bluuebunny/crossref_metadata_2025 huggingface dataset:
EVALUATION OF HEPATOBILIARY SCINTIGRAPHY AS AN INDICATOR OF HEPATIC FUNCTION IN DOMESTIC PIGEONS (COLUMBA LIVIA) BEFORE AND AFTER EXPOSURE TO ETHYLENE GLYCOL
Authors: HADLEY TARAH L. , DANIEL GREGORY B. , ROTSTEIN DAVID S. , AVENELL JAMES S. , ZAGAYA NANCY , JONES MICHAEL P.
Published: February 2007

This study investigated the use of quantitative hepatobiliary scintigraphy to assess liver function in 14 white Carneaux pigeons (Columba livia). Liver scintigraphy using 99mTc‐mebrofenin was performed and liver function was quantified using deconvolutional analysis and the area under the normalized heart time–activity curve as previously described in the dog and horse. Liver biopsies were performed in all birds before and after toxin‐induced liver damage with ethylene glycol. Before the induction of liver disease, all biopsy specimens showed varying degrees of granulomatous inflammation. After ethylene glycol administration, hepatic lesions were scored and compared with scintigraphic findings. Scintigraphic results showed a significant decrease (P=0.04) in hepatic function using the area under the normalized time–activity curve. There was good correlation between the overall histologic score posttoxin exposure and scintigraphic measures of liver function (P<0.03). Based upon these preliminary results, the area under the heart time–activity curve can determine hepatic extraction as a measure of hepatic parenchymal cell function. The results also showed that worsening hepatic cellular function correlated with increased histologic damage to the liver. The use of hepatobiliary scintigraphy using 99mTc‐mebrofenin to determine liver function in pigeons has not been previously reported. Additional studies are warranted to evaluate the application of this technique in clinical patients and to establish the sensitivity of this technique.
Interactions of imidazoline ligands with the active site of purified monoamine oxidase A
Authors: Jones Tadeusz Z. E. , Giurato Laura , Guccione Salvatore , Ramsay Rona R.
Published: February 2007

The two forms of monoamine oxidase, monoamine oxidase A and monoamine oxidase B, have been associated with imidazoline‐binding sites (type 2). Imidazoline ligands saturate the imidazoline‐binding sites at nanomolar concentrations, but inhibit monoamine oxidase activity only at micromolar concentrations, suggesting two different binding sites [Ozaita A, Olmos G, Boronat MA, Lizcano JM, Unzeta M & García‐Sevilla JA (1997)Br J Pharmacol121,901–912]. When purified human monoamine oxidase A was used to examine the interaction with the active site, inhibition by guanabenz, 2‐(2‐benzofuranyl)‐2‐imidazoline and idazoxan was competitive with kynuramine as substrate, givingKivalues of 3 µm, 26 µmand 125 µm, respectively. Titration of monoamine oxidase A with imidazoline ligands induced spectral changes that were used to measure the binding affinities for guanabenz (19.3 ± 3.9 µm) and 2‐(2‐benzofuranyl)‐2‐imidazoline (49 ± 8 µm). Only one type of binding site was detected. Agmatine, a putative endogenous ligand for some imidazoline sites, reduced monoamine oxidase A under anaerobic conditions, indicating that it binds close to the flavin in the active site. Flexible docking studies revealed multiple orientations within the large active site, including orientations close to the flavin that would allow oxidation of agmatine.
Unconventional translation initiation of human trypsinogen 4 at a CUG codon with an N‐terminal leucine
Authors: Németh Attila L. , Medveczky Péter , Tóth Júlia , Siklódi Erika , Schlett Katalin , Patthy András , Palkovits Miklós , Ovádi Judit , Tõkési Natália , Németh Péter , Szilágyi László , Gráf László
Published: February 2007

Summary Chromosomal rearrangements apparently account for the presence of a primate‐specific gene (protease serine 3) in chromosome 9. This gene encodes, as the result of alternative splicing, both mesotrypsinogen and trypsinogen 4. Whereas mesotrypsinogen is known to be a pancreatic protease, neither the chemical nature nor biological function of trypsinogen 4 has been explored previously. The trypsinogen 4 sequence contains two predicted translation initiation sites: an AUG site that codes for a 72‐residue leader peptide on Isoform A, and a CUG site that codes for a 28‐residue leader peptide on Isoform B. We report studies that provide evidence for the N‐terminal amino acid sequence of trypsinogen 4 and the possible mechanism of expression of this protein in human brain and transiently transfected cells. We raised mAbs against a 28‐amino acid synthetic peptide representing the leader sequence of Isoform B and against recombinant trypsin 4. By using these antibodies, we isolated and chemically identified trypsinogen 4 from extracts of both post mortem human brain and transiently transfected HeLa cells. Our results show that Isoform B, with a leucine N terminus, is the predominant (if not exclusive) form of the enzyme in post mortem human brain, but that both isoforms are expressed in transiently transfected cells. On the basis of our studies on the expression of a series of trypsinogen 4 constructs in two different cell lines, we propose that unconventional translation initiation at a CUG with a leucine, rather than a methionine, N terminus may serve as a means to regulate protein expression.
RCAN1‐1L is overexpressed in neurons of Alzheimer's disease patients
Authors: Harris Cathryn D. , Ermak Gennady , Davies Kelvin J. A.
Published: February 2007

