bluuebunny/crossref_metadata_2025 huggingface dataset:
Biochemical and Phylogenetic Characterization of a Novel Diaminopimelate Biosynthesis Pathway in Prokaryotes Identifies a Diverged Form of
ll
-Diaminopimelate Aminotransferase
Authors: Hudson André O. , Gilvarg Charles , Leustek Thomas
Published: March 2008

A variant of the diaminopimelate (DAP)-lysine biosynthesis pathway uses an
ll
-DAP aminotransferase (DapL, EC 2.6.1.83) to catalyze the direct conversion of
l
-2,3,4,5-tetrahydrodipicolinate to
ll
-DAP. Comparative genomic analysis and experimental verification of DapL candidates revealed the existence of two diverged forms of DapL (DapL1 and DapL2). DapL orthologs were identified in eubacteria and archaea. In some species the corresponding
dapL
gene was found to lie in genomic contiguity with other
dap
genes, suggestive of a polycistronic structure. The DapL candidate enzymes were found to cluster into two classes sharing approximately 30% amino acid identity. The function of selected enzymes from each class was studied. Both classes were able to functionally complement
Escherichia coli dapD
and
dapE
mutants and to catalyze
ll
-DAP transamination, providing functional evidence for a role in DAP/lysine biosynthesis. In all cases the occurrence of
dapL
in a species correlated with the absence of genes for
dapD
and
dapE
representing the acyl DAP pathway variants, and only in a few cases was
dapL
coincident with
ddh
encoding
meso
-DAP dehydrogenase. The results indicate that the DapL pathway is restricted to specific lineages of eubacteria including the
Cyanobacteria
,
Desulfuromonadales
,
Firmicutes
,
Bacteroidetes
,
Chlamydiae
,
Spirochaeta
, and
Chloroflexi
and two archaeal groups, the
Methanobacteriaceae
and
Archaeoglobaceae.
Chlamydia pneumoniaeGroEL1 Protein Is Cell Surface Associated and Required for Infection of HEp-2 Cells
Authors: Wuppermann Frederik N. , Mölleken Katja , Julien Marion , Jantos Christian A. , Hegemann Johannes H.
Published: March 2008

Chlamydia pneumoniaeis an important obligate intracellular pathogen that replicates within an inclusion in the eukaryotic cell. The initial event of a chlamydial infection is the adherence to and subsequent uptake of the infectious elementary bodies (EBs) by the human cell. These processes require yet-unidentified bacterial and eukaryotic surface proteins. The GroEL1 protein, which exhibits a very strong antigenicity and in vitro can activate various eukaryotic cells, is a potential pathogenicity factor. We localized the protein during the infection process and found it in the inclusion but outside the chlamydial particles. GroEL1 was also localized on the surface of EBs, and the protein could be washed off the EBs. Latex beads coated with recombinantly produced GroEL1 (rGroEL1) bound in a dose-dependent manner to HEp-2 cells. Likewise, GroEL1, when expressed and displayed on the yeast cell surface, mediated adhesion to HEp-2 cells. Interestingly, the homologous GroEL2 and GroEL3 proteins showed no adhesive properties. Incubation of primary umbilical vein endothelial cells with soluble GroEL1 and GroEL1-coated latex beads activated the translocation of the general transcription factor NF-κB into the nucleus. Finally, preincubation of HEp-2 cells with rGroEL1 significantly reduced subsequent infection withC. pneumoniae, although adhesion of infectious bacteria to eukaryotic cells was not affected. Taken together, these data support a role for extracellular GroEL1 in the establishment of the chlamydial infection.
Characterization of the Periplasmic Domain of MotB and Implications for Its Role in the Stator Assembly of the Bacterial Flagellar Motor
Authors: Kojima Seiji , Furukawa Yukio , Matsunami Hideyuki , Minamino Tohru , Namba Keiichi
Published: March 2008

MotA and MotB are integral membrane proteins that form the stator complex of the proton-driven bacterial flagellar motor. The stator complex functions as a proton channel and couples proton flow with torque generation. The stator must be anchored to an appropriate place on the motor, and this is believed to occur through a putative peptidoglycan-binding (PGB) motif within the C-terminal periplasmic domain of MotB. In this study, we constructed and characterized an N-terminally truncated variant of
Salmonella enterica
serovar Typhimurium MotB consisting of residues 78 through 309 (MotB
C
). MotB
C
significantly inhibited the motility of wild-type cells when exported into the periplasm. Some point mutations in the PGB motif enhanced the motility inhibition, while an in-frame deletion variant, MotB
C
(Δ197-210), showed a significantly reduced inhibitory effect. Wild-type MotB
C
and its point mutant variants formed a stable homodimer, while the deletion variant was monomeric. A small amount of MotB was coisolated only with the secreted form of MotB
C
-His
6
by Ni-nitrilotriacetic acid affinity chromatography, suggesting that the motility inhibition results from MotB-MotB
C
heterodimer formation in the periplasm. However, the monomeric mutant variant MotB
C
(Δ197-210) did not bind to MotB, suggesting that MotB
C
is directly involved in stator assembly. We propose that the MotB
C
dimer domain plays an important role in targeting and stable anchoring of the MotA/MotB complex to putative stator-binding sites of the motor.
Twitching Motility Is Essential for Virulence in
Dichelobacter nodosus
Authors: Han Xiaoyan , Kennan Ruth M. , Davies John K. , Reddacliff Leslie A. , Dhungyel Om P. , Whittington Richard J. , Turnbull Lynne , Whitchurch Cynthia B. , Rood Julian I.
Published: March 2008

