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.     

The nitrogen cycle in a ‘Terra Firme’ rainforest on oxisol in the Amazon territory of Venezuela

Standing stocks and fluxes of nitrogen, including nitrogen fixation and denitrification, were measured in a tropical rainforest on Oxisol in the Amazon Territory of Venezuela. The standing stock of nitrogen was comparable to that of temperate forests, but was higher than that in an adjacent forest on Spodosol. Fluxes were higher than in forests in the temperate zone, but lower than in another tropical forest on more fertile soil. Even though nitrogen was abundant, this does not mean that nitrogen could not be limiting to agriculture if the forest is cleared and the land cultivated. The nitrogen fixing and nitrogen conserving mechanisms are dependent upon the structure of the undisturbed forest, and destruction of the forest would eventually decrease the input of nitrogen to the soil.     

Gene fusion vectors based on the gene for staphylococcal protein A

Two plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusion, have been constructed. These vectors will allow fusion of any gene to the protein A gene, thus giving hybrid proteins which can be purified, in a one-step procedure, by IgG affinity chromatography. As an example of the practical use of such vectors, the protein A gene has been fused to the lacZ gene of Escherichia coli. E. coli strains containing such plasmids produce hybrid proteins with both IgG binding and β-galactosidase activities. The hybrid protein(s) can be immobilized on IgG-Sepharose by its protein A moiety with high efficiency without losing its enzymatic activity and they can be eluted from the column by competitive elution with pure protein A. The fused protein(s) also binds to IgG-coated microtiter wells which means that the in vivo product can be used as an enzyme conjugate in ELISA tests.     

Differential degradation of a recombinant albumin-binding receptor in Escherichia coli.

The degradation in Escherichia coli of the recombinant serum-albumin-binding receptor derived from streptococcal protein G was investigated using a dual-affinity fusion approach. The proteolytic degradation of the receptor was characterized when fused to human proinsulin and human secretin. Several cleavages occurred at sequences not normally regarded as proteolytically sensitive, such as the dipeptide sequences Ile-Gly, Val-Ser and Ser-Ala. Depending on the fusion partner, large differences in the degradation of the albumin-binding domain were observed. Thus, susceptibility to proteolysis of a recombinant protein can be affected by a neighbouring domain.     

Semi-automated solid-phase DNA sequencing.

Increasing the efficiency of DNA sequencing necessitates the development of systems which reduce the need for manual operations by integrating template preparation, sequencing reactions, product separation and detection. A semi-automated system, whereby PCR-amplified biotinylated genomic or plasmid DNA is immobilized on streptavidin-coated magnetic beads, has been developed.     

Expression of subregion and haplotype preference for H-2Kk restricted cytotoxic T-cell responses in vivo and at the level of the precursor frequencies

Several strains of mice bearing the H-2K^k allele were found to generate in vivo strong CTL responses against TNP-haptenated syngeneic cells, while several other strains of mice were found to generate comparably weak or no responses. C3H × DBA/2)F1 mice (H-2^k × H-2^d) and A/J mice with the recombinant haplotype $\text{H-2}{}^{\mathrm{<mtext>k</mtext><mtext>d</mtext>}}$ generated CTL responses in vivo that were completely restricted toward the H-2^k haplotype or the K end of the $\text{H-2}{}^{\mathrm{<mtext>k</mtext><mtext>d</mtext>}}$ haplotype, respectively. The CTL activity of C3H × DBA/2)F1 and A/J mice against haptenated H-2^k targets was found to be more than 25-fold higher than the CTL activity on H-2^d targets. The CTL responses in vitro under macroculture conditions showed, on the other hand, only a 3- to 6-fold higher cytotoxic activity against the haptenated H-2^k targets as compared with haptenated allogeneic or H-2^d targets; and limiting dilution experiments in microcultures revealed that the CTL precursor frequencies were only 2- to 3-fold smaller for TNP-haptenated H-2^d or haptenated allogeneic targets than for haptenated H-2^k target cells. This indicated that sufficient numbers of H-2^d-restricted and allorestricted CTL precursors were actually present in these strains, but did not develop detectable cytotoxic activity in vivo. The exceptional property of the H-2^k haplotype is, therefore, only partly determined by a difference in the CTL precursor frequencies, and to the larger extent determined at the level of the activation of the CTL response.