bluuebunny/crossref_metadata_2025 huggingface dataset:
A Temperature-based Controller for a Shape Memory Alloy Actuator
Authors: Elahinia Mohammad H. , Ashrafiuon Hashem , Ahmadian Mehdi , Tan Hanghao
Published: May 2005

This paper presents a robust nonlinear control that uses a state variable estimator for control of a single degree of freedom rotary manipulator actuated by shape memory alloy (SMA) wire. A model for SMA actuated manipulator is presented. The model includes nonlinear dynamics of the manipulator, a constitutive model of the shape memory alloy, and the electrical and heat transfer behavior of SMA wire. The current experimental setup allows for the measurement of only one state variable which is the angular position of the arm. Due to measurement difficulties, the other three state variables, arm angular velocity and SMA wire stress and temperature, cannot be directly measured. A model-based state estimator that works with noisy measurements is presented based on the extended Kalman filter (EKF). This estimator estimates the state vector at each time step and corrects its estimation based on the angular position measurements. The estimator is then used in a nonlinear and robust control algorithm based on variable structure control (VSC). The VSC algorithm is a control gain switching technique based on the arm angular position (and velocity) feedback and EKF estimated SMA wire stress and temperature. Using simulation it is shown that the state vector estimates help reduce or avoid the undesirable and inefficient overshoot problem in SMA one-way actuation control.
The Case for Hydrogen in a Carbon Constrained World
Authors: Berry Gene D. , Aceves Salvador M.
Published: May 2005

Unlike other fuels, hydrogen (H2) can be generated and consumed without generating carbon dioxide (CO2). This creates both significant engineering challenges and unsurpassed ecological advantages for H2 as a fuel, while enabling an inexhaustible (closed) global fuel cycle based on the cleanest, most abundant, natural, and elementary substances: H2, O2, and H2O. If generated using light, heat, and/or electrical energy from solar, wind, fission, or (future) fusion power sources, H2 becomes a versatile, storable, and universal carbonless energy carrier, a necessary element for future global energy system(s) aimed at being free of air and water pollution, CO2, and other greenhouse gases. The case for hydrogen rests fundamentally on the need to eliminate pollution and stabilize Earth’s atmosphere and climate system.
BordetellaAlcS Transporter Functions in Alcaligin Siderophore Export and Is Central to Inducer Sensing in Positive Regulation of Alcaligin System Gene Expression
Authors: Brickman Timothy J. , Armstrong Sandra K.
Published: May 2005

Bordetella pertussisandBordetella bronchiseptica, which are respiratory mucosal pathogens of mammals, produce and utilize the siderophore alcaligin to acquire iron in response to iron starvation. A predicted permease of the major facilitator superfamily class of membrane efflux pumps, AlcS (synonyms, OrfX and Bcr), was reported to be encoded within the alcaligin gene cluster. In this study,alcSnull mutants were found to be defective in growth under iron starvation conditions, in iron source utilization, and in alcaligin export.transcomplementation using clonedalcSgenes ofB. pertussisorB. bronchisepticarestored the wild-type phenotype to thealcSmutants. Although the levels of extracellular alcaligin measured inalcSstrain culture fluids were severely reduced compared with the wild-type levels,alcSmutants had elevated levels of cell-associated alcaligin, implicating AlcS in alcaligin export. Interestingly, a ΔalcAmutation that eliminated alcaligin production suppressed the growth defects ofalcSmutants. This suppression and the alcaligin production defect were reversed bytranscomplementation of the ΔalcAmutation in the double-mutant strain, confirming that the growth-defective phenotype ofalcSmutants is associated with alcaligin production. In analcA::mini-Tn5 lacZ1operon fusion strain background, analcSnull mutation resulted in enhanced AlcR-dependent transcriptional responsiveness to alcaligin inducer; conversely, AlcS overproduction blunted the transcriptional response to alcaligin. These transcription studies indicate that the alcaligin exporter activity of AlcS is required to maintain appropriate intracellular alcaligin levels for normal inducer sensing and responsiveness necessary for positive regulation of alcaligin system gene expression.
The
Campylobacter jejuni
Response Regulator, CbrR, Modulates Sodium Deoxycholate Resistance and Chicken Colonization
Authors: Raphael Brian H. , Pereira Sonia , Flom Gary A. , Zhang Qijing , Ketley Julian M. , Konkel Michael E.
Published: May 2005

Two-component regulatory systems play a major role in the physiological response of bacteria to environmental stimuli. Such systems are composed of a sensor histidine kinase and a response regulator whose ultimate function is to affect the expression of target genes. Response regulator mutants of
Campylobacter jejuni
strain F38011 were screened for sensitivity to sodium deoxycholate. A mutation in Cj0643, which encodes a response regulator with no obvious cognate histidine kinase, resulted in an absence of growth on plates containing a subinhibitory concentration of sodium deoxcholate (1%, wt/vol). In broth cultures containing 0.05% (wt/vol) sodium deoxycholate, growth of the mutant was significantly inhibited compared to growth of the
C. jejuni
F38011 wild-type strain. Complementation of the
C. jejuni cbrR
mutant in
trans
restored growth in both broth and plate cultures supplemented with sodium deoxycholate. Based on the phenotype displayed by its mutation, we designated the gene corresponding to Cj0643 as
cbrR
(
Campylobacter b
ile
r
esistance
r
egulator). While the MICs of a variety of bile salts and other detergents for the
C. jejuni cbrR
mutant were lower, no difference was noted in its sensitivity to antibiotics or osmolarity. Finally, chicken colonization studies demonstrated that the
C. jejuni cbrR
mutant had a reduced ability to colonize compared to the wild-type strain. These data support previous findings that bile resistance contributes to colonization of chickens and establish that the response regulator, CbrR, modulates resistance to bile salts in
C. jejuni
.
Characterization of Two New Aminopeptidases in
Escherichia coli
Authors: Zheng Yu , Roberts Richard J. , Kasif Simon , Guan Chudi
Published: May 2005

