bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene expression profiling reveals novel TGFβ targets in adult lung fibroblasts
Authors: Renzoni Elisabetta A , Abraham David J , Howat Sarah , Shi-Wen Xu , Sestini Piersante , Bou-Gharios George , Wells Athol U , Veeraraghavan Srihari , Nicholson Andrew G , Denton Christopher P , Leask Andrew , Pearson Jeremy D , Black Carol M , Welsh Kenneth I , du Bois Roland M
Published: January 2005

Background

Transforming growth factor beta (TGFβ), a multifunctional cytokine, plays a crucial role in the accumulation of extracellular matrix components in lung fibrosis, where lung fibroblasts are considered to play a major role. Even though the effects of TGFβ on the gene expression of several proteins have been investigated in several lung fibroblast cell lines, the global pattern of response to this cytokine in adult lung fibroblasts is still unknown.

Methods

We used Affymetrix oligonucleotide microarrays U95v2, containing approximately 12,000 human genes, to study the transcriptional profile in response to a four hour treatment with TGFβ in control lung fibroblasts and in fibroblasts from patients with idiopathic and scleroderma-associated pulmonary fibrosis. A combination of the Affymetrix change algorithm (Microarray Suite 5) and of analysis of variance models was used to identify TGFβ-regulated genes. Additional criteria were an average up- or down- regulation of at least two fold.

Results

Exposure of fibroblasts to TGFβ had a profound impact on gene expression, resulting in regulation of 129 transcripts. We focused on genes not previously found to be regulated by TGFβ in lung fibroblasts or other cell types, including nuclear co-repressor 2, SMAD specific E3 ubiquitin protein ligase 2 (SMURF2), bone morphogenetic protein 4, and angiotensin II receptor type 1 (AGTR1), and confirmed the microarray results by real time-PCR. Western Blotting confirmed induction at the protein level of AGTR1, the most highly induced gene in both control and fibrotic lung fibroblasts among genes encoding for signal transduction molecules.

Upregulation of AGTR1 occurred through the MKK1/MKK2 signalling pathway. Immunohistochemical staining showed AGTR1 expression by lung fibroblasts in fibroblastic foci within biopsies of idiopathic pulmonary fibrosis.

Conclusions

This study identifies several novel TGFβ targets in lung fibroblasts, and confirms with independent methods the induction of angiotensin II receptor type 1, underlining a potential role for angiotensin II receptor 1 antagonism in the treatment of lung fibrosis.
Coccidioides posadasii infection alters the expression of pulmonary surfactant proteins (SP)-A and SP-D
Authors: Awasthi Shanjana , Magee D Mitchell , Coalson Jacqueline J
Published: January 2005

Background

Coccidioidomycosis or Valley Fever is caused by Coccidioides in Southwest US and Central America. Primary pulmonary infection is initiated by inhalation of air-borne arthroconidia. Since, lung is the first organ that encounters arthroconidia, different components of the pulmonary innate immune system may be involved in the regulation of host defense. Pulmonary surfactant proteins (SP)-A and SP-D have been recognized to play an important role in binding and phagocytosis of various microorganisms, but their roles in Coccidioides infection are not known.

Methods

In this study, we studied the changes in amounts of pulmonary SP-A, SP-D and phospholipid in murine model of Coccidioides posadasii infection, and binding of SP-A and SP-D to Coccidioidal antigens. Mice were challenged intranasally with a lethal dose of C. posadasii (n = 30 arthroconidia) and bronchoalveolar lavage fluid (BALF) samples were collected on day 10, post infection. In another group of animals, mice were immunized with protective formalin killed spherule (FKS) vaccine prior to infection. The concentrations of BALF SP-A, SP-D, total phospholipid were measured using enzyme linked immunosorbent assay and biochemical assays.

Results

We found that in lavage fluid samples of C. posadasii infected mice, the concentrations of total phospholipid, SP-A and SP-D were 17 % (SEM 3.5, p < 0.001), 38 % (SEM 5.8, p < 0.001) and 4 % (SEM 1.3, p < 0.001) of those in lavage fluid samples of non-infected control mice, respectively. However, the concentrations of SP-A and SP-D remained unchanged in BALF samples of C. posadasii protected mice after immunization with FKS vaccine. Also, we found that both SP-A and SP-D bind to Coccidiodal antigens.

Conclusion

Our results suggest that the C. posadasii infection perturbs the pulmonary SP-A, SP-D, and phospholipids, potentially enabling the disease progression and promoting fungal dissemination.
Modulation of epithelial sodium channel (ENaC) expression in mouse lung infected with Pseudomonas aeruginosa
Authors: Dagenais André , Gosselin Diane , Guilbault Claudine , Radzioch Danuta , Berthiaume Yves
Published: January 2005

Background

The intratracheal instillation of Pseudomonas aeruginosa entrapped in agar beads in the mouse lung leads to chronic lung infection in susceptible mouse strains. As the infection generates a strong inflammatory response with some lung edema, we tested if it could modulate the expression of genes involved in lung liquid clearance, such as the α, β and γ subunits of the epithelial sodium channel (ENaC) and the catalytic subunit of Na+-K+-ATPase.

