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High-pressure freezing (HPF) has been generally accepted as the most reliable method for cryofixation of biological samples, yielding a deep vitreous freezing. In recent cell biology, mammalian cultured cells are widely used, but HPF of cultured cell monolayer has not reached its full potential. In this study, we developed a new reliable device for HPF of cultured cell monolayer by using a 10-microm-thin stainless disc both as culture plate and specimen carrier. We describe the practical procedure, and demonstrate fine structures of HeLa cells cultured and cryofixed on the stainless discs as results.

The annular ligament across the stapediovestibular joint connects the stapes footplate and the vestibular window and plays an important role in the sound conductive system of the ear. In this study, we investigated the distribution of extracellular matrix components in the ligament by histochemical methods at light and electron microscopic levels. As results, light microscopic immunohistochemistry of fibrillin and 36-kDa microfibril-associated glycoprotein (MAGP-36) showed intense immunoreactivities in the annular ligament between the stapes footplate and vestibular window. In addition, the histochemical localization of hyaluronic acid by using biotinylated hyaluronic acid-binding protein (HABP) clarifi ed the presence of hyaluronic acid in the annular ligament. At the electron microscopic level, the immunogold labeling of fibrillin showed intense labeling on the periphery of the electron-dense mantle. Furthermore, the labeling of fibrillin was preferentially seen on the fibrous components among the electronlucent amorphous substance. The immunogold labeling of MAGP-36 was seen on the electron-dense mantle and scattered on the electron-lucent amorphous substance. The gold labeling with biotinylated HABP clearly showed a distribution of hyaluronic acid throughout the amorphous space in the ligament. The present results provide a histochemical profile of the annular ligament of the rat stapediovestibular joint that may provide clues to elucidation of pathological changes in the ligaments and conductive hearing loss in humans.

It is clinicopathologically important to elucidate the cell kinetics for the maintenance of normal gastric epithelium. In a rat gastric mucosa isolated after stimulation, a number of cells were exfoliated into the gastric lumen of the pit region. The present study was undertaken to clarify the origin of exfoliated cells and their histochemical profiles by taking the advantages of cryotechniques. As results, most of the exfoliated cells were identified as pit-parietal cells labeled with both peanut-lectin and anti-H+/K+-ATPase antibody. Quantitative analysis verified a time-dependent increase in the number of exfoliated cells in the gastric mucosa isolated after stimulation. The exfoliated cells exhibited a diffuse intracellular staining for E-cadherin, suggesting a dissociation of the adhesion molecule prior to the cell exfoliation. It should be noted that most of the exfoliated cells were negative to the apoptotic markers (TUNEL staining and caspase-3). Ultrastructurally, autophagosome-like structures consisting of H+/K+-ATPase positive membranes were frequently seen in the exfoliated pit-parietal cells. In addition, the pit-parietal cell exfoliation was accompanied by sealing of their basal portion with the cytoplasmic processes of adjacent surface mucous cells. The present morphological findings provide a new insight into the cell kinetics in the gastric epithelium in vitro.

Mucin, a major component of mucus, is a highly O-glycosylated, high-molecular-mass glycoprotein extensively involved in the physiology of gastrointestinal mucosa. To detect and characterize mucins derived from site-specific mucous cells, we developed a monoclonal antibody, designated PGM34, by immunizing a mouse with purified pig gastric mucin. The reactivity of PGM34 with mucin was inhibited by periodate treatment of the mucin, but not by trypsin digestion. This suggests that PGM34 recognizes the carbohydrate portion of mucin. To determine the epitope, oligosaccharide-alditols obtained from pig gastric mucin were fractionated by successive gel-filtration, ion-exchange, and normal-phase HPLC, and tested for reactivity with PGM34. Two purified oligosaccharide-alditols that reacted with PGM34 were obtained. Their structures were determined by NMR spectroscopy as Fucα1–2Galβ1–4GlcNAc(6SO3H)β1–6(Fucα1–2Galβ1–3)GalNAc-ol and Fucα1–2Galβ1–4GlcNAc(6SO3H)β1–6(Galβ1–3)GalNAc-ol. None of the defucosylated or desulfated forms of these oligosaccharides reacted with PGM34. Thus, the epitope of PGM34 was determined as the Fucα1–2Galβ1–4GlcNAc(6SO3H)β- sequence. Immunohistochemical examination of rat gastrointestinal tract showed that PGM34 stained surface mucous cells close to the generative cell zone in the gastric fundus and goblet cells in the small intestine, but only slightly stained antral mucous cells in the stomach. These data, taken together, show that PGM34 is a very useful tool for elucidating the role of mucins with characteristic sulfated oligosaccharides.

Mucosubstance histochemical study of 33 cholesteatoma tissues was performed to clarify the distribution and character of mucin in the perimatrix. Mean density of glandular cysts was 0.18 per mm(2). Mean frequency of ruptured cysts was 0.16 per cyst. Glandular cysts as well as hollow spaces in the perimatrix were filled with sulfomucin and sialomucin. Fragments of mucin were found in some macrophages and multinucleated giant cells. Since phagocytosis is host defenses attempt, the process indicates that mucin in the perimatrix is a cause of inflammation. Sialomucin infiltrated in the subepidermis where the epidermis formed papillary proliferation without an apparent sign of inflammation. Six glandular cysts were found in the matrix and the debris. They may have been eliminated from the perimatrix as a sequel to cholesteatoma growth. These findings suggest that embedded mucosa in the perimatrix may play a crucial role in pathogenesis of acquired cholesteatoma.

