STABILITY OF ISOLATED MITOTIC APPARATUS DURING STORAGE*

Mitotic apparatus (MA) were isolated from sea urchin zygotes using various isolation procedures, and various properties of the isolated MA were studied and compared. MA isolated using hexylene glycol had birefringences which depended on the pH of the isolation medium, the lower the pH the higher the MA birefringence. The stability of the MA Birefringence also depended on the pH of the hexylene glycol isolation medium (the lower the pH the slower the rate of decay of birefringence), as did the final birefringence reached after prolonged storage. MA isolated using glycerol-dimethylsulphoxide (MTME) had much more stable birefringence than MA isolated using hexylene glycol; their birefringence decay rates were about 1000 times slower than those of MA isolated using hexylene glycol. Birefringence which remained after extraction of MA with H₂O, or 0.5 M KC1 was also studied; the results depended on the MA isolation medium, on the medium the MA were stored in, and on the amount of time the MA were stored after isolation, as described in detail in the text. These results are discussed, and it is suggested that several components (including, perhaps, oriented ribosomes) contribute to birefringence of isolated MA.     

THE MITOTIC APPARATUS ISOLATED IN GLYCEROL-CONTAINING MEDIUM

The mitotic apparatus of the sea urchin egg was isolated at 30°C in an isolation medium containing glycerol which is known to stabilize microtubules. After isolation in the 1 m glycerol-isolation medium, the mitotic apparatus was stabilized on addition of glycerol to a final concentration of 3 to 4 m. Without the addition, the chromosomes were disjoined from the spindle and the interzonal region between separating chromosomes was fragile resulting in separation of half spindles. Lowering the temperature of the isolation medium to 20°C or below, the isolation procedure allowed to isolate spindles. The isolated spindle behaved in a manner similar to the mitotic apparatus on the effect of glycerol concentration.
The glycerol-mitotic apparatus contained tubulin which was extractable with the isolation medium containing Ca ions or an organic mercurial. Tubulin was also extracted upon lowering the temperature to 0°C in the presence of GTP. Addition of KCl to a final concentration of 0.6 m immediately dispersed the mitotic apparatus. The extract revealed a colchicine binding of 0.001 mole per 105,000 × g of protein. The colchicine binding complex was found to have a molecular weight of 105,000. The DEAE Sephadex column chromatography of the KCl extract allowed to elute tubulin fraction which bound 0.1 mole colchicine per 105,000 × g of protein. The mitotic apparatus tubulin was shown to contain α and β subunits with mobilities quite identical with those of brain tubulin subunits. The molecular weights of the α and β subunits were 55,000 ± 1,000 and 51,000 ± 1,000, respectively.     

ULTRASTRUCTURE AND BIREFRINGENCE OF THE ISOLATED MITOTIC APPARATUS OF MARINE EGGS

Isolated mitotic apparatuses (MA) of clam and sea urchin eggs were investigated by polarizing and electron microscopy. Examination of fixed MA in oils of different refractive index revealed that at least 90% of the retardation of isolated MA is due to positive, form birefringence, the remaining retardation deriving from positive, intrinsic birefringence. Electron micrographs reveal the isolated MA to be composed of microtubules, ribosome-like particles, and a variety of vesicles. In the clam MA the number of vesicles and ribosome-like particles relative to the number of microtubules is much lower than in the sea urchin MA. In clam MA this allows form and intrinsic birefringence to be related directly to microtubules. The relation of birefringence to microtubules in isolated sea urchin MA is more complex since ribosome-like particles adhere to microtubules, are oriented by them, and are likely to contribute to the form birefringence of the isolated MA. However, comparison of values of retardation for clam and sea urchin MA, indicates that the major part of the birefringence in sea urchin MA is also due to microtubules. The interpretation of the structures giving rise to birefringence in the MA of the living cells is likely to be even more complex since masking substances, compression, or tension on the living MA may alter the magnitude or sign of the birefringence.     

