Thrombin activates the hypoxia-inducible factor-1 signaling pathway in vascular smooth muscle cells: Role of the p22(phox)-containing NADPH oxidase.

The heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1) is activated under hypoxic conditions, resulting in the upregulation of its target genes plasminogen activator inhibitor-1 (PAI-1) and vascular endothelial growth factor (VEGF). PAI-1 and VEGF are also induced in response to vascular injury, which is characterized by the activation of platelets and the coagulation cascade as well as the generation of reactive oxygen species (ROS). However, it is not known whether HIF-1 is also stimulated by thrombotic factors. We investigated the role of thrombin, platelet-associated growth factors, and ROS derived from the p22(phox)-containing NADPH oxidase in the activation of HIF-1 and the induction of its target genes PAI-1 and VEGF in human vascular smooth muscle cells (VSMCs). Thrombin, platelet-derived growth factor-AB (PDGF-AB), and transforming growth factor-beta(1) (TGF-beta(1)) upregulated HIF-1alpha protein in cultured and native VSMCs. This response was accompanied by nuclear accumulation of HIF-1alpha as well as by increased HIF-1 DNA-binding and reporter gene activity. The thrombin-induced expression of HIF-1alpha, PAI-1, and VEGF was attenuated by antioxidant treatment as well as by transfection of p22(phox) antisense oligonucleotides. Inhibition of p38 mitogen-activated protein kinase and phosphatidylinositol-3-kinase significantly decreased thrombin-induced HIF-1alpha, PAI-1, and VEGF expression. These findings demonstrate that the HIF-1 signaling pathway can be stimulated by thrombin and platelet-associated growth factors and that a redox-sensitive cascade activated by ROS derived from the p22(phox)-containing NADPH oxidase is crucially involved in this response.

Strategy for the characterization of autoantigens in autoimmune diseases. Investigation of the target antigens of antimitochondrial antibodies by radioimmunoassay, immunoblotting, monoclonal antibodies and affinity chromatography.

We present a strategy to characterize specific antigen/autoantibody systems using polyclonal sera from patients as a probe and crude antigenic preparations such as mitoplasts. Sera from patients with primary biliary cirrhosis (PBC) are characterized by at least one of two specific subtypes of antimitochondrial antibodies (AMA), anti-p48 or anti-p62. Immunoblotting of such sera with mitoplast preparations derived from human, rat and rabbit livers revealed three proteins of approximately 27, 48 and 68 kDa as target antigens. On the basis of the molecular weight of these antigens we were able to purify them by elution from preparative SDS gels. Immunization of NZB mice with the high molecular weight component (the 68 kDa antigen from human liver mitoplasts) elicited a monoclonal antibody. The 68 kDa protein was then isolated by affinity chromatography and may well represent the prime target antigen of anti-p62 antimitochondrial antibodies. This experimental approach could be applied to protein target antigens of other autoantibodies.

Role of the oral calcium-loading test with measurement of intact parathyroid hormone in the diagnosis of symptomatic subtle primary hyperparathyroidism.

BACKGROUND: This study was designed to assess the diagnostic value of the oral calcium tolerance test with measurement of intact parathyroid hormone by the immunoradiometric assay (IRMA PTH) in the diagnosis of primary hyperparathyroidism in patients with symptoms who have minimal, intermittent, or no elevation of the levels of total calcium and/or intact PTH. METHODS: After baseline levels of IRMA PTH and total calcium were measured, an oral calcium load of 1000 mg elemental calcium was administered to 10 patients with hyperparathyroidism and 18 normal control subjects. Total calcium and IRMA PTH levels were measured at 30, 60, and 120 minutes after the oral calcium load was administered. RESULTS: The mean suppression of the baseline level of IRMA PTH in the patients with hyperparathyroidism was 83.7% +/- 6.5% (mean +/- 1 SEM), but the levels of the normal control subjects fell significantly (p < 0.05) lower to 58.8% +/- 3.7% (mean +/- 1 SEM). CONCLUSIONS: This study suggests that the oral calcium tolerance test may be a valuable adjunct in confirming the diagnosis of primary hyperparathyroidism in patients with symptoms who have minimal, intermittent, or no elevation of the levels of total calcium and/or IRMA PTH:

New calixarene-bonded stationary phases in high-performance liquid chromatography: comparative studies on the retention behavior and on influences of the eluent.

