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1.  Dose-dependent, therapeutic potential of angiotensin-(1–7) for the treatment of pulmonary arterial hypertension 
Pulmonary Circulation  2015;5(4):649-657.
The effects of the heptapeptide angiotensin-(1–7) (Ang-(1–7)), via its receptor Mas, oppose many of the effects of the classic angiotensin II signaling pathway, and pharmacological exploitation of this effect is currently actively pursued for a wide range of cardiovascular, neoplastic, or immunological disorders. On the basis of its vasodilatory and antiproliferative properties, Ang-(1–7) has consequentially also been proposed as a novel therapeutic strategy for the treatment of pulmonary arterial hypertension (PAH). In this study, we tested the effectiveness of Ang-(1–7) and its stable, cyclic analog cAng-(1–7) over a range of doses for their therapeutic potential in experimental PAH. In the monocrotaline (MCT) rat model of PAH, Ang-(1–7) or cAng-(1–7) were injected in doses of 30, 100, 300, or 900 μg kg−1 day−1, and effects on pulmonary hemodynamics and vascular remodeling were assessed. Five weeks after MCT injection, right ventricular systolic pressure (RVSP) was significantly reduced for 3 dose groups treated with Ang-(1–7) (100, 300, and 900 μg kg−1 day−1) and for all dose groups treated with cAng-(1–7), as compared to untreated controls, yet the total reduction of RVSP was <50% at best and thus markedly lower than that with a positive treatment control with ambrisentan. Medial-wall thickness in pulmonary arterioles was only slightly reduced, without reaching significance, for any of the tested Ang-(1–7) compounds and doses. The reported moderate attenuation of PAH does not confirm the previously postulated high promise of this strategy, and the therapeutic usefulness of Ang-(1–7) may be limited in PAH relative to that in other cardiovascular diseases.
doi:10.1086/683696
PMCID: PMC4671739  PMID: 26697172
pulmonary hypertension; vascular remodeling; renin-angiotensin system
2.  Hemodynamic Effects of the Non-Peptidic Angiotensin-(1-7) Agonist AVE0991 in Liver Cirrhosis 
PLoS ONE  2015;10(9):e0138732.
Background & Aims
Although in cirrhosis with portal hypertension levels of the vasoconstrictor angiotensin II are increased, this is accompanied by increased production of angiotensin (Ang)-(1–7), the endogenous ligand of the Mas receptor (MasR), which blunts hepatic fibrosis and decreases hepatic vascular resistance. Therefore, we investigated the effects of the non-peptidic Ang-(1–7) agonist, AVE0991, in experimental cirrhosis.
Methods
Cirrhosis was induced by bile duct ligation (BDL) or carbon tetrachloride (CCl4) intoxication. The coloured microsphere technique assessed portal and systemic hemodynamic effects of AVE0991 in vivo. Hepatic expression of eNOS, p-eNOS, iNOS, JAK2, ROCK and p-Moesin were analyzed by western blots. Activities of ACE and ACE2 were investigated fluorometrically. Moreover, fibrosis was assessed in BDL rats receiving AVE0991.
Results
In vivo, AVE0991 decreased portal pressure (PP) in both rat models of cirrhosis. Importantly, systemic effects were not observed. The hepatic effects of AVE0991 were based on upregulation of vasodilating pathways involving p-eNOS and iNOS, as well as by downregulation of the vasoconstrictive pathways (ROCK, p-Moesin). Short-term treatment with AVE0991 decreased the activity of ACE2, long-term treatment did not affect hepatic fibrosis in BDL rats.
Conclusions
The non-peptidic agonist of Ang-(1–7), AVE0991, decreases portal pressure without influencing systemic pressure. Thus, although it does not inhibit fibrosis, AVE0991 may represent a promising new therapeutic strategy for lowering portal pressure.
doi:10.1371/journal.pone.0138732
PMCID: PMC4583473  PMID: 26406236
3.  Optimization of ethylene glycol production from (d)-xylose via a synthetic pathway implemented in Escherichia coli 
Background
Ethylene glycol (EG) is a bulk chemical that is mainly used as an anti-freezing agent and a raw material in the synthesis of plastics. Production of commercial EG currently exclusively relies on chemical synthesis using fossil resources. Biochemical production of ethylene glycol from renewable resources may be more sustainable.
Results
Herein, a synthetic pathway is described that produces EG in Escherichia coli through the action of (d)-xylose isomerase, (d)-xylulose-1-kinase, (d)-xylulose-1-phosphate aldolase, and glycolaldehyde reductase. These reactions were successively catalyzed by the endogenous xylose isomerase (XylA), the heterologously expressed human hexokinase (Khk-C) and aldolase (Aldo-B), and an endogenous glycolaldehyde reductase activity, respectively, which we showed to be encoded by yqhD. The production strain was optimized by deleting the genes encoding for (d)-xylulose-5 kinase (xylB) and glycolaldehyde dehydrogenase (aldA), and by overexpressing the candidate glycolaldehyde reductases YqhD, GldA, and FucO. The strain overproducing FucO was the best EG producer reaching a molar yield of 0.94 in shake flasks, and accumulating 20 g/L EG with a molar yield and productivity of 0.91 and 0.37 g/(L.h), respectively, in a controlled bioreactor under aerobic conditions.
