bluuebunny/crossref_metadata_2025_split huggingface dataset:
Impact of
mutS
Inactivation on Foreign DNA Acquisition by Natural Transformation in
Pseudomonas stutzeri
Authors: Meier Petra , Wackernagel Wilfried 
Published: December 2004

ABSTRACT

In prokaryotic mismatch repair the MutS protein and its homologs recognize the mismatches. The
mutS
gene of naturally transformable
Pseudomonas stutzeri
ATCC 17587 (genomovar 2) was identified and characterized. The deduced amino acid sequence (859 amino acids; 95.6 kDa) displayed protein domains I to IV and a mismatch-binding motif similar to those in MutS of
Escherichia coli
. A
mutS
::
aac
mutant showed 20- to 163-fold-greater spontaneous mutability. Transformation experiments with DNA fragments of
rpoB
containing single nucleotide changes (providing rifampin resistance) indicated that mismatches resulting from both transitions and transversions were eliminated with about 90% efficiency in
mutS
+
. The
mutS
+
gene of strain ATCC 17587 did not complement an
E. coli
mutant but partially complemented a
P. stutzeri
JM300 mutant (genomovar 4). The declining heterogamic transformation by DNA with 0.1 to 14.6% sequence divergence was partially alleviated by
mutS
::
aac
, indicating that there was a 14 to 16% contribution of mismatch repair to sexual isolation. Expression of
mutS
+
from a multicopy plasmid eliminated autogamic transformation and greatly decreased heterogamic transformation, suggesting that there is strong limitation of MutS in the wild type for marker rejection. Remarkably,
mutS
::
aac
altered foreign DNA acquisition by homology-facilitated illegitimate recombination (HFIR) during transformation, as follows: (i) the mean length of acquired DNA was increased in transformants having a net gain of DNA, (ii) the HFIR events became clustered (hot spots) and less dependent on microhomologies, which may have been due to topoisomerase action, and (iii) a novel type of transformants (14%) had integrated foreign DNA with no loss of resident DNA. We concluded that in
P. stutzeri
upregulation of MutS could enforce sexual isolation and downregulation could increase foreign DNA acquisition and that MutS affects mechanisms of HFIR.
Glutathione Plays a Fundamental Role in Growth and Symbiotic Capacity of
Sinorhizobium meliloti
Authors: Harrison Judith , Jamet Alexandre , Muglia Cecilia I. , Van de Sype Ghislaine , Aguilar O. Mario , Puppo Alain , Frendo Pierre 
Published: December 2004

ABSTRACT

Rhizobia form a symbiotic relationship with plants of the legume family to produce nitrogen-fixing root nodules under nitrogen-limiting conditions. We have examined the importance of glutathione (GSH) during free-living growth and symbiosis of
Sinorhizobium meliloti
. An
S. meliloti
mutant strain (SmgshA) which is unable to synthesize GSH due to a gene disruption in
gshA
, encoding the enzyme for the first step in the biosynthesis of GSH, was unable to grow under nonstress conditions, precluding any nodulation. In contrast, an
S. meliloti
strain (SmgshB) with
gshB
, encoding the enzyme involved in the second step in GSH synthesis, deleted was able to grow, indicating that γ-glutamylcysteine, the dipeptide intermediate, can partially substitute for GSH. However, the SmgshB strain showed a delayed-nodulation phenotype coupled to a 75% reduction in the nitrogen fixation capacity. This phenotype was linked to abnormal nodule development. Both the SmgshA and SmgshB mutant strains exhibited higher catalase activity than the wild-type
S. meliloti
strain, suggesting that both mutant strains are under oxidative stress. Taken together, these results show that GSH plays a critical role in the growth of
S. meliloti
and during its interaction with the plant partner.
Molecular Analysis of the
Vibrio cholerae
Type II Secretion ATPase EpsE
Authors: Camberg Jodi L. , Sandkvist Maria 
Published: December 2004