The stapes footplate articulates with the vestibular window through the annular ligament. This articulation is known as the stapediovestibular joint (SVJ). We investigated the ultrastructure of adult rat SVJ and report here on the characteristic ultrastructure of the corresponding annular ligament. Transmission electron microscopy showed that this annular ligament comprises thick ligament fibers consisting of a peripheral mantle of microfibrils and an electron-lucent central amorphous substance that is regularly arranged in a linear fashion, forming laminated structures parallel to the horizontal plane of the SVJ. Scanning electron microscopy revealed that transverse microfibrils cross the thick ligament fibers, showing a lattice-like structure. The annular ligament was vividly stained with elastica van Gieson's stain and the Verhoeff's iron hematoxylin method. Staining of the electron-lucent central amorphous substance of the thick ligament fibers by the tannate-metal salt method revealed an intense electron density. These results indicate that the annular ligament of the SVJ is mainly composed of mature elastic fibers.

We previously isolated a monoclonal antibody named PH2 that inhibits phosphatidylserine-mediated phagocytosis of apoptotic cells by macrophages [C. Fujii, A. Shiratsuchi, J. Manaka, S. Yonehara, Y. Nakanishi. Cell Death Differ. 8 (2001) 1113-1122]. We report here the identification of the cognate antigen. A protein bound by PH2 in Western blotting was identified as the 170-kDa subunit of eukaryotic translation initiation factor 3 (eIF3 p170/eIF3a). When eIF3a was expressed in a culture cell line as a protein fused to green fluorescence protein, the fusion protein was detected at the cell surface only after the induction of apoptosis. The same phenomenon was seen when the localization of endogenous eIF3a was determined using anti-eIF3a antibody, and eIF3a seemed to be partially degraded during apoptosis. Furthermore, bacterially expressed N-terminal half of eIF3a fused to glutathione S-transferase bound to the surface of macrophages and inhibited phagocytosis of apoptotic cells by macrophages when it was added to phagocytosis reactions. These results collectively suggest that eIF3a translocates to the cell surface upon apoptosis, probably after partial degradation, and bridges apoptotic cells and macrophages to enhance phagocytosis.

High-pressure freezing (HPF) has been accepted generally as the most reliable method for cryoimmobilization of biological samples. However, the depth of vitreous freezing in biological samples was less than expected, probably because of the poor thermal conductivity with high water contents. In this study, we introduce a new assembly of the specimen carrier using a 10-microm thin stainless foil for the specimen chamber cover in the HPF technique and describe the fine structure of rat gastric glands processed with the assembly. A low-magnification view of the gastric surface region showed a well-preserved morphology in which the vitreous freezing reached deeper than 100-microm from the freezing face. The present results prove that the 10-microm thin stainless foil is useful in HPF, providing deep vitrification in biological samples.

Wilson disease is a genetic disorder characterized by the accumulation of copper in the body due to a defect of biliary copper excretion. Although the Wilson disease gene has been cloned, the cellular localization of the gene product (ATP7B) has not been fully clarified. Therefore, the precise physiological action of ATP7B is still unknown. We examined the distribution of ATP7B using an anti-ATP7B antibody, green fluorescent protein (GFP)-ATP7B (GFP-ATP7B) and ATP7B-DsRed in various cultured cells. Intracellular organelles were visualized by fluorescence microscopy. The distribution of ATP7B was compared with that of Rab7 and Niemann-Pick C1 (NPC1), proteins that localize in the late endosomes. U18666A, which induces the NPC phenotype, was used to modulate the intracellular vesicle traffic. GFP-ATP7B colocalized with various late endosome markers including Rab7 and NPC1 but not with Golgi or lysosome markers. U18666A induced the formation of late endosome-lysosome hybrid organelles, with GFP-ATP7B localized with NPC1 in these structures. We have confirmed that ATP7B is a late endosome-associated membrane protein. ATP7B appears to translocate copper from the cytosol to the late endosomal lumen, thus participating in biliary copper excretion via lysosomes. Thus, defective copper ATPase activity of ATP7B in the late endosomes appears to be the main defect of Wilson disease.

The stapes footplate articulates the vestibular window (stapediovestibular joint; SV joint). The SV joint is known as crucial region of otosclerosis. To shed a light on pathogenesis of otosclerosis, we have examined the histochemical characteristics of the rat ossicular joint cartilage. Interestingly, glycoconjugate histochemistry revealed that the cartilage matrix components of the SV joint were significantly different from those of the other ossicular joints, i.e. incudomalleolar (IM) and incudostapedial (IS) joints. Alcian blue, pH 2.5 and high-iron diamine procedures vividly stained the cartilage matrices of the IM and IS joints, while those of the SV joint were faintly stained. Chondroitin sulfate immunohistochemistry revealed intense labeling of the cartilage of the IM and IS joints, while the immunolabeling of the SV joint was negligible. On the contrary, keratan sulfate immunolabeling was discernible on the cartilage matrices of the SV joint, but not on those of the IM and IS joints. Type II collagen immunolabeling revealed that the marginal region of the IM and IS joint cartilage matrices was devoid of the immunostaining, while the SV joint cartilage matrices were uniformly labeled with this antibody. In this study, we have succeeded in demonstrating the histochemical characteristics of the rat SV joint cartilage matrices of which is quite different from those of the other ossicular joints.

Gastric ulcers in humans are notoriously chronic and recurring lesions. Although the average individual who undergoes no treatments requires many years for healing, most studies on the healing process of the experimentally induced ulcers have mainly focused on the early stages. Natural history of the ulcer healing has not been completely revealed. We have undertaken long-term investigation up to the 150th day after the cryo-injury to shed light on the natural history of the ulcer healing process compared with developmental changes of postnatal fundic glands. By the 30th day, restitutive gastric glands were mostly seen to cover the ulcer lesions, where well-developed gland-type mucous cells, showing Griffonia simplicifolia agglutinin (GSA)-II labeling, appeared to occupy the basal portion. Most of the bromodeoxyuridine-labeled cells were superimposed on the GSA-II-positive cell zone, forming the proliferative zone. By the 150th day, the restitutive glands were complete, with all epithelial components and topology of the normal fundic glands. The process of the ulcer healing was quite compatible with the developmental changes of the postnatal fundic glands. These results imply that the regeneration of gastric epithelium during the ulcer healing follows pathways linked to the ontogenetic course of the fundic gland.

