bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Solar UV Radiation on Morphology and Photosynthesis of Filamentous Cyanobacterium
Arthrospira platensis
Authors: Wu Hongyan , Gao Kunshan , Villafañe Virginia E. , Watanabe Teruo , Helbling E. Walter
Published: September 2005

To study the impact of solar UV radiation (UVR) (280 to 400 nm) on the filamentous cyanobacterium
Arthrospira
(
Spirulina
)
platensis
, we examined the morphological changes and photosynthetic performance using an indoor-grown strain (which had not been exposed to sunlight for decades) and an outdoor-grown strain (which had been grown under sunlight for decades) while they were cultured with three solar radiation treatments: PAB (photosynthetically active radiation [PAR] plus UVR; 280 to 700 nm), PA (PAR plus UV-A; 320 to 700 nm), and P (PAR only; 400 to 700 nm). Solar UVR broke the spiral filaments of
A. platensis
exposed to full solar radiation in short-term low-cell-density cultures. This breakage was observed after 2 h for the indoor strain but after 4 to 6 h for the outdoor strain. Filament breakage also occurred in the cultures exposed to PAR alone; however, the extent of breakage was less than that observed for filaments exposed to full solar radiation. The spiral filaments broke and compressed when high-cell-density cultures were exposed to full solar radiation during long-term experiments. When UV-B was screened off, the filaments initially broke, but they elongated and became loosely arranged later (i.e., there were fewer spirals per unit of filament length). When UVR was filtered out, the spiral structure hardly broke or became looser. Photosynthetic O
2
evolution in the presence of UVR was significantly suppressed in the indoor strain compared to the outdoor strain. UVR-induced inhibition increased with exposure time, and it was significantly lower in the outdoor strain. The concentration of UV-absorbing compounds was low in both strains, and there was no significant change in the amount regardless of the radiation treatment, suggesting that these compounds were not effectively used as protection against solar UVR. Self-shading, on the other hand, produced by compression of the spirals over adaptive time scales, seems to play an important role in protecting this species against deleterious UVR. Our findings suggest that the increase in UV-B irradiance due to ozone depletion not only might affect photosynthesis but also might alter the morphological development of filamentous cyanobacteria during acclimation or over adaptive time scales.
Membrane Damage and Microbial Inactivation by Chlorine in the Absence and Presence of a Chlorine-Demanding Substrate
Authors: Virto R. , Mañas P. , Álvarez I. , Condon S. , Raso J.
Published: September 2005

The relationship between cell inactivation and membrane damage was studied in two gram-positive organisms,
Listeria monocytogenes
and
Bacillus subtilis
, and two gram-negative organisms,
Yersinia enterocolitica
and
Escherichia coli
, exposed to chlorine in the absence and presence of 150 ppm of organic matter (Trypticase soy broth).
L. monocytogenes
and
B. subtilis
were more resistant to chlorine in distilled water. The addition of small amounts of organic matter to the chlorination medium drastically increased the resistance of both types of microorganisms, but this effect was more marked in
Y. enterocolitica
and
E. coli
. In addition, the survival curves for these microorganisms in the presence of organic matter had a prolonged shoulder. Sublethal injury was not detected under most experimental conditions, and only gram-positive cells treated in distilled water showed a relevant degree of injury. The exposure of bacterial cells to chlorine in distilled water caused extensive permeabilization of the cytoplasmic membrane, but the concentrations required were much higher than those needed to inactivate cells. Therefore, there was no relationship between the occurrence of membrane permeabilization and cell death. The addition of organic matter to the treatment medium stabilized the cytoplasmic membrane against permeabilization in both the gram-positive and gram-negative bacteria investigated. Exposure of
E. coli
cells to the outer membrane-permeabilizing agent EDTA increased their sensitivity to chlorine and caused the shoulders in the survival curves to disappear. Based on these observations, we propose that bacterial envelopes could play a role in cell inactivation by modulating the access of chlorine to the key targets within the cell.
Novel Method for Detection of Butanolides in
Streptomyces coelicolor
Culture Broth, Using a His-Tagged Receptor (ScbR) and Mass Spectrometry
Authors: Yang Yung-Hun , Joo Hwang-Soo , Lee Kwangwon , Liou Kwang-Kyung , Lee Hei-Chan , Sohng Jae-Kyung , Kim Byung-Gee
Published: September 2005

