Jun-ichi Kawano

A protein-specific monoclonal antibody to rat liver beta 1-->4 galactosyltransferase and its application to immunohistochemistry.

We have produced a new protein-specific monoclonal antibody (MAb) to rat liver beta 1-->4 galactosyltransferase. This MAb, GTL2, was selected as the most reactive IgG to a periodate-treated antigen. Antigen and protein specificities of GTL2 were verified by immunoblotting of a non-glycosylated recombinant protein of human galactosyltransferase and enzymatically deglycosylated rat galactosyltransferase. Using GTL2, an immunohistochemical study was done in rat liver, epididymis, and salivary glands. Intense staining was observed in Golgi areas of epididymal duct epithelial cells, and submandibular and sublingual acinar cells. Hepatocytes showed weaker staining. Immunoelectron microscopic observation revealed that the staining was exclusively localized in trans-Golgi membranes of these cells.

Characterization of rat and human steroid sulfatases

Rat and human steroid sulfatases were purified from liver and placenta, respectively, by the same procedure. The rat and human enzymes were solubilized with Triton X-100, and purified by immunoaffinity chromatography with a monoclonal antibody showing high binding activities to both the enzymes. They were further purified by high-pressure anion-exchange chromatography to compare their structural and catalytic properties. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that both enzymes had a molecular weight of 62,000. The enzymes had similar amino acid compositions and amino-terminal amino acid sequences. Significant differences of the optimum pH, Michaelis constant and maximum velocity were observed between these enzymes. The optimum pH of each enzyme varied from 6.0 to 8.0, depending on substrates and with or without Triton X-100. In detergent-free media, steroid sulfates competitively inhibited the ability of these enzymes to hydrolyze 4-nitrophenyl sulfate. In media containing Triton X-100, on the other hand, the inhibition types of the steroid sulfates on the hydrolyzing activities of the rat and human enzymes were noncompetitive- and mixed-types, respectively.

Subcellular localization of N-acetylglucosaminide beta 1-4 galactosyltransferase revealed by immunoelectron microscopy.

We prepared a monoclonal antibody (MAb) against N-acetylglucosaminide beta 1----4 galactosyltransferase purified from F9 embryonal carcinoma cells. The MAb recognized the protein portion of the enzyme, since it inhibited galactosyltransferase activity, reacted with the enzyme both from F9 cells and from bovine milk, and did not exhibit anti-carbohydrate activity. Using this MAb, we studied the subcellular localization of the enzyme by immunoelectron microscopy. Intense staining was observed in trans-Golgi stacks within testicular interstitial cells and mucous neck cells, confirming the specificity of the immunological reaction. Cell surface galactosyltransferase was detected in the following regions: cultured cells such as F9 embryonal carcinoma cells, testicular interstitial cells, seminiferous tubule epithelial cells, Sertoli cells, the head of the epididymal sperm, epididymal epithelial cells, and apical surfaces of epithelial cells in the fundic gland and of intestinal goblet cells. The use of Triton X-100 intensified the cell surface immunoreactivity, and in certain cases the mode of distribution of the cell surface enzyme was different from that described in previous reports. In addition, nuclear envelopes of cultured cells were distinctly stained. The possible significance of the latter finding is discussed in relation to recent advances in nuclear localization of glycoproteins.

Bromodeoxyuridine-immunohistochemistry on cellular differentiation and migration in the fundic gland of Xenopus laevis during development

