Ganglioside-Induced Neuritogenesis: Verification That Gangliosides Are the Active Agents, and Comparison of Molecular Species

Abstract: Gangliosides were previously reported to induce neuritogenesis in primary neuronal cultures and in some neurally derived cell lines. Because isolated gangliosides usually contain variable quantities of peptides, we investigated the possibility the neurite-stimulating activity could be caused by these contaminants. Ganglioside preparations from bovine brain and other sources were subjected to a three-step purification procedure that eliminated at least 95% of the contaminating peptides. These purfied preparations retained their capacity to induce extensive neurite growth in neuro-2A murine neuroblastoma. Proteolytic digestion and a number of additional procedures were used to reduce residual contamination further without loss of activity. Several crude ganglioside samples had negative effects on neurite development until freed of theri inhibitory factors, which were derived from the tissue and/or introduced during laboratory operations. This was particularly evident for bovine white matter gangliosides whose activity increased in proportion to peptide removal. When carefully purified, virtually all of 11 different gangliosides tested were highly active, with the possible exception of GM4, which demonstrated only moderate activity in a limited number of tests. All of the neutral glycolipids tested, as well as sulfatides and free sialic acid, were inactive.     

Assembly reconciliation

MOTIVATION: Many genomes are sequenced by a collaboration of several centers, and then each center produces an assembly using their own assembly software. The collaborators then pick the draft assembly that they judge to be the best and the information contained in the other assemblies is usually not used. METHODS: We have developed a technique that we call assembly reconciliation that can merge draft genome assemblies. It takes one draft assembly, detects apparent errors, and, when possible, patches the problem areas using pieces from alternative draft assemblies. It also closes gaps in places where one of the alternative assemblies has spanned the gap correctly. RESULTS: Using the Assembly Reconciliation technique, we produced reconciled assemblies of six Drosophila species in collaboration with Agencourt Bioscience and The J. Craig Venter Institute. These assemblies are now the official (CAF1) assemblies used for analysis. We also produced a reconciled assembly of Rhesus Macaque genome, and this assembly is available from our website http://www.genome.umd.edu. AVAILABILITY: The reconciliation software is available for download from http://www.genome.umd.edu/software.htm     

Peptide ligands that bind IgM antibodies and block interaction with antigen.

We have selected a peptide-display phage library on IgM Abs and identified a panel of phage-expressing peptides that bind to IgM Abs in general, but not to Abs of other classes. A synthetic peptide corresponding to one of the displayed peptide sequences also binds to IgM Abs. The peptides bind to both soluble pentameric Abs and to monomeric cell-surface IgM. The phage-displayed and synthetic peptides inhibit the binding of IgM Abs to Ag. These peptides may create confounding artifacts when IgM Abs are used for epitope mapping studies. Nonetheless, the peptides may have both experimental and therapeutic utility.     

Lamellar to tubular conformational changes in the endoplasmic reticulum of the retinal pigment epithelium of the newt,Notophthalmus viridescens

Summary The retinal pigment epithelium (RPE) of the newt (Notophthalmus viridescens) was examined ultrastructurally under both in-vivo and in-vitro conditions. Five distinct conformations of smooth endoplasmic reticulum (SER), two lamellar and three tubular, were observed. The two lamellar conformations included myeloid bodies, which have previously been described (Yorke and Dickson 1984), and fenestrated SER. The latter appeared as layers of flattened or curved cisternae which were penetrated by fenestrations. Fenestrated SER became indistinguishable from the highly branched and convoluted random-tubular SER through the formation of an intermediate configuration (tubular sheets). The remaining tubular SER conformations appeared to arise from random-tubular SER through a progressive reduction in branching and a straightening of individual tubules. Fascicular SER was represented by the hexagonal organization of straight, unbranched tubules into bundles (fascicles). Spiral SER consisted of a similar hexagonal arrangement, but the unbranched tubules spiralled about one another. Neighbouring tubules in areas of spiral SER were also joined together by pairs of electrondense bars. Although lamellar (especially myeloid bodies) and random-tubular configurations of the SER were common features in vivo, fascicular and spiral SER were primarily conformations encountered in vitro. Conditions favouring bilayer lipid phases also appear to facilitate the formation of both myeloid bodies and fascicular SER. These conditions included increased duration of incubation, low (<20° C) incubation temperatures, and Ca²⁺-free incubations with EGTA. Random-tubular SER was most prevalent in media supplemented with fetal calf serum and also after warmer (30° C) incubation temperatures. We speculate that the different conformations of SER observed in the newt RPE may be due, in part, to lipid phase transitions within the membranes of this organelle. However, the specific formation of fascicular and spiral SER may also involve some additional factor, possibly a protein.Supported by grant # MT-5039 from the Medical Research Council of Canada to DHD     

Uptake and release of possible false transmitter amino acids by rat brain tissue

—The uptake of l [¹⁴C]glutamine by a crude isolated nerve ending fraction of rat brain was found to be linear with time for at least 5 min, profoundly temperature-dependent, apparently half-saturated at a substrate concentration of 0·26 mm , partially inhibited by dinitrophenol and ouabain and elevated [K⁺], weakly Na⁺-dependent, poorly inhibited by drugs which block uptake of biogenic amines and more strongly inhibited by glutamic acid (IC₅₀= 0·5mm ) than by aspartic acid, GABA, glycine or methionine. The [¹⁴C]glutamine taken up appeared to be associated with nerve endings and was released by membrane-disruption; about 20 per cent was associated with free mitochondria. Glutamine, δ-aminolevulinic acid and several other amino acids were poor inhibitors of [³H]GABA-uptake; δ-aminolevulinic acid was a poor inhibitor of [³H]glutamine-uptake, whereas glutamine was a moderately effective competitive inhibitor (K_i= 1 mm ). [¹⁴C]glutamine and [³H]GABA were released from brain slices by electrical stimulation or 50 mm K⁺, while labeled δ-aminolevulinic acid, leucine, urea, amphetamine and tyramine were poorly released. [¹⁴C]glutamine was not released by unlabeled glutamate or several aromatic amines. We conclude that the neuropsychiatric features of porphyria are not likely due to a ‘false transmitter’ role for δ-aminolevulinic acid although such a role for glutamine in hepatic encephalopathy or other neuropsychiatric diseases should be considered.