Bovine fetus-specific serum proteins. Purification and characterization of alpha1-fetoprotein and immunological identification of alpha2- and beta-fetoproteins.

Bovine alpha1-fetoprotein was isolated from fetal calf serum by successive procedures of concanavalin A-Sepharose chromatography, DEAE-Sephadex chromatography, SP-Sephadex chromatography and preparative disc polyacrylamide gel electrophoresis. The bovine alpha1-fetoprotein preparation was considered homogeneous by several physicochemical and immunochemical criteria. Bovine alpha1-fetoprotein has a molecular weight of 68 000 with an amino acid composiotn similar to that of other mammalian alhpa1-fetoprotein. In addition, bovine alpha1-fetoprotein was shown to exist as two distinct variants on the basis of carbohydrate heterogeneity. alpha2-Fetoprotein and a new beta-fetoprotein were immunologically identified in fetal calf serum. These fetoproteins, like alpha1-fetoprotein, were not detectable in non-pregnant cow serum by immunoelectrophoresis.     

Purification and immunohistochemical localization of rat liver glutathione peroxidase

Glutathione peroxidase was purified from the rat liver to give a single protein band in polyacrylamide gel electrophoresis. Rabbits were immunized with this purified enzyme, and a highly specific anti-glutathione peroxidase antiserum was obtained. Using this antibody, an immunohistochemical technique (the indirect method of peroxidase-labeled antibody) was applied to study the localization of the enzyme in the liver cells.On immunohistochemical observation, glutathione peroxidase was localized exclusively in the cytoplasm of hepatocytes, and a stronger ‘immuno-staining’ was exhibited in the peripheries of the hepatic lobules than in the central zone.     

Fluorescent analog of OSW-1 and its cellular localization

OSW-1 is a steroidal saponin, which has emerged as an attractive anticancer agent with highly cancer cell selective activity. A fluorescent analog was prepared from the natural product to analyze its cellular uptake and localization. We found that the fluorescent analog is rapidly internalized into cells and is primarily distributed in endoplasmic reticulum and Golgi apparatus.     

Histochemical evaluation of mouse and rat kidneys with lectin-horseradish peroxidase conjugates

Paraffin sections of mouse and rat kidney were stained with a battery of ten lectin-horseradish peroxidase conjugates and lectin binding was correlated with the ultrastructural distribution of periodate-reactive sugar residues as determined by the periodic acid-thiocarbohydrazide-silver proteinate technique. Various segments of the uriniferous tubule in both species showed differential affinity for labelled lectins. Significant differences were also evident between comparable tubular segments in mouse and rat kidneys. Neutral glycoconjugates containing terminal beta-galactose and terminal alpha-N-acetylgalactosamine were prevalent on the luminal surface of the proximal convoluted tubule in the rat, but alpha-N-acetylgalactosamine was absent in this site in the mouse. In both species, terminal N-acetylglucosamine was abundant in the brush border of proximal straight tubules but absent in proximal convolutions. Fucose was demonstrated in both proximal and distal segments of mouse kidney tubules but only in the distal nephron and collecting ducts in the rat. Lectin staining revealed striking heterogeneity in the structure and distribution of cellular glycoconjugates. Such cellular heterogeneity was previously unrecognizable with earlier histochemical methods. The marked cellular heterogeneity observed with several lectin-conjugates in distal convoluted tubules and collecting ducts of both species raises a prospect that lectins can provide specific markers for intercalated and principal cells in the mammalian kidney. Glycoconjugates containing terminal sialic acid and penultimate beta-galactose were present on vascular endothelium in both rodent kidneys, as were terminal alpha-galactose residues; but both species lacked reactivity for Ulex europeus I lectin in contrast to human vascular endothelial cells. The constant binding pattern of lectin conjugates allows convenient and precise differentiation of renal tubular segments and should prove valuable in the study of changes in kidney morphology promoted by experimental manipulation or pathologic changes.     

Complete cDNA sequence and chromosomal localization of mouse alpha 1-antitrypsin

A cDNA encoding the complete open reading frame of murine alpha 1-antitrypsin has been cloned and sequenced. The nucleic acid and predicted amino acid sequences show homology to human alpha 1-antitrypsin and demonstrate the preservation of critical structural determinants for intracellular targeting, carbohydrate attachment, and catalytic function. The alpha 1-antitrypsin gene locus (Aat) has been localized on murine chromosome 12E----F by in situ hybridization.     

Purification and immunoelectron microscopic localization of cellular glutathione peroxidase in rat hepatocytes: quantitative analysis by postembedding method

To measure quantitatively the intracellular distribution of cellular glutathione peroxidase (GPX) in rat hepatocytes, ultrathin sections were stained by a postembedding immunogold technique. GPX had a specific activity of 1670 Units/mg protein, and was purified 2050-fold from rat liver by means of heat denaturation, ammonium sulfate fractionation, and a series of chromatographic procedures including thiol-Sepharose 4B. The purified GPX was shown to be electrophoretically pure, and was a homotetramer of 22 kDa subunits. Monospecific polyclonal antibodies were raised in rabbits by immunization. By immunoblot analysis, both the light mitochondrial the and cytosolic fractions of rat liver homogenate gave a single band with an identical mobility to that of the purified enzyme. Under the light microscope, hepatocytes showed nuclear staining and granular cytoplasmic staining, corresponding to certain intracellular structures. The labeling density (number of gold particles/m²) for GPX obtained by immunoelectron microscopy was 11.9 in the nuclei, 19.6 in mitochondria, 3.32 in peroxisomes, 1.95 in lysosomes, and 9.81 in the cytoplasmic matrix. These results suggest that cellular GPX is present in various compartments of rat hepatocytes, and that the GPX occurs in relatively higher amounts in mitochondria.     

