bluuebunny/crossref_metadata_2025 huggingface dataset:
High-Level Molecular Diversity of Copper-Zinc Superoxide Dismutase Genes among and within Species of Arbuscular Mycorrhizal Fungi
Authors: Corradi Nicolas , Ruffner Beat , Croll Daniel , Colard Alexandre , Horák Ales , Sanders Ian R.
Published: February 2009

In the ecologically important arbuscular mycorrhizal fungi (AMF),Sod1encodes a functional polypeptide that confers increased tolerance to oxidative stress and that is upregulated inside the roots during early steps of the symbiosis with host plants. It is still unclear whether its expression is directed at scavenging reactive oxygen species (ROS) produced by the host, if it plays a role in the fungus-host dialogue, or if it is a consequence of oxidative stress from the surrounding environment. All these possibilities are equally likely, and molecular variation at theSod1locus can possibly have adaptive implications for one or all of the three mentioned functions. In this paper, we analyzed the diversity of theSod1gene in six AMF species, as well as 14Glomus intraradicesisolates from a single natural population. By sequencing this locus, we identified a large amount of nucleotide and amino acid molecular diversity both among AMF species and individuals, suggesting a rapid divergence of its codons. TheSod1gene was monomorphic within each isolate we analyzed, and quantitative PCR strongly suggest this locus is present as a single copy inG. intraradices. Maximum-likelihood analyses performed using a variety of models for codon evolution indicated that a number of amino acid sites most likely evolved under the regime of positive selection among AMF species. In addition, we found that some isolates ofG. intraradicesfrom a natural population harbor very divergent orthologousSod1sequences, and our analysis suggested that diversifying selection, rather than recombination, was responsible for the persistence of this molecular diversity within the AMF population.
Quantitative Measurement of Varicella-Zoster Virus Infection by Semiautomated Flow Cytometry
Authors: Gates Irina V. , Zhang Yuhua , Shambaugh Cindy , Bauman Meredith A. , Tan Charles , Bodmer Jean-Luc
Published: February 2009

Varicella-zoster virus (VZV; human herpesvirus 3) is the etiological cause of chickenpox and, upon reactivation from latency, zoster. Currently, vaccines are available to prevent both diseases effectively. A critical requirement for the manufacturing of safe and potent vaccines is the measurement of the biological activity to ensure proper dosing and efficacy, while minimizing potentially harmful secondary effects induced by immunization. In the case of live virus-containing vaccines, such as VZV-containing vaccines, biological activity is determined using an infectivity assay in a susceptible cellular host in vitro. Infectivity measurements generally rely on the enumeration of plaques by visual inspection of an infected cell monolayer. These plaque assays are generally very tedious and labor intensive and have modest throughput and high associated variability. In this study, we have developed a flow cytometry assay to measure the infectivity of the attenuated vaccine strain (vOka/Merck) of VZV in MRC-5 cells with improved throughput. The assay is performed in 96-well tissue culture microtiter plates and is based on the detection and quantification of infected cells expressing VZV glycoproteins on their surfaces. Multiple assay parameters have been investigated, including specificity, limit of detection, limit of quantification, range of linear response, signal-to-noise ratio, and precision. This novel assay appears to be in good concordance with the classical plaque assay results and therefore provides a viable, higher-throughput alternative to the plaque assay.
NovelCoprinopsis cinereaPolyesterase That Hydrolyzes Cutin and Suberin
Authors: Kontkanen Hanna , Westerholm-Parvinen Ann , Saloheimo Markku , Bailey Michael , Rättö Marjaana , Mattila Ismo , Mohsina Marzia , Kalkkinen Nisse , Nakari-Setälä Tiina , Buchert Johanna
Published: February 2009

Three cutinase gene-like genes from the basidiomyceteCoprinopsis cinerea(Coprinus cinereus) found with a similarity search were cloned and expressed inTrichoderma reeseiunder the control of an induciblecbh1promoter. The selected transformants of all three polyesterase constructs showed activity withp-nitrophenylbutyrate, used as a model substrate. The most promising transformant of the cutinase CC1G_09668.1 gene construct was cultivated in a laboratory fermentor, with a production yield of 1.4 g liter−lpurified protein. The expressed cutinase (CcCUT1) was purified to homogeneity by immobilized metal affinity chromatography exploiting a C-terminal His tag. The N terminus of the enzyme was found to be blocked. The molecular mass of the purified enzyme was determined to be around 18.8 kDa by mass spectrometry. CcCUT1 had higher activity on shorter (C2to C10) fatty acid esters ofp-nitrophenol than on longer ones, and it also exhibited lipase activity. CcCUT1 had optimal activity between pH 7 and 8 but retained activity over a wide pH range. The enzyme retained 80% of its activity after 20 h of incubation at 50°C, but residual activity decreased sharply at 60°C. Microscopic analyses and determination of released hydrolysis products showed that the enzyme was able to depolymerize apple cutin and birch outer bark suberin.
Characterization ofrrdA, a TetR Family Protein Gene Involved in the Regulation of Secondary Metabolism inStreptomyces coelicolor
Authors: Ou Xijun , Zhang Bo , Zhang Lin , Zhao Guoping , Ding Xiaoming
Published: February 2009

