AFP Synthesis Research Articles

Computational prediction of antifungal peptides via Chou's PseAAC and SVM.

With the increase in immunocompromised patients in the recent years, fungal infections have emerged as new and serious threat in hospitals. This, and the insufficiency of current antifungal therapies alongside their toxic effects on patients, has led to the increased interest in seeking new antifungal peptides. In the present study, we have developed a prediction method for screening of antifungal peptides. For this, we have chosen Chou's pseudo amino acid composition (PseAAC) to translate peptide sequences into numeric values. Thus, the SVM classifier was performed for binomial classification of antifungal peptides. The performance of the classifier was evaluated via ten-fold cross-validation and an independent dataset. For further validation of the model developed, 22 P24-derived peptides were predicted using the classifier and in vitro assays were performed on the three peptides with the highest prediction score. The results showed that the PseAAC <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mo>+</mml:mo></mml:math> SVM method is able to predict AFPs with ACC of 94.76%. In vitro results also validate the SEN and SPC of the classifier. The results suggest that the computational approach used in this study is highly efficient for prediction of antifungal peptides, which can save time and money in AFP screening and synthesis of novel peptides.

Modification of Histone Acetylation Facilitates Hepatic Differentiation of Human Bone Marrow Mesenchymal Stem Cells

The multi-potentiality of mesenchymal stem cells makes them excellent options for future tissue engineering and clinical therapy, including liver injury. In this study, we investigated the effects of valproic acid (VPA), a direct inhibitor of histone deacetylase (HDAC), on the hepatic differentiation of human bone marrow mesenchymal stem cells (BMMSCs). The cells were found to differentiate into a more homogeneous hepatocyte-like population when pretreated with 5 mM VPA for 72 h. The expression of liver-specific markers was significantly upregulated in the VPA-treated group at the mRNA and protein levels. VPA treatment also significantly enhanced the hepatic functions of the differentiated cells, including glycogen storage, cytochrome P450 activity, AFP and ALB synthesis, and urea production. Further analysis showed that treatment with 5 mM of VPA for 72 h greatly improved the histones H3 and H4 acetylation. These results demonstrated that VPA could considerably improve the hepatic differentiation of human BMMSCs, probably because the chromatin-acetylated state changes upon VPA treatment through its HDAC inhibitory effect. Thus, this study provides a direct research model for producing human hepatocytes for clinical purposes.

Requirement of 3D extracellular network for maintenance of mature hepatocyte morphology and suppression of alpha-fetoprotein synthesis in vitro

It has been previously shown that adult mouse hepatocytes when co-cultured with rat liver-derived cell lines IAR-2 and IAR-20 can form organotypic hepatocyte islands consisting of cuboidal cells with expressed cell polarity and domain-specific localization of plasma-membrane proteins. The synthesis of alpha-fetoprotein, a fetal-specific antigen, was almost completely suppressed in these islands. It was noticed that organotypic islands were surrounded by fibrils and often covered with a roof of supporting cells. Here we demonstrate that the formation of adult mouse hepatocyte islands in co-cultures depends on the creation of 3D ECM scaffold formed by co-operative activity of hepatocytes and supporting partners. Noteworthy, the collagen type I network was observed exclusively around organotypic islands with complete suppression of alpha-fetoprotein synthesis. In contrast to non-transformed cells, IAR-2 cells, transformed either spontaneously or by transfection with mutant human N-ras Asn12 cDNA, were completely unable to support in co-culture the differentiated morphology of hepatocytes and to suppress AFP synthesis. Transformed IAR-2 cells were able to produce most of the main ECM components except collagen type I, but failed to form extracellular fibrils both in pure culture and in co-culture with hepatocytes. Together, the data show that 3D ECM scaffold (possibly based on collagen type I) plays an important role in maintaining the differentiated morphology of hepatocytes and suppression of alpha-fetoprotein synthesis in vitro.

Biological Significance of AFP Expression in Liver Injury Induced by CCL4.