At least two different isoforms of RCAN1 mRNA are expressed in neuronal cells in normal human brain. Although RCAN1 mRNA is elevated in brain regions affected by Alzheimer's disease, it is not known whether the disease affects neuronal RCAN1, or if other cell types (e.g. astrocytes or microglia) are affected. It is also unknown how many protein isoforms are expressed in human brain and whether RCAN1 protein is overexpressed in Alzheimer's disease. We explored the expression of both RCAN1‐1 and RCAN1‐4 mRNA isoforms in various cell types in normal and Alzheimer's disease postmortem samples, using the combined technique of immunohistochemistry and in situ hybridization. We found that both exon 1 and exon 4 are predominantly expressed in neuronal cells, and no significant expression of either of the exons was observed in astocytes or microglial cells. This was true in both normal and Alzheimer's disease brain sections. We also demonstrate that RCAN1‐1 mRNA levels are approximately two‐fold higher in neurons from Alzheimer's disease patients versus non‐Alzheimer's disease controls. Using western blotting, we now show that there are three RCAN1 protein isoforms expressed in human brain: RCAN1‐1L, RCAN1‐1S, and RCAN1‐4. We have determined that RCAN1‐1L is expressed at twice the level of RCAN1‐4, and that there is very minor expression of RCAN1‐1S. We also found that the RCAN1‐1L protein is overexpressed in Alzheimer's disease patients, whereas RCAN1‐4 is not. From these results, we conclude that RCAN1‐1 may play a role in Alzheimer's disease, whereas RCAN1‐4 may serve another purpose.
Diagnosing appendicular osteosarcoma with ultrasound‐guided fine‐needle aspiration: 36 cases
Authors: Britt T. , Clifford C. , Barger A. , Moroff S. , Drobatz K. , Thacher C. , Davis G.
Published: February 2007

Objectives: To determine the ability to obtain diagnostic cytology samples from appendicular bone lesions using ultrasound‐guided needle aspirations. Secondary objectives were to compare cytological evaluations with histopathological results and to determine the utility of staining malignant mesenchymal cells for the presence of alkaline phosphatase.

Methods: Aspirations from 36 aggressive appendicular bone lesions with histological diagnoses were included in the study. Ultrasound was used to guide the needle to the medullary cavity or the adjacent soft tissue mass. The smears stained with Wright‐Giemsa and nitroblue tetrazolium chloride/5‐bromo‐4‐chloro‐3‐indoyl phosphate toluidine salt (NBT/BCIP) were examined.

Results: A diagnostic sample was obtained in 32 of the 36 cases. Of the 32 diagnostic samples, cytology indicated sarcoma, with a sensitivity of 97 per cent (confidence interval: 83 to 100 per cent) and a specificity of 100 per cent (confidence interval: 16 to 100 per cent). When a diagnosis of sarcoma was made on cytology, alkaline phosphatase staining indicated osteosarcoma, with a sensitivity of 100 per cent (confidence interval: 87 to 100 per cent).

Clinical Significance: The results of this study indicate that ultrasound‐guided needle aspiration of aggressive bone lesions is a viable technique for identifying malignant mesenchymal cells and for diagnosing sarcomas. It is cost‐effective and minimally invasive. Furthermore, identifying alkaline‐phosphatase‐negative malignant mesenchymal cells from a bone aspiration may rule out osteosarcoma, whereas alkaline‐phosphatase‐positive malignant mesenchymal cells are suggestive of osteosarcoma.
Treatment of tibial fractures with plates using minimally invasive percutaneous osteosynthesis in dogs and cats
Authors: Schmökel H. G. , Stein S. , Radke H. , Hurter K. , Schawalder P.
Published: February 2007

Objectives:The aim of the here described case series was to develop and evaluate the minimally invasive percutaneous osteosynthesis for the plate fixation of tibial fractures in dogs and cats.

Methods:Six dogs and four cats with shaft fractures of the tibia were treated using minimally invasive percutaneous osteosynthesis. Follow‐up radiographs four to six weeks after fracture fixation were evaluated for fracture healing. For the long‐term follow‐up (minimum 2·4 years), owners were contacted by phone to complete a questionnaire.

Results:All fractures healed without the need for a second procedure. Follow‐up radiographs obtained after four to six weeks in seven cases showed advanced bony healing with callus formation and filling of the fracture gaps with calcified tissue in all seven. All the patients had a good to excellent long‐term result with full limb function. The time needed for regaining full limb use was two to three months.

ClinicalSignificance:Minimally invasive percutaneous osteosynthesis seems to be a useful technique for the treatment of tibial shaft fractures in dogs and cats.
Substitute Geometry of the Features of Size: Applications to Multidimensional Features
Authors: Portman V. , Shuster V. , Rubenchik Y. , Shneor Y.
Published: February 2007

The problems of the substitute geometry for features of size are considered and an algorithm for synthesis of the substitute features (SF) is developed. Three and only three classes of surfaces are proved to have an incomplete set of position and orientation deviations within the SF equation: cylinders with any directrix, surfaces of revolution with any meridian, and helical surfaces with any profile. The form accuracy of multidimensional features relating to these classes is considered: ellipsoid of revolution, epitrochoidal cylinder, and Archimedean screw. The deterministic consideration is accompanied by evaluation of the uncertainty of the standard assessments of the geometric accuracy and capacity of the computing procedure.
Three-Dimensional Verification of Geometric Tolerances With the “Fitting Gauge” Model
Authors: Pairel Eric
Published: February 2007

