Biomedical subjects

B Wiesner

Publications and source records attributed to B Wiesner.

At least 19 recordsLinked to original sources

PubMed2006

[Spontaneous pneumomediastinum in asthma].

A pneumomediastinum is characterized by free air within the mediastinum. In most cases it is caused by an injury of the thorax. A spontaneous pneumomediastinum (sPM) occurred without any recognizable cause. By a sudden intraalveolar hypertension (caused by cough or physical exertion) a damage of alveoli is followed by diffusion of air along the bronchovascular sheats to the mediastinal soft tissue. sPM is a rare complication of asthma especially in young men. We demonstrate a young asthmatic patient with sPM first diagnosed as an allergic reaction to draw the attention to this rare but differential diagnostic important entity. sPM is reported in connection with drug inhalation abuse as a more frequent problem within the last years.

Adult↗

PubMed2006

Multicentre evaluation of performance of C-reactive protein analysis over a one-year period.

This communication deals with a longitudinal evaluation of C-reactive protein (CRP) analysis during a one-year period using a single lot of liquid control sera (3 levels) (BIOREF-CRP levels 1, 2 and 3) in different laboratories. A total of 652 sets of data were returned from 20 participating laboratories using 13 different reagent-measuring device combinations. The use of the control materials was defined in a standard operating procedure. Data was returned to the organizers on a monthly basis and questions could be asked or problems presented during the evaluation period. Although the performance of different reagents varied, the control materials were shown to be stable over the whole of the evaluation period when stored at 4-7 degrees C in a refrigerator/cold room. Typical problems were encountered, examples of which are presented here in graphical and tabular form.

C-Reactive Protein↗

PubMed2003

Effects of kinins on mammalian spermatozoa and the impact of peptidolytic enzymes.

Effects of kinins, mainly bradykinin (Bk), and other components of the kallikrein-kinin system on sperm motility and further fertility-related functions have been described repeatedly. However, reported data are in part controversial and the mechanism of kinin effects on sperm motility is not yet understood. In the present report we describe a significant promoting effect of Bk on sperm motility at subnanomolar concentrations. This effect was stabilized and even increased by suppression of Bk hydrolysis in semen samples. As sperm membrane-bound angiotensin-converting enzyme and neutral metalloendopeptidase are mainly involved in Bk hydrolysis, an effective cocktail of enzyme inhibitors promoting the sperm motility consists of phosphoramidon and lisinopril (both at 10-7 m). The effects of Bk on sperm cells are not mediated by the B2 Bk receptor. Using several biochemical, molecular and genetic methods we could not detect any Bk receptor on spermatozoa.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2003

[Pulmonary varicosity, a rare reason of solitary nodule].

Pulmonary varicosity is a very rare entity. Beside congenital varices, most combined with other malformations of the pulmonary vessels, varicosities are described above all in connection with mitral insufficiency. Most cases with pulmonary varicosity are diagnosed by chance, because of absence of any symptoms or only slight symptoms respectively. We demonstrate our own case with respect to the diagnostic requirements (computerized tomography, multi-slice CT) and the differential diagnostic aspects.

Aged↗

PubMed2003

Energy metabolism and intracellular pH in boar spermatozoa.

The effect of energy metabolism on intracellular pH was studied in boar spermatozoa using nuclear magnetic resonance (NMR) spectroscopy and confocal microscopy with the pH-sensitive dye seminaphthorhodafluor (SNARF-1). Freshly ejaculated spermatozoa had a high adenylate energy charge (AEC=0.8), which decreased to 0.6 under aerobic conditions and to 0.2 under anaerobic conditions. Correspondingly, no ATP resonances but high AMP resonance were visible in (31)P-NMR-spectra of the spermatozoa. When an artificial oxygen buffer (Fluosol) and a purpose-built air supply system were used during (31)P-NMR data acquisition, ATP resonances reappeared whereas the AMP resonance disappeared. Boar spermatozoa kept under aerobic conditions have intracellular compartments that differ markedly in pH, as demonstrated by both (31)P-NMR spectroscopy and confocal microscopy. Using confocal microscopy, the midpiece of the flagellum in which all mitochondria are located was identified as an acidic compartment (pH(i-mp) 6.7). The intracellular pH of both the head (pH(i-h)) and the long principal piece of the flagellum (pH(i-pp)) were 7.2 and, thus, only slightly below the extracellular pH (pH(e) 7.3). Storage of spermatozoa in a glucose-free medium at 15 degrees C when they are immotile slowly shifted the pH(i-mp) from 6.7 to 6.9 within 20 h, whereas pH(i-h) and pH(i-pp) remained unchanged (pH 7.1-7.2). When glucose was present in the medium, all visible compartments of the spermatozoa as well as the medium were acidified to pH 6.2 within 20 h. Under these conditions a resonance at 4.8 mg kg(-1) appeared representing glycerol 3-phosphate.

