bluuebunny/crossref_metadata_2025 huggingface dataset:
Inactivation of a Human Norovirus Surrogate by High-Pressure Processing: Effectiveness, Mechanism, and Potential Application in the Fresh Produce Industry
Authors: Lou Fangfei , Neetoo Hudaa , Chen Haiqiang , Li Jianrong
Published: December 2010

Fresh produce is often a high-risk food for norovirus contamination because it can become contaminated at both preharvest and postharvest stages and it undergoes minimal or no processing. Currently, there is no effective method to eliminate the viruses from fresh produce. This study systematically investigated the effectiveness of high-pressure processing (HPP) on inactivating murine norovirus (MNV-1), a surrogate for human norovirus, in aqueous medium and fresh produce. We demonstrated that MNV-1 was effectively inactivated by HPP. More than a 5-log-PFU/g reduction was achieved in all tested fresh produce when it was pressurized at 400 MPa for 2 min at 4°C. We found that pressure, pH, temperature, and food matrix affected the virus survival in foods. MNV-1 was more effectively inactivated at 4°C than at 20°C in both medium and fresh produce. MNV-1 was also more sensitive to HPP at neutral pH than at acidic pH. We further demonstrated that disruption of viral capsid structure, but not degradation of viral genomic RNA, is the primary mechanism of virus inactivation by HPP. However, HPP does not degrade viral capsid protein, and the pressurized capsid protein was still antigenic. Overall, HPP had a variable effect on the sensorial quality of fresh produce, depending on the pressure level and type of product. Taken together, HPP effectively inactivated a human norovirus surrogate in fresh produce with a minimal impact on food quality and thus can provide a novel intervention for processing fruits intended for frozen storage and related products such as purees, sauces, and juices.
Quantitative PCR Coupled with Melt Curve Analysis for Detection of SelectedPseudo-nitzschiaspp. (Bacillariophyceae) from the Northwestern Mediterranean Sea
Authors: Andree Karl B. , Fernández-Tejedor Margarita , Elandaloussi Laurence M. , Quijano-Scheggia Sonia , Sampedro Nagore , Garcés Esther , Camp Jordi , Diogène Jorge
Published: December 2010

The frequency and intensity ofPseudo-nitzschiaspp. blooms along the coast of Catalonia have been increasing over the past 20 years. As species from this genus that are documented as toxigenic have been found in local waters, with both toxic and nontoxic species cooccurring in the same bloom, there is a need to develop management tools for discriminating the difference. Currently, differentiation of toxic and nontoxic species requires time-consuming electron microscopy to distinguish taxonomic features that would allow identification as to species, and cryptic species can still remain misidentified. In this study, cells ofPseudo-nitzschiafrom clonal cultures isolated from seawater were characterized to their species identity using scanning electron microscopy, and subsamples of each culture were used to create an internal transcribed spacer 1 (ITS-1), 5.8S, and ITS-2 ribosomal DNA database for development of species-specific quantitative PCR (qPCR) assays. Once developed, these qPCR assays were applied to field samples collected over a 2-year period in Alfaques Bay in the northwestern Mediterranean Sea to evaluate the possibility of a comprehensive surveillance for allPseudo-nitzschiaspp. using molecular methods to supplement optical microscopy, which can discern taxonomy only to the genus level within this taxon. TotalPseudo-nitzschiacell density was determined by optical microscopy from water samples collected weekly and compared to results obtained from the sum of eightPseudo-nitzschiaspecies-specific qPCR assays using duplicate samples. Species-specific qPCR followed by melt curve analysis allowed differentiation of amplicons and identification of false positives, and results correlated well with the totalPseudo-nitzschiacell counts from optical microscopy.
Analyses of both the
alkB
Gene Transcriptional Start Site and
alkB
Promoter-Inducing Properties of
Rhodococcus
sp. Strain BCP1 Grown on
n
-Alkanes
Authors: Cappelletti M. , Fedi S. , Frascari D. , Ohtake H. , Turner R. J. , Zannoni D.
Published: December 2010

Rhodococcus
sp. strain BCP1, known for its capacity to grow on short-chain
n
-alkanes (C
2
to C
7
) and to cometabolize chlorinated solvents, was found to also utilize medium- and long-chain
n
-alkanes (C
12
to C
24
) as energy and carbon sources. To examine this feature in detail, a chromosomal region which includes the
alkB
gene cluster encoding a non-heme di-iron monooxygenase (
alkB
), two rubredoxins, and one rubredoxin reductase was cloned from the BCP1 genome. Furthermore, the activity of the
alkB
gene promoter (P

alkB

) was examined in the presence of gaseous, liquid, and solid
n
-alkanes along with intermediates of the putative
n
-alkane degradation pathway. A recombinant plasmid, pTP
alkB
LacZ, was constructed by inserting the
lacZ
gene downstream of P

alkB

, and it was used to transform
Rhodococcus
sp. strain BCP1. Measurements of β-galactosidase activity showed that P

alkB

is induced by C
6
to C
22
n
-alkanes. Conversely, C
2
to C
5
and >C
22
n
-alkanes and alkenes, such as hexene, were not inducers of
alkB
expression. The effects on P

alkB

expression induced by alternative carbon sources along with putative products of
n
-hexane metabolism were also evaluated. This report highlights the great versatility of
Rhodococcus
sp. strain BCP1 and defines for the first time the
alkB
gene transcriptional start site and the
alkB
promoter-inducing capacities for substrates different from
n
-alkanes in a
Rhodococcus
strain.
Efficient Homofermentative
l
-(+)-Lactic Acid Production from Xylose by a Novel Lactic Acid Bacterium,
Enterococcus mundtii
QU 25
Authors: Abdel-Rahman Mohamed Ali , Tashiro Yukihiro , Zendo Takeshi , Hanada Katsuhiro , Shibata Keisuke , Sonomoto Kenji
Published: December 2010

