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Biomedical subjects

U Pohl

Publications and source records attributed to U Pohl.

At least 37 records · Page 2Linked to original sources
PubMed2000

Pitfalls of using lucigenin in endothelial cells: implications for NAD(P)H dependent superoxide formation.

Since an increased endothelial superoxide formation plays an important role in the pathogenesis of endothelial dysfunction its specific detection is of particular interest. The widely used superoxide probe lucigenin, however, has been reported to induce superoxide under certain conditions, especially in the presence of NADH. This raises questions as to the conclusion of a NAD(P)H oxidase as the major source of endothelial superoxide. Using independent methods, we showed that lucigenin in the presence of NADH leads to the production of substantial amount of superoxide (approximately 15-fold of control) in endothelial cell homogenates. On the other hand, these independent methods revealed that endothelial cells without lucigenin still produce superoxide in a NAD(P)H-dependent manner. This was blocked by inhibitors of the neutrophil NADPH oxidase diphenyleniodonium and phenylarsine oxide. Our results demonstrate that a NAD(P)H-dependent oxidase is an important source for endothelial superoxide but the latter, however, cannot be measured reliably by lucigenin.

Acridines↗
PubMed2000

Antisense oligonucleotides against cytochrome P450 2C8 attenuate EDHF-mediated Ca(2+) changes and dilation in isolated resistance arteries.

Using a novel vessel culture technique in combination with antisense oligonucleotide transfection, we tested whether the endothelium-derived hyperpolarizing factor (EDHF) is a cytochrome P450 (CYP)-related compound. Isolated resistance arteries from hamster gracilis muscle (n=19) were perfused and exposed to antisense (As), sense (S), or scrambled (Scr) oligonucleotides against the coding region of CYP2C8/9, an isoform expressed in endothelial cells. Thereafter, NO- and prostaglandin-independent, EDHF-mediated vascular responses associated with hyperpolarization [i.e., decrease in smooth muscle calcium (Fura 2) and vasodilation] were studied after the application of acetylcholine (ACh). These EDHF-mediated responses were markedly attenuated (by 70%) by As- but not by S- or Scr-oligonucleotide treatment. However, the responses to norepinephrine (0.3 micromol/l), the NO donor sodium nitroprusside (1 micromol/l), and the K(Ca) channel activator NS1619 (100 micromol/l) were unaltered. As treatment, which specifically targeted the endothelial layer (as assessed by confocal microscopy), had no inhibitory effect on increases in endothelial calcium to ACh. It is concluded that a CYP2C8/9-related isoform functions as an EDHF synthase in hamster resistance arteries and that a product of this enzyme is an EDHF, or at least an integral part of the signaling cascade leading to EDHF-mediated responses.-Bolz, S.-S., Fisslthaler, B., Pieperhoff, S., de Wit, C., Fleming, I., Busse, R., Pohl, U. Antisense oligonucleotides against cytochrome P450 2C8 attenuate EDHF-mediated Ca(2+) changes and dilation in isolated resistance arteries.

Acetylcholine↗
PubMed2000

Intact endothelial and smooth muscle function in small resistance arteries after 48 h in vessel culture.

Long-term culture of resistance vessels allows introduction of molecular biology techniques for use in microvascular research. The aim of the present study was to establish a culture protocol that preserved vascular integrity and function in microvessels for 48 h in culture. Skeletal muscle resistance arteries were excised from the hamster gracilis muscle. Segments were assigned to immediate functional tests or to vessel culture, during which segments were perfused and superfused at a transmural pressure of 45 mmHg with Leibovitz (L15) medium containing 15% fetal calf serum and antibiotics for 48 h. Cultured and freshly isolated vessels showed similar levels of spontaneous tone, myogenic responses, changes in smooth muscle intracellular calcium (Ca(i)(2+)) (fura 2), and vascular diameter (video microscopy) in response to 0.3 M norepinephrine and similar concentration-response curves for acetylcholine (endothelium dependent, +/-N(omega)-nitro-L-arginine) and sodium nitroprusside (endothelium independent). Measurements of endothelial Ca(i)(2+) revealed similar acetylcholine-induced increases in endothelial Ca(i)(2+) in both groups. It is concluded that vascular function can be preserved while maintaining vessels in culture. Thus it is possible to utilize protocols that require long-term treatment.

Animals↗
PubMed2000

Analysis and modeling of the treatment process characterizing the cooperation within multi-professional treatment teams.

