bluuebunny/crossref_metadata_2025 huggingface dataset:
GvpE- and GvpD-mediated transcription regulation of the p-gvp genes encoding gas vesicles in Halobacterium salinarum
Authors: Hofacker Annette , Schmitz Kerstin-Maike , Cichonczyk Alexander , Sartorius-Neef Simone , Pfeifer Felicitas
Published: June 2004

The transcription of the 14 p-gvp genes involved in gas vesicle formation of Halobacterium salinarum PHH1 is driven by the four promoters pA, pD, pF and pO. The regulation of these promoters was investigated in Haloferax volcanii transformants with respect to the endogenous regulatory proteins GvpE and GvpD. Northern analyses demonstrated that the transcription derived from the pA and pD promoters was enhanced by GvpE, whereas the activities of the pF and pO promoters were not affected. Similar results were obtained using promoter fusions with the bgaH reporter gene encoding an enzyme with β-galactosidase activity. The largest amount of specific β-galactosidase activity was determined for pA-bgaH transformants, followed by pF-bgaH and pD-bgaH transformants. The presence of GvpE resulted in a severalfold induction of the pA and pD promoter, whereas the pF promoter was not affected. A lower GvpE-induced pA promoter activity was seen in the presence of GvpD in the pA-bgaH/DEex transformants, suggesting a function of GvpD in repression. To determine the DNA sequences involved in the GvpE-mediated activation, a 50-nucleotide region of the pA promoter was investigated by 4-nucleotide scanning mutagenesis. Some of these mutations affected the basal transcription, especially mutations in the region of the TATA box and the putative BRE sequence element, and also around position −10. Mutant E, harbouring a sequence with greater identity to the consensus BRE element, showed a significantly enhanced basal promoter activity compared to wild-type. Mutations not affecting basal transcription, but yielding a reduced GvpE-mediated activation, were located immediately upstream of BRE. These results suggested that the transcription activation by GvpE is in close contact with the core transcription machinery.
Inference for the Proportional Hazards Model with Misclassified Discrete‐Valued Covariates
Authors: Zucker David M. , Spiegelman Donna
Published: June 2004

Summary. We consider the Cox proportional hazards model with discrete‐valued covariates subject to misclassification. We present a simple estimator of the regression parameter vector for this model. The estimator is based on a weighted least squares analysis of weighted‐averaged transformed Kaplan–Meier curves for the different possible configurations of the observed covariate vector. Optimal weighting of the transformed Kaplan–Meier curves is described. The method is designed for the case in which the misclassification rates are known or are estimated from an external validation study. A hybrid estimator for situations with an internal validation study is also described. When there is no misclassification, the regression coefficient vector is small in magnitude, and the censoring distribution does not depend on the covariates, our estimator has the same asymptotic covariance matrix as the Cox partial likelihood estimator. We present results of a finite‐sample simulation study under Weibull survival in the setting of a single binary covariate with known misclassification rates. In this simulation study, our estimator performed as well as or, in a few cases, better than the full Weibull maximum likelihood estimator. We illustrate the method on data from a study of the relationship between trans‐unsaturated dietary fat consumption and cardiovascular disease incidence.
Analyzing Time‐to‐Event Data in a Clinical Trial When an Unknown Proportion of Subjects Has Experienced the Event at Entry
Authors: Balasubramanian Raji , Lagakos Stephen W.
Published: June 2004

Summary.  In some clinical trials, where the outcome is the time until development of a silent event, an unknown proportion of subjects who have already experienced the event will be unknowingly enrolled due to the imperfect nature of the diagnostic tests used to screen potential subjects. For example, commonly used diagnostic tests for evaluating HIV infection status in infants, such as DNA PCR and HIV Culture, have low sensitivity when given soon after infection. This can lead to the inclusion of an unknown proportion of HIV‐infected infants into clinical trials aimed at the prevention of transmission from HIV‐positive mothers to their infants through breastfeeding. The infection status of infants at the end of the trial, when they are more than a year of age, can be determined with certainty. For those infants found to be infected with HIV at the end of the trial, it cannot be determined whether this occurred during the study or whether they were already infected when they were enrolled. In these settings, estimates of the cumulative risk of the event by the end of the study will overestimate the true probability of event during the study period and hypothesis tests comparing two or more intervention strategies can also be biased. We present inference methods for the distribution of time until the event of interest in these settings, and investigate issues in the design of such trials when there is a choice of using both imperfect and perfect diagnostic tests.
Measuring the Relative Information in Allele‐Sharing Linkage Studies
Authors: Nicolae Dan L. , Kong Augustine
Published: June 2004

Summary. In the context of allele‐sharing methods, this article investigates ways of measuring the information in the marker datarelativeto the amount of information that would have been available if the identity‐by‐descent (IBD) process were known. Such measures are needed to decide whether new markers can substantially modify the evidence for excess sharing. We propose new measures that take advantage of the properties of the exponential model introduced byKong and Cox(1997,American Journal of Human Genetics61,1179–1188). These measures are related to Fisher Information and hence are also efficiency measures. Large‐sample and small‐sample properties of the new and previously proposed measures of information are examined.
Modeling Microarray Data Using a Threshold Mixture Model
Authors: Kauermann Göran , Eilers Paul
Published: June 2004

