bluuebunny/crossref_metadata_2025 huggingface dataset:
The calcium‐binding protein ALG‐2 promotes endoplasmic reticulum exit site localization and polymerization of Trk‐fused gene (TFG) protein
Authors: Kanadome Takashi , Shibata Hideki , Kuwata Keiko , Takahara Terunao , Maki Masatoshi
Published: November 2016

Apoptosis‐linked gene 2 (ALG‐2), which is a gene product of PDCD6, is a 22‐kDa Ca2+‐binding protein. Accumulating evidence points to a role for ALG‐2 as a Ca2+‐responsive adaptor protein. On binding to Ca2+, ALG‐2 undergoes a conformational change that facilitates its interaction with various proteins. It also forms a homodimer and heterodimer with peflin, a paralog of ALG‐2. However, the differences in cellular roles for the ALG‐2 homodimer and ALG‐2/peflin heterodimer are unclear. In the present study, we found that Trk‐fused gene (TFG) protein interacted with the ALG‐2 homodimer. Immunostaining analysis revealed that TFG and ALG‐2 partially overlapped at endoplasmic reticulum exit sites (ERES), a platform for COPII‐mediated protein transport from the endoplasmic reticulum. Time‐lapse live‐cell imaging demonstrated that both green fluorescent protein‐fused TFG and mCherry‐fused ALG‐2 are recruited to ERES after thapsigargin treatment, which raises intracellular Ca2+ levels. Furthermore, overexpression of ALG‐2 induced the accumulation of TFG at ERES. TFG has an ALG‐2‐binding motif and deletion of the motif decreased TFG binding to ALG‐2 and shortened its half‐life at ERES, suggesting a critical role for ALG‐2 in retaining TFG at ERES. We also demonstrated, by in vitro cross‐linking assays, that ALG‐2 promoted the polymerization of TFG in a Ca2+‐dependent manner. Collectively, the results suggest that ALG‐2 acts as a Ca2+‐sensitive adaptor to concentrate and polymerize TFG at ERES, supporting a potential role for ALG‐2 in COPII‐dependent trafficking from the endoplasmic reticulum.
The Impact of Flipped Online Kanji Instruction on Written Vocabulary Learning for Introductory and Intermediate Japanese Language Students
Authors: Mori Yoshiko , Omori Motoko , Sato Kumi
Published: November 2016

This study explored the pedagogical benefits of flipped online kanji (i.e., Chinese characters used in Japanese) instruction integrated into college‐level introductory and intermediate Japanese language courses. Using a quasi‐experimental mixed design, the investigation looked at the effects of two instructional approaches: (1) a flip approach in which students learned new characters using online materials prior to class, and (2) the conventional (nonflip) approach in which in‐class lectures were followed by printed exercises. During the 2014–2015 academic year, a total of 46 students who were enrolled in introductory or intermediate courses learned 9 to 12 sets of 10 new kanji characters per semester through one lesson per week. Half of the lessons were taught in the flip format, and the other half were taught in the nonflip format. The results showed that introductory students scored better on postlesson tests in the flip condition than in the nonflip condition only for fall 2014 but not for spring 2015, whereas no statistically significant difference was observed for intermediate students. However, the students in both courses reported that online exercises helped them improve their kanji knowledge. Consequently, a substantial portion of kanji instruction can be shifted to self‐paced, semi‐structured online learning with no negative impact on instructional quality.
Novel translocation intermediate allows re‐evaluation of roles of ATP, proton motive force and SecG at the late stage of preprotein translocation
Authors: Nishiyama Ken‐ichi , Tokuda Hajime
Published: November 2016

Presecretory proteins such as pOmpA are translocated across the inner membrane of Escherichia coli by Sec translocase powered by ATP and proton motive force (PMF). Translocation activity has been determined by protease protection assaying in vitro. We identified a new translocation intermediate at a late stage, which was protected by proteinase K (PK), but became PK sensitive upon urea extraction. At a late stage of pOmpA translocation driven by PMF in the presence of a nonhydrolyzable ATP analogue, the PK‐protected materials arose, but were pulled back upon urea extraction, indicating that completion of translocation requires ATP hydrolysis. When inverted membrane vesicles prepared from secG‐null strain (ΔSecG IMV) were used in the absence of PMF, the translocation intermediate was accumulated. When the ATP concentration was low in the absence of PMF, the translocation intermediate was also accumulated. Imposition of PMF in the presence of a low ATP concentration caused recovery of pOmpA translocation and resistance to urea extraction for SecG+ IMV, but not for ΔSecG IMV. Thus, analysis of the late translocation intermediate showed that two of three constituents, physiological concentration of ATP, PMF and SecG, are required for the catalytic cycle of preprotein translocation, that is, completion and subsequent initiation of translocation.
Reconstruction of body cavity volume in terrestrial tetrapods
Authors: Clauss Marcus , Nurutdinova Irina , Meloro Carlo , Gunga Hanns‐Christian , Jiang Duofang , Koller Johannes , Herkner Bernd , Sander P. Martin , Hellwich Olaf
Published: November 2016