Conclusions
We have demonstrated the feasibility to produce EG from (d)-xylose via a synthetic pathway in E. coli at approximately 90 % of the theoretical yield.
Electronic supplementary material
The online version of this article (doi:10.1186/s12934-015-0312-7) contains supplementary material, which is available to authorized users.
doi:10.1186/s12934-015-0312-7
PMCID: PMC4559361  PMID: 26336892
Synthetic metabolic pathway; Ethylene glycol; Xylose; Metabolic engineering; Escherichia coli
4.  Animal Models and “Omics” Technologies for Identification of Novel Biomarkers and Drug Targets to Prevent Heart Failure 
BioMed Research International  2015;2015:212910.
It is now accepted that heart failure (HF) is a complex multifunctional disease rather than simply a hemodynamic dysfunction. Despite its complexity, stressed cardiomyocytes often follow conserved patterns of structural remodelling in order to adapt, survive, and regenerate. When cardiac adaptations cannot cope with mechanical, ischemic, and metabolic loads efficiently or become chronically activated, as, for example, after infection, then the ongoing structural remodelling and dedifferentiation often lead to compromised pump function and patient death. It is, therefore, of major importance to understand key events in the progression from a compensatory left ventricular (LV) systolic dysfunction to a decompensatory LV systolic dysfunction and HF. To achieve this, various animal models in combination with an “omics” toolbox can be used. These approaches will ultimately lead to the identification of an arsenal of biomarkers and therapeutic targets which have the potential to shape the medicine of the future.
doi:10.1155/2015/212910
PMCID: PMC4508378  PMID: 26236717
5.  Bacteriophage-Based Latex Agglutination Test for Rapid Identification of Staphylococcus aureus 
Journal of Clinical Microbiology  2014;52(9):3394-3398.
Rapid diagnosis is essential for the management of Staphylococcus aureus infections. A host recognition protein from S. aureus bacteriophage phiSLT was recombinantly produced and used to coat streptavidin latex beads to develop a latex agglutination test (LAT). The diagnostic accuracy of this bacteriophage-based test was compared with that of a conventional LAT, Pastorex Staph-Plus, by investigating a clinical collection of 86 S. aureus isolates and 128 coagulase-negative staphylococci (CoNS) from deep tissue infections. All of the clinical S. aureus isolates were correctly identified by the bacteriophage-based test. While most of the CoNS were correctly identified as non-S. aureus isolates, 7.9% of the CoNS caused agglutination. Thus, the sensitivity of the bacteriophage-based LAT for identification of S. aureus among clinical isolates was 100%, its specificity was 92.1%, its positive predictive value (PPV) was 89.6%, and its negative predictive value (NPV) was 100%. The sensitivity, specificity, PPV, and NPV of the Pastorex LAT for the identification of S. aureus were 100%, 99.2%, 98.9%, and 100%, respectively. Among the additionally tested 35 S. aureus and 91 non-S. aureus staphylococcal reference and type strains, 1 isolate was false negative by each system; 13 and 8 isolates were false positive by the bacteriophage-based and Pastorex LATs, respectively. The ability of the phiSLT protein to detect S. aureus was dependent on the presence of wall teichoic acid (WTA) and corresponded to the production of ribitol phosphate WTA, which is found in most S. aureus clones but only a small minority of CoNS. Bacteriophage-based LAT identification is a promising strategy for rapid pathogen identification. Finding more specific bacteriophage proteins would increase the specificity of this novel diagnostic approach.
doi:10.1128/JCM.01432-14
PMCID: PMC4313167  PMID: 25031449
6.  Developmental stage-dependent metabolic regulation during meiotic differentiation in budding yeast 
BMC Biology  2014;12:60.
Background
The meiotic developmental pathway in yeast enables both differentiation of vegetative cells into haploid spores that ensure long-term survival, and recombination of the parental DNA to create genetic diversity. Despite the importance of proper metabolic regulation for the supply of building blocks and energy, little is known about the reprogramming of central metabolic pathways in meiotically differentiating cells during passage through successive developmental stages.
Results
Metabolic regulation during meiotic differentiation in budding yeast was analyzed by integrating information on genome-wide transcriptional activity, 26 enzymatic activities in the central metabolism, the dynamics of 67 metabolites, and a metabolic flux analysis at mid-stage meiosis. Analyses of mutants arresting sporulation at defined stages demonstrated that metabolic reprogramming is tightly controlled by the progression through the developmental pathway. The correlation between transcript levels and enzymatic activities in the central metabolism varies significantly in a developmental stage-dependent manner. The complete loss of phosphofructokinase activity at mid-stage meiosis enables a unique setup of the glycolytic pathway which facilitates carbon flux repartitioning into synthesis of spore wall precursors during the co-assimilation of glycogen and acetate. The need for correct homeostasis of purine nucleotides during the meiotic differentiation was demonstrated by the sporulation defect of the AMP deaminase mutant amd1, which exhibited hyper-accumulation of ATP accompanied by depletion of guanosine nucleotides.
