Synthesis Of Serum Proteins Research Articles

Synthesis of serum proteins by cultures of chick embryo yolk sac endodermal cells

Endodermal cells were isolated from yolk sacs of 3-day chick embryos and cultured for 6 days in Eagle's minimal essential media plus 10% fetal calf serum. During this period cells rapidly lost their ability to synthesize DNA as judged by [ 3H]thymidine incorporation into DNA. In spite of this loss of DNA synthesis serum protein synthesis and secretion remained at a constant 45% of total protein synthesis and secretion. This was determined by immunoprecipitation of culture media using antibodies directed against embryonic chick serum proteins. Media were also analyzed for the synthesis and secretion of specific serum proteins using polyacrylamide gel electrophoresis. The relative synthesis and secretion of the individual serum proteins followed that previously observed in ovo with the exception of α-globulin-a which became undetectable. When culture media were supplemented with ovalbumin or insulin the relative synthesis and secretion of certin specific serum proteins were altered. However, analysis of these same media samples showed that the total amounts of serum protein synthesis and secretion were unaffected.

A fine structural and cytochemical study of the rhesus monkey yolk sac: endoderm and mesothelium.

This study has examined the fine structure and some cytochemical characteristics of the endodermal and mesothelial cells of the rhesus monkey yolk sac between 25 and 66 days of gestation. The endodermal cells were characterized by abundant granular endoplasmic reticulum, some agranular endoplasmic reticulum, a well-developed Golgi apparatus, and numerous large mitochondria. During the earlier part of the period studied, endodermal cells had a few acid phosphatase and arylsulfatase-positive lysosomes and moderate numbers of catalase-positive microperoxisomes. During the later stages of development, large granules (believed to be lysosomes) with a heterogeneous content were numerous in the cytoplasm. Mesothelial cells showed fewer development changes. Throughout this period they were usually flattened cells with long microvilli, small mitochondria, and limited amounts of granular endoplasmic reticulum. The mesothelial cells had acid phosphatase reaction product in the Golgi region and occasional large vesicles, but were negative for arylsulfatase and catalase. One specimen was incubated at 37 degrees C in the presence of horseradish peroxidase in order to examine endocytosis. Both the mesothelial cells and endodermal cells internalized the peroxidase into a variety of cytoplasmic vesicles. Based on their cytology, the endodermal cells may function in the synthesis of serum proteins during this period, as has been suggested in other species. They may also be involved in lipid metabolism. The mesothelial cells appeared less synthetically active, but evidence suggested that they may be involved in collagen and extracellular matrix production. The endocytic activity displayed by both cell types may indicate a role in fluid and metabolite transfer across the epithelia. The cytology of both cell types was very similar to that described for human yolk sacs, suggesting that the rhesus monkey may be a useful species in which to study the maturation of yolk sac function.

Synthesis of larval serum proteins 1 and 2 of Drosophila melanogaster by third instar fat body

Larval serum proteins (LSPs) 1 and 2 of Drosophila melanogaster have been shown to accumulate in third instar larval haemolymph ( Roberts et al., 1977). In the present paper the major third instar larval tissues have been assayed for LSP synthesis. Polypeptides radiolabelled in organ culture have been analyzed by SDS-polyacrylamide gel electrophoresis and by immunoelectrophoresis. Fat body was shown to be the major source of LSPs 1 and 2 and the two proteins were synthesized by all regions of third instar fat body.

Thermal trauma-induced changes in the synthesis of rat serum proteins

1. 1. Infliction of non-lethal scalding was found to cause an increase in the rate of incorporation of 35S-methionine into albumin and globulin fractions of rat serum proteins, the increase being remarkably higher for α- and β-globulins than for albumin and γ-globulins 2. 2. The most pronounced enhancement was associated with the 60- and 70-kd constituents of β-globulins, the 34-, 55- and 136-kd polypeptides of α-2-globulins and the 50-, 60-, 125- and 145-kd polypeptides of α-1-globulins. 3. 3. The response of serum proteins to the heat shock was similar, though the changes were significantly less pronounced.

Serum protein synthesis by somatic cell hybrids.

