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81.
The two membrane segments of leader peptidase partition one by one into the lipid bilayer via a Sec/YidC interface 下载免费PDF全文
We have addressed the mechanism of insertion of both transmembrane segments (TMs) of leader peptidase, a double-spanning protein, into the Escherichia coli inner membrane. Using photo-crosslinking, the first TM (H1) was shown to insert at a Sec-translocon/YidC interface in a fixed orientation. H1 lost its contacts with the Sec-translocon and gained access to lipids near YidC soon after complete exposure outside the ribosome. Following lipid integration, it moved away from the Sec/YidC insertion site. The second TM (H2) inserted and interacted with SecY and YidC in a similar transient fashion. The data are consistent with a linear integration model in which the TMs of polytopic inner membrane proteins move one by one from a Sec/YidC insertion site into the lipid bilayer. We propose that YidC assists the lipid partitioning of single TMs. 相似文献
82.
Novel chimeric immunomodulatory compounds containing short CpG oligodeoxyribonucleotides have differential activities in human cells 总被引:1,自引:0,他引:1 下载免费PDF全文
Marshall JD Hessel EM Gregorio J Abbate C Yee P Chu M Van Nest G Coffman RL Fearon KL 《Nucleic acids research》2003,31(17):5122-5133
Immunostimulatory DNA sequences (ISS) containing CpG motifs induce interferon-α (IFN-α) and interferon-γ (IFN-γ) from human peripheral blood mononuclear cells and stimulate human B cells to proliferate and produce IL-6. We studied the motif and structural requirements for both types of activity using novel chimeric immunomodulatory compounds (CICs), which contain multiple heptameric ISS connected by non-nucleoside spacers in both linear and branched configurations. We found that the optimal motifs and structure for IFN-α production versus B cell activation differed. IFN-α production was optimal for CICs containing the sequences 5′-TCGXCGX and 5′-TCGXTCG, where X is any nucleotide. The presentation of multiple copies of these heptameric ISS with free 5′-ends via long, hydrophilic spacers, such as hexaethylene glycol, significantly enhanced the induction of IFN-α. Conversely, human B cell activity was predominately dependent on ISS motif, with 5′-TCGTXXX and 5′-AACGTTC being the most active sequences. Thus, we found CICs could be ‘programmed’ for IFN-α production or B cell activation as independent variables. Additionally, CICs with separate human- and mouse-specific motifs were synthesized and these were used to confirm in vivo activity in mice. CICs may offer unique advantages over conventional ISS because identification of the optimal motifs, spacers and structures for different biological properties allows for the assembly of CICs exhibiting a defined set of activities tailored for specific clinical applications. 相似文献
83.
Transgenic plants expressing resistance to herbivorous insects may represent a safe and sustainable pest control alternative if they do not interfere with the natural enemies of target pests. Here we examined interactions between oryzacystatin I (OCI), a proteinase inhibitor from rice genetically engineered into potato (Solanum tuberosum cv. Kennebec, line K52) to increase resistance to insect herbivory, and the insect predator Perillus bioculatus. This stinkbug is a relatively specialized predator of caterpillars and leaf-beetle larvae, and may also include plant sap in its predominantly carnivorous diet. One of its preferred prey is Colorado potato beetle (Leptinotarsa decemlineata), a major target of insect resistance development for potato field crops. Gelatin/sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that a major fraction of proteinase (gelatinase) activity in P. bioculatus extracts is OCI-sensitive. Among five gelatinolytic bands detected, the slowest-moving one (proteinase I) was inhibited strongly by purified OCI expressed in Escherichia coli or by OCI-transgenic potato extracts, while three other proteinases were partly sensitive to these treatments. There was also evidence of slight inhibition of proteinase I by untransformed potato foliage, suggesting the presence of a natural inhibitor related to OCI at low level in potato foliage. Interestingly, only about 50% of the maximum potential activity of proteinase I was recovered in extracts of P. bioculatus feeding on L. decemlineata larval prey on a diet of OCI-potato foliage, indicating that the predator was sensitive to OCI in the midgut of its prey. However, P. bioculatus on OCI-prey survived, grew and developed normally, indicating ability to compensate prey-mediated exposure to the OCI inhibitor. Confinement of P. bioculatus to potato foliage provided no evidence that potato plant-derived nutrition is a viable alternative to predation, restriction to potato foliage in fact being inferior to free water for short-term survival of nonfeeding first-instar larvae. These results support the view that OCI, an effective inhibitor of a substantial fraction of digestive enzymatic potential in P. bioculatus, should not interfere with its predation potential when expressed in potato plants fed to its prey at a maximum level of approximately 0.8% of total soluble proteins in mature foliage. 相似文献
84.