Thanks to the “fitting gauge” conceptual model, developed in our lab, any geometric tolerance can be interpreted in the form of a virtual three-dimensional gauge, which is able to be assembled with the part to be inspected. From a file containing the sampled points of the part to inspect, the experimental software, using this conceptual model, permits one to build the virtual gauge defined by the geometric tolerance and to check that it can be assembled and adjusted, according to a precise order, with clouds of points representing the part. Checking the geometric tolerances is thus strictly in conformity with their standardized meaning and it is extremely simplified.
3D Digitizing Strategy Planning Approach Based on a CAD Model
Authors: Derigent William , Chapotot Emilie , Ris Gabriel , Remy Sébastien , Bernard Alain
Published: February 2007

The objective of this paper is to describe a new method to determine the three-dimensional (3D) digitizing strategy for a computer aided design (CAD) known mechanical part by using a plane laser sensor. The Research Centre for Automatic control in Nancy, France has initiated a project to create an automatic 3D digitizing system. Previous papers focused on the visibility determinations using spherical geometry in a three axis environment. Here, the latest developments are presented. They enable one to improve this approach by taking into account a five axis environment. Using a new approach based on the Minkovsky operations to calculate the visibility of the different face of the B-Rep model of the part, the minimum set of directions required to entirely digitize the part is computed.
Multimodal Industrial Inspection and Analysis
Authors: Singh Prabhjot , Wu Yanyan , Kaucic Robert , Chen Jiaqin , Little Francis
Published: February 2007

Diagnosis of complex engineering systems requires the use of multiple sensor sources to acquire information. In this paper we present a survey of multimodal data acquisition systems for nondestructive testing (NDT) and engineering analysis. We begin with a summary of the relative strengths and weaknesses of individual NDT modalities. Thereafter we present existing multimodal inspection hardware systems that use complementary NDT sensors. The advantages of such multimodal data acquisition over conventional single modality sensors in inspection and analysis are highlighted. Possible approaches to fuse complementary multimodal sensor data are discussed. We conclude with possible directions for the future development of multimodal inspection systems.
Tolerance Analysis of Free-Form Surfaces in Composite Material
Authors: Polini Wilma , Marrocu M. , D’Ambrosio L.
Published: February 2007

The aim of this work is to present a method for the tolerance analysis of an assembly involving free-form surfaces based on dimensional and geometric tolerances. The great effort of the present work is to deal with dimensional and geometric tolerances applied to free-form surfaces. The case of study is the flaperon, a structural component of the aircraft, that is constituted by one skin cover, eight ribs, and one spar. The ten components of the flaperon are connected by adhesive, in order to overcome the problems due to the rivets use. Adhesive thickness between the faced parts to connect should be as constant as possible in order to obtain the right structural connection. The influence of the tolerances applied to the ten components of the flaperon on the value of the gap at the interfaces among each couple of components has been deeply investigated by means of the proposed method. Moreover, the influence of the tolerances on the aerodynamic performances of the flaperon has also been analyzed.
Measurement Techniques for the Inspection of Porosity Flaws on Machined Surfaces
Authors: Steiner D. , Katz R.
Published: February 2007

Continuous improvement of product quality is essential to the success of manufacturing enterprises. In-process inspection plays an important role in improving product quality control of mass produced products. The change that has occurred in traditional mass production, i.e., the change from dedicated manufacturing to reconfigurable and flexible manufacturing, has increased the demand for computer integrated manufacturing (CIM). Automated inspection serves as a key component of CIM, for example, detecting and measuring surface flaws. Today, inspection of surface flaws is mainly performed on sheet metal, paper, glass, rags, etc. These objects are flat in nature and do not include 3D features or edges of features. However, structural parts which are produced using casting process are complicated and are composed of a large number of features. When these parts are machined (flat milled) porosity defects arise in addition to surface flaws such as scratches and texture defects. Porosity defects arise on the surface when the cutting tool cuts through an air bubble (pore void) created during casting. In many applications, inspection and accurate measurement of porosity flaws, within the capabilities of the measuring device, is essential and crucial to determine the ability of the product to function well. In this paper we present a technique for measuring the correct size of a pore using a low value threshold, to avoid false detection, miss detection and underestimation of pore size. Also we present two algorithms for detecting edge-connected pores which are pores on the edge of features. Detection is done without the use of a feature data base. One of the edge-connected pore detection algorithms uses basic morphological operations for determining the locations of the pores, while the other algorithm uses curve analysis for the same purpose. The feasibility of the proposed porosity measurement technique and edge-connected pore detection algorithms is demonstrated and validated on the joint face of an engine cylinder head.
Application of Regression Spline to Reverse Modeling
Authors: Moroni Giovanni , Rasella Marco
Published: February 2007

When dealing with inspection or reverse modeling, the problem of free-form curves and surfaces reconstruction has to be faced starting from a set of measured points. Because in point sampling the acquisition error is unavoidable, curves and surfaces fitting should be based on a rigorous diagnostic phase. We consider statistical regression analysis in which, treating error as a variable of the problem, we distinguish between the systematic behavior of measured points and noise in the reconstruction of curves and surfaces. The model we introduce for a regression based free-form reconstruction is the so-called regression spline. It is a well known model in the literature, with a consolidated theory and applications in fields such as chemical, econometric, and biomedical. Our purpose is to discuss the application of this powerful and flexible approach in a reverse modeling environment.
Computational Metrology for the Design and Manufacture of Product Geometry: A Classification and Synthesis
Authors: Srinivasan Vijay
Published: February 2007