Two genes in the
Escherichia coli
genome,
ypdE
and
ypdF
, have been cloned and expressed, and their products have been purified. YpdF is shown to be a metalloenzyme with Xaa-Pro aminopeptidase activity and limited methionine aminopeptidase activity. Genes homologous to
ypdF
are widely distributed in bacterial species. The unique feature in the sequences of the products of these genes is a conserved C-terminal domain and a variable N-terminal domain. Full or partial deletion of the N terminus in YpdF leads to the loss of enzymatic activity. The conserved C-terminal domain is homologous to that of the methionyl aminopeptidase (encoded by
map
) in
E. coli
. However, YpdF and Map differ in their preference for the amino acid next to the initial methionine in the peptide substrates. The implication of this difference is discussed.
ypdE
is the immediate downstream gene of
ypdF
, and its start codon overlaps with the stop codon of
ypdF
by 1 base. YpdE is shown to be a metalloaminopeptidase and has a broad exoaminopeptidase activity.
Levels and Activity of the
Pseudomonas putida
Global Regulatory Protein Crc Vary According to Growth Conditions
Authors: Ruiz-Manzano Ana , Yuste Luis , Rojo Fernando
Published: May 2005

The global regulatory protein Crc is involved in the repression of several catabolic pathways for sugars, hydrocarbons, and nitrogenated and aromatic compounds in
Pseudomonas putida
and
Pseudomonas aeruginosa
when other preferred carbon sources are present in the culture medium (catabolite repression), therefore modulating carbon metabolism. We have analyzed whether the levels or the activity of Crc is regulated. Crc activity was followed by its ability to inhibit the induction by alkanes of the
P. putida
OCT plasmid alkane degradation pathway when cells grow in a complete medium, where the effect of Crc is very strong. The abundance of
crc
transcripts and the amounts of Crc protein were higher under repressing conditions than under nonrepressing conditions. The presence of
crc
on a high-copy-number plasmid considerably increased Crc levels, but this impaired its ability to inhibit the alkane degradation pathway. Crc shows similarity to a family of nucleases that have highly conserved residues at their catalytic sites. Mutation of the corresponding residues in Crc (Asp
220
and His
246
) led to proteins that can inhibit induction of the alkane degradation pathway when present at normal or elevated levels in the cell. Repression by these mutant proteins occurred only under repressing conditions. These results suggest that both the amounts and the activity of Crc are modulated and support previous proposals that Crc may form part of a signal transduction pathway. Furthermore, the activity of the mutant proteins suggests that Crc is not a nuclease.
Iron-Responsive Regulation of theHelicobacter pyloriIron-Cofactored Superoxide Dismutase SodB Is Mediated by Fur
Authors: Ernst Florian D. , Homuth Georg , Stoof Jeroen , Mäder Ulrike , Waidner Barbara , Kuipers Ernst J. , Kist Manfred , Kusters Johannes G. , Bereswill Stefan , van Vliet Arnoud H. M.
Published: May 2005

Maintaining iron homeostasis is a necessity for all living organisms, as free iron augments the generation of reactive oxygen species like superoxide anions, at the risk of subsequent lethal cellular damage. The iron-responsive regulator Fur controls iron metabolism in many bacteria, including the important human pathogenHelicobacter pylori, and thus is directly or indirectly involved in regulation of oxidative stress defense. Here we demonstrate that Fur is a direct regulator of theH. pyloriiron-cofactored superoxide dismutase SodB, which is essential for the defense against toxic superoxide radicals. Transcription of thesodBgene was iron induced inH. pyloriwild-type strain 26695, resulting in expression of the SodB protein in iron-replete conditions but an absence of expression in iron-restricted conditions. Mutation of thefurgene resulted in constitutive, iron-independent expression of SodB. RecombinantH. pyloriFur protein bound with low affinity to thesodBpromoter region, but addition of the iron substitute Mn2+abolished binding. The operator sequence of the iron-free form of Fur, as identified by DNase I footprinting, was located directly upstream of thesodBgene at positions −5 to −47 from the transcription start site. The direct role of Fur in regulation of theH. pylori sodBgene contrasts with the small-RNA-mediatedsodBregulation observed inEscherichia coli. In conclusion,H. pyloriFur is a versatile regulator involved in many pathways essential for gastric colonization, including superoxide stress defense.
Characterization of Pathways Dependent on the
uvsE
,
uvrA1
, or
uvrA2
Gene Product for UV Resistance in
Deinococcus radiodurans
Authors: Tanaka Masashi , Narumi Issay , Funayama Tomoo , Kikuchi Masahiro , Watanabe Hiroshi , Matsunaga Tsukasa , Nikaido Osamu , Yamamoto Kazuo
Published: May 2005

