Tyrosyl Phosphorylation Of Proteins Research Articles

Is there a Role for Sodium Orthovanadate in the Treatment of Diabetes?

Diabetes is a metabolic disorder, whose incidences are increasing day by day. Various classes of anti-diabetic drugs are clinically approved by the Food and Drug Administration (FDA) for the treatment of diabetes mellitus, but unfortunately, none of them is able to treat this condition. Thus, the exploration of novel mechanistic pathways of existing molecules may help to develop more safe and effective anti-diabetic agents. Sodium orthovanadate is a well known common laboratory agent used to preserve the protein tyrosyl phosphorylation state of the protein. The data related to sodium orthovanadate and diabetes mellitus has been collected from Pubmed. Various reports have indicated the potential of sodium orthovanadate as Protein Tyrosine Phosphatase (PTP1B) inhibitors which play an important role in the pathogenesis of diabetes. However, safety of Sodium orthovanadate is still questionable. The sodium orthovanadate could be developed as an anti-diabetic agent. However, further studies are required to confirm its safety profile in the treatment of diabetes mellitus before starting a clinical trial.

Sodium orthovanadate inhibits proliferation and triggers apoptosis in oral squamous cell carcinoma in vitro

Sodium orthovanadate (SOV) is a general inhibitor of tyrosine phosphatases, a large family of enzymes that catalyze the removal of phosphate groups from tyrosine residues. SOV is commonly used in the laboratory to preserve the protein tyrosyl phosphorylation state of proteins under study. It has shown promising antineoplastic activity in some human cancer cell lines; this effect has not been fully investigated in head and neck squamous cell carcinoma. In this study, the effect of SOV on cell growth, proliferation, viability, and apoptosis was assessed in Cal27 cells, an oral squamous cell carcinoma (OSCC) cell line. SOV exhibited dose-dependent inhibition of cell growth and decrease in cell viability and colony formation. The IC50 values for treatment lasting 72 h and 7 days were 25 and 10 µM, respectively. The cytotoxic effect of the drug was associated with poly(ADP-ribose)polymerase cleavage detected by immunoblot. Flow cytometry of Cal27 cells stained with annexinV-FITC and propidium iodide showed a dose-dependent increase in apoptosis that reached approximately 40% at 25 µM SOV. These findings demonstrate that SOV has in vitro antiproliferative and proapoptotic effect on OSCC cells.

Effects of Sulfur Amino Acids on Tyrosyl or Serine/Threonine Phosphorylation and Translocation of Cytosolic Compounds to Cell Membrane in Stimulus-treated Human Neutrophils

We investigated the effects of various sulfur amino acids on the phosphorylation of proteins and the translocation of cytosolic compounds to cell membrane in stimulus-treated human neutrophils using specific monoclonal antibodies. D,L-homocysteine and D,L-homocysteine-thiolactone enhanced fMLP-induced tyrosyl phosphorylation of proteins and the translocation of p47phox, p67phox, and rac to the cell membrane in a concentration-dependent manner. L-cystathionine, NAc-L-cysteine and carboxymethylcysteine suppressed the tyrosyl phophorylation and translocation of cytosolic compounds to the cell membrane. L-cystathionine, L-cysteine and NAc-L-cysteine suppressed PMA-induced serine/threonine phosphorylation and the translocation of cytosolic compounds to the cell membrane. L-cysteine, NAc-L-cysteine and D,L-homocysteine enhanced AA-induced serine/threonine phosphorylation and the translocation of cytosolic compounds to the cell membrane, but L-cystathionine had opposite effects. These results indicated that the effects of sulfur amino acids on tyrosyl or serine/threonine phosphorylation and the translocation of p47phox, p67phox, and rac to the cell membrane in the stimulus-treated human neutrophils were in parallel with those of the stimulus-induced superoxide generation reported in previous paper. L-cysteine, D,L-homocysteine and L-cystathionine weakly inhibited lipid peroxidation, but the other sulfur amino acids tested had no effect.

