Tatsuo Suganuma

Role of ATP7B in biliary copper excretion in a human hepatoma cell line and normal rat hepatocytes

BACKGROUND & AIMS Wilsons disease is a genetic disorder characterized by the accumulation of copper in the body caused by a defect of biliary copper excretion. The Wilsons disease gene has been cloned; however, the precise localization of the gene product (ATP7B) and its role in biliary copper excretion have not been clarified. METHODS We constructed a chimeric protein between green fluorescent protein (GFP) and ATP7B (GFP-ATP7B) and expressed it in a human hepatoma cell line (Huh7) and isolated rat hepatocytes. The Golgi apparatus, late endosomes, lysosomes, and bile canaliculus were visualized by fluorescence microscopy. Brefeldin A and nocodazole were used to redistribute the Golgi proteins. Bafilomycin A1 was used to analyze the association between GFP-ATP7B and the late endosomes. RESULTS GFP-ATP7B colocalized with rhodamine-dextran and late endosome markers but not with the Golgi markers, lysosome markers, or a tight junction protein. Brefeldin A and nocodazole redistributed the Golgi proteins, but they did not affect the distribution of ATP7B. CONCLUSIONS Although it is widely believed that ATP7B is located at the Golgi apparatus, its main localization is in late endosomes. ATP7B seems to translocate copper from the cytosol to the late endosomal lumen, thus participating in biliary copper excretion via lysosomes.

Roles of ARFRP1 (ADP-ribosylation factor-related protein 1) in post-Golgi membrane trafficking

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.

The Wilson Disease Protein ATP7B Resides in the Late Endosomes with Rab7 and the Niemann-Pick C1 Protein

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.

Three-dimensional regular arrangement of the annular ligament of the rat stapediovestibular joint

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 Giesons stain and the Verhoeffs 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.

Exfoliation of gastric pit-parietal cells into the gastric lumen associated with a stimulation of isolated rat gastric mucosa in vitro: a morphological study by the application of cryotechniques

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.

Confirmation of mucin in lymphatic vessels of acquired cholesteatoma

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.

Histochemical demonstration of mucin in lymphatic vessels of human middle ear cholesteatoma

SummaryWe investigated the possible existence of mucin in lymphatic vessels in cholesteatoma perimatrix using the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method. Histochemical staining distinguished two types of lymphatic vessels, one of which contained PA-TCH-SP reacting substance showing a loose mesh-like appearance. Connective tissue was edematous around this vessel and was infiltrated by abundant round cells. The second type of lymphatic vessel did not contain PA-TCH-SP reacting substance and few round cells were seen infiltrating tissue around this vessel. Gland-like structures of mucous epithelium in the perimatrix were heavily stained by the PA-TCH-SP method. Secretory granules of the mucous epithelium and its luminal content had a loose mesh-like appearance. Since contents of the gland-like structures may leak through the chinks of epitehlial cells into subepidermal connective tissues, the resultant inflow of mucin into the lymphatic vessels may then cause inflammation of the cholesteatoma perimatrix.

A Novel Missense Mutation in the Thyroid Peroxidase Gene, R175Q, Resulting in Insufficient Cell Surface Enzyme in Two Siblings

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).