A computational analysis of substrate binding strength by phosphorylase kinase and protein kinase A

Protein kinases exhibit various degrees of substrate specificity. The large number of different protein kinases in the eukaryotic proteomes makes it impractical to determine the specificity of each enzyme experimentally. To test if it were possible to discriminate potential substrates from non-substrates by simple computational techniques, we analysed the binding enthalpies of modelled enzyme-substrate complexes and attempted to correlate it with experimental enzyme kinetics measurements. The crystal structures of phosphorylase kinase and cAMP-dependent protein kinase were used to generate models of the enzyme with a series of known peptide substrates and non-substrates, and the approximate enthalpy of binding assessed following energy minimization. We show that the computed enthalpies do not correlate closely with kinetic measurements, but the method can distinguish good substrates from weak substrates and non-substrates. Copyright (C) 2002 John Wiley Sons, Ltd.

Surface miscibility of EPC/DOTAP/DOPE in binary and ternary mixed monolayers

Surface pressure (pi)-molecular area (A) curves were used to characterize the packing of pseudo-ternary mixed Langmuir monolayers of egg phosphatidylcholine (EPC), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and L-alpha-dioleoyl phosphatidylethanolamine (DOPE). This pseudo-ternary mixture EPC/DOPE/DOTAP has been successfully employed in liposome formulations designed for DNA non-viral vectors. Pseudo-binary mixtures were also studied as a control. Miscibility behavior was inferred from pi-A curves applying the additivity rule by calculating the excess free energy of mixture (Delta G(Exc)). The interaction between the lipids was also deduced from the surface compressional modulus (C(s)(-1)). The deviation from ideality shows dependence on the lipid polar head type and monolayer composition. For lower DOPE concentrations, the forces are predominantly attractive. However, if the monolayer is DOPE rich, the DOTAP presence disturbs the PE-PE intermolecular interaction and the net interaction is then repulsive. The ternary monolayer EPC/DOPE/DOTAP presented itself in two configurations, modulated by the DOPE content, in a similar behavior to the DOPE/DOTAP monolayers. These results contribute to the understanding of the lipid interactions and packing in self-assembled systems associated with the in vitro and in vivo stability of liposomes. (C) 2010 Elsevier B.V. All rights reserved.

Structural effects of lipophilic methotrexate conjugates on model phospholipid biomembranes

Lipophilic conjugates of the antitumor drug methotrexate (MTX) with lipoamino acids (LAAs) have been previously described as a tool to enhance MTX passive entrance into cells, overcoming a form of transport resistance which makes tumour cells insensitive to the antimetabolite. A knowledge of the mechanisms of interaction of such lipophilic derivatives with cell membranes could be useful for planning further lipophilic MTX derivatives with an optimal antitumour activity. To this aim, a calorimetric study was undertaken using a biomembrane model made from synthetic 1,2-dipalmitoyl-glycero-3-phosphocholine (DPPC) multilamellar liposomes. The effects of MTX and conjugates on the phase transition of liposomes were investigated using differential scanning calorimetry. The interaction of pure MTX with the liposomes was limited to the outer part of the phospholipid bilayers, due to the polar nature of the drug. Conversely, its lipophilic conjugates showed a hydrophobic kind of interaction, perturbing the packing order of DPPC bilayers. In particular, a reduction of the enthalpy of transition from the gel to the liquid crystal phase of DPPC membranes was observed. Such an effect was related to the structure and mole fraction of the conjugates in the liposomes. The antitumour activity of MTX conjugates was evaluated against cultures of a CCRF-CEM human leukemic T-cell line and a related MTX resistant sub-line. The in vitro cell growth inhibitory activity was higher for bis(tetradecyl) conjugates than for both the other shorter- and longer-chain derivatives. The biological effectiveness of the various MTX derivatives correlated very well with the thermotropic effects observed on the phase transition of DPPC biomembranes. (C), 2001 Elsevier Science B.V All rights reserved.