Methods

Pseudomonas aeruginosa entrapped in agar beads were instilled in the lung of resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) mouse strains. The mRNA expression of ENaC and Na+-K+-ATPase subunits was tested in the lung by Northern blot following a 3 hours to 14 days infection.

Results

The infection of the different mouse strains evoked regulation of α and β ENaC mRNA. Following Pseudomonas instillation, the expression of αENaC mRNA decreased to a median of 43% on days 3 and 7 after infection and was still decreased to a median of 45% 14 days after infection (p < 0.05). The relative expression of βENaC mRNA was transiently increased to a median of 241%, 24 h post-infection before decreasing to a median of 43% and 54% of control on days 3 and 7 post-infection (p < 0.05). No significant modulation of γENaC mRNA was detected although the general pattern of expression of the subunit was similar to α and β subunits. No modulation of α1Na+-K+-ATPase mRNA, the catalytic subunit of the sodium pump, was recorded. The distinctive expression profiles of the three subunits were not different, between the susceptible and resistant mouse strains.

Conclusions

These results show that Pseudomonas infection, by modulating ENaC subunit expression, could influence edema formation and clearance in infected lungs.
Identification of α-type subunits of the Xenopus 20S proteasome and analysis of their changes during the meiotic cell cycle
Authors: Wakata Yuka , Tokumoto Mika , Horiguchi Ryo , Ishikawa Katsutoshi , Nagahama Yoshitaka , Tokumoto Toshinobu
Published: January 2005

Background

The 26S proteasome is the proteolytic machinery of the ubiquitin-dependent proteolytic system responsible for most of the regulated intracellular protein degradation in eukaryotic cells. Previously, we demonstrated meiotic cell cycle dependent phosphorylation of α4 subunit of the 26S proteasome. In this study, we analyzed the changes in the spotting pattern separated by 2-D gel electrophoresis of α subunits during Xenopus oocyte maturation.

Results

We identified cDNA for three α-type subunits (α1, α5 and α6) of Xenopus, then prepared antibodies specific for five subunits (α1, α3, α5, α6, and α7). With these antibodies and previously described monoclonal antibodies for subunits α2 and α4, modifications to all α-type subunits of the 26S proteasome during Xenopus meiotic maturation were examined by 2D-PAGE. More than one spot for all subunits except α7 was identified. Immunoblot analysis of 26S proteasomes purified from immature and mature oocytes showed a difference in the blots of α2 and α4, with an additional spot detected in the 26S proteasome from immature oocytes (in G2-phase).

Conclusions

Six of α-type subunits of the Xenopus 26S proteasome are modified in Xenopus immature oocytes and two subunits (α2 and α4) are modified meiotic cell cycle-dependently.
CLOE: Identification of putative functional relationships among genes by comparison of expression profiles between two species
Authors: Pellegrino Maurizio , Provero Paolo , Silengo Lorenzo , Di Cunto Ferdinando
Published: January 2005

Background

Public repositories of microarray data contain an incredible amount of information that is potentially relevant to explore functional relationships among genes by meta-analysis of expression profiles. However, the widespread use of this resource by the scientific community is at the moment limited by the limited availability of effective tools of analysis. We here describe CLOE, a simple cDNA microarray data mining strategy based on meta-analysis of datasets from pairs of species. The method consists in ranking EST probes in the datasets of the two species according to the similarity of their expression profiles with that of two EST probes from orthologous genes, and extracting orthologous EST pairs from a given top interval of the ranked lists. The Gene Ontology annotation of the obtained candidate partners is then analyzed for keywords overrepresentation.

Results

We demonstrate the capabilities of the approach by testing its predictive power on three proteomically-defined mammalian protein complexes, in comparison with single and multiple species meta-analysis approaches. Our results show that CLOE can find candidate partners for a greater number of genes, if compared to multiple species co-expression analysis, but retains a comparable specificity even when applied to species as close as mouse and human. On the other hand, it is much more specific than single organisms co-expression analysis, strongly reducing the number of potential candidate partners for a given gene of interest.

Conclusions

CLOE represents a simple and effective data mining approach that can be easily used for meta-analysis of cDNA microarray experiments characterized by very heterogeneous coverage. Importantly, it produces for genes of interest an average number of high confidence putative partners that is in the range of standard experimental validation techniques.
A new mixture model approach to analyzing allelic-loss data using Bayes factors
Authors: Desai Manisha , Emond Mary J
Published: January 2005

Background

Allelic-loss studies record data on the loss of genetic material in tumor tissue relative to normal tissue at various loci along the genome. As the deletion of a tumor suppressor gene can lead to tumor development, one objective of these studies is to determine which, if any, chromosome arms harbor tumor suppressor genes.

Results

We propose a large class of mixture models for describing the data, and we suggest using Bayes factors to select a reasonable model from the class in order to classify the chromosome arms. Bayes factors are especially useful in the case of testing that the number of components in a mixture model is n0 versus n1. In these cases, frequentist test statistics based on the likelihood ratio statistic have unknown distributions and are therefore not applicable. Our simulation study shows that Bayes factors favor the right model most of the time when tumor suppressor genes are present. When no tumor suppressor genes are present and background allelic-loss varies, the Bayes factors are often inconclusive, although this results in a markedly reduced false-positive rate compared to that of standard frequentist approaches. Application of our methods to three data sets of esophageal adenocarcinomas yields interesting differences from those results previously published.

