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- PublicationExpression and tissue distribution of the mRNAs encoding the human thromboxane A2 receptor (TP) alpha and beta isoformsThe human (h) TXA2 receptor (TP), a G protein-coupled receptor, exists as two isoforms, TPalpha and TPbeta, which arise by alternative mRNA splicing and differ exclusively in their carboxyl terminal cytoplasmic regions. In this study, a reverse transcriptase - polymerase chain reaction (RT-PCR) based strategy was developed to examine the expression of the TPs in tissues of physiologic relevance to TXA2. Although most of the 17 different cell / tissue types examined expressed both TP isoforms, the liver hepatoblastoma HepG2 cell line was found to exclusively express TPalpha mRNA. In most cell types, TPalpha mRNA predominated over TPbeta mRNA. Moreover, although the levels of TPalpha mRNA expression were similar in most of the cell / tissue types examined, extensive differences in the levels of TPbeta mRNA were observed. Consequently, the relative expression of TPalpha : TPbeta mRNA varied considerably due to extensive differences in TPbeta mRNA expression. Most strikingly, primary HUVEC’s were found to express: (i) low levels of TPbeta and (ii) approximately 6- fold greater levels of TPalpha than TPbeta . These data were confirmed in the spontaneously transformed HUVEC derived ECV304 cell line. Expression of TP mRNAs in the various tissue / cells correlated with protein expression, as assessed by radioligand binding using the selective TP antagonist [3H] SQ29,548.
421Scopus© Citations 110 - PublicationInvestigation of the role of the carboxyl terminal tails of the alpha and beta isoforms of the human thromboxane A2 receptor (TP) in mediating receptor : effector couplingWe have investigated the functional coupling of alpha and beta isoforms of the human thromboxane A2 receptor (TP) to Galpha16 and Galpha12 members of the Gq and G12 families of heterotrimeric G proteins in human embryonic kidney (HEK) 293 cell lines HEK.alpha10 or HEK.beta3, stably over-expressing TPalpha and TPbeta, respectively. Moreover, using HEK.TP-328 cells which over-expresses a variant of TP truncated at the point of divergence of TPalpha and TPbeta we investigated the requirement of the C-tail per se in mediating G protein coupling and effector activation. Both TPalpha and TPbeta couple similarly to Galpha16 to affect increases in IP3 and mobilization of intracellular calcium ([Ca2+]i) in response to the TP agonist U46619. Whilst both TP isoforms mediated [Ca2+]i mobilization in cells co-transfected with Galpha12, neither receptor generated corresponding increases in IP3 indicating that the Galpha12 mediated increases in [Ca2+]i do not involve PLC activation. Verapamil, an inhibitor of voltage dependent Ca2+ channels reduced [Ca2+]i mobilization in TPalpha and TPbeta cells co-transfected with Galpha12 to approximately 40% of that mobilized in its absence whereas 3,4,5-trimethyloxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), an antagonist of intracellular Ca2+ release, had no effect on [Ca2+]i mobilization by either receptor isoform co-transfected with Galpha12. Despite the lack of differential coupling specificity by TPalpha and TPbeta, TP-328 signaled more efficiently in the absence of a co-transfected G protein compared to the wild type receptors but, on the other hand, displayed an impaired ability to couple to co-transfected Galpha11, Galpha12 or Galpha16 subunits. In studies investigating the role of the C-tail in influencing coupling to the effector adenylyl cyclase, similar to TPalpha but not TPbeta, TP-328 coupled to GalphaS, leading to increased cAMP, rather than to Galphai. Whereas TP-328 signaled more efficiently in the absence of co-transfected G protein compared to the wild type TPalpha co-transfection of Galphas did not augment cAMP generation by TP-328. Hence, from these studies involving the wild type TPalpha, TPbeta and TP-328, we conclude that the C-tail sequences of TP are not a major determinant of G protein coupling specificity to Galpha11 and Galpha16 members of the Gq family or to Galpha12; it may play a role in determining GS versus Gi coupling and may act as a determinant of coupling efficiency.
