Depolarization-Induced Increase in Surface Binding and Internalization of 125I-Nerve Growth Factor by PC 12 Pheochromocytoma Cells

The binding and internalization of 125I-nerve growth factor (NGF) by PC12 pheochromocytoma cells was studied as a function of extracellular potassium concentration. Both surface-bound and internalized fractions of 125I-NGF associated with the cells under depolarizing conditions (50 mM K+) increased to 144 +/- 28% (average +/- SEM, six different cell preparations) and to 176 +/- 12% (n = 6), respectively, of those observed at 6.0 mM K+. Scatchard-type analysis of the data indicates increased sites for the binding and internalization of iodinated NGF by the cells. Similar enhancement was observed for cells treated with NGF as well. This voltage-dependent phenomenon was reversible, and also observed in the presence of veratridine. Moreover, withdrawal of extracellular Ca2+ abolished high K+-induced modulation of 125I-NGF binding and internalization, indicating that this effect may be mediated by Ca2+.     

Lack of association and linkage between HLA and familial polyposis coli

Summary We investigated possible association of and linkage between HLA and familial polyposis coli (FPC). In 182 individuals from 66 pedigrees of FPC and 108 individuals from a normal population, HLA-A,-B, and-C antigens were determined. When the frequencies of HLA antigens in 66 unrelated patients and in normal controls were compared, no association of FPC with HLA was observed. For the linkage analysis, HLA haplotypes of 17 affected sib pairs were investigated by the affected sib pair method. The number of pairs which shared two, one, and no haplotypes identical by descent was not significantly different from the number expected with random occurrence (P>0.95). Finally, seven families were analyzed using Morton's sequential test. A maximum lod score of-0.056 at a recombination fraction of 0.4, and a lod of-3.089 at a recombination fraction of 0.05 were obtained. Therefore, there is neither an association of nor linkage between FPC and HLA.     

1H- and13C-NMR spectroscopic studies of lipid extracts of the red algaGracilaria longa

Lipid extracts of the red algaGracilaria longa were studied by¹H- and¹³C-NMR spectroscopy. Peaks in the¹³C-NMR spectra attributable to sterols, chlorophylls and carotenoids allowed free and acylated cholesterol, chlorophylla and lutein to be identified as the most abundant components of these classes. A content of 0.5 ± 0.1 μmoles of total cholesterol/g wet alga was estimated from the¹H-NMR spectrum, which also allowed the determination of the phosphatidylcholine/total lipid molar ratio (9.5 ± 0.5%). The¹³C-NMR spectroscopic experiments provided information on the position of the double bonds on the fatty acid residues. A comparison between NMR spectra of lipid extracts obtained for wet and dried alga showed that the alga undergoes both a dramatic peroxidation and some glycolipid degradation during the drying process.     

Modulation of the urokinase receptor in human colon cell lines by N,N-dimethylformamide

The present study documents the effect of the planar, polar differentiation promoter N,N-dimethylformamide (DMF) on urokinase binding to colon carcinoma cells. Exposure of the colon carcinoma cell lines to the agent resulted in enhanced specific binding of radioactive urokinase to all cells tested. Insulin binding to the cells was, however, unaffected by DMF. A DMF exposure period of 45 h was required to observe maximum urokinase binding to two representative cell lines FET and RKO. Optimal stimulation of both cell lines occurred with 0.8% DMF. Scatchard analysis revealed the dissociation constants to be unchanged by the agent with the increased binding of radioactive plasminogen activator reflecting an up-regulation of binding sites. In this regard, the cell line RKO upon exposure to DMF, displayed approx. 700,000 receptors/cell, the highest value published, to date, for any cell line.     

Cloning and sequence analysis of theMaackia amurensis haemagglutinin cDNA

Maackia amurensis haemagglutinin (MAH) is a leguminous lectin which preferentially binds to a cluster of sialylatedO-linked carbohydrate chains (Konami Y, Yamamoto K, Osawa T, Irimura T (1994)FEBS Lett 342:334–38). In the present study a 950 bp cDNA clone encoding MAH was isolated from a cDNA library constructed from germinatedMaackia amurensis seeds. From the nucleotide sequence, MAH was predicted to consist of 285 amino acid residues containing a signal peptide of 29 amino acids. The results also confirmed our previous findings from the amino acid sequence analysis, which indicated that two highly conserved amino acid residues in all other well-known leguminous lectins were replaced in MAH. These residues were lysine-105 and aspartic acid-135. The corresponding amino acid residues in other leguminous lectins were glycine and asparagine, respectively. These differences were due to the presence of nucleotides AAA and GAT in place of AAT/C and GGA/T.Abbreviations MAH Maackia amurensis haemagglutinin.     

