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111.
112.
The structure and dynamics of the R6 human insulin hexamer are investigated by two- and three-dimensional homonuclear 1H-NMR spectroscopy. The R6 hexamer, stabilized by Zn2+ and phenol, provides a model of an allosteric protein assembly and is proposed to mimic aspects of receptor recognition. Despite the large size of the assembly (36 kDa), its extreme thermal stability permits high-resolution spectra to be observed at 55 degrees C. Each spin system is represented uniquely, implying either 6-fold symmetry or fast exchange among allowed protomeric conformations. Dramatic changes in chemical shifts and long-range nuclear Overhauser enhancements (NOEs) are observed relative to the spectra of insulin monomers. Complete sequential assignment is obtained and demonstrates native secondary structure with distinctive R-state N-terminal extension of the B-chain alpha-helix (residues B1 to B19). The distance-geometry structure of an R-state promoter is similar to those of R6 crystal structures. Specific long-range intra- and intersubunit NOEs, assigned by stepwise analysis of engineered insulin monomer and dimers, demonstrate that tertiary and quaternary contacts are also similar. Although the hexamer is well-ordered in solution, binding of phenol to an internal cavity occurs within milliseconds, implying the existence of "gatekeeper" residues whose flexibility provides a portal of entry and release. Changes in 1H-NMR chemical shifts on hexamer assembly are readily rationalized by analysis of aromatic ring-currents and provide sensitive probes for sites of protein-protein interaction and phenol binding. Our results provide a foundation for the interaction and phenol binding. Our results provide a foundation for the studies of insulin analogues (such as "designed" insulins of therapeutic interest) under conditions of clinical formulation and for the investigation of the effects of protein assembly on the dynamics of individual protomers. 相似文献
113.
Engels M.; Jacoby E.; Kruger P.; Schlitter J.; Wollmer A. 《Protein engineering, design & selection : PEDS》1992,5(7):669-677
The transition of insulin between its crystallographically definedstates T and R is connected with considerable change even ofbackbone structure: the N-terminal B chain (residues B1 B8) refolds from extended conformation in T into helical inR, and vice versa. Although hitherto observed only in hexamersthe transition of the monomer was adequate for developing andtesting the method of targeted energy minimization(TEM), capable of coping with conformational changes of suchextent at moderate computational expenditure. The simulationis performed in a predetermined number of steps consisting oftwo atomic displacements each, one by force in the directionof the target structure, the second by energy minimization releasingthe constraint caused in the first. The transition pathway isrepresented by the string of energy minimized transient structures.Due to the directedness of the algorithm the simulated pathwayfor R T is not the reversal of that for T R. It is, therefore,not pretended that the minimum energy pathway was identified.In the T R direction the N-terminal B chain first swivels whileremaining largely stretched and then winds up extending thepre-existing helix B9B19. The A chain advances into thespace abandoned and withdraws from it in the R T simulation.In the latter the extended helix first kinks at B8/B9, and thenthe B1 B8 segment is unwound and stretched. The helical H-bondsof that segment are formed late in T R and are maintained duringalmost half of R T. The AN helix is less stable and more involvedin the transitions than helix AC. The two pathways seem plausiblefrom both the energetic and geometric points of view. Knowledgeof them will be of value to suggest mutations to test them byexperimental evidence. 相似文献
114.
P. Sood M. Zachut I. Dekel H. Dube S. Jacoby U. Moallem 《Journal of dairy science》2017,100(11):9372-9381
The varied and elusive etiology of repeat breeding (RB) in dairy cows necessitates evaluation of oocytes and follicles, which have not previously been assessed together. Accordingly, we evaluated characteristics of preovulatory follicles and the competence of oocytes in control (CTL) and RB cows. The estrous cycles of 35 cows (18 CTL and 17 RB) were synchronized using PGF2α and estrus detection. Cows with a corpus luteum were treated with PGF2α and, 14 to 15 d after a visible behavioral estrus, they were administered a second PGF2α, followed 48 h later by follicular fluid (FF) aspiration of the preovulatory follicles. Estradiol (E2)-active preovulatory follicles did not differ in diameter between the 2 groups of cows. However, FF of RB cows had higher E2 concentrations than that of CTL cows: 1,854.9 and 1,073.6 ng/mL, respectively, but similar androstenedione and progesterone concentrations. In the second part of the study, 14 consecutive ovum pick-ups (OPU) were performed in 5 CTL and 5 RB cows, at 3- to 4-d intervals. The RB and CTL cows did not differ in average numbers of follicles available per cow per session (7.1 and 7.3, respectively), oocyte recovery rates (42.2 and 44.1%, respectively), or cleavage rates (57.6 and 63.4%, respectively), but blastocyst production was markedly less in RB than in CTL cows (12.5 and 29.2%, respectively). We conclude that part of the RB cows' etiology occurs at an earlier phase of folliculogenesis, thereby impairing oocyte competence, and subsequently reducing the probability of normal fertilization, which diminish embryo vitality and development. 相似文献
115.
116.
