Reductive metabolism of ascorbic acid in the central nervous system

Rat and feline brain and feline spinal cord were examined for the presence of semidehydroascorbate reductase (EC 1.6.5.4) and dehydroascorbate reductase (EC 1.8.5.1). Semidehydroascorbate reductase (SDAR), as monitored by both ascorbyl radical-dependent nicotinamide adenine dinucleotide (NADH) oxidase activity and NADH-dependent ascorbyl radical quenching, was present in all tissues studied. Rat cerebrum exhibited the highest levels and feline spinal cord the lowest. SDAR activity was about twice as high in feline cerebral cortex as in underlying white matter, and paralleled ascorbic acid levels. Subcellular fractionation of rat cerebrum localized most SDAR in a large granular fraction. In contrast, dehydroascorbate reductase was not detectable in any of the tissues examined. The results suggest that semidehydroascorbate reductase is the major enzyme catalyzing the regeneration of reduced ascorbic acid in the central nervous system.     

Expression of DA11, a neuronal-injury-induced fatty acid binding protein,coincides with axon growth and neuronal differentiation during central nervous system development

DA11 is the first fatty acid binding protein (FABP) for which gene expression has been shown to be upregulated following neuronal injury in the adult peripheral nervous system. To understand better the potential regulatory role(s) of this unique FABP in axonal growth and neuronal differentiation, we undertook a temporal and spatial study of DA11 gene expression in the developing rat central nervous system (CNS). Transient upregulation of DA11 mRNA and protein levels in CNS tissues were quantified by Northern blot hybridization and Western immunoblot analyses at different developmental ages. Homogenates of embryonic and neonatal cerebral cortex, cerebellum, brainstem, and hippocampal tissues contained 100-fold more DA11 mRNA and protein than corresponding adult tissues. Significant increase in DA11 mRNA was observed as early as embryonic day (E) 14 in cerebral cortex and cerebellum and E19 in brain stem and hippocampus. Postnatal levels of DA11 remained elevated through postnatal day (P) 10 in cerebral cortex, P14 in brain stem and hippocampus, and P20 in cerebellum. Localization of DA11-like immunoreactivity to specific CNS tissues, cell types, and intracellular compartments at P9 revealed a spatial pattern of neuronal expression different than that reported for other FABPs. DA11 protein was detected in the nucleus, cytoplasm, axons, and dendrites of differentiating neurons in cerebral cortex, hippocampus, cerebellum, brain stem, spinal cord, and olfactory bulb. The strong association of DA11 gene expression with development throughout the CNS suggests that this unique FABP plays an important role in axonal growth and neuronal differentiation in many different neuronal populations. J. Neurosci. Res. 48:551–562, 1997. © 1997 Wiley-Liss Inc.     

Immunohistochemical investigation of γ-aminobutyric acid ontogeny and transient expression in the central nervous system of Xenopus laevis tadpoles

The ontogeny of the γ-aminobutyric acid (GABA)-positive neurons in the brain of Xenopus laevis tadpoles was investigated by means of immunohistochemistry, using specific antibodies both against GABA and its biosynthetic enzyme, glutamate decarboxylase (GAD). The results obtained with the two antisera were comparable. The GABA system differentiates very early during development. At stages 35/36, numerous GABA-positive neurons were seen throughout the prosencephalon and formed two main bilateral clusters within the lateral walls of the forebrain that ran caudally toward the hindbrain. Other GABA-immunolabeled cell bodies, together with a conspicuous network of GABAergic fibers, were seen in the posterior hypothalamus. In the spinal cord, the lateral marginal zone was GABA-positive, as were Rohon-Beard neurons, interneurons, and Kolmer-Agdhur cells. A very rich GABA innervation was observed in the pars intermedia of the pituitary. At stage 50, plentiful immunopositive neurons and fibers were found in the telencephalic hemispheres, the diencephalon, and the mesencephalon (optic tectum and tegmentum). By stage 54, the number of GABA-immunoreactive neurons in the posterior hypothalamus had decreased, so that, at stage 58, there were very few GABA-labeled cell bodies in the dorsolateral walls of the infundibulum, despite a strong GABAergic innervation within the median eminence and the pars intermedia. From stage 58 to stage 66, the distribution pattern was very similar to that described in the adult X. laevis and in other amphibian species. These results point to transient GABA expression within the hypothalamus, possibly related to either 1) a naturally occurring cell death or 2) a phenotypic switch. © 1996 Wiley-Liss, Inc.     

