bluuebunny/crossref_metadata_2025 huggingface dataset:
A Set of Activators and Repressors Control Peripheral Glucose Pathways in
Pseudomonas putida
To Yield a Common Central Intermediate
Authors: del Castillo Teresa , Duque Estrella , Ramos Juan L.
Published: February 2008

Pseudomonas putida
KT2440 channels glucose to the central Entner-Doudoroff intermediate 6-phosphogluconate through three convergent pathways. The genes for these convergent pathways are clustered in three independent regions on the host chromosome. A number of monocistronic units and operons coexist within each of these clusters, favoring coexpression of catabolic enzymes and transport systems. Expression of the three pathways is mediated by three transcriptional repressors, HexR, GnuR, and PtxS, and by a positive transcriptional regulator, GltR-2. In this study, we generated mutants in each of the regulators and carried out transcriptional assays using microarrays and transcriptional fusions. These studies revealed that HexR controls the genes that encode glucokinase/glucose 6-phosphate dehydrogenase that yield 6-phosphogluconate; the genes for the Entner-Doudoroff enzymes that yield glyceraldehyde-3-phosphate and pyruvate; and
gap-1
, which encodes glyceraldehyde-3-phosphate dehydrogenase. GltR-2 is the transcriptional regulator that controls specific porins for the entry of glucose into the periplasmic space, as well as the
gtsABCD
operon for glucose transport through the inner membrane. GnuR is the repressor of gluconate transport and gluconokinase responsible for the conversion of gluconate into 6-phosphogluconate. PtxS, however, controls the enzymes for oxidation of gluconate to 2-ketogluconate, its transport and metabolism, and a set of genes unrelated to glucose metabolism.
Bile Salts and Glycine as Cogerminants for
Clostridium difficile
Spores
Authors: Sorg Joseph A. , Sonenshein Abraham L.
Published: February 2008

Spore formation by
Clostridium difficile
is a significant obstacle to overcoming hospital-acquired
C. difficile
-associated disease. Spores are resistant to heat, radiation, chemicals, and antibiotics, making a contaminated environment difficult to clean. To cause disease, however, spores must germinate and grow out as vegetative cells. The germination of
C. difficile
spores has not been examined in detail. In an effort to understand the germination of
C. difficile
spores, we characterized the response of
C. difficile
spores to bile. We found that cholate derivatives and the amino acid glycine act as cogerminants. Deoxycholate, a metabolite of cholate produced by the normal intestinal flora, also induced germination of
C. difficile
spores but prevented the growth of vegetative
C. difficile
. A model of resistance to
C. difficile
colonization mediated by the normal bacterial flora is proposed.
A New Model of Pneumococcal Lipoteichoic Acid Structure Resolves Biochemical, Biosynthetic, and Serologic Inconsistencies of the Current Model
Authors: Seo Ho Seong , Cartee Robert T. , Pritchard David G. , Nahm Moon H.
Published: February 2008

Lipoteichoic acid (LTA) is an essential bacterial membrane polysaccharide (cell wall component) that is attached to the membrane via a lipid anchor. According to the currently accepted structure of pneumococcal LTA, the polysaccharide is comprised of several repeating units, each of which starts with glucose and ends with ribitol, with the lipid anchor predicted to be Glc(β1→3)AATGal(β1→3)Glc(α1→3)-acyl
2
Gro, where AATGal is 2-acetamido-4-amino-2,4,6-trideoxy-
d
-galactose. However, this lipid anchor has not been detected in pneumococcal membranes. Furthermore, the currently accepted structure does not explain the Forssman antigen properties of LTA and predicts a molecular weight for LTA that is larger than its actual observed molecular weight. To resolve these problems, we used mass spectrometry to analyze the structure of LTA isolated from several pneumococcal strains. Our study found that the R36A pneumococcal strain produces LTA that is more representative of pneumococci than that previously characterized from the R6 strain. Analysis of LTA fragments obtained after hydrofluoric acid and nitrous treatments showed that the fragments were consistent with an LTA nonreducing terminus consisting of GalNAc(α1→3)GalNAc(β1→, which is the minimal structure for the Forssman antigen. Based on these data, we propose a revised model of LTA structure: its polysaccharide repeating unit begins with GalNAc and ends with AATGal, and its lipid anchor is Glc(α1→3)-acyl
2
Gro, a common lipid anchor found in pneumococcal membranes. This new model accurately predicts the observed molecular weights. The revised model should facilitate investigation of the relationship between LTA's structure and its function.
Elucidation of an Alternate Isoleucine Biosynthesis Pathway in
Geobacter sulfurreducens
Authors: Risso Carla , Van Dien Stephen J. , Orloff Amber , Lovley Derek R. , Coppi Maddalena V.
Published: February 2008

The central metabolic model for
Geobacter sulfurreducens
included a single pathway for the biosynthesis of isoleucine that was analogous to that of
Escherichia coli
, in which the isoleucine precursor 2-oxobutanoate is generated from threonine.
13
C labeling studies performed in
G. sulfurreducens
indicated that this pathway accounted for a minor fraction of isoleucine biosynthesis and that the majority of isoleucine was instead derived from acetyl-coenzyme A and pyruvate, possibly via the citramalate pathway. Genes encoding citramalate synthase (GSU1798), which catalyzes the first dedicated step in the citramalate pathway, and threonine ammonia-lyase (GSU0486), which catalyzes the conversion of threonine to 2-oxobutanoate, were identified and knocked out. Mutants lacking both of these enzymes were auxotrophs for isoleucine, whereas single mutants were capable of growth in the absence of isoleucine. Biochemical characterization of the single mutants revealed deficiencies in citramalate synthase and threonine ammonia-lyase activity. Thus, in
G. sulfurreducens
, 2-oxobutanoate can be synthesized either from citramalate or threonine, with the former being the main pathway for isoleucine biosynthesis. The citramalate synthase of
G. sulfurreducens
constitutes the first characterized member of a phylogenetically distinct clade of citramalate synthases, which contains representatives from a wide variety of microorganisms.
The Nitric Oxide (NO)-Sensing Repressor NsrR of
Neisseria meningitidis
Has a Compact Regulon of Genes Involved in NO Synthesis and Detoxification
Authors: Heurlier Karin , Thomson Melanie J. , Aziz Naveed , Moir James W. B.
Published: February 2008

