Imaging tests, tumor marker (TM) screening, and biochemical tests provide a definitive diagnosis of hepatocellular carcinoma (HCC). However, some patients with HCC may present TM-negative results, warranting a need for developing more sensitive and accurate screening biomarkers. Various diseases exhibit increased blood levels of bile acids, biosynthesized from cholesterol in the liver, and they have been associated with HCC. Herein, we analyzed plasma bile acids using liquid chromatography/tandem mass spectrometry and integrated them with conventional biomarkers to develop a diagnostic screening model for HCC. Plasma samples were obtained from patients diagnosed with chronic hepatitis, hepatic cirrhosis (HC), and HCC. A QTRAP 6500 mass spectrometer and a Nexera liquid chromatograph with a YMC-Triart C18 analytical column were used. The mobile phase A was a 20 mmol/L ammonium formate solution, and mobile phase B was a methanol/acetonitrile mixture (1:1, v/v) with 20 mmol/L ammonium formate. After determining the concentrations of 32 bile acids, statistical analysis and diagnostic screening model development were performed. Plasma concentrations of bile acids differed between sample groups, with significant differences observed between patients with HC and HCC. By integrating bile acid results with conventional biochemical tests, a potential diagnostic screening model for HCC was successfully developed. Future studies should increase the sample size and analyze the data in detail to verify the diagnostic efficacy of the model.

To evaluate the correlation of high levels [>2.0 multiples of median (MoM)] of amniotic fluid alpha-fetoprotein (AFAFP) in midtrimester with abnormal fetal outcome.

We retrospectively studied 6245 pregnant women with singleton pregnancy who had undergone amniocentesis between 15 and 27 weeks' gestation at Mackay Memorial Hospital between January 2014 and June 2020. Fifty-five cases had high AFAFP levels (>2.0 MoM). We investigated the abnormal fetal outcomes.

Among the fifty-five cases with high AFAFP levels (>2.0 MoM), thirty (54.5%) had fetal chromosomal abnormalities, major structural abnormalities, and/or adverse obstetric events. Eight cases (14.5%) had chromosomal abnormalities including trisomy 21 (3 cases), trisomy 18 (3 cases), mosaic trisomy 18 (1 cases), and mosaic ring 13 (1 case). Seventeen cases (30.9%) had major structural abnormalities including abdominal wall defect (6 cases) and central nervous system (5 cases), gastrointestinal tract (3 cases), cardiovascular (2 cases), and genitourinary tract (2 cases) abnormalities. Fifteen cases (27%) had adverse obstetric events, including preterm delivery (5 cases), intrauterine fetal demise (4 cases), small for gestational age (4 cases), preeclampsia (4 cases), gestational diabetes mellitus (2 cases), gestational hypertension (1 case), preterm prelabor rupture of membrane (1 case), prolonged labor (1 case), and preterm uterine contraction (1 case).

A high AFAFP level (>2.0 MoM) in midtrimester can be associated with abnormal fetal outcome, including chromosomal abnormalities, major structural abnormalities, and adverse obstetric events. Women with a prenatal diagnosis of high AFAFP levels (>2.0 MoM) should be alerted of the possibility of abnormal fetal outcomes, and further detailed genetic studies and serial sonographic examinations are recommended.

Alpha-fetoprotein (AFP) is an oncofetal glycoprotein that has been used as a tumor marker for hepatocellular carcinoma (HCC) in combination with ultrasound and other imaging modalities. Its utility is limited because of both low sensitivity and specificity, and discrepancies among the different methods of measurements. Moreover, its accuracy varies according to patient characteristics and the AFP cut-off values used. Combination of AFP with novel biomarkers such as AFP-L3, Golgi specific membrane protein (GP73) and des-gamma-carboxyprothrombin significantly improved its accuracy in detecting HCC. Increased AFP level could also signify severity of hepatic destruction and subsequent regeneration and is commonly observed in patients with acute and chronic liver conditions and cirrhosis. Hereditary and other non-hepatic disorders can also cause AFP elevation.

Aim: Natural killer cell receptor group 2D (NKG2D) plays an important role in the immune regulation of tumors. We speculate that DNA methylation are involved in the regulation of NKG2D gene. Methods: We investigated the methylation status of the NKG2D promoter in peripheral blood mononuclear cells of hepatocellular carcinoma (HCC) patients, chronic hepatitis B patients and healthy controls by methylation-specific PCR and the mRNA expression level was examined by real-time quantitative PCR. Results: The methylation frequency of NKG2D promoter in HCC patients was higher than that of chronic hepatitis B patients and healthy controls. NKG2D promoter methylation has a good predictive value for HCC diagnosis. Conclusion: NKG2D promoter methylation can be used as a noninvasive marker for detecting HCC.