Serum protein synthesis by rodent hepatomaa x fibroblast, hepatom a x teratocarcinoma, and hepatoma x hepatoma somatic cell hybrids was analyzed by Laurell (rocket) and crossed immunoelectrophoresis. With the hepatoma x fibroblast hybrids, of the nine serum proteins investigated, only transferrin was synthesized by the hybrids (with the exception of one hybrid clone which made albumin). Rat hepatoma (MHC) x mouse teratocarcinoma hybrids did not synthesize transferrin, but one of three clones did produce albumin. Rat hepatopma (Faza) x mouse hepatoma (Hepa 1) hybrids synthesized reduced amounts of albumin and transferrin, compared with the parental hepatoma cell lines. From these studies transferrin synthesis appears to be under different controls from the other serum proteins examined.

Synthesis of serum and cytosol vitamin D-binding proteins by rat liver and kidney.

The synthesis of vitamin D-binding proteins in rat was examined using liver and kidney slices, isolated hepatocytes, and isolated renal tubules. Rat liver synthesized both serum vitamin D-binding protein (DBP) and a tissue DBP-binding component and secreted serum DBP. Rat kidney also synthesized a tissue DBP-binding component and cytosol vitamin D-binding protein (CDBP) which was immunologically related to serum DBP, but secretion of renal CDBP was not observed. Renal CDBP had a higher molecular weight (approximately 65,000) than that of serum DBP (54,000) and was heat-labile, while serum DBP was heat-stable (60 degrees C, 60 min). Renal CDBP was considered to exist in cytosol, forming a complex with a tissue DBP-binding component. These results indicate that liver is a site of synthesis of serum DBP and that CDBP, which is immunologically related to but physicochemically different from serum DBP, is synthesized in situ in kidney.

Use of hepatocytes in primary culture for biochemical studies on liver functions.

Rat parenchymal hepatocytes isolated with collagenase were cultured as monolayers in Williams medium E supplemented with calf serum. Freshly isolated cells showed very low activities of various liver functions, and they had to be cultured for 6-24 h to allow recovery of these functions. Insulin and dexamethasone greatly increased cell viability in primary. After culture for 24 h, these cells showed various liver functions as seen in vivo and responded well to various added hormones and amino acids. The concentrations of amino acids in the medium regulated synthesis of serum proteins and insulin stimulated lipogenesis, which in turn regulated synthesis of lipoproteins. Insulin also stimulated glycogen synthesis and the stimulation was parallel with the number of insulin receptors. Glucagon stimulated glycogenolysis and its stimulation involved the function of the cytoskeleton. Glucagon and dexamethasone induced various enzymes of amino acid catabolism, such as tryptophan oxygenase, tyrosine aminotransferase and serine dehydratase. These inductions were inhibited by insulin or catecholamine. The effect of catecholamine was due to its alpha-adrenergic action. The beta-action of isoproterenol was low in freshly isolated cells, but increased during culture of the cells. Acquirement of hormonal responses during neonatal development can be studied in this culture system. Mature hepatocytes in culture are usually quiescent, but when insulin and epidermal growth factor were added, DNA synthesis by the cells increased markedly and they showed density-dependent growth. In this culture system, serum could be omitted for 2 days when the dishes were coated with fibronectin without appreciable change of functions, but serum was needed for longer culture of the cells. A factor that increased cell survival was found in serum and in pituitary gland. These results show that hepatocytes in primary culture are a simple and useful system for studies of liver functions in vitro and related works were also reviewed.

Stimulation of albumin synthesis in mouse hepatoma cells by Bt 2cAMP: Cell cycle specificity

