GLYCOGEN METABOLISM AND CHANGES IN THE ACTIVITIES OF PHOSPHORYLASE, PHOSPHOFRUCTOKINASE AND PYRUVATE KINASE DURING DEVELOPMENT OF SEA URCHIN EGGS

The glucose and glycogen contents of sea urchin eggs and embryos were measured enzymatically. Unfertilized eggs of Hemicentrotus pulcherrimus and Anthocidaris crassispina contain about 20.9 and 24.4 μg of glycogen per mg protein, respectively. As for glucose, unfertilized eggs of Hemicentrotus and Anthocidaris contain about 0.7 and 1.9 μg per mg protein, respectively. Glycogen consumption during embryonic development differs with different species of sea urchins. In Anthocidaris , glycogen decreases significantly after fertilization. The oxidation of glucose and glycogen accounts for about 50% of oxygen consumed until the early blastula stage in this species. The contribution ratio of glucose and glycogen to the overall energy pool becomes less than 10% at later stages. In Hemicentrotus , however, the glycogen content remains unchanged until the early blastula stage and thereafter decreases. The importance of glucose and glycogen as an energy fuel seems little throughout the development of Hemicentrotus. Activities of phosphorylase (EC 2.4.1.1), phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40) were measured at various embryonic stages in both species of sea urchins. The difference between two species in the consumption of glucose and glycogen can not be elucidated by the differences in the activities of these enzymes.     

CHANGES DURING DEVELOPMENT OF MOUSE BRAIN IN THE ACTIVITIES AND SUBCELLULAR DISTRIBUTIONS OF CREATINE AND ADENYLATE KINASES

Abstract— The activities and electrophoretic patterns of creatine (CK) and adenylate (AK) phosphokinases were determined in the mitochondrial and high speed supernatant fractions of developing and adult mouse brain. The CK activity of the cytosol increases three-fold between 9 and 19 days of age and four-fold by 29 days. Mitochondrial CK activity increases from birth to reach a maximum four-fold that at birth by 19 days of age. The developmental changes in soluble AK activity parallels that of CK. Although the increase in CK activity of the mitochondria exceeds that of the mitochondrial proteins during the period of rapid brain development, mitochondrial AK specific activity changes little. No major changes were found during development in the electrophoretic patterns of proteins with CK or AK activity in the supernatant and mitochondrial fractions. The changes in mitochondrial CK and AK activity parallel the change from a predominantly glycolytic to an aerobic mode of brain metabolism and support the concept that these enzymes play an important role in mitochondrial energy metabolism. Soluble CK and AK activity increase concomitant with neuronal maturation and the associated greatly increased need for rapidly available high energy phosphate.     

人肌肌酸激酶在SDS溶液中失活与构象变化的比较研究

应用紫外差吸收光谱、荧光发射光谱、ＣＤ先谱等监测手段,研究了ＳＤＳ溶液滴定人肌肌酸激酶时的构象与活力变化的关系。结果表明酶的活力丧失先于以紫外差吸收先谱、荧先发射谱和巯基暴露数目所监测到的构象变化。ＳＤＳ滴定时引起的酶的荧光发射光谱的变化在低滴定度阶段随着ＳＤＳ滴定量的增加,荧光强度下降,发射峰位红移,当ＳＤＳ浓度达到２．１ｍｍｏｌ／Ｌ时,荧光强度增大,继续增加ＳＤＳ滴定量,荧光强度又降低,发射峰位红移直至终态。紫外差吸收光谱随着ＳＤＳ溶液的加入,２８１ｎｍ．２８７ｎｍ和２９２ｎｍ的负差吸收峰增大。ＣＤ光谱结果表明在本实验所用的ＳＤＳ浓度范围内,ＳＤＳ对人肌肌酸激酶的二级结构几乎没有影响。上述结果支持了酶的活性部位构象柔性的观点。     

