Evidence for Multiple, Local Functions of Ciliary Neurotrophic Factor (CNTF) in Retinal Development: Expression of CNTF and Its Receptor and In Vitro Effects on Target Cells

Abstract: There is increasing, although largely indirect, evidence that neurotrophic factors not only function as target-derived survival factors for projection neurons, but also act locally to regulate developmental processes. We studied the expression of ciliary neurotrophic factor (CNTF) and the CNTF-specific ligand-binding α-subunit of the CNTF receptor complex (CNTFRα) in the rat retina, a well-defined CNS model system, and CNTF effects on cultured retinal neurons. Both CNTF and CNTFRα (mRNA and protein) are expressed during phases of retinal neurogenesis and differentiation. Retina-specific Müller glia are immunocytochemically identified as the site of CNTF production and CNTFRα-expressing, distinct neuronal cell types as potential CNTF targets. Biological effects on corresponding neurons in culture further support the conclusion that locally supplied CNTF plays a regulatory role in the development of various retinal cell types including ganglion cells and interneurons.     

Immunocytochemical colocalization of adhesive proteins with clathrin in human blood platelets: further evidence for coated vesicle-mediated transport of von Willebrand factor,fibrinogen and fibronectin

Coated membranes and vesicles play an important role in receptor-mediated endocytosis and intracellular trafficking in various cell types, and are also present in blood platelets. Platelets take up certain proteins from the blood plasma, such as von Willebrand factor and fibrinogen, and these substances are transferred to storage granules. The receptors for these plasma proteins on the platelet plasma membrane have been well characterized, but morphological evidence for their transport to the storage granules is not yet available. In an attempt to clarify this aspect, we employed postembedding immunocytochemistry on platelets embedded in the acrylic resin LR White. Clathrin as the major coat component of coated vesicles was localized in the cytoplasm, on the plasmic faces of -granules and the open canalicular system, and on the plasmic face of the plasma membrane. Colocalizations of the adhesive proteins, von Willebrand factor, fibrinogen and fibronectin, with clathrin could be observed at the same typical locations as coated vesicles were seen in Araldite-embedded material. These colocalizations have not been reported to date and furnish further evidence for a coated vesicle-mediated transport of blood plasma-derived adhesive proteins from their receptors on the outer plasma membrane to the -granules.     

Molecular cloning and functional expression of bacteriophage PK1E-encoded endoneuraminidase Endo NE

Homopolymeric α-2,8-linked sialic acid (PSA) has been found as a capsular component of sepsis- and meningitis-causing bacterial pathogens, and on eukaryotic cells as a post-translational modification of the neural cell adhesion molecule (NCAM). The polysaccharide is specifically recognized and degraded by a phage-encoded enzyme, the endo-N-acetylneuraminidase E (Endo NE). Endo NE therefore has become a valuable tool in the study of bacterial pathogenesis and eukaryotic morphogenesis. In this report we describe the molecular cloning of Endo NE and the expression of a functionally active recombinant enzyme. The cloned DNA sequence (2436 bp) encodes a polypeptide of 811 amino acids, which at the 5′ end contains a totally conserved neuraminidase motif. Expressed in Escherichia coli, the enzyme migrates as a single band of approximately 74 kDa in SDS-PAGE. A central domain of 669 amino acid residues is about 90% homologous to the recently cloned Endo NF. Both phage-induced lysis of bacteria and the catalysis of PSA degradation by the recombinant enzyme are efficiently inhibited by a polyclonal antiserum raised against the intact phage particle. The C-terminal region seems to be essential to enzymatic functions, as truncation of 32 amino acids outside the homology domain completely abolishes Endo NE activity. Our data also indicate that the 38 kDa protein, previously assumed to be a subunit of the Endo NE holoenzyme, is the product of a separate gene locus and is not necessary for in vitro depolymerase activity.     

Thyroglobulin structure-function: the effect of iodination on the structure of human thyroglobulin

Thyroglobulin of very low iodine content has been prepared from a single non-toxic human goitre. The initial iodine content of the protein (0.038%) has been increased to levels of 0.16% and 0.85% by in vitro treatment with thyroid peroxidase and the resulting proteins studied with respect to their intrinsic fluorescence, circular dichroism spectra and binding of the hydrophobic probe 1,8-anilinonaphthalene sulfonic acid (ANS). While significant differences were observed between levels of iodination in both the ANS binding and intrinsic fluorescence of the thyroglobulin, no significant differences in the near and far UV circular dichroism spectra of the protein as a function of iodine content were observed. These data suggest that, the iodination of thyroglobulin effects specific areas of the protein without significant disruption of its overall secondary structure.     

Familial pericentric inversion of the X chromosome [inv(X)(p11q28)]

A familial pericentric inversion of the X chromosome [46,X,inv(X)(p11q28)] and [46,inv(X)(p11q28), Y] is reported. The carriers of the inv(X) presented no clinical symptoms. Either the inverted or the normal X chromosome may be late replicating.     

Convenient procedures for the biosynthesis, isolation, and isotope labeling of cytochalasins.

Efforts to improve small-scale yields of useful cytochalasins by fermentation resulted in selection of an enriched aflatoxin medium which increased yields by fivefold over those reported in the literature. With Helminthosporium dematoideum and Zygosporium masonii in stationary culture for 3 weeks, cytochalasins B and D were obtained in quantities approaching 700 and 500 mg/liter, respectively. It appears that the critical component in this growth medium is factors associated with whole wheat. By using these procedures, coupled with improvements in isolation, supplementation with two radioactive phenylalanine species readily produced [14C]- or [3H]cytochalasin B. Oxidation of carrier-free radioactive cytochalasin B to cytochasasin A readily provided this labeled congener as well. The isotopic ocnversion of precursor to crystalline products that met analytical criteria ranged from 2 to 4% of the administered radioactivity.     

Terminal uridylyl transferase of Vigna unguiculata: purification and characterization of an enzyme catalyzing the addition of a single UMP residue to the 3''-end of an RNA primer.

An enzyme which catalyzes the addition of a single UMP residue from UTP to the 3'-end of an RNA primer and which is referred to as terminal uridylyl transferase (TUT) has been extensively purified from the membrane fraction of vigna unguiculata leaves. The purification procedure involved (i) solubilization by cation depletion (ii) DEAE-Sepharose CL-6B column chromatography (iii) affinity chromatography of poly(U)-Sepharose 4B and (iv) glycerol gradient centrifugation. The molecular weight of the native enzyme was approximately 50,000 as determined by velocity sedimentation. Under conditions that were optimal for UMP-incorporation (5 mM Mg2+, low salt, 30 degrees C) TUT displayed a marked specificity for UTP as substrate, was unable to incorporate deoxyribonucleoside triphosphates and required a single-stranded oligo- or polyribonucleotide as primer. When oligoA20, tRNAasp of E. coli or alfalfa mosaic virus RNA 4 were used as primers at various substrate to primer ratio's, the vast majority of the product appeared to consist of primer molecules elongated with a single UMP residue as shown by polyacrylamide gelelectrophoresis and nearest neighbour analysis. We believe TUT to be a novel enzyme which has not been reported before and which may be a feasible tool in RNA sequencing as it enables the specific 3'-terminal labeling of RNA molecules.