bluuebunny/crossref_metadata_2025 huggingface dataset:
The Leucine-Responsive Regulatory Protein, Lrp, Modulates Microcin J25 Intrinsic Resistance inEscherichia coliby Regulating Expression of the YojI Microcin Exporter
Authors: Socías Sergio B. , Vincent Paula A. , Salomón Raúl A.
Published: December 2008

ManyEscherichia coliK-12 strains display an intrinsic resistance to the peptide antibiotic microcin J25. In this study, we present results showing that the leucine-responsive regulatory protein, Lrp, is involved in this phenotype by acting as a positive regulator of YojI, a chromosomally encoded efflux pump which expels microcin out of cells. Exogenous leucine antagonizes the effect of Lrp, leading to a diminished expression of the pump and an increased susceptibility to microcin J25.
IscR Controls Iron-Dependent Biofilm Formation inEscherichia coliby Regulating Type I Fimbria Expression
Authors: Wu Yun , Outten F. Wayne
Published: December 2008

Biofilm formation is a complex developmental process regulated by multiple environmental signals. In addition to other nutrients, the transition metal iron can also regulate biofilm formation. Iron-dependent regulation of biofilm formation varies by bacterial species, and the exact regulatory pathways that control iron-dependent biofilm formation are often unknown or only partially characterized. To address this gap in our knowledge, we examined the role of iron availability in regulating biofilm formation inEscherichia coli. The results indicate that biofilm formation is repressed under low-iron conditions inE. coli. Furthermore, a key iron regulator, IscR, controls biofilm formation in response to changes in cellular Fe-S homeostasis. IscR regulates the FimE recombinase to control expression of type I fimbriae inE. coli. We propose that iron-dependent regulation of FimE via IscR leads to decreased surface attachment and biofilm dispersal under iron-limiting conditions.
Functional Mapping of
Brucella abortus
Cyclic β-1,2-Glucan Synthase: Identification of the Protein Domain Required for Cyclization
Authors: Guidolin L. Soledad , Ciocchini Andrés E. , Iñón de Iannino Nora , Ugalde Rodolfo A.
Published: December 2008

Cyclic β-1,2-glucans (CβG) are periplasmic homopolysaccharides that have been shown to play an important role in several symbiotic and pathogenic relationships. Cyclic β-1,2-glucan synthase (Cgs), the enzyme responsible for the synthesis of CβG, is an integral membrane polyfunctional protein that catalyzes the four enzymatic activities (initiation, elongation, phosphorolysis, and cyclization) required for the synthesis of CβG. Recently, we have identified the glycosyltransferase and the β-1,2-glucooligosaccharide phosphorylase domains of
Brucella abortus
Cgs. In this study, we performed large-scale linker-scanning mutagenesis to gain further insight into the functional domains of Cgs. This analysis allowed us to construct a functional map of the enzyme and led to the identification of the minimal region required for the catalysis of initiation and elongation reactions. In addition, we identified the Cgs region (residues 991 to 1544) as being the protein domain required for cyclization and demonstrated that upon cyclization and releasing of the CβG, one or more glucose residues remain attached to the protein intermediate that serves as a primer for the next round of CβG synthesis. Finally, our results indicate that the overall control of the degree of polymerization of CβG is the result of a balance between elongation, phosphorolysis, and cyclization reactions.
Leucine-Responsive Regulatory Protein (Lrp) Acts as a Virulence Repressor inSalmonella entericaSerovar Typhimurium
Authors: Baek Chang-Ho , Wang Shifeng , Roland Kenneth L. , Curtiss Roy
Published: December 2008

Leucine-responsive regulatory protein (Lrp) is a global gene regulator that influences expression of a large number of genes including virulence-related genes inEscherichia coliandSalmonella. No systematic studies examining the regulation of virulence genes by Lrp have been reported inSalmonella. We report here that constitutive expression of Lrp [lrp(Con)] dramatically attenuatesSalmonellavirulence while anlrpdeletion (Δlrp) mutation enhances virulence. Thelrp(Con) mutant caused pleiotropic effects that include defects in invasion, cytotoxicity, and colonization, whereas the Δlrpmutant was more proficient at these activities than the wild-type strain. We present evidence that Lrp represses transcription of key virulence regulator genes—hilA,invF, andssrA—inSalmonellapathogenicity island 1 (SPI-1) and 2 (SPI-2), by binding directly to their promoter regions, PhilA, PinvF, and PssrA. In addition, Western blot analysis showed that the expression of the SPI-1 effector SipA was reduced in thelrp(Con) mutant and enhanced in the Δlrpmutant. Computational analysis revealed putative Lrp-binding consensus DNA motifs located in PhilA, PinvF, and PssrA. These results suggest that Lrp binds to the consensus motifs and modulates expression of the linked genes. The presence of leucine enhanced Lrp binding to PinvFin vitro and the addition of leucine to growth medium decreased the level ofinvFtranscription. However, leucine had no effect on expression ofhilAandssrAor on cellular levels of Lrp. In addition, Lrp appears to be an antivirulence gene, since the deletion mutant showed enhanced cell invasion, cytotoxicity, and hypervirulence in BALB/c mice.
Growth Deficiencies ofNeisseria meningitidis pfsandluxSMutants Are Not Due to Inactivation of Quorum Sensing
Authors: Heurlier Karin , Vendeville Agnès , Halliday Nigel , Green Andrew , Winzer Klaus , Tang Christoph M. , Hardie Kim R.
Published: December 2008