Addition of 1 m m dibutyryl cyclic AMP (Bt 2cAMP) to cultures of mouse hepatoma cells, Hepa, specifically stimulates the synthesis of serum proteins including albumin. This stimulation is accompanied by an inhibition of cell proliferation. We have investigated these phenomena in synchronous cultures of Hepa. Proliferation of Hepa was arrested by isoleucine starvation. Synchronous growth was initiated by addition of complete growth medium or complete growth medium supplemented with 1 m m Bt 2cAMP. S phase and mitosis were estimated by determinations of [ 3H]thymidine incorporation and by cell numbers. The rate of albumin synthesis relative to total protein synthesis was measured by pulse labeling cultures for 30 min with [ 3H]leucine and comparing amounts of immunoprecipitable label with trichloroacetic acid-precipitable label. Treatment of synchronous cultures with Bt 2cAMP did not alter the duration of S phase or the onset of mitosis. The relative rate of albumin synthesis in Bt 2cAMP-treated culture began increasing after mitosis. The timing of the Bt 2cAMP stimulation of albumin synthesis was further investigated by adding Bt 2cAMP to cultures of Hepa at various times after the initiation of synchronous growth. The relative rate of albumin synthesis was then measured at a fixed postmitotic time. An increased relative rate of albumin synthesis was observed only in cultures exposed to Bt 2cAMP before or during S phase. Thus the postmitotic increase in the synthesis of albumin requires the presence of Bt 2cAMP during S phase.

The effects of repeated thermal injury on rat liver and serum protein synthesis rates

1. 1. The effects of single and repeated thermal injury on liver cells and blood serum proteins metabolism were investigated in experiments performed on rats. 2. 2. During the first three postburn days amino acid incorporation into nuclear and mitochondrial proteins proceeded at an increased rats, whereas the effects of the repeated injury dependent on time interval elapsed between two injuries.

Protein synthesis with membrane-bound polysomes and albumin messenger RNA from livers of mutant mice.

Investigation of deficiencies in serum protein synthesis resulting from deletion-mutations at the albino locus in mice was continued using in vitro conditions. Previous work showed that although total protein synthesis was only slightly lower in livers from albinos, newly synthesized protein appearing in plasma was 22% of that in controls. It was thought that the disorganized endoplasmic reticulum and Golgi apparatus, characteristic for the liver (and kidney) of these mutants, might be responsible for the observed deficiencies. In the present study membrane-bound polysomes isolated from the livers of newborn albinos were 55% (c3H/c3H strain) and 62% (c14CoS strain) as efficient as those from normal littermates in incorporating radioactive leucine into protein in a cell-free system. These differences could not be eliminated by the addition of excess liver mRNA, exogenous soluble factors or by the exchange of cell sap between albino and control polysomes. In an earlier study albino liver slices synthesized only 13% (or 17% per mg of total protein synthesized) as much albumin as controls. We have now found that the level of albumin poly(A)+-RNA isolated from albino livers assayed with a reticulocyte lysate, was almost as high (85%) as in controls. It was concluded that the very low level of albumin in albino livers did not result from a deficiency of albumin mRNA. Whether the rate-limiting step in synthesis of albumin in mutant livers is at the level of translation or processing for secretion requires further investigation.

Effects of cyclic AMP on the kinetics of serum protein synthesis in cultured mouse hepatoma cells

The mouse hepatoma cell line, Hepa, was cultured in the presence of either 1 mM N 6, O 2′ -dibutyryl cyclic AMP (Bt 2cAMP), 0.5 mM 3-isobutyl-1-methylxanthine, or 1 μg/ml cholera toxin. The synthesis and secretion of albumin, α-fetoprotein, and transferrin were elevated above controls by 24 h reaching two- to fourfold stimulations within 72 h. These effects were reversible and were specific for the serum proteins. The stimulation of serum protein synthesis was accompanied by a decrease in the rate of cell proliferation. Protein synthetic parameters were analyzed in Hepa cells 72 h after exposure to N 6,O 2′ -dibutyryl cyclic AMP. The cellular rate of albumin synthesis was increased fourfold and the relative rate of albumin synthesis was increased approximately threefold. N 6,O 2′ -Dibutyryl cyclic AMP did not affect the size distribution of either total Hepa polyribosomes or of albumin-synthesizing polyribosomes. The elongation rate on total mRNA and on albumin mRNA was decreased by approximately 40%. These results indicate that the rate of initiation of total Hepa mRNA and of albumin mRNA also decreased by 40%. The nonspecific nature of the N 6,O 2′ -dibutyryl cyclic AMP effect on Hepa protein synthetic parameters must be due to an alteration in the level of a common substrate, perhaps ATP. The specific threefold increase in the relative rate of albumin synthesis with no alteration in polyribosome sizes requires a threefold increase in the relative amount of functional albumin mRNA in Hepa cells. This prediction was confirmed by cell-free translation of Hepa polyribosomes.

