Postnatal Development of Synaptic Glycine Receptors in Normal and Hyperglycinemic Rats

The postnatal development of glycine synaptic receptors has been studied. Strychnine binding to the synaptic membrane fraction is very low at birth, increases thereafter, and reaches adult values at the 15th day in the brain, and at the 30th day in the spinal cord. Throughout postnatal development, there are more glycine receptors in the spinal cord than in the brain. The development of receptors in the spinal cord displays a pattern similar to that reported previously for the glycine reuptake system in spinal cord slices and in the activity of spinal cord glycine synthase. In rats with experimental hyperglycinemia strychnine binding to spinal cord glycine receptors increases much more rapidly, reaching a level 1.5 times the control value by day 10. When the hyperglycinemia was induced after the 10th postnatal day, however, no effect on the glycine receptors was observed. This increased number of receptors could be explained by an effect of glycine on the synaptic stabilisation process. No changes in the KD for strychnine were observed either during postnatal development or in hyperglycinemic rats. The KD remained approximately 10 nM in the spinal cord and 50 nM in the brain. Results are discussed with respect to the ontogeny of glycinergic synapses and the pathogenesis of nonketotic hyperglycinemia.     

Molecular Characterization of GP50: A Neuron-Specific, Synaptic-Enriched Glycoprotein

The molecular properties of the neuron-specific, synaptic-enriched glycoprotein GP50 have been investigated with the aid of the monoclonal antibody MabSM-GP50. GP50 immunoreactivity was detected in the brains of the frog, trout, pigeon, snake, rabbit, mouse, cow, and human, although variation in quantity and electrophoretic mobility of the immunoreactive protein between species was apparent. Deglycosylation of synaptic membranes (SMs) with endoglycosidase H, peptide:N-glycosidase F, trifluoromethane-sulfonic acid, and alkaline sodium borohydride indicated that GP50 is associated primarily, if not exclusively, with high-mannose and/or hybrid-type oligosaccharides and lacks complex N-linked and O-linked sugar chains. GP50 remained associated with the membrane fraction following extraction of SMs with alkaline sodium carbonate, was partially (55%) present in the detergent phase following the phase partitioning of SMs in the presence of Triton X-114, and was resistant to proteolytic digestion with trypsin when present as a component of intact membranes. Taken together, these results indicate that GP50 is an integral component of the SM. Sucrose gradient centrifugation of Triton X-100 extracts of SMs or of forebrain and cerebellar homogenates resolved GP50 into two fractions with sedimentation coefficients of 3.6S and 7.3S, which accounted for 45 and 55% of the total, respectively. The 7.3S form occurred exclusively in the aqueous phase following partitioning with Triton X-114, whereas the 3.6S species was found in both the aqueous and detergent phases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impaired Development of Rat Cerebellum Induced by Neonatal Injection of the Glycoprotein Synthesis Inhibitor, Tunicamycin

The effects of the protein glycosylation inhibitor tunicamycin on the postnatal development of the rat cerebellum were examined in vivo. Tunicamycin (0.2 micrograms) was injected intracranially into 1-day-old rats. Inhibition of glycosylation of the macromolecules in the cerebellum by tunicamycin treatment was suggested by a reduced incorporation of [3H]glucosamine into the trichloroacetic acid (TCA)-insoluble fraction. The tunicamycin treatment did not affect gain in body weight significantly. However the cerebellar weight was significantly reduced by 30-40% compared with that of the controls. Development of GABAergic and cholinergic innervations in the hypoplastic cerebellum was examined by measuring the activities of glutamate decarboxylase (GAD) and choline acetyltransferase (ChAT). The specific activity and the total activity of GAD were significantly reduced in the tunicamycin-treated cerebellum. In contrast the specific activity of ChAT was significantly increased, whereas the total activity of ChAT per cerebellum was identical with that of the controls. These results suggest that the intracranial injection of tunicamycin affects the postnatal development of rat cerebellum, such as GABAergic and cholinergic innervations.     

