FMRFamide-like neuropeptides as putative ligands of the latrophilin-like HC110-R from Haemonchus contortus.

The latrophilin-like receptor HC110-R of the parasitic nematode Haemonchus contortus has been previously identified as a target for the novel anthelmintic drug emodepside, but the natural ligand(s) remained completely unknown to date. Here, we investigate 11 different FMRFamide-like neuropeptides as putative ligands by surface plasmon resonance with an immobilized recombinant 54kDa aminoterminal fragment of HC110-R as an interaction partner. AF1, AF10 and PF2 exhibit binding with low affinities as indicated by a K(d) of 11 microM for AF1, 52 microM for AF10 and 583 microM for PF2. Our data indicate that AF1, AF10, and PF2 are putative natural ligands of HC110-R presumably involved in the control of pharyngeal pumping of nematode worms.

Genomic organization of the cadmium-inducible tandem repeat 25-kDa metallothionein of the oligochaete worm Enchytraeus buchholzi.

The terrestric oligochaete worm Enchytraeus buchholzi survives in cadmium (Cd)-polluted environments by aid of its Cd-inducible 25 kDa cysteine-rich protein (CRP). Here, we analyze promoter and structure of the crp gene and compare its relationship to MT genes. The crp gene, approximately 12 kbp long, consists of 10 exons with exons 2 to 9 encoding eight almost identical repeats of predominantly 31 amino acids of the CRP. The introns of the crp gene contain various repetitive elements including retrotransposon-like sequences. The 683-bp promoter of the non-constitutive crp gene exhibits a much higher basal activity than the mouse MT-II promoter in HepG2 cells. Essential for crp promoter activity is the distal region (-683/-521) with a GC box and the proximal region (-308/-8) with the four MREa, b, c, d and AP-1, -2, -3 elements, whereas the central portion (-521/-309) with CAAT box, CRE and a XRE causes promoter repression. The TATA box-, MREc- and the AP-2, -3-containing region are required for high crp promoter activity. Our data support the view that the crp gene is a unique MT-gene and has evolved by exon duplications from a MT-like ancestral gene.

Cyclooctadepsipeptides--an anthelmintically active class of compounds exhibiting a novel mode of action.

There are three major classes of anthelmintics for veterinary use: the benzimidazoles/prebenzimidazoles, the tetrahydropyrimidines/imidazothiazoles, and the macrocyclic lactones. In nematodes, there are five targets for the existing anthelmintics: the nicotinergic acetylcholine receptor which is the target of tetrahydropyrimidines/imidazothiazoles and indirectly that of the acetylcholineesterase inhibitors; the GABA receptor which is the target of piperazine, the glutamate-gated chloride channel as the target of the macrocyclic lactones, and beta-tubulin as the target of prebenzimidazoles/benzimidazoles. All these anthelmintics are now in serious danger because of the worldwide spread of resistant nematodes in sheep, cattle, horses and pigs. The class of cyclooctadepsipeptides has entered the scene of anthelmintic research in the early 1990s. PF1022A, the first anthelmintically active member, is a natural compound from the fungus Mycelia sterilia that belongs to the microflora of the leaves of the Camellia japonica. PF1022A contains 4 N-Methyl-L-leucines, 2 D-lactic acids and 2-D-phenyllactic acids arranged as a cyclic octadepsipeptide with an alternating L-D-L-configuration. Emodepside is a semisynthetic derivative of PF1022A with a morpholine ring at each of the two D-phenyllactic acids in para position. The anthelmintic activity is directed against gastrointestinal nematodes in chicken, mice, rats, meriones, dogs, cats, sheep, cattle and horses. Moreover, emodepside is active against Trichinella spiralis larvae in muscles, microfilariae and preadult filariae and Dictyocaulus viviparus. PF1022A and emodepside are fully effective against benzimidazole-, levamisole or ivermectin-resistant nematodes in sheep and cattle. In Ascaris suum both cyclooctadepsipeptides lead to paralysis indicating a neuropharmacological action of these compounds. Using a PF1022A-ligand immunoscreening of a cDNA library from Haemonchus contortus a cDNA clone of 3569 base pairs could be identified. This clone codes for a novel 110 kDa heptahelical transmembrane receptor, named HC110R. Database- and phylogenetic analysis reveals that this receptor is a homolog to B0457.1 from Caenorhabditis elegans and has significant similarity to latrophilins from human, cattle and rat. HC110R is located in the plasma membrane and in lysosomes and endosomes. Alpha-latrotoxin, the poison of the black widow spider, binds at a 54 kDa aminoterminal fragment of HC110R. After binding a Ca2+-influx into HEK293 cells is induced which can be blocked by EGTA, Cd2+ or nifedipin. PF1022A or emodepside also bind to this 54 kDa aminoterminal region of HC110R and interact with the functional responses of alpha-latrotoxin. In C. elegans antibodies against the C-or N-terminus of HC110R bind to the B0457.1 protein located in the pharynx. Electrophysiological studies reveal that emodepside inhibits pharyngeal pumping of the nematodes in a concentration dependent way with an IC(50) value of about 4 nM. Thus, it is tempting to speculate that emodepside exerts its action on nematodes via a latrophilin-like receptor which might have an important regulatory function on pharyngeal pumping.

