rhIL-11诱导转录因子GATA-1和NF-E2在Dami细胞的表达

目的探讨重组人白细胞介素11（rhIL-11）对巨核细胞白血病细胞株Dami内转录因子GATA-1和NF-E2表达的调节作用。方法用rhIL-11（50ng／ml）刺激Dami细胞1、2、4h,Western blot和RT-PCR法分析rhIL-11诱导Dami细胞GATA-1、NF-E2mRNA表达水平。结果rhIL-11可诱导Dami细胞GATA-1、NF-E2mRNA表达,1h后作用下降。结论rhIL-11可能通过诱导GATA-1、NF-E2表达调控巨核细胞生成。     

High-level erythroid expression of human alpha-globin genes in transgenic mice.

The human alpha 1-globin gene was fused downstream of two erythroid-specific DNase I super-hypersensitive sites that are normally located upstream of the human beta-globin locus. This construct was injected into fertilized mouse eggs, and expression was analyzed in 16-day fetal livers and brains. All 11 fetuses that contained intact copies of the transgene expressed correctly initiated human alpha-globin mRNA in the erythroid fetal liver but not in brain. Levels of expression ranged from 4% to 337% of endogenous mouse beta-globin mRNA. A human alpha-globin construct that did not contain super-hypersensitive sites was not expressed. These results demonstrate that human beta-globin locus activation sequences can stimulate high levels of human alpha-globin gene expression in erythroid tissue of transgenic mice. The results also provide a foundation for experiments designed to coexpress human alpha- and beta-globin genes in transgenic mice and suggest a feasible approach for production of a mouse model for human sickle cell disease.     

Isolation of cDNA encoding the human NF-E2 protein.

The human homolog of mouse NF-E2 was isolated from the K562 cell line and found to encode a member of the basic leucine-zipper family of DNA-binding regulatory proteins. The deduced amino acid sequence of the mouse and human proteins exhibited near identity. Comparison to the related protein, Nrf1, revealed significant homologies at isolated regions, particularly within the basic domain, suggesting that NF-E2 and Nrf1 are members of a distinct subfamily of basic leucine-zipper proteins that share similar DNA-binding properties. High levels of human NF-E2 mRNA were observed in human erythroleukemic cell lines examined. Extensive survey of human tissue samples found NF-E2 expression not limited to erythropoeitic organs. Expression in the colon and testis suggests that NF-E2 may participate in the regulation of genes other than globin.     

A common mutant EcoRI restriction endonuclease site in the 5'' flanking portion of the human alpha-globin gene.

A mutant EcoRI endonuclease restriction site has been identified in 3 of 37 Black subjects and in 2 sibs of one of these persons. This mutation was not encountered in 13 Whites. It is located approximately 6 kilobases "inside" the normal site in the 5' flanking sequence of the alpha-globin chain complex. The shortened alpha-globin gene-bearing segment produced in the EcoRI digest produces a restriction map similar to that observed for the common alpha-globin gene deletion observed in the Black population. However, the restriction map with BamHI is normal, confirming that all four alpha loci are present.     

Prostaglandin F2alpha binding sites in human corpora lutea.

The specific binding of 3H-prostaglandin (PG) F2alpha to homogenates of human corpora lutea of the cycle and ectopic pregnancy was examined. Corpora lutea of ectopic pregnancy bound significantly (P less than 0.01) higher amounts of added 3H-PGF2alpha than those of the luteal phase of the menstrual cycle. The 3H-PGF2alpha binding sites in corpora lutea of ectopic pregnancy were further characterized. The specific 3H-PGF2alpha binding to all corpora lutea was biphasic: all contained sites of 10(-8)M Kd, two also had sites of Kd greater than 10(-8)M while the other contained sites of 10(-9)M Kd. PGs competed for 3H-PGF2alpha binding in the following order: PGF2alpha greater than 15(S)15 methyl PGF2alpha greater than PGF1alpha greater than PGE2 greater than PGE1 greater than PGB1 greater than PGA1. Binding was time and temperature dependent; maximum binding was obtained by 1 h at 22 C; AT 38 C, the initial binding was high but rapidly declined after 30 min of incubation. A cationic requirement for 3H-PGF2ALPHA binding is suggested by the findings that the addition of EDTA severely reduced the binding which was reversed by concomittant addition of Ca+ to the medium. Preincubation of homogenates with proteolytic enzymes drastically reduced the binding, suggesting that the binding sites are protein in nature.     

