bluuebunny/crossref_metadata_2025 huggingface dataset:
Cysteine Residues in the Transmembrane Regions of M13 Procoat Protein Suggest that Oligomeric Coat Proteins Assemble onto Phage Progeny
Authors: Nagler Christof , Nagler Gisela , Kuhn Andreas
Published: January 2007

The M13 phage assembles in the inner membrane of
Escherichia coli
. During maturation, about 2,700 copies of the major coat protein move from the membrane onto a single-stranded phage DNA molecule that extrudes out of the cell. The major coat protein is synthesized as a precursor, termed procoat protein, and inserts into the membrane via a Sec-independent pathway. It is processed by a leader peptidase from its leader (signal) peptide before it is assembled onto the phage DNA. The transmembrane regions of the procoat protein play an important role in all these processes. Using cysteine mutants with mutations in the transmembrane regions of the procoat and coat proteins, we investigated which of the residues are involved in multimer formation, interaction with the leader peptidase, and formation of M13 progeny particles. We found that most single cysteine residues do not interfere with the membrane insertion, processing, and assembly of the phage. Treatment of the cells with copper phenanthroline showed that the cysteine residues were readily engaged in dimer and multimer formation. This suggests that the coat proteins assemble into multimers before they proceed onto the nascent phage particles. In addition, we found that when a cysteine is located in the leader peptide at the −6 position, processing of the mutant procoat protein and of other exported proteins is affected. This inhibition of the leader peptidase results in death of the cell and shows that there are distinct amino acid residues in the M13 procoat protein involved at specific steps of the phage assembly process.
Quorum-Sensing Regulation of Adhesion in
Serratia marcescens
MG1 Is Surface Dependent
Authors: Labbate Maurizio , Zhu Hua , Thung Leena , Bandara Rani , Larsen Martin R. , Willcox Mark D. P. , Givskov Michael , Rice Scott A. , Kjelleberg Staffan
Published: January 2007

Serratia marcescens
is an opportunistic pathogen and a major cause of ocular infections. In previous studies of
S. marcescens
MG1, we showed that biofilm maturation and sloughing were regulated by
N
-acyl homoserine lactone (AHL)-based quorum sensing (QS). Because of the importance of adhesion in initiating biofilm formation and infection, the primary goal of this study was to determine whether QS is important in adhesion to both abiotic and biotic surfaces, as assessed by determining the degree of attachment to hydrophilic tissue culture plates and human corneal epithelial (HCE) cells. Our results demonstrate that while adhesion to the abiotic surface was AHL regulated, adhesion to the HCE cell biotic surface was not. Type I fimbriae were identified as the critical adhesin for non-QS-mediated attachment to the biotic HCE cell surface but played no role in adhesion to the abiotic surface. While we were not able to identify a single QS-regulated adhesin essential for attachment to the abiotic surface, four AHL-regulated genes involved in adhesion to the abiotic surface were identified. Interestingly, two of these genes,
bsmA
and
bsmB
, were also shown to be involved in adhesion to the biotic surface in a non-QS-controlled fashion. Therefore, the expression of these two genes appears to be cocontrolled by regulators other than the QS system for mediation of attachment to HCE cells. We also found that QS in
S. marcescens
regulates other potential cell surface adhesins, including exopolysaccharide and the outer membrane protein OmpX. We concluded that
S. marcescens
MG1 utilizes different regulatory systems and adhesins in attachment to biotic and abiotic surfaces and that QS is a main regulatory pathway in adhesion to an abiotic surface but not in adhesion to a biotic surface.
Functional Characterization of the Initiation Enzyme of S-Layer Glycoprotein Glycan Biosynthesis in
Geobacillus stearothermophilus
NRS 2004/3a
Authors: Steiner Kerstin , Novotny René , Patel Kinnari , Vinogradov Evgenij , Whitfield Chris , Valvano Miguel A. , Messner Paul , Schäffer Christina
Published: January 2007

The glycan chain of the S-layer glycoprotein of
Geobacillus stearothermophilus
NRS 2004/3a is composed of repeating units [→2)-α-
l
-Rha
p-
(1→3)-β-
l
-Rha
p
-(1→2)-α-
l
-Rha
p
-(1→], with a 2-
O
-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain, a core saccharide composed of two or three α-
l
-rhamnose residues, and a β-
d
-galactose residue as a linker to the S-layer protein. In this study, we report the biochemical characterization of WsaP of the S-layer glycosylation gene cluster as a UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase that primes the S-layer glycoprotein glycan biosynthesis of
Geobacillus stearothermophilus
NRS 2004/3a. Our results demonstrate that the enzyme transfers in vitro a galactose-1-phosphate from UDP-galactose to endogenous phosphoryl-polyprenol and that the C-terminal half of WsaP carries the galactosyltransferase function, as already observed for the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WbaP from
Salmonella enterica
. To confirm the function of the enzyme, we show that WsaP is capable of reconstituting polysaccharide biosynthesis in WbaP-deficient strains of
Escherichia coli
and
Salmonella enterica
serovar Typhimurium.
DRS Is Far Less Divergent than Streptococcal Inhibitor of Complement of Group A Streptococcus
Authors: Sagar Vivek , Kumar Rajesh , Ganguly Nirmal K. , Menon Thangam , Chakraborti Anuradha
Published: January 2007

When 100 group A streptococcus isolates were screened,
drs
, a variant of
sic
, was identified in
emm12
and
emm55
isolates. Molecular characterization showed that the
drs
gene sequence is highly conserved, unlike the
sic
gene sequence. However, the variation in gene size observed was due to the presence of extra internal repeat sequences.
The Essential GTPase YqeH Is Required for Proper Ribosome Assembly in
Bacillus subtilis
Authors: Uicker William C. , Schaefer Laura , Koenigsknecht Mark , Britton Robert A.
Published: January 2007

