Effects of roziglitazone and interactions with growth-regulating factors in ventricular cell hypertrophy

Chronic administration of thiazolidinediones might predispose to cardiac hypertrophy. The aim was to investigate direct effects of rosiglitazone in rat ventricular cardiomyocytes maintained in vitro (24 h). Rosiglitazone (=10-5 M) did not increase protein synthesis and produced small inconsistent increases in cellular protein. In the presence of serum (10% v/v), but not insulin-like growth factor (IGF-1, 10-8 M) or insulin (1 U/ml), an interaction with rosiglitazone to stimulate protein synthesis was observed. The hypertrophic responses to noradrenaline (5×10-6 M), PMA (10-7 M) and ET-1 (10-7 M) were not attenuated by rosiglitazone. Rosiglitazone (10-7 M) did not influence protein synthesis in response to insulin (1 U/ml) and elevated glucose (2.5×10-2 M) alone or in combination, but attenuated the increase in protein mass observed in response to elevated glucose alone. In re-differentiated cardiomyocytes, a model of established hypertrophy, rosiglitazone (10-8 M–10-6 M) increased protein synthesis. Together, these data indicate that rosiglitazone does not initiate cardiomyocyte hypertrophy directly in vitro. However, during chronic administration, the interaction of rosiglitazone with locally-derived growth-regulating factors may make a modest contribution to cardiac remodelling and influence the extent of compensatory hypertrophy of the compromised rat heart.

Quantifying Cell Binding Kinetics Mediated By Surface-Bound Blood Type B Antigen To Immobilized Antibodies

Cell adhesion is crucial to many biological processes, such as inflammatory responses, tumor metastasis and thrombosis formation. Recently a commercial surface plasmon resonance (SPR)-based BIAcore biosensor has been extended to determine cell binding mediated by surface-bound biomolecular interactions. How such cell binding is quantitatively governed by kinetic rates and regulating factors, however, has been poorly understood. Here we developed a novel assay to determine the binding kinetics of surface-bound biomolecular interactions using a commercial BIAcore 3000 biosensor. Human red blood cells (RBCs) presenting blood group B antigen and CM5 chip bearing immobilized anti-B monoclonal antibody (mAb) were used to obtain the time courses of response unit, or sensorgrams, when flowing RBCs over the chip surface. A cellular kinetic model was proposed to correlate the sensorgrams with kinetic rates. Impacts of regulating factors, such as cell concentration, flow duration and rate, antibody-presenting level, as well as pH value and osmotic pressure of suspending medium were tested systematically, which imparted the confidence that the approach can be applied to kinetic measurements of cell adhesion mediated by surface-bound biomolecular interactions. These results provided a new insight into quantifying cell binding using a commercial SPR-based BIAcore biosensor.

Surface-Bound Selectin-Ligand Binding Is Regulated By Carrier Diffusion

Two-dimensional (2D) kinetics of receptor-ligand interactions governs cell adhesion in many biological processes. While the dissociation kinetics of receptor-ligand bond is extensively investigated, the association kinetics has much less been quantified. Recently receptor-ligand interactions between two surfaces were investigated using a thermal fluctuation assay upon biomembrane force probe technique (Chen et al. in Biophys J 94:694-701, 2008). The regulating factors on association kinetics, however, are not well characterized. Here we developed an alternative thermal fluctuation assay using optical trap technique, which enables to visualize consecutive binding-unbinding transition and to quantify the impact of microbead diffusion on receptor-ligand binding. Three selectin constructs (sLs, sPs, and PLE) and their ligand P-selectin glycoprotein ligand 1 were used to conduct the measurements. It was indicated that bond formation was reduced by enhancing the diffusivity of selectin-coupled carrier, suggesting that carrier diffusion is crucial to determine receptor-ligand binding. It was also found that 2D forward rate predicted upon first-order kinetics was in the order of sPs > sLs > PLE and bond formation was history-dependent. These results further the understandings in regulating association kinetics of surface-bound receptor-ligand interactions.

