THE NUTRITION OF ANIMAL TISSUES CULTIVATED IN VITRO : IV. AMINO ACID REQUIREMENTS OF CHICK EMBRYONIC HEART FIBROBLASTS

1. The amino acid requirements of freshly explanted chick embryonic heart tissues cultivated in completely synthetic media have been determined, employing a nutritional depletion technique. Arginine, histidine, lysine, tyrosine, tryptophan, phenylalanine, cystine, methionine, threonine, leucine, and valine were found to be essential. Serine, isoleucine, glycine, and glutamine were found to be non-essential. Glutamic acid, aspartic acid, α-alanine, proline, and hydroxyproline were found to be inhibitory in this test system. 2. A total amino acid level of approximately 100 mg. per cent was found to be optimal and DL-amino acids were found to be non-toxic, unless used in high concentrations. 3. A comparison has been made of the amino acid requirements of various types of tissue cultures, of the chick, and of man and certain differences in these requirements have been discussed.     

Isolation and partial characterization of intestinal calcium-binding proteins from the cow, pig, horse, guinea pig, and chick.

1. Intestinal calcium-binding proteins have been isolated in high purity from mucosal tissue of the cow, pig, horse, guinea pig, and chick. The proteins from all species exhibit rapid, although not identical, electrophoretic mobilities and possesses high affinities for calcium. 2. The intestinal calcium-binding proteins of mammalian origin exhibit a molecular size of approx. 11 000 by calibrated gel filtration and 9000 on the basis of amino acid composition. The analogous chick protein was found to be about 27 000-28 000 molecular weight by these methods. 3. The amino acid composition of each intestinal calcium-binding protein has been determined and indicates a considerable degree of similarity, especially among the mammalian species. 4. Immunoassay procedures have failed to show any species cross-reactivity when tested against antiserum prepared in response to either the bovine or chick intestinal calcium-binding protein.     

Amino acid requirements and peptidase activities of Streptococcus salivarius subsp. thermophilus

The aim of this work was to investigate the relationship between amino acid requirements and peptidase enzyme systems in three Streptococcus salivarius subsp. thermophilus strains. A synthetic medium without nitrogen components and a milk (RD milk) without its non-protein nitrogen fraction were prepared with different mixtures of amino acids. The strains showed different amino acid requirements. Some amino acids proved to be essential, some were required, while others did not affect growth. In the synthetic medium, only leucine and glutamic acid were essential for growth. In RD milk, the amino acid requirements were found to be lower, with only the absence of glutamic acid causing complete inhibition of growth. Relationships between aminopeptidase activities of the strains and their amino acid requirements were observed. Strains with higher amino acid requirements were also found to express a wider range of peptidases.     

Molecular structure and functional testing of human L1CAM: an interspecies comparison.

The rodent, avian, and insect L1-like cell adhesion molecules are members of the immunoglobulin superfamily that have been implicated in axon growth. We have isolated an L1-like molecule from human brain and found that it also supports neurite growth in vitro. We have also cloned and sequenced the entire coding region of human L1CAM and found that it shows a very high degree of homology to mouse L1cam, with 92% identity at the amino acid level. This similarity suggests that L1CAM is an important molecule in normal human nervous system development and nerve regeneration. Overall, there is substantially less homology to chick Ng-CAM; they are 40% identical at the amino acid level but many regions are highly conserved. Comparison of the sequences from human, mouse, chick, and Drosophila indicates that the L1 immunoglobulin domain 2 and fibronectin type III domain 2 are strongly conserved and thus are likely functionally important.     

