Neurite outgrowth in response to transfected N-CAM changes during development and is modulated by polysialic acid

We have used monolayers of control 3T3 cells and 3T3 cells transfected with a cDNA encoding human N-CAM as a culture substrate for embryonic chick retinal ganglion cells (RGCs). At embryonic day 6 (E6), but not at E11, RGCs extended longer neurites on monolayers of N-CAM-transfected cells. This loss of RGC responsiveness was not associated with substantial changes in the level of N-CAM expression on RGC growth cones. The neurite outgrowth response from E6 RGCs could be inhibited by removal of N-CAM from the monolayer, by removal of alpha 2-8-linked polysialic acid from neuronal N-CAM, or by antibodies that bind exclusively to chick (neuronal) N-CAM. In contrast, the response was not dependent on neuronal beta 1 integrin function. These data provide substantive evidence for a homophilic binding mechanism directly mediating N-CAM-dependent neurite outgrowth, and suggest that changes in polysialic acid expression on neuronal N-CAM may modulate N-CAM-dependent axonal growth during development.     

Ultrastructural localisation of oestrogen receptor in breast cancer cell nuclei

Summary A method of localising oestrogen receptor in nuclei of breast cancer cells by the protein A-gold technique is described. Mast cell granules were found to take up uncoated particles of the gold sol selectively.     

Characterization of bovine aortic protein kinase C with histone and platelet protein P47 as substrates.

A Ca2+- and phospholipid-dependent protein kinase (protein kinase C) was partially purified from the media of bovine aortas by chromatography on DEAE-Sephacel and phenyl-Sepharose. Enzyme activity was characterized with both histone and a 47 kDa platelet protein (P47) as substrates, because the properties of protein kinase C can be modified by the choice of substrate. Both phosphatidylserine and Ca2+ were required for kinase activity. With P47 as substrate, protein kinase C had a Ka for Ca2+ of 5 microM. Addition of diolein to the enzyme assay caused a marked stimulation of activity, especially at low Ca2+ concentrations, but the Ka for Ca2+ was shifted only slightly, to 2.5 microM. With histone as substrate, the enzyme had a very high Ka (greater than 50 microM) for Ca2+, which was substantially decreased to 3 microM-Ca2+ by diolein. A Triton X-100 mixed-micelle preparation of lipids was also utilized to assay protein kinase C with histone as the substrate. Under these conditions kinase activity was almost totally dependent on the presence of diolein; again, diolein caused a large decrease in the Ka for Ca2+, from greater than 100 microM to 2.5 microM. The increased sensitivity of protein kinase C to Ca2+ with P47 rather than histone, and the ability of diacylglycerol to activate protein kinase C without shifting the Ka for Ca2+, when P47 is the substrate, illustrate that the mechanism of protein kinase C activation is influenced by the exogenous substrate used to assay the enzyme.     

Autophosphorylation of smooth-muscle caldesmon.

Caldesmon, a major actin- and calmodulin-binding protein of smooth muscle, has been implicated in regulation of the contractile state of smooth muscle. The isolated protein can be phosphorylated by a co-purifying Ca2+/calmodulin-dependent protein kinase, and phosphorylation blocks inhibition of the actomyosin ATPase by caldesmon [Ngai & Walsh (1987) Biochem. J. 244, 417-425]. We have examined the phosphorylation of caldesmon in more detail. Several lines of evidence indicate that caldesmon itself is a kinase and the reaction is an intermolecular autophosphorylation: (1) caldesmon (141 kDa) and a 93 kDa proteolytic fragment of caldesmon can be separated by ion-exchange chromatography: both retain caldesmon kinase activity, which is Ca2+/calmodulin-dependent; (2) chymotryptic digestion of caldesmon generates a Ca2+/calmodulin-independent form of caldesmon kinase; (3) caldesmon purified to electrophoretic homogeneity retains caldesmon kinase activity, and elution of enzymic activity from a fast-performance-liquid-chromatography ion-exchange column correlates with caldesmon of Mr 141,000; (4) caldesmon is photoaffinity-labelled with 8-azido-[alpha-32P]ATP; labelling is inhibited by ATP, GTP and CTP, indicating a lack of nucleotide specificity; (5) caldesmon binds tightly to Affi-Gel Blue resin, which recognizes proteins having a dinucleotide fold. Autophosphorylation of caldesmon occurs predominantly on serine residues (83.3%), with some threonine (16.7%) and no tyrosine phosphorylation. Autophosphorylation is site-specific: 98% of the phosphate incorporated is recovered in a 26 kDa chymotryptic peptide. Complete tryptic/chymotryptic digestion of this phosphopeptide followed by h.p.l.c. indicates three major phosphorylation sites. Caldesmon exhibits a high degree of substrate specificity: apart from autophosphorylation, brain synapsin I is the only good substrate among many potential substrates examined. These observations indicate that caldesmon may regulate its own function (inhibition of the actomyosin ATPase) by Ca2+/calmodulin-dependent autophosphorylation. Furthermore, caldesmon may regulate other cellular processes, e.g. neurotransmitter release, through the Ca2+/calmodulin-dependent phosphorylation of other proteins such as synapsin I.     

