bluuebunny/crossref_metadata_2025 huggingface dataset:
Evolutionary Genetics of a New Pathogenic
Escherichia
Species:
Escherichia
albertii
and Related
Shigella
boydii
Strains
Authors: Hyma Katie E. , Lacher David W. , Nelson Adam M. , Bumbaugh Alyssa C. , Janda J. Michael , Strockbine Nancy A. , Young Vincent B. , Whittam Thomas S.
Published: January 2005

A bacterium originally described as
Hafnia
alvei
induces diarrhea in rabbits and causes epithelial damage similar to the attachment and effacement associated with enteropathogenic
Escherichia
coli
. Subsequent studies identified similar
H. alvei
-like strains that are positive for an intimin gene (
eae
) probe and, based on DNA relatedness, are classified as a distinct
Escherichia
species,
Escherichia
albertii
. We determined sequences for multiple housekeeping genes in five
E. albertii
strains and compared these sequences to those of strains representing the major groups of pathogenic
E. coli
and
Shigella
. A comparison of 2,484 codon positions in 14 genes revealed that
E. albertii
strains differ, on average, at ∼7.4% of the nucleotide sites from pathogenic
E. coli
strains and at 15.7% from
Salmonella
enterica
serotype Typhimurium. Interestingly,
E. albertii
strains were found to be closely related to strains of
Shigella
boydii
serotype 13 (
Shigella
B13), a distant relative of
E. coli
representing a divergent lineage in the genus
Escherichia
. Analysis of homologues of intimin (
eae
) revealed that the central conserved domains are similar in
E. albertii
and
Shigella
B13 and distinct from those of
eae
variants found in pathogenic
E. coli
. Sequence analysis of the cytolethal distending toxin gene cluster (
cdt
) also disclosed three allelic groups corresponding to
E. albertii
,
Shigella
B13, and a nontypeable isolate serologically related to
S. boydii
serotype 7. Based on the synonymous substitution rate, the
E. albertii
-
Shigella
B13 lineage is estimated to have split from an
E. coli
-like ancestor ∼28 million years ago and formed a distinct evolutionary branch of enteric pathogens that has radiated into groups with distinct virulence properties.
TheErwinia chrysanthemiType III Secretion System Is Required for Multicellular Behavior
Authors: Mee-Ngan Yap , Yang Ching-Hong , Barak Jeri D. , Jahn Courtney E. , Charkowski Amy O.
Published: January 2005

Enterobacterial animal pathogens exhibit aggregative multicellular behavior, which is manifested as pellicles on the culture surface and biofilms at the surface-liquid-air interface. Pellicle formation behavior requires production of extracellular polysaccharide, cellulose, and protein filaments, known as curli. Protein filaments analogous to curli are formed by many protein secretion systems, including the type III secretion system (TTSS). Here, we demonstrate thatErwinia chrysanthemi, which does not carry curli genes, requires the TTSS for pellicle formation. These data support a model where cellulose and generic protein filaments, which consist of either curli or TTSS-secreted proteins, are required for enterobacterial aggregative multicellular behavior. Using this assay, we found thathrpY, which encodes a two-component system response regulator homolog, is required for activity ofhrpS, which encodes a σ54-dependent enhancer-binding protein homolog. In turn,hrpSis required for activity of the sigma factor homologhrpL, which activates genes encoding TTSS structural and secreted proteins. Pellicle formation was temperature dependent and pellicles did not form at 36°C, even though TTSS genes were expressed at this temperature. We found that cellulose is a component of theE. chrysanthemipellicle but that pellicle formation still occurs in a strain with an insertion in a cellulose synthase subunit homolog. Since the TTSS, but not the cellulose synthase subunit, is required forE. chrysanthemipellicle formation, this inexpensive assay can be used as a high throughput screen for TTSS mutants or inhibitors.
The
hrpK
Operon of
Pseudomonas syringae
pv. tomato DC3000 Encodes Two Proteins Secreted by the Type III (Hrp) Protein Secretion System: HopB1 and HrpK, a Putative Type III Translocator
Authors: Petnicki-Ocwieja Tanja , van Dijk Karin , Alfano James R.
Published: January 2005

Pseudomonas syringae
is a gram-negative bacterial plant pathogen that is dependent on a type III protein secretion system (TTSS) and the effector proteins it translocates into plant cells for pathogenicity. The
P. syringae
TTSS is encoded by
hrp-hrc
genes that reside in a central region of a pathogenicity island (Pai). Flanking one side of this Pai is the exchangeable effector locus (EEL). We characterized the transcriptional expression of the open reading frames (ORFs) within the EEL of
P. syringae
pv. tomato DC3000. One of these ORFs, PSPTO1406 (
hopB1
) is expressed in the same transcriptional unit as
hrpK
. Both HopB1 and HrpK were secreted in culture and translocated into plant cells via the TTSS. However, the translocation of HrpK required its C-terminal half. HrpK shares low similarity with a putative translocator, HrpF, from
Xanthomonas campestris
pv. vesicatoria. DC3000 mutants lacking HrpK were significantly reduced in disease symptoms and multiplication in planta, whereas DC3000
hopB1
mutants produced phenotypes similar to the wild type. Additionally,
hrpK
mutants were reduced in their ability to elicit the hypersensitive response (HR), a programmed cell death associated with plant defense. The reduced HR phenotype exhibited by
hrpK
mutants was complemented by
hrpK
expressed in bacteria but not by HrpK transgenically expressed in tobacco, suggesting that HrpK does not function inside plant cells. Further experiments identified a C-terminal transmembrane domain within HrpK that is required for HrpK translocation. Taken together, HopB1 is a type III effector and HrpK plays an important role in the TTSS and is a putative type III translocator.
The
bkdR
Gene of
Streptomyces coelicolor
Is Required for Morphogenesis and Antibiotic Production and Encodes a Transcriptional Regulator of a Branched-Chain Amino Acid Dehydrogenase Complex
Authors: Sprusansky Ondrej , Stirrett Karen , Skinner Deborah , Denoya Claudio , Westpheling Janet
Published: January 2005