Streptomycesnot only exhibits complex morphological differentiation but also produces a plethora of secondary metabolites, particularly antibiotics. To improve our general understanding of the complex network of undecylprodigiosin (Red) biosynthesis regulation, we used an in vivo transposition system to identify novel regulators that influence Red production inStreptomyces coelicolorM145. Using this screening system, we obtained 25 Red-deficient mutants. Twenty-four of these mutants had a transposon inserted in the previously described Red biosynthetic gene cluster and produced different amounts of another secondary metabolite, actinorhodin (Act). One mutant was shown to have an insertion in a different region of the chromosome upstream of the previously uncharacterized generrdA(regulator ofredD, sco1104), which encodes a putative TetR family transcription factor. Compared with wild-type strain M145, therrdAnull mutant exhibited increased Red production and decreased Act production. A high level ofrrdAexpression resulted in a severe reduction in Red production and Act overproduction. Reverse transcription-PCR analysis showed that RrdA negatively regulated Red production by controllingredDmRNA abundance, while no change was observed at the transcript level of the Act-specific activator gene,actII-orf4. The effects on Act biosynthesis might arise from competition for precursors that are common to both pathways.
High-Throughput Bioluminescence-Based Mutant Screening Strategy for Identification of Bacterial Virulence Genes
Authors: Karsi Attila , Gülsoy Nagihan , Corb Erin , Dumpala Pradeep R. , Lawrence Mark L.
Published: February 2009

A high-throughput bioluminescence screening procedure for identification of virulence genes in bacteria was developed and applied to the fish pathogenEdwardsiella ictaluri. A random transposon mutant library expressing bioluminescence was constructed and robotically arrayed on 384-well plates. Mutants were cultivated and mixed with catfish serum and neutrophils in 96-well plates, and bioluminescence was used to detect mutants that are more susceptible to killing by these host factors. The virulence and vaccine efficacy of selected mutants were determined in channel catfish. Transposon insertion sites in 13 mutants attenuated in the natural host were mapped to theE. ictalurigenome. Ten unique genes were mutated, including genes encoding a negative regulator of sigmaE activity, a glycine cleavage system protein, tricarboxylic acid cycle enzymes, an O polysaccharide biosynthesis enzyme, proteins encoded on the native plasmid pEI1, and a fimbrial chaperon protein. Three of these mutants were found to have potential as live attenuated vaccines. This study demonstrates a novel application of bioluminescence to identify bacterial genes required for host resistance; as a result, efficacious and genetically defined live attenuated vaccine candidates were developed.
A Vaccine against Rumen Methanogens Can Alter the Composition of Archaeal Populations
Authors: Williams Yvette J. , Popovski Sam , Rea Suzanne M. , Skillman Lucy C. , Toovey Andrew F. , Northwood Korinne S. , Wright André-Denis G.
Published: February 2009

The objectives of this study were to formulate a vaccine based upon the different species/strains of methanogens present in sheep intended to be immunized and to determine if a targeted vaccine could be used to decrease the methane output of the sheep. Two 16S rRNA gene libraries were used to survey the methanogenic archaea in sheep prior to vaccination, and methanogens representing five phylotypes were found to account for >52% of the different species/strains of methanogens detected. A vaccine based on a mixture of these five methanogens was then formulated, and 32 sheep were vaccinated on days 0, 28, and 103 with either a control or the anti-methanogen vaccine. Enzyme-linked immunosorbent assay analysis revealed that each vaccination with the anti-methanogen formulation resulted in higher specific immunoglobulin G titers in plasma, saliva, and rumen fluid. Methane output levels corrected for dry-matter intake for the control and treatment groups were not significantly different, and real-time PCR data also indicated that methanogen numbers were not significantly different for the two groups after the second vaccination. However, clone library data indicated that methanogen diversity was significantly greater in sheep receiving the anti-methanogen vaccine and that the vaccine may have altered the composition of the methanogen population. A correlation between 16S rRNA gene sequence relatedness and cross-reactivity for the methanogens (
R
2
= 0.90) also exists, which suggests that a highly specific vaccine can be made to target specific strains of methanogens and that a more broad-spectrum approach is needed for success in the rumen. Our data also suggest that methanogens take longer than 4 weeks to adapt to dietary changes and call into question the validity of experimental results based upon a 2- to 4-week acclimatization period normally observed for bacteria.
Ser67Asp and His68Asp Substitutions in
Candida parapsilosis
Carbonyl Reductase Alter the Coenzyme Specificity and Enantioselectivity of Ketone Reduction
Authors: Zhang Rongzhen , Xu Yan , Sun Ying , Zhang Wenchi , Xiao Rong
Published: February 2009

A short-chain carbonyl reductase (SCR) from
Candida parapsilosis
catalyzes an anti-Prelog reduction of 2-hydroxyacetophenone to (
S
)-1-phenyl-1,2-ethanediol (PED) and exhibits coenzyme specificity for NADPH over NADH. By using site-directed mutagenesis, the mutants were designed with different combinations of Ser67Asp, His68Asp, and Pro69Asp substitutions inside or adjacent to the coenzyme binding pocket. All mutations caused a significant shift of enantioselectivity toward the (
R
)-configuration during 2-hydroxyacetophenone reduction. The S67D/H68D mutant produced (
R
)-PED with high optical purity and yield in the NADH-linked reaction. By kinetic analysis, the S67D/H68D mutant resulted in a nearly 10-fold increase and a 20-fold decrease in the
k
cat
/

K
m

value when NADH and NADPH were used as the cofactors, respectively, but maintaining a
k
cat
value essentially the same with respect to wild-type SCR. The ratio of

