bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of FimW, FimY, and FimZ in Regulating the Expression of Type I Fimbriae in
Salmonella enterica
Serovar Typhimurium
Authors: Saini Supreet , Pearl Jeffrey A. , Rao Christopher V.
Published: February 2009

Type I fimbriae in
Salmonella enterica
serovar Typhimurium are surface appendages that facilitate binding to eukaryotic cells. Expression of the
fim
gene cluster is known to be regulated by three proteins—FimW, FimY, and FimZ—and a tRNA encoded by
fimU
. In this work, we investigated how these proteins and tRNA coordinately regulate
fim
gene expression. Our results indicate that FimY and FimZ independently activate the P

fimA

promoter which controls the expression of the
fim
structural genes. FimY and FimZ were also found to strongly activate each other's expression and weakly activate their own expression. FimW was found to negatively regulate
fim
gene expression by repressing transcription from the P

fimY

promoter, independent of FimY or FimZ. Moreover, FimW and FimY interact within a negative feedback loop, as FimY was found to activate the P

fimW

promoter. In the case of
fimU
, the expression of this gene was not found to be regulated by FimW, FimY, or FimZ. We also explored the effect of
fim
gene expression on
Salmonella
pathogenicity island 1 (SPI1). Our results indicate that FimZ alone is able to enhance the expression of
hilE
, a known repressor of SPI1 gene expression. Based on our results, we were able to propose an integrated model for the
fim
gene circuit. As this model involves a combination of positive and negative feedback, we hypothesized that the response of this circuit may be bistable and thus a possible mechanism for phase variation. However, we found that the response was continuous and not bistable.
ClpXP Degrades SsrA-Tagged Proteins inStreptococcus pneumoniae
Authors: Ahlawat Sarita , Morrison Donald A.
Published: February 2009

Bacterial proteins that are abnormally truncated due to incomplete mRNA or the presence of rare codons are extended by an SsrA tag during ribosome rescue in atrans-translation process important for maintaining protein quality. InEscherichia coli, the SsrA-tagged proteins become the target of the Tsp, Lon, FtsH, ClpXP, and ClpAP proteases. Here we show that degradation of model SsrA-tagged proteins inStreptococcus pneumoniaedepends primarily or exclusively on ClpXP in vivo. In addition, we show theE. coliSsrA tag is also a target ofS. pneumoniaeClpXP in vivo, even though the N-terminal portions of the tags differ significantly between the two species, suggesting there may be no adaptor protein for SsrA inS. pneumoniae.
SleC Is Essential for Cortex Peptidoglycan Hydrolysis during Germination of Spores of the Pathogenic Bacterium
Clostridium perfringens
Authors: Paredes-Sabja Daniel , Setlow Peter , Sarker Mahfuzur R.
Published: February 2009

Clostridial spore germination requires degradation of the spore's peptidoglycan (PG) cortex by cortex-lytic enzymes (CLEs), and two
Clostridium perfringens
CLEs, SleC and SleM, degrade cortex PG in vitro. We now find that only SleC is essential for cortex hydrolysis and viability of
C. perfringens
spores.
C. perfringens sleC
spores did not germinate completely with nutrients, KCl, or a 1:1 chelate of Ca
2+
and dipicolinic acid (Ca-DPA), and the colony-forming efficiency of
sleC
spores was 10
3
-fold lower than that of wild-type spores. However,
sleC
spores incubated with various germinants released most of their DPA, although slower than wild-type or
sleM
spores, and DPA release from
sleC sleM
spores was very slow. In contrast, germination and viability of
sleM
spores were similar to that of wild-type spores, although
sleC sleM
spores had 10
5
-fold-lower viability. These results allow the following conclusions about
C. perfringens
spore germination: (i) SleC is essential for cortex hydrolysis; (ii) although SleM can degrade cortex PG in vitro, this enzyme is not essential; (iii) action of SleC alone or with SleM can accelerate DPA release; and (iv) Ca-DPA does not trigger spore germination by activation of CLEs.
Identification of Major Histocompatibility Complex Restriction and Anchor Residues of Foot-and-Mouth Disease Virus-Derived Bovine T-Cell Epitopes
Authors: Gerner Wilhelm , Hammer Sabine E. , Wiesmüller Karl-Heinz , Saalmüller Armin
Published: February 2009

Despite intensive research on the identification of T-cell epitopes in cattle after foot-and-mouth disease virus (FMDV) infection during the last 20 years, knowledge of major histocompatibility complex (MHC) restriction and anchor residues of such epitopes is still sparse. Therefore, as a first step, we tested lymphocytes from two experimentally FMDV serotype A24-vaccinated and -challenged cattle for recognition of FMDV-derived pentadecapeptides in proliferation assays. Two epitopes were identified: amino acid residues 66 to 80 within the structural protein 1D and amino acid residues 22 to 36 within the structural protein 1A. The latter epitope was recognized by lymphocytes from both cattle. Peptide-specific proliferation was caused by a response of CD4+T helper cells as identified by carboxyfluorescein diacetate succinimidyl ester proliferation assays. Having identified one epitope that was recognized by two cattle, we hypothesized that these animals should have common MHC class II alleles. Cloning and sequencing of DRB3, DQA, and DQB alleles revealed that both animals possessed DQA allele 22021 and DQB allele 1301 but had no common DRB3 allele. A parallel analysis of amino acid residues involved in MHC presentation by peptides with alanine substitutions showed that the amino acid residues in positions 5 and 9 within the pentadecapeptide representing the 1A epitope were important for MHC binding in both cattle. These data indicate that the epitope located on FMDV protein 1A can be presented by MHC class II DQ molecules encoded by DQA allele 22021 and DQB allele 1301 and present the first evidence of the binding motif of this particular DQ molecule.
Role of Natural Killer Cells in a Cohort of Elite Suppressors: Low Frequency of the Protective KIR3DS1 Allele and Limited Inhibition of Human Immunodeficiency Virus Type 1 Replication In Vitro
Authors: O'Connell Karen A. , Han Yefei , Williams Thomas M. , Siliciano Robert F. , Blankson Joel N.
Published: February 2009