Dissolution of sucrose crystals in the anhydrous sorbitol melt

The dissolution of a sugar (sucrose as a model) with higher melting point was studied in a molten food polyol (sorbitol as a model) with lower melting point, both in anhydrous state. A DSC and optical examination revealed the dissolution of anhydrous sucrose crystals (mp 192 degreesC) in anhydrous sorbitol (mp 99 degreesC) liquid melt. The sucrose-sorbitol crystal mixtures at the proportions of 10, 30, 60, 100 and 150 g of sucrose per 100 g of sorbitol were heat scanned in a DSC to above melting endotherm of sorbitol but well below the onset temperature of melting of sucrose at three different temperatures 110, 130 and 150 degreesC. The heat scanning modes used were with or without isothermal holding. The dissolution of sucrose in the sorbitol liquid melt was manifested by an increase in the glass transition temperature of the melt and corresponding decrease in endothermic melting enthalpy of sucrose. At given experimental conditions, as high as 25 and 85% of sucrose dissolved in the sorbitol melt during 1 h of isothermal holding at 110 and 150 degreesC, respectively. Optical microscopic observation also clearly showed the reduction in the size of sucrose crystals in sorbitol melt during the isothermal holding at those temperatures. (C) 2003 Elsevier Science Ltd. All rights reserved.

A kinetic study of the thermally initiated free radical reactions of styrene with octanedithiol as a model for the reactions of multifunctional thiols with divinylbenzene

The kinetics of chain reactions of octanedithiol with styrene, thermally initiated with TX29B50 (a 50:50 wt% solution of TX29 diperoxy initiator in a phthalate plasticizer), have been studied over a range of initiator concentrations, a range of mixture formulations and a range of temperatures. This system has been investigated as a model system for the reactions of polyfunctional thiols with divinyl benzene. The reactions have been shown to follow first-order kinetics for both the thiol and the ene species and to be characterized by a dependence on the initiator concentration to the power of one half. The kinetic rate parameters have been shown to adhere to Arrhenius behaviour. A kinetic model for the chain reactions for this system has been proposed. (C) 2003 Society of Chemical Industry.

Study on solubility of pharmaceutical compounds in ionic liquids

Dissertation to obtain the degree of master in Chemical and Biochemical Engineering

Comparative effects of sediments contaminated by carcinogenic and non-carcinogenic PAHs in Dicentrarchus labrax: a semi-quantitative histopathological approach

Dissertação para obtenção do Grau de Mestre em Engenharia do Ambiente

X-ray diffraction, thermal analysis, and Raman scattering study of K2BeF4 and comparation to other member of the (beta)-K2SO4 family with ferroelectric -paraelectric transition

Thermal analysis, powder diffraction, and Raman scattering as a function of the temperature were carried out on K2BeF4. Moreover, the crystal structure was determined at 293 K from powder diffraction. The compound shows a transition from Pna21 to Pnam space group at 921 K with a transition enthalpy of 5 kJ/mol. The transition is assumed to be first order because the compound shows metastability. Structurally and spectroscopically the transition is similar to those observed in (NH4)2SO4, which suggests that the low-temperature phase is ferroelectric. In order to confirm it, the spontaneous polarization has been computed using an ionic model.

X-ray diffraction, thermal analysis, and Raman scattering study of K2BeF4 and comparation to other member of the (beta)-K2SO4 family with ferroelectric -paraelectric transition

Thermal analysis, powder diffraction, and Raman scattering as a function of the temperature were carried out on K2BeF4. Moreover, the crystal structure was determined at 293 K from powder diffraction. The compound shows a transition from Pna21 to Pnam space group at 921 K with a transition enthalpy of 5 kJ/mol. The transition is assumed to be first order because the compound shows metastability. Structurally and spectroscopically the transition is similar to those observed in (NH4)2SO4, which suggests that the low-temperature phase is ferroelectric. In order to confirm it, the spontaneous polarization has been computed using an ionic model.