Distribution of steroid sulfatase was studied immunohistochemically as well as enzyme-histochemically in rat cornea and retina. The immunostaining with a monoclonal antibody specific to the enzyme revealed that the enzyme protein was abundantly distributed in inner segments of photoreceptor cells and in retinal pigment epithelial cells. Other parts of the retina contained less amounts of the enzyme. The cornea showed no specific immunostaining. Electron microscopic immunohistochemical studies revealed that the enzyme molecules were localized in endoplasmic reticula and nuclear envelopes of the retinal cells. Distribution patterns resulted from the enzyme-histochemical studies were comparable to the immunohistochemical ones. The enzyme activity, however, was detected not only in these retinal cells, but also in corneal cells, including corneal epithelial, stroma, and endothelial cells. No regional difference on the enzyme activity was shown in the cornea.

A new tumor cell line (SUIT-4) derived from ascites of a patient with carcinoma of the pancreas has been established in tissue culture and in nude mice, and maintained for over 7 years. In tissue culture, the cells grew as a confluent monolayer with piling up of cells in some areas. The population doubling time during the exponential phase of the cell growth was 43.9 h in vitro. Chromosome count ranged from 63 to 68 with a modal number of 67. Subcutaneous injection of cultured cells into the flanks of nude mice resulted in tumor formation with a doubling time of 88.8 h. Histopathologically, xenografts in nude mice were moderately differentiated tubular adenocarcinoma, and the tumor cells showed spontaneous metastasis to the regional lymph nodes in 6 of 21 nude mice and to the lung in 4 of 21. Transmission electron microphotographs confirmed the ductal cell origin of the carcinoma and revealed that the cells had abundant mitochondria and lysosomes. SUIT-4 cells released carcinoembryonic antigen (3.08 × 102 ng/1 × 106 cells/24 h) and carbohydrate antigen 19-9 (4.75 × 104 U/1 × 106 cells/24 h) during exponential cell growth in vitro. Reverse transcriptase-polymerase chain reaction studies revealed that SUIT-4 cells expressed matrix metalloproteinases 1, 3, 7, 10 and 14.

BACKGROUND: Three siblings with goitre and latent to mild hypothyroidism were suspected of having thyroid peroxidase (TPO) abnormality. Direct sequencing of their genomic DNAs showed two novel mutations of the TPO gene, one of which was G1687T (Gly533Cys; exon 9) and the other 1808-13del (Asp574/Leu575del; exon 10). The two mutations were compound heterozygous, as the former was found in their father's DNA as heterozygous, and the latter was found in DNA from their mother, also as heterozygous. As Gly533 and Asp574/Leu575 were well-conserved amino acids in the peroxidase superfamily, Gly533Cys- and Asp574/Leu575del-TPOs were thought to be affected structurally or functionally. In expression studies using CHO-Kl cells and mRNAs introduced with individual mutations, both mutated TPO proteins were expressed at the same molecular size as wild-type TPO and had enzyme activity, although Gly533Cys-TPO was slightly lower in efficiency of expression and more degenerative than wild-type TPO. METHODS: We examined the localization of both mutated TPOs. Gly533Cys-TPO was located on the endoplasmic reticulum (ER) and nuclear envelope but not on the plasma membrane, whereas Asp574/Leu575del-TPO was located not only on the ER and nuclear envelope but also on the plasma membrane, as wild-type TPO. Nevertheless, only one point differed between Asp574/Leu575del- and wild-type TPOs: the mutated TPO was expressed on the plasma membrane surface at less than half the rate of wild-type TPO. RESULTS: Gly533Cys-TPO synthesized almost no thyroid hormone because of its defective localization on the apical membrane surface of thyrocytes, whereas Asp574/Leu575del-TPO performed thyroid hormone synthesis at a rate of less half that of wild-type TPO. In cotransfection experiments using three combinations of wild-type and G1687T-mRNAs, wild-type and 1808-13del-mRNAs, and G1687T-, 1808-13del-mRNAs, the three kinds of mRNAs were considered to have no influence on cell surface TPO expression of another mRNA when a 50%-maximal amount of each mRNA was transfected. When a larger amount of each mRNA was transfected, the former two combinations showed the level of cell surface TPO expression obtained from the saturating amount of wild-type mRNA, whereas the last combination of mutated mRNAs covered only about half of the expression level. CONCLUSION: Defective thyroid hormone production resulting from the abnormal TPOs was at a level that caused latent hypothyroidism when the patients were born. With their growth, thyroid hormone volume gradually became inadequate and their thyroid gland enlarged compensatorily.

OBJECTIVE: Thyroid peroxidase (TPO) deficiency is one of the causes of thyroid dyshormonogenesis, because TPO plays a key role in thyroid hormone biosynthesis. To determine the frequency and pattern of TPO abnormalities, we have been screening TPO genes of patients with congenital goitrous hypothyroidism. SUBJECTS AND METHODS: TPO genes of a patient with congenital goitrous hypothyroidism and her parents were directly sequenced, and two novel missense mutations (R665W and G771R) were found. The former was derived from her father and the latter from her mother. R665 and G771 were well conserved in the peroxidase superfamily. When mRNAs containing each of the mutations were transfected into CHO-K1 cells, each cell showed faint TPO enzyme activity. However, immunofluorescence and immunoelectron microscopic analyses revealed that neither of the mutated TPOs reached the plasma membrane. CONCLUSIONS: Two novel missense mutations in the TPO gene were found. TPO proteins encoded by these mutated alleles showed abnormal cellular localization; namely, localization on the plasma membrane was disturbed. The loss of plasma membrane localization in mutated TPOs brought about the iodide organification defect, which was diagnosed as congenital hypothyroidism.