Natural killer (NK) cells are associated with the innate immune response and are important in many viral infections. Recent studies indicate that NK cells can control human immunodeficiency virus type 1 (HIV-1) replication. We studied the effect of NK cells on HIV-1 replication in a subpopulation of HIV-1-infected individuals termed elite suppressors (ES) or elite controllers. These patients maintain a clinically undetectable viral load without treatment and thus provide a fascinating cohort in which to study the immunological response to HIV-1. Using an autologous system, we analyzed the effects of NK cells and CD8
+
T cells on viral replication in CD4
+
T lymphoblasts. Although we had postulated that NK cells of ES would be highly effective at controlling viral replication, we found that NK cells from some, but not all, ES were capable of inhibiting replication in the presence of interleukin-2, and the inhibition was less robust than that mediated by CD8
+
T cells. Additionally, we examined whether particular alleles of the KIR receptors, specifically KIR3DS1 and KIR3DL1, or allele-ligand combinations correlated with the control of HIV-1 replication by NK cells and whether any specific KIR alleles were overrepresented in ES. Our ES cohort did not differ from the general population with respect to the frequency of individual KIR. However, of the eight ES studied, the four exhibiting the most NK cell-mediated control of viral replication also had the fewest activating KIR and were haplotype A. Thus, the strong NK cell-mediated inhibition of viral replication is not necessary for the immunological control of HIV-1 in all ES.
Nipah Virus Edits Its P Gene at High Frequency To Express the V and W Proteins
Authors: Kulkarni Sachin , Volchkova Valentina , Basler Christopher F. , Palese Peter , Volchkov Viktor E. , Shaw Megan L.
Published: February 2009

Nipah virus (NiV) is predicted to encode four proteins from its P gene (P, V, W, and C) via mRNA editing and an alternate open reading frame. By use of specific antibodies, the expression of the V, W, and C proteins in NiV-infected cells has now been confirmed. Analysis of the P-gene transcripts shows a ratio of P:V:W mRNA of 1:1:1, but this differs over time, with greater proportions of V and W transcripts observed as the infection progresses. Eighty-two percent of transcripts are edited, with up to 11 G insertions observed. This exceptionally high editing frequency ensures expression of the V and W proteins.
Mortality and cancer incidence in workers in two Australian prebake aluminium smelters
Authors: Sim M R , Monaco A Del , Hoving J L , MacFarlane E , McKenzie D , Benke G , de Klerk N , Fritschi L
Published: February 2009

Background:

Exposures in the aluminium production industry have been declining and this study aimed to investigate cancer and mortality in two Australian prebake aluminium smelters.

Methods:

The cohort included 4396 males who had worked in a prebake smelter for at least 3 months. They were matched against the National Death Index and the National Cancer Statistics Clearing House to obtain cause of death and type of cancer. Standardised Mortality Ratios (SMRs) and Standardised Incidence Ratios (SIRs) were calculated for the whole cohort, for production or maintenance work categories and for duration of employment categories.

Results:

The SMRs for all causes, circulatory, respiratory and injury deaths were at or below expected. Mesothelioma was the only significantly increased cause of death (SMR 3.52, 95% CI 1.47 to 8.46). Death from prostate cancer in production workers was elevated (SMR 2.39, 95% CI 1.29 to 4.44) and in those who had worked for more than 20 years in production or maintenance jobs (SMR 3.67, 95% CI 1.53 to 8.81). There were statistically significant excesses of incident stomach cancer, mesothelioma, and kidney cancer cases, while the SIR for melanoma was significantly reduced. There was no significant trend for duration of exposure for any type of incident cancer.

Conclusions:

This study found no overall excess of mortality or cancer, but incident mesothelioma and kidney cancer risks were elevated. The lack of excess risk for lung or bladder cancer or deaths from respiratory disease may be related to the different level and pattern of exposure between Søderberg and prebake smelters.
Philip Morris involvement in the development of an air quality laboratory in El Salvador
Authors: Kummerfeldt C E , Barnoya J , Bero L
Published: February 2009

Background:

The tobacco industry has organised research institutions to generate misleading data on indoor air quality, including second-hand smoke exposure and health effects.

Objectives:

To describe tobacco industry involvement in the organisation and financial support of an air quality research laboratory in El Salvador.

Methods:

Tobacco industry documents on the internet were systematically searched from August 2007 to February 2008 for air quality studies undertaken in El Salvador, and laboratory personnel were interviewed.

Results:

Philip Morris sought to establish a network of air quality laboratories throughout Latin America. In El Salvador, in 1997, through Tabacalera de El Salvador (a subsidiary of Philip Morris) and the Salvadoran Foundation for Economic Development (FUSADES), the industry organised an air quality research laboratory. FUSADES was part of the industry’s Latin American Scientific Network, which consisted of doctors hired as consultants who would send air samples from their research to FUSADES. Philip Morris Scientific Affairs personnel hired LabStat, a Canadian-based laboratory, to provide technical assistance to FUSADES (train and assist the laboratory in air quality measurements). In addition, the Washington-based HMS Group successfully implemented a plan to upgrade the laboratory and obtain international certifications. HMS Group also assisted in searching for sustainable funding for FUSADES, including seeking funds from international aid for Hurricane Mitch.

