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

Sphingolipids,particularly gangliosides,are enriched in neuronal membranes where they have been implicated as mediators of various regulatory events. We recently provided evidence that sphingolipid synthesis is necessary to maintain neuronal growth by demonstrating that in hippocampal neurons,inhibition of ceramide synthesis by Fumonisin B1 (FB1) disrupted axonal outgrowth (Harel,R. and Futerman,A. H. (1993) J. Biol. Chem. 268,14476-14481). We now analyze further the relationship between neuronal growth and sphingolipid metabolism by examining the effect of an inhibitor of glucosylceramide synthesis,D-threo-1-phenyl-2-decanoylamino-3-morpholino-1- propanol (PDMP) and by examining the effects of both FB1 and PDMP at various stages of neuronal development. No effects of FB1 or PDMP were observed during the first 2 days in culture,but by day 3 axonal morphology was significantly altered,irrespective of the time of addition of the inhibitors to the cultures. Cells incubated with FB1 or PDMP had a shorter axon plexus and less axonal branches. FB1 appeared to cause a retraction of axonal branches between days 2 and 3,although long term incubation had no apparent effect on neuronal morphology or on the segregation of axonal or dendritic proteins. In contrast,incubation of neurons with conduritol B-epoxide,an inhibitor of glucosylceramide degradation,caused an increase in the number of axonal branches and a corresponding increase in the length of the axon plexus. A direct correlation was observed between the number of axonal branch points per cell and the extent of inhibition of either sphingolipid synthesis or degradation. These results suggest that sphingolipids play an important role in the formation or stabilization of axonal branches. View Publication

An in vivo transplantation system has been used to evaluate the developmental capacities of specific mouse mammary epithelial cell populations. Specifically,mouse mammary epithelial cells with distinctly limited developmental potentials have been identified using this procedure. Two distinct epithelial cell progenitors have been identified by experiments designed to determine whether basal lobular and ductal phenotypes could develop independently under conditions imposed by a limiting dilution. The prediction that these separate epithelial progenitors must exist was based upon the results from transplantation experiments carried out in epithelium-divested mammary fat pads of syngeneic mice with mammary epithelium from two different transgenic mouse models. The results presented here demonstrate the following points: 1) lobular,i.e. secretory,progenitor cells are present as distinct entities among the mammary epithelial cells found in immature virgin female mice; 2) similarly,ductal epithelial progenitors are present within the same population; 3) lobular progenitors are present in greater numbers,although both cell populations are extremely small; 4) as expected,some inocula produce outgrowths with simultaneous development of both lobular and ductal phenotypes--it is not known whether this indicates cooperative interaction between the two epithelial progenitors or signals the presence of a third progenitor type capable of producing both ductular and lobular committed daughters; 5) these findings have important consequences in the design of experiments aimed at testing the effects of known and putative mammary oncogenes and tumor suppressor genes,using techniques which include cellular transformation in vitro followed by in vivo cultivation and evaluation. View Publication

