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Biomedical subjects

M Böhm

Publications and source records attributed to M Böhm.

At least 307 records · Page 17Linked to original sources
PubMed1995

Myocardial Gi alpha-protein levels in patients with hypertensive cardiac hypertrophy, ischemic heart disease and cardiogenic shock.

OBJECTIVE: Increased inhibitory G-protein alpha-subunits (Gi alpha) have been reported to be related to adenylyl cyclase desensitization in the failing human heart. In order to investigate whether this cellular alteration occurs already at the stage of hypertensive cardiac hypertrophy or in catecholamine-refractory cardiogenic shock, Gi alpha levels were studied in myocardial samples from patients with hypertensive cardiac hypertrophy, coronary heart disease without heart failure and from patients with cardiogenic shock on high-dose catecholamine therapy as well as from patients without evidence of heart disease. METHODS: Gi alpha was quantified with pertussis-toxin-catalyzed 32P-ADP-ribosylation and with a radioimmunoassay in myocardial samples from patients within 16 h of death. The radioimmunoassay was constructed with recombinant G-protein alpha-subunits (rGi alpha 1) from transformed E. coli harbouring the full-length cDNA of Gi alpha 1, iodinated peptide 125I-KENLKDCGLF and immunoprecipitating antiserum (MB 1) raised against the synthetic peptide (KENLKDCGLF) in rabbits. RESULTS: Pertussis toxin substrates and immunochemical Gi alpha remained stable up to 80 h following storage at room temperature in myocardium obtained during cardiac transplantation. Gs alpha, adenylyl cyclase, beta-adrenoceptors and inhibitory receptors were not stable and could not be determined. Increases in myocardial Gi alpha of 65-82% of both pertussis toxin substrates and immunologically quantified Gi alpha were observed in hypertensive cardiac hypertrophy. Catecholamine therapy in patients who died of catecholamine-refractory shock increased myocardial Gi alpha by 225% compared to myocardium from patients with coronary heart disease without heart failure and without catecholamine therapy or compared to control myocardium. CONCLUSION: These findings provide evidence than an increase in myocardial Gi alpha-proteins could be of relevance in pathological conditions other than chronic heart failure. Since an increase in Gi alpha levels already occurs in hypertensive cardiac hypertrophy, it could play a role in contributing to the development of contractile dysfunction and heart failure in later stages of the syndrome. Finally, an increase in Gi alpha could be one mechanism contributing to catecholamine refractoriness in shock. This could provide a target for pharmacological treatment in this condition.

Autoradiography↗
PubMed1994

Frequent allelic deletion at a novel locus on chromosome 5 in human lung cancer.

Frequent allelic deletions in tumor cells are indicative of the inactivation of tumor suppressor genes. Recently, we isolated the single-copy sequence del-27 (I. Wieland, M. Böhm, and S. Bogatz, Proc. Natl. Acad. Sci. USA, 89: 9705-9709, 1992). Here we show that del-27 detects a restriction fragment length polymorphism that allows examination for loss of heterozygosity (LOH) in tumor specimens. LOH at the del-27 locus occurred in 57% (4 of 7) of the informative lung carcinomas independent of the histopathological differentiation grade. LOH for exon 11 of the APC gene occurred in 71% (5 of 7) of the informative cases but was not associated with LOH at the del-27 locus. The del-27 sequence was localized to chromosome 5p13-5q14, proximal to the MCC/APC region, using a somatic cell hybrid panel. Together with our previous finding that del-27 is deleted homozygously in a lung carcinoma cell line, these results suggest that del-27 is linked closely to a novel putative tumor suppressor gene.

Alleles↗
PubMed1994

Antiadrenergic effect of carbachol but not of adenosine on contractility in the intact human ventricle in vivo.

