The application of strand invasion phenomenon,directed by peptide nucleic acid (PNA) and single‐stranded DNA binding protein (SSB) for the recognition of specific sequences of human endogenous retroviral HERV‐W family

The HERV‐W family of human endogenous retroviruses represents a group of numerous sequences that show close similarity in genetic composition. It has been documented that some members of HERV‐W–derived expression products are supposed to play significant role in humans' pathology, such as multiple sclerosis or schizophrenia. Other members of the family are necessary to orchestrate physiological processes (eg, ERVWE1 coding syncytin‐1 that is engaged in syncytiotrophoblast formation). Therefore, an assay that would allow the recognition of particular form of HERV‐W members is highly desirable. A peptide nucleic acid (PNA)–mediated technique for the discrimination between multiple sclerosis‐associated retrovirus and ERVWE1 sequence has been developed. The assay uses a PNA probe that, being fully complementary to the ERVWE1 but not to multiple sclerosis‐associated retrovirus (MSRV) template, shows high selective potential. Single‐stranded DNA binding protein facilitates the PNA‐mediated, sequence‐specific formation of strand invasion complex and, consequently, local DNA unwinding. The target DNA may be then excluded from further analysis in any downstream process such as single‐stranded DNA‐specific exonuclease action. Finally, the reaction conditions have been optimized, and several PNA probes that are targeted toward distinct loci along whole HERV‐W env sequences have been evaluated. We believe that PNA/single‐stranded DNA binding protein–based application has the potential to selectively discriminate particular HERV‐W molecules as they are at least suspected to play pathogenic role in a broad range of medical conditions, from psycho‐neurologic disorders (multiple sclerosis and schizophrenia) and cancers (breast cancer) to that of an auto‐immunologic background (psoriasis and lupus erythematosus).     

Isomeric lipid signatures reveal compartmentalized fatty acid metabolism in cancer

The cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular FAs and their de novo synthesis. Given that oxidation of de novo synthesized FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates; however, hitherto, all FAs have been considered part of a common pool. To probe potential metabolic partitioning of cellular FAs, cancer cells were supplemented with stable isotope-labeled FAs. Structural analysis of the resulting glycerophospholipids revealed that labeled FAs from uptake were largely incorporated to canonical (sn-) positions on the glycerol backbone. Surprisingly, labeled FA uptake also disrupted canonical isomer patterns of the unlabeled lipidome and induced repartitioning of n-3 and n-6 PUFAs into glycerophospholipid classes. These structural changes support the existence of differences in the metabolic fates of FAs derived from uptake or de novo sources and demonstrate unique signaling and remodeling behaviors usually hidden from conventional lipidomics.     

Rapid Migration of Inositol Phospholipids with Axonally Transported Substances in the Rabbit Optic Pathway

Following an intraocular injection of myo-[2-3H]inositol, the axonal transport of labelled water-soluble substances and inositol phospholipids was investigated. Evidence was obtained for a rapid axonal transport of a relatively small amount of labelled inositol phospholipids. In contrast to other axonally transported phospholipids, there was no significant accumulation of labelled, rapidly transported inositol phospholipids in the nerve terminal region at later time intervals following the isotope administration.     

The role of the lipid matrix in the biosynthesis of dolichyl-linked oligosaccharides

The enzymes in the dolichol pathway are membrane-proteins that utilize a combination of hydrophilic and extremely hydrophobic substrates. The enzymes in this pathway that have been purified and characterized to any extent have either been shown to be stabilized by mixed phospholipid/detergent micelles, or else require a lipid matrix for catalytic activity. Further understanding of the mechanisms of these essential enzymes may require developing methods for the reconstitution of the glycosyltransferases and their hydrophobic substrates in appropriate lipid matrices. Abbreviations: CHO, Chinese hamster ovary; Dol, dolichol; DAG, diacylglycerol; DOPC, dioleolylphosphatidylcholine; DOPE, dioleolyphosphatidylethanolamine; ER, endoplasmic reticulum; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol This revised version was published online in November 2006 with corrections to the Cover Date.     

Endocytosis of α1-acid glycoprotein variants and of neoglycoproteins containing mannose derivatives by a mouse hybridoma cell line (2C11–12). Comparison with mouse peritoneal macrophages

Macrophages from various origins are known to express membrane lectins that mediate the endocytosis of mannose-bearing glycoconjugates. Most macrophage tumor cell-lines lack such receptors. In this paper we show by flow cytometry analysis that a newly generated macrophage hybridoma (2C_11–12), which displays several macrophage characteristics, also expresses mannose membrane lectins, resulting in the internalization of fluoresceinylated neoglycoproteins into acidic compartments.Thioglycolate elicited mouse peritoneal macrophages and the 2C_11–12 hybridomas were compared by flow cytometry with regard to the binding and endocytosis of ₁-acid glycoprotein (AGP) variants separated by affinity chromatography on immobilized concanavalin A. AGP C eluted specifically with methyl -mannopyranoside, which contains two bi-antennary oligosaccharides, was endocytosed as mannosylated serum albumin (Man-BSA). In both types of macrophages, the fluoresceinylated ligands were internalized in acidic compartments as demonstrated by the fluorescence intensity increase upon monensin post-incubation. However the behaviour of the internalized ligands was found to be quite different. AGP C and Man-BSA were rapidly degraded by thioglycolate elicited peritoneal macrophages and excreted in the medium as small peptide fragments; conversely they remained a longer time in the 2C_11–12 hybridoma.     

