bluuebunny/crossref_metadata_2025 huggingface dataset:
Enterobacterial Common Antigen Integrity Is a Checkpoint for Flagellar Biogenesis inSerratia marcescens
Authors: Castelli María E. , Fedrigo Griselda V. , Clementín Ana L. , Ielmini M. Verónica , Feldman Mario F. , Véscovi Eleonora García
Published: November 2007

Serratia marcescensstrains are ubiquitous bacteria isolated from environmental niches, such as soil, water, and air, and also constitute emergent nosocomial opportunistic pathogens. Among the numerous extracellular factors thatS. marcescensis able to produce, the PhlA phospholipase is the only described exoprotein secreted by the flagellar apparatus while simultaneously being a member of the flagellar regulon. To gain insight into the regulatory mechanism that couples PhlA and flagellar expression, we conducted a generalized insertional mutagenesis and screened for PhlA-deficient strains. We found that three independent mutations in theweccluster, which impaired the assembly of enterobacterial common antigen (ECA), provoked the inhibition of PhlA expression. Swimming and swarming assays showed that in these strains, motility was severely affected. Microscopic examination and flagellin immunodetection demonstrated that a strong defect in flagellum expression was responsible for the reduced motility in thewecmutant strains. Furthermore, we determined that in the ECA-defective strains, the transcriptional cascade that controls flagellar assembly was turned off due to the down-regulation offlhDCexpression. These findings provide a new perspective on the physiological role of the ECA, providing evidence that inS. marcescens, its biosynthesis conditions the expression of the flagellar regulon.
Dam Methyltransferase Is Required for Stable Lysogeny of the Shiga Toxin (Stx2)-Encoding Bacteriophage 933W of Enterohemorrhagic
Escherichia coli
O157:H7
Authors: Murphy Kenan C. , Ritchie Jennifer M. , Waldor Matthew K. , Løbner-Olesen Anders , Marinus M. G.
Published: November 2007

Shiga toxin 2 (Stx2), one of the principal virulence factors of enterohemorrhagic
Escherichia coli
, is encoded by 933W, a lambda-like prophage. 933W prophage induction contributes to Stx2 production, and here, we provide evidence that Dam methyltransferase is essential for maintenance of 933W lysogeny. Our findings are consistent with the idea that the 933W prophage has a relatively low threshold for induction, which may promote Stx2 production during infection.
A Novel Caffeine Dehydrogenase in
Pseudomonas
sp. Strain CBB1 Oxidizes Caffeine to Trimethyluric Acid
Authors: Yu Chi Li , Kale Yogesh , Gopishetty Sridhar , Louie Tai Man , Subramanian Mani
Published: November 2007

A unique heterotrimeric caffeine dehydrogenase was purified from
Pseudomonas
sp. strain CBB1. This enzyme oxidized caffeine to trimethyluric acid stoichiometrically and hydrolytically, without producing hydrogen peroxide. The enzyme was not NAD(P)
+
dependent; coenzyme Q
0
was the preferred electron acceptor. The enzyme was specific for caffeine and theobromine and showed no activity with xanthine.
The Amino Acid Valine Is Secreted in Continuous-Flow Bacterial Biofilms
Authors: Valle Jaione , Da Re Sandra , Schmid Solveig , Skurnik David , D'Ari Richard , Ghigo Jean-Marc
Published: November 2007

Biofilms are structured communities characterized by distinctive gene expression patterns and profound physiological changes compared to those of planktonic cultures. Here, we show that many gram-negative bacterial biofilms secrete high levels of a small-molecular-weight compound, which inhibits the growth of onlyEscherichia coliK-12 and a rare few other natural isolates. We demonstrate both genetically and biochemically that this molecule is the amino acid valine, and we provide evidence that valine production within biofilms results from metabolic changes occurring within high-density biofilm communities when carbon sources are not limiting. This finding identifies a natural environment in which bacteria can encounter high amounts of valine, and we propose that in-biofilm valine secretion may be the long-sought reason for widespread but unexplained valine resistance found in most enterobacteria. Our results experimentally validate the postulated production of metabolites that is characteristic of the conditions associated with some biofilm environments. The identification of such molecules may lead to new approaches for biofilm monitoring and control.
Carbon Catabolite Repression of Type IV Pilus-Dependent Gliding Motility in the Anaerobic Pathogen
Clostridium perfringens
Authors: Mendez Marcelo , Huang I-Hsiu , Ohtani Kaori , Grau Roberto , Shimizu Tohru , Sarker Mahfuzur R.
Published: November 2007