Mitochondria, Calcium Regulation, and Acute Glutamate Excitotoxicity in Cultured Cerebellar Granule Cells

Abstract: Exposure of cultured cerebellar granule cells to 100 µ M glutamate plus glycine in the absence of Mg²⁺ causes calcium loading of the in situ mitochondria and is excitotoxic, as demonstrated by a collapse of the cellular ATP/ADP ratio, cytoplasmic Ca²⁺ deregulation (the failure of the cell to maintain a stable cytoplasmic free Ca²⁺ concentration), and extensive cell death. Glutamate-evoked Ca²⁺ deregulation is exacerbated by the mitochondrial respiratory chain inhibitor rotenone. Cells maintained by glycolytic ATP, i.e., in the presence of the mitochondrial ATP synthase inhibitor oligomycin, remain viable for several hours but are still susceptible to glutamate; thus, disruption of mitochondrial ATP synthesis is not a necessary step in glutamate excitotoxicity. In contrast, the combination of rotenone (or antimycin A) plus oligomycin, which collapses the mitochondrial membrane potential, therefore preventing mitochondrial Ca²⁺ transport, allows glutamate-exposed cells to maintain a high ATP/ADP ratio while accumulating little ⁴⁵Ca²⁺ and maintaining a low bulk cytoplasmic free Ca²⁺ concentration determined by fura-2. It is concluded that mitochondrial Ca²⁺ accumulation is a necessary intermediate in glutamate excitotoxicity, whereas the decreased Ca²⁺ flux into cells with depolarized mitochondria may reflect a feedback inhibition of the NMDA receptor mediated by localized Ca²⁺ accumulation in a microdomain accessible to the mitochondria.     

Developmental Expression and Functional Characterization of the 4C5 Antigen in the Postnatal Cerebellar Cortex

Abstract: The monoclonal antibody 4C5 recognizes a neuron-specific surface antigen (4C5 antigen) in the CNS and PNS of the rat. In the present study we investigated the expression of 4C5 antigen in the developing cerebellum of the rat and the functional role of this molecule during cerebellar morphogenesis. Immunoblotting and immunohistochemistry in sections of cerebellar cortex revealed an age-dependent decrease in the expression of the 4C5 antigen. In cerebellar primary cell cultures, 4C5 immunoreactivity was detected both on granule and on Purkinje neurons. Granule cell migration was inhibited in cerebellar explants derived from 8-day-old rats and cultured for 2 days in the presence of antibodies against the 4C5 antigen. Electron microscope immunocytochemistry revealed that in 8-day-old rat cerebellum, 4C5 immunoreactivity was localized on the cell bodies of granule neurons in the external and internal granular layers and on parallel fibers in the developing molecular layer as well as at contact sites between these cellular elements. It was not detected on Bergmann glia. These results suggest strongly that the 4C5 antigen is involved in granule cell migration during cerebellar development, possibly via neuron-neuron interactions.     

β-Dystroglycan: Subcellular Localisation in Rat Brain and Detection of a Novel Immunologically Related, Postsynaptic Density-Enriched Protein

Abstract: The distribution of a glycoprotein component of the muscle dystrophin complex, β-dystroglycan, has been determined in subcellular fractions of adult rat forebrain. The results show that β-dystroglycan is enriched in several membrane fractions, including synaptic membranes, but in marked contrast to dystrophin is not detectable in the postsynaptic density fraction. The antiserum also recognises a second molecular species of apparent molecular mass of 164 kDa which is highly enriched in the postsynaptic density fraction. Preabsorption of the antiserum with the antigen (a 22-mer peptide corresponding to the C-terminal sequence of rabbit skeletal muscle β-dystroglycan) abolished reactivity against both β-dystroglycan and the 164-kDa postsynaptic density-enriched protein, confirming that the two species are immunologically related. Enzymatic removal of N-linked oligosaccharide lowered the apparent molecular mass of β-dystroglycan by 3 kDa but did not alter the mass of the 164-kDa species.     

