekt von Ascorbate unabhängig von der Stabilisierung von BH4 ist. Stattdessen scheint Ascorbate schnell die Protein Phosphatase 2A (PP2A) zu inhibieren und anschließend aktivierende AMP-aktivierte Protein Kinase (AMPK) und eNOS Phosphorylierungsmuster zu verstärken. Mehrere Lignane, die aus den Wurzeln der entzündungshemmenden Pflanze Krameria lappacea isoliert wurden, wurden auf eine mögliche eNOS aktivierende oder NO erhöhende Wirkung untersucht. Unter den untersuchten Lignanen war nur DPPB (2-(2,4-dihydroxyphenyl)-5-(E)-propenylbenzofuran) in der Lage, die NO Freisetzung zu steigern. Wir konnten zeigen, dass DPPB die eNOS Aktivität durch Steigern des intrazellulären Ca2+ Levels und Ca2+/CaM-abhängige Kinase Kinase β (CaMKKβ) und AMPK Signaltransduktion erhöht.The endothelium serves as a main regulator of vascular homeostasis by maintaining the balance between vasodilation and vasoconstriction, inhibition and stimulation of smooth muscle cell proliferation and migration, and thrombogenesis and fibrinolysis. Disruption of this balance is generally referred to as endothelial dysfunction, which is a key event in the development of atherosclerosis, the main pathology underlying cardiovascular diseases. A dysfunctional endothelium is mainly characterized by reduced endothelium-dependent vasodilation due to impaired endothelial nitric oxide synthase (eNOS) activity or decreased availability of its synthesis product, nitric oxide (NO). Several natural products, found in the daily diet or in phytomedical preparations, have previously been discussed as modulators of eNOS function. Identification of so far unknown natural products, influencing NO availability and the elucidation of their mechanism of action provide valuable information for possible future therapeutic applications. In this work we investigated the molecular mechanism underlying an increased eNOS activity elicited by two selected compounds, ascorbate and DPPB (2-(2,4-dihydroxyphenyl)-5-(E)-propenylbenzofuran). Previous studies have shown that ascorbate (vitamin C) can stabilize and regenerate the essential eNOS cofactor tetrahydrobiopterin (BH4) after long-term treatment, thereby preventing the uncoupling of eNOS. Additionally, infusions of ascorbate were able to rapidly increase endothelium-dependent vasodilation in patients with different pathological conditions. In this study we found that the fast effect of ascorbate on eNOS activity is independent on BH4 stabilization. Instead, ascorbate was shown to rapidly enhance eNOS activity by inhibition of protein phosphatase 2A (PP2A), followed by an increase in activating AMP-activated protein kinase (AMPK) and eNOS phosphorylation patterns. Several lignans isolated from the roots of the anti-inflammatory medicinal plant Krameria lappacea were investigated for their influence on eNOS activity and endothelial NO release. Among these compounds only DPPB was able to increase endothelial NO release. We could show that DPPB increases eNOS activity via raising intracellular Ca2+ levels and increasing the Ca2+/CaM-dependent kinase kinase β (CaMKKβ) and AMPK signaling

Outcome measures used in the evaluation of gluteal tendinopathy: a scoping review protocol

Protocol for a Scoping Revie

Lignan Derivatives from Krameria lappacea Roots Inhibit Acute Inflammation in Vivo and Pro-inflammatory Mediators in Vitro

The roots of Krameria lappacea are used traditionally against oropharyngeal inflammation. So far, the astringent and antimicrobial properties of its proanthocyanidin constituents are considered to account for the anti-inflammatory effect. The aim of the present study was to characterize pharmacologically a lipophilic extract of K. lappacea roots and several isolated lignan derivatives (111) in terms of their putative anti-inflammatory activity. The dichloromethane extract (ID50 77 \u3bcg/cm2) as well compounds 111 (ID50 0.310.60 \u3bcmol/cm2) exhibited topical antiedematous properties comparable to those of indomethacin (ID50 0.29 \u3bcmol/cm2) in a mouse ear in vivo model. Two of the most potent compounds, 2-(2-hydroxy-4-methoxyphenyl)-5-(3-hydroxypropyl)benzofuran (5) and (+)-conocarpan (7), were studied regarding their time-dependent edema development and leukocyte infiltration up to 48 h after croton oil-induced dermatitis induction, and they showed activity profiles similar to that of hydrocortisone. In vitro studies of the isolated lignan derivatives demonstrated the inhibition of NFkB, cyclooxygenase-1 and -2, 5-lipoxygenase, and microsomal prostaglandin E2 synthase-1 as well as antioxidant properties, as mechanisms possibly contributing to the observed in vivo effects. The present findings not only support the ethnopharmacological use of K. lappacea roots but also reveal that the isolated lignan derivatives contribute strongly to the anti-inflammatory activity of this herbal drug

Impact of Trans-Resveratrol-Sulfates and -Glucuronides on Endothelial Nitric Oxide Synthase Activity, Nitric Oxide Release and Intracellular Reactive Oxygen Species

