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Biomedical subjects

C P Shen

Publications and source records attributed to C P Shen.

18 recordsLinked to original sources
PubMed2006

Search for invisible decays of eta and eta' in J/psi --> phi eta and phi eta'.

Using a data sample of 58 x 10(6) J/psi decays collected with the Beijing Spectrometer II detector at the Beijing Electron Positron Collider, searches for invisible decays of eta and eta' in J/psi to phi eta and phi eta' are performed. The phi signals, which are reconstructed in K+K- final states, are used to tag the eta and eta' decays. No signals are found for the invisible decays of either eta or eta', and upper limits at the 90% confidence level are determined to be 1.65 x 10(-3) for the ratio B(eta-->invisible)/B(eta --> gamma gamma) and 6.69 x 10(-2) for B(eta' --> invisible)/B(eta' --> gammagamma). These are the first searches for eta and eta' decays into invisible final states.

Journal Article↗
PubMed2006

Observation of a near-threshold enhancement in the omega(phi) mass spectrum from the doubly OZI-suppressed decay J/psi-->gamma(omega)phi.

An enhancement near threshold is observed in the omega(phi) invariant mass spectrum from the doubly Okubo-Zweig-Iizuka-suppressed decays of J/psi-->gamma(omega)phi, based on a sample of 5.8 x 10(7) J/psi events collected with the BESII detector. A partial wave analysis shows that this enhancement favors JP=0+, and its mass and width are M=1812(+19)(-26)(stat)+/-18(syst) MeV/c2 and Gamma=105+/-20(stat)+/-28(syst) MeV/c2. The product branching fraction is determined to be B(J/psi-->gammaX)B(X-->omega(phi))=[2.61+/-0.27(stat)+/-0.65(syst)]x10(-4).

Journal Article↗
PubMed1998

Miranda as a multidomain adapter linking apically localized Inscuteable and basally localized Staufen and Prospero during asymmetric cell division in Drosophila.

Neuroblasts in the developing Drosophila CNS asymmetrically localize the cell fate determinants Numb and Prospero as well as prospero RNA to the basal cortex during mitosis. The localization of Prospero requires the function of inscuteable and miranda, whereas prospero RNA localization requires inscuteable and staufen function. We demonstrate that Miranda contains multiple functional domains: an amino-terminal asymmetric localization domain, which interacts with Inscuteable, a central Numb interaction domain, and a more carboxy-terminal Prospero interaction domain. We also show that Miranda and Staufen have similar subcellular localization patterns and interact in vitro. Furthermore, miranda function is required for the asymmetric localization of Staufen. Miranda localization is disrupted by the microfilament disrupting agent latrunculin A. Our results suggest that Miranda directs the basal cortical localization of multiple molecules, including Staufen and prospero RNA, in mitotic neuroblasts in an actin-dependent manner.

Actins↗
PubMed1998

Notch inhibition of E47 supports the existence of a novel signaling pathway.

E47 is a widely expressed transcription factor that activates B-cell-specific immunoglobulin gene transcription and is required for early B-cell development. In an effort to identify processes that regulate E47, and potentially B-cell development, we found that activated Notch1 and Notch2 effectively inhibit E47 activity. Only the intact E47 protein was inhibited by Notch-fusion proteins containing isolated DNA binding and activation domains were unaffected-suggesting that Notch targets an atypical E47 cofactor. Although overexpression of the coactivator p300 partially reversed E47 inhibition, results of several assays indicated that p300/CBP is not a general target of Notch. Notch inhibition of E47 did not correlate with its ability to activate CBF1/RBP-Jkappa, the mammalian homolog of Suppressor of Hairless, a protein that associates physically with Notch and defines the only known Notch signaling pathway in drosophila. Importantly, E47 was inhibited independently of CBF1/RPB-Jkappa by Deltex, a second Notch-interacting protein. We provide evidence that Notch and Deltex may act on E47 by inhibiting signaling through Ras because (i) full E47 activity was found to be dependent on Ras and (ii) both Notch and Deltex inhibited GAL4-Jun, a hybrid transcription factor whose activity is dependent on signaling from Ras to SAPK/JNK.

