Persyn, a member of the synuclein family, has a distinct pattern of expression in the developing nervous system

The synucleins are a unique family of small intracellular proteins that have recently attracted considerable attention because of their involvement in human neurodegenerative diseases. We have cloned a new member of the synuclein family called persyn. In contrast to other synucleins, which are presynaptic proteins of CNS neurons, persyn is a cytosolic protein that is expressed predominantly in the cell bodies and axons of primary sensory neurons, sympathetic neurons, and motoneurons. Northern blotting, in situ hybridization, Western blotting, and immunohistochemistry revealed that persyn mRNA and protein are expressed in these neurons from the earliest stages of axonal outgrowth and are maintained at a high level throughout life. Persyn also becomes detectable in evolutionary recent regions of the brain by adulthood.     

Developmentally regulated gene expression of thrombomodulin in postimplantation mouse embryos

Embryonic lethality of thrombomodulin-deficient mice has indicated an essential role for this regulator of blood coagulation in murine development. Here, the embryonic expression pattern of thrombomodulin was defined by surveying beta-galactosidase activity in a mouse strain in which the reporter gene was placed under the regulatory control of the endogenous thrombomodulin promoter via homologous recombination in embryonic stem cells. The murine trophoblast was identified as a previously unrecognized anatomical site where TM expression is conserved between humans and mice and may exert a critical function during postimplantation development. Targeted reporter gene expression in mesodermal precursors of the endothelial cell lineage defined thrombomodulin as an early marker of vascular differentiation. Analysis of the thrombomodulin promoter in differentiating ES cells and in transgenic mice provided evidence for a disparate and cell type-specific gene regulatory control mechanism in the parietal yolk sac. The thrombomodulin promoter as defined in this study will allow the targeting of gene expression to the parietal yolk sac of transgenic mice and the initiation of investigations into the role of parietal endoderm in placental function.     

Autoantigen Ku in the brain. Developmentally regulated expression and subcellular localization

A double-stranded DNA end-binding factor with high levels of expression in brain and testis of adult mice was identified as the Ku protein, earlier described as an autoantigen in connective tissue diseases and found to be essential for recombination of the immunoglobulin genes and DNA repair. High Ku levels were found in the cerebellum and pituitary gland, lower levels in the hippocampus, hypothalamus and white matter structures. Ku levels were much higher in embryonic rat brain than in the adult brain, suggesting a role of the Ku protein in brain development. In embryonic rat brain, Ku was associated with cell nuclei, but was predominantly located in the cytosol in the adult rat cerebellum and hippocampus. The abundant expression of Ku in the brain suggests the involvement of Ku autoantibodies in the pathogenesis of neuropsychiatric complications in connective tissue diseases.     

Developmentally regulated expression of interleukin-1 beta by peri-implantation conceptuses in swine

Interleukin-1 beta (IL-1 beta) is one of the critical inflammatory cytokines involved in many physiological processes. This study investigated the temporal expression of IL-1 beta in pig conceptuses. A human IL-1 beta cDNA probe and a anti-human IL-1 beta antibody were used to examine IL-1 beta gene and protein expression in pig conceptuses on days 11, 12, 13, 14, 15, 20, 30, 45, 60, 75, 90, 105 and 112 of pregnancy using Northern, Slot and Western blot analyses. A human cell line (A375.S2) was used to determine IL-1 activity in pig conceptuses. High levels of IL-1 beta mRNA were detected in days 11, 12 and 13 conceptuses. IL-1 beta protein was also detected in conceptuses on days 11, 12, 13, and 14, but not in conceptuses recovered after day 15 of pregnancy. IL-1 beta biological activity was demonstrated in days 11 and 12 conceptus homogenates, but not in homogenates of days 112 allantochorion. Low levels of IL-1 beta mRNA were detected by Northern blot analysis in Day 15 conceptuses, endometrium and myometrium only when poly(A+) RNA was used. The production of IL-1 beta by peri-implantation pig conceptuses was temporally associated with maternal recognition of pregnancy. The results suggest that conceptus IL-1 beta may be important for conceptus development and establishment of pregnancy.     