水痘-带状疱疹病毒糖蛋白E在昆虫细胞中的表达、鉴定及免疫原性分析

目的:利用昆虫-杆状表达系统建立表达和纯化分泌形式的水痘-带状疱疹病毒(varicellazoster virus,VZV)糖蛋白gE的方法,并鉴定其理化性质及免疫原性。方法:利用Gibson assembly同源重组试剂盒快速构建重组质粒pFastbac-VZV gE。在sf9昆虫细胞中鉴定序列优化前后表达量的差异,并在High FiveTM细胞中大量表达。通过Ni-NTA亲和层析方法得到高纯度gE蛋白,之后通过酶联免疫吸附试验等验证了其理化性质,并进行小鼠免疫分析其免疫原性。结果:构建了pFastbac-VZV g E1/2重组质粒,经过PCR及双酶切鉴定后均为阳性克隆。使用BAC/PAC试剂盒提取Bacmid转染sf9细胞制备杆状病毒,经Western blot检测,sf9细胞开始表达gE蛋白且随着病毒代次升高g E蛋白表达量增加。序列优化后表达量明显增加,但是大部分以胞内形式存在。通过Ni-NTA一步亲和层析获得纯度较高的gE蛋白,并与VZV单抗9C8具有较好的反应性。通过免疫小鼠产生高滴度抗体,且免疫荧光结果显示其血清可以与天然病毒上的gE抗原结合。结论:成功获得了杆状表达系统表达的gE蛋白并且纯化和鉴定了蛋白质的性质及免疫原性,为进一步研发具有自主产权的VZV亚单位疫苗研究奠定了基础。     

狂犬病病毒糖蛋白在大肠埃希菌中的表达及鉴定

目的表达狂犬病病毒糖蛋白（GP）,用于狂犬病疫苗免疫抗体评估和狂犬病病毒糖蛋白功能的研究。方法采用分析软件,分析其可能的抗原表位,利用PCR方法扩增狂犬病病毒SRV9疫苗株G蛋白抗原位点区域基因,PCR产物经EcoRI和SalI双酶切后,插入大肠埃希菌表达载体pGEX-6P-1,构建重组表达质粒pGEX-6P-1/G87a和pGEX-6P-1/G100a。将重组质粒转化大肠埃希菌BL21感受态细胞中,在IPTG诱导下表达目的蛋白,进行SDS-PAGE分析。表达蛋白进行电洗脱纯化和Western blot鉴定分析。结果成功构建了pGEX-6P-1/G87a和pGEX-6P-1/G100a表达质粒,序列分析表明,插入片段大小分别为1314 bp和1275 bp。SDS-PAGE分析结果证明,在大肠埃希菌系统中成功表达了狂犬病病毒部分糖蛋白,表达的融合蛋白含有GST标签,大小分别约为74×103和73×103。Western blot鉴定结果表明,表达产物有抗原特异性并能与狂犬病病毒抗血清反应。结论利用大肠埃希菌表达系统成功表达了狂犬病病毒部分糖蛋白,表达产物有良好的反应原性。     

北京中医药大学基础医学院

目的:通过对更年期抑郁症模型大鼠行为学以及学习记忆的观察,探讨中药复方更逍遥冲剂的作用机制 方法:SD雌性大鼠40只,随机分为假手术组、更年期抑郁症模型组、中药中剂量组、中药大剂量组和西药对照组,观察开野实验中大鼠活动度、强迫游泳中大鼠的不动时间、以及通道式水迷宫检测大鼠学习记忆能力.结果:模型大鼠经过21d治疗后,开野实验水平积分、垂直积分明显增加,强迫游泳不动时间缩短,水迷宫实验游出时间缩短,错误次数减少.结论:更逍遥冲剂能够明显的改善更年期抑郁症大鼠的行为学评分,并具有改善其学习记忆能力的作用.     

