Minor Alternative Pathways Research Articles

Ethanol Metabolism in the Liver, the Induction of Oxidant Stress, and the Antioxidant Defense System.

The liver metabolizes ethanol through three enzymatic pathways: alcohol dehydrogenase (ADH), cytochrome p450 (also called MEOS), and catalase. Alcohol dehydrogenase class I (ADH1) is considered the most important enzyme for the metabolism of ethanol, MEOS and catalase (CAT) are considered minor alternative pathways. However, contradicting experiments suggest that the non-ADH1 pathway may have a greater relevance for the metabolism of ethanol than previously thought. In some conditions, ethanol is predominately metabolized to acetaldehyde via cytochrome P450 family 2 (CYP2E1), which is involved in the generation of reactive oxygen species (ROS), mainly through electron leakage to oxygen to form the superoxide (O2•−) radical or in catalyzed lipid peroxidation. The CAT activity can also participate in the ethanol metabolism that produces ROS via ethanol directly reacting with the CAT-H2O2 complex, producing acetaldehyde and water and depending on the H2O2 availability, which is the rate-limiting component in ethanol peroxidation. We have shown that CAT actively participates in lactate-stimulated liver ethanol oxidation, where the addition of lactate generates H2O2, which is used by CAT to oxidize ethanol to acetaldehyde. Therefore, besides its known role as a catalytic antioxidant component, the primary role of CAT could be to function in the metabolism of xenobiotics in the liver.

Pest categorisation of naturally-spreading psorosis.

The EFSA Panel on Plant Health performed a pest categorisation of naturally‐spreading psorosis of citrus for the European Union. Naturally‐spreading psorosis is poorly defined, because the status of both the disease and its causal agent(s) is uncertain. However, Citrus psorosis virus (CPsV) is a well‐ characterised Ophiovirus that is systematically associated with the psorosis disease and therefore considered to be its causal agent. Efficient diagnostics are available for CPsV. It is present in at least three EU MS. Naturally‐spreading psorosis is currently regulated by Directive 2000/29/EC, while CPsV is not explicitly mentioned in this Directive. CPsV has the potential to enter, establish and spread in the EU territory. However, the main pathway for entry is closed by the existing legislation so that entry is only possible through minor alternative pathways. Plants for planting are the major means of spread while there are uncertainties on the existence and efficiency of a natural spread mechanism. CPsV introduction and spread in the EU would have negative consequences on the EU citrus industry. Of the criteria evaluated by EFSA to qualify as a Union quarantine pest or as a Union regulated non‐quarantine pest (RNQP), Naturally‐spreading psorosis does not meet the criterion of being a well characterised pest or disease. As it is not explicitly mentioned in the legislation, it is unclear whether CPsV meets the criterion of being currently regulated or under official control. It meets, however, all the RNQP criteria. The key uncertainties of this categorisation concern: (1) the causal role of CPsV in the psorosis disease as well as elements of its biology and epidemiology, (2) the exact nature of the Naturally‐spreading psorosis syndrome and the identity of its causal agent and, consequently, (3) whether CPsV should be considered as being covered by the current legislation.

Pest categorisation of Citrus tristeza virus (non-European isolates).

The Panel on Plant Health performed a pest categorisation of non‐European isolates of Citrus tristeza virus (CTV) for the EU territory. CTV is a well characterised virus for which efficient detection assays are available. It is transmitted by vegetative multiplication of infected hosts and by aphid vectors. The most efficient one, Toxoptera citricida, has limited EU presence but another one, Aphis gossypii, is broadly distributed. CTV is reported from a range of countries outside the EU and EU isolates are present in seven of the eight citrus‐growing member states. Non‐EU isolates are not known to occur in the EU and therefore do not meet one of the criteria for being a Union regulated non‐quarantine pest. The natural host range of CTV is restricted to Citrus, Fortunella and Poncirus species. CTV non‐EU isolates are listed in Annex IIAI of Directive 2000/29/EC and the main pathway for entry, plants for planting, is closed by the existing legislation. CTV isolates may therefore only enter through minor alternative pathways. They have the potential to subsequently spread through plants for planting and through the action of aphid vectors. CTV non‐EU isolates are able to cause severe symptoms on a range of citrus crops that EU isolates do not induce. Overall, non‐EU CTV isolates meet all the criteria evaluated by EFSA to qualify as Union quarantine pests. The main knowledge gaps and uncertainties concern (1) the status of Rutaceae species other than Citrus, Fortunella and Poncirus as natural hosts for CTV; (2) the potential undetected presence of non‐EU CTV isolates in the EU and in particular the prevalence and biological properties of CTV isolates that may be present in ornamental citrus; and (3) the inability of EU CTV isolates apparently related to non‐European stem pitting (SP) isolates to cause SP in sweet orange.

