An ultralow-noise superconducting radio-frequency ion trap for frequency metrology with highly charged ions.

We present a novel ultrastable superconducting radio-frequency (RF) ion trap realized as a combination of an RF cavity and a linear Paul trap. Its RF quadrupole mode at 34.52 MHz reaches a quality factor of Q ≈ 2.3 × 105 at a temperature of 4.1 K and is used to radially confine ions in an ultralow-noise pseudopotential. This concept is expected to strongly suppress motional heating rates and related frequency shifts that limit the ultimate accuracy achieved in advanced ion traps for frequency metrology. Running with its low-vibration cryogenic cooling system, electron-beam ion trap, and deceleration beamline supplying highly charged ions (HCIs), the superconducting trap offers ideal conditions for optical frequency metrology with ionic species. We report its proof-of-principle operation as a quadrupole-mass filter with HCIs and trapping of Doppler-cooled 9Be+ Coulomb crystals.

An electron beam ion trap and source for re-acceleration of rare-isotope ion beams at TRIUMF.

Electron beam driven ionization can produce highly charged ions (HCIs) in a few well-defined charge states. Ideal conditions for this are maximally focused electron beams and an extremely clean vacuum environment. A cryogenic electron beam ion trap fulfills these prerequisites and delivers very pure HCI beams. The Canadian rare isotope facility with electron beam ion source-electron beam ion sources developed at the Max-Planck-Institut für Kernphysik (MPIK) reaches already for a 5 keV electron beam and a current of 1 A with a density in excess of 5000 A/cm2 by means of a 6 T axial magnetic field. Within the trap, the beam quickly generates a dense HCI population, tightly confined by a space-charge potential of the order of 1 keV times the ionic charge state. Emitting HCI bunches of ≈107 ions at up to 100 Hz repetition rate, the device will charge-breed rare-isotope beams with the mass-over-charge ratio required for re-acceleration at the Advanced Rare IsotopE Laboratory (ARIEL) facility at TRIUMF. We present here its design and results from commissioning runs at MPIK, including X-ray diagnostics of the electron beam and charge-breeding process, as well as ion injection and HCI-extraction measurements.

[Structure and process quality of quality-assured mamma diagnostics in Schleswig-Holstein]. / Struktur- und Prozessqualität in der qualitätsgesicherten Mammadiagnostik in Schleswig-Holstein.

PURPOSE: To demonstrate the structure and process quality of quality-assured mamma diagnostics (QuaMaDi) by means of quality indicators as defined in the European Guidelines for Quality Assurance in Breast Cancer Screening and Diagnosis and in the National Guideline on Early Detection of Cancer in Germany. Furthermore, spatial differences and changes in the chronological sequence were analyzed. MATERIALS AND METHODS: We used administrative data as documented in the time period 2006 - 2009 in QuaMaDi in Schleswig-Holstein (SH), Germany, and analyzed quality indicators as defined in the abovementioned guidelines (absolute and relative frequencies, 95 % confidence intervals). RESULTS: Each year approximately 6 % of all women age 20 or older living in SH are examined using QuaMaDi. Only minor differences regarding age and clinical data were seen between the patients in the four regions of SH. Reference values for the quality indicators are largely reached (i. e., proportion of women with breast density ACR 3 or 4 plus additional ultrasound = 96.2 %; proportion with repeated mammography = 0.2 %). Spatial differences are only minor. In the chronological sequence, quality indicators improve, if they did not reach the reference values in the beginning, or indicate a high and constant quality. CONCLUSION: With regard to those quality indicators that were computable, reference values as defined in the guidelines were reached in 9 of 12 cases. In one case the difference between the observed value and the reference values is system-immanent and in another case the difference is less than four percentage points (reference value 90 %).

Donor treatment with a PHD-inhibitor activating HIFs prevents graft injury and prolongs survival in an allogenic kidney transplant model.