晚成性哺乳动物体温调节能力的胎后发育

按照体温调节能力的发育情况,哺乳动物的生长发育可分为早成性、晚成性和未成熟性3类。本文主要综述了晚成性哺乳动物体温调节能力的发育特点。这类动物的幼体出生时一般身体裸露,热传导率较高,产热能力较差,不能进行有效的体温调节。当环境温度低于热中性区时,单独的个体不能维持较高的恒定体温。但晚成性幼体也具有一定的体温调节能力,当受低温刺激时,即使新生幼体也会具有增加代谢率的反应;同时结合亲体关怀和幼体之间的聚群效应以及巢的保温作用等方式,仍能使其体温维持在一个较高的水平。晚成性幼体生理性产热的不足,也可看作是一种有利的特点,这样可以减少能量在体温调节方面的消耗,从而增加用于生长发育的能量。文章最后对可能的发展方向进行了展望。     

Postnatal Development and Possible Ligand Function of the CNS-Specific Membrane Glycoprotein CNSgp 130

CNSgp 130 is a CNS-specific membrane glycoprotein present in large amounts in the adult mammalian CNS. Using immunohistological techniques, we demonstrated that CNSgp130 is not detectable in the rat cerebellum at birth, and does not appear in the cerebellum until the tenth day of postnatal life. It is expressed first in the white matter of the cerebellar folia, and subsequently (by day 14) it is expressed also in the molecular layer. Expression in the granular layer is not seen until the 18th day of postnatal life, by which time the adult pattern of expression is established. CNSgp130 is also not detectable in the cerebrum at birth. However, it is expressed weakly but diffusely in the cerebrum by the fourth day of life. By the 10th day, there is strong expression in the cerebrum, in marked contrast to its virtual absence from the cerebellum at this stage. By quantitative absorption analysis, CNSgp 130 was undetectable on the day of birth, and increased steadily to 80% of adult values by the 22nd day of postnatal life. Binding studies with pure CNSgp130 demonstrated a Pronase-sensitive ligand in adult chicken brain. This ligand was absent from neonatal rat brain and non-CNS tissues.     

Postnatal Development of Thiamine Metabolism in Rat Brain

The activities of thiamine diphosphatase (TDPase), thiamine triphosphatase (TTPase), and thiamine pyrophosphokinase and the contents of thiamine and its phosphate esters were determined in rat brain cortex, cerebellum, and liver from birth to adulthood. Microsomal TTPase activity in the cerebral cortex and cerebellum increased from birth to 3 weeks, whereas that in the liver did not change during postnatal development. Microsomal TDPase activity in the cerebral cortex showed a transient increase at 1-2 weeks, but that in the cerebellum did not change during development. In contrast to the activity of the brain enzyme, that of liver microsomal TDPase increased stepwise after birth. Thiamine pyrophosphokinase activity in the cerebellum increased from birth to 3 weeks and then decreased, whereas that in the cerebral cortex and liver showed less change during development. TDP and thiamine monophosphate (TMP) levels increased after birth and plateaued at 3 weeks whereas TTP and thiamine levels showed little change during development in the cerebral cortex and cerebellum. The contents of thiamine and its phosphate esters in the liver showed more complicated changes during development. It is concluded that thiamine metabolism in the brain changes during postnatal development in a different way from that in the liver and that the development of thiamine metabolism differs among brain regions.     

Biochemical Characterisation and Localization in Brain of a Human Brain-Leucocyte Membrane Glycoprotein Recognised by a Monoclonal Antibody

Abstract: The F10-44-2 monoclonal antibody was originally shown to interact with a determinant found predominantly in human brain and leucocytes. In this study we demonstrate by quantitative absorption analysis with homogenates of the head of the caudate nucleus, putamen, thalamus, cerebral grey matter, cerebral white matter, corpus callosum and cerebellar folia that the determinant is restricted to the white matter of the CNS. Immunofluorescence studies on frozen sections of the above brain subregions confirm the absorption analyses, showing staining only of white matter. In addition, and unexpectedly, we found very bright staining around blood vessels, particularly in the cerebellum. Biochemical studies established that the molecule in white matter bearing the F10-44-2 determinants is a sialylated membrane glycoprotein with an apparent molecular weight of 90,000, which is similar to but slightly smaller than the T lymphocyte form of the antigen. Developmental studies comparing 16-week foetal and adult cerebrum showed a fivefold increase in F10-44-2 antigen content. Thus, in the human CNS, the F10-44-2 antigen is a medium-sized glycoprotein which is restricted to white matter and shows a marked increase in concentration during development. No such molecule has been described previously.     

