Nicotinamide deamidase released by neuroblastoma cell cultures.

Abstract— An enzymic activity which catalyses the deamidation of nicotinamide to nicotinic acid has been found in the growth media of neuroblasts and glioblasts cultivated in vitro. Some properties of the crude nicotinamide deamidase from the growth medium of neuroblastoma M1 cells have been studied.     

Lineage, migration, and morphogenesis of longitudinal glia in the Drosophila CNS as revealed by a molecular lineage marker

Previous studies described three different classes of glial cells in the developing CNS of the early Drosophila embryo that prefigure and ensheath the major CNS axon tracts. Among these are 6 longitudinal glial cells on each side of each segment that overlie the longitudinal axon tracts. Here we use transformant lines carrying a P element containing a 130 bp sequence from the fushi tarazu gene in front of the lacZ reporter gene to direct beta-galactosidase expression in the longitudinal glia. Using this molecular lineage marker, we show that 1 of the "neuroblasts" in each hemisegment is actually a glioblast, which divides once symmetrically, in contrast to the typical asymmetric neuroblast divisions, producing 2 glial cells, which migrate medially and divide to generate the 6 longitudinal glial cells. As with neuroblasts, mutations in Notch and other neurogenic genes lead to supernumerary glioblasts. The results indicate that the glioblast is similar to other neuroblasts; however, the positionally specified fate of this blast cell is to generate a specific lineage of glia rather than a specific family of neurons.     

Progenitor properties of symmetrically dividing Drosophila neuroblasts during embryonic and larval development

Asymmetric cell division generates two daughter cells of differential gene expression and/or cell shape. Drosophila neuroblasts undergo typical asymmetric divisions with regard to both features; this is achieved by asymmetric segregation of cell fate determinants (such as Prospero) and also by asymmetric spindle formation. The loss of genes involved in these individual asymmetric processes has revealed the roles of each asymmetric feature in neurogenesis, yet little is known about the fate of the neuroblast progeny when asymmetric processes are blocked and the cells divide symmetrically. We genetically created such neuroblasts, and found that in embryos, they were initially mitotic and then gradually differentiated into neurons, frequently forming a clone of cells homogeneous in temporal identity. By contrast, larval neuroblasts with the same genotype continued to proliferate without differentiation. Our results indicate that asymmetric divisions govern lineage length and progeny fate, consequently generating neural diversity, while the progeny fate of symmetrically dividing neuroblasts depends on developmental stages, presumably reflecting differential activities of Prospero in the nucleus.     

The novel gene glaikit, is expressed during neurogenesis in the Drosophila melanogaster embryo

A novel gene glaikit (gkt) has been identified which is expressed in the delaminating neuroblasts of the D. melanogaster embryonic central nervous system. At the earliest stages of embryonic development the expression of glaikit was ubiquitous, but by the time the neuroblasts are delaminating gkt expression became restricted to neuroblasts and a few ganglion mother cells. The gkt gene has no characterized homologues and encodes no previously described protein motifs. There are, however, evolutionary conserved predicted genes present in S. pombe, S. cerevisiae and C. elegans. Ectopic neuroblasts induced in either Notch or Delta mutant backgrounds also showed expression of glaikit.     

Morphometric and electron microscopic studies of the secretory granules of neurosecretory cells of the supraoptic and paraventricular nuclei of white rats and mice]

The morphometrical and electron microscopic analysis of secretory granules in the perikaryons of neurosecretory cells of the supraoptic and paraventricular nuclei in male rats and mice has shown than in the cells of these nuclei in both species of animals there occur secretory granules of the same kind and size. Therefore this method fails to determine which of them contain oxytocin and which of them contain vasorpressin. The neurosecretory granules located in the Golgi apparatus zone are of a less size and have more osmiophilic cnetral material than the granules localized on the periphery which mainly have granular central material and are of a greater size. The distinctions in the size and type of secretory granules are associated with certain stages of their "maturation". Granular particles appear to be "swallen", more active forms of storing neurohormones. The presence of larger granular particles in the supraoptic nucleus of mice allows to suggest greater reactivity of this nucleus than in rats which is likely to be associated with a higher ability of mice, as compared with rats, to adaptate to disturbances in water-salt metabolism.     

