THE SUBCELLULAR LOCALIZATION OF ACETYLCHOLINESTERASE AND ITS MOLECULAR FORMS IN PIG CEREBRAL CORTEX

Abstract— The distribution of acetylcholinesterase among the subcellular fractions of pig cerebral cortex was determined. The crude mitochondrial and microsomal fractions obtained by differential centrifugation accounted for 75% of the enzyme, with the remainder divided between the crude nuclear and soluble fractions.
The occurrence and distribution of the multiple molecular forms of AChE was the same in all four fractions with the dominant species of molecular weights 350,000, 270,000 and 60,000. Further purification of the mitochondrial fraction by density gradient centrifugation gave a series of membrane fractions with very similar multiple forms. The one possible exception was the fraction containing the purified synaptosomal membranes where one band of mol wt 270,000 predominated, although the other molecular weight entities were present. The electrophoretic pattern of AChE present in the fractionated microsomes was the same as in the crude preparation. The content and pattern of the multiple molecular forms of AChE was therefore the same in all fractions of pig brain, apart from that containing the purified synaptosomal membranes.     

SUBCELLULAR LOCALIZATION OF NEWLY-FORMED [3H]ACETYLCHOLINE IN RAT CEREBRAL CORTEX IN VITRO

—Slices from rat brain cortex were incubated for either 5 or 60 min in a medium containing [³H]choline and 4·7 or 25 mm -KCl. Bioassayable ACh and labelled ACh were determined in the incubation medium, in the total tissue homogenate and in subcellular fractions. Raising the KCl concentration from 4·7 to 25 mm stimulated the release and synthesis of total and of labelled ACh. In medium containing 25 mm -KCl the amounts of ACh decreased in the tissue and in the nerve ending cytoplasm, but remained constant in the synaptic vesicles. After incubation in 25 mm -KCl medium the ACh in the vesicles was labelled to the same extent as the cytoplasmic ACh but after incubation in 4·7 mm -KCl medium vesicular ACh was labelled less than cytoplasmic ACh. During 5 min incubation in medium containing 25 mm -KCl the ratio of labelled to total ACh was much higher in the medium than in the homogenate, the vesicles or the cytoplasm. During the last 15 min of the 60 min incubation the ratio of labelled to total ACh in the medium was still higher than that in the tissue fractions, but less so than during the 5 min incubation. It is concluded that the vesicular and cytoplasmic fractions are not identical with the store in the tissue from which newly-synthesized ACh is preferentially released.     

CHARACTERIZATION AND LOCALIZATION OF ACID HYDROLASE ACTIVITY IN THE SYNAPTOSOMAL FRACTION FROM RAT CEREBRAL CORTEX

Synaptosomes were prepared from the cerebral cortex of adult rats by a rapid technique of centrifugation in a Ficoll-sucrose discontinuous gradient. The synaptosomal fraction contained 40 per cent of the total gradient activity of acid α-naphthyl phosphatase (EC 3.1.3.2). Quantitative electron microscopy of this fraction revealed rare, typical, extrasynaptosomal dense body lysosomes. pH-activity profiles of free and Triton X-100 (total) activities were prepared for α-naphthyl phosphatase, β-glucuronidase (EC 3.2.1.31), β-galactosidase (EC 3.2.1.23), arylsulfatase (EC 3.1.6.1) and N-acetylglucosaminidase (EC 3.2.1.30). The ratios of total to free activity varied in the order: arylsulfatase > β-galactosidase > β-glucuronidase > N-acetylglucosaminidase > acid phosphohydrolase. Incubation of synaptosomal fractions at pH 5 and 37°C produced significant activation of β-galactosidase and N-acetylglucosaminidase but no activation of cryptic lactate dehydrogenase (EC 1.1.1.27). Hyposmotic suspension and subfractionation of the synaptosomal fraction produced considerable solubilization of lactate dehydrogenase, arylsulfatase and β-galactosidase but only partial liberation of α-naphthyl phosphatase, the remainder being associated with synaptosomal membrane fragments. Incomplete equilibrium sedimentation of synaptosomes in a continuous sucrose gradient (0·55-1·5 M) provided a broad lactate dehydrogenase and Na + K ATPase (EC 3.6.1.4) peak (peak I) at low sucrose densities. β-Glucuronidase, β-glucosidase and α-naphthyl phosphatase were significantly present in peak I. Conversely, N-acetylglucosaminidase, arylsulphatase and β-galactosidase were predominantly located in denser particles sedimenting through 1·2 M sucrose (peak II). Electron microscopy confirmed the heterogeneity of this second peak and the presence of numerous extrasynapto-somal dense body lysosomes.     