Alpha-fetoprotein (AFP) is a major mammalian embryo-specific and tumor-associated protein that is also present in small quantities in adults at normal conditions. Discovery of the phenomenon of AFP biosynthesis in carcinogenesis by G. Abelev and Yu. Tatarinov 50 years ago, in 1963, provoked intensive studies of this protein. AFPs of some mammalian species were isolated, purified and physico-chemically and immunochemically characterized. Despite the significant success in study of AFP, its three-dimensional structure, mechanisms of receptor binding along with a structure of the receptor itself and, what is the most important, its biological role in embryo- and carcinogenesis remain still obscure. Due to difficulties linked with methodological limitations, research of AFP was to some extent extinguished by the 1990 s. However, over the last decade a growing number of investigations of AFP and its usage as a tumor-specific biomarker have been observed. This was caused by the use of new technologies, primarily, computer-based and genetic engineering approaches in studying of this very important oncodevelopmental protein. Our review summarizes efforts of different scientific groups throughout the world in studying AFP for 50 years with emphasis on detailed description of recent achievements in this field.

Synthesis of most of the plasma proteins is one of the main functions of the hepatocytes. Albumin synthesis is quantitatively the most abundant. In the present study we investigated albumin- and alpha-fetoprotein-gene-expression, and the function of the secretory apparatus during rat liver development. To this purpose we used the method of radioactive biosynthetic labeling of newly synthesized albumin and alpha-fetoprotein (AFP) to monitor the secretory capacity of endodermal cells derived from ventral foregut region (embryonic day 10, E10), and of embryonic and fetal hepatoblasts. Synthesis and secretion of albumin and AFP were already detected in the low numbered ventral foregut endodermal cells; fibrinogen synthesis was detectable in the E12 hepatoblasts, which were in higher number. The whole secretory machinery was functional from the earliest stages of liver development, and the speed of secretion was comparable with that of the adult hepatocytes. There was almost 4-fold increase of hepatoblasts cell volume in fetal stage compared with embryonic stage. The model used suggests that the hepatocyte secretory apparatus is already functional before the emergence of the liver bud. This is the first comparative report to analyze the hepatocyte secretory function, cell proliferation and cell volume during liver development.

Alpha-fetoprotein (AFP) is a tumor-associated embryonic molecule whose precise biological function remains unclear. A complete definition of the physiological activities of this oncofetal protein has been severely limited, until now, by the lack of a purification procedure appropriate to obtain pure AFP in appreciable amount. The present report describes a purification procedure extremely rapid and simple and takes advantage of the well-known fact that AFP contains copper. We have developed a single-step purification procedure by immobilized copper-chelate affinity chromatography using the culture medium from human hepatoblastoma cell line HepG2 grown in the absence of serum. This method yields AFP at high purity and high yield. Purified AFP amino acid sequence, molecular mass, carbohydrate structure, and copper content were found to be in line with previous studies. Moreover, we found that the purified AFP has superoxide dismutase activity with efficiency similar to that of the native Cu, Zn SODs at physiological pH. This result may provide further support to the idea that AFP is a bifunctional protein, acting in cellular defence against oxidative stress both as a copper buffer and as a superoxide radical scavenger.

Alpha-fetoprotein (AFP) is a major mammalian oncofetal protein, which is also present in small quantities in adults. It is a member of the albuminoid gene superfamily, which consists of AFP, serum albumin, vitamin D binding protein, and alpha-albumin (afamin). Although physicochemical and immunological properties of AFP have been well-studied, its biological role in embryo- and carcinogenesis and in adult organisms as well as mechanisms underlying its functioning remain unclear. During the recent decades, the biological role of AFP has been evaluated by identification of its functionally important sites. Comparison of primary structure of AFP and some physiologically active proteins revealed similarity of some polypeptide regions. This has been used for prediction of AFP functions (i.e., its multifunctionality). Localization of functionally important sites followed by determination of their amino acid composition and type of biological activity has provided valuable information for structural-functional mapping of AFP. Some peptide fragments of AFP have been synthesized and tested for biological activity. This review summarizes data on structural-functional interrelationships. We also describe functionally important AFP sites found by various groups during the last decade of structural-functional mapping of AFP with experimentally confirmed and putative biologically active sites.

In this study we describe two patients with congenital absence of alpha-fetoprotein (AFP). The pathological examination results, including an immunohistochemical stain, which define qualitatively the levels of AFP detected by the biochemical studies and the comparative genomic hybridization (CGH) are enclosed. A description of the suggested functions of AFP and the means of its production are set forth. An explanation is suggested for the lack of symptoms in a newborn with undetectable levels of AFP and the mechanism by which this condition might occur.

Alpha-fetoprotein (AFP) serum levels in man have long been utilized as a tumor marker and as a birth defect screening agent in the clinical laboratory. Although the physiological role of AFP has remained obscure, the stereotypic carrier/transport function of a fetal counterpart to albumin has been attributed to this oncofetal protein. However, reports from a multitude of investigators in the last decade have provided a rationale to reconsider and extend the biological role of AFP to include cell growth modulation during development. Previously, a leucine zipper-like (heptad) motif, which mimicked that found in the steroid/thyroid receptor superfamily, was postulated for portions of the third domain of AFP. The present report proposes the presence of additional potential heterodimerization sites for the nuclear receptor superfamily members and other growth factors in the second and third domains of human AFP.