To clarify whether α−fetoprotein (AFP) expression in acute liver injury is related to hepatic regeneration, we measured serum AFP and GOT in rats with CCl4-induced liver injury, and investigated proliferating activity of AFP−positive cells using a combined technique of immunocytochemistry and autoradiography of 3H−thymidine. Serum AFP reached a peak just after that of GOT. AFP−immunoreactivity was found in hepatocytes bordering necrotic area around central veins and ultrastructural findings showed apoptotic phenomenon in AFP−positive cells. The combined technique showed the presence of 3H-thymidine grains in hepatocytes bordering the necrotic area, but the grains were not simultaneously seen in AFP-positive cells. Our findings suggest that AFP synthesis is related rather with cell death than with regeneration after severe hepatitis induced by CCl4.

Cellular aspects of alpha-fetoprotein reexpression in tumors

The cellular basis of AFP synthesis in normal development, liver regeneration, hepatocarcinogenesis and in tumors is discussed in the review. The attempt is made to interprete the production of AFP by germ cell and liver tumors as a consequence of their origin from the cell types producing AFP in normal conditions. Thus, AFP in germ cell tumors is explained by the development of the yolk sac visceral endoderm (YSVE) in teratocarcinomas, since YSVE is the first site of AFP synthesis in the embryo. The next site of AFP production is embryonal hepatoblast and just hepatoblastomas are the maximal producers of AFP among liver cancers. The reason for AFP resumption in hepatocellular carcinomas (HCC) is not yet clear. This problem is discussed in the light of possible role of oval cells in the HCC origin and the concept of the two states of the mature hepatocyte, associated and non-associated with AFP production. The crucial role of extracellular matrix in the control of AFP-producing state of hepatocyte is emphasized.

Antogonistic effects of retinoic acid and triiodothyronine in the expression of corticoid-binding globulin (CBG) by cultured fetal hepatocytes

Cultures of rat fetal hepatocytes were used to investigate the effects and interplay of triiodothyronine (T 3) and retinoic acid (RA) in the regulation of gene expression of CBG, compared to that of α-fetoprotein (AFP). The cultured cells showed cytological features typical to hepatocytes and actually synthesized CBG and AFP, as evidenced from in situ hybridization with specific radioactive and cell mRNA levels disappeared with a half-life of about 2 days, thereby reflecting the decrease previously seen in hepatic CBG mRNA contents during embryonic life. The K d values for CBG binding were unchanged under these conditions. Culturing of hepatocytes in the presence of T 3 resulted in dose-dependent stimulations of both medium CBG and cell mRNA levels, with an EC 50 concentration of about 10 −9 M. In sharp contrast, RA caused a reduction in CBG biosynthesis (IC 50 = 1.7 × 10 −7 M) and, in addition, antagonized the stimulatory influence of T 3. Under the same experimental conditions, AFP synthesis failed to be affected in a similar fashion. We conclude that thyroid hormones and RA directly act on hepatocytes to specifically regulate the expression of CBG in an antagonistic way.

Inhibitory effect of prostaglandin Δ 12-PGJ2 on cell proliferation and α-fetoprotein expression in HuH-7 human hepatoma cells

9-deoxy-Δ 9, Δ 12-13,14-dihydro-prostaglandin D2 (Δ 12-PGJ2) is a potent inhibitor of proliferation of tumor cells. In the present study, the effect of Δ 12-PGJ2 on the α-fetoprotein (AFP) and the albumin gene expression was analyzed in HuH-7 human hepatoma cells. Δ 12-PGJ2 inhibited the cell growth and reduced the medium AFP concentrations dose-dependently. To determine whether this decline of AFP depends only on the relative decrease in cell numbers by Δ 12-PGJ2, or is in part, due to the decrease in the cellular AFP synthesis by Δ 12-PGJ2, Northern blot analysis was performed in this study. By Northern blotting, it was shown that Δ 12-PGJ2 caused a marked reduction in the levels of the AFP mRNA and the albumin mRNA. In contrast, the level of the β-actin mRNA was not changed by Δ 12-PGJ2. In the transient chloramphnicol acetyltransferase plasmid transfection experiments, Δ 12-PGJ2 did not suppress the AFP enhancer activity, which possibly regulates both the AFP and the albumin gene expression in HuH-7 hepatoma cells, but resulted in the selective repression of the AFP and the albumin promoter activity. These results suggest that Δ 12-PGJ2 suppresses not only cell growth but also expression of the AFP gene and the albumin gene at the transcriptional level in human hepatoma cells.

