scholarly journals Fgr but not Syk tyrosine kinase is a target for beta2 integrin-induced c-Cbl-mediated ubiquitination in adherent human neutrophils

2003 ◽  
Vol 370 (2) ◽  
pp. 687-694 ◽  
Author(s):  
Fredrik MELANDER ◽  
Tommy ANDERSSON ◽  
Karim DIB
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Src Kinase ◽  
E3 Ligase ◽  
In Vitro Assay ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
Vitro Assay ◽  
Β2 Integrin

An early and critical event in β2 integrin signalling during neutrophil adhesion is activation of Src tyrosine kinases and Syk. In the present study, we report Src kinase-dependent β2 integrin-induced tyrosine phosphorylation of Cbl occurring in parallel with increased Cbl-associated tyrosine kinase activity. These events concurred with activation of Fgr and, surprisingly, also with dissociation of this Src tyrosine kinase from Cbl. Moreover, the presence of the Src kinase inhibitor PP1 in an in vitro assay had only a limited effect on the Cbl-associated kinase activity. These results suggest that an additional active Src-dependent tyrosine kinase associates with Cbl. The following observations imply that Syk is such a kinase: (i) β2 integrins activated Syk in a Src-dependent manner, (ii) Syk was associated with Cbl much longer than Fgr was, and (iii) the Syk inhibitor piceatannol (3,4,3′,5′-tetrahydroxy-trans-stilbene) abolished the Cbl-associated kinase activity in an in vitro assay. Effects of the mentioned interactions between these two kinases and Cbl may be related to the finding that Cbl is a ubiquitin E3 ligase. Indeed, we detected β2 integrin-induced ubiquitination of Fgr that, similar to the phosphorylation of Cbl, was abolished in cells pretreated with PP1. However, the ubiquitination of Fgr did not cause any apparent degradation of the protein. In contrast with Fgr, Syk was not modified by the E3 ligase. Thus Cbl appears to be essential in β2 integrin signalling, first by serving as a matrix for a subsequent agonist-induced signalling interaction between Fgr and Syk, and then by mediating ubiquitination of Fgr which possibly affects its interaction with Cbl.

scholarly journals Effect of Massoia (Massoia aromatica Becc.) Bark on the Phagocytic Activity of Wistar Rat Macrophages

2018 ◽  
Author(s):  
Triana Hertiani ◽  
Agustinus Yuswanto ◽  
Sylvia Utami Tunjung Pratiwi ◽  
Harlyanti Mashar
Keyword(s):  
Phagocytic Activity ◽  
Wistar Rats ◽  
In Vitro Assay ◽  
Phagocytic Index ◽  
Dependent Manner ◽  
Vitro Assay ◽  
Macrophage Cells ◽  
In Vivo Assay

Massoia (Massoia aromatica Becc., Lauraceae) bark has been widely used as a component of traditional Indonesian medicine. The indigenous people boil or steam the bark for traditional applications. Our preliminary research revealed the potency of Massoia essential oil and its major compound, C-10 Massoialactone as potential immunomodulator in vitro. However, no scientific evidence regarding its in vivo effects is available. Therefore, this study evaluated the potential immunomodulatory effects of Massoia bark infusion on the nonspecific immune response (phagocytosis) of Wistar rats. The aqueous extract of Massoia bark was obtained by boiling pulverized bark in water, and the C-10 massoialactone content of the extract was determined through Thin Layer Chromatography (TLC) densitometry. For the in vitro assay, macrophages were treated with the freeze-dried infusion at the concentrations of 2.5, 5, 10, 20, or 40 μg/mL media. For the in vivo assay, 2-month-old male Wistar rats were divided into 5 groups. The baseline group received distilled water at the dose of 1 mL/100 g BW with the immunostimulant herbal product “X” administered as the positive control at the dose of 0.54 mL/rat. The treatment groups received the infusion at a dose of 100, 300, or 500 mg/100 g BW. Treatments were given orally every day for 14 days. The ability of macrophage cells to phagocyte latex was determined as phagocytic index (PI) and was observed under microscopy with 300 macrophages. The in vitro study revealed that the phagocytic activity of the infusion-treated macrophages significantly increased in comparison with that of the control macrophages in a concentration-dependent manner. Among all treatment concentrations, the concentration of 40 μg/ml provided the highest activity with a PI value of 70.51% ± 1.11%. The results of the in vivo assay confirmed those of the in vitro assay. The results of the present study indicate that Massoia bark can increase the phagocytic activity of rat macrophage cells. Its potential as a naturally derived immunomodulatory agent requires further study.

