A scanning electron microscopic study of IAA-induced tumors in bean (Phaseolus vulgaris L.) embryos

A scanning electron microscopic study of indole-3-acetic acid (IAA) induced tumour in the hypocotyl region of bean embryos shows a distinct morphological, structural and topographical change from the non-treated bean embryos. The IAA-induced tumour surface, in the hypocotyl region, shows distinct cell enlargement, some cellular proliferation in the parenchymatous tissue, total destruction of the epidermis and stomata and some variation in trichome structures. In dole-3-acetic acid inhibits the normal growth of the epicotyl, and, as age progresses, adventitious roots appear all over the surface. When IAA-depletion occurs, epicotyl growth resumes, which indicates that this tumour formation in bean embryos is an IAA-dependent tumour.     

Distribution of diamine oxidase activity and polyamine pattern in bean and soybean seedlings at different stages of germination

Diamine oxidase (DAO, EC 1.4.3.6.) activity and polyamine content were measured in the shoot apex, leaves, epicotyl, cotyledons, hypocotyl and roots of light-grown bean ( Phaseolus vulgaris L. cv. Lingot) and soybean ( Glycine max L. cv. Sakai) seedlings at 3 different stages of germination (5, 8 and 14 days) as well as in embryos and cotyledons from soaked seeds. No DAO activity was detected in embryos and cotyledons of either plants. In bean seedlings DAO activity was only detectable in the shoot apex, primary leaves and cotyledons, while in soybean the activity was only detectable in the hypocotyl and roots. During seedling growth, in both plants, a different pattern of DAO activity was observed. In both species spermidine and spermine were the most abundant polyamines in embryos and cotyledons. Cadaverine, absent in bean, was only detected in soybean embryos. In the seedlings of both plants, increasing gradients of putrescine, spermidine and spermine from base to shoot apex were found. A high concentration of cadaverine was present in soybean hypocotyls and roots. A possible correlation between DAO activity and the endogenous content of the preferential substrate is discussed in relation to the possible involvement of the enzyme in regulating the cellular level of polyamines.     

Modulation in protein phosphorylation during G2 phase of the cell cycle in two-cell mouse embryos

The protein phosphorylation activities in extracts were assayed for 2-cell mouse embryos at three stages of the G2 phase of the cell cycle. The 2-cell embryos were unique in having a prolonged G2 phase and so easily staged at early G2 (EG2), middle G2 (MG2) and late G2 (LG2) by timing the embryo isolation from pregnant mice. The embryo extracts were used both as sources of protein kinases and their substrates. The phosphoproteins of the extracts were labelled with [gamma-32P]ATP and separated by electrophoresis on SDS-polyacrylamide gels. The present study revealed that protein phosphorylation increased 3-6-fold during the progression of 2-cell embryos from EG2 to LG2 and the level of protein phosphorylation at any stages was greatly decreased by the presence of cAMP. Thus, the protein phosphorylation system of 2-cell mouse embryos seems to differ from those reported systems in mammals in its negative dependence on cAMP.     

Adenosine 3′,5′-cyclic monophosphate-binding protein in sea urchin eggs and embryos

Adenosine 3′,5′-cyclic monophosphate (cAMP) binds to high-molecular substances which are probably proteins, in homogenates of sea urchin eggs and embryos. The bound cAMP is exchangeable. Optimal pH for the binding capacity of the proteins with cAMP is 4.0, and is shifted to 5.0 in the presence of 5 mM caffeine. The level of bound cAMP increases steeply during 10 minutes of incubation. This is then followed by a less steep increase. The level of bound cAMP decreases in the presence of NaCl. The dissociation onstant between cAMP and the proteins in homogenates of unfertilized and ertilized eggs is about 10 nM, and the value in the embryos at the gastrula stage is lower than that in the unfertilized egg homogenate.     

