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A Hildebrandt

Publications and source records attributed to A Hildebrandt.

At least 37 records · Page 2Linked to original sources
PubMed1995

Chamber-specific expression of human myocardial proteins detected by two-dimensional gel electrophoresis.

High resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), followed by computer-assisted image analysis (PDQUEST) was used to screen atrial and ventricular protein patterns for quantitative and qualitative differences in protein expression. Myocardial proteins from left ventricular (LV) and right atrial (RA) samples from end-stage, failing explanted hearts and from a healthy donor heart (control) were separated by 2-D large gel electrophoresis. Ten RA versus ten LV gels from explanted dilated cardiomyopathic (DCM) hearts were analyzed for quantitative differences in their spot patterns. Of the 197 spots matched to every gel, 40 spots differed significantly in intensity between RA and LV for DCM patients. A larger number of atrial and ventricular gels (20 RA, 20 LV) from DCM patients and from a healthy donor heart (4 RA, 4 LV gels) were analyzed for qualitative differences in protein expression. Three protein spots (SSP 1120: M(r)/pI:20.5 kDa/4.6; SSP 1119: M(r)/pI:20.6 kDa/4.5; SSP 0117:M(r)/pI:20.7/ < 4.5) that are present in all RA gels for DCM patients are absent in all LV gels. Two protein spots (SSP 0112: M(r)/pI:17.2 kDa,/ < 4.4; SSP 0114:M(r)/pI:17.6 kDa/ < 4.4) occur only in all LV gels but not in the RA gels. These five qualitatively differing spots are identical in DCM patients and in the healthy donor heart. Some of the differing spots were internally sequenced and identified as myosin light chain isoforms (myosin light chain 2, atrial; myosin light chain 2, ventricular; myosin light chain 1, atrial) with the Protein Identification Resource (PIR) accession numbers A44451, S03708, A30881, respectively. Additionally, phosphoglycerate mutase (PIR: JQ0750) and ATP synthase alpha chain (PIR: S17193) were identified. Thus, quantitative and qualitative differences between atrial and ventricular protein patterns were identified by 2-D PAGE. A characteristic distribution of myosin light chains between atrial and ventricular human myocardium was found using our approach.

Amino Acid Sequence↗
PubMed1995

Reversal of multidrug resistance by novel cyclosporin A analogues and the cyclopeptolide SDZ 214-103 biosynthesized in vitro.

It was shown that cyclopeptolide SDZ 214-103 (10 microM) is more active in rhodamine-123 accumulation in actinomycin-D-resistant human lymphoma cells CCRF/ACTD400 than cyclosporin A (10 microM), but equipotent in the doxorubicin-resistant Friend erythroleukemia cell line F4-6/ADR. In F4-6/ADR cells, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay showed comparable cytotoxic effects of doxorubicin at various concentrations in the presence of SDZ 214-103 and cyclosporin A. For the other novel cyclosporin A analogues minor multidrug-resistance-modulating potency was demonstrated. At equipotent modulating doses of verapamil (10 microM) and cyclosporin A (10 microM) in the MTT assay regarding doxorubicin cytotoxicity, cyclosporin A was efficient in the rhodamine-123-uptake assay while verapamil was not active when identical incubation times were used.

Cyclosporins↗
PubMed1994

Human cardiac fibroblasts express an angiotensin receptor with unusual binding characteristics which is coupled to cellular proliferation.

To assess the cellular localization of angiotensin receptors in human heart, we isolated human cardiac fibroblasts from explanted end-stage failing human hearts. As judged by an immunofluorescence assay, the isolated cells consisted of 96% of fibroblasts. Using receptor binding studies, we could identify a single angiotensin binding site on these cells, with a Kd of 0.6nM and a Bmax of 1.5 fmol/mg protein. Inhibiting concentrations 50 for Ang II and Ang I/II (1-7) were 40nM and 10nM, respectively. Stimulation with Ang II (100 nM) and Ang I/II (1-7) (100nM) led to cellular proliferation which could not be inhibited by Losartan or PD 123319. These results suggest that human cardiac fibroblasts express an as yet unknown angiotensin receptor-subtype.

Angiotensin II↗
PubMed1994

Characterization of myocardial protein composition in dilated cardiomyopathy by two-dimensional gel electrophoresis.

