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961.
Stirling PC Bakhoum SF Feigl AB Leroux MR 《Nature structural & molecular biology》2006,13(10):865-870
Molecular chaperones have evolved diverse tertiary and quaternary structures to stabilize non-native polypeptides and facilitate their transition to the native state. Indeed, different families of chaperones lack sequence similarity, and few are represented ubiquitously in all three domains of life. Despite their discrete evolutionary paths, recent crystal structures reveal that many chaperones use seemingly convergent strategies to bind non-native proteins. This crystallographic evidence shows, or strongly suggests, that chaperones including prefoldin, Skp, trigger factor, Hsp40 and Hsp90 have clamp-like structural features used to grip substrate proteins. We explore the notion that clamp-like structures are evolutionarily favored by both ATP-dependent and ATP-independent molecular chaperones. Presumably, clamps present a multivalent binding surface ideal for protecting unstable protein conformers until they reach the native state or are transferred to another component of the folding machinery. 相似文献
962.
Anton Russell Rosabelle Samuel Verena Klejna Michael H. J. Barfuss Barbara Rupp Mark W. Chase 《Annals of botany》2010,106(1):37-56
Background and Aims
Here evidence for reticulation in the pantropical orchid genus Polystachya is presented, using gene trees from five nuclear and plastid DNA data sets, first among only diploid samples (homoploid hybridization) and then with the inclusion of cloned tetraploid sequences (allopolyploids). Two groups of tetraploids are compared with respect to their origins and phylogenetic relationships.Methods
Sequences from plastid regions, three low-copy nuclear genes and ITS nuclear ribosomal DNA were analysed for 56 diploid and 17 tetraploid accessions using maximum parsimony and Bayesian inference. Reticulation was inferred from incongruence between gene trees using supernetwork and consensus network analyses and from cloning and sequencing duplicated loci in tetraploids.Key Results
Diploid trees from individual loci showed considerable incongruity but little reticulation signal when support from more than one gene tree was required to infer reticulation. This was coupled with generally low support in the individual gene trees. Sequencing the duplicated gene copies in tetraploids showed clearer evidence of hybrid evolution, including multiple origins of one group of tetraploids included in the study.Conclusions
A combination of cloning duplicate gene copies in allotetraploids and consensus network comparison of gene trees allowed a phylogenetic framework for reticulation in Polystachya to be built. There was little evidence for homoploid hybridization, but our knowledge of the origins and relationships of three groups of allotetraploids are greatly improved by this study. One group showed evidence of multiple long-distance dispersals to achieve a pantropical distribution; another showed no evidence of multiple origins or long-distance dispersal but had greater morphological variation, consistent with hybridization between more distantly related parents. 相似文献963.
Optimization of square-wave electroporation for transfection of porcine fetal fibroblasts 总被引:2,自引:0,他引:2
Jason W. Ross Jeffrey J. Whyte Jianguo Zhao Melissa Samuel Kevin D. Wells Randall S. Prather 《Transgenic research》2010,19(4):611-620
Development of a transgenic porcine biomedical research model requires effective delivery of DNA into the donor cell followed
by selection of genetically modified somatic cell lines to be used for nuclear transfer. The objective of the current study
was 2-fold: (1) to compare the effectiveness of a single 1 ms pulse of different voltages (V; 100, 150, 200, 250, 300, 350)
and multiple 1 ms pulses (1, 2, 3, 4 or 5) at 300 V for delivery and expression of super-coiled GFP vector in surviving cells
of three fetal fibroblast cell lines, and (2) to determine the ability of these electroporation parameters to produce stably
transfected fibroblast colonies following G418 selection. Cell line (P < 0.001) and voltage (P < 0.001) affected DNA delivery into the cell as assessed by GFP expression while survival at 24 h was affected by voltage
(P < 0.001) and not by cell line (P = 0.797). Using a single pulse while increasing voltage resulted in the percentage of GFP expressing cells increasing from
3.2 ± 0.8% to 43.0 ± 3.4% while survival decreased from 90.5 ± 8.0% to 44.8 ± 2.0%. The number of pulses at 300 V significantly
affected survival (P < 0.001) and GFP expression (P < 0.001). Survival steadily decreased following 1–5 pulses from 63.2 ± 6.3% to 3.0 ± 0.3% with GFP expression of surviving
cells increasing from 35.6 ± 2.67% to 71.4 ± 6.1%. Electroporation of a selectable marker at a 1:1 copy number ratio to a
co-electroporated transgene resulted in 83% of G418 resistant colonies also being PCR positive for the secondary transgene.
These electroporation conditions, specifically, three 1 ms pulses of 300 V to 200 μL of 1 × 106 cells/mL in the presence of 12.5 μg DNA/mL effectively introduced DNA into somatic cells. The utilization of these conditions
produced numerous transgenic fibroblast colonies following G418 selection that when used for somatic cell nuclear transfer
resulted in the production of live offspring. 相似文献
964.