The genome of a radiation-resistant bacterium,
Deinococcus radiodurans
, contains one
uvsE
gene and two
uvrA
genes,
uvrA1
and
uvrA2
. Using a series of mutants lacking these genes, we determined the biological significance of these components to UV resistance. The UV damage endonuclease (UvsE)-dependent excision repair (UVER) pathway and UvrA1-dependent pathway show some redundancy in their function to counteract the lethal effects of UV. Loss of these pathways does not cause increased sensitivity to UV mutagenesis, suggesting either that these pathways play no function in inducing mutations or that there are mechanisms to prevent mutation other than these excision repair pathways. UVER efficiently removes both cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) from genomic DNA. In contrast, the UvrA1 pathway does not significantly contribute to the repair of CPDs but eliminates 6-4PPs. Inactivation of
uvrA2
does not result in a deleterious effect on survival, mutagenesis, or the repair kinetics of CPDs and 6-4PPs, indicating a minor role in resistance to UV. Loss of
uvsE
,
uvrA1
, and
uvrA2
reduces but does not completely abolish the ability to eliminate CPDs and 6-4PPs from genomic DNA. The result indicates the existence of a system that removes UV damage yet to be identified.
Structural Comparison of Ten Serotypes of Staphylocoagulases in
Staphylococcus aureus
Authors: Watanabe S. , Ito T. , Takeuchi F. , Endo M. , Okuno E. , Hiramatsu K.
Published: May 2005

Staphylocoagulase detection is the hallmark of a
Staphylococcus aureus
infection. Ten different serotypes of staphylocoagulases have been reported to date. We determined the nucleotide sequences of seven staphylocoagulase genes (
coa
) and their surrounding regions to compare structures of all 10 staphylocoagulase serotypes, and we inferred their derivations. We found that all staphylocoagulases are comprised of six regions: signal sequence, D1 region, D2 region, central region, repeat region, and C-terminal sequence. Amino acids at both ends, 33 amino acids in the N terminal (the signal sequences and the seven N-terminal amino acids in the D1 region) and 5 amino acids in the C terminal, were exactly identical among the 10 serotypes. The central regions were conserved with identities between 80.6 and 94.1% and similarities between 82.8 and 94.6%. Repeat regions comprising tandem repeats of 27 amino acids with a 92% identity on average were polymorphic in the number of repeats. On the other hand, D1 regions other than the seven N-terminal amino acids and D2 regions were less homologous, with diverged identities from 41.5 to 84.5% and 47.0 to 88.9%, respectively, and similarities from 53.5 to 88.7% and 56.8 to 91.9%, respectively, although the predicted prothrombin-binding sites were conserved among them. In contrast, flanking regions of
coa
were highly homologous, with nucleotide identities of more than 97.1%. Phylogenetic relations among
coa
did not correlate with those among the flanking regions or housekeeping genes used for multilocus sequence typing. These data indicate that
coa
could be transmitted to
S. aureus
, while the less homologous regions in
coa
presumed to be responsible for different antigenicities might have evolved independently.
Expression and Site-Directed Mutagenesis of the Lactococcal Abortive Phage Infection Protein AbiK
Authors: Fortier Louis-Charles , Bouchard Julie D. , Moineau Sylvain
Published: May 2005

Abortive infection mechanisms ofLactococcus lactisform a heterogeneous group of phage resistance systems that act after early phage gene expression. One of these systems, AbiK, aborts infection of the three most prevalent lactococcal phage groups of the dairy industry. In this study, it is demonstrated that the antiphage activity depends on the level of expression of theabiKgene and on the presence of a reverse transcriptase (RT) motif in AbiK. TheabiKgene was shown to be part of an operon that includes two additional open reading frames, with one of these encoding a phage-related transcriptional repressor named Orf4. Expression of AbiK is driven by two promoters, PabiKand Porf3, the latter being repressed by Orf4 in vivo. Binding of the purified Orf4 to the Porf3promoter was demonstrated in vitro by gel retardation assays. The N-terminal half of the deduced AbiK protein possesses an RT motif that was modified by site-directed mutagenesis. Conservative mutations in key positions resulted in the complete loss of the resistance phenotype. These data suggest that an RT activity might be involved in the phage resistance activity of AbiK. A model for the mode of action of AbiK is proposed.
Purification and Characterization of Allophanate Hydrolase (AtzF) from
Pseudomonas
sp. Strain ADP
Authors: Shapir Nir , Sadowsky Michael J. , Wackett Lawrence P.
Published: May 2005

AtzF, allophanate hydrolase, is a recently discovered member of the amidase signature family that catalyzes the terminal reaction during metabolism of
s
-triazine ring compounds by bacteria. In the present study, the
atzF
gene from
Pseudomonas
sp. strain ADP was cloned and expressed as a His-tagged protein, and the protein was purified and characterized. AtzF had a deduced subunit molecular mass of 66,223, based on the gene sequence, and an estimated holoenzyme molecular mass of 260,000. The active protein did not contain detectable metals or organic cofactors. Purified AtzF hydrolyzed allophanate with a
k
cat
/

K
m

of 1.1 × 10
4
s
−1
M
−1
, and 2 mol of ammonia was released per mol allophanate. The substrate range of AtzF was very narrow. Urea, biuret, hydroxyurea, methylcarbamate, and other structurally analogous compounds were not substrates for AtzF. Only malonamate, which strongly inhibited allophanate hydrolysis, was an alternative substrate, with a greatly reduced
k
cat
/

