Effects of Gangliosides GM1 and GD1 a on GAP-43 Phosphorylation and Dephosphorylation in Isolated Growth Cones

Abstract: Phosphorylation of the nervous system-specific protein GAP-43 in growth cones in vivo increases as the growth cones near their targets, at a time when the gangliosides GM1 and GD1a are being accumulated in the growth cone membrane, thus raising the possibility that the gangliosides could modulate GAP-43 behavior. We used a subcellular fraction of intact isolated growth cones to show that both GM1 and GD1a affected the calcium- dependent posttranslational regulation of GAP-43 in several similar ways. Both gangliosides induced rapid incorporation of phosphate into GAP-43; however, the induction was undetectable with our antibody 2G12 that is specific for kinase C-phosphorylated GAP-43. Furthermore, neither ganglioside stimulated kinase C activity in isolated growth cones, suggesting that the rapid Phosphorylation may not be on Ser⁴¹, the kinase C site. However, both gangliosides did induce a slower accumulation of GAP-43 phosphorylated on Ser⁴¹, apparently by inhibiting a phosphatase. Finally, calcium-dependent proteolysis of GAP-43 was also stimulated by both GM1 and GD1a. In contrast, GD1a, but not GM1, caused the redistribution of GAP-43 into the isolated growth cone cytoskeleton. The results demonstrate that both gangliosides can modulate the calcium-dependent regulation of GAP-43.     

Characterization of gp93, a Novel, Highly Heterogeneous Glycoprotein Present in Growth Cone Membranes

Abstract: gp93 was first described in growth cones from fetal rat brain as a 90–97-kDa glycoprotein family that binds wheat-germ agglutinin and consists of at least 12 different isoelectric variants (pl range ∼4.9–6.4). Of particular interest is that different sets of gp93 variants are expressed in growth cones isolated from different brain regions. The preparation of a polyclonal antibody to gp93 allowed further characterization of this glycoprotein. The carbohydrate groups of gp93 were partially characterized by digestion with different glycosidases. The results indicate that most or all oligosaccharide units are N-linked (asparagine-linked) and contain sialic acid. Two-dimensional polyacrylamide gel electrophoresis and western blot with anti-gp93 show that deglycosylated gp93 is an only slightly heterogeneous polypeptide of 66 kDa, indicating that gp93 heterogeneity is due, primarily or exclusively, to differential glycosylation. Analysis of the tissue distribution in fetal rat showed gp93 to be highly enriched in the brain. Immunoblots and immunostaining of cross sections of developing cerebellum revealed that gp93 is developmentally regulated in this tissue, associated primarily with growing parallel fibers and Purkinje dendrites. Immunostaining of neurons in culture shows significant amounts of gp93 in elongating neurites and growth cones. Our results indicate that gp93 is a developmentally regulated glycoprotein of the brain that is most prominent in growth cones and growing neurites and that appears to be glycosylated differentially by different neurons.     

Estimation of fungal biomass in the decaying cones ofPinus densiflora

Fungal biomass in the decaying cones ofPinus densiflora was investigated. Leaching, immobilization and mobilization phases were recognized in the decomposition process of cones. Fungal biomass was estimated by the agar-film technique, using a conversion factor of 0.62 mg dry wt. mm⁻³ of hyphal volume to biomass and a factor of 2.5 for in-efficiencency of homogenization. The fungal biomass was 4.9±2.1 (mean±S.D.) mg dry wt. g⁻¹ dry matter in the cones on the tree, 11±6 mg g⁻¹ in the leaching phase, 19±7 mg g⁻¹ in the immobilization phase and 30±15 mg g⁻¹ in the mobilization phase. It significantly increased after cones had lain on the forest floor, and also in the immobilization phase. The latter result suggests that the fungal biomass contributed to the immobilization of nitrogen in the decomposition process. The ratio of ergosterol content to fungal biomass in the cones was 2.9–8.8 μg mg⁻¹ dry wt., lying in the range of 2–16 μg mg⁻¹ reported for mycelia. This suggested that the estimate of fungal biomass was reasonable. Reduction in this ratio with the dry weight loss in the cones suggested that the proportion of relatively active fungal biomass decreased with the progress of decomposition.     

Characterization of a motif for specific binding to hyaluronan in chicken SPACR

The chicken sialoprotein associated with cones and rods (SPACR) binds to hyaluronan (HA) in the interphotoreceptor matrix space, but the motif for HA binding is still unknown. This study was conducted to determine the critical site required for specific binding to HA. Western blotting study showed that SPACR binds biotinylated HA, and this interaction was specifically inhibited by unlabeled HA. A series of GST fusion proteins covering whole SPACR was prepared, and reactivity with HA was individually screened to narrow down the region for the binding. Further, putative HA-binding motif found near the carboxyl-terminus of SPACR was mutated by site-directed mutagenesis to identify the critical binding site. Finally, we showed that native SPACR derived from retina similarly binds to HA-affinity column under both reducing and non-reducing conditions. These results revealed that the specific putative HA-binding motif is located near the carboxyl-terminus of chicken SPACR, and suggested that a structural integrity such as folded structure is not largely involved in the HA binding.     

