Role of membrane microdomains in PTH-mediated down-regulation of NaPi-IIa in opossum kidney cells

BACKGROUND: Parathyroid hormone (PTH) rapidly down-regulates type IIa sodium-dependent phosphate transporter (NaPi-IIa) via an endocytic pathway. Since the relationship between PTH signaling and NaPi-IIa endocytosis has not been explored, we investigated the role of membrane microdomains in this process. METHODS: We examined the submembrane localization of NaPi-IIa in opossum kidney (OK-N2) cells that stably expressed human NaPi-IIa, and searched for a PTH-induced specific phosphorylating substrate on their membrane microdomains by immunoblotting with specific antibody against phospho substrates of protein kinases. RESULTS: We found that NaPi-IIa was primarily localized in low-density membrane (LDM) domains of the plasma membrane; PTH reduced the levels of immunoreactive NaPi-IIa in these domains. Furthermore, PTH activated both protein kinase A (PKA) and protein kinase Calpha (PKCa) and increased the phosphorylation of 250 kD and 80 kD substrates; this latter substrate was identified as ezrin, which a member of the ezrin-radixin-moesin (ERM) protein family. In response to PTH, ezrin was phosphorylated by both PKA and PKC. Dominant negative ezrin blocked the reduction in NaPi-IIa expression in the LDM domains that was induced by PTH. CONCLUSION: These data suggest that NaPi-IIa and PTH-induced phosphorylated proteins that include ezrin are compartmentalized in LDM microdomains. This compartmentalization may play an important role in the down-regulation of NaPi-IIa via endocytosis.     

A Candidate for Cancer Gene Therapy: MIP-lα Gene Transfer to an Adenocarcinoma Cell Line Reduced T\imorigenicity and Induced Protective Immunity in Immunocompetent Mice

Purpose. To evaluate the possibility of cancer gene therapy by the gene delivery of chemokine, the effects of human macrophage inflammatory protein l (hu-MIP-l), murine-macrophage inflammatory protein l (mu-MIP-l), and human-interleukin 8 (hu-IL-8) on tumor progression and immunization were studied. Methods. Cachexia-inducing and highly tumorigenic adenocarcinoma cells (cell line colon 26, clone 20) were transfected with either a control plasmid, hu-MIP-l, mu-MIP-l, or hu-IL-8 expression vector. The production of hu-MIP-1 reached >1.5 ng/ml in vitro when transfectant cells were cultured at a cell density of 2 × 10⁵ cells in 7 ml for 3 days. Immunocompetent BALB/c mice were inoculated into the footpad with the tumor cells, and then primary tumor growth, morphological analyses, and tumor immunogenicity were studied. Results. The secretion of hu-MIP-l, mu-MIP-l, and hu-IL-8 did not affect the growth rate in vitro. Reduced tumorigenicities in vivo were observed in transfected cells with hu-MIP-l and mu-MIP-l. Morphologic observation of the site of inoculation of cells transfected with hu-MIP-l showed infiltration of macrophages and neutrophils on the 5th day after the inoculation. Mice that had rejected cells transfected with hu-MIP-l gene were immune to a subsequent challenge with the parental cells. Conclusions. The rejection of the cells depends on cytolysis and generates potent and long lasting antitumor immunity. These data suggest that tumor cells transfected with the MIP-l gene might be useful as an effective therapy for the treatment of certain tumors.     

Colonic lipoma with florid vascular proliferation and nodule-aggregating appearance related to repeated intussusception

A unique case of repeatedly intussuscepted colonic lipoma mimicking an epithelial tumor in a 50-year-old man is reported. The tumor was located in the ascending colon and was approximately 5 cm in diameter. Colonoscopic and barium-enema examinations suggested a huge epithelial tumor because of its nodule-aggregating appearance. In contrast, computed tomography examination showed a fatty element in the core of the lesion. The biopsy specimens suggested a primary angiomatous lesion because of its pronounced vascular proliferation. Because the presumed diagnoses based on the examinations were different, the preoperative diagnosis was not confirmed. The tumor was composed of intramural lipoma with a multiple polypoid mucosa overlay. This lesion was unique in that the lipoma appeared to be within the muscularis propria and the multiple polypoid appearance of its covering mucosa. The mucosal changes including florid vascular proliferation, fibromuscular obliteration and epithelial regeneration suggested a reparative process, with ischemic damage due to the effects of intussusception being the most likely event. It should be kept in mind that even a simple lipoma can have a unique appearance reminiscent of epithelial tumor when it repeatedly experiences intussusception.     

