Electric field-induced orientation of L- and DL-phosphatidylcholine bilayers

Membrane orientation induced by an alternating electric field has been examined for the L-enantiomer and racemic dipalmitoylphosphatidylcholine (DPPC) bilayers. The orientation effect was measured by bending curvature of hairpin-like deformation of the multilamellar cylindrical tubes with varying field-strength, frequency and tube size. It has been observed that both L- and DL-DPPC tubes are similar in the profiles of field-strength dependence and frequency dependence on the curvature deformation, but different in the deformed curvatures. DL-DPPC tubes deform largely as compared with L-DPPC tubes. The square of the deformed curvature of DL-DPPC tubes is larger than that of L-DPPC by about 37% on average. The result indicates that the racemic membrane is responsive to the electric field as compared with the L-enantiomer membrane. This suggests that a hybrid arrangement of head groups of the racemic lipid leads an effective response of the membrane due to the head group orientation.     

Oral supplementation with a combination of l-citrulline and l-arginine rapidly increases plasma l-arginine concentration and enhances NO bioavailability

Background

Chronic supplementation with l-citrulline plus l-arginine has been shown to exhibit anti-atherosclerotic effects. However, the short-term action of this combination on the nitric oxide (NO)–cGMP pathway remains to be elucidated. The objective of the present study was to investigate the acute effects of a combination of oral l-citrulline and l-arginine on plasma l-arginine and NO levels, as well as on blood circulation.

Methods

Rats or New Zealand white rabbits were treated orally with l-citrulline, or l-arginine, or a combination of each at half dosage. Following supplementation, plasma levels of l-arginine, NO_x, cGMP and changes in blood circulation were determined sequentially.

Results

l-Citrulline plus l-arginine supplementation caused a more rapid increase in plasma l-arginine levels and marked enhancement of NO bioavailability, including plasma cGMP concentrations, than with dosage with the single amino acids. Blood flow in the central ear artery in rabbits was also significantly increased by l-citrulline plus l-arginine administration as compared with the control.

Conclusion

Our data show for the first time that a combination of oral l-citrulline and l-arginine effectively and rapidly augments NO-dependent responses at the acute stage. This approach may have clinical utility for the regulation of cardiovascular function in humans.     

d-Polyglutamine Amyloid Recruits l-Polyglutamine Monomers and Kills Cells

Polyglutamine (polyQ) amyloid fibrils are observed in disease tissue and have been implicated as toxic agents responsible for neurodegeneration in expanded CAG repeat diseases such as Huntington's disease. Despite intensive efforts, the mechanism of amyloid toxicity remains unknown. As a novel approach to probing polyQ toxicity, we investigate here how some cellular and physical properties of polyQ amyloid vary with the chirality of the glutamine residues in the polyQ. We challenged PC12 cells with small amyloid fibrils composed of either l- or d-polyQ peptides and found that d-fibrils are as cytotoxic as l-fibrils. We also found using fluorescence microscopy that both aggregates effectively seed the aggregation of cell-produced l-polyQ proteins, suggesting a surprising lack of stereochemical restriction in seeded elongation of polyQ amyloid. To investigate this effect further, we studied chemically synthesized d- and l-polyQ in vitro. We found that, as expected, d-polyQ monomers are not recognized by proteins that recognize l-polyQ monomers. However, amyloid fibrils prepared from d-polyQ peptides can efficiently seed the aggregation of l-polyQ monomers in vitro, and vice versa. This result is consistent with our cell results on polyQ recruitment but is inconsistent with previous literature reports on the chiral specificity of amyloid seeding. This chiral cross-seeding can be rationalized by a model for seeded elongation featuring a “rippled β-sheet” interface between seed fibril and docked monomers of opposite chirality. The lack of chiral discrimination in polyQ amyloid cytotoxicity is consistent with several toxicity mechanisms, including recruitment of cellular polyQ proteins.     