Structure of a cDNA for the pro alpha 2 chain of human type I procollagen. Comparison with chick cDNA for pro alpha 2(I) identifies structurally conserved features of the protein and the gene

Nucleotide sequences were determined for cloned cDNAs encoding for more than half of the pro alpha 2 chain of type I procollagen from man. Comparisons with previously published data on homologous cDNAs from chick embryos made it possible to examine evolution of the gene in two species which have diverged for 250-300 million years. The amino acid sequence of the alpha-chain domain supported previous indications that there is a strong selective pressure to maintain glycine as every third amino acid and to maintain a prescribed distribution of charged amino acids. However, there is little apparent selective pressure on other amino acids. The amino acid sequence of the C-propeptide domain showed less divergence than the alpha-chain domain. The 5' end or N terminus of the human C-propeptide, however, contained an insert of 12 bases coding for 4 amino acids not found in the chick C-propeptide. About 100 amino acid residues from the N terminus, two residues found in the chick sequence were missing from the human. In the second half of the C-propeptide, there was complete conservation of a 37 amino acid sequence and conservation of 50 out of 51 amino acids in the same region, an observation which suggested that the region serves some special purpose such as directing the association of one pro alpha 2(I) C-propeptide with two pro alpha 1(I) C-propeptides so as to produce the heteropolymeric structure of type I procollagen. In addition, comparison of human and chick DNAs for pro alpha 2(I) revealed three different classes of conservation of nucleotide sequence which have no apparent effect on the structure of the protein: a preference for U on the third base position of codons for glycine, proline, and alanine; a high degree of nucleotide conservation in the 51 amino acid highly conserved region of the C-propeptide; a high degree of nucleotide conservation in the 3'-noncoding region. These three classes of nucleotide conservation may reflect unusual features of collagen genes, such as their high GC content or their highly repetitive coding sequences.     

Effect of hyperosmolarity on the activity of amino acid transport system L in avian fibroblasts

The transport of selected neutral amino acids known as good substrates of amino acid transport System L has been studied in chick embryo fibroblasts exposed for 4 hours to hyperosmolar culture medium. The activity of the L system, as measured by initial rates of L-phenylalanine uptake, increased in hyperosmolarity treated cells when determined before any cell depletion of intracellular amino acids. This effect was lost after depletion but reappeared after reloading the cells with pertinent substrates of System L. This transport activity appeared to be related to the internal level of amino acids capable of exchange through System L. In hyperosmolarity-treated chick embryo fibroblasts a higher level of System L substrates was obtained during the reloading phase in comparison to control cells. This expanded amino acid pool reflected an increased activity of transport System A, an agency of amino acid mediation known to enlarge its capacity following a hyperosmolar treatment of chick embryo fibroblasts (see Tramacere et al., 1984). L-Methionine, a preferred substrate of both A and L systems, appeared to be involved in the coupling between the activity of amino acid transport Systems A and L in these cells.     

Globin synthesis in fibroblasts fused with erythroblasts. I. Avian fibroblasts.

Heterokaryons of chick embryo erythroblasts fused with other avian fibroblasts were studied with regard to globin production. After the incorporation of radioactive amino acids, soluble proteins were separated on SDS-urea polyacrylamide gels. There was a striking increase in radioactivity above background in the globin region from lysates of fusion cultures when compared with fibroblast cultures. This was maximal at 24 hours after fusion, and then declined. Electrophoresis on acid-or alkaline-urea gels further identified the material as globin chains. Tryptic digestion and fingerprinting revealed methionine-labeled peptides characteristic of chick embryo erythroblast globin. An apparent stimulation of globin chain synthesis by heterokaryons compared to erythroblasts was found to be due to a difference in the specific activity of the precursor amino acid pools in the different cell types.     

Molecular cloning of bovine and chick nerve growth factor (NGF): delineation of conserved and unconserved domains and their relationship to the biological activity and antigenicity of NGF.

