One hundred and seventy-five isolates of the pathogenic bacterium Listeria monocytogenes recovered from human clinical (blood and cerebrospinal fluid), animal, and environmental sources in Europe, North America, and elsewhere were analyzed electrophoretically for allelic variation at 16 genetic loci encoding metabolic enzymes. Forty-five distinctive allele profiles (electrophoretic types, ETs) were distinguished, among which mean genetic diversity per locus (H) was 0.424. Cluster analysis of a matrix of genetic distances between paired ETs revealed two primary phylogenetic divisions of the species separated at a distance of 0.54. ETs in division I were presented by strains of serotypes 4b, 1/2b, and 4a, whereas strains of ETs in division II were of serotypes 1/2a and 1/2c. Human and animal isolates did not represent distinctive subsets of ETs. The occurrence of linkage disequilibrium between enzyme loci and the widespread distribution of certain ETs indicate that the genetic structure of L. monocytogenes is clonal. One clone, marked by ET1, caused major epidemics of human disease in western Switzerland in the period 1983-1987 and in Los Angeles County, California, in 1985, both of which were attributed to contamination of soft cheese. ET 1 is closely related to the clone (ET7) that caused two large outbreaks of listeriosis in Massachusetts in 1979 and 1983.

Activation of autocrine and paracrine signalling by endothelin 1 (ET1) binding to its receptors elicits pleiotropic effects on tumour cells and on the host microenvironment. This activation modulates cell proliferation, apoptosis, migration, epithelial-to-mesenchymal transition, chemoresistance and neovascularization, thus providing a strong rationale for targeting ET1 receptors in cancer. In this Review, we discuss the advances in our understanding of the diverse biological roles of ET1 in cancer and describe the latest preclinical and clinical progress that has been made using small-molecule antagonists of ET1 receptors that inhibit ET1-driven signalling.

Increased cyclic GMP from enhanced synthesis or suppressed catabolism (e.g. PDE5 inhibition by sildenafil, SIL) activates protein kinase G (PKG) and blunts cardiac pathological hypertrophy. Suppressed calcineurin (Cn)-NFAT (nuclear factor of activated T-cells) signaling appears to be involved, though it remains unclear how this is achieved. One potential mechanism involves activation of Cn/NFAT by calcium entering via transient receptor potential canonical (TRPC) channels (notably TRPC6). Here, we tested the hypothesis that PKG blocks Cn/NFAT activation by modifying and thus inhibiting TRPC6 current to break the positive feedback loop involving NFAT and NFAT-dependent TRPC6 upregulation. TRPC6 expression rose with pressure-overload in vivo, and angiotensin (ATII) or endothelin (ET1) stimulation in neonatal and adult cardiomyocytes in vitro. 8Br-cGMP and SIL reduced ET1-stimulated TRPC6 expression and NFAT dephosphorylation (activity). TRPC6 upregulation was absent if its promoter was mutated with non-functional NFAT binding sites, whereas constitutively active NFAT triggered TRPC6 expression that was not inhibited by SIL. PKG phosphorylated TRPC6, and both T70 and S322 were targeted. Both sites were functionally relevant, as 8Br-cGMP strongly suppressed current in wild-type TRPC6 channels, but not in those with phospho-silencing mutations (T70A, S322A or S322Q). NFAT activation and increased protein synthesis stimulated by ATII or ET1 was blocked by 8Br-cGMP or SIL. However, transfection with T70A or S322Q TRPC6 mutants blocked this inhibitory effect, whereas phospho-mimetic mutants (T70E, S322E, and both combined) suppressed NFAT activation. Thus PDE5-inhibition blocks TRPC6 channel activation and associated Cn/NFAT activation signaling by PKG-dependent channel phosphorylation.

The Caenorhabditis elegans mutation e873, which results in a recessive uncoordinated phenotype (formerly named Unc-72) and which had been isolated after 32P treatment (Brenner 1974), has now been found to act as a crossover suppressor and to be associated with a translocation between linkage groups (LG's) III and V. The translocation has been named, eT1(III; V); eT1 acts as a dominant crossover suppressor for both the right half of LGIII and the left half of LGV, providing a balancer for a total of 39 map units. The uncoordinated e873 phenotype has been shown to be a consequence of an inactive unc-36III gene. It was possible to demonstrate that, in translocation heterozygotes, eT1 chromosomes marked with either sma-3 or dpy-11 segregate from normal LGIII, while those marked with bli-5, sma-2 or unc-42 segregate from normal LGV. Since bli-5 and sma-2 are normally on LGIII, and dpy-11 is normally on LGV, it is concluded that: (a) eT1 is a reciprocal translocation; (b) there is a breakpoint between sma-3 and sma-2 in LGIII (the region containing unc-36) and one between dpy-11 and unc-42 in LGV; (c) there is no dominant centromere between sma-2 and bli-5 on LGIII, since in eT1 these genes are not linked to a LGIII centromere. Similarly, it is highly unlikely that there is a centromere to the left of dpy-11 on LGV. The new gene order in eT1 was determined by measuring recombination rates between markers in eT1 homozygotes. It is concluded that the new order is: dpy-1 sma-3 (break) dpy-11 unc-60, and bli-5 sma-2 (break) unc-42 unc-51.--This is the first analysis of a C. elegans translocation with respect to reciprocity, breakpoints and new gene order.

