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11.
Tse AK Wan CK Shen XL Zhu GY Cheung HY Yang M Fong WF 《Experimental cell research》2007,313(8):1722-1734
1,25-dihydroxyvitamin D(3) (VD(3)) induces differentiation in a number of leukemia cell lines and under various conditions is able to either stimulate or inhibit nuclear factor kappa B (NF-kappaB) activity. Here we report a time-dependent biphasic regulation of NF-kappaB in VD(3)-treated HL-60 leukemia cells. After VD(3) treatment there was an early approximately 4 h suppression and a late 8-72 h prolonged reactivation of NF-kappaB. The reactivation of NF-kappaB was concomitant with increased IKK activities, IKK-mediated IkappaBalpha phosphorylation, p65 phosphorylation at residues S276 and S536, p65 nuclear translocation and p65 recruitment to the NF-kappaB/vitamin D responsive element promoters. In parallel with NF-kappaB stimulation, there was an up-regulation of NF-kappaB controlled inflammatory and anti-apoptotic genes such as TNFalpha, IL-1beta and Bcl-xL. VD(3)-triggered reactivation of NF-kappaB was associated with PI3K/Akt phosphorylation. PI3K/Akt antagonists suppressed VD(3)-stimulated IkappaBalpha phosphorylation as well as NF-kappaB-controlled gene expression. The early approximately 4 h VD(3)-mediated NF-kappaB suppression coincided with a prolonged increase of IkappaBalpha protein which require de novo protein synthesis, lasted for as least 72 h and was insensitive to MAPK, IKK or PI3K/Akt inhibitors. Our data suggest a novel biphasic regulation of NF-kappaB in VD(3)-treated leukemia cells and our results may have provided the first molecular explanation for the contradictory observations reported on VD(3)-mediated immune-regulation. 相似文献
12.
Modulation of nuclear internalization of Tat peptides by fluorescent dyes and receptor-avid peptides
The nuclear internalization of biomolecules by Tat peptide provides a mechanism to deliver drugs to cells. However, translocation of molecular imaging probes to the nucleus may induce undesirable mutagenesis. To assess the feasibility of retaining its cell permeating effect without nuclear translocation, Tat-peptide was conjugated with a somatostatin receptor (STR)-avid ligand (Oct) and labeled with fluorescent dyes. The results show that Tat-Oct-5-FAM (fluorescein 5'-carboxylic acid) remained in the cytoplasm of STR-positive AR42J cells. Co-incubation of Tat-Oct-5-FAM with ATP induced nuclear translocation. These data suggest that both dye and Oct-STR endocytosis complex could modulate nuclear internalization of Tat peptides. 相似文献
13.
Maines MD Miralem T Lerner-Marmarosh N Shen J Gibbs PE 《The Journal of biological chemistry》2007,282(11):8110-8122
Human biliverdin reductase (hBVR), a dual specificity kinase (Ser/Thr/Tyr) is, as protein kinase C (PKC) betaII, activated by insulin and free radicals (Miralem, T., Hu, Z., Torno, M. D., Lelli, K. M., and Maines, M. D. (2005) J. Biol. Chem. 280, 17084-17092; Lerner-Marmarosh, N., Shen, J., Torno, M. D., Kravets, A., Hu, Z., and Maines, M. D. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7109-7114). Here, by using 293A cells co-transfected with pcDNA3-hBVR and PKC betaII plasmids, we report the co-immunoprecipitation of the proteins and co-purification in the glutathione S-transferase (GST) pulldown assay. hBVR and PKC betaII, but not the reductase and PKC zeta, transphosphorylated in assay systems supportive of activity of only one of the kinases. PKC betaII K371R mutant protein ("kinase-dead") was also a substrate for hBVR. The reductase increased the Vmax but not the apparent Km values of PKC betaII for myelin basic protein; activation was independent of phospholipids and extended to the phosphorylation of S2, a PKC-specific substrate. The increase in substrate phosphorylation was blocked by specific inhibitors of conventional PKCs and attenuated by sihBVR. The effect of the latter could be rescued by subsequent overexpression of hBVR. To a large extent, the activation was a function of the hBVR N-terminal chain of valines and intact ATP-binding site and the cysteine-rich C-terminal segment. The cobalt protoporphyrin-activated hBVR phosphorylated a threonine in a peptide corresponding to the Thr500 in the human PKC betaII activation loop. Neither serine nor threonine residues in peptides corresponding to other phosphorylation sites of the PKC betaII nor PKC zeta activation loop-derived peptides were substrates. The phosphorylation of Thr500 was confirmed by immunoblotting of hBVR.PKC betaII immunocomplex. The potential biological relevance of the hBVR activation of PKC betaII was suggested by the finding that in cells transfected with the PKC betaII, hBVR augmented phorbol myristate acetate-mediated c-fos expression, and infection with sihBVR attenuated the response. Also, in cells overexpressing hBVR and PKC betaII, as well as in untransfected cells, upon treatment with phorbol myristate acetate, the PKC translocated to the plasma membrane and co-localized with hBVR. hBVR activation of PKC betaII underscores its potential function in propagation of signals relayed through PKCs. 相似文献
14.