Gabriel Navarrete-Vázquez Sergio Hidalgo-Figueroa Mariana Torres-Piedra Jorge Vergara-Galicia Julio Cesar Rivera-Leyva Samuel Estrada-Soto Ismael León-Rivera Berenice Aguilar-Guardarrama Yolanda Rios-Gómez Rafael Villalobos-Molina Maximiliano Ibarra-Barajas 《Bioorganic & medicinal chemistry》2010,18(11):3985-3991
A series of 1H-benzo[d]imidazole analogues of Pimobendan, substituted at position 5 with either –CF3 or –NO2, were synthesized using a short synthetic route. All the nitro derivatives were potent, and exhibited a concentration- and partial endothelium-dependent vasorelaxant effects, with EC50s <5 μM. 2-Methoxy-4-[5-nitro-1H-benzo[d]imidazol-2-yl]phenol (compound 13) was the most potent derivative of the series, showing an EC50 value of 1.81 μM and Emax of 91.7% for ex vivo relaxant response in intact aortic rings, resulting in a 2.5-fold higher activity compared to Pimobendan. The closely related 5-CF3 analogue (compound 8), was 19 times less potent than 13. The antihypertensive activity of compound 13 was evaluated at doses of 25, 50 and 100 mg kg?1, using spontaneously hypertensive rats (SHR), showing a statistically significant dose-dependent effect. 相似文献
965.
Samuel T. Turvey Leigh A. Barrett Tom Hart Ben Collen Hao Yujiang Zhang Lei Zhang Xinqiao Wang Xianyan Huang Yadong Zhou Kaiya Wang Ding 《Proceedings. Biological sciences / The Royal Society》2010,277(1697):3139-3147
Geographical range contraction is a fundamental ecological characteristic of species population decline, but relatively little investigation has been conducted into general trends in the dynamic properties of range collapse. The Yangtze River dolphin or baiji (Lipotes vexillifer), probably the first large mammal species to have become extinct in over 50 years, was believed to have experienced major range collapse during its decline through progressive large-scale range contraction and fragmentation. This range-collapse model is challenged by a new dataset of 406 baiji last-sighting records collected from across the baiji''s historical range during an interview survey of Yangtze fishing communities. Although baiji regional abundance may have varied across its range, analyses of the extensive new sighting series provide comprehensive evidence that baiji population decline was not associated with any major contraction in geographical range across the middle–lower Yangtze drainage, even in the decade immediately before probable global extinction of the species. Extinction risk in baiji was therefore seemingly not related to evidence of range collapse. Baiji apparently underwent large-scale periodic and seasonal movements across their range, and we propose that range contraction and fragmentation may not be general biogeographic characteristics for declining populations of mobile species in connected landscapes. 相似文献
966.
Kensuke Nakata Samuel Zschokke 《Proceedings. Biological sciences / The Royal Society》2010,277(1696):3019-3025
Almost all spiders building vertical orb webs face downwards when sitting on the hubs of their webs, and their webs exhibit an up–down size asymmetry, with the lower part of the capture area being larger than the upper. However, spiders of the genus Cyclosa, which all build vertical orb webs, exhibit inter- and intraspecific variation in orientation. In particular, Cyclosa ginnaga and C. argenteoalba always face upwards, and C. octotuberculata always face downwards, whereas some C. confusa face upwards and others face downwards or even sideways. These spiders provide a unique opportunity to examine why most spiders face downwards and have asymmetrical webs. We found that upward-facing spiders had upside-down webs with larger upper parts, downward-facing spiders had normal webs with larger lower parts and sideways-facing spiders had more symmetrical webs. Downward-facing C. confusa spiders were larger than upward- and sideways-facing individuals. We also found that during prey attacks, downward-facing spiders ran significantly faster downwards than upwards, which was not the case in upward-facing spiders. These results suggest that the spider''s orientation at the hub and web asymmetry enhance its foraging efficiency by minimizing the time to reach prey trapped in the web. 相似文献
967.
Seth H Sheldon Joseph J Gard Samuel J Asirvatham 《Indian pacing and electrophysiology journal》2010,10(8):357-371
Premature ventricular contractions (PVCs) and non-sustained ventricular tachycardia (NSVT) are frequently encountered and a marker of electrocardiomyopathy. In some instances, they increase the risk for sustained ventricular tachycardia, ventricular fibrillation, and sudden cardiac death. While often associated with a primary cardiomyopathy, they have also been known to cause tachycardia-induced cardiomyopathy in patients without preceding structural heart disease. Medical therapy including beta-blockers and class III anti-arrhythmic agents can be effective while implantable cardiac defibrillators (ICD) are indicated in certain patients. Radiofrequency ablation (RFA) is the preferred, definitive treatment in those patients that improve with anti-arrhythmic therapy, have tachycardia-induced cardiomyopathy, or have certain subtypes of PVCs/NSVT. We present a review of PVCs and NSVT coupled with case presentations on RFA of fascicular ventricular tachycardia, left-ventricular outflow tract ventricular tachycardia, and Purkinje arrhythmia leading to polymorphic ventricular tachycardia. 相似文献
968.