Antiperoxidation activity of triterpenoids from rhizome of Anemone raddeana

Four triterpenoid compounds hederacolchiside E (1), hederasaponin B (2), raddeanoside 20 (3) and raddeanoside 21 (4) were isolated from ethanol extracts of rhizome of Anemone raddeana Regel. The effects of these triterpenoids on superoxide generation, tyrosyl phosphorylation of proteins and translocation of cytosolic compounds, such as p47(phox), p67(phox) and Rac to the cell membrane in human neutrophils was investigated. The superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was slightly suppressed by hederasaponin B, raddeanoside 20 and raddeanoside 21 in a concentration dependent manner. The superoxide generation induced by arachidonic acid (AA) was suppressed by hederasaponin B and raddeanoside 21 significantly. fMLP- and AA-induced tyrosyl phosphorylation and translocation of the cytosolic proteins: p47(phox), p67(phox), and Rac to the cell membrane were suppressed in parallel with the suppression of stimulus-induced superoxide generation.

Tumor Suppressive Protein Gene Associated with Retinoid-Interferon-Induced Mortality (GRIM)-19 Inhibits src-Induced Oncogenic Transformation at Multiple Levels

Interferons (IFNs) inhibit the growth of infectious pathogens and tumor development. Although IFNs are potent tumor suppressors, they modestly inhibit the growth of some human solid tumors. Their weak activity against such tumors is augmented by co-treatment with differentiation-inducing agents such as retinoids. Previous studies from our laboratory identified a novel gene product, gene associated with retinoid-interferon-induced mortality (GRIM)-19, as an IFN/all-trans retinoic acid-induced growth suppressor. However, the mechanisms of its growth suppressive actions are unclear. The src-family of tyrosine kinases is important regulators of various cell growth responses. Mutational activation of src causes cellular transformation by altering transcription and cytoskeletal properties. In this study, we show that GRIM-19 suppresses src-induced cellular transformation in vitro and in vivo by down-regulating the expression of a number of signal transducer and activator of transcription-3 (STAT3)-dependent cellular genes. In addition, GRIM-19 inhibited the src-induced cell motility and metastasis by suppressing the tyrosyl phosphorylation of focal adhesion kinase, paxillin, E-cadherin, and gamma-catenin. Effects of GRIM-19 on src-induced cellular transformation are reversible in the presence of specific short hairpin RNA, indicating its direct effect on transformation. GRIM-19-mediated inhibition of the src-induced tyrosyl phosphorylation of cellular proteins, such as focal adhesion kinase and paxillin, seems to occur independently of the STAT3 protein. GRIM-19 had no significant effect on the cellular transformation induced by other oncogenes such as myc and Ha-ras. Thus, GRIM-19 not only blocks src-induced gene expression through STAT3 but also the activation of cell adhesion molecules.

UNC-51-like kinase regulation of fibroblast growth factor receptor substrate 2/3

UNC-51-like kinases (ULK) are members of an evolutionarily conserved sub-family of ubiquitously expressed serine/threonine-specific protein kinases. Here we report that fibroblast growth factor receptor substrate (FRS) 2/3 are novel ULK2 carboxy-terminal domain interacting proteins. FRS2/3 are homologs that function as adaptor proteins to mediate signaling of multiple receptor tyrosine kinases. ULK2 interacts with the phospho-tyrosine binding (PTB) domain of FRS2/3. We demonstrate that siRNA targeting ULK2 in mouse P19 cells results in elevated FGFR1 mediated FRS3 and SHP2 tyrosyl phosphorylation. In addition, RNAi-mediated decrease in ULK2 causes increased interaction between FGFR1 and FRS3. ULK2 phosphorylates FRS2/3 in vitro, suggesting that ULK2 mediated phosphorylation may be a mechanism of FRS2/3 regulation. The data presented support a model in which ULK2, by interaction with FRS2/3 and inhibition of SynGAP, functions to negatively regulate tyrosyl phosphorylation of signaling proteins downstream of FGFR1.

