Comparison of aerobic granulation in SBR and continuous-flow plants.

Up to now, aerobic granulation of activated sludge is only realised in SBRs, where the discontinuous feed and sedimentation allow the formation of dense granules with excellent settling properties. However, aerobic granulation in continuous-flow plants (CFP) is gaining more and more interest in order to exploit the advantages of these excellent sludge properties to construct compact and efficient WWTP. Within the scope of this project, a SBR and CFP were operated in parallel to investigate the aerobic granulation of activated sludge and to compare the biomass in terms of their structure and settling behavior. CFP operation included two experimental phases with different reactor designs. The use of synthetic wastewater during phase I led to a biomass with a SVI of 42 ml g-1, whereby the SVI declined only to 85 ml g-1 in the second phase and the use of municipal sewage. After the start-up period, microscopic images of the biomass from CFP comprised small compact granules with a high flocculent fraction. Particle size distribution for phase II confirm, that 72% of the particles had a size over 200 µm. A strong correlation was observed between the appearance of NOx-N in the first reactor and the SVI. The results illustrate, that the anaerobic conditions during feeding are essential to keep stable granules.

The influence of temperature and SRT on high-solid digestion of municipal sewage sludge.

The influence of temperature and solids retention time (SRT) on high-solid digestion of municipal sewage sludge was investigated in laboratory-scale reactors. Digestion with high-solid concentration reduces the required digestion volume and is advantageous for urban areas. The experimental conditions comprised total suspended solids (TSS) in digested sludge between 4.0 and 4.6%, temperatures in a range of 33 to 41 °C and the SRT between 10 and 25 d. High-solid digestion operates with increased NH4-N concentrations released from organic compounds. The anaerobic process can be limited by high NH4-N concentration and toxic NH3. In this study a stable digestion was observed up to 2,000 mg L(-1) NH4-N and 75 mg L(-1) NH3. Volatile suspended solids (VSS) and chemical oxygen demand removal was 53% and 57% respectively. However, digestion with 10 d SRT led to a declined VSS removal of 49%. The removal at 41 and 37 °C showed minor differences, while reduced NH4-N release and reduced methane production were observed at 33 °C. For economic reasons, high-solid digestion at 41 °C is not recommended, but will not impair VSS removal. The outcomes of this study confirm that digestion with up to 7.8% TSS in the feed is feasible for the tested temperatures and SRT down to 15 d.

The fungicide Mancozeb induces metacaspase-dependent apoptotic cell death in Saccharomyces cerevisiae BY4741.

Mancozeb (MZ), a mixture of ethylene-bis-dithiocarbamate manganese and zinc salts, is one of the most widely used fungicides in agriculture. Toxicologic studies in mammals and mammalian cells indicate that this fungicide can cause neurological and cytological disorders, putatively associated with pro-oxidant and apoptotic effects. Yeast adaptation to sub-inhibitory concentrations of MZ has been correlated with oxidative response, proteins degradation, and energy metabolism, and its main effect on yeast has been attributed to its high reactivity with thiol groups in proteins. Herein, we show that acute MZ treatments on aerobic exponentially growing yeast of wild type (BY4741) and deletion mutant strains, coupled with multiplex flow cytometry analysis, conclusively demonstrated that MZ displays the typical features of pro-oxidant activity on Saccharomyces, elevating mitochondrial ROS, and causing hyper-polarization of mitochondrial membranes leading to apoptosis. A drastic reduction of cellular viability associated with the maintenance of cell membrane integrity, as well as phosphatidyl serine externalization on yeast cells exposed to MZ, also supports an apoptotic mode of action. Moreover, abrogation of the apoptotic response in yca1 deficient mutants indicates that metacaspase-1 is involved in the programmed cell death mechanism induced by MZ in yeast.

Two histone deacetylases, FfHda1 and FfHda2, are important for Fusarium fujikuroi secondary metabolism and virulence.