Human Metapneumovirus SH and G Glycoproteins Inhibit Macropinocytosis-Mediated Entry into Human Dendritic Cells and Reduce CD4 + T Cell Activation

Human metapneumovirus (HMPV) is a major etiologic agent of respiratory disease worldwide. HMPV reinfections are common in healthy adults and children, suggesting that the protective immune response to HMPV is incomplete and short-lived. We used gene-deletion viruses to evaluate the role of the attachment G and small hydrophobic SH glycoproteins on virus uptake by primary human monocyte-derived dendritic cells (MDDC) in vitro and on subsequent MDDC maturation and activation of autologous T cells. HMPV with deletion of G and SH (ΔSHG) exhibited increased infectivity but had little effect on MDDC maturation. However, MDDC stimulated with ΔSHG induced increased proliferation of autologous Th1-polarized CD4(+) T cells. This effect was independent of virus replication. Increased T cell proliferation was strictly dependent on contact between virus-stimulated MDDC and CD4(+) T cells. Confocal microscopy revealed that deletion of SH and G was associated with an increased number of immunological synapses between memory CD4(+) T cells and virus-stimulated MDDC. Uptake of HMPV by MDDC was found to be primarily by macropinocytosis. Uptake of wild-type (WT) virus was reduced compared to that of ΔSHG, indicative of inhibition by the SH and G glycoproteins. In addition, DC-SIGN-mediated endocytosis provided a minor alternative pathway that depended on SH and/or G and thus operated only for WT. Altogether, our results show that SH and G glycoproteins reduce the ability of HMPV to be internalized by MDDC, resulting in a reduced ability of the HMPV-stimulated MDDC to activate CD4(+) T cells. This study describes a previously unknown mechanism of virus immune evasion. Human metapneumovirus (HMPV) is a major etiologic agent of respiratory disease worldwide. HMPV reinfections are common in healthy adults and children, suggesting that the protective immune response to HMPV is incomplete and short-lived. We found that HMPV attachment G and small hydrophobic SH glycoproteins reduce the ability of HMPV to be internalized by macropinocytosis into human dendritic cells (DC). This results in a reduced ability of the HMPV-stimulated DC to activate Th1-polarized CD4(+) T cells. These results contribute to a better understanding of the nature of incomplete protection against this important human respiratory virus, provide new information on the entry of HMPV into human cells, and describe a new mechanism of virus immune evasion.

3D Domain Swapping Provides a Minor Alternative Refolding Pathway for Ribonuclease A

The C-terminal β-hairpin of RNase A contains a turn with a cis Asn113-Pro114 peptide bond. Pioneering pulsed HX experiments have shown that the C-terminal β-hairpin forms early during refolding. This is puzzling since the Asn113-Pro114 bond is predominately trans at this stage and this conformation destabilizes the native monomer. RNase A, when refolded at high concentration, forms a series of 3D domain-swapped oligomers. In the oligomers formed by C-terminal β-strand swapping, Asn113-Pro114 is trans and permits the formation of a new intersubunit β-sheet. We hypothesize that oligomeric species with trans Asn113-Pro114 may form during refolding. Such species could account for the HX results while comfortably accommodating Asn113-Pro114 in the trans conformation. Here, we test this hypothesis by employing chromatographic methods to detect oligomers forming in refolding conditions and find significant amounts of dimer. We propose that a 3D domain-swapped dimeric intermediate provides a minor alternative pathway for RNase A refolding.