Long-term survival of renal allografts depends on the chronic immune response and is probably influenced by the initial injury caused by ischemia and reperfusion. Hypoxia-inducible transcription factors (HIFs) are essential for adaptation to low oxygen. Normoxic inactivation of HIFs is regulated by oxygen-dependent hydroxylation of specific prolyl-residues by prolyl-hydroxylases (PHDs). Pharmacological inhibition of PHDs results in HIF accumulation with subsequent activation of tissue-protective genes. We examined the effect of donor treatment with a specific PHD inhibitor (FG-4497) on graft function in the Fisher-Lewis rat model of allogenic kidney transplantation (KTx). Orthotopic transplantation of the left donor kidney was performed after 24 h of cold storage. The right kidney was removed at the time of KTx (acute model) or at day 10 (chronic model). Donor animals received a single dose of FG-4497 (40 mg/kg i.v.) or vehicle 6 h before donor nephrectomy. Recipients were followed up for 10 days (acute model) or 24 weeks (chronic model). Donor preconditioning with FG-4497 resulted in HIF accumulation and induction of HIF target genes, which persisted beyond cold storage. It reduced acute renal injury (serum creatinine at day 10: 0.66 +/- 0.20 vs. 1.49 +/- 1.36 mg/dL; P < 0.05) and early mortality in the acute model and improved long-term survival of recipient animals in the chronic model (mortality at 24 weeks: 3 of 16 vs. 7 of 13 vehicle-treated animals; P < 0.05). In conclusion, pretreatment of organ donors with FG-4497 improves short- and long-term outcomes after allogenic KTx. Inhibition of PHDs appears to be an attractive strategy for organ preservation that deserves clinical evaluation.

Role of mesothelial cell-derived granulocyte colony-stimulating factor in interleukin-17-induced neutrophil accumulation in the peritoneum.

Recent studies suggest that peritoneal CD4(+) T lymphocytes may control recruitment of polymorphonuclear leukocytes (PMN) during peritonitis by an interleukin-17 (IL-17)-dependent mechanism. IL-17 and granulocyte colony-stimulating factor (G-CSF) have been proposed to form an axis that regulates PMN transmigration. Here we report on the role of G-CSF released by human peritoneal mesothelial cells (HPMCs) in IL-17A-mediated peritoneal PMN accumulation. In vitro exposure of HPMCs to IL-17A resulted in a time- and dose-dependent release of G-CSF. This effect was related to the induction of G-CSF mRNA and mediated through the nuclear factor-kappaB (NF-kappaB) pathway. The novel observation was that IL-17A-stimulated NF-kappaB activation in HPMCs followed a biphasic profile, with an early induction (45 min), followed by the return to basal levels (90 min), and a delayed induction (3 h). Tumor necrosis factor alpha synergistically amplified IL-17A-induced G-CSF production by enhanced NF-kappaB activation and through stabilization of G-CSF mRNA. Intraperitoneal (i.p.) administration of IL-17A in Balb/c mice resulted in increased local levels of G-CSF and selective PMN accumulation. Administration of anti-G-CSF blocking antibody before IL-17A injection significantly reduced the IL-17A-triggered PMN infiltration. This effect occurred despite increased i.p. levels of PMN-specific chemokines KC and macrophage inflammatory protein-2 seen in animals treated with anti-G-CSF antibody. These data demonstrate that the mesothelium-derived G-CSF plays an important role in IL-17A-induced PMN recruitment into the peritoneum.

Expression of hypoxia-inducible transcription factors in developing human and rat kidneys.

Early kidney development is associated with the coordinated branching of the renal tubular and vascular system and hypoxia has been proposed to be a major regulatory factor in this process. Under low oxygen levels, the hypoxia-inducible transcription factor (HIF) regulates the expression of genes involved in angiogenesis, erythropoiesis and glycolysis. To investigate the role of HIF in kidney development, we analyzed the temporal and spatial expression of the oxygen regulated HIF-1alpha and -2alpha subunits at different stages of rat and human kidney development. Using double-staining procedures, localization of the HIF target geneproducts vascular endothelial growth factor (VEGF) and endoglin was studied in relation to HIFalpha. In both species, we found marked nuclear expression of HIF-1alpha in medullary and cortical collecting ducts and in glomerular cells. In contrast, HIF-2alpha was expressed in interstitial and peritubular cells podocytes of the more mature glomeruli. After completion of glomerulogenesis and nephrogenesis, HIF-1alpha and -2alpha were no longer detectable. The HIF-target gene VEGF colocalized with HIF-1alpha protein in glomeruli and medullary collecting ducts. HIF-2alpha colocalized with the endothelium-associated angiogenic factor, endoglin. Both HIFalpha isoforms are activated in the developing kidney in a cell-specific and temporally controlled manner, indicating a regulatory role of oxygen tension in nephrogenesis. HIF-1alpha seems to be primarily involved in tubulogenesis and HIF-2alpha in renal vasculogenesis. Both isoforms are found in glomerulogenesis, potentially having synergistic effects.