Alpha-fetoprotein (AFP) is a major serum protein produced during fetal development. Experimental findings suggest that AFP has antiestrotrophic activity and that it can be developed as a therapeutic agent to treat existing estrogen-dependent breast cancer or to prevent premalignant foci from developing into breast cancer. The antiestrotrophic activity of AFP was reported to be localized to a peptide consisting of amino acids 447-480, a 34-mer peptide termed P447. A series of parsings and substitutions of amino acids in the P447 sequence was intended to identify the shortest analog which retained antiestrotrophic activity. Peptides related to P447 were generated using solid phase peptide synthesis. Several shorter peptides, including an 8-mer called P472-2 (amino acids 472-479, peptide sequence EMTPVNPG), retained activity, whereas peptides shorter than eight amino acid residues were inactive. The dose-related antiestrotrophic activity of AFP-derived peptides was determined in an immature mouse uterine growth assay that measures their ability to inhibit estradiol-stimulated uterine growth. In this assay, the maximal inhibitory activities exhibited by peptide P472-2 (49%), by peptide P447 (45%), and by intact AFP (35-45%) were comparable. The octapeptide P472-2 was also active against estradiol-stimulated growth of T47D human breast cancer cells in culture. These data suggest that peptide P472-2 is the minimal sequence in AFP, which retains the antiestrotrophic activity found with the full-length molecule. The synthetic nature and defined structure of this 8-mer peptide suggest that it can be developed into a new drug which opposes the action of estrogen, perhaps including the promotional effects of estradiol in the development of human breast cancer.

Alpha-fetoprotein (AFP) isolated from rodent amniotic fluid or human cord sera, upon incubation with a molar excess of estradiol, is converted to a form which inhibits estrogen-stimulated tissue growth. The purpose of this study was to determine whether recombinant human AFP produced in an E. coli expression system retained this function. The recombinant protein was similar to the natural protein isolated from pooled human cord sera in all functional aspects evaluated. It was detected by monoclonal and polyclonal antibodies to the natural protein. Following exposure to estradiol, it was converted to an inhibitor of estrogen-stimulated growth of immature mouse uterus yielding a dose/response curve similar to that of the natural protein. It inhibited the growth of estrogen-dependent (MCF-7) but not estrogen-independent (MDA-MB-231) breast cancer xenografts with the same schedule dependency and resultant histological changes as the natural protein. Availability of large quantities of homogeneous, biologically active recombinant human AFP will facilitate further studies of structure/function, mechanism, and therapeutic potential of this agent as a regular of breast cancer growth.

Among the proteins that comprise the albumin family, alpha-fetoprotein (AFP) is the only member which exhibits immunoregulatory properties. However, some investigations have argued that AFP-mediated immunosuppression is not an inherent property of the molecule itself, but is instead, hypothesized to be either a function of a low molecular weight inhibitor bound to AFP or to a post-translational modification of the protein. AFP cannot be isolated from natural sources in quantities sufficient for the detailed biochemical and functional analyses required to resolve these issues. We have therefore produced recombinant forms of the protein (rAFP) by cloning the cDNA's for mouse and human AFP in both eukaryotic and prokaryotic expression systems. As described in this report, we were able to abundantly express rAFP's in bacterial, baculovirus and yeast expression systems. Recombinant proteins derived from each expression system were recognized by polyclonal and monoclonal anti-AFP antibodies as determined by immunoblot analysis. Pure recombinant protein samples, as characterized by polyacrylamide gel analyses, N-terminal sequencing and FPLC and HPLC chromatography, were evaluated for their immunoregulatory properties in murine and human in vitro immunological assays. The results of these studies establish that rAFP is functionally equivalent to natural fetal derived AFP molecules. Importantly, the data reported here demonstrate that AFP-mediated immunoregulation is an activity intrinsic to the molecule itself and cannot be attributed to either putative non-covalently bound moieties or to post-translational modifications such as glycosylation and sialylation. These studies provide a basis for initiating detailed investigations into the potential clinical usefulness of AFP as an immunotherapeutic agent.

Alpha-fetoprotein (AFP) is an oncofetal protein classified in a super-family together with albumin and Vitamin-D binding (Gc) protein which present as globular proteins comprised of three domains. Several subdomain regions on AFP have been previously proposed to serve as dimerization interfaces for nuclear receptors or perhaps other co-factors/inhibitors. The cellular uptake and internalization of AFP together with its subcellular compartmentalization is now well documented in a variety of cell types. A myriad of reports have emerged which have detected, identified, and characterized various binding proteins associated with AFP in different cellular compartments. However, the literature is devoid of any attempts to summarize, categorize, and relate these proteins to the various physiological activities attributed to this fetal protein. It is conceivable that AFP could interact and/or bind cytoplasmic chaperone proteins that normally escort nuclear factors or transcription co-factors through the cytoplasm toward organelle interfaces. A dual concept proposing binding or escort proteins for AFP together with subdomain dimerization interfaces on the AFP molecule can be reconciled into a composite hypothesis to formulate a rationale for the growth regulating properties ascribed to AFP during the last decade. Thus, AFP might serve as a modulator/modifier of various cell growth regulatory pathways during embryonic and fetal development in vertebrates.