Justice JP Borchers MT Crosby JR Hines EM Shen HH Ochkur SI McGarry MP Lee NA Lee JJ 《American journal of physiology. Lung cellular and molecular physiology》2003,284(1):L169-L178
A strategy to deplete eosinophils from the lungs of ovalbumin (OVA)-sensitized/challenged mice was developed using antibody-mediated depletion. Concurrent administration [viz. the peritoneal cavity (systemic) and as an aerosol to the lung (local)] of a rat anti-mouse CCR3 monoclonal antibody resulted in the abolition of eosinophils from the lung such that the airway lumen was essentially devoid of eosinophils. Moreover, perivascular/peribronchial eosinophil numbers were reduced to levels indistinguishable from saline-challenged animals. This antibody-mediated depletion was not accompanied by effects on any other leukocyte population, including, but not limited to, T cells and mast cells/basophils. In addition, no effects were observed on other underlying allergic inflammatory responses in OVA-treated mice, including OVA-specific immunoglobulin production as well as T cell-dependent elaboration of Th2 cytokines. The ablation of virtually all pulmonary eosinophils in OVA-treated mice (i.e., without concurrent effects on T cell activities) resulted in a significant decrease in mucus accumulation and abolished allergen-induced airway hyperresponsiveness. These data demonstrate a direct causative relationship between allergen-mediated pulmonary pathologies and eosinophils. 相似文献
85.
Rapid genome divergence at orthologous low molecular weight glutenin loci of the A and Am genomes of wheat 总被引:19,自引:0,他引:19 下载免费PDF全文
Wicker T Yahiaoui N Guyot R Schlagenhauf E Liu ZD Dubcovsky J Keller B 《The Plant cell》2003,15(5):1186-1197
To study genome evolution in wheat, we have sequenced and compared two large physical contigs of 285 and 142 kb covering orthologous low molecular weight (LMW) glutenin loci on chromosome 1AS of a diploid wheat species (Triticum monococcum subsp monococcum) and a tetraploid wheat species (Triticum turgidum subsp durum). Sequence conservation between the two species was restricted to small regions containing the orthologous LMW glutenin genes, whereas >90% of the compared sequences were not conserved. Dramatic sequence rearrangements occurred in the regions rich in repetitive elements. Dating of long terminal repeat retrotransposon insertions revealed different insertion events occurring during the last 5.5 million years in both species. These insertions are partially responsible for the lack of homology between the intergenic regions. In addition, the gene space was conserved only partially, because different predicted genes were identified on both contigs. Duplications and deletions of large fragments that might be attributable to illegitimate recombination also have contributed to the differentiation of this region in both species. The striking differences in the intergenic landscape between the A and A(m) genomes that diverged 1 to 3 million years ago provide evidence for a dynamic and rapid genome evolution in wheat species. 相似文献
86.
1-15N-L-Tryptophan (1-15N-L-Trp) was synthesized from 15N-aniline by a Sandmeyer reaction, followed by cyclization to isatin, reduction to indole with LiAlH4, and condensation of the 15N-indole with L-serine, catalyzed by tryptophan synthase. 1-15N-L-Trp was complexed with wild-type tryptophan synthase and beta-subunit mutants, betaK87T, betaD305A, and betaE109D, in the absence or presence of the allosteric ligands sodium chloride and disodium alpha-glycerophosphate. The enzyme complexes were observed by 15N-heteronuclear single-quantum coherence nuclear magnetic resonance (15N-HSQC NMR) spectroscopy for the presence of 1-15N-L-Trp bound to the beta-active site. No 15N-HSQC signal was detected for 1-15N-L-Trp in 10 mm triethanolamine hydrochloride buffer at pH 8. 1-15N-L-Trp in the presence of wild-type tryptophan synthase in the absence or presence of 50 mm sodium chloride showed a cross peak at 10.25 ppm on the 1H axis and 129 ppm on the 15N axis as a result of reduced solvent exchange for the bound 1-15N-L-Trp, consistent with formation of a closed conformation of the active site. The addition of disodium alpha-glycerophosphate produced a signal twice as intense, suggesting that the equilibrium favors the closed conformation. 15N-HSQC NMR spectra of betaK87T and betaE109D mutant Trp synthase with 1-15N-L-Trp showed a similar cross peak either in the presence or absence of disodium alpha-glycerophosphate, indicating the preference for a closed conformation for these mutant proteins. In contrast, the betaD305A Trp synthase mutant only showed a 15N-HSQC signal in the presence of disodium alpha-glycerophosphate. Thus, this mutant Trp synthase favored an open conformation in the absence of disodium alpha-glycerophosphate but was able to form a closed conformation in the presence of disodium alpha-glycerophosphate. Our results demonstrate that the 15N-HSQC NMR spectra of 1-15N-L-Trp bound to Trp synthase can be used to determine the conformational state of mutant forms in solution rapidly. In contrast, UV-visible spectra of wild-type and mutant Trp synthase in the presence of L-Trp with NaCl and/or disodium alpha-glycerophosphate are more difficult to interpret in terms of altered conformational equilibria. 相似文献
87.
