Analysis of acidic compounds using capillary electrochromatography

Capillary electrochromatography, CEC, is a hybrid of CE and HPLC and is rapidly gaining interest as a potential complementary technique. This paper provides an overview of literature concerning the separation of acidic compounds by CEC which fall into three distinct groups. These groups are those performed using capillaries packed with novel or unique stationary phases designed for CEC, and a smaller group where standard HPLC stationary phases packings such as ODS has been used. The third group involves the use of surface coated capillaries. This paper reviews the separation of acidic compounds by CEC and also includes a number of novel applications to illustrate the separation approaches and the analytical performance possible.     

Evaluating the role of root citrate exudation as a mechanism of aluminium resistance in maize genotypes

Organic anion exudation by roots as a mechanism of aluminium (Al) resistance has been intensively studied lately. In the present study, we evaluated qualitative and quantitative aspects of root exudation of organic anions in maize genotypes of distinct sensitivity to Al in response to Al exposure. Maize seedlings were grown axenically in nutrient solution and root exudates were collected along the whole seminal root axis for a short period (4 h) using a divided-root-chamber technique. In root exudates collected from 10-mm long root apices, citrate accounted for 67% of the total organic anions found, followed by malate (29%), trans-aconitate (3%), fumarate (<1%), and cis-aconitate (1%). Rates of citrate exudation from root apices of two genotypes with differential resistance to Al were consistently higher in the Al resistant one, differing by a factor of 1.7 – 3.0 across a range of external Al concentrations. Furthermore, relative Al resistance of eight maize genotypes correlated significantly well with their citrate exudation rate measured at 40 M Al. Higher exudation rates were accompanied by a less inhibited root elongation. The exudation of citrate along the longitudinal axis of fully developed seminal roots showed a particular pattern: citrate was exuded mainly in the regions of root apices, either belonging to the main root or to the lateral roots in the most basal part of the main root. The involvement of citrate in a mechanism of Al resistance is evaluated in terms of protection of the root from the effects of excess Al on root elongation and on nutrient uptake along a root axis showing distinct sites of citrate exudation.     

Molecular characterization of the renal organic anion transporter 1

Organic anions of diverse chemical structures are secreted in renal proximal tubules. The first step in secretion, uptake of organic anions across the basolateral membrane of tubule cells, is mediated for the polyspecific organic anion transporter 1 (OAT1), which exchanges extracellular organic anions for intracellular α-ketoglutarate or glutarate. OAT1 orthologs cloned from various species show 12 putative transmembrane domains and possess several sites for potential post-translational modification. The gene for the human OAT1 is located on chromosome 11q13.1 and is composed of 10 exons. Alternative splicing within exon 9 gives rise to four variants, two of which (OAT1-1 and OAT1-2) are functional. Following heterologous expression in Xenopus laevis oocytes, flounder renal OAT1 transported p-aminohippurate, glutarate, several diuretics, and the nephrotoxic agent ochratoxin A. Two cationic amino acid residues, lysine 394 and arginine 478, were found to be important for interaction with glutarate. Anionic neurotransmitter metabolites and the heavy-metal chelator, 2,3-dimercaptopropane sulfonate, interacted with the rabbit renal OAT1, which is expressed in kidneys and the retina.     