Protein kinase D (PKD) is a serine/threonine kinase with emerging myocardial functions; in skinned adult rat ventricular myocytes (ARVMs), recombinant PKD catalytic domain phosphorylates cardiac troponin I at Ser22/Ser23 and reduces myofilament Ca(2+) sensitivity. We used adenoviral gene transfer to determine the effects of full-length PKD on protein phosphorylation, sarcomere shortening and [Ca(2+)](i) transients in intact ARVMs. In myocytes transduced to express wild-type PKD, the heterologously expressed enzyme was activated by endothelin 1 (ET1) (5 nmol/L), as reflected by PKD phosphorylation at Ser744/Ser748 (PKC phosphorylation sites) and Ser916 (autophosphorylation site). The ET1-induced increase in cellular PKD activity was accompanied by increased cardiac troponin I phosphorylation at Ser22/Ser23; this measured approximately 60% of that induced by isoproterenol (10 nmol/L), which activates cAMP-dependent protein kinase (PKA) but not PKD. Phosphorylation of other PKA targets, such as phospholamban at Ser16, phospholemman at Ser68 and cardiac myosin-binding protein C at Ser282, was unaltered. Furthermore, heterologous PKD expression had no effect on isoproterenol-induced phosphorylation of these proteins, or on isoproterenol-induced increases in sarcomere shortening and relaxation rate and [Ca(2+)](i) transient amplitude. In contrast, heterologous PKD expression suppressed the positive inotropic effect of ET1 seen in control cells, without altering ET1-induced increases in relaxation rate and [Ca(2+)](i) transient amplitude. Complementary experiments in "skinned" myocytes confirmed reduced myofilament Ca(2+) sensitivity by ET1-induced activation of heterologously expressed PKD. We conclude that increased myocardial PKD activity induces cardiac troponin I phosphorylation at Ser22/Ser23 and reduces myofilament Ca(2+) sensitivity, suggesting that altered PKD activity in disease may impact on contractile function.

The human intestine is colonised with highly diverse and individually defined microbiota, which likely has an impact on the host well-being. Drivers of the individual variation in the microbiota compositions are multifactorial and include environmental, host and dietary factors. We studied the impact of the host secretor status, encoded by fucosyltransferase 2 (FUT2) -gene, on the intestinal microbiota composition. Secretor status determines the expression of the ABH and Lewis histo-blood group antigens in the intestinal mucosa. The study population was comprised of 14 non-secretor (FUT2 rs601338 genotype AA) and 57 secretor (genotypes GG and AG) adult individuals of western European descent. Intestinal microbiota was analyzed by PCR-DGGE and for a subset of 12 non-secretor subjects and 12 secretor subjects additionally by the 16S rRNA gene pyrosequencing and the HITChip phylogenetic microarray analysis. All three methods showed distinct clustering of the intestinal microbiota and significant differences in abundances of several taxa representing dominant microbiota between the non-secretors and the secretors as well as between the FUT2 genotypes. In addition, the non-secretors had lower species richness than the secretors. The soft clustering of microbiota into enterotypes (ET) 1 and 3 showed that the non-secretors had a higher probability of belonging to ET1 and the secretors to ET3. Our study shows that secretor status and FUT2 polymorphism are associated with the composition of human intestinal microbiota, and appears thus to be one of the key drivers affecting the individual variation of human intestinal microbiota.

OBJECTIVE

The aim of this study was to evaluate echocardiography-based indices of myocardial function and markers of vascular inflammation and endothelial dysfunction in the early phases of severe sepsis.

METHODS

Forty-five adult patients (67% women; age 51 ± 18 years; Acute Physiology and Chronic Health Disease Classification System II score, 23 ± 7) admitted to the intensive care unit up to 24 hours after fulfilling criteria for severe sepsis or septic shock were studied. Clinical, laboratorial (endothelin 1 [ET1], vascular cellular adhesion molecule 1), and echocardiographic data were collected within the first 24 hours and again 72 hours and 7 days after admission.