3T3 Cells↗
PubMed1998

Synovial fibroblasts and the sphingomyelinase pathway: sphingomyelin turnover and ceramide generation are not signaling mechanisms for the actions of tumor necrosis factor-alpha.

The activation of sphingomyelinase and the generation of ceramide has been proposed to mediate tumor necrosis factor-alpha (TNF-alpha)-induced nuclear factor (NF)-kappaB activation through its second messenger ceramide. Ceramide may also be an important regulator of cell growth, senescence, and apoptosis. Aberrant cell proliferation and apoptosis have been implicated in the rampant fibroblast proliferation and pannus formation characteristic of rheumatoid arthritis. However, the role of TNF-alpha and the sphingomyelinase pathway in the process have not been determined. The objective of this study was to determine whether TNF-alpha activates the sphingomyelin pathway in human synovial fibroblasts (HSF) and the potential role of ceramide in HSF proliferation and apoptosis. Cultured human synovial fibroblasts were stimulated with exogenous TNF-alpha, sphingomyelinase, and ceramide. Apoptosis was assessed by cell morphology and annexin V labeling. NF-kappaB and stress kinase pathway activation were determined by immunoblotting techniques. Sphingomyelinase activation was determined by quantitation of sphingomyelin and ceramide radioactivity in [14C]serine-prelabeled HSF cells. The addition of TNF-alpha (50 ng/ml) to HSF did not elicit detectable sphingomyelinase activation. TNF-alpha was shown to activate NF-kappaB (p65 translocation and degradation of IkappaBalpha) and the stress kinase pathway (phosphorylation of ATF-2, p38, and c-jun). In contrast, exogenous ceramide had no effect on these signaling pathways nor did ceramide stimulate the generation of interleukin-6 or interleukin-8. High concentrations of ceramide (> or =25 micromol/L) were cytotoxic, whereas lower concentrations of ceramide inhibited cell cycle progression. Thus, although TNF-alpha stimulates the NF-kappaB and stress kinase pathways in HSF, these effects of TNF-alpha are not associated with sphingomyelinase turnover or induction of apoptosis.

Apoptosis↗
PubMed1997

Miranda is required for the asymmetric localization of Prospero during mitosis in Drosophila.

Asymmetric division of Drosophila neuroblasts, sensory organ precursor cells, and cells in the procephalic neurogenic region involves the segregation of Numb and Prospero proteins into one of the two daughter cells. We have isolated a novel gene, miranda, based on the ability of its gene product to interact with the Prospero asymmetric localization domain. miranda expression coincides spatially and temporally with asymmetric cell divisions and asymmetric localization of Prospero. Miranda protein is localized asymmetrically, along with Prospero, to the basal cell membrane during mitosis. Loss of miranda gene function abolishes asymmetric Prospero localization during mitosis. The asymmetric localization of Miranda protein requires inscuteable. Our results suggest that miranda functions downstream of inscuteable and works as an adapter that connects Prospero to the basal cell membrane during asymmetric cell division.

Amino Acid Sequence↗
PubMed1996

E47 activates the Ig-heavy chain and TdT loci in non-B cells.

The E2A proteins, E12 and E47, are basic helix-loop-helix (bHLH) proteins essential for the B-cell lineage. Initially identified as immunoglobulin enhancer-binding proteins, they have also been shown to activate immunoglobulin enhancer-based reporters in transient transfection assays. Here, we examine the relationship between E2A DNA binding activity and activation of the endogenous, chromosomal immunoglobulin heavy chain (IgH) locus. Using sterile I(mu) transcription as an indicator of IgH enhancer activity, we see a direct correlation between E2A DNA binding activity and I(mu) transcription in stable BxT hybrids. We also observe a 1000-fold stimulation of endogenous I(mu) transcription in fibroblasts that express high levels of E47 and less stimulation in cells that express E12. By contrast, none of the other IgH enhancer-binding proteins tested (E2-2, Pu.1, Oct-2, OCA-B, TFE3 and USF) were able to activate I(mu) transcription. E47 overexpression also resulted in transcriptional activation of the endogenous gene encoding TdT, indicating that it, too, is a target of E2A proteins early in the B-cell lineage. Our results indicate that E2A proteins have the distinctive property of activating silent, chromatin-embedded B-cell-specific genes, underscoring their crucial role in B-cell development.