人肌肌酸激酶胍变性时的失活与构象变化的比较研究

应用二阶导数光谱、紫外差吸收光谱和荧光光谱等监测手段,研究了人肌肌酸激酶在盐酸胍溶液中的构象变化。二阶导数光谱结果表明,若以6M盐酸胍中肌酸激酶酪氨酸残基的暴露程度为100％,则天然酶酪氨酸残基的暴露程度只有2％。而紫外差吸收光谱和荧光光谱的变化与兔肌肌酸激酶的结果相似。比较不同胍浓度下人肌肌酸激酶的失活与构象变化,表明酶的失活先于构象变化。同时还测定了不同浓度胍溶液中人肌酶的失活与构象变化的速度常数。结果表明以几种方法测定的构象变化均为单相的一级过程,而酶的失活却呈现了由快慢两相组成的一级反应过程。比较同浓度胍溶液中的失活速度与构象变化速度,发现酶失活的快相反应速度常数比构象变化的速度常数大1—2个数量级,慢相速度常数与构象变化速度常数相近。上述结果进一步支持了酶的活性部位构象柔性的观点。     

HETEROCHRONIC DEVELOPMENTAL PLASTICITY IN LARVAL SEA URCHINS AND ITS IMPLICATIONS FOR EVOLUTION OF NONFEEDING LARVAE

Preexisting developmental plasticity in feeding larvae may contribute to the evolutionary transition from development with a feeding larva to nonfeeding larval development. Differences in timing of development of larval and juvenile structures (heterochronic shifts) and differences in the size of the larval body (shifts in allocation) were produced in sea urchin larvae exposed to different amounts of food in the laboratory and in the field. The changes in larval form in response to food appear to be adaptive, with increased allocation of growth to the larval apparatus for catching food when food is scarce and earlier allocation to juvenile structures when food is abundant. This phenotypic plasticity among full siblings is similar in direction to the heterochronic evolutionary changes in species that have greater nutrient reserves within the ova and do not depend on particulate planktonic food. This similarity suggests that developmental plasticity that is adaptive for feeding larvae also contributes to correlated and adaptive evolutionary changes in the transition to nonfeeding larval development. If endogenous food supplies have the same effect on morphogenesis as exogenous food supplies, then changes in genes that act during oogenesis to affect nutrient stores may be sufficient to produce correlated adaptive changes in larval development.     

CHANGES IN CALCIUM CONCENTRATIONS OF SERUM AND COELOMIC FLUID OF THE BULLFROG, RANA CATESBEIANA, DURING LARVAL DEVELOPMENT AND METAMORPHOSIS

The serum calcium levels of bullfrog tadpoles (stage 26 to 33) and adults are higher than those of the coelomic fluid. The serum levels increase gradually from stage 26 (7.6 mg/100 ml) to stage 30 (8.4 mg/100 ml), and then sharply to stage 33 (10.5 mg/100 ml), while the coelomic fluid levels increase from 7.1 to 8.7 mg/100 ml during this period. Only minor differences are found in serum and coelomic fluid sodium levels among larval stages with the exception of a temporary decrease during metamorphic climax.
These results suggest that the adult type of regulation of serum calcium concentrations is established during larval development and is fully achieved after the completion of metamorphosis. The control mechanism for serum calcium may be different from that for coelomic fluid.     

棉铃虫4龄幼虫的摄食行为和中肠蛋白酶活性的变化

通过测定棉铃虫4龄幼虫,每隔12 h的摄食量、体重净增量以及丝氨酸蛋白酶活性的变化,揭示了它的取食行为及中肠蛋白酶活性的变化规律。结果表明:棉铃虫4龄幼虫取食及消化呈有规律的变化,蜕皮后4龄初,摄食量、体重净增量、类胰蛋白酶、类胰凝乳蛋白酶和总蛋白酶活性均逐渐增加;进入中期(36～48 h),各项指标达到最高值;其后,随着摄食量的下降,各项指标逐渐下降。同时, 连续观察幼虫取食高峰期的取食行为6 h,结果表明: 其间一次连续取食的平均时间为(3.6±0.4) min/次, 总平均取食时间为(62.4±8.9) min,约占整个时间的1/6。该文还对棉铃虫4龄幼虫不同时段取食行为与蛋白酶活性变化之间的相关性及调控机制作了初步讨论。     

