Polymerase chain reaction amplification of genomic fragments of bovine herpesvirus-1

Especial conditions were developed for the amplification of five DNA segments from US region of BHV-1 by polymerase chain reaction. In order to eliminate most nonspecific products it was found that addition of three cosolvents DMSO, glycerol and NP 40 was a simple method for increasing the specificity of amplification.

CO2:n käyttö puhdistuksessa ja inerttinä kaasuna öljynjalostusympäristössä

Tutkimuksen tavoitteena on löytää CO2:lle puhdistus- ja inertointikohteita öljynjalostusympäristöstä. CO2:na käytettäisiin Porvoon vetylaitokselta sivutuotteena tulevaa CO2:a. Vetylaitokselta saatava CO2-virta ei ole riittävän puhdasta käytettäväksi suoraan pesuissa ja inertoinnissa. CO2:n eri olomuotoja voidaan käyttää puhdistuksessa. Tutkimuksen lähtökohtana olleen ylikriittisen CO2:n tehokkuus perustuu sen liuottavuuteen. Huonosti liukenevien aineiden liukoisuus ylikriittiseen CO2:in paranee lisäaineiden ja pinta-aktiivisten aineiden käytöllä. Kiinteä CO2 jäädyttää ja poistaa epäpuhtauden sublimoitumisesta aiheutuvan paineaallon voimasta. Kuivajääpuhdistus soveltuu parhaiten tasaisten pintojen puhdistamiseen. Ylikriittisellä CO2:lla onnistuu nykyisellä teknologialla vain pienien kappaleiden puhdistaminen. Kuivajääpuhdistuksen toimivuutta kokeiltiin käytännössä Neste Oilin Porvoon jalostamolla hyvin tuloksin. Tasaisilta pinnoilta saatiin poistetuksi bitumia ja rasvakerros. Käyttökustannusvertailussa osoittautui ylikriittistä CO2:a käyttävä laitteisto halvemmaksi ja kuivajääpuhallus kalliimmaksi kuin konventionaaliset menetelmät. Säiliöiden paineistamiseen ja inertointiin käytetään yleisesti N2:ä. N2:llä inertoitavia kohteita voitaisiin korvata CO2:lla. CO2:n käyttöä rajoittavia seikkoja on hinta ja sen reaktiivisuus alkalimetallien kanssa. Vertailtaessa näiden kahden liukoisuuksia hiilivetyihin osoittautui CO2 monin kerroin liukoisemmaksi. Tämän ominaisuuden ansiosta CO2 voisi olla hyvä väliaine laitteiden hiilivetyvapaaksi saattamisessa.

The Hydrophobic interaction: modeling hydrophobic interactions and aggregation of non-polar particles in aqueous solutions

