A hydrogen-bonded network is observed above the hemes in all of the high-resolution crystal structures of cytochrome oxidases. It includes water and a pair of arginines, R481 and R482 (Rhodobacter ...sphaeroides numbering), that interact directly with heme a and the heme a 3 propionates. The hydrogen-bonded network provides potential pathways for proton release. The arginines, and the backbone peptide bond between them, have also been proposed to form part of a facilitated electron transfer route between CuA and heme a. Our studies show that mutations of R482 (K, Q, and A) and R481 (K) retain substantial activity and are able to pump protons, but at somewhat reduced rates and stoichiometries. A slowed rate of electron transfer from cytochrome c to CuA suggests a change in the orientation of cytochrome c binding in all but the R to K mutants. The mutant R482P is more perturbed in its structure and is altered in the redox potential difference between heme a and CuA: +18 mV for R482P and +46 mV for the wild type (heme a − CuA). The electron transfer rate between CuA and heme a is also altered from 93000 s-1 in the wild type to 50 s-1 in the oxidized R482P mutant, reminiscent of changes observed in a CuA-ligand mutant, H260N. In neither case is the ∼2000-fold change in the rate accounted for by the altered redox potentials, suggesting that both cause a major modification in the path or reorganization energy of electron transfer.
Software Dynamic Translation (SDT) systems are used for program instrumentation, dynamic optimization, security, intrusion detection, and many other uses. As noted by many researchers, a major source ...of SDT overhead is the execution of code which is needed to translate an indirect branch's target address into the address of the translated destination block. This paper discusses the sources of indirect branch (IB) overhead in SDT systems and evaluates several techniques for overhead reduction. Measurements using SPEC CPU2000 show that the appropriate choice and configuration of IB translation mechanisms can significantly reduce the IB handling overhead. In addition, cross-architecture evaluation of IB handling mechanisms reveals that the most efficient implementation and configuration can be highly dependent on the implementation of the underlying architecture.
This article compares intergroup and intragroup clinical and morphologic findings in patients with ischemic cardiomyopathy (IC), idiopathic dilated cardiomyopathy (IDC), and dilated hypertrophic ...cardiomyopathy (HC) undergoing cardiac transplantation (CT). Few previous publications have described findings in native hearts explanted at the time of CT. The explanted heart in 92 patients having CT was examined in uniform manner with particular attention to the sizes of the ventricular cavities and the presence of and extent of ventricular scarring. Of the 92 hearts examined, 47 had IC, 35 had IDC, and 10 had dilated HC. Although considerable degrees of intragroup variation occurred, the mean degree of left ventricular dilatation was similar among the patients with IC, IDC, and dilated HC. All patients with IC had left ventricular free wall scarring more extensive than that involving the ventricular septum, but the intragroup variation in the amounts of scarring was considerable. Nine of the 10 patients with dilated HC also had ventricular wall scarring, but it was more extensive in the ventricular septum than in the left ventricular free wall and involvement of the right ventricular wall also was present. Eight (23%) of the 35 IDC patients also had grossly visible ventricular scars but they were small and only 1 of the 8 had coronary narrowing and that was not in the distribution of the scarring. Narrowing of 1 or more epicardial coronary arteries >75% in cross-sectional area by plaque was present in all 47 IC patients, in 8 of the 35 IDC patients (7 had no ventricular scars), and in none of the 10 dilated HC patients. Coronary angiography was the major clinical tool allowing separation of the IC, IDC, and HC patients. Coronary angiography did not detect narrowing in any of the 8 patients with IDC who were found to have coronary narrowing on anatomic study. Thus, among patients with IC, IDC, and dilated HC having CT, distinctive anatomic features allow separation of patients with IC, IDC, and dilated HC, but within each group considerable variation in left ventricular cavity size and extent of ventricular scarring occurs.
