Intracellular homeostasis for zinc is achieved through the coordinate regulation of specific transporters engaged in zinc influx, efflux, and intracellular compartmentalization. We have identified a novel mammalian zinc transporter, zinc transporter 5 (ZnT-5), by virtue of its similarity to ZRC1, a zinc transporter of Saccharomyces cerevisiae, a member of the cation diffusion facilitator family. Human ZnT-5 (hZnT-5) cDNA encodes a 765-amino acid protein with 15 predicted membranespanning domains. hZnT-5 was ubiquitously expressed in all tested human tissues and abundantly expressed in the pancreas. In the human pancreas, hZnT-5 was expressed abundantly in insulin-containing ␤ cells that contain zinc at the highest level in the body. The hZnT-5 immunoreactivity was found to be associated with secretory granules by electron microscopy. The hZnT-5-derived zinc transport activity was detected using the Golgi-enriched vesicles prepared from hZnT-5-induced HeLa/hZnT-5 cells in which exogenous hZnT-5 expression is inducible by the Tet-on gene regulation system. This activity was dependent on time, temperature, and concentration and was saturable. Moreover, zinc at a high concentration (10 mM) inhibited the growth of yeast expressing hZnT-5. These results suggest that ZnT-5 plays an important role for transporting zinc into secretory granules in pancreatic ␤ cells.

We isolated cDNA of the mouse homologue of the src-suppressed C kinase substrate (SSeCKS) and analyzed the effects of lipopolysaccharide (LPS) injection on the tissue expression pattern of this protein. Northern blotting analysis showed that SSeCKS mRNA was expressed abundantly in the testis but at undetectable levels in other tissues of untreated control mice. Intraperitoneal administration of LPS strongly induced SSeCKS mRNA expression in the lung, heart, liver, spleen, kidney, lymph node, adrenal gland, and pituitary gland, as well as in the brain. In lung and spleen, the SSeCKS mRNA levels increased almost 10-fold at 1 hr after LPS injection and persisted at high levels until 4 hr. Both in situ hybridization and immunohistochemical studies revealed that LPS administration conspicuously elevated expression of SSeCKS mRNA and protein in vascular endothelial cells of several organs. Ectopic expression of SSeCKS caused loss of cytoplasmic F-actin fibers in the mouse endothelial cell line LEII. These results indicate that SSeCKS is one of the major LPS-responsive proteins and may participate in alteration of cytoskeletal architecture in endothelial cells during inflammation.

In a previous in situ hybridization study, we demonstrated the mRNA expression of napsin, an aspartic protease of the pepsin family, in the kidney, lung, and lymphoid organs of mice. However, findings on the cellular localization of napsin at the protein level are controversial, and no information on the subcellular localization is available. The present immunohistochemical study revealed the cellular and subcellular localization of napsin in mice and rats, and also analyzed the influences of chemical-induced proteinuria on the renal expression of this enzyme in rats. Immunohistochemistry using a polyclonal antibody against mouse napsin showed that napsin immunoreactivity was noticeable in lysosomes of renal proximal tubule cells and in lamellar bodies of pulmonary type II alveolar cells. In the lung, immunoreactivity was also found in lysosomes of alveolar macrophages and on the surface of type I alveolar cells; the immunoreactivities in these cells may be due to the uptake and adhesion of napsin secreted from type II alveolar cells, since they did not express napsin mRNA. Conversely, immunoreactivity for napsin was undetectable in B lymphocytes with intense mRNA expression. In puromycin- or doxorubicin-induced proteinuria, napsin mRNA expression was markedly elevated in renal proximal tubules, showing characteristic distribution patterns. Immunostaining of kidneys with proteinuria showed intense immunoreactivity for napsin in congested and enlarged lysosomes, called protein absorption droplets. These results indicate that napsin functions as a lysosomal protease and is involved in protein catabolism in renal proximal tubules.