K
m

of 21 s
−1
M
−1
. Data suggested that the AtzF catalytic cycle proceeds through a covalent substrate-enzyme intermediate. AtzF reacts with malonamate and hydroxylamine to generate malonohydroxamate, potentially derived from hydroxylamine capture of an enzyme-tethered acyl group. Three putative catalytically important residues, one lysine and two serines, were altered by site-directed mutagenesis, each with complete loss of enzyme activity. The identity of a putative serine nucleophile was probed using phenyl phosphorodiamidate that was shown to be a time-dependent inhibitor of AtzF. Inhibition was due to phosphoroamidation of Ser189 as shown by liquid chromatography/matrix-assisted laser desorption ionization mass spectrometry. The modified residue corresponds in sequence alignments to the nucleophilic serine previously identified in other members of the amidase signature family. Thus, AtzF affects the cleavage of three carbon-to-nitrogen bonds via a mechanism similar to that of enzymes catalyzing single-amide-bond cleavage reactions. AtzF orthologs appear to be widespread among bacteria.
Novel Molecular Features of the Fibrolytic Intestinal Bacterium
Fibrobacter intestinalis
Not Shared with
Fibrobacter succinogenes
as Determined by Suppressive Subtractive Hybridization
Authors: Qi Meng , Nelson Karen E. , Daugherty Sean C. , Nelson William C. , Hance Ioana R. , Morrison Mark , Forsberg Cecil W.
Published: May 2005

Suppressive subtractive hybridization was conducted to identify unique genes coding for plant cell wall hydrolytic enzymes and other properties of the gastrointestinal bacterium
Fibrobacter intestinalis
DR7 not shared by
Fibrobacter succinogenes
S85. Subtractive clones from
F. intestinalis
were sequenced and assembled to form 712 nonredundant contigs with an average length of 525 bp. Of these, 55 sequences were unique to
F. intestinalis.
The remaining contigs contained 764 genes with BLASTX similarities to other proteins; of these, 80% had the highest similarities to proteins in
F. succinogenes
, including 30 that coded for carbohydrate active enzymes. The expression of 17 of these genes was verified by Northern dot blot analysis. Of genes not exhibiting BLASTX similarity to
F. succinogenes
, 30 encoded putative transposases, 6 encoded restriction modification genes, and 45% had highest similarities to proteins in other species of gastrointestinal bacteria, a finding suggestive of either horizontal gene transfer to
F. intestinalis
or gene loss from
F. succinogenes
. Analysis of contigs containing segments of two or more adjacent genes revealed that only 35% exhibited BLASTX similarity and were in the same orientation as those of
F. succinogenes
, indicating extensive chromosomal rearrangement. The expression of eight transposases, and three restriction-modification genes was confirmed by Northern dot blot analysis. These data clearly document the maintenance of carbohydrate active enzymes in
F. intestinalis
necessitated by the preponderance of polysaccharide substrates available in the ruminal environment. It also documents substantive changes in the genome from that of
F. succinogenes
, which may be related to the introduction of the array of transposase and restriction-modification genes.
Role of π Dimers in Coupling (“Handcuffing”) of Plasmid R6K's γ
ori
Iterons
Authors: Kunnimalaiyaan Selvi , Inman Ross B. , Rakowski Sheryl A. , Filutowicz Marcin
Published: May 2005

One proposed mechanism of replication inhibition in iteron-containing plasmids (ICPs) is “handcuffing,” in which the coupling of origins via iteron-bound replication initiator (Rep) protein turns off origin function. In minimal R6K replicons, copy number control requires the interaction of plasmid-encoded π protein with the seven 22-bp iterons of the γ origin of replication. Like other related Rep proteins, π exists as both monomers and dimers. However, the ability of π dimers to bind iterons distinguishes R6K from most other ICPs, where only monomers have been observed to bind iterons. Here, we describe experiments to determine if monomers or dimers of π protein are involved in the formation of handcuffed complexes. Standard ligation enhancement assays were done using π variants with different propensities to bind iterons as monomers or dimers. Consistent with observations from several ICPs, a hyperreplicative variant (π·P106L
∧
F107S) exhibits deficiencies in handcuffing. Additionally, a novel dimer-biased variant of π protein (π·M36A
∧
M38A), which lacks initiator function, handcuffs iteron-containing DNA more efficiently than does wild-type π. The data suggest that π dimers mediate handcuffing, supporting our previously proposed model of handcuffing in the γ
ori
system. Thus, dimers of π appear to possess three distinct inhibitory functions with respect to R6K replication: transcriptional autorepression of π expression, in
cis
competition (for origin binding) with monomeric activator π, and handcuffing-mediated inhibition of replication in
trans
.
Alteration of the Fatty Acid Profile of
Streptomyces coelicolor
by Replacement of the Initiation Enzyme 3-Ketoacyl Acyl Carrier Protein Synthase III (FabH)
Authors: Li Yongli , Florova Galina , Reynolds Kevin A.
Published: May 2005

The first elongation step of fatty acid biosynthesis by a type II dissociated fatty acid synthases is catalyzed by 3-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII, FabH). This enzyme, encoded by the
fabH
gene, catalyzes a decarboxylative condensation between an acyl coenzyme A (CoA) primer and malonyl-ACP. In organisms such as
Escherichia coli
, which generate only straight-chain fatty acids (SCFAs), FabH has a substrate preference for acetyl-CoA. In streptomycetes and other organisms which produce a mixture of both SCFAs and branched-chain fatty acids (BCFAs), FabH has been shown to utilize straight- and branched-chain acyl-CoA substrates. We report herein the generation of a
Streptomyces coelicolor
mutant (YL/ecFabH) in which the chromosomal copy of the
fabH
gene has been replaced and the essential process of fatty acid biosynthesis is initiated by plasmid-based expression of the
E. coli
FabH (bearing only 35% amino acid identity to the
Streptomyces
enzyme). The YL/ecFabH mutant produces predominantly SCFAs (86%). In contrast, BCFAs predominate (∼70%) in both the
S. coelicolor
parental strain and
S. coelicolor
YL/sgFabH (a Δ
fabH
mutant carrying a plasmid expressing the
Streptomyces glaucescens
FabH). These results provide the first unequivocal evidence that the substrate specificity of FabH observed in vitro is a determinant of the fatty acid made in an organism. The YL/ecFabH strain grows significantly slower on both solid and liquid media. The levels of FabH activity in cell extracts of YL/ecFabH were also significantly lower than those in cell extracts of YL/sgFabH, suggesting that a decreased rate of fatty acid synthesis may account for the observed decreased growth rate. The production of low levels of BCFAs in YL/ecFabH suggests either that the
E. coli
FabH is more tolerant of different acyl-CoAs substrates than previously thought or that there is an additional pathway for initiation of BCFA biosynthesis in
Streptomyces coelicolor
.
The ExsA Protein of
Bacillus cereus
Is Required for Assembly of Coat and Exosporium onto the Spore Surface
Authors: Bailey-Smith Karen , Todd Sarah J. , Southworth Thomas W. , Proctor John , Moir Anne
Published: May 2005