3T3 Cells↗
PubMed1996

Phosphorylation of E47 as a potential determinant of B-cell-specific activity.

The E2A gene encodes two basic helix-loop-helix proteins designated E12 and E47. Although these proteins are widely expressed, they are required only for the B-lymphocyte lineage where DNA binding is mediated distinctively by E47 homodimers. By studying the properties of deltaE47, an N-terminal truncation of E47, we provide evidence that phosphorylation may contribute to B-cell-specific DNA binding by E47. Two serines N terminal to the deltaE47 basic helix-loop-helix domain were found to be phosphorylated in a variety of cell types but were hypophosphorylated in B cells. Phosphorylating these serines in vitro inhibited DNA binding by deltaE47 homodimers but not by deltaE47-containing heterodimers, such as deltaE47:MyoD. These results argue that hypophosphorylation may be a prerequisite for activity of E47 homodimers in B cells, suggesting the use of an inductive (nonstochastic) step in early B-cell development.

3T3 Cells↗
PubMed1996

Microvascular endothelial cells from E-selectin-deficient mice form tubes in vitro.

Studies of capillary morphogenesis and angiogenesis in vitro have suggested a role for E-selectin (CD62E) in the process of differentiation into tube-like structures. Recent studies by our group and others have demonstrated that mice lacking E-selectin because of germline inactivation of the E-selectin gene by gene targeting are viable and fertile, without apparent deficiencies in vascular development. Murine lung endothelial cells from wild-type and E-selectin-deficient animals were isolated using an activation-dependent sterile sorting method, and differentiation into tube-like structures on sparse fibronectin, Matrigel, and collagen gels was compared. Both types of murine lung endothelial cells spontaneously organized to form multicellular tubes and extensive anastomotic networks. There were no major differences in either the time course of development or the general appearance of the multicellular cords or tube-like structures formed by murine lung endothelial cells from wild-type or E-selectin-deficient mice, although different patterns were observed on different extracellular matrices. These studies, thus, demonstrate that E-selectin is not required for morphogenesis of 3-dimensional vascular structures in vitro.

Animals↗
PubMed1995

B-cell-specific DNA binding by an E47 homodimer.

B cells express a unique E-box-binding activity that contains basic helix-loop-helix (bHLH) proteins encoded by the E2A gene. E2A proteins play a central role in immunoglobulin gene transcription and are also required for the generation of the B-lymphocyte lineage. In muscle, E2A proteins bind DNA as heterodimers with muscle-specific bHLH partners, such as MyoD and myogenin, and these heterodimers are thought to be both necessary and sufficient for muscle determination in cultured cells. Our results indicate that in B cells, the bHLH partners for E2A proteins are not B-cell-restricted proteins, but are the E2A proteins themselves. UV cross-linking, gel purification, and the analysis of "forced heterodimers" indicate that BCF1 is primarily a homodimer of the E2A protein E47. Since E47 is widely expressed, our results argue for a difference in the inherent DNA-binding properties of the E47 protein in B cells and may help explain the restricted B-lineage defect observed in E2A-deficient mice.

Amino Acid Sequence↗
PubMed1995

Activation-dependent isolation and culture of murine pulmonary microvascular endothelium.