Histone modifications are crucial for the regulation of secondary metabolism in various filamentous fungi. Here we studied the involvement of histone deacetylases (HDACs) in secondary metabolism in the phytopathogenic fungus Fusarium fujikuroi, a known producer of several secondary metabolites, including phytohormones, pigments, and mycotoxins. Deletion of three Zn(2+)-dependent HDAC-encoding genes, ffhda1, ffhda2, and ffhda4, indicated that FfHda1 and FfHda2 regulate secondary metabolism, whereas FfHda4 is involved in developmental processes but is dispensable for secondary-metabolite production in F. fujikuroi. Single deletions of ffhda1 and ffhda2 resulted not only in an increase or decrease but also in derepression of metabolite biosynthesis under normally repressing conditions. Moreover, double deletion of both the ffhda1 and ffhda2 genes showed additive but also distinct phenotypes with regard to secondary-metabolite biosynthesis, and both genes are required for gibberellic acid (GA)-induced bakanae disease on the preferred host plant rice, as Δffhda1 Δffhda2 mutants resemble the uninfected control plant. Microarray analysis with a Δffhda1 mutant that has lost the major HDAC revealed differential expression of secondary-metabolite gene clusters, which was subsequently verified by a combination of chemical and biological approaches. These results indicate that HDACs are involved not only in gene silencing but also in the activation of some genes. Chromatin immunoprecipitation with the Δffhda1 mutant revealed significant alterations in the acetylation state of secondary-metabolite gene clusters compared to the wild type, thereby providing insights into the regulatory mechanism at the chromatin level. Altogether, manipulation of HDAC-encoding genes constitutes a powerful tool to control secondary metabolism in filamentous fungi.

[Is the determination of the defibrillation threshold in patients with an implantable cardioverter-defibrillator still required?]. / Wird die Bestimmung der Defibrillationsschwelle bei Patienten mit implantierbarem Kardioverter/Defibrillator noch benötigt?

BACKGROUND: Intraoperative testing of implantable cardioverter-defibrillators (ICDs) is time consuming and associated with risks. In the present study, we elucidated whether the initial implantation of an ICD with high energy output makes intraoperative defibrillation threshold testing (DFTT) unnecessary even though antiarrhythmic (AA) therapy is needed in the future. METHODS: A total of 111 patients (94 men, 17 women) receiving an ICD with subsequent AA therapy (mexiletine, amiodarone, sotalol, flecainide) were analyzed retrospectively. DFT was performed during ICD implantation and after AA drug therapy. In a second step, DFT results from the study cohort were analyzed for implantation of virtual ICDs with either low (≤ 30 J, LOD), intermediate (34 J, IOD), or high energy output (36 J, HOD). RESULTS: In the study cohort, all patients reached the safety margin (SM) of 10 J between DFT and maximal shock energy of the ICD. After loading of AA agents, 6 patients (12%) with a LOD, 3 patients (11%) with an IOD, and 3 (13%) patients with a HOD failed the 10 J SM. Using virtual ICDs, 6 (5.5%) patients with a LOD, 1 patient (1%) with an IOD, and no patients with a HOD would have failed the 10 J SM. After loading of AA agents, 18 patients (16%) with a virtual LOD, 12 patients (10.8%) with an IOD, and still 9 patients (8%) with a HOD would have failed the 10 J SM. CONCLUSION: Our results demonstrate that the 10 J SM would have been achieved intraoperatively in all patients with virtual HOD ICDs. Thus, determination of the DFT during implantation does not seem to be obligatory. However, in patients receiving AA agents, DFT testing is still required.

Pathology, natural history and treatment of abdominal aortic aneurysms.

With increasing age of the population and improvement of diagnostic tools, the incidence of abdominal aortic aneurysms (AAA) has been rising steadily. Despite an improvement in operative and interventional treatment options, AAA is the cause of death in 1-3% of men over 65 years of age in industrial countries, mostly due to rupture [1]. Therefore, routine screening for AAA by ultrasonography has been postulated in the past: a 60 year old man with an abdominal aortic diameter of less than 3 cm has a life-time risk of developing AAA close to zero. However, routine screening has not been found to be cost effective. Despite of the results of two well-designed studies, the limits of AAA qualifying the patient for surgery or intervention in contrast to conservative treatment is still a matter of debate. The present review article summarizes the current knowledge of the pathology, incidence, risks, natural course as well as symptoms and current treatment strategies of AAA on the basis of the recent literature.

Expression of nitric oxide related enzymes in coronary heart disease.