We would like to introduce several aspects of the analysis and modeling of the treatment process characterizing the cooperation within multi-professional treatment teams. We will determine what is meant by a treatment process in order to then look at five views and four levels of their description. We will introduce possible methods for surveying and describing it. Currently an extensive analysis of the current state of the treatment process and of the weaknesses is underway in the Department of Child and Adolescent Psychiatry of the Heidelberg University Medical Center.

Computer Simulation↗
PubMed1999

Morphological and functional effects of antisense RNA to the deleted in colorectal carcinoma (DCC) gene in a pancreatic carcinoma cell line.

To investigate the role of the deleted in colorectal carcinoma gene (DCC) in cells of pancreatic origin (MiaPaCa-2) we established cell lines stably expressing DCC antisense RNA. Expression of DCC antisense RNA led to striking alterations in the MiaPaCa-2 cell line. Antisense transfectants had nearly lost adherence and had acquired a spherical morphology. The ordered structure of actin bundles in the parental cell line had been lost largely in DCC antisense RNA expressing cell clones. Moreover, the antisense DCC transfected cells displayed a decreased growth rate, a decrease of cells in G1 phase and an accumulation in S phase of the cell cycle. These heavily altered characteristics of MiaPaCa-2 cells expressing DCC antisense RNA point to a yet unknown role for DCC in an important intracellular pathway.

Carcinoma, Ductal, Breast↗
PubMed1999

The 67-kDa laminin-binding protein is involved in shear stress-dependent endothelial nitric-oxide synthase expression.

It has been suggested that the mechanical forces acting on endothelial cells may be sensed in part by cell-matrix connections. We therefore studied the role of different matrix proteins, in particular laminin I, on a shear stress-dependent endothelial response, namely nitric-oxide synthase (eNOS) expression. Primary porcine aortic endothelial cells were seeded onto glass plates either noncoated (NC cells) or precoated with fibronectin (FN cells), laminin (LN cells), or collagen I (CN cells). Western blots were used to detect differences in the final matrix composition of these cells. A shear stress of 16 dyn/cm2 was applied for 6 h. Only LN cells showed detectable amounts of laminin I in their underlying matrix when they reached confluence. They reacted with a 2-fold increase of eNOS expression (n = 16, p < 0.001) to the exposure of shear stress, which went along with enhanced eNOS protein and NO release. In contrast, neither FN cells (n = 9) nor NC cells (n = 13) showed a significant increase of eNOS expression under shear stress. The increase in CN cells was borderline (1.4-fold; n = 9, p < 0.05) and was not associated with an increase of eNOS protein. The shear-induced increase in eNOS expression of LN cells was abolished by the peptide YIGSR, which blocks the cellular binding to laminin I via a 67-kDa laminin-binding protein, whereas a control peptide (YIGSK) had no effect. The induction of eNOS expression by shear stress is stimulated by an interaction of endothelial cells with laminin which is, at least in part, mediated by a 67-kDa laminin-binding protein.

Animals↗
PubMed1999

The human glia maturation factor-gamma gene: genomic structure and mutation analysis in gliomas with chromosome 19q loss.

Human glia maturation factor-gamma (hGMF-gamma) is a recently identified gene that may be involved in glial differentiation, neural regeneration, and inhibition of tumor cell proliferation. The gene maps to the long arm of chromosome 19 at band q13.2, a region that is frequently deleted in human malignant gliomas and is thus suspected to harbor a glioma tumor suppressor gene. Given the putative role of hGMF-gamma in cell differentiation and proliferation and its localization to chromosome 19q13, this gene is an interesting candidate for the chromosome 19q glioma tumor suppressor gene. To evaluate this possibility, we determined the genomic structure of human hGMF-gamma and performed mutation screening in a series of 41 gliomas with and without allelic loss of chromosome 19q. Mutations were not detected, which suggests that hGMF-gamma is not the chromosome 19q glioma suppressor gene. However, the elucidation of the genomic structure of hGMF-gamma may prove useful in future investigations of hGMF-gamma in the normal adult and developing human nervous system.

Adult↗
PubMed1999

Mobilizing a health professional's workstation--results of an evaluation study.