Summary. An important goal of microarray studies is the detection of genes that show significant changes in expression when two classes of biological samples are being compared. We present an ANOVA‐style mixed model with parameters for array normalization, overall level of gene expression, and change of expression between the classes. For the latter we assume a mixing distribution with a probability mass concentrated at zero, representing genes with no changes, and a normal distribution representing the level of change for the other genes. We estimate the parameters by optimizing the marginal likelihood. To make this practical, Laplace approximations and a backfitting algorithm are used. The performance of the model is studied by simulation and by application to publicly available data sets.
Multiple Testing to Establish Superiority/Equivalence of a New Treatment Compared with k Standard Treatments for Unbalanced Designs
Authors: Kwong Koon Shing , Cheung Siu Hung , Chan Wai Sum
Published: June 2004

Summary.  In clinical studies, multiple superiority/equivalence testing procedures can be applied to classify a new treatment as superior, equivalent (same therapeutic effect), or inferior to each set of standard treatments. Previous stepwise approaches (Dunnett and Tamhane, 1997, Statistics in Medicine16, 2489–2506; Kwong, 2001, Journal of Statistical Planning and Inference97, 359–366) are only appropriate for balanced designs. Unfortunately, the construction of similar tests for unbalanced designs is far more complex, with two major difficulties: (i) the ordering of test statistics for superiority may not be the same as the ordering of test statistics for equivalence; and (ii) the correlation structure of the test statistics is not equi‐correlated but product‐correlated. In this article, we seek to develop a two‐stage testing procedure for unbalanced designs, which are very popular in clinical experiments. This procedure is a combination of step‐up and single‐step testing procedures, while the familywise error rate is proved to be controlled at a designated level. Furthermore, a simulation study is conducted to compare the average powers of the proposed procedure to those of the single‐step procedure. In addition, a clinical example is provided to illustrate the application of the new procedure.
Cutpoint Selection for Categorizing a Continuous Predictor
Authors: O'Brien Sean M.
Published: June 2004

Summary.  This article presents a new approach for choosing the number of categories and the location of category cutpoints when a continuous exposure variable needs to be categorized to obtain tabular summaries of the exposure effect. The optimum categorization is defined as the partition that minimizes a measure of distance between the true expected value of the outcome for each subject and the estimated average outcome among subjects in the same exposure category. To estimate the optimum partition, an efficient nonparametric estimate of the unknown regression function is substituted into a formula for the asymptotically optimum categorization. This new approach is easy to implement and it outperforms existing cutpoint selection methods.
Small‐Sample Inference for the Comparison of Means of Log‐Normal Distributions
Authors: Gill Paramjit S.
Published: June 2004

Summary.  We propose a likelihood‐based test for comparing the means of two or more log‐normal distributions, with possibly unequal variances. A modification to the likelihood ratio test is needed when sample sizes are small. The performance of the proposed procedures is compared with the F‐ratio test using Monte Carlo simulations.
The Analysis of Placement Values for Evaluating Discriminatory Measures
Authors: Sullivan Pepe Margaret , Cai Tianxi
Published: June 2004

Summary. The idea of using measurements such as biomarkers, clinical data, or molecular biology assays for classification and prediction is popular in modern medicine. The scientific evaluation of such measures includes assessing the accuracy with which they predict the outcome of interest. Receiver operating characteristic curves are commonly used for evaluating the accuracy of diagnostic tests. They can be applied more broadly, indeed to any problem involving classification to two states or populations (D= 0 or 1). We show that the ROC curve can be interpreted as a cumulative distribution function for the discriminatory measureYin the affected population(D= 1)afterYhas been standardized to the distribution in the reference population(D= 0). The standardized values are called placement values. If the placement values have a uniform(0, 1) distribution, thenYis not discriminatory, because its distribution in the affected population is the same as that in the reference population. The degree to which the distribution of the standardized measure differs from uniform(0, 1) is a natural way to characterize the discriminatory capacity ofYand provides a nontraditional interpretation for the ROC curve. Statistical methods for making inference about distribution functions therefore motivate new approaches to making inference about ROC curves. We demonstrate this by considering the ROC‐GLM regression model and observing that it is equivalent to a regression model for the distribution of placement values. The likelihood of the placement values provides a new approach to ROC parameter estimation that appears to be more efficient than previously proposed methods. The method is applied to evaluate a pulmonary function measure in cystic fibrosis patients as a predictor of future occurrence of severe acute pulmonary infection requiring hospitalization. Finally, we note the relationship between regression models for the mean placement value and recently proposed models for the area under the ROC curve which is the classic summary index of discrimination.
Post‐transplant lymphoproliferative disorder limited to the skin
Authors: Samolitis Nancy J. , Bharadwaj Jayaram S. , Weis John R. , Harris Ronald M.
Published: June 2004

Background:  Post‐transplant lymphoproliferative disorder (PTLD) is a condition affecting immunosuppressed transplant patients and has a variety of clinical presentations. It is rarely found in the skin, and cases of PTLD in the skin are usually linked with lymph node or other organ involvement.