γ-Butyrolactone derivative molecules in
Streptomyces
play a crucial role in cell density control, secondary metabolism, and cell differentiation. As their synthesis level in the cell is very low compared to those of similar
N
-acyl homoserine lactone molecules from gram-negative bacteria, it is very hard to analyze them even with several hundredfold concentration of the culture broth. We have developed a very quick and easy detection method using an affinity capture technique with His-tagged receptor proteins and electrospray tandem mass spectrometry. Using
Streptomyces coelicolor
as a model system, SCB1 was detected from only 100 ml of the culture broth after solvent extraction. This method can be further applied to detection and quantitative analysis of butanolides and inhibitor screening of the receptor molecules.
Survival of
Campylobacter jejuni
in Water: Effect of Grazing by the Freshwater Crustacean
Daphnia carinata
(Cladocera)
Authors: Schallenberg M. , Bremer P. J. , Henkel S. , Launhardt A. , Burns C. W.
Published: September 2005

Environmental studies of the human-pathogenic bacterium
Campylobacter jejuni
have focused on linking distributions with potential sources. However, in aquatic ecosystems, the abundance of
C. jejuni
may also be regulated by predation. We examine the potential for grazing by the freshwater planktonic crustacean
Daphnia carinata
to reduce the survival of
C. jejuni
. We use a system for measuring grazing and clearance rates of
D. carinata
on bacteria and demonstrate that
D. carinata
can graze
C. jejuni
cells at a rate of 7% individual
−1
h
−1
under simulated natural conditions in the presence of an algal food source. We show that passage of
C. jejuni
through the
Daphnia
gut and incorporation into fecal material effectively reduces survival of
C. jejuni
. This is the first evidence to suggest that grazing by planktonic organisms can reduce the abundance of
C. jejuni
in natural waters. Biomanipulation of planktonic food webs to enhance
Daphnia
densities offers potential for reducing microbial pathogen densities in drinking water reservoirs and recreational water bodies, thereby reducing the risk of contracting water-borne disease.
Phylogenetic Characterization of Virulence and Resistance Phenotypes of
Pseudomonas syringae
Authors: Hwang Michael S. H. , Morgan Robyn L. , Sarkar Sara F. , Wang Pauline W. , Guttman David S.
Published: September 2005

Individual strains of the plant pathogenic bacterium
Pseudomonas syringae
vary in their ability to produce toxins, nucleate ice, and resist antimicrobial compounds. These phenotypes enhance virulence, but it is not clear whether they play a dominant role in specific pathogen-host interactions. To investigate the evolution of these virulence-associated phenotypes, we used functional assays to survey for the distribution of these phenotypes among a collection of 95
P. syringae
strains. All of these strains were phylogenetically characterized via multilocus sequence typing (MLST). We surveyed for the production of coronatine, phaseolotoxin, syringomycin, and tabtoxin; for resistance to ampicillin, chloramphenicol, rifampin, streptomycin, tetracycline, kanamycin, and copper; and for the ability to nucleate ice at high temperatures via the ice-nucleating protein INA. We found that fewer than 50% of the strains produced toxins and significantly fewer strains than expected produced multiple toxins, leading to the speculation that there is a cost associated with the production of multiple toxins. None of these toxins was associated with host of isolation, and their distribution, relative to core genome phylogeny, indicated extensive horizontal genetic exchange. Most strains were resistant to ampicillin and copper and had the ability to nucleate ice, and yet very few strains were resistant to the other antibiotics. The distribution of the rare resistance phenotypes was also inconsistent with the clonal history of the species and did not associate with host of isolation. The present study provides a robust phylogenetic foundation for the study of these important virulence-associated phenotypes in
P. syringae
host colonization and pathogenesis.
Identification of 9,17-Dioxo-1,2,3,4,10,19-Hexanorandrostan-5-oic Acid, 4-Hydroxy-2-Oxohexanoic Acid, and 2-Hydroxyhexa-2,4-Dienoic Acid and Related Enzymes Involved in Testosterone Degradation in
Comamonas testosteroni
TA441
Authors: Horinouchi Masae , Hayashi Toshiaki , Koshino Hiroyuki , Kurita Tomokazu , Kudo Toshiaki
Published: September 2005

