Developmental changes in prolyl hydroxylase activity and protein in chick embryo.

Changes in prolyl hydroxylase activity and immunoreactive protein were studied in various chick embryo tissues during the embryonic development. Both the enzyme activity and the amoung of immunoreactive protein increased till the 16th day of development and declined thereafter in all tissues studied. Comparison of the enzyme activity to the content of the total immuno-reactive protein indicated that there are distinct differences in the degree of enzyme activity between different chick embryo tissues, and in the same tissue between different stages of embryonic development. The highest relative enzyme activities were found in cartilage and skin, in which about 60% of the enzyme was active on the 16th day of development and only 20-30% was active on the 20th day of development; the lowest values were observed in spleen and large vessels, in which below 10% of the enzyme protein was in the active form on the 20th day of development Gel filtration studies demonstrated that in cartilage of 16-day-old chick embryos about 60% of the total immunoreactive enzyme in the tissue was present in the form of active prolylhydroxylase tetramer, whereas on the 20th day of development only 30% of the enzyme protein in cartilage was in the tetramer form. By contrast, in large vessels of the 16-day-old chick embryos, essentially all the enzyme was in the form of prolyl hydroxylase monomers.     

Developmental changes of amp-deaminase activity in chick heart muscle

• 1.1. The developmental changes of 5'AMP-deaminase activity were studied in the hearts of 7–21 days chick embryos, 1–58 days chicks and in adult hen.
• 2.2. The activity of 5'AMP-deaminase in the embryonic and adult hen cardiac muscle were similar and amounted about 20 nmoles NH₃× min⁻¹ per mg protein. The activity dropped about fourfold at hatching, remained low for 18 days of postnatal development and increased slowly reaching its embryonic value in the heart of adult hen.
• 3.3. The changes of 5'AMP-deaminase were not correlated with the development of cardiac myocytes as measured by morphometry and myosin ATP-ase activity.

     

Developmental changes in the activity of membrane-bound gamma-glutamyl transpeptidase and in the sialylation of synaptosomal membranes from the chick embryonic brain.

gamma-Glutamyl transpeptidase (GGT) is a membrane-bound sialoglycoprotein. The developmental changes in GGT activity and in sialic acid content were determined in a crude synaptosomal membrane fraction from the cerebral hemispheres of the chick embryo between days 11 and 19 of incubation. The GGT activity increased almost eightfold during the examined developmental period, while sialic acid content rose significantly only between days 11 and 15. Cortical administered on day 13 significantly increased GGT activity. On the other hand, the content of membrane bound sialic acid was not substantially affected. The value of the GGT apparent Michaelis constant (Kmapp) for gamma-glutamyl-p-nitroanilide in the presence of 20 mmol.l-1 glycylglycine was 1.5 mmol.l-1 and cortisol did not influence it. However, Vmax was increased by this hormone. The affinity of GGT to concanavalin A (ConA) did not change during development. Neither the administration of cortisol nor neuroaminidase treatment had any effect on the interaction of GGT with ConA. Desialylation of crude synaptosomal fraction did not change GGT activity. The results presented here suggest no developmental nor functional relationship between the activity of GGT and the level of sialylation in synaptosomal membranes from the cerebral hemispheres of the chick embryo.     

Developmental changes in the activity of phosphatidylethanolamine N-methyltransferases in rat brain.

