搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

Protein modification by small ubiquitin-related modifier proteins (SUMOs) controls diverse cellular functions. Dysregulation of SUMOylation or deSUMOylation processes has been implicated in the development of cancer and neurodegenerative diseases. However,no small-molecule inhibiting protein SUMOylation has been reported so far. Here,we report inhibition of SUMOylation by ginkgolic acid and its analog,anacardic acid. Ginkgolic acid and anacardic acid inhibit protein SUMOylation both in vitro and in vivo without affecting in vivo ubiquitination. Binding assays with a fluorescently labeled probe showed that ginkgolic acid directly binds E1 and inhibits the formation of the E1-SUMO intermediate. These studies will provide not only a useful tool for investigating the roles of SUMO conjugations in a variety of pathways in cells,but also a basis for the development of drugs targeted against diseases involving aberrant SUMOylation. View Publication

The nonreceptor tyrosine kinase c-Src is frequently over-expressed or hyperactivated in various human cancers and contributes to cancer progression in cooperation with up-regulated growth factor receptors. However,Src-selective anticancer drugs are still in clinical trials. To identify more effective inhibitors of c-Src-mediated cancer progression,we developed a new screening platform using Csk-deficient cells that can be transformed by c-Src. We found that purvalanol A,developed as a CDK inhibitor,potently suppressed the anchorage-independent growth of c-Src-transformed cells,indicating that the activation of CDKs contributes to the c-Src transformation. We also found that purvalanol A suppressed the c-Src activity as effectively as the Src-selective inhibitor PP2,and that it reverted the transformed morphology to a nearly normal shape with less cytotoxicity than PP2. Purvalanol A induced a strong G2-M arrest,whereas PP2 weakly acted on the G1-S transition. Furthermore,when compared with PP2,purvalanol A more effectively suppressed the growth of human colon cancer HT29 and SW480 cells,in which Src family kinases and CDKs are activated. These findings demonstrate that the coordinated inhibition of cell cycle progression and tyrosine kinase signaling by the multi-selective purvalanol A is effective in suppressing cancer progression associated with c-Src up-regulation. View Publication

New strategies that augment T cell responses are required to broaden the therapeutic arsenal against cancer. CD96,TIGIT,and CD226 are receptors that bind to a communal ligand,CD155,and transduce either inhibitory or activating signals. The function of TIGIT and CD226 is established,whereas the role of CD96 remains ambiguous. Using a panel of engineered antibodies,we discovered that the T cell stimulatory activity of anti-CD96 antibodies requires antibody cross-linking and is potentiated by Fc$\gamma$ receptors. Thus,soluble Fc silent" anti-CD96 antibodies failed to stimulate human T cells whereas the same antibodies were stimulatory after coating onto plastic surfaces. Remarkably the activity of soluble anti-CD96 antibodies was reinstated by engineering the Fc domain to a human IgG1 isotype and it was dependent on antibody trans-cross-linking by Fc$\gamma$RI. In contrast neither human IgG2 nor variants with increased Fc$\gamma$ receptor IIB binding possessed stimulatory activity. Anti-CD96 antibodies acted directly on T cells and augmented gene expression networks associated with T cell activation leading to proliferation cytokine secretion and resistance to Treg suppression. Furthermore CD96 expression correlated with survival in HPV+ head and neck squamous cell carcinoma and its cross-linking activated tumor-infiltrating T cells thus highlighting the potential of anti-CD96 antibodies in cancer immunotherapy." View Publication

The receptor for advanced glycation end products (RAGE) is produced either as a transmembrane or soluble form (sRAGE). Substantial evidence supports a role for RAGE and its ligands in disease. sRAGE is reported to be a competitive,negative regulator of membrane RAGE activation,inhibiting ligand binding. However,some reports indicate that sRAGE is associated with inflammatory disease. We sought to define the biological function of sRAGE on inflammatory cell recruitment,survival,and differentiation in vivo and in vitro. To test the in vivo impact of sRAGE,the recombinant protein was intratracheally administered to mice,which demonstrated monocyte- and neutrophil-mediated lung inflammation. We also observed that sRAGE induced human monocyte and neutrophil migration in vitro. Human monocytes treated with sRAGE produced proinflammatory cytokines and chemokines. Our data demonstrated that sRAGE directly bound human monocytes and monocyte-derived macrophages. Binding of sRAGE to monocytes promoted their survival and differentiation to macrophages. Furthermore,sRAGE binding to cells increased during maturation,which was similar in freshly isolated mouse monocytes compared with mature tissue macrophages. Because sRAGE activated cell survival and differentiation,we examined intracellular pathways that were activated by sRAGE. In primary human monocytes and macrophages,sRAGE treatment activated Akt,Erk,and NF-kappaB,and their activation appeared to be critical for cell survival and differentiation. Our data suggest a novel role for sRAGE in monocyte- and neutrophil-mediated inflammation and mononuclear phagocyte survival and differentiation. View Publication