Effect of Agkistroden blomhoffi (mamushi) on superoxide generation in human neutrophils

Background The purpose of this study is to investigate the effect of Agkistroden blomhoffi (mamushi) aqueous extract on stimulus-induced superoxide generation in human neutrophils. Methods Superoxide generation was assayed by measuring the reduction of ferricytochrome C ( cyt c) using a dual-beam spectrophotometer. The phosphorylation of neutrophil proteins and the translocation of cytosolic compounds to the cell membrane were detected using specific monoclonal antibodies. Results Mamushi aqueous extract markedly suppressed fMLP-induced superoxide generation in a concentration-dependent manner. fMLP-induced translocation of cytosolic compounds p47 phox, p67 phox and Rac to the cell membrane was suppressed in parallel to the suppression of superoxide generation. The fractions with MW below 10 kDa dose-dependently suppressed tyrosyl phosphorylation of 45.0, 77.6 and 99.5 kDa proteins in fMLP-treated human neutrophils; the inhibition of tyrosyl phosphorylation was in parallel to that of the fMLP-induced translocation of p47 phox to the cell membrane and superoxide generation. Conclusions Mamushi aqueous extract markedly suppressed fMLP-induced superoxide generation in human neutrophils and also suppressed the translocation of cytosolic compounds to the cell membrane and tyrosyl phosphorylation of proteins. The efficacious components are the fractions with MW below 10 kDa. O-phosphoserylethanolamine, a new dipeptide isolated from mamushi, also suppressed fMLP-induced superoxide generation in human neutrophils in a dose-dependent manner.

Crosstalk between Src and major vault protein in epidermal growth factor‐dependent cell signalling

Vaults are highly conserved, ubiquitous ribonucleoprotein (RNP) particles with an unidentified function. For the three protein species (TEP1, VPARP, and MVP) and a small RNA that comprises vault, expression of the unique 100-kDa major vault protein (MVP) is sufficient to form the basic vault structure. To identify and characterize proteins that interact with the Src homology 2 (SH2) domain of Src and potentially regulate Src activity, we used a pull-down assay using GST-Src-SH2 fusion proteins. We found MVP as a Src-SH2 binding protein in human stomach tissue. Interaction of Src and MVP was also observed in 253J stomach cancer cells. A subcellular localization study using immunofluorescence microscopy shows that epidermal growth factor (EGF) stimulation triggers MVP translocation from the nucleus to the cytosol and perinuclear region where it colocalizes with Src. We found that the interaction between Src and MVP is critically dependent on Src activity and protein (MVP) tyrosyl phosphorylation, which are induced by EGF stimulation. Our results also indicate MVP to be a novel substrate of Src and phosphorylated in an EGF-dependent manner. Interestingly, purified MVP inhibited the in vitro tyrosine kinase activity of Src in a concentration-dependent manner. MVP overexpression downregulates EGF-dependent ERK activation in Src overexpressing cells. To our knowledge, this is the first report of MVP interacting with a protein tyrosine kinase involved in a distinct cell signalling pathway. It appears that MVP is a novel regulator of Src-mediated signalling cascades.

Inhibition of vessel permeability by TNP-470 and its polymer conjugate, caplostatin

Angiogenesis inhibitors, such as TNP-470 and the nontoxic HPMA copolymer-TNP-470 (caplostatin), are emerging as a class of anticancer drugs. We report that TNP-470 and caplostatin inhibit vascular hyperpermeability of tumor blood vessels as well as that induced in mouse skin by different mediators. Treatment with TNP-470 or angiostatin for 3 days was sufficient to reduce permeability of tumor blood vessels, delayed-type hypersensitivity, and pulmonary edema induced by IL-2. TNP-470 also inhibited VPF/VEGF-induced phosphorylation of VEGFR-2, calcium influx, and RhoA activation in endothelial cells. These results identify an activity of TNP-470, that of inhibiting vessel hyperpermeability. This activity likely contributes to TNP-470's antiangiogenic effect and suggests that caplostatin can be used in the treatment of cancer and inflammation.

