Investigation of the content of alpha-fetoprotein and serum albumin in the vitreous body of the eye of human embryos

The content of serum albumin and alpha-fetoprotein in the vitreous body of the eyes of human fetuses from the 16th through the 24th week was investigated. It was detected that albumin and alpha-fetoprotein in the vitreous body of human eyes are presented in equal molar concentrations in the 16th week. There is 1.5-fold increased concentration of alpha-fetoprotein in comparison to albumin during the 17th week. After the 17th week, there was a reduction in the concentration of both proteins. It was reported that cyanine dye, used for detection of albumin, does not interact with alpha-fetoprotein.     

Control of albumin and alpha-fetoprotein expression in rat liver and in some transplantable hepatocellular carcinomas.

Albumin and alpha-fetoprotein production by rat liver and by four selected transplantable hepatocellular carcinomas is compared to the messenger RNA present in these tissues. Albumin and alpha-fetoprotein were measured by radioimmunoassay of serum concentration, immunofluorescence, and in vitro incorporation of labeled amino acids into proteins specifically precipitated by antisera. The number of mRNA molecules per cell was calculated from the hybridization of specific cDNA probes to polysomal mRNA and by translational activity of polysomal RNA in a wheat germ system. The amount of albumin and alpha-fetoprotein produced by the different tissues is directly related to the number of functional mRNA molecules per cell for each protein.     

Effect of 5-azacytidine on rat liver alpha-fetoprotein gene expression

Neonatal rats given 5-azacytidine intraperitoneally (30 micrograms/animal/day) on days 1-5 postpartum had 55% lower serum alpha-fetoprotein levels on day 6 compared to saline injected controls. On day 14, alpha-fetoprotein levels were 4-fold lower in 5-azacytidine treated animals. Cytosol alpha-fetoprotein was proportionately reduced. There were no significant changes in liver to body weight ratio, total serum protein, and both serum and cytosol albumin levels. The molecular basis for decreased serum alpha-fetoprotein levels was found to be a reduced concentration of alpha-fetoprotein mRNA in the livers of 5-azacytidine injected animals. These results are discussed with respect to the effects of 5-azacytidine on DNA methylation and cell differentiation.     

Effects of various factors on the growth and function of a human hepatoblastoma cell line, HuH-6--an approach for serum-free cell culture

The effects on a human hepatoblastoma cell line (HuH-6) of serum and Ca2+ were investigated. A higher concentration of serum reduced the production of alpha-fetoprotein, whereas Ca2+ enhanced that of both albumin and alpha-fetoprotein. In view of these facts, a serum-free medium using RPMI-1640 supplemented with lactalbumin hydrolysate, epidermal growth factor, hydrocortisone, insulin, chrelatoxine, glucagone and selenium was then developed. Collagen type IV was superior to collagen type I, laminin, fibronectin and plastic in supporting the growth of HuH-6 in the serum-free medium. Higher amounts of both albumin and alpha-fetoprotein were secreted in HuH-6 cultured in serum-free medium than in serum-supplemented medium.     

Immunochemical purification and characterization of ovine alpha-fetoprotein.

Ovine alpha-fetoprotein was successfully isolated from fetal sheep serum by using rabbit anti-ovine alpha-fetoprotein linked to an agarose immunoadsorbent column. Antibody used in this affinity chromatography column was produced by immunizing a rabbit with highly purified alpha-fetoprotein-antibody complex to yield a monospecific antiserum to ovine alpha-fetoprotein. Following affinity chromatography, alpha-fetoprotein was further purified by preparative polyacrylamide disc gel electrophoresis ultimately yielding a 105-fold purification. The purified alpha-fetoprotein was homogeneous on analytical polyacrylamide disc gel electrophoresis. Ovine alpha-fetoprotein was found to be immunochemically related to human alpha-fetoprotein and to exhibit a molecular weight and amino acid composition similar to other mammalian alpha-fetoproteins.     

