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A Zilberstein

Publications and source records attributed to A Zilberstein.

At least 37 records · Page 2Linked to original sources
PubMed1994

Inhibition of IL-1 release from human monocytes and suppression of streptococcal cell wall and adjuvant-induced arthritis in rats by an extract of Tripterygium wilfordii Hook.

1. It was investigated whether an extract of Tripterygium wilfordii Hook f (TW) inhibits IL-1 production by monocytes and suppresses the development of IL-1-dependent arthritis induced in rats with streptococcal cell wall and adjuvant. 2. TW preferentially inhibited IL-1 alpha and IL-1 beta production by bacterial lipopolysaccharide (LPS)-stimulated human monocytes with IC50 of approximately 1 microgram/ml. 3. Oral administration of TW dose-dependently suppressed joint swelling and structural damage in streptococcal cell wall-induced arthritis (ED50 = 20 mg/kg/day) and in adjuvant-induced arthritis (ED50 = 46 mg/kg/day for developing and 8 mg/kg/day for established arthritis).

Animals↗
PubMed1993

Expression of glnA in the cyanobacterium Synechococcus sp. strain PCC 7942 is initiated from a single nif-like promoter under various nitrogen conditions.

The glnA mRNA, encoding glutamine synthetase, is differentially accumulated in the cyanobacterium Synechococcus sp. strain PCC 7942 in media containing different nitrogen sources. With the different nitrogen compounds, transcription of glnA initiated at a single site located -146 nucleotides upstream of the translation start site of the gene. A similarity of the nif-like promoter of the glnA gene of Anabaena sp. strain PCC 7120 and a binding-site sequence for the Synechococcus sp. strain PCC 7942 transcription regulator, NtcA, were found upstream of the transcription initiation site.

Amino Acid Sequence↗
PubMed1992

Inhibition of breast cancer cell growth in vitro by a tyrosine kinase inhibitor.

Human breast cancer cell proliferation is regulated by growth factors that bind to receptors with intrinsic tyrosine kinase (TK) activity, including the epidermal growth factor (EGF) receptor. To determine whether inhibition of receptor TK activity inhibits tumor growth, we studied the effects of a tyrosine kinase inhibitor, RG-13022, on cultured human breast cancer cells. RG-13022 represents a class of compounds which have been shown to inhibit preferentially the TK activity of the EGF receptor in a cell-free system and also to inhibit EGF-stimulated growth of cultured cells. RG-13022 significantly inhibited EGF-stimulated autophosphorylation of its receptor in two breast cancer cell lines that have abundant, although not amplified, EGF receptor content (MDA-231 and T47D). RG-13022 also inhibited EGF-stimulated DNA synthesis and proliferation of T47D and MCF-7 breast cancer cells in a reversible and dose-dependent manner. Inhibition was observed at 0.1 microM, and it was maximal at 10 microM. The effect was rapid (within 3 h), persisted for 18 h, and was partially reversed by 24 h at 1 microM. At 5 microM, inhibition persisted for more than 50 h. Inhibitory effects were also observed in a panel of estrogen receptor-positive and estrogen receptor-negative breast cancer cell lines. RG-13022 inhibited not only EGF-induced growth but also growth stimulated by insulin, insulin-like growth factor I, insulin-like growth factor II, or transforming growth factor alpha. RG-13022 also totally blocked estrogen-stimulated phosphorylation of the EGF receptor, as well as estrogen-induced cell proliferation, suggesting that functioning TK pathways are required for estrogen action. The TK inhibitor RG-13022 is a potent inhibitor of hormonally regulated growth of human breast cancer. Tyrosine kinase inhibitors have the potential of providing a new strategy for the "endocrine therapy" of breast cancer.

Breast Neoplasms↗
PubMed1992

Inhibition kinetics and selectivity of the tyrosine kinase inhibitor erbstatin and a pyridone-based analogue.

