bluuebunny/crossref_metadata_2025 huggingface dataset:
GAS2
and
GAS4
, a Pair of Developmentally Regulated Genes Required for Spore Wall Assembly in
Saccharomyces cerevisiae
Authors: Ragni Enrico , Coluccio Alison , Rolli Eleonora , Rodriguez-Peña José Manuel , Colasante Gaia , Arroyo Javier , Neiman Aaron M. , Popolo Laura
Published: December 2006

The
GAS
multigene family of
Saccharomyces cerevisiae
is
composed of five paralogs (
GAS1
to
GAS5
).
GAS1
is the only one of these genes that has been
characterized to date. It encodes a
glycosylphosphatidylinositol-anchored protein functioning as aβ
(1,3)-glucan elongase and required for proper cell wall
assembly during vegetative growth. In this study, we characterize the
roles of the
GAS2
and
GAS4
genes. These genes are
expressed exclusively during sporulation. Their mRNA levels showed a
peak at 7 h from induction of sporulation and then decreased.
Gas2 and Gas4 proteins were detected and reached maximum levels between
8 and 10 h from induction of sporulation, a time roughly
coincident with spore wall assembly. The double null
gas2
gas4
diploid mutant showed a severe reduction in the
efficiency of sporulation, an increased permeability of the spores to
exogenous substances, and production of inviable spores, whereas the
single
gas2
and
gas4
null diploids were similar to
the parental strain. An analysis of spore ultrastructure indicated that
the loss of Gas2 and Gas4 proteins affected the proper attachment of
the glucan to the chitosan layer, probably as a consequence of the lack
of coherence of the glucan layer. The ectopic expression of
GAS2
and
GAS4
genes in a
gas1
null mutant
revealed that these proteins are redundant versions of Gas1p
specialized to function in a compartment at a pH value close to
neutral.
Endoplasmic Reticulum Retention Signal-Dependent Glycylation of the Hsp70/Grp170-Related Pgp1p in
Tetrahymena
Authors: Xie Rong , Clark Kathleen M. , Gorovsky Martin A.
Published: December 2006

Glycylation is an uncommon posttranslational modification. It has been found that tubulin glycylation is essential for cell survival in
Tetrahymena
. Here we describe
PGP1
, a
Tetrahymena
gene encoding an Hsp70 homologue that is a novel glycylated protein. Pgp1p is a conserved glycoprotein that localizes within the lumen of the endoplasmic reticulum (ER). We demonstrate that
PGP1
is essential for viability and present evidence that both glycosylation and ER retention are necessary but not sufficient for glycylation.
Development of Signature-Tagged Mutagenesis in
Burkholderia pseudomallei
To Identify Genes Important in Survival and Pathogenesis
Authors: Cuccui J. , Easton A. , Chu K. K. , Bancroft G. J. , Oyston P. C. F. , Titball R. W. , Wren B. W.
Published: December 2006

Burkholderia pseudomallei
, the causative agent of melioidosis, is an important human pathogen in Southeast Asia and northern Australia for which a vaccine is unavailable. A panel of 892 double signature-tagged mutants was screened for virulence using an intranasal BALB/c mouse model of infection. A novel DNA tag microarray identified 33 mutants as being attenuated in spleens, while 6 were attenuated in both lungs and spleens. The transposon insertion sites in spleen-attenuated mutants revealed genes involved in several stages of capsular polysaccharide biosynthesis and DNA replication and repair, a putative oxidoreductase, ABC transporters, and a lipoprotein that may be important in intercellular spreading. The six mutants identified as missing in both lungs and spleens were found to have insertions in
recA
involved in the SOS response and DNA repair; putative auxotrophs of leucine, threonine,
p
-aminobenzoic acid, and a mutant with an insertion in
aroB
causing auxotrophy for aromatic compounds were also found. Murine challenge studies revealed partial protection in BALB/c mice vaccinated with the
aroB
mutant. The refined signature-tagged mutagenesis approach developed in this study was used to efficiently identify attenuating mutants from this highly pathogenic species and could be applied to other organisms.
Functional Analysis of Nine Putative Chemoreceptor Proteins inSinorhizobium meliloti
Authors: Meier Veronika M. , Muschler Paul , Scharf Birgit E.
Published: December 2006

The genome of the symbiotic soil bacteriumSinorhizobium meliloticontains eight genes coding for methyl-accepting chemotaxis proteins (MCPs) McpS to McpZ and one gene coding for a transducer-like protein, IcpA. Seven of the MCPs are localized in the cytoplasmic membrane via two membrane-spanning regions, whereas McpY and IcpA lack such hydrophobic regions. The periplasmic regions of McpU, McpV, and McpX contain the small-ligand-binding domain Cache. In addition, McpU possesses the ligand-binding domain TarH. By probing gene expression withlacZfusions, we have identifiedmcpUandmcpXas being highly expressed. Deletion of any one of the receptor genes caused impairments in the chemotactic response toward most organic acids, amino acids, and sugars in a swarm plate assay. The data imply that chemoreceptor proteins inS. melilotican sense more than one class of carbon source and suggest that many or all receptors work as an ensemble. Tactic responses were virtually eliminated for a strain lacking all nine receptor genes. Capillary assays revealed three important sensors for the strong attractant proline: McpU, McpX, and McpY. Receptor deletions variously affected free-swimming speed and attractant-induced chemokinesis. Noticeably, cells lackingmcpUwere swimming 9% slower than the wild-type control. We infer that McpU inhibits the kinase activity of CheA in the absence of an attractant. Cells lacking one of the two soluble receptors were impaired in chemokinetic proficiency by more than 50%. We propose that the internal sensors, IcpA and the PAS domain containing McpY, monitor the metabolic state ofS. meliloti.
Weak Rolling Adhesion Enhances Bacterial Surface Colonization
Authors: Anderson Brett N. , Ding Albert M. , Nilsson Lina M. , Kusuma Kaoru , Tchesnokova Veronika , Vogel Viola , Sokurenko Evgeni V. , Thomas Wendy E.
Published: December 2006