Comamonas testosteroni
TA441 utilizes testosterone via aromatization of the A ring followed by
meta
-cleavage of the ring. The product of the
meta
-cleavage reaction, 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid, is degraded by a hydrolase, TesD. We directly isolated and identified two products of TesD as 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and (2
Z
,4
Z
)-2-hydroxyhexa-2,4-dienoic acid. The latter was a pure 4
Z
isomer. 2-Hydroxyhexa-2,4-dienoic acid was converted by a hydratase, TesE, and the product isolated from the reaction solution was identified as 2-hydroxy-4-hex-2-enolactone, indicating the direct product of TesE to be 4-hydroxy-2-oxohexanoic acid.
Dependence of Arbuscular-Mycorrhizal Fungi on Their Plant Host for Palmitic Acid Synthesis
Authors: Trépanier Martin , Bécard Guillaume , Moutoglis Peter , Willemot Claude , Gagné Serge , Avis Tyler J. , Rioux Jacques-André
Published: September 2005

Lipids are the major form of carbon storage in arbuscular-mycorrhizal fungi. We studied fatty acid synthesis by
Glomus intraradices
and
Gigaspora rosea
. [
14
C]Acetate and [
14
C]sucrose were incorporated into a synthetic culture medium to test fatty acid synthetic ability in germinating spores (
G. intraradices
and
G. rosea
), mycorrhized carrot roots, and extraradical fungal mycelium (
G. intraradices
). Germinating spores and extraradical hyphae could not synthesize 16-carbon fatty acids but could elongate and desaturate fatty acids already present. The growth stimulation of germinating spores by root exudates did not stimulate fatty acid synthesis. 16-Carbon fatty acids (16:0 and 16:1) were synthesized only by the fungi in the mycorrhized roots. Our data strongly suggest that the fatty acid synthase activity of arbuscular-mycorrhizal fungi is expressed exclusively in the intraradical mycelium and indicate that fatty acid metabolism may play a major role in the obligate biotrophism of arbuscular-mycorrhizal fungi.
Evidence for Aceticlastic Methanogenesis in the Presence of Sulfate in a Gas Condensate-Contaminated Aquifer
Authors: Struchtemeyer Christopher G. , Elshahed Mostafa S. , Duncan Kathleen E. , McInerney Michael J.
Published: September 2005

The anaerobic metabolism of acetate was studied in sediments and groundwater from a gas condensate-contaminated aquifer in an aquifer where geochemical evidence implicated sulfate reduction and methanogenesis as the predominant terminal electron-accepting processes. Most-probable-number tubes containing acetate and microcosms containing either [2-
14
C]acetate or [U-
14
C]acetate produced higher quantities of CH
4
compared to CO
2
in the presence or absence of sulfate.
14
CH
4
accounted for 70 to 100% of the total labeled gas in the [
14
C]acetate microcosms regardless of whether sulfate was present or not. Denaturing gradient gel electrophoresis of the acetate enrichments both with and without sulfate using
Archaea
-specific primers showed identical predominant bands that had 99% sequence similarity to members of
Methanosaetaceae
. Clone libraries containing archaeal 16S rRNA gene sequences amplified from sediment from the contaminated portion of the aquifer showed that 180 of the 190 clones sequenced belonged to the
Methanosaetaceae
. The production of methane and the high frequency of sequences from the
Methanosaetaceae
in acetate enrichments with and without sulfate indicate that aceticlastic methanogenesis was the predominant fate of acetate at this site even though sulfate-reducing bacteria would be expected to consume acetate in the presence of sulfate.
Improved System for Construction and Analysis of Single-Copy β-Galactosidase Operon Fusions in
Yersinia enterocolitica
Authors: Maxson Michelle E. , Darwin Andrew J.
Published: September 2005

