Protein-mediated isolation of plasmid DNA by a zinc finger-glutathione S-transferase affinity linker

The sequence-specific affinity chromatographic isolation of plasmid DNA from crude lysates of E. coli DH5α fermentations is addressed. A zinc finger-GST fusion protein that binds a synthetic oligonucleotide cassette containing the appropriate DNA recognition sequence is described. This cassette was inserted into the Smal site of pUC19 to enable the affinity isolation of the plasmid. It is shown that zinc finger-GST fusion proteins can bind both their DNA recognition sequence and a glutathione-derivatized solid support simultaneously. Furthermore, a simple procedure for the isolation of such plasmids from clarified cell lysates is demonstrated. Cell lysates were clarified by cross-flow Dean vortex microfiltration, and the permeate was incubated with zinc finger-GST fusion protein. The resulting complex was adsorbed directly onto glutathione-Sepharose. Analysis of the glutathione-eluted complex showed that plasmid DNA had been recovered, largely free from contamination by genomic DNA or bacterial cell proteins. © 2002 Wiley Periodicals, Inc.

A cross-national investigation into the marketing department's influence within the firm:toward initial empirical generalizations

An ongoing strong debate within the marketing discipline concerns the role of marketing within the firm. It has been frequently reported that the marketing function is in a deep decline. Marketing executives and academics alike are interested in the antecedents of this decline and potential performance consequences of this decline. Recent academic research have started investigations on this important topic. Using studies in single countries innovativeness and accountability of the marketing department has been reported as major antecedents of the influence of the marketing department within the organization. Academic research, however, does not provide convincing evidence for a direct link between this influence and business performance. Instead it shows that market orientation is a crucial intervening variable, as marketing department influence is positively related market orientation, which subsequently positively related to business performance. As noted prior research, however, studies firms in single countries. In this article we execute a cross-national study on the antecedents and performance consequences of marketing department influence in order to derive initial empirical generalizations. This study is executed in seven Western-oriented countries, including USA, UK, The Netherlands, Germany, Sweden, Israel and Australia. The study heavily builds on the framework developed in the 2009 Journal of Marketing article of Verhoef and Leeflang. This framework is tested per country and subsequently meta-analytic tests are used to derive initial empirical generalizations. An important empirical generalization is that innovativeness, the customer-connecting capabilities, and accountability of the marketing department are positively related to marketing department influence. Interestingly, a second initial generalization is that creativity of the marketing negative induces less influence. Our results also show a third empirical generalization in that firms having a CEO with a marketing background tend to have more influential marketing departments. Confirming prior research a fourth initial empirical generalization is that MD influence measures and market orientation are positively related. Market orientation is subsequently positively related to business performance. Our most important generalization is, however, that MD influence is positively related to business performance. Hence, beyond striving to become market oriented, firms should also aim to have strong marketing departments. These departments can create a stronger focus on the customer and can also coordinate marketing efforts. In order to become more influential marketing departments should: (1) acquire innovative capabilities, (2) be more connected to customers, (3) invest in accountability, and (4) be careful with be careful being too creative.

Creative self-efficacy and individual creativity in teams:cross-level interactions with team informational resources

We propose a cross-level perspective on the relation between creative self-efficacy and individual creativity in which team informational resources, comprising both shared “knowledge of who knows what” (KWKW) and functional background diversity, benefit the creativity of individuals more with higher creative self-efficacy. To test our hypotheses, we conducted a multi-level study with 176 employees working in 34 research and development teams of a multinational company in 4 countries. In support of our hypotheses, the link between creative self-efficacy and individual creativity was more positive with greater shared KWKW, and this interactive effect was pronounced for teams of high rather than low functional background diversity. We discuss implications for the study of creative self-efficacy in team contexts. (PsycINFO Database Record (c) 2012 APA, all rights reserved).

Dietary analysis and patterns of nutritional supplement use in normal and age-related macular disease affected subjects:a prospective cross-sectional study

Background: Poor diet is thought to be a risk factor for many diseases, including age-related macular disease (ARMD), which is the leading cause of blind registration in those aged over 60 years in the developed world. The aims of this study were 1) to evaluate the dietary food intake of three subject groups: participants under the age of 50 years without ARMD (U50), participants over the age of 50 years without ARMD (O50), and participants with ARMD (AMD), and 2) to obtain information on nutritional supplement usage. Methods: A prospective cross-sectional study designed in a clinical practice setting. Seventy-four participants were divided into three groups: U50; 20 participants aged < 50 years, from 21 to 40 (mean ± SD, 37.7 ± 10.1 years), O50; 27 participants aged > 50 years, from 52 to 77 (62.7 ± 6.8 years), and ARMD; 27 participants aged > 50 years with ARMD, from 55 to 79 (66.0 ± 5.8 years). Participants were issued with a three-day food diary, and were also asked to provide details of any daily nutritional supplements. The diaries were analysed using FoodBase 2000 software. Data were input by one investigator and statistically analysed using Microsoft Excel for Microsoft Windows XP software, employing unpaired t-tests. Results: Group O50 consumed significantly more vitamin C (t = 3.049, p = 0.005) and significantly more fibre (t = 2.107, p = 0.041) than group U50. Group ARMD consumed significantly more protein (t = 3.487, p = 0.001) and zinc (t = 2.252, p = 0.029) than group O50. The ARMD group consumed the highest percentage of specific ocular health supplements and the U50 group consumed the most multivitamins. Conclusions: We did not detect a deficiency of any specific nutrient in the diets of those with ARMD compared with age- and gender-matched controls. ARMD patients may be aware of research into use of nutritional supplementation to prevent progression of their condition.

