Mitochondrial dysfunction in a cell model of thyroid oncocytoma

The role of mitochondrial dysfunction in cancer has long been a subject of great interest. In this study, such dysfunction has been examined with regards to thyroid oncocytoma, a rare form of cancer, accounting for less than 5% of all thyroid cancers. A peculiar characteristic of thyroid oncocytic cells is the presence of an abnormally large number of mitochondria in the cytoplasm. Such mitochondrial hyperplasia has also been observed in cells derived from patients suffering from mitochondrial encephalomyopathies, where mutations in the mitochondrial DNA(mtDNA) encoding the respiratory complexes result in oxidative phosphorylation dysfunction. An increase in the number of mitochondria occurs in the latter in order to compensate for the respiratory deficiency. This fact spurred the investigation into the presence of analogous mutations in thyroid oncocytic cells. In this study, the only available cell model of thyroid oncocytoma was utilised, the XTC-1 cell line, established from an oncocytic thyroid metastasis to the breast. In order to assess the energetic efficiency of these cells, they were incubated in a medium lacking glucose and supplemented instead with galactose. When subjected to such conditions, glycolysis is effectively inhibited and the cells are forced to use the mitochondria for energy production. Cell viability experiments revealed that XTC-1 cells were unable to survive in galactose medium. This was in marked contrast to the TPC-1 control cell line, a thyroid tumour cell line which does not display the oncocytic phenotype. In agreement with these findings, subsequent experiments assessing the levels of cellular ATP over incubation time in galactose medium, showed a drastic and continual decrease in ATP levels only in the XTC-1 cell line. Furthermore, experiments on digitonin-permeabilised cells revealed that the respiratory dysfunction in the latter was due to a defect in complex I of the respiratory chain. Subsequent experiments using cybrids demonstrated that this defect could be attributed to the mitochondrially-encoded subunits of complex I as opposed to the nuclearencoded subunits. Confirmation came with mtDNA sequencing, which detected the presence of a novel mutation in the ND1 subunit of complex I. In addition, a mutation in the cytochrome b subunit of complex III of the respiratory chain was detected. The fact that XTC-1 cells are unable to survive when incubated in galactose medium is consistent with the fact that many cancers are largely dependent on glycolysis for energy production. Indeed, numerous studies have shown that glycolytic inhibitors are able to induce apoptosis in various cancer cell lines. Subsequent experiments were therefore performed in order to identify the mode of XTC-1 cell death when subjected to the metabolic stress imposed by the forced use of the mitochondria for energy production. Cell shrinkage and mitochondrial fragmentation were observed in the dying cells, which would indicate an apoptotic type of cell death. Analysis of additional parameters however revealed a lack of both DNA fragmentation and caspase activation, thus excluding a classical apoptotic type of cell death. Interestingly, cleavage of the actin component of the cytoskeleton was observed, implicating the action of proteases in this mode of cell demise. However, experiments employing protease inhibitors failed to identify the specific protease involved. It has been reported in the literature that overexpression of Bcl-2 is able to rescue cells presenting a respiratory deficiency. As the XTC-1 cell line is not only respiration-deficient but also exhibits a marked decrease in Bcl-2 expression, it is a perfect model with which to study the relationship between Bcl-2 and oxidative phosphorylation in respiratory-deficient cells. Contrary to the reported literature studies on various cell lines harbouring defects in the respiratory chain, Bcl-2 overexpression was not shown to increase cell survival or rescue the energetic dysfunction in XTC-1 cells. Interestingly however, it had a noticeable impact on cell adhesion and morphology. Whereas XTC-1 cells shrank and detached from the growth surface under conditions of metabolic stress, Bcl-2-overexpressing XTC-1 cells appeared much healthier and were up to 45% more adherent. The target of Bcl-2 in this setting appeared to be the actin cytoskeleton, as the cleavage observed in XTC-1 cells expressing only endogenous levels of Bcl-2, was inhibited in Bcl-2-overexpressing cells. Thus, although unable to rescue XTC-1 cells in terms of cell viability, Bcl-2 is somehow able to stabilise the cytoskeleton, resulting in modifications in cell morphology and adhesion. The mitochondrial respiratory deficiency observed in cancer cells is thought not only to cause an increased dependency on glycolysis but it is also thought to blunt cellular responses to anticancer agents. The effects of several therapeutic agents were thus assessed for their death-inducing ability in XTC-1 cells. Cell viability experiments clearly showed that the cells were more resistant to stimuli which generate reactive oxygen species (tert-butylhydroperoxide) and to mitochondrial calcium-mediated apoptotic stimuli (C6-ceramide), as opposed to stimuli inflicting DNA damage (cisplatin) and damage to protein kinases(staurosporine). Various studies in the literature have reported that the peroxisome proliferator-activated receptor-coactivator 1(PGC-1α), which plays a fundamental role in mitochondrial biogenesis, is also involved in protecting cells against apoptosis caused by the former two types of stimuli. In accordance with these observations, real-time PCR experiments showed that XTC-1 cells express higher mRNA levels of this coactivator than do the control cells, implicating its importance in drug resistance. In conclusion, this study has revealed that XTC-1 cells, like many cancer cell lines, are characterised by a reduced energetic efficiency due to mitochondrial dysfunction. Said dysfunction has been attributed to mutations in respiratory genes encoded by the mitochondrial genome. Although the mechanism of cell demise in conditions of metabolic stress is unclear, the potential of targeting thyroid oncocytic cancers using glycolytic inhibitors has been illustrated. In addition, the discovery of mtDNA mutations in XTC-1 cells has enabled the use of this cell line as a model with which to study the relationship between Bcl-2 overexpression and oxidative phosphorylation in cells harbouring mtDNA mutations and also to investigate the significance of such mutations in establishing resistance to apoptotic stimuli.