Previous experiments with purified mouse and bovine nerve growth factor (NGF) have shown that the biological activities of these two NGFs are identical, whereas the immunological cross-reactivity of antibodies produced against the two NGF molecules is very limited. This observation, together with the fact that antibodies to mouse NGF do not affect the development of sympathetic and sensory neurons in chick embryos, suggests that the domain of the NGF molecules responsible for the biological action has been highly conserved during evolution, whereas other domains determining the immunological properties were under less rigorous evolutionary constraint. The nucleotide sequences of bovine and chick NGF were determined from a cDNA clone prepared from mRNA of bovine seminal vesicles and from cloned chick genomic DNA, and the amino acid sequences deduced therefrom were compared with the available sequences of mouse and human NGF. All six cysteine residues were conserved in agreement with the previous finding that the biological activity of NGF is conformation-dependent requiring intact disulfide bridges. Amino acid changes are mainly confined to hydrophilic regions expected to be potential antigenic determinants, thus providing an explanation for the poor immunological cross-reactivities between the different NGFs. One single hydrophilic region is conserved in all NGFs and this region could be involved in the biological activity. The carboxy termini of bovine and chick NGF differ from that of mouse NGF, the changes in the amino acid sequences suggest that chick and bovine NGF are probably not processed by the gamma-subunit and that no 7S complex can be formed as in the mouse submandibular gland.     

3H]kainic acid binding sites in chick cerebellar membranes

1. A total particulate fraction of chick cerebellar membranes, obtained by a simple method, has been found to specifically bind [3H]kainic acid. Non-neuronal tissue, like chick liver, does not show any appreciable specific binding under the same experimental conditions. 2. Specific [3H]kainic acid binding to chick cerebellar membranes increases linearly with tissue concentration, reaches the binding equilibrium almost instantaneously and is pH and temperature dependent. 3. Specifically bound [3H]kainic acid is displaced by suitable concentrations of unlabelled kainic acid, L-glutamic acid and other excitatory amino acid analogues, both agonist and antagonist. This pharmacological pattern agrees with the general pharmacological properties of kainic acid receptors. 4. Saturation kinetic studies of kainic acid binding sites show one single binding mode with an apparent dissociation constant KD = 278 nM and a maximum number of binding sites of 187 pmoles/mg of protein. 5. In view of the mentioned data and the high amount of receptor sites found in chick cerebellar membranes, as compared with related values in rat cerebellum, we suggest that these receptors play a different physiological role or that they have a different cellular localization in chick and rat cerebellum.     

The Cloning and Characterization of Chick Tyrosinase from a Novel Embryonic cDNA Library

Very little is known about the genes involved in the regulation of avian skin and feather pigmentation. In mammals, two gene families have been identified as being important for the regulation of melanin biosynthesis. To isolate the avian equivalents of these families, we have generated an embryonic chick melanocyte cDNA library. Neural crest cells from 500 black chick embryos were cultured under conditions supportive of melanocyte differentiation and proliferation. A cDNA library was constructed and screened with a mouse tyrosinase cDNA probe. Nineteen clones were obtained, seven of which cross-hybridized to a mouse tyrosinase cDNA on Southern blots. The longest of these clones, B8.3 (1.9 kb), was sequenced and found to share 99.7% nucleotide and 99.8% amino acid sequence homology to a reported chick tyrosinase cDNA. Both Northern blot analysis andin situhybridization demonstrated that clone B8.3 was expressed in the retinal pigment epithelium of chick embryos. Our results suggest therefore that the cDNA library described here may allow the cloning of novel melanogenic genes.     

Ketone body utilization in duodenum. Differential effect of fasting on lipogenesis from acetoacetate and 3-hydroxybutyrate

The effect of fasting and refeeding on oxidation, lipogenesis and amino acid synthesis from ketone bodies has been studied in neonatal chick duodenal mucosa. Oxidation and amino acid synthesis were higher from acetoacetate and were stimulated by fasting from both 3-hydroxybutyrate and acetoacetate. On the contrary, lipogenesis was always higher from 3-hydroxybutyrate and fasting reduced lipogenesis rate from acetoacetate (by 66%) but not from 3-hydroxybutyrate. Results suggests the existence of a cytosolic fast-dependent acetoacetyl-CoA synthetase in chick duodenal mucosa which is involved in phospholipid synthesis.     