Although it is generally assumed that herbivores have more voluminous body cavities due to larger digestive tracts required for the digestion of plant fiber, this concept has not been addressed quantitatively. We estimated the volume of the torso in 126 terrestrial tetrapods (synapsids including basal synapsids and mammals, and diapsids including birds, non‐avian dinosaurs and reptiles) classified as either herbivore or carnivore in digital models of mounted skeletons, using the convex hull method. The difference in relative torso volume between diet types was significant in mammals, where relative torso volumes of herbivores were about twice as large as that of carnivores, supporting the general hypothesis. However, this effect was not evident in diapsids. This may either reflect the difficulty to reliably reconstruct mounted skeletons in non‐avian dinosaurs, or a fundamental difference in thebauplanof different groups of tetrapods, for example due to differences in respiratory anatomy. Evidently, the condition in mammals should not be automatically assumed in other, including more basal, tetrapod lineages. In both synapsids and diapsids, large animals showed a high degree of divergence with respect to the proportion of their convex hull directly supported by bone, with animals like elephants orTriceratopshaving a low proportion, and animals such as rhinoceros having a high proportion of bony support. The relevance of this difference remains to be further investigated.
Percutaneous PDA Closure in Extremely Low Birth Weight Babies
Authors: Narin Nazmi , Pamukcu Ozge , Baykan Ali , Sunkak Suleyman , Ulgey Ayse , Uzum Kazim
Published: November 2016

Aim

Patent Ductus Arteriosus is an important cause of morbidity and mortality in preterms. As birth weight decrease, risks increase. Main aim of our study is to emphasize the effectiveness and safety of percutaneous PDA closure even in extremely low birth infants.

Materials and Methods

In our center between the dates June 2014–June 2016, PDA of 10 patients less than 1,000 gr were closed percutaneously. To the best of our knowledge this study includes the largest cohort of infants less than 1,000 g in the literature, that PDA of those were percutaneously closed.

Results

Symptomatic patients, less than 1,000 gr having PDA were included in the study. All have 3 times medical therapy for PDA closure but it did not work. PDA was decided to be contributor of this medical state of them. The mean patient age was 19.5 ± 7.2 days. The median weight was 950 (842–983) gr. Mean gestational age was: 26.3 ± 0.63 weeks. Mean PDA diameter was 1.9 ± 0.41 mm. Morphology of PDA:6 of them were conical and 4 of them were tubular. In all patients ADOII‐AS device were used for PDA closure via venous route. No major complications were reported. Left pulmonary arterial stenosis was detected in 1 patient who was resolved in 6 months duration.

Conclusion

We want to emphasize that in experienced centers percutaneous closure of PDA can be an alternative to surgery even in the extremely low birth weight babies.
How Dentists Read a Technique Article: A Grounded Theory Investigation
Authors: Chambers David W. , Lyon Lucinda J.
Published: November 2016

Purpose

To conduct an empirical investigation using qualitative techniques of the way dentists engage in the process of reading a technique‐oriented journal article and what they pay attention to in the process.

Materials and Methods

Grounded theory was used to identify how dentists read an article describing the fabrication of an interim prosthesis in the esthetic zone. Twenty‐one experienced practitioners were videotaped, and their verbatim reflections were coded. The sequence of attending to various features of the paper was noted.

Results

Ninety‐five percent of readers voiced specific, multiple attempts to identify or refine the main purpose of the article as they processed the material. All readers engaged in various activities to navigate through the article, including skipping and backtracking, and none “read” the article straight through. All readers also made repeated observations about the relevance of the technique to their personal practice situation. Eighty percent used some form of “distancing,” whereby the content and value of the article were accepted, but the reader reserved the privilege of not being bound by the results because of technical, sponsorship, or methodological issues that “might be present.” The quality of photographs was accepted as a proxy for the quality of technical work performed.

Conclusions

Dentists actively customized the reading of a journal article that described a technical procedure. They imposed a non‐linear structure for absorbing information and a standard of personal relevance, and, while accepting the results, created reasons for not necessarily having to accept them as applicable. The approach clinicians use in reading a procedural article may be different from the structure writers use in preparing a paper.
Two Virtuous Actions Cannot both be Completed
Authors: Ing Michael D. K.
Published: November 2016

This essay highlights an alternative tradition of understanding value conflicts in early Confucian thought. In contrast to a prominent position among interpreters that argues for the resolvability or harmonization of conflicting values, I argue that some early Confucians conceptualized value conflicts as irresolvable. In other words, when meaningful aspects of a situation come into tension with each other and values are threatened to be either left unfulfilled or harmed, early Confucians put forth a variety of views. Some believed that all values could be tended to as long as one had the moral imagination of a sage, whereas others saw the world as a place where irreconcilable conflicts between values could occur for even the most profound people. Within this diversity, I take up a view about irresolvable value conflicts where the confrontation with a deep value conflict leaves behind a moral remainder that can mar even the character of a sage.
Ethnography and Jewish Ethics
Authors: Raucher Michal S.
Published: November 2016

This essay offers a Jewish approach to ethnography in religious ethics. Following the work of other ethnographers working in religious ethics, I explore how an ethnographic account of reproductive ethics among Haredi (ultra‐Orthodox) Jewish women in Jerusalem enhances and improves Jewish ethical discourse. I argue that ethnography should become an integral part of Jewish ethics for three reasons. First, with a contextual approach to guidance and application of law and norms, an ethnographic approach to Jewish ethics parallels the way ethical decisions are made on a daily basis in Jewish communities. Second, ethnography bolsters the voices of those involved in ethical discourse. Third, ethnography facilitates the bridge between local ethical questions and global ethical discourse.
The Buddhist Roots of Watsuji Tetsurô's Ethics of Emptiness
Authors: Sevilla Anton Luis
Published: November 2016