U Rosenschein A Roth T Rassin S Basan S Laniado HI Miller 《Canadian Metallurgical Quarterly》1997,95(6):1411-1416
BACKGROUND: It has been demonstrated that therapeutic ultrasound effects ultrasound thrombolysis by selectively disrupting the fibrin matrix of the thrombus. This study was conducted to evaluate the clinical feasibility of percutaneous transluminal coronary ultrasound thrombolysis in acute myocardial infarction (AMI). METHODS AND RESULTS: Consecutive patients (n = 15) with evidence of anterior AMI and Thrombolysis in Myocardial Infarction (TIMI) grade 0 or 1 flow in the left anterior descending artery underwent coronary ultrasound thrombolysis. Angiographic follow-up was performed after 10 minutes and 12 to 24 hours. Ultrasound induced successful reperfusion (TIMI grade 3 flow) in 87% of the patients. Adjunct percutaneous transluminal coronary angioplasty (PTCA) after ultrasound thrombolysis produced a final residual stenosis of 20 +/- 12% as determined by quantitative coronary angiographic analysis. There were no adverse angiographic signs or clinical effects during the procedure. There was no change in the degree of flow in any of the patients at the 12- to 24-hour angiograms. During hospitalization, 1 patient had recurrent ischemia on the fifth day after the procedure, and emergent catheterization revealed occlusion at the treatment site. The patient was successfully treated with PTCA. CONCLUSIONS: These results suggest that ultrasound thrombolysis has the potential to be a safe and effective catheter-based therapeutic modality in reperfusion therapy for patients with AMI and other clinical conditions associated with intracoronary thrombosis. 相似文献
117.
Examined the contrast between spontaneous and directed recognition by using the flanker paradigm. It was reasoned that spontaneous recognition of a flanking word would be reflected by the influence that word had on recognition of a target word. In a first experiment, when attention was divided at test, recognition decisions were made more rapidly when flanker and target words were congruent, rather than incongruent, with regard to the response they dictated. In later experiments, the authors attempted to specify factors that influence spontaneous recognition of a flanking word and examined the effects of number of prior presentations and physical similarity between study and test. To anticipate, the nature of the results leads to questioning whether recognition is ever truly spontaneous. The findings are discussed in relation to the relativity of automaticity (O. Neumann, 1984). (PsycINFO Database Record (c) 2010 APA, all rights reserved) 相似文献
118.
C Cordon-Cardo A Koff M Drobnjak P Capodieci I Osman SS Millard PB Gaudin M Fazzari ZF Zhang J Massague HI Scher 《Canadian Metallurgical Quarterly》1998,90(17):1284-1291
BACKGROUND: The p27KIP1 gene, whose protein product is a negative regulator of the cell cycle, is a potential tumor suppressor gene; however, no tumor-specific mutations of this gene have been found in humans. This study was undertaken to identify and to assess potential alterations of p27KIP1 gene expression in patients with benign prostatic hyperplasia (BPH) and patients with prostate cancer. METHODS: We analyzed 130 prostate carcinomas from primary and metastatic sites, as well as prostate samples from normal subjects and from patients with BPH. Immunohistochemistry and in situ hybridization were used to determine the levels of expression and the microanatomical localization of p27 protein and messenger RNA (mRNA), respectively. Immunoblotting and immunodepletion assays were performed on a subset of the prostate tumors. Associations between alterations in p27KIP1 expression and clinicopathologic variables were evaluated with a nonparametric test. The Kaplan-Meier method and the logrank test were used to compare disease-relapse-free survival. Prostate tissues of p27Kip1 null (i.e., knock-out) and wild-type mice were also evaluated. RESULTS: Normal human prostate tissue exhibited abundant amounts of p27 protein and high levels of p27KIP1 mRNA in both epithelial cells and stromal cells. However, p27 protein and p27KIP1 mRNA were almost undetectable in epithelial cells and stromal cells of BPH lesions. Furthermore, p27Kip1 null mice developed enlarged (hyperplastic) prostate glands. In contrast to BPH, prostate carcinomas were found to contain abundant p27KIP1 mRNA but either high or low to undetectable levels of p27 protein. Primary prostate carcinomas expressing lower levels of p27 protein appeared to be biologically more aggressive (two-sided P = .019 [Cox regression analysis]). CONCLUSIONS/IMPLICATIONS: On the basis of these results, we infer that loss of p27Kip1 expression in the human prostate may be causally linked to BPH and that BPH is not a precursor to prostate cancer. 相似文献
119.
R McShane K Gedling B Kenward R Kenward T Hope R Jacoby 《Canadian Metallurgical Quarterly》1998,13(8):556-563
BACKGROUND: Patients with dementia who go out unaccompanied are at risk of accidents or getting lost. It is not known whether they could benefit from electronic tracking devices or whether such devices are practically feasible. METHOD: The likely demand for an electronic tracking device was assessed by means of a telephone survey of a convenience sample of 99 carers. The practical feasibility of a tracking system was assessed in 24 patients with dementia. RESULTS: The telephone survey suggested that 20% of patients were at continuing risk of traffic accidents and 45% were at continuing risk of getting lost. About 7% could have benefited from using the device at the time of survey and a further 11% could have benefited at an earlier point in their illness. In the feasibility study, only nine patients consistently used the device. In two patients, it was successfully used in a search. One patient was injured by a passing vehicle when he had got lost out of range of the device. A major barrier to using the device was recognizing the risk of getting lost before it happened. CONCLUSION: Significant numbers of patients are at risk. Electronic tracking devices may occasionally be useful in carefully selected cases. 相似文献
120.