The activated methyl cycle (AMC) is a central metabolic pathway used to generate (and recycle) several important metabolites and enable methylation. Pfs and LuxS are considered integral components of this pathway because they convertS-adenosylhomocysteine (SAH) toS-ribosylhomocysteine (SRH) andS-ribosylhomocysteine to homocysteine (HCY), respectively. The latter reaction has a second function since it also generates the precursor of the quorum-sensing molecule autoinducer 2 (AI-2). By demonstrating that there was a complete lack of AI-2 production inpfsmutants of the causative agent of meningitis and septicemia,Neisseria meningitidis, we showed that the Pfs reaction is the sole intracellular source of the AI-2 signal. Analysis oflacZreporters and real-time PCR experiments indicated thatpfsis expressed constitutively from a promoter immediately upstream, and careful study of thepfsmutants revealed a growth defect that could not be attributed to a lack of AI-2. Metabolite profiling of the wild type and of apfsmutant under various growth conditions revealed changes in the concentrations of several AMC metabolites, particularly SRH and SAH and under some conditions also HCY. Similar studies established that anN. meningitidis luxSmutant also has metabolite pool changes and growth defects in line with the function of LuxS downstream of Pfs in the AMC. Thus, the observed growth defect ofN. meningitidis pfsandluxSmutants is not due to quorum sensing but is probably due to metabolic imbalance and, in the case ofpfsinactivation, is most likely due to toxic accumulation of SAH.
The
Escherichia coli
K5 Capsule Is Not Synthesized in a Protected Compartment within the Cytoplasm
Authors: Hudson Thomas , Goldrick Marie , Roberts Ian S.
Published: December 2008

The intracellular expression of the K5 lyase enzyme, which degrades the K5 polysaccharide, decreased cell surface expression of the
Escherichia coli
K5 capsule. This indicates that biosynthesis of K5 polysaccharide in the cytoplasm is accessible to the action of K5 lyase and is not synthesized within a protected cytoplasmic compartment.
The R1162 Mob Proteins Can Promote Conjugative Transfer from Cryptic Origins in the Bacterial Chromosome
Authors: Meyer Richard
Published: December 2008

The mobilization proteins of the broad-host-range plasmid R1162 can initiate conjugative transfer of a plasmid from a 19-bp locus that is partially degenerate in sequence. Such loci are likely to appear by chance in the bacterial chromosome and could act as cryptic sites for transfer of chromosomal DNA when R1162 is present. The R1162-dependent transfer of chromosomal DNA, initiated from one such potential site in
Pectobacterium atrosepticum
, is shown here. A second active site was identified in
Escherichia coli
, where it is also shown that large amounts of DNA are transferred. This transfer probably reflects the combined activity of the multiple cryptic origins in the chromosome. Transfer of chromosomal DNA due to the presence of a plasmid in the cytoplasm describes a previously unrecognized potential for the exchange of bacterial DNA.
The
Lactobacillus plantarum ftsH
Gene Is a Novel Member of the CtsR Stress Response Regulon
Authors: Fiocco Daniela , Collins Michael , Muscariello Lidia , Hols Pascal , Kleerebezem Michiel , Msadek Tarek , Spano Giuseppe
Published: December 2008

FtsH proteins have dual chaperone-protease activities and are involved in protein quality control under stress conditions. Although the functional role of FtsH proteins has been clearly established, the regulatory mechanisms controlling
ftsH
expression in gram-positive bacteria remain largely unknown. Here we show that
ftsH
of
Lactobacillus plantarum
WCFS1 is transiently induced at the transcriptional level upon a temperature upshift. In addition, disruption of
ftsH
negatively affected the growth of
L. plantarum
at high temperatures. Sequence analysis and mapping of the
ftsH
transcriptional start site revealed a potential operator sequence for the CtsR repressor, partially overlapping the −35 sequence of the
ftsH
promoter. In order to verify whether CtsR is able to recognize and bind the
ftsH
promoter, CtsR proteins of
Bacillus subtilis
and
L. plantarum
were overproduced, purified, and used in DNA binding assays. CtsR from both species bound specifically to the
ftsH
promoter, generating a single protein-DNA complex, suggesting that CtsR may control the expression of
L. plantarum ftsH
. In order to confirm this hypothesis, a Δ
ctsR
mutant strain of
L. plantarum
was generated. Expression of
ftsH
in the Δ
ctsR
mutant strain was strongly upregulated, indicating that
ftsH
of
L. plantarum
is negatively controlled by CtsR. This is the first example of an
ftsH
gene controlled by the CtsR repressor, and the first of the low-G+C gram-positive bacteria where the regulatory mechanism has been identified.
Functional Differences of Two Distinct Catalases in
Mesorhizobium loti
MAFF303099 under Free-Living and Symbiotic Conditions
Authors: Hanyu Masaki , Fujimoto Hanae , Tejima Kouhei , Saeki Kazuhiko
Published: December 2008

