THE DISTRIBUTION OF ACETYL-CoA IN SPECIFIC AREAS OF THE CNS OF THE RAT AS MEASURED BY A MODIFICATION OF A RADIO-ENZYMATIC ASSAY FOR ACETYLCHOLINE AND CHOLINE1

A rapid and sensitive enzymatic assay for measuring picomole quantities of acetyl-CoA, acetylcholine (ACh), and choline from the same tissue extract has been developed. After ACh and choline were extracted into 15% 1 N formic acid/85% acetone, the pellet was further extracted with 5% trichloroacetic acid (TCA) to remove the remaining acetyl-CoA. The two extraction solvents were pooled and lipids, organic solvents, and TCA were removed first by a heptane-chloroform wash followed by an ether extraction. In the acetyl-CoA assay, endogenous ACh and choline were removed by extractions with sodium tetraphenylboron in butenenitrile prior to the enzymatic reactions. The acetyl-CoA remaining in the aqueous phase was then converted enzymatically to labelled ACh in the presence of [Me-¹⁴C]choline using choline acetyltransferase. The unreacted labelled precursor was converted to choline phosphate by the enzyme choline kinase. The [¹⁴C]ACh formed from acetyl-CoA was extracted into sodium tetraphenylboron in butenenitrile and a portion of the organic phase containing the [¹⁴C]ACh was counted in a scintillation counter. Acetylcholine and choline were assayed from the same tissue extracts by a modification of the procedure by SHEA & APRISON (1973). Acetyl-CoA levels in rat whole brain when killed by the near-freezing procedure were found to be 5.50 ± 0.2 nmol/g. The content of acetyl-CoA was the same whether the rats were killed by the near-freezing method or by total freezing in liquid nitrogen. The levels of acetyl-CoA did not change with time after death when the tissue was maintained at a temperature of ?10°C. In the same tissue extracts from rat whole brain killed by the near-freezing method, the content of ACh was 20.6 ± 0.7 nmol/g and choline 58.2 ± 1.2 nmol/g. Although reproducible, the level reported for choline is high when assayed under this condition. The content of choline however after total freezing was found to be 25.2 ± 2.0 nmol/g. The sensitivity (d. p. m. of sample twice blank) is 10 pmol for the acetyl-CoA assay and 25 pmol for the ACh and choline assays. The regional distribution of these three compounds in the brain of rats as well as the content of acetyl-CoA in heart, liver and kidney are presented.     

芒柄花素的放射免疫测定法

本文建立了芒柄花素(formononetin)的放射免疫测定法(RIA)。该方法利用芒柄花素的衍生物作为同位素示踪物,获得的针对芒柄花素—7-O—羧甲基—牛血清白蛋白(BSA—F)的抗血清对芒柄花素具有高的亲和性(Ka=6.8×10~9 L/M)。方法的检出限量为3.5±1.24 ng(n=13),检出范围为3.5—100 ng。用此方法测定红三叶草(Trifolium pratense L.)中的芒柄花素含量,批内、批间的平均误差分别为6.5％和11.9％,加样回收率平均为98.5％。红三叶草中芒柄花素含量为0.1305±0.495％(n=12)。南京地区夏季杂牧草饲料中芒柄花素含量为0.0062±0.030％(n=6)。并用此方法测定了饲喂红三叶草的湖羊血清中芒柄花素的血浓-时间曲线图。     

促肾上腺皮质激素释放激素放射免疫测定法的建立及其应用

用本室制备的兔抗羊促肾上腺皮质激素释放激素（ＣＲＨ）多克隆抗体和￣（１２５）Ｉ标记的Ｔｙｒ°－ｒＣＲＨ建立了ＣＲＨ放射免疫测定法。并用该方法测定了人胎盘组织和大鼠脑及胸腺组织ＣＲＨ含量。所得抗体与ｒＣＲＨ的亲合常数Ｋａ＝５．３×１０￣９Ｌ／ｍｏｌ,与促肾上腺皮质激素、血管紧张素Ｉ、β－内啡肽、生长激素释放因子、血管活性肠肽、胆囊收缩素、加压素等１８种肽没有交叉反应。￣（１２５）Ｉ－ｒＣＲＨ的比放射性为４１６３ｋＢｑ／μｇ。建成的ＣＲＨ放射免疫测定法取代比率为２．２９,灵敏度为４０ｐｇ,ＥＤ＿（５０）约为６５０ｐｇ,百分精密度和百分准确度分别为３．８４％和１０．３６％。用该法所测人胎盘和大鼠组织ＣＲＨ含量的结果表明,分娩胎盘组织ＣＲＨ含量远高于妊娠６─７周者,约相当于后者的１５倍;大鼠组织ＣＲＨ含量以下丘脑最高,垂体次之,然后依次是海马、胸腺和大脑皮质;且下丘脑和垂体的ＣＲＨ含量雄鼠明显高于雌鼠。     