Bacterial adhesion to and subsequent colonization of surfaces are the first steps toward forming biofilms, which are a major concern for implanted medical devices and in many diseases. It has generally been assumed that strong irreversible adhesion is a necessary step for biofilm formation. However, some bacteria, such as
Escherichia coli
when binding to mannosylated surfaces via the adhesive protein FimH, adhere weakly in a mode that allows them to roll across the surface. Since single-point mutations or even increased shear stress can switch this FimH-mediated adhesion to a strong stationary mode, the FimH system offers a unique opportunity to investigate the role of the strength of adhesion independently from the many other factors that may affect surface colonization. Here we compare levels of surface colonization by
E. coli
strains that differ in the strength of adhesion as a result of flow conditions or point mutations in FimH. We show that the weak rolling mode of surface adhesion can allow a more rapid spreading during growth on a surface in the presence of fluid flow. Indeed, an attempt to inhibit the adhesion of strongly adherent bacteria by blocking mannose receptors with a soluble inhibitor actually increased the rate of surface colonization by allowing the bacteria to roll. This work suggests that (i) a physiological advantage to the weak adhesion demonstrated by commensal variants of FimH bacteria may be to allow rapid surface colonization and (ii) antiadhesive therapies intended to prevent biofilm formation can have the unintended effect of enhancing the rate of surface colonization.
Intracellular Copper Does Not Catalyze the Formation of Oxidative DNA Damage in
Escherichia coli
Authors: Macomber Lee , Rensing Christopher , Imlay James A.
Published: December 2006

Because copper catalyzes the conversion of H
2
O
2
to hydroxyl radicals in vitro, it has been proposed that oxidative DNA damage may be an important component of copper toxicity. Elimination of the copper export genes,
copA
,
cueO
, and
cusCFBA
, rendered
Escherichia coli
sensitive to growth inhibition by copper and provided forcing circumstances in which this hypothesis could be tested. When the cells were grown in medium supplemented with copper, the intracellular copper content increased 20-fold. However, the copper-loaded mutants were actually less sensitive to killing by H
2
O
2
than cells grown without copper supplementation. The kinetics of cell death showed that excessive intracellular copper eliminated iron-mediated oxidative killing without contributing a copper-mediated component. Measurements of mutagenesis and quantitative PCR analysis confirmed that copper decreased the rate at which H
2
O
2
damaged DNA. Electron paramagnetic resonance (EPR) spin trapping showed that the copper-dependent H
2
O
2
resistance was not caused by inhibition of the Fenton reaction, for copper-supplemented cells exhibited substantial hydroxyl radical formation. However, copper EPR spectroscopy suggested that the majority of H
2
O
2
-oxidizable copper is located in the periplasm; therefore, most of the copper-mediated hydroxyl radical formation occurs in this compartment and away from the DNA. Indeed, while
E. coli
responds to H
2
O
2
stress by inducing iron sequestration proteins, H
2
O
2
-stressed cells do not induce proteins that control copper levels. These observations do not explain how copper suppresses iron-mediated damage. However, it is clear that copper does not catalyze significant oxidative DNA damage in vivo; therefore, copper toxicity must occur by a different mechanism.
Bacteroides fragilis
Synthesizes a DNA Invertase Affecting both a Local and a Distant Region
Authors: Roche-Hakansson Hazeline , Chatzidaki-Livanis Maria , Coyne Michael J. , Comstock Laurie E.
Published: December 2006

The activity of a fourth conserved tyrosine site-specific recombinase (Tsr) of
Bacteroides fragilis
was characterized. Its gene,
tsr19
, is adjacent to
mpi
, encoding the global DNA invertase regulating capsular polysaccharide biosynthesis. Unlike the other described Tsrs of
B. fragilis
, Tsr19 brings about inversion of two DNA regions, one local and one located distantly.
Characterization of Colony Morphology Variants Isolated fromStreptococcus pneumoniaeBiofilms
Authors: Allegrucci Magee , Sauer Karin
Published: December 2006

In this study, we report the isolation of colony morphology variants fromStreptococcus pneumoniaeserotype 3 biofilms. The colony variants differed in colony size (large, medium, and small) and their mucoid appearance on blood agar. The small nonmucoid variant (SCV) emerged during the initial attachment stage ofS. pneumoniaebiofilm formation and dominated over the course of biofilm growth. Mucoid variants appeared at later biofilm developmental stages. The reduction in colony size/mucoidy correlated with a decrease in capsule production and an increase in initial attachment. The large mucoid variant formed flat unstructured biofilms, failed to aggregate in liquid culture, and adhered poorly to solid surfaces. In contrast, SCVs autoaggregated in liquid culture, hyperadhered to solid surfaces, and formed biofilms with significant three-dimensional structure, mainly in the form of microcolonies. The variants showed similar antibiotic resistance/susceptibility based on a modified Kirby-Bauer test and when grown as biofilms. However, antimicrobial treatment ofS. pneumoniaebiofilms altered the colony variant's distribution and mainly affected the most interior areas of biofilm microcolonies. To further explore the nature of the variants, the capsule biosynthetic operon (cps3DSUM) was explored in greater detail. The genetic analysis indicated that the emergence of nonmucoid variants was due to a deletion comprisingcps3DSUas well as additional genes upstream of thecps3operon. Overall, our findings suggest that in vitro biofilm formation ofS. pneumoniaeserotype 3 coincides with the emergence of colony variants with distinct genotypic and phenotypic characteristics.
Specific Binding of D1 Protein Degradation Products to the
psbAI
Promoter in
Synechococcus
sp. Strain PCC 7942
Authors: Stelljes Christian , Koenig Friederike
Published: December 2006