Background & Aims: Tissue recruitment of dendritic cells (DCs) is essential for antigen presentation. This study aimed to examine cellular and molecular mechanisms for DC recruitment to the liver. Methods: Purified rat DCs were injected into circulation and their traffics were analyzed in normal and Kupffer cell–depleted rats by intravital confocal microscopy and immunohistology. Affinities of DCs to sinusoidal cells were examined by a cell-binding assay. DC precursor recruitment was induced by particulate injection. Results: Both DC precursors and DCs at the antigen-transporting stage could be recruited to the liver, and their majority initially showed a selective binding to Kupffer cells. In the Kupffer cell–depleted rats, DCs could neither be recruited to the liver nor adhere to sinusoidal walls. Pretreatment with varied monosaccharides showed that sugar residues consisting of N -acetylgalactosamine were necessary for this binding. The binding was calcium-dependent, implying the C-type lectin involvement. Furthermore, DCs could endocytose N -acetylgalactosamine polymers in a receptor-specific manner. Conclusions: The DC–Kupffer cell binding through N -acetylgalactosamine–specific C-type lectin-like receptors is crucial for DC recruitment to the liver. Rat DCs at least partly possess receptors for endocytosis of galactosylated antigens. These DC receptors as well as Kupffer cell lectins are presumably responsible for this binding. GASTROENTEROLOGY 2001;121:1460-1472

High-pressure freezing/freeze substitution followed by Lowicryl K4M embedding provided an excellent morphology and antigenicity of the gastric glands, as well as the intraluminal fluid contents. Taking advantage of this, we histochemically investigated the secretory dynamics of the zymogenic contents in rat gastric gland, with special references to phospholipase A(2) (PLA(2)) and phospholipase Cgamma1 (PLCgamma1). The combination of immunogold labeling and KMnO4-uranyl acetate-lead citrate staining for zymogenic contents clearly demonstrated the rapid diffusion of PLA(2) molecules from the exocytosed zymogenic contents into the mucinous contents in gastric glandular lumens. In contrast, the exocytosed PLCgamma1 molecules remained within the zymogenic contents in the glandular lumens. These findings indicated the distinction between the exocytosed PLA(2) and PLCgamma1 in their diffusion rate. In addition, the mucinous contents surrounding the exocytosed zymogenic contents were intensely labeled with Griffonia simplicifolia II lectin which specifically recognizes the mucin of mucous neck cells. Interestingly, some of the PLA(2) immunolabeling on the mucinous contents was associated with the apical membranes of gastric epithelial cells, especially that of parietal cells. The secretory dynamics of the zymogenic contents in rat gastric glands, including their interaction with the mucinous contents are discussed.

Ghrelin, a novel GH-releasing acylated peptide, was recently isolated from rat stomach. It stimulated the release of GH from the anterior pituitary through the GH secretagogue receptor (GHS-R). Ghrelin messenger RNA and the peptide are present in rat stomach, but its cellular source has yet to be determined. Using two different antibodies against the N- and C-terminal regions of rat ghrelin, we identified ghrelin-producing cells in the gastrointestinal tracts of rats and humans by light and electron microscopic immunohistochemistry and in situ hybridization combined with immunohistochemistry. Ghrelin-immunoreactive cells, which are not enterochromaffin-like cells, D cells, or enterochromaffin cells, accounted for about 20% of the endocrine cell population in rat and human oxyntic glands. Rat ghrelin was present in round, compact, electron-dense granules compatible with those of X/A-like cells whose hormonal product and physiological functions have not previously been clarified. The localization, population, and ultrastructural features of ghrelin-producing cells (Gr cells) indicate that they are X/A-like cells. Ghrelin also was found in enteric endocrine cells of rats and humans. Using two RIAs for the N- and C-terminal regions of ghrelin, we determined its content in the rat gastrointestinal tract. Rat ghrelin was present from the stomach to the colon, with the highest content being in the gastric fundus. Messenger RNAs of ghrelin and GHS-R also were found in these organs. Ghrelin probably functions not only in the control of GH secretion, but also in the regulation of diverse processes of the digestive system. Our findings provide clues to additional, as yet undefined, physiological functions of this novel gastrointestinal hormone.

The fine structure of tuft cells in the main excretory duct of rat submandibular gland was investigated using the high pressure freezing and freeze substitution (HPF-FS) method and compared with that seen with both conventional chemical fixation (CF) method and en bloc treatment with ruthenium red. Some MEDs also were subjected to histochemistry for lectins. The apical vesicles and tubules of tuft cells observed by TEM after the HPF-FS method were different in shape from those treated by CF. With the first method, these vesicles and tubules, which may represent sections of a tubular system, appeared more slender and filled with a material of moderate density. A prominent glycocalyx covering the microvillar plasma membrane was observed in tuft cells processed both with the HPF-FS method and with ruthenium red. The surface of microvilli and the tubulo-vesicular structures of these cells exhibited the same soybean agglutinin (SBA) reactivity, suggesting a relationship between them.

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.

We report here the characterization of gp27 (hp24γ3), a glycoprotein of the p24 family of small and abundant transmembrane proteins of the secretory pathway. Immunoelectron and confocal scanning microscopy show that at steady state, gp27 localizes to thecis side of the Golgi apparatus. In addition, some gp27 was detected in COPI- and COPII-coated structures throughout the cytoplasm. This indicated cycling that was confirmed in three ways. First, 15°C temperature treatment resulted in accumulation of gp27 in pre-Golgi structures colocalizing with anterograde cargo. Second, treatment with brefeldin A caused gp27 to relocate into peripheral structures positive for both KDEL receptor and COPII. Third, microinjection of a dominant negative mutant of Sar1p trapped gp27 in the endoplasmic reticulum (ER) by blocking ER export. Together, this shows that gp27 cycles extensively in the early secretory pathway. Immunoprecipitation and coexpression studies further revealed that a significant fraction of gp27 existed in a hetero-oligomeric complex. Three members of the p24 family, GMP25 (hp24α2), p24 (hp24β1), and p23 (hp24δ1), coprecipitated in what appeared to be stochiometric amounts. This heterocomplex was specific. Immunoprecipitation of p26 (hp24γ4) failed to coprecipitate GMP25, p24, or p23. Also, very little p26 was found coprecipitating with gp27. A functional requirement for complex formation was suggested at the level of ER export. Transiently expressed gp27 failed to leave the ER unless other p24 family proteins were coexpressed. Comparison of attached oligosaccharides showed that gp27 and GMP25 recycled differentially. Only a very minor portion of GMP25 displayed complex oligosaccharides. In contrast, all of gp27 showed modifications by medial and trans enzymes at steady state. We conclude from these data that a portion of gp27 exists as hetero-oligomeric complexes with GMP25, p24, and p23 and that these complexes are in dynamic equilibrium with individual p24 proteins to allow for differential recycling and distributions.