Methods:  We report a case of plasmacytoid PTLD that is limited to the skin. A 63‐year‐old man with a history of cardiac transplant presented with exophytic tumors involving the lower extremity. The diagnosis and classification of the various forms of PTLD are discussed.

Results:  Histology, immunohistochemical stains, and in situ hybridization revealed an aggressive plasmacytoid tumor that was Epstein–Barr virus positive. The patient's tumors resolved with decreased immunosuppression and localized radiation.

Conclusion:  This case is unusual for several reasons including involvement limited to the skin, presentation 15 years following transplant, and plasmacytoid phenotype of the tumor. This disorder will likely be seen by dermatologists and dermatopathologists with the increasing use of immunosuppressive medications in the dermatologist's patient population.
CTLA‐4 gene polymorphism and its association with Graves’ disease in the Lebanese population
Authors: Nakkash‐Chmaisse H. , Makki R. F. , Abdelhamid E. , Fakhoury H. , Salti N. N. , Salti I.
Published: June 2004

Graves’ disease is an organ‐specific autoimmune disease that has a female predominance. It is probably the result of a complex interaction of genetic and environmental factors. This disease is characterized by immune system activation, evidenced by elevated serum thyroid‐specific autoantibodies and lymphocytic infiltration of the target organ (the thyroid gland), associated with raised levels of circulating activated T lymphocytes. Several reports have demonstrated genetic linkage and association between the genetic markers of the CTLA‐4 gene on chromosome 2q33 and Graves’ disease. In order to confirm this association in the Lebanese population, a bi‐allelic A/G polymorphism at position 49 of CTLA‐4 exon 1 was studied in 34 patients with Graves’ disease, and in 38 healthy individuals, using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis. The results showed a significant increase in allele and genotype frequencies in patients with Graves’ disease compared to controls. This suggests that the CTLA‐4 gene might play a role in the development of Graves’ disease in the Lebanese population.
Biogeographical patterns of genetic differentiation in dung beetles of the genusTrypocopris(Coleoptera, Geotrupidae) inferred from mtDNA and AFLP analyses
Authors: Carisio Loredana , Cervella Piero , Palestrini Claudia , DelPero Massimiliano , Rolando Antonio
Published: June 2004

Aim To examine the phylogeography and population structure of three dung beetle species of the genusTrypocopris(Coleoptera, Geotrupidae). We wanted to test whether genetic differences and genealogies among populations were in accordance with morphologically described subspecies and we aimed to establish times of divergence among subspecies to depict the appropriate temporal framework of their phylogeographical differentiation. We also wished to investigate the historical demographic events and the relative influences of gene flow and drift on the distribution of genetic variability of the different populations.

Location Europe (mostly Italy).

Methods We collected adult males from dung pats from 15 Italian localities over the period 2000–2002. For sequence analysis, some dried specimens from Albania, Croatia, Slovakia and Spain were also used. We applied cytochrome oxidase I mitochondrial DNA sequencing and the amplified fragment length polymorphism (AFLP) technique to determine whether phylogeographical patterns within the three species support the proposed hypotheses of subspecies designations, and to detect further structure among populations that might mediate diversification.

Results and main conclusions The results show a high concordance between the distribution of mtDNA variation and the main morphological groups recognized as subspecies, which thus may represent independent evolutionary units. The degree of mitochondrial divergence suggests that speciation events occurred during the Pliocene, while diversification of the main subspecific lineages took place in the Pleistocene, fromc.0.3 to 1.5 Ma. Mitochondrial and nuclear data also reveal that there is phylogeographical structuring among populations within each of the main groups and that both contemporary and historical processes determined this pattern of genetic structure. Geographical populations form monophyletic clades in both phylogenetic and network reconstructions. Despite the high levels of intrapopulational diversity,FSTvalues indicate moderate but significant genetic differentiation among populations, and a Bayesian clustering analysis of the AFLP data clearly separates the geographical populations. Nucleotide and gene diversity estimates reveal interspecific differences in the degree of diversification among populations that may be related to the different ecological requirements of the three species.
Distraction produces an increase in pain‐evoked anterior cingulate activity
Authors: Dowman Robert
Published: June 2004

This study examined the effects of distraction on pain‐evoked activity in the anterior cingulate cortex (ACC). Twenty‐eight healthy adults were given painful electrical stimulation of the sural nerve during an attend condition, where they rated the subjective magnitude of each electrical stimulus, and during a distraction condition, where they performed an arithmetic distraction task. The magnitude of the pain‐evoked ACC activity was estimated from the dipole source localization analysis of the somatosensory evoked potential. Subjective pain ratings were smaller and pain‐evoked ACC activity was larger during the distraction condition than during the attend condition. Recent regional cerebral blood flow studies have also reported a distraction‐related increase in pain‐evoked ACC activity. Our results confirm these reports, and verify that the distraction effect specifically involves pain‐evoked ACC activity. The cognitive demands of the distraction task present the possibility that the pain‐evoked ACC activity might be involved, at least in part, in response competition and/or orienting attention toward painful stimuli.
Cycle-Resolved NO Measurements in a Two-Stroke Large-Bore Natural Gas Engine
Authors: Puzinauskas Paulius V. , Olsen Daniel B. , Willson Bryan D.
Published: June 2004