The D1:1 protein and its potentially occurring degradation products were overexpressed in
Escherichia coli
. Protein-DNA interaction is shown for the promoter region of
psbAI
. The D1:1 degradation products may be involved in transcription regulation of
psbAI
by binding in the promoter region. Additionally, C-terminal fragments of the D1 protein bind to a sequence with similarity to
isiB
, a gene which encodes a flavodoxin-like protein.
An Actin Homolog of the Archaeon
Thermoplasma acidophilum
That Retains the Ancient Characteristics of Eukaryotic Actin
Authors: Hara Futoshi , Yamashiro Kan , Nemoto Naoki , Ohta Yoshinori , Yokobori Shin-ichi , Yasunaga Takuo , Hisanaga Shin-ichi , Yamagishi Akihiko
Published: December 2006

Actin, a central component of the eukaryotic cytoskeleton, plays a crucial role in determining cell shape in addition to several other functions. Recently, the structure of the archaeal actin homolog Ta0583, isolated from the archaeon
Thermoplasma acidophilum
, which lacks a cell wall, was reported by Roeben et al. (J. Mol. Biol.
358:
145-156, 2006). Here we show that Ta0583 assembles into bundles of filaments similar to those formed by eukaryotic actin. Specifically, Ta0583 forms a helix with a filament width of 5.5 nm and an axial repeating unit of 5.5 nm, both of which are comparable to those of eukaryotic actin. Eukaryotic actin shows a greater resemblance to Ta0583 than to bacterial MreB and ParM in terms of polymerization characteristics, such as the requirement for Mg
2+
, critical concentration, and repeating unit size. Furthermore, phylogenetic analysis also showed a closer relationship between Ta0583 and eukaryotic actin than between MreB or ParM and actin. However, the low specificity of Ta0583 for nucleotide triphosphates indicates that Ta0583 is more primitive than eukaryotic actin. Taken together, our results suggest that Ta0583 retains the ancient characteristics of eukaryotic actin.
On Torque and Tumbling in Swimming
Escherichia coli
Authors: Darnton Nicholas C. , Turner Linda , Rojevsky Svetlana , Berg Howard C.
Published: December 2006

Bacteria swim by rotating long thin helical filaments, each driven at its base by a reversible rotary motor. When the motors of peritrichous cells turn counterclockwise (CCW), their filaments form bundles that drive the cells forward. We imaged fluorescently labeled cells of
Escherichia coli
with a high-speed charge-coupled-device camera (500 frames/s) and measured swimming speeds, rotation rates of cell bodies, and rotation rates of flagellar bundles. Using cells stuck to glass, we studied individual filaments, stopping their rotation by exposing the cells to high-intensity light. From these measurements we calculated approximate values for bundle torque and thrust and body torque and drag, and we estimated the filament stiffness. For both immobilized and swimming cells, the motor torque, as estimated using resistive force theory, was significantly lower than the motor torque reported previously. Also, a bundle of several flagella produced little more torque than a single flagellum produced. Motors driving individual filaments frequently changed directions of rotation. Usually, but not always, this led to a change in the handedness of the filament, which went through a sequence of polymorphic transformations, from normal to semicoiled to curly 1 and then, when the motor again spun CCW, back to normal. Motor reversals were necessary, although not always sufficient, to cause changes in filament chirality. Polymorphic transformations among helices having the same handedness occurred without changes in the sign of the applied torque.
Role of mRNA Stability in Growth Phase Regulation of Gene Expression in the Group A Streptococcus
Authors: Barnett Timothy C. , Bugrysheva Julia V. , Scott June R.
Published: December 2006

The impressive disease spectrum ofStreptococcus pyogenes(the group A streptococcus [GAS]) is believed to be determined by its ability to modify gene expression in response to environmental stimuli. Virulence gene expression is controlled tightly by several different transcriptional regulators in this organism. In addition, expression of most, if not all, GAS genes is determined by a global mechanism dependent on growth phase. To begin an analysis of growth-phase regulation, we compared the transcriptome 2 h into stationary phase to that in late exponential phase of a serotype M3 GAS strain. We identified thearctranscript as more abundant in stationary phase in addition to thesagandsdatranscripts that had been previously identified. We found that in stationary phase, the stability ofsagA,sda, andarcTtranscripts increased dramatically. We found that polynucleotide phosphorylase (PNPase [encoded bypnpA]) is rate limiting for decay ofsagAandsdatranscripts in late exponential phase, since the stability of these mRNAs was greater in apnpAmutant, while stability of control mRNAs was unaffected by this mutation. Complementation restored the wild-type decay rate. Furthermore, in apnpAmutant, thesagAmRNA appeared to be full length, as determined by Northern hybridization. It seems likely that mRNAs abundant in stationary phase are insensitive to the normal decay enzyme(s) and instead require PNPase for this process. It is possible that PNPase activity is limited in stationary phase, allowing persistence of these important virulence factor transcripts at this phase of growth.
Repression of the Inner Membrane Lipoprotein NlpA by Rns in EnterotoxigenicEscherichia coli
Authors: Bodero Maria D. , Pilonieta M. Carolina , Munson George P.
Published: December 2006