Enterococcus mundtii
QU 25, a newly isolated lactic acid bacterium, efficiently metabolized xylose into
l
-lactate. In batch fermentations, the strain produced 964 mM
l
-(+)-lactate from 691 mM xylose, with a yield of 1.41 mol/mol xylose consumed and an extremely high optical purity of ≥99.9% without acetate production.
Role of the Cell Wall Microenvironment in Expression of a Heterologous SpaP-S1 Fusion Protein by
Streptococcus gordonii
Authors: Davis Elisabeth , Kennedy Dustin , Halperin Scott A. , Lee Song F.
Published: December 2010

The charge density in the cell wall microenvironment of Gram-positive bacteria is believed to influence the expression of heterologous proteins. To test this, the expression of a SpaP-S1 fusion protein, consisting of the surface protein SpaP of
St
r
eptococcus mutans
and a pertussis toxin S1 fragment, was studied in the live vaccine candidate bacterium
Streptococcus gordonii
. Results showed that the parent strain PM14 expressed very low levels of SpaP-S1. By comparison, the
dlt
mutant strain, which has a mutation in the
dlt
operon preventing
d
-alanylation of the cell wall lipoteichoic acids, and another mutant strain, OB219(pPM14), which lacks the LPXTG major surface proteins SspA and SspB, expressed more SpaP-S1 than the parent. Both the
dlt
mutant and the OB219(pPM14) strain had a more negatively charged cell surface than PM14, suggesting that the negative charged cell wall played a role in the increase in SpaP-S1 production. Accordingly, the addition of Ca
2+
, Mg
2+
, and K
+
, presumably increasing the positive charge of the cell wall, led to a reduction in SpaP-S1 production, while the addition of bicarbonate resulted in an increase in SpaP-S1 production. The level of SpaP-S1 production could be correlated with the level of PrsA, a peptidyl-prolyl
cis/trans
isomerase, in the cells. PrsA expression appears to be regulated by the cell envelope stress two-component regulatory system LiaSR. The results collectively indicate that the charge density of the cell wall microenvironment can modulate heterologous SpaP-S1 protein expression in
S. gordonii
and that this modulation is mediated by the level of PrsA, whose expression is regulated by the LiaSR two-component regulatory system.
Development of an Aerosol Surface Inoculation Method for
Bacillus
Spores
Authors: Lee Sang Don , Ryan Shawn P. , Snyder Emily Gibb
Published: December 2010

A method was developed to deposit
Bacillus subtilis
spores via aerosolization onto various surface materials for biological agent decontamination and detection studies. This new method uses an apparatus coupled with a metered dose inhaler to reproducibly deposit spores onto various surfaces. A metered dose inhaler was loaded with
Bacillus subtilis
spores, a surrogate for
Bacillus anthracis
. Five different material surfaces (aluminum, galvanized steel, wood, carpet, and painted wallboard paper) were tested using this spore deposition method. This aerosolization method deposited spores at a concentration of more than 10
7
CFU per coupon (18-mm diameter) with less than a 50% coefficient of variation, showing that the aerosolization method developed in this study can deposit reproducible numbers of spores onto various surface coupons. Scanning electron microscopy was used to probe the spore deposition patterns on test coupons. The deposition patterns observed following aerosol impaction were compared to those of liquid inoculation. A physical difference in the spore deposition patterns was observed to result from the two different methods. The spore deposition method developed in this study will help prepare spore coupons via aerosolization fast and reproducibly for bench top decontamination and detection studies.
Specific Real-Time PCR for Simultaneous Detection and Identification of
Legionella pneumophila
Serogroup 1 in Water and Clinical Samples
Authors: Mérault N. , Rusniok C. , Jarraud S. , Gomez-Valero L. , Cazalet C. , Marin M. , Brachet E. , Aegerter P. , Gaillard J. L. , Etienne J. , Herrmann J. L. , Lawrence C. , Buchrieser C.
Published: December 2010

Legionella pneumophila
, a bacterium that replicates within aquatic amoebae and persists in the environment as a free-living microbe, is the causative agent of Legionnaires' disease. Among the many
Legionella
species described,
L. pneumophila
is associated with 90% of human disease, and within the 15 serogroups (Sg),
L. pneumophila
Sg1 causes more than 84% of Legionnaires' disease worldwide. Thus, rapid and specific identification of
L. pneumophila
Sg1 is of the utmost importance for evaluation of the contamination of collective water systems and the risk posed. Previously we had shown that about 20 kb of the 33-kb locus carrying the genes coding for the proteins involved in lipopolysaccharide biosynthesis (LPS gene cluster) by
L. pneumophila
was highly specific for Sg1 strains and that three genes (
lpp0831
,
wzm
, and
wzt
) may serve as genetic markers. Here we report the sequencing and comparative analyses of this specific region of the LPS gene cluster in
L. pneumophila
Sg6, -10, -12, -13, and -14. Indeed, the
wzm
and
wzt
genes were present only in the Sg1 LPS gene cluster, which showed a very specific gene content with respect to the other five serogroups investigated. Based on this observation, we designed primers and developed a classical and a real-time PCR method for the detection and simultaneous identification of
L. pneumophila
Sg1 in clinical and environmental isolates. Evaluation of the selected primers with 454
Legionella
and 38 non-
Legionella
strains demonstrated 100% specificity. Sensitivity, specificity, and predictive values were further evaluated with 209 DNA extracts from water samples of hospital water supply systems and with 96 respiratory specimens. The results showed that the newly developed quantitative Sg1-specific PCR method is a highly specific and efficient tool for the surveillance and rapid detection of high-risk
L. pneumophila
Sg1 in water and clinical samples.
Real-Time Solvent Tolerance Analysis ofPseudomonassp. Strain VLB120ΔC Catalytic Biofilms
Authors: Halan Babu , Schmid Andreas , Buehler Katja
Published: December 2010

