Biomedical subjects
Publications and source records attributed to M Eberhard.
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BACKGROUND: The recently discovered Jak/STAT signal transduction pathway is associated with cytokine or growth factor receptors; whether members of the G protein-coupled receptor superfamily also activate this pathway is not yet clear. As a first member, the angiotensin (AT)1A receptor has been demonstrated to phosphorylate Jak and STAT proteins. Bombesin, a neurotransmitter and growth factor in many cells and tissues, activates its G protein-coupled receptor and in addition phosphorylates proteins that might be members of the Jak/STAT family. This study investigated whether bombesin- or angiotensin-mediated growth effects are associated with STAT protein activation. METHODS: Functional receptors were characterized using ligand-binding studies, second-messenger activation and determination of ligand-mediated growth effects. STAT protein activation was analysed by electrophoretic mobility shift assay (EMSA) using labelled DNA response elements recognizing all known STAT proteins. RESULTS: Functional bombesin receptors mediating mitogenic effects were demonstrated on Swiss 3T3 fibroblasts, human melanoma cells (A375-6) and primary human lung fibroblasts; however, bombesin-related STAT protein activation was not observed by EMSA. Interferon-alpha typically activated a STAT1-STAT2-p48 heterotrimer, as well as STAT1-3 hetero- and homodimers in human melanoma cells and significantly inhibited growth of this cell line in vitro. Functional AT1A receptors on primary rat cardiac fibroblasts mediated angiotensin-stimulated growth effects but, in contrast to recently published data, did not activate any known STAT protein. CONCLUSION: Interferon alpha-stimulated growth inhibition is mediated by activation of the Jak/STAT pathway, whereas bombesin or AT1A receptor-mediated effects on cellular proliferation do not involve phosphorylation of STAT proteins.
Bombesin was originally isolated from amphibian skin, whereas its mammalian counterpart, gastrin-releasing peptide (GRP), was first identified in the nervous system of the gastrointestinal tract. Whether GRP is present in the human skin is not known. Bombesin-like peptides are also known to modulate growth. We therefore investigated whether human melanoma cell lines express functional GRP-preferring bombesin receptors and whether they alter growth or other specific cellular functions of these tumour cells. GRP receptor mRNA was found in HBL, D-10, Me-28 and A375-6 cell lines, but only A375-6 cells express a large number of high-affinity binding sites for [125I]-[Tyr4] bombesin (K(d) 0.31 +/- 0.04 nmol L(-1), 3880 +/- 429 binding sites per cell). Bombesin dose-dependently increased cytosolic calcium, but did not alter interleukin (IL) 1beta-induced reduction of cell viability or IL-6 secretion, both A375-6-specific cell functions. Growth of A375-6 cells was not altered by bombesin or the specific GRP receptor antagonist BIM26226 as measured by [3H]-thymidine incorporation or methylene blue assay, whereas insulin alone or in combination with other potential growth factors dose-dependently stimulated growth of these cells. The newly characterized GRP-preferring bombesin receptors on highly malignant human melanoma cells could initiate studies of growth effects on solid tumours or in vivo scanning using radiolabelled tracers.
The relationship between the Ca2+ transport of platelet endoplasmic reticulum and hypertension was analyzed in 17 untreated patients exhibiting various degrees of hypertension. Each patient underwent a 24-h recording of ambulatory blood pressure. Platelets from patients were permeabilized with saponin and the rate of ATP-driven thapsigargin-sensitive Ca2+ uptake determined using the fluorescent Ca2+ indicator fluo-3. No relationship between blood pressure (systolic, diastolic, day, night) and the rate of Ca2+ uptake into the sacroplasmic reticulum of platelets was found. A weak but insignificant correlation between Ca2+ uptake and the heart rate was noted. Therefore, the increase in cytosolic Ca2+ of platelets in hypertension may not be due to changes of the activity of Ca2+ uptake into the sacroplasmic reticulum.
