Each of 314 strains of Pseudomonas aeruginosa recovered from 87 French cystic fibrosis (CF) patients was typed by multilocus enzyme electrophoresis to investigate the genetic diversity, the relatedness and the molecular epidemiology of strains isolated from cases of chronic pulmonary colonization. Comparison of allele profiles at 18 enzyme loci identified 17 electrophoretic types (ETs). Of the 314 isolates, 290 (92%) were either ET1 (n = 127) or ET2 (n = 163), which differed only at the shikimate dehydrogenase (SKD) locus. The mean genetic diversity (H) was 0.138. These results suggest that there is cross-colonization between patients and/or that two predominant groups of strains are able to colonize French CF patients. Sequential isolates collected from 18 patients during a period of 12-28 months were analysed to assess genomic variability and its relationship to clinical outcome. Six patients were colonized by a stable strain. For the others, double infections or changes in colonization over time were observed. No relationships were detected between the clinical outcome and the persistence of stable isolates, the emergence of transient superinfecting variants, the presence of multiple ETs or the shift of ET during the monitoring.

The endothelial cell-derived peptide endothelin 1 (ET1) stimulates cell proliferation and differentiated functions of human osteoblastic cells (HOC), and HOC constitutively express the endothelin A receptor (ETRA). Therefore, ET1 may play an important role in the regulation of bone cell metabolism. As glucocorticoids (GC) exert a profound influence on bone metabolism and increase the effects of ET1 on bone cell metabolism in vitro, the effects of GC on ETRA expression in HOC were investigated. Dexamethasone (DEX) increased ETRA mRNA levels in a dose- and time-dependent fashion. The effects of dexamethasone, prednisolone, and deflazacort on the increase of ETRA mRNA levels correlate positively with their binding affinity to the GC receptor. Scatchard analysis of ET1 binding data to HOC revealed that DEX increased the binding capacity for ET1 from 25,300 to 62,800 binding sites per osteoblastic cell, leading to an enhanced mitogenic effect of ET1 on HOC after preincubation with DEX. Transiently transfected primary HOC with a reporter gene construct, containing the 5'-flanking region of the ETRA gene fused to luciferase gene, showed a promoter-dependent expression of the reporter gene and the induction of reporter gene expression by DEX treatment. Total RNA extracts of femoral head biopsies with osteonecrotic lesions from GC-treated patients showed threefold higher ETRA mRNA levels compared with extracts of bone biopsies from patients with traumatically induced osteonecrosis and coxarthrosis. Furthermore, GC treatment increased plasma ET1 levels by 50% compared with pretreatment values. These findings suggest that GC induced upregulation of ETRA, and ET1 plasma levels enhance ET1's anabolic action on bone cell metabolism. Increased ET1 concentrations may also impair bone perfusion by vasoconstriction in a metabolically activated skeletal region.

The development of a bone metastasis involves interactions between the tumor cells, the bone marrow microenvironment and the bone cells themselves. A better understanding of the pathophysiological changes occurring in bone metastasis can be obtained from histopathological examination of invaded specimens. This review focuses on the main molecular mechanisms implied in the localization and growth of malignant cells in the bone marrow. The corresponding histologic developmental stages are illustrated both in osteolytic (or mixed metastasis) or in the osteosclerotic forms by histological analysis, immunohistochemistry and microcomputed tomographic analysis of bone samples. In both cases, the malignant cells find a "fertile soil" in the bone marrow microenvironment. They use the growth factors released by bone cells for the coupling between osteoclasts/osteoblasts to promote their own development. In turn, they elaborate a variety of cytokines that can promote osteoclastogenesis (PTHrP, IL-1, IL-6…) or on the contrary, other growth factors that can boost the osteoblastic activity (ET1, IGFs). A "vicious circle" occurs between the malignant cells and the bone cells leading to the radiological expression of the metastasis.

The rice pathogen Fusarium fujikuroi is well known for its ability to produce the plant hormones gibberellins (GAs). However, the majority of closely related Fusarium species is unable to produce GAs although the GA gene cluster is present in their genomes. In this study, we analyzed five orchid-associated Fusarium isolates for their capacity to produce GAs. Four of them did not produce any GAs and were shown not to contain any GA biosynthetic genes. However, the fifth isolate, which has been identified as F. proliferatum based on five molecular markers, produced significant amounts of GAs in contrast to previously characterized F. proliferatum strains. We focused on the molecular characterization of two GA-specific genes, ggs2 and cps/ks, both inactive in F. proliferatum strain D-02945. Complementation of a F. fujikuroi Deltaggs2 mutant with the ET1 ggs2 gene fully restored GA biosynthesis, confirming that the orchid-associated isolate contains an active gene copy. A possible correlation between GA production and their role in plant-fungal interactions is discussed.

