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971.
Characterization of a novel member of the DOK family that binds and modulates Abl signaling 下载免费PDF全文
A novel member of the p62(dok) family of proteins, termed DOKL, is described. DOKL contains features of intracellular signaling molecules, including an N-terminal PH (pleckstrin homology) domain, a central PTB (phosphotyrosine binding) domain, and a C-terminal domain with multiple potential tyrosine phosphorylation sites and proline-rich regions, which might serve as docking sites for SH2- and SH3-containing proteins. The DOKL gene is predominantly expressed in bone marrow, spleen, and lung, although low-level expression of the RNA can also be detected in other tissues. DOKL and p62(dok) bind through their PTB domains to the Abelson tyrosine kinase in a kinase-dependent manner in both yeast and mammalian cells. DOKL is phosphorylated by the Abl tyrosine kinase in vivo. In contrast to p62(dok), DOKL lacks YxxP motifs in the C terminus and does not bind to Ras GTPase-activating protein (RasGAP) upon phosphorylation. Overexpression of DOKL, but not p62(dok), suppresses v-Abl-induced mitogen-activated protein (MAP) kinase activation but has no effect on constitutively activated Ras- and epidermal growth factor-induced MAP kinase activation. The inhibitory effect requires the PTB domain of DOKL. Finally, overexpression of DOKL in NIH 3T3 cells inhibits the transforming activity of v-Abl. These results suggest that DOKL may modulate Abl function. 相似文献
972.
An extracellular signal-regulated kinase 1- and 2-dependent program of chromatin trafficking of c-Fos and Fra-1 is required for cyclin D1 expression during cell cycle reentry 下载免费PDF全文
Mitogens activate cell signaling and gene expression cascades that culminate in expression of cyclin D1 during the G(0)-to-G(1) transition of the cell cycle. Using cell cycle arrest in response to oxidative stress, we have delineated a dynamic program of chromatin trafficking of c-Fos and Fra-1 required for cyclin D1 expression during cell cycle reentry. In serum-stimulated lung epithelial cells, c-Fos was expressed, recruited to chromatin, phosphorylated at extracellular signal-regulated kinase 1- and 2 (ERK1,2)-dependent sites, and degraded prior to prolonged recruitment of Fra-1 to chromatin. Immunostaining showed that expression of nuclear c-Fos and that of cyclin D1 are mutually exclusive, whereas nuclear Fra-1 and cyclin D1 are coexpressed as cells traverse G(1). Oxidative stress prolonged the accumulation of phospho-ERK1,2 and phospho-c-Fos on chromatin, inhibited entry of Fra-1 into the nucleus, and blocked cyclin D1 expression. After induction of the immediate-early gene response in the presence of oxidative stress, inhibition of ERK1,2 signaling promoted degradation of c-Fos, recruitment of Fra-1 to chromatin, and expression of cyclin D1. Our data indicate that termination of nuclear ERK1,2 signaling is required for an exchange of Fra-1 for c-Fos on chromatin and initiation of cyclin D1 expression at the G(0)-to-G(1) transition of the cell cycle. 相似文献
973.
In the past decades, a large number of studies in mammalian cells have revealed that processing of glycoproteins is compartmentalized into several subcellular organelles that process N-glycans to generate complex-type oligosaccharides with terminal N -acetlyneuraminic acid. Recent studies also suggested that processing of N-glycans in insect cells appear to follow a similar initial pathway but diverge at subsequent processing steps. N-glycans from insect cell lines are not usually processed to terminally sialylated complex-type structures but are instead modified to paucimannosidic or oligomannose structures. These differences in processing between insect cells and mammalian cells are due to insufficient expression of multiple processing enzymes including glycosyltransferases responsible for generating complex-type structures and metabolic enzymes involved in generating appropriate sugar nucleotides. Recent genomics studies suggest that insects themselves may include many of these complex transferases and metabolic enzymes at certain developmental stages but expression is lost or limited in most lines derived for cell culture. In addition, insect cells include an N -acetylglucosaminidase that removes a terminal N -acetylglucosamine from the N-glycan. The innermost N -acetylglucosamine residue attached to asparagine residue is also modified with alpha(1,3)-linked fucose, a potential allergenic epitope, in some insect cells. In spite of these limitations in N-glycosylation, insect cells have been widely used to express various recombinant proteins with the baculovirus expression vector system, taking advantage of their safety, ease of use, and high productivity. Recently, genetic engineering techniques have been applied successfully to insect cells in order to enable them to produce glycoproteins which include complex-type N-glycans. Modifications to insect N-glycan processing include the expression of missing glycosyltransferases and inclusion of the metabolic enzymes responsible for generating the essential donor sugar nucleotide, CMP- N -acetylneuraminic acid, required for sialylation. Inhibition of N -acetylglucosaminidase has also been applied to alter N-glycan processing in insect cells. This review summarizes current knowledge on N-glycan processing in lepidopteran insect cell lines, and recent progress in glycoengineering lepidopteran insect cells to produce glycoproteins containing complex N-glycans. 相似文献
974.