Recent work with bacteria and eukaryotes has shown that GTPases play important roles in ribosome assembly. Here we show that the essential GTPase YqeH is required for proper 70S ribosome formation and 30S subunit assembly/stability in
Bacillus subtilis
.
The W-Beijing Lineage of
Mycobacterium tuberculosis
Overproduces Triglycerides and Has the DosR Dormancy Regulon Constitutively Upregulated
Authors: Reed Michael B. , Gagneux Sebastien , DeRiemer Kathryn , Small Peter M. , Barry Clifton E.
Published: January 2007

The Beijing family of
Mycobacterium tuberculosis
strains has been associated with epidemic spread and an increased likelihood of developing drug resistance. The characteristics that predispose this family to such clinical outcomes have not been identified, although one potential candidate, the phenolic glycolipid PGL-tb, has been shown to mediate a fulminant lethal disease in mice and rabbits due to lipid-mediated immunosuppression. However, PGL-tb is not uniformly expressed throughout the Beijing lineage and may not be the only unique virulence trait associated with this family. In an attempt to define phenotypes common to all Beijing strains, we interrogated a carefully selected set of isolates representing the five extant lineages of the Beijing family. Comparison of lipid production in this set revealed that all Beijing strains accumulated large quantities of triacylglycerides in in vitro aerobic culture. This accumulation was found to be coincident with upregulation of Rv3130c, whose product was previously characterized as a triacylglyceride synthase. Rv3130c is a member of the DosR-controlled regulon of
M. tuberculosis
, and further examination revealed that several members of this regulon were upregulated throughout this strain family. The upregulation of the DosR regulon may confer an adaptive advantage for growth in microaerophilic or anaerobic environments encountered by the bacillus during infection and thus may be related to the epidemiological phenomena associated with this important strain lineage.
Regulatory Role of PopN and Its Interacting Partners in Type III Secretion ofPseudomonas aeruginosa
Authors: Yang Hongjing , Shan Zhiying , Kim Jaewha , Wu Weihui , Lian Wei , Zeng Lin , Xing Laijun , Jin Shouguang
Published: January 2007

The type III secretion system (T3SS) ofPseudomonas aeruginosaplays a significant role in pathogenesis. We have previously identified type III secretion factor (TSF), which is required for effective secretion of the type III effector molecules, in addition to the low calcium signal. TSF includes many low-affinity high-capacity calcium binding proteins, such as serum albumin and casein. A search for the TSF binding targets on the bacterial outer membrane resulted in identification of PopN, a component of the T3SS that is readily detectable on the bacterial cell surface. PopN specifically interacts with Pcr1, and bothpopNandpcr1mutants have a constitutive type III secretion phenotype, suggesting that the two proteins form a complex that functions as a T3SS repressor. Further analysis of thepopNoperon genes resulted in identification of protein-protein interactions between Pcr1 and Pcr4 and between Pcr4 and Pcr3, as well as between PopN and Pcr2 in the presence of PscB. UnlikepopNandpcr1mutants,pcr3andpcr4mutants are totally defective in type III secretion, while apcr2mutant exhibits reduced type III secretion. Interestingly, PopN, Pcr1, Pcr2, and Pcr4 are all secreted in a type III secretion machinery-dependent manner, while Pcr3 is not. These findings imply that these components have important regulatory roles in controlling type III secretion.
Distinct Functions of the Two Specificity Determinants in Replication Initiation of Plasmids ColE2-P9 and ColE3-CA38
Authors: Aoki Kazuteru , Shinohara Miki , Itoh Tateo
Published: January 2007

The plasmid ColE2-P9 Rep protein specifically binds to the cognate replication origin to initiate DNA replication. The replicons of the plasmids ColE2-P9 and ColE3-CA38 are closely related, although the actions of the Rep proteins on the origins are specific to the plasmids. The previous chimera analysis identified two regions, regions A and B, in the Rep proteins and two sites, α and β, in the origins as specificity determinants and showed that when each component of the region A-site α pair and the region B-site β pair is derived from the same plasmid, plasmid DNA replication is efficient. It is also indicated that the replication specificity is mainly determined by region A and site α. By using an electrophoretic mobility shift assay, we demonstrated that region B and site β play a critical role for stable Rep protein-origin binding and, furthermore, that 284-Thr in this region of the ColE2 Rep protein and the corresponding 293-Trp of the ColE3 Rep protein mainly determine the Rep-origin binding specificity. On the other hand, region A and site α were involved in the efficient unwinding of several nucleotide residues around site α, although they were not involved in the stable binding of the Rep protein to the origin. Finally, we discussed how the action of the Rep protein on the origin involving these specificity determinants leads to the plasmid-specific replication initiation.
Controlled Expression of
nif
and
isc
Iron-Sulfur Protein Maturation Components Reveals Target Specificity and Limited Functional Replacement between the Two Systems
Authors: Dos Santos Patricia C. , Johnson Deborah C. , Ragle Brook E. , Unciuleac Mihaela-Carmen , Dean Dennis R.
Published: January 2007