OBJECTIVES: The purpose of this study was to investigate the antiadrenergic effects of adenosine and carbachol on beta-adrenoceptor-stimulated human ventricular contractility in vivo. In addition, the antiadrenergic effects of adenosine and carbachol were compared in vitro. BACKGROUND: Adenosine is reported to exhibit an antiadrenergic negative inotropic response in the beta-adrenergic-stimulated ventricular myocardium in vitro. The effect of adenosine is similar to the antiadrenergic effect of m-cholinoceptor stimulation in vitro. METHODS: The inotropic response in vivo was assessed in seven healthy volunteers by M-mode echocardiography and simultaneous blood pressure monitoring. It was calculated as the increase in the rate-corrected velocity of circumferential fiber shortening and in the systolic pressure/dimension ratio. All volunteers received pretreatment with 450 mg of dipyridamole/day for 48 h. In addition, the effects of adenosine and carbachol in the presence of 0.03 mumol/liter of isoproterenol on cumulative concentration-response curves of isolated, electrically driven human ventricular muscle strips were compared in vitro (n = 13). RESULTS: The positive inotropic response to continuous infusion of 20 ng/kg per min of isoproterenol (increase of rate-corrected velocity of circumferential fiber shortening [10.2 +/- 2.1% x square root of beats/min per ms] and increase of systolic pressure/dimension ratio 1.09 +/- 0.3 mm Hg/mm) was significantly (p < 0.01) reduced by 3.6 micrograms/kg body weight of intravenous carbachol (4.2 +/- 1.2% x square root of beats/min per ms, 0.21 +/- 0.18 mm Hg/mm) but not by 50 micrograms/kg of intravenous adenosine (8.2 +/- 3.1% x square root of beats/min per ms, 1.35 +/- 0.42 mm Hg/mm), although adenosine induced a significant negative dromotropic effect. In vitro comparison of force of contraction with cumulative concentration-response curves in the presence of 0.03 mumol/liter of isoproterenol demonstrated an EC50 value (concentration producing half-maximal effect) for adenosine 466 times higher than that for carbachol (65.3 vs. 0.14 mumol/liter, p < 0.001). CONCLUSIONS: In contrast to carbachol, adenosine does not attenuate the catecholamine-induced increase in contractility in the human ventricle in vivo. These differences between the A1-adenosine receptor- and m-cholinoceptor-mediated effects could be due to fewer A1-adenosine receptors or a less efficient receptor-effector coupling, or both.

Adenosine↗
PubMed1994

The amido black assay: a simple and quantitative multipurpose test of adhesion, proliferation, and cytotoxicity in microplate cultures of keratinocytes (HaCaT) and other cell types growing adherently or in suspension.

A new multipurpose cell micro-assay has been developed, using the protein dye amido black 10B as an indicator of cell numbers in 96-well plates. The assay is reliable, rapidly performed and can be combined with morphological evaluation and photography of stained cells. It permits investigations of various cell types including the human keratinocyte line HaCaT and subclones, mouse 3T3 fibroblasts and myeloma cells X63-Ag8.653. Briefly, cells are fixed by formaldehyde or glutaraldehyde and, following aspiration of fixative and non-adherent cells, are stained by amido black at pH 3.5. The protein-bound dye is completely eluted by NaOH and is scanned in a microplate reader at 620 nm against 405 nm or 750 nm. Non-adherent and semi-adherent cells are assayed by centrifugation of plates before fixation. The assay revealed a good linear correlation between absorbance of amido black, cell count and DNA content within the range 1000-64,000 HaCaT cells/well. The slope of the regression line varied with different cell types. Experiments with HaCaT cells and its c-Ha-ras oncogene-transfected subclones demonstrated the suitability of the assay for optimizing culture conditions, dose-response studies and for the screening and quantification of cell adhesion to extracellular matrix molecules. The assay was also used to evaluate cytotoxicity of drugs such as hexadecylphosphocholine, target cell killing in co-cultures with interleukin-2-activated lymphocytes, and the testing of hybridoma antibodies for their biological effects on proliferation and adhesion. The assay is highly reproducible, sensitive, independent of cellular aggregation and economic for multiple applications.

3T3 Cells↗
PubMed1994

Radioimmunochemical quantification of Gi alpha in right and left ventricles from patients with ischaemic and dilated cardiomyopathy and predominant left ventricular failure.