Malgré son importance dans notre vie de tous les jours, certaines propriétés de l?eau restent inexpliquées. L'étude des interactions entre l'eau et les particules organiques occupe des groupes de recherche dans le monde entier et est loin d'être finie. Dans mon travail j'ai essayé de comprendre, au niveau moléculaire, ces interactions importantes pour la vie. J'ai utilisé pour cela un modèle simple de l'eau pour décrire des solutions aqueuses de différentes particules. Récemment, l?eau liquide a été décrite comme une structure formée d?un réseau aléatoire de liaisons hydrogènes. En introduisant une particule hydrophobe dans cette structure à basse température, certaines liaisons hydrogènes sont détruites ce qui est énergétiquement défavorable. Les molécules d?eau s?arrangent alors autour de cette particule en formant une cage qui permet de récupérer des liaisons hydrogènes (entre molécules d?eau) encore plus fortes : les particules sont alors solubles dans l?eau. A des températures plus élevées, l?agitation thermique des molécules devient importante et brise les liaisons hydrogènes. Maintenant, la dissolution des particules devient énergétiquement défavorable, et les particules se séparent de l?eau en formant des agrégats qui minimisent leur surface exposée à l?eau. Pourtant, à très haute température, les effets entropiques deviennent tellement forts que les particules se mélangent de nouveau avec les molécules d?eau. En utilisant un modèle basé sur ces changements de structure formée par des liaisons hydrogènes j?ai pu reproduire les phénomènes principaux liés à l?hydrophobicité. J?ai trouvé une région de coexistence de deux phases entre les températures critiques inférieure et supérieure de solubilité, dans laquelle les particules hydrophobes s?agrègent. En dehors de cette région, les particules sont dissoutes dans l?eau. J?ai démontré que l?interaction hydrophobe est décrite par un modèle qui prend uniquement en compte les changements de structure de l?eau liquide en présence d?une particule hydrophobe, plutôt que les interactions directes entre les particules. Encouragée par ces résultats prometteurs, j?ai étudié des solutions aqueuses de particules hydrophobes en présence de co-solvants cosmotropiques et chaotropiques. Ce sont des substances qui stabilisent ou déstabilisent les agrégats de particules hydrophobes. La présence de ces substances peut être incluse dans le modèle en décrivant leur effet sur la structure de l?eau. J?ai pu reproduire la concentration élevée de co-solvants chaotropiques dans le voisinage immédiat de la particule, et l?effet inverse dans le cas de co-solvants cosmotropiques. Ce changement de concentration du co-solvant à proximité de particules hydrophobes est la cause principale de son effet sur la solubilité des particules hydrophobes. J?ai démontré que le modèle adapté prédit correctement les effets implicites des co-solvants sur les interactions de plusieurs corps entre les particules hydrophobes. En outre, j?ai étendu le modèle à la description de particules amphiphiles comme des lipides. J?ai trouvé la formation de différents types de micelles en fonction de la distribution des regions hydrophobes à la surface des particules. L?hydrophobicité reste également un sujet controversé en science des protéines. J?ai défini une nouvelle échelle d?hydrophobicité pour les acides aminés qui forment des protéines, basée sur leurs surfaces exposées à l?eau dans des protéines natives. Cette échelle permet une comparaison meilleure entre les expériences et les résultats théoriques. Ainsi, le modèle développé dans mon travail contribue à mieux comprendre les solutions aqueuses de particules hydrophobes. Je pense que les résultats analytiques et numériques obtenus éclaircissent en partie les processus physiques qui sont à la base de l?interaction hydrophobe.<br/><br/>Despite the importance of water in our daily lives, some of its properties remain unexplained. Indeed, the interactions of water with organic particles are investigated in research groups all over the world, but controversy still surrounds many aspects of their description. In my work I have tried to understand these interactions on a molecular level using both analytical and numerical methods. Recent investigations describe liquid water as random network formed by hydrogen bonds. The insertion of a hydrophobic particle at low temperature breaks some of the hydrogen bonds, which is energetically unfavorable. The water molecules, however, rearrange in a cage-like structure around the solute particle. Even stronger hydrogen bonds are formed between water molecules, and thus the solute particles are soluble. At higher temperatures, this strict ordering is disrupted by thermal movements, and the solution of particles becomes unfavorable. They minimize their exposed surface to water by aggregating. At even higher temperatures, entropy effects become dominant and water and solute particles mix again. Using a model based on these changes in water structure I have reproduced the essential phenomena connected to hydrophobicity. These include an upper and a lower critical solution temperature, which define temperature and density ranges in which aggregation occurs. Outside of this region the solute particles are soluble in water. Because I was able to demonstrate that the simple mixture model contains implicitly many-body interactions between the solute molecules, I feel that the study contributes to an important advance in the qualitative understanding of the hydrophobic effect. I have also studied the aggregation of