To evaluate the idea of a multifunctional mobile pen computer to support information processing tasks of health care professionals we carried out a field study. On a tablet-sized mobile pen computer we set up a health professional workstation, MEDIAS/WIN, with most of the application systems that are presently used by health professionals at Heidelberg University Medical Center. Our experiences during the study, involving 11 physicians, nine nurses and four computer scientists showed that it's not enough to make existing applications mobile most user interfaces have to be adapted for pen input. The presented system served as a valuable basis for study purposes but was not suitable for use in clinical routine. Patient data of the current clinical case and medical knowledge in the form of reference lists were found to be most important for mobile usage. Desirable functionality also included mobile access to electronic mail, electronic ordering of meals and some documentation tasks, e.g. the documentation of diagnoses and orders for diagnostic and therapy.

Electronic Data Processing↗
PubMed1999

Endothelium-derived hyperpolarizing factor but not NO reduces smooth muscle Ca2+ during acetylcholine-induced dilation of microvessels.

1. We hypothesized that nitric oxide (NO) and the endothelium-dependent hyperpolarizing factor (EDHF) may dilate microvessels by different cellular mechanisms, namely Ca2+-desensitization versus decrease in intracellular free calcium. 2. Effects of acetylcholine (ACh) and the NO donors sodium nitroprusside (SNP, 0.1 - 10 micromol l(-1)) and S-Nitroso-N-acetyl-D, L-penicillamine (SNAP, 0.01 - 10 micromol l-1) on intracellular calcium ([Ca2+]i, fura 2) and vascular diameter (videomicroscopy) were studied in isolated resistance arteries from hamster gracilis muscle (194+/-12 microm) pretreated with indomethacin and norepinephrine. Membrane potential changes were determined using 1, 3-dibutylbarbituric acid trimethineoxonol (DiBAC4(3)). 3. ACh (0.1 and 1 micromol l-1)-induced dilations were associated with a [Ca2+]i decrease (by 13+/-3 and 32+/-4%) and hyperpolarization of vascular smooth muscle (VSM, by 12+/-1% at 1 micromol l-1 ACh). Nomega-nitro-L-arginine (L-NA, 30 micromol l(-1)) partially inhibited the dilation but did not affect VSM [Ca2+]i decreases or hyperpolarization. In contrast, the KCa channel inhibitors tetrabutylammonium (TBA, 1 mmol l(-1)) and charybdotoxin (ChTX, 1 micromol l(-1)) abolished the ACh-induced [Ca2+]i decrease and the hyperpolarization in VSM while a significant dilation remained (25 and 40%). This remaining dilation was abolished by L-NA. ChTX did not affect [Ca2+]i increase and hyperpolarization in endothelial cells. SNP- or SNAP-induced dilations were not associated with decreases in VSM [Ca2+]i or hyperpolarization although minor transient decreases in VSM [Ca2+]i were observed at high concentrations. 4. These data suggest that ACh-induced dilations in microvessels are predominantly mediated by a factor different from NO and PGI2, presumably EDHF. EDHF exerts dilation by activation of KCa channels and a subsequent decrease in VSM [Ca2+]i, NO dilates the microvessels in a calcium-independent manner.

Acetylcholine↗
PubMed1999

Homozygous deletions of the CDKN2C/p18INK4C gene on the short arm of chromosome 1 in anaplastic oligodendrogliomas.

Allelic deletions of the short arm of chromosome 1 are common in oligodendrogliomas and have been correlated with chemosensitivity and better prognosis in patients with high-grade oligodendrogliomas. In these tumors, 1p loss is also inversely related to deletions of the CDKN2A gene on 9p, which encodes the key cell cycle regulatory molecule p16INK4A. Because the CDKN2C gene, which encodes the homologous p18INK4C cell cycle regulatory protein, maps to chromosomal band 1p32, CDKN2C is an attractive candidate for the oligodendroglioma suppressor gene on chromosome 1. To evaluate this possibility, we studied 39 high-grade oligodendrogliomas for homozygous deletions and point mutations of the CDKN2C gene, as well as for allelic loss of 1p. Although no mutations were detected in the CDKN2C coding region, two tumors had homozygous deletions that involved CDKN2C. Interestingly, these cases did not have CDKN2A gene deletions. Coupled with the recent report of rare point mutations of CDKN2C in oligodendrogliomas, these findings suggest that CDKN2C inactivation may be oncogenic in a small percentage of human oligodendrogliomas.

Anaplasia↗
PubMed1999

Pentobarbital-sensitive EDHF comediates ACh-induced arteriolar dilation in the hamster microcirculation.

