Effects of α-tocopherol analogs on lysosome membranes and fatty acid monolayers

The surface pressures of α-tocopherol analogs, fatty acids, and their mixtures were measured in their spread monolayers at an air—water interface. The surface pressure—area isotherms for the mixed monolayers of α-tocopherol and either stearic acid, oleic acid or linoleic acid deviated positively from those calculated on the basis of the additivity rule, and the magnitude depended on the length of the phytyl side chain in α-tocopherol and on the degree of unsaturation of the fatty acid chains. Lysosome membranes of mouse liver were stabilized by addition of α-tocopherol. A decrease in the length of the phytyl side chain in α-tocopherol reduced its ability to stabilize lysosome membranes. A good correlation was obtained between the extent of stabilizing activity of α-tocopherol analogs on lysosome membranes and the degree of positive deviation of the surface pressure for their mixtures with fatty acids.     

Effects of Hofmeister ions on the α-helical structure of proteins

The molecular conformation of proteins is sensitive to the nature of the aqueous environment. In particular, the presence of ions can stabilize or destabilize (denature) protein secondary structure. The underlying mechanisms of these actions are still not fully understood. Here, we combine circular dichroism (CD), single-molecule Förster resonance energy transfer, and atomistic computer simulations to elucidate salt-specific effects on the structure of three peptides with large α-helical propensity. CD indicates a complex ion-specific destabilization of the α-helix that can be rationalized by using a single salt-free computer simulation in combination with the recently introduced scheme of ion-partitioning between nonpolar and polar peptide surfaces. Simulations including salt provide a molecular underpinning of this partitioning concept. Furthermore, our single-molecule Förster resonance energy transfer measurements reveal highly compressed peptide conformations in molar concentrations of NaClO₄ in contrast to strong swelling in the presence of GdmCl. The compacted states observed in the presence of NaClO₄ originate from a tight ion-backbone network that leads to a highly heterogeneous secondary structure distribution and an overall lower α-helical content that would be estimated from CD. Thus, NaClO₄ denatures by inducing a molten globule-like structure that seems completely off-pathway between a fully folded helix and a coil state.     

Effects of macromolecular crowding on the structural stability of human α-lactalbumin

The folding of protein, an important process for protein to fulfill normal functions, takes place in crowded physiological environments. α-Lactalbumin, as a model system for protein-folding studies, has been used extensively because it can form stable molten globule states under a range of conditions. Here we report that the crowding agents Ficoll 70, dextran 70, and polyethylene glycol (PEG) 2000 have different effects on the structural stability of human α-lactalbumin (HLA) represented by the transition to a molten globule state: dextran 70 dramatically enhances the thermal stability of Ca(2+)-depleted HLA (apo-HLA) and Ficoll 70 enhances the thermal stability of apo-HLA to some extent, while PEG 2000 significantly decreases the thermal stability of apo-HLA. Ficoll 70 and dextran 70 have no obvious effects on trypsin degradation of apo-HLA but PEG 2000 accelerates apo-HLA degradation by trypsin and destabilizes the native conformation of apo-HLA. Furthermore, no interaction is observed between apo-HLA and Ficoll 70 or dextran 70, but a weak, non-specific interaction between the apo form of the protein and PEG 2000 is detected, and such a weak, non-specific interaction could overcome the excluded-volume effect of PEG 2000. Our data are consistent with the results of protein stability studies in cells and suggest that stabilizing excluded-volume effects of crowding agents can be ameliorated by non-specific interactions between proteins and crowders.     

Integrating the intrinsic conformational preferences of noncoded α-amino acids modified at the peptide bond into the noncoded amino acids database

Recently, we reported a database (Noncoded Amino acids Database; http://recerca.upc.edu/imem/index.htm) that was built to compile information about the intrinsic conformational preferences of nonproteinogenic residues determined by quantum mechanical calculations, as well as bibliographic information about their synthesis, physical and spectroscopic characterization, the experimentally established conformational propensities, and applications (Revilla-López et al., J Phys Chem B 2010;114:7413-7422). The database initially contained the information available for α-tetrasubstituted α-amino acids. In this work, we extend NCAD to three families of compounds, which can be used to engineer peptides and proteins incorporating modifications at the--NHCO--peptide bond. Such families are: N-substituted α-amino acids, thio-α-amino acids, and diamines and diacids used to build retropeptides. The conformational preferences of these compounds have been analyzed and described based on the information captured in the database. In addition, we provide an example of the utility of the database and of the compounds it compiles in protein and peptide engineering. Specifically, the symmetry of a sequence engineered to stabilize the 3(10)-helix with respect to the α-helix has been broken without perturbing significantly the secondary structure through targeted replacements using the information contained in the database.     