Cycle-resolved NO data were acquired from a Cooper-Bessemer GMV 4TF two-stroke engine to better understand and quantify large bore natural gas engine NOx emission. The cycle resolved NO data were extracted separately from the engine’s cylinder two and four exhaust ports and taken simultaneously with cycle resolved pressure traces, conventional steady-state emission measurements and a variety of additional performance and diagnostic data. The test variables were intake manifold boost pressure, ignition method and ignition timing. Relationships between individual cycle pressure traces and the NO produced by that cycle were investigated. Furthermore, mass-averaged NO values were calculated and integrated in order to compare with average exhaust emissions from a steady-state analyzer and combustion pressure characteristics. The steady measurements revealed that NO and NO2 emissions respond differently to the test variables. The mass averaged cycle-resolved NO values compare well with the steady exhaust emission measurements and exhibit strong correlations with peak pressure and crank angle location of peak pressure.
A Model for Droplet Vaporization for Use in Gasoline and HCCI Engine Applications
Authors: Ra Youngchul , Feitz Rolf D.
Published: June 2004

A model for unsteady droplet vaporization is presented that considers the droplet temperature range from flash-boiling conditions to normal evaporation. The theory of continuous thermodynamics was used to model the properties and compositions of multicomponent fuels such as gasoline. In order to model the change of evaporation rate from normal to boiling conditions more realistically, an unsteady internal heat flux model and a new model for the determination of the droplet surface temperature is proposed. An explicit form of the equation to determine the heat flux from the surrounding gas mixture to the droplet-gas interface was obtained from an approximate solution of the quasi-steady energy equation for the surrounding gas mixture, with the inter-diffusion of fuel vapor and the surrounding gas taken into account. The model was applied to calculate evaporation processes of droplets for various ambient temperatures and droplet temperatures.
A Mathematical Model for Schlieren and Shadowgraph Images of Transient Expanding Spherical Thin Flames
Authors: Parsinejad Farzan , Matlo Matyas , Metghalchi Mohamad
Published: June 2004

Optical behavior of spherical flames is investigated using both Schlieren and shadowgraph methods. A mathematical model has been developed to predict the intensity of refracted light beams interacting with a transient expanding thin flame. Experimental facilities have been built to visualize transient expanding spherical flames. The facilities include a cylindrical chamber with two end glasses for optical observation. Shadowgraph and Schlieren pictures of flame propagation have been taken using a high-speed charged coupled device camera. Experimental results are in very good agreement with those predicted by the theoretical model. Schlieren and shadowgraph techniques have also been used to view smooth, cracked and cellular flames; these techniques will be useful in future in studies to determine the stability of propagating flame.
Combustion Structures in Lifted Ethanol Spray Flames
Authors: Marley Stephen K. , Welle Eric J. , Lyons Kevin M.
Published: June 2004

The development of a double flame structure in lifted ethanol spray flames is visualized using OH planar laser-induced fluorescence (PLIF). While the OH images indicate a single reaction zone exists without co-flow, the addition of low-speed co-flow facilitates the formation of a double flame structure that consists of two diverging flame fronts originating at the leading edge of the reaction zone. The outer reaction zone burns steadily in a diffusion mode, and the strained inner flame structure is characterized by both diffusion and partially premixed combustion exhibiting local extinction and re-ignition events.
Pre-integrated Nonequilibrium Combustion-Response Mapping for Gas Turbine Emissions
Authors: Korakianitis T. , Dyer R. , Subramanian N.
Published: June 2004

In gas turbine combustion the gas dynamic and chemical energy release mechanisms have comparable time scales, so that equilibrium chemistry is inadequate for predicting species formation (emissions). In current practice either equilibrium chemical reactions are coupled with experimentally derived empirical equations, or time-consuming computations are used. Coupling nonequilibrium chemistry, fluid dynamic, and initial and boundary condition equations results in large sets of numerically stiff equations; and their time integration demands enormous computational resources. The response modeling approach has been used successfully for large reaction sets. This paper makes two new contributions. First it shows how pre-integration of the heat release maps eliminates the stiffness of the equations. This is a new modification to the response mapping approach, and it performs satisfactorily for non-diffusion systems. Second the theoretical framework is further extended to predict species formation in cases with diffusion, which is applicable to gas turbine combustion systems and others. The methodology to implement this approach to reacting systems, and to gas turbine combustion, is presented. The benefits over other reaction-mapping techniques are discussed.
Neural Network Emulation of a Magnetically Suspended Rotor
Authors: Escalante A. , Guzma´n V. , Parada M. , Medina L. , Diaz S. E.
Published: June 2004

The use of magnetic bearings in high speed/low friction applications is increasing in industry. Magnetic bearings are sophisticated electromechanical systems, and modeling magnetic bearings using standard techniques is complex and time consuming. In this work a neural network is designed and trained to emulate the operation of a complete system (magnetic bearing, PID controller, and power amplifiers). The neural network is simulated and integrated into a virtual instrument that will be used in the laboratory both as a teaching and a research tool. The main aims in this work are: (1) determining the minimum amount of artificial neurons required in the neural network to emulate the magnetic bearing system, (2) determining the more appropriate ANN training method for this application, and (3) determining the errors produced when a neural network trained to emulate system operation with a balanced rotor is used to predict system response when operating with an unbalanced rotor. The neural network is trained using as input the position data from the proximity sensors; neural network outputs are the control signals to the coil amplifiers.
Dynamic Response of a Hydrodynamic Thrust Bearing-Mounted Rotor
Authors: Shiau T. N. , Hsu W. C. , Chang J. R.
Published: June 2004