It is unclear to what extent the endothelium-derived hyperpolarizing factor (EDHF) contributes to the control of microcirculatory blood flow in vivo. We analyzed, by intravital microscopy in hamster muscles, the potential role of EDHF along the vascular tree under stimulated (ACh) or basal conditions. Experiments were performed in conscious as well as anesthetized (pentobarbital, urethan) animals. Additionally, cellular effects of the potential EDHF were studied in isolated small arteries. In pentobarbital-anesthetized animals, treatment with Nomega-nitro-L-arginine (L-NNA; 30 micromol/l) and indomethacin (3 micromol/l) reduced the dilation in response to 10 micromol/l ACh from 60 +/- 6 to 20 +/- 4%. This nitric oxide/prostaglandin-independent dilation (NPID), which was of a similar magnitude in large and small arterioles, was abolished by potassium depolarization or charybdotoxin (ChTX, 1 micromol/l) but not by glibenclamide. In conscious animals, NPID amounted to 33 +/- 3%. The inhibitor of the P-450 monooxygenase 17-octadecynoic acid (ODYA) reduced NPID further to 9 +/- 4%. ChTX abolished the NPID and also reduced basal diameters (by -11 +/- 3%). The induction of anesthesia with pentobarbital reduced NPID (to 12 +/- 6%), whereas urethan anesthesia was without effect. Pentobarbital also reduced the ACh-induced hyperpolarization of vascular smooth muscle in isolated arteries, whereas ChTX abolished it. This study suggests that a considerable part of the ACh dilation in the microcirculation is mediated by EDHF, which also contributes to the control of basal tone in conscious animals. The direct inhibitory effect of pentobarbital and ODYA supports the idea that "microcirculatory" EDHF is a product of the cytochrome P-450 pathway. The role of EDHF might be underestimated in pentobarbital-anesthetized animals.

Acetylcholine↗
PubMed1999

p53 enhances BAK and CD95 expression in human malignant glioma cells but does not enhance CD95L-induced apoptosis.

The temperature-sensitive murine p53val135 mutant was introduced into 3 human malignant glioma cell lines to examine the effects of the p53 status on BCL-2 family protein expression, CD95 expression, and sensitivity to CD95 ligand (CD95L)-induced apoptosis. p53val135 behaves as a dominant negative mutant at 38.5 degrees C but assumes p53 wild-type properties. In order to dissect (i) specific effects of wild-type versus mutant p53, and (ii) transdominant-negative versus gain-of-function effects of mutant p53, we included glioma cell lines with functional wild-type (LN-229), mutant (LN-18) or deleted (LN-308) p53 genes. Wild-type, but not mutant, p53val135 promoted G2/M arrest and accumulation of BAK protein in all cell lines. The levels of other BCL-2 family members including BAX, BCL-2, BCL-X or MCL-1 were not consistently modulated by mutant or wild-type p53val135. Wild-type, but not mutant, p53val135 enhanced CD95 expression in all cell lines. CD95L-evoked caspase 3 activity was unaffected by wild-type p53 in all cell lines. Unexpectedly, mutant p53val135 differentially modulated caspase 3 activity in a gain-of-function fashion in that caspase 3 activity induced by CD95L was enhanced in LN-229 and LN-308 cells but reduced in LN-18 cells. Yet, mutant p53val135 enhanced the sensitivity to CD95L in LN-18 cells, had no effect in LN-229 cells, and decreased the sensitivity of LN-308 cells. Corresponding to the unaltered CD95L-evoked caspase 3 activity, wild-type p53val135 had no major effect on CD95L-induced apoptosis, except for a moderate sensitization of LN-229 cells but only when protein synthesis was inhibited. Thus, wild-type p53 induces BAK and CD95 expression in human glioma cells without enhancing their susceptibility to CD95-mediated apoptosis, and mutant p53 modulates CD95L-evoked apoptotic signalling in a gain-of-function fashion up-stream and down-stream of caspase 3 activation.

Animals↗
PubMed1999

Characterization of Tu-2449, a glioma cell line derived from a spontaneous tumor in GFAP-v-src-transgenic mice: comparison with established murine glioma cell lines.