THE SUBCELLULAR LOCALIZATION OF HISTIDINE DECARBOXYLASE IN VARIOUS REGIONS OF RAT BRAIN

Abstract— The subcellular distribution of histidine decarboxylase (assayed by two different isotopic methods) and several biochemical markers (lactate dehydrogenase, DOPA decarboxylase and protein) was determined in rat cerebral cortex. After differential centrifugation, the enzyme activity was found mainly in the crude mitochondrial and soluble fractions. Further separation of the former on discontinuous sucrose gradients showed that the particulate histidine decarboxylase (HD) was found in the synaptosomal fraction. After osmotic shock, HD activity appeared in the supernatant fraction suggesting that a major portion of the enzyme is localized in the cytoplasm of cortical nerve endings. By analogy with other brain amines, this finding, together with the presence of histamine in synaptic vesicles (K ataoka and de R obertis , 1967), can be taken as further support for the hypothesis of a role as neurotransmitter for histamine.
Various brain regions were homogenized under conditions leading to synaptosome formation. The distribution of HD between 'particulate' and soluble fractions differed from one region to the other, but did not give any clear-cut indication of regions rich in cell bodies or nerve terminals.     

FACTORS AFFECTING THE TURNOVER OF CEREBRAL GLYCOGEN AND LIMIT DEXTRIN IN VIVO

The turnover of cerebral glycogen in mice has been investigated by using [U-¹⁴C]glucose as a precursor. The time required for turnover of total glycogen and limit dextrin has been determined in normal animals and animals given phenobarbital or hydrocortisone. In all 3 groups, the turnover time for limit dextrin was twice that of total glycogen. Phenobarbital increased the time for turnover of total glycogen and limit dextrin approximately 2-fold, whereas hydrocortisone diminished the turnover time of both fractions to one-half. The accumulation of glycogen during phenobarbital anesthesia (2·5-fold) is attributed to the decrease in rate of phosphorolysis rather than elevated glycogenesis. The ratio of phosphorylase a to total phosphorylase was significantly decreased in the brains of phenobarbital-treated mice, while the ratio of glycogen synthetase I to total synthetase activity was not affected. The administration of hydrocortisone had no effect on either the phosphorylase or synthetase of mouse brain. A mathematical model was devised to determine the rate constants for incorporation of labelled glucose into brain glycogen and the subsequent loss of radioactivity. Metabolite levels and enzyme activities have been correlated with the observed changes in glycogen turnover.     

THE TURNOVER AND SUBCELLULAR LOCALIZATION OF THE CHICK FOREBRAIN SMALL NUCLEAR RNAs

Analysis of 2 day old chick forebrain RNA on polyacrylamide gels revealed the presence of the 29S, 18S, 5.5s and 5s rRNAs, 4S tRNA and the small mol wt nuclear RNA (snRNA) species K, L, C, DD and G. The turnover of these molecules was studied following the intracerebral injection of [³H]uridine into 2 day old animals. The specific activities of these molecules declined over a 23 day time course and all displayed first-order decay kinetics. Apparent half-lives (in days) for 29S (7). 18S (S), L (24), C (6.5), DD′ (8.5), 5.5S (7), 5S (13), G (6) and 4S (7) were obtained. The subcellular localization of the snRNAs was studied in 5 day old chick forebrain. The snRNAs accounted for almost 17% of nuclear RNA, in contrast to their low levels in whole forebrain (1.2%). This was due to an approx 20-fold nuclear enrichment of C, DD′, G′ and H. Relatively low levels of K and L were present within the nuclear fraction. However, K and L, and to a lesser extent C and DD, were readily detected within the post-microsomal supernatant. Production of a pH 5 enzyme fraction from this supernatant resulted in a 2-3-fold enrichment of K, L, C and DD′ with respect to tRNA. The polysome containing fractions also displayed significant levels of L with about 1 mol of L per 30 of 5S rRNA. A new method for the determination of the subcellular distribution of the snRNAs is described. It depends upon the purity of the nuclear, microsomal and cell sap subfractions rather than upon total recoveries from such fractions. Utilizing the relative amounts of these RNA species within whole forebrain and the major subfractions, distributions (as a percentage within each fraction) were obtained. The observation of significant levels of K, L, C and DD within the cytoplasmic fractions raises some doubts about previous suggestions of an exclusively nuclear role for these molecules. It is proposed that the snRNAs may be involved in a multistage interaction within the process of eukaryotic gene expression.     