CHANGES IN NUCLEAR HISTONES DURING FERTILIZATION, AND EARLY EMBRYONIC DEVELOPMENT IN THE PULMONATE SNAIL, Helix aspersa

Calf thymus histories comprising two fractions, one rich in lysine, the other having roughly equal amounts of lysine and arginine, Loligo testes histones rich in arginine, and salmine, are compared with respect to their amino acid compositions, and their staining properties when the proteins are fixed on filter paper. The three types of basic proteins; somatic, arginine-rich spermatid histones, and protamine can be distinguished on the following basis. Somatic and testicular histones stain with fast green or bromphenol blue under the same conditions used for specific staining of histones in tissue preparations. The former histones lose most or all of their stainability after deamination or acetylation. Staining of the arginine-rich testicular histones remains relatively unaffected by this treatment. Protamines do not stain with fast green after treatment with hot trichloracetic acid, but are stained by bromphenol blue or eosin after treatment with picric acid. These methods provide a means for the characterization of nuclear basic proteins in situ. Their application to the early developmental stages of Helix aspersa show the following: After fertilization the protamine of the sperm is lost, and is replaced by faintly basic histones which differ from adult histones in their inability to bind fast green, and from protamines, by both their inability to bind eosin, and their weakly positive reaction with bromphenol blue. These "cleavage" histones are found in the male and female pronuclei, the early polar body chromosomes, and the nuclei of the cleaving egg and morula stages. During gastrulation, the histone complement reverts to a type as yet indistinguishable from that of adult somatic cells.     

胡子鲶的胚胎和幼鱼发育的研究

胡子鲶(Clarias fuscus)的胚胎和幼鱼的发育过程与蟾胡子鲶(C.batrachus)类似,器官分化时间和大小比例则有一定的差异。胡子鲶卵呈球形,富含卵黄,卵径1.7—1.9毫米;出膜仔鱼全长4.8—5.1毫米,比蟾胡子鲶约大1/3;幼鱼卵黄囊被吸收消失的时间比蟾胡子鲶迟1—2天。心脏的出现和搏动与开始出现血液循环的间隔时间只有3—5小时;耳石的出现几乎和心脏的出现处在同一时间内,与白鲢有较大的差异。在仔鱼前期居维氏管明显,但出现的时间比蟾胡子鲶稍迟些。仔鱼出膜形式与蟾胡子鲶不同,也与一般鱼类出膜形式不同。仔鱼是以腹部卵黄囊顶破卵膜,以卵黄囊先出膜,一般鱼类多数是以头部或尾部先出膜的。在水温28.5—31℃的条件下,从受精到孵化出膜的时间为28小时25分;幼鱼期共历时12—15天。       