K
d

(dissociation constant) values between NADH and NADPH for the S67D/H68D mutant and SCR were 0.28 and 1.9 respectively, which indicates that the S67D/H68D mutant has a stronger preference for NADH and weaker binding for NADPH. Moreover, the S67D/H68D enzyme exhibited a secondary structure and melting temperature similar to the wild-type form. It was also found that NADH provided maximal protection against thermal and urea denaturation for S67D/H68D, in contrast to the effective protection by NADP(H) for the wild-type enzyme. Thus, the double point mutation S67D/H68D successfully converted the coenzyme specificity of SCR from NADP(H) to NAD(H) as well as the product enantioselectivity without disturbing enzyme stability. This work provides a protein engineering approach to modify the coenzyme specificity and enantioselectivity of ketone reduction for short-chain reductases.
In Situ Hydrogen and Nitrous Oxide as Indicators of Concomitant Fermentation and Denitrification in the Alimentary Canal of the EarthwormLumbricus terrestris
Authors: Wüst Pia K. , Horn Marcus A. , Drake Harold L.
Published: February 2009

The earthworm gut is a unique microzone in aerated soils that has been proposed to selectively stimulate ingested soil microorganisms by its in situ conditions, which include anoxia, high water content, a near-neutral pH, and high concentrations of organic compounds. The central objective of this study was to resolve potential links between in situ conditions and anaerobic microbial activities during the gut passage ofLumbricus terrestris. Both H2and N2O were emitted by living earthworms, and in situ microsensor analyses revealed both H2and N2O in the O2-free gut center. The highest H2concentrations occurred in foregut and midgut regions, whereas the highest N2O concentrations occurred in crop/gizzard and hindgut regions. Thus, H2-producing fermentations were more localized in the foregut and midgut, whereas denitrification was more localized in the crop/gizzard and hindgut. Moisture content, total carbon, and total nitrogen were highest in the foregut and decreased from the anterior to posterior end of the gut. Nitrite, ammonium, and iron(II) concentrations were highest in the crop/gizzard and decreased from the anterior to posterior end of the alimentary canal. Concentrations of soluble organic compounds were indicative of distinct fermentation processes along the alimentary canal, with maximal concentrations of organic acids (e.g., acetate and butyrate) occurring in the midgut. These findings suggest that earthworms (i) contribute to the terrestrial cycling of carbon and nitrogen via anaerobic microbial activities in the alimentary canal and (ii) constitute a mobile source of reductant (i.e., emitted H2) for microbiota in aerated soils.
Pea Aphid as both Host and Vector for the Phytopathogenic Bacterium
Pseudomonas syringae
Authors: Stavrinides John , McCloskey Jodi K. , Ochman Howard
Published: February 2009

Aphids are widespread agricultural pests that are capable of disseminating plant viral diseases; however, despite coming into frequent contact with epiphytic bacteria, aphids are considered to have no role in bacterial transmission. Here, we demonstrate the ability of pea aphids to vector the phytopathogen
Pseudomonas syringae
pv. syringae B728a (PsyB728a). While feeding on plants colonized by epiphytic bacteria, aphids acquire the bacteria, which colonize the digestive tract, multiply, and are excreted in the aphid honeydew, resulting in inoculation of the phyllosphere with up to 10
7
phytopathogenic bacteria per cm
2
. Within days of ingesting bacteria, aphids succumb to bacterial sepsis, indicating that aphids serve as an alternative, nonplant host for PsyB728a. The related strain
Pseudomonas syringae
pv. tomato DC3000 is >1,000-fold less virulent than PsyB728a in the pea aphid, suggesting that PsyB728a possesses strain-specific pathogenicity factors that allow it to exploit aphids as hosts. To identify these factors, we performed a mutagenesis screen and recovered PsyB728a mutants that were hypovirulent, including one defective in a gene required for flagellum formation and motility. These interactions illustrate that aphids can also vector bacterial pathogens and that even seemingly host-restricted pathogens can have alternative host specificities and lifestyles.
Differences in Hydrogenase Gene Expression between
Methanosarcina acetivorans
and
Methanosarcina barkeri
Authors: Guss Adam M. , Kulkarni Gargi , Metcalf William W.
Published: February 2009

Methanosarcina acetivorans
C2A encodes three putative hydrogenases, including one cofactor F
420
-linked (
frh
) and two methanophenazine-linked (
vht
) enzymes. Comparison of the amino acid sequences of these putative hydrogenases to those of
Methanosarcina barkeri
and
Methanosarcina mazei
shows that each predicted subunit contains all the known residues essential for hydrogenase function. The DNA sequences upstream of the genes in
M. acetivorans
were aligned with those in other
Methanosarcina
species to identify conserved transcription and translation signals. The
M. acetivorans vht
promoter region is well conserved among the sequenced
Methanosarcina
species, while the second
vht
-type homolog (here called
vhx
) and
frh
promoters have only limited similarity. To experimentally determine whether these promoters are functional in vivo, we constructed and characterized both
M. acetivorans
and
M. barkeri
strains carrying reporter gene fusions to each of the
M. acetivorans
and
M. barkeri
hydrogenase promoters. Generally, the
M. acetivorans
gene fusions are not expressed in either organism, suggesting that
cis
-acting mutations inactivated the
M. acetivorans
promoters. The
M. barkeri
hydrogenase gene fusions, on the other hand, are expressed in both organisms, indicating that
M. acetivorans
possesses the machinery to express hydrogenases, although it does not express its own hydrogenases. These data are consistent with specific inactivation of the
M. acetivorans
hydrogenase promoters and highlight the importance of testing hypotheses generated by using genomic data.
A Widely Conserved Gene Cluster Required for Lactate Utilization inBacillus subtilisand Its Involvement in Biofilm Formation
Authors: Chai Yunrong , Kolter Roberto , Losick Richard
Published: February 2009