Distinct interactions between Ca2+/calmodulin and neurotransmitter stimulation of adenylate cyclase in striatum and hippocampus

1. Ca2+ and cAMP both act as intracellular second messengers of receptor activation. In neuronal tissue, Ca2+ acting via calmodulin can elevate cAMP levels. This regulation by Ca2+ provides a means whereby the elevation of intracellular [Ca2+] might modulate cAMP generation. 2. In the present studies, the impact of the Ca2+/calmodulin regulation on receptor-mediated stimulation of activity is compared in striatum and hippocampus--regions of differing sensitivity to Ca2+/camodulin. Ca2+/calmodulin stimulated striatal and hippocampal adenylate cyclase activity by 1.4- and 2.7-fold respectively, while dopamine and vasoactive intestinal peptide (VIP) stimulated the enzyme activity of these respective regions by 1.3- and 2-fold. 3. In the presence of Ca2+/calmodulin, the dopamine dose-response curve in the striatum was shifted upward, without alteration of the slope of the curve or of the maximal stimulation of activity elicited by dopamine. In the hippocampus, the ability of VIP to stimulate adenylate cyclase activity was reduced by the presence of calmodulin. 4. The dose dependence of these actions of calmodulin was examined. In the striatum, the stimulation of adenylate cyclase activity by 0.1 to 0.3 microM calmodulin obscured dopamine stimulation, while 1 to 10 microM was additive with the dopamine stimulation. In the hippocampus, all concentrations of calmodulin (0.1 to 10 microM) reduced VIP-mediated stimulation of enzyme activity. 5. These data suggest that the ratio of calmodulin-sensitive to calmodulin-insensitive adenylate cyclase activity varies in different rat brain regions and that, in those regions in which this ratio is low (e.g., rat striatum and most peripheral systems), calmodulin- and receptor-mediated activation of adenylate cyclase activity will be additive, while in those systems in which this ratio is high (e.g., most of the central nervous system), calmodulin will reduce receptor-mediated stimulation of enzyme activity.     

Reversible conversion of coenzyme F420 to the 8-OH-AMP and 8-OH-GMP esters,F390-A and F390-G,on oxygen exposure and reestablishment of anaerobiosis in Methanobacterium thermoautotrophicum

Intracellular levels of F₃₉₀ (AMP and GMP adducts of the 5-deazaflavin cofactor F₄₂₀) in Methanobacterium thermoautotrophicum were analysed after gasing fermenter cultures with several consecutive cycles of substrate gas and gas mixtures containing 5% oxygen. No F₃₉₀ was detected in growing cells, hydrogen starved cells and CO₂ starved cells prior to O₂ contamination. Also, no F₃₉₀ was found in hydrogen depleted cells after O₂ treatment. Exposure of exponentially growing cells and CO₂ starved cells to oxygen lead to the formation of F₃₉₀ species; the increase in the detected amount of F₃₉₀ was coupled to a decrease of the F₄₂₀ level. As soon as anaerobiosis was reestablished F₃₉₀ cofactors were degraded and growth proceeded. Independent of the physiological condition of Methanobacterium thermoautotrophicum methanopterin was formed upon O₂ exposure. After normal growth conditions were restored the level of detected methanopterin decreased again.     

Alternative splicing generates a secreted form of N-CAM in muscle and brain

A number of different membrane associated isoforms of the neural cell adhesion molecule (N-CAM) have previously been identified. Here the structure of a novel secreted isoform of N-CAM is established by analysis of a cDNA corresponding to an N-CAM mRNA from human skeletal muscle. The mRNA incorporates a novel sequence block into the extracellular domain, which introduces an in-frame stop codon and thus prematurely terminates the coding sequence, generating a truncated N-CAM polypeptide. Analysis of genomic clones indicates that the inserted sequence is present as a discrete exon within the human N-CAM gene, and Northern analysis shows it to be associated specifically with a 5.2 kb mRNA species from skeletal muscle and brain. Stable transfectants expressing the secreted isoform accumulate it in the cytoplasm and release it to the culture medium. In contrast, cells transfected with cDNA encoding lipid-tailed N-CAM express it predominantly at the cell surface. The existence of a secreted isoform may further expand the spectrum of N-CAM function beyond its known involvement in intercellular adhesion to extracellular matrix interactions.     

Isolation and characterization of rabbit gastrin

The heptadecapeptide form the rabbit gastrin was extracted from 16 rabbit antra and purified by a combination of DEAE Sephadex, C-18 SEP PAK cartridges, fast performance liquid chromatography (FPLC) and reverse phase high pressure liquid chromatography (HPLC) steps. After the HPLC purification, a sharp, single peak of gastrin-like immunoreactivity was detected that had the same absorption to immunoreactivity ratio as human gastrin. An amino terminal pyrrolidone carboxylic acid blocking group was removed by incubation with pyrrolidone carboxylic peptidase. The amino acid analysis, microsequence analysis and mass spectrometry all confirmed the structure of rabbit gastrin being pQGPWLQEEEEAYGWMDFamide. This sequence is identical to human gastrin-17 except for glutamine in position 6 which replaces glutamate in human gastrin. Both sulfated and unsulfated rabbit gastrin-17 were characterized by mass spectrometry.