During microtubule depolymerization, the central, juxtanuclear Golgi apparatus scatters to multiple peripheral sites. We have tested here whether such scattering is due to a fragmentation process and subsequent outward tracking of Golgi units or if peripheral Golgi elements reform through a novel recycling pathway. To mark the Golgi in HeLa cells, we stably expressed the Golgi stack enzyme N-acetylgalactosaminyltransferase-2 (GalNAc-T2) fused to the green fluorescent protein (GFP) or to an 11–amino acid epitope, VSV-G (VSV), and the trans/TGN enzyme β1,4-galactosyltransferase (GalT) fused to GFP. After nocodazole addition, time-lapse microscopy of GalNAc-T2–GFP and GalT–GFP revealed that scattered Golgi elements appeared abruptly and that no Golgi fragments tracked outward from the compact, juxtanuclear Golgi complex. Once formed, the scattered structures were relatively stable in fluorescence intensity for tens of minutes. During the entire process of dispersal, immunogold labeling for GalNAc-T2–VSV and GalT showed that these were continuously concentrated over stacked Golgi cisternae and tubulovesicular Golgi structures similar to untreated cells, suggesting that polarized Golgi stacks reform rapidly at scattered sites. In fluorescence recovery after photobleaching over a narrow (FRAP) or wide area (FRAP-W) experiments, peripheral Golgi stacks continuously exchanged resident proteins with each other through what appeared to be an ER intermediate. That Golgi enzymes cycle through the ER was confirmed by microinjecting the dominant-negative mutant of Sar1 (Sar1pdn) blocking ER export. Sar1pdn was either microinjected into untreated or nocodazole-treated cells in the presence of protein synthesis inhibitors. In both cases, this caused a gradual accumulation of GalNAc-T2–VSV in the ER. Few to no peripheral Golgi elements were seen in the nocodazole-treated cells microinjected with Sar1pdn. In conclusion, we have shown that Golgi-resident glycosylation enzymes recycle through the ER and that this novel pathway is the likely explanation for the nocodazole-induced Golgi scattering observed in interphase cells.

Characterization of gastric Na+/I- symporter (NIS) of the rat was carried out. Sequencing of the open reading frame of gastric NIS mRNA showed only three nucleotide changes when compared with FRTL-5 NIS cDNA, and two of these changes led to amino acid changes. The results of Northern blot analysis showed that abundant NIS mRNA was expressed in the stomach when compared with other organs. Western blot analysis using gastric mucosa and FRTL-5 lysates detected the difference in molecular weight between FRTL-5 and gastric mucosa lysates, suggesting abnormal posttranslational modification of gastric NIS protein. Immunohistochemically, gastric NIS protein was located in the cornification layer of the stratified squamous epithelium of the pars proventricularis and in parietal cells and on the apical border of surface epithelial cells of the pars glandularis. Gastric NIS protein was present in tubulovesicular structures and lysosomes in parietal cells by immunoelectron microscopy. Gastric NIS protein exists to trap I- from the gastric lumen, except in parietal cells. Results indicated that a very large amount of gastric NIS mRNA is expressed to be translated, whereas only a small amount of immature gastric NIS protein is detected. This may indicate that immature gastric NIS protein rapidly degrades to peptides.

Membrane-bound forms of β1-4 galactosyltransferase (GalTase) are distributed in trans Golgi cisternae and plasma membrane, while a soluble form of the enzyme also exists in extracellular fluid. In epididymis, the soluble form has been found in luminal fluid and suggested to play a role on sperm maturation. In the present paper, regional distributions of the membrane-bound and soluble forms of GalTase were studied in rat epididymis. Immunohistochemical studies showed that a very high concentration of GalTase was localized in Golgi apparatuses of epididymal duct epithelium from initial segment to intermediate caput, although much lower amounts of GalTase were in efferent ducts, distal caput, corpus, and cauda. The same distribution pattern was also shown by biochemical assays of the microsomal GalTase activity. On the other hand, assays of the soluble GalTase activity in epididymal fluids revealed a relatively uniform distribution pattern. Even in initial segment and caput epididymis, only low levels of the activity were detected in the fluid.

We demonstrated anionic sites on the lateral wall of cochlear duct and Reissner's membrane (RM) of ICR mice by Lowicryl K4M resin post-embedding and poly-L-lysine-colloidal gold conjugate (PL-CG) as a polycationic probe. The basement membrane and endolymphatic cell surface of RM were labeled with PL-CG pH 2.5 and pH 1.0. However, the perilymphatic cell surface was not labeled. PL-CG pH 2.5 and pH 1.0 strongly labeled the endolymphatic surface of the spiral prominence epithelium (SP), whereas the endolymphatic surface of the marginal cell (MC) in the stria vascularis was not labeled. Pre-digestion with several glycosidases eliminated PL-CG labeling. Our result suggests that an anionic charge located on the basement membrane of RM is largely due to the presence of heparan sulfate, chondroitin sulfate, and hyaluronic acid. An anionic charge on the endolymphatic cell surface of RM was mainly dependent on the presence of heparan sulfate. An anionic charge on the SP epithelium was caused to a substantial degree by chondroitin sulfate. We obtained histochemical evidence that the glycoconjugate content of the MC surface was quite different from that of the endolymphatic cell surface of RM and SP. We also identified RM-MC and SP-MC junctions at the ends of the stria vascularis between the marginal cells and the other endolymphatic epithelial cells of the cochlear duct.

