bluuebunny/crossref_metadata_2025 huggingface dataset:
Design and Application of Highly Responsive Fluorescence Resonance Energy Transfer Biosensors for Detection of Sugar in LivingSaccharomyces cerevisiaeCells
Authors: Ha Jae-Seok , Song Jae Jun , Lee Young-Mi , Kim Su-Jin , Sohn Jung-Hoon , Shin Chul-Soo , Lee Seung-Goo
Published: September 2007

A protein sensor with a highly responsive fluorescence resonance energy transfer (FRET) signal for sensing sugars in livingSaccharomyces cerevisiaecells was developed by combinatorial engineering of the domain linker and the binding protein moiety. Although FRET sensors based on microbial binding proteins have previously been created for visualizing various sugars in vivo, such sensors are limited due to a weak signal intensity and a narrow dynamic range. In the present study, the length and composition of the linker moiety of a FRET-based sensor consisting of CFP-linker1-maltose-binding protein-linker2-YFP were redesigned, which resulted in a 10-fold-higher signal intensity. Molecular modeling of the composite linker moieties, including the connecting peptide and terminal regions of the flanking proteins, suggested that an ordered helical structure was preferable for tighter coupling of the conformational change of the binding proteins to the FRET response. When the binding site residue Trp62 of the maltose-binding protein was diversified by saturation mutagenesis, the Leu mutant exhibited an increased binding constant (82 μM) accompanied by further improvement in the signal intensity. Finally, the maltose sensor with optimized linkers was redesigned to create a sugar sensor with a new specificity and a wide dynamic range. When the optimized maltose sensors were employed as in vivo sensors, highly responsive FRET images were generated from real-time analysis of maltose uptake ofSaccharomyces cerevisiae(baker's yeast).
Mucosa-Associated Bacterial Diversity in Relation to Human Terminal Ileum and Colonic Biopsy Samples
Authors: Ahmed Shakil , Macfarlane George T. , Fite Alemu , McBain Andrew J. , Gilbert Peter , Macfarlane Sandra
Published: September 2007

Little is known about bacterial communities that colonize mucosal surfaces in the human gastrointestinal tract, but they are believed to play an important role in host physiology. The objectives of this study were to investigate the compositions of these populations in the distal small bowel and colon. Healthy mucosal tissue from either the terminal ileum (
n
= 6) or ascending (
n
= 8), transverse (
n
= 8), or descending colon (
n
= 4) of 26 patients (age, 68.5 ± 1.2 years [mean ± standard deviation]) undergoing emergency resection of the large bowel was used to study these communities. Mucosa-associated eubacteria were characterized by using PCR-denaturing gradient gel electrophoresis (DGGE), while real-time PCR was employed for quantitative analysis. Mucosal communities were also visualized in situ using confocal laser scanning microscopy. DGGE banding profiles from all the gut regions exhibited at least 45% homology, with five descending colon profiles clustering at ca. 75% concordance. Real-time PCR showed that mucosal bacterial population densities were highest in the terminal ileum and that there were no significant differences in overall bacterial numbers in different parts of the colon. Bifidobacterial numbers were significantly higher in the large bowel than in the terminal ileum (
P
= 0.006), whereas lactobacilli were more prominent in the distal large intestine (
P
= 0.019).
Eubacterium rectale
(
P
= 0.0004) and
Faecalibacterium prausnitzii
(
P
= 0.001) were dominant in the ascending and descending colon. Site-specific colonization in the gastrointestinal tract may be contributory in the etiology of some diseases of the large intestine.
Enhancement of the Stability of a Prolipase from
Rhizopus oryzae
toward Aldehydes by Saturation Mutagenesis
Authors: Di Lorenzo Mirella , Hidalgo Aurelio , Molina Rafael , Hermoso Juan A. , Pirozzi Domenico , Bornscheuer Uwe T.
Published: September 2007

A prolipase from
Rhizopus oryzae
(proROL) was engineered in order to increase its stability toward lipid oxidation products such as aldehydes with the aim of improving its performance in oleochemical industries. Out of 22 amino acid residues (15 Lys and 7 His) prone to react with aldehydes, 6 Lys and all His residues (except for the catalytic histidine) were chosen and subjected to saturation mutagenesis. In order to quickly and reliably identify stability mutants within the resulting libraries, active variants were prescreened by an activity staining method on agar plates. Active mutants were expressed in
Escherichia coli
Origami in a 96-well microtiterplate format, and a stability test using octanal as a model deactivating agent was performed. The most stable histidine mutant (H201S) conferred a stability increase of 60%, which was further enhanced to 100% by combination with a lysine mutant (H201S/K168I). This increase in stability was also confirmed for other aldehydes. Interestingly, the mutations did not affect specific activity, as this was still similar to the wild-type enzyme.
Real-Time Quantitative PCR Measurement of Ileal
Lactobacillus salivarius
Populations from Broiler Chickens To Determine the Influence of Farming Practices
Authors: Harrow Sally A. , Ravindran Velmurugu , Butler Ruth C. , Marshall John W. , Tannock Gerald W.
Published: September 2007