A type II phosphatidylinositol 4-kinase associates with T cell receptor ζ chain in Con A stimulated splenic lymphocytes through tyrosyl phosphorylation-dependent mechanisms

T cells show rapid reorganization of cytoskeleton in response to antigenic stimulation. The molecular mechanisms by which TCR-CD3 regulates actin cytoskeleton are not well defined. Here we show that a type II PtdIns 4-kinase associates with cytoskeletal fraction in splenic lymphocytes in response to Con A. Protein tyrosyl phosphorylation of type II PtdIns 4-kinase appears to be the mechanism for its association with cytoskeleton. Over-lay blots suggest that the enzyme binds to TCR-CD3 ζ chain in the cytoskeletal fraction. Anti-TCR-CD3 ζ antibodies competitively inhibit PtdIns 4-kinase association with TCR-CD3 ζ chain. Immunodepletion of TCR-CD3 ζ decreases PtdIns 4-kinase activity in the cytoskeletal fraction with a concomitant increase in PtdIns 4-kinase activity in anti-TCR-CD3 ζ immunoprecipitates. We propose that the association of type II PtdIns 4-kinase with TCR-CD3 ζ chain may bring the enzyme into close proximity of actin and a possible regulation of actin polymerization through localized production of PtdIns4 P and PtdIns(4,5) P 2.

Suppression of ARG kinase activity by STI571 induces cell cycle arrest through up-regulation of CDK inhibitor p18/INK4c.

ARG is a tyrosine kinase closely related to ABL, which is oncogenic when fused to the transcriptional repressor ETV6 (ETS translocation variant 6). In this study, we investigated the growth-inhibitory effect of STI571 (signal transduction inhibitor number 571) on ETV6/ARG-expressing cells and its molecular mechanisms using HT93A, a cell line derived from a patient with AML-M3 carrying t(1;12). STI571 effectively suppressed overall tyrosyl phosphorylation of intracellular proteins including ETV6/ARG fusion protein, as well as the growth of HT93A cells with an IC(50) of 200 nM. The growth inhibition was primarily because of cell cycle arrest at G1 phase when cells were treated with 100 nM STI571 for 48 h, and apoptosis was induced after longer exposure (72 h) or by a higher dose (1000 nM). STI571 increased the amount of p18/INK4c after 2 h of culture, when the cell cycle pattern was not yet affected, but not that of other CDK inhibitors (CKI). p18/INK4c was more abundant in G1-enriched fractions than in S- and G2/M-enriched fractions of STI571-treated HT93A cells, suggesting that the upregulation of p18/INK4c expression correlates with the cell cycle arrest. Treatment of HT93A cells with antisense oligonucleotides against the Ink4c gene abrogated the growth inhibition by STI571. These results suggest that leukemogenesis by an aberrant ARG kinase involves the suppression of p18/INK4c, which is ubiquitously expressed and considered the major CKI in hematopoietic stem cells. STI571 can be an effective drug for the treatment of leukemias with deregulated ARG kinase activity.

Glycoprotein Ib-mediated platelet activation. A signalling pathway triggered by thrombin.

Platelet activation by thrombin plays a major role in the development of haemostasis and thrombosis. Thrombin activates human platelets by cleaving the N-terminal region of G-protein-coupled protease-activated receptors (PARs). On the other hand, the platelet membrane glycoprotein GPIb acts as a thrombin-binding site and promotes platelet activation by low thrombin concentrations. We present here new evidence in favour of a thrombin receptor function for GPIb. We have selected conditions in which thrombin-GPIb interactions were enhanced by thrombin immobilization. Activation was studied independently of PAR cleavage by using active-site-blocked thrombin. We show that immobilized, proteolytically inactive thrombin induces platelet adhesion and spreading, dense granule secretion and integrin alphaIIbbeta3-dependent platelet-platelet interactions. The pathway must be dependent on GPIb because it is deficient in platelets from a patient with Bernard Soulier syndrome and inhibited by a monoclonal antibody to GPIb (SZ2) or by an excess of glycocalicin. Secreted ADP plays a major role in GPIb-dependent thrombin-induced platelet activation which is, in addition, regulated by cAMP concentration. Thrombin-induced GPIb-dependent platelet activation leads to tyrosyl phosphorylation of several proteins. Inhibition of platelet-platelet interactions and protein tyrosine phosphorylations by inhibitors of phosphatidylinositol 3-kinases and protein kinase C implies that activation of the latter are important steps of the GPIb-coupled signalling pathway triggered by thrombin.