Secondary transport of amino acids by membrane vesicles derived from lactic acid bacteria

Lactococci are fastidious bacteria which require an external source of amino acids and many other nutrients. These compounds have to pass the membrane. However, detailed analysis of transport processes in membrane vesicles has been hampered by the lack of a suitable protonmotive force (pmf)-generating system in these model systems. A membrane-fusion procedure has been developed by which pmf-generating systems can be functionally incorporated into the bacterial membrane. This improved model system has been used to analyze the properties of amino acid transport systems in lactococci. Detailed studies have been made of the specificity and kinetics of amino acid transport and also of the interaction of the transport systems with their lipid environment. The properties of a pmf-independent, arginine-catabolism specific transport system in lactococci will be discussed.Abbreviations pmf protonmotive force - transmembrane electrical potential - pH transmembrane pH gradient - PE phosphatidylethanolamine - PC phosphatidylcholine Paper adapted from a treatise Secondary Transport of Amino Acids by Membrane Vesicles Derived from Lactic Acid Bacteria and awarded the Kluyver Prize 1988 by the Netherlands Society of Microbiology.     

Acyl-CoA: 1-acyl-glycero-3-phosphoethanolamine O-acyltransferase and liver plasma membrane fluidity

Investigations have been carried out on the influence of membrane lipid composition and physical state on acyl-CoA: 1-acyl-glycerol-3-phosphoethanolamine O-acyltransferase activity in rat liver plasma membranes. The lipid composition of the membranes was modified either by way of lipid transfer proteins or by partial delipidation with exogenous phospholipases and subsequent enrichment of the membranes with different phospholipids. The results indicated that membrane rigidification by enrichment of the membranes with DPPC or SM reduced the transfer of oleic and palmitic acid to lysophosphatidylethanolamine, whereas all phospholipids inducing membrane fluidization lead to acyltransferase activation. The eventual role of membrane fluidity in the deacylation-reacylation cycle is discussed.     

Spontaneous release of endogenous aspartate and glutamate from rat striatal slices is increased following destruction of local neurons by ibotenic acid

We sought to determine in rat striatum whether the release of neurotransmitter amino acids aspartate (Asp), glutamate (Glu) and gamma-aminobutyric acid (GABA) were affected by local neurons. To do so, unilateral microinjections of ibotenic acid, an excitotoxin that destroys local neurons without affecting fibers of passage, were made into the striatum. Release of endogenous amino acids from lesioned and intact striatal slices were measured by HPLC one week later. The effectiveness and specificity of the lesion were confirmed by measuring the enzyme activity associated with extrinsic dopamine neurons (tyrosine hydroxylase; 111±14%), intrinsic GABA neurons (glutamic acid decarboxylase; 19±7%) and intrinsic acetylcholine neurons (choline acetyltransferase; 37±10%). Destruction of local striatal neurons markedly attenuated the release of GABA (41±12% of control) elicited by depolarization with K⁺ (35 mM), but did not significantly reduce the K⁺-evoked release of Asp (80±17%) and Glu (92±8%). However, spontaneous release of Asp and Glu was significantly greater than that observed in unlesioned tissue (159±18% and 209±27%, respectively), while the spontaneous release of GABA was not significantly reduced (75±43%). Although release of the neurotransmitter amino acids Asp, Glu and GABA were affected by the lesion, the release of the non-neurotransmitter amino acid tyrosine was unaffected. These data are consistent with the hypotheses that: 1) the predominant source of releasable stores of endogenous Asp and Glu in the striatum arises from extinsic neurons, and 2) that the spontaneous release of Asp and Glu from axon terminals in the striatum may be regulated, at least in part, by local inhibitory neurons.     

Lithium Selectively Inhibits Muscarinic Receptor-Stimulated Inositol Tetrakisphosphate Accumulation in Mouse Cerebral Cortex Slices

The in vitro effects of Li on agonist- and depolarization-stimulated accumulation of inositol phosphates were determined in mouse cerebral cortex slices. Of the agents examined, only the cholinergic agonist carbachol produced a significant accumulation of inositol tetrakisphosphate (InsP4) in the absence of Li. Lithium at 5 mM enhanced the accumulation of inositol monophosphate (InsP1) and inositol bisphosphate (InsP2) due to all the stimuli used and potentiated inositol trisphosphate (InsP3) accumulation due to histamine and noradrenaline, although at lower Li concentrations, carbachol-stimulated InsP3 accumulation was reduced. Li also enhanced InsP4 accumulation in the presence of noradrenaline, histamine, and elevated KCl level but, in marked contrast, reduced carbachol-stimulated InsP4 accumulation with an IC50 of 100 microM. There was a significant time delay between the initiation of carbachol stimulation and the beginning of the InsP4 inhibition due to Li. The phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate did not mimic the effects of Li. The results suggest that muscarinic receptor-mediated InsP4 production might be one of the targets for the therapeutic action of Li.