The expression of the inner membrane protein NlpA is repressed by the enterotoxigenicEscherichia coli(ETEC) virulence regulator Rns, a member of the AraC/XylS family. The Rns homologs CfaD from ETEC and AggR from enteroaggregativeE. colialso repress expression ofnlpA. In vitro DNase I and potassium permanganate footprinting revealed that Rns binds to a site overlapping the start codon ofnlpA, preventing RNA polymerase from forming an open complex atnlpAp. A second Rns binding site between positions −152 and −195 relative to thenlpAtranscription start site is not required for repression. NlpA is not essential for growth ofE. coliunder laboratory conditions, but it does contribute to the biogenesis of outer membrane vesicles. As outer membrane vesicles have been shown to contain ETEC heat-labile toxin, the repression ofnlpAmay be an indirect mechanism through which the virulence regulators Rns and CfaD limit the release of toxin.
The rate of tension recovery in cardiac muscle correlates with the relative residual tension prevailing after restretch
Authors: Campbell Kenneth S. , Holbrook Anastasia M.
Published: December 2006

Isolated cardiac muscles generate tension more quickly at higher levels of Ca2+ activation. We investigated the molecular mechanisms underlying this effect in permeabilized rat myocardial preparations by measuring the rate of tension recovery following brief shortening/restretch perturbations. Separate series of experiments used Ca2+-activating solutions with different pH values (pH 6.75, 7.00, and 7.25) and different phosphate (Pi) concentrations (0, 2.5, and 5.0 mM added Pi) to modulate the recovery kinetics. Subsequent analysis showed that the rate of tension recovery correlated ( P < 0.001) with the relative residual tension, that is, the minimum tension measured immediately after restretch normalized to the steady-state isometric tension for the experimental condition. This new finding suggests that the rate at which cardiac muscles develop force increases with the proportion of cross bridges bound to the thin filament and is strong evidence of cooperative contractile activation.
Heart failure attenuates muscle metaboreflex control of ventricular contractility during dynamic exercise
Authors: Sala-Mercado Javier A. , Hammond Robert L. , Kim Jong-Kyung , McDonald Phillip J. , Stephenson Larry W. , O'Leary Donal S.
Published: December 2006

Underperfusion of active skeletal muscle elicits a reflex pressor response termed the muscle metaboreflex (MMR). In normal dogs during mild exercise, MMR activation causes large increases in cardiac output (CO) and mean arterial pressure (MAP); however, in heart failure (HF) although MAP increases, the rise in CO is virtually abolished, which may be due to an impaired ability to increase left ventricular contractility (LVC). The objective of the present study was to determine whether the increases in LVC seen with MMR activation during dynamic exercise in normal animals are abolished in HF. Conscious dogs were chronically instrumented to measure CO, MAP, and left ventricular (LV) pressure and volume. LVC was calculated from pressure-volume loop analysis [LV maximal elastance ( Emax) and preload-recruitable stroke work (PRSW)] at rest and during mild and moderate exercise under free-flow conditions and with MMR activation (via partial occlusion of hindlimb blood flow) before and after rapid ventricular pacing-induced HF. In control experiments, MMR activation at both workloads [mild exercise (3.2 km/h) and moderate exercise (6.4 km/h at 10% grade)] significantly increased CO, Emax, and PRSW. In contrast, after HF was induced, CO, Emax, and PRSW were significantly lower at rest. Although CO increased significantly from rest to exercise, Emax and PRSW did not change. In addition, MMR activation caused no significant change in CO, Emax, or PRSW at either workload. We conclude that MMR causes large increases in LVC in normal animals but that this ability is abolished in HF.
Pulmonary artery smooth muscle cells from normal subjects and IPAH patients show divergent cAMP-mediated effects on TRPC expression and capacitative Ca2+entry
Authors: Zhang Shen , Patel Hemal H. , Murray Fiona , Remillard Carmelle V. , Schach Christian , Thistlethwaite Patricia A. , Insel Paul. A. , Yuan Jason X.-J.
Published: December 2006

Pulmonary vascular remodeling due to overgrowth of pulmonary artery smooth muscle cells (PASMC) is a major cause for the elevated vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Increased cytosolic Ca2+concentration, resulting from enhanced capacitative Ca2+entry (CCE) and upregulated transient receptor potential (TRP) channel expression, is involved in stimulating PASMC proliferation. The current study was designed to determine the impact of cAMP, a second messenger that we hypothesized would blunt aspects of PASMC activity, as a possible contributor to IPAH pathophysiology. Short-term (30 min) pretreatment with forskolin (FSK; 10 μM), a direct activator of adenylyl cyclase, in combination with the cyclic nucleotide phosphodiesterase inhibitor isobutylmethylxanthine (IBMX; 200 μM), attenuated CCE in PASMC from normal subjects, patients without pulmonary hypertension (NPH), and patients with IPAH. The FSK-mediated CCE inhibition was independent of protein kinase A (PKA), because the PKA inhibitor H89 negligibly affected the decrease in CCE produced by cAMP. By contrast, longer (4 h) treatment with FSK (with IBMX) attenuated CCE in normal and NPH PASMC but enhanced CCE in IPAH PASMC. This enhancement of CCE was abolished by PKA inhibition and associated with an upregulation of TRPC3. In addition, cAMP increased TRPC1 mRNA expression in IPAH (but not in normal or NPH) PASMC, an effect blunted by H89. Furthermore, iloprost, a prostacyclin analog that increases cAMP, downregulated TRPC3 expression in IPAH PASMC and FSK-mediated cAMP increase inhibited IPAH PASMC proliferation. Although a rapid rise in cellular cAMP decreases CCE by a PKA-independent mechanism, sustained cAMP increase inhibits CCE in normal and NPH PASMC but increases CCE via a PKA-dependent pathway in IPAH PASMC. The divergent effect of cAMP on CCE parallels effects on TRPC expression. The results suggest that the combined use of a PKA inhibitor and cAMP-elevating drugs may provide a novel approach for treatment of IPAH.
Characterization of a human surfactant protein A1 (SP-A1) gene-specific antibody; SP-A1 content variation among individuals of varying age and pulmonary health
Authors: Tagaram Hephzibah Rani S. , Wang Guirong , Umstead Todd M. , Mikerov Anatoly N. , Thomas Neal J. , Graff Gavin R. , Hess Joseph C. , Thomassen Mary Jane , Kavuru Mani S. , Phelps David S. , Floros Joanna
Published: December 2006