Effect of adenylyl cyclase activation on intracellular and extracellular cAMP and cGMP in preimplantation cattle blastocysts

The effects of direct and indirect activation of adenylyl cyclase on the production of intracellular and extracellular cAMP and cGMP by 13- to 16-day-old cattle embryos were determined. Embryos were incubated for 2 h in a Krebs Ringer bicarbonate medium containing the phosphodiesterase inhibitor isobutyl-methylxanthine, to which stimulating agents forskolin (100 mumol l-1), cholera toxin (2 micrograms ml-1), or both were added. Total (intra- and extracellular) basal cAMP and cGMP concentrations ranged from 6.65 +/- 0.895 to 3.4 +/- 0.708 fmol microgram-1 protein in 13-day-old embryos and from 4.05 +/- 1.151 to 0.19 +/- 0.041 fmol microgram-1 protein in 16-day-old embryos. Forskolin induced an increase (P < 0.001) in cAMP that ranged from 5.4-fold on day 13 to 2.7-fold on day 16, whereas cholera toxin induced an increase (P < 0.001) that ranged from 30-fold at day 13 to 21-fold at day 16, similar to the effect of forskolin and cholera toxin combined. Individually, forskolin and cholera toxin had no effect on cGMP concentrations, but together they induced an increase (P < 0.05). cAMP (P < 0.01) and cGMP (P < 0.001) concentrations decreased with embryo age from day 13 to day 16 for all treatments; the decrease was greater for cGMP than cAMP (5-24-fold versus 1.6-3.3-fold, respectively). It is concluded that inducible adenylyl cyclase is present in 13- to 16-day-old cattle embryos and that the embryos secrete cAMP and cGMP into the incubation medium. In addition, basal and inducible concentrations of cAMP and cGMP decrease with embryo age from day 13 to day 16. These observations indicate that cAMP and cGMP may have a role in the rapid embryonic cell proliferation that occurs at this time or in signalling to the endometrium.     

Embryonic cAMP and developmental potential in Drosophila melanogaster

Summary Measurements of cAMP in early embryos of Drosophila melanogaster demonstrate that the dunce gene plays a major role, and the rutabaga gene a secondary role, in maternal regulation of embryonic cAMP content. Studying the double mutant combination, we find that variability in elevated cAMP content between individual embryos is associated with a wide variability in developmental potential. Embryos with about five times the normal cAMP content define a threshold between apparently normal and abnormal development. Measurements of cAMP content in anterior and posterior halves of embryos indicate that the posterior embryonic region, which is developmentally more sensitive to the effects of elevated cAMP than the anterior region, does not contain more cAMP than the anterior region. The variety of developmental defects observed is discussed in relation to possible targets of cAMP action. Offprint requests to: J.A. Kiger, Jr     

Cyclic nucleotides of human and animal glial tumors

Studies on the level of cyclic nucleotides (cAMP and cGMP) in human and animal glial tumours showed that the content of both nucleotides, especially that of cAMP, decreases in all the tumours. The cAMP/cGMP ratio also drops down. Concurrently it appears to be the most consistent parameter of nucleotide metabolism both in brain tissue and in human or animal glial tumours. The growing tumour affects cAMP and cGMP metabolism not only in the involved but also in the other hemisphere. No principal differences between human and animal tumours have been revealed in the content of cyclic nucleotides and its variation in tumour tissue.     

The involvement of cAMP signaling pathway in axis specification in Xenopus embryos

The cAMP signaling system has been postulated to be involved in embryogenesis of many animal species, however, little is known about its role in embryonic axis formation in vertebrates. In this study, the role of the cAMP signaling pathway in patterning the body plan of the Xenopus embryo was investigated by expressing and activating the exogenous human 5-hydroxytryptamine type 1a receptor (5-HT(1a)R) which inhibits adenylyl cyclase through inhibitory G-protein in embryos in a spatially- and temporally-controlled manner. In embryos, ventral, but not dorsal expression and stimulation of this receptor during blastula and gastrula stages induced secondary axes but were lacking anterior structures. At the molecular level, 5-HT(1a)R stimulation induced expression of the dorsal mesoderm marker genes, and downregulated expression of the ventral markers but had no effect on expression of the pan mesodermal marker gene in ventral marginal zone explants. In addition, ventral expression and stimulation of the receptor partially restored dorsal axis of UV-irradiated axis deficient embryo. Finally, the total mass of cAMP differs between dorsal and ventral regions of blastula and gastrula embryos and this is regulated in a temporally-specific manner. These results suggest that the cAMP signaling system may be involved in the transduction of ventral signals in patterning early embryos.     

