bluuebunny/crossref_metadata_2025_split huggingface dataset:
Global structural rearrangement of the cell penetrating ribonuclease colicin E3 on interaction with phospholipid membranes
Authors: Mosbahi Khédidja , Walker Daniel , James Richard , Moore Geoffrey R. , Kleanthous Colin 
Published: February 2006

AbstractNuclease type colicins and related bacteriocins possess the unprecedented ability to translocate an enzymatic polypeptide chain across the Gram‐negative cell envelope. Here we use the rRNase domain of the cytotoxic ribonuclease colicin E3 to examine the structural changes on its interaction with the membrane. Using phospholipid vesicles as model membranes we show that anionic membranes destabilize the nuclease domain of the rRNase type colicin E3. Intrinsic tryptophan fluorescence and circular dichroism show that vesicles consisting of pure DOPA act as a powerful protein denaturant toward the rRNase domain, although this interaction can be entirely prevented by the addition of salt. Binding of E3 rRNase to DOPA vesicles is an endothermic process (ΔH = 24 kcal mol−1), reflecting unfolding of the protein. Consistent with this, binding of a highly destabilized mutant of the E3 rRNase to DOPA vesicles is exothermic. With mixed vesicles containing anionic and neutral phospholipids at a ratio of 1:3, set to mimic the charge of the Escherichia coli inner membrane, destabilization of E3 rRNase is lessened, although the melting temperature of the protein at pH 7.0 is greatly reduced from 50°C to 30°C. The interaction of E3 rRNase with 1:3 DOPA:DOPC vesicles is also highly dependent on both ionic strength and temperature. We discuss these results in terms of the likely interaction of the E3 rRNase and the related E9 DNase domains with the E. coli inner membrane and their subsequent translocation to the cell cytoplasm.
Genetic selection for protein solubility enabled by the folding quality control feature of the twin‐arginine translocation pathway
Authors: Fisher Adam C. , Kim Woojin , Delisa Matthew P. 
Published: February 2006

AbstractOne of the most vexing problems facing structural genomics efforts and the biotechnology enterprise in general is the inability to efficiently produce functional proteins due to poor folding and insolubility. Additionally, protein misfolding and aggregation has been linked to a number of human diseases, such as Alzheimer's. Thus, a robust cellular assay that allows for direct monitoring, manipulation, and improvement of protein folding could have a profound impact. We report the development and characterization of a genetic selection for protein folding and solubility in living bacterial cells. The basis for this assay is the observation that protein transport through the bacterial twin‐arginine translocation (Tat) pathway depends on correct folding of the protein prior to transport. In this system, a test protein is expressed as a tripartite fusion between an N‐terminal Tat signal peptide and a C‐terminal TEM1 β‐lactamase reporter protein. We demonstrate that survival of Escherichia coli cells on selective medium expressing a Tat‐targeted test protein/β‐lactamase fusion correlates with the solubility of the test protein. Using this assay, we isolated solubility‐enhanced variants of the Alzheimer's Aβ42 peptide from a large combinatorial library of Aβ42 sequences, thereby confirming that our assay is a highly effective selection tool for soluble proteins. By allowing the bacterial Tat pathway to exert folding quality control on expressed target protein sequences, we have generated a powerful tool for monitoring protein folding and solubility in living cells, for molecular engineering of solubility‐enhanced proteins or for the isolation of factors and/or cellular conditions that stabilize aggregation‐prone proteins.
Exploring the interaction between the protein kinase A catalytic subunit and caveolin‐1 scaffolding domain with shotgun scanning, oligomer complementation, NMR, and docking
Authors: Levin Aron M. , Coroneus John G. , Cocco Melanie J. , Weiss Gregory A. 
Published: February 2006