Regulators of fetal liver differentiation in vitro

Seventeen-day-old fetal rat hepatocytes were employed to examine factors required to promote differentiation in vitro. In the absence of effectors, primary fetal hepatocytes dedifferentiated, as characterized by the rapid decline in synthesis of fetal α-fetoprotein (AFP), albumin, and transferrin. On the other hand, cells maintained in the presence of glucocorticoid hormone produced high levels of albumin and transferrin. Glucocorticoid could not prevent the decline in fetal AFP synthesis, but induced synthesis of the 65K variant AFP—the major AFP species produced by adult rat liver. Fetal hepatocytes maintained in the presence of 8-bromo-cAMP (8-BrcAMP), or methyl isobutyl xanthine (MIX), an agent that increases intracellular cAMP levels, synthesized high levels of fetal AFP and albumin but reduced levels of transferrin. Both glucocorticoid and 8-BrcAMP or MIX induced expression of adult liver-specific genes such as tyrosine aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK), suggesting that these fetal hepatocytes have matured. Cells maintained in the presence of glucocorticoid hormone and MIX (or 8-BrcAMP) contained more albumin, TAT, and PEPCK mRNAs and synthesized increased amounts of the 65K variant AFP than those with either agent alone. However, the glucocorticoid/MIX cells produced intermediate levels of the fetal AFP and transferrin. Our data indicate that both glucocorticoid hormone and cAMP are necessary for optimal differentiation of fetal hepatocytes in vitro.

Methylation of the alphafetoprotein gene in cell populations isolated from rat livers during carcinogenesis

We examined the methylation pattern and organization of the AFP gene in whole livers and in isolated cell populations purified from livers of rats fed a carcinogenic diet which interferes with DNA methylation. Using restriction endonuclease digestion, we find no differences in methylation pattern and overall organization of the AFP gene in oval cells (AFP-producers) and hepatocytes (non-producers) isolated at the early stages of carcinogenesis. Our studies indicate that in cell populations which produce AFP as well as in cells which are not active in AFP synthesis, the majority of the CCGG sites of the AFP gene are extensively methylated. In addition, we describe the existence of polymorphism in the AFP and albumin genes of Sprague-Dawley rats.

Structural changes in the cytoskeleton in regenerating mouse liver cells

After CCl/sub 4/ poisoning induced in rats poisoning centrilobular necroses formed in the liver during the next 24 h. Single a-feto protein-containing cells appeared onnthe second day of regeneration. By the end of the 2nd day a perinecrotic layer of cells containing AFP was formed. There is a definite correlation between loss of biliary capillary antigen, the appearance of bundles of prekeratin and actin, and expression of AFP synthesis. It is possible to include all these features in a single marker ocmplex of ''embronalization'' of the hepatocyte.

The lack of estrogen control of rodent alphafetoprotein levels

AbstractAlphafetoprotein (AFP) binding of estrogens is thought to protect fetal and neonatal rats from the toxic effects of high maternal estrogen levels. Since the plasma concentrations of several other steroid binding proteins are ligand regulated, we tested the hypothesis that estrogens regulate plasma AFP levels. A batch Sephadex equilibrium binding assay and Scatchard analysis were used to quantitate AFP in plasma of both adult mice (C3H/HeN MTV−) chronically fed diethylstilbestrol (DES) or estradiol (E2) and neonatal rats (CD) injected daily with E2 for the first 15 days of life. Estrogen treatment of adults did not increase AFP above the nondetectable adult levels, nor did it alter the plasma disappearance curve of neonatal AFP. In both adults and neonates, other estrogen responses were noted, demonstrating that the failure of biologically active estrogens to alter AFP levels is not due to insufficient amounts of estrogen. We suggest that estrogens do not alter patterns of AFP synthesis and degradation in the rat, a finding which contrasts with the general observation that steroids regulate the levels of their binding proteins.

The presence of intervening sequences in the alpha-fetoprotein gene of the mouse.

The presence of intervening sequences in the alpha-fetoprotein gene of the mouse.