A real-time quantitative PCR assay targeting a 16S-23S intergenic spacer region sequence was devised to measure the sizes of populations of
Lactobacillus salivarius
present in ileal digesta collected from broiler chickens. This species has been associated with deconjugation of bile salts in the small bowel and reduced broiler productivity. The assay was tested as a means of monitoring the sizes of
L. salivarius
populations from broilers fed diets with different compositions, maintained at different stocking densities, or given the antimicrobial drugs bacitracin and monensin in the feed. Stocking densities did not influence the numbers of
L. salivarius
cells in the ileum. A diet containing meat and bone meal reduced the size of the
L. salivarius
population relative to that of chickens given the control diet, as did administration of bacitracin and monensin in the feed. These changes in the target bacterial population were associated with improved broiler weight gain.
Microarray Analysis of
Escherichia coli
Strains from Interstitial Beach Waters of Lake Huron (Canada)
Authors: Kon T. , Weir S. C. , Trevors J. T. , Lee H. , Champagne J. , Meunier L. , Brousseau R. , Masson L.
Published: September 2007

DNA microarray analyses revealed that clusters of repetitive extragenic palindromic PCR-related
Escherichia coli
isolates were isogenic only within interstitial Lake Huron beach water samples and not in surrounding waters. This suggested that adaptation and growth occurred within the interstitial water sites tested. All isolates were nonpathogenic, and three lake isolates possessed tetracycline resistance genes.
Identification of
opdA
, a Gene Involved in Biodegradation of the Endocrine Disrupter Octylphenol
Authors: Porter A. W. , Hay A. G.
Published: September 2007

Octylphenol (OP) is an estrogenic detergent breakdown product. Structurally similar nonylphenols are transformed via type II
ispo
substitution, resulting in the production of hydroquinone and removal of the branched side chain. Nothing is known, however, about the gene(s) encoding this activity. We report here on our efforts to clone the gene(s) encoding OP degradation activity from
Sphingomonas
sp. strain PWE1, which we isolated for its ability to grow on OP. A fosmid library of PWE1 DNA yielded a single clone, aew4H12, which accumulated a brown polymerization product in the presence of OP. Sequence analysis of loss-of-function transposon mutants of aew4H12 revealed a single open reading frame,
opdA
, that conferred OP degradation activity.
Escherichia coli
subclones expressing
opdA
caused OP disappearance, with the concomitant production of hydroquinone and 2,4,4-trimethyl-1-pentene as well as small amounts of 2,4,4-trimethyl-2-pentanol. These metabolites are consistent with a type II
ipso
substitution reaction, the same mechanism described for nonylphenol biodegradation in other sphingomonads. Based on
opdA
's sequence homology to a unique group of putative flavin monooxygenases and the recovery of hydroxylated OP intermediates from
E. coli
expressing
opdA
, we conclude that this gene encodes the observed type II
ipso
substitution activity responsible for the initial step in OP biodegradation.
Comparative Transcriptome Analysis of
Methylibium petroleiphilum
PM1 Exposed to the Fuel Oxygenates Methyl
tert
-Butyl Ether and Ethanol
Authors: Hristova Krassimira R. , Schmidt Radomir , Chakicherla Anu Y. , Legler Tina C. , Wu Janice , Chain Patrick S. , Scow Kate M. , Kane Staci R.
Published: September 2007

High-density whole-genome cDNA microarrays were used to investigate substrate-dependent gene expression of
Methylibium petroleiphilum
PM1, one of the best-characterized aerobic methyl
tert
-butyl ether (MTBE)-degrading bacteria. Differential gene expression profiling was conducted with PM1 grown on MTBE and ethanol as sole carbon sources. Based on microarray high scores and protein similarity analysis, an MTBE regulon located on the megaplasmid was identified for further investigation. Putative functions for enzymes encoded in this regulon are described with relevance to the predicted MTBE degradation pathway. A new unique dioxygenase enzyme system that carries out the hydroxylation of
tert
-butyl alcohol to 2-methyl-2-hydroxy-1-propanol in
M. petroleiphilum
PM1 was discovered. Hypotheses regarding the acquisition and evolution of MTBE genes as well as the involvement of IS elements in these complex processes were formulated. The pathways for toluene, phenol, and alkane oxidation via toluene monooxygenase, phenol hydroxylase, and propane monooxygenase, respectively, were upregulated in MTBE-grown cells compared to ethanol-grown cells. Four out of nine putative cyclohexanone monooxygenases were also upregulated in MTBE-grown cells. The expression data allowed prediction of several hitherto-unknown enzymes of the upper MTBE degradation pathway in
M. petroleiphilum
PM1 and aided our understanding of the regulation of metabolic processes that may occur in response to pollutant mixtures and perturbations in the environment.
Genomic Diversity in
Staphylococcus xylosus
Authors: Dordet-Frisoni Emilie , Dorchies Géraud , De Araujo Cécilia , Talon Régine , Leroy Sabine
Published: September 2007

Staphylococcus xylosus
is a commensal of the skin of humans and animals and a ubiquitous bacterium naturally present in food. It is one of the major starter cultures used for meat fermentation, but a few strains could potentially be hazardous and are related to animal opportunistic infections. To better understand the genetic diversity of
S. xylosus
intraspecies, suppressive and subtractive hybridization (SSH) was carried out with the
S. xylosus
C2a strain, a commensal of human skin, used as the driver for three tester strains, S04002 used as a starter culture, S04009 isolated from cow mastitis, and 00-1747, responsible for mouse dermatitis. SSH revealed 122 tester-specific fragments corresponding to 149 open reading frames (ORFs). A large proportion of these ORFs resembled genes involved in specific metabolisms. Analysis of the distribution of the tester-specific fragments in 20
S. xylosus
strains of various origins showed that the
S. xylosus
species could be divided into two clusters with one composed only of potentially hazardous strains. The genetic content diversity of this species is colocalized in a region near the origin of replication of the chromosome. This region of speciation previously observed in the
Staphylococcus
genus corresponded in
S. xylosus
species to a strain-specific region potentially implicated in ecological fitness.
Evaluation of Two DNA Template Preparation Methods for Post-Immunomagnetic Separation Detection of
Cryptosporidium parvum
in Foods and Beverages by PCR
Authors: Frazar Christian D. , Orlandi Palmer A.
Published: September 2007