An investigation of dopaminergic metabolites in the striatum and in the substantia nigra in vivo utilising radiolabelled L-DOPA and high performance liquid chromatography: a new approach in the search for transmitter metabolites

Although the major routes of dopamine metabolism seem to be established, at least in terminal regions such as the striatum, it is important to search for previously unknown metabolites and to investigate the relevance of previously suggested minor alternative pathways. An urgent issue is to verify and quantify the transformation of dopamine to putative toxic species, another is to further explore metabolism of dopamine located in cell bodies/dendrites, e.g. in the substantia nigra. We have developed a new method in order to widen the search for alternative metabolites of dopamine. The method is based on systemic injection of tritiated L-DOPA to rats in vivo. Brain tissue was homogenised and centrifuged and the resulting supernatant fractioned following passage through a liquid chromatography system. The radioactivity of each fraction was measured using a scintillation system. By identifying fractions containing major catecholamines and metabolites, according to a standard solution, novel metabolites can be searched for in the remaining fractions. It was possible to obtain sufficient radioactivity in separate fractions of supernatant of homogenised tissue, even from such a small brain nucleus as substantia nigra. Radioactivity was obtained in those fractions that contained the major catecholamines and their metabolites, as well as in other fractions where it may represent previously unknown metabolites of L-DOPA/dopamine. The method was used to evaluate the possibility that cytochrome P450 2E1 is involved in the metabolism of dopamine in the substantia nigra. Significant changes in the radioactivity pattern were induced by inhibition of the enzyme but conclusions about whether cytochrome P450 2E1 is involved in the metabolism of dopamine or not requires further studies. The method can be used to study the metabolism of dopamine and can be extended, by using other radiolabelled precursors, also to evaluate metabolism of other transmitters, e.g. serotonin.

Alternative pathways of sterol synthesis in yeast: Use of C 27 sterol tracers to study aberrant double-bond migrations and evaluate their relative importance

Yeast produce traces of aberrant sterols by minor alternative pathways, which can become significant when normal metabolism is blocked by inhibitors or mutations. We studied sterols generated in the absence of the Δ 8–Δ 7 isomerase (Erg2p) or Δ 5 desaturase (Erg3p) by incubating three mutant strains of Saccharomyces cerevisiae with 5α-cholest-8-en-3β-ol, 8-dehydrocholesterol (Δ 5,8 sterol), or isodehydrocholesterol (Δ 6,8 sterol), together with the corresponding 3 α- 3 H isotopomer. Nine different incubations gave altogether 16 sterol metabolites, including seven Δ 22E sterols formed by action of the yeast C-22 desaturase (Erg5p). These products were separated by silver-ion high performance liquid chromatography (Ag +-HPLC) and identified by gas chromatography–mass spectrometry, nuclear magnetic resonance spectroscopy, and radio-Ag +-HPLC. When Δ 8–Δ 7 isomerization was blocked, exogenous Δ 8 sterol underwent desaturation to Δ 5,8, Δ 6,8, and Δ 8,14 sterols. Formation of Δ 5,8 sterol was strongly favored over Δ 6,8 sterol, but both pathways are essentially dormant under normal conditions of sterol synthesis. The Δ 5,8 sterol was metabolically almost inert except for Δ 22 desaturation, whereas the Δ 6,8 sterol was readily converted to Δ 5,7, Δ 5,7,9(11), and Δ 7,9(11) sterols. The combined results indicate aberrant metabolic pathways similar to those in mammalian systems. However, Δ 5,7 sterol undergoes only slight isomerization or desaturation in yeast, an observation that accounts for the lower levels of Δ 5,8 and Δ 5,7,9(11) sterols in wild-type yeast compared to Smith–Lemli–Opitz individuals.