A complete separation of myo-inositol 1,4,5-[4,5-(32)P]trisphosphate prepared from human erythrocytes, and myo-[2-3H]inositol 1,3,4-trisphosphate prepared from carbachol-stimulated rat parotid glands [Irvine, Letcher, Lander & Downes (1984) Biochem. J. 223, 237-243], was achieved by anion-exchange high-performance liquid chromatography. This separation technique was then used to study the metabolism of these two isomers of inositol trisphosphate in carbachol-stimulated rat parotid glands. Fragments of glands were pre-labelled with myo-[2-3H]inositol, washed, and then stimulated with carbachol. At 5s after stimulation a clear increase in inositol 1,4,5-trisphosphate was detected, with no significant increase in inositol 1,3,4-trisphosphate. After this initial lag however, inositol 1,3,4-phosphate rose rapidly; by 15s it predominated over inositol 1,4,5-trisphosphate, and continued to rise so that after 15 min it was at 10-20 times the radiolabelling level of the 1,4,5-isomer. In contrast, after the initial rapid rise (maximal within 15s), inositol 1,4,5-trisphosphate levels declined to near control levels after 1 min and then rose again very gradually over the next 15 min. When a muscarinic blocker (atropine) was added after 15 min of carbachol stimulation, inositol 1,4,5-trisphosphate levels dropped to control levels within 2-3 min, whereas inositol 1,3,4-trisphosphate levels took at least 15 min to fall, consistent with the kinetics observed earlier for total parotid inositol trisphosphates [Downes & Wusteman (1983) Biochem. J. 216, 633-640]. Phosphatidylinositol bisphosphate (PtdInsP2) from stimulated and control cells were degraded chemically to inositol trisphosphate to seek evidence for 3H-labelled PtdIns(3,4)P2. No evidence could be obtained that a significant proportion of PtdInsP2 was this isomer; in control tissues it must be less than 5% of the total PtdInsP2 radiolabelled by myo-[2-3H]inositol. These data indicate that, provided that inositol 1,4,5-trisphosphate is studied independently of inositol 1,3,4-trisphosphate, the former shows metabolic characteristics consistent with its proposed role as a second messenger for calcium mobilization. The metabolic profile of inositol 1,3,4-trisphosphate is entirely different, and its function and source remain unclear.     

Developmental changes in glycosaminoglycans,collagen and collagenase activity in embryonic chick skin

In order to study remodelling of connective tissue during development, changes in glycosaminoglycans, collagen and collagenase activity in embryonic chick skin at various stages have been studied.Collagen content in the skin increased rapidly during days 14 to 18, then leveled off until hatching. Prior to the increase of collagen deposition in the skin, a sharp decrease in chondroitin sulfate was observed between days 11 and 14, while dermatan sulfate increased almost 4 fold during days 12 to 14, then increased steadily until hatching. Hyaluronic acid decreased progressively during the stages investigated (days 11 to 20).At the same stage as the rate of collagen deposition in the tissue became maximal (day 16), the amount of dialyzable hydroxyproline showed a maximum indicating that an increased rate of collagen deposition in the tissue was accompanied by accelerated collagenolysis.Culture of skin from various stages of embryonic development revealed that 16 day old tissue was potentially capable of secreting the highest levels of collagenase. This collagenase was mostly inactive against soluble collagen and collagen fibrils but could be activated by 3 M NaSCN treatment.     

Developmental changes in glycosaminoglycans, collagen and collagenase activity in embryonic chick skin.

In order to study remodeling of connective tissue during development, changes in glycosaminoglycan, collagen and collagenase activity in embryonic chick skin at various stages have been studied. Collagen content in the skin increased rapidly during days 14 to 18, then leveled off until hatching. Prior to the increase of collagen deposition in the skin, a sharp decrease in chondroitin sulfate was observed between days 11 and 14, while dermatan sulfate increased almost 4 fold during days 12 to 14, then increased steadily until hatching. Hyaluronic acid decreased progressively during the stages investigated (days 11 to 20). At the same stage as the rate of collagen deposition in the tissue became maximal (day 16), the amount of dialyzable hydroxyproline showed a maximum, indicating that an increased rate of collagen deposition in the tissue was accompanied by accelerated collagenolysis. Culture of skin from various stages of embryonic development revealed that 16 day old tissue was potentially capable of secreting the highest levels of collagenase. This collagenase was mostly inactive against soluble collagen and collagen fibrils but could be activated by 3 M NaSCN treatment.     

Developmental changes in the activity and substrate specificities of mouse brain monoamine oxidase

Developmental changes in monoamine oxidase (MAO) activity in the mouse brain were investigated with the substrates -phenylethylamine (PEA), tryptamine, and 5-hydroxytryptamine (5-HT). In the newborn brain, MAO activity towards PEA was found to be much lower than the adult and to be inhibited by clorgyline in a double-sigmoidal fashion. The inhibition curve shifted to a single-sigmoidal pattern with age. MAO activity towards 5-HT as substrate was inhibited by 90% and in a single-sigmoidal manner by clorgyline throughout the postnatal life. Lineweaver-Burk plots with PEA as substrate presented two linear lines (apparentK _m: 28.6 and 4.1 M) for the newborn and one line (apparentK _m: 11.4 M) for the adult, respectively. The plot with highK _m value for the newborn brain disappeared in a clorgyline-treated preparation. These findings suggest that age-dependent alterations in the ratio of MAO-A/MAO-B activity affect the substrate specificity of the enzyme.     