Cryptosporidium parvum
oocysts were recovered by immunomagnetic separation from six artificially contaminated foods. Two DNA isolation methods were subsequently evaluated by PCR. The FTA Concentrator-PS filter provided rapid and reproducible detection, although variability increased at lower inoculum levels (88% and 15% detection in high- and low-inoculum-level samples, respectively). Total DNA extraction generated consistent results at all oocyst levels but resulted in longer analysis time (100% and 59% detection in high- and low-inoculum-level samples, respectively). Also reflected in this study was that the matrix played an important role in the ability to recover oocysts, as sample turbidity, pH, and PCR inhibitors all influenced detection.
Cyclic AMP-Protein Kinase A and Snf1 Signaling Mechanisms Underlie the Superior Potency of Sucrose for Induction of Filamentation in
Saccharomyces cerevisiae
Authors: Van de Velde Sam , Thevelein Johan M.
Published: September 2007

Under specific environmental conditions, the yeast
Saccharomyces cerevisiae
can undergo a morphological switch to a pseudohyphal growth pattern. Pseudohyphal differentiation is generally studied upon induction by nitrogen limitation in the presence of glucose. It is known to be controlled by several signaling pathways, including mitogen-activated protein kinase, cyclic AMP-protein kinase A (cAMP-PKA), and Snf1 kinase pathways. We show that the alpha-glucoside sugars maltose and maltotriose, and especially sucrose, are more potent inducers of filamentation than glucose. Sucrose even induces filamentation in nitrogen-rich media and in the
mep2
Δ
/mep2
Δ ammonium permease mutant on ammonium-limiting medium. We demonstrate that glucose also inhibits filamentation by means of a pathway parallel to the cAMP-PKA pathway. Deletion of
HXK2
shifted the pseudohyphal growth pattern on glucose to that of sucrose, while deletion of
SNF4
abrogated filamentation on both sugars, indicating a negative role of glucose repression and a positive role for Snf1 activity in the control of filamentation. In all strains and in all media, sucrose induction of filamentation is greatly diminished by deletion of the sucrose/glucose-sensing G-protein-coupled receptor Gpr1, whereas it has no effect on induction by maltose and maltotriose. The competence of alpha-glucoside sugars to induce filamentation is reflected in the increased expression of the cell surface flocculin gene
FLO11
. In addition, sucrose is the only alpha-glucoside sugar capable of rapidly inducing
FLO11
expression in a Gpr1-dependent manner, reflecting the sensitivity of Gpr1 for this sugar and its involvement in rapid sucrose signaling. Our study identifies sucrose as the most potent nutrient inducer of pseudohyphal growth and shows that glucose inactivation of Snf1 kinase signaling is responsible for the lower potency of glucose.
Compromised Outer Membrane Integrity in
Vibrio cholerae
Type II Secretion Mutants
Authors: Sikora Aleksandra E. , Lybarger Suzanne R. , Sandkvist Maria
Published: September 2007

The type II secretion (T2S) system of
Vibrio cholerae
is a multiprotein complex that spans the cell envelope and secretes proteins important for pathogenesis as well as survival in different environments. Here we report that, in addition to the loss of extracellular secretion, removal or inhibition of expression of the T2S genes,
epsC-N
, results in growth defects and a broad range of alterations in the outer membrane that interfere with its barrier function. Specifically, the sensitivity to membrane-perturbing agents such as bile salts and the antimicrobial peptide polymyxin B is increased, and periplasmic constituents leak out into the culture medium. As a consequence, the σ
E
stress response is induced. Furthermore, due to the defects caused by inactivation of the T2S system, the Δ
eps
deletion mutant of
V. cholerae
strain N16961 is incapable of surviving the passage through the infant mouse gastrointestinal tract. The growth defect and leaky outer membrane phenotypes are suppressed when the culture medium is supplemented with 5% glucose or sucrose, although the
eps
mutants remain sensitive to membrane-damaging agents. This suggests that the sugars do not restore the integrity of the outer membrane in the
eps
mutant strains per se but may provide osmoprotective functions.
Chemotactic Control of the Two Flagellar Systems of
Rhodobacter sphaeroides
Is Mediated by Different Sets of CheY and FliM Proteins
Authors: del Campo Ana Martínez , Ballado Teresa , de la Mora Javier , Poggio Sebastian , Camarena Laura , Dreyfus Georges
Published: September 2007

Rhodobacter sphaeroides
expresses two different flagellar systems, a subpolar flagellum (fla1) and multiple polar flagella (fla2). These structures are encoded by different sets of flagellar genes. The chemotactic control of the subpolar flagellum (fla1) is mediated by three of the six different CheY proteins (CheY6, CheY4, or CheY3). We show evidence that CheY1, CheY2, and CheY5 control the chemotactic behavior mediated by fla2 flagella and that RSP6099 encodes the fla2 FliM protein.
Probing the Antiprotease Activity of λCIII, an Inhibitor of theEscherichia coliMetalloprotease HflB (FtsH)
Authors: Halder Sabyasachi , Datta Ajit Bikram , Parrack Pradeep
Published: September 2007

