CHICK BRAIN CREATINE KINASE ONTOGENY BEFORE THE ADVENT OF CREATINE AND INSULIN

The ontogeny of brain creatine kinase (CK) was studied during chick embryo development. The cytosolic activity increased 270% in 10 h from the 2nd to the 3rd days of incubation; this was followed by a plateau phase throughout development and at the end of incubation there appeared to be another increase of cytosolic and mitochondrial CK activities. Therefore, early embryonic chick brain CK is another‘constitutive’enzyme like the early embryonic chick heart CK since creatine has not been enzymatically detected in the embryo until day 4 of incubation. Insulin does not appear to stimulate the early increase of brain CK activity since the hormone is not present in the embryo until day 5 of incubation. It is likely that CK increase is associated with neuronal multiplication at early stages and possibly to neuronal maturation before hatching.     

Alcohol dehydrogenase activity in the developing chick embryo

Before day 9 of incubation, chick embryos contain no measurable alcohol dehydrogenase (ADH) activity. Following day 9 of incubation, chick embryo liver ADH activity increases as a linear function of liver mass. A single dose of ethanol given at the start of incubation is cleared only slowly prior to day 9 of incubation but is completely cleared by day 13. Chick embryo liver ADH has two detectable isozymes throughout development. The percentage contribution of each isozyme to total ADH activity does not change significantly during development. The Km apparent of chick liver ADH is significantly increased shortly after hatching relative to the Km apparent of embryonic ADH. Ethanol exposure during incubation has no effect on the development of ADH activity or isozyme distribution.     

Free ADP levels in transgenic mouse liver expressing creatine kinase. Effects of enzyme activity, phosphagen type, and substrate concentration

ADP is an important regulator of hepatic metabolism. Despite its importance the level of free ADP in the liver remains controversial. Recently, we engineered transgenic mice which express high levels of creatine kinase in liver. The reaction catalyzed by creatine kinase was assumed to be at equilibrium and used to calculate a free ADP level of 0.059 mumol/g wet weight. In this report we test the equilibrium assumption by studying the free ADP level as a function of enzyme activity or substrate content. Over a 5-fold range of creatine kinase activity, from 150-800 mumol/min/g wet weight, there was no change in the free ADP level. The average value of ADP for these mice was 0.061 +/- 0.016 mumol/g wet weight. Similarly, altering hepatic creatine content from 1.6 to 30 mumol/g wet weight had no effect on the calculated total free ADP level. The average value of ADP for the creatine levels was 0.048 +/- 0.015 mumol/g wet weight. Finally, the free ADP level was calculated using the equilibrium with cyclocreatine rather than creatine as substrate. The equilibrium of the reaction with cyclocreatine lies 30 times more toward phosphorylation than does the equilibrium with creatine. A free ADP level of 0.063 +/- 0.031 mumol/g wet weight was calculated using cyclocreatine. This value is not different from that found with creatine. These results show that the equilibrium assumption used to calculate free ADP levels in transgenic mouse liver is valid, and the presence of creatine kinase does not affect ADP levels.     

Phosphorylation of the triadin cytoplasmic domain by CaM protein kinase in rabbit fast-twitch muscle sarcoplasmic reticulum

Identification of estrogenresponsive genes is important to understand the molecular mechanisms of estrogen action. Suppression subtractive hybridization was employed to screen estrogenresponsive genes in chick liver. A single injection of estrogen into 6weekold chick induced upregulation of several known genes encoded for yolk proteins, such as Vitellogenin I and II and very low density lipoprotein II (apo-VLDL II). One novel sequence displayed a dramatic change (3fold increase) in response to estrogen treatment. This cDNA fragment was extended and the resultant sequence was analyzed. Translated amino acid sequence was 90, 88, 83 and 87% identical to the Larginine:glycine amidinotransferase of pig, rat, frog and human, respectively. The sequence has a conservative catalytic site of Larginine:glycine amidinotransferase. The expression pattern of this gene in organs is consistent with previous reports of Larginine:glycine amidinotransferase in chick. Thus, this clone represented the chicken Larginine:glycine amidinotransferase. It appeared that estrogeninduced alteration of arginine:glycine amidinotransferase was not dependent on protein synthesis, because concurrent administration of cycloheximide did not affect the estrogenmediated expression pattern. This is the first study demonstrating that Larginine:glycine amidinotransferase is a target of the estrogen receptor.     

