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, alpha-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.     

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.     

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.     

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.     

The Role of Dispensable Amino Acids for the Maximum Growth of Chick

Chicks were fed on the purified diets of which amino acid pattern was modeled after whole egg protein and crude protein content was 21.1%, changing the dietary ratio of indispensable amino acid nitrogen to dispensable amino acid nitrogen (I/D ratio) from 1/1.5 to 3/1 at regular intervals. The balances among amino acids in each indispensable and dispensable group of test diets were kept the same pattern as that of the whole egg, respectively. Optimum I/D ratio for normal chick growth was estimated to be in the range of between 1/1 and 1.5/1, because feed efficiency was the highest at the I/D ratio 1/1 and growth rate was the highest at the I/D ratio 1.5/1.

Chicks were killed and the serum was collected at the end of the experiment. It was shown that the I/D ratios of free amino acid in the serum of chicks were strongly influenced by that of diet.

White Leghorn chicks fed on the Scott’s reference amino acid diet grew as well as those fed on a conventional chick starter. Nitrogen retention of the former was a little less than that of the latter, but the amount of carcass fat of the former was almost twice as much as the latter.

Growth rate of chick was considerably reduced, when glutamic acid which is the only dispensable amino acid in the Scott’s diet was replaced by a mixture of glutamic acid, aspartic acid, alanine and serine, nitrogen content being kept at constant. Sufficient amount of glutamic acid in the Scott’s diet seems to be essential for the maximum growth of chick.     

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.     

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.     

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.     

THE EXTENT OF AXOPLASMIC TRANSPORT DURING DEVELOPMENT, DETERMINED BY MIGRATION OF VARIOUS RADIOACTIVELY-LABELLED MATERIALS

Abstract— Several isotopic precursors have been monocularly injected into chick embryos and into day-old or 15-day-old chicks. After various intervals, the incorporation of various isotopes into acid insoluble material within the retina of the injected eye and within the optic lobes, was determined. Radioactive proline and fucose were used as precursors of protein and glycoprotein respectively while uridine was used as an RNA precursor. The proportion of rapidly migrating proteins and glycoproteins was reduced during maturation. The extent of RNA migrating also appeared to decline during development. The proportion of synthesized protein that was transported was relatively constant and independent of the amino acid used. Around 30 per cent of retinally synthesized glycoprotein migrated distally and this migrating material appeared to contain very few sialic acid residues. A considerable amount of retinally synthesized gangliosides also appeared rapidly in the distal regions of the optic nerve.     

STUDIES OF THE INTER-RELATIONSHIP BETWEEN CEREBROSPINAL FLUID AND PLASMA AMINO ACID CONCENTRATIONS IN NORMAL INDIVIDUALS

Abstract— —The concentration of free amino acids has been determined in lumbar CSF in 37 fasting normal subjects. The values obtained have been compared with the concentration of the same amino acids measured in venous plasma collected simultaneously and with ventricular CSF amino acid concentrations. Twenty-three amino acids have been identified and quantitated in CSF and plasma. Trace quantities of eight other amino acids have been also detected.
The concentration of 13 amino acids in CSF has been shown to be directly related to the plasma concentration. No such relationship was noted for the other 7 amino acids. Significant variations in the concentration of individual amino acids relating to both age and sex have been noted. A large number of unidentified ninhydrin positive compounds have been found in CSF. Preliminary studies have identified one of these as ɛ-aminobutyric acid (GABA).     

Chick kidney ferredoxin: Complementary DNA cloning and vitamin D effects on mRNA levels

Vertebrate ferredoxin is non-heme iron-sulfur protein found in steroideogenic tissues that serves as an electron shuttle in mitochondrial mixed function oxidase systems such as the 25-hydroxyvitamin D₃-1α-hydroxylase. A 2530-bp chick kidney ferredoxin cDNA was cloned, and the association between ferredoxin mRNA levels and the regulation of 1α-hydroxylase activity by vitamin D status was examined. The cDNA sequence indicates that the chick kidney mitochondrial mixed function oxidases use the same ferredoxin as do those in the chick testis and that the chick ferredoxin shares greater than 92% amino acid homology with mammalian ferredoxins. Southern blot analysis of genomic DNA indicates that there is a single copy of the ferredoxin gene present in the chick genome. Three species of mRNA, 1.8, 3.5 and 5.5 kb, were identified by Northern analysis. Slot blot analysis of poly A⁺ RNA from kidneys of vitamin D-deficient or -replete chicks indicates a 40% induction of ferredoxin message levels in the vitamin D-deficient chick kidney. This suggests that gene regulation of ferredoxin may be part of the mechanism of regulation for 25-hydroxyvitamin D₃-1α-hydroxylase activity in the chick kidney.     

Synthesis and intracellular localization of chick acid α-glucosidase in chick erythrocyte-human fibroblast heterokaryons : A model system for the study of lysosomal enzyme synthesis

The synthesis and localization of chick acid α-glucosidase has been studied in chick erythrocyte-human fibroblast heterokaryons. Monospecific antibodies raised against purified chick liver acid α-glucosidase were used. It was found that the acid α-glucosidase in the heterokaryons is of chick origin, and is localized in the same lysosomes as the human lysosomal enzymes. It is concluded that chick erythrocyte-human fibroblast heterokaryons provide a useful model system for the study of lysosomal enzyme synthesis and routing.     