We report that catabolism ofl-lactate inBacillus subtilisdepends on the previously uncharacterizedyvfV-yvfW-yvbY(herein renamedlutABC) operon, which is inferred to encode three iron-sulfur-containing proteins. The operon is under the dual control of a GntR-type repressor (LutR, formerly YvfI) and the master regulator for biofilm formation SinR and is induced during growth in response tol-lactate. Operons with high similarity tolutABCare present in the genomes of a variety of gram-positive and gram-negative bacteria, raising the possibility that LutABC is a widely conserved and previously unrecognized pathway for the utilization ofl-lactate or related metabolites.
Rrp2, a σ
54
-Dependent Transcriptional Activator of
Borrelia burgdorferi
, Activates
rpoS
in an Enhancer-Independent Manner
Authors: Blevins Jon S. , Xu Haijun , He Ming , Norgard Michael V. , Reitzer Larry , Yang X. Frank
Published: February 2009

Rrp2 is the sole σ
54
-dependent transcriptional activator present in the
Borrelia burgdorferi
genome. We showed that recombinant Rrp2 binds to DNA in a sequence-nonspecific manner. During infection, Rrp2 activates σ
54
-dependent
rpoS
expression without an apparent upstream enhancer element commonly associated with other σ
54
-dependent transcriptional activators.
A Conserved α-Helix Essential for a Type VI Secretion-Like System of
Francisella tularensis
Authors: Bröms Jeanette E. , Lavander Moa , Sjöstedt Anders
Published: February 2009

Francisella tularensis
harbors genes with similarity to genes encoding components of a type VI secretion system (T6SS) recently identified in several gram-negative bacteria. These genes include
iglA
and
iglB
encoding IglA and IglB, homologues of which are conserved in most T6SSs. We used a yeast two-hybrid system to study the interaction of the Igl proteins of
F. tularensis
LVS. We identified a region of IglA, encompassing residues 33 to 132, necessary for efficient binding to IglB, as well as for IglAB protein stability and intramacrophage growth. In particular, residues 103 to 122, overlapping a highly conserved α-helix, played an absolutely essential role. Point mutations within this domain caused modest defects in IglA-IglB binding in the yeast
Saccharomyces cerevisiae
but markedly impaired intramacrophage replication and phagosomal escape, resulting in severe attenuation of LVS in mice. Thus, IglA-IglB complex formation is clearly crucial for
Francisella
pathogenicity. This interaction may be universal to type VI secretion, since IglAB homologues of
Yersinia pseudotuberculosis, Pseudomonas aeruginosa, Vibrio cholerae, Salmonella enterica
serovar Typhimurium, and
Escherichia coli
were also shown to interact in yeast, and the interaction was dependent on preservation of the same α-helix. Heterologous interactions between nonnative IglAB proteins further supported the notion of a conserved binding site. Thus, IglA-IglB complex formation is clearly crucial for
Francisella
pathogenicity, and the same interaction is conserved in other human pathogens.
HtaA Is an Iron-Regulated Hemin Binding Protein Involved in the Utilization of Heme Iron in
Corynebacterium diphtheriae
Authors: Allen Courtni E. , Schmitt Michael P.
Published: February 2009

Many human pathogens, including
Corynebacterium diphtheriae
, the causative agent of diphtheria, use host compounds such as heme and hemoglobin as essential iron sources. In this study, we examined the
Corynebacterium hmu
hemin transport region, a genetic cluster that contains the
hmuTUV
genes encoding a previously described ABC-type hemin transporter and three additional genes, which we have designated
htaA
,
htaB
, and
htaC
. The
hmu
gene cluster is composed of three distinct transcriptional units. The
htaA
gene appears to be part of an iron- and DtxR-regulated operon that includes
hmuTUV
, while
htaB
and
htaC
are transcribed from unique DtxR-regulated promoters. Nonpolar deletion of either
htaA
or the
hmuTUV
genes resulted in a reduced ability to use hemin as an iron source, while deletion of
htaB
had no effect on hemin iron utilization in
C. diphtheriae
. A comparison of the predicted amino acid sequences of HtaA and HtaB showed that they share some sequence similarity, and both proteins contain leader sequences and putative C-terminal transmembrane regions. Protein localization studies with
C. diphtheriae
showed that HtaA is associated predominantly with the cell envelope when the organism is grown in minimal medium but is secreted during growth in nutrient-rich broth. HtaB and HmuT were detected primarily in the cytoplasmic membrane fraction regardless of the growth medium. Hemin binding studies demonstrated that HtaA and HtaB are able to bind hemin, suggesting that these proteins may function as cell surface hemin receptors in
C. diphtheriae
.
Self-paced exercise is less physically challenging than enforced constant pace exercise of the same intensity: influence of complex central metabolic control
Authors: Lander P J , Butterly R J , Edwards A M
Published: February 2009

Objective:

To examine whether self-pacing reduces the physiological challenge of performing 5000 m rowing ergometry exercise in comparison with a matched-intensity exercise condition in which a constant effort pacing strategy is enforced.

Methods:

Nine healthy well-trained male participants volunteered to participate in three 5000 m rowing conditions (two submaximal and one maximal conditions) in an individualised order. In the submaximal conditions, participants were required to (1) perform 5000 m at a constant rating of perceived exertion (RPE 15-Hard) (SubRPE) or (2) perform 5000 m at an enforced constant pace equivalent to the mean power output (PO) of the SubRPE condition (SubEXT). A maximal condition (MaxTT) was included to disguise the purpose of the study and to facilitate an element of randomisation in the test sequence. Dynamic intratest responses were assessed every 30s: PO, Vo2, iEMG, core (Tc) and skin temperatures (Tsk).

