技术资料

Butein,a plant polyphenol,was shown to be a specific protein tyrosine kinase inhibitor. This compound inhibited not only the epidermal growth factor (EGF)-stimulated auto-phosphotyrosine level of EGF receptor in HepG2 cells but also tyrosine-specific protein kinase activities of EGF receptor (IC50 = 65 microM) and p60c-src (IC50 = 65 microM) in vitro. The inhibition was competitive to ATP and non-competitive to the phosphate acceptor,poly (Glu,Ala,Tyr) 6:3:1 for EGF receptor tyrosine kinase. In contrast,butein non-significantly inhibited the activities of serine- and threonine-specific protein kinases,such as protein kinase C (PKC) and cAMP-dependent protein kinase (PKA). View Publication

1. Polymorphonuclear leukocytes (PMN) may contribute to the pathogenesis of acute coronary heart disease (CHD). 2. Epidemiological and laboratory evidence suggests that red wine,by virtue of its polyphenolic constituents,may be more effective than other alcoholic beverages in reducing the risk of CHD mortality. 3 The aim of the present study was to investigate the effects of trans-resveratrol (3,4',5-trihydroxy-trans-stilbene),a polyphenol present in most red wines,on functional and biochemical responses of PMN,upon in vitro activation. 4. trans-Resveratrol exerted a strong inhibitory effect on reactive oxygen species produced by PMN stimulated with 1 microM formyl methionyl leucyl phenylalamine (fMLP) (IC50 1.3+/-0.13 microM,mean+/-s.e.mean),as evaluated by luminol-amplified chemiluminescence. 5. trans-Resveratrol prevented the release of elastase and beta-glucuronidase by PMN stimulated with the receptor agonists fMLP (1 microM,IC50 18.4+/-1.8 and 31+/-1.8 microM),and C5a (0.1 microM,IC50 41.6+/-3.5 and 42+/-8.3 microM),and also inhibited elastase and beta-glucuronidase secretion (IC50 37.7+/-7 and 25.4+/-2.2 microM) and production of 5-lipoxygenase metabolites leukotriene B4 (LTB4),6-trans-LTB4 and 12-trans-epi-LTB4 (IC50 48+/-7 microM) by PMN stimulated with the calcium ionophore A23187 (5 microM). 6. trans-Resveratrol significantly reduced the expression and activation of the beta2 integrin MAC-1 on PMN surface following stimulation,as revealed by FACS analysis of the binding of an anti-MAC-1 monoclonal antibody (MoAb) and of the CBRM1/5 MoAb,recognizing an activation-dependent epitope on MAC-1. Consistently,PMN homotypic aggregation and formation of mixed cell-conjugates between PMN and thrombin-stimulated fixed platelets in a dynamic system were also prevented by transresveratrol. 7. These results,indicating that trans-resveratrol interferes with the release of inflammatory mediators by activated PMN and down-regulates adhesion-dependent thrombogenic PMN functions,may provide some biological plausibility to the protective effect of red wine consumption against CHD. View Publication