Adenovirus-mediated overexpression and stimulation of the human angiotensin II type 2 receptor in porcine cardiac fibroblasts does not modulate proliferation, collagen I mRNA expression and ERK1/ERK2 activity, but inhibits protein tyrosine phosphatases.

The contribution of the angiotensin (Ang) II type 2 receptor (AT2R) to cardiac hypertrophy is still controversial. Here we examined the effect of overexpressing the human AT2R in cultured porcine cardiac fibroblasts (pFib) on proliferation, procollagen I mRNA expression, and - as putatively underlying signal-transduction pathways - on mitogen-activated protein kinase ERK1/ERK2 and phosphotyrosine phosphatase activities. As quantitated by 125I-(Sar1,Ile8)-Ang II binding, transduction of cardiac fibroblasts with the adenoviral AT2R expression vector led to a six- to tenfold higher AT2 than endogenous Ang II type 1 receptor (AT1R) expression. The overexpressed AT2R had the same apparent molecular mass as the endogenous AT2R in rat PC12W cells. Proliferation was not significantly lower in AT2R expressing pFib than in antisense-transduced controls (TA2) upon stimulation with Ang II (AT2R 110.5+/-4.8% vs. TA2 110.2+/-5.5%), Ang II plus the AT1R blocker Irbesartan (97.1+/-1.4% vs. 108.0+/-5.0; P=0.052) and the partial AT2R antagonist CGP42112 at the agonistic concentration of 50 nM (92.1+/-2.7% vs. 99.8+/-3.1%; P=0.053). Procollagen Ialpha2 (COL1A2) mRNA levels were quantitated by (a) northern blot analysis and (b) reverse transcriptase polymerase chain reaction. COL1A2/GAPDH (a) and COL1A2/beta-actin (b) ratios revealed no differences between AT2R-transduced fibroblasts and antisense controls when stimulated with Ang II (1 microM, 24 h) plus Irbesartan and 10 ng/ml transforming growth factor beta1. Ang II stimulation of the endogenous AT1R increased extracellular signal regulated kinase 1/2 activities. This response was reduced by Irbesartan, but PD123319 had no effect. Time course and magnitude of Ang II stimulated ERK1/ERK2 activation was identical in AT2R-transduced and control cells. Also, neither simultaneous nor Ang II pre-stimulation, suggested to induce gene expression of the MAP kinase phosphatase 1, modulated phorbol myristate acetate-stimulated ERK1/ERK2 activation in AT2R-transduced pFib, in AT2R-transduced human umbilical vein endothelial cells, and in PC12W cells. By the use of a tyrosine phosphatase assay we observed an inhibition of phosphotyrosine phosphatase activity by 30.8% (P=0.009, n=5) after 5 min Ang II stimulation of AT2R-expressing pFib. Immunoprecipitation-tyrosine phosphatase assays revealed that inhibition of phosphotyrosine phosphatase 1B, which regulates insulin signaling, contributed to this effect. In conclusion, stimulation of the overexpressed human AT2R in porcine cardiac fibroblasts inhibited tyrosine phosphatase activity but had no significant effect on fibroblast functions related to cardiac fibrosis. It is conceivable that possible antifibrotic AT2R effects are species specific and/or require the interaction between fibroblasts and cardiomyocytes, probably via paracrine factors, or mechanical load.

Activation of the cardiac renin-angiotensin system and increased myocardial collagen expression in human aortic valve disease.