SUBCELLULAR DISTRIBUTION OF NORADRENALINE IN GUINEA-PIG CEREBRAL CORTEX AND SPLEEN

Abstract— The distribution of noradrenaline (NA) in subcellular fractions of guinea-pig cerebral cortex and spleen was determined by differential and density gradient centrifugation. Of the primary fractions, the microsomal fraction from both tissues was enriched in NA, that of the spleen having the higher specific activity. Microsomal fractions were therefore placed on gradients and NA determined in the subfractions since these fractions appeared suitable preparations in which to search for discrete populations of vesicles. So that the non-occluded micro-particulate bound noradrenaline (MPBNA) content of gradient subfractions could be measured, [³H]NA was used to control for the diffusion and or adsorption of free NA, and occluded lactate dehydrogenase was used to estimate the amount of entrapped MPBNA and soluble NA. Non-occluded MPBNA on gradients from microsomal fractions of cerebral cortex formed a single peak mainly in subfraction F (0.6-0.8 m -sucrose). Spleen microsomal fractions, however yielded two peaks of MPBNA. one in sub-fractions D to G (0.4-1.0 m -sucrose) and the other in sub-fraction J (1.4 m -sucrosc); electron microscopy showed that the latter subfraction contained large vesicles.
Since there were unexpectedly small amounts of MPBNA in microsomal subfractions D and E of cerebral cortex, the synaptosome fraction was investigated. Following water treatment of synaptosomes. MPBNA formed a peak in subfraction E (0.4-0.6 m -sucrose) with smaller amounts in subfractions D and F (0.4 and 0.6 0.8 m -sucrose).     

IN VIVO METHYLATION AND TURNOVER OF RAT BRAIN HISTONES

Abstract— The turnover of the different histone components from brain nuclei was studied after the administration of l -[³H]lysine and l -[¹⁴C-methyl]methionine to newborn rats. The radioactivities of the different histone subfractions as well as other proteins were determined over a 280-day period. Biphasic type decay curves (³H and ¹⁴C) were obtained for total brain histones and all the subfractions. From 6 to 40 days of age the half life of total brain histones was 19 days. After reaching brain maturity the half life was 132 days. The lysine rich histone (F₁) was found to turnover the fastest of all the histones, having half lives of 13 and 112 days, respectively. The decay curve for the slightly lysine rich histones (F_2a2, F_2b) gave half lives of 25 days up to 40 days of age and 189 days after reaching brain maturity. The arginine rich histones (F_2a1, F₃) gave a half life of 32 days up to 40 days of age, while no turnover was observed after maturity. The turnover rates of the methyl groups and/or methionyl residues did not vary significantly from the turnover rates of the lysyl residues in the F₂ and F₃ histones. The lysine-rich histones did not contain significant amounts of methionyl residues or methyl groups.
Amino acid analysis of the brain histones revealed that about 3·6 per cent of the lysyl residues in the slightly lysine rich histones were methylated, mainly as ε-N-dimethyllysine. About 13 per cent of the lysyl residues in the arginine rich histones were methylated, mainly as ε-N-monomethyllysine and ε-N-dimethyllysine.     

