Cellular immunolocalization of alpha-fetoprotein in rat liver

Increased synthesis of alpha-fetoprotein (AFP) was induced in rat liver by the administration of 3'-methyl-4-dimethyl-aminoazobenzene. The indirect immunoperoxidase technique was used to detect AFP. Cellular localization of AFP was studied using a number of different fixation procedures. Serial sections stained with immunoglobulin served to determine the extent of diffusion of serum proteins into liver cells during fixation. Background staining was minimized when Lillie's neutral buffered formalin plus acetic acid was used as the fixative. After 3'-methyl-4-dimethylaminoazobenzene ingestion, bile duct cell proliferation occurred. The serum AFP was positive in all rats after 17 days on the diet. In rats with AFP-positive sera the immunohistochemical reaction in mature hepatocytes was positive while bile duct cells and small hepatocytes were negative for AFP.     

Cellular and subcellular immunolocalization of L-glutamate decarboxylase in rat pancreatic islets

The cellular and subcellular distribution of L-glutamate decarboxylase (GAD), the biosynthetic enzyme for gamma-aminobutyric acid (GABA), was determined immunohistochemically in rat pancreatic islet using light and electron microscopic techniques. The cellular distribution of GAD was determined at the light microscopic level using an elution/re-staining protocol and a computerized digital image processing technique. At this level of resolution, immunofluorescent GAD was observed to be co-localized with immunofluorescent insulin in the islet B-cells and absent in both the A-cells, which contained glucagon, and the D-cells, which contained somatostatin. Subcellular localization of GAD was determined using an electron microscopic, colloidal gold post-embedding protocol and was compared to insulin immunoreactivity in serial sections of the same B-cell. In the same islet B-cell, GAD immunoreactivity appeared predominantly in the extragranular cytoplasm, whereas insulin immunoreactivity was associated with the secretory granules. Quantitative analysis of GAD immunoreactivity in the B-cell revealed 15.3 +/- 1.8 gold particles/micron2 in the cytoplasm, 1.7 +/- 0.2 gold particles/micron2 in the secretory granules, and 0.4 +/- 0.4 gold particles/micron2 in the mitochondria. The results of this study, localization of the biosynthetic enzyme for GABA to the B-cell cytoplasmic compartment and its absence in the secretory granules which contain insulin, are compatible with the hypothesis that GABA functions as an intracellular mediator of B-cell activity.     

Differences in the binding of thyroid hormones and indoles by rat alpha 1-fetoprotein and serum albumin

The transport of small molecules in the blood, normally assured by serum albumin in the adult, is not well known in the fetus since the albumin concentration is low in fetal serum and inversely related to the alpha 1-fetoprotein concentration. In order to investigate whether rat alpha 1-fetoprotein might be a fetal counterpart to albumin, the binding properties of these two proteins have been compared with respect to a series of molecules of biological importance, especially during fetal development: thyroid hormones and indole analogues. Though high-affinity binding of thyroxine was found with both rat alpha 1-fetoprotein and albumin, a significant difference in the number of binding sites for this hormone was found with the two proteins. Further, while rat serum albumin strongly bound L-tryptophan and indolyl-3-acetic acid (Ka approximately equal to 10(5) M-1), rat alpha 1-fetoprotein did not bind any of the indoles tested. These results are discussed with respect to the physiological and pharmacological significance of the transport role of these proteins.     

Ligand properties of diethylstilbestrol: studies with purified native and fatty acid-free rat alpha 1-fetoprotein and albumin.

We report the equilibrium binding parameters for the interactions of the estrogen analogue diethylstilbestrol (DES) with highly purified rat alpha 1-fetoprotein (AFP) and serum albumin preparations. At 25 degrees C and pH 7.4, an association constant (Ka) of about 1.5 X 10(6)M-1 and 2 sites/mole are measured with the DES-AFP system, whereas for the DES-albumin interaction, we find a Ka of approximately 2 X 10(5)M-1 and about 11 sites/mole of protein. The removal of fatty acids from pure AFP causes a reversible 3 fold increase of the number of DES binding sites; the same delipidation procedure applied to albumin slightly diminishes its DES binding parameters. We also demonstrate the capability of DES to displace competitively estradiol-17 beta (E2) from its high affinity sites on the estrophilic rat AFP. Finally, the binding behaviour of the two serum proteins towards the synthetic estrogen is compared to their interaction with the natural hormones. The physiological and pharmacological relevance of these data is discussed.     

Fluorescence studies of the interactions of serum albumin and rat alpha1-fetoprotein with aflatoxin B1. Specificity and binding parameters.

The interaction of bovine serum albumin and rat alpha1-fetoprotein with aflatoxin B1 has been followed by the fluorescence quenching of the protein in presence of the ligand. The binding parameters (n, number of sites and Kd, dissociation constant) have been determined for the bovine serum albumin-alflatoxin B1 system: n = 3.5 and Kd = 3.1 +/- 0.5 . 10(-5) M and for the alpha-fetoprotein-aflatoxin system: n = 4 and Kd = 3.7 +/-0.5 . 10(-5) M. The competition of anilino-naphthalene-sulfonate and aflatoxin B1 for the same hydrophobic sites on bovine serum albumin has been demonstrated. The fluorescence quenching of various proteins (lysozymes, egg-albumin, gamma-globulin) by aflatoxin B1 have shown that there is not a strict specificity of aflatoxin towards proteins.     