The inhibition mechanisms of the epidermal growth factor (EGF) receptor tyrosine kinase and the cAMP-dependent kinase activities by erbstatin and its analogue, RG 14921, were studied by kinetic analysis. Both compounds were slow-binding inhibitors of the EGF receptor kinase. Erbstatin inhibited the EGF receptor kinase as a partial competitive inhibitor with respect to both ATP and the peptide substrate, suggesting that it binds at a site distinct from the ATP and peptide binding sites of the enzyme, and thus lowers the binding affinities of the enzyme for both substrates. In contrast, the analogue RG 14921 inhibited EGF receptor kinase activity as a non-competitive inhibitor with respect to both ATP and the peptide substrate. The distinct modes of inhibition by structurally related compounds suggest a dynamic and possibly extended structure of the catalytic center of the kinase domain of the receptor. Erbstatin and RG 14921 exerted similar effects on cAMP-dependent protein kinase activity. In this system, both compounds displayed potent inhibition and acted by a mode of competitive inhibition with respect to ATP and non-competitive with the peptide substrate.

Adenosine Triphosphate↗
PubMed1992

Modified kinetics of platelet-derived growth factor-induced Ca2+ increases in NIH-3T3 cells overexpressing phospholipase C gamma 1.

The effects of platelet-derived growth factor (PDGF) on cytosolic free Ca2+ concentration ([Ca2+]i) and inositol phosphates were studied in NIH-3T3 fibroblasts transfected with cDNA for phospholipase C gamma 1 (PLC gamma 1) to yield a 7-fold overexpression of this enzyme, compared with cells containing normal levels of PLC gamma 1. In a study published recently [Margolis, Zilberstein, Franks, Felder, Kremer, Ullrich, Rhee, Skorecki & Schlessinger (1990) Science 248, 607-610] it was reported that this overexpression of PLC gamma 1 caused a specific potentiation of the inositol phosphate response to PDGF, but this was not associated with an enhancement of the [Ca2+]i response. In the present study, measurements of the time course and isomeric profile of PDGF-induced inositol phosphate formation demonstrated that the initial rate of Ins(1,4,5)P3 formation was also enhanced in the PLC gamma 1-overexpressing cells, yielding a 10-fold greater increase at 1 min compared with the parental NIH-3T3 cells. By contrast, bradykinin-induced phosphoinositide metabolism was unchanged in PLC gamma 1-transfected cells. Measurements of [Ca2+]i in cell populations and single cells showed a significant latent period following PDGF addition prior to the [Ca2+]i increases in both cell lines, which decreased in a dose-dependent manner with increasing PDGF concentration. The duration of the latent period was decreased and the maximal rate of [Ca2+]i rise was increased in the PLC gamma 1-overexpressing cells at all doses of PDGF examined. In single-cell measurements these cells also responded to PDGF with a greater peak amplitude of [Ca2+]i. Both intracellular Ca2+ mobilization and Ca2+ influx across the plasma membrane were enhanced in the PLC gamma 1-overexpressing cells. There was no difference between the two cell lines in either the latency or the magnitude of the [Ca2+]i increases induced by bradykinin. These data provide further evidence that PLC gamma 1 is responsible for the PDGF-induced stimulation of Ins(1,4,5)P3 formation. Moreover, in contrast to earlier conclusions, the modified kinetics of the [Ca2+]i changes in PLC gamma 1-overexpressing cells suggest that Ins(1,4,5)P3 does play a predominant second messenger role in the PDGF-induced [Ca2+]i increases. The data also indicate that the latent period may be a function of the time required to reach a threshold level of Ins(1,4,5)P3, rather than an intrinsic property of the PDGF receptor.

Animals↗
PubMed1991

Antigen- and ionophore-induced signal transduction in rat basophilic leukemia cells involves protein tyrosine phosphorylation.