Conclusion:

Air quality studies that have used the FUSADES laboratory should be carefully interpreted, given the support that this laboratory received from Philip Morris.
UNIFORM HAZARD SPECTRA BASED ON PARK-ANG DAMAGE INDEX
Authors: DATTA DEBARATI , GHOSH SIDDHARTHA
Published: February 2009

The primary emphases in the performance-based seismic design (PBSD) philosophy are in the accounting for uncertainties in seismic demand/capacity and in the better quantification of seismic damage using suitable inelastic damage parameters. Uniform hazard spectra (UHS) provide probabilistic information regarding a seismic demand on a single degree oscillator for a specific site. UHS are very good tools for probabilistic hazard estimation as intended in PBSD. In the present work, UHS are generated for Park-Ang damage index of an elastic-perfectly plastic oscillator. Park-Ang damage index takes into account the effects of both displacement ductility demand and hysteretic energy demand in low-cycle-fatigue, and therefore is a demand parameter suitable for PBSD. The UHS are generated for a specific site using artificially generated ground motions. Two types of UHS plot are illustrated. A correlation between the probability of exceedance (of certain target damage index) and stated level of structural capacity is also established. Information provided by the UHS are proposed to be used, with the aid of equivalent systems, in the development of a reliability-based seismic design framework considering Park-Ang damage index as the seismic demand parameter.
AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H+-ATPase accumulation in epididymal clear cells
Authors: Hallows Kenneth R. , Alzamora Rodrigo , Li Hui , Gong Fan , Smolak Christy , Neumann Dietbert , Pastor-Soler Núria M.
Published: February 2009

Acidic luminal pH and low [HCO3−] maintain sperm quiescent during maturation in the epididymis. The vacuolar H+-ATPase (V-ATPase) in clear cells is a major contributor to epididymal luminal acidification. We have shown previously that protein kinase A (PKA), acting downstream of soluble adenylyl cyclase stimulation by alkaline luminal pH or HCO3−, induces V-ATPase apical membrane accumulation in clear cells. Here we examined whether the metabolic sensor AMP-activated protein kinase (AMPK) regulates this PKA-induced V-ATPase apical membrane accumulation. Immunofluorescence labeling of rat and non-human primate epididymides revealed specific AMPK expression in epithelial cells. Immunofluorescence labeling of rat epididymis showed that perfusion in vivo with the AMPK activators 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) or A-769662 induced a redistribution of the V-ATPase into subapical vesicles, even in the presence of a luminal alkaline (pH 7.8) buffer compared with that of controls perfused without drug. Moreover, preperfusion with AICAR blocked the PKA-mediated V-ATPase translocation to clear cell apical membranes induced by N 6-monobutyryl-cAMP (6-MB-cAMP). Purified PKA and AMPK both phosphorylated V-ATPase A subunit in vitro. In HEK-293 cells [32P]orthophosphate in vivo labeling of the A subunit increased following PKA stimulation and decreased following RNA interference-mediated knockdown of AMPK. Finally, the extent of PKA-dependent in vivo phosphorylation of the A subunit increased with AMPK knockdown. In summary, our findings suggest that AMPK inhibits PKA-mediated V-ATPase apical accumulation in epididymal clear cells, that both kinases directly phosphorylate the V-ATPase A subunit in vitro and in vivo, and that AMPK inhibits PKA-dependent phosphorylation of this subunit. V-ATPase activity may be coupled to the sensing of acid-base status via PKA and to metabolic status via AMPK.
Alterations to enteric neural signaling underlie secretory abnormalities of the ileum in experimental colitis in the guinea pig
Authors: Hons Ian M. , Burda Joshua E. , Grider John R. , Mawe Gary M. , Sharkey Keith A.
Published: February 2009

Inflammatory bowel diseases (IBD) can involve widespread gastrointestinal dysfunction, even in cases in which inflammation is localized to a single site. The underlying pathophysiology of dysfunction in noninflamed regions is unclear. We examined whether colitis is associated with altered electrogenic ion transport in the ileal mucosa and/or changes in the properties of ileal submucosal neurons. Colitis was induced by administration of trinitrobenzene sulfonic acid (TNBS), and the uninflamed ileum from animals was examined 3, 7, and 28 days later. Electrogenic ion transport was assessed in Ussing chambers. Intracellular microelectrode recordings were used to examine the neurophysiology of the submucosal plexus of the ileum in animals with colitis. Noncholinergic secretion was reduced by 33% in the ileum from animals 7 days after the induction of colitis. The epithelial response to vasoactive intestinal peptide (VIP) was unaltered in animals with colitis, but the response to carbachol was enhanced. Slow excitatory synaptic transmission was dramatically reduced in VIP-expressing, noncholinergic secretomotor neurons. This change was detected as early as 3 days following TNBS treatment. No changes to fast synaptic transmission or the number of VIP neurons were observed. In addition, cholinergic secretomotor neurons fired more action potentials during a given stimulus, and intrinsic primary afferent neurons had broader action potentials in animals with colitis. These findings implicate changes to enteric neural circuits as contributing factors in inflammation-induced secretory dysfunction at sites proximal to a localized inflammatory insult.
Effect of the artificial sweetener, sucralose, on gastric emptying and incretin hormone release in healthy subjects
Authors: Ma Jing , Bellon Max , Wishart Judith M. , Young Richard , Blackshaw L. Ashley , Jones Karen L. , Horowitz Michael , Rayner Christopher K.
Published: February 2009

The incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), play an important role in glucose homeostasis in both health and diabetes. In mice, sucralose, an artificial sweetener, stimulates GLP-1 release via sweet taste receptors on enteroendocrine cells. We studied blood glucose, plasma levels of insulin, GLP-1, and GIP, and gastric emptying (by a breath test) in 7 healthy humans after intragastric infusions of 1) 50 g sucrose in water to a total volume of 500 ml (∼290 mosmol/l), 2) 80 mg sucralose in 500 ml normal saline (∼300 mosmol/l, 0.4 mM sucralose), 3) 800 mg sucralose in 500 ml normal saline (∼300 mosmol/l, 4 mM sucralose), and 4) 500 ml normal saline (∼300 mosmol/l), all labeled with 150 mg 13C-acetate. Blood glucose increased only in response to sucrose ( P < 0.05). GLP-1, GIP, and insulin also increased after sucrose ( P = 0.0001) but not after either load of sucralose or saline. Gastric emptying of sucrose was slower than that of saline ( t50: 87.4 ± 4.1 min vs. 74.7 ± 3.2 min, P < 0.005), whereas there were no differences in t50 between sucralose 0.4 mM (73.7 ± 3.1 min) or 4 mM (76.7 ± 3.1 min) and saline. We conclude that sucralose, delivered by intragastric infusion, does not stimulate insulin, GLP-1, or GIP release or slow gastric emptying in healthy humans.
Physiological roles of A1and A2Aadenosine receptors in regulating heart rate, body temperature, and locomotion as revealed using knockout mice and caffeine
Authors: Yang Jiang-Ning , Chen Jiang-Fan , Fredholm Bertil B.
Published: February 2009

Heart rate (HR), body temperature (Temp), locomotor activity (LA), and oxygen consumption (O2C) were studied in awake mice lacking one or both of the adenosine A1or A2Areceptors (A1R or A2AR, respectively) using telemetry and respirometry, before and after caffeine administration. All parameters were lower during day than night and higher in females than males. When compared with wild-type (WT) littermates, HR was higher in male A1R knockout (A1RKO) mice but lower in A2ARKO mice and intermediate in A1-A2AR double KO mice. A single dose of an unselective β-blocker (timolol; 1 mg/kg) abolished the HR differences between these genotypes. Deletion of A1Rs had little effect on Temp, whereas deletion of A2ARs increased it in females and decreased it in males. A1-A2ARKO mice had lower Temp than WT mice. LA was unaltered in A1RKO mice and lower in A2ARKO and A1-A2ARKO mice than in WT mice. Caffeine injection increased LA but only in mice expressing A2AR. Caffeine ingestion also increased LA in an A2AR-dependent manner in male mice. Caffeine ingestion significantly increased O2C in WT mice, but less in the different KO mice. Injection of 30 mg/kg caffeine decreased Temp, especially in KO mice, and hence in a manner unrelated to A1R or A2AR blockade. Selective A2Bantagonism had little or no effect. Thus A1R and A2AR influence HR, Temp, LA, and O2C in mice in a sex-dependent manner, indicating effects of endogenous adenosine. The A2AR plays an important role in the modulation of O2C and LA by acute and chronic caffeine administration. There is also evidence for effects of higher doses of caffeine being independent of both A1R and A2AR.
Translocon closure to Ca2+leak in proliferating vascular smooth muscle cells
Authors: Amer Mohamed S , Li Jing , O'Regan David J. , Steele Derek S. , Porter Karen E. , Sivaprasadarao Asipu , Beech David J.
Published: February 2009

Vascular smooth muscle cells have a proliferative phenotype that is important in vascular development, adaptation, and disease. Intracellular calcium handling is thought to play pivotal roles in determining the properties of these cells, and thus previously unrecognized mechanisms for transmembrane calcium movement are of potential interest. An unsolved question is the mechanism of constitutive (passive) calcium leak from the intracellular stores. Studies of other cell types have suggested that the translocon is a calcium leak pathway. Here we investigated the contribution of the translocon in proliferating vascular smooth muscle cells. Calcium leak into the cytoplasm was measured using fura-2, and protein synthesis was measured using radioactive methionine. Puromycin, emetine, and anisomycin are chemicals that inhibit protein synthesis, acting via the translocon; all three agents strongly inhibited protein synthesis in the smooth muscle cells within 1 h. Puromycin, which opens the translocon, evoked a transient increase in cytoplasmic calcium that was similar in amplitude to the calcium rise evoked by thapsigargin. The puromycin effect was abolished by thapsigargin. The treatment of cells for 1 h with emetine or anisomycin, which close the translocon, inhibited the calcium release evoked by puromycin but not the calcium release evoked by extracellular ATP, endothelin-1, or the calcium ionophore ionomycin. Thapsigargin-evoked calcium rises were slightly suppressed by emetine but unaffected by puromycin or anisomycin. The data suggest that the translocon has the capacity to act as a calcium leak pathway in the ribosomal endoplasmic reticulum but that it is normally closed and lacks relevance to physiological calcium leak mechanisms.
Cardiac systolic and diastolic function during whole body heat stress
Authors: Brothers R. Matthew , Bhella Paul S. , Shibata Shigeki , Wingo Jonathan E. , Levine Benjamin D. , Crandall Craig G.
Published: February 2009