Effects of micromolar concentrations of manganese, copper, and zinc on α1-adrenoceptor-mediating contraction in rat aorta

To determine the influences of the Mn, Cu, and Zn on α₁-adrenoceptor (AR)-mediated vasoconstriction, we investigated their effects on vasoconstriction produced by the α₁-AR agonist phenylephrine in isolated rings of rat thoracic aorta. The cumulative concentration-contraction curves for phenylephrine were obtained in the absence and presence of Mn (0.3, 1, 3 μM), Cu (1, 10, 16 μM), and Zn (0.3, 1, 10 μM). Mn, Cu, and Zn each inhibited phenylephrine-mediated contraction in a dose-dependent manner. The maximal phenylephrine-induced contraction was significantly reduced by the pretreatment of the arterial rings with 10 and 16 μM Cu (p<0.05). The results suggest that variations in the plasma concentrations of metal might lead to changes in α₁-AR-mediated constrictive response.     

The Effects of Molecular Crowding on the Amyloid Fibril Formation of α-Lactalbumin and the Chaperone Action of α-Casein

Amyloid fibrils arise from the slow aggregation of intermediately folded protein states. In this study the kinetics of the protein fibril formation of α-lactalbumin and its prevention by αS-casein in the presence and absence of the crowding agent, dextran (68 kDa), have been compared using a thioflavin T binding assay. It was found that αS-casein, a molecular chaperone found in bovine milk, is a potent in vitro inhibitor of α-lactalbumin fibrillization. The effect of αS-casein in preventing fibril formation was significant, although less than it is in the absence of the crowding agent, dextran. The interaction between the chaperone and the α-lactalbumin and structural change in the target protein are also shown using intrinsic fluorescence intensity, an ANS binding assay, CD spectroscopy and size-exclusion HPLC. In summary, α-casein interacts with α-lactalbumin and prevents amyloid formation but not as well as it does when the crowding agent, dextran, not present.     

Effects of thiazole analogs of vitamin B1 on neuromuscular transmission and α-latrotoxin-induced transmitter release in skeletal muscles

Thiazole analogs of vitamin B₁ 3-decyloxycarbonylmethyl-4-methyl-5-(2-hydroxyethyl)thiazole chloride (DMHT) and 3-decyloxycarbonylmethyl-4-methylthiazole chloride (DMT) suppress quantum transmitter release from nerve terminals in the frog skeletal muscle. Intraperitoneal administration of these compounds to mice suppresses behavioral motor activity, diminishes motor coordination, and suppresses the corazol-induced seizures. Application of DMHT reduces the -latrotoxin-induced massive transmitter release from nerve terminals in the frog skeletal muscle and suppresses latrotoxin-induced seizures in mice. In model experiments, DMHT blocks Ca²⁺ entry through the ion channels formed by -latrotoxin in a bilayer lipid membrane. It has been suggested that the effectiveness of DMHT and DMT is determined by the presence of a thiazole cycle in their molecules that, among all endogenous biologically active compounds, is possessed only by vitamin B₁ and its metabolites.Neirofiziologiya/Neurophysiology, Vol. 27, No. 5/6, pp. 368–374, September–December, 1995.     

Effects of α-mangostin on mitochondrial energetic metabolism

Although α-mangostin prevents from toxicity associated to oxidative stress, it also promotes apoptotic cell death in cancer cells. Such effects have been associated with mitochondrial membrane depolarization and cytochrome c release. Therefore, the aim of this work was to analyze the potentially harmful effect of this natural compound on relevant parameters of mitochondrial function from normal tissue. Our results showed that α-mangostin protected mitochondria from peroxidative damage, but at high concentration, it acted as an uncoupler, reduced dramatically ADP-stimulated respiration and inhibited the activity of respiratory complex IV, making mitochondria prone to permeability transition, which is a mitochondrial player on cell fate.     

Effects of α-bungarotoxin on acetylcholine-induced response at the Helix neuronal membrane

The effects of -bungarotoxin (-BT) on two patterns of acetylcholine (ACh)-induced response differing in desensitization rate were investigated in isolated mollusk neurons using intracellular dialysis and concentration clamping techniques. It was found that -BT depressed both types of ACh-induced response — a reversible action in the majority of experiments performed. It also exerted a blocking effect on ACh-induced currents dependent on the presence of albumin, although albumin itself produced no noticeable change in ACh-induced response. Concentration dependence of -BT-induced blockade on both types of currents evoked by 1 and 10 µM ACh was investigated. The -BT concentrations producing a 50% suppression of the current evoked by 1 µM ACh were calculated by approximating concentration plots as (13.85±1.25)×10^–9 and (5.56±1.0)×10^–8 g/ml for type A and B cells respectively.Institute of Experimental Biology. Academy of Sciences of the Armenian SSR, Erevan. Translated from Neirofiziologiya, Vol. 21, No. 6, pp. 729–735, November–December, 1989.     