Products from the degradation of the branched-chain amino acids valine, leucine, and isoleucine contribute to the production of a number of important cellular metabolites, including branched-chain fatty acids, ATP and other energy production, cell-cell signaling for morphological development, and the synthesis of precursors for polyketide antibiotics. The first nonreversible reactions in the degradation of all three amino acids are catalyzed by the same branched-chain α-keto acid dehydrogenase (BCDH) complex. Actinomycetes are apparently unique among bacteria in that they contain two separate gene clusters, each of which encodes a BCDH enzyme complex. Here, we show that one of these clusters in
Streptomyces coelicolor
is regulated, at least in part, at the level of transcription by the product of the
bkdR
gene. The predicted product of this gene is a protein with similarity to a family of proteins that respond to leucine and serve to activate transcription of amino acid utilization operons. Unlike most other members of this class, however, the
S. coelicolor bkdR
gene product serves to repress transcription, suggesting that the branched-chain amino acids act as inducers rather than coactivators of transcription. BkdR likely responds to the presence of branched-chain amino acids. Its role in transcriptional regulation may be rationalized by the fact that transition from vegetative growth to aerial mycelium production, the first stage of morphological development in these complex bacteria, is coincident with extensive cellular lysis generating abundant amounts of protein that likely serve as the predominant source of carbon and nitrogen for metabolism. We suggest that
bkdR
plays a key role in the ability of
Streptomyces
species to sense nutrient availability and redirect metabolism for the utilization of branched-chain amino acids for energy, carbon, and perhaps even morphogen synthesis. A null mutant of
bkdR
is itself defective in morphogenesis and antibiotic production, suggesting that the role of the
bkdR
gene product may be more global than specific nutrient utilization.
The Cpx Envelope Stress Response Affects Expression of the Type IV Bundle-Forming Pili of EnteropathogenicEscherichiacoli
Authors: Nevesinjac Anna Z. , Raivio Tracy L.
Published: January 2005

The Cpx envelope stress response mediates adaptation to potentially lethal envelope stresses inEscherichiacoli. The two-component regulatory system consisting of the sensor kinase CpxA and the response regulator CpxR senses and mediates adaptation to envelope insults believed to result in protein misfolding in this compartment. Recently, a role was demonstrated for the Cpx response in the biogenesis of P pili, attachment organelles expressed by uropathogenicE. coli. CpxA senses misfolded P pilus assembly intermediates and initiates increased expression of both assembly and regulatory factors required for P pilus elaboration. In this report, we demonstrate that the Cpx response is also involved in the expression of the type IV bundle-forming pili of enteropathogenicE. coli(EPEC). Bundle-forming pili were not elaborated from an exogenous promoter inE. colilaboratory strain MC4100 unless the Cpx pathway was constitutively activated. Further, an EPECcpxRmutant synthesized diminished levels of bundle-forming pili and was significantly affected in adherence to epithelial cells. Since type IV bundle-forming pili are very different from chaperone-usher-type P pili in both form and biogenesis, our results suggest that the Cpx envelope stress response plays a general role in the expression of envelope-localized organelles with diverse structures and assembly pathways.
Biochemical Activities of the
absA
Two-Component System of
Streptomyces coelicolor
Authors: Sheeler Nancy L. , MacMillan Susan V. , Nodwell Justin R.
Published: January 2005

The AbsA1 sensor kinase and its cognate response regulator AbsA2 are important regulators of antibiotic synthesis in
Streptomyces coelicolor
. While certain point mutations in
absA1
reduce or eliminate the synthesis of several antibiotics, null mutations in these genes bring about enhanced antibiotic synthesis. We show here that AbsA1, which is unusual in sequence and structure, is both an AbsA2 kinase and an AbsA2∼P phosphatase. The half-life of AbsA2∼P in solution is 68.6 min, consistent with a role in maintaining a relatively stable state of transcriptional repression or activation. We find that mutations in the
absA
locus that enhance antibiotic synthesis impair AbsA2 kinase activity and that mutations that repress antibiotic synthesis impair AbsA2∼P phosphatase activity. These results support a model in which the phosphorylation state of AbsA2 is determined by the balance of the kinase and phosphatase activities of AbsA1 and where AbsA2∼P represses antibiotic biosynthetic genes either directly or indirectly.
Structural and Genetic Characterization of Enterohemorrhagic
Escherichia coli
O145 O Antigen and Development of an O145 Serogroup-Specific PCR Assay
Authors: Feng Lu , Senchenkova Sof'ya N. , Tao Jiang , Shashkov Alexander S. , Liu Bin , Shevelev Sergei D. , Reeves Peter R. , Xu Jianguo , Knirel Yuriy A. , Wang Lei
Published: January 2005

Enterohemorrhagic
Escherichia coli
O145 strains are emerging as causes of hemorrhagic colitis and hemolytic uremic syndrome. In this study, we present the structure of the
E. coli
O145 O antigen and the sequence of its gene cluster. The O145 antigen has repeat units containing three monosaccharide residues: 2-acetamido-2-deoxy-
d
-glucose (GlcNAc), 2-acetamidoylamino-2,6-dideoxy-
l
-galactose, and
N
-acetylneuraminic acid. It is very closely related to
Salmonella enterica
serovar Touera and
S. enterica
subsp.
arizonae
O21 antigen. The
E. coli
O145 gene cluster is located between the JUMPStart sequence and the
gnd
gene and consists of 15 open reading frames. Putative genes for the synthesis of the O-antigen constituents, for sugar transferase, and for O-antigen processing were annotated based on sequence similarities and the presence of conserved regions. The putative genes located in the
E. coli
O145 O-antigen gene cluster accounted for all functions expected for synthesis of the structure. An
E. coli
O145 serogroup-specific PCR assay based on the genes
wzx
and
wzy
was also developed by screening
E. coli
and
Shigella
isolates of different serotypes.
SigB-Dependent In Vitro Transcription ofprfAand Some Newly Identified Genes ofListeria monocytogenesWhose Expression Is Affected by PrfA In Vivo
Authors: Rauch Marcus , Luo Qin , Müller-Altrock Stefanie , Goebel Werner
Published: January 2005

Recent studies have identified several new genes inListeria monocytogeneswhich are positively or negatively affected by PrfA and grouped into three classes (E. Milohanic et al., Mol. Microbiol.47:1613-1625, 2003). In vitro transcription performed with promoters of some class III genes showed strict SigB-dependent but PrfA-independent transcription initiation. Transcription starting at theprfApromoter PprfA2was also optimal with SigB-loaded RNA polymerase, suggesting a direct link between SigB- and PrfA-dependent gene expression.
The Royal Society of Medicine
Authors: Hunting P
Published: January 2005

With a membership of just over 17 500 the Royal Society of Medicine is the largest medical society in Britain. Its ancestor, the Medical and Chirurgical Society of London, was founded in 1805 when a band of malcontents seceded from the Medical Society of London. By the charter of 1834 the Society became the Royal Medical and Chirurgical Society, which amalgamated with over a dozen other societies to create the Royal Society of Medicine in 1907. It occupies number 1 Wimpole Street, a monumental building that has been redeveloped for the Society’s bicentenary in 2005.
National review of urology outpatient practice in the UK
Authors: Gilmore P E , Shackley D C , Clarke N W , Betts C D , O’Flynn K J
Published: January 2005

Objective

To audit the current UK outpatient workload and compare this to the national standards as set out by the British Association of Urological Surgeons (BAUS) in A Quality Urological Service for Patients in the New Millennium published in October 2000.

Participants

520 UK (NHS) and 21 Republic of Ireland (non-NHS) consultant urologists registered with BAUS in 2000.

Main outcome measures

Extent to which consultant urologists are able to comply with guidelines set out by their specialist association, the BAUS and by the Royal College of Surgeons of England.

Results

The questionnaire return rate was 61% (318/520; regional range 42%–75%). The median “routine” clinics/week was two (1–5) with a mean of 13 (1–40) new and 26 (7–80) follow ups. Fifteen percent (49/318) of consultants worked alone in clinic; of the remainder assistance included specialist registrar 67% (212/318), staff grade/associate specialist 32% (102/318), senior house officer 53% (172/318), and pre-registration house officer 2% (7/318). Only 21% (66/318; regional range 0%–46%) of responding consultants followed the BAUS recommendations for outpatient workload/manpower.

Conclusions

A minority of consultants are able to adhere to the outpatient workload guidelines as set out by BAUS council in 2000. In addition, there appears to be significant variations within and between training regions. Development of this project into a regional audit tool may allow intraregional guideline formation governing hospital outpatient workload.
AN SVM-BASED INCREMENTAL LEARNING ALGORITHM FOR USER ADAPTATION OF SKETCH RECOGNITION
Authors: PENG BINBIN , LIU WENYIN , LIU YIN , HUANG GUANGLIN , SUN ZHENGXING , JIN XIANGYU
Published: January 2005

User adaptation is a critical problem in the design of human-computer interaction systems. Many pattern recognition problems, such as handwriting/sketching recognition and speech recognition, are user dependent, since different users' handwritings, drawing styles, and accents are different. Therefore, the classifiers for these problems should provide the functionality of user adaptation so as to let each particular user experience better recognition accuracy according to his input habit/style. However, the user adaptation functionality requires the classifiers to have the incremental learning ability, by which the classifiers can adapt to the user quickly without too much computation cost. In this paper, an SVM-based incremental learning algorithm is presented to solve this problem for sketch recognition. Our algorithm utilizes only the support vectors instead of all the historical samples, and selects some important samples from all newly added samples as training data. The importance of a sample is measured according to its distance to the hyper-plane of the SVM classifier. Theoretical analysis, experimentation, and evaluation of our algorithm in our online graphics recognition system SmartSketchpad, are presented to show the effectiveness of this algorithm. According to our experiments, this algorithm can reduce both the training time and the required storage space for the training dataset to a large extent with very little loss of precision.
FORWARD AND BACKWARD CHAIN-CODE REPRESENTATION FOR MOTION PLANNING OF CARS
Authors: SHIH FRANK Y. , WU YI-TA , CHEN BRIAN L. C.
Published: January 2005

The path-planning problem is presented to show a car of any desired shape moving from a starting position to a destination in a finite space with arbitrarily shaped obstacles in it. In this paper, a new chain-code representation is developed to record the motion path when forward and backward movements are allowed. By placing the smooth turning-angle constraint, we can obtain more realistic results to the actual motion of cars. Meanwhile, by combining rotational mathematical morphology and distance transformation, we can obtain the shortest collision-free path. As soon as the distance map and the collision-free codes have been established offline, the shortest paths of cars starting from any location toward the destination can be promptly obtained online. Experimental results show that our algorithm works successfully in different conditions. We also extend our algorithm to the automated parallel parking and three-dimensional path planning.
GENETIC OPTIMIZATION OF RADIAL BASIS PROBABILISTIC NEURAL NETWORKS
Authors: ZHAO WEN-BO , HUANG DE-SHUANG , DU JI-YAN , WANG LI-MING
Published: January 2005

This paper discusses using genetic algorithms (GA) to optimize the structure of radial basis probabilistic neural networks (RBPNN), including how to select hidden centers of the first hidden layer and to determine the controlling parameter of Gaussian kernel functions. In the process of constructing the genetic algorithm, a novel encoding method is proposed for optimizing the RBPNN structure. This encoding method can not only make the selected hidden centers sufficiently reflect the key distribution characteristic in the space of training samples set and reduce the hidden centers number as few as possible, but also simultaneously determine the optimum controlling parameters of Gaussian kernel functions matching the selected hidden centers. Additionally, we also constructively propose a new fitness function so as to make the designed RBPNN as simple as possible in the network structure in the case of not losing the network performance. Finally, we take the two benchmark problems of discriminating two-spiral problem and classifying the iris data, for example, to test and evaluate this designed GA. The experimental results illustrate that our designed GA can significantly reduce the required hidden centers number, compared with the recursive orthogonal least square algorithm (ROLSA) and the modified K-means algorithm (MKA). In particular, by means of statistical experiments it was proved that the optimized RBPNN by our designed GA, have still a better generalization performance with respect to the ones by the ROLSA and the MKA, in spite of the network scale having been greatly reduced. Additionally, our experimental results also demonstrate that our designed GA is also suitable for optimizing the radial basis function neural networks (RBFNN).
Contribution of defects in glucose uptake to carbohydrate intolerance in liver cirrhosis: assessment during physiological glucose and insulin concentrations
Authors: Nielsen Michael F. , Caumo Andrea , Aagaard Niels Kristian , Chandramouli Visvanathan , Schumann William C. , Landau Bernard R. , Schmitz Ole , Vilstrup Hendrik
Published: January 2005

It is well established that subjects with liver cirrhosis are insulin resistant, but the contribution of defects in insulin secretion and/or action to glucose intolerance remains unresolved. Healthy individuals and subjects with liver cirrhosis were studied on two occasions: 1) an oral glucose tolerance test was performed, and 2) insulin secretion was inhibited and glucose was infused in a pattern and amount mimicking the systemic delivery rate of glucose after a carbohydrate meal. Insulin was concurrently infused to mimic a healthy postprandial insulin profile. Postabsorptive glucose concentrations were equal (5.36 ± 0.12 vs. 5.40 ± 0.25 mmol/l, P = 0.89), despite higher insulin ( P < 0.01), C-peptide ( P < 0.01), and free fatty acid ( P = 0.05) concentrations in cirrhotic than in control subjects. Endogenous glucose release (EGR; 11.50 ± 0.50 vs. 11.73 ± 1.00 μmol·kg−1·min−1, P = 0.84) and the contribution of gluconeogenesis to EGR (6.60 ± 0.47 vs. 6.28 ± 0.64 μmol·kg−1·min−1, P = 0.70) were unaltered by cirrhosis. A minimal model recently developed for the oral glucose tolerance test demonstrated an impaired insulin sensitivity index ( P < 0.05), whereas the β-cell response to glucose was unaltered ( P = 0.72). During prandial glucose and insulin infusions, the integrated glycemic response was greater in cirrhotic than in control subjects ( P < 0.05). EGR decreased promptly and comparably in both groups, but glucose disappearance was insufficient at the prevailing glucose concentration ( P < 0.05). Moreover, identical rates of [3-3H]glucose infusion produced higher tracer concentrations in cirrhotic than in control subjects ( P < 0.05), implying a defect in glucose uptake. In conclusion, carbohydrate intolerance in liver cirrhosis is determined by insulin resistance and the ability of glucose to stimulate insulin secretion. During prandial glucose and insulin concentrations, EGR suppression was unaltered, but glucose uptake was impaired, which demonstrates that intolerance can be ascribed to a defect in glucose uptake, rather than abnormalities in glucose production or β-cell function. Although insulin secretion ameliorates glucose intolerance, impaired glucose uptake during physiological glucose and insulin concentrations produces marked and sustained hyperglycemia, despite concurrent abnormalities in glucose production or insulin secretion.
Effect of repeated cycles of acute esophagitis and healing on esophageal peristalsis, tone, and length
Authors: Zhang X. , Geboes K. , Depoortere I. , Tack J. , Janssens J. , Sifrim D.
Published: January 2005

Severe esophagitis is associated with motor abnormalities in the esophageal body and lower esophageal sphincter. Reflux disease involves repeated episodes of mucosal inflammation and spontaneous or treatment-induced healing. The aims of this study were 1) to further assess changes induced by acute esophagitis on esophageal peristalsis, tone, and shortening and 2) to assess the effect of repeated sequences of acute esophagitis-healing on these motor parameters. Experiments were performed on adult cats. Esophageal manometry and barostat were performed before, 24 h after, and every 7 days after intraesophageal acid perfusion (0.1 N HCl, 80 min). Esophageal length was measured during manometry, and compliance of the esophageal body was assessed with barostat. The identical protocol was performed 8 and 16 wk after the first acid perfusion. The degree of esophageal mucosal damage was evaluated by endoscopy, histopathology, and myeloperoxidase activity. Acid perfusion induced severe esophagitis. At 24 h, distal peristaltic contractions disappeared, lower esophageal sphincter pressure was reduced by 60%, the esophagus length was 1–2 cm shorter, and esophageal compliance was reduced by 30%. Most parameters recovered in 4 wk. Subsequent repeated acute injuries induced similar endoscopic esophagitis but a different pattern of inflammatory infiltration and fibrosis in the mucosa and muscle layers, resulting in milder motor disturbances. Acute experimental esophagitis provokes severe but reversible hypomotility. Spaced repeated acute injuries provoke milder motor effects, suggesting an adaptive response.
Effects of extreme hemodilution with hemoglobin-based O2 carriers on microvascular pressure
Authors: Cabrales Pedro , Tsai Amy G. , Winslow Robert M. , Intaglietta Marcos
Published: January 2005

A surface-modified polyethylene glycol-conjugated human hemoglobin (MP4) and αα-cross-linked human hemoglobin (ααHb) were used to restore oxygen carrying capacity in conditions of extreme hemodilution (hematocrit 11%) in the hamster window model preparation. Changes in microvascular function were analyzed in terms of effects on capillary pressure and functional capillary density (FCD). MP4, at 1.0 ± 0.2 g/dl blood concentration, significantly lowered mean arterial pressure (MAP) below baseline (99.6 ± 7.6 mmHg) to 82.4 ± 6.9 mmHg ( P < 0.05) and decreased of FCD to 70 ± 9%. ααHb caused a greater recovery in MAP to 94.4 ± 6.2 mmHg and lowered FCD to 62 ± 8%. However, differences between ααHb and MP4 in FCD were not statistically significant. Capillary pressures were in the ranges of 17–21 mmHg for MP4 and 15–19 mmHg for ααHb, with both significantly lower than baseline ( P < 0.05). Pressure in 80-μm-diameter arterioles was significantly increased with ααHb relative to MP4 ( P < 0.05). These results were compared with previous findings on the relation between capillary pressure and FCD; they supported the concept of a relationship between FCD and capillary pressure. Measurement of changes in arteriolar diameter, microvascular blood flow, and FCD show that there was no statistical difference between using ααHb and MP4 in extreme hemodilution. Microvascular resistance in arterioles with a diameter range of 70–80 μm showed an increase relative to control with ααHb, whereas MP4 caused a decrease.
Direct action of T3on phosphorylation potential in the sheep heart in vivo
Authors: Portman Michael A. , Qian Kun , Krueger Julia , Ning Xue-Han
Published: January 2005

Thyroid acting through ligand binding to nuclear receptors modifies myocardial respiratory kinetics and oxidative phosphorylation in the heart. Direct nongenomic action of thyroid hormone on high-energy phosphate concentrations and respiratory kinetics has never been proven in vivo but might be responsible for observed changes in oxygen utilization efficiency immediately after triiodothyronine (T3) administration. We tested the hypothesis that T3directly and rapidly modifies myocardial high-energy phosphate concentrations and phosphorylation potential in vivo. Anesthetized sheep (age 28–40 days) thyroidectomized shortly after birth (Thy) and euthyroid age-matched controls (Con) underwent median sternotomy and received T3infusion (0.8 μg/kg), followed by epinephrine infusion to increase myocardial oxygen consumption (MV̇o2).31P magnetic resonance spectra were monitored via a surface coil over the left ventricle. T3increased phosphocreatine (PCr)/ATP and decreased ADP in Thy animals without causing a change in MV̇o2. T3produced no changes in high-energy phosphates in Con animals. T3did not modify the PCr/ATP or ADP response to epinephrine and elevation in MV̇o2in either group. Cardiac mitochondria isolated from Thy and Con animals showed no change in respiratory rate or ADP/ATP exchange efficiency after T3incubation. T3infusion in a hypothyroid state decreases ADP concentration, thereby altering the equilibrium between phosphorylation potential and myocardial respiratory rate. These T3-induced effects are not due to changes in ADP/ATP exchange efficiency through action at the adenine nucleotide translocator but may be due to T3mediation of substrate utilization, confirmed in other models.
Role of WNK kinases in regulating tubular salt and potassium transport and in the development of hypertension
Authors: Gamba Gerardo
Published: January 2005

A recently discovered family of protein kinases is responsible for an autosomal-dominant disease known as Gordon's syndrome or pseudohypoaldosteronism type II (PHA-II) that features hyperkalemia and hyperchloremic metabolic acidosis, accompanied by hypertension and hypercalciuria. Four genes have been described in this kinase family, which has been named WNK, due to the absence of a key lysine in kinase subdomain II (with no K kinases). Two of these genes, WNK1 and WNK4 located in human chromosomes 12 and 17, respectively, are responsible for PHA-II. Immunohystochemical analysis revealed that WNK1 and WNK4 are predominantly expressed in the distal convoluted tubule and collecting duct. The physiological studies have shown that WNK4 downregulates the activity of ion transport pathways expressed in these nephron segments, such as the apical thiazide-sensitive Na+-Cl−cotransporter and apical secretory K+channel ROMK, as well as upregulates paracellular chloride transport and phosphorylation of tight junction proteins such as claudins. In addition, WNK4 downregulates other Cl−influx pathways such as the basolateral Na+-K+-2Cl−cotransporter and Cl−/HCO3−exchanger. WNK4 mutations behave as a loss of function for the Na+-Cl−cotransporter and a gain of function when it comes to ROMK and claudins. These dual effects of WNK4 mutations fit with proposed mechanisms for developing electrolyte abnormalities and hypertension in PHA-II and point to WNK4 as a multifunctional regulator of diverse ion transporters.
Towards optimization of selective photothermolysis: prothrombotic pharmaceutical agents as potential adjuvants in laser treatment of port wine stains
Authors: Beek Johan F. , Moldovan Nicanor I. , van der Horst Chantal M. A. M. , van Gemert Martin J. C. , Heger Michal
Published: January 2005

For the past two decades much research on selective photothermolysis of port wine stain vasculature has been devoted to optimizing laser parameters. Unfortunately, 60% of patients still respond suboptimally to laser therapy, despite significant innovations in treatment strategies and laser technology. Here we present a novel treatment approach based on combining selective photothermolysis with the administration of prothrombotic and/or anti-fibrinolytic pharmaceutical agents, with the aim of enhancing vaso-occlusion and post-treatment remodelling in difficult-to-target vessels. A hypercoagulable state of blood will instill laser-induced occlusive thrombosis in a wider array of vessel diameters at greater dermal depths, whereby larger vascular segments will ultimately undergo the chronic inflammatory processes that result in blood volume reduction, and thus lesional blanching. With thrombosis as a primary trigger for these inflammatory processes, we have extrapolated the thresh-old damage profile that is required for clinically relevant thrombus formation. Consequently, a recently proposed model of thrombus organization, in which recanalization is associated with endothelial progenitor cell-mediated neovasculogenesis, is elaborated in the framework of lesional blanching and juxtaposed to angiogenic reconstruction of affected dermal vasculature. Since neovasculogenesis and angiogenesis are regulated by the degree of vaso-occlusion and corollary drop in local oxygen tension, both can be manipulated by the administration of procoagulant pharmaceuticals. Lastly, in an effort to optimally balance selective photothermolysis with pharmacokinetics and clinical safety, the use of a gold nanoshell drug delivery system, in which the procoagulant drugs are encapsulated by a wavelength-modulated, gold-coated polymer matrix, is proposed. We have termed this modality site-specific pharmaco-laser therapy.
The spacer domain of ADAMTS13 contains a major binding site for antibodies in patients with thrombotic thrombocytopenic purpura
Authors: Luken Brenda M. , Turenhout Ellen A.M. , Hulstein Janine J.J. , Van Mourik Jan A. , Fijnheer Rob , Voorberg Jan
Published: January 2005

Thrombotic thrombocytopenic purpura (TTP) is a microangiopathy often associated with a severely decreased activity of ADAMTS13. In plasma of the majority of patients withTTP, antibodies are present that inhibit the vonWillebrand factor (VWF) processing activity of ADAMTS13.We describe a sensitive assay that monitors binding of recombinant ADAMTS13 to immobilized IgG derived from patient plasma. Analysis of fifteen patients with TTP and severely reduced ADAMTS13 activity revealed that in all patients antibodies directed toADAMTS13 were present. Levels of anti-ADAMTS13 antibodies varied considerably among patients, specific antibody levels in plasma range from less than 100 ng/ml to over 1 μg/ml. Longitudinal analysis in three patients revealed that anti-ADAMTS13 antibody levels declined with different kinetics. For further characterization of anti- ADAMTS13 antibodies, we prepared a series of recombinan fragments corresponding to the various ADAMTS13 domains. All seven TTP plasma samples tested, showed reactivity of antibodies towards a fragment consisting of the disintegrin/ TSR1/cysteine-rich/spacer domains. In one patient, we also observed reactivity towards the TSR2–8 repeats. No binding of antibodies to propeptide, metalloprotease and CUB domains was detected. To further delineate the binding site in the disintegrin/ TSR1/cysteine-rich/spacer region, we prepared additional ADAMTS13 fragments. Antibodies directed towards the cysteine- rich/spacer fragment were found in all plasma samples analyzed. No antibodies reacting with the disintegrin/TSR1 domains were detected. A recombinant fragment comprising the spacer domain was recognized by all patients samples analyzed, suggesting that the 130-amino-acid spacer domain harbors a major binding site for anti-ADAMTS-13 antibodies.
Identification and characterization of the plasma kallikrein-kinin system inhibitor, haemaphysalin, from hard tick, Haemaphysalis longicornis
Authors: Kato Noriko , Okayama Takahide , Isawa Haruhiko , Yuda Masao , Chinzei Yasuo , Iwanaga Shiroh
Published: January 2005

The plasma kallikrein-kinin system inhibitor, haemaphysalin, from the hard tick, Haemaphysalis longicornis, was identified. It was found that haemaphysalin inhibited activation of the plasma kallikrein-kinin system by interfering with reciprocal activation between factor XII and prekallikrein. It did not, however, inhibit amidolytic activities of factor XIIa and kallikrein. Direct binding assay indicated that factor XII/XIIa and high molecular weight kininogen (HK) are the target molecules of haemaphysalin, and that Zn2+ ions are involved in the interactions of haemaphysalin with these target molecules. This suggests that haemaphysalin interacts with target molecules by recognizing their conformational changes induced by Zn2+ ions. Furthermore, haemaphysalin interacted with the fibronectin type II domain and domain D5, the cell binding domains of factor XII and HK, respectively. This finding suggests that haemaphysalin interferes with the association of factor XII and the prekallikrein-HK complex with a biologic activating surface by binding to these cell-binding domains, leading to inhibition of the reciprocal activation between factor XII and prekallikrein.
Ultrasound elastography for the age determination of venous thrombi
Authors: Barbera Letterio , Muth-Werthmann Dajana , Siebers Stefan , Ermert Helmut , Philippou Stathis , Mumme Achim , Geier Bruno
Published: January 2005

The exact age determination of venous thrombi is important if thrombolytic therapy or surgical thrombectomy is considered. Clinical symptoms as well as duplex-ultrasound and phlebography are unreliable in this respect and do not allow an exact age estimation. Ultrasound elastography can provide information about the elastic properties of thrombi. Since thrombus elasticity decreases with age due to the organisation process, it should be possible to use elastography to stage the degree of organisation and thereby determine the age of venous thrombi. Experimental venous thrombi aging 1, 3, 6, 9, 12 and 15 days were created in a porcine model by laparoscopic ligation of the infrarenal Vena cava in combination with transfemoral infusion of thrombin. The thrombosed iliac veins were explanted and embedded in gelatine, after that they underwent examination by ultrasound elastography. In addition, histological evaluation of the thrombi was performed. Elastography demonstrated a decline in thrombus elasticity between days 6 and 12 with the 12-day-old thrombi being about 3 times harder then the 6-dayold thrombi. This correlated with the histological findings, which demonstrated a marked increase in fibroblast and collagen production in the clots during this time, with the 12– and 15-day thrombi showing signs of advanced organisation. In conclusion, in an experimental setting, ultrasound elastography was helpful in determining the exact age of venous thrombi. The differences in elasticity were most pronounced between days 6 and 12, which is also the most relevant time frame when considering invasive therapies in human venous thrombosis.
Roles of the multifunctional glycoprotein, emmprin (basigin; CD147), in tumour progression
Authors: Yan Li , Zucker Stanley , Toole Bryan P.
Published: January 2005

Emmprin (basigin;CD147) is a widely distributed cell surface glycoprotein that belongs to the Ig superfamily and is highly enriched on the surface of malignant tumour cells. Emmprin is involved in numerous physiological and pathological systems and exhibits several molecular and cellular characteristics, but a major function of emmprin is stimulation of synthesis of several matrix metalloproteinases. In tumours, emmprin most likely stimulates matrix metalloproteinase production in stromal fibroblasts and endothelial cells as well as in tumour cells themselves by a mechanism involving homophilic interactions between emmprin molecules on apposing cells or on neighbouring cells after membrane vesicle shedding. Membrane-associated cofactors, including caveolin-1 and annexin II, regulate emmprin activity. Emmprin induces angiogenesis via stimulation of VEGF production, invasiveness via stimulation of matrix metalloproteinase production and multidrug resistance via hyaluronan-mediated up-regulation of ErbB2 signaling and cell survival pathway activities. Although the detailed mechanisms whereby it regulates these numerous phenomena are not yet known, it is clear that emmprin is a major mediator of malignant cell behavior.
The urokinase plasminogen activator and its receptor
Authors: Alfano Daniela , Franco Paola , Vocca Immacolata , Gambi Nadia , Pisa Viviana , Mancini Alessandro , Caputi Mario , Carriero Maria Vincenza , Iaccarino Ingram , Stoppelli Maria Patrizia
Published: January 2005

The urinary-type plasminogen activator, or uPA, controls matrix degradation through the conversion of plasminogen into plasmin and is regarded as the critical trigger for plasmin generation during cell migration and invasion, under physiological and pathological conditions (such as cancer metastasis).The proteolytic activity of uPA is responsible for the activation or release of several growth factors and modulates the cell survival/apoptosis ratio through the dynamic control of cell-matrix contacts. The urokinase receptor (uPAR), binding to the EGF-like domain of uPA, directs membrane-associated extracellular proteolysis and signals through transmembrane proteins, thus regulating cell migration, adhesion and cytoskeletal status. However, recent evidence highlights an intricate relationship linking the uPA/uPAR system to cell growth and apoptosis.
Poly(ADP-ribosyl)ation, a molecular switch of transcription, shows an attractive relationship with urokinase expression
Authors: Caldini Riccardo , Rosso Mario Del , Chevanne Marta
Published: January 2005

Poly(ADP-ribosyl)ation is a posttranslational modification of proteins that consists in the transfer of ADP-ribose units from NAD+ onto protein acceptors to form long and branched polymers. PARP activity is stimulated either by genotoxic stimuli or by environmental factors. The negative charged polymers alter functional activity of several proteins involved in genome stability, gene expression, cell proliferation and differentiation. Increasing evidence supports the view that PARP, for its crucial position in DNA repair and DNA transcription, influences cell survival not only during tissue injure, but also in environmental homeostasis modification. Therefore, it may be considered a molecular switch in the control of transcription, eventually leading to the choice of cell for life and death. This review summarizes the recent findings on PARP activity and special emphasis is given to its role in urokinase-type plasminogen activator upregulation.
Gallic Acid Antagonizes P-Selectin–Mediated Platelet–Leukocyte Interactions
Authors: Appeldoorn Chantal C.M. , Bonnefoy Arnaud , Lutters Bianca C.H. , Daenens Kim , van Berkel Theo J.C. , Hoylaerts Marc F. , Biessen Erik A.L.
Published: January 2005

Background—

Current paradigm attributes the low incidence of cardiovascular disorders in Mediterranean countries despite a high saturated fat intake, the “French paradox,” to the antioxidant capacity of red wine polyphenols. Conceivably, other antiinflammatory pathways may contribute to at least a similar extent to the atheroprotective activity of these polyphenols. We have investigated whether gallic acid (GA), an abundant red wine polyphenol, modulates the activity of P-selectin, an adhesion molecule that is critically involved in the recruitment of inflammatory cells to the vessel wall and thus in atherosclerosis.

Methods and Results—

GA potently inhibited the binding of a peptide antagonist (IC
50
, 7.2 μmol/L) and biotin-PAA-Le
a
-SO
3
H, an established high-affinity ligand, to P-selectin (IC
50
, 85 μmol/L). Under dynamic flow conditions, GA markedly and dose dependently attenuated the rolling of monocytic HL60 cells over P-selectin-transfected Chinese hamster ovary cells (EC
50
, 14.5 μmol/L) while increasing the velocity of P-selectin-dependent rolling of human blood leukocytes over a platelet monolayer. In vivo tests established that GA administration to normolipidemic C57/Bl6 and aged atherosclerotic apolipoprotein E–deficient mice impaired the baseline rolling of conjugates between activated platelets and circulating monocytes over femoral vein endothelium, as judged by online video microscopy (ED
50
, 1.7±0.3 and 1.5±0.4 mg · kg
−1
· h
−1
, respectively).

Conclusions—

Our findings provide a solid mechanistic foundation through which GA intervenes in major inflammatory pathobiologies by binding and antagonizing P-selectin.
Impaired Flow-Mediated Dilation and Risk of Restenosis in Patients Undergoing Coronary Stent Implantation
Authors: Patti Giuseppe , Pasceri Vincenzo , Melfi Rosetta , Goffredo Costanza , Chello Massimo , D’Ambrosio Andrea , Montesanti Rosamaria , Di Sciascio Germano
Published: January 2005

Background—

Impaired endothelial function is a key event in the atherosclerosis process and predicts future cardiovascular events in subjects with and without coronary artery disease (CAD). We performed the first prospective study evaluating whether early measurement of brachial artery endothelium-dependent dilation (flow-mediated dilation [FMD]) after coronary stenting could predict occurrence of in-stent-restenosis.

Methods and Results—

The study population included 136 patients with single-vessel CAD undergoing percutaneous coronary intervention (PCI) with stenting and at least 6 months of follow-up. All patients underwent ultrasound detection of brachial artery reactivity 30 days after PCI; FMD was investigated before and after 5 minutes of occlusion of the brachial artery, and nitroglycerin-mediated dilation was investigated before and after administration of sublingual nitrates. Clinical in-stent restenosis was demonstrated in 20 patients (15%), whereas 116 patients (85%) remained free of signs or symptoms of recurrent ischemia. FMD was significantly impaired in patients with restenosis versus those without restenosis (percent diameter variation 4.6±5.8% versus 9.5±6.6%,
P
=0.002); moreover, 4% of patients with FMD ≥7% (median value) developed in-stent restenosis versus 28% of those with FMD <7% (
P
=0.0001). On multivariate analysis, FMD was the strongest predictor of restenosis (OR 4.5, 95% CI 2.4 to 12.0); conversely, nitroglycerin-mediated dilation did not independently predict the risk of restenosis (OR 2.4, 95% CI 0.8 to 6.3).

Conclusions—

This is the first prospective study indicating that impaired FMD independently predicts occurrence of in-stent restenosis in patients undergoing PCI. Early evaluation of endothelial function after stenting may represent a useful screening tool to stratify patients according to future risk of restenosis.
CD4+CD25+ regulatory T cells control induction of autoimmune hemolytic anemia
Authors: Mqadmi Amina , Zheng Xiaoying , Yazdanbakhsh Karina
Published: January 2005

Autoimmune hemolytic anemia (AIHA) is the result of increased destruction of red blood cells (RBCs) due to the production of autoantibodies, and it can be life-threatening. To study the mechanisms that trigger AIHA, we used the Marshall-Clarke and Playfair model of murine AIHA, in which mice repeatedly immunized with rat RBCs develop erythrocyte autoantibodies as well as rat-specific alloantibodies. We analyzed the role of CD25+ T-regulatory subsets in controlling AIHA in C57/Bl6 mice using antibody depletion studies. Treatment with anti-CD25 antibody but not isotype control prior to immunization with rat RBCs increased the incidence of AIHA from 30% to 90%. Adoptive transfer of purified splenic population of CD4+CD25+ but not CD4+CD25- cells from immunized mice into naive recipients prevented the induction of autoantibody production. Altogether, our data establish a critical role for CD4+CD25+ cells for control of AIHA, which may help to establish therapeutic strategies for treatment of AIHA.
Changes in extracranial arteries in obstructive sleep apnoea
Authors: Schulz R. , Seeger W. , Fegbeutel C. , Hüsken H. , Bödeker R. H. , Tillmanns H. , Grebe M.
Published: January 2005

Obstructive sleep apnoea (OSA) is linked with increased cardiovascular morbidity and mortality, possibly through an enhancement of atherosclerotic vascular changes. Up to now, however, only a few studies have tried to evaluate the occurrence of atherosclerosis in patients with OSA.

In the present study, ultrasonography of the large extracranial vessels was performed in a group of consecutively admitted OSA patients (n = 35) and a control group of non-OSA patients (n = 35). Common carotid artery-intima media thickness (CCA-IMT) was measured at the far wall of both proximal carotid arteries. Furthermore, the presence of plaques and stenoses of the extracranial vessels was determined. All measurements were carried out blinded to the status of the patients.

In the OSA group, CCA-IMT was significantly increased when compared with the non-OSA patients and was related to the degree of nocturnal hypoxia. Additionally, the formation of plaques was more pronounced and extracranial vessel stenosis was more common in the OSA patients.

In conclusion, these findings are in favour of an independent influence of obstructive sleep apnoea on atherosclerotic changes of the arterial wall, and represent further strong arguments for obstructive sleep apnoea being a risk factor on its own for the emergence of cardiovascular disease.