脊髓和周围神经可溶性酸性蛋白质的研究

 利用微型双向电泳、SDS电泳、免疫印迹法、DEAE-Sephadex色谱、高效液相色谱及氨基酸分析等方法,对牛脊髓(中枢神经)和马尾神经(周围神经)的可溶性酸性蛋白质进行了研究。结果表明在牛脊髓和马尾神经中有钙调素(CaM)、S-100蛋白和神经元特异烯醇化酶(NSE)等可溶性酸性蛋白质存在;脊髓中这些酸性蛋白质的含量远较马尾神经为高。     

COMPARATIVE STUDIES ON THE MYELIN PROTEINS OF BOVINE PERIPHERAL NERVE AND SPINAL CORD

Abstract— (1) Two myelin fractions of bovine peripheral nerve and spinal cord have been studied comparatively. Cholesterol as well as cerebroside content per mg of protein in the peripheral nerve myelin was less than that in the spinal cord myelin, while no significant difference in the total phospholipid content was noted.
(2) The basic proteins in myelin fractions were quantitatively estimated by disc gel electrophoresis. Around one-fourth of the total myelin protein in the bovine peripheral nerve was a basic protein with a mobility of 1.07 relative to lysozyme by Reisfeld's disc gel electrophoresis.
(3) The myelin proteins in the peripheral nerve were less completely solubilized than those of the spinal cord by treatment with deoxycholate as well as by Triton-salt solution. The protein fractions obtained from the peripheral nerve myelin by techniques similar to that for obtaining the proteolipids from the spinal cord myelin, contained different types of protein.
(4) 2',3'-Cyclic nucleotide 3'-phosphohydrolase activity in the peripheral nerve myelin was only one tenth of that in the spinal cord myelin. The Triton-salt insoluble fraction showed remarkable high activity among subfractions of the spinal cord myelin.
(5) By immunological studies, it may be concluded that an antigenic substance for experimental allergic neuritis was localized in the peripheral nerve myelin, but not in its basic protein.     

IMMUNOCHEMICAL AND BIOCHEMICAL STUDIES DEMONSTRATING THE IDENTITY OF A BOVINE SPINAL CORD PROTEIN (SCP) AND A BASIC PROTEIN OF BOVINE PERIPHERAL MYELIN (BF)

A protein extracted from bovine peripheral myelin (BF) and a protein extracted from bovine spinal cord (SCP) have been shown to be identical: the proteins cross-react immunochemicaliy with each other but not with highly purified CNS myelin basic protein. Neither BF nor SCP have anti-encephalitogenic activity. Their electrophoretic behavior is the same at three different pH values. Their apparent molecular weight by sodium dodecyl sulfate-gel electrophoresis is 13,800 ± 550. The amino acid compositions of the proteins are essentially identical. BF and SCP each contain 2 cysteine residues and have valine at the C terminus. The 23 major tryptic peptides are identical on peptide maps. Circular dichroic analyses yield essentially identical curves, which, when computed by best-fit curve analysis, indicate that each has 0%α helix and a large percentage of β structure.     

THE ISOLATION OF GAMMA-GLUTAMYL PEPTIDES FROM MONKEY BRAIN

Abstract— The isolation and identification of the following gamma-glutamyl peptides from the brain of Macaca irus and Cercopithecus ethiops is reported: glutamyl-glutamine, glutamyl-glycine, glutamyl-alanine, glutamyl-valine, glutamyl-isoleucine, glutamyl-glutamate, glutamyl-serine and glutamyl-alanyl-glycine.     

SIMULTANEOUS ISOLATION OF PURIFIED MICROSOMAL and MYELIN FRACTIONS FROM RAT SPINAL CORD

Abstract— The fraction that sediments between 2 × 10⁵ g -min and 6 × 10⁶ g -min from dilute dispersions of rat brain in 0.32 m -sucrose is a microsomal fraction with very little contamination by myelin. A crude microsomal fraction prepared in the same way from rat spinal cord contains more myelin than microsomes. Centrifugation of the crude microsomal fraction in 0.85 m -sucrose gave a floating fraction, an infranatant fraction (purified microsomes) and a small pellet. The purified microsomes contained very little myelin as judged by electron microscopy and polyacrylamide gel electrophoresis. The lipid composition resembled that of spinal cord myelin except that the purified microsomes contained relatively less cholesterol and ethanolamine plasmalogens. The content of galactolipids was much greater in spinal cord microsomes than in brain microsomes. The spinal cord CDP-ethanol-amine:diglyceride ethanolaminephosphotransferase activity (EC 2.7.8.1) was concentrated in the purified microsomes.
A spinal cord myelin fraction isolated from the 2 × 10⁵ g -min pellet was quite pure as judged by electron microscopy, enzyme activities and polyacrylamide gel electrophoresis. No NADPH-cyto-chrome c reductase activity (EC 1.6.2.3) could be detected in the purified myelin. The ethanolaminephosphotransferase specific activity was about 5% of that found in the purified microsomal fraction. The protein content was 25% by weight for spinal cord myelin and 31% for brain myelin. Of the total spinal cord 2',3'-cyclic nucleotide-3'-phosphohydrolase activity, 16% was lost from the crude myelin during purification, 21% was recovered in the purified myelin, and 11% was found in the floating fraction from the crude microsomes. The purified myelin and microsomal fractions from spinal cord were relatively pure. Additional myelin was recovered in the floating fraction from the crude microsomes.     