270Scopus© Citations 44 - PublicationThromboxane A2 signalling in humans : a ‘tail’ of two receptorsSince its discovery in 1975, we now have a wealth of knowledge relating to the biochemical, pharmacological and physiologic actions of thromboxane (TX) A2 and its related metabolites. These molecular insights have been greatly expedited by the molecular cloning and characterisation of a complementary (c) DNA for the human TXA receptor, now termed T Prostanoid or TP receptor, from a megakaryocytic / placental cDNA library in 1991 and later through the discovery of a cDNA encoding a second isoform of the human TP receptor in 1994. The requirement for two TP receptors in primates, but not in other species thus far investigated, is unclear but points to potential species-specific physiologic differences. In this review, I will describe some recent advances in the research field of TXA2/TP receptor signalling, focussing particularly on studies pertaining to the human TP receptor isoforms.
769Scopus© Citations 84 - PublicationThromboxane A2 receptor mediated activation of the mitogen activated protein kinase cascades in human uterine smooth muscle cellsBoth thromboxane (TX) A2 and 8-epi prostaglandin (PG) F2alpha have been reported to stimulate mitogenesis of vascular smooth muscle (SM) in a number of species. However, TXA2 and 8-epiPGF2alpha mediated mitogenic signalling have not been studied in detail in human vascular SM. Thus, using the human uterine ULTR cell line as a model, we investigated TXA2 receptor (TP) mediated mitogenic signalling in cultured human vascular SM cells. Both the TP agonist U46619 and 8-epiPGF2alpha elicited time and concentration dependent activation of the extracellular signal regulated kinase (ERK)s and c-Jun N-terminal kinase (JNK)s in ULTR cells. Whereas the TP antagonist SQ29,548 abolished U46619-mediated signalling, it only partially inhibited 8-epiPGF2alpha mediated ERK and JNK activation in ULTR cells. Both U46619 and 8-epiPGF2alpha induced ERK activations were inhibited by the protein kinase (PK) C, PKA and phosphoinositide 3-kinase inhibitors GF 109203X, H-89 and wortmannin, respectively, but were unaffected by pertussis toxin. In addition, U46619 mediated ERK activation in ULTR cells involves transactivation of the EGF receptor. In humans, TXA2 signals through two distinct TP isoforms. In investigating the involvement of the TP isoforms in mitogenic signalling, both TPalpha and TPbeta, independently directed U46619 and 8-epiPGF2alpha mediated ERK and JNK activation in human embryonic kidney (HEK) 293 cells over-expressing the individual TP isoforms. However, in contrast to that which occurred in ULTR cells, SQ29,548 abolished 8-epiPGF2alpha mediated ERK and JNK activation through both TPalpha and TPbeta in HEK 293 cells providing further evidence that 8-epiPGF2alpha may signal through alternative receptors, in addition to the TPs, in human uterine ULTR cells.