OBJECTIVE

To characterize the functional effect of endothelin-1 (ET1) and endothelin-3 (ET3), immunohistochemically localize ET1-like immunoreactivity, and measure the tissue levels of immunoreactive endothelin (irET) in canine genitourinary (GU) tissues.

METHODS

Canine GU tissues were characterized by measuring ET1 levels using a RIA, immunohistochemical staining of ET1 and isometric tension studies.

RESULTS

Immunoreactive endothelin was present, to varying degrees, in the vas deferens, ureter, prostate, bladder and urethra. Functionally, ET1 demonstrated the typical concentration response characteristics in the canine bladder base, bladder body, and prostate. The maximal tension (Emax) measured following ET1 challenge was approximately 20-fold greater in the bladder body (0.67 +/- 0.21 g/mm.2) and bladder base (0.48 +/- 0.18 g/mm.2) as compared to the prostate 0.04 +/- 0.001 g/mm.2 The Emax of ET3 in the bladder body (0.31 +/- 0.12 g/mm.2) and bladder base (0.19 +/- 0.08 g/mm.2) was significantly lower than the corresponding Emax of ET1. No measurable contractile response was elicited by ET3 in the canine prostate. Immunohistochemical staining localized the ET-like immunoreactivity to the glandular epithelium of the prostate and the transitional epithelium of the bladder.

CONCLUSIONS

Endothelins are ubiquitous in the canine lower GU tract with predominant localization to the epithelial elements. Endothelins are also functionally active in canine GU tissues, but the specific role of endothelins in the physiology and pathophysiology of GU tissues requires further investigation.

The authors investigated the effects of endothelin-1 (ET1) on inositol trisphosphate (IP3) production, 1, 2-diacylglycerol (DAG) formation, measured as phosphatidic acid (PA), cAMP formation, and contraction in iris sphincter of different mammalian species. They found that ET1 is a potent agonist for IP3 production, DAG formation, and contraction in rabbit, dog, cat, and pig iris sphincters, and for cAMP formation in all species that were investigated--rabbit, dog, cat, pig, bovine, monkey, and human sphincters. In the bovine model, ET1 induced cAMP formation in a dose-dependent manner, with an EC50 of 28 nM. This is the first report that showed an effect of the peptide on the adenylate cyclase system. In rabbit sphincter, ET1 induced a significant increase in IP3 production by 30 sec and reached a 6-fold level more than control within 1 and 5 min. ET1-stimulated IP3 production is dose dependent with an EC50 of 45 nM, this value is about 100- and 56-fold lower than those we reported for substance P and carbachol, respectively. ET1 also increased 32P labeling of PA more than 6-fold; and in rabbit sphincter, ET1 is a more potent agonist in contracting the sphincter than in contracting the dilator (the EC50 values for sphincter and dilator were 46 and 120 nM, respectively). L-type Ca2+ channels are not involved in IP3- and contraction responses because several blockers of these channels did not affect the ET1-induced responses, implying that in the iris sphincter, ET1 elicits the physiologic response through the G protein activation of phospholipase C and/or adenylate cyclase and not through the activation of voltage-dependent Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelin B receptor (ET(B)R) is a G protein-coupled receptor (GPCR) specific for endothelin peptides (including endothelin-1, ET1), which mediates a variety of key physiological functions in normal tissues, such as modulation of vasomotor tone, tissue differentiation, or cell proliferation. Moreover, ET(B)R, overexpressed in various cancer cells including melanoma, has been implicated in the growth and progression of tumors, as well as in controlling T cell homing to tumors. To gather information on receptor structure and function, antibodies are generally considered choice molecular probes, but generation of such reagents against the native conformation of GPCRs is a real technical challenge. Here, we show that electroporation-aided genetic immunization, coupled to cardiotoxin pretreatment, is a simple and very efficient method to raise large amounts of polyclonal antibodies highly specific for native human ET(B)R (hET(B)R), as assessed by both flow cytometry analysis of different stably transfected cell lines and a new and rapid cell-based enzyme-linked immunosorbent assay that we also describe. The antibodies recognized two major epitopes on hET(B)R, mapped within the N-terminal extracellular domain. They were used to reveal hET(B)R on membranes of three different human melanoma cell lines, by flow cytometry and confocal microscopy, a method that we show is more relevant than mRNA polymerase chain reaction in assessing receptor expression. In addition, ET-1 partially competed with antibodies for receptor binding. The strategy described here, thus, efficiently generated new immunological tools to further analyze the role of ET(B)R under both normal and pathological conditions, including cancers. Above all, it can now be used to raise monoclonal antibodies against hET(B)R and, more generally, against GPCRs that constitute, by far, the largest reservoir of potential pharmacological targets.