Role of GABA-ergic brain systems in the activating effect of diazepam

AFP-containing hepatocytes were shown to lose the membrane antigen, localized in the region of bile capillaries, due to the mouse liver regeneration induced by CCl4 and paracetamol poisoning. Both in the liver regeneration and early postnatal ontogenesis the cessation of the AFP synthesis in hepatocytes coincides with the appearance of the bile capillary antigen on the cell surface. It is suggested that the AFP synthesis cessation is the result of the arrangement of cell contacts characteristic of the definitive liver balk.

Immunofluorescent Localization of Alpha‐fetoprotein Synthesis in the Endodermal Sinus of Rat Placenta

Specific immunofluorescence staining for AFP of the rat yolk sac showed that the synthesis was confined to the visceral layer of the yolk sac endoderm. The AFP synthesis took place not only in the extraplacental yolk sac as demonstrated by Gitlin in 1967, but also in the endodermal sinus structures within the placenta. No immunofluorescence staining was found of Reichert's membrane close to the parietal layer of the yolk sac. These findings are in accordance with the earlier demonstrated AFP synthesis in the visceral layer of the endodermal sinus structures in the tumors.

α‐Fetoprotein Synthesis in Yolk Sac Tumor: Sex‐dependent Production of α‐Fetoprotein by Transplantable Rat Yolk Sac Tumor

Transplantable rat yolk sac tumor cells produce α‐fetoprotein (AFP) and their ability to synthesize AFP is maintained for generations in female rats. However, when they are transplanted in male rats, the AFP production decreases markedly.To examine this sex dependency in AFP production, experiments at cellular and subcellular levels were carried out. In cell incubation studies, yolk sac tumor cells maintained in female rat (YST‐F cells) synthesized more AFP than yolk sac tumor cells maintained in male rat (YST‐M cells). Using cytosol RNAs prepared from YST‐F and YST‐M cells, AFP production was studied in cell‐free protein synthesizing system derived from wheat germ.In this system, cytosol RNA from both YST‐F and YST‐M cells directed AFP synthesis. But the amount of AFP synthesized was smaller in the presence of RNA from YST‐M cells. From these results, it was suggested that the reduced AFP synthesis by YST‐M cells is, at least partly, due to a quantitative decrease in their cytosol messenger RNA coded for AFP.

Cellular basis of induced alpha-fetoprotein synthesis by hepatocytes of adult mouse after hepatotoxic injury and partial hepatectomy.

AbstractThe dynamics of α‐fetoprotein (AFP) production by hepatocytes following different experimental manoeuvres that are known to induce regeneration of the liver was studied in young adult mice by simultaneous identification of dividing cells arrested during mitosis and of cells containing immuno‐histochemically detectable AFP. After 70% hepatectomy AFP was produced by a few, largely periportally located hepatocytes, following waves of brisk and active cell division. Carbon tetrachloride (CCl4)‐induced hepatic injury, on the other hand, led to participation of a relatively larger number of cells in early AFP synthesis preceding a less extensive cell proliferation. Initially, AFP‐producing cells were located in the peripheral part of the lobule but later they were more centrally placed, most often surrounding the zone of necrosis. Cells in mitosis often contained large amounts of AFP. CCl4 administration 48 h after 70% hepatectomy resulted in extensive AFP production by approximately a quarter of all surviving liver cells, although the concurrent rate of cell division was low. Intoxication with dimethylnitrosamine showed a pattern identical to that of hepatectomy but both cell division and AFP production were of comparatively lower magnitude. It is concluded that at least two distinct modes exist for augmented synthesis of AFP by hepatocytes of adult animals. In one, pre‐existent cells are stimulated to produce the protein before and irrespective of cell division while in the other, small amounts are synthesized by newly generated cells. Combination of the two mechanisms results in high levels of AFP production by stimulation of a large number of young hepatocytes.