Protection against reactive oxygen species (ROS) is important for legume-nodulating rhizobia during the establishment and maintenance of symbiosis, as well as under free-living conditions, because legume hosts might assail incoming microbes with ROS and because nitrogenase is extremely sensitive to ROS. We generated mutants of two potential catalase genes in
Mesorhizobium loti
MAFF303099 to investigate their physiological significance. Biochemical results indicated that genes with the locus tags mlr2101 and mlr6940 encoded a monofunctional catalase and a bifunctional catalase-peroxidase, respectively, that were named
katE
and
katG
. Under free-living conditions, the
katG
mutant demonstrated an extended generation time and elevated sensitivity to exogenous H
2
O
2
, whereas the
katE
mutant exhibited no generation time extension and only a slight increase in sensitivity to exogenous H
2
O
2
. However, the
katE
mutant showed a marked decrease in its survival rate during the stationary phase. With regard to symbiotic capacities with
Lotus japonicus
, the
katG
mutant was indistinguishable from the wild type; nevertheless, the mutants with disrupted
katE
formed nodules with decreased nitrogen fixation capacities (about 50 to 60%) compared to those formed by the wild type. These mutant phenotypes agreed with the expression profiles showing that transcription of
katG
, but not
katE
, was high during the exponential growth phase and that transcription levels of
katE
versus
sigA
were elevated during stationary phase and were approximately fourfold higher in bacteroids than mid-exponential-phase cells. Our results revealed functional separation of the two catalases, as well as the importance of KatE under conditions of strong growth limitation.
Essential Role for the BacA Protein in the Uptake of a Truncated Eukaryotic Peptide in
Sinorhizobium meliloti
Authors: Marlow Victoria L. , Haag Andreas F. , Kobayashi Hajime , Fletcher Vivien , Scocchi Marco , Walker Graham C. , Ferguson Gail P.
Published: December 2008

The inner membrane BacA protein is essential for the establishment of chronic intracellular infections by
Sinorhizobium meliloti
and
Brucella abortus
within plant and mammalian hosts, respectively. In their free-living state,
S. meliloti
and
B. abortus
mutants lacking BacA have reductions in their outer membrane lipid A very-long-chain fatty acid (VLCFA) contents and exhibit low-level resistance to the glycopeptide bleomycin in comparison to their respective parent strains. In this paper we investigate the hypothesis that BacA is involved in peptide uptake in
S. meliloti
. We determined that an
S. meliloti
Δ
bacA
mutant is completely resistant to a truncated form of the eukaryotic peptide Bac7, Bac7(1-16), and this phenotype appears to be independent of its lipid A alteration. Subsequently, we discovered that BacA and/or
Escherichia coli
SbmA is essential for fluorescently labeled Bac7(1-16) uptake in
S. meliloti
. Given that there are hundreds of root nodule-specific peptides within the legume host, our data suggest that BacA-mediated peptide uptake could play a central role in the chronic infection process of
S. meliloti
. However, since we determined that two symbiotically defective
S. meliloti bacA
site-directed mutants (with the Q193G and R389G mutations, respectively) with known reductions in their lipid A VLCFA contents are still capable of peptide uptake, these findings suggest that BacA-dependent peptide uptake cannot fully account for the essential role of BacA in the legume symbiosis. Further, they provide evidence that the BacA function that leads to the
S. meliloti
lipid A VLCFA modification plays a key role in the chronic infection of legumes.
Plasma levels of PBEF/Nampt/visfatin are decreased in patients with liver cirrhosis
Authors: de Boer Jan Freark , Bahr Matthias J. , Böker Klaus H. W. , Manns Michael P. , Tietge Uwe J. F.
Published: December 2008

Liver cirrhosis is a catabolic disease associated with a high incidence of insulin resistance and diabetes mellitus. Pre-B cell colony-enhancing factor/ nicotinamide phosphoribosyltransferase/visfatin has been characterized as a novel adipokine with a potential role in glucose metabolism and nicotinamide dinucleotide (NAD) generation. We studied plasma levels and metabolic relevance of visfatin in 19 patients with cirrhosis and 19 body mass index-, age-, and sex-matched controls. In addition, hepatic mRNA expression was assessed by qPCR in livers of seven patients with cirrhosis and four controls. Circulating visfatin was 78% lower in cirrhotics ( P < 0.001) and decreased with worsening of the clinical stage of liver disease. Hepatic visfatin secretion decreased with clinical stage ( P < 0.05) and reduced liver function ( P = 0.01). Consistent with these data, hepatic visfatin mRNA expression was significantly lower in cirrhotic livers ( P < 0.05). Circulating visfatin in cirrhosis was correlated with body cell mass ( r = 0.72, P < 0.01) as well as with body fat mass ( r = 0.53, P < 0.05) but not with plasma glucose, insulin, the degree of insulin resistance, or whole body glucose oxidation rates. Higher visfatin levels were associated with higher hepatic glucose production ( r = 0.53, P < 0.05) and also with a higher arterial ketone body ratio (KBR) ( r = 0.48, P < 0.05), an indicator of increased hepatic NAD generation. In conclusion, circulating visfatin levels are significantly decreased in liver cirrhosis, presumably attributable to decreased hepatic expression and production. Plasma visfatin in cirrhosis is not associated with insulin resistance but correlates with hepatic glucose production and the arterial KBR, indicating a potential link between the NAD-generating properties of visfatin and metabolism.
Posttranslational regulation of Abcc2 expression by SUMOylation system
Authors: Minami Satoko , Ito Kousei , Honma Masashi , Ikebuchi Yuki , Anzai Naohiko , Kanai Yoshikatsu , Nishida Tamotsu , Tsukita Sachiko , Sekine Shuichi , Horie Toshiharu , Suzuki Hiroshi
Published: December 2008