315Scopus© Citations 68 - PublicationProstaglandin D2 receptor-mediated desensitization of the alpha isoform of the human thromboxane A2 receptorThromboxane (TX) A2 and prostaglandin (PG) D2 mediate opposing actions in platelets and in vascular and non-vascular smooth muscle. Here, we investigated the effects of stimulation of the PGD2 receptor (DP) on signaling by the TXA2 receptor (TP) expressed in human platelets and in human embryonic kidney (HEK) 293 cells over-expressing the individual TPalpha and TPbeta isoforms. In platelets, the selective DP agonist BW245C abolished TP-mediated mobilization of intracellular calcium ([Ca2+]i) and inhibited platelet aggregation in response to the TXA2 mimetic U46619. DP-mediated desensitization of TP signaling in platelets was prevented by pre-treatment with the cAMP-dependent PKA inhibitor, H-89, but was unaffected by the PKC inhibitor GF 109203X. In HEK 293 cells signaling by TPalpha, but not TPbeta, was subject to DP mediated desensitization in a PKA dependent, PKC independent manner. U46619-induced signaling by TP-328, a truncated variant of TP containing only those residues common to TPalpha and TPbeta, was insensitive to prior DP stimulation indicating that the carboxyl terminal tail of TPalpha contains the target site(s) for DP-mediated desensitization. Mutation of Ser329 to Ala329 within a consensus PKA site in TPalpha rendered the mutant TPalphaS329A insensitive to BW245C-mediated desensitization. Whole cell phosphorylation assays established that TPalpha, but not TPbeta or TPalphaS329A, was subject to DP-mediated phosphorylation and that TPalpha phosphorylation was blocked by the PKA inhibitor H-89. These data establish that TPalpha, but not TPbeta, is subject to DP mediated cross desensitization, which occurs through direct PKA mediated phosphorylation of TPalpha at Ser329.
260Scopus© Citations 29 - PublicationThe role of N-linked glycosylation in determining the surface expression, G protein interaction and effector coupling of the alpha isoform of the human thromboxane A2 receptorIn humans, thromboxane (TX) A2 signals through two TXA2 receptor (TP) isoforms, termed TPalpha and TPbeta, that diverge exclusively within their carboxyl terminal cytoplasmic domains. The amino terminal extracellular region of the TPs contains two highly conserved Asn (N)-linked glycosylation sites at Asn4 and Asn16. Whilst it has been established that impairment of N-glycosylation of TPalpha significantly affects ligand binding/intracellular signalling, previous studies did not ascertain whether N-linked glycosylation was critical for ligand binding per se or whether it was required for the intracellular trafficking and the functional expression of TPalpha on the plasma membrane (PM). In the current study, we investigated the role of N-linked glycosylation in determining the functional expression of TPalpha, by assessment of its ligand binding, G-protein coupling and intracellular signalling properties, correlating it with the level of antigenic TPalpha protein expressed on the PM and/or retained intracellularly. From our data, we conclude that N-glycosylation of either Asn4 or Asn16 is required and sufficient for expression of functionally active TPalpha on the PM while the fully non-glycosylated TPalphaN4,N16-Q4,Q16 is almost completely retained within the endoplasmic reticulum and remains functionally inactive, failing to associate with its coupling G protein Galphaq and, in turn, failing to mediate phospholipase Cbeta activation.
445Scopus© Citations 23 - PublicationSynthetic peroxisome proliferator-activated receptor gamma agonists rosiglitazone and troglitazone suppress transcription by promoter 3 of the human thromboxane A2 receptor gene in human erythroleukemia cellsThe human thromboxane (TX)A2 receptor (TP) gene encodes two TP isoforms, TPalpha and TP beta that are regulated by distinct promoters designated promoter (Prm) 1 and Prm3, respectively. Previous studies established that 15d-delta 12,14-prostaglandin J2 (15d-PGJ2) selectively inhibits Prm3 activity and TP beta expression through a peroxisome proliferator-activated receptor (PPAR)gamma mechanism without affecting Prm1 activity or TPalpha expression in human megakaryocytic erythroleukemia (HEL) 92.1.7 cells. Herein, we investigated the effect of synthetic thiazolidinedione (TZD) PPARgamma ligands rosiglitazone and troglitazone on TP gene expression in HEL cells. Like 15d-PGJ2, both TZDs suppressed Prm3 activity, TPbeta mRNA expression and TP-mediated calcium mobilization without affecting Prm1 or TPalpha mRNA expression. However, unlike 15d-PGJ2, both TZDs mediated their PPARgamma-dependent effects through trans-repression of an activator protein-1 (AP-1) element, a site previously found to be critical for basal Prm3 activity. These data provide further evidence for the role of PPARgamma in regulating the human TP gene; they highlight further differences in TPalpha and TPbeta expression/regulation and point to essential differences between natural and synthetic PPARgamma agonists in mediating those effects.