Treatment of rat basophilic leukemia cells (RBL-2H3) with antigen or ionophore leads to an increase in cellular protein tyrosine phosphorylation. Three major proteins of molecular mass of 72, 92, and 110 kDa are targeted by antigen and a 110-kDa species by ionophore, A23187. The antigen- and ionophore-induced tyrosine phosphorylation responses are dose-dependent and correlate with increases in serotonin release from activated cells. The presence of extracellular Ca2+ is required to sustain the antigen- and ionophore-stimulated tyrosine phosphorylation as well as mediator release. A protein tyrosine kinase inhibitor, RG 50864, differentially inhibits the antigen-stimulated tyrosine phosphorylation in the decreasing order of 72, 91, and 110-kDa proteins. The compound inhibition of the 72-kDa protein tyrosine phosphorylation correlates with that of serotonin release. In ionophore-stimulated cells, the inhibition of the 110-kDa protein tyrosine phosphorylation and serotonin release by RG 50864 occurs in parallel. These results suggest that the 72- and 110-kDa phosphoproteins may represent the respective regulators of serotonin release in antigen- and ionophore-activated cells. The 110-kDa tyrosine phosphorylated proteins from antigen- and ionophore-stimulated cells exhibit identical electrophoretic mobility and V8 protease-generated phosphopeptide maps, suggesting that these two proteins may be the same. These results provide new evidence that both the stimulatory actions of antigen and ionophore on mediator release are mediated through enhanced protein tyrosine phosphorylation in RBL-2H3 cells. Significantly, the present study suggests the presence of multiple tyrosine phosphorylation signaling pathways in RBL cells and that their selective utility may be determined by the nature of the stimulus.

Animals↗
PubMed1991

Kinetic analysis of the inhibition of the epidermal growth factor receptor tyrosine kinase by Lavendustin-A and its analogue.

Lavendustin-A was reported to be a potent tyrosine kinase inhibitor of the epidermal growth factor (EGF) receptor (Onoda, T., Iinuma, H., Sasaki, Y., Hamada, M., Isshibi, K., Naganawa, H., Takeuchi, T., Tatsuta, K., and Umezawa, K. (1989) J. Nat. Prod. 52, 1252-1257). Its inhibition kinetics was studied in detail using the baculovirus-expressed recombinant intracellular domain of the EGF receptor (EGFR-IC). Lavendustin-A (RG 14355) is a slow and tight binding inhibitor of the receptor tyrosine kinase. The pre-steady state kinetic analysis demonstrates that the inhibition corresponds to a two-step mechanism in which an initial enzyme-inhibitor complex (EI) is rapidly formed followed by a slow isomerization step to form a tight complex (EI*). The dissociation constant for the initial rapid forming complex is 370 nM, whereas the overall dissociation constant is estimated to be less than or equal to 1 nM. The difference between the two values is due to the tight binding nature of the inhibitor to the enzyme in EI*. The kinetic analysis using a preincubation protocol to pre-equilibrate the enzyme with the inhibitor in the presence of one substrate showed that Lavendustin-A is a hyperbolic mixed-type inhibitor with respect to both ATP and the peptide substrate, with a major effect on the binding affinities for both substrates. An analogue of Lavendustin-A (RG 14467) showed similar inhibition kinetics to that of Lavendustin-A. The results of the pre-steady state analysis are also consistent with the proposed two-step mechanism. The dissociation constant for the initial fast forming complex in this case is 3.4 microM, whereas the overall dissociation constant is estimated to be less than or equal to 30 nM. It is a partial (hyperbolic) competitive inhibitor with respect to ATP. Its inhibition is reduced to different extents by different peptide substrates, when the peptide is added to the enzyme simultaneously with the inhibitor. When studied with the least protective peptide, K1 (a peptide containing the major autophosphorylation site of the EGF receptor), RG 14467 acts as a hyperbolic noncompetitive inhibitor with respect to the peptide.

Adenosine Triphosphate↗
PubMed1991

The antiproliferative effects of tyrosine kinase inhibitors tyrphostins on a human squamous cell carcinoma in vitro and in nude mice.