Type IV fimbriae are essential virulence factors of
Dichelobacter nodosus
, the principal causative agent of ovine foot rot. The
fimA
fimbrial subunit gene is required for virulence, but
fimA
mutants exhibit several phenotypic changes and it is not certain if the effects on virulence result from the loss of type IV fimbria-mediated twitching motility, cell adherence, or reduced protease secretion. We showed that mutation of either the
pilT
or
pilU
gene eliminated the ability to carry out twitching motility. However, the
pilT
mutants displayed decreased adhesion to epithelial cells and reduced protease secretion, whereas the
pilU
mutants had wild-type levels of extracellular protease secretion and adherence. These data provided evidence that PilT is required for the type IV fimbria-dependent protease secretion pathway in
D. nodosus
. It was postulated that sufficient fimbrial retraction must occur in the
pilU
mutants to allow protease secretion, but not twitching motility, to take place. Although no cell movement was detected in a
pilU
mutant of
D. nodosus
, aberrant motion was detected in an equivalent mutant of
Pseudomonas aeruginosa
. These observations explain how in
D. nodosus
protease secretion can occur in a
pilU
mutant but not in a
pilT
mutant. In addition, virulence studies with sheep showed that both the
pilT
and
pilU
mutants were avirulent, providing evidence that mutation of the type IV fimbrial system affects virulence by eliminating twitching motility, not by altering cell adherence or protease secretion.
Expression of the Gonococcal Global Regulatory Protein Fur and Genes Encompassing the Fur and Iron Regulon during In Vitro and In Vivo Infection in Women
Authors: Agarwal Sarika , Sebastian Shite , Szmigielski Borys , Rice Peter A. , Genco Caroline A.
Published: March 2008

The ferric uptake regulatory protein, Fur, functions as a global regulatory protein of gene transcription in the mucosal pathogen
Neisseria gonorrhoeae
. We have shown previously that several
N. gonorrhoeae
Fur-repressed genes are expressed in vivo during mucosal gonococcal infection in men, which suggests that this organism infects in an iron-limited environment and that Fur is expressed under these conditions. In this study we have demonstrated expression of the gonococcal
fur
gene in vitro, in human cervical epithelial cells, and in specimens from female subjects with uncomplicated gonococcal infection. In vitro studies confirmed that the expression of the gonococcal
fur
gene was repressed during growth under iron-replete growth conditions but that a basal level of the protein was maintained. Using GFP transcriptional fusions constructed from specific Fur binding sequences within the
fur
promoter/operator region, we determined that this operator region was functional during
N. gonorrhoeae
infection of cervical epithelial cells. Furthermore, reverse transcription-PCR analysis, as well as microarray analysis, using a custom
Neisseria
Fur and iron regulon microarray revealed that several Fur- and iron-regulated genes were expressed during
N. gonorrhoeae
infection of cervical epithelial cells. Microarray analysis of specimens obtained from female subjects with uncomplicated gonococcal infection corroborated our in vitro findings and point toward a key role of gonococcal Fur- and iron-regulated genes in gonococcal disease.
Deficiency of the Rgg Regulator Promotes H2O2Resistance, AhpCF-Mediated H2O2Decomposition, and Virulence inStreptococcus pyogenes
Authors: Pulliainen Arto Tapio , Hytönen Jukka , Haataja Sauli , Finne Jukka
Published: March 2008

Streptococcus pyogenes(group A streptococcus [GAS]), a catalase-negative gram-positive bacterium, is aerotolerant and survives H2O2exposures that kill many catalase-positive bacteria. The molecular basis of the H2O2resistance is poorly known. Here, we demonstrate that serotype M49 GAS lacking the Rgg regulator is more resistant to H2O2and also decomposes more H2O2than the parental strain. Subgenomic transcriptional profiling and genome-integrated green fluorescent protein reporters showed that a bicistronic operon, a homolog of theStreptococcus mutans ahpCFoperon, is transcriptionally up-regulated in the absence of Rgg. Phenotypic assays withahpCFoperon knockouts demonstrated that the gene products decompose H2O2and protect GAS against peroxide stress. In a murine intraperitoneal-infection model, Rgg deficiency increased the virulence of GAS, although in anahpCF-independent manner. Rgg-mediated repression of H2O2resistance is divergent from the previously characterized peroxide resistance repressor PerR. Moreover, Rgg-mediated repression of H2O2resistance is inducible by cellular stresses of diverse natures—ethanol, organic hydroperoxide, and H2O2. Rgg is thus identified as a novel sensoregulator of streptococcal H2O2resistance with potential implications for the virulence of the catalase-negative GAS.
Sustained Axenic Metabolic Activity by the Obligate Intracellular Bacterium
Coxiella burnetii
Authors: Omsland Anders , Cockrell Diane C. , Fischer Elizabeth R. , Heinzen Robert A.
Published: March 2008

