Fluorescent probes in model membranes. II. monolayer studies of 2,2′-(vinylenedi-p-phenylene)bisbenzoxazole, d-3-aminodesoxyequilenin and n-octadecylnaphthyl-2-amino-6-sulfonic acid with host-lipid tetradecanoic acid

Film studies at the air-water interface have been carried out for pure films of 2,2′-(vinylenedi-p-phenylene)bisbenzoxazole (VPBO), d-3-aminodesoxy-equlenin (EQ) and N-octadecylnapthyl-2-amino-6-sulfonic acid (ONS), and for mixed films with tetradecanoic acid for the first two fluorescent probes. Pure film isotherms indicate highly rigid non-monomolecular films for both VPBO and EQ, revealing the presence of strong intermolecular forces. In mixed films with tetradecanoic acid VPBO rapidly segregates with resultant film loss over a wide concentration range. EQ, however, can be stabilized by the host-lipid at low concentrations. This, coupled with an ability to only slightly affect the host-lipid liquid-condensed/liquid-expanded phase change, suggests that EQ can be regarded as “non-perturbing” and should be retained in condensed lipid phases.ONS, because of its unusual polar headgroup, resembled hexadecanoic acid more than octadecanoic acid. While difficulties in spreading ONS precluded the study of mixed films, the indications are that it would be a satisfactory expanded lipid state probe if mixing can be brought about.     

Reduction of dehydroascorbic acid at low pH

Ascorbic acid and dehydroascorbic acid are unstable in aqueous solution in the presence of copper and iron ions, causing problems in the routine analysis of vitamin C. Their stability can be improved by lowering the pH below 2, preferably with metaphosphoric acid. Dehydroascorbic acid, an oxidised form of vitamin C, gives a relatively low response on the majority of chromatographic detectors, and is therefore routinely determined as the increase of ascorbic acid formed after reduction. The reduction step is routinely performed at a pH that is suboptimal for the stability of both forms. In this paper, the reduction of dehydroascorbic acid with tris-[2-carboxyethyl] phosphine (TCEP) at pH below 2 is evaluated. Dehydroascorbic acid is fully reduced with TCEP in metaphosphoric acid in less than 20 min, and yields of ascorbic acid are the same as at higher pH. TCEP and ascorbic acid formed by reduction, are more stable in metaphosphoric acid than in acetate or citrate buffers at pH 5, in the presence of redox active copper ions. The simple experimental procedure and low probability of artefacts are major benefits of this method, over those currently applied in a routine assay of vitamin C, performed on large number of samples.     

Energization of amino acid transport in energy-depleted Ehrlich cells and plasma membrane vesicles

We redirect attention to contributions to the energization of the active transport of amino acids in the Ehrlich cell, beyond the known energization by down-gradient comigration of Na⁺, beyond possible direct energization by coupling to ATP breakdown, and beyond known energization by exchange with prior accumulations of amino acids. We re-emphasize the uphill operation of System L, and by prior depletion of cellular amino acids show that this system must receive energy beyond that made available by their coupled exodus. After this depletion the Na⁺-independent accumulation of the norbornane amino acid, 2-aminobicycloheptane-2-carboxylic acid becomes strongly subject to stimulation by incubation with glucose. Energy transfer between Systems A and L through the mutual substrate action of ordinary amino acids was minimized although not entirely avoided by the use of amino acid analogs specific to each system.When 2,4-dinitrophenol was included in the depleting treatment, and pyruvate, phenazine methosulfate, or glucose used for restoration, recovery of uptake of the norbornane amino acid was independent of external Na⁺ or K⁺ levels. Restoration of the uptake of 2-(methylamino)isobutyric acid was, however, decreased by omission of external K⁺. Contrary to an earlier finding, restoration of uptake of each of these amino acids was associated with distinct and usually correlated rises in cellular ATP levels. ATP addition failed to stimulate exodus of the norbornane amino acid from plasma membrane vesicles, although either NADH or phenazine methosulfate did stimulate exodus. ATP production and use is thus associated with transport energization, although evidence for a direct role failed to appear.     

Plasticity of glutamatergic control of striatal acetylcholine release in experimental parkinsonism: opposite changes at group-II metabotropic and NMDA receptors

To investigate whether adaptive changes of glutamatergic transmission underlie dysfunction of the cholinergic system in experimental parkinsonism, the effects of group-II metabotropic glutamate and NMDA receptor ligands on acetylcholine release was studied in striatal slices and synaptosomes obtained from naive rats, 6-hydroxydopamine hemi-lesioned rats and 6-hydroxydopamine hemi-lesioned rats chronically treated with levodopa (L-DOPA) plus benserazide (non-dyskinetic). Group-II metabotropic glutamate receptor agonists LY354740, DCG-IV and L-CCG-I inhibited the electrically-evoked endogenous acetylcholine release from slices, while NMDA facilitated it. LY354740 also inhibited K+-evoked acetylcholine release from synaptosomes. LY354740-induced inhibition was prevented by the group-II metabotropic glutamate receptor antagonist LY341495. In hemi-parkinsonian rats, sensitivity towards LY354740 was reduced while that to NMDA was enhanced in the lesioned (denervated) compared with unlesioned striatum. Moreover, dizocilpine inhibited acetylcholine release in the lesioned compared with unlesioned striatum. Chronic treatment with L-DOPA normalized sensitivity towards glutamatergic agonists. We conclude that striatal dopamine denervation results in plastic changes at group-II metabotropic glutamate and NMDA receptors that may shift glutamatergic control of acetylcholine release towards facilitation. From a clinical perspective, L-DOPA and NMDA antagonists appear effective in counteracting overactivity of striatal cholinergic interneurones associated with Parkinson's disease.     