The human surfactant protein A (SP-A) locus consists of two functional genes (SP-A1, SP-A2) with gene-specific products exhibiting qualitative and quantitative differences. The aim here was twofold: 1) generate SP-A1 gene-specific antibody, and 2) use this to assess gene-specific SP-A content in the bronchoalveolar lavage fluid (BALF). An SP-A1-specific polyclonal antibody (hSP-A1_Ab68-88_Col) was raised in chicken, and its specificity was determined by immunoblot and ELISA using mammalian Chinese hamster ovary (CHO) cell-expressed SP-A1 and SP-A2 variants and by immunofluorescence with stably transfected CHO cell lines expressing SP-A1 or SP-A2 variants. SP-A1 content was evaluated according to age and lung status. A gradual decrease ( P < 0.05) in SP-A1/SP-A ratio was observed in healthy subjects (HS) with increased age, although no significant change was observed in total SP-A content among age groups. Total SP-A and SP-A1 content differed significantly between alveolar proteinosis (AP) patients and HS, with no significant difference observed in SP-A1/SP-A ratio between AP and HS. The cystic fibrosis (CF) ratio was significantly higher compared with AP, HS, and noncystic fibrosis (NCF), even though SP-A1 and total SP-A were decreased in CF compared with most of the other groups. The ratio was higher in culture-positive vs. culture-negative samples from CF and NCF ( P = 0.031). A trend of an increased ratio was observed in culture-positive CF (0.590 ± 0.10) compared with culture-positive NCF (0.368 ± 0.085). In summary, we developed and characterized an SP-A1 gene-specific antibody and used it to identify gene-specific SP-A content in BALFs as a function of age and lung health.
IL-17 enhances IL-1β-mediated CXCL-8 release from human airway smooth muscle cells
Authors: Dragon Stéphane , Rahman Muhammad Shahidur , Yang Jie , Unruh Helmut , Halayko Andrew J. , Gounni Abdelilah Soussi
Published: December 2006

Recent studies into the pathogenesis of airway disorders such as asthma have revealed a dynamic role for airway smooth muscle cells in the perpetuation of airway inflammation via secretion of cytokines and chemokines. In this study, we evaluated whether IL-17 could enhance IL-1β-mediated CXCL-8 release from human airway smooth muscle cells (HASMC) and investigated the upstream and downstream signaling events regulating the induction of CXCL-8. CXCL-8 mRNA and protein induction were assessed by real-time RT-PCR and ELISA from primary HASMC cultures. HASMC transfected with site-mutated activator protein (AP)-1/NF-κB CXCL-8 promoter constructs were treated with selective p38, MEK1/2, and phosphatidylinositol 3-kinase (PI3K) inhibitors to determine the importance of MAPK and PI3K signaling pathways as well as AP-1 and NF-κB promoter binding sites. We demonstrate IL-17 induced and synergized with IL-1β to upregulate CXCL-8 mRNA and protein levels. Erk1/2 and p38 modulated IL-17 and IL-1β CXCL-8 promoter activity; however, IL-1β also activated the PI3K pathway. The synergistic response mediating CXCL-8 promoter activity was dependent on both MAPK and PI3K signal transduction pathways and required the cooperation of AP-1 and NF-κB cis-acting elements upstream of the CXCL-8 gene. Collectively, our observations indicate MAPK and PI3K pathways regulate the synergy of IL-17 and IL-1β to enhance CXCL-8 promoter activity, mRNA induction, and protein synthesis in HASMC via the cooperative activation of AP-1 and NF-κB trans-acting elements.
Collagen I and thrombin activate MMP-2 by MMP-14-dependent and -independent pathways: implications for airway smooth muscle migration
Authors: Henderson Neil , Markwick Lee J. , Elshaw Shona R. , Freyer Anette M. , Knox Alan J. , Johnson Simon Richard
Published: December 2006