Developmentally regulated spontaneous activity in the embryonic chick retina

Even before birth and the onset of sensory experience, neural activity plays an important role in shaping the vertebrate nervous system. In the embryonic chick visual system, activity in the retina before vision has been implicated in the refinement of retinotopic maps, the elimination of transient projections, and the survival of a full complement of neurons. In this study, we report the detection of a physiological substrate for these phenomena: waves of spontaneous activity in the ganglion cell layer of the embryonic chick retina. The activity is robust and highly patterned, taking the form of large amplitude, rhythmic, and wide-ranging waves of excitation that propagate across the retina. Activity waves are most prominent and organized between embryonic days 13-18, coinciding with the developmental period during which retinal axons refine their connections in their targets. The spatial and temporal features of the patterns observed are consistent with the role of activity patterns in shaping eye-specific projections and retinotopic maps but inconsistent with the hypothesis that they specify lamina-specific projections in the tectum. Antagonists of glutamatergic and glycinergic transmission and of gap junctional communication suppress spontaneous activity, whereas antagonists to GABAergic transmission potentiate it. Based on these results, we propose that spontaneous activity in the ganglion cells is regulated by chemical inputs from both bipolar and amacrine cells and by gap junctional coupling involving ganglion cells.     

Cloning, expression and chromosomal localization of Wnt-13, a novel member of the Wnt gene family

The Wnt genes, encoding structurally-related secreted glycoproteins, are implicated in mammary carcinogenesis induced by mouse mammary tumor virus. In search of the Wnt gene(s) expressed in human gastric cancer, a WTGC1 cDNA fragment sharing 66.9% amino-acid homology with human and mouse Wnt-2 was isolated by degenerate polymerase chain reaction. The human gene corresponding to WTGC1 was designated as Wnt-13 and overlapping Wnt-13 cDNAs were cloned. Nucleotide sequence analysis indicated that the Wnt-13 gene encodes the protein of 372 amino acids, including a signal peptide, two potential N-glycosylation sites and 24 cystein residues highly conserved among members of the Wnt gene family. The Wnt-13 mRNA of 2.5 kb in size was detected in heart, brain, placenta, lung, prostate, testis, ovary, small intestine and colon of adult human and also in brain, lung and kidney of fetal human. Among various cancer cell lines, the Wnt-13 mRNA was detected in HeLa (cervical cancer), MKN28 and MKN74 (gastric cancer). The Wnt-13 gene has been mapped to human chromosome 1p13. These results suggest that the Wnt-13 gene may be involved in normal human development or differentiation as well as in human carcinogenesis.     

Developmentally regulated changes in the glycoproteins of the equine embryonic capsule

The embryonic capsule, which covers the equine blastocyst after it loses its zona pellucida, is composed of mucin-like glycoproteins. In the present study, we investigated both macroscopic and molecular changes in the capsule during development. The weight of the capsule increased from day 11-12 of pregnancy and reached a maximum at about day 18, coinciding with the time during which the conceptus migrates extensively throughout the uterus. The sialic acid content of the capsule declined markedly from about day 16, the time of conceptus 'fixation' in the uterus, which suggests a unique developmentally regulated mechanism for the control of embryo mobility. These results lead us to propose that the capsule may have an anti-adhesion function in the developing conceptus, and that this effect could be regulated by the sugar side chains of the capsular glycoproteins. The glycosylation characteristics of the blastocyst coverings also underwent changes at about day 9 of pregnancy, which may be related to loss of the zona pellucida. An anti-capsule monoclonal antibody was raised and shown to recognize a tissue-specific antigen present only on the capsule and trophoblast. This antigen was present on the trophoblastic cells soon after the blastocyst is formed, reached a maximum concentration at about day 18, and was absent after day 22, coinciding with the disappearance of the capsule. Immunohistochemical studies indicate that the mucin-like capsular glycoproteins are secreted, at least in major part, by the trophoblast.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmentally regulated changes in the cell surface architecture of Leishmania parasites

The cell surface of Leishmania parasites is coated by a highly unusual glycocalyx which varies markedly during the parasite life cycle. The predominant molecule on the extracellular promastigote (sandfly) stage is a complex lipophosphoglycan (LPG), which together with a number of GPI-anchored proteins and a family of low molecular weight glycoinositolphospholipids (GIPLs), forms a morphologically distinct protective coat over the plasma membrane. The structure of the LPG has been shown to vary in different species and during promastigote development in the sandfly. This polymorphism is thought to be important in allowing Leishmania parasites to colonize a range of insect hosts, and in facilitating the regulated migration of promastigotes along the sandfly alimentary canal. Stage-specific changes in LPG are also involved in preadapting promastigotes to life in the mammalian host. This complex glycocalyx coat is absent from the amastigote stage that proliferates in the phagolysosomes of mammalian macrophages, as the expression of both the LPG and GPI-anchored proteins is massively down-regulated. Instead, the plasma membrane of amastigotes is coated by a densely packed layer of parasite-derived GIPLs and host-derived glycosphingolipids. We propose that the down-regulation of the promastigote macromolecules and the acquisition of host glycolipids by amastigotes represents an important strategy to avoid detection by specific and non-specific components of the immune system.     