Watsuji Tetsurô (1889–1960) is famous for having constructed a systematic socio‐political ethics on the basis of the idea of emptiness. This essay examines his 1938 essay “The Concept of ‘Dharma’ and the Dialectics of Emptiness in Buddhist Philosophy” and the posthumously published The History of Buddhist Ethical Thought (based on lectures given in the 1920s), in order to clarify the Buddhist roots of his ethics. It aims to answer two main questions which are fundamentally linked: “Which way does Watsuji's legacy turn: toward totalitarianism or toward a balanced theory of selflessness?” and “Is Watsuji's systematic ethics Buddhist?” In order to answer these questions, this essay discusses Watsuji's view of dharma, dependent arising, and morality in Hīnayāna Buddhism. It then proceeds to Watsuji's fine‐tuning of the concept of emptiness in Mādhyamika and Yogācāra Buddhism. Finally, this essay shows how Watsuji's modernist Buddhist theory connects to his own systematic ethical theory. These two theories share a focus on non‐duality, negation, and emptiness. But they differ in their accounts of the relations between the individual and the community, between the “is” and the “ought,” and between hermeneutics and transcendence. These findings give us hints as to Watsuji's origins, pitfalls, and possibilities.
Entry Bar as Surreptitious Deportation? Zapret na v'ezd in Russian Immigration Law and Practice: A Comparative Perspective
Authors: Kubal Agnieszka
Published: November 2016

Since 2013, a three-year entry bar (zapret na v'ezd) has been issued in Russia to migrants with a record of two or more administrative offenses. This article examines the sociolegal characteristics of zapret na v'ezd by situating it in a global, comparative perspective, vis-à-vis the legal developments in the areas of deportation and removal in the United States and the United Kingdom. This article argues that the Russian entry bar law experienced a shift, established by other migration-receiving jurisdictions, from controlling the migration process to controlling the social conduct of migrants, toward an increased reliance on deportability as a form of post-entry control of the migrant population. At a broader level, I aim to shed more light on the migration governance processes in Russia—the third largest destination of migrants worldwide—by moving away from the intellectually dead-end explanations that consider Russia as a deviant exception.
Understanding China's Court Mediation Surge: Insights from a Local Court
Authors: Li Yedan , Kocken Joris , van Rooij Benjamin
Published: November 2016

This article seeks to understand how reported mediation rates in Chinese courts are produced and what they actually signify. It analyzes data obtained through prolonged fieldwork at a court in central China. The article finds that the court has directly responded to central level mediation incentives by enhancing its overall mediation rate. It has done so strategically by seeking the highest increase using the fullest discretion in the mediation incentive structure and seeking to optimize the highest rate at the lowest cost and risk to the court. This has undermined the objectives of the central level incentives toward mediation, while also drawing the courts' scarce resources away toward unnecessary mediation practices, in part far removed from the courtroom. The article concludes by drawing out broader theoretical conclusions about how information asymmetries, discretion, and goal displacement play out in hierarchical control structures of authoritarian courts.
Invasive Melanoma in a Patient with Congenital Ichthyosiform Erythroderma
Authors: Jaju Prajakta , Novoa Roberto A. , Swetter Susan M. , Sarin Kavita Y.
Published: November 2016

We describe the case of a 26‐year‐old woman with a history of congenital ichthyosiform erythroderma (CIE) who initially presented with a stage IIA amelanotic melanoma on her forearm that was surgically excised. We also review the literature on CIE‐associated skin cancers and discuss the possible contribution of ichthyosis to the risk of cutaneous malignancies. Our findings emphasize the importance of close lifelong skin cancer screening in individuals with CIE and highlight the unique malignancy risk of these individuals.
Water Policy in a Time of Climate Change: Coping with Complexity
Authors: Skinner Rob M.
Published: November 2016

The unpredictable impacts of climate change, combined with significant social and economic changes, mean that policy analysis is becoming inordinately more complex. Coping with this increasing complexity while engaging with a full range of stakeholders and the community requires a new approach to leadership and governance. Water planners (and others involved in the planning processes) need new skills in active listening and constructive cultural behavior, and all agencies need to recognize that thinking and acting only in the interests of their own silo of responsibilities will inevitably produce suboptimal outcomes. Leadership focused on constructive behavior that recognizes and rewards generosity of spirit across disciplines and between organizations is the foundation of this new approach.
Mathematical Analyses and Numerical Simulations for Forward Extrusion of Circular, Square, and Rhomboidal Sections From Round Billets Through Streamlined Dies
Authors: Hussein Ahmed Waleed , Kadhim Abdulkareem Jalil
Published: November 2016

In the present investigation, an analysis using three-dimensional upper-bound method based on continuous velocity filed has been carried out for the extrusion of circular, square, and rhomboidal sections from round billets. The die surface representation in the present work could easily be applied to the extrusion of many different shapes just by defining the functions that describe the entry and exit sections and putting them into the general formulation. The die profile was tested for the third- and fifth-order polynomial functions. The extrusion process is also simulated using the finite element code, ansys (V 14.0), in order to assist the mathematical solution and to show the stress and strain distributions for the products when the strain hardening effect is taking into the account. Effects of friction, shape complexity, reduction of area, and die length on the extrusion pressure were also investigated. The results obtained in this work were compared with the theoretical results of other workers and found to be in highly compatible.
Novel Device For Removal of Blot Clot Obstructions in Drainage Tubes
Authors: Thompson Scott M. , Thiels Cornelius A. , Aho Johnathon M.
Published: November 2016