Growth of
Coxiella burnetii
, the agent of Q fever, is strictly limited to colonization of a viable eukaryotic host cell. Following infection, the pathogen replicates exclusively in an acidified (pH 4.5 to 5) phagolysosome-like parasitophorous vacuole. Axenic (host cell free) buffers have been described that activate
C. burnetii
metabolism in vitro, but metabolism is short-lived, with bacterial protein synthesis halting after a few hours. Here, we describe a complex axenic medium that supports sustained (>24 h)
C. burnetii
metabolic activity. As an initial step in medium development, several biological buffers (pH 4.5) were screened for
C. burnetii
metabolic permissiveness. Based on [
35
S]Cys-Met incorporation,
C. burnetii
displayed optimal metabolic activity in citrate buffer. To compensate for
C. burnetii
auxotrophies and other potential metabolic deficiencies, we developed a citrate buffer-based medium termed complex
Coxiella
medium (CCM) that contains a mixture of three complex nutrient sources (neopeptone, fetal bovine serum, and RPMI cell culture medium). Optimal
C. burnetii
metabolism occurred in CCM with a high chloride concentration (140 mM) while the concentrations of sodium and potassium had little effect on metabolism. CCM supported prolonged de novo protein and ATP synthesis by
C. burnetii
(>24 h). Moreover,
C. burnetii
morphological differentiation was induced in CCM as determined by the transition from small-cell variant to large-cell variant. The sustained in vitro metabolic activity of
C. burnetii
in CCM provides an important tool to investigate the physiology of this organism including developmental transitions and responses to antimicrobial factors associated with the host cell.
Putative Quorum-Sensing Regulator BlxR ofBrucella melitensisRegulates Virulence Factors Including the Type IV Secretion System and Flagella
Authors: Rambow-Larsen Amy A. , Rajashekara Gireesh , Petersen Erik , Splitter Gary
Published: March 2008

Brucella melitensisis an intracellular pathogen that establishes a replicative niche within macrophages. While the intracellular lifestyle ofBrucellais poorly understood and few virulence factors have been identified, components of a quorum-sensing pathway inBrucellahave recently been identified. The LuxR-type regulatory protein, VjbR, and anN-acylhomoserine lactone signaling molecule are both involved in regulating expression of thevirB-encoded type IV secretion system. We have identified a second LuxR-type regulatory protein (BlxR) inBrucella. Microarray analysis of ablxRmutant suggests that BlxR regulates the expression of a number of genes, including those encoding the type IV secretion system and flagella. Confirming these results, deletion ofblxRinB. melitensisreduced the transcriptional activities of promoters for thevirBoperon, flagellar genes, and another putative virulence factor gene,bopA. Furthermore, our data suggested that both BlxR and VjbR are positively autoregulated and cross-regulate the expression of each other. TheblxRdeletion strain exhibited reduced growth in macrophages, similar to that observed for avjbRdeletion strain. However, unlike thevjbRdeletion, theblxRdeletion did not fully attenuate virulence in mice. More strikingly, bioluminescent imaging revealed that dissemination of theblxRmutant was similar to that of wild-typeB. melitensis, while thevjbRmutant was defective for systemic spread in IRF-1−/−mice, suggesting that these regulators are not functionally redundant but that they converge in a common pathway regulating bacterial processes.
The Extracellular Death Factor: Physiological and Genetic Factors Influencing Its Production and Response inEscherichia coli
Authors: Kolodkin-Gal Ilana , Engelberg-Kulka Hanna
Published: March 2008

Gene pairs specific for a toxin and its antitoxin are called toxin-antitoxin modules and are found on the chromosomes of many bacteria. The most studied of these modules isEscherichia coli mazEF, in whichmazFencodes a stable toxin, MazF, andmazEencodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. In a previous report from this laboratory, it was shown thatmazEF-mediated cell death is a population phenomenon requiring a quorum-sensing peptide called theextracellulardeathfactor (EDF). EDF is the linear pentapeptide NNWNN (32). Here, we further confirm that EDF is a signal molecule in a mixed population. In addition, we characterize some physiological conditions and genes required for EDF production and response. Furthermore, stress response and the gene specifying MazEF, the Zwf (glucose-6-phosphate dehydrogenase) gene, and the protease ClpXP are critical in EDF production. Significant strain differences in EDF production and response explain variations in the induction ofmazEF-mediated cell death.
Campylobacter jejuniCsrA Mediates Oxidative Stress Responses, Biofilm Formation, and Host Cell Invasion
Authors: Fields Joshua A. , Thompson Stuart A.
Published: March 2008

The putative global posttranscriptional regulatorcsrAwas mutated inCampylobacter jejuni81-176. ThecsrAmutant was attenuated in surviving oxidative stress. CsrA also contributed to biofilm formation and adherence to and invasion of INT407 intestinal epithelial cells, suggesting a regulatory role for CsrA inC. jejunipathogenesis.
ThesimOperon Facilitates the Transport and Metabolism of Sucrose Isomers inLactobacillus caseiATCC 334
Authors: Thompson John , Jakubovics Nicholas , Abraham Bindu , Hess Sonja , Pikis Andreas
Published: March 2008

Inspection of the genome sequence ofLactobacillus caseiATCC 334 revealed two operons that might dissimilate the five isomers of sucrose. To test this hypothesis, cells ofL. caseiATCC 334 were grown in a defined medium supplemented with various sugars, including each of the five isomeric disaccharides. Extracts prepared from cells grown on the sucrose isomers contained high levels of two polypeptides withMrs of ∼50,000 and ∼17,500. Neither protein was present in cells grown on glucose, maltose or sucrose. Proteomic, enzymatic, and Western blot analyses identified the ∼50-kDa protein as an NAD+- and metal ion-dependent phospho-α-glucosidase. The oligomeric enzyme was purified, and a catalytic mechanism is proposed. The smaller polypeptide represented an EIIA component of the phosphoenolpyruvate-dependent sugar phosphotransferase system. Phospho-α-glucosidase and EIIA are encoded by genes at the LSEI_0369 (simA) and LSEI_0374 (simF) loci, respectively, in a block of seven genes comprising thesucroseisomermetabolism (sim) operon. Northern blot analyses provided evidence that three mRNA transcripts were up-regulated during logarithmic growth ofL. caseiATCC 334 on sucrose isomers. InternalsimAandsimFgene probes hybridized to ∼1.5- and ∼1.3-kb transcripts, respectively. A 6.8-kb mRNA transcript was detected by both probes, which was indicative of cotranscription of the entiresimoperon.
Human RNA “Rumor” Viruses: the Search for Novel Human Retroviruses in Chronic Disease
Authors: Voisset Cécile , Weiss Robin A. , Griffiths David J.
Published: March 2008