Biofilms are ubiquitous surface-associated microbial communities embedded in an extracellular polymeric (EPS) matrix, which gives the biofilm structural integrity and strength. It is often reported that biofilm-grown cells exhibit enhanced tolerance toward adverse environmental stress conditions, and thus there has been a growing interest in recent years to use biofilms for biotechnological applications. We present a time- and locus-resolved, noninvasive, quantitative approach to study biofilm development and its response to the toxic solvent styrene.Pseudomonassp. strain VLB120ΔC-BT-gfp1 was grown in modified flow-cell reactors and exposed to the solvent styrene. Biofilm-grown cells displayed stable catalytic activity, producing (S)-styrene oxide continuously during the experimental period. The pillar-like structure and growth rate of the biofilm was not influenced by the presence of the solvent. However, the cells experience severe membrane damage during styrene treatment, although they obviously are able to adapt to the solvent, as the amount of permeabilized cells decreased from 75 to 80% down to 40% in 48 h. Concomitantly, the fraction of concanavalin A (ConA)-stainable EPS increased, substantiating the assumption that those polysaccharides play a major role in structural integrity and enhanced biofilm tolerance toward toxic environments. Compared to control experiments with planktonic grown cells, thePseudomonasbiofilm adapted much better to toxic concentrations of styrene, as nearly 65% of biofilm cells were not permeabilized (viable), compared to only 7% in analogous planktonic cultures. These findings underline the robustness of biofilms under stress conditions and its potential for fine chemical syntheses.
Lag Phase of
Salmonella enterica
under Osmotic Stress Conditions
Authors: Zhou K. , George S. M. , Métris A. , Li P. L. , Baranyi J.
Published: December 2010

Salmonella enterica
serovar Typhimurium was grown at salt concentrations ranging from 0.5 to 7.5% in minimal medium with and without added osmoprotectant and in a rich medium. In minimal medium, the cells showed an initial decline period, and consequently the definition of the lag time of the resultant log count curve was revised. The model of Baranyi and Roberts (Int. J. Food Microbiol. 23:277-294, 1994) was modified to take into account the initial decline period, based on the assumption that the log count curve of the total population was the sum of a dying and a surviving-then-growing subpopulation. The lag time was defined as the lag of the surviving subpopulation. It was modeled by means of a parameter quantifying the biochemical work the surviving cells carry out during this phase, the “work to be done.” The logarithms of the maximum specific growth rates as a function of the water activity in the three media differed only by additive constants, which gave a theoretical basis for bias factors characterizing the relationships between different media. Models for the lag and the “work to be done” as a function of the water activity showed similar properties, but in rich medium above 5% salt concentrations, the data showed a maximum for this work. An accurate description of the lag time is important to avoid food wastage, which is an issue of increasing significance in the food industry, while maintaining food safety standards.
Metallic Copper as an Antimicrobial Surface
Authors: Grass Gregor , Rensing Christopher , Solioz Marc
Published: December 2010

Bacteria, yeasts, and viruses are rapidly killed on metallic copper surfaces, and the term “contact killing” has been coined for this process. While the phenomenon was already known in ancient times, it is currently receiving renewed attention. This is due to the potential use of copper as an antibacterial material in health care settings. Contact killing was observed to take place at a rate of at least 7 to 8 logs per hour, and no live microorganisms were generally recovered from copper surfaces after prolonged incubation. The antimicrobial activity of copper and copper alloys is now well established, and copper has recently been registered at the U.S. Environmental Protection Agency as the first solid antimicrobial material. In several clinical studies, copper has been evaluated for use on touch surfaces, such as door handles, bathroom fixtures, or bed rails, in attempts to curb nosocomial infections. In connection to these new applications of copper, it is important to understand the mechanism of contact killing since it may bear on central issues, such as the possibility of the emergence and spread of resistant organisms, cleaning procedures, and questions of material and object engineering. Recent work has shed light on mechanistic aspects of contact killing. These findings will be reviewed here and juxtaposed with the toxicity mechanisms of ionic copper. The merit of copper as a hygienic material in hospitals and related settings will also be discussed.
Genomewide Expression Profile Analysis of the Candida glabrata Pdr1 Regulon
Authors: Caudle Kelly E. , Barker Katherine S. , Wiederhold Nathan P. , Xu Lijing , Homayouni Ramin , Rogers P. David
Published: December 2010

