Isolation of a cDNA clone for chick intestinal apolipoprotein AI (Apo-AI) and its use for detecting apo-AI mRNA expression in several chick tissues

Three cDNA clones for chick apolipoprotein AI (Apo-AI), the major protein component of plasma high-density lipoproteins, have been isolated. The identity of the clones has been established first by screening a cDNA library in the pEX1 expression vector with anti-Apo-AI antibodies, second by Western blot analysis of the proteins expressed by positive clones. The use of the clone containing the largest, presumably full-size, cDNA insert (apo5C12) in molecular hybridization experiments confirms that apo-AI mRNA is expressed mainly in chick small intestine and liver. Furthermore, we provide evidence that brain, heart and skeletal muscle also synthesize significant amounts of apo-AI mRNA. The Southern-blot hybridization pattern of the restriction-enzyme-digested chick DNA with the apo5C12 DNA is consistent with there being a single copy of the apo-AI gene.     

Plasma lipoproteins, tissue cholesterol overload, and skeletal muscle apolipoprotein A-I synthesis in the developing chick

In the present study we investigated the changes of plasma lipids, lipoproteins, and tissue lipids that occur during the late embryonic life (5 days before hatching) and the postnatal period (0, 2, 7, 14, and 30 days after hatching) of the chick. The chick emerges from the egg with extreme hypercholesterolemia associated with a high level of cholesterol-rich VLDL + IDL. The density gradient profile of plasma lipoproteins showed that the concentrations of VLDL + IDL and LDL decreased during the first week of postnatal life, whereas HDL concentration increased sharply around hatching and remained stable afterwards. All plasma lipoprotein classes of the newborn chick (2 days from hatching) were enriched in cholesterol and cholesteryl esters; 2 weeks after hatching, the relative amount of cholesterol and cholesteryl esters decreased. In the newborn chick, plasma VLDL + IDL consisted of two populations of cholesteryl ester-rich lipoproteins: the main one (designated apoB-VLDL) contained apoB and no apoA-I; the other (designated apoA-I-VLDL) contained predominantly apoA-I. In the newborn chick there was an accumulation of free and esterified cholesterol in the liver and, to a lesser extent, in the skeletal muscle. These cholesterol deposits were depleted 2 to 7 days after hatching. The depletion in skeletal muscle was preceded by and associated with a striking increase in the synthesis of apoA-I in this tissue, as demonstrated by immunological methods and apoA-I mRNA measurements. In addition, apoA-I-containing HDL were secreted in vitro by explants of skeletal muscle of the newborn chick. The synthesis of apoA-I in the skeletal muscle decreased to the level found in the adult animal 1 week after hatching. It is likely that the rise of HDL and apoA-I in plasma observed 1-2 days after hatching reflects the production of apoA-I-containing HDL by skeletal muscle. We suggest that the cholesterol overload in skeletal muscle might stimulate the production of apoA-I which, in turn, would promote the removal of cholesterol from this tissue. The hypothesis that metabolic stimuli play a role in inducing apoA-I synthesis in skeletal muscle is supported by the observation that feeding the newborn chick a diet rich in proteins and lipids and free of carbohydrates delays the fall of apoA-I mRNA which normally occurs 1 week after hatching.     

Ontogenically regulated expression of metallothionein and its messenger RNA in chick liver

By electrophoretic and immunological assay the concentration of hepatic metallothionein in new born chick liver was found to be ontogenically modulated, reaching a peak accumulation per gram liver in fourth day of hatching and declining below the detection limit after second week postnatal. The protein was undetectable upto second week of incubation in egg-embryonic stage. The concentration of metallothionein mRNA shows drastic change during first few days after hatching. The greatest accumulation of metallothionein mRNA was detected in the one day new born chicks, which declined rapidly there after, and reduced to a barely detectable level. Metallothionein was also detected in the in vitro translated product of one day neonatal chick hepatic poly(A+) RNA by S-cysteine labelling and immunoprecipitation. The naturally occurring new-born chick liver metallothionein was found to be a zinc-metallothionein and the concentration of hepatic zinc in new-born chick was found to undergo drastic modulation during development, unlike some other chick tissues. Endogenous zinc ion mobilization can thus play a significant role in the developmental regulation of chick metallothionein expression.     