In order to identify alterations in the myocardial protein pattern that characterize dilated cardiomyopathy (DCM), we compared, by two-dimensional gel electrophoresis, right atrial protein patterns from five patients with DCM and four with normal left ventricular function (two gels per patient). Using computer assisted analysis (PDQUEST, 4.1) we found reproducible protein patterns in the 18 gels (23 x 30 cm, pH 4-9, molecular weight 10-150 kDa). In the two gels from the same patient, 91% of proteins were identical in their position in the pattern and the relative intensities of these protein species correlated with r = 0.85. Three hundred and two +/- 50 protein species were found in several gels, 186 in all 18 gels. Seven proteins in the DCM group were decreased in their relative intensity by > 100%, six were increased by > 100%. Significant quantitative differences between DCM and control patients were found for 25 protein species. Based on seven external marker proteins, a pH and molecular weight value could be calculated for each protein. So far, 30 protein species have been identified by antibodies, amino acid analysis or sequencing procedures. From the 25 proteins that are significantly different between DCM and controls, three have been identified. Expression of the mitochondrial creatine kinase and alpha cristallin B chain was significantly increased in DCM; the malate dehydrogenase family was also significantly decreased in DCM. Two-dimensional electrophoresis appears to be a powerful method for the detection of disease-associated alterations in the myocardial protein pattern.

Adult↗
PubMed1994

Myocardial cyclic AMP and norepinephrine content in human heart failure.

Impaired production of myocardial cyclic adenosine monophosphate (cAMP) is thought to contribute to contractile dysfunction in end stage heart failure, but myocardial cAMP content has not yet been evaluated in heart failure patients in comparison with controls. We therefore measured the myocardial content of cAMP by radioimmunoassay in endomyocardial biopsies from patients in different stages of heart failure and in controls and correlated it with biochemical and functional parameters. The myocardial content of norepinephrine was determined by HPLC in the same biopsies in order to assess if the myocardium studied was affected by heart failure. Myocardial cAMP (in fmol.microgram-1 non-collagen protein) in 20 patients with heart failure (LVEF: 27 +/- 8%, cAMP: 5.8 +/- 2.0) was unchanged in comparison with eight controls (LVEF: 64 +/- 4.7%, cAMP, 4.9 +/- 2.1). In contrast, myocardial norepinephrine (in pg.microgram-1 non-collagen protein) in the same biopsies was significantly reduced in heart failure (4.0 +/- 3.0) in comparison with the same controls (11.5 +/- 3.0, P < 0.0002). Plasma cAMP in 20 heart failure patients (22.0 +/- 4.2 pmol.l-1) was not different from controls(22.0 +/- 7.8), whereas plasma norepinephrine was increased (heart failure: 460 +/- 257 pg.ml-1, controls 182 +/- 49, P < 0.001). Myocardial cAMP levels are indistinguishable from controls in human heart failure and therefore do not contribute to a further characterization of the cardiac adrenergic system in these patients. This is most likely due to the impossibility of obtaining biopsies with truly unstimulated adenylyl cyclase activity.

Adult↗
PubMed1994

Isolation and characterization of macrovascular and microvascular endothelial cells from human hearts.

In vivo models to investigate mechanisms of local hemostasis in the macro- and microvascular coronary circulation are not available. Therefore, we established a culture system of human macro- and microvascular endothelial cells with high cellular yield and high endothelial cell purity. Microvascular endothelial cells from human hearts were isolated by enzymatic treatment of cardiac muscle preparations obtained during heart transplantation. The isolated microvessels were used to start cultures that were subsequently separated and purified from contaminating nonendothelial cells by paramagnetic beads linked to the lectin Ulex europaeus agglutinin I. Macrovascular endothelial cells were isolated from epicardial coronary arteries and purified by paramagnetic beads as well. With this method high purity (< 2% nonendothelial cells) was achieved as judged from fluorescence-activated cell sorting. Immunochemistry demonstrated the expression of several typical endothelial markers. The two endothelial cell types displayed functional heterogeneity in respect to bradykinin degradation and plasminogen activator inhibitor-1 activity. Thus the ability to selectively isolate and culture human macro- and microvascular cardiac endothelial cells provides a valuable tool to systematically investigate endothelial function in human hearts.

Bradykinin↗
PubMed1994

Tissue kallikrein activity and kinin release in human endothelial cells.