METABOLISM OF HEXOSES IN RAT CEREBRAL CORTEX SLICES

Abstract—

• 1 The metabolism of two ¹⁴C-labelled hexoses and one hexose analogue, viz. mannose, fructose and glucosamine, has been compared with that of glucose for slices of rat cerebral cortex incubated in vitro.
• 2 The metabolism of [U-¹⁴C]mannose was essentially identical to that of glucose; oxygen consumption and CO₃ production were similar and maximal at a substrate concentration of 2·75 mM. Incorporation of label into lactate, aspartate, glutamate and GABA was similar for the two substrates at 5·5 mM substrate concentration.
• 3 With [U-¹⁴C]fructose, maximal oxygen consumption and CO₃ production were obtained at a substrate concentration of 11 mM. At 5·5 mM, incorporation into lactate was 5 per cent, into glutamate and GABA 30 per cent, into alanine 63 per cent and into aspartate 152 per cent of that from glucose. Increasing substrate concentration to 27·5 mm was without effect on incorporation into amino acids from glucose and raised incorporation from fructose into glutamate, GABA and alanine to a level similar to that found with glucose; at the higher substrate concentration aspartate incorporation from fructose was 200 per cent and lactate 42 per cent of that with glucose. Unlabelled fructose was without effect on incorporation of radioactivity from [3-¹⁴C]pyruvate into CO₂ or amino acids; it increased incorporation into lactate by 36 per cent. Unlabelled glucose diminished incorporation into CO₂ from [U-¹⁴C]fructose to 35 per cent; incorporation into lactate was stimulated 178 per cent at 5·5 mM fructose; at 27·5 mM it was diminished to 75 per cent.
• 4 By comparison with [1-¹⁴C]glucose, incorporation of radioactivity from [1-¹⁴C]-glucosamine into lactate, CO₂, alanine, GABA and glutamine was very low; incorporation into aspartate was similar to glucose. Thus the metabolism of glucosamine resembled that of fructose. Glucosamine-1-phosphate, glucosamine-6-phosphate, and an unidentified metabolite, all accumulated.

     

OXIDATIVE METABOLISM OF THE CEREBRAL CORTEX OF THE RAT IN SEVERE INSULIN-INDUCED HYPOGLYCAEMIA

—Measurements were made of organic phosphates, carbohydrate substrates, amino acids and ammonia in the cerebral cortex, as well as of cerebral blood flow and of cerebral metabolic rate for oxygen and glucose in rats that developed an isoelectric EEG pattern (‘coma’) during insulin-induced hypoglycaemia. The results were compared to those obtained in control animals, as well as in hypoglycaemic animals with an EEG pattern of slow waves and polyspikes. In animals with slow waves and polyspikes, there was a decrease in all citric acid cycle intermediates except succinate and oxaloacetate, and a decrease in the pool size of intermediates. In animals that had an isoelectric EEG for 5–15 min, there were further decreases in citrate, isocitrate, α-ketoglutarate, malate and fumarate, but since the concentration of succinate (and oxaloacetate) increased, the pool size remained the same. In isoelectric animals, the results revealed extensive utilization of amino acids by both transamination and deamination reactions. However, since glycogen had disappeared and the amino acid pattern was constant after the first 5 min of isoelectric EEG, further oxidation must have occurred at the expense of non-carbohydrate, non-amino acid substrates. There were two- to three-fold increases in cerebral blood flow in animals with slow waves and polyspikes and in animals with isoelectric EEG, and no decrease in the cerebral metabolic rate for oxygen. Since less than half of the oxygen consumption could be accounted for in terms of glucose extraction, the data indicate that severe hypoglycaemia is associated with extensive oxidation of endogenous substrates other than carbohydrates and free acids.     

癫痫大鼠大脑皮质及海马神经元NOS的变化

给大白鼠侧脑室注射马桑内酯（Ｃｏｒｉａｒｉａ Ｌａｃｔｏｎｅ, ＣＬ）（１７５×１０－ ２ｍ ｏｌ／Ｌ２μｌ）后可诱发癫痫,用ＮＡＤＰＨｄ 组织化学方法观察大脑皮质及海马ＮＯＳ阳性神经元的变化, 结果： 大脑皮质ＮＯＳ阳性神经元数目逐渐增加, 至２ｈ 达高峰, 与生理盐水组相比差异具有非常显著性意义（Ｐ＜ ００１）, 随着ＣＬ作用时间延长ＮＯＳ反应由弱变强;海马区ＮＯＳ阳性神经元２ｈ 时才出现染色明显加深。对体外培养的大脑皮质及海马神经元用ＣＬ （２５×１０－ ５ｍ ｏｌ／Ｌ） 作用１／２ｈ、１ｈ、２ｈ、４ｈ 后ＮＯＳ阳性神经元均未见明显增加。     