ABSTRACT

The
type II secretion system is a macromolecular assembly that
facilitates the extracellular translocation of folded proteins in
gram-negative bacteria. EpsE, a member of this secretion system in
Vibrio cholerae
, contains a nucleotide-binding motif
composed of Walker A and B boxes that are thought to participate in
binding and hydrolysis of ATP and displays structural homology
to other transport ATPases. Here we demonstrate that purified
EpsE is an Mg
2+
-dependent ATPase and define
optimal conditions for the hydrolysis reaction. EpsE displays
concentration-dependent activity, which may suggest that the active
form is oligomeric. Size exclusion chromatography showed that the
majority of purified EpsE is monomeric; however, detailed analyses of
specific activities obtained following gel filtration revealed the
presence of a small population of active oligomers. We further report
that EpsE binds zinc through a tetracysteine motif near its carboxyl
terminus, yet metal displacement assays suggest that zinc is not
required for catalysis. Previous studies describing interactions
between EpsE and other components of the type II secretion pathway
together with these data further support the hypothesis that EpsE
functions to couple energy to the type II apparatus, thus enabling
secretion.
pH Regulates Genes for Flagellar Motility, Catabolism, and Oxidative Stress inEscherichia coliK-12
Authors: Maurer Lisa M. , Yohannes Elizabeth , Bondurant Sandra S. , Radmacher Michael , Slonczewski Joan L. 
Published: December 2004

ABSTRACTGene expression profiles ofEscherichia coliK-12 W3110 were compared as a function of steady-state external pH. Cultures were grown to an optical density at 600 nm of 0.3 in potassium-modified Luria-Bertani medium buffered at pH 5.0, 7.0, and 8.7. For each of the three pH conditions, cDNA from RNA of five independent cultures was hybridized to AffymetrixE.coliarrays. Analysis of variance with an α level of 0.001 resulted in 98% power to detect genes showing a twofold difference in expression. Normalized expression indices were calculated for each gene and intergenic region (IG). Differential expression among the three pH classes was observed for 763 genes and 353 IGs. Hierarchical clustering yielded six well-defined clusters of pH profiles, designated Acid High (highest expression at pH 5.0), Acid Low (lowest expression at pH 5.0), Base High (highest at pH 8.7), Base Low (lowest at pH 8.7), Neutral High (highest at pH 7.0, lower in acid or base), and Neutral Low (lowest at pH 7.0, higher at both pH extremes). Flagellar and chemotaxis genes were repressed at pH 8.7 (Base Low cluster), where the cell's transmembrane proton potential is diminished by the maintenance of an inverted pH gradient. High pH also repressed the proton pumps cytochrome o (cyo) and NADH dehydrogenases I and II. By contrast, the proton-importing ATP synthase F1Foand the microaerophilic cytochromed(cyd), which minimizes proton export, were induced at pH 8.7. These observations are consistent with a model in which high pH represses synthesis of flagella, which expend proton motive force, while stepping up electron transport and ATPase components that keep protons inside the cell. Acid-induced genes, on the other hand, were coinduced by conditions associated with increased metabolic rate, such as oxidative stress. All six pH-dependent clusters included envelope and periplasmic proteins, which directly experience external pH. Overall, this study showed that (i) low pH accelerates acid consumption and proton export, while coinducing oxidative stress and heat shock regulons; (ii) high pH accelerates proton import, while repressing the energy-expensive flagellar and chemotaxis regulons; and (iii) pH differentially regulates a large number of periplasmic and envelope proteins.
Cold Shock of a Hyperthermophilic Archaeon:
Pyrococcus furiosus
Exhibits Multiple Responses to a Suboptimal Growth Temperature with a Key Role for Membrane-Bound Glycoproteins
Authors: Weinberg Michael V. , Schut Gerrit J. , Brehm Scott , Datta Susmita , Adams Michael W. W. 
Published: December 2004