Results:

There was no difference between performance times of the two submaximal trials. The mean PO represented 83.83 (SD 8.88)% (SubRPE) and 83.40 (8.84)% (SubEXT) of the mean MaxTT power output. Tc(SubRPE:38.46 (0.23)°C, SubEXT:38.72 (0.36)°C; p<0.01), post-test BLa (SubRPE:5.24 (2.18), SubEXT:6.19 (2.51) mmol/l; p<0.05) and iEMG (p<0.05) were significantly elevated in SubEXT compared with SubRPE. There were no differences in the dynamics of HR or Vo2between SubEXT and SubRPE. The intratest stroke-to-stroke variability of power output was significantly greater in the SubRPE condition compared with SubEXT (p<0.01).

Conclusions:

Enforced constant paced exercise presents a significantly greater physiological challenge than self-paced exercise. The ability to dynamically self-pace effort via manipulations of power output during exercise is an important behavioural response to homeostatic challenges and thus forms an integral part of a complex central regulatory process.
Overweight, obesity and risk of work disability: a cohort study of construction workers in Germany
Authors: Claessen H , Arndt V , Drath C , Brenner H
Published: February 2009

Objectives:

Although obesity and permanent work disability impose a great burden on the individual and are very costly for society, data on the impact of being overweight on occupational disability are sparse, especially in men who work hard physically. The aim of this study was to investigate the association of body mass index (BMI) with work disability among construction workers.

Methods:

The association between BMI and work disability was examined during a mean follow-up period of 10.8 years in a cohort of 16 875 male construction workers in Württemberg, Germany, who participated in routine occupational health examinations from 1986 to 1992. Hazard ratios were calculated with normal weight (20.0–22.4 kg/m2) as reference using the Cox proportional hazards model, after adjustment for potential confounding factors.

Results:

Overall, a U-shaped association of BMI with all-cause work disability (total number = 3064 cases) was observed, with the lowest risk of disabilities at BMI levels between 25 and 27.4 kg/m2. Strong positive associations were observed between BMI and work disability due to osteoarthritis or cardiovascular diseases, whereas BMI was inversely related to work disability due to cancer, even after exclusion of the first 3 years of follow-up.

Conclusions:

Moderate overweight is not associated with increased risk of work disability among construction workers, but obesity increases the risk of work disability due to osteoarthritis and cardiovascular disease.
Unstable angina following anaphylaxis
Authors: Ameratunga R , Webster M , Patel H
Published: February 2009

A 55-year-old woman developed unstable angina following an episode of severe anaphylaxis which was treated with 0.5 mg intramuscular epinephrine (adrenaline). The exact cause of her ongoing unstable angina was uncertain but may have reflected either vasospasm superimposed upon an area of atherosclerotic coronary disease, or vasospasm induced plaque rupture. Four weeks later, she had a second episode of anaphylaxis, and suffered a cardiac arrest after receiving a bolus of intravenous epinephrine. This case illustrates the importance of careful assessment of patients after anaphylaxis, both to prevent recurrence and to treat complications from the episode. It highlights the dangers of intravenous epinephrine in treating anaphylaxis outside anaesthetic and intensively monitored settings.
The Chemical Biology of Protein Phosphorylation
Authors: Tarrant Mary Katherine , Cole Philip A.
Published: February 2009

The explosion of scientific interest in protein kinase-mediated signaling networks has led to the infusion of new chemical methods and their applications related to the analysis of phosphorylation pathways. We highlight some of these chemical biology approaches across three areas. First, we discuss the development of chemical tools to modulate the activity of protein kinases to explore kinase mechanisms and their contributions to phosphorylation events and cellular processes. Second, we describe chemical techniques developed in the past few years to dissect the structural and functional effects of phosphate modifications at specific sites in proteins. Third, we cover newly developed molecular imaging approaches to elucidate the spatiotemporal aspects of phosphorylation cascades in live cells. Exciting advances in our understanding of protein phosphorylation have been obtained with these chemical biology approaches, but continuing opportunities for technological innovation remain.
Tumor necrosis factor-alpha impairs intestinal phosphate absorption in colitis
Authors: Chen Huacong , Xu Hua , Dong Jiali , Li Jing , Ghishan Fayez K.
Published: February 2009

Phosphate homeostasis is critical for many physiological functions. Up to 85% of phosphate is stored in bone and teeth. The remaining 15% is distributed in cells. Phosphate absorption across the brush-border membrane (BBM) of enterocytes occurs mainly via a sodium-dependent pathway, which is mediated by type IIb sodium-phosphate cotransporters (NaPi-IIb). Patients of inflammatory bowel diseases (IBDs) suffer not only from diarrhea and nutrient malabsorption but also from bone loss. About 31–59% of patients with IBD develop bone disorders. Since the intestine is a primary location for dietary phosphate absorption, it is logical to postulate that there is an inverse relationship between gastrointestinal disorders and phosphate transport, which, in turn, contributes to bone disorders observed in patients with IBD. Phosphate absorption and NaPi-IIb expression was studied with BBM vesicles isolated from trinitrobenzene sulphonic acid (TNBS) animals as well as in Caco-2 cells. The mechanism of TNF-α downregulation of NaPi-IIb expression was investigated by luciferase assay, gel mobility shift assay (GMSA), and coimmunoprecipitation. Intestinal phosphate absorption mediated by NaPi-IIb was reduced both in TNBS colitis and in TNF-α-treated cells. Transient transfection indicated that TNF-α inhibits NaPi-IIb expression by reducing NaPi-IIb basal promoter activity. GMSAs identified NF1 protein as an important factor in TNF-α-mediated NaPi-IIb downregulation. Signaling transduction study and coimmunoprecipitation suggested that TNF-α interacts with EGF receptor to activate ERK1/2 pathway. Intestinal phosphate absorption mediated by NaPi-IIb protein is reduced in colitis. This inhibition is mediated by the proinflammatory cytokine TNF-α through a novel molecular mechanism involving TNF-α/EGF receptor interaction.
Differential involvement of COX1 and COX2 in the vasculopathy associated with the α-galactosidase A-knockout mouse
Authors: Park James L. , Shu Liming , Shayman James A.
Published: February 2009