A duodenally absorbable CXC chemokine receptor 4 antagonist,KRH-1636, exhibits a potent and selective anti-HIV-1 activity

A low molecular weight nonpeptide compound, KRH-1636, efficiently blocked replication of various T cell line-tropic (X4) HIV type 1 (HIV-1) in MT-4 cells and peripheral blood mononuclear cells through the inhibition of viral entry and membrane fusion via the CXC chemokine receptor (CXCR)4 coreceptor but not via CC chemokine receptor 5. It also inhibited binding of the CXC chemokine, stromal cell-derived factor 1alpha, to CXCR4 specifically and subsequent signal transduction. KRH-1636 prevented monoclonal antibodies from binding to CXCR4 without down-modulation of the coreceptor. The inhibitory effect against X4 viral replication by KRH-1636 was clearly reproduced in the human peripheral blood lymphocytesevere combined immunodeficiency mouse system. Furthermore, this compound was absorbed into the blood after intraduodenal administration as judged by anti-HIV-1 activity and liquid chromatography MS in the plasma. Thus, KRH-1636 seems to be a promising agent for the treatment of HIV-1 infection.     

The usefulness of three-dimensional imaging in the diagnosis and treatment of clinically ambiguous gingival swelling

We evaluated and treated a 54-year-old woman with gingival swelling. Conventional intraoral and panoramic radiography did not provide sufficient information for either determining the cause of gingival swelling or planning treatment of clinical symptoms. The 3D Accuitomo XYZ Slice View Tomograph (3DX) is a compact dental computed tomography device that allowed for accurate identification and optimal treatment of the causes of gingival swelling. At four years after treatment, 3DX radiographs showed no abnormalities in treated teeth or healing of surrounding structures. We conclude that high-resolution 3D images obtained with 3DX promise to be very effective for diagnosing oral diseases and determining effective treatment.     

Clinical results of commissure plication annuloplasty for mitral regurgitation in children

Purpose  We evaluated the clinical results of commissure plication annuloplasty for mitral regurgitation (MR) in children. Methods  Twenty-eight patients underwent a valve repair with commissure plication annuloplasty for MR from 1988 to 2005. The mean age was 2.7 ± 3.3 years. Several appropriate techniques were combined (cleft closure in 5 patients, chordal shortening in 2 patients, artificial chordal replacement in 4 patients, leaflet fixation in 2 patients, and so on). The mean follow-up period was 6.2 years. Results  There was one operative death (3.6%) and no late deaths. Two patients underwent a second repair 19 and 23 months after their initial repairs. The actuarial freedom from the reoperation rate was 90.4% ± 0.6% at 10 years. The freedom from moderate MR or more was shown to decrease over time, 87.8% ± 0.7% at 5 years and 78.0% ± 11.0% at 10 years. Furthermore, the 10-year freedom from mild MR or more was 56.5% ± 11.9%. A progression of MR was seen. Most of the residual or recurrent MR cases weighed less than 10 kg at operation. Conclusions  The combination of commissure plication annuloplasty and several appropriate techniques provided adequate results for MR in children. Since a progression of MR was observed, a careful follow-up is therefore needed in such cases.     

Human skin melanocyte migration towards stromal cell‐derived factor‐1α demonstrated by optical real‐time cell mobility assay: modulation of their chemotactic ability by α‐melanocyte‐stimulating hormone

To identify potential regulators of normal human melanocyte behaviour, we have developed an in vitro human melanocyte migration assay, using the optically accessible, real‐time cell motility assay device TAXIScan. Coating of the glass surface with an extracellular matrix that served as scaffolding molecule was essential to demonstrate efficient melanocyte migration. Among several chemokines tested, stromal cell‐derived factor (SDF)‐1α/CXCL12 was the most effective driver of human normal skin melanocytes. Incubation of melanocytes with α‐melanocyte‐stimulating hormone (MSH) before the assay specifically enhanced CXCR4 expression and consequently chemotaxis towards SDF‐1α/CXCL12. These results suggest that α‐MSH acts on melanocytes to produce melanin as well as stimulates the cells to migrate to the site where they work through CXCR4 up‐regulation, which is a new dynamic mode of action of α‐MSH on melanocyte physiology.