During a whole body heat stress, stroke volume is either maintained or slightly elevated despite reduced ventricular filling pressures and central blood volume, suggestive of improved cardiac diastolic and/or systolic function. Heat stress improves cardiac systolic and diastolic function in patients with congestive heart failure, although it remains unknown whether similar responses occur in healthy individuals, which is the hypothesis to be tested. Nine male volunteers underwent a whole body heat stress. Echocardiographic indexes of diastolic and systolic function were performed following a supine resting period, and again following an increase in internal temperature of ∼1.0°C via passive heat stress. Despite previous reports of heat stress-induced decreases in ventricular filling pressures and central blood volume, no changes in indexes of diastolic function were identified during heating [i.e., unchanged early diastolic mitral annular tissue velocity (E′), mitral inflow during the early diastolic phase (E), the E/E′ ratio, and isovolumetric relaxation time]. Heat stress increased late diastolic septal ( P = 0.03) and lateral ( P = 0.01) mitral annular tissue velocities (A′), mitral inflow velocity during atrial contraction ( P < 0.001), and the relative contribution of atrial contraction to left ventricular filling during diastole ( P = 0.01), all indicative of improved atrial systolic function. Furthermore, indexes of ventricular systolic function were increased by heat stress [i.e., increased septal ( P = 0.001) and lateral ( P = 0.01) mitral annular systolic velocities and isovolumic acceleration at the septal ( P = 0.03) and lateral ( P < 0.001) mitral annulus]. These data are suggestive of improved atrial and ventricular systolic function by the heat stress. Together these data support previous findings, which used the less precise measure of ejection fraction, that heat stress improves indexes of systolic function, while diastolic function is maintained.
Differential regulation of angiotensin-(1-12) in plasma and cardiac tissue in response to bilateral nephrectomy
Authors: Ferrario Carlos M. , Varagic Jasmina , Habibi Javad , Nagata Sayaka , Kato Johji , Chappell Mark C. , Trask Aaron J. , Kitamura Kazuo , Whaley-Connell Adam , Sowers James R.
Published: February 2009

We examined the effects of 48 h bilateral nephrectomy on plasma and cardiac tissue expression of angiotensin-(1-12) [ANG-(1-12)], ANG I, and ANG II in adult Wistar-Kyoto rats to evaluate functional changes induced by removing renal renin. The goal was to expand the evidence of ANG-(1-12) being an alternate renin-independent, angiotensin-forming substrate. Nephrectomy yielded divergent effects on circulating and cardiac angiotensins. Significant decreases in plasma ANG-(1-12), ANG I, and ANG II levels postnephrectomy accompanied increases in cardiac ANG-(1-12), ANG I, and ANG II concentrations compared with controls. Plasma ANG-(1-12) decreased 34% following nephrectomy, which accompanied 78 and 66% decreases in plasma ANG I and ANG II, respectively ( P < 0.05 vs. controls). Contrastingly, cardiac ANG-(1-12) in anephric rats averaged 276 ± 24 fmol/mg compared with 144 ± 20 fmol/mg in controls ( P < 0.005). Cardiac ANG I and ANG II values were 300 ± 15 and 62 ± 7 fmol/mg, respectively, in anephric rats compared with 172 ± 8 fmol/mg for ANG I and 42 ± 4 fmol/mg for ANG II in controls ( P < 0.001). Quantitative immunofluorescence revealed significant increases in average grayscale density for cardiac tissue angiotensinogen, ANG I, ANG II, and AT1 receptors of WKY rats postnephrectomy. Faint staining of cardiac renin, unchanged by nephrectomy, was associated with an 80% decrease in cardiac renin mRNA. These changes were accompanied by significant increases in p47phox, Rac1, and Nox4 isoform expression. In conclusion, ANG-(1-12) may be a functional precursor for angiotensin peptide formation in the absence of circulating renin.
Rho-kinase-mediated suppression of KDR current in cerebral arteries requires an intact actin cytoskeleton
Authors: Luykenaar Kevin D. , El-Rahman Rasha Abd , Walsh Michael P. , Welsh Donald G.
Published: February 2009

This study examined the role of the actin cytoskeleton in Rho-kinase-mediated suppression of the delayed-rectifier K+ (KDR) current in cerebral arteries. Myocytes from rat cerebral arteries were enzymatically isolated, and whole cell KDR currents were monitored using conventional patch-clamp electrophysiology. At +40 mV, the KDR current averaged 19.8 ± 1.6 pA/pF (mean ± SE) and was potently inhibited by UTP (3 × 10−5 M). This suppression was observed to depend on Rho signaling and was abolished by the Rho-kinase inhibitors H-1152 (3 × 10−7 M) and Y-27632 (3 × 10−5 M). Rho-kinase was also found to concomitantly facilitate actin polymerization in response to UTP. We therefore examined whether actin dynamics played a role in the ability of Rho-kinase to suppress KDR current and found that actin disruption using either cytochalasin D (1 × 10−5 M) or latrunculin A (1 × 10−8 M) prevented current modulation. Consistent with our electrophysiological observations, both Rho-kinase inhibition and actin disruption significantly attenuated UTP-induced depolarization and constriction of cerebral arteries. We propose that UTP initiates Rho-kinase-mediated remodeling of the actin cytoskeleton and consequently suppresses the KDR current, thereby facilitating the depolarization and constriction of cerebral arteries.
Advanced methods for quantification of infarct size in mice using three-dimensional high-field late gadolinium enhancement MRI
Authors: Bohl Steffen , Lygate Craig A. , Barnes Hannah , Medway Debra , Stork Lee-Anne , Schulz-Menger Jeanette , Neubauer Stefan , Schneider Jurgen E.
Published: February 2009

