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211.
Wilson TJ Ouellet J Zhao ZY Harusawa S Araki L Kurihara T Lilley DM 《RNA (New York, N.Y.)》2006,12(6):980-987
RNA catalysis is important in the processing and translation of RNA molecules, yet the mechanisms of catalysis are still unclear in most cases. We have studied the role of nucleobase catalysis in the hairpin ribozyme, where the scissile phosphate is juxtaposed between guanine and adenine bases. We show that a modified ribozyme in which guanine 8 has been substituted by an imidazole base is active in both cleavage and ligation, with ligation rates 10-fold faster than cleavage. The rates of both reactions exhibit bell-shaped dependence on pH, with pK(a) values of 5.7 +/- 0.1 and 7.7 +/- 0.1 for cleavage and 6.1 +/- 0.3 and 6.9 +/- 0.3 for ligation. The data provide good evidence for general acid-base catalysis by the nucleobases. 相似文献
212.
Aeromonas hydrophila transports extracellular protein toxins via the type II secretion system, an export mechanism comprised of numerous proteins that spans both the inner and outer membranes. Two components of this secretion system, ExeA and ExeB, form a complex in the inner membrane that functions to locate and/or assemble the ExeD secretin in the outer membrane. In the studies reported here, two-codon insertion mutagenesis of exeA revealed that an insertion at amino acid 495 in the C-terminal region of ExeA did not alter ExeAB complex formation yet completely abrogated its involvement in ExeD secretin assembly and thus rendered the bacteria secretion negative. In silico analysis of protein motifs with similar amino acid profiles revealed that this amino acid is located within a putative peptidoglycan (PG) binding motif in the periplasmic domain of ExeA. Substitution mutations of three highly conserved amino acids in the motif were constructed. In cells expressing each of these mutants, the ability to assemble the ExeD secretin or secrete aerolysin was lost, while ExeA retained the ability to form a complex with ExeB. In in vivo cross-linking experiments, wild-type ExeA could be cross-linked to PG, whereas the three substitution mutants of ExeA could not. These data indicate that PG binding and/or remodelling plays a role in the function of the ExeAB complex during assembly of the ExeD secretin. 相似文献
213.
The expansion of CD8(+) T cells in response to Ag can be characterized as either dependent or independent of CD4(+) T cells. The factors that influence this dichotomy are poorly understood but may be dependent upon the degree of inflammation associated with the Ag. Using dendritic cells derived from MHC class II-deficient mice to avoid interaction with CD4(+) T cells in vivo, we have compared the immunogenicity of peptide-pulsed dendritic cells stimulated with molecules associated with infection to those stimulated via CD40. In the absence of CD4(+) T cell help, the expansion of primary CD8(+) T cells after immunization with TNF-alpha- or poly(I:C)-stimulated dendritic cells was minimal. In comparison, LPS- or CpG-stimulated dendritic cells elicited substantial primary CD8(+) T cell responses, though not to the same magnitude generated by immunization with CD40L-stimulated dendritic cells. Remarkably, mice immunized with any stimulated dendritic cell population generated fully functional recall CD8(+) T cells without the aid of CD4(+) T cell help. The observed hierarchy of immunogenicity was closely correlated with the expression of CD70 (CD27L) on the stimulated dendritic cells, and Ab-mediated blockade of CD70 substantially prevented the CD4(+) T cell-independent expansion of primary CD8(+) T cells. These results indicate that the expression of CD70 on dendritic cells is an important determinant for helper-dependence of primary CD8(+) T cell expansion and provide an explanation for the ability of a variety of pathogens to stimulate primary CD8(+) T cell responses in the absence of CD4(+) T cells. 相似文献
214.