Functionalization of polyoxomolybdates: the example of nitrosyl derivatives

Nitrosyl derivatives of polyoxomolybdates have been synthesized and characterized by X-ray diffraction. Most of them contain the Mo^II(NO)³⁺ unit and their structures are related to the following structural types: Lindqvist, Keggin and decatungstate [W₁₀O₃₂]^4–. Reductive nitrosylation of (NBu₄)₄[-Mo₈O₂₆] by hydroxylamine in methanol yields (NBu₄)₂[Mo₅O₁₃(OMe)₄(NO){Na(MeOH)}]. 3MeOH, which is a versatile reagent yielding a variety of derivatives (i) by the transformation of [Mo₅O₁₃(OMe)₄(NO)]^3– into [Mo₆O₁₈(NO)]^3– in acetonitrile, (ii) by the formation of [PMo₁₂O₃₉(NO)]^4– by reaction of [Mo₅O₁₃(OMe)₄(NO)]^3– with [PMO₁₂O₄₀]^3– in basic condition and (iii) by the formation of mixed valence Mo^VI/Mo^V/Mo^II decamolybdates [Mo₁₀O₂₄(OMe)₇(NO)]^2–, [Mo₁₀O₂₅(OMe)₆(NO)]^– and [Mo₁₀O₂₀(OMe)₉(NO)₃]^2– by chemical reduction of [Mo₅O₁₃(OMe)₄(NO)]^3–; Mo^II is localized while Mo^V are delocalized in the first two species but localized in the third. The unique ligating properties of [Mo₅O₁₃(OMe)₄(NO)]^3– have been documented: this species acts as a tetradentate ligand in [Ce{Mo₅O₁₃(OMe)₄(NO)}₂]^2–, a symmetrically tetraligating ligand in [Rh₂Cp*₂(-Br){-Mo₅O₁₃(OMe)₄(NO)}] and a bidentate ligand in [Mo₅O₁₃(OMe)₄(NO){RhCp*(H₂O)}]^–. Some polyoxomolybdates of the type [Mo₅(NO)₂O₁₂{RC(NH₂)NHO}₂{RC(NH)NO}₂]^2–, which contain the Mo⁰(NO) ₂ ²⁺ unit, have also been characterized.     

Influence of polysaccharides on neutrophil function: Specific antagonists suggest a model for cooperative saccharide-associated inhibition of immune complex-triggered superoxide production

We have previously shown that certain monoisaccharides (N-acfetyl-D-glucosamine and mannose) could cooperativly inhabit the ability of neutrophils to release superoxide anions in response to immuule complexes. To test the possible orgins of the cooperative inhibition of superoxide releaqe,m we have examined the effect of a panel of particular β-glucan and hyaluronan triggered superoxide pelease from neutrophils, other polysaccharides including chitin and mannan were without effeat. Both chitin and mannan, but not other polysaccharides, inhibited the immune complex-mediated qtimulation of superoxide peleaqe in a dose-dependent fashion, In sharp contrast to the coopepative inhibition mediated by monosaacharides, chiting and mannan exhitbted Hill coefficients of 1. This inhibition of superoxide production was not due to simple blockage of Fc receotirs since fluorescent immune complexes bound equlally well to neutrophils in the presence of mannan of chitin as shown by equfluorescence microscopy and quantitative fluorometpy. Furthermore, this inhibition of superoxide release was lot observed when neutrophils were qtimulated with phorbol myristate aaetate and ionophore A23187 or Hyaluronan. Therefope, the secific inhibition of superoxide production by mannan and hitin aould lot be explailed bu eithep peceptor blockage or by some nolspecific effects on cells. We suggest that there molicules interdere with a step in transmembrace qignalling, presumably involving the intergrin CR3. The obserted Hill Cofficients suggest the possibility that one polysaccharide may simultaniouslybind to two monosaccharide bindine sites yielding a Hill coefficient of 1, wheras individual monosacaharides seperately bind vielding a Hill coefficient of 2.     