ABSTRACT

The hyperthermophilic archaeon,
Pyrococcus furiosus
, was grown on maltose near its optimal growth temperature, 95°C, and at the lower end of the temperature range for significant growth, 72°C. In addition, cultures were shocked by rapidly dropping the temperature from 95 to 72°C. This resulted in a 5-h lag phase, during which time little growth occurred. Transcriptional analyses using whole-genome DNA microarrays representing 2,065 open reading frames (ORFs) in the
P. furiosus
genome showed that cells undergo three very different responses at 72°C: an early shock (1 to 2 h), a late shock (5 h), and an adapted response (occurring after many generations at 72°C). Each response involved the up-regulation in the expression of more than 30 ORFs unique to that response. These included proteins involved in translation, solute transport, amino acid biosynthesis, and tungsten and intermediary carbon metabolism, as well as numerous conserved-hypothetical and/or membrane-associated proteins. Two major membrane proteins were evident after one-dimensional sodium dodecyl sulfate-gel analysis of cold-adapted cells, and staining revealed them to be glycoproteins. Their cold-induced expression evident from the DNA microarray analysis was confirmed by quantitative PCR. Termed CipA (PF0190) and CipB (PF1408), both appear to be solute-binding proteins. While the archaea do not contain members of the bacterial cold shock protein (Csp) family, they all contain homologs of CipA and CipB. These proteins are also related phylogenetically to some cold-responsive genes recently identified in certain bacteria. The Cip proteins may represent a general prokaryotic-type cold response mechanism that is present even in hyperthermophilic archaea.
Ler Is a Negative Autoregulator of theLEE1Operon in EnteropathogenicEscherichia coli
Authors: Berdichevsky Tatiana , Friedberg Devorah , Nadler Chen , Rokney Assaf , Oppenheim Amos , Rosenshine Ilan 
Published: December 2004

ABSTRACTEnteropathogenicEscherichia coli(EPEC) causes severe diarrhea in young children. Essential for colonization of the host intestine is the LEE pathogenicity island, which comprises a cluster of operons encoding a type III secretion system and related proteins. TheLEE1operon encodes Ler, which positively regulates many EPEC virulence genes in the LEE region and elsewhere in the chromosome. We found that Ler acts as a specific autorepressor ofLEE1transcription. We further show that Ler specifically binds upstream of theLEE1operon in vivo and in vitro. A comparison of the Ler affinities to different DNA regions suggests that the autoregulation mechanism limits the steady-state level of Ler to concentrations that are just sufficient for activation of theLEE2andLEE3promoters and probably other LEE promoters. This mechanism may reflect the need of EPEC to balance maximizing the colonization efficiency by increasing the expression of the virulence genes and minimizing the immune response of the host by limiting their expression. In addition, we found that the autoregulation mechanism reduces the cell-to-cell variability in the levels ofLEE1expression. Our findings point to a new negative regulatory circuit that suppresses the noise and optimizes the expression levels oflerand otherLEE1genes.
Expression and Characterization of a Soluble, Active Form of the Jaagsiekte Sheep Retrovirus Receptor, Hyal2
Authors: Vigdorovich Vladimir , Strong Roland K. , Miller A. Dusty 
Published: December 2004

ABSTRACTRetrovirus entry into cells is mediated by specific interactions between virus envelope glycoproteins and cell surface receptors. Many of these receptors contain multiple membrane-spanning regions, making their purification and study difficult. The jaagsiekte sheep retrovirus (JSRV) receptor, hyaluronidase 2 (Hyal2), is a glycosylphosphatidylinositol (GPI)-anchored molecule containing no peptide transmembrane regions, making it an attractive candidate for study of retrovirus entry. Further, the hyaluronidase activity reported for human Hyal2, combined with its broad expression pattern, may point to a critical function of Hyal2 in the turnover of hyaluronan, a major extracellular matrix component. Here we describe the properties of a soluble form of human Hyal2 (sHyal2) purified from a baculoviral expression system. sHyal2 is a 54-kDa monomer with weak hyaluronidase activity compared to that of the known hyaluronidase Spam1. In contrast to a previous report indicating that Hyal2 cleaved hyaluronan to a limit product of 20 kDa and was active only at acidic pH, we find that sHyal2 is capable of further degradation of hyaluronan and is active over a broad pH range, consistent with Hyal2 being active at the cell surface where it is normally localized. Interaction of sHyal2 with the JSRV envelope glycoprotein was analyzed by viral inhibition assays, showing >90% inhibition of transduction at 28 nM sHyal2, and by surface plasmon resonance, revealing a remarkably tight specific interaction with a dissociation constant (KD) of 32 ± 1 pM. In contrast to results obtained with avian retroviruses, purified receptor was not capable of promoting transduction of cells that do not express the virus receptor.
Transforming The "Market-Model University": Environmental Philosophy, Citizenship And The Recovery of the Humanities
Authors: Scott Dane 
Published: December 2004

