bluuebunny/crossref_metadata_2025 huggingface dataset:
PseudomonasDiversity in Crude-Oil-Contaminated Intertidal Sand Samples Obtained after thePrestigeOil Spill
Authors: Mulet Magdalena , David Zoyla , Nogales Balbina , Bosch Rafael , Lalucat Jorge , García-Valdés Elena
Published: December 2010

The Galicia seashore, in northwestern Spain, was one of the shorelines affected by thePrestigeoil spill in November 2002. The diversity of autochthonousPseudomonaspopulations present at two beaches (Carnota municipality) was analyzed using culture-independent and culture-dependent methods. The first analysis involved the screening of anrpoDgene library. The second involved the isolation of 94Pseudomonasstrains that were able to grow on selective media by direct plating or after serial enrichments on several carbon sources: biphenyl, gentisate, hexadecane, methylnaphthalene, naphthalene, phenanthrene, salicylate, xylene, and succinate. Eight denitrifyingPseudomonasstrains were also isolated by their ability to grow anaerobically with nitrate. The calculated coverage index forPseudomonasspecies was 89% when clones and isolates were considered together, and there were 29 phylospecies detected. The most abundant were members of the speciesP. stutzeri,P.putida,P. anguilliseptica, andP. oleovorans. Thirty-one isolates could not be identified at the species level and were considered representatives of 16 putative novelPseudomonasspecies. One isolate was considered representative of a novelP. stutzerigenomovar. Concordant results were obtained when the diversities of the cloned DNA library and the cultured strains were compared. The clone library obtained by therpoDPCR method was a useful tool for evaluatingPseudomonascommunities and also for microdiversity studies ofPseudomonaspopulations.
Bacteriophage Ecology in
Escherichia coli
and
Pseudomonas aeruginosa
Mixed-Biofilm Communities
Authors: Kay Matthew K. , Erwin Thomas C. , McLean Robert J. C. , Aron Gary M.
Published: December 2010

Phage therapy is being reexamined as a strategy for bacterial control in medical and other environments. As microorganisms often live in mixed populations, we examined the effect of
Escherichia coli
bacteriophage λW60 and
Pseudomonas aeruginosa
bacteriophage PB-1 infection on the viability of monoculture and mixed-species biofilm and planktonic cultures. In mixed-species biofilm communities,
E. coli
and
P. aeruginosa
maintained stable cell populations in the presence of one or both phages. In contrast,
E. coli
planktonic populations were severely depleted in coculture in the presence of λW60. Both
E. coli
and
P. aeruginosa
developed phage resistance in planktonic culture; however, reduced resistance was observed in biofilm communities. Increased phage titers and reduced resistance in biofilms suggest that phage can replicate on susceptible cells in biofilms. Infectious phage could be released from mixed-culture biofilms upon treatment with Tween 20 but not upon treatment with chloroform. Tween 20 and chloroform treatments had no effect on phage associated with planktonic cells, suggesting that planktonic phage were not cell or matrix associated. Transmission electron microscopy showed bacteriophage particles to be enmeshed in the extracellular polymeric substance component of biofilms and that this substance could be removed by Tween 20 treatment. Overall, this study demonstrates how mixed-culture biofilms can maintain a reservoir of viable phage and bacterial populations in the environment.
The
yctCBA
Operon of
Yersinia ruckeri
, Involved in
In Vivo
Citrate Uptake, Is Not Required for Virulence
Authors: Navais Roberto , Méndez Jessica , Reimundo Pilar , Pérez-Pascual David , Gómez Esther , Guijarro José A.
Published: December 2010

A three-gene operon, named
yctCBA
(

Y

ersinia

c

itrate

t

ransporter), induced by citrate and repressed by glucose was identified from a previously selected
in vivo
-induced (
ivi
) clone in the fish pathogen
Yersinia
ruckeri
. Interestingly, despite being an
ivi
clone, the drastic growth reduction of the
yctC
mutant in the presence of citrate, and the relatively high content of this compound in rainbow trout serum, the operon was not required for virulence.
Production of Secretory and Extracellular N-Linked Glycoproteins inEscherichia coli
Authors: Fisher Adam C. , Haitjema Charles H. , Guarino Cassandra , Çelik Eda , Endicott Christine E. , Reading Craig A. , Merritt Judith H. , Ptak A. Celeste , Zhang Sheng , DeLisa Matthew P.
Published: December 2010

TheCampylobacter jejuni pglgene cluster encodes a complete N-linked protein glycosylation pathway that can be functionally transferred intoEscherichia coli. In this system, we analyzed the interplay between N-linked glycosylation, membrane translocation and folding of acceptor proteins in bacteria. We developed a recombinant N-glycan acceptor peptide tag that permits N-linked glycosylation of diverse recombinant proteins expressed in the periplasm of glycosylation-competentE. colicells. With this “glycosylation tag,” a clear difference was observed in the glycosylation patterns found on periplasmic proteins depending on their mode of inner membrane translocation (i.e., Sec, signal recognition particle [SRP], or twin-arginine translocation [Tat] export), indicating that the mode of protein export can influence N-glycosylation efficiency. We also established that engineered substrate proteins targeted to environments beyond the periplasm, such as the outer membrane, the membrane vesicles, and the extracellular medium, could serve as substrates for N-linked glycosylation. Taken together, our results demonstrate that theC. jejuniN-glycosylation machinery is compatible with distinct secretory mechanisms inE. coli, effectively expanding the N-linked glycome of recombinantE. coli. Moreover, this simple glycosylation tag strategy expands the glycoengineering toolbox and opens the door to bacterial synthesis of a wide array of recombinant glycoprotein conjugates.
Identification of a Tyrosine Decarboxylase Gene (
tdcA
) in
Streptococcus thermophilus
1TT45 and Analysis of Its Expression and Tyramine Production in Milk
Authors: La Gioia Federica , Rizzotti Lucia , Rossi Franca , Gardini Fausto , Tabanelli Giulia , Torriani Sandra
Published: December 2010

