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R Kaufmann

Publications and source records attributed to R Kaufmann.

At least 145 records · Page 8Linked to original sources
PubMed1998

The mechanism of melanocyte dendrite formation: the impact of differentiating keratinocytes.

In human epidermis one dendritic melanocyte interacts with about 36 keratinocytes and supplies them with melanin. In contrast to the vivo situation melanocytes in culture are far less dendritic. In the present study different culture systems were tested in order to observe the mechanism of melanocyte dendrite formation. In particular, we focused on the role of keratinocytes in this process. Time lapse studies revealed that only differentiated keratinocytes enhance melanocyte dendricity. Differentiated keratinocytes form connected cell sheets, which attach to part of the melanocyte plasma membrane. By contraction and retraction of keratinocyte units, new dendrites were drawn out from the melanocytes. Melanocytes remain passive during this process, which is indicated by the observation that sometimes extended dendrites could not withstand the tension and shear.

Calcium↗
PubMed1998

A note on interactions between temperature, viscosity, body size and swimming energetics in fish larvae

In a previous study, it was shown that at a given speed the larvae of a species of freshwater fish, the Danube bleak Chalcalburnus chalcoides, expended considerably more metabolic energy at 15 degreesC than at 20 degreesC. We applied hydromechanical arguments to our previous data in order to determine whether the higher cost of swimming at the lower temperature might be due to the effects of viscous forces. However, even under the unrealistic assumption of the larvae swimming in the viscous regime at Reynolds numbers as high as 2000, we show here that hydromechanical forces cannot explain the high energy cost of swimming at 15 degreesC. Instead, we offer a new hypothesis that the 'two-gear system' of the swimming muscles operating in juvenile and adult fish is not yet functional in the larvae, with the consequence that, when these fish are swimming at high speeds in cold water, the muscle fibres have to operate over an increasingly inefficient range of shortening velocities.

Journal Article↗
PubMed1998

Prion rods contain small amounts of two host sphingolipids as revealed by thin-layer chromatography and mass spectrometry.

Sphingolipids were detected in prions, the agents of transmissible spongiform encephalopathies. The analysis was carried out on highly purified, infectious prion rods, which are composed mainly of insoluble aggregates of the N-terminally truncated prion protein, so-called PrP 27-30. Lipid classes were quantified by high performance thin-layer chromatography with a detection limit of 25-50 ng per lipid class. Matrix-assisted laser desorption/ionization mass spectrometry was applied for the first time to lipid analysis in complex biological samples. A newly developed preparation technique improved the sensitivity to 1-20 pg per molecular species. Only the sphingolipids, galactosylceramide and sphingomyelin, were consistently observed in chloroform/methanol (2:1 v/v) extracts of prion rods. The molar ratio of PrP to the sphingolipids was between 2:1 and 40:1, depending on the purity of the prion preparation. The same lipids were also present in the low density fraction of a gradient centrifugation of prion-rods after sonication in 0.2% SDS. From the two alternatives, that the sphingolipids are either required for prion function or are relics from the cellular location of PrP in caveolae, the second alternative appears more plausible since the preparation of highest specific infectivity contained the lowest amount of sphingolipids.

Animals↗
PubMed1998

Intermediates of prothrombin activation induce intracellular calcium mobilization in rat aortic smooth muscle cells.

While effects of alpha-thrombin have been well characterized in different cell types, the biological function of intermediates of prothrombin activation is still undefined. Meizothrombin could be shown to be a potent agonist for vascular contraction which seems to be mediated by an interaction with the vascular smooth muscle. To explore this effect at intracellular signaling level, we used rat aortic smooth muscle cells and investigated the effect of the intermediates formed by cleavage of human prothrombin with ecarin, the prothrombin activator from Echis carinatus venom, on mobilization of free intracellular calcium. The ecarin-activated prothrombin induced very rapidly transient calcium mobilization in SMC's comparable to that observed with alpha-thrombin. We conclude that meizothrombin/meizothrombin desF1 (MT/MT desF1) are the most likely candidates for this effect. Furthermore, our results suggest the involvement of PAR-1-type thrombin receptors in MT/MT desF1-induced calcium signaling in rat aortic smooth muscle cells.