AbstractThe techniques of phage‐displayed homolog shotgun scanning, oligomer complementation, NMR secondary structure analysis, and computational docking provide a complementary suite of tools for dissecting protein–protein interactions. Focusing these tools on the interaction between the catalytic sub‐unit of protein kinase A (PKAcat) and caveolin‐1 scaffolding domain (CSD) reveals the first structural model for the interaction. Homolog shotgun scanning varied each CSD residue as either a wild‐type or a homologous amino acid. Wild‐type to homolog ratios from 116 different homologous CSD variants identified side‐chain functional groups responsible for precise contacts with PKAcat. Structural analysis by NMR assigned an α‐helical conformation to the central residues 84– 97 of CSD. The extensive mutagenesis data and NMR secondary structure information provided constraints for developing a model for the PKAcat–CSD interaction. Addition of synthetic CSD to phage‐displayed CSD resulted in oligomer complementation, or enhanced binding to PKAcat. Together with previous experiments examining the interaction between CSD and endothelial nitric oxide synthase (eNOS), the results suggest a general oligomerization‐dependent enhancement of binding between signal transducing enzymes and caveolin‐1.
Monitoring protein aggregation during thermal unfolding in circular dichroism experiments
Authors: Benjwal Sangeeta , Verma Shikha , Röhm Klaus‐Heinrich , Gursky Olga 
Published: February 2006

AbstractThermal unfolding monitored by spectroscopy or calorimetry is widely used to determine protein stability. Equilibrium thermodynamic analysis of such unfolding is often hampered by its irreversibility, which usually results from aggregation of thermally denatured protein. In addition, heat‐induced protein misfolding and aggregation often lead to formation of amyloid‐like structures. We propose a convenient method to monitor in real time protein aggregation during thermal folding/ unfolding transition by recording turbidity or 90° light scattering data in circular dichroism (CD) spectroscopic experiments. Since the measurements of turbidity and 90° light scattering can be done simultaneously with far‐ or near‐UV CD data collection, they require no additional time or sample and can be directly correlated with the protein conformational changes monitored by CD. The results can provide useful insights into the origins of irreversible conformational changes and test the linkage between protein unfolding or misfolding and aggregation in various macromolecular systems, including globular proteins and protein–lipid complexes described in this study, as well as a wide range of amyloid‐forming proteins and peptides.
Cooperative hydrogen bond interactions in the streptavidin–biotin system
Authors: Hyre David E. , Le Trong Isolde , Merritt Ethan A. , Eccleston John F. , Green N. Michael , Stenkamp Ronald E. , Stayton Patrick S. 
Published: February 2006

AbstractThe thermodynamic and structural cooperativity between the Ser45– and D128–biotin hydrogen bonds was measured by calorimetric and X‐ray crystallographic studies of the S45A/D128A double mutant of streptavidin. The double mutant exhibits a binding affinity ∼2 × 107 times lower than that of wild‐type streptavidin at 25°C. The corresponding reduction in binding free energy (ΔΔG) of 10.1 kcal/mol was nearly completely due to binding enthalpy losses at this temperature. The loss of binding affinity is 11‐fold greater than that predicted by a linear combination of the single‐mutant energetic perturbations (8.7 kcal/mol), indicating that these two mutations interact cooperatively. Crystallographic characterization of the double mutant and comparison with the two single mutant structures suggest that structural rearrangements at the S45 position, when the D128 carboxylate is removed, mask the true energetic contribution of the D128–biotin interaction. Taken together, the thermodynamic and structural analyses support the conclusion that the wild‐type hydrogen bond between D128–OD and biotin–N2 is thermodynamically stronger than that between S45–OG and biotin–N1.
The Health Promotion of Aging Workers from the Perspective of Occupational Health Professionals
Authors: Naumanen Paula 
Published: February 2006

ABSTRACT
Objectives:
This study defines and describes, from the perspective of occupational health professionals, (a) the aging worker, (b) health promotion for aging workers, (3) employees' health‐promoting activities, (4) workplace health promotion factors, (5) occupational health promotion activities, (6) copartners supporting the health promotion, and (7) the impact of health promotion activities for older workers. A model is proposed, based on these issues. Design: A cross‐sectional, qualitative design was used to analyze e‐mailed essays. Content analysis of the essays yielded themes that were validated by an independent research colleague. Sample: Ten group essays from 16 occupational health professionals who had participated in the Health Promotion Project of Aging Workers in Northern Karelia, Finland were received. Results: An aging employee was defined as a mature worker. Health promotion was defined as early prevention of the influence of health hazards. Workers can improve their health through living habits, positive attitude, personal relationships, and education. Individual work arrangements, good work atmosphere, and appreciative leadership were essential factors for the health of workers. Occupational health professionals supported workers by health examinations, workplace visits, counseling, and organizing activities. Conclusions: The impacts of health promotion activities were better health, productivity, and work satisfaction.
Assessment of the validity of a population pharmacokinetic model for epirubicin
Authors: Ralph Lorraine D. , Sandstrom Marie , Twelves Chris , Dobbs Nicola A. , Thomson Alison H. 
Published: February 2006