AbstractThis paper addresses the problem of the declining status of philosophy and the humanities in higher education. These events have given rise to discussions of the "corporatization" of higher education and the dominance of the "Market-Model University." In the Market-Model University the chief goals are to teach marketable skills and develop marketable innovations. In responding to these developments, my thesis is that practical philosophy, exemplified by environmental philosophy, off ers direction for positive reforms that will revitalize philosophy and the humanities. (While my comments focus on philosophy, I believe much of what is said can apply to other fields in the humanities.) More specifically, they point to a "Citizen-Model University" as a positive alternative to the Market-Model. In the Citizen-Model University the chief goal is education for "citizenship". This focus in education will allow democratic societies to better meet the challenges of the twenty-first century. Such challenges are increasingly characterized by difficult decisions involving environmental problems (e.g., global warming) and technological developments (e.g., biotechnology). These kinds of problems cannot be left to the "invisible hand" of the market. They require democratic citizens capable of moral deliberation, leading to intelligent choices.
Catching the "Third Wave" of Democratization?: Debating Political Party Effectiveness in Africa Since 1980
Authors: McMahon Edward R. 
Published: December 2004

AbstractMany observers believe that multi-party democracy increasingly represents the inevitable future of governance around the world, including Africa. Some countries such as South Africa, Ghana, Kenya, and Senegal have in fact made remarkable progress in instituting and moving toward consolidation of democratic systems. There has also been a history on the continent, however, of political systems that place de facto or de jure legal constraints on the ability of political parties to function. In fact, in recent years many African leaders have only grudgingly permitted multi-party politics under donor pressure. There remains a current of underlying skepticism toward political parties, and arguments exist against multi-party politics. This paper identifies and explains five key arguments. It then critiques them and determines that while individual elements of these arguments may have some validity, the conclusion that is drawn, i.e.that party activity should be constrained, if not prohibited, is not consonant with democratic governance. The final section presents suggestions of how weaknesses in political party functioning could be addressed without placing limits on the ability of parties to play their legitimate role in a democratic political process.
A Physically Motivated Modification of the Strain Equivalence Approach
Authors: Olsson Mattias , Ristinmaa Matti 
Published: December 2004

A phenomenological thermodynamic approach used for describing elasto-plasticity coupled with isotropic damage is considered. Using this approach it was recently shown by the authors that certain unwanted features exist in the plasticity-induced damage theories. This paper gives a suggestion on how to solve these issues. Based on physical arguments a simple modification of the postulate of strain equivalence is suggested. To show the effects of the modification, the von Mises plasticity model coupled with isotropic damage is used as a prototype model in the derivations. Both analytical considerations and simulations of a strain-controlled uniaxial model are performed. The results reveal that the modification of the postulate of strain equivalence gives promising results. In contrast to the postulates of strain equivalence and (complementary) energy equivalence, the present formulation predicts the elastic strain to vanish when failure takes place.
Laser Fusion to Impart Cohesion to Textured Filament Yarns
Authors: Acar M. , Dudeney W. L. , Jones J. , Jackson M. R. , Malalasekera W. 
Published: December 2004

The conventional intermingling process uses a high-speed air jet, positioned in the path of the yarn being textured; to create knot-like intermittent entangled nodes (‘nips’ or ‘tacks’) in the textured thermoplastic continuous multi-filament yarn. This paper reports a proof-of-concept research into the use of a pulse laser to fuse such yarns at discrete points at regular intervals along its length to impart cohesion at required frequencies. Pulse frequency and duration, hence the heat intensity, are varied to determine a suitable range of operating conditions. Results from a range of polyester and nylon yarns show that fusing textured filament yarn to create nips is feasible using a pulse laser as an alternative to intermingling by air jets. The multifilament yarn is also modeled to compute the heat flow in the yarn when heated by a pulse laser. This model can be used, as a tool to estimate the heat energy required to fuse a given textured yarn.
Myeloid-related proteins 8 and 14 induce a specific inflammatory response in human microvascular endothelial cells
Authors: Viemann Dorothee , Strey Anke , Janning Annette , Jurk Kerstin , Klimmek Kerstin , Vogl Thomas , Hirono Keiichi , Ichida Fukiko , Foell Dirk , Kehrel Beate , Gerke Volker , Sorg Clemens , Roth Johannes 
Published: December 2004