Conclusions
Our systems-level analysis shows that reprogramming of the central metabolism during the meiotic differentiation is controlled at different hierarchical levels to meet the metabolic and energetic needs at successive developmental stages.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-014-0060-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s12915-014-0060-x
PMCID: PMC4176597  PMID: 25178389
Differentiation; Flux analysis; Meiosis; Metabolic reprogramming; Metabolome; Systems biology; Transcriptome; Yeast
7.  The German Aortic Valve Registry (GARY): in-hospital outcome 
European Heart Journal  2013;35(24):1588-1598.
Background
Aortic stenosis is a frequent valvular disease especially in elderly patients. Catheter-based valve implantation has emerged as a valuable treatment approach for these patients being either at very high risk for conventional surgery or even deemed inoperable. The German Aortic Valve Registry (GARY) provides data on conventional and catheter-based aortic procedures on an all-comers basis.
Methods and results
A total of 13 860 consecutive patients undergoing repair for aortic valve disease [conventional surgery and transvascular (TV) or transapical (TA) catheter-based techniques] have been enrolled in this registry during 2011 and baseline, procedural, and outcome data have been acquired. The registry summarizes the results of 6523 conventional aortic valve replacements without (AVR) and 3464 with concomitant coronary bypass surgery (AVR + CABG) as well as 2695 TV AVI and 1181 TA interventions (TA AVI). Patients undergoing catheter-based techniques were significantly older and had higher risk profiles. The stroke rate was low in all groups with 1.3% (AVR), 1.9% (AVR + CABG), 1.7% (TV AVI), and 2.3% (TA AVI). The in-hospital mortality was 2.1% (AVR) and 4.5% (AVR + CABG) for patients undergoing conventional surgery, and 5.1% (TV AVI) and AVI 7.7% (TA AVI).
Conclusion
The in-hospital outcome results of this registry show that conventional surgery yields excellent results in all risk groups and that catheter-based aortic valve replacements is an alternative to conventional surgery in high risk and elderly patients.
doi:10.1093/eurheartj/eht381
PMCID: PMC4065384  PMID: 24022003
Aortic stenosis; Surgery; Catheter-based valve replacement; GARY
9.  Genetic Deficiency in Neprilysin or Its Pharmacological Inhibition Initiate Excessive Stress-Induced Alcohol Consumption in Mice 
PLoS ONE  2012;7(11):e50187.
Both acquired and inherited genetic factors contribute to excessive alcohol consumption and the corresponding development of addiction. Here we show that the genetic deficiency in neprilysin [NEP] did not change the kinetics of alcohol degradation but led to an increase in alcohol intake in mice in a 2-bottle-free-choice paradigm after one single stress stimulus (intruder). A repetition of such stress led to an irreversible elevated alcohol consumption. This phenomenon could be also observed in wild-type mice receiving an orally active NEP inhibitor. We therefore elucidated the stress behavior in NEP-deficient mice. In an Elevated Plus Maze, NEP knockouts crossed more often the area between the arms, implicating a significant stronger stress response. Furthermore, such animals showed a decreased locomotor activity under intense light in a locomotor activity test, identifying such mice to be more responsive in aversive situations than their wild-type controls. Since the reduction in NEP activity itself does not lead to significant signs of an altered alcohol preference in mice but requires an environmental stimulus, our findings build a bridge between stress components and genetic factors in the development of alcoholism. Therefore, targeting NEP activity might be a very attractive approach for the treatment of alcohol abuse in a society with increasing social and financial stress.
doi:10.1371/journal.pone.0050187
PMCID: PMC3503995  PMID: 23185571
10.  Inter-individual variance and cardiac cycle dependency of aortic root dimensions and shape as assessed by ECG-gated multi-slice computed tomography in patients with severe aortic stenosis prior to transcatheter aortic valve implantation: is it crucial for correct sizing? 
To evaluate the inter-individual variance and the variability of the aortic root dimensions during the cardiac cycle by computed tomography (CT) in patients with severe aortic stenosis prior to transcatheter aortic valve implantation (TAVI). Fifty-six patients (m/w = 16/40, 81 ± 6.8 years), scheduled for a transapical aortic valve implantation with available preprocedural ECG-gated CT were retrospectively included. The evaluation included sizing of the aortic annulus and the aortic sinus, measurements of the coronary topography, aortic valve planimetry and scoring of calcification. The new defined aortic annulus sphericity ratio revealed a mostly elliptical shape with increasing diastolic deformation. The calculated effective diameter (ED), determined from the annulus’ lumen area, turned out to be the parameter least affected from cardiac cycle changes while systolic and diastolic annulus dimensions and shape (diameter and area) differed significantly (p < 0.001). In about 70 % of the patients with relevant paravalvular leaks the finally implanted prosthesis was too small according to the CT based calculated ED. The ostial height of the coronaries showed a high variability with a critical minimum range <5 mm. The degree of the aortic calcification did not have an influence on the aortic annulus deformation during the cardiac cycle, but on the occurrence of paravalvular leaks. The aortic root anatomy demonstrated a high inter-individual variability and cardiac cycle dependency. These results must be strongly considered during the patient evaluation prior to TAVI to avoid complications. The systolic effective diameter, as measured by ECG-gated CT, represents an appropriate parameter for sizing the aortic annulus.
doi:10.1007/s10554-012-0123-4
PMCID: PMC3608865  PMID: 22986896
Transcatheter; Percutaneous; Aortic valve; Implantation; Computed tomography; Effective diameter
11.  Cationic Amphiphilic Drugs Are Potent Inhibitors of Yeast Sporulation 
PLoS ONE  2012;7(8):e42853.
