bluuebunny/crossref_metadata_2025 huggingface dataset:
Helping the public ‘Discover Health’ in their local library. Providing health information in public libraries: a partnership approach in Scotland
Authors: Henry Elspeth , Marley Lesley
Published: May 2004

A partnership between the Specialist Health Promotion Service of NHS Tayside and Dundee City Council developed a project, ‘Discover Health’, to bring health information to the public. The two main formats used were traditional leaflets and Internet access via the People's Network, both available in public libraries. The steering group for the project was made up of staff from both organisations. The NHS side secured the funding; the Council side provided the accommodation and information and communication technologies. The partnership worked well, and the project is continuing, with expansion in the pipeline for other areas within Tayside. The various aspects of the functioning of the partnership are discussed. A literature review of similar initiatives is included.
Collision Detection and Part Interaction Modeling to Facilitate Immersive Virtual Assembly Methods
Authors: Kim Chang E. , Vance Judy M.
Published: May 2004

Realistic part interaction is an important component of an effective virtual assembly application. Both collision detection and part interaction modeling are needed to simulate part-to-part and hand-to-part interactions. This paper examines several polygonal-based collision detection packages and compares their usage for virtual assembly applications with the Voxmap PointShell (VPS) software developed by the Boeing Company. VPS is a software developer’s toolkit for real-time collision and proximity detection, swept-volume generation, dynamic animation, and 6 degree-of-freedom haptics which is based on volumetric collision detection and physically based modeling. VPS works by detecting interactions between two parts: a dynamic object moving in the virtual environment, and a static object defined as a collection of all other objects in the environment. The method was found to provide realistic collision detection and physically-based modeling interaction, with good performance at the expense of contact accuracy. Results from several performance tests on VPS are presented. This paper concludes by presenting how VPS has been implemented to handle multiple dynamic part collisions and two-handed assembly using the 5DT dataglove in a projection screen virtual environment.
Cellular Stoichiometry of the Components of the Chemotaxis Signaling Complex
Authors: Li Mingshan , Hazelbauer Gerald L.
Published: June 2004

The chemotactic sensory system of
Escherichia coli
comprises membrane-embedded chemoreceptors and six soluble chemotaxis (Che) proteins. These components form signaling complexes that mediate sensory excitation and adaptation. Previous determinations of cellular content of individual components provided differing and apparently conflicting values. We used quantitative immunoblotting to perform comprehensive determinations of cellular amounts of all components in two
E. coli
strains considered wild type for chemotaxis, grown in rich and minimal media. Cellular amounts varied up to 10-fold, but ratios between proteins varied no more than 30%. Thus, cellular stoichiometries were almost constant as amounts varied substantially. Calculations using those cellular stoichiometries and values for in vivo proportions of core components in complexes yielded an in vivo stoichiometry for core complexes of 3.4 receptor dimers and 1.6 CheW monomers for each CheA dimer and 2.4 CheY, 0.5 CheZ dimers, 0.08 CheB, and 0.05 CheR per complex. The values suggest a core unit of a trimer of chemoreceptor dimers, a dimer (or two monomers) of kinase CheA, and two CheW. These components may interact in extended arrays and, thus, stoichiometries could be nonintegral. In any case, cellular stoichiometries indicate that CheY could be bound to all signaling complexes and this binding would recruit essentially the entire cellular complement of unphosphorylated CheY, and also that phosphatase CheZ, methylesterase CheB, and methyltransferase CheR would be present at 1 per 2, per 14, and per 20 core complexes, respectively. These characteristic ratios will be important in quantitative treatments of chemotaxis, both experimental and theoretical.
Identification of
mdoD
, an
mdoG
Paralog Which Encodes a Twin-Arginine-Dependent Periplasmic Protein That Controls Osmoregulated Periplasmic Glucan Backbone Structures
Authors: Lequette Yannick , Ödberg-Ferragut Carmen , Bohin Jean-Pierre , Lacroix Jean-Marie
Published: June 2004

Osmoregulated periplasmic glucans (OPGs) of
Escherichia coli
are anionic and highly branched oligosaccharides that accumulate in the periplasmic space in response to low osmolarity of the medium. The glucan length, ranging from 5 to 12 glucose residues, is under strict control. Two genes that form an operon,
mdoGH
, govern glucose backbone synthesis. The new gene
mdoD
, which appears to be a paralog of
mdoG
, was characterized in this study. Cassette inactivation of
mdoD
resulted in production of OPGs with a higher degree of polymerization, indicating that OpgD, the
mdoD
product (according to the new nomenclature), controls the glucose backbone structures. OpgD secretion depends on the Tat secretory pathway. Orthologs of the
mdoG
and
mdoD
genes are found in various proteobacteria. Most of the OpgD orthologs exhibit a Tat-dependent secretion signal, while most of the OpgG orthologs are Sec dependent.
A Homologue ofaliBIs Found in the Capsule Region of NonencapsulatedStreptococcus pneumoniae
Authors: Hathaway Lucy J. , Meier Patricia Stutzmann , Bättig Patrick , Aebi Suzanne , Mühlemann Kathrin
Published: June 2004