Clostridium perfringens
is an anaerobic, gram-positive, spore-forming bacterium responsible for the production of severe histotoxic and gastrointestinal diseases in humans and animals. In silico analysis of the three available genome-sequenced
C. perfringens
strains (13, SM101, and ATCC13124) revealed that genes that encode flagellar proteins and genes involved in chemotaxis are absent. However, those strains exhibit type IV pilus (TFP)-dependent gliding motility. Since carbon catabolite regulation has been implicated in the control of different bacterial behaviors, we investigated the effects of glucose and other readily metabolized carbohydrates on
C. perfringens
gliding motility. Our results demonstrate that carbon catabolite regulation constitutes an important physiological regulatory mechanism that reduces the proficiencies of the gliding motilities of a large number of unrelated human- and animal-derived pathogenic
C. perfringens
strains. Glucose produces a strong dose-dependent inhibition of gliding development without affecting vegetative growth. Maximum gliding inhibition was observed at a glucose concentration (1%) previously reported to also inhibit other important behaviors in
C. perfringens
, such as spore development. The inhibition of gliding development in the presence of glucose was due, at least in part, to the repression of the genes
pilT
and
pilD
, whose products are essential for TFP-dependent gliding proficiency. The inhibitory effects of glucose on
pilT
and
pilD
expression were under the control of the key regulatory protein CcpA (
c
atabolite
c
ontrol
p
rotein A). The deficiency in CcpA activity of a
ccpA
knockout
C. perfringens
mutant strain restored the expressions of
pilT
and
pilD
and gliding proficiency in the presence of 1% glucose. The carbon catabolite repression of the gliding motility of the
ccpA
mutant strain was restored after the introduction of a complementing plasmid harboring a wild-type copy of
ccpA
. These results point to a central role for CcpA in orchestrating the negative effect of carbon catabolite regulation on
C. perfringens
gliding motility. Furthermore, we discovered a novel positive role for CcpA in
pilT
and
pilD
expression and gliding proficiency in the absence of catabolite regulation. Carbon catabolite repression of gliding motility and the dual role of CcpA, either as repressor or as activator of gliding, are analyzed in the context of the different social behaviors and diseases produced by
C. perfringens
.
A Novel Role for Enzyme I of the
Vibrio cholerae
Phosphoenolpyruvate Phosphotransferase System in Regulation of Growth in a Biofilm
Authors: Houot Laetitia , Watnick Paula I.
Published: November 2007

Glucose is a universal energy source and a potent inducer of surface colonization for many microbial species. Highly efficient sugar assimilation pathways ensure successful competition for this preferred carbon source. One such pathway is the phosphoenolpyruvate phosphotransferase system (PTS), a multicomponent sugar transport system that phosphorylates the sugar as it enters the cell. Components required for transport of glucose through the PTS include enzyme I, histidine protein, enzyme IIA
Glc
, and enzyme IIBC
Glc
. In
Escherichia coli
, components of the PTS fulfill many regulatory roles, including regulation of nutrient scavenging and catabolism, chemotaxis, glycogen utilization, catabolite repression, and inducer exclusion. We previously observed that genes encoding the components of the
Vibrio cholerae
PTS were coregulated with the
vps
genes, which are required for synthesis of the biofilm matrix exopolysaccharide. In this work, we identify the PTS components required for transport of glucose and investigate the role of each of these components in regulation of biofilm formation. Our results establish a novel role for the phosphorylated form of enzyme I in specific regulation of biofilm-associated growth. As the PTS is highly conserved among bacteria, the enzyme I regulatory pathway may be relevant to a number of biofilm-based infections.
Determinants for the Subcellular Localization and Function of a Nonessential SEDS Protein
Authors: Real Gonçalo , Fay Allison , Eldar Avigdor , Pinto Sérgio M. , Henriques Adriano O. , Dworkin Jonathan
Published: November 2007

TheBacillus subtilisSpoVE integral membrane protein is essential for the heat resistance of spores, probably because of its involvement in spore peptidoglycan synthesis. We found that an SpoVE-yellow fluorescent protein (YFP) fusion protein becomes localized to the forespore during the earliest stages of engulfment, and this pattern is maintained throughout sporulation. SpoVE belongs to a well-conserved family of proteins that includes the FtsW and RodA proteins ofB. subtilis. These proteins are involved in bacterial shape determination, although their function is not known. FtsW is necessary for the formation of the asymmetric septum in sporulation, and we found that an FtsW-YFP fusion localized to this structure prior to the initiation of engulfment in a nonoverlapping pattern with SpoVE-cyan fluorescent protein. Since FtsW and RodA are essential for normal growth, it has not been possible to identify loss-of-function mutations that would greatly facilitate analysis of their function. We took advantage of the fact that SpoVE is not required for growth to obtain point mutations in SpoVE that block the development of spore heat resistance but that allow normal protein expression and targeting to the forespore. These mutant proteins will be invaluable tools for future experiments aimed at elucidating the function of members of the SEDS (“shape, elongation, division, and sporulation”) family of proteins.
The GntR-Like Regulator TauR Activates Expression of Taurine Utilization Genes in
Rhodobacter capsulatus
Authors: Wiethaus Jessica , Schubert Britta , Pfänder Yvonne , Narberhaus Franz , Masepohl Bernd
Published: November 2007

Rhodobacter capsulatus
can efficiently grow with taurine as the sole sulfur source. The products of the
tpa
-
tauR
-
xsc
gene region are essential for this activity. TauR, a MocR-like member of the GntR superfamily of transcriptional regulators, activates
tpa
transcription, as shown by analysis of wild-type and
tauR
mutant strains carrying a
tpa
-
lacZ
reporter fusion. Activation of the
tpa
promoter requires taurine but is not inhibited by sulfate, which is the preferred sulfur source. TauR directly binds to the
tpa
promoter, as demonstrated by DNA mobility shift assays. As expected for a transcriptional activator, the TauR binding site is located upstream of the transcription start site, which has been determined by primer extension. Site-directed promoter mutations reveal that TauR binds to direct repeats, an unusual property that has to date been shown for only one other member of the MocR subfamily, namely, GabR from
Bacillus subtilis
. In contrast, all other members of the GntR family analyzed so far bind to inverted repeats.
Processing of
as
-
48ABC
RNA in AS-48 Enterocin Production by
Enterococcus faecalis
Authors: Fernández Matilde , Sánchez-Hidalgo Marina , García-Quintáns Nieves , Martínez-Bueno Manuel , Valdivia Eva , López Paloma , Maqueda Mercedes
Published: November 2007

Enterocin AS-48 production and immunity characters are encoded by 10 genes (
as
-