Inducible α-glucosidase of Mucor rouxii: Effects of dimorphism on the development of the wall-bound activity

Some properties of the inducible α-glucosidase system of Mucor rouxii were investigated. This enzymatic activity was induced after resuspending glucose-grown cells in a maltose-supplemented medium. The wall-bound activity of α-glucosidase was determined by using intact cells in the enzymatic assay; this activity represented from 80 to 90% of the total activity present in the induced cells. The addition of glucose before, or during, the induction period repressed α-glucosidase synthesis. α-Glucosidase induction was tested under aerobic and anaerobic conditions. It was found that the enzyme synthesis and the appearance of wall-bound activity were not affected by changing the gaseous environment. On the other hand, it was observed that anaerobically grown yeast-like cells were much less efficient than aerobic mycelia to develop wall-bound α-glucosidase activity. This could explain earlier observations about the incapacity of M. rouxii to utilize maltose as a substrate for anaerobic growth. This idea was strengthened by the fact that, if an anaerobic culture was induced to develop under a mycelial morphology by adding to the medium the chemical agent EDTA, these cells also acquired the capacity to grow on maltose and concomitantly possessed wall-bound α-glucosidase activity. The relevance of the structure of the cell wall on the capacity of M. rouxii to metabolize maltose is discussed.     

Effects of cultivation conditions on the production of heterologous α-galactosidase by Kluyveromyces lactis

Growth conditions relevant for the large-scale production of heterologous proteins with yeasts were studied on a laboratory scale. A strain of Kluyveromyces lactis, containing 15 copies of an expression cassette encoding guar -galactosidase integrated into its ribosomal DNA, was used as a model. By using urea as a nitrogen source, it was possible to produce active extracellular -galactosidase in shake-flask cultures grown on a defined mineral medium. Inclusion of urea instead of ammonium sulphate prevented unwanted acidification of cultures. With urea-containing mineral medium, enzyme production in shake flasks was comparable to that in complex media containing peptone. In contrast, the presence of peptone was required to achieve high productivity in chemostat cultures. The low productivity in chemostat cultures growing on mineral media was not due to loss oft the expression cassette, since addition of peptone to such cultures resulted in an immediate high rate of -galactosidase production. The discrepancy between the behaviour of shake-flask and chemostat cultures during growth on mineral medium illustrates the necessity of physiological studies for the scalling-up of heterologous protein production from laboratory to production scale.     

Effects of Calcium Ion on the Catalytic Activity of α-Chymotrypsin in Organic Solvents

Addition of small amounts of calcium ion markedly accelerated the transesterification of N-acetyl-l-tyrosine methyl ester to its ethyl ester by the catalysis of α-chymotrypsin in organic solvents. Maximum increase of the reaction rate was about 12-fold in the presence of 25 μm of calcium ion in ethanol. The rate increase was strongly dependent on calcium ion concentration and nature of organic solvents. Esterification of N-acetyl-l-tyrosine and hydrolysis of N-acetyl-l-tyrosine ethyl ester by α-chymotrypsin in organic solvents were also accelerated by calcium ion. The reactions obeyed Michaelis–Menten kinetics, and the acceleration of the reactions was due to the increase in k_cat.     

Can the stereochemical outcome of glycosylation reactions be controlled by the conformational preferences of the glycosyl donor?

Previous static and dynamical density functional theory studies of the 2,6-di-O-acetyl-3,4-O-isopropylidene-D-galactopyranosyl cations and their methanol adducts has led to an hypothesis that these cations exist in two families of conformers characterized as (2)S(O) and B(2,5), respectively. These families differ by ring inversion, each with its own reactivity. New calculations on the 2,6-di-O-acetyl-3,4-di-O-methyl-D-galactopyranosyl cation confirmed these trends. Removing the isopropylidene group allows more flexibility, but two families of conformers can be discerned with the monocyclic oxocarbenium ions in the E(3) conformation and the bicyclic dioxolenium ions in the (4)H(5) conformation. Attack on the beta-face of these monocyclic cations is favored by hydrogen bonding and the anomeric effect. The experimentally observed high beta-stereoselectivity of mannopyranosyl donors and high alpha-stereoselectivity of glucopyranosyl donors with the 4,6-O-benzylidene protecting groups can be rationalized assuming that the trans-fused 1,3-dioxane ring allows population of only one family of conformers. The combination of hydrogen bonding and conformational changes of the pyranose ring in response to the C-5[bond]O-5[bond]C-1[bond]C-2 torsion angle changes are identified as key factors in stereoselectivity. Based on these observations a strategy to design face discriminated glycosyl donors that exist predominantly in only one family of conformers is proposed.     