Temporal onset of synapsin I gene expression coincides with neuronal differentiation during the development of the nervous system

The spinal trigeminal nucleus is involved in the transmission of orofacial sensory information. Neither the distribution of the neuromessenger, nitric oxide, within the trigeminal system nor the possible relationship of this simple gas with trigeminothalamic neurons has been carefully studied. Using immunocytochemical (against nitric oxide synthase) and histochemical (NADPH-diaphorase staining) techniques, we have found that nitric oxide neurons and processes are more prominent in the nucleus caudalis and the dorsomedial aspect of the nucleus oralis than in other spinal trigeminal regions. To study the relationship of nitric oxide to trigeminothalamic neurons and intertrigeminal interneurons of the spinal trigeminal nucleus, spinal trigeminal neurons were retrogradely labeled with fluorogold by thalamic injections or by injections into the junction of the nucleus interpolaris and nucleus caudalis. Medullary sections were subsequently processed with NADPH-diaphorase histochemistry. None of the diaphorase-stained neurons in the spinal trigeminal nucleus was found to contain fluorogold; however, some diaphorase-stained processes were found in close proximity to trigeminothalamic neurons. Following spinal trigeminal nucleus injections, many diaphorasestained neurons were found to contain fluorogold, especially in the nucleus caudalis, suggesting that nitric oxide-containing neurons in the spinal trigeminal nucleus are intertrigeminal interneurons. Collectively, these data indicate that nitric oxide is most prominent in interneurons located in nucleus caudalis and that these interneurons give rise to processes that appose trigeminothalamic neurons, raising the possibility that they may indirectly influence orofacial nociceptive processing at the level of the spinal trigeminal nucleus via nitric oxide production. © 1994 Wiley-Liss, Inc.     

Extensive peroxynitrite activity during progressive stages of central nervous system inflammation

Nitric oxide (NO) production has been associated with disease activity in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). This free radical can be transformed by superoxide to peroxynitrite, an extremely toxic oxidant which causes lipid peroxidation. In addition, peroxynitrite nitrates tyrosine residues, resulting in nitrotyrosine, which can be identified immunohistochemically. The results of this study indicate that peroxynitrite is formed very early during EAE development, correlating with clinical disease activity. Nitrotyrosine-positive cells display a widespread distribution in brain and spinal cord during severe disease and are associated with both perivascular infiltrates and parenchymal sites. Double-staining procedures demonstrated that a subpopulation of CD11b-positive cells (macrophages/microglia) reacted with nitrotyrosine antibodies. Immunostaining for inducible NO synthase demonstrated a similar distribution as nitrotyrosine staining. These experiments indicate that peroxynitrite is formed during progressive stages of disease activity.     

Dynamic expression of de novo DNA methyltransferases Dnmt3a and Dnmt3b in the central nervous system

To explore the role of DNA methylation in the brain, we examined the expression pattern of de novo DNA methyltransferases Dnmt3a and Dnmt3b in the mouse central nervous system (CNS). By comparing the levels of Dnmt3a and Dnmt3b mRNAs and proteins in the CNS, we showed that Dnmt3b is detected within a narrow window during early neurogenesis, whereas Dnmt3a is present in both embryonic and postnatal CNS tissues. To determine the precise pattern of Dnmt3a and Dnmt3b gene expression, we carried out X-gal histochemistry in transgenic mice in which the lacZ marker gene is knocked into the endogenous Dnmt3a or Dnmt3b gene locus (Okano et al. [1999] Cell 99:247-257). In Dnmt3b-lacZ transgenic mice, X-gal-positive cells are dispersed across the ventricular zone of the CNS between embryonic days (E) 10.5 and 13.5 but become virtually undetectable in the CNS after E15.5. In Dnmt3a-lacZ mice, X-gal signal is initially observed primarily in neural precursor cells within the ventricular and subventricular zones between E10.5 and E17.5. However, from the newborn stage to adulthood, Dnmt3a X-gal signal was detected predominantly in postmitotic CNS neurons across all the regions examined, including olfactory bulb, cortex, hippocampus, striatum, and cerebellum. Furthermore, Dnmt3a signals in CNS neurons increase during the first 3 weeks of postnatal development and then decline to a relatively low level in adulthood, suggesting that Dnmt3a may be of critical importance for CNS maturation. Immunocytochemistry experiments confirmed that Dnmt3a protein is strongly expressed in neural precursor cells, postmitotic CNS neurons, and oligodendrocytes. In contrast, glial fibrillary acidic protein-positive astrocytes exhibit relatively weak or no Dnmt3a immunoreactivity in vitro and in vivo. Our data suggest that whereas Dnmt3b may be important for the early phase of neurogenesis, Dnmt3a likely plays a dual role in regulating neurogenesis prenatally and CNS maturation and function postnatally.     