48ABCC
1
DD
1
EFGH

) of the pMB2 plasmid from the
Enterococcus faecalis
S-48 strain. Among these,
as
-
48A
, encoding the AS-48 peptide, and the
as
-
48BC
genes constitute a cluster required for AS-48 biogenesis and full immunity. In this study, the levels of expression of this cluster have been altered by insertion and site-directed mutagenesis as well as by expression coupled to
trans
complementation. Phenotypic studies of the mutants have indicated cotranscription of the three genes and revealed that the inactivation of
as
-
48B
prevents the production of AS-48, thus confirming its essentiality in AS-48 biogenesis. These studies have also supported the involvement of
as
-
48C
in enterocin immunity. In addition, they established that the intergenic region between the
as
-
48A
and
as
-
48B
genes is decisive for AS-48 expression, since a 3-bp substitution, which should disrupt a potential 47-nucleotide complex secondary structure, resulted in a hypoproducing phenotype. Transcriptional analyses of the
E. faecalis
wild-type and mutant strains supports the possibility that the
as
-
48ABC
genes are transcribed from the P

A

promoter located upstream of
as
-
48A
. Moreover, analysis and bioinformatic predictions of RNA folding indicate that
as
-
48ABC
mRNA is processed at the secondary structure located between
as
-
48A
and
as
-
48B
. Thus, synthesis of the AS-48 peptide appears to be controlled at the posttranscriptional level and is uncoupled from
as
-
48BC
translation. This mechanism of genetic regulation has not been previously described for the regulation of bacteriocin expression in enterococci.
PhaP Is Involved in the Formation of a Network on the Surface of Polyhydroxyalkanoate Inclusions in
Cupriavidus necator
H16
Authors: Dennis Douglas , Sein Vicki , Martinez Edgar , Augustine Brian
Published: November 2007

Polyhydroxyalkanoate (PHA) inclusions are polymeric storage inclusions formed in some bacterial species when carbon levels are high but levels of another essential nutrient, such as nitrogen, are low. Though much is known about PHA synthesis, little is known about inclusion structure. In this study, atomic force microscopy (AFM) was employed to elucidate the structure of PHA inclusions at the nanoscale level, including the characterization of different layers of structure. AFM data suggest that underneath the inclusion envelope, there is a 2- to 4-nm-thick network layer that resides on top of a harder layer that is likely to be a crystalline lamellar polymer. The network is comprised of ∼20-nm-wide linear segments and junctions that are typically formed by the joining of three to four of the linear segments. In some cases, ∼50-nm globular structures that are raised ∼1 to 2 nm above the network are present at the junctions. These globular structures always have a central pore that is ∼15 nm in diameter. To determine if the major surface protein of PHA inclusions, PhaP, is involved in the structure of this network, inclusions from
Cupriavidus necator
H16 Δ
phaP
were examined. No network structure was detected. Instead, apparently random globular structures were found on the surfaces of the inclusions. When PhaP levels were reconstituted in this strain by the addition of
phaP
on a plasmid, the network was also reconstituted, albeit in a slightly different arrangement from that of the wild-type network. We conclude that PhaP participates in the formation of the inclusion network.
Primary Culture of
Mycobacterium ulcerans
from Human Tissue Specimens after Storage in Semisolid Transport Medium
Authors: Eddyani Miriam , Debacker Martine , Martin Anandi , Aguiar Julia , Johnson Christian R. , Uwizeye Cécile , Fissette Krista , Portaels Françoise
Published: November 2007

Tissue specimens collected from patients with clinically suspected Buruli ulcer treated in two Buruli ulcer treatment centers in Benin between 1998 and 2004 were placed in semisolid transport medium and transported at ambient temperature for microbiological analysis at the Institute of Tropical Medicine in Antwerp, Belgium. The impact of the delay before microbiological analysis on primary culture of
Mycobacterium ulcerans
was investigated. The length of storage in semisolid transport medium varied from 6 days to 26 weeks. Of the 1,273 tissue fragments positive for
M. ulcerans
DNA by an IS
2404
-specific PCR, 576 (45.2%) yielded positive culture results. The sensitivity of direct smear examination was 64.6% (822/1,273 tissue fragments). The median time required to obtain a positive culture result was 11 weeks. Positive cultures were obtained even from samples kept for more than 2 months at ambient temperatures. Moreover, there was no reduction in the viability of
M. ulcerans
, as detected by culture, when specimens remained in semisolid transport medium for long periods of time (up to 26 weeks). We can conclude that the method with semisolid transport medium is very robust for clinical specimens from patients with Buruli ulcer that, due to circumstances, cannot be analyzed in a timely manner. This transport medium is thus very useful for the confirmation of a diagnosis of Buruli ulcer with specimens collected in the field.
Analysis of Toxigenic
Corynebacterium ulcerans
Strains Revealing Potential for False-Negative Real-Time PCR Results
Authors: Cassiday Pamela K. , Pawloski Lucia C. , Tiwari Tejpratap , Sanden Gary N. , Wilkins Patricia P.
Published: November 2007

Diphtheria surveillance depends on the rapid and reliable recognition of the toxin gene in
Corynebacterium diphtheriae
. Real-time PCR is a rapid tool to confirm the presence of the diphtheria toxin gene (
tox
) in an isolate or specimen. We report that some toxigenic
Corynebacterium ulcerans
strains show atypical results in a real-time PCR for
tox
.
Shiga Toxin Gene-Containing
Escherichia coli
from Cattle and Diarrheic Children in the Pastoral Systems of Southwestern Uganda
Authors: Majalija Samuel , Segal Heidi , Ejobi Francis , Elisha B. Gay
Published: November 2007

