PRODUCTION OF 1-METHYLADENINE INDUCED BY CONCANAVALIN A IN STARFISH FOLLICLE CELLS

Concanavalin A (Con A) was found to induce maturation of oocytes with follicular envelopes in the starfish, Asterina pectinifera . Treating a Con A sample with 85% ethanol and heat revealed that the maturation-inducing activity of the sample was not due to possible contamination with 1-methyladenine, but to Con A itself. However, Con A had little maturation inducing effect on isolated oocytes from which the follicular envelope had been removed, suggesting that its effect is indirect and probably mediated by the follicle cells. When follicle cells were incubated in seawater containing Con A, a maturation-inducing substance was found to have been produced in the incubation medium. This was purified and identified as 1-methyladenine. Therefore it is concluded that Con A has the same capacity as GSS, a gonad-stimulating peptide hormone of neural origin, to induce production of the maturation-inducing substance. Other plant lectins such as phytohemagglutinin P and wheat germ agglutinin had little effect in inducing production of 1-methyladenine in follicle cells.     

EFFECTS OF METHIONINE AND S-ADENOSYLMETHIONINE ON PRODUCTION OF 1-METHYLADENINE IN STARFISH FOLLICLE CELLS

Incubation of follicle cells of the starfish ( Asterias amurensis ) with a gonad-stimulating hormonal peptide (GSS) leads to the production of 1-methyladenine. the trigger of oocyte maturation. Addition of L-methionine to the incubation medium promotes the production of 1-methyladenine. This study shows that S -adenosylmethionine also enhances the production of 1-methyladenine in such an incubation mixture. However, in the absence of GSS, addition of S -adenosylmethionine failed to produce an appreciable amount of 1-methyladenine. When an homogenate of isolated follicle cells was incubated, a certain amount of 1-methyladenine was produced, whether or not GSS was present. Addition of S -adenosylmethionine to the incubation mixture of follicle homogenate enhanced the production of 1-methyladenine. Although addition of adenosine triphosphate (ATP) to such an incubation mixture had little effect in producing 1-methyladenine, it exerted a promoting effect on 1-methyladenine production when S -adenosyl-methionine was present. These results suggest that methionine, through its active form, S -adenosylmethionine, acts as a donor of methyl group in the formation of 1-methyladenine.     

BINDING OF CONCANAVALIN A TO THE SURFACE OF STARFISH FOLLICLE CELLS AND PRODUCTION OF 1-METHYLADENINE

Starfish follicle cells, treated with concanavalin A (Con A), continued to produce 1-methyl-adenine (1-MeAde), an inducer of starfish oocyte maturation, after rinsing with artificial seawater (ASW). On the other hand, they ceased to produce the substance if treated with methyl α-Dmannoside (αMM). These cells produced again 1-MeAde when re-stimulated with Con A after removal of αMM. An optical study with fluorescein revealed that Con A bound to the cells was not dissociated by rinsing with ASW, but was removed if the cells were treated with αMM. These results suggest that continuous binding of Con A to the surface of the follicle cells is essential for the production of 1-MeAde.     

重组人卵泡刺激素在昆虫细胞中的表达

为了研究在昆虫细胞中表达重组人卵泡刺激素,我们以人胎盘组织提取的染色体DNA为模板,利用重叠PCR方法扩增出hFSHβ亚基的cDNA的编码区。将此cDNA克隆入核型多角体病毒(AcNPV)非融合蛋白基因表达载体pVL1393,我们得到了表达载体pVL1393-hFSHβ,然后与BaculoGold~(TM)线性杆状病毒DNA共转染昆虫细胞SF9,经多次扩增后获得高滴度的重组病毒AcNPV-hFSHβ。将此重组病毒感染昆虫细胞,我们得到了在胞浆中表达的hFSHβ亚基,Western blot显示分子量大约为21kDa。以重组病毒AcNPV-hFSHβ与AcNPV-hCGα一同感染昆虫细胞得到了具有分泌性的重组hFSH异二聚体,在非还原的条件下Western blot显示分子量大约为33kDa。     