Conventional methods to quantify infarct size after myocardial infarction in mice are not ideal, requiring either tissue destruction for histology or relying on nondirect measurements such as wall motion. We therefore implemented a fast, high-resolution method to directly measure infarct size in vivo using three-dimensional (3D) late gadolinium enhancement MRI (3D-LGE). Myocardial T1 relaxation was quantified at 9.4 Tesla in five mice, and reproducibility was tested by repeat imaging after 5 days. In a separate set of healthy and infarcted mice ( n = 8 of each), continuous T1 measurements were made following intravenous or intraperitoneal injection of a contrast agent (0.5 μmol/g gadolinium-diethylenetriamine pentaacetic acid). The time course of T1 contrast development between viable and nonviable myocardium was thereby determined, with optimal postinjection imaging windows and inversion times identified. Infarct sizes were quantified using 3D-LGE and compared with triphenyltetrazolium chloride histology on day 1 after infarction ( n = 8). Baseline myocardial T1 was highly reproducible: the mean value was 952 ± 41 ms. T1 contrast peaked earlier after intravenous injection than with intraperitoneal injection; however, contrast between viable and nonviable myocardium was comparable for both routes ( P = 0.31), with adequate contrast remaining for at least 60 min postinjection. Excellent correlation was obtained between infarct sizes derived from 3D-LGE and histology ( r = 0.91, P = 0.002), and Bland-Altman analysis indicated good agreement free from systematic bias. We have validated an improved 3D MRI method to noninvasively quantify infarct size in mice with unsurpassed spatial resolution and tissue contrast. This method is particularly suited to studies requiring early quantification of initial infarct size, for example, to measure damage before intervention with stem cells.
Endothelial heparanase secretion after acute hypoinsulinemia is regulated by glucose and fatty acid
Authors: Wang Fang , Kim Min Suk , Puthanveetil Prasanth , Kewalramani Girish , Deppe Sylvia , Ghosh Sanjoy , Abrahani Ashraf , Rodrigues Brian
Published: February 2009

Following diabetes, the heart increases its lipoprotein lipase (LPL) at the coronary lumen by transferring LPL from the cardiomyocyte to the endothelial lumen. We examined how hyperglycemia controls secretion of heparanase, the enzyme that cleaves myocyte heparan sulphate proteoglycan to initiate this movement. Diazoxide (DZ) was used to decrease serum insulin and generate hyperglycemia. A modified Langendorff technique was used to separate coronary from interstitial effluent, which were assayed for heparanase and LPL. Within 30 min of DZ, interstitial heparanase increased, an effect that closely mirrored an augmentation in interstitial LPL. Endothelial cells were incubated with palmitic acid (PA) or glucose, and heparanase secretion was determined. PA increased intracellular heparanase, with no effect on secretion of this enzyme. Unlike PA, glucose dose-dependently lowered endothelial intracellular heparanase, which was strongly associated with increased heparanase activity in the incubation medium. Preincubation with cytochalasin D or nocodazole prevented the high glucose-induced depletion of intracellular heparanase. Our data suggest that following hyperglycemia, translocation of LPL from the cardiomyocyte cell surface to the apical side of endothelial cells is dependent on the ability of the fatty acid to increase endothelial intracellular heparanase followed by rapid secretion of this enzyme by glucose, which requires an intact microtubule and actin cytoskeleton.
Recent advances in genetic predisposition to clinical acute lung injury
Authors: Gao Li , Barnes Kathleen C.
Published: February 2009

It has been well established that acute lung injury (ALI), and the more severe presentation of acute respiratory distress syndrome (ARDS), constitute complex traits characterized by a multigenic and multifactorial etiology. Identification and validation of genetic variants contributing to disease susceptibility and severity has been hampered by the profound heterogeneity of the clinical phenotype and the role of environmental factors, which includes treatment, on outcome. The critical nature of ALI and ARDS, compounded by the impact of phenotypic heterogeneity, has rendered the amassing of sufficiently powered studies especially challenging. Nevertheless, progress has been made in the identification of genetic variants in select candidate genes, which has enhanced our understanding of the specific pathways involved in disease manifestation. Identification of novel candidate genes for which genetic association studies have confirmed a role in disease has been greatly aided by the powerful tool of high-throughput expression profiling. This article will review these studies to date, summarizing candidate genes associated with ALI and ARDS, acknowledging those that have been replicated in independent populations, with a special focus on the specific pathways for which candidate genes identified so far can be clustered.
Maternal exposure to endotoxin delays alveolarization during postnatal rat lung development
Authors: Cao Lei , Wang Jinxia , Tseu Irene , Luo Daochun , Post Martin
Published: February 2009

Maternal bacterial infections adversely affect lung development by crossing the placental barrier and infecting the developing fetus. The underlying mechanism negatively affecting pulmonary development remains unknown. Herein, we investigated whether a systemic maternal infection affects postnatal inflammation and alveolar development. Pregnant rats were injected with 2.5 mg/kg LPS on day 20 and 21 (term = 22 days). Postnatal (PN0–21) mRNA and protein expression of cytokines (IL-1β, IL-6, IL-10, CXCL1/2, TNFα) and genes implicated in alveologenesis [tropoelastin, lysyl oxidase (LOX), lysyl oxidase-like (LOXL)1, tenascin-C (TNC), fibulin 5, vascular endothelial growth factor (VEGF-A), VEGF receptor (VEGFR)2, VEGFR1, platelet-derived growth factor (PDGF)A, PDGFB, and PDGFRα] were quantified by real-time PCR and beadlyte technology. Lung transcript and protein levels of IL-1β, IL-6, and CXCL1/2 were significantly greater in LPS-exposed pups than those of control pups at PN0, 2, 6, 10, and 14. Bronchoalveolar lavage fluid (BALF) of LPS-exposed animals contained significantly more macrophages at PN2 and 14 than BALF of control pups. Morphometric analysis revealed that LPS-exposed animals had fewer and larger alveoli, fewer secondary septa, and decreased peripheral vessel density when compared with control pups. This morphological delay in alveolar development disappeared after PN14. Tropoelastin, LOXL1, VEGF, VEGFR2, and PDGFRα mRNA expression of LPS-exposed animals was significantly greater than those of control animals in PN2–14 lungs. TNC, LOX, fibulin 5, VEGFR1, PDGFA, and PDGFB expression was not affected by maternal LPS exposure. Together, the data demonstrate that maternal exposure to endotoxin results in a prolonged pulmonary inflammation postnatally, altered gene expression of molecules implicated in alveologenesis, and delayed morphological maturation of the lung.
Cellular mechanisms of Cl−transport in trout gill mitochondrion-rich cells
Authors: Parks Scott K. , Tresguerres Martin , Goss Greg G.
Published: February 2009