Postnatal Development of Cholinergic Enzymes and Receptors in Mouse Brain

The developmental profiles for the cholinergic enzymes acetylcholinesterase and choline acetyltransferase, and the muscarinic and nicotinic receptors were determined in whole mouse brain. The enzyme activities (per milligram of protein) increased steadily from birth, reaching adult levels at 20 days of age. These increases were primarily due to increases in Vmax. Muscarinic receptor numbers, measured by [3H]quinuclidinyl benzilate binding, also increased from birth to 25 days of age. Brain nicotinic receptors were measured with the ligands L-[3H]nicotine and alpha-[125I]-bungarotoxin. Neonatal mouse brain had approximately twice the number of alpha-bungarotoxin binding sites found in adult mouse brain. Binding site numbers rose slightly until 10 days of age, after which they decreased to adult values, which were reached at 25 days of age. The nicotine binding site was found in neonatal brain at concentrations comparable to those at the alpha-bungarotoxin site followed by a steady decline in nicotine binding until adult values were reached. Thus, brain nicotinic and muscarinic systems develop in totally different fashions; the quantity of muscarinic receptors increases with age, while the quantity of nicotinic receptors decreases. It is conceivable that nicotinic receptors play an important role in directing the development of the cholinergic system.     

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.     

Identification of a gp63 surface glycoprotein in Leishmania tarentolae

The promastigote stage of most if not all Leishmania species possesses an abundant surface glycoprotein of 63 kDa (gp63) that has protease activity. We show that the lizard parasite Leishmania tarentolae appears to lack the surface protease activity. L. tarentolae does, however, possess an approximately 63-kDa molecule that is antigenically cross-reactive with the L. major gp63. Additionally, the genome of L. tarentolae contains sequences that hybridise at high stringency to a L. major gp63 gene probe.     

Evidence that a Cerebellum-Enriched, Synaptic Junction Glycoprotein Is Related to Fodrin and Resists Extraction with Triton in a Calcium-Dependent Manner

Abstract: Subcellular fractions from rat cerebellum and other tissues were examined for the presence of a 240K glycoprotein, designated GP-A. Previous results have shown that GP-A is enriched in cerebellum synaptic junction (SJ) fractions when compared to parent synaptic plasma membrane (SPM) fractions and is not detected in forebrain SPM or SJ fractions. In the present studies, GP-A was not detected in myelin, mitochondria, purified nuclei, or cytosolic fractions from cerebellum, but was present in microsomal fractions. GP-A is partially soluble in the non-ionic detergent Triton X-100 and is completely soluble when cerebellum SPMs are treated with the ionic detergent N-lauryl sarcosinate. The solubilization of GP-A from cerebellum membranes was shown to be a function of bound calcium ions, e.g., pretreating SPMs with 100 μM-1mM Ca²⁺ decreased the solubility of GP-A in Triton by approximately threefold. GP-A is a major concanavalin A (Con A)-binding glycoprotein in cerebellum SJ fractions and migrates on sodium dodecyl sulfate (SDS) gels with a slower relative mobility than the 235K/ 230K fodrin doublet. Comparisons between purified fodrin and the 235K/230K doublet in cerebellum and fore-brain synaptic fractions by two-dimensional peptide mapping indicated that they were identical. The Con A-binding property of GP-A was exploited to purify it by affinity chromatography with agarose-Con A. Peptide mapping comparisons between affinity-purified GP-A and GP-A in SPM and SJ fractions indicated that GP-A in synaptic fractions is apparently homogeneous. Peptide map comparisons between GP-A and 235K fodrin polypeptide indicated that these two synaptic components are highly related (50% of their respective peptides are shared). The 235K fodrin polypeptide in SJs reacted with anti-fodrin antisera on Western blots; however, GP-A failed to cross-react. These observations, together with results from previous studies, indicate that GP-A is highly enriched in cerebellum compared to other neuronal and nonneural tissues. Moreover, GP-A is enriched in SJs relative to SPM fractions, is related to fodrin, and is most likely a cell-surface glycoprotein at asymmetric synapses in cerebellum. GP-A may be involved in neuronal recognition or synaptic transmission in the cerebellum. The important role of calcium in synaptic transmission, together with the decreased solubility of GP-A in Triton that results from micromolar concentrations of calcium, suggest that GP-A may play a role in stabilizing cerebellar synaptic junctions.     

