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Escherichia coli carbamoyl-phosphate synthetase (CPSase) is comprised of a 40-kDa glutaminase (GLN) and a 120-kDa synthetase (CPS) subunit. The CPS subunit consists of two homologous domains, CPS.A and CPS.B, which catalyze the two different ATP-dependent partial reactions involved in carbamoyl phosphate synthesis. Sequence similarities and controlled proteolysis experiments suggest that the CPS subdomains consist, in turn, of three subdomains, designated A1, A2, A3 and B1, B2, B3 for CPS.A and CPS.B, respectively. Previous studies of individually cloned CPS.A and CPS.B from E. coli and mammalian CPSase have shown that homologous dimers of either of these “half-molecules” could catalyze all three reactions involved in ammonia-dependent carbamoyl phosphate synthesis. Four smaller recombinant proteins were made for this study as follows: 1) A1-A2 in which the A3 subdomain was deleted from CPS.A, 2) B1-B2 lacking subdomain B3 of CPS.B, 3) the A2 subdomain of CPS.A, and 4) the B2 subdomain of CPS.B. When associated with the GLN subunit, A1-A2 and B1-B2 had both glutamine- and ammonia-dependent CPSase activities comparable to the wild-type protein. In contrast, the 27-kDa A2 and B2 recombinant proteins, which represent only 17% of the mass of the parent protein, were unable to use glutamine as a nitrogen donor, but the ammonia-dependent activity was enhanced 14–16-fold. The hyperactivity suggests that A2 and B2 are the catalytic subdomains and that A1 and B1 are attenuation domains which suppress the intrinsically high activity and are required for the physical association with the GLN subunit.

DC magnetron sputter deposition of copper thin films is evaluated for resistivity, conformality, etc., in this work. Sputter deposition is attractive as an alternative to chemical vapor deposition because sputtering is a well-characterized and cost effective technique which is already widely implemented. Conformal copper deposition may be feasible since copper reflow at temperatures much lower than the melting point of 1083°C has been observed by a number of workers. In this case, an attractive approach is sputter deposition of copper at a sufficiently high temperature so that the need for a separate reflow anneal is eliminated. In the present work, it was found that better conformality is achieved with lower deposition rate, higher pressure, lower sputter power, and lower bias voltage. However, low sputter power may affect adhesion of the copper film to an underlying barrier layer such as TiN, and, of course, the associated low deposition rate affects overall throughput undesirably. Good gap filling of 1:1 aspect ratios with a 0.5 μm opening is achievable with copper sputtering, but a practical manufacturing process remains to be demonstrated since a high defect density has been observed in copper conductors fabricated with conformal sputtering and chemical-mechanical polishing.

Recently arrived and settled Salvadoran refugees and Anglo-Canadians in London, Ontario were compared with respect to psychological distress, quality of life, and life satisfaction. A matched sample of 60 participants in each group completed a demographic questionnaire, the Brief Symptom Inventory, a shortened version of the Quality of Life Questionnaire, and the Satisfaction with Life Scale in either English or Spanish. Significant group differences were obtained on measures of quality of life and life satisfaction, but not of psychological distress. The results are discussed in relation to findings with other refugee groups. Cette etude compare la detresse psychologique, la qualite de vie et la satisfaction par rapport aux conditions de vie chez refugies du Salvador et chez des anglo-canadiens recemment arrives et installes depuis quelques annees dans la ville de London, Ontario. Un echantillon apparie de 60 participants par groupe (recemment arrives versus installes depuis quelques annees) completait e...

Escherichia coli carbamyl-phosphate synthetase consists of two subunits that act in concert to synthesize carbamyl phosphate. The 40-kDa subunit is an amidotransferase (GLN subunit) that hydrolyzes glutamine and transfers ammonia to the 120-kDa synthetase subunit (CPS subunit). The enzyme can also catalyze ammonia-dependent carbamyl phosphate synthesis if provided with exogenous ammonia. In mammalian cells, homologous amidotransferase and synthetase domains are carried on a single polypeptide chain called CAD. Deletion of the 29-residue linker that bridges the GLN and CPS domains of CAD stimulates glutamine-dependent carbamyl phosphate synthesis and abolishes the ammonia-dependent reaction (Guy, H. I., and Evans, D. R. (1997) J. Biol. Chem. 272, 19906-19912), suggesting that the deletion mutant is trapped in a closed high activity conformation. Since the catalytic mechanisms of the mammalian and bacterial proteins are the same, we anticipated that similar changes in the function of the E. coli protein could be produced by direct fusion of the GLN and CPS subunits. A construct was made in which the intergenic region between the contiguous carA and carB genes was deleted and the sequences encoding the carbamyl-phosphate synthetase subunits were fused in frame. The resulting fusion protein was activated 10-fold relative to the native protein, was unresponsive to the allosteric activator ornithine, and could no longer use ammonia as a nitrogen donor. Moreover, the functional linkage that coordinates the rate of glutamine hydrolysis with the activation of bicarbonate was abolished, suggesting that the protein was locked in an activated conformation similar to that induced by the simultaneous binding of all substrates.

Health Promotion, Wellness Programs, Quality of Life and the Marketing of PsychologyDAVID R. EVANSThe University of Western OntarioAbstractThis paper focusses on fifteen years of research into the definition of quality of life and the factors that influence it. The initial phase of the research involving the development of the Quality of Life Questionnaire and the identification of general and specific factors that impact on life quality is considered. As governments and companies seek to reduce health costs, attention has turned to the potential impact of health promotion programs, and wellness programs on health costs. Hence, a second aim of this paper is to discuss the application of the research to health promotion and the development of wellness programs. A strategy to enhance quality of life using the Quality of Life Questionnaire, and group interventions to enhance specific personality characteristics are described. Finally, the current research program is used as a case study to demonstrate some of the factors that contribute to the invisibility of psychology.In the past few decades there has been considerable interest in health promotion both here in Canada (Epp, 1986), in the United States (United States Department of Health, Education, and Welfare, 1979) and Internationally (World Health Organization, 1984). Health promotion is the process of assisting people to move toward a state of optimal health (Alonzo, 1993; Epp, 1986). There has been increased emphasis on the development of health promotion programs since the member states of the World Health Organization adopted the goal of "Health for All by the Year 2000" (Kickbusch, 1987). Several authors have equated the goal of health promotion with the enhancement of quality of life and the achievement of optimal well - being (Alonzo, 1993; Elias & Murphy, 1986; Epp, 1986; Herbert, 1995; Sullivan, 1991). Hence, it can be argued that one avenue to the development of health promotion programs is the development of programs to enhance quality of life (Evans, 1994, 1995, July).THE DEFINITION AND MEASUREMENT OF QUALITY OF LIFEThe first step in any research program is to define the central constructs and to develop valid measures of the constructs. The definition of quality of life has been quite elusive despite the extensive literature and research in the area (Evans, 1994; Romney, & Evans, 1996; Romney, Jenkins, & Bynner, 1992, Faden, & Leplege, 1992). Quality of life has been used interchangeably with such terms as well - being, psychological well - being, happiness, life satisfaction, positive and negative affect, and the good life (Cheng, 1988, Evans, 1994, George, 1992). In an effort to develop a behaviourally based multidimensional measure of quality of life Evans and his colleagues developed the Quality of Life Questionnaire (Evans, Burns, Robinson, & Garrett, 1985; Evans, & Cope, 1989). The methodology utilized in developing the questionnaire was the rational - empirical approach advocated by Jackson (1970). The Quality of Life Questionnaire is comprised of 15 twelve item scales designed to assess material well - being, physical well - being, personal growth, marital relations, parent - child relations, extended family relations, extrafamilial relations, altruistic behaviour, political behaviour, job characteristics, occupational relations, job satisfiers, creative/aesthetic behaviour, sports activity, and vacation behaviour. A scoring procedure permits the derivation of a prorated Quality of Life Score based on those scales which are relevant to the individual (Evans, & Cope, 1989). A paper and pencil, and a computer version of the questionnaire are available.Evans (1994) reported preliminary data on the relationship between a range of global quality of life measures, including the Quality of Life Questionnaire (Evans, & Cope, 1989), the Perceived Quality of Life Scales (Pellizzari, 1992), the Positive and Negative Affect Scale (Watson, Clark, & Tellegen, 1988), and the Satisfaction with Life Scale (Diener, Emmons, Larsen, & Griffin,1985). …

