Intermolecular Interactions of Noble-Gas-Containing Species

The importance of intermolecular interactions to chemistry, physics, and biology is difficult to overestimate. Without intermolecular forces, condensed phase matter could not form. The simplest way to categorize different types of intermolecular interactions is to describe them using van der Waals and hydrogen bonded (H-bonded) interactions. In the H-bond, the intermolecular interaction appears between a positively charged hydrogen atom and electronegative fragments and it originates from strong electrostatic interactions. H-bonding is important when considering the properties of condensed phase water and in many biological systems including the structure of DNA and proteins. Vibrational spectroscopy is a useful tool for studying complexes and the solvation of molecules. Vibrational frequency shift has been used to characterize complex formation. In an H-bonded system A∙∙∙H-X (A and X are acceptor and donor species, respectively), the vibrational frequency of the H-X stretching vibration usually decreases from its value in free H-X (red-shift). This frequency shift has been used as evidence for H-bond formation and the magnitude of the shift has been used as an indicator of the H-bonding strength. In contrast to this normal behavior are the blue-shifting H-bonds, in which the H-X vibrational frequency increases upon complex formation. In the last decade, there has been active discussion regarding these blue-shifting H-bonds. Noble-gases have been considered inert due to their limited reactivity with other elements. In the early 1930 s, Pauling predicted the stable noble-gas compounds XeF6 and KrF6. It was not until three decades later Neil Bartlett synthesized the first noble-gas compound, XePtF6, in 1962. A renaissance of noble-gas chemistry began in 1995 with the discovery of noble-gas hydride molecules at the University of Helsinki. The first hydrides were HXeCl, HXeBr, HXeI, HKrCl, and HXeH. These molecules have the general formula of HNgY, where H is a hydrogen atom, Ng is a noble-gas atom (Ar, Kr, or Xe), and Y is an electronegative fragment. At present, this class of molecules comprises 23 members including both inorganic and organic compounds. The first and only argon-containing neutral chemical compound HArF was synthesized in 2000 and its properties have since been investigated in a number of studies. A helium-containing chemical compound, HHeF, was predicted computationally, but its lifetime has been predicted to be severely limited by hydrogen tunneling. Helium and neon are the only elements in the periodic table that do not form neutral, ground state molecules. A noble-gas matrix is a useful medium in which to study unstable and reactive species including ions. A solvated proton forms a centrosymmetric NgHNg+ (Ng = Ar, Kr, and Xe) structure in a noble-gas matrix and this is probably the simplest example of a solvated proton. Interestingly, the hypothetical NeHNe+ cation is isoelectronic with the water-solvated proton H5O2+ (Zundel-ion). In addition to the NgHNg+ cations, the isoelectronic YHY- (Y = halogen atom or pseudohalogen fragment) anions have been studied with the matrix-isolation technique. These species have been known to exist in alkali metal salts (YHY)-M+ (M = alkali metal e.g. K or Na) for more than 80 years. Hydrated HF forms the FHF- structure in aqueous solutions, and these ions participate in several important chemical processes. In this thesis, studies of the intermolecular interactions of HNgY molecules and centrosymmetric ions with various species are presented. The HNgY complexes show unusual spectral features, e.g. large blue-shifts of the H-Ng stretching vibration upon complexation. It is suggested that the blue-shift is a normal effect for these molecules, and that originates from the enhanced (HNg)+Y- ion-pair character upon complexation. It is also found that the HNgY molecules are energetically stabilized in the complexed form, and this effect is computationally demonstrated for the HHeF molecule. The NgHNg+ and YHY- ions also show blue-shifts in their asymmetric stretching vibration upon complexation with nitrogen. Additionally, the matrix site structure and hindered rotation (libration) of the HNgY molecules were studied. The librational motion is a much-discussed solid state phenomenon, and the HNgY molecules embedded in noble-gas matrices are good model systems to study this effect. The formation mechanisms of the HNgY molecules and the decay mechanism of NgHNg+ cations are discussed. A new electron tunneling model for the decay of NgHNg+ absorptions in noble-gas matrices is proposed. Studies of the NgHNg+∙∙∙N2 complexes support this electron tunneling mechanism.