674 - PublicationHomologous desensitization of signalling by the beta isoform of the human thromboxane A2 receptorThromboxane (TX) A2 is a potent stimulator of platelet activation/aggregation and smooth muscle contraction and contributes to a variety of pathologies within the vasculature. In this study, we investigated the mechanism whereby the cellular responses to TXA2 mediated through the TPbeta isoform of the human TXA2 receptor (TP) are dynamically regulated by examining the mechanism of agonist-induced desensitization of intracellular signalling and second messenger generation by TPbeta. It was established that TPbeta is subject to profound agonist-induced homologous desensitization of signalling (intracellular calcium mobilization and inositol 1,3,5 trisphosphate generation) in response to stimulation with the TXA2 mimetic U46619 and this occurs through two key mechanisms: TPbeta undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, protein kinase (PK)C mechanism whereby Ser145 within intracellular domain (IC)2 has been identified as the key phospho-target. In addition, TPbeta also undergoes more profound and sustained agonist-induced desensitization involving G protein-coupled receptor kinase (GRK)2/3-phosphorylation of both Ser239 and Ser357 within its IC3 and carboxyl-terminal C-tail domains, respectively. Inhibition of phosphorylation of either Ser239 or Ser357, through site directed mutagenesis, impaired desensitization while mutation of both Ser239 and Ser357 almost completely abolished desensitization of signalling, GRK phosphorylation and beta-arrestin association, thereby blocking TPbeta internalization. These data suggest a model whereby agonist-induced PKC phosphorylation of Ser145 partially impairs TPbeta signalling while GRK2/3 phosphorylation at both Ser239 and Ser357 within its IC3 and C-tail domains, respectively, sterically inhibits G-protein coupling, profoundly desensitizing signalling, and promotes beta-arrestin association and, in turn, facilitates TPbeta internalization.
151Scopus© Citations 26 - PublicationIn Silico Promoter Analysis can Predict Genes of Functional Relevance in Cell Proliferation: Validation in a Colon Cancer ModelSpecific combinations of transcription-factor binding sites in the promoter regions of genes regulate gene expression, and thus key functional processes in cells. Analysis of such promoter regions in specific functional contexts can be used to delineate novel disease-associated genes based on shared phenotypic properties. The aim of this study was to utilize promoter analysis to predict cell proliferation-associated genes and to test this method in colon cancer cell lines. We used freely-available bioinformatic techniques to identify cell-proliferation-associated genes expressed in colon cancer, extract a shared promoter module, and identify novel genes that also contain this module in the human genome. An EGRF/ETSF promoter module was identified as prevalent in proliferation-associated genes from a colon cancer cDNA library. We detected 30 other genes, from the known promoters of the human genome, which contained this proliferation-associated module. This group included known proliferation-associated genes, such as HERG1 and MCM7, and a number of genes not previously implicated in cell proliferation in cancer, such as TSPAN3, Necdin and APLP2. Suppression of TSPAN3 and APLP2 by siRNA was performed and confirmed by RT-PCR. Inhibition of these genes significantly inhibited cell proliferation in colon cancer cell lines. This study demonstrates that promoter analysis can be used to identify novel cancer-associated genes based on shared functional processes.