Development of antioxidant capacity in tissues of the chick embryo

The concentrations of vitamin E, vitamin A, selenium, reduced glutathione and the activities of glutathione peroxidase and superoxide dismutase were determined in the yolk, yolk sac membrane, liver and brain of the developing chick embryo. The changes in the concentrations of vitamins E and A in the yolk and liver during development were consistent with the occurrence of a preferential transfer of vitamin A from the yolk to the embryo before day 14 of incubation, whereas the main period of vitamin E transfer occurred later, during the last week of incubation. The concentrations of reduced glutathione in the yolk sac membrane, liver and brain were similar at all developmental stages studied. However, the levels of the other measured antioxidant systems were very much higher in the liver than in the brain. Thus, in the newly hatched chick, the levels of vitamin E, vitamin A, selenium, glutathione peroxidase and superoxide dismutase were, respectively, 58.0-, 174.7-, 3.6-, 4.0- and 4.7- fold higher in the liver than in the brain when expressed on the basis of tissue fresh weight.     

Changes of creatine concentration during development of chick embryo

Changes of creatine concentration during development of chick embryo. Acta physiol. pol., 1985, 36 (3): 208-215. Investigations were carried out on embryos of Star Cross chicken after 6, 8, 10, 12, 14, 16, 18 and 20 days of incubation. It was found that the concentration of total creatine increased from 0.18 to 0.67 mg/g of embryo weight. The increase of creatine concentration at the time of development of the embryo was proportional to the increase in nitrogen concentration, and the share of creatine nitrogen in the pool of total nitrogen was about 1% throughout the whole period of embryonal development and during the first two days after hatching. The amount of creatine in fresh egg and in the yolk sac of the newly hatched chicken was about 1.5 mg. It was estimated that chick embryos during their development synthesized, on the average, 18 mg (6 mMol) of creatine. The course of changes in creatine concentration in the developing chick embryo is very similar to the course of changes in the rate of heat production.     

Relationship between changes in free cholesterol and pyrophosphomevalonate decarboxylase activity during myelination

The patterns of cholesterol content in chick brain and liver were studied during embryonic development and compared with the variations in the specific activities of mevalonate-activating enzymes during the same period. Total cholesterol content in both embryonic chick brain and liver increased during incubation. The relative percentage of free cholesterol was always maintained over 85% in brain, while in liver this percentage decreased to less than 10% during the later days of incubation. A straight parallelism was observed between free cholesterol and pyrophosphomevalonate decarboxylase activity in the embryonic brain. On the other hand, the hepatic decarboxylase exhibited a lower specific activity than in brain and did not show significant variations throughout the same period of incubation. Changes in brain pyrophosphomevalonate decarboxylase activity were more pronounced than those observed in both mevalonate kinase and phosphomevalonate kinase activities, in spite of that the specific activity of decarboxylase was the lowest of the three mevalonate-activating enzymes, suggesting that this reaction is one rate-limiting step for cholesterogenesis during myelination.     

Rhythmic contractions in chick amnio-yolk sac and snake amnion during embryogenesis

The rhythmic movements of fetal membranes in chick and reptile embryos were studied to explore the developmental role of the extra-embryonic motor activity. In the snakes Lamprophis fuliginosus and Elaphe radiata, rhythmic contractions of amnion inside the developing egg were recorded from the 11th incubation day until pre-hatching stages (ca. day 60-72). The duration of these contractions averaged 2.02+/-0.27 min. The frequency ranged from 2 to 6 per 10 min and averaged 4.61+/-0.57 per 10 min. A tendency of frequency to increase toward the end of embryogenesis was observed. Lowering the temperature from 28 to 20 degrees C significantly decreased the frequency of amnion contractions to 2.85+/-0.91 per 10 min. The isolated snake amnion retained its capacity for spontaneous contraction. Noradrenaline inhibited, acetylcholine stimulated and serotonin did not affect the rhythmic activity of the isolated snake amnion. Similar effects were found when these agents were applied into the snake amniotic cavity. In the chick, yolk sac rhythmic contractions were recorded from the fifth until the 12th incubation days. The duration of these contractions ranged from 15 to 60 s, their frequency averaged 11.8+/-3.18 per 10 min and depended on temperature. The low temperature threshold was approximately 30 degrees C. After surgical removal of the amnion and embryo, the yolk sac continued contracting inside the egg. The yolk sac rhythmic contractions likely participate in the space movement of the embryo inside the egg during embryogenesis.     

Enzymes involved in adenine nucleotide metabolism of developing chick embryo liver.