The CIII protein encoded by the temperate coliphage lambda acts as an inhibitor of the ubiquitousEscherichia colimetalloprotease HflB (FtsH). This inhibition results in the stabilization of transcription factor λCII, thereby helping the phage to lysogenize the host bacterium. λCIII, a small (54-residue) protein of unknown structure, also protects σ32, another specific substrate of HflB. In order to understand the details of the inhibitory mechanism of CIII, we cloned and expressed the protein with an N-terminal six-histidine tag. We also synthesized and studied a 28-amino-acid peptide, CIIIC, encompassing the central 14 to 41 residues of CIII that exhibited antiproteolytic activity. Our studies show that CIII exists as a dimer under native conditions, aided by an intersubunit disulfide bond, which is dispensable for dimerization. Unlike CIII, CIIIC resists digestion by HflB. While CIII binds to HflB, it does not bind to CII. On the basis of these results, we discuss various mechanisms for the antiproteolytic activity of CIII.
l-Rhamnose Transport Is Sugar Kinase (RhaK) Dependent inRhizobium leguminosarumbv. trifolii
Authors: Richardson Jason S. , Oresnik Ivan J.
Published: September 2007

Strains ofRhizobium leguminosarumwhich are unable to catabolizel-rhamnose, a methyl-pentose sugar, are compromised in the ability to compete for nodule occupancy versus wild-type strains. Previous characterization of the 11-kb region necessary for the utilization of rhamnose identified a locus carrying catabolic genes and genes encoding the components of an ABC transporter. Genetic evidence suggested that the putative kinase RhaK carried out the first step in the catabolism of rhamnose. Characterization of this kinase led to the observation that strains carrying rhamnose kinase mutations were unable to transport rhamnose into the cell. The absence of a functional rhamnose kinase did not stop the transcription and translation of the ABC transporter components. By developing an in vitro assay for RhaK activity, we have been able to show that (i) RhaK activity is consistent with RhaK phosphorylating rhamnose and (ii) biochemical activity of RhaK is necessary for rhamnose transport.
A Wall of Funnels Concentrates Swimming Bacteria
Authors: Galajda Peter , Keymer Juan , Chaikin Paul , Austin Robert
Published: September 2007

Randomly moving but self-propelled agents, such as
Escherichia coli
bacteria, are expected to fill a volume homogeneously. However, we show that when a population of bacteria is exposed to a microfabricated wall of funnel-shaped openings, the random motion of bacteria through the openings is rectified by tracking (trapping) of the swimming bacteria along the funnel wall. This leads to a buildup of the concentration of swimming cells on the narrow opening side of the funnel wall but no concentration of nonswimming cells. Similarly, we show that a series of such funnel walls functions as a multistage pump and can increase the concentration of motile bacteria exponentially with the number of walls. The funnel wall can be arranged along arbitrary shapes and cause the bacteria to form well-defined patterns. The funnel effect may also have implications on the transport and distribution of motile microorganisms in irregular confined environments, such as porous media, wet soil, or biological tissue, or act as a selection pressure in evolution experiments.
Amino Acid Toxicities of
Escherichia coli
That Are Prevented by Leucyl-tRNA Synthetase Amino Acid Editing
Authors: Karkhanis Vrajesh A. , Mascarenhas Anjali P. , Martinis Susan A.
Published: September 2007

Leucyl-tRNA synthetase (LeuRS) has evolved an editing function to clear misactivated amino acids. An
Escherichia coli
-based assay was established to identify amino acids that compromise the fidelity of LeuRS and translation. Multiple nonstandard as well as standard amino acids were toxic to the cell when LeuRS editing was inactivated.
How Moist Heat Kills Spores of
Bacillus subtilis
Authors: Coleman William H. , Chen De , Li Yong-qing , Cowan Ann E. , Setlow Peter
Published: September 2007

Populations of
Bacillus subtilis
spores in which 90 to 99.9% of the spores had been killed by moist heat gave only two fractions on equilibrium density gradient centrifugation: a fraction comprised of less dense spores that had lost their dipicolinic acid (DPA), undergone significant protein denaturation, and were all dead and a fraction with the same higher density as that of unheated spores. The latter fraction from heat-killed spore populations retained all of its DPA, but ≥98% of the spores could be dead. The dead spores that retained DPA germinated relatively normally with nutrient and nonnutrient germinants, but the outgrowth of these germinated spores was significantly compromised, perhaps because they had suffered damage to some proteins such that metabolic activity during outgrowth was greatly decreased. These results indicate that DPA release takes place well after spore killing by moist heat and that DPA release during moist-heat treatment is an all-or-nothing phenomenon; these findings also suggest that damage to one or more key spore proteins causes spore killing by moist heat.
Dissection of 16S rRNA Methyltransferase (KsgA) Function in
Escherichia coli
Authors: Inoue Koichi , Basu Soumit , Inouye Masayori
Published: September 2007