Serum albumin levels decrease during pregnancy while the concentration of most other maternal serum proteins of hepatic origin remain stable or increase. In a study of 289 women, most maternal characteristics such as race, age, smoking, a history of previous low birth-weight, infant sex and gestational age at delivery were not related to maternal serum albumin levels at 18 or 30 weeks' gestational age. The degree of maternal obesity significantly correlated with the concentration of albumin. There was a significant negative correlation in individual women between maternal serum levels of albumin and alpha-fetoprotein, with high levels of maternal serum alpha-fetoprotein predicting lower levels of albumin. We hypothesize that there may be a negative feedback effect of alpha-fetoprotein of fetal origin on the maternal production of albumin during pregnancy.

AFP or SA immobilized on nitrocellulose membranes (AFP-NC or SA-NC) were used as affinity matrices to purify cell membrane proteins with affinity for AFP (AFP-BP) and for SA (SA-BP) from membrane-enriched extracts of Raji cells (a B-lymphoma cell line), as well as for normal resting and activated peripheral blood lymphocytes (PBMC). SDS-PAGE and ligand blotting assays showed that AFP-BP and SA-BP isolated from Raji cells are probably identical molecules. They consisted of two sets of polypeptides of 31 kDa and 18 kDa. The glycoprotein nature of isolated 31 kDa and 18 kDa peptides was suggested by positive staining with Schiff's reagent, and amino-acid analysis revealed similar amino-acid composition for the two glycoproteins. In human PHA-activated PBMC, only the 18 kDa polypeptide was identified and isolated as AFP-BP or SA-BP. As in Raji cells, this 18 kDa polypeptide, isolated by affinity for AFP or for SA, appeared to be the same molecule. Contrary to Raji cells and activated PBMC, no proteins with an affinity for AFP or for SA were identified or isolated in resting PBMC. These observations strongly suggest that the isolated 31 kDa and 18 kDa glycoproteins are probably AFP receptors previously demonstrated in several neoplastic and normal cells undergoing growth and/or differentiation; indeed, they were identical to albumin-binding proteins described by others.

alpha-Fetoprotein (AFP) is a well-known tumor marker of hepatocellular carcinoma (HCC). Monoclonal antibodies against AFP possessing specific binding ability to HCC are potential candidates for immunoscintigraphy and immunotherapy. A new monoclonal antibody against AFP (0325-6-9) was isolated. Its specificity and targeting tumor ability were characterized by enzyme-linked immunosorbent assay (ELISA), cell immunostain and complement killing. These results suggest that 0325-6-9 is specific to hepatoma cells. The nucleotide sequences of variable regions of 0325-6-9 were determined by M13 dideoxynucleotide sequencing method. With the information of nucleotide sequence, this antibody then could be modified by recombinant technology for its usage in in vivo diagnosis and immunotherapy.

Alphafetoprotein (AFP), an oncofetal antigen, plays very important roles in the early embryonic life and oncogenesis. Under various physiological and pathological conditions AFP exhibits microheterogeneity, probably as a result of differential expression of its epitopes. To analyse the epitopes we have developed a panel of monoclonal antibodies against human AFP purified by a new and efficient method using an immunoadsorbent consisting of polyclonal antibodies immobilized on cyanogen bromide activated Sepharose. Clones producing antibodies of various isotypes, e.g. IgG1, IgG2a, IgG2b, IgA and IgM have been subcloned and characterized. The antibodies showed high avidity for AFP (with half-maximal binding concentrations between 0.012 and 3.87 nM). Mutual inhibition efficiencies of a panel of 14 monoclonal antibodies were determined by RIA. Based on these inhibition data a computer program was used to group these antibodies with respect to their "epitope specificity distance". As a result of this grouping, clones have been identified which can recognize at least five different epitopes on AFP. This panel of antibodies may be very useful for analysis of the epitopic variation of AFP under various physiological and pathological conditions.