Quanhua?Xing Binran?Zhao Ke?Xu Hehua?Yang Xia?Liu Songwen?Wang Demin?Jin Longping?Yuan Bin?WangEmail author 《Plant Molecular Biology Reporter》2004,22(2):155-164
New rice lines, restorer line RB207 and maintainer line Yewei B, with better agronomic traits were separately developed from
variant progeny of R207 (rice restorer line) and V20B (rice maintainer line) through transformation of genomic DNA ofEchinochloa crusgalli (C4 plant) andOryza minuta, respectively. The phenotypes of the variant lines were apparently different from those of the receptors. Yewei B had stronger
tolerance to high temperature than did V20B. The number of spikelets per panicle and the 1000-grain weight of RB207 increased
by 40% over those of R207. The results of amplified fragment length polymorphism (AFLP) analysis indicated that the polymorphism
rates were both 4.4% between genomes of the variant lines and their receptors. Results demonstrated that special DNA segments
fromE. crusgalli andO. minuta might integrate into the genome of cultivated rice and could be stably passed on. The study further shows that transformation
of genomic DNA of distant relatives is an effective approach for creating new rice germ plasm.
These authors contributed equally to this work. 相似文献
975.
Although Alzheimer's disease pathologically affects the brain, familial Alzheimer's disease associated mutations of beta-amyloid precursor protein and presenilin are ubiquitously expressed and therefore aberrant intracellular signals, separate from but similar to, the brain may be expected. Here, we report selective down regulation of the serine/threonine kinase, Akt/PKB, concurrent with elevated endogenous GSK3beta kinase activity in familial Alzheimer's disease beta-amyloid precursor protein expressing human embryonic kidney (HEK) and familial Alzheimer's disease presenilin lymphoblast cells. Further, familial Alzheimer's disease presenilin in the human lymphoblast was associated with beta-catenin destabilization. Moreover, limited immunohistochemistry analysis reveals Akt/PKB in a subset of neurofibrillary tangles where GSK3beta and tau have been reported to co-localize, suggesting a possible Akt/GSK3beta and tau interaction in vivo. Our data suggest that familial Alzheimer's disease mutants of beta-amyloid precursor protein and presenilin signal, at least in part, through the Akt/GSKbeta pathway and that Akt/GSK3beta-mediated signalling may contribute to the underlying Alzheimer's disease pathogenesis induced by familial Alzheimer's disease mutants. 相似文献
976.
Huijsdens XW Linskens RK Koppes J Tang YL Meuwissen SG Vandenbroucke-Grauls CM Savelkoul PH 《FEMS immunology and medical microbiology》2004,41(1):79-84
In many animal species different intestinal Helicobacter species have been described and a few species are associated with intestinal infection. In humans, the only member of the Helicobacter family which is well described in literature is Helicobacter pylori. No other Helicobacter-associated diseases have definitely been shown in humans. We developed a sensitive quantitative PCR to investigate whether Helicobacter species DNA can be detected in the human gastrointestinal tract. We tested gastric biopsies (including biopsies from H. pylori positive persons), intestinal mucosal biopsies and fecal samples from healthy persons, and intestinal mucosal biopsies from patients with inflammatory bowel disease (IBD) for the presence of Helicobacter species. All gastric biopsies, positive for H. pylori by culture, were also positive in our newly developed PCR. No Helicobacter species were found in the mucosal biopsies from patients with IBD (n = 50) nor from healthy controls (n = 25). All fecal samples were negative. Our study suggests that Helicobacter species, other than H. pylori, are not present in the normal human gastrointestinal flora and our results do not support a role of Helicobacter species in IBD. 相似文献
977.
Hemoglobin cross-linked with small molecular modifiers turns out to be more stable. Modifications of proteins with polyethylene glycol (PEG) have been proven to enlarge the molecular size of proteins, to prolong their retention time in the circulation as well as blunt immune reactions. In the present study, the optimal conditions for porcine hemoglobin (pHb) modification with bis (3, 5-dibromosalicyl) fumarate (DBBF) and PEG were evaluated. The derivative of DBBF cross-linked pHb (DBBF-pHb) showed improved oxygen affinity and the ability to resist the dissociation of the alpha2beta2 tetramer compared with the natural protein. DBBF-pHb was then bound to the activated PEG. The results indicated that the pHb modified with DBBF and PEG had more stable tetrameric conformation with a molecular weight of 107000. Their oxygen half-saturation pressure (P50) is around 3.33 kPa, which approximates the physiological P50 of human red blood cells. Both routine and reinforced immunizing methods were adopted to study the immunogenicity of modified products and the results showed that the products had very low immunogenicity evaluated by enzyme-linked immunoadsordent assay (ELISA). Somewhat beneficial effects were shown in the treatment of hemorrhagic shock where modified hemoglobin solutions were used as resuscitation fluids in the hemorrhagic shock Sprague-Dawley (SD) rats model. 相似文献
978.