AimsThe aim of this study was to evaluate a population model for epirubicin clearance using internal and external validation techniques.MethodsJackknife samples were used to identify outliers in the population dataset and individuals influencing covariate selection. Sensitivity analyses were performed in which serum aspartate transaminase (AST) values (a covariate in the population model) or epirubicin concentrations were randomly changed by ±10%. Cross‐validation was performed five times, on each occasion using 80% of the data for model development and 20% to assess the performance of the model. External validation was conducted by assessing the ability of the population model to predict concentrations and clearances in a separate group of 79 patients.ResultsStructural parameter estimates from all jackknife samples were within 7.5% of the final population estimates and examination of log likelihood values indicated that the selection of AST in the final model was not due to the presence of outliers. Alteration of AST or epirubicin concentrations by ± 10% had a negligible effect on population parameter estimates and their precision. In the cross‐validation analysis, the precision of clearance estimates was better in patients with AST concentrations >150 U l−1. In the external validation, epirubicin concentrations were over‐predicted by 81.4% using the population model and clearance values were also poorly predicted (imprecision 43%).ConclusionsThe results of internal validation of population pharmacokinetic models should be interpreted with caution, especially when the dataset is relatively small.
Efficacy of needle‐free administration of recombinant human growth hormone in adults with growth hormone deficiency
Authors: Pereira Alberto M. , van der Klaauw Agatha A. , Koppeschaar Hans P. F. , Smit Jan W. A. , van Thiel Sjoerd W. , van Doorn Jaap , Biermasz Nienke R. , Roelfsema Ferdinand , Romijn Johannes A. 
Published: February 2006

AimNeedle‐free administration of recombinant human growth hormone (rhGH) is effective in the treatment of growth hormone deficiency (GHD) in children, but has not been studied in adult patients. Therefore, we evaluated the efficacy of needle‐free administration of rhGH in adults with GHD.MethodsInsulin‐like growth factor‐I (IGF‐I) concentrations were compared in newly diagnosed patients with GHD (n = 21) and in patients previously treated by subcutaneous injection of rhGH (switchers, n = 34), at baseline, 12 months and 24 months.ResultsIn the new patients, IGF‐I standard deviation scores (SDS) increased from − 1.82 ± 0.46 to + 0.75 ± 0.33 at 12 months and to + 0.65 ± 0.41 at 24 months (P ≤ 0.001 vs. baseline). In switchers, IGF‐I SDS remained unchanged with values of + 0.98 ± 0.32 at baseline, + 0.87 ± 0.23 at 12 months and + 0.73 ± 0.29 at 24 months (P = 0.696 vs. baseline). In new patients, the rhGH dose was 0.46 ± 0.03 mg day−1 at 12 months and 0.47 ± 0.03 mg day−1 at 24 months. In switchers, the rhGH dose was 0.53 ± 0.04 mg day−1 at baseline (s.c. injection), 0.52 ± 0.03 mg day−1 at 12 months and 0.48 ± 0.03 mg day−1 at 24 months (NS between the different time points). There was no difference in the dose of rhGH at 12 and 24 months between the two groups. Side‐effects were generally minor and consisted of local tissue reactions.ConclusionAdministration of rhGH by needle‐free, transdermal injection is effective in maintaining IGF‐I concentrations in the normal range for age in adults with GHD, and is as effective as traditional subcutaneous injection of rhGH.
Quantitative prediction ofin vivoinhibitory interactions involving glucuronidated drugs fromin vitrodata: the effect of fluconazole on zidovudine glucuronidation
Authors: Uchaipichat Verawan , Winner Leanne K. , Mackenzie Peter I. , Elliot David J. , Williams J Andrew , Miners John O. 
Published: February 2006