Shiga toxin-producing
Escherichia coli
(STEC) strains from cattle and diarrheic children in a pastoralist community in Uganda were investigated. The STEC strains belonged to a variety of different serogroups, and 70% of the strains were positive for the intimin gene,
eae
. STEC strains from two of the children were closely related to bovine strains.
Protease Activity, Self Interaction, and Small Interfering RNA Binding of the Silencing Suppressor P1b fromCucumber Vein Yellowing Ipomovirus
Authors: Valli Adrian , Dujovny Gabriela , García Juan Antonio
Published: November 2007

The RNA silencing pathway mediated by small interfering RNAs (siRNAs) plays an important antiviral role in eukaryotes. To counteract this defense barrier, a large number of plant viruses express proteins with RNA silencing suppression activity. Recently, it was reported that the ipomovirusCucumber vein yellowing virus(CVYV), which lacks the typical silencing suppressor of members of the familyPotyviridae, i.e., HCPro, has a duplicated P1 coding sequence and that the downstream P1 copy, named P1b, has silencing suppression activity. In this study, we provide experimental evidence that P1b is a serine protease that self-cleaves at its C terminus but that its proteolytic activity is not essential for silencing suppression. In contrast, a putative zinc finger and a conserved basic motif in the N-terminal region of the protein are required for efficient silencing suppression. In vitro gel filtration-fast protein liquid chromatography and in vivo bimolecular fluorescence complementation assays showed that P1b binds itself to form oligomeric structures and that the zinc finger-like motif is essential for the self interaction. Moreover, we observed that CVYV P1b forms complexes with synthetic siRNAs, and this ability correlated with both silencing suppression activity and enhancement ofPotato virus Xpathogenicity in a mutational analysis. Together, these results suggest that CVYV P1b resembles potyviral HCPro and other viral proteins in interfering RNA silencing by preventing siRNA loading into the RNA-induced silencing complex.
JC Virus Agnoprotein Inhibits In Vitro Differentiation of Oligodendrocytes and Promotes Apoptosis
Authors: Merabova Nana , Kaniowska Dorota , Kaminski Rafal , Deshmane Satish L. , White Martyn K. , Amini Shohreh , Darbinyan Armine , Khalili Kamel
Published: November 2007

Productive infection of oligodendrocytes, which are responsible for the formation of myelin sheath in the central nervous system, with the human neurotropic virus JC virus (JCV) causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). In addition to encoding T antigen and the capsid proteins, which are produced at the early and late phases of the infection cycle, respectively, JCV encodes a small regulatory protein named agnoprotein that is important for successful completion of the virus life cycle. Here we used bipotential CG-4 cells to examine the impact of agnoprotein on oligodendrocyte differentiation and survival in the absence of JCV lytic infection. We demonstrate that the expression of agnoprotein delayed the formation of complex outgrowth networks of the cells during oligodendrocyte differentiation. These alterations were accompanied by high levels of DNA damage, induction of proapoptotic proteins, and suppression of prosurvival signaling. Accordingly, apoptosis was significantly increased upon the induction of CG-4 cells toward differentiation in cells expressing agnoprotein. These observations provide the first evidence for the possible involvement of agnoprotein, independent from its role in viral replication, in a series of biological events that may contribute to the pathological features seen in PML lesions.
Disease severity, self-reported experience of workplace discrimination and employment loss during the course of chronic HIV disease: differences according to gender and education
Authors: Dray-Spira R , Gueguen A , Lert F ,   the VESPA Study Group*
Published: November 2007

Objectives:

Evidence for the existence of a harmful effect of chronic disease on employment status has been provided. Although this effect of chronic illness on employment has been reported to be higher among the groups with the lowest position on the labour market, the mechanisms of such inequalities are poorly understood. The present study aimed at investigating social inequalities in the chances of maintaining employment during the course of HIV infection and at examining the correlates of such inequalities.

Methods:

The authors used data from a national representative sample of people living with HIV in France (ANRS-EN12-VESPA survey). Retrospective information on social trajectory and disease characteristics from the time of HIV diagnosis was available. The risk of employment loss associated with indicators of disease severity and HIV-related workplace discrimination was computed over time since HIV diagnosis according to sociodemographic and occupational factors, using Cox proportional hazards models.

Results:

Among the 478 working-age participants diagnosed as being HIV-infected in the era of multitherapies and employed at the time of HIV diagnosis, 149 experienced employment loss. After adjusting for sociodemographic and occupational factors, disease severity and self-reported HIV-related discrimination at work were significantly associated with the risk of employment loss in a socially-differentiated manner: advancement in HIV disease was associated with an increased risk of employment loss among women (HR 4.45, 95% CI 2.10 to 9.43) but not among men; self-reported experience of HIV-related discrimination at work was associated with an increased risk of employment loss among individuals with a primary/secondary educational level (HR 8.85, 95% CI 3.68 to 21.30) but not among those more educated.