Identification of beta-adrenergic binding sites in rabbit myometrium.

Rabbit uterine muscle may contract or relax with adrenergic stimulation depending on the hormonal milieu. This difference in contractile activity has been shown to be due to alteration of adrenergic response between alpha-adrenergic (contraction) and beta-adrenergic (relaxation). When rabbits are treated with estrogen followed by progesterone, norepinephrine produces myometrial relaxation. This effect is blocked by propranolol, indicating that it is mediated by beta receptor activation. A subcellular preparation of this myometrium has adenylate cyclase activity that can be stimulated by isoproterenol + guanyl-5'-yl-imidodi-phosphate (Gpp(NH)p). The radioligand [125I]iodohydroxybenzylpindolol binds to the same preparation. The binding is rapid, 80% maximal in 10 min, and readily reversible (t1/2 = 5 min). The binding is high affinity (Kd = 0.12 nM), low capacity (15 fmol/mg protein), and is to a single class of binding sites. Binding is competed for stereoselectively by beta adrenergic agonists and antagonists. The competition of beta adrenergic agonists for binding, isoproterenol = ritodrine greater than epinephrine greater than norepinephrine, is consistent with interactions at a beta2-adrenergic receptor.     

乙型肝炎病毒前S1蛋白反式激活蛋白2结合蛋白1基因的克隆及其反式调节基因的筛选

目的 克隆HBV前S1蛋白反式激活蛋白2结合蛋白1(PS1TP2BP1)基因,并筛选与其相互作用的反式激活基因.方法 应用聚合酶链反应(PCR)扩增PS1TP2BP1基因,鉴定正确后再将其亚克隆到真核表达载体pcDNATM3.1/myc-His A上;以真核表达质粒pcDNATM3.1/mycHis A-PS1TP2BP1转染HepG2细胞,构建cDNA消减文库;进行测序及同源性分析.结果 从HepG2细胞来源的cDNA中扩增出PS1TP2BP1基因,并成功进行TA克隆,酶切、测序均正确后,成功构建真核表达重组体.消减文库扩增后得到35个阳性克隆,经菌落PCR分析,得到15个含200～1000 bp插入片段的菌落.对所得片段测序,并进行间源性分析,显示15种已知基因编码蛋白可能是PS1TP2BP1反式激活靶基因.结论 成功克隆PS1TP2BP1,并构建了PS1TP2BP1反式激活基因差异表达的cDNA消减文库,为今后进一步分析、研究病毒蛋白的致病机制奠定了基础.     

Identification of human neutralization-inducing regions of the human immunodeficiency virus type 1 envelope glycoproteins.

Four major neutralizing regions of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein were identified and characterized with a panel of 80 HIV-1 antibody-positive human sera. Levels of neutralizing antibodies against the HIV-1 strains IIIB, SF2, and RF were compared with reactivity in ELISAs against peptides that correspond to certain regions of the HIV-1 envelope. A correlation between high neutralizing activity and strong seroreactivity against specific peptides suggested that the corresponding regions might be involved in neutralization. This was further substantiated by using peptides to inhibit neutralization by a panel of 10 HIV-1 antibody-positive sera. The positions of three neutralizing sites, defined earlier mostly by antisera from animals, were confirmed in the present study. Human sera thus recognize the strain-specific third variable region of gp120 (amino acids 304-318), the C-terminal end of gp120 (amino acids 489-508), and the conserved region in the intracellular part of gp41 (amino acids 732-746). It is likely that these different regions mediate help rather than self-sufficient neutralization. Furthermore, a human neutralizing region was detected in a conserved part of gp41 (amino acids 647-671). Accordingly, neutralizing antibodies directed to this region were found to be cross-reactive between HIV-1 strains. Peptides corresponding to these four regions were able to inhibit neutralization mediated by serum from HIV-1 antibody-positive individuals. These results indicate that this conserved B-cell epitope of the HIV-1 envelope elicits a virus-neutralizing antibody response during natural infection in humans and may therefore be considered for inclusion in a vaccine against HIV-1.     