We have analyzed the extent of regulation by the nitric oxide (NO)-sensitive repressor NsrR from
Neisseria meningitidis
MC58, using microarray analysis. Target genes that appeared to be regulated by NsrR, based on a comparison between an
nsrR
mutant and a wild-type strain, were further investigated by quantitative real-time PCR, revealing a very compact set of genes, as follows:
norB
(encoding NO reductase),
dnrN
(encoding a protein putatively involved in the repair of nitrosative damage to iron-sulfur clusters),
aniA
(encoding nitrite reductase),
nirV
(a putative nitrite reductase assembly protein), and
mobA
(a gene associated with molybdenum metabolism in other species but with a frame shift in
N. meningitidis
). In all cases, NsrR acts as a repressor. The NO protection systems
norB
and
dnrN
are regulated by NO in an NsrR-dependent manner, whereas the NO protection system cytochrome
c
′ (encoded by
cycP
) is not controlled by NO or NsrR, indicating that
N. meningitidis
expresses both constitutive and inducible NO protection systems. In addition, we present evidence to show that the anaerobic response regulator FNR is also sensitive to NO but less so than NsrR, resulting in complex regulation of promoters such as
aniA
, which is controlled by both FNR and NsrR:
aniA
was found to be maximally induced by intermediate NO concentrations, consistent with a regulatory system that allows expression during denitrification (in which NO accumulates) but is down-regulated as NO approaches toxic concentrations.
Dissection of Ammonium Uptake Systems inCorynebacterium glutamicum: Mechanism of Action and Energetics of AmtA and AmtB
Authors: Walter Britta , Küspert Melanie , Ansorge Daniel , Krämer Reinhard , Burkovski Andreas
Published: February 2008

Corynebacterium glutamicumhas two different Amt-type proteins. While AmtB has a low substrate affinity and is not saturable up to 3 mM methylammonium, AmtA has a high substrate affinity and mediates saturable, membrane potential-dependent transport, resulting in a high steady-state accumulation of methylammonium, even in the absence of metabolic trapping.
Identification and Characterization of an Archaeon-Specific Riboflavin Kinase
Authors: Mashhadi Zahra , Zhang Hong , Xu Huimin , White Robert H.
Published: February 2008

The riboflavin kinase inMethanocaldococcus jannaschiihas been identified as the product of the MJ0056 gene. Recombinant expression of the MJ0056 gene inEscherichia coliled to a large increase in the amount of flavin mononucleotide (FMN) in theE. colicell extract. The unexpected features of the purified recombinant enzyme were its use of CTP as the phosphoryl donor and the absence of a requirement for added metal ion to catalyze the formation of FMN. Identification of this riboflavin kinase fills another gap in the archaeal flavin biosynthetic pathway. Some divalent metals were found to be potent inhibitors of the reaction. The enzyme represents a unique CTP-dependent family of kinases.
Functional Characterization and Mutagenesis of the Proposed Behavioral Sensor TlpD of
Helicobacter pylori
Authors: Schweinitzer Tobias , Mizote Tomoko , Ishikawa Naohiro , Dudnik Alexey , Inatsu Sakiko , Schreiber Sören , Suerbaum Sebastian , Aizawa Shin-Ichi , Josenhans Christine
Published: February 2008

Helicobacter pylori
requires flagellar motility and chemotaxis to establish and maintain chronic infection of the human stomach. The pH gradient in the stomach mucus is essential for bacterial orientation and guides the bacterium toward a narrow layer of the mucus, suggesting that
H. pylori
is capable of energy sensing or taxis. In the present study,
H. pylori
wild-type behavior in a temporal swimming assay could be altered by electron transport inhibitors, indicating that a connection between metabolism and behavior exists. In order to elucidate mechanisms of behavioral responses of
H. pylori
related to energy sensing, we investigated the phenotypes of single and multiple mutants of the four proposed chemotaxis sensor proteins. All sensor mutants were motile, but they diverged in their behavior in media supporting different energy yields. One proposed intracellular sensor, TlpD, was crucial for behavioral responses of
H. pylori
in defined media which did not permit growth and led to reduced bacterial energy levels. Suboptimal energetic conditions and inhibition of electron transport induced an increased frequency of stops and direction changes in the wild type but not in
tlpD
mutants. Loss of metabolism-dependent behavior in
tlpD
mutants could be reversed by complementation but not by electron donors bypassing the activity of the electron transport chain, in contrast to the case for the wild type. TlpD, which apparently lacks transmembrane domains, was detected both in the bacterial cytoplasm and at the bacterial periphery. The proposed energy sensor TlpD was found to mediate a repellent tactic response away from conditions of reduced electron transport.
Lethality of suicide methods
Authors: Elnour A A , Harrison J
Published: February 2008

Objectives:

To (a) quantify the lethality of suicide methods used in Australia in the period 1 July 1993 to 30 June 2003, (b) examine method-specific case fatality by age and sex, and (c) identify changes in case fatality during the study period.

Methods:

Two sources of data on episodes of self-harm in Australia were used, mortality and hospital separation data. Double counting of cases recorded in both sources was controlled by omitting fatal hospital cases from estimates of episodes of self-harm.

Results:

Overall case fatality was 12%. For each suicide method, case fatality was higher in males and older age groups. Firearms were the most lethal suicide means (90%) followed by hanging (83%). Rates of suicide involving firearms declined over time, and those involving hanging rose. Case fatality for firearm cases changed little over time, but declined for self-harm by hanging/suffocation, poisoning, sharp objects, and crashing a motor vehicle.

