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Isolation, characterization and synthesis of alpha-foetoprotein from neonatal-rat skin.

ABSTRACT: Monospecific anti-[rat alpha-foetoprotein(alpha-FP)] immunoglobulin G was coupled to CNBr-activated Sepharose-4B (4.5 mg/ml packed volume of gel) to yield an adsorbent. The immunoaffinity column was used to isolate alpha-FP from neonatal-rat skin. Purified skin alpha-FP was found to be immunologically and electrophoretically similar to serum alpha-FP. It yielded a single band with mol.wt. 68000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. However, on polyacrylamide-gel electrophoresis under non-denaturing conditions, the alpha-FP displayed slow- and fast-moving variants similar to those observed in serum alpha-FP. A Scatchard plot of oestradiol binding to the alpha-FP yielded an association constant of 2.5 X 10(9)M-1 by dextran-coated-charcoal and 0.75 X 10(8)M-1 by Sephadex-gel-filtration procedures respectively. Skin explants from newborn rats were found to incorporate [14C]leucine into immunoprecipitable intracellular alpha-FP. Cycloheximide inhibited the synthesis of alpha-FP in skin explant culture. Our results indicate that newborn-rat skin contains alpha-FP that is similar to serum alpha-FP and which may arise in neonatal-rat skin as a result of synthesis in situ.

SUBMITTER: Mujoo K

PROVIDER: S-EPMC1152490 | biostudies-other | 1983 Oct

REPOSITORIES: biostudies-other

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Project description:1. Rat alpha-foetoprotein, an oestrogen-binding foetal globulin, was isolated in large quantities from amniotic fluid and serum by preparative electrophoresis on polyacrylamide slab gels or by chromatography on an immunoadsorbent column. Subsequently the two electrophoretic forms of this protein were separated by electrophoresis on the same medium. 2. Both forms were found to show identical binding with oestradiol. From the extrinsic fluorescence of the bound dye 8-anilinonaphthalene-1-sulphonic acid it was shown that the polarity of the binding site is practically identical for both forms. One residue of tryptophan was determined for both forms. The two electrophoretic variants display the same amount of secondary structure as demonstrated by circular dichroism. 3. The affinity of total alpha-foetoprotein for oestradiol as a function of pH was studied by using a Sephadex G-25 gel-equilibration method. Maximal binding occurred at pH8.5. Only a fractional number of binding sites per molecule could be measured at pH7.4, whereas at higher pH the number of sites was very close to unity. There was no significant effect of pH on the value of the association constant (K(a)=4.3x10(7)+/-1.2x10(7)m(-1)). 4. Displacement experiments of bound labelled oestradiol with various steroids have permitted investigation of the specificity of alpha-foetoprotein. This foetal globulin binds rather strongly compounds that display the rigid structure of the oestratriene skeleton (oestradiol, oestrone). Diminished binding for diethylstilboestrol and a diethylstilboestrol affinity label was observed. No binding was measured with a more flexible structure such as hexoestrol [4,4'-(1,2-diethylethane-1,2-diyl)bisphenol]. 5. Chemical modification of cysteine residues of alpha-foetoprotein with two alkylating reagents [iodoacetic acid and 8-[N-(iodoacetylaminoethyl)amino]naphthalene-1-sulphonic acid] has very little effect on the oestrogen binding. It is suggested that the oestrogen-binding site does not contain a cysteine residue. From the kinetics of alkylation and from the fluorescence properties of the chemically bound thiol reagent 8-[N-(iodoacetylaminoethyl)amino]naphthalene-1-sulphonic acid], it was demonstrated that the very-slow-reacting thiol group is probably located in a non-polar region of the molecule.

Project description:ObjectivesThis study aimed to compare two abbreviated MRI (AMRI) protocols to complete MRI for HCC detection: non-contrast (NC)-AMRI without/with alpha foetoprotein (AFP) and dynamic contrast-enhanced (Dyn)-AMRI.MethodsThis retrospective single-center study included 351 patients (M/F: 264/87, mean age: 57y) with chronic liver disease, who underwent MRI for HCC surveillance between 2014 and 2020. Two reconstructed AMRI sets were obtained based on complete MRI: NC-AMRI (T2-weighted imaging (WI) + diffusion-WI) and Dyn-AMRI (T2-WI + dynamic T1-WI) and were assessed by 2 radiologists who reported all suspicious lesions, using LI-RADS/adapted LI-RADS classification. The reference standard was based on all available patient data. Inter-reader agreement was assessed and MRI diagnostic performance was compared to the reference standard.ResultsThe reference standard demonstrated 83/351 HCC-positive patients (prevalence: 23.6%, median size: 22 mm, and positive MRIs: 83/631). Inter-reader agreement was substantial for all sets. Sensitivities of Dyn-AMRI and complete MRI (both 92.8%) were similar, higher than NC-AMRI (72.3%, p < 0.001). Specificities were not different between sets. NC-AMRI + AFP (92.8%) had similar sensitivity to Dyn-AMRI and complete MRI. In patients with small size HCCs (≤ 2 cm), sensitivities of Dyn-AMRI (85.3%) and complete MRI (88.2%) remained similar (p = 0.564), also outperforming NC-AMRI (52.9%, p < 0.05). NC-AMRI + AFP had similar sensitivity (88.2%) to Dyn-AMRI and complete MRI (p = 0.706 and p = 1, respectively).ConclusionsDyn-AMRI has similar diagnostic performance to complete MRI for HCC detection, while both outperform NC-AMRI, especially for small size HCCs. NC-AMRI + AFP demonstrates similar sensitivity to Dyn-AMRI and complete MRI.Clinical relevance statementDue to the low sensitivity of ultrasound for hepatocellular screening, new screening methods are needed. Abbreviated MRI (AMRI) is a candidate, especially non-contrast AMRI with serum alpha foetoprotein as the acquisition time is low, without the need for contrast medium injection.Key points• Dynamic contrast-enhanced abbreviated MRI using extracellular gadolinium-based contrast agent and complete MRI have similar diagnostic performance for hepatocellular carcinoma detection in an at-risk population. • Non-contrast abbreviated MRI with alpha foetoprotein has similar diagnostic performance to dynamic contrast-enhanced abbreviated MRI and complete MRI, including when considering small size hepatocellular carcinoma ≤ 2 cm. • Non-contrast abbreviated MRI and dynamic contrast-enhanced abbreviated MRI can be performed in 7 and 10 min, excluding patient setup time.

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Isolation, characterization and synthesis of alpha-foetoprotein from neonatal-rat skin.

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