The structure of the O-specific polysaccharide from Ralstonia pickettii

The following structure of the Ralstonia pickettii have been determined using NMR and chemical methods: -->4)-alpha-D-Rha-(1-->4)-alpha-L-GalNAcA-(1-->3)-beta-D-BacNAc-(1-->.     

Partitioning of liquid-ordered/liquid-disordered membrane microdomains induced by the fluidifying effect of 2-hydroxylated fatty acid derivatives

Cellular functions are usually associated with the activity of proteins and nucleic acids. Recent studies have shown that lipids modulate the localization and activity of key membrane-associated signal transduction proteins, thus regulating the cell's physiology. Membrane Lipid Therapy aims to reverse cell dysfunctions (i.e., diseases) by modulating the activity of membrane signaling proteins through regulation of the lipid bilayer structure. The present work shows the ability of a series of 2-hydroxyfatty acid (2OHFA) derivatives, varying in the acyl chain length and degree of unsaturation, to regulate the membrane lipid structure. These molecules have shown greater therapeutic potential than their natural non-hydroxylated counterparts. We demonstrated that both 2OHFA and natural FAs induced reorganization of lipid domains in model membranes of POPC:SM:PE:Cho, modulating the liquid-ordered/liquid-disordered structures ratio and the microdomain lipid composition. Fluorescence spectroscopy, confocal microscopy, Fourier transform infrared spectroscopy and differential detergent solubilization experiments showed a destabilization of the membranes upon addition of the 2OHFAs and FAs which correlated with the observed disordering effect. The changes produced by these synthetic fatty acids on the lipid structure may constitute part of their mechanism of action, leading to changes in the localization/activity of membrane proteins involved in signaling cascades, and therefore modulating cell responses.     

Evidence that Fe2+ complexes of 3-aminopicolinate and 3-mercaptopicolinate activate and inhibit phosphoenolpyruvate carboxykinase

3-Mercaptopicolinic acid is known to be an inhibitor of phosphoenolpyruvate carboxykinase and 3-aminopicolinic acid permits Fe²⁺ to activate the enzyme. The potency of mercaptopicolinate is increased by incubating the enzyme with Fe²⁺ prior to assaying for activity. In the present work, the average combining ratio of either pyridine carboxylate with Fe²⁺ at pH 7.5 was determined to be 2:1 when measured by the method of continuous variation of Job or by elemental analysis of the isolated pyridine carboxylate-Fe²⁺ complexes. The ratio of 3-mercaptopicolinate or 3-aminopicolinate to Fe²⁺ that caused the greatest inhibition or activation of purified phosphoenolpyruvate carboxykinase was 2:1. In the absence of Fe²⁺, neither pyridine carboxylate altered the activity of the enzyme. These results indicate that the two pyridine carboxylates can interact with phosphoenolpyruvate carboxykinase as Fe²⁺ coordination complexes.     

5-Hydroxylevulinic acid,a new intermediate in the biosynthesis of protoanemonin

Administration of 5-hydroxy[1-¹⁴C]-and [4-¹⁴C]levulinic acid to Helleborus foetidus led to the isolation of [1-¹⁴C]- and [4-¹⁴C]protoanemonin, respectively. There was also incorporation of radioactivity into the four glucosides ranunculin, isoranunculin, ranuncoside and ranunculoside. Acid hydrolysis of radioactive ranuncoside gave labelled 5-hydroxylevulinic acid (HKV). A study of the incorporation of various ¹⁴C-labelled tracers into protoanemonin suggested that HKV is formed in higher plants by a new reduction of 2-ketoglutarate (2-KG) without free 4,5-dioxovalerate (DOVA) as an intermediate. A scheme for the biosynthesis of the antibiotic protoanemonin and its glucosidic precursors is proposed. It is shown that 5-(β-d-glucopyranosyloxy)levulinic acid could be the genuine precursor of all the compounds studied.     

