Daughterless coordinates somatic cell proliferation,differentiation and germline cyst survival during follicle formation in Drosophila

During Drosophila oogenesis two distinct stem cell populations produce either germline cysts or the somatic cells that surround each cyst and separate each formed follicle. From analyzing daughterless (da) loss-of-function, overexpression and genetic interaction phenotypes, we have identified several specific requirements for da(+) in somatic cells during follicle formation. First, da is a critical regulator of somatic cell proliferation. Also, da is required for the complete differentiation of polar and stalk cells, and elevated da levels can even drive the convergence and extension that is characteristic of interfollicular stalks. Finally, da is a genetic regulator of an early checkpoint for germline cyst progression: Loss of da function inhibits normally occurring apoptosis of germline cysts at the region 2a/2b boundary of the germarium, while da overexpression leads to postmitotic cyst degradation. Collectively, these da functions govern the abundance and diversity of somatic cells as they coordinate with germline cysts to form functional follicles.     

Biphasic activation of Aurora-A kinase during the meiosis I- meiosis II transition in Xenopus oocytes

Xenopus Aurora-A (also known as Eg2) is a member of the Aurora family of mitotic serine/threonine kinases. In Xenopus oocytes, Aurora-A phosphorylates and activates a cytoplasmic mRNA polyadenylation factor (CPEB) and therefore plays a pivotal role in MOS translation. However, hyperphosphorylation and activation of Aurora-A appear to be dependent on maturation-promoting factor (MPF) activation. To resolve this apparent paradox, we generated a constitutively activated Aurora-A by engineering a myristylation signal at its N terminus. Injection of Myr-Aurora-A mRNA induced germinal vesicle breakdown (GVBD) with the concomitant activation of MOS, mitogen-activated protein kinase, and MPF. Myr-Aurora-A-injected oocytes, however, appeared to arrest in meiosis I with high MPF activity and highly condensed, metaphase-like chromosomes but no organized microtubule spindles. No degradation of CPEB or cyclin B2 was observed following GVBD in Myr-Aurora-A-injected oocytes. In the presence of progesterone, the endogenous Aurora-A became hyperphosphorylated and activated at the time of MPF activation. Following GVBD, Aurora-A was gradually dephosphorylated and inactivated before it was hyperphosphorylated and activated again. This biphasic pattern of Aurora-A activation mirrored that of MPF activation and hence may explain meiosis I arrest by the constitutively activated Myr-Aurora-A.     

Vaccination-induced protection of lambs against the parasitic nematode Haemonchus contortus correlates with high IgG antibody responses to the LDNF glycan antigen

Lambs respond to vaccination against bacteria and viruses but have a poor immunological response to nematodes. Here we report that they are protected against the parasitic nematode Haemonchus contortus after vaccination with excretory/secretory (ES) glycoproteins using Alhydrogel as an adjuvant. Lambs immunized with ES in Alhydrogel and challenged with 300 L3 larvae/kg body weight had a reduction in cumulative egg output of 89% and an increased percentage protection of 54% compared with the adjuvant control group. Compared to the adjuvant dimethyl dioctadecyl ammonium bromide, Alhydrogel induced earlier onset and significantly higher ES- specific IgG, IgA, and IgE antibody responses. In all vaccinated groups a substantial proportion of the antibody response was directed against glycan epitopes, irrespective of the adjuvant used. In lambs vaccinated with ES in Alhydrogel but not in any other group a significant increase was found in antibody levels against the GalNAcbeta1,4 (Fucalpha1,3)GlcNAc (fucosylated LacdiNAc, LDNF) antigen, a carbohydrate antigen that is also involved in the host defense against the human parasite Schistosoma mansoni. In lambs the LDNF-specific response increased from the first immunization onward and was significantly higher in protected lambs. In addition, an isotype switch from LDNF-specific IgM to IgG was induced that correlated with protection. These data demonstrate that hyporesponsiveness of lambs to H. contortus can be overcome by vaccination with ES glycoproteins in a strong T-helper 2 type response-inducing aluminum adjuvant. This combination generated high and specific antiglycan antibody responses that may contribute to the vaccination-induced protection.     

