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

Culture materials and grains contaminated with certain isolates of Fusarium moniliforme cause equine leucoencephalomalacia,porcine pulmonary edema syndrome,and liver cancer in rats. The causative agents are thought to be a family of compounds called fumonisins,which bear considerable structural similarity to the long-chain (sphingoid) base backbones of sphingolipids. Incubation of rat hepatocytes with fumonisins inhibited incorporation of [14C]serine into the sphingosine moiety of cellular sphingolipids with an IC50 of 0.1 microM for fumonisin B1. In contrast,fumonisin B1 increased the amount of the biosynthetic intermediate sphinganine,which suggests that fumonisins inhibit the conversion of [14C]sphinganine to N-acyl-[14C]sphinganines,a step that is thought to precede introduction of the 4,5-trans double bond of sphingosine (Merrill,A.H.,Jr. and Wang,E. (1986) J. Biol. Chem. 261,3764-3769). In agreement with this mechanism,fumonisin B1 inhibited the activity of sphingosine N-acyltransferase (ceramide synthase) in rat liver microsomes with 50% inhibition at approximately 0.1 microM and reduced the conversion of [3H]sphingosine to [3H]ceramide by intact hepatocytes. As far as we are aware,this is the first discovery of a naturally occurring inhibitor of this step of sphingolipid metabolism. These findings suggest that disruption of the de novo pathway of sphingolipid biosynthesis may be a critical event in the diseases that have been associated with consumption of fumonisins. View Publication

Serotonergic axons from the raphe nuclei in the brainstem project to every region of the brain,where they make connections through their extensive terminal arborizations. This serotonergic innervation contributes to various normal behaviors and psychiatric disorders. The protocadherin-alpha (Pcdha) family of clustered protocadherins consists of 14 cadherin-related molecules generated from a single gene cluster. We found that the Pcdhas were strongly expressed in the serotonergic neurons. To elucidate their roles,we examined serotonergic fibers in a mouse mutant (Pcdha(Delta CR/Delta CR)) lacking the Pcdha cytoplasmic region-encoding exons,which are common to the gene cluster. In the first week after birth,the distribution pattern of serotonergic fibers in Pcdha(Delta CR/Delta CR) mice was similar to wild-type,but by 3 weeks of age,when the serotonergic axonal termini complete their arborizations,the distribution of the projections was abnormal. In some target regions,notably the globus pallidus and substantia nigra,the normally even distribution of serotonin axonal terminals was,in the mutants,dense at the periphery of each region,but sparse in the center. In the stratum lacunosum-molecular of the hippocampus,the mutants showed denser serotonergic innervation than in wild-type,and in the dentate gyrus of the hippocampus and the caudate-putamen,the innervation was sparser. Together,the abnormalities suggested that Pcdha proteins are important in the late-stage maturation of serotonergic projections. Further examination of alternatively spliced exons encoding the cytoplasmic tail showed that the A-type (but not the B-type) cytoplasmic tail was essential for the normal development of serotonergic projections. View Publication

Recombinant human GM-CSF has been expressed as a fusion protein in E. coli in the form of inclusion bodies. Using denaturing agents,acid cleavage and sulfitolysis,the biologically inactive GM-CSF protein could be highly purified and additionally renaturated under suitable reoxidizing conditions. The thorough repair of the two disulfide bridges could be confirmed by sequencing fragments obtained by tryptic digestion. Refolding of the molecule has been studied by CD spectrometry and identity by Western blotting and SDS-PAGE analysis. As could be demonstrated,full biological activity (colony-forming assay with fresh human bone marrow cells) was restored during renaturation of the GM-CSF protein. Further proof of biological equivalence of the E. coli-derived protein with a yeast-derived biologically active rh GM-CSF has been published elsewhere. View Publication