Conclusions

Our results indicate that Bayes factors are useful for analyzing allelic-loss data.
Optimized LOWESS normalization parameter selection for DNA microarray data
Authors: Berger John A , Hautaniemi Sampsa , Järvinen Anna-Kaarina , Edgren Henrik , Mitra Sanjit K , Astola Jaakko
Published: January 2005

Background

Microarray data normalization is an important step for obtaining data that are reliable and usable for subsequent analysis. One of the most commonly utilized normalization techniques is the locally weighted scatterplot smoothing (LOWESS) algorithm. However, a much overlooked concern with the LOWESS normalization strategy deals with choosing the appropriate parameters. Parameters are usually chosen arbitrarily, which may reduce the efficiency of the normalization and result in non-optimally normalized data. Thus, there is a need to explore LOWESS parameter selection in greater detail.

Results and discussion

In this work, we discuss how to choose parameters for the LOWESS method. Moreover, we present an optimization approach for obtaining the fraction of data points utilized in the local regression and analyze results for local print-tip normalization. The optimization procedure determines the bandwidth parameter for the local regression by minimizing a cost function that represents the mean-squared difference between the LOWESS estimates and the normalization reference level. We demonstrate the utility of the systematic parameter selection using two publicly available data sets. The first data set consists of three self versus self hybridizations, which allow for a quantitative study of the optimization method. The second data set contains a collection of DNA microarray data from a breast cancer study utilizing four breast cancer cell lines. Our results show that different parameter choices for the bandwidth window yield dramatically different calibration results in both studies.

Conclusions

Results derived from the self versus self experiment indicate that the proposed optimization approach is a plausible solution for estimating the LOWESS parameters, while results from the breast cancer experiment show that the optimization procedure is readily applicable to real-life microarray data normalization. In summary, the systematic approach to obtain critical parameters in the LOWESS technique is likely to produce data that optimally meets assumptions made in the data preprocessing step and thereby makes studies utilizing the LOWESS method unambiguous and easier to repeat.
An SVD-based comparison of nine whole eukaryotic genomes supports a coelomate rather than ecdysozoan lineage
Authors: Stuart Gary W , Berry Michael W
Published: January 2005

Background

Eukaryotic whole genome sequences are accumulating at an impressive rate. Effective methods for comparing multiple whole eukaryotic genomes on a large scale are needed. Most attempted solutions involve the production of large scale alignments, and many of these require a high stringency pre-screen for putative orthologs in order to reduce the effective size of the dataset and provide a reasonably high but unknown fraction of correctly aligned homologous sites for comparison. As an alternative, highly efficient methods that do not require the pre-alignment of operationally defined orthologs are also being explored.

Results

A non-alignment method based on the Singular Value Decomposition (SVD) was used to compare the predicted protein complement of nine whole eukaryotic genomes ranging from yeast to man. This analysis resulted in the simultaneous identification and definition of a large number of well conserved motifs and gene families, and produced a species tree supporting one of two conflicting hypotheses of metazoan relationships.

Conclusions

Our SVD-based analysis of the entire protein complement of nine whole eukaryotic genomes suggests that highly conserved motifs and gene families can be identified and effectively compared in a single coherent definition space for the easy extraction of gene and species trees. While this occurs without the explicit definition of orthologs or homologous sites, the analysis can provide a basis for these definitions.
Predicting binding sites of hydrolase-inhibitor complexes by combining several methods
Authors: Sen Taner Z , Kloczkowski Andrzej , Jernigan Robert L , Yan Changhui , Honavar Vasant , Ho Kai-Ming , Wang Cai-Zhuang , Ihm Yungok , Cao Haibo , Gu Xun , Dobbs Drena
Published: January 2005

Background

Protein-protein interactions play a critical role in protein function. Completion of many genomes is being followed rapidly by major efforts to identify interacting protein pairs experimentally in order to decipher the networks of interacting, coordinated-in-action proteins. Identification of protein-protein interaction sites and detection of specific amino acids that contribute to the specificity and the strength of protein interactions is an important problem with broad applications ranging from rational drug design to the analysis of metabolic and signal transduction networks.

Results

In order to increase the power of predictive methods for protein-protein interaction sites, we have developed a consensus methodology for combining four different methods. These approaches include: data mining using Support Vector Machines, threading through protein structures, prediction of conserved residues on the protein surface by analysis of phylogenetic trees, and the Conservatism of Conservatism method of Mirny and Shakhnovich. Results obtained on a dataset of hydrolase-inhibitor complexes demonstrate that the combination of all four methods yield improved predictions over the individual methods.