The epidemiology, phylogeny, and biology of nonencapsulatedStreptococcus pneumoniaeare largely unknown. Increased colonization capacity and transformability are, however, intriguing features of these pneumococci and play an important role. Twenty-seven nonencapsulated pneumococci were identified in a nationwide collection of 1,980 nasopharyngeal samples and 215 blood samples obtained between 1998 and 2002. On the basis of multilocus sequence typing and capsule region analysis we divided the nonencapsulated pneumococci into two groups. Group I was closely related to encapsulated strains. Group II had a clonal population structure, including two geographically widespread clones able to cause epidemic conjunctivitis and invasive diseases. Group II strains also carried a 1,959-bp homologue ofaliB(aliB-like ORF 2) in the capsule region, which was highly homologous to a sequence in the capsule region ofStreptococcus mitis. In addition, strains of the two major clones in group II had an additional sequence,aliB-like ORF 1 (1,968 to 2,004 bp), upstream ofaliB-like ORF 2. Expression ofaliB-like ORF 1 was detected by reverse transcription-PCR, and the corresponding RNA was visualized by Northern blotting. A gene fragment homologous tocapNof serotypes 33 and 37 suggests that group II strains were derived from encapsulated pneumococci some time ago. Therefore, loss of capsule expression in vivo was found to be associated with the importation of one or twoaliBhomologues in some nonencapsulated pneumococci.
Methylation-Independent Aerotaxis Mediated by the
Escherichia coli
Aer Protein
Authors: Bibikov Sergei I. , Miller Andrew C. , Gosink Khoosheh K. , Parkinson John S.
Published: June 2004

Aer is a membrane-associated protein that mediates aerotactic responses in
Escherichia coli
. Its C-terminal half closely resembles the signaling domains of methyl-accepting chemotaxis proteins (MCPs), which undergo reversible methylation at specific glutamic acid residues to adapt their signaling outputs to homogeneous chemical environments. MCP-mediated behaviors are dependent on two specific enzymes, CheR (methyltransferase) and CheB (methylesterase). The Aer signaling domain contains unorthodox methylation sites that do not conform to the consensus motif for CheR or CheB substrates, suggesting that Aer, unlike conventional MCPs, might be a methylation-independent transducer. Several lines of evidence supported this possibility. (i) The Aer protein was not detectably modified by either CheR or CheB. (ii) Amino acid replacements at the putative Aer methylation sites generally had no deleterious effect on Aer function. (iii) Aer promoted aerotactic migrations on semisolid media in strains that lacked all four of the
E. coli
MCPs. CheR and CheB function had no influence on the rate of aerotactic movements in those strains. Thus, Aer senses and signals efficiently in the absence of deamidation or methylation, methylation changes, methylation enzymes, and methyl-accepting chemotaxis proteins. We also found that chimeric transducers containing the PAS-HAMP sensing domain of Aer joined to the signaling domain and methylation sites of Tar, an orthodox MCP, exhibited both methylation-dependent and methylation-independent aerotactic behavior. The hybrid Aear transducers demonstrate that methylation independence does not emanate from the Aer signaling domain but rather may be due to transience of the cellular redox changes that are thought to trigger Aer-mediated behavioral responses.
Functional Dissection of
Escherichia coli
Trigger Factor: Unraveling the Function of Individual Domains
Authors: Kramer G. , Rutkowska A. , Wegrzyn R. D. , Patzelt H. , Kurz T. A. , Merz F. , Rauch T. , Vorderwülbecke S. , Deuerling E. , Bukau B.
Published: June 2004

In
Escherichia coli
, the ribosome-associated chaperone Trigger Factor (TF) promotes the folding of newly synthesized cytosolic proteins. TF is composed of three domains: an N-terminal domain (N), which mediates ribosome binding; a central domain (P), which has peptidyl-prolyl
cis/trans
isomerase activity and is involved in substrate binding in vitro; and a C-terminal domain (C) with unknown function. We investigated the contributions of individual domains (N, P, and C) or domain combinations (NP, PC, and NC) to the chaperone activity of TF in vivo and in vitro. All fragments comprising the N domain (N, NP, NC) complemented the synthetic lethality of Δ
tig
Δ
dnaK
in cells lacking TF and DnaK, prevented protein aggregation in these cells, and cross-linked to nascent polypeptides in vitro. However, Δ
tig
Δ
dnaK
cells expressing the N domain alone grew more slowly and showed less viability than Δ
tig
Δ
dnaK
cells synthesizing either NP, NC, or full-length TF, indicating beneficial contributions of the P and C domains to TF's chaperone activity. In an in vitro system with purified components, none of the TF fragments assisted the refolding of denatured
d
-glyceraldehyde-3-phosphate dehydrogenase in a manner comparable to that of wild-type TF, suggesting that the observed chaperone activity of TF fragments in vivo is dependent on their localization at the ribosome. These results indicate that the N domain, in addition to its function to promote binding to the ribosome, has a chaperone activity per se and is sufficient to substitute for TF in vivo.
Quorum Sensing Regulates Type III Secretion inVibrio harveyiandVibrio parahaemolyticus
Authors: Henke Jennifer M. , Bassler Bonnie L.
Published: June 2004

In a process known as quorum sensing, bacteria communicate with one another by producing, releasing, detecting, and responding to signal molecules called autoinducers.Vibrio harveyi, a marine pathogen, uses two parallel quorum-sensing circuits, each consisting of an autoinducer-sensor pair, to control the expression of genes required for bioluminescence and a number of other target genes. Genetic screens designed to discover autoinducer-regulated targets inV. harveyihave revealed genes encoding components of a putative type III secretion (TTS) system. Using transcriptional reporter fusions and TTS protein localization studies, we show that the TTS system is indeed functional inV. harveyiand that expression of the genes encoding the secretion machinery requires an intact quorum-sensing signal transduction cascade. The newly completed genome of the closely related marine bacteriumVibrio parahaemolyticus, which is a human pathogen, shows that it possesses the genes encoding both of theV. harveyi-like quorum-sensing signaling circuits and that it also has a TTS system similar to that ofV. harveyi. We show that quorum sensing regulates TTS inV. parahaemolyticus. Previous reports connecting quorum sensing to TTS in enterohemorrhagic and enteropathogenicEscherichia colishow that quorum sensing activates TTS at high cell density. Surprisingly, we find that at high cell density (in the presence of autoinducers), quorum sensing represses TTS inV. harveyiandV. parahaemolyticus.
Detection and Characterization of Conjugative Degradative Plasmids in Xenobiotic-Degrading
Sphingomonas
Strains
Authors: Basta Tamara , Keck Andreas , Klein Joachim , Stolz Andreas
Published: June 2004