A 16S rRNA methyltransferase, KsgA, identified originally in
Escherichia coli
is highly conserved in all living cells, from bacteria to humans. KsgA orthologs in eukaryotes possess functions in addition to their rRNA methyltransferase activity.
E. coli
Era is an essential GTP-binding protein. We recently observed that KsgA functions as a multicopy suppressor for the cold-sensitive cell growth of an
era
mutant [Era(E200K)] strain (Q. Lu and M. Inouye, J. Bacteriol.
180
:5243-5246, 1998). Here we observed that although KsgA(E43A), KsgA(G47A), and KsgA(E66A) mutations located in the
S
-adenosylmethionine-binding motifs severely reduced its methyltransferase activity, these mutations retained the ability to suppress the growth defect of the Era(E200K) strain at a low temperature. On the other hand, a KsgA(R248A) mutation at the C-terminal domain that does not affect the methyltransferase activity failed to suppress the growth defect. Surprisingly,
E. coli
cells overexpressing wild-type KsgA, but not KsgA(R248A), were found to be highly sensitive to acetate even at neutral pH. Such growth inhibition also was observed in the presence of other weak organic acids, such as propionate and benzoate. These chemicals are known to be highly toxic at acidic pH by lowering the intracellular pH. We found that KsgA-induced cells had increased sensitivity to extreme acid conditions (pH 3.0) compared to that of noninduced cells. These results suggest that
E. coli
KsgA, in addition to its methyltransferase activity, has another unidentified function that plays a role in the suppression of the cold-sensitive phenotype of the Era(E200K) strain and that the additional function may be involved in the acid shock response. We discuss a possible mechanism of the KsgA-induced acid-sensitive phenotype.
One Perturbation of the Mother Cell Gene Regulatory Network Suppresses the Effects of Another during Sporulation of
Bacillus subtilis
Authors: Wang Lijuan , Perpich John , Driks Adam , Kroos Lee
Published: September 2007

In the mother cell of sporulating
Bacillus subtilis
, a regulatory network functions to control gene expression. Four transcription factors act sequentially in the order σ
E
, SpoIIID, σ
K
, GerE. σ
E
and σ
K
direct RNA polymerase to transcribe different regulons. SpoIIID and GerE are DNA-binding proteins that activate or repress transcription of many genes. Several negative regulatory loops add complexity to the network. First, transcriptionally active σ
K
RNA polymerase inhibits early sporulation gene expression, resulting in reduced accumulation of σ
E
and SpoIIID late during sporulation. Second, GerE represses
sigK
transcription, reducing σ
K
accumulation about twofold. Third, SpoIIID represses
cotC
, which encodes a spore coat protein, delaying its transcription by σ
K
RNA polymerase. Partially circumventing the first feedback loop, by engineering cells to maintain the SpoIIID level late during sporulation, causes spore defects. Here, the effects of circumventing the second feedback loop, by mutating the GerE binding sites in the
sigK
promoter region, are reported. Accumulation of pro-σ
K
and σ
K
was increased, but no spore defects were detected. Expression of σ
K
-dependent reporter fusions was altered, increasing the expression of
gerE-lacZ
and
cotC-lacZ
and decreasing the expression of
cotD-lacZ
. Because these effects on gene expression were opposite those observed when the SpoIIID level was maintained late during sporulation, cells were engineered to both maintain the SpoIIID level and have elevated
sigK
expression late during sporulation. This restored the expression of σ
K
-dependent reporters to wild-type levels, and no spore defects were observed. Hence, circumventing the second feedback loop suppressed the effects of perturbing the first feedback loop. By feeding information back into the network, these two loops appear to optimize target gene expression and increase network robustness. Circumventing the third regulatory loop, by engineering cells to express
cotC
about 2 h earlier than normal, did not cause a detectable spore defect.
Functional Characterization of Bacterial Oligosaccharyltransferases Involved in O-Linked Protein Glycosylation
Authors: Faridmoayer Amirreza , Fentabil Messele A. , Mills Dominic C. , Klassen John S. , Feldman Mario F.
Published: September 2007

Protein glycosylation is an important posttranslational modification that occurs in all domains of life. Pilins, the structural components of type IV pili, are O glycosylated inNeisseria meningitidis,Neisseria gonorrhoeae, and some strains ofPseudomonas aeruginosa. In this work, we characterized theP. aeruginosa1244 andN. meningitidisMC58 O glycosylation systems inEscherichia coli. In both cases, sugars are transferred en bloc by an oligosaccharyltransferase (OTase) named PglL inN. meningitidisand PilO inP. aeruginosa. We show that, like PilO, PglL has relaxed glycan specificity. Both OTases are sufficient for glycosylation, but they require translocation of the undecaprenol-pyrophosphate-linked oligosaccharide substrates into the periplasm for activity. Whereas PilO activity is restricted to short oligosaccharides, PglL is able to transfer diverse oligo- and polysaccharides. This functional characterization supports the concept that despite their low sequence similarity, PilO and PglL belong to a new family of “O-OTases” that transfer oligosaccharides from lipid carriers to hydroxylated amino acids in proteins. To date, such activity has not been identified for eukaryotes. To our knowledge, this is the first report describing recombinant O glycoproteins synthesized inE. coli.
Local and Global Regulators Linking Anaerobiosis tocupAFimbrial Gene Expression inPseudomonas aeruginosa
Authors: Vallet-Gely Isabelle , Sharp Josh S. , Dove Simon L.
Published: September 2007