三疣梭子蟹UHRF1基因在胚胎、幼体和性腺发育过程中的表达分析

为探讨泛素样含PHD和环指域蛋白1(UHRF1)基因在三疣梭子蟹(Portunus trituberculatus)发育过程中的作用, 实验采用SMART RACE方法, 克隆了三疣梭子蟹UHRF1(PtUHRF1)基因。该基因cDNA全长为2849 bp, 开放阅读框(ORF)为2298 bp, 预测其编码1个含有765个氨基酸的蛋白质。结构域分析显示, 该蛋白质包含UBL、PHD、TTD、SRA、RING finger 5个功能结构域。同源分析表明, 三疣梭子蟹PtUHRF1的氨基酸序列与其他物种有较高的同源性。qRT-PCR结果显示, PtUHRF1基因在三疣梭子蟹所有组织中均有表达, 但在精巢中表达量显著高于其他组织。该基因在胚胎和幼体发育不同时期表达差异显著, 在受精卵中的表达量最高, 并显著高于胚胎发育其他时期, 是多细胞时期表达量的2.5倍。PtUHRF1基因在性腺发育不同时期表达存在显著差异, 在卵巢II期表达量达到峰值, 之后逐渐下降; 在精巢Ⅰ期的表达量最高, 随着精巢发育逐渐下降。实验结果表明, PtUHRF1参与了三疣梭子蟹胚胎、幼体和性腺发育调控, 为进一步深入研究该基因在三疣梭子蟹及甲壳动物生长发育中的作用提供参考。     

MULTIPLE FORMS OF β -GALACTOSIDASE AND THE CHANGES IN ITS PATTERN DURING DEVELOPMENT OF DICTYOSTELIUM DISCOIDEUM

Four forms of β-galactosidase (EC 3.2.1.23) were separated from cell extracts of Dictyostelium discoideum by polyacrylamide gel electrophoresis. Changes in the pattern of multiple forms of this enzyme during differentiation and dedifferentiation were studied. The band closest to the anode (Band 1) existed throughout development, but the other three bands appeared and disappeared at certain stages. One of those bands was specific for the vegetative stage (Band 3), and another for the morphogenetic stages (Band 2). The last one predominantly appeared during dedifferentiation of disaggregated slug cells (Band 4), although it was slightly detected during culmination. During the process of dedifferentiation, the increase in activity of Band 4 coincided with the decrease in Band 2. After the completion of dedifferentiation, Band 4 also disappeared, only Band 1 remaining. These multiple forms were not only electrophoretically separable but different in their sensitivities to various inhibitors. All forms of the enzyme were localized in subcellular particles, probably in lysosomes and phagosomes, and the relative activities in these two fractions varied during development.     

真水狼蛛胚胎发育过程中形态和主要化学物质含量的变化

采用常规石蜡切片和液体石蜡透明卵壳的方法,系统地观察了真水狼蛛的胚胎早期,体节期,胚胎速转期和幼虫期4个发育阶段的形态特征和发育过程,并测定了胚胎发育过程中卵的内主要化学物质含量的变化,在28度时,真水狼蛛的卵从产卵到孵化共需144小时,其中胚胎早期42小时,体节期33小时,胚胎逆转期27小时,在胚胎逆转期后进入前幼虫期,前幼虫期42小时,真水狼蛛的胚胎逆转现象很明显,表明真水狼蛛的进化程度较高,在胚胎发育过程中,卵的含水量,含脂量和卵重在胎发育24hr后开始下降,卵的蛋白质含量在48小时后也开始下降,含糖量下降不明显。     

大鳞副泥鳅仔稚鱼生长与消化酶活性变化

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METABOLISM DURING EMBRYONIC AND METAMORPHIC DEVELOPMENT OF INSECTS

1. For species of insects that deposit their eggs on foliage, as Leptinotarsa decemlineata, Crioceris asparagi, and Anasa tristis, the rates of metabolism as measured by the CO₂ output or the oxygen intake indicate, during embryonic development, a short formative period, followed by a very active extended growth. Those species that deposit their eggs in the soil, as Cotinis nitida and Popillia japonica, show that the formative period is greatly extended and the growth period similarly lengthened. 2. It has been shown from metabolism experiments that intensive histolysis occurs during the prepupal period and becomes less intensive during pupal development. 3. Metabolism experiments show a greater amount of energy change during embryonic development as compared to the energy developed during metamorphosis. This is shown by the greater CO₂ output and by the oxygen intake. 4. Low respiratory quotients, varying from 0.42 to 0.71, have been obtained during the embryonic and pupal development of insects, resembling similar low quotients obtained with hibernating forms. 5. Changes from a neutral to an acid reaction (pH 6.8 to 5.9) have been observed to take place in some species during prepupal and early pupal development. As metamorphosis is completed a converse change occurs (pH 5.9 to 6.8). Changes in pH from an acid to neutral reaction were also observed to take place in some species, as Cotinis nitida and Hylemyia cilicrura, during embryonic development.     