RESULTS

Intrahospital mortality was 33% (15 deaths). Left ventricular (LV) dysfunction (LV ejection fraction <55%) was identified in 15 (33%) patients, whereas right ventricular (RV) dysfunction (RV tissue Doppler peak systolic velocity [RV-Sm] <12 cm/s) was present in 14 (30%) patients. LogET1 was increased in patients with LV dysfunction (2.3 ± 0.6 vs 1.8 ± 0.4 pg/mL; P = .01) and RV dysfunction (2.5 ± 0.5 vs 1.8 ± 0.4 pg/mL; P < .001) and had negative correlations with LV ejection fraction (r = -0.50; P = .002) and RV-Sm (r = -0.67; P < .001). Left ventricular end-diastolic diameter, RV-Sm, and diastolic dysfunction were able to discriminate survivors from nonsurvivors, and the combination of these parameters identified groups of very low and high risk.

CONCLUSIONS

Both LV and RV systolic dysfunctions are prevalent in severe sepsis, being directly associated with markers of endothelial dysfunction. Left ventricular nondilation, RV dysfunction, and diastolic dysfunction seem related to poor prognosis in this scenario.

We previously established a gamma-ray dose-response curve for recessive lethal events (lethals) captured over the eT1 balancer. In this paper we analyze the nature of lethal events produced, with a frequency of 0.04 per eT1 region, at a dose of 1500 r. To do so, we developed a protocol that, in the absence of cytogenetics, allows balanced lethals to be analyzed for associated chromosomal rearrangements. A set of 35 lethal strains was chosen for the analysis. Although the dosage was relatively low, a large number of multiple-break events were observed. The fraction of lethals associated with rearrangements was found to be 0.76. Currently most X- and gamma-ray dosages used for mutagenesis in C. elegans are 6000-8000 r. From our data we conservatively estimated that 43% of rearrangements induced with 8000 r would be accompanied by additional chromosome breaks in the genome. With 1500 r the value was 5%. The 35 lethals studied were derived from 875 screened F1's. Among these lethals there were (1) at least two unc-36 duplications, (2) at least four translocations, (3) at least six deficiencies of chromosome V (these delete about 90% of the unc-60 to unc-42 region) and (4) several unanalyzed rearrangements. Thus, it is possible to recover desired rearrangements at reasonable rates with a dose of only 1500 r. We suggest that the levels of ionizing radiation employed in most published C. elegans studies are excessive and efforts should be made to use reduced levels in the future.

There is accumulating evidence that endothelin-1 plays an important role in vascular pathophysiology. Our objective was to examine whether molecular variations at the endothelin-1 locus were involved in susceptibility to myocardial infarction and variation in blood pressure. The entire coding sequence and 1.4 kb of the 5' flanking region were screened. Five polymorphisms were detected, which were genotyped in the ECTIM (Etude Cas-Témoin de l'Infarctus du Myocarde) Study, a multicenter study comparing 648 male patients who had survived a myocardial infarction and 760 population-based controls. The polymorphisms were not associated with myocardial infarction, nor did they contribute to blood pressure levels in the population at large. However, a G/T polymorphism predicting an Lys/Asn change (ET1/C198) strongly interacted (P<0.001) with body mass index in the determination of blood pressure levels. There was a steeper increase of blood pressure with body mass index in carriers of the T allele than in GG homozygotes. As a consequence, the T allele was associated with an increase of blood pressure in overweight subjects (body mass index>>/=26 kg/m2), while no significant effect was observed in lean subjects (body mass index <26 kg/m2). To determine whether this finding could be replicated, the ET1/C198 was genotyped in the Glasgow Heart Scan Study, a population-based study including 619 men and 663 women. Subjects homozygous for the T allele had higher resting blood pressure levels than others (P<0.05). A similar interaction between the T allele and body mass index was observed on the maximum blood pressure achieved during a treadmill exercise test (P<0.001). In conclusion, results from 2 independent studies suggest that the ET1/C198 polymorphism is associated with blood pressure levels in overweight people.

From 10,900 F1 progeny of ethyl methanesulfonate (EMS)-mutagenized Caenorhabditis elegans nematodes, we isolated 194 lethal mutations on the left arm of LGV, a region balanced by the reciprocal translocation of eT1. The analysis of 166 of those mutations resulted in the identification of one deficiency and alleles of 78 genes including 38 new genes, thus increasing the number of identified essential genes to 101. We estimate that there are a minimum of 120 essential genes in this region, which comprises approximately 7% of the recombinational distance, although only about 4.2% of the genes, in C. elegans. We calculate that there are a minimum of 2850 essential genes in the genome. The left arm of LGV has two recombinational gene clusters separated by a high-recombination and/or essential gene-sparse region. One gene in this region, let-330, is the largest EMS target on the left arm of LGV, with twice as many alleles (16) as the next most EMS-mutable genes, let-332 and rol-3. Another gene in the sparse region, lin-40, and the region near lin-40 are major targets for Tc1 mobilization-induced mutagenesis. The analysis of essential genes in large regions should help to define C. elegans in terms of all its genes and aid in the understanding of the relationship of genome structure to genome function.