For investigating the dynamic response of a hydrodynamic thrust bearing-mounted flexible rotor, the dynamic characteristic data of thrust bearings for high surface velocities are applied for constructing the equation of motion for the rotor system, which is modeled with the finite element method. Based on the short bearing approximation and the π film cavitation model of the nonlinear oil-film forces, the dynamic responses are investigated using direct numerical integration with a variable order solver based on the numerical differentiation formulas. Harmonic, quasi-periodic, and chaotic motions are predicted for a range of spin speeds of the rotor. Poincare´ maps of predicted rotor trajectories are also examined. It shows that spin speeds of the rotor and the oil film force coefficient might promote undesirable nonsynchronous vibrations.
The Identification of the Unbalance of a Flexible Rotating Machine From a Single Rundown
Authors: Lees A. W. , Sinha J. K. , Friswell M. I.
Published: June 2004

The reliable estimation of a flexible foundation model and the state of unbalance (both amplitude and phase) of a turbogenerator from machine rundown measured vibration data is an active research area. Earlier studies on the estimation of both these quantities used the whole frequency range of the rundown as a single band. However, such an identification may be inaccurate for large flexible foundations having many modes in the rundown frequency range. For reliable identification, the whole frequency range has to be divided into a number of frequency bands and the frequency-dependent foundation models have to be estimated together with the unbalance. This paper combines the unbalance estimation with the split frequency range for the foundation model, and the highlights the limitations observed during estimation of foundation models and the state of unbalance. It is shown that the accuracy of the method may be enhanced by judicious choice of a weighting function. Having established the method in simulation, experimental data from a 3-m long test rig, with four journal bearings, is used to test the method. The approach seems to give reliable estimates of the machine unbalance.
Effect of Inlet Air Cooling by Ice Storage on Unit Sizing of a Gas Turbine Cogeneration Plant
Authors: Yokoyama Ryohei , Ito Koichi
Published: June 2004

In the commercial sector, heat and power demands peak in the summer daytime because of high space cooling demands, and cogeneration plants are required to produce maximum heat and power to meet their demands. However, gas turbine cogeneration plants have the disadvantage of decreases in maximum power output in the summer daytime, which reduces the availability of gas turbines. One of the ways to avoid the aforementioned disadvantage is to cool inlet air and augment maximum power output. In addition, one of the ways for inlet air cooling is to make ice by driving electric compression refrigerators using off-peak power generated during the nighttime, store it in ice banks, and use its heat for inlet air cooling during the on-peak period. The objective of this paper is to investigate the effect of inlet air cooling by ice storage on the unit sizing and cost of a gas turbine cogeneration plant. An optimal unit sizing method based on the mixed-integer linear programming is used to rationally determine equipment capacities and operational strategies of the plant. A numerical study is conducted, in which the gas turbine cogeneration plants with and without inlet air cooled by ice storage are compared with each other, and the effect of inlet air cooling on the equipment capacities as well as the annual total cost and its items is clarified.
Millimeter-Scale, Micro-Electro-Mechanical Systems Gas Turbine Engines
Authors: Epstein Alan H.
Published: June 2004

The confluence of market demand for greatly improved compact power sources for portable electronics with the rapidly expanding capability of micromachining technology has made feasible the development of gas turbines in the millimeter-size range. With airfoil spans measured in 100’s of microns rather than meters, these “microengines” have about 1 millionth the air flow of large gas turbines and thus should produce about one millionth the power, 10–100 W. Based on semiconductor industry-derived processing of materials such as silicon and silicon carbide to submicron accuracy, such devices are known as micro-electro-mechanical systems (MEMS). Current millimeter-scale designs use centrifugal turbomachinery with pressure ratios in the range of 2:1 to 4:1 and turbine inlet temperatures of 1200–1600 K. The projected performance of these engines are on a par with gas turbines of the 1940s. The thermodynamics of MEMS gas turbines are the same as those for large engines but the mechanics differ due to scaling considerations and manufacturing constraints. The principal challenge is to arrive at a design which meets the thermodynamic and component functional requirements while staying within the realm of realizable micromachining technology. This paper reviews the state of the art of millimeter-size gas turbine engines, including system design and integration, manufacturing, materials, component design, accessories, applications, and economics. It discusses the underlying technical issues, reviews current design approaches, and discusses future development and applications.
Characterization and Heterologous Gene Expression of a Novel Esterase fromLactobacillus caseiCL96
Authors: Choi Young J. , Miguez Carlos B. , Lee Byong H.
Published: June 2004

