MEASUREMENT OF NEURON-SPECIFIC (NSE) AND NON-NEURONAL (NNE) ISOENZYMES OF ENOLASE IN RAT, MONKEY AND HUMAN NERVOUS TISSUE

Four double antibody solid-phase radioimmunoassay systems are described for the measurement of neuron-specific enolase (NSE) and non-neuronal enolase (NNE) from rat, monkey and human brain tissue. NSE and NNE are antigenically distinct, making their respective assays specific. The levels of neuronal and non-neuronal enolase (an enolase recently shown to be localized in glial cells) are determined in various regions of rat, monkey and human nervous system. Both neuronal and glial enolases are major proteins of brain tissue with each representing about 1.5% of total brain soluble protein. NSE levels are highest and NNE levels lowest in brain areas having a high proportion of grey matter, such as the cerebral cortex. The reverse is true for areas high in white matter, such as the pyramidal tract and the corpus callosum. Peripheral nervous system levels of NSE are much lower than those of brain with the spinal cord intermediate between the two. Radioimmunological and immunocytochemical data show that neuron-specific enolase is also present in neuroendocrine cells located in non-nervous tissue, which include pinealocytes, parafollicular cells of the thyroid, adrenal medullary chromaffin cells, glandular cells of the pituitary and Islet of Langerhans cells in the pancreas. Unlike neurons, these cells also contain non-neuronal enolase in high amounts.     

Transient expression of vanilloid receptor subtype 1 in rat cardiomyocytes during development

Vanilloid receptor subtype 1 (VR1) is a non-selective cation channel detected on sensory neurons that is sensitive to capsaicin, the main pungent ingredient of hot chili pepper. Capsaicin application to neonatal animals causes cardiorespiratory distress, and this susceptibility to capsaicin changes during early postnatal life. This prompted us to hypothesise that in addition to its known neuronal localisation, VR1 is also expressed by non-neuronal cells of the heart during development. This issue was addressed in the rat heart during pre- and postnatal development by means of RT-PCR, western blot and immunohistochemistry. VR1-mRNA was transiently expressed from E14 to P30 but absent from adult hearts. Translation into protein was verified by western blotting. Immunohistochemistry proved that VR1 protein was localised in cardiomyocytes during those developmental stages at which mRNA was detected. In conclusion, VR1 is not neuron-specific but is transiently expressed on cardiomyocytes during ontogeny thereby pointing to a developmental role of this cation channel.     

Neuron-specific recombination by Cre recombinase inserted into the murine tau locus

To determine the neuronal function of genes in vivo, the neuron-specific deletion of a target gene in animals is required. Tau, a microtubule-associated protein, is expressed abundantly in neurons but scarcely in glias and other tissues. Therefore, to generate mice that express Cre recombinase in neurons, we inserted Cre recombinase into the tau locus. By crossing these tau-Cre mice with ROSA26 lacZ reporter mice, we observed Cre recombinase activity in the neurons from most of the central nervous system, but not in glias nor in non-neuronal tissues. This neuronal-specific activity appeared during embryogenesis. We further crossed tau-Cre mice with rab8 ‘floxed’ mice, and showed that the recombination was nearly complete in the brain, but incomplete or non-detectable in other tissues. Thus, tau-Cre knockin mouse is a useful tool for studying the neuronal function of a gene in vivo.     

Binding and cytotoxic effects of Clostridium botulinum type A, C1 and E toxins in primary neuron cultures from foetal mouse brains

Binding of purified Clostridium botulinum type A, C1 and E toxins to cultured cells was studied by an immunocytochemical method. Type A and C1 toxins bound strongly to neuron cultures prepared from brains of foetal mice, but binding of type E toxin was weak. None of the toxin types bound to the feeder layer, composed of non-neuronal cells. The heavy-chain component of the type C1 toxin bound to neurons, but the light chain component did not. Type C1 toxin also bound only to cell lines of neuronal origin. When type C1 toxin [final concentration 4 x 10(2) LD50 (10 ng) per well] was added to primary neuron cultures in 96-well plates, degeneration of neuronal processes and rounding of neuronal somas were observed, but type A and E toxins did not produce such changes. The binding and cytotoxic activities of type C1 toxin were blocked by heat treatment (80 degrees C for 30 min) or by preincubation of the toxin with polyclonal anti-C1 IgG and some of the monoclonal antibodies which neutralized the toxin activity in mice. In the neuronal processes treated with C1 toxin, many degenerated mitochondria, membranous dense bodies and vesicles were observed by electron microscopy; these ultrastructural changes were similar to those of Wallerian degeneration in vivo.     