The kininogenase, tissue kallikrein (EC 3.4.21.8), has been identified in different blood vessels. The enzyme was mainly found in vascular smooth muscle cells. It is not known whether it is present and functionally active in vascular endothelial cells. The following study investigates the presence of tissue kallikrein in endothelial cells from human umbilical veins and pulmonary arteries. Tissue kallikrein was demonstrated in three ways: 1) by immunostaining in endothelial cells; 2) by measurement of tissue kallikrein activity using a colorimetric assay; 3) by the measurement of kinin release in intact and homogenised endothelial cells with a radioimmunoassay. Immunostaining demonstrated the presence of tissue kallikrein in endothelial cells from human umbilical veins and endothelial cells from human pulmonary arteries. Tissue kallikrein-like activity, measured by the degradation of D-Val-cyclohexyl-Ala-Arg-4-nitraniline, was 3.57 +/- 0.5 mU/10(6) endothelial cells from human umbilical veins and 7.52 +/- 0.84 mU/10(6) endothelial cells from human pulmonary arteries. Intracellular kinin concentrations were 424 +/- 83 pg/10(6) cells in endothelial cells from human umbilical veins and 576 +/- 146 pg/10(6) cells in endothelial cells from human pulmonary arteries, and they increased in a time-dependent manner after homogenisation. The increase was abolished by aprotinin (1000 kIU), an inhibitor of tissue kallikrein in both cell types. Addition of exogenous kallikrein (5 mU) to homogenised cells led to a five fold increase of kinin concentrations after five minutes, indicating a sufficient resource of cellular kininogen. Removal of extracellularly bound kininogen by washing with dextran sulphate (100 mg/l) resulted in an approximately 75% reduction of the cellular kinin release.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1994

Dilated cardiomyopathy: computer-assisted analysis of endomyocardial biopsy protein patterns by two-dimensional gel electrophoresis.

In order to identify disease-associated alterations in the myocardial protein patterns in dilated cardiomyopathy, we used 2-dimensional gel electrophoresis to analyse the proteins of endomyocardial biopsies from patients and controls. Proteins (150 micrograms) from biopsies (1-3 mg wet weight) were first separated by isoelectric focusing, then applied to large 2-dimensional gels. A computer-assisted system (PDQUEST) was used for spot detection, quantification and comparison of 2-dimensional protein patterns. From a single endomyocardial biopsy about 1000 different protein species were resolved. The spot pattern was influenced by the concentration of protein during sample preparation, by the amount of protein loaded onto the gels and by the development time of silver staining. Variances of spot position in the first and second dimension and in the long diagonals were less than 5%. Coefficients of variance for the spot quantities in 8 gels were 16 +/- 8%. Contaminating blood proteins could be identified in the biopsy patterns. Computer-assisted comparison between cardiomyopathy (n = 5) and controls (n = 5) over the whole gel revealed that 55 protein spots were increased 100%, 27 protein spots decreased 100%. Four proteins showed significant quantitative differences between the cardiomyopathic hearts and controls. Fourteen proteins were identified by amino acid analysis or microsequencing. An isoelectric point and molecular mass grid was laid over the whole gel based on these identified protein species, resulting in approximate isoelectric point values and molecular masses for all other protein species. Thus, myocardial 2-dimensional protein patterns obtained from endomyocardial biopsies can be used for the characterization of cardiac diseases.

Adult↗
PubMed1993

A highly conserved repetitive sequence from Physarum polycephalum contains nucleotide arrangements similar to replicator sequences.

An interspersed repetitive sequence from Physarum polycephalum has been cloned and analysed. The 394 bp sequence is highly conserved and contains several homopolymeric (dA)-(dT) tracts capable of forming bent DNA structures and a 10/11 match to the yeast-ARS-consensus sequence. The repetition frequency of the described sequence is about 3000 to 7000, a number that would fit with the distribution of replicator segments in Physarum.

Animals↗
PubMed1993

Effect of angiotensin-converting-enzyme inhibition on bradykinin metabolism by vascular endothelial cells.

The degradation of bradykinin by angiotensin-converting-enzyme (ACE) activity in cultured human endothelial cells was studied by direct measurement of bradykinin and by its effect on the release of endothelium-derived relaxing factors. The half-life of exogenous bradykinin (10,000 pg/ml) was calculated from the decay of the bradykinin concentration as 46 +/- 2 min in cell monolayers, 133 +/- 15 min in conditioned medium, and 24 +/- 2 min in homogenates. Most of the bradykinin-degrading activity in cell monolayers could be inhibited in a concentration-dependent manner by the ACE inhibitors lisinopril, ramiprilat, and captopril. Bradykinin-degrading activity was released into the culture medium containing one-fourth of the bradykinin-degrading activity found in the presence of cell monolayers. In cell homogenates higher unspecific bradykinin-degrading activities were present. The functional consequence of bradykinin degradation was demonstrated by the potentiating effect of ramiprilat on the generation of endothelium-derived relaxing factors nitric oxide and prostacyclin from endothelial cells. The study supports the concept of increased vasodilatory effects of bradykinin during ACE inhibition.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1990

A purified cellular extract accelerates the cell cycle in Physarum polycephalum.