神经降压素的放射免疫测定

本工作通过戊二醛将神经降压素(NT)连接到甲状腺球蛋白(TG)分子上,免疫家兔,获得了具有较高滴度,良好亲和力和较好特异性的抗神经降压素血清。使用此抗血清建立放射免疫测定,检测大鼠垂体和部分脑区NT免疫活性物质含量,结果与国外文献相近。     

A RADIOIMMUNOASSAY FOR THE DETECTION OF DELTA CRYSTALLIN IN CHICK LENS DIFFERENTIATION

Delta crystallin was isolated from 10–13 day chick embryo lens fiber cells. The lens fiber cell extract was isoelectrically precipitated at pH 5.1 to remove alpha and beta crystallins. Further purification by filtration through Sephadex G-150 and then acrylamide gel electrophoresis yielded a single, homogeneous preparation of delta crystallin, as characterized by gel electrophoresis. This purified delta crystallin was injected into rabbits to produce a potent antiserum to chick lens delta crystallin. The purified delta crystallin was iodinated with ¹²⁵Iodine, using the chloramine-T procedure. A radioimmunoassay for delta crystallin was then developed, using the principles of competitive protein binding analysis. The radioimmunoassay developed here had a minimum sensitivity of 50 nanograms, and effectively ranged to 1000 nanograms.
Developing lens rudiments from early chick embryos, beginning from 24 hr incubation up to 72 hr were examined at 6 hr intervals. All determinations from 24 hr through the 48 hr sample showed less than 10 nanograms per 100 lens rudiments. This was below the effective minimum detection limits of the assay. The first accumulation of delta crystallin was detected in the 54 hr sample, and increased thereafter.     

A SPECIFIC HYDROXYLAMINE-FERRIC CHLORIDE REACTION FOR HISTOCHEMICAL LOCALIZATION OF PECTIN

An alkaline solution of hydroxylamine is caused to react with the methyl esters of pectin present in sections of plant tissues to produce hydroxamic acids which, after acidification with hydrochloric acid, yield a red color upon addition of 2.5% ferric chloride solution. The test is specific and preferable to the use of ruthenium red for localization of pectin in the compound middle lamella. Subjecting sections to methylation and using reagents made up in 60% alcohol increases the intensity and sharpness of the color formation.     

A TECHNIQUE FOR THE STUDY OF ACETYLCHOLINE TURNOVER IN MOUSE BRAIN IN VIVO

Abstract— —A method to measure the rate of acetylcholine turnover in mouse brain in vivo has been developed. It is based on the formation of labelled acetylcholine from intravenously injected labelled choline. The isotopic dilution of choline in the brain has been measured by assaying endogenous choline in the brain by an enzymatic method using tritium-labelled acetyl-CoA and purified choline acetyltransferase.
The rate of acetylcholine turnover in the brain could be calculated at 50 n-moles acetylcholine/g/min in conscious mice. In anaesthetized mice and in mice treated with oxotremorine, a decrease of acetylcholine turnover to about 10 n-moles/g/min was found.     

A RADIOIMMUNOASSAY FOR MYELIN BASIC PROTEIN AND ITS USE FOR QUANTITATIVE MEASUREMENTS

—A specific radioimmunoassay (RIA) for myelin basic protein is described which is sensitive to 10⁻⁹ g of basic protein. The amount of basic protein detected in isolated myelin by the RIA and by SDS-gel electrophoresis and spectrophotometric quantitation agree to within experimental error. In contrast to isolated myelin, the major portion of the basic protein in fresh tissue is not accessible to its antibody. It is shielded from its antibody in a complex which is disrupted by heat, organic solvents, and various detergents. Maximum antibody binding was obtained with tissue heated to 100°C for 10 min. It is possible to calculate that the RIA quantitatively detects basic protein in boiled tissue. Boiled adult rat brain contains approximately 2·5 μg of basic protein/mg wet wt of cerebral cortex. The antibody to basic protein has no capacity to bind non-neural tissues.     