CD93 and GIPC expression and localization during central nervous system inlfammation

CD93 and GAIP-interacting protein, C termius （GIPC） have been shown to interactively alter phagocytic processes of immune cells. CD93 and GIPC expression and localization during cen-tral nervous system inflammation have not yet been reported. In this study, we established a rat model of brain inlfammation by lipopolysaccharide injection to the lateral ventricle. In the brain of rats with inlfammation, western blots showed increased CD93 expression that decreased over time. GIPC expression was unaltered. Immunohistochemistry demonstrated extensive distribution of CD93 expression mainly in cell membranes in the cerebral cortex. After lipopoly-saccharide stimulation, CD93 expression increased and then reduced, with distinct staining in the cytoplasm and nucleus. Double immunolfuorescence staining in cerebral cortex of normal rats showed that CD93 and GIPC widely expressed in resting microglia and neurons. CD93 was mainly expressed in microglial and neuronal cell membranes, while GIPC was expressed in both cell membrane and cytoplasm. In the cerebral cortex at 9 hours after model establishment, CD93-immunoreactive signal diminished in microglial membrane, with cytoplasmic transloca-tion and aggregation detected. GIPC localization was unaltered in neurons and microglia. These results are the ifrst to demonstrate CD93 participation in pathophysiological processes of central nervous system inlfammation.     

Postsynaptic and differential localization to neuronal subtypes of protocadherin beta16 in the mammalian central nervous system

The formation of synapses is dependent on the expression of surface adhesion molecules that facilitate correct recognition, stabilization and function. The more than 60 clustered protocadherins (Pcdhα, Pcdhβ and Pcdhγ) identified in human and mouse have attracted considerable attention because of their clustered genomic organization and the potential role of α- and γ-Pcdhs in allocating a neuronal surface code specifying synaptic connectivity. Here, we investigated whether β-Pcdhs also contribute to these processes. By performing RT-PCR, we found a striking parallel onset of expression of many β-Pcdhs around the onset of neurogenesis and wide expression in the central nervous system. We generated antibodies specific to Pcdhb16 and showed localization of Pcdhb16 protein in the adult mouse cerebellum, hippocampus and cerebral cortex. Analysing the mouse retina in detail revealed localization of Pcdhb16 to specific cell types and, importantly, subsets of synapses. We show that Pcdhb16 localizes predominantly to postsynaptic compartments and the comparison with Pcdhb22 implies differential localization and functions of individual β-Pcdhs in the mammalian central nervous system. Moreover, we provide evidence for a role of β-Pcdhs in the outer segments and connecting cilia of photoreceptors. Our data show for the first time that β-Pcdhs also localize to specific neuronal subpopulations and synapses, providing support for the hypothesis that clustered Pcdhs are candidate genes for the specification of synaptic connectivity and neuronal networks.     

Giant cell angiitis of the central nervous system with atypical presentation

Giant cell angiitis of the CNS is an uncommon form of vasculitis. Neurological manifestations, both of the peripheral and CNS, are common. The most frequent manifestations are visual loss and stroke. Hemorrhagic onset is uncommon. Most cases have a fatal outcome and a tissue diagnosis is rarely established in life. We describe an unusual case of giant cell angiitis beginning as a hemorrhagic tumoral‐like lesion. The results of the histological and ultrastructural analysis have also been reported. Our case illustrates that giant cell angiitis should be considered as a cause of intracerebral hemorrhage, particularly when associated with a relapsing and remitting disease of the CNS.     