Conclusions:

Chronic HIV disease affects the chances of maintaining employment in a socially-differentiated manner, resulting in increasing inequalities regarding workforce participation. Disease severity and workplace HIV-related discrimination, particularly affecting the employment status of the most socioeconomically disadvantaged, may play a major role.
Differential dependence of stretch and shear stress signaling on caveolin-1 in the vascular wall
Authors: Albinsson Sebastian , Nordström Ina , Swärd Karl , Hellstrand Per
Published: November 2007

The role of caveolae in stretch- versus flow-induced vascular responses was investigated using caveolin 1-deficient [knockout (KO)] mice. Portal veins were stretched longitudinally for 5 min (acute) or 72 h (organ culture). Basal ERK1/2 and Akt phosphorylation were increased in organ-cultured KO veins, as were protein synthesis and vessel wall cross sections. Stretch stimulated acute phosphorylation of ERK1/2 and long-term phosphorylation of focal adhesion kinase (FAK) and cofilin but did not affect Akt phosphorylation. Protein synthesis, and particularly synthesis of smooth muscle differentiation markers, was increased by stretch. These effects did not differ in portal veins from KO and control mice, which also showed the same contractile response to membrane depolarization and inhibition by the Rho kinase inhibitor Y-27632. KO carotid arteries had increased wall cross sections and responded to pressurization (120 mmHg) for 1 h with increased ERK1/2 but not Akt phosphorylation, similar to control arteries. Shear stress by flow for 15 min, on the other hand, increased phosphorylation of Akt in carotids from control but not KO mice. In conclusion, caveolin 1 contributes to low basal ERK1/2 and Akt activity and is required for Akt-dependent signals in response to shear stress (flow) but is not essential for trophic effects of stretch (pressure) in the vascular wall.
Structural and functional determinants in the S5-P region of HCN-encoded pacemaker channels revealed by cysteine-scanning substitutions
Authors: Au Ka-Wing , Siu Chung-Wah , Lau Chu-Pak , Tse Hung-Fat , Li Ronald A.
Published: November 2007

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are responsible for the membrane pacemaker current that underlies the spontaneous generation of bioelectrical rhythms. However, their structure-function relationship is poorly understood. Previously, we identified several pore residues that influence HCN gating properties and proposed a pore-to-gate mechanism. Here, we systematically introduced cysteine-scanning substitutions into the descending portion of the P loop (residues 339–345) of HCN1-R (where R is resistance to sulfhydryl-reactive agents) channels, in which all endogenous cysteines except C303 have been removed or replaced. F339C, K340C, A341C, M342C, S343C, and M345C did not produce functional currents. Interestingly, the loss of function phenotype of F339C could be rescued by the reducing agent dithiothreitol (DTT). H344C but not HCN1-R and DTT-treated F339C channels were sensitive to blockade by divalent Cd2+ (current with 100 μM Cd2+/control current at −140 mV = 67.6 ± 2.9%, 109.3 ± 3.1%, and 103.8 ± 1.7%, respectively). Externally applied methanethiosulfate ethylammonium, a covalent sulfhydryl-reactive compound, irreversibly modified H344C by reducing the current at −140 mV (to 43.7 ± 6.5%), causing a hyperpolarizing steady-state activation shift (change in half-activation voltage: ∼6 mV) and decelerated gating kinetics (by up to 3-fold). Based on these results, we conclude that pore residues 339–345 are important determinants of the structure-function properties of HCN channels and that the side chain of H344 is externally accessible.
Muscle-specific overexpression of IGF-I improves E-C coupling in skeletal muscle fibers from dystrophic mdx mice
Authors: Schertzer Jonathan D. , Poel Chris van der , Shavlakadze Thea , Grounds Miranda D. , Lynch Gordon S.
Published: November 2007

Duchenne muscular dystrophy (DMD) is a lethal X-linked disease caused by the absence of functional dystrophin. Abnormal excitation-contraction (E-C) coupling has been reported in dystrophic muscle fibers from mdx mice, and alterations in E-C coupling components may occur as a direct result of dystrophin deficiency. We hypothesized that muscle-specific overexpression of insulin-growth factor-1 (IGF-I) would reduce E-C coupling failure in mdx muscle. Mechanically skinned extensor digitorum longus muscle fibers from mdx mice displayed a faster decline in depolarization-induced force responses (DIFR); however, there were no differences in sarcoplasmic reticulum (SR)-mediated Ca2+ resequestration or in the properties of the contractile apparatus when compared with nondystrophic controls. The rate of DIFR decline was restored to control levels in fibers from transgenic mdx mice that overexpressed IGF-I in skeletal muscle ( mdx/IGF-I mice). Dystrophic muscles have a lower transcript level of a specific dihydropyridine receptor (DHPR) isoform, and IGF-I-mediated changes in E-C coupling were associated with increased transcript levels of specific DHPR isoforms involved in Ca2+ regulation. Importantly, IGF-I overexpression also increased the sensitivity of the contractile apparatus to Ca2+. The results demonstrate that IGF-I can ameliorate fundamental aspects of E-C coupling failure in dystrophic muscle fibers and that these effects are important for the improvements in cellular function induced by this growth factor.
TRPV1 receptor signaling mediates afferent nerve sensitization during colitis-induced motility disorders in rats
Authors: De Schepper H. U. , De Man J. G. , Ruyssers N. E. , Deiteren A. , Van Nassauw L. , Timmermans J.-P. , Martinet W. , Herman A. G. , Pelckmans P. A. , De Winter B. Y.
Published: November 2007