We report a significantly improved system for studying single-copy
lacZ
operon fusions in
Yersinia enterocolitica
: a simple procedure for the stable integration of
lacZ
operon fusions into the
ara
locus and a strain with a deletion mutation that abolishes the low level of endogenous β-galactosidase activity.
Viable
Blastocystis
Cysts in Scottish and Malaysian Sewage Samples
Authors: Suresh K. , Smith H. V. , Tan T. C.
Published: September 2005

Blastocystis
cysts were detected in 38% (47/123) (37 Scottish, 17 Malaysian) of sewage treatment works. Fifty percent of influents (29% Scottish, 76% Malaysian) and 28% of effluents (9% Scottish, 60% Malaysian) contained viable cysts. Viable cysts, discharged in effluent, provide further evidence for the potential for waterborne transmission of
Blastocystis
.
Advantage Provided by Iron for
Escherichia coli
Growth and Cultivability in Drinking Water
Authors: Appenzeller Brice M. R. , Yañez Carolina , Jorand Frederic , Block Jean-Claude
Published: September 2005

The presence of iron, used both as a nutrient and as an electron acceptor, was demonstrated to give an advantage to
Escherichia coli
bacteria in drinking water. Slight additions of ferrous sulfate to water with initial low iron concentrations led to a significant increase in the number of
E. coli
bacteria. The presence of ferric oxide in water under anaerobic conditions increased bacterial cultivability.
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

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

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

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

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

srfAis 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

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

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

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.
NSR STRING IN AdS3 BACKGROUND: NONLOCAL CHARGES
Authors: MAHARANA JNANADEVA
Published: September 2005

We consider NSR superstring in AdS3⊗S3⊗R4 background. The action is expressed in terms of the variables on the group manifolds SL (2, R) and SU(2) as (1+1)-dimensional σ-models with Wess–Zumino terms, associated with AdS3 and S3 respectively. R4 is the flat Euclidean space. We show the existence of classical nonlocal conserved currents in the superspace formulation for the nonlinear σ-model with WZ term in the context of NSR string. We propose Ward identities utilizing existence of the nonlocal conserved currents.
AN "ACCIDENTAL" SYMMETRY OPERATOR FOR THE DIRAC EQUATION IN THE COULOMB POTENTIAL
Authors: KHACHIDZE TAMARI T. , KHELASHVILI ANZOR A.
Published: September 2005

On the basis of the generalization of the theorem about K-odd operators (K is the Dirac's operator), certain linear combination is constructed, which appears to commute with the Dirac Hamiltonian for Coulomb field. This operator coincides with the Johnson and Lippmann operator and is closely connected to the familiar Laplace–Runge–Lenz vector. Our approach guarantees not only derivation of Johnson–Lippmann operator, but simultaneously commutativity with the Dirac Hamiltonian.
Induction of cellular prion protein gene expression by copper in neurons
Authors: Varela-Nallar Lorena , Toledo Enrique M. , Larrondo Luis F. , Cabral Ana L. B. , Martins Vilma R. , Inestrosa Nibaldo C.
Published: September 2005