Retroviruses are an important group of pathogens that cause a variety of diseases in humans and animals. Four human retroviruses are currently known, including human immunodeficiency virus type 1, which causes AIDS, and human T-lymphotropic virus type 1, which causes cancer and inflammatory disease. For many years, there have been sporadic reports of additional human retroviral infections, particularly in cancer and other chronic diseases. Unfortunately, many of these putative viruses remain unproven and controversial, and some retrovirologists have dismissed them as merely “human rumor viruses.” Work in this field was last reviewed in depth in 1984, and since then, the molecular techniques available for identifying and characterizing retroviruses have improved enormously in sensitivity. The advent of PCR in particular has dramatically enhanced our ability to detect novel viral sequences in human tissues. However, DNA amplification techniques have also increased the potential for false-positive detection due to contamination. In addition, the presence of many families of human endogenous retroviruses (HERVs) within our DNA can obstruct attempts to identify and validate novel human retroviruses. Here, we aim to bring together the data on “novel” retroviral infections in humans by critically examining the evidence for those putative viruses that have been linked with disease and the likelihood that they represent genuine human infections. We provide a background to the field and a discussion of potential confounding factors along with some technical guidelines. In addition, some of the difficulties associated with obtaining formal proof of causation for common or ubiquitous agents such as HERVs are discussed.
Early improvement in cardiac tissue perfusion due to mesenchymal stem cells
Authors: Schuleri Karl H. , Amado Luciano C. , Boyle Andrew J. , Centola Marco , Saliaris Anastasios P. , Gutman Matthew R. , Hatzistergos Konstantinos E. , Oskouei Behzad N. , Zimmet Jeffrey M. , Young Randell G. , Heldman Alan W. , Lardo Albert C. , Hare Joshua M.
Published: March 2008

The underlying mechanism(s) of improved left ventricular function (LV) due to mesenchymal stem cell (MSC) administration after myocardial infarction (MI) remains highly controversial. Myocardial regeneration and neovascularization, which leads to increased tissue perfusion, are proposed mechanisms. Here we demonstrate that delivery of MSCs 3 days after MI increased tissue perfusion in a manner that preceded improved LV function in a porcine model. MI was induced in pigs by 60-min occlusion of the left anterior descending coronary artery, followed by reperfusion. Pigs were assigned to receive intramyocardial injection of allogeneic MSCs (200 million, ∼15 injections) ( n = 10), placebo ( n = 6), or no intervention ( n = 8). Resting myocardial blood flow (MBF) was serially assessed by first-pass perfusion magnetic resonance imaging (MRI) over an 8-wk period. Over the first week, resting MBF in the infarct area of MSC-treated pigs increased compared with placebo-injected and untreated animals [0.17 ± 0.03, 0.09 ± 0.01, and 0.08 ± 0.01, respectively, signal intensity ratio of MI to left ventricular blood pool (LVBP); P < 0.01 vs. placebo, P < 0.01 vs. nontreated]. In contrast, the signal intensity ratios of the three groups were indistinguishable at weeks 4 and 8. However, MSC-treated animals showed larger, more mature vessels and less apoptosis in the infarct zones and improved regional and global LV function at week 8. Together these findings suggest that an early increase in tissue perfusion precedes improvements in LV function and a reduction in apoptosis in MSC-treated hearts. Cardiac MRI-based measures of blood flow may be a useful tool to predict a successful myocardial regenerative process after MSC treatment.
Chronic hypoxia increases fetoplacental vascular resistance and vasoconstrictor reactivity in the rat
Authors: Jakoubek Vít , Bíbová Jana , Herget Jan , Hampl Václav
Published: March 2008

An increase in fetoplacental vascular resistance caused by hypoxia is considered one of the key factors of placental hypoperfusion and fetal undernutrition leading to intrauterine growth restriction (IUGR), one of the serious problems in current neonatology. However, although acute hypoxia has been shown to cause fetoplacental vasoconstriction, the effects of more sustained hypoxic exposure are unknown. This study was designed to test the hypothesis that chronic hypoxia elicits elevations in fetoplacental resistance, that this effect is not completely reversible by acute reoxygenation, and that it is accompanied by increased acute vasoconstrictor reactivity of the fetoplacental vasculature. We measured fetoplacental vascular resistance as well as acute vasoconstrictor reactivity in isolated perfused placentae from rats exposed to hypoxia (10% O2) during the last week of a 3-wk pregnancy. We found that chronic hypoxia shifted the relationship between perfusion pressure and flow rate toward higher pressure values (by ∼20%). This increased vascular resistance was refractory to a high dose of sodium nitroprusside, implying the involvement of other factors than increased vascular tone. Chronic hypoxia also increased vasoconstrictor responses to angiotensin II (by ∼75%) and to acute hypoxic challenges (by >150%). We conclude that chronic prenatal hypoxia causes a sustained elevation of fetoplacental vascular resistance and vasoconstrictor reactivity that are likely to produce placental hypoperfusion and fetal undernutrition in vivo.
Locations of ectopic beats coincide with spatial gradients of NADH in a regional model of low-flow reperfusion
Authors: Kay Matthew , Swift Luther , Martell Brian , Arutunyan Ara , Sarvazyan Narine
Published: March 2008