Lipid composition of rat liver microsomes under various experimental conditions

Cholesterol and phospholipid content, and phospholipid composition (sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethaolamine) were assayed in rat liver microsomes during regeneration, foetal development and pregnancy. Cholesterol was assayed using Liebermann-Buchard reagent; the phospholipid extract was separated by thin-layer chromatography. While in pregnancy no changes were observed, during foetal development and liver regeneration there was a significative decrease of cholesterol/phospholipid ratio, and of phosphatidylcholine content. Moreover, in developing liver microsomes, there is also a significative increase of sphingomyelin and phosphatidylserine + phosphatidylinositol.     

Effect of fixation on immunolocalization of transferrin and albumin in the liver of the adult rat

The influence of the fixation procedure on the localization of albumin and transferrin in adult rat liver has been carried out using an indirect immunoperoxidase technique at the light and electron microscopic levels. Perfusion and immersion fixations with different concentrations of paraformaldehyde (with or without addition of glutaraldehyde) have been investigated. According to the mode of fixation (perfusion versus immersion) and the concentration of the fixative, the number of albumin and transferrin containing hepatocytes could vary from 10% to 100%, and different labeling patterns could be observed at the electron microscopic level. For the same concentration of fixative, a perfusion fixation induces a less intense labeling than an immersion fixation. Thus similar results are obtained after immersion fixation in 6% paraformaldehyde + 0.25% glutaraldehyde or after perfusion fixation in 4% paraformaldehyde + 0.025% glutaraldehyde. Similar data are noticed after immersion fixation in 4% paraformaldehyde or after perfusion fixation in 1% paraformaldehyde + 0.025% glutaraldehyde. Moreover, perfusion fixation induced a more fine cell structure preservation than immersion fixations and avoided the appearance of zones of fixation.     

Mouse alpha 1-fetoprotein and albumin. A comparison of their binding properties with estrogen and fatty acid ligands

The binding of estradiol-17 beta (E2), diethylstilbestrol (DES), and polyene fatty acids, in particular arachidonate (C20:4), to alpha 1-fetoprotein (alpha-FP) and albumin purified from mouse embryo sera was studied using equilibrium dialysis and electrophoretic techniques. E2, arachidonate, and DES all bind to alpha-FP, but with decreasing strength. E2 is a high affinity, low capacity ligand (Ka approximately 0.8 X 10(8) M-1 and approximately 0.3 sites/mol of alpha-FP at 25 degrees C); arachidonate is a weaker ligand disposing of more sites (Ka approximately 0.3 X 10(7) M-1 and 4-5 sites/mol of alpha-FP); the binding of DES is of comparatively low affinity and capacity (Ka approximately 0.2 X 10(7) M-1 and n approximately 0.7/mol of alpha-FP). In spite of different structures and equilibrium parameters, E2, DES, and arachidonate are able to compete with each other for binding to the fetoprotein. The C22:4 and C22:6 fatty acids are also efficient concentration-dependent inhibitors of E2 or DES binding. Albumin binds the fatty acids and DES, but equilibrium parameters are different from those of alpha-FP. In particular, arachidonate is a better ligand for albumin, where it interacts with at least two classes of apparent sites (Ka1 approximately 0.3 X 10(8) M-1 and n1 approximately 1; Ka2 approximately 0.2 X 10(7) M-1 and n2 approximately 30). In contrast to alpha-FP, albumin virtually does not bind E2. Also, no competition could be demonstrated between DES and fatty acid ligands for binding to albumin. None of the studied interactions, with either albumin or alpha-FP, was modified even by high doses of bilirubin. The possible functions of the various binding activities present in fetal sera in the process of growth are discussed.     

Individual alpha peak frequency variability and reproducibility in various experimental conditions

The test-retest study of the individual alpha peak frequency (IAPF) variability was provided in eyes closed and eyes open conditions in standard (8-12 Hz) and individual alpha band range in different brain areas in 96 healthy male subjects (aged 25-40). The pairwise comparisons showed that IAPF means are not different in the left and right hemisphere sites, in eyes closed and eyes open conditions, in standard and individual alpha range. Computed in the first 5 s after opening eyes fronto-central area IAPF mean was lower than in eyes closed condition. IAPF computed in individual alpha range in posterior area in eyes closed condition had the minimal variability and was the most reproducible in test retest investigation.     

The immunolocalization of ACTH and alpha MSH in human and rat pituitaries.

Human and rat pituitaries were investigated immunohistochemically for ACTH and alpha MSH activity by means of the enzyme-labeling technique. In rat pituitaries cells present in both the anterior and intermediate lobes were reactive with the anti-ACTH antibodies, the cells from the intermediate lobe were also reactive with anti-alpha MSH antibodies. In human pituitaries, ACTH-immunoreactivity was found in cells from the anterior lobe and cells invading the posterior lobe. In 5 out of 15 pituitaries ACTH-immunoreactive cells located at or invading the posterior lobe were also reactive with the anti-alpha MSH antibodies. It is concluded that the human pituitary cells that invade the posterior lobe represent a population which is at least immunohistochemically identical with the intermediate lobe cells of the rat. The ACTH-immunoreactivity of intermediate lobe cells may be explained by the presence of a corticotropin-like intermediate lobe peptide (CLIP) which has been suggested to be a prohormonal fragment of alpha MSH.