Adenylate cyclase activity and cyclic nucleotide content in human adrenal tumors under Icenko-Cushing syndrome

The adenylate cyclase activity and cyclic nucleotide content in excised human adrenal tumours (Icenko-Cushing syndrome) were determined. The experimental data were compared to those obtained for hyperplastic adrenals. All adrenal tumours under study revealed a decreased cAMP level, an increased cGMP level and a resulting decrease of the cAMP/cGMP ratio. In malignant adrenal tumours the adenylate cyclase activity was sharply increased in comparison with that in hyperplastic adrenals. In the majority of malignant tumours the adenylate cyclase response to ACTH was either altogether absent or sharply decreased. In benign adrenal tumours the basal activity of the enzyme was unchanged and the enzyme response to ACTH was essentially normal. The decrease of adenylate cyclase response to ACTH in malignant tumours is apparently not due to the impaired catalytic activity of the enzyme, since its response to stimulation by sodium fluoride remains unaffected. In some tumours (one malignant and two benign ones) a non-specific stimulation of adenylate cyclase by hormones, which are not natural activators of the enzyme was observed. It was assumed that these changes are due to the damage of hormonal receptors in adrenal tumours.     

Role of the testa in preventing cellular rupture during imbibition of legume seeds

Studies with the seeds of soybean, navy bean, pea, and peanut were made to determine the extent of leakage of intracellular enzymes during imbition. Embryos with intact testae from all four species were found to leak detectable activities of either intracellular enzymes of the cytosol (glucose-6-phosphate dehydrogenase) or enzymes found in both the cytosol and organelles (malate dehydrogenase, glutamate dehydrogenase, glutamate oxaloacetate transaminase, and NADP-isocitrate dehydrogenase) after 6 hours imbition at 25 C. Pea and peanut embryos with testae leaked considerably lower levels of activity for these enzymes than did those of soybean and bean. Leakage of mitochondrial marker enzymes (fumarase, cytochrome c oxidase, and adenylate kinase) was not detected from embryos with testae, suggesting that a differential diffusion of intracellular components out of cells occurred. Soybean and bean embryos without testae leaked high, and proportionally (per cent dry seed basis) similar, levels of all cytosol, cytosol-organelle, and mitochondrial marker enzymes and protein during imbibition, indicating that cell membranes were not differential to leakage and that they had ruptured. Pea and peanut embryos without testae leaked detectable activities of all cytosol and cytosol-organelle enzymes, although fumarase was the only detectable mitochondrial marker enzyme leaked, suggesting that some degree of differential leakage may have occurred in these species. The outermost layers of embryo cells of seeds without testae of all four species absorbed and sequestered the nonpermeating pigment Evan's blue after 5 to 15 minutes imbibition, indicating that membranes had ruptured. This occurred to a much lesser extent in seeds with intact testae. Both soybean and bean embryos without testae were observed to disintegrate during imbibition, whereas those of pea and peanut did not. These data indicate that seeds of certain legumes are susceptible to cellular rupture during imbibition when seed coats are damaged or missing.     

The participation of serotonin in regulating cyclic nucleotide levels in early sea urchin embryos

Abnormal cleavage, decrease in the intracellular cAMP and cGMP content and a trend for increase of extracellular cAMP content were observed in sea urchin embryos incubated with KIuR-14 serotoninolytic substance. The addition of serotonin leads to normalization of cleavage and cAMP and cGMP content. It suggests serotonin-specific effect of KIuR-14 and functional relations between serotonin and cyclic nucleotides.     

