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71.
RNA-directed construction of structurally complex and active ligase ribozymes through recombination 总被引:1,自引:0,他引:1 下载免费PDF全文
RNA-directed recombination can be used to catalyze a disproportionation reaction among small RNA substrates to create new combinations of sequences. But the accommodation of secondary and tertiary structural constraints in the substrates by recombinase ribozymes has not been explored. Here, we show that the Azoarcus group I intron can recombine oligoribonucleotides to construct class I ligase ribozymes, which are catalytically active upon synthesis. The substrate oligonucleotides, ranging in size from 58 to 104 nucleotides (nt), along with the 152-nt ligase ribozymes they reconstitute, can contain significant amounts of secondary structure. However, substrate recognition by the Azoarcus ribozyme depends on the existence of a single accessible CAU triplet for effective recombination. A biphasic temperature reaction profile was designed such that the sequential recombination/ligation events could take place in a thermocycler without human intervention. A temperature-dependent pH shift of the reaction buffer contributes to the success of the net reaction. When the substrate for the ligase ribozyme is introduced into the reaction mixture, as much as 11% can be observed being converted to product by the recombined ligase in the same reaction vessel. Recombination followed by ligation can also occur under isothermal conditions at 37 degrees C. Tertiary structure formation of the ligase upon construction can provide some protection from cleavage by the Azoarcus ribozyme when compared to the constituent substrates. These data suggest that RNA-directed recombination can, in fact, articulate complex ribozymes, and that there are logical rules that can guide the optimal placement of the CAU recognition sequence. 相似文献
72.
Hipp MS Patel CN Bersuker K Riley BE Kaiser SE Shaler TA Brandeis M Kopito RR 《The Journal of cell biology》2012,196(5):573-587
Pathognomonic accumulation of ubiquitin (Ub) conjugates in human neurodegenerative diseases, such as Huntington's disease, suggests that highly aggregated proteins interfere with 26S proteasome activity. In this paper, we examine possible mechanisms by which an N-terminal fragment of mutant huntingtin (htt; N-htt) inhibits 26S function. We show that ubiquitinated N-htt-whether aggregated or not-did not choke or clog the proteasome. Both Ub-dependent and Ub-independent proteasome reporters accumulated when the concentration of mutant N-htt exceeded a solubility threshold, indicating that stabilization of 26S substrates is not linked to impaired Ub conjugation. Above this solubility threshold, mutant N-htt was rapidly recruited to cytoplasmic inclusions that were initially devoid of Ub. Although synthetically polyubiquitinated N-htt competed with other Ub conjugates for access to the proteasome, the vast majority of mutant N-htt in cells was not Ub conjugated. Our data confirm that proteasomes are not directly impaired by aggregated N-terminal fragments of htt; instead, our data suggest that Ub accumulation is linked to impaired function of the cellular proteostasis network. 相似文献
73.
Agatha A. van der Klaauw Sophie Croizier Edson Mendes de Oliveira Lukas K.J. Stadler Soyoung Park Youxin Kong Matthew C. Banton Panna Tandon Audrey E. Hendricks Julia M. Keogh Susanna E. Riley Sofia Papadia Elana Henning Rebecca Bounds Elena G. Bochukova Vanisha Mistry Stephen O’Rahilly Richard B. Simerly I. Sadaf Farooqi 《Cell》2019,176(4):729-742.e18
74.
Research on the human-nonhuman animal bond has focused primarily on its advantages to the human. The purpose of this study is to investigate behaviors of caregivers (owners) of companion animals (pets) and to examine the relationship between such behaviors and scores on a pet attachment scale. Participants were 501 largely nontraditional (older, married, employed full-time) college students living with a pet dog or cat. The study categorized owner behaviors as essential, standard, enriched, or luxury care. Almost all participants reported engaging in essential care behaviors, with numbers declining from category to category. Pet attachment scores appeared related to standard and enriched care behaviors but not to essential care. Too few participants reported doing luxury care behaviors to link them to attachment. The results suggest that even pet owners reporting low attachment provide beneficial care and attention to their pets and that pet attachment may be of limited use when looking at the benefit of the human-animal bond to the companion animal. 相似文献
75.