Adenosine Diphosphate↗

PubMed2002

Cellular uptake of antisense oligonucleotides after complexing or conjugation with cell-penetrating model peptides.

The uptake by mammalian cells of phosphorothioate oligonucleotides was compared with that of their respective complexes or conjugates with cationic, cell-penetrating model peptides of varying helix-forming propensity and amphipathicity. An HPLC-based protocol for the synthesis and purification of disulfide bridged conjugates in the 10-100 nmol range was developed. Confocal laser scanning microscopy (CLSM) in combination with gel-capillary electrophoresis and laser induced fluorescence detection (GCE-LIF) revealed cytoplasmic and nuclear accumulationin all cases. The uptake differences between naked oligonucleotides and their respective peptide complexes or conjugates were generally confined to one order of magnitude. No significant influence of the structural properties of the peptide components upon cellular uptake was found. Our results question the common belief that the increased biological activity of oligonucleotides after derivatization with membrane permeable peptides may be primarily due to improved membrane translocation.

Amino Acid Sequence↗

PubMed2001

Astrocytes induce manganese superoxide dismutase in brain capillary endothelial cells.

Astrocytes induce blood-brain barrier (BBB) properties in brain endothelial cells (EC)*O(2)*, generated in blood and EC, opens the BBB. Hence, high activity of superoxide dismutase (SOD) is a prerequisite for normal BBB function. Therefore, the influence of rat astrocytes on the expression of manganese (Mn)SOD in rat EC was investigated in two coculture models of the BBB, allowing either exchange of soluble factors or additionally cellular contacts. Activity, protein content and mRNA expression of endothelial MnSOD were significantly increased in both coculture models in comparison to monoculture by soluble astrocytic factors, such as cytokines. High activity of endothelial MnSOD may be considered as a further essential property of the BBB, which is induced and maintained by astrocytes.

Animals↗

PubMed2001

An inhibitory role of Rho in the vasopressin-mediated translocation of aquaporin-2 into cell membranes of renal principal cells.

Vasopressin regulates water reabsorption in renal collecting duct principal cells by a cAMP-dependent translocation of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the cell membrane. In the present work primary cultured inner medullary collecting duct cells were used to study the role of the proteins of the Rho family in the translocation of AQP2. Clostridium difficile toxin B, which inhibits all members of the Rho family, Clostridium limosum C3 toxin, which inactivates only Rho, and the Rho kinase inhibitor, Y-27632, induced both depolymerization of actin stress fibers and AQP2 translocation in the absence of vasopressin. The data suggest an inhibitory role of Rho in this process, whereby constitutive membrane localization is prevented in resting cells. Expression of constitutively active RhoA induced formation of actin stress fibers and abolished AQP2 translocation in response to elevation of intracellular cAMP, confirming the inhibitory role of Rho. Cytochalasin D induced both depolymerization of the F-actin cytoskeleton and AQP2 translocation, indicating that depolymerization of F-actin is sufficient to induce AQP2 translocation. Thus Rho is likely to control the intracellular localization of AQP2 via regulation of the F-actin cytoskeleton.

Amides↗

PubMed2001

Cell volume kinetics of adherent epithelial cells measured by laser scanning reflection microscopy: determination of water permeability changes of renal principal cells.

The water channel aquaporin-2 (AQP2), a key component of the antidiuretic machinery in the kidney, is rapidly regulated by the antidiuretic hormone vasopressin. The hormone exerts its action by inducing a translocation of AQP2 from intracellular vesicles to the cell membrane. This step requires the elevation of intracellular cyclic AMP. We describe here a new method, laser scanning reflection microscopy (LSRM), suitable for determining cellular osmotic water permeability coefficient changes in primary cultured inner medullary collecting duct (IMCD) cells. The recording of vertical-reflection-mode x-z-scan section areas of unstained, living IMCD cells proved useful and valid for the investigation of osmotic water permeability changes. The time-dependent increases of reflection-mode x-z-scan section areas of swelling cells were fitted to a single-exponential equation. The analysis of the time constants of these processes indicates a twofold increase in osmotic water permeability of IMCD cells after treatment of the cells both with forskolin, a cyclic AMP-elevating agent, and with Clostridium difficile toxin B, an inhibitor of Rho proteins that leads to depolymerization of F-actin-containing stress fibers. This indicates that both agents lead to the functional insertion of AQP2 into the cell membrane. Thus, we have established a new functional assay for the study of the regulation of the water permeability at the cellular level.