Characterization of Ion-Exchange Fibers for Controlled Drug Delivery

Increasing attention has been focused on methods that deliver pharmacologically active compounds (e.g. drugs, peptides and proteins) in a controlled fashion, so that constant, sustained, site-specific or pulsatile action can be attained. Ion-exchange resins have been widely studied in medical and pharmaceutical applications, including controlled drug delivery, leading to commercialisation of some resin based formulations. Ion-exchangers provide an efficient means to adjust and control drug delivery, as the electrostatic interactions enable precise control of the ion-exchange process and, thus, a more uniform and accurate control of drug release compared to systems that are based only on physical interactions. Unlike the resins, only few studies have been reported on ion-exchange fibers in drug delivery. However, the ion-exchange fibers have many advantageous properties compared to the conventional ion-exchange resins, such as more efficient compound loading into and release from the ion-exchanger, easier incorporation of drug-sized compounds, enhanced control of the ion-exchange process, better mechanical, chemical and thermal stability, and good formulation properties, which make the fibers attractive materials for controlled drug delivery systems. In this study, the factors affecting the nature and strength of the binding/loading of drug-sized model compounds into the ion-exchange fibers was evaluated comprehensively and, moreover, the controllability of subsequent drug release/delivery from the fibers was assessed by modifying the conditions of external solutions. Also the feasibility of ion-exchange fibers for simultaneous delivery of two drugs in combination was studied by dual loading. Donnan theory and theoretical modelling were applied to gain mechanistic understanding on these factors. The experimental results imply that incorporation of model compounds into the ion-exchange fibers was attained mainly as a result of ionic bonding, with additional contribution of non-specific interactions. Increasing the ion-exchange capacity of the fiber or decreasing the valence of loaded compounds increased the molar loading, while more efficient release of the compounds was observed consistently at conditions where the valence or concentration of the extracting counter-ion was increased. Donnan theory was capable of fully interpreting the ion-exchange equilibria and the theoretical modelling supported precisely the experimental observations. The physico-chemical characteristics (lipophilicity, hydrogen bonding ability) of the model compounds and the framework of the fibrous ion-exchanger influenced the affinity of the drugs towards the fibers and may, thus, affect both drug loading and release. It was concluded that precisely controlled drug delivery may be tailored for each compound, in particularly, by choosing a suitable ion-exchange fiber and optimizing the delivery system to take into account the external conditions, also when delivering two drugs simultaneously.

Biophysical Characterization of Oxidized Phospholipids : Implications for Altered Membrane Structure and Function

The present study aims to elucidate the modifications in the structure and functionality of the phospholipid matrix of biological membranes brought about by free radical-mediated oxidative damage of its molecular constituents. To this end, the surface properties of two oxidatively modified phospholipids bearing an aldehyde or carboxyl function at the end of truncated sn-2 acyl chain were studied using a Langmuir balance. The results obtained reveal both oxidized species to have a significant impact on the structural dynamics of phospholipid monolayers, as illustrated by the progressive changes in force-area isotherms with increasing mole fraction of the oxidized lipid component. Moreover, surface potential measurements revealed considerable modifications in the electric properties of oxidized phospholipid containing monolayers during film compression, suggesting a packing state-controlled reorientation of the intramolecular electric dipoles of the lipid headgroups and acyl chains. Based on the above findings, a model describing the conformational state of oxidized phospholipid molecules in biological membranes is proposed, involving the protrusion of the acyl chains bearing the polar functional groups out from the hydrocarbon phase to the surrounding aqueous medium. Oxidative modifications alter profoundly the physicochemical properties of unsaturated phospholipids and are therefore readily anticipated to have important implications for their interactions with membrane-associating molecules. Along these lines, the carboxyl group bearing lipid was observed to bind avidly the peripheral membrane protein cytochrome c. The binding was reversed following increase in ionic strength or addition of polyanionic ATP, thus suggesting it to be driven by electrostatic interactions between cationic residues of the protein and the deprotonated lipid carboxyl exposed to the aqueous phase. The presence of aldehyde function bearing oxidized phospholipid was observed to enhance the intercalation of four antimicrobial peptides into phospholipid monolayers and liposomal bilayers. Partitioning of the peptides to monolayers was markedly attenuated by the aldehyde scavenger methoxyamine, revealing it to be mediated by the carbonyl moiety possibly through efficient hydrogen bonding or, alternatively, formation of covalent adduct in form of a Schiff base between the lipid aldehydes and primary amine groups of the peptide molecules. Lastly, both oxidized phospholipid species were observed to bind with high affinity three small membrane-partitioning therapeutic agents, viz. chlorpromazine, haloperidol, and doxorubicin. In conclusion, the results of studies conducted using biomimetic model systems support the notion that oxidative damage influences the molecular architecture as well as the bulk physicochemical properties of phospholipid membranes. Further, common polar functional groups carried by phospholipids subjected to oxidation were observed to act as molecular binding sites at the lipid-water interface. It is thus plausible that oxidized phospholipid species may elicit cellular level effects by modulating integration of various membrane-embedded and surface-associated proteins and peptides, whose conformational state, oligomerization, and functionality is known to be controlled by highly specific lipid-protein interactions and proper physical state of the membrane environment.