Conclusions:

This study (Australia) and two others (USA) show differences in method-specific lethality by gender and age. This study adds the finding of changes in lethality over time. Understanding of suicidality in populations, on which prevention efforts depend, requires explanation of these findings.
Conspicuity and bicycle crashes: preliminary findings of the Taupo Bicycle Study
Authors: Thornley S J , Woodward A , Langley J D , Ameratunga S N , Rodgers A
Published: February 2008

Objective:

To describe the methods, characteristics of participants, and report on the preliminary findings of a longitudinal study of cyclists.

Design:

Web-based survey to establish a cohort of cyclists.

Setting:

Participants in the largest mass-participation bicycle event in New Zealand, the Wattyl Lake Taupo Cycle Challenge.

Participants:

2469 riders who had enrolled online in the 2006 Wattyl Lake Taupo Cycle Challenge.

Main outcome measures:

Self-reported crashes in preceding 12 months.

Results:

Of 5653 eligible riders, 2469 (44%) completed the study questionnaire. Mean age was 44 years, 73% were male, and the average number of kilometers cycled per week in the preceding 12 months was 130. The annual incidence of crashes leading to injury that disrupted usual daily activities for at least 24 h was 0.5 per cyclist/year. About one-third of these crashes resulted in presentation to a health professional. The mean number of days absent from work attributable to bicycle crashes was 0.39 per cyclist/year. After adjustment for potential confounders and exposure (kilometers cycled per year), the rate of days off work from bicycle crash injury was substantially lower among riders who reported always wearing fluorescent colors (multivariate incidence rate ratio 0.23, 95% CI 0.09 to 0.59).

Conclusions:

Low cyclist conspicuity may increase the risk of crash-related injury and subsequent time off work. Increased use of high-visibility clothing is a simple intervention that may have a large impact on the safety of cycling.
Case-control study of acute renal failure in patients with cystic fibrosis in the UK
Authors: Smyth A , Lewis S , Bertenshaw C , Choonara I , McGaw J , Watson A
Published: February 2008

Background:

There has been a recent increase in the number of reported cases of acute renal failure (ARF) in cystic fibrosis (CF). A case-control study was conducted to determine the factors which are associated with an increased risk of ARF.

Methods:

24 cases of confirmed ARF were identified in patients with CF from 20 UK CF centres presenting between 1997 and 2004. Using the UK CF database, sex- and age-matched controls were identified. Risk factors were analysed by conditional logistic regression and Mantel-Haenszel analysis.

Results:

21 of the 24 patients with ARF had received an aminoglycoside at the time of their episode of ARF or in the preceding week compared with only 3 of 42 controls during the same time period (OR 81.8, 95% CI 4.7 to 1427, p<0.001). In the year before the episode of ARF, significantly more cases than controls had received gentamicin (19/24 cases vs 1/42 controls, p<0.001). The numbers receiving tobramycin were similar (9/24 cases vs 16/42 controls, p = 0.9). A known risk factor for renal impairment (prior renal disease, acute dehydration or long-term treatment with a nephrotoxic drug) was present in 18/24 cases and 7/42 controls (OR 24.0, 95% CI 3.1 to 186.6, p = 0.002).

Conclusions:

In patients with CF the use of an intravenous aminoglycoside is a risk factor for ARF; gentamicin is more nephrotoxic than tobramycin. Most patients who develop ARF have a risk factor which necessitates withholding aminoglycosides or more closely monitoring their use.
TGF-β mediatesPTENsuppression and cell motility through calcium-dependent PKC-α activation in pancreatic cancer cells
Authors: Chow Jimmy Y. C. , Dong Hui , Quach Khai T. , Van Nguyen Phuoc Nam , Chen Kevin , Carethers John M.
Published: February 2008

Transforming growth factor-β (TGF-β) suppresses growth via the TGF-β-SMAD pathway but promotes growth in cancer cells with disrupted SMAD signaling and corresponds to an invasive phenotype. TGF-β also downregulates the tumor suppressor PTEN that is rarely mutated in sporadic pancreatic cancer; this downregulation may mediate cell proliferation and invasiveness, but the mechanism is unknown. Here, we examined whether TGF-β modulation of PTEN was mediated by protein kinase C (PKC). We have previously demonstrated that SMAD4-null BxPc-3 pancreatic cancer cells treated with TGF-β1 (10 ng/ml) suppressed PTEN expression and increased cell proliferation. TGF-β-treated cells were examined for PKC activation and its coupling to PTEN expression, utilizing pharmacological and knockdown methods. Calcium mobilization and cell migration were also examined. In BxPc-3 cells, only two PKC isoforms were activated by TGF-β, and PTEN downregulation by TGF-β was specifically mediated by PKC-α. In parallel, TGF-β rapidly induced an increase in cytoplasmic free calcium from intracellular stores, consistent with subsequent PKC-α activation. The TGF-β-induced increase in cell migration was blocked by knockdown of PKC-α. Thus calcium-dependent PKC-α mediates TGF-β-induced transcriptional downregulation of PTEN, and this pathway promotes cell migration in a SMAD4-null environment. The TGF-β-PKC-α-PTEN cascade may be a key pathway for pancreatic cancer cells to proliferate and metastasize.
Vagal tone dominates autonomic control of mouse heart rate at thermoneutrality
Authors: Swoap S. J. , Li C. , Wess J. , Parsons A. D. , Williams T. D. , Overton J. M.
Published: February 2008