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.     

Glycine-serine interrelations in the chick.

Glycine and serine are involved in numerous important functions in the body in addition to protein synthesis. Glycine is synthesized by higher animals; however, the rate is not adequate to support maximal growth of the chick. Studies indicate that chicks fed a glycine-serine free crystalline amino acid diet grow at approximately 80% the rate of chicks fed the same diet supplemented with glycine. An equimolar quantity of L-serine has been shown to support equal chick performance as glycine, thus indicating that serine conversion is adequate to meet the dietary need for glycine. The serine-glycine interconversion is catalyzed by the folic acid containing enzyme sering hydroxymethyltransferase, and a deficiency of this vitamin decreases the effectiveness of serine in meeting the chick's dietary need for glycine. Studies with chicks fed normal and high levels of a crystalline amino acid mixture devoid of glycine and serine indicate that the chick has the metabolic potential to synthesize more of these two amino acids than should be required for normal growth. The observed dietary response to glycine or serine or both indicates, however, that this synthetic potential is not being utilized.     

Purification and properties of an 18-kilodalton, 1,25-dihydroxyvitamin D3 modulated protein from embryonic chick intestine

An 18,000-dalton protein (pI = 5.1) shown previously to be modulated by 1,25-dihydroxyvitamin D3 was purified to allow its further characterization. This protein from embryonic chick intestine was shown to comigrate during two-dimensional electrophoresis with an abundant protein from the intestine of 4-week-old chickens. The protein was purified from 4-week chick intestine and analyzed for amino acid composition, and 28 amino acids of its N-terminal sequence were determined. The N-terminal amino acid sequence had significant homology to cellular retinol binding protein II, an intestinal protein that has been recently sequenced. The purified 18-kilodalton protein was shown to bind retinol by fluorescence spectrophotometry. This 18-kilodalton protein is dramatically changed by 1,25-dihydroxyvitamin D3 in the chick embryonic organ culture system. Therefore, further study of it may lead to a better understanding of vitamin A and D interaction and how 1,25-dihydroxyvitamin D3 acts through proteins to stimulate intestinal calcium and phosphate transport.     

Isolation of tropoelastin a from lathyritic chick aortae.

Tropoelastin a was isolated from lathyritic chick aortae by using severe denaturing conditions for the initial extraction. The amino acid composition of this new species of tropoelastin is elastin-like in its high proportion of proline, glycine, alanine and valine. However, it differs from authentic tropoelastin b in containing a higher percentage of polar amino acids and cysteine residues. In addition, the amount of proline hydroxylation is 3 times higher than that found in chick tropoelastin b.     

Extracellular matrix metabolism by chondrocytes. 7. Evidence that L-glutamine is an essential amino acid for chondrocytes and other connective tissue cells

The incorporation of [3H]glycine into acid-insoluble protein and of [3H]acetate into glycosaminoglycans by cultured chick chondrocytes was stimulated by the addition of L-glutamine to the incubation medium. The effect of exogenous L-glutamine on protein synthesis was studied further by examining changes in the sedimentation patterns on sucrose gradients of ribosomes isolated from chondrocytes incubated in presence and absence of L-glutamine. It was found that the absence of L-glutamine caused a disaggregation of polyribosomes that was revered by the addition of this amino acid to the culture medium. No detectable glutamine synthetase activity could be measured in avian articular cartilage. These results indicate that L-glutamine is an essential amino acid for cartilage in that an extracellular supply of this amino acid is required for the maintenance of protein and glycosaminoglycan synthesis. A dependence of L-glutamine was also demonstrated for other avain connective tissues.     

Characterization of an associated microfibril protein through recombinant DNA techniques.

