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

The growth factors that drive the division and differentiation of stem cells during early human embryogenesis are unknown. The secretion of endorphins,progesterone (P(4)),human chorionic gonadotropin,17beta-estradiol,and gonadotropin-releasing hormone by trophoblasts that lie adjacent to the embryoblast in the blastocyst suggests that these pregnancy-associated factors may directly signal the growth and development of the embryoblast. To test this hypothesis,we treated embryoblast-derived human embryonic stem cells (hESCs) with ICI 174,864,a delta-opioid receptor antagonist,and RU-486 (mifepristone),a P(4) receptor competitive antagonist. Both antagonists potently inhibited the differentiation of hESC into embryoid bodies,an in vitro structure akin to the blastocyst containing all three germ layers. Furthermore,these agents prevented the differentiation of hESC aggregates into columnar neuroectodermal cells and their organization into neural tube-like rosettes as determined morphologically. Immunoblot analyses confirmed the obligatory role of these hormones; both antagonists inhibited nestin expression,an early marker of neural precursor cells normally detected during rosette formation. Conversely,addition of P(4) to hESC aggregates induced nestin expression and the formation of neuroectodermal rosettes. These results demonstrate that trophoblast-associated hormones induce blastulation and neurulation during early human embryogenesis. View Publication

Histone lysine methylation is an important epigenetic mark that regulates gene expression and chromatin organization. G9a and G9a-like protein (GLP) are euchromatin-associated methyltransferases that repress transcription by methylating histone H3 Lys9. BIX-01294 was originally identified as a G9a inhibitor during a chemical library screen of small molecules and has previously been used in the generation of induced pluripotent stem cells. Here we present the crystal structure of the catalytic SET domain of GLP in complex with BIX-01294 and S-adenosyl-L-homocysteine. The inhibitor is bound in the substrate peptide groove at the location where the histone H3 residues N-terminal to the target lysine lie in the previously solved structure of the complex with histone peptide. The inhibitor resembles the bound conformation of histone H3 Lys4 to Arg8,and is positioned in place by residues specific for G9a and GLP through specific interactions. View Publication

Stochastic processes and imprinting,along with genetic factors,lead to monoallelic or allele-biased gene expression. Stochastic monoallelic expression fine-tunes information processing in immune cells and the olfactory system,and imprinting plays an important role in development. Recent studies suggest that both stochastic events and imprinting may be more widespread than previously considered. We are interested in allele-biased gene expression occurring in the brain because parent-of-origin effects suggestive of imprinting appear to play a role in the transmission of schizophrenia (SZ) and autism spectrum disorders (ASD) in some families. In addition,allele-biased expression could help explain monozygotic (MZ) twin discordance and reduced penetrance. The ability to study allele-biased expression in human neurons has been transformed with the advent of induced pluripotent stem cell (iPSC) technology and next generation sequencing. Using transcriptome sequencing (RNA-Seq) we identified 801 genes in differentiating neurons that were expressed in an allele-biased manner. These included a number of putative SZ and ASD candidates,such as A2BP1 (RBFOX1),ERBB4,NLGN4X,NRG1,NRG3,NRXN1,and NLGN1. Overall,there was a modest enrichment for SZ and ASD candidate genes among those that showed evidence for allele-biased expression (chi-square,p = 0.02). In addition to helping explain MZ twin discordance and reduced penetrance,the capacity to group many candidate genes affecting a variety of molecular and cellular pathways under a common regulatory process - allele-biased expression - could have therapeutic implications. View Publication

The chaperone GRP78/Dna K is conserved throughout evolution down to prokaryotes. The GRP78 inhibitor OSU-03012 (AR-12) interacted with sildenafil (Viagra) or tadalafil (Cialis) to rapidly reduce GRP78 levels in eukaryotes and as a single agent reduce Dna K levels in prokaryotes. Similar data with the drug combination were obtained for: HSP70,HSP90,GRP94,GRP58,HSP27,HSP40 and HSP60. OSU-03012/sildenafil treatment killed brain cancer stem cells and decreased the expression of: NPC1 and TIM1; LAMP1; and NTCP1,receptors for Ebola/Marburg/Hepatitis A,Lassa fever,and Hepatitis B viruses,respectively. Pre-treatment with OSU-03012/sildenafil reduced expression of the coxsakie and adenovirus receptor in parallel with it also reducing the ability of a serotype 5 adenovirus or coxsakie virus B4 to infect and to reproduce. Similar data were obtained using Chikungunya,Mumps,Measles,Rubella,RSV,CMV,and Influenza viruses. OSU-03012 as a single agent at clinically relevant concentrations killed laboratory generated antibiotic resistant E. coli and clinical isolate multi-drug resistant N. gonorrhoeae and MRSE which was in bacteria associated with reduced Dna K and Rec A expression. The PDE5 inhibitors sildenafil or tadalafil enhanced OSU-03012 killing in N. gonorrhoeae and MRSE and low marginally toxic doses of OSU-03012 could restore bacterial sensitivity in N. gonorrhoeae to multiple antibiotics. Thus,Dna K and bacterial phosphodiesterases are novel antibiotic targets,and inhibition of GRP78 is of therapeutic utility for cancer and also for bacterial and viral infections. View Publication

