OBJECTIVESTo compare expressions of tyrosine-phosphorylated proteins in different hepatocellular carcinoma cell lines with different metastasis potential and to screen key molecules associated with HCC metastasis and recurrence.

METHODSUsing two-dimensional electrophoresis, Western blotting and MALDI-TOF-MS/MS, we analyzed tyrosine-phosphorylated protein profiles of Hep3B, MHCC97L and MHCC97H, HCC cell lines with different metastasis potentials.

RESULTS10 spots were detected in Hep3B, 19 in MHCC97L and 17 in MHCC97H. Seventeen significantly different phosphotyrosine proteins in gel were identified by MALDI-TOF-MS/MS, including Annexin I.

CONCLUSIONThe changed expression of tyrosine-phosphorylated proteins is associated with HCC metastasis and recurrence.

Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Electrophoresis, Gel, Two-Dimensional ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Neoplasm Metastasis ; Neoplasm Proteins ; analysis ; Phosphotyrosine ; analysis

The 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a protein of unknown function in vivo, is abundantly expressed in myelinating glia in two isoforms, CNP1 and CNP2. In this study, immunoblot analysis showed that CNP1 is the major isoform in adult forebrain, and that both isoforms are included in the postsynaptic density (PSD) fraction and tyrosine-phosphorylated at the basal level. However, subcellular distribution and detergent extraction data showed that CNP is nonspecifically associated with the PSD fraction. Immunocytochemistry revealed that CNP is detected, in a weak but punctate pattern, in dissociated rat hippocampal neurons of 3 days to 2 weeks in vitro. The CNP-positive punctae were distributed throughout soma and dendrites, and distinct from PSD95-positive ones. Immunoblot analysis indicated that CNP is also expressed in neuronal stem cell lines, HiB5 and F11. Interestingly, in addition to the known two isoforms, a new CNP isoform of MW 45 kDa was expressed in these cell lines and was the major type of isoform in F11 cells. Taken together, our data suggest that CNP is expressed in the early stage of in vitro development and nonspecifically included in the adult rat PSD fraction.
2',3'-Cyclic-Nucleotide Phosphodiesterases/*metabolism ; Aging/physiology ; Animals ; Cells, Cultured ; Hippocampus/cytology/metabolism ; Immunohistochemistry ; Nerve Tissue Proteins/*metabolism ; Neurons/metabolism ; Phosphotyrosine/metabolism ; Prosencephalon/cytology/*metabolism ; Rats ; Rats, Sprague-Dawley ; Substrate Specificity

OBJECTIVETo explore the specificity of anti-phosphotyrosine monoclonal antibody PY20 in bcr-abl+ cells and its possible clinical applications.

METHODSBcr-abl cell lines( K562, MEG-01) and bcr-abl- cells lines( Jurkat, MCF-7 )were stained with PY20. Phosphotyrosine protein of K562 and MEG-01 cells was detected by flow cytometry before and after treatment with imatinib. Phosphotyrosine protein in bone marrow cells from 49 patients with chronic myeloid leukemia (CML), Ph+ acute lymphoblastic leukemia(Ph(+) -ALL), Ph- ALL, acute myeloid leukemia (AML-M1, M2, M3, M5, FAB classification), chronic lymphocytic leukemia (CML) and 3 normal donor. Positive cells over 5% of total cells was considered positive cases for phosphotyrosine protein. The level of tyrosine phosphorylation was determined by median fluorescence intensity (MFI).

RESULTSBcr-abl cell lines and marrow cells from 10 CML patients and 8 ALL patients were all PY20-positive, while bcr-abl- cell lines and marrow cells from 18 leukemia patients and 3 normal donor were all PY20-negative. MFI decreased remarkably after blocked by imatinib in K562 cells and MEG-01 cells. The positive cell percent of marrow cells from 10 newly diagnosed CML patients and 9 imatinib-sensitive CML patients was (54.20 +/- 19.82)% and (14.84 +/- 6.17)% (P < 0.05), while that of 2 cases of imatinib-resistant was 64.3% and 57.2%. There was significant difference of MFI between imatinib-resistant patients and imatinib-sensitive patients (99.42 +/- 4.87 vs 46.41 +/- 4.67, P < 0.01).

CONCLUSIONPY20 monoclonal antibody is highly specific for bcr-abl+ cells. It might be useful in rapid detection of bcr-abl+ cells and sensitivity to imatinib of CML patients.