The dendritic cell-specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni egg antigens and recognizes the glycan antigen Lewis x

Schistosoma mansoni soluble egg antigens (SEAs) are crucially involved in modulating the host immune response to infection by S. mansoni. We report that human dendritic cells bind SEAs through the C-type lectin dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN). Monoclonal antibodies against the carbohydrate antigens Lewisx (Lex) and GalNAcbeta1-4(Fucalpha1-3)GlcNAc (LDNF) inhibit binding of DC-SIGN to SEAs, suggesting that these glycan antigens may be critically involved in binding. In a solid-phase adhesion assay, DC-SIGN-Fc binds polyvalent neoglycoconjugates that contain the Lex antigen, whereas no binding was observed to Galbeta1-4GlcNAc, and binding to neoglycoconjugates containing only alpha-fucose or oligosaccharides with a terminal alpha1-2-linked fucose is low. These data indicate that binding of DC-SIGN to Lex antigen is fucose-dependent and that adjacent monosaccharides and/or the anomeric linkage of the fucose are important for binding activity. Previous studies have shown that DC-SIGN binds HIV gp120 that contains high-mannose-type N-glycans. Site-directed mutagenesis within the carbohydrate recognition domain (CRD) of DC-SIGN demonstrates that amino acids E324 and E347 are involved in binding to HIV gp120, Lex, and SEAs. By contrast, mutation of amino acid Val351 abrogates binding to SEAs and Lex but not HIV gp120. These data suggest that DC-SIGN recognizes these ligands through different (but overlapping) regions within its CRD. Our data imply that DC-SIGN not only is a pathogen receptor for HIV gp120 but may also function in pathogen recognition by interaction with the carbohydrate antigens Lex and possibly LDNF, which are found on important human pathogens, such as schistosomes and the bacterium Helicobacter pylori.     

Identification of a novel UDP-Glc:GlcNAc beta1-->4-glucosyltransferase in Lymnaea stagnalis that may be involved in the synthesis of complex-type oligosaccharide chains

Several studies suggest, that the snail Lymnaea stagnalis contains glycoproteins whose oligosaccharide side chains have structural features not commonly found in mammalian glycoproteins. In this study, prostate glands of L. stagnalis were incubated in media containing either [(3)H]-mannose, [(3)H]-glucosamine, or [(3)H]-galactose, and the metabolically radiolabeled protein-bound oligosaccharides were analyzed. The newly synthesized diantennary-like complex-type asparagine-linked chains contained a considerable amount of glucose, next to mannose, GlcNAc, fucose, galactose, and traces of GalNAc. Since glucose has not been found before as a constituent of diantennary N-linked glycans as far as we know, we assayed the prostate gland of L. stagnalis for a potential glucosyltransferase activity involved in the biosynthesis of such structures. We report here, that the prostate gland of L. stagnalis contains a beta1-->4-glucosyltransferase activity that transfers glucose from UDP-glucose to acceptor substrates carrying a terminal N-acetylglucosamine. The enzyme prefers substrates carrying a terminal GlcNAc that is beta6 linked to a Gal or a GalNAc, structures occurring in O-linked glycans, or a GlcNAc that is beta2 linked to mannose, as is present in N-linked glycans. Based on combined structural and enzymatic data, we propose that the novel beta1-->4-gluco-syltransferase present in the prostate gland may be involved in the biosynthesis of Glcbeta1-->4GlcNAc units in complex-type glycans, in particular in N-linked diantennary glycans.     

Muscular sarcocystosis in coyotes from Oklahoma

In a recent survey in Oklahoma (USA), 52 free-ranging coyotes were examined for the presence of sarcocysts. Two of these coyotes were found infected with sarcocysts in skeletal muscle. By light microscopy, the cyst wall was thin and smooth. Ultrastructurally, the cyst wall had minute villar protrusions. The sarcocysts were 14.4 to 50.4 microm wide and 46.8 to 99 microm long. This is the first report of Sarcocystis sp. sarcocysts in the skeletal muscle of coyotes.     

Naturally occurring and experimentally transmitted Hepatozoon americanum in coyotes from Oklahoma

Twenty free-ranging coyotes (Canis latrans) in Oklahoma (USA) were examined for the presence of naturally occurring infections with Hepatozoon americanum and to determine if bone lesions attributable to H. americanum were present. Although eight of the 20 free-ranging coyotes were found to be naturally infected with H. americanum, no bone lesions were detected. In addition, two coyote pups were exposed to H. americanum oocysts collected from experimentally infected ticks and the course of the resulting infection was followed. Both experimentally infected coyotes developed hepatozoonosis detectable by specific muscle lesions beginning 4 wk after exposure. Bone lesions were detected grossly and histologically at necropsy. Histologic evidence of periosteal bone proliferation ranged from segmental areas of plump hypercellularity and thickening of the periosteum, with minor degrees of osteogenesis, to extensive proliferation of woven bone and periosteal hypercellularity and thickening. Nymphal Amblyomma maculatum that fed on one of the experimentally infected coyote pups became infected and mature H. americanum oocysts were recovered when the ticks molted to adults. These results demonstrate that coyotes in some parts of Oklahoma are naturally infected with H. americanum, that experimentally infected coyotes can develop clinical disease, including characteristic bone lesions, and that A. maculatum nymphs can acquire infections by feeding on them.     