The Effect of the B Subunit of Cholera Toxin on the Action of Nerve Growth Factor on PC12 Cells

Abstract: Exogenous gangliosides, especially ganglioside GM1 (GM1), seem to potentiate the action of nerve growth factor (NGF). We have examined the possible regulation of the NGF signaling pathway in PC12 cells by the B subunit of cholera toxin (CTB), which binds to endogenous GM1 specifically and with a high affinity. CTB treatment (1 μg/ml) enhanced NGF-induced neurite outgrowth from PC12 cells, NGF-induced activation of ribosomal protein S6 kinase, and NGF-induced stimulation of trk phosphorylation. CTB plus NGF also caused a greater inhibition of [³H]-thymidine incorporation into DNA than did NGF alone. These enhancing effects of CTB were blocked by the presence of cytochalasin B in the culture medium but were not affected by the presence of colchicine or by the depletion of Ca²⁺ in the medium. ¹²⁵I-NGF binding experiments revealed that CTB treatment did not affect the specific binding of NGF to the cells. These results strongly suggest that the binding of cell surface GM1 by CTB modulates the pathway of intracellular signaling initiated by NGF and that the association of CTB with a cytoskeletal component is essential for these effects.     

Unique tissue distribution of a mouse macrophage C-type lectin

We examined mouse tissue for the expression of macrophage galactose/N-acetylgalactosamine-specificC-type lectin using a rat monoclonal antibody (mAb) specificfor this lectin (mAb LOM-14). The binding of mAb LOM-14 wasdetected in detergent extracts from tissue by means of immunoblottinganalysis. It was shown that this mAb did not cross-react withmouse hepatic lectins, a structural homologue. The macrophagelectin was widely distributed among various mouse tissues asjudged by the affinity isolation followed by the immunochemicaldetection. The exceptions were brain, liver, kidney, small intestine,and peripheral blood. Extracts from these organs exhibited,at best, very weak signals upon mAb LOM-14 binding, despitethe presence of cells expressing macrophage markers. The mostintense signal was observed in the extract from skin, suggestingthat cells expressing this lectin are abundant in skin. Thetissues shown to contain this lectin were further investigatedby immunohistochemical staining of the sections. Cells weredistributed in the connective tissue and in the interstice,particularly the dermis and subcutaneous layer of skin. Cellslocalized in the epithelium of skin (epidermis) or other epitheliathat we examined were not stained. Perivascular localizationof cells stained with mAb LOM-14 was also demonstrated in cardiacand skeletal muscle tissues. Immunoelectron microscopy revealedthe presence of this lectin along the rough endoplasmic reticulum.In conclusion, the distribution of C-type lectin specific forgalactose/N-acetylgalactosamine in mice was unique. The connectivetissue-specific distribution should provide important informationon the biological role of this lectin. lectin macrophage calcium-type lectin connective tissue     

Purification and characterisation of β-N-acetylhexosaminidase from the butter clam, saxidomus purpuratus

A β-N-acetylhexosaminidase [EC 3.2.1.30] has been purified ～98-fold from an extract of the digestive organs of Saxidomus purpuratus by using ammonium sulfate fractionation, and chromatography on Toyopearl HW-50, CM-cellulose, and Sepharose 4B. The purified enzyme, the molecular weight of which was estimated to be ～66,000 by gel filtration, was composed of two sub-units of molecular weight 30,000 as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The purified enzyme had a pH optimum of 3.8 and an optimum temperature of 55°, and its activity was enhanced ～2-fold in the presence of 0.1m sodium chloride. The Michaelis constants toward p-nitrophenyl 2-acetamido-2-deoxy-β-d-glucoside and -galactoside were 1.2 × 10^?4 and 1.3 × 10^?4m, respectively.     

Differential Responses of the Phosphorylation of Ribosomal Protein S6 to Nerve Growth Factor and Epidermal Growth Factor in PC 12 Cells

Previous studies from this laboratory have shown that the phosphorylation of the S6 protein of the ribosomes is catalyzed by at least two different and separable kinase activities in PC12 cells. One of these activities is increased by treatment of the cells with nerve growth factor, the other by treatment of the cells with epidermal growth factor. The present work shows that these two factors stimulate the phosphorylation of S6 with quite different kinetics, and that both the number of phosphates incorporated into S6 and the phosphopeptide pattern of S6 are different in cells treated with nerve growth factor than in cells treated with epidermal growth factor. The characteristics of the nerve growth factor-sensitive S6 kinase and of the epidermal growth factor-sensitive kinase were also clearly different. Substrate specificity and inhibitor studies indicated that neither was identical to cyclic AMP-dependent kinase, kinase C, or the calcium/calmodulin-dependent kinases. However, two major phosphopeptides produced by S6 phosphorylation in nerve growth factor-treated cells were also seen on phosphorylation of S6 by cyclic AMP-dependent kinase in vitro. In addition, when rat liver 40S ribosomal subunits were pretreated with cyclic AMP-dependent kinase in vitro, the action of the nerve growth factor-sensitive S6 kinase was increased about twofold.