Actins↗

PubMed2000

Evidence for an amphipathicity independent cellular uptake of amphipathic cell-penetrating peptides.

The cellular uptake of a peptide set derived from membrane-permeable alpha-helical amphipathic peptides by stepwise alterations of structure forming propensity and charge was studied by confocal laser scanning microscopy (CLSM) combined with HPLC. For CLSM monitoring, an online protocol was employed that avoided bias of the uptake results by washout. Using this protocol, extensive fluorescence, approaching the intensity of the external peptide, was observed in the cytosol and nucleus within minutes in all cases, irrespective of the degree of amphipathicity. HPLC analyses of the cell lysates revealed the unmetabolized peptides to be the predominant source of the intracellular fluorescence. Significant amphipathicity-dependent differences became apparent only after washing the peptide-loaded cells, reflecting the effects of amphipathicity on resistance to wash out. Exposure of the cells to the peptides at 37 and 0 degrees C led to similar results, indicating the nonendocytic character of the uptake. With a view to practical applications, the results of the present study open the possibility of exploiting nonamphipathic peptides as vectors for translocating polar compounds into the cell interior, which would circumvent substantial obstacles currently connected with the use of amphipathic vector peptides, such as membrane toxicity and low solubility. Moreover, differences in the uptake of several members of the investigated peptide series into different cell types present a promising basis for the design of cell-type specific vector peptides.

Amino Acid Sequence↗

PubMed1999

Protein kinase A anchoring proteins are required for vasopressin-mediated translocation of aquaporin-2 into cell membranes of renal principal cells.

The antidiuretic hormone arginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells by inducing a cAMP-dependent translocation of water channels (aquaporin-2, AQP-2) from intracellular vesicles into the apical cell membranes. In subcellular fractions from primary cultured rat inner medullary collecting duct (IMCD) cells, enriched for intracellular AQP-2-bearing vesicles, catalytic protein kinase A (PKA) subunits and several protein kinase A anchoring proteins (AKAPs) were detected. In nonstimulated IMCD cells the majority of AQP-2 staining was detected intracellularly but became mainly localized within the cell membrane after stimulation with AVP or forskolin. Quantitative analysis revealed that preincubation of the cells with the synthetic peptide S-Ht31, which prevents the binding between AKAPs and regulatory subunits of PKA, strongly inhibited AQP-2 translocation in response to forskolin. Preincubation of the cells with the PKA inhibitor H89 prior to forskolin stimulation abolished AQP-2 translocation. In contrast to H89, S-Ht31 did not affect the catalytic activity of PKA. These data demonstrate that not only the activity of PKA, but also its tethering to subcellular compartments, are prerequisites for cAMP-dependent AQP-2 translocation.

Animals↗

PubMed1999

Structural requirements for cellular uptake of alpha-helical amphipathic peptides.

The structure of the cell-permeable alpha-helical amphipathic model peptide FLUOS-KLALKLALKALKAALKLA-NH2 (I) was modified stepwise with respect to its helix parameters hydrophobicity, hydrophobic moment and hydrophilic face as well as molecular size and charge. Cellular uptake and membrane destabilizing activity of the resulting peptides were studied using aortic endothelial cells and HPLC combined with CLSM. With the exceptions that a reduction of molecule size below 16 amino acid residues and the introduction of a negative net charge abolished uptake, none of the investigated structural parameters proved to be essential for the passage of these peptides across the plasma membrane. Membrane toxicity also showed no correlation to any of the parameters investigated and could be detected only at concentrations higher than 2 microM. These results implicate helical amphipathicity as the only essential structural requirement for the entry of such peptides into the cell interior, in accord with earlier studies. The pivotal role of helical amphipathicity was confirmed by uptake results obtained with two further pairs of amphipathic/non-amphipathic 18-mer peptides with different primary structure, net charge and helix parameters from I. The amphipathic counterparts were internalized into the cells to a comparable extent as I, whereas no cellular uptake could be detected for the non-amphipathic analogues. The mode of uptake remains unclear and involves both temperature-sensitive and -insensitive processes, indicating non-endocytic contributions.