OBJECTIVES: We sought to determine whether the cardiac renin-angiotensin system (RAS) is activated in human aortic valve disease depending on left ventricular function, and we analyzed the concomitant regulation of the extracellular matrix components. BACKGROUND: In animal models with pressure or volume load, activation of the cardiac RAS increases fibrosis. In human aortic valve disease, the ventricular collagen protein content is increased, but only scarce data on the activation state of the cardiac RAS and its effects on collagen and fibronectin messenger ribonucleic acid (mRNA) are available. METHODS: In left ventricular biopsies from patients with aortic valve stenosis (AS) and aortic valve regurgitation and from control subjects, we quantitated mRNAs for angiotensin-converting enzyme (ACE), chymase, transforming growth factor-beta1 (TGF-beta1), collagen I, collagen III and fibronectin by reverse-transcription polymerase chain reaction. Proteins were localized by immunohistochemistry; ACE activity was determined by high performance liquid chromatography; and TGF-beta protein by quantitative enzyme immunoassay. RESULTS: Protein, ACE and TGF-beta1 mRNA were significantly increased in patients with AS and AR (1.5- to 2.1-fold) and correlated with each other. The increase occurred also in patients with normal systolic function. Collagen I and III and fibronectin mRNAs were both upregulated about twofold in patients with AS and AR. In AS, collagen and fibronectin mRNA expression levels were positively correlated with left ventricular end-diastolic pressure and inversely with left ventricular ejection fraction (LVEF). CONCLUSIONS: In human hearts, pressure and volume overload increases cardiac ACE and TGF-beta1 in the early stages. This activation of the cardiac RAS may contribute to the observed increase in collagen I and III and fibronectin mRNA expression. The increase in extracellular matrix already exists in patients with a normal LVEF, and it increases with functional impairment.

Angiotensin II directly increases transforming growth factor beta1 and osteopontin and indirectly affects collagen mRNA expression in the human heart.

OBJECTIVES: Angiotensin II (Ang II) induces fibroblast proliferation and collagen synthesis in the myocardium, but its precise mechanisms of action in human hearts are still unknown. Therefore, we investigated whether Ang II directly affects the collagen mRNA content in the human myocardium and in isolated human cardiac fibroblasts or whether the growth factors TGFbeta-1 and osteopontin are involved in this process. METHODS AND RESULTS I: In a first set of experiments, the direct effect of Ang II on collagen I, TGFbeta-1 and osteopontin mRNA content in fresh samples of human atrial myocardium was determined by the use of a short stimulation period. After 4 h, Ang II-stimulated atrial samples gave a significantly higher expression of both TGFbeta-1 (183+/-21% of control, p<0.05) and osteopontin mRNA (275+/-58%, p<0.02) than the controls. In contrast, the expression of collagen I mRNA was unchanged (95+/-8%). Stimulation with TGFbeta-1 led to an increase in collagen I and III mRNA (127+/-10%, p<0.05; 140+/-15%, p<0.02). METHODS AND RESULTS II: In a second protocol, to assess the effects of longer stimulation periods, we determined the effects of Ang II and its potential mediator TGFbeta-1 on collagen I, III and fibronectin mRNA expression and on proliferation of cultured human cardiac fibroblasts. Ang II caused a dose-dependent stimulation of proliferation but did not affect collagen I, II or fibronectin mRNA content after 24 h. In contrast, TGFbeta-1 stimulation significantly increased collagen I and III mRNA expression (124+/-5% and 128+/-5%, p<0.002). CONCLUSIONS: In the human heart, Ang II does not directly increase collagen or fibronectin mRNA, but it does increase TGFbeta-1 and osteopontin mRNA expression. Since TGFbeta-1 induces collagen I and III mRNA in atrial samples and in isolated cardiac fibroblasts, it may represent a necessary mediator of the Ang II effects in the human heart.

Analysis and functional characterization of alternatively spliced angiotensin II type 1 and 2 receptor transcripts in the human heart.