Conclusions

We developed a consensus method for predicting protein-protein interface residues by combining sequence and structure-based methods. The success of our consensus approach suggests that similar methodologies can be developed to improve prediction accuracies for other bioinformatic problems.
A database for G proteins and their interaction with GPCRs
Authors: Elefsinioti Antigoni L , Bagos Pantelis G , Spyropoulos Ioannis C , Hamodrakas Stavros J
Published: January 2005

Background

G protein-coupled receptors (GPCRs) transduce signals from extracellular space into the cell, through their interaction with G proteins, which act as switches forming hetero-trimers composed of different subunits (α,β,γ). The α subunit of the G protein is responsible for the recognition of a given GPCR. Whereas specialised resources for GPCRs, and other groups of receptors, are already available, currently, there is no publicly available database focusing on G Proteins and containing information about their coupling specificity with their respective receptors.

Description

gpDB is a publicly accessible G proteins/GPCRs relational database. Including species homologs, the database contains detailed information for 418 G protein monomers (272 Gα, 87 Gβ and 59 Gγ) and 2782 GPCRs sequences belonging to families with known coupling to G proteins. The GPCRs and the G proteins are classified according to a hierarchy of different classes, families and sub-families, based on extensive literature searchs. The main innovation besides the classification of both G proteins and GPCRs is the relational model of the database, describing the known coupling specificity of the GPCRs to their respective α subunit of G proteins, a unique feature not available in any other database. There is full sequence information with cross-references to publicly available databases, references to the literature concerning the coupling specificity and the dimerization of GPCRs and the user may submit advanced queries for text search. Furthermore, we provide a pattern search tool, an interface for running BLAST against the database and interconnectivity with PRED-TMR, PRED-GPCR and TMRPres2D.

Conclusions

The database will be very useful, for both experimentalists and bioinformaticians, for the study of G protein/GPCR interactions and for future development of predictive algorithms. It is available for academics, via a web browser at the URL: http://bioinformatics.biol.uoa.gr/gpDB
A novel method for prokaryotic promoter prediction based on DNA stability
Authors: Kanhere Aditi , Bansal Manju
Published: January 2005

Background

In the post-genomic era, correct gene prediction has become one of the biggest challenges in genome annotation. Improved promoter prediction methods can be one step towards developing more reliable ab initio gene prediction methods. This work presents a novel prokaryotic promoter prediction method based on DNA stability.

Results

The promoter region is less stable and hence more prone to melting as compared to other genomic regions. Our analysis shows that a method of promoter prediction based on the differences in the stability of DNA sequences in the promoter and non-promoter region works much better compared to existing prokaryotic promoter prediction programs, which are based on sequence motif searches. At present the method works optimally for genomes such as that of Escherichia coli, which have near 50 % G+C composition and also performs satisfactorily in case of other prokaryotic promoters.

Conclusions

Our analysis clearly shows that the change in stability of DNA seems to provide a much better clue than usual sequence motifs, such as Pribnow box and -35 sequence, for differentiating promoter region from non-promoter regions. To a certain extent, it is more general and is likely to be applicable across organisms. Hence incorporation of such features in addition to the signature motifs can greatly improve the presently available promoter prediction programs.
Activation of α1A-adrenergic receptor promotes differentiation of rat-1 fibroblasts to a smooth muscle-like phenotype
Authors: Saeed Abdelwahab E , Parmentier Jean-Hugues , Malik Kafait U
Published: January 2005

Background

Fibroblasts, as connective tissue cells, are able to transform into another cell type including smooth muscle cells. α1A-adrenergic receptor (α1A-AR) stimulation in rat-1 fibroblasts is coupled to cAMP production. However, the significance of an increase in cAMP produced by α1A-AR stimulation on proliferation, hypertrophy and differentiation in these cells is not known.

Results

Activation of the α1A-AR in rat-1 fibroblasts by phenylephrine (PE) inhibited DNA synthesis by 67% and blocked the re-entry of 81% of the cells into S phase of the cell cycle. This cell cycle blockage was associated with hypertrophy characterized by an increase in protein synthesis (64%) and cell size. Elevation of cAMP levels decreased both DNA and protein synthesis. Inhibition of adenylyl cyclase or protein kinase A reversed the antiproliferative effect of cAMP analogs but not PE; the hypertrophic effect of PE was also not altered. The functional response of rat-1 cells to PE was accompanied by increased expression of cyclin-dependent kinase (Cdk) inhibitors p27kip1 and p21cip1/waf1, which function as negative regulators of the cell cycle. Stimulation of α1A-AR also upregulated the cell cycle regulatory proteins pRb, cyclin D1, Cdk 2, Cdk 4, and proliferating cell nuclear antigen. The antiproliferative effect of PE was blocked by p27kip1 antisense but not sense oligonucleotide. PE also promoted expression of smooth muscle cell differentiation markers (smooth muscle alpha actin, caldesmon, and myosin heavy chain) as well as the muscle development marker MyoD.

Conclusions

Stimulation of α1A-AR promotes cell cycle arrest, hypertrophy and differentiation of rat-1 fibroblasts into smooth muscle-like cells and expression of negative cell cycle regulators by a mechanism independent of the cAMP/PKA signaling pathway.
MAB21L2, a vertebrate member of the Male-abnormal 21 family, modulates BMP signaling and interacts with SMAD1
Authors: Baldessari Danila , Badaloni Aurora , Longhi Renato , Zappavigna Vincenzo , Consalez G Giacomo
Published: January 2005

Background

Through in vivo loss-of-function studies, vertebrate members of the Male abnormal 21 (mab-21) gene family have been implicated in gastrulation, neural tube formation and eye morphogenesis. Despite mounting evidence of their considerable importance in development, the biochemical properties and nature of MAB-21 proteins have remained strikingly elusive. In addition, genetic studies conducted in C. elegans have established that in double mutants mab-21 is epistatic to genes encoding various members of a Transforming Growth Factor beta (TGF-beta) signaling pathway involved in the formation of male-specific sensory organs.