The aa(3)-type cytochrome c oxidase of Rhodobacter sphaeroides, a proteobacterium of the alpha subgroup, is structurally similar to the core subunits of the terminal oxidase in the mitochondrial ...electron transport chain. Subunit I, the product of the coxI gene, normally binds two heme A molecules. A deletion of cox10, the gene for the farnesyltransferase required for heme A synthesis, did not prevent high level accumulation of subunit I in the cytoplasmic membrane. Thus, subunit I can be expressed and stably inserted into the cytoplasmic membrane in the absence of heme A. Aposubunit I was purified via affinity chromatography to a polyhistidine tag. Copurification of subunits II and III with aposubunit I indicated that assembly of the core oxidase complex occurred without the binding of heme A. In addition to formation of the apooxidase containing all three large structural proteins, CoxI-II and CoxI-III heterodimers were isolated from cox10 deletion strains harboring expression plasmids with coxI and coxII or with coxI and coxIII, respectively. This demonstrated that subunit assembly of the apoenzyme was not an inherently ordered or sequential process. Thus, multiple paths must be considered for understanding the assembly of this integral membrane metalloprotein complex.
Antimitotic chemotherapeutic agents target tubulin, the major protein in mitotic spindles. Tubulin isotype composition is thought to be both diagnostic of tumor progression and a determinant of the ...cellular response to chemotherapy. This implies that there is a difference in isotype composition between normal and tumor tissues.
To determine whether such a difference occurs in breast tissues, total tubulin was fractionated from lysates of paired normal and tumor breast tissues, and the amounts of beta-tubulin classes I + IV, II, and III were measured by competitive enzyme-linked immunosorbent assay (ELISA). Only primary tumor tissues, before chemotherapy, were examined. Her2/neu protein amplification occurs in about 30% of breast tumors and is considered a marker for poor prognosis. To gain insight into whether tubulin isotype levels might be correlated with prognosis, ELISAs were used to quantify Her2/neu protein levels in these tissues.
Beta-tubulin isotype distributions in normal and tumor breast tissues were similar. The most abundant beta-tubulin isotypes in these tissues were beta-tubulin classes II and I + IV. Her2/neu levels in tumor tissues were 5-30-fold those in normal tissues, although there was no correlation between the Her2/neu biomarker and tubulin isotype levels.
These results suggest that tubulin isotype levels, alone or in combination with Her2/neu protein levels, might not be diagnostic of tumorigenesis in breast cancer. However, the presence of a broad distribution of these tubulin isotypes (for example, 40-75% beta-tubulin class II) in breast tissue, in conjunction with other factors, might still be relevant to disease progression and cellular response to antimitotic drugs.
Cytochrome
c oxidase moves both electrons and protons in its dual role as a terminal electron acceptor and a contributor to the proton motive force which drives the formation of ATP. Although the ...sequence of electron transfer events is well-defined, the correlated mechanism and routes by which protons are translocated across the membrane are not. A recent model Michel, Proc. Natl. Acad. Sci. USA 95 (1998) 12819 offers a detailed molecular description of when and how protons are translocated through the protein to the outside, which contrasts with previous models in several respects. This article reviews the behavior of site-directed mutants of
Rhodobacter sphaeroides cytochrome
c oxidase in the context of these different models. Studies of the internally located lysine 362 on the K channel and aspartate 132 on the D channel, indicate that D132, but not K362, is connected to the exterior region. Analysis of the externally located arginine pair, 481 and 482, and the Mg/Mn ligands, histidine 411 and aspartate 412, which are part of the hydrogen-bonded network that includes the heme propionates, indicates that alterations in this region do not strongly compromise proton pumping, but do influence the pH dependence of overall activity and the control of activity by the pH gradient. The results are suggestive of a region of ‘sequestered’ protons: beyond a major energetic gate, but selectively responsive to the external environment.