An increase of Gi alpha-related pertussis toxin substrates has been observed in the failing myocardium. In order to quantify the protein expression of Gi alpha directly, we developed a fast radioimmunoassay using the iodinated synthetic peptide 125I-KENLKDCGLF. beta-adrenoceptors were studied with 125I-cyanopindolol binding for comparison. Immunoblot experiments using recombinant G-protein alpha-subunits showed that DS4 immunostained the G-protein alpha-subunits with a rank order of potency rGi alpha 1 = rGi alpha 2 > rGo alpha >> rGi alpha 3. The G-protein alpha-subunits recognized by DS4 in human ventricular membranes comigrated with rGi alpha 1 and rGi alpha 2. The radioimmunoassay had a sensitivity of 2.5 micrograms/ml transducin alpha with an interassay variation of less than 10%. The non-labelled peptide selectively competed with the myocardial 40 kDa membrane protein for binding to the antiserum DS4. Radioimmunochemical quantification of Gi alpha from cardiac membranes showed that in left ventricular membranes (LV) from dilated cardiomyopathy (DCM), there was an increase of Gi alpha by 138.5% when related to mg protein and 135% when related to 3H-ouabain binding sites as membrane marker. In LV from ischaemic cardiomyopathy (ICM), the increase was smaller (58.4%) when related to mg protein compared to the increase of Gi alpha when related to 3H-ouabain binding sites as membrane marker (155% v NF). In contrast, in the right ventricles (RV) there was no increase of Gi alpha in ICM or DCM. The numbers of beta-adrenoceptors were reduced in RV and LV of both, ICM and DCM. It is concluded that the radioimmunoassay may become an important tool for studying the expression of Gi alpha-protein levels and changes thereof in pathological conditions. The amount of immunodetectable Gi alpha-proteins is increased in failing LV due to DCM and ICM but not in RV, while beta-adrenoceptor down-regulation occurred in RV and LV in both conditions. These findings might indicate that the liability of the LV but not of RV to express Gi alpha-proteins may be increased in predominant LV heart failure. Alternatively, the underlying mechanism, e.g. sympathetic activation, may be regulated locally in the failing heart producing different changes in adjacent chambers.

Adolescent↗
PubMed1994

Titin, myosin light chains and C-protein in the developing and failing human heart.

We investigated relative amounts of titin, myosin light chains (MLC), C-protein, and myosin heavy chains (MHC) in the functionally intact contractile apparatus of embryonic, adult normal, and adult failing human left ventricle by SDS polyacrylamide gel electrophoresis and Western blot analysis. The amount of titin and the titin-MHC ratio was significantly (P < 0.05) lower in the embryonic and adult failing human compared to the adult normal fibres. Additionally, we found a protein band having a lower molecular weight but the same immunoreactivity as native titin (fast-migrating titin; FM-titin) in the failing heart only. The MLC-1/MLC-2 ratio was about 1.0 in normal and failing hearts but about 2.0 in the embryonic heart. Relative amount of C-protein was the same in normal and failing fibres but lower in the embryonic ventricles. Length-tension ratio of chemically skinned fibres prepared from terminally failing hearts was impaired compared to normal heart fibres. We conclude that both the reduced titin/MHC ratio and the expression of a structurally different titin form may be involved in the impaired contractile function of the terminally failing human heart.

Carrier Proteins↗
PubMed1994

Differences of E-cadherin expression levels and patterns in human lung cancer.

Fifty-two lung carcinomas obtained at surgical resection were examined by immunofluorescence for their expression levels and patterns of the calcium-dependent intercellular adhesion molecule E-cadherin. In well-differentiated squamous cell and adenocarcinomas expression of E-cadherin was confined to the lateral cell border, similar to the expression level and pattern of normal lung tissue. The E-cadherin level was reduced and the expression pattern was spotty or diffuse in moderately and poorly differentiated squamous cell carcinomas and in small cell carcinomas of the lung. Also, most metastases resected had a reduced level and an altered pattern of E-cadherin expression. In contrast, no such correlation was found in adenocarcinomas of the lung. This indicates that different cellular mechanisms are responsible in the progression of squamous cell carcinomas versus adenocarcinomas of the lung.