This study sought to examine the relationship between MUC1 expression at the deepest invasive portion,invasive/metastatic potential,and prognosis of colorectal cancer in relation to cellular proliferation. MUC1 expression was detected immunohistochemically using KL-6 antibody (anti-MUC1 monoclonal antibody) in 100 surgically resected specimens of advanced colorectal cancer. Distinct staining of the luminal surfaces,defined as positive immunoreactive (IR)-MUC1 expression,was seen in more than 30% of the tumor cells at the deepest invasive portion. The proliferating cell nuclear antigen labeling index (PCNA-LI) was also examined in the same areas. IR-MUC1 expression was detected in 71 (71%) of 100 lesions. Lesions with lymphatic or venous invasion showed a significantly higher incidence of IR-MUC1 expression than those without lymphatic or venous invasion (80 vs. 42% and 82 vs. 61%,respectively). Lesions with lymph node metastasis showed a significantly higher incidence of IR-MUC1 expression than those without lymph node metastasis (88 vs. 53%). Lesions with liver metastasis showed a significantly higher incidence of IR-MUC1 expression than those without liver metastasis (92 vs. 59%). Dukes' stage was also significantly correlated with IR-MUC1 expression. The incidence of IR-MUC1 expression did not significantly differ with regard to histologic subclassification and depth of invasion. There was no significant correlation between IR-MUC1 expression and the PCNA-LI. IR-MUC1 expression at the deepest invasive portion revealed a significant correlation with prognosis; furthermore,in patients with better differentiated lesions,in those with lesions confined to muscularis propria or subserosa (subadventitial) invasion,in those with Dukes' B and C,or in those undergoing curative resection,IR-MUC1 expression significantly correlated with prognosis. Patients with high PCNA-LI lesions showed a significantly poorer prognosis than those with low PCNA-LI lesions. Only in patients undergoing curative resection,patients with IR-MUC1-positive and high PCNA-LI lesions showed a significantly poorer prognosis than those with IR-MUC1-negative and low PCNA-LI lesions. The significant risk factors in the order of poorer prognosis in patients undergoing curative resection by the multivariate analysis were the histologic grade (moderately-poorly,poorly or mucinous adenocarcinomas),IR-MUC1 expression,and lymph node metastasis. These results indicate that IR-MUC1 expression is an important predictor of the metastatic potential and the prognosis of colorectal cancer,independent of histologic grade,depth of invasion or cellular proliferative activity. Combined analysis of IR-MUC1 and histologic grade,and combined expression of IR-MUC1 and PCNA at the deepest invasive portion are especially useful in predicting colorectal cancer prognosis. View Publication

Simultaneous ex vivo expansion of different progenitor cell types may be beneficial for cord blood (CB) transplantation,to overcome a potential limitation due to restricted cell numbers. Therefore,1.5 x 10(6) CD34+ cells isolated from fresh or thawed CB samples were inoculated in a large-scale stirred suspension bioreactor and cultured in the presence of Flt3-L,SCF and IL-3. At days 0,7,10,14,21 and 28,the spinner cultures were analyzed for viable cells,colony-forming cells (CFC),including erythroid burst-forming unit (BFU-E),granulocyte-macrophage colony-forming unit (CFU-GM) and granulocyte-erythrocyte-megakaryocyte-monocyte colony-forming unit (CFU-GEMM) as well as long-term culture-initiating cells (LTC-IC). Expansion of thawed CD34+ cells resulted in a substantial amplification of total cells (maximal at day 28: 154 +/- 132-fold),CFC (maximal at day 14: 45 +/- 36-fold),CFU-GM (maximal at day 14: 88 +/- 85-fold),CFU-GEMM (maximal at day 7: 4 +/- 2-fold) and of LTC-IC (maximal at day 10: 8 +/- 3-fold). There was no significant difference between fresh and thawed CD34+ cells. These results demonstrate that simultaneously committed progenitors as well as the more immature CFU-GEMM and LTC-IC can be substantially amplified from CD34+-enriched CB samples in large-scale stirred suspension cultures within 7-14 days without exhausting the proliferative potential and,thus,it may be possible to improve CB transplantation by ex vivo generated cells. View Publication

Human blastocyst-derived,pluripotent cell lines are described that have normal karyotypes,express high levels of telomerase activity,and express cell surface markers that characterize primate embryonic stem cells but do not characterize other early lineages. After undifferentiated proliferation in vitro for 4 to 5 months,these cells still maintained the developmental potential to form trophoblast and derivatives of all three embryonic germ layers,including gut epithelium (endoderm); cartilage,bone,smooth muscle,and striated muscle (mesoderm); and neural epithelium,embryonic ganglia,and stratified squamous epithelium (ectoderm). These cell lines should be useful in human developmental biology,drug discovery,and transplantation medicine. View Publication