Insulin is known to cause an increase in endothelin-1 (ET-1) receptors in vascular smooth muscle cells (SMCs), but the effect of insulin-like growth factor 1 (IGF-1) on ET-1 receptor expression is not known. We therefore carried out the present study to determine the effect of IGF-1 on the binding of ET-1 to, and ET type A receptor (ETAR) expression and ET-1-induced 3H-thymidine incorporation in, vascular SMCs. In serum-free medium, IGF-1 treatment increased the binding of 125I-ET-1 to SMC cell surface ET receptors from a specific binding of 20.1%+/-3.1% per mg of protein in control cells to 45.1%+/-8.6% per mg of protein in cells treated with IGF-1 (10 nM). The effect of IGF-1 was dose-related, with a significant effect (1.4-fold) being seen at 1 nM. The minimal time for IGF-1 treatment to be effective was 30 min and the maximal effect was reached at 6 h. Immunoblotting analysis showed that ETAR expression in IGF-1-treated cells was increased by 1.7-fold compared to controls. Levels of ETAR mRNA measured by the RT-PCR method and Northern blotting were also increased by 2-fold in IGF-1-treated SMCs. These effects of IGF-1 were abolished by cycloheximide or genistein. Finally, ET-1-stimulated thymidine uptake and cell proliferation were enhanced by IGF-1 treatment, with a maximal increase of 3.2-fold compared to controls. In conclusion, in vascular SMCs, IGF-1 increases the expression of the ET-1 receptor in a dose- and time-related manner. This effect is associated with increased thymidine uptake and involves tyrosine kinase activation and new protein synthesis. These findings support the role of IGF-1 in the development of atherosclerotic, hypertensive, and diabetic vascular complications. 相似文献
215.
Kinetics of iron release from ferric binding protein (FbpA): mechanistic implications in bacterial periplasm-to-cytosol Fe3+ transport 总被引:1,自引:0,他引:1
The ferric binding protein (FbpA) transports iron across the periplasmic space of certain Gram-negative bacteria and is an important component involved in iron acquisition by pathogenic Neisseria spp. (Neisseria gonorrheae and Neisseria meningitidis). Previous work has demonstrated that the synergistic anion, required for tight Fe(3+) sequestration by FbpA, also plays a key role in inserting Fe(3+) into the FbpA binding site. Here, we investigate the iron release process from various forms of holo-FbpA, Fe(3+)FbpA-X, during the course of a chelator competition reaction using EDTA and Tiron. Fe(3+)FbpA-X represents the protein assembly complex with different synergistic anions, X = PO(4)(3)(-) and NTA. Stepwise mechanisms of Fe(3+) release are proposed on the basis of kinetic profiles of these chelator competition reactions. Fe(3+)FbpA-PO(4) and Fe(3+)FbpA-NTA react differently with EDTA and Tiron during the Fe(3+)-exchange process. EDTA replaces PO(4)(3)(-) and NTA from the first coordination shell of Fe(3+) and acts as a synergistic anion to give a spectroscopically distinguishable intermediate, Fe(3+)FbpA-EDTA, prior to pulling Fe(3+) out of the protein. Tiron, on the other hand, does not act as a synergistic anion but is a more efficient competing chelator as it removes Fe(3+) from FbpA at rate much faster than EDTA. These results reaffirm the contribution of the synergistic anion to the FbpA iron transport process as the anion, in addition to playing a facilitative role in iron binding, appears to have a "gatekeeper" role, thereby modulating the Fe(3+) release process. 相似文献
216.
217.
Brenna A. McLeod Moira W. Brown Timothy R. Frasier Bradley N. White 《Conservation Genetics》2010,11(1):339-345
Low levels of genetic variability identified within the North Atlantic right whale (Eubalaena glacialis), when compared to the Southern right whale (E. australis) and other large whales, have been suggested to result from population reductions due to whaling. Previous genetic analysis of 218 whale bones from sixteenth century Basque whaling sites in the western North Atlantic revealed only a single right whale bone. We determined the genotypes of 27 microsatellite loci using DNA isolated from this bone. All alleles from the historic specimen occur in the extant western North Atlantic population and both the probability of identity of the specimen and the number of heterozygous loci are similar to that in the extant population. Assessments of how genetically different the historical population might have been suggest genetic characteristics have not changed substantially over four centuries of whaling. 相似文献
218.