ThecupAgene cluster ofPseudomonas aeruginosaencodes components and assembly factors of a putative fimbrial structure that enable this opportunistic pathogen to form biofilms on abiotic surfaces. InP. aeruginosathe control ofcupAgene expression is complex, with the H-NS-like MvaT protein functioning to repress phase-variable (on/off) expression of the operon. Here we identify four positive regulators ofcupAgene expression, including three unusual regulators encoded by thecgrABCgenes and Anr, a global regulator of anaerobic gene expression. We show that thecupAgenes are expressed in a phase-variable manner under anaerobic conditions and that thecgrgenes are essential for this expression. We show further thatcgrgene expression is negatively controlled by MvaT and positively controlled by Anr and anaerobiosis. Expression of thecupAgenes therefore appears to involve a regulatory cascade in which anaerobiosis, signaled through Anr, stimulates expression of thecgrgenes, resulting in a concomitant increase incupAgene expression. Our findings thus provide mechanistic insight into the regulation ofcupAgene expression and identify anaerobiosis as an inducer of phase-variablecupAgene expression, raising the possibility that phase-variable expression of fimbrial genes important for biofilm formation may occur inP. aeruginosapersisting in the largely anaerobic environment of the cystic fibrosis host lung.
Flavin-Dependent Thymidylate Synthase ThyX Activity: Implications for the Folate Cycle in Bacteria
Authors: Leduc Damien , Escartin Frédéric , Nijhout H. Frederik , Reed Michael C. , Liebl Ursula , Skouloubris Stéphane , Myllykallio Hannu
Published: September 2007

Although flavin-dependent ThyX proteins show thymidylate synthase activity in vitro and functionally complement
thyA
defects in heterologous systems, direct proof of their cellular functions is missing. Using insertional mutagenesis of
Rhodobacter capsulatus thyX
, we constructed the first defined
thyX
inactivation mutant. Phenotypic analyses of the obtained mutant strain confirmed that
R. capsulatus
ThyX is required for de novo thymidylate synthesis. Full complementation of the
R. capsulatus thyX
::
spec
strain to thymidine prototrophy required not only the canonical thymidylate synthase ThyA but also the dihydrofolate reductase FolA. Strikingly, we also found that addition of exogenous methylenetetrahydrofolate transiently inhibited the growth of the different
Rhodobacter
strains used in this work. To rationalize these experimental results, we used a mathematical model of bacterial folate metabolism. This model suggests that a very low dihydrofolate reductase activity is enough to rescue significant thymidylate synthesis in the presence of ThyX proteins and is in agreement with the notion that intracellular accumulation of folates results in growth inhibition. In addition, our observations suggest that the presence of flavin-dependent thymidylate synthase X provides growth benefits under conditions in which the level of reduced folate derivatives is compromised.
PARALLEL ALGORITHMS FOR CELLULAR MODELS SIMULATION
Authors: JAFER SHAFAGH , WAINER GABRIEL A.
Published: September 2007

DEVS is a sound formal modeling and simulation (M&S) framework based on generic dynamic system concepts. Cell-DEVS is a formalism for cell-shaped models based on DEVS. This work presents a new simulation technique for execution of DEVS and Cell-DEVS models in parallel environments. These techniques are modifications to the original Time Warp mechanism offered by WARPED kernel. Time Warp functionalities are revised to include two new algorithms namely, Local Rollback Frequency Model (LRFM) and Global Rollback Frequency Model (GRFM). The resulting simulator is used as new simulation engine for CD++, an M&S toolkit that implements DEVS and Cell-DEVS theories. The results obtained allowed us to achieve considerable speedups due to the reductions that LRFM and GRFM protocols perform on number of rollbacks and anti-messages.
HOW SOLUTION DENSITY AFFECTS THE FINDING OF SPATIALLY ROBUST SOLUTIONS
Authors: DRZADZEWSKI GRZEGORZ , WINEBERG MARK
Published: September 2007

The common definition for robust solutions considers a solution robust if it remains optimal (or near optimal) when the parameters defining the fitness function are perturbed. We call this parameter robustness or temporal robustness. In this paper we propose an alternate definition for robustness, which we call spatial or solution robustness, if both the solution and the neighbourhood around the solution has high fitness. With this definition, we created a set of functions with useful properties to allow for the testing of solution robustness. We then focus on the effect of the precision (density) of the search space and find that it has a drastic effect on both the number of solutions and their quality.
REDUCTION BY HYDROGEN OF VANADIUM IN PHOSPHATE AND VANADATE LEAD APATITES: AN ESR STUDY
Authors: IKRAM M. , AHMED H. , MENDES PIETRO , MIR F. A. , BASHIR A. , PAULA A. , ROSSI A. M. , EON J. G.
Published: September 2007

A solid solution of phosphate and vanadate lead apatites Pb 10( PO 4)6-x( VO 4)x( OH )2 (x = 0.5, 1.0, 2.0, 3.0, and 6.0) were reduced with hydrogen gas at temperature 300°C for one hour. All these compounds were investigated by XRD and ESR spectroscopy. Only one reduced oxidation state, V 4+, was detected. Two types of species-like isolated vanadium (+ 4) and clusters of V 4+ were observed. The EPR powder spectrum computer simulations were performed using the program Simfonia from Bruker. Spin Hamiltonian parameters were also calculated. As the concentrations of vanadium ions increase to 2.0, the intensity of the ESR lines increases, and x = 3.0 the intensity of the ESR line decreases sharply. After this concentration, i.e. x > 3.0, there is again an increase in the intensity. The variation of the line shape and intensity of the ESR spectra can be attributed to increases in the hopping rate of the charge carriers (polarons) in different apatite compositions.
A VERY LOW NOISE, HIGH STABILITY, VOLTAGE REFERENCE FOR HIGH SENSITIVITY NOISE MEASUREMENTS
Authors: CIOFI C. , CANNATÀ' G. , SCANDURRA G. , MERLINO R.
Published: September 2007

