Ecto-Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor

Abstract: The phosphorylation of surface proteins by ectoprotein kinase has been proposed to play a role in mechanisms underlying neuronal differentiation and their responsiveness to nerve growth factor (NGF). PC 12 clones represent an optimal model for investigating the mode of action of NGF in a homogeneous cell population. In the present study we obtained evidence that PC12 cells possess ectoprotein kinase and characterized the endogenous phosphorylation of its surface protein substrates. PC12 cells maintained in a chemically defined medium exhibited phosphorylation of proteins by [γ-³²P]ATP added to the medium at time points preceding the intracellular phosphorylation of proteins in cells labeled with ³²P_i. This activity was abolished by adding apyrase or trypsin to the medium but was not sensitive to addition of an excess of unlabeled P_i. As also expected from ecto-protein kinase activity, PC12 cells catalyzed the phosphorylation of an exogenous protein substrate added to the medium, dephospho-α-casein, and this activity competed with the endogenous phosphorylation for extracellular ATP. Based on these criteria, three protein components migrating in sodium dodecyl sulfate gels with apparent molecular weights of 105K, 39K, and 20K were identified as exclusive substrates of ecto-protein kinase in PC12 cells. Of the phosphate incorporated into these proteins from extracellular ATP, 75–87% was found in phosphothreonine. The phosphorylation of the 39K protein by ecto-protein kinase did not require Mg²⁺, implicating this activity in the previously demonstrated regulation of Ca²⁺-dependent, high-affinity norepinephrine uptake in PC12 cells by extracellular ATP. The protein kinase inhibitor K-252a inhibited both intra- and extracellular protein phosphorylation in intact PC12 cells. Its hydrophilic analogue K-252b, had only minimal effects on intracellular protein phosphorylation but readily inhibited the phosphorylation of specific substrates of ecto-protein kinase in PC12 cells incubated with extracellular ATP, suggesting the involvement of ecto-protein kinase in the reported inhibition of NGF-induced neurite extension by K-252b. Preincubation of PC12 cells with 50 ng/ml of NGF for 5 min stimulated the activity of ecto-protein kinase toward all its endogenous substrates. Exposure of PC12 cells to the same NGF concentration for 3 days revealed another substrate of ecto-protein kinase, a 53K protein, whose surface phosphorylation is expressed only after NGF-induced neuronal differentiation. In the concentration range (10–100 μM) at which 6-thioguanine blocked NGF-promoted neurite outgrowth in PC12 cells, 6-thioguanine effectively inhibited the phosphorylation of specific proteins by ecto-protein kinase. This study provides the basis for continued investigation of the involvement of ecto-protein kinase and its surface protein substrates in neuronal differentiation, neuritogenesis, and synaptogenesis.     

ANTIVIRAL ACTIVITY OF CULTURED BLUE-GREEN ALGAE (CYANOPHYTA)1

Lipophilic and hydrophilic extracts from approximately 600 strains of cultured cyanophytes, representing some 300 species, were examined for antiviral activity against three pathogenic viruses. Approximately 10% of the cultures produced substances that caused significant reduction in cytopathic effect normally associated with viral infection. The screening program identified the order Chroococcales as commonly producing antiviral agents.     