We studied the origins of ectopic beats during low-flow reperfusion after acute regional ischemia in excised rat hearts. The left anterior descending coronary artery was cannulated. Perfusate was delivered to the cannula using an high-performance liquid chromatography pump. This provided not only precise control of flow rate but also avoided mechanical artifacts associated with vessel occlusion and deocclusion. Optical mapping of epicardial transmembrane potential served to identify activation wavefronts. Imaging of NADH fluorescence was used to quantify local ischemia. Our experiments suggest that low-flow reperfusion of ischemic myocardium leads to a highly heterogeneous ischemic substrate and that the degree of ischemia between adjacent patches of tissue changes in time. In contrast to transient ectopic activity observed during full-flow reperfusion, persistent ectopic arrhythmias were observed during low-flow reperfusion. The origins of ectopic beats were traceable to areas of high spatial gradients of changes in NADH fluorescence caused by low-flow reperfusion.
Adenosine-mediated alteration of vascular reactivity and inflammation in a murine model of asthma
Authors: Ponnoth Dovenia S. , Nadeem Ahmed , Mustafa S. Jamal
Published: March 2008

Chronic respiratory disorders such as asthma are believed to be associated with adverse cardiovascular events. We hypothesize that asthmatic inflammation translates into systemic inflammation and alters vascular responses where adenosine (AD) plays an important role. Therefore, this study investigated the effects of aerosolized AD, used to elevate lung AD levels, on vascular reactivity and inflammation in our allergic mouse model of asthma. Balb/c mice were divided into four groups: control (Con), Con + aerosolized AD (Con + AD), allergen sensitized and challenged (Sen), and Sen + aerosolized AD (Sen + AD). The animals were sensitized with ragweed (200 μg ip) on days 1 and 6, followed by 1% ragweed aerosol challenges from days 11 to 13. On day 14, the Con + AD and Sen + AD groups received a single AD aerosol challenge (6 mg/ml) for 2 min, followed by the collection of the aorta and plasma on day 15. Organ bath experiments showed concentration-dependent aortic relaxations to AD in the Con and Con + AD groups, which were impaired in the Sen and Sen + AD groups. Real-time PCR data showed changes in aortic AD receptors (ARs), with the expression of A1ARs upregulated, whereas the expression of A2ARs and endothelial nitric oxide synthase genes were downregulated, resulting in an impairment of vasorelaxation in the Sen and Sen + AD groups. The A1AR antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) reversed the impairment in vasorelaxation observed in the Sen and Sen + AD groups, whereas the A2BAR antagonist alloxazine inhibited vasorelaxation in all groups. Allergen challenge caused systemic inflammation in allergic mice, with AD aerosol further enhancing it as determined by the inflammatory cytokines profile in plasma. In conclusion, asthmatic mice showed altered vascular reactivity and systemic inflammation, with AD aerosol further exacerbating these effects.
Loading effect of fibroblast-myocyte coupling on resting potential, impulse propagation, and repolarization: insights from a microstructure model
Authors: Jacquemet Vincent , Henriquez Craig S.
Published: March 2008

The numerous nonmyocytes present within the myocardium may establish electrical connections with myocytes through gap junctions, formed naturally or as a result of a cell therapy. The strength of the coupling and its potential impact on action potential characteristics and conduction are not well understood. This study used computer simulation to investigate the load-induced electrophysiological consequences of the coupling of myocytes with fibroblasts, where the fibroblast resting potential, density, distribution, and coupling strength were varied. Conduction velocity (CV), upstroke velocity, and action potential duration (APD) were analyzed for longitudinal and transverse impulse propagation in a two-dimensional microstructure tissue model, developed to represent a monolayer culture of cardiac cells covered by a layer of fibroblasts. The results show that 1) at weak coupling (<0.25 nS), the myocyte resting potential was elevated, leading to CV up to 5% faster than control; 2) at intermediate coupling, the myocyte resting potential elevation saturated, whereas the current flowing from the myocyte to the fibroblast progressively slowed down both CV and upstroke velocity; 3) at strong couplings (>8 nS), all of the effects saturated; and 4) APD at 90% repolarization was usually prolonged by 0–20 ms (up to 60–80 ms for high fibroblast density and coupling) by the coupling to fibroblasts. The changes in APD depended on the fibroblast resting potential. This complex, coupling-dependent interaction of fibroblast and myocytes also has relevance to the integration of other nonmyocytes in the heart, such as those used in cellular therapies.
Serum thyroid hormone levels may not accurately reflect thyroid tissue levels and cardiac function in mild hypothyroidism
Authors: Liu Yingheng , Redetzke Rebecca A. , Said Suleman , Pottala James V. , de Escobar Gabriella Morreale , Gerdes A. Martin
Published: March 2008