Prion diseases are caused by the conformational transition of the native α-helical cellular prion protein (PrPC) into a β-sheet pathogenic isoform. However, the normal physiological function of PrPC remains elusive. We report herein that copper induces PrPC expression in primary hippocampal and cortical neurons. PrPC induced by copper has a normal glycosylation pattern, is proteinase K-sensitive and reaches the cell surface attached by a glycosyl phosphatidylinositol anchor. Immunofluorescence analysis revealed that copper induces PrPC levels in the cell surface and in an intracellular compartment that we identified as the Golgi complex. In addition, copper induced the activity of a reporter vector driven by the rat PrPC gene ( Prnp) promoter stably transfected into PC12 cells, whereas no effect was observed in glial C6 clones. Also cadmium, but not zinc or manganese, upregulated Prnp promoter activity in PC12 clones. Progressive deletions of the promoter revealed that the region essential for copper modulation contains a putative metal responsive element. Although electrophoretic mobility shift assay demonstrated nuclear protein binding to this element, supershift analysis showed that this is not a binding site for the metal responsive transcription factor-1 (MTF-1). The MTF-1-independent transcriptional activation of Prnp is supported by the lack of Prnp promoter activation by zinc. These findings demonstrate that Prnp expression is upregulated by copper in neuronal cells by an MTF-1-independent mechanism, and suggest a metal-specific modulation of Prnp in neurons.
Histamine-mediated increases in cytosolic [Ca2+] involve different mechanisms in human pulmonary artery smooth muscle and endothelial cells
Authors: Mauban Joseph R. H. , Wilkinson Katherine , Schach Christian , Yuan Jason X.-J.
Published: September 2005

Agonist stimulation of human pulmonary artery smooth muscle cells (PASMC) and endothelial cells (PAEC) with histamine showed similar spatiotemporal patterns of Ca2+ release. Both sustained elevation and oscillatory patterns of changes in cytosolic Ca2+ concentration ([Ca2+]cyt) were observed in the absence of extracellular Ca2+. Capacitative Ca2+ entry (CCE) was induced in PASMC and PAEC by passive depletion of intracellular Ca2+ stores with 10 μM cyclopiazonic acid (CPA; 15–30 min). The pyrazole derivative BTP2 inhibited CPA-activated Ca2+ influx, suggesting that depletion of CPA-sensitive internal stores is sufficient to induce CCE in both PASMC and PAEC. The recourse of histamine-mediated Ca2+ release was examined after exposure of cells to CPA, thapsigargin, caffeine, ryanodine, FCCP, or bafilomycin. In PASMC bathed in Ca2+-free solution, treatment with CPA almost abolished histamine-induced rises in [Ca2+]cyt. In PAEC bathed in Ca2+-free solution, however, treatment with CPA eliminated histamine-induced sustained and oscillatory rises in [Ca2+]cyt but did not affect initial transient increase in [Ca2+]cyt. Furthermore, treatment of PAEC with a combination of CPA (or thapsigargin) and caffeine (and ryanodine), FCCP, or bafilomycin did not abolish histamine-induced transient [Ca2+]cyt increases. These observations indicate that 1) depletion of CPA-sensitive stores is sufficient to cause CCE in both PASMC and PAEC; 2) induction of CCE in PAEC does not require depletion of all internal Ca2+ stores; 3) the histamine-releasable internal stores in PASMC are mainly CPA-sensitive stores; 4) PAEC, in addition to a CPA-sensitive functional pool, contain other stores insensitive to CPA, thapsigargin, caffeine, ryanodine, FCCP, and bafilomycin; and 5) although the CPA-insensitive stores in PAEC may not contribute to CCE, they contribute to histamine-mediated Ca2+ release.
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

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 >30-fold in cells from patients with Fanconi anemia (P < 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

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

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

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 >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

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

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

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.
Positive Contribution of Pathogenic Mutations in the Mitochondrial Genome to the Promotion of Cancer by Prevention from Apoptosis
Authors: Shidara Yujiro , Yamagata Kumi , Kanamori Takashi , Nakano Kazutoshi , Kwong Jennifer Q. , Manfredi Giovanni , Oda Hideaki , Ohta Shigeo
Published: September 2005