The effect of cyclic nucleotides on the sensitivity of early mouse embryos to biogenic monoamine antagonists]

The effect of lipophilic cAMP analogs on the sensitivity of preimplantation mouse embryos of two strains to cytotoxic serotonin and adrenalin antagonists was studied. Dioctanoyl-cAMP significantly decreased the sensitivity of embryos to inmecarb and cyproheptadine: experimental embryos developed to the stage of morula or blastocyst, in contrast to control embryos incubated without this protector. A somewhat weaker effect was observed in experiments with propranolol: embryos incubated in the propranolol-containing medium after the addition of dioctanoyl-cAMP were capable of one to two cleavage divisions. 8-bromomonobutyryl-cAMP partially suppressed the inhibitory effect of cyproheptadine and did not affect the sensitivity of embryos to propranolol. These data suggest cAMP involvement in the regulatory activity of neurotransmitters in the early mouse embryos.     

Elevation of Cyclic AMP-Dependent Protein Kinase Activity during Migration of Primary Mesenchyme Cell in Sand Dollar Blastulae

Concetration of intracellular cyclic AMP (cAMP), and activities of adenylate cyclase and cAMP-dependent protein kinase were examined in swimming and mesenchyme blastulae and primary mesenchyme cells (PMCs) of the sand dollar, Clypeaster japonicus , respectively. In mesenchyme blastulae, the concentration of cAMP increased 45% from that in swimming blastulae. PMCs contained a concentration of cAMP 40% higher than that in whole embryos at the mesenchyme blastula stage. The activity of adenylate cyclase in mesenchyme blastulae was 100% higher than that in swimming blastulae. The activites of cAMP-dependent protein kinase in whole embryos at the above two developmental stages, on the other hand, were quite similar to each other. However, in PMCs the activity of the enzyme was conspicuously higher than that in these embryos, and it reached 190% higher than that in these embryos. Inhibition of cAMP-dependent protein kinase activity by a synthetic inhibitor, H8, caused severe inhibition of PMC migration but it did not exert any effect on PMC ingression. These results suggest that the cAMP-dependent protein kinase activity is involved in PMC migration, but not in PMC ingression.     

激光预处理可保护蚕豆细胞免受UV—B辐射的损伤

当蚕豆的胚被 He- Ne激光 (632 .8nm,1 .63J· mm- 2 )照射 5min或被 CO2 激光 (1 0 60nm,2 .53J· mm- 2 )照射 1 min后 ,将其置入 Knop营养液中进行恒温培养。当蚕豆的上胚轴长到大约 3cm时 ,在光背景 (PAR)为 70μmol· m- 2 · s- 1条件下 ,分别用 1 .0 2、3.0 3、4.52 k J· m- 2 的 UV- B辐射蚕豆的上胚轴 7h。根据蚕豆丙二醛 (MDA)、抗坏血酸 (As A)和 UV- B吸收物的含量变化 ,来测试激光对 UV- B照射蚕豆的上胚轴的保护作用。结果显示 :激光预处理可保护蚕豆上胚轴对 UV- B辐射的作用。与对照组 (没有用 UV- B或激光照射 )、UV- B单独照射组比较 ,在激光预处理的条件下 ,MDA的含量明显减少 ,As A和 UV- B吸收化合物的含量增加。如先用激光处理 ,然后再用 UV- B辐射 ,UV- B吸收物的含量将比单独用激光和 UV-B处理获得更好的改善。从而认为 ,激光预处理能增强植物对 UV- B的抵抗力。     

Increase in levels of cyclic AMP during avian limb chondrogenesis in vitro.