A novel esterase gene (estI) ofLactobacillus caseiCL96 was localized on a 3.3-kb BamHI DNA fragment containing an open reading frame (ORF) of 1,800 bp. The ORF ofestIwas isolated by PCR and expressed inEscherichia coli, the methylotrophic bacteriumMethylobacterium extorquens, and the methylotrophic yeastPichia pastorisunder the control of T7, methanol dehydrogenase (PmxaF), and alcohol oxidase (AOX1) promoters, respectively. The amino acid sequence of EstI indicated that the esterase is a novel member of the GHSMG family of lipolytic enzymes and that the enzyme contains a lipase-like catalytic triad, consisting of Ser325, Asp516, and His558.E. coliBL21(DE3)/pLysS containingestIexpressed a novel 67.5-kDa protein corresponding to EstI in an N-terminal fusion with the S · tag peptide. The recombinantL. caseiCL96 EstI protein was purified to electrophoretic homogeneity in a one-step affinity chromatography procedure on S-protein agarose. The optimum pH and temperature of the purified enzyme were 7.0 and 37°C, respectively. Among the pNP (p-nitrophenyl) esters tested, the most selective substrate was pNP-caprylate (C8), withKmandkcatvalues of 14 ± 1.08 μM and 1,245 ± 42.3 S−1, respectively.
Biofilm Development and Cell Death in the Marine Bacterium
Pseudoalteromonas tunicata
Authors: Mai-Prochnow Anne , Evans Flavia , Dalisay-Saludes Doralyn , Stelzer Sacha , Egan Suhelen , James Sally , Webb Jeremy S. , Kjelleberg Staffan
Published: June 2004

The newly described green-pigmented bacterium
Pseudoalteromonas tunicata
(D2) produces target-specific inhibitory compounds against bacteria, algae, fungi, and invertebrate larvae and is frequently found in association with living surfaces in the marine environment. As part of our studies on the ecology of
P. tunicata
and its interaction with marine surfaces, we examined the ability of
P. tunicata
to form biofilms under continuous culture conditions within the laboratory.
P. tunicata
biofilms exhibited a characteristic architecture consisting of differentiated microcolonies surrounded by water channels. Remarkably, we observed a repeatable pattern of cell death during biofilm development of
P. tunicata
, similar to that recently reported for biofilms of
Pseudomonas aeruginosa
(J. S. Webb et al., J. Bacteriol.
185:
4585-4595, 2003). Killing and lysis occurred inside microcolonies, apparently resulting in the formation of voids within these structures. A subpopulation of viable cells was always observed within the regions of killing in the biofilm. Moreover, extensive killing in mature biofilms appeared to result in detachment of the biofilm from the substratum. A novel 190-kDa autotoxic protein produced by
P. tunicata
, designated AlpP, was found to be involved in this biofilm killing and detachment. A Δ
alpP
mutant derivative of
P. tunicata
was generated, and this mutant did not show cell death during biofilm development. We propose that AlpP-mediated cell death plays an important role in the multicellular biofilm development of
P. tunicata
and subsequent dispersal of surviving cells within the marine environment.
Engineering the
nifH
Promoter Region and Abolishing Poly-β-Hydroxybutyrate Accumulation in
Rhizobium etli
Enhance Nitrogen Fixation in Symbiosis with
Phaseolus vulgaris
Authors: Peralta Humberto , Mora Yolanda , Salazar Emmanuel , Encarnación Sergio , Palacios Rafael , Mora Jaime
Published: June 2004

Rhizobium etli
, as well as some other rhizobia, presents nitrogenase reductase (
nifH
) gene reiterations. Several
R. etli
strains studied in this laboratory showed a unique organization and contained two complete
nifHDK
operons (copies a and b) and a truncated
nifHD
operon (copy c). Expression analysis of
lacZ
fusion demonstrated that copies a and b in strain CFN42 are transcribed at lower levels than copy c, although this copy has no discernible role during nitrogen fixation. To increase nitrogenase production, we constructed a chimeric
nifHDK
operon regulated by the strong
nifHc
promoter sequence and expressed it in symbiosis with the common bean plant (
Phaseolus vulgaris
), either cloned on a stably inherited plasmid or incorporated into the symbiotic plasmid (pSym). Compared with the wild-type strain, strains with the nitrogenase overexpression construction assayed in greenhouse experiments had, increased nitrogenase activity (58% on average), increased plant weight (32% on average), increased nitrogen content in plants (15% at 32 days postinoculation), and most importantly, higher seed yield (36% on average), higher nitrogen content (25%), and higher nitrogen yield (72% on average) in seeds. Additionally, expression of the chimeric
nifHDK
operon in a poly-β-hydroxybutyrate-negative
R. etli
strain produced an additive effect in enhancing symbiosis. To our knowledge, this is the first report of increased seed yield and nutritional content in the common bean obtained by using only the genetic material already present in
Rhizobium
.
Novel Expression System for Large-Scale Production and Purification of Recombinant Class IIa Bacteriocins and Its Application to Piscicolin 126
Authors: Gibbs Gerard M. , Davidson Barrie E. , Hillier Alan J.
Published: June 2004