IMMUNOCHEMICAL STUDIES ON THE BRAIN SPECIFIC PROTEIN

Abstract— In the soluble brain proteins of various species-man, ox, cat, rabbit, rat, mouse, hen, snake, frog and fish–there is a protein group which migrates more slowly than Moore's S-100 protein and faster than the albumin fraction on disc electrophoresis. The protein group is absent from any organs other than brain, and has a different pattern and number of fractions in different species. Immunochemically, the protein fraction group of the mammalian brains shows some common and identical distinctive antigenic determinants compared with the brain protein of the other animals-hen, snake, frog and fish. The protein group was designated the 'SPR' proteins, which were separated to 'P_II,', 'P_III', 'P_IV' and 'P_v' fractions. Common antigenic determinants are found in these fractions. The protein group is found in human brain in larger amounts in grey matter than in white matter and in small amounts in the cellular nuclei of human and bovine brain.     

THE DIRECT ISOLATION OF THE MITOTIC APPARATUS

A method for isolating the mitotic apparatus from dividing sea urchin eggs without the use of ethyl alcohol or of detergents is described. In the present method, the eggs are dispersed directly in a medium containing 1 M (to 1.15 M) sucrose, 0.15 M dithiodiglycol, and 0.001 M Versene at pH 6, releasing the visibly intact mitotic apparatus. The method is designed for studies of enzyme activities, lipid components, and the variables affecting the stability of the apparatus.     

SELECTIVE EXTRACTION OF ISOLATED MITOTIC APPARATUS : Evidence That Typical Microtubule Protein Is Extracted by Organic Mercurial

Mitotic apparatus isolated from sea urchin eggs has been treated with meralluride sodium under conditions otherwise resembling those of its isolation. The treatment causes a selective morphological disappearance of microtubules while extracting a major protein fraction, probably consisting of two closely related proteins, which constitutes about 10% of mitotic apparatus protein. Extraction of other cell particulates under similar conditions yields much less of this protein. The extracted protein closely resembles outer doublet microtubule protein from sea urchin sperm tail in properties considered typical of microtubule proteins: precipitation by calcium ion and vinblastine, electrophoretic mobility in both acid and basic polyacrylamide gels, sedimentation coefficient, molecular weight, and, according to a preliminary determination, amino acid composition. An antiserum against a preparation of sperm tail outer doublet microtubules cross-reacts with the extract from mitotic apparatus. On the basis of these findings it appears that microtubule protein is selectively extracted from isolated mitotic apparatus by treatment with meralluride, and is a typical microtubule protein.     

FURTHER STUDIES ON CELL DIVISION WITHOUT MITOTIC APPARATUS IN SEA URCHIN EGGS

A large quantity of paraffin oil, sucrose solution, or sea water was injected into the eggs of the heart urchin Clypeaster japonicus shortly before the onset of the first cleavage. The injected oil became spherical, pushing the mitotic apparatus aside. The sucrose solution mixed with the protoplasm and caused disintegration of the mitotic apparatus, and the sea water formed a vacuole at the center of the cell. In all these cases, cleavage may take place almost normally in spite of the absence of the mitotic apparatus or its displacement within the cell. In some eggs, furrowing may take place when more than fifty per cent of the endoplasm has been replaced with sea water before onset of cleavage.     

SOME IMMUNOCHEMICAL CHARACTERISTICS OF W1 AND W2 WOLFGRAM PROTEINS ISOLATED FROM RAT BRAIN MYELIN

Starting from a chloroform-methanol (2: 1 v/v) insoluble pellet of rat brain myelin, two pure proteins W1 and W2 were isolated by sodium dodecylsulphate preparative polyacrylamide gel electrophoresis. Their amino acid composition was compared. Antibodies against these proteins were prepared in rabbits. It was found that the two antigens have common antigenic similarities. The presence of one precipitin line of identity when myelin or isolated W1 and W2 from different animals were tested, led to the conclusion that there was no species specificity. The importance of the availability of such antisera is discussed.     