HAPLOID PARTHENOGENETIC SPOROPHYTES OF LAMINARIA JAPONICA (PHAEOPHYCEAE)1

Parthenogenetic sporophytes were obtained from three strains of Laminaria japonica Areschoug. These sporophytes grew to maturity in the sea, producine spores that all grew into female gametophytes. These female gametophytes gave rise to another generation of parthenogenetic sporophytes during the next year, so that by the year 1990 parthenogenetic sporophytes had been cultivated for 12, 9, and 7 generations, respectively, for the three strains. When female gametophytes from parthenogenetic sporophytes were combined with normal male gametophytes, normal sporophytes that reproduced and gave rise to both female and male gametophytes were obtained. The parthenogenetic sporophytes were shorter and narrower than the normal sporophytes of the same strain. Chromosome counts on mature sporophytes showed that normal sporophytes (from fertilized eggs) were diploid (2n = approximately 40) and that the spores they produced were haploid (n = approximately 20), while nuclei from both somatic and sporangial cells in parthenogenetic sporophytes were haploid. All gametophytes were haploid. Young sporophytes derived from cultures with both female and male gametophytes were diploid, while young, sporophytes obtained from female gametophytes from parthenogenetic sporophytes had haploid, diploid, or polyploidy chromosome numbers. Polyploidy was associated with abnormal cell shapes. The presence of haploid parthenogenetic sporophytes should be use in breeding kelp strains with useful characteristics, since the sporophyte phenotype is expressed from a haploid genotype which can be more readily selected.     

Familial mediterranean fever (FMF) in Moroccan Jews: Demonstration of a founder effect by extended haplotype analysis

Familial Mediterranean fever (FMF) is an autosomal recessive disease causing attacks of fever and serositis. The FMF gene (designated “MEF”) is on 16p, with the gene order 16cen–D16S80–MEF–D16S94–D16S283–D16S291–16pter. Here we report the association of FMF susceptibility with alleles at D16S94, D16S283, and D16S291 among 31 non-Ashkenazi Jewish families (14 Moroccan, 17 non-Moroccan). We observed highly significant associations at D16S283 and D16S291 among the Moroccan families. For the non-Moroccans, only the allelic association at D16S94 approached statistical significance. Haplotype analysis showed that 18/25 Moroccan FMF chromosomes, versus 0/21 noncarrier chromosomes, bore a specific haplotype for D16S94–D16S283–D16S291. Among non-Moroccans this haplotype was present in 6/26 FMF chromosomes versus 1/28 controls. Both groups of families are largely descended from Jews who fled the Spanish Inquisition. The strong haplotype association seen among the Moroccans is most likely a founder effect, given the recent origin and genetic isolation of the Moroccan Jewish community. The lower haplotype frequency among non-Moroccan carriers may reflect differences both in history and in population genetics.     

BRCA1 maps proximal to D17S579 on chromosome 17q21 by genetic analysis

Previous studies have demonstrated linkage between early-onset breast cancer and ovarian cancer and genetic markers on chromosome 17q21. These markers define the location of a gene (BRCA1) which appears to be inherited as an autosomal dominant susceptibility allele. We analyzed five families with multiple affected individuals for evidence of linkage to the BRCA1 region. Two of the five families appear to be linked to BRCA1. One apparently linked family contains critical recombinants, suggesting that the gene is proximal to the marker D17S579 (Mfd188). These findings are consistent with the maximum-likelihood position estimated by the Breast Cancer Linkage Consortium and with recombination events detected in other linked families. Linkage analysis was greatly aided by PCR-based analysis of paraffin-embedded normal breast tissue from deceased family members, demonstrating the feasibility and importance of this approach. One of the two families with evidence of linkage between breast cancer and genetic markers flanking BRCA1 represents the first such family of African-American descent to be reported in detail.     

Numerical and chemical classification ofStreptosporangium and some related actinomycetes

One hundred and seventeen streptosporangia from soil were compared with marker strains of the familyStreptosporangiaceae for many phenotypic properties. The data were examined using the Jaccard, pattern and simple matching coefficients with clustering achieved using average, complete and single linkage algorithms. Particular confidence was placed in the product of the pattern, average linkage analysis given the sharp definition of aggregate groups and clusters and a combination of low test error and high cophenetic correlation values. The test strains were assigned to five aggregate groups that were equated with the generaStreptosporangium (group A),Microbispora (group B),Planobispora andPlanomonospora (Group C),Kutzneria (neéStreptosporangium viridogriseum (group D), andMicrotetraspora (group E). The streptosporangia, both isolates and marker strains, were assigned to 5 major, 7 minor and 18 single membered clusters. Representative streptosporangia examined for chemical markers were characterised by the presence ofmeso-diaminopimelic acid in whole-organism hydrolysates, complex mixtures of straight- and branched chain fatty acids, di- and tetrahydrogenated menaquinones as predominant isoprenologues, and complex polar lipid patterns containing diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides and uncharacterised components. The chemical and numerical data support the taxonomic integrity of the validly described species ofStreptosporangium and suggest that the genus is markedly underspeciated.     

