Glutamine Transport in Mouse Cerebral Astrocytes

Abstract: We measured initial influx and exchange of [¹⁴C]glutamine in primary astrocyte cultures in the presence and absence of Na⁺. Kinetic analysis of transport in Na⁺-free solution indicated two saturable Na⁺-independent components, one of which was identifiable functionally as system L₁ transport. In the presence of Na⁺, multiple hyperbolic components were not resolvable from the kinetic data. Nevertheless, other evidence supported participation by at least three Na⁺-dependent neutral amino acid transporters (systems A, ASC, and N). System A transport of glutamine was usually absent or minimal, based on lack of inhibition by α-(methylamino)isobutyric acid. However, vigorous system A-mediated transport emerged after derepression by substrate deprivation. Participation by system ASC was indicated by trans-acceleration of Na⁺-dependent uptake, preferential inhibition of an Li⁺-intolerant component of uptake by cysteine, and inhibition by cysteine of a component resistant to inhibition by histidine and α-(methylamino)isobutyric acid. Because nonsaturable transport of glutamine appeared negligible, and system L transport of glutamine was suppressed in the presence of Na⁺, low-affinity system ASC transport may be the major route of export of glutamine from astrocytes. At 700 µ M glutamine, the primary uptake route was system N transport, identified on the basis of selective inhibition by histidine and asparagine, pH sensitivity, and tolerance of Li⁺ in place of Na⁺.     

Characterisation of a potential biomarker of phospholipidosis from amiodarone-treated rats

A novel and relatively simple analytical method for the separation, characterisation and semi-quantitation of phospholipids (PLs) from extracts of complex biological samples has been developed. This methodology allows PL extracts from cells and tissues to be analysed by liquid chromatography (LC) coupled to electrospray ionisation mass spectrometry (ESI-MS). Complex mixtures of PLs were separated on a high-performance liquid chromatography (HPLC) system using 0.5% ammonium hydroxide in methanol/water/hexane/formate mixture with UV detection at 205 nm. Identification and structural characterisation of molecular species were carried out utilising ESI-MS and MS/MS in the negative ion mode.The abnormal accumulation of PLs (phospholipidosis) was induced in male Sprague-Dawley rats by administration of the cationic amphiphilic drug (CAD), amiodarone. Analysis of the PL profile of liver and lung tissues, lymphocytes and serum from treated rats was carried out using this analytical procedure (LC-ESI/MS/MS). Differences in PL profiles between treated and untreated animals were highlighted by principal component analysis (PCA). This led to the selection of a potential metabolic marker of phospholipidosis (PLD) identified as a lyso-bis-phosphatidic acid (LBPA) derivative, also known as bis(monoglycero)phosphate (BMP). This PL was absent in control animals but was present in quantifiable amounts in all samples from amiodarone-treated rats.     

Molecular basis of ion permeability in a voltage‐gated sodium channel

Voltage‐gated sodium channels are essential for electrical signalling across cell membranes. They exhibit strong selectivities for sodium ions over other cations, enabling the finely tuned cascade of events associated with action potentials. This paper describes the ion permeability characteristics and the crystal structure of a prokaryotic sodium channel, showing for the first time the detailed locations of sodium ions in the selectivity filter of a sodium channel. Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios (Na⁺ ≈ Li⁺ ≫ K⁺, Ca²⁺, Mg²⁺) measured for these channels. In an E178D selectivity filter mutant constructed to have altered ion selectivities, the sodium ion binding site nearest the extracellular side is missing. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and are associated with side chains of the selectivity filter residues, rather than polypeptide backbones.     

Design and synthesis of pyridone inhibitors of non-nucleoside reverse transcriptase

Next generation NNRTIs are sought which possess both broad spectrum antiviral activity against key mutant strains and a high genetic barrier to the selection of new mutant viral strains. Pyridones were evaluated as an acyclic conformational constraint to replace the aryl ether core of MK-4965 (1) and the more rigid indazole constraint of MK-6186 (2). The resulting pyridone compounds are potent inhibitors of HIV RT and have antiviral activity in cell culture that is superior to other next generation NNRTI’s.     

Effects of a metalloporphyrinic peroxynitrite decomposition catalyst,ww-85, in a mouse model of spinal cord injury

The aim of the present study was to assess the effect of a metalloporphyrinic peroxynitrite decomposition catalyst, ww-85, in the pathophysiology of spinal cord injury (SCI) in mice. Spinal cord trauma was induced by the application of vascular clips to the dura via a four-level T5–T8 laminectomy. SCI in mice resulted in severe trauma characterized by oedema, neutrophil infiltration, production of inflammatory mediators, tissue damage and apoptosis. ww-85 treatment (30–300 µg/kg, i.p. 1 h after the SCI) significantly reduced in a dose-dependent manner: (1) the degree of spinal cord inflammation and tissue injury, (2) neutrophil infiltration (myeloperoxidase activity), (3) nitrotyrosine formation and PARP activation, (4) pro-inflammatory cytokines expression, (5) NF-κB activation and (6) apoptosis. Moreover, ww-85 significantly ameliorated the recovery of limb function (evaluated by motor recovery score) in a dose-dependent manner. The results demonstrate that ww-85 treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma.     