In this paper we demonstrate that by exploiting the non linear characteristic of low noise PN junction diodes, a very low noise, high stability voltage reference can be obtained starting from a conventional solid state series voltage reference. In order to obtain such a result, a series connection of N identical diodes is supplied in the forward region of the I-V characteristic by means of a proper resistance. While the DC voltage drop across the diodes can be a large fraction of the voltage supplied by the reference, the noise introduced by the reference itself is reduced by a much larger factor because of the low value of the small signal equivalent resistance of the diodes. In its simplest implementation, such a voltage source would suffer from a relatively high temperature dependence of the supplied voltages because of the intrinsic properties of PN junctions. However, by resorting to a proper temperature control circuit, high stability can be obtained. As an example, by employing an AD586 voltage reference and with N = 4, a 2.560 V reference has been obtained with a stability over temperature better that 50 μ V /° C and a voltage noise as low as 2 × 10−15, 6 × 10−17 and 1.5 × 10−17 V 2/ Hz at 100 mHz, 1 Hz and for frequencies larger than 10 Hz, respectively.
Skeletal muscle VEGF gradients in peripheral arterial disease: simulations of rest and exercise
Authors: Ji James W. , Mac Gabhann Feilim , Popel Aleksander S.
Published: September 2007

VEGF is a key promoter of angiogenesis and a major target of proangiogenic therapy for peripheral arterial disease (PAD). Greater understanding of VEGF angiogenic signaling and guidance by gradients for new capillaries will aid in developing new proangiogenic therapies and improving existing treatments. However, in vivo measurements of VEGF concentration gradients at the cell scale are currently impossible. We have developed a computational model to quantify VEGF distribution in extensor digitorum longus skeletal muscle using measurements of VEGF, VEGF receptor (VEGFR), and neuropilin-1 (NRP1) expression in an experimental model of rat PAD. VEGF is secreted by myocytes, diffuses through and interacts with extracellular matrix and basement membranes, and binds VEGFRs and NRP1 on endothelial cell surfaces of blood vessels. We simulate the effects of increased NRP1 expression and of therapeutic exercise training on VEGF gradients, receptor signaling, and angiogenesis. Our study predicts that angiogenic therapy for PAD may be achieved not only through VEGF upregulation but also through modulation of VEGFRs and NRP1. We predict that expression of 104NRP1/cell can increase VEGF binding to receptors by 1.7-fold (vs. no NRP1); in nonexercise-trained muscle with PAD, angiogenesis is hindered due to limited VEGF upregulation, signaling, and gradients; in exercise-trained muscle, VEGF signaling is enhanced by upregulation of VEGFRs and NRP1, and VEGF signaling is strongest within the first week of exercise therapy; and hypoxia-induced VEGF release is important to direct angiogenesis towards unperfused tissue.
Endothelial PI 3-kinase activity regulates lymphocyte diapedesis
Authors: Nakhaei-Nejad Maryam , Hussain Amer M. , Zhang Qiu-Xia , Murray Allan G.
Published: September 2007

Lymphocyte recruitment to sites of inflammation involves a bidirectional series of cues between the endothelial cell (EC) and the leukocyte that culminate in lymphocyte migration into the tissue. Remodeling of the EC F-actin cytoskeleton has been observed after leukocyte adhesion, but the signals to the EC remain poorly defined. We studied the dependence of peripheral blood lymphocyte transendothelial migration (TEM) through an EC monolayer in vitro on EC phosphatidylinositol 3-kinase (PI 3-kinase) activity. Lymphocytes were perfused over cytokine-activated EC using a parallel-plate laminar flow chamber. Inhibition of EC PI 3-kinase activity using LY-294002 or wortmannin decreased lymphocyte TEM (48 ± 6 or 34 ± 7%, respectively, vs. control; mean ± SE; P < 0.05). Similarly, EC knockdown of the p85α regulatory subunit of PI 3-kinase decreased lymphocyte transmigration. Treatment of EC with jasplakinolide to inhibit EC F-actin remodeling also decreased lymphocyte TEM to 24 ± 10% vs. control ( P < 0.05). EC PI 3-kinase inhibition did not change the strength of lymphocyte adhesion to the EC or formation of the EC “docking structure” after intercellular adhesion molecule-1 ligation, whereas this was inhibited by jasplakinolide treatment. A similar fraction of lymphocytes migrated on control or LY-294002-treated EC and localized to interendothelial junctions. However, lymphocytes failed to extend processes below the level of vascular endothelial (VE)-cadherin on LY-294002-treated EC. Together these observations indicate that EC PI 3-kinase activity and F-actin remodeling are required during lymphocyte diapedesis and identify a PI 3-kinase-dependent step following initial separation of the VE-cadherin barrier.
Heterogeneity ofIK1in the mouse heart
Authors: Panama Brian K. , McLerie Meredith , Lopatin Anatoli N.
Published: September 2007

Previous studies have shown that cardiac inward rectifier potassium current ( IK1) channels are heteromers of distinct Kir2 subunits and suggested that species- and tissue-dependent expression of these subunits may underlie variability of IK1. In this study, we investigated the contribution of the slowly activating Kir2.3 subunit and free intracellular polyamines (PAs) to variability of IK1in the mouse heart. The kinetics of activation was measured in Kir2 concatemeric tetramers with known subunit stoichiometry. Inclusion of only one Kir2.3 subunit to a Kir2.1 channel led to an approximate threefold slowing of activation kinetics, with greater slowing on subsequent additions of Kir2.3 subunits. Activation kinetics of IK1in both ventricles and both atria was found to correspond to fast-activating Kir2.1/Kir2.2 channels, suggesting no major contribution of Kir2.3 subunits. In contrast, IK1displayed significant variation in both the current density and inward rectification, suggesting involvement of intracellular PAs. The total levels of PAs were similar across the mouse heart. Measurements of the free intracellular PAs in isolated myocytes, using transgenically expressed Kir2.1 channels as PA sensors, revealed “microheterogeneity” of IK1rectification as well as lower levels of free PAs in atrial myocytes compared with ventricular cells. These findings provide a quantitative explanation for the regional heterogeneity of IK1.
Inhibition of MMP-2 gene expression with small interfering RNA in rabbit vascular smooth muscle cells
Authors: Hlawaty Hanna , San Juan Aurélie , Jacob Marie-Paule , Vranckx Roger , Letourneur Didier , Feldman Laurent J.
Published: September 2007

