Polyribosomes and protein synthesis in after-ripening pear seeds

Polyribosomes have been isolated from pear seeds which show a capacity for in vitro protein synthesis. Translational capacity increased during stratification with a peak after 13 days of stratification. This increase was greatly diminished in seeds which were incubated at 25°C (“warm stratification”). The peak in protein synthetic activity coincides with the period of stratification required for normal development of excised embryos.     

Rates of ribosomal protein and total protein synthesis during Drosophila early embryogenesis

Embryos at various stages of early development from 1.5 to 5 hr after oviposition were made permeable with octane and labeled for 1 hr with [³H]phenylalanine. Measurements of the rate of incorporation of [³H]phenylalanine into ribosomal proteins and total protein were made using these synchronized Drosophila embryos. The rate of synthesis of those ribosomal proteins incorporated into ribosomes increases until 3 to 4 hr after fertilization (550 pg/embryo-hr) then declines later in embryonic development. The rate of total protein synthesis is maximal as early during embryonic development as could be measured. During the period between 1.5 and 2.5 hr after fertilization this rate is 9.4 ng/embryo-hr and then also declines. The synthesis of ribosomal proteins accounts for a substantial portion (4.5%–8.9%) of total protein synthesis in early embryos. These results indicate that ribosome formation is a significant activity during the earliest stages of Drosophila development.     

Control of protein synthesis during blastocyst formation in the mouse.

In order to evaluate the dependence of the embryo on new mRNA synthesis during the period leading to blastulation, quantitative and qualitative aspects of protein synthesis in developing mouse morulae were investigated using α-amanitin, an inhibitor of RNA polymerase II. Only 1 of 423 early morulae cultured for 27 hr in the presence of 11 μg/ml α-amanitin cavitated, although most progressed as far as fully compacted morulae. About two-thirds of the untreated embryos cavitated during the same period. Incorporation of [³⁵S]methionine into protein was measured at 3- or 4-hr intervals over a 24-hr period and showed a two- to fivefold increase in control embryos. This increase was blocked in the α-amanitin-treated group although initial levels of incorporation were maintained. Total uptake of the amino acid appeared to be unaffected by the inhibitor. RNA synthesis, as measured by [³H]uridine incorporation over the same period, was reduced by between 5 and 52%, and the preblastulation surge in RNA synthesis was also blocked by α-amanitin. Two-dimensional polyacrylamide gel electrophoresis of labeled polypeptides synthesized by the embryos after 24-hr incubation in the presence or absence of the inhibitor revealed three distinct classes of polypeptide. The majority of polypeptides continued to be synthesized in the presence of α-amanitin whereas a small number of polypeptides, the synthesis of which would normally have increased during the development of the morula to the blastocyst, were prevented from doing so. A few polypeptides which normally cease to be synthesized over this period continued to be synthesized in the presence of α-amanitin. It is concluded that, while most of the proteins detectable at the morula stage are synthesized on mRNA templates of relatively long translational life, the general surge in protein synthesis, including the increased synthesis of a few species of polypeptide, are dependent on continuous translational activity.     

tRNA methyltransferases during embryogenesis in Musca domestica

Activity and diversity of the tRNA methyltransferases were examined during embryogenesis of the housefly, Musca domestica. A rapid rise in the activity of the tRNA methyltransferases was observed during the first 3 hr of embryogenesis. Activity increased slowly until the tenth hour of embryogenesis and then declined until hatching at 12 hr. The greatest diversity of tRNA methyltransferases, as indicated by extent of methylation, existed at 6 hr of embryogenesis; the least diversity was observed in 1-hr embryos, while 12-hr embryos showed intermediate levels. Inhibition of embryonic tRNA methyltransferases at high concentrations of enzyme was observed in all extracts examined.     

In vitro protein synthesis during germination and vernalization in winter wheat embryos

Protein synthesis during germination at 24?C and vernalizationat 4?C in winter wheat embryos were investigated with a cell-freesystem. During germination, the capacity for protein synthesisincreased in the early stage between 12 and 36 hr of imbibitionthen declined to a final low level between 48 and 72 hr. Thistransition was due to quantitative changes of the activitiesof ribosomal and supernatant fractions in the early stage andmainly to those of the supernatant fraction in the later stage.During vernalization, the capacity for protein synthesis continuedto decline over 15 to 60 days at 4?C. This transition was dueto the change in activity of the supernatant fraction; the activityof the ribosomal fraction was nearly constant. Electrophoretic analysis of in vitro products indicated thatthe high molecular weight proteins present in 12-hr embryoshad disappeared in 48-hr germinated wheat embryos and that theproducts in 24- and 36-hr embryos were types intermediate betweenthose of 12- and 48-hr embryos. The products in each vernalizedembryo resembled those in 24- and 36-hr germinated embryos.Therefore, it was concluded that the mRNA species for translationchanged during germination and vernalization in winter wheatembryos. (Received January 20, 1977; )     

