bluuebunny/crossref_metadata_2025_split huggingface dataset:
Presence of Two Different Active
nirS
Nitrite Reductase Genes in a Denitrifying
Thauera
sp. from a High-Nitrate-Removal-Rate Reactor
Authors: Etchebehere Claudia , Tiedje James 
Published: September 2005

ABSTRACT

The
nirS
nitrite reductase genes were studied in two strains (strains 27 and 28) isolated from two denitrifying reactors and characterized as
Thauera
according to their 16S rRNA gene sequences. Strain 28 contains a single
nirS
sequence, which is related to the
nirS
of
Thauera mechernichensis
, and strain 27 contains two
nirS
sequences; one is similar to the
nirS
sequence from
Thauera mechernichensis
(gene 2), but the second one (gene 8) is from a separate clade with
nirS
from
Pseudomonas stutzeri
,
Azoarcus
species,
Alcaligenes faecalis
, and other
Thauera
species. Both genes were expressed, but gene 8 was constitutively expressed while gene 2 was positively regulated by nitrate.
Dual Luciferase Assay System for Rapid Assessment of Gene Expression in
Saccharomyces cerevisiae
Authors: McNabb David S. , Reed Robin , Marciniak Robert A. 
Published: September 2005

ABSTRACT

A new reporter system has been developed for quantifying gene expression in the yeast
Saccharomyces cerevisiae
. The system relies on two different reporter genes,
Renilla
and firefly luciferase, to evaluate regulated gene expression. The gene encoding
Renilla
luciferase is fused to a constitutive promoter (
PGK1
or
SPT15
) and integrated into the yeast genome at the
CAN1
locus as a control for normalizing the assay. The firefly luciferase gene is fused to the test promoter and integrated into the yeast genome at the
ura3
or
leu2
locus. The dual luciferase assay is performed by sequentially measuring the firefly and
Renilla
luciferase activities of the same sample, with the results expressed as the ratio of firefly to
Renilla
luciferase activity (Fluc/Rluc). The yeast dual luciferase reporter (DLR) was characterized and shown to be very efficient, requiring approximately 1 minute to complete each assay, and has proven to yield data that accurately and reproducibly reflect promoter activity. A series of integrating plasmids were generated that contain either the firefly or
Renilla
luciferase gene preceded by a multicloning region in two different orientations and the three reading frames to make possible the generation of translational fusions. Additionally, each set of plasmids contains either the
URA3
or
LEU2
marker for genetic selection in yeast. A series of S288C-based yeast strains, including a two-hybrid strain, were developed to facilitate the use of the yeast DLR assay. This assay can be readily adapted to a high-throughput platform for studies requiring numerous measurements.
Functional Conservation and Specialization among Eukaryotic Anti-Silencing Function 1 Histone Chaperones
Authors: Tamburini Beth A. , Carson Joshua J. , Adkins Melissa W. , Tyler Jessica K. 
Published: September 2005

ABSTRACT

Chromatin disassembly and reassembly, mediated by histone chaperones such as anti-silencing function 1 (Asf1), are likely to accompany all nuclear processes that occur on the DNA template. In order to gain insight into the functional conservation of Asf1 across eukaryotes, we have replaced the budding yeast Asf1 protein with
Drosophila
Asf1 (dAsf1) or either of the two human Asf1 (hAsf1a and hAsf1b) counterparts. We found that hAsf1b is best able to rescue the growth defect of
Saccharomyces cerevisiae
lacking Asf1. Moreover, dAsf1 and hAsf1b but not hAsf1a can replace the role of yeast Asf1 in protecting against replicational stress and activating the
PHO5
gene, while only hAsf1a can replace the role of Asf1 in protecting against double-stranded-DNA-damaging agents. Furthermore, it appears that the interaction between Asf1 and the DNA damage checkpoint protein Rad53 is not required for Asf1's role in maintaining genomic integrity. In addition to indicating the functional conservation of the Asf1 proteins across species, these studies suggest distinct roles for the two human Asf1 proteins.
Transcript Profiling Reveals Novel Marker Genes Involved in Fruiting Body Formation in
Tuber borchii
Authors: Gabella Silvia , Abbà Simona , Duplessis Sebastien , Montanini Barbara , Martin Francis , Bonfante Paola 
Published: September 2005