The role of mitochondrial dysfunction in cancer has been a subject of great interest and much ongoing investigation. Although most cancer cells harbor somatic mutations in mitochondrial DNA (mtDNA), the question of whether such mutations contribute to the promotion of carcinomas remains unsolved. Here we used trans-mitochondrial hybrids (cybrids) containing a common HeLa nucleus and mtDNA of interest to compare the role of mtDNA against the common nuclear background. We constructed cybrids with or without a homoplasmic pathogenic point mutation at nucleotide position 8,993 or 9,176 in the mtDNA ATP synthase subunit 6 gene (MTATP6) derived from patients with mitochondrial encephalomyopathy. When the cybrids were transplanted into nude mice, the MTATP6 mutations conferred an advantage in the early stage of tumor growth. The mutant cybrids also increased faster than wild type in culture. To complement the mtDNA mutations, we transfected a wild-type nuclear version of MTATP, whose codons were converted to the universal genetic codes containing a mitochondrial target sequence, into the nucleus of cybrids carrying mutant MTATP6. The restoration of MTATP slowed down the growth of tumor in transplantation. Conversely, expression of a mutant nuclear version of MTATP6 in the wild-type cybrids declined respiration and accelerated the tumor growth. These findings showed that the advantage in tumor growth depended upon the MTATP6 function but was not due to secondary nuclear mutations caused by the mutant mitochondria. Because apoptosis occurred less frequently in the mutant versus wild-type cybrids in cultures and tumors, the pathogenic mtDNA mutations seem to promote tumors by preventing apoptosis.
Osterix, a Transcription Factor for Osteoblast Differentiation, Mediates Antitumor Activity in Murine Osteosarcoma
Authors: Cao Ying , Zhou Zhichao , de Crombrugghe Benoit , Nakashima Kazuhisa , Guan Hui , Duan Xiaoping , Jia Shu-Fang , Kleinerman Eugenie S.
Published: September 2005

Osterix is a novel zinc finger–containing transcription factor that is essential for osteoblast differentiation and bone formation. We hypothesized that osterix might have a role in osteosarcoma tumor growth and metastasis. Northern blot analysis showed that the mRNA level of osterix was decreased in two mouse osteosarcoma cell lines compared with its level in normal mouse osteoblasts. Osterix expression was also decreased in three human osteosarcoma cell lines. Transfection of the osx gene into the mouse osteosarcoma cells inhibited tumor cell growth in vitro and in vivo and significantly reduced tumor incidence, tumor volume, and lung metastasis following intratibial injection. Osterix expression was also associated with decreased osteolysis. Using an in vitro migration assay, osterix suppressed the migration of tumor cells to lung extracts. These results suggest that osterix expression may play a role in osteosarcoma tumor growth and metastasis.
Differential Effects of Neuropeptide Y on the Growth and Vascularization of Neural Crest–Derived Tumors
Authors: Kitlinska Joanna , Abe Ken , Kuo Lydia , Pons Jennifer , Yu Muchieh , Li Lijun , Tilan Jason , Everhart Lindsay , Lee Edward W. , Zukowska Zofia , Toretsky Jeffrey A.
Published: September 2005

Neuropeptide Y (NPY) is a sympathetic neurotransmitter recently found to be potently angiogenic and growth promoting for endothelial, vascular smooth muscle and neuronal cells. NPY and its cognate receptors, Y1, Y2 and Y5, are expressed in neural crest–derived tumors; however, their role in regulation of growth is unknown. The effect of NPY on the growth and vascularization of neuroendocrine tumors was tested using three types of cells: neuroblastoma, pheochromocytoma, and Ewing's sarcoma family of tumors (ESFT). The tumors varied in expression of NPY receptors, which was linked to differential functions of the peptide. NPY stimulated proliferation of neuroblastoma cells via Y2/Y5Rs and inhibited ESFT cell growth by Y1/Y5-mediated apoptosis. In both tumor types, NPY receptor antagonists altered basal growth levels, indicating a regulatory role of autocrine NPY. In addition, the peptide released from the tumor cells stimulated endothelial cell proliferation, which suggests its paracrine angiogenic effects. In nude mice xenografts, exogenous NPY stimulated growth of neuroblastoma tumors, whereas it increased apoptosis and reduced growth of ESFT. However, in both tumors, NPY treatment led to an increase in tumor vascularization. Taken together, this is the first report of NPY being a growth-regulatory factor for neuroendocrine tumors, acting both by autocrine activation of tumor cell proliferation or apoptosis and by angiogenesis. NPY and its receptors may become targets for novel approaches in the treatment of these diseases, directed against both tumor cell proliferation and angiogenesis.