1. This paper describes the changes in the activity of adenylate deaminase, adenylate and inosinate phosphatase, and adenosine deaminase in the developing chick embryo liver. 2. The adenylate and inosinate phosphatase and adenosine deaminase activity appears considerably higher in chick embryo liver with respect to other chick embryo tissues previously examined. 3. During development the control exerted by ATP on AMP breakdown undergoes variations. Consequently, in the first period of incubation AMP is degraded by the direct pathway (AMP-IMP) and in the last period of incubation by the indirect pathway (AMP-adenosine). In the intermediate period (from the 12th to the 15th day of incubation) both pathways may be followed. 4. The ability to synthesize purine nucleotides through "salvage pathway" seems to be acquired by embryonic liver at least at the 15th day.     

Emoxipin as an inhibitor of angiogenesis]

The effect of emoxypin on angiogenesis in rabbit cornea in aseptic inflammation induced by intracorneal implantation of a piece of quartz and on the development of the vessels of the chick embryo yolk sac was studied. 1% emoxypin pipetted thrice a day for 10-14 days inhibited corneal neovascularization and reduced the formation of new blood vessels. We observed an inhibitory effect on the development of vascular bed of the embryo yolk sac on incubation hour 64-72. The drug affected neither general growth of the embryos no the number of somites.     

Higher homolog and N-ethyl analog of creatine as synthetic phosphagen precursors in brain, heart, and muscle, repressors of liver amidinotransferase, and substrates for creatine catabolic enzymes

Tissues of chicks fed 5% N-methyl-3-guanidinopropionate (N-amidino-N-methyl-beta-alanine) for 12 days accumulated the following amounts of free plus phosphorylated derivatives as mumol/g, wet weight: brain, 5.5; heart, 7.3; leg muscle, 21.0; and breast muscle, 24.4. Since total creatine levels remained nearly the same in brain, N-methyl-3-guanidinopropionate-P provided brain with a supplemental reservoir of high energy phosphate. Tissues of rats fed 2% N-ethylguanidinoacetate (N-amidino-N-ethylglycine) accumulated large amounts of N-ethylguanidinoacetate-P, which has thermodynamic properties similar to creatine-P and is the kinetically most reactive synthetic phosphagen yet described. N-Ethylguanidinoacetate derivatives replaced creatine derivatives mole-for-mole, and the fraction of synthetic to total phosphagen after 19 days was 60% in heart, 54% in slow oxidative muscle, 42% in fast glycolytic muscles, and 22% in brain. N-Ethylguanidinoacetate served as a false end product co-repressor of liver arginine:glycine amidinotransferase levels in both chicks and chick embryos; N-methyl-3-guanidinopropionate and N-propylguanidinoacetate were relatively inactive. Creatinine amidohydrolase reversibly cyclized both N-ethylguanidinoacetate and N-propylguanidinoacetate with even lower Km values than for creatine derivatives, but it did not react significantly with N-methyl-3-guanidinopropionate, 3-guanidinopropionate, or 1-carboxy-methyl-2-imino-imidazolidine (cyclocreatine). Creatine amidinohydrolase also hydrolyzed N-acetimidoylsarcosine, but was relatively unreactive toward N-ethylguanidinoacetate, N-methyl-3-guanidinopropionate, 3-guanidinopropionate, and cyclocreatine. Amidinohydrolase can therefore be used to remove interfering creatine in assays of tissues for coexisting N-ethylguanidinoacetate or N-methyl-3-guanidinopropionate. Assays are now available to follow changes during metabolic stresses of any combination or all of the following phosphagens accumulated by the same tissue: creatine-P, N-ethylguanidinoacetate-P, cyclocreatine-P, N-methyl-3-guanidinopropionate-P, and homocyclocreatine-P.     

Vitamin A economy of the developing chick embryo and of the freshly hatched chick

1. The changes in the net amounts of retinol, retinyl esters and retinal in both the developing chick embryo and the newly hatched chick were investigated. The embryo requires about 68nmol of the vitamin for its growth, whereas the baby chick requires about 108nmol during the first 7 days after hatching. 2. Retinal was present in the egg in fairly high concentrations at the beginning of the incubation but it virtually disappeared from the extra-embryonic tissue after day 17 of incubation. It was not found in the liver of the embryo or of the newly hatched chick up until day 7.     