Enzymes involved in the metabolism nitric oxide (NO) and reactive oxygen species (ROS) may play a role for the decreased availability of NO in atherosclerosis. We, therefore, hypothesized that the pattern of gene expression of these enzymes is altered in atherosclerosis. Myocardial tissue from patients with coronary heart disease (CHD) or without CHD (control group) was investigated. The level of enzymes related to NO/ROS metabolism was determined both at mRNA level and protein level by rt-PCR, real-time PCR, and western blot. The expression of NOS1-3 (synthesis of NO), arginase1 (reduction of L-arginine), p22phox (active subunit of NADPH oxidase), GTPCH (rate limiting enzyme for tetrahydrobiopterin), SOD1-3 (scavengers of superoxide anions), PRTMT1-3, and DDAH2 (involved in the metabolism of ADMA) was determined. All enzymes were found to be expressed in human myocardium. NOS isoforms were decreased in CHD in protein level, but only the downregulation of NOS3 expression reached statistical significance. The expression of PRMT1 and PRMT3 was increased. In addition, the expression of DDAH2 was reduced, both theoretically leading to an increase of ADMA concentration. SOD3 was downregulated in tissue from patients with CHD. Taken together, in myocardial tissue from patients with atherosclerosis, the expression of genes increasing ADMA levels is enhanced in contrast to a reduced expression of genes promoting NO synthesis. These results may contribute to the explanation of increased oxidative stress in atherosclerosis on the level of gene expression.

Upregulation of ID protein by growth and differentiation factor 5 (GDF5) through a smad-dependent and MAPK-independent pathway in HUVSMC.

GDF5 (growth and differentiation factor five), a member of the TGF-beta superfamily, binds specifically to BMPR1b, BMPR2 and ACTR2a receptors forming a heterodimeric complex, thereby inducing phosphorylation of smad1, 5, 8 and translocation to the nucleus. ID1 (inhibitor of differentiation or DNA binding) is essential for G1 to S phase transition inhibiting DNA binding thereby playing an important role in the control of differentiation, proliferation and angiogenesis. The objective of this study was, therefore, to characterize the signal transduction pathway of GDF5, especially the involvement of ID1, in human umbilical vein smooth muscle cells (HUVSMC). We observed the expression of BMPR1a, BMPR1b, BMPR2, ACTR2a, smad1, smad 5, ID1, ID2 and ID3 in HUVSMC. Application of GDF5 upregulated ID1 and ID3 expression by involvement of the smad signaling pathway. GDF5 caused phorsphorylation of smad1 followed by upregulation of ID1 and ID3. Co-incubation with anti-GDF5 prevented these effects. GDF5 significantly inhibited phosphorylation of p38 MAPK and induced phosphorylation of ERK. The specific inhibitor of p38 MAPK or ERK, SB203580 or U0126 did not induce ID protein expression. Smad1 siRNA transfection inhibited the upregulation of ID protein. GDF5 had chemotactic activity in HUVSMC; this effect was partly blocked by transfection of smad1 or ID1 siRNA. Our results indicate that GDF5 induces ID1 and ID3 in HUVSMC by a smad-dependent, MAPK-independent pathway. GDF5 binds to specific receptors, thereby inducing phosphorylation and translocation of smad1 to the nucleus where it is involved in the regulation of transcription. Since ID1 has been shown to be crucial for cell cycle control, we propose that GDF5 could be involved in the process of angiogenesis.

Coronary fistula of right coronary artery to vena cava superior and ectasia of pulmonary artery.

We report on a patient with coronary heart disease, a coronary fistula of right coronary artery to vena cava superior and pulmonary hypertension. Combined with coronary artery revascularization, the coronary fistula was closed successfully.

[Cardiomyopathy associated with uncontrolled self medication of anabolic steroids]. / Kardiomyopathie assoziiert mit unkontrollierter Selbstmedikation anaboler Steroide.

Though doping has become increasingly ostracized in the context of professional sports, an enormous number of unrecorded cases must be assumed in semi-professional competitive sports as well as in popular sports. This holds especially true for those forms of sports which are done in order to obtain a well-proportioned, athletic, healthy looking body. This case report describes a formerly healthy young man who had to be urgently admitted to an intensive care unit due to severe myocardial pump failure. As anamnestic information was insufficient and inadequate, the taking of anabolic steroids in high doses was proven, as their metabolites could be detected by urine analysis. Until now, myocardial contractile dysfunction has persisted for more than twelve months after the initial admission. Though other diagnoses which might have led to this impaired myocardial contractile performance have been excluded, cardiomyopathy associated with the taking of anabolic steroids must be assumed. Even in non-professional and public sports, a widespread abuse of doping substances exists. Hence, cardiomyopathy associated with the misuse of anabolic steroids has to be considered especially in young, formerly healthy patients.