A Structural Domain Mediates Attachment of Ethanolamine Phosphoglycerol to Eukaryotic Elongation Factor 1A in Trypanosoma brucei

Ethanolamine phosphoglycerol (EPG) represents a protein modification that so far has only been found in eukaryotic elongation factor 1A (eEF1A). In mammals and plants, EPG is covalently attached to two conserved glutamate residues located in domains II and III of eEF1A. In contrast, Trypanosoma brucei eEF1A contains a single EPG attached to Glu362 in domain III. The sequence and/or structural requirements for covalent linkage of EPG to eEF1A have not been determined for any organism. Using a combination of biosynthetic labelling of parasites with tritiated ethanolamine and mass spectrometry analyses, we demonstrate that replacement of Glu362 in T. brucei eEF1A by site-directed mutagenesis prevents EPG attachment, whereas single or multiple amino acid substitutions around the attachment site are not critical. In addition, by expressing a series of eEF1A deletion mutants in T. brucei procyclic forms, we demonstrate that a peptide consisting of 80 amino acids of domain III of eEF1A is sufficient for EPG attachment to occur. Furthermore, EPG addition also occurs if domain III of eEF1A is fused to a soluble reporter protein. To our knowledge, this is the first report addressing amino acid sequence, or structure, requirements for EPG modification of eEF1A in any organism. Using T. brucei as a model organism, we show that amino acid substitutions around the modification site are not critical for EPG attachment and that a truncated version of domain III of eEF1A is sufficient to mediate EPG addition.     

Tissue culture studies; identification of components and synthetic replacements for the active fraction of chick embryo extract ultrafiltrate

Some of the compounds in the active fraction of ultrafiltrates of chick embryo extract have been identified as taurine, serine, glutamic acid, xanthine, uracil, glucose-6-phosphate, glucose, ferrous iron, and inorganic phosphate. Based on the identity of these compounds a synthetic replacement for the ultrafilterable portion of chick embryo extract has been devised. There is an additional nutritional requirement that can be met by vitamin B₁₂. Folic acid appears to be beneficial to the system though the requirements of this or any of the above compounds except vitamin B₁₂ remain for future research. The low nitrogen content of the isolated fraction and the synthetic mixture suggests that the main nutrition of chick cells in roller tube cultures is derived from the non-dialyzable portion of the medium.     

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.     

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.     

Osmoregulation of amino acid transport activity in cultured fibroblasts

The effect of exposure of chick embryo cells to increasing concentrations of Na⁺ in the culture medium on the subsequent amino acid transport as determined at physiological osmolarity was investigated in detail. It was found that the hyperosmolar treatment stimulated amino acid transport in a dose-dependent manner up to 200 mM Na⁺. Changes were measurable as early as 1 h after altering Na⁺ and reached a maximum after 4 h, remaining constant thereafter. The maintenance of this effect required continuous exposure of the cell to high Na⁺ in the culture medium. Hyperosmolarity-mediated increases in amino acid transport activity by system A have been detected with l-proline and l-alanine. Transport activities of systems ASC and L did not change appreciably after exposure of the cells to high Na⁺. Inhibition of protein synthesis by cycloheximide or RNA synthesis by actinomycin D (actD) prevented these uptake changes. Kinetic analysis indicated that the stimulation of the activity of transport system A by high Na⁺ treatment occurred through a mechanism affecting V_max rather than K_m.     

COMPARATIVE PEPTIDE MAPPING AND ISOELECTRIC FOCUSING OF ISOLATED SUBUNITS FROM CHICK EMBRYO BRAIN TUBULIN

The α- and β-subunits of chick embryo brain tubulin have been isolated under denaturing conditions and compared with respect to their molecular weight, amino acid composition, tryptic peptide maps, amide content and isoelectric focusing properties. An 8 M-Urea-containing polyacrylamide gel system with varying acrylamide concentrations was used for calculation of the retardation coefficients (K_R) of the tubulin subunits. A molecular weight of 53,000 was estimated for each subunit by comparison to K_R values for standard proteins. Amide contents of approx 41% of the carboxyl groups of α-tubulin and 48% of the carboxyl groups of β-tubulin were calculated using the average PI value, the pK_intrinsic for the ionizable side chains of the amino acids and the amino acid composition of each subunit. Comparative peptide maps of trypsin digested α- and β-tubulin demonstrated 16 peptides unique to each subunit and 23 peptides which comigrate. Both subunits give rise to multiple species on electrofocusing gels. The average isoelectric points for the α- and β-subunits are 5.4 and 5.2, respectively.     

THE NUTRITION OF ANIMAL TISSUES CULTIVATED IN VITRO : III. USE OF A DEPLETION TECHNIQUE FOR DETERMINING SPECIFIC NUTRITIONAL REQUIREMENTS

1. Cultivation of freshly explanted chick embryonic heart tissues in Hanks's salt solution for 3 to 4 days has been shown to create a general state of nutritional deficiency in the cultures. Provided the depletion is not prolonged beyond 4 days, the cultures subsequently revive and survive to a normal period in synthetic medium M 150. 2. Paper chromatographic studies on the culture medium have shown that the amino acid metabolism of the depleted cultures is restored to a normal pattern within a few days in medium M 150. 3. By the use of the nutritional deficiency technique, a coenzyme A requirement for this type of culture has been established. 4. The application of these findings to tissue cell nutrition and the possible hazards of using serum, or other uncharacterized additions, either to establish cultures, or as part of the experimental medium, are discussed.