Evidence for microbial Fe(III) reduction in anoxic, mining-impacted lake sediments (Lake Coeur d'Alene, Idaho)

Mining-impacted sediments of Lake Coeur d'Alene, Idaho, contain more than 10% metals on a dry weight basis, approximately 80% of which is iron. Since iron (hydr)oxides adsorb toxic, ore-associated elements, such as arsenic, iron (hydr)oxide reduction may in part control the mobility and bioavailability of these elements. Geochemical and microbiological data were collected to examine the ecological role of dissimilatory Fe(III)-reducing bacteria in this habitat. The concentration of mild-acid-extractable Fe(II) increased with sediment depth up to 50 g kg(-1), suggesting that iron reduction has occurred recently. The maximum concentrations of dissolved Fe(II) in interstitial water (41 mg liter(-1)) occurred 10 to 15 cm beneath the sediment-water interface, suggesting that sulfidogenesis may not be the predominant terminal electron-accepting process in this environment and that dissolved Fe(II) arises from biological reductive dissolution of iron (hydr)oxides. The concentration of sedimentary magnetite (Fe(3)O(4)), a common product of bacterial Fe(III) hydroxide reduction, was as much as 15.5 g kg(-1). Most-probable-number enrichment cultures revealed that the mean density of Fe(III)-reducing bacteria was 8.3 x 10(5) cells g (dry weight) of sediment(-1). Two new strains of dissimilatory Fe(III)-reducing bacteria were isolated from surface sediments. Collectively, the results of this study support the hypothesis that dissimilatory reduction of iron has been and continues to be an important biogeochemical process in the environment examined.     

Noncovalent association of P-selectin glycoprotein ligand-1 and minimal determinants for binding to P-selectin

P-selectin glycoprotein ligand-1 (PSGL-1) is a disulfide-bonded, homodimeric mucin ( approximately 250 kDa) on leukocytes that binds to P-selectin on platelets and endothelial cells during the initial steps in inflammation. Because it has been proposed that only covalently dimerized PSGL-1 can bind P-selectin, we investigated the factors controlling dimerization of PSGL-1 and re-examined whether covalent dimers are required for binding its P-selectin. Recombinant forms of PSGL-1 were created in which the single extracellular Cys (Cys(320)) was replaced with either Ser (C320S-PSGL-1) or Ala (C320A-PSGL-1). Both recombinants migrated as monomeric species of approximately 120 kDa under both nonreducing and reducing conditions on SDS-polyacrylamide gel electrophoresis. P-selectin bound similarly to cells expressing either wild type or mutated forms of PSGL-1 in both flow cytometric and rolling adhesion assays. Unexpectedly, chemical cross-linking studies revealed that both C320S- and C320A-PSGL-1 noncovalently associate in the plasma membrane and cross-linking generates dimeric species. Chimeric recombinants of PSGL-1 in which the transmembrane domain in PSGL-1 was replaced with the transmembrane domain of CD43 (CD43TMD-PSGL-1) could not be chemically cross-linked, suggesting that residues within the transmembrane domain of PSGL-1 are required for noncovalent association. Cells expressing CD43TMD-PSGL-1 bound P-selectin. To further address the ability of P-selectin to bind monomeric derivatives of PSGL-1, intact HL-60 cells were trypsin-treated, which generated a soluble approximately 25-kDa NH(2)-terminal fragment of PSGL-1 that bound to immobilized P-selectin. Because N-glycosylation of PSGL-1 hinders trypsin cleavage, a recombinant form of PSGL-1 was generated in which all three potential N-glycosylation sites were mutated (DeltaN-PSGL-1). Cells expressing DeltaN-PSGL-1 bound P-selectin, and trypsin treatment of the cells generated NH(2)-terminal monomeric fragments (<10 kDa) of PSGL-1 that bound to P-selectin. These results demonstrate that Cys(320)-dependent dimerization of PSGL-1 is not required for binding to P-selectin and that a small monomeric fragment of PSGL-1 is sufficient for P-selectin recognition.