Alpha-Fetoprotein (AFP) is a product of specific fetal tissues and of neoplastic cells of hepatocyte or germ cell origin in adults. This protein belongs to a gene family that is phylogenetically most closely related to serum albumin. Its primary, secondary, and tertiary structural aspects appear similar to the three-domain concept proposed for the latter protein. The primary sequence of AFP departs most widely from serum albumin in the first 135 amino acid residues, with about 42% of the remaining 590 residues of the human proteins being identical. Some evidence exists that there are limited sequence differences in the AFP of a given animal species. AFP shows considerable charge heterogeneity that appears to relate mostly to its glycoid moiety. The proteins of some species such as the rat show more pronounced heterogeneities than that of humans. The variations in extent and type of glycosylations are evidenced by differences in the binding to various lectins. These interactions are being extensively explored in attempts to differentiate the sources of the protein produced by various normal and neoplastic cells and may provide valuable diagnostic methods. AFP, like serum albumin, shows relatively strong binding affinities for a variety of ligands. The most notable difference is the strong preferential binding of polyunsaturated fatty acids by AFP. This protein may play a role in transporting these substances to developing and to malignant cells. Various agents affect the synthesis of this protein both by specific fetal tissues and by neoplastic cells. Marked differences in the responses of cells, particularly those of neoplastic types, are indicative of variations in the genetic factors responsible for control of its synthesis. The subject of the genomic repression of the synthesis of AFP seen in fetal life upon maturation of the liver and the reoccurrence of synthesis upon malignant conversion of hepatocytes and of certain germ cells are of particular interest. The regulation of the closely related AFP and albumin genes is providing a powerful and attractive model to examine molecular events in the activation and inactivation of specific genes during development and in oncogenic processes. Extensive measurements of AFP during pregnancy and in the course of neoplasias, notably hepatoma, are being made to aid in following changes in such developments. Various specific physiological roles for this protein are also being proposed. One of these is its possible action in the regulation of immune processes.(ABSTRACT TRUNCATED AT 400 WORDS)

The N-terminal native sequence tripeptide of alpha-fetoprotein, L-threonyl-L-leucyl-L-histidine N-methylamide, was synthesized and its interaction with Cu(II) ions was investigated by potentiometric titration at 25 degrees C in 0.15 M-NaCl and by visible-absorption, e.p.r. and n.m.r. spectroscopy. Analyses of the results in the pH range 4-10 indicated the presence of multiple complex species in solution: MHL, MH-2L, MHL2, ML2 and MH-1L2, where M, H and L represent metal ion, proton and ligand anion respectively. Only the species MH-2L and MH-1L2 are present in significant amounts at physiological pH. The results of the visible-absorption spectroscopy are consistent with the findings of species distribution that MH-2L is the major complex species detected above physiological pH that has the spectral characteristics of lambda max. = 523 nm and epsilon max. = 98 M-1.cm-1. The nine superhyperfine lines in e.p.r. spectra of the major species MH-2L strongly support the co-ordination of four nitrogen atoms by Cu(II). Both 1H- and 13C-n.m.r. studies suggest that the species MH-2L is a square-planar complex. The results from the equilibrium-dialysis experiments showed that this peptide is able to compete with albumin for Cu(II) ions. At equimolar concentrations of albumin and the peptide, about 52% of the Cu(II) was bound to the peptide. The possibility that alpha-fetoprotein plays an important role as the Cu(II)-transport protein in fetal life is discussed.

A solid phase immunoassay was used to evaluate the levels of serum and cerebrospinal fluid of alphafetoprotein and carcinoembryonic antigen in 33 individuals treated for backache and headache with no evidence of organic neurological disease, 19 patients with primary CNS tumor (benign or malignant) and 22 with CNS metastasis from a solid tumor. AFP serum and CSF levels were found in trace amounts or slightly elevated not exceeding normal limits in all groups. Patients with CNS metastasis were found to have statistically significant higher CEA levels (both in serum and CSF) than the control group, and the patients with primary brain tumors. Patients with leptomeningeal dissemination had statistically significantly higher CEA CSF levels than did patients with primary tumors, and patients with parenchymal metastasis.

By use of different restriction enzymes sensitive to internal cytosine methylation (HpaII, AvaI, HhaI) we have analysed the methylation patterns of albumin and AFP genes in tissues and cell lines with high (liver, yolk sac, hepatoma cell lines), low (fetal and neonatal kidney) or undetectable (spleen, JF1 fibroblasts) expression of either gene. We show that expression of the AFP gene is associated to the demethylation of a whole region or domain extending from -4 to +3 Kb. Moreover, demethylation of a site located at the upstream limit of this domain appears to be correlated with the commitment of the cell type to synthesize AFP. As concerns the albumin gene, we show that the domain in which demethylation is correlated with active gene transcription in hepatoma cell lines has different borders than in tissue. This difference might be related to the different amounts of mRNA synthesized or to an alteration in gene regulation in tumor cells. Finally, we show that low expression of albumin and AFP genes in fetal and neonatal kidney is not correlated with domain demethylation, suggesting that the regulatory mechanisms of expression of these genes are different in kidney as compared with liver.