The chromatographic behavior of six calixarene-bonded stationary phases is reported. Varying analyte selectivities (i.e., for phenols, substituted aromatics, polycyclic aromatic hydrocarbons, barbituric acid derivatives, xanthines) exist as a function of the ring-size of the calix[n]arenes (n=4, 6, 8) and the substitution at the "upper rim" with para-tert.-butyl groups. Although eluents with unusually high proportions of water were used, a comparison with conventional reversed-phase (RP) columns shows a predominantly reversed-phase character with remarkable selectivities of these phases. The influences of several organic solvents on retention variations of solutes are compared for RP-C18, phenyl and calixarene phases.

[Phantom studies using echo contrast media to improve the Doppler color sonographic imaging of the superficial femoral artery in the adductor canal]. / Phantomuntersuchungen mit echokontrastgebenden Substanzen zur Verbesserung der farb-doppler-sonographischen Darstellung der A. femoralis superficialis im Adduktorenkanal.

The adductor canal was simulated using 2.6 cm muscular tissue and 2 fasciae to analyse the limits of colour-coded Doppler sonography (angiodynography) in this region. Defects in the spectral signal cause a significant underestimation of mean, peak systolic and peak diastolic (backflow) velocities and of calculated blood flow. Furthermore the pulsatility index is overestimated and the colour-coded visualisation of the arteries is almost lost. For the most part, these changes can be compensated by administration of a sonographic contrast agent (SH U 454). A minimum of 9 mg microbubbles/ml blood is required. Nevertheless, the adjustment of system controls (e.g. transducer power) becomes more difficult and an ideal setting impossible.

[Phantom studies of the value of color-coded Doppler sonography in arterial occlusive disease of the lower extremities]. / Phantomuntersuchungen zur Wertigkeit der farbkodierten Doppler-Sonographie bei der arteriellen Verschlusskrankheit der unteren Extremitäten.

A new practically orientated phantom for evaluating Doppler Duplex equipment was developed and used to determine the possibilities and limits of color coded Doppler Sonography (Angiodynography) in the diagnosis of arterial occlusive disease of the lower extremity. The problems that may occur in quantifying flow in these arteries are analyzed. The influence of superimposed slices of various soft-tissues on flow quantification, on the spectral waves and on color coded visualisation of the arteries and the possibility to compensate those changes by the use of sonographic contrast agents are discussed.

Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures.

Nicotinic acetylcholine receptors play important roles in numerous cognitive processes as well as in several debilitating central nervous system (CNS) disorders. In order to fully elucidate the diverse roles of nicotinic acetylcholine receptors in CNS function and dysfunction, a detailed knowledge of their cellular and subcellular localizations is essential. To date, methods to precisely localize nicotinic acetylcholine receptors in the CNS have predominantly relied on the use of anti-receptor subunit antibodies. Although data obtained by immunohistology and immunoblotting are generally in accordance with ligand binding studies, some discrepancies remain, in particular with electrophysiological findings. In this context, nicotinic acetylcholine receptor subunit-deficient mice should be ideal tools for testing the specificity of subunit-directed antibodies. Here, we used standard protocols for immunohistochemistry and western blotting to examine the antibodies raised against the alpha3-, alpha4-, alpha7-, beta2-, and beta4-nicotinic acetylcholine receptor subunits on brain tissues of the respective knock-out mice. Unexpectedly, for each of the antibodies tested, immunoreactivity was the same in wild-type and knock-out mice. These data imply that, under commonly used conditions, these antibodies are not suited for immunolocalization. Thus, particular caution should be exerted with regards to the experimental approach used to visualize nicotinic acetylcholine receptors in the brain.