Glyceraldehyde 3-phosphate dehydrogenase protein and mRNA are both differentially expressed in adult chickens but not chick embryos

We have determined the 679 nucleotide sequence of a cDNA clone which, by hybridization-translation experiments, corresponds to a 36K chick brain protein. Our studies provide a partial amino acid sequence for this protein, identifying it as chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Antisera raised against purified chicken GAPDH reacted with a 36K protein present in chick brain extracts and estimated to be the fourth most prevalent protein, as determined by either Coomassie Blue staining or by in vitro translation of chick brain mRNA. The amounts of GAPDH mRNA in chick brain, liver and muscle and adult chicken brain are similar, whereas the relative amount of adult chicken muscle GPDH mRNA is greatly elevated and that of adult liver lowered. The GAPDH protein levels showed a similar variation between tissues, suggesting that the levels of GAPDH protein are largely regulated by the amount of available GAPDH mRNA. The chicken GAPDH clone does not hybridize to rat mRNA, even though GAPDH is one of the most evolutionarily conserved proteins, indicating that selection pressures are heavier at the primary protein sequence level than at the nucleic acid sequence level for this gene, a situation contrasting to that of the tubulins.     

Activities of the enzymes of ketone body metabolism in the developing chick

The concentration of ketone bodies in the blood of the developing chick prior to and just after hatching were higher than those found in the adult. The activities of 3-oxo acid-CoA transferase and acetoacetyl-CoA thiolase in the heart, leg and pectoral muscle before and after hatching were higher than those of the adult. The activity of 3-hydroxybutyrate dehydrogenase increased constantly during incubation and after hatching in all three muscle tissues. In the liver the activities of the enzymes of ketone body synthesis increased during incubation and after hatching. It is suggested that the liver could provide fuel to the extrahepatic tissues of the developing chick and ketone bodies could contribute as fuel for oxidation in the skeletal muscle of the newly hatched bird.     

Analysis of the mRNA coding for the chick vitamin D-induced calbindin and its regulation by 1,25-dihydroxyvitamin D3

We have used specific cloned cDNA probes generated from the mRNA coding for the vitamin D-induced 28,000-Da chick intestinal calcium binding protein (calbindin) to study the hormonal regulation of the expression of this mRNA by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. The calbindin-mRNA has been analyzed in chicken intestinal poly(A)+ mRNA samples as well as other chicken tissues by "Northern" blot analysis. There exists a predominant mRNA species of approximately 2000 nucleotides and two minor cross-hybridizing species that are nearly equivalent in proportion; their sizes are approximately 2600 and 3100 nucleotides. All three mRNA species are nonexistent in the chick intestine in the absence of vitamin D3 intake. However, all three mRNA species begin to accumulate at the same time in the chick intestine following the administration of the hormonally active metabolite of vitamin D3, 1,25-(OH)2D3. This response in the intestine is very similar to other steroid hormone-regulated gene products. All three mRNA species exist in the cell cytoplasm and are present on soluble polysome complexes, suggesting that all three are engaged in protein synthesis. Examination of other chick tissues (both vitamin D-deficient and -replete) reveals a close association between mRNA expression and previously observed calbindin expression. Each tissue is unique in the steady-state level of expression of the calbindin-mRNAs.     

Two types of asymmetric acetylcholinesterase in chick hindlimb muscle: Developmental profiles,in Vivo and in cell culture,and recovery after inactivation

1. We have analyzed the behavior of two types of asymmetric molecular forms (A forms) of acetylcholinesterase (AChE) during development of chick hindlimb muscle, in vivo and in cell culture, and upon irreversible inactivation of peroneal muscle AChE with diisopropylfluorophosphate (DFP) in vivo. 2. In agreement with previous developmental studies on chick muscle, globular forms of AChE (G forms) are predominant in chick hindlimb at early embryonic ages, being gradually replaced by A forms as hatching (and, therefore, onset of locomotion) approaches. Of the two A-form types, AI appears and accumulates significantly earlier than AII, so that A/G and II/I ratios higher than 1 are attained only at about hatching time. 3. Cultures prepared from 11-day chick embryo hindlimb myoblasts express both types of A forms, with a combined activity of 27% of total AChE after 12 days in culture. AI forms appear again earlier and are much more abundant than type II asymmetric species through the life span of cultures. 4. All AChE activity in the peroneal muscle is irreversibly inactivated by injection of DFP in vivo. The recovery of A forms follows the same sequence described for normal development, with a delayed and slower recovery of AII forms as compared with AI. 5. Several hypotheses involving tail polypeptides or tissue target molecules, or posttranslational interconversion, are proposed to help explain the earlier appearance and accumulation of AI forms in chick muscle.     