Postembryonic development of the mushroom bodies in the ant, Camponotus japonicus

Mushroom bodies (MB) are insect brain centers involved in learning and other complex behaviors and they are particularly large in ants. We describe the larval and pupal development of the MB in the carpenter ant, Camponotus japonicus. Based on morphological cues, we characterized the stages of preimaginal development of worker ants. We then describe morphological changes and neurogenesis underlying the MB development. Kenyon cells are produced in a proliferation cluster formed by symmetrical division of MB neuroblasts. While the duration of larval instars shows great individual variation, MB neuroblasts increase in number in each successive larval instar. The number of neuroblasts increases further during prepupal stages and peaks during early pupal stages. It decreases rapidly, and then neurogenesis generally ceases during the mid pupal stage (P4). In contrast to the larval period, the MB development of individuals is highly synchronized with physical time throughout metamorphosis. We show that carpenter ants (C. japonicus) have approximately half as many MB neuroblasts than are found in the honey bee Apis mellifera. Mature MBs of carpenter ants and honey bees reportedly comprise almost the same number of neurons. We therefore suggest that the MB neuroblasts in C. japonicus divide more often in order to produce a final number of MB neurons similar to that of honey bees.     

Inhibition by neuroblastoma growth inhibitory factor of ascites-type neuroblastoma cell growth in coculture with normal glioblasts

A new ascites type neuroblastoma clone (NAs-1), which is characteristic both in anchorage-independent growth and catecholaminergic functions, attached on the monolayer culture of glioblasts and was subjected to morphological differentiation including the extrusion of neuronal processes. Other conventional neuroblastoma cells (Neuro2a, NS-20Y, and N1E-115) as well as NAs-1 in cocultured with normal glioblasts underwent a decrease in cell growth rates and DNA synthesis under the effect of the neuroblastoma growth inhibitory factor (NGIF) produced by glioblasts. After their NGIF production had been reduced by u.v. irradiation, glioblasts lost the growth-inhibitory and differentiation-promoting effects in coculture with NAs-1. The supplement of NGIF into u.v.-treated glioblasts restored the dose-dependent growth inhibition of NAs-1. The addition of nerve growth factor into the coculture system brought about neither the marked effect on growth inhibition of NAs-1 nor the morphological differentiation. The results imply a direct function of NGIF on the paracrine regulation of neuroblastoma cell growth in the coculture with normal glioblasts.     

The age of olfactory bulb neurons in humans

Continuous turnover of neurons in the olfactory bulb is implicated in several key aspects of olfaction. There is a dramatic decline postnatally in the number of migratory neuroblasts en route to the olfactory bulb in humans, and it has been unclear to what extent the small number of neuroblasts at later stages contributes new neurons to the olfactory bulb. We have assessed the age of olfactory bulb neurons in humans by measuring the levels of nuclear bomb test-derived (14)C in genomic DNA. We report that (14)C concentrations correspond to the atmospheric levels at the time of birth of the individuals, establishing that there is very limited, if any, postnatal neurogenesis in the human olfactory bulb. This identifies a fundamental difference in the plasticity of the human brain compared to other mammals.     

Time of origin of cells in the histiogenesis of the spinal cord]

In order to establish the moment of appearance of neuroblasts and ectoglia of the spinal cord the autoradiographic study with the use of H3-thymidine and C14-thmidine injected to pregnant mice with the intervals between injections 121/2 or 24 hours was undertaken. It was establised that spinal neurons were removed from the nervous tube beginning from the 10th up to 13th days of embroyogenesis. The motoneurons of the anterior horn were the first to appear (10th-12th days), the neurons of the intermideate zone were the next to appear (11th - 12th days) and the last were the neurons of posterior horn (13th day). Beginning from the 13th day of embryogenesis there appeared the ectoglia which migrated following meurblasts two days later. The saturation of the grey matter with glial cells and the saturation of the white matter with Schwann cells was brought about by means of additional multiplication at the site of the glioblasts removed from the nervous tube. The main function of the matrix layer neuroepithelium of the nervous tube as a provider of cells to the spinal cord terminated on the 15th day of embryogenesis.     