The ABC transporters
Candida glabrata
Cdr1 (CgCdr1), CgPdh1, and CgSnq2 are known to mediate azole resistance in the pathogenic fungus
C. glabrata
. Activating mutations in
CgPDR1
, a zinc cluster transcription factor, result in constitutive upregulation of these ABC transporter genes but to various degrees. We examined the genomewide gene expression profiles of two matched azole-susceptible and -resistant
C. glabrata
clinical isolate pairs. Of the differentially expressed genes identified in the gene expression profiles for these two matched pairs, there were 28 genes commonly upregulated with
CgCDR1
in both isolate sets including
YOR1
,
LCB5
,
RTA1
,
POG1
,
HFD1
, and several members of the
FLO
gene family of flocculation genes. We then sequenced
CgPDR1
from each susceptible and resistant isolate and found two novel activating mutations that conferred increased resistance when they were expressed in a common background strain in which
CgPDR1
had been disrupted. Microarray analysis comparing these reengineered strains to their respective parent strains identified a set of commonly differentially expressed genes, including
CgCDR1
,
YOR1
, and
YIM1
, as well as genes uniquely regulated by specific mutations. Our results demonstrate that while CgPdr1 activates a broad repertoire of genes, specific activating mutations result in the activation of discrete subsets of this repertoire.
Global Analysis of Protein Palmitoylation in African Trypanosomes
Authors: Emmer Brian T. , Nakayasu Ernesto S. , Souther Christina , Choi Hyungwon , Sobreira Tiago J. P. , Epting Conrad L. , Nesvizhskii Alexey I. , Almeida Igor C. , Engman David M.
Published: December 2010

Many eukaryotic proteins are posttranslationally modified by the esterification of cysteine thiols to long-chain fatty acids. This modification, protein palmitoylation, is catalyzed by a large family of palmitoyl acyltransferases that share an Asp-His-His-Cys Cys-rich domain but differ in their subcellular localizations and substrate specificities. In
Trypanosoma brucei
, the flagellated protozoan parasite that causes African sleeping sickness, protein palmitoylation has been observed for a few proteins, but the extent and consequences of this modification are largely unknown. We undertook the present study to investigate
T. brucei
protein palmitoylation at both the enzyme and substrate levels. Treatment of parasites with an inhibitor of total protein palmitoylation caused potent growth inhibition, yet there was no effect on growth by the separate, selective inhibition of each of the 12 individual
T. brucei
palmitoyl acyltransferases. This suggested either that
T. brucei
evolved functional redundancy for the palmitoylation of essential palmitoyl proteins or that palmitoylation of some proteins is catalyzed by a noncanonical transferase. To identify the palmitoylated proteins in
T. brucei
, we performed acyl biotin exchange chemistry on parasite lysates, followed by streptavidin chromatography, two-dimensional liquid chromatography-tandem mass spectrometry protein identification, and QSpec statistical analysis. A total of 124 palmitoylated proteins were identified, with an estimated false discovery rate of 1.0%. This palmitoyl proteome includes all of the known palmitoyl proteins in procyclic-stage
T. brucei
as well as several proteins whose homologues are palmitoylated in other organisms. Their sequences demonstrate the variety of substrate motifs that support palmitoylation, and their identities illustrate the range of cellular processes affected by palmitoylation in these important pathogens.
Regulation of Central Metabolism Genes of
Mycobacterium tuberculosis
by Parallel Feed-Forward Loops Controlled by Sigma Factor E (σ
E
)
Authors: Datta Pratik , Shi Lanbo , Bibi Nazia , Balázsi Gábor , Gennaro Maria Laura
Published: December 2010

Cells respond to external stimuli through networks of regulatory interactions. The human pathogen
Mycobacterium tuberculosis
responds to stress encountered during infection by arresting multiplication and implementing critical metabolic changes that lead to or sustain the nonreplicative state. Much of this differentiation program is recapitulated when
M. tuberculosis
cultures are subjected to gradual oxygen depletion
in vitro
. Here we report that hypoxic induction of critical central metabolism genes in the glyoxylate shunt (
icl1
) and in the methylcitrate cycle (
gltA1
) involves both global and local regulators. The global regulators are accessory sigma factors σ
B
for
icl1
and σ
E
for
gltA1
. The local regulators are the products of two paralogous genes mapping at positions adjacent to the corresponding effector gene or operon. We call these genes
lrpI
and
lrpG
(for

l

ocal

r

egulatory

p

rotein of
icl1
and
gltA1
). We also found that (i) each sigma factor controls the corresponding local regulator, (ii) both global and local regulators are required for effector gene induction, and (iii) the occurrence of sigma factor control of effector gene induction is independent of its control over the corresponding local regulator. Together, these data indicate that induction of
icl1
and
gltA1
utilizes parallel feed-forward loops with an AND input function. Both feed-forward loops are affected by σ
E
, since this sigma factor is part of the
gltA1
loop and controls
sigB
in the
icl1
loop. Feed-forward loops may critically contribute to the cellular developmental program associated with
M. tuberculosis
dormancy.
Upregulation of MetC Is Essential for
d
-Alanine-Independent Growth of an
alr/dadX
-Deficient
Escherichia coli
Strain
Authors: Kang Lishan , Shaw Allan C. , Xu Daqi , Xia Wenjuan , Zhang Jingyuan , Deng Jianhui , Wöldike Helle F. , Liu Yun , Su Jing
Published: December 2010

d
-Alanine is a central component of the cell wall in most prokaryotes.
d
-Alanine synthesis in
Escherichia coli
is carried out by two different alanine racemases encoded by the
alr
and
dadX
genes. Deletion of
alr
and
dadX
from the
E. coli
genome results in a
d
-alanine auxotrophic phenotype. However, we have observed growth of prototrophic phenotypic revertants during routine culturing of a
d
-alanine auxotrophic strain. We present a detailed comparison of the proteome and transcriptome profiles of the
d
-alanine auxotroph and a prototrophic revertant strain. Most noticeably, a general upregulation of genes involved in methionine synthesis in the revertant strain was detected. The appearance of the revertant phenotype was genetically linked to point mutations in the methionine repressor gene (
metJ
). Our results reveal an alternative metabolic pathway which can supply essential
d
-alanine for peptidoglycan synthesis of
alr
- and
dadX
-deficient
E. coli
mutants and provide evidence for significant alanine racemase coactivity of the
E. coli
cystathionine beta-lyase (MetC).
Defining the Plasmid-Borne Restriction-Modification Systems of the Lyme Disease SpirocheteBorrelia burgdorferi
Authors: Rego Ryan O. M. , Bestor Aaron , Rosa Patricia A.
Published: December 2010