hydrophobic particles in aqueous solutions in the presence of cosolvents. Here I have demonstrated that the important features of the destabilizing effect of chaotropic cosolvents on hydrophobic aggregates may be described within the same two-state model, with adaptations to focus on the ability of such substances to alter the structure of water. The relevant phenomena include a significant enhancement of the solubility of non-polar solute particles and preferential binding of chaotropic substances to solute molecules. In a similar fashion, I have analyzed the stabilizing effect of kosmotropic cosolvents in these solutions. Including the ability of kosmotropic substances to enhance the structure of liquid water, leads to reduced solubility, larger aggregation regime and the preferential exclusion of the cosolvent from the hydration shell of hydrophobic solute particles. I have further adapted the MLG model to include the solvation of amphiphilic solute particles in water, by allowing different distributions of hydrophobic regions at the molecular surface, I have found aggregation of the amphiphiles, and formation of various types of micelle as a function of the hydrophobicity pattern. I have demonstrated that certain features of micelle formation may be reproduced by the adapted model to describe alterations of water structure near different surface regions of the dissolved amphiphiles. Hydrophobicity remains a controversial quantity also in protein science. Based on the surface exposure of the 20 amino-acids in native proteins I have defined the a new hydrophobicity scale, which may lead to an improvement in the comparison of experimental data with the results from theoretical HP models. Overall, I have shown that the primary features of the hydrophobic interaction in aqueous solutions may be captured within a model which focuses on alterations in water structure around non-polar solute particles. The results obtained within this model may illuminate the processes underlying the hydrophobic interaction.<br/><br/>La vie sur notre planète a commencé dans l'eau et ne pourrait pas exister en son absence : les cellules des animaux et des plantes contiennent jusqu'à 95% d'eau. Malgré son importance dans notre vie de tous les jours, certaines propriétés de l?eau restent inexpliquées. En particulier, l'étude des interactions entre l'eau et les particules organiques occupe des groupes de recherche dans le monde entier et est loin d'être finie. Dans mon travail j'ai essayé de comprendre, au niveau moléculaire, ces interactions importantes pour la vie. J'ai utilisé pour cela un modèle simple de l'eau pour décrire des solutions aqueuses de différentes particules. Bien que l?eau soit généralement un bon solvant, un grand groupe de molécules, appelées molécules hydrophobes (du grecque "hydro"="eau" et "phobia"="peur"), n'est pas facilement soluble dans l'eau. Ces particules hydrophobes essayent d'éviter le contact avec l'eau, et forment donc un agrégat pour minimiser leur surface exposée à l'eau. Cette force entre les particules est appelée interaction hydrophobe, et les mécanismes physiques qui conduisent à ces interactions ne sont pas bien compris à l'heure actuelle. Dans mon étude j'ai décrit l'effet des particules hydrophobes sur l'eau liquide. L'objectif était d'éclaircir le mécanisme de l'interaction hydrophobe qui est fondamentale pour la formation des membranes et le fonctionnement des processus biologiques dans notre corps. Récemment, l'eau liquide a été décrite comme un réseau aléatoire formé par des liaisons hydrogènes. En introduisant une particule hydrophobe dans cette structure, certaines liaisons hydrogènes sont détruites tandis que les molécules d'eau s'arrangent autour de cette particule en formant une cage qui permet de récupérer des liaisons hydrogènes (entre molécules d?eau) encore plus fortes : les particules sont alors solubles dans l'eau. A des températures plus élevées, l?agitation thermique des molécules devient importante et brise la structure de cage autour des particules hydrophobes. Maintenant, la dissolution des particules devient défavorable, et les particules se séparent de l'eau en formant deux phases. A très haute température, les mouvements thermiques dans le système deviennent tellement forts que les particules se mélangent de nouveau avec les molécules d'eau. A l'aide d'un modèle qui décrit le système en termes de restructuration dans l'eau liquide, j'ai réussi à reproduire les phénomènes physiques liés à l?hydrophobicité. J'ai démontré que les interactions hydrophobes entre plusieurs particules peuvent être exprimées dans un modèle qui prend uniquement en compte les liaisons hydrogènes entre les molécules d'eau. Encouragée par ces résultats prometteurs, j'ai inclus dans mon modèle des substances fréquemment utilisées pour stabiliser ou déstabiliser des solutions aqueuses de particules hydrophobes. J'ai réussi à reproduire les effets dûs à la présence de ces substances. De plus, j'ai pu décrire la formation de micelles par des particules amphiphiles comme des lipides dont la surface est partiellement hydrophobe et partiellement hydrophile ("hydro-phile"="aime l'eau"), ainsi que le repliement des protéines dû à l'hydrophobicité, qui garantit le fonctionnement correct des processus biologiques de notre corps. Dans mes études futures je poursuivrai l'étude des solutions aqueuses de différentes particules en utilisant les techniques acquises pendant mon travail de thèse, et en essayant de comprendre les propriétés physiques du liquide le plus important pour notre vie : l'eau.

