Fuminori Tanabe

Mechanism of inhibitory effect of glycyrrhizin on replication of human immunodeficiency virus (HIV)

Glycyrrhizin (GL) achieved a dose-dependent inhibition of the replication of human immunodeficiency virus type 1 (HIV-1) in MOLT-4 (clone No. 8) cells within the concentration range of 0.075 to 0.6 mM. Within this concentration range, GL also effected a dose-dependent reduction in the protein kinase C (PKC) activity of MOLT-4 (clone No. 8) cells. A well-known PKC inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), also proved inhibitory to HIV-1 replication in MOLT-4 (clone No. 8) cells. PKC inhibition may thus be considered as one of the mechanisms by which GL inhibits HIV-1 replication. In addition, GL may also owe its anti-HIV-1 activity, at least in part, to an interference with virus-cell binding, since the compound at 1.2 mM partially inhibited the adsorption of radiolabeled HIV-1 particles to MT-4 cells. At this concentration GL also suppressed giant cell formation induced by co-culturing MOLT-4 (clone No. 8) cells with MOLT-4/HTLV-IIIB cells, whereas the PKC inhibitor H-7 failed to do so.

Tumor necrosis factor enhances replication of human immunodeficiency virus (HIV) in vitro

The effect of tumor necrosis factor (TNF) on the replication of human immunodeficiency virus type 1 (HIV-1) was investigated in several T4 lymphocyte cell lines. TNF markedly enhanced the cytopathogenicity of HIV-1, virion-associated reverse transcriptase (RT) activity in the cell culture supernatant, and viral antigen expression in MOLT-4 cells as early as 3 days after HIV-1 infection. A slight increase in RT activity was also observed in the supernatant of H9 cell cultures exposed to TNF. However, TNF did not increase either RT activity in MT-4 cell supernatants or viral antigen expression in HUT-78 cells. Thus, TNF is able to stimulate the replication of HIV-1 in de novo infected T4 cells although not all T4 cells seem to be sensitive to this stimulatory effect.

Rapid down-regulation of protein kinase C in (chediak-higashi syndrome) beige mouse by phorbol ester

Protein kinase C(PKC) activity in macrophages and polymorphonuclear leukocytes was assayed in beige mouse, the model of Chediak-Higashi syndrome, control C57BL/6 and the heterozygous (+/bg) mice. Regarding enzyme activity in the cytosolic and membrane fractions of these cells, there was no difference between beige mouse and the control. After short-term activation by TPA, the translocated membrane-bound PKC activity in beige mouse decreased rapidly compared with that in control mouse. However, the cytosolic PKC activity decreased at just the same pace as the control. The change in [3H] PDBu binding paralleled the changes in PKC activity. An increase in Ca2+/phospholipid-independent protein kinase by TPA was notable in the membrane fraction of beige mouse. The increase in the kinase activity was abolished and the PKC activity recovered to normal level by the addition of calpain inhibitor, leupeptin, to the incubation of cells along with TPA. Therefore, these findings suggest that a rapid decrease in membrane-bound PKC activity in beige mouse by TPA stimulation is associated with calpain.

Abnormal down‐regulation of PKC is responsible for giant granule formation in fibroblasts from CHS (beige) mice—a thiol proteinase inhibitor, E‐64‐d, prevents giant granule formation in beige fibroblasts

We have previously reported that the abnormally rapid down‐regulation of protein kinase C (PKC) activity is responsible for the cellular dysfunction in natural killer (NK) cells and polymorphonuclear leukocytes (PMNs) from Chediak‐Higashi syndrome (beige) mice. In this report, we examined whether the down‐regulation of PKC is associated with giant granule formation in fibroblasts from beige mice. In cultured beige fibroblasts, the membrane‐bound PKC activity declined significantly after phorbol ester stimulation. We found that E‐64‐d, which is a thiol proteinase inhibitor and protects PKC from calpain‐mediated proteolysis, reversed the declined PKC activity and prevented giant granule formation in beige fibroblasts. Moreover, E‐64‐d corrected the reduced lysosomal elastase and cathepsin G activity in beige fibroblasts. In contrast, specific PKC inhibitors, chelerythrin and calphostin C, promoted giant granule formation in normal fibroblasts. We also demonstrate that ceramide production is enhanced in beige fibroblasts and is involved in the rapid down‐regulation of PKC. These results suggest that the accelerated breakdown of PKC observed in beige fibroblasts is caused by enhanced ceramide production and is also responsible for giant granule formation. J. Leukoc. Biol. 67: 749–755; 2000.

The thiol proteinase inhibitors improve the abnormal rapid down-regulation of protein kinase C and the impaired natural killer cell activity in (Chediak-Higashi syndrome) beige mouse

Protein kinase C (PKC) is essential in intracellular signal transduction for various cell functions including natural killer (NK) cell activity. This enzyme is hydrolysed by calpain, which is Ca2+-dependent thiol proteinase. We showed here that in NK activity-deficient beige (bg/bg) mouse, the model of Chediak-Higashi syndrome, the translocated membrane-bound PKC activity declined rapidly in NK cell-enriched lymphocytes after TPA stimulation. However, the rapid decline was abolished by the pretreatment of cells with leupeptin (a thiol and serine proteinase inhibitor) or E64 (a thiol proteinase inhibitor). Furthermore, these reagents improved the impaired NK cell activity in beige mouse whereas they did not affect NK cell activity in C57BL/6 (+/+) and the heterozygous (+/bg) mice. Meanwhile, TPA stimulation induced only low levels in NK cytotoxic factors (NKCF) release from beige NK cells, but these reagents augmented the lowered NKCF release. These results suggest that the improvement of impaired NK cell activity in beige mouse by the thiol proteinase inhibitors may be due to the elimination of abnormal rapid down-regulation of PKC, resulting in the augmentation of the lowered PKC activity.