Autoradiographic localization and analysis of endothelin-1 binding sites in human synovial tissue.

OBJECTIVE. To determine the localization of endothelin binding sites and immunoreactivity in human synovial tissues. METHODS. Quantitative in vitro autoradiographic and immunohistochemical techniques were used to localize and characterize 125I-labeled endothelin-1 (125I-ET-1) binding sites and endothelin-like immunoreactivity in sections of rheumatoid, osteoarthritic, and normal synovium. RESULTS. Specific 125I-ET-1-binding sites, characteristic of the ETA receptor, were localized to the media of synovial blood vessels in all 3 groups. No difference was found in vascular binding site density in rheumatoid and osteoarthritic synovium. Endothelin-like immunoreactivity was localized to endothelial cells in blood vessels displaying 125I-ET-1 binding sites. CONCLUSION. We conclude that endothelin may act locally, modulating synovial perfusion and exacerbating hypoxia in chronic arthritis.     

Genomic remnants of alpha-globin genes in the hemoglobinless antarctic icefishes.

Alone among piscine taxa, the antarctic icefishes (family Channichthyidae, suborder Notothenioidei) have evolved compensatory adaptations that maintain normal metabolic functions in the absence of erythrocytes and the respiratory oxygen transporter hemoglobin. Although the uniquely "colorless" or "white" condition of the blood of icefishes has been recognized since the early 20th century, the status of globin genes in the icefish genomes has, surprisingly, remained unexplored. Using alpha- and beta-globin cDNAs from the antarctic rockcod Notothenia coriiceps (family Nototheniidae, suborder Notothenioidei), we have probed the genomes of three white-blooded icefishes and four red-blooded notothenioid relatives (three antarctic, one temperate) for globin-related DNA sequences. We detect specific, high-stringency hybridization of the alpha-globin probe to genomic DNAs of both white- and red-blooded species, whereas the beta-globin cDNA hybridizes only to the genomes of the red-blooded fishes. Our results suggest that icefishes retain inactive genomic remnants of alpha-globin genes but have lost, either through deletion or through rapid mutation, the gene that encodes beta-globin. We propose that the hemoglobinless phenotype of extant icefishes is the result of deletion of the single adult beta-globin locus prior to the diversification of the clade.     

Identification of endothelial cell binding sites on the laminin gamma 1 chain

The laminins belong to a family of trimeric basement membrane glycoproteins with multiple domains, structures, and functions. Endothelial cells bind laminin-1 and form capillary-like structures when plated on a laminin-1-rich basement membrane matrix, Matrigel. Laminin-1 is composed of 3 chains, alpha1, beta1, and gamma1. Because laminin-1 is known to contain multiple biologically active sites, we have screened 156 synthetic overlapping peptides spanning the entire laminin gamma1 chain for potential angiogenic sequences. Only 7 of these peptides, designated as C16, C25, C30, C38, C64, C75, and C102, disrupted the formation of capillary-like structures by human umbilical vein endothelial cells on Matrigel. Dose-response experiments in the presence of 50 to 200 microg/mL showed that tube formation was prevented by most peptides at 150 and 200 microg/mL, except for C16, which showed strong activity at all concentrations. Active peptides promoted vessel sprouting from aorta rings and angiogenesis in the chick chorioallantoic membrane assay. In addition, the active peptides also promoted endothelial cell adhesion to dishes coated with 0.1 microg of peptide and inhibited attachment to laminin-1 but not to plastic or fibronectin. Four of the active peptides, C25, C38, C75, and C102, may have cell-type specificity with endothelial cells, since they did not promote PC12 neurite outgrowth or adhesion of B16-F10 melanoma and human submandibular gland cells. These results suggest that specific laminin gamma1-chain peptides have angiogenic activity with potential therapeutic applications.