Asymmetrical flow field-flow fractionation in the study of water-soluble macromolecules

Asymmetrical flow field-flow fractionation (AsFlFFF) was constructed, and its applicability to industrial, biochemical, and pharmaceutical applications was studied. The effect of several parameters, such as pH, ionic strength, temperature and the reactants mixing ratios on the particle sizes, molar masses, and the formation of aggregates of macromolecules was determined by AsFlFFF. In the case of industrial application AsFlFFF proved to be a valuable tool in the characterization of the hydrodynamic particle sizes, molar masses and phase transition behavior of various poly(N-isopropylacrylamide) (PNIPAM) polymers as a function of viscosity and phase transition temperatures. The effect of sodium chloride salt and the molar ratio of cationic and anionic polyelectrolytes on the hydrodynamic particle sizes of poly (methacryloxyethyl trimethylammonium chloride) and poly (ethylene oxide)-block-poly (sodium methacrylate) and their complexes were studied. The particle sizes of PNIPAM polymers, and polyelectrolyte complexes measured by AsFlFFF were in agreement with those obtained by dynamic light scattering. The molar masses of PNIPAM polymers obtained by AsFlFFF and size exclusion chromatography agreed also well. In addition, AsFlFFF proved to be a practical technique in thermo responsive behavior studies of polymers at temperatures up to about 50 oC. The suitability of AsFlFFF for biological, biomedical, and pharmaceutical applications was proved, upon studying the lipid-protein/peptide interactions, and the stability of liposomes at different temperatures. AsFlFFF was applied to the studies on the hydrophobic and electrostatic interactions between cytochrome c (a basic peripheral protein) and anionic lipid, and oleic acid, and sodium dodecyl sulphate surfactant. A miniaturized AsFlFFF constructed in this study was exploited in the elucidation of the effect of copper (II), pH, ionic strength, and vortexing on the particle sizes of low-density lipoproteins.

Quantum Chemical Studies of Intermolecular Interactions

This thesis studies the intermolecular interactions in (i) boron-nitrogen based systems for hydrogen splitting and storage, (ii) endohedral complexes, A@C60, and (iii) aurophilic dimers. We first present an introduction of intermolecular interactions. The theoretical background is then described. The research results are summarized in the following sections. In the boron-nitrogen systems, the electrostatic interaction is found to be the leading contribution, as 'Coulomb Pays for Heitler and London' (CHL). For the endohedral complex, the intermolecular interaction is formulated by a one-center expansion of the Coulomb operator 1/rab. For the aurophilic attraction between two C2v monomers, a London-type formula was derived by fully accounting for the anisotropy and point-group symmetry of the monomers.