It is generally accepted that cardiac sympathetic tone dominates the control of heart rate (HR) in mice. However, we have recently challenged this notion given that HR in the mouse is responsive to ambient temperature (Ta) and that the housing Tais typically 21–23°C, well below the thermoneutral zone (∼30°C) of this species. To specifically test the hypothesis that cardiac sympathetic tone is the primary mediator of HR control in the mouse, we first examined the metabolic and cardiovascular responses to rapid changes in Tato demonstrate the sensitivity of the mouse cardiovascular system to Ta. We then determined HR in 1) mice deficient in cardiac sympathetic tone (“β-less” mice), 2) mice deficient in cardiac vagal tone [muscarinic M2receptor ( M2R−/−) mice], and 3) littermate controls. At a Taof 30°C, the HR of β-less mice was identical to that of wild-type mice (351 ± 11 and 363 ± 10 beats/min, respectively). However, the HR of M2R−/−mice was significantly greater (416 ± 7 beats/min), demonstrating that vagal tone predominates over HR control at this Ta. When these mice were calorically restricted to 70% of normal intake, HR fell equally in wild-type, β-less, and M2R−/−mice (ΔHR = 73 ± 9, 76 ± 3, and 73 ± 7 beats/min, respectively), suggesting that the fall in intrinsic HR governs bradycardia of calorically restricted mice. Only when the Tawas relatively cool, at 23°C, did β-less mice exhibit a HR (442 ± 14 beats/min) that was different from that of littermate controls (604 ± 10 beats/min) and M2R−/−mice (602 ± 5 beats/min). These experiments conclusively demonstrate that in the absence of cold stress, regulation of vagal tone and modulation of intrinsic rate are important determinants of HR control in the mouse.
TGF-β signaling promotes survival and repair in rat alveolar epithelial type 2 cells during recovery after hyperoxic injury
Authors: Buckley S. , Shi W. , Barsky L. , Warburton D.
Published: February 2008

Hyperoxic rats treated with inosine during oxygen exposure have increased levels of active transforming growth factor (TGF)-β in the bronchoalveolar lavage (BAL), yet alveolar epithelial type 2 cells (AEC2) isolated from these animals demonstrate less hyperoxia-induced DNA damage and increased expression of active Smad2. To determine whether TGF-β1 signaling per se protected AEC2 against hyperoxic damage, freshly isolated AEC2 from hyperoxic rats were incubated with TGF-β1 for 24 h and assayed for DNA damage by fluorescein-activated cell sorter analysis of TdT-mediated dUTP nick end labeling. TGF-β1 was protective over a concentration range similar to that in BAL of inosine-treated hyperoxic animals (50–5,000 pg/ml). TGF-β1 also augmented hyperoxia-induced DNA repair activity and cell migration, stimulated autocrine secretion of fibronectin, accelerated closure of a monolayer scratch wound, and restored hyperoxia-depleted VEGF secretion by AEC2 to normoxic levels. The TGF-β receptor type I activin-like kinase-4, -5, and -7 inhibitor peptide SB-505124 abolished the protective effect of TGF-β on hyperoxic DNA damage and increased TdT-mediated dUTP nick end labeling in normoxic cells. These data suggest that endogenous TGF-β-mediated Smad signaling is required for AEC2 homeostasis in vitro, while exogenous TGF-β1 treatment of hyperoxia-damaged AEC2 results in a cell that is equipped to survive, repair, migrate, secrete matrix, and induce new blood vessel formation more efficiently than AEC2 primed by hyperoxia alone.
Hepatocyte growth factor regulates cyclooxygenase-2 expression via β-catenin, Akt, and p42/p44 MAPK in human bronchial epithelial cells
Authors: Lee Young H. , Suzuki Yuichiro J. , Griffin Autumn J. , Day Regina M.
Published: February 2008

Hepatocyte growth factor (HGF) is upregulated in response to lung injury and has been implicated in tissue repair through its antiapoptotic and proliferative activities. Cyclooxygenase-2 (COX-2) is an inducible enzyme in the biosynthetic pathway of prostaglandins, and its activation has been shown to play a role in cell growth. Here, we report that HGF induces gene transcription of COX-2 in human bronchial epithelial cells (HBEpC). Treatment of HBEpC with HGF resulted in phosphorylation of the HGF receptor (c-Met), activation of Akt, and upregulation of COX-2 mRNA. Adenovirus-mediated gene transfer of a dominant negative (DN) Akt mutant revealed that HGF increased COX-2 mRNA in an Akt-dependent manner. COX-2 promoter analysis in luciferase reporter constructs showed that HGF regulation required the β-catenin-responsive T cell factor-4 binding element (TBE). The HGF activation of the COX-2 gene transcription was blocked by DN mutant of β-catenin or by inhibitors that blocked activation of Akt. Inhibition of p42/p44 MAPK pathway blocked HGF-mediated activation of β-catenin gene transcription but not Akt activation, suggesting that p42/p44 MAPK acts in a parallel mechanism for β-catenin activation. We also found that inhibition of COX-2 with NS-398 blocked HGF-induced growth in HBEpC. Together, the results show that the HGF increases COX-2 gene expression via an Akt-, MAPK-, and β-catenin-dependent pathway in HBEpC.
Effects of decorin and biglycan on human airway smooth muscle cell proliferation and apoptosis
Authors: D'Antoni Michelle L. , Torregiani Chiara , Ferraro Pasquale , Michoud Marie-Claire , Mazer Bruce , Martin James G. , Ludwig Mara S.
Published: February 2008

Proteoglycans (PG) are altered in the asthmatic airway wall. Because PGs are known to affect cell proliferation and apoptosis, we hypothesized that alterations in PG might influence the airway smooth muscle (ASM) hyperplasia observed in the asthmatic airway. Human ASM cells were seeded on plastic or plates coated with decorin (Dcn), biglycan (Bgn), or collagen type I (Col I) (1, 3, and 10 μg/ml). Cells were stimulated with platelet-derived growth factor (PDGF), and cell number was assessed at 0, 48, and 96 h. Cell proliferation was measured by bromodeoxyuridine (BrdU) incorporation and apoptosis by annexin V and propidium iodide staining at 48 h post-PDGF stimulation. A significant decrease in cell number was observed with cells seeded on Dcn (10 μg/ml) at 0, 48, and 96 h ( P < 0.01). Dcn induced both decreases in BrdU incorporation and increases in annexin V staining ( P < 0.05). Bgn decreased cell number at time 0 only ( P < 0.05) and affected neither proliferation nor apoptosis. Col I (10 μg/ml) caused a significant increase in cell number at 48 and 96 h ( P < 0.01). Adding exogenous Dcn (1–30 μg/ml) to the medium had no effect on cell number. Exposing Dcn-coated matrices to chondroitinase ABC, an enzyme that degrades glycosaminoglycan side chains, reversed the Dcn-induced decrease in cell number. These studies demonstrate that different PGs have variable effects on ASM cell proliferation and apoptosis. Recently described decreases in Dcn in the asthmatic airway wall could potentially permit more exuberant ASM growth.
Adding protein to a carbohydrate supplement provided after endurance exercise enhances 4E-BP1 and RPS6 signaling in skeletal muscle
Authors: Morrison Paul J. , Hara Daisuke , Ding Zhenping , Ivy John L.
Published: February 2008