Increased proinflammatory mediators and ECM deposition are key features of the airways in asthma. Matrix metalloproteinases (MMPs) are produced by airway smooth muscle (ASM) cells and have multiple roles in inflammation and tissue remodeling. We hypothesized that components of the asthmatic airway would stimulate MMP production and activation by ASM and contribute to airway remodeling. We measured human ASM-derived MMP mRNA, protein, and activity by real-time RT-PCR, zymography, Western blotting, and MMP activity assay. Collagen I and thrombin caused a synergistic increase in MMP-2 protein and total MMP activity but paradoxically decreased MMP-2 mRNA. Additionally, collagen I activated MMP-2 in culture supernatants independent of the cell surface. Together, collagen I and thrombin strongly enhanced MMP-14 mRNA and protein but had no effect individually, suggesting increased MMP-14, the activating protease for MMP-2, may be partially responsible for MMP-2 activation. Furthermore, collagen I reduced tissue inhibitor of metalloproteinase-2 protein (TIMP-2). We examined the role of MMPs in functions of ASM related to airway remodeling and found migration and proliferation were MMP dependent, whereas adhesion and apoptosis were not. Ilomastat inhibited migration by 25%, which was also inhibited by TIMPs 1–4 and increased by the MMP-2 activator thrombin. These in vitro findings suggest that the environment within the airways of patients with asthma enhances MMP-2 and -14 protein and activity by a complex interaction of transcriptional and posttranscriptional mechanisms, which may contribute to ASM migration.
Identification of poly(ADP-ribose)polymerase-1 and Ku70/Ku80 as transcriptional regulators of S100A9 gene expression
Authors: Grote Jens , König Simone , Ackermann Doreen , Sopalla Claudia , Benedyk Malgorzata , Los Marek , Kerkhoff Claus
Published: December 2006

Background

S100 proteins, a multigenic family of non-ubiquitous cytoplasmic Ca2+-binding proteins, have been linked to human pathologies in recent years. Dysregulated expression of S100 proteins, including S100A9, has been reported in the epidermis as a response to stress and in association with neoplastic disorders. Recently, we characterized a regulatory element within the S100A9 promotor, referred to as MRE that drives the S100A9 gene expression in a cell type-specific, activation- and differentiation-dependent manner (Kerkhoff et al. (2002) J. Biol. Chem. 277, 41879–41887).

Results

In the present study, we investigated transcription factors that bind to MRE. Using the MRE motif for a pull-down assay, poly(ADP-ribose)polymerase-1 (PARP-1) and the heterodimeric complex Ku70/Ku80 were identified by mass spectrometry and confirmed by chromatin immunoprecipitation. Furthermore, TPA-induced S100A9 gene expression in HaCaT keratinocytes was blocked after the pharmacologic inhibition of PARP-1 with 1,5-isoquinolinediol (DiQ).

Conclusion

The candidates, poly(ADP-ribose)polymerase-1 (PARP-1) and the heterodimeric complex Ku70/Ku80, are known to participate in inflammatory disorders as well as tumorgenesis. The latter may indicate a possible link between S100 and inflammation-associated cancer.
On the activity loss of hydrolases in organic solvents: II. a mechanistic study of subtilisin Carlsberg
Authors: Castillo Betzaida , Bansal Vibha , Ganesan Ashok , Halling Peter , Secundo Francesco , Ferrer Amaris , Griebenow Kai , Barletta Gabriel
Published: December 2006

Background

Enzymes have been extensively used in organic solvents to catalyze a variety of transformations of biological and industrial significance. It has been generally accepted that in dry aprotic organic solvents, enzymes are kinetically trapped in their conformation due to the high-energy barrier needed for them to unfold, suggesting that in such media they should remain catalytically active for long periods. However, recent studies on a variety of enzymes demonstrate that their initial high activity is severely reduced after exposure to organic solvents for several hours. It was speculated that this could be due to structural perturbations, changes of the enzyme's pH memory, enzyme aggregation, or dehydration due to water removal by the solvents. Herein, we systematically study the possible causes for this undesirable activity loss in 1,4-dioxane.

Results

As model enzyme, we employed the protease subtilisin Carlsberg, prepared by lyophilization and colyophilization with the additive methyl-β-cyclodextrin (MβCD). Our results exclude a mechanism involving a change in ionization state of the enzyme, since the enzyme activity shows a similar pH dependence before and after incubation for 5 days in 1,4-dioxane. No apparent secondary or tertiary structural perturbations resulting from prolonged exposure in this solvent were detected. Furthermore, active site titration revealed that the number of active sites remained constant during incubation. Additionally, the hydration level of the enzyme does not seem to affect its stability. Electron paramagnetic resonance spectroscopy studies revealed no substantial increase in the rotational freedom of a paramagnetic nitroxide inhibitor bound to the active site (a spin-label) during incubation in neat 1,4-dioxane, when the water activity was kept constant using BaBr2 hydrated salts. Incubation was also accompanied by a substantial decrease in Vmax/KM.

Conclusion

These results exclude some of the most obvious causes for the observed low enzyme storage stability in 1,4-dioxane, mainly structural, dynamics and ionization state changes. The most likely explanation is possible rearrangement of water molecules within the enzyme that could affect its dielectric environment. However, other mechanisms, such as small distortions around the active site or rearrangement of counter ions, cannot be excluded at this time.
CESAR: conventional ventilatory support vs extracorporeal membrane oxygenation for severe adult respiratory failure
Authors: Peek Giles J , Clemens Felicity , Elbourne Diana , Firmin Richard , Hardy Pollyanna , Hibbert Clare , Killer Hilliary , Mugford Miranda , Thalanany Mariamma , Tiruvoipati Ravin , Truesdale Ann , Wilson Andrew
Published: December 2006

Background

An estimated 350 adults develop severe, but potentially reversible respiratory failure in the UK annually. Current management uses intermittent positive pressure ventilation, but barotrauma, volutrauma and oxygen toxicity can prevent lung recovery. An alternative treatment, extracorporeal membrane oxygenation, uses cardio-pulmonary bypass technology to temporarily provide gas exchange, allowing ventilator settings to be reduced. While extracorporeal membrane oxygenation is proven to result in improved outcome when compared to conventional ventilation in neonates with severe respiratory failure, there is currently no good evidence from randomised controlled trials to compare these managements for important clinical outcomes in adults, although evidence from case series is promising.