沙冬青叶肉细胞中一种特殊内含物的发育

用透射电镜观察了沙冬青（Ａｍ ｍ ｏｐｉｐｔａｎｔｈｕｓｍ ｏｎｇｏｌｉｃｕｓ）叶肉细胞中一种电子密度很高的近似椭圆形的特殊内含物的发育． 它始于中央液泡外侧,起初只是少量的泡状成分和电子密度很高的物质,然后两种成分逐渐增多,并随液泡膜内吞形成突起,不断伸向液泡中央,有的突起占据了液泡很大一部分体积． 接着突起中的泡状成分开始解体,电子密度很高的物质越来越多,直至充满整个突起． 当突起继续内伸时,它的尾部不断收缩变小,最后完全脱离液泡膜而游离在中央液泡里面． 这种内含物一般只出现在严冬季节,里面含有大量的脂．     

FORMATION OF A PRESPORE SPECIFIC STRUCTURE FROM A MITOCHONDRION DURING DEVELOPMENT OF THE CELLULAR SLIME MOLD DICTYOSTELIUM DISCOIDEUM

The origin of a unique vacuole (PSV), which was specifically present in the prespore cell of the cellular slime mold Dictyostelium discoideum. was investigated electronmicroscopically. A considerable number of PSV-mitochondrion complexes was found in the intermediate fraction between a pure PSV and a pure mitochondria fractions, which were obtained by isopicnic centrifugation of cellular components of the prespore cell. Similar complexes were also observed in the differentiating prespore cells. Furthermore, the activity of succinic dehydrogenase, a typical mitochondrial enzyme was found cytochemically to be localized in the PSV as well as in mitochondria. From these results, it was concluded that the PSV was formed from the mitochondrion through some intermediate steps.     

A JOINT INDEX FOR THE INTENSITY OF SEX‐SPECIFIC SELECTION

A growing body of experimental and field data shows that selective pressures often differ between males and females. Surprisingly, to date, little attempt has been made to formalize a metric expressing the relative behavior of directional selection in the two sexes. We propose an index that describes the extent to which concordant or antagonistic selection is operating between the sexes for a given trait. This joint index could prove especially useful for the study of intralocus sexual conflict and the evolution of sexual dimorphism, providing a common scale to directly compare different traits within or among taxonomic levels, and allowing an assessment on how common sexually antagonistic selection might be in extant populations.     

DEVELOPMENT OF A LEXICON FOR THE DESCRIPTION OF PEANUT FLAVOR

A lexicon of terms to describe desirable as well as undesirable flavors in peanuts has been developed. The lexicon and an intensity rating scale was developed by a 13 member panel of flavor and peanut specialists representing industry and the USDA-Agricultural Research Service. This system is intended to provide definitive, common terminology for use in communicating differences in peanut flavor variables among all phases of peanut research and industry.     

DEVELOPMENT OF A NEW BIOSENSOR FOR DETERMINATION OF CATALASE ACTIVITY

Catalase is one of the major antioxidant enzymes that catalyzes the hydrolysis of H₂O₂. The aim of this study was to suggest a new method for the assay of catalase activity. For this purpose, an amperometric biosensor based on glucose oxidase for determination of catalase activity was developed. Immobilization of glucose oxidase was made by a cross-linking method with glutaraldehyde on a Clark-type electrode (dissolved oxygen probe). Optimization and characterization properties of the biosensor were studied and determination of catalase activity in defined conditions was investigated in artificial serum solution. The results were compared with a reference method.     

CONTROL OF ACETYLCHOLINE SYNTHESIS—THE INHIBITION OF CHOLINE ACETYLTRANSFERASE BY ACETYLCHOLINE

Abstract— The inhibition of choline acetyltransferase by acetylcholine in vitro occurs at a concentration of 10 m m and increases progressively to 45 per cent at a concentration of 100 m m . The inhibition is competitive for choline and noncompetitive for acetyl-CoA. It is suggested that the synthesis of acetylcholine may be controlled by its accumulation in synaptic vesicles.     