Background Dihydroorotase (DHO) is a zinc metalloenzyme, although the number of active site zinc ions has been controversial. E. coli DHO was initially thought to have a mononuclear metal center, but the subsequent X-ray structure clearly showed two zinc ions, α and β, at the catalytic site. Aquifex aeolicus DHO, is a dodecamer comprised of six DHO and six aspartate transcarbamoylase (ATC) subunits. The isolated DHO monomer, which lacks catalytic activity, has an intact α-site and conserved β-site ligands, but the geometry of the second metal binding site is completely disrupted. However, the putative β-site is restored when the complex with ATC is formed and DHO activity is regained. Nevertheless, the X-ray structure of the complex revealed a single zinc ion at the active site. The structure of DHO from the pathogenic organism, S. aureus showed that it also has a single active site metal ion. Results Zinc analysis showed that the enzyme has one zinc/DHO subunit and the addition of excess metal ion did not stimulate catalytic activity, nor alter the kinetic parameters. The metal free apoenzyme was inactive, but the full activity was restored upon the addition of one equivalent of Zn2+ or Co2+. Moreover, deletion of the β-site by replacing the His180 and His232 with alanine had no effect on catalysis in the presence or absence of excess zinc. The 2.2 Å structure of the double mutant confirmed that the β-site was eliminated but that the active site remained otherwise intact. Conclusions Thus, kinetically competent A. aeolicus DHO has a mononuclear metal center. In contrast, elimination of the putative second metal binding site in amidohydrolyases with a binuclear metal center, resulted in the abolition of catalytic activity. The number of active site metal ions may be a consideration in the design of inhibitors that selectively target either the mononuclear or binuclear enzymes.

Laser produced plasma (LPP) systems have been developed as the primary approach for use in EUV scanner light sources for optical imaging of circuit features at 20nm nodes and beyond. This paper provides a review of development progress and productization status for LPP extreme-ultra-violet (EUV) sources with performance goals targeted to meet specific requirements from ASML. We present the latest results on power generation and collector protection for sources in the field operating at 10W nominal power and in San Diego operating in MOPA (Master Oscillator Power Amplifier) Prepulse mode at higher powers. Semiconductor industry standards for reliability and source availability data are provided. In these proceedings we show results demonstrating validation of MOPA Prepulse operation at high dose-controlled power: 40 W average power with closed-loop active dose control meeting the requirement for dose stability, 55 W average power with closed-loop active dose control, and early collector protection tests to 4 billion pulses without loss of reflectivity.

The amidotransferase domain (GLNase) of mammalian carbamyl-phosphate synthetase II hydrolyzes glutamine and transfers ammonia to the synthetase domain where carbamyl phosphate is formed in a three-step reaction sequence. The synthetase domain consists of two homologous subdomains, CPS.A and CPS.B. Recent studies suggest that CPS.A catalyzes the initial ATP dependent-activation of bicarbonate, whereas CPS.B uses a second ATP to form carbamyl phosphate. To establish the function of these substructural elements, we have cloned and expressed the mammalian protein and its subdomains in Escherichia coli. Recombinant CPSase (GLNase-CPS.A-CPS.B) was found to be fully functional. Two other proteins were made; the first consisted of only GLNase and CPS.A, whereas the second lacked CPS.A and had the GLNase domain fused directly to CPS.B. Remarkably, both proteins catalyzed the entire series of reactions involved in glutamine-dependent carbamyl phosphate synthesis. The stoichiometry, like that of the native enzyme, was 2 mol of ATP utilized per mol of carbamyl phosphate formed. GLN-CPS.B is allosterically regulated, whereas GLN-CPS.A was insensitive to effectors, a result consistent with evidence showing that allosteric effectors bind to CPS.B. These properties are not peculiar to the mammalian protein, because the separately cloned CPS.A subdomain of the E. coli enzyme was also found to catalyze carbamyl phosphate synthesis. Gel filtration chromatography and chemical cross-linking studies showed that these molecules are dimers, a structural organization that may be a prerequisite for the overall reaction. Thus, the homologous CPS.A and CPS.B subdomains are functionally equivalent, although in the native enzyme they may have different functions resulting from their juxtaposition relative to the other components in the complex.

Deposition of nickel silicide nanowires has been achieved in the temperature range of 320 to 420 °C by decomposition of silane on nickel surfaces. The substrates consisted of Ni foils and thin Ni films (∼10–100 nm) evaporated on 1-μm-thick layers of SiO2 predeposited on Si wafers. Nanowire growth between two metal pads was achieved with aid of an electric field. It was found that thinner diameter nanowires were produced at low temperatures and that the density of the nanowires was dependent on the reactor pressure. The current–voltage relationship of these nanowires has also been examined.

The use of high aspect ratio contact structures with electroplated gold interconnects on state-of-the-art bipolar integrated circuits requires very high performance diffusion barriers. In the present work, these are obtained by adding nitrogen to the sputtering ambient and optimizing the substrate temperature during deposition. This results in performance not obtainable previously with this material system.

Dihydroorotase (DHOase, EC 3.5.2.3) catalyzes the reversible cyclization of carbamyl aspartate to form dihydroorotate, the third step in de novo pyrimidine biosynthesis. In mammals this activity is carried by the zinc-containing domain of the 243 kDa multifunctional protein CAD. We have replaced conserved residues in the cloned 46 kDa DHOase domain by site-directed mutagenesis. Mutants His1471Ala and His1473Ala lacked catalytic activity, judging by their failure to complement a DHOase-deficient Escherichia coli strain, and were unable to coordinate the active site zinc ion in zinc blotting experiments. This result confirmed earlier predictions. A mutant protein in which the third suspected zinc ligand was changed, Glu1512Asn, had a kcat similar to that of the intact CAD molecule and a Km similar to that of the wild-type recombinant DHOase, observations that argue against a role for glutamate 1512 in catalysis. Mutant His1590Asn had no measurable catalytic activity. This histidine residue was tentatively identified as the third zinc ligand by the failure of the mutant to bind the full complement of zinc in atomic absorption measurements. Mutant His1690Asn had a kcat 34-fold lower and a Km 9-fold higher than those of wild-type recombinant. The kinetic parameters of the mutant His1642Asn were also altered, but to a lesser extent. Diethyl pyrocarbonate (DEPC) was shown previously to inactivate mammalian DHOase. Spectroscopic studies and [14C]DEPC incorporation demonstrated that the loss of activity is associated with the modification of approximately two histidine residues located at or near the active site.(ABSTRACT TRUNCATED AT 250 WORDS)