Immunohistochemical analysis of in vivo patterns of TRAF-3 expression, a member of the TNF receptor-associated factor family

An immunohistochemical approach was used to explore the in vivo expression of TNF receptor-associated factor 3 (TRAF-3), a putative signaling protein that binds to the cytosolic domains of CD30, CD40, and lymphotoxin-beta receptors. TRAF-3 immunostaining was detected in many types of cells throughout the human body. TRAF-3 immunostaining was only rarely present in thymocytes but was found in the thymic epithelioreticular cells. Lymphocytes in the bone marrow were also typically TRAF-3 immunonegative, whereas myeloid progenitor cells and megakaryocytes were often TRAF-3 positive. Peripheral blood lymphocytes were mostly TRAF-3 immunonegative, while granulocytes were TRAF-3 immunopositive. Monocytes were strongly immunostained for TRAF-3, but macrophages in nodes typically contained little or no TRAF-3 immunoreactivity. Some lymphocytes within the germinal centers of secondary lymphoid follicles in normal and reactive nodes were TRAF-3 immunopositive, as were occasional interfollicular lymphocytes in the T cell regions of these organs, but most lymphocytes appeared to be TRAF-3 immunonegative or stained only weakly. Plasma cells, however, were strongly TRAF-3 positive. Stimulation of PBLs with anti-CD3 Ab induced marked increases in the steady state levels of TRAF-3 protein in vitro as determined by immunoblotting, while levels of TRAF-2 were unchanged, implying a dynamic regulation of TRAF-3 expression. The findings establish for the first time the cell type- and differentiation-specific patterns of expression of a member of the TRAF family of proteins.     

Cloning and expression of a novel, tissue specifically expressed member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family

We report the cloning and expression of the fifth member of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family. Degenerate polymerase chain reaction amplification and hybridization screening of a rat sublingual gland (RSLG) cDNA library were used to identify a novel isoform termed ppGaNTase-T5. Conceptual translation of the cDNA reveals a uniquely long stem region not observed for other members of this enzyme family. Recombinant proteins expressed transiently in COS7 cells displayed transferase activity in vitro. Relative activity and substrate preferences of ppGaNTase-T5 were compared with previously identified isoforms (ppGaNTase-T1, -T3, and -T4); ppGaNTase-T5 and -T4 glycosylated a restricted subset of peptides whereas ppGaNTase-T1 and -T3 glycosylated a broader range of substrates. Northern blot analysis revealed that ppGaNTase-T5 is expressed in a highly tissue-specific manner; abundant expression was seen in the RSLG, with lesser amounts of message in the stomach, small intestine, and colon. Therefore, the pattern of expression of ppGaNTase-T5 is the most restricted of all isoforms examined thus far. The identification of this novel isoform underscores the diversity and complexity of the family of genes controlling O-linked glycosylation.     

Cloning, sequencing, and recombinant expression of the porcine inhibitor of carbonic anhydrase: a novel member of the transferrin family

The plasma from many vertebrates contains a component that specifically binds and inhibits carbonic anhydrase II with nanomolar affinity. Amino-terminal sequencing of pICA, the previously identified 79-kDa carbonic anhydrase inhibitor isolated from porcine plasma [Roush, E. D., & Fierke, C. A. (1992) Biochemistry 31, 12536-12542], and sequencing of four proteolytic fragments of pICA revealed that each of the partial sequences has 40-80% sequence identity with members of the transferrin protein family. We describe here the isolation of a full-length cDNA clone of pICA from a lambda gt11 porcine liver cDNA library. Heterologous expression of this cDNA clone in a Pichia pastoris expression system led to the secretion into the medium of 5 mg/L of a 79-kDa protein that specifically reacts with anti-pICA antibodies and binds tightly to a carbonic anhydrase-Sepharose affinity column. Pairwise sequential alignment of pICA with various transferrins reveals an amino acid identity as high as 64% and predicts that 16 transferrin disulfide bonds are conserved. However, despite these structural similarities, the properties of pICA are distinct from the properties of transferrin. pICA exhibits a significantly decreased affinity for iron that can be attributed to the loss of one of the eight amino acids that coordinate iron in the transferrins as well as both of the arginine residues responsible for anion binding. In addition, the antigenic determinants of pICA and the transferrins are not identical. These data imply that pICA, along with saxiphilin, is a member of a diverse superfamily of transferrin-like proteins with functions other than iron binding.     