Oligosaccharide alterations of rat glioblast membrane-bound glycoproteins during differentiation induced by glia maturation factor

Differentiation of normal glioblasts was induced by glia maturation factor (GMF), and the structural change in the oligosaccharide chains of the plasmalemmal glycoproteins was investigated. After the glycopeptides obtained by trypsin treatment of the intact cells had been digested with pronase, the resulting glycopeptides were separated into 4 fractions by gel filtration. The first 2 fractions were found to contain mainly N-glycosidically linked glycopeptides, and the last 2, O-linked oligosaccharides. There were a variety of N-linked oligosaccharides whose apparent molecular weights were greater than that of isomaltoheptaose. As compared to those, O-linked oligosaccharides were fewer in type and lower in molecular weight. The N-linked oligosaccharides corresponding to isomaltohepta- decaose and larger saccharide chains augmented in differentiated glioblasts, whereas the N-linked oligosaccharides smaller than isomaltoheptade- caose decreased. The turnover rate of the high molecular weight oligosaccharides was faster than that of other membrane oligosaccharides, and was accelerated by GMF treatment. The content of an O-linked oligosaccharide fraction increased after GMF treatment.     

RNA biosynthesis during differentiation of various cell types of chicken embryo in cerebral hemispheres

Summary During the development of the chick embryo from the 6th to the 15th day of incubation, the cell types in cerebral hemispheres undergo differentiation. During this period the indifferent cells of the germinal layer migrate away from the neural cavity to form the mantle layer. These cells differentiate into neuroblasts and spongioblasts.RNA biosynthesis is very active in the cells of the germmal layer of the young embryos. From the 10th day on, it decreased becoming very weak in the 15-days old embryos. The RNA is stored in the nucleus and its passage to cytoplasm is very slow.In 6 and 8-days old embryos the RNA biosynthesis in the mantle layer is not very active but increases during embryonic development as the germinal cells differentiate. The biosynthesis is always more intense in the neuroblasts than in the spongioblasts. The RNA is stored in the nucleus and its passage to cytoplasm is slow in the young neuroblasts and the spongioblasts. The formation of Nissl bodies in neuroblasts and the differentiation of neuroblasts into neurons, which corresponds to the development of axons and dendrites, both are accompanied by an activation of the RNA passage from the nucleus into the cytoplasm.With the technical assistance of A. Brossard.     

Proliferation pattern of postembryonic neuroblasts in the brain of Drosophila melanogaster.

The spatio-temporal proliferation pattern of postembryonic neuroblasts in the central brain region of the supra-esophageal ganglion of Drosophila melanogaster was studied by labeling DNA replicating cells with 5-bromo-2'-deoxyuridine (BrdU). There are five proliferating neuroblasts per hemisphere in larvae just after hatching: one in the ventro-lateral, and the other four in the postero-dorsal region of the brain. Dividing neuroblasts increase during the late first-late second instar larval stages, reaching a plateau of about 85 neuroblasts per hemisphere. Most neuroblasts cease dividing 20-30 hr after puparium formation (APF), while only four in the postero-dorsal region continue making progenies until 85-90 hr APF. The four distinct neuroblasts proliferating in the early larval and late pupal stages are identical; they lie in the cortex above the calyces of the mushroom bodies (corpora pedunculata), proliferating over a period twice as long as that for the other neuroblasts. Their daughter neurons project into the mushroom body neuropile, and hence are likely to be the Kenyon cells. The cell-cycle period of the four neuroblasts (named mushroom body neuroblasts: MBNbs) is rather constant (1.1-1.5 hr) during the mid larval-early pupal stages and is longer before and after that. The total number of the MBNb progenies made throughout the embryonic and postembryonic development was estimated to be 800-1200 per hemisphere.     

The Relative Concentration of Solids in the Nucleolus, Nucleus, and Cytoplasm of the Developing Nerve Cell of the Chick

Growing and differentiating nerve cells of the fifth cranial ganglion of the chick embryo were studied by several means. During the period of 70 hours to 11 days of incubation (Hamburger-Hamilton stages 19 to 37) average cell mass increased more than 4.5 times while cells changed from relatively undifferentiated neuroblasts to morphologically characteristic nerve cells with long processes. By making simplifying assumptions about thickness of nucleus and nucleolus, relative to cytoplasmic thickness, it was possible to calculate solute concentration of nucleus and nucleolus relative to that of the cytoplasm from measurements of optical retardations through living cells. Differences in relative solute concentration were observed in nucleolus, cytoplasm, and nucleoplasm in the approximate ratio 1.2:1.0:0.8, respectively. The ratio remained essentially constant during the growth period examined despite the fact that the cell components grow at markedly different rates. This suggests that solid concentrations are physical characteristics of nucleus, nucleolus, and cytoplasm which are maintained even during rapid growth and differentiation. By cytochemical means it was demonstrated that mass increase in the nucleus is not associated with increase in deoxyribonucleic acid. Both ribonucleic acid and protein are in greater concentration in nucleolus and cytoplasm than in the nucleoplasm. Electron microscopy shows interruptions in the nuclear envelope as well as an approximately even distribution of electron density in nucleus and cytoplasm. It is pointed out that consistent differences in solid concentration can exist on either side of the nuclear envelope even though it contains "pores." Implications of these data are discussed.     