The restriction-modification (R-M) systems of many bacteria present a barrier to the stable introduction of foreign DNA. The Lyme disease spirocheteBorrelia burgdorferihas two plasmid-borne putative R-M genes,bbe02andbbq67, whose presence limits transformation by shuttle vector DNA fromEscherichia coli. We show that both thebbe02andbbq67loci in recipientB. burgdorferilimit transformation with shuttle vector DNA fromE. coli, irrespective of itsdam,dcm, orhsdmethylation status. However, plasmid DNA purified fromB. burgdorferitransformed naïveB. burgdorferimuch more efficiently than plasmid DNA fromE. coli, particularly when thebbe02andbbq67genotypes of theB. burgdorferiDNA source matched those of the recipient. We detected adenine methylation of plasmid DNA prepared fromB. burgdorferithat carriedbbe02andbbq67. These results indicate that thebbe02andbbq67loci ofB. burgdorferiencode distinct R-M enzymes that methylate endogenous DNA and cleave foreign DNA lacking the same sequence-specific modification. Our findings have basic implications for horizontal gene transfer amongB. burgdorferistrains with distinct plasmid contents. Further characterization and identification of the nucleotide sequences recognized by BBE02 and BBQ67 will facilitate efficient genetic manipulation of this pathogenic spirochete.
Specific Role of the Cyanobacterial PipX Factor in the Heterocysts of
Anabaena
sp. Strain PCC 7120
Authors: Valladares Ana , Rodríguez Virginia , Camargo Sergio , Martínez-Noël Giselle M. A. , Herrero Antonia , Luque Ignacio
Published: December 2010

The PipX factor is a regulatory protein that seems to occur only in cyanobacteria. In the filamentous, heterocyst-forming
Anabaena
sp. strain PCC 7120, open reading frame (ORF)
asr0485
, identified as the
pipX
gene, is expressed mainly under conditions of combined-nitrogen deprivation dependent on the global N regulator NtcA and the heterocyst-specific regulator HetR. Primer extension and 5′ rapid amplification of cDNA ends (RACE) analyses detected three transcription start points corresponding to a canonical NtcA-activated promoter (to which direct binding of NtcA was observed), an NtcA- and HetR-dependent promoter, and a consensus-type promoter, the last with putative −35 and −10 determinants. Activation of
pipX
took place in cells differentiating into heterocysts at intermediate to late stages of the process. Accordingly, disruption of
pipX
led to impaired diazotrophic growth, reduced nitrogenase activity, and impaired activation of the nitrogenase structural genes. The nitrogenase activity of the mutant was low under oxic conditions, likely resulting from inefficient protection against oxygen. In line with this, the activation of the
coxB2A2C2
and
coxB3A3C3
operons, encoding heterocyst-specific terminal respiratory oxidases responsible for internal oxygen removal, was deficient in the
pipX
mutant. Therefore, the
Anabaena
PipX factor shows a spatiotemporal specificity contributing to normal heterocyst function, including full activation of the nitrogenase structural genes and genes of the nitrogenase-protective features of the heterocyst.
Phenotypic and Genotypic Evidence for
l
-Fucose Utilization by
Campylobacter jejuni
Authors: Muraoka Wayne T. , Zhang Qijing
Published: December 2010

Campylobacter jejuni
remains among the leading causes of bacterial food-borne illness. The current understanding of
Campylobacter
physiology suggests that it is asaccharolytic and is unable to catabolize exogenous carbohydrates. Contrary to this paradigm, we provide evidence for
l
-fucose utilization by
C. jejuni
. The fucose phenotype, shown in chemically defined medium, is strain specific and linked to an 11-open reading frame (ORF) plasticity region of the bacterial chromosome. By constructing a mutation in
fucP
(encoding a putative fucose permease), one of the genes in the plasticity region, we found that this locus is required for fucose utilization. Consistent with their function in fucose utilization, transcription of the genes in the locus is highly inducible by fucose. PCR screening revealed a broad distribution of this genetic locus in strains derived from various host species, and the presence of this locus was consistently associated with fucose utilization. Birds inoculated with the
fucP
mutant strain alone were colonized at a level comparable to that by the wild-type strain; however, in cocolonization experiments, the mutant was significantly outcompeted by the wild-type strain when birds were inoculated with a low dose (10
5
CFU per bird). This advantage was not observed when birds were inoculated at a higher inoculum dose (10
8
CFU per bird). These results demonstrated a previously undescribed substrate that supports growth of
C. jejuni
and identified the genetic locus associated with the utilization of this substrate. These findings substantially enhance our understanding of the metabolic repertoire of
C. jejuni
and the role of metabolic diversity in
Campylobacter
pathobiology.
Characterization and Manipulation of the Pathway-Specific Late Regulator AlpW RevealsStreptomyces ambofaciensas a New Producer of Kinamycins
Authors: Bunet Robert , Song Lijiang , Mendes Marta Vaz , Corre Christophe , Hotel Laurence , Rouhier Nicolas , Framboisier Xavier , Leblond Pierre , Challis Gregory L. , Aigle Bertrand
Published: December 2010