Antibodies, Monoclonal ; analysis ; Bone Marrow Cells ; metabolism ; Flow Cytometry ; Fusion Proteins, bcr-abl ; analysis ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism ; Leukemia, Myeloid, Acute ; metabolism ; Phosphotyrosine ; analysis ; immunology

We investigated the co-stimulatory role of a cell-surface protein, CD99. Co-ligation of CD99 and suboptimal CD3 induced T-cell activation to a level comparable to that obtained with optimal CD3 or CD3+CD28. We also noted concomitant enhancement of the earliest T-cell receptor (TCR) signaling events. In addition, co-ligation of CD99 and CD3 led to translocation of TCR complexes into the lipid raft, without concomitant migration of CD99 to the raft, and consequent enhancement of TCR zeta-mediated signal 1. These data demonstrate the unique properties of CD99 co-stimulation that distinguish this molecule from CD28 and other raft-resident co-stimulatory factors.
Antigens, CD/*immunology ; Antigens, CD3/immunology ; Cell Adhesion Molecules/*immunology ; Down-Regulation ; Humans ; Jurkat Cells ; Lymphocyte Activation/*immunology ; Membrane Microdomains/*immunology ; Membrane Proteins/*immunology ; Phosphorylation ; Phosphotyrosine/*metabolism ; Protein Transport ; Receptors, Antigen, T-Cell/*immunology ; T-Lymphocytes/*immunology

The objective of this study was to investigate the specificity of detecting the phosphotyrosine level with anti-phosphotyrosine monoclonal antibody PY20 for diagnosis and prognosis of patients with chronic myeloid leukemia (CML) and the possibility of its clinical application. The positive rate of PY20 in 28 newly diagnosed CML patients was detected by flow cytometry using anti-PY20 antibody, the bcr-abl fusion gene was detected by nested RT-PCR, the Ph chromosome was measured by R-banding cytogenetic analysis, and the coincidence of PY20 positive rate with results of bcr-abl fusion gene and Ph chromosome detection was compared. In addition, the positive rate of PY20, the changes of bcr-abl fusion gene and Ph chromosome were determined in follow up 7 CML patients after allo-hematopoietic stem cell transplantation. The results indicated that the positive rates of PY20 in 28 newly diagnosed CML patients in groups of chronic phase (CP), accelerated phase (AP), and blast phase (BP) were (40.31% +/- 1.22)%, (77.28 +/- 1.14)% and (78.12 +/- 1.32)% respectively. The positive rate of PY20 in CP was lower than that in AP and BP (p < 0.05). There was no difference in positive rate of PY20 between AP and BP (p > 0.05). PY20 expression level of leukocytes from peripheral blood and bone marrow showed no difference (p < 0.05). The positive rates of PY20 in patients with CR, PR and NR were (15.56% +/- 1.51)%, (38.73% +/- 2.31)% and (60.43% +/- 2.04)% respectively. The positive and negative coincidence between PY20 and RT-PCR was 92.31% and 95.45% respectively. The positive and negative coincidence between PY20 and Ph Chromosome in newly diagnosed patients was 88.46% and 95.46% respectively. Ph chromosome and PY20 were all negative in 7 CML patients after allo-HSCT. Bcr-abl fusion gene was negative persistently in 5 patients, but in the other 2 patients, the fusion gene was persistently positive. In conclusion, the detection of the level of phosphotyrosine in CML cells has high sensitivity and specificity. The results of PY20 cell positive rate combined with detection of bcr/abl fusion gene and Ph chromosome might be useful in diagnosis as a good index of monitoring.
Adolescent ; Adult ; Antibodies, Monoclonal ; chemistry ; Female ; Flow Cytometry ; Fusion Proteins, bcr-abl ; genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; metabolism ; Male ; Middle Aged ; Philadelphia Chromosome ; Phosphotyrosine ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Young Adult

OBJECTIVETo study the potential signal pathway involved in pathogenesis of hypertrophic scar formation.

METHODSThe samples of scar were obtained from patients with burn wound scars 6 - 28 months post-burn, while the samples of normal control skin came from the donor site of the same patients. Immunohistochemistry and light microscopy techniques were used to identify the expression of epidermal growth factor receptor (EGFR) and phosphotyrosine proteins (p-Tyr), as well as the phosphorylation of signal transducer and activator of transcription 3 (Stat3) in both hypertrophic scars (n = 6) and normal skin (n = 6).

RESULTSSignificant differences were observed in the p-Tyr and EGFR positive expression keratinocytes both in hypertrophic scars and normal skin. The expression of p-Tyr, EGFR and Stat3 protein was greater in hypertrophic scars than in normal skin. However, there was no significant difference in p-Stat3 expression between scar tissues and normal skin.