Cross-linking of collagen I by tissue transglutaminase provides a promising biomaterial for promoting bone healing

Transglutaminases (TGs) stabilize proteins by the formation of ε(γ-glutamyl)lysine cross-links. Here, we demonstrate that the cross-linking of collagen I (COL I) by tissue transglutaminase (TG2) causes an alteration in the morphology and rheological properties of the collagen fibers. Human osteoblasts (HOB) attach, spread, proliferate, differentiate and mineralize more rapidly on this cross-linked matrix compared to native collagen. When seeded on cross-linked COL I, HOB are more resistant to the loss of cell spreading by incubation with RGD containing peptides and with α1, α2 and β1 integrin blocking antibodies. Following adhesion on cross-linked collagen, HOB show increased phosphorylation of the focal adhesion kinase, and increased expression of β1 and β3 integrins. Addition of human bone morphogenetic protein to HOB seeded on TG2 cross-linked COL I enhanced the expression of the differentiation marker bone alkaline phosphatase when compared to cross-linked collagen alone. In summary, the use of TG2-modified COL I provides a promising new scaffold for promoting bone healing. © 2014 Springer-Verlag.

Chain-link based HVDC voltage source converter using current injection

VSC converters are becoming more prevalent for HVDC applications. Two circuits are commercially available at present, a traditional six-switch, PWM inverter, implemented using series connected IGBTs - ABBs HVDC Light®, and the other a modular multi-level converter (MMC) - Siemens HVDC-PLUS. This paper presents an alternative MMC topology, which utilises a novel current injection technique, and exhibits several desirable characteristics.

The application of counter immunoelectrophoresis (CIE) in ocular protein studies. Part II:kinin activity in the lens wearing eye

The kinin family are a group of bioactive peptides that are closely involved in the modulation of vascular inflammation and local injury. We have demonstrated here, for the first time, a link between kinin activity and contact lens wear. Protein extracts from daily and extended wear etafilcon A, Group IV, Acuvue lenses (Vistakon), were analysed by counter immunoelectrophoresis. In this way, kinin activity associated with contact lens wear was detected. High molecular weight kininogen was used as the marker protein. In contrast, no kinin activity was detected in the non-lens wearing normal eye. © 2002 British Contact Lens Association. Published by Elsevier Science Ltd. All rights reserved.

The use of erythrocytic and animal models in the study of protein phosphorylation

Phosphorylation processes are common post-transductional mechanisms, by which it is possible to modulate a number of metabolic pathways. Proteins are highly sensitive to phosphorylation, which governs many protein-protein interactions. The enzymatic activity of some protein tyrosine-kinases is under tyrosine-phosphorylation control, as well as several transmembrane anion-fluxes and cation exchanges. In addition, phosphorylation reactions are involved in intra and extra-cellular 'cross-talk' processes. Early studies adopted laboratory animals to study these little known phosphorylation processes. The main difficulty encountered with these animal techniques was obtaining sufficient kinase or phosphatase activity suitable for studying the enzymatic process. Large amounts of biological material from organs, such as the liver and spleen were necessary to conduct such work with protein kinases. Subsequent studies revealed the ubiquity and complexity of phosphorylation processes and techniques evolved from early rat studies to the adaptation of more rewarding in vitro models. These involved human erythrocytes, which are a convenient source both for the enzymes, we investigated and for their substrates. This preliminary work facilitated the development of more advanced phosphorylative models that are based on cell lines. © 2005 Elsevier B.V. All rights reserved.

Modulation of glucagon receptor pharmacology by Receptor Activity-modifying Protein-2 (RAMP2)

The glucagon and glucagon-like peptide-1 (GLP-1) receptors play important, opposing roles in regulating blood glucose levels. Consequently, these receptors have been identified as targets for novel diabetes treatments. However, drugs acting at the GLP-1 receptor, whilst having clinical efficacy, have been associated with severe adverse side-effects and targeting of the glucagon receptor has yet to be successful. Here we use a combination of yeast reporter assays and mammalian systems, to provide a more complete understanding of glucagon receptor signaling considering the effect of multiple ligands, association with the receptor-interacting protein, receptor activity modifying protein-2 (RAMP2) and individual G protein α-subunits. We demonstrate that RAMP2 alters both ligand selectivity and G protein preference of the glucagon receptor. Importantly, we also uncover novel cross-reactivity of therapeutically used GLP-1 receptor ligands at the glucagon receptor that is abolished by RAMP2 interaction. This study reveals the glucagon receptor as a previously unidentified target for GLP-1 receptor agonists and highlights a role for RAMP2 in regulating its pharmacology. Such previously unrecognized functions of RAMPs highlight the need to consider all receptor-interacting proteins in future drug development.