HISTOCHEMICAL LOCALIZATION OF ENZYME ACTIVITIES IN ROOT MERISTEM CELLS

Avers , Charlotte J. (Douglass Coll., Rutgers—The State University, New Brunswick, N. J.) Histochemical localization of enzyme activities in root meristem cells. Amer. Jour. Bot. 48(2): 137–143. Illus. 1961,—Particle counts were made in epidermal cells of the root meristem of 2 grasses after exposure of living seedlings to various substrates involved in dehydrogenation reactions. Hydrolytic enzyme activities also were recorded for 1 of these species. The mean number of particles stained with Janus green B was about 90 for each species, but significantly lower counts were obtained in all the dehydrogenase tests. With lactate, pyruvate, glutamate, citrate, and isocitrate as substrates, Phleum cells showed about 25% of the Janus green count, while Panicum cells were about 33% active. These substrates are known to be oxidized by DPN-linked enzymes. The succinic dehydrogenase counts were about 50% of the Janus green total, and 60–70% particle activity was recorded with hexose-phosphate substrates which are probably oxidized in TPN-mediated reactions. In Phleum, particulate activity occurred in the adenosine triphosphatase and aryl sulfatase tests, but a non-particulate distribution characterized 5-nucleotidase activity. The particle counts in the ATPase tests were not significantly different from the Janus green counts, but the 85% particle activity in the aryl sulfatase tests was significantly lower than the Janus green results. These intracellular distributions were compared with those obtained by various authors using biochemical and histochemical techniques and were found to be in close agreement. It was suggested that the evidence indicated intracellular differentiation of at least one kind of cellular organelle, which in all probability was mitochondria.     

MECHANICAL PROPERTIES OF STARFISH OOCYTES*

The stiffness of the starfish oocyte was determined from the degree of deformation when it was compressed by a definite force between a pair of parallel plates. The deformation of the oocyte increases during continued application of a constant force, indicating visco-elasticity of the cell. A cyclic change in stiffness of the oocyte accompanying meiotic divisions was found: the stiffness of the oocyte decreases during early stage of meiotic division, increases before the onset of the first polar body formation, then decreases, increases again before the onset of the second polar body formation, and decreases thereafter. Deuteration causes increase in stiffness of the oocyte.     

MESENCHYME CELLS IN STARFISH DEVELOPMENT: EFFECT OF TUNICAMYCIN ON THEIR DIFFERENTIATION, MIGRATION AND FUNCTION

The effect of tunicamycin, an inhibitor of protein glycosylation, on starfish development was investigated. Specific developmental events such as 1) bulging of the archenteron tip, 2) migration of mesenchyme cells, 3) formation of coelomic pouches and 4) mouth formation, are inhibited in the presence of this drug. These events are discussed in connection with differentiation, migration and function of mesenchyme cells. The possibility is discussed that tunicamycin exerts its effect by interfering with de novo synthesis of a cell surface factor(s) supporting dynamic cell surface activities.     

大鳞副泥鳅肠道黏液细胞及消化酶活性分布特征研究

采用阿利新兰-碘酸雪夫氏反应(AB-PAS)染色法及酶学方法研究了大鳞副泥鳅成熟个体肠道各段黏液细胞分布及消化酶活性。结果表明, 大鳞副泥鳅肠道黏液细胞分为Ⅰ、Ⅱ、Ⅲ和Ⅳ 4种类型。前肠至后肠, 黏液细胞数量逐渐减少。前肠主要分布Ⅲ和Ⅳ混合型黏液细胞, 后肠则以Ⅱ和Ⅳ型酸性黏液细胞为主。肠道胰蛋白酶活性显著高于淀粉酶和脂肪酶。且后肠消化酶活性显著低于前肠和中肠。根据黏液细胞及消化酶活性分布特点, 表明大鳞副泥鳅属于杂食性鱼类, 前肠为其主要的消化吸收场所, 后肠中性黏液细胞的数量较少以及消化酶活性较低, 表明其对食物的消化吸收功能较弱, 与其为辅助呼吸功能的特点相关。     