Sex-related differences in the expression of chick enterocyte butyrylcholinesterase during embryonic and post-hatching development

Summary The butyrylcholinesterase activity of chick enterocytes was studied from day 15 in ovo up to day 90 after hatching. The activities detected in both sexes at the level of jejuno-ileum change in a parallel manner, but the activity is always higher in the female than in the male during embryonic development. After hatching, the differences are less apparent although the study of the enzyme distribution along the intestine showed sex-related variations, mainly at the level of the anterior and middle parts of jejuno-ileum in the young adult. Analysis of butyrylcholinesterase by sucrose gradient centrifugation allowed to identify two globular soluble species (G₁ and G₄ forms). The G₄/(G₁ + G₄) ratio decreases during the development but this variation in the female does not parallel that observed in the male. Besides, the molecular form distribution along the intestine, studied after hatching, differs according to the sex. Taken together our results lead to hypothesize that the ontogeny and the regulation of the chick enterocyte butyrylcholinesterase depend on hormones.Abbreviations AChE Acetylcholinesterase - BuChE Butyrylcholinesterase     

Regulation of apolipoprotein A1 synthesis in avian muscles

Until recently, liver and intestinal mucosa were believed to be the sole sites of synthesis of apolipoprotein A1 (Apo-A1), the major protein component of serum high density lipoprotein particles. We recently showed (Shackelford, J.E., and Lebherz, H.G. (1983) J. Biol. Chem. 258, 7175-7180) that chick breast muscle also synthesizes and secretes Apo-A1 but does so at high rates only around the time of hatching. In the present work, we investigate the regulation of synthesis of Apo-A1 in chicken muscles. 1) The primary translation product encoded for by muscle Apo-A1 mRNA is about 2600 daltons larger than the mature serum protein which is consistent with the idea that, like its mammalian liver counterpart, chick muscle Apo-A1 mRNA codes for an NH2-terminal extension (prepro segment) which is 24 amino acids long. 2) The developmentally regulated rise and fall in muscle Apo-A1 synthesis which occurs around the time of hatching is associated with a large accumulation followed by depletion of Apo-A1 mRNA molecules during this period. 3) Reinitiation of Apo-A1 synthesis to high levels in mature breast muscle occurred in vivo following surgical denervation and in vitro by maintaining breast muscle explants for several days in defined culture media. 4) Cardiac, but not smooth, muscles also synthesize and secrete Apo-A1 at high levels around the time of hatching. These and other observations are discussed in terms of possible regulatory "signals" which may control Apo-A1 synthesis in avian muscles.     

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.     

Developmental changes in gene expressions of beta-carotene cleavage enzyme and retinoic acid synthesizing enzymes in the chick duodenum

Vitamin A is derived from provitamin A carotenoids, mainly beta-carotene, by beta-carotene 15,15'-monooxygenase (BCMO1; EC 1.13.11.21). We previously reported that chick duodenal BCMO1 activity increased abruptly just after hatching. In this study, we further investigated mechanisms and physiological roles of the postnatal induction of BCMO1 expression in the chick duodenum. We showed that BCMO1 mRNA levels increased in the chick duodenum during postnatal period after hatching, but remain unchanged in the chick liver throughout the perinatal period. Serum hydrocortisone (HC) levels were also increased after hatching. Moreover, HC-administered chicks showed an enhancement of duodenal BCMO1 mRNA during the perinatal period. We further analyzed the developmental gene expression patterns of three types of retinoic acid (RA) synthesizing enzymes in the chick duodenum. Among them, retinal dehydrogenase 1 (RALDH1) mRNA levels in the chick duodenum increased during the postnatal period, indicating a similar developmental expression pattern to that of BCMO1. These results suggest that the postnatal induction of BCMO1 gene expression in the chick duodenum may be caused by the elevation of serum HC levels and may contribute to the RALDH1-mediated RA synthetic pathway.     

Developmental changes in vitamin A level and lack of retinyl palmitate in chick lungs

1. Developmental changes in retinol and retinyl palmitate contents in lungs of chick embryos and posthatch chicks were investigated. 2. Remarkable changes in the lung retinol levels were found during development of chicks. Embryonic lungs 5 days prior to hatching contained the highest content of retinol. The level then declined rapidly and was lowest on 1 day before hatching. 3. Its level then rose substantially within 7 days after hatching. 4. No retinyl palmitate in chick lungs was detectable at any of the developmental stages examined, nor even in adult hen. 5. Serum retinol level changed in parallel with the lung retinol. 6. The patterns of changes in liver retinol and retinyl palmitate were remarkably different from that occurring in the lung retinol. In chick embryonic livers, the levels of them were low, followed by a rapid increase after hatching. 7. The high level and its rapid decrease of lung retinol content during development of chick embryos may be functionally connected with retinol action in embryonic lungs for cellular differentiation and maturation.     