Methods/Design

The aim of the randomised controlled trial of Conventional ventilatory support vs extracorporeal membrane oxygenation for severe adult respiratory failure (CESAR) is to assess whether, for patients with severe, but potentially reversible, respiratory failure, extracorporeal membrane oxygenation will increase the rate of survival without severe disability ('confined to bed' and 'unable to wash or dress') by six months post-randomisation, and be cost effective from the viewpoints of the NHS and society, compared to conventional ventilatory support. Following assent from a relative, adults (18–65 years) with severe, but potentially reversible, respiratory failure (Murray score ≥ 3.0 or hypercapnea with pH < 7.2) will be randomised for consideration of extracorporeal membrane oxygenation at Glenfield Hospital, Leicester or continuing conventional care in a centre providing a high standard of conventional treatment. The central randomisation service will minimise by type of conventional treatment centre, age, duration of high pressure ventilation, hypoxia/hypercapnea, diagnosis and number of organs failed, to ensure balance in key prognostic variables. Extracorporeal membrane oxygenation will not be available for patients meeting entry criteria outside the trial. 180 patients will be recruited to have 80% power to be able to detect a one third reduction in the primary outcome from 65% at 5% level of statistical significance (2-sided test). Secondary outcomes include patient morbidity and health status at 6 months.

Discussion

Analysis will be based on intention to treat. A concurrent economic evaluation will also be performed to compare the costs and outcomes of both treatments.
Reactive oxygen species induce expression of vascular endothelial growth factor in chondrocytes and human articular cartilage explants
Authors: Fay Jakob , Varoga Deike , Wruck Christoph J , Kurz Bodo , Goldring Mary B , Pufe Thomas
Published: December 2006

Vascular endothelial growth factor (VEGF) promotes cartilage-degrading pathways, and there is evidence for the involvement of reactive oxygen species (ROS) in cartilage degeneration. However, a relationship between ROS and VEGF has not been reported. Here, we investigate whether the expression of VEGF is modulated by ROS.

Aspirates of synovial fluid from patients with osteoarthritis (OA) were examined for intra-articular VEGF using ELISA. Immortalized C28/I2 chondrocytes and human knee cartilage explants were exposed to phorbol myristate acetate (PMA; 0–20 μg/ml), which is a ROS inducer, or 3-morpholino-sydnonimine hydrochloride (SIN-1; 0–20 μM), which is a ROS donor. The levels of VEGF protein and nitric oxide (NO) production were determined in the medium supernatant, using ELISA and Griess reagent, respectively. Gene expression of VEGF-121 and VEGF-165 was determined by splice variant RT-PCR. Expression of VEGF and VEGF receptors (VEGFR-1 and VEGFR-2) was quantified by real-time RT-PCR.

Synovial fluid from OA patients revealed markedly elevated levels of VEGF. Common RT-PCR revealed that the splice variants were present in both immortalized chondrocytes and cartilage discs. In immortalized chondrocytes, stimulation with PMA or SIN-1 caused increases in the levels of VEGF, VEGFR-1 and VEGFR-2 mRNA expression. Cartilage explants produced similar results, but VEGFR-1 was only detectable after stimulation with SIN-1. Stimulation with PMA or SIN-1 resulted in a dose-dependent upregulation of the VEGF protein (as determined using ELISA) and an increase in the level of NO in the medium.

Our findings indicate ROS-mediated induction of VEGF and VEGF receptors in chondrocytes and cartilage explants. These results demonstrate a relationship between ROS and VEGF as multiplex mediators in articular cartilage degeneration.
Scenario Formulation of Stochastic Linear Programs and the Homogeneous Self-Dual Interior-Point Method
Authors: Sun Jie , Liu Xinwei
Published: December 2006

We consider a homogeneous self-dual interior-point algorithm for solving multistage stochastic linear programs. The algorithm is particularly suitable for the so-called “scenario formulation” of the problem, whose constraint system consists of a large block-diagonal matrix together with a set of sparse nonanticipativity constraints. Due to this structure, the major computational work required by the homogeneous self-dual interior-point method can be split into three steps, each of which is highly decomposable. Numerical results on some randomly generated problems and a multistage production-planning problem are reported.
Analyzing Document-Duplication Effects on Policies for Browser and Proxy Caching
Authors: Tan Yong , Ji Yonghua , Mookerjee Vijay S.
Published: December 2006

Browser and proxy-server caching are effective and relatively inexpensive methods of improving Web performance. Most existing research considers caching to occur independently at the browser and the proxy server. When the browser and the proxy-server cache independently, documents may get duplicated across the two levels. This paper analyzes the impact of document duplication on the performance of several browser-proxy caching policies. We first derive an exact expression and an accurate approximation for the delay under a joint browser-proxy caching policy in which no duplication is permitted. This policy is compared to a base or benchmark policy in which caching occurs independently at the two levels, and hence, duplication of documents is freely permitted. We next propose a more general caching policy in which a controlled amount of duplication is permitted. This policy is analyzed and an exact expression and an approximate expression for performance are derived. Finally, a simulation study is performed to confirm the accuracy of the theoretical results and extend these results for situations that are difficult to analyze mathematically.
Maximally Violated Mod-p Cuts for the Capacitated Vehicle-Routing Problem
Authors: Reinelt Gerhard , Wenger Klaus M.
Published: December 2006