Purine metabolism in germinating wheat embryos

1. Both the acid-soluble fraction and the nucleic acid fraction of wheat embryos were extensively labelled after incubation for 6hr. in the presence of [8-(14)C]adenine. Subsequent incubation in the absence of labelled adenine resulted in no loss of radioactivity to the medium during a 48hr. period. Radioautography indicated that during this period there was a continuous increase in the radioactivity present in the acid-insoluble fractions of the root and leaf tissues relative to that present in the coleorhiza and coleoptile. 2. During incubation at 25 degrees there was a 26-fold increase in the activity of 3'-nucleotidase between 4hr. and 24hr.; the activities of enzymes hydrolysing AMP and IMP increased to a smaller extent. The activities of adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase increased three- to five-fold during incubation at 25 degrees for 24hr. 3. Adenosine kinase, inosine phosphorylase and 5-phosphoribosyl pyrophosphate synthetase activities were high in extracts from dry embryos and did not increase during 48hr. at 25 degrees . 4. The increase in 3'-nucleotidase activity was prevented by cycloheximide, cryptopleurine or incubation at 4 degrees , but not by actinomycin D; these treatments did not depress the activity of the other enzymes measured. 5. The results are discussed in relation to RNA translocation within the wheat embryo during germination.     

Size and specific activity of the UTP pool and overall rates of RNA synthesis in early mouse embryos

Mouse embryos from the one-cell to the blastocyst stage were cultured for 2 hr in the presence of 5 μM [³H]uridine or 10 μM [³H]adenosine, and the size and specific activity of the UTP and ATP pools were determined by an Escherichia coli RNA polymerase assay using synthetic poly(dA-dT) as template. The total UTP pool increased in size and specific activity with development from 0.05 pmole (0.06% labeled) in the one-cell stage to 0.54 pmole (27% labeled) in the blastocyst stage. The total ATP pool remained relatively constant in size at about 1 pmole/embryo, but increased in specific activity from 2.6 to 52% from one-cell to blastocyst. The turnover of the [³H]UTP pool was also examined under pulse-chase conditions in eight-cell and morula-stage embryos. The UTP pool decayed with approximately first-order kinetics up to 20 hr of chase, but the rate of decay was slower in eight-cell embryos (t_0.5 = 5.5 hr) than in morulae (t_0.5 = 2.8 hr). The observed specific activities of the UTP pools were used to calculate the overall rates of uridine incorporation into acid-precipitable material during early development. The rate of uridine incorporation per embryo increased from 3.6 × 10^?3 pmole/2 hr in the two-cell embryo to 1.8 × 10^?1 pmole/2 hr in the blastocyst. The rate of RNA synthesis per cell over a 2-hr period was estimated at 2.5 pg in the two- to four-cell embryo, 5 pg in the eight-cell, and 10 pg in the morula-early blastocyst.     

Augmentation of mitochondrial oxidative metabolism in lung tissue during recovery of animals from acute ozone exposure

After O₃-mediated lung injury in rats (3 ppm O₃ exposure for 4 hr) recovery was studied in terms of alteration in lung mitochondrial oxidative metabolism. As judged from O₂ consumption, succinate oxidation in lung homogenate exhibited a 20% (P < 0.05) decrease at 0 hr but attained the control rate (0.6 μmole O₂/min/lung) within 12 hr and the peak rate (55% over control, P < 0.001) within 48 hr of recovery. Thereafter, the rate plateaued and at about the fifth day began to decline, exhibiting only a 15% (P < 0.05) increase over control after 21 days. The half-life for duration of this augmentation appeared to be 10 days. During recovery, the yield of isolated mitochondria was increasingly greater for exposed lungs relative to control (viz., 25–30% increase after 96 hr) as viewed from mitochondrial packed volume and protein content. Mitochondria from exposed lungs exhibited a 17–24% (P < 0.05) increase in activity (per mg of protein) for oxidation of 2-oxoglutarate, succinate, glycerol-1-phosphate, and ascorbate-Wurster's blue. The over-all augmentation of O₂ consumption observed in exposed rat lungs, therefore, would be attributable primarily to increase in population of mitochondria. Enhanced mitochondrial metabolism might serve as an index for assessing the repair process of injured lung.     