Expression levels of angiotensin II type 1 and type 2 receptors (AT1, AT2) vary at different cardiac localizations and are regulated in cardiac diseases. Differential splicing of the 5' untranslated exons of the primary AT1 mRNA transcripts may modulate translational efficiency and thus receptor expression. We therefore searched for AT1 and AT2 mRNA splice patterns specific to chamber localization or to cardiac performance and analyzed their effect on protein expression in transfection experiments. The exon composition of the AT1 and AT2 mRNA transcripts in normal and diseased human hearts were analyzed using a reverse transcription polymerase chain reaction followed by HPLC quantitation of the amplificates. We compared atrial (n=18) and ventricular (n=28) samples and endomyocardial biopsies (n=10) from patients with normal and severely impaired cardiac function and one donor heart, which was not used for transplantation. AT1 transcripts with the exon composition 1/2/5 and 1/5 represented about 93-98% of all AT1 mRNAs; transcript 1/2/3/5 represented 8% in the atria and 2% in ventricles. Since exon 2 reduces translational efficiency in vitro, the ratios of transcripts with and without exon 2, (1/2/5+1/2/3/5) to (1/5), were compared. These were 1.24+/-0.07 in normal atria, 0.96+/-0.09 in atria from failing hearts (P<0.05), 0.68 in the left ventricle of the donor heart, and 0.58+/-0.03 in failing left ventricles. Endomyocardial biopsy specimens showed significant differences between controls and heart failure (controls 0.63+/-0.04 vs. heart failure 0.52+/-0.02, P<0.05). Of the two identified AT2 transcripts, mRNA 1/2/3 was the most abundant in the human heart (92%). Luciferase reporter gene assays were performed to test the effect of the various 5' untranslated regions (5' UTRs) on protein expression. Among the constructs which contained the AT1 promoter/AT1 5' UTRs the plasmid Ex 1/2/5 exhibited 27% lower luciferase activity than Ex 1/5 (n=24, P<0.001), and Ex 1/2/3/5 expressed only 35.9% of Ex 1/5 activity (P<0.001). Among the reporter gene plasmids with the AT2 promoter/AT2 5' UTRs the construct Ex 1/2/3 expressed a 31% lower luciferase activity than Ex 1/3 (n=20, P<0.001). In conclusion, alternative splicing may represent a mechanism of ATR regulation in vivo. In the human heart, AT1 splice patterns differ distinctly between atria and ventricles and to a lesser degree between controls and failing hearts. This may lead to differences in AT1 mRNA translation into protein in the various cardiac areas and under different pathophysiological conditions.

Characterization of two distinct mechanisms for induction of apoptosis in human vascular endothelial cells.

Tissue homeostasis is fundamentally influenced by the functional integrity and state of endothelial cells. Survival and death of endothelial cells are encountered in cardiovascular disease and may, moreover, affect and determine the development of atherosclerosis and restenosis following intracoronary therapeutical interventions. Apoptosis was studied in cultured human umbilical vein endothelial cells (HUVEC) to investigate the regulation of endothelial cell death following serum/growth factor depletion as well as incubation with actinomycin-D. Apoptosis was verified by DNA fragmentation and quantified by fluorescence activated cell sorting (FACS) analysis after TdT-mediated deoxyuridine-triphosphate nick end-labeling (TUNEL). An ELISA was used for detecting intracytoplasmatic nucleosomes. Untreated HUVEC showed 16+/-6% TUNEL positive cells after 24 hours as analyzed by FACS. Serum/growth factor depletion increased apoptosis by 79+/-7%, while 50 ng/ml of the pro-apoptotic drug actinomycin-D induced comparable effects (72+/-11%). Apoptosis by serum/ growth factor depletion could be blocked completely by the anti-apoptotic agent cycloheximide (2 microg/ml), but was ineffective in blocking actinomycin-D-induced apoptosis. Pyrrolidine dithiocarbamate (PDTC) also acted as an anti-apoptotic agent by blocking apoptosis induced by actinomycin-D, but had no effect on apoptosis induced by factor depletion. Thus, two independent mechanisms for regulation of apoptosis are suggested to be present in human vascular endothelial cells.

Efficient transcription of the human angiotensin II type 2 receptor gene requires intronic sequence elements.

To investigate mechanisms of human angiotensin II type 2 receptor (hAT2) gene regulation we functionally characterized the promoter and downstream regions of the gene. 5'-Terminal deletion mutants from -1417/+100 to -46/+100 elicited significant but low functional activity in luciferase reporter gene assays with PC12W cells. Inclusion into the promoter constructs of intron 1 and the transcribed region of the hAT2 gene up to the translation start enhanced luciferase activity 6.7+/-1.6-fold and 11.6+/-1.7-fold (means+/-S.E.M.) respectively, whereas fusion of the promoter to the spliced 5' untranslated region of hAT2 cDNA did not, which indicated an enhancement caused by intronic sequence elements. Reverse transcriptase-mediated PCR confirmed that the chimaeric hAT2-luciferase mRNA was regularly spliced in PC12W cells. A Northern blot analysis of transfected cells showed levels of luciferase mRNA expression consistent with the respective enzyme activities. Mapping of intron 1 revealed that a 12 bp sequence in the centre of the intron was required for the increase in promoter activity, whereas the 5' adjacent intronic region mediated a decrease in luciferase activity. Mutation of the 12 bp region led to altered protein binding and markedly decreased luciferase activity. Cloned into a promoterless luciferase vector, a 123 bp intron 1 fragment was able to direct reporter gene expression to the same activity as occurred in conjunction with the 5' flanking region. These results indicate that sequence elements in intron 1 are necessary for efficient transcription of hAT2. In reporter gene assays, intron 1 might by itself function as a promoter and initiate transcription from an alternative start point.