To examine the role of both endurance exercise and nutrient supplementation on the activation of mRNA translation signaling pathways postexercise, rats were subjected to a 3-h swimming protocol. Immediately following exercise, the rats were provided with a solution containing either 23.7% wt/vol carbohydrates (CHO), 7.9% wt/vol protein (Pro), 31.6% wt/vol (23.7% wt/vol CHO + 7.9% wt/vol Pro) carbohydrates and Pro (CP), or a placebo (EX). The rats were then killed at 0, 30, and 90 min postexercise, and phosphorylation states of mammalian target of rapamycin (mTOR), ribosomal S6 kinase (p70S6K), ribosomal protein S6 (rpS6), and 4E-binding protein 1 (4E-BP1), were analyzed by immunoblot analysis in the red and white quadriceps muscle. Results demonstrated that rat groups provided with any of the three nutritional supplements (CHO, Pro, CP) transiently increased the phosphorylation states of mTOR, 4E-BP1, rpS6, and p70S6Kcompared with EX rats. Although CHO, Pro, and CP supplements phosphorylated mTOR and p70S6Kafter exercise, only CP elevated the phosphorylation of rpS6 above all other supplements 30 min postexercise and 4E-BP1 30 and 90 min postexercise. Furthermore, the phosphorylation states of 4E-BP1 ( r2= 0.7942) and rpS6 ( r2= 0.760) were highly correlated to insulin concentrations in each group. These results suggest that CP supplementation may be most effective in activating the mTOR-dependent signaling pathway in the postprandial state postexercise, and that there is a strong relationship between the insulin concentration and the activation of enzymes critical for mRNA translation.
Alterations in Gemin5 Expression Contribute to Alternative mRNA Splicing Patterns and Tumor Cell Motility
Authors: Lee Jong Heun , Horak Christine E. , Khanna Chand , Meng Zhaojing , Yu Li Rong , Veenstra Timothy D. , Steeg Patricia S.
Published: February 2008

The role of Gemin5 in alternative mRNA splicing, tumor cell motility, and proteomic instability was investigated. Isotope Capture Affinity Tag proteomic analysis was conducted on MDA-MB-435 tumor cells transfected with either a control vector (C-100) or the Nm23-H1 metastasis suppressor (H1-177). Ingenuity pathway analysis revealed that RNA posttranscriptional processing was the most prominent class of differentially expressed proteins. Within this category, overexpression of Acinus1, Poly(a) binding protein, HNRPA2B1, Bop1, and Gemin5 was confirmed in less metastatic H1-177 cells. Overexpression of the latter four proteins was also observed in the lower metastatic antisense Ezrin transfectant of a murine osteosarcoma model system, confirming the general relevance of the trends. Gemin5, a component of the spliceosomal complex, was chosen for further study. Analysis of global mRNA splicing by SpliceArray chips revealed that 16 genes were differentially spliced in C-100 compared with H1-177 cells; transient transfection of gemin5 into C-100 cells restored the splice pattern to that of H1-177 cells. Alternative splicing patterns for the engulfment and cell motility 1 and thrombospondin 4 genes were confirmed by semiquantitative reverse transcription-PCR. Gemin5 overexpression coordinately reduced C-100 cell motility by 50%, and siRNA-mediated reduction of Gemin5 expression increased the motility of H1-177 cells by 2-fold (P < 0.004). The data provide the first demonstration that alterations in the expression of a spliceosome protein can effect both specific splicing events and tumor cell motility. The data also show that changes in mRNA splicing patterns accompany metastatic progression, which may contribute to proteome instability. [Cancer Res 2008;68(3):639–44]
Inhibition of Endoplasmic Reticulum Stress–Induced Apoptosis of Melanoma Cells by the ARC Protein
Authors: Chen Li Hua , Jiang Chen Chen , Watts Ralph , Thorne Rick F. , Kiejda Kelly A. , Zhang Xu Dong , Hersey Peter
Published: February 2008

We have shown previously that most melanoma cell lines are insensitive to endoplasmic reticulum (ER) stress–induced apoptosis, but resistance can be reversed through activation of caspase-4 by inhibition of the MEK/ERK pathway. We report in this study that apoptosis was induced by the ER stress inducer thapsigargin or tunicamycin via a caspase-8–mediated pathway in the melanoma cell line Me1007, although the MEK/ERK pathway was activated in this cell line. The high sensitivity of Me1007 to ER stress–induced apoptosis was associated with low expression levels of the apoptosis repressor with caspase recruitment domain (ARC) protein that was expressed at relatively high levels in the resistant melanoma cell lines. Transfection of cDNA encoding ARC into Me1007 cells inhibited both caspase-8 activation and apoptosis induced by thapsigargin or tunicamycin. In contrast, inhibition of ARC by small interfering RNA knockdown sensitized the resistant melanoma cell lines to ER stress–induced apoptosis, which was inhibitable by blockage of caspase-8 activation. Both exogenous and endogenous ARC seemed to predominantly locate to the cytoplasm and mitochondria and could be coimmunoprecipitated with caspase-8. Taken together, ER stress can potentially activate multiple apoptosis signaling pathways in melanoma cells in a context-dependent manner. Whereas the MEK/ERK signaling pathway plays an important role in inhibiting ER stress–induced caspase-4 activation, ARC seems to be critical in blocking activation of casapse-8 in melanoma cells subjected to ER stress. [Cancer Res 2008;68(3):834–42]
Antiangiogenic Therapy with Mammalian Target of Rapamycin Inhibitor RAD001 (Everolimus) Increases Radiosensitivity in Solid Cancer
Authors: Manegold Philipp C. , Paringer Carmen , Kulka Ulrike , Krimmel Klaus , Eichhorn Martin E. , Wilkowski Ralf , Jauch Karl-Walter , Guba Markus , Bruns Christiane J.
Published: February 2008