ISOLATION AND CHARACTERIZATION OF GLYCOSAMINOGLYCANS IN PERIPHERAL NERVE AND SPINAL CORD OF MONKEY

—The isolation of uronic acid-containing glycosaminoglycans from peripheral nerve and spinal cord of monkey was done by combining the cetyl pyridinium procedure and DEAE-Sephadex column chromatography. The constituent analyses of the isolated GAG-fractions indicated that hyaluronic acid, chondroitin-4-sulphate, chondroitin-6-sulphate, heparan sulphate and a testicular hyaluronidase-resistant galactosamine-containing GAG were present in both tissues. Hyaluronic acid was the predominant GAG (63 per cent) in both tissues and its level was much higher than in brain. Chondroitin-4-sulphate constituted 16 per cent in both tissues. The levels of heparan sulphate and hyaluronidase-resistant galactosamine-containing GAG in these tissues were much lower than in brain. The results indicate that the patterns of GAGs in peripheral nerve and spinal cord of monkey are similar but differ from that of brain.     

PURIFICATION AND COMPARISON OF 2'',3''-CYCLIC NUCLEOTIDE 3''-PHOSPHOHYDROLASES FROM BOVINE BRAIN AND SPINAL CORD

The membrane-bound enzyme 2′,3′-cyclic nucleotide 3′-phosphohydrolase has been purified from acetone powders of bovine white matter and spinal cord. Affinity chromatography on AMP-Sepharose has been used as the final step in the chromatographic purifications. The yield was about 3 mg of purified enzyme per 100 g of tissue in each instance. The enzymes from the two sources were indistinguishable by chromatography, gel electrophoresis, and amino acid analysis; the enzyme from spinal cord, however, has shown a specific activity of 225 units/mg compared to 342 units/mg for the enzyme from white matter. Both proteins had a molecular weight of 100,000 by gel filtration and 50,000 by sodium dodecyl sulfate-gel electrophoresis under reducing conditions. The properties of the enzyme, including amino acid composition determined on the purified soluble protein and on the protein purified by sodium dodecyl sulfate-gel electrophoresis, were those of a basic hydrophobic protein.     

BULK ISOLATION OF LARGE VENTRAL SPINAL NEURONS

—Cell bodies of spinal neurons can be isolated in bulk fractions, using methods developed for neurons from brain. The technique described here includes sieving steps which allow one to purify and concentrate selectively the larger neuronal perikarya (50 μm or more), most of which could be alpha motoneurons. An average of 170,000 large neurons can be obtained from 50 g of trimmed bovine ventral enlargements. Roughly 6% of the total large neurons in the ventral grey matter are recovered, and 85% of the isolated neurons are larger than 50 μm in their smallest dia. Non-neuronal contaminants, mostly capillary fragments, account for 19% of the total particles in the fraction. Each isolated neuron contains an average of 9690 pg protein and 1353 pg RNA and hydrolyses 2·2 pmol of acetylthiocholine/min. Pretreatment of the spinal tissue with collagenase before cell isolation is also evaluated here.     

辣椒素引起脊髓P物质释放及其对血压的影响

为进一步研究脊髓 P 物质(SP)在调节心血管活动中的作用,在大鼠脊髓蛛网膜下腔注射(ith)辣淑素(cap),以刺激脊髓 SP 能神经末梢释放 SP,结果引起血浆去甲肾上腺素(NA)和肾上腺素(AD)含量增高,及具有剂量依赖性的动脉血压上升,心率升高。ith 具有高度特异性的 SP 受体拮抗剂或 SP 抗血清均可阻断 cap 引起的升压效应,免疫组化测定也观察到注入的cap 剂量越大,脊髓胸段 SP 样免疫阳性反应物的致密度越低,这些观察结果支持 cap 可以引起脊髓内 SP 的释放的说法。在第一颈段(C_1)横断脊髓后 ith cap 所引起的升压效应与完整动物 ith cap 的升压效应无显著差异。以上结果提示脊髓 SP 能神经末梢释放的 SP 可以通过交感肾上腺髓质系统引起心血管兴奋效应,SP 可能是引起交感节前神经元兴奋的神经递质。     

THE RELEASE OF AMINO ACIDS FROM THE HEMISECTED SPINAL CORD DURING STIMULATION

Abstract— Isolated frog or toad hemicords were incubated for 40 min with either [¹⁴C]glycine, [³H]GABA, l -[¹⁴C]glutamate. l -[¹⁴C]aspartate, l -[¹⁴C]serine, l [¹⁴C]threonine or l -[³H]leucine, and the release of these compounds from the cord was measured under resting conditions and during electrical stimulation. Stimulation of spinal roots produced no significant change in the efflux of any of the compounds tested. Direct stimulation of the rostral cord however, produced a large increase in the efflux of [¹⁴C]glycine, [³H]GABA, l -[¹⁴C]glutamate and l -[¹⁴C]aspartate. These increased effluxes were calcium dependent, the effects of stimulation being reduced in a calcium-free, or magnesium-supplemented (10 mM) medium. Stimulation failed to produce an increase in the efflux of l -[¹⁴C]serine, l -[¹⁴C]threonine, l -[¹⁴H]leucine, [¹⁴C]mannitol or [¹⁴C]urea. These results are consistent with the suggestions that glycine, GABA, glutamate and aspartate may be synaptic transmitters in the spinal cord.     

