技术资料

Phosphatidylinositol (PtdIns) 4-kinases catalyze the synthesis of PtdIns-4-P,the immediate precursor of PtdIns-4,5-P2. Here we report the cloning of a novel,ubiquitously expressed PtdIns 4-kinase (PI4Kbeta). The 2.4-kilobase pair cDNA encodes a putative translation product of 801 amino acids which shows greatest homology to the yeast PIK1 gene. The recombinant protein exhibits lipid kinase activity when expressed in Escherichia coli,and specific antibodies recognize a 110-kDa PtdIns 4-kinase in cell lysates. The biochemical properties of PI4Kbeta are characteristic of a type III enzyme. Interestingly,both recombinant PI4Kbeta and the endogenous protein are inhibited by 150 nM wortmannin,suggesting that we have cloned the previously described PtdIns 4-kinase that is responsible for regulating the synthesis of agonist-sensitive pools of polyphosphoinositides (Nakanishi,S.,Catt,J. K.,and Balla,T. (1995) Proc. Natl. Acad. Sci. U. S. A. 92,5317-5321). View Publication

A cDNA clone coding for a functional human prostanoid FP receptor has been isolated from a uterus cDNA library. The human FP receptor consists of 359 amino acid residues with a predicted molecular mass of 40,060,and has the seven putative transmembrane domains characteristic of G-protein-coupled receptors. Challenge of Xenopus oocytes expressing the FP receptor with 10 nM of either prostaglandin (PG) F2 alpha or the selective FP-receptor agonist fluprostenol resulted in an elevation in intracellular Ca2+. Radioreceptor binding studies using membranes prepared from mammalian COS cells transfected with the FP receptor cDNA showed that the rank order of potency for prostaglandins and prostaglandin analogs in competition for [3H]PGF2 alpha specific binding sites was as predicted for the FP receptor,with PGF2 alpha approximately fluprostenol textgreater PGD2 textgreater PGE2 textgreater U46619 textgreater iloprost. In summary,we have cloned the human prostanoid FP receptor which is functionally coupled to the Ca2+ signalling pathway. View Publication

We have cloned the FP receptor from rat corpus luteum and human uterus cDNA libraries,respectively. The coding DNA sequence in the rat cDNA is 1101 bp and is similar to the mouse cDNA coding for a receptor protein of 366 amino acids. The human sequence shows a 5 bp deficiency in the 3' region,truncating the coding sequence to 359 amino acids. Northern blot analysis indicates highest expression in the ovary. Cell lines have been established giving stable expression of the FP receptor. Activation of the cloned FP receptor gave an increase in intracellular calcium,indicating signaling via phospholipase C-mediated phosphoinositide turnover. Using [3H]PGF2 alpha,binding of PGs showed the rank order of fluprostenol textgreater PhXA70 textgreater PGF2 alpha textgreater or = PhXA85 textgreater PGD2 textgreater PGE2. View Publication

In the present study,we describe the cloning and sequence analysis of rat IL-3. Two different mRNA isoforms were isolated after transfection of COS cells with the cytokine genomic sequences. One of the isoforms has been predicted before by Cohen et al. (1986),and the other one is identical except that it encodes a protein with an insertion of three amino acids at position 56. As names for the two isoforms,we propose IL-3alpha for the predicted and IL-3beta for the novel molecule. IL-3beta mRNA was detected as the predominant isoform in rat lymphocytes in vivo. High levels of the cytokine were obtained after infection of human cells (A549) with a recombinant adenovirus harboring rIL-3beta cDNA (IG.Ad.CMV.IL-3beta). The biological properties of the IL-3beta protein were tested in a FDC-P1 proliferation assay and in a hematopoietic progenitor colony forming assay. To assess in-vivo bioactivity,lysed 293 cells containing IG.Ad.CMV.rIL-3beta virus were injected subcutaneously into F344 rats. Stimulation of hematopoiesis and leucocytosis were observed during the treatment. After subcutaneous injections of the lysed adeno-producer cells in mice,the only effect observed was a cellular infiltration at the site of injection,confirming the poor cross-reactivity between the two species. The biological properties in vitro and in vivo demonstrate that the cDNA sequences of IL-3beta presented here encode active rat IL-3 protein. View Publication