CONCLUSIONDifferent tyrosine kinase activity occurs in hypertrophic scars and normal cutaneous tissues. Initially, varied expression of EGFR is due to different ligand stimulations. However, phosphotyrosine protein and Stat3 are subsequently activated through phosphorylation. In scar tissues, although EGFR has an intrinsic tyrosine kinase activity when activated by EGFR correlated ligand, phosphorylation of Stat3 showed no significant changes. Therefore, cellular signal pathways are induced by EGFR, which might play a role in hypertrophic scar pathogenesis.

Adult ; Cicatrix, Hypertrophic ; metabolism ; DNA-Binding Proteins ; analysis ; Female ; Humans ; Immunohistochemistry ; Male ; Phosphorylation ; Phosphotyrosine ; analysis ; Receptor, Epidermal Growth Factor ; analysis ; STAT3 Transcription Factor ; Skin ; chemistry ; Trans-Activators ; analysis ; Wound Healing

STATs (signal transducers and activators of transcription) are proteins with dual functions: signal transducers in the cytoplasm and transcriptional activators in the nucleus. STAT proteins act as transcription factors activated by phosphorylation on its tyrosine residues upon stimulation by various cytokines. The phosphorylated STAT molecules then form homo- or heterodimers through SH2-mediated interaction and translocate into the nucleus to activate the transcription of various target genes. STAT5 recognizes the interferon-gamma activated site TTCNNNGAA (GAS sequence) in the promoter region of the beta-casein gene. Except for prolactin-dependent beta-casein production in mammary gland cells, the biological consequences of STAT5a activation in various systems are not clear. Here we showed that STAT5a was phosphorylated 10 min after desferrioxamine (DFO) treatment, and reached a maximum induction at 4 h in mammary epithelial cells (HC11) and transfected COS-7 cells. Under hypoxic conditions (2% O2), a maximal phosphorylation of STAT5a was observed within 6 h. EMSA (electrophoretic mobility shift assay) showed that DFO or hypoxia enhanced the binding activities of STAT5a DNA to beta-casein gene promoter in mammary epithelial cells (HC11) and transfected COS-7 cells. These results showed that DFO or hypoxia induces tyrosine phosphorylation of STAT5a and also increases the binding activity of STAT5a DNA in mammary epithelial cells. Our data suggest that the STAT5 may act as a mediator in hypoxia-mediated gene expression.
Animals ; Anoxia/*genetics/*metabolism ; Caseins/genetics ; Cell Line ; DNA/genetics/metabolism ; DNA-Binding Proteins/*metabolism ; Deferoxamine/pharmacology ; Epithelial Cells/drug effects/*metabolism ; Gene Expression Regulation ; Mammary Glands, Animal/cytology/*metabolism ; Mice ; Phosphorylation/drug effects ; Phosphotyrosine/metabolism ; Promoter Regions (Genetics)/genetics ; Protein Binding ; Response Elements/*genetics ; Support, Non-U.S. Gov't ; Trans-Activators/*metabolism

Diverse signaling pathways have been proposed to regulate store-operated calcium entry (SOCE) in a wide variety of cell types. However, it still needs to be determined if all of these known pathways operate in a single cell type. In this study, we examined involvement of various signaling molecules in SOCE using human fibroblast cells (HSWP). Bradykinin (BK)-stimulated Ca2+ entry, previously shown to be via SOCE, is enhanced by the addition of vanadate, an inhibitor of tyrosine phosphatases. Furthermore, SOCE is regulated by cytochrome P-450, as demonstrated by the fact that the products of cytochrome P-450 activity (14,15 EET) stimulated SOCE while econazole, an inhibitor of cytochrome P450, suppressed BK-stimulated Ca2+ entry. In contrast, Ca2+ entry was unaffected by the guanylate cyclase inhibitor LY83583, or the membrane permeant cyclic GMP analog 8-bromo-cyclic GMP (8-Br-cGMP). Neither nitric oxide donors nor phorbol esters affected BK-stimulated Ca2+ entry. SOCE in HSWP cells is primarily regulated by tyrosine phosphorylation and the cytochrome P-450 pathway, but not by cyclic GMP, nitric oxide, or protein kinase C. Thus, multiple pathways do operate in a single cell type leading to the activation of Ca2+ entry and some of these signaling pathways are more prominently involved in regulating calcium entry in different cell types.
Vanadates/pharmacology ; Tetradecanoylphorbol Acetate/pharmacology ; Protein-Tyrosine-Phosphatase/*metabolism ; Phosphotyrosine/metabolism ; Phosphorylation/drug effects ; Nitric Oxide/metabolism ; Humans ; Fibroblasts ; Epidermal Growth Factor/pharmacology ; Enzyme Inhibitors/pharmacology ; Econazole/pharmacology ; Cytochrome P-450 Enzyme System/antagonists & inhibitors/*metabolism ; Cyclic GMP/analogs & derivatives/metabolism ; Cells, Cultured ; Calcium Channels/*metabolism ; Calcium/metabolism ; Bradykinin/pharmacology