Many human tumors of epithelial origin contain cells overexpressing the epidermal growth factor (EGF) receptor, and there is convincing evidence that cancer cell growth is correlated with the loss of the normal regulation of the EGF receptor signal transduction pathway. Some cancers are clearly dependent on activation of the EGF receptor for their proliferation. Recently, a class of compounds, tyrphostins, which inhibit the protein tyrosine kinase activity of the growth factor receptor, have been described. In this report, we have examined the antiproliferative effects of potent new tyrphostins on a well-characterized human squamous cell carcinoma in vitro and in vivo. We found that two of these compounds (RG-13022 and RG-14620) suppressed not only EGF-stimulated cancer cell proliferation in vitro but also tumor growth in nude mice. RG-13022 also increased the life span of these tumor-bearing nude mice. When administered to tumor-bearing nude mice together with monoclonal antibodies to the EGF receptor at a suboptimal dose which had no effect alone, inhibition of tumor growth was markedly enhanced. These data suggest that tyrphostins have potential as anticancer agents.

Animals↗
PubMed1991

PDGF stimulation of inositol phospholipid hydrolysis requires PLC-gamma 1 phosphorylation on tyrosine residues 783 and 1254.

PDGF binding to its receptor promotes the association with and stimulates the phosphorylation of PLC-gamma 1 at tyrosine and serine residues. Also, PDGF induces an increase in the hydrolysis of inositol phospholipids by PLC. How PDGF activates PLC was investigated by substituting phenylalanine for tyrosine at PLC-gamma 1 phosphorylation sites 771, 783, and 1254 and expressing the mutant enzymes in NIH 3T3 cells. Phenylalanine substitution at Tyr-783 completely blocked the activation of PLC by PDGF, whereas mutation at Try-1254 inhibited and mutation at Tyr-771 enhanced the response. Like the wild type, PLC-gamma 1 substituted with phenylalanine at Tyr-783 became associated with the PDGF receptor and underwent phosphorylation at serine residues in response to PDGF. These results suggest that PLC-gamma 1 is the PLC isozyme that mediates PDGF-induced inositol phospholipid hydrolysis, that phosphorylation on Tyr-783 is essential for PLC-gamma 1 activation. These results provide direct evidence that growth factor receptors activate the function of intracellular protein by tyrosine phosphorylation.

Animals↗
PubMed1991

Autophosphorylation of the intracellular domain of the epidermal growth factor receptor results in different effects on its tyrosine kinase activity with various peptide substrates. Phosphorylation of peptides representing Tyr(P) sites of phospholipase C-gamma.

The effect of autophosphorylation on the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) is not well understood. We previously demonstrated that phospholipase C-gamma physically associates with the EGF-activated EGFR, but not with a kinase-negative mutant of the EGFR, and, moreover, that only the tyrosine-phosphorylated EGFR is able to associate with phospholipase C-gamma. We have now investigated the effect of autophosphorylation on the tyrosine kinase activity of the EGFR by employing the purified kinase-active intracellular domain of the EGFR (EGFR-IC) produced by a baculovirus expression system. Synthetic peptides, including ones which contain the individual major tyrosine phosphorylation sites of phospholipase C-gamma, were used as substrates. We found that the extensively prephosphorylated EGFR-IC exhibited similar reaction kinetics to the unphosphorylated EGFR-IC when angiotensin II was used as a nonspecific substrate. In contrast there was a clear stimulation of kinase activity due to autophosphorylation of the EGFR-IC when peptides representing either the major autophosphorylation site of the EGFR or the EGFR phosphorylation sites of phospholipase C-gamma were used as substrates. However, the modes of stimulation for these peptides differed. The binding affinity (Km) for the unphosphorylated EGFR-IC for the peptide containing Tyr-771 of phospholipase C-gamma was relatively poor compared with other peptides, but improved 5-6-fold when the EGFR-IC was prephosphorylated. On the other hand, autophosphorylation improved the reaction velocity (Vm) of the phosphorylation of other peptides by 2-3-fold, with little or no increase in affinity. These results suggest that autophosphorylation of the EGFR may induce a conformational change of its kinase domain which enhances its kinase activity with exogenous substrates and may induce association with phospholipase C-gamma by increasing its affinity to a domain containing Tyr-771.