Purpose: Radiotherapy exerts direct antivascular effects in tumors and also induces a proangiogenic stress response in tumor cells via the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway. Therefore, the combination of radiotherapy and antiangiogenic therapy with mTOR inhibitor RAD001 (Everolimus) might exert additive/synergistic effects on tumor growth.

Experimental Design: Effects of radiation combined with mTOR inhibitor RAD001 were studied on proliferation of murine colon cancer CT-26, human pancreatic cancer L3.6pl, and human umbilical vascular endothelial cells in vitro. In vivo tumor growth of subcutaneous colon cancer CT 26 and orthotopic pancreatic cancer L3.6pl was assessed after fractionated radiotherapy (5 × 2 or 5 × 4 Gy) with or without the addition of the mTOR inhibitor RAD001. RAD001 (1.5 mg/kg/d) was administered until the end of experiments beginning before or after radiotherapy.

Results: A single dose of 2 Gy reduced in vitro proliferation of L3.6pl (−16%), CT-26 (−70%), and human umbilical vascular endothelial cells (HUVEC; −72%). The mTOR inhibitor RAD001 (10 ng/mL) suppressed proliferation of HUVEC (−83%), L3.6pl (−8%), and CT-26 (−82%). Combination of even low concentrations of 0.01 ng/mL RAD001 and 0.25 Gy radiation significantly reduced proliferation of HUVECs (−57%), whereas additive effects of RAD001 and radiation on tumor cells were seen only at the highest concentrations tested. In vivo, RAD001 introduced before radiotherapy (5 × 2 Gy) improved tumor growth control in mice (L3.6pl: 326 mm3 versus 1144 mm3; CT-26: 210 mm3 versus 636 mm3; P < 0.05 versus control). RAD001 turned out to possess a dose-modifying effect on radiotherapy.

Conclusion: Endothelial cells seem to be most sensitive to combination of mTOR inhibition and radiotherapy. Additive tumor growth delay using the mTOR inhibitor RAD001 and radiotherapy in vivo therefore might rely on combined antiangiogenic and antivascular effects.
Inhibition of IκB Kinase Subunit 2 in Cutaneous T-Cell Lymphoma Down-Regulates Nuclear Factor-κB Constitutive Activation, Induces Cell Death, and Potentiates the Apoptotic Response to Antineoplastic Chemotherapeutic Agents
Authors: Sors Aurore , Jean-Louis Francette , Bégué Elodie , Parmentier Laurent , Dubertret Louis , Dreano Michel , Courtois Gilles , Bachelez Hervé , Michel Laurence
Published: February 2008

Purpose: A key molecular feature of cutaneous T-cell lymphomas (CTCL) is the constitutive activation of the nuclear factor-κB (NF-κB) transcription factor. We investigated in vitro the effects on CTCL survival and chemoresistance of a specific inhibition of IκB kinase subunit 2 (IKK2).

Experimental Design: Selective IKK2 inhibition was carried out by transfection of SeAx and MyLa CTCL lines with an inactive form of IKK2 and by exposing these lines and tumor cells from 10 patients with Sézary syndrome (SS) to AS602868, a new IKK2 inhibitor. The constitutive nuclear translocation of NF-κB was analyzed by electrophoretic mobility shift assay and confocal microscopy. Apoptosis was determined by Annexin V/propidium iodide–positive staining and mitochondrial transmembrane potential alterations as well as poly(ADP-ribose)polymerase cleavage. The expression of Bcl-2 family oncoproteins and survivin was studied by immunoblotting.

Results: Specific IKK2 inhibition resulting from transfection or from incubation with AS602868 allowed a down-regulation of NF-κB transcriptional activity. As shown by electrophoretic mobility shift assay and apoptosis assays, AS602868 down-regulated the nuclear translocation of NF-κB and induced a potent apoptotic response in CTCL lines and in tumor cells from patients with SS while preserving the viability of both peripheral blood lymphocytes from healthy donors and of nonmalignant T cells from SS patients. Moreover, CTCL death induction by conventional antineoplastic agents etoposide and vincristine was potentiated by AS602868. Finally, AS602868-induced apoptosis of CTCL cells was associated with an up-regulation of Bax dimers and a decrease of survivin.

Conclusion: These results indicate that IKK2 inhibition represents a promising strategy for the treatment of advanced stages of CTCL.
A Single-Nucleotide Polymorphism in the Aromatase Gene Is Associated with the Efficacy of the Aromatase Inhibitor Letrozole in Advanced Breast Carcinoma
Authors: Colomer Ramon , Monzo Mariano , Tusquets Ignasi , Rifa Juli , Baena José M. , Barnadas Agusti , Calvo Lourdes , Carabantes Francisco , Crespo Carmen , Muñoz Montserrat , Llombart Antonio , Plazaola Arrate , Artells Rosa , Gilabert Monstsrrat , Lloveras Belen , Alba Emilio
Published: February 2008

Purpose: To evaluate the efficacy of treatment with the aromatase inhibitor letrozole in breast cancer patients segregated with respect to DNA polymorphisms of the aromatase gene CYP19.