In the present study the level of cAMP was measured during in vitro chondrogenesis of wing mesenchyme of stage 24 chick embryos and was found to increase significantly from 6.3 pmol/mg protein at the end of the first day of culture to 9.7 pmol/mg protein on the second day, when chondrogenic expression is first detected by the appearance of an Alcian blue staining extracellular matrix. Nonchondrogenic cultures derived from wings of stage 19 embryos had a lower level of cAMP (4.4 +/- 0.07 pmol/mg protein). The level of cAMP in intact wings was 4.5 +/- 0.4 pmol/mg protein and did not change between stages 19 through 25. The correlatin between increased levels of cAMP and the onset of chondrogenesis is consistent with a role of cAMP in the expression of differentiated functions in chondrocytes, as well as in some other cell types.     

High-performance liquid chromatographic identification and quantitation of cyclic adenosine 3':5'-monophosphate in higher (Phaseolus vulgaris L.) and lower (Chlorella sp.) plants

Cyclic adenosine 3':5'-monophosphate (cAMP) was extracted from Phaseolus vulgaris L. cv. Limburg seedlings and Chlorella sp. The cAMP was purified by charcoal adsorption, polyvinylpyrrolidone (PVP) and Dowex 50 W × 8 column chromatography and by preparative high-performance liquid chromatography (HPLC). Quantitation was achieved by combining phosphodiesterase (PDE) treatment with analytical HPLC (reversed phase ion-pair partition chromatography) and cAMP-dependent protein kinase activation. This provided a very specific, accurate and sensitive assay for cAMP determinations. The cAMP content found in Chlorella (70–90 pmol/g dry wt) was comparable with previous reports using other quantitation methods, whereas the endogenous concentration found in bean seedlings (92 pmol/g dry wt) was considerably lower than previously reported data.     

THE ONTOGENY OF DOPAMINE-DEPENDENT INCREASE OF ADENOSINE 3'',5''-CYCLIC MONOPHOSPHATE IN THE CHICK RETINA

The adenosine 3′,5′-cyclic monophosphate level of chick embryonic retina changes during the course of development. In retinas from 6- to 15-day-old embryos the cAMP level is approximately 7 pmol/mg protein. A sharp 3-fold increase is observed between the 16th and 18th embronic day and remains constant thereafter. A dopamine-dependent increase in cAMP of the chick retina is already present in 7-day-old embryos, and by the 8th embryonic day maximal response is attained. Glutamate promotes a 2-fold stimulation. Carbachol, γ-aminobutyric acid and glycine do not cause any significant change in the level of cAMP of the embryonic tissue. Guanosine 3′,5′-cyclic monophosphate also accumulates during development. Its concentration is approx 0.5 pmol/mg protein from the 8th to the 14th embryonic day, then increases gradually until the 19th day of development when the level observed is approx 14 pmol/mg protein.     

Effect of cAMP on choline uptake and phosphatidylcholine biosynthesis in cultured chick heart cells

The effect of an analogue of cAMP on the uptake and metabolism of choline in the heart was studied in isolated cardiac cells. The cells were obtained from 7-day-old chick embryos and maintained in culture. The effects of cAMP were studied using the dibutyryl cAMP analogue and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. After a 2-h incubation with [3H]choline, about 85% of the label was recovered in phosphocholine, with most of the rest in phospholipid. During a subsequent chase incubation, [3H]phosphocholine was transferred to phosphatidylcholine with little accumulation in CDP-choline. This suggests the rate-limiting step for the conversion of phosphocholine to phosphatidylcholine in these cells is the synthesis of CDP-choline. cAMP decreased the incorporation of choline into phosphatidylcholine, but did not change the flux of metabolites through the step catalyzed by CTP:phosphocholine cytidylyltransferase. cAMP had little effect on choline uptake at low (1-25 microM) extracellular choline concentrations, but significantly (p less than 0.05) decreased choline uptake at higher (37.5-50 microM) extracellular choline concentrations. Thus, cardiac cells take up and metabolize choline to phosphocholine, with CTP:phosphocholine cytidylyltransferase being the rate-limiting step in phosphatidylcholine biosynthesis. cAMP decreases [3H]choline uptake and its subsequent incorporation into phosphocholine and phospholipid. However, the metabolism of choline within the cell is unaffected.