In this study, a tyrosine decarboxylase gene (
tdcA
) was identified in 1 among 83
Streptococcus thermophilus
strains tested. Its sequence, nearly identical to that of a
tdcA
of
Lactobacillus curvatus
, indicated a horizontal gene transfer event. Transcription in milk and the formation of critical levels of tyramine were observed in the presence of tyrosine.
Lactobacillus plantarum
Extracellular Chitin-Binding Protein and Its Role in the Interaction between Chitin, Caco-2 Cells, and Mucin
Authors: Sánchez Borja , González-Tejedo Carmen , Ruas-Madiedo Patricia , Urdaci María C. , Margolles Abelardo
Published: December 2010

In the present work, we describe the adhesion capabilities of a recombinant
Lactococcus lactis
strain producing an extracellular protein from
Lactobacillus plantarum
. Our results show that this protein may offer the bacterium a mechanism to bind to
N
-acetylglucosamine-containing polymers, such as human mucins, present in different environments.
Neutrophilic Iron-Oxidizing “
Zetaproteobacteria
” and Mild Steel Corrosion in Nearshore Marine Environments
Authors: McBeth Joyce M. , Little Brenda J. , Ray Richard I. , Farrar Katherine M. , Emerson David
Published: December 2010

Microbiologically influenced corrosion (MIC) of mild steel in seawater is an expensive and enduring problem. Little attention has been paid to the role of neutrophilic, lithotrophic, iron-oxidizing bacteria (FeOB) in MIC. The goal of this study was to determine if marine FeOB related to
Mariprofundus
are involved in this process. To examine this, field incubations and laboratory microcosm experiments were conducted. Mild steel samples incubated in nearshore environments were colonized by marine FeOB, as evidenced by the presence of helical iron-encrusted stalks diagnostic of the FeOB
Mariprofundus ferrooxydans
, a member of the candidate class “
Zetaproteobacteria.
” Furthermore,
Mariprofundus
-like cells were enriched from MIC biofilms. The presence of
Zetaproteobacteria
was confirmed using a
Zetaproteobacteria
-specific small-subunit (SSU) rRNA gene primer set to amplify sequences related to
M. ferrooxydans
from both enrichments and
in situ
samples of MIC biofilms. Temporal
in situ
incubation studies showed a qualitative increase in stalk distribution on mild steel, suggesting progressive colonization by stalk-forming FeOB. We also isolated a novel FeOB, designated
Mariprofundus
sp. strain GSB2, from an iron oxide mat in a salt marsh. Strain GSB2 enhanced uniform corrosion from mild steel in laboratory microcosm experiments conducted over 4 days. Iron concentrations (including precipitates) in the medium were used as a measure of corrosion. The corrosion in biotic samples (7.4 ± 0.1 mM) was significantly higher than that in abiotic controls (5.0 ± 0.1 mM). These results have important implications for the role of FeOB in corrosion of steel in nearshore and estuarine environments. In addition, this work shows that the global distribution of
Zetaproteobacteria
is far greater than previously thought.
Modeling of Pathogen Survival during Simulated Gastric Digestion
Authors: Koseki Shige , Mizuno Yasuko , Sotome Itaru
Published: December 2010

The objective of the present study was to develop a mathematical model of pathogenic bacterial inactivation kinetics in a gastric environment in order to further understand a part of the infectious dose-response mechanism. The major bacterial pathogens
Listeria monocytogenes
,
Escherichia coli
O157:H7, and
Salmonella
spp. were examined by using simulated gastric fluid adjusted to various pH values. To correspond to the various pHs in a stomach during digestion, a modified logistic differential equation model and the Weibull differential equation model were examined. The specific inactivation rate for each pathogen was successfully described by a square-root model as a function of pH. The square-root models were combined with the modified logistic differential equation to obtain a complete inactivation curve. Both the modified logistic and Weibull models provided a highly accurate fitting of the static pH conditions for every pathogen. However, while the residuals plots of the modified logistic model indicated no systematic bias and/or regional prediction problems, the residuals plots of the Weibull model showed a systematic bias. The modified logistic model appropriately predicted the pathogen behavior in the simulated gastric digestion process with actual food, including cut lettuce, minced tuna, hamburger, and scrambled egg. Although the developed model enabled us to predict pathogen inactivation during gastric digestion, its results also suggested that the ingested bacteria in the stomach would barely be inactivated in the real digestion process. The results of this study will provide important information on a part of the dose-response mechanism of bacterial pathogens.
Interaction between the Candida albicans High-Osmolarity Glycerol (HOG) Pathway and the Response to Human β-Defensins 2 and 3
Authors: Argimón Silvia , Fanning Saranna , Blankenship Jill R. , Mitchell Aaron P.
Published: December 2010

Human β-defensins 2 and 3 are small cationic peptides with antimicrobial activity against the fungal pathogen
Candida albicans
. We found that
hog1
and
pbs2
mutants were hypersensitive to treatment with these peptides, pointing to a role of the high-osmolarity glycerol (HOG) pathway in the response to defensin-induced cell injury.
The Fungal Type II Myosin in Penicillium marneffei, MyoB, Is Essential for Chitin Deposition at Nascent Septation Sites but Not Actin Localization
Authors: Cánovas David , Boyce Kylie J. , Andrianopoulos Alex
Published: December 2010

