Drug resistance in veterinary helminths

At present, there is no effective alternative to chemical control of parasitic helminths where livestock are grazed intensively. Resistance to anthelmintics has become a major problem in veterinary medicine, and threatens both agricultural income and animal welfare. The molecular and biochemical basis of this resistance is not well understood. The lack of reliable biological and molecular tests means that we are not able to follow the emergence and spread of resistance alleles and clinical resistance as well as we need. This review summarizes some of the recent findings on resistance mechanisms, puts forward some recommendations for limiting its impact and suggests some priorities for research in this area.     

The Cu chaperone CopZ is required for Cu homeostasis in Rhodobacter capsulatus and influences cytochrome cbb3 oxidase assembly

Cu homeostasis depends on a tightly regulated network of proteins that transport or sequester Cu, preventing the accumulation of this toxic metal while sustaining Cu supply for cuproproteins. In Rhodobacter capsulatus, Cu‐detoxification and Cu delivery for cytochrome c oxidase (cbb₃‐Cox) assembly depend on two distinct Cu‐exporting P_1B‐type ATPases. The low‐affinity CopA is suggested to export excess Cu and the high‐affinity CcoI feeds Cu into a periplasmic Cu relay system required for cbb₃‐Cox biogenesis. In most organisms, CopA‐like ATPases receive Cu for export from small Cu chaperones like CopZ. However, whether these chaperones are also involved in Cu export via CcoI‐like ATPases is unknown. Here we identified a CopZ‐like chaperone in R. capsulatus, determined its cellular concentration and its Cu binding activity. Our data demonstrate that CopZ has a strong propensity to form redox‐sensitive dimers via two conserved cysteine residues. A ΔcopZ strain, like a ΔcopA strain, is Cu‐sensitive and accumulates intracellular Cu. In the absence of CopZ, cbb₃‐Cox activity is reduced, suggesting that CopZ not only supplies Cu to P_1B‐type ATPases for detoxification but also for cuproprotein assembly via CcoI. This finding was further supported by the identification of a ~150 kDa CcoI‐CopZ protein complex in native R. capsulatus membranes.     

Effect of 17β-estradiol and flavonoids on the regulation of expression of newly identified oestrogen responsive genes in a rat raphe nuclei-derived cell line

Due to the health risks attributed to perimenopausal hormone therapy, phytoestrogens such as flavonoids are receiving widespread attention to help alleviate menopausal symptoms, including hormone-driven mood disorders. Based on our previous reporter gene study regarding their transactivational activity in raphe nuclei cells from a brain region involved in regulation of mood disturbances, we herein study their effects on the regulation of expression of 17β-estradiol (E2)-regulated genes. DNA microarray was used to globally assess E2-induced gene expression in RNDA cells, a rat raphe nuclei-derived cellular model expressing oestrogen receptor β. Out of 212 regulated genes, six were selected for verification and as endpoints for the effect of flavonoids on the regulation of mRNA expression in proliferating as well as differentiating RNDA cells. Under proliferative conditions, E2 up-regulated mRNA expression of Cml-5, Sox-18 and Krt-19. Similar effects were observed in response to 8-prenylnaringenin (8-PN), genistein (GEN), daidzein (DAI) and equol (EQ). In line with E2, mRNA expression of Nefm and Zdhhc-2 was down-regulated following 8-PN, GEN, DAI, EQ and naringenin treatment. No regulation was observed on Slc6a4 mRNA expression in response to E2 or the flavonoids in proliferating RNDA cells. When cells were shifted to conditions promoting differentiation, changes in cell morphology, in mRNA expression levels and in responsiveness towards E2 and the tested flavonoids were noticed. These expression studies additionally highlighted some of the genes as markers for RNDA cellular differentiation. RNDA cells should prove useful to elucidate molecular and cellular mechanisms of exogenous oestrogen receptor ligands with neural cell populations.     

Biochemical characterization of human 3-methylglutaconyl-CoA hydratase and its role in leucine metabolism

The metabolic disease 3-methylglutaconic aciduria type I (MGA1) is characterized by an abnormal organic acid profile in which there is excessive urinary excretion of 3-methylglutaconic acid, 3-methylglutaric acid and 3-hydroxyisovaleric acid. Affected individuals display variable clinical manifestations ranging from mildly delayed speech development to severe psychomotor retardation with neurological handicap. MGA1 is caused by reduced or absent 3-methylglutaconyl-coenzyme A (3-MG-CoA) hydratase activity within the leucine degradation pathway. The human AUH gene has been reported to encode for a bifunctional enzyme with both RNA-binding and enoyl-CoA-hydratase activity. In addition, it was shown that mutations in the AUH gene are linked to MGA1. Here we present kinetic data of the purified gene product of AUH using different CoA-substrates. The best substrates were (E)-3-MG-CoA (V(max) = 3.9 U.mg(-1), K(m) = 8.3 microM, k(cat) = 5.1 s(-1)) and (E)-glutaconyl-CoA (V(max) = 1.1 U.mg(-1), K(m) = 2.4 microM, k(cat) = 1.4 s(-1)) giving strong evidence that the AUH gene encodes for the major human 3-MG-CoA hydratase in leucine degradation. Based on these results, a new assay for AUH activity in fibroblast homogenates was developed. The only missense mutation found in MGA1 phenotypes, c.719C>T, leading to the amino acid exchange A240V, produces an enzyme with only 9% of the wild-type 3-MG-CoA hydratase activity.     

The families of kinases removing the Ca2+ releasing second messenger Ins(1,4,5)P3

The formation and degradation of the second messenger D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] are of great metabolic importance, because of its role in the mediation of calcium release from intracellular stores. The concentration of Ins(1,4,5)P3 in the cell is regulated by three signaling enzymes: phospholipase C isoforms release Ins(1,4,5)P3 from the plasma membrane by hydrolysis of phosphatidyl inositol 4,5-bisphosphate, whereas inositol phosphate 5-phosphatases remove it by dephosphorylation and a group of inositol phosphate kinases eliminate it by further phosphorylation at its 3- or 6-hydroxy group. The latter group is formed by the three isoforms of Ins(1,4,5)P3 3-kinase (IP3K) and inositol phosphate multikinase. In this article the tissue specific gene expression, molecular structure, role in calcium oscillations, regulation by calcium calmodulin, by phosphorylation and by intracellular localization of the IP3K isoforms are discussed. Another important aspect is the evolution of diverse inositol phosphate metabolizing enzymes from a eukaryotic founder by different mechanisms of gene diversification. Finally the role of IPMK in calcium signaling will be elucidated in more detail.