Binding of bilirubin by bovine and human alpha-fetoprotein.

alpha-Fetoprotein, a fetal protein associated with certain tumors, was found to bind bilirubin. Addition of human or bovine alpha-fetoprotein to bilirubin solutions enhanced the light absorbance of bilirubin and shifted its maximum. Bovine alpha-fetoprotein caused a marked shift towards shorter wavelengths, while human alpha-fetoprotein gave a slight red shift. The spectral changes were used to study the characteristics of the binding of bilirubin by bovine alpha-fetoprotein. These studies indicated the presence of one binding site/molecule of alpha-fetoprotein with an association constant of about 1.1 . 10(6) M-1. A difference between the spectral changes brought about by alpha-fetoprotein and albumin allowed comparison of their relative affinities for bilirubin. The spectrum approximated the average between the spectra induced by the two proteins when the ratio of bovine alpha-fetoprotein to bovine albumin was 6.3 : 1, and of the human proteins 21 : 1, respectively. These results show that alpha-fetoprotein from two species binds bilirubin with an affinity somewhat lower than that of albumin. Binding of bilirubin by alpha-fetoprotein is in agreement with the recent demonstration of structural homology between alpha-fetoprotein and albumin. Whether alpha-fetoprotein plays a role in the metabolism of bilirubin or other degradation products of heme remains to be investigated.     

Determination of the distribution of fatty acids and diethylstilbestrol between serum albumin and alpha-fetoprotein by concanavalin A affinity chromatography

The distribution of fatty acids and diethylstilbestrol between serum albumin and alpha-fetoprotein was measured in vitro by a new method based on the separation of the two proteins by virtue of the binding specificity of concanavalin A for the carbohydrate moiety of alpha-fetoprotein. Human and bovine proteins were investigated. It was found that palmitate and oleate were distributed almost equally between albumin and alpha-fetoprotein, while docosahexaenoate and diethylstilbestrol bound preferentially to alpha-fetoprotein even at an albumin: alpha-fetoprotein ratio of 10:1. The results confirm the binding specificity of alpha-fetoprotein for polyunsaturated fatty acids and also show that alpha-fetoprotein binds diethylstilbestrol much more strongly than albumin does. This suggests that alpha-fetoprotein may play a role in the fetal uptake of diethylstilbestrol.     

Concentrations of major plasma proteins in serum and whole-tissue extracts of porcine fetuses during development.

In extracts from fetuses up to 32 days of gestation, the major serum proteins were fetuin, alpha-fetoprotein and alpha 1-antitrypsin, but albumin was not detected. The concentration of all proteins rose with age until 40-50 days of gestation; and then the serum concentration of alpha-fetoprotein (2.9 mg ml-1), alpha 1-antitrypsin (4.4 mg ml-1) and transferrin (2.6 mg ml-1) fell progressively to about 1 mg ml-1 at birth, whereas those of fetuin, albumin and alpha 1-acid glycoprotein increased. The patterns of serum proteins in fetuses at about the middle of gestation were similar in extracts and sera. At birth, the major proteins were alpha 1-acid glycoprotein and fetuin, which accounted for 45 and 18% of serum proteins, respectively. Albumin represented only 7% of serum proteins at this age. For most of the second gestational period, the six quantified proteins accounted for about 85% of total serum proteins. In early gestation, a significant proportion of serum proteins was intracellular.     

Isolation and characterization of alpha-fetoprotein from the mouse hepatoma BW7756.