Medical drains frequently become obstructed requiring removal or re-intervention. Currently, there is no widely accepted practice for maintaining drain patency. A simple device to remove obstructions from medical drains may have significant benefit. We manufactured a prototype drain clot extractor (DCE) for extracting obstructions. Fresh nonanticoagulated swine blood was instilled within drainage tubes of 16, 20, and 32 French (n = 2 for each size) to generate a 4 cm blood clot. The operator was then equipped with a 0.035 in. guidewire (GW) and given 2.5 min to attempt removal while blinded. Similarly, the operator was equipped with the DCE. Volumes removed were determined by volume displacement and by drain size and method of removal. Comparison of DCE to GW by analysis of variance and means comparison using Tukey were used for statistical analysis. The DCE facilitated mechanical removal of obstructions for 16, 20, and 32 French drainage tubes. Volumes removed: 16 French (GW mean 10% volume, DCE mean 70% volume p = 0.02); 20 French (GW mean 12.5% volume, DCE mean 75% volume p = 0.01); 32 French (GW mean 37.5% volume, DCE mean 100% volume p = 0.01). DCE volume removal had no dependence on drain size (p > 0.05). This device may have utility for reducing morbidity from early drain discontinuation or replacement. Further in vivo and clinical studies are needed to determine the safety, feasibility, and efficacy of this device as tool for removing medical drainage obstructions.
Deterministic and Stochastic Model Order Reduction for Vibration Analyses of Structures With Uncertainties
Authors: Yang Ji , Faverjon Béatrice , Peters Herwig , Marburg Steffen , Kessissoglou Nicole
Published: November 2016

To reduce the computational effort using polynomial chaos expansion to predict the dynamic characteristics of structures with several uncertain parameters, hybrid techniques combining stochastic finite element analysis with either deterministic or stochastic model order reduction (MOR) are developed. For the deterministic MOR, the Arnoldi-based Krylov subspace technique is implemented to reduce the system matrices of the finite element model. For the stochastic MOR, a stochastic reduced basis method is implemented in which the structural modal and frequency responses are approximated by a small number of basis vectors using stochastic Krylov subspace. To demonstrate the computational efficiency of each reduced stochastic finite element model, variability in the natural frequencies and frequency responses of a simply supported flexible plate randomized by uncertain geometrical and material parameters is examined. Results are compared with both Monte Carlo (MC) simulations and nonreduced stochastic models. Using the reduced models, the effects of the individual uncertain parameters as well as the combined uncertainties on the dynamic characteristics of the plate are examined.
A New Incremental Harmonic Balance Method With Two Time Scales for Quasi-Periodic Motions of an Axially Moving Beam With Internal Resonance Under Single-Tone External Excitation
Authors: Huang J. L. , Zhu W. D.
Published: November 2016

Quasi-periodic motion is an oscillation of a dynamic system characterized by m frequencies that are incommensurable with one another. In this work, a new incremental harmonic balance (IHB) method with only two time scales, where one is one of the m frequencies, referred to as a fundamental frequency, and the other is an interval distance of two adjacent frequencies, is proposed for quasi-periodic motions of an axially moving beam with three-to-one internal resonance under single-tone external excitation. It is found that the interval frequency of every two adjacent frequencies, located around the fundamental frequency or one of its integer multiples, is fixed due to nonlinear coupling among resonant vibration modes. Consequently, only two time scales are used in the IHB method to obtain all incommensurable frequencies of quasi-periodic motions of the axially moving beam. The present IHB method can accurately trace from periodic responses of the beam to its quasi-periodic motions. For periodic responses of the axially moving beam, the single fundamental frequency is used in the IHB method to obtain solutions. For quasi-periodic motions of the beam, the present IHB method with two time scales is used, along with an amplitude increment approach that includes a large number of harmonics, to determine all the frequency components. All the frequency components and their corresponding amplitudes, obtained from the present IHB method, are in excellent agreement with those from numerical integration using the fourth-order Runge–Kutta method.
Effect of Bolts on Flow and Heat Transfer in a Rotor–Stator Disk Cavity
Authors: Mohamed Sulfickerali Noor , Chew John W. , Hills Nicholas J.
Published: November 2016

Previous studies have indicated some differences between steady computational fluid dynamics (CFD) predictions of flow in a rotor–stator disk cavity with rotating bolts compared to measurements. Recently, time-dependent CFD simulations have revealed the unsteadiness present in the flow and have given improved agreement with measurements. In this paper, unsteady Reynolds averaged Navier–Stokes (URANS) 360 deg model CFD calculations of a rotor–stator cavity with rotor bolts were performed in order to better understand the flow and heat transfer within a disk cavity previously studied experimentally by other workers. It is shown that the rotating bolts generate unsteadiness due to wake shedding which creates time-dependent flow patterns within the cavity. At low throughflow conditions, the unsteady flow significantly increases the average disk temperature. A systematic parametric study is presented giving insight into the influence of number of bolts, mass flow rate, cavity gap ratio, and the bolts-to-shroud gap ratio on the time-dependent flow within the cavity.
Design of Hybrid Hydrostatic/Hydrodynamic Journal Bearings for Optimum Self-Compensation Under Misaligning External Loads
Authors: Martinez Esparza L. F. , Cervantes de Gortari J. G. , Chicurel Uziel E. J.
Published: November 2016