Amino Acid Sequence↗
PubMed1991

Variability in Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Small Subunits and Carboxylation Activity in Fern Gametophytes Grown under Different Light Spectra.

Two distinct ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (SSU) populations were observed in Pteris vittata gametophytes grown under different illumination conditions. Exposure of the fern gametophytes to continuous red light (R) resulted in Rubisco SSUs that were not recognized by polyclonal antibodies raised against SSUs from spinach. Unlike the R-induced SSUs, blue light (B) induced SSUs were well recognized. This difference in SSU composition also reflected in Rubisco activity. In vitro, B-induced Rubisco exhibits a significantly higher carboxylation activity as compared to the R-induced Rubisco. Approximately a two- to threefold increase in the V(max) value of the B-induced carboxylase as compared to the R-induced one was measured. It thus seems very likely that certain domains in the SSU molecule affect enzyme activity.

Journal Article↗
PubMed1991

Epidermal growth factor (EGF) stimulates association and kinase activity of Raf-1 with the EGF receptor.

Raf-1 serine- and threonine-specific protein kinase is transiently activated in cells expressing the epidermal growth factor (EGF) receptor upon treatment with EGF. The stimulated EGF receptor coimmunoprecipitates with Raf-1 kinase and mediates protein kinase C-independent phosphorylation of Raf-1 on serine residues. Hyperphosphorylated Raf-1 has lower mobility on sodium dodecyl sulfate gels and has sixfold-increased activity in immunocomplex kinase assay with histone H1 or Raf-1 sequence-derived peptide as a substrate. Raf-1 activation requires kinase-active EGF receptor; a point mutant lacking tyrosine kinase activity in inactive in Raf-1 coupling and association. It is noteworthy that tyrosine phosphorylation of c-Raf-1 induced by EGF was not detected in these cells. These observations suggest that Raf-1 kinase may act as an important downstream effector of EGF signal transduction.

Amino Acid Sequence↗
PubMed1991

Induction of neurite outgrowth by v-src mimics critical aspects of nerve growth factor-induced differentiation.

PC12 cells treated with nerve growth factor (NGF) or infected with Rous sarcoma virus differentiate into sympathetic, neuronlike cells. To compare the differentiation programs induced by NGF and v-src, we have established a PC12 cell line expressing a temperature-sensitive v-src protein. The v-src-expressing PC12 cell line was shown to elaborate neuritic processes in a temperature-inducible manner, indicating that the differentiation process was dependent on the activity of the v-src protein. Further characterization of this cell line, in comparison with NGF-treated PC12 cells, indicated that the events associated with neurite outgrowth induced by these two agents shared features but could be distinguished by others. Both NGF- and v-src-induced neurite outgrowths were reversible. In addition, NGF and v-src could prime PC12 cells for NGF-induced neurite outgrowth, and representative early and late NGF-responsive genes were also induced by v-src. However, unlike NGF-induced neurite growth, v-src-induced neurite outgrowth was not blocked at high cell density. A comparison of phosphotyrosine containing-protein profiles showed that v-src and NGF each increase tyrosine phosphorylation of multiple cellular proteins. There was overlap in substrates; however, both NGF-specific and v-src-specific tyrosine phosphorylations were observed. One protein which was found to be phosphorylated in both the NGF- and v-src-induced PC12 cells was phospholipase C-gamma 1. Taken together, these results suggest that v-src's ability to function as an inducing agent may be a consequence of its ability to mimic critical aspects of the NGF differentiation program and raise the possibility that Src-like tyrosine kinases are involved in mediating some of the events triggered by NGF.

Cell Count↗
PubMed1991

Tyrphostins inhibit PDGF-induced DNA synthesis and associated early events in smooth muscle cells.