Current Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) using short-read sequencing strategies resolve expressed Ab transcripts with limited resolution of the C region. In this article,we present the near-full-length AIRR-seq (FLAIRR-seq) method that uses targeted amplification by 5' RACE,combined with single-molecule,real-time sequencing to generate highly accurate (99.99%) human Ab H chain transcripts. FLAIRR-seq was benchmarked by comparing H chain V (IGHV),D (IGHD),and J (IGHJ) gene usage,complementarity-determining region 3 length,and somatic hypermutation to matched datasets generated with standard 5' RACE AIRR-seq using short-read sequencing and full-length isoform sequencing. Together,these data demonstrate robust FLAIRR-seq performance using RNA samples derived from PBMCs,purified B cells,and whole blood,which recapitulated results generated by commonly used methods,while additionally resolving H chain gene features not documented in IMGT at the time of submission. FLAIRR-seq data provide,for the first time,to our knowledge,simultaneous single-molecule characterization of IGHV,IGHD,IGHJ,and IGHC region genes and alleles,allele-resolved subisotype definition,and high-resolution identification of class switch recombination within a clonal lineage. In conjunction with genomic sequencing and genotyping of IGHC genes,FLAIRR-seq of the IgM and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles,28 (87%) of which were previously uncharacterized. Together,these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV,IGHD,IGHJ,and IGHC gene diversity for the most comprehensive view of bulk-expressed Ab repertoires to date. View Publication

Induced pluripotent stem cell (iPSC) derived endothelial cells (iECs) have emerged as a promising tool for studying vascular biology and providing a platform for modelling various vascular diseases,including those with genetic origins. Currently,primary ECs are the main source for disease modelling in this field. However,they are difficult to edit and have a limited lifespan. To study the effects of targeted mutations on an endogenous level,we generated and characterized an iPSC derived model for venous malformations (VMs). CRISPR-Cas9 technology was used to generate a novel human iPSC line with an amino acid substitution L914F in the TIE2 receptor,known to cause VMs. This enabled us to study the differential effects of VM causative mutations in iECs in multiple in vitro models and assess their ability to form vessels in vivo. The analysis of TIE2 expression levels in TIE2L914F iECs showed a significantly lower expression of TIE2 on mRNA and protein level,which has not been observed before due to a lack of models with endogenous edited TIE2L914F and sparse patient data. Interestingly,the TIE2 pathway was still significantly upregulated and TIE2 showed high levels of phosphorylation. TIE2L914F iECs exhibited dysregulated angiogenesis markers and upregulated migration capability,while proliferation was not affected. Under shear stress TIE2L914F iECs showed reduced alignment in the flow direction and a larger cell area than TIE2WT iECs. In summary,we developed a novel TIE2L914F iPSC-derived iEC model and characterized it in multiple in vitro models. The model can be used in future work for drug screening for novel treatments for VMs.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10456-024-09925-9. View Publication

Genetic variants in the X‐chromosomal gene coding for the calcium−/calmodulin‐dependent serine protein kinase (CASK) are associated with a neurodevelopmental disorder. CASK is a member of the membrane‐associated guanylate kinase (MAGUK) family of proteins. It acts as a scaffold at presynaptic sites,as a regulator of the transport of glutamate receptors,and as a transcriptional regulator. The PDZ domain of CASK has been reported to bind to presynaptic cell adhesion molecules such as Neurexin1‐3,CNTNAP2,SynCAM and SALM1. Structural analyses of related MAGUKs indicate that the canonical SH3 and GK domains combine with the PDZ domain to form the so‐called PSG supramodule. Conserved aromatic residues (Y723 and W914) flanking the GK domain contribute to the formation of a dimeric structure of two PSG modules,which is required for high‐affinity binding to the type 2 PDZ ligand motif of,for example,Neurexin. Here we identify previously uncharacterized patient variants in the SH3 domain of CASK (I672V; P673L),which alter the intermolecular binding pocket for Y723. Both variants interfere with the binding of Neurexin‐1β,in a manner similar to the previously reported Y723C variant. Intriguingly,binding to the type 1 PDZ ligand of the cell adhesion molecule SALM1 is not altered. Using a set of highly selective patient variants,we show that the binding of SALM1 to CASK is actually not mediated by the CASK PDZ domain or the PSG supramodule,but depends on other type 1 PDZ domain‐containing proteins such as SAP97 and Veli,which associate with CASK through its L27 domains. Our data underline the relevance of an intact PSG tandem of CASK for human health. View Publication