A systematic survey for the presence of plasmids in 17 different xenobiotic-degrading
Sphingomonas
strains was performed. In almost all analyzed strains, two to five plasmids with sizes of about 50 to 500 kb were detected by using pulsed-field gel electrophoresis. A comparison of plasmid preparations untreated or treated with S1 nuclease suggested that, in general,
Sphingomonas
plasmids are circular. Hybridization experiments with labeled gene probes suggested that large plasmids are involved in the degradation of dibenzo-
p
-dioxin, dibenzofuran, and naphthalenesulfonates in
S. wittichii
RW1,
Sphingomonas
sp. HH69, and
S. xenophaga
BN6, respectively. The plasmids which are responsible for the degradation of naphthalene, biphenyl, and toluene by
S. aromaticivorans
F199 (pNL1) and of naphthalenesulfonates by
S. xenophaga
BN6 (pBN6) were site-specifically labeled with a kanamycin resistance cassette. The conjugative transfer of these labeled plasmids was attempted with various bacterial strains as putative recipient strains. Thus, a conjugative transfer of plasmid pBN6 from
S. xenophaga
BN6 to a cured mutant of strain BN6 and to
Sphingomonas
sp. SS3 was observed. The conjugation experiments with plasmid pNL1 suggested a broader host range of this plasmid, because it was transferred without any obvious structural changes to
S. yanoikuyae
B1,
Sphingomonas
sp. SS3, and
S. herbicidovorans
. In contrast, major plasmid rearrangements were observed in the transconjugants after the transfer of plasmid pNL1 to
Sphingomonas
sp. HH69 and of pBN6 to
Sphingomonas
sp. SS3. No indications for the transfer of a
Sphingomonas
plasmid to bacteria outside of the
Sphingomonadaceae
were obtained.
Role of the Anti-Sigma Factor SpoIIAB in Regulation of σ
G
during
Bacillus subtilis
Sporulation
Authors: Serrano Mónica , Neves Alexandre , Soares Cláudio M. , Moran Charles P. , Henriques Adriano O.
Published: June 2004

RNA polymerase sigma factor σ
F
initiates the prespore-specific program of gene expression during
Bacillus subtilis
sporulation. σ
F
governs transcription of
spoIIIG
, encoding the late prespore-specific regulator σ
G
. However, transcription of
spoIIIG
is delayed relative to other genes under the control of σ
F
, and after synthesis, σ
G
is initially kept in an inactive form. Activation of σ
G
requires the complete engulfment of the prespore by the mother cell and expression of the
spoIIIA
and
spoIIIJ
loci. We screened for random mutations in
spoIIIG
that bypassed the requirement for
spoIIIA
for the activation of σ
G
. We found a mutation (
spoIIIGE156K
) that resulted in an amino acid substitution at position 156, which is adjacent to the position of a mutation (E155K) previously shown to prevent interaction of SpoIIAB with σ
G
. Comparative modelling techniques and in vivo studies suggested that the
spoIIIGE156K
mutation interferes with the interaction of SpoIIAB with σ
G
. The σ
GE156K
isoform restored σ
G
-directed gene expression to
spoIIIA
mutant cells. However, expression of
sspE
-
lacZ
in the
spoIIIA spoIIIGE156K
double mutant was delayed relative to completion of the engulfment process and was not confined to the prespore. Rather, β-galactosidase accumulated throughout the entire cell at late times in development. This suggests that the activity of σ
GE156K
is still regulated in the prespore of a
spoIIIA
mutant, but not by SpoIIAB. In agreement with this suggestion, we also found that expression of
spoIIIGE156K
from the promoter for the early prespore-specific gene
spoIIQ
still resulted in
sspE-lacZ
induction at the normal time during sporulation, coincidently with completion of the engulfment process. In contrast, transcription of
spoIIIGE156K
, but not of the wild-type
spoIIIG
gene, from the mother cell-specific
spoIID
promoter permitted the rapid induction of
sspE-lacZ
expression. Together, the results suggest that SpoIIAB is either redundant or has no role in the regulation of σ
G
in the prespore.
The Last Gene of the
fla/che
Operon in
Bacillus subtilis
,
ylxL
, Is Required for Maximal σ
D
Function
Authors: Werhane H. , Lopez P. , Mendel M. , Zimmer M. , Ordal G. W. , Márquez-Magaña L. M.
Published: June 2004

ylxL
was found to be the last gene of the
fla/che
operon in
Bacillus subtilis
and is cotranscribed with the gene for the flagellum-specific alternate sigma factor, σ
D
. The
ylxL
gene was disrupted by insertional mutagenesis, and the resultant mutant strain was found to be compromised for σ
D
-dependent functions.
The Acidic Nature of the CcmG Redox-Active Center Is Important for CytochromecMaturation inEscherichia coli
Authors: Edeling Melissa A. , Ahuja Umesh , Heras Begoña , Thöny-Meyer Linda , Martin Jennifer L.
Published: June 2004