The outermost layer of spores of the
Bacillus cereus
family is a loose structure known as the exosporium. Spores of a library of Tn
917
-LTV1 transposon insertion mutants of
B. cereus
ATCC 10876 were partitioned into hexadecane; a less hydrophobic mutant that was isolated contained an insertion in the
exsA
promoter region. ExsA is the equivalent of SafA (YrbA) of
Bacillus subtilis
, which is also implicated in spore coat assembly; the gene organizations around both are identical, and both proteins contain a very conserved N-terminal cortex-binding domain of ca. 50 residues, although the rest of the sequence is much less conserved. In particular, unlike SafA, the ExsA protein contains multiple tandem oligopeptide repeats and is therefore likely to have an extended structure. The
exsA
gene is expressed in the mother cell during sporulation. Spores of an
exsA
mutant are extremely permeable to lysozyme and are blocked in late stages of germination, which require coat-associated functions. Two mutants expressing differently truncated versions of ExsA were constructed, and they showed the same gross defects in the attachment of exosporium and spore coat layers. The protein profile of the residual exosporium harvested from spores of the three mutants—two expressing truncated proteins and the mutant with the original transposon insertion in the promoter region—showed some differences from the wild type and from each other, but the major exosporium glycoproteins were retained. The
exsA
gene is extremely important for the normal assembly and anchoring of both the spore coat and exosporium layers in spores of
B. cereus
.
Structure-Based Site-Directed Mutagenesis of the UDP-MurNAc-Pentapeptide-Binding Cavity of the FemX Alanyl Transferase from
Weissella viridescens
Authors: Maillard Antoine P. , Biarrotte-Sorin Sabrina , Villet Régis , Mesnage Stéphane , Bouhss Ahmed , Sougakoff Wladimir , Mayer Claudine , Arthur Michel
Published: May 2005

Weissella viridescens
FemX (FemX
Wv
) belongs to the Fem family of nonribosomal peptidyl transferases that use aminoacyl-tRNA as the amino acid donor to synthesize the peptide cross-bridge found in the peptidoglycan of many species of pathogenic gram-positive bacteria. We have recently solved the crystal structure of FemX
Wv
in complex with the peptidoglycan precursor UDP-MurNAc-pentapeptide and report here the site-directed mutagenesis of nine residues located in the binding cavity for this substrate. Two substitutions, Lys36Met and Arg211Met, depressed FemX
Wv
transferase activity below detectable levels without affecting protein folding. Analogues of UDP-MurNAc-pentapeptide lacking the phosphate groups or the C-terminal
d
-alanyl residues were not substrates of the enzyme. These results indicate that Lys36 and Arg211 participate in a complex hydrogen bond network that connects the C-terminal
d
-Ala residues to the phosphate groups of UDP-MurNAc-pentapeptide and constrains the substrate in a conformation that is essential for transferase activity.
A Multidomain Fusion Protein inListeria monocytogenesCatalyzes the Two Primary Activities for Glutathione Biosynthesis
Authors: Gopal Shubha , Borovok Ilya , Ofer Amos , Yanku Michaela , Cohen Gerald , Goebel Werner , Kreft Jürgen , Aharonowitz Yair
Published: May 2005

Glutathione is the predominant low-molecular-weight peptide thiol present in living organisms and plays a key role in protecting cells against oxygen toxicity. Until now, glutathione synthesis was thought to occur solely through the consecutive action of two physically separate enzymes, γ-glutamylcysteine ligase and glutathione synthetase. In this report we demonstrate thatListeria monocytogenescontains a novel multidomain protein (termed GshF) that carries out complete synthesis of glutathione. Evidence for this comes from experiments which showed that in vitro recombinant GshF directs the formation of glutathione from its constituent amino acids and the in vivo effect of a mutation in GshF that abolishes glutathione synthesis, results in accumulation of the intermediate γ-glutamylcysteine, and causes hypersensitivity to oxidative agents. We identified GshF orthologs, consisting of a γ-glutamylcysteine ligase (GshA) domain fused to an ATP-grasp domain, in 20 gram-positive and gram-negative bacteria. Remarkably, 95% of these bacteria are mammalian pathogens. A plausible origin for GshF-dependent glutathione biosynthesis in these bacteria was the recruitment by a GshA ancestor gene of an ATP-grasp gene and the subsequent spread of the fusion gene between mammalian hosts, most likely by horizontal gene transfer.
Structures of the Iron-Sulfur Flavoproteins fromMethanosarcina thermophilaandArchaeoglobus fulgidus
Authors: Andrade Susana L. A. , Cruz Francisco , Drennan Catherine L. , Ramakrishnan Vijay , Rees Douglas C. , Ferry James G. , Einsle Oliver
Published: May 2005