A method to design hybrid hydrostatic/hydrodynamic journal bearings, with the criterion of optimized self-compensation under misaligning loads, is presented. An analysis considering laminar and turbulent flow of a Newtonian incompressible lubricant between the bearing and a misaligned shaft, with restricted lubricant supply to each recess, is discussed. The mathematical model considers the modified steady-state Reynolds lubrication equation, an exact function for the local bearing radial clearance with a misaligned shaft, the continuity integral–differential equations at the recess limits, and boundary conditions at the cavitation zone and outer limits. The finite-difference method was used, and a modular computer program was developed. The procedure follows a univariate search to determine the optimum size and position of recesses and therefore obtain the design with the maximum reactive moment under misaligning loads. A validation of the model was obtained comparing the results with experimental and calculated data from the literature. Results for a 4 + 4 LBP hybrid bearing design are presented.
Material Selection Issues for a Nozzle Guide Vane Against Service-Induced Failure
Authors: Wu Xijia , Zhang Zhong , Jiang Leiyong , Patnaik Prakash
Published: November 2016

Nozzle guide vanes (NGV) of gas turbine engines are the first components to withstand the impingement of hot combustion gas and therefore often suffer thermal fatigue failures in service. A lifting analysis is performed for the NGV of a gas turbine engine using the integrated creep–fatigue theory (ICFT). With the constitutive formulation of inelastic strain in terms of mechanism-strain components such as rate-independent plasticity, dislocation glide-plus-climb, and grain boundary sliding (GBS), the dominant deformation mechanisms at the critical locations are thus identified quantitatively with the corresponding mechanism-strain component. The material selection scenarios are discussed with regards to damage accumulated during take-off and cruise. The interplay of those deformation mechanisms in the failure process is elucidated such that an “optimum” material selection solution may be achieved.
Mask registration and lithography platform portability for nitride fin-based field effect transistors prototyping
Authors: Suh Jae Woo , Babb Michael E. , Principe Edward L. , Harris Harlan Rusty
Published: November 2016

The authors report on the integration of a hybrid lithography process for AlGaN/GaN high-electron-mobility nanoscaled, fin-based field effect transistors with an Au-free contact. The integration consists of electron beam lithography (EBL) and optical photolithography to minimize prototyping cost and time while maximizing resolution and device array density. I-line direct photolithography is performed using standard photoresist, while EBL is accomplished using 2% polymethyl methacrylate for high resolution of fin structure patterning. Au-free contact is characterized using linear transmission line method (TLM) and is deposited along with registration marks via electron beam evaporation. The registration marks and TLM structures are used for alignment in the EBL process. Details of the device fabrication process and electrical results are discussed and show possible parallel processing of GaN and Si devices.
D-arabinose metabolism in Escherichia coli B: induction and cotransductional mapping of the L-fucose-D-arabinose pathway enzymes
Authors: Elsinghorst E A , Mortlock R P
Published: November 2016

D-Arabinose is degraded by Escherichia coli B via some of the L-fucose pathway enzymes and a D-ribulokinase which is distinct from the L-fuculokinase of the L-fucose pathway. We found that L-fucose and D-arabinose acted as the apparent inducers of the enzymes needed for their degradation. These enzymes, including D-ribulokinase, appeared to be coordinately regulated, and mutants which constitutively synthesized the L-fucose enzymes also constitutively synthesized D-ribulokinase. In contrast to D-arabinose-positive mutants of E. coli K-12, in which L-fuculose-1-phosphate and D-ribulose-1-phosphate act as inducers of the L-fucose pathway, we found that these intermediates did not act as inducers in E. coli B. To further characterize the E. coli B system, some of the L-fucose-D-arabinose genes were mapped by using bacteriophage P1 transduction. A transposon Tn10 insertion near the E. coli B L-fucose regulon was used in two- and three-factor reciprocal crosses. The gene encoding D-ribulokinase, designated darK, was found to map within the L-fucose regulon, and the partial gene order was found to be Tn10-fucA-darK-fucI-fucK-thyA.
Signal transduction in chemotaxis to oxygen in Escherichia coli and Salmonella typhimurium
Authors: Shioi J , Tribhuwan R C , Berg S T , Taylor B L
Published: November 2016

Pathways previously proposed for sensory transduction in chemotaxis to oxygen (aerotaxis) involved either (i) cytochrome o, the electron transport system, and proton motive force or (ii) enzyme IIGlucose and the phosphoenolpyruvate:carbohydrate phosphotransferase system for active transport. This investigation distinguished between these possibilities. Aerotaxis was absent in a cyo cyd strain of Escherichia coli that lacked both cytochrome o and cytochrome d, which are the terminal oxidases for the branched electron transport system in E. coli. Aerotaxis, measured by either a spatial or temporal assay, was normal in E. coli strains that had a cyo+ or cyd+ gene or both. The membrane potential of all oxidase-positive strains was approximately -170 mV in aerated medium at pH 7.5. Behavioral responses to changes in oxygen concentration correlated with changes in proton motive force. Aerotaxis was normal in ptsG and ptsI strains that lack enzyme IIGlucose and enzyme I, respectively, and are deficient in the phosphotransferase system. A cya strain that is deficient in adenylate cyclase also had normal aerotaxis. We concluded that aerotaxis was mediated by the electron transport system and that either the cytochrome d or the cytochrome o branch of the pathway could mediate aerotaxis.
Site-specific integration and expression of a developmental promoter in Myxococcus xanthus
Authors: Li S F , Shimkets L J
Published: November 2016