Endothelin 1 (ET-1) and its receptors, ETA and ETB, play important roles in regulating renal function and blood pressure, and these components are expressed in sensory nerves. Activation of transient receptor potential vanilloid (TRPV) 1 channels expressed in sensory nerves innervating the renal pelvis enhances afferent renal nerve activity (ARNA), diuresis, and natriuresis. We tested the hypothesis that ET-1 increases ARNA via activation of ETB, whereas ETA counterbalances ETB in wild-type (WT) but not TRPV1-null mutant mice. ET-1 alone or with BQ123, an ETA antagonist, perfused into the left renal pelvis increased ipsilateral ARNA in WT but not in TRPV1-null mutant mice, and ARNA increases were greater in the latter. [Ala1, 3,11,15]-endothelin 1, an ETB agonist, increased ARNA that was greater than that induced by ET-1 in WT mice only. [Ala1, 3,11,15]-endothelin 1-induced increases in ARNA were abolished by chelerythrine, a protein kinase C inhibitor, but not by H89, a protein kinase A inhibitor. Chelerythrine, H89, and BQ788, an ETB antagonist, did not affect ARNA triggered by capsaicin in WT mice. Substance P release from the renal pelvis was increased by [Ala1, 3,11,15]-endothelin 1 in WT mice only, and the increase was abolished by chelerythrine but not by H89. Chelerythrine, H89, and BQ788 did not affect capsaicin-induced substance P release. Our data show that ET1 increases ARNA via activation of ETB, whereas ETA counterbalances ETB in WT but not in TRPV1-null mutant mice, suggesting that TRPV1 mediates ETB-dependent increases in ARNA, diuresis, and natriuresis possibly via the protein kinase C pathway.

BACKGROUND

Cortical injections of the vasoconstrictor endothelin-1 (ET1) have widely been used to induce focal circumscribed ischemic lesions in the motor cortex of rodents in the context of stroke recovery studies. In order to apply this model correctly, it is essential to understand the time course of regional flow changes and of the development of penumbra and infarction.

METHODS

Multitracer micro-PET of ET1 focal ischemia in rats was performed using [11C]-flumazenil ([11C]FMZ) as a flow- and viability tracer and [18F]-fluoromisonidazole ([18F]FMISO) as hypoxia marker in order to characterize the physiological time-course of this model. Nine adult Sprague-Dawley rats received stereotaxic injections of ET1 into the right primary motor cortex, 3 served as controls. PET imaging was started 2, 3 and 20 h after the last ET1 injection. Histology was obtained at the end of the scans. Standardized uptake value ratios reflecting cerebral blood flow (CBF), [11C]FMZ-binding and [18F]FMISO-retention were calculated for the region of hypoperfusion and the normoperfused cortex.

RESULTS

CBF in the hypoperfused cortex was significantly reduced (p < 0.01) at 5 h (0.58 ± 0.025), 6 h (0.54 ± 0.043) and 23 h (0.66 ± 0.024) compared to controls (1.00 ± 0.011) and moderately reduced (p < 0.05) in the remainder of the affected hemisphere at 5 h (0.93 ± 0.036). [11C]FMZ-binding was within the control range at all time points. Significant [18F]FMISO-retention (1.16 ± 0.091, p < 0.05) was observed only after 6 h in the ischemic core that later turned into infarct.

CONCLUSIONS

ET1 injections yield reproducible, slowly developing ischemic lesions with constant levels of hypoperfusion. This multitracer micro-PET study suggests that the ET1 model is appropriate for inducing chronic circumscribed ischemic lesions but seems to be less suited for studying acute stroke pathophysiology.

The type VI secretion system (T6SS) is a widespread molecular weapon deployed by many bacterial species to target eukaryotic host cells or rival bacteria. Using a dynamic injection mechanism, diverse effectors can be delivered by T6SS directly into recipient cells. Here, we report a new family of T6SS effectors encoded by extended Hcps carrying diverse toxin domains. Bioinformatic analyses revealed that these Hcps with C-terminal extension toxins, designated as Hcp-ET, exist widely in the Enterobacteriaceae. To verify our findings, Hcp-ET1 was tested for its antibacterial effect, and showed effective inhibition of target cell growth via the predicted HNH-DNase activity by T6SS-dependent delivery. Further studies showed that Hcp-ET2 mediated interbacterial antagonism via a Tle1 phospholipase (encoded by DUF2235 domain) activity. Notably, comprehensive analyses of protein homology and genomic neighborhoods revealed that Hcp-ET3-4 is fused with 2 toxin domains (Pyocin S3 and Colicin-DNase) C-terminally, and its encoding gene is followed 3 duplications of the cognate immunity genes. However, some bacteria encode a separated hcp-et3 and an orphan et4 (et4O1) genes caused by a termination-codon mutation in the fusion region between Pyocin S3 and Colicin-DNase encoding fragments. Our results demonstrated that both of these toxins had antibacterial effects. Further, all duplications of the cognate immunity protein contributed to neutralize the DNase toxicity of Pyocin S3 and Colicin, which has not been reported previously. In conclusion, we propose that Hcp-ET proteins are polymorphic T6SS effectors, and thus present a novel encoding pattern of T6SS effectors.