BACKGROUND The epidermal growth factor receptor (EGFR) and Notch signaling pathways have been implicated in self-renewal of normal breast stem cells. We investigated the involvement of these signaling pathways in ductal carcinoma in situ (DCIS) of the breast. METHODS Samples of normal breast tissue (n = 15),pure DCIS tissue of varying grades (n = 35),and DCIS tissue surrounding an invasive cancer (n = 7) were used for nonadherent (i.e.,mammosphere) culture. Mammosphere cultures were treated at day 0 with gefitinib (an EGFR inhibitor),DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester) (a gamma-secretase inhibitor),or Notch 4-neutralizing antibody. Mammosphere-forming efficiency (MFE) was calculated by dividing the number of mammospheres of 60 microm or more formed by the number of single cells seeded and is expressed as a percentage. The Notch 1 intracellular domain (NICD) was detected immunohistochemically in paraffin-embedded DCIS tissue from 50 patients with at least 60 months of follow-up. All statistical tests were two-sided. RESULTS DCIS had a greater MFE than normal breast tissue (1.5% versus 0.5%,difference = 1%,95% confidence interval [CI] = 0.62% to 1.25%,Ptextless.001). High-grade DCIS had a greater MFE than low-grade DCIS (1.6% versus 1.09%,difference = 0.51%,95% CI = 0.07% to 0.94%,P = .01). The MFE of high-grade DCIS treated with gefitinib in the absence of exogenous EGF was lower than that of high-grade DCIS treated with mammosphere medium lacking gefitinib and exogenous EGF (0.56% versus 1.36%,difference 0.8%,95% CI = 0.33% to 1.4%,P = .004). Increased Notch signaling as detected by NICD staining was associated with recurrence at 5 years (P = .012). DCIS MFE was reduced when Notch signaling was inhibited using either DAPT (0.89% versus 0.51%,difference = 0.38%,95% CI = 0.2% to 0.6%,Ptextless.001) or a Notch 4-neutralizing antibody (0.97% versus 0.2%,difference = 0.77%,95% CI = 0.52% to 1.0%,Ptextless.001). CONCLUSION We describe a novel primary culture technique for DCIS. Inhibition of the EGFR or Notch signaling pathways reduced DCIS MFE. View Publication

BACKGROUND: The mitomycins are natural products that contain a variety of functional groups,including aminobenzoquinone- and aziridine-ring systems. Mitomycin C (MC) was the first recognized bioreductive alkylating agent,and has been widely used clinically for antitumor therapy. Precursor-feeding studies showed that MC is derived from 3-amino-5-hydroxybenzoic acid (AHBA),D-glucosamine,L-methionine and carbamoyl phosphate. A genetically linked AHBA biosynthetic gene and MC resistance genes were identified previously in the MC producer Streptomyces lavendulae NRRL 2564. We set out to identify other genes involved in MC biosynthesis. RESULTS: A cluster of 47 genes spanning 55 kilobases of S. lavendulae DNA governs MC biosynthesis. Fourteen of 22 disruption mutants did not express or overexpressed MC. Seven gene products probably assemble the AHBA intermediate through a variant of the shikimate pathway. The gene encoding the first presumed enzyme in AHBA biosynthesis is not,however,linked within the MC cluster. Candidate genes for mitosane nucleus formation and functionalization were identified. A putative MC translocase was identified that comprises a novel drug-binding and export system,which confers cellular self-protection on S. lavendulae. Two regulatory genes were also identified. CONCLUSIONS: The overall architecture of the MC biosynthetic gene cluster in S. lavendulae has been determined. Targeted manipulation of a putative MC pathway regulator led to a substantial increase in drug production. The cloned genes should help elucidate the molecular basis for creation of the mitosane ring system,as well efforts to engineer the biosynthesis of novel natural products. View Publication

Molluscum contagiosum poxvirus (MCV) type 1 and type 2 encode two chemokine-like proteins MC148R1 and MC148R2. It is believed that MC148R proteins function by blocking the inflammatory response. However,the mechanism of the proposed biological activities of MC148R proteins and the role of the additional C-terminal cysteines that do not exist in other chemokines are not understood. Here,we demonstrated in two different assay systems that His-tagged MC148R1 displaces the interaction between CXCL12α and CXCR4. The N-terminal cysteines but not the additional C-terminal cysteines modulate this displacement. His-tagged MC148R1 blocked both CXCL12α-mediated and MIP-1α-mediated chemotaxis. In contrast,MC148R2 blocked MIP-1α-mediated but not CXCL12α-mediated chemotaxis. Immunoprecipitation by antibodies to MC148R1 or CXCL12α followed by immunoblotting and detection by antibodies to the other protein demonstrated physical interaction of His-tagged CXCL12α and His-tagged MC148R1. Interaction with chemokines might mask the receptor interaction site resulting in decreased binding and impairment of the biological activities. View Publication