Smoothelin is an indicator of reversible phenotype modulation of smooth muscle cells in balloon-injured rat carotid arteries.

Restenosis is the major obstacle interfering with a successful long-term outcome of balloon angioplasty. Neointima formation following endothelial injury is the result of phenotype modulation and proliferation of smooth muscle cells (SMC). To characterize these time-dependent changes, a rat balloon injury model of carotid artery restenosis was assessed. We applied monoclonal antibodies recognizing desmin, sm-alpha-actin and smoothelin, a novel marker specific for the differentiated phenotype of SMC. Neointima formation could be seen from day 7 after injury onwards. During early phases, the number of smoothelin-positive cells in the media was decreased compared with uninjured controls. Smoothelin staining was absent in the neointima during formation. Increased levels of smoothelin in both media and neointima were observed at days 28 and 56, correlating with a decrease in proliferation as assessed by Ki-67 antigen staining. No such changes were observed for desmin and sm-alpha-actin. Following balloon injury, SMC in both the media and the neointima underwent an early, reversible dedifferentiation, followed by proliferation. The novel SMC-specific marker protein smoothelin can be used to monitor this SMC (de)differentiation in neointima and media. These findings support the pivotal role of SMC phenotype modulation in neointima formation and restenosis.

Physiologic hyperinsulinemia enhances human skeletal muscle perfusion by capillary recruitment.

Despite intensive study, the relation between insulin's action on blood flow and glucose metabolism remains unclear. Insulin-induced changes in microvascular perfusion, independent from effects on total blood flow, could be an important variable contributing to insulin's metabolic action. We hypothesized that modest, physiologic increments in plasma insulin concentration alter microvascular perfusion in human skeletal muscle and that these changes can be assessed using contrast-enhanced ultrasound (CEU), a validated method for quantifying flow by measurement of microvascular blood volume (MBV) and microvascular flow velocity (MFV). In the first protocol, 10 healthy, fasting adults received insulin (0.05 mU. kg(-1). min(-1)) via a brachial artery for 4 h under euglycemic conditions. At baseline and after insulin infusion, MBV and MFV were measured by CEU during continuous intravenous infusion of albumin microbubbles with intermittent harmonic ultrasound imaging of the forearm deep flexor muscles. In the second protocol, 17 healthy, fasting adults received a 4-h infusion of either insulin (0.1 mU. kg(-1). min(-1), n = 9) or saline (n = 8) via a brachial artery. Microvascular volume was assessed in these subjects by an alternate CEU technique using an intra-arterial bolus injection of albumin microbubbles at baseline and after the 4-h infusion. With both protocols, muscle glucose uptake, plasma insulin concentration, and total blood flow to the forearm were measured at each stage. In protocol 2 subjects, tissue extraction of 1-methylxanthine (1-MX) was measured as an index of perfused capillary volume. Caffeine, which produces 1-MX as a metabolite, was administered to these subjects before the study to raise plasma 1-MX levels. In protocol 1 subjects, insulin increased muscle glucose uptake (180%, P < 0.05) and MBV (54%, P < 0.01) and decreased MFV (-42%, P = 0.07) in the absence of significant changes in total forearm blood flow. In protocol 2 subjects, insulin increased glucose uptake (220%, P < 0.01) and microvascular volume (45%, P < 0.05) with an associated moderate increase in total forearm blood flow (P < 0.05). Using forearm 1-MX extraction, we observed a trend, though not significant, toward increasing capillary volume in the insulin-treated subjects. In conclusion, modest physiologic increments in plasma insulin concentration increased microvascular blood volume, indicating altered microvascular perfusion consistent with a mechanism of capillary recruitment. The increases in microvascular (capillary) volume (despite unchanged total blood flow) indicate that the relation between insulin's vascular and metabolic actions cannot be fully understood using measurements of bulk blood flow alone.

Branched chain amino acids activate messenger ribonucleic acid translation regulatory proteins in human skeletal muscle, and glucocorticoids blunt this action.