Many agents are active in multiple myeloma,but the majority of patients relapse. This clinical pattern suggests most cancer cells are eliminated,but cells with the clonogenic potential to mediate tumor regrowth are relatively chemoresistant. Our previous data suggested that CD138(+) multiple myeloma plasma cells cannot undergo long-term proliferation but rather arise from clonogenic CD138(neg) B cells. We compared the relative sensitivity of these distinct cell types to clinical antimyeloma agents and found that dexamethasone,lenadilomide,bortezomib,and 4-hydroxycyclophosphamide inhibited CD138(+) multiple myeloma plasma cells but had little effect on CD138(neg) precursors in vitro. We further characterized clonogenic multiple myeloma cells and stained cell lines using the Hoechst side population and Aldefluor assays. Each assay identified CD138(neg) cells suggesting that they possess high drug efflux capacity and intracellular drug detoxification activity. We also found that multiple myeloma cells expressing the memory B-cell markers CD20 and CD27 could give rise to clonogenic multiple myeloma growth in vitro and engraft immunodeficient nonobese diabetes/severe combined immunodeficient mice during both primary and secondary transplantation. Furthermore,both the side population and Aldefluor assays were capable of identifying circulating clonotypic memory B-cell populations within the peripheral blood of multiple myeloma patients. Our results suggest that circulating clonotypic B-cell populations represent multiple myeloma stem cells,and the relative drug resistance of these cells is mediated by processes that protect normal stem cells from toxic injury. View Publication

Gas gangrene caused by Clostridium perfringens type A infection is a highly lethal infection of soft tissue characterized by rapid spread of tissue necrosis. This tissue destruction is related to profound attenuation of blood flow accompanied by formation of platelet-leukocyte aggregates in the blood vessels. Several studies have identified $\alpha$-toxin,which has both sphingomyelinase and phospholipase C activities,as a major virulence factor in the aggregate formation via activation of the platelet gpIIbIIIa. Here,we show that $\alpha$-toxin greatly and rapidly increases plasma membrane localization of CD11b,which binds to the platelet gpIIbIIIa via fibrinogen,in mouse neutrophils. Interestingly,short-term treatment of $\alpha$-toxin has little effect on gene expression profiles in neutrophils,and the toxin does not change the total protein expression levels of CD11b in whole cell lysates. The following analysis demonstrated that CD11b localizes to intracellular vesicles in intact cells,but the localization changed to the cytoplasmic membrane in $\alpha$-toxin-treated cells. These results suggest that CD11b is recruited to the cytoplasmic membrane by $\alpha$-toxin. Previously,we reported that $\alpha$-toxin promotes the formation of ceramide by its sphingomyelinase activity in mouse neutrophils. Interestingly,a synthetic cell-permeable ceramide analog,C2-ceramide,increases plasma membrane localization of CD11b,suggesting that ceramide production by $\alpha$-toxin recruits CD11b to the cytoplasmic membrane to promote platelet-leukocyte aggregation. Together,our results illustrate that the increase of cell membrane CD11b expression by $\alpha$-toxin might be crucial for the pathogenesis of C. perfringens to promote formation of platelet-leukocyte aggregates,leading to rapid tissue necrosis due to ischemia. View Publication

Peripheral serotonin (5-hydroxytryptamine: 5-HT) synthesized in the intestine by enterochromaffin cells (ECs),plays an important role in the regulation of peristaltic of the gut,epithelial secretion and promotes the development and maintenance of the enteric neurons. Recent studies showed that the indigenous gut microbiota modulates 5-HT signalling and that ECs use sensory receptors to detect dietary and microbiota-derived signals from the lumen to subsequently transduce the information to the nervous system. We hypothesized that Clostridium ramosum by increasing gut 5-HT availability consequently contributes to high-fat diet-induced obesity. Using germ-free mice and mice monoassociated with C. ramosum,intestinal cell lines and mouse organoids,we demonstrated that bacterial cell components stimulate host 5-HT secretion and program the differentiation of colonic intestinal stem progenitors toward the secretory 5-HT-producing lineage. An elevated 5-HT level regulates the expression of major proteins involved in intestinal fatty acid absorption in vitro,suggesting that the presence of C. ramosum in the gut promotes 5-HT secretion and thereby could facilitates intestinal lipid absorption and the development of obesity. View Publication

The ability of human embryonic stem cells (hESCs) to differentiate into essentially all somatic cell types has made them a valuable tool for studying human development and has positioned them for broad applications in toxicology,regenerative medicine,and drug discovery. This unit describes a protocol for the large-scale expansion and maintenance of hESCs in vitro. hESC cultures must maintain a balance between the cellular states of pluripotency and differentiation; thus,researchers must use care when growing these technically demanding cells. The culture system is based largely on the use of a proprietary serum-replacement product and basic fibroblast growth factor (bFGF),with mouse embryonic fibroblasts as a feeder layer. These conditions provide the basis for relatively inexpensive maintenance and expansion of hESCs,as well as their engineered counterparts,human induced pluripotent stem cells (hiPSCs). View Publication