Aldehyde dehydrogenase (ALDH) activity has been implicated in multiple biological and biochemical pathways and has been used to identify potential cancer stem cells. Our main hypothesis is that ALDH activity may be a lung cancer stem cell marker. Using flow cytometry,we sorted cells with bright (ALDH(br)) and dim (ALDH(lo)) ALDH activity found in H522 lung cancer cell line. We used in vitro proliferation and colony assays as well as a xenograft animal model to test our hypothesis. Cytogenetic analysis demonstrated that the ALDH(br) cells are indeed a different clone,but when left in normal culture conditions will give rise to ALDH(lo) cells. Furthermore,the ALDH(br) cells grow slower,have low clonal efficiency,and give rise to morphologically distinct colonies. The ability to form primary xenografts in NOD/SCID mice by ALDH(br) and ALDH(lo) cells was tested by injecting single cell suspension under the skin in each flank of same animal. Tumor size was calculated weekly. ALDH1A1 and ALDH3A1 immunohistochemistry (IHC) was performed on excised tumors. These tumors were also used to re-establish cell suspension,measure ALDH activity,and re-injection for secondary and tertiary transplants. The results indicate that both cell types can form tumors but the ones from ALDH(br) cells grew much slower in primary recipient mice. Histologically,there was no significant difference in the expression of ALDH in primary tumors originating from ALDH(br) or ALDH(lo) cells. Secondary and tertiary xenografts originating from ALDH(br) grew faster and bigger than those formed by ALDH(lo) cells. In conclusion,ALDH(br) cells may have some of the traditional features of stem cells in terms of being mostly dormant and slow to divide,but require support of other cells (ALDH(lo)) to sustain tumor growth. These observations and the known role of ALDH in drug resistance may have significant therapeutic implications in the treatment of lung cancer. View Publication

The purpose of this study is to review the development of BRAF inhibitors,with emphasis on the trials conducted with dabrafenib (GSK2118436) and the evolving role of dabrafenib in treatment for melanoma patients. Fifty percent of cutaneous melanomas have mutations in BRAF,resulting in elevated activity of the mitogen-activated protein kinase signaling pathway. Dabrafenib inhibits the mutant BRAF (BRAF(mut)) protein in melanomas with BRAF(V600E) and BRAF(V600K) genotypes. BRAF(V600E) metastatic melanoma patients who receive dabrafenib treatment exhibit high clinical response rates and compared with dacarbazine chemotherapy,progression-free survival. Efficacy has also been demonstrated in BRAF(V600K) patients and in those with brain metastases. Dabrafenib has a generally mild and manageable toxicity profile. Cutaneous squamous cell carcinomas and pyrexia are the most significant adverse effects. Dabrafenib appears similar to vemurafenib with regard to efficacy but it is associated with less toxicity. It is expected that new combinations of targeted drugs,such as the combination of dabrafenib and trametinib (GSK1120212,a MEK inhibitor),will provide higher response rates and more durable clinical benefit than dabrafenib monotherapy. View Publication

Bacterial lipopeptides (bLPs) are increasingly used as adjuvants to activate cell-mediated immune responses to foreign Ags. To explore mechanisms whereby bLPs adjuvant T cell responses,we stimulated human PBMCs with bLPs. We found that bLPs stimulate T cells to proliferate and produce IFN-gamma in an accessory cell-dependent manner and in the absence of exogenous protein Ags. The ability of bLPs to stimulate T cell proliferation was Toll-like receptor 2 dependent and required IL-12,interaction with costimulatory molecules,and MHC proteins. Our data suggest that bLPs adjuvant adaptive Th1 responses by enhancing Ag presentation of endogenous peptides. View Publication

Bone morphogenetic protein (BMP) signals coordinate developmental patterning and have essential physiological roles in mature organisms. Here we describe the first known small-molecule inhibitor of BMP signaling-dorsomorphin,which we identified in a screen for compounds that perturb dorsoventral axis formation in zebrafish. We found that dorsomorphin selectively inhibits the BMP type I receptors ALK2,ALK3 and ALK6 and thus blocks BMP-mediated SMAD1/5/8 phosphorylation,target gene transcription and osteogenic differentiation. Using dorsomorphin,we examined the role of BMP signaling in iron homeostasis. In vitro,dorsomorphin inhibited BMP-,hemojuvelin- and interleukin 6-stimulated expression of the systemic iron regulator hepcidin,which suggests that BMP receptors regulate hepcidin induction by all of these stimuli. In vivo,systemic challenge with iron rapidly induced SMAD1/5/8 phosphorylation and hepcidin expression in the liver,whereas treatment with dorsomorphin blocked SMAD1/5/8 phosphorylation,normalized hepcidin expression and increased serum iron levels. These findings suggest an essential physiological role for hepatic BMP signaling in iron-hepcidin homeostasis. View Publication

Small-molecule inhibitors targeting growth factor receptors have failed to show efficacy for brain cancers,potentially due to their inability to achieve sufficient drug levels in the CNS. Targeting non-oncogene tumor co-dependencies provides an alternative approach,particularly if drugs with high brain penetration can be identified. Here we demonstrate that the highly lethal brain cancer glioblastoma (GBM) is remarkably dependent on cholesterol for survival,rendering these tumors sensitive to Liver X receptor (LXR) agonist-dependent cell death. We show that LXR-623,a clinically viable,highly brain-penetrant LXRα-partial/LXRβ-full agonist selectively kills GBM cells in an LXRβ- and cholesterol-dependent fashion,causing tumor regression and prolonged survival in mouse models. Thus,a metabolic co-dependency provides a pharmacological means to kill growth factor-activated cancers in the CNS. View Publication