A LewisX Glycoprotein Screen Identifies the Low Density Lipoprotein Receptor-related Protein 1 (LRP1) as a Modulator of Oligodendrogenesis in Mice

In the developing and adult CNS multipotent neural stem cells reside in distinct niches. Specific carbohydrates and glycoproteins are expressed in these niche microenvironments which are important regulators of stem cell maintenance and differentiation fate. LewisX (LeX), also known as stage-specific embryonic antigen-1 or CD15, is a defined carbohydrate moiety expressed in niche microenvironments of the developing and adult CNS. LeX-glycans are involved in stem cell proliferation, migration, and stemness. A few LeX carrier proteins are known, but a systematic analysis of the targets of LeX glycosylation in vivo has not been performed so far. Using LeX glycosylation as a biomarker we aimed to discover new glycoproteins with a potential functional relevance for CNS development. By immunoaffinity chromatography we enriched LeX glycoproteins from embryonic and postnatal mouse brains and used one-dimensional nLC-ESI-MS/MS for their identification. We could validate phosphacan, tenascin-C, and L1-CAM as major LeX carrier proteins present in vivo. Furthermore, we identified LRP1, a member of the LDL receptor family, as a new LeX carrier protein expressed by mouse neural stem cells. Surprisingly, little is known about LRP1 function for neural stem cells. Thus, we generated Lrp1 knock-out neural stem cells by Cre-mediated recombination and investigated their properties. Here, we provide first evidence that LRP1 is necessary for the differentiation of neural stem cells toward oligodendrocytes. However, this function is independent of LeX glycosylation.     

Postnatal Development of Noradrenaline and 3-Methoxy-4-Hydroxyphenylethyleneglycol Sulphate Levels in Rat Brain Regions

Abstract: Developmental changes in brain levels of noradrenaline (NA) and 3-methoxy-4-hydroxyphenylethyleneglycolsulphate (MHPG-SO₄) were studied in rats. In most brain regions, MHPG-SO₄ level rapidly increased to approach or exceed adult levels at the time of weaning, while NA levels increased more gradually and reached adult levels following weaning, Pharmacological studies showed that the MHPG-SO₄ level in the neonatal brain reflects the degradation of released NA. The developmental characteristics of noradrenergic neurons in eight discrete brain regions are discussed.     

幼年菲菊头蝠形态和声音发育

2006年5月31日—7月20日于桂林市郊积水洞对菲菊头蝠(Rhinolophus pusillus)幼蝠形态和叫声发育进行了研究。方差分析结果显示,幼蝠在出生后近3周内体重和前臂长增长迅速,随后增长速率变缓,且体重和前臂长增长与年龄均显著相关(年龄与体重：P=0.025;年龄与前臂长：P=0.042)。幼蝠叫声时程波动大,且均长于成年个体叫声时程,时程与年龄呈显著相关性。幼蝠在出生后3周内的叫声特征为频谱结构多样、频率波动大、谐波较多,在第3周后叫声多为FM-CF-FM叫声;幼蝠叫声第1谐波频率与年龄增长显著相关,而第二谐波频率与年龄相关性较弱;幼蝠在35天后主频率已经接近成年蝙蝠叫声频率;且前臂长与幼蝠叫声频率呈显著线性关系。