Acetone enhances the direct analysis of procyanidin- and prodelphinidin-based condensed tannins in lotus species by the butanol-HCl-iron assay

The butanol-HCl spectrophotometric assay is widely used for quantifying extractable and insoluble condensed tannins (CT, syn. proanthocyanidins) in foods, feeds, and foliage of herbaceous and woody plants, but the method underestimates total CT content when applied directly to plant material. To improve CT quantitation, we tested various cosolvents with butanol-HCl and found that acetone increased anthocyanidin yields from two forage Lotus species having contrasting procyanidin and prodelphinidin compositions. A butanol-HCl-iron assay run with 50% (v/v) acetone gave linear responses with Lotus CT standards and increased estimates of total CT in Lotus herbage and leaves by up to 3.2-fold over the conventional method run without acetone. The use of thiolysis to determine the purity of CT standards further improved quantitation. Gel-state 13C and 1H–13C HSQC NMR spectra of insoluble residues collected after butanol-HCl assays revealed that acetone increased anthocyanidin yields by facilitating complete solubilization of CT from tissue.

Modulation of the activity and selectivity of the immobilized lipases by surfactants and solvents

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Enzyme engineering of aminotransferases for improved activity and thermostability

The production by biosynthesis of optically active amino acids and amines satisfies the pharmaceutical industry in its demand for chiral building blocks for the synthesis of various pharmaceuticals. Among several enzymatic methods that allow the synthesis of optically active aminoacids and amines, the use of minotransferase is a promising one due to its broad substrate specificity and no requirement for external cofactor regeneration. The synthesis of chiral compounds by aminotransferases can be done either by asymmetric synthesis starting from keto acids or ketones, and by kinetic resolution starting from racemic aminoacids or amines. The asymmetric synthesis of substituted (S)-aminotetralin, an active pharmaceutical ingredient (API), has shown to have two major factors that contribute to increasing the cost of production. These factors are the raw material cost of biocatalyst used to produce it and product loss during biocatalyst separation. To minimize the cost contribution of biocatalyst and to minimize the loss of product, two routes have been chosen in this research: 1. To engineer the aminotransferase biocatalyst to have greater specific activity, and 2. Improve the engineering of the process by immobilization of biocatalyst in calcium alginate and addition of cosolvents. An (S)-aminotransferase (Mutant CNB03-03) was immobilized, not as purified enzyme but as enzyme within spray dried cells, in calcium alginate beads and used to produce substituted (S)-aminotetralin at 50 °C and pH 7 in experiments where the immobilized biocatalyst was recycled. Initial rate of reaction for cycle 1 (6 hr duration) was determined to be 0.258 mM/min, for cycle 2 (20 hr duration) it decreased by ~50% compared to cycle 1, and for cycle 3 (20 hr duration) it decreased by ~90% compared to cycle 1 (immobilized preparation consisted of 50 mg of spray dried cells per gram of calcium alginate). Conversion to product for each cycle decreased as well, from 100% in cycle 1 (About 50 mM), 80% in cycle 2, and 30% after cycle 3. This mutant was determined to be deactivated at elevated temperatures during the reaction cycle and was not stable enough to allow multiple cycles in its immobilized form. A new mutant aminotransferase was isolated by applying error-prone polymerase chain reaction (PCR) on the gene coding for this enzyme and screening/selection: CNB04-01. This mutant showed a significant improvement in thermostability in comparison to CNB03-03. The new mutant was immobilized and tested under similar reaction conditions. Initial rate remained fairly constant (0.2 mM/min) over four cycles (each cycle with a duration of about 20 hours) with the mutant retaining almost 80% of initial rate in the fourth cycle. The final product concentrations after each cycle did not decrease during recycle experiments. Thermostability of CNB04-01 was much improved compared to CNB03-03. Under the same reaction conditions as stated above, the addition of co-solvents was studied in order to increase substituted tetralone solubility. Toluene and sodium dodecylsulfate (SDS) were used. SDS at 0.01% (w/v) allowed four recycles of the immobilized spray dried cells of CNB04-01, always reaching higher product concentration (80-85 mM) than the system with toluene at 3% (v/v) -70 mM-. The long term activity of immobilized CNB04-01 in a system with SDS 0.01% (w/v) at 50 °C, pH 7 was retained for three cycles (20 to 24 hours each one), reaching always final product concentration between 80-85 mM, but dropping precipitously in the fourth cycle to a final product concentration of 50 mM. Although significant improvement of immobilization on productivity and stability were observed using CNB04-01, another observation demonstrated the limitations of an immobilization strategy on reducing process costs. After analyzing the results of this experiment it was seen that a sudden drop occurred on final product concentration after the third recycle. This was due to product accumulation inside the immobilized preparation. In order to improve the economics of the process, research was focused on developing a free enzyme with an even higher activity, thus reducing raw material cost as well as improving biomass separation. A new enzyme was obtained (CNB05-01) using error-prone PCR and screening using as a template the gene derived from the previous improved enzyme. This mutant was determined to have 1.6 times the initial rate of CNB04-01 and had a higher temperature optimum (55°). This new enzyme would allow reducing enzyme loading in the reaction by five-fold compared to CNB03-03, when using it at concentration of one gram of spray dried cells per liter (completing the reaction after 20-24 hours). Also this mutant would allow reducing process time to 7-8 hours when used at a concentration of 5 grams of spray dried cells per liter compared to 24 hours for CNB03-03, assuming that the observations shown before are scalable. It could be possible to improve the economics of the process by either reducing enzyme concentration or reducing process time, since the production cost of the desired product is primarily a function of both enzyme concentration and process time.