Meiosis is a highly regulated developmental process that occurs in all eukaryotes that engage in sexual reproduction. Previous epidemiological work shows that male and female infertility is rising and environmental factors, including pollutants such as organic solvents, are thought to play a role in this phenomenon. To better understand how organic compounds interfere with meiotic development, the model organism Saccharomyces cerevisiae was exposed to 446 bioactive molecules while undergoing meiotic development, and sporulation efficiency was quantified employing two different high-throughput assays. 12 chemicals were identified that strongly inhibited spore formation but did not interfere with vegetative growth. Many of these chemicals are known to bind to monoamine-receptors in higher eukaryotes and are cationic amphiphilic drugs. A detailed analysis of one of these drugs, tripelennamine, revealed that it induces sporulation-specific cytotoxicity and a strong inhibition of meiotic M phase. The drug, however, only mildly interfered with pre-meiotic DNA synthesis and the early meiotic transcriptional program. Chemical-genomic screening identified genes involved in autophagy as hypersensitive to tripelennamine. In addition, we found that growing and sporulating yeast cells heterozygous for the aminophospholipid translocase, NEO1, are haploinsufficient in the presence of the drug.
doi:10.1371/journal.pone.0042853
PMCID: PMC3414501  PMID: 22905177
13.  Transapical aortic valve implantation - The Leipzig experience 
Annals of Cardiothoracic Surgery  2012;1(2):129-137.
Background
Transcatheter aortic valve implantation (TAVI) represents a significant development in the treatment of high risk patients with aortic stenosis. As one of the first centers to perform transapical TAVI (taTAVI), we herein review our five-year experience with this technique.
Methods
All patients undergoing taTAVI with an Edwards Sapien valve at the Leipzig Heart Center between 2006 and 2011 (n=439) were analysed. Data was drawn from a prospective database and retrospectively analysed. The learning curve was reviewed by means of descriptive statistics as well as cumulative sum failure analysis (CUSUM). All results are presented in compliance with Valve Academic Research Consortium (VARC) criteria.
Results
The mean patient age was 81.5±6.4 years and 64.0% were female. The mean logistic EuroSCORE and STS risk of mortality were 29.7%±15.7% and 11.4%±7.6%, respectively. Procedural success was 90.2%. Stroke occurred in 2.1% of patients intra-operatively and a further 2.1% suffered stroke during their hospital stay. Mean transvalvular gradient was 9.0±3.9 mmHg and effective valve orifice area 1.3±0.6 cm2. Moderate or greater aortic insufficiency was present in 5.7% of patients and remained stable during follow up. Overall survival was 90% at 30 days, 73% at 1 year, 68% at 2 years, 58% at 3 years, 53% at 4 years, and 44% at 5 years. CUSUM analysis revealed a definitive learning curve regarding the occurrence of major complications, with a progressive improvement after the initial 150 cases.
Conclusion
TaTAVI has become a routine approach for high risk patients with symptomatic severe aortic stenosis. Although taTAVI is a safe procedure with reproducible results, future research should focus on methods of reducing known complications and the associated learning curve for this procedure.
doi:10.3978/j.issn.2225-319X.2012.06.09
PMCID: PMC3741744  PMID: 23977483
Transcatheter aortic valve implantation (TAVI); transapical TAVI (taTAVI); aortic stenosis
15.  Distribution of Cardiac Stem Cells in the Human Heart 
ISRN Cardiology  2012;2012:483407.
Introduction. The existence of human cardiac stem cells (hCSC) and their regenerative capacity are not fully defined. The aim of this study was to identify and analyse the distribution of hCSCs by flow cytometry (FCM). Methods. Tissue samples from the left ventricle (LV) and the appendages of the right atrium (RA) and left atrium (LA) were taken during cardiac surgery. Mononuclear cells (MNCs) were isolated, labelled for the stem-cell-marker c-kit and hematopoietic-lineage markers and analysed by FCM. Results. HCSCs could be isolated from the RA, LA, and LV without significant quantitative difference between both atria (A) (RA 4.80 ± 1.76% versus LA 4.99 ± 1.69% of isolated MNCs, P = 0.922). The number of hCSCs was significantly higher in both atria compared to the left ventricle (A 4.90 ± 1.29% versus LV 0.62 ± 0.14% of isolated MNCs, P = 0.035). Conclusion. The atria contain a higher concentration of hCSC than the left ventricle. HCSCs located in the atria could serve as an endogenous source for heart regeneration.
doi:10.5402/2012/483407
PMCID: PMC3312548  PMID: 22462025
16.  Effects of ACE2 Inhibition in the Post-Myocardial Infarction Heart 
Journal of cardiac failure  2010;16(9):777-785.
There is evidence that angiotensin-converting enzyme 2 (ACE2) is cardioprotective. To assess this in the post-myocardial infarction (MI) heart, we treated adult male Sprague-Dawley rats with either placebo (PL) or C16, a selective ACE2 inhibitor, following permanent coronary artery ligation or sham operation.