The nitrogen-fixing organism
Azotobacter vinelandii
contains at least two systems that catalyze formation of [Fe-S] clusters. One of these systems is encoded by
nif
genes, whose products supply [Fe-S] clusters required for maturation of nitrogenase. The other system is encoded by
isc
genes, whose products are required for maturation of [Fe-S] proteins that participate in general metabolic processes. The two systems are similar in that they include an enzyme for the mobilization of sulfur (NifS or IscS) and an assembly scaffold (NifU or IscU) upon which [Fe-S] clusters are formed. Normal cellular levels of the Nif system, which supplies [Fe-S] clusters for the maturation of nitrogenase, cannot also supply [Fe-S] clusters for the maturation of other cellular [Fe-S] proteins. Conversely, when produced at the normal physiological levels, the Isc system cannot supply [Fe-S] clusters for the maturation of nitrogenase. In the present work we found that such target specificity for IscU can be overcome by elevated production of NifU. We also found that NifU, when expressed at normal levels, is able to partially replace the function of IscU if cells are cultured under low-oxygen-availability conditions. In contrast to the situation with IscU, we could not establish conditions in which the function of IscS could be replaced by NifS. We also found that elevated expression of the Isc components, as a result of deletion of the regulatory
iscR
gene, improved the capacity for nitrogen-fixing growth of strains deficient in either NifU or NifS.
Regulators of Expression of the Oligopeptide Permease A Proteins of
Borrelia burgdorferi
Authors: Medrano Melisa S. , Ding Yanpeng , Wang Xing-Guo , Lu Peng , Coburn Jenifer , Hu Linden T.
Published: January 2007

Borrelia burgdorferi
undergoes an infectious cycle that requires adaptation to different hosts and marked differences in environment.
B. burgdorferi
copes with its different environments by regulating the expression of proteins required for survival in specific settings. The
B. burgdorferi
oligopeptide permease (Opp) is one of only a few transporters encoded by the
B. burgdorferi
genome. Opp proteins in other bacteria serve multiple environmental adaptation functions.
B. burgdorferi
appears to broaden the usage of this transporter by utilizing five different substrate binding proteins (OppA proteins) that interact with the integral membrane components of the transporter. Expression of the OppA proteins is individually regulated and may play different roles in adaptation to host environments. Very little is known about the mechanisms used by
B. burgdorferi
to regulate the expression of different OppA proteins. Here we show that the alternative sigma factors, RpoS and RpoN, regulate the expression of
oppA5
but not that of other
oppA
genes. Using a reporter assay with
Escherichia coli
and gel shift binding assays, we also show that the
B. burgdorferi
BosR/Fur homologue interacts with the
oppA4
promoter and that another candidate transcription factor, EbfC, interacts with the
oppA5
promoter. Binding to the promoters was confirmed by gel shift assays. Expression of BosR/Fur in its different hosts does appear to parallel the expression of
oppA4
. A better understanding of the factors involved in gene regulation in
B. burgdorferi
will help to identify coregulated proteins that may cooperate to allow the organism to survive in a specific environment.
Positive Regulation of
fur
Gene Expression via Direct Interaction of Fur in a Pathogenic Bacterium,
Vibrio vulnificus
Authors: Lee Hyun-Jung , Bang So Hyun , Lee Kyu-Ho , Park Soon-Jung
Published: January 2007

In pathogenic bacteria, the ability to acquire iron, which is mainly regulated by the ferric uptake regulator (Fur), is essential to maintain growth as well as its virulence. In
Vibrio vulnificus
, a human pathogen causing gastroenteritis and septicemia,
fur
gene expression is positively regulated by Fur when the iron concentration is limited (H.-J. Lee et al., J. Bacteriol. 185:5891-5896, 2003). Footprinting analysis revealed that an upstream region of the
fur
gene was protected by the Fur protein from DNase I under iron-depleted conditions. The protected region, from −142 to −106 relative to the transcription start site of the
fur
gene, contains distinct AT-rich repeats. Mutagenesis of this repeated sequence resulted in abolishment of binding by Fur. To confirm the role of this
cis
-acting element in Fur-mediated control of its own gene in vivo,
fur
expression was monitored in
V. vulnificus
strains using a transcriptional fusion containing the mutagenized Fur-binding site (
fur
mt
::
luxAB
). Expression of
fur
mt
::
luxAB
showed that it was not regulated by Fur and was not influenced by iron concentration. Therefore, this study demonstrates that
V. vulnificus
Fur acts as a positive regulator under iron-limited conditions by direct interaction with the
fur
upstream region.
Conditional Mutation of an Essential Putative Glycoprotease Eliminates Autolysis in
Staphylococcus aureus
Authors: Zheng Li , Yu Chuanxin , Bayles Kenneth , Lasa Iñigo , Ji Yinduo
Published: January 2007

Our previous studies demonstrated that a putative
Staphylococcus aureus
glycoprotease (Gcp) is essential for bacterial survival, indicating that Gcp may be a novel target for developing antibacterial agents. However, the biological function of Gcp is unclear. In order to elucidate the reason that Gcp is required for growth, we examined the role of Gcp in bacterial autolysis, which is an important biological process for bacterial growth. Using both a
spacp
-regulated
gcp
expression strain and a TetR-regulated
gcp
antisense expression strain, we found that the down-regulation of
gcp
expression can effectively inhibit Triton X-100-induced lysis, eliminate penicillin- and vancomycin-caused cell lysis, and dramatically increase tolerance to hydrolases. Moreover, we determined whether resistance to lysis is due to a defect in murein hydrolase activity by using a zymogram analysis. The results showed that the cell lysate of a down-regulated
gcp
expression mutant displayed several bands of decreased murein hydrolytic activity. Furthermore, we explored the potential mechanism of Gcp's involvement in autolysis and demonstrated that Gcp may function independently from several key autolysins (Atl, LytM, and LytN) and regulators (ArlRS, Mgr/Rat, and CidA). Taken together, the above results indicate that the essential Gcp is involved in the modification of substrates of murein hydrolases as well as in the regulation of expression and/or activity of some murein hydrolases, which, in turn, may play important roles in bacterial viability.
The Symbiosis Regulator CbrA Modulates a Complex Regulatory Network Affecting the Flagellar Apparatus and Cell Envelope Proteins
Authors: Gibson Katherine E. , Barnett Melanie J. , Toman Carol J. , Long Sharon R. , Walker Graham C.
Published: January 2007

