技术资料

Luminal and myoepithelial cells have been separated from normal adult human breast epithelium using fluorescence activated cell sorting. Their isolation was based on the exclusive expression of two surface antigens,epithelial membrane antigen (EMA) and the common acute lymphoblastic leukaemia antigen (CALLA/CD10/neutral endopeptidase 24.11). Sorted luminal and myoepithelial cells displayed distinctively different morphologies when maintained in monolayer culture,differences which were enhanced by the addition of hydrocortisone,insulin and cholera toxin to the culture medium. The EMA-positive cells formed an attenuated monolayer with indistinct cell boundaries while CALLA-positive cells,by contrast,formed tightly packed arrays of refractile cells. The distribution of the cell type-specific markers cytokeratin 18 (luminal cells) and smooth muscle alpha-actin (myoepithelial cells) indicated that the sorted populations were approximately 98% pure. However,a significant minority (approximately 15%) of sorted luminal cells consistently expressed the basal-cell marker cytokeratin 14 in culture. A marked difference was noted in the proliferative behaviour of the two types of sorted cells,with myoepithelial cells dividing rapidly in response to the humoural additives,in contrast to the luminal cells which proliferated slowly. Both types of sorted cells could be cloned in the presence of feeder layers of mouse fibroblasts. Clones of luminal and myoepithelial cells were also distinctive; all spread" luminal clones were similar in appearance to each other� View Publication

Costimulatory signals are critical to T cell activation,but how their effects are mediated remains incompletely characterized. Here,we demonstrate that locally produced C5a and C3a anaphylatoxins interacting with their G protein-coupled receptors (GPCRs),C5aR and C3aR,on APCs and T cells both upstream and downstream of CD28 and CD40L signaling are integrally involved in T cell proliferation and differentiation. Disabling these interactions reduced MHC class II and costimulatory-molecule expression and dramatically diminished T cell responses. Importantly,impaired T cell activation by Cd80-/-Cd86-/- and Cd40-/- APCs was reconstituted by added C5a or C3a. C5aR and C3aR mediated their effects via PI-3 kinase-gamma-dependent AKT phosphorylation,providing a link between GPCR signaling,CD28 costimulation,and T cell survival. These local paracrine and autocrine interactions thus operate constitutively in naive T cells to maintain viability,and their amplification by cognate APC partners thus is critical to T cell costimulation. View Publication

CD24 is a signal-transducing molecule on the surfaces of most human B cells that can modulate their response to activation signals by antagonizing IL-induced differentiation into antibody-forming cells and inducing proliferation in combination with signals generated by Ag receptors. A cDNA that directs the expression of CD24 on the surfaces of transfected COS cells was cloned by its homology to a cDNA encoding the murine M1/69-J11d heat stable Ag. The CD24 cDNA encodes a mature peptide of only 31 to 35 amino acids that is extensively glycosylated and is attached to the outer surface of the plasma membrane by a glycosyl phosphatidylinositol lipid anchor. Although CD24 is structurally similar to M1/69-J11d,and the two Ag appear to have a common genetic ancestry,the homology of CD24 to the M1/69-J11d Ag is confined to a small cluster of amino acids comprising potential N-linked glycosylation sites. Combined with the differences in expression patterns of the human and murine Ag,this indicates that CD24 and M1/69-J11d may not have equivalent functional roles in lymphoid development. The novel structure of CD24 suggests that signaling could be triggered by the binding of a lectin-like ligand to the carbohydrates projecting from the CD24 peptide,and transduced through the release of second messengers derived from the glycosyl phosphatidylinositol membrane anchor of CD24. View Publication

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We have resolved B220+ IgM- B-lineage cells in mouse bone marrow into four fractions based on differential cell surface expression of determinants recognized by S7 (leukosialin,CD43),BP-1,and 30F1 (heat stable antigen). Functional differences among these fractions can be correlated with Ig gene rearrangement status. The largest fraction,lacking S7,consists of pre-B cells whereas the others,expressing S7,include B lineage cells before pre-B. These S7+ fractions,provisionally termed Fr. A,Fr. B,and Fr. C,can differentiate in a stromal layer culture system. Phenotypic alteration during such culture suggests an ordering of these stages from Fr. A to Fr. B to Fr. C and thence to S7- pre-B cells. Using polymerase chain reaction amplification with pairs of oligonucleotide primers for regions 5' of JH1,DFL16.1,and Jk1,we find that the Ig genes of Fr. A are in germline configuration,whereas Fr. B and C are pro-B cell stages with increasing D-J rearrangement,but no V-D-J. Finally,functional analysis demonstrates that the proliferative response to IL-7,an early B lineage growth factor,is restricted to S7+ stages and,furthermore,that an additional,cell contact-mediated signal is essential for survival of Fr. A. View Publication