The amidotransferase or glutaminase (GLNase) domain of mammalian carbamyl phosphate synthetase (CPSase), part of the 243-kDa CAD polypeptide, consists of a carboxyl half that is homologous to all trpG-type amidotransferases and an amino half unique to the carbamyl phosphate synthetases. The two halves of the mammalian GLNase domain have been cloned separately, expressed in Escherichia coli, and purified. The 21-kDa carboxyl half, the catalytic subdomain, is extraordinarily active. The k is 347-fold higher and the K is 40-fold lower than the complete GLNase domain. Unlike the GLNase domain, the catalytic subdomain does not form a stable hybrid complex with the E. coli CPSase synthetase subunit. Nevertheless, titration of the synthetase subunit with the catalytic subdomain partially restores glutamine-dependent CPSase activity. The 19-kDa amino half, the interaction subdomain, binds tightly to the E. coli CPSase large subunit. Thus, the GLNase domain consists of two subdomains which can autonomously fold and function. The catalytic subdomain weakly interacts with the synthetase domain and has all of the residues necessary for catalysis. The interaction subdomain is required for complex formation and also attenuates the intrinsically high activity of the catalytic subdomain and, thus, may be a key element of the interdomain functional linkage.

FLASH (FLICE-associated huge protein or CASP8AP2) is a large multifunctional protein that is involved in many cellular processes associated with cell death and survival. It has been reported to promote apoptosis, but we show here that depletion of FLASH in HT1080 cells by siRNA interference can also accelerate the process. As shown previously, depletion of FLASH halts growth by down-regulating histone biosynthesis and arrests the cell cycle in S-phase. FLASH knockdown followed by stimulating the cells with Fas ligand or anti-Fas antibodies was found to be associated with a more rapid cleavage of PARP, accelerated activation of caspase-8 and the executioner caspase-3 and rapid progression to cellular disintegration. As is the case for most anti-apoptotic proteins, FLASH was degraded soon after the onset of apoptosis. Depletion of FLASH also resulted in the reduced intracellular levels of the anti-apoptotic proteins, MCL-1 and the short isoform of cFLIP. FLASH knockdown in HT1080 mutant cells defective in p53 did not significantly accelerate Fas mediated apoptosis indicating that the effect was dependent on functional p53. Collectively, these results suggest that under some circumstances, FLASH suppresses apoptosis.

We have employed first-principles density-functional calculations to study the electronic characteristics of graphene functionalized by metal-bis-arene and metal-carbonyl molecules. It is shown that functionalization with M-bis-arene (M(C6H6)@gr, M=Ti, V, Cr, Mn, Fe) molecules leads to an opening in the band gap of graphene (up to 0.81eV for the Cr derivative), and functionalization with M-carbonyl (M(CX)3@gr, X=O,N; M= Cr, Mn, Fe, Co) up to one 1eV for M=Cr and X=O, and therefore transforms graphene from a semi-metal to a semiconductor. The band gap induced by attachment of a metal atom topped by a functionalizing group is attributed to modification of π-conjugation and depends on the concentration of functionalizing molecules, metal’s and moiety’s electronic structure. This approach offers a means of tailoring the band structure of graphene and potentially its applications for future electronic devices.

Carbamyl-phosphate synthetases from different organisms have similar catalytic mechanisms and amino acid sequences, but their structural organization, sub-unit structure, and mode of regulation can be very different. Escherichia coli carbamyl-phosphate synthetase (CPSase), a monofunctional protein consisting of amido-transferase and synthetase subunits, is allosterically inhibited by UMP and activated by NH3, IMP, and ornithine. In contrast, mammalian CPSase II, part of the large multifunctional polypeptide, CAD, is inhibited by UTP and activated by 5-phosphoribosyl-1-pyrophosphate (PRPP). Previous photoaffinity labeling studies of E. coli CPSase showed that allosteric effectors bind near the carboxyl-terminal end of the synthetase subunit. This region of the molecule may be a regulatory subdomain common to all CPSases. An E. coli mammalian hybrid CPSase gene has been constructed and expressed in E. coli. The hybrid consists of the E. coli CPSase synthetase catalytic subdomains, residues 1-900 of the 1073 residue polypeptide, fused to the amino-terminal end of the putative 190-residue regulatory subdomain of the mammalian protein. The hybrid CPSase had normal activity, but was no longer regulated by the prokaryotic allosteric effectors. Instead, the glutamine- and ammonia-dependent CPSase activities and both ATP-dependent partial reactions were activated by PRPP and inhibited by UTP, indicating that the binding sites of both of these ligands are located in a regulatory region at the carboxyl-terminal end of the CPSase domain of CAD. The apparent ligand dissociation constants and extent of inhibition by UTP are similar in the hybrid and the wild type mammalian protein, but PRPP binds 4-fold more weakly to the hybrid. The allosteric ligands affected the steady state kinetic parameters of the hybrid differently, suggesting that while the linkage between the catalytic and regulatory subdomains has been preserved, there may be qualitative differences in interdomain signal transmission. Nevertheless, switching prokaryotic and eukaryotic allosteric controls argues for remarkable conservation of structure and regulatory mechanisms in this family of proteins.

The multifunctional protein CAD catalyzes the first three steps in de novo pyrimidine biosynthesis in mammalian cells. Glutamine-dependent carbamyl-phosphate synthetase (CPSase), aspartate transcarbamylase, and dihydroorotase activities are carried by a 243-kDa polypeptide chain that is organized into discrete functional domains connected by interdomain linkers. One of the connecting chain segments, the DA linker bridging the dihydroorotase and aspartate transcarbamylase domains, is unusually long (109 residues) and conserved in length in all eukaryotic species. A plasmid (pCK-CAD10) that encodes the entire 243-kDa polypeptide was constructed and expressed in Escherichia coli. The recombinant protein was purified to homogeneity by ion exchange and gel filtration chromatography. The purified protein had kinetic parameters that were close to those obtained for native CAD. Moreover, the CPSase activity was allosterically regulated. Gel filtration showed that the recombinant protein had the same molecular mass as native CAD. Thus, this complex mammalian protein is expressed and folds correctly in bacterial cells and, despite its extreme protease sensitivity, can be isolated intact. A deletion mutant that lacked the DA linker was then constructed. The kinetic parameters of the mutant protein were, for the most part, unaltered, showing that the DA linker is not essential for the proper folding or optimal functioning of the individual domains. However, a significant decrease in the thermal stability of the CPSase domain suggested that the linker helps to stabilize the complex. Moreover, the channeling of carbamyl phosphate, determined by measuring the extent to which the exogenously added intermediate could dilute the endogenous carbamyl phosphate pool, was appreciably reduced when the DA linker was removed. Thus, although the domains function autonomously, some of the linkers are important for interdomain interactions in CAD.