Gravitropic plant growth regulation and ethylene: an unsought cardinal coordinate for a disused model.

According to the Cholodny-Went hypothesis, gravitropic differential growth is brought about by the redistribution of auxin (indolyl-3-acetic acid, IAA). We reinvestigated the relevance of different auxins and studied the role of ethylene in hypocotyls of sunflower and shoots and roots of rye and maize seedlings. Incubation of coleoptiles and of sunflower hypocotyls in solutions of IAA and dichlorophenoxyacetic acid as well as naphthylacetic acid resulted in a two- to threefold length increase compared to water controls. In spite of this pronounced general effect on elongation growth, gravi-curvature was similar to water controls. In contrast to this, inhibition of ethylene synthesis by aminoethoxyvinylglycine prevented differential growth of both hypocotyls and coleoptiles and of roots of maize. In horizontally stimulated maize roots growing on surfaces, inhibition of ethylene perception by methylcyclopropene inhibited roots to adapt growth to the surface, resulting in a lasting vertical orientation of the root tips. This effect is accompanied by up- and down-regulation of a number of proteins as detected by two-dimensional matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Together the data query the regulatory relevance of IAA redistribution for gravitropic differential growth. They corroborate the crucial regulatory role of ethylene for gravitropic differential growth, both in roots and coleoptiles of maize as well as in hypocotyls.

[Leo-insulins in the treatment of diabetes. Experiences from an internal medicine practice]. / Leo-Insuline in der Diabetes-Behandlung. Erfahrungen im Rahmen einer internistischen Praxis.

An account is given on the long-term control of 27 unselected diabetics treated with Leo insulins in private internal medical practice. Both primary adjustments in recently manifested juvenile diabetes and change-over from other insulins or oral antidiabetics were carried out as outpatient treatment. Thorough training of the patient, which is a prerequisite to successful cooperation between the diabetic and his physician, is just as important as regular metabolic check-ups. The therapeutic results was considerably improved using Leo insulins. While 26 out of 27 patients were inadequately compensated before the beginning of therapy, the subsequent observation revealed very good adjustment in 11, good adjustment in 3 and satisfactory adjustment in 4 patients.

[Principles of quantitative color Doppler ultrasound of pelvic and leg arteries. Normal sample]. / Grundlagen der quantitativen Farbdopplersonographie der Arterien des Beckens und der Beine. Normalkollektiv.

Statistical analysis of a group of 25 healthy individuals, examined via angiodynography, shows a large range of scatter for all measured parameters in pelvic and lower extremity arteries. Blood flow and velocity values, systolic acceleration and vessel diameter decrease whereas the pulsatility index rises from the centre towards the periphery. Problem areas for morphological and quantitative evaluation are the abdominal aorta, pelvic vessels, superficial femoral artery in the distal adductor canal and the fibular artery. Nearly all vessels showed tri- or multiphasic spectral patterns. Biphasic waveform, however, may be normal in lower extremity arteries. The data yielded by colour-coded Doppler sonography were comparable to results obtained with other procedures.

Induction of the plasminogen activator inhibitor-1 gene expression by mild hypoxia via a hypoxia response element binding the hypoxia-inducible factor-1 in rat hepatocytes.

Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of both tissue-type and urokinase-type plasminogen activators. The balance between plasminogen activators and PAI-1 plays an important role in several physiological and pathophysiological processes such as atherosclerosis or thrombosis. Because these conditions are associated with hypoxia, it was the aim of the present study to investigate the influence of low O(2) tension on the expression of PAI-1 mRNA and protein using primary cultured rat hepatocytes as a model system. We found that PAI-1 mRNA and protein were induced by mild hypoxia (8% O(2)). The hypoxia-dependent PAI-1 mRNA induction was transcriptionally regulated because it was inhibited by actinomycin D (ActD). Luciferase (LUC) reporter gene constructs driven by about 800 bp of the 5'-flanking region of the rat PAI-1 gene were transiently transfected into primary rat hepatocytes; mild hypoxia caused a 3-fold induction, which was mediated by the PAI-1 promoter region -175/-158 containing 2 putative hypoxia response elements (HRE) binding the hypoxia-inducible factor (HIF-1). Mutation of the HRE-1 (-175/-168) or HRE-2 (-165/-158) also abolished the induction by mild hypoxia. Cotransfection of a HIF-1alpha vector and the PAI-1-LUC constructs, as well as gel shift assays, showed that the HRE-2 of the PAI-1 promoter was most critical for induction by hypoxia and HIF-1 binding. Thus, PAI-1 induction by mild hypoxia via a HIF-1 binding HRE in the rat PAI-1 promoter appears to be the mechanism causing the increase in PAI-1 in many clinical conditions associated with O(2) deficiency.