Spring and autumn reproduction of Calanus sinicus in the Yellow Sea

The fecundity, hatching success and naupliar survival of Calanus sinicus were studied in the Yellow Sea during research cruises in April and October 2006, with emphasis on the regulation of reproduction. During both cruises, the egg production rate (EPR) showed large spatial variations (0 to 25.4 eggs female(-1) d(-1)), generally coinciding with the food availability. In April, the abundant phytoplankton and ciliates in the study area supported active reproduction, which would probably initiate the annual population development. In October, females remained immature in the Yellow Sea Cold Water Mass, likely due to the unfavorable environment (poor food and low temperature). However, reproduction and recruitment were high in the neritic region, which may explain the local population recovery in late autumn. Hatching success varied markedly among stations in April (4 to 85%), whereas it was high overall in the neritic region during October (> 90%). Based on the potential recruitment rates, the spring recruitment would be more important for the annual population dynamics, Female gonad maturity, body size and lipid reserve were examined in relation to fecundity. Regression analyses suggested that the reproductive index (defined as the proportion of females with mature gonads) could be an indicator for the EPR of C. sinicus. Among the regulating factors, external food (ciliates and phytoplankton) seems essential for reproduction, whereas inner lipid reserve may mainly serve metabolic needs. Moreover, fecundity is positively related to body size but independent of temperature, which might exert indirect influences on reproduction.

Brain Cholinergic and Dopaminergic Functions in Streptozotocin Induced Diabetic Rats: Effects of Curcumin and Vitamin D3 Supplementation

Department of Biotechnology, Cochin University of Science and Technology

Relative importance of transpiration rate and leaf morphological traits for the regulation of leaf temperature

Urban greening solutions such as green roofs help improve residents’ thermal comfort and building insulation. However, not all plants provide the same level of cooling. This is partially due to differences in plant structure and function, including different mechanisms that plants employ to regulate leaf temperature. Ranking of multiple leaf/plant traits involved in the regulation of leaf temperature (and, consequently, plants’ cooling ‘service’) is not well understood. We therefore investigated the relative importance of water loss, leaf colour, thickness and extent of pubescence for the regulation of leaf temperature, in the context of species for semi-extensive green roofs. Leaf temperature were measured with an infrared imaging camera in a range of contrasting genotypes within three plant genera (Heuchera, Salvia and Sempervivum). In three glasshouse experiments (each evaluating three or four genotypes of each genera) we varied water availability to the plants and assessed how leaf temperature altered depending on water loss and specific leaf traits. Greatest reductions in leaf temperature were closely associated with higher water loss. Additionally, in non-succulents (Heuchera, Salvia), lighter leaf colour and longer hair length (on pubescent leaves) both contributed to reduced leaf temperature. However, in succulent Sempervivum, colour/pubescence made no significant contribution; leaf thickness and water loss rate were the key regulating factors. We propose that this can lead to different plant types having significantly different potentials for cooling. We suggest that maintaining transpirational water loss by sustainable irrigation and selecting urban plants with favourable morphological traits is the key to maximising thermal benefits provided by applications such as green roofs.

Interference of the blood clot on granulation tissue formation after tooth extraction. Histomorphological study in rats.

The interference of a blood clot in the first postoperative hours of dental extraction wounds was studied in rats. Sixty male albino rats were divided into two groups: Group I, immediately after extraction of right maxillary incisor the gingival mucosa was approximated and sutured; Group II, after 6 to 8 minutes postoperatively the blood clot was removed with saline irrigation and absorbent paper cones. The mucosa was then approximated and sutured. Six animals in each group were sacrificed after 12 hours, 1, 4, 7 and 10 days. There was a profound delay in healing in Group II since, although a new blood clot was later formed, it was not organized. The quality and the constitution, maintenance and retraction of the clot are the regulating factors in connective tissue formation during alveolar healing.