Bone marrow participates in the biosynthesis of human transcobalamin II

Studies concerning the site of synthesis of the vitamin B12 binding serum protein, transcobalamin II, have been done in various mammalian animals. The actual site of biosynthesis in man has not yet been defined. The finding that bone marrow derived cells release apo- transcobalamin II in the mouse led us to examine the genetic patterns of transcobalamin II in man, both before and after marrow transplantation. A gradual but incomplete transformation of the recipient's transcobalamin II type into donor's type, corresponding to 75% or less of the total activity, was registered in 4 cases. Surprisingly, persistent host-type TC II, in spite of different donor type, was observed in 4 further marrow recipients. We conclude that hematopoietic cells transferred with the transplanted marrow participate in the biosynthesis of human transcobalamin II.

Bone Marrow Participates in the Biosynthesis of Human Transcobalamin II

Studies concerning the site of synthesis of the vitamin B12 binding serum protein, transcobalamin II, have been done in various mammalian animals. The actual site of biosynthesis in man has not yet been defined. The finding that bone marrow derived cells release apo-transcobalamin II in the mouse led us to examine the genetic patterns of transcobalamin II in man, both before and after marrow transplantation. A gradual but incomplete transformation of the recipient's transcobalamin II type into donor's type, corresponding to 75% or less of the total activity, was registered in 4 cases. Surprisingly, persistent host-type TC II, in spite of different donor type, was observed in 4 further marrow recipients. We conclude that hematopoietic cells transferred with the transplanted marrow participate in the biosynthesis of human transcobalamin II.

Yolk sac endoderm: Exclusive site of serum protein synthesis in the early chick embryo

In order to determine which cell type or types synthesized serum proteins, yolk sacs from 4-day chick embryos were manually separated into ectoderm, mesoderm, and endoderm and incubated in the presence of radioactive valine. Analysis of the incubation media by polyacrylamide gel electrophoresis as well as by immunoprecipitation showed that all serum proteins were synthesized exclusively by the cells of the endoderm. These included transferrin and three embryo-specific serum proteins: α-globulin a, α-globulin b, and prealbumin.

Synthesis of serum proteins by the posthaematopoietic feline yolk sac.

The feline yolk sac persists even after the end of its haematopoietic phase with prominent ER-cisternae in the endoderm suggesting biosynthetic capacity. Therefore, yolk sac explants from the 54th day and 57th day were incubated with [3H]-L-leucine in order to study its protein biosynthesis. Newly synthesized proteins were discovered in sliced SDS-polyacrylamide gels by the use of scintillation technique and identified by molecular weight determination and isoelectrofocusing, also using stained electropherograms of unlabeled tissue, serum, and marker proteins. The highest radioactive incorporations were found in 69,000--70,000 dalton proteins and interpreted as serum albumin and alpha-fetoprotein. The autoradiography revealed that the cytoplasm of the endoderm is the site of the most active biosynthesis of proteins which were obviously stored in the ER-cisternae for some time. The yolk sac fluid proteins are almost exclusively serum proteins, although in a very low concentration. We regard a large-scale formation of serum proteins in the yolk sac endoderm as the cause of this organ's very late regression in the cat.

Growth control of normal and transformed cells

Both serum factors and protein synthesis are required for normal cell growth. Swiss 3T3 cells require the serum growth factors insulin and EGF (epidermal growth factor) during the initial part of the G1 period, until they pass a restriction point about 2 h before the initiation of DNA synthesis. Concentrations of cycloheximide that inhibit protein synthesis by as much as 70% dramatically lengthen the cell cycle before the restriction point, while the cell cycle after the restriction point remains nearly constant. These results are consistent with a model in which labile proteins are required for transit of cells past the serum-sensitive restriction point. The relation of these findings to the growth control of transformed cells is discussed.