Separation of bivalent anti-T cell immunotoxin from Pichia pastoris glycoproteins by borate anion exchange

A major problem encountered in the large-scale purification of the bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(G4S), from Pichia pastoris supernatants was the presence of host glycoproteins exhibiting similar charge, size, and hydrophobicity characteristics. We overcame this problem by employing borate anion exchange chromatography. The borate anion has an affinity for carbohydrates and imparts negative charges to these structures. We found that at a concentration of sodium borate between 50 and 100 mM, the nonglycosylated immunotoxin did not bind to Poros 50 HQ anion exchanger resin, but glycoproteins, including aggregates related to the immunotoxin, did. By using this property of the immunotoxin in the presence of sodium borate, we successfully developed a 3-step purification procedure: (i) Butyl-650M hydrophobic interaction chromatography, (ii) Poros 50 HQ anion exchange chromatography in the presence of borate, and (iii) HiTrap Q anion exchange chromatography. The final preparation exhibited a purity of greater than 98% and a yield of greater than 50% from the supernatant. Previously, boronic acid resins have been used to separate glycoproteins from proteins. However, combining borate anion with conventional anion exchange resins accomplishes the separation of the immunotoxin from glycoproteins and eliminates the need to evaluate nonstandard resins with respect to good manufacturing practice guidelines.     

The pharmacological actions of 5-hydroxytryptamine,FMRF-amide and substance P and their possible occurrence in the heart of the snail Helix aspersa L.

The actions of 5-hydroxytryptamine (5-HT), FMRF-amide and substance P were tested on the isolated heart of Helix aspersa. All three compounds were found to produce positive inotropic and chronotropic effects, the order of potency being 5-HT > substance P > FMRF-amide. However, whereas the responses to 5-HT and FMRF-amide were maximal within a few seconds, the response to substance P had a longer latency. Two other similar undecapeptides, eledoisin and physalaemin, were also tested and were found to have very similar actions to those of substance P. The effects of 5-HT and FMRF-amide could be separated using the 5-HT blockers methysergide and ketanserin, which had relatively little effect on the response to FMRF-amide.Immunohistochemical staining was carried out on sections through the Helix auricle and ventricle for 5-HT, FMRF-amide and substance P. Substantial 5-HT-like and substance P-like immunoreactivity was observed, apparently concentrated in nerve endings, but the level of FMRF-amide-like immunoreactivity was considerably lower. The fluorescence produced by all three compounds was evenly distributed throughout the heart tissue. HPLC analysis of heart tissue extract demonstrated a high level of 5-HT (about 8 μg/g wet weight) but a negligible catecholamine content.     

Artificial hybrid protein containing a toxic protein fragment and a cell membrane receptor-binding moiety in a disulfide conjugate. II. Biochemical and biologic properties of diphtheria toxin fragment A-S-S-human placental lactogen.

The biochemical and biologic properties of a purified disulfide conjugate of diphtheria toxin fragment A and human placental lactogen (toxin A-hPL) have been studied by (a) assaying the ADP-ribosyltransferase activity of the intact conjugate, (b) assaying the binding of the intact conjugate to mammary gland plasma membrane lactogenic receptors, and (c) assaying the effect of the conjugate on the rate of protein synthesis in rabbit mammary gland explants maintained in organ culture. The toxin A-hPL conjugate retains one-third of the NAD+:EF-2 ADP-ribosyltransferase activity of toxin A, and 26% of the hPL-binding activity to lactogenic receptors. Binding activity was demonstrated by radioreceptor assay and by assaying toxin A activity bound to membranes which was competitively displaced by excess hPL. Since the toxin A-hPL conjugate retained activities of its separate subunits, it could be regarded as a structural analogue of nicked diphtheria toxin with replacement of the original membrane-binding chain by another binding chain that is specific for lactogenic receptor. However, the conjugate failed to inhibit protein synthesis in organ-cultured mammary gland explants, although these were sensitive to native diphtheria toxin and could bind hPL. It is concluded from these results that the toxin A-hPL conjugate does not act as a functional analogue of diphtheria toxin with altered receptor specificity, and that the hPL receptor cannot mediate the entry of toxin A or toxin A-hPL from membrane-bound conjugate into the cytosol site of action of toxin A.