Modelo cinético estocástico para a transcrição considerando colisões entre as moléculas de RNA polimerase

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Physiological Studies on Candida albicans

Candida albicans is a common opportunistic, dimorphic human fungal pathogen. One of its virulence factors is the morphological switch between yeasts and hyphal or pseudohyphal forms, which can invade tissues and cause damage. Our studies focus on factors regulating pseudohyphae and epigenetic modifications of C. albicans. Regulating factors of pseudohyphae are aromatic alcohols and high phosphate. At low concentrations, exogenous aromatic alcohols induced pseudohyphae, as did high phosphate. For addressing the pathways involved in inducing pseudohyphae by aromatic alcohols or high phosphate, we used mutants defective in cAMP dependent PKA pathway (efg1/efg1), MAP kinase pathway (cph1/cph1), or both (cph1/cph1/efg1/efg1). These mutants failed to produce either hyphae or pseudohyphae in the presence of aromatic alcohols; but high phosphate still stimulated pseudohyphae. Gcn4, a transcription activator of more than 500 amino acid related genes, is turned-on in response to amino acid starvation. The accumulation of aromatic alcohols sends nitrogen starvation signals, which inhibit eIF2B, which in turn derepresses Gcn4p. High phosphate also induces pseudohyphae by derepressing Gcn4p, although the pathways involved are still unknown. In sum, aromatic alcohols and high phosphate induce pseudohyphae by derepressing Gcn4. In this study we found a novel posttranslational histone modification in C. albicans, which is biotinylation. Western blot and Mass spectrometry techniques were used to find that Histones H2B and H4 were biotinylated at every condition tested such as yeast vs. hyphae, aerobic growth vs. anaerobic growth, rich medium vs. defined medium. In C. albicans lysines K8, K11 in histone H4 and lysines K17, K18, K31 in histone H2B are biotin attachment sites as found using mass spectrometry. Biotin was also found to enhance the germ tube formation of C. albicans. Germ tube formation assays with biotin-starved cells as inoculum showed low percent of germ tubes (1-5%). Addition of biotin to the media showed 100% germ tubes. Biotinylation of histones were not detected from biotin-starved cells. Appendix-A details work related to Farnesol quantification assays in several strains of C.albicans and Ceratocystis ulmi, and growth studies of class E VPS strains of Saccharomyces Cerevisiae. Adviser: Kenneth W. Nickerson

Oxylipins biosynthesis in Lactobacillus helveticus in the presence of oxidative stress

This PhD thesis is aimed at studying the possible pathways and the mechanisms that can trigger oxylipins biosynthesis, and particularly that of short chain aldehydes and alcohols, in Lactobacillus helveticus, also in the presence of oxidative stress, using a totally labelled linoleic acid as precursor. In plants and fungi these molecules, involved in defence mechanisms against pathogens and in communication systems, derive from the oxidation of cellular unsaturated fatty acids (UFAs) and their accumulation is associated with stress exposure. Since some oxylipins are produced also by lactobacilli, it is possible to hypothesize that a metabolic pathway from UFAs to oxylipins, similar to what happens in plants and fungi, is present also in lactic acid bacteria. The results obtained pointed out that some volatile molecules are the result of UFAs catabolism, since they appear only when cells are incubated in their presence. Labelled linoleic acid is integrated in the membrane and subsequently transformed into aldehydes and alcohols, whose extent and carbon atoms number depend on stress exposure. The enzymes responsible for this metabolic pathway in plants and fungi (e.g. lipoxygenase, dioxygenase) seem to be absent in Lactobacillus helveticus and in other lactobacilli. Proteomic analyses show the over expression of many proteins, including thioredoxin reductase (part of the bacterial oxidative defence system), mainly in cells grown with linoleic acid without oxidative stress exposure, confirming that linoleic acid itself induces oxidative stress. 6 general oxidoreductases (class including dioxygenases and peroxidase) were found and therefore a deeper investigation on them could be productive in elucidating all steps involved in oxylipins biosynthesis in bacteria. Due to the multiple role of oxylipins (flavouring agents, antimicrobial compounds and interspecific signalling molecules) the identification of genes involved and regulating factors should have an important biotechnological impact, also allowing the overproduction of selected bioactive molecules.