The trpG-type amidotransferases, a homologous but structurally diverse family of molecules, catalyze glutamine hydrolysis to supply ammonia for many biosynthetic reactions. The amidotransferase or glutaminase (GLNase) domain of mammalian carbamyl phosphate synthetase (CPSase), part of a 243-kDa polypeptide that initiates de novo pyrimidine biosynthesis, has been cloned and expressed in Escherichia coli. Complementation studies showed that a functional protein was produced in vivo which could provide ammonia for carbamyl phosphate synthesis by the host CPSase synthetase subunit. The recombinant 38-kDa protein was identified by immunoblotting, but when purified to homogeneity, had marginal glutaminase activity. Titration of the E. coli CPSase synthetase subunit with the mammalian GLNase domain resulted in the formation of a fully active 1:1 stoichiometric stable complex which catalyzed the glutamine-dependent overall reaction. The hybrid, isolated by gel filtration, had kinetic parameters (KGLNm = 102 microM, KATPm = 1.8 mM, kcat = 5.7 s-1) similar to those of the native E. coli CPSase. Thus, the amidotransferase activity of mammalian CPSase is carried by an autonomous domain which folds independently. However, optimal catalytic activity requires association of the glutaminase and synthetase domains. The conservation of this linkage in the mammalian E. coli hybrid suggests that the subunit interfaces must be nearly identical in the eukaryotic and prokaryotic proteins.

A real-time, label free assay was developed for microbial detection, utilizing double-stranded DNA targets and employing the next generation of an impedimetric sensor array platform designed by Sharp Laboratories of America (SLA). Real-time curves of the impedimetric signal response were obtained at fixed frequency and voltage for target binding to oligonucleotide probes attached to the sensor array surface. Kinetic parameters of these curves were analyzed by the integrated data analysis package for signal quantification. Non-specific binding presented a major challenge for assay development, and required assay optimization. For this, differences were maximized between binding curve kinetic parameters for probes binding to complementary targets versus non-target controls. Variables manipulated for assay optimization included target concentration, hybridization temperature, buffer concentration, and the use of surfactants. Our results showed that (i) different target–probe combinations required optimization of specific sets of variables; (ii) for each assay condition, the optimum range was relatively narrow, and had to be determined empirically; and (iii) outside of the optimum range, the assay could not distinguish between specific and non-specific binding. For each target–probe combination evaluated, conditions resulting in good separation between specific and non-specific binding signals were established, generating high confidence in the SLA impedimetric dsDNA assay results.

We have employed first-principles density-functional calculations to study the electronic characteristics of covalently functionalized graphene by metal-bis-arene chemistry. It is shown that functionalization with M-bis-arene (M=Ti, V, Cr, Mn, Fe) molecules leads to an opening in the band gap of graphene (up to 0.81eV for the Cr derivative), and as a result, transforms it from a semi-metal to a semiconductor. The band gap induced by attachment of a metal atom topped by a benzene ring is attributed to modification of π-conjugation and depends on the concentration of functionalizing molecules. This approach offers a means of tailoring the band structure of graphene and potentially its applications for future electronic devices.

The two main, low frequency radio components discovered at Neptune by the Planetary Radio Astronomy experiment carried aboard Voyager 2 have a well defined periodicity at about 16.1 hour. By analyzing all the available data, i.e. about 60 days around the closest approach for the 'Burst' component and 15 days for the 'Smooth' component, we determine, for each component, the best estimate of the radio period. We conclude that the two estimates are not statistically different. While the two kinds of radio emissions have very different characteristics (in particular their frequency ranges and beaming properties), and probably correspond to different emission processes, their modulation is very likely due to the rotation of the planetary magnetic field tied to the core of the planet, as it has already been assumed for the other giant planets. The deduced estimate of the sidereal rotation period of Neptune is 16.108 +/- 0.006 (or 16h06.5m +/- 0.04 m).

In three studies that employed community-based samples the relationship between personality, marital, and job-related factors and quality of life was examined. Study 1 indicated that hardiness and self-esteem were important components of overall quality of life. The marital communication skills of expressiveness and intimacy were identified as major aspects of overall quality of life in the second study. In Study 3, satisfaction with various job characteristics was related to overall quality of life. These studies start to provide definition to the quality of life concept in terms of personality characteristics, skills, and beliefs that have potential for modification.

The relationships among coping responses, social support, personality factors, and psychological well-being in adults with acute leukemia in remission were studied. The psychological well-being measure was related to quality of life. Forty persons (21 male, 19 female), average age 47 years and average time since diagnosis of 24 months, completed demographic questions, the PRF-E, the Symptom Distress Scale, a Coping Responses inventory, the modified Social Support Questionnaire, and the General Behavior Inventory. An R of .80 was obtained between psychological well-being and Endurance, Affiliation, Cognitive Structure, Autonomy, and Nurturance. Findings were related to fighting spirit and confronting coping style, concepts associated with psychological well-being and longevity in cancer patients.

Mammalian dihydroorotase (DHOase) is part of a large multidomain protein called CAD, which initiates the first three steps in the de novo pyrimidine biosynthetic pathway. DHOase activity is carried out by a 44-kDa structural domain which could be isolated in active form from elastase digests. A core domain the same size as monofunctional dihydroorotases was defined, although the domain borders were uncertain. Two recombinants were overexpressed in Escherichia coli. The first encoded the core domain with 55 and 13 residues added to the amino and carboxyl ends, respectively, and was expressed in insoluble form. The recombinant protein was refolded from urea into a soluble form which was resistant to protease digestion but was catalytically inactive. In contrast, the proteolytic fragment from CAD could be unfolded and refolded with recovery of 40-100% catalytic activity. The second construct, which approximated the proteolytic fragment, had 21 residues on the amino end and 65 residues on the carboxyl end of the core domain. A 46-kDa soluble protein was expressed at 4% of the total soluble protein. The recombinant protein was catalytically active and had the expected amino-terminal sequence. The protein was purified to homogeneity. Dihydroorotase saturation curves gave a Km = 41.9 +/- 3.5 microM and a kcat = 2.79 +/- 0.06 s-1, parameters that were similar to those obtained for the proteolytic fragment. The Km was 6-fold higher and the kcat 2-fold lower than the values obtained for the parent protein, which suggests that interdomain interactions stabilize the active conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Ethics for the Practice of Psychology in Canada (Revised and Expanded Edition), by Derek Truscott and Kenneth H. Crook. Edmonton, Alberta: University of Alberta Press. 2013, 236 Pages (ISBN 978-0-88864-652-1, CAN $49.95)Reviewed by DAVID R. EVANSDOI: 10.1037/a0035674Psychologists-whether practitioners, researchers or aca- demics-encounter ethical issues in their work regularly if not daily. Hence, there is a need for a system of ethics to assist them with the myriad ethical issues they encounter. The Canadian Psychological Association Code of Ethics for Psychologists (CPA Code of Ethics) provides such a system of ethics. Over the years of teaching ethics to graduate students, the majority indicated that the CPA Code of Ethics was the most useful approach to solving ethical issues that they encountered, rather than a prescriptive approach that had limitations. I feel this preamble is important in placing the Truscott and Crook book in context. The authors base much of the discussion of the many topics they cover in relation to the principles and ethical prin- ciples of the CPA Code of Ethics. Given its reliance on the CPA Code of Ethics, its readability, and its thoroughness, the book is an excellent textbook or reference book for senior undergrad- uates and entry-level graduate students, as well as for practi- tioners wishing to renew their acquaintance with the range of ethical questions they encounter in their practice.The opening paragraph of the Introduction quickly makes the case for the importance of a knowledge of ethics to the profession of psychology. After defining ethics, the authors distinguish be- tween descriptive and prescriptive ethics, and then place ethics in comparison to practice standards and the law. Next the authors argue that behaving as an ethical professional goes beyond a set of rules because "ethical standards and expectations evolve and change" (p. xx), and further the rules cannot anticipate every ethical dilemma. In the last part of the Introduction the authors outline the various learning devices they provide to help the reader get the most out of the book. These include discussion questions, reflective journaling, ethical case studies, and recommended read- ing.The first three chapters focus on ethical systems, professional standards, and legal expectations. An important focus in the first chapter is to place our professional ethics in the context of four foundational ethical systems: Teleology, Deontology, Rational Ethics, and Virtue Ethics. The description of the four systems and their evaluation is very brief and may leave readers who are unfamiliar with ethical theories a bit confounded. Following this discussion, the authors provide a history of the CPA Code of Ethics for Psychologists: its history, and the four hierarchical principles that are central to the code. The second chapter is excellent, providing a succinct, generic description of the provin- cial and territorial provisions that regulate the profession of psy- chology. Topics covered in the chapter include: entrance stan- dards, professional standards, and practice guidelines; the importance of ethics committees and provincial and territorial discipline committees in maintaining professional accountability; the anatomy of the provincial complaints process; and discipline hearings. Finally, in the chapter, the authors stress the responsi- bility placed on psychologists to regulate themselves, and to re- spond to unethical or unprofessional behaviour on the part of colleagues. In the third chapter the authors provide information about the legal system so that psychologists can avoid behaviours that might "prompt others to seek legal remediation" (p. 41). They describe the legal system as adversarial, visible and remedial, and they go on to distinguish between criminal and civil law. Next, they consider the important components of negligence in terms of duty of care, reasonable care, causation, and the plaintiff's con- duct. …