We have studied Cl−transport mechanisms in freshwater rainbow trout gill mitochondrion-rich (MR) cells using intracellular pH (pHi) imaging. Scanning electron microscopy demonstrated maintenance of cellular polarity in isolated MR cells. MR cell subtypes were identified by Na+introduction to the bath, and Cl−transport mechanisms were subsequently examined. Cl−-free exposure resulted in an alkalinization of pHiin both MR cell subtypes, which was dependent on HCO3−in the bath and inhibited by 1 mM DIDS. Recovery of pHifrom an acidified state in Na+-free conditions was also DIDS sensitive. These results are the first functional evidence for Cl−/HCO3−exchangers in fish gill MR cells. A direct switch from NaCl to Cl−-free conditions caused a pHiacidification in a subset of MR cells, which was enhanced in the absence of HCO3−. The acidification was replaced by an alkalinization when Cl−removal was performed in the presence of NPPB (500 μM) or EIPA (500 μM). Finally, we found that the Na+-induced alkalinization of pHifound in a previous study is inhibited by EIPA. This inhibitor profile's results suggest the presence of a Cl−-dependent Na+/H+exchange mechanism.
Charge-to-substrate ratio during organic cation uptake by rat OCT2 is voltage dependent and altered by exchange of glutamate 448 with glutamine
Authors: Schmitt Bernhard M. , Gorbunov Dmitry , Schlachtbauer Peter , Egenberger Brigitte , Gorboulev Valentin , Wischmeyer Erhard , Müller Thomas , Koepsell Hermann
Published: February 2009

Uptake of substrate and electric charge was measured simultaneously in voltage-clamped Xenopus laevis oocytes expressing rat organic cation transporter 2 (rOCT2). At 0 mV, saturating substrate concentrations induced uptake of more positive elementary charges than monovalent organic cations, with charge-to-substrate ratios of 1.5 for guanidinium+, 3.5 for tetraethylammonium+, and 4.0 for 1-methyl-4-phenylpyridinium+. At negative holding potentials, the charge-to-substrate ratios decreased toward unity. At 0 mV, charge-to-substrate ratios higher than unity were observed at different extracellular pH and after replacement of extracellular Na+, K+, Ca2+, Mg2+, and/or Cl−. Charge-to-substrate ratios were not influenced by intracellular succinate2− or glutarate2−. The effects of membrane potential and ion substitution strongly suggest that the surplus of transported positive charge is not generated by passive ion permeabilities. Rather, we hypothetize that small cations are taken up together with organic cation substrates whereas the outward reorientation of the empty transporter is electroneutral. Nonselective cotransport of small cations was supported by the three-dimensional structures of rOCT2 in its inward-facing and outward-facing conformations, which we determined by homology modeling based on known corresponding structures of H+-lactose permease of E. coli, and by functional analysis of OCT mutants. In our model, the innermost cavity of the outward-open binding cleft is negatively charged by Glu448 and Asp475, whereas the inward-open innermost cavity is electroneutral, containing Asp379, Asp475, Lys215, and Arg440. Substitution of Glu448 by glutamine reduced the charge-to-TEA+ ratio at 0 mV to unity. The observed charge excess associated with organic cation uptake into depolarized cells may contribute to tubular damage in renal failure.
Glomerular sieving coefficient of serum albumin in the rat: a two-photon microscopy study
Authors: Tanner George A.
Published: February 2009

Recent studies of the sieving of serum albumin in the rat kidney using a two-photon microscope suggested that the glomerular sieving coefficient (GSC) of albumin is 0.034, much higher than earlier micropuncture determinations. In the present study, we critically evaluated the use of the two-photon microscope to measure the GSC of albumin in the Munich-Wistar rat in vivo. The albumin GSC averaged 0.004 (SD 0.004), n = 34 glomeruli, when determined with a Zeiss two-photon microscope system and 0.002 (SD 0.002), n = 5, when determined with an Olympus two-photon microscope system. These values are close to the lower limit of detection of GSC, which we estimate to be ∼0.001–0.003. We identified several factors that were likely responsible for the higher albumin GSCs reported earlier using two-photon microscopy. These include animal conditions (i.e., low glomerular filtration rate) and failure to recognize the role of out-of-focus fluorescence in contaminating the fluorescence signal from the urinary space of Bowman's capsule. We observed that hypothermia plus dehydration or a low blood pressure led to an increased albumin GSC. High levels of illumination (high laser outputs) resulted in a falsely elevated albumin GSC. Use of external, instead of internal, photodetectors resulted in an exaggerated albumin GSC because of greater collection of out-of-focus fluorescence. In conclusion, the albumin concentration in the glomerular filtrate of the normal rat, determined by two-photon microscopy, is exceedingly low (5–10 mg/dl).
Diabetic kidney lesions of GIPRdntransgenic mice: podocyte hypertrophy and thickening of the GBM precede glomerular hypertrophy and glomerulosclerosis
Authors: Herbach Nadja , Schairer Irene , Blutke Andreas , Kautz Sabine , Siebert Angela , Göke Burkhard , Wolf Eckhard , Wanke Ruediger
Published: February 2009