A series of intercellular signals are involved in the regulation of gene expression during fruiting body formation of Myxococcus xanthus. Mutations which block cell interactions, such as csgA (formerly known as spoC), also prevent expression of certain developmentally regulated promoters. csgA+ cells containing Tn5 lac omega DK4435, a developmentally regulated promoter fused to lacZ, began synthesizing lacZ mRNA 12 to 18 h into the developmental cycle. beta-Galactosidase specific activity increased about 12 h later. Neither lacZ mRNA nor beta-galactosidase activity was detected in a developing csgA mutant containing omega DK4435. The developmental promoter and its fused lacZ reporter gene were cloned into a pBR322-derived plasmid vector containing a portion of bacteriophage Mx8. These plasmids preferentially integrated into the M. xanthus chromosome by site-specific recombination at the bacteriophage Mx8 attachment site and maintained a copy number of 1 per chromosome. The integrated plasmids were relatively stable, segregating at a frequency of 0.0007% per generation in the absence of selection. The cloned and integrated promoter behaved like the native promoter, expressing beta-galactosidase at the proper time during wild-type development and failing to express the enzyme during development of a csgA mutant. The overall level of beta-galactosidase expression in merodiploid cells containing one native promoter and one promoter fused to lacZ was about half that of cells containing a single promoter fused to lacZ. These results suggest that the timing of developmentally regulated gene expression is largely independent of the location of this gene within the chromosome. Furthermore, they show that site-specific recombination can be a useful tool for establishing assays for promoter or gene function in M. xanthus.
Genetics and regulation of enterobactin genes in Shigella flexneri
Authors: Schmitt M P , Payne S M
Published: November 2016

Although Shigella flexneri possesses the genes for two siderophore systems, enterobactin and aerobactin, the enterobactin system is only rarely utilized. To investigate the regulation of enterobactin expression in S. flexneri, all of the genes specifically required for synthesis and transport of enterobactin were cloned from both an expressing (Ent+) and a nonexpressing (Ent-) strain. Notable differences between the cloned genes included endonuclease restriction site changes and the presence of an IS1 element in the Ent- DNA. Southern hybridization revealed that this IS1 element, present at the 3' end of the entF gene, is conserved at this location in different strains and serotypes of Ent- S. flexneri. The Ent- cloned genes were tested for their ability to complement the defect in 11 different Escherichia coli enterobactin mutants. The Ent- genes fully complemented nine mutants but failed to complement the entF mutant AN117 and only partially complemented the entE mutant AN93. Whole-cell RNA isolated from E. coli and the Shigella strains was hybridized to 32P-labeled DNA containing the entB gene or a fragment carrying a portion of the entF gene. E. coli and the Ent+ Shigella strains exhibited derepression of transcription of these genes in low-iron media. Transcription in the Ent- strain remained repressed regardless of iron concentration. Expression of the entB and entF genes was also examined in an Ent- Shigella fur mutant. Expression of entF was only partially derepressed and entB remained fully repressed at all iron concentrations, suggesting that factors other than Fur are responsible for the repression of these enterobactin genes in the Ent- Shigella strains.
Selenium increases hydrogenase expression in autotrophically cultured Bradyrhizobium japonicum and is a constituent of the purified enzyme
Authors: Boursier P , Hanus F J , Papen H , Becker M M , Russell S A , Evans H J
Published: November 2016

We have investigated the effect of added selenite on autotrophic growth and the time course of hydrogen oxidation derepression in Bradyrhizobium japonicum 122DES cultured in a medium purified to remove selenium compounds. In addition, hydrogenase was purified to near homogeneity and examined for the specific incorporation of Se into the enzyme. The addition of Se at 0.1 microM significantly increased total cell protein and hydrogenase specific activity of harvested cells. Also, the addition of SeO3(2-) enhanced the time course of hydrogenase derepression by 133%, whereas VO3, AsO2(2-), SO2(2-), and TeO3(2-) failed to substantially affect hydrogenase derepression. During the final chromatographic purification of hydrogenase, a striking coincidence in peaks of protein content, Se radioactivity, and hydrogenase activity of fractions was obtained. The total Se content expressed per milligram of protein increased manyfold during the purification procedure. The mean Se content of the purified hydrogenase was 0.56 +/- 0.13 mol of Se per mol of enzyme. These results indicate that Se is an important element in the H2 metabolism of B. japonicum and that hydrogenase from B. japonicum is a seleno protein.
Highly repetitive tRNA(Pro)-tRNA(His) gene cluster from Photobacterium phosphoreum
Authors: Giroux S , Beaudet J , Cedergren R
Published: November 2016