Sinorhizobium meliloti
participates in a nitrogen-fixing symbiosis with legume plant host species of the genera
Medicago
,
Melilotus
, and
Trigonella
. We recently identified an
S. meliloti
two-component sensory histidine kinase, CbrA, which is absolutely required to establish a successful symbiosis with
Medicago sativa
(K. E. Gibson, G. R. Campbell, J. Lloret, and G. C. Walker, J. Bacteriol. 188:4508-4521, 2006). In addition to having a symbiotic defect, the
cbrA
::Tn
5
mutant also has free-living phenotypes that suggest a cell envelope perturbation. Because the bases for these phenotypes are not well understood, we undertook an identification of CbrA-regulated genes. We performed a microarray analysis and compared the transcriptome of the
cbrA
::Tn
5
mutant to that of the wild type. Our global analysis of gene expression identified 162 genes that are differentially expressed in the
cbrA
::Tn
5
mutant, including those encoding proteins involved in motility and chemotaxis, metabolism, and cell envelope function. With regard to those genes with a known role in symbiosis, we observed increased expression of nine genes with overlapping functions in bacterial invasion of its host, which suggests that the mutant could be competent for invasion. Since these CbrA-repressed genes are vital to the invasion process, it appears that down-regulation of CbrA activity is important at this stage of nodule development. In contrast, our previous work showed that CbrA is required for bacteria to establish themselves within the host as nitrogen-fixing symbionts. Therefore, we propose a model in which CbrA functions as a developmental switch during symbiosis.
Functional Characterization of the Gene Cluster from
Pseudomonas syringae
pv. phaseolicola NPS3121 Involved in Synthesis of Phaseolotoxin
Authors: Aguilera Selene , López-López Karina , Nieto Yudith , Garcidueñas-Piña Rogelio , Hernández-Guzmán Gustavo , Hernández-Flores José Luis , Murillo Jesús , Alvarez-Morales Ariel
Published: January 2007

Pseudomonas syringae
pv. phaseolicola is the causal agent of halo blight disease of beans (
Phaseolus vulgaris
L.), which is characterized by water-soaked lesions surrounded by a chlorotic halo resulting from the action of a non-host-specific toxin known as phaseolotoxin. This phytotoxin inhibits the enzyme ornithine carbamoyltransferase involved in arginine biosynthesis. Different evidence suggested that genes involved in phaseolotoxin production were clustered. Two genes had been previously identified in our laboratory within this cluster:
argK
, which is involved in the immunity of the bacterium to its own toxin, and
amtA
, which is involved in the synthesis of homoarginine. We sequenced the region around
argK
and
amtA
in
P. syringae
pv. phaseolicola NPS3121 to determine the limits of the putative phaseolotoxin gene cluster and to determine the transcriptional pattern of the genes comprising it. We report that the phaseolotoxin cluster (Pht cluster) is composed of 23 genes and is flanked by insertion sequences and transposases. The mutation of 14 of the genes within the cluster lead to a Tox
−
phenotype for 11 of them, while three mutants exhibited low levels of toxin production. The analysis of fusions of selected DNA fragments to
uidA
, Northern probing, and reverse transcription-PCR indicate the presence of five transcriptional units, two monocistronic and three polycistronic; one is internal to a larger operon. The site for transcription initiation has been determined for each promoter, and the putative promoter regions were identified. Preliminary results also indicate that the gene product of
phtL
is involved in the regulation of the synthesis of phaseolotoxin.
SOS Regulation of the Type III Secretion System of EnteropathogenicEscherichia coli
Authors: Mellies Jay L. , Haack Kenneth R. , Galligan Derek C.
Published: January 2007

Genomes of bacterial pathogens contain and coordinately regulate virulence-associated genes in order to cause disease. EnteropathogenicEscherichia coli(EPEC), a major cause of watery diarrhea in infants and a model gram-negative pathogen, expresses a type III secretion system (TTSS) that is encoded by the locus of enterocyte effacement (LEE) and is necessary for causing attaching and effacing intestinal lesions. Effector proteins encoded by the LEE and in cryptic prophage are injected into the host cell cytoplasm by the TTTS apparatus, ultimately leading to diarrhea. The LEE is comprised of multiple polycistronic operons, most of which are controlled by the global, positive regulator Ler. Here we demonstrated that theLEE2andLEE3operons also responded to SOS signaling and that this regulation was LexA dependent. As determined by a DNase I protection assay, purified LexA protein bound in vitro to a predicted SOS box located in the divergent, overlappingLEE2/LEE3promoters. Expression of thelexA1allele, encoding an uncleavable LexA protein in EPEC, resulted in reduced secretion, particularly in the absence of the Ler regulator. Finally, we obtained evidence that the cryptic phage-locatednleAgene encoding an effector molecule is SOS regulated. Thus, we demonstrated, for the first time to our knowledge, that genes encoding components of a TTSS are regulated by the SOS response, and our data might explain how a subset of EPEC effector proteins, encoded in cryptic prophages, are coordinately regulated with the LEE-encoded TTSS necessary for their translocation into host cells.
Functional Characterization and Membrane Topology of
Escherichia coli
WecA, a Sugar-Phosphate Transferase Initiating the Biosynthesis of Enterobacterial Common Antigen and O-Antigen Lipopolysaccharide
Authors: Lehrer Jason , Vigeant Karen A. , Tatar Laura D. , Valvano Miguel A.
Published: January 2007