AimsUsing the fluconazole–zidovudine (AZT) interaction as a model, to determine whether inhibition of UDP–glucuronosyltransferase (UGT) catalysed drug metabolismin vivocould be predicted quantitatively fromin vitrokinetic data generated in the presence and absence bovine serum albumin (BSA).MethodsKinetic constants for AZT glucuronidation were generated using human liver microsomes (HLM) and recombinant UGT2B7, the principal enzyme responsible for AZT glucuronidation, as the enzyme sources with and without fluconazole.Kivalues were used to estimate the decrease in AZT clearancein vivo.ResultsAddition of BSA (2%) to incubations decreased theKmvalues for AZT glucuronidation by 85–90% for the HLM (923 ± 357 to 91 ± 9 µm) and UGT2B7 (478–70 µm) catalysed reactions, with little effect onVmax. Fluconazole, which was shown to be a selective inhibitor of UGT2B7, competitively inhibited AZT glucuronidation by HLM and UGT2B7. Like theKm, BSA caused an 87% reduction in theKifor fluconazole inhibition of AZT glucuronidation by HLM (1133 ± 403 to 145 ± 36 µm) and UGT2B7 (529 to 73 µm).Kivalues determined for fluconazole using HLM and UGT2B7 in the presence (but not absence) of BSA predicted an interactionin vivo. The predicted magnitude of the interaction ranged from 41% to 217% of the reported AUC increase in patients, depending on the value of thein vivofluconazole concentration employed in calculations.ConclusionsKivalues determined under certain experimental conditions may quantitatively predict inhibition of UGT catalysed drug glucuronidationin vivo.
An ex vivo evaluation of a new root canal irrigation technique with intracanal aspiration
Authors: Fukumoto Y. , Kikuchi I. , Yoshioka T. , Kobayashi C. , Suda H. 
Published: February 2006

AbstractAim  To evaluate the effectiveness of a new root canal irrigation technique with intracanal aspiration in removing the smear layer and to assess irrigant extrusion ex vivo.Methodology  Thirty‐five instrumented canals of extracted human canine teeth that had been resected apically by removing 3 mm of the root tip were divided into one control and four experimental groups of seven teeth each. The roots were fixed in a plastic case and surrounded with normal saline agar coloured with 1% acid red. No irrigation was performed in the control teeth. Each root canal in the experimental groups was irrigated with 9 mL of 14% ethylenediaminetetraacetic acid for 3 min, and then with 6 mL of 6% sodium hypochlorite (NaOCl) for 2 min. In the intracanal aspiration technique, the irrigant was delivered from the tip of an injection needle placed 12 mm from the apical root‐end and an aspiration needle that was connected to a Root ZXTM® apex locator placed 2 and 3 mm short of the apical root‐end in groups 1 and 2, respectively. In the conventional method, the tip of an injection needle used for delivery of the irrigant and as an active electrode was placed 2 and 3 mm short of the apical root‐end in groups 3 and 4, respectively, the tip of the aspiration needle was placed 12 mm from the apical root‐end in these groups. The readings of the Root ZXTM® during irrigation were recorded. The cleanliness of the canal was evaluated by scoring smear layer from scanning electron microscopy (SEM) images of the canal. Extrusion of NaOCl was detected by measuring the discoloured area of the agar around the apical root‐end. The data obtained were statistically analysed by one‐way anova, the Kruskal–Wallis test and Friedman's test.Results  In the SEM study, the canals in groups 1–3 were significantly cleaner than those in the control and group 4 (P < 0.05). The mean Root ZXTM® readings in groups 1–3 were approximately ‘0.5’. The discoloured area in group 3 was significantly larger than the other groups (P < 0.05).Conclusions  Irrigation using the intracanal aspiration technique allowed more effective removal of the smear layer than that performed by the conventional method in an apically resected canine tooth. The intracanal aspiration technique produced limited extrusion of the irrigant beyond the apical foramen.
Longitudinal study of periapical and endodontic status in a Danish population
Authors: Kirkevang L.‐L. , Væth M. , Hörsted‐Bindslev P. , Wenzel A. 
Published: February 2006