BACKGROUND

Intermedin (IMD), a novel peptide related to calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM), may have localized actions as a modulator of cardiac function. The aim of the study is to explore the effect of IMD on angiotensin II (Ang II) and endothelin-1 (ET-1) induced hypertrophy in ventricular myocytes of neonatal rat and to try to elucidate the possible mechanism.

METHODS

Neonatal rat cardiomyocytes were cultured in serum-free medium with and without AngII (1 micromol/L) or ET-1 (60 micromol/L) in the presence and absence of IMD (1 micromol/L). Hypertrophic responses (including cell surface area, alpha-actin, and beta-myosin heavy chain mRNA expression) and cardiomyocyte expression of NADPH oxidase gp91phox were determined.

RESULTS

Ang II induced increases in cardiomyocyte size to 305 +/- 32 microm2 (n = 198, p < 0.05, at 48 hours), alpha-actin expression to 4 +/- 2.8-fold (n = 6, p < 0.05, at 48 hours) and beta-myosin heavy chain expression to 11 +/- 4.8-fold (n = 6, p < 0.05, at 48 hours), and expression of the gp91phox subunit of NADPH oxidase to 29.4 +/- 12.7-fold (n = 6, p < 0.05, at 48 hours). These effects were all significantly inhibited by IMD; cardiomyocyte size, alpha-actin expression, beta-myosin heavy chain expression, and gp91phox expression were reduced to 265 +/- 32 microm2 (n = 374, p < 0.05), 3.0 +/- 1.7-fold (n = 6, p < 0.05), 8.7 +/- 4.9-fold (n = 6, p < 0.05), 3.9 +/- 3-fold (n = 6, p < 0.05), respectively. IMD also significantly inhibited ET1-induced increases in cardiomyocyte size and superoxide generation.

CONCLUSIONS

IMD exerts an antihypertrophic effect on neonatal cardiomyocytes by reduced levels of superoxide, suggesting that an antioxidant action contributes to the antihypertrophic actions of IMD.

BACKGROUND

The aim of the present study was to evaluate the effects of hyperbaric oxygenation on lipid peroxidation, on the release of circulating cytokines (TNFa, IL6, IL1b) and endothelin-1 (ET1).

METHODS

METHODS

single arm, prospective study.

METHODS

ICU hyperbaric division of a University Hospital.

METHODS

fifteen healthy volunteers (10 male and 5 female, mean age 32+/-7 years) studied during hyperbaric oxygenation divided at random into two groups: group A (7 subjects) and group B (8 subjects).

METHODS

Both groups were consecutively pressurized at 2 atmospheres (2 atm abs) and 2.8 atm abs, with a constant descending rate of 1 m/min; in accordance with the experimental design, group A breathed pure oxygen continuously through facial masks and group B breathed chamber air during pressurization.

METHODS

Twenty millilitres of blood were drawn from all individuals at the following times: 1) basal, before HBO; 2) after 10 min at 2 atm abs; 3) after 10 min at 2.8 atm abs; 4) 30 min after the end of HBO. In all collected samples thiobarbituric reacting substances were evaluated, using the spectrophotometric technique, IL1 TNF and IL6 serum levels by ELISA and endothelin 1 plasma levels by radioimmunoassay.

RESULTS

In both groups, TBARS levels showed a twofold increase (p<0.05) in relation to the baseline, during and after hyperbaric oxygenation. Serum IL6 and IL1b values did not significantly change over the study in any of the volunteers. TNFa amounts significantly increased (p<0.05) during HBO, at 2 atm abs and 2.8 atm abs in both groups, with almost twofold increments. ET1 plasma values increased (p<0.05) in all volunteers during and after HBO: at 2 atm abs (range 7 to 24 pg/ml), 2.8 atm abs (range 7 to 19 pg/ml) and 30 min after (range 8 to 17 pg/ml) in relation to baseline (range 4 to 12 pg/ml). All the studied compounds had a similar trend in the two groups.

CONCLUSIONS

Hyperbaric oxygenation in healthy volunteers can induce not only lipid peroxidation, but also liberation of compounds such as TNFa and endothelins, no matter whether pure oxygen is breathed or not. These results suggest that the phenomenon behind this release might be leukocyte activation as induced by HBO. The possible role of ET1 in determining vasoconstriction occurring during HBO is also suggested.