Tyrphostins are low-molecular-weight synthetic inhibitors of protein tyrosine kinase, which block cell proliferation. Since platelet-derived growth factor (PDGF) is thought to figure prominently in disorders of vascular smooth muscle cells (VSMC), such as atherosclerosis, hypertension, and restenosis, we examined whether tyrphostins would inhibit PDGF-induced mitogenesis in VSMC. In this communication, we demonstrate that tyrphostins with the benzenemalononitrile nucleus inhibited PDGF-dependent growth of VSMC as well as PDGF-dependent DNA synthesis in these cells, with the concentrations for 50% inhibition ranging from 0.04 to 9 microM. Up to 30-fold higher tyrphostin concentrations were required to inhibit serum-stimulated DNA synthesis of VSMC. The effect of the tyrphostins is reversible, since on their removal a normal proliferative response to PDGF was resumed. Tyrphostins also inhibited PDGF-receptor autophosphorylation and PDGF-induced phosphorylation of intracellular substrates, including the phosphorylation of phospholipase C-gamma, with a potency ratio similar to their antimitogenic activity. The expression of c-fos mRNA, a mitogenic nuclear signal, was also reduced in PDGF-stimulated VSMC treated with tyrphostins at concentrations which inhibit PDGF-induced mitogenesis. It is concluded that tyrphostins are potent reversible inhibitors of PDGF-induced mitogenesis which act by inhibiting the tyrosine kinase activity of the PDGF receptor and the subsequent signaling cascade. Tyrphostins may be useful in the study and treatment of VSMC proliferation disorders.

Animals↗
PubMed1990

Effect of phospholipase C-gamma overexpression on PDGF-induced second messengers and mitogenesis.

Platelet-derived growth factor (PDGF) stimulates phospholipase C (PLC) activity and the phosphorylation of the gamma isozyme of PLC (PLC-gamma) in vitro and in living cells. The role of PLC-gamma in the phosphoinositide signaling pathway was addressed by examining the effect of overexpression of PLC-gamma on cellular responses to PDGF. Overexpression of PLC-gamma correlated with PDGF-induced tyrosine phosphorylation of PLC-gamma and with PDGF-induced breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2). However, neither bradykinin- nor lysophosphatidic acid-induced phosphoinositide metabolism was enhanced in the transfected cells, suggesting that the G protein-coupled phosphoinositide responses to these ligands are mediated by other PLC isozymes. The enhanced PDGF-induced generation of inositol trisphosphate (IP3) did not enhance intracellular calcium signaling or influence PDGF-induced DNA synthesis. Thus, enzymes other than PLC-gamma may limit PDGF-induced calcium signaling and DNA synthesis. Alternatively, PDGF-induced calcium signaling and DNA synthesis may use biochemical pathways other than phosphoinositide metabolism for signal transduction.

Animals↗
PubMed1990

The tyrosine phosphorylated carboxyterminus of the EGF receptor is a binding site for GAP and PLC-gamma.

Phospholipase C-gamma (PLC-gamma) and GTPase activating protein (GAP) are substrates of EGF, PDGF and other growth factor receptors. Since either PLC-gamma or GAP also bind to the activated receptors it was suggested that their SH2 domains are mediating this association. We attempted to delineate the specific region of the EGF receptor that is responsible for the binding, utilizing EGF receptor mutants, PLC-gamma, and a bacterially expressed TRP E fusion protein containing the SH2 domains of GAP. As previously shown, tyrosine autophosphorylation of the wild-type receptor wsa crucial in mediating the association and in agreement, a kinase negative EGF receptor could bind PLC-gamma or TRP E GAP SH2, but only when cross tyrosine phosphorylated by an active EGF receptor kinase. The importance of autophosphorylation for association was confirmed by demonstrating that a carboxy-terminal deletion of the EGFR missing four autophosphorylation sites bound these proteins poorly. To study the role of EGF receptor autophosphorylation further, a 203 amino acid EGF receptor fragment was generated with cyanogen bromide that contained all known tyrosine autophosphorylation sites. This fragment bound both TRP E GAP SH2 and PLC-gamma but only when tyrosine phosphorylated. This data localizes a major binding site for SH2 domain containing proteins to the carboxy-terminus of the EGF receptor and points to the importance of tyrosine phosphorylation in mediating this association.

Animals↗