Mark A. Arbing Samuel K. Handelman Alexandre P. Kuzin Grégory Verdon Chi Wang Min Su Francesca P. Rothenbacher Mariam Abashidze Mohan Liu Jennifer M. Hurley Rong Xiao Thomas Acton Masayori Inouye Gaetano T. Montelione Nancy A. Woychik John F. Hunt 《Structure (London, England : 1993)》2010,18(8):996-1010
969.
Herpesviruses enter cells by membrane fusion either at the plasma membrane or in endosomes, depending on the cell type. Glycoprotein B (gB) is a conserved component of the multiprotein herpesvirus fusion machinery and functions as a fusion protein, with two internal fusion loops, FL1 and FL2. We determined the crystal structures of the ectodomains of two FL1 mutants of herpes simplex virus type 1 (HSV-1) gB to clarify whether their fusion-null phenotypes were due to global or local effects of the mutations on the structure of the gB ectodomain. Each mutant has a single point mutation of a hydrophobic residue in FL1 that eliminates the hydrophobic side chain. We found that neither mutation affected the conformation of FL1, although one mutation slightly altered the conformation of FL2, and we conclude that the fusion-null phenotype is due to the absence of a hydrophobic side chain at the mutated position. Because the ectodomains of the wild-type and the mutant forms of gB crystallized at both low and neutral pH, we were able to determine the effect of pH on gB conformation at the atomic level. For viruses that enter cells by endocytosis, the low pH of the endosome effects major conformational changes in their fusion proteins, thereby promoting fusion of the viral envelope with the endosomal membrane. We show here that upon exposure of gB to low pH, FL2 undergoes a major relocation, probably driven by protonation of a key histidine residue. Relocation of FL2, as well as additional small conformational changes in the gB ectodomain, helps explain previously noted changes in its antigenic and biochemical properties. However, no global pH-dependent changes in gB structure were detected in either the wild-type or the mutant forms of gB. Thus, low pH causes local conformational changes in gB that are very different from the large-scale fusogenic conformational changes in other viral fusion proteins. We propose that these conformational changes, albeit modest, play an important functional role during endocytic entry of HSV. 相似文献
970.
William S. Mason Chen Liu Carol E. Aldrich Samuel Litwin Matthew M. Yeh 《Journal of virology》2010,84(16):8308-8315
Chronic hepatitis B virus (HBV) infections are associated with persistent immune killing of infected hepatocytes. Hepatocytes constitute a largely self-renewing population. Thus, immune killing may exert selective pressure on the population, leading it to evolve in order to survive. A gradual course of hepatocyte evolution toward an HBV-resistant state is suggested by the substantial decline in the fraction of infected hepatocytes that occurs during the course of chronic infections. Consistent with hepatocyte evolution, clones of >1,000 hepatocytes develop postinfection in the noncirrhotic livers of chimpanzees chronically infected with HBV and of woodchucks infected with woodchuck hepatitis virus (W. S. Mason, A. R. Jilbert, and J. Summers, Proc. Natl. Acad. Sci. U. S. A. 102:1139-1144, 2005; W. S. Mason et al., J. Virol. 83:8396-8408, 2009). The present study was carried out to determine (i) if extensive clonal expansion of hepatocytes also occurred in human HBV carriers, particularly in the noncirrhotic liver, and (ii) if clonal expansion included normal-appearing hepatocytes, not just hepatocytes that appear premalignant. Host DNA extracted from fragments of noncancerous liver, collected during surgical resection of hepatocellular carcinoma (HCC), was analyzed by inverse PCR for randomly integrated HBV DNA as a marker of expanding hepatocyte lineages. This analysis detected extensive clonal expansion of hepatocytes, as previously found in chronically infected chimpanzees and woodchucks. Tissue sections were stained with hematoxylin and eosin (H&E), and DNA was extracted from the adjacent section for inverse PCR to detect integrated HBV DNA. This analysis revealed that clonal expansion can occur among normal-appearing human hepatocytes.Transient hepatitis B virus (HBV) infections, which generally last <6 months, do not cause cirrhosis and cause only minor increases in the risk of hepatocellular carcinoma (HCC) (3, 46). Chronic infections, typically lifelong, can cause cirrhosis and HCC (3). Of the ∼350 million HBV carriers now alive, ca. 60 million will die prematurely of cirrhosis and/or HCC. Cirrhosis, which usually develops late in infection, is a significant risk factor for HCC. Early reports stated that most HCCs occur on a background of cirrhosis. However, later studies suggested that as many as 50% of HCCs may occur in noncirrhotic liver (4), that is, in patients in whom the progression of liver disease still appears rather mild. Thus, liver damage that appears severe by histologic examination is not a prerequisite