The ATP-binding cassette transporter family C 2 (Abcc2) is a member of efflux transporters involved in the biliary excretion of organic anions from hepatocytes. Posttranslational regulation of Abcc2 has been implicated, although the molecular mechanism is not fully understood. In the present study, we performed yeast two-hybrid screening to identify novel protein(s) that particularly interacts with the linker region of Abcc2 located between the NH2-terminal nucleotide binding domain and the last membrane-spanning domain. The screening resulted in the identification of a series of small ubiquitin-like modifier (SUMO)-related enzymes and their substrates. In yeast experiments, all of these interactions were abolished by substituting the putative SUMO consensus site in the linker region (IKKE) in Abcc2 to IRKE. In vitro SUMOylation experiments confirmed that the Abcc2 linker was a substrate of Ubc9-mediated SUMOylation. It was also found that the IKKE sequence is the target of SUMOylation, since a mutant with IKKE is substituted by IRKE was not SUMOylated. Furthermore, we demonstrated for the first time that Abcc2, endogenously expressed in rat hepatoma-derived McARH7777 cells, is SUMOylated. Suppression of endogenous Ubc9 by small interfering RNA resulted in a selective 30% reduction in Abcc2 protein expression in the postnuclear supernatant, whereas subcellular localization of Abcc2 confirmed by semiquantitative immunofluorescence analysis was minimally affected. This is the first demonstration showing the regulation of ABC transporter expression by SUMOylation.
Liquiritigenin, a flavonoid aglycone from licorice, has a choleretic effect and the ability to induce hepatic transporters and phase-II enzymes
Authors: Kim Young Woo , Kang Hee Eun , Lee Myung Gull , Hwang Se Jin , Kim Sang Chan , Lee Chang Ho , Kim Sang Geon
Published: December 2008

Liquiritigenin (LQ), an active component of licorice, has an inhibitory effect on LPS-induced inhibitory nitric oxide synthase expression. This study investigated the effects of LQ on choleresis, the expression of hepatic transporters and phase-II enzymes, and fulminant hepatitis. The choleretic effect and the pharmacokinetics of LQ and its glucuronides were monitored in rats. After intravenous administration of LQ, the total area under the plasma concentration-time curve of glucuronyl metabolites was greater than that of LQ in plasma, which accompanied elevations in bile flow rate and biliary excretion of bile acid, glutathione, and bilirubin. The expressions of hepatocellular transporters and phase-II enzymes were assessed by immunoblots, real-time PCR, and immunohistochemistry. In the livers of rats treated with LQ, the protein and mRNA levels of multidrug resistance protein 2 and bile salt export pump were increased in the liver, which was verified by their increased localizations in canalicular membrane. In addition, LQ treatment enhanced the expression levels of major hepatic phase-II enzymes. Consistent with these results, LQ treatments attenuated galactosamine/LPS-induced hepatitis in rats, as supported by decreases in the plasma alanine aminotransferase, liver necrosis, and plasma TNF-α. These results demonstrate that LQ has a choleretic effect and the ability to induce transporters and phase-II enzymes in the liver, which may be associated with a hepatoprotective effect against galactosamine/LPS. Our findings may provide insight into understanding the action of LQ and its therapeutic use for liver disease.
Brief oral stimulation, but especially oral fat exposure, elevates serum triglycerides in humans
Authors: Mattes Richard D.
Published: December 2008

Oral exposure to dietary fat results in an early initial spike, followed by a prolonged elevation, of serum triglycerides in humans. The physiological and pathophysiological implications remain unknown. This study sought to determine the incidence of the effect, the required fat exposure duration, and its reliability. Thirty-four healthy adults participated in four to six response-driven trials held at least a week apart. They reported to the laboratory after an overnight fast, a catheter was placed in an antecubital vein, and a blood sample was obtained. Participants then ingested 50 g of safflower oil in capsules with 500 ml of water within 15 min to mimic a high fat meal but without oral fat exposure. Blood was collected 0, 10, 20, 30, 40, 50, 60, 120, 240, 360, and 480 min after capsule ingestion with different forms (full fat, nonfat, none) and durations of oral fat exposures (10 s, 5 min, 20 min, and/or 2 h). A triglyceride response (increase of triglyceride >10 mg/dl within 30 min) was observed in 88.2%, 70.5%, and 50% of participants with full-fat, nonfat, and no oral exposure, respectively. Test-retest reliability was 75% with full-fat exposure but only 45.4% with nonfat exposure. Full-fat and nonfat exposures led to comparable significant elevations of triglyceride over no oral stimulation with 10-s exposures, but full fat led to a greater rise than nonfat with 20 min of exposure. These data indicate that nutritionally relevant oral fat exposures reliably elevate serum triglyceride concentrations in most people.
Novel insights of the gastric gland organization revealed by chief cell specific expression of moesin
Authors: Zhu Lixin , Hatakeyama Jason , Zhang Bing , Makdisi Joy , Ender Cody , Forte John G.
Published: December 2008

ERM (ezrin, radixin, and moesin) proteins play critical roles in epithelial and endothelial cell polarity, among other functions. In gastric glands, ezrin is mainly expressed in acid-secreting parietal cells, but not in mucous neck cells or zymogenic chief cells. In looking for other ERM proteins, moesin was found lining the lumen of much of the gastric gland, but it was not expressed in parietal cells. No significant radixin expression was detected in the gastric glands. Moesin showed an increased gradient of expression from the neck to the base of the glands. In addition, the staining pattern of moesin revealed a branched morphology for the gastric lumen. This pattern of short branches extending from the glandular lumen was confirmed by using antibody against zonula occludens-1 (ZO-1) to stain tight junctions. With a mucous neck cell probe (lectin GSII, from Griffonia simplicifolia) and a chief cell marker (pepsinogen C), immunohistochemistry revealed that the mucous neck cells at the top of the glands do not express moesin, but, progressing toward the base, mucous cells showing decreased GSII staining had low or moderate level of moesin expression. The level of moesin expression continued to increase toward the base of the glands and reached a plateau in the base where chief cells and parietal cells abound. The level of pepsinogen expression also increased toward the base. Pepsinogen C was located on cytoplasmic granules and/or more generally distributed in chief cells, whereas moesin was exclusively expressed on the apical membrane. This is a clear demonstration of distinctive cellular expression of two ERM family members in the same tissue. The results provide the first evidence that moesin is involved in the cell biology of chief cells. Novel insights on gastric gland morphology revealed by the moesin and ZO-1 staining provide the basis for a model of cell maturation and migration within the gland.
Evidence for unloading arterial baroreceptors during low levels of lower body negative pressure in humans
Authors: Fu Qi , Shibata Shigeki , Hastings Jeffrey L. , Prasad Anand , Palmer M. Dean , Levine Benjamin D.
Published: December 2008