Effects of α-Fetoprotein on Murine Immune Responses

Abstract Because of the current interest in the possible “immunosuppressive” effects of α-fetoprotein a systematic study of the effect of normal sera, amniotic fluid (AmF), sera from hepatoma-bearing animals (Tu sera), and AFP isolated by immunoabsorption from AmF or Tu sera on various models of the immune response of the mouse was carried out. Whereas some in vitro reactions were inhibited by AFP, other in vitro systems were only weakly inhibited, unaffected, or even enhanced by the addition of AFP. AFP by itself was mitogenic for murine lymphoid cells, inhibited the LPS mitogenic response 3-fold, but did not affect the response to Con A. Very high concentrations of AFP partially inhibited primary in vitro plaque-forming cell (PFC) responses to sheep red blood cells (SRBC). The secondary in vitro PFC response to keyhole limpet hemocyanin (KLH) conjugated to trinitrophenol (TNP) was inhibited 2- to 4-fold. The induction of cytotoxic effector cells directed against P815 mastocytoma cells was increased 9-fold in the presence of AFP. There was no significant difference between the effect of AmFAFP and TuAFP on any of the in vitro reactions studied. Normal and tumor sera produced a similar reduction in secondary PFC responses in spite of a 6000-fold difference in AFP concentration. The in vivo PFC response to SRBC in mice bearing a transplantable hepatoma was greater than that of normal mice even though the serum AFP concentration of the tumor-bearing mice was 1000 times normal. In addition, no AFP synthesis or coating of lymphocytes was observed during the course of a graft-vs-host or mixed lymphocyte reactions. Therefore, although some effects on immune phenomena occur upon addition of serum proteins and AFP, these data do not support a general immunosuppressive role for AFP in the mouse.

POSTNATAL DECLINE OF SERUM ALPHA FETOPROTEIN (AFP) CONCENTRATION

The concentration of serum AFP of 35 newborn infants was measured serially during the first 3 to 12 weeks of life. In general, the concentration at birth was found to be inversely related to gestational age. However, the wide range of values makes the use of the serum AFP concentration at birth of little value in establishing the duration of gestation. The individual curves of decline of serum AFP concentration are classified into 3 groups. Group I has a rapid regular fall with a half-life of 4.5 ± 0.8 days. The 14 infants in this group are AGA term and AGA preterm with uncomplicated postnatal courses. Group II has a slow regular fall with a half-life of 8 to 14 days. This group is made up of 2 very small AGA preterm infants with marked respiratory difficulties and 1 SGA preterm infant. Group III is characterized by serial values which decline irregularly with spikes and plateaus so that the half-life cannot be determined. Of the 18 members of this group, 1 AGA term infant had asphyxia and seizures, and 17 preterm infants had a variety of problems including SGA, sepsis, severe RDS and shock. The curves of Groups II and III may be the result of continued postnatal AFP synthesis by the liver in response to chemical, circulatory or toxic hepatic injury. Several infants in this group had prolonged hyperbilirubinemia and 1 infant had marked cholestasis with hepatic failure.

Detection of alpha-foetoprotein in mouse liver differentiated hepatocytes before their progression through S phase.

α-FOETOPROTEIN (AFP) is a serum protein present in large amounts during embryonic life and preserved in only nanogram quantities in adult mammals1. Increased levels of AFP are seen in adults in association with two forms of tumours—teratocarcinomas and hepatocarcinomas—and also during liver regeneration2. In early development AFP is synthesised in the yolk sac and in the liver2,3 where it has been demonstrated by immunofluorescence in the majority of the hepatocytes4. Detailed aspects of AFP synthesis by hepatocytes, however, remain to be established. It has been suggested that it is synthesised by a definite population of hepatocytes5, by one of the stages in their differentiation6,7, or by liver cell precursors8. Sufficient data to decide between these alternatives are not presently available. We describe here the immunohistochemical localisation of AFP in regenerating mouse livers, in which DNA-synthesising cells have been identified by thymidine incorporation. AFP was found in a minority of adult differentiated hepatocytes, many of which had not progressed through S phase. Thus DNA synthesis is not a prerequisite for the induction of AFP synthesis in the adult liver.

Tissue sites of alpha fetoprotein synthesis by the rat during pregnancy and hepatoma growth.

The tissue sites of alpha1 fetoprotein (AFT) synthesis by the rat during gestation and hepatoma growth were determined by specific incorporation of a radiolabeled amino acid precursor into AFP by tissue cultures in vitro. During gestation, AFP were produced by the yolk sac, the fetal liver, and in small amounts by the fetal gastrointestinal tract; there was no synthesis by maternal rat tissues. During growth of a transplantable hepatoma, only the hepatoma tissue synthesized AFP; the nontumor tissue of the host contained AFP but did not produce it.