Antecedents and consequences of percieved effective managerial coaching:an individual, team-level and cross-level examination

The major contribution of the study is the identification of a positive link between perceived effective managerial coaching (PEMC) and team task performance and also, the examination of PEMC adopting a multilevel research design and incorporating dual-source data. Specifically, drawing on social psychology, the thesis aims at developing and testing a comprehensive conceptual framework of the antecedents and consequences of PEMC for knowledge workers. The model takes into consideration intrapersonal, interpersonal and team-level characteristics, which relate to PEMC and, subsequently associate with important work outcomes. In this regard, the thesis identifies PEMC as a practice of dual nature in that it may be experienced not only as a one-on-one workplace developmental interaction, but also as a managerial practice that is experienced by each member of a team for co-ordination purposes. Adopting a cross-sectional survey research design, the hypotheses are tested in three organisations in Greece and the UK. In particular, hierarchical linear modelling of 191 employees nested in 60 teams yields that employees’ learning goal orientation (LGO) and high-quality exchanges between an employee and a manager (LMX) are positively related to effective MC, while a manager’s LGO moderates the relationship between employees’ LGO and PEMC. In turn, PEMC, as a one-on-one practice, is related to cognitive outcomes, such as information sharing, while as a shared team practice is related also to behavioural outcomes, including individual and team performance. Overall, the study contributes to a growing body of coaching and management literature that acknowledges PEMC as a core managerial practice.

How education should link with career paths

How good is your pharmacy practice? And what does “good” look like? Take a look back at the education and training you have received in your career to date. Has it stood you in good stead? Certainly there is a need to establish a model of professional education and development that produces good pharmacists — you should be able to demonstrate your competence regardless of your sector of work. It may help if we move away from the view that excellence in pharmacy practice is primarily defined by where you practise and the kind of job that you do. The Modernising Pharmacy Careers programme’s aspirations to integrate the undergraduate degree with the preregistration training year are bold and to be applauded — provided the outcome delivers changes that are more than superficial. The new model needs to deliver greater integration of education with practice, while retaining an adequate science base. Theory should be put into the context of practice-based, cross-sector learning needs and opportunities. For example, is the classroom really the best environment in which to learn dispensing? Pharmacokinetic theory could be put into context through creatively designed work placements. And it might make more sense to learn patient counselling in a community pharmacy, and so on. Is it resources we lack to make this happen? Or do we lack the collective will to be imaginative, to be radical and to conceive new approaches to professional education? Implicit in this new approach to education is the expectation that pharmacists should teach and mentor and, conversely, that those who teach should also engage in relevant practice. University education must produce graduates whose knowledge and competence are useful to employers. Moreover, graduates must be adequately prepared to enter any area of the profession endowed with professional self-confidence (something, arguably, that needs further development within the pharmacy psyche). Of course, becoming qualified is just the beginning. Post-qualification, pharmacists need structured career paths that foster this professional confidence, support learning and ensure recognition. To this end, the Royal Pharmaceutical Society-led professional curriculum group is working to define knowledge, skills and experience for all areas of advanced practice. More than ever, the profession needs to adopt a culture of learning, teaching and practice research that is unified. The question is: how do we move away from merely collecting qualifications (trophies) to developing meaningful careers? Pharmacists in all areas and at all levels of the profession need to consider their own willingness to make this shift. The RPS and MPC are leading the way, but are we following?

Investigating the ability of antibodies to recognize specific oxidized protein epitopes

There is a growing awareness that inflammatory diseases have an oxidative pathology, which can result in specific oxidation of amino acids within proteins. Antibody-based techniques for detecting oxidative posttranslational modifications (oxPTMs) are often used to identify the level of protein oxidation. There are many commercially available antibodies but some uncertainty to the potential level of cross reactivity they exhibit; moreover little information regarding the specific target epitopes is available. The aim of this work was to investigate the potential of antibodies to distinguish between select peptides with and without oxPTMs. Two peptides, one containing chlorotyrosine (DY-Cl-EDQQKQLC) and the other an unmodified tyrosine (DYEDQQKQLC) were synthesized and complementary anti-sera were produced in sheep using standard procedures. The anti-sera were tested using a half-sandwich ELISA and the anti-serum raised against the chloro-tyrosine containing peptide showed increased binding to the chlorinated peptide, whereas the control anti-serum bound similarly to both peptides. This suggested that antibodies can discriminate between similar peptide sequences with and without an oxidative modification. A peptide (STSYGTGC) and its variants with chlorotyrosine or nitrotyrosine were produced. The anti-sera showed substantially less binding to these alternative peptides than to the original peptides the anti-sera were produced against. Work is ongoing to test commercially available antibodies against the synthetic peptides as a comparison to the anti-sera produced in sheep. In conclusion, the antisera were able to distinguish between oxidatively modified and unmodified peptides, and two different sequences around the modification site.