Administration of 3 microg dexamethasone at day 8 of incubation into the allantoic cavity resulted in various embryopathies in embryos by the 15th day of incubation. Chicken alpha-foetoprotein (chAFP) and serum albumin concentrations, when examined at the 12th day of incubation, were below the corresponding controls (P<0.01), whereas total serum protein was only slightly lower, On day 15 chAFP concentrations were similar to those of the controls, serum albumin values were markedly reduced and total serum protein was significantly decreased. The results suggested a selective reduction of serum albumin synthesis in the 15-day-old chick embryo. Following administration of 2 IU insulin on the first day of incubation, into the yolk sac, one third of the embryos developed embryopathies. ChAFP, serum albumin and whole serum protein values were unchanged on either the 12th day or 15th day of incubation. Inoculation of 100 microg adrenaline into the allantoic cavity on the sixth day of incubation resulted in embryopathies in only a few instances. ChAFP total serum protein values were not affected. However, serum albumin concentrations were significantly lower on the 12th day, but not on the 15th. The responses to L-thyroxine were both time- and dose-dependent. Inoculation of 2 microg thyroxine on the second day of incubation resulted in few embryopathies. ChAFP levels were significantly lower on the 12th and 15th day of incubation. In contrast, serum albumin and total serum protein concentrations were normal at both times. Similar results were obtained when 2 microg of thyroxine were administered at the third day of incubation. Thus in order to inhibit selectively chAFP synthesis, administration of 2 microg thyroxine on day 2 or 3 of incubation is suggested. When 10 microg thyroxine were injected on day 8 of incubation, 80% of the embryos showed embryopathies on day 12 of incubation. ChAFP and serum albumin levels were markedly depressed. The concentration of total serum protein was significantly decreased on day 12 but less so, though still significantly, on day 15. These results demonstrate the possibility to influence selectively chAFP and serum albumin synthesis, thereby facilitating studies on chemically induced teratological, including pathophysiological, processes in the avian embryo.

1. Antisera to native or unfolded glutathione S-transferase from human liver recognize either antigen but do not recognize native or unfolded glutathione S-transferase from human placenta. 2. Antisera to native or unfolded glutathione S-transferase from placenta recognize either antigen but do not recognize native or unfolded glutathione S-transferase from liver. 3. Antisera to unfolded human serum albumin crossreacts with unfolded alpha-fetoprotein but does not recognize unfolded glutathione S-transferase.

Using a characterized human vitamin D binding protein (DBP) cDNA probe and a panel of rodent X human somatic cell hybrids, we established the chromosomal location of the structural gene for DBP on human chromosome 4. In situ hybridization of 3H-labeled DBP cDNA to human metaphase chromosomes confirmed this assignment and allowed regional localization to bands 4q11-4q13. A restriction fragment length polymorphism associated with the DBP gene that should prove useful in future linkage studies was identified with the enzyme BamHI.

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.

Alpha-Fetoprotein (AFP) is a major serum glycoprotein during embryonic and early postnatal life. A number of diverse biologic functions have been attributed to AFP, including osmotic and carrier function and immunosuppressive activity. In this study we demonstrate that AFP selectively stimulates in vitro proliferation of two distinct subsets of adult murine bone marrow cells. One population of AFP-reactive bone marrow cells expresses surface receptors for soybean agglutinin (SBA) lectin. SBA+ bone marrow cells are resistant to cytotoxic pretreatment with T-cell-specific antisera and are not retained on Ig-anti-Ig affinity columns. The absence of conventional T- and B-cell markers, coupled with the presence of SBA receptors, suggests that AFP-activated non-T bone marrow cells may belong to an immature set of B lymphocytes. A second population of AFP-responsive bone marrow cells expresses the Thy-1+ Lyt 1+2- phenotype characteristic of conventional mature adult T helper cells. The potential physiological relevance of the mitogenic effects of AFP on bone marrow cells with respect to immunoregulatory processes in the fetal/newborn environments is discussed.

The drug-binding properties of human alpha-foetoprotein (alpha FP) were investigated by a fluorescence-spectral method. Human alpha FP was shown to bind to albumin's site I marker (warfarin, phenylbutazone), site II marker (L-tryptophan), but not site III marker (cholic acid, digoxin). The binding of human alpha FP towards lower alcohols was examined, and this binding seems to depend partly on the hydrophobicity of the ligands. The binding of human alpha FP is discussed in comparison with human serum albumin or rat alpha FP.

Using a computer program designed to detect evolutionary relationships between proteins, I find that residues 72-110 of the mature sequence of human tissue-type plasminogen activator (t-PA) and 39 residues at the carboxy terminus of human albumin have a comparison score that is 8.8 standard deviation units higher than that obtained with a comparison of randomized sequences of these proteins. The probability (p) of getting this score by chance is approximately 10(-18), indicating that part of t-PA and albumin are derived from a common ancestor. I also find that alpha-fetoprotein, a relative of albumin is related to t-PA. Part of this region on t-PA has been previously shown to be related to epidermal growth factor. t-PA, albumin, alpha-fetoprotein, and epidermal growth factor have diverse biological activities. The finding that these proteins are related suggests some new approaches for studying their functions.

We find that gossypol, a male anti-fertility compound, is a reversible competitive inhibitor of estrogen binding to rat alpha-fetoprotein (AFP). The Kd of gossypol for rat AFP is 1.75 microM, which is similar to gossypol's affinity for lactate dehydrogenase isozyme X, the putative site where gossypol exerts its anti-fertility effects. Reacting sodium cyanoborohydride with gossypol reduces its affinity for AFP, showing that intact aldehyde groups on gossypol are important for binding to rat AFP and indicating that gossypol is specifically inter-acting with a nucleophilic site on AFP that influences estrogen binding.