Effects of fasting and diabetes on some enzymes and transport of glutamate in cortex slices or synaptosomes from rat brain

Phosphate-activated glutaminase (PAG) and glutamic acid decarboxylase (GAD) were assayed in homogenates and synaptosomes obtained from starved (48 hr or 120 hr) and diabetic (streptozotocin) rat brain cortex. Glutamine synthetase (GS) was assayed in homogenates, microsomal and soluble fractions, from brain cortex of similarly treated rats.l-Glutamate uptake and exit rates were determined in cortex slices and synaptosomes under the same conditions. The specific activity (s.a.) of PAG, a glutamate producing enzyme, decreased (50%) in the homogenate after 120-hr starvation. In synaptosomes it decreased (25%) only after 48-hr starvation. The s.a of GAD and GS, which are glutamate-consuming enzymes, were progressively increased with time of starvation, reaching 39% and 55% respectively after 120 hr. GS in the microsomes or the soluble fraction and GAD in the synaptosomes showed no change in s.a. under these conditions. Diabetes increased (40%) microsomal GS s.a. and decreased GAD s.a. (18%) in the homogenate. Thel-glutamate uptake rate was decreased (48%) by diabetes in slices but not in synaptosomes. It is suggested that a) enzymes of the glutamate system respond differently in different subcellular fractions towards diabetes or deprivation of food and b) diabetes may affect the uptake system in glial cells but not in neurons.Abbreviations used AET 2-aminoethylisourethonium bromide - GAD glutamic acid decarboxylase - GS glutamine synthetase - GSH glutathione - PAG phosphate-activated glutaminase - PLP pyridoxal phosphate - r.c.f. relative centrifugal force - s.a. specific activity     

Platelet activating factor (PAF) production by mouse embryos in vitro and its effect on embryonic metabolism

Factors affecting the production of platelet activating factor (PAF) by mouse embryos during culture in vitro were investigated. Detectable levels of embryo-derived PAF were produced within 1-4 hr with maximum PAF activity being observed after 6 hr of culture in vitro. The amount of PAF detected in media after 24 hr of culture of two-cell embryos was equivalent to 12.8 ng PAF/embryo. However, differences in activity were apparent with increased time in culture. Reduced synthesis of PAF during culture in vitro was supported by the observation that morulae stage embryos collected fresh from the reproductive tract displayed more PAF activity than morulae resulting from the 48 hr culture of two-cell embryos. In addition to determining production characteristics of PAF by embryos, we also show that the production of CO2 from carbon-1 position of lactate is positively correlated with the ability of embryos to develop during subsequent culture in vitro and therefore could be used as a measure of embryo viability. Furthermore, culture of embryos in media supplemented with PAF resulted in an increase in lactate utilization demonstrating a direct effect of PAF on the embryo. As PAF is produced by preimplantation embryos, an autocoid role of PAF in regulating embryo development is implicated. Therefore, the reduced production of PAF by embryos in vitro may explain the decreased viability of embryos commonly observed following their culture in vitro.     

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。     

Alterations in enzyme and cytochrome profiles of Rana catesbeiana liver organelles during thyroxine-induced metamorphosis. Changes in membrane-localized phosphohydrolases, oxidoreductases, and cytochrome levels in response to in vivo thyroxine administration.

A primary objective of the present study has been to determine the changes which occur in Rana catesbeiana liver organelle membranes during thyroxine-induced metamorphosis. To this end, enzyme and cytochrome profiles were determined for mitochondria, microsomes, and nuclear membrane fractions isolated from livers of R. catesbeiana tadpoles which had been fasted for 6 days at 15 +/- 0.5 degrees and then immersed in thyroxine, 2.6 X 10(-8) M, for periods of up to 12 days at 23.5 +/- 0.4 degrees. The ratio of total succinate-cytochrome c reductase activity in the initial homogenate fraction to the total activity of this mitochondrial "marker" enzyme recovered in the final mitochondrial fraction remained constant, approximately 0.5, throughout the course of thyroxine treatment; however, after a 3- to 4-day latency the mitochondrial protein mass recovered per unit mass of initial homogenate protein was found to increase significantly (approximately 2-fold by Day 10 of thyroxine treatment). A similar increase was also observed in the yield of microsomal, but not nuclear membrane, protein mass as a function of thyroxine treatment. Prolonged thyroxine treatment (12 days) resulted in approximately 50% decreases in tadpole liver homogenate and microsomal NADH-cytochrome c reductase specific activities; in contrast, mitochondrial and nuclear membrane NADH-cytochrome c reductase specific activities were not altered under the same conditions. In addition, homogenate and microsomal NADPH-cytochrome c reductase specific activities were found to have increased significantly after 12 days of thyroxine treatment; however, the specific activity of NADPH-cytochrome c reductase in the mitochondrial fraction was unchanged. It was also observed that thyroxine treatment resulted in increases in homogenate and microsomal glucose-6-phosphatase specific activities, whereas the mitochondrial as well as nuclear membrane glucose-6-phosphatase specific activities remained unchanged. Furthermore, in contrast to homogenate and mitochondrial monoamine oxidase specific activities, which decreased 30 and 40%, respectively, as a consequence of thyroxine treatment (12 days), the succinate-cytochrome c reductase and oligomycin-sensitive Mg2+ ATPase specific activities determined for these fractions increased significantly. In all instances, changes as a result of thyroxine treatment in membrane-localized homogenate or organelle enzyme specific activities were apparent only after a 3- to 4-day initial latent period. The in vitro effects of thyroxine (10(-10) - 10(-5) M) on the membrane-localized enzyme activities examined in this study were either negligible or, as in the case of mitochondrial succinate-cytochrome c reductase and microsomal NADH-cytochrome c reductase, opposite to the changes observed in response to in vivo thyroxine treatment, with the exception of microsomal NADPH-cytochrome c reductase activity which was enhanced approximately 2-fold by 10(-5) M thyroxine...     