In disperse solution, “osmotic stress” is a restricted case of preferential interactions

In the practice of “osmotic stress,” the effect of excluded cosolvents on a biochemical equilibrium is interpreted as the number of water molecules participating in the reaction. This action is attributed to lowering of solvent water activity by the cosolvent. This concept of osmotic stress in disperse solution is erroneous: (i) A cosolvent cannot be both excluded and inert, i.e., noninteracting, because exclusion requires a positive free energy change; (ii) a decrease in water activity alone by addition of solute cannot affect an equilibrium when the reacting surface is in contact with the solvent; and (iii) osmotic stress in disperse solution is a restricted case of preferential interactions; the reaction is driven by the free energy of cosolvent exclusion, and the derived number of water molecules is solely a measure of the mutual perturbations of the chemical potentials of the cosolvent and the protein.

Cell membrane fluctuations are regulated by medium macroviscosity: Evidence for a metabolic driving force

Extracellular fluid macroviscosity (EFM), modified by macromolecular cosolvents as occurs in body fluids, has been shown to affect cell membrane protein activities but not isolated proteins. In search for the mechanism of this phenomenon, we examined the effect of EFM on mechanical fluctuations of the cell membrane of human erythrocytes. The macroviscosity of the external medium was varied by adding to it various macromolecules [dextrans (70, 500, and 2,000 kDa), polyethylene glycol (20 kDa), and carboxymethyl-cellulose (100 kDa)], which differ in size, chemical nature, and in their capacity to increase fluid viscosity. The parameters of cell membrane fluctuations (maximal amplitude and half-width of amplitude distribution) were diminished with the elevation of solvent macroviscosity, regardless of the cosolvent used to increase EFM. Because thermally driven membrane fluctuations cannot be damped by elevation of EFM, the existence of a metabolic driving force is suggested. This is supported by the finding that in ATP-depleted red blood cells elevation of EMF did not affect cell membrane fluctuations. This study demonstrates that (i) EFM is a regulator of membrane dynamics, providing a possible mechanism by which EFM affects cell membrane activities; and (ii) cell membrane fluctuations are driven by a metabolic driving force in addition to the thermal one.