WecA is an integral membrane protein that initiates the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharide (LPS) by catalyzing the transfer of
N
-acetylglucosamine (GlcNAc)-1-phosphate onto undecaprenyl phosphate (Und-P) to form Und-P-P-GlcNAc. WecA belongs to a large family of eukaryotic and prokaryotic prenyl sugar transferases. Conserved aspartic acids in putative cytoplasmic loops 2 (Asp90 and Asp91) and 3 (Asp156 and Asp159) were targeted for replacement mutagenesis with either glutamic acid or asparagine. We examined the ability of each mutant protein to complement O-antigen LPS synthesis in a
wecA
-deficient strain and also determined the steady-state kinetic parameters of the mutant proteins in an in vitro transfer assay. Apparent

K
m

and
V
max
values for UDP-GlcNAc, Mg
2+
, and Mn
2+
suggest that Asp156 is required for catalysis, while Asp91 appears to interact preferentially with Mg
2+
, possibly playing a role in orienting the substrates. Topological analysis using the substituted cysteine accessibility method demonstrated the cytosolic location of Asp90, Asp91, and Asp156 and provided a more refined overall topological map of WecA. Also, we show that cells expressing a WecA derivative C terminally fused with the green fluorescent protein exhibited a punctate distribution of fluorescence on the bacterial surface, suggesting that WecA localizes to discrete regions in the bacterial plasma membrane.
Activity patterns, blood lactate concentrations and ratings of perceived exertion during a professional singles tennis tournament
Authors: Mendez-Villanueva Alberto , Fernandez-Fernandez Jaime , Bishop David , Fernandez-Garcia Benjamin , Terrados Nicolas
Published: January 2007

Objective:To examine the game characteristics and physiological (ie, blood lactate concentration) and perceptual (ie, rating of perceived exertion, RPE) responses during actual tennis competition in professional performers.

Methods:Eight trained and internationally ranked (Association of Tennis Professionals rankings) male tennis players were studied during singles matches (best of three sets) played on an outdoor clay court surface during a professional, invitational tournament. Blood lactate concentrations (n = 53) and RPE (n = 113) were determined at selected changeovers during the game. The variables describing the characteristics of the matches, (a) duration of rallies (DRs); (b) rest time (RT); (c) effective playing time (EPT); and (d) shots per rally (SR), were determined from video recordings.

Results:The mean (SD) values for the match-play activity variables were DR 7.5 (7.3) s, RT 16.2 (5.2) s, EPT 21.5 (4.9%), SR 2.7 (2.2) shots. Average blood lactate concentration and RPE values were 3.8 (2.0) mmol/l and 13 (2). Blood lactate concentrations and RPE values were significantly higher (p<0.01) in service games than in receiving games. Both blood lactate concentration and RPE values were significantly correlated with SR and DR (r = 0.80 to 0.28; p<0.001).

Conclusions:Blood lactate concentrations and RPE were found to be influenced by the characteristics of the match and the playing situation (ie, serving or returning). These specific situations might be used to alter the overload training stimulus during tennis on-court practice.
High incidence of exercise-induced bronchoconstriction in triathletes of the Swiss national team
Authors: Knöpfli Bruno H , Luke-Zeitoun Mona , von Duvillard Serge P , Burki Adrian , Bachlechner Christian , Keller Heidi
Published: January 2007

Aim:To assess the progression of bronchial reactivity (BR) and incidence of bronchial hyperreactivity (BH), exercise-induced bronchoconstriction (EIB) and asthma in triathletes over 2 years.

Methods:Subjects were seven athletes from the Swiss national triathlon team (mean (SD) age 24.3 (4.8) years), who initially were not asthmatic, not treated with antiasthmatic medication, and who had performed at international level for more than 3 consecutive years (2001–2003). To assess BR, BH and EIB, subjects ran on a 400 m track for 8 min at intensities equal to the anaerobic threshold. Tests were conducted in ambient temperatures of 4.4 (2.8)°C, –8.8 (2.4)°C and 3.6 (1.5)°C, and humidity of 52 (16)%, 83 (13)% and 93 (2)%. Forced expiratory volume in 1 s (FEV1) was measured before and at 2, 5, 10 and 15 min after EIB, and 5 min after inhalation of a β2 agonist. Two methods were used to calculate the incidence: (1) the standard assessment; (2) extrapolation of the decrease in FEV1to the BH limit.

Results:BR increased significantly in the seven athletes (FEV1: year, p = 0.04; year × EIB, p = 0.002; EIB p<0.001). Within 2 years, BR had increased significantly and even reached BH in some athletes. Three athletes exhibited BH. After extrapolation of the decrease in FEV1in all seven athletes, the limit of 10% by definition for BH was determined to occur within 1.77–4.81 years, resulting in 21–57% of athletes with newly developed BH per year.

Conclusion:Athletes develop EIB quickly, a rate of increase 195–286 times that of the normal rate for development of asthma.
Proximal biceps rupture: management of an unusual injury in an arm wrestler
Authors: Pratt D A , Tennent T D
Published: January 2007

Arm wrestling may cause unusual injuries, which may require operative repair in the sporting individual. Injury to the proximal biceps as a consequence of arm wrestling has not been reported previously. The diagnosis and treatment of a 36-year-old man who sustained a proximal biceps rupture while arm wrestling and his operative management are described.
Jamestown Canyon virus detection in human tissue specimens
Authors: Zheng Huachuan , Murai Yoshihiro , Hong Mei , Nakanishi Yuko , Nomoto Kazuhiro , Masuda Shinji , Tsuneyama Koichi , Takano Yasuo
Published: January 2007

Aim:To clarify the advantages and disadvantages of different detection methods for Jamestown Canyon virus (JCV) in human tissue specimens.