BACKGROUND

Shigellosis remains a serious public health problem and an important cause of morbidity and mortality worldwide. The aim of this study was to characterize fliC and the genetic relatedness of Shigella spp. isolated during a one-year period from children in a suspected outbreak in Tehran, Iran.

RESULTS

Fifty Shigella spp. were isolated from 3779 stool samples of children with diarrhea (prevalence rate: 1.32%). Among the isolates, 92% were characterized as Shigella sonnei, while 6% and 2% were identified as S. flexneri and S. boydii, respectively. S. dysenteriae was not recovered from the patients. All isolates were negative for fliC except for Shigella standard strains. The enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) profiles allowed differentiating the 50 isolates into 5 ERIC types, which were grouped into five clusters (ET1-ET5). Computer-assisted clustering of the strains showed a high degree of similarity among the isolates.

CONCLUSIONS

In conclusion, given the clonal correlation of the Shigella strains isolated in this study and the lack of fliC among them, we propose that probably a single or limited fliC-defected Shigella clone spread and caused the outbreak.

Macrorestriction analysis by pulsed-field gel electrophoresis was used to assess the diversity of strains within the epidemic-associated electrophoretic type 1 (ET1) clone of Listeria monocytogenes. For this purpose, a total of 144 isolates from Switzerland shown by multilocus enzyme electrophoresis to belong to the ET1 were examined. These isolates were subtyped by macrorestriction analysis using the enzymes ApaI and SmaI and field inversion gel electrophoresis. Among these 144 isolates, 45 were isolated in human listeriosis cases of the postepidemic period of 1988 to 1993 and 44 were isolated in animal listeriosis cases of the same period. Forty-seven isolates were from the epidemic period of 1983 to 1987, and eight additional isolates were from cattle from two different farms. Twenty-nine different subtypes could be identified among the 144 isolates tested. Five major subtypes were found more frequently than the others during the postepidemic period, both in humans and in animals. Two of these subtypes had been previously implicated in outbreaks of listeriosis, thus suggesting that particular pulsed-field gel electrophoresis subtypes may be frequently associated with disease in humans and animals. Two of these frequent subtypes were also suspected to be related to small clusters of listeriosis cases during the postepidemic period. The results obtained by typing epidemiologically related isolates from different animals within the same farms and from different body sites of a given patient confirmed the potential of macrorestriction analysis for epidemiological studies restricted to short periods of time and to small number of isolates. The analysis of 47 isolates related to the Swiss listeriosis epidemic period of 1983 to 1987 and the use of Southern blotting and hybridization experiments show that the interpretation of relatedness between isolates presenting slightly different macrorestriction patterns may be more complex than commonly accepted. In such cases, careful interpretation of the potential molecular mechanisms leading to the differences observed between patterns is necessary.

OBJECTIVE

Medicinal plants are increasingly being projected as suitable alternative sources of antiviral agents. The development of a suitable in vitro pharmacodynamic screening technique could contribute to rapid identification of potential bioactive plants and also to the standardization and/or pharmacokinetic-pharmacodynamic profiling of the bioactive components.

RESULTS

Recombinant viral vectors (lentiviral, retroviral and adenoviral) transferring the firefly luciferase gene were constructed and the inhibition of viral vector infectivity by various concentrations of plant extracts was evaluated in HeLa or Hep2 cells by measuring the changes in luciferase activity. Cytotoxicity of the extracts was evaluated in parallel on HeLa or Hep2 cells stably expressing luciferase. Amongst the 15 extracts screened, only the methanol (ME) and the ethyl acetate (ET) fractions of the lichen, Ramalina farinacea specifically reduced lentiviral and adenoviral infectivity in a dose-dependent manner. Further, chromatographic fractionation of ET into four fractions (ET1-ET4) revealed only ET4 to be selectively antiviral with an IC50 in the 20 microg ml(-1) range. Preliminary mechanistic studies based on the addition of the extracts at different time points in the viral infection cycle (kinetic studies) revealed that the inhibitory activity was highest if extract and vectors were preincubated prior to infection, suggesting that early steps in the lentiviral or adenoviral replication cycle could be the major target of ET4. Inhibition of wild-type HIV-1 was also observed at a 10-fold lower concentration of the extract.

CONCLUSIONS

The vector-based assay is a suitable in vitro pharmacodynamic evaluation technique for antiviral medicinal plants. The technique has successfully demonstrated the presence of antiviral principles in R. farinacea.

CONCLUSIONS

Potential anti-HIV medicinal plants could rapidly be evaluated with the reported vector-based technique. The lichen, R. farinacea could represent a lead source of antiviral substances and is thus worthy of further studies.