24 - PublicationExpression of genes for bone morphogenetic proteins BMP-2, BMP-4 and BMP-6 in various parts of the human skeleton(BioMed Central, 2007-12-27)
; ; ; ; ; ; BACKGROUND: Differences in duration of bone healing in various parts of the human skeleton are common experience for orthopaedic surgeons. The reason for these differences is not obvious and not clear.METHODS: In this paper we decided to measure by the use of real-time RT-PCR technique the level of expression of genes for some isoforms of bone morphogenetic proteins (BMPs), whose role is proven in bone formation, bone induction and bone turnover. Seven bone samples recovered from various parts of skeletons from six cadavers of young healthy men who died in traffic accidents were collected. Activity of genes for BMP-2, -4 and -6 was measured by the use of fluorescent SYBR Green I.RESULTS: It was found that expression of m-RNA for BMP-2 and BMP-4 is higher in trabecular bone in epiphyses of long bones, cranial flat bones and corpus mandibulae then in the compact bone of diaphyses of long bones. In all samples examined the expression of m-RNA for BMP-4 was higher than for BMP-2.CONCLUSION: It was shown that m-RNA for BMP-6 is not expressed in the collected samples at all. It is postulated that differences in the level of activation of genes for BMPs is one of the important factors which determine the differences in duration of bone healing of various parts of the human skeleton.253Scopus© Citations 32 - PublicationMetabotropic Receptor-Activated Calcium Increases and Store-Operated Calcium Influx in Mouse Müller Cells(Association for Research in Vision and Opthalmology, 2008-07)
; ; ; Purpose: Metabotropic receptor agonists that signal through Gq-coupled pathways increase Ca2+ in mammalian Müller cells by release from intracellular stores and Ca2+ influx pathways that have not been well described. The authors examined the involvement of voltage-dependent and non–voltage-dependent Ca2+ channels in metabotropic muscarinic receptor-activated Ca2+ increases and store-operated Ca2+ influx in cultured mouse Müller cells. Methods: Intracellular Ca2+ was measured using fluorescence imaging with the ratiometric dye fura-2. Currents were recorded using the whole-cell patch-clamp recording method. mRNA and protein were identified using reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemical approaches. Results: The muscarinic receptor agonist carbachol (3–20 μM) produced increases in Ca2+ that were blocked by the muscarinic receptor antagonists atropine and pirenzepine. RT-PCR confirmed mRNA for metabotropic M1 muscarinic receptors. Depletion of Ca2+ stores by the sarcoplasmic/endoplasmic Ca2+ ATPase (SERCA) inhibitors thapsigargin and cyclopiazonic acid or the inhibition of phospholipase C occluded the carbachol-activated increase in Ca2+. Carbachol-activated Ca2+ increases in Müller cells were enhanced by the diacylglycerol derivative 1-oleyl-2-acetyl-sn-glycerol and were blocked by transient receptor potential (TRP) channel blockers Gd3+, La3+, 2-APB, and flufenamic acid. Both muscarinic receptor activation and thapsigargin treatment depleted Ca2+ stores and produced Ca2+ entry that was attenuated by La3+, 2-APB, Gd3+, and flufenamic acid. mRNA and protein for TRPC1 and TRPC6 were present in mouse Müller cells, and carbachol activated a Gd3+-sensitive, TRP-like cation channel. Conclusions: Metabotropic muscarinic receptor-activated Ca2+ increases in mouse Müller cells require the release of Ca2+ from intracellular stores and the activation of Ca2+ entry that involves TRP-like cation channels but is independent of voltage-dependent Ca2+ channels.427Scopus© Citations 21 - PublicationDifferential regulation of RhoA-mediated signaling by the TPalpha and TPbeta isoforms of the human thromboxane A2 receptor : independent modulation of TPalpha signaling by prostacyclin and nitric oxide(Elsevier, 2008-08)
; ; ; In humans, thromboxane (TX) A2 signals through the TPalpha and TPbeta isoforms of the TXA2 receptor that exhibit common and distinct roles. For example, Gq/phospholipase (PL)Cbeta signaling by TPalpha is directly inhibited by the vasodilators prostacyclin and nitric oxide (NO) whereas that signaling by TPbeta is unaffected. Herein, we investigated whether TPalpha and/or TPbeta regulate G12/Rho activation and whether that signaling might be differentially regulated by prostacyclin and/or NO. Both TPalpha and TPbeta independently regulated RhoA activation and signaling in clonal cells over-expressing TPalpha or TPbeta and in primary human aortic smooth muscle cells (1o AoSMCs). While RhoA- signaling by TPalpha was directly impaired by prostacyclin and NO through protein kinase (PK)A- and PKG-dependent phosphorylation, respectively, signaling by TPbeta was not directly affected by either agent. Collectively, while TPalpha and TPbeta contribute to RhoA activation, our findings support the hypothesis that TPalpha is involved in the dynamic regulation of haemostasis and vascular tone, such as in response to prostacyclin and NO. Conversely, the role of TPbeta in such processes remains unsolved. Data herein provide essential new insights into the physiologic roles of TPalpha and TPbeta and, through studies in AoSMCs, reveal an additional mode of regulation of VSM contractile responses by TXA2.551Scopus© Citations 31 - PublicationRecycling of the human prostacyclin receptor is regulated through a direct interaction with Rab11a GTPase(Elsevier, 2008-12)