Activation of the extracellular signal-regulated kinase (ERK) pathway has been shown to occur in monocytes following stimulation with LPS. However,the importance of this event for monocyte function is not clear. To address this issue,we used the novel MAP/ERK kinase (MEK) inhibitor,U0126. Stimulation of monocytes with LPS resulted in activation of the mitogen-activated protein kinase (MAPK) family members ERK,Jun NH2-terminal kinase (JNK),and p38. Treatment of monocytes with LPS in the presence of U0126 blocked the activation of ERK1 and ERK2. However,the activation of Jun NH2-terminal kinase and p38 family members was not affected by the compound,confirming the selectivity of U0126. To examine the effects of MEK inhibition on monocyte function,we measured production of the cytokines IL-1,IL-8,and TNF,as well as PGE2. Monocytes treated with LPS in the presence of U0126 failed to release IL-1,IL-8,TNF,or PGE2. The failure to secrete IL-1 and TNF was due to decreased levels of mRNA. These results demonstrate that activation of MEK/ERK is critical for cytokine and PGE2 production by monocytes in response to LPS. View Publication

Abnormalities in mucin-type glycoprotein expression have been documented in a variety of cancers,identifying these molecules as targets for immunologically based therapies and prognostic/diagnostic assays. We examined the expression of the membrane-bound MUC1 mucin in normal,histologically atypical,and neoplastic lung to determine its potential contribution to lung carcinogenesis. In vivo,intense MUC1 immunoreactivity was present in normal type II pneumocytes as well as in a range of atypical lesions derived from type II cells and textgreater60% of primary and metastatic non-small cell lung cancers. Expression was not associated with altered survival,although it was highly correlated with the adenocarcinoma histology. A carcinogenesis model using 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone-exposed hamsters revealed that MUC1 mRNA increased prior to the histological appearance of tumors. In vitro studies using MUC1 expressing non-small cell lung cancer cell lines revealed that differentiation away from a type II cell lineage was associated with dramatic down-regulation of MUC1. We propose that MUC1 is a powerful new marker for the type II pneumocyte cell lineage that allows us to follow the type II pneumocyte lineage during the process of lung carcinogenesis. View Publication

SU5416,a novel synthetic compound,is a potent and selective inhibitor of the Flk-1/KDR receptor tyrosine kinase that is presently under evaluation in Phase I clinical studies for the treatment of human cancers. SU5416 was shown to inhibit vascular endothelial growth factor-dependent mitogenesis of human endothelial cells without inhibiting the growth of a variety of tumor cells in vitro. In contrast,systemic administration of SU5416 at nontoxic doses in mice resulted in inhibition of subcutaneous tumor growth of cells derived from various tissue origins. The antitumor effect of SU5416 was accompanied by the appearance of pale white tumors that were resected from drug-treated animals,supporting the antiangiogenic property of this agent. These findings support that pharmacological inhibition of the enzymatic activity of the vascular endothelial growth factor receptor represents a novel strategy for limiting the growth of a wide variety of tumor types. View Publication

Efficient gene transfer into human hematopoietic stem cells (HSCs) is an important goal in the study of the hematopoietic system as well as for gene therapy of hematopoietic disorders. A lentiviral vector based on the human immunodeficiency virus (HIV) was able to transduce human CD34+ cells capable of stable,long-term reconstitution of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. High-efficiency transduction occurred in the absence of cytokine stimulation and resulted in transgene expression in multiple lineages of human hematopoietic cells for up to 22 weeks after transplantation. View Publication

The adult rat hippocampus contains fibroblast growth factor 2-responsive stem cells that are self-renewing and have the ability to generate both neurons and glia in vitro,but little is known about the molecular events that regulate stem cell differentiation. Hippocampus-derived stem cell clones were used to examine the effects of retinoic acid (RA) on neuronal differentiation. Exposure to RA caused an immediate up-regulation of NeuroD,increased p21 expression,and concurrent exit from cell cycle. These changes were accompanied by a threefold increase in the number of cells differentiating into immature neurons. An accompanying effect of RA was to sustain or up-regulate trkA,trkB,trkC,and p75NGFR expression. Without RA treatment,cells were minimally responsive to neurotrophins (NTs),whereas the sequential application of RA followed by brain-derived neurotrophic factor or NT-3 led to a significant increase in neurons displaying mature y-a-minobutyric acid,acetylcholinesterase,tyrosine hydroxylase,or calbindin phenotypes. Although NTs promoted maturation,they had little effect on the total number of neurons generated,suggesting that RA and neurotrophins acted at distinct stages in neurogenesis. RA first promoted the acquisition of a neuronal fate,and NTs subsequently enhanced maturation by way of RA-dependent expression of the Trk receptors. In combination,these sequential effects were sufficient to stimulate stem cell-derived progenitors to differentiate into neurons displaying a variety of transmitter phenotypes. View Publication