Developmental change in the glycosaminoglycan composition of the chick embryo brain

GAGs content was examined in chick embryo brain starting from the 9th day of incubation to the 4th post-hatching day. Chondroitin 6-sulfate, hyaluronate and heparan sulfate were recovered at any developmental stage examined. C6-S was the main GAG (except on the 15th day), while HS was the least represented. The highest differences in the relative amounts of GAGs are observed on the 9th day. C6-S shows high developmental relative changes, while HA and HS exhibit a similar pattern.     

Developmental changes in collagen glycosyltransferase activities in chick embryos

The developmental pattern of collagen galactosyltransferase and collagen glucosyltransferase activities was determined in chick embryos between the 4th and 21st day of growth. Both enzyme activities increased up to the 16th day and decreased thereafter in whole chick embryos and in most tissues studied. The highest collagen glycosyltransferase activities were found in the leg tendons of the 16-day-old embryos, and the activities found in cartilage were higher than those noted in either skin or skull, indicating that the the activities of the collagen glycosyltransferases may play a part in the regulation of the carbohydrate content of the collagen synthesized by a given tissue. The changes observed in the collagen glycosyltransferase activities agree with previous data on the development of prolyl and lysyl hydroxylase activities and also with findings on collagen turnover in the developing chick embryo.     

Developmental changes in the activity and cellular localization of hepatic casein kinase II in the rat

Abstract The activity and cellular localization of hepatic casein kinase II(CKII) was examined during late fetal development in the rat. Cultured fetal hepatocytes displayed constitutive CKII activity which was not further activated by growth factor exposure. Similarly, fetal liver CKII showed approximately fivefold greater activity than adult liver. The fetal hepatic activity was, to a large degree, localized to a nuclear fraction. Postnuclear cytosol preparations from fetal and adult liver showed similar CKII activity. In all cases, FPLC ion exchange chromatography followed by Western immunoblotting showed that immunoreactive CKII coincided with kinase activity. However, Parallel determinations of CKII activity and immunoreactive CKII levels showed a higher(five-to sixfold) CKII specific activity in nuclear extracts compared to cytosol. In summary, fetal hepatic CKII demonstrates coincident nuclear localization and activation. We hypothesize that the regulation of hepatic CKII is relevant to the mitogen-independent proliferation displayed by fetal rat hepatocytes. © Wiley-Liss, Inc.     

Developmental changes in the calcium currents in embryonic chick ventricular myocytes

Summary Using the patch-clamp technique, we recorded whole-cell calcium current from isolated cardiac myocytes dissociated from the apical ventricles of 7-day and 14-day chick embryos. In 70% of 14-day cells after 24 hr in culture, two component currents could be separated from totalI _Ca activated from a holding potential (V _h) of –80 mV. L-type current (I _L) was activated by depolarizing steps fromV _h –30 or –40 mV. The difference current (I _T) was obtained by subtractingI _L, fromI _Ca.I _T could also be distinguished pharmacologically fromI _L in these cells.I _T was selectively blocked by 40–160 m Ni²⁺, whereasI _L was suppressed by 1 m D600 or 2 m nifedipine. The Ni²⁺-resistant and D600-resistant currents had activation thresholds and peak voltages that were near those ofI _T andI _L defined by voltage threshold, and resembled those in adult mammalian heart. In 7-day cells,I _T andI _L could be distinguished by voltage threshold in 45% (S cells), while an additional 45% of 7-day cells were nonseparable (NS) by activation voltage threshold. Nonetheless, in mostNS cells,I _Ca was partly blocked by Ni²⁺ and by D600 given separately, and the effects were additive when these agents were given together. Differences among the cells in the ability to separateI _T andI _L by voltage threshold resulted largely from differences in the position of the steady-state inactivation and activation curves along the voltage axis. In all cells at both ages in which the steady-state inactivation relation was determined with a double-pulse protocol, the half-inactivation potential (V _1/2) of the Ni²⁺-resistant currentI _L averaged –18 mV. In contrast,V _1/2 of the Ni²⁺-sensitiveI _T was –60 mV in 14-day cells, –52 mV in 7-dayS cells, and –43 mV in 7-day NS cells. The half-activation potential was near –2 mV forI _L at both ages, but that ofI _T was –38 mV in 14-day and –29 mV in 7-day cells. Maximal current density was highly variable from cell to cell, but showed no systematic differences between 7-day and 14-day cells. These results indicate that the main developmental change that occurs in the components ofI _Ca is a negative shift with, embryonic age in the activation and inactivation relationships ofI _T along the voltage axis.     