Rats with experimental colitis suffer from impaired gastric emptying (GE). We previously showed that this phenomenon involves afferent neurons within the pelvic nerve. In this study, we aimed to identify the mediators involved in this afferent hyperactivation. Colitis was induced by trinitrobenzene sulfate (TNBS) instillation. We determined GE, distal front, and geometric center (GC) of intestinal transit 30 min after intragastric administration of a semiliquid Evans blue solution. We evaluated the effects of the transient receptor potential vanilloid type 1 (TRPV1) antagonists capsazepine (5–10 mg/kg) and N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropyrazine-1(2 H)carboxamide (BCTC; 1–10 mg/kg) and the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP-(8-37) (150 μg/kg). To determine TRPV1 receptor antagonist sensitivity, we examined their effect on capsaicin-induced relaxations of isolated gastric fundus muscle strips. Immunocytochemical staining of TRPV1 and RT-PCR analysis of TRPV1 mRNA were performed in dorsal root ganglion (DRG) L6–S1. TNBS-induced colitis reduced GE but had no effect on intestinal motility. Capsazepine reduced GE in controls but had no effect in rats with colitis. At doses that had no effects in controls, BCTC and CGRP-(8-37) significantly improved colitis-induced gastroparesis. Capsazepine inhibited capsaicin-induced relaxations by 35% whereas BCTC completely abolished them. TNBS-induced colitis increased TRPV1-like immunoreactivity and TRPV1 mRNA content in pelvic afferent neuronal cell bodies in DRG L6–S1. In conclusion, distal colitis in rats impairs GE via sensitized pelvic afferent neurons. We provided pharmacological, immunocytochemical, and molecular biological evidence that this sensitization is mediated by TRPV1 receptors and involves CGRP release.
Acute exposure to a moderate strength static magnetic field reduces edema formation in rats
Authors: Morris Cassandra E. , Skalak Thomas C.
Published: November 2007

External application of static magnetic fields (SMF), used specifically for the treatment of inflammatory conditions such as soft tissue injuries, has recently become popular as a complementary and/or alternative therapy with minimal investigation into efficacy or mechanism. Localized inflammation was induced via injection of inflammatory agents λ-carrageenan (CA) or histamine into rat hindpaws, alone or in conjunction with pharmacological agents, resulting in a spatially and temporally defined inflammatory reaction. Application of a 10- or 70-mT, but not a 400-mT, SMF for 15 or 30 min immediately following histamine-induced edema resulted in a significant, 20–50% reduction in edema formation. In addition, a 2-h, 70-mT field application to CA-induced edema also resulted in significant (33–37%) edema reduction. Field application before injection or at the time of maximal edema did not influence edema formation or resolution, respectively. Together, these results suggest the existence of a therapeutic threshold of SMF strength (below 400 mT) and a temporal dependence of efficacy. Administration of pharmacological agents directed at nitric oxide signaling and L-type Ca2+ channel dynamics in conjunction with SMF treatment and histamine-induced edema revealed that the potential mechanism of SMF action may be via modulation of vascular tone through effects on L-type Ca2+ channels in vascular smooth muscle cells.
Characterization of agonist-induced vasoconstriction in mouse pulmonary artery
Authors: Xu Minlin , Platoshyn Oleksandr , Makino Ayako , Dillmann Wolfgang H. , Akassoglou Katerina , Remillard Carmelle V. , Yuan Jason X.-J.
Published: November 2007

In recent years, transgenic mouse models have been developed to examine the underlying cellular and molecular mechanisms of lung disease and pulmonary vascular disease, such as asthma, pulmonary thromboembolic disease, and pulmonary hypertension. However, there has not been systematic characterization of the basic physiological pulmonary vascular reactivity in normal and transgenic mice. This represents an intellectual “gap”, since it is important to characterize basic murine pulmonary vascular reactivity in response to various contractile and relaxant factors to which the pulmonary vasculature is exposed under physiological conditions. The present study evaluates excitation- and pharmacomechanical-contraction coupling in pulmonary arteries (PA) isolated from wild-type BALB/c mice. We demonstrate that both pharmaco- and electromechanical coupling mechanisms exist in mice PA. These arteries are also reactive to stimulation by α1-adrenergic agonists, serotonin, endothelin-1, vasopressin, and U-46619 (a thromboxane A2analog). We conclude that the basic vascular responsiveness of mouse PA is similar to those observed in PA of other species, including rat, pig, and human, albeit on a different scale and to varying amplitudes.
Caveolin-1 and caveolin-3 form heterooligomeric complexes in atrial cardiac myocytes that are required for doxorubicin-induced apoptosis
Authors: Volonte Daniela , McTiernan Charles F. , Drab Marek , Kasper Michael , Galbiati Ferruccio
Published: November 2007

Caveolae are 50- to 100-nm invaginations of the plasma membrane. Caveolins are the structural protein components of caveolar membranes. The caveolin gene family is composed of three members: caveolin-1, caveolin-2, and caveolin-3. Caveolin-1 and caveolin-2 are coexpressed in many cell types, including adipocytes, endothelial cells, epithelial cells, and fibroblasts. In contrast, caveolin-3 expression is essentially restricted to skeletal and smooth muscle cells as well as cardiac myocytes. While the interaction between caveolin-1 and caveolin-2 has been documented previously, the reciprocal interaction between endogenous caveolin-1 and caveolin-3 and their functional role in cell types expressing both isoforms have yet to be identified. Here we demonstrate for the first time that caveolin-1 and caveolin-3 are coexpressed in mouse and rat cardiac myocytes of the atria but not ventricles. We also found that caveolin-1 and caveolin-3 can interact and form heterooligomeric complexes in this cell type. Doxorubicin is an effective anticancer agent, but its use is limited by the possible development of cardiotoxicity. Using caveolin-1- and caveolin-3-null mice, we show that both caveolin-1 and caveolin-3 expression are required for doxorubicin-induced apoptosis in the atria through activation of caspase 3. Together, these results bring new insight into the functional role of caveolae and suggest that caveolin-1/caveolin-3 heterooligomeric complexes may play a key role in chemotherapy-induced cardiotoxicity in the atria.
Stimulation of NTS A1adenosine receptors differentially resets baroreflex control of regional sympathetic outputs
Authors: Scislo Tadeusz J. , Ichinose Tomoko K. , O'Leary Donal S.
Published: November 2007