d-Glucose- and d-mannose-based antimetabolites. Part 2. Facile synthesis of 2-deoxy-2-halo-d-glucoses and -d-mannoses

Modified d-glucose and d-mannose analogs are potentially clinically useful metabolic inhibitors. Biological evaluation of 2-deoxy-2-halo analogs has been impaired by limited availability and lack of efficient methods for their preparation. We have developed practical synthetic approaches to 2-deoxy-2-fluoro-, 2-chloro-2-deoxy-, 2-bromo-2-deoxy-, and 2-deoxy-2-iodo derivatives of d-glucose and d-mannose that exploit electrophilic addition reactions to a commercially available 3,4,6-tri-O-acetyl-d-glucal.     

Formation of l-quebrachitol from d-bornesitol in leaves of Acer pseudoplatanus

d-Bornesitol and l-quebrachitol have been found in the leaves of Acer pseudoplatanus L. The results of incorporation studies using labeled myo-inositol-¹⁴C, l-inositol-¹⁴C and d-bornesitol-¹⁴C indicate that l-quebrachitol is produced by epimerization of d-bornesitol. In Artemisia vulgaris, however, the precursor of l-quebrachitol is l-inositol.     

A high-throughput assay for screening l- or d-amino acid specific aminotransferase mutant libraries

Aminotransferases are pyridoxal phosphate-dependent enzymes whose potential for the biocatalytic production of enantiopure amino acids is increasingly recognized. Because of this, there is a growing interest in engineering them to alter their substrate specificity and to increase their catalytic activity. Here, we report the development of a high-throughput assay for screening α-ketoglutarate-dependent aminotransferase mutant libraries. To achieve this, we exploited the l-glutamate dehydrogenase coupled assay that has previously been shown to allow for aminotransferase activity to be monitored in vitro. We adapted this assay to allow screening of mutant libraries of either l- or d-amino acid specific aminotransferases in a continuous fashion. This assay requiring clarified cell lysates is reproducible, rapid, and sensitive because it allowed for the identification of a catalytically active mutant of Bacillus sp. YM-1 d-amino acid aminotransferase displaying a decrease in k_cat/K_M of more than two orders of magnitude. In addition, this assay allowed us to discover a mutant of Escherichia coli branched-chain amino acid aminotransferase, F36W, which is approximately 60-fold more specific toward the natural substrate l-leucine than l-phenylalanine as compared with wild type. This result demonstrates the potential of our assay for the discovery of mutant aminotransferases displaying altered substrate specificity, an important goal of enzyme engineering.     

A study of l-leucine, l-phenylalanine and l-alanine transport in the perfused rat mammary gland: possible involvement of LAT1 and LAT2

The transport of l-leucine, l-phenylalanine and l-alanine by the perfused lactating rat mammary gland has been examined using a rapid, paired-tracer dilution technique. The clearances of all three amino acids by the mammary gland consisted of a rising phase followed by a rapid fall-off, respectively, reflecting influx and efflux of the radiotracers. The peak clearance of l-leucine was inhibited by BCH (65%) and d-leucine (58%) but not by l-proline. The inhibition of l-leucine clearance by BCH and d-leucine was not additive. l-leucine inhibited the peak clearance of radiolabelled l-leucine by 78%. BCH also inhibited the peak clearance of l-phenylalanine (66%) and l-alanine (33%) by the perfused mammary gland. Lactating rat mammary tissue was found to express both LAT1 and LAT2 mRNA. The results suggest that system L is situated in the basolateral aspect of the lactating rat mammary epithelium and thus probably plays a central role in neutral amino acid uptake from blood. The finding that l-alanine uptake by the gland was inhibited by BCH suggests that LAT2 may make a significant contribution to neutral amino acid uptake by the mammary epithelium.     

