S Hakomori

Inhibition of chemotactic motility and trans-endothelial migration of human neutrophils by sphingosine 1-phosphate

In previous studies, we reported that sphingosine 1‐phosphate (Sph‐1‐P) inhibits the chemotactic motility of some cancer cell lines such as mouse melanoma cells, as well as human smooth muscle cells, at a very low concentration, as demonstrated by a transwell migration assay method (Proc. Natl. Acad. Sci. USA 89, 9698, 1992; J. Cell Biol. 130, 193, 1995). In this study, we investigated the effect of Sph‐1‐P on the chemotactic motility and invasiveness of human neutrophils, utilizing three different assay systems: (a) a transwell migration assay where IL‐8 or fLMP was added as a chemotactic factor, (b) a phagokinetic assay with gold colloids, and (c) a trans‐endothelial migration assay with human umbilical vein endothelial cells (HUVECs) plated on collagen layers. We found that among various sphingosine derivatives, Sph‐1‐P specifically inhibited the IL‐8‐ or fLMP‐induced chemotactic migration of neutrophils at concentrations below 1 μM. Phagokinetic activity of neutrophils was also suppressed by Sph‐1‐P, but more moderately than by the PKC inhibitory sphingosine analog, trimethylsphingosine. Finally, Sph‐1‐P inhibited trans‐endothelial migration and invasiveness of neutrophils into HUVEC‐covered collagen layers, whereas no effect on their adhesion to HUVECs was observed. These observations strongly suggest that Sph‐1‐P can act as a specific and effective motility regulator of human neutrophils, raising the possibility of future applications of Sph‐1‐P, or its analogs, as anti‐inflammatory agents regulating invasive migration of neutrophils through endothelial layers at injured vascular sites.

Sphingosine-1-phosphate Inhibits Haptotactic Motility by Overproduction of Focal Adhesion Sites in B16 Melanoma Cells through EDG-Induced Activation of Rho

Abstract: We investigated the molecular mechanism by which Sph‐1‐P affects the FN‐dependent haptotactic motility of serum‐starved mouse melanoma B16/F10 cells. We found that EDG‐5‐induced Rho activation followed by enhanced tyrosine phosphorylation of FAK and paxillin, and β1‐integrin activation leading to overexpression of focal adhesion sites, as well as increment of stress fiber formation, must be the molecular basis of inhibition of haptotactic cell motility by Sph‐1‐P.

Molecular detection of disseminated cancer cells in the peripheral blood and expression of sialylated antigens in colon cancers

To improve the survival rate of patients with colon cancer, liver metastases must be eradicated in a clinically occult state. This study was designed to find a predictor for potential liver metastases or micrometastases in colon cancer.

Glycosphingolipid antigens from Leishmania (L.) amazonensis amastigotes. Binding of anti- glycosphingolipid monoclonal antibodies in vitro and in vivo

Specific glycosphingolipid antigens of Leishmania (L.) amazonensis amastigotes reactive with the monoclonal antibodies (MoAbs) ST-3, ST-4 and ST-5 were isolated, and their structure was partially elucidated by negative ion fast atom bombardment mass spectrometry. The glycan moieties of five antigens presented linear sequences of hexoses and N-acetylhexosamines ranging from four to six sugar residues, and the ceramide moieties were found to be composed by a sphingosine d18:1 and fatty acids 24:1 or 16:0. Affinities of the three monoclonal antibodies to amastigote glycosphingolipid antigens were also analyzed by ELISA. MoAb ST-3 reacted equally well with all glycosphingolipid antigens tested, whereas ST-4 and ST-5 presented higher affinities to glycosphingolipids with longer carbohydrate chains, with five or more sugar units (slow migrating bands on HPTLC). Macrophages isolated from footpad lesions of BALB/c mice infected with Leishmania (L.) amazonensis were incubated with MoAb ST-3 and, by indirect immunofluorescence, labeling was only detected on the parasite, whereas no fluorescence was observed on the surface of the infected macrophages, indicating that these glycosphingolipid antigens are not acquired from the host cell but synthesized by the amastigote. Intravenous administration of 125I-labeled ST-3 antibody to infected BALB/c mice showed that MoAb ST-3 accumulated significantly in the footpad lesions in comparison to blood and other tissues.