Resveratrol (3,5,4\u27-trihydroxy-trans-stilbene) is a polyphenolic natural product mainly present in grape skin, berries and peanuts. In the vasculature resveratrol is thought to boost endothelial function by increasing endothelial nitric oxide synthase (eNOS) expression, by enhancing eNOS activity, and by reduction of reactive oxygen species (ROS) levels. Recent studies show that dietary resveratrol is metabolized in the liver and intestine into resveratrol-sulfate and -glucuronide derivatives questioning the relevance of multiple reported mechanistic in vitro data on resveratrol. In this study, we compare side by side different physiologically relevant resveratrol metabolites (resveratrol sulfates- and -glucuronides) and their parent compound in their influence on eNOS enzyme activity, endothelial NO release, and intracellular ROS levels. In contrast to resveratrol, none of the tested resveratrol metabolites elevated eNOS enzyme activity and endothelial NO release or affected intracellular ROS levels, leaving the possibility that not tested metabolites are active and able to explain in vivo findings

ICON 2020—International Scientific Tendinopathy Symposium Consensus:the development of a core outcome set for gluteal tendinopathy

Gluteal tendinopathy (GT) is common and can be debilitating and challenging to manage. A lack of condition specific and appropriate outcome measures compromise evidence synthesis for treatment and limits clinical guideline development. Our objective was to develop a core outcome measurement set for GT (COS-GT). Participants were patients with GT and expert health professionals (HPs). A scoping review identified measures used in GT research, which were mapped to the nine International Scientific Tendinopathy Symposium Consensus core domains, and included in two surveys of HPs. The first survey identified the feasible and true measures for each domain. The second survey refined the list which a patient focus group then considered. Meeting online, HPs reached consensus (agreement ≥70%) on the most appropriate COS-GT measures. 34 HPs and seven patients were recruited. 57 measures were mapped to the nine core domains. Six measures did not proceed past survey one. Of those that progressed, none had adequate clinimetric properties for a COS-GT. Thus, participants decided on interim measures: the global rating of change, pain at night, time to pain onset with single limb stance, pain with stair walking, pain self-efficacy and hip abduction strength. HP participants additionally recommended that pain over the last week, the European Quality of Life-5 dimensions-5 levels and the Victorian Institute of Sport Assessment-Gluteal be considered in clinical trials, as they currently provide best easures of the relevant tendinopathy domains. In conclusion this interim COS-GT should guide outcome measure selection in clinical practice and future research trials in patients with GT.</p

Silymarin Constituents Enhance ABCA1 Expression in THP-1 Macrophages

Silymarin is a hepatoprotective mixture of flavonolignans and flavonoids extracted from the seeds of milk thistle (Silybum marianum L. Gaertn). This study investigates the effect of major bioactive constituents from silymarin, silybin A, silybin B, isosilybin A, isosilybin B, silydianin, silychristin, isosilychristin, and taxifolin, on the expression of ABCA1, an important cholesterol efflux transporter, in THP-1-derived macrophages. Four of the studied compounds, isosilybin A, silybin B, silychristin and isosilychristin, were found to significantly induce ABCA1 protein expression without affecting cell viability. Moreover, isosilybin A, a partial PPARγ agonist, was found to promote cholesterol efflux from THP-1 macrophages in a concentration-dependent manner. These findings first show ABCA1 protein up-regulating activity of active constituents of silymarin and provide new avenues for their further study in the context of cardiovascular disease

Erythrodiol, an Olive Oil Constituent, Increases the Half-Life of ABCA1 and Enhances Cholesterol Efflux from THP-1-Derived Macrophages

Cholesterol efflux (ChE) from macrophages is an initial step of reverse cholesterol transport (RCT). The ATP-binding cassette transporter A1 (ABCA1) is a key transporter for ChE and its increased expression is regarded to attenuate atherosclerosis. Thus, the identification and characterization of molecules raising ABCA1 and thereby stimulating ChE is of pharmacological relevance. In this study, we tested dietary compounds from olive oil for their capacity of enhancing cellular ABCA1 protein level. We identified erythrodiol (Olean-12-ene-3β,28-diol) as an ABCA1 stabilizer and revealed its positive influence on ChE in THP-1-derived human macrophages. Among the nine tested compounds from olive oil, erythrodiol was the sole compound raising ABCA1 protein level (at 10 μM). None of the tested compounds impaired viability of THP-1 macrophages from 5 to 20 μM as determined by resazurin conversion. Western blot analyses of key membrane transporters contributing to ChE showed that the protein level of ABCG1 and scavenger receptor class B member 1 (SR-B1) remain unaffected by erythrodiol. Besides, erythrodiol (10 μM) did not influence the mRNA level of ABCA1, ABCG1, and SR-B1, as determined by quantitative reverse transcription PCR, but significantly inhibited the degradation of ABCA1 as evident by an increased half-life of the protein in the presence of cycloheximide, an inhibitor of de novo protein synthesis. Therefore, erythrodiol promotes ChE from THP-1-derived human macrophages by stabilizing the ABCA1 protein. This bioactivity makes erythrodiol a good candidate to be further explored for therapeutic or preventive application in the context of atherosclerosis