SummaryCellular differentiation and migration in the fundic glands of adult and larval Xenopus laevis have been examined using bromodeoxyuridine-immunohistochemistry. In the adult fundic gland, cumulative labeling with bromodeoxyuridine revealed a proliferative cell zone between the surface mucous cells and mucous neck cells, in what is referred to as the neck portion of the gland. The labeling-index of mucous neck cells had rapidly increased by week-5. The labeling-index of oxynticopeptic cells showed a more delayed increase until week-7, coincident with the decrease in the labeling of mucous neck cells. In the immature fundic glands of larvae, the labeled proliferating cells were randomly distributed throughout the developing gastric mucosa. During metamorphosis, the labeling-index of immature epithelial cells was highest at stage 63. Following administration of bromodeoxyurdine at this, stage, there was no significant loss of labeled epithelial cells during the metamorphosing period. Furthermore, there was no significant difference in the labeling-indices among the epithelial cells, such as surface mucous cells/generative cells, mucous neck cells, and oxynticopeptic cells, 7 days after administration. Cellular differentiation and migration pathways of epithelial cells in the fundic gland of adult X. laevis and its larvae are discussed.

A simple contrast enhancement by potassium permanganate oxidation for Lowicryl K4M ultrathin sections prepared by high pressure freezing/freeze substitution

A simple contrast enhancement method is presented for Lowicryl K4M ultrathin sections prepared by high pressure freezing/freeze substitution. The sections were treated with an acidified potassium permanganate oxidizing solution followed by uranyl acetate and lead citrate staining. The method, designated KMnO4–UA/Pb staining, provided a much greater contrast in electron microscopy than conventional UA/Pb staining. In detail, the visibility of plasma membrane was especially improved and the nuclear heterochromatin, mitochondria and cytoplasmic ribosomes showed an adequate increase in electron density. In the mucous cells of rat Brunners glands, the Golgi cisternae were well defined with the KMnO4–UA/Pb staining. Interestingly, the membranes of the intermediate compartments were moderately reactive to the KMnO4–UA/Pb staining, whereas the cis and the trans compartments were only faintly stained. It should be emphasized that the KMnO4 oxidation following colloidal gold labelling did not cause a remarkable reduction of immunogold labelling and the enhanced contrast helped us to examine the gold particles with high accuracy. This contrast enhancement method is highly promising, with the potential to become a useful tool for histochemical investigation, including immunocytochemistry with the Lowicryl K4M ultrathin sections prepared by high pressure freezing/freeze substitution techniques.

A monoclonal antibody to rat liver arylsulfatase C and its application in immunohistochemistry

We purified arylsulfatase C from rat liver microsomes and prepared a monoclonal antibody (P42C2) to the purified enzyme. By SDS-PAGE and immunoblotting analysis using P42C2, the molecular weight of the purified enzyme and of the enzyme in liver and kidney microsomes were estimated at 62,000 daltons. P42C2 caused little inhibition of arylsulfatase C activity, and was bound only slightly to liver microsomes. Localization of arylsulfatase C was studied at the light and electron microscopic level by the indirect immunoperoxidase method using P42C2. In rat liver, arylsulfatase C was detected mainly in the hepatocytes, and less frequently in endothelial cells, Kupffers cells, and Itos cells. In rat kidney, strong staining was observed in the straight portions of the proximal tubules. The podocytes, interstitial cells, endothelial cells, and epithelial cells of Henles thin limbs were stained faintly. By electron microscopy, arylsulfatase C was found localized on the membranes of the endoplasmic reticulum and nuclear envelopes in these cells. These immunohistochemical findings agree with the localization demonstrated by an enzyme-histochemical method which we had previously developed.

Fluid Dynamics of the Excretory Flow of Zymogenic and Mucin Contents in Rat Gastric Gland Processed by High-pressure Freezing/Freeze Substitution

The high-pressure freezing/freeze substitution technique followed by Lowi-cryl K4M embedding provided an excellent ultrastructure and retention of antigenicity of rat gastric glands as well as the intraluminal fluid contents. By taking this advantage, we histochemically investigated the excretory flow of the zymogenic and mucin contents in rat gastric glandular lumen at the ultrastructural level. The combination of KMnO4-UA/Pb staining for zymogenic contents and Griffonia simplicifolia agglutinin-II (GSA-II) labeling for mucous neck cell (MNC) mucin distinguished the exocytosed zymogenic contents from the MNC mucin in the glandular lumen. Interestingly, at the base and neck regions, the zymogenic contents showed a droplet-like appearance, forming a distinct interface with the MNC mucin. At the pit region, the GSA-II labeling demonstrated restricted paths, designated as MNC mucous channels, which flowed into the surface mucous gel layer. It should be noted that the interface between exocytosed zymogenic contents and MNC mucin disappeared, and that the zymogenic contents merged into the MNC mucous channels. At the top pit region, the surface mucous gel layer showed laminated arrays of three types of gastric mucins. On the basis of these ultrastructural findings, we propose a model of the excretory flow in rat gastric gland.