Protein Synthesis in Yolk Sac Tumor: Histochemical Studies of Human and Rat Yolk Sac Tumor

Synthesis of various serum proteins, such as albumin, prealbumin, α1‐antitrypsin, transferrin and α‐fetoprotein, by human and rat yolk sac tumors was studied by indirect immunofluorescence and immunoperoxidase techniques. These serum proteins were found to be present in the cytoplasm of tumor cells forming the lining of vitelline cysts, as well as Schiller‐Duval bodies and vacuolated meshwork on histological sections. Two of these tumors were cultured. Immunopathological studies revealed that albumin, prealbumin, α1‐antitrypsin, transferrin and α‐fetoprotein were found in the cytoplasm of these cultured tumor cells. The immunofluorescence of these proteins was also found on the PAS‐positive granules, which existed intra‐ and extra‐cellularly.The ability to synthesize these serum proteins of these tumor cells was not affected by tissue culture and was maintained during subcloning.It was demonstrated by electron microscope ferritin antibody technique that α‐fetoprotein was mainly synthesized on the ribosomes of the rough endoplasmic reticula of rat yolk sac tumor cells.

Effect of Low Lysine Diet on Rat Protein Metabolism

Prolonged lysine deficiency resulted in inhibition of growth of the rat and in lowered levels of albumin and beta-globulins, but not of total liver proteins. The apparent incorporation of [3H]lysine into total and chromosomal liver proteins, however, was increased in lysine deficient rats relative to pair-fed controls. The concentration of free lysine in neither serum nor liver was altered in lysine deficient rats. The specific radioactivity of liver free lysine 2 hours after administration of [3H]lysine was noticeably elevated in the deficient rats. However, when the pool of liver free lysine is considered, synthesis of all proteins studied was inhibited. The largest inhibitory effect was seen in serum albumin and total liver protein synthesis. Inhibition of snythesis of nuclear chromatin protein was less pronounced. The data indicate an adaptive mechanism operates to preserve liver lysine in rats fed lysine deficient diets for a prolonged period. In spite of retention of lysine, the synthesis of liver and serum proteins was inhibited.

Effect of tunicamycin on the secretion of serum proteins by primary cultures of rat and chick hepatocytes. Studies on transferrin, very low density lipoprotein, and serum albumin

Using rat or chick hepatocyte monolayers, we have studied the effect of tunicamycin, a specific inhibitor of protein glycosylation, on the synthesis and secretion of serum proteins. Tunicamycin inhibited glucosamine incorporation into rat liver transferrin and the apoprotein B chain of chick liver very low density lipoprotein (VLDL) by 75 to 90%. In contrasts, amino acid incorporation into these two glycoproteins, as well as into the normally unglycosylated proteins, rat serum albumin and apoprotein A of chick liver VLDL, was decreased by only 10 to 25% in the presence of the antibiotic. Despite the inhibitory effect of tunicamycin on glycosylation, secretion of all four proteins was virtually unimpaired. Thus, the carbohydrate moieties of rat liver transferrin or apoprotein B of chick liver VLDL do not appear to play an essential role in the secretion process.

Alpha-fetoprotein, prealbumin, albumin, alpha-l-antitrypsin and transferrin as diagnostic and therapeutic markers for endodermal sinus tumors

According to Gitlin, alpha-fetoprotein (AFP), albumin, prealbumin, alpha-1-antitrypsin and transferrin are normal products of the human yolk sac. They are expected to reappear in human endodermal sinus tumor (yolk sac tumor). The synthesis of alpha-fetoprotein and other serum proteins by human endodermal sinus tumor was studied in the culture cells and in the tumor tissue transplanted into nude mice. The results gave evidences of synthesis of some of these proteins including alpha-fetoprotein and alpha-1-antitrypsin. Serum concentrations of these proteins were studied in eight children having endodermal sinus tumors. Serum AFP levels were abnormally high in all cases, whereas concentrations of other serum proteins were almost within normal ranges. This might be simply reflected by the fact that pre-albumin, albumin, alpha-1-antitrypsin, and transferrin are already present in large quantities in sera of normal subjects while alpha-fetoprotein is present only in a negligible quantity. Alpha-fetoprotein, as a diagnostic and therapeutic marker of endodermal sinus tumor, showed good correlation to the tumor growth. Serum AFP concentrations declined almost to 0 ng/ml with a half-life of 4 days when surgical removal was complete, whereas serum AFP decreased only to 100-200 ng/ml with radiation and chemotherapy alone.