The increasing use of advanced measurement tools and technology in industry over the past 30 years has ushered in a new set of challenging computational problems. These problems can be broadly classified as fitting and filtering of discrete geometric data collected by measurements made on manufactured products. Collectively, they define the field of computational metrology for the design specification, production, and verification of product geometry. The fitting problems can be posed and solved as optimization problems; they involve both continuous and combinatorial optimization problems. The filtering problems can be unified under convolution problems, which include convolutions of functions as well as convolutions of sets. This paper presents the status of research and standardization efforts in computational metrology, with an emphasis on its classification and synthesis.
Form Defects Tolerancing by Natural Modes Analysis
Authors: Samper Serge , Formosa Fabien
Published: February 2007

The form defects quality needs methods to express allowable deviations. We propose a new language for form defects expression. This one is based on natural mode shapes of a discretized feature. The finite element method is used in order to compute those modes. Then a basis of defects is built with the natural modes. A defect is projected in this basis and thus the coordinates (modal coefficient) represent it. Hence, tolerancing is possible, by limiting those coordinates. The methods proposed in the literature can be applied on elementary geometries or there is a need to express the set of possible features (explicit geometry). Our method is versatile because it is based on the discretization of the feature (implicit geometry). The modal tolerancing method proposes two ways to express specifications of form defects: (1) The spectral tolerancing shows the modal coordinates and their limits in a bar chart graph by drawing the limits. In this method, we can see the decomposition of the measured feature and express tolerancing on each coordinate. (2) When a specification needs to link coordinates, we propose the modal domain method. An inclusion test of the feature coordinates gives the result of the metrology. Those methods are presented in an example.
Adaptive Range Sampling Using a Stochastic Model
Authors: Devir Zvi , Lindenbaum Michael
Published: February 2007

We consider the task of sequential point sampling for three-dimensional structure reconstruction and focus on terrestrial topographic mapping using a laser range scanner. Both the sampling and the reconstruction rely on a stochastic model of the sampled object. We describe several algorithms for sequential point sampling including a new adaptive algorithm that is specifically designed for mechanical devices and produces grid-like sampling patterns. Experimental results verify that relying on the stochastic model indeed leads to efficient sampling associated with accurate surface reconstruction.
Methods and Artifacts for Comparison of Scanning CMM Performance
Authors: Farooqui Sami A. , Morse Edward P.
Published: February 2007

Coordinate measuring machines (CMMs) with continuous-contact scanning capabilities are experiencing more and more use in a wide variety of discrete-part manufacturing industries. Many users of these CMMs, if asked, will say that their metrology requirements include high density scans at high speeds with high accuracy. These requirements are in conflict, as there will be some point at which the accuracy of the scanned data begins to decrease with an increase in the scanning speed. This paper addresses the effects of scanning speed on the performance of different CMMs using contact sensors. The use of a family of artifacts is proposed in order to evaluate the relative scanning performance as a function of scanning speed and the direction of the scan within the CMM volume. The artifacts that are developed for these tests have a sinusoidal waveform superimposed on a flat surface. This paper describes a series of experiments that utilize these artifacts and assess the ability of these tests to capture how a scanning CMM will perform when measuring actual parts.
Comparative High-Density Microarray Analysis of Gene Expression during Growth of
Lactobacillus helveticus
in Milk versus Rich Culture Medium
Authors: Smeianov Vladimir V. , Wechter Patrick , Broadbent Jeffery R. , Hughes Joanne E. , Rodríguez Beatriz T. , Christensen Tove K. , Ardö Ylva , Steele James L.
Published: February 2007

Lactobacillus helveticus
CNRZ32 is used by the dairy industry to modulate cheese flavor. The compilation of a draft genome sequence for this strain allowed us to identify and completely sequence 168 genes potentially important for the growth of this organism in milk or for cheese flavor development. The primary aim of this study was to investigate the expression of these genes during growth in milk and MRS medium by using microarrays. Oligonucleotide probes against each of the completely sequenced genes were compiled on maskless photolithography-based DNA microarrays. Additionally, the entire draft genome sequence was used to produce tiled microarrays in which noninterrupted sequence contigs were covered by consecutive 24-mer probes and associated mismatch probe sets. Total RNA isolated from cells grown in skim milk or in MRS to mid-log phase was used as a template to synthesize cDNA, followed by Cy3 labeling and hybridization. An analysis of data from annotated gene probes identified 42 genes that were upregulated during the growth of CNRZ32 in milk (
P
< 0.05), and 25 of these genes showed upregulation after applying Bonferroni's adjustment. The tiled microarrays identified numerous additional genes that were upregulated in milk versus MRS. Collectively, array data showed the growth of CNRZ32 in milk-induced genes encoding cell-envelope proteinases, oligopeptide transporters, and endopeptidases as well as enzymes for lactose and cysteine pathways, de novo synthesis, and/or salvage pathways for purines and pyrimidines and other functions. Genes for a hypothetical phosphoserine utilization pathway were also differentially expressed. Preliminary experiments indicate that cheese-derived, phosphoserine-containing peptides increase growth rates of CNRZ32 in a chemically defined medium. These results suggest that phosphoserine is used as an energy source during the growth of
L. helveticus
CNRZ32.
Characterization of Growing Microorganisms in Soil by Stable Isotope Probing with H
2
18
O
Authors: Schwartz Egbert
Published: February 2007