OBJECTIVE: Endothelial-like vascular progenitor cells (VPCs) can be collected in peripheral blood stem cell (PBSC) products that are used in hematopoietic stem cell transplantation (HSCT). The association between VPCs in PBSC products and transplant-related toxicity caused by high-dose chemo/radiotherapy was assessed to identify potential mediators of vascular repair. MATERIALS AND METHODS: PBSC grafts in 29 patients (mean age: 48 years; range,20-67 years) undergoing autologous HSCT were analyzed using a cell culture assay for VPC cluster formation in fibronectin-coated dishes in serum-rich angiogenic conditions. Transplant toxicity was estimated using total length of hospital stay (LOS) following HSCT and the Seattle criteria for transplant-related organ toxicity for 8 organ systems (grade 0-4). RESULTS: LOS following graft reinfusion was lower (14.7 vs 20.0 days,p = 0.002) and the mean number of organs with any toxicity (1.0 vs 2.4,p = 0.016) or with toxicity grade textgreater or = 2 was reduced (0.2 vs 1.6 organs,p = 0.007) in patients with high graft VPC content (n = 10,textgreater2.0 x 10(3) VPCs/kg) compared with reduced VPC content (n = 19,textless or = 2.0 x 10(3) VPCs/kg). An association between graft CD34(+) levels and LOS or organ toxicity was not observed. In addition,graft VPC levels were independent of graft CD34 counts,peripheral blood monocytes and hemoglobin levels,age,and disease (p = NS). CONCLUSION: PBSC products enriched for VPCs are associated with reduced toxicity following HSCT. Identifying specific factors that contribute to high graft VPC levels is needed. View Publication

Cdx1,Cdx2,and Cdx4 comprise the caudal-like Cdx gene family in mammals,whose homologues regulate hematopoietic development in zebrafish. Previously,we reported that overexpression of Cdx4 enhances hematopoietic potential from murine embryonic stem cells (ESCs). Here we compare the effect of ectopic Cdx1,Cdx2,and Cdx4 on the differentiation of murine ESC-derived hematopoietic progenitors. The 3 Cdx genes differentially influence the formation and differentiation of hematopoietic progenitors within a CD41(+)c-kit(+) population of embryoid body (EB)-derived cells. Cdx1 and Cdx4 enhance,whereas Cdx2 strongly inhibits,the hematopoietic potential of CD41(+)ckit(+) EB-derived cells,changes that are reflected by effects on hematopoietic lineage-specific and Hox gene expression. When we subject stromal cell and colony assay cultures of EB-derived hematopoietic progenitors to ectopic expression of Cdx genes,Cdx4 dramatically enhances,whereas Cdx1 and Cdx2 both inhibit hematopoietic activity,probably by blocking progenitor differentiation. These data demonstrate distinct effects of Cdx genes on hematopoietic progenitor formation and differentiation,insights that we are using to facilitate efforts at in vitro culture of hematopoietic progenitors from ESC. The behavior of Cdx genes in vitro suggests how derangement of these developmental regulators might contribute to leukemogenesis. View Publication

A heparin-binding mitogen was isolated from conditioned medium of human embryonic lung fibroblasts. It exhibited broad target-cell specificity whose pattern was distinct from that of any known growth factor. It rapidly stimulated tyrosine phosphorylation of a 145-kDa protein in responsive cells,suggesting that its signaling pathways involved activation of a tyrosine kinase. Purification identified a major polypeptide with an apparent molecular mass of 87 kDa under reducing conditions. Partial amino acid sequence analysis and cDNA cloning revealed that it was a variant of hepatocyte growth factor,a mitogen thought to be specific for hepatic cells and structurally related to plasminogen. Recombinant expression of the cDNA in COS-1 cells established that it encoded the purified growth factor. Its site of synthesis and spectrum of targets imply that this growth factor may play an important role as a paracrine mediator of the proliferation of melanocytes and endothelial cells,as well as cells of epithelial origin. View Publication

Mobilization of bone marrow-derived endothelial progenitor cells (EPC) might explain exercise-induced improvement of endothelial function. We assessed whether a maximal exercise bout could alter the number of circulating EPC in healthy subjects and whether this effect is related to their cardiovascular risk profile. Additionally,we investigated possible mediators of this effect,namely nitric oxide (NO) bioavailability and vascular endothelial growth factor (VEGF) release. Healthy subjects (group 1,n = 11; group 2,n = 14) performed a symptom-limited cardiopulmonary exercise test on a bicycle ergometer. Numbers of CD34+/kinase insert domain receptor (KDR)+ cells were determined by flow-cytometric analysis,either after magnetic separation of CD34+ cells (group 1) or starting from whole blood (group 2). Serum concentrations of VEGF and NO metabolites were measured by using ELISA. Following exercise,EPC increased by 76% (15.4 +/- 10.7 cells/ml vs. 27.2 +/- 13.7 cells/ml; P = 0.01) in group 1 and by 69% in group 2 (30.9 +/- 14.6 cells/ml vs. 52.5 +/- 42.6 cells/ml; P = 0.03). The increase in EPC correlated positively with LDL and total cholesterol/HDL ratio and negatively with peak oxygen consumption and oxygen consumption at anaerobic threshold. VEGF levels increased with exercise,with a strong trend toward significance (P = 0.055). NO levels remained unchanged. The present study demonstrates that a maximal bout of exercise induces a significant shift in CD34+ cells toward CD34+/KDR+ cells. This response was larger in subjects with a less favorable lipid profile. View Publication