Cytochromecbiogenesis inEscherichia coliis a complex process requiring at least eight genes (ccmABCDEFGH). One of these genes,ccmG, encodes a thioredoxin-like protein with unusually specific redox activity. Here, we investigate the basis for CcmG function and demonstrate the importance of acidic residues surrounding the redox-active center.
A Mutant Allele of
rpoD
Results in Increased Conversion of Aminoimidazole Ribotide to Hydroxymethyl Pyrimidine in
Salmonella enterica
Authors: Dougherty Michael J. , Downs Diana M.
Published: June 2004

An allele of
rpoD
(
rpoD1181
) that results in increased synthesis of the pyrimidine moiety of thiamine in
Salmonella enterica
was identified. The S508Y substitution caused by
rpoD1181
is analogous to the S506F derivative of the
Escherichia coli
protein. The properties of this
E
.
coli
mutant protein have been well characterized in vitro. Identification of a metabolic phenotype caused by the
rpoD1181
allele of
S
.
enterica
allows past in vitro results to be incorporated in continuing efforts to understand cellular processes that are integrated with the thiamine biosynthetic pathway.
RISK MEASUREMENT OF INVESTMENTS IN THE SATELLITE RING OF A CORE-SATELLITE PORTFOLIO: TRADITIONAL VERSUS ALTERNATIVE APPROACHES
Authors: TILL HILARY
Published: May 2004

This paper provides a risk framework for fiduciaries by considering using a core-satellite approach to investing. While the article mainly covers the additional risk measurement techniques, which are needed when investing in hedge funds, its recommendations are also relevant for other investments that have default, devaluation, and/or liquidity risks associated with them. Also, while the article's focus is on quantitative techniques, we note that a fiduciary must also understand the economic basis for each investment's returns.
The Bacterial Cytoskeleton and Its PutativeRole in Membrane Vesicle Formation Observedin a Gram-Positive Bacterium Producing Starch-Degrading Enzymes
Authors: Mayer Frank , Gottschalk Gerhard
Published: May 2004

Bacteria may possess various kinds of cytoskeleton. In general, bacterial cytoskeletons may play a role in the control and preservation of the cell shape. Such functions become especially evident when the bacteria do not possess a true wall and are nevertheless elongated (e.g. Mycoplasma spp.) or under extreme cultivation conditions whereby loss of the entire bacterial cell wall takes place. Bacterial cytoskeletons may control and preserve the cell shape only if a number of preconditions are fulfilled. They should be present not only transiently, but permanently, they should be located as a lining close to the inner face of the cytoplasmic membrane, enclosing the entire cytoplasm, and they should comprise structural elements (fibrils) crossing the inner volume of the cell in order to provide the necessary stability for the lining. Complete loss of the cell wall layers had earlier been observed to occur during extensive production of bacterial starch-degrading enzymes in an optimized fermentation process by a Gram-positive bacterium. Even under these conditions, the cells had maintained their elongated shape and full viability. Which of the various kinds of bacterial cytoskeleton might have been responsible for shape preservation? Only one of them, the primary or basic cytoskeleton turns out to fulfil the necessary preconditions listed above. Its structural features now provided a first insight into a possible mechanism of formation of membrane blebs and vesicles as observed in the Gram-positive eubacterium Thermoanaerobacterium thermosulfurogenes EM1, and the putative role of the cytoskeletal web in this process.
Enhancement of Translation Initiation by A/T-Rich Sequences Downstream of the Initiation Codon in Escherichia coli
Authors: Qing Guoliang , Xia Bing , Inouye Masayori
Published: May 2004

The region located downstream of the initiation codon constitutes part of the translation initiation signal, significantly affecting the level of protein expression in E. coli. In order to determine its influence on translation initiation, we inserted random 12-base sequences downstream of the initiation codon of the lacZ gene. A total of 119 random clones showing higher β-galactosidase activities than the control lacZ gene were isolated and subsequently sequenced. Analysis of these clones revealed that their insertion sequences are strikingly rich in A and T, but poor in G, with no consensus sequences among them. Toeprinting experiments and polysome profile analysis confirmed that the A/T-rich sequences enhance translation at the level of initiation. Collectively, the present data demonstrate that A/T richness of the region following the initiation codon plays a significant role in E. coli gene expression.
Molecular Genetic Analyses of Potential β-Galactosidase Genes in Xanthomonas campestris
Authors: Yang Tsuey-Ching , Hu Rouh-Mei , Hsiao Yi-Min , Weng Shu-Fen , Tseng Yi-Hsiung
Published: May 2004

Xanthomonas campestris pv. campestris, which displays no significant β-1,4-D-galactopyranosidase activity, has three annotated β-galactosidase genes in the sequenced genome, designated galA, galB and galC herein. GalA and GalB are similar to glycosyl hydrolase (GH) family 2 enzymes, including Escherichia coli LacZ. galA and galB cannot express detectable activity even after being cloned in-frame and driven by the vector’s promoter. GalC is a GH35 enzyme homologous to the Xanthomonas axonopodis pv. manihotis Bga. The latter cleaves β,1-3-linked galactose 1,000 times faster than β,1-4-linked galactose and is not responsible for lactose utilization. In X. campestris pv. campestris cells, GalC is readily detectable by Western blotting, and the levels can be increased by cloning the gene under the control of the vector’s promoter. Results of insertional mutation, transcriptional fusion assay and Western blotting indicated that galC, clustered with several GH genes, is cotranscribed with the upstream gene(s) and is expressed constitutively. Xc17L is a previously isolated mutant with elevated β-galactosidase activity and a greatly improved ability to grow on lactose. Results of DNA sequencing of Xc17L galA, galB and galC, enzyme assays of galA, galB and galC mutants derived from Xc17L, and Western blotting of GalC in Xc17L indicated that the three β-galactosidase genes do not encode the elevated β-galactosidase activity in Xc17L. The presence of a fourth β-galactosidase gene is proposed.
Analysis of fruE, a Novel Developmental Gene of Myxococcus xanthus
Authors: Akiyama Takuya , Komano Teruya
Published: May 2004