BACKGROUND

Measurement of prohormones representing different pathophysiological pathways could enhance risk stratification in patients with community-acquired pneumonia (CAP) and other lower respiratory tract infections (LRTI).

METHODS

We assessed clinical parameters and five biomarkers, the precursor levels of adrenomedullin (ADM), endothelin-1 (ET1), atrial-natriuretic peptide (ANP), anti-diuretic hormone (copeptin), and procalcitonin in patients with LRTI and CAP enrolled in the multicenter ProHOSP study. We compared the prognostic accuracy of these biomarkers with the pneumonia severity index (PSI) and CURB65 (Confusion, Urea, Respiratory rate, Blood pressure, Age 65) score to predict serious complications defined as death, ICU admission and disease-specific complications using receiver operating curves (ROC) and reclassification methods.

RESULTS

During the 30 days of follow-up, 134 serious complications occurred in 925 (14.5%) patients with CAP. Both PSI and CURB65 overestimated the observed mortality (X2 goodness of fit test: P = 0.003 and 0.01). ProADM or proET1 alone had stronger discriminatory powers than the PSI or CURB65 score or any of either score components to predict serious complications. Adding proADM alone (or all five biomarkers jointly) to the PSI and CURB65 scores, significantly increased the area under the curve (AUC) for PSI from 0.69 to 0.75, and for CURB65 from 0.66 to 0.73 (P < 0.001, for both scores). Reclassification methods also established highly significant improvement (P < 0.001) for models with biomarkers if clinical covariates were more flexibly adjusted for. The developed prediction models with biomarkers extrapolated well if evaluated in 434 patients with non-CAP LRTIs.

CONCLUSIONS

Five biomarkers from distinct biologic pathways were strong and specific predictors for short-term adverse outcome and improved clinical risk scores in CAP and non-pneumonic LRTI. Intervention studies are warranted to show whether an improved risk prognostication with biomarkers translates into a better clinical management and superior allocation of health care resources.

BACKGROUND

NCT00350987.

BACKGROUND

Endothelin-1 (ET1) is dysregulated in pulmonary hypertension (PH). It may be important in the pathobiology of congenital diaphragmatic hernia (CDH).

OBJECTIVE

We hypothesized that ET1 levels in the first month would be higher in infants with CDH who subsequently expired or were discharged on oxygen (poor outcome). We further hypothesized that ET1 levels would be associated with concurrent severity of PH.

METHODS

We sampled plasma at 24 to 48 hours, and 1, 2, and 4 weeks of age in 40 prospectively enrolled newborns with CDH. We performed echocardiograms to estimate pulmonary artery pressure at less than 48 hours of age and weekly to 4 weeks. PH was classified in relationship to systemic blood pressure (SBP): less than 2/3 SBP, 2/3 SBP-systemic is related to pressure, or systemic-to-suprasystemic pressure.

RESULTS

ET1 levels at 1 and 2 weeks were higher in infants with poor outcome compared with infants discharged on room air (median and interquartile range: 27.2 [22.6, 33.7] vs. 19.1 [16.1, 29.5] pg/ml, P = 0.03; and 24.9 [17.6, 39.5] vs. 17.4 [13.7, 21.8] pg/ml, P = 0.01 at 1 and 2 weeks, respectively). Severity of PH was significantly associated with increasing ET1 levels at 2 weeks (16.1 [13.7, 21.8], 21.0 [17.4, 31.1], and 23.6 [21.9, 39.5] pg/ml for increasing PH class, P = 0.03). Increasing severity of PH was also associated with poor outcome at that time (P = 0.001).

CONCLUSIONS

Infants with CDH and poor outcome have higher plasma ET1 levels and severity of PH than infants discharged on room air. Severity of PH is associated with ET1 levels.