[The value of angiodynography in follow-up of surgical vascular prostheses]. / Wertigkeit der Angiodynographie zur Verlaufskontrolle chirurgischer Gefässprothesen.

Apart from the proximal anastomosis, aortoiliac/-femoral bypass is accessible to angiodynography. The spectral waves can mostly be derived and analyzed very well. Femoropopliteal grafts can be demonstrated over the whole length, including the anastomoses if they are not located in the adductor (Hunter's) canal. Pathological findings such as stenosis or aneurysms can be easily detected. Because of a large range of scatter, blood velocity or flow are no suitable parameter to determine functioning and prognosis of the bypass. A triphasic spectral waveform in the graft, the failing of aneurysms, stenosis or turbulent blood flow in the anastomosis, a high "Pulsatility Index" and a short systolic blood flow acceleration distal to the bypass, proved to be more important parameters, that can be easily determined via colour coded Doppler sonography.

Pulmonary gas exchange and central hemodynamics at rest and during exercise in patients with chronic obstructive pulmonary disease.

Right-heart catheterization and ergometry with arterial and mixed venous blood gas analysis were performed in 27 patients with a wide range of chronic obstructive pulmonary diseases. The purpose of the examination was to evaluate the risk in patients for lung surgery or to detect additional heart diseases. Patients who developed exertional hypoxia (group 1) were compared with others who did not (group 2). In all patients the steady-state maximal workload was determined by ventilatory dysfunction. Both groups had normal values for mixed venous pO2 and normal increase of the circulatory parameters during exercise. The patients with exertional hypoxia differed from the others in that they showed no decrease of venous admixture and alveolar-arterial oxygen gradient. In addition, these patients had increasing pCO2 values at rest compared with exercise, indicating alveolar hypoventilation and ventilation-perfusion mismatching. Because of the good correlation of the absolute values of FEV1 (forced expiratory volume in 1 s) with pulmonary artery pressures, parameters of gas exchange and working capacity, this lung function parameter seems to have a central role in predicting the functional state of patients with chronic obstructive disease. Ergometry and blood gas analysis should be performed in addition because these values cannot be predicted with the calculated postoperative FEV1.

[The value of color Doppler ultrasound in follow-up of percutaneous transluminal angioplasty of the thigh area]. / Wertigkeit der Farbdopplersonographie zur Verlaufskontrolle nach perkutaner transluminaler Angioplastie der Oberschenkeletage.

The value of colour-coded Doppler sonography to evaluate results of percutaneous transluminal angioplasty of the superficial femoral artery was tested in 26 patients. Because of good accessibility, morphology may be demonstrated similar to intravenous DSA. However, accurate evaluation of dilatation success also requires haemodynamic measurements. Numerous parameters were determined one day before, one day after and one month after angioplasty at various points of the extremity (dilated segment, popliteal artery, lower leg arteries). Results were compared with those of normal individuals. Because of the large range of scatter and overlapping between normal and pathological values, only a few parameters can be used as valuable indicators for successful PTA. Spectral wave form becomes bi- or triphasic pattern and due to this the Pulsatility Index rises distal to the dilated area. An increasing systolic acceleration rate, peak systolic velocity and prestenotic Pulsatility Index have excellent predictive value. Using a Damping Factor, the later walking range may be predicted.