Preferred hydrolysis of methyl d-pyroglutamates (5-carbomethoxy-2-pyrrolidones) by α-chymotrypsin

Methyl d-pyroglutamate, d-III-A, is hydrolyzed by α-chymotrypsin, and the l-enantiomer is not. Methyl α-methylpyroglutamate, III-B, and methyl α-benzylpyroglutamate, III-C, are hydrolyzed by α-chymotrypsin with d-preference based on the glutamic structure, l-preference based on the alanine and phenylalanine structures. γ-Carbomethoxy-γ-valerolactone, IV, is hydrolyzed without stereoselectivity. Kinetics of hydrolysis of the substrates are reported. Modes of association of the substrates with the active site and the relevance of these results to the reactive orientation of the α-acylamido and hydrolyzing groups of noncyclized substrates are discussed.     

L-Galactosaccharinic acids: new saccharinic acids formed by calcium hydroxide treatment of L-sorbose

The new saccharinic acids, α- and β-L-galactosaccharinic acids (2-C-methyl-L-xylonic and 2-C-methyl-L-lyxonic acids), have been synthesized. The same acids were isolated, as their lactones, from the syrup obtained by treatment of L-sorbose with calcium hydroxide. The acids were produced in comparable amounts, and the factors that might regulate the stereoselectivity of the benzylic acid type of rearrangement are discussed.     

Single-step bioconversion for the preparation of l-gulose and l-galactose

Both carbohydrate monomers l-gulose and l-galactose are rarely found in nature, but are of great importance in pharmacy R&D and manufacturing. A method for the production of l-gulose and l-galactose is described that utilizes recombinant Escherichia coli harboring a unique mannitol dehydrogenase. The recombinant E. coli system was optimized by genetic manipulation and directed evolution of the recombinant protein to improve conversion. The resulting production process requires a single step, represents the first readily scalable system for the production of these sugars, is environmentally friendly, and utilizes inexpensive reagents, while producing l-galactose at 4.6 g L⁻¹ d⁻¹ and l-gulose at 0.90 g L⁻¹ d⁻¹.     

Metabolic studies of rat liver plasma membranes using d-[l-C]glucosamine

The metabolism of plama membranes of rat liver cells was studied using d-[l-¹⁴C]glucosamine. The labelling of plasma membranes occurred more slowly than that of microsomes, reaching a maximum at about 3 h after injection compared to 1.5 h for microsomes, and the radioactivity decayed with a half-life of 37 h which is close to the value obtained using [guanidino-¹⁴C]arginine to label proteins. Hexosamine and sialic acid of plasma membranes were found to metabolize at practically equal rates.     

Attraction, deformation and contact of membranes induced by low frequency electric fields

The force of attraction between erythrocyte ghosts induced by low frequency electric fields (60 Hz) was measured as a function of the intermembrane separation. It varied from 10(-14) N for separation of the order of the cell diameter to 10(-12) N for close approach and contact in 20 mM sodium phosphate buffers (conductivity 260 mS/m, pH 8.5). For large separations the interaction force followed a dependence on separation as predicted for dipole-dipole interactions. For small separation an empirical formula was obtained. The membranes deformed at close approach (less than 1 microns) before making contact. The contact area increased with time until reaching the final equilibrium state. The ghosts separated reversibly after switching off the electric field. The membrane tension induced by the ghost interaction at contact was estimated to be of the order of 0.1 mN/m. These first quantitative measurements of the force/separation dependence for intermembrane interactions induced by low frequency electric fields indicate that attractive forces, membrane deformation and contact area of cells depend strongly on intermembrane separation and field strength. The quantitative relationship between them are important for measuring membrane surface and mechanical properties, intermembrane forces and understanding mechanisms of membrane adhesion, instability and fusion in electric fields and in general.     