Coronary artery ligation resulting in MI between 25–50% of the left ventricular (LV) circumference caused substantial cardiac remodeling. Daily C16 administration from post-operative days 2 to 28 at a dose that inhibited myocardial ACE2 activity was associated with a significant increase in MI size and reduction in LV % fractional shortening. Treatment with C16 did not significantly affect post-MI increases in LV end-diastolic dimension but did inhibit increases in wall thickness and fibrosis in non-infarcted LV. On post-operative day 7, C16 had no significant effect on the increased level of apoptosis in the infarct and border zones nor did it significantly affect capillary density surrounding the MI. It did, however, significantly reduce the number of c-kit+ cells in the border region.
These findings support the notion that ACE2 exerts cardioprotective effects by preserving jeopardized cardiomyocytes in the border zone. The reduction in hypertrophy and fibrosis with C16, however, suggests that ACE2 activity has diverse effects on post-MI remodeling.
doi:10.1016/j.cardfail.2010.04.002
PMCID: PMC2929397  PMID: 20797602
Angiotensin-converting enzyme 2 (ACE2); C16; renin-angiotensin system; c-kit+ stem cells; cardiac remodeling
17.  Candidate Agtr2 influenced genes and pathways identified by expression profiling in the developing brain of Agtr2−/y mice 
Genomics  2009;94(3):188-195.
Intellectual disability (ID) is a common developmental disability observed in one to three percent of the human population. A possible role for the Angiotensin II type 2 receptor (AGTR2) in brain function, affecting learning, memory, and behavior, has been suggested in humans and rodents. Mice lacking the Agtr2 gene (Agtr2−/y) showed significant impairment in their spatial memory and exhibited abnormal dendritic spine morphology. To identify Agtr2 influenced genes and pathways, we performed whole genome microarray analysis on RNA isolated from brains of Agtr2−/y and control male mice at embryonic day 15 (E15) and postnatal day one (P1). The gene expression profiles of the Agtr2−/y brain samples were significantly different when compared to profiles of the age-matched control brains. We identified 62 differently expressed genes (p ≤ 0.005) at E15 and in P1 brains of the Agtr2−/y mice. We verified the differential expression of several of these genes in brain samples using quantitative RT-PCR. Differentially expressed genes encode molecules involved in multiple cellular processes including microtubule functions associated with dendritic spine morphology. This study provides insight into Agtr2 influenced candidate genes and suggests that expression dysregulation of these genes may modulate Agtr2 actions in the brain that influences learning and memory.
doi:10.1016/j.ygeno.2009.05.011
PMCID: PMC3164574  PMID: 19501643
Learning and memory; Intellectual Disability; Dendritic spine; Expression profiling; Agtr2
18.  Cardiac phenotype and angiotensin II levels in AT1a, AT1b, and AT2 receptor single, double, and triple knockouts 
Cardiovascular Research  2010;86(3):401-409.
Aims
Our aim was to determine the contribution of the three angiotensin (Ang) II receptor subtypes (AT1a, AT1b, AT2) to coronary responsiveness, cardiac histopathology, and tissue Ang II levels using mice deficient for one, two, or all three Ang II receptors.
Methods and results
Hearts of knockout mice and their wild-type controls were collected for histochemistry or perfused according to Langendorff, and kidneys were removed to measure tissue Ang II. Ang II dose-dependently decreased coronary flow (CF) and left ventricular systolic pressure (LVSP), and these effects were absent in all genotypes deficient for AT1a, independently of AT1b and AT2. The deletion of Ang II receptors had an effect neither on the morphology of medium-sized vessels in the heart nor on the development of fibrosis. However, the lack of both AT1 subtypes was associated with atrophic changes in the myocardium, a reduced CF and a reduced LVSP. AT1a deletion alone, independently of the presence or absence of AT1b and/or AT2, reduced renal Ang II by 50% despite a five-fold rise of plasma Ang II. AT1b deletion, on top of AT1a deletion (but not alone), further decreased tissue Ang II, while increasing plasma Ang II. In mice deficient for all three Ang II receptors, renal Ang II was located only extracellularly.
Conclusion
The lack of both AT1 subtypes led to a baseline reduction of CF and LVSP, and the effects of Ang II on CF and LVSP were found to be exclusively mediated via AT1a. The lack of AT1a or AT1b does not influence the development or maintenance of normal cardiac morphology, whereas deficiency for both receptors led to atrophic changes in the heart. Renal Ang II levels largely depend on AT1 binding of extracellularly generated Ang II, and in the absence of all three Ang II receptors, renal Ang II is only located extracellularly.
doi:10.1093/cvr/cvq004
PMCID: PMC2868177  PMID: 20071356
Angiotensin II; G protein-coupled receptors; Genetically modified animals
20.  High-Protein Diet in Lactation Leads to a Sudden Infant Death-Like Syndrome in Mice 
PLoS ONE  2011;6(3):e17443.
Background
It is well accepted that reduced foetal growth and development resulting from maternal malnutrition are associated with a number of chronic conditions in later life. On the other hand such generation-transcending effects of over-nutrition and of high-protein consumption in pregnancy and lactation, a proven fact in all developed societies, are widely unknown. Thus, we intended to describe the generation-transcending effects of a high-protein diet, covering most relevant topics of human life like embryonic mortality, infant death, and physical health in later life.