A new approach to characterize growing microorganisms in environmental samples based on labeling microbial DNA with H
2
18
O is described. To test if sufficient amounts of
18
O could be incorporated into DNA to use water as a labeling substrate for stable isotope probing,
Escherichia coli
DNA was labeled by cultivating bacteria in Luria broth with H
2
18
O and labeled DNA was separated from [
16
O]DNA on a cesium chloride gradient. Soil samples were incubated with H
2
18
O for 6, 14, or 21 days, and isopycnic centrifugation of the soil DNA showed the formation of two bands after 6 days and three bands after 14 or 21 days, indicating that
18
O can be used in the stable isotope probing of soil samples. DNA extracted from soil incubated for 21 days with H
2
18
O was fractionated after isopycnic centrifugation and DNA from 17 subsamples was used in terminal restriction fragment length polymorphism (TRFLP) analysis of bacterial 16S rRNA genes. The TRFLP patterns clustered into three groups that corresponded to the three DNA bands. The fraction of total fluorescence contributed by individual terminal restriction fragments (TRF) to a TRFLP pattern varied across the 17 subsamples so that a TRF was more prominent in only one of the three bands. Labeling soil DNA with H
2
18
O allows the identification of newly grown cells. In addition, cells that survive but do not divide during an incubation period can also be characterized with this new technique because their DNA remains without the label.
Isolation of
Methylophaga
spp. from Marine Dimethylsulfide-Degrading Enrichment Cultures and Identification of Polypeptides Induced during Growth on Dimethylsulfide
Authors: Schäfer Hendrik
Published: February 2007

Dimethylsulfide (DMS)-degrading enrichment cultures were established from samples of coastal seawater, nonaxenic
Emiliania huxleyi
cultures, and mixed marine methyl halide-degrading enrichment cultures. Bacterial populations from a broad phylogenetic range were identified in the mixed DMS-degrading enrichment cultures by denaturing gradient gel electrophoresis (DGGE). Sequences of dominant DGGE bands were similar to those of members of the genera
Methylophaga
and
Alcanivorax
. Several closely related
Methylophaga
strains were obtained that were able to grow on DMS as the carbon and energy source.
Roseobacter
-related populations were detected in some of the enrichment cultures; however, none of the
Roseobacter
group isolates that were tested were able to grow on DMS. Oxidation of DMS by
Methylophaga
sp. strain DMS010 was not affected by addition of the inhibitor chloroform or methyl
tert
-butyl ether, suggesting that DMS metabolism may occur by a route different from those described for
Thiobacillus
species and other unidentified marine isolates. Addition of DMS and methanethiol to whole-cell suspensions of strain DMS010 induced oxygen uptake when strain DMS010 was grown on DMS but not in cells grown on methanol. The apparent

K
m

s of strain DMS010 for DMS and for methanethiol were 2.1 and 4.6 μM, respectively, when grown on DMS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the biomass of strain DMS010 and analysis of peptide bands by mass spectrometry techniques and N-terminal sequencing provided the first insight into the identity of polypeptides induced during growth on DMS. These included XoxF, a homolog of the large subunit of methanol dehydrogenase for which a biological role has not been identified previously.
Development of a Real-Time PCR Probe for Quantification of the Heterotrophic Dinoflagellate
Cryptoperidiniopsis brodyi
(Dinophyceae) in Environmental Samples
Authors: Park Tae-Gyu , de Salas Miguel F. , Bolch Christopher J. S. , Hallegraeff Gustaaf M.
Published: February 2007

A TaqMan format real-time PCR probe was developed against the internal transcribed spacer 2 ribosomal DNA region for the specific detection and quantification of
Cryptoperidiniopsis brodyi
in environmental samples. The assay specificity was confirmed by testing against related dinoflagellates and verified by sequencing PCR amplicons from natural water samples. Phylogenetic analysis of the sequenced environmental samples also showed that this assay is specific to
C. brodyi
. The
C. brodyi
-specific assay was used in conjunction with
Pfiesteria piscicida-
and
Pfiesteria shumwayae
-specific real-time PCR assays to investigate the temporal variations of
C. brodyi
,
P. piscicida
, and
P. shumwayae
abundance in the Derwent estuary, Tasmania. The 18-month field survey from November 2004 to April 2006 revealed that
C. brodyi
occurred in all seasons at very low densities, mostly below 25 cells liter
−1
, with higher abundance (maximum, 112 cells liter
−1
) in April and May.
P. piscicida
was detected only once, in May 2005 at 60 cells liter
−1
.
P. shumwayae
was not detected during the survey.
Analysis of Homothallic
Saccharomyces cerevisiae
Strain Mating during Must Fermentation
Authors: Ambrona Jesús , Ramírez Manuel
Published: February 2007

