Kazuhiro Ohmi

Simple and rapid purification of brevin.

Brevin or plasma gelsolin, a calcium dependent actin-binding and actin-severing protein, was purified from bovine plasma by a very rapid and simple procedure; ammonium sulfate fractionation and only one step of anion exchange column chromatography by a convenient use. It takes only 24 hrs to complete all the procedure. The purity of brevin prepared by this method was more than 95% on SDS-PAGE and total recovery was much better than previous preparation methods. This brevin preparation has about 8 isomers on 2-D PAGE and strong severing activity on F-actin under electron microscopic observation.

Globotriaosyl ceramide (CD77/Gb3) in the glycolipid-enriched membrane domain participates in B-cell receptor–mediated apoptosis by regulating Lyn kinase activity in human B cells

The role of CD77 expressed on a fraction of germinal center B cells, also known as glycosphyngolipid Gb3, and as a functional receptor for Shiga toxins (Stx) in B-cell receptor (BCR)-mediated apoptosis was investigated. Using Stx1-sensitive Burkitts lymphoma Ramos cells as an in vitro model of CD77(+) germinal center B cells, intracellular signaling events mediated by either Stx1 or anti-CD77 antibody were examined immunobiochemically and immunocytologically. We observed prompt activation of Lyn and Syk kinases leading to increased binding of these proteins to surface IgM (sIgM) in Ramos cells after Stx1 treatment. We also observed microscopic colocalization of CD77 and sIgM after stimulation with Stx1. Along with the synergism between the cross-linking of CD77 and that of sIgM in their effect on apoptosis induction, it was highly probable that CD77 cross-linking induces activation of the BCR signaling cascade. Analysis using sucrose density gradient centrifugation suggested that Stx1 binding to CD77 induced recruitment and activation of Lyn in the glycolipid-enriched membrane (GEM) fractions. Once activated, however, Lyn seemed to acquire an increased detergent solubility and moved outside of the GEM fractions. This study describes the participation of the GEM domain in BCR-signaling cascade and suggests a possible role of CD77 as a regulator of BCR-induced apoptosis in human B cells.

Effect of K252a, a protein kinase inhibitor, on the proliferation of vascular smooth muscle cells.

In the growth arrested cultures of bovine carotid smooth muscle, K252a (10 - 100 ng/ml), a protein kinase inhibitor with wide spectrum suppressed the cell proliferation induced by TPA and increase of serum. K252a was more potent in the antiproliferative activity than H7, a C-kinase-specific inhibitor, but less than staurosporine, another wide-spectrum protein kinase inhibitor. Since C-kinase plays an important role in the signal transduction leading to the cell proliferation and K252a inhibits C-kinase in vitro, the antiproliferative effect of K252a to carotid smooth muscle cells is likely to be exerted through c-kinase dependent pathway.

Prominent immunogenicity of monosialosyl galactosylgloboside, carrying a stage-specific embryonic antigen-4 (SSEA-4) epitope in the ACHN human renal tubular cell line-a simple method for producing monoclonal antibodies against detergent-insoluble microdomains/raft.

The binding of Shiga toxin (Stx) to Gb3Cer† in detergent-insoluble microdomains (DIM)/raft of the ACHN human renal tubular cell line causes the temporal activation of the Src-family kinase Yes [1]. As a strategy for examining signaling mechanisms in DIM/raft, monoclonal antibodies (MAbs) are reliable tools for characterizing the constituent molecules in these microdomains. Thus, we employed DIM/raft suspensions of ACHN cells as an immunogen to develop MAbs. Simply subcutaneous injections of ACHN DIM/raft could elevate the serum titer after several boosts. The first screening was performed using dot-blot immunostaining with culture supernatants on a polyvinylidene difluoride (PVDF) membrane, on which DIM/raft or their chloroform/methanol (C/M) (2:1, v/v) extracts were dot-blotted. The next screening was performed by flowcytometric analysis of ACHN cells treated with or without a permeabilizing reagent. Many of the clones (21/31 clones=68%) thus obtained were also found to recognize to lipid fractions of the DIM/raft. Strikingly, all of the 21 clones that reacted to the lipid fraction were found to recognize monosialosyl galactosylgloboside (MSGG) or GL7, which carries the SSEA-4 epitope. Using DIM/raft as immunogens may enable us to easily obtain MAbs for glycolipids.

Distribution of a gelsolin-like 74,000 mol. wt protein in neural and endocrine tissues

A Ca2(+)-dependent actin binding protein with a molecular weight of 74,000, was purified from bovine adrenal medulla by using deoxyribonuclease I affinity chromatography followed by ion-exchange chromatography and gel filtration. This protein broke actin filaments into fragments and promoted nucleation of actin polymerization in a Ca2(+)-dependent manner as effectively as gelsolin. Proteolytic and immunological comparison with gelsolin which is widely distributed actin-severing protein, indicated that the 74,000 mol. wt protein is a distinct protein, but its domain structure resembles that of gelsolin. Immunoblotting using antibody against this protein showed a tissue-specific distribution. The protein was detected in various endocrine, neuroendocrine and nervous tissues, but not in muscle tissues and plasma which contained relatively large amounts of cytoplasmic and plasma gelsolin. This fact might indicate that this actin-severing protein is involved in the regulation of the secretory process of endocrine and nervous tissues. In the exocytotic process regulated by Ca2+, this protein probably plays a role to free secretory organelles like vesicles from the cytoskeletal network, mainly F-actin, which prevents the movement of secretory vesicles in the resting state.