Surfactant coated aerosol powders and their properties

The hygroscopic growth of aerosols is an important factor effecting particle size. The consequence of the hygroscopic growth of pharrnaceutical aerosols is a change in their deposition characteristics, such that there is an increase in the total amount deposited in the lung. In this study the hygroscopic growth of disodium fluorescein (DF) aerosol powders was investigated by coating the powders with lauric and capric acids. The coating procedure was carried out in dichloromethane and chloroform, which acted as cosolvents for the fatty acids. An assessment of the extent and the nature of the coating was carried out. The qualitative assessment of the coating was achieved by infra-red spectroscopy, electronscanning chemical analysis and scanning electron microscopy. The quantitative analysis was carried out by differential refractometry, ultra-violet spectroscopy and gas liquid chromatography. These powders were generated under conditions approaching those in the lung, of 97 % relative humidity and 37"C. Coated and uncoated DF aerosol powders were introduced into a controlled temperature and relative humidity apparatus, designed and constructed for the investigation of hygroscopic growth in these studies. A vertical spinning disc device was used to generate the powders. Under conditions of controlled temperature and relative humidity mentioned, the growth ratio of disodium fluorescein alone was 1.45 compared with 1.68, for a nominal coating of DF with lauric acid of 0.12 gg-1, 1.0 for a nominal lauric acid coating of 0.2 gg-1, and 1.02 for a nominal capric acid coating of 0.18 gg-1. The range of control of hygroscopic growth of these aerosols has implications for the deposition of these preparations in the respiratory tract. These implications are discussed in the light of the current knowledge of the effects of hygroscopic growth on the deposition of pharmaceutical and environmental aerosols. A series of experiments in which pulmonary ventilation using a simple radioaerosol generator and delivery system are reported showing that particle size determination may be used to aid the design of diagnostic aerosol generators.

Azidoprofen as a soft anti-flammatory agent for the topical treatment of Psoriasis

Azidoprofen {2-(4-azidophenyl)propionic acid; AZP}, an azido-substituted arylalkanoic acid, was investigated as a model soft drug candidate for a potential topical non-steroidal anti-inflammatory agent (NSAIA). Reversed-phase high performance liquid chromatography (HPLC) methods were developed for the assay of AZP, a series of ester analogues and their· degradation products. 1H-NMR spectroscopy was also employed as an analytical method in selected cases. Reduction of the azido-group to the corresponding amine has been proposed as a potential detoxification mechanism for compounds bearing this substituent. An in vitro assay to measure the susceptibility of azides towards reduction was developed using dithiothreitol as a model reducing agent. The rate of reduction of AZP was found to be base-dependent, hence supporting the postulated mechanism of thiol-mediated reduction via nucleophilic attack by the thiolate anion. Prodrugs may enhance topical bioavailability through the manipulation of physico-chemical properties of the parent drug. A series of ester derivatives of AZP were investigated for their susceptibility to chemical and enzymatic hydrolysis, which regenerates the parent acid. Use of alcoholic cosolvents with differing alkyl functions to that of the ester resulted in transesterification reactions, which were found to be enzyme-mediated. The skin penetration of AZP was assessed using an in vitro hairless mouse skin model, and silastic membrane in some cases. The rate of permeation of AZP was found to be a similar magnitude to that of the well established NSAIA ibuprofen. Penetration rates were dependent on the vehicle pH and drug concentration when solutions were employed. In contrast, flux was independent of pH when suspension formulations were used. Pretreatment of the skin with various enhancer regimes, including oleic acid and azone in propylene glycol, promoted the penetration of AZP. An intense IR absorption due to the azide group serves as a highly diagnostic marker, enabling azido compounds to be detected in the outer layers of the· stratum corneum following their application to skin, using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). This novel application enabled a non-invasive examination of the percutaneous penetration enhancement of a model azido compound in vivo in man, in the presence of the enhancer oleic acid.