A DNA fragment comprising the four tRNA gene sequences of the Escherichia coli argT locus hybridized with two Sau3A-generated DNA fragments from the vibrio Photobacterium phosphoreum (ATCC 11040). Detailed sequence analysis of the longer fragment shows the following gene organization: 5'-promoter-tRNA(Pro)-tRNAPro-tRNA(Pro)-tRNA(His)-tRNA(Pro)-tRNA(Pro)- tRNA(His)-tRNA(Pro)-five pseudogenes derived from the upstream tRNAPro interspersed by putative Rho-independent terminators. This sequence demonstrates the presence of highly repetitive, tandem tRNA genes in a bacterial genome. Furthermore, a stretch of 304 nucleotides from this cluster was found virtually unchanged in the other (shorter) fragment which was previously sequenced. The two clusters together contain eight tRNA(Pro) pseudogenes and eight fully intact tRNA(Pro) genes, an unusually high number for a single eubacterial isoacceptor tRNA. These results show that the organization of some tRNA operons is highly variable in eubacteria.
Duplication of a 2,4-dichlorophenoxyacetic acid monooxygenase gene in Alcaligenes eutrophus JMP134(pJP4)
Authors: Perkins E J , Lurquin P F
Published: November 2016

The Alcaligenes eutrophus JMP134 plasmid pJP4 contains genes necessary for the complete degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 3-chlorobenzoic acid. tfdA encodes 2,4-D monooxygenase, the initial enzyme in the 2,4-D catabolic pathway. The tfdA locus has recently been localized to a region on pJP4 13 kilobases away from a cluster of five genes, tfdB to tfdF, which encode the enzymes responsible for the further degradation of 2,4-D to chloromaleylacetic acid (W.R. Streber, K. N. Timmis, and M. H. Zenk, J. Bacteriol. 169:2950-2955, 1987). A second, dissimilar locus on pJP4, tfdAII, has been observed which encodes 2,4-D monooxygenase activity. Gas chromatographic analysis of the 2,4-D metabolites of A. eutrophus harboring pJP4 or subclones thereof localized tfdAII to within a 9-kilobase SstI fragment of pJP4 which also carries the genes tfdBCDEF. This fragment was further characterized in Escherichia coli by deletion and subcloning analysis. A region of 2.5 kilobases, adjacent to tfdC, enabled E. coli extracts to degrade 2,4-D to 2,4-dichlorophenol. Hybridization under low-stringency conditions was observed between tfdA and tfdAII, signifying that the 2,4-D monooxygenase gene was present as two related copies on pJP4.
Genetic organization and regulation of proteins associated with production of syringotoxin by Pseudomonas syringae pv. syringae
Authors: Morgan M K , Chatterjee A K
Published: November 2016

Many strains of Pseudomonas syringae pv. syringae produce one of two low-molecular-weight, peptide-containing phytotoxins, either syringomycin (SR) or syringotoxin (ST). An analysis of Tn5-induced ST-mutants revealed alterations in the presence of two large proteins (ca. 470 and 435 kilodaltons). Apparent truncated forms of the 470 (ST1)- or 435 (ST2)-kilodalton proteins were observed in some mutants. Mapping of the Tn5 insertions and size determinations of truncated proteins suggested that both ST1 and ST2 are in the same transcriptional unit, with ST1 being directly upstream of ST2. When an ST-producing strain of P. syringae pv. syringae was grown under toxin-inducing conditions, ST1 and ST2 were first detected at the end of the exponential phase of growth, which was when the first accumulation of ST was observed. High iron levels were essential for efficient ST production. At concentrations of FeCl3 of between 0.2 and 5 microM, the amount of toxin accumulated was almost directly proportional to the iron concentrations. The amount of ST1 and ST2 present showed a corresponding increase in response to iron concentrations. Our genetic and physiological data implicate ST1 and ST2 in the biosynthesis of syringotoxin.
Energy-dependent, high-affinity transport of nickel by the acetogen Clostridium thermoaceticum
Authors: Lundie L L , Yang H C , Heinonen J K , Dean S I , Drake H L
Published: November 2016

The nickel transport system of Clostridium thermoaceticum was investigated with 63NiCl2 and an anaerobic microfiltration transport assay. Transport was optimal at pH 7 to pH 7.5 and 65 degrees C and decreased in the presence of metabolic uncouplers and inhibitors. Exogenous nickel was concentrated 3,000-fold over the apparent nickel concentration gradient during typical transport assays. Stored cellular energy appeared to provide a short-term energy source to power nickel transport, and starvation experiments demonstrated external energy source stimulation of nickel translocation. The apparent Km and Vmax for nickel transport by carbon monoxide-dependent chemolithotrophic cells approximated 3.2 microM Ni and 400 pmol of Ni transported per min per mg of cells (dry weight), respectively. Magnesium, calcium, cobalt, iron, manganese, and zinc did not inhibit the transport of nickel.
An enhancer of DNA replication
Authors: Gennaro M L , Novick R P
Published: November 2016

cmp, a nucleotide sequence element in the plasmid pT181 of Staphylococcus aureus, acts as an enhancer of DNA replication. When cmp is present on an unrelated vector along with the pT181 origin of replication, it increases the ability of the linked pT181 origin to compete with a coresident pT181 plasmid for the initiator protein RepC. cmp is contained within a 156-base-pair segment, and its deletion from pT181 reduces by twofold the frequency of plasmid replication under derepressed conditions. The enhancer sequence contains a locus of DNA bending, and enhancer activity decreases with distance from the replication origin.
Interference between Rhizobium meliloti and Rhizobium trifolii nodulation genes: genetic basis of R. meliloti dominance
Authors: Debellé F , Maillet F , Vasse J , Rosenberg C , de Billy F , Truchet G , Dénarié J , Ausubel F M
Published: November 2016