家兔发热时脑脊液和血浆中精氨酸加压素含量的变化及禁水的抗热作用

本实验观察了发热家兔脑脊液(CSF)和血浆中精氨酸加压素(AVP)含量的变化及禁水对家兔内毒素(ET)发热效应的影响。实验结果表明:1.隔区注射AVP可明显抑制家兔ET性发热效应;2.发热组家兔CSF和血浆中AVP含量较正常组明显降低;3.禁水可明显对抗家兔ET性发热效应,其抗热作用与CSF和血浆中AVP含量升高有关;4.禁水也可使正常家兔体温水平下移。上述实验结果提示,AVP可能是家兔体内一种内源性退热物质,同时在正常体温调节中也可能发挥一定的作用。     

RELATIONS BETWEEN THE FEEDING AND RADIO-SENSITIVITY CYCLES AND THE ECDYSONE CYCLE IN BOMBYX MORI, AT THE END OF EMBRYONIC DEVELOPMENT AND DURING THE FIRST FOUR LARVAL INSTARS

The cycle of ecdysone secretion in Bombyx mori was determined at the end of embryonic development and during the first four larval instars by means of radio-immunological titration (RIA). There is a relationship between this cycle and the radio-sensitivity cycle of this insect. This radio-sensitivity (tested through the percentage of abnormal ecdysis and of permanent larvae) is minimum when the hormone rate is important during X-irradiation; the effect is maximum when the hormonal rate is minimum. The fall of the ecdysone rate during the first food-intakes entails a reverse radio-sensitivity.     

CHANGES IN GLUCOSE OXIDATION DURING GROWTH OF EMBRYONIC HEART CELLS IN CULTURE

A rapid and convenient method has been utilized to investigate glucose oxidation during growth of chick embryo heart cells in tissue culture. Primary isolates of chick embryo heart cells showed exponential growth when plated at low densities and exhibited density-inhibited growth as cultures became confluent. The density-dependent growth inhibition of chick embryo heart cells is associated with a marked decrease in the specific activity of glucose oxidation to CO₂. This decrease in glucose oxidation was observed as density increased as either a function of time in culture or as related to initial plating density. The decrease in ¹⁴CO₂ production associated with density-dependent inhibition of growth is due to a marked decrease in activity of the pentose phosphate pathway.     

CHANGES IN ACTIVITIES OF THYMIDYLATE SYNTHASE AND DIHYDROFOLATE REDUCTASE IN SEA URCHIN EGGS AFTER FERTILIZATION

Thymidylate synthase activity in sea urchin eggs increases just after fertilization and decreases 30 min later. Then, cyclic variation in the activity occurs in association with the cleavage cycle. Dihydrofolate reductase activity in fertilized eggs is almost the same as in unfertilized eggs and shows no marked change within 3 hr after fertilization. Aminopterin, an analogue of dihydrofolate, inhibits dihydrofolate reductase, and arrests cleavage. On incubation in sea water containing aminopterin (20-100μM) from the time of fertilization, the development of Clypeaster and Pseudocentrotus eggs was arrested at the 32–64 cell stage, and that of Anthocidaris eggs was arrested at the morula stage. Dihydrofolate (100μM) counteracts the inhibitory effect of aminopterin on egg cleavage. Thymidine at concentrations above 10μM also prevents inhibition by aminopterin. Other deoxyribonucleosides at concentrations of 10μM to 100μM do not affect inhibition of egg cleavage by aminopterin. Deoxyadenosine at concentrations above 5 mM inhibits egg cleavage, but other deoxyribonucleosides have no effect.