Matrix metalloproteinase-2 (MMP-2) is constitutively expressed in vascular smooth muscle cells (VSMCs). Using small interfering RNA (siRNA), we evaluated the effect of MMP-2 inhibition in VSMCs in vitro and ex vivo. Rabbit VSMCs were transfected in vitro with 50 nmol/l MMP-2 siRNA or scramble siRNA. Flow cytometry and confocal microscopy showed cellular uptake of siRNA in ∼80% of VSMCs. MMP-2 mRNA levels evaluated by real-time RT-PCR, pro-MMP-2 activity from conditioned culture media evaluated by gelatin zymography, and VSMC migration were reduced by 44 ± 19%, 43 ± 14%, and 36 ± 14%, respectively, in MMP-2 siRNA-transfected compared with scramble siRNA-transfected VSMCs ( P < 0.005 for all). Ex vivo MMP-2 siRNA transfection was performed 2 wk after balloon injury of hypercholesterolemic rabbit carotid arteries. Fluorescence microscopy showed circumferential siRNA uptake in neointimal cells. Gelatin zymography of carotid artery culture medium demonstrated a significant decrease of pro-MMP-2 activity in MMP-2 siRNA-transfected compared with scramble siRNA-transfected arteries ( P < 0.01). Overall, our results demonstrate that in vitro MMP-2 siRNA transfection in VSMCs markedly inhibits MMP-2 gene expression and VSMC migration and that ex vivo delivery of MMP-2 siRNA in balloon-injured arteries reduces pro-MMP-2 activity in neointimal cells, suggesting that siRNA could be used to modify arterial biology in vivo.
Sex-related effects on diabetes-induced alterations in calcium release in the rat heart
Authors: Yaras Nazmi , Tuncay Erkan , Purali Nuhan , Sahinoglu Babur , Vassort Guy , Turan Belma
Published: September 2007

The present study was designed to determine whether the properties of local Ca2+release and its related regulatory mechanisms might provide insight into the role of sex differences in heart functions of control and streptozotocin-induced diabetic adult rats. Left ventricular developed pressure, the rates of pressure development and decay (±dP/d t), basal intracellular Ca2+level ([Ca2+]i), and spatiotemporal parameters of [Ca2+]itransients were found to be similar in male and female control rats. However, spatiotemporal parameters of Ca2+sparks in cardiomyocytes isolated from control females were significantly larger and slower than those in control males. Diabetes reduced left ventricular developed pressure to a lower extent in females than in males, and the diabetes-induced depressions in both +dP/d t and −dP/d t were less in females than in males. Diabetes elicited a smaller reduction in the amplitude of [Ca2+]itransients in females than in males, a smaller reduction in sarcoplasmic reticulum-Ca2+load, and less increase in basal [Ca2+]i. Similarly, the elementary Ca2+events and their control proteins were clearly different in both sexes, and these differences were more marked in diabetes. Diabetes-induced depression of the Ca2+spark amplitude was significantly less in females than in matched males. Levels of cardiac ryanodine receptors (RyR2) and FK506-binding protein 12.6 in control females were significantly higher than those shown in control males. Diabetes induced less RyR2 phosphorylation and FK506-binding protein 12.6 unbinding in females. Moreover, total and free sulfhydryl groups were significantly less reduced, and PKC levels were less increased, in diabetic females than in diabetic males. The present data related to local Ca2+release and its related proteins describe some of the mechanisms that may underlie sex-related differences accounting for females to have less frequent development of cardiac diseases.
Heart rate recovery and heart rate complexity following resistance exercise training and detraining in young men
Authors: Heffernan Kevin S. , Fahs Christopher A. , Shinsako Kevin K. , Jae Sae Young , Fernhall Bo
Published: September 2007

The purpose of this study was to examine heart rate recovery (HRR) and linear/nonlinear heart rate variability (HRV) before and after resistance training. Fourteen young men (25.0 ± 1.1 yr of age) completed a crossover design consisting of a 4-wk time-control period, 6 wk of resistance training (3 days/wk), and 4 wk of detraining. Linear HRV was spectrally decomposed using an autoregressive approach. Nonlinear dynamics of heart rate complexity included sample entropy (SampEn) and Lempel-Ziv entropy (LZEn). HRR was calculated from a graded maximal exercise test as maximal heart rate attained during the test minus heart rate at 1 min after exercise (HRR). There was no change in SampEn, LZEn, or HRR after the time-control portion of the study ( P > 0.05). SampEn ( P < 0.05), LZEn ( P < 0.05), and HRR ( P < 0.05) increased after resistance training and returned to pretraining values after detraining. There was no change in spectral measures of HRV at any time point ( P > 0.05). These findings suggest that resistance exercise training increases heart rate complexity and HRR after exercise but has no effect on spectral measures of HRV in young healthy men. These autonomic changes regress shortly after cessation of training.