Developmental regulation of intracellular and surface androgen receptors in T cells.

Increasing information indicates that testosterone actions on cells are mediated not only through the classical intracellular androgen receptor (iAR), but also through membrane androgen receptors (mAR) on cell surfaces. Here, we investigate the expression pattern of mAR and iAR in thymic T cells, which is compared with that of splenic T cells. Thymic T cells are testosterone-sensitive in vivo, i.e. treatment of female C57BL/10 mice with testosterone for 3 weeks decreased the total number of thymic T cells by approximately 90%. The percentage of CD4(-) CD8(-) T cells increased, whereas that of the subsequent CD4(+) CD8(+) T cells was diminished. Flow cytometry and confocal laser scanning microscopy (CLSM) with different anti-iAR antibodies localized iAR predominantly in the cytoplasm, but not on the surface of thymic T cells. The iAR are functionally active since the iAR are induced by testosterone to translocate from cytoplasm to nucleus, and they bind the testosterone analogue 3H-R1881 with high affinity (K(d) approximately 2.2 nM) and saturable capacity (approximately 10,000 binding sites per cell) as determined by Scatchard analysis. By contrast, the impeded ligand testosterone-BSA-FITC (T-BSA-FITC) did not bind to the surface of thymic T cells. In accordance, testosterone was unable to induce any rapid rise in the intracellular free Ca(2+) concentration of Fura-2 loaded thymocytes. This indicates that thymic T cells do not express any significant amounts of mAR. Conversely, splenic T cells express functionally active mAR, whereas their expressed iAR are not functional in the genomic pathway. Our results support the view of a delicately balanced developmental regulation of iAR and mAR in T cells.

Human ortholog to mouse gene imap38 encoding an ER-localizable G-protein belongs to a gene family clustered on chromosome 7q32-36.

Inducibility of the mouse gene imap38 in the spleen has been recently described to correlate with resistance to Plasmodium chabaudi malaria. Here, we characterize the human ortholog gene himap1. The HIMAP1 34 kDa protein is localizable at the endoplasmic reticulum in transfected cells. It contains a GTP-binding domain, but it does not bind GTP, in contrast to mouse IMAP38. The himap1 gene belongs to a gene family clustered on chromosome 7q32-36 within a region highly syntenic to the mouse imap38 locus on chromosome 6B. The himap genes 1, 2, 3, and 4 display a conserved intron/exon structure. The mRNA of the himap1 gene is predominantly expressed in the spleen, in lymph nodes to a lesser extent, and only at very low levels in diverse cancer cell lines. In accordance, imap-like genes in mice and plants are associated with proliferative and apoptotic events suggesting a role in the control of cell death/survival.

Cadmium resistance conferred to yeast by a non-metallothionein-encoding gene of the earthworm Enchytraeus.

The earthworm Enchytraeus is able to survive in cadmium (Cd)-polluted environments. Upon Cd exposure, the worms express a gene encoding the putative non-metallothionein 25-kDa cysteine-rich protein (CRP), which contains eight repeats with highly conserved cysteines in Cys-X-Cys and Cys-Cys arrangements exhibiting 36-53% identities to the 6-7-kDa metallothioneins of different organisms. Here, we demonstrate that the CRP protein confers a highly Cd-resistant phenotype to a Cd-hypersensitive yeast strain. Cd resistance increases with increasing numbers of expressed CRP repeats, but even one 3-kDa CRP repeat still mediates Cd resistance. Site-directed mutagenesis reveals that each single cysteine within a given repeat is important for Cd resistance, though to a different extent. However, replacement of other conserved amino acids such as Pro(136) and Asp(196) at the CRP repeat junctions does not affect Cd resistance. Our data indicate (i) that the non-metallothionein CRP protein is able to detoxify Cd and (ii) that this is dependent on the availability of sulfhydryl groups of the conserved cysteines.