The complete primary structure of a new extracellular protein associated with elastic fiber microfibrils was determined by recombinant DNA techniques. Antiserum to insoluble bovine ocular zonule protein was used to screen a lambda gt11 cDNA expression library constructed from whole chick embryo poly(A)+ RNA. The cDNAs encoding immunoreactive fusion polypeptides were then used to rescreen the library by plaque hybridization. Nucleotide sequencing of overlapping cDNA clones revealed an open translation reading frame of 1326 bases beginning at an initiation start sequence and ending at a stop codon. The contiguous cDNA sequence contains a 3'-untranslated region of 563 bases with a possible polyadenylation site 16 bases upstream from the poly(A) tail. Primer extension of chick aortic mRNA taken together with the sequence data, reveals a 5'-untranslated region of 95 bases extending upstream from the translation start site. Northern blot analyses indicated that the isolated cDNA hybridized with a 2.1-kilobase mRNA in preparations of whole chick embryo and chick embryonic aortic, heart, and muscle RNAs. The initial translation protein encoded by the cDNA is 53,932 kDa and possesses a hydrophilic amino acid composition with glutamic acid comprising 22% of the total amino acid residues. Antiserum was elicited to a synthetic peptide sequence (14 amino acids) encoded within the deduced protein primary structure. Western blots of extracted proteins from chick embryonic aortae cultured in the presence of beta-aminopropionitrile showed that the medium and a mild salt extract contained an immunoreactive protein possessing an apparent molecular mass of 58,000 whereas harsh denaturants extracted a 32,000-kDa protein. Pulse-chase experiments using radiolabeled lysine showed that the newly synthesized 58,000-kDa protein was chased into a 32,000-kDa protein within a 2-24-h period. Immunoelectron microscopy of tissue sections from chick aortae, bovine nuchal ligament, and human ocular zonules showed that the peptide-elicited antibody localized specifically to ultrastructurally definable microfibril structures.     

Amphibian cells in culture. I. Nutritional studies.

Nutritional requirements of amphibian cells in culture were studied for the purpose of modifying a minimal medium in which frog cells could proliferate and which could be used for obtaining drug-resistant and auxotrophic variants. The serum, purine, CO2, and amino acid requirements for ICR 2A (a Rana pipiens haploid cell strain) have been investigated employing two different media: L-15, a nonbicarbonate, amino acid-buffered medium and Eagle's MEM, a bicarbonate-buffered medium. In this paper we present evidence to support the following conclusions: (1) With L-15 as the base medium, 10% fetal calf serum (FCS) supports optimal cell growth during exponential phase. Calf serum, whole, dialyzed, or heat-inactivated, cannot substitute for FCS and, in fact, is inhibitory. (2) Purines are required by ICR 2A cells only if grown in a nonbicarbonate-buffered medium, since the cells under these conditions cannot produce enough endogenous CO2 to support de novo purine synthesis. (3) In addition to the amino acids considered essential for mammalian cells in culture, ICR 2A cells depend upon exogenous asparagine. Glutamine and/or aspartic acid cannot replace the asparagine requirement. However, ICR 2A cells do utilized exogenous glutamine as an oxidative substrate.     

Identification of a novel isoform of the retinoic acid receptor gamma expressed in the mouse embryo.

Retinoic acid is known to have profound effects on developmental processes. It has been implicated as a putative morphogen in the developing chick limb bud and regenerating amphibian limb blastema and has been demonstrated to have powerful teratogenic effects in mammals, including humans. Recently, three specific retinoic acid receptors (RARs), RAR alpha, -beta, and -gamma, were identified and shown to be members of the steroid receptor superfamily. We report the identification of a novel RAR gamma isoform, mRAR gamma B, which differs from the previously described mouse RAR gamma at its amino terminus. In addition, we show that both RAR gamma isoforms are expressed maximally at midgestation in structures known to be affected adversely by retinoic acid administration to pregnant mice. Multiple RAR isoforms, each of which may play a unique or combinatorial role as a regulator of mammalian development, are thus expressed in the mouse embryo.