Dihydroorotase (DHOase) catalyzes the formation of dihydroorotate in the de novo pyrimidine biosynthetic pathway. The gene encoding the type I DHOase from the hyperthermophilic bacterium Aquifex aeolicus has been cloned in Escherichia coli with a polyhistidine affinity tag appended to the amino-terminal end and sequenced. The recombinant protein was expressed at high levels and could be purified readily in a single step by Ni(2+) affinity chromatography. Both native and selenomethionine-labeled proteins were crystallized using the hanging-drop vapor-diffusion technique. Screens of the purified protein identified several conditions that yielded crystals; however, the best crystals were obtained using 1 M Li(2)SO(4), 10 mM NiCl(2), 100 mM Tris acetate pH 8.5 as the precipitant. Well formed diamond-shaped crystals appeared within 1 d and continued to grow over several weeks to about 0.5 mm in the largest dimension. The crystals diffract to 1.7 A and belong to space group C2, with unit-cell parameters a = 119.8, b = 88.0, c = 55.2 A, beta = 99.0 degrees and a mosaic spread of 0.6 degrees. There is one DHOase monomer in the asymmetric unit.

A recent proteomics study identified FAM129B or MINERVA as a target of the MAP kinase (Erk1/2) signaling cascade in human melanoma cells. Phosphorylation of the protein was found to promote cell invasion and the dissociation of the protein from the cell-cell junctions. Suppression of apoptosis during metastasis is a prerequisite for the survival and spread of cancer cells. During apoptosis, the adherens junctions are disassembled as the dying cell retracts, and new contacts are formed between normal neighboring cells. In this study, we show that FAM129B was cytosolic in exponentially growing HeLa cells but was translocated to the adherens junctions where it colocalized with β-catenin whenever contact between two or more cells was established. Silencing the FAM129B gene expression by specific siRNAs did not induce apoptosis or inhibit the growth of HeLa cells. However, when apoptosis was induced by exposure to TNFα/cycloheximide or other apoptotic signaling molecules, the onset of apoptosis was accelerated 3–4-fold when FAM129B was depleted. Annexin V binding, the inactivation of the DNA repair enzyme, poly(ADP-ribose) polymerase, and the activation of the caspases occurred more rapidly in the cells lacking FAM129B. The rapid induction of apoptosis in FAM129B knockdown cells was reversed by co-transfection with recombinant FAM129B, indicating that its effect on apoptosis was specific. As apoptosis proceeded, FAM129B was degraded and disappeared from the plasma membrane. Thus, one crucial facet of the mechanism by which FAM129B promotes cancer cell invasion is likely to be the suppression of apoptosis.

CAD is a multidomain protein that catalyzes the first three steps in mammalian de novo pyrimidine biosynthesis. The 243-kDa polypeptide consists of four functional domains; glutamine amidotransferase (GLNase), carbamyl phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase), and dihydroorotase (DHOase). Controlled proteolysis of hamster CAD was found to cleave the molecule into 18 fragments which successively accumulate and disappear during the course of digestion. Each fragment was isolated and partially sequenced to determine its location in the polypeptide chain. Proteolysis was found to usually occur at the junctions between the domains and sub-domains identified by sequence homology. All proteases of low to moderate specificity cleaved the molecule in a similar fashion. The rate of proteolysis widely varied and the interdomain regions were not always accessible to proteases. Each of the major functional domains is postulated to consist of subdomains. The duplicated halves of the CPSase domain (116 kDa) have a homologous structure consisting of 11-, 25-26-, and 21-22-kDa subdomains. Prolonged digestion cleaved the DHOase domain (36.6 kDa) into two stable species suggesting that this region is comprised of 11.5- and 15.0-kDa subdomains. Similarly, proteolysis of the 21-kDa catalytic subdomain of the GLNase domain (40 kDa) indicated a bilobal structure consisting of 12.3- and 8.5-kDa chain segments. The connecting region between the two ATCase subdomains (16.4 and 18 kDa) was not cleaved. Copurification of many of the domains showed that they remain associated by noncovalent interactions even after the connecting segments have been cleaved. The chain segments, the linkers, which connect the domains and subdomains were conserved in length but not in sequence, were predicted to be relatively hydrophilic and flexible but did not show a tendency to assume a particular secondary structure. These studies provide a more detailed map of the structural organization of the CAD polypeptide.

CA-125 and carcinoembryonic antigen (CEA) are two biomarkers present in blood that can indicate the presence of ovarian cancer. They can also be used, both in conjunction with each other and independently, to determine the effectiveness of the treatment being meted for the disease. A label-free multiplexed interdigitated electrode array (IDEA) immunosensor was developed to detect both CA-125 and CEA in buffer solution at levels typically seen in patients with ovarian cancer . Electrochemical impedance spectroscopy was used to measure the increase in impedance when a binding event occurred between the target antigen and its specific antibody that was anchored to the surface of an interdigitated electrode array. CA-125 was detected in concentrations as low as 10units/mL and as high as 80units/mL. CEA was detected in concentrations as low as 1pg/mL and as high as 10μg/mL.

CAD is a 243-kDa multidomain polypeptide which catalyzes the first three steps in mammalian de novo pyrimidine biosynthesis. The largest cDNA clone obtained thus far, pCAD142 (Shigesada, K., Stark, G.R., Maley, J. A., Niswander, L. A., and Davidson, J. N. (1985) Mol. Cell. Biol. 5, 1735), lacks the 5' end of the mRNA which encodes the amino terminus of CAD. To clone this missing segment, a synthetic oligonucleotide complementary to pCAD142 and poly(A)+ RNA template, isolated from a Syrian hamster cell line which overproduces the CAD mRNA, were used for cDNA synthesis. The resulting clone pKB11, which has a 1369-base pair (bp) cDNA insert, overlapping pCAD142 by 781 bp, was identified by hybridization methods and sequence analysis and found to contain the entire cDNA sequence for the amino end of the CAD polypeptide. The deduced amino acid sequence is homologous to seven carbamyl phosphate synthetases. Primer extension, oligonucleotide-directed RNase H digestion, and RNA sequencing indicated that pKB11 extends to within 68 bases of the 5' end of the CAD mRNA. This conclusion was confirmed by Northern blotting analysis of the 5'-flanking region of CAD gene. The probable 3' end of an unidentified gene which codes for a 1-kilobase (kb) transcript was identified immediately upstream of the CAD gene. Northern analysis using probes complementary to the region between the CAD and the 1-kb genes detected the presence of a small transcript of less than 300 nucleotides. The sequence revealed three potential translation initiation sites raising the possibility of more than one CAD translation product. The major translation start codon was identified as the first ATG in pKB11 by sequence homology, in vitro transcription and translation, and protein studies. Starting from this ATG within pKB11, the clone encodes a 143-residue domain of unknown function. This study completes the determination of the primary structure of the CAD polypeptide. The CAD mRNA is 7.5 kb in length and has 6675 bp of coding sequence and about 200 bp and 600 bp of untranslated sequence at the 5' and 3' ends, respectively.