The blood-cochlear barrier, which consists of the molecular size and ionic charge barriers, is known to play an important role in production and absorption of inner ear fluids. In this study, we employed poly-L-lysine colloidal gold conjugates (PL-CG) in combination with Lowicryl K4M resin to demonstrate anionic sites in blood capillaries of the cochlear duct. Male ICR mice weighing 30-40 g with a positive Preyer's reflex were used. The basement membranes of blood capillaries of the stria vascularis and the spiral ligament were successfully labeled with PL-CG pH 2.5. The luminal surface of capillaries in the stria vascularis and the spiral ligament intensely reacted with PL-CG pH 2.5. However, PL-CG pH 1.0 stained only the basement membrane of the spiral ligament. Predigestion with several glycosidases nearly eliminated PL-CG labeling. Anionic charge located on the luminal surface of the endothelial cell was mainly caused by the presence of sialic acid. On the contrary, anionic charge of the basement membrane was caused in a substantial degree by chondroitin and heparan sulfate-rich glycosaminoglycans. We obtained histochemical evidence that blood capillaries of the stria vascularis differ from those of the spiral ligament.

The interaction between the extracellular matrix and human tumor-cell clones S2-013 and S2-020, derived from a pancreatic cancer cell line (SUIT-2), was examined in vitro, using various cell differentiation-promoting matrices in two- and three-dimensional cultures. S2-013 cells (well-differentiated tubular adenocarcinoma in xenografts in nude mice) cultured in Matrigel formed glandular structures. Ultrastructural observation revealed a morphological polarity of cells and a distinct basal lamina. On the other hand, S2-020 cells (poorly differentiated tubular adenocarcinoma in xenografts) cultured in Matrigel formed neither glandular structures nor a basal lamina, but only cell aggregates. The morphology of these two sublines cultured in Matrigel expressed the histological degree of differentiation which they presented in nude mice. In contrast, in type I collagen gel, S2-013 cells formed glandular structures without a basal lamina, and in soft agar, they were able to form neither glandular structures nor a basal lamina. S2-020 cells cultured in type I collagen gel or soft agar formed the same simple cell aggregates as in Matrigel. Matrices used in a three-dimensional culture influenced the degree of differentiation in S2-013 cells but had no effect on the morphological differentiation in S2-020 cells. To detect the factors which induce basal lamina formation, S2-013 cells were cultured on a microporous membrane coated with extracellular matrix components such as laminin, type IV collagen, and fibronectin. S2-013 cells formed a basal lamina only on the laminin. These cell lines may be useful in investigating the mechanisms regulating the formation of glandular structures and basal lamina.

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.

Mucosubstance histochemistry of human middle ear cholesteatoma revealed that sialomucins are abundant and sulfomucins present in small amounts in the glandlike structures of the cholesteatoma perimatrix. Based on the study, various glycoproteins were injected into the dermis of the external ear canal and infiltrated into the tympanic membranes of guinea pigs. Tympanic membranes were obtained 7 days later and light- and electron-microscopically studied. Injection of a sialomucin from bovine submaxillary gland resulted in marked proliferation of epidermis and degeneration of the lamina propria. Asialomucin prepared from sialomucin by hydrolysis produced mild thickening of the epidermis but the lamina propria was not degenerated. Fetuin and bovine serum albumin did not cause proliferation of the epidermis. Cholesterol granuloma formed in some of the specimens injected with sialomucin. The presence of sialomucin in cholesteatoma perimatrix and these experimental studies using tympanic membranes of guinea pigs suggests that sialomucins participate in the proliferation of epidermis and degeneration of subepidermal connective tissue in human middle ear cholesteatoma.

Summary The goal of specimen preparation for transmission electron microscopy is to obtain high-quality ultra-thin sections with which we can correlate cellular structure to physiological function. In this study, we newly developed a capsule-supporting ring that can be useful for resin embedding of glass-mounted specimens. The present device allowed us to re-embed a semi-thin section on a microscope slide into a resin block not only for efficient ultra-thin sectioning but also for a correlative light and electron microscopy. Similar to epoxy resins for morphological observations, semi-thin sections of low-viscosity hydrophilic resins, such as Lowicryl series, can be re-embedded into the resin, which can be useful for cytochemical gold labelling. A further application of the present device improved flat embedding of cultured cells on glass cover slips for electron microscopy, preserving in situ sub-cellular structures close to their native state. We practically describe the use of capsule-supporting ring and demonstrate representative micrographs as results.

Mucous neck cells (MNCs) of the fundic gland are phylogenetically thought to have first appeared in amphibians. We studied the origin and differentiation of MNCs in fundic glands of Xenopus laevis. By means of lectin histochemical methods using Griffonia simplicifolia agglutinin-II (GSA-II), MNCs were detected specifically in fundic glands of adult X. laevis. Mucous granules of MNCs were labeled by GSA-II-colloidal gold (CG) staining. Other cells such as surface mucous cells (SMCs), oxynticopeptic cells (OPCs), and endocrine cells did not react to GSA-II. Ulex europaeus agglutinin-I specifically stained OPCs, but not MNCs and SMCs. During the morphogenetic period of the stomach in metamorphosing larvae, GSA-II reactive cells randomly appeared in various portions of the underdeveloped fundic glands and then rapidly localized in the neck portion. At this time, newly appearing mucous granules of MNC type were labeled by GSA-II-CG. Two types of cells intermediate to MNCs and SMCs and intermediate to MNCs and OPCs were observed in the larval gastric region. Cells intermediate to MNCs and OPCs were also found in adults. In these cells, mucous granules of MNC type were labeled by GSA-II-CG, but mucous granules of SMC type and zymogen-like granules did not react to GSA-II. These observations suggest that GSA-II is a useful marker in studying the differentiation of MNCs and their precursors regardless of species differentiation.

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.