Cytokinesis is essential for proliferative growth but also plays equally important roles during morphogenesis and development. The human pathogen
Penicillium marneffei
is capable of dimorphic switching in response to temperature, growing in a multicellular filamentous hyphal form at 25°C and in a unicellular yeast form at 37°C.
P. marneffei
also undergoes asexual development at 25°C to produce multicellular differentiated conidiophores. Thus,
P. marneffe
i exhibits cell division with and without cytokinesis and division by budding and fission, depending on the cell type. The type II myosin gene,
myoB
, from
P. marneffei
plays important roles in the morphogenesis of these cell types. Deletion of
myoB
leads to chitin deposition defects at sites of cell division without perturbing actin localization. In addition to aberrant hyphal cells, distinct conidiophore cell types are lacking due to malformed septa and nuclear division defects. At 37°C, deletion of
myoB
prevents uninucleate yeast cell formation, instead producing long filaments resembling hyphae at 25°C. The Δ
myoB
cells also often lyse due to defects in cell wall biogenesis. Thus, MyoB is essential for correct morphogenesis of all cell types regardless of division mode (budding or fission) and defines differences between the different types of growth.
A Single Mating-Type Locus Composed of Homeodomain Genes Promotes Nuclear Migration and Heterokaryosis in the White-Rot Fungus Phanerochaete chrysosporium
Authors: James Timothy Y. , Lee Maria , van Diepen Linda T. A.
Published: December 2010

The white-rot basidiomycete fungusPhanerochaete chrysosporium(Agaricomycetes) is a model species that produces potent wood-degrading enzymes. The mating system of the species has been difficult to characterize due to its cryptic fruiting habit and lack of clamp connections in the heterokaryotic phase. By exploiting the draft genome sequence, we reevaluated the mating system ofP. chrysosporiumby studying the inheritance and segregation of putative mating-type gene homologues, the homeodomain transcription factor genes (MAT-A) and the pheromone receptors (MAT-B). A pattern of mating incompatibility and fructification consistent with a bipolar system with a singleMATlocus was observed, but the rejection response was much weaker than that seen in other agaricomycete species, leading to stable heterokaryons with identicalMATalleles. The homeodomain genes appear to comprise the singleMATlocus because they are heterozygous in wild strains and hyperpolymorphic at the DNA sequence level and promote aspects of sexual reproduction, such as nuclear migration, heterokaryon stability, and basidiospore formation. The pheromone receptor loci that might constitute aMAT-Blocus, as in many other Agaricomycetes, are not linked to theMAT-Alocus and display low levels of polymorphism. This observation is inconsistent with a bipolar mating system that includes pheromones and pheromone receptors as mating-type determinants. The partial uncoupling of nuclear migration and mating incompatibility in this species may be predicted to lead to parasexual recombination and may have contributed to the homothallic behavior observed in previous studies.
Fungal Skn7 Stress Responses and Their Relationship to Virulence
Authors: Fassler Jan S. , West Ann H.
Published: December 2010

The histidine kinase-based phosphorelay has emerged as a common strategy among bacteria, fungi, protozoa, and plants for triggering important stress responses and interpreting developmental cues in response to environmental as well as chemical, nutritional, and hormone signals. The absence of this type of signaling mechanism in animals makes the so-called “two-component” pathway an attractive target for development of antimicrobial agents. The best-studied eukaryotic example of a two-component pathway is the SLN1 pathway in
Saccharomyces cerevisiae
, which responds to turgor and other physical properties associated with the fungal cell wall. One of the two phosphoreceiver proteins known as response regulators in this pathway is Skn7, a highly conserved stress-responsive transcription factor with a subset of activities that are dependent on SLN1 pathway phosphorylation and another subset that are independent. Interest in Skn7as a determinant in fungal virulence stems primarily from its well-established role in the oxidative stress response; however, the involvement of Skn7 in maintenance of cell wall integrity may also be relevant. Since the cell wall is crucial for fungal survival, structural and biosynthetic proteins affecting wall composition and signaling pathways that respond to wall stress are likely to play key roles in virulence. Here we review the molecular and phenotypic characteristics of different fungal Skn7 proteins and consider how each of these properties may contribute to fungal virulence.
Regulation of the CgPdr1 Transcription Factor from the Pathogen Candida glabrata
Authors: Paul Sanjoy , Schmidt Jennifer A. , Moye-Rowley W. Scott
Published: December 2010

Candida glabrata
is an opportunistic human pathogen that is increasingly associated with candidemia, owing in part to the intrinsic and acquired high tolerance the organism exhibits for the important clinical antifungal drug fluconazole. This elevated fluconazole resistance often develops through gain-of-function mutations in the zinc cluster-containing transcriptional regulator
C. glabrata
Pdr1 (CgPdr1). CgPdr1 induces the expression of an ATP-binding cassette (ABC) transporter-encoding gene, Cg
CDR1
.
Saccharomyces cerevisiae
has two CgPdr1 homologues called ScPdr1 and ScPdr3. These factors control the expression of an ABC transporter-encoding gene called Sc
PDR5
, which encodes a homologue of Cg
CDR1
. Loss of the mitochondrial genome (ρ
0
cell) or overexpression of the mitochondrial enzyme ScPsd1 induces Sc
PDR5
expression in a strictly ScPdr3-dependent fashion. ScPdr3 requires the presence of a transcriptional Mediator subunit called Gal11 (Med15) to fully induce Sc
PDR5
transcription in response to ρ
0
signaling. ScPdr1 does not respond to either ρ
0
signals or ScPsd1 overproduction. In this study, we employed transcriptional fusions between CgPdr1 target promoters, like Cg
CDR1
, to demonstrate that CgPdr1 stimulates gene expression via binding to elements called pleiotropic drug response elements (PDREs). Deletion mapping and electrophoretic mobility shift assays demonstrated that a single PDRE in the Cg
CDR1
promoter was capable of supporting ρ
0
-induced gene expression. Removal of one of the two ScGal11 homologues from
C. glabrata
caused a major defect in drug-induced expression of Cg
CDR1
but had a quantitatively minor effect on ρ
0
-stimulated transcription. These data demonstrate that CgPdr1 appears to combine features of ScPdr1 and ScPdr3 to produce a transcription factor with chimeric regulatory properties.
Regulation of
nleA
in Shiga Toxin-Producing
Escherichia coli
O84:H4 Strain 4795/97
Authors: Schwidder Maike , Hensel Michael , Schmidt Herbert
Published: December 2010