Methods:Specimens of lung and gastric carcinomas, and normal lung tissue, gastric mucosa, and tonsil were examined forT-antigen,VPand agnoprotein of JCV by nested PCR, Southern blotting and sequencing. JCV load targetingT-antigenwas evaluated by real-time PCR, and JCV existence morphologically by immunohistochemistry, in-situ hybridisation (ISH) and PCR. For these experiments, the JCI cell line (JCV cultured neuroblastoma cell line) was employed as positive control.

Results:In lung and gastric carcinomas,T-antigen,VPandagnoproteinof JCV could be detected by nested PCR whose products were confirmed by Southern blots and sequencing. With real-time PCR, frozen samples of gastric carcinomas gave better detection of JCV than their corresponding paraffin-embedded tissues (p<0.05). The positive rate of JCV was high in lung carcinoma, compared with normal lung tissue (p<0.05). It was the same for JCV copies in gastric carcinoma (p<0.05). Only the positive control exhibited JCV in the nucleus by ISH and immunohistochemistry. In-situ PCR showed that JCV genomic DNA was located in the nucleus of the carcinoma cell, some alveolar epithelial cells, and tonsil lymphocytes. In ISH and PCR, NBT/BCIP colouring was stronger than Fuchsin.

Conclusions:Nested PCR whose amplicons should be confirmed by Southern blot and sequencing was a comparatively sensitive approach to detect JCV genomic DNA in human non-neural tissues. Real-time PCR might be employed to quantify copy number of JCV. In-situ PCR was a good method to observe the JCV location in cells, given appropriate modulation of amplification cycles. Combinations of various approaches will be adopted to explore the oncogenic roles of JCV in malignancies.
My approach to intraductal lesions of the prostate gland
Authors: Pickup M , Van der Kwast T H
Published: January 2007

The morphologically heterogeneous (intra)ductal lesions of the prostate frequently present a diagnostic challenge, particularly when found within prostate needle biopsies. By current convention, all high-grade intra-acinar and intraductal neoplastic lesions of prostatic origin fall under the diagnostic umbrella term: prostatic intraepithelial neoplasm (PIN). Although a long-standing contentious issue, some lesions currently adhering to the diagnostic criteria of PIN may actually represent the intraductal spread of (generally high grade) invasive cancer. Illustrating this fact, the well-described ductal subtype of prostatic adenocarcinoma is frequently associated with conventional-type acinar adenocarcinoma, and has a tendency to propagate within adjacent intact prostatic ducts. Clearly, the misdiagnosis of lesions representing invasive disease as preinvasive has the potential for unfavourable clinical sequelae. As yet, however, many of these lesions have escaped the establishment of reliable morphologic criteria or immunohistochemical differentiation for diagnosis. By defining stringent architectural and cytonuclear features specific for each of these lesions, it may be feasible to separate potentially sinister lesions from the subset of traditional (preinvasive) PIN lesions with limited clinical urgency. This review discusses the (intra)ductal lesions of the prostate, along with their differential diagnoses. Given the current state of knowledge, a pragmatic approach to their effective reporting is outlined, taking into consideration the clinical implications, as well as current guidelines for treatment and follow-up.
Evidence for enhanced eNOS function in coronary microvessels during the second window of protection
Authors: Kim Song-Jung , Zhang Xiaoping , Xu Xiaobin , Chen Alice , Gonzalez Joaquin B. , Koul Sharat , Vijayan Kalpana , Crystal George J. , Vatner Stephen F. , Hintze Thomas H.
Published: January 2007

Nitric oxide (NO) derived from endothelial NO synthase (NOS) (eNOS) has been identified as a trigger for the second window of protection (SWOP), but its role as a mediator during the SWOP is a matter of debate. Eighteen mongrel dogs were chronically instrumented to measure left ventricular function, coronary blood flow, and wall thickening. Myocardial preconditioning was induced by 10 min coronary artery occlusion. After 24 h of reperfusion (during the SWOP), the hearts were excised. Coronary microvessels were isolated and incubated in presence of 1) the endothelium-dependent agonists carbachol and bradykinin, 2) the calcium ionophore A23187, and 3) the angiotensin-converting enzyme (ACE) inhibitors enalaprilat and ramiprilat. Nitrite, a metabolite of NO, was measured. Under baseline conditions, nitrite production in microvessels from SWOP was 30% higher than that from normal (96 ± 4 vs. 74 ± 3 pmol/mg, P < 0.01, respectively). Nitrite production in response to carbachol, bradykinin, and A23187 was also enhanced in microvessels from SWOP ( P < 0.05). These enhanced responses were abolished by NG-nitro-l-arginine methyl ester (l-NAME) or the endothelial receptor-specific antagonists atropine and HOE-140. The level of eNOS protein in the SWOP myocardium was twofold higher than that in the non-SWOP myocardium. Nitrite production in response to the ACE inhibitors was greater in microvessels from SWOP. These effects were blocked by l-NAME, HOE-140, or dichloroisocoumarin (which inhibits kinin formation). We found that a brief ischemic episode induced delayed, enhanced NO production in coronary microvessels and an upregulation of eNOS protein. These findings suggest that eNOS is a mediator during the SWOP. The ability of ACE inhibitors to enhance NO release during the SWOP points to an additional clinical application for these drugs.
β1-Adrenoreceptor activation contributes to ischemia-reperfusion damage as well as playing a role in ischemic preconditioning
Authors: Spear Joseph F. , Prabu Subbuswamy K. , Galati Domenico , Raza Haider , Anandatheerthavarada Hindupur K. , Avadhani Narayan G.
Published: January 2007