全反式维甲酸抑制MDM2基因在胶质瘤细胞SHG-44中的表达

目的 探讨全反式维甲酸对胶质瘤细胞SHG-44中MDM2基因表达的影响,为进一步研究脑胶质瘤的进展机制及基因治疗提供依据.方法 分别利用cDNA微阵列与Western blot技术分析在10μmol/L全反式维甲酸(all-transretinoic acid,ATRA)处理前后的胶质瘤SHG-44细胞MDM2基因和蛋白的差异表达应用免疫组化链霉菌抗生物素蛋白-过氧化酶(Streptavidin-Peroxidase,SP)法检测Ⅱ级与Ⅳ级胶质瘤标本MDM2蛋白的表达.随机选择数个差异基因进行Northern杂交实验,以验证cDNA微阵列的结果.结果 应用cDNA微阵列检测发现,MDM2基因在ATRA处理与未处理的SHG-44细胞之间表达量的比值为0.37,提示ATRA可抑制MDM2基因在SHG-44中的表达.该结果进一步得Northern杂交实验结果的支持.Western blot分析结果显示10μmol/L ATRA处理前后胶质瘤SHG-44细胞之间MDM2蛋白的相对表达量分别为21.40±0.58和14.02±0.35(t=24.728,P=0.000),提示MDM2蛋白在SHG-44中的表达受到ATRA抑制.Ⅱ级和Ⅳ级胶质瘤标本MDM2蛋白的阳性表达率分别为24.00%(6/25)和56.52%(13/23)(X2=5.298,P=0.021),MDM2蛋白的表达随胶质瘤恶性程度的增高而增加.结论 ATRA可抑制SHG-44胶质瘤细胞中MDM2基因的表达,MDM2基因的表达水平与胶质瘤的演进有关.     

Developmental and spatial expression pattern of alpha-taxilin in the rat central nervous system

Alpha-taxilin has been identified as a binding partner of syntaxin family members and thus has been proposed to function in syntaxin-mediated intracellular vesicle trafficking. However, the lack of detailed information concerning the cellular and subcellular localization of alpha-taxilin impedes an understanding of the role of this protein. In the present study, we characterized alpha-taxilin-expressing cells in the rat CNS with a specific antibody. During embryonic development, alpha-taxilin was prominently expressed in nestin-positive neural stem cells in vivo and in vitro. As CNS development proceeded, the alpha-taxilin expression level was rapidly down-regulated. In the postnatal CNS, alpha-taxilin expression was almost confined to the neuronal lineage, with the highest levels of expression in motor neurons within the brainstem nuclei and spinal cord and in primary sensory neurons in mesencephalic trigeminal nucleus. At the cellular level, alpha-taxilin was preferentially located in Nissl substance-like structures with a tigroid or globular morphology within the soma and proximal to dendrites, but it was excluded from terminals. Combined staining with propidium iodide demonstrated that alpha-taxilin distribution overlapped with the cytoplasmic compartment enriched in RNA species, suggesting a close association of alpha-taxilin with actively translating ribosomes or polysomes in neurons. In agreement with this, a recent study indicated the preferential binding of alpha-taxilin to the nascent polypeptide-associated complex (alphaNAC), a dynamic component of the ribosomal exit tunnel in eukaryotic cells. Taken together, these findings suggest that alpha-taxilin plays multiple roles in the generation and maintenance of neurons through modulation of the NAC-mediated translational machinary and/or the syntaxin-mediated vesicle traffic in the soma.     

Clinical experience with linezolid for the treatment of central nervous system infections

Linezolid, an oxazolidinone, exhibits bacteriostatic activity against virtually all Gram-positive bacteria and even covers atypical organisms like mycobacteria and Nocardia. However, little is known about its effectiveness for central nervous system (CNS) infections. We report on our good experience with linezolid for the treatment of CNS infections in 10 patients, amongst whom three were caused by mycobacteria. While six of our patients clinically improved during linezolid therapy even after failure of various antibiotics, it was unsuccessful in one case. Side-effects were only mild gastrointestinal problems in one patient after long term-treatment, which however led to the cessation of therapy. Linezolid appears to be a safe alternative to vancomycin for therapy-resistant CNS infections because of its good CSF penetration and few side-effects.     