The link between thyroid dysfunction and cardiovascular diseases has been recognized for more than 100 years. Although overt hypothyroidism leads to impaired cardiac function and possibly heart failure, the cardiovascular consequences of borderline low thyroid function are not clear. Establishment of a suitable animal model would be helpful. In this study, we characterized a rat model to study the relationship between cardiovascular function and graded levels of thyroid activity. We used rats with surgical thyroidectomy and subcutaneous implantation of slow release pellets with three different T4 doses for 3 wk. In terminal experiments, cardiac function was evaluated by echocardiograms and hemodynamics. Myocardial arteriolar density was also quantified morphometrically. Thyroid hormone levels in serum and heart tissue were determined by RIA assays. Thyroidectomy alone led to cardiac atrophy, severe cardiac dysfunction, and a dramatic loss of arterioles. The low T4 dose normalized serum T3 and T4 levels, but cardiac tissue T3 and T4 remained below normal. Low-dose T4 failed to prevent cardiac atrophy or restore cardiac function and arteriolar density to normal values. All cardiac function parameters and myocardial arteriolar density were normalized with the middle dose of T4, whereas the high dose produced hyperthyroidism. Our results show that thyroid hormones are important regulators of cardiac function and myocardial arteriolar density. This animal model will be useful in studying the pathophysiological consequences of mild thyroid dysfunction. Results also suggest that cardiac function may provide valuable supplemental information in proper diagnosis of mild thyroid conditions.
Reversal of elastase-induced pulmonary emphysema and promotion of alveolar epithelial cell proliferation by simvastatin in mice
Authors: Takahashi Saeko , Nakamura Hidetoshi , Seki Makoto , Shiraishi Yoshiki , Yamamoto Miyuki , Furuuchi Momoyo , Nakajima Takahiro , Tsujimura Shuko , Shirahata Toru , Nakamura Miho , Minematsu Naoto , Yamasaki Motohiro , Tateno Hiroki , Ishizaka Akitoshi
Published: March 2008

Besides lowering cholesterol, statins exert multiple effects, such as anti-inflammatory activity and improvement of endothelial cell function. We examined whether simvastatin (SS) protects against the development of elastase-induced pulmonary emphysema in mice by using mean linear intercepts of alveoli (Lm) as a morphometric parameter of emphysema. After injection of intratracheal elastase on day 0, C57BL/6 mice were treated daily with SS (SS+ group) or PBS (SS− group) for 2 wk. A 21% decrease in Lm on day 7 was observed in the SS+ group vs. the SS− group. Anti-inflammatory effects of SS were observed as a decrease in percentage of neutrophils up to day 3, and in hydroxyproline concentration on day 3, in bronchoalveolar lavage fluid (BALF). SS also increased the number of proliferating cell nuclear antigen (PCNA)-positive alveolar epithelial cells between days 3 and 14. To confirm the role of statins in promoting proliferation of alveolar cells, mice were treated with SS (SS+) vs. PBS (SS−) for 12 days, starting 3 wk after elastase administration. After SS treatment, Lm decreased by 52% and PCNA-positive alveolar epithelial cells increased compared with the SS− group. Concentrations of vascular endothelial growth factor in BALF and endothelial nitric oxide synthase protein expression in pulmonary vessels tended to be higher in the SS+ group vs. the SS− group in this protocol. In conclusion, SS inhibited the development of elastase-induced pulmonary emphysema in mice. This therapeutic effect was due not only to anti-inflammation but also to the promotion of alveolar epithelial cell regeneration, partly mediated by restoring endothelial cell functions.
Regulation of angiopoietin expression by bacterial lipopolysaccharide
Authors: Mofarrahi Mahroo , Nouh Thamir , Qureshi Salman , Guillot Loic , Mayaki Dominique , Hussain Sabah N. A.
Published: March 2008

Angiopoietins are ligands for Tie-2 receptors and play important roles in angiogenesis and inflammation. While angiopoietin-1 (Ang-1) inhibits inflammatory responses, angiopoietin-2 (Ang-2) promotes cytokine production and vascular leakage. In this study, we evaluated in vivo and in vitro effects of Escherichia coli lipopolysaccharides (LPS) on angiopoietin expression. Wild-type C57/BL6 mice were injected with saline (control) or E. coli LPS (20 mg/ml ip) and killed 6, 12, and 24 h later. The diaphragm, lung, and liver were excised and assayed for mRNA and protein expression of Ang-1, Ang-2, and Tie-2 protein and tyrosine phosphorylation. LPS injection elicited a severalfold rise in Ang-2 mRNA and protein levels in the three organs. By comparison, both Ang-1 and Tie-2 levels in the diaphragm, liver, and lung were significantly attenuated by LPS administration. In addition, Tie-2 tyrosine phosphorylation in the lung was significantly reduced in response to LPS injection. In vitro exposure to E. coli LPS elicited cell-specific changes in Ang-1 expression, with significant induction in Ang-1 expression being observed in cultured human epithelial cells, whereas significant attenuation of Ang-1 expression was observed in response to E. coli LPS exposure in primary human skeletal myoblasts. In both cell types, E. coli LPS elicited substantial induction of Ang-2 mRNA, a response that was mediated in part through NF-κB. We conclude that in vivo endotoxemia triggers functional inhibition of the Ang-1/Tie-2 receptor pathway by reducing Ang-1 and Tie-2 expression and inducing Ang-2 levels and that this response may contribute to enhanced vascular leakage in sepsis.
Inhibition of Urinary Bladder Carcinogenesis by Broccoli Sprouts
Authors: Munday Rex , Mhawech-Fauceglia Paulette , Munday Christine M. , Paonessa Joseph D. , Tang Li , Munday John S. , Lister Carolyn , Wilson Paula , Fahey Jed W. , Davis Warren , Zhang Yuesheng
Published: March 2008