[Value of Doppler color ultrasonography in diagnosis of arterial occlusive disease of the lower extremity. A direct comparison with percutaneous angiography]. / Wertigkeit der Farbdopplersonographie in der Diagnostik der arteriellen Verschlusskrankheit der unteren Extremität. Ein direkter Vergleich mit der perkutanen Angiographie.

UNLABELLED: A Direct Comparison with Percutaneous Angiography: AIM: To compare colour-coded Doppler sonography (CCDS) with conventional angiography in severe occlusive vascular disease of the lower limb. METHODS: In 55 patients 1141 vessel segments were evaluated, 700 of them with atheromatous plaques, 270 with stenoses, 208 with occlusions and 6 with aneurysms. RESULTS: Deeper-seated vessels such as the abdominal aorta, the pelvic arteries, the superficial femoral artery at the level of the adductor canal and parts of the lower leg arteries are less accessible for direct CCDS. Many pathological changes however can be diagnosed indirectly by changes in the spectral wave form distal to the lesion. In superficial vascular segments (the common femoral artery, the profunda femoris artery, the superficial femoral artery above the adductor canal and the popliteal artery) image quality was excellent, more pathological changes were found, and the degree of stenosis was better estimated in comparison to angiography. CONCLUSION: The value of CCDS in patients with intermittent claudication is limited to those who have been examined with angiography e.g. before angioplasty, to follow-up examinations after vascular dilatation or surgery and to supplementary visualisation after angiography especially in readily accessible (superficial) vascular segments.

Mapping of a binding site for ATP within the extracellular region of the Torpedo nicotinic acetylcholine receptor beta-subunit.

Using 2,8,5'-[3H]ATP as a direct photoaffinity label for membrane-bound nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata, we have identified a binding site for ATP in the extracellular region of the beta-subunit of the receptor. Photolabeling was completely inhibited in the presence of saturating concentrations of nonradioactive ATP, whereas neither the purinoreceptor antagonists suramin, theophyllin, and caffeine nor the nAChR antagonists alpha-bungarotoxin and d-tubocurarine affected the labeling reaction. Competitive and noncompetitive nicotinic agonists and Ca2+ increased the yield of the photoreaction by up to 50%, suggesting that the respective binding sites are allosterically linked with the ATP site. The dissociation constant KD of binding of ATP to the identified site on the nAChR was of the order of 10(-4) M. Sites of labeling were found in the sequence regions Leu11-Pro17 and Asp152-His163 of the nAChR beta-subunit. These regions may represent parts of a single binding site for ATP, which is discontinuously distributed within the primary structure of the N-terminal extracellular domain. The existence of an extracellular binding site for ATP confirms, on the molecular level, that this nucleotide can directly act on nicotinic receptors, as has been suggested from previous electrophysiological and biochemical studies.

Membrane protein subunit fractionation by means of inverse pore gradient elution polyacrylamide gel electrophoresis.

We report here the preparative scale isolation of the four subunits of the nicotinic acetylcholine receptor (nAChR) applying short inverse pore gradient SDS gels on an elution-PAGE apparatus. The nAChR subunits are of similar molecular weights (alpha, 50.2 kDa; beta, 53.7 kDa; gamma, 56.3 kDa; delta, 57.6 kDa) and isoelectric point (approx 5.5) and share the typical properties of amphiphatic membrane proteins that are difficult to separate by chromatographic procedures. Preparative PAGE, which has proved to be the method of choice for nAChR-subunit fractionation, however, is time-consuming and achieves only moderate resolutions yielding dilute fractions. We present here the fractionation of a membrane preparation of Torpedo electric organ (2 mg of total protein) on short gels (2 cm) with linear or concave inverse pore gradients. All of the four subunits are isolated in homogeneous fractions of approx 70 micrograms/ml within only 4 h. Compared to the standard method, this means a 50% reduction in separation times with threefold higher concentrated protein fractions. We also give the theoretical explanation and experimental validation of the improved features resulting from short inverse-gradient polyacrylamide gels.