BINDING OF MUSCARONE BY EXTRACTS OF HOUSEFLY BRAIN: RELATIONSHIP TO RECEPTORS FOR ACETYLCHOLINE

The supernatant fluid fraction isolated by centrifugation from a homogenate of heads of houseflies bound muscarone reversibly, with a binding constant of 2·4 × 10^?6m and an amount of 70 nmol/g of protein. Studies with 28 drugs demonstrated strong blockade by the 17 cholinergic agents, suggesting both nicotinic and muscarinic characteristics in the mode of binding. The binding appeared to involve acetylcholine receptors.     

A METHOD FOR DETERMINATION OF ACETYLCHOLINE BY SLOW PYROLYSIS COMBINED WITH MASS FRAGMENTOGRAPHY ON A PACKED CAPILLARY COLUMN

A new procedure is described for the estimation of ACh by pyrolysis-gas chromatography/ mass spectrometry. ACh iodide and the iodides of ACh-d₁₆ or propionylcholine are slowly demethylated at 250°C by pyrolysis on the tip of a glass probe after insertion into the heated entrance of a glass tube leading to a packed capillary column. The volatile tertiary amines are then carried by helium to the column and trapped in its initial part which is kept at about 60°C. After 2–3 min the chromatography is started when the amines are released by heating this part to the ambient temperature in the oven (165°C). Peaks due to demethylated ACh and propionylcholine are well separated. The limit of detection is about 0.3pmol. After pyrolysis of mixtures of ACh and either ACh-d₁₆ or propionylcholine the peak amplitudes are linearly related to the doses.     

高原鼠兔下丘脑促肾上腺皮质激素释放因子的放射免疫测定

高原鼠兔已被我们选定为研究高原低氧适应机制的模型动物。我们过去的研究从器官水平乃至细胞和亚细胞水平均已证明该动物对高原低氧是不敏感的,属于高原低氧完全适应型动物。在研究下丘脑神经肽对高原低氧神经内分泌系的调控作用时,我们用大 鼠下丘脑促肾上腺皮质激素释放因子(Corticotropin releasing factor,CRF)的抗原、抗体放射免疫方法测定高原鼠免下丘脑CRF水平,获得了满意的结果,其灵敏度范围为3—200微微克/管,批间和批内变异系数分别为2.69％和7.24％。高原鼠兔下丘脑正中隆起处(Median eminence,ME)的提取物等的稀释液显示出与合成的大鼠CRF及其抗体间放射免疫反应的标准曲线,有很好的平行关系。高原鼠兔和大鼠ME处CRF的水平用此法测定分别为10.13±3.05和17.22±3.88微微克/毫克蛋白。大鼠ME处CRF水平随模拟海拔高度的增加而降低,而高原鼠兔不变化。随着肾上腺双侧切除,下丘脑ME之CRF下降,血浆皮质酮水平急剧下降,本结果提示,高原鼠兔的下丘脑确实含有与大鼠相类似结构与组成的CRF。此测定方法的确立,为研究高原鼠兔低氧神经内分泌适应机理,开拓了新路。     

用于目标序列染色体定位的镜鲤BAC-FISH体系构建

为了构建用于镜鲤(Cyprinus carpio var. specularis)特定基因组序列染色体定位的实验体系, 在细菌人工染色体(Bacterial Artificial Chromosome, BAC)文库筛选池中对已知短序列基因组片段进行PCR扩增, 筛选出包含目标序列的BAC克隆, 提取BAC质粒进行缺刻平移标记制备探针, 开展荧光原位杂交(Fluorescence in situ hybridization, FISH)实验。通过对染色体片前处理、BAC质粒探针制备、C0t-1 DNA封闭基因组重复序列、预杂交、荧光染料选择、信号放大等一系列实验条件和方法的探索优化, 成功实现了目标序列在镜鲤有丝分裂中期染色体上的定位。定位对象既包括在染色体上有单一位点的序列, 如斑马鱼微卫星标记Z6884和Z4268, 也包括在染色体上有多个位点的重复序列, 如黄河鲤性别相关标记CCmf1。来自斑马鱼同一条染色体上的两个微卫星标记被分别定位于镜鲤不同染色体上, 为鲤鱼染色体数目加倍的进化假设提供了一项直接实验证据, 同时将现有遗传连锁图谱与染色体对应起来, 可作为染色体识别和细胞遗传学图谱构建的依据。黄河鲤性别相关重复序列被定位于不少于四条染色体上, 为性别决定相关基因的筛查提供了研究线索。这一BAC-FISH实验体系将成为鲤细胞遗传学图谱构建、基因组进化和比较基因组学研究中的重要研究工具。       