113Cd nmr study of the metal cluster structure of human liver metallothionein

Cadmium-113 nuclear magnetic resonance (¹¹³Cd nmr) was used to elucidate the structural properties of the cadmium binding sites in human liver metallothionein. The isotopically labeled ¹¹³Cd-metallothionein was prepared by the in vitro exchange of the native metals (greater than 94% zinc) for ¹¹³CdCl₂ during isolation. The two isoproteins, MT-1 and MT-2, showed ¹¹³Cd nmr resonances in the chemical shift range 610–670 ppm. The multiplet structure of the resonances is due to two bond scalar interactions between adjacent ¹¹³Cd ions linked by cysteine thiolate ligands. Homonuclear ¹¹³Cd decoupling experiments allowed the determination of the metal cluster structure, which, similar to the rabbit liver metallothionein, consists of a four- and a three-metal cluster designated cluster A and cluster B, respectively. Chemical shift similarities in the ¹¹³Cd nmr spectra of the human, rabbit and calf liver MT-1 and MT-2 are observed, especially for cluster A. Small variations in chemical shifts are explained in terms of differences in the primary structure between the two human isoproteins.     

Natural selection for 2,4,5-trichlorophenoxyacetic acid mineralizing bacteria in agent orange contaminated soil

Agent Orange contaminated soils were utilized in direct enrichment culture studies to isolate 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 2,4-dichlorophenoxyacetic acid (2,4-D) mineralizing bacteria. Two bacterial cultures able to grow at the expense of 2,4,5-T and/or 2,4-D were isolated. The 2,4,5-T degrading culture was a mixed culture containing two bacteria, Burkholderia species strain JR7B2 and Burkholderia species strain JR7B3. JR7B3 was able to metabolize 2,4,5-T as the sole source of carbon and energy, and demonstrated the ability to affect metabolism of 2,4-D to a lesser degree. Strain JR7B3 was able to mineralize 2,4,5-T in pure culture and utilized 2,4,5-T in the presence of 0.01 yeast extract. Subsequent characterization of the 2,4-D degrading culture showed that one bacterium, Burkholderiaspecies strain JRB1, was able to utilize 2,4-D as a sole carbon and energy source in pure culture. Polymerase chain reaction (PCR) experiments utilizing known genetic sequences from other 2,4-D and 2,4,5-T degrading bacteria demonstrated that these organisms contain gene sequences similar to tfdA, B, C, E, and R (Strain JRB1) and the tftA, C, and E genes (Strain JR7B3). Expression analysis confirmed that tftA, C, and E and tfdA, B, and C were transcribed during 2,4,5-T and 2,4-D dependent growth, respectively. The results indicate a strong selective pressure for 2,4,5-T utilizing strains under field condition.     

The antitumor agent 3-bromopyruvate has a short half-life at physiological conditions

Clinical research is currently exploring the validity of the anti-tumor candidate 3-bromopyruvate (3-BP) as a novel treatment for several types of cancer. However, recent publications have overlooked rarely-cited earlier work about the instability of 3-BP and its decay to 3-hydroxypyruvate (3-HP) which have obvious implications for its mechanism of action against tumors, how it is administered, and for precautions when preparing solutions of 3-BP. This study found the first-order decay rate of 3-BP at physiological temperature and pH has a half-life of only 77 min. Lower buffer pH decreases the decay rate, while choice of buffer and concentration do not affect it. A method for preparing more stable solutions is also reported.     

Selection of Small Peptides, Inhibitors of Translation

Identification of small molecular weight compounds targeting specific sites in the ribosome can accelerate development of new antibiotics and provide new tools for ribosomal research. We demonstrate here that antibiotic-size short peptides capable of inhibiting protein synthesis can be selected by using specific elements of ribosomal RNA as a target. The ‘h18’ pseudoknot encompassing residues 500-545 of the small ribosomal subunit RNA was used as a target in screening a heptapeptide phage-display library. Two of the selected peptides could efficiently interfere with both bacterial and eukaryotic translation. One of these inhibitory peptides exhibited a high-affinity binding to the isolated small ribosomal subunit (K_d of 1.1 μM). Identification of inhibitory peptides that likely target a specific rRNA structure may pave new ways for validating new antibiotic sites in the ribosome. The selected peptides can be used as a tool in search of novel site-specific inhibitors of translation.     

Functional characterization of two microsomal fatty acid desaturases from Jatropha curcas L.

Linoleic acid (LA, C18:2) and α-linolenic acid (ALA, C18:3) are polyunsaturated fatty acids (PUFAs) and major storage compounds in plant seed oils. Microsomal ω-6 and ω-3 fatty acid (FA) desaturases catalyze the synthesis of seed oil LA and ALA, respectively. Jatropha curcas L. seed oils contain large proportions of LA, but very little ALA. In this study, two microsomal desaturase genes, named JcFAD2 and JcFAD3, were isolated from J. curcas. Both deduced amino acid sequences possessed eight histidines shown to be essential for desaturases activity, and contained motif in the C-terminal for endoplasmic reticulum localization. Heterologous expression in Saccharomyces cerevisiae and Arabidopsis thaliana confirmed that the isolated JcFAD2 and JcFAD3 proteins could catalyze LA and ALA synthesis, respectively. The results indicate that JcFAD2 and JcFAD3 are functional in controlling PUFA contents of seed oils and could be exploited in the genetic engineering of J. curcas, and potentially other plants.