Petrogenetic evolution of Uralian-Alaskan-type mafic-ultramafic complexes in the southern and middle Urals, Russia

The PhD thesis at hand consists of three parts and describes the petrogenetic evolution of Uralian-Alaskan-type mafic ultramafic complexes in the Ural Mountains, Russia. Uralian-Alaskan-type mafic-ultramafic complexes are recognized as a distinct class of intrusions. Characteristic petrologic features are the concentric zonation of a central dunite body grading outward into wehrlite, clinopyroxenite and gabbro, the absence of orthopyroxene and frequently occurring platinum group element (PGE) mineralization. In addition, the presence of ferric iron-rich spinel discriminates Uralian-Alaskan-type complexes from most other mafic ultramafic rock assemblages. The studied Uralian-Alaskan-type complexes (Nizhnii Tagil, Kytlym and Svetley Bor) belong to the southern part of a 900 km long, N–S-trending chain of similar intrusions between the Main Uralian Fault to the west and the Serov-Mauk Fault to the east. The first chapter of this thesis studies the evolution of the ultramafic rocks tracing the compositional variations of rock forming and accessory minerals. The comparison of the chemical composition of olivine, clinopyroxene and chromian spinel from the Urals with data from other localities indicates that they are unique intrusions having a characteristic spinel and clinopyroxene chemistry. Laser ablation-ICPMS (LA-ICPMS ) analyses of trace element concentrations in clinopyroxene are used to calculate the composition of their parental melt which is characterized by enriched LREE (0.5-5.2 prim. mantle) and other highly incompatible elements (U, Th, Ba, Rb) relative to the HREE (0.25-2.0 prim. mantle). A subduction-related geotectonic setting is indicated by a positive anomaly for Sr and negative anomalies for Ti, Zr and Hf. The mineral compositions monitor the evolution of the parental magmas and decipher differences between the studied complexes. In addition, the observed variation in LREE/HREE (for example La/Lu = 2-24) can be best explained with the model of an episodically replenished and erupted open magma chamber system with the extensive fractionation of olivine, spinel and clinopyroxene. The data also show that ankaramites in a subduction-related geotectonic setting could represent parental magmas of Uralian-Alaskan-type complexes. The second chapter of the thesis discusses the chemical variation of major and trace elements in rock-forming minerals of the mafic rocks. Electron microprobe and LA-ICPMS analyses are used to quantitatively describe the petrogenetic relationship between the different gabbroic lithologies and their genetic link to the ultramafic rocks. The composition of clinopyroxene identifies the presence of melts with different trace element abundances on the scale of a thin section and suggests the presence of open system crustal magma chambers. Even on a regional scale the large variation of trace element concentrations and ratios in clinopyroxene (e.g. La/Lu = 3-55) is best explained by the interaction of at least two fundamentally different magma types at various stages of fractionation. This requires the existence of a complex magma chamber system fed with multiple pulses of magmas from at least two different coeval sources in a subduction-related environment. One source produces silica saturated Island arc tholeiitic melts. The second source