Animals↗

PubMed1999

p53 mutations and codon 213 polymorphism of p53 in lung cancers of former uranium miners.

PURPOSE: There is a high prevalence of G-->T transversions of p53 in lung cancers of smokers. One study has reported a special "hotspot" mutation at codon 249 of p53 in lung cancers of former uranium miners. The aim of our study was to look for mutational spectra of p53 in former German uranium miners with lung cancers. METHODS: We investigated 16 patients with lung cancer who had worked as uranium miners in Germany and 13 lung cancer patients without a mining history of the same region. By means of the polymerase chain reaction and sequencing we looked for mutations in exons 5 7 of the p53 gene. RESULTS: We could not find any suggestion of hotspot mutations. The only G-->T mutation in former uranium miners was detected in the only nonsmoker. In 3 patients (19% of the total) we found a codon 213/3 polymorphism. CONCLUSIONS: The results indicate that G-->T transversions do not seem to be very common mutations in p53 in lung cancers probably caused by radiation. Therefore, p53 may be mutated early in lung cancer development if radiation exposure is a critical factor in carcinogenesis. In accordance with studies of thyroid cancer patients in the Chernobyl region, our results may indicate an overrepresentation of codon 213/3 polymorphism in p53 in radiation-caused cancers.

Codon↗

PubMed1999

Measurement of intracellular Ca2+ changes using novel caged cyclic nucleotides and confocal laser scanning microscopy.

The intention of this study is to explore the applicability of confocal microscopy in conjunction with the use of caged cyclic nucleotide derivatives. The methodological potential of UV laser confocal microscopy has been assessed. It is shown that illumination of a single cell or a small area of a single cell is possible, whereby the intracelluar Ca2+ signal is measured at illuminated and non-illuminated cells. Such measurements do not have a high time resolution because of the specific system parameters. However, with an N2 pulse laser (not part of the standard microscope set-up), Ca2+ signals with a time resolution of around 100 ms have been measured. This facilitates investigation of the kinetics of Ca2+ influx. Intracellular Ca2+ measurements at HEK293 and sperm cells have been made here. For sperm cells the advantages of confocal microscopy are best evidenced in conjunction with the use of caged cyclic nucleotides; a cyclic nucleotide-gated Ca2+ influx at the tail of these cells has thereby been demonstrated for the first time.

Animals↗

PubMed1999

p53 protein, EGF receptor, and anti-p53 antibodies in serum from patients with occupationally derived lung cancer.

The oncogene product epidermal growth factor receptor (EGF-R), the tumour suppressor gene product p53 and anti-p53 antibodies are detectable in the serum of certain cancer patients. Increased levels of some of these products were reported in lung cancer patients after occupational asbestos exposure and after exposure to polycyclic aromatic hydrocarbons or vinylchloride. In the first step, this study investigated the possible diagnostic value of serum EGF-R, p53-protein and anti-p53 antibodies, measured by an enzyme-linked immunosorbent assay, in lung tumour patients. In addition to being investigated on a molecular epidemiological basis, these parameters were examined as biomarkers of carcinogenesis, especially with regard to asbestos incorporation effects or of radon-induced lung cancers. Also, a possible effect of cigarette smoking and age dependence were studied. A total of 116 male patients with lung or pleural tumours were examined. The histological classification was four small-cell cancers, six large-cell cancers, 32 adenocarcinomas, 47 squamous carcinomas, 12 mixed lung carcinomas, five diffuse malignant mesotheliomas and ten lung metastasis of extrapulmonary tumours. Twenty-two lung cancers and all mesotheliomas were related to asbestos, 22 lung cancers were related to ionizing radiation and 61 patients had cigarette smoke-related lung cancer. Besides these patients 50 male patients with non-malignant lung or pleural diseases were included; of the latter eight subjects suffered from asbestosis. Controls were 129 male subjects without any lung disease. No significantly elevated or decreased serum values for p53 protein, EGF-R, or anti-p53 antibodies as a function of histological tumour type, age, or degree and type of exposure (asbestos, smoking, ionizing radiation) could be found. The utility of p53-protein, EGF-R and anti-p53 antibodies as routine biomarkers for screening occupationally derived lung cancers is limited.

Adenocarcinoma↗

PubMed1999

Polarized expression of the vasopressin V2 receptor in Madin-Darby canine kidney cells.