The multifunctional protein CAD catalyzes the first three steps in pyrimidine biosynthesis in mammalian cells, including the synthesis of carbamyl phosphate from bicarbonate, MgATP and glutamine. The Syrian hamster CAD glutaminase (GLNase) domain, a trpG-type amidotransferase, catalyzes glutamine hydrolysis in the absence of MgATP and bicarbonate (Km = 95 microM and kcat = 0.14 s-1). Unlike E. coli carbamyl phosphate synthetase (Wellner, V.P., Anderson, P.M., and Meister, A. (1973) Biochemistry 12, 2061-2066), a stable thioester intermediate did not accumulate when the mammalian enzyme was incubated with glutamine. However, a covalent adduct could be isolated when the protein was denatured in acid. The steady state concentration of the intermediate increased with increasing glutamine concentration to nearly one mole per mole of enzyme with half saturation at 105 microM, close to the Km value for glutamine. The adduct formed at the active site of the glutaminase domain. The rate of breakdown of the intermediate (k4), determined directly, was 0.17 s-1 and the rate of formation (k3) was estimated as 0.52 s-1. In the absence of MgATP and bicarbonate, k4 = kcat indicating that the decomposition of the intermediate is the rate-limiting step. The intermediate was chemically and kinetically competent, and the glutamine dissociation constant (330 microM) and rate constants were consistent with steady state kinetics and accurately predicted the steady state concentration of the intermediate. These studies suggest a mechanism similar to the cysteine proteases such as recently proposed by Mei and Zalkin (Mei, B., and Zalkin, H. (1989) J. Biol. Chem. 264, 16613-16619) who identified a catalytic triad in glutamine phosphoribosyl-5'-pyrophosphate amidotransferase, a purF-type enzyme. MgATP and bicarbonate increased kcat of the glutaminase reaction 14-fold by accelerating both the rate of formation and the rate of breakdown of the intermediate, and prevented the accumulation of the intermediate; however, the Km value for glutamine was not significantly altered. The instability of the thioester intermediate leads to appreciable hydrolysis of glutamine in the absence of the other substrates. However, bicarbonate alone spares glutamine by increasing the Km and Ks of glutamine to 600 and 8960 microM, respectively, thus reducing kcat/Km 3-fold when MgATP is limiting. In the absence of MgATP and bicarbonate, ammonia decreased the rate of hydrolysis and the accumulation of the thioester intermediate indicating that ammonia had direct access to the thioester at the GLNase domain active site.(ABSTRACT TRUNCATED AT 400 WORDS)

The article entitled "Empirically Supported Treatments in Psychology: Implications for Canadian Professional Psychology" is well written and makes a number of important observations and recommendations that are important for professional psychology in Canada. However, the article raises a number of important issues that need urgent consideration if the health related(f.1) branches of professional psychology in Canada are to expand and thrive. As suggested by the title of this commentary the evidence-based movement is too little and too late. It is too little, in that considerably more, and much more complex evidence for what professional psychologists do is required. It is too late, in that both in Canada and the United States professional psychologists have suffered considerable set backs in both the economic and popularity domains.Statutory or Common law in Canada requires that, when practitioners are obtaining informed consent to embark on a procedure with a client, they must disclose the nature of the procedure, the associated risks and benefits, and the outcome expected (Evans, 1997). In addition, there is a requirement to inform clients about alternatives to treatment, including refusal of treatment, and the related risks and benefits of these alternatives. For example, suppose a practitioner proposed to treat a client for anxiety in the work setting using the counselling approach outlined by Evans, Hearn, Uhlemann, and Ivey (1998). He or she would be expected to state: the specifics of the treatment, including number of sessions; the alternatives to treatment; and the risks and benefits associated with each option and nontreatment. The latter prescription would be very difficult to comply with, simply because there is insufficient research completed to speak with any confidence on many of these requirements. This is but one example of a broad range of treatment proposals professional psychologists make to their clients daily, which are fraught with a comparable lack of research backing. A similar argument could be made from the vantage point of forensic psychologists, who must have similar research support for the conclusions they draw concerning the results of assessment and treatment. Hence, the evidence-based movement, while an important first step, does not go far enough.The evidence base for the practices of psychologists needed to be established decades ago to offset the recent, forced exodus of professional psychologists from human service settings on the one hand, and the failure of psychology to thrive, on the other hand. The public is informed daily of new procedures that have the potential to ameliorate heart disease, cancer, and a range of other disorders. Such is not the case for psychological disorders. Along with other information about psychology, the public and politicians need to hear of research about psychological interventions, if the current lack of knowledge about psychologists and what they do is to be offset. Given psychology is inherent in every aspect of human behaviour, the potential for professional psychology is vast. However, this potential has not materialized, and, if anything, advances made in professional psychology are being rolled back. Hence, the conclusion that the evidence-based movement is perhaps too late.There are two factors working against the development of an expanded evidence base for professional psychology. These factors involve economics and research strategy. Behind each announcement in the media of a medical advance is a vast amount of money, usually in the millions, if not billions of dollars by the time the procedure becomes routine. Secondly, there is a research strategy, such as that outlined by Evans (1997), which encourages both the required basic research, and the important applied research. Using such a research strategy, basic research could be translated into the day-to-day practice of psychologists with the evidence-based information required to obtain appropriate informed consent. …

Pt, Ir, Au and few other precious metals have highly conducive electrical and chemical properties; hence have been widely used in pH sensors and bimolecular sensing applications. The chief objective of this research is to highlight and demonstrate the advantages that Iridium Oxide (IrOx) nanowires offer over these competing metals in improving the performance metrics of biomolecular sensing. Iridium oxide has very good conductivity and very high charge storing capacity, and hence has an ability to detect very small changes in the surface charge. Nanowires have an ideal morphology to crowd protein molecules and highly increase the surface area of interaction. Higher area of interaction along with iridium oxide's high intrinsic physical adsorption rate, strongly enhance the rate of immobilization of biomolecules and hence enabling high sensitivity detection. Inflammatory protein, C-Reactive protein (CRP) that is a biomarker for cardiovascular disease was used as the model biomolecule for this study.