produces silica undersaturated, ultra-calcic, alkaline melts. Taken these data collectively, the mixing of the two different parental magmas is the dominant petrogenetic process explaining the observed chemical variations. The results further imply that this is an intrinsic feature of Uralian-Alaskan-type complexes and probably of many similar mafic-ultramafic complexes world-wide. In the third chapter of this thesis the major element composition of homogeneous and exsolved spinel is used as a petrogenetic indicator. Homogeneous chromian spinel in dunites and wehrlites monitors the fractionation during the early stages of the magma chamber and the onset of clinopyroxene fractionation as well as the reaction of spinel with interstitial liquid. Exsolved spinel is present in mafic and ultramafic rocks from all three studied complexes. Its composition lies along a solvus curve which defines an equilibrium temperature of 600°C, given that spinel coexists with olivine. This temperature is considered to be close to the temperature of the host rocks into which the studied Uralian-Alaskan-type complexes intruded. The similarity of the exsolution temperatures in the different complexes over a distance of several hundred kilometres implies a regional tectonic event that terminated the exsolution process. This event is potentially associated with the final exhumation of the Uralian-Alaskan-type complexes along the Main Uralian Fault and the Serov-Mauk Fault in the Uralian fold belt.

Ability of the PM3 Quantum Mechanical Method to Model Intermolecular Hydrogen Bonding between Neutral Molecules

The PM3 semiempirical quantum-mechanical method was found to systematically describe intermolecular hydrogen bonding in small polar molecules. PM3 shows charge transfer from the donor to acceptor molecules on the order of 0.02-0.06 units of charge when strong hydrogen bonds are formed. The PM3 method is predictive; calculated hydrogen bond energies with an absolute magnitude greater than 2 kcal mol-' suggest that the global minimum is a hydrogen bonded complex; absolute energies less than 2 kcal mol-' imply that other van der Waals complexes are more stable. The geometries of the PM3 hydrogen bonded complexes agree with high-resolution spectroscopic observations, gas electron diffraction data, and high-level ab initio calculations. The main limitations in the PM3 method are the underestimation of hydrogen bond lengths by 0.1-0.2 for some systems and the underestimation of reliable experimental hydrogen bond energies by approximately 1-2 kcal mol-l. The PM3 method predicts that ammonia is a good hydrogen bond acceptor and a poor hydrogen donor when interacting with neutral molecules. Electronegativity differences between F, N, and 0 predict that donor strength follows the order F > 0 > N and acceptor strength follows the order N > 0 > F. In the calculations presented in this article, the PM3 method mirrors these electronegativity differences, predicting the F-H- - -N bond to be the strongest and the N-H- - -F bond the weakest. It appears that the PM3 Hamiltonian is able to model hydrogen bonding because of the reduction of two-center repulsive forces brought about by the parameterization of the Gaussian core-core interactions. The ability of the PM3 method to model intermolecular hydrogen bonding means reasonably accurate quantum-mechanical calculations can be applied to small biologic systems.