The complete genome of the bacterium Erwinia tasmaniensis strain Et1/99 consisting of a 3.9 Mb circular chromosome and five plasmids was sequenced. Strain Et1/99 represents an epiphytic plant bacterium related to Erwinia amylovora and E. pyrifoliae, which are responsible for the important plant diseases fire blight and Asian pear shoot blight, respectively. Strain Et1/99 is a non-pathogenic bacterium and is thought to compete with these and other bacteria when occupying the same habitat during initial colonization. Genome analysis revealed tools for colonization, cellular communication and defence modulation, as well as genes coding for the synthesis of levan and a not detected capsular exopolysaccharide. Strain Et1/99 may secrete indole-3-acetic acid to increase availability of nutrients provided on plant surfaces. These nutrients are subsequently accessed and metabolized. Secretion systems include the hypersensitive response type III pathway present in many pathogens. Differences or missing parts within the virulence-related factors distinguish strain Et1/99 from pathogens such as Pectobacterium atrosepticum and the related Erwinia spp. Strain Et1/99 completely lacks the sorbitol operon, which may also affect its inability to invade fire blight host plants. Erwinia amylovora in contrast depends for virulence on utilization of sorbitol, the dominant carbohydrate in rosaceous plants. The presence of other virulence-associated factors in strain Et1/99 indicates the ancestral genomic background of many plant-associated bacteria.

In myocardium, the 90-kDa ribosomal S6 kinase (RSK) is activated by diverse stimuli and regulates the sarcolemmal Na(+)/H(+) exchanger through direct phosphorylation. Only limited information is available on other cardiac RSK substrates and functions. We evaluated cardiac myosin-binding protein C (cMyBP-C), a sarcomeric regulatory phosphoprotein, as a potential RSK substrate. In rat ventricular myocytes, RSK activation by endothelin 1 (ET1) increased cMyBP-C phosphorylation at Ser(282), which was inhibited by the selective RSK inhibitor D1870. Neither ET1 nor D1870 affected the phosphorylation status of Ser(273) or Ser(302), cMyBP-C residues additionally targeted by cAMP-dependent protein kinase (PKA). Complementary genetic gain- and loss-of-function experiments, through the adenoviral expression of wild-type or kinase-inactive RSK isoforms, confirmed RSK-mediated phosphorylation of cMyBP-C at Ser(282). Kinase assays utilizing as substrate wild-type or mutated (S273A, S282A, S302A) recombinant cMyBP-C fragments revealed direct and selective Ser(282) phosphorylation by RSK. Immunolabeling with a Ser(P)(282) antibody and confocal fluorescence microscopy showed RSK-mediated phosphorylation of cMyBP-C across the C-zones of sarcomeric A-bands. In chemically permeabilized mouse ventricular muscles, active RSK again induced selective Ser(282) phosphorylation in cMyBP-C, accompanied by significant reduction in Ca(2+) sensitivity of force development and significant acceleration of cross-bridge cycle kinetics, independently of troponin I phosphorylation at Ser(22)/Ser(23). The magnitudes of these RSK-induced changes were comparable with those induced by PKA, which phosphorylated cMyBP-C additionally at Ser(273) and Ser(302). We conclude that Ser(282) in cMyBP-C is a novel cardiac RSK substrate and its selective phosphorylation appears to regulate cardiac myofilament function.

BACKGROUND

The genus Erwinia includes plant-associated pathogenic and non-pathogenic Enterobacteria. Important pathogens such as Erwinia amylovora, the causative agent of fire blight and E. pyrifoliae causing bacterial shoot blight of pear in Asia belong to this genus. The species E. tasmaniensis and E. billingiae are epiphytic bacteria and may represent antagonists for biocontrol of fire blight. The presence of genes that are putatively involved in virulence in E. amylovora and E. pyrifoliae is of special interest for these species in consequence.

RESULTS

Here we provide the complete genome sequences of the pathogenic E. pyrifoliae strain Ep1/96 with a size of 4.1 Mb and of the non-pathogenic species E. billingiae strain Eb661 with a size of 5.4 Mb, de novo determined by conventional Sanger sequencing and next generation sequencing techniques. Genome comparison reveals large inversions resulting from homologous recombination events. Furthermore, comparison of deduced proteins highlights a relation of E. billingiae strain Eb661 to E. tasmaniensis strain Et1/99 and a distance to E. pyrifoliae for the overall gene content as well as for the presence of encoded proteins representing virulence factors for the pathogenic species. Pathogenicity of E. pyrifoliae is supposed to have evolved by accumulation of potential virulence factors. E. pyrifoliae carries factors for type III secretion and cell invasion. Other genes described as virulence factors for E. amylovora are involved in the production of exopolysaccharides, the utilization of plant metabolites such as sorbitol and sucrose. Some virulence-associated genes of the pathogenic species are present in E. tasmaniensis but mostly absent in E. billingiae.

CONCLUSIONS

The data of the genome analyses correspond to the pathogenic lifestyle of E. pyrifoliae and underlines the epiphytic localization of E. tasmaniensis and E. billingiae as a saprophyte.