Regional distribution of steroid sulfatase was studied in the rat epididymal duct by enzyme-histochemical, immuno-histochemical and biochemical methods. A large amount of the enzyme was localized in the proximal part of the caput. This specific localization resulted from regional differences in the amount of the enzyme in principal cells, major constituents of the epithelium. In principal cells the amount of the enzyme, largest in the proximal part of the caput, drastically decreased caudally. In the corpus and cauda, principal cells possessed trace amounts of the enzyme, while clear cells had a large amount. Distribution of the enzyme generally agreed with that of estradiol binding sites.

Microparticles (MPs) are believed to play an important role in inflammatory diseases such as rheumatoid arthritis (RA). Leukocytapheresis (LCAP) is one of the options available for the treatment of RA. We analyzed the levels of MPs in RA, by flow cytometry, especially in relation to the effect of LCAP. Twenty female patients with RA were recruited into this study. Six of the 20 patients with RA further received LCAP. Plasma levels of platelet-derived MPs were high in patients with RA and are correlated with disease activity. LCAP significantly improved RA in all six patients. The numbers of platelet-derived MPs significantly decreased after the first session of LCAP, which was probably due to direct removal by LCAP. Mean numbers of platelet-derived MPs after four sessions of LCAP markedly decreased. The numbers of granulocyte-derived MPs, which are suggested to have an anti-inflammatory effect, were markedly increased after the first session of LCAP. These data suggest that removal of platelet-derived MPs and increase of granulocyte-derived MPs are novel mechanisms of LCAP as effective treatment in RA.

Mucin, a major component of mucus, is a highly O-glycosylated, high-molecular-mass glycoprotein extensively involved in the physiology of gastrointestinal mucosa. To detect and characterize mucins derived from site-specific mucous cells, we developed a monoclonal antibody, designated PGM34, by immunizing a mouse with purified pig gastric mucin. The reactivity of PGM34 with mucin was inhibited by periodate treatment of the mucin, but not by trypsin digestion. This suggests that PGM34 recognizes the carbohydrate portion of mucin. To determine the epitope, oligosaccharide-alditols obtained from pig gastric mucin were fractionated by successive gel-filtration, ion-exchange, and normal-phase HPLC, and tested for reactivity with PGM34. Two purified oligosaccharide-alditols that reacted with PGM34 were obtained. Their structures were determined by NMR spectroscopy as Fucalpha1-2Galbeta1-4GlcNAc(6SO(3)H)beta1-6(Fucalpha1-2Galbeta1-3)GalNAc-ol and Fucalpha1-2Galbeta1-4GlcNAc(6SO(3)H)beta1-6(Galbeta1-3)GalNAc-ol. None of the defucosylated or desulfated forms of these oligosaccharides reacted with PGM34. Thus, the epitope of PGM34 was determined as the Fucalpha1-2Galbeta1-4GlcNAc(6SO(3)H)beta- sequence. Immunohistochemical examination of rat gastrointestinal tract showed that PGM34 stained surface mucous cells close to the generative cell zone in the gastric fundus and goblet cells in the small intestine, but only slightly stained antral mucous cells in the stomach. These data, taken together, show that PGM34 is a very useful tool for elucidating the role of mucins with characteristic sulfated oligosaccharides.

To elucidate a functional transformation of gastric parietal cells, we have newly developed an isolated rat gastric mucosa model whose parietal cells exhibited a reverting process from the active to the resting state of acid secretion. Briefly, the parietal cells were treated with cimetidine following prior stimulation of acid secretion in the model, and cryofixed by plunge freezing for light microscopy or high-pressure freezing for electron microscopy. As a result, immunohistochemistry of H + /K + -ATPase demonstrated a progressive translocation of H + /K + -ATPase from the apical to the cytoplasmic region. The ultrastructure of parietal cells at 5 min in the reverting phase was quite similar to that of maximally stimulated one. However, the apical microvilli of intracellular canaliculi (IC) changed bulbous by degrees, resulted in complete occlusion of IC at 60 min in the reverting phase. The apical membranes were subsequently internalized into the cytoplasm forming unique penta-laminar membranes. Interestingly, at 90 min in the reverting phase, the penta-laminar membranes formed a number of multilamellar autophagosomes that were intensely labeled for H + /K + -ATPase. Then, the parietal cells exhibited well-developed Golgi apparatus and lysosomal compartments involving the multilamellar membranes at 105 min, and mostly reverted to their resting conformation at 120 min in the reverting phase. Corresponding to the ultrastructural changes of microvilli, the immunohistochemistry of ezrin showed a dissociation of ezrin from the apical region at 30 min in the reverting phase. The present findings provide new insights into the functional transformation in gastric parietal cells reverting to their resting conformation.

Cryofixation is currently accepted as the best initial fixation step to preserve not only the fine structure but also the antigenicity of biological samples. To elucidate the functional transformation of gastric parietal cells, we have newly developed an in vitro experimental model, named the isolated gastric mucosa. In this study, acid secretion of the parietal cell was stimulated with histamine or inhibited with cimetidine, and the samples were cryofixed by plunge freezing for light microscopy or high-pressure freezing for electron microscopy. As a result, the organization of glandular cells was well-preserved and quite similar to freshly excised rat gastric mucosa for at least 2 h after isolation. Immunohistochemistry of H+/K+-ATPase demonstrated a translocation of H+/K+-ATPase from the cytoplasm to the apical membrane associated with histamine-stimulation. In cimetidine-treated mucosa, most of the parietal cells were morphologically in the resting state, showing numerous tubulovesicles in their cytoplasm. In contrast, histamine-stimulated parietal cells exhibited well-developed intracellular canaliculi lined with long microvilli. To the best of our knowledge, the present study is first to demonstrate an electron micrograph that strongly suggests a membrane fusion between the tubulovescile and the apical membrane. Moreover, a stimulation-associated translocation of ezrin was clearly shown from the cytoplasm to the apical region, corresponding to apical microvilli development in the isolated gastric mucosa model. We here describe the preparation of the isolated rat gastric mucosa model, which provides new insights into the functional transformation of parietal cells by the application of cryotechniques.