四川清风藤根皮中三萜成分的研究

从四川清风藤（Ｓａｂｉａ ｓｃｈｕｍ ａｎｎｉａｎａ Ｄｉｅｌｓ）的根皮分离到５ 个三萜化合物,根据理化常数、波谱分析及配合衍生物制备,分别鉴定为：３－氧,Δ１１,１３（１８）－齐墩果二烯（１）、３,１１－二氧,Δ１２－齐墩果烯（２）、３β－羟基,Δ１１,１３（１８）－齐墩果二烯（３）、３－氧,１１α－羟基,Δ１２－齐墩果烯（４）和３β,１１α－二羟基,Δ１２－齐墩果烯（５）。其中,４为新化合物     

罗非鱼绿色气球菌的鉴定及致病性研究

研究首次报道了从广东茂名市茂南区患病罗非鱼体内分离的两特殊细菌菌株Mnlv1 和Mnlv2, 通过细菌形态学、培养条件比较、生理生化特征及16S rRNA 基因检测等鉴定了该病原, 采用三种不同的感染方式对罗非鱼实施人工感染以期探讨该菌对罗非鱼的致病强度及致病条件, 同时也比较了两菌株对29 种不同的抗生素的敏感性差异。结果表明: 两菌株均为绿色气球菌(Aerococcus viridans), 革兰氏染色阳性(G+), 溶血性。感染实验结果显示, 该菌株对罗非鱼具有较强的致病性, 并且随着环境胁迫作用的加强, 鱼体发病死亡率增高。药敏试验结果显示, 两菌株为对头孢曲松、米诺四环素、诺氟沙星等15 种药物高度敏感, 对麦迪霉素、壮观霉素和新霉素3 种药物中度敏感, 对红霉素、乙酰螺旋霉素、苯唑青霉素等10 种药物不敏感。研究将为罗非鱼绿色气球菌病原的诊断及防治提供理论基础。       

PROTEIN SYNTHESIS IN FRACTIONS FROM ISOLATED BRAIN CELL NUCLEI

Abstract— 1. The incorporation in vivo and in vitro of isotopically labelled leucine into fractions of nuclear proteins from young and adult rat brain was investigated.
2. During post-natal cerebral maturation, the ability of nuclei from brain cells to synthesize proteins decreased. The specific activities of all the fractions of nuclear protein were highest in 3-day-old rats and declined thereafter. Nuclei from adult brain cells exhibited only 10 per cent of the activity found in nuclei from brain cells of 3-day-old rats.
3. The 'residual protein' fraction was most rapidly labelled, peak activity being reached within 30 min after injection. In vitro , the 'residual protein' fraction attained maximum activity within 40 min.
4. The specific activity of the chromatin acidic proteins (HCl-insoluble) was considerably higher than that of the histones both in vivo and in vitro. Histones were the most inert of all the nuclear protein fractions studied.
The possible functional significance of the various protein fractions during the process of cerebral maturation and in the adult brain is discussed.     

周围神经43kD蛋白免疫化学研究

目的：制图周围神经43kD蛋白单克隆抗体,并检测该蛋白在正常及损伤坐骨神经中的表达,方法：实验用SDS－聚丙烯酰胺胺凝胶电泳系统,从周围神经中分离回收43kD蛋白作为抗原,免疫BALB／c小鼠,通过杂交瘤技术和点膜印迹法检测,获得分泌识别43kD蛋白的单克隆抗体的杂交瘤细胞株,以Westernblot方法检测单克隆抗体的特异性,并检测43kD蛋白在正常坐骨神经及损伤坐骨神经远侧端中的表达。结果：经检测获得了识别43kD蛋白的单克隆抗体,Westernblot显示在正常大鼠坐骨神经与损伤后2周的坐骨神经远侧端组织电泳图谱43kD处均出现特异的阳性反应条带,在损伤神经中43kD蛋白阳性反应产物着色较深。结论：43kD蛋白具有独特的免疫化学特性,在正常与损伤坐骨神经中的有表达,在损伤坐骨神经中表达更强。     

小麦叶绿体中CTK结合蛋白的结合活性研究

叶绿体中存在着与细胞分裂素(CTK)专一结合的蛋白质。这一蛋白与6-苄氨基嘌呤(6 BA)的亲和力很强,解离常数达3.7×10~(-8)mol/L。最大结合量为10.7 pmol 6 BA/mg蛋白,Seatchard分析表明只有一类结合位点。不同的叶绿体纯化步骤对CTK结合蛋白的活性有不同的影响,分离步骤少而快速的差速离心法可以得到具有较高结合活性的叶绿体。叶绿体经分离纯化后,低温保存时的结合活性较稳定,-20℃以下可以较长期保存。用EDTA预处理叶绿体,不降低CTK结合蛋白对6 BA的结合活性,而用高浓度的NaCl处理,可以使叶绿体结合6BA的能力明显下降。这说明EDTA不能使CTK结合蛋白从叶绿体膜系统表面解离,而高浓度NaCl则有这种可能性。     