OBJECTIVE: Establish a reproducible method for the isolation and cultivation of murine pulmonary microvascular endothelium. To this end, we exploited the localized pattern of microvascular endothelial activation induced in vivo by inflammatory stimuli to isolate a subpopulation of endothelium for in vitro study. METHODS: Immunohistochemical analyses of the pulmonary vasculature of mice treated systemically with gram-negative bacterial endotoxin (LPS) demonstrated selective expression of VCAM-1 (CD106) in the endothelial lining of small collecting veins, venules, septal capillaries, and, infrequently, small arteries, which was not observed in control mice. Single cell suspensions prepared by enzymatic dissociation of peripheral lobular tissues dissected from the lungs of LPS-stimulated mice were incubated with a phycoerythrin-conjugated antimouse VCAM-1 monoclonal antibody (MK 1.91). Cells expressing this antigen were isolated by sterile fluorescence-activated cell sorting (FACS). Positive cell populations were collected and cultured for 1-2 weeks. When confluent, these primary cultures were further FACS enriched for endothelium, positively selecting for cells incorporating a fluorescent derivative of acetylated low density lipoprotein (Di-I-Ac-LDL). RESULTS: The resulting population of cells (mouse lung endothelial cells, MLEC) were uniformly positive for the endothelial markers von Willebrand factor, thrombomodulin, and Dil-Ac-LDL uptake. MLEC readily formed tube-like structures when cultured on Matrigel and spontaneously demonstrated a sprouting phenotype on fibronectin or collagen matrices. MLEC retained responsiveness to cytokines (IL-1 alpha, IL-1 beta, TNF alpha, IFN gamma) up to at least eight passages from primary culture and demonstrated upregulation of E-selectin (CD62E) and P-selectin (CD62P) mRNA as early as 2 hr after LPS stimulation. Characteristic temporal expression patterns of cell surface E-selectin (maximal at 4 hr and declining toward baseline by 24 hr), VCAM-1 (maximal at 6-8 hr and remaining elevated for 24-48 hr), and ICAM-1 (maximal at 6-8 hr and maintained at 24 hr) were observed when cultured MLEC were treated with recombinant murine TNF alpha or recombinant human (rh) IL-1 alpha or rhIL-1 beta. The rolling, adhesion, and transmigration of human polymorphonuclear leukocytes was markedly increased on cytokine-activated MLEC monolayers under defined flow conditions. CONCLUSION: The strategy of activation-dependent isolation allows for the reproducible selection of a specific subset of microvascular endothelial cells for in vitro study. This experimental approach should further facilitate study of the functional heterogeneity of endothelium and its pathophysiologic dysfunction.

Animals↗
PubMed1995

Flavonoids inhibit cytokine-induced endothelial cell adhesion protein gene expression.

Treatment of human endothelial cells with cytokines such as interleukin-1, tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma induces the expression of specific leukocyte adhesion molecules on the endothelial cell surface. Interfering with either leukocyte adhesion or adhesion protein upregulation is an important therapeutic target as evidenced by the potent anti-inflammatory actions of neutralizing antibodies to these ligands in various animal models and in patients. In the present study we report that cotreatment of human endothelial cells with certain hydroxyflavones and flavanols blocks cytokine-induced ICAM-1, VCAM-1, and E-selectin expression on human endothelial cells. One of the most potent flavones, apigenin, exhibited a dose- and time-dependent, reversible effect on adhesion protein expression as well as inhibiting adhesion protein upregulation at the transcriptional level. Apigenin also inhibited IL-1 alpha-induced prostaglandin synthesis and TNF-alpha-induced IL-6 and IL-8 production, suggesting that the hydroxyflavones may act as general inhibitors of cytokine-induced gene expression. Although apigenin did not inhibit TNF-alpha-induced nuclear translocation of NF-kappa B(p50(NFKB1)/p65(RelA)) we found this flavonoid did inhibit TNF-alpha induced beta-galactosidase activity in SW480 cells stably transfected with a beta-galactosidase reporter construct driven by four NF-kappa B elements, suggesting an action on NF-kappa B transcriptional activation. Adhesion of leukocytes to cytokine-treated endothelial cells was blocked in endothelial cells cotreated with apigenin. Finally, apigenin demonstrated potent anti-inflammatory activity in carrageenan induced rat paw edema and delayed type hypersensitivity in the mouse. We conclude that flavonoids offer important therapeutic potential for the treatment of a variety of inflammatory diseases involving an increase in leukocyte adhesion and trafficking.

Base Sequence↗
PubMed1994

Constitutively expressed Oct-2 prevents immunoglobulin gene silencing in myeloma x T cell hybrids.