Patients and Methods: Postmenopausal patients (n = 67) with hormone receptor–positive metastatic breast cancer were treated with the aromatase inhibitor letrozole. PCR allelic discrimination was used to examine three single-nucleotide polymorphisms (SNP) in DNA obtained from breast carcinoma tissue. Two SNPs analyzed (rs10046 and rs4646) were located in the 3′ untranslated region and one (rs727479) was in the intron of the aromatase CYP19 gene. The primary end point of treatment efficacy was time to progression (TTP).

Results: Median age was 62 years and median number of metastatic sites was 2. Observed allelic SNP frequencies were rs10046, 71%; rs4646, 46%; and rs727479, 63%. Of the 67 patients, 65 were evaluable for efficacy. Median TTP was 12.1 months. We observed no relationship between TTP and the rs10046 or rs727479 variants. In contrast, we found that TTP was significantly improved in patients with the rs4646 variant, compared with the wild-type gene (17.2 versus 6.4 months; P = 0.02).

Conclusion: In patients with hormone receptor–positive metastatic breast cancer treated with the aromatase inhibitor letrozole, the presence of a SNP in the 3′ untranslated region of the CYP19 aromatase gene is associated with improved treatment efficacy. Testing for the CYP19 rs4646 SNP as a predictive tool for breast cancer patients on antiaromatase therapy deserves prospective evaluation.
The role of TRPV6 in breast carcinogenesis
Authors: Bolanz Katrin A. , Hediger Matthias A. , Landowski Christopher P.
Published: February 2008

TRPV6 is an endothelial calcium entry channel that is strongly expressed in breast adenocarcinoma tissue. In this study, we further confirmed this observation by analysis of breast cancer tissues, which indicated that TRPV6 mRNA expression was up-regulated between 2-fold and 15-fold compared with the average in normal breast tissue. Whereas TRPV6 is expressed in the cancer tissue, its role as a calcium channel in breast carcinogenesis is poorly understood. Therefore, we investigated how TRPV6 affects the viability, apoptosis, and calcium transport in the breast cancer cell line T47D. Hormones can also affect the tumor development; hence, we determined the effects of estradiol, progesterone, and 1,25-vitamin D on TRPV6 transcription. Interestingly, the estrogen receptor antagonist tamoxifen reduced expression of TRPV6 and is able to inhibit its calcium transport activity (IC50, 7.5 μmol/L). The in vitro model showed that TRPV6 can be regulated by estrogen, progesterone, tamoxifen, and 1,25-vitamin D and has a large influence on breast cancer cell proliferation. Moreover, the effect of tamoxifen on cell viability was enhanced when TRPV6 expression was silenced with small interfering RNA. TRPV6 may be a novel target for the development of calcium channel inhibitors to treat breast adenocarcinoma expressing TRPV6. [Mol Cancer Ther 2008;7(2):271–9]
In vivooptical imaging of human lymphoma xenograft using a library-derived peptidomimetic against α4β1 integrin
Authors: Peng Li , Liu Ruiwu , Andrei Mirela , Xiao Wenwu , Lam Kit S.
Published: February 2008

Increasing literature suggests that cell adhesion molecule α4β1 integrin plays a pivotal role in autoimmune diseases and cancer development. Noninvasive visualization of α4β1 integrin in vivo will facilitate the understanding of its involvement in disease progression and development of targeted therapies. Due to the lack of high-affinity targeting ligands, molecular imaging of α4β1 integrin is much less explored than that of αvβ3 and αvβ5 integrins. We have recently reported using the one bead–one compound combinatorial library method to identify a peptidomimetic, LLP2A, that preferentially binds to activated α4β1 integrin. Here, we described the use of LLP2A-Cy5.5 conjugate as an in vivo optical imaging probe in a human lymphoma xenograft model. This univalent LLP2A-Cy5.5 conjugate retained the binding activity and specificity to α4β1 integrin as shown by cell binding assays using α4β1-positive Molt-4 T-leukemia cells. The subcutaneous Molt-4 tumor was clearly visualized from 1 to 24 h after tail vein injection of the conjugate. Direct imaging and confocal microscopic examination of excised tumors and organs confirmed the accumulation of LLP2A in tumors and revealed very little or no uptake in normal organs except for lymph nodes. Kidney uptake was high when the whole organ was scanned but it was negative when examined microscopically, suggesting that LLP2A bound to the renal tubules loosely. Tumor uptake of LLP2A-Cy5.5 conjugate was blocked by excess unlabeled LLP2A. This study showed that the combinatorial chemical library-derived peptidomimetic LLP2A can be easily developed into an optical imaging probe for noninvasively monitoring of activated α4β1 integrin in vivo. [Mol Cancer Ther 2008;7(2):432–7]
Renin-Angiotensin System Gene Polymorphisms and Atrial Fibrillation: A Regression Approach for the Detection of Gene-Gene Interactions in a Large Hospitalized Population
Authors: Tsai Chia-Ti , Hwang Juey-Jen , Chiang Fu-Tien , Wang Yi-Chih , Tseng Chuen-Den , Tseng Yung-Zu , Lin Jiunn-Lee
Published: February 2008

Objectives: To test the association between renin-angiotensin system gene variants and atrial fibrillation (AF) using a regression approach. Methods: A total of 1,236 consecutive patients (227 with AF and 1,009 with normal sinus rhythm as controls) were recruited. Angiotensin-converting enzyme (ACE) gene I/D polymorphism; T174M, M235T, G–6A, A–20C, G–152A and G–217A polymorphisms of the angiotensinogen (AGT) gene, and A1166C polymorphism of the angiotensin II type I receptor (AT1R) gene were genotyped. We used a regression approach based on a generalized linear model to evaluate haplotype effects and to detect gene-gene interactions by incorporating interaction terms in the model. Results: In single-locus analyses, no locus was associated with AF. After adjustment for AF risk factors, we found significant differences in the global AGT gene haplotype profile (the global score statistic = 30.364, p = 0.001) and individual haplotype frequencies between AF patients and controls. Furthermore, significant 2-way gene-gene interactions between ACE I/D polymorphism and AGT gene haplotypes and between AT1R A1166C polymorphism and AGT gene haplotypes, and 3-way interaction between ACE I/D, AT1R A1166C and AGT gene haplotypes were detected. Conclusions: These results are compatible with the concept of multilocus and multigene effects in determining the risk of complex diseases such as AF, which would be missed with conventional single-locus approaches.
PARP-1 Inhibition Prevents Oxidative and Nitrosative Stress–Induced Endothelial Cell Death via Transactivation of the VEGF Receptor 2
Authors: Mathews Marlene T. , Berk Bradford C.
Published: February 2008