Long-Term Production of Ergot Peptides by Immobilized Claviceps purpurea in Semicontinuous and Continuous Culture

The semicontinuous and continuous production of pharmaceutically useful ergot peptides with immobilized Claviceps purpurea could be demonstrated. A key aspect was the presence of high concentrations of CaCl₂ (96.9 mM) to give marked prolongation of the productive phase, and cultivation in a bubble column reactor became possible. Restriction of the phosphate supply avoided an otherwise problematic massive increase of outgrowing hyphae.     

MONOPHYLY OF ANEUPLOID ASTRAGALUS (FABACEAE): EVIDENCE FROM NUCLEAR RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER SEQUENCES

Evolutionary relationships within Astragalus L. (Fabaceae) were inferred from nucleotide sequence variation in nuclear ribosomal DNA of both New World and Old World species. The internal transcribed spacer regions (ITS) of 18S–26S nuclear ribosomal DNA from representatives of 26 species of Astragalus, three species of Oxytropis DC., and two outgroup taxa were analyzed by polymerase chain reaction amplification and direct DNA sequencing. The length of the ITS 1 region within these taxa varied from 221 to 231 bp, while ITS 2 varied in length from 207 to 217 bp. Of the aligned, unambiguous positions, approximately 34% were variable in each spacer region. In pairwise comparisons among Astragalus species and outgroup taxa, sequence divergence at these sites ranged from 0 to 18.8% in ITS 1 and from 0 to 21.7% in ITS 2. Parsimony analyses of these sequences resulted in a well-resolved phylogeny that is highly concordant with previous cytogenetic and chloroplast DNA evidence for a major phylogenetic division in the genus. These data suggest that the New World aneuploid species of Astragalus form a monophyletic but morphologically cryptic group derived from euploid species of Old World (Eurasian) origin, which are consequently paraphyletic.     

Intramolecular interference effects in dynamic light scattering from rigid rings

A formula for the apparent diffusion coefficient [D_app(κ)] of a rigid ring is derived from the exact first cumulant of its dynamic structure factor. D_app(κ) is expressed in terms of the ring radius, the diffusion coefficients (D_zz and D_xx) for translation parallel and perpendicular, respectively, to the symmetry axis, and the diffusion coefficients (D and D) for rotation around the symmetry and transverse axes, respectively. D_app(κ) exhibits oscillations as a function of the scattering vector k , which depend on D and the anisotropy of translational diffusion (D_zz ? D_xx). The maxima in D_app(κ) are associated with minima in the static structure factor S(κ, 0), which are due to destructive intramolecular interference. The oscillations in D_app(κ) result from periodic variations in the relative intensities of scattered light from different orientations of the ring, which manifest the various motions to different extents. The orientations contributing most to the scattered intensity are those that exhibit the least destructive interference and consequently contribute most to the decay of the dynamic structure factor. © 1993 John Wiley & Sons, Inc.     

CONSERVATION OF MOLECULAR PREPATTERNS DURING THE EVOLUTION OF CUTICLE MORPHOLOGY IN DROSOPHILA LARVAE

We are using patterns of cuticle specialization in Drosophila larvae as models to investigate the molecular, genetic, and developmental bases of morphological evolution. Members of the virilis species group differ markedly from one another in the distribution of hairs on the dorsal surface of first instar larvae. In particular, characteristic bands of hairs cover about 20% of each trunk segment in some species but about 70% in others. These major types do not correlate with recently proposed phylogenetic relationships, suggesting that similar phenotypes have arisen independently in different lineages. The patterns of expression of several genes that control or reflect intrasegmental patterning are indistinguishable in species with very different cuticle morphologies. We conclude that, in this case, morphology probably has evolved via altered response to a conserved molecular prepattern.