ABSTRACT

cDNA arrays were used to explore mechanisms controlling fruiting body development in the truffle
Tuber borchii.
Differences in gene expression were higher between reproductive and vegetative stage than between two stages of fruiting body maturation. We suggest hypotheses about the importance of various physiological processes during the development of fruiting bodies.
The H2O2Stress-Responsive Regulator PerR Positively RegulatessrfAExpression inBacillus subtilis
Authors: Hayashi Kentaro , Ohsawa Taku , Kobayashi Kazuo , Ogasawara Naotake , Ogura Mitsuo 
Published: September 2005

ABSTRACTsrfAis an operon required for the synthesis of surfactin and the development of genetic competence inBacillus subtilis.We observed that the expression ofsrfAis downregulated upon treatment with H2O2. Thus, we examined the involvement of several oxidative stress-responsive transcription factors insrfAexpression. Our DNA microarray analysis revealed that the H2O2stress-responsive regulator PerR is required forsrfAexpression. This was confirmed bylacZfusion analysis. A ComX feeding assay and epistatic analyses revealed that the role of PerR insrfAexpression is independent of other known regulators ofsrfAexpression, namely,comQXP,rapC, andspx. Gel mobility shift and footprint assays revealed that PerR binds directly to two tandemly arranged noncanonical PerR boxes located in the upstream promoter region ofsrfA. A transcriptionalsrfA-lacZfusion lacking both PerR boxes showed diminished and PerR-independent expression, indicating that the PerR boxes we identified function as positiveciselements forsrfAtranscription.
Mutational Analysis of
Escherichia coli
Heat Shock Transcription Factor Sigma 32 Reveals Similarities with Sigma 70 in Recognition of the −35 Promoter Element and Differences in Promoter DNA Melting and −10 Recognition
Authors: Kourennaia Olga V. , Tsujikawa Laura , deHaseth Pieter L. 
Published: September 2005

ABSTRACT

Upon the exposure of
Escherichia coli
to high temperature (heat shock), cellular levels of the transcription factor σ
32
rise greatly, resulting in the increased formation of the σ
32
holoenzyme, which is capable of transcription initiation at heat shock promoters. Higher levels of heat shock proteins render the cell better able to cope with the effects of higher temperatures. To conduct structure-function studies on σ
32
in vivo, we have carried out site-directed mutagenesis and employed a previously developed system involving σ
32
expression from one plasmid and a β-galactosidase reporter gene driven by the σ
32
-dependent
groE
promoter on another in order to monitor the effects of single amino acid substitutions on σ
32
activity. It was found that the recognition of the −35 region involves similar amino acid residues in regions 4.2 of
E. coli
σ
32
and σ
70
. Three conserved amino acids in region 2.3 of σ
32
were found to be only marginally important in determining activity in vivo. Differences between σ
32
and σ
70
in the effects of mutation in region 2.4 on the activities of the two sigma factors are consistent with the pronounced differences between both the amino acid sequences in this region and the recognized promoter DNA sequences.
Mutation at the “Exit Gate” of the
Salmonella
Gyrase A Subunit Suppresses a Defect in the Gyrase B Subunit
Authors: Blanc-Potard Anne-Béatrice , Labesse Gilles , Figueroa-Bossi Nara , Bossi Lionello 
Published: September 2005

ABSTRACT

In
Salmonella enterica
serovar Typhimurium, an S431P substitution in the B subunit of gyrase (allele
gyrB651
) confers resistance to nalidixic acid and causes reduced DNA superhelicity and hypersensitivity to novobiocin. Selection for novobiocin resistance allowed isolation of a mutation in the
gyrA
gene (allele
gyrA659
), a T467S substitution, which partially suppresses the supercoiling defect of
gyrB651
. Modeling analysis suggests that this mutation acts by destabilizing the GyrA bottom dimer interface. This is the first example of a
gyrA
mutation that compensates for a
gyrB
defect.
RasA Is Required for
Myxococcus xanthus
Development and Social Motility
Authors: Pham Vinh D. , Shebelut Conrad W. , Mukherjee Bipasha , Singer Mitchell 
Published: September 2005

ABSTRACT

An insertion in the
rasA
gene entirely blocked developmental aggregation and sporulation in
Myxococcus xanthus
while also reducing swarm expansion on a 0.3% agar surface. Data presented here demonstrate that
rasA
is required for extracellular fibril formation and social gliding motility.
A bootstrap method to avoid the effect of concurvity in generalised additive models in time series studies of air pollution
Authors: Figueiras Adolfo , Roca-Pardiñas Javier , Cadarso-Suárez Carmen 
Published: September 2005