Men with erectile dysfunction (ED) frequently have a disproportionate burden of comorbid vascular disorders including atherosclerotic disease. We investigated whether scheduled tadalafil is better than on-demand (OD) in improving endothelium-dependent vasodilatation of cavernous arteries in men with ED and whether this effect is also exerted on markers of endothelial function. We did an open-label, randomized, crossover study including 20 male outclinic patients aged 18 years or older (mean age 54 years) who had at least a 3-month history of ED of any severity or etiology. Tadalafil (20 mg) on alternate days (ADs) or OD was administered for 4 weeks. Primary end points were variations of basal inflow (peak systolic velocity (PSV)) and flow-mediated dilatation (FMD) of cavernous arteries compared with baseline at penile Duplex ultrasound. Secondary end points were variations of Q13-SIEDY scores regarding morning erections and of markers of endothelial function, that is, vascular cell adhesion molecule (VCAM), intercellular cell adhesion molecule, endothelin-1 (ET-1), insulin and C-reactive protein (CRP). PSVs and FMD were higher after AD treatment when compared with OD and baseline, respectively (P=0.0001), and improvements were maintained from 2 weeks after discontinuation (P<0.005). Patients receiving tadalafil AD experienced a significant improvement of morning erections as compared to AD treatment (P<0.0001); ET1, VCAM and CRP showed a robust decrease after chronic vs OD regimes (P<0.05), with concomitant increase in insulin levels (P<0.05), without any variation in blood pressure and other laboratory parameters. Chronic but not OD tadalafil improves endothelial function with sustained effects from its discontinuation. Chronic treatment also produces a dramatic increase in morning erections, which determines better oxygenation to the penis, thus providing a rationale for vascular rehabilitation.

The vasoactive peptide endothelin 1 (ET1) is encoded by a well characterized gene located on human chromosome 6. Recently, two human genomic fragments were isolated which potentially encode related vasoconstrictor peptides, endothelin 2 (ET2) and endothelin 3 (ET3) (Inoue, A., Yanagisawa, M., Kimura, S., Kasuya, Y., Miyauchi, T., Goto, K., and Masaki, T. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2863-2867). Inoue et al. were unable to detect transcripts of the ET2 and ET3 genes and observed ET1 gene expression exclusively in endothelial cells. In this study, we document transcription of the ET3 gene by isolating from a hypothalamic cDNA library DNA clones complementary to human ET3 mRNA. ET3 mRNA encodes a 238-amino acid precursor that includes ET3 and a 15-amino acid homologous segment, the ET3-like sequence. On the basis of DNA isolated from human-mouse somatic hybrid cell lines, we assigned the ET3 gene to human chromosome 20. The ET3 and ET1 genes are, therefore, not genetically linked. RNA blot hybridization with restriction fragments derived from cDNAs revealed that the ET3 and ET1 genes are both expressed in lung, pancreas, and spleen. Cultured endothelial cells and cardiac tissues express the ET1 but not the ET3 gene. Observations that genes encoding endothelin-related peptides are expressed in a variety of human tissues suggest that these peptides may participate in complex vasoregulatory mechanisms.

To determine the organ distribution of production of the three endothelin (ET) isopeptides, we have developed three ribonuclease protection assays specific for the messenger RNAs (mRNAs) of rat ETs 1, 2, and 3.12 organs from adult Sprague-Dawley rats were examined: heart, lung, liver, spleen, kidney, stomach, small intestine, large intestine, testis, muscle, salivary gland, and brain. The mRNA for ET1 was five times more abundant in the lung than in any other organ studied, moderate expression was seen in the large intestine, and lower levels of mRNA were detected in each of the other organs examined. ET2 was expressed at high level in both large and small intestine and at low level in stomach, muscle, and heart, but ET2 mRNA could not be detected elsewhere. ET3 mRNA was found in all organs, particularly in small intestine, lung, kidney, and large intestine. Because of reports suggesting that ETs might be involved in the hypoperfusion and hypofiltration observed in postischemic kidneys, we have also studied levels of mRNA in kidneys that had previously been subjected to 25 or 45 min of clamping of the renal pedicle. At 6 h after 45 min of ischemia, ET1 mRNA increased to a peak of 421 +/- 69% (mean +/- SEM, n = 3) of that in a standard renal RNA preparation. By contrast, ET3 mRNA decreased in the postischemic organ, falling to a value of 19 +/- 2% of standard at the same time point. The effects of ischemia on ET1 and ET3 mRNAs were long-lasting, with elevation of ET1 and depression of ET3 persisting for days. ET2 mRNA remained undetectable throughout. These findings (a) support a role for ET1 in postischemic renal vascular phenomena and (b) demonstrate a situation in which the expression of ET isoforms is clearly subject to differential regulation.

BACKGROUND

Erwinia pyrifoliae is a newly described necrotrophic pathogen, which causes fire blight on Asian (Nashi) pear and is geographically restricted to Eastern Asia. Relatively little is known about its genetics compared to the closely related main fire blight pathogen E. amylovora.