Bioinformatische, genomweite Analyse genexpressionsregulierender Faktoren in Lebergewebe

Für eine Reihe einzelner genetischer Faktoren und Promotorelemente wurde in der Vergangenheit eine Regulation der Genexpression in der Leber (und auch in anderen Geweben) gezeigt. Mit der Verfügbarkeit des gesamten humanen Genoms sowie dessen Expressionsdaten in großen Microarray- und SAGE-Datenbanken bietet sich die Möglichkeit, solche Regulationsmechanismen in großem, genomweitem Maßstab zu untersuchen. Dabei geht diese Arbeit der Frage nach, ob es übergeordnete, eine Expression speziell in der Leber fördernde oder hemmende Faktoren gibt oder ob jedes Gen von einer unabhängigen Kombination von Faktoren reguliert wird, in dessen Summe die Expression des individuellen Gens in der Leber am stärksten ist. Sollten sich übergeordnete, eine Expression in der Leber stimulierende Faktoren finden, wären diese interessant für die Entwicklung neuer Behandlungskonzepte bei Lebererkrankungen. Zur Untersuchung dieser Fragestellung wurden aus einem Affymetrix Microarray Datenset für 12 Gewebe die Expressiondaten von insgesamt jeweils 15.472 Genen extrahiert. In einem zweiten Schritt wurden zusätzlich die Promotorsequenzen der einzelnen zugehörigen Gene, definiert als eine 1000 bp Region upstream des Transkriptionsstarts, in dieselbe Datenbank abgelegt. Die Promotorsequenzen wurden über den PromotorScan-Algorithmus analysiert. Auf diese Weise wurden Transkriptionsfaktorbindungsstellen auf 7042 der Promotoren identifiziert. Es fand sich eine Gesamtzahl von 241.984 Transkriptionsfaktorbindungsstellen. Anhand der Microarray-Expressionsdaten wurde die Gesamtgruppe der verfügbaren Gene und Promotoren in zwei Gruppen unterteilt, nämlich in die Gruppe der Gene, deren Expression in der Leber deutlich am höchsten gefunden wurde und in die Gruppe der Gene, die in anderen Geweben am höchsten exprimiert waren. Jeder potentiell bindende Transkriptionsfaktor wurde auf unterschiedliches Vorkommen in diesen beiden Gruppen hin untersucht. Dies geschah unter der Vorstellung, dass übergeordnete Faktoren, die eine Expression in der Leber stimulieren in der Gruppe der Gene, die in der Leber am höchsten exprimiert sind, verhältnismäßig wesentlich häufiger zu finden sein könnten. Eine solches häufigeres Vorkommen ließ sich jedoch für keinen einzigen Faktor nachweisen. Transkriptionsfaktorbindungsstellen sind typischerweise zwischen 5 und 15 bp lang. Um auszuschließen, dass mit dem verwendeten PromotorScan-Algorithmus Transkriptionsfaktorbindungsstellen, die bisher nicht bekannt sind, nicht übersehen wurden, wurden die Häufigkeit sämtlicher möglicher 8 bp (48) und 10 bp (410) Nukleotid-Kombinationen in diesen Promotoren untersucht. Biologisch relevante Unterschiede fanden sich zwischen den beiden Gruppen nicht. In gleicher Weise wurde auch die Bedeutung von TATA-Boxen untersucht. TATA-Boxen kommt bei der Transkriptionsinitiierung eine wichtige Rolle zu, indem über sie die Bindung des initialen Transkriptionskomplexes vermittelt wird. Insgesamt 1033 TATA-Boxen wurden ebenfalls mittels PromotorScan vorausgesagt. Dabei waren 57 auf Promotoren von Genen, die in der Leber überexprimiert waren und 976 auf Promotoren von Genen, die in anderen Geweben überexprimiert waren. Der Vergleich dieser beiden Gruppen ließ keine signifikant unterschiedliche Häufigkeit an TATA-Boxen erkennen. Im weiteren wurde die Bedeutung von CpG-Islands für eine potentiell differentielle Regulation untersucht. Insgesamt wurden 8742 CpG-Islands in einem Bereich von bis zu 5 kb upstream des Transkriptionsstarts identifiziert, 364 davon auf Promotoren von Genen, die am höchsten in der Leber exprimiert waren, 8378 auf Promotoren von Genen, die in anderen Geweben am höchsten exprimiert waren. Signifikante Unterschiede in der Verteilung von CpG-Islands auf Promotoren dieser beiden Gengruppen ließen sich nicht nachweisen. Schließlich wurden die RNA- und Proteinsequenzen des Transkriptoms und Proteoms hinsichtlich ihrer Zusammensetzung aus einzelnen Nukleotiden bzw. Aminosäuren analysiert. Auch hierbei fanden sich keine signifikanten Unterschiede in der Verteilung zwischen beiden Gengruppen. Die Zusammenschau der Ergebnisse zeigt, dass die Regulation der einzelnen Gene im Lebergewebe im wesentlichen individuell erfolgt. Im Rahmen der vorgelegten bioinformatischen Analysen fanden sich keine übergeordneten genetischen „Leberfaktoren“, die speziell eine Expression von Genen in der Leber stimulieren. Neue therapeutische Ansätze, die auf eine Regulation der Genexpression in der Leber zielen, werden somit auch weiterhin auf die Beeinflussung individueller Gene fokussiert bleiben.