Recent experiments involving disruption of the Oct-2 gene have shown that this largely B cell-restricted transcription factor is not required in the early stages of B cell development. However, B cells that lack Oct-2 may be blocked from differentiation past the surface immunoglobulin-positive stage. To identify a possible function for Oct-2 in the late stage immunoglobulin-secreting cell, we have used the method of somatic cell fusion. When the immunoglobulin-producing myeloma MPC11 is fused to a T lymphoma, Oct-2 production ceases, as does the expression of immunoglobulin, J chain, and several other B cell-specific gene products. In the present study, we show that by preventing the loss of Oct-2 in the hybrid cells, we can preserve expression of all other tested B cell-specific genes. These results establish a central role for Oct-2 in maintaining the genetic program of the immunoglobulin-secreting plasmacyte.

Animals↗
PubMed1993

Regulation of the expression of intercellular adhesion molecule 1 in cultured human endothelial cells derived from rheumatoid synovium.

OBJECTIVE: To examine the regulation of intercellular adhesion molecule 1 (ICAM-1) in human synovial microvascular endothelial cells (HSE) and human umbilical vein endothelial cells (HUVE) upon exposure to a variety of agents. METHODS: Cultured endothelial cells were treated with various cytokines alone and in combination. The expression of ICAM-1 was evaluated at several levels, including an investigation of messenger RNA (mRNA) and surface protein expression. RESULTS: Treatment of HSE with interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor alpha (TNF alpha) resulted in minimal increases in ICAM-1 expression, in contrast to findings with HUVE. Incubation of HUVE or HSE with IL-1 or TNF in combination with interferon-gamma (IFN gamma) greatly potentiated the increase in ICAM-1 surface expression. The synergistic effect of IFN gamma and TNF was confirmed by several methods, including a cell-based enzyme-linked immunosorbent assay, fluorescence-activated cell sorting, immunofluorescence staining, and determination of mRNA levels. IFN gamma also augmented the actions of several other agonists on HSE, i.e., IL-4, lipopolysaccharide, and TNF beta/lymphotoxin. Immunoprecipitation of TNF alpha + IFN gamma-stimulated, 125I-labeled HSE cells with anti-ICAM-1 revealed a single 90-kd band, similar in size to ICAM-1 from HUVE treated in an identical manner. Unexpectedly, IFN gamma alone was a potent stimulus for HSE ICAM-1 mRNA synthesis, but was relatively ineffective in HUVE. CONCLUSION: These studies indicate that IFN gamma plays a critical synergistic role in the regulation of ICAM-1 expression in human synovial endothelial cells.

Adolescent↗
PubMed1991

Lectin binding to gp60 decreases specific albumin binding and transport in pulmonary artery endothelial monolayers.

The effect of albumin binding to cultured bovine pulmonary artery endothelial cell (BPAEC) monolayers on the transendothelial flux of 125I-labelled bovine serum albumin (BSA) was examined to determine its possible role on albumin transcytosis. The transport of 125I-BSA tracer across BPAEC grown on gelatin- and fibronectin-coated filters (0.8 microns pore diam.) was affected by the presence of unlabelled BSA in the medium in that transendothelial 125I-BSA permeability decreased, reaching a 40% reduction at BSA concentrations equal to or greater than 5 mg/ml. BSA binding to BPAEC monolayers was saturated at concentration of 10 mg/ml with an apparent binding affinity of 6 x 10(-7) M. In contrast, gelatin added to the medium altered neither 125I-BSA binding nor transport. Several lectins were tested for their ability to inhibit 125I-BSA binding and transport. One lectin, Ricinus communis (RCA), reduced 125I-BSA binding by 70% and transport by 40%. Other lectins, Ulex europaeus, Triticum vulgare, and Glycine max decreased neither 125I-BSA binding nor transport. The reduction of 125I-BSA transport by RCA was not observed in the presence of saturating levels of BSA, indicating that RCA influenced only the albumin-dependent component of transport. RCA, but not other lectins, precipitated a 60 kDa plasmalemmal glycoprotein from cell lysates of surface radioiodinated BPAEC monolayers. This 60 kDa glycoprotein appears to be the equivalent of gp60 identified previously as an albumin binding glycoprotein in rat microvascular endothelium. In summary, approximately 40% of albumin transport across BPAEC monolayers is dependent on albumin binding. This component of albumin transport is inhibited by 80% by the binding of RCA to gp60. These results suggest that binding of albumin to gp60 on pulmonary artery endothelial cell membrane is a critical determinant of transendothelial albumin flux involving mechanisms such as plasmalemmal vesicular transcytosis.