Methods
Female mice received control food (21% protein) or were fed a high protein diet (42% protein) during mating. After fertilisation, females stayed on their respective diet until weaning. At birth, pups were put to foster mothers who were fed with standard food or with HP diet. After weaning, control diet was fed to all mice. All offspring were monitored up to 360 days after birth. We determined glucose-tolerance and measured cardiovascular parameters using a tip-catheter. Finally, abdominal fat amount was measured.
Results and Conclusions
We identified a worried impact of high-protein diet during pregnancy on dams' body weight gain, body weight of newborns, number of offspring, and also survival in later life. Even more important is the discovery that high-protein diet during lactation caused a more than eight-fold increase in offspring mortality. The observed higher newborn mortality during lactation is a hitherto non-described, unique link to the still incompletely understood human sudden infant death syndrome (SIDS). Thus, although offspring of lactating mothers on high-protein diet might have the advantage of lower abdominal fat within the second half of life, this benefit seems not to compensate the immense risk of an early sudden death during lactation. Our data may implicate that both pregnant women and lactating mothers should not follow classical high-protein diets.
doi:10.1371/journal.pone.0017443
PMCID: PMC3052301  PMID: 21408058
21.  New Function for an Old Enzyme: NEP Deficient Mice Develop Late-Onset Obesity 
PLoS ONE  2010;5(9):e12793.
Background
According to the World Health Organization (WHO) there is a pandemic of obesity with approximately 300 million people being obese. Typically, human obesity has a polygenetic causation. Neutral endopeptidase (NEP), also known as neprilysin, is considered to be one of the key enzymes in the metabolism of many active peptide hormones.
Methodology/Principal Findings
An incidental observation in NEP-deficient mice was a late-onset excessive gain in body weight exclusively from a ubiquitous accumulation of fat tissue. In accord with polygenetic human obesity, mice were characterized by deregulation of lipid metabolism, higher blood glucose levels, with impaired glucose tolerance. The key role of NEP in determining body mass was confirmed by the use of the NEP inhibitor candoxatril in wild-type mice that increased body weight due to increased food intake. This is a peripheral and not a central NEP action on the switch for appetite control, since candoxatril cannot cross the blood-brain barrier. Furthermore, we demonstrated that inhibition of NEP in mice with cachexia delayed rapid body weight loss. Thus, lack in NEP activity, genetically or pharmacologically, leads to a gain in body fat.
Conclusions/Significance
In the present study, we have identified NEP to be a crucial player in the development of obesity. NEP-deficient mice start to become obese under a normocaloric diet in an age of 6–7 months and thus are an ideal model for the typical human late-onset obesity. Therefore, the described obesity model is an ideal tool for research on development, molecular mechanisms, diagnosis, and therapy of the pandemic obesity.
doi:10.1371/journal.pone.0012793
PMCID: PMC2940827  PMID: 20862277
22.  Gene Deletion of the Kinin Receptor B1 Attenuates Cardiac Inflammation and Fibrosis During the Development of Experimental Diabetic Cardiomyopathy 
Diabetes  2009;58(6):1373-1381.
OBJECTIVE
Diabetic cardiomyopathy is associated with increased mortality in patients with diabetes. The underlying pathology of this disease is still under discussion. We studied the role of the kinin B1 receptor on the development of experimental diabetic cardiomyopathy.
RESEARCH DESIGN AND METHODS
We utilized B1 receptor knockout mice and investigated cardiac inflammation, fibrosis, and oxidative stress after induction of streptozotocin (STZ)-induced diabetes. Furthermore, the left ventricular function was measured by pressure-volume loops after 8 weeks of diabetes.
RESULTS
B1 receptor knockout mice showed an attenuation of diabetic cardiomyopathy with improved systolic and diastolic function in comparison with diabetic control mice. This was associated with a decreased activation state of the mitogen-activated protein kinase p38, less oxidative stress, as well as normalized cardiac inflammation, shown by fewer invading cells and no increase in matrix metalloproteinase-9 as well as the chemokine CXCL-5. Furthermore, the profibrotic connective tissue growth factor was normalized, leading to a reduction in cardiac fibrosis despite severe hyperglycemia in mice lacking the B1 receptor.
CONCLUSIONS
These findings suggest that the B1 receptor is detrimental in diabetic cardiomyopathy in that it mediates inflammatory and fibrotic processes. These insights might have useful implications on future studies utilizing B1 receptor antagonists for treatment of human diabetic cardiomyopathy.
doi:10.2337/db08-0329
PMCID: PMC2682670  PMID: 19276445
23.  Control of ATP homeostasis during the respiro-fermentative transition in yeast 
Respiring Saccharomyces cerevisiae cells respond to a sudden increase in glucose concentration by a pronounced drop of their adenine nucleotide content. Transient accumulation of the purine salvage pathway intermediate inosine accounts for the apparent loss of adenine nucleotides.Inosine formation in response to perturbations of cellular energy balance depends on the presence of a fermentable carbon source. Under respiratory conditions, AMP accumulates instead and no inosine is formed.Conversion of AXPs into inosine is facilitated by AMP deaminase, Amd1, and IMP-specific 5'-nucleotidase, Isn1. Inosine recycling into the AXP pool is facilitated by the purine nucleoside phosphorylase, Pnp1, and joint action of the phosphoribosyltransferases, Hpt1 and Xpt1.Impaired inosine formation results in altered metabolite pool dynamics in response to glucose addition, but does not change glycolytic flux. However, mutants blocked in inosine formation exhibit delayed growth acceleration after glucose addition.