RESULTS

The genome of the type strain of E. pyrifoliae strain DSM 12163T, was sequenced using both 454 and Solexa pyrosequencing and annotated. The genome contains a circular chromosome of 4.026 Mb and four small plasmids. Based on their respective role in virulence in E. amylovora or related organisms, we identified several putative virulence factors, including type III and type VI secretion systems and their effectors, flagellar genes, sorbitol metabolism, iron uptake determinants, and quorum-sensing components. A deletion in the rpoS gene covering the most conserved region of the protein was identified which may contribute to the difference in virulence/host-range compared to E. amylovora. Comparative genomics with the pome fruit epiphyte Erwinia tasmaniensis Et1/99 showed that both species are overall highly similar, although specific differences were identified, for example the presence of some phage gene-containing regions and a high number of putative genomic islands containing transposases in the E. pyrifoliae DSM 12163T genome.

CONCLUSIONS

The E. pyrifoliae genome is an important addition to the published genome of E. tasmaniensis and the unfinished genome of E. amylovora providing a foundation for re-sequencing additional strains that may shed light on the evolution of the host-range and virulence/pathogenicity of this important group of plant-associated bacteria.

A fusion protein consisting of the endothelin B (ET(B)) receptor and the enhanced green fluorescent protein (EGFP) in conjunction with Cyanin3- or fluorescein-conjugated endothelin 1 (Cy3-ET1, Fluo-ET1) was used to investigate the ligand-mediated internalization of the ET(B) receptor. The ET(B) receptor and the ET(B)/EGFP fusion protein displayed very similar pharmacological properties when expressed in Chinese hamster ovary cells. The integrity of the fusion protein was verified by low temperature PAGE analysis of the (125)I-ET1-bound ET(B) receptor and the (125)I-ET1-bound ET(B)/EGFP fusion protein. Fluorescence microscopy of Chinese hamster ovary cells expressing the ET(B)/EGFP fusion protein demonstrated strong signals at the plasma membrane. On addition of Cy3-ET1, internalization of ligand and receptor occurred within 5 min via a sucrose-sensitive (i.e., clathrin-mediated) pathway. On further incubation, ET(B)/EGFP and Cy3-ET1 fluorescences were found in the perinuclear region, colocalized with fluorescent low density lipoproteins, a marker of the late endosomal/lysosomal pathway, but not with fluorescent transferrin, a marker of the recycling pathway. No dissociation of Cy3-ET1 from the receptor was seen within 4 h. Using (125)I-ET1 or Cy3-ET1, binding sites were again demonstrable at the cell surface within 2 h. The reappearance of binding sites was abolished by prior treatment of the cells with cycloheximide, an inhibitor of protein synthesis. The data demonstrate that the ligand-occupied ET(B) receptor is internalized; however, it does not recycle like most of the G protein-coupled receptors but is sorted to the late endosomal/lysosomal pathway in a manner similar to that of the family of protease-activated receptors.

A strong negative correlation between polyphenols consumption and coronary heart disease has been extensively documented. These results prompted investigations on the mechanisms responsible for polyphenols effects in cardiovascular disease. The aim of this work was to investigate in apoE KO mice the effect of P183/1 (a mixture of cathechin, caffeic acid and resveratrol) on atherosclerosis and gene expression patterns in the vascular wall. ApoE KO mice were fed a diet supplemented with P183/1, 40 and 160 mg/kg body weight/day for 8 weeks. The supplementation with the high dose of P183/1 significantly reduced the presence of atherosclerotic plaque by 40 and 36% in the aortic sinus and in the ascending aorta, respectively. This reduction was associated with a reduced expression of markers for macrophages, lymphocytes (both Th1 and Th2) and of MCP-1, MIP-1alpha, MIP-1beta, CCR1, CCR2 and ET1 in the vascular wall. In conclusion, P183/1 supplementation significantly decreases atherosclerosis in ApoE KO mice by affecting inflammatory cells recruitment and expression of pro-inflammatory chemokines in the vascular wall.