Piscicolin 126 is a class IIa bacteriocin isolated from
Carnobacterium piscicola
JG126 that exhibits strong activity against
Listeria monocytogenes
. The gene encoding mature piscicolin 126 (m-
pisA
) was cloned into an
Escherichia coli
expression system and expressed as a thioredoxin-piscicolin 126 fusion protein that was purified by affinity chromatography. Purified recombinant piscicolin 126 was obtained after CNBr cleavage of the fusion protein followed by reversed-phase chromatography. Recombinant piscicolin 126 contained a single disulfide bond and had a mass identical to that of native piscicolin 126. This novel bacteriocin expression system generated approximately 26 mg of purified bacteriocin from 1 liter of
E. coli
culture. The purified recombinant piscicolin 126 acted by disruption of the bacterial cell membrane.
Morphological, Chemical, and Genetic Diversity of Tropical Marine Cyanobacteria
Lyngbya
spp. and
Symploca
spp. (
Oscillatoriales
)
Authors: Thacker Robert W. , Paul Valerie J.
Published: June 2004

Although diverse natural products have been isolated from the benthic, filamentous cyanobacterium
Lyngbya majuscula
, it is unclear whether this chemical variation can be used to establish taxonomic relationships among disparate collections. We compared morphological characteristics, secondary-metabolite compositions, and partial 16S ribosomal DNA (rDNA) sequences among several collections of
L. majuscula
Gomont,
Lyngbya
spp., and
Symploca
spp. from Guam and the Republic of Palau. The morphological characteristics examined were cell length, cell width, and the presence or absence of a calyptra. Secondary metabolites were analyzed by two-dimensional thin-layer chromatography. Each collection possessed a distinct cellular morphology that readily distinguished
Lyngbya
spp. from
Symploca
spp. Each collection yielded a unique chemotype, but common chemical characteristics were shared among four collections of
L. majuscula
. A phylogeny based on secondary-metabolite composition supported the reciprocal monophyly of
Lyngbya
and
Symploca
but yielded a basal polytomy for
Lyngbya
. Pairwise sequence divergence among species ranged from 10 to 14% across 605 bp of 16S rDNA, while collections of
L. majuscula
showed 0 to 1.3% divergence. Although the phylogeny of 16S rDNA sequences strongly supported the reciprocal monophyly of
Lyngbya
and
Symploca
as well as the monophyly of
Lyngbya bouillonii
and
L. majuscula
, genetic divergence was not correlated with chemical and morphological differences. These data suggest that 16S rDNA sequence analyses do not predict chemical variability among
Lyngbya
species. Other mechanisms, including higher rates of evolution for biosynthetic genes, horizontal gene transfer, and interactions between different genotypes and environmental conditions, may play important roles in generating qualitative and quantitative chemical variation within and among
Lyngbya
species.
Evidence for the Presence of
Simkania negevensis
in Drinking Water and in Reclaimed Wastewater in Israel
Authors: Kahane Simona , Platzner Nizhia , Dvoskin Bella , Itzhaki Amir , Friedman Maureen G.
Published: June 2004

Simkania negevensis
is a recently discovered chlamydia-like intracellular microorganism which has been associated with bronchiolitis in infants and with community-acquired pneumonia in adults; a high seroprevalence of antibodies to the microorganism has been found in various population groups.
S. negevensis
can be grown in various cell lines as well as in free-living amoebae such as
Acanthamoeba polyphaga
. In this study, evidence for the existence of
Simkania
or
Simkania
-like microorganisms in drinking water and in reclaimed wastewater is presented for the first time. Detection of the microorganism was made possible by the development of a specific and sensitive filter membrane immunoassay and was confirmed by PCR detection of microbial DNA in the water samples. The common presence of
S. negevensis
in water sources together with the high seroprevalence of antibodies to it and early age of acquisition of infection may implicate water as a source of infection. The possible significance of this finding for public health and for municipal water testing and treatment needs to be further examined.
Evaluation of a Cocktail of Three Bacteriophages for Biocontrol of
Escherichia coli
O157:H7
Authors: O'Flynn G. , Ross R. P. , Fitzgerald G. F. , Coffey A.
Published: June 2004

Escherichia coli
O157:H7 is an endemic pathogen causing a variety of human diseases including mild diarrhea, hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura. This study concerns the exploitation of bacteriophages as biocontrol agents to eliminate the pathogen
E. coli
O157:H7. Two distinct lytic phages (e11/2 and e4/1c) isolated against a human strain of
E. coli
O157:H7, a previously isolated lytic phage (pp01), and a cocktail of all three phages were evaluated for their ability to lyse the bacterium in vivo and in vitro. Phage e11/2, pp01, and the cocktail of all three virulent phages resulted in a 5-log-unit reduction of pathogen numbers in 1 h at 37°C. However, bacteriophage-insensitive mutants (BIMs) emerged following the challenge. All tested BIMs had a growth rate which approximated that of the parental O157 strain, although many of these BIMs had a smaller, more coccoid cellular morphology. The frequency of BIM formation (10
−6
CFU) was similar for e11/2, pp01, and the phage cocktail, while BIMs insensitive to e4/1c occurred at the higher frequency (10
−4
CFU). In addition, BIMs commonly reverted to phage sensitivity within 50 generations. In an initial meat trial experiment, the phage cocktail completely eliminated
E. coli
O157:H7 from the beef meat surface in seven of nine cases. Given that the frequency of BIM formation is low (10
−6
CFU) for two of the phages, allied to the propensity of these mutants to revert to phage sensitivity, we expect that BIM formation should not hinder the use of these phages as biocontrol agents, particularly since low levels of the pathogen are typically encountered in the environment.
Virus-Binding Proteins Recovered from Bacterial Culture Derived from Activated Sludge by Affinity Chromatography Assay Using a Viral Capsid Peptide
Authors: Sano Daisuke , Matsuo Takahiro , Omura Tatsuo
Published: June 2004