Protein kinase A (PKA) activation has been implicated in early-phase ischemic preconditioning. We recently found that during ischemia PKA activation causes inactivation of cytochrome- c oxidase (CcO) and contributes to myocardial damage due to ischemia-reperfusion. It may be that β-adrenergic stimulation during ischemia via endogenous catecholamine release activates PKA. Thus β-adrenergic stimulation may mediate both myocardial protection and damage during ischemia. The present studies were designed to determine the role of the β1-adrenergic receptor (β1-AR) in myocardial ischemic damage and ischemic preconditioning. Langendorff-perfused rabbit hearts underwent 30-min ischemia by anterior coronary artery ligation followed by 2-h reperfusion. Occlusion-reperfusion damage was evaluated by delineating the nonperfused volume of myocardium at risk and volume of myocardial necrosis after 2-h reperfusion. In some hearts ischemic preconditioning was accomplished by two 5-min episodes of global low-flow ischemia separated by 10 min before coronary occlusion-reperfusion. Orthogonal electrocardiograms were recorded, and coronary flow was monitored by a drip count. Three hearts from each experimental group were used to determine mitochondrial CcO and aconitase activities. Two-hour reperfusion after occlusion caused an additional decrease in CcO activity vs. that after 30-min occlusion alone. Blocking the β1-AR during occlusion-reperfusion reversed CcO activity depression and preserved myocardium at risk for necrosis. Similarly, mitochondrial aconitase activity exhibited a parallel response after occlusion-reperfusion as well as for the other interventions. Furthermore, classic ischemic preconditioning had no effect on CcO depression. However, blocking the β1-AR during preconditioning eliminated the cardioprotection. If the β1-AR was blocked after preconditioning, the myocardium was preserved. Interestingly, in both of the latter cases the depression in CcO activity was reversed. Thus the β1-AR plays a dual role in myocardial ischemic damage. Our findings may lead to therapeutic strategies for preserving myocardium at risk for infarction, especially in coronary reperfusion intervention.
Validation of a novel noninvasive cardiac index of left ventricular contractility in patients
Authors: Zhong Liang , Tan Ru-San , Ghista Dhanjoo N. , Ng Eddie Yin-Kwee , Chua Leok-Poh , Kassab Ghassan S.
Published: January 2007

Although there are several excellent indexes of myocardial contractility, they require accurate measurement of pressure via left ventricular (LV) catheterization. Here we validate a novel noninvasive contractility index that is dependent only on lumen and wall volume of the LV chamber in patients with normal and compromised LV ejection fraction (LVEF). By analysis of the myocardial chamber as a thick-walled sphere, LV contractility index can be expressed as maximum rate of change of pressure-normalized stress (dσ*/d tmax, where σ* = σ/P and σ and P are circumferential stress and pressure, respectively). To validate this parameter, dσ*/d tmax was determined from contrast cine-ventriculography-assessed LV cavity and myocardial volumes and compared with LVEF, dP/d tmax, maximum active elastance ( Ea,max), and single-beat end-systolic elastance [ Ees(SB)] in 30 patients undergoing clinically indicated LV catheterization. Patients with different tertiles of LVEF exhibit statistically significant differences in dσ*/d tmax. There was a significant correlation between dσ*/d tmax and dP/d tmax (dσ*/d tmax = 0.0075dP/d tmax − 4.70, r = 0.88, P < 0.01), Ea,max (dσ*/d tmax = 1.20 Ea,max + 1.40, r = 0.89, P < 0.01), and Ees(SB) [dσ*/d tmax = 1.60 Ees(SB) + 1.20, r = 0.88, P < 0.01]. In 30 additional individuals, we determined sensitivity of the parameter to changes in preload (intravenous saline infusion, n = 10 subjects), afterload (sublingual glyceryl trinitrate, n = 10 subjects), and increased contractility (intravenous dobutamine, n = 10 patients). We confirmed that the index is not dependent on load but is sensitive to changes in contractility. In conclusion, dσ*/d tmax is equivalent to dP/d tmax, Ea,max, and Ees(SB) as an index of myocardial contractility and appears to be load independent. In contrast to other measures of contractility, dσ*/d tmax can be assessed with noninvasive cardiac imaging and, thereby, should have more routine clinical applicability.
Macromolecular transport in heart valves. III. Experiment and theory for the size distribution of extracellular liposomes in hyperlipidemic rabbits
Authors: Zeng Zhongqing , Nievelstein-Post Patricia , Yin Yongyi , Jan Kung-Ming , Frank Joy S. , Rumschitzki David S.
Published: January 2007

The heart valve leaflets of 29-day cholesterol-fed rabbits were examined by ultrarapid freezing without conventional chemical fixation/processing, followed by rotary shadow freeze-etching. This procedure images the leaflets' subendothelial extracellular matrix in extraordinary detail, and extracellular lipid liposomes, from 23 to 220 nm in diameter, clearly appear there. These liposomes are linked to matrix filaments and appear in clusters. Their size distribution shows 60.7% with diameters 23–69 nm, 31.7% between 70 and 119 nm, 7.3% between 120 and 169 nm, and 0.3% between 170 and 220 nm (superlarge) and suggests that smaller liposomes can fuse into larger ones. We couple our model from Part II of this series (Zeng Z, Yin Y, Jan KM, Rumschitzki DS. Am J Physiol Heart Circ Physiol 292: H2671–H2686, 2007) for lipid transport into the leaflet to the nucleation-polymerization model hierarchy for liposome formation proposed originally for aortic liposomes to predict liposome formation/growth in heart valves. Simulations show that the simplest such model cannot account for the observed size distribution. However, modifying this model by including liposome fusing/merging, using parameters determined from aortic liposomes, leads to predicted size distributions in excellent agreement with our valve data. Evolutions of both the liposome size distribution and total liposome mass suggest that fusing becomes significant only after 2 wk of high lumen cholesterol. Inclusion of phagocytosis by macrophages limits the otherwise monotonically increasing total liposome mass, while keeping the excellent fit of the liposome size distribution to the data.
Erythropoietin promotes survival of primary human endothelial cells through PI3K-dependent, NF-κB-independent upregulation of Bcl-xL
Authors: Zhande Rachel , Karsan Aly
Published: January 2007