Expression of ACE mRNA in the human atrial myocardium is not dependent on left ventricular function, ACE inhibitor therapy, or the ACE I/D genotype.

The activity of the cardiac renin-angiotensin system is altered in human heart failure, but the regulatory mechanisms are unknown. We analyzed whether angiotensin-converting enzyme (ACE) mRNA expression in heart failure is altered in the atrial myocardium, and whether a correlation exists between atrial ACE mRNA expression and the parameters of left ventricular function. We also investigated whether the use of ACE inhibitors or the ACE I/D genotype modulates the atrial ACE mRNA content. For this purpose patients who were to undergo routine cardiac surgery were selected in a prospective manner according to left ventricular function and ACE inhibitor therapy. Samples of atrial myocardium were taken, and ACE mRNA expression was determined by internally standardized reverse transcription polymerase chain reaction. Atrial ACE mRNA expression did not differ in patients with left ventricular ejection fraction higher than 55% (2423+/-199 copies/ng RNA) and those with a value less than 45% (2661+/-143 copies/ng RNA, n.s.). ACE mRNA expression also did not differ in patients using ACE inhibitors (2585+/-175 copies/ng RNA) and those not using ACE inhibitors (2476+/-185 copies/ng RNA). Furthermore, atrial ACE mRNA expression was not affected by the ACE genotype (DD 2573+/-203, ID 2472+/-215, II 2563+/-249 copies/ng RNA). We conclude that the regulation of atrial ACE mRNA expression occurs predominantly by local mechanical or para- or autocrine factors.

"5 A Day" achievement badge for urban boy scouts: formative evaluation results.

BACKGROUND: Certain cancers are more common among African Americans (AA). Fruit and vegetables (F&V) reduce cancer risk, but Americans, and African Americans in particular, do not meet the "5 A Day" goal. Scouting organizations, particularly urban Boy Scout groups that target inner-city youth, provide promising channels for nutritional behavioral change programs. METHODS: Focus groups were conducted with urban Boy Scouts and their parents to identify factors influencing F&V consumption and evaluate potential intervention activities. Twenty-four-hour dietary recalls were collected from 85 area Boy Scouts. A national data set was used to obtain values for F&V consumption by African American and European American (boys age 0-16). RESULTS: Vegetable preferences were low and a negative peer influence for vegetables was reported. The group has limited food-preparation skills, but both parents and scouts reported that F&V were available in their homes. Use of goal setting and use of problem-solving techniques were limited. The local scouts' mean F&V intake was 3.2 servings per day. Ethnic differences in F&V consumption were identified in the national data. CONCLUSIONS: Based on these results and previous interventions in schools, an overall structure for the intervention was developed to include eight weekly troop sessions and two camping sessions, parent newsletters, seven weekly home badge assignments, and ten comic books.

Protein kinase C-dependent regulation of the human AT1 promoter in vascular smooth muscle cells.

The expression level of angiotensin II (ANG II) type 1 receptors (AT1) determines the magnitude of ANG II signaling in vascular smooth muscle cells (VSMC). AT1 mRNA expression in cultured bovine VSMC increased twofold after 8 h of protein kinase C (PKC) activation with phorbol 12-myristate 13-acetate (PMA), whereas stimulation with forskolin did not alter the AT1 mRNA level. The expression of AT1 promoter/exon 1 [-513/+92 base pairs (bp)] luciferase constructs transfected into VSMC increased 2.4-fold with PMA stimulation. In-gel kinase assays demonstrated rapid phosphorylation of mitogen-activating protein kinases (MAPK) ERK1 and ERK2 by PMA. Electrophoretic gel mobility shift assays showed sequence-specific binding of nuclear proteins from PMA-activated VSMC, identified as activator protein 1 (AP-1) complex in competition assays, to a radiolabeled AT1-promoter fragment (-368/-399 bp). Recombinant AP-1 binds in a sequence-specific manner to the -386/-399-bp region. Site-specific mutagenesis destroying the AP-1 site, the adjacent polyoma enhancer activator 3 element, or both sites simultaneously indicated that both sites together are necessary and sufficient to control basal and PMA-induced activation of the human AT1 promoter in transfected VSMC. The capability of the phorbol ester PMA to activate the human AT1 promoter in VSMC via an AP-1 element suggests a prominent role for PKC/MAPK and Ets proteins in AT1 regulation.