Kawana K Quayle AJ Ficarra M Ibana JA Shen L Kawana Y Yang H Marrero L Yavagal S Greene SJ Zhang YX Pyles RB Blumberg RS Schust DJ 《The Journal of biological chemistry》2007,282(10):7368-7375
Chlamydia trachomatis is an obligate intracellular pathogen that can persist in the urogenital tract. Mechanisms by which C. trachomatis evades clearance by host innate immune responses are poorly described. CD1d is MHC-like, is expressed by epithelial cells, and can signal innate immune responses by NK and NKT cells. Here we demonstrate that C. trachomatis infection down-regulates surface-expressed CD1d in human penile urethral epithelial cells through proteasomal degradation. A chlamydial proteasome-like activity factor (CPAF) interacts with the CD1d heavy chain, and CPAF-associated CD1d heavy chain is then ubiquitinated and directed along two distinct proteolytic pathways. The degradation of immature glycosylated CD1d was blocked by the proteasome inhibitor lactacystin but not by MG132, indicating that degradation was not via the conventional proteasome. In contrast, the degradation of non-glycosylated CD1d was blocked by lactacystin and MG132, consistent with conventional cellular cytosolic degradation of N-linked glycoproteins. Immunofluorescent microscopy confirmed the interruption of CD1d trafficking to the cell surface, and the dislocation of CD1d heavy chains into both the cellular cytosol and the chlamydial inclusion along with cytosolic CPAF. C. trachomatis targeted CD1d toward two distinct proteolytic pathways. Decreased CD1d surface expression may help C. trachomatis evade detection by innate immune cells and may promote C. trachomatis persistence. 相似文献
15.
The major group B coxsackievirus (CVB) receptor is a component of the epithelial tight junction (TJ), a protein complex that regulates the selective passage of ions and molecules across the epithelium. CVB enters polarized epithelial cells from the TJ, causing a transient disruption of TJ integrity. Here we show that CVB does not induce major reorganization of the TJ, but stimulates the specific internalization of occludin-a TJ integral membrane component-within macropinosomes. Although occludin does not interact directly with virus, depletion of occludin prevents CVB entry into the cytoplasm and inhibits infection. Both occludin internalization and CVB entry require caveolin but not dynamin; both are blocked by inhibitors of macropinocytosis and require the activity of Rab34, Ras, and Rab5, GTPases known to regulate macropinocytosis. Thus, CVB entry depends on occludin and occurs by a process that combines aspects of caveolar endocytosis with features characteristic of macropinocytosis. 相似文献
16.
17.
Mei-Qin Liu Xin Shen Wei-Lun Yin Cun-Fu Lu 《植物学报(英文版)》2007,49(3):382-385
T-A cloning takes advantage of the unpaired adenosyl residue added to the 3' terminus of amplified DNAs by Taq and other thermostable DNA polymerase and uses a Ilnearlzed plasmld vector with a protruding 3' thymldylate residue at each of Its 3' termini to clone polymerase chain reaction (PCR)-derived DNA fragments. It Is a simple, reliable, and efficient Ilgatlon-dependent cloning method for PCR products, but the drawback of variable cloning efficiency occurs during application. In the present work, the relationship between variable T-A cloning efficiency and the different 5' end nucleotlde base of primers used In PCR amplification was studied. The results showed that different cloning efficiency was obtained with different primer pairs containing A, T, C and G at the 5' terminus respectively. The data shows that when the 5' end base of primer pair was adenosyl, more white colonies could be obtained In cloning the corresponding PCR product In comparison with other bases. And the least white colonies were formed when using the primer pair with 5' cytldylate end. The gluanylate end primers resulted In almost the same cloning efficiency In the white colonies amount as the thymldylate end primer did, and this efficiency was much lower than that of adenosyl end primers. This presumably is a consequence of variability In 3'dA addition to PCR products mediated by Taq polymerase. Our results offer instructions for primer design for researchers who choose T-A cloning to clone PCR products. 相似文献
18.