Iron-sulfur flavoproteins (ISF) constitute a widespread family of redox-active proteins in anaerobic prokaryotes. Based on sequence homologies, their overall structure is expected to be similar to that of flavodoxins, but in addition to a flavin mononucleotide cofactor they also contain a cubane-type [4Fe:4S] cluster. In order to gain further insight into the function and properties of ISF, the three-dimensional structures of two ISF homologs, one from the thermophilic methanogenMethanosarcina thermophilaand one from the hyperthermophilic sulfate-reducing archaeonArchaeoglobus fulgidus, were determined. The structures indicate that ISF assembles to form a tetramer and that electron transfer between the two types of redox cofactors requires oligomerization to juxtapose the flavin mononucleotide and [4Fe:4S] cluster bound to different subunits. This is only possible between different monomers upon oligomerization. Fundamental differences in the surface properties of the two ISF homologs underscore the diversity encountered within this protein family.
Surface Structure and Nanomechanical Properties of
Shewanella putrefaciens
Bacteria at Two pH values (4 and 10) Determined by Atomic Force Microscopy
Authors: Gaboriaud Fabien , Bailet Sidney , Dague Etienne , Jorand Frédéric
Published: May 2005

The nanomechanical properties of gram-negative bacteria (
Shewanella putrefaciens
) were investigated in situ in aqueous solutions at two pH values, specifically, 4 and 10, by atomic force microscopy (AFM). For both pH values, the approach force curves exhibited subsequent nonlinear and linear regimens that were related to the progressive indentation of the AFM tip in the bacterial cell wall, including a priori polymeric fringe (nonlinear part), while the linear part was ascribed to compression of the plasma membrane. These results indicate the dynamic of surface ultrastructure in response to changes in pH, leading to variations in nanomechanical properties, such as the Young's modulus and the bacterial spring constant.
A Gene Encoding
l
-Methionine γ-Lyase Is Present in
Enterobacteriaceae
Family Genomes: Identification and Characterization of
Citrobacter freundii
l
-Methionine γ-Lyase
Authors: Manukhov Ilya V. , Mamaeva Daria V. , Rastorguev Sergei M. , Faleev Nicolai G. , Morozova Elena A. , Demidkina Tatyana V. , Zavilgelsky Gennadii B.
Published: May 2005

Citrobacter freundii
cells produce
l
-methionine γ-lyase when grown on a medium containing
l
-methionine. The nucleotide sequence of the hybrid plasmid with a
C. freundii
EcoRI insert of about 3.0 kbp contained two open reading frames, consisting of 1,194 nucleotides and 1,296 nucleotides, respectively. The first one (denoted
megL
) encoded
l
-methionine γ-lyase. The enzyme was overexpressed in
Escherichia coli
and purified. The second frame encoded a protein belonging to the family of permeases. Regions of high sequence identity with the 3′-terminal part of the
C. freundii megL
gene located in the same regions of
Salmonella enterica
serovar Typhimurium,
Shigella flexneri
,
E. coli
, and
Citrobacter rodentium
genomes were found.
Quorum Sensing Negatively Controls Type III Secretion Regulon Expression in
Pseudomonas aeruginosa
PAO1
Authors: Bleves Sophie , Soscia Chantal , Nogueira-Orlandi Patricia , Lazdunski Andrée , Filloux Alain
Published: May 2005

A systematic analysis of the type III secretion (T3S) genes of
Pseudomonas aeruginosa
strain PAO1 revealed that they are under quorum-sensing control. This observation was supported by the down-regulation of the T3S regulon in the presence of RhlR-C
4
HSL and the corresponding advanced secretion of ExoS in a
rhlI
mutant.
Evolution of Subspecies of
Francisella tularensis
Authors: Svensson Kerstin , Larsson Pär , Johansson Daniel , Byström Mona , Forsman Mats , Johansson Anders
Published: May 2005

Analysis of unidirectional genomic deletion events and single nucleotide variations suggested that the four subspecies of
Francisella tularensis
have evolved by vertical descent. The analysis indicated an evolutionary scenario where the highly virulent
F. tularensis
subsp.
tularensis
(type A) appeared before the less virulent
F. tularensis
subsp.
holarctica
(type B). Compared to their virulent progenitors, attenuated strains of
F. tularensis
exhibited specific unidirectional gene losses.
Non-malignant consequences of decreasing asbestos exposure in the Brazil chrysotile mines and mills
Authors: Bagatin E , Neder J A , Nery L E , Terra-Filho M , Kavakama J , Castelo A , Capelozzi V , Sette A , Kitamura S , Favero M , Moreira-Filho D C , Tavares R , Peres C , Becklake M R
Published: May 2005

Aims:To investigate the consequences of improvement in the workplace environment over six decades (1940–96) in asbestos miners and millers from a developing country (Brazil).

Methods:A total of 3634 Brazilian workers with at least one year of exposure completed a respiratory symptoms questionnaire, chest radiography, and a spirometric evaluation. The study population was separated into three groups whose working conditions improved over time: group I (1940–66, n = 180), group II (1967–76, n = 1317), and group III (1977–96, n = 2137).