for HCC.Interestingly, during chronic HBV infections there is, in the face of persistent viremia, a decline over time in the fraction of infected hepatocytes, from 100% to as little as a few percent (5, 12-14, 16, 17, 22, 23, 27, 34, 37, 38). Along with HCC, this is perhaps the most surprising and unexplained outcome of chronic infection. The timing of this decline has not been systematically studied, but it is presumably gradual, occurring over years or decades, and dependent on persistent, albeit low-level, killing of infected hepatocytes by antiviral cytotoxic T lymphocytes (CTLs) (20). It is believed that the liver is largely a closed, self-renewing population. Such a population might be expected to evolve under any strong or persistent selective pressure. In HBV-infected patients, the earliest and most persistent selective pressure is immune killing of infected hepatocytes, which should initially constitute the entire hepatocyte population. Persistent killing of HBV-infected hepatocytes could lead to clonal expansion of mutant or epigenetically altered hepatocytes that had lost the ability to support infection and that were not, therefore, targeted by antiviral CTLs.Such a selective pressure may explain why foci of altered hepatocytes (FAH) and HCC are typically virus negative (1, 6, 11, 26, 29, 31, 35, 40, 41, 44). Normal or preneoplastic hepatocytes (e.g., in FAH) that have evaded the host immune response should undergo clonal expansion, because their death rate is lower than that of surrounding hepatocytes, even if they do not have a higher growth rate. Indeed, clonal expansion of hepatocytes has been detected, in the absence of cirrhosis, in woodchucks chronically infected with woodchuck hepatitis virus (WHV) (19) and in chimpanzees chronically infected with HBV (21). The presence of discrete foci of normal-appearing but virus-negative hepatocytes in chronically infected woodchuck livers (39) suggested, but did not prove, that normal-appearing hepatocytes that had lost the ability to support virus replication might clonally expand.The purpose of the present study was, therefore, to determine if normal-appearing hepatocytes undergo clonal expansion. To address this issue, we focused on noncirrhotic livers, because hepatocyte appearance and organization in many cirrhotic nodules are often considered to indicate premalignancy (7, 24, 25, 44), and this, together with the cellular environment in the cirrhotic liver, may explain why as many as 50% of cirrhotic nodules have been found to be made up of clonally expanded hepatocytes (2, 18, 24, 25, 28, 44). In older HBV patients, cirrhosis, the result of cumulative scarring due to ongoing tissue injury, presumably produces an evolutionary pressure on the hepatocyte population due to restricted blood flow and altered hepatic architecture.Clonal expansion was detected by assaying for integrated HBV DNA by inverse PCR (19, 21). Because integration occurs at random sites in host DNA, each integration event provides a unique genetic marker for the cell in which it occurred, and for any daughter cells. Thus, the clonal expansion of these tagged hepatocytes can be measured by determining how many times a given virus-cell DNA junction is repeated in a liver fragment. Analysis of fragments of nontumorous liver from noncirrhotic HCC patients revealed that at least 1% of hepatocytes are present as clones of >1,000 cells. Examination of 5-μm-thick sections of paraffin-embedded livers from the same patients revealed that clonally expanded hepatocytes were present in liver sections lacking preneoplastic lesions or changes. Therefore, normal-appearing hepatocytes must have undergone clonal expansion. Although clonal expansion was detected by analysis of integrated HBV DNA, the expansion did not appear to be due to the site of integration of the viral DNA into host DNA.These results are consistent with the hypothesis that immune selection and the later emergence of liver cirrhosis, with altered lobular organization and restricted blood flow, may constitute the two major selective pressures on the hepatocyte population that culminate in hepatocellular carcinoma. More-direct proof of the role, if any, of immune selection in hepatocyte evolution and HCC will require, first of all, an assay with a greater ability to detect clonally expanded hepatocytes. The present approach is limited by a number of factors, including a need for integration near a particular restriction endonuclease cleavage site in host DNA and for conservation of particular viral sequences so that the integrated DNA can be amplified using the PCR primers chosen. These issues may explain why the fraction of clonally expanded hepatocytes reported here is much less than that suggested by histologic data showing that more than 50% of hepatocytes appear negative for virus replication in long-term carriers. Further dissection of this issue will also require localization and determination of the virologic status of hepatocyte clones present in tissue sections. 相似文献