Background:In recent years a great number of studies have applied generalised additive models (GAMs) to time series data to estimate the short term health effects of air pollution. Lately, however, it has been found that concurvity—the non-parametric analogue of multicollinearity—might lead to underestimation of standard errors of the effects of independent variables. Underestimation of standard errors means that for concurvity levels commonly present in the data, the risk of committing type I error rises by over threefold.Methods:This study developed a conditional bootstrap methology that consists of assuming that the outcome in any observation is conditional upon the values of the set of independent variables used. It then tested this procedure by means of a simulation study using a Poisson additive model. The response variable of this model is a function of an unobserved confounding variable (that introduces trend and seasonality), real black smoke data, and temperature. Scenarios were created with different coefficients and degrees of concurvity.Results:Conditional bootstrap provides confidence intervals with coverages close to nominal (95%), irrespective of the degree of concurvity, number of variables in the model or magnitude of the coefficient to be estimated (for example, for a concurvity of 0.85, bootstrap confidence interval coverage is 95% compared with 71% in the case of the asymptotic interval obtained directly with S-plus gam function).Conclusions:The bootstrap method avoids the problem of concurvity in time series studies of air pollution, and is easily generalised to non-linear dose-risk effects. All bootstrap calculations described in this paper can be performed using S-Plus gam.boot software.
A Quantitative Measurement of the Human Somatic Mutation Rate
Authors: Araten David J. , Golde David W. , Zhang Rong H. , Thaler Howard T. , Gargiulo Lucia , Notaro Rosario , Luzzatto Lucio 
Published: September 2005

Abstract
The mutation rate (μ) is a key biological feature of somatic cells that determines risk for malignant transformation, and it has been exceedingly difficult to measure in human cells. For this purpose, a potential sentinel is the X-linked PIG-A gene, because its inactivation causes lack of glycosylphosphatidylinositol-linked membrane proteins. We previously found that the frequency (f) of PIG-A mutant cells can be measured accurately by flow cytometry, even when f is very low. Here we measure both f and μ by culturing B-lymphoblastoid cell lines and first eliminating preexisting PIG-A mutants by flow sorting. After expansion in culture, the frequency of new mutants is determined by flow cytometry using antibodies specific for glycosylphosphatidylinositol-linked proteins (e.g., CD48, CD55, and CD59). The mutation rate is then calculated by the formula μ = f/d, where d is the number of cell divisions occurring in culture. The mean μ in cells from normal donors was 10.6 × 10−7 mutations per cell division (range 2.4 to 29.6 × 10−7). The mean μ was elevated &gt;30-fold in cells from patients with Fanconi anemia (P &lt; 0.0001), and μ varied widely in ataxia-telangiectasia with a mean 4-fold elevation (P = 0.002). In contrast, μ was not significantly different from normal in cells from patients with Nijmegen breakage syndrome. Differences in μ could not be attributed to variations in plating efficiency. The mutation rate in man can now be measured routinely in B-lymphoblastoid cell lines, and it is elevated in cancer predisposition syndromes. This system should be useful in evaluating cancer risk and in the design of preventive strategies.
Expression Profiling of Homocysteine Junction Enzymes in the NCI60 Panel of Human Cancer Cell Lines
Authors: Zhang Wen , Braun Aaron , Bauman Zachary , Olteanu Horatiu , Madzelan Peter , Banerjee Ruma 
Published: September 2005

Abstract
Methionine metabolism provides two key cellular reagents: S-adenosylmethionine and glutathione, derived from the common intermediate, homocysteine. A majority of cancer cells exhibit a methionine-dependent phenotype whereby they are unable to grow in medium in which methionine is replaced by its precursor, homocysteine. Additionally, CpG island hypermethylation of tumor suppressor gene promoters is observed in a background of global hypomethylation in cancerous cells. In this study, we have profiled the expression levels of the homocysteine junction enzymes, methionine synthase (MS), MS reductase (MSR), and cystathionine β-synthase (CBS) in the NCI60 panel of cancer cell lines. The doubling time of non–small lung cell cancer lines, which exhibit the lowest levels of MS within the panel, was significantly correlated with expression of MS. The ratio of MS to MSR varied over a 5-fold range in the different cell types, which may modulate methionine synthesis. Interestingly, markedly reduced CBS expression was seen in the methionine-dependent prostate cancer cell line, PC-3, but not in the methionine-independent cell line, DU-145. However, neither provision of the transsulfuration pathway product, cysteine, nor overexpression of CBS rescued the growth impairment, indicating that reduced CBS was not responsible for the methionine-dependent phenotype in this cell line.
Trimodal Cancer Treatment: Beneficial Effects of Combined Antiangiogenesis, Radiation, and Chemotherapy
Authors: Huber Peter E. , Bischof Marc , Jenne Jürgen , Heiland Sabine , Peschke Peter , Saffrich Rainer , Gröne Hermann-Josef , Debus Jürgen , Lipson Kenneth E. , Abdollahi Amir 
Published: September 2005