Results:Respiratory symptoms were significantly related to spirometric abnormalities, smoking, and latency time. Breathlessness, in particular, was also associated with age, pleural abnormality and increased cumulative exposure to asbestos fibres. The odds ratios (OR) for parenchymal and/or non-malignant pleural disease were significantly lower in groups II and III compared to group I subjects (0.29 (0.12–0.69) and 0.19 (0.08–0.45), respectively), independent of age and smoking status. Similar results were found when groups were compared at equivalent latency times (groups IvII: 30–45 years; groups IIvIII: 20–25 years). Ageing, dyspnoea, past and current smoking, and radiographic abnormalities were associated with ventilatory impairment. Lower spirometric values were found in groups I and II compared to group III: lung function values were also lower in higher quartiles of latency and of cumulative exposure in these subjects.

Conclusions:Progressive improvement in occupational hygiene in a developing country is likely to reduce the risk of non-malignant consequences of dust inhalation in asbestos miners and millers.
Atrazine in municipal drinking water and risk of low birth weight, preterm delivery, and small-for-gestational-age status
Authors: Villanueva C M , Durand G , Coutté M-B , Chevrier C , Cordier S
Published: May 2005

Background:Atrazine is a herbicide used extensively worldwide. Bioassays have shown that it is embryotoxic and embryolethal. Evidence of adverse reproductive outcomes from exposure in the general population is sparse.

Aims:To evaluate the association between atrazine levels in municipal drinking water and the following adverse reproductive outcomes: increased risk of preterm delivery, low birth weight (LBW), and small-for-gestational-age (SGA) status.

Methods:A total of 3510 births that took place from 1 October 1997, to 30 September 1998 were analysed. Atrazine measurements were available for 2661 samples from water treatment plants over the past decade. A seasonal pattern was identified, with atrazine peaking from May to September. The geometric mean of the atrazine level for this period was calculated for each water distribution unit and merged with the individual data by municipality of residence.

Results:Atrazine levels in water were not associated with an increased risk of LBW or SGA status and were slightly associated with prematurity. There was an increased risk of SGA status in cases in which the third trimester overlapped in whole or in part with the May–September period, compared with those in which the third trimester occurred totally from October to April (OR = 1.37, 95% CI 1.04 to 1.81). If the entire third trimester took place from May to September, the OR was 1.54 (95% CI 1.11 to 2.13).

Conclusions:Low levels of atrazine, a narrow exposure range, and limitations in the exposure assessment partly explain the lack of associations with atrazine. Findings point to the third trimester of pregnancy as the potential vulnerable period for an increased risk of SGA birth. Exposures other than atrazine and also seasonal factors may explain the increased risk.
Urinary mercury concentrations associated with dental restorations in adult women aged 16–49 years: United States, 1999–2000
Authors: Dye B A , Schober S E , Dillon C F , Jones R L , Fryar C , McDowell M , Sinks T H
Published: May 2005

Background:Mercury amalgam dental restorations have been used by dentists since the mid 19th century and issues on safety continue to be periodically debated within the scientific and public health communities. Previous studies have reported a positive association between urine mercury levels and the number of dental amalgams, but this relation has never been described in a nationally representative sample in the United States.

Aims and Methods:Using household interview, dietary interview, dental examination, and laboratory data from the 1999–2000 National Health and Nutrition Examination Survey (NHANES), the association between mercury concentrations and dental restorations was examined in US women of reproductive age.

Results:In women of childbearing age, approximately 13% of all posterior dental surfaces were restored with amalgams and the average urinary mercury level in women was low (1.34 μg/l). It is estimated that an increase of 1.8 μg/l in the log transformed values for mercury in urine would occur for each 10 dental surfaces restored with amalgam.

Conclusions:Although the findings do not address the important issues of adverse health effects at low thresholds of mercury exposure, they do provide important reference data that should contribute significantly to the ongoing scientific and public health policy debate on the use of dental amalgams in the USA.
Serum markers of collagen metabolism: construction workers compared to sedentary workers
Authors: Kuiper J I , Verbeek J H A M , Everts V , Straub J P , Frings-Dresen M H W
Published: May 2005

Background:Evaluation of causal relations between physical load and musculoskeletal disorders is hampered by the lack of knowledge as to the biological relevance of different loading parameters and the large variability between individuals. As indicators of molecular changes in the extracellular matrices of structures of the musculoskeletal system, biomarkers of collagen metabolism may provide important information on biological effects of physical load. The carboxyterminal propeptide of type I collagen (PICP) is a serum marker of synthesis and the carboxyterminal telopeptide region of type I collagen (CTx) reflects degradation of type I collagen.

Aims:To explore the feasibility of biomarkers of type I collagen metabolism as measures of the effects of physical load at tissue level.

Methods:Serum concentrations of PICP and CTx were assessed in a group of male construction workers involved in heavy manual materials handling (n = 47) and in a group of male sedentary workers (n = 49).

Results:Serum concentrations of both PICP and CTx seemed to be related to heavy physical work. The ratio PICP/CTx, illustrative of the effective metabolic changes, did not differ between the two groups.