Amino Acid Sequence↗
PubMed1997

Thrombin receptor activation results in calcium signaling and protein kinase C-dependent stimulation of DNA synthesis in HEp-2g laryngeal carcinoma cells.

BACKGROUND: Recently, the expression of the "tethered ligand" thrombin receptor in carcinosarcoma and melanoma cells has been shown. However, the role of the thrombin receptor in tumor cell metabolism still is undefined. METHODS: In this article, the "tethered ligand" thrombin receptor was identified on human epidermoid carcinoma cells (HEp-2g cell line) by using immunofluorescence studies with a monoclonal antithrombin receptor antibody and radioligand binding. Furthermore, the effects of alpha-thrombin and thrombin receptor activating peptides (TRAP)-6 on calcium mobilization, protein kinase C (PKC) translocation, and DNA synthesis were estimated. RESULTS: Pharmacologic characterization using [3H]TRAP-6 as a radioligand demonstrated a single class of high affinity binding sites (dissociation constant [KD] = 7.2 +/- 2.2 x 10(-7) M) and a binding capacity of 27 +/- 3.4 fmol/mg protein. The function of these binding sites was demonstrated by alpha-thrombin- and TRAP-6-induced mobilization of free intracellular calcium, and translocation of PKC from cytosol to cell membrane. Moreover, alpha-thrombin and TRAP-6 induced an increase in [3H]thymidine incorporation in HEp-2g cells that could be blocked by the PKC inhibitor bisindolylmaleimide. CONCLUSIONS: To the authors' knowledge, the results of this study demonstrate for the first time functional thrombin receptors in epidermoid carcinoma cells. The thrombin receptor appears to be involved in growth regulation in HEp-2g cells by a PKC-dependent mechanism.

Calcium↗
PubMed1997

High-performance liquid chromatography and laser desorption/ionization mass spectrometry of retinyl esters.

Laser desorption/ionization mass spectrometry (LDI-MS) at 337 nm laser wavelength was used to analyze retinol and several long-chain fatty acid esters of retinol. Employing this ionization technique helped to overcome the inherent problems resulting from thermal instability of retinyl esters which render this group of compounds rather difficult for standard ionization techniques. Mass spectra were recorded with a linear time-of-flight instrument in positive ion mode. Under these conditions, retinyl esters formed radical molecular ions (M.+) and in addition fragmented by elimination of the fatty acyl chain to uniformly form a peak at m/z = 269 u. The elimination of carbon dioxide was also observed in the spectra. The method is suitable to identify specific retinyl esters in complex mixtures of these compounds. A gradient reversed-phase high-performance liquid chromatography (HPLC) is described for the separation of retinol and 15 related fatty acid esters within 28 min. HPLC was applied to separate retinyl esters from rat liver extracts. The LDI mass spectrum of the collected HPLC fraction of rat liver extract showed the molecular parent ions of retinyl myristate, pentadecanoate, palmitoleate, palmitate, heptadecanoate, linoleate, oleate, stearate, and 3,4-didehydroretinyl palmitate. LDI-MS was found to be more appropriate than matrix-assisted laser desorption/ionization for the described analytical task.

Animals↗
PubMed1997

Novel bradykinin signalling events in PC-12 cells: stimulation of the cAMP pathway leads to cAMP-mediated translocation of protein kinase Cepsilon.