Matrix metalloproteinase-2 and -9 secretion by the equine ovary during follicular growth and prior to ovulation 总被引:3,自引:0,他引:3
Riley SC Thomassen R Bae SE Leask R Pedersen HG Watson ED 《Animal reproduction science》2004,81(3-4):329-339
Profound hormonally controlled tissue remodelling occurs in the equine ovary for follicle growth and development, and also for the alteration in follicle shape directed towards the ovulation fossa, the site where ovulation occurs. The aim of this study was to examine the spatial and temporal regulation of matrix metalloproteinases (MMP)-2 and MMP-9, important enzymes in tissue remodelling, during follicle growth, and ovulation. Using gelatin substrate zymography, we measured these MMPs in follicular fluid of large anovulatory follicles collected during spring transition, early dominant follicles (> 23 mm), and at oestrus in follicles approximately 3 days prior to ovulation, and post-hCG treatment when ovulation was predicted in approximately 4 h. The most abundant activity detected in follicular fluid was MMP-2, although there were no changes in secretion or activation in association with ovulation. The activity of MMP-9 was detected in lower amounts, with no changes prior to ovulation, although it decreased significantly (P < 0.05) post-hCG treatment. At oestrus, when different regions of the ovary were maintained in explant culture for 24 h, there were no significant changes in either MMP-2 or MMP-9 secretion by stromal tissues collected at the ovarian fossa, adjacent to the preovulatory follicle but away from the fossa, and a further site remote from the preovulatory follicle. Over this same time period, follicular progesterone (P < 0.01) and oestradiol (P < 0.05) increased significantly, although oestradiol tended to decrease after hCG administration. These findings indicate that MMP-2 and MMP-9 are not key acute regulators for the changes in follicle shape immediately prior to ovulation. 相似文献
76.
Medical school graduates from 1986 to 1988 and current residents in 12 family practice residency programs in the Northwest (N = 302) were surveyed to identify important factors in the recruitment process for their first postresidency placement. The study sought to compare the recruitment practices of rural communities and urban sites. Specific questions addressed in the study concerned sources of information about practice opportunities, stage in training when job search was initiated, factors related to unsuccessful site visits, and activities scheduled in the visit. Results indicated that referrals from faculty were the most valued source of information. Most job searches were initiated in the first 6 months of the third year in training. An unreceptive physician community and a reluctant spouse or partner were substantial problems for residents making site visits to rural communities. Rural sites tended to provide a broader mix of professional and personal activities during the visit. 相似文献
77.
Matthew R. First Steven A. Policastro Matthew J. Strom Scott C. Riley Stephanie H. Robbins-Wamsley Lisa A. Drake 《Biofouling》2014,30(6):685-693
A volumetric approach for determining the fouling burden on surfaces is presented, consisting of a 3D camera imaging system with fine (5?μm) resolution. Panels immersed in an estuary on the southwest coast of Florida, USA were imaged and the data were used to quantify seasonal changes in the biofouling community. Test panels, which were submerged in seawater for up to one?year, were analyzed before and after gentle scrubbing to quantify the biovolume of the total fouling community (ie soft and hard organisms) and the hard fouling community. Total biofouling ranged from 0.01 to 1.16?cm3 cm?2 throughout the immersion period; soft fouling constituted 22–87% of the total biovolume. In the future, this approach may be used to inform numerical models of fluid–surface interfaces and to evaluate, with high resolution, the morphology of fouling organisms in response to antifouling technologies. 相似文献
78.