Hydrodynamic Characterization of Bile Salt Host-Guest Complexes by Pulsed Field Gradient NMR Spectroscopy

The work described herein is aimed at understanding primary and secondary aggregation of bile salt micelles and how micelles can perform chiral recognition of binapthyl analytes. Previous work with cholate and deoxycholate using micellar electrokinetic chromatography (MEKC) and nuclear magnetic resonance (NMR) has provided insightinto cholate and deoxycholate micelle formation, especially with respect to the critical micelle concentration (CMC). Chiral separations of the model analyte, 1,1â??-binaphthyl-2,2â??-diyl hydrogen phosphate (BNDHP), via cholate (C) and deoxycholate (DC) mediated MEKC separataions previously have shown the DC CMC to be 7-10 mM andthe cholate CMC at 14 mM at ph 12. A second model analyte,1,1â??-binaphthol (BN), was also previously investigated to probe micellar structure, but the MEKC data for this analyte implied a higher CMC, which may be interpreted as secondary aggregation. Thiswork extends the investigation of bile salts to include pulsed field gradient spin echo (PFGSE) NMR experiments being used to gain information about the size and degree of polydispersity of cholate and deoxycholate micelles. Concentrations of cholate below 10mM show a large variation in effective radius likely due to the existence of transient preliminary aggregates. The onset of the primary micelle shows a dramatic increase in effective radius of the micelle in cholate and deoxycholate. In the region of expectedsecondary aggregation a gradual increase of effective radius was observed with cholate; deoxycholate showed a persistent aggregate size in the secondary micelle region that is modulated by the presence of an analyte molecule. Effective radii of cholate anddeoxycholate (individually) were compared with and without R- and S-BNDHP in order to observe the effective radius difference of micelles with and without analyte present. The presence of S-BNDHP consistently resulted in a larger effective aggregate radius incholate and deoxycholate, confirming previous data of the S-BNDHP interacting more with the micelle than R-BNDHP. In total, various NMR techniques, like diffusion NMR can be used to gain a greater understanding of the bile salt micellization process and chiral resolution.

Endothelial cell protection and complement inhibition in xenotransplantation: a novel in vitro model using whole blood

BACKGROUND: Studying the interactions between xenoreactive antibodies, complement and coagulation factors with the endothelium in hyperacute and acute vascular rejection usually necessitates the use of in vivo models. Conventional in vitro or ex vivo systems require either serum, plasma or anti-coagulated whole blood, making analysis of coagulation-mediated effects difficult. Here a novel in vitro microcarrier-based system for the study of endothelial cell (EC) activation and damage, using non-anticoagulated whole blood is described. Once established, the model was used to study the effect of the characterized complement- and coagulation inhibitor dextran sulfate (DXS, MW 5000) for its EC protective properties in a xenotransplantation setting. METHODS: Porcine aortic endothelial cells (PAEC), grown to confluence on microcarrier beads, were incubated with non-anticoagulated whole human blood until coagulation occurred or for a maximum of 90 min. PAEC-beads were either pre- or co-incubated with DXS. Phosphate buffered saline (PBS) experiments served as controls. Fluid phase and surface activation markers for complement and coagulation were analyzed as well as binding of DXS to PAEC-beads. RESULTS: Co- as well as pre-incubation of DXS, followed by washing of the beads, significantly prolonged time to coagulation from 39 +/- 12 min (PBS control) to 74 +/- 23 and 77 +/- 20 min, respectively (P < 0.005 vs. PBS). DXS treatment attenuated surface deposition of C1q, C4b/c, C3b/c and C5b-9 without affecting IgG or IgM deposition. Endothelial integrity, expressed by positivity for von Willebrand Factor, was maintained longer with DXS treatment. Compared with PBS controls, both pre- and co-incubation with DXS significantly prolonged activated partial thromboplastin time (>300 s, P < 0.05) and reduced production of thrombin-antithrombin complexes and fibrinopeptide A. Whilst DXS co-incubation completely blocked classical pathway complement activity (CH50 test) DXS pre-incubation or PBS control experiments showed no inhibition. DXS bound to PAEC-beads as visualized using fluorescein-labeled DXS. CONCLUSIONS: This novel in vitro microcarrier model can be used to study EC damage and the complex interactions with whole blood as well as screen ''endothelial protective'' substances in a xenotransplantation setting. DXS provides EC protection in this in vitro setting, attenuating damage of ECs as seen in hyperacute xenograft rejection.