Pin?YangEmail author Rui?Ren Maolin?Guo Aixin?Song Xiangli?Meng Caixia?Yuan Qinghua?Zhou Huili?Chen Zhenhai?Xiong Xiaoli?Gao 《Journal of biological inorganic chemistry》2004,9(4):495-506
The development of artificial nucleases that hydrolyze DNA or RNA is of great interest in molecular biology, biotechnology, and medicine. We now report that a magnesium(II) complex of diethylenetriamine (Mg-dien) can effectively promote the double-stranded cleavage of plasmid DNA and the dideoxynucleotide dApdA under physiological conditions of pH and temperature. Experiments performed in the presence of hydrogen peroxide, radical scavengers, or under rigorously anaerobic conditions indicate that DNA cleavage mediated by Mg-dien occurs via a hydrolytic path. Mg-dien efficiently hydrolyzes supercoiled pBR322 DNA and the pseudo-first-order rate constant at 37 degrees C and pH 8.0 is estimated to be 1.60 h(-1). The dinucleotide dApdA hydrolysis, with Mg-dien at 170 microM, shows a rate enhancement factor of ca. 5 x 10(8). 1H and 31P(1H) NMR studies show that Mg-dien effectively hydrolyzes 5'-dAMP to give deoxyadenosine and inorganic phosphate. While Mg2+ has been found at the catalytic sites of many natural nucleases, Mg-dien appears to be the first synthetic Mg2+-containing system capable of hydrolyzing dideoxynucleotides and DNA and thus may provide a simple model system to assist mechanistic studies of naturally occurring nucleases. 相似文献
979.
We analyzed carbohydrate chains of human, bovine, sheep, and rat alpha1-acid glycoprotein (AGP) and found that carbohydrate chains of AGP of different animals showed quite distinct variations. Human AGP is a highly negatively charged acidic glycoprotein (pKa = 2.6; isoelectic point = 2.7) with a molecular weight of approximately 37,000 when examined by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and contains di-, tri-, and tetraantennary carbohydrate chains. Some of the tri- and tetraantennary carbohydrate chains are substituted with a fucose residue (sialyl Lewis x type structure). In sheep AGP, mono- and disialo-diantennary carbohydrate chains were abundant. Tri- and tetrasialo-triantennary carbohydrate chains were also present as minor oligosaccharides, and some of the sialic acid residues were substituted with N-glycolylneuraminic acid. In rat AGP, very complex mixtures of disialo-carbohydrate chains were observed. Complexity of the disialo-oligosaccharides was due to the presence of N, O-acetylneuraminic acids. Triantennary carbohydrate chains carrying N,O-acetylneuraminic acid were also observed as minor component oligosaccharides. We found some novel carbohydrate chains containing both N-acetylneuraminic acid and N-glycolylneuraminic acid in bovine AGP. Interestingly, triantennary carbohydrate chains were hardly detected in bovine AGP, but diantennary carbohydrate chains with tri- or tetrasialyl residues were abundant. Furthermore the major sialic acid in these carbohydrate chains was N-glycolylneuraminic acid. It should be noted that these sialic acids are attached to multiple sites of the core oligosaccharide and are not present as disialyl groups. 相似文献
980.
Lee H Lin CI Liao JJ Lee YW Yang HY Lee CY Hsu HY Wu HL 《American journal of physiology. Cell physiology》2004,287(6):C1657-C1666
Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S-1-P) are both low molecular weight lysophospholipid (LPL) ligands that are recognized by the Edg family of G protein-coupled receptors. In endothelial cells, these two ligands activate Edg receptors, resulting in cell proliferation and cell migration. The intercellular adhesion molecule-1 (ICAM-1, CD54) is one of many cell adhesion molecules belonging to the immunoglobulin superfamily. This study showed that LPA and S-1-P enhance ICAM-1 expression at both the mRNA and protein levels in human umbilical cord vein endothelial cells (HUVECs). This enhanced ICAM-1 expression in HUVECs was first observed at 2 h postligand treatment. Maximal expression appeared at 8 h postligand treatment, as detected by flow cytometry and Western blotting. Furthermore, the effects of S-1-P on ICAM-1 expression were shown to be concentration dependent. Prior treatment of HUVECs with pertussis toxin, a specific inhibitor of Gi, ammonium pyrrolidinedithiocarbamate and BAY 117082, inhibitors of the nuclear factor (NF)-B pathway, or Clostridium difficile toxin B, an inhibitor of Rac, prevented the enhanced effect of LPL-induced ICAM-1 expression. However, pretreatment of HUVECs with exoC3, an inhibitor of Rho, had no effect on S-1-P-enhanced ICAM-1 expression. In a static cell-cell adhesion assay system, pretreatment of LPL enhanced the adhesion between HUVECs and U-937 cells, a human mononucleated cell line. The enhanced adhesion effect could be prevented by preincubation with a functional blocking antibody against human ICAM-1. These results suggest that LPLs released by activated platelets might enhance interactions of leukocytes with the endothelium through a Gi-, NF-B-, and possibly Rac-dependent mechanism, thus facilitating wound healing and inflammation processes. lysophosphatidic acid; sphingosine 1-phosphate; inflammation; intercellular adhesion molecule-1; nuclear factor-B; human umbilical cord vein endothelial cells 相似文献