Angiotensin II (AII) has been implicated as an important factor in the pathophysiology of heart diseases. Following the recent identification of two subtypes of the AII receptor in cardiac tissue of animals, we investigated the possible occurrence of these, or similar, subtypes in human atrial tissue. In right-atrial tissue from patients undergoing heart surgery, we determined the AII-receptor profile in receptor binding studies, using [125I]-angiotensin as radioligand and subtypes to identify and quantify AII-receptor subpopulations. In 35 patients (23 requiring coronary bypasses, 10 valvular surgery and two combined coronary and valvular surgery), the left-ventricular ejection fraction was determined in the preoperative phase, and right- and left-atrial pressure during surgery. In membranes of human right atria, AII receptors are present in high density (median: Bmax = 294 fmol.mg-1 protein, range: 111-2073) and two different subtypes can be distinguished. Type-1 receptors (AT1) accounted for 33 +/- 10% of the population whereas type-2 receptors (AT2) made up 67 +/- 10% of the population. There was no correlation between any of the measured cardiac functions and total AII-receptor density or receptor affinity. However, the percentage of AT1 receptors was higher in the atria of patients with normal right-atrial pressure; left-ventricular ejection fraction was positively and right-atrial pressure inversely correlated with the percentage of AT1 receptors (r = 0.740 and -0.901, respectively; P < 0.001, for both). Moreover, the percentage of AT2 receptors was directly correlated with the levels of left-atrial pressure (r = 0.853; P < 0.001). It is concluded that the ratio of AT1 to AT2 receptors correlates well with right-atrial pressure and left-ventricular function. This is a first indication of a possible involvement of AII-receptor subtypes in the pathophysiology of cardiac dysfunctions.
Putative domain--domain interactions of the monomeric bifunctional enzyme indoleglycerol phosphate synthase:phosphoribosyl anthranilate isomerase from Escherichia coli were probed by separating the domains on the gene level and expressing them as monofunctional proteins. The engineered monofunctional enzymes were found to be stable, monomeric proteins with virtually full catalytic activity. In addition, binding of indolyglycerol phosphate to the active site of indoleglycerol phosphate synthase and binding of reduced 1-[(2-carboxyphenyl)amino]-1-deoxyribulose 5-phosphate, a competitive inhibitor of both indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase, were almost identical in both the mono- and bifunctional enzymes. Furthermore, no association between the monofunctional enzymes was found, neither in vitro, by sedimentation and gel filtration experiments, nor in vivo, by coexpression of the domains in the same cell. Thus, no selective advantages of the bifunctional enzyme from Escherichia coli over the respective monofunctional enzymes were found on a functional level. However, the phosphoribosyl anthranilate isomerase domain appears to stabilize the indoleglycerol phosphate synthase domain of the bifunctional enzyme from Escherichia coli by interactions that seem to subtly influence the kinetics of ligand binding.
Data from steady state and transient kinetics show that the functional phosphoribosyl anthranilate isomerase domain of the naturally bifunctional enzyme from Escherichia coli has properties similar to those of its artificially excised domain. The naturally monofunctional enzyme from Saccharomyces cerevisiae has significantly higher values of both kcat and kcat/KMPRA. The primary product of a single turnover of phosphoribosylanthranilate is fluorescent, but it slowly isomerizes to the nonfluorescent stable product. The latter is the competent substrate of indoleglycerol phosphate synthase, which catalyzes the subsequent step of tryptophan biosynthesis. The isomerization is characterized by a monoexponential decay independent of phosphoribosyl anthranilate isomerase. Due to a tentative assignment of the fluorescent, primary product and the nonfluorescent, stable product to an enol and a keto compound, respectively, tryptophan biosynthesis appears to be rate-limited by an uncatalyzed enol/keto tautomerization. A formal kinetic mechanism of the reaction catalyzed by phosphoribosyl anthranilate isomerase is proposed that is consistent with the combined enzymic and ligand binding properties of the three variants of phosphoribosyl anthranilate isomerase.
