No / The vitrification solutions used in the cryopreservation of biological samples aim to minimize the deleterious formation of ice by dehydrating cells and promoting the formation of the glassy state of water. They contain a mixture of different cryoprotective agents (CPAs) in water, typically polyhydroxylated alcohols and/or dimethyl sulfoxide (DMSO), which can damage cell membranes. Molecular dynamics simulations have been used to investigate the behavior of pure DPPC, pure DOPC, and mixed DOPC-β-sitosterol bilayers solvated in a vitrification solution containing glycerol, ethylene glycol, and DMSO at concentrations that approximate the widely used plant vitrification solution 2. As in the case of solutions containing a single CPA, the vitrification solution causes the bilayer to thin and become disordered, and pores form in the case of some bilayers. Importantly, the degree of thinning is, however, substantially reduced compared to solutions of DMSO containing the same total CPA concentration. The reduction in the damage done to the bilayers is a result of the ability of the polyhydroxylated species (especially glycerol) to form hydrogen bonds to the lipid and sterol molecules of the bilayer. A decrease in the amount of DMSO in the vitrification solution with a corresponding increase in the amount of glycerol or ethylene glycol diminishes further its damaging effect due to increased hydrogen bonding of the polyol species to the bilayer headgroups. These findings rationalize, to our knowledge for the first time, the synergistic effects of combining different CPAs, and form the basis for the optimization of vitrification solutions. / Australian Research Council linkage grant No. LP0884027; Alcoa Australia Ltd.; BHP Billiton Worsley Alumina Pty. Ltd.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

No description available.

Tuberculosis (TB) affects nearly a third of the world’s population and has been termed a ‘Global Emergency’ by the WHO. The emergence of multi/extensively drug resistant (M/XDR) strains of Mycobacterium tuberculosis (Mtb), the causative agent of TB, and the increasing incidences of azole drug resistant sterol demethylases (CYP51) from pathogenic fungi has propelled studies to understand mechanisms of azole drug resistance on the drug target CYP51. Since Mtb is devoid of a sterol biosynthetic pathway, the presence and study of CYP51B1 and 19 other Cytochrome P450s in its genome is important to clarify host-pathogen mechanism of infection and the potential of using azole drugs to treat TB. In this study, CYP51B1 from Mtb was used as the model enzyme to study CYP51 mutants from Candida albicans fluconazole-resistant clinical strains. By protein engineering methods, F89H, L100F, S348F, G388S and R391K CYP51B1 mutants were made and azole drug binding properties were investigated using stopped-flow kinetics and static equilibrium methods. Dissociation constant (Kd) values were derived for a range of commercially available azole drugs by fitting the equilibrium binding data to a hyperbolic equation. Kd values for stopped-flow kinetics were derived by plotting observed binding rates (kobs) across different azole drug concentrations against time, followed by fitting multiple kobs data to a linear equation to derive azole drug de-binding (koff) and binding (kon) rate constants – the Kd was obtained by koff/kon. Extinction coefficient for heme b content in mutants and Wild Type (WT) CYP51B1 were an average of ɛ419 = 96.1 mM-1 cm-1. Biochemical characterisation of the mutants were carried out using established experiments on CYP51 – reduction of Fe(III)-heme to Fe(II)-heme, NO binding to Fe(III)-heme, rates of CO-Fe(II) adduct formation and rates of collapse of the P450 to P420 species in the presence of CO and estriol with redox partners from Mtb. In order to elucidate the effects of the above mutations on the iron-heme catalytic region, electron paramagnetic resonance (EPR) experiments were carried out with and without azole drugs. Circular dichroism (CD), differential scanning calorimetry (DSC) and multi-angled laser light scattering (MALLS) analysis confirmed that F89H, R391K and L100F mutants were stable and homogeneous. Crystallogenesis was successful for the above mentioned mutants and atomic structures were obtained for all mutants and WT CYP51B1 (in ligand-bound and substrate-free forms), except for S348F and G388S mutants which were expressed as inclusion bodies and 60% holoenzyme, respectively. Reconstituted catalytic assays to determine the sterol demethylating propensity of the mutants were carried out using redox partners from Mtb or E. coli, and with lanosterol and dihydrolanosterol as the surrogate substrates. Redox potentiometry showed similar potentials to WT for all mutants except for the G388S mutant which was relatively positive (–102 mV). Redox cycling experiments followed by EPR analysis for mutants and WT resulted in a novel P450 high-spin species at g value 5.84 (80 %) which gradually collapsed to the initial low spin state over 48 h. Expression trials were concurrently carried out on two other Mtb P450 genes – CYP123 (Rv0744c) and CYP136 (Rv3059) products of which may have similar functions to CYP51B1 or may share similar redox partners. CYP123 is located on the same operon as CYP51B1 while CYP136 has a 29% sequence identity to another CYP51 from a marine slime bacterium. Although further work is necessary, in this study CYP123 was expressed totally as inclusion bodies while CYP136 was expressed as soluble apoprotein fused with trigger factor chaperone.