Epitope specificity and isotype of monoclonal anti-D antibodies dictate their ability to inhibit phagocytosis of opsonized platelets

Blood ◽  
2007 ◽  
Vol 110 (4) ◽  
pp. 1359-1361 ◽  
Author(s):  
Mimi Kjaersgaard ◽  
Rukhsana Aslam ◽  
Michael Kim ◽  
Edwin R. Speck ◽  
John Freedman ◽  
...  
Keyword(s):  
Immune Globulin ◽  
In Vitro Assay ◽  
Dependent Manner ◽  
Hemolytic Disease ◽  
Thrombocytopenic Purpura ◽  
Vitro Assay ◽  
Autoimmune Thrombocytopenic Purpura ◽  
Epitope Specificity ◽  
Primary Indication

Abstract Rh immune globulin (WinRho SDF; Cangene, Mississauga, ON, Canada) is an effective treatment for autoimmune thrombocytopenic purpura; however, maintaining a sustained supply for its use in autoimmune thrombocytopenic purpura and its primary indication, hemolytic disease of the newborn, makes the development of alternative reagents desirable. We compared Rh immune globulin and 6 human monoclonal anti-D antibodies (MoAnti-D) with differing isotypes and specificities for their ability to opsonize erythrocytes and inhibit platelet phagocytosis in an in vitro assay. Results demonstrated that opsonization of erythrocytes with Rh immune globulin significantly (P < .001) reduced phagocytosis of fluorescently labeled opsonized platelets in an Fc-dependent manner. Of the MoAnti-D that shared specificity but differed in isotype, only IgG3 antibodies could significantly (P < .001) inhibit platelet phagocytosis. In contrast, 2 MoAnti-D shared isotypes and differed in specificity; however, only one could significantly (P < .001) inhibit platelet phagocytosis. The results suggest that MoAnti-D epitope specificity and isotypes are critical requirements for optimal inhibition of opsonized platelet phagocytosis.

Early mouse endoderm is patterned by soluble factors from adjacent germ layers

Development ◽  
2000 ◽  
Vol 127 (8) ◽  
pp. 1563-1572 ◽  
Author(s):  
J.M. Wells ◽  
D.A. Melton
Keyword(s):  
Primitive Streak ◽  
In Vitro Assay ◽  
Specific Gene ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Germ Layers ◽  
Vitro Assay ◽  
Organ Specific ◽  
Early Mouse

Endoderm that forms the respiratory and digestive tracts is a sheet of approximately 500–1000 cells around the distal cup of an E7.5 mouse embryo. Within 2 days, endoderm folds into a primitive gut tube from which numerous organs will bud. To characterize the signals involved in the developmental specification of this early endoderm, we have employed an in vitro assay using germ layer explants and show that adjacent germ layers provide soluble, temporally specific signals that induce organ-specific gene expression in endoderm. Furthermore, we show that FGF4 expressed in primitive streak-mesoderm can induce the differentiation of endoderm in a concentration-dependent manner. We conclude that the differentiation of gastrulation-stage endoderm is directed by adjacent mesoderm and ectoderm, one of the earliest reported patterning events in formation of the vertebrate gut tube.

Proline-Rich Tyrosine Kinase-2 (PYK2): A New Target for the Treatment of Hematopoietic Neoplasms with TEL-FGFR3 Fusion or FGFR3 Active Mutation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3360-3360
Author(s):  
Daisuke Okamura ◽  
Fumiharu Yagasaki ◽  
Tomoya Maeda ◽  
Maho Ishikawa ◽  
Itsuro Jinnai ◽  
...  
Keyword(s):  
Cell Growth ◽  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Myeloma Cell ◽  
Kinase Assay ◽  
G1 Arrest ◽  
Dependent Manner ◽  
Tyrosine Kinase 2