Low levels (i.e., ≤20 mmHg) of lower body negative pressure (LBNP) have been utilized to unload “selectively” cardiopulmonary baroreceptors in humans, since steady-state mean arterial pressure and heart rate (HR) have been found unchanged at such levels. However, transient reductions in blood pressure (BP), followed by reflex compensation, may occur without detection, which could unload arterial baroreceptors. The purposes of this study were to test the hypothesis that the arterial baroreflex is engaged even during low levels of LBNP and to determine the time course of changes in hemodynamics. Fourteen healthy individuals (age range 20–54 yr) were studied. BP (Portapres and Suntech), HR (ECG), pulmonary capillary wedge pressure (PCWP) or pulmonary artery diastolic pressure (PDP) and right atrial pressure (RAP) (Swan-Ganz catheter) and hemodynamics (Modelflow) were recorded continuously at baseline and −15- and −30-mmHg LBNP for 6 min each. Application of −15-mmHg LBNP resulted in rapid and sustained falls in RAP and PCWP or PDP, progressive decreases in cardiac output and stroke volume, followed subsequently by transient reductions in both systolic and diastolic BP, which were then restored through the arterial baroreflex feedback mechanism after ∼15 heartbeats. Additional studies were performed in five subjects using even lower levels of LBNP, and this transient reduction in BP was observed in three at −5- and in all at −10-mmHg LBNP. The delay for left ventricular stroke volume to fall at −15-mmHg LBNP was about 10 cardiac cycles. An increase in systemic vascular resistance was detectable after 20 heartbeats during −15-mmHg LBNP. Steady-state BP and HR remained unchanged during mild LBNP. However, BP decreased, while HR increased, at −30-mmHg LBNP. These results suggest that arterial baroreceptors are consistently unloaded during low levels (i.e., −10 and −15 mmHg) of LBNP in humans. Thus “selective” unloading of cardiopulmonary baroreceptors cannot be presumed to occur during these levels of mild LBNP.
Hyperoxia-induced NF-κB activation occurs via a maturationally sensitive atypical pathway
Authors: Wright Clyde J. , Zhuang Tiangang , La Ping , Yang Guang , Dennery Phyllis A.
Published: December 2008

NF-κB activation is exaggerated in neonatal organisms after oxidant and inflammatory insults, but the reason for this and the downstream effects are unclear. We hypothesized that specific phosphorylation patterns of IκBα could account for differences in NF-κB activation in hyperoxia-exposed fetal and adult lung fibroblasts. After exposure to hyperoxia (>95% O2), nuclear NF-κB binding increased in fetal, but not adult, lung fibroblasts. Unique to fetal cells, phosphorylation of IκBα on tyrosine 42, rather than serine 32/36 as seen in TNF-α-exposed cells, preceded NF-κB nuclear translocation. In fetal cells stably transfected with an NF-κB-driven luciferase reporter, hyperoxia significantly suppressed reporter activity, in contrast to increased reporter activity after TNF-α incubation. Targeted gene profiling analysis showed that hyperoxia resulted in decreased expression of multiple genes, including proapoptotic factors. Transfection with a dominant-negative IκBα (Y42F), which cannot be phosphorylated on tyrosine 42, resulted in upregulation of multiple proapoptotic genes. In support of this finding, caspase-3 activity and DNA laddering were specifically increased in fetal lung fibroblasts expressing Y42F after exposure to hyperoxia. These data demonstrate a unique pathway of NF-κB activation in fetal lung fibroblasts after exposure to hyperoxia, whereby these cells are protected against apoptosis. Activation of this pathway in fetal cells may prevent the normal pattern of fibroblast apoptosis necessary for normal lung development, resulting in aberrant lung morphology in vivo.
Effects of artificial gravity during bed rest on bone metabolism in humans
Authors: Smith S. M. , Zwart S. R. , Heer M. A. , Baecker N. , Evans H. J. , Feiveson A. H. , Shackelford L. C. , LeBlanc A. D.
Published: December 2008

We report results from a study designed to explore the utility of artificial gravity (AG) as a countermeasure to bone loss induced by microgravity simulation. After baseline testing, 15 male subjects underwent 21 days of 6° head-down bed rest to simulate the deconditioning associated with spaceflight. Eight of the subjects underwent 1 h of centrifugation (AG; 1 Gzat the heart, 2.5 Gzat the feet) each day for 21 days, whereas seven of the subjects served as untreated controls (Con). Blood and urine were collected before, during, and after bed rest for bone marker determinations. Bone mineral density (BMD) and bone mineral content (BMC) were determined by dual-energy X-ray absorptiometry and peripheral quantitative computerized tomography before and after bed rest. Urinary excretion of bone resorption markers increased during bed rest, but the AG and Con groups did not differ significantly. The same was true for serum C-telopeptide. During bed rest, bone alkaline phosphatase (ALP) and total ALP tended to be lower in the AG group ( P = 0.08, P = 0.09). Neither BMC nor BMD changed significantly from the pre-bed rest period in AG or Con groups, and the two groups were not significantly different. However, when AG and Con data were combined, there was a significant ( P < 0.05) effect of time for whole body total BMC and total hip and trochanter BMD. These data failed to demonstrate efficacy of this AG prescription to prevent the changes in bone metabolism observed during 3 wk of bed rest.
Regulatory mechanisms of skeletal muscle protein turnover during exercise
Authors: Rose Adam J. , Richter Erik A.
Published: December 2008