Adenocarcinoma↗
PubMed1994

Differences of E-cadherin expression levels and patterns in primary and metastatic human lung cancer.

Normal lung epithelium and 52 lung carcinomas obtained at surgical resection were examined by immunofluorescence for their expression levels and patterns of the calcium-dependent intercellular adhesion molecule E-cadherin. In dysplastic lung tissue and in well-differentiated squamous cell and adenocarcinomas, expression of E-cadherin was confined to the lateral cell border, similar to the expression level and pattern of normal lung tissue. The E-cadherin level was reduced and expression pattern was spotty or diffuse in moderately and poorly differentiated squamous cell and in small cell carcinomas of the lung. Most metastases resected also had a reduced level and an altered pattern of E-cadherin expression. In contrast, no such correlation was found in adenocarcinomas of the lung. This indicates that different cellular mechanisms are responsible in the progression of squamous cell carcinomas and adenocarcinomas of the lung.

Adenocarcinoma↗
PubMed1994

Changes in cAMP formation in mononuclear leukocytes of heart and renal transplant recipients.

In mononuclear leukocytes (MNL) of renal transplant recipients treated with cyclosporine A and prednisone, an increase of basal cAMP generation has been observed. In order to characterize the mechanisms underlying changes of cAMP generation in patients who were treated with immunosuppressives following heart transplantation, we investigated the beta-adrenoceptor--G protein--adenylate cyclase signal transduction cascade in heart transplant recipients and for comparison in renal transplant recipients as well as controls. Basal cAMP formation in MNL was elevated in heart transplant recipients by 272% and in renal transplant recipients by 148% compared to controls. Following beta-adrenoceptor stimulation with isoprenaline, cAMP formation in MNL of heart transplant recipients was similar to the controls, but was enhanced in renal transplant recipients to 138%. Investigation of beta-adrenoceptor density on MNL as a possible cause for increased cAMP formation revealed similar receptor numbers in controls and in cardiac or renal transplant recipients. Furthermore, the increase of the beta-adrenoceptor density on MNL, which is observed following infusion of isoprenaline, was similar in controls and heart transplant recipients. The amount of pertussis- and cholera toxin substrates was the same in heart transplant recipients as in controls. In contrast, MNL of renal transplant recipients showed a marked increase of Gs alpha by 45% and a smaller albeit significant increase of Gi alpha by 15%, as judged by cholera toxin and pertussis toxin labeling, respectively. Investigation of inotropic parameters by echocardiography under control conditions and during the infusion of increasing concentrations of isoprenaline revealed no difference in the basal contractility and the inotropic response to beta-adrenergic stimulation in controls and heart transplant recipients. It is concluded that changes of G-protein expression are involved in the increase of the cAMP-generation in MNL of heart transplant recipients. These alterations in MNL cannot be taken as a model of cellular function in the transplanted heart, but it is reasonable to suggest that elevations of cAMP formation in MNL may contribute to the immunosuppressive effects of the treatment with cyclosporine A or corticosteroids, the mechanism of which could be an alteration of Gs alpha or the catalyst in renal transplant recipients and the catalyst in heart transplant recipients which occurs without any changes of beta-adrenoceptors.

Adenylate Cyclase Toxin↗
PubMed1994

Lysis of allogeneic and autologous melanoma cells by IL-7-induced lymphokine-activated killer cells.