Characterization of the ebp(fm) pilus-encoding operon of Enterococcus faecium and its role in biofilm formation and virulence in a murine model of urinary tract infection.

We recently identified 15 genes encoding putative surface proteins with features of MSCRAMMs and/or pili in the Enterococcus faecium TX0016 (DO) genome, including four predicted pilus-encoding gene clusters; we also demonstrated that one of these, ebpABC(fm), is transcribed as an operon, that its putative major pilus subunit, EbpC(fm) (also called pilB), is polymerized into high molecular weight complexes, and that it is enriched among clinical E. faecium isolates. Here, we created a deletion of the ebpABC(fm) operon in an endocarditis-derived E. faecium strain (TX82) and showed, by a combination of whole-cell ELISA, flow cytometry, immunoblot and immunogold electron microscopy, that this deletion abolished EbpC(fm) expression and eliminated EbpC(fm)-containing pili from the cell surface. However, transcription of the downstream sortase, bps(fm), was not affected. Importantly, the ebpABC(fm) deletion resulted in significantly reduced biofilm formation (p < 0.0001) and initial adherence (p < 0.0001) versus the wild-type; both were restored by complementing ebpABC(fm) in trans, which also restored cell surface expression of EbpC(fm) and pilus production. Furthermore, the deletion mutant was significantly attenuated in two independent mixed infection mouse urinary tract experiments, i.e., outnumbered by the wild-type in kidneys (p = 0.0003 and < 0.0001, respectively) and urinary bladders (p = 0.0003 and = 0.002). In conclusion, we have shown that the ebpABC(fm) locus encodes pili on the E. faecium TX82 cell surface and provide the first evidence that pili of this emerging pathogen are important for its ability to form biofilm and to cause infection in an ascending UTI model.

Attractant and repellent signaling conformers of sensory rhodopsin-transducer complexes.

Attractant and repellent signaling conformers of the dual-signaling phototaxis receptor sensory rhodopsin I and its transducer subunit (SRI-HtrI) have recently been distinguished experimentally by the opposite connection of their retinylidene protonated Schiff bases to the outwardly located periplasmic side and inwardly located cytoplasmic side. Here we show that the pK(a) of the outwardly located Asp76 counterion in the outwardly connected conformer is lowered by approximately 1.5 units from that of the inwardly connected conformer. The pK(a) difference enables quantitative determination of the relative amounts of the two conformers in wild-type cells and behavioral mutants prior to photoexcitation, comparison of their absorption spectra, and determination of their relative signaling efficiency. We have shown that the one-photon excitation of the SRI-HtrI attractant conformer causes a Schiff base connectivity switch from inwardly connected to outwardly connected states in the attractant signaling photoreaction. Conversely, a second near-UV photon drives the complex back to the inwardly connected conformer in the repellent signaling photoreaction. The results suggest a model of the color-discriminating dual-signaling mechanism in which phototaxis responses (his-kinase modulation) result from the photointerconversion of the two oppositely connected SRI-HtrI conformers by one-photon and two-photon activation. Furthermore, we find that the related repellent phototaxis SRII-HtrII receptor complex has an outwardly connected retinylidene Schiff base like the repellent signaling forms of the SRI-HtrI complex, indicating the general applicability of macro conformational changes, which can be detected by the connectivity switch, to phototaxis signaling by sensory rhodopsin-transducer complexes.