Yeast cells are exposed to strongly fluctuating nutrient concentrations in their natural environment, which requires rapid and efficient adaptation through rearrangements on all levels of their metabolism. The quantitative understanding of these adaptation processes represents the basis for a directed optimization of the microorganism to suit the needs of biochemical production processes that often impose non-uniform or harsh cultivation conditions (Lara et al, 2006), or require a redirection of metabolic fluxes to improve productivity (Bailey, 1991). In this context, controlled perturbation experiments represent a valuable tool, as they allow studying the transition from one defined physiological state to another under well-characterized conditions. Measurements of metabolite pool dynamics and enzymatic activities in response to different perturbations enable the quantitative mathematical analysis of glycolytic dynamics, which is ultimately meant to discriminate the impact of allosteric regulation and changes in the enzymatic make-up of the cell on the overall metabolic response (Rizzi et al, 1997; Teusink et al, 2000; Daran-Lapujade et al, 2007; van den Brink et al, 2008).
A long-standing problem in the context of these studies was the apparent loss of adenine nucleotides, which immediately follows the relief from glucose limitation (Theobald et al, 1997; Kresnowati et al, 2006). In this study, we showed that the transient accumulation of the purine salvage pathway (PSP) metabolites, IMP and inosine, account for the loss of AXP nucleotides. The pathway for inosine formation and recycling was identified, and the interplay between different pathways during the respiro-fermentative transition is schematically summarized in Figure 10. The presence of a quickly metabolizable sugar causes accumulation of the phosphorylated metabolites F1,6P, T6P and G3P, which results in a transient imbalance of ATP-consuming and ATP-regenerating reactions, and provokes a drop in both ATP and intracellular phosphate (Pi) concentrations. The joint action of fast ATP consumption and the Adk1 reaction results in the net-production of AMP. The accumulation of AMP, however, is prevented by Amd1 and Isn1, which readily convert AMP via IMP into inosine. Recycling of inosine into IMP is facilitated by Pnp1 and the concomitant action of Hpt1 and Xpt1, with Hpt1 having the predominant role. We suggest that inosine formation serves to prevent unscheduled AMP accumulation, and to store AXP nucleotides in a metabolically ‘neutral' form until re-equilibration of glycolysis eventually allows recovery of ATP levels. Recycling of inosine via IMP represents an energy-saving way to replenish the AXP pool, as de novo synthesis of IMP starting from PRPP requires 4 ATP molecules, whereas IMP production from inosine and PRPP is an ATP-neutral process (Figure 10).
In contrast to the behavior observed in the presence of fermentable carbon sources, neither IMP nor inosine were formed in response to perturbations of the cellular energy balance under respiratory conditions. Instead, the drop in ATP level caused a concomitant increase in AMP concentration. Therefore, we conclude that the conversion of AXP nucleotides into inosine represents a specific short-term metabolic response to the perturbation of cellular energy homeostasis, which is controlled by the presence of a fermentable carbon source (or a sugar derivative that can undergo rapid phosphorylation). The discrimination between AMP accumulation and AMP-to-inosine conversion seems to be controlled by Amd1 activity. At the present stage of investigation, we cannot conclude whether regulation of Amd1 is brought about by allosteric control or by posttranslational modification. However, given that inorganic phosphate is a potent inhibitor of Amd1 (Merkler et al, 1989) and intracellular phosphate concentration transiently drops when cells become exposed to fermentable carbon sources (our data, and e.g. Hohmann et al, 1996), phosphate availability is likely to have a pivotal role in the regulation of AXP pool size and inosine formation.
The impact of defective AXP cycling on the global metabolic response to glucose addition was tested under conditions in which respiration was inhibited by antimycin A. The amd1 mutant, in which adenine nucleotide cycling was completely blocked, showed the strongest deviations from the wild-type behavior. In this mutant, adenylic energy charge exhibited a strong drop after glucose addition, and recovered much slower than in wild-type cells. Furthermore, deletion of AMD1 resulted in strong accumulation of AMP and pronounced changes in the dynamics of trehalose-6-phosphate, glycerol-3-phosphate, and PRPP after glucose addition (Figure 6).
Despite pronounced changes in metabolite pool dynamics caused by the deletion of AMD1, no alterations in the production of the fermentative end products, ethanol and glycerol, nor in the consumption of glucose were observed (Figure 9B). However, the amd1 and isn1 mutant strains showed delayed growth acceleration after glucose addition (Figure 9C). The observation of unaltered fermentation capacity but concomitant delay in growth acceleration in the amd and isn1 mutants points to the possibility that regulation of glycolysis is not the major target of AMP. Indeed, our study revealed a delayed increase of PRPP concentration in the amd1 strain after glucose addition. The PRPP protein has a central role as a precursor for purine nucleotide de novo synthesis and the synthesis of amino acids (Vavassori et al, 2005). Hence, limitation of this important precursor may negatively affect growth. In addition, AMP accumulation may have an important signaling function acting, for example, through the cAMP/PKA pathway (Thevelein et al, 2005; Zaman et al, 2009) and/or via Snf1 (Celenza and Carlson, 1984). However, experimental evidence to support this potential link is missing. Thus, the assumption of a signaling role of AMP remains speculative, although intriguing, when asking for the actual function of AXP-to-inosine conversion during the respiro-fermentative transition.