Results

Through a gain-of-function approach, we analyze the interaction of Mab21l2 with a TGF-beta signaling pathway in early vertebrate development. We show that the vertebrate mab-21 homolog Mab21l2 antagonizes the effects of Bone Morphogenetic Protein 4 (BMP4) overexpression in vivo, rescuing the dorsal axis and restoring wild-type distribution of Chordin and Xvent2 transcripts in Xenopus gastrulae. We show that MAB21L2 immunoprecipitates in vivo with the BMP4 effector SMAD1, whilst in vitro it binds SMAD1 and the SMAD1-SMAD4 complex. Finally, when targeted to an heterologous promoter, MAB21L2 acts as a transcriptional repressor.

Conclusions

Our results provide the first biochemical and cellular foundation for future functional studies of mab-21 genes in normal neural development and its pathological disturbances.
Identification of cardiac malformations in mice lacking Ptdsrusing a novel high-throughput magnetic resonance imaging technique
Authors: Schneider Jürgen E , Böse Jens , Bamforth Simon D , Gruber Achim D , Broadbent Carol , Clarke Kieran , Neubauer Stefan , Lengeling Andreas , Bhattacharya Shoumo
Published: January 2005

Background

Congenital heart defects are the leading non-infectious cause of death in children. Genetic studies in the mouse have been crucial to uncover new genes and signaling pathways associated with heart development and congenital heart disease. The identification of murine models of congenital cardiac malformations in high-throughput mutagenesis screens and in gene-targeted models is hindered by the opacity of the mouse embryo.

Results

We developed and optimized a novel method for high-throughput multi-embryo magnetic resonance imaging (MRI). Using this approach we identified cardiac malformations in phosphatidylserine receptor (Ptdsr) deficient embryos. These included ventricular septal defects, double-outlet right ventricle, and hypoplasia of the pulmonary artery and thymus. These results indicate that Ptdsr plays a key role in cardiac development.

Conclusions

Our novel multi-embryo MRI technique enables high-throughput identification of murine models for human congenital cardiopulmonary malformations at high spatial resolution. The technique can be easily adapted for mouse mutagenesis screens and, thus provides an important new tool for identifying new mouse models for human congenital heart diseases.
The genetics of ray pattern variation in Caenorhabditis briggsae
Authors: Baird Scott Everet , Davidson Cynthia R , Bohrer Justin C
Published: January 2005

Background

How does intraspecific variation relate to macroevolutionary change in morphology? This question can be addressed in species in which derived characters are present but not fixed. In rhabditid nematodes, the arrangement of the nine bilateral pairs of peripheral sense organs (rays) in tails of males is often the most highly divergent character between species. The development of ray pattern involves inputs from hometic gene expression patterns, TGFβ signalling, Wnt signalling, and other genetic pathways. In Caenorhabditis briggsae, strain-specific variation in ray pattern has provided an entrée into the evolution of ray pattern. Some strains were fixed for a derived pattern. Other strains were more plastic and exhibited derived and ancestral patterns at equal frequencies.

Results

Recombinant inbred lines (RILs) constructed from crosses between the variant C. briggsae AF16 and HK104 strains exhibited a wide range of phenotypes including some that were more extreme than either parental strain. Transgressive segregation was significantly associated with allelic variation in the C. briggsae homolog of abdominal B, Cb-egl-5. At least two genes that affected different elements of ray pattern, ray position and ray fusion, were linked to a second gene, mip-1. Consistent with this, the segregation of ray position and ray fusion phenotypes were only partially correlated in the RILs.

Conclusions

The evolution of ray pattern has involved allelic variation at multiple loci. Some of these loci impact the specification of ray identities and simultaneously affect multiple ray pattern elements. Others impact individual characters and are not constrained by covariance with other ray pattern elements. Among the genetic pathways that may be involved in ray pattern evolution is specification of anteroposterior positional information by homeotic genes.
Climate change and recent genetic flux in populations of Drosophila robusta
Authors: Levitan Max , Etges William J
Published: January 2005

Background

Studied since the early 1940's, chromosomal polymorphisms in the deciduous woods species Drosophila robusta have been characterized by well-defined latitudinal, longitudinal, and elevational clines, but – until at least ten years ago – stable, local population frequencies. Recent biogeographical analyses indicate that D. robusta invaded North America from southeast Asia and has persisted in eastern temperate forests for at least 20–25 my without speciating. The abundant chromosome polymorphisms found across the range of D. robusta are thus likely to be relatively ancient, having accumulated over many well known climatic cycles in North America. Sufficient long-term data are now available such that we can now gauge the rate of these evolutionary changes in natural populations due to environmental change.