Animals↗
PubMed1991

Repression of immunoglobulin enhancers by the helix-loop-helix protein Id: implications for B-lymphoid-cell development.

It has been proposed that the helix-loop-helix (HLH) protein Id serves as a general antagonist of cell differentiation by inhibiting bHLH (HLH with an adjacent stretch of basic amino acids) proteins specifically required for developmental programs (such as MyoD). We show here that ectopic expression of Id represses in vivo activity of the bHLH protein E2-5 (encoded by the E2A gene) and of both the immunoglobulin heavy-chain (IgH) and kappa-light-chain gene enhancers to which E2-5 binds. Id does not affect the activity of the bHLH-zip protein, TFE3, which also binds these enhancers. We examined a large panel of B-cell lines that represent different stages of lymphoid development and found only two that express Id mRNA. The cell lines Ba/F3 and LyD9 have been categorized previously as early B-lymphoid-cell progenitors. Unlike their more mature B-lymphoid-cell counterparts, Ba/F3 and LyD9 cells do not express I mu sterile transcripts, which are indicative of IgH enhancer activity. Moreover, Ba/F3-derived nuclear extracts lack E2-box-binding activity, indicating the absence of free bHLH proteins, and transfected Ba/F3 cells fail to support the activity of the IgH enhancer. Hence, expression of Id correlates inversely with bHLH protein activity and enhancer function in vivo. These results suggest that Id may play a role early in B-lymphoid-cell development to regulate transcription of the IgH locus.

3T3 Cells↗
PubMed1990

Lectin analysis of common glycoproteins detected on the surface of continuous microvascular endothelium in situ and in culture: identification of sialoglycoproteins.

For many years, molecular interactions with vascular endothelium have been studied in vitro on cultured endothelial cells. Yet, it is clear that the different environmental conditions in vivo vs. in vitro may cause phenotypic drift and altered expression of cell surface molecules. In this study, we identify several endothelial surface proteins of similar apparent molecular mass by radioiodination of cultured microvascular cells and by intravascular radioiodination of rat heart endothelium in situ. The radioiodinated surface polypeptides detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (followed by autoradiography) were subjected to lectin affinity chromatography in order to provide an additional screen for identifying common surface glycoproteins and a means for partial characterization of their glycans. With a battery of 18 lectins, seven major (gp140, gp120, gp100, gp85, gp75, gp60, gp47) and 6 minor (gp330, gp300, gp180, gp160, gp150, gp42) glycoproteins were identified on the cultured cells each with a different lectin binding profile. The lectin binding profiles of many endothelial glycoproteins in situ were similar to those of their counterparts in culture. A common set of seven major glycoproteins with the same apparent molecular masses was found in situ as well as in vitro. These common glycoproteins were characterized further using both sialidase digestion and sequential lectin affinity chromatography of cell lysates. Most of the glycoproteins appear to have both complex-type N-linked and O-linked glycans except for gp60 with only O-linked glycans, gp47 with only complex N-linked sugars, and gp42 with only simple N-linked sugars. A subset of sialoglycoproteins (gp140, gp120, gp100, gp60, gp47) was identified. One of them, gp120, is podocalyxin based on immunoprecipitation with specific antiserum and another one, gp60, is a recently identified albumin binding protein on the surface of cultured microvascular endothelial cells. This study shows that gp60 is indeed present on the surface of endothelium in situ and that it is a sialoglycoprotein with typical O-linked glycans. It is apparent that the continuous type of microvascular endothelium can indeed express in culture and in situ a common set of major glycoproteins.

Animals↗