Association of endodermal sinus tumour,testicular teratoma and alpha1-fetoprotein.

An endodermal sinus tumour in the retroperitoneal region was associated with the presence of alpha1-fetoprotein (AFP) in the patient''s serum. At autopsy a simple cystic teratoma of the right testicle was also found. The association of these two tumours has been reported before. The classification of these malignant germ-cell tumours and an understanding of their evolution may be aided by the discovery that AFP is often found in the patient''s serum.     

Collateral immunoprecipitation and localization of cellular antigens using monoclonal antibody--gold conjugates

Colloidal gold--protein complexes have been used to precipitate iodinated cell surface glycoproteins from cellular lysates. Both direct and 'sandwich' techniques with monoclonal antibodies were used. The same reagents were employed for immunolocalization of the antigenic determinants at the SEM level. The immunoprecipitation had a low background and was efficient. No washing of the precipitate was necessary. The gold-antibody precipitation (GAP) technique thus bridges the gap between immunocytochemistry and biochemistry.     

Highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody conjugates for enzyme immunoassays

The periodate-mediated conjugation of horseradish peroxidase to antibody is one of the most popular methods to prepare conjugates for enzyme immunoassays of antigens or corresponding antibodies. A very simple method to obtain peroxidase, which is both about five times cheaper than the rather expensive commercial preparations and has a significant higher activity, is reported. Moreover, the conjugation method was critically investigated and considerably simplified. Conjugates thus obtained are about three times more active than the best obtained with the original method.     

Rat alpha1-acid glycoprotein. Purification and immunological estimation of its serum concentration.

A rapid method is described for the preparation of serum alpha1-acid glycoprotein from rats with inflammation induced with turpentine oil injection. The protein obtained by two purification steps, batchwise adsorption with DEAE-cellulose followed by chromatography on CM-cellulose, was proved to be native alpha1-acid glycoprotein in a high degree of purity by electrophoretical, immunological, ultracentrifugal and carbohydrate analysis. The monospecific and potent antiserum to this protein was prepared by immunizing rabbits with the desialyzed material emulsified with Freund's incomplete adjuvant. Using purified alpha1-acid glycoprotein and its specific antiserum, the concentration of alpha1-acid glycoprotein in rat serum was determined by single radial immunodiffusion. Abnormally high levels of its concentration (5-6 times higher than the control) were observed in inflammatory and tumor bearing rats.     

Purification of turnip peroxidase and its kinetic properties

Peroxidase from turnip roots was purified using metal affinity chromatography up to a specific activity of 337 units/mg protein with 3.02 RZ and 63.5% recovery. After purification, the enzyme showed 2-3 bands on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme was found to be 37-39 kD with matrix assisted laser desorption ionization mass spectrometer (MALDI-MS). The enzyme showed maximum activity in phosphate buffer, pH 6.0, and lowest activity in borate buffer at the same pH. The Km of the enzyme was found to be 7.07 x 104 mM. Turnip peroxidase also contains an iron moiety which is found to be about 0.28%. The enzyme showed 50% inhibition of its specific activity with ethylene diamine tetraacetic acid (EDTA).     

Immunohistochemical localization of ornithine aminotransferase in normal rat tissues by Fab'-horseradish peroxidase conjugates

Immunohistochemical localization of ornithine aminotransferase (L-ornithine: 2-oxo-acid aminotransferase, EC 2.6.1.13), a mitochondrial enzyme whose hereditary absence induces gyrate atrophy of the choroid and retina, was elucidated by a direct immunoperoxidase method using Fab'-horseradish peroxidase conjugates. In immunodiffusion studies, the antibodies raised with the re-crystallized enzyme were highly specific to ornithine aminotransferase. To show localization of ornithine aminotransferase in normal rat tissues, clear immunohistochemical staining of this enzyme through the inner mitochondrial membrane in paraffin sections was achieved with Fab'-horseradish peroxidase conjugates. Strong immunoreactivity was present in cerebral neurons, hepatocytes, and epithelial cells of renal tubuli, gut mucous membranes, and ocular tissues. Specific distribution of ornithine aminotransferase was found in ependymal cell groups: namely, epithelial cells of the choroid plexus, pigmented and nonpigmented epithelial cells of the ciliary body. and Müller cells and pigment epithelium of the retina.     

Carcinoembryonic antigen and alpha1-fetoprotein in ulcerative colitis and regional enteritis

Sera were collected from 108 patients with inflammatory bowel disease and assayed for carcinoembryonic antigen (CEA) and alpha₁-fetoprotein (AFP). Seven (14%) of 51 patients with ulcerative colitis had a positive test for CEA and one of these had associated carcinoma of the colon. Ten (19%) of 52 patients with regional enteritis were also seropositive. The sera of 4 (9%) of 47 patients with ulcerative colitis and 2 (5%) of 41 patients with regional enteritis contained small amounts of AFP. Of two unclassified patients one had a positive CEA and the other a positive AFP. No serum was positive for both CEA and AFP. In addition, multiple samples were available for sequential analysis in eight CEA-positive patients but there was no apparent relationship between seropositivity and disease activity. Continued follow-up is now in progress to determine the significance of detectable fetal antigen levels in inflammatory bowel disease.     

Alcohol dehydrogenase in the mouse epididymis. Genetic variation and cellular localization

The cellular localization of alcohol dehydrogenase (ADH) in the mouse epididymis was investigated using differential substrate specificities and genetic variation as a means of distinguishing these enzymes histochemically in tissue sections. ADH-C2 exhibited high activity in BALB/c epididymis and was observed as a discrete zone within duct epithelial cells near the nuclei. This isozyme exhibited no detectable activity in C57BL/6J epididymis extracts or histochemical sections.