Genetic instability and genome renewal may cause loss of heterozygosity (LOH) in homothallic wine yeasts (
Saccharomyces cerevisiae
), leading to the elimination of the recessive lethal or deleterious alleles that decrease yeast fitness. LOH was not detected in genetically stable wine yeasts during must fermentation. However, after sporulation, the heterozygosity of the new yeast population decreased during must fermentation. The frequency of mating between just-germinated haploid cells from different tetrads was very low, and the mating of haploid cells from the same ascus was favored because of the physical proximity. Also, mating restriction between haploid cells from the same ascus was found, leading to a very low frequency of self spore clone mating. This mating restriction slowed down the LOH process of the yeast population, maintaining the heterozygote frequency higher than would be expected assuming a fully random mating of the haploid yeasts or according to the Mortimer genome renewal proposal. The observed LOH occurs because of the linkage of the locus
MAT
to the chromosome III centromere, without the necessity for self spore clone mating or the high frequency of gene conversion and rapid asymmetric LOH observed in genetically unstable yeasts. This phenomenon is enough in itself to explain the high level of homozygosis found in natural populations of wine yeasts. The LOH process for centromere-linked markers would be slower than that for the nonlinked markers, because the linkage decreases the frequency of newly originated heterozygous yeasts after each round of sporulation and mating. This phenomenon is interesting in yeast evolution and may cause important sudden phenotype changes in genetically stable wine yeasts.
Generation by a Widely Applicable Approach of a Hybrid Dioxygenase Showing Improved Oxidation of Polychlorobiphenyls
Authors: Cámara Beatriz , Seeger Michael , González Myriam , Standfuß-Gabisch Christine , Kahl Silke , Hofer Bernd
Published: February 2007

Recently, a sequence-based approach has been developed for the fast isolation and characterization of class II aryl-hydroxylating dioxygenase activities (S. Kahl and B. Hofer, Microbiology
149:
1475-1481, 2003). It comprises the PCR amplification of segments of alpha subunit genes of unknown sequence that encode the catalytic center and their fusion with sequences of the
bphA
gene cluster of
Burkholderia xenovorans
LB400. One of the resulting chimeric enzymes, harboring the core segment of a dioxygenase from
Pseudomonas
sp. strain B4-Magdeburg, has now been characterized with respect to the oxidation of chlorobiphenyls (CBs). Its substrate and product specificities differed favorably from those of the parental dioxygenase of strain LB400. The hybrid possessed a higher regiospecificity and yielded less unproductive dioxygenations at
meta
and
para
carbons. It attacked
ortho
-,
meta
-, and
para
-chlorinated rings with comparable efficiencies. It gave significantly higher yields in
ortho
,
meta
-dioxygenation of recalcitrant congeners containing a doubly
ortho
-chlorinated ring. While the parental enzyme yielded mainly unproductive
meta
,
para
dioxygenation of 2,5,4′-CB, the hybrid predominantly converted this congener into an
ortho
,
meta
-dioxygenated product. The subsequent enzymes of the LB400 catabolic pathway were able to transform most of the metabolites formed by the novel dioxygenase, indicating that the substrate ranges of these biocatalysts are not adapted to that of their initial pathway enzyme. Some of the catabolites, however, were identified as problematic for further degradation. Our results demonstrate that the outlined approach can successfully be applied to obtain novel dioxygenase specificities that favorably complement or supplement known ones.
Genetic Characterization of 2,4,6-Trichlorophenol Degradation in
Cupriavidus necator
JMP134
Authors: Sánchez M. A. , González B.
Published: February 2007

The degradation pathway of 2,4,6-trichlorophenol (2,4,6-TCP), a hazardous pollutant, in the aerobic bacterium
Cupriavidus necator
JMP134(pJP4) (formerly
Ralstonia eutropha
JMP134) is encoded by the
tcp
genes. These genes are located in a genetic context,
tcpRXABCYD
, which resembles a putative catabolic operon. In this work, these gene sequences were individually disrupted and mutant strains were evaluated for their ability to grow on or degrade 2,4,6-TCP. The
tcpX
and
tcpA
mutants completely failed to degrade this compound. Although the
tcpC
mutant was also unable to grow on 2,4,6-TCP, it still transformed this chlorophenol to 6-chlorohydroquinol. In contrast, the
tcpD
mutant grew on 2,4,6-TCP, suggesting the presence of additional maleylacetate reductase-encoding genes. Five other open reading frames encoding maleylacetate reductases, in addition to the
tcpD
gene, were found in the genome of
C. necator
, and two of them provide this function in the
tcpD
mutant. The
tcpR
gene, encoding a putative LysR-type transcriptional regulator, was disrupted, and this mutant strain completely failed to grow on 2,4,6-TCP. Transcriptional fusion studies demonstrated that TcpR activates the expression of the
tcp
genes, responding specifically to 2,4,6-TCP. The transcriptional start of the
tcp
operon was mapped, and a putative σ
70
-type promoter was identified.
Ferrous Iron- and Sulfur-Induced Genes in
Sulfolobus metallicus
Authors: Bathe Stephan , Norris Paul R.
Published: February 2007