Myxococcus xanthus is a gram-negative soil bacterium that undergoes multicellular development upon nutrient starvation. In the present study, a TnV insertion developmental mutation, Ω773, of M. xanthus was analyzed. The TnV Ω773 insertion was found to be located within a novel developmental gene, fruE. The FruE protein is composed of 140 amino acid residues and bears an N-terminal signal peptide. The amino acid sequence of FruE shared no significant similarity with any other known protein in the databases. The fruE mutant displayed a development-delayed phenotype. The formation of tightly aggregated mounds in the fruE mutant was slower than that in the wild-type strain. The initiation of spore production in the fruE mutant was delayed by 12 h in comparison to the wild-type strain, and the process of spore formation was more asynchronous than that of the wild-type strain. The transcription initiation sites of the fruE gene were located 81 bp (P1) and 57 bp (P2) upstream of the fruE initiation codon. Although both promoters were active during vegetative growth and development, the P1 promoter was more active during development and the P2 promoter was more active during vegetative growth. The expression of the fruE gene increased to a peak at 6 h poststarvation and then decreased. The decrease in fruE expression was not observed in the D and E signal mutants.
Bioinformatic Analyses of the Bacterial L-Ascorbate Phosphotransferase System Permease Family
Authors: Hvorup Rikki , Chang Abraham B. , Saier Jr. Milton H.
Published: May 2004

The tripartite L-ascorbate permease of Escherichia coli is the first functionally characterized member of a large family of enzyme II complexes (SgaTBA, encoding enzymes IIC, IIB and IIA) of the bacterial phosphotransferase system (PTS). We here report bioinformatic analyses of these proteins. Forty-five homologous systems from a wide variety of bacteria were identified, but no homologues were found in archaea or eukaryotes. These systems fell into five structural types: (1) IIC, IIB and IIA are encoded by distinct genes; (2) IIC and IIB are encoded by distinct genes, but the IIA-encoding gene is absent; (3) IIC and IIB are encoded by a fused gene, but IIA is a distinct gene product; (4) IIA and IIB are fused, but IIC is encoded by a distinct gene, and (5) IIC and IIB are encoded by distinct genes, but IIA is fused to a transcriptional regulator. Phylogenetic analyses revealed that gene fusion/splicing events have occurred repeatedly during the evolutionary divergence of family members, although no evidence for shuffling of constituents between systems was obtained. The SgaTBA family proved to be distantly related to the GatCBA family of PTS permeases, and this family was also analyzed. In contrast to the SgaTBA family, no gene splicing/fusion has occurred during the evolutionary divergence of GatCBA family members as each domain is always encoded by a distinct gene. However, GatC homologues were identified in organisms that lack other PTS proteins, suggesting a transport mechanism not coupled to substrate phosphorylation. Topological analyses suggest that in contrast to all other PTS permeases, IIC proteins of the Sga and Gat families exhibit 12 transmembrane α-helical segments and are distantly related to secondary carriers. Like many secondary carriers, GatC (IIC) homologues could be shown to have arisen by an ancient intragenic duplication event. These results suggest that the Sga and Gat families of PTS permeases comprise a small superfamily in which the transmembrane IIC domains evolved independently of all other known PTS permeases.
Inhibitory Effects of Metabolite I of Erdosteine on the Generation of Nitric Oxide and Peroxynitrite Chemiluminescence by Human Neutrophils
Authors: Dal Sasso Monica , Culici Maria , Bianchi Tiziana , Fonti Elena , Braga Pier Carlo
Published: May 2004

Polymorphonuclear neutrophils (PMNs) can generate superoxide anions and nitric oxide (NO), which is not only an important mediator of various cellular activities, but can also react with superoxide anions to produce peroxynitrite anions (ONOO–). Peroxynitrite is a potent and potentially toxic oxidant that damages various types of biomolecules. It preferentially mediates the oxidation of thiolic groups in protein and non-protein molecules, thus altering their functions. The aim of this study was to examine whether, in addition to its ability to reduce the respiratory bursts of human PMNs, the SH metabolite I (Met I) of erdosteine, can interfere with NO and NO-derived peroxynitrite production, thus extending its antioxidant activity. This was done by means of the luminol amplified chemiluminescence (LACL), which has been widely used to detect the production of reactive oxidant species (ROS) by PMNs under various conditions. At 5 and 10 µg/ml, Met I significantly reduced LACL after fMLP and PMA stimulation. When L-Arg was added to the reaction medium, as a NO donor, the chemiluminescence of fMLP increased by up to 67% and that of PMA by up to 132%, but was once again significantly reduced by 5 and 10 µg/ml of Met I. In a cell-free system, the use of linsidomine (SIN-1) makes it possible to investigate the behavior of LACL induced by peroxynitrite release, which was significantly reduced by Met I concentrations ranging from 1.25 to 10 µg/ml. Our findings indicate that Met I, a molecule with a SH group, reacts with ROS, NO and NO-derived peroxynitrite, and has both antioxidant and scavenging activity. This is of interest for the strategy of protecting against damage induced by radical species in the pulmonary cell environment, in which they can induce a phlogogenic loop, and suggests that adding exogenous thiols may be useful in antagonizing the toxic effects of reactive molecules on endogenous thiols.
Antitussive Profile of the NOP Agonist Ro-64-6198 in the Guinea Pig
Authors: McLeod Robbie L. , Jia Yanlin , Fernandez Xiomara , Parra Leonard E. , Wang Xin , Tulshian Deen B. , Kiselgof Eugenia J. , Tan Zheng , Fawzi Ahmad B. , Smith-Torhan April , Zhang Hongtao , Hey John A.
Published: May 2004