Gangaraju Rajashekhar Ahmed Ramadan Chandrika Abburi Breedge Callaghan Dmitry O. Traktuev Carmella Evans-Molina Raj Maturi Alon Harris Timothy S. Kern Keith L. March 《PloS one》2014,9(1)
Diabetic retinopathy (DR) is the leading cause of blindness in working-age adults. Early stage DR involves inflammation, vascular leakage, apoptosis of vascular cells and neurodegeneration. In this study, we hypothesized that cells derived from the stromal fraction of adipose tissue (ASC) could therapeutically rescue early stage DR features. Streptozotocin (STZ) induced diabetic athymic nude rats received single intravitreal injection of human ASC into one eye and saline into the other eye. Two months post onset of diabetes, administration of ASC significantly improved “b” wave amplitude (as measured by electroretinogram) within 1–3 weeks of injection compared to saline treated diabetic eyes. Subsequently, retinal histopathological evaluation revealed a significant decrease in vascular leakage and apoptotic cells around the retinal vessels in the diabetic eyes that received ASC compared to the eyes that received saline injection. In addition, molecular analyses have shown down-regulation in inflammatory gene expression in diabetic retina that received ASC compared to eyes that received saline. Interestingly, ASC were found to be localized near retinal vessels at higher densities than seen in age matched non-diabetic retina that received ASC. In vitro, ASC displayed sustained proliferation and decreased apoptosis under hyperglycemic stress. In addition, ASC in co-culture with retinal endothelial cells enhance endothelial survival and collaborate to form vascular networks. Taken together, our findings suggest that ASC are able to rescue the neural retina from hyperglycemia-induced degeneration, resulting in importantly improved visual function. Our pre-clinical studies support the translational development of adipose stem cell-based therapy for DR to address both retinal capillary and neurodegeneration. 相似文献
219.
Karen L. Baab Sarah E. Freidline Steven L. Wang Timothy Hanson 《American journal of physical anthropology》2010,141(1):97-115
Variation in cranial robusticity among modern human populations is widely acknowledged but not well‐understood. While the use of “robust” cranial traits in hominin systematics and phylogeny suggests that these characters are strongly heritable, this hypothesis has not been tested. Alternatively, cranial robusticity may be a response to differences in diet/mastication or it may be an adaptation to cold, harsh environments. This study quantifies the distribution of cranial robusticity in 14 geographically widespread human populations, and correlates this variation with climatic variables, neutral genetic distances, cranial size, and cranial shape. With the exception of the occipital torus region, all traits were positively correlated with each other, suggesting that they should not be treated as individual characters. While males are more robust than females within each of the populations, among the independent variables (cranial shape, size, climate, and neutral genetic distances), only shape is significantly correlated with inter‐population differences in robusticity. Two‐block partial least‐squares analysis was used to explore the relationship between cranial shape (captured by three‐dimensional landmark data) and robusticity across individuals. Weak support was found for the hypothesis that robusticity was related to mastication as the shape associated with greater robusticity was similar to that described for groups that ate harder‐to‐process diets. Specifically, crania with more prognathic faces, expanded glabellar and occipital regions, and (slightly) longer skulls were more robust than those with rounder vaults and more orthognathic faces. However, groups with more mechanically demanding diets (hunter‐gatherers) were not always more robust than groups practicing some form of agriculture. Am J Phys Anthropol, 2010. © 2009 Wiley‐Liss, Inc. 相似文献
220.
Hutchinson AB Patel P Sansom SL Farnham PG Sullivan TJ Bennett B Kerndt PR Bolan RK Heffelfinger JD Prabhu VS Branson BM 《PLoS medicine》2010,7(9):e1000342