Membrane properties of structurally modified ceramides : effects on lipid lateral distribution and sphingomyelin-interactions in artificial bilayer membranes

Ceramides comprise a class of sphingolipids that exist only in small amounts in cellular membranes, but which have been associated with important roles in cellular signaling processes. The influences that ceramides have on the physical properties of bilayer membranes reach from altered thermodynamical behavior to significant impacts on the molecular order and lateral distribution of membrane lipids. Along with the idea that the membrane physical state could influence the physiological state of a cell, the membrane properties of ceramides have gained increasing interest. Therefore, membrane phenomena related to ceramides have become a subject of intense study both in cellular as well as in artificial membranes. Artificial bilayers, the so called model membranes, are substantially simpler in terms of contents and spatio-temporal variation than actual cellular membranes, and can be used to give detailed information about the properties of individual lipid species in different environments. This thesis focuses on investigating how the different parts of the ceramide molecule, i.e., the N-linked acyl chain, the long-chain sphingoid base and the membrane-water interface region, govern the interactions and lateral distribution of these lipids in bilayer membranes. With the emphasis on ceramide/sphingomyelin(SM)-interactions, the relevance of the size of the SMhead group for the interaction was also studied. Ceramides with methylbranched N-linked acyl chains, varying length sphingoid bases, or methylated 2N (amide-nitrogen) and 3O (C3-hydroxyl) at the interface region, as well as SMs with decreased head group size, were synthesized and their bilayer properties studied by calorimetric and fluorescence spectroscopic techniques. In brief, the results showed that the packing of the ceramide acyl chains was more sensitive to methyl-branching in the mid part than in the distal end of the N-linked chain, and that disrupting the interfacial structure at the amide-nitrogen, as opposed to the C3-hydroxyl, had greater effect on the interlipid interactions of ceramides. Interestingly, it appeared that the bilayer properties of ceramides could be more sensitive to small alterations in the length of the long-chain base than what was previously reported for the N-linked acyl chain. Furthermore, the data indicated that the SM-head group does not strongly influence the interactions between SMs and ceramides. The results in this thesis illustrate the pivotal role of some essential parts of the ceramide molecules in determining their bilayer properties. The thesis provides increased understanding of the molecular aspects of ceramides that possibly affect their functions in biological membranes, and could relate to distinct effects on cell physiology.

The use of protein structure/activity relationships in the rational design of stable particulate delivery systems

The recombinant heat shock protein (18 kDa-hsp) from Mycobacterium leprae was studied as a T-epitope model for vaccine development. We present a structural analysis of the stability of recombinant 18 kDa-hsp during different processing steps. Circular dichroism and ELISA were used to monitor protein structure after thermal stress, lyophilization and chemical modification. We observed that the 18 kDa-hsp is extremely resistant to a wide range of temperatures (60% of activity is retained at 80ºC for 20 min). N-Acylation increased its ordered structure by 4% and decreased its ß-T1 structure by 2%. ELISA demonstrated that the native conformation of the 18 kDa-hsp was preserved after hydrophobic modification by acylation. The recombinant 18 kDa-hsp resists to a wide range of temperatures and chemical modifications without loss of its main characteristic, which is to be a source of T epitopes. This resistance is probably directly related to its lack of organization at the level of tertiary and secondary structures.

Chemokines in the cerebrospinal fluid of patients with active and stable relapsing-remitting multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the human central nervous system. Although its etiology is unknown, the accumulation and activation of mononuclear cells in the central nervous system are crucial to its pathogenesis. Chemokines have been proposed to play a major role in the recruitment and activation of leukocytes in inflammatory sites. They are divided into subfamilies on the basis of the location of conserved cysteine residues. We determined the levels of some CC and CXC chemokines in the cerebrospinal fluid (CSF) of 23 relapsing-remitting MS patients under interferon-ß-1a therapy and 16 control subjects using ELISA. MS patients were categorized as having active or stable disease. CXCL10 was significantly increased in the CSF of active MS patients (mean ± SEM, 369.5 ± 69.3 pg/mL) when compared with controls (178.5 ± 29.1 pg/mL, P < 0.05). CSF levels of CCL2 were significantly lower in active MS (144.7 ± 14.4 pg/mL) than in controls (237.1 ± 16.4 pg/mL, P < 0.01). There was no difference in the concentration of CCL2 and CXCL10 between patients with stable MS and controls. CCL5 was not detectable in the CSF of most patients or controls. The qualitative and quantitative differences of chemokines in CSF during relapses of MS suggest that they may be useful as a marker of disease activity and of the mechanisms involved in the pathogenesis of the disease.