The lysosomal storage disorder Fabry disease is characterized by excessive globotriaosylceramide (Gb3) accumulation in major organs such as the heart and kidney. Defective lysosomal α-galactosidase A (Gla) is responsible for excessive Gb3 accumulation, and one cell sensitive to the effects of Gb3 accumulation is vascular endothelium. Endothelial dysfunction is associated with Fabry disease and excessive cellular Gb3. We previously demonstrated that excessive vascular Gb3 in a mouse model of Fabry disease, the Gla-knockout ( Gla−/0) mouse, results in abnormal vascular function, which includes abnormal endothelium-dependent contractions, a vascular phenomenon known to involve cyclooxygenase (COX). Therefore, we hypothesized that the vasculopathy in the Gla knockout mouse may be due to a vasoactive COX-derived product. To test this hypothesis, vascular reactivity experiments were performed in aortic rings from wild-type ( Gla+/0) and Gla−/0 mice in the presence and absence of specific and nonspecific COX inhibitors. Specific inhibition of COX1 or COX2 in endothelium-intact rings from Gla−/0 mice decreased overall phenylephrine contractility compared with untreated Gla−/0 rings, whereas COX inhibitors had no effect on contractility in endothelium-denuded rings. Nonspecific inhibition of COX with indomethacin (10 μmol/l) or COX1 inhibition with valeryl salicylate (3 mmol/l) improved endothelial function in rings from Gla−/0 mice, but COX2 inhibition with NS-398 (1 μmol/l) further increased endothelial dysfunction in rings from Gla−/0 mice. These results suggest that, in the Gla−/0 mice, COX1 and COX2 activity are increased and localized in the endothelium, producing vasopressor and vasorelaxant products, which contribute to the Fabry-related vasculopathy.
Calcium influx-dependent differential actions of superoxide and hydrogen peroxide on microvessel permeability
Authors: Zhou Xueping , Wen Ke , Yuan Dong , Ai Ling , He Pingnian
Published: February 2009

Our previous study demonstrated that reactive oxygen species (ROS) released from activated blood cells contribute significantly to the increased microvessel permeability during inflammation. This study aims to define the individual roles of hydrogen peroxide (H2O2) and superoxide in ROS-induced increases in permeability and endothelial intracellular Ca2+concentration ([Ca2+]i) in individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity ( Lp). Endothelial [Ca2+]iwas measured in fura-2 AM-loaded microvessels. Perfusing microvessels with superoxide generated by hypoxanthine and xanthine oxidase (HX/XO) induced immediate and transient increases in Lp. The mean peak value, which occurred within 5 min of HX/XO exposure, was 4.3 ± 0.6 times that of the control. In contrast, the perfusion of H2O2(100 and 500 μM) caused no immediate increases in Lp. A significant Lpincrease, 3.6 ± 0.6 times the control value, occurred 30 min after the perfusion of H2O2at 500 μM. The perfusion of H2O2at 100 or 500 μM for 1 h increased Lpto 6.6 ± 0.9 and 11.3 ± 3.6 times the control value, respectively. The increased endothelial [Ca2+]iin HX/XO or H2O2perfused vessels was correlated with the time course of the increases in Lp. Inhibiting Ca2+influx by LaCl3prevented the permeability increase induced by HX/XO or H2O2. These results demonstrated differential actions of superoxide and H2O2on microvessel permeability and endothelial [Ca2+]i. Superoxide-induced permeability increases were immediate and transient, whereas H2O2-induced permeability increases were progressive, demonstrating concentration and time dependence. Ca2+influx plays an essential role in both superoxide and H2O2-induced permeability increases.
Mechanical work and energetic analysis of eccentric cardiac remodeling in a volume overload heart failure in rats
Authors: Takewa Yoshiaki , Chemaly Elie R. , Takaki Miyako , Liang Li Fan , Jin Hongwei , Karakikes Ioannis , Morel Charlotte , Taenaka Yoshiyuki , Tatsumi Eisuke , Hajjar Roger J.
Published: February 2009

Eccentric cardiac remodeling seen in dilated cardiomyopathy or regurgitant valvular disease is a well-known process of heart failure progression, but its mechanoenergetic profile has not been yet established. We made a volume overload (VO) heart failure model in rats and for the first time investigated left ventricular (LV) mechanical work and energetics in cross-circulated whole heart preparations. Laparotomy was performed in 14 Wistar male rats, and abdominal aortic-inferior vena caval shunt was created in seven rats (VO group). Another seven rats underwent a sham operation without functional shunt (Sham group). LV dimensions changes were followed with weekly transthoracic echocardiography. Three months after surgery, we measured LV pressure and volume and myocardial O2 consumption in isolated heart cross circulation. LV internal dimensions in both systolic and diastolic phases were significantly increased in the VO group versus the Sham group ( P < 0.05). LV pressure was markedly decreased in the VO group versus in the Sham group ( P < 0.05). LV end-systolic pressure-volume relation shifted downward, and myocardial O2 consumption related to Ca2+ handling significantly decreased. The contractile response to Ca2+ infusion was attenuated. Nevertheless, the increase in Ca2+ handling-related O2 consumption per unit change in LV contractility in the VO group was significantly higher than that in the Sham group ( P < 0.05). The levels of sarco(endo)plasmic reticulum Ca2+-ATPase 2a protein were reduced in the VO group ( P < 0.01). In conclusion, VO failing rat hearts had a character of marked contractile dysfunction accompanied with less efficient energy utilization in the Ca2+ handling processes. These results suggest that restoring Ca2+ handling in excitation-contraction coupling would improve the contractility of the myocardium after eccentric cardiac remodeling.
Phenotypical transitions and Ca2+activation properties in human muscle fibers: effects of a 60-day bed rest and countermeasures
Authors: Mounier Yvonne , Tiffreau Vincent , Montel Valérie , Bastide Bruno , Stevens Laurence
Published: February 2009

