Ligand modulation of [35S]GTPgammaS binding at human alpha(2A), alpha(2B) and alpha(2C) adrenoceptors

Affinities and efficacies of chemically diverse ligands--some of them used as clinical agents--were examined, employing [3H]RX821,002 and [35S]GTPgammaS binding assays, respectively, at human (h) cloned, halpha(2A), halpha(2B) and halpha(2C) adrenoceptors (AR) expressed in Chinese hamster ovary (CHO) cells. As compared to noradrenaline (NA, efficacy defined as 100%), the majority of the 13 agonists tested generally behaved as partial agonists. Amongst 18 antagonists, pK(B) and pK(i) values, which were highly correlated for each alpha(2)-AR subtype, failed to reveal any strikingly selective agents. Inverse agonist properties were not detected for any antagonist, consistent with a lack of constitutive activity suggested by the monophasic inhibition of [35S]GTPgammaS binding by GTPgammaS. These data should facilitate interpretation of experimental and clinical actions of adrenergic agonists. Moreover, they emphasize the continuing need for alpha(2)-AR subtype-selective antagonists in order to define further the roles and therapeutic relevance of halpha(2A)-, halpha(2B)-, and halpha(2C)-AR.     

The two enantiospecific dichlorprop/alpha-ketoglutarate-dioxygenases from Delftia acidovorans MC1--protein and sequence data of RdpA and SdpA

Two alpha-ketoglutarate-dependent dioxygenases carrying enantiospecific activity for the etherolytic cleavage of racemic phenoxypropionate herbicides [(RS)-2-(2,4-dichlorophenoxy)propionate and (RS)-2-(4-chloro-2-methylphenoxy)propionate] from Delftia acidovorans MC1 were characterized with respect to protein and sequence data. The (S)-phenoxypropionate/alpha-ketoglutarate-dioxygenase (SdpA) appeared as a monomeric enzyme with a molecular weight of 32 kDa in the presence of SDS. N-terminal sequences revealed relationship to alpha-ketoglutarate-dependent taurine dioxygenase (TauD) and to 2,4-dichlorophenoxyacetate/alpha-ketoglutarate-dioxygenase (TfdA). The (R)-phenoxypropionate/alpha-ketoglutarate-dioxygenase (RdpA) referred to 36 kDa in the presence of SDS and to 108 kDa under native conditions. Internal sequences of fragments obtained after digestion made evident relationship to TfdA and TauD. Two-dimensional electrophoretic separation resulted in the resolution of up to 3 individual spots with almost identical molecular weights but different isoelectric points with both RdpA and SdpA. The structural differences of these isoenzyme forms are not yet clear.     

Functional dissection of the Drosophila modifier of variegation Su(var)3-7

An increase in the dose of the heterochromatin-associated Su(var)3-7 protein of Drosophila augments the genomic silencing of position-effect variegation. We have expressed a number of fragments of the protein in flies to assign functions to the different domains. Specific binding to pericentric heterochromatin depends on the C-terminal half of the protein. The N terminus, containing six of the seven widely spaced zinc fingers, is required for binding to bands on euchromatic arms, with no preference for pericentric heterochromatin. In contrast to the enhancing properties of the full-length protein, the N terminus half has no effect on heterochromatin-dependent position-effect variegation. In contrast, the C terminus moiety suppresses variegation. This dominant negative effect on variegation could result from association of the fragment with the wild type endogenous protein. Indeed, we have found and mapped a domain of self-association in this C-terminal half. Furthermore, a small fragment of the C-terminal region actually depletes pericentric heterochromatin from endogenous Su(var)3-7 and has a very strong suppressor effect. This depletion is not followed by a depletion of HP1, a companion of Su(var)3-7. This indicates that Su(var)3-7 does not recruit HP1 to heterochromatin. We propose in conclusion that the association of Su(var)3-7 to heterochromatin depends on protein-protein interaction mediated by the C-terminal half of the sequence, while the silencing function requires also the N-terminal half containing the zinc fingers.     

A germline-specific gap junction protein required for survival of differentiating early germ cells

Germ cells require intimate associations and signals from the surrounding somatic cells throughout gametogenesis. The zero population growth (zpg) locus of Drosophila encodes a germline-specific gap junction protein, Innexin 4, that is required for survival of differentiating early germ cells during gametogenesis in both sexes. Animals with a null mutation in zpg are viable but sterile and have tiny gonads. Adult zpg-null gonads contain small numbers of early germ cells, resembling stem cells or early spermatogonia or oogonia, but lack later stages of germ cell differentiation. In the male, Zpg protein localizes to the surface of spermatogonia, primarily on the sides adjacent to the somatic cyst cells. In the female, Zpg protein localizes to germ cell surfaces, both those adjacent to surrounding somatic cells and those adjacent to other germ cells. We propose that Zpg-containing gap junctional hemichannels in the germ cell plasma membrane may connect with hemichannels made of other innexin isoforms on adjacent somatic cells. Gap junctional intercellular communication via these channels may mediate passage of crucial small molecules or signals between germline and somatic support cells required for survival and differentiation of early germ cells in both sexes.     