The HOX family of homeobox genes plays an important role in normal and malignant hematopoiesis. Dysregulated HOX gene expression profoundly effects the proliferation and differentiation of hematopoietic stem cells (HSCs) and committed progenitors,and aberrant activation of HOX genes is a common event in human myeloid leukemia. HOXB6 is frequently overexpressed in human acute myeloid leukemia (AML). To gain further insight into the role of HOXB6 in hematopoiesis,we overexpressed HOXB6 in murine bone marrow using retrovirus-mediated gene transfer. We also explored structure-function relationships using mutant HOXB6 proteins unable to bind to DNA or a key HOX-binding partner,pre-B-cell leukemia transcription factor-1 (PBX1). Additionally,we investigated the potential cooperative interaction with myeloid ecotropic viral integration site 1 homolog (MEIS1). In vivo,HOXB6 expanded HSCs and myeloid precursors while inhibiting erythropoiesis and lymphopoiesis. Overexpression of HOXB6 resulted in AML with a median latency of 223 days. Coexpression of MEIS1 dramatically shortened the onset of AML. Cytogenetic analysis of a subset of HOXB6-induced AMLs revealed recurrent deletions of chromosome bands 2D-E4,a region frequently deleted in HOXA9-induced AMLs. In vitro,HOXB6 immortalized a factor-dependent myelomonocytic precursor capable of granulocytic and monocytic differentiation. These biologic effects of HOXB6 were largely dependent on DNA binding but independent of direct interaction with PBX1. View Publication

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage embryos. They can maintain an undifferentiated state indefinitely and can differentiate into derivatives of all three germ layers,namely ectoderm,endoderm and mesoderm. Although much progress has been made in the propagation and differentiation of ES cells,induction of photoreceptors has generally required coculture with or transplantation into developing retinal tissue. Here,we describe a protocol for generating retinal cells from ES cells by stepwise treatment with defined factors. This method preferentially induces photoreceptor and retinal pigment epithelium (RPE) cells from mouse and human ES cells. In our protocol,differentiation of RPE and photoreceptors from mouse ES cells requires 28 d and the differentiation of human ES cells into mature RPE and photoreceptors requires 120 and 150 d,respectively. This differentiation system and the resulting pluripotent stem cell-derived retinal cells will facilitate the development of transplantation therapies for retinal diseases,drug testing and in vitro disease modeling. It will also improve our understanding of the development of the central nervous system,especially the eye. View Publication

The extrafollicular (EF) plasmablast response to self-antigens that contain Toll-like receptor (TLR) ligands is prominent in murine lupus models and some bacterial infections,but the inhibitors and activators involved have not been fully delineated. Here,we used two conventional dendritic cell (cDC) depletion systems to investigate the role of cDCs on a classical TLR-dependent autoreactive EF response elicited in rheumatoid-factor B cells by DNA-containing immune complexes. Contrary to our hypothesis,cDC depletion amplified rather than dampened the EF response in Fas-intact but not Fas-deficient mice. Further,we demonstrated that cDC-dependent regulation requires Fas and Fas ligand (FasL) expression by T cells,but not Fas expression by B cells. Thus,cDCs activate FasL-expressing T cells that regulate Fas-expressing extrafollicular helper T (Tefh) cells. These studies reveal a regulatory role for cDCs in B cell plasmablast responses and provide a mechanistic explanation for the excess autoantibody production observed in Fas deficiency. View Publication

The action of retinoids on gene regulation is mediated by three distinct nuclear retinoic acid receptor (RAR) subtypes called RAR alpha,beta and gamma. Since RAR gamma is predominantly expressed in adult skin,specific ligands for this subtype could (i) represent valuable tools to evaluate the biological role of RAR gamma in skin and (ii) provide therapeutic entities with a higher therapeutic index at lower teratogenic risk. Using in vitro binding studies and a functional transactivation assay,we have identified three compounds with high RAR gamma selectivity. View Publication