Phospholipid cytochrome c interactions

The ability of the peripherally associated membrane protein cytochrome c (cyt c) to bind phospholipids in vitro was studied using fluorescence spectroscopy and large unilamellar liposomes. Previous work has shown that cyt c can bind phospholipids using two distinct mecha- nisms and sites, the A-site and the C-site. This binding is mediated by electrostatic or hydrophobic interactions, respectively. Here, we focus on the mechanism underlying these interactions. A chemically modified cyt c mutant Nle91 was used to study the ATP-binding site, which is located near the evolutionarily invariant Arg 91 on the protein surface. This site was also demonstrated to mediate phospholipid binding, possibly by functioning as a phospholipid binding site. Circular dichroism spectroscopy, time resolved fluorescence spectroscopy of zinc- porphyrin modified [Zn2+-heme] cyt c and liposome binding studies of the Nle91 mutant were used to demonstrate that ATP induces a conformational change in membrane- bound cyt c. The ATP-induced conformational changes were mediated by Arg 91 and were most pronounced in cyt c bound to phospholipids via the C-site. It has been previously reported that the hydrophobic interaction between phospho- lipids and cyt c (C-site) includes the binding of a phospholipid acyl chain inside the protein. In this mechanism, which is known as extended phospholipid anchorage, the sn-2 acyl chain of a membrane phospholipid protrudes out of the membrane surface and is able to bind in a hydrophobic cavity in cyt c. Direct evidence for this type of bind- ing mechanism was obtained by studying cyt c/lipid interaction using fluorescent [Zn2+- heme] cyt c and fluorescence quenching of brominated fatty acids and phospholipids. Under certain conditions, cyt c can form fibrillar protein-lipid aggregates with neg- atively charged phospholipids. These aggregates resemble amyloid fibrils, which are involved in the pathogenesis of many diseases. Congo red staining of these fibers con- firmed the presence of amyloid structures. A set of phospholipid-binding proteins was also found to form similar aggregates, suggesting that phospholipid-induced amyloid formation could be a general mechanism of amyloidogenesis.

Moonlighting Proteins of Lactobacillus crispatus : Extracellular Localization, Cell Wall Anchoring and Interactions with the Host

Moonlighting functions have been described for several proteins previously thought to localize exclusively in the cytoplasm of bacterial or eukaryotic cells. Moonlighting proteins usually perform conserved functions, e. g. in glycolysis or as chaperonins, and their traditional and moonlighting function(s) usually localize to different cell compartments. The most characterized moonlighting proteins in Grampositive bacteria are the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which function in bacteria-host interactions, e. g. as adhesins or plasminogen receptors. Research on bacterial moonlighting proteins has focused on Gram-positive bacterial pathogens, where many of their functions have been associated with bacterial virulence. In this thesis work I show that also species of the genus Lactobacillus have moonlighting proteins that carry out functions earlier associated with bacterial virulence only. I identified enolase, GAPDH, glutamine synthetase (GS), and glucose-6-phosphate isomerase (GPI) as moonlighting proteins of Lactobacillus crispatus strain ST1 and demonstrated that they are associated with cell surface and easily released from the cell surface into incubation buffer. I also showed that these lactobacillar proteins moonlight either as adhesins with affinity for basement membrane and extracellular matrix proteins or as plasminogen receptors. The mechanisms of surface translocation and anchoring of bacterial moonlighting proteins have remained enigmatic. In this work, the surface localization of enolase, GAPDH, GS and GPI was shown to depend on environmental factors. The members of the genus Lactobacillus are fermentative organisms that lower the ambient pH by producing lactic acid. At acidic pH enolase, GAPDH, GS and GPI were associated with the cell surface, whereas at neutral pH they were released into the buffer. The release did not involve de novo protein synthesis. I showed that purified recombinant His6-enolase, His6-GAPDH, His6-GS and His6-GPI reassociate with cell wall and bind in vitro to lipoteichoic acids at acidic pH. The in-vitro binding of these proteins localizes to cell division septa and cell poles. I also show that the release of moonlighting proteins is enhanced in the presence of cathelicidin LL- 37, which is an antimicrobial peptide and a central part of the innate immunity defence. I found that the LL-37-induced detachment of moonlighting proteins from cell surface is associated with cell wall permeabilization by LL-37. The results in this thesis work are compatible with the hypothesis that the moonlighting proteins of L. crispatus associate to the cell wall via electrostatic or ionic interactions and that they are released into surroundings in stress conditions. Their surface translocation is, at least in part, a result from their release from dead or permeabilized cells and subsequent reassociation onto the cell wall. The results of this thesis show that lactobacillar cells rapidly change their surface architecture in response to environmental factors and that these changes influence bacterial interactions with the host.