Abstract
It has been suggested that chemotherapy and radiotherapy could favorably be combined with antiangiogenesis in dual anticancer strategy combinations. Here we investigate the effects of a trimodal strategy consisting of all three therapy approaches administered concurrently. We found that in vitro and in vivo, the antiendothelial and antitumor effects of the triple therapy combination consisting of SU11657 (a multitargeted small molecule inhibitor of vascular endothelial growth factor and platelet-derived growth factor receptor tyrosine kinases), Pemetrexed (a multitargeted folate antimetabolite), and ionizing radiation were superior to all single and dual combinations. The superior effects in human umbilical vein endothelial cells and tumor cells (A431) were evident in cell proliferation, migration, tube formation, clonogenic survival, and apoptosis assays (sub-G1 and caspase-3 assessment). Exploring potential effects on cell survival signaling, we found that radiation and chemotherapy induced endothelial cell Akt phosphorylation, but SU11657 could attenuate this process in vitro and in vivo in A431 human tumor xenografts growing s.c. on BALB/c nu/nu mice. Triple therapy further decreased tumor cell proliferation (Ki-67 index) and vessel count (CD31 staining), and induced greater tumor growth delay versus all other therapy regimens without increasing apparent toxicity. When testing different treatment schedules for the A431 tumor, we found that the regimen with radiotherapy (7.5 Gy single dose), given after the institution of SU11657 treatment, was more effective than radiotherapy preceding SU11657 treatment. Accordingly, we found that SU11657 markedly reduced intratumoral interstitial fluid pressure from 8.8 ± 2.6 to 4.2 ± 1.5 mm Hg after 1 day. Likewise, quantitative T2-weighed magnetic resonance imaging measurements showed that SU11657-treated mice had reduced intratumoral edema. Our data indicates that inhibition of Akt signaling by antiangiogenic treatment with SU11657 may result in: (a) normalization of tumor blood vessels that cause prerequisite physiologic conditions for subsequent radio/chemotherapy, and (b) direct resensitization of endothelial cells to radio/chemotherapy. We conclude that trimodal cancer therapy combining antiangiogenesis, chemotherapy, and radiotherapy has beneficial molecular and physiologic effects to emerge as a clinically relevant antitumor strategy.
Reversible Conversion of Immortal Human Cells from Telomerase-Positive to Telomerase-Negative Cells
Authors: Kumakura Shin-ichi , Tsutsui Takeo W. , Yagisawa Junko , Barrett J. Carl , Tsutsui Takeki 
Published: September 2005

Abstract
Immortal cell lines and tumors maintain their telomeres via the telomerase pathway or via a telomerase-independent pathway, referred to as alternative lengthening of telomeres (ALT). Here, we show the reversible conversion of the human papillomavirus type 16 E6-induced immortal human fibroblasts E6 Cl 6 from telomerase-positive (Tel+) to telomerase-negative (Tel−) cells. Tel+ cells converted spontaneously to Tel− cells that reverted to Tel+ cells following treatment with trichostatin A (TSA) and/or 5-aza-2′-deoxycytidine (5-AZC), which induced the reversion from complete to partial methylation of the CpG islands of the human telomerase reverse transcriptase (hTERT) promoter in Tel− E6 Cl 6 cells. Tel− E6 Cl 6 cells lacked the phenotypes characteristic of ALT cell lines such as very long and heterogenous telomeres and ALT-associated promyelocytic leukemia nuclear bodies (APB) but grew for &gt;240 population doublings (PD) after they became telomerase negative. The ratios of histone H3 (H3) lysine (K) 9 methylation to each of H3-K4 methylation, H3-K9 acetylation, and H3-K14 acetylation of the chromatin containing the hTERT promoter in Tel− E6 Cl 6 cells and ALT cell lines were greater than those in Tel+ cells and decreased following treatment with TSA and/or 5-AZC, inversely corresponding to telomerase activity. Our findings suggest the possibility that human tumors may be able to reversibly interconvert their telomere maintenance phenotypes by chromatin structure-mediated regulation of hTERT expression.
Oncogenic K-RAS Is Required to Maintain Changes in Cytoskeletal Organization, Adhesion, and Motility in Colon Cancer Cells
Authors: Pollock Claire B. , Shirasawa Senji , Sasazuki Takehiko , Kolch Walter , Dhillon Amardeep S. 
Published: September 2005