Isothiocyanates are a well-known class of cancer chemopreventive agents, and broccoli sprouts are a rich source of several isothiocyanates. We report herein that dietary administration to rats of a freeze-dried aqueous extract of broccoli sprouts significantly and dose-dependently inhibited bladder cancer development induced by N-butyl-N-(4-hydroxybutyl) nitrosamine. The incidence, multiplicity, size, and progression of bladder cancer were all inhibited by the extract, while the extract itself caused no histologic changes in the bladder. Moreover, inhibition of bladder carcinogenesis by the extract was associated with significant induction of glutathione S-transferase and NAD(P)H:quinone oxidoreductase 1 in the bladder, enzymes that are important protectants against oxidants and carcinogens. Isothiocyanates are metabolized to dithiocarbamates in vivo, but dithiocarbamates readily dissociate to isothiocyanates. We found that >70% of the isothiocyanates present in the extract were excreted in the urine as isothiocyanate equivalents (isothiocyanates + dithiocarbamates) in 12 h after a single p.o. dose, indicating high bioavailability and rapid urinary excretion. In addition, the concentrations of isothiocyanate equivalents in the urine of extract-treated rats were 2 to 3 orders of magnitude higher than those in plasma, indicating that the bladder epithelium, the major site of bladder cancer development, is most exposed to p.o. dosed isothiocyanate. Indeed, tissue levels of isothiocyanate equivalents in the bladder were significantly higher than in the liver. In conclusion, broccoli sprout extract is a highly promising substance for bladder cancer prevention and the isothiocyanates in the extract are selectively delivered to the bladder epithelium through urinary excretion. [Cancer Res 2008;68(5):1593–600]
Small Molecular Weight Variants of p53 Are Expressed in Human Melanoma Cells and Are Induced by the DNA-Damaging Agent Cisplatin
Authors: Avery-Kiejda Kelly A. , Zhang Xu Dong , Adams Luke J. , Scott Rodney J. , Vojtesek Borivoj , Lane David P. , Hersey Peter
Published: March 2008

Purpose: Metastatic melanoma is largely unresponsive to DNA-damaging chemotherapy agents, although WTp53 is frequently detected. Several isoforms of p53 have been discovered, some of which inhibit p53 function. We therefore examined whether p53 isoforms were present in melanoma and whether they may contribute to aberrant p53 function in melanoma.

Experimental Design: We studied the expression and subcellular localization of p53 and its isoforms in a panel of human melanoma cell lines using Western blot, two-dimensional electrophoresis, and reverse transcription-PCR. We also characterized the relationship between the expression of p53, p53 isoforms, and p53 target genes following treatment with the DNA-damaging agent cisplatin.

Results: We report that p53β and Δ40p53 were expressed in the majority of melanoma cell lines at the mRNA level, but were absent or expressed at low levels in fibroblasts and melanocytes, suggesting that their expression may play a role in melanoma development. Analysis by two-dimensional gel electrophoresis revealed that p53β was expressed at the protein level in melanoma cells. Both p53 and the small molecular weight forms of p53 were aberrantly expressed between the nuclear and cytosolic fractions of melanoma cell lines, compared with normal fibroblasts. Treatment with cisplatin had differential effects on WTp53 and the small molecular weight form of p53 that were cell line dependent. Δ40p53 was shown to inhibit, whereas p53β was shown to enhance, p53-dependent transcription of p21 and PUMA.

Conclusions: p53β and Δ40p53 are expressed in melanoma and this may have important implications for understanding resistance of melanoma to DNA-damaging chemotherapy.
Molecular, Pathologic, and Clinical Features of Early-Onset Endometrial Cancer: Identifying Presumptive Lynch Syndrome Patients
Authors: Walsh Michael D. , Cummings Margaret C. , Buchanan Daniel D. , Dambacher Wendy M. , Arnold Sven , McKeone Diane , Byrnes Rebecca , Barker Melissa A. , Leggett Barbara A. , Gattas Michael , Jass Jeremy R. , Spurdle Amanda B. , Young Joanne , Obermair Andreas
Published: March 2008

Purpose: A woman with early-onset endometrial cancer (EC) may represent the “sentinel” cancer event in a Lynch syndrome kindred. The aim of this study was to determine the incidence of Lynch syndrome in a series of young-onset EC, and to identify molecular, clinical, and pathologic features that may alert clinicians to the presence of this disorder.

Experimental Design: Patients with EC, ages ≤50 years, were identified from the Queensland Centre for Gynaecological Cancer. Tumor sections underwent histopathology review and were immunostained for mismatch repair proteins. Tumor DNA was tested for microsatellite instability and methylation of MLH1. Patients were conservatively classified as presumptive Lynch syndrome if their tumors showed loss of at least one mismatch repair protein and were negative for methylation of MLH1. Personal and family history of cancer was reviewed where available.

Results: Presumptive Lynch syndrome was seen in 26 of 146 (18%) tumors. These tumors were more likely to be poorly differentiated, International Federation of Gynecology and Obstetrics stage II and above, have tumor-infiltrating lymphocytes, have higher mitotic rate, and have deeper myometrial invasion (P < 0.05). Lynch syndrome cases were more likely to be associated with a positive family history when analyzed for Amsterdam criteria II, diagnosis of a Lynch syndrome spectrum cancer in at least one first-degree relative, and family history of any cancer (P < 0.05).

