Latent dystrophic subcutaneous calcification in patients with chronic venous insufficiency.

Dystrophic calcification in the skin occurs in association with a variety of disorders. To determine the association between subcutaneous calcification and chronic venous insufficiency, X-ray examinations were performed in 20 patients with chronic venous insufficiency and in 20 control subjects to detect latent calcification in their lower legs. Of the 20 patients, 13 (65%) had subcutaneous calcification, and the prevalence appeared to increase with disease duration, while only 4 control subjects (20%) had minimal calcification. Two types of calcification were identified based on their radiographic features: punctate and trabecular/reticular types. Patients with trabecular/reticular calcification had longer disease duration and more severe clinical scores than patients with punctate calcification. None of the control subjects had trabecular/reticular types of calcification. The identification of the presence and progression of latent calcification in the lower legs is useful, and may be necessary for the long-term management of chronic venous insufficiency, since calcification of skin tissues impedes wound healing and can be a risk factor for refractory ulcers.

Involvement of slingshot in the Rho-mediated dephosphorylation of ADF/cofilin during Xenopus cleavage.

ADF/cofilin is a key regulator for actin dynamics during cytokinesis. Its activity is suppressed by phosphorylation and reactivated by dephosphorylation. Little is known, however, about regulatory mechanisms of ADF/cofilin function during formation of contractile ring actin filaments. Using Xenopus cycling extracts, we found that ADF/cofilin was dephosphorylated at prophase and telophase. In addition, constitutively active Rho GTPase induced dephosphorylation of ADF/cofilin in the egg extracts. This dephosphorylation was inhibited by Na(3)VO (4) but not by other conventional phosphatase-inhibitors. We cloned a Xenopus homologue of Slingshot phosphatase (XSSH), originally identified in Drosophila and human as an ADF/cofilin phosphatase, and raised antibody specific for the catalytic domain of XSSH. This inhibitory antibody significantly suppressed the Rho-induced dephosphorylation of ADF/cofilin in extracts, suggesting that the dephosphorylation at telophase is dependent on XSSH. XSSH bound to actin filaments with a dissociation constant of 0.4 microM, and the ADF/cofilin phosphatase activity was increased in the presence of F-actin. When latrunculin A, a G-actin-sequestering drug, was added to extracts, both Rho-induced actin polymerization and dephosphorylation of ADF/cofilin were markedly inhibited. Jasplakinolide, an actin-stabilizing drug, alone induced actin polymerization in the extracts and lead to dephosphorylation of ADF/cofilin. These results suggest that Rho-induced dephosphorylation of ADF/cofilin is dependent on the XSSH activation that is caused by increase in the amount of F-actin induced by Rho signaling. XSSH colocalized with both actin filaments and ADF/cofilin in the actin patches formed on the surface of the early cleavage furrow. Injection of inhibitory antibody blocked cleavage of blastomeres. Thus, XSSH may reorganize actin filaments through dephosphorylation and reactivation of ADF/cofilin at early stage of contractile ring formation.

Transduction of HOXD3-antisense into human melanoma cells results in decreased invasive and motile activities.

Homeobox genes regulate sets of genes that determine cellular fates in embryonic morphogenesis and maintenance of adult tissue architecture by regulating cellular motility and cell-cell interactions. Our previous studies showed that a specific member, HOXD3, when overexpressed, enhanced cell motility and invasiveness of human lung cancer A549 cells (Hamada et al. Int. J. Cancer 2001; 93: 516-25 [19]). In the present study, we investigated the roles of HOXD3 in motile and invasive behavior of human malignant melanoma cells. Of seven melanoma cell lines examined here, six cell lines expressed the HOXD3 gene, whereas normal melanocytes did not. We transduced the HOXD3-antisense gene expression vector into two cell lines (A375M and MMIV). The cell transduced with the HOXD3-antisense gene showed reduced in vitro invasion of Matrigel. The transduction of the HOXD3-antisense gene also decreased cell spreading, haptotactic activity to vitronectin and laminin-1, and phagokinetic activity. To find the difference of gene expression between the HOXD3-antisense-transduced A375M cells and the control A375MNeo2 cells, we carried out cDNA microarray analysis. The results of the microarray analysis indicated that the increased expression of cdc42-interacting protein 4, KIAA0554 and tropomyosin 1, which are all associated with the cytoskeletal system, may be involved in the reduction of motile and invasive activity by the HOXD3-antisense gene transduction.

Analysis of the two p97/VCP/Cdc48p proteins of Caenorhabditis elegans and their suppression of polyglutamine-induced protein aggregation.

A class of inherited neurodegenerative diseases including Huntington's disease is caused by polyglutamine (polyQ) expansion in the responsible proteins. Pathology is typically associated with polyQ expansions of greater than 40 residues, and the longer the length of the expansion, the earlier the onset of disease. It has been reported that p97/VCP/Cdc48p, a member of AAA family of proteins, can bind to longer polyQ tracts. In Caenorhabditis elegans, two p97/VCP/Cdc48p homologues, C41C4.8 and C06A1.1, have been identified. Our results indicate that these p97/VCP/Cdc48p homologues have essential but redundant functions in C. elegans. To provide a model system for investigating the molecular basis of pathogenesis, we have expressed polyQ expansions fused to green fluorescent protein in the body wall muscle cells of C. elegans. When the repeats are longer than 40, discrete cytoplasmic aggregates are formed and these appear at an early stage of embryogenesis. The formation of aggregates was partially suppressed by co-expression of either C41C4.8 or C06A1.1. These results suggest that these p97/VCP/Cdc48p homologues, AAA chaperones, may play a protective role in polyQ aggregation.