Abstract Constitutive activation of Fibroblast Growth Factor 3 (FGFR3) tyrosine kinase have been identified in various human cancers and have been reported to play an important role in some hematopoietic neoplasms. We have previously reported that TEL-FGFR3 in a patient with peripheral T-cell Lymphoma and AML conferred IL-3 independency to Ba/F3 cells and activates PLCγ, PK3K, STAT3, STAT5, MAPK through its constitutive tyrosine kinase activity in TEL-FGFR3 transfected Ba/F3 cells (TF-V5). In KMS-11, human multiple myeloma cell line which expresses constitutively active mutant FGFR3, activations of PI3K and STAT3 pathways have been reported. However, little is known about how FGFR3 tyrosine kinase (TK) activates these downstream molecules. Here, we show that PYK2, a member of focal adhesion kinases, plays a pivotal role for the activation of PI3K, STAT3 and STAT5 in FGFR3 oncogenic pathways, and is a candidate for therapeutic target. PP1/PP2, a kinase inhibitor of SRC and PYK2, inhibited the cell growth of TF-V5 and KMS-11 cells in a dose-dependent manner (IC50=15μM, 25μM respectively), not affecting the cell growth of IL-3 dependent Ba/F3 cells. Another specific SRC inhibitor did not affect the cell growth of TF-V5 and KMS-11 cells. TEL-FGFR3 transfection to Ba/F3 cells led to the overexpression of PYK2 but not FAK. Expression and phosphorylation of PYK2 were identified in KMS-11 cells. Immunoprecipitation analysis using FGFR3 TK inhibitor SU5402 showed that the activation of PYK2 which was recruited to FGFR3 was dependent on the kinase activity of FGFR3. The cell growth of TF-V5 was completely inhibited at the concentration of PP1/PP2(30μM), which inhibited auto-phosphorylation of PYK2. PP1/PP2 suppressed the activation of PI3K-ATK pathway and decreased expression of C-MYC, inducing G1-arrest of TF-V5. PP1/PP2 induced intrinsic apoptosis of TF-V5 and did not affect activation of BAX but decrease expression of BCL-2 and BCL-XL through inactivation of STAT3 and STAT5. PP1/PP2 also inhibited the activation of PI3K and STAT3 in KMS-11 cells, inducing G1-arrest and apoptosis. PP1/PP2 inhibited tyrosine kinase of PYK2 mesured by in vitro kinase assay (IC50=23μM, 13μM, respectively). Further PYK2 C-terminus Associated Protein (PAP) siRNA expression plasmid significantly decreased the proliferation of TF-V5 but not mock transfected Ba/F3 cells. Our data demonstrates that PYK2 is an attractive molecular target for FGFR3 associated hematopoietic neoplasm.

Peroxynitrite inhibits enterocyte proliferation and modulates Src kinase activity in vitro

2003 ◽  
Vol 285 (5) ◽  
pp. G861-G869 ◽  
Author(s):  
Douglas A. Potoka ◽  
Jeffrey S. Upperman ◽  
Xiao-Ru Zhang ◽  
Joshua R. Kaplan ◽  
Seth J. Corey ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Src Kinase ◽  
Dependent Manner ◽  
Gut Barrier ◽  
Enterocyte Proliferation ◽  
Barrier Failure ◽  
Enterocyte Apoptosis ◽  
Gut Barrier Failure

Overproduction of nitric oxide (NO) or its toxic metabolite, peroxynitrite (ONOO-), after endotoxemia promotes gut barrier failure, in part, by inducing enterocyte apoptosis. We hypothesized that ONOO-may also inhibit enterocyte proliferation by disrupting the Src tyrosine kinase signaling pathway, thereby blunting repair of the damaged mucosa. We examined the effect of ONOO-on enterocyte proliferation and Src kinase activity. Sprague-Dawley rats were challenged with LPS or saline, whereas intestinal epithelial cell line cells were treated with ONOO-or decomposed ONOO-in vitro. Enterocyte proliferation in vivo and in vitro was measured by 5-bromo-2′-deoxyuridine (BrdU) or [3H]thymidine incorporation. Src kinase activity in cell lysates was determined at various times. LPS challenge in vivo and ONOO-treatment in vitro inhibited enterocyte proliferation. ONOO-treatment blunted the activity of Src and its downstream target, focal adhesion kinase, in a time-dependent manner. ONOO-blocked mitogen (FBS, EGF)-induced enterocyte proliferation and Src phosphorylation while increasing Src nitration. Thus ONOO-may promote gut barrier failure not only by inducing enterocyte apoptosis but also by disrupting signaling pathways involved in enterocyte proliferation.

scholarly journals Activation of phosphatidylinositol-3 kinase by ligation of the interleukin-7 receptor is dependent on protein tyrosine kinase activity

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1579-1586 ◽  
Author(s):  
H Dadi ◽  
S Ke ◽  
CM Roifman
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Enzyme Activation ◽  
Dependent Manner ◽  
Protein Tyrosine ◽  
Tyrosine Residues ◽  
Interleukin 7 ◽  
Phosphatidylinositol 3

Abstract Ligation of the interleukin-7 receptor (IL-7R) results in a rapid phosphorylation of tyrosine residues on multiple substrates. In addition, we have recently shown that the IL-7R mediates activation of phosphatidylinositol-3 (PI-3) kinase. Because PI-3 kinase activity can be immunoprecipitated with antiphosphotyrosine antibodies in most receptor systems studied, it has been examined that either PI-3 kinase or an associated protein become tyrosine-phosphorylated after ligand binding. We studied here the possibility that PI-3 kinase, which is directly linked to mitogenic responses in growth factor receptors, is tyrosine-phosphorylated after stimulation of the IL-7R. Using anti-p85 alpha or anti-p85 beta antibodies raised against the p85 subunit of PI- 3 kinase for immunoprecipitation and subsequent blotting with antiphosphotyrosine clearly shows that IL-7-stimulated human precursor cells contain both p85 alpha and p85 beta proteins phosphorylated on tyrosine residues. Specific protein tyrosine kinase inhibitors such as tyrphostin AG-490 block total cell lysate phosphorylation and tyrosine phosphorylation on p85. Similar concentrations of this inhibitor also block in vitro and in vivo PI-3 kinase activity suggesting that this enzyme activation is dependent on the phosphorylation event of p85. In addition, AG-490 blocks IL-7-mediated proliferation in a dose-dependent manner, suggesting a link between the early events of PI-3 kinase phosphorylation and activation with IL-7R-induced cell growth.