Molecular biology of angiotensin receptors and their role in human cardiovascular disease.

The actions of angiotensin II in the cardiovascular system are transmitted by two known and possibly some unknown angiotensin receptor types. AT1 and AT2 both correspond to G-protein-coupled receptors with seven hydrophobic transmembrane domains, several N-glycosylation sites and a potential G-protein binding site. Cloning of coding regions and promoter sequences contributed to the understanding of receptor protein function and regulation. Angiotensin receptors with atypical binding properties for the known AT1- and AT2-specific ligands are expressed on human cardiac fibroblasts and in the human ulcrus. In several animal models, receptors with high affinity for angiotensin (1-7) have been described. AT1 stimulation is mediated by the generation of phospholipid-derived second messengers, activation of protein kinase C, the MAPkinase pathway and of immediate early genes. Recently, phosphorylation and dephosphorylation of tyrosine kinases have been associated with AT1- and AT2-mediated signal transduction. ATR are regulated by phosphorylation, internalization, modification of transcription rate and mRNA stability. Regulation is highly cell and organ specific and includes upregulation of ATR in some pathophysiological situations where the renin angiotensin system is activated. Whereas the function of AT1 in the cardiovascular system is relatively well established, there is little information regarding the role of AT2. Recent hypotheses suggest an antagonism between AT1 and AT2 at the signal transduction and the functional level. Transgenic animal models, particularly with targeted disruption of the AT1 and AT2 genes, suggest the contribution of both genes to blood pressure regulation. Genetic polymorphisms have been described in the AT1 and AT2 gene or neighbored regions and are used to analyze the association between gene defects and cardiovascular diseases. AT1 antagonists are now being introduced into the treatment of hypertension and potentially heart failure, and more interesting pharmacological developments are expected from the ongoing basic studies.

Subtype 2 and atypical angiotensin receptors in the human heart.

Angiotensin receptors have been described in the human heart and are suspected to play a central role in remodeling after myocardial infarction and in cardiac hypertrophy. Two subtypes, AT1 and AT2, have so far been described in humans, with AT2 being the dominant subtype in human atria. We have now determined subtype numbers and distribution by binding in ventricular myocardium from patients with end-stage heart failure. We found about 50-80% of subtype AT2 in the right and left ventricles from patients with end-stage heart failure due to coronary artery disease and cardiomyopathy, indicating that AT2 is the dominant angiotensin receptor subtype in the whole human heart. To determine the cellular localization of angiotensin receptors in human myocardium in addition to the known localization on myocytes, smooth muscle cells and endothelial cells, we investigated cardiac fibroblasts. They express an angiotensin receptor with yet incompletely understood binding characteristics which is coupled to proliferation and DNA synthesis. As AT2 is the dominant angiotensin receptor subtype in human heart, we cloned the complete mRNA sequence by a rapid amplification of cDNA ends (RACE) procedure and thereafter the promoter sequence from a human genomic library. Once the sequence of the mRNA and thus exon 1 was obtained by the RACE-PCR, a probe was constructed for the most 5' region of exon 1 and used for screening of a human genomic DNA bank. After cutting of the positive clones with EcoR1 and Not1, a 4000 bp fragment hybridized with the probe and was further sequenced. A functional AT2 promoter, with > 90% homology with the mouse promoter and 35% homology with the human AT1 promoter containing numerous cis-acting sequences for basal (TFIID) and inducible (AP-1, PEA-3, CBF) transcription factors in the first 1000 bp was identified.

Characterization of the manganese superoxide dismutase cDNA and gene from the human parasite Onchocerca volvulus.