Solvation-Driven Charge Transfer and Localization in Metal Complexes

In any physicochemical process in liquids, the dynamical response of the solvent to the solutes out of equilibrium plays a crucial role in the rates and products: the solvent molecules react to the changes in volume and electron density of the solutes to minimize the free energy of the solution, thus modulating the activation barriers and stabilizing (or destabilizing) intermediate states. In charge transfer (CT) processes in polar solvents, the response of the solvent always assists the formation of charge separation states by stabilizing the energy of the localized charges. A deep understanding of the solvation mechanisms and time scales is therefore essential for a correct description of any photochemical process in dense phase and for designing molecular devices based on photosensitizers with CT excited states. In the last two decades, with the advent of ultrafast time-resolved spectroscopies, microscopic models describing the relevant case of polar solvation (where both the solvent and the solute molecules have a permanent electric dipole and the mutual interaction is mainly dipole−dipole) have dramatically progressed. Regardless of the details of each model, they all assume that the effect of the electrostatic fields of the solvent molecules on the internal electronic dynamics of the solute are perturbative and that the solvent−solute coupling is mainly an electrostatic interaction between the constant permanent dipoles of the solute and the solvent molecules. This well-established picture has proven to quantitatively rationalize spectroscopic effects of environmental and electric dynamics (time-resolved Stokes shifts, inhomogeneous broadening, etc.). However, recent computational and experimental studies, including ours, have shown that further improvement is required. Indeed, in the last years we investigated several molecular complexes exhibiting photoexcited CT states, and we found that the current description of the formation and stabilization of CT states in an important group of molecules such as transition metal complexes is inaccurate. In particular, we proved that the solvent molecules are not just spectators of intramolecular electron density redistribution but significantly modulate it. Our results solicit further development of quantum mechanics computational methods to treat the solute and (at least) the closest solvent molecules including the nonperturbative treatment of the effects of local electrostatics and direct solvent−solute interactions to describe the dynamical changes of the solute excited states during the solvent response.

The two -state model of receptor activation: The agonist and the efficacy

The Two State model describes how drugs activate receptors by inducing or supporting a conformational change in the receptor from “off” to “on”. The beta 2 adrenergic receptor system is the model system which was used to formalize the concept of two states, and the mechanism of hormone agonist stimulation of this receptor is similar to ligand activation of other seven transmembrane receptors. Hormone binding to beta 2 adrenergic receptors stimulates the intracellular production of cyclic adenosine monophosphate (cAMP), which is mediated through the stimulatory guanyl nucleotide binding protein (Gs) interacting with the membrane bound enzyme adenylylcyclase (AC). ^ The effects of cAMP include protein phosphorylation, metabolic regulation and transcriptional regulation. The beta 2 adrenergic receptor system is the most well known of its family of G protein coupled receptors. Ligands have been scrutinized extensively in search of more effective therapeutic agents at this receptor as well as for insight into the biochemical mechanism of receptor activation. Hormone binding to receptor is thought to induce a conformational change in the receptor that increases its affinity for inactive Gs, catalyzes the release of GDP and subsequent binding of GTP and activation of Gs. ^ However, some beta 2 ligands are more efficient at this transformation than others, and the underlying mechanism for this drug specificity is not fully understood. The central problem in pharmacology is the characterization of drugs in their effect on physiological systems, and consequently, the search for a rational scale of drug effectiveness has been the effort of many investigators, which continues to the present time as models are proposed, tested and modified. ^ The major results of this thesis show that for many b2 -adrenergic ligands, the Two State model is quite adequate to explain their activity, but dobutamine (+/−3,4-dihydroxy-N-[3-(4-hydroxyphenyl)-1-methylpropyl]- b -phenethylamine) fails to conform to the predictions of the Two State model. It is a weak partial agonist, but it forms a large amount of high affinity complexes, and these complexes are formed at low concentrations much better than at higher concentrations. Finally, dobutamine causes the beta 2 adrenergic receptor to form high affinity complexes at a much faster rate than can be accounted for by its low efficiency activating AC. Because the Two State model fails to predict the activity of dobutamine in three different ways, it has been disproven in its strictest form. ^