Effects of all-trans retinoic acid on metabolic gene expression in the glioma cell line SHG-44

BACKGROUND： Genetic abnormalities and changes in gene expression have been shown in various grades of glioma. However, the relationship between gene expression patterns and pathways related to malignant transformation of glioma remains poorly understood. OBJECTIVE： To screen differentially expressed genes between normal and all-trans retinoic acid-treated glioma cell line SHG-44 cells with a complementary DNA （cDNA） microarray. DESIGN, TIME AND SETTING： The genomics, in vitro study was performed at the Laboratory of Neurobiology, Third Military Medical University of Chinese PLA, China from January to October 2007. MATERIALS： The glioma cell line SHG-44 was provided by the Third Military Medical University of Chinese PLA. AII-trans retinoic acid was purchased from Sigma, USA. cDNA microarray was purchased from City University of Hong Kong. METHODS： The glioma cell line SHG-44 was treated with 10 μmol/L all-trans retinoic acid for 3 days Differentiation-related genes were determined using cDNA microarray. MAIN OUTCOME MEASURES： Gene expression patterns were compared between normal and all-trans retinoic acid-treated SHG-44 cells. Differentially expressed genes were randomly selected and determined by Northern blot analysis. RESULTS： Northern blot analysis revealed downregulated RPL 13 gene expression and upregulated SOD2 gene expression, which was identical to cDNA microarray results. Five differentially expressed genes （TPI1, BPGM, ALDOA, LDHA, and RRM1） were shown to be involved in cell metabolism, in six metabolic pathways. Four differentially expressed genes （TPI1, BPGM, ALDOA, and LDHA） were associated with carbohydrate metabolism, such as fructose metabolism, pyruvic acid metabolism, pentose phosphate pathway, glycolysis, and gluconeogenesis. One differentially expressed gene （RRM1） was correlated with purine and pyrimidine metabolism. CONCLUSION： Five metabolic genes （TPI1, BPGM, ALDOA, LDHA, and RRM1）, which participate in cell carbohydrate and nucleotide metabolism, were shown to closely correlate with glioma development.     

Segmental differentiation in the leech central nervous system: proposed segmental homologs of the heart accessory neurons.

As part of an on-going study of segmental differentiation in the central nervous system (CNS) of the leech Hirudo medicinalis, a search was made for putative segmental homologs of the heart accessory (HA) neurons, which exist exclusively as a bilateral pair in the ganglia of the fifth and sixth body segments. As it is not yet feasible to obtain adequate cell lineage information in H. medicinalis, potential homologs of the HA neurons were determined using morphological, immunohistochemical, and electrophysiological criteria. Among cells in other body ganglia with somata in the same locations as HA neurons, a pair was found having extensive morphological and physiological similarities to HA neurons. These we have called HA-like (HAL) neurons. Adult HA and HAL neurons have closely related patterns of primary branching, in terms of shape, intraganglionic pathways taken, and extraganglionic projections. The number, location, and relative thickness of branches are also similar among these cells. In embryos 10 to 11 days old, HA and HAL neurons have virtually identical branching patterns, with primary and secondary branches of nearly uniform caliber. Differences in branch thickness develop gradually; by embryonic day 20, they resemble those found in adult neurons. Two features found to differ between HA and HAL neurons were the cell body diameter (larger for the HA cells) and the expression of antigens recognized by the monoclonal antibody Laz1-1 (absent at a detectable level in the HA neurons). At a physiological level, the HA and HAL neurons showed action potentials of similar size and shape, as well as inhibitory synaptic inputs from a common source, the heart interneurons (HN). The observations presented here suggest that there is a common developmental origin for the HA and HAL neurons, and hence that their fates are positionally determined by as yet unknown factors.     

Evidence of normal mitosis with complete cytokinesis in central nervous system neurons during sustained depolarization with ouabain

Ouabain-induced depolarization of culture-matured spinal cord neurons from chick embryos causes the activation of DNA synthesis with subsequent mitotic nuclear division in a large fraction of the cells. Many of the activated neurons do not complete cytokinesis, and the binucleate cells thus formed have served as a convenient index for assaying the effectiveness of mitogenesis induction. However, the fact that the frequency of neurons induced to initiate DNA synthesis is consistently much larger than the frequency of binucleate neurons ultimately observed suggested the possibility that the difference may represent activated neurons that do complete cytokinesis, but go undetected in the binucleate assay cultures. This possibility was explored in the present study by determining the respective frequencies of induced neurons entering mitotic metaphase and completing mitotic telophase, and comparing them with the frequency of binucleate neurons ultimately formed. The results provide further evidence that normal mitogenic and mitotic processes are induced in central nervous system neurons by the depolarization treatment, and indicate that between 40 and 60% of the neurons which complete telophase subsequently undergo complete cytokinesis.