The endothelin peptides constitute a family of potent vasoconstrictor molecules. Endothelin-1 (ET1) is secreted by vascular endothelial cells and may have a role in the regulation of vascular tone. To better understand the function of ET1, we have investigated the transcriptional regulation of the ET1 gene. Utilizing reporter gene transfection experiments, we have previously identified two promoter regions, located at base pairs -148 to -117 (Region A) and -117 to -98 (Region B) of the ET1 gene. Both regions are necessary for high level ET1 transcription in endothelial cells. A nuclear protein binding to the GATA motif in Region A has been identified and proven to be necessary for expression of the ET1 gene. However, the cis-acting sequences and their cognate binding proteins for Region B have not been investigated. To identify protein binding motifs in Region B we performed DNase I footprinting and gel mobility shift assays using a DNA fragment encoding base pairs -204 to -94 of the ET1 gene. Results from these studies indicated that the AP1 consensus sequence (GTGACTAA) in Region B as the only protein-binding motif. Site-directed mutagenesis of the ET1 AP1 site resulted in a 30-fold reduction in promoter activity, establishing the functional significance of this sequence. Additional experiments investigated the role of Jun and Fos in ET1 transcription. By employing antisera to Jun and Fos in gel mobility shift assays, both of these proteins were identified as endothelial cell nuclear proteins binding to the ET1 AP1 sequence. In trans-activation experiments, we showed that cotransfection of c-fos and c-jun expression plasmids markedly increased the transcription rate of chloramphenicol acetyltransferase reporter plasmids containing three synthetic ET1 AP1 sites. Taken together, these data indicate the importance of the AP1 recognition sequence, and the role of Fos and Jun proteins in the regulation of ET1 gene transcription.

Protein kinase A (PKA) is an important effector enzyme commonly activated by cAMP. The present study focuses on our finding that the vasoactive peptide endothelin-1 (ET1), whose signaling is not coupled to cAMP production, stimulates PKA in two independent cellular models. Using an in vivo assay for PKA activity, we found that ET1 stimulated PKA in HeLa cells overexpressing ET1 receptors and in aortic smooth muscle cells expressing endogenous levels of ET1 receptors. In these cell models, ET1 did not stimulate cAMP production, indicating a novel mechanism for PKA activation. The ET1-induced activation of PKA was found to be dependent on the degradation of inhibitor of kappaB, which was previously reported to bind and inhibit PKA. ET1 potently stimulated the nuclear factor-kappaB pathway, and this effect was inhibited by overexpression of the inhibitor of kappaB dominant negative mutant (IkappaBalpham) and by treatment with the proteasome inhibitor MG-132. Importantly, IkappaBalpham and MG-132 had similar inhibitory effects on ET1-induced activation of PKA without affecting G(s)-mediated activation of PKA or ET1-induced phosphorylation of mitogen-activated protein kinase. Finally, another vasoactive peptide, angiotensin II, also stimulated PKA in a cAMP-independent manner in aortic smooth muscle cells. These findings suggest that cAMP-independent activation of PKA might be a general response to vasoactive peptides.

BACKGROUND

The DLX gene family encodes for homeobox transcription factors involved in the control of morphogenesis and tissue homeostasis. Their expression can be regulated by Endothelin1 (ET1), a peptide associated with breast cancer invasive phenotype. Deregulation of DLX gene expression was found in human solid tumors and hematologic malignancies. In particular, DLX4 overexpression represents a possible prognostic marker in ovarian cancer. We have investigated the role of DLX genes in human breast cancer progression.

METHODS

MDA-MB-231 human breast carcinoma cells were grown in vitro or injected in nude mice, either subcutaneously, to mimic primary tumor growth, or intravenously, to mimic metastatic spreading. Expression of DLX2, DLX5 and DLX6 was assessed in cultured cells, either treated or not with ET1, tumors and metastases by RT-PCR. In situ hybridization was used to confirm DLX gene expression in primary tumors and in lung and bone metastases. The expression of DLX2 and DLX5 was evaluated in 408 primary human breast cancers examining the GSE1456 and GSE3494 microarray datasets. Kaplan-Meier estimates for disease-free survival were calculated for the patients grouped on the basis of DLX2/DLX5 expression.

RESULTS

Before injection, or after subcutaneous growth, MDA-MB-231 cells expressed DLX2 but neither DLX5 nor DLX6. Instead, in bone and lung metastases resulting from intravenous injection we detected expression of DLX5/6 but not of DLX2, suggesting that DLX5/6 are activated during metastasis formation, and that their expression is alternative to that of DLX2. The in vitro treatment of MDA-MB-231 cells with ET1, resulted in switch from DLX2 to DLX5 expression. By data mining in microarray datasets we found that expression of DLX2 occurred in 21.6% of patients, and was significantly correlated with prolonged disease-free survival and reduced incidence of relapse. Instead, DLX5 was expressed in a small subset of cases, 2.2% of total, displaying reduced disease-free survival and high incidence of relapse which was, however, non-significantly different from the other groups due to the small size of the DLX+ cohort. In all cases, we found mutually exclusive expression of DLX2 and DLX5.

CONCLUSIONS

Our studies indicate that DLX genes are involved in human breast cancer progression, and that DLX2 and DLX5 genes might serve as prognostic markers.