Placental growth and development is crucial for successful pregnancy. The aim of this study was to characterize the activity and localization of the matrix metalloproteinase 2 (MMP-2) and MMP-9, which are capable of degrading basement membrane collagen (predominantly collagen type IV), and their endogenous tissue inhibitor of matrix metalloproteinases (TIMPs), in amniotic fluid and in the developing ovine placenta. Cell deletion by apoptosis during placental development was also examined. Zymography with gelatin as substrate indicated that MMP-2 (72 kDa gelatinase A; predominantly latent form) was present in increasing amounts in amniotic fluid from day 70 of gestation to labour (days 140-145), and MMP-9 (92 kDa gelatinase B; predominantly latent form) was detectable from day 125 to labour; there was no increase in MMP-2 or -9 in labour. A broad range of TIMPs was detected in amniotic fluid; the molecular masses corresponded to TIMP-1, -2 and -3. Immunohistochemical techniques localized MMP-2, MMP-9 and TIMP-3 in the sheep placenta, predominantly in the trophoblast layer in uninucleate, but not binucleate, cells. However, MMP-2 and -9 activated proteins in placental homogenates were low throughout pregnancy. Apoptosis was identified by morphological criteria and also by TdT-mediated dUTP nick end labelling. Apoptosis was present in discrete regions in the placenta, predominantly in trophoblast cells near the tips and the basal regions of the fetomaternal interdigitations. During pregnancy the sheep placenta becomes more complex and the area of the fetomaternal interface increases. MMP-2 and -9 are likely to be involved in breaking down basement membranes to allow cell migration during this process. It is suggested that digestion of supporting extracellular matrix may trigger apoptosis and in some way increase the branching pattern in the villi. 相似文献
79.
Sean P. Riley Jennifer L. Patterson Samantha Nava Juan J. Martinez 《Cellular microbiology》2014,16(6):849-861
Bacteria of the genus Rickettsia are transmitted from arthropod vectors and primarily infect cells of the mammalian endothelial system. Throughout this infectious cycle, the bacteria are exposed to the deleterious effects of serum complement. Using Rickettsia conorii, the etiologic agent of Mediterranean spotted fever (MSF), as a model rickettsial species, we have previously demonstrated that this class of pathogen interacts with human factor H to mediate partial survival in human serum. Herein, we demonstrate that R. conorii also interacts with the terminal complement complex inhibitor vitronectin (Vn). We further demonstrate that an evolutionarily conserved rickettsial antigen, Adr1/RC1281, interacts with human vitronectin and is sufficient to mediate resistance to serum killing when expressed at the outer‐membrane of serum sensitive Escherichia coli. Adr1 is an integral outer‐membrane protein whose structure is predicted to contain eight membrane‐embedded β‐strands and four ‘loop’ regions that are exposed to extracellular milieu. Site‐directed mutagenesis of Adr1 revealed that at least two predicted ‘loop’ regions are required to mediate resistance to complement‐mediatedkilling and vitronectin acquisition. These results demonstrate that rickettsial species have evolved multiple mechanisms to evade complement deposition and that evasion of killing in serum is an evolutionarily conserved virulence attribute for this genus of obligate intracellular pathogens. 相似文献
80.
Besselsen DG Franklin CL Livingston RS Riley LK 《ILAR journal / National Research Council, Institute of Laboratory Animal Resources》2008,49(3):277-290
Rodent parvoviruses, Helicobacter spp., murine norovirus, and several other previously unknown infectious agents have emerged in laboratory rodents relatively recently. These agents have been discovered serendipitously or through active investigation of atypical serology results, cell culture contamination, unexpected histopathology, or previously unrecognized clinical disease syndromes. The potential research impact of these agents is not fully known. Infected rodents have demonstrated immunomodulation, tumor suppression, clinical disease (particularly in immunodeficient rodents), and histopathology. Perturbations of organismal and cellular physiology also likely occur. These agents posed unique challenges to laboratory animal resource programs once discovered; it was necessary to develop specific diagnostic assays and an understanding of their epidemiology and transmission routes before attempting eradication, and then evaluate eradication methods for efficacy. Even then management approaches varied significantly, from apathy to total exclusion, and such inconsistency has hindered the sharing and transfer of rodents among institutions, particularly for genetically modified rodent models that may not be readily available. As additional infectious agents are discovered in laboratory rodents in coming years, much of what researchers have learned from experiences with the recently identified pathogens will be applicable. This article provides an overview of the discovery, detection, and research impact of infectious agents recently identified in laboratory rodents. We also discuss emerging syndromes for which there is a suspected infectious etiology, and the unique challenges of managing newly emerging infectious agents. 相似文献