Purification of alpha-fetoprotein from mouse hepatoma BW7756 extracts was performed using ammonium sulfate precipitations, gel filtration, ion-exchange chromatography and isoelectric focusing. These procedures produced a 5.6% yield of alpha-fetoprotein with 96% purity. Polyacrylamide slab gel electrophoresis, extended agarose electrophoresis and immunoelectrophoresis demonstrated that mouse hepatoma alpha-fetoprotein migrated at pH 8.6 as a rapid alpha1, or postalbumin globulin. Crossed antibody electrophoresis of the agarose zone containing alpha-fetoprotein failed to demonstrate microheterogeneity. Molecular weight analysis of the mouse hepatoma alpha-fetoprotein on a calibrated Sephadex G-200 column yielded a value of 72 000-73 000 for the native protein. Sodium dodecyl sulfate gel electrophoresis subsequently demonstrated a single polypeptide chain with a molecular weight of 72 000. Amino acid analysis showed the alpha-fetoprotein to be an acidic protein dominated by hydrophobic residues. The total carbohydrate content was 5.5%, and 3 mol of sialic acid were detected per mol of alpha-fetoprotein. Although neutral sugars were the principal class present, galactosamine was the most abundant single sugar detected.     

Linoleate and linolenate desaturation by rat hepatoma cells

Morris 7777 rat hepatoma cells in culture possess high delta 6 and delta 5 desaturase activities over linolenic acid added to the medium as albumin or alpha-fetoprotein complexes. After 2 hours incubation with [1-14C] linolenic acid (7 microM), around 40% of the radioactivity was recovered in other polyene fatty acids, mainly pentaenes. After 24 hours incubation with this substrate the polyene derivatives raised to more than 60%. However, [1-14C] linoleic acid was poorly converted to other polyene fatty acids. Linoleic acid up to 58 microM concentration in the medium do not inhibited linolenic acid desaturation. Long-term supplementation with 50 microM linoleic or linolenic acid, which modified the fatty acid profile of hepatoma lipids, enhanced the desaturase activities against linoleic acid. Desaturase activities were not affected by the fatty acid protein carrier, alpha-fetoprotein or albumin.     

Synthesis and characterization of a recombinant fragment of human alpha-fetoprotein with antigenic selectivity versus albumin

A DNA sequence coding for human alpha-fetoprotein amino acid sequence 38-119 was synthesized and cloned in a bacterial expression vector. The alpha-fetoprotein sequence was selected as the least homologous to albumin, since the two proteins have an overall amino acid identity of approximately 38%. A chimeric protein was obtained which was purified by preparative electrophoresis and characterized in its primary structure by fast atom bombardment mass spectometry. About 70% of the alpha-fetoprotein sequence was physically mapped and found to correspond to the amino acids encoded in the synthetic gene. The use of this recombinant protein allowed the selection of monoclonal antibodies recognizing both the recombinant fragment and native alpha-fetoprotein. These antibodies should allow the development of an immunoassay for alpha-fetoprotein with absolute selectivity versus albumin. This might result in more sensitive clinical determinations, avoiding the possibility of cross-reactions.     

Effect of alpha fetoprotein on the processes of reparative regeneration of the skin and tubular bones]

The influence of alpha-fetoprotein of man, dog and cat on the processes of reparative regeneration of the skin and long tubular bones was studied. alpha-fetoprotein was shown to stimulate regenerative processes by forming young connective tissue, but had no strong species specificity. Commercial preparation of human alpha-fetoprotein proved to possess maximum physiological activity in comparison with other alpha-fetoproteins under study.     

Studies on human alpha-fetoprotein. Isolation and characterization of monomeric and polymeric forms and amino-terminal sequence analysis.