Interactions of natural products with β-lactoglobulins, members of the lipocalin family

Natural products constitute an important source of new drugs. The bioavailability of the drugs depends on their absorption, distribution, metabolism and elimination. To achieve good bioavailability, the drug must be soluble in water, stable in the gastrointestinal tract and palatable. Binding proteins may improve the solubility of drug compounds, masking unwanted properties, such as bad taste, bitterness or toxicity, transporting or protecting these compounds during processing and storage. The focus of this thesis was to study the interactions, including ligand binding and the effect of pH and temperature, of bovine and reindeer β-lactoglobulin (βLG) with such compounds as retinoids, phenolic compounds as well as with compounds from plant extracts, and to investigate the transport properties of the βLG-ligand complex. To examine the binding interactions of different ligands to βLG, new methods were developed. The fluorescence binding method for the evaluation of ligand binding to βLG was miniaturized from a quartz cell to a 96-well plate. A method of ultrafiltration sampling combined with high-performance liquid chromatography was developed to assess the binding of compounds from extracts. The interactions of phenolic compounds or retinoids and βLG were investigated using the 96-well plate method. The majority of flavones, flavonols, flavanones and isoflavones and all of the retinoids included were shown to bind to bovine and reindeer βLG. Phenolic compounds, contrary to retinol, were not released at acidic pH. Those results suggest that βLG may have more binding sites, probably also on the surface of βLG. An extract from Camellia sinensis (L.) O. Kunze (black tea), Urtica dioica L. (nettle) and Piper nigrum (black pepper) were used to evaluate whether βLG could bind compounds from plant extracts. Piperine from P. nigrum was found to bind tightly and rutin from U. dioica weakly to βLG. No components from C. sinensis bound to βLG in our experiment. The uptake and membrane permeation of bovine and reindeer βLG, free and bound with retinol, palmitic acid and cholesterol, were investigated using Caco-2 cell monolayers. Both bovine and reindeer βLG were able to cross the Caco-2 cell membrane. Free and βLG-bound retinol and palmitic acid were transported equally, whereas cholesterol could not cross the Caco-2 cell monolayer free or bound to βLG. Our results showed that βLG can bind different natural product compounds, but cannot enhance transport of retinol, palmitic acid or cholesterol through Caco-2 cells. Despite this, βLG, as a water-soluble binding protein, may improve the solubility of natural compounds, possibly protecting them from early degradation and transporting some of them through the stomach. Furthermore, it may decrease their bad or bitter taste during oral administration of drugs or in food preparations. βLG can also enhance or decrease the health benefits of herbal teas and food preparations by binding compounds from extracts.