Many Shiga toxin-producing
Escherichia coli
(STEC) strains express a type III secretion system (TTSS) encoded by the locus of enterocyte effacement (LEE). Using the TTSS, STEC is able to inject effector proteins directly into eukaryotic host cells, where they cause characteristic attaching and effacing (A/E) lesions. In addition to the LEE-encoded effectors, a number of non-LEE-encoded effectors, located on phage-associated elements, have been described. One of them, the
n
on-
L
EE-encoded
e
ffector
A
(NleA), is widely distributed among pathogenic
E. coli
. In this study, we investigated the influence of environmental conditions on the expression of the phage-encoded effector
nleA
gene (designated
nleA
4795
) present in STEC O84:H4 strain 4795/97. We demonstrated that a particular NaCl concentration and starvation stress increase the activity of the
nleA
4795
promoter. Moreover, several regulators that control
nleA
4795
expression were identified. The involvement of the LEE regulators Ler, GrlA, and GrlR show that
nleA
4795
is integrated in the LEE regulation circuit. Furthermore, the binding of Ler to sequences upstream of
nleA
4795
underlined these findings.
DifA, a Methyl-Accepting Chemoreceptor Protein-Like Sensory Protein, Uses a Novel Signaling Mechanism to Regulate Exopolysaccharide Production inMyxococcus xanthus
Authors: Xu Qian , Black Wesley P. , Nascimi Heidi M. , Yang Zhaomin
Published: December 2010

DifA is a methyl-accepting chemotaxis protein (MCP)-like sensory transducer that regulates exopolysaccharide (EPS) production inMyxococcus xanthus. Here mutational analysis and molecular biology were used to probe the signaling mechanisms of DifA in EPS regulation. We first identified the start codon of DifA experimentally; this identification extended the N terminus of DifA for 45 amino acids (aa) from the previous bioinformatics prediction. This extension helped to address the outstanding question of how DifA receives input signals from type 4 pili without a prominent periplasmic domain. The results suggest that DifA uses its N-terminus extension to sense an upstream signal in EPS regulation. We suggest that the perception of the input signal by DifA is mediated by protein-protein interactions with upstream components. Subsequent signal transmission likely involves transmembrane signaling instead of direct intramolecular interactions between the input and the output modules in the cytoplasm. The basic functional unit of DifA for signal transduction is likely dimeric as mutational alteration of the predicted dimeric interface of DifA significantly affected EPS production. Deletions of 14-aa segments in the C terminus suggest that the newly defined flexible bundle subdomain in MCPs is likely critical for DifA function because shortening of this bundle can lead to constitutively active mutations.
A Caffeyl-Coenzyme A Synthetase Initiates Caffeate Activation prior to Caffeate Reduction in the Acetogenic Bacterium
Acetobacterium woodii
Authors: Hess Verena , Vitt Stella , Müller Volker
Published: December 2010

The anaerobic acetogenic bacterium
Acetobacterium woodii
couples the reduction of caffeate with electrons derived from hydrogen to the synthesis of ATP by a chemiosmotic mechanism using sodium ions as coupling ions, but the enzymes involved remain to be established. Previously, the electron transfer flavoproteins EtfA and EtfB were found to be involved in caffeate respiration. By inverse PCR, we identified three genes upstream of
etfA
and
etfB
:
carA
,
carB
, and
carC. carA
encodes a potential coenzyme A (CoA) transferase,
carB
an acyl-CoA synthetase, and
carC
an acyl-CoA dehydrogenase.
carA
, -
B
, and -
C
are located together with
etfA/carE
and
etfB/carD
on one polycistronic message, indicating that CarA, CarB, and CarC are also part of the caffeate respiration pathway. The genetic data suggest an initial ATP-dependent activation of caffeate by CarB. To prove the proposed function of CarB, the protein was overproduced in
Escherichia coli
, and the recombinant protein was purified. Purified CarB activates caffeate to caffeyl-CoA in an ATP- and CoA-dependent reaction. The enzyme has broad pH and temperature optima and requires K
+
for activity. In addition to caffeate, it can use ρ-coumarate, ferulate, and cinnamate as substrates, with 50, 15, and 9%, respectively, of the activity obtained with caffeate. Expression of the
car
operon is induced not only by caffeate, ρ-coumarate, ferulate, and cinnamate but also by sinapate. There is no induction by ρ-hydroxybenzoate or syringate.
Canonical Protein Splicing of a Class 1 Intein That Has a Class 3 Noncanonical Sequence Motif
Authors: Reitter Julie N. , Mills Kenneth V.
Published: December 2010

A
Thermobifida fusca
intein has two characteristics of class 3 inteins: a noncontiguous covariant Trp-Cys-Thr triplet and a Ser flanking its C terminus. However, it has Cys at position one, characteristic of class 1 inteins. Splicing does not require the internal Cys, which may instead coordinate the active site. Therefore, the intein is class 1.
Complete Genome Sequence of
Burkholderia rhizoxinica
, an Endosymbiont of
Rhizopus microsporus
Authors: Lackner Gerald , Moebius Nadine , Partida-Martinez Laila , Hertweck Christian
Published: December 2010

