Tsutomu Oinuma

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.

CALNUC (nucleobindin) is localized in the Golgi apparatus in insect cells

A mouse monoclonal antibody 12B1 was raised against Golgi fractions from Sf21 insect cells and selected as Golgi-specific by immunostaining of the cells. The antigen was purified from the cells by immunoaffinity chromatography with the monoclonal antibody, and its N-terminal and internal amino acid sequences were determined. Based on the partial amino acid sequences, cDNA encoding the antigen protein was cloned and sequenced. The amino acid sequence deduced from the cDNA nucleotide sequence showed a homology to those of CALNUC family proteins, CALNUC (or nucleobindin, a calcium-binding Golgi protein with DNA-binding activity) and protein NEFA (a cell surface protein with DNA-binding, EF-hand, and acidic domains). The insect protein had two EF-hand loops at the same sites as the mammalian CALNUC family proteins, but had no leucine zipper which the mammalian homologues commonly have. An electron microscopic immunoperoxidase study demonstrated that the insect protein was localized in the cis-Golgi cisternae and cis-Golgi networks. Since this localization is identical to that of mammalian CALNUC, the insect protein was considered to be a homologue of CALNUC rather than that of NEFA. Assays involving proteinase K digestion, sodium carbonate extraction and Triton X-114 extraction revealed that the insect CALNUC-like protein was a soluble protein tightly associated with the luminal surface of Golgi membranes as reported for mammalian CALNUC. The insect protein was also shown to have calcium-binding activity as does mammalian CALNUC. These data verify that the insect protein is CALNUC. The existence of CALNUC in insect cells suggests that CALNUC is an essential calcium-binding Golgi protein in a wide range of the animal kingdom. A phylogenetic tree analysis, however, suggested that NEFA was derived from CALNUC long after the segregation of a mammalian ancestor from an insect ancestor.

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.

Multistratified Expression of Polysialic Acid and Its Relationship to VAChT-containing Neurons in the Inner Plexiform Layer of Adult Rat Retina

We investigated the localization of polysialic acid (PSA), neural cell adhesion molecule (NCAM), and vesicular acetylcholine transporter (VAChT) in adult rat retina by using immunofluorescence with a confocal laser scanning microscope. Western blot analysis showed a typical broad smear of PSA and isoforms of NCAM (120, 140, and 180 kD). PSA immunofluorescence revealed multistratification in the inner plexiform layer (IPL). Dual immunostaining for PSA and NCAM exhibited the selective co-expression of PSA and NCAM on Müller cells. Moreover, dual immunolabeling for PSA and VAChT completely separated the five strata in the IPL. Strata 1, 3, and 5 were immunoreactive for PSA and Strata 2 and 4 for VAChT. These results suggest the possibility that PSA molecules on Müller cells are spatially related to ON and OFF retinal channels in the IPL.

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.

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.

A monoclonal antibody against insect CALNUC recognizes the prooncoprotein EWS specifically in mammalian cells. Immunohistochemical and biochemical studies of the antigen in rat tissues.

Abstract. A monoclonal antibody against insect CALNUC was shown to recognize an 85-kDa nuclear protein specifically in mammalian cells. Amino acid sequencing of the protein purified from rat liver revealed it to be EWS, a prooncoprotein for Ewing sarcomas and related tumors. Using the antibody, distribution of EWS was studied in rat tissues fixed with 4% paraformaldehyde by immunohistochemical methods. On thaw-fixed cryosections or those of perfusion-fixed tissues, almost all cell nuclei showed the specific staining. In immersion-fixed tissues, the staining unexpectedly disappeared in particular tissues (kidney cortex, liver, etc.), although it was recovered by autoclaving the cryosections. Western blotting also demonstrated the ubiquitous expression of EWS in the tissues. In extracts from the liver, the 85-kDa band rapidly disappeared in a Ca2+-dependent manner, but never in the testis. The antigen was very labile in kidney homogenates even without Ca2+. Biochemical studies with digoxigenin-labeled EWS showed that the Ca2+-dependent disappearance was associated with upward mobility shifts of EWS. These suggested that EWS was ubiquitously expressed in rat tissues, and that the antigen was masked in particular tissues during the immersion fixation.