Transfer of an IncP plasmid carrying the Rhizobium meliloti nodFE, nodG, and nodH genes to Rhizobium trifolii enabled R. trifolii to nodulate alfalfa (Medicago sativa), the normal host of R. meliloti. Using transposon Tn5-linked mutations and in vitro-constructed deletions of the R. meliloti nodFE, nodG, and nodH genes, we showed that R. meliloti nodH was required for R. trifolii to elicit both root hair curling and nodule initiation on alfalfa and that nodH, nodFE, and nodG were required for R. trifolii to elicit infection threads in alfalfa root hairs. Interestingly, the transfer of the R. meliloti nodFE, nodG, and nodH genes to R. trifolii prevented R. trifolii from infecting and nodulating its normal host, white clover (Trifolium repens). Experiments with the mutated R. meliloti nodH, nodF, nodE, and nodG genes demonstrated that nodH, nodF, nodE, and possibly nodG have an additive effect in blocking infection and nodulation of clover.
Inhibition of cell-cell interactions in Myxococcus xanthus by congo red
Authors: Arnold J W , Shimkets L J
Published: November 2016

The function of molecules associated with the cell surface may be determined by examining the phenotype of cells treated with inhibitors specific to these cell surface molecules. This strategy was used to examine the function of the major Congo red receptor of the myxobacterium Myxococcus xanthus, which has a developmental cycle that involves social interactions among cells. A class of social motility mutations (A+ S-), known as dsp, may inhibit the same subcellular component as Congo red because the phenotype of wild-type cells which had been treated with Congo red resembled in several ways the phenotype of the Dsp mutants. First, Congo red inhibited agglutination of wild-type cells, whereas Dsp cells were incapable of agglutinating, even in the absence of Congo red. Second, Congo red inhibited fruiting body formation by wild-type cells and reduced the yield of myxospores. Untreated Dsp cells were unable to form fruiting bodies and produced few myxospores. Third, Congo red reduced the rate of wild-type gliding motility to a level comparable to that of untreated Dsp cells, but did not inhibit the A motility of Dsp cells. Finally, binding studies showed that Dsp cells lacked the major Congo red receptor. Wild-type cells bound Congo red with an apparent association constant of 2.4 X 10(5) M-1, while Dsp cells bound it with an apparent association constant of 8.5 X 10(3) M-1. Binding of Congo red to wild-type cells was saturated in less than 10 min and was reversible when excess Congo red was removed. These results suggest that the Congo red receptors are controlled by the S motility system and that these receptors are involved in cell cohesion, social motility, and fruiting body formation.
Characteristics of an RNA polymerase population isolated from Bacillus subtilis late in sporulation
Authors: Cummings C W , Haldenwang W G
Published: November 2016

The sigma-factor composition of Bacillus subtilis RNA polymerase alters during endospore formation. The best-documented change is the appearance of a major sporulation-specific sigma factor (sigma epsilon), which is an RNA polymerase subunit readily detected at 2 to 4 h into the 8-h sporulation process. To determine the nature of the RNA polymerase in differentiating cells after the period of sigma epsilon abundance, we isolated RNA polymerase from cells that were harvested at 6 h after the onset of sporulation. Highly purified fractions of RNA polymerase from these cells contained at least six proteins which cosedimented with core RNA polymerase (beta beta' alpha 2) during glycerol gradient centrifugation. Most of these proteins were in the size range of 20,000 to 29,000 daltons, although one 90,000-dalton protein was also evident. None of the putative RNA polymerase subunits were present in quantities similar to that observed for sigma epsilon during its period of prominence in the cell but instead resembled the minor vegetative-cell sigma factors in abundance. In vitro transcriptions using cloned B. subtilis DNAs as templates revealed at least two novel transcriptional activities in the enzyme that was isolated from cells at 6 h after the onset of sporulation but absent in an RNA polymerase preparation extracted from cells at 4 h after the onset of sporulation. One of these activities was reconstituted by the addition of a 25,000 to 27,000-dalton protein fraction to core RNA polymerase.
Molecular cloning and high-level expression of a bromoperoxidase gene from Streptomyces aureofaciens Tü24
Authors: van Pée K H
Published: November 2016

A bromoperoxidase gene was cloned from Streptomyces aureofaciens Tü24 into Streptomyces lividans TK64 by using the promoter-probe vector pIJ486. Subcloning of DNA from the original, unstable clone allowed the gene to be localized to a 1.7-kilobase (kb) fragment of DNA. Southern blotting showed that the cloned 1.7-kb insert hybridized to a 4.3-kb fragment in an SstI digest of S. aureofaciens Tü24 total DNA. The 1.7-kb insert was shown to code for a protein with the electrophoretic properties of the subunits of the nonheme bromoperoxidase isolated from S. aureofaciens Tü24. The protein produced by S. lividans TK64 transformed with pHM621, which contained an 8.0-kb insert, was shown to be identical to the S. aureofaciens Tü24 bromoperoxidase in terms of its electrophoretic mobility on denaturing and nondenaturing polyacrylamide gels and its NH2-terminal amino acid sequence. The bromoperoxidase was overproduced (up to 180 times) by S. lividans TK64 containing pHM621. Based on the heat stability of the S. aureofaciens Tü24 bromoperoxidase, a new and simple purification procedure with very high yields was developed.