Switching resistance of a metal/oxide/metal structure (Pt/PrxCa1-xMnO3/Pt) upon the stimulation of electric pulse has triggered vast research interests and activities for next generation resistive RAM application. Continued from an earlier paper [1] that studied the mechanism of switching resistance, this paper extends to the switching endurance discussion using admittance spectra. Experimental data indicated that there exist interfacial dipoles (or states) at metal/oxide interfaces. Switching resistance comes from the change of interfacial dipoles. However, those interfacial dipoles are all meta-stable indicating the problem of long term switching endurance. We have tested many metal/PCMO contact combinations and characterized those contacts with basic memory criteria: bit separation, data retention, switch endurance, switching speed, and readout limitations. Among them, TiN/PCMO showed large bit separation, excellent data retention, and fast switching speed, but failed long term switching endurance test. Furthermore, the poor switch endurance is discussed using energy-well diagram. Therefore, a material system providing bi-stable states with reasonably large free energy separations is a must for this type of RRAM application. These energy levels can come from either structure or electrochemical potential differences at the metal/oxide interfaces.

Rotational energy relaxation cross sections, σR , for nitrogen in dilute atomic gases (He, Ne, Ar, and Xe) are calculated by classical trajectory simulations for a variety of simple potentials and from the Boltzmann equation for hard convex bodies in the single impulse collision approximation. The single impulse approximation, which ignores multiple impulse (chattering) collisions, is reasonably adequate to describe linear and angular momentum relaxation, but not rotational energy relaxation. For the light noble gases, the hard body derived results together with the small chattering corrections suffice to fit σR . It is also the case that chattering collisions markedly decrease σR from the single impulse approximation value for Xe–N2 in the hard‐body model. However, the value of σR experimentally measured and calculated by Kistemaker and de Vries using a soft potential is considerably less than that obtained from hard‐body models including chattering. Addition of a square well attractive potential of the ...

• This study examined whether the subjective impression of memory function might differentiate healthy elderly subjects from patients with memory complaints, and whether memory complaints differed between patients with and without a dementing illness. Both self-assessment and relatives' responses on a new memory questionnaire differentiated patient groups from control subjects. The relatives' form measuring deterioration in memory function over time identified dementing individuals from those with nondementing causes for their memory complaints. Factor analysis indicated that patients' memory complaints correlated with depression rather than objective memory performance, while relatives' ratings correlated with objective memory scores, not depression. Stepwise discriminant function analyses showed that objective memory testing greatly improved specificity but not sensitivity of the subjective memory questionnaire alone.

Glutamine-dependent carbamoyl-phosphate synthetase (EC 6.3.5.5) catalyzes the first step in de novo pyrimidine biosynthesis. The mammalian enzyme is part of a 240-kDa multifunctional protein which also has the second (aspartate carbamoyltransferase, EC 2.1.3.2), and third (dihydroorotase, EC 3.5.2.3) activities of the pathway. Shigesada et al. (Shigesada, K., Stark, G.R., Maley, J.A., and Davidson, J.N. (1985) Mol. Cell Biol. 175, 1-7) produced a truncated cDNA clone from a Syrian hamster cell line that contained most of the coding region for this protein. We have completed sequencing this clone, known as pCAD142. The cDNA insert contained all of the coding region for the glutaminase (GLN) and carbamyl phosphate synthetase (CPS) domains but lacked a short amino-terminal segment. By comparing the primary structure of the mammalian chimera to monofunctional proteins we have identified the borders of the functional domains. The GLN domain is 21 kDa, close to the size of the functionally similar polypeptide products of the Escherichia coli pabA and hisH genes. The domain has the three regions of homology common to trpG-type glutamine amidotransferases, as well as a fourth region specific to the carbamyl phosphate synthetases. The CPSase domain is similar to other reported CPSases in size (120 kDa), primary structure (37-67% amino acid identity), and homology between its amino and carboxyl halves. Analysis of the nucleotide and amino acid sequence identities among the various carbamyl phosphate synthetases suggests that the gene fusion which joined the GLN and CPS domains was an early event in the evolution of eukaryotic organisms and that the Saccharomyces cerevisiae enzyme consisting of separate subunits arose by defusion from an ancestral multifunctional protein.

Mammalian DHOase (S-dihydroorotate amidohydrolase, EC 3.5.2.3) is part of a large multifunctional protein called CAD, which also has a carbamoyl-phosphate synthetase [carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.5.5] and aspartate transcarbamoylase (carbamoyl-phosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) activities. We sequenced selected restriction fragments of a Syrian hamster CAD cDNA. The deduced amino acid sequence agreed with the sequence of tryptic peptides and the amino acid composition of the DHOase domain isolated by controlled proteolysis of CAD. Escherichia coli transformed with a recombinant plasmid containing the cDNA segment 5' to the aspartate transcarbamoylase coding region expressed a polypeptide recognized by DHOase domain-specific antibodies. Thus, the order of domains within the polypeptide is NH2-carbamoyl-phosphate synthetase-DHO-aspartate transcarbamoylase-COOH. The 334-residue DHOase domain has a molecular weight of 36,733 and a pI of 6.1. A fragment of CAD having DHOase activity that was isolated after trypsin digestion has extensions on both the NH2 (18 residues) and COOH (47-65 residues) termini of this core domain. Three of five conserved histidines are within short, highly conserved regions that may participate in zinc binding. Phylogenetic analysis clustered the monofunctional and fused DHOases separately. Although these families may have arisen by convergent evolution, we favor a model involving DHOase gene duplication and insertion into an ancestral bifunctional locus.

The results of liver transplantation are now well established in terms of graft and patient survival, but there is surprisingly little data on the quality of life attained. The authors mailed questionnaires to 32 consecutive adult liver recipients to assess their quality of life. Thirty-one patients (14 males, 17 females) with a mean age of 37 years (range 16 to 55), responded (97%). The mean time since transplantation was 19 months (range three to 50). Eighty percent of the respondents functioned at normal or near normal levels as measured by the Karnofsky Performance Index. Sixty-five per cent (20 patients) indicated they were currently able to live and function as they did before they became ill with liver disease. The respondents' scores were similar to normative scores on all of the following measures: life satisfaction, well being, and general affect (Campbell); and material well being, personal growth, marital relations, family relations and friendships (Evans). It is concluded that liver transplantation restores physical, mental and social well being in most patients with endstage liver disease.

Serine/Threonine kinases participate in complex, interacting signaling pathways in eukaryotes, prokaryotes, and archae. While most organisms contain many different kinases, the extreme hyperthermophile, Aquifex aeolicus encodes a single hypothetical Ser/Thr kinase. A gene homologous to eukaryotic protein phosphatases overlaps the kinase gene by a single base pair. The putative kinase, AaSTPK and phosphatase, AaPPM, were cloned and expressed in E. coli, purified to homogeneity and found to be functional. AaSTPK is a 34-kDa monomer that can use MgATP, MnATP, or MnGTP as co-substrates, although MgATP appears to be the preferred substrate. AaSTPK was autophosphorylated on a threonine residue and was dephosphorylated by AaPPM. AaPPM phosphatase is homologous to the PPM sub-family of Ser/Thr phosphatases and was stimulated by MnCl2 and CoCl2 but not MgCl2. AaSTPK also phosphorylated one threonine residue on the carbamoyl phosphate synthetase, CPS.A subunit. Carbamoyl phosphate synthetase reconstituted with phosphorylated CPS.A had unaltered catalytic activity but allosteric inhibition by UMP and activation by the arginine intermediate, ornithine, were both appreciably attenuated. These changes in allosteric regulation would be expected to activate pyrimidine biosynthesis by releasing the constraints imposed on carbamoyl phosphate synthetase activity by UMP and uncoupling the regulation of pyrimidine and arginine biosynthesis. CPS.A was also dephosphorylated by AaPPM. Aquifex aeolicus occupies the lowest branch on the prokaryotic phylogenetic tree. The Thr/Ser kinase, its cognate phosphatase and a protein substrate may be elements of a simple signaling pathway, perhaps the most primitive example of this mode of regulation described thus far.