Construção de um calorímetro isotérmico diferencial de alta sensibilidade e baixo custo

The high cost of sensitivity commercial calorimeters may represent an obstacle for many calorimetric research groups. This work describes the construction and calibration of a batch differential heat conduction calorimeter with sample cells volumes of about 400 μL. The calorimeter was built using two small high sensibility square Peltier thermoelectric sensors and the total cost was estimated to be about US$ 500. The calorimeter was used to study the excess enthalpy of solution of binary mixtures of liquids, as a function of composition, for the following binary systems of solvents: water + 1,4-dioxane or + dimethylsulfoxide at 298,2 ± 0,5 K.

Magnetic and transport properties of LaMnO3+δ, La1-xCaxMnO3, La1-xCaxMn1-yFeyO3 and La1-xSrxMn1-yFeyO3

Interest to hole-doped mixed-valence manganite perovskites is connected to the ‘colossal’ magnetoresistance. This effect or huge drop of the resistivity, ρ, in external magnetic field, B, attains usually the maximum value near the ferromagnetic Curie temperature, TC. In this thesis are investigated conductivity mechanisms and magnetic properties of the manganite perovskite compounds LaMnO3+, La1-xCaxMnO3, La1-xCaxMn1-yFeyO3 and La1- xSrxMn1-yFeyO3. When the present work was started the key role of the phase separation and its influence on the properties of the colossal magnetoresistive materials were not clear. Our main results are based on temperature dependencies of the magnetoresistance and magnetothermopower, investigated in the temperature interval of 4.2 - 300 K in magnetic fields up to 10 T. The magnetization was studied in the same temperature range in weak (up to 0.1 T) magnetic fields. LaMnO3+δ is the parent compound for preparation of the hole-doped CMR materials. The dependences of such parameters as the Curie temperature, TC, the Coulomb gap, Δ, the rigid gap, γ, and the localization radius, a, on pressure, p, are observed in LaMnO3+δ. It has been established that the dependences above can be interpreted by increase of the electron bandwidth and decrease of the polaron potential well when p is increased. Generally, pressure stimulates delocalization of the electrons in LaMnO3+δ. Doping of LaMnO3 with Ca, leading to La1-xCaxMnO3, changes the Mn3+/Mn4+ ratio significantly and brings an additional disorder to the crystal lattice. Phase separation in a form of mixture of the ferromagnetic and the spin glass phases was observed and investigated in La1- xCaxMnO3 at x between 0 and 0.4. Influence of the replacement of Mn by Fe is studied in La0.7Ca0.3Mn1−yFeyO3 and La0.7Sr0.3Mn1−yFeyO3. Asymmetry of the soft Coulomb gap and of the rigid gap in the density of localized states, small shift of the centre of the gaps with respect to the Fermi level and cubic asymmetry of the density of states are obtained in La0.7Ca0.3Mn1−yFeyO3. Damping of TC with y is connected to breaking of the double-exchange interaction by doping with Fe, whereas the irreversibility and the critical behavior of the magnetic susceptibility are determined by the phase separation and the frustrated magnetic state of La0.7Sr0.3Mn1−yFeyO3.

Physiological evaluation of heat stress in gestating sows under different housing systems in bedding and concrete floor

The purpose of this paper was to observe the use of bedding (wood shavings) in physiological variables that indicate thermal stress in gestating sows. The experiment was conducted in order to evaluate the effect of two types of floor (concrete and wood shavings). Worse microclimatic conditions were observed in bedding systems (P<0.05), with an increase in temperature and enthalpy of 1.14 ºC and 2.37 kJ.kg dry air-1, respectively. The floor temperature at the dirty area was higher in the bedding presence in comparison to its absence. In spite of the worse microclimatic conditions in the bedding, the rectal temperature did not differ significantly (P>0.05) but the skin surface temperature was higher in the bedding systems. The same occurred with the respiratory rates. The physical characteristics of the floor material influenced the rate of heat loss by conductance. Estimated values were 35.04 and 7.99 W m-2 for the conductive heat loss between the animal and floor for treatments with or without bedding, respectively. The use of bedding in sow rearing has a negative impact on microclimatic conditions, what implies in thermoregulatory damages.