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The Ca2+ antagonist mibefradil at supratherapeutic concentrations induced a sustained increase of cytosolic Ca2+ in cultured rat cardiac fibroblasts and human platelets which lack sensitivity to K+ depolarization and Ca2+ channel block by verapamil or other Ca2+ antagonists. At concentrations above 10 microM, mibefradil elevated substantially cytosolic [Ca2+] without affecting the peak level of agonist-induced Ca2+ transients. These Ca2+-mobilizing actions of 10 or 100 microM mibefradil stand in contrast to the Ca2+ antagonism and relaxation of vascular muscle at 1 microM concentrations. Since a substantial part of mibefradil-induced increase in cytosolic Ca2+ was independent of extracellular Ca2+, and in order to define better the mechanism of Ca2+ increase, we exposed permeabilized cultured rat cardiac fibroblasts and human platelets to mibefradil at concentrations sufficiently high to identify covert effects. In permeabilized fibroblasts or platelets mibefradil at concentrations above 10 microM activated dose-dependent Ca2+ release from intracellular Ca2+ stores. Verapamil had no effect at concentrations of up to 100 microM. Mibefradil-induced Ca2+ release was not affected by ryanodine, thapsigargin, removal of ATP or dithioerythreitol, indicating that neither Ca2+ - nor disulfide reagent-induced Ca2+ release were involved and that mibefradil did not release Ca2+ by inhibition of the Ca2+-ATPase pump of endoplasmic reticulum. The rate, but not the amplitude, of mibefradil-induced Ca2+ release is increased up to fourfold in the presence of pentosan polysulphate or heparin, two potent inhibitors of inositol 1,4,5-trisphosphate-induced Ca2+ release. Depletion of Ca2+ stores of permeabilized cells inositol 1,4,5-trisphosphate in the presence of thapsigargin completely blocked mibefradil-induced Ca2+ release, and depletion of Ca2+ stores by mibefradil prevented further Ca2+ release by inositol 1,4,5-trisphosphate. Mibefradil at supratherapeutic concentrations (> or = microM) thus mobilized Ca2+ from an inositol 1,4,5-trisphosphate-sensitive Ca2+ pool in cultured rat cardiac fibroblasts and human platelets.
1. Although angiotensin II (ANG II) has been identified as a key factor in the development of cardiac hypertrophy and remodelling, the role of its degradation fragment ANG II (3-8), angiotensin IV (ANG IV), is unknown. The presence of ANG IV in the blood circulation as well as the identification of ANG IV receptors in the heart and other organs indicates that ANG IV may act as a peptide hormone. 2. ANG IV receptors were characterized by binding of 125I-ANG IV to membranes of cultured rabbit cardiac fibroblasts. Incorporation of [3H]thymidine, [3H]uridine and [3H]leucine into DNA, RNA and proteins, respectively, was determined to analyse the growth effects of ANG IV, ANG II and the combination of both peptides. 3. ANG IV displaces 125I-ANG IV bound to membranes of rabbit cardiac fibroblasts with high affinity, whereas ANG II receptor-specific ligands ([Sar1,Ile8]ANG II, losartan, CGP 42 112 A) do not. 125I-ANG IV binds to a single class of binding site with a dissociation constant (Kd) of 4.87 +/- 0.11 nmol/l. The density of ANG IV receptors (Bmax) is 371 +/- 8.3 fmol/mg of protein. 125I-ANG IV binding is not markedly affected in the presence of the non-hydrolysable GTP analogue GTP gamma S, whereas binding of 125I-ANG II is reduced. 4. In quiescent cells, a 24 h exposure of ANG IV (100 nmol/l) increased rates of thymidine and uridine incorporation by 127% and 246%, respectively. A small but statistically insignificant increase in leucine incorporation was observed under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
Autosomal dominant arrhythmogenic right ventricular cardiomyopathy (ARVD, MIM 107970) is one of the major causes of juvenile sudden death. We have previously assigned the disease locus to chromosome 14q23-q24. Here we report on a novel variant of ARVD, which is transmitted associated to 1q42-q43 and is characterized by a concealed form, showing effort-induced polymorphic tachycardias. Since both loci ARVD1 and ARVD2 map in proximity of alpha-actinin genes, the possible implication of these myofibrillar proteins in the pathogenesis of ARVD is discussed. Two additional ARVD families, tested with markers of chromosomes 1q42-q43 and 14q23-q24, failed to show linkage, providing evidence of further genetic heterogeneity.