Previously we showed that pressor and differential regional sympathoexcitatory responses (adrenal > renal ≥ lumbar) evoked by stimulation of A1adenosine receptors located in the nucleus of the solitary tract (NTS) were attenuated/abolished by baroreceptor denervation or blockade of glutamatergic transmission in the NTS, suggesting A1receptor-elicited inhibition of glutamatergic transmission in baroreflex pathways. Therefore we tested the hypothesis that stimulation of NTS A1adenosine receptors differentially inhibits/resets baroreflex responses of preganglionic adrenal (pre-ASNA), renal (RSNA), and lumbar (LSNA) sympathetic nerve activity. In urethane-chloralose-anesthetized male Sprague-Dawley rats ( n = 65) we compared baroreflex-response curves (iv nitroprusside and phenylephrine) evoked before and after bilateral microinjections into the NTS of A1adenosine receptor agonist ( N6-cyclopentyladenosine, CPA; 0.033–330 pmol/50 nl). CPA evoked typical dose-dependent pressor and differential sympathoexcitatory responses and similarly shifted baroreflex curves for pre-ASNA, RSNA, and LSNA toward higher mean arterial pressure (MAP) in a dose-dependent manner; the maximal shifts were 52.6 ± 2.8, 48.0 ± 3.6, and 56.8 ± 6.7 mmHg for pre-ASNA, RSNA, and LSNA, respectively. These shifts were not a result of simple baroreceptor resetting because they were two to three times greater than respective increases in baseline MAP evoked by CPA. Baroreflex curves for pre-ASNA were additionally shifted upward: the maximal increases of upper and lower plateaus were 41.8 ± 16.4% and 45.3 ± 8.7%, respectively. Maximal gain (%/mmHg) measured before vs. after CPA increased for pre-ASNA (3.0 ± 0.6 vs. 4.9 ± 1.3), decreased for RSNA (4.1 ± 0.6 vs. 2.3 ± 0.3), and remained unaltered for LSNA (2.1 ± 0.2 vs. 2.0 ± 0.1). Vehicle control did not alter the baroreflex curves. We conclude that the activation of NTS A1adenosine receptors differentially inhibits/resets baroreflex control of regional sympathetic outputs.
Intergenic transcription and developmental regulation of cardiac myosin heavy chain genes
Authors: Haddad Fadia , Qin Anqi X. , Bodell Paul W. , Jiang Weihua , Giger Julia M. , Baldwin Kenneth M.
Published: November 2007

Cardiac myosin heavy chain (MHC) gene expression undergoes a rapid transition from β- to α-MHC during early rodent neonatal development (0–21 days of age). Thyroid hormone (3,5,3′-triiodothyronine, T3) is a major player in this developmental shift; however, the exact mechanism underlying this transition is poorly understood. The goal of this study was to conduct a more thorough analysis of transcriptional activity of the cardiac MHC gene locus during the early postnatal period in the rodent, in order to gain further insight on the regulation of cardiac MHC genes. We analyzed the expression of α- and β-MHC at protein, mRNA, and pre-mRNA levels at birth and 7, 10, 15, and 21 days after birth in euthyroid and hypothyroid rodents. Using novel technology, we also analyzed RNA expression across the cardiac gene locus, and we discovered that the intergenic (IG) region between the two cardiac genes possesses bidirectional transcriptional activity. This IG transcription results in an antisense RNA product as described previously, which is thought to exert an inhibitory effect on β-MHC gene transcription. On the second half of the IG region, sense transcription occurs, resulting in expression of a sense IG RNA that merges with the α-MHC pre-mRNA. This sense IG RNA transcription was detected in the α-MHC gene promoter, approximately −1.8 kb relative to the α-MHC transcription start site. Both sense and antisense IG RNAs were developmentally regulated and responsive to a hypothyroid state ( 11 , 14 ). This novel observation provides more complexity to the cooperative regulation of the two genes, suggesting the involvement of epigenetic processes in the regulation of cardiac MHC gene locus.
The neurovascular dysfunction induced by angiotensin II in the mouse neocortex is sexually dimorphic
Authors: Girouard H. , Lessard A. , Capone C. , Milner T. A. , Iadecola C.
Published: November 2007

Women are less susceptible to the cerebrovascular complications of hypertension, such as a stroke and vascular dementia. The mechanism of such protection may be related to a reduced vulnerability of women to the cerebrovascular actions of hypertension. To test this hypothesis, we used a model of hypertension based on infusion of angiotensin II (ANG II), an octapeptide that plays a key role in hypertension and produces cerebrovascular dysregulation. Cerebral blood flow (CBF) was monitored by laser-Doppler flowmetry in anesthetized (urethane-chloralose) C57BL/6J male and female mice equipped with a cranial window. ANG II administration (0.25 μg·kg−1·min−1 iv × 30–45 min) elevated arterial pressure equally in both sexes but attenuated the CBF increase induced by whisker stimulation or by the endothelium-dependent vasodilator acetylcholine (ACh) in male but not in female mice. The administration of ANG II for 7 days (2.74 mg·kg−1·day−1), using osmotic minipumps, also attenuated these cerebrovascular responses in male, but not female, mice. The reduced susceptibility to the effect of ANG II in female mice was abolished by ovariectomy and reinstated by estrogen administration to ovariectomized mice. Administration of estrogen to male mice abolished the ANG II-induced attenuation of CBF responses. We conclude that female mice are less susceptible to the cerebrovascular dysregulation induced by ANG II, an effect related to estrogen. Such protection from the deleterious cerebrovascular effects of hypertension may play a role in the reduced vulnerability to the cerebrovascular complications of hypertension observed in women.
Impact of ozone exposure on the phagocytic activity of human surfactant protein A (SP-A) and SP-A variants
Authors: Mikerov Anatoly N. , Umstead Todd M. , Gan Xiaozhuang , Huang Weixiong , Guo Xiaoxuan , Wang Guirong , Phelps David S. , Floros Joanna
Published: November 2007