As a critical tumor suppressor,p53 is inactivated in human cancer cells by somatic gene mutation or disruption of pathways required for its activation. Therefore,it is critical to elucidate the mechanism underlying p53 activation after genotoxic and cellular stresses. Accumulating evidence has indicated the importance of posttranslational modifications such as acetylation in regulating p53 stability and activity. However,the physiological roles of the eight identified acetylation events in regulating p53 responses remain to be fully understood. By employing homologous recombination,we introduced various combinations of missense mutations (lysine to arginine) into eight acetylation sites of the endogenous p53 gene in human embryonic stem cells (hESCs). By determining the p53 responses to DNA damage in the p53 knock-in mutant hESCs and their derivatives,we demonstrate physiological importance of the acetylation events within the core domain (K120 and K164) and at the C-terminus (K370/372/373/381/382/386) in regulating human p53 responses to DNA damage. View Publication

UNLABELLED: Human bocavirus 1 (HBoV1) is a single-stranded DNA parvovirus that causes lower respiratory tract infections in young children worldwide. In this study,we identified novel splice acceptor and donor sites,namely,A1' and D1',in the large nonstructural protein (NS1)-encoding region of the HBoV1 precursor mRNA. The novel small NS proteins (NS2,NS3,and NS4) were confirmed to be expressed following transfection of an HBoV1 infectious proviral plasmid and viral infection of polarized human airway epithelium cultured at an air-liquid interface (HAE-ALI). We constructed mutant pIHBoV1 infectious plasmids which harbor silent mutations (sm) smA1' and smD1' at the A1' and D1' splice sites,respectively. The mutant infectious plasmids maintained production of HBoV1 progeny virions at levels less than five times lower than that of the wild-type plasmid. Importantly,the smA1' mutant virus that does not express NS3 and NS4 replicated in HAE-ALI as effectively as the wild-type virus; however,the smD1' mutant virus that does not express NS2 and NS4 underwent an abortive infection in HAE-ALI. Thus,our study identified three novel NS proteins,NS2,NS3,and NS4,and suggests an important function of the NS2 protein in HBoV1 replication in HAE-ALI. IMPORTANCE: Human bocavirus 1 infection causes respiratory diseases,including acute wheezing in infants,of which life-threatening cases have been reported. In vitro,human bocavirus 1 infects polarized human bronchial airway epithelium cultured at an air-liquid interface that mimics the environment of human lower respiratory airways. Viral nonstructural proteins are often important for virus replication and pathogenesis in infected tissues or cells. In this report,we identified three new nonstructural proteins of human bocavirus 1 that are expressed during infection of polarized human bronchial airway epithelium. Among them,we proved that one nonstructural protein is critical to the replication of the virus in polarized human bronchial airway epithelium. The creation of nonreplicating infectious HBoV1 mutants may have particular utility in vaccine development for this virus. View Publication

Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function,developmental pathways,and disease mechanisms. Here,we develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi,in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain,can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors,cardiomyocytes,and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn),CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types,dissect developmental pathways,and model disease. View Publication

Identification and quantification of the characteristics of stem cell preparations is critical for understanding stem cell biology and for the development and manufacturing of stem cell based therapies. We have developed image analysis and visualization software that allows effective use of time-lapse microscopy to provide spatial and dynamic information from large numbers of human embryonic stem cell colonies. To achieve statistically relevant sampling,we examined textgreater 680 colonies from 3 different preparations of cells over 5 days each,generating a total experimental dataset of 0.9 terabyte (TB). The 0.5 Giga-pixel images at each time point were represented by multi-resolution pyramids and visualized using the Deep Zoom Javascript library extended to support viewing Giga-pixel images over time and extracting data on individual colonies. We present a methodology that enables quantification of variations in nominally-identical preparations and between colonies,correlation of colony characteristics with Oct4 expression,and identification of rare events. View Publication