The genome sequence ofStreptomyces ambofaciens, a species known to produce the congocidine and spiramycin antibiotics, has revealed the presence of numerous gene clusters predicted to be involved in the biosynthesis of secondary metabolites. Among them, the type II polyketide synthase-encodingalpcluster was shown to be responsible for the biosynthesis of a compound with antibacterial activity. Here, by means of a deregulation approach, we gained access to workable amounts of the antibiotics for structure elucidation. These compounds, previously designated as alpomycin, were shown to be known members of kinamycin family of antibiotics. Indeed, a mutant lacking AlpW, a member of the TetR regulator family, was shown to constitutively produce kinamycins. Comparative transcriptional analyses showed that expression ofalpV, the essential regulator gene required for activation of the biosynthetic genes, is strongly maintained during the stationary growth phase in thealpWmutant, a stage at whichalpVtranscripts and thereby transcripts of the biosynthetic genes normally drop off. Recombinant AlpW displayed DNA binding activity toward specific motifs in the promoter region of its own gene and that ofalpVandalpZ. These recognition sequences are also targets for AlpZ, the γ-butyrolactone-like receptor involved in the regulation of thealpcluster. However, unlike that of AlpZ, the AlpW DNA-binding ability seemed to be insensitive to the signaling molecules controlling antibiotic biosynthesis. Together, the results presented in this study revealS. ambofaciensto be a new producer of kinamycins and AlpW to be a key late repressor of the cellular control of kinamycin biosynthesis.
Mental health and unintentional injurers: results from the national co-morbidity survey replication
Authors: Connorton Ellen , Miller Matthew , Perry Melissa J , Hemenway David
Published: December 2010

Objective

To examine whether unintentionally injuring others is associated with subsequent mental illness, and whether mental illness is a risk factor for unintentionally injuring others.

Methods

Onset of first psychiatric diagnoses was compared with onset of first unintentional injuring. Multivariate logistic regression estimated the association between unintentional injuring and lifetime prevalence of mental illness, specifically of Diagnostic and Statistical Manual of Mental Disorders IV disorders associated with trauma exposure among respondents of part II of the National Co-morbidity Survey Replication a household survey of 5692 US adults. Analyses controlled for age, sex, race and having been injured in a serious accident.

Results

Of 5692 respondents, 110 reported unintentionally causing death or injury to another person. Unintentionally injuring others was a risk factor for subsequent mental health problems. Multivariate regression results showed an increased risk of subsequent depression (OR 3.1, CI 1.7 to 5.7), anxiety (OR 3.3, CI 1.6 to 6.6), posttraumatic stress disorder (PTSD) (OR 6.6, CI 3.1 to 14.0), alcohol use (OR 3.8, CI 1.9 to 7.3) and drug use (OR 8.0, CI 4.1 to 15.3). Conversely, mental health problems were a risk factor for unintentionally injuring another person. Multivariate regression results showed an increased risk of unintentional injuring among those with a prior diagnosis of depression, PTSD, alcohol use, and drug use.

Conclusions

After injuring, unintentional injurers are likely to experience depression, anxiety, PTSD and drug or alcohol abuse/dependence compared with non-injurers. Those diagnosed with depression, anxiety, PTSD, alcohol or drug or alcohol abuse/dependence are more likely to cause serious injuries to others.
iNOS expression in vascular resident macrophages contributes to circulatory dysfunction of splanchnic vascular smooth muscle contractions in portal hypertensive rats
Authors: Kajita Masahiro , Murata Takahisa , Horiguchi Kazuhide , Iizuka Masateru , Hori Masatoshi , Ozaki Hiroshi
Published: December 2010

Portal hypertension, a major complication of cirrhosis, is caused by both increased portal blood flow due to arterial vasodilation and augmented intrahepatic vascular resistance due to sinusoidal constriction. In this study, we examined the possible involvement of resident macrophages in the tone regulation of splanchnic blood vessels using bile duct ligated (BDL) portal hypertensive rats and an in vitro organ culture method. In BDL cirrhosis, the number of ED2-positive resident macrophages increased by two- to fourfold in the vascular walls of the mesenteric artery and extrahepatic portal vein compared with those in sham-operated rats. Many ED1-positive monocytes were also recruited into this area. The expression of inducible nitric oxide (NO) synthase (iNOS) mRNA was increased in the vascular tissues isolated from BDL rats, and accordingly, nitrate/nitrite production was increased. Immunohistochemistry revealed that iNOS was largely expressed in ED1-positive and ED2-positive cells. We further analyzed the effect of iNOS expression on vascular smooth muscle contraction using an in vitro organ culture system. iNOS mRNA expression and nitrate production significantly increased in vascular tissues (without endothelium) incubated with 1 μg/ml lipopolysaccharide (LPS) for 6 h. Immunohistochemistry indicated that iNOS was largely expressed in ED2-positive resident macrophages. α-Adrenergic-stimulated contractility of the mesenteric artery was greatly suppressed by LPS treatment and was restored by NG-nitro-l-arginine methyl ester (NO synthase inhibitor); in contrast, portal vein contractility was largely unaffected by LPS. Sodium nitroprusside (NO donor) and 8-bromo-cGMP showed greater contractile inhibition in the mesenteric artery than in the portal vein with decreasing myosin light chain phosphorylation. In the presence of an α-adrenergic agonist, the mesenteric artery cytosolic Ca2+level was greatly reduced by sodium nitroprusside; however, the portal vein Ca2+level was largely unaffected. These results suggest that the induction of iNOS in monocytes/macrophages contributes to a hypercirculatory state in the cirrhosis model rat in which the imbalance of the responsiveness of visceral vascular walls to NO (mesenteric artery >> portal vein) may account for the increased portal venous flow in portal hypertension.
Role of arachidonic acid lipoxygenase metabolites in acetylcholine-induced relaxations of mouse arteries
Authors: Gauthier Kathryn M. , Goldman Daniel H. , Aggarwal Nitin T. , Chawengsub Yuttana , Falck J. R. , Campbell William B.
Published: December 2010