Muscle biopsies were taken from soleus and vastus lateralis before and after a 60-day bed rest (BR) to examine expression changes in the regulatory proteins of the thin filament and in contractile function. Twenty-four women separated in three groups were submitted to BR or a combined protocol of resistance and aerobic exercises during BR or received a supplementation of amino acids during BR. Ca2+-tension relationships were established in single skinned fibers identified by their myosin heavy chain and troponin C isoform expressions. Expression patterns of regulatory proteins were analyzed on muscle pieces. For both muscles, BR produced similar decreases in slow and fast fiber diameters but larger decreases in P0maximal forces in slow than in fast fibers. Specific forces were decreased in slow soleus and vastus fibers, which displayed a reduction in Ca2+affinity. These changes were accompanied by slow-to-fast transitions in regulatory proteins, with troponins C and T appearing as sensitive markers of unloading. Exercises prevented the changes in fiber diameters and forces and counteracted most of the slow-to-fast transitions. The nutrition program had a morphological beneficial effect on slow fibers. However, these fibers still presented decreases in specific P0after BR. Phenotypical transitions due to BR were not prevented by amino acids. Finally, in vastus lateralis muscle, BR induced a decrease in O-glycosylation level that was prevented by exercise and attenuated by nutrition. In conclusion, this study has addressed for the first time in women the respective efficiencies of two countermeasures associated with BR on muscle properties and regulatory protein expression.
The effect of remifentanil on respiratory variability, evaluated with dynamic modeling
Authors: Mitsis G. D. , Governo R. J. M. , Rogers R. , Pattinson K. T. S.
Published: February 2009

Opioid drugs disrupt signaling in the brain stem respiratory network affecting respiratory rhythm. We evaluated the influence of a steady-state infusion of a model opioid, remifentanil, on respiratory variability during spontaneous respiration in a group of 11 healthy human volunteers. We used dynamic linear and nonlinear models to examine the effects of remifentanil on both directions of the ventilatory loop, i.e., on the influence of natural variations in end-tidal carbon dioxide (PetCO2) on ventilatory variability, which was assessed by tidal volume (Vt) and breath-to-breath ventilation (i.e., the ratio of tidal volume over total breath time Vt/Ttot), and vice versa. Breath-by-breath recordings of expired CO2and respiration were made during a target-controlled infusion of remifentanil for 15 min at estimated effect site (i.e., brain tissue) concentrations of 0, 0.7, 1.1, and 1.5 ng/ml, respectively. Remifentanil caused a profound increase in the duration of expiration. The obtained models revealed a decrease in the strength of the dynamic effect of PetCO2variability on Vt (the “controller” part of the ventilatory loop) and a more pronounced increase in the effect of Vt variability on PetCO2(the “plant” part of the loop). Nonlinear models explained these dynamic interrelationships better than linear models. Our approach allows detailed investigation of drug effects in the resting state at the systems level using noninvasive and minimally perturbing experimental protocols, which can closely represent real-life clinical situations.
Strength training combined with plyometric jumps in adults: sex differences in fat-bone axis adaptations
Authors: Guadalupe-Grau A. , Perez-Gomez J. , Olmedillas H. , Chavarren J. , Dorado C. , Santana A. , Serrano-Sanchez J. A. , Calbet J. A. L.
Published: February 2009

Leptin and osteocalcin play a role in the regulation of the fat-bone axis and may be altered by exercise. To determine whether osteocalcin reduces fat mass in humans fed ad libitum and if there is a sex dimorphism in the serum osteocalcin and leptin responses to strength training, we studied 43 male (age 23.9 2.4 yr, mean ± SD) and 23 female physical education students (age 23.2 ± 2.7 yr). Subjects were randomly assigned to two groups: training (TG) and control (CG). TG followed a strength combined with plyometric jumps training program during 9 wk, whereas the CG did not train. Physical fitness, body composition (dual-energy X-ray absorptiometry), and serum concentrations of hormones were determined pre- and posttraining. In the whole group of subjects (pretraining), the serum concentration of osteocalcin was positively correlated ( r = 0.29–0.42, P < 0.05) with whole body and regional bone mineral content, lean mass, dynamic strength, and serum-free testosterone concentration ( r = 0.32). However, osteocalcin was negatively correlated with leptin concentration ( r = −0.37), fat mass ( r = −0.31), and the percent body fat ( r = −0.44). Both sexes experienced similar relative improvements in performance, lean mass (+4–5%), and whole body (+0.78%) and lumbar spine bone mineral content (+1.2–2%) with training. Serum osteocalcin concentration was increased after training by 45 and 27% in men and women, respectively ( P < 0.05). Fat mass was not altered by training. Vastus lateralis type II MHC composition at the start of the training program predicted 25% of the osteocalcin increase after training. Serum leptin concentration was reduced with training in women. In summary, while the relative effects of strength training plus plyometric jumps in performance, muscle hypertrophy, and osteogenesis are similar in men and women, serum leptin concentration is reduced only in women. The osteocalcin response to strength training is, in part, modulated by the muscle phenotype (MHC isoform composition). Despite the increase in osteocalcin, fat mass was not reduced.
Arginase I–Producing Myeloid-Derived Suppressor Cells in Renal Cell Carcinoma Are a Subpopulation of Activated Granulocytes
Authors: Rodriguez Paulo C. , Ernstoff Marc S. , Hernandez Claudia , Atkins Michael , Zabaleta Jovanny , Sierra Rosa , Ochoa Augusto C.
Published: February 2009