ABSTRACT. A new cell line (CoMS) was established from a 3-year-old male mongrel dog with mast cell tumor of the oral mucosa. CoMS cells grow in suspension with a doubling time of 27.0 ± 0.7 hr. The cytoplasmic granules were formalin-sensitive, showed diverse appearances in their ultrastructural findings and contained heparin proteoglycan and neutral protease chymase. Calcium ionophore A23187, substance P and concanavalin A caused significant histamine release from CoMS cells, while compound 48/80 failed to release histamine. This cell line will make an available source for studies on canine mast cell tumors. KEY WORDS: canine, cell line, mast cell tumor.J. Vet. Med. Sci. 63(9): 1031-1034, 2001 Mast cell tumor (MCT) is one of the common neoplasms in dogs, accounting for 7% to 21% of all skin tumors and 11% to 27% of all malignant skin tumors [19]. There are still some dilemmas caused by its unpredictable biological behaviors with regard to diagnosis and treatment of MCT [9]. Studies on several neoplastic cell lines of human or rodent mast cells such as HMC-1, P-815 and RBL-2H3 have provided many informations about mast cell biology [17,18,20]. There will be anatomical, biochemical, immunological and pharmacological differences between human, rodent and canine mast cells. Available cell lines of canine MCT are therefore required for the research on this disease.In this paper, we describe a successful establishment of a new canine MCT cell line named CoMS in continuous culture and report morphology, histamine release activity and granular components such as proteases and heparin of CoMS cells.CoMS cell line was obtained from a mucosal mass in the lower lip of a 3-year-old male mongrel dog with MCT. The dog was referred to the Veterinary Teaching Hospital of Hokkaido University with massive swelling of a left lower lip and both cervical lymph nodes. The clinical managements were consisted of surgical excision, radiotherapy and chemotherapy. In spite of these treatments, the dog died of deterioration of general conditions 56 days after the first admission. Multiple gastroduodenal ulcers and multiple distant metastases to the internal organs such as the liver, spleen, kidneys and lungs but no metastasis to the skin were observed at necropsy.The specimen of CoMS collected at surgery was initially maintained in vivo passage in 5-week-old female C.B-17 severe combined immunodeficiency (SCID) mice (Clea Lab., Tokyo, Japan) by subcutaneous implantation, then the tumor tissues excised from the mice were used for establishment of the cell line in vitro.These tumor tissues were minced finely in RPMI 1640 medium containing 2 mg/ml sodium bicarbonate, 25 mM Hepes buffer, 100 U/ml penicillin and 100 µg/ml streptomycin, filtrated through a stainless mesh and washed two times in phosphate buffered saline. The isolated cells were then suspended and incubated in the above RPMI 1640 medium with 10% heat inactivated fetal calf serum (FCS) at 37°C in a humidified atmosphere of 5% CO 2 and were passaged every 4 to 6 day. The cell line n...

Kidney-derived aspartic protease-like protein (KAP), initially identified in the mouse kidney, is a novel aspartic protease exclusively expressed in the lung and spleen as well as the kidney. Its orthologues have been identified in the human and rat, and termed napsin. We performed in situ hybridization analysis to determine the cellular expression of napsin mRNA in the kidney, lung, and lymphatic organs of adult mice and to demonstrate, for the first time, its expression patterns in ontogeny. In the adult mouse kidney, extremely intense signals for napsin mRNA were observed in the proximal straight and convoluted tubules, in agreement with a previous study. The first signals for napsin mRNA during nephrogenesis occurred selectively in mesonephric tubules at embryonic day 13, and in metanephric tubules from embryonic day 14. In the lung, a distribution restricted to type II alveolar cells or their precursors was found from embryonic day 15, at the onset of type II cell differentiation, to the adult stage. In the spleen, the mRNA was expressed in lymph nodules of the white pulp and the marginal zone-namely, B-lymphocyte-rich regions from postnatal day 0 to adult. The lymph node and Peyer's patch displayed similar expression patterns, but T cell-dependent areas in these organs and the thymus lacked such signals. These findings suggest that mouse napsin possesses crucial functional roles not only in the kidney but also in the lung and lymphatic tissues, even during fetal stages.