Genes of
Sulfolobus metallicus
that appeared to be upregulated in relation to growth on either ferrous iron or sulfur were identified using subtractive hybridization of cDNAs. The genes upregulated during growth on ferrous iron were found in a cluster, and most were predicted to encode membrane proteins. Quantitative reverse transcription-PCR of cDNA showed upregulation of most of these genes during growth on ferrous iron and pyrite compared to results during growth on sulfur. The highest expression levels observed included those for genes encoding proteins with similarities to cytochrome
c
oxidase subunits and a CbsA-like cytochrome. The genes identified here that may be involved in oxidation of ferrous iron by
S. metallicus
are termed
fox
genes. Of three available genomes of
Sulfolobus
species (
S. tokodaii
,
S. acidocaldarius
, and
S. solfataricus
), only that of
S. tokodaii
has a cluster of highly similar open reading frames, and only
S. tokodaii
of these three species was also able to oxidize ferrous iron. A gene encoding sulfur oxygenase-reductase was identified as the source of the dominant transcript in sulfur-grown cells of
S. metallicus
, with the predicted protein showing high identities to the previously described examples from
S. tokodaii
and species of
Acidianus
.
Gene Targeting in Gram-Negative Bacteria by Use of a Mobile Group II Intron (“Targetron”) Expressed from a Broad-Host-Range Vector
Authors: Yao Jun , Lambowitz Alan M.
Published: February 2007

Mobile group II introns (“targetrons”) can be programmed for insertion into virtually any desired DNA target with high frequency and specificity. Here, we show that targetrons expressed via anm-toluic acid-inducible promoter from a broad-host-range vector containing an RK2 minireplicon can be used for efficient gene targeting in a variety of gram-negative bacteria, includingEscherichia coli,Pseudomonas aeruginosa, andAgrobacterium tumefaciens. Targetrons expressed from donor plasmids introduced by electroporation or conjugation yielded targeted disruptions at frequencies of 1 to 58% of screened colonies in theE. coli lacZ,P. aeruginosa pqsAandpqsH, andA. tumefaciens aopBandchvIgenes. The development of this broad-host-range system for targetron expression should facilitate gene targeting in many bacteria.
The
MAT1
Locus of
Histoplasma capsulatum
Is Responsive in a Mating Type-Specific Manner
Authors: Bubnick Meggan , Smulian A. George
Published: February 2007

Recombination events associated with sexual replication in pathogens may generate new strains with altered virulence.
Histoplasma capsulatum
is a mating-competent, pathogenic fungus with two described phenotypic mating types, + and −. The mating (
MAT
) locus of
H. capsulatum
was identified to facilitate molecular studies of mating in this organism. Through syntenic analysis of the
H. capsulatum
genomic sequence databases, a
MAT1-1
idiomorph region was identified in
H. capsulatum
strains G217B and WU24, and a
MAT1-2
idiomorph region was identified in the strain G186AR. A mating type-specific PCR assay was developed, and two clinical isolates of opposite genotypic mating type, UH1 and VA1, were identified. A known − mating type strain, T-3-1 (ATCC 22635), was demonstrated to be of
MAT1-2
genotypic mating type. The clinical isolates UH1 and VA1 were found to be mating compatible and also displayed mating-type-dependent regulation of the
MAT
transcription factors in response to extracts predicted to contain mating pheromones. These studies support a role for the identified
MAT1
locus in determining mating type in
H. capsulatum
.
Basic Helix-Loop-Helix Transcription Factor Heterocomplex of Yas1p and Yas2p Regulates Cytochrome P450 Expression in Response to Alkanes in the Yeast
Yarrowia lipolytica
Authors: Endoh-Yamagami Setsu , Hirakawa Kiyoshi , Morioka Daisuke , Fukuda Ryouichi , Ohta Akinori
Published: February 2007

The expression of the
ALK1
gene, which encodes cytochrome P450, catalyzing the first step of alkane oxidation in the alkane-assimilating yeast
Yarrowia lipolytica
, is highly regulated and can be induced by alkanes. Previously, we identified a
cis
-acting element (alkane-responsive element 1 [ARE1]) in the
ALK1
promoter. We showed that a basic helix-loop-helix (bHLH) protein, Yas1p, binds to ARE1 in vivo and mediates alkane-dependent transcription induction. Yas1p, however, does not bind to ARE1 by itself in vitro, suggesting that Yas1p requires another bHLH protein partner for its DNA binding, as many bHLH transcription factors function by forming heterodimers. To identify such a binding partner of Yas1p, here we screened open reading frames encoding proteins with the bHLH motif from the
Y. lipolytica
genome database and identified the
YAS2
gene. The deletion of the
YAS2
gene abolished the alkane-responsive induction of
ALK1
transcription and the growth of the yeast on alkanes. We revealed that Yas2p has transactivation activity. Furthermore, Yas1p and Yas2p formed a protein complex that was required for the binding of these proteins to ARE1. These findings allow us to postulate a model in which bHLH transcription factors Yas1p and Yas2p form a heterocomplex and mediate the transcription induction in response to alkanes.
Regulators of the
Bacillus subtilis cydABCD
Operon: Identification of a Negative Regulator, CcpA, and a Positive Regulator, ResD
Authors: Puri-Taneja Ankita , Schau Matthew , Chen Yinghua , Hulett F. Marion
Published: February 2007