; ; ; ; ; ; The human prostacyclin receptor (hIP) undergoes agonist-induced internalization but the mechanisms regulating its intracellular trafficking and/or recycling to the plasma membrane are poorly understood. Herein, we conducted a yeast-two-hybrid screen to identify proteins interacting with the carboxyl terminal (C)-tail domain of the hIP and discovered a novel interaction with Rab11a. This interaction was confirmed by co-immunoprecipitations in mammalian HEK293 and was augmented by cicaprost stimulation. The hIP co-localized to Rab11-containing recycling endosomes in both HEK293 and endothelial EA.hy 926 cells in a time dependent manner following cicaprost stimulation. Moreover, over-expression of Rab11a significantly increased recycling of the hIP, while the dominant negative Rab11S25N impaired that recycling. Conversely, while the hIP co-localized to Rab4-positive endosomes in response to cicaprost, ectopic expression of Rab4a did not substantially affect overall recycling nor did Rab4a directly interact with the hIP. The specific interaction between the hIP and Rab11a was dependent on a 22 amino acid (Val299 – Gln320) sequence within its C-tail domain and was independent of isoprenylation of the hIP. This study elucidates a critical role for Rab11a in regulating trafficking of the hIP and has identified a novel Rab11 binding-domain (RBD) within its C-tail domain that is both necessary and sufficient to mediate interaction with Rab11a.548Scopus© Citations 29 - PublicationComputational modelling of cancerous mutations in the EGFR/ERK signalling pathway(Springer (Biomed Central Ltd.), 2009)
; ; ; Background: The Epidermal Growth Factor Receptor (EGFR) activated Extracellular-signal Regulated Kinase (ERK) pathway is a critical cell signalling pathway that relays the signal for a cell to proliferate from the plasma membrane to the nucleus. Deregulation of the EGFR/ERK pathway due to alterations affecting the expression or function of a number of pathway components has long been associated with numerous forms of cancer. Under normal conditions, Epidermal Growth Factor (EGF) stimulates a rapid but transient activation of ERK as the signal is rapidly shutdown. Whereas, under cancerous mutation conditions the ERK signal cannot be shutdown and is sustained resulting in the constitutive activation of ERK and continual cell proliferation. In this study, we have used computational modelling techniques to investigate what effects various cancerous alterations have on the signalling flow through the ERK pathway. Results: We have generated a new model of the EGFR activated ERK pathway, which was verified by our own experimental data. We then altered our model to represent various cancerous situations such as Ras, B-Raf and EGFR mutations, as well as EGFR overexpression. Analysis of the models showed that different cancerous situations resulted in different signalling patterns through the ERK pathway, especially when compared to the normal EGF signal pattern. Our model predicts that cancerous EGFR mutation and overexpression signals almost exclusively via the Rap1 pathway, predicting that this pathway is the best target for drugs. Furthermore, our model also highlights the importance of receptor degradation in normal and cancerous EGFR signalling, and suggests that receptor degradation is a key difference between the signalling from the EGF and Nerve Growth Factor (NGF) receptors. Conclusion: Our results suggest that different routes to ERK activation are being utilised in different cancerous situations which therefore has interesting implications for drug selection strategies. We also conducted a comparison of the critical differences between signalling from different growth factor receptors (namely EGFR, mutated EGFR, NGF, and Insulin) with our results suggesting the difference between the systems are large scale and can be attributed to the presence/absence of entire pathways rather than subtle difference in individual rate constants between the systems.322Scopus© Citations 46 - PublicationOxygen-mediated regulation of biofilm development is controlled by the alternative sigma factor sigma(B) in Staphylococcus epidermidisUsing a modified rotating-disk reactor to sparge oxygen to Staphylococcus epidermidis cultures, we found that oxygen negatively regulates biofilm development by influencing the activity of {sigma}B. Under anaerobic conditions, increased {sigma}B activity activates icaADBC, which encodes enzymes responsible for polysaccharide intercellular adhesin synthesis, by repressing transcription of the negative regulator icaR.