Aurora kinases are essential for chromosomal segregation and cell division and thereby important for maintaining the proper genomic integrity. There are three classes of aurora kinases in humans: A,B,and C. Aurora kinase A is frequently overexpressed in various cancers. The link of the overexpression and tumorigenesis is yet to be understood. By employing virtual screening,we have found that anacardic acid,a pentadecane aliphatic chain containing hydroxylcarboxylic acid,from cashew nut shell liquid could be docked in Aurora kinases A and B. Remarkably,we found that anacardic acid could potently activate the Aurora kinase A mediated phosphorylation of histone H3,but at a similar concentration the activity of aurora kinase B remained unaffected in vitro. Mechanistically,anacardic acid induces the structural changes and also the autophosphorylation of the aurora kinase A to enhance the enzyme activity. This data thus indicate anacardic acid as the first small-molecule activator of Aurora kinase,which could be highly useful for probing the function of hyperactive (overexpressed) Aurora kinase A. View Publication

The neurosphere assay can detect and expand neural stem cells (NSCs) and progenitor cells,but it cannot discriminate between these two populations. Given two assays have purported to overcome this shortfall,we performed a comparative analysis of the distribution and frequency of NSCs and progenitor cells detected in 400 mum coronal segments along the ventricular neuraxis of the adult mouse brain using the neurosphere assay,the neural colony forming cell assay (N-CFCA),and label-retaining cell (LRC) approach. We observed a large variation in the number of progenitor/stem cells detected in serial sections along the neuraxis,with the number of neurosphere-forming cells detected in individual 400 mum sections varying from a minimum of eight to a maximum of 891 depending upon the rostral-caudal coordinate assayed. Moreover,the greatest variability occurred in the rostral portion of the lateral ventricles,thereby explaining the large variation in neurosphere frequency previously reported. Whereas the overall number of neurospheres (3730 +/- 276) or colonies (4275 +/- 124) we detected along the neuraxis did not differ significantly,LRC numbers were significantly reduced (1186 +/- 188,7 month chase) in comparison to both total colonies and neurospheres. Moreover,approximately two orders of magnitude fewer NSC-derived colonies (50 +/- 10) were detected using the N-CFCA as compared to LRCs. Given only 5% of the LRCs are cycling (BrdU+/Ki-67+) or competent to divide (BrdU+/Mcm-2+),and proliferate upon transfer to culture,it is unclear whether this technique selectively detects endogenous NSCs. Overall,caution should be taken with the interpretation and employment of all these techniques. View Publication

Our previous studies have shown that z-guggulsterone,a constituent of Indian Ayurvedic medicinal plant Commiphora mukul,inhibits the growth of human prostate cancer cells by causing apoptosis. We now report a novel response to z-guggulsterone involving the inhibition of angiogenesis in vitro and in vivo. The z-guggulsterone treatment inhibited capillary-like tube formation (in vitro neovascularization) by human umbilical vein endothelial cells (HUVEC) and migration by HUVEC and DU145 human prostate cancer cells in a concentration- and time-dependent manner. The z- and E-isomers of guggulsterone seemed equipotent as inhibitors of HUVEC tube formation. The z-guggulsterone-mediated inhibition of angiogenesis in vitro correlated with the suppression of secretion of proangiogenic growth factors [e.g.,vascular endothelial growth factor (VEGF) and granulocyte colony-stimulating factor],down-regulation of VEGF receptor 2 (VEGF-R2) protein level,and inactivation of Akt. The z-guggulsterone-mediated suppression of DU145 cell migration was increased by knockdown of VEGF-R2 protein level. Ectopic expression of constitutively active Akt in DU145 cells conferred protection against z-guggulsterone-mediated inhibition of cell migration. Oral gavage of 1 mg z-guggulsterone/d (five times/wk) to male nude mice inhibited in vivo angiogenesis in DU145-Matrigel plug assay as evidenced by a statistically significant decrease in tumor burden,microvessel area (staining for angiogenic markers factor VIII and CD31),and VEGF-R2 protein expression. In conclusion,the present study reveals that z-guggulsterone inhibits angiogenesis by suppressing the VEGF-VEGF-R2-Akt signaling axis. Together,our results provide compelling rationale for further preclinical and clinical investigation of z-guggulsterone for its efficacy against prostate cancer. View Publication