Levels of cerebral amyloid, presumably β-amyloid (Abeta), toxicity and the incidence of cortical and subcortical ischemia increases with age. However, little is known about the severe pathological condition and dementia that occur as a result of the comorbid occurrence of this vascular risk factor and Abeta toxicity. Clinical studies have indicated that small ischemic lesions in the striatum are particularly important in generating dementia in combination with minor amyloid lesions. These cognitive deficits are highly likely to be caused by changes in the cortex. In this study, we examined the viability and morphological changes in microglial and neuronal cells, gap junction proteins (connexin43) and neuritic/axonal retraction (Fer Kinase) in the striatum and cerebral cortex using a comorbid rat model of striatal injections of endothelin-1 (ET1) and Abeta toxicity. The results demonstrated ventricular enlargement, striatal atrophy, substantial increases in β-amyloid, ramified microglia and increases in neuritic retraction in the combined models of stroke and Abeta toxicity. Changes in connexin43 occurred equally in both groups of Abeta-treated rats, with and without focal ischemia. Although previous behavioral tests demonstrated impairment in memory and learning, the visual discrimination radial maze task did not show significant difference, suggesting the cognitive impairment in these models is not related to damage to the dorsolateral striatum. These results suggest an insight into the relationship between cortical/striatal atrophy, pathology and functional impairment.

Subepithelial fibroblasts of rat duodenal villi were cultured and the physiological characteristics were studied using fura-2 fluorescence. The intracellular calcium concentration (Ca2+i) responded to various substances, i.e., endothelins (ET1 and ET3), substance P, serotonin, angiotensin II, ATP, and bradykinin. The Ca2+i responses to ET1 >> 0.1 nM) and ET3 >> 1 nM) were transient and sometimes followed oscillations that consisted of an initial Ca2+ release from the intracellular store and a sustained Ca2+ influx. Simultaneously with Ca2+i measurement, changes in the cell shape were monitored using fluorescence intensity upon 360-nm excitation. Stellate cells (with thick cell body and slender processes), formed as a result of 1 mM dibutyryl(Bt2)-cAMP treatment, began to change immediately after the short-term application of the endothelin and became flat about 20 min later. This process was not affected by the depletion of extracellular Ca2+ or by the treatment with BAPTA acetoxymethyl ester that completely suppressed the Ca2+i response. Substance P >> 100 nM) increased Ca2+i, but did not induce any morphological changes. The conversion of the shape from flat to stellate, induced by Bt2cAMP treatment, was not accompanied by any Ca2+i change. BQ-123, a specific blocker of the ETA-type receptor, did not block either Ca2+i change or shape conversion at low (100 nM) concentration. The results indicated that shape conversion in subepithelial fibroblasts did not require any Ca2+i response. Our findings regarding the characteristics of subepithelial fibroblasts in intestinal villi imply a functional similarity to astrocytes in the brain.

OBJECTIVE

Bosentan, an endothelin (ET) ETA-ETB receptors antagonist, is an effective therapy for idiopathic pulmonary arterial hypertension (PAH) and for PAH related to connective tissue disease (CTD). The aim of this study was to evaluate the behaviour of ET-1 and brain natriuretic peptide (BNP) venous plasma levels during a 6-month dual ET-1 receptor blockade and the potential influence of baseline ET-1 venous plasma levels on the clinical efficacy of bosentan.

METHODS

Twenty-five patients with PAH (idiopathic n=16, CTD n=9) in WHO functional class II-III were included in this study. After initial evaluation, patients' WHO class, 6-minute walking-test (6MWT), ET-1 and BNP venous plasma levels were assessed at baseline and after 6-month bosentan therapy. To evaluate whether the ET-1 levels could influence the clinical response to bosentan, data were analyzed for the whole population which was stratified according to high and low ET-1 plasma levels (on the basis of the baseline median value of ET-1 plasma: Gr.1<18.7 pg/ml, Gr.2>18.7 pg/ml).

RESULTS

Study population included patients with moderate-severe PAH. After 6-month of treatment we observed a significant increase in 6MWT distance (from 435+/-85) m to 467+/-77 m, p>0.001) and an improvement in WHO class (from 2.4+/-0.5 to 2+/-0.6 p>0.01), with a significant decrease in BNP (from 87+/-33 pg/ml to 67+/-41 pg/ml, p=0.006) and a trend towards lower ET-1 plasma levels (from 17.7+/-5 pg/ml to 16+/-6 pg/ml, p=ns). Improvement in effort tolerance (Delta distance) was not correlated to modification in ET-1 (DeltaET-1) and BNP (DeltaBNP) plasma levels, while we found a significant correlation between DeltaET-1 and DeltaBNP (r=0.63, p=0.0006). Analyzing the subpopulation, Gr.2 patients were older (Gr.1: 41+/-10 years vs Gr.2: 50+/-9 years, p=0.04), had less effort capacity (6MWT distance, Gr.1: 469+/-76 m, vs Gr.2: 398+/-82 m, p=0.03), and showed a trend towards higher BNP values (Gr.1: 82+/-41 pg/ml vs Gr.2: 92+/-23 pg/ml, p=0.051), but no significant differences in pulmonary hemodynamics. After the 6-month treatment both groups showed a significant improvement in 6MWT (Gr.1: +32+/-24 m, Gr.2: +32+/-21 m p=0.05) without differences between groups. WHO class had a trend towards lower class (Gr.1: -0.5+/-0.5, Gr.2: -0.3+/-0.4 p=0.15) in both groups. BNP plasma levels showed a significant decrease only in Gr.2 (Gr.1: -6+/-41 pg/ml, Gr.2: -34+/-19 pg/ml p=0.02); similarly ET-1 plasma levels showed a trend towards a decrease only in Gr.2 (Gr.1: 0.2+/-4.6 pg/ml, Gr.2: -3.8+/-6.6 pg/ml p=0.09).