Effect of five flavonoid compounds isolated from leaves of Diospyros kaki on stimulus-induced superoxide generation and tyrosyl phosphorylation of proteins in human neutrophils

Background: The crude drug “kaki-yô” is a traditional medicine used in Japan as a hypotensive drug. Methods: The effect of five flavonoid compounds isolated from the leaves of Diospyros kaki was investigated on the stimulus-induced superoxide generation and phosphorylation of tyrosine residues of protein in human neutrophils. The five compounds examined were kaempferol 3- O-β- d-galactopyranoside (TR), kaempferol 3- O-β- d-glucopyranoside (AS), isorhamnetin 3- O-β- d-glucopyranoside (IS), quercetin 3- O-β- d-galactopyranoside (HY), quercetin 3- O-β- d-glucopyranosyl-(6→1)-α- l-rhamnopyranoside (RU). Results: When the cells were preincubated with these five compounds, the superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was significantly suppressed in a concentration-dependent manner. The arachidonic acid (AA)-induced superoxide generation was suppressed by TR, AS, HY and RU. On the other hand, the superoxide generation was weakly enhanced by IS in low concentration (5–20 μmol/l), but was suppressed in high concentration (50 μmol/l). The superoxide generation induced by phorbol 12-myristate 13-acetate (PMA) suppressed the TR, IS, HY and RU, but AS gave no effect. When the cells were incubated with fMLP in the presence of TR, IS and RU, fMLP-induced tyrosyl phosphorylation of 45-kDa proteins of the cells was dose-dependently suppressed in parallel to the suppression of fMLP-induced superoxide generation. These five flavonoids showed almost no hemolytic effect even at a concentration of 500 μmol/l. Conclusion: Flavonoid compounds suppressed stimulus-induced superoxide generation and tyrosyl phosphorylation and may have pharmaceutical application.

Effect of three triterpenoids, lupeol, betulin, and betulinic acid on the stimulus-induced superoxide generation and tyrosyl phosphorylation of proteins in human neutrophils

Background: The roots of Anemone raddeana are used in Chinese folk medicine for curing rheumatism and neuralgia. Methods: The three triterpenoids lupeol, betulin and betulinic acid were isolated from ethanol extracts of the roots of A. raddeana. The effect of these triterpenoids on superoxide generation and tyrosyl phosphorylation of proteins in human neutrophils was investigated. Results: The superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was significantly suppressed by betulin and lupeol depending on the concentration of the triterpenoids. The suppressive effect of betulinic acid was low. The phorbol 12-myristate 13-acetate (PMA)-induced superoxide generation was suppressed by betulin in a concentration-dependent manner, but not by lupeol and betulinic acid. In contrast, the superoxide generation induced by arachidonic acid (AA) was suppressed by lupeol, while betulin and betulinic acid weakly enhanced the AA-induced superoxide generation. Lupeol and betulin suppressed tyrosyl phosphorylation of a 45.0-kDa protein in fMLP-treated human neutrophils in parallel to the suppression of fMLP-induced superoxide generation, but betulinic acid did not. Lupeol, betulin and betulinic acid showed no hemolytic effect even at a concentration of 500 μmol/l. Conclusions: Lupeol and betulin suppress superoxide generation by preventing tyrosyl phosphorylation of a 45.0-kDa protein in human neutrophils, and may have pharmaceutical applications.

Interleukin-17 induces src/MAPK cascades activation in human renal epithelial cells.