The manganese-containing superoxide dismutase (MnSOD) is a major component of the cellular defence mechanisms against the toxic effects of the superoxide radical. Within the framework of studies on anti-oxidant enzymes and their protective role in the human parasitic nematode Onchocerca volvulus, sequences encoding the MnSOD were isolated and examined in this study. Degenerate primers were designed based upon conserved regions of MnSOD sequences from other organisms, and were used in PCR on reverse-transcribed O. volvulus total RNA and genomic DNA to identify partial cDNA and genomic DNA fragments encoding the O. volvulus MnSOD (OvMnSOD). The genomic DNA PCR product was used to screen an O. volvulus adult worm lambda unizap II cDNA library and the nucleotide sequence of the longest clone determined. The complete 5'-end of the OvMnSOD cDNA was obtained using the rapid amplification of cDNA ends (RACE) procedure with O. volvulus total RNA and was found to possess a spliced leader sequence at the 5'-terminus. The deduced primary sequence encodes a 25 kDa protein, which has the conserved residues required for enzyme activity and metal binding. The 24 N-terminal amino acids encoded by the OvMnSOD cDNA comprise a putative mitochondrial transit peptide. The OvMnSOD gene was also isolated from an O. volvulus adult worm lambda fix II genomic library, a restriction map was constructed and the nucleotide sequence determined. The OvMnSOD gene was found to possess five exons and four introns with consensus splice-site junctions. Potential regulatory elements were identified in the 5' genomic flanking sequence. Southern-blot analysis with total worm genomic DNA indicates a single-copy gene, with a restriction pattern consistent with that of the isolated gene.

[Angiotensin receptors--organ and subtype specific regulation in cardiovascular diseases by modulation of the renin-angiotensin system. Studies of the rat model and in human myocardium]. / Angiotensinrezeptoren--Organ- und subtypspezifische Regulation bei kardiovaskulären Erkrankungen und durch Modulation des Renin-Angiotensin-Systems. Untersuchungen am Rattenmodell und im menschlichen Myokard.

So far, two angiotensin receptor subtypes, called AT1 and AT2, have been described in an animal model and in human. AT1 mediates almost all known effects of angiotensin II and its gene sequence and regulation is well studied. In contrast, only few data on function and regulation of AT2 are available. The complete mRNA sequence of AT2 has only recently been cloned and sequenced. The angiotensin receptors' receptor density and subtype distribution is organ specific. In the rat, lowest densities are found in the myocardium, followed by kidney, liver, adrenal medulla and cortex. The percentage of AT1 in the different organs amounts to 80, 85, 90, 57 and 10%. Angiotensin receptor subtypes have also been quantitated in human myocardium. There, the relatively unknown subtype AT2 dominates (67%). Myocardial receptor density is low, amounting to about 11 fmol/mg protein corresponding to 1/20-1/50 of the density of beta-adrenergic receptors. Angiotensin receptors in the human heart are present on cardiac fibroblasts and induce proliferation of these cells. Blockade of the renin angiotensin system by ACEI and AT1 antagonists in the rat downregulates angiotensin receptors in liver, kidney and adrenals to about 50% in an organ- and subtype specific manner, whereas cyclosporin A upregulates receptors twice. In end-stage human heart failure, but not in early stages, angiotensin receptors are downregulated to 1/3 of control values. Regulator mechanisms at transcriptional level have been elucidated by reporter gene assays; PMA, an activator of proteinkinase C, stimulates the transcription of the AT1 gene. The organ- and subtypespecific regulation of angiotensin receptors by pharmacological agents and/or cardiovascular diseases can contribute to the understanding of these drugs and of the pathophysiology of the corresponding diseases.

Characterization of enzymatically active Onchocerca volvulus Cu/Zn superoxide dismutase expressed in Escherichia coli.

The Onchocerca volvulus superoxide dismutase was expressed in Escherichia coli, using a protocol designed to produce the native enzyme rather than a fusion protein. The recombinant O. volvulus superoxide dismutase (rOVSOD) was found in the cytosol of the disrupted bacteria and represented > 10% of the total bacterial protein. The enzyme was purified to homogeneity using DEAE-Sepharose chromatography, followed by phenyl-Sepharose chromatography. The rOVSOD was enzymatically active which was demonstrated by its reactivity with O2.- produced either by the xanthine-xanthine oxidase system or by stimulated eosinophils. The specific activity was determined to be 4668 U mg-1. This activity could be blocked by rabbit antiserum raised against the rOVSOD. The maximal activity was obtained upon supplementation of the bacterial growth media and enzyme buffer with copper and zinc ions. Activity characteristics in the presence of inhibitors was also characteristic of a Cu/Zn superoxide dismutase. The rOVSOD has an apparent subunit molecular mass of 16,000 in SDS-PAGE. The active enzyme behaves as a dimer of 32 kDa as determined by gel filtration.