Glial fibrillary acidic protein (GFAP)-v-src transgenic mice develop spontaneous gliomas with a high incidence of malignant progression. We characterize the first glial cell line derived from v-src transgenic mice, Tu-2449 in comparison with a virally induced murine glioma, SRB-10, and a spontaneous murine glioma, P497. Doubling times were lowest, as clonogenicity in soft agar was highest for Tu-2449, and to a lesser degree, Tu-2449 cells formed spheroids and showed migratory behaviour and invaded fetal rat brain aggregates. BCL-2 and BAX expression were lower in Tu-2449 and P497 than in SRB-10 cells. Only Tu-2449 cells accumulated p53 protein in response to genotoxic stress. Tu-2449 and SRB-10 cells that showed low CD95 expression were resistant to CD95 ligand (CD95L)-induced apoptosis unless coexposed to CD95L and inhibitors of RNA or protein synthesis. A chemosensitivity profile revealed Tu-2449 to be rather chemoresistant. Tu-2449 thus displays growth characteristics and patterns of resistance to apoptosis similar to those of other mouse and human glioma cell lines and may therefore become a valuable tool to evaluate new therapies for malignant gliomas in vitro and in vivo.

Animals↗
PubMed1998

Myogenic effects enhance norepinephrine constriction: inhibition by nitric oxide and felodipine.

Myogenic, pressure-induced vasoconstriction may amplify the effects of circulating vasoconstrictors. Through intravital microscopy in cremaster arterioles (31 to 115 microm diameter), the relative contribution of myogenic responses (MR) to norepinephrine (NE)-induced constriction and the inhibitor potency of nitric oxide (NO) or a Ca2+ entry blocker (CEB), felodipine (F), were examined. In 24 anesthetized hamsters, a vessel occluder was placed around the aorta to control cremaster vessel inflow pressure (IP). NE infusion increased blood pressure (by 50 +/- 2 mm Hg) and induced significant constriction (24% +/- 9%) in small arterioles (< 65 microm) only. The constriction, which was not altered by adrenergic blockade, was dependent on the actual IP and was abolished when the IP increase was blocked. NO synthase (NOS) blockade unmasked a significant MR in large arterioles. F inhibited the MR predominantly in large vessels. In isolated microvessels, F completely blocked the pressure-induced Ca2+ increase and MR. We conclude that circulating NE constricts muscle arterioles mainly by a myogenic mechanism. NO effectively opposes MR in larger arterioles, thus restricting MR and vasoconstrictor reinforcement to a small section of the vasculature being tightly controlled by metabolic signals. MR, which otherwise would impair adjustment of peripheral resistance, is reduced by CEB predominantly in larger arterioles, similar to NO.

Animals↗
PubMed1998

Nitric oxide opposes myogenic pressure responses predominantly in large arterioles in vivo.

A myogenic vasoconstriction may amplify the effects of circulating vasoconstrictors. In cremaster arterioles, the contribution of a myogenic component to the constriction on intravenous infusion of norepinephrine (NE) or angiotensin II (Ang II) was studied. Second, the role of endothelium-derived nitric oxide (NO) in the control of these myogenic constrictions and its site of action in the resistance vascular bed was investigated. In 30 anesthetized (pentobarbital) hamsters, the cremaster was prepared for intravital microscopy, and a pneumatic vessel occluder was placed around the aorta to vary blood pressure in the hindquarter of the animal. Intravenous infusion of NE (0.5 nmol/min) increased the systemic blood pressure by 52+/-2 mm Hg. Simultaneously, constrictions of up to 33+/-6% were observed in the small arterioles (SAs; maximal inner diameter, 36 to 65 microm). The constrictions were not significantly altered by a local adrenergic blockade but were abolished when the pressure elevation in the cremaster arterioles was blocked by partial occlusion of the abdominal aorta. Diameters in large arterioles (LAs; maximal inner diameter, 65 to 127 microm), however, did not change significantly on NE infusion. Similar responses in the arterioles were observed when the local pressure was increased stepwise from 60 to 120 mm Hg by partial opening of the aortic occluder. However, after treatment of the cremaster tissue with the inhibitor of the NO synthase, N(G)-nitro-L-arginine (L-NNA, 30 micromol/L), a significant pressure-induced constriction of up to 16+/-3% occurred in LAs, whereas the magnitude of the constriction in SAs remained unchanged. L-NNA also abolished the increases in blood flow that were observed with increments in pressure in control animals. Similar results were obtained when Ang II was used to increase blood pressure. We conclude that a myogenic constriction of SAs contributes markedly to the overall response of cremaster arterioles to circulating vasoconstrictors. NO effectively opposes the myogenic response in LAs, thus preventing myogenic constrictions in a vascular region where constriction cannot be fully controlled by metabolic dilation. If this attenuating effect of NO on myogenic constriction also takes place in other organs, it might be a decisive mechanism in controlling changes of total peripheral vascular resistance elicited by vasoconstrictors.

Abdomen↗