Burkholderia rhizoxinica
is an intracellular symbiont of the phytopathogenic fungus
Rhizopus microsporus
. The vertically transmitted endosymbiont not only delivers the antimitotic macrolide rhizoxin to its host but is also essential for vegetative spore formation of the fungus. To shed light on the genetic equipment of this model organism, we sequenced the whole genome of
B. rhizoxinica
HKI 0454, thus providing the first genomic insight into an intracellular mutualist of a fungal species. The 3.75-Mb genome consists of a chromosome and two strain-specific plasmids. The primary metabolism appears to be specialized for the uptake of fungal metabolites. Besides the rhizoxin biosynthesis gene cluster, there are 14 loci coding for nonribosomal peptide synthetase (NRPS) assembly lines, which represent novel targets for genomic mining of cryptic natural products. Furthermore, the endosymbionts are equipped with a repertoire of virulence-related factors, which can now be studied to elucidate molecular mechanisms underlying bacterial-fungal interaction.
Genome Sequence of
Lactobacillus amylovorus
GRL1112
Authors: Kant Ravi , Paulin Lars , Alatalo Edward , de Vos Willem M. , Palva Airi
Published: December 2010

Lactobacillus amylovorus
is a common member of the normal gastrointestinal tract (GIT) microbiota in pigs. Here, we report the genome sequence of
L. amylovorus
GRL1112, a porcine feces isolate displaying strong adherence to the pig intestinal epithelial cells. The strain is of interest, as it is a potential probiotic bacterium.
Complete Genome Sequence of
Streptococcus thermophilus
Strain ND03
Authors: Sun Zhihong , Chen Xia , Wang Jicheng , Zhao Wenjing , Shao Yuyu , Wu Lan , Zhou Zhemin , Sun Tiansong , Wang Lei , Meng He , Zhang Heping , Chen Wei
Published: December 2010

Streptococcus thermophilus
strain ND03 is a Chinese commercial dairy starter used for the manufacture of yogurt. It was isolated from naturally fermented yak milk in Qinghai, China. We present here the complete genome sequence of ND03 and compare it to three other published genomes of
Streptococcus thermophilus
strains.
Zeb1 and twist are more commonly expressed in metastatic than primary lung tumours and show inverse associations with claudins
Authors: Merikallio Heta , Kaarteenaho Riitta , Pääkkö Paavo , Lehtonen Siri , Hirvikoski Pasi , Mäkitaro Riitta , Harju Terttu , Soini Ylermi
Published: December 2010

Background

Zeb1 and twist regulate expression of genes which take part in epitheliomesenchymal transition (EMT). Claudins are tight junctional proteins regulating polarity and paracellular permeability of epithelial cells.

Aims and methods

To study Zeb1 and twist in 289 primary and 54 metastatic lung tumours and their relation to expression of claudins 1–5 and 7 by immunohistochemistry.

Results

Metastatic tumours showed a significantly lower expression of zeb1 and twist (33.0% and 38.0% vs 5.0% and 14.5%, p<0.001 for both) and they also had a significantly lower expression of claudin 1 (p<0.001), claudin 4 (p<0.001), claudin 5 (p=0.001) and claudin 7 (p<0.001) than did primary tumours. Primary lung tumours showed a strong inverse association between zeb1 and claudin 1 (p=0.024) and claudin 2 (p=0.003). For twist an inverse association was found with claudin 5 (p=0.007). In metastatic tumours zeb1 was inversely associated with claudin 7 (p=0.050) and twist with claudin 5 (p=0.025). Overexpression of claudin 5 was associated with decreased survival (p=0.017). For zeb1 or twist, no association with survival was observed in primary or metastatic tumours.

Conclusions

According to protein expression, involvement of zeb1 and twist in EMT in primary lung tumours is relatively infrequent. Carcinomas metastatic to the lung showed a significantly higher expression of these transcriptional factors than primary lung tumours, indicating their probable importance in the metastatic process. Zeb1 and twist were inversely associated with several claudins, indicating a role in their down-regulation.
Lung cancer and occupational exposures other than cotton dust and endotoxin among women textile workers in Shanghai, China
Authors: Checkoway H , Ray R M , Lundin J I , Astrakianakis G , Seixas N S , Camp J E , Wernli K J , Fitzgibbons E D , Li W , Feng Z , Gao D L , Thomas D B
Published: December 2010

Objectives

Numerous epidemiological studies of lung cancer among textile workers worldwide consistently indicate reduced risks related to cotton dust exposure, presumably due to endotoxin contamination. Our objective was to investigate associations with other exposures potentially related to lung cancer, including wool and synthetic fibre dusts, formaldehyde, silica, dyes and metals, that have only been studied to a limited extent in the textile industry.

Methods

We conducted a case–cohort study nested within a cohort of 267 400 women textile workers in Shanghai, China. We compared work assignments and exposure histories of 628 incident lung cancer cases, diagnosed during 1989–1998, with those of a reference subcohort of 3188 workers. We reconstructed exposures with a job–exposure matrix developed specifically for textile factories. Cox proportional hazards modelling was applied to estimate age/smoking-adjusted relative risks (hazard ratios) and risk gradients associated with job assignments and specific agents other than cotton dust and endotoxin.

Results

No associations were observed for lung cancer with wool, silk or synthetic fibre dusts, or with most other agents. However, increased risks, although statistically imprecise, were noted for ≥10 years’ exposures to silica (adjusted HR 3.5, 95% CI 1.0 to 13) and ≥10 years’ exposures to formaldehyde (adjusted HR 2.1, 95% CI 0.4 to 11).