Objective—

PARP-1, a DNA base repair enzyme, is activated by DNA breaks induced by oxidative (ROS) and nitrosative (RNS) stress. By consuming NAD
+
, PARP-1 activation can lead to ATP depletion and cell death. Studies suggest that inhibiting PARP-1 activity can attenuate pathologies associated with vascular smooth muscle and endothelial dysfunction. PARP-1 inhibition can also activate the prosurvival serine/threonine kinase, Akt. Vascular endothelial growth factor (VEGF) regulates endothelial cell survival via Akt activation downstream of VEGF receptor 2 (VEGFR2) activation. Here we investigated the hypothesis that PARP-1 inhibition protects human umbilical vein endothelial cells (HUVECs) from ROS- and RNS-induced cell death by limiting NAD
+
depletion and by activating a prosurvival signaling pathway via VEGFR2 phosphorylation.

Methods and Results—

We activated PARP-1 in HUVECs by treatment with hydrogen peroxide (H
2
O
2
) and peroxynitrite (ONOO
−
). Both depleted HUVECs of NAD
+
and ATP, processes that were limited by the PARP-1 inhibitor, PJ34. ONOO
−
and H
2
O
2
-induced cell death and apoptosis were attenuated in cells treated with PJ34 or PARP-1 siRNA. PARP-1 inhibition increased Akt, BAD, and VEGFR2 phosphorylation in HUVECs and in PJ34-treated rabbit aortas. The VEGFR2-specific tyrosine kinase inhibitor SU1498 decreased PARP-1 inhibition-mediated phosphorylation of VEGFR2 and Akt, and also reversed survival effects of PJ34. Finally, PARP-1 inhibition protected cells from death induced by serum starvation, evidence for a role in cell survival independent of energy protection.

Conclusions—

PARP-1 inhibition prevents ROS- and RNS-induced HUVEC death by maintaining cellular energy in the form of NAD
+
and ATP, and also by activating a survival pathway via VEGFR2, Akt, and BAD phosphorylation.
Vascular Calcifications in Homozygote Familial Hypercholesterolemia
Authors: Awan Z. , Alrasadi K. , Francis G.A. , Hegele R.A. , McPherson R. , Frohlich J. , Valenti D. , de Varennes B. , Marcil M. , Gagne C. , Genest J. , Couture P.
Published: February 2008

Background—

Patients with homozygous familial hypercholesterolemia (hmzFH) attributable to LDL receptor gene mutations have shown a remarkable increase in survival over the last 20 years. Early onset coronary heart disease (CHD) and calcific aortic valve stenosis are the major complications of this disorder. We now report extensive premature calcification of the aorta in patients with hmzFH.

Methods and Results—

We examined 25 hmzFH patients from Canada; mean age was 32 years (range 5 to 54), and mean baseline cholesterol before treatment was 19±5 mmol/L (737±206 mg/dL). Aortic calcification was quantified using computed tomography (CT). An elevated mean calcium score was found in patients by age 20 and correlated with age (r
2
=0.53,
P
=0.001). One quarter (24%) of patients underwent aortic valve surgery.

Conclusions—

We document premature severe aortic calcifications in all adult hmzFH patients studied. These presented considerable surgical management challenges. Strategies to identify and monitor aortic calcification in hmzFH by noninvasive techniques are required, as are clinical trials to determine whether additional or more intensive therapies will prevent the progression of such calcifications. Whether vascular calcifications in hmzFH subjects are related to sustained increases in LDL-C levels or to other mechanisms, such as abnormal osteoblast activity, remains to be determined.
Cerebral Proliferative Angiopathy
Authors: Lasjaunias Pierre L. , Landrieu Pierre , Rodesch Georges , Alvarez Hortensia , Ozanne Augustin , Holmin Staffan , Zhao Wen-Yuan , Geibprasert Sasikhan , Ducreux Dennis , Krings Timo
Published: February 2008

Background and Purpose—

The purpose of this article is to describe “cerebral proliferative angiopathy” (CPA) as a clinical entity, which may be regarded as separate from “classical” brain AVMs in angioarchitecture, natural history, clinical presentation, and, therefore, treatment and which can be discerned from other cerebral AVMs by characteristic imaging features.

Methods—

In a prospectively entered databank encompassing 1434 patients with brain AVMs, a subgroup of 49 patients harboring specific angiographic characteristics were identified. Their charts and imaging films were retrospectively reviewed.

Results—

We found a preponderance of CPA in young (mean age: 22) females (67%). Clinical symptoms were seizures, disabling headaches, and stroke-like symptoms; hemorrhagic presentations were exceptional. On cross-sectional imaging, CPA demonstrated as a diffuse network of densely enhancing vascular spaces with intermingled normal brain parenchyma. The discrepancy between the large size of the nidus and the small shunting volume, the absence of flow-related aneurysms, the presence of diffuse angiogenesis (eg, transdural supply, progressive arterial occlusion), and the small calibre of a multitude of feeding arteries and draining veins were the angiographic hallmarks of this disease.

Conclusion—

The diffuse angiogenetic activity is presumably related to reduced perinidal perfusion and subsequent chronic cortical ischemia. Natural history demonstrates a low risk of hemorrhage. CPA may be regarded as a separate clinical entity different to “classical” cerebral AVMs, because normal brain is interspersed with the abnormal vascular channels increasing the risk of neurological deficit in aggressive treatments, which in the light of the natural history does not seem to be indicated.