Wolff B Billich A Brunowsky W Herzig G Lindley I Nussbaumer P Pursch E Rabeck C Winkler G 《Analytical biochemistry》2003,318(2):276-284
Steroid sulfatase (STS; E.C. 3.1.6.2) is an enzyme involved in the local production of estrogens and androgens in target organs. Inhibitors of steroid sulfatase activity are considered novel therapeutic agents for the treatment of different pathologic conditions, including cancers of breast, endometrium, and prostate and disorders of the pilosebaceous unit. Evaluation of steroid sulfatase inhibition in cells up to now has been a cumbersome process, involving the extraction of a radioactive cleavage product into organic solvents. Here, we describe a rapid, nonradioactive cellular assay in microtiter plate format, using 4-methylumbelliferyl sulfate as a substrate. The reaction product, 4-methylumbelliferone, is read in a fluorescence microtiter plate reader. Several cell lines were assayed for sulfatase activity. To increase the sensitivity of the assay, we developed a Chinese hamster ovary (CHO) cell line stably transfected with a cDNA encoding the human steroid sulfatase. The steroid sulfatase activity in transfected cells correlated with the presence of the enzyme in these cells, as determined by immunofluorescence. For most STS inhibitors tested, including estrone-3-O-sulfamate, the results from the CHO cellular assay were in good agreement with those from a standard cell-free assay. 相似文献
88.
Leukemia inhibitory factor induces apoptosis of the mammary epithelial cells and participates in mouse mammary gland involution 总被引:5,自引:0,他引:5
Schere-Levy C Buggiano V Quaglino A Gattelli A Cirio MC Piazzon I Vanzulli S Kordon EC 《Experimental cell research》2003,282(1):35-47
Leukemia inhibitory factor (LIF) is a multifunctional glycoprotein that displays multiple biological activities in different cell types, but to date there has been no report on its expression in the normal mammary gland. In this study we found that LIF is expressed at low but detectable levels in postpubertal, adult virgin, and pregnant mouse mammary glands. However, LIF expression drops after parturition to become almost undetectable in lactating glands. Interestingly, LIF expression shows a steep increase shortly after weaning that is maintained for the following 3 days. During this period, known as the first stage of mammary gland involution, the lack of suckling induces local factors that cause extensive epithelial cell death. It has been shown that Stat3 is the main factor in signaling the initiation of apoptosis, but the mechanism of its activation remains unclear. Herein, we show that LIF expression in the gland is induced by milk stasis and not by the decrease of circulating lactogenic hormones after weaning. Implantation of LIF containing pellets in lactating glands results in a significant increase in epithelium apoptosis. In addition, this treatment also induces Stat3 phosphorylation. We conclude that LIF regulated expression in the mouse mammary gland may play a relevant role during the first stage of mammary gland involution. Our results also show that LIF-induced mammary epithelium apoptosis could be mediated, at least partially, by Stat3 activation. 相似文献
89.
Moncoq K Broutin I Larue V Perdereau D Cailliau K Browaeys-Poly E Burnol AF Ducruix A 《FEBS letters》2003,554(3):240-246
Grb14 belongs to the Grb7 family of adapter proteins and was identified as a negative regulator of insulin signal transduction. Its inhibitory effect on the insulin receptor kinase activity is controlled by a newly discovered domain called PIR. To investigate the biochemical and biophysical characteristics of this new domain, we cloned and purified recombinant PIR-SH2, PIR, and SH2 domains. The isolated PIR and PIR-SH2 domains were physiologically active and inhibited insulin-induced reinitiation of meiosis in the Xenopus oocytes system. However, NMR experiments on (15)N-labelled PIR revealed that it did not present secondary structure. These results suggest that the PIR domain belongs to the growing family of intrinsically unstructured proteins. 相似文献
90.
Three highly mutable loci of the wall-less pathogens Mycoplasma bovis, Mycoplasma pulmonis and Mycoplasma agalactiae undergo high-frequency genomic rearrangements and generate extensive antigenic variation of major surface lipoproteins. Adjacent to each locus, an open reading frame exists as a single chromosomal copy and is predicted to encode a site-specific DNA recombinase exhibiting high homology to the recombinases XerD of Escherichia coli and CodV of Bacillus subtilis. Each of the mycoplasmal proteins are members of the lambda integrase family of tyrosine site-specific recombinases and likely mediates site-specific DNA inversions observed within the adjacent, variable loci. 相似文献