Tankyrase,a protein with homology to ankyrins and to the catalytic domain of poly(adenosine diphosphate-ribose) polymerase (PARP),was identified and localized to human telomeres. Tankyrase binds to the telomeric protein TRF1 (telomeric repeat binding factor-1),a negative regulator of telomere length maintenance. Like ankyrins,tankyrase contains 24 ankyrin repeats in a domain responsible for its interaction with TRF1. Recombinant tankyrase was found to have PARP activity in vitro,with both TRF1 and tankyrase functioning as acceptors for adenosine diphosphate (ADP)-ribosylation. ADP-ribosylation of TRF1 diminished its ability to bind to telomeric DNA in vitro,suggesting that telomere function in human cells is regulated by poly(ADP-ribosyl)ation. View Publication

IL-33,a new member of the IL-1 family,has been described as an important inducer of Th2 cytokines and mediator of inflammatory responses. In this study,we demonstrate that murine basophils sorted directly from the bone marrow,without prior exposure to IL-3 or Fc(epsilon)R cross-linking,respond to IL-33 alone by producing substantial amounts of histamine,IL-4,and IL-6. These cells express ST2 constitutively and generate a cytokine profile that differs from their IL-3-induced counterpart by a preferential production of IL-6. In vivo,IL-33 promotes basophil expansion in the bone marrow (BM) through an indirect mechanism of action depending on signaling through the beta(c) chain shared by receptors for IL-3,GM-CSF,and IL-5. IL-3 can still signal through its specific beta(IL-3) chain in these mutant mice,which implies that it is not the unique growth-promoting mediator in this setup,but requires IL-5 and/or GMCSF. Our results support a major role of the latter growth factor,which is readily generated by total BM cells as well as sorted basophils in response to IL-33 along with low amounts of IL-3. Furthermore,GM-CSF amplifies IL-3-induced differentiation of basophils from BM cells,whereas IL-5 that is also generated in vivo,affects neither their functions nor their growth in vitro or in vivo. In conclusion,our data provide the first evidence that IL-33 not only activates unprimed basophils directly,but also promotes their expansion in vivo through induction of GM-CSF and IL-3. View Publication

Human monoclonal antibodies against RSV have high potential for use as prophylaxis or therapeutic molecules,and they also can be used to define the structure of protective epitopes for rational vaccine design. In the past,however,isolation of human monoclonal antibodies was difficult and inefficient. Here,we describe contemporary methods for activation and proliferation of primary human memory B cells followed by cytofusion to non-secreting myeloma cells by dielectrophoresis to generate human hybridomas secreting RSV-specific monoclonal antibodies. We also provide experimental methods for screening human B cell lines to obtain RSV-specific lines,especially lines secreting neutralizing antibodies. View Publication

In the present investigation,we studied the modulating effects of several tea catechins and bioflavonoids on DNA methylation catalyzed by prokaryotic SssI DNA methyltransferase (DNMT) and human DNMT1. We found that each of the tea polyphenols [catechin,epicatechin,and (-)-epigallocatechin-3-O-gallate (EGCG)] and bioflavonoids (quercetin,fisetin,and myricetin) inhibited SssI DNMT- and DNMT1-mediated DNA methylation in a concentration-dependent manner. The IC(50) values for catechin,epicatechin,and various flavonoids ranged from 1.0 to 8.4 microM,but EGCG was a more potent inhibitor,with IC(50) values ranging from 0.21 to 0.47 microM. When epicatechin was used as a model inhibitor,kinetic analyses showed that this catechol-containing dietary polyphenol inhibited enzymatic DNA methylation in vitro largely by increasing the formation of S-adenosyl-L-homocysteine (a potent noncompetitive inhibitor of DNMTs) during the catechol-O-methyltransferase-mediated O-methylation of this dietary catechol. In comparison,the strong inhibitory effect of EGCG on DNMT-mediated DNA methylation was independent of its own methylation and was largely due to its direct inhibition of the DNMTs. This inhibition is strongly enhanced by Mg(2+). Computational modeling studies showed that the gallic acid moiety of EGCG plays a crucial role in its high-affinity,direct inhibitory interaction with the catalytic site of the human DNMT1,and its binding with the enzyme is stabilized by Mg(2+). The modeling data on the precise molecular mode of EGCG's inhibitory interaction with human DNMT1 agrees perfectly with our experimental finding. View Publication