Molecular Mechanisms of Androgen Receptor Interactions

The androgen receptor (AR) mediates the effects of the male sex-steroid hormones (androgens), testosterone and 5?-dihydrotestosterone. Androgens are critical in the development and maintenance of male sexual characteristics. AR is a member of the steroid receptor ligand-inducible transcription factor family. The steroid receptor family is a subgroup of the nuclear receptor superfamily that also includes receptors for the active forms of vitamin A, vitamin D3, and thyroid hormones. Like all nuclear receptors, AR has a conserved modular structure consisting of a non-conserved amino-terminal domain (NTD), containing the intrinsic activation function 1, a highly conserved DNA-binding domain, and a conserved ligand-binding domain (LBD) that harbors the activation function 2. Each of these domains plays an important role in receptor function and signaling, either via intra- and inter-receptor interactions, interactions with specific DNA sequences, termed hormone response elements, or via functional interactions with domain-specific proteins, termed coregulators (coactivators and corepressors). Upon binding androgens, AR acquires a new conformational state, translocates to the nucleus, binds to androgen response elements, homodimerizes and recruits sequence-specific coregulatory factors and the basal transcription machinery. This set of events is required to activate gene transcription (expression). Gene transcription is a strictly modulated process that governs cell growth, cell homeostasis, cell function and cell death. Disruptions of AR transcriptional activity caused by receptor mutations and/or altered coregulator interactions are linked to a wide spectrum of androgen insensitivity syndromes, and to the pathogenesis of prostate cancer (CaP). The treatment of CaP usually involves androgen depletion therapy (ADT). ADT achieves significant clinical responses during the early stages of the disease. However, under the selective pressure of androgen withdrawal, androgen-dependent CaP can progress to an androgen-independent CaP. Androgen-independent CaP is invariably a more aggressive and untreatable form of the disease. Advancing our understanding of the molecular mechanisms behind the switch in androgen-dependency would improve our success of treating CaP and other AR related illnesses. This study evaluates how clinically identified AR mutations affect the receptor s transcriptional activity. We reveal that a potential molecular abnormality in androgen insensitivity syndrome and CaP patients is caused by disruptions of the important intra-receptor NTD/LBD interaction. We demonstrate that the same AR LBD mutations can also disrupt the recruitment of the p160 coactivator protein GRIP1. Our investigations reveal that 30% of patients with advanced, untreated local CaP have somatic mutations that may lead to increases in AR activity. We report that somatic mutations that activate AR may lead to early relapse in ADT. Our results demonstrate that the types of ADT a CaP patient receives may cause a clustering of mutations to a particular region of the receptor. Furthermore, the mutations that arise before and during ADT do not always result in a receptor that is more active, indicating that coregulator interactions play a pivotal role in the progression of androgen-independent CaP. To improve CaP therapy, it is necessary to identify critical coregulators of AR. We screened a HeLa cell cDNA library and identified small carboxyl-terminal domain phosphatase 2 (SCP2). SCP2 is a protein phosphatase that directly interacts with the AR NTD and represses AR activity. We demonstrated that reducing the endogenous cellular levels of SCP2 causes more AR to load on to the prostate specific antigen (PSA) gene promoter and enhancer regions. Additionally, under the same conditions, more RNA polymerase II was recruited to the PSA promoter region and overall there was an increase in androgen-dependent transcription of the PSA gene, revealing that SCP2 could play a role in the pathogenesis of CaP.

Implications of co-twin dependence for twins' social interactions, mental health and alcohol use : A follow-up study of Finnish twins from adolescence to early adulthood

Objective: The aim of the present study was to examine co-twin dependence and its impact on twins' social contacts, leisure-time activities and psycho-emotional well-being. The role of co-twin dependence was also examined as a moderator of genetic and environmental influences on alcohol use in adolescence and in early adulthood. Methods: The present report is based on the Finnish Twin Cohort Study (FinnTwin16), a population-based study of five consecutive birth cohorts of Finnish twins born in the years 1975-1979. Baseline assessments were collected through mailed questionnaires, within two months of the twins' sixteenth birthday yielding replies from 5563 twin individuals. All respondent twins were sent follow-up questionnaires at ages of 17, 18½, and in early adulthood, when twins were 22-27 years old. Measures: The questionnaires included a survey of health habits and attitudes, a symptom checklist and questions about twins' relationships with parents, peers and co-twin. Measures used were twins' self-reports of their own dependence and their co-twin's dependence at age 16, reports of twins' leisure-time activities and social contacts, alcohol use, psychological distress and somatic symptoms both in adolescence and in early adulthood. Results: In the present study 25.6% of twins reported dependence on their co-twin. There were gender and zygosity differences in dependence, females and MZ twins were more likely to report dependence than males and DZ twins. Co-twin dependence can be viewed on one hand as an individual characteristic, but on the other hand as a pattern of dyadic interaction that is mutually regulated and reciprocal. Most of the twins (80.7%) were either concordantly co-twin dependent or concordantly co-twin independent. The associations of co-twin dependence with twins' social interactions and psycho-emotional characteristics were relatively consistent both in adolescence and in early adulthood. Dependence was related to higher contact frequency and a higher proportion of shared leisure-time activities between twin siblings at the baseline and the follow-up. Additionally co-twin dependence was associated with elevated levels of psycho-emotional distress and somatic complaints, especially in adolescence. In the framework of gene-environment interaction, these results suggest that the genetic contribution to individual differences in drinking patterns is dependent on the nature of the pair-wise relationship of twin siblings. Conclusions: The results of this study indicate that co-twin dependence is a genuine feature of the co-twin relationship and shows the importance of studying the impact of various features of co-twin relationships on individual twins' social and psycho-emotional life and well-being. Our study also offers evidence that differences in inter-personal relationships contribute to the effects of genetic propensities.