In order to assess the potential of interleukin 7 (IL-7) as an immunotherapeutic agent in human melanoma, we have evaluated the in vitro activity of IL-7-induced lymphokine-activated killer (LAK) cells from patients with advanced melanoma against allogeneic and autologous melanoma cells. Peripheral blood lymphocytes (PBLs) from 14 patients with stage III melanoma were isolated and incubated in the presence of 1,000 U ml-1 IL-7 and 100 U ml-1 IL-2 for comparison. LAK-cell activity was determined by a 24 h cytotoxicity assay using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. The activity of IL-7-induced LAK cells against two allogeneic melanoma cell lines was 32.7% (+/- 17.9) against SK-Mel-37 and 38.1% (+/- 12.5) against SK-Mel-23 at an effector-to-target (E/T) ratio of 20:1. The activity of IL-2-induced LAK cells was significantly higher against SK-Mel-37 (78 +/- 24.6%) and against SK-Mel-23 (73.5 +/- 19.7%). IL-7 and suboptimal doses of IL-2 (10 U ml-1) were found to have a co-stimulatory on lymphocyte proliferation as well as on LAK activity. Against autologous melanoma cells, the activity of IL-7- and IL-2-induced LAK cells did not differ significantly (55.8 +/- 25.6% versus 68.7 +/- 21.7% respectively). In two patients, IL-7-induced LAK-cell activity against autologous melanoma cells exceeded even that of IL-2 significantly (67% vs 35% and 95% vs 82%). Levels of tumour necrosis factor alpha (TNF-alpha) in the supernatants of LAK-cell cultures generated by IL-7 were lower than those of IL-2-generated LAK-cell cultures. These results suggest that IL-7 is a potential alternative to immunotherapy with IL-2 in terms of efficacy and possible side-effects and encourages pilot studies with IL-7 in melanoma patients.

Adult↗
PubMed1994

Influence of halothane on the effect of cAMP-dependent and cAMP-independent positive inotropic agents in human myocardium.

Volatile anaesthetics have a variety of effects on the myocardium, namely a negative inotropic effect and a catecholamine sensitizing effect. The present study was designed to see if the hydrocarbon anaesthetics interact specifically with subcellular targets of the myocardial cell, by examining the effects of halothane in the presence of positive inotropic agents with different mechanisms of action. Experiments were performed in isolated electrically driven left ventricular preparations (1 Hz, 37 degrees C, Ca2+ 1.8 mmol litre-1) from human hearts obtained at cardiac surgery. The concentration-response curves of noradrenaline, milrinone, BayK 8644 and Ca2+ were investigated in the absence and in the presence of halothane. Halothane enhanced the efficacy of noradrenaline and milrinone but not of Ca2+ or BayK 8644. The potency of milrinone was also increased by halothane, whereas the potency of BayK 8644 was decreased and those of noradrenaline and Ca2+ were unchanged. Halothane differentially influences the effects of agents with different positive inotropic mechanisms. This experimental approach can be taken as a functional method to localize the mechanisms of action of the inhalation anaesthetics in human myocardium, namely sensitization of cAMP formation and interaction with L-type Ca2+ channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

cAMP concentrations, cAMP dependent protein kinase activity, and phospholamban in non-failing and failing myocardium.

OBJECTIVE: Several signal transduction defects such as a reduction of myocardial cAMP formation and an altered intracellular Ca2+ handling have been observed in the failing human myocardium. The aim of the study was to obtain data on changes beyond cAMP formation involving cAMP dependent protein kinase and its substrates. METHODS: cAMP dependent protein kinase activity and cAMP concentrations were measured in the particulate and soluble fraction of failing human hearts (ischaemic, and dilated cardiomyopathy) and non-failing donor hearts. Phospholamban was quantified by cAMP dependent phosphorylation using 32P-ATP as substrate and on western blots using a monoclonal antibody. RESULTS: cAMP concentrations were reduced in the particulate fraction in both ischaemic and dilated cardiomyopathy and in the soluble fraction in dilated cardiomyopathy, but there was no difference in cAMP dependent protein kinase activity. Both phospholamban levels and cAMP dependent phosphorylation of phospholamban were similar in non-failing myocardium and in both ischaemic and dilated cardiomyopathy. CONCLUSIONS: These findings show that the reduction of cAMP formation is the predominant alteration in heart failure, but cAMP dependent protein kinase and phospholamban are evidently unchanged.

Adenosine Triphosphatases↗