大鼠脊髓不完全性损伤后前角运动神经元的酶细胞化学改变

以改良Ａｌｅｎ氏法造成Ｗｉｓｔａｒ大鼠不完全性脊髓损伤,采用神经学功能评分法评定大鼠运动功能,应用定量酶细胞化学方法观察脊髓前角运动神经元内乙酰胆碱酯酶（ＡＣｈＥ）和酸性磷酸酶（ＡｃＰ）活性变化。结果显示：１．脊髓损伤后大鼠运动功能障碍,随后逐渐恢复。２．前角运动神经元内ＡＣｈＥ活性减弱、ＡｃＰ活性增强;随后酶活性呈逐渐恢复,四周时ＡＣｈＥ活性基本恢复正常。结果说明：大鼠脊髓不完全性损伤后运动功能变化与前角运动神经元的功能状态具有较强的相关性;前角运动神经元在不完全性脊髓损伤运动功能恢复中起重要作用。     

INHIBITION OF GERMINAL VESICLE BREAKDOWN AND POLAR BODY FORMATION BY NICOTINAMIDE IN STARFISH AND SURF CLAM OOCYTES*

Nicotinamide inhibited both germinal vesicle breakdown (GVBD) and polar body formation (PBF) in surf clam and starfish oocytes. In the surf clam nicotinamide at 0.3 mM completely blocked PBF in the fertilized oocytes. For blockage of GVBD higher concentration was required. In the starfish, nicotinamide (30 mM) prevented PBF but not GVBD, when added 7 min after the commencement of 1-methyladenine (1-MeAde) administration. These results suggest that PBF is blocked by nicotinamide independent of its effect on GVBD. In the case of starfish, NAD⁺was more effective than nicotinamide in inhibiting oocyte maturation. Nicotinamide also blocked GVBD induced by microinjection of the cytoplasm containing maturation-promoting factor (MPF) obtained from 1-MeAde-treatcd oocytes. These results suggest that nicotinamide prevents the action of MPF rather than inhibiting the interaction of 1-McAde with cell membrane or the induction of MPF.     

EFFECT OF LOCAL APPLICATION OF 1-METHYLADENINE ON THE SITE OF POLAR BODY FORMATION IN STARFISH OOCYTE*

Mechanism by which the site of polar body formation is determined in starfish oocytes was investigated in relation to the action of 1-methyladenine (1-MeAde). Local staining with Nile Blue of Asterina pectinifera oocytes revealed that there exists a prospective site of polar body formation (PSPBF) on the nearest surface to the position of germinal vesicle. The site of polar body formation was found to shift to some extent from PSPBF toward the area locally applied with 1-MeAde, suggesting that the actual site of polar body formation is not determined yet at the germinal vesicle stage. Oocytes whose germinal vesicles had been shifted by centrifugation from PSPBF to the opposite surface before the commencement of germinal vesicle breakdown (GVBD) (less than 15 min after 1-MeAde treatment), failed to form polar bodies, whereas oocytes centrifuged after commencement of GVBD (20 min after 1-MeAde treatment) did form polar bodies where their fading germinal vesicles had reached by centrifugation. In the oocytes which failed to form polar bodies by centrifugation, an aster was observed near PSPBF of each oocyte. When inseminated, every oocyte treated with 1-MeAde developed normally irrespectively of the mode of polar body formation including the site and the occurrence, and the animal pole of every larva was derived from PSPBF.     