Possible involvement of microfilaments in protein kinase C translocation

We investigated the role of microfilaments in stimulus-induced translocation of protein kinase C (PKC) in polymorphonuclear leukocytes (PMNs) from C57BL/6 mice. Cytochalasin B and dihydrocytochalasin B almost completely inhibited PKC translocation induced by either TPA or Ca2+ ionophore after pretreatment of cells for 30 min. In addition, ML-9, a potent inhibitor of Ca2+/calmodulin-dependent myosin light chain kinase which regulate microfilament contraction, and a calmodulin antagonist W-7, also inhibited PKC translocation. These findings suggest the possibility that microfilaments are involved in the translocation of PKC.

Antithetical effects of hemicellulase-treated Agaricus blazei on the maturation of murine bone-marrow-derived dendritic cells

We report the effects of hemicellulase‐treated Agaricus blazei (ABH) on the maturation of bone‐marrow‐derived dendritic cells (BMDCs). ABH activated immature BMDCs, inducing up‐regulation of surface molecules, such as CD40, CD80 and major histocompatibility complex class I antigens, as well as inducing allogeneic T‐cell proliferation and T helper type 1 cell development. However, unlike lipopolysaccharide (LPS), ABH did not stimulate the BMDCs to produce proinflammatory cytokines, such as interleukin‐12 (IL‐12) p40, tumour necrosis factor‐α, or IL‐1β. In addition, ABH suppressed LPS‐induced DC responses. Pretreatment of DCs with ABH markedly reduced the levels of LPS‐induced cytokine secretion, while only slightly decreasing up‐regulation of the surface molecules involved in maturation. ABH also had a significant impact on peptidoglycan‐induced or CpG oligodeoxynucleotide‐induced IL‐12p40 production in DCs. The inhibition of LPS‐induced responses was not associated with a cytotoxic effect of ABH nor with an anti‐inflammatory effect of IL‐10. However, ABH decreased NF‐κB‐induced reporter gene expression in LPS‐stimulated J774.1 cells. Interestingly, DCs preincubated with ABH and then stimulated with LPS augmented T helper type 1 responses in culture with allogeneic T cells as compared to LPS‐stimulated but non‐ABH‐pretreated DCs. These observations suggest that ABH regulates DC‐mediated responses.

Thiol proteinase inhibitors reverse the increased protein kinase C down-regulation and concanavalin A cap formation in polymorphonuclear leukocytes from chediak-higashi syndrome (Beige) mouse

Protein kinase C (PKC) plays an essential role in intracellular signal transduction for various cell functions, including concanavalin A (Con A)‐induced cap formation. This enzyme is known to be proteolysed by calpain, which is a Ca2+ ‐dependent thiol proteinase. As reported previously, in polymorphonuclear leukocytes (PMNs) from beige mouse, the model of Chediak‐Higashi syndrome, Con A‐induced cap formation significantly increased compared with that in normal mouse. However, after pretreatment of beige PMNs with the thiol proteinase inhibitors leupeptin or E‐64, the capping decreased to normal levels. Meanwhile, Con A‐induced the translocation of PKC from the cytosolic to membrane fraction within 5 min in both mice, which is essential to the activation of this enzyme. However, after the translocation, an abnormal rapid decline in membrane‐bound PKC activity was noted in beige mouse PMNs. Both leupeptin and E‐64 also corrected the rapid decline in PKC activity observed in the beige mouse. These findings suggest that the normalization of Con A cap formation in beige mouse PMNs by the thiol proteinase inhibitors is associated with the correction of abnormality in PKC activity.

A potent inhibitor of protein kinase C inhibits natural killer activity

A potent inhibitor of protein kinase C(PKC), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), dose-dependently inhibited natural killer (NK) activity in large granular lymphocytes (LGL) pretreated at 37 degrees C for 30 min. However, neither N-(2-aminoethyl)-5-isoquinolinesulfonamide dihydrochloride (H-9), which inhibits more effectively cyclic nucleotide-dependent protein kinases than other kinases, nor N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA1004), which was used as a control for H-7, reduced NK activity. The inhibitor effect of H-7 was not due to changes in effector cell viability or target cell binding. We also found that H-7 suppresses PKC activity in both the cytosol and membrane fractions of LGL. From these findings, PKC is considered to play an essential role in the lytic mechanism of NK cell-mediated cytolysis.

Modulation of protein kinase C activity during inhibition of tumor cell growth by IFN-β and -γ

Abstract We investigated the effects of human interferon(IFN)-β and -γ on protein kinase C activity in human HEp-2 and KHm-14 tumor cells during IFN-induced inhibition of cell growth. Cytosolic protein kinase C activity in both cell lines was strikingly decreased following treatment with either IFN-β or -γ. In the particulate fraction, IFN-γ decreased protein kinase C activity within 1 hr but it reappeared after 24 hr, whereas IFN-β decreased the activity during the inhibition of cell growth. Furthermore, phorbol-12,13-dibutyrate(PDBu)-binding activity was altered in parallel with the changes in protein kinase C activity induced by the IFNs. In summary, we showed that IFN-β and γ cause long-term modulation of protein kinase C activity in these cultured tumor cells.