Sterol is an abundant component of eukaryotic cell membranes and is thought to influence membrane properties such as permeability, fluidity and microdomain formation. Drosophila is an excellent model system in which to study functional requirements for membrane sterol because, although it does not synthesize sterol, it nevertheless requires sterols to complete development. Moreover, Drosophila normally incorporates sterols into cell membranes. Thus, dietary sterol depletion can be used to specifically reduce membrane sterol levels. In contrast, vertebrates do synthesize cholesterol. In this way, sterol depletion in vertebrates demand the use of approaches such as chemical extractions, drug treatments or genetic manipulation which are prone to have side effects. We have controlled the level and type of dietary sterol available to developing Drosophila larvae in order to investigate the requirement for sterol in cell membranes, and to distinguish it from the function of sterol as a precursor for signaling molecules. Strikingly, we show that membrane sterol levels can be reduced 6-fold in most tissues without affecting cell or larval viability. Larvae respond to sterol depletion by arresting their growth and development, and by increasing the level of specific sphingolipid variants that promote survival when sterol is scarce. Thus, non-sterol lipids are able to substitute for sterols in the maintenance of basic membrane biophysical properties required for life. Despite this, Drosophila larvae regulate their growth to maintain membrane sterol levels within tight limits. The existence of this novel membrane sterol-dependent growth control mechanism indicates an important role for bulk membrane sterol in the tissue specific functions of differentiated cells.

Sterols play many important roles in physiology, including maintaining cell membrane integrity, and producing vitamin D and steroid hormones. Recent studies implicate sterol metabolism in the host innate immune response. Previous work, based on transcriptional profiling studies of mouse cytomegalovirus (MCMV) infection of primary bone-marrow-derived macrophages (BMDM, MΦ), uncovered a previously uncharacterized role of interferon in regulating the cholesterol pathway. Notably, Toll-like receptor (TLR) induced interferon modulates the suppression of SREBP2 (Sterol Regulatory Element-Binding Protein 2) activation, the master transcription factor for sterol biosynthesis. This finding resulted in the downregulation of the sterol biosynthesis pathway. However, how interferon is molecularly linked to sterol metabolism, and what part of the pathway mediates the antiviral effect remains unknown. The central hypothesis of the thesis is that the antiviral effect of interferon is in part mediated by secondary sterol metabolites and the dependency of viral replication on the host mevalonate branch of the sterol biosynthesis pathway. To test this hypothesis, my studies have examined the components of the host sterol pathway and their respective roles in influencing viral replication. Paradigmatically, I used MCMV and BMDM to explore the host- metabolic-virus interactions. Specifically, my findings address the question of how MCMV replication depends on the sterol biosynthesis pathway, and how the pathway is modulated by interferon as an antiviral response. In Chapter 2, the importance of the sterol biosynthesis pathway for viral replication was investigated using a combination of gene silencing and pharmacological inhibitors. These studies demonstrated that resistance to viral infection through suppressing the cholesterol pathway is not due to a requirement of the virus for cholesterol itself, but instead involves the mevalonate-isoprenoid arm of the pathway. This branch of the pathway chemically links lipids to specific host proteins (protein prenylation). These results suggest a new role for the mevalonate arm during viral infection. In Chapter 3, I examined what part of the sterol pathway mediates the antiviral effects. Oxysterols are natural modulators of sterol biosynthesis, and are produced by the oxidation of cholesterol by the enzyme cholesterol hydroxylase. Oxysterol suppression of SREBP2 activation leads to transcriptional repression of the sterol biosynthesis pathway. Additionally, oxysterols also modulate cholesterol homeostasis through cholesterol efflux. My studies led to identifing cholesterol-25-hydroxylase (Ch25h) as an interferon-stimulated gene (ISG). CH25H oxidizes cholesterol to produce a soluble oxysterol metabolite, 25-hydroxycholesterol (25-HC). Treatment of cells with 25-HC resulted in antiviral effects against MCMV and MHV-68. 