Role of outgrowth endothelial cells for applications in tissue engineering

Co-culture systems, consisting of outgrowth endothelial cells (OEC) and primary osteoblasts (pOB), represent a prom¬ising instrument to mimick the natural conditions in bone repair processes and provide a new concept to develop constructs for bone replacement. Furthermore, co-culture of OEC and pOB could provide new insights into the molecular and cellular mechanisms that control essential processes during bone repair. The present study described several advantages of the co-culture of pOB and OEC for bone tissue engineering applications, including beneficial effects on the angiogenic activation of OEC, as well as on the assembly of basement membrane matrix molecules and factors involved in vessel maturation and stabilization. The ongoing angiogenic process in the co-culture system proceeded during the course of co-cultivation and correlated with the upregulation of essential angiogenic factors, such as VEGF, angiopoietins, basement membrane molecules and mural cell-specific markers. Furthermore the co-culture system appeared to maintain osteogenic differentiation capacity.rnrnAdditional treatment of co-cultures with growth factors or morphogens might accelerate and improve bone formation and furthermore could be useful for potential clinical applications. In this context, the present study highlights the central role of the morphogen, sonic hedgehog, which has been shown to affect angiogenic activation as well as osteogenic differentiation in the co-culture model of OEC and pOB. Treatment of co-cultures with sonic hedgehog resulted in an increased formation of microvessel-like structures as early as after 24 hours. This proangiogenic effect was induced by the upregulation of the proangiogenic factors, VEGF, angiopoietin1 and angiopoietin 2. In contrast to treatment with a commonly used proangiogenic agent, VEGF, Shh stimulation induced an increased expression of factors associated with vessel maturation and stabilization, mediated through the upregulation of growth factors that are strongly involved in pericyte differentiation and recruitment, including PDGF-BB and TGFbeta. In addition, Shh treatment of co-cultures also resulted in an upregulation of osteogenic differentiation markers like alkaline phosphatase, osteocalcin, osteonectin and osteopontin, as well as an increased matrix calcification. This was a result of upregulation of the osteogenic differentiation regulating factors, BMP2 and RUNX2 which could be assessed in response to Shh treatment. rn

Die Regulation des Enzyms Paraoxonase-2 sowie dessen Einfluss auf die Hämatopoese