油松种子园球果产量近期预测方法的研究

用可见半面树冠法和信息段法,对陇县八渡油松种子园球果近期产量进行了预测,建立了预测模型,并验证其预测结果精度高,可靠性强。其中信息段法更适合油松种子园球果产量近期预测预报。     

Regulated exocytosis of an H+/myo-inositol symporter at synapses and growth cones

Phosphoinositides, synthesized from myo-inositol, play a critical role in the development of growth cones and in synaptic activity. As neurons cannot synthesize inositol, they take it up from the extracellular milieu. Here, we demonstrate that, in brain and PC12 cells, the recently identified H(+)/myo-inositol symporter HMIT is present in intracellular vesicles that are distinct from synaptic and dense-core vesicles. We further show that HMIT can be triggered to appear on the cell surface following cell depolarization, activation of protein kinase C or increased intracellular calcium concentrations. HMIT cell surface expression takes place preferentially in regions of nerve growth and at varicosities and leads to increased myo-inositol uptake. The symporter is then endocytosed in a dynamin-dependent manner and becomes available for a subsequent cycle of stimulated exocytosis. HMIT is thus expressed in a vesicular compartment involved in activity-dependent regulation of myo-inositol uptake in neurons. This may be essential for sustained signaling and vesicular traffic activities in growth cones and at synapses.     

Tau Protein Function in Axonal Formation

Tau protein is a predominantly neuronal microtubule-associated protein that is enriched in axons and is capable of promoting microtubule assembly and stabilization. In the present article we review some of the key experiments directed to obtain insights about tau protein function in developing neurons. Aspects related to whether or not tau has essential, unique, or complementary functions during axonal formation are discussed.     

Cultivation of particle-associated heterotrophic bacteria during a spring phytoplankton bloom in the North Sea

Seawater contains free-living and particle-attached bacteria. Only a small fraction is cultivable on plates. As free-living and particle-associated bacteria differ in their physiological traits, their cultivability on plates may coincide with particle association. Using filtration and Imhoff sedimentation cones, particles were collected during a spring phytoplankton bloom off Helgoland (North Sea) in order to obtain particle-associated bacteria as inocula. Direct dilution plating resulted in 526 strains from 3 µm filtration retentates and 597 strains from settled particles. Motile Gammaproteobacteria from the genera Pseudoalteromonas, Shewanella, Psychrobacter, Vibrio and Colwellia, as well as particle-attached Flavobacteriia affiliating with the genera Tenacibaculum and Gramella, were frequently isolated. As a result, a diverse collection comprised of 266 strains was deposited. Two strains were most likely to represent novel genera and 78 strains were probably novel species. Recently, a high-throughput cultivation study from the same site using seawater as an inoculum had retrieved 271 operational phylogenetic units (OPUs) that represented 88% of the 4136 characterized strains at the species level. A comparison of 16S rRNA gene sequences revealed that the collection obtained matched 104 of the 271 seawater OPUs at the species level and an additional 113 at the genus level. This large overlap indicated a significant contribution of particle-associated bacteria to the cultivable microbiome from seawater. The presence of 49 genera not identified in the larger seawater study suggested that sample fractionation was an efficient strategy to cultivate rare members of the planktonic microbiome. The diverse collection of heterotrophic bacteria retrieved in this study will be a rich source for future studies on the biology of particle-associated bacteria.     

Sequence and localization of an ultraviolet (sws1) opsin in the retina of the Japanese sardine Sardinops melanostictus (Teleostei: Clupeiformes)

A full‐length complementary (c)DNA encoding ultraviolet (UV)‐sensitive opsin (sws1) was isolated from the retina of the Japanese sardine Sardinops melanostictus. The sws1 phylogenetic tree showed a sister group relationship with the Cypriniformes, following the ray‐finned fish phylogeny. By expressing reconstituted opsin in vitro, it was determined that the maximum absorbance spectrum (λ_max) of sws1 is around 382 nm, being intermediate in position between two subtypes of sws1 pigment that are UV sensitive (λ_max = 355–380 nm) and violet sensitive (λ_max = 388–455 nm), which have been reported to date. The ocular media transmitted >20% transmittance of light in the range of 360–600 nm. In situ hybridization analyses revealed that sws1 messenger (m)RNA is localized in a central single cone surrounded by four double cones in a square mosaic. The square mosaic occupies the ventro‐temporal quadrant of the retina and the in situ hybridization signals were dominant in this area suggesting that the fish may use UV vision when looking upward. Based on these results, considerable significances of potential UV sensitivity, in relation to characteristic habits of S. melanostictus, are discussed.