Results

Recent local collections have revealed significant changes in the frequencies of several chromosomal forms. New data presented here extend the range of these changes to six states, three in the northeastern United States and three west of the Mississippi River. These data reinforce recent directional changes in which the frequencies of three gene arrangements have reached percentage levels typical of distant southern populations consistent with regional climatic changes. Another gene arrangement has been steadily decreasing in frequency at a number of the sites studied. Meteorological records from 1945 to 2003 indicate temperature increases at all study sites, particularly average minimum air temperatures.

Conclusions

Observation of parallel genetic flux suggests that these long-term temporal frequency shifts in widely disparate populations of D. robusta are evolutionary responses to environmental change. Since these chromosomes are known to be sensitive to ambient temperature, regional climatic shifts associated with global warming are likely to be responsible.
The Prader-Willi syndrome murine imprinting center is not involved in the spatio-temporal transcriptional regulation of the Necdin gene
Authors: Watrin Françoise , Le Meur Elodie , Roeckel Nathalie , Ripoche Marie-Anne , Dandolo Luisa , Muscatelli Françoise
Published: January 2005

Background

The human Prader-Willi syndrome (PWS) domain and its mouse orthologue include a cluster of paternally expressed genes which imprinted expression is co-ordinately regulated by an imprinting center (IC) closely associated to the Snurf-Snrpn gene. Besides their co-regulated imprinted expression, two observations suggest that the spatio-temporal expression of these genes could also be co-regulated. First, the PWS genes have all been reported to be expressed in the mouse nervous system. Second, Snurf-Snrpn and its associated IC are the most ancient elements of the domain which later acquired additional functional genes by retrotransposition. Although located at least 1.5 megabases from the IC, these retroposons acquired the same imprinted regulation as Snurf-Snrpn. In this study, we ask whether the IC, in addition to its function in imprinting, could also be involved in the spatio-temporal regulation of genes in the PWS domain.

Results

We compared the expression pattern of Snurf-Snrpn and C/D-box small nucleolar RNAs (snoRNAs) MBII-85 and MBII-52 to the expression pattern of the two evolutionary related retroposons Ndn and Magel2, in the developing mouse embryo. We show that these genes have highly similar expression patterns in the central nervous system, suggesting that they share a common central nervous system-specific regulatory element. Among these genes, Ndn and Magel2 display the most similar expression patterns. Using transgenic mice containing the Ndn and Magel2 genes, we show that the transgenic Ndn gene whereas not imprinted is correctly expressed. Search for DNase I hypersensitive sites in the Ndn-Magel2 genomic region and comparative genomic analyses were performed in order to identify potential transcriptional cis-regulatory elements.

Conclusions

These results strongly suggest that paternally expressed genes of the PWS domain share a common central nervous system-specific regulatory element. We proposed that this regulatory element could co-localize with the IC. However, we demonstrate that the IC, if required for imprinted regulation, is not involved in the spatio-temporal regulation of distantly located retrotransposed genes such as the Ndn gene in the PWS domain.
Characterization of the chicken inward rectifier K+ channel IRK1/Kir2.1 gene
Authors: Mutai Hideki , Kenyon Lawrence C , Locke Emily , Kikuchi Nami , Oberholtzer John Carl
Published: January 2005

Background

Inward rectifier potassium channels (IRK) contribute to the normal function of skeletal and cardiac muscle cells. The chick inward rectifier K+ channel cIRK1/Kir2.1 is expressed in skeletal muscle, heart, brain, but not in liver; a distribution similar but not identical to that of mouse Kir2.1. We set out to explore regulatory domains of the cIRK1 promoter that enhance or inhibit expression of the gene in different cell types.

Results

We cloned and characterized the 5'-flanking region of cIRK1. cIRK1 contains two exons with splice sites in the 5'-untranslated region, a structure similar to mouse and human orthologs. cIRK1 has multiple transcription initiation sites, a feature also seen in mouse. However, while the chicken and mouse promoter regions share many regulatory motifs, cIRK1 possesses a GC-richer promoter and a putative TATA box, which appears to positively regulate gene expression. We report here the identification of several candidate cell/tissue specific cIRK1 regulatory domains by comparing promoter activities in expressing (Qm7) and non-expressing (DF1) cells using in vitro transcription assays.

Conclusion

While multiple transcription initiation sites and the combinatorial function of several domains in activating cIRK1 expression are similar to those seen in mKir2.1, the cIRK1 promoter differs by the presence of a putative TATA box. In addition, several domains that regulate the gene's expression differentially in muscle (Qm7) and fibroblast cells (DF1) were identified. These results provide fundamental data to analyze cIRK1 transcriptional mechanisms. The control elements identified here may provide clues to the tissue-specific expression of this K+ channel.
16S rRNA gene based analysis of Enterobacter sakazakii strains from different sources and development of a PCR assay for identification
Authors: Lehner Angelika , Tasara Taurai , Stephan Roger
Published: January 2005

Background

E. sakazakii is considered to be an opportunistic pathogen, implicated in food borne diseases causing meningitis or enteritis especially in neonates and infants. Cultural standard identification procedures for E. sakazakii include the observation of yellow pigmentation of colonies and a positive "Equation missing"glucosidase activity. Up to now, only one PCR system based on a single available 16S rRNA gene sequence has been published for E. sakazakii identification. However, in our hands a preliminary evaluation of this system to a number of target and non-target strains showed significant specificity problems of this system. In this study full-length 16S rRNA genes of thirteen E. sakazakii strains from food, environment and human origin as well as the type strain ATCC 51329 were sequenced. Based on this sequence data a new specific PCR system for E. sakazakii was developed and evaluated.