DEVELOPMENT OF A LEXICON FOR BEEF FLAVOR IN INTACT MUSCLE

ABSTRACT

A lexicon describing the flavor characteristics of beef across different cuts, grades, and cooking temperatures and methods was developed. Four major cuts of the United States Department of Agriculture (USDA) quality grade beef were cooked to five endpoint temperatures using braising, broiling (oven broiling and electric charbroiling), roasting and grilling (indoor and outdoor grilling). Six highly trained panelists identified and defined a total of 38 aroma and flavor characteristics in 176 beef samples. Beef identity, brown/roasted, bloody/serumy, metallic, fat‐like, overall sweet, sour aromatics and five tastes were present in practically all samples. Other attributes were present only in certain samples, depending on either the sample group or the cooking method/endpoint temperature combination used. This lexicon potentially offers the beef industry a standard tool to identify and quantify flavor attributes as impacted by temperature, cooking method, aging process, storage time, diet regime, packaging, USDA quality grades, etc.

PRACTICAL APPLICATIONS

Until now, the beef industry's main focus has been to assess beef tenderness and juiciness, with an emphasis on ways to improve beef texture. Meat companies and academic institutions have been using the 1995 American Meat Science Association guidelines to assess the flavor of beef, which are not comprehensive. Recent work has focused on flavor, and the industry needs a standardized flavor lexicon that can be used for many projects. It is important for the industry to be able to systematically identify and quantify flavor attributes that drive consumer acceptance.     

A MODEL FOR DEVELOPMENT AND EVOLUTION OF COMPLEX MORPHOLOGICAL STRUCTURES

How 'complex' or composite morphological structures like the mammalian craniomandibular region arise during development and how they are altered during evolution are two major unresolved questions in biology. Herein, we have described a model for the development and evolution of complex morphological structures. The model assumes that natural selection acts upon an array of phenotypes generated by variation in a variety of underlying genetic and epigenetic controlling factors. Selection refines the integration of the various morphogenetic components during ontogeny in order to produce a functioning structure and to adapt the organisms to differing patterns of environmental heterogeneity. The model was applied to the development and evolution of the mammalian mandible (which is used as a paradigm of complex morphological structures). The embryology of the mandible was examined in detail in order to identify the fundamental developmental units which are necessary to assemble the final morphological structure. The model is quite general since equivalent units exist for the development of many other biological structures. This model could be applied to many other developing morphological structures as well as other groups of organisms. For example, it can be applied to cell parameters during Drosophila development (Atchley, 1987). The model as discussed in this paper assumes that morphological changes in the mandible result from evolutionary changes in its underlying developmental units. The developmental units relate to characteristics of cellular condensations which are produced from the differentiation of embryonic neural crest cells. The developmental units include: the number of stem cells in preskeletal condensations (n), the time of initiation of condensation formation (t), the fraction of cells that is mitotically active within a condensation (f), the rate of division of these cells (r), and their rate of cell death (d). These units and their derivative structures are discussed in terms of types of tissue differentiation (chondrogenesis, osteogenesis, primary/secondary osteogenesis, intramembranous/endochondral ossification) and growth properties of major morphological regions of the mandible. Variation in these five units provides the developmental basis for ontogenetic and phylogenetic modification of mandibular morphology. We have discussed how these developmental units are influenced by (a) the cell lineage from which they arise, (b) epithelial-mesenchymal (inductive tissue) interactions, (c) regulation of cell differentiation, and (d) extrinsic factors such as muscles, teeth and hormones. Evidence was provided that variation in mandibular morphology is heritable, subject to modification by natural selection, and that divergence among different genetic stocks has apparently occurred through changes in these developmental units and their derivative structures.(ABSTRACT TRUNCATED AT 400 WORDS)