Human albumin and alpha-fetoprotein are partitioned in an aqueous two-phase system composed of 10% (w/w) Dextran and 7.5% (w/w) poly(ethylene glycol). When a small amount of poly(ethylene glycol) is replaced by Cibacron Blue F3G-A-liganded poly(ethylene glycol) the partition coefficient, K, of albumin increases by the factor of about 4000 whereas the K value of alpha-fetoprotein undergoes only a small change. The change of the partition coefficient in a logarithmic scale induced by increasing dye-polymer concentrations turned out as a useful measure for the affinity of albumin and alpha-fetoprotein to the dyes. The effect of pH and salt concentration on the affinity partition of albumin and alpha-fetoprotein is demonstrated. The partition of the two proteins in presence of Cibacron Blue F3G-A-liganded poly(ethylene glycol) is compared with seven other triazine dye-poly(ethylene glycol) derivatives.

Conformational transitions of HAFP in the pH-range 2-12 were studied by fluorescence spectroscopy, fluorescence polarization measurements, circular dichroism and hydrophobic chromatography in order to compare molecular architecture of HAFP and that of human serum albumin. It was found that HAFP has a remarkably hydrophilic exposed molecular surface at neutral pH and possesses extensive hydrophobic binding sites located in crevices. Conformational changes occur in HAFP in the acid and alkaline pH regions; extensive hydrophobic areas in HAFP are exposed by both acid and alkaline transitions. The alpha-helix contents of HAFP were determined as 67% at pH 7.6, 47% at pH 2.11.

Analbuminemic rats, which lack albumin synthesis in the liver, have been shown to have a defect in splicing of albumin mRNA precursors. Change in serum alpha-fetoprotein concentration during the neonatal period in these mutant rats was compared with change of their gene expression of albumin. In analbuminemic rats the serum alpha-fetoprotein concentration at birth was almost the same as that of normal rats, and its concentration gradually decreased to a nondetectable level within 3 to 4 weeks after birth, as in normal rats, without increase in serum albumin concentration. In spite of the absence of increase in serum albumin, increase in transcripts of the albumin gene was observed. The level of "albumin mRNA precursors" in nuclei of the liver was less than 0.05 ng/micrograms of nuclear RNA at birth, but increased to 1 ng/micrograms within 3 weeks after birth in normal rats. Although the extent of increase was less, a similar switch-on of the albumin gene was observed in analbuminemic rats in the developmental period. These data clearly indicated that even in analbuminemic rats, coordinated regulation of the alpha-fetoprotein and albumin genes took place at a transcriptional level during development.

The binding of hemin to human alpha-fetoprotein has been estimated by means of fluorescence and spectrophotometric titration. Spectrophotometric titration discloses one strong binding site for hemin with an association constant of 1.5 X 10(7) M-1. The binding causes a shift of the absorption maximum to a higher wavelength and a rise in the molar absorption coefficient. Fluorescence reveals that the binding of hemin to human AFP quenches the protein fluorescence, which changes in character from a tryptophan type to a tyrosine type. As postulated by our results, the binding of hemin to human AFP is similar to the binding of hemin to HSA.

Angiotensinogen was synthesized by cells derived from the Reuber H35 rat hepatoma. Independent clones produced similar amounts of angiotensinogen, which corresponded to about four times more than expected for normal hepatocytes. The protein was secreted rapidly but could be visualized within cells using immunofluorescence. For one clone, it is shown that maximal angiotensinogen synthesis occurred during mid-exponential growth. Somatic cell genetics techniques have been used to investigate the regulation of angiotensinogen expression. Eleven clones of dedifferentiated variant hepatoma cells that failed to produce most or all of the liver specific proteins analyzed including albumin fell into two groups: Seven clones produced only 1-3% as much angiotensinogen as the differentiated clones, and four showed a reduction to 10-30%. Clones of the latter class were the only ones among the eleven analyzed that retained the potential to give rise to revertants, showing restoration of the differentiated state. All revertants fully restored angiotensinogen production, but only some of them re-expressed albumin. Somatic hybrids between differentiated hepatoma cells and one of the variants showed a substantial reduction in angiotensinogen production, whereas for some clones, albumin synthesis was fully maintained. These results show that regulation of the expression of angiotensinogen and of a second serum protein, albumin, was independent and that angiotensinogen synthesis was a faithful indicator of the general differentiation profile of all classes of clones.