Mechanisms of 5-aminolevulinic acid uptake at the choroid plexus

5-Aminolevulinic acid (5-ALA) is a precursor of porphyrins and heme that has been implicated in the neuropsychiatric symptoms associated with porphyrias. It is also being used clinically to delineate malignant gliomas. The blood-CSF barrier may be an important interface for 5-ALA transport between blood and brain as in vivo studies have indicated 5-ALA is taken up by the choroid plexuses whereas the normal blood-brain barrier appears to be relatively impermeable. This study examines the mechanisms of 5-[(3)H]ALA uptake into isolated rat lateral ventricle choroid plexuses. Results suggest that there are two uptake mechanisms. The first was a Na(+)-independent uptake system that was pH dependent (being stimulated at low pH). Uptake was inhibited by the dipeptide Gly-Gly and by cefadroxil, an alpha-amino-containing cephalosporin. These properties are the same as the proton-dependent peptide transporters PEPT1 and PEPT2, which have recently been shown to transport 5-ALA in frog oocyte expression experiments. Choroid plexus uptake was not inhibited by captopril, a PEPT1 inhibitor, suggesting PEPT2-mediated uptake. The presence of PEPT2 and absence of PEPT1 in the choroid plexus were confirmed by western blotting. The second potential mechanism was both Na(+) and HCO(3)(-) dependent and appears to be an organic anion transporter, although it is possible that removal of Na(+) and HCO(3)(-) may indirectly affect PEPT2 by affecting intracellular pH. The presence of PEPT2 and a putative Na(+)/HCO(3)(-)-dependent organic anion transporter is important not only for an understanding of 5-ALA movement between blood and brain but also because these transporters may affect the distribution of a number of drugs between blood and CSF.     

Endogenous expression of liver-specific drug transporters for organic anions in the rat hepatocytoma fusion cell line HPCT-1E3

HPCT-1E3 cells, a fusion cell line between primary rat hepatocytes and Fao Reuber hepatoma cells H35, are immortalized hybrid cells with many phenotypic properties of liver parenchyma including phase I and II metabolism and bile acid secretion. Selective elimination of endogenous compounds and drugs by the liver involves transport proteins that complementarily mediate uptake and efflux in co-operation with metabolism, but the study of this function is limited by the unavailability of an integrated in vitro model. Therefore, we investigated the expression of some important liver-specific import and export carrier proteins for organic anions in this cell line. RT-PCR analysis indicated gene expression of Oat2, Oatplal, Oatpla4, Oatplb2, Rfc-1/MTX-1, FOLR, Mrp1-6, mdr1, and Lrp. Uptake and efflux as well as inhibition studies confirmed the functional activity of Oat, Oatp, Rfc-1, Mrp, and Mdr carriers. In conclusion, the hepatocyte-like HPCT-1E3 cell line shows endogenous expression of all liver-specific carrier proteins for organic anions and may hence represent a valuable in vitro model for the study of transport phenomena and their regulation in hepatocytes.     

Thiol-bearing synthetic peptides retain the antioxidant activity of apolipoproteinA-I(Milano)

Apolipoprotein(apo)A-I(Milano) (R173C) and apoA-I(Paris) (R151C) are rare cysteine variants of wild-type (WT) apoA-I that possess novel antioxidant properties on phospholipid surfaces. Yet, the two variants differ in their ability to inhibit lipid peroxidation. In this study, we used synthetic peptides (18mers) to investigate the structural basis for the difference in antioxidant activity between apoA-I(Milano) and apoA-I(Paris). A peptide (aa 167-R173C-184) based on the amphipathic alpha helix harboring the R173C mutation inhibited superoxide anion-mediated oxidation of phospholipid in a dose-dependent manner, but it failed to directly quench superoxide anions in aqueous solution, indicating that the peptide acted at the level of phospholipid to inhibit lipid peroxidation just like the full-length cysteine variant. Peptide 145-R151C-162 based on the helical segment containing R151C exhibited the same capacity as peptide 167-R173C-184 to inhibit lipid peroxidation. Thus, the difference in antioxidant activity between apoA-I(Milano) and apoA-I(Paris) was not governed by the primary amino acid sequence of their individual amphipathic alpha helices, rather contextual constraints within the full-length variants set the difference in antioxidant activity. Cysteine-free peptides were weak inhibitors of lipid peroxidation. These results suggest that thiol-bearing helical peptides based on apoA-I(Milano) may be useful to combat inflammatory related diseases.