Erythropoietin (EPO) regulates the production of red blood cells primarily by preventing apoptosis of erythroid progenitors. More recently, however, EPO has emerged as a major cytoprotective cytokine in several nonhemopoietic tissues in the setting of stress or injury. The underlying mechanisms of the protective responses of EPO have not been fully defined. Here we show that EPO triggers a phosphatidylinositol 3-kinase-(PI3K)-dependent survival pathway that counteracts endothelial cell death. The protection conferred by PI3K relies on the subsequent induction of Bcl-xL, a prosurvival member of the Bcl-2 protein family. In addition, EPO counteracts the upregulation of the pro-apoptotic BH3-only protein BIM, which is induced by serum withdrawal. EPO also activates extracellular signal-regulated kinase 1 and 2 (ERK1/2), which are involved in a Bcl-xL-independent cytoprotective pathway. EPO caused a prolonged activation of nuclear factor (NF)-κB, which was blocked by inhibition of PI3K, but not by inhibition of mitogen-activated protein (MAP)/ERK kinase (MEK), suggesting that EPO-activated NF-κB requires PI3K activity. However, the activation of the NF-κB pathway was not required for the ability of EPO to counteract endothelial apoptosis. Thus EPO promotes survival of endothelial cells through PI3K-dependent Bcl-xL-induction and BIM regulation, as well as through a separate mechanism involving the ERK pathway.
Aortic stiffness and pulse pressure amplification in Wistar-Kyoto and spontaneously hypertensive rats
Authors: Cosson Emmanuel , Herisse Monique , Laude Dominique , Thomas Frédérique , Valensi Paul , Attali Jean-Raymond , Safar Michel E. , Dabire Hubert
Published: January 2007

In humans, increased body weight and arterial stiffness are significantly associated, independently of blood pressure (BP) level. The finding was never investigated in rodents devoid of metabolic disorders as spontaneously hypertensive rats (SHR). Using simultaneous catheterization of proximal and distal aorta, we measured body weight, intra-arterial BP, heart rate and their variability (spectral analysis), aortic pulse wave velocity (PWV), and systolic and pulse pressure (PP) amplifications in unrestrained conscious Wistar-Kyoto (WKY) rats and SHR between 6 and 24 wk of age. Aortic proximal systolic and diastolic pressure, PP, and mean BP were significantly higher in SHR than in WKY rats and increased significantly with age (with the exception of PP). PP amplification increased with age but did not differ between strains. PWV was significantly associated with heart rate variability. PWV was significantly higher (via two-way variance analysis) in SHR than in WKY rats (strain effect) and increased markedly with age in both strains (age effect). Adjustment of PWV to mean BP attenuated markedly both the age and the strain effects. After adjustment for body weight, either alone or associated with mean BP, the age effect was not more significant, but the strain effect was markedly enhanced. In conscious unanesthetized SHR and WKY rats, aortic stiffness is consistently associated with body weight independent of age and mean BP. An intervention study should consider in the objectives systolic BP and PP amplifications measured in conscious animals, central control of body weight, and autonomic nervous system.
Role of lipoprotein-associated lysophospholipids in migratory activity of coronary artery smooth muscle cells
Authors: Damirin Alatangaole , Tomura Hideaki , Komachi Mayumi , Liu Jin-Peng , Mogi Chihiro , Tobo Masayuki , Wang Ju-Qiang , Kimura Takao , Kuwabara Atsushi , Yamazaki Yuji , Ohta Hideo , Im Doon-Soon , Sato Koichi , Okajima Fumikazu
Published: January 2007

The migration of vascular smooth muscle cells (SMCs) is a hallmark of the pathogenesis of atherosclerosis and restenosis after angioplasty. Plasma low-density lipoprotein (LDL), but not high-density lipoprotein (HDL), induced the migration of human coronary artery SMCs (CASMCs). Among bioactive lipids postulated to be present in LDL, lysophosphatidic acid (LPA) appreciably mimicked the LDL action. In fact, the LDL-induced migration was markedly inhibited by pertussis toxin, an LPA receptor antagonist Ki-16425, and a small interfering RNA (siRNA) targeted for LPA1receptors. Moreover, LDL contains a higher amount of LPA than HDL does. HDL markedly inhibited LPA- and platelet-derived growth factor (PDGF)-induced migration, and sphingosine 1-phosphate (S1P), the content of which is about fourfold higher in HDL than in LDL, mimicked the HDL action. The inhibitory actions of HDL and S1P were suppressed by S1P2receptor-specific siRNA. On the other hand, the degradation of the LPA component of LDL by monoglyceride lipase or the antagonism of LPA receptors by Ki-16425 allowed LDL to inhibit the PDGF-induced migration. The inhibitory effect of LDL was again suppressed by S1P2receptor-specific siRNA. In conclusion, LPA/LPA1receptors and S1P/S1P2receptors mediate the stimulatory and inhibitory migration response to LDL and HDL, respectively. The balance of not only the content of LPA and S1P in lipoproteins but also the signaling activity between LPA1and S1P2receptors in the cells may be critical in determining whether the lipoprotein is a positive or negative regulator of CASMC migration.