Results

By phylogenetic analysis of the new full-length 16S rRNA gene sequence data obtained we could show the presence of a second phylogenetic distinct lineage within the E. sakazakii species. The newly developed 16S rRNA gene targeting PCR system allows identification of E. sakazakii strains from both lineages. The assay's ability to correctly identify different E. sakazakii isolates as well as to differentiate E. sakazakii from other closely related Enterobacteriaceae species and other microorganisms was shown on 75 target and non-target strains.

Conclusion

By this study we are presenting a specific and reliable PCR identification system, which is able to correctly identify E. sakazakii isolates from both phylogenetic distinct lines within the E. sakazakii species. The impact of this second newly described phylogenetic line within the E. sakazakii species in view of clinical and food safety aspects need further investigation.
A genomic island present along the bacterial chromosome of the Parachlamydiaceae UWE25, an obligate amoebal endosymbiont, encodes a potentially functional F-like conjugative DNA transfer system
Authors: Greub Gilbert , Collyn François , Guy Lionel , Roten Claude-Alain
Published: January 2005

Background

The genome of Protochlamydia amoebophila UWE25, a Parachlamydia-related endosymbiont of free-living amoebae, was recently published, providing the opportunity to search for genomic islands (GIs).

Results

On the residual cumulative G+C content curve, a G+C-rich 19-kb region was observed. This sequence is part of a 100-kb chromosome region, containing 100 highly co-oriented ORFs, flanked by two 17-bp direct repeats. Two identical gly-tRNA genes in tandem are present at the proximal end of this genetic element. Several mobility genes encoding transposases and bacteriophage-related proteins are located within this chromosome region. Thus, this region largely fulfills the criteria of GIs. The G+C content analysis shows that several modules compose this GI. Surprisingly, one of them encodes all genes essential for F-like conjugative DNA transfer (traF, traG, traH, traN, traU, traW, and trbC), involved in sex pilus retraction and mating pair stabilization, strongly suggesting that, similarly to the other F-like operons, the parachlamydial tra unit is devoted to DNA transfer. A close relatedness of this tra unit to F-like tra operons involved in conjugative transfer is confirmed by phylogenetic analyses performed on concatenated genes and gene order conservation. These analyses and that of gly-tRNA distribution in 140 GIs suggest a proteobacterial origin of the parachlamydial tra unit.

Conclusions

A GI of the UWE25 chromosome encodes a potentially functional F-like DNA conjugative system. This is the first hint of a putative conjugative system in chlamydiae. Conjugation most probably occurs within free-living amoebae, that may contain hundreds of Parachlamydia bacteria tightly packed in vacuoles. Such a conjugative system might be involved in DNA transfer between internalized bacteria. Since this system is absent from the sequenced genomes of Chlamydiaceae, we hypothesize that it was acquired after the divergence between Parachlamydiaceae and Chlamydiaceae, when the Parachlamydia-related symbiont was an intracellular bacteria. It suggests that this heterologous DNA was acquired from a phylogenetically-distant bacteria sharing an amoebal vacuole. Since Parachlamydiaceae are emerging agents of pneumonia, this GI might be involved in pathogenicity. In future, conjugative systems might be developed as genetic tools for Chlamydiales.
Extended-Spectrum β-lactamase (ESBL) producing Enterobacter aerogenes phenotypically misidentified as Klebsiella pneumoniae or K. terrigena
Authors: Claeys Geert , De Baere Thierry , Wauters Georges , Vandecandelaere Patricia , Verschraegen Gerda , Muylaert An , Vaneechoutte Mario
Published: January 2005

Background

Enterobacter aerogenes and Klebsiella pneumoniae are common isolates in clinical microbiology and important as producers of extended spectrum β-lactamases (ESBL). The discrimination between both species, which is routinely based on biochemical characteristics, is generally accepted to be straightforward. Here we report that genotypically unrelated strains of E. aerogenes can be misidentified as K. pneumoniae by routine laboratories using standard biochemical identification and using identification automates.

Results

Ten clinical isolates, identified as K. pneumoniae or K. terrigena with the routinely used biochemical tests and with API-20E, were identified as E. aerogenes by tDNA-PCR – an identification that was confirmed by 16S rRNA gene sequencing for five of these isolates. Misidentification also occurred when using the automated identification systems Vitek 2 and Phoenix, and was due to delayed positivity for ornithine decarboxylase and motility. Subculture and prolonged incubation resulted in positive results for ornithine decarboxylase and for motility. It could be shown by RAPD-analysis that the E. aerogenes strains belonged to different genotypes.