AbstractAim  To describe and discuss changes in periapical and endodontic status in a general Danish population.Methodology  In 1997, 616 randomly selected individuals had a full‐mouth radiographic survey taken. In 2003, 77% of the participants attended for a new full‐mouth radiographic examination. Information on endodontic treatment and periapical status was obtained. The periapical index (PAI) was used to assess apical periodontitis (AP).Results  More participants had root filling(s) and AP in 2003 than in 1997. More teeth had AP and/or root fillings in 2003. Fewer of the root‐filled teeth (RFT) had AP in 2003. Less than 3% of the teeth without root fillings (NRFT) that in 1997 had no AP, developed AP and/or received a root filling. Of the NRFT which in 1997 had AP, more than 35% still had AP and no root filling in 2003. Approximately, 30% of the NRFT with AP in 1997 received a root filling. Of the teeth that received a root filling, 40% had healed, whereas 60% had not. Approximately, 25% of the NRFT that in 1997 had AP had been extracted. In 1997 there were 618 RFT, 314 of the RFT had no AP in 1997. Almost 20% of the RFT that in 1997 were periapically sound, developed AP. Of the 304 RFT with AP in 1997, approximately 30% had healed, in 60% AP persisted, and 10% were extracted.Conclusions  The present study indicates that caution must be exercised when statements on the outcome of root canal treatment are made based on the cross‐sectional studies.
A simple mounting model for consistent determination of the accuracy and repeatability of apex locators
Authors: ElAyouti A. , Löst C. 
Published: February 2006

AbstractAim  To develop a precise and simple mounting model (MM) for evaluating apex locators and to compare the repeatability of the MM with a conventional visual method (VM).Methodology  Electronic working length determination was performed in 32 maxillary central incisors using two methods: (i) the MM method and (ii) a conventional VM. The MM utilizes a micrometer to determine the distance travelled by the measuring file during working length determination. In the VM, the length of the measuring file (representing the working length) is determined visually using a caliper and a microscope at 6× magnification. Each measurement was repeated once. The repeatability of each method was evaluated by calculating the coefficient of repeatability.Results  The coefficient of repeatability, which includes 95% of the differences between repeated measurements, was 0.04 mm for MM compared with 0.9 mm for VM. The measurement error of MM was significantly lower than VM (0.02 and 0.4 mm respectively). There was a statistically significant difference between the means of absolute difference in repeated measurements: MM 0.01 mm (95% confidence interval (CI): 0.01; 0.02 mm) compared with VM 0.4 mm (95% CI: 0.3; 0.5 mm).Conclusion  The new MM had superior repeatability in comparison with the conventional method where visual interpretation is a source of inaccurate measurement.
Reinforcement effect of polyethylene fibre in root‐filled teeth: comparison of two restoration techniques
Authors: Belli S. , Erdemir A. , Yildirim C 
Published: February 2006

AbstractAim  To compare in vitro two different fibre placement techniques on reinforcement of root‐filled molar teeth with mesio‐occluso‐distal (MOD) cavities.Methodology  Fifty sound extracted human mandibular molars were used (n = 10). Group 1 served as control. From group 2–5, endodontic access and standard MOD cavities were prepared. Following root canal treatment, group 2 was kept unrestored. In groups 3 and 4, the teeth were restored with composite resin (AP‐X; Kuraray, Japan). A groove was prepared on occlusal surface of the finished restorations in group 4 from buccal to lingual direction. Polyethylene woven fibre (Ribbond; Seattle, WA, USA) was inserted in the groove in combination with flowable resin that was cured for 20 s and covered with composite resin. Cavity surfaces were covered with flowable resin in group 5, and polyethylene fibre was placed into the bed of resin in a buccal to lingual direction before the composite restoration was placed. All specimens were stored in 100% humidity at 37 °C for 24 h. Compressive loading of the teeth was performed by a universal testing machine at a crosshead speed of 0.5 mm min−1 until failure. The data were recorded in Newton and submitted to anova and Tukey post hoc test.Results  The mean load necessary to fracture the samples in each group was: G1: 1671.57 ± 131.54a; G2: 375.21 ± 34.30b; G3: 749.47 ± 124.54c; G4: 1224.36 ± 132.17d; G5: 926.88 ± 118.28e. Different superscript letters demonstrate significant difference between groups.Conclusions  Polyethylene fibre use over or under MOD composite restorations significantly increased fracture strength. However, when the fibre was placed on the occlusal surface of the restoration in buccal to lingual direction, significantly higher fracture resistance was observed.