Diabetic nephropathy is the leading cause of end-stage renal disease and the largest contributor to the total cost of diabetes care. Rodent models are excellent tools to gain more insight into the pathogenesis of diabetic nephropathy. In the present study, we characterize the age-related sequence of diabetes-associated kidney lesions in GIPRdntransgenic mice, a novel mouse model of early-onset diabetes mellitus. Clinical-chemical analyses as well as qualitative and quantitative morphological analyses of the kidneys of GIPRdntransgenic animals and nontransgenic littermate controls were performed at 3, 8, 20, and 28 wk of age. Early renal changes of transgenic mice consisted of podocyte hypertrophy, reduced numerical volume density of podocytes in glomeruli, and homogenous thickening of the glomerular basement membrane, followed by renal and glomerular hypertrophy as well as mesangial expansion and matrix accumulation. At 28 wk of age, glomerular damage was most prominent, including advanced glomerulosclerosis, tubulointerstitial lesions, and proteinuria. Real-time PCR demonstrated increased glomerular expression of Col4a1, Fn1, and Tgfb1. Immunohistochemistry revealed increased mesangial deposition of collagen type IV, fibronectin, and laminin. The present study shows that GIPRdntransgenic mice exhibit renal changes that closely resemble diabetes-associated kidney alterations in humans. Data particularly from male transgenic mice indicate that podocyte hypertrophy is directly linked to hyperglycemia, without the influence of mechanical stress. GIPRdntransgenic mice are considered an excellent new tool to study the mechanisms involved in onset and progression of diabetic nephropathy.
Reproducibility of endurance capacity and V̇o2peak in male Sprague-Dawley rats
Authors: Copp Steven W. , Davis Robert T. , Poole David C. , Musch Timothy I.
Published: February 2009

The rat model of treadmill running is an invaluable tool for the investigation of experimentally and pathologically induced alterations in exercise performance. Interpretation of such data often presumes knowledge of the within-rat reproducibility of performance measures; however, the literature is bereft of this information. We tested the hypothesis that within-rat exercise endurance capacity and peak O2 uptake (V̇o2peak) are highly reproducible across five measurements spanning ∼5 wk when assessed with treadmill performance protocols. Male Sprague-Dawley rats ( n = 13) performed five graded exercise tolerance tests to fatigue and five maximal exercise tests on a motor-driven treadmill for determination of endurance capacity and V̇o2peak, respectively. There were no differences ( P = 0.47) in average time to fatigue among any of the five exercise tolerance tests (average range 45.9–52.1 min), and the average within-rat coefficient of variation (CV) over the five runs was 0.13. There were no differences ( P > 0.05) among the average CVs from any consecutive weekly exercise tolerance tests (range of 4 CVs 0.06–0.10). As expected with the increase in body mass, relative V̇o2peak decreased (average range from 80.1 to 75.7 ml·min−1·kg−1, P < 0.05) throughout the five maximal exercise tests. However, there were no differences ( P = 0.63) in the average within-rat CVs among any consecutive V̇o2peak tests (range of 4 CVs 0.03–0.04), and the average within-rat CV for all five tests was 0.06. The present data obtained from the protocols described herein demonstrate that within-rat measurements of endurance capacity and V̇o2peak are highly reproducible. These results have significant implications for improving and refining exercise testing and experimental designs.
Glutamate Transporters Prevent Excessive Activation of NMDA Receptors and Extrasynaptic Glutamate Spillover in the Spinal Dorsal Horn
Authors: Nie Hui , Weng Han-Rong
Published: February 2009

Activation of N-methyl-d-aspartate (NMDA) receptors in the spinal dorsal horn neurons is a key process related to sensory transmission, neural plasticity, and pathogenesis of pain. In this study, we investigated how activation of NMDA receptors in spinal substantia gelatinosa neurons is regulated by glutamate re-uptake through glutamate transporters located in the astrocytic and neuronal plasma membranes. Using visualized whole cell patch recording techniques, NMDA excitatory postsynaptic currents evoked by graded peripheral inputs in spinal substantia gelatinosa neurons of spinal slices from young adult rats were analyzed before and after combined inhibition of glial and neuronal glutamate transporters by d-threo-β-benzyloxyaspartate (TBOA). Blockade of glutamate transporters increased the number and duration of NMDA receptors activated by weak and by strong primary afferent inputs as well as by exogenous glutamate. The enhancement in activation of NMDA receptors induced by TBOA was greater in neurons that have weaker synaptic input at baseline. Impaired glutamate uptake increased the open probability of NMDA channels and caused glutamate spillover outside the active synapses, leading to activation of extrasynaptic NMDA receptors and/or receptors located in neighboring synapses. Finally, blockade of glutamate transporters resulted in an increased proportion of NR2B subunit activation induced by peripheral input, and this increase was further augmented by stronger afferent input. These data indicate that glutamate transporters regulate spatiotemporal and intensity coding for sensory input and prevent excessive activation of glutamate receptors in the spinal dorsal horn. It is suggested that remedying dysfunctional glutamate transporters may be a potential new avenue to prevent the pathogenesis of pain.