This paper makes a contribution to the branch and cut approach to the capacitated vehicle-routing problem (CVRP). In the CVRP, the demands of a set of customers have to be met at minimum total travel cost using vehicles of identical capacity based at a single depot. The potential of maximally violated mod-p cutting-planes (Caprara et al. 2000) for the CVRP is investigated via a computational study. The foundation of the assessment is formed by classes of problem-specific constraints taken from the literature. In several separation algorithms for the CVRP, it is advantageous to shrink inclusionwise maximal minimum-weight cuts in support graphs as preprocessing. It is mentioned how a partition of the set of customers into such mincuts can be computed in a fast and elegant way using the mincut algorithm of Hao and Orlin (1994). Interestingly, maximally violated mod-p cuts, which are general-purpose cuts of Chvátal-Gomory type, stand comparison with problem-specific cuts for the CVRP and they are clearly useful on top of such cuts. The first-time proven optimal solution of the CVRP instance B-n68-k9 is reported. The computation used a branching strategy with far lookahead and relied on maximally violated mod-p cuts. This paper on maximally violated cuts belongs to a set of papers originating from Applegate et al. (1995) where a separation of maximally violated combs for the traveling-salesman problem (TSP) is suggested.
FBP: A Frontier-Based Tree-Pruning Algorithm
Authors: Huo Xiaoming , Kim Seoung Bum , Tsui Kwok-Leung , Wang Shuchun
Published: December 2006

A frontier-based tree-pruning algorithm (FBP) is proposed. The new method has an order of computational complexity comparable to cost-complexity pruning (CCP). Regarding tree pruning, it provides a full spectrum of information: namely, (1) given the value of the penalization parameter λ, it gives the decision tree specified by the complexity-penalization approach; (2) given the size of a decision tree, it provides the range of the penalization parameter λ, within which the complexity-penalization approach renders this tree size; (3) it finds the tree sizes that are inadmissible—no matter what the value of the penalty parameter is, the resulting tree based on a complexity-penalization framework will never have these sizes. Simulations on real data sets reveal a “surprise:” in the complexity-penalization approach, most of the tree sizes are inadmissible. FBP facilitates a more faithful implementation of cross validation (CV), which is favored by simulations. Using FBP, a stability analysis of CV is proposed.
A Unified Framework for Numerically Inverting Laplace Transforms
Authors: Abate Joseph , Whitt Ward
Published: December 2006

We introduce and investigate a framework for constructing algorithms to invert Laplace transforms numerically. Given a Laplace transform

\hat{f}

of a complex-valued function of a nonnegative real-variable, f, the function f is approximated by a finite linear combination of the transform values; i.e., we use the inversion formula

f(t) \approx f_n (t) \equiv \frac{1}{t} \sum_{k = 0}^{n}\omega_{k}\hat{f}\biggl(\frac{\alpha_{k}}{t}\biggr),\quad 0 < t < \infty,

where the weights ωk and nodes αk are complex numbers, which depend on n, but do not depend on the transform

\hat{f}

or the time argument t. Many different algorithms can be put into this framework, because it remains to specify the weights and nodes. We examine three one-dimensional inversion routines in this framework: the Gaver-Stehfest algorithm, a version of the Fourier-series method with Euler summation, and a version of the Talbot algorithm, which is based on deforming the contour in the Bromwich inversion integral. We show that these three building blocks can be combined to produce different algorithms for numerically inverting two-dimensional Laplace transforms, again all depending on the single parameter n. We show that it can be advantageous to use different one-dimensional algorithms in the inner and outer loops.
Achieving Success in Large Projects: Implications from a Study of ERP Implementations
Authors: Ferratt Thomas W. , Ahire Sanjay , De Prabuddha
Published: December 2006

Executives in charge of large projects must decide how to spend their energies, even though typically they are not trained to manage such projects. We have derived two implications for managers based on prior research: adhere to the fundamentals of project management and unearth the best practices for large-project success. Through a study of more than 70 enterprise-resource-planning (ERP) projects, we have investigated our hypothesis that greater success in implementation is related to greater adoption of the best practices. For most of the participants in our study, our hypothesized model holds. For some, however, careful deviation from this model also proved successful. Additional implications we have derived include recommendations to specify a model of the project outcomes, understand the factors that make a project large and risky, and include a focus on managing large projects in executive education and development.
Montgomery County’s Public Health Service Uses Operations Research to Plan Emergency Mass Dispensing and Vaccination Clinics
Authors: Aaby Kay , Herrmann Jeffrey W. , Jordan Carol S. , Treadwell Mark , Wood Kathy
Published: December 2006

To curb outbreaks of contagious diseases, county health departments must set up and operate clinics to dispense medications and vaccines. Carefully planning these clinics in advance of such an event is difficult and important. We developed and implemented operations research models to improve clinic planning for the Montgomery County (Maryland) Public Health Services. They include discrete-event simulation models and capacity-planning and queueing-system models. We validated these models using data that we collected during full-scale simulations of disease outbreaks. We also developed guidelines for the physical design of clinics based on general queueing principles and our own experiences.
Supply Chain Collaboration Through Shared Capacity Models
Authors: Shirodkar Shamin , Kempf Karl
Published: December 2006

Collaboration between companies in a supply chain has been the subject of a great deal of discussion supported by a surprisingly small number of documented successful implementations. To help Intel to procure production materials, we applied multiple mathematical optimization models, each facilitated by shared capacity models developed in collaboration with material suppliers. One decision tool supports the weekly process of ordering materials. A second tool forecasts capacity rearrangements that suppliers need to consider over the next few quarters. Supplier capacity expansion spanning the next few years is the focus of a third tool. All parties have realized productivity and profitability gains using this set of integrated tools.