Raft.1, a monoclonal antibody raised against the raft microdomain, recognizes G-protein β1 and 2, which assemble near nucleus after Shiga toxin binding to human renal cell line

Raft microdomains are glycolipid-enriched microdomain scaffolding molecules involved in signal transduction. The binding of Shiga toxin to globotriaosyl ceramide in raft microdomains of the human renal tubular cell line ACHN causes temporal activation of Src-kinase Yes. To study the downstream signaling mechanism proceeding to the activation of Yes, we raised monoclonal antibodies (MAbs) against raft microdomains. The MAbs were screened on the basis of, first, binding to raft microdomains with dot-blot immunostaining, second, intracellular localization of the epitope by flowcytometry after permeabilization, and third, translocation of the antigen molecules after Stx treatment by immunohistochemical staining. Raft.1 MAb bound to the molecules that accumulated to the particular region near the nucleus after Stx treatment. Two-dimensional Western blotting and matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis revealed that the antigen molecule is GTP binding protein β subunits 1 and 2 (Gβ1 and 2). That Raft.1 recognized Gβ1 and 2 was further confirmed by the reactivity to recombinant Gβ1 and 2 proteins. To our knowledge, this is the first report of production of a MAb recognizing Gβ1 and 2. Because Gβ1 and 2 are highly conserved all through organisms and are deeply involved in signal transduction, Raft.1 is expected to be utilized frequently in research.

Acceleration of actin polymerization and rapid microfilament reorganization in cultured hepatocytes by cyclochlorotin, a hepatotoxic cyclic peptide.

Cyclochlorotin (= chloropeptide, CC) is a hepatotoxic mycotoxin of Penicillium islandicum Sopp. The effect of CC on actin polymerization was examined by the measurement of changes in fluorescence intensity using pyrene-labeled actin and high shear viscosity. In the presence of CC, the time course of actin polymerization was accelerated in a dose dependent manner (2.5 ng/ml-2.5 microg/ml), without affecting the final level of viscosity. CC exerted a strong stabilizing effect on actin, enabling it to maintain its filamentous form in the presence of members of actin-binding proteins, including those of the gelsolin family prepared from hepatocytes. Microscopic observation revealed that in cultured hepatocytes, 1.0 microg/ml of CC induced bleb formation and changes in the microfilament. These observations indicated that after contact of the hepatocyte with CC, the following events were probable. The toxin passed through the cell membrane by a transport system and immediately reacted with the actin-actin binding proteins underlying the lipid bilayer. Bleb formation and hepatotoxicity were thus induced.

Inhibition of Shiga Toxin Cytotoxicity in Human Renal Cortical Epithelial Cells by Nitrobenzylthioinosine

Nitrobenzylthioinosine (NBTI), a nucleoside-transport inhibitor, has been found to possess the ability to prevent the cytotoxic action of Shiga toxin (Stx) 1 in human renal cortical epithelial cells (HRCECs), thereby protecting HRCECs from cell death. Further examination revealed that NBTI does not affect either the binding or the endocytosis of Stx1 but alters the intracellular transport of Stx1. Generally, endocytosed Stx1 is thought to be transported from endosomes to the endoplasmic reticulum. In NBTI-treated cells, however, the endocytosed Stx1 is delivered to an early endosome, but no further transportation occurs. Moreover, Stx1 is rapidly excreted from NBTI-treated HRCECs, preventing the accumulation of Stx1. Investigation of the NBTI-mediated protection mechanism against Stx cytotoxicity may provide insights into the analysis of Stx-mediated cell damage and lead to improvements in therapeutic approaches for diseases caused by Stx.

Analysis of the interaction of reserpine with actin by the photoaffinity labelling method

The interaction of reserpine with one of the cytoskeletal proteins, actin, was analyzed by the photoaffinity labelling method using [3H]reserpine. Reserpine bound sufficiently to G- or oligomeric actin, but hardly to F-actin under the same experimental conditions. This result could be explained if reserpine binds to a specific region of the G-actin molecule that is responsible for actin-actin interactions. It is concordant with this idea that [3H]reserpine bound only to specific proteolytic fragments of actin. When reserpine was mixed with crude extracts of two kinds of tissues, chicken gizzard smooth muscle and bovine adrenal medulla, it bound to the 42 kDa protein of sodium dodecyl sulfate-polyacrylamide gel electrophoresis in both cases. Its molecular size and abundance suggest strongly that this 42 kDa protein is actin. The binding of reserpine to actin was inhibited by dopamine in a dose-dependent manner. These results suggest that actin could be one of the target molecules of reserpine.

The proliferative response of p53 knock-out mouse-derived vascular smooth muscle cell line, P53LMAC01, to PDGF, when compared with human aortic smooth muscle cells.

To develop an in vitro experimental model of vascular smooth muscle cell hyperplasia, a major feature in chronic cardiac rejection, we studied a novel vascular smooth muscle cell line, P53LMAC01 (AC01), which was established from aortic smooth muscles of p53 knock-out mice, to determine its response to a platelet-derived growth factor (PDGF) and to Cyclosporin A (CsA). The responses were compared with those of human aortic smooth muscle cells (AOSMC). The AC01 exhibited a distinct proliferative response to PDGF similar to that of AOSMC under serum-free conditions. 10 ng/ml of PDGF-BB increased by a factor of 4.5 and PDGF-AB doubled the thymidine uptake, but PDGF-AA caused only a slight increase. The proliferation was markedly inhibited by 10(-6) M of CsA but less affected by 10(-7) M. These results indicate that the AC01 cell line could provide a convenient experimental system for investigating chronic rejection in vitro and that the system might work as a screening model of agents for treating transplant-related arteriosclerosis.