Branched chain amino acids (BCAA) are particularly effective anabolic agents. Recent in vitro studies suggest that amino acids, particularly leucine, activate a signaling pathway that enhances messenger ribonucleic acid translation and protein synthesis. The physiological relevance of these findings to normal human physiology is uncertain. We examined the effects of BCAA on the phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (eIF4E-BP1) and ribosomal protein S6 kinase (p70(S6K)) in skeletal muscle of seven healthy volunteers. We simultaneously examined whether BCAA affect urinary nitrogen excretion and forearm skeletal muscle protein turnover and whether the catabolic action of glucocorticoids could be mediated in part by inhibition of the action of BCAA on the protein synthetic apparatus. BCAA infusion decreased urinary nitrogen excretion (P < 0.02), whole body phenylalanine flux (P < 0.02), plasma phenylalanine concentration (P < 0.001), and improved forearm phenylalanine balance (P = 0.03). BCAA also increased the phosphorylation of both eIF4E-BP1 (P < 0.02) and p70(S6K) (P < 0.03), consistent with an action to activate the protein synthetic apparatus. Dexamethasone increased plasma phenylalanine concentration (P < 0.001), prevented the BCAA-induced anabolic shift in forearm protein balance, and inhibited their action on the phosphorylation of p70(S6K). We conclude that in human skeletal muscle BCAA act directly as nutrient signals to activate messenger ribonucleic acid translation and potentiate protein synthesis. Glucocorticoids interfere with this action, and that may be part of the mechanism by which they promote net protein catabolism in muscle.

Phosphorylation of cytokeratin 8 and 18 in human vascular smooth muscle cells of atherosclerotic lesions and umbilical cord vessels.

Expression of cytokeratins (CK) is considered a hallmark of the state of epithelial differentiation. CK also occur in certain vascular smooth muscle cells (VSMC), inferring an association with a less differentiated phenotype. Recently, CK posttranslational modification was shown to occur in epithelial cells in stress, mitosis or apoptosis. The aim of this study was to determine potential CK phosphorylation patterns in human VSMC. Tissue samples of normal peripheral and coronary arteries, atherosclerotic lesions and umbilical cord vessels were evaluated by immunofluorescence microscopy applying antibodies specific for cytokeratins 8 and 18, specific cytokeratin phosphorylation sites, Ki-67-antigen as a proliferation marker and nick end labeling (TUNEL) to detect apoptosis. All samples contained cytokeratin-positive VSMC but diverse phosphorylation patterns. The C-terminal serine 431 of cytokeratin 8 (CK8Ser-431) was phosphorylated in the vast majority of CK-expressing VSMC of coronary artery lesions. Only a subset of these cells demonstrated phosphorylation of CK18Ser-33 or, to an even lesser extent, CK8Ser-73. DNA fragmentation occurred predominantly in samples containing cells with phosphorylated CK8Ser-431 domains. In contrast, occluded peripheral lesions exhibited little or no phosphorylation. Neonatal VSMC in umbilical cord vessels contain abundant phosphorylated CK domains, again predominantly CK8Ser-431, but also CK18Ser-33. Again, only single cells were found to be proliferating or to contain DNA fragmentation. Thus, abundant CK phosphorylation in VSMC of atherosclerotic lesions suggests a specific functional response to cell stress and a possible relation to apoptosis.

Accuracy of the hemocue portable glucose analyzer in a large nonhomogeneous population.

Several studies have reported inconsistent results between HemoCue (HC) whole blood glucose measurements compared to plasma glucose. We selected a large patient population with diverse pathologies and healthy volunteers to evaluate HC. For this comparison, whole blood glucose concentration was measured using HC and referenced to laboratory plasma glucose. The population (n = 512) included healthy volunteers, diabetics, and patients with heart failure, liver failure, renal failure, renal and liver transplant, and other chronic diseases. Patients were on a wide variety of medications, vitamins, and food supplements. Venous blood samples were collected in tubes containing potassium oxalate and sodium fluoride. Comparison of the results was made using the method of Bland and Altman and ANOVA at three selected glucose ranges. The glucose measurement ([HC + laboratory]/2) ranges were 24-75, 76-129, and 130-404 mg/dL. A positive bias for all three glucose ranges was observed: 38 +/- 17 mg/dL for the high glucose group compared to 24 +/- 9 mg/dL and 22 +/- 10 mg/dL for the middle and low groups, respectively. In the high glucose group 90% of the values were within 10% (R = 0.97) of the laboratory reference values compared to 81% and 55% in the normal and low glucose groups, respectively. HC glucose measurements were generally within two SD from the laboratory plasma reference. HC consistently yielded lower whole blood glucose measurements than plasma with the largest differences seen in the low glucose range (29%). HC measured more consistently at the higher glucose concentrations and was 16% lower than plasma, although the mean absolute error was highest for that range. No significant effects in the bias could be attributed to disease while possible effects from instrument modifications by the manufacturer remain uncertain.