In the rat pheochromocytoma cell line PC-12, bradykinin (BK) stimulated phosphatidylinositol hydrolysis by 4-5-fold and, additionally, intracellular cAMP accumulation by approx. 1.6-fold. EC50 values for BK were 3 nM and 2 nM respectively. The BK-induced increase in cAMP accumulation was paralleled by a 1.6-fold increase in protein kinase A (PKA) activity. The time course of BK-stimulated inositol phosphate formation was rapid (t1/2<1 min), whereas the BK-induced cAMP accumulation was lagging (t1/2 approx. 6 min). The effect of BK on the cAMP pathway was independent of pertussis toxin, excluding an indirect stimulation of adenylate cyclase via betagamma-complexes from Gi or Go proteins. Two different protein kinase C (PKC) inhibitors, bisindolylmaleimide and Ro 31-820, failed to prevent BK-induced cAMP accumulation, and exclude PKC as mediator of BK action on adenylate cyclase. In contrast, the stimulatory effect of BK on cAMP accumulation was completely abolished by two calmodulin antagonists, chlorpromazine and ophiobolin, suggesting an indirect, Ca2+/calmodulin-mediated effect of BK on the cAMP pathway. In addition, exposure of PC-12 cells to BK resulted in a translocation of the PKC isoforms alpha, delta, epsilon and zeta displaying different kinetics. The BK-induced translocations of the PCDs alpha and delta were rapid and biphasic, whereas the PKCs epsilon and zeta revealed a slower and slightly transient translocation in response to BK. The BK-elicited translocation of PKCepsilon, but not that of the PKCs alpha, delta and zeta, was prevented by two different inhibitors of adenylate cyclase, 2',5'-dideoxyadenosine and MDL-12,330A, as well as the PKA inhibitor adenosine 3':5'-monophosphothioate. These findings suggest that the BK-induced translocation of novel (n)PKCepsilon is mediated via the cAMP pathway. Since nPKCepsilon appears to regulate neurite outgrowth in PC-12 cells [Hundke, McMahon, Dadgar and Messing (1995) J. Biol. Chem. 270, 30134-30140] our results provide evidence for a novel signalling mechanism that might be involved in BK-induced neuronal differentiation of PC-12 cels.

Adenylate Cyclase Toxin↗
PubMed1997

IgG-antibodies to CNS proteins in patients with multiple sclerosis.

The CSF and sera of 185 patients with multiple sclerosis (MS), 130 patients with other inflammatory diseases of the CNS (OID) and 50 patients with spinal disc syndrome (controls) were investigated for IgG-antibodies to CNS proteins by SDS-PAGE and immunoblotting and by isoelectric focusing combined with affinity blotting. IgG-antibodies to CNS proteins in serum (immunoblotting) were detected in 18 patients with MS (10%), in 29 patients with OID (22%) and in four controls (8%). Intrathecal synthesis of IgG-antibodies to CNS proteins was demonstrated in 11 patients with MS (6%), in 37 patients with OID (28%) and in none of the controls. In 4/11 patients with MS intrathecally produced antibodies were shown to be specific for glial fibrillary acidic protein (GFAP). Of patients with MS, 180 displayed oligoclonal IgG-bands in the CSF. Specificity of these bands for CNS proteins was demonstrated only in 2/180 specimens (1%). These findings indicate, that in most patients with MS oligoclonal IgG-bands in the CSF do not contain relevant amounts of antibodies to CNS proteins.

Enzyme-Linked Immunosorbent Assay↗
PubMed1997

A new glycosylated lipopeptide incorporated into the cell wall of a smooth variant of Gordona hydrophobica.