Differential processing of mammalian l-histidine decarboxylase enzymes

In the mammalian species studied so far, the l-histidine decarboxylase (HDC) enzyme responsible for histamine biosynthesis has been shown to undergo post-translational processing. The processing is best characterized for the mouse enzyme, where di-asparate DD motifs mediate the production of active ∼55 and ∼60 kDa isoforms from the ∼74 kDa precursor in a caspase-9 dependent manner. The identification of conserved di-aspartate motifs at similar locations in the rat and human HDC protein sequences has led to proposals that these may represent important processing sites in these species also. Here we used transfected Cos7 cells to demonstrate that the rat and human HDC proteins undergo differential processing compared to each other, and found no evidence to suggest that conserved di-aspartate motifs are required absolutely for processing in this cell type. Instead we identified SKD and EEAPD motifs that are important for caspase-6 dependent production of ∼54 and ∼59 kDa isoforms in the rat and human proteins, respectively. The addition of staurosporine, which is known to pharmacologically activate caspase enzymes, increased processing of the human HDC protein. We propose that caspase-dependent processing is a conserved feature of mammalian HDC enzymes, but that proteolysis may involve different enzymes and occur at diverse sites and sequences.     

The orientation of d-β-hydroxybutyrate dehydrogenase in the mitochondrial inner membrane

d-β-Hydroxybutyrate dehydrogenase of beef heart mitochondria is a lipid-requiring enzyme, bound to the inner membrane. The orientation of this enzyme in the membrane has been studied by comparing the characteristics of the enzyme in mitochondria and ‘inside-out’ submitochondrial vesicles. We observe that the enzymic activity is (1) latent in intact mitochondria; (2) relatively stable to trypsin digestion in mitochondria but rapidly inactivated in submitochondrial vesicles by this treatment; and (3) released more rapidly from submitochondrial vesicles by phospholipase A₂ digestion than from mitochondria. Conclusive evidence that d-β-hydroxybutyrate dehydrogenase is localized on the matrix face of the mitochondrial inner membrane is provided by the correlation that the enzyme is released from submitochondrial vesicles before the membrane becomes leaky to cytochrome $\text{c}$. The arrangement of d-β-hydroxybutyrate dehydrogenase in the membrane is discussed within a generalized classification of the orientation of proteins in membranes. The evidence indicates that d-β-hydroxybutyrate dehydrogenase is an amphipathic molecule and as such is inlaid in the membrane, i.e. the enzyme is partially inserted into the hydrophobic milieu of the membrane, with the polar, functional end extending into the aqueous milieu.     

l-Lactate generates hydrogen peroxide in purified rat liver mitochondria due to the putative l-lactate oxidase localized in the intermembrane space

In order to ascertain whether and how mitochondria can produce hydrogen peroxide (H₂O₂) as a result of l-lactate addition, we monitored H₂O₂ generation in rat liver mitochondria and in submitochondrial fractions free of peroxisomal and cytosolic contamination. We found that H₂O₂ is produced independently on the respiratory chain with 1:1 stoichiometry with pyruvate, due to a putative flavine-dependent l-lactate oxidase restricted to the intermembrane space. The l-lactate oxidase reaction shows a hyperbolic dependence on l-lactate concentration and is inhibited by NAD⁺ in a competitive manner, being the enzyme different from the l-lactate dehydrogenase isoenzymes as shown by their pH profiles.     

Phosphorylation of mouse serine racemase regulates d-serine synthesis

Serine racemase (SR) catalyses the synthesis of the transmitter/neuromodulator d-serine, which plays a major role in synaptic plasticity and N-methyl d-aspartate receptor neurotoxicity. We now report that SR is phosphorylated at Thr71 and Thr227 as revealed by mass spectrometric analysis and in vivo phosphorylation assays. Thr71 phosphorylation was observed in the cytosolic and membrane-bound SR while Thr227 phosphorylation was restricted to the membrane fraction. The Thr71 site has a motif for proline-directed kinases and is the main phosphorylation site of SR. Experiments with a phosphorylation-deficient SR mutant indicate that Thr71 phosphorylation increases SR activity, suggesting a novel mechanism for regulating d-serine production.     