Cryofixation processing is an excellent method to improve the retention of adrenomedullin antigenicity

Abstract. We reappraised the precise immunohistochemical localization of adrenomedullin (AM) by means of the combined use of the catalyzed signal amplification (CSA) system and plunge freezing (PF)/freeze substitution (FS) for light microscopy or high-pressure freezing (HPF)/FS for electron microscopy, focusing on the rat adrenal gland and heart. In the case of adrenal glands, the PF processing showed that almost all medullary cells were intensively immunoreactive, while the cortical cells showed weak immunoreaction. In the heart, almost all cardiac muscle cells of the atria were also vividly stained with the PF/FS and the CSA enhancement. On the contrary, traces of immunoreactions were seen in most of the ventricular cells. These results are consistent with the previous reports of AM radioimmunoassays and the expression of AM mRNA. However, the chemical fixation processing revealed heterogeneous immunostaining in the atrial and ventricular myocardium as well as the adrenal medulla. Intensity of the immunostaining in the chemically fixed tissues was not likely to correspond with that of AM radioimmunoassays. The HPF/FS processing clearly demonstrated the immunogold labeling on secretory granules of adrenal medullary cells as well as cardiac muscle cells of the right auricles. Immunogold labeling intensity of the cryofixed specimens was 3- to 25-fold higher than that of the chemically fixed ones.

Human colorectal carcinoma-specific glycoconjugates detected by pokeweed mitogen lectin.

Pokeweed mitogen (PWM) lectin, known to bind branched poly-N-acetyllactosamines, has a highly selective affinity for human colorectal carcinomas. We performed light microscopic (LM) histochemistry with PWM lectin on paraffin sections of human colorectal tissues. In histological sections, normal mucosae and adenomas with mild dysplasia exhibited negative reaction (0/10, 0/13, respectively) with or without neuraminidase pre-digestion, whereas adenomas with moderate dysplasia showed a small increase in PWM lectin reactivity after neuraminidase digestion (4/23). In contrast, we observed a high incidence of positive reactivity in colorectal carcinoma without neuraminidase pre-digestion (38/44). After digestion with neuraminidase, there was increased reactivity of colorectal carcinomas in situ (7/12) and invasive carcinomas (13/32). These results imply that human colorectal carcinomas consistently contain substantial amounts of PWM-reactive branched poly-N-acetyllactosamine glycoconjugates structures. We also compared the staining patterns of PWM lectin and monoclonal antibodies (MAb) directed to Lewis X (LeX) or Lewis Y (LeY) antigen. PWM lectin reactivity was largely confined to the apical membranes of carcinoma tissues. MAb-LeX or MAb-LeY immunoreactivity was seen on the apical membranes and in the cytoplasm of both adenomas and carcinomas. Therefore, histochemical studies with this lectin should be useful for identification of carcinoma tissues and analysis of glycoconjugates associated with colorectal carcinoma.

Regional distribution of arylsulfatase C and estrone-sulfate sulfatase activities in rat brain and hypophysis

Regional distributions of arylsulfatase C and estrone-sulfate sulfatase activities were studied in rat brain and hypophysis by both histochemical and biochemical methods. Both methods showed that high activities of both enzymes were localized in pineal gland, choroid plexus, and adenohypophysis. Ultracytochemical techniques visualized the arylsulfatase C activity in the endoplasmic reticulum and the nuclear envelope of pineal cells, ependymal cells, and some types of cells of the adenohypophysis.