COMPARISON OF LEVELS OF ADENOSINE 3'',5''-MONOPHOSPHATE IN HIGHLY PURIFIED AND MIXED PRIMARY CULTURES OF NEURONS AND NON-NEURONAL CELLS FROM EMBRYONIC CHICK SYMPATHETIC GANGLIA

Primary cultures containing ≥99% neurons, ≥99% non-neuronal cells (glia), or both cell types were prepared from the sympathetic ganglia of 12-day chick embryos. Levels of cyclic AMP in the non-neuronal cells (～14 pmol/mg protein) were approximately 3-fold higher than levels in the neurons (～4 pmol/mg protein). Mixed cultures had concentrations of cyclic AMP which fell between the values measured for pure neuronal and pure non-neuronal cultures. The measured cyclic AMP values of mixed cultures were indistinguishable from values predicted by summing the expected contributions of the neurons and non-neuronal cells. Thus, contact between the neurons and non-neuronal cells in these mixed cultures did not appear to alter the level of cyclic AMP in either cell type. Neuronal-glial interactions, such as the specific neuronal stimulation of non-neuronal cell proliferation, occurred independently of any changes in the level of cyclic AMP in the mixed cultures. Cell density was varied in both pure and mixed cultures, and both cyclic AMP concentrations and amounts of [³H]thymidine incorporation into DNA were measured. The cyclic AMP content of the non-neuronal cells varied inversely with cell density. [³H]Thymidine incorporation was independent of cell density in both neuronal and non-neuronal cultures. Parallel density-dependent decreases in cyclic AMP concentration and [³H]thymidine incorporation were observed in mixed cultures as cell density was increased. The data suggest that there is no relationship between changes in rate of non-neuronal cell proliferation and cyclic AMP levels in these cultures.     

Expression of a specific neuronal protein, 14-3-2, during in vitro differentiation of neuroblastoma cells

Expression of the neuronal marker 14-3-2 or NSE (neuron-specific enolase) has been studied during in vitro differentiation of cells in culture. The 14-3-2 protein of neuroblastoma cells is immunologically identical with that found in mouse brain extract. The lack of detectable 14-3-2 in cultures of non-neuronal lines shows that this protein, as has been already shown in vivo, is also a specific marker of neurons in vitro. The presence of 14-3-2 in a differentiated hypothalamic clone—but not in its presumptive precursor—indicates selective initial derepression of 14-3-2. Moreover, modulation of the amount of 14-3-2 already present in dividing neuroblastoma cells is related to the confluent phase of growth or morphological differentiation of neuroblasts. Both mechanisms may be related to the mechanisms underlying initial differentiation and subsequent maturation of neurons in vivo. In dividing neuroblastoma cells modulation of the basal level of 14-3-2 is not necessarily associated with expression of the morphological differentiation, but seems generally concomitant with an arrest of cell division.     

粉剂防治粘虫Pseudaletia separata (Walker)研究

粘虫Pseudaletia separata(Walker)是粮食作物的毁灭性害虫,也是全国农业发展纲要(修正草案)所规定要消灭的八大害虫之一。粘虫在大发生时为害面积很广,虫体发育迅速,食量很大,过去多采用人工器械捕打办法,但不能澈底解决问题,尤其在作物密植的情况下,捕打也发生了困难,常因捕打时将作物践踏而引起损失,因此要求在发生时应用化学防治方法,以达到迅速消灭为害之目的。     

Zebrafish Rohon-Beard Neuron Development: Cdk5 in the Midst

Cyclin-dependent kinase 5 (cdk5) is a proline-directed serine/threonine kinase that is activated mostly by association with its activators, p35 and p39. Initially projected as a neuron-specific kinase, cdk5 is expressed ubiquitously and its kinase activity solely depends on the presence of its activators, which are also found in some non-neuronal tissues. As a multifunctional protein, cdk5 has been linked to axonogenesis, cell migration, exocytosis, neuronal differentiation and apoptosis. Cdk5 plays a critical role in functions other than normal physiology, especially in neurodegeneration. Its contribution to both normal physiological as well as pathological processes is mediated by its specific substrates. Cdk5-null mice are embryonically lethal, therefore making it difficult to study precisely what cdk5 does to the nervous system at early stages of development, be it neuron development or programmed cell death. Zebrafish model system bypasses the impediment, as it is amenable to reverse genetics studies. One of the functions that we have followed for the cdk5 ortholog in zebrafish in vivo is its effect on the Rohon-Beard (RB) neurons. RB neurons are the primary sensory spinal neurons that die during the first two days of zebrafish development eventually to be replaced by the dorsal root ganglia (DRG). Based on ours studies and others’, here we discuss possible mechanisms that may be involved in cdk5’s role in RB neuron development and survival.