Conclusions

Exposures to silica and formaldehyde, although not widespread among the cohort, may have increased lung cancer risk. Silica is an established human lung carcinogen, whereas there is only weak prior evidence supporting an association with formaldehyde. Both exposures warrant consideration as potential lung carcinogens in textile manufacturing.
Spectrum of tardive syndromes: clinical recognition and management
Authors: Bhidayasiri Roongroj , Boonyawairoj Suthida
Published: December 2010

Tardive syndrome (TS) refers to a group of delayed onset disorders characterised by abnormal movements and caused by dopamine receptor blocking agents (DRBAs). Classical tardive dyskinesia is a specific type of oro-buccal-lingual dyskinesia. However, TS may exist in other forms—for example, stereotypy, dystonia, and akathisia—and frequently occur in combination. The onset typically is insidious and after reaching its maximum severity it often stabilises. Frequently reported risk factors are age, dose and duration of neuroleptic exposure, the use of conventional DRBAs, and co-existing mood disorders. This review highlights the broad spectrum of TS, not limited to classical tardive dyskinesia, as well as the clues for its recognition. Despite challenges in the treatment of TS, dictated by the different phenomenology, severity of TS and the need for ongoing neuroleptic treatment, the authors provide evidence based recommendations for patient management, which is not restricted to only withdrawal of the offending neuroleptics or the selection of an alternative medication, such as clozapine. In a minority of cases with significant functional disability, symptomatic or suppressive treatments should be considered. Recently, there has been a resurgence of stereotactic pallidal surgery for the treatment of TS. Although the efficacy of both pallidotomy and pallidal deep brain stimulation in dystonia has been encouraging, the evidence is still limited.
Calcitonin gene-related peptide facilitates revascularization during hindlimb ischemia in mice
Authors: Mishima Toshiaki , Ito Yoshiya , Hosono Kanako , Tamura Yukio , Uchida Yasushi , Hirata Mitsuhiro , Suzsuki Tatsunori , Amano Hideki , Kato Shintaro , Kurihara Yukiko , Kurihara Hiroki , Hayashi Izumi , Watanabe Masahiko , Majima Masataka
Published: December 2010

It is known that the neural system plays a fundamental role in neovascularization. A neuropeptide, calcitonin gene-related peptide (CGRP), is widely distributed in the central and peripheral neuronal systems. However, it remains to be elucidated the role of CGRP in angiogenesis during ischemia. The present study examined whether endogenous CGRP released from neuronal systems facilitates revascularization in response to ischemia using CGRP knockout mice (CGRP−/−). CGRP−/− or their wild-type littermates (CGRP+/+) were subjected to unilateral hindlimb ischemia. CGRP−/− exhibited impaired blood flow recovery from ischemia and decreased capillary density expressed in terms of the number of CD-31-positive cells in the ischemic tissues compared with CGRP+/+. In vivo microscopic studies showed that the functional capillary density in CGRP−/− was reduced. Hindlimb ischemia increased the expression of pro-CGRP mRNA and of CGRP protein in the lumbar dorsal root ganglia. Lack of CGRP decreased mRNA expression of growth factors, including CD31, vascular endothelial growth factor-A, basic fibroblast growth factor, and transforming growth factor-β, in the ischemic limb tissue. The application of CGRP enhanced the mRNA expression of CD31 and VEGF-A in human umbilical vein endothelial cells (HUVECs) and fibroblasts. Subcutaneous infusion of CGRP8–37, a CGRP antagonist, using miniosmotic pumps delayed angiogenesis and reduced the expression of proangiogenic growth factors during hindlimb ischemia. These results indicate that endogenous CGRP facilitates angiogenesis in response to ischemia. Targeting CGRP may provide a promising approach for controlling angiogenesis related to pathophysiological conditions.
Detrimental effects of thyroid hormone analog DITPA in the mouse heart: increased mortality with in vivo acute myocardial ischemia-reperfusion
Authors: Hassan Talukder M. A. , Yang Fuchun , Nishijima Yoshinori , Chen Chun-An , Xie Lin , Mahamud Shouvik D. , Kalyanasundaram Anuradha , Bonagura John D. , Periasamy Muthu , Zweier Jay L.
Published: December 2010

There is emerging evidence that treatment with thyroid hormone (TH) can improve postischemic cardiac function. 3,5-Diiodothyropropionic acid (DITPA), a TH analog, has been proposed to be a safer therapeutic agent than TH because of its negligible effects on cardiac metabolism and heart rate. However, conflicting results have been reported for the cardiac effects of DITPA. Importantly, recent clinical trials demonstrated no symptomatic benefit in patients with DITPA despite some improved hemodynamic and metabolic parameters. To address these issues, dose-dependent effects of DITPA were investigated in mice for baseline cardiovascular effects and postischemic myocardial function and/or salvage. Mice were treated with subcutaneous DITPA at 0.937, 1.875, 3.75, or 7.5 mg·kg−1·day−1for 7 days, and the results were compared with untreated mice for ex vivo and/or in vivo myocardial ischemia-reperfusion (I/R). DITPA had no effects on baseline body temperature, body weight, or heart rate; however, it mildly increased blood pressure. In isolated hearts, baseline contractile function was significantly impaired in DITPA-pretreated mice; however, postischemic recovery was comparable between untreated and DITPA-treated groups. In vivo baseline cardiac parameters were significantly affected by DITPA, with increased ventricular dimensions and decreased contractile function. Importantly, DITPA-treated mice demonstrated high prevalence of fatal cardiac rhythm abnormalities during in vivo ischemia and/or reperfusion. There were no improvements in myocardial infarction and postischemic fractional shortening with DITPA. Myocardial sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), phospholamban (PLB), and heat shock protein (HSP) levels remained unchanged with DITPA treatment. Thus DITPA administration impairs baseline cardiac parameters in mice and can be fatal during in vivo acute myocardial I/R.
Early experimental hypertension preserves the myocardial microvasculature but aggravates cardiac injury distal to chronic coronary artery obstruction
Authors: Urbieta Caceres Victor Hugo , Lin Jing , Zhu Xiang-Yang , Favreau Frederic D. , Gibson Matthew E. , Crane John A. , Lerman Amir , Lerman Lilach O.
Published: December 2010