Developmentally regulated and spatially restricted antigens of radial glial cells

Radial glial cells, present in many parts of the embryonic vertebrate central nervous system (CNS), have been implicated in the guidance of neuroblasts from the ventricular zone to their laminar destinations. Moreover, radial glial cells may be progenitors of some CNS neurons and glia. To gain new insight into the structure and development of these cells, we have generated and characterized a panel of monoclonal antibodies that recognize radial glial cells of the chick optic tectum. Mice were immunized with homogenates of embryonic day (E) 10 tectum, and antibodies were analyzed by immunofluorescence and immunoblotting. We describe here three pairs of antibodies. 1) H5 and a previously generated antibody, R5 (Dr?ger et al., J. Neurosci. 4:2025, 1984), stain the whole extent of the radial glial cell from E7 to E20. In cultures prepared from E10 tecta, both stain a filamentous meshwork in glial cells but not in neurons. On immunoblots, both recognize a protein of approximately 52 kD that is closely related (or identical) to vimentin. 2) H28 and H29 stain radial glia between E7 and E14, but not later. Moreover, H28 and H29 staining is markedly more intense in the ventricular and intermediate zones than in the laminae of the tectal plate. Both of these antibodies recognize an intracellular epitope in cultured glial cells and a protein of approximately 35 kD on immunoblots. 3) H2 and H27 recognize antigens concentrated in the most superficial processes and endfeet of radial glia in late (E16-E20) embryos. They stain distinct structures in cultured glia, suggesting that they recognize distinct antigens. H27 recognizes a protein of approximately 29 kD on immunoblots. Thus antibodies H5 and R5 are good markers of radial glial cells at all stages, whereas the others define antigens that are developmentally regulated and localized to discrete domains. Together, these antibodies can be used to study temporal and spatial specializations of radial glia.     

Cloning and expression analysis of a novel salicylate suppressible gene, Hs-CUL-3, a member of cullin/Cdc53 family

By using a mRNA differential display technique to search for salicylate suppressible genes, we identified a cDNA in human foreskin fibroblasts, which by GenBankTM DNA data base search shows sequence homology to the recently reported cullin/Cdc53 (CUL) family genes, especially CUL-3. We have cloned the full-length human CUL-3 (Hs-CUL-3) cDNA. It encodes a 768-amino acid polypeptide and has a predicted molecular weight of 88,939. The amino acid sequence of Hs-CUL-3 shows 46% homology to that of its Caenorhabditis elegans ortholog, Ce-CUL-3, and 27 and 23% to that of Hs-CUL-1 and Hs-CUL-2, respectively. Northern blot analysis showed that phorbol 12-myristate 13-acetate increased the expression of Hs-CUL-3 mRNA in a concentration- and time-dependent manner, and this increase was inhibited by sodium salicylate. Hs-CUL-3 widely expressed in human tissues and its expression in cultured COLO205 colon cancer cells was increased when compared with that in normal colon cells. It is likely that Hs-CUL-3 is involved in cell proliferation control.     

Developmentally regulated extinction of Ly-49 receptor expression permits maturation and selection of NK1.1+ T cells

Clonally distributed inhibitory receptors negatively regulate natural killer (NK) cell function via specific interactions with allelic forms of major histocompatibility complex (MHC) class I molecules. In the mouse, the Ly-49 family of inhibitory receptors is found not only on NK cells but also on a minor (NK1.1+) T cell subset. Using Ly-49 transgenic mice, we show here that the development of NK1.1+ T cells, in contrast to NK or conventional T cells, is impaired when their Ly-49 receptors engage self-MHC class I molecules. Impaired NK1.1+ T cell development in transgenic mice is associated with a failure to select the appropriate CD1-reactive T cell receptor repertoire. In normal mice, NK1.1+ T cell maturation is accompanied by extinction of Ly-49 receptor expression. Collectively, our data imply that developmentally regulated extinction of inhibitory MHC-specific receptors is required for normal NK1.1+ T cell maturation and selection.