25-HC was found to have no effects on MCMV entry into the host cell, but rather mediated inhibition of viral gene transcription. In addition, 25-HC-specific antiviral effect partially involved the suppression of the isoprenoid pathway, rather than cholesterol efflux. This work uncovered a physiological role for 25-HC as a sterol-lipid effector of an innate immune pathway. The antiviral activity of 25-HC in a lipid replete condition was found to occur at a concentration higher than the concentration required to inhibit SREBP2 activation. This implies that the antiviral effects of 25-HC is independent of SREBP2 in sterol replete conditions. Conversely, the antiviral action of 25-HC was signifi enhanced in cells under sterol-depleted conditions, suggesting that the antiviral effect of 25- HC is likely mediated through multiple processes involving SREBP2 dependent and independent mechanisms. These sterol dependent and independent mechanisms are examined in Chapter 4, using pathway expression profiling and pharmacological synergy studies. These studies showed that 25-HC suppression of the isoprenoid synthetic pathway is crucial in controlling infection, but also highlighted that other 25-HC dependent antiviral mechanisms are likely to exist. The inhibition of the mevalonate-isoprenoid arm by statins and 25-HC clearly demonstrated that MCMV replication dependents on protein prenylation. Chapter 5 investigation showed that either chemical inhibition of geranylgeranylation of host proteins or limiting mevalonate production led to restriction of MCMV replication. Importantly, through a series of systematic loss of function siRNA screenings demonstrated that specific host RabGTPases mediating vesicular transport pathways play vital roles in the replication and the assembly of the virus. This finding provides new mechanistic insights in to the dependency of cytomegalovirus replication on the host cell trafficking pathways and lays the groundwork for further definition of this important aspect of host-viral interactions. In summary, the overall findings of this research support the original hypothesis, by highlighting the importance of the host mevalonate-isoprenoid pathway, and provide further definition of the mechanisms and components linking sterol metabolism with interferon mediated antiviral effect.

Sterol is an abundant component of eukaryotic cell membranes and is thought to influence membrane properties such as permeability, fluidity and microdomain formation. Drosophila is an excellent model system in which to study functional requirements for membrane sterol because, although it does not synthesize sterol, it nevertheless requires sterols to complete development. Moreover, Drosophila normally incorporates sterols into cell membranes. Thus, dietary sterol depletion can be used to specifically reduce membrane sterol levels. In contrast, vertebrates do synthesize cholesterol. In this way, sterol depletion in vertebrates demand the use of approaches such as chemical extractions, drug treatments or genetic manipulation which are prone to have side effects. We have controlled the level and type of dietary sterol available to developing Drosophila larvae in order to investigate the requirement for sterol in cell membranes, and to distinguish it from the function of sterol as a precursor for signaling molecules. Strikingly, we show that membrane sterol levels can be reduced 6-fold in most tissues without affecting cell or larval viability. Larvae respond to sterol depletion by arresting their growth and development, and by increasing the level of specific sphingolipid variants that promote survival when sterol is scarce. Thus, non-sterol lipids are able to substitute for sterols in the maintenance of basic membrane biophysical properties required for life. Despite this, Drosophila larvae regulate their growth to maintain membrane sterol levels within tight limits. The existence of this novel membrane sterol-dependent growth control mechanism indicates an important role for bulk membrane sterol in the tissue specific functions of differentiated cells.