Skeletal muscle protein turnover is a relatively slow metabolic process that is altered by various physiological stimuli such as feeding, fasting, and exercise. During exercise, catabolism of amino acids contributes very little to ATP turnover in working muscle. With regard to protein turnover, there are now consistent data from tracer studies in rodents and humans showing that global protein synthesis is blunted in working skeletal muscle. Whether there is altered skeletal muscle protein breakdown during exercise remains unclear. The blunting of protein synthesis is believed to be mediated by suppressed mRNA translation initiation and elongation steps involving, but not limited to, changes in eukaryotic initiation factor 4E binding protein 1 and eukaryotic elongation factor 2 phosphorylation (eEF2), respectively. Evidence is provided that upstream signaling to translation factors is mediated by signaling downstream of changes in intracellular Ca2+and energy turnover. In particular, a signaling cascade involving Ca2+/calmodulin-eEF2 kinase-eEF2 is implicated. The possible functional significance of altered protein turnover in working skeletal muscle during exercise is discussed. Further work with available and new techniques will undoubtedly reveal the functional significance and signaling mechanisms behind changes in skeletal muscle protein turnover during exercise.
Can O2Dysregulation Induce Premature Aging?
Authors: Douglas Robert M. , Haddad Gabriel G.
Published: December 2008

Chronic intermittent or episodic hypoxia, as occurs during a number of disease states, can have devastating effects, and prolonged exposure to this hypoxia can result in cell injury or cell death. Indeed, intermittent hypoxia activates a number of signaling pathways that are involved in oxygen sensing, oxidative stress, metabolism, catecholamine biosynthesis, and immune responsiveness. The cumulative effect of these processes over time can undermine cell integrity and lead to a decline in function. Furthermore, the ability to respond adequately to various stressors is hampered, and this is traditionally defined as premature aging or senescence. This review highlights recent advances in our understanding of the cellular and molecular mechanisms that are involved in the response to intermittent hypoxia and the potential interplay among various pathways that may accelerate the aging process.
Engineered GPCRs as Tools to Modulate Signal Transduction
Authors: Pei Ying , Rogan Sarah C. , Yan Feng , Roth Bryan L.
Published: December 2008

Different families of G-protein-coupled receptors (GPCRs) have been engineered to provide exclusive control over the activation of these receptors and thus to understand better the consequences of their signaling in vitro and in vivo. These engineered receptors, named RASSLs (receptors activated solely by synthetic ligands) and DREADDs (designer receptors exclusively activated by designer drugs), are insensitive to their endogenous ligands but can be activated by synthetic drug-like compounds. Currently, the existing RASSLs and DREADDs cover the Gi, Gq, and Gssignaling pathways. These modified GPCRs can be utilized as ideal tools to study GPCR functions selectively in specific cellular populations.
Interleukin-6 Overexpression Induces Pulmonary Hypertension
Authors: Steiner M. Kathryn , Syrkina Olga L. , Kolliputi Narasaish , Mark Eugene J. , Hales Charles A. , Waxman Aaron B.
Published: December 2008

Inflammatory cytokine interleukin (IL)-6 is elevated in the serum and lungs of patients with pulmonary artery hypertension (PAH). Several animal models of PAH cite the potential role of inflammatory mediators. We investigated role of IL-6 in the pathogenesis of pulmonary vascular disease. Indices of pulmonary vascular remodeling were measured in lung-specific IL-6–overexpressing transgenic mice (Tg
+
) and compared to wild-type (Tg
−
) controls in both normoxic and chronic hypoxic conditions. The Tg
+
mice exhibited elevated right ventricular systolic pressures and right ventricular hypertrophy with corresponding pulmonary vasculopathic changes, all of which were exacerbated by chronic hypoxia. IL-6 overexpression increased muscularization of the proximal arterial tree, and hypoxia enhanced this effect. It also reproduced the muscularization and proliferative arteriopathy seen in the distal arteriolar vessels of PAH patients. The latter was characterized by the formation of occlusive neointimal angioproliferative lesions that worsened with hypoxia and were composed of endothelial cells and T-lymphocytes. IL-6–induced arteriopathic changes were accompanied by activation of proangiogenic factor, vascular endothelial growth factor, the proproliferative kinase extracellular signal-regulated kinase, proproliferative transcription factors c-MYC and MAX, and the antiapoptotic proteins survivin and Bcl-2 and downregulation of the growth inhibitor transforming growth factor-β and proapoptotic kinases JNK and p38. These findings suggest that IL-6 promotes the development and progression of pulmonary vascular remodeling and PAH through proproliferative antiapoptotic mechanisms.
The Safety of Fiberoptic Endoscopic Evaluation of Swallowing in Acute Stroke Patients
Authors: Warnecke Tobias , Teismann Inga , Oelenberg Stefan , Hamacher Christina , Ringelstein E. Bernd , Schäbitz Wolf R. , Dziewas Rainer
Published: December 2008

Background and Purpose—

Fiberoptic endoscopic evaluation of swallowing (FEES) is an excellent method for the accurate examination of swallowing function in the acute phase of stroke. The present study investigates the safety of FEES related to patients characteristics in a setting of acute stroke care.