A cell wall component of a smooth variant of Gordona hydrophobica 1775/15 was isolated and purified, and its structure was determined by various chemical methods, including chemical synthesis of part structures, Edman degradation, gas chromatography/mass spectrometry analysis, matrix-assisted laser desorption ionization-post-source decay (MALDI-PSD) tandem mass spectrometry, and 1H and 13C NMR using one- and two-dimensional, homo- and heteronuclear correlated spectroscopy. The cell wall component was found to be a (mono-) glycosylated peptidolipid (GPL) consisting of a tridecapeptide interlinked by a beta-hydroxylated fatty acid (3-hydroxyeicosanoic acid, 20:0 (3-OH)) to form a cyclic lactone ring structure. The main fraction of GPL, for which we propose the name gordonin, was identified as 3-hydroxyeicosanoyl-L-seryl-L-phenylalanyl-L-seryl- L-seryl-D-alanyl-L -(O-beta-D-glucopyranosyl)-threonyl-glycyl-D-leucyl-L-valyl-L-seryl-L -phenylalanyl-glycyl-L-valyl lactone. The other GPLs constitute structural variations within the nature of the beta-hydroxylated fatty acid (20:0(3-OH) versus 22:1(3-OH)) in a ratio of about 1:0.9 as well as within one amino acid (D-Leu versus L-Phe) in about 30%. Sequence information was obtained in part by Edman degradation as well as gas chromatography/mass spectrometry analysis of di- and tripeptide fragments. However, the complete amino acid sequence could only be established by MALDI-PSD from the linear molecule, i.e. after ring opening of the lactone. In contrast, rough variants of G. hydrophobica 1775/15 lack these peptidolipids or synthesize them to a much lesser extent indicating that gordonin contributes significantly to the physicochemical character of the cell surface.

Actinomycetales↗
PubMed1997

Identification of endogenous Des-Arg9-[Hyp3]-bradykinin in human plasma with post-source-decay matrix-assisted laser desorption/ionization mass spectrometry.

In the present study, the purification and sequence analysis of a peptide extracted from human plasma is described. After three chromatographic steps in one of the plasma fractions, a molecular mass of 920 Da was determined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. Further analysis with post-source-decay (PSD)-MALDI-MS revealed an amino acid sequence arg-pro(pro-gly+16)-phe-ser-pro-phe. With the data given from the spectrum, it was impossible to clarify the partial sequence (pro-gly+16). A comparison with a data base suggested that the sequence is very similar to des-arg9bradykinin (arg-pro-pro-gly-phe-ser-pro-phe). But many fragment masses, including the mass of the parent ion of the unknown peptide, are 16 Da larger than the corresponding fragment masses of des-arg9-bradykinin. The interpretation of the fragment pattern does not allow determination of the position of the amino acid that is 16 Da larger. With automated Edman sequence analysis of the fraction of interest, three more major component peptides in this fraction were observed, which were not detected with MALDI-MS. Nevertheless, with the help of the Edman sequence data it was possible to identify the unknown amino acid in the PSD-MALDI-MS sequence as hydroxyproline at the third position. In conclusion, the existence of des-arg9[hyp3]-bradykinin (arg-pro-hyp-gly-phe-ser-pro-phe) in human plasma is described. Furthermore, the study demonstrates that PSD-MALDI-MS and automated Edman sequence analysis complement each other if single unknown peptides from mixtures are to be sequenced.

Bradykinin↗
PubMed1997

Cholecystokinin B-type receptor signaling is involved in human pancreatic cancer cell growth.

Cholecystokinin (CCK) is known to stimulate pancreatic cancer cell growth, but no detailed CCK receptor subtype characterization and investigation of CCK receptor-mediated cellular responses in human pancreatic cancer cells have been reported thus far. In this study, CCK binding sites were identified in human pancreatic cancer cells (MIA-PaCa-2) using radioligand binding studies. Pharmacological characterization demonstrated a single class of high-affinity CCK sites on MIA-PaCa-2 cells (326 +/- 18 pM, receptor density 16.9 +/- 2.3 fmol/mg protein). These CCK binding sites displayed a typical CCKB binding profile as shown in competition studies by using different CCK-related compounds and non-peptide CCK antagonists discriminating between CCKA and CCKB sites. CCKB receptor-connected effector systems have been characterized in MIA-PaCA-2 cells, and their involvement in CCK-8S-induced proliferative effects on MIA-PaCa-2 cells has been demonstrated.

Calcium↗
PubMed1997

Isolation and structural elucidation of two pyrokinins from the retrocerebral complex of the American cockroach.