scholarly journals Constitutive Association of BRCA1 and c-Abl and Its ATM-Dependent Disruption after Irradiation

2002 ◽  
Vol 22 (12) ◽  
pp. 4020-4032 ◽  
Author(s):  
Nicolas Foray ◽  
Didier Marot ◽  
Voahangy Randrianarison ◽  
Nicole Dalla Venezia ◽  
Didier Picard ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Direct Interaction ◽  
Cell Cycle Checkpoint ◽  
Dependent Manner ◽  
Tyrosine Kinase Activity ◽  
Abl Tyrosine Kinase ◽  
C Terminus

ABSTRACT BRCA1 plays an important role in mechanisms of response to double-strand breaks, participating in genome surveillance, DNA repair, and cell cycle checkpoint arrests. Here, we identify a constitutive BRCA1-c-Abl complex and provide evidence for a direct interaction between the PXXP motif in the C terminus of BRCA1 and the SH3 domain of c-Abl. Following exposure to ionizing radiation (IR), the BRCA1-c-Abl complex is disrupted in an ATM-dependent manner, which correlates temporally with ATM-dependent phosphorylation of BRCA1 and ATM-dependent enhancement of the tyrosine kinase activity of c-Abl. The BRCA1-c-Abl interaction is affected by radiation-induced modification to both BRCA1 and c-Abl. We show that the C terminus of BRCA1 is phosphorylated by c-Abl in vitro. In vivo, BRCA1 is phosphorylated at tyrosine residues in an ATM-dependent, radiation-dependent manner. Tyrosine phosphorylation of BRCA1, however, is not required for the disruption of the BRCA1-c-Abl complex. BRCA1-mutated cells exhibit constitutively high c-Abl kinase activity that is not further increased on exposure to IR. We suggest a model in which BRCA1 acts in concert with ATM to regulate c-Abl tyrosine kinase activity.

scholarly journals Activation of phosphatidylinositol-3 kinase by ligation of the interleukin-7 receptor is dependent on protein tyrosine kinase activity

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1579-1586 ◽  
Author(s):  
H Dadi ◽  
S Ke ◽  
CM Roifman
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Enzyme Activation ◽  
Dependent Manner ◽  
Protein Tyrosine ◽  
Tyrosine Residues ◽  
Interleukin 7 ◽  
Phosphatidylinositol 3

Ligation of the interleukin-7 receptor (IL-7R) results in a rapid phosphorylation of tyrosine residues on multiple substrates. In addition, we have recently shown that the IL-7R mediates activation of phosphatidylinositol-3 (PI-3) kinase. Because PI-3 kinase activity can be immunoprecipitated with antiphosphotyrosine antibodies in most receptor systems studied, it has been examined that either PI-3 kinase or an associated protein become tyrosine-phosphorylated after ligand binding. We studied here the possibility that PI-3 kinase, which is directly linked to mitogenic responses in growth factor receptors, is tyrosine-phosphorylated after stimulation of the IL-7R. Using anti-p85 alpha or anti-p85 beta antibodies raised against the p85 subunit of PI- 3 kinase for immunoprecipitation and subsequent blotting with antiphosphotyrosine clearly shows that IL-7-stimulated human precursor cells contain both p85 alpha and p85 beta proteins phosphorylated on tyrosine residues. Specific protein tyrosine kinase inhibitors such as tyrphostin AG-490 block total cell lysate phosphorylation and tyrosine phosphorylation on p85. Similar concentrations of this inhibitor also block in vitro and in vivo PI-3 kinase activity suggesting that this enzyme activation is dependent on the phosphorylation event of p85. In addition, AG-490 blocks IL-7-mediated proliferation in a dose-dependent manner, suggesting a link between the early events of PI-3 kinase phosphorylation and activation with IL-7R-induced cell growth.

scholarly journals In vitro assay for cyclin-dependent kinase activity in yeast

1998 ◽  
Vol 3 (1) ◽  
pp. 5-8
Author(s):  
Christine N. Tennyson ◽  
Vivien Measday ◽  
Brenda J. Andrews
Keyword(s):  
Kinase Activity ◽  
In Vitro Assay ◽  
Cyclin Dependent Kinase ◽  
Vitro Assay
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