A SIMPLE CASE OF SALT ANTAGONISM IN STARFISH EGGS

The jelly surrounding the eggs of the starfish, Asterias forbesi, is insoluble in normal sea water, but rapidly swells and dissolves when the eggs are washed in a pure isotonic solution of NaCl. In the presence of a small proportion of CaCl₂ this solvent and disintegrative action of the NaCl solution is entirely prevented, and in the mixed solution the jelly exhibits the same insolubility and other properties as in normal sea water. 2. This action of CaCl₂ in preventing the dissolution of the jelly runs parallel with its action in preventing certain definite effects of the pure NaCl solution on the living egg (agglutination, cytolytic action, membrane formation, prevention of maturation). 3. The inference is that the essential factor in these and similar antagonistic and protective actions is the formation of solid water-insoluble colloidal salts (e.g., soaps and proteinates) of calcium (or other metal) with the structural colloids of the protoplasm. Apparently the presence of a certain proportion of such compounds is necessary to the structural stability of the living protoplasm, and especially to the water-insolubility and semipermeability of its external layer or plasma membrane. When the cell is immersed in the pure NaCl solution, water-soluble Na compounds are substituted for the insoluble Ca compounds which normally provide the necessary insolubility and coherence, and disintegration results.     

MECHANICAL PROPERTIES OF THE CELL SURFACE IN STARFISH EGGS

The mechanical properties of the cell surface of the starfish egg at various stages of maturation have been investigated using the cell elastimeter. When constant negative pressure was applied to a part of the cell with a micropipette closely in contact with it, it bulged out, and the bulge rapidly increased at first and then gradually reached a steady value within one min. The relation between the deformation of the cell surface (i.e. degree of bulging) and applied negative pressure was almost linear in both oocytes at the germinal vesicle stage and mature eggs. The surface force and the elasticity value: i.e., the product of the elastic modulus of the surface membrane (layer) and its thickness, were determined from the relation between the deformation and the negative pressure. The elasticity value was about 5 times the surface force in both oocytes at the germinal vesicle stage and mature eggs. When maturation of the oocyte was induced by 1-methyladenine, the stiffness of the cell surface decreased shortly before the breakdown of the germinal vesicle. The stiffness transiently increased at the time of formation of the first and second polar-bodies.     

A HISTOCHEMICAL ENZYME KINETIC SYSTEM APPLIED TO THE TRYPSIN-LIKE AMIDASE AND ESTERASE ACTIVITY IN HUMAN MAST CELLS

A method for the determination of enzyme kinetic constants V_m, K_m, and K_i in a histochemical system has been devised. As a substitute for the reciprocal of the reaction velocity, the times necessary to reach a fixed amount of end product (the initial visible color) in a tissue site at various substrate concentrations are plotted, according to the method of Lineweaver and Burk, against the reciprocal of the substrate concentrations. The technique as applied to trypsin-like esterase and amidase activities in human mast cells indicates that a single enzyme or closely related enzymes in this site are responsible for the hydrolysis of both the amide and ester substrates and that typical trypsin substrates act as competitive inhibitors of their hydrolysis. Parallel biochemical studies were performed to evaluate the effect of certain aspects of the experimental histochemical method on a purified homospecific enzyme. The relative kinetic constants derived by the histochemical method afford a further means of characterizing enzymic activity in a histochemical system.     

IN VITRO BEHAVIOR OF GUINEA PIG ARTERIAL AND VENOUS ENDOTHELIAL CELLS *

Techniques to clone endothelial cells from adult guinea pig artery and vein are described. Cloned arterial and venous endothelial cells differed in their in vitro behavior. The differences between these two types of endothelial cells grown under identical conditions are: (1) venous endothelial cells have a greator mitotic potential than the arterial cells, (2) arterial endothelial cells have a much greator affinity to the substratum than venous endothelial cells, and (3) arterial endothelial cells exhibit a marked propensity to deposit lipid in their cytoplasm; whereas, the venous endothelial cells do not reveal lipid droplets. Labeled ³H-oleic acid added to the nutrient medium was incorporated into the lipid deposits found in the arterial endothelial cells. Both types of endothelial cells bound antibody prepared against smooth muscle myosin. The observed difference in the in vitro behavior of these two types of endothelial cells may be a reflection of their intrinsic characteristics.