On the basis of this study, we put forward the implication of adenine nucleotide cycling through the PSP in energy homeostasis in yeast. Answering the question whether this pathway is also active in humans could potentially contribute to a better understanding of metabolic processes that control the metabolic transition from respiratory to respiro-fermentative energy supply in muscle upon heavy exercise. In particular, it may help to understand conflicting results on the impaired physical performance of individuals carrying an AMP deaminase dysfunction under these conditions (Tarnopolsky et al, 2001; Fischer et al, 2007).
Respiring Saccharomyces cerevisiae cells respond to a sudden increase in glucose concentration by a pronounced drop of their adenine nucleotide content ([ATP]+[ADP]+[AMP]=[AXP]). The unknown fate of ‘lost' AXP nucleotides represented a long-standing problem for the understanding of the yeast's physiological response to changing growth conditions. Transient accumulation of the purine salvage pathway intermediate, inosine, accounted for the apparent loss of adenine nucleotides. Conversion of AXPs into inosine was facilitated by AMP deaminase, Amd1, and IMP-specific 5′-nucleotidase, Isn1. Inosine recycling into the AXP pool was facilitated by purine nucleoside phosphorylase, Pnp1, and joint action of the phosphoribosyltransferases, Hpt1 and Xpt1. Analysis of changes in 24 intracellular metabolite pools during the respiro-fermentative growth transition in wild-type, amd1, isn1, and pnp1 strains revealed that only the amd1 mutant exhibited significant deviations from the wild-type behavior. Moreover, mutants that were blocked in inosine production exhibited delayed growth acceleration after glucose addition. It is proposed that interconversion of adenine nucleotides and inosine facilitates rapid and energy-cost efficient adaptation of the AXP pool size to changing environmental conditions.
doi:10.1038/msb.2009.100
PMCID: PMC2824524  PMID: 20087341
ATP homeostasis; metabolic regulation; purine nucleotide metabolism; respiro-fermentative transition; yeast
24.  Angiotensin-(1–7) and the G Protein-Coupled Receptor Mas Are Key Players in Renal Inflammation 
PLoS ONE  2009;4(4):e5406.
Angiotensin (Ang) II mediates pathophysiologial changes in the kidney. Ang-(1–7) by interacting with the G protein-coupled receptor Mas may also have important biological activities.
In this study, renal deficiency for Mas diminished renal damage in models of renal insufficiency as unilateral ureteral obstruction and ischemia/reperfusion injury while the infusion of Ang-(1–7) to wild-type mice pronounced the pathological outcome by aggravating the inflammatory response. Mas deficiency inhibited NF-κB activation and thus the elevation of inflammation-stimulating cytokines, while Ang-(1–7) infusion had proinflammatory properties in experimental models of renal failure as well as under basal conditions. The Ang-(1–7)-mediated NF-κB activation was Mas dependent but did not involve Ang II receptors. Therefore, the blockade of the NF-κB-activating properties of the receptor Mas could be a new strategy in the therapy of failing kidney.
doi:10.1371/journal.pone.0005406
PMCID: PMC2672164  PMID: 19404405
25.  Improved Learning and Memory in Aged Mice Deficient in Amyloid β-Degrading Neutral Endopeptidase 
PLoS ONE  2009;4(2):e4590.
Background
Neutral endopeptidase, also known as neprilysin and abbreviated NEP, is considered to be one of the key enzymes in initial human amyloid-β (Aβ) degradation. The aim of our study was to explore the impact of NEP deficiency on the initial development of dementia-like symptoms in mice.
Methodology/Principal Findings
We found that while endogenous Aβ concentrations were elevated in the brains of NEP-knockout mice at all investigated age groups, immunohistochemical analysis using monoclonal antibodies did not detect any Aβ deposits even in old NEP knockout mice. Surprisingly, tests of learning and memory revealed that the ability to learn was not reduced in old NEP-deficient mice but instead had significantly improved, and sustained learning and memory in the aged mice was congruent with improved long-term potentiation (LTP) in brain slices of the hippocampus and lateral amygdala. Our data suggests a beneficial effect of pharmacological inhibition of cerebral NEP on learning and memory in mice due to the accumulation of peptides other than Aβ degradable by NEP. By conducting degradation studies and peptide measurements in the brain of both genotypes, we identified two neuropeptide candidates, glucagon-like peptide 1 and galanin, as first potential candidates to be involved in the improved learning in aged NEP-deficient mice.
Conclusions/Significance
Thus, the existence of peptides targeted by NEP that improve learning and memory in older individuals may represent a promising avenue for the treatment of neurodegenerative diseases.
doi:10.1371/journal.pone.0004590
PMCID: PMC2643003  PMID: 19240795

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