Conclusions

Clinical E. aerogenes isolates can be easily misidentified as Klebsiella due to delayed positivity for ornithine decarboxylase and motility. The phenomenon may be widespread, since it was shown to occur among genotypically unrelated strains from different hospitals and different isolation dates. A useful clue for correct identification is the presence of an inducible β-lactamase, which is highly unusual for K. pneumoniae. In several instances, the use of genotypic techniques like tDNA-PCR may circumvent problems of phenotypic identification.
Antitumor effects of two bisdioxopiperazines against two experimental lung cancer models in vivo
Authors: Lu Da-Yong , Xu Bin , Ding Jian
Published: January 2005

Background

Probimane (Pro), an anti-cancer agent originating in China, was derived from razoxane (ICRF-159, Raz), a drug created in Britain, specifically targeting at cancer metastasis and as a cardioprotectant of anthrocyclines. Pro and Raz are bisdioxopiperazine compounds. In this work, we evaluated the anti-tumor and anti-metastatic effects of Pro and Raz in vivo against two lung tumor models, one of murine origin (Lewis lung carcinoma, LLC) and one of human origin (LAX-83).

Results

After determining the lethal dosage of Pro and Raz, we assessed and compared the inhibitory effects of Pro and Raz against primary tumor growth and metastatic occurrences of LLC at the dosage of LD5. Pro and Raz were active against primary tumor growth and significantly inhibited pulmonary metastasis of LLC at same dose-ranges (inhibitory rates > 90 %). Both Raz and Pro were effective in 1, 5, and 9 day administration schedules. Three different schedules of Raz and Pro were effective against the primary tumor growth of LLC (35–50 %). The synergistic anticancer effect of Raz with bleomycin (Ble) (from 41.3 % to 73.3 %) was more obvious than those with daunorubicin (Dau) (from 33.1 % to 56.3 %) in the LLC tumor model. Pro was also seen to have synergistic anti-cancer effects with Ble in the LLC model. Both Raz and Pro inhibited the growth of LAX 83 in a statistically significant manner.

Conclusions

These data suggest that both Raz and Pro may have anti-tumor potentiality and Raz and Pro have combinative effects with Ble or Dau. The potential targets of bisdioxopiperazines may include lung cancers, especially on tumor metastasis. The anti-cancer effects of Raz and Pro can be increased with the help of other anticancer drugs.
Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from Coleus forskohliiBriq
Authors: Engprasert Surang , Taura Futoshi , Kawamukai Makoto , Shoyama Yukihiro
Published: January 2005

Background

Isopentenyl diphosphate (IPP), a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP) synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase gene in Coleus forskohlii Briq.

Results

The open reading frame for full-length GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of C. forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases. Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of C. forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in Escherichia coli harboring pACCAR25ΔcrtE from Erwinia uredovora and plasmid carrying C. forskohlii GGPP synthase. These results suggested that cDNA encoded functional GGPP synthase. Furthermore, C. forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots.

Conclusion

This investigation proposed that forskolin was synthesised via a non-mevalonate pathway. GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots.
Endotracheal tube cuff pressure in three hospitals, and the volume required to produce an appropriate cuff pressure
Authors: Sengupta Papiya , Sessler Daniel I , Maglinger Paul , Wells Spencer , Vogt Alicia , Durrani Jaleel , Wadhwa Anupama
Published: January 2005

Background

Cuff pressure in endotracheal (ET) tubes should be in the range of 20–30 cm H2O. We tested the hypothesis that the tube cuff is inadequately inflated when manometers are not used.

Methods

With IRB approval, we studied 93 patients under general anesthesia with an ET tube in place in one teaching and two private hospitals. Anesthetists were blinded to study purpose. Cuff pressure in tube sizes 7.0 to 8.5 mm was evaluated 60 min after induction of general anesthesia using a manometer connected to the cuff pilot balloon. Nitrous oxide was disallowed. After deflating the cuff, we reinflated it in 0.5-ml increments until pressure was 20 cmH2O.

Results

Neither patient morphometrics, institution, experience of anesthesia provider, nor tube size influenced measured cuff pressure (35.3 ± 21.6 cmH2O). Only 27% of pressures were within 20–30 cmH2O; 27% exceeded 40 cmH2O. Although it varied considerably, the amount of air required to achieve a cuff pressure of 20 cmH2O was similar with each tube size.

Conclusion

We recommend that ET cuff pressure be set and monitored with a manometer.
A suite of web applications to streamline the interdisciplinary collaboration in secondary data analyses
Authors: Pietrobon Ricardo , Guller Ulrich , Martins Henrique , Menezes Andreia P , Higgins Laurence D , Jacobs Danny O
Published: January 2005

Background

We describe a system of web applications designed to streamline the interdisciplinary collaboration in outcomes research.

Description

The outcomes research process can be described as a set of three interrelated phases: design and selection of data sources, analysis, and output. Each of these phases has inherent challenges that can be addressed by a group of five web applications developed by our group. QuestForm allows for the formulation of relevant and well-structured outcomes research questions; Research Manager facilitates the project management and electronic file exchange among researchers; Analysis Charts facilitate the communication of complex statistical techniques to clinicians with varying previous levels of statistical knowledge; Literature Matrices improve the efficiency of literature reviews. An outcomes research question is used to illustrate the use of the system.

Conclusions

The system presents an alternative to streamline the interdisciplinary collaboration of clinicians, statisticians, programmers, and graduate students.