In der vorliegenden Arbeit wurden durch den Einsatz von drei unabhängigen Methoden Proteine und Faktoren identifiziert, die die PON2-mRNA-Expression beeinflussen. Anhand der erhaltenen Faktoren wurden verstärkt solche ausgewählt, die eine Rolle in der Tumorbiologie spielen. Unter Verwendung verschiedener Zellmodelle wurde schließlich der Effekt dieser Faktoren auf die PON2-Expression analysiert. Die Ergebnisse dieser Arbeit zeigen, dass die PON2-Expression in K562-Zellen durch den PI3K / Akt-Signalweg, der in vielen Tumoren übermäßig aktiviert vorliegt, gesteigert wird. Auch eine Beteiligung des Wnt / β-Catenin-Signalweges kann nicht ausgeschlossen werden. Pharmakologische Inhibitoren von GSK-3β, einer Kinase die in beiden Signalwegen involviert ist, führt zu einer Steigerung der PON2-Expression durch den Transkriptionsfaktor LEF-1. Des Weiteren wurde gezeigt, dass die Familie der FoxO-Transkriptionsfaktoren an der Regulation der PON2-Expression in K562-Zellen beteiligt sind, wenn gleich es für die jeweiligen FoxO-Isoformen Unterschiede gibt.rnIm Hinblick auf die Assoziation von PON2 mit Leukämien wurde anhand eines PON2-/--Mausmodells, der Einfluss von PON2 auf die Hämatopoese untersucht. Dabei wurden signifikante Unterschiede in einigen Stammzellkompartimenten festgestellt. Ferner scheint PON2 die Entwicklung von Erythrozyten und Thrombozyten zu beeinflussen. Dies äußert sich in einer offensichtlichen Splenomegalie, zumindest bei alten weiblichen PON2-/--Mäusen.rnAbschließend wurde zur Generierung eines konditionalen PON2-Überexpressionsmausmodells erfolgreich ein Gene-Targeting-Vektor entwickelt. Durch eine gewebe-, zeit- und zellspezifische Steigerung der PON2-Expression ist es möglich, den Effekt einer PON2-Überexpression im Hinblick auf verschiedene Erkrankungen zu untersuchen.rnBisher war wenig über die Regulation des humanen PON2 bekannt. Die Ergebnisse der vorliegenden Arbeit zeigen erstmals, durch welche Signalwege und Transkriptionsfaktoren PON2 in Leukämiezellen reguliert wird. Im Hinblick auf die Rolle von PON2 in der Tumorbiologie ist es erstmals möglich, die PON2-Expression gezielt durch die Inhibition bzw. Aktivierung der PON2-regulierenden Faktoren zu beeinflussen, und damit neue Wege in der Krebstherapie zu beschreiten. rn

Reproductive investment and seedling survival of the mast-fruiting rain forest tree, Microberlinia bisulcata A. chev

In the southern part of Korup National Park, Cameroon, the mast fruiting tree Microberlinia bisulcata occurs as a codominant in groves of ectomycorrhizal Caesalpiniaceae within a mosaic of otherwise species-rich lowland rain forest. To estimate the amount of carbon and nutrients invested in reproduction during a mast fruiting event, and the consequential seed and seedling survival, three related field studies were made in 1995. These provided a complete seed and seedling budget for the cohort. Seed production was estimated by counting woody pods on the forest floor. Trees produced on average 26,000 (range 0-92,000) seeds/tree, with a dry mass of 16.6 kg/tree. Seeds were contained in woody pods of mass 307 kg/tree. Dry mass production of pods and seeds was 1034 kg ha(-1), equivalent to over half (55%) of annual leaf litterfall for this species, and contained 13% of the nitrogen and 21% of the phosphorus in annual leaf litterfall. Seed and young-seedling mortality was investigated with open quadrats and cages to exclude vertebrate predators, at two distances from the parent tree. The proportion of seeds on the forest floor which disappeared in the first 6 wk after dispersal was 84%, of which 26.5% was due to likely vertebrate removal, 36% to rotting, and 21.5% to other causes. Vertebrate predation was greater close to the stem than 5 m beyond the crown (41 vs 12% of seeds disappearing) where the seed shadow was less dense. Previous studies have demonstrated an association between mast years at Korup and high dry-season radiation before flowering, and have shown lower leaf-litterfall phosphorus concentrations following mast fruiting. The emerging hypothesis is that mast fruiting is primarily imposed by energy limitation for fruit production, but phosphorus supply and vertebrate predation are regulating factors. Recording the survival of naturally-regenerating M. bisulcata seedlings (6-wk stage) showed that 21% of seedlings survived to 31 mo. A simple three-stage recruitment model was constructed. Mortality rates were initially high and peaked again in each of the next two dry seasons, with smaller peaks in the two intervening wet seasons, these latter coinciding with annual troughs in radiation. The very poor recruitment of M. bisulcata trees in Korup, demonstrated in previous investigations, appears not to be due to a limitation in seed or young-seedling supply, but rather by factors operating at the established-seedling stage.