Surfactant protein A (SP-A) enhances phagocytosis of Pseudomonas aeruginosa. SP-A1 and SP-A2 encode human (h) SP-A; SP-A2 products enhance phagocytosis more than SP-A1. Oxidation can affect SP-A function. We hypothesized that in vivo and in vitro ozone-induced oxidation of SP-A (as assessed by its carbonylation level) negatively affects its function in phagocytosis (as assessed by bacteria cell association). To test this, we used P. aeruginosa, rat alveolar macrophages (AMs), hSP-As with varying levels of in vivo (natural) oxidation, and ozone-exposed SP-A2 (1A, 1A0) and SP-A1 (6A2, 6A4) variants. SP-A oxidation levels (carbonylation) were measured; AMs were incubated with bacteria in the presence of SP-A, and the phagocytic index was calculated. We found: 1) the phagocytic activity of hSP-A is reduced with increasing levels of in vivo SP-A carbonylation; 2) in vitro ozone exposure of hSP-A decreases its function in a dose-dependent manner as well as its ability to enhance phagocytosis of either gram-negative or gram-positive bacteria; 3) the activity of both SP-A1 and SP-A2 decreases in response to in vitro ozone exposure of proteins with SP-A2 being affected more than SP-A1. We conclude that both in vivo and in vitro oxidative modifications of SP-A by carbonylation reduce its ability to enhance phagocytosis of bacteria and that the activity of SP-A2 is affected more by in vitro ozone-induced oxidation. We speculate that functional differences between SP-A1 and SP-A2 exist in vivo and that the redox status of the lung microenvironment differentially affects function of SP-A1 and SP-A2.
Regulatory role for nucleosome assembly protein-1 in the proliferative and vasculogenic phenotype of pulmonary endothelium
Authors: Clark Jennifer , Alvarez Diego F. , Alexeyev Mikhail , King Judy A. C. , Huang Lan , Yoder Mervin C. , Stevens Troy
Published: November 2007

Pulmonary microvascular endothelial cells (PMVECs) are enriched with progenitor cells that underlie their rapid proliferation and vasculogenic capacity. However, the molecular basis for such an enhanced growth potential is unknown. Nucleosome assembly protein-1 (NAP1), and its related family of proteins, have been incriminated in control of cell growth in a range of species. We therefore sought to determine whether NAP1 contributes to the rapid proliferation and vasculogenesis that is observed in PMVECs. NAP1 was expressed at a high level in two fast-growing cell types, including PMVECs and resident microvascular endothelial progenitor cells that were selected from PMVECs, whereas it was expressed at a low level in slow-growing pulmonary artery endothelial cells (PAECs). Heterologous NAP1 expression increased the growth potential of PAECs, whereas inhibiting NAP1 expression reduced the growth potential of PMVECs. Despite its impact on endothelial cell growth, NAP1 did not influence the expression of endothelial cell-selective markers (PECAM-1, VE-cadherin, von Willebrand factor), and it did not influence cell type-specific lectin binding criterion; PMVECs interact with Griffonia simplicifolia lectin, whereas PAECs interact with Helix pomatia lectin. PMVECs possess a higher basal transelectrical resistance than do PAECs, indicative of their more restrictive barrier property. Changing NAP1 expression did not normalize this basal barrier function between PMVECs and PAECs. To examine whether the growth-promoting actions of NAP1 influence blood vessel formation, endothelial cells were mixed into Matrigel and subcutaneously implanted. PMVECs generated eightfold more blood vessels than did PAECs over a 10-day time course. Heterologous NAP1 expression in PAECs increased the number of blood vessels formed by this cell type, where blood vessel growth approached that seen with PMVECs. Thus, our findings indicate that NAP1 functions as an important regulator of the proliferative and vasculogenic endothelial cell phenotype without globally impacting endothelial cell phenotype specification.
Applicability of recent methods used to estimate spontaneous baroreflex sensitivity to resting mice
Authors: Laude Dominique , Baudrie Véronique , Elghozi Jean-Luc
Published: November 2007

Short-term blood pressure (BP) variability is limited by the arterial baroreflex. Methods for measuring the spontaneous baroreflex sensitivity (BRS) aim to quantify the gain of the transfer function between BP and pulse interval (PI) or the slope of the linear relationship between parallel BP and PI changes. These frequency-domain (spectral) and time-domain (sequence) techniques were tested in conscious mice equipped with telemetric devices. The autonomic relevance of these indexes was evaluated using pharmacological blockades. The significant changes of the spectral bandwidths resulting from the autonomic blockades were used to identify the low-frequency (LF) and high-frequency (HF) zones of interest. The LF gain was 1.45 ± 0.14 ms/mmHg, with a PI delay of 0.5 s. For the HF gain, the average values were 2.0 ± 0.19 ms/mmHg, with a null phase. LF and HF bands were markedly affected by atropine. On the same 51.2-s segments used for cross-spectral analysis, an average number of 26.4 ± 2.2 slopes were detected, and the average slope in resting mice was 4.4 ± 0.5 ms/mmHg. Atropine significantly reduced the slopes of the sequence method. BRS measurements obtained using the sequence technique were highly correlated to the spectral estimates. This study demonstrates the applicability of the recent methods used to estimate spontaneous BRS in mice. There was a vagal predominance in the baroreflex control of heart rate in conscious mice in the present conditions.