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24795号 / 医博第4987号 / 新制||医||1066(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小林 恭, 教授 波多野 悦朗, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p> Cellular cholesterol homeostasis is a fundamental and highly regulated process. Transcription factors known as sterol regulatory element binding proteins (SREBP) are responsible for the expression of many genes involved in the uptake and biosynthesis of cholesterol. SREBP activation and lipid dysregulation has been associated with cellular endoplasmic reticulum (ER) stress and the activation of the unfolded protein response (UPR). Our lab has previously reported a relationship between ER stress and SREBP activation causing lipid dysregulation and hepatic steatosis. This project was designed to elucidate the mechanism of ER stress-induced SREBP activation and determine its relationship with cellular pathologies associated with ER stress and lipid accumulation. My research has examined the mechanism by which ER stress activates SREBP-2 in various cell lines, including epithelial and macrophage cells. This research revealed that (1) ER stress-induced SREBP-2 activation is not dependent on caspases and occurs through the conventional sterol-mediated proteolytic pathway; (2) the mechanism of ER stress-induced SREBP-2 activation is sensitive to changes in ER calcium; (3) ER stress is associated with SREBP-2 activation and lipid dysregulation in a model of renal injury; and ( 4) ER stress-induced SREBP activation in vitro is not associated with lipid accumulation in macrophage foam cells. </P> <p> This project has also offered me the opportunity to further enhance our understanding of the mechanism by which ER stress causes SREBP activation in a sterolindependent manner. </P> / Thesis / Doctor of Philosophy (PhD)

Apple scab (causal organism: Venturia inaequalis) is an economically devastating disease of apples that is predominantly controlled with fungicides. Of the chemical classes currently available, the sterol-inhibiting (SI) and strobilurin (QoI) fungicides are the most commonly used. Recent observations indicate that V. inaequalis populations in Virginia have developed resistance to myclobutanil and other SIs. However, little is known about the frequency and distribution of SI and QoI resistance in Virginia's scab populations. The first objective of this research was to evaluate V. inaequalis populations in Virginia for SI and QoI resistance. Fungal isolates were collected from experimental orchards at the Alson H. Smith Jr., Agricultural Research and Extension Center (AHS AREC) and from commercial orchards in Virginia and Maryland. Sensitivities were determined by assessing colony growth at 19°C on potato dextrose agar (PDA) amended with 0 or 1.0 µg ml-1 of myclobutanil (SI) (N=87) or trifloxystrobin (QoI) (N=25) at 28 days. A range of fungicide sensitivity was observed for both chemical classes. The second objective of this research was to monitor the temporal dynamics of SI resistance over five sequential field seasons. To monitor shoot growth, neon rubber bands were placed over actively growing shoot tips following myclobutanil application or sample collection. Fungal isolates were collected from the same trees from 2007 through 2010 (N=176) and compared with isolates collected from wild apple seedlings (N=3). A continuum of SI resistance was observed for each year, and the V. inaequalis population exhibited a baseline shifted toward reduced sensitivity. The third objective of this research was to examine the spatial distribution of SI fungicide resistance within the tree canopy in a lower-density orchard (less than 150 trees A-1). Leaves collected from larger trees (>8m) in a lower-density orchard at the AHS AREC were analyzed for manganese deposition, pre- and post-mancozeb application. Fungal isolates (N=105) were collected from several locations within the canopy in replicated trees in the same orchard. Weather sensors also monitored the microclimates within those tree canopies. Spray deposition, microclimate and SI resistance were influenced by canopy location. The fourth objective of this research was to investigate potential SI resistance mechanisms. Previously classified isolates were screened for point mutations within the CYP51A1 gene (Appendix C), differences in polymorphic bands (alleles) (Appendix D), and differences in metabolism of myclobutanil (Appendix E). The consensus sequences for the CYP51A1 gene were identical for all isolates tested (N=9), and results from amplified fragment length polymorphism experiment (N=82) were inconclusive. There were, however, significant differences among incubation time and myclobutanil concentration in the bioassay (N=11). Our results indicate that myclobutanil is still an effective compound for control of apple scab in many areas of Virginia. / Ph. D.