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.     

Nucleotide sequence of the gene, protein purification and characterization of the pSC101-encoded tetracycline resistance-gene-repressor

The nucleotide sequence of the pSC101-encoded tetracycline repressor gene (tetR) was confirmed. The deduced amino acid sequence is compared to that of other repressor proteins. To overproduce the repressor protein, tetR was placed under the control of bacteriophage lambda promoter pL. Tet repressor protein was purified to homogeneity and shown to bind specifically to two tet operators and also to tetracycline (Tc). The inducer function of Tc is demonstrated by the loss of the specific binding between the tet operator DNA and the Tet repressor-Tc complex.     

Methionyl-transfer ribonucleic acid deficiency during G1 arrest of Saccharomyces cerevisiae.

The mesl- mutants of Saccharomyces cerevisiae cease division and accumulate in the G1 interval of the cell cycle when deprived of methionine or shifted from 23 to 36 degrees C in the presence of methionine. Synchronous cell cycle arrest results from a deficiency of charged methionyl-transfer ribonucleic acid (methionyl-tRNAMet) as shown by direct measurement of the in vivo pools of methionine, S-adenosylmethionine, and methionyl-tRNAMet. The deficiency of methionyl-tRNAMet in these cells is the consequence of a lesion in a single gene, mes1. mes1 appears to be the structural gene for the methionyl-tRNA synthetase because some revertants of this mutation exhibited a thermolabile methionyl-tRNA synthetase in vitro. A sufficient hypothesis to explain these and previous results is that the control of cell division by S. cerevisiae in response to nutrient limitation is mediated through aminoacyl-tRNA or subsequent steps in protein biosynthesis.     

Effect of bombesin upon plasma somatostatin-like immunoreactivity, insulin and glucagon in normal and chemically sympathectomized dogs

The present study was designed to determine the effects of intravenously infused bombesin (10 ng/kg/min) upon basal and postprandial plasma somatostatin-like immunoreactivity (SLI), glucagon, insulin and triglyceride levels in normal (n = 12) and chemically sympathectomized (n = 11) dogs. Basal plasma SLI, glucagon and insulin levels rose significantly during the infusion of bombesin in the normal dogs, and this was not altered by chemical sympathectomy. Bombesin infusion enhanced the postprandial SLI response, while attenuating the postprandial glucagon response by 50% and the insulin response in the early postprandial phase of the meal. Sympathectomy did not significantly alter the basal levels of these polypeptides, but augmented the postprandial plasma SLI response during the first 90 min, and reduced the postprandial glucagon response during the infusion of bombesin. The postprandial insulin response was not affected by sympathectomy. In both normal and chemically sympathectomized dogs the rise in postprandial triglyceride levels was attenuated by bombesin infusion.     

Angiotensin-like activity in resistance vessels

Summary Angiotensin II (ANG II) was localized immunocytochemically in kidney and various other organs of the chinese hamster. In the kidney ANG II-like activity was found in the epitheloid cells of the juxtaglomerular apparatus as well as in the media i.e. the smooth muscle cells of arcuate and interlobular arteries and afferent arterioles. ANG II-like activity was also observed in the medial muscle cells of resistance vessels in other organs and tissues such as submandibular gland and brown adipose tissue. The site of synthesis of ANG II needs to be investigated but the data point to the possibility of an intracellular function of ANG II in smooth muscle cells of blood vessels.These studies were supported by the Deutsche Forschungsgemeinschaft within the SFB 90 Cardiovasculäres System     

Comparison of 35S and biotin as labels for in situ hybridization: use of an HPV model system.

Colorimetric in situ hybridization is a method of potential importance in diagnosis and research. The largest criticism of the method has been a perceived loss of sensitivity compared with autoradiographic techniques. Our more positive experience with automation of colorimetric in situ hybridization led us to undertake a direct comparison of the sensitivity of 35S- and biotin-labeled probes. Serial sections of formalin-fixed, paraffin-embedded cell pellets from four human cervical carcinoma cell lines with known copies of HPV (CaSki, 400-600 copies HPV 16; HeLa, 10-50 copies HPV 18; SiHa, 1-2 copies HPV 16; HTB31, no known copies HPV) were hybridized with protocols optimized for autoradiographic or colorimetric detection. Both methods gave comparable results, with differences in each technique seen at the limits of sensitivity. The 1-2 copies of HPV 16 per SiHa cell can be detected with both methods; however, grain counting is required for interpretation of the autoradiographic result. This degree of sensitivity for colorimetric in situ hybridization in formalin-fixed, paraffin-embedded material is achieved through careful optimization of probe size and labeling, adequate tissue digestion, and removal of background. Autoradiography may be preferred in situations where quantitation is required, but colorimetric detection retains the advantages of speed, potential for automation, and improved localization of signal with comparable sensitivity.