ADP-ribosylation factor (ARF)-related protein 1 (ARFRP1) is a small GTPase with significant similarity to the ARF family. However, little is known about the function of ARFRP1 in mammalian cells, although knockout mice of its gene are embryonic lethal. In the present study, we demonstrate that ARFRP1 is associated mainly with the trans-Golgi compartment and the trans-Golgi network (TGN) and is an essential regulatory factor for targeting of Arl1 and GRIP domain-containing proteins, golgin-97 and golgin-245, onto Golgi membranes. Furthermore, we show that, in concert with Arl1 and GRIP proteins, ARFRP1 is implicated in the Golgi-to-plasma membrane transport of the vesicular stomatitis virus G protein as well as in the retrograde transport of TGN38 and Shiga toxin from endosomes to the TGN.

We have investigated the antioxidant effect of adrenomedullin (AM) on endothelial function in the Dahl salt-sensitive (DS) rat hypertension model. Dahl salt-resistant (DR) and DS rats were fed an 8% NaCl diet. In addition, the DS rats were subcutaneously infused with either saline or recombinant human AM for 4 weeks. Although systolic blood pressures measured weekly in AM- and saline-infused rats did not significantly differ, aortic O2*- levels were significantly (P0.01) higher in the latter. Likewise, both endothelial nitric oxide synthase (eNOS) mRNA and protein were significantly higher in saline-infused DS rats. Infusion of AM reduced both O2*- and eNOS expression to levels comparable to those seen in DR rats. AM infusion also upregulated the gene expression of guanosine-5'-triphosphate cyclohydrolase I and downregulated the expression of p22(phox), suggesting that AM increased the NOS coupling and bioavailability of NO. AM possesses significant antioxidant properties that improve endothelial function.

Intestinal intraepithelial lymphocytes (IEL) that reside at basolateral site regulate the proliferation and differentiation of epithelial cells (EC) for providing a first line of host defense in intestine. However, it remains unknown how IEL interact and communicate with EC. Here, we show that IEL express junctional molecules like EC. We identified mRNA expression of the junctional molecules in IEL such as zonula occludens (ZO)-1, occludin and junctional adhesion molecule (JAM) (tight junction), beta-catenin and E-cadherin (adherens junction), and connexin26 (gap junction). IEL constitutively expressed occludin and E-cadherin at protein level, while other T cells in the thymus, spleen, liver, mesenteric lymph node, and Peyer's patches did not. Gammadelta IEL showed higher level of these expressions than alphabeta IEL. The expression of occludin was augmented by anti-CD3 Ab stimulation. These results suggest the possibility of a novel role of IEL concerning epithelial barrier and communication between IEL and EC.

Abundant inflammatory cells infiltrate in the advancing front of the cholesteatoma perimatrix towards the middle ear mucosa. However, the cause of inflammation is not yet clear. The middle ear mucosa is often embedded in the cholesteatoma perimatrix. We hypothesized that the embedded mucosa is the cause of inflammation. Surgical specimens obtained from 20 cases of acquired cholesteatoma were used for the study. Lymphatic vessels were stained by the immunohistochemical method using the antibody against podoplanin. Mucin was simultaneously stained by alcian blue. The results of our examination were the following: (1) the presence of infiltrating mucin in the perimatrix; (2) the degeneration and reduction of lymphatic vessels; (3) the accumulation of inflammatory cells around morbid lymphatic vessels. Based on our findings, cholesteatoma can be defined as an inflammation affected by infiltrating mucin that escaped from embedded mucosa in the perimatrix.

Thyroid peroxidase (TPO) abnormality is one of the causes of congenital hypothyroidism. Two missense mutations were found as a compound heterozygous mutation in two siblings with congenital goitrous hypothyroidism. One of these mutations, G614A (R175Q), was a novel mutation. Characterization of the novel mutation and a cotransfection experiment with two mutated TPO mRNAs were carried out. G614A-mRNA introduced into CHO-K1 cells expressed TPO protein with the same molecular weight as that of wild-type mRNA. The R175Q-TPO was thought to possess enzyme activity. In terms of localization, a very small amount of mutated TPO was expressed on the plasma membrane of CHO-K1 cells. This plasma membrane expression of R175Q-TPO was insufficient to perform thyroid hormone synthesis, but was markedly different from R665W-TPO. When G614A- and C2083T-mRNAs were cotransfected, cell surface TPO-positive cells were only 13.1% in contrast to 54.4% for wild-type mRNA. The low positivity and intensity of cell surface TPO suggested that in the patients' thyroids thyroid hormone synthesis was hardly performed. The congenital hypothyroidism of the patients was thought to be a result of the mutations of the TPO gene (G614A/C2083T).

Intermediate filaments are one of the three major cytoskeletons. Some roles of intermediate filaments in cellular functions have emerged based on various diseases associated with mutations of cytokeratins. However, the precise functions of intermediate filament are still unclear. To resolve this, we manipulated intermediate filaments of cultured cells by expressing a mutant cytokeratin. Arginine 89 of cytokeratin18 plays an important role in intermediate filament assembly. The expression of green fluorescent protein-tagged cytokeratin18 arg89cys induced aggregations and loss of the intermediate filament network composed of cytokeratins in liver-derived epithelial cells, Huh7 and OUMS29, but only induced the formation of cytokeratin aggregates and did not affect the intermediate filament network of endogenous vimentin in HEK293. The expression of this mutant affected the distribution of Golgi apparatus and the reassembly of Golgi apparatus after perturbations by nocodazole or brefeldin A in both Huh7 and OUMS29, but not in HEK293. Our data show that loss of the original intermediate filament network, but not the existence of cytokeratin aggregates, induces redistribution of the Golgi apparatus. The original intact intermediate filament network is necessary for the organization of Golgi apparatus. Cell Motil. Cytoskeleton 57:37–52, 2004. © 2003 Wiley-Liss, Inc.