Nobel metals in contact with perovskite metal oxides, Pr1−xCaxMnO3 (PCMO) for example, have shown switching resistance values, with a couple of orders of magnitude difference, upon the stimulation of electrical pulses. In this paper, the Pt/PCMO/Pt structures were made through e-beam evaporation (Pt electrodes) and RF sputtering (PCMO films) for the switching mechanism study. Specially designed experiments along with extensive electrical characterizations were performed on the Pt/PCMO/Pt structure. The existence of a contact resistance, or an interfacial layer, between Pt electrodes and PCMO was evident by simply measuring initial resistance (R0) of the stack against PMCO film thickness. Temperature dependence of the R0 and the time-bias tests were used to study the transport mechanism in the bulk of PCMO and the interfacial layer. Above a threshold voltage, the transport changes from mainly electronic to ionic conduction especially at the interfaces, causing electrode polarization. The transient characteristics of Pt/PCMO/Pt stack, i.e. the response to the pulsing in the time domain, were characterized in the frequency domain instead through the admittance spectroscopy measurements. The temperature dependence of Cole-Cole plots were used to study the polarized interfacial layer or interface dipole polarization (IDP). This IDP layer is the origin of contact resistance and also responsible for the uni-polar long-short switching because the calculated relaxation time constants of the IDP corresponding to low resistive state (LRS) and high resistive state (HRS) were similar to the experimental values. Therefore, the so-called bi-stable resistive states are just two different IDP states: one is much leakier than the other.

CAD is a multifunctional protein that initiates and regulates mammalian de novo pyrimidine biosynthesis. The activation of the pathway required for cell proliferation is a consequence of the phosphorylation of CAD Thr-456 by mitogen-activated protein (MAP) kinase. Although most of the CAD in the cell was cytosolic, cell fractionation and fluorescence microscopy showed that Thr(P)-456 CAD was primarily localized within the nucleus in association with insoluble nuclear substructures, including the nuclear matrix. CAD in resting cells was cytosolic and unphosphorylated. Upon epidermal growth factor stimulation, CAD moved to the nucleus, and Thr-456 was found to be phosphorylated. Mutation of the CAD Thr-456 and inhibitor studies showed that nuclear import is not mediated by MAP kinase phosphorylation. Two fluorescent CAD constructs, NLS-CAD and NES-CAD, were prepared that incorporated strong nuclear import and export signals, respectively. NLS-CAD was exclusively nuclear and extensively phosphorylated. In contrast, NES-CAD was confined to the cytoplasm, and Thr-456 remained unphosphorylated. Although alternative explanations can be envisioned, it is likely that phosphorylation occurs within the nucleus where much of the activated MAP kinase is localized. Trapping CAD in the nucleus had a minimal effect on pyrimidine metabolism. In contrast, when CAD was excluded from the nucleus, the rate of pyrimidine biosynthesis, the nucleotide pools, and the growth rate were reduced by 21, 36, and 60%, respectively. Thus, the nuclear import of CAD appears to promote optimal cell growth. UMP synthase, the bifunctional protein that catalyzes the last two steps in the pathway, was also found in both the cytoplasm and nucleus.

Efficient end point detection (EPD) helps prevent numerous Chemical Mechanical Polishing (CMP) process defects such as dishing, erosion, excessive over-polish etc. During the CMP of Shallow Trench Isolation (STI) structures, the process should be effectively stopped at the buried Si3N4 layer to prevent any/all of the aforementioned process defects. In this research, novel in-situ metrology technique that used the real time tracking of Coefficient of Friction (COF) data during polishing was employed for EPD during STI-CMP. The experiments were performed on the CETR CP-4 CMP Tester using 1“X 1” sample coupons having a 2 ‘mu;m pitch STI pattern. The COF signal during polishing is a characteristic signature of the given process parameters and conditions. The in-situ EPD was based on the principle that the COF values are strongly dependent upon the surface of the materials that are being polished. Extended polishing to polish the underlying layer after the process end point was reached, can give an estimate of the polishing slurry selectivity with respect to the buried layer. The ex-situ surface characterization of coupons was performed at different points of the process using Atomic Force Microscopy (AFM). The repeatability and reliability of this technique was evaluated after carrying out multiple tests at similar process conditions. The demonstrated methodology can also be implemented for characterization of polishing pads and slurries besides STI-CMP process optimization.

Friction in the CMP process is a subject of ongoing interest. The sources of friction can be the viscosity of the slurry between the pad and wafer, as well as the physical contact between the pad asperities and the wafer. The contact between the pad and the wafer can be direct or also through slurry particles trapped between the pad asperity and the wafer. We measure the friction between the wafer and the pad by measuring the CMP table current and subtracting out the background level. This is done for different concentrations of silica abrasive between 0 and 13%, as well as for different pressures and table speeds. Two widely different types of silica particles used in CMP are spherical colloid particles and aggregated fumed particles. We also compare the resultant friction behavior by comparing the results for both types of particles. For different abrasive contributions, the friction measurements are compared to the CMP removal rates. This evaluation is also done with a stationary wafer carrier, so that the observed removal rate at a specific point on the wafer is proportional to the local pressure on the wafer. The wafer carrier used for these tests uses a gimbal design. In this case the pressure at the leading edge of the wafer is larger than that at the trailing edge. From these studies we find that, for gimbaled systems with grooved pads, the friction is primarily caused by pad-asperity contact, and the addition of slurry particles does significantly change the friction interaction in the CMP process. Further, the non-uniformity in friction across the wafer is controlled by the pad surface structure and mechanical design of the CMP tool.

Dihydroorotase (DHOase) catalyzes the reversible condensation of carbamoyl aspartate to form dihydroorotate in de novo pyrimidine biosynthesis. The enzyme from Aquifex aeolicus, a hyperthermophilic organism of ancient lineage, was cloned and expressed in Escherichia coli. The purified protein was found to be a 45-kDa monomer containing a single zinc ion. Although there is no other DHOase gene in the A. aeolicus genome, the recombinant protein completely lacked catalytic activity at any temperature tested. However, DHOase formed an active complex with aspartate transcarbamoylase (ATCase) from the same organism. Whereas the k(cat) of 13.8 +/- 0.03 s(-1) was close to the value observed for the mammalian enzyme, the K (m)for dihydroorotate, 3.03 +/- 0.05 mM was 433-fold higher. Gel filtration and chemical cross-linking showed that the complex exists as a 240-kDa hexamer (DHO(3)-ATC(3)) and a 480-kDa duodecamer (DHO(6)-ATC(6)) probably in rapid equilibrium. Complex formation protects both DHOase and ATCase against thermal degradation at temperatures near 100 degrees C where the organism grows optimally. These results lead to the reclassification of both enzymes: ATCase, previously considered a Class C homotrimer, now falls into Class A, whereas the DHOase is a Class 1B enzyme. CD spectroscopy indicated that association with ATCase does not involve a significant perturbation of the DHOase secondary structure, but the visible absorption spectrum of a Co(2+)-substituted DHOase is appreciably altered upon complex formation suggesting a change in the electronic environment of the active site. The association of DHOase with ATCase probably serves as a molecular switch that ensures that free, uncomplexed DHOase in the cell remains inactive. At pH 7.4, the equilibrium ratio of carbamoyl aspartate to dihydroorotate is 17 and complex formation may drive the reaction in the biosynthetic direction.