343Scopus© Citations 28 - PublicationFrequency modulation atomic force microscopy in ambient environments utilizing robust feedback tuning(American Institute of Physics, 2009-02-02)
; ; ; Frequency modulation atomic force microscopy (FM-AFM) is rapidly evolving as the technique of choice in the pursuit of high resolution imaging of biological samples in ambient environments. The enhanced stability afforded by this dynamic AFM mode combined with quantitative analysis enables the study of complex biological systems, at the nanoscale, in their native physiological environment. The operational bandwidth and accuracy of constant amplitude FM-AFM in low Q environments is heavily dependent on the cantilever dynamics and the performance of the demodulation and feedback loops employed to oscillate the cantilever at its resonant frequency with a constant amplitude. Often researchers use ad hoc feedback gains or instrument default values that can result in an inability to quantify experimental data. Poor choice of gains or exceeding the operational bandwidth can result in imaging artifacts and damage to the tip and/or sample. To alleviate this situation we present here a methodology to determine feedback gains for the amplitude and frequency loops that are specific to the cantilever and its environment, which can serve as a reasonable "first guess", thus making quantitative FM-AFM in low Q environments more accessible to the nonexpert. This technique is successfully demonstrated for the low Q systems of air (Q∼40) and water (Q∼1). In addition, we present FM-AFM images of MC3T3-E1 preosteoblast cells acquired using the gains calculated by this methodology demonstrating the effectiveness of this technique.472Scopus© Citations 28 - PublicationTranscriptional regulation of the human prostacyclin receptor gene is dependent on Sp1, PU.1 and Oct-1 in megakaryocytes and endothelial cellsProstacyclin plays a central role in haemostasis, inflammation and nociception. However, the factors regulating expression of the prostacyclin receptor (IP) gene in humans, or in other species, have not been identified. Herein it was sought to identify the key trans-acting factors and cis-acting elements regulating IP expression in the megakaryoblastic human erythroleukemia (HEL) 92.1.7 and the vascular endothelial EA.hy 926 cell lines. Using deletion and genetic reporter analyses, the essential core promoter, termed PrmIP, was localized to -1022 to -895 proximal to the transcription initiation site, while an upstream repressor region, localized to -1502 to -1271, was also identified. Bioinformatic analysis revealed evolutionary conserved Sp1, PU.1 and Oct-1 sites within the core PrmIP and disruption of those elements each led to substantial reductions in PrmIP-directed gene expression in both HEL and EA.hy 926 cells. Electrophoretic mobility shift assays (EMSAs) and supershift assays established that Sp1, PU.1 and Oct-1 can bind to elements within the core promoter in vitro while chromatin immunoprecipitiation (ChIP) assays confirmed their specific binding to chromatin in vivo. Furthermore, combination mutations of the Sp1, PU.1 and Oct-1 elements revealed that they act independently to co-regulate basal transcription of the IP gene while ectopic expression of each of the trans-acting factors led to substantial increases in PrmIP-directed gene expression and IP mRNA expression in both HEL and EA.hy 926 cells. While EMSA and antibody supershift assays established that the Ets family member Fli1, but not Ets-1, is capable of binding to the PU.1 element within PrmIP in vitro, ChIP analysis established that neither Fli1 nor Ets-1 bind to that element in vivo. Collectively, these data provide critical insights into the transcriptional regulation of the IP gene in human megakaryocytic and endothelial cells, identifying Sp1, PU.1 and Oct-1 as the critical factors involved in its basal regulation in humans.