THE AMPHOTERIC PROPERTIES OF SOME AMINO-ACIDS AND PEPTIDES

The titration curves of solutions of glycine, alanine, α-ammo-butyric acid, leucine, glycyl-glycine, alanyl-glycine, alanyl-alanine, acetone, acetamide, urea, acetic acid, and aceturic acid were determined and some of the relations as dependent upon the chemical structures discussed. The isoelectric points of some of the amphoteric electrolytes were found experimentally. The definition of isoelectric point, its theoretical significance, and method of calculation were considered in some detail.     

上海郊区黑斑蛙皮中两个新肽的分离和结构鉴定

<正> 黑斑蚌(Rana nigromaculata)皮的甲醇提取物经碱性氧化铝层析,所得50％乙醇洗脱液冻干后用反相HPLC分离得到18组峰。其中第9—10组峰与第15组峰进一步统化得9-10-10(Fig.1)与15-5-2(Fig.2)。按文献方法所得氨基酸组成分析结果分别是:     

PATTERNS OF AMINO ACID RELEASE FROM NERVE-ENDINGS ISOLATED FROM SPINAL CORD AND MEDULLA

The metabolic properties of synaptosomes prepared from the crude mitochondrial and crude nuclear fractions of the medulla/spinal cord were studied. They showed similar properties, glycine being enriched in the latter. The respiration and glycolysis rates were similar to the cortical synaptosomes previously studied. A major difference from cortical synaptosomes was the enrichment of glycine. Medulla/spinal cord synaptosome suspensions and beds responded metabolically to electrical pulses; respiration and lactate production increased by 50 and 25 per cent respectively. Differential release of glutamate, aspartate, GABA and glycine occurred during both electrical stimulation, and when potassium in the medium was increased. Omitting calcium and adding EGTA greatly reduced this response with both forms of stimulation. The electrically induced release of GABA was completely reversible whilst that of aspartate and glycine was only partially reversible. The electrically stimulated release of glycine and other amino acids was reduced in synaptosomes prepared from rats treated intramuscularly with tetanus toxin 15 hr before death. No action of the toxin was seen on synaptosomes incubated with tetanus toxin after preparation.     

嗜盐隐杆藻胞外多糖的分离、纯化及理化特性

嗜盐隐杆藻（Ａｐｈａｎｏｔｈｅｃｅ ｈａｔｏｐｈｙｔｉｃａ）培养液经离心,浓缩、透析、有机溶剂沉淀得胞外多糖（Ｅｘｏｐｏｌｙｓａｃｃｈａｒｉｄｅｓ,ＥＰＳ）粗品,经ＤＥＡＥ－纤维素二次柱层析纯化得ＥＰＳ精品。葡聚糖Ｇ－２００凝胶过滤表明其为单一组份。对其进行理化测试并对各组分进行定量分析,多糖、已糖醛酸、硫酸根含量分别为４０．９６％２３．２７％和３４．４６％,元素分析你测得Ｃ、Ｈ、Ｎ、Ｓ含量分别为     

脊髓内源性物质对脊髓神经元在体外存活的影响

神经元在体外的存活是衡量一种营养因子有无神经营养作用的重要指标之一。我们用人胚制备脊髓提取液,并用Centricon(Millipo-re)将粗提取液分成<10KD、10-30KD及>30KD三种组份,研究了粗提取液及这三种组份对体外培养中的脊髓神经元存活的影响,结果表明加粗提取液及<10KD的实验组比对照组活性要好,表现在线粒体中琥珀酸脱氢酶活性高(MTT法),神经元中NSE活性高(NSE-ELISA法)及细胞生长合成的总蛋白的量高等方面。但以<10KD组份对细胞的促活作用最强,与对照组相比有显著性差异。以上结果显示人胚脊髓中存在对脊髓神经元有促进存活的物质。     

新生大鼠脊髓切片运动神经元的电生理参数测定

用微电极技术对新生大鼠脊髓横切薄片运动神经元(MN)进行细胞内记录,测得静息电位为-62±4mV(n=26),膜电阻为67±31MΩ,时间常数3.8±1.6ms,动作电位幅度68±7mV(n=26),阈电位-50±8mV,超射值6±4mV。灌流谷氨酸(1～30mmol/L)诱导伴膜电阻降低的缓慢去极化反应,5-羟色胺(50μmol/L)介导伴膜电导降低的电压依赖性内向电流。结果表明新生大鼠脊髓切片MN的细胞内生物电记录是一种稳定可靠的电生理学和药理学研究方法。