Histone lysine methylation has important roles in the organization of chromatin domains and the regulation of gene expression. To analyze its function and modulate its activity,we screened for specific inhibitors against histone lysine methyltransferases (HMTases) using recombinant G9a as the target enzyme. From a chemical library comprising 125,000 preselected compounds,seven hits were identified. Of those,one inhibitor,BIX-01294 (diazepin-quinazolin-amine derivative),does not compete with the cofactor S-adenosyl-methionine,and selectively impairs the G9a HMTase and the generation of H3K9me2 in vitro. In cellular assays,transient incubation of several cell lines with BIX-01294 lowers bulk H3K9me2 levels that are restored upon removal of the inhibitor. Importantly,chromatin immunoprecipitation at several G9a target genes demonstrates reversible reduction of promoter-proximal H3K9me2 in inhibitor-treated mouse ES cells and fibroblasts. Our data identify a biologically active HMTase inhibitor that allows for the transient modulation of H3K9me2 marks in mammalian chromatin. View Publication

We have previously reported the genetic correction of Huntington's disease (HD) patient-derived induced pluripotent stem cells using traditional homologous recombination (HR) approaches. To extend this work,we have adopted a CRISPR-based genome editing approach to improve the efficiency of recombination in order to generate allelic isogenic HD models in human cells. Incorporation of a rapid antibody-based screening approach to measure recombination provides a powerful method to determine relative efficiency of genome editing for modeling polyglutamine diseases or understanding factors that modulate CRISPR/Cas9 HR. View Publication

Human pluripotent stem (hPS) cells are a potential source of cells for medical therapy and an ideal system to study fate decisions in early development. However,hPS cells cultured in vitro exhibit a high degree of heterogeneity,presenting an obstacle to clinical translation. hPS cells grow in spatially patterned colony structures,necessitating quantitative single-cell image analysis. We offer a tool for analyzing the spatial population context of hPS cells that integrates automated fluorescent microscopy with an analysis pipeline. It enables high-throughput detection of colonies at low resolution,with single-cellular and sub-cellular analysis at high resolutions,generating seamless in situ maps of single-cellular data organized by colony. We demonstrate the tool's utility by analyzing inter- and intra-colony heterogeneity of hPS cell cycle regulation and pluripotency marker expression. We measured the heterogeneity within individual colonies by analyzing cell cycle as a function of distance. Cells loosely associated with the outside of the colony are more likely to be in G1,reflecting a less pluripotent state,while cells within the first pluripotent layer are more likely to be in G2,possibly reflecting a G2/M block. Our multi-scale analysis tool groups colony regions into density classes,and cells belonging to those classes have distinct distributions of pluripotency markers and respond differently to DNA damage induction. Lastly,we demonstrate that our pipeline can robustly handle high-content,high-resolution single molecular mRNA FISH data by using novel image processing techniques. Overall,the imaging informatics pipeline presented offers a novel approach to the analysis of hPS cells that includes not only single cell features but also colony wide,and more generally,multi-scale spatial configuration. View Publication

Pulmonary metastasis of breast cancer requires recruitment and expansion of T-regulatory cells (Treg) that promote escape from host protective immune cells. However,it remains unclear precisely how tumors recruit Tregs to support metastatic growth. Here we report the mechanistic involvement of a unique and previously undescribed subset of regulatory B cells. These cells,designated tumor-evoked Bregs (tBreg),phenotypically resemble activated but poorly proliferative mature B2 cells (CD19(+) CD25(High) CD69(High)) that express constitutively active Stat3 and B7-H1(High) CD81(High) CD86(High) CD62L(Low) IgM(Int). Our studies with the mouse 4T1 model of breast cancer indicate that the primary role of tBregs in lung metastases is to induce TGF-$\beta$-dependent conversion of FoxP3(+) Tregs from resting CD4(+) T cells. In the absence of tBregs,4T1 tumors cannot metastasize into the lungs efficiently due to poor Treg conversion. Our findings have important clinical implications,as they suggest that tBregs must be controlled to interrupt the initiation of a key cancer-induced immunosuppressive event that is critical to support cancer metastasis. View Publication