Evaluation of anti-Wnt/β-catenin signaling agents by pGL4-TOP transfected stable cells with a luciferase reporter system

Refractory and relapsed leukemia is a major problem during cancer therapy, which is due to the aberrant activation of Wnt/β-catenin signaling pathway. Activation of this pathway is promoted by wingless (Wnt) proteins and induces co-activator β-catenin binding to lymphoid enhancer factor (LEF)/T-cell factor protein (TCF). To provide a convenient system for the screening of anti-Wnt/β-catenin agents, we designed a bi-functional pGL4-TOP reporter plasmid that contained 3X β-catenin/LEF/TCF binding sites and a selectable marker. After transfection and hygromycin B selection, HEK 293-TOP and Jurkat-TOP stable clones were established. The luciferase activity in the stable clone was enhanced by the recombinant Wnt-3A (rWnt-3A; 100-400 ng/mL) and GSK3β inhibitor (2’Z,3’E)-6-bromoindirubin-3’-oxime (BIO; 5 µM) but was inhibited by aspirin (5 mM). Using this reporter model, we found that norcantharidin (NCTD; 100 µM) reduced 80% of rWnt-3A-induced luciferase activity. Furthermore, 50 µM NCTD inhibited 38% of BIO-induced luciferase activity in Jurkat-TOP stable cells. Employing ³H-thymidine uptake assay and Western blot analysis, we confirmed that NCTD (50 µM) significantly inhibited proliferation of Jurkat cells by 64%, which are the dominant β-catenin signaling cells and decreased β-catenin protein in a concentration-dependent manner. Thus, we established a stable HEK 293-TOP clone and successfully used it to identify the Wnt/β-catenin signaling inhibitor NCTD.

Diastolic function is associated with quality of life and exercise capacity in stable heart failure patients with reduced ejection fraction

Exercise capacity and quality of life (QOL) are important outcome predictors in patients with systolic heart failure (HF), independent of left ventricular (LV) ejection fraction (LVEF). LV diastolic function has been shown to be a better predictor of aerobic exercise capacity in patients with systolic dysfunction and a New York Heart Association (NYHA) classification ≥II. We hypothesized that the currently used index of diastolic function E/e' is associated with exercise capacity and QOL, even in optimally treated HF patients with reduced LVEF. This prospective study included 44 consecutive patients aged 55±11 years (27 men and 17 women), with LVEF<0.50 and NYHA functional class I-III, receiving optimal pharmacological treatment and in a stable clinical condition, as shown by the absence of dyspnea exacerbation for at least 3 months. All patients had conventional transthoracic echocardiography and answered the Minnesota Living with HF Questionnaire, followed by the 6-min walk test (6MWT). In a multivariable model with 6MWT as the dependent variable, age and E/e' explained 27% of the walked distance in 6MWT (P=0.002; multivariate regression analysis). No association was found between walk distance and LVEF or mitral annulus systolic velocity. Only normalized left atrium volume, a sensitive index of diastolic function, was associated with decreased QOL. Despite the small number of patients included, this study offers evidence that diastolic function is associated with physical capacity and QOL and should be considered along with ejection fraction in patients with compensated systolic HF.

From Escherichia coli heat-stable enterotoxin to mammalian endogenous guanylin hormones

The isolation of heat-stable enterotoxin (STa) from Escherichia coli and cholera toxin from Vibrio cholerae has increased our knowledge of specific mechanisms of action that could be used as pharmacological tools to understand the guanylyl cyclase-C and the adenylyl cyclase enzymatic systems. These discoveries have also been instrumental in increasing our understanding of the basic mechanisms that control the electrolyte and water balance in the gut, kidney, and urinary tracts under normal conditions and in disease. Herein, we review the evolution of genes of the guanylin family and STa genes from bacteria to fish and mammals. We also describe new developments and perspectives regarding these novel bacterial compounds and peptide hormones that act in electrolyte and water balance. The available data point toward new therapeutic perspectives for pathological features such as functional gastrointestinal disorders associated with constipation, colorectal cancer, cystic fibrosis, asthma, hypertension, gastrointestinal barrier function damage associated with enteropathy, enteric infection, malnutrition, satiety, food preferences, obesity, metabolic syndrome, and effects on behavior and brain disorders such as attention deficit, hyperactivity disorder, and schizophrenia.