Cardiac alpha-actin in smooth muscle cells: detection in umbilical cord vessels and in atherosclerotic lesions.

Phenotypic modulation of smooth muscle cells (SMC) is a key event during the development of atherosclerotic and restenotic lesions. During this process, the composition of the cytoskeleton is substantially altered, with changes predominantly in actin expression reflecting a shift from smooth muscle alpha-actin to the non-muscle beta-isoform. We now demonstrate that yet another actin isoform, cardiac alpha-actin, is synthesized, de novo, in SMC of various atherosclerotic lesions. Using a highly specific monoclonal antibody against cardiac alpha-actin, we analyzed and compared the accumulation of this actin isoform in diverse SMC by immunofluorescence microscopy and immunoblotting. As expected, cardiac alpha-actin was present in human myocardium but not in healthy SMC of adult aorta, coronary arteries, trabeculae of the spleen, colon, stomach or skeletal muscle. Interestingly, the presence of cardiac alpha-actin was detected in umbilical cord vessels, human myometrium, in atherosclerotic coronary lesions and atherosclerotic lesions from peripheral vascular disease. The distribution of cardiac alpha-actin often paralleled that of cytokeratins 8 and 18, intermediate filament proteins typically found in dedifferentiated SMC. Taken together, the data presented here illustrate the expression of cardiac alpha-actin to be limited to either fetal vessels or those vessels or tissue having suffered damage or atrophy, outside its 'native' environment in the heart. The demonstration of cardiac alpha-actin in SMC of umbilical cord vessels and in atherosclerotic lesions but not in apparently healthy vessels supports the notion that SMC in atherosclerotic lesions exhibit a dedifferentiated phenotype.

Endothelin-1 and smooth muscle cells: induction of jun amino-terminal kinase through an oxygen radical-sensitive mechanism.

Endothelin-1 (ET-1) has been proposed to contribute to atherogenesis and plaque rupture in coronary heart disease through activation of mitogen-activated protein kinases (MAPKs) in smooth muscle cells (SMCs). Reactive oxygen species (ROS) have been shown to be important signal transduction molecules in SMCs. Thus, the present study aimed to assess the role of ROS in ET-1-mediated activation of c-Jun amino-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2. Rat SMCs were exposed to ET-1 over time at concentrations from 10(-6) to 10(-10) mol/L, and MAPK activity was quantified. Activation of JNK and ERK was observed with a maximum stimulation at 10(-7) mol/L ET-1. JNK and ERK were activated by ET-1 binding to a single receptor (ET-1A) but differed in their downstream mechanisms: only JNK activation was sensitive to the radical scavenger N-acetylcysteine and diphenylene iodonium, an inhibitor of NADPH oxidase, indicating a role for ROS. The downstream MAPK effector and proinflammatory transcription factor, the activator protein-1 complex, was maximally activated 2 hours after the addition of ET-1. It was mainly composed of the JNK substrate c-Jun, and activation was also dependent on ROS formation. We suggest that plaque activation by ET-1 can be mediated through ROS. It can be hypothesized that the clinical benefit of antioxidants in the treatment of atherogenesis may partially depend on neutralization of ET-1-mediated ROS production.

JNK and c-Jun but not ERK and c-Fos are associated with sustained neointima-formation after balloon injury.