Arachidonic acid (AA) metabolites function as EDHFs in arteries of many species. They mediate cyclooxygenase (COX)- and nitric oxide (NO)-independent relaxations to acetylcholine (ACh). However, the role of AA metabolites as relaxing factors in mouse arteries remains incompletely defined. ACh caused concentration-dependent relaxations of the mouse thoracic and abdominal aorta and carotid, femoral, and mesentery arteries (maximal relaxation: 57 ± 4%, 72 ± 4%, 82 ± 3%, 80 ± 3%, and 85 ± 3%, respectively). The NO synthase inhibitor nitro-l-arginine (l-NA; 30 μM) blocked relaxations in the thoracic aorta, and l-NA plus the COX inhibitor indomethacin (10 μM) inhibited relaxations in the abdominal aorta and carotid, femoral, and mesenteric arteries (maximal relaxation: 31 ± 10%, 33 ± 5%, 41 ± 8%, and 73 ± 3%, respectively). In mesenteric arteries, NO- and COX-independent relaxations to ACh were inhibited by the lipoxygenase (LO) inhibitors nordihydroguaiaretic acid (NDGA; 10 μM) and BW-755C (200 μM), the K+channel inhibitor apamin (1 μM), and 60 mM KCl and eliminated by endothelium removal. They were not altered by the cytochrome P-450 inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (20 μM) or the epoxyeicosatrienoic acid antagonist 14,15-epoxyeicosa-5( Z)-enoic acid (10 μM). AA relaxations were attenuated by NDGA or apamin and eliminated by 60 mM KCl. Reverse-phase HPLC analysis revealed arterial [14C]AA metabolites that comigrated with prostaglandins, trihydroxyeicosatrienoic acids (THETAs), hydroxyepoxyeicosatrienoic acids (HEETAs), and hydroxyeicosatetraenoic acids (HETEs). Epoxyeicosatrienoic acids were not observed. Mass spectrometry confirmed the identity of 6-keto-PGF1α, PGE2, 12-HETE, 15-HETE, HEETAs, 11,12,15-THETA, and 11,14,15-THETA. AA metabolism was blocked by NDGA and endothelium removal. 11( R),12( S),15( S)-THETA relaxations (maximal relaxation: 73 ± 3%) were endothelium independent and blocked by 60 mM KCl. Western immunoblot analysis and RT-PCR of the aorta and mesenteric arteries demonstrated protein and mRNA expression of leukocyte-type 12/15-LO. Thus, in mouse resistance arteries, 12/15-LO AA metabolites mediate endothelium-dependent relaxations to ACh and AA.
CytochromeP-450 3A13 and endothelin jointly mediate ductus arteriosus constriction to oxygen in mice
Authors: Baragatti Barbara , Ciofini Enrica , Scebba Francesca , Angeloni Debora , Sodini Daria , Luin Stefano , Ratto Gian Michele , Ottaviano Virginia , Pagni Eleonora , Paolicchi Aldo , Nencioni Simona , Coceani Flavio
Published: December 2010

The fetal ductus arteriosus (DA) contracts to oxygen, and this feature, maturing through gestation, is considered important for its closure at birth. We have previously obtained evidence of the involvement of cytochrome P-450, possibly of the 3A subfamily (CYP3A), in oxygen sensing and have also identified endothelin (ET)-1 as the attendant effector for the contraction. Here, we examined comparatively wild-type (WT) and CYP3A-null ( Cyp3a−/−) mice for direct validation of this concept. We found that the CYP3A subfamily is represented only by CYP3A13 in the WT DA. CYP3A13 was also detected in the DA by immunofluorescence microscopy, being primarily colocalized with the endoplasmic reticulum in both endothelial and muscle cells. However, a distinct signal was also evident in the plasma membrane. Isolated DAs from term WT animals developed a sustained contraction to oxygen with transient contractions superimposed. Conversely, no tonic response occurred in Cyp3a−/−DAs, whereas the phasic response persisted unabated. Oxygen did not contract the preterm WT DA but caused a full-fledged contraction after retinoic acid (RA) treatment. RA also promoted an oxygen contraction in the Cyp3a−/−DA. However, responses of RA-treated WT and Cyp3a−/−mice differed in that only the former abated with ET-1 suppression. This implies the existence of an alternative target for RA responsible for the oxygen-induced contraction in the absence of CYP3A13. In vivo, the DA was constricted in WT and Cyp3a−/−newborns, although with a tendency to be less narrowed in the mutant. We conclude that oxygen acts primarily through the complex CYP3A13 (sensor)/ET-1 (effector) and, in an accessory way, directly onto ET-1. However, even in the absence of CYP3A13, the DA may close postnatally thanks to the contribution of ET-1 and the likely involvement of compensating mechanism(s) identifiable with an alternative oxygen-sensing system and/or the withdrawal of relaxing influence(s) operating prenatally.
Chronic heart failure reduces Akt phosphorylation in human skeletal muscle: relationship to muscle size and function
Authors: Toth Michael J. , Ward Kimberly , van der Velden Jos , Miller Mark S. , VanBuren Peter , LeWinter Martin M. , Ades Philip A.
Published: December 2010