We have previously shown that N/OFQ, the endogenous peptide ligand for the ‘opioid-like’ NOP receptor, inhibits cough in guinea pigs and cats. In the present study we sought to continue our characterization of the cough-suppressant effects of NOP stimulation by profiling the pulmonary and antitussive effects of a novel non-peptide NOP agonist, Ro-64-6198, in guinea pigs. In receptor-binding assays, we confirmed that Ro-64-6198 selectively binds to NOP receptors over other opioid receptors. The Ki values for Ro-64-6198 at NOP, MOP, KOP and DOP receptors was 0.3, 36, 214 and 3,787 nmol/l, respectively. In GTPγS-binding assays, Ro-64-6198 displayed >900-fold functional selectivity at NOP relative to MOP receptors. We evaluated the effects of Ro-64-6198 (3 and 10 µmol/l) in isolated guinea pig nodose ganglia cells on the increases in intracellular Ca2+ concentration evoked by capsaicin stimulation (1 × 10–8–1 × 10–6 mol/l). Similar to previously reported data with N/OFQ, Ro-64-6198 (3 and 10 µmol/l) significantly attenuated Ca2+ responses in nodose ganglia cells produced by exposure to capsaicin. The effect of Ro-64-6198 (3 µmol/l) on capsaicin-induced intracellular Ca2+ responses was blocked by the NOP antagonist, J113397 (3 µmol/l). In guinea pig in vivo studies, aerosolized capsaicin (10–300 µmol/l) produced a dose-dependent increase in cough number. Ro-64-6198 given i.p. significantly inhibited cough due to capsaicin (300 µmol/l) exposure. In a duration study we found that the maximum antitussive effect (42 ± 8% inhibition) of Ro-64-6198 (3 mg/kg) was observed at 1 h after i.p. administration. Also at 1 h after administration, Ro-64-6198 (0.003–3.0 mg/kg, i.p.) produced a dose-dependent inhibition of cough. The antitussive effect of Ro-64-6198 (3 mg/kg, i.p.) was blocked by J113397 (12 mg/kg, i.p.) but not by the classical opioid antagonist naltrexone (10 mg/kg, i.p.). Although the antitussive action of Ro-64-6198 may be mediated by a central and/or a peripheral site of action, we hypothesize that selective oral NOP agonists that do not penetrate the blood-brain barrier may provide a novel approach for the treatment of cough. Moreover, because these drugs do not interact at MOP receptors, they may be devoid of codeine-like side effects such as respiratory depression, sedation, constipation or proclivities for addictive liabilities.
Comparative Effects of Quinapril with Enalapril in Rats with Heart Failure
Authors: Watanabe Kenichi , Ma Meilei , Wen Juan , Tachikawa Hitoshi , Kodama Makoto , Aizawa Yoshifusa , Yamaguchi Kenichi , Takahashi Toshihiro
Published: May 2004

The cardioprotective properties of angiotensin-converting enzyme (ACE) inhibitors, quinapril and enalapril were studied in a rat model of heart failure. Seventy-five rats were divided into five groups and administered quinapril or enalapril at 2 and 20 mg/kg/day (groups Q2, Q20, E2 and E20) or vehicle alone (group V, all groups n = 15). Although both ACE inhibitors improved survival rate and ventricular function in a dose-dependent manner, the left ventricular end-diastolic pressure and expression level of transforming growth factor-β1 mRNA were the lowest in group Q20. These results suggest that quinapril may confer greater protection than enalapril against injury from the renin-angiotensin system in heart failure.
The role of homologous recombination repair in the formation of chromosome aberrations
Authors: Griffin C.S. , Thacker J.
Published: May 2004

The repair of DNA double strand breaks by homologous recombination can occur by at least two pathways: a Rad51-dependent pathway that is predominantly error free, and a Rad51-independent pathway (single strand annealing, SSA) that is error prone. In theory, chromosome exchanges can result from (mis)repair by either pathway. Both repair pathways will involve a search for homologous sequence, leading to co-localization of chromatin. Genes involved in homologous recombination repair (HRR) have now been successfully knocked out in mice and the role of HRR in the formation of chromosome exchanges, particularly after ionising radiation, is discussed in the light of new evidence.
Repair rates of R-band, G-band and C-band DNA in murine and human cultured cells
Authors: Sanders M.H. , Bates S.E. , Wilbur B.S. , Holmquist G.P.
Published: May 2004