Methods—

A prospective study of FEES-associated complications was carried out in 300 acute stroke patients over a 1-year period. A neurologist and a speech-language pathologist of the stroke unit team performed FEES within a mean time interval of 1.9±0.8 days after stroke onset. A closely meshed monitoring of cardiovascular parameters was done during each examination. A discomfort rating was obtained from the patients.

Results—

In none of the 300 subjects any airway comprise, decrease in the level of consciousness, symptomatic bradycardia/tachycardia, laryngospasm, or epistaxis requiring special treatment was observed. The incidence of self-limiting nosebleeds was 6% and did not significantly differ in relation to major stroke types (ischemic versus hemorrhagic), acute treatment strategy (thrombolysis versus no thrombolysis), or secondary prevention regime (anticoagulant therapy versus antiplatelet drugs). Whereas no alterations in diastolic blood pressure were noted, statistically significant changes in systolic blood pressure, heart rate, and oxygen saturation occurred. However, these alterations did not cause any severe adverse event and were clinically judged as being mild. The assessment of comfort revealed an excellent tolerance of FEES in >80% of patients.

Conclusion—

This study demonstrates that FEES is a well-tolerated and safe method to assess swallowing function when performed by a speech-language pathologist and a neurologist in a stroke unit setting.
Methylphenidate-induced impulsivity: pharmacological antagonism by β-adrenoreceptor blockade
Authors: Milstein JA , Dalley JW , Robbins TW
Published: December 2008

Noradrenaline—dopamine interactions mediate increases in locomotor activity, development of sensitisation and subjective effects of psychostimulant drugs. However, the modulatory effects of noradrenaline on psychostimulant-induced impulsivity are less clear. This article examined the relative roles of noradrenaline and dopamine in the modulation of methylphenidate-induced impulsive responding in rats performing the 5-choice serial reaction time task. Experiment 1 examined the systemic antagonism of methylphenidate-induced impulsivity with either propranolol, a β-adrenoreceptor blocker, or prazosin, an α1-adrenoreceptor antagonist, which antagonises the locomotor activating effects of amphetamine. Propranolol completely abolished methylphenidate-induced impulsivity. This effect was centrally rather than peripherally mediated, as nadolol, a peripheral β-blocker failed to affect methylphenidate-induced premature responding. Prazosin partially attenuated the methylphenidate-mediated increase in premature responding. A second experiment examined the effects of selective anti-DβH saporin-induced cortical noradrenaline depletion on methylphenidate-induced impulsivity. Contrary to the effects of β-adrenoreceptor blockade, cortical noradrenergic depletion did not alter methylphenidate-induced impulsivity. Other experiments examined the comparative effects of selective dopamine and serotonin receptor blockade. D4 dopamine receptor blockade with systemically administered L-745,870 also attenuated methylphenidate-induced impulsivity. The other antagonists had no effect on methylphenidate-induced impulsivity. Taken together, these studies provide evidence for a modulatory role of β-adrenoreceptors on methylphenidate-induced impulsive responding.
Acute lung inflammation and ventilator-induced lung injury caused by ATP via the P2Y receptors: an experimental study
Authors: Matsuyama Hiroki , Amaya Fumimasa , Hashimoto Soshi , Ueno Hiroshi , Beppu Satoru , Mizuta Mitsuhiko , Shime Nobuaki , Ishizaka Akitoshi , Hashimoto Satoru
Published: December 2008

Background

Extracellular adenosine 5'-triphosphate (ATP) is an endogenous signaling molecule involved in multiple biological phenomena, including inflammation. The effects of extracellular ATP in the lung have not been fully clarified. This study examined 1) the biological roles of extracellular ATP in the pathogenesis of lung inflammation and 2) the possibility of involvement of extracellular ATP in mechanical ventilation-induced lung injury.

Methods

The effects of intratracheal ATP on lung permeability, edema or lung inflammation were assessed by measurements of the lung wet-to-dry weight ratio and lung permeability index, immunohistochemistry and expression of key cytokines by real-time polymerase chain reaction. The ATP concentration in broncho-alveolar lavage (BAL) fluid from mice mechanically ventilated was measured by luciferin-luciferase assay. The suppressive effects of a P2 receptor antagonist on ventilator-induced lung inflammation were also examined.

Results

ATP induced inflammatory reactions in the lung mainly via the ATP-P2Y receptor system. These reactions were alleviated by the co-administration of a specific P2 receptor antagonist. Mechanical ventilation with a large tidal volume caused lung inflammation and increased the ATP concentration in BAL fluid. P2 receptor antagonism partially mitigated the inflammatory effects of large tidal volume ventilation.