Leucine-rich repeat C4 protein is involved in nervous tissue development and neurite outgrowth, and induction of glioma cell differentiation 总被引:1,自引:0,他引:1
Wu M Huang H Chen Q Li D Zheng Z Xiong W Zhou Y Li X Zhou M Lu J Shen S Li G 《Acta biochimica et biophysica Sinica》2007,39(10):731-738
LRRC4, leucine-rich repeat C4 protein, has been identified in human (GenBank accession No. AF196976), mouse (GenBank accession No. DQ177325), rat (GenBank accession No. DQ119102) and bovine (GenBank accession No. DQ 164537) with identical domains. In terms of their similarity, the genes encoding LRRC4 in these four mammalian species are orthogs and therefore correspond to the same gene entity. Based on previous research, and using in situ hybridization, we found that LRRC4 had the strongest expression in hippocampal CA1 and CA2, the granule cells of the dentate gyrus region, the mediodoral thalamic nucleus, and cerebella Purkinje cell layers. Using a P19 cell model, we also found that LRRC4 participates in the differentiation of neuron and glia cells. In addition, extracellular proteins containing both an LRR cassette and immunoglobulin domains have been shown to participate in axon guidance. Our data from neurite outgrowth assays indicated that LRRC4 promoted neurite extension of hippocampal neurons, and induced differentiation of glioblastoma U251 cells into astrocyte-like cells, confirmed by morphology observation and glial fibrillary acidic protein expression. 相似文献
19.
Zhou X Wang J Liu Q Shen J Zheng M Zhang Y 《Acta biochimica et biophysica Sinica》2007,39(12):939-946
Sperm maturation, including the acquisition of motility and the full ability to fertilize oocyte, occurs during its transit through the dynamic environment of the epididymis. However, the roles of many genes involved in the process of sperm maturation still remain to be found. Based on an expressed sequence tag named imds-60, which was first found in uterus but is highly expressed in epididymis, the full-length cDNA sequence of imds-60 with a complete open reading frame was obtained in mouse epididymis by GenBank searching, polymerase chain reaction-based procedures, and 5'- and 3'-rapid amplification of cDNA ends. This protein was predicted to have an N-terminal signal peptide and a C-terminal DNase I-like domain with nine transmembrane motifs in the middle part of the protein. Northern blot analysis showed that the mRNA of imds-60 was highly expressed in epididymis but at a rather lower level in uterus, seminal vesicle gland, and stomach. Further study revealed that the mRNA of imds-60 is only expressed in corpus and cauda regions of epididymis, not in caput. It is regulated partially by androgen and peaked in male mice aged from 3 weeks to adult. The imds-60 protein might play an important role in cell communication during sperm maturation. 相似文献
20.
Tao M Wang L Wendt-Pienkowski E George NP Galm U Zhang G Coughlin JM Shen B 《Molecular bioSystems》2007,3(1):60-74
The tallysomycins (TLMs) belong to the bleomycin (BLM) family of antitumor antibiotics. The BLM biosynthetic gene cluster has been cloned and characterized previously from Streptomyces verticillus ATCC 15003, but engineering BLM biosynthesis for novel analogs has been hampered by the lack of a genetic system for S. verticillus. We now report the cloning and sequencing of the TLM biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 and the development of a genetic system for S. hindustanus, demonstrating the feasibility to manipulate TLM biosynthesis in S. hindustanus by gene inactivation and mutant complementation. Sequence analysis of the cloned 80.2 kb region revealed 40 open reading frames (ORFs), 30 of which were assigned to the TLM biosynthetic gene cluster. The TLM gene cluster consists of nonribosomal peptide synthetase (NRPS) genes encoding nine NRPS modules, a polyketide synthase (PKS) gene encoding one PKS module, genes encoding seven enzymes for deoxysugar biosynthesis and attachment, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The involvement of the cloned gene cluster in TLM biosynthesis was confirmed by inactivating the tlmE glycosyltransferase gene to generate a TLM non-producing mutant and by restoring TLM production to the DeltatlmE::ermE mutant strain upon expressing a functional copy of tlmE. The TLM gene cluster is highly homologous to the BLM cluster, with 25 of the 30 ORFs identified within the two clusters exhibiting striking similarities. The structural similarities and differences between TLM and BLM were reflected remarkably well by the genes and their organization in their respective biosynthetic gene clusters. 相似文献