Repair of cyclobutane pyrimidine dimers (CPDs) in cultured neonatal human fibroblasts and in Mus spretus × M. castaneus F1 neonatal skin fibroblasts was analyzed after UVC-irradiation by cleavage with T4 endonuclease V cyclopyrimidine dimer glycosylase, alkaline-agarose gel electrophoresis, and Southern blotting. The blots were sequentially probed with 32P-labeled Alu, or B2, to preferentially illuminate R-band DNA, by L1 to preferentially illuminate G-band DNA, and by satellite DNA to illuminate C-band DNA. These three different DNA populations showed slightly different global nucleotide excision repair rates that are in the order of speed, R-band DNA > G-band DNA > C-band DNA. Fibroblasts from out-bred neonatal mice and humans showed similar band-specific repair rate ratios and the global repair rate of murine fibroblasts was almost as rapid as that of the human fibroblasts. The mass distribution of the human Alu-probed signal was further analyzed. Gel mobility data was fitted to a logistic equation to include all Mr values. Hypothetical distributions of DNA randomly cleaved to a particular number-average molecular weight were fit to the logistic gel mobility function to determine how such a randomly cleaved distribution of a particular cleavage frequency would be displayed along the experimental gel. This revealed a rapidly repaired kinetic fraction that represented 17% of the Alu-probed signal (R-band DNA), almost none of the L1 probed signal (G-band DNA), and reflects transcription coupled repair of active genes. The remaining Alu-probed DNA showed a random distribution of UVC-induced CPDs throughout all stages of global nucleotide excision repair. The Alu-probed CPDs disappeared with an excellent fit to first order kinetics and with a half-life of seven hours.
Progress towards understanding the nature of chromatid breakage
Authors: Bryant P.E. , Gray L.J. , Peresse N.
Published: May 2004

The wide range of sensitivities of stimulated T-cells from different individuals to radiation-induced chromatid breakage indicates the involvement of several low penetrance genes that appear to link elevated chromatid breakage to cancer susceptibility. The mechanisms of chromatid breakage are not yet fully understood. However, evidence is accumulating that suggests chromatid breaks are not simply expanded DNA double-strand breaks (DSB). Three models of chromatid breakage are considered. The classical breakage-first and the Revell “exchange” models do not accord with current evidence. Therefore a derivative of Revell’s model has been proposed whereby both spontaneous and radiation-induced chromatid breaks result from DSB signaling and rearrangement processes from within large looped chromatin domains. Examples of such rearrangements can be observed by harlequin staining whereby an exchange of strands occurs immediately adjacent to the break site. However, these interchromatid rearrangements comprise less than 20% of the total breaks. The rest are thought to result from intrachromatid rearrangements, including a very small proportion involving complete excision of a looped domain. Work is in progress with the aim of revealing these rearrangements, which may involve the formation of inversions adjacent to the break sites. It is postulated that the disappearance of chromatid breaks with time results from the completion of such rearrangements, rather than from the rejoining of DSB. Elevated frequencies of chromatid breaks occur in irradiated cells with defects in both nonhomologous end-joining (NHEJ) and homologous recombination (HR) pathways, however there is little evidence of a correlation between reduced DSB rejoining and disappearance of chromatid breaks. Moreover, at least one treatment which abrogates the disappearance of chromatid breaks with time leaves DSB rejoining unaffected. The I-SceI DSB system holds considerable promise for the elucidation of these mechanisms, although the break frequency is relatively low in the cell lines so far derived. Techniques to study and improve such systems are under way in different cell lines. Clearly, much remains to be done to clarify the mechanisms involved in chromatid breakage, but the experimental models are becoming available with which we can begin to answer some of the key questions.
Mechanisms and consequences of methylating agent-induced SCEs and chromosomal aberrations: a long road traveled and still a far way to go
Authors: Kaina B.
Published: May 2004

Since the milestone work of Evans and Scott, demonstrating the replication dependence of alkylation-induced aberrations, and Obe and Natarajan, pointing to the critical role of DNA double-strand breaks (DSBs) as the ultimate trigger of aberrations, the field has grown extensively. A notable example is the identification of DNA methylation lesions provoking chromosome breakage (clastogenic) effects, which made it possible to model clastogenic pathways evoked by genotoxins. Experiments with repair-deficient mutants and transgenic cell lines revealed both O6-methylguanine (O6MeG) and N- methylpurines as critical lesions. For SN2 agents such as methyl- methanesulfonate (MMS), base N-methylation lesions are most critical, likely because of the formation of apurinic sites blocking replication. For SN1 agents, such as N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O6-methylguanine (O6MeG) plays the major role both in recombination and clastogenicity in the post-treatment cell cycle, provided the lesion is not pre-replicatively repaired by O6-methylguanine-DNA methyltransferase (MGMT). The conversion probability of O6MeG into SCEs and chromosomal aberrations is estimated to be about 30:1 and >10,000:1 respectively, indicating this mispairing pro-mutagenic lesion to be highly potent in inducing recombination giving rise to SCEs. O6MeG needs replication and mismatch repair to become converted into a critical secondary genotoxic lesion. Here it is proposed that this secondary lesion can be tolerated by a process termed recombination bypass. This process is supposed to be important in the tolerance of lesions that can not be processed by translesion synthesis accomplished by low-fidelity DNA polymerases. Recombination bypass results in SCEs and might represent an alternative pathway of tolerance of non-instructive lesions. In the case of O6MeG-derived secondary lesions, recombination bypass appears to protect against cell killing since SCEs are already induced with low, non-toxic doses of MNNG. Saturation of lesion tolerance by recombination bypass or translesion synthesis may cause block of DNA replication leading to DSBs at stalled replication forks, which result in chromatid-type aberrations. Along with this model, several putative consequences of methylation-induced aberrations will be discussed such as cell death by apoptosis as well its role in tumor promotion and progression.
Human fibroblasts expressing hTERT show remarkable karyotype stability even after exposure to ionizing radiation
Authors: Pirzio L.M. , Freulet-Marrière M.-A. , Bai Y. , Fouladi B. , Murnane J.P. , Sabatier L. , Desmaze C.
Published: May 2004