Plasmodia of the myxomycete Physarum polycephalum (strain Cl) were collected at different times during the cell cycle and extracts were prepared from homogenates using a buffer optimized for microinjection into plasmodial veins. These extracts were injected into plasmodia during the first 3 h of the cell cycle. The time of the following mitosis was monitored and compared with that of the buffer-injected controls. Extracts of plasmodia homogenized 45 min before late telophase accelerated the onset of mitosis in the injected plasmodium up to 70 min, i.e., an advance of 10-14% compared to the 8- to 10-h cell cycle duration of the controls. The accelerating activity vanished completely after heating, freezing, or protease digestion, thus indicating the peptide nature of the active agent. Purification of the active compound by means of gel filtration revealed a molecular mass of about 2500 Da. The active portion of the extract was further fractionated by HPLC and the activity determined in a single peak.

Animals↗
PubMed1990

15-Deoxyspergualin for induction of graft nonreactivity after cardiac and renal allotransplantation in primates.

In order to assess the immunosuppressive potentials of 15-deoxyspergualin (15-DS) in a preclinical experiment, heterotopic cardiac (n = 27, group I) and classic renal (n = 25, group II) allotransplantations were performed in Chacma baboons. The following immunosuppressive regimens were applied: Groups IB and IIB were treated with 15-DS alone (4 mg/kg/day) for p.o. days 0-9. Groups IC and IIC were treated with cyclosporine A (10-40 mg/kg/day) for p.o. days 0-30. Groups ID and IID received a combination of 15-DS (for p.o. days 0-9) and CsA (for p.o. days 0-30). Groups IA and IIA served as control and received no medication. The mean graft survival was 11.0 days for group IA, 28.2 days for group IB (P less than 0.05; IB vs. IA), 32.4 days for group IC, and 43.1 days for group ID (P less than 0.025; ID vs. IA). After renal transplantation, the corresponding figures were 12.3 days for group IIA, 8.5 days for group IIB, 30.4 days for group IIC and 148.9 days for group IID (P less than 0.025; IID vs. IIA). After cardiac and renal transplantation, acute rejection was the main cause of graft failure. Treatment-related side effects, mainly gastrointestinal complications, were observed only in primates, who were treated with 15-DS alone. After cardiac transplantation, permanent graft non-reactivity was not achieved, but a delayed rejection occurred within a mean of 21.8 days after immunosuppression had been stopped. Following renal transplantation, graft nonreactivity was also not achieved in groups IIB and IIC. In group IID, however, 4 of 8 animals (50%) were graft-tolerant 340, 256, 244, and 164 days after treatment discontinuation. Thus, the combination of 15-DS and CsA led to a significant prolongation of graft survival in both groups. Long-term nonreactivity was achieved only after renal transplantation, when initially treated with 15-DS and CsA.

Animals↗
PubMed1989

Nuclear polyphosphate as a possible source of energy during the sporulation of Physarum polycephalum.

31P NMR spectroscopic analysis of the polyphosphate pool in cellular and nuclear extracts of Physarum polycephalum demonstrates that plasmodia and cysts contain inorganic polyphosphates with an average chain length of about 100 phosphates. However, only during sporulation are these high-molecular-weight polyphosphates degraded to a lower molecular weight corresponding to an average chain length of about 10 phosphates. Since polyphosphates are degraded even in the presence of a sufficiently large pool of inorganic phosphate, produced by intracellular injection, we conclude that the degradation of polyphosphates serves in supplying energy for biosynthesis during sporulation rather than in increasing the availability of phosphate.

Cell Nucleus↗
PubMed1989

Heart transplantation--the treatment of choice for patients with end-stage ischaemic heart disease.

Between December, 1967 and August, 1988, 147 heart transplants (64 orthotopic, 68 heterotopic procedures; 15 heart-and-lung replacements) were performed on 128 patients. In the majority of the recipients, dilated cardiomyopathy or end-stage ischaemic heart disease was diagnosed. From 1985 to the present, 70 transplants (45 orthotopic, 11 heterotopic and 14 heart-lung) took place. Seventeen of these patients (mean age 46.6 years) suffered from end-stage disabling (NYHA IV) coronary artery disease; in each case the angiogram verified severe stenosis or occlusion of the three main coronary artery systems. Their histories revealed a total of 22 previous myocardial infarctions; 8 patients needed a total of 9 surgical revascularization procedures. The left ventricular ejection fraction (LVEF) ranged from 9% to 24% (mean 15.3%). Before transplantation three patients required intraaortic balloon pump (IABP) support. Fourteen of the 17 patients are at present still alive with post operative periods ranging from 8 weeks to 3.5 years (the actuarial 90-days and 1-year-survival rates being respectively 91.7% and 81.5%). Twelve of the patients are in NYHA class I; 2 are in class II. Three late deaths occurred: one from pneumocystic carinii/cytomegalovirus pneumonia, a second from atypical pneumonia and a third from chronic graft rejection. Radionuclide ventricular studies demonstrated postoperative left ventricular ejection fractions ranging from 54%-81% (mean 71%).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