Conclusion: Presumptive Lynch syndrome was identified in 18% of early-onset EC. A risk of this magnitude would argue for routine immunohistochemical testing of tumors in patients diagnosed with EC at or before the age of 50 years.
Inpatient Brain MRI for New-Onset Seizures: Utility and Cost Effectiveness
Authors: Rauch Daniel A. , Carr Emily , Harrington John
Published: March 2008

Inpatient magnetic resonance imaging for evaluation of new-onset afebrile seizures can extend hospital stay, can add cost, and may not affect acute management. The study was to assess cost and impact on acute management of inpatient brain magnetic resonance imagings performed as part of new-onset seizure evaluation. Retrospective chart review of brain magnetic resonance imagings on pediatric inpatients admitted for new-onset afebrile seizure has been carried out. Seventy-five patients were identified. Seventy-one of the patients (95%) stayed an additional day for the magnetic resonance imaging. Thirteen had abnormal results. No patient had treatment changes based upon the magnetic resonance imaging results. If these magnetic resonance imagings had been done as an outpatient, the savings to the hospital could have been $215 575. Thus, obtaining an inpatient brain magnetic resonance imaging for evaluation of new-onset seizures did not lead to acute changes in management and added to facility costs and length of stay. Scheduling a magnetic resonance imaging as an outpatient could achieve cost savings without compromising care.
Gang Membership and Drug Involvement
Authors: Bjerregaard Beth
Published: March 2008

Previous research has consistently demonstrated a relationship between gang membership and involvement in illegal substances. In addition, researchers have noted that gang members are frequently more heavily involved in drug sales, which often lead to increases in violent behaviors. Most of this research, however, is either cross-sectional or ethnographic in nature, and therefore does not reveal the causal nature of these relationships. This research attempts to establish the temporal ordering of these relationships while controlling for a variety of relevant variables and to determine whether the relationships between drug involvement and violence differ for gang members versus nongang members. The findings indicate that gang membership is weakly associated with drug involvement, including both usage and sales. This involvement, however, does not appear to be related to assaults. Results suggest that gang membership is not determinative of drug involvement among a national random sample of youth.
Assessing the Race–Crime and Ethnicity–Crime Relationship in a Sample of Serious Adolescent Delinquents
Authors: Piquero Alex R. , Brame Robert W.
Published: March 2008

Official record studies consistently show that Blacks exhibit higher levels of involvement in criminal offending than Whites do. Although self-report studies suggest somewhat lower levels of Black overrepresentation in criminal offending activity (especially with less serious forms of crime), there appears to be considerable evidence that Blacks are disproportionately involved in serious crime. Yet most of this evidence is based on data from broad cross-sections of the general population. To date, there is little evidence on which to base inferences about the relationship between race and criminal involvement within serious offender populations. In this article, the authors use both official record and self-report data on samples of serious adolescent offenders in Philadelphia and Phoenix to reach a better understanding of the relationship between race and criminal activity. The analysis suggests that consistent race differences of the kind normally seen in the criminological literature are not evident in our sample of serious offenders.
Inmate Recidivism as a Measure of Private Prison Performance
Authors: Spivak Andrew L. , Sharp Susan F.
Published: March 2008

The growth of the private corrections industry has elicited interest in the comparative performance of state and private prisons. One way to measure the service quality of private prisons is to examine inmates' postrelease performance. Current empirical evidence is limited to four studies, all conducted in Florida. This analysis replicates and adds to the Florida measures in a different state and enhances previous methods. It uses data for a large cohort of Oklahoma state prison inmates released between 1997 and 2001. Controlling for known covariates, multivariate survival analysis revealed comparative rates of reincarceration for inmates in multiple exposure and comparison groups. These results are unique among prior studies on this topic; private prison inmates had a greater hazard of recidivism in all eight models tested, six of which were statistically significant. Finding no empirical support for claims of superior service from private corrections, the authors discuss policy implications and prospects for future research.
Sex Crimes, Children, and Pornography
Authors: Mears Daniel P. , Mancini Christina , Gertz Marc , Bratton Jake
Published: March 2008

“Get tough” approaches for responding to sex crimes have proliferated during the past decade. Child pornography in particular has garnered attention in recent years. Policy makers increasingly have emphasized incarceration as a response to such crime, including accessing child pornography. Juxtaposed against such efforts is a dearth of knowledge about “get tough” policies for responding to sex crimes, particularly those targeting children, and how most appropriately to respond to such crimes. The authors examine data from a national telephone survey of Americans to explore views toward sex crimes, with a special emphasis on crimes against children. The findings indicate the public supports tough responses to child sex crimes, but they also support treatment of sex offenders. Also, despite views that incarceration is an appropriate response to possessing child pornography, several social and demographic cleavages in such support exist. The authors discuss these findings and their implications for policy and research.
Reporting Error in Household Gun Ownership in the 2000 General Social Survey
Authors: Legault Richard L.
Published: March 2008

The use of surveys is one of the few ways to determine the extent and character of firearms ownership in the United States. The findings from such surveys have implications for both firearms research and firearms policy. Using data from the 2000 General Social Survey, the author examines the possibility of error in the reporting of household firearm ownership. Strong and significant differences in ownership reporting are found between married men and women, especially for those who were not socialized in part of a traditional “gun culture.”
The Impact of Information on Death Penalty Support, Revisited
Authors: Lambert Eric G. , Camp Scott D. , Clarke Alan , Jiang Shanhe
Published: March 2008

In 1972, former Supreme Court Justice Marshall postulated that the public was uninformed about the death penalty and information would change their support for it. There is some indication that information about the death penalty may change people’s level of support. This study re-examines data used by Lambert and Clarke (2001). Using multivariate analyses, the impact that information has on death penalty support is tested, along with level of prior knowledge about the death penalty, personal characteristics (gender, age, political affiliation, race, being a criminal justice major, academic level), and religious factors. The results suggest that information on both deterrence and innocence leads to a reduction in death penalty support and views on the death penalty. Furthermore, the results suggest that the information presented may have varying effects among different subgroups of people.