大鼠颌下腺及其离体培养细胞脱氢表雄酮（DHEA）的免疫组织化学定位

用免疫组织化学ABC法,研究了颌下腺及无血清培养的颌下腺上皮细胞DHEA的定位,结果显示,大鼠颌下腺的浆液性腺泡的上皮细胞及各级导管上皮细胞均呈DHEA免疫反应阳性,无血清培养腺上皮细胞也呈DHEA免疫反应阳性,阳性物质分布于胞质,胞核呈阴性反应,此结果提示：大鼠颌下腺能自身合成DHEA,DHEA对消化功能可能具有重要的调节作用。     

TURNOVER OF PROTEIN PHOSPHORUS IN RESPIRING SLICES OF GUINEA PIG CEREBRAL CORTEX: CELLULAR LOCALIZATION OF PHOSPHOPROTEIN SENSITIVE TO ELECTRICAL STIMULATION

Abstract— In experiments designed to localize the increased turnover of phosphoprotein-P which occurs in respiring brain slices as a result of electrical stimulation, a cell separation procedure was used to prepare a fraction enriched in neuronal cell bodies from incubated slices labelled with [³²P]phosphate. Labelled phosphoprotein was found to be twice as concentrated in the neuron-enriched fraction as in other fractions. Electrical stimulation for 10 s increased the rate of incorporation of [³²P]phosphate into phosphoproteins in the neuron-enriched fraction by 25 per cent ( P < 0.05), but had no effect on incorporation into a partially purified glial fraction contaminated with neuropil and cell debris.     

大鼠体内气管损伤修复过程及气管干细胞的定位研究

目的观察大鼠体内气管损伤修复过程,进行气管干细胞的定位.方法应用氟尿嘧啶(5-FU)诱发在体气管上皮损伤,动态观察修复过程;对损伤后气管上皮细胞行Hoechst33342荧光染色,并用RT-PCR法检测ABC转运蛋白ABCG2/bcrp1基因.结果1.5-FU作用30min后大鼠气管上皮细胞绝大部分脱落,可见少量间隔分布的类似裸核的细胞呈钉状位于基底膜上,免疫组化检测增殖细胞核抗原阴性,证明为G0期细胞.其中部分细胞Hoechst33342染色阴性,为侧群(side population,SP)细胞.2.将5-FU 去除3-6h后,上皮细胞形态变为扁平,9-12h 后细胞变为立方,细胞数目逐渐增多,24h上皮细胞数更多,连接成片,可见纤毛,48h 接近恢复假复层纤毛柱状上皮.3.RT-PCR检测ABCG2/Bcrp1阳性反应产物长度为272bp.结论5-FU打击后,残余的G0期气管上皮细胞中含有干细胞.     

SUBCELLULAR DISTRIBUTION OF PYRUVATE KINASE (EC 2.7.1.40) IN CEREBRAL CORTEX

—The subcellular distribution of pyruvate kinase (EC 2.7.1.40) in the cerebral cortex of the rat was studied. The enzyme, which had been previously reported in the cytoplasm, was found to be present in synaptosomal, microsomal and mitochondrial fractions as well. The activity of the enzyme in the synaptosomal fraction was localized predominantly in the synaptosomal membrane and was not dissociated by repeated washing or recentri-fuging in a sucrose gradient. Some kinetic parameters of the membrane-associated pyruvate kinase were measured.     

TOPOGRAPHICAL AND SUBCELLULAR LOCALIZATION OF CHOLINE ACETYLTRANSFERASE IN RAT HIPPOCAMPAL REGION

—Choline acetyltransferase (ChAc) was localized in discrete layers in hippocampus regio superior and in area dentata. The highest activity in hippocampus was found in a narrow infrapyramidal zone, but high activities were also observed in the rest of stratum oriens and in stratum pyramidale. In area dentata the highest activities were found in a narrow supragranular zone and in hilus fasciae dentatae. The localization corresponded very closely to that of acetylcholinesterase. The main part of ChAc activity was confined to the synaptosome fraction. The results are compatible with the view that pyramidal and granular cells are excited by cholinergic boutons, mainly located on the basal or apical dendrites near the somata.