Interleukin-17 (IL-17) is a T cell derived pro-inflammatory cytokine exhibiting multiple biological activities in a variety of cells. In our previous study, we found that IL-17 expressed early on borderline change of renal allograft rejection by Banff classification both in rat renal allograft model and human renal specimens. Renal epithelial cells (RECs) are the important targets in renal allograft rejection. The purpose of this study was to explore the signalling pathways by which human interleukin-17 (hIL-17) contributes to renal allograft rejection by inducing IL-6, IL-8 and MCP-1 expression in human renal epithelial cells (hRECs). Using reverse transcriptase-polymerase chain reaction (RT-PCR), immunoprecipitation and western blot analysis, we report that the early signalling events triggered by the hIL-17 involved tyrosyl phosphorylation of proteins and increased the levels of IL-6, IL-8 and MCP-1 in a dose-dependent manner. Tyrosyl phosphorylation of proteins was induced by IL-17 in 1 min and peaked in 5 min. Further, IL-17 induced the phosphorylation of src kinase and mitogen-activated protein (MAP) kinase. Using a specific src kinase inhibitor, PP2, to treat the hRECs before hIL-17 stimulation, we found that PP2 not only inhibited the phosphorylation of src kinase but also inhibited IL-6, IL-8 and MCP-1 mRNA expression, in a dose-dependent manner. These findings provide the first evidence that the mechanism of IL-17 signalling involves src/MAPK cascades activation.

G120K-PEG, a human GH antagonist, decreases GH signal transduction in the liver of mice

After receptor binding, growth hormone (GH) induces GH receptors (GHR) dimerization and JAK2 is activated after its association with a dimerized GHR, stimulating the tyrosyl phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-2 and Shc proteins. G120K-PEG, a GH antagonist is produced by a mutation that blocks GH action by preventing the GHR dimerization. This study shows that the inhibitory effect of G120K-PEG was maximal with a GH:G120K-PEG ratio of 1:100, as no increase in JAK2 tyrosyl phosphorylation was observed with this dose of GH. When the dose of GH was increased and with a GH:G120K-PEG ratio of 1:10 some tyrosyl phosphorylation of JAK2 could be observed. Additionally, GH-induced IRS-1, IRS-2 and SHC tyrosyl phosphorylation was inhibited ∼50% at equimolar concentrations of the antagonist of GH and almost abolished with a GH:G120K-PEG ratio of 1:100. The results clearly show that G120K-PEG inhibits GH signal transduction in mouse liver.

Effect of S-(1,2-dicarboxyethyl) glutathione and S-(1,2-dicarboxyethyl) cysteine on the stimulus-induced superoxide generation and tyrosyl phosphorylation of proteins in human neutrophils.

We investigated the effects of S-(1,2-dicarboxyethyl) glutathione (DCEG) and S-(1,2-dicarboxyethyl) cysteine (DCEC) on the stimulus-induced superoxide generation and tyrosyl phosphorylation of proteins in human neutrophils. When the cells were preincubated with DCEG, the superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was enhanced in concentration-dependent manner but DCEC showed no effect. The influence of DCEG and DCEC on phorbol 12-myristate 13-acetate-induced superoxide generation showed no effect. On the other hand, the superoxide generation induced by arachidonic acid was markedly suppressed by DCEG and DCEC in concentration-dependent manner. The suppression of DCEG was more effective than that of DCEC. The superoxide generation induced by fMLP in the DCEG-treated cells was suppressed by genistein. DCEG enhanced tyrosyl phosphorylation of 80.0 kDa, 60.0 kDa, and 45.0 kDa proteins in human neutrophils. The tyrosyl phosphorylation of 80.0 kDa, 60.0 kDa, and 45.0 kDa proteins was suppressed by genistein. The enhancement of tyrosyl phosphorylation of these proteins in the DCEG-treated cells was parallel to the enhancement of the fMLP-induced superoxide generation.