PROTEIN SYNTHESIS IN ISOLATED NUCLEI FROM ADULT RAT BRAIN

Nuclei from adult rat brains isolated with isotonic sucrose were incubated with [³H]leucine and later purified by centrifugation through hypertonic sucrose solutions. It was found that under these conditions, tritiated leucine was incorporated into TCA precipitable material. Protein synthesis was impaired if the nuclei were treated with the nonionic detergent Triton X-100 or hypertonic sucrose. The presence of puromycin or cycloheximide markedly inhibited the incorporation of the radioactive amino acid. Actinomycin D and RNase did not have any effect on the incorporation. Autoradiography indicated the presence of labelled material within the nuclei and not in cytoplasmic contaminants. Glial nuclei were more actively involved in protein synthesis than neuronal nuclei.     

IMMUNOCHEMICAL STUDY ON THE SPECIES SPECIFICITY OF SPERM-BINDING PROTEIN FROM THE SURFACE OF THE SEA URCHIN EGG*

Immunological species specificity of sperm-binding protein from eggs of the 4 sea urchin species, Hemicentrotus pulcherrimus, Pseudocentrotus depressus, Anthocidaris crassispina and Temnopleurus toreumaticus, was examined by means of double immuno-diffusion technique in agar. Ca-soluble fraction of sperm-binding protein which is considered to be responsible for initial sperm-egg bonding at fertilization, has species-specific antigenic component. Correlations in antigenic constituents among the 4 species are described.     

灵芝—共生真菌的发酵液化学成分研究

从灵芝一共生真菌(Calcarisporium arbuscula Preuss)的发酵液中分离得到7个化合物,经波谱分析确定其结构分别为zythiostromic acid A(1),zythiostromolide(2),7,8-二甲基苯并[g]蝶啶-2,4(1H,3H)-二酮(7,8-dimethylbenzo[g]pteridine-2,4(1H,3H)-dione,3),麦角甾醇(ergosterol,4),琥珀酸(butane-dioic acid,5),狼毒甲素(5,5'-[oxybis(methylene)]bis-2-furancarboxaldehyde,6),5-羟甲基糠醛(5-hydrox-ylmethyl-2-furancarboxaldehyde,7).1,2,3首次从齿梗孢属真菌中分离得到.     

STUDIES OF NATIVE GLYCOGEN ISOLATED FROM SYNCHRONIZED TETRAHYMENA PYRIFORMIS (HSM)

Native glycogen was isolated from Tetrahymena pyriformis (HSM) by isopycnic centrifugation in cesium chloride density gradients. A density of 1.62 to 1.65 was isopycnic for glycogen. Most of the banded glycogen existed as 35 to 40 mµ particles which had a sedimentation coefficient of 214. These particles were composed of aggregates of 2 to 3 mµ spherical particles. Extraction of glycogen with hot alkali reduced the sedimentation coefficient of native glycogen from 214 to 64.7 and the particle diameter from approximately 40 to 20 mµ and smaller. Cell division was synchronized by a repetitive 12-hour temperature cycle, and glycogen was measured at several times during the cell cycle. The temperature cycle consisted of 9.5 hours at 12°C and 2.5 hours at 27°C. Approximately 90 per cent of the cells divided during the last 1.5 hours of the warm period. The carbohydrate/protein ratio of cells at the end of the cold period was 0.27 and was reduced slightly during the warm period. Glucose was incorporated into glycogen during both periods, although the rate of incorporation was greater during the warm period. No preferential incorporation on the basis of particle size was noted. Incorporation was measured in both native glycogen and KOH-extracted glycogen. Tetrahymena glycogen is compared with rat liver glycogen previously isolated by similar procedures, and the significance of using combined rate-zonal and isopycnic centrifugation for isolating native glycogen is discussed.