Growth factor stimulation induces Y783 phosphorylation of phosphoinositide-specific PLC-gamma1, and the subsequent activation of this enzyme in a cellular signaling cascade. Previously, we showed that a double point mutation, Y509A/F510A, of PLC-gamma1, abolished interactions with translational elongation factor 1-alpha. Here, we report that the Y509A/F510A mutant PLC-gamma1 displayed extremely high levels of Y783 phosphorylation and enhanced catalytic activity, compared to wild-type PLC-gamma1, upon treatment of COS7 cells with EGF. In quiescent COS7 cells, the Y509A/F510A mutant PLC-gamma1 exhibited a constitutive hydrolytic activity, whereas the wild-type counterpart displayed a basal level of activity. Upon treatment of COS7 cells with EGF, the Y783F mutation in Y509A/F510A PLC-gamma1 (Y509A/F510A/Y783F triple mutant) cells also led to an enhanced catalytic activity, whereas Y783F mutation alone displayed a basal level of activity. Our results collectively suggest that the Y509A/F510A mutant is more susceptible to receptor tyrosine kinase-induced Y783 phosphorylation than is wild-type PLC-gamma1, but no longer requires Y783 phosphorylation step for the Y509A/F510A mutant PLC-gamma1 activation in vivo.
Amino Acid Substitution/drug effects/*genetics ; Animals ; COS Cells ; Cercopithecus aethiops ; Enzyme Activation/drug effects ; Epidermal Growth Factor/*pharmacology ; Hydrolysis/drug effects ; Mutant Proteins/metabolism ; Phosphatidylinositols/*metabolism ; Phospholipase C gamma/*genetics/metabolism ; Phosphorylation/drug effects ; Phosphotyrosine/*metabolism ; Point Mutation/*genetics ; Rats

OBJECTIVETo investigate the effect of Jiaotai Pill (, JTP) at different constitutional proportions on insulin signaling through phosphatidylinositol 3-kinase (PI3K) pathway in the skeletal muscle of diabetic rats.

METHODSThe rat model of type 2 diabetes mellitus (T2DM) was established by intravenous injection of a small dose of streptozotoein plus high fat diet feeding. JTP at the same dosage of cinnamon and the increasing dosage of Coptis chinensis was administered to diabetic rats for nine weeks respectively. Plasma glucose and insulin levels were assayed. The expressions of proteins were determined by Western blot method.

RESULTSAll the three formulations of JTP decreased plasma glucose and fasting insulin levels as well as increased the protein expressions of insulin receptor β (InsRβ) subunit, insulin receptor substrate-1 (IRS-1), PI3K p85 subunit and glucose transporter 4 (GLUT4) in skeletal muscle. Meanwhile, JTP increased the tyrosine phosphorylation of InsRβ subunit and IRS-1, and reduced the serine phosphorylation of IRS-1 in skeletal muscle. Interestingly, the effect of JTP on improving insulin sensitivity was not dose-dependent. In contrast, JTP containing the least amount of Coptis chinensis exhibited the best effect.

CONCLUSIONJTP at different constitutional proportions attenuates the development of diabetes in a rat model of T2DM. The mechanism might be associated with enhancing insulin signaling through PI3K pathway in the skeletal muscle.

Animals ; Body Weight ; drug effects ; Diabetes Mellitus, Experimental ; drug therapy ; enzymology ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Glucose Tolerance Test ; Glucose Transporter Type 4 ; metabolism ; Homeostasis ; drug effects ; Insulin ; metabolism ; Insulin Receptor Substrate Proteins ; metabolism ; Insulin Resistance ; Male ; Muscle, Skeletal ; drug effects ; enzymology ; metabolism ; pathology ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; Phosphotyrosine ; metabolism ; Protein Subunits ; metabolism ; Rats ; Rats, Wistar ; Receptor, Insulin ; metabolism ; Signal Transduction ; drug effects