Coronary artery disease is a leading cause of death. Hypertension (HT) increases the incidence of cardiac events, but its effect on cardiac adaptation to coexisting coronary artery stenosis (CAS) is unclear. We hypothesized that concurrent HT modulates microvascular function in chronic CAS and aggravates microvascular remodeling and myocardial injury. Four groups of pigs ( n = 6 each) were studied: normal, CAS, HT, and CAS+HT. CAS and HT were induced by placing local irritant coils in the left circumflex coronary artery and renal artery, respectively. Six weeks later multidetector computerized tomography (CT) was used to assess systolic and diastolic function, microvascular permeability, myocardial perfusion, and responses to adenosine in the “area at risk.” Microvascular architecture, inflammation, and fibrosis were then explored in cardiac tissue. Basal myocardial perfusion was similarly decreased in CAS and CAS+HT, but its response to adenosine was significantly more attenuated in CAS. Microvascular permeability in CAS+HT was greater than in CAS and was accompanied by amplified myocardial inflammation, fibrosis, and microvascular remodeling, as well as cardiac systolic and diastolic dysfunction. On the other hand, compared with normal, micro-CT-derived microvascular (20–200 μm) transmural density decreased in CAS but not in HT or CAS+HT. We conclude that the coexistence of early renovascular HT exacerbated myocardial fibrosis and vascular remodeling distal to CAS. These changes were not mediated by loss of myocardial microvessels, which were relatively preserved, but possibly by exacerbated myocardial inflammation and fibrosis. HT modulates cardiac adaptive responses to CAS and bears cardiac functional consequences.
Hydrogen sulfide and cerebral microvascular tone in newborn pigs
Authors: Leffler Charles W. , Parfenova Helena , Basuroy Shyamali , Jaggar Jonathan H. , Umstot Edward S. , Fedinec Alexander L.
Published: December 2010

Hydrogen sulfide (H2S) is a gaseous signaling molecule that appears to be involved in numerous biological processes, including regulation of blood pressure and vascular tone. The present study is designed to address the hypothesis that H2S is a functionally significant, endogenous dilator in the newborn cerebrovascular circulation. In vivo experiments were conducted using newborn pigs with surgically implanted, closed, cranial windows. Topical application of H2S concentration-dependently (10−6 to 2 × 10−4 M) dilated pial arterioles. This dilation was blocked by glibenclamide (10−6 M). l-Cysteine, the substrate of the H2S-producing enzymes cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS), also dilated pial arterioles. The dilation to l-cysteine was blocked by the CSE inhibitor d,l-propargylglycine (PPG, 10 mM) but was unaffected by the CBS inhibitor amino-oxyacetate (AOA, 1 mM). Western blots detected CSE, but not CBS, in cerebral microvessels, whereas CBS is detected in brain parenchyma. Immunohistological CSE expression is predominantly vascular while CBS is expressed mainly in neurons and astrocytes. l-Cysteine (5 mM) increased H2S concentration in cerebrospinal fluid (CSF), measured by GC-MS, from 561 ± 205 to 2,783 ± 818 nM before but not during treatment with PPG (1,030 ± 70 to 622 ± 78 nM). Dilation to hypercapnia was inhibited by PPG but not AOA. Hypercapnia increased CSF H2S concentration from 763 ± 243 to 4,337 ± 1789 nM before but not during PPG treatment (357 ± 178 vs. 425 ± 217 nM). These data show that H2S is a dilator of the newborn cerebral circulation and that endogenous CSE can produce sufficient H2S to decrease vascular tone. H2S appears to be a physiologically significant dilator in the cerebral circulation.
Large-conductance Ca2+-activated K+channel β1-subunit knockout mice are not hypertensive
Authors: Xu Hui , Garver Hannah , Galligan James J. , Fink Gregory D.
Published: December 2010

Large-conductance Ca2+-activated K+(BK) channels are composed of pore-forming α-subunits and accessory β1-subunits that modulate Ca2+sensitivity. BK channels regulate arterial myogenic tone and renal Na+clearance/K+reabsorption. Previous studies using indirect or short-term blood pressure measurements found that BK channel β1-subunit knockout (BK β1-KO) mice were hypertensive. We evaluated 24-h mean arterial pressure (MAP) and heart rate in BK β1-KO mice using radiotelemetry. BK β1-KO mice did not have a higher 24-h average MAP when compared with wild-type (WT) mice, although MAP was ∼10 mmHg higher at night. The dose-dependent peak declines in MAP by nifedipine were only slightly larger in BK β1-KO mice. In BK β1-KO mice, giving 1% NaCl to mice to drink for 7 days caused a transient (5 days) elevation of MAP (∼5 mmHg); MAP returned to pre-saline levels by day 6. BK β1-KO mesenteric arteries in vitro demonstrated diminished contractile responses to paxilline, increased reactivity to Bay K 8644 and norepinephrine (NE), and maintained relaxation to isoproterenol. Paxilline and Bay K 8644 did not constrict WT or BK β1-KO mesenteric veins (MV). BK β1-subunits are not expressed in MV. The results indicate that BK β1-KO mice are not hypertensive on normal or high-salt intake. BK channel deficiency increases arterial reactivity to NE and L-type Ca2+channel function in vitro, but the L-type Ca2+channel modulation of MAP is not altered in BK β1-KO mice. BK and L-type Ca2+channels do not modulate murine venous tone. It appears that selective loss of BK channel function in arteries only is not sufficient to cause sustained hypertension.
New concepts of IL-10-induced lung fibrosis: fibrocyte recruitment and M2 activation in a CCL2/CCR2 axis
Authors: Sun Lei , Louie Marisa C. , Vannella Kevin M. , Wilke Carol A. , LeVine Ann Marie , Moore Bethany B. , Shanley Thomas P.
Published: December 2010