Expression of Maternally and Embryonically Derived Hypoxanthine Phosphoribosyl Transferase (Hprt) Activity in Mouse Eggs and Early Embryos

X-chromosome activity in early mouse development has been studied by a gene dosage method that involves measuring the activity level of the X-linked enzyme hypoxanthine phosphoribosyl transferase (HPRT) in single eggs and embryos from XO females and from females heterozygous for In(X)1H, a paracentric inversion of the X chromosome. The HPRT activity in oocytes increased threefold over a 24-hr period beginning after ovulation. Afterward, the activity plateaued in unfertilized eggs but continued to increase for at least 66 hr in presumed OY embryos. Both before and after ovulation, the level of activity in unfertilized eggs from In(X)/X females was twice that from XO females, and the distributions of activity in eggs for both sets of females remained unimodal. Beginning with the two-cell stage, distributions of activity for embryos from In(X)/X females were trimodal, which is evidence for embryonic activity. It is proposed that activation of a maternal mRNA or proenzyme is responsible for the HPRT activity increase in oocytes and early embryos and is supplemented by dosage-dependent activity of the embryonic Hprt gene as early as the two-cell stage.     

Protein synthesis in the early stages of cardiac hypertrophy

Cardiac hypertrophy, induced in rats by either tri-iodothyronine or isoproterenol, administered daily for 7 days, was monitored using several parameters. Both treatments increased RNA concentrations 24 hr after the first injection, while heart weight increased following 2 injections to 46% above control after 7 days. Cardiac protein synthetic activity, as determined by the rate of peptidyl-puromycin formation, was increased by both tri-iodothyronine and isoproterenol 24 hr after a single injection, implying an increase in the number of functional ribosomes. RNA activity (the rate of peptidyl-puromycin formation per unit RNA) remained constant, suggesting that neither accelerated rates of initiation or translation nor increased activation of pre-existing, non-translating ribosomes was involved in the observed increase in protein synthetic activity. In contrast, constant infusion of [14C] tyrosine indicated no change in protein synthetic rate 24 hr after a single tri-iodothyronine injection and decreased protein synthetic rate after isoproterenol injection. It is concluded that the use of [3H]puromycin to estimate protein synthetic activity may be a more sensitive procedure for detecting early changes in protein synthesis in cardiac hypertrophy than constant isotope infusion, owing to the problems associated with determining the precise precursor pool for protein synthesis in this latter method.     

Mitochondrial component of the phosphorylcreatine shuttle is enhanced during rat heart perinatal development

Aerobic metabolism is enhanced during perinatal heart development in parallel with increased cardiac function. The mitochondrial component of the phosphorylcreatine shuttle is important in providing energy for contraction and was examined in weanling and adult rat left ventricle. Creatine kinase activity was enhanced in tissue homogenate and purified cardiac myocytes of adults. Mitochondrial analyses attribute this enhancement to increased creatine kinase activity per milligram mitochondrial protein. Other enzymatic markers of mitochondrial function are not enhanced in activity during perinatal heart growth. The unique response of creatine kinase points to the shuttle mechanism and of mitochondrial creatine kinase, in particular, as a major contributor to heart functional regulation.     

Rates of synthesis of major classes of RNA in Drosophila embryos.