The contamination of water environments by pathogenic viruses has raised concerns about outbreaks of viral infectious diseases in our society. Because conventional water and wastewater treatment systems are not effective enough to inactivate or remove pathogenic viruses, a new technology for virus removal needs to be developed. In this study, the virus-binding proteins (VBPs) in a bacterial culture derived from activated sludge were successfully recovered. The recovery of VBPs was achieved by applying extracted crude proteins from a bacterial culture to an affinity column in which a custom-made peptide of capsid protein from the poliovirus type 1 (PV1) Mahoney strain (H
2
N-DNPASTTNKDKL-COOH) was immobilized as a ligand. VBPs exhibited the ability to adsorb infectious particles of PV1 Sabin 1 as determined by enzyme-linked immunosorbent assay. The evaluation of surface charges of VBPs with ion-exchange chromatography found that a majority of VBP molecules had a net negative charge under the conditions of affinity chromatography. On the other hand, a calculated isoelectric point implied that the viral peptide in the affinity column was also charged negatively. As a result, the adsorption of the VBPs to the viral peptide in the affinity column occurred with a strong attractive force that was able to overcome the electrostatic repulsive force. Two-dimensional electrophoresis revealed that the isolated VBPs include a number of proteins, and their molecular masses were widely distributed but smaller than 100 kDa. Amino acid sequences of N termini of five VBPs were determined. Homology searches for the N termini against all protein sequences in the National Center for Biotechnology Information (NCBI) database showed that the isolated VBPs in this study were newly discovered proteins. These VBPs that originated with bacteria in activated sludge might be stable, because they are existing in the environment of wastewater treatments. Therefore, a virus removal technology utilizing VBPs as viral adsorbents can be developed, since it is possible to replicate VBPs by protein cloning techniques.
Distribution of RuBisCO Genotypes along a Redox Gradient in Mono Lake, California
Authors: Giri Bruno J. , Bano Nasreen , Hollibaugh James T.
Published: June 2004

Partial sequences of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) (EC 4.1.1.39) genes were retrieved from samples taken along a redox gradient in alkaline, hypersaline Mono Lake, Calif. The form I gene (
cbbL
) was found in all samples, whereas form II (
cbbM
) was not retrieved from any of the samples. None of the RuBisCO sequences we obtained were closely related (nucleotide similarity, <90%) to sequences in the database. Some could be attributed to organisms isolated from the lake (
Cyanobium
) or appearing in enrichment cultures. Most (52%) of the sequences fell into in one clade, containing sequences that were identical to sequences retrieved from an enrichment culture grown with nitrate and sulfide, and another clade contained sequences identical to those retrieved from an arsenate-reducing, sulfide-oxidizing enrichment.
Effects of Quaternary-Ammonium-Based Formulations on Bacterial Community Dynamics and Antimicrobial Susceptibility
Authors: McBain Andrew J. , Ledder Ruth G. , Moore Louise E. , Catrenich Carl E. , Gilbert Peter
Published: June 2004

Quaternary ammonium compounds (QACs) are widely used as adjuncts to hygiene in domestic cleaning products. Current concern that the increased use of such biocides in consumer products might contribute to the emergence of antibiotic resistance has led us to examine the effects of a QAC-containing domestic cleaning fluid on the population dynamics and antimicrobial susceptibility of domestic sink drain biofilm communities. QAC susceptibilities of numerically dominant, culturable drain bacteria (15 genera, 17 species) were determined in vitro before and after repeated QAC exposure (14 passages). A fully characterized drain microcosm was then exposed to short-term (12 days) and long-term (3 months) dosing with a QAC-containing domestic detergent (QD). QAC exposure of isolated cultures caused both increases (three species) and circa twofold decreases (six species) in QAC susceptibility. The susceptibility ofRalstoniasp. was considerably decreased following 14 consecutive QAC passages. Control drain microcosm biofilms maintained dynamic stability, as evidenced by culture and denaturing gradient gel electrophoresis (DGGE) analysis. Bacterial population densities were largely unaffected during short-term exposure to use levels of QD, although 50% QD caused circa 10-fold viability reductions. DGGE analysis supported these observations; identified the major microcosm genera asPseudomonas,Pseudoalteromonas,Erwinia, andEnterobacter, and showed that aeromonads increased in abundance under 10 to 50% QD. Long-term exposure of the microcosms to QD did not significantly alter the pattern of antimicrobial susceptibility. These data demonstrate the recalcitrance of domestic drain biofilms toward QAC and that although repeated QAC exposure of drain isolates in pure culture results in susceptibility change in some test bacteria, such changes do not necessarily occur within complex communities.