A mass spectrometric study of some pesticides /|nK. S. Subramanian. -- 260 St. Catharines, Ont. : [s. n.],

The fragmentation processes in the mass spectra of a series of organophosphorus, organochlorine, thio and dithiocarbamate as well as a number of miscellaneous pesticides have been studied i n detail by using the Bendix timeof- flight, MS-12 single-focussing and MS-30 double-focussing mass spectrometers. Interpretation of all the spectra have been presented; their mode s of dissociation elucidated, aided by metastable transitions wherever possible and the structures of the various f ragmentation species postulated wherever f easible. The fragmentation mechanisms are based on the concepts of inductive, resonance and steric ef~ects. Multiple bond cleavages accompanied by simultaneous bond formation and rearrangement reactions involving cycli c t r ansition states have clarified t he formation of various ions . Due emphasis has been placed on the effect of the functional groups or substituents in altering the mass spectral behaviour of the pesticides as they form the basis for the identifi cation of the otherwise identical pesticides. The organophosphorus pesticides which have been studied include i) the phosphates (eg: DDVP and Phosdrin ); ii) phosphorothionates (eg: Parathion, 0-2, 4 dichloro phenyl 0, O-diethyl thionophosphate); iii) phosphorothioites (eg: Tributyl phosphorotrithioite); i V) phosphorothioates (eg: Ethion) and v) phosphorodithioates (eg: Carbophenolthion). Cleavages and rearrangements of the ester moiety dominate the spectrum of phosdrin while that of DDVP is + dominated by t he fragmentation modes of the (OH30)2P=0 + moiety. Fragmentation §f the (CH30)2P=S characterises the spectrum of (OH30)2"P -Cl while cleavages of the + (C2H50 )2P=S species mark the spectra of parathion and 0-2, 4- di chlorophenyl O, O-diethyl thiophosphate. The 0(, cl eavages of the thioether f unction rather than + cleavages of the (C2H50)2P=S signify the spectrum of carbophenolthion. Tributyl phosphorotrithioite behaves more like an aliphatic hydrocarbon than like the corresponding phosphites. The isopropyl and butyl esters of 2, 4 dichlorophenoxy acetic acid show cleavage and rearrangement ions typical of an ester. In spite of its structural similari ty to pp' - DDT and pp' - DDD, Kalthane has a completely different mass spectral behaviour due to the influence of its hydroxyl function. The thiocarbamate pesticides studied include Eptam and Perbulate. Both are structurally similar but having different alkyl substituents on nitrogen and sulphur. This structurQlsimilarity leads to similar types of (N-C), (O-S) and (S-alkyl cleavages). However, perbulate differs from Eptam in showing a rearrangement ion at mle 161 and in forming an isocyanate ion as the base peak. In Eptam the base peak i s the alkyl ion. The dithiocarbamate, Vegadex, resembles the thiocarbamates in undergoing simple cleavages but it differs from them in having a weak parent ion; in the formation of its base peak and in undergoing a series of rearrangement reactions. The miscellaneous pesticides studied include 1-Naphthalene acetic aCid- methyl ester, Fiperonyl butoxide and Allethrin. The ester i s stable to electron impact and shows only fewer ions. Piper onyl butoxide, a polyether, shows characteristics of an et her, alcohol and aldehyde . Allethrin is regarded as an ester of the type R-C-O-R1 with n R being a substituted cyclopr opane moiety and o Rt, a substituted cyclopentenone mOiety. Accordingly it shows cleavage ions typical of an aliphatic ester and undergoes bond ruptures of the cyclic moieties to give unusual ions. Its base peak is an odd electron ion, quite contrary to expectations.

Development of thermally stable versions of the Burgess Reagent : approaches to the chemoenzymatic total synthesis of morphine

The present studies describe our recent work on expanding the use of the Burgess reagent and its reaction with oxiranes. Several new variants of the Burgess reagent and its chiral auxiliary version were evaluated for their thermal stability by NMR spectroscopy. Three new versions of the reagent were synthesized and their stability was determined. The reactivity of all five Burgess reagents was compared in a dehydration reaction and reactions with epoxides and diols. Progress toward a chemoenzymatic synthesis of morphine is also included in this report. The synthesis began with the whole cell oxidation of bromobenzene by Escherichia coli JMI09(pDTG601). The preparation of several precursors for a key step involving the lohnson-Claisen rearrangement and progress toward the total synthesis are described.

Exact Multivariate Tests of Asset Pricing Models with Stable Asymmetric Distributions

In this paper, we propose exact inference procedures for asset pricing models that can be formulated in the framework of a multivariate linear regression (CAPM), allowing for stable error distributions. The normality assumption on the distribution of stock returns is usually rejected in empirical studies, due to excess kurtosis and asymmetry. To model such data, we propose a comprehensive statistical approach which allows for alternative - possibly asymmetric - heavy tailed distributions without the use of large-sample approximations. The methods suggested are based on Monte Carlo test techniques. Goodness-of-fit tests are formally incorporated to ensure that the error distributions considered are empirically sustainable, from which exact confidence sets for the unknown tail area and asymmetry parameters of the stable error distribution are derived. Tests for the efficiency of the market portfolio (zero intercepts) which explicitly allow for the presence of (unknown) nuisance parameter in the stable error distribution are derived. The methods proposed are applied to monthly returns on 12 portfolios of the New York Stock Exchange over the period 1926-1995 (5 year subperiods). We find that stable possibly skewed distributions provide statistically significant improvement in goodness-of-fit and lead to fewer rejections of the efficiency hypothesis.