We have been successful in labeling to high specific activity (3 × 10⁵ dpm/μg) the RNA synthesized by large numbers of Drosophila embryos. Embryos of various developmental stages were rendered permeable with octane and labeled with [³H]uridine for 1 hr. At each stage the total dpm incorporated into RNA and the specific activity of the UTP pool were measured and used to calculate the absolute rate of RNA synthesis per embryo. This rate increases during embryonic development, from 1 pmole UTP/hr at 2 hr after oviposition to 6 pmoles UTP/hr at 15 hr. The rates of synthesis of nuclear and cytoplasmic poly(A)^? and poly(A)⁺ RNAs were determined by analyzing the fractionated RNAs from each stage by sucrose gradient sedimentation. There is a significant activation of nuclear RNA synthesis at the blastoderm stage (approximately 2 hr after oviposition). After blastoderm, the rates of synthesis of nuclear and cytoplasmic poly(A)^? and poly(A)⁺ RNA per embryo increase continuously; the rate of synthesis of each of these classes per nucleus, however, remains fairly constant. After making corrections for turnover during the labeling period, we find that the rates of synthesis of the major classes of RNA per nucleus at the gastrula stage are: cytoplasmic poly(A)⁺ RNA, 0.06 fg/nucleus-min; hnRNA, 0.86 fg/nucleus-min; and ribosomal RNA, 0.46 fg/nucleus-min. These rates are compared to rates of RNA synthesis in sea urchin embryos.     

Morphology and ultrastructure of the serosal cells (teratocytes) in Cardiochiles nigriceps Viereck (Hymenoptera : Braconidae) embryos

The morphogenetic changes of the serosal membrane during embryonic development of Cardiochiles nigriceps Viereck (Hymenoptera : Braconidae) were investigated. Eggs observed soon after oviposition into the natural host Heliothis virescens (F.) (Lepidoptera, Noctuidae) showed a transparent chorion and a uniform texture. After 5 hr, the embryo exhibited a distinct granular appearance and by 12 hr attained the germ band stage. A serosal membrane originated from the anterior pole of the embryo between 14 and 15 hr after the egg was laid, eventually forming with the cells both in the anterior and posterior pole a continuous envelope around the developing embryo.Ultrastructural observations revealed that the serosal cells in contact with the abdominal region of the embryo, beginning 24–25 hr after oviposition, formed a syncytium. However, the syncytial tissue did not extend to the cells around the head and thorax. The serosal cells at both embryo poles increased in size without losing their structural organization, and developed into teratocytes when the larva hatched. In contrast, the serosal cells surrounding the body of the embryo persisted longer on the head and thorax region of the newly hatched larva, while the syncytial tissue degraded more rapidly after hatching.In vitro rearing experiments showed that C. nigriceps embryos removed from parasitized host larvae just before and just after serosa formation, hatched only when the medium used was formulated with the addition of fetal bovine serum. Embryos did not develop or hatch when placed in a serum-free medium. Once the syncytium deriving from the serosal membrane became evident, embryos readily developed and hatched in serumfree media. The results of this study seem to suggest that the serosal embryonic membrane could have a nutritional role for the developing parasitoid embryo.     

Effects of oxygen toxicity on early development of mouse embryos.

To examine the effects of oxygen toxicity on embryonic development, mouse pronuclear embryos were cultured under low oxygen conditions with or without superoxide dismutase (SOD), and the blastulation rate was compared with that of embryos cultured under standard conditions. The blastulation rate of mouse pronuclear embryos cultured under standard conditions was only 1.5% (2/131). This rate was increased significantly, to 28.5% (43/151), when the embryos were cultured under low oxygen conditions; and to 31.0% (35/113) when SOD (500 micrograms/ml) was added to the medium under standard conditions; the rate was increased to 75.2% (115/153) when the embryos were cultured under low oxygen conditions in the presence of SOD. The minimum effective concentration of SOD in the culture medium was 50 micrograms/ml under conditions of 5% O2. The blastulation rate was significantly decreased after 1-hr exposure of pronuclear embryos to room atmospheric oxygen concentration (20% O2), and subsequent culture under 5% O2 with SOD did not result in an improved blastulation rate. Culture with SOD under 5% O2 promoted the development of two-cell stage embryos to the blastocyst stage. When two-cell stage embryos were collected 48 hr after hCG and cultured for 66 hr, their blastulation rate was similar to that of embryos collected from mice 114 hr after hCG. These results suggested that embryonic development in vitro is greatly affected by atmospheric oxygen throughout the early embryonic stages and that this harmful effect can be prevented by culturing embryos under low oxygen conditions and in the presence of SOD.