Ca2+ and Mg2+ binding to rat parvalbumin was measured by means of the fluorescent Ca2+ indicator fluo-3 using a method developed earlier [Eberhard, M. & Erne, P. (1991) Eur. J. Biochem. 202, 1333-1338]. We demonstrate that rat parvalbumin contains two equivalent Ca2+/Mg2+ binding sites and that Ca2+ and Mg2+ compete for the same sites. Dissociation constants (Kd) for Ca2+ and Mg2+ in Hepes buffer containing 150 mM K+ at 35 degrees C and pH 7.2 are 11.0 +/- 1.8 nM and 41 +/- 8 microM, respectively. At an ionic strength below 0.2 M, Kd values of Ca2+ binding to rat parvalbumin are approximately proportional to the ion concentration. Kd values of Ca2+ binding were found to be about fourfold larger in the presence of Na+ as compared with K+, indicating that Na+ distinctly influences Ca2+ binding to rat parvalbumin. Both Ca2+ and Mg2+ binding to parvalbumin are exothermic whereas Ca2+ and Mg2+ binding to fluo-3 are endothermic entropy-driven processes.
In many developing countries health data are insufficient. These data are needed to get valid information to make decisions about health programmes. Baseline surveys are in terms of economy and resources a method which is fruitful. These baseline surveys should not only be performed at country and province level. Baseline surveys at district level is needed to monitor local needs. The present baseline survey was focus on immunization, diarrhoea incidence and diarrhoea treatment, sanitation and children nutrition status. 388 households were visited in a random 30 cluster baseline survey in Choma district of Zambia. It revealed that 79% of the children aged 12-23 months was fully immunized and the drop-out rate in polio and DPT vaccination routines was as low as 9 and 12% respectively. The incidence of diarrhoea disease was high and estimated at 4.8 episodes per child per year. ORS and SSS were used as treatment in 52% and 15% of all cases. More than 90% of mothers would seek help at RHCs or PHCs in case of diarrhoea. About 2/3 of the households had no safe water supply and 2/3 no pitlatrine. Only a few had a refuse pit. The nutrition status of children were assessed by using upper arm circumference measurements. Only about 40% of the children age 12-23 month and about 75% of children aged 24-59 months respectively were adequately nourished. The district had in this survey a fair immunization programme but intensified sanitary and nutritional programmes seem justified. Periodic baseline surveys with 2-3 years interval is recommended to determine the impact and future direction of the programme.
Metabolism of inositol 1,4,5-trisphosphate (IP3) and IP3-induced release of Ca2+ was analyzed in permeabilized human platelets. Both rapid Ca2+ release and hydrolysis of IP3 to inositol 1,4-bisphosphate (IP2) was observed after addition of IP3 to permeabilized, Ca(2+)-loaded platelets. In the absence of ATP or in the presence of inhibitors of the Ca(2+)-ATPase of the endoplasmic reticulum, no release of Ca2+ and little hydrolysis of IP3 occurs, indicating a coupling between the Ca2+ gradient across the membrane of the IP3-sensitive Ca2+ store and conversion of IP3 to IP2. In addition, the rapid recovery of the sensitivity of the IP3-sensitive Ca2+ store after successive additions of IP3 (increment detection) appears to be associated with hydrolysis of IP3.
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A sensitive and rapid assay of Ca2+ binding to proteins was developed, based on the competition of Ca2+ binding to the protein of interest and fluo-3, a fluorescent Ca2+ indicator. Ca2+ binding to fluo-3 and bovine alpha-lactalbumin was analyzed at ten different pH values and a range of Na+ and K+ concentrations. We demonstrate that the binding constants of alpha-lactalbumin, determined by means of the competition assay and using intrinsic protein fluorescence, are the same within experimental error. The dissociation constant of the alpha-lactalbumin--Ca2+ complex in 50 mM Hepes containing 150 mM Na+ at pH 7.4 and 25 degrees C, was found to be 123 +/- 2 nM and 103 +/- 43 nM when determined by the competition assay and intrinsic protein fluorescence, respectively. Binding of Ca2+ to alpha-lactalbumin did not depend on pH in the range 6.6-8.4 and was differently affected by Na+ and K+. EDTA-agarose, a chelating chromatography material, was synthesized and used to remove Ca2+ from buffer and protein solutions. The total concentration of Ca2+ in 50 mM Hepes, containing 150 mM Na+ at pH 7.4, was lowered to 119 +/- 13 nM and the number of Ca2+ bound/molecule alpha-lactalbumin was lowered to 0.069 +/- 0.006. No interaction between fluo-3 and alpha-lactalbumin could be discerned from spectral analysis and fluorescence anisotropy measurements.