Microdissection Studies on the Polarity of Unequal Division in Grasshopper Neuroblasts: II. Cell Division in Binucleate Neuroblasts

The grasshopper neuroblast divides unequally to produce two types of cells: a large daughter neuroblast that contains a doughnut-shaped nucleus and repeats unequal division with definite polarity, and a small daughter ganglion cell that has a spherical nucleus with low mitotic activity. Binucleate neuroblasts were induced by preventing cytokinesis in the course of microdissection experiments, and subsequent divisions were traced to analyze the factors that determine the polarity of unequal division.
In binucleate neuroblasts, both daughter chromosome groups developed into neuroblast-type nuclei. Mitosis of the two nuclei proceeded synchronously. Although the axes of the two mitotic apparatuses formed at late prophase were random in direction, they became parallel with the original division axis at metaphase. The two mitotic apparatuses shifted simultaneously toward the ganglion cell side during anaphase, just as in normal neuroblasts, and the binucleate cell divided unequally. These findings showed that the poearity of unequal division is strictly maintained in grasshpper neuroblasts, even when they contain two nuclei.     

Changes in the cerebral cortex in the progeny of DOCA-dependent females]

Methods of light microscopy and morphometrical analysis were used for studying semithin sections of the antero-parietal portions of the cortex of neonatal rats from mothers treated by desoxycorticosterone acetate (DOCA) during the last 7 (II group) of last 14 days (III group) of gestation. Animals of the III group were given greater doses of DOCA. Experimental neonatal rats had greater absolute and relative masses of the cerebrum and the thicker cortex: in the II group at the expense of enlargement of neurons, greater amount of glioblasts and the volume of neuropile; in the III group--at the expense of still greater neuron sizes, enlargement of glioblasts and their greater number, as well as the growth of the neuropile volume. In the neocortex of the II group of animals processes of proliferation of glioblasts were more pronounced, greater doses and amount of actions of DOCA (III group) being followed by processes of reinforced differentiation of nerve and glial cells. Experimental animals were found to have greater amount of microgliocytes, hypertrophy of endotheliocyte and pericyte nuclei. Symptoms of blood stagnation and perivascular edema were very rarely noted in neonatal rats of the III group.     

LeX is expressed by principle progenitor cells in the embryonic nervous system, is secreted into their environment and binds Wnt-1

LeX/SSEA1/CD15 is an extracellular matrix-associated carbohydrate expressed by ES cells and by adult neural and bone marrow stem cells. It is important for cell adhesion, compaction and FGF2 responses of early embryonic stem cells; however, its function at later stages is not clear. We now show that LeX is expressed by primary mouse neural progenitor cells, including neural stem cells, neuroblasts and glioblasts, but not by their more differentiated products. LeX distinguishes highly proliferative cells even in the primitive neuroepithelium, demonstrating heterogeneity in cell potential before radial glia arise. At later stages, LeX expressing progenitors are frequently radial in morphology. Surface LeX expression can be used to enrich neural stem and progenitor cells from different CNS regions throughout development by FACS. We found that LeX expression is particularly strong in neural regions with prolonged neurogenesis, e.g., the olfactory epithelium, hippocampus, basal forebrain and cerebellum. These regions also express high levels of the growth factors FGF8 and/or Wnt-1. We show here that LeX-containing molecules in the developing nervous system bind Wnt-1. Our findings suggest that LeX, which is present on the surface of principle neural progenitors and secreted into their extracellular niche, may bind and present growth factors important for their proliferation and self-renewal.     

Biological effects of bovine glia maturation factor on glial cells in culture

Optimal bioassay conditions for bovine glia maturation factor (GMF) were determined among glial cells from normal glioblasts to glioma cells. Rat glioblasts 4–8 days after subculture show the highest response to GMF with regard to morphological transformation and mitogenic activity. Bovine GMF enhances DNA synthesis of rat glioblasts at 12 hr after stimulation; maximum incorporation of [methyl-³H]thymidine was detected at 18 hr. GMF increases twofold the saturation density of rat glioblasts but does not alter that of C6 astrocytoma cells. The apparent inhibition of mitogenic activity of high doses of GMF is seen in both normal and malignant glial cells.