Oskar anchoring restricts pole plasm formation to the posterior of the Drosophila oocyte

Localization of the maternal determinant Oskar at the posterior pole of Drosophila melanogaster oocyte provides the positional information for pole plasm formation. Spatial control of Oskar expression is achieved through the tight coupling of mRNA localization to translational control, such that only posterior-localized oskar mRNA is translated, producing the two Oskar isoforms Long Osk and Short Osk. We present evidence that this coupling is not sufficient to restrict Oskar to the posterior pole of the oocyte. We show that Long Osk anchors both oskar mRNA and Short Osk, the isoform active in pole plasm assembly, at the posterior pole. In the absence of anchoring by Long Osk, Short Osk disperses into the bulk cytoplasm during late oogenesis, impairing pole cell formation in the embryo. In addition, the pool of untethered Short Osk causes anteroposterior patterning defects, owing to the dispersion of pole plasm and its abdomen-inducing activity throughout the oocyte. We show that the N-terminal extension of Long Osk is necessary but not sufficient for posterior anchoring, arguing for multiple docking elements in Oskar. This study reveals cortical anchoring of the posterior determinant Oskar as a crucial step in pole plasm assembly and restriction, required for proper development of Drosophila melanogaster.     

Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier

Members of the Dr family of adhesins of Escherichia coli recognize as a receptor the Dr(a) blood-group antigen present on the complement regulatory and signalling molecule, decay-accelerating factor (DAF). One member of this family, the Dr haemagglutinin, also binds to a second receptor, type IV collagen. Structure/function information regarding these adhesins has been limited and domains directly involved in the interaction with DAF have not been determined. We devised a strategy to identify amino acids in the Dr haemagglutinin that are specifically involved in the interaction with DAF. The gene encoding the adhesive subunit, draE, was subjected to random mutagenesis and used to complement a strain defective for its expression. The resulting mutants were enriched and screened to obtain those that do not bind to DAF, but retain binding to type IV collagen. Individual amino acid changes at positions 10, 63, 65, 75, 77, 79 and 131 of the mature DraE sequence significantly reduced the ability of the DraE adhesin to bind DAF, but not collagen. Over half of the mutants obtained had substitutions within amino acids 63-81. Analysis of predicted structures of DraE suggest that these proximal residues may cluster to form a binding domain for DAF.     

Glycosylation of the hepatitis C virus envelope protein E1 occurs posttranslationally in a mannosylphosphoryldolichol-deficient CHO mutant cell line

The addition of N-linked glycans to a protein is catalyzed by oligosaccharyltransferase, an enzyme closely associated with the translocon. N-glycans are believed to be transferred as the protein is being synthesized and cotranslationally translocated in the lumen of the endoplasmic reticulum. We used a mannosylphosphoryldolichol-deficient Chinese hamster ovary mutant cell line (B3F7 cells) to study the temporal regulation of N-linked core glycosylation of hepatitis C virus envelope protein E1. In this cell line, truncated Glc(3)Man(5)GlcNAc(2) oligosaccharides are transferred onto nascent proteins. Pulse-chase analyses of E1 expressed in B3F7 cells show that the N-glycosylation sites of E1 are slowly occupied until up to 1 h after protein translation is completed. This posttranslational glycosylation of E1 indicates that the oligosaccharyltransferase has access to this protein in the lumen of the endoplasmic reticulum for at least 1 h after translation is completed. Comparisons with the N-glycosylation of other proteins expressed in B3F7 cells indicate that the posttranslational glycosylation of E1 is likely due to specific folding features of this acceptor protein.     

Formin-2, polyploidy,hypofertility and positioning of the meiotic spindle in mouse oocytes

Successful reproduction in mammals requires a competent egg, which is formed during meiosis through two assymetrical cell divisions. Here, we show that a recently identified formin homology (FH) gene, formin-2 (Fmn2), is a maternal-effect gene that is expressed in oocytes and is required for progression through metaphase of meiosis I. Fmn2(-/-) oocytes cannot correctly position the metaphase spindle during meiosis I and form the first polar body. We demonstrate that Fmn2 is required for microtubule-independent chromatin positioning during metaphase I. Fertilization of Fmn2(-/-) oocytes results in polyploid embryo formation, recurrent pregnancy loss and sub-fertility in Fmn2(-/-) females. Injection of Fmn2 mRNA into Fmn2-deficient oocytes rescues the metaphase I block. Given that errors in meiotic maturation result in severe birth defects and are the most common cause of chromosomal aneuploidy and pregnancy loss in humans, studies of Fmn2 may provide a better understanding of infertility and birth defects.