CONCLUSIONS

Our data confirm that bosentan is an effective therapy for patients with PAH. Its clinical efficacy (effort tolerance and NYHA) seems to be independent from baseline venous ET1 plasma levels. Bosentan therapy seems to elicit different patterns in ET-1 and BNP plasma levels, with decrease of the peptides only in patients with higher activation of the systemic endothelin system. Further studies are warranted to explore the potential impact of baseline ET-1 levels on the long-term effects (clinical worsening) of bosentan therapy.

We have developed a sensitive and selective radioimmunoassay for porcine/human endothelin (ET1). The assay has a detection limit of 0.62 pg/tube and exhibits no cross-reactivity to atrial natriuretic peptide, arginine vasopressin, or angiotensin II. Procedures were developed for extraction of endothelin from human plasma samples and samples of buffer from endothelial cell incubations using C18 Sep-Pak extraction cartridges. The mean recovery following extraction was approximately 80%. Both bovine and porcine aortic endothelial cells were found to produce immunoreactive endothelin (IR-ET) with porcine cells producing 4.7 +/- 1.1 ng of IR-ET/mg cell protein after 6 hours. Human plasma samples were extracted, assayed and found to contain a mean concentration of 2.0 +/- 0.4 pg/ml of IR-ET.

1H NMR studies on the nonselective endothelin receptor agonist sarafotoxin SRTb have identified a helix between residues Asp 8 and His 16, and a beta-turn involving residues Cys 3 to Met 6; however, the biologically important C-terminal five residues were found to be conformationally variable. The average RMSD, measured for the final 43 refined structures to the average structure over residues 1-16, was 0.78 +/- 0.18 A for the backbone atoms and 1.39 +/- 0.22 A for all atoms. The torsion angles Cys 3 psi/Lys 4 theta, Thr 7 psi/Asp 8 theta and Gln 17 theta were identified as sites of conformational variability. Differences were found between the structures in the bicyclic loop region for SRTb and those published for ET1, another nonselective receptor agonist, which may explain the observed differences in potency of these peptides. The conformation of an ETB receptor-specific agonist, IRL 1620, which lacks the N-terminal seven residues and the two intrachain disulfides, was found by NMR and circular dichroism spectroscopy to be predominantly random coil, despite the fact that its affinity for the ETB receptor almost equals that of ET1. However, close analysis of the NMR results indicated the presence of turn-like structures, or a nascent helix, in the part of the sequence corresponding to the helical region in the parent peptides. These results suggest that the helical conformation may be required for ligand binding to the ETB receptor as well as to the ETA receptor.

Streptococcus pneumoniae serotype 5 is the third most common capsular type causing invasive diseases in children younger than 5 years in Latin America. Preliminary data on Colombian serotype 5 isolates indicated a common clonal origin associated with resistance to tetracycline (TET) and chloramphenicol (CHL). We studied 172 S. pneumoniae serotype 5 invasive isolates from Argentina, Brazil, Colombia, Guatemala, Mexico, and Uruguay and confirmed the presence of the Colombia(5)-19 clone throughout Latin America. Fifteen subtypes of a pulsed-field gel electrophoresis pattern and 4 electrophoretic types (ET) were obtained. Most of the isolates from different geographical regions belonged to pattern A (34.3%), subtype A5 (41.9%), and ET1 (91.1%). The A pattern (n = 59) was resistant to TET and had variable resistance to CHL; it was present in Brazil (10.2%), Colombia (78%), Guatemala (8.5%), and Mexico (3.4%). Subtype A5 with variable susceptibility to TET and sensitive to CHL was found in Argentina (29.2%), Mexico (8.3%), and Uruguay (62.5%). Subtypes A1-A4, A7-A8, and A9-A11 (closely related to A) also shared ET1, while subtype A6 was assigned to ET1, ET2, and ET3. Eleven subtypes (n = 21) were found to be specific for one country each. In summary, the S. pneumoniae serotype 5 isolates from Latin American are genetically closely related but show different patterns of antibiotic resistance, probably as a result of horizontal transfer.