Abstract
RAS oncogenes are thought to play a role at multiple stages of tumorigenesis. The role and mechanisms by which RAS oncogenes maintain the transformed state of human cancer cells are poorly understood. Here, we have studied the role of oncogenic K-RAS in maintaining cytoskeletal disruption, cell adhesion and motility in metastatic colon carcinoma cells. Targeted deletion of K-RASG13D from HCT116 colon carcinoma cells restored their ability to assemble stress fibers and focal adhesions/complexes, accompanied by increased cell-matrix adhesion and reduced motility. We further show that oncogenic K-Ras induces high Rho activity, but uncouples Rho from stress fiber formation. This uncoupling required the maintenance of high levels of the activator protein-1 family member, Fra-1, via a mitogen-activated protein/extracellular signal-regulated kinase–dependent pathway. We also show that PI3-kinase signaling is required for the motility of HCT116 cells downstream of oncogenic K-Ras. Our findings suggest that mutated K-RAS oncogenes are essential for maintenance of the transformed and invasive phenotype of human colon cancer cells.
Circumventing Multidrug Resistance in Cancer by β-Galactoside Binding Protein, an Antiproliferative Cytokine
Authors: Ravatn Roald , Wells Valerie , Nelson Leigh , Vettori David , Mallucci Livio , Chin Khew-Voon 
Published: September 2005

Abstract
We report here that β-galactoside binding protein (βGBP), an antiproliferative cytokine which can program cancer cells to undergo apoptosis, exhibits equal therapeutic efficacy against cancer cells that display diverse mechanisms of drug resistance and against their parental cells. The mechanisms of drug resistance in the cancer cells that we have examined include overexpression of P-glycoprotein, increased efficiency of DNA repair, and altered expression and mutation in the topoisomerase I and II enzymes. We also report that βGBP exerted its effect by arresting the cells in S phase prior to the activation of programmed cell death. The uniquely similar profile of response to βGBP by these drug-resistant cells and their parental cells extends the therapeutic potential of this cytokine in the treatment of cancers and offers a promising alternative to patients whose tumors are refractory to the currently available cadre of chemotherapeutic agents.
Supra-additive Growth Inhibition by a Celecoxib Analogue and Carboxyamido-triazole Is Primarily Mediated through Apoptosis
Authors: Winters Mary E. , Mehta Arpita I. , Petricoin Emanuel F. , Kohn Elise C. , Liotta Lance A. 
Published: September 2005

Abstract
Combination studies of celecoxib and chemotherapeutic agents suggest that combining cyclooxygenase-2 inhibitors with other agents may have supra-additive or synergistic effects on tumor growth inhibition. Carboxyamido-triazole (CAI), a voltage-independent calcium channel inhibitor, has been shown to induce growth inhibition and apoptosis in cancer cells. We found that continuous exposure to cytostatic doses of CAI and LM-1685, a celecoxib analogue, reduced the proliferation and survival of seven human cancer cell lines by at least one log (P ≤ 0.001) over either agent alone. To explore the mechanism of action of this combination, we further studied the effects of LM-1685/CAI on CCL-250 colorectal carcinoma cells. We found that the supra-additive antiproliferative effects occurred throughout a range of LM-1685 doses (5-25 μmol/L) and paralleled a decrease in COX-2 activity as measured by prostaglandin E2 production. In these cells, treatment with LM-1685/CAI suppressed the extracellular signal-regulated kinase pathway within the first hour but ultimately results in high, sustained activation of ERK over a 9-day period (P = 0.0005). Suppression of cyclin D1 and phospho-AKT, and cleavage of caspase-3 and PARP were concomitant with persistent ERK activation. Addition of PD98059, a MEK-1 inhibitor, suppressed ERK activation and significantly but incompletely reversed these signaling events and apoptosis. Flow cytometry experiments revealed that the CAI/LM-1685 combination induced a 3-fold increase in apoptosis over control (P = 0.005) in 3 days. We show that the combination of CAI and LM-1685 produces a cytotoxic effect by suppressing proliferation and triggering apoptosis.