A quantitative trait locus (QTL) controlling HbF levels has previously been mapped to chromosome 6q23 in an Asian-Indian kindred with beta thalassemia and heterocellular hereditary persistence of fetal hemoglobin (HPFH). Five protein-coding genes,ALDH8A1,HBS1L,cMYB,AHI1,and PDE7B reside in this 1.5-megabase (Mb) candidate interval of 6q23. To direct sequencing efforts we compared the expression profiles of these 5 genes between 12 individuals with elevated and 14 individuals with normal HbF levels during adult erythropoiesis by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). Two genes,cMYB and HBS1L,demonstrated simultaneous transcriptional down-regulation in individuals with elevated HbF levels. Transfection of K562 cells encoding human cDNA of cMYB and HBS1L genes showed that,although overexpression of ectopic cMYB inhibited gamma-globin gene expression,overexpression of HBS1L had no effect. Low levels of cMYB were associated with low cell expansions,accelerated erythroid maturation,and higher number of macrophages in erythroid cell culture. These observations suggest that differences in the intrinsic levels of cMYB may account for some of the variation in adult HbF levels. The possible mechanism of cMYB influencing gamma- to beta-globin switching is discussed. View Publication

Embryonic stem cell (ESC) identity and self-renewal is maintained by extrinsic signaling pathways and intrinsic gene regulatory networks. Here,we show that three members of the Ccr4-Not complex,Cnot1,Cnot2,and Cnot3,play critical roles in maintaining mouse and human ESC identity as a protein complex and inhibit differentiation into the extraembryonic lineages. Enriched in the inner cell mass of blastocysts,these Cnot genes are highly expressed in ESC and downregulated during differentiation. In mouse ESCs,Cnot1,Cnot2,and Cnot3 are important for maintenance in both normal conditions and the 2i/LIF medium that supports the ground state pluripotency. Genetic analysis indicated that they do not act through known self-renewal pathways or core transcription factors. Instead,they repress the expression of early trophectoderm (TE) transcription factors such as Cdx2. Importantly,these Cnot genes are also necessary for the maintenance of human ESCs,and silencing them mainly lead to TE and primitive endoderm differentiation. Together,our results indicate that Cnot1,Cnot2,and Cnot3 represent a novel component of the core self-renewal and pluripotency circuitry conserved in mouse and human ESCs. View Publication

Which isoforms of apolipoprotein E (apoE) we inherit determine our risk of developing late-onset Alzheimer’s Disease (AD),but the mechanism underlying this link is poorly understood. In particular,the relevance of direct interactions between apoE and amyloid-β (Aβ) remains controversial. Here,single-molecule imaging shows that all isoforms of apoE associate with Aβ in the early stages of aggregation and then fall away as fibrillation happens. ApoE-Aβ co-aggregates account for ~50% of the mass of diffusible Aβ aggregates detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. We show how dynamic interactions between apoE and Aβ tune disease-related functions of Aβ aggregates throughout the course of aggregation. Our results connect inherited APOE genotype with the risk of developing AD by demonstrating how,in an isoform- and lipidation-specific way,apoE modulates the aggregation,clearance and toxicity of Aβ. Selectively removing non-lipidated apoE4-Aβ co-aggregates enhances clearance of toxic Aβ by glial cells,and reduces secretion of inflammatory markers and membrane damage,demonstrating a clear path to AD therapeutics. Subject terms: Protein aggregation,Nanoscale biophysics View Publication

The aim is to investigate the clinical implications of the Oct-4 and Nestin protein in human breast cancers. A total of 346 cases including 26 fresh and 320 paraffin-embedded tumor tissues were selected for characterizing the frequency of CD44(+)CD24(-) tumor cells by flow cytometry and the differential expression of the stem cell-related genes between CD44(+)CD24(-) and non-CD44(+)CD24(-) tumor cells was analyzed by PCR Array and immunofluorescence. In comparison with the non-CD44(+)CD24(-) tumor cells,the CD44(+)CD24(-),particularly for those with high percentage of Oct-4(+) and Nestin(+),tumor cells had higher tumorigenicity by forming mammospheres in vitro. More importantly,42 (13.125%) out of 320 tumor tissues were positive for Oct-4 and Nestin staining. Universal analysis and multivariate analysis revealed that the expression of Oct-4 and Nestin was associated significantly with younger age,pathogenic degrees,lymph node metastasis and triple-negative breast cancer independently (P textless 0.05) as well as shorter survival (P = 0.001). Oct-4 and Nestin were important regulators of the development of breast cancer,and Oct-4 and Nestin may be used as predictors for the prognosis of breast cancers. View Publication