Von Neumann-Morgenstern Stable Sets in Matching Problems

The following properties of the core of a one well-known: (i) the core is non-empty; (ii) the core is a lattice; and (iii) the set of unmatched agents is identical for any two matchings belonging to the core. The literature on two-sided matching focuses almost exclusively on the core and studies extensively its properties. Our main result is the following characterization of (von Neumann-Morgenstern) stable sets in one-to-one matching problem only if it is a maximal set satisfying the following properties : (a) the core is a subset of the set; (b) the set is a lattice; (c) the set of unmatched agents is identical for any two matchings belonging to the set. Furthermore, a set is a stable set if it is the unique maximal set satisfying properties (a), (b) and (c). We also show that our main result does not extend from one-to-one matching problems to many-to-one matching problems.

Étude d'un variant de la toxine STb produite par Escherichia coli

Les E. coli entérotoxinogènes (ETEC) sont souvent la cause de diarrhée post-sevrage chez le porc. Deux types d’entérotoxines sont retrouvées chez les ETEC, soit les thermolabiles, comme la toxine LT, et les thermostables, comme EAST-1, STa et STb. Cette dernière est composée de 48 acides aminés et est impliquée dans la pathologie causée par les ETEC. Pour la première fois un variant de la toxine STb fut découvert dans une étude. Nous avons alors émis l’hypothèse qu’il y a présence de variants dans la population de souches ETEC du Québec. Dans les 100 souches STb+ analysées, 23 possédaient le gène de la toxine avec une variation dans la séquence génétique : l’asparagine était présente en position 12 remplaçant ainsi l’histidine. Une corrélation entre la présence du variant et la présence de facteurs de virulence retrouvés dans ces 100 souches ETEC étudiées a été effectuée. Ce variant semble fortement associé à la toxine STa puisque toutes les souches variantes ont hybridé avec le gène codant pour cette dernière. Étant donné sa présence répandue dans la population de souches ETEC du Québec, nous avons de plus émis l’hypothèse que ce variant a des caractéristiques biologiques altérées par rapport à la toxine sauvage. L’analyse par dichroïsme circulaire a montré que le variant et la toxine sauvage ont une structure secondaire ainsi qu’une stabilité similaires. Par la suite, l’attachement au récepteur de la toxine, le sulfatide, a été étudié par résonnance plasmonique de surface (biacore). Le variant a une affinité au sulfatide légèrement réduite comparativement à la toxine sauvage. Puisque l’internalisation de la toxine fut observée dans une étude précédente et qu’elle semble liée à la toxicité, nous avons comparé l’internalisation du variant et de la toxine sauvage à l’intérieur des cellules IPEC-J2. L’internalisation du variant dans les cellules est légèrement supérieure à l’internalisation de la toxine sauvage. Ces résultats suggèrent que le variant est biochimiquement et structurellement comparable à la toxine sauvage.

The Effects of the Length of the Period of Commitment on the Size of Stable International Environmental Agreements

This paper extends the standard model of self-enforcing dynamic international environmental agreements by allowing the length of the period of commitment of such agreements to vary as a parameter. It analyzes the pattern of behavior of the size of stable coalitions, the stock of pollutant and the emission rate as a function of the length of the period of commitment. It is shown that the length of the period of commitment can have very significant effects on the equilibrium. Three distinct intervals for the length of the period of commitment are identified, across which the equilibrium and its dynamic behavior differ considerably. Whereas for sufficiently high values of the period of commitment only self-enforcing agreements of two countries are possible, for sufficiently low such values full cooperation can be generated. Lengths of periods of commitment between those two thresholds are characterized by an inverse relationship between the length of commitment and the membership size of the agreement. This suggests that considerable attention should be given to the determination of the length of such international agreements.

Copper Doped Methylene Blue Sensitized Poly(vinyl alcohol)–Acrylamide Films for Stable Diffraction Efficiency

Copper doped methylene blue sensitized poly(vinyl alcohol) (MBPVA)–acrylamide films were fabricated to improve the storage life of recorded gratings. The films were fabricated using gravity settling method and the copper chloride concentration was optimized as 3:18 10 3 mol/l for a dye concentration of 6:2 10 4 mol/l. The gratings recorded on the optimized film constitution could be stored for months with stable diffraction efficiency (24%) without any chemical or thermal fixing techniques. The resolution of the material is found to be unaffected with the addition of copper chloride.