BACKGROUND: Formation of neointima after balloon angioplasty is regulated via inducible transcription factors (ITF), such as c-Jun and c-Fos, depending on mitogen activated protein (MAP) kinases, which have been shown to be activated after balloon injury. The precise localization of activated MAP-kinases and concomitant expression of transcription factors in the vessel wall remains to be elucidated. We have now studied the localization and time-dependent expression of MAP-kinases together with corresponding ITFs in the rat carotid angioplasty model. DESIGN: Animals were sacrificed at 0. 5, 6 and 24 h and 3, 5, 7, 14 and 28 days after injury. Cryocut sections were stained using antibodies directed against c-Jun, phosphorylated c-Jun, c-Fos, c-Jun amino-terminal kinase (JNK), extracellular signal related kinase (ERK), von Willebrand factor, ki67 antigen, and alpha-actin. RESULTS: c-Jun expression was strongly induced in smooth muscle cells (SMC) 30 min after injury and remained upregulated for 24 h, thereafter dropping to basal levels at day 3. Re-expression was observed at day 7 and 14 but not day 28. Expression patterns of JNK and phosphorylated c-Jun were highly congruent to that of c-Jun. In contrast, c-Fos expression was restricted to 30 min and, less pronounced than c-Jun and JNK, was visible after 7 days. Also, its expression was congruent with the presence of ERK. CONCLUSIONS: These findings demonstrate a clear association between MAP-kinases and their transcription factor substrates in vivo with a predominant association of JNK and c-Jun with sustained SMC proliferation.

Physiological hyperinsulinemia stimulates p70(S6k) phosphorylation in human skeletal muscle.

Using tracer methods, insulin stimulates muscle protein synthesis in vitro, an effect not seen in vivo with physiological insulin concentrations in adult animals or humans. To examine the action of physiological hyperinsulinemia on protein synthesis using a tracer-independent method in vivo and identify possible explanations for this discrepancy, we measured the phosphorylation of ribosomal protein S6 kinase (P70(S6k)) and eIF4E-binding protein (eIF4E-BP1), two key proteins that regulate messenger ribonucleic acid translation and protein synthesis. Postabsorptive healthy adults received either a 2-h insulin infusion (1 mU/min.kg; euglycemic insulin clamp; n = 6) or a 2-h saline infusion (n = 5). Vastus lateralis muscle was biopsied at baseline and at the end of the infusion period. Phosphorylation of P70(S6k) and eIF4E-BP1 was quantified on Western blots after SDS-PAGE. Physiological increments in plasma insulin (42 +/- 13 to 366 +/- 36 pmol/L; P: = 0.0002) significantly increased p70(S6k) (P: < 0.01), but did not affect eIF4E-BP1 phosphorylation in muscle. Plasma insulin declined slightly during saline infusion (P: = 0.04), and there was no change in the phosphorylation of either p70(S6k) or eIF4E-BP1. These findings indicate an important role of physiological hyperinsulinemia in the regulation of p70(S6k) in human muscle. This finding is consistent with a potential role for insulin in regulating the synthesis of that subset of proteins involved in ribosomal function. The failure to enhance the phosphorylation of eIF4E-BP1 may in part explain the lack of a stimulatory effect of physiological hyperinsulinemia on bulk protein synthesis in skeletal muscle in vivo.

Expression of monocyte chemoattractant protein-1 cDNA in vascular smooth muscle cells: induction of the synthetic phenotype: a possible clue to SMC differentiation in the process of atherogenesis.

In the arterial wall, smooth muscle cells (SMC) normally exist in a quiescent, differentiated state, representing the contractile phenotype. During the development of atherosclerosis SMC change towards the synthetic phenotype going along with proliferation, chemotactic response and increased monocyte binding. Expression of monocyte chemoattractant protein-1 (MCP-1), a potent chemoattractant for monocytes, has been shown to be among the earliest events in atherogenesis. We investigated the effect of MCP-1 on differentiated and dedifferentiated SMC. Differentiation of SMC was induced using Matrigel as a matrix for cultivation. MCP-1 was expressed in SMC by means of a recombinant adenovirus. Expression of MCP-1 led to dedifferentiation of SMC as demonstrated by induction of cytokeratin 18, a marker for the synthetic phenotype. Concurrently, migration was only detectable in MCP-1 expressing cells, whereas SMC infected with a control virus, coding for the nuclear-targeted lacZ gene showed no migration. The expression of intercellular adhesion molecule-1 (ICAM-1) could be demonstrated in synthetic SMC and was induced after infection of differentiated cells with recombinant adenovirus, coding for MCP-1 (AdMCP-1). Expression of ICAM-1 was associated with a tenfold higher monocyte binding compared to lacZ infected cells. Our data suggest that MCP-1 plays an important role for SMC in the functional switch from the contractile to the synthetic phenotype in the course of atherogenesis.