L-Ascorbic acid biosynthesis

Biosynthesis of L-ascorbate (vitamin C) occurs by different pathways in plants and mammals. Yeast contain D-erythroascorbate, a C5 analog of ascorbate. UDP-D-glucuronic acid is the precursor in mammals. Loss of UDP forms glucuronic acid/glucuronolactone. Reduction of these at C-1 then forms L-gulonic acid/L-gulono-1,4-lactone. The lactone is oxidized by a microsomal L-gulono-1,4-lactone oxidase to ascorbate. Only the L-gulono-1,4-lactone oxidase has been purified and cloned, and very little is known about the properties of the other enzymes. Plants form ascorbate from GDP-D-mannose via GDP-L-galactose, L-galactose, and L-galactono-1,4-lactone. The final oxidation of L-galactono-1,4-lactone to ascorbate is catalyzed by a mitochondrial L-galactono-1,4-lactone dehydrogenase located on the inner membrane and using cytochrome c as electron acceptor. GDP-mannose pyrophosphorylase and L-galactono-1,4-lactone dehydrogenase have been cloned. Yeast synthesizes D-erythroascorbate from D-arabinose and D-arabinono-1,4-lactone in a pathway analogous to that in plants. The plant, mammalian, and yeast aldonolactone oxidase/dehydrogenases that catalyze the last step in each pathway have significant sequence homology. L-Gulono-1,4-lactone oxidase is mutated and not expressed in animals, such as primates, that have lost ascorbate biosynthesis capacity. Assessment of the literature reveals that little is known about many of the enzymes involved in ascorbate biosynthesis or about the factors controlling flux through the pathways. There is also a possibility that minor alternative pathways exist in plants and mammals.

Reaction sequences for (Na + + K +)-dependent ATPase hydrolytic activities: new quantitative kinetic models

To delineate better the reaction sequence of the (Na + + K +)-ATPase and illuminate properties of the active site, kinetic data were fitted to specific quantitative models. For the (Na + + K +)-ATPase reaction, double-reciprocal plots of velocity against ATP (in the millimolar range), with a series of fixed KCl concentrations, are nearly parallel, in accord with the ping pong kinetics of ATP binding at the low-affinity sites only after P i release. However, contrary to requirements of usual formulations, P i is not a competitor toward ATP. A new steady-state kinetic model accommodates these data quantitatively, requiring that under usual assay conditions most of the enzyme activity follows a sequence in which ATP adds after P i release, but also requiring a minor alternative pathway with ATP adding after K + binds but before P i release. The fit to the data also reveals that P i binds nearly as rapidly to E 2 · K · ATP as to E 2 · K, whereas ATP binds quite slowly to E 2 · P · K: the site resembles a cul-de-sac with distal ATP and proximal P i sites. For the K +-nitrophenyl phosphatase reaction also catalyzed by this enzyme, the apparent affinities for both substrate and P i (as inhibitor) decrease with higher KCl concentrations, and both P i and TNP-ATP appear to be competitive inhibitors toward substrate with 10 mM KCl but noncompetitive inhibitors with 1 mM KCl. These data are accommodated quantitatively by a steady-state model allowing cyclic hydrolytic activity without obligatory release of K +, and with exclusive binding of substrate vs. either P i or TNP-ATP. The greater sensitivity of the phosphatase reaction to both P i and arsenate is attributable to the weaker binding by the occuluded-K + enzyme form occurring in the (Na + + K +)-ATPase reaction sequence. The steady-state models are consistent with cyclical interconversion of high- and low-affinity substrate sites accompanying E 1/E 2 transitions, with distortion to low-affinity sites altering not only affinity and route of access but also separating the adenine- and phosphate-binding regions, the latter serving in the E 2 conformation as the active site for the phosphatase reaction.

ChemInform Abstract: ELECTROCHEMICAL REDUCTION OF TETRAPHENYLPHOSPHONIUM CATION IN LOW ACIDITY MEDIA

AbstractDie Reduktion wird polarographisch, coulometrisch und durch cyclische Voltammetrie verfolgt und im präparativen Maßstab durchgeführt.

Electrochemical reduction of tetraphenylphosphonium cation in low acidity media

The electrochemical reduction on mercury of P + φ 4 in low acidity solvents is studied using polarography, cyclic voltammetry, coulometry and preparative-scale electrolysis. Triphenylphosphine oxide is a major product besides triphenylphosphine and benzene. The reduction process involves the cleavage of a PC bond leading to an unstable carbanion which reacts on the residual water to give OH −. Pφ 3O is then formed by reaction of OH − on the starting P + φ 4. The formation of a small amount of biphenyl through a one-electron process is a distinct but minor alternative pathway.