Human alpha-fetoprotein has been isolated from the serum and ascitic fluid of a patient with hepatoma by a combination of immunoadsorbent column chromatography and Sephadex G-150 gel filtration. Human alpha-fetoprotein is a sialylated glycoprotein with an estimated molecular weight of 67 500, composed of a single-chain polypeptide of approximately 580 amino acid residues and 3.6% carbohydrate. It is a negatively charged protein with an acid isoelectric point (pH 4.57). In addition to the monomeric form of alpha-fetoprotein, we have identified human alpha-fetoprotein polymers, including dimeric and trimeric forms, which dissociate to the monomer only upon exposure to disulfide-reducing reagents, implying that their formation is dependent upon intermolecular disulfide bonds. These polymers are found in human alpha-fetoprotein isolated by isoelectric focusing in both the major (pI 4.57) and minor (pI 5.2) alpha-fetoprotein fractions. The first 17 residues of the NH2-terminal amino acid sequence of the hepatoma-derived human alpha-fetoprotein have been identified. Fetal alpha-fetoprotein is indistinguishable from hepatoma alpha-fetoprotein by several criteria, including immunoelectrophoresis, acryalmide gel electrophoresis, and proclivity for dimerization.     

Alpha-fetoprotein and albumin in experimentally-induced exencephaly in the rat.

Alpha-fetoprotein and albumin were quantified in the sera and amniotic fluids from control, Vitamin A-treated non-exencephalic and Vitamin A-treated exencephalic rat fetuses. Exencephaly was associated with amniotic fluid alpha-fetoprotein concentrations which were significantly elevated over those of Vitamin A-treated non-exencephalic and of untreated fetuses. Amniotic fluid albumin concentrations also were higher in the exencephalic fetuses than in the non-exencephalic fetuses. Serum alpha-fetoprotein and albumin concentrations were lower in the exencephalic than in the non-exencephalic fetuses. The results are cosistent with simple diffusion across a defective barrier as the cause of elevated amniotic fluid alpha-fetoprotein concentrations in the presence of open neural tube defects. This experimental model of neural tube defects result in changes in amniotic fluid alpha-fetoprotein similar to those changes found in human amniotic fluid alpha-fetoprotein concentrations in the presence of neural tube defects.     

alpha-Fetoprotein synthesis in transformed fetal rat liver cells

We have reported that transformed fetal liver cells produced a variant alpha-fetoprotein of 65K that differed from the mature alpha-fetoprotein of 69K and 73K in the polypeptide backbone. In the present study, we demonstrated that the biosynthetic pathway of the variant alpha-fetoprotein differed from that of the mature alpha-fetoprotein. The 65K variant was synthesized first as a preprotein of 49.5K which was processed to a polypeptide of 59K in the presence of microsomal membranes. The latter was the precursor of the variant alpha-fetoprotein found in cells and medium of transformed fetal liver cells. The 65K alpha-fetoprotein was encoded by a mRNA of 16S while mature AFP was encoded by a mRNA of 20S.     

Immunochemical identification of human placental organ specific alpha2-globulin and its concentration in amniotic fluid]

An organospecific alpha2-globulin of human placenta was identified with the aid of immunochemical analysis. This antigen differed immunologically from the alpha2- and beta1-globulins of pregnancy, alpha-fetoprotein, placental lactogen, and chorionic gonadotropin. High level of the antigen revealed was found in the placental tissue and the amniotic fluid at the early terms of pregnancy, but its concentration sharply decreased by the time of labour.     

Cytoplasmic and nuclear receptors of estradiol in the hypothalamus and cerebral cortex of female rats in the neonatal period

The content of estradiol receptors (E2) was studied in the cytoplasmic and nuclear fractions of the female rat hypothalamus and cortex during neonatal development. In the cytosol the E2-binding proteins, having a high capacity, include both true estradiol receptors and proteins identical with alpha-fetoprotein. True E2 receptors were found in the nuclear fraction: their concentration underwent almost no changes in hypothalamus and decreased from the 1st to the 5th day of postnatal development in cortex.     

Strain and sex differences in serum alpha-fetoprotein levels in Mus musculus

Normal levels of circulating serum alpha-fetoprotein (AFP) in different inbred strains of mice, as determined by radioimmunoassay, are reported. These strains show different and characteristic values for the mean serum AFP concentration, suggesting genetic control of the serum level of this protein. Furthermore, within each strain a difference in serum levels of the protein is observed between sexes; males have a significantly higher serum AFP concentration.