Hematopoietic stem cells in mice during embryonic development preceding the establishment of liver hematopoiesis]

We studied the time course of appearance of CFUs (7-8 days old) in embryos of (C57B1/6 x CBA)F1 mice from the 8th day of embryonic development. Significant amounts of CFUs could be detected from the 10th day of development, initially in the body of the embryo from the stage of 30-33 pairs of somites, then in the yolk sac and still later, from the stage of about 40 pairs of somites, in liver anlage. CFUs could not be reliably detected until the 9th day of development either in the embryo itself or in the yolk sac. However, after incubation of nine day old embryos for four days in organ culture, such cultures contained CFUs. CFUs could be found in significant levels in embryos explanted from the embryos at the stage no earlier than 24 pairs of somites. When the yolk sac and the embryo were cultivated separately, CFUs could also be detected, however, the removal of liver primordium from the embryo did not influence the amount of CFUs in its body. CFUs were not found in cultures of liver primordium from nine day old embryos. Thus, we can detect pre-CFUs in 9 day old embryos at the stage 25-28 pairs of somites using the system of organ culture; at the same time CFUs cannot be found in intact embryos of the same age. These data provide evidence that before the establishment of liver hemopoiesis precursors of CFUs are located both in the yolk sac and in the embryo outside rudimentary liver. However, our results do not provide any data for the conclusion about the primary source of pre-CFUs in the mouse embryo.     

Functional expression of phosphagen kinase systems confers resistance to transient stresses in Saccharomyces cerevisiae by buffering the ATP pool

Phosphagen kinase systems provide different advantages to tissues with high and fluctuating energy demands, in particular an efficient energy buffering system. In this study we show for the first time functional expression of two phosphagen kinase systems in Saccharomyces cerevisiae, which does not normally contain such systems. First, to establish the creatine kinase system, in addition to overexpressing creatine kinase isoenzymes, we had to install the biosynthesis pathway of creatine by co-overexpression of L-arginine:glycine amidinotransferase and guanidinoacetate methyltransferase. Although we could achieve considerable creatine kinase activity, together with more than 3 mM intracellular creatine, this was not sufficient to confer an obvious advantage to the yeast under the specific stress conditions examined here. Second, using arginine kinase, we successfully installed an intracellular phosphagen pool of about 5 mM phosphoarginine. Such arginine kinase-expressing yeast showed improved resistance under two stress challenges that drain cellular energy, which were transient pH reduction and starvation. Although transient starvation led to 50% reduced intracellular ATP concentrations in wild-type yeast, arginine kinase overexpression stabilized the ATP pool at the pre-stress level. Thus, our results demonstrate that temporal energy buffering is an intrinsic property of phosphagen kinases that can be transferred to phylogenetically very distant organisms.     

Purification, characterization, and ontogeny of acetyl-CoA carboxylase isozyme of chick embryo brain.

Acetyl-CoA carboxylase catalyzes the first committed step in the synthesis of fatty acids. Because fatty acids are required during myelination in the developing brain, it was proposed that the level of acetyl-CoA carboxylase may be highest in embryonic brain. The presence of acetyl-CoA carboxylase activity was detected in chick embryo brain. Its activity varied with age, showing a peak in the 17-18-day-old embryo and decreasing thereafter. The enzyme, affinity-purified from 18-day-old chick embryo brain, appeared as a major protein band on polyacrylamide electrophoresis gels in the presence of sodium dodecyl sulfate (Mr 265,000), indistinguishable from the 265 kDa isozyme of liver acetyl-CoA carboxylase. It had significant activity (Sp act = 1.1 mumol/min per mg protein) in the absence of citrate. There was a maximum stimulation of only 25% in the presence of citrate. Dephosphorylation using [acetyl-CoA carboxylase] phosphatase 2 did not result in activation of the enzyme. Palmitoyl-CoA (0.1 mM) and malonyl-CoA (1 mM) inhibited the activity to 95% and 71%, respectively. Palmitoylcarnitine, however, did not show significant inhibition. The enzyme was inhibited (greater than 95%) by avidin; however, avidin did not show significant inhibition in the presence of excess biotin. The enzyme was also inhibited (greater than 90%) by antibodies against liver acetyl-CoA carboxylase. An immunoblot or avidin-blot detected only one protein band (Mr 265,000) in preparations from chick embryo brain or adult liver. These observations suggest that acetyl-CoA carboxylase is present in embryonic brain and that the enzyme appears to be similar to the 265 kDa isozyme of liver.     

