Masaharu Kamo | PubMed Health

PubMed2005

Further application of a two-step heparin affinity chromatography method using divalent cations as eluents: purification and identification of membrane-bound heparin binding proteins from the mitochondrial fraction of HL-60 cells.

Membrane proteins were obtained from the mitochondrial fraction of HL-60 cells by solubilization with octyl glucoside and bound to heparin-gels. Bound proteins were successively eluted with solutions containing increasing concentrations of Mg(2+) in the first and increasing concentrations of Ca(2+) in the second chromatography. After SDS-PAGE and subsequent N-terminal amino acid analysis of proteins on each band, 13 proteins were identified. Fifteen out of the 37 proteins analysed were modified at their N-termini. These results show that this two-step affinity chromatography method using divalent cations as eluents can be applied to a variety of membranes for the isolation of specific proteins.

Amino Acid Sequence↗

PubMed2004

Mechanism of Fas-mediated cell death and its enhancement by TNF-alpha in human salivary gland adenocarcinoma cell line HSG.

Fas-mediated cell death in a human salivary gland adenocarcinoma cell line (HSG) was induced by treatment of the cells with agonistic anti-Fas antibody (CH-11), and this cell death was enhanced by pretreatment with tumor necrosis factor alpha (TNF-alpha). The mode of cell death was apoptosis, because it was accompanied by caspase activation and the cleavage of poly(ADP-ribose) polymerase. The TNF-alpha treatment of the cells increased the expression of Fas, which was accompanied by the activation of nuclear factor kappaB (NFkappaB). These results suggest that the enhancement of the apoptosis caused by TNF-alpha resulted from increased sensitivity of the HSG cells to CH-11-mediated apoptosis due to induction of Fas protein by TNF-alpha via the activation of NFkappaB. In order to elucidate the apoptosis signaling pathway, we examined the effect of various caspase inhibitors on the apoptosis induced by CH-11. Fas-mediated apoptosis of HSG cells was slightly inhibited by the caspase-9 inhibitor although it was mainly inhibited by that for caspase-8. Based on this finding, we consider CH-11-induced apoptosis in HSG cells to be mainly mediated by the type I death signaling pathway that is caused by a caspase cascade initiated by the activation of caspase-8 at the death-inducing signaling complex (DISC).

Adenocarcinoma↗