Column chromatography was used to investigate the interaction of human alpha-fetoprotein and albumin with different immobilized dyes. Binding of alpha-fetoprotein to the dye conjugates was studied at pH 7.0. Between 56 and 93% of the total alpha-fetoprotein applied to the column was recovered in the break-through fractions of the respective runs. Of all the dyes, Cibacron Blue F3G-A adsorbs alpha-fetoprotein most strongly. This interaction clearly depends on the degree of dye substitution of the gel. A relatively weak interaction exists between alpha-fetoprotein and immobilized Procion Red HE-3B. This is used in the purification of alpha-fetoprotein by negative chromatography resulting in a 16.6-fold enrichment of this protein. Human albumin binds tightly to immobilized Cibacron Blue F3G-A as well as to Cibacron Brilliant Blue FBR-P and shows a lower affinity to Procion Blue MX-R. Procion Red dyes, which are structurally different from Cibacron Blue F3G-A are also capable of interacting with serum albumin. The results obtained are discussed in terms of the present theoretical conceptions about dye-protein interactions.

The main features of the oncodevelopmental biology of alpha 1-fetoprotein (AFP) are reviewed. Progress made in the molecular biology of AFP gene regulation is discussed and we present our recent data on the mechanisms of AFP suppression by glucocorticoid hormones. The relationship between AFP gene transcription and cell replication is examined, and it is suggested that the degree of methylation of the AFP gene (or of co-methylated regulatory DNA sequences) conditions its response to hormones.

Hepatocytes can be maintained in culture for periods of a few hours to many days. This review summarizes the metabolic characteristics of these cultures and describes their use in studying the regulation of plasma protein synthesis. Hormones selectively stimulate the synthesis of certain proteins. Cortisol stimulates the synthesis of fibrinogen and other acute-phase proteins; whereas, insulin stimulates albumin synthesis. In the latter case insulin increases the rate of a nuclear process. Mediators elaborated by leukocytes stimulate acute-phase protein synthesis in hepatocytes. Plasmin-generated fibrin peptides stimulate fibrinogen synthesis via a leukocytic mediator. Lipoprotein synthesis is stimulated by fatty acids and is inhibited by albumin and other macromolecules. These and other processes are susceptible to detailed analysis using sub-cellular fractions (mRNA, nuclei, transcription factors, etc.) isolated from hepatocytes. Studies on fetal or embryonic hepatocytes and hepatomas are yielding information on the regulation of secretory protein synthesis during development and following neoplastic transformation.

An extract of the livers of normal chickens (N) and chickens (Eb) infected with avian erythroblastosis virus (EbV) contained a small molecular weight protein (SMWP, mol. wt 11,000). When the extract was not dehaeminized, SMWP in agarose electrophoresis was shown to have peroxidase activity probably due to the presence of haem. When this non-dehaeminized extract was chromatographed on Con-A Sepharose neither SMWP nor an antigen (EbAg) present in Eb livers were retained. The association of EbAg with SMWP is still unexplained. Immunoelectrophoresis shows a reaction of identity between chicken SMWP and serum albumin. Chicken SMWP is thus not a beta 2-microglobulin. The finding of an immunological relationship between SMWP and albumin confirms the biochemical homology of SMWP with serum albumin in terms of amino acid residues. It has been suggested that SMWP may be a precursor or fragment of albumin but the possibility of its being a distinct entity, a microalbumin, should not be discounted.

Mice were immunized with a purified preparation of human alpha-foetoprotein (AFP) and their spleen cells were hybridized with mouse myeloma cells. Eleven hybridoma cell lines secreting antibody to AFP were obtained. These antibodies were classified into two groups on the basis of different antigenic determinants they recognized. Seven cell lines produced antibodies directed to one determinant of AFP (determinant a), while the remaining 4 produced antibodies to another determinant (determinant b). A native AFP molecule bore one each of a and b determinants which were accessible by monoclonal antibodies; it was detected by a sandwich-type solid-phase radioimmunoassay only when antibody to a was used for coating wells of the microtitre plate and antibody to b as radiolabelled reagent, or vice versa. The presence of two different antigenic determinants on AFP was further confirmed by a chemical modification. When AFP was reduced in the presence of 2 mM dithiothreitol and then alkylated, determinant b was completely destroyed, but the determinant a remained unaffected. Furthermore, reduced and alkylated AFP was detected by the radioimmunoassay employing antibody to a both for coating wells and as radiolabelled reagent, thereby indicating that it bore two determinant as, one of which had been unaccessible in the native AFP.

The nucleotide sequence of a recombinant DNA clone, containing a partial mRNA sequence for human alpha-fetoprotein (AFP) in the plasmid vector pBR322, has been determined. Two regions of the cloned nucleotide sequence were found to agree with published amino acid sequences of two cyanogen bromide peptides derived from human AFP. Examination of the amino acid sequence, deduced from the cloned portion of the mRNA coding region, reveals extensive homology with the third domain of the human serum albumin molecule. A total of 44% (56/128) amino acids and 54% (207/384) nucleotides are identical in the two structures. The landmark cysteine residues are found in the same positions in both polypeptide chains, presumably forming the same disulfide bridges in AFP as those found in the albumin. The sequence homology reinforces the evidence that human AFP and albumin constitute a gene family, in analogy to the same family found in rodents. A comparison of the human and rodent sequence data suggests that the rate of molecular evolution has been faster for AFP than for albumin.