The propagation of pluripotent mouse embryonic stem (ES) cells depends on signals transduced through the cytokine receptor subunit gp130. Signalling molecules activated downstream of gp130 in ES cells include STAT3,the protein tyrosine phosphatase SHP-2,and the mitogen-activated protein kinases,ERK1 and ERK2. A chimaeric receptor in which tyrosine 118 in the gp130 cytoplasmic domain was mutated did not engage SHP-2 and failed to activate ERKs. However,this receptor did support ES cell self-renewal. In fact,stem cell colonies formed at 100-fold lower concentrations of cytokine than the unmodified receptor. Moreover,altered ES cell morphology and growth were observed at high cytokine concentrations. These indications of deregulated signalling in the absence of tyrosine 118 were substantiated by sustained activation of STAT3. Confirmation that ERK activation is not required for self-renewal was obtained by propagation of pluripotent ES cells in the presence of the MEK inhibitor PD098059. In fact,the growth of undifferentiated ES cells was enhanced by culture in PD098059. Thus activation of ERKs appears actively to impair self-renewal. These data imply that the self-renewal signal from gp130 is a finely tuned balance of positive and negative effectors. View Publication

PP1 has previously been described as an inhibitor of the Src-family kinases p56(Lck) and FynT. We have therefore decided to use PP1 to determine the functional role of Src in platelet-derived growth factor (PDGF)-induced proliferation and migration of human coronary artery smooth muscle cells (HCASMCs). A synthetic protocol for PP1/AGL1872 has been developed,and the inhibitory activity of PP1/AGL1872 against Src was examined. PP1/AGL1872 potently inhibited recombinant p60(c-src) in vitro and Src-dependent tyrosine phosphorylation in p60(c-srcF572)-transformed NIH3T3 cells. PP1/AGL1872 also potently inhibited PDGF-stimulated migration of HCASMCs,as determined in the modified Boyden chamber,as well as PDGF-stimulated proliferation of HCASMCs. Surprisingly,in addition to inhibition of Src kinase,PP1/AGL1872 was found to inhibit PDGF receptor kinase in cell-free assays and in various types of intact cells,including HCASMCs. PP1/AGL1872 did not inhibit phosphorylation of the vascular endothelial growth factor receptor KDR (VEGF receptor-2; kinase-insert domain containing receptor) in cell-free assays as well as in intact human coronary artery endothelial cells. In line with the insensitivity of KDR,PP1/AGL1872 had only a weak effect on vascular endothelial growth factor-stimulated migration of human coronary artery endothelial cells. On treatment of cells expressing different receptor tyrosine kinases,the activities of the epidermal growth factor receptor,fibroblast growth factor receptor-1,and insulin-like growth factor-1 receptor were resistant to PP1/AGL1872,whereas PDGF alpha-receptor was susceptible,albeit to a lesser extent than PDGF beta-receptor. These data suggest that the previously described tyrosine kinase inhibitor PP1/AGL1872 is not selective for the Src family of tyrosine kinases. It is also a potent inhibitor of the PDGF beta-receptor kinase but is not a ubiquitous tyrosine kinase inhibitor. PP1/AGL1872 inhibits migration and proliferation of HCASMCs probably by interference with 2 distinct tyrosine phosphorylation events,creating a novel and potent inhibitory principle with possible relevance for the treatment of pathological HCASMC activity,such as vascular remodeling and restenosis. View Publication