AbstractMyeloid-related protein 8 (MRP8) and MRP14, S100 proteins secreted by activated phagocytes, bind specifically to endothelial cells. The endothelial response to MRP8/MRP14, however, is unknown. Using oligonucleotide microarray analysis, we show for the first time that MRP8/MRP14 induce a thrombogenic, inflammatory response in human microvascular endothelial cells by increasing the transcription of proinflammatory chemokines and adhesion molecules and by decreasing the expression of cell junction proteins and molecules involved in monolayer integrity. All changes on the gene expression level could be confirmed using biochemical and functional assays. We demonstrated that the expression of MRP8/MRP14 closely correlated with the inflammatory activity in systemic vasculitis, confirming the important role of these proteins for distinct inflammatory reactions in endothelia. MRP8/MRP14 may represent novel targets for anti-inflammatory strategies.
A balance between tissue factor and tissue factor pathway inhibitor is required for embryonic development and hemostasis in adult mice
Authors: Pedersen Brian , Holscher Todd , Sato Yuichiro , Pawlinski Rafal , Mackman Nigel 
Published: December 2004

AbstractInactivation of the murine tissue factor (TF) gene or tissue factor pathway inhibitor 1 (TFPI) gene results in embryonic lethality, indicating that both are required for embryonic development. We have shown that expression of low levels of TF from a transgene (hTF) rescues TF-null embryos. However, low-TF mice (mTF–/–/hTF+) have hemostatic defects in the uterus, placenta, heart, and lung. In this study, we hypothesized that the death of TFPI–/– embryos was due to unregulated TF/FVIIa activity and that the hemostatic defects in low-TF mice were due to insufficient TF expression. Therefore, we attempted to rescue TFPI–/– embryos by reducing TF expression, and to restore hemostasis in low-TF mice by abolishing TFPI expression. Intercrossing TFPI+/–/mTF+/–/hTF+/– mice generated close to the expected number of TFPI–/–/low-TF mice at weaning age from 128 offspring, indicating rescue of TFPI–/– embryos from embryonic lethality. Conversely, a decrease in TFPI levels dose-dependently prolonged the survival of low-TF mice and rescued the hemorrhagic defects in the lung and placenta but not in the heart or uterus. These results indicate that the correct balance between TF and TFPI in different organs is required to maintain hemostasis during embryonic development and in adult mice.
Effect of Single-Agent Rituximab Given at the Standard Schedule or As Prolonged Treatment in Patients With Mantle Cell Lymphoma: A Study of the Swiss Group for Clinical Cancer Research (SAKK)
Authors: Ghielmini Michele , Schmitz Shu-Fang Hsu , Cogliatti Sergio , Bertoni Francesco , Waltzer Ursula , Fey Martin F. , Betticher Daniel C. , Schefer Hubert , Pichert Gabriella , Stahel Rolf , Ketterer Nicolas , Bargetzi Mario , Cerny Thomas 
Published: December 2004

Purpose To evaluate the effect of single-agent rituximab given at the standard or a prolonged schedule in patients with newly diagnosed, or refractory or relapsed mantle cell lymphoma (MCL). Patients and Methods After induction treatment with the standard schedule (375 mg/m2 weekly × 4), patients who were responding or who had stable disease at week 12 from the start of treatment were randomly assigned to no further treatment (arm A) or prolonged rituximab administration (375 mg/m2) every 8 weeks for four times (arm B). Results The trial enrolled 104 patients. After induction, clinical response was 27% with 2% complete responses. Among patients with detectable t(11;14)-positive cells in blood and bone marrow at baseline, four of 20, and one of 14, respectively, became polymerase chain-reaction–negative after induction. Anemia was the only adverse predictor of response in the multivariate analysis. After a median follow-up of 29 months, response rate and duration of response were not significantly different between the two schedules in 61 randomly assigned patients. Median event-free survival (EFS) was 6 months in arm A versus 12 months in arm B; the difference was not significant (P = .1). Prolonged treatment seemed to improve EFS in the subgroup of pretreated patients (5 months in arm A v 11 months in arm B; P = .04). Thirteen percent of patients in arm A and 9% in arm B presented with grade 3 to 4 hematologic toxicity. Conclusion Single-agent rituximab is active in MCL, but the addition of four single doses at 8-week intervals does not seem to significantly improve response rate, duration of response, or EFS after treatment with the standard schedule.