The sequence of chick alpha-actinin reveals homologies to spectrin and calmodulin

We have sequenced a cDNA, isolated from a chick embryo fibroblast lambda gt11 library, that encodes all 887 amino acids of alpha-actinin. Sequence from 10 different peptides from chick smooth muscle alpha-actinin was found to match that derived from the cDNA. The deduced protein sequence can be divided into three distinct domains: (a) the N-terminal 240 amino acid contains a highly conserved region (compared with Dictyostelium alpha-actinin) which probably represents the actin-binding domain, (b) amino acids 270-740 contain four repeats of a spectrin-like sequence, and (c) the C-terminal sequence contains two EF-hand Ca2+-binding sites. Each of these sites is defective in at least one oxygen-containing Ca2+-chelating amino acid side chain, suggesting that they are nonfunctional. Southern blots suggest that the alpha-actinin cDNA described here hybridizes to only one gene in chicken. Northern blots reveal only one size class of mRNA in fibroblasts and smooth muscle, but no hybridizing species could be detected in skeletal muscle poly(A+) RNA. The results are consistent with the view that smooth and skeletal muscle alpha-actinins are encoded by separate genes, which are considerably divergent.     

Development of lipidic composition of neonatal chick liver and intestine microsomes: changes induced by cholesterol feeding

Changes in cholesterol and phospholipid content of chick liver and intestine microsomes were studied throughout the two first weeks of life. Differences observed throughout postnatal development were mainly due to the free cholesterol. Cholesterol feeding resulted in a clear increase of the amounts of both free and esterified cholesterol. Phospholipid content of chick liver and intestine microsomes did not change significantly after hatching. Phosphatidylcholine and phosphatidylethanolamine were found to be the major phospholipids. Although the amount of each phospholipid could be affected by cholesterol feeding, its relative percentage did not change by this treatment.     

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.     

Synthesis and secretion of apolipoprotein A1 by chick breast muscle

The present work shows that chick breast muscles synthesize and secrete a protein which is very similar to chicken serum apolipoprotein A1 (Apo-A1), the major protein constituent of serum "high density" lipoprotein particles. This conclusion is based on the following observations. 1) When chick breast muscle explants were incubated in the presence of radioactive amino acids, a labeled protein of the same size as serum Apo-A1 (Mr approximately equal to 27,000) accumulated in the incubation media; 2) the muscle-derived secretory protein and serum Apo-A1 generated the same size distribution of peptide fragments following digestion with Staphylococcus aureus V8 protease; and 3) antibodies raised against serum Apo-A1 specifically precipitated the radioactive muscle secretory protein. The newly secreted muscle-derived Apo-A1 was associated with lipid, as judged by its "flotation" behavior during centrifugation of the labeled incubation media in the presence of 0.2 g/ml of sodium bromide; this observation suggests that muscle explants secreted Apo-A1 molecules as part of lipoprotein particles or that these Apo-A1 molecules became associated with lipid shortly after their secretion. The present work, together with the very recent report by Blue et al. (Blue, M.L., Ostapchuk, P., Gordon, J.S., and Williams, D.L. (1982) J. Biol. Chem. 257, 11151-11159) demonstrate that avian tissues other than liver and intestinal mucosa synthesize and secrete Apo-A1. Results of short term amino acid incorporation experiments showed that chick breast muscles synthesize Apo-A1 at high rates only during the terminal stages of embryonic development and early stages of postembryonic maturation. Around the time of hatching, the relative rate of synthesis of Apo-A1 by chick breast muscle was found to be higher than in liver, a documented major site of synthesis of this apolipoprotein. Possible physiological implications of the present work will be considered.     

Synthesis of sterols by chick brain and liver during embryonic development

The evolution throughout embryonic development of the rate at which acetate was converted into sterols was studied in chick brain and liver. Acetate incorporation (nmol/h/g tissue) was clearly higher in brain than in liver and sharply decreased with the age of embryo. Cholesterol and desmosterol were the major sterols formed from acetate by chick embryo brain, followed by lanosterol and squalene. No desmosterol was found in chick embryo liver, organ where cholesterol was the major sterol synthesized. In brain, the relative percentage of cholesterol increased throughout embryonic development reaching more than 50% at hatching, while the percentage of desmosterol decreased during the same period and represented at hatching only about 10–15% of the total nonsaponifiable fraction. The relative percentages of lanosterol and squalene did not change significantly throughout the period assayed. In liver, the percentage of cholesterol increased until 19 days but sharply decreased at hatching.