Myeloid-derived suppressor cells (MDSC) producing arginase I are increased in the peripheral blood of patients with renal cell carcinoma (RCC). MDSC inhibit T-cell function by reducing the availability of l-arginine and are therefore considered an important tumor escape mechanism. We aimed to determine the origin of arginase I–producing MDSC in RCC patients and to identify the mechanisms used to deplete extracellular l-arginine. The results show that human MDSC are a subpopulation of activated polymorphonuclear (PMN) cells expressing high levels of CD66b, CD11b, and VEGFR1 and low levels of CD62L and CD16. In contrast to murine MDSC, human MDSC do not deplete l-arginine by increasing its uptake but instead release arginase I into the circulation. Activation of normal PMN induces phenotypic and functional changes similar to MDSC and also promotes the release of arginase I from intracellular granules. Interestingly, although activation of normal PMN usually ends with apoptosis, MDSC showed no increase in apoptosis compared with autologous PMN or PMN obtained from normal controls. High levels of VEGF have been shown to increase suppressor immature myeloid dendritic cells in cancer patients. Treatment of RCC patients with anti-VEGF antibody bevacizumab, however, did not reduce the accumulation of MDSC in peripheral blood. In contrast, the addition of interleukin-2 to the treatment increased the number of MDSC in peripheral blood and the plasma levels of arginase I. These results may provide new insights on the mechanisms of tumor-induced anergy/tolerance and may help explain why some immunotherapies fail to induce an antitumor response. [Cancer Res 2009;69(4):1553–60]
Effect of Long-Term Homocysteine Reduction with B Vitamins on Arterial Wall Inflammation Assessed by Fluorodeoxyglucose Positron Emission Tomography: A Randomised Double-Blind, Placebo-Controlled Trial
Authors: Potter Kathleen , Lenzo Nat , Eikelboom John W. , Arnolda Leonard F. , Beer Christopher , Hankey Graeme J.
Published: February 2009

Background: Homocysteine may promote atherosclerosis by exacerbating inflammatory processes within the arterial wall. B-vitamin supplements reduce total plasma homocysteine concentrations (tHcy), but it is not known whether the treatment also reduces arterial wall inflammation. We used 18F-fluorodeoxygluose positron emission tomography (18F-FDG PET) to investigate whether long-term homocysteine-lowering treatment alters arterial wall inflammation in patients with a history of ischemic stroke. Methods: 30 stroke patients were randomly assigned to B-vitamin therapy (folic acid 2 mg, vitamin B6 25 mg and vitamin B12 0.5 mg) or placebo in a double-blind clinical trial. After a mean treatment period of 4.0 ± 0.7 years, all subjects had tHcy, carotid intima-medial thickness (CIMT) and flow-mediated dilation (FMD) of the brachial artery measured and underwent an 18F-FDG PET scan. Standardised uptake values (SUV) were measured at six sites in the carotid, femoral and aortic arteries. Areas of locally increased tracer uptake in the arterial wall (‘hot spots’) were also identified and counted. Results: Long-term B-vitamin treatment significantly reduced tHcy compared with placebo (8.4 μmol/l, 95% confidence interval, CI, 7.2–9.6 vs. 11.6 μmol/l, 95% CI 10.0–13.4, p = 0.002). The treatment did not affect mean arterial SUV (2.0 ± 0.3 vitamins vs. 2.1 ± 0.3 placebo, p = 0.65) or the number of hot spots (n = 1.1 ± 1.0 vitamins vs. n = 1.2 ± 1.0 placebo, p = 0.65). There was no significant correlation between mean arterial SUV and CIMT or FMD. Conclusions: These results suggest that a long-term Hcy reduction with B vitamins does not affect arterial wall inflammation assessed by 18F-FDG PET.
The Relationship between Hematoma Iron Content and Perihematoma Edema: An MRI Study
Authors: Lou Min , Lieb Kathrin , Selim Magdy
Published: February 2009

Background: Iron neurotoxicity has been linked to delayed neuronal injury and edema formation after intracerebral hemorrhage (ICH). We have previously shown that serum ferritin, an indicator of body iron load, correlates with the relative perihematoma edema volume (RPHEV) on days 3–4 after ICH. We undertook this study to directly examine the relationship between in vivo brain and hematoma iron content, measured by MRI, and RPHEV. Methods: We retrospectively reviewed prospectively collected clinical and laboratory data from 36 consecutive patients with acute spontaneous lobar ICH who had MRI performed within 2–4 days of ICH onset. We measured hematoma and edema volumes, and the signal intensity on T2-weighted images (T2SI), as an estimate of iron content, in the hematoma and contralateral globus pallidus (GP). We calculated the RPHEV and T2SI in the hematoma and GP, relative to T2SI in the frontal deep white matter which contains negligible iron, to estimate the hematoma and brain iron load. We used Spearman correlation coefficient to determine the association of relative T2SI of the hematoma and GP with RPHEV. Results: We found a significant inverse correlation between the relative T2SI in the hematoma (r = –0.75, p < 0.001) and to a lesser extent in the GP (r = –0.34, p = 0.04) and the RPHEV. Conclusions: Our findings suggest that in vivo brain and hematoma iron content, as measured by MRI, is linked to perihematoma edema after ICH, and provide further support to existing preclinical evidence linking iron-mediated toxicity to delayed neuronal injury after ICH.