Conclusion

Our observations suggest that the ATP-P2Y receptor system is partially involved in the pathogenesis of ventilator-induced lung injury.
Thyrotropin-Independent Induction of Thyroid Endoderm from Embryonic Stem Cells by Activin A
Authors: Ma Risheng , Latif Rauf , Davies Terry F.
Published: December 2008

To model the differentiation of thyroid epithelial cells, we examined embryoid bodies derived from undifferentiated murine embryonic stem cells treated with activin A to induce endoderm differentiation, the germ layer from which thyroid cells occur. The resulting endodermal cells were then further exposed to TSH and/or IGF-I for up to 21 d. Oct-4 and REX1 expression, required to sustain stem cell self-renewal and pluripotency, were appropriately down-regulated, whereas GATA-4, and α-fetoprotein, both endodermal-specific markers, increased as the embryonic stem cells were exposed to activin A. By d 5 culture, TSH receptor (TSHR) and sodium iodide symporter (NIS) gene and protein expression were markedly induced. Cells isolated by the fluorescence-activated cell sorter simultaneously expressed not only TSHR and NIS proteins but also PAX8 mRNA, an expression pattern unique to thyroid cells and expected in committed thyroid progenitor cells. Such expression continued until d 21 with no influence seen by the addition of TSH or IGF-I. The sequence of gene expression changes observed in these experiments demonstrated the emergence of definitive thyroid endoderm. The activin A induction of thyroid-specific markers, NIS and TSHR, occurred in the absence of TSH stimulation, and, therefore, the emergence of thyroid endoderm in vitro paralleled the emergence of thyroid cells in TSHR-knockout mice. Activin A is clearly a major regulator of thyroid endoderm.
Minireview: Liver X Receptor β: Emerging Roles in Physiology and Diseases
Authors: Gabbi Chiara , Warner Margaret , Gustafsson Jan-Åke
Published: December 2008

Liver X receptors, LXRα and LXRβ, are nuclear receptors belonging to the large family of transcription factors. After activation by oxysterols, LXRs play a central role in the control of lipid and carbohydrate metabolism as well as inflammation. The role of LXRα has been extensively studied, particularly in the liver and macrophages. In the liver it prevents cholesterol accumulation by increasing bile acid synthesis and secretion into the bile through ATP-binding cassette G5/G8 transporters, whereas in macrophages it increases cholesterol reverse transport. The function of LXRβ is still under investigation with most of the current knowledge coming from the study of phenotypes of LXRβ−/− mice. With these mice new emerging roles for LXRβ have been demonstrated in the pathogenesis of diseases such as amyotrophic lateral sclerosis and chronic pancreatitis. The present review will focus on the abnormalities described so far in LXRβ−/− mice and the insight gained into the possible roles of LXRβ in human diseases.
Pharmacokinetic and pharmacodynamic profile of a new sustained-release GH formulation, LB03002, in children with GH deficiency
Authors: Peter Ferenc , Savoy Conrad , Ji Hyi-Jeong , Juhasz Mihaly , Bidlingmaier Martin , Saenger Paul
Published: December 2008

Objective

LB03002 is a novel, sustained-release recombinant human GH, developed for once-a-week s.c. injection. To evaluate the suitability for long-term GH replacement therapy in children with GH deficiency (GHD), the present study assessed the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of LB03002 at three doses.

Study design and patients

The randomised, comparator-controlled, assessor-blinded, phase II study assessed 37 (24 boys, 13 girls) pre-pubertal, GH-naïve children with GHD, in 11 European centres, for PK and PD analyses. GH, IGF1 and IGFBP3 concentrations were measured following the last daily GH dose and the first and 13th once-a-week administration of LB03002 at doses of 0.2, 0.5 or 0.7 mg/kg.

Results

GH Cmax values after the three doses of LB03002 were increased up to fourfold, with a clear dose proportionality. For each LB03002 dose, GH area under the concentration versus time curve did not increase from the first to 13th (month 3) administration, indicating no accumulation of circulating GH. IGF1 Cmax showed a progressive increase during LB03002 administration. Conversely, IGFBP3 showed a rapid increase in Cmax. IGF1 SDS were fully normalised after 3 months of treatment, whereas IGFBP3 SDS were already in the normal range for all the three LB03002 dosages after 1 week.

Conclusions

At the doses used, LB03002 has a suitable profile for long-term treatment to promote growth in children with GHD. The quantitative changes in IGF1 and IGFBP3 indicate adequate stimulation of the IGF system by LB03002 and the pattern of increase is comparable with that seen in GHD children in a standard IGF1 generation test using daily GH.
Molecular Assemblies on Insulating Ultrathin Films Analyzed by NC‐AFM and KPFM
Authors: Glatzel Thilo , Zimmerli Lars , Meyer Ernst
Published: December 2008

Recently porphyrin‐based molecules have proven to be promising candidates to form self‐assembled structures on insulating surfaces. Their close similarity to hemoglobin and chlorophyll‐based molecules makes them promising candidates for functional building parts in molecular electronics. To analyze their opto‐electronic activity, new measurement methods have to be applied. We investigated the formation of such molecular assemblies on ultrathin insulating films by means of a room temperature non‐contact atomic force microscope (NC‐AFM) in combination with a Kelvin probe force microscope (KPFM). As a substrate we used a Cu(111) surface, where either ultrathin KBr or NaCl films are deposited. On those surfaces the evaporated cyano‐porphyrins are forming one‐ and two‐dimensional structures that are comparable to the ones observed on bulk alkali halides. The simultaneously measured surface potential clearly shows a material contrast between the molecules and the insulating films. Finally, we succeeded in obtaining variations of the surface potential induced by the molecular assemblies at the molecular level.
Modeling of NC‐AFM Imaging of Alkanethiols on the Au (111) Surface
Authors: Sushko Maria L. , Kalampokidis Alexandros , Gal Andrei Y. , Shluger Alexander L.
Published: December 2008

We investigate the mechanisms of contrast formation in NC‐AFM imaging of self‐assembled monolayers of alkanethiols on the Au (111) surface. Comparing the potential models with implicit and explicit electrostatics, we demonstrate that, similar to the imaging of polar solid surfaces, the electrostatic interaction plays the central role in contrast formation. Careful comparison of several tip models showed that the model of a clean SiO2 tip gives the closest agreement with the experimental data.