BACKGROUND: The vasopressin V2 receptor is expressed in the polarized principal cell of the renal collecting duct. Inactivating mutations of the vasopressin V2 receptor gene cause X-linked nephrogenic diabetes insipidus (NDI). Most of the mutant V2 receptors show transport defects, as analyzed in non-polarized cells, but data pertaining to polarized cells have not previously been presented. METHODS: Madin-Darby canine kidney cell (MDCK) II clones stably expressing c-myc-tagged human V2 receptors were characterized for [3H]-arginine vasopressin (AVP)-binding and AVP-sensitive adenylyl cyclase activity. The V2 receptors were immunocytochemically localized using the tyramide signal amplification technique in conjunction with an anti-c-myc antibody. RESULTS: The introduction of the c-myc epitope at the N- or C-terminus did not affect the functional properties of the V2 receptor expressed in MDCK II clones. However, the use of standard immunofluorescence methodology for these MDCK II clones yielded only weak signals. With the tyramide signal amplification technique, strong signals were obtained, showing the V2 receptor to be mainly localized within the lateral and, to a minor extent, apical membrane. In MDCK II clones stably expressing the c-myc-tagged V2 receptor NDI mutant L44P, fluorescent signals were found exclusively within the cell. CONCLUSION: The wild-type V2 receptor is expressed mainly in the lateral membrane, whereas the L44P mutant is completely retained within the cell. In conjunction with tyramide signal amplification, MDCK II cells constitute a suitable model for the analysis of transport-defective mutants of the V2 receptor.

Adenylyl Cyclases↗

PubMed1999

[CT-assisted transthoracic puncture of pulmonary lesions and mediastinal infiltrations. A retrospective study of 300 patients].

With this retrospective study we evaluated the results and the complications of our 343 CT-guided transthoracic biopsies, which we performed in 300 patients with localised and infiltrative lesions of the lung and the mediastinum during 4 years, using the VacuCut-needle in 96% of our cases. In 256 pts. bronchoscopy without final diagnosis was performed before transthoracic biopsy. In 219 pts. (73.0%) we had positive results. In 209 pts. with malignancies the positive rate was 73.7%. In patients with benign processes the positive rate was less (67.8%). Out of 32 pts, suffering from mediastinal lesions 29 had malignancies. In 79.3% positive results were obtained. The rate of complications was low (9%) in 311 biopsies of pulmonary lesions, most were pneumothoraces (7.7%). Haemorrhages occurred in 4 pts. (1.3%) 5 pts. (1.6%) required drainage because of pneumothorax. On the base of our good results and the low complication rate we recommend CT-guided transthoracic aspiration biopsy with the VacuCut-needle as a useful and accurate diagnostic technique.

Adult↗

PubMed1998

Polarized cell surface expression of the green fluorescent protein-tagged vasopressin V2 receptor in Madin Darby canine kidney cells.

We have analyzed the polarized cell surface expression of the G protein-coupled vasopressin V2 receptor (V2 receptor) in Madin-Darby canine kidney (MDCK) epithelial cells by both conventional cell surface biotinylation assays and laser scanning microscopy of green fluorescent protein (GFP)-tagged receptors. Cell surface biotinylation assays with stably transfected filter-grown cells expressing alkaline phosphatase (PhoA)-tagged receptors demonstrated that the V2 receptor is located predominantly basolaterally at steady state, while minor amounts are expressed apically. Laser scanning microscopy of filter- and glass-grown MDCK cells stably transfected with a GFP-tagged V2 receptor confirmed that the receptor is expressed mainly basolaterally; within the basolateral compartment, however, the receptor was confined to the lateral subdomain. The results obtained with the GFP-tagged receptor are thus consistent with and refine those from the biotinylation assay, which does not discriminate lateral from basal membrane regions. Our data indicate that the GFP methodology may effectively supplement cell surface biotinylation assays in future studies of polarized receptor transport. We finally show that microinjection of a plasmid encoding the GFP-tagged V2 receptor into the nucleus of MDCK cells led to the same results as experiments with stably transfected cells. However, since there was no need for selecting stably transfected cell lines, the experiments were complete within hours. The microinjection technique thus constitutes a powerful single cell technique to study the intracellular transport of G protein-coupled receptors. The methodology may be applicable to any cell type, even to tissue-derived, primary cultured cells; coinjection of transport-regulating compounds should also be possible.

Alkaline Phosphatase↗