Effect of sarsasapogenin and its derivatives on the stimulus coupled responses of human neutrophils

Methods: The effects of three sapogenins (sarsasapogenin, tigogenin and hecogenin) on the stimulus-induced superoxide generation and protein tyrosyl phosphorylation in human neutrophils were investigated. Results: When the cells were preincubated with sapogenin, three sapogenins dose-dependently suppressed the superoxide generations induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA), respectively. In both cases, their effects were in the order: sarsasapogenin>tigogenin>hecogenin. While sarsasapogenin suppressed the superoxide generation induced by arachidonic acid (AA) as well, the superoxide generation was scarcely suppressed by tigogenin and significantly enhanced by hecogenin. In parallel to their effects on the superoxide generation, the three sapogenins dose-dependently suppressed the fMLP-induced and PMA-induced tyrosyl phosphorylations of 45 kDa protein in neutrophils, respectively. Conclusions: Of the sapogenins tested, sarsasapogenin may have the most clinical use as it suppresses superoxide generation.

Effect of six flavonoid compounds from Ixeris sonchifolia on stimulus-induced superoxide generation and tyrosyl phosphorylation in human neutrophils

Background: Ixeris sonchifolia (Bge.) Hance is an herbal medication used in China as an analgesic. Methods: The effect of six flavonoid compounds isolated from Ixeris sonchifolia (Bge.) Hance on stimulus-induced superoxide generation and phosphorylation of tyrosine residues of protein in human neutrophils was investigated. The six compounds examined were luteolin 7-glucuronide methylester (LGME), luteolin 7-glucuronide ethylester (LGEE), luteolin 7-glucoside (LG), luteolin 7-glucopyranosyl-(1→6)-glucoside (LGG6), luteolin 7-glucopyranosyl-(1→2)-glucoside (LGG2) and apigenin 7-glucoside (AG). Results: When the cells were preincubated with these six flavonoids, the superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was significantly suppressed in a concentration-dependent manner. These flavonoids also suppressed the superoxide generation induced by arachidonic acid (AA). The rate of suppression by these flavonoids was AG>LG, LGG6, LGEE, LGG2>LGME. In case of the superoxide generation induced by phorbol 12-myristate 13-acetate (PMA), LG, LGG6 and AG suppressed the superoxide generation but LGME, LGEE and LGG2 gave no effect. When the cells were incubated with fMLP in the presence of LGME, LGEE and AG, fMLP-induced tyrosyl phosphorylation of 45-kDa proteins of the cells was dose-dependently suppressed in parallel to the suppression of fMLP-induced superoxide generation. Conclusion: Flavonoids suppress tyrosine phosphorylase in a dose-dependent manner, and may have pharmacoceutical applications.

Effect of Three Flavonoids, 5,7,3′,4′-Tetrahydroxy-3-methoxy Flavone, Luteolin, and Quercetin, on the Stimulus-Induced Superoxide Generation and Tyrosyl Phosphorylation of Proteins in Human Neutrophil

The effect of three flavonoids, 5,7,3′,4′-tetrahydoxy-3-methoxy flavone (THMF), luteolin, and quercetin, on the stimulus-induced superoxide generation and tyrosyl phosphorylation of proteins in human neutrophils were investigated. When the cells were preincubated with these flavonoids, the superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was significantly suppressed, showing a dependence on amounts of the flavonoid. The suppressing effect of the flavonoid was THMF > luteolin > quercetin. These flavonoids also suppressed the superoxide generation induced by phorbol 12-myristate 13-acetate. In this case also, THMF was more effective than luteolin and quercetin. On the other hand, the superoxide generation induced by arachidonic acid was markedly suppressed by quercetin. The suppressing effect was quercetin ⪢ THMF > luteolin. THMF, luteolin, and quercetin significantly suppressed tyrosyl phosphorylation of 80.1-, 58.0-, and 45.0-kDa proteins in fMLP-treated human neutrophils. The suppression depended on the concentration of the flavonoids, and the inhibition of tyrosyl phosphorylation was in parallel to that of the fMLP-induced superoxide generation, respectively. While luteolin and quercetin showed a weak hemolytic activity at 2.5 mM, THMF showed almost no hemolytic activity even at 5 mM, suggesting an advantage of THMF for its clinical use.