An ethanol-tolerant mutant, ET1, was isolated by an enrichment method from Escherichia coli JM109. Strains JM109 and ET1 were transformed with expression vector pZY507bc containing Zymomonas mobilis alcohol dehydrogenase II (adhB) and pyruvate decarboxylase (pdc) genes, resulting in an ethanol-sensitive recombinant strain JMbc and an ethanol-tolerant recombinant strain, ET1bc. Alcohol dehydrogenase and pyruvate decarboxylase activities were 24 and 32% lower, respectively, in JMbc than in ET1bc. ET1bc fermented 10% (w/v) xylose to give 39.4 g ethanol/l (77%, theoretical yield), a 1.3-fold increase compared with the ethanol-sensitive strain JMbc.

OBJECTIVE

Endothelin 1 (ET1) is one of a newly discovered family of potent naturally occurring vasoconstrictors produced by the endothelium. A few publications indicated that the peptide may have a role in idiopathic Raynaud's phenomenon and Raynaud's phenomenon secondary to connective tissue disease. The aim of this study was to compare serum endothelin concentrations in people with vibration induced white finger (VWF) with those of controls exposed to vibration, and unexposed (pure) controls.

METHODS

Male volunteers from a stonemasonry, two quarries, and an insurance company were classified by questionnaire and clinical examination into men with VWF (cases, n = 31), exposed controls (n = 22), or pure controls (n = 36). All subjects were asked to provide two venous blood specimens: a baseline sample after a period of warm equilibration (30 minutes seated in a warm room and 20 minutes with both hands immersed in a water bath at 37 degrees C); and again after cold challenge (both hands immersed in a water bath at 6 degrees C for six minutes). Serum concentrations of the 21 amino acid peptide endothelin ET1-21 were measured by radioimmunoassay.

RESULTS

Baseline concentrations of ET1-21 were found to be lower in cases (mean = 12.2 pmol/l) than in the two control groups (mean = 14.7 pmol/l in exposed controls; mean = 14.3 pmol/l in pure controls). Among cases there was a broad inverse relation between severity, as measured by the Griffin blanching score, and baseline ET1-21 (Spearman rank correlation coefficient -0.58, P < 0.001). Cold challenge provoked an overall rise in ET1-21 in all groups, but larger and significant mean absolute and percentage rises were found in cases (4.1 pmol/l and 54%) than in the control groups (2.6 pmol/l and 21% in exposed controls; 1.5 pmol/l and 20% in pure controls). Similar but more obvious differences occurred when controls were compared with those cases who gave a more severe history of disease (Griffin blanching score>> or = 24) and those cases found to blanch after cold challenge. In these case subsets baseline ET1-21 was nearly 50% lower than for controls and a four and a half to fivefold greater percentage rise in ET1-21 occurred upon cold challenge. Differences were significant. Close matching for age and smoking did not alter the principal findings. No significant differences, whether in baseline or cold response, were found between unexposed and exposed controls.

CONCLUSIONS

Baseline findings seem to contradict various published series and attempts are made to reconcile the differences. It is suggested that a lower baseline ET1-21 in cases may result from a disease compensation mechanism or damage effect. The large relative rise in serum ET1-21 when cases are cold challenged may contribute directly or indirectly to vasospasm, but a simple mechanism is unlikely and interpretation is limited by the absence of measurements of forearm blood flow.

OBJECTIVE

To analyze the relationship between QTc interval and cardiovascular risk factors in women with polycystic ovary syndrome (PCOS).

METHODS

Study group included 119 PCOS women (age: 32.2+/-5.2 years) and the control group 64 age-matched healthy women; they all underwent QT interval measurement, and plasma levels of high-sensitivity CRP (hsCRP), endothelin-1 (ET1), insulin, and testosterone determinations.

RESULTS

In PCOS women hsCRP (2.35+/-2.14 mg/L vs. 1.01+/-1.28 mg/L; P=0.04), ET1 (23.6+/-10.3 ng/L vs. 7.7+/-15.9 ng/L; P=0.01), and insulin (16.5+/-7.8 mIU/L vs. 11.8+/-10.7 mIU/L; P=0.03) levels were significantly higher, and QTc interval significantly shorter than in controls (401+/-61 ms vs. 467+/-61 ms; P=0.007). In 67 (56%) PCOS patients with a short QTc interval (<400 ms), plasma testosterone levels were significantly higher than in PCOS women with normal QTc interval (2.3+/-2.1 nmol/L vs. 1.4+/-1.7 nmol/L; P=0.02).

CONCLUSIONS

In patients with polycystic ovary syndrome increased testosterone levels may attenuate the effects of coronary risk factors.