Metabolism of cholesterol in the tissues and blood of the chick embryo

Three artificially inseminated laying White Leghorn hens were given 35-50 micro c of cholesterol-4-(14)C intravenously. Their subsequently produced eggs contained cholesterol-(14)C-labeled yolks. Some of the fertilized eggs were analyzed for cholesterol content and radioactivity. Other eggs were incubated until hatching. The specific activity of the cholesterol contained in the serum and tissues of newly hatched chicks was determined and compared with that of yolk sac, which was taken as representative of egg yolk cholesterol before its metabolic transfer into the chick embryo. The specific activities of cholesterol in intestine, liver, serum, heart, and skeletal muscle and the whole chick were 95-98% of that in yolk sac, but that of brain cholesterol was only 11% of this value. These results indicate that whereas most of the cholesterol in the chick originated from the egg yolk, cholesterol biosynthesis was active in the brain and provided about 90% of its cholestero content. Newly hatched chicks were found to be hyperlipemic compared with older chicks and had fatty livers with a high cholesterol content. Desmosterol was found in 9- and 15-day old chick embryos but not in the newly hatched chicks, in which the only sterol was cholesterol.     

Effect of some antineoplastics on metabolic heme pathway

1. The porphyrinogenic ability of several antineoplastics used in the therapy of the different cancers was evaluated. The action of cyclophosphamide, busulfan and 5-fluorouracil on the amount and nature of the accumulated hepatic porphyrins and on the activity of delta-aminolaevulinate synthase (ALA-S), were estimated at different doses and times of drug treatment in 17-day-old chick embryos. 2. It was observed that cyclophosphamide produces a significant increase in the accumulation of hepatic porphyrins at different doses as well as in the activity of the ALA-S, at all the incubation times. Cyclophosphamide alters the pattern of porphyrins accumulated in the liver, where a coproporphyrin: protoporphyrin ratio higher than in the controls can be observed. 3. Busulfan increased the hepatic porphyrins accumulated in the liver but to a lesser degree than cyclophosphamide. 4. 5-Fluorouracil did not modify the hepatic porphyrin content when it was administered at doses up to 40 mg/embryo. 5. When the embryos were injected with busulfan or 5-fluorouracil no significant differences were observed in the activity of ALA-S up to 11 hr of incubation. 6. These results indicate that cyclophosphamide has a remarkable porphyrinogenic capacity in chick embryo while busulfan, notwithstanding the fact that it alters the haem pathway, it does so to a degree that does not impair the regulation of ALA-S activity. Fluorouracil seems to be non porphyrinogenic in this system, up to 40 mg/embryo.     

Suppression and stimulation of DNA synthesis by ADP-ribosylation of nuclear proteins from adult hen and chick embryo liver.

The stimulation of DNA synthesis by ADP-ribosylation of nuclear proteins was observed in chick embryo liver nuclei. In contrast, a significant decrease in template activity was detected in hen liver nuclei treated with NAD. When a 0.35 M NaCl extract from embryo, but not adult, liver nuclei was treated with NAD and then combined with either adult or embryo liver nuclear residue, the ability to activate the template was greatly enhanced. These results indicate that in the chick embryo liver, the ADP-ribosylation of the nuclei plays an important role as a regulator of DNA synthesis.     

Studies on the endogenous phospholipids of chick embryo myocardium and their in vitro hydrolysis by endogenous phospholipases during embryogenesis

The phospholipid profiles of the myocardium (from 10- and 18-day old chick embryos and 13-day old chick) and their in vitro response to the endogenous lipolytic enzymes (mainly of the phospholipase group) at pH 7.4 and 38 degrees C for 60 min were analyzed by TLC technology and densitometry. Cardiolipin (CL) was shown to be one of the major phospholipids of the chick embryo myocardium and its concentration increased as the chick embryo advanced in development. Monolysocardiolipin (MLCL) was produced subsequent to in vitro incubation of whole tissue homogenates in all myocardia studied as well as a concurrent reduction in CL. This deacylation of CL increased in magnitude as the chick embryo advanced in development indicating its age relatedness. The level of phosphatidyl ethanolamine (PE) plasmalogen was also high in all myocardia studied. Lyso alkenyl PE (LPE) was produced subsequent to in vitro incubation and its level increased as the chick embryo advanced in development, indicating PLA(2) action on the sn-2 fatty acid of PE. Phosphatidyl choline (PC) plasmalogen was also present in the chick embryo myocardium and its level increased gradually as the chick embryo advanced in development. In contrast, yolk-sac membrane contains very minute amounts of CL and PE. No PC was detected and no LPE was formed following in vitro incubation. The yolk of the unfertilized chicken egg has no CL and has very minute amounts of PE, no PC and no lysophospholipids were detected following in vitro incubation in all samples analyzed.