Electrokinetic studies of the testosterone-aqueous d-glucose interface

Electro-osmosis and streaming-potential measurements were made across a testosterone-plug membrane, using water and aqueous solutions of d-glucose as permeants. The electrophoretic velocity of testosterone particles dispersed in these solutions was also measured, experiments being confined to the range where linear flux-force relationships hold. Phenomenological coefficients were evaluated by using these linear relations, and the results analyzed inthe light of the thermodynamics of irreversible processes. Saxen's relationship holds between electro-osmosis and streaming potential. Concentration dependence of the various phenomenological coefficients was also examined. Cross-phenomenological coefficients were found to decrease with increase in the concentration of d-glucose solutions. The results are explained on the basis of strong hydrogen-bonding between d-glucose and the surrounding water molecules. Such membrane parameters as pore size, average number of pores, and the membrane constant were evaluated. Electro-osmotic and electrophoretic data were used to estimate the zeta potential, in order to characterize the membrane-permeant interface. The dependence of the zeta potential on the concentration was also examined.     

The influence of selected O-alkyl derivatives of cyclodextrins on the enzymatic decomposition of l-tryptophan by l-tryptophan indole-lyase

A series of O-alkyl derivatives of cyclodextrin: heksakis[2,3,6-tri-O-(2′-methoxyethyl)]-α-cyclodextrin; heksakis(2,3-di-O-methyl)-α-cyclodextrin; heptakis(2,3-di-O-methyl)-β-cyclodextrin; heksakis[2,3-di-O-methyl-6-O-(2′-methoxyethyl)]-α-cyclodextrin; heptakis[2,3-di-O-methyl-6-O-(2′-methoxyethyl)]-β-cyclodextrin; heksakis[2,3-di-O-(2′-methoxyethyl)]-α-cyclodextrin and heptakis[2,3-di-O-(2′-methoxyethyl)]-β-cyclodextrin have been synthesized. Purity and composition of the obtained substances were examined. The cyclodextrin derivatives listed above as well as (2-hydroxypropyl)-α-cyclodextrin and (2-hydroxypropyl)-β-cyclodextrin, the two commercially available ones, have been investigated as the additives in the course of enzymatic decomposition of l-tryptophan by l-tryptophan indole-lyase. It has been found that each of cyclodextrin derivatives causes the inhibition of enzymatic process, both competitive and non-competitive. The competitive inhibition is connected with the formation of inclusion complexes between cyclodextrins and l-tryptophan, related to the geometry of these complexes. The mechanism of the non-competitive inhibition is not so evident; it could be related to the formation of the cyclodextrin complexes on the surface of the enzyme, leading to the change in the flexibility of the enzyme molecule.     

l-carnitine supplementation as a potential antioxidant therapy for inherited neurometabolic disorders

In recent years increasing evidence has emerged suggesting that oxidative stress is involved in the pathophysiology of a number of inherited metabolic disorders. However the clinical use of classical antioxidants in these diseases has been poorly evaluated and so far no benefit has been demonstrated. l-Carnitine is an endogenous substance that acts as a carrier for fatty acids across the inner mitochondrial membrane necessary for subsequent beta-oxidation and ATP production. Besides its important role in the metabolism of lipids, l-carnitine is also a potent antioxidant (free radical scavenger) and thus may protect tissues from oxidative damage. This review addresses recent findings obtained from patients with some inherited neurometabolic diseases showing that l-carnitine may be involved in the reduction of oxidative damage observed in these disorders. For some of these diseases, reduced concentrations of l-carnitine may occur due to the combination of this compound to the accumulating toxic metabolites, especially organic acids, or as a result of protein restricted diets. Thus, l-carnitine supplementation may be useful not only to prevent tissue deficiency of this element, but also to avoid oxidative damage secondary to increased production of reactive species in these diseases. Considering the ability of l-carnitine to easily cross the blood–brain barrier, l-carnitine supplementation may also be beneficial in preventing neurological damage derived from oxidative injury. However further studies are required to better explore this potential.