Physiological potential of Brachiaria brizantha cv. Marandu seeds kept in contact with phosphatic fertilizers

The effect of mixture of seeds of Brachiaria brizantha, cv. Marandu, with different sources, granulometry, and phosphatic fertilizer doses during various periods of exposure on the physiological potential of the seeds has been assessed. The treatments consisted in seed exposure during periods of 0, 3, 6, 12, 24, 36, 48, 72, 96, and 120 h to the following fertilizers: ground granulated single superphosphate (SS), and powdered (SSp); and ground granulated ammonium monophosphate (AMP), at doses of 40 and 80 kg P2O5 ha-1. Tests of germination, tetrazolium, moisture content, and vigor (first count, electrical conductivity, emergence, emergence speed, and fresh mass of seedlings) were performed. It has been concluded that seed physiological potential of B. brizantha cv. Marandu is reduced with increase on the exposure period to phosphatic fertilizer. Such effect, however, is dependent on the product source, granulometry, and dose. SSp was the most harmful to seeds, followed by SSp and AMP, respectively. Moreover, considering a 60% germination rate as acceptable, it may be inferred that seeds can be kept in contact with AMP and SSp, in dose of 80 kg of P2O5 ha-1, respectively, for periods of 71.2 and 16.2 hours.

Elucidation of a mechanism of cell lysis by chorhexidine : a biophysical approach

Chlorhexidine is an effective antiseptic used widely in disinfecting products (hand soap), oral products (mouthwash), and is known to have potential applications in the textile industry. Chlorhexidine has been studied extensively through a biological and biochemical lens, showing evidence that it attacks the semipermeable membrane in bacterial cells. Although extremely lethal to bacterial cells, the present understanding of the exact mode of action of chlorhexidine is incomplete. A biophysical approach has been taken to investigate the potential location of chlorhexidine in the lipid bilayer. Deuterium nuclear magnetic resonance was used to characterize the molecular arrangement of mixed phospholipid/drug formulations. Powder spectra were analyzed using the de-Pake-ing technique, a method capable of extracting both the orientation distribution and the anisotropy distribution functions simultaneously. The results from samples of protonated phospholipids mixed with deuterium-labelled chlorhexidine are compared to those from samples of deuterated phospholipids and protonated chlorhexidine to determine its location in the lipid bilayer. A series of neutron scattering experiments were also conducted to study the biophysical interaction of chlorhexidine with a model phospholipid membrane of DMPC, a common saturated lipid found in bacterial cell membranes. The results found the hexamethylene linker to be located at the depth of the glycerol/phosphate region of the lipid bilayer. As drug concentration was increased in samples, a dramatic decrease in bilayer thickness was observed. Differential scanning calorimetry experiments have revealed a depression of the DMPC bilayer gel-to-lamellar phase transition temperature with an increasing drug concentration. The enthalpy of the transition remained the same for all drug concentrations, indicating a strictly drug/headgroup interaction, thus supporting the proposed location of chlorhexidine. In combination, these results lead to the hypothesis that the drug is folded approximately in half on its hexamethylene linker, with the hydrophobic linker at the depth of the glycerol/phosphate region of the lipid bilayer and the hydrophilic chlorophenyl groups located at the lipid headgroup. This arrangement seems to suggest that the drug molecule acts as a wedge to disrupt the bilayer. In vivo, this should make the cell membrane leaky, which is in agreement with a wide range of bacteriological observations.

X-ray diffraction, thermal analysis, and Raman scattering study of K2BeF4 and comparation to other member of the (beta)-K2SO4 family with ferroelectric -paraelectric transition

Thermal analysis, powder diffraction, and Raman scattering as a function of the temperature were carried out on K2BeF4. Moreover, the crystal structure was determined at 293 K from powder diffraction. The compound shows a transition from Pna21 to Pnam space group at 921 K with a transition enthalpy of 5 kJ/mol. The transition is assumed to be first order because the compound shows metastability. Structurally and spectroscopically the transition is similar to those observed in (NH4)2SO4, which suggests that the low-temperature phase is ferroelectric. In order to confirm it, the spontaneous polarization has been computed using an ionic model.