549Scopus© Citations 14 - PublicationBioorganometallic Chemistry: A Key To New Chemotherapy?6-Substituted fulvenes are interesting and easily accessible starting materials for the synthesis of novel substituted titanocenes via reductive dimerisation, carbolithiation or hydridolithiation reactions, which are followed by a transmetallation reaction with titanium tetrachloride in the latter two cases. Depending on the substitution pattern, these titanocenes prove to be bioorganometallic anticancer drugs, which have significant potential against advanced or metastatic renal-cell cancer. Patients bearing these stages of kidney cancer have a poor prognosis so far and therefore real progress in the area of metal-based anticancer drugs may come from this simple and effective synthetic approach.
67 - PublicationToggle switches, pulses and oscillations are intrinsic properties of the Src activation/deactivation cycleSrc-family kinases (SFKs) play a pivotal role in growth factor signaling, mitosis, cell motility and invasiveness. In their basal state, SFKs maintain a closed autoinhibited conformation, where the Src homology 2 domain interacts with an inhibitory phosphotyrosine in the C-terminus. Activation involves dephosphorylation of this inhibitory phosphotyrosine, followed by intermolecular autophosphorylation of a specific tyrosine residue in the activation loop. The spatiotemporal dynamics of SFK activation controls cell behavior, yet these dynamics remain largely uninvestigated. In the present study, we show that the basic properties of the Src activation/deactivation cycle can bring about complex signaling dynamics, including oscillations, toggle switches and excitable behavior. These intricate dynamics do not require imposed external feedback loops and occur at constant activities of Src inhibitors and activators, such as C-terminal Src kinase and receptor-type protein tyrosine phosphatases. We demonstrate that C-terminal Src kinase and receptor-type protein tyrosine phosphatase underexpression or their simultaneous overexpression can transform Src response patterns into oscillatory or bistable responses, respectively. Similarly, Src overexpression leads to dysregulation of Src activity, promoting sustained self-perpetuating oscillations. Distinct types of responses can allow SFKs to trigger different cell-fate decisions, where cellular outcomes are determined by the stimulation threshold and history. Our mathematical model helps to understand the puzzling experimental observations and suggests conditions where these different kinetic behaviors of SFKs can be tested experimentally.
478Scopus© Citations 27 - PublicationRapid depletion of dissolved oxygen in 96 well microtitre plate Staphylococcus epidermidis biofilm assays promotes biofilm development and is influenced by inoculum cell concentrationBiofilm-related research using 96-well microtiter plates involves static incubation of plates indiscriminate of environmental conditions, making oxygen availability an important variable which has not been considered to date. By directly measuring dissolved oxygen concentration over time we report here that dissolved oxygen is rapidly consumed in Staphylococcus epidermidis biofilm cultures grown in 96-well plates irrespective of the oxygen concentration in the gaseous environment in which the plates are incubated. These data indicate that depletion of dissolved oxygen during growth of bacterial biofilm cultures in 96-well plates may significantly influence biofilm production. Furthermore higher inoculum cell concentrations are associated with more rapid consumption of dissolved oxygen and higher levels of S. epidermidis biofilm production. Our data reveal that oxygen depletion during bacterial growth in 96-well plates may significantly influence biofilm production and should be considered in the interpretation of experimental data using this biofilm model.
871Scopus© Citations 17