Effects of water content on the enantioselectivity of α-chymotrypsin catalysis in organic media

Summary The -chymotrypsin-catalyzed transesterification between N-trifluoroacetyl-DL-phenylalanine trifluoroethyl ester and 1-propanol was carried out in a variety of organic solvents. The addition of small quantities of water enhanced both the rate of reaction and enantioselectivity. A high enantioselectivity was achieved in ethyl acetate (E = 120), diethyl ether (86), or acetonitrile (60). The competing hydrolysis became significant at water content higher than 0.5% (w/w).     

Effects of phospholipids on the antioxidant activity of α-tocopherol in the singlet oxygen oxidation of canola oil

This study evaluated the effects of phosphatidylcholine (PC) or phosphatidylethanolamine (PE) on the antioxidative activity of α-tocopherol during oxidation of canola oil by singlet oxygen at 10°C for seven hours. Singlet oxygen was produced by chlorophyll b (4 ppm) under 1,700 lux. The oxidation of oil was evaluated by headspace oxygen consumption by gas chromatography and peroxide values (POVs). Concentrations of PC, PE, chlorophyll, and α-tocopherol were determined by HPLC. PC and PE protected chlorophyll from degradation, but they accelerated the degradation of α-tocopherol under singlet oxygen. Contents of PC and PE did not change for seven hours under singlet oxygen. α-Tocopherol significantly lowered POV and headspace oxygen consumption of canola oil under singlet oxygen, and its antioxidant activity was increased by the co-presence of PC and PE. PC and PE increased chemical quenching of singlet oxygen by tocopherol in decreasing the oil oxidation.     

Effects of Lanthionine Ketimine-5-Ethyl Ester on the α-Synucleinopathy Mouse Model

Neurochemical Research - Potentially druggable mechanisms underlying synaptic deficits seen in Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are under intense interrogations. In...     

Effects of succinylacetone on methyl α-d-glucoside uptake by the rat renal tubule

Succinylacetone, a catabolic end-product of tyrosine, is excreted in large quantities in urine from individuals with hereditary tyrosinemia and the Fanconi syndrome. Succinylacetone inhibits rat renal tubular concentrative uptake of the glucose transport analogue, methyl α-d-glucoside, in a noncompetitive and reversible fashion. This compound also depresses oxygen consumption by the rat renal tubule without fine structural damage to mitochondria. It is concluded that succinylacetone may be a useful probe in elucidation of the biochemical mechanism underlying the human Fanconi syndrome.     

Effects of Suramin on the ATP- and α,β-Methylene-ATP-induced Constriction of the Rabbit Ear Artery

Abstract

The isolated rabbit ear artery contains both dilation-mediating P_2Y-receptors and constriction-mediating P_2X-receptors. Suramin antagonizes the effects of ATP at either receptor.     

Effects of α-tocopherol on the lipid peroxidation and fluidity of porcine intestinal brush-border membranes

The effect of α-tocopherol on the lipid fluidity of porcine intestinal brush-border membranes was studied using pyrene as a fluorescent probe. Addition of α-tocopherol to the medium decreased fluorescence intensity and lifetime, but increased the fluorescence polarization of pyrene-labeled membranes. β-, γ-, and δ-Tocopherols gave no appreciable effect on the fluorescence intensity and polarization of the complex. The apparent dissociation constant (3.1 ± 0.12 μM) of the interaction of α-tocopherol with the membranes, estimated from the change in the fluorescence intensity with varying concentrations of α-tocopherol, was in good agreement with the concentration required to cause the half-maximal inhibition of lipid peroxidation of the membranes performed by incubation with 100 μM ascorbic acid and 10 μM Fe²⁺. Decrease of the slope in the thermal Perrin plot of the polarization of pyrene-labeled membranes by α-tocopherol suggests that the movement of pyrene molecules in the membranes is restricted by binding of the tocopherol. This interpretation was confirmed by an increased harmonic mean of the rotational relaxation time of the dye molecules in the membranes from 10.9 ± 0.16 to 18.5 ± 0.51 μs after addition of 25 μM α-tocopherol to the medium. The perturbation of lipid phase in the membranes induced by α-tocopherol was also suggested from a decreased quenching rate constant of pyrene fluorescence in the membranes for Tl⁺. Based on these results, the effect of α-tocopherol on the lipid fluidity of the membranes is discussed.