Genetic manipulation of the aa 3-type cytochrome c oxidase of Rhodobacter sphaeroides was used to determine the minimal structural subunit associations required for the assembly of the heme A and ...copper centers of subunit I. In the absence of the genes for subunits II and III, expression of the gene for subunit I in Rb. sphaeroides allowed purification of a form of free subunit I (subunit I a ) that contained a single heme A. No copper was present in this protein, indicating that the heme a 3−CuB active site was not assembled. In cells expressing the genes for subunits I and II, but not subunit III, two oxidase forms were synthesized that were copurified by histidine affinity chromatography and separated by anion-exchange chromatography. One form was a highly active subunit I−II oxidase containing a full complement of structurally normal metal centers. This shows that association of subunit II with subunit I is required for stable formation of the active site in subunit I. In contrast, subunit III is not required for the formation of any of the metal centers or for the production of an oxidase with wild-type activity. The second product of the cells lacking subunit III was a large amount of a free form of subunit I that appeared identical to subunit I a . Since significant amounts of subunit I a were also isolated from wild-type cells, it is likely that subunit I a will be present in any preparation of the aa 3-type oxidase isolated via an affinity tag on subunit I.
In recent years, analysts have been adding considerable detail to their finite element models in order to replicate actual physical behaviour with greater accuracy. One such detail is the connections ...between parts. Meshing of such models, although not quick, can be done without much difficulty. What can be difficult, however, is accurately capturing the interaction of all of the parts that compose the connection. Friction plays an important role in that interaction. Therefore, this paper discusses the fundamentals of friction modelling between solid elements in LS-DYNA. However, the discussion is applicable to most nonlinear finite element analysis codes used for crashworthiness analysis. Understanding the details behind friction modelling will improve an analyst's modelling skills and thus, lead to quicker and better designs.
Two recessive alleles of ERG10 and three temperature-sensitive recessive alleles of HMG1 (3-hydroxy-3-methyl-glutaryl-CoA reductase isoenzyme 1) were isolated in a screen for mevalonate auxotrophs in ...Saccharomyces cerevisiae. The essential, single-copy ERG10 gene was cloned by complementation of the temperature-sensitive phenotype of erg10-21. The 1,194-base pair continuous open reading frame, encoding a 398-amino acid polypeptide with a calculated molecular mass of 41,681 daltons, was demonstrated to encode cytoplasmic aceto-acetyl-CoA thiolase. Acetoacetyl-CoA thiolase activity corresponded to the number of copies of ERG10 present in cell extracts, and null alleles of ERG10 produced no detectable acetoacetyl-CoA thiolase enzyme activity. The deduced amino acid sequence was 40-95% identical to acetoacetyl-CoA thiolases from other organisms. This identity included the active site cysteines located at amino acids 91 and 384 in the Erg10 protein.
Modeling slip base mechanisms Hiser, N R; Reid, J D
International journal of crashworthiness,
01/2005, Letnik:
10, Številka:
5
Journal Article
Recenzirano
Slip base mechanisms are commonly used in breakaway devices for the roadside safety industry; for example, the base of structures such as sign posts, luminaire poles, and guardrail system posts are ...often slip base mechanisms. Due to the breakaway feature of the slip base, it has considerable potential for reducing the amount of crash resistance, and thus occupant injury, when vehicles inadvertently leave the roadway and strike these objects. The objective of this study was to develop improved methods of modeling slip base structures using nonlinear, finite element analysis. Two bolt preloading techniques were developed and evaluated, including one method which utilized a discrete spring element, and a second method which utilized pre-stressed bolt shaft elements. Both methods produced desired bolt preload, as well as acceptable slip behavior when impacted in a simple shear scenario. However, when the impact conditions became more severe, the model using pre-stressed solid elements was significantly more accurate. The stress-based clamping model was then incorporated into a new cable guardrail system model. Performance of the slip base model was acceptable in both end-on impact and length-of-need impact simulations. Thus, the model was deemed appropriate for use in further development and analysis of existing cable systems, as well as alternate slip base applications.
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