Karl-Jürgen Halbhuber

Modern laser scanning microscopy in biology, biotechnology and medicine

Laser microscopic techniques currently used in morphology and cell biology represent highly sensitive tools for detecting biomolecules within their natural environment. Use of the fluorescence-, reflectance- and transmission modes of confocal laser scanning microscopes (CLSM) equipped with He-Ne- and Ar+-ion lasers for CeIV and DAB based detection of endogenous or immunobound enzymatic activities in tissue sections (vibratome, cryostat, paraffin and semithin plastic sections) opens a wide range of interesting new possibilities in cellular and molecular biology. Increased resolution power, blur-free confocal imaging, higher sensitivity, optical sectioning capability and 3D-image analysis provide a large quantity of valuable information about biological objects specimens. The new infrared multiphoton laser scanning microscopy (NIR-LSM) is increasingly becoming the optical tool of choice for (a) fluorescence imaging of cellular and subcellular components with high spatial and temporal resolution, (b) fluorescence resonance energy transfer between physiologically relevant molecular species involving protein-protein interactions, (c) nanoprocessing within living cells and tissues, with varied applications in (d) photochemistry and (e) medical diagnostics as well. Both, CLSM and NIR-LSM as modern microscopical strategies are indispensable in basic research and will prove to be invaluable for clinical diagnostic studies and therapy in the near future.

Met receptor tyrosine kinase transactivation is involved in proteinase-activated receptor 2-mediated hepatocellular carcinoma cell invasion

The expression of proteinase-activated receptor (PAR)(2) in human hepatocellular carcinoma (HCC) was established by reverse transcription-polymerase chain reaction, confocal immunofluorescence and electron microscopy in permanent cell lines, primary HCC cell cultures and HCC tumor tissue. Stimulation of HCC cells with trypsin and the PAR(2)-selective activating peptide, 2-furoyl-LIGRLO-NH(2), increased cell invasion across Matrigel. Both effects were blocked by a PAR(2)-selective pepducin antagonist peptide (pal-PAR(2)) and by PAR(2) silencing with specific small interfering RNA (siRNA). PAR(2)-initiated HCC cell invasion was also blocked by inhibiting the hepatocyte growth factor receptor (Met receptor tyrosine kinase) with the receptor-targeted kinase inhibitors, SU 11274 and PHA 665752, or by downregulation of Met with specific siRNA. The involvement of Met in PAR(2)-mediated HCC invasive signaling was further supported by the finding that treatment of HCC cells with trypsin or the PAR(2)-selective agonist peptide, 2-furoyl-LIGRLO-NH(2), stimulated Met activation-phosphorylation. In addition, Met-dependent stimulation of p42/p44 mitogen-activated protein Kinases was found to be critical for the PAR(2)-Met receptor tyrosine kinase-invasive signaling axis in HCC cells. Our study establishes an important link between the PAR(2) and Met receptor tyrosine kinase signaling in promoting HCC cell invasion.

Characterization of iron oxide nanoparticles adsorbed with cisplatin for biomedical applications

The aim of this study was to characterize the behaviour of cisplatin adsorbed magnetic nanoparticles (cis-MNPs) for minimal invasive cancer treatments in preliminary in vitro investigations. Cisplatin was adsorbed to magnetic nanoparticles (MNPs) by simple incubation. For stability determinations, cis-MNPs were incubated in dH(2)O, phosphate-buffered saline (PBS) and fetal calf serum (FCS) at 4-121 degrees C up to 20 weeks. Hydrodynamic diameters were measured using laser diffraction. The extent of cisplatin linkage was determined by atomic absorption spectrometry. The magnetite core size was assessed by vibrating sample magnetometry and transmission electron microscopy. The specific loss power (SLP) was measured in an alternating magnetic field. Our results showed that a maximum of 10.3 +/- 1.6 (dH(2)O), 10 +/- 1.6 (PBS) and 13.4 +/- 2.2 (FCS) mg cisplatin g(-1) Fe could be adsorbed to MNPs. With hyperthermal (42 degrees C) or thermal ablative (60 degrees C) temperatures, used for therapeutic approaches, cisplatin did not desorb from cis-MNPs in dH(2)O during incubation times of 180 or 30 min, respectively. In PBS and FCS, cisplatin amounts adsorbed to MNPs decreased rapidly to approximately 50% and 25% at these temperatures. This cisplatin release will be necessary for successful chemotherapeutic activity and should increase the therapeutic effect of magnetic heating treatment in medicinal applications. The hydrodynamic diameters of MNPs or cis-MNPs were around 70 nm and magnetization data showed superparamagnetic behaviour. The obtained mean core diameter was around 12 nm. The SLP of the sample was calculated to be 75.5 +/- 1.6 W g(-1). In conclusion, cis-MNPs exhibit advantageous features for a facilitated desorption of cisplatin in biological media and the heating potential is adequate for hyperthermic treatments. Therefore, even though further detailed investigations are still necessary, tentative use in local tumour therapies aiming at a specific chemotherapeutic release in combination with magnetic heating seems to be feasible in the long term.

Effects of prostaglandin D2 on tetrodotoxin-resistant Na+ currents in DRG neurons of adult rat

&NA; Tetrodotoxin‐resistant (TTX‐R) Na+ channels play a key role in the generation of action potentials in nociceptive dