bluuebunny/crossref_metadata_2025 huggingface dataset:
Conjugal TOL Transfer from
Pseudomonas putida
to
Pseudomonas aeruginosa
: Effects of Restriction Proficiency, Toxicant Exposure, Cell Density Ratios, and Conjugation Detection Method on Observed Transfer Efficiencies
Authors: Arango Pinedo Catalina , Smets Barth F.
Published: January 2005

The effects of restriction proficiency and premating exposure to toxicants on conjugal transfer of the TOL plasmid between
Pseudomonas
spp. was investigated by examinations of filter matings. A
Pseudomonas putida
KT2442-derived strain carrying a
gfp
-tagged variant of the TOL plasmid was used as a donor, and both restriction-deficient (PAO1162N) and -proficient (PAO2002N)
Pseudomonas aeruginosa
strains were used as recipients. The in situ enumeration of conjugation events allowed us to obtain frequency estimates that were unbiased by transconjugant growth or plasmid retransfer. We observed a strong dependence of the plasmid transfer frequency on the initial donor-to-recipient ratio of surface matings, which invalidated the use of mass action-based plasmid transfer kinetic estimators. Careful control of the initial parental cell densities permitted evaluations of the true effects of restriction proficiency and toxicant exposure on TOL transfer. At standard donor-to-recipient ratios (10
−3
for PAO1162N and 2 × 10
1
for PAO2002N) and total cell densities (10
5
cells/mm
2
for PAO1162N and 10
6
cells/mm
2
for PAO2002N), plasmid transfer frequencies without toxicant exposure were approximately 10
−7
(events/mm
2
)
−1
for PAO1162N and 10
−11
(events/mm
2
)
−1
for PAO2002N based on in situ observations of conjugation events. The enumeration of transconjugants via selective plating yielded transfer frequencies that were up to 1 order of magnitude lower. Premating exposure to sodium dodecyl sulfate (1 to 10 mM) significantly increased the transfer frequency for the restriction-proficient strain PAO2002N (
P
< 0.05) but not for the restriction-deficient strain PAO1162N. On the other hand, premating exposure to ethanol, toluene, or phenol had no positive effect on the plasmid transfer frequency. Clearly, restriction proficiency provides a strong barrier to interspecific transfer of the TOL plasmid, and this barrier was only marginally attenuated by recipient exposure to toxicants within the ranges examined.
First Record of the Rare SpeciesAeromonas culicicolafrom a Drinking Water Supply
Authors: Figueras M. J. , Suarez-Franquet A. , Chacón M. R. , Soler L. , Navarro M. , Alejandre C. , Grasa B. , Martínez-Murcia A. J. , Guarro J.
Published: January 2005

We describe the recovery of the rare speciesAeromonas culicicola, so far known only in mosquitoes in India, from a drinking water supply in Spain. Typing, using enterobacterial repetitive intergenic consensus-PCR, revealed that the 27 new isolates belonged to 3 very closely related strains. These strains were genetically identified by 16S rRNA gene sequencing. Spanish strains differed from the mosquito strains in three nucleotide positions. The AHCYTOEN gene was present in these water strains, which may have a public health significance.
Byssochlamys nivea
as a Source of Mycophenolic Acid
Authors: Puel Olivier , Tadrist Souria , Galtier Pierre , Oswald Isabelle P. , Delaforge Marcel
Published: January 2005

Byssochlamys
species are responsible for spoilage and degradation of fruits and silages and can also produce the mycotoxin patulin. We analyzed secondary metabolite production by
Byssochlamys nivea
. Mycophenolic acid and its precursors, 5-methylorsellinic acid and 5,7-dihydroxy-4-methylphthalide, were identified in all of the
B. nivea
strains that we examined.
Purification and Characterization of a Novel Class IIa Bacteriocin, Piscicocin CS526, from Surimi-Associated
Carnobacterium piscicola
CS526
Authors: Yamazaki Koji , Suzuki Minako , Kawai Yuji , Inoue Norio , Montville Thomas J.
Published: January 2005

The bacteriocin piscicocin CS526 was inactivated by proteolytic enzymes, was stable at 100°C for 30 min, had a pH range of 2 to 8, and was active against
Enterococcus
,
Listeria
,
Pediococcus
, and
Leuconostoc
. The N-terminal sequence was YGNG

L

, not the YGNG

V

consensus motif common in class IIa bacteriocins (alternate residues underlined). The molecular mass of piscicocin CS526, which had a bactericidal mode of action, was ∼4,430 Da.
Effect of Culture Conditions on Microorganism Identification by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry
Authors: Valentine Nancy , Wunschel Sharon , Wunschel David , Petersen Catherine , Wahl Karen
Published: January 2005

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has been used to identify bacteria based upon protein signatures. This research shows that while some different proteins are produced by vegetative bacteria when they are cultured in different growth media, positive identification with MALDI-TOF MS is still possible with the protocol established at the Pacific Northwest National Laboratory (K. H. Jarman, S. T. Cebula, A. J. Saenz, C. E. Petersen, N. B. Valentine, M. T. Kingsley, and K. L. Wahl, Anal. Chem.
72:
1217-1223, 2000). A core set of small proteins remain constant under at least four different culture media conditions and blood agar plates, including minimal medium M9, rich media, tryptic soy broth (TSB) or Luria-Bertani (LB) broth, and blood agar plates, such that analysis of the intact cells by matrix-assisted laser desorption/ionization mass spectrometry allows for consistent identification.
Novel Keratinase from
Bacillus subtilis
S14 Exhibiting Remarkable Dehairing Capabilities
Authors: Macedo Alexandre J. , da Silva Walter O. Beys , Gava Renata , Driemeier David , Henriques João Antonio Pêgas , Termignoni Carlos
Published: January 2005

We report the isolation of a keratinolytic-producing
Bacillus subtilis
strain and the characterization of the exceptional dehairing properties of its subtilisin-like keratinase. This enzyme can be an alternative to sodium sulfide, the major pollutant from tanneries, and may completely replace it. Its unique nonactivity upon collagen enhances its industrial potential.
Use of Artificial Neural Networks To Accurately Identify
Cryptosporidium
Oocyst and
Giardia
Cyst Images
Authors: Widmer Kenneth W. , Srikumar Deepak , Pillai Suresh D.
Published: January 2005

Cryptosporidium parvum
and
Giardia lamblia
are protozoa capable of causing gastrointestinal diseases. Currently, these organisms are identified using immunofluorescent antibody (IFA)-based microscopy, and identification requires trained individuals for final confirmation. Since artificial neural networks (ANN) can provide an automated means of identification, thereby reducing human errors related to misidentification, ANN were developed to identify
Cryptosporidium
oocyst and
Giardia
cyst images. Digitized images of
C. parvum
oocysts and
G. lamblia
cysts stained with various commercial IFA reagents were used as positive controls. The images were captured using a color digital camera at 400× (total magnification), processed, and converted into a binary numerical array. A variety of “negative” images were also captured and processed. The ANN were developed using these images and a rigorous training and testing protocol. The
Cryptosporidium
oocyst ANN were trained with 1,586 images, while
Giardia
cyst ANN were trained with 2,431 images. After training, the best-performing ANN were selected based on an initial testing performance against 100 images (50 positive and 50 negative images). The networks were validated against previously “unseen” images of 500
Cryptosporidium
oocysts (250 positive, 250 negative) and 282
Giardia
cysts (232 positive, 50 negative). The selected ANNs correctly identified 91.8 and 99.6% of the
Cryptosporidium
oocyst and
Giardia
cyst images, respectively. These results indicate that ANN technology can be an alternate to having trained personnel for detecting these pathogens and can be a boon to underdeveloped regions of the world where there is a chronic shortage of adequately skilled individuals to detect these pathogens.
Rectal Carriage of Enterohemorrhagic
Escherichia coli
O157 in Slaughtered Cattle
Authors: Low J. Christopher , McKendrick Iain J. , McKechnie Caroline , Fenlon David , Naylor Stuart W. , Currie Carol , Smith David G. E. , Allison Lesley , Gally David L.
Published: January 2005

Escherichia coli
O157:H7 is an important cause of diarrhea, hemorrhagic colitis, and potentially fatal human illness. Cattle are considered a primary reservoir of infection, and recent experimental evidence has indicated that the terminal rectum is the principal site of bacterial carriage. To test this finding in naturally colonized animals, intact rectum samples from 267 cattle in 24 separate lots were obtained immediately after slaughter, and fecal material and mucosal surfaces were cultured for
E. coli
O157 by direct and enrichment methods. Two locations, 1 and 15 cm proximal to the recto-anal junction, were tested. In total, 35 animals were positive for
E. coli
O157 at at least one of the sites and 232 animals were negative as determined by all tests. The frequency of isolation and the numbers of
E. coli
O157 cells were higher at the site closer to the recto-anal junction, confirming our previous experimental findings. We defined low- and high-level carriers as animals with
E. coli
O157 levels of <1 × 10
3
CFU g
−1
or <1 × 10
3
CFU ml
−1
and animals with
E. coli
O157 levels of ≥1 × 10
3
CFU g
−1
or ≥1 × 10
3
CFU ml
−1
in feces or tissues, respectively. High-level carriage was detected in 3.7% of the animals (95% confidence interval, 1.8 to 6.8%), and carriage on the mucosal surface of the terminal rectum was associated with high-level fecal excretion. In summary, our results support previous work demonstrating that the mucosal epithelium in the bovine terminal rectum is an important site for
E. coli
O157 carriage in cattle. The data also support the hypothesis that high-level fecal shedding (≥1 × 10
3
CFU g of feces
−1
) of enterohemorrhagic
E. coli
O157 results from colonization of this site.
An uncommon cause of back pain in pregnancy
Authors: Vadivelu R , Green T P , Bhatt R
Published: January 2005

Back pain is a major cause of disability worldwide. A case of a 29 year old Caucasian woman with low back pain secondary to an uncommon benign condition called osteitis condensans ilii is presented and the literature is reviewed. Limited information in the literature combined with poor awareness across the specialty and among the primary care physicians leads to an extensive investigation and misdiagnosis of this benign condition. This report emphasis the salient features and the differential diagnosis in diagnosing and managing this rare condition.
Treatment of scabies: newer perspectives
Authors: Karthikeyan K
Published: January 2005

Scabies is a common dermatological problem managed by general physicians, family physicians, and dermatologists. Scabies manifests itself in various forms in different individuals. The treatment of scabies has undergone developments with the evolution of new treatment strategies and antiectoparasitic drugs. The advent of drugs such as permethrin and ivermectin has revolutionised treatment. Management of scabies not only includes prescribing the drugs but also educating patients properly about the method of application of the drug. Treatment failures may result for various reasons and must be managed appropriately.
Active inflammatory bowel disease and coronary artery dissection
Authors: Srinivas M , Basumani P , Muthusamy R , Wheeldon N
Published: January 2005

The case of a young woman with flare-up of Crohn’s disease, who had an acute myocardial infarction due to the spontaneous dissection of the left anterior descending coronary artery, is reported. The literature on this rare condition is reviewed and a mechanism postulated for spontaneous coronary artery dissection in inflammatory bowel disease.
Irreversible sensorineural hearing loss due to clarithromycin
Authors: Coulston J , Balaratnam N
Published: January 2005

Clarithromycin is a commonly used advanced generation macrolide. This case study reviews a case of an 81 year old woman who developed sensorineural deafness in the right ear after the start of low dose oral clarithromycin for an infective exacerbation of chronic obstructive pulmonary disease. Despite cessation of this drug after only three days, the sensorineural deafness was found to be irreversible.

Reversible sensorineural deafness secondary to macrolides has previously been described and evidence in the literature shows that a dose related phenomenon occurs. Research has indicated that transient dysfunction of the outer hair cells could be the possible mechanism.

In this case, however, the patient experienced an irreversible sensorineural deafness associated with the start of low dose oral clarithromycin. This is a side effect profile that has not previously been reported.
Neurological channelopathies
Authors: Graves T D , Hanna M G
Published: January 2005

Ion channels are membrane-bound proteins that perform key functions in virtually all human cells. Such channels are critically important for the normal function of the excitable tissues of the nervous system, such as muscle and brain. Until relatively recently it was considered that dysfunction of ion channels in the nervous system would be incompatible with life. However, an increasing number of human diseases associated with dysfunctional ion channels are now recognised. Such neurological channelopathies are frequently genetically determined but may also arise through autoimmune mechanisms. In this article clinical, genetic, immunological, and electrophysiological aspects of this expanding group of neurological disorders are reviewed. Clinical situations in which a neurological channelopathy should enter into the differential diagnosis are highlighted. Some practical guidance on how to investigate and treat this complex group of disorders is also included.
PPARγ-mediated insulin sensitization: the importance of fat versus muscle
Authors: Kintscher Ulrich , Law Ronald E.
Published: January 2005

Peroxisome proliferator-activated receptor-γ (PPARγ) is a nuclear hormone receptor that functions as a transcriptional regulator in a variety of tissues. PPARγ activation, e.g., through binding of the synthetic glitazones or thiazolidinediones (TZD), results in a marked improvement in type 2 diabetic patients of insulin and glucose parameters resulting from an improvement of whole body insulin sensitivity. The role of different metabolic tissues (fat, skeletal muscle, liver) in mediating PPARγ function in glucose and insulin homeostasis is still unclear. Recently, the function of PPARγ in adipose tissue and skeletal muscle has been intensively characterized by using targeted deletion of PPARγ in those tissues. In those studies, adipose PPARγ has been identified as an essential mediator for the maintainance of whole body insulin sensitivity. Two major mechanisms have been described. 1) Adipose PPARγ protects nonadipose tissue against excessive lipid overload and maintains normal organ function (liver, skeletal muscle); and 2) adipose PPARγ guarantees a balanced and adequate production of secretion from adipose tissue of adipocytokines such as adiponectin and leptin, which are important mediators of insulin action in peripheral tissues. In contrast to studies in adipose-specific PPARγ-deficient mice, the data in muscle-specific PPARγ−/−mice demonstrate that whole body insulin sensitivity is, at least in part, relying on an intact PPARγ system in skeletal muscle. Finally, these early and elegant studies using tissue-specific PPARγ knockout mouse models pinpoint adipose tissue as the major target of TZD-mediated improvement of hyperlipidemia and insulin sensitization.
Luminal adenosine stimulates chloride secretion through A1receptor in mouse jejunum
Authors: Ghanem Esam , Lövdahl Cecilia , Daré Elisabetta , Ledent Catherine , Fredholm Bertil B. , Boeynaems Jean-Marie , Van Driessche Willy , Beauwens Renaud
Published: January 2005

Adenosine is known to stimulate chloride secretion by mouse jejunum. Whereas the receptor on the basolateral side is believed to be A2B, the receptor involved in the luminal effect of adenosine has not been identified. We found that jejuna expressed mRNA for all adenosine receptor subtypes. In this study, we investigated the stimulation of chloride secretion by adenosine in jejuna derived from mice lacking the adenosine receptors of A1(A1R) and A2A(A2AR) or control littermates. The jejunal epithelium was mounted in a Ussing chamber, and a new method on the basis of impedance analysis was used to calculate the short-circuit current ( Isc) values. Chloride secretion was assessed by the Iscafter inhibition of the sodium-glucose cotransporter by adding phloridzin to the apical bathing solution. The effect of apical adenosine on chloride secretion was lost in jejuna from mice lacking the A1R. There was no difference in the response to basolaterally applied adenosine or to apical forskolin. Furthermore, in jejuna from control mice, the effect of apical adenosine was also abolished in the presence of 8-cyclopentyl-1,3-dipropylxanthine, a specific A1R antagonist. Responses to adenosine were identical in jejuna from control and A2AR knockout mice. This study demonstrates that A1R (and not A2AR) mediates the enhancement of chloride secretion induced by luminal adenosine in mice jejunum.
Acute colitis induced by dextran sulfate sodium progresses to chronicity in C57BL/6 but not in BALB/c mice: correlation between symptoms and inflammation
Authors: Melgar Silvia , Karlsson Agneta , Michaëlsson Erik
Published: January 2005

Exposure to dextran sulfate sodium (DSS) induces acute colitis, which is normally resolved after DSS removal. To study chronicity, mice are typically subjected to three to five cycles of weekly DSS exposures, each followed by a 1- to 2-wk rest period. Here, we describe a novel and convenient way of inducing chronic, progressive colitis by a single exposure to DSS. C57BL/6 mice exposed to DSS for 5 days developed acute colitis that progressed to severe chronic inflammation. The plasma haptoglobin levels remained high during the chronic phase, showing that the inflammation was active. Surprisingly, the mice regained their original weight along with the progression of colitis, and the only apparent symptom was loose feces. Histopathological changes 4 wk after DSS removal were dense infiltrates of mononuclear cells, irregular epithelial structure, and persistent deposits of collagen. A progressive production of the cytokines IL-1β, IL-12 p70, and IL-17 correlated with the extensive cellular infiltration, whereas high IFN-γ production was mainly found late in the chronic phase. Similar to C57BL/6 mice, BALB/c mice exposed to 5 days of DSS developed acute colitis as previously described. The acute colitis was accompanied by elevated plasma levels of haptoglobin and increased colonic levels of IL-1α/β, IL-6, IL-18, and granulocyte colony-stimulating factor. However, soon after DSS removal, BALB/c mice recovered and were symptom free within 2 wk and completely recovered 4 wk after DSS removal in terms of histopathology, haptoglobin levels, and local cytokine production. In summary, these data stress the effect of genetic background on the outcome of DSS provocation. We believe that the present protocol to induce chronic colitis in C57BL/6 mice offers a robust model for validating future therapies for treatment of inflammatory bowel disease.
Identification of differentially expressed genes in response to dietary iron deprivation in rat duodenum
Authors: Collins James F. , Franck Christina A. , Kowdley Kris V. , Ghishan Fayez K.
Published: January 2005

We sought to identify novel genes involved in intestinal iron absorption by inducing iron deficiency in rats during postnatal development from the suckling period through adulthood. We then performed comparative gene chip analyses (RAE230A and RAE230B chips; Affymetrix) with cRNA derived from duodenal mucosa. Real-time PCR was used to confirm changes in gene expression. Genes encoding the apical iron transport-related proteins [ divalent metal transporter 1 (DMT1) and duodenal cytochrome b] were strongly induced at all ages studied, whereas increases in mRNA encoding the basolateral proteins iron-regulated gene 1 and hephaestin were observed only by real-time PCR. In addition, transferrin receptor 1 and heme oxygenase 1 were induced. We also identified induction of novel genes not previously associated with intestinal iron transport. The Menkes copper ATPase (ATP7a) and metallothionein were strongly induced at all ages studied, suggesting increased copper absorption by enterocytes during iron deficiency. We also found significantly increased liver copper levels in 7- to 12-wk-old iron-deficient rats. Also upregulated at most ages examined were the sodium-dependent vitamin C transporter, tripartite motif protein 27, aquaporin 4, lipocalin-interacting membrane receptor, and the breast cancer-resistance protein (ABCG2). Some genes also showed decreased expression with iron deprivation, including several membrane transporters, metabolic enzymes, and genes involved in the oxidative stress response. We speculate that dietary iron deprivation leads to increased intestinal copper absorption via DMT1 on the brush-border membrane and the Menkes copper ATPase on the basolateral membrane. These findings may thus explain copper loading in the iron-deficient state. We also demonstrate that many other novel genes may be differentially regulated in the setting of iron deprivation.
Recurrent networks of submucous neurons controlling intestinal secretion: a modeling study
Authors: Chambers Jordan D. , Bornstein Joel C. , Sjövall Henrik , Thomas Evan A.
Published: January 2005

Secretomotor neurons, immunoreactive for vasoactive intestinal peptide (VIP), are important in controlling chloride secretion in the small intestine. These neurons form functional synapses with other submucosal VIP neurons and transmit via slow excitatory postsynaptic potentials (EPSPs). Thus they form a recurrent network with positive feedback. Intrinsic sensory neurons within the submucosa are also likely to form recurrent networks with positive feedback, provide substantial output to VIP neurons, and receive input from VIP neurons. If positive feedback within recurrent networks is sufficiently large, then neurons in the network respond to even small stimuli by firing at their maximum possible rate, even after the stimulus is removed. However, it is not clear whether such a mechanism operates within the recurrent networks of submucous neurons. We investigated this question by performing computer simulations of realistic models of VIP and intrinsic sensory neuron networks. In the expected range of electrophysiological properties, we found that activity in the VIP neuron network decayed slowly after cessation of a stimulus, indicating that positive feedback is not strong enough to support the uncontrolled firing state. The addition of intrinsic sensory neurons produced a low stable firing rate consistent with the common finding that basal secretory activity is, in part, neurogenic. Changing electrophysiological properties enables these recurrent networks to support the uncontrolled firing state, which may have implications with hypersecretion in the presence of enterotoxins such as cholera-toxin.
Characterization of pancreas-projecting rat dorsal motor nucleus of vagus neurons
Authors: Browning Kirsteen N. , Coleman F. Holly , Travagli R. Alberto
Published: January 2005

The electrophysiological and morphological properties of rat dorsal motor nucleus of the vagus (DMV) neurons innervating the pancreas were examined by using whole cell patch clamp recordings from brain stem slices and postfixation morphological reconstructions of Neurobiotin-filled neurons. Recordings were made from 178 DMV neurons whose projections had been identified by previous apposition of the fluorescent neuronal tracer DiI to the body of the pancreas. DMV neurons projecting to the pancreas had an input resistance of 434 ± 14 MΩ, an action potential duration of 3 ± 0.1 ms, and an afterhyperpolarization of 18 ± 0.4 mV amplitude and 108 ± 7 ms time constant of decay; these electrophysiological properties resembled those of gastric-projecting neurons but were significantly different from those of intestinal-projecting neurons. Interestingly, 14 of 178 pancreas-projecting neurons showed the presence of a slowly developing afterhyperpolarization whose presence was not reported in DMV neurons projecting to any other gastrointestinal area. The morphological characteristics of pancreas-projecting neurons (soma area 274 ± 12 μm2; soma diameter of 25 ± 0.7 μm; soma form factor 0.74 ± 0.01; segments 9.7 ± 0.41), however, were similar to those of intestinal- but differed from those of gastric-projecting neurons. In summary, these results suggest that pancreas-projecting rat DMV neurons are heterogeneous with respect to some electrophysiological and morphological properties. These differences might underlie functional differences in the vagal modulation of pancreatic functions.
Shear stress inhibits smooth muscle cell migration via nitric oxide-mediated downregulation of matrix metalloproteinase-2 activity
Authors: Garanich Jeffrey S. , Pahakis Manolis , Tarbell John M.
Published: January 2005

Vascular smooth muscle cell (SMC) migration is a hallmark of intimal hyperplasia (IH), the progression of which is affected by hemodynamic conditions at the diseased site. The realization that SMCs are exposed to blood flow in both denuded vessels (direct blood flow) and intact vessels (interstitial blood flow) motivated this study of the effects of fluid flow shear stress (SS) on SMC migration. Rat aortic SMCs were seeded onto Matrigel-coated cell culture inserts, and their migratory activity toward PDGF-BB when exposed to SS in a rotating disk apparatus was quantified. Four hours of either 10 or 20 dyn/cm2SS significantly inhibited SMC migration to the bottom side of the insert. This inhibition was associated with downregulation of SMC matrix metalloproteinase (MMP)-2 activation. Four hours of 10 dyn/cm2SS also drastically increased SMC production of NO. A NO synthase inhibitor ( NG-nitro-l-arginine methyl ester; 100 μM) abolished the shear-induced increase in SMC NO production as well as the inhibition of migration and MMP-2 activity. A NO donor ( S-nitroso- N-acetyl-penicillamine; 500 μM) suppressed SMC migration via the reduction of both total and active MMP-2 levels. Addition of 10 μM MMP-2 inhibitor I to inserts significantly reduced SMC migration. Western blots showed no effect of 4 h of 20 dyn/cm2SS on SMC production of PDGF-AA, another chemical known to suppress SMC migration. Thus it appears that SS acts to suppress SMC migration by upregulating the cellular production of NO, which in turn inhibits MMP-2 activity.
A2Badenosine receptor mediates human chorionic vasoconstriction and signals through arachidonic acid cascade
Authors: Donoso M. Verónica , López Rodrigo , Miranda Ramiro , Briones René , Huidobro-Toro J. Pablo
Published: January 2005

Because adenosine is a vascular tone modulator, we examined the effect of adenosine and congeners in the vascular reactivity of isolated human placental vessels and in perfused cotyledons. We characterized its vasomotor action and tentatively identified the receptor subtypes and their intracellular signaling mechanisms. We recorded isometric tension from the circular layer of chorionic vessel rings maintained under 1.5 g of basal tension or precontracted with KCl. The relative order of potency of adenosine and structural analogs is consistent with the expression of A2Breceptors, 5′-( N-ethylcarboxamido)adenosine (NECA) being the most potent. The maximal contraction ranged from 45% to 60% of the KCl standard response, except for an A2Areceptor agonist that did not exceed 15%. Consistently, NECA was 100-fold more potent than adenosine to raise the perfusion pressure of ex vivo perfused cotyledons. In contrast, a selective A3receptor agonist relaxed precontracted rings of chorionic vessels. Whereas a selective A3receptor antagonist was ineffective to antagonize adenosine-induced contraction, A2or A1receptor antagonists reduced adenosine-induced vasoconstriction concentration-dependently. Denudation of the endothelial layer reduced adenosine- and NECA-induced contractions by 50–70%. Furthermore, indomethacin reduced adenosine- or NECA-induced contractions concentration-dependently in intact and endothelium-denuded rings. A thromboxane receptor antagonist blocked adenosine- and NECA-induced contractions in intact and endothelium-denuded rings, suggesting the involvement of an arachidonic acid metabolite as the mediator of the vasoconstriction. We propose that adenosine A2Breceptors mediate the adenosine-induced contraction vasomotor effect in human chorionic vessels and that this involves synthesis of a thromboxane receptor activator or a related prostanoid.
Platelet-activating factor induces cardioprotection in isolated rat heart akin to ischemic preconditioning: role of phosphoinositide 3-kinase and protein kinase C activation
Authors: Penna Claudia , Alloatti Giuseppe , Cappello Sandra , Gattullo Donatella , Berta Giovanni , Mognetti Barbara , Losano Gianni , Pagliaro Pasquale
Published: January 2005

Ischemic preconditioning (IP) is a cardioprotective mechanism against myocellular death and cardiac dysfunction resulting from reperfusion of the ischemic heart. At present, the precise list of mediators involved in IP and the pathways of their mechanisms of action are not completely known. The aim of the present study was to investigate the role of platelet-activating factor (PAF), a phospholipid mediator that is known to be released by the ischemic-reperfused heart, as a possible endogenous agent involved in IP. Experiments were performed on Langendorff-perfused rat hearts undergoing 30 min of ischemia followed by 2 h of reperfusion. Treatment with a low concentration of PAF (2 × 10−11 M) before ischemia reduced the extension of infarct size and improved the recovery of left ventricular developed pressure during reperfusion. The cardioprotective effect of PAF was comparable to that observed in hearts in which IP was induced by three brief (3 min) periods of ischemia separated by 5-min reperfusion intervals. The PAF receptor antagonist WEB-2170 (1 × 10−9 M) abrogated the cardioprotective effect induced by both PAF and IP. The protein kinase C (PKC) inhibitor chelerythrine (5 × 10−6 M) or the phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 (5 × 10−5 M) also reduced the cardioprotective effect of PAF. Western blot analysis revealed that following IP treatment or PAF infusion, the phosphorylation of PKC-ε and Akt (the downstream target of PI3K) was higher than that in control hearts. The present data indicate that exogenous applications of low quantities of PAF induce a cardioprotective effect through PI3K and PKC activation, similar to that afforded by IP. Moreover, the study suggests that endogenous release of PAF, induced by brief periods of ischemia and reperfusion, may participate to the triggering of the IP of the heart.
Regional myocardial work by strain Doppler echocardiography and LV pressure: a new method for quantifying myocardial function
Authors: Urheim Stig , Rabben Stein Inge , Skulstad Helge , Lyseggen Erik , Ihlen Halfdan , Smiseth Otto A.
Published: January 2005

There is a need for better methods to quantify regional myocardial function. In the present study, we investigated the feasibility of quantifying regional function in terms of a segmental myocardial work index as derived from strain Doppler echocardiography (SDE) and invasive pressure. In 10 anesthetized dogs, we measured left ventricular (LV) pressure by micromanometer and myocardial longitudinal strains by SDE and sonomicrometry. The regional myocardial work index (RMWI) was calculated as the area of the pressure-strain loop. As a reference method for strain, we used sonomicrometry. By convention, the loop area was assigned a positive sign when the pressure-strain coordinates rotated counterclockwise. Measurements were done at baseline and during volume loading and left anterior descending coronary artery (LAD) occlusion, respectively. There was a good correlation between RMWI calculated from strain by SDE and strain by sonomicrometry ( y = 0.73 x + 0.21, r = 0.82, P < 0.01). Volume loading caused an increase in RMWI from 1.3 ± 0.2 to 2.2 ± 0.1 kJ/m3 ( P < 0.05) by SDE and from 1.5 ± 0.3 to 2.7 ± 0.3 kJ/m3 ( P = 0.066) by sonomicrometry. Short-term ischemia (1 min) caused a decrease in RMWI from 1.3 ± 0.2 to 0.3 ± 0.04 kJ/m3 ( P < 0.05) and from 1.3 ± 0.3 to 0.5 ± 0.2 kJ/m3 ( P < 0.05) by SDE and sonomicrometry, respectively. In the nonischemic ventricle and during short-term ischemia, the pressure-strain loops rotated counterclockwise, consistent with actively contracting segments. Long-term ischemia (3 h), however, caused the pressure-strain loop to rotate clockwise, consistent with entirely passive segments, and the loop areas became negative, −0.2 ± 0.1 and −0.1 ± 0.03 kJ/m3 ( P < 0.05) by SDE and sonomicrometry, respectively. A RMWI can be estimated by SDE in combination with LV pressure. Furthermore, the orientation of the loop can be used to assess whether the segment is active or passive.
Sympathoexcitation by central ANG II: Roles for AT1 receptor upregulation and NAD(P)H oxidase in RVLM
Authors: Gao Lie , Wang Wei , Li Yu-Long , Schultz Harold D. , Liu Dongmei , Cornish Kurtis G. , Zucker Irving H.
Published: January 2005

Chronic heart failure is often associated with sympathoexcitation and blunted arterial baroreflex function. These phenomena have been causally linked to elevated central ANG II mechanisms. Recent studies have shown that NAD(P)H oxidase-derived reactive oxygen species (ROS) are important mediators of ANG II signaling and therefore might play an essential role in these interactions. The aims of this study were to determine whether central subchronic infusion of ANG II in normal animals has effects on O2− production and expression of NAD(P)H oxidase subunits as well as ANG II type 1 (AT1) receptors in the rostral ventrolateral medulla (RVLM). Twenty-four male New Zealand White rabbits were divided into four groups and separately received a subchronic intracerebroventricular infusion of saline alone, ANG II alone, ANG II with losartan, and losartan alone for 1 wk. On day 7 of intracerebroventricular infusion, mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) values were recorded, and arterial baroreflex sensitivity was evaluated while animals were in the conscious state. We found that ANG II significantly increased baseline RSNA (161.9%; P < 0.05), mRNA and protein expression of AT1 receptors (mRNA, 66.7%; P < 0.05; protein, 85.1%; P < 0.05), NAD(P)H oxidase subunits (mRNA, 120.0–200.0%; P < 0.05; protein, 90.9–197.0%; P < 0.05), and O2− production (83.2%; P < 0.05) in the RVLM. In addition, impaired baroreflex control of HR (Gainmax reduced by 48.2%; P < 0.05) and RSNA (Gainmax reduced by 53.6%; P < 0.05) by ANG II was completely abolished by losartan. Losartan significantly decreased baseline RSNA (−49.5%; P < 0.05) and increased baroreflex control of HR (Gainmax increased by 64.8%; P < 0.05) and RSNA (Gainmax increased by 67.9%; P < 0.05), but had no significant effects on mRNA and protein expression of AT1 receptor and NAD(P)H oxidase subunits and O2− production in the RVLM. These data suggest that in normal rabbits, NAD(P)H oxidase-derived ROS play an important role in the modulation of sympathetic activity and arterial baroreflex function by subchronic central treatment of exogenous ANG II via AT1 receptors.
Effects of nociceptive stimuli on the pulmonary circulation in the ovine fetus
Authors: Houfflin-Debarge V. , Delelis A. , Jaillard S. , Larrue B. , Deruelle P. , Ducloy A. S. , Puech F. , Storme L.
Published: January 2005

The fetus is able to exhibit a stress response to painful events, and stress hormones have been shown to modulate pulmonary vascular tone. At birth, the increased level of stress hormones plays a significant role in the adaptation to postnatal life. We therefore hypothesized that pain may alter pulmonary circulation in the perinatal period. The hemodynamic response to subcutaneous injection of formalin, which is used in experimental studies as nociceptive stimulus, was evaluated in chronically prepared, fetal lambs. Fetal lambs were operated on at 128 days gestation. Catheters were placed into the ascending aorta, superior vena cava, and main pulmonary artery. An ultrasonic flow transducer was placed around the left pulmonary artery. Three subcutaneous catheters were placed in the lambs' limb. The hemodynamic responses to subcutaneous injection of formalin, to formalin after fetal analgesia by sufentanil, and to sufentanil alone were recorded. Cortisol and catecholamine concentrations were also measured. Pulmonary vascular resistances (PVR) increased by 42% ( P < 0.0001) after formalin injection. Cortisol increased by 54% ( P = 0.05). During sufentanil infusion, PVR did not change significantly after formalin. Cortisol increased by 56% ( P < 0.05). PVR did not change during sufentanil infusion. Norepinephrine levels did not change during any of the protocols. Our results indicate that nociceptive stimuli may increase the pulmonary vascular tone. This response is not mediated by an increase in circulating catecholamine levels. Analgesia prevents this effect. We speculate that this pulmonary vascular response to nociceptive stimulation may explain some hypoxemic events observed in newborn infants during painful intensive care procedures.
Inflammatory processes in muscle injury and repair
Authors: Tidball James G.
Published: January 2005

Modified muscle use or injury can produce a stereotypic inflammatory response in which neutrophils rapidly invade, followed by macrophages. This inflammatory response coincides with muscle repair, regeneration, and growth, which involve activation and proliferation of satellite cells, followed by their terminal differentiation. Recent investigations have begun to explore the relationship between inflammatory cell functions and skeletal muscle injury and repair by using genetically modified animal models, antibody depletions of specific inflammatory cell populations, or expression profiling of inflamed muscle after injury. These studies have contributed to a complex picture in which inflammatory cells promote both injury and repair, through the combined actions of free radicals, growth factors, and chemokines. In this review, recent discoveries concerning the interactions between skeletal muscle and inflammatory cells are presented. New findings clearly show a role for neutrophils in promoting muscle damage soon after muscle injury or modified use. No direct evidence is yet available to show that neutrophils play a beneficial role in muscle repair or regeneration. Macrophages have also been shown capable of promoting muscle damage in vivo and in vitro through the release of free radicals, although other findings indicate that they may also play a role in muscle repair and regeneration through growth factors and cytokine-mediated signaling. However, this role for macrophages in muscle regeneration is still not definitive; other cells present in muscle can also produce the potentially regenerative factors, and it remains to be proven whether macrophage-derived factors are essential for muscle repair or regeneration in vivo. New evidence also shows that muscle cells can release positive and negative regulators of inflammatory cell invasion, and thereby play an active role in modulating the inflammatory process. In particular, muscle-derived nitric oxide can inhibit inflammatory cell invasion of healthy muscle and protect muscle from lysis by inflammatory cells in vivo and in vitro. On the other hand, muscle-derived cytokines can signal for inflammatory cell invasion, at least in vitro. The immediate challenge for advancing our current understanding of the relationships between muscle and inflammatory cells during muscle injury and repair is to place what has been learned in vitro into the complex and dynamic in vivo environment.
Comparison of Hsc70 orthologs from polar and temperate notothenioid fishes: differences in prevention of aggregation and refolding of denatured proteins
Authors: Place Sean P. , Hofmann Gretchen E.
Published: January 2005

Although a great deal is known about the cellular function of molecular chaperones in general, very little is known about the effect of temperature selection on the function of molecular chaperones in nonmodel organisms. One major unanswered question is whether orthologous variants of a molecular chaperone from differential thermally adapted species vary in their thermal responses. To address this issue, we utilized a comparative approach to examine the temperature interactions of a major cytosolic molecular chaperone, Hsc70, from differently thermally adapted notothenioids. Using in vitro assays, we measured the ability of Hsc70 to prevent thermal aggregation of lactate dehydrogenase (LDH). We further compared the capacity of Hsc70 to refold chemically denatured LDH over the temperature range of −2 to +45°C. Hsc70 purified from the temperate species exhibited greater ability to prevent the thermal denaturation of LDH at 55°C compared with Hsc70 from the cold-adapted species. Furthermore, Hsc70 from the Antarctic species lost the ability to competently refold chemically denatured LDH at a lower temperature compared with Hsc70 from the temperate species. These data indicate the function of Hsc70 in notothenioid fishes maps onto their thermal history and that temperature selection has acted on these molecular chaperones.
Effects of increasing doses of activated recombinant factor VII on haemostatic parameters in swine
Authors: Ryan Kathy L. , Delgado Angel V. , Martinez Raul S. , Uscilowicz John M. , Cortez Douglas S. , Martinowitz Uri , Pusateri Anthony E.
Published: January 2005

This study examined dose-response relationships between activated recombinant factorVII (rFVIIa) and (1) in vivo haemostasis and (2) in vitro measures of coagulation and platelet function. Anesthetized swine were used. Ear bleeding time (BT) was measured and blood was sampled following increasing doses of rFVIIa (0, 90, 180, 360 and 720 μg/kg; n = 6) or saline (n = 6). BT was not altered by rFVIIa. Prothrombin time (PT) using standard or pig-specific methods was decreased by rFVIIa. Activated clotting time (ACT) was decreased by rFVIIa. Thromboelastography using collagen (COLL) or pig thromboplastin (p-ThP) as agonist demonstrated shorter reaction times, shortened time to reach maximum velocity of clot formation, and increased α -angle in the presence of rFVIIa. rFVIIa dosing increased maximum velocity of clot formation when p-ThP was used to initiate the reaction but not when COLL was used. rFVIIa at the highest concentration increased maximum amplitude when COLL was used to initiate the reaction. Platelet aggregation was not altered by rFVIIa. Following completion of the dose escalation phase, a severe liver injury was produced. rFVIIa altered neither blood loss nor survival time following injury but improved mean arterial pressure. A small increase in systemic thrombin-antithrombin III complex occurred after administration of rFVIIa at doses of 180 μg/kg and above. However, there was no histological evidence of intravascular coagulation after rFVIIa administration. In summary, rFVIIa activity was detectable in vitro but did not change haemostasis in normal swine.
The ABO blood group genotype and factor VIII levels as independent risk factors for venous thromboembolism
Authors: Tirado Isabel , Soria José , Oliver Arturo , Martínez-Sánchez Elisabeth , Vallvé Cristina , Borrell Monserrat , Urrutia Teresa , Fontcuberta Jordi , Mateo José
Published: January 2005

Factor VIII (FVIII), von Willebrand factor (vWF) and the ABO blood groups have been associated with thrombosis. The ABO locus has functional effects on vWF and FVIII levels and is genetically correlated with FVIII, vWF and thrombosis. We carried out a case-control study to assess the role of FVIII, vWF and ABO types on thrombotic risk. We analyzed 250 patients with venous thrombosis and 250 unrelated controls. FVIII, vWF and other factors related to thrombophilia were measured, ABO groups were analyzed by genotyping. FVIII and vWF were higher in non-O individuals. Group O was more frequent in the controls (44.3% v 23.3%; difference 21.1%; 95% CI: 13.0–29.3%) and Group A in patients (59.2% v. 41.5%; difference 17.7%, 95% CI: 9.1–26.4%). Individuals carrying the A1
allele had a higher risk of thrombosis (OR 2.6; 95% CI, 1.8–3.8). The risk attributed to vWF disappeared after adjusting for the ABO group. Patients with FVIII above the 90th percentile had a high thrombotic risk (adjusted OR 3.7; 95%CI, 2.1–6.5), and a high risk of recurrence (OR 2.3; 95%CI: 1.3–4.1). In conclusion, high FVIII levels and non-O blood groups, likely those with the A1
allele, are independent risk factors for venous thromboembolism and should be considered in evaluating of thrombophilia.
Fluvastatin increases the expression of adhesion molecules, monocyte chemoattractant protein-1 and tissue factor in HUVEC stimulated by patient IgG fractions containing antiphospholipid antibodies
Authors: Dunoyer-Geindre Sylvie , Dimitrova Yordanka , Fish Richard J. , Satta Nathalie , Reber Guido , Kruithof Egbert K.O. , de Moerloose Philippe
Published: January 2005

The presence of antiphospholipid antibodies (APLA) is associated with an increased risk of recurrent thrombosis and pregnancy loss. APLA are able to activate endothelial cells (EC) and induce an increase in the expression of inflammatory marker proteins, such as leukocyte adhesion molecules, tissue factor or the monocyte chemoattractant protein-1 (MCP-1). Our objective was to investigate the effect of statins on EC activation induced by APLA in vitro. IgG was purified from the plasma of six patients with APLA and from healthy controls. EC were incubated with patient IgG or with control IgG, in the presence or absence of 5μM of fluvastatin, and expression of the leukocyte adhesion molecules, VCAM-1 and E-selectin, analyzed by flow cytometry and by quantitative reverse transcriptase-PCR (QRT-PCR).The expression of tissue factor and the chemokine MCP-1 was analyzed by QRT-PCR alone. Incubation of EC with patient IgG increased the expression of VCAM-1, E-selectin, tissue factor and MCP-1. Prior treatment of the cells with fluvastatin further increased the expression of these proteins. The fluvastatin effect was reversed by co-incubation with mevalonate or geranylgeranylpyrophosphate and mimicked by the geranylgeranyl transferase inhibitor GGTI-286. Our results show that in cultured human EC, statins increase the extent of inflammatory activation induced by APLA. This effect appears to be mediated by an inhibitory effect of statins on one or more geranylgeranylated protein(s).