By monitoring the contractile activity of the hyperneural muscle of the American cockroach in vitro two peptides were isolated from the retrocerebral complex of the American cockroach. Three purification steps using reversed-phase high performance liquid chromatography on C-18 columns containing trifluoroacetic acid or heptafluorobutyric acid as organic modifiers were sufficient to achieve homogeneous peptide preparations. The structures of both peptides were elucidated by a combination of Edman degradation and mass spectrometry which yielded the following structures: His-Thr-Ala-Gly Phe-Ile-Pro-Arg-Leu-NH2 (Pea-PK-1) and Ser-Pro-Pro-Phe-Ala-Pro-Arg-Leu-NH2 (Pea-PK-2). The C-terminal sequence Phe-X-Pro-Arg-Leu-NH2 characterized the peptides as members of the insect pyrokinin family. The synthetic peptides were shown to have the same retention times as the natural peptides. The occurrence of both peptides in the retrocerebral complex suggests a physiological role as neurohormones. The effects of the synthetic pyrokinis were clearly distinguishable in their actions on the hyperneural muscle. Regarding the threshold concentrations, Pea-PK-2 was only 0.3% as active as Pea-PK-1.

Amino Acid Sequence↗
PubMed1997

Thrombin has a bimodal effect on glioma cell growth.

Using rat glioma C6 cells as a model, we have found a bimodal effect of alpha-thrombin on cell growth. In C6 cells treated with alpha-thrombin at concentrations from 0.02 nM to 1.0 nM, inhibition of cell proliferation was noted. Because the thrombin receptor agonist peptide TRAP-6 also induced inhibition of cell proliferation and the thrombin receptor antagonist peptide T1 prevented the inhibitory effect of alpha-thrombin on C6 glioma cell growth, thrombin receptor involvement in antiproliferative action of alpha-thrombin in C6 glioma cells is highly likely. However, stimulation of cell proliferation observed when C6 cells were treated with alpha-thrombin at higher doses (> 1.0 nM) seems to be mediated by as yet undefined thrombin receptor-independent biochemical mechanisms.

Animals↗
PubMed1997

Subcutaneously pedicled island flaps.

Subcutaneously pedicled island flaps (SPIF) are very mobile local flaps bringing into the surgical defect skin of similar texture and color to that of the recipient site. SPIF flaps are eminently simple to create: an island of skin is incised until the fatty tissue is visualized and the flap connections to surrounding tissue are carefully disconnected without damaging the nutrient vessels. The viability of such flaps is excellent. If appropriately performed, side effects such as pincushioning are rare.

Esthetics↗
PubMed1997

Candida albicans suppresses transcription of melanogenesis enzymes in cultured melanocytes.

Human skin can be colonized by different yeasts that may have an impact on skin pigmentation. In order to study this effect normal human melanocytes were cultured with different yeasts. Reverse transcription polymerase chain reaction (RT-PCR) analysis gives evidence that Candida albicans suppresses the transcription of melanogenesis enzymes.

Aldose-Ketose Isomerases↗
PubMed1997

The implantable fuzzy controlled Helmholtz-left ventricular assist device: first in vitro testing.

To perform first experimental tests for validation of a new left ventricular assist device (LVAD) with a high efficiency energy converter, a new pump design and a novel type of perfusion control, a functional labtype, were manufactured. With a stroke volume of 65 ml, a total pump housing volume of 450 ml (including valves and connectors), and a weight of 430 g, it is one of the smallest and lightest implantable pulsatile electromechanical LVADs. Pulsatile operation is generated by a special reduction and displacement gear which transforms a uniform rotational movement of a sensorless, electronically commutated DC motor into a translatory pusher plate movement. A prolonged duration for filling (60% of the cycle time) supports full-empty pumping and consequently a high overall pump efficiency. Active adaptation of output flow to organ perfusion demand is achieved by changing the rotational speed of the motor by means of a sensorless fuzzy controller, which detects preload and afterload induced effects at the motor current input. First in vitro test results obtained within a circulatory mock loop that simulates physiological preloads and afterloads are presented. They comprise preload sensitivity and the function of the novel perfusion controller as well as preload and afterload related flow data. The results prove the feasability of the energy conversion with the novel gear and control concept for an implantable electromechanical pulsatile LVAD.

Animals↗