Ectopic expression of telomerase results in an immortal phenotype in various types of normal cells, including primary human fibroblasts. In addition to its role in telomere lengthening, telomerase has now been found to have various functions, including the control of DNA repair, chromatin modification, and the control of expression of genes involved in cell cycle regulation. The investigations on the long-term effects of telomerase expression in normal human fibroblast highlighted that these cells show low frequencies of chromosomal aberrations. In this paper, we describe the karyotypic stability of human fibroblasts immortalized by expression of hTERT. The ectopic overexpression of telomerase is associated with unusual spontaneous as well as radiation-induced chromosome stability. In addition, we found that irradiation did not enhance plasmid integration in cells expressing hTERT, as has been reported for other cell types. Long-term studies illustrated that human fibroblasts immortalized by telomerase show an unusual stability for chromosomes and for plasmid integration sites, both with and without exposure to ionizing radiation. These results confirm a role for telomerase in genome stabilisation by a telomere-independent mechanism and point to the possibility for utilizing hTERT-immortalized normal human cells for the study of gene targeting.
Mechanisms of formation of chromosomal aberrations: insights from studies with DNA repair-deficient cells
Authors: Palitti F.
Published: May 2004

In order to understand the mechanisms of formation of chromosomal aberrations, studies performed on human syndromes with genomic instability can be fruitful. In this report, the results from studies in our laboratory on the importance of the transcription-coupled repair (TCR) pathway on the induction of chromosomal damage and apoptosis by ultraviolet light (UV) are discussed. UV61 cells (hamster homologue of human Cockayne’s syndrome group B) deficient in TCR showed a dramatic increase in the induction of chromosomal aberrations and apoptosis following UV treatment. At relatively low UV doses, the induction of chromosomal aberrations preceded the apoptotic process. Chromosomal aberrations probably lead to apoptosis and most of the cells had gone through an S phase after the UV treatment before entering apoptosis. At higher doses of UV, the cells could go into apoptosis already in the G1 phase of the cell cycle. Abolition of TCR by treatment with α-amanitin (an inhibitor of RNA polymerase II) in the parental cell line AA8 also resulted in the induction of elevated chromosomal damage and apoptotic response similar to the one observed in UV61 cells treated with UV alone. This suggests that the lack of TCR is responsible for the increased frequencies of chromosomal aberrations and apoptosis in UV61 cells. Hypersensitivity to the induction of chromosomal damage by inhibitors of antitopoisomerases I and II in Werner’s syndrome cells is also discussed in relation to the compromised G2 phase processes involving the Werner protein.
DNA damage processing and aberration formation in plants
Authors: Schubert I. , Pecinka A. , Meister A. , Schubert V. , Klatte M. , Jovtchev G.
Published: May 2004

Various types of DNA damage, induced by endo- and exogenous genotoxic impacts, may become processed into structural chromosome changes such as sister chromatid exchanges (SCEs) and chromosomal aberrations. Chromosomal aberrations occur preferentially within heterochromatic regions composed mainly of repetitive sequences. Most of the preclastogenic damage is correctly repaired by different repair mechanisms. For instance, after N-methyl-N-nitrosourea treatment one SCE is formed per >40,000 and one chromatid-type aberration per ∼25 million primarily induced O6-methylguanine residues in Vicia faba. Double-strand breaks (DSBs) apparently represent the critical lesions for the generation of chromosome structural changes by erroneous reciprocal recombination repair. Usually two DSBs have to interact in cis or trans to form a chromosomal aberration. Indirect evidence is at hand for plants indicating that chromatid-type aberrations mediated by S phase-dependent mutagens are generated by post-replication (mis)repair of DSBs resulting from (rare) interference of repair and replication processes at the sites of lesions, mainly within repetitive sequences of heterochromatic regions. The proportion of DSBs yielding structural changes via misrepair has still to be established when DSBs, induced at predetermined positions, can be quantified and related to the number of SCEs and chromosomal aberrations that appear at these loci after DSB induction. Recording the degree of association of homologous chromosome territories (by chromosome painting) and of punctual homologous pairing frequency along these territories during and after mutagen treatment of wild-type versus hyperrecombination mutants of Arabidopsis thaliana, it will be elucidated as to what extent the interphase arrangement of chromosome territories becomes modified by critical lesions and contributes to homologous reciprocal recombination. This paper reviews the state of the art with respect to DNA damage processing in the course of aberration formation and the interphase arrangement of homologous chromosome territories as a structural prerequisite for homologous rearrangements in plants.
DNA and telomeres: beginnings and endings
Authors: Bailey S.M. , Goodwin E.H.
Published: May 2004

How a cell deals with its DNA ends is a question that returns us to the very beginnings of modern telomere biology. It is also a question we are still asking today because it is absolutely essential that a cell correctly distinguishes between natural chromosomal DNA ends and broken DNA ends, then processes each appropriately – preserving the one, rejoining the other. Effective end-capping of mammalian telomeres has a seemingly paradoxical requirement for proteins more commonly associated with DNA double strand break (DSB) repair. Ku70, Ku80, DNA-PKcs (the catalytic subunit of DNA-dependent protein kinase), Xrcc4 and Artemis all participate in DSB repair through nonhomologous end-joining (NHEJ). Somewhat surprisingly, mutations in any of these genes cause spontaneous chromosomal end-to-end fusions that maintain large blocks of telomeric sequence at the points of fusion, suggesting loss or failure of a critical terminal structure, rather than telomere shortening, is at fault. Nascent telomeres produced via leading-strand DNA synthesis are especially susceptible to these end-to-end fusions, suggesting a crucial difference in the postreplicative processing of telomeres that is linked to their mode of replication. Here we will examine the dual roles played by DNA repair proteins. Our review of this rapidly advancing field primarily will focus on mammalian cells, and cannot include even all of this. Despite these limitations, we hope the review will serve as a useful gateway to the literature, and will help to frame the major issues in this exciting and rapidly progressing field. Our apologies to those whose work we are unable to include.