The
cydABCD
operon of
Bacillus subtilis
encodes products required for the production of cytochrome
bd
oxidase. Previous work has shown that one regulatory protein, YdiH (Rex), is involved in the repression of this operon. The work reported here confirms the role of Rex in the negative regulation of the
cydABCD
operon. Two additional regulatory proteins for the
cydABCD
operon were identified, namely, ResD, a response regulator involved in the regulation of respiration genes, and CcpA, the carbon catabolite regulator protein. ResD, but not ResE, was required for full expression of the
cydA
promoter in vivo. ResD binding to the
cydA
promoter between positions −58 and −107, a region which includes ResD consensus binding sequences, was not enhanced by phosphorylation. A
ccpA
mutant had increased expression from the full-length
cydA
promoter during stationary growth compared to the wild-type strain. Maximal expression in a
ccpA
mutant was observed from a 3′-deleted
cydA
promoter fusion that lacked the Rex binding region, suggesting that the effect of the two repressors, Rex and CcpA, was cumulative. CcpA binds directly to the
cydA
promoter, protecting the region from positions −4 to −33, which contains sequences similar to the CcpA consensus binding sequence, the
cre
box. CcpA binding was enhanced upon addition of glucose-6-phosphate, a putative cofactor for CcpA. Mutation of a conserved residue in the
cre
box reduced CcpA binding 10-fold in vitro and increased
cydA
expression in vivo. Thus, CcpA and ResD, along with the previously identified
cydA
regulator Rex (YdiH), affect the expression of the
cydABCD
operon. Low-level induction of the
cydA
promoter was observed in vivo in the absence of its regulatory proteins, Rex, CcpA, and ResD. This complex regulation suggests that the
cydA
promoter is tightly regulated to allow its expression only at the appropriate time and under the appropriate conditions.
Comparative Analysis of
hmuO
Function and Expression in
Corynebacterium
Species
Authors: Kunkle Carey A. , Schmitt Michael P.
Published: February 2007

We have constructed defined deletions in the
hmuO
gene from
Corynebacterium diphtheriae
and
Corynebacterium ulcerans
and show that the
C. ulcerans hmuO
mutation results in a significant reduction in hemoglobin-iron utilization, whereas in
C. diphtheriae
strains, deletion of
hmuO
caused no or only partial reduction in the utilization of heme as an iron source. We also show that expression from the
C. ulcerans hmuO
promoter exhibits minimal regulation by iron and heme whereas transcription from the
C. diphtheriae hmuO
promoter shows both significant iron repression and heme-dependent activation. These findings indicate that variability in HmuO function and expression exists among
Corynebacterium
species.
Interaction of Related Tn
916
-Like Transposons: Analysis of Excision Events Promoted by Tn
916
and Tn
5386
Integrases
Authors: Rice Louis B. , Carias Lenore L. , Hutton-Thomas Rebecca , Rudin Susan
Published: February 2007

In recent work, we described the excision of a large genomic region from
Enterococcus faecium
D344R in which the sequence from “joint” regions suggested that excision resulted from the interaction of conjugative transposon Tn
916
and the related mobile element Tn
5386
. In the present study, we examined the ability of integrases and integrase-excisase combinations from Tn
916
and Tn
5386
to promote the excision of constructs consisting of the termini of Tn
916
, Tn
5386
, and the VanB mobile element Tn
5382
. Integrases alone from either Tn
916
or Tn
5386
promoted the circularization of constructs from the three different transposons, even when the different termini used in the constructs were discordant in their transposon of origin. The termini of Tn
916
and Tn
5382
found in all joints were consistent with previously identified Tn
916
and Tn
5382
termini. Substantial variation was seen in the integrase terminus of Tn
5386
used to form joints, regardless of the integrase that was responsible for circularization. Variability was observed in joints formed from Tn
5386
constructs, in contrast to joints observed with the termini of Tn
916
or Tn
5382
. The coexpression of excisase yielded some variability in the joint regions observed. These data confirm that integrases from some Tn
916
-like elements can promote circularization with termini derived from heterologous transposons and, as such, could promote excision of large genomic regions flanked by such elements. These findings also raise interesting questions about the sequence specificities of the C terminals of Tn
916
-like integrases, which bind to the ends and facilitate strand exchange.
Defective Ribonucleoside Diphosphate Reductase Impairs Replication Fork Progression inEscherichia coli
Authors: Guarino Estrella , Jiménez-Sánchez Alfonso , Guzmán Elena C.
Published: February 2007

The observed lengthening of theCperiod in the presence of a defective ribonucleoside diphosphate reductase has been assumed to be due solely to the low deoxyribonucleotide supply in thenrdA101mutant strain. We show here that thenrdA101mutation induces DNA double-strand breaks at the permissive temperature in arecB-deficient background, suggesting an increase in the number of stalled replication forks that could account for the slowing of replication fork progression observed in thenrdA101strain in a Rec+context. These DNA double-strand breaks require the presence of the Holliday junction resolvase RuvABC, indicating that they have been generated from stalled replication forks that were processed by the specific reaction named “replication fork reversal.” Viability results supported the occurrence of this process, as specific lethality was observed in thenrdA101 recBdouble mutant and was suppressed by the additional inactivation ofruvABC. None of these effects seem to be due to the limitation of the deoxyribonucleotide supply in thenrdA101strain even at the permissive temperature, as we found the same level of DNA double-strand breaks in thenrdA+strain growing under limited (2-μg/ml) or under optimal (5-μg/ml) thymidine concentrations. We propose that the presence of an altered NDP reductase, as a component of the replication machinery, impairs the progression of the replication fork, contributing to the lengthening of theCperiod in thenrdA101mutant at the permissive temperature.