IL-10 is most commonly recognized as an anti-inflammatory cytokine possessing immunosuppressive effects necessary for regulated resolution of proinflammation. However, its role in the development of fibrosis during inflammatory resolution has not been clear. Few prior studies have linked IL-10 with the inhibition of fibrosis principally on the basis of regulating inflammation thought to be driving fibroproliferation. In contrast, in a model of long-term overexpression of IL-10, we observed marked induction of lung fibrosis in mice. The total cell number retrieved by bronchoalveolar lavage (BAL) increased 10-fold in the IL-10 overexpression (IL-10 OE) mice, with significant infiltration of T and B lymphocytes and collagen-producing cells. The presence of increased fibrocytes, isolated from collagenase-digested lungs, was identified by flow cytometry using dual staining of CD45 and collagen 1. Quantitative PCR analysis on an array of chemokine/chemokine receptor genes showed that receptor CCR2 and its ligand, CCL2, were highly upregulated in IL-10 OE mice, suggesting that IL-10-induced fibrocyte recruitment was CCL2/CCR2 specific. Given the prior association of alternatively activated (M2) macrophages with development of fibrosis in other disease states, we also examined the effect of IL-10 OE on the M2 macrophage axis. We observed significantly increased numbers of M2 macrophages in both BAL and whole lung tissue from the IL-10 OE mice. Administration of rabbit anti-CCL2 antiserum to IL-10 OE mice for three consecutive weeks significantly decreased fibrosis as evidenced by lung hydroxyproline content, compared with mice that received preimmune rabbit serum. These results indicate that overexpression of IL-10 induces fibrosis, in part, by fibrocyte recruitment and M2 macrophage activation, and likely in a CCL2/CCR2 axis.
TGF-β regulates Nox4, MnSOD and catalase expression, and IL-6 release in airway smooth muscle cells
Authors: Michaeloudes Charalambos , Sukkar Maria B. , Khorasani Nadia M. , Bhavsar Pankaj K. , Chung Kian Fan
Published: December 2010

Reactive oxygen species (ROS) are generated as a result of normal cellular metabolism, mainly through the mitochondria and peroxisomes, but their release is enhanced by the activation of oxidant enzymes such as NADPH oxidases or downregulation of endogenous antioxidant enzymes such as manganese-superoxide dismutase (MnSOD) and catalase. Transforming growth factor-β (TGF-β), found to be overexpressed in airway smooth muscle (ASM) from asthmatic and chronic obstructive pulmonary disease patients, may be a pivotal regulator of abnormal ASM cell (ASMC) function in these diseases. An important effect of TGF-β on ASMC inflammatory responses is the induction of IL-6 release. TGF-β also triggers intracellular ROS release in ASMCs by upregulation of NADPH oxidase 4 (Nox4). However, the effect of TGF-β on the expression of key antioxidant enzymes and subsequently on oxidant/antioxidant balance is unknown. Moreover, the role of redox-dependent pathways in the mediation of the proinflammatory effects of TGF-β in ASMCs is unclear. In this study, we show that TGF-β induced the expression of Nox4 while at the same time inhibiting the expression of MnSOD and catalase. This change in oxidant/antioxidant enzymes was accompanied by elevated ROS levels and IL-6 release. Further studies revealed a role for Smad3 and phosphatidyl-inositol kinase-mediated pathways in the induction of oxidant/antioxidant imbalance and IL-6 release. The changes in oxidant/antioxidant enzymes and IL-6 release were reversed by the antioxidants N-acetyl-cysteine (NAC) and ebselen through inhibition of Smad3 phosphorylation, indicating redox-dependent activation of Smad3 by TGF-β. Moreover, these findings suggest a potential role for NAC in preventing TGF-β-mediated pro-oxidant and proinflammatory responses in ASMCs. Knockdown of Nox4 using small interfering RNA partially prevented the inhibition of MnSOD but had no effect on catalase and IL-6 expression. These findings provide novel insights into redox regulation of ASM function by TGF-β.
Persistent rhinitis and epithelial remodeling induced by cyclic ozone exposure in the nasal airways of infant monkeys
Authors: Carey Stephan A. , Ballinger Carol A. , Plopper Charles G. , McDonald Ruth J. , Bartolucci Alfred A. , Postlethwait Edward M. , Harkema Jack R.
Published: December 2010

Children chronically exposed to high levels of ozone (O3), the principal oxidant pollutant in photochemical smog, are more vulnerable to respiratory illness and infections. The specific factors underlying this differential susceptibility are unknown but may be related to air pollutant-induced nasal alterations during postnatal development that impair the normal physiological functions (e.g., filtration and mucociliary clearance) serving to protect the more distal airways from inhaled xenobiotics. In adult animal models, chronic ozone exposure is associated with adaptations leading to a decrease in airway injury. The purpose of our study was to determine whether cyclic ozone exposure induces persistent morphological and biochemical effects on the developing nasal airways of infant monkeys early in life. Infant (180-day-old) rhesus macaques were exposed to 5 consecutive days of O3[0.5 parts per million (ppm), 8 h/day; “1-cycle”] or filtered air (FA) or 11 biweekly cycles of O3(FA days 1– 9; 0.5 ppm, 8 h/day on days 10– 14; “11-cycle”). The left nasal passage was processed for light microscopy and morphometric analysis. Mucosal samples from the right nasal passage were processed for GSH, GSSG, ascorbate (AH2), and uric acid (UA) concentration. Eleven-cycle O3induced persistent rhinitis, squamous metaplasia, and epithelial hyperplasia in the anterior nasal airways of infant monkeys, resulting in a 39% increase in the numeric density of epithelial cells. Eleven-cycle O3also induced a 65% increase in GSH concentrations at this site. The persistence of epithelial hyperplasia was positively correlated with changes in GSH. These results indicate that early life ozone exposure causes persistent nasal epithelial alterations in infant monkeys and provide a potential mechanism for the increased susceptibility to respiratory illness exhibited by children in polluted environments.