Patients with chronic heart failure (HF) frequently lose muscle mass and function during the course of the disease. A reduction in anabolic stimuli to the muscle has been put forth as a potential mechanism underlying these alterations. The present study examined the hypothesis that skeletal muscle tissue from HF patients would show reduced IGF-1 expression and phosphorylation of signaling molecules downstream of receptor activation. To isolate the unique effect of HF on these variables, we limited the confounding effects of muscle disuse and/or acute disease exacerbation by recruiting controls ( n = 11) with similar physical activity levels as HF patients ( n = 11) and by testing patients at least 6 mo following any bouts of disease exacerbation/hospitalization. IGF-1 expression in skeletal muscle was similar between patients and controls. Despite this, HF patients were characterized by reduced levels of phospho-Akt/Akt (S473; −43%; P < 0.05), whereas no differences were found in total Akt protein content or phospho- or total protein content of mammalian target of rapamycin (mTOR; S2448), glycogen synthase kinase-3β (GSK-3β; S9), eukaryotic translation initiation factor 4E binding protein-1 (eIF4E-BP; T37/46), p70 ribosomal S6 kinase (p70 S6K; T389), or eIF2Bε (S540). Reduced phospho-Akt/Akt levels and phospho-mTOR/mTOR were related to decreased skeletal muscle myosin protein content ( r = 0.602; P < 0.02) and knee extensor isometric torque ( r = 0.550; P < 0.05), respectively. Because patients and controls were similar for age, muscle mass, and physical activity, we ascribe the observed alterations in Akt phosphorylation and its relationship to myosin protein content to the unique effects of the HF syndrome.
Nitric oxide-mediated vasodilation becomes independent of β-adrenergic receptor activation with increased intensity of hypoxic exercise
Authors: Casey Darren P. , Curry Timothy B. , Wilkins Brad W. , Joyner Michael J.
Published: December 2010

Hypoxic vasodilation in skeletal muscle at rest is known to include β-adrenergic receptor-stimulated nitric oxide (NO) release. We previously reported that the augmented skeletal muscle vasodilation during mild hypoxic forearm exercise includes β-adrenergic mechanisms. However, it is unclear whether a β-adrenergic receptor-stimulated NO component exists during hypoxic exercise. We hypothesized that NO-mediated vasodilation becomes independent of β-adrenergic receptor activation with increased exercise intensity during hypoxic exercise. Ten subjects (7 men, 3 women; 23 ± 1 yr) breathed hypoxic gas to titrate arterial O2 saturation to 80% while remaining normocapnic. Subjects performed two consecutive bouts of incremental rhythmic forearm exercise (10% and 20% of maximum) with local administration (via a brachial artery catheter) of propranolol (β-adrenergic receptor inhibition) alone and with the combination of propranolol and nitric oxide synthase inhibition [ NG-monomethyl-l-arginine (l-NMMA)] under normoxic and hypoxic conditions. Forearm blood flow (FBF, ml/min; Doppler ultrasound) and blood pressure [mean arterial pressure (MAP), mmHg; brachial artery catheter] were assessed, and forearm vascular conductance (FVC, ml·min−1·100 mmHg−1) was calculated (FBF/MAP). During propranolol alone, the rise in FVC (Δ from normoxic baseline) due to hypoxic exercise was 217 ± 29 and 415 ± 41 ml·min−1·100 mmHg−1 (10% and 20% of maximum, respectively). Combined propranolol-l-NMMA infusion during hypoxic exercise attenuated ΔFVC at 20% (352 ± 44 ml·min−1·100 mmHg−1; P < 0.001) but not at 10% (202 ± 28 ml·min−1·100 mmHg−1; P = 0.08) of maximum compared with propranolol alone. These data, when integrated with earlier findings, demonstrate that NO contributes to the compensatory vasodilation during mild and moderate hypoxic exercise; a β-adrenergic receptor-stimulated NO component exists during low-intensity hypoxic exercise. However, the source of the NO becomes less dependent on β-adrenergic mechanisms as exercise intensity increases.
EGFR-Homing dsRNA Activates Cancer-Targeted Immune Response and Eliminates Disseminated EGFR-Overexpressing Tumors in Mice
Authors: Shir Alexei , Ogris Manfred , Roedl Wolfgang , Wagner Ernst , Levitzki Alexander
Published: January 2011

Purpose: The cause of most cancer deaths is incurable dissemination of cancer cells into vital organs. Current systemic therapies for disseminated cancers provide limited efficacy and are often accompanied by toxic side effects. We have recently shown that local application of epidermal growth factor receptor (EGFR)–targeted polyinosine-cytosine (polyIC) eradicates preestablished EGFR-overexpressing tumors. Here we show for the first time the high efficiency of systemic application of polyIC/melittin-polyethyleneimine-polyethyleneglycol-EGF (polyIC/MPPE) in combination with human immune cells.

Experimental design: Cancer-targeted activation of immune cells was examined in vitro and in vivo following transfection with polyIC/MPPE. The therapeutic efficiency of the strategy was then examined on disseminated EGFR-overexpressing tumors grown in severe combined immunodeficient (SCID) mice.

Results: Intravenous delivery of polyIC/MPPE followed by intraperitoneal injection of peripheral blood mononuclear cells induced the complete cure of SCID mice with preestablished disseminated EGFR-overexpressing tumors, with no adverse toxic effects. The immune cells and the cytokines they produce are localized to the tumor site of the treated animal and contribute decisively to the demise of the tumor cells. The immune system homes to the tumors, due to the chemokines produced by the internalized polyIC.

Conclusion: The EGFR-homing vector loaded with polyIC can be used to treat and possibly cure patients with disseminated EGFR-overexpressing tumors. The possibility of adopting this strategy to treat other tumors that express a protein capable of ligand induced internalization is discussed. Clin Cancer Res; 17(5); 1033–43. ©2010 AACR.