Developmental changes in internal structure of chick heart plasma membrane

Although changes in electrophysiologically measurable membrane properties of chick embryo cardiac plasma membrane have been repeatedly documented during embryonic development, ultrastructural techniques were heretofore too insensitive to detect developmental changes in internal structure of this membrane. We report here significant structural changes detected by applying the quantitative analysis of Kordylewski, Karrison, and Page (Amer. J. Physiol. 245, H992-H997, 1983 and 248, H297-H304, 1985) to stereo imaged electron microscopic negatives of glutaraldehyde-fixed chick embryo hearts, freeze fractured and photographed with a goniometer stage. Between Hamburger-Hamilton stages 12+ (about 48 hr incubation) and 24 (about 96 hr incubation), plasmalemmal P-face particle density of ventricular myocytes increased from 2228 +/- 139 to 3063 +/- 109 (P less than 0.01); thereafter, measurements at stages 30, 37, 40, and 45 (7, 11, 15, and 19 days incubation) showed a slower significant linear increase which gave a least-squares line with a slope of 41 +/- 13 particles/day (P less than 0.01). Just before hatching, (stage 45) the value of 3762 +/- 234 was similar to, though slightly smaller than, the values of 4122 +/- 153 (8 days after hatching) and 4281 +/- 218 (adult chicken). These results indicate striking stage-dependent changes in the population of integral membrane proteins (channels, carriers, receptors, etc.), especially marked during early embryogenesis.     

Developmental changes in mouse brain polyamine metabolism

Mouse brain ornithine decarboxylase (ODC) activity is high at the time of birth, whereas S-adenosyl-l-methionine decarboxylase (SAM-DC) activity is low. ODC activity, and putrescine, spermidine and spermine concentrations decline rapidly during postnatal development to the low level characteristic of mature brains, while SAM-DC activity behaves in the opposite manner. The fluctuations in mouse brain polyamine metabolism are in accord with those found in the rat. The apparentK_m values of ODC and SAM-DC for their substrates decline parallel with the decrease of substrate and product concentrations during ontogeny suggesting substrate and/or product dependent regulation of polyamine synthesis in the developing brain.     

Developmental changes in the subcellular localization of R-cadherin in chick retinal pigment epithelium

 It has been reported that in the chick embryonic retina, N-cadherin first appears at the very early stages and is subsequently substituted by R-cadherin at the middle to late stages of development. To examine the role of R-cadherin in the morphogenesis of chick retinal pigment epithelium (RPE), the distribution of this adhesion molecule was studied by immunofluorescence cytochemistry and immunoelectron microscopy from embryonic day (E) 6 to hatching. R-cadherin immunoreactivity was detected at E6, and was strongest at E12–13. During these stages, R-cadherin was expressed uniformly on the lateral plasma membranes of RPE cells in contact with each other. Thereafter, R-cadherin immunoreactivity was markedly decreased, with intense immunoreactivity restricted to zonulae adherentes in latero-apical regions at E16. R-cadherin immunoreactivity was no longer detectable in the newly hatched chick RPE, even though morphologically well developed zonulae adherentes were present in latero-apical regions. No immunoreactivity was detected on the apical side facing the neural retina or on the basal side facing the basal lamina at any stage of development. These findings indicate that R-cadherin plays an important role as a major cadherin subtype in the morphogenesis of chick embryo RPE, and is involved initially in non-specific cell-cell adhesions, and subsequently in the formation and maintenance of developing zonulae adherentes. Accepted: 11 April 1997