Conclusions:The higher turnover rate but similar effective synthesis may be indicative of an increased type I collagen content in the connective tissues as a result of adaptive remodelling in response to years of exposure to physical load. Further validation of these biomarkers is required with respect to dose-response relations and temporal associations between exposure to back load and biomarker concentrations.
DESINGULARIZATION OF QUASIPLURISUBHARMONIC FUNCTIONS
Authors: GUEDJ VINCENT
Published: May 2005

Let T be a positive closed current of bidegree (1,1) on a compact complex surface. We show that for all ε > 0, one can find a finite composition of blow-ups π such that π*T decomposes as the sum of a divisorial part and a positive closed current whose Lelong numbers are all less than ε.
NEW SAMPLING METHOD TO IMPROVE THE SFDR OF WIDE BANDWIDTH ADC DEDICATED TO NEXT GENERATION WIRELESS TRANSCEIVER
Authors: EL-SANKARY KAMAL , ASSI ALI , SAWAN MOHAMAD
Published: May 2005

Modern wireless communication standards that support high rates of voice and video streaming need high-speed Analog-to-Digital Converters (ADCs) with wide Spurious-Free Dynamic Range (SFDR). Conventional time-interleaved ADCs suffer from spurious components that seriously affect the SFDR. In this paper, we present the mathematical background describing the effect of randomizing the samples among the interleaved ADCs and we propose a digitally oriented method based on this analysis to randomize the mismatches among the ADC channels. Analyses and simulations show the effectiveness of the proposed approach in multi-channel ADCs with arbitrary bit resolution, channel's number and sampling rate. For a 10-bit 500 MS/s ADC, the SFDR achieved using the proposed randomizing method can be as wide as 75 dB, which is an enhancement of more than 26 dB comparing to the conventional time interleaved ADC.
A COMPUTATIONAL-RAM (C-RAM) ARCHITECTURE FOR REAL-TIME MESH-BASED VIDEO MOTION TRACKING PART 1: MOTION ESTIMATION
Authors: SAYED MOHAMMED , BADAWY WAEL
Published: May 2005

This paper presents a new Computational-RAM (C-RAM) architecture for real-time mesh-based video motion tracking. The motion tracking consists of two operations: mesh-based motion estimation and compensation. The proposed motion estimation architecture is presented in Part 1 and the proposed motion compensation architecture is presented in Part 2. The motion estimation architecture stores two frames and computes motion vectors for a regular triangular mesh structure as defined by MPEG-4 Part 2.1 The motion estimation architecture uses the block-matching algorithm (BMA) to estimate the vertical and horizontal motion vectors for each mesh node. Parallel and pipelined implementations have been used to overcome the huge computational requirements of the motion estimation process. The two frames are stored in embedded S-RAMs generated with Virage™ Memory Compiler. The proposed motion estimation architecture has been prototyped, simulated and synthesized using the TSMC 0.18 μm CMOS technology. At 100 MHz clock frequency, the proposed architecture processes one CIF video frame (i.e., 352×288 pixels) in 1.48 ms, which means it can process up to 675 frames per second. The core area of the proposed motion estimation architecture is 24.58 mm2 and it consumes 46.26 mW.
LOW POWER FULL SEARCH BLOCK MATCHING MOTION ESTIMATION VLSI ARCHITECTURES
Authors: ELGAMEL MOHAMED A. , BAYOUMI MAGDY A. , SHAMS AHMED M. , ZAVIDOVIQUE BERTRAND
Published: May 2005

Power consumption is very critical for portable video applications. During compression, the motion estimation unit consumes the largest portion of power since it performs a huge amount of computation. Different low power architectures for implementing the full-search block-matching (FSBM) motion estimation are discussed. Also, architectural enhancements to further reduce the power consumed during FSBM motion estimation without sacrificing throughput or optimality are presented. The proposed approach achieves these power savings by disabling portions of the architecture that perform unnecessary computations. A comparison between the different architectures including our enhancements and others is presented using simulation and analytical analysis. Different benchmarks are used to test and compare the discussed architectures. Analytical and simulation results show the effectiveness of the enhancements.
LOW LEVEL HARDWARE COMPRESSION FOR MULTI-GIGABIT NETWORKS
Authors: PAPAEFSTATHIOU IOANNIS
Published: May 2005

As it has already been proved, link layer compression is very effective when used in packet networks. In particular, packet compression is especially useful when encryption is applied to the network packets. Encrypting the packets causes the data to be random in nature, and thus no compression can be applied after it. It is believed that low level encryption will be applied to the vast majority of Internet Protocol (IP) networks in the near future, and thus a large number of very sophisticated encryption devices have already been manufactured. Based on these facts, we claim that hardware devices that can compress network streams at link speed (and perform the compression just before the encryption), will also be widely used in the future networks. In this paper, we present such a hardware compressor/decompressor core that can work at speeds up to 10 Gb/s, it is fairly inexpensive and can very easily be plugged into an existing network node without causing any side effects. We additionally examine the performance against complexity tradeoffs of such compressor/decompressor devices. Finally, it is claimed that compression devices with throughput ranging from 0.5 to 10 Gb/s can be efficiently implemented based on the reference architecture.
NOVEL DESIGN AND FPGA IMPLEMENTATION OF DA-RNS FIR FILTERS
Authors: WANG WEI , SWAMY M. N. S. , AHMAD M. O.
Published: May 2005

Field programmable gate array (FPGA)-based digital signal processing has been widely used in multimedia applications. By combining distributed arithmetic (DA) and residue number system (RNS) in such designs, efficient area, speed and power efficiency can be achieved. In this paper, we propose novel techniques for the design and FPGA implementation of DA-RNS finite impulse response (FIR) filters. By introducing a novel low-cost moduli set and its selection method, efficient modulo arithmetic units inside the subfilters are designed. Then, a new residue-to-binary conversion algorithm, a so-called modified DA Chinese remainder theorem, is derived to reduce the modulo operations and provide an efficient residue-to-binary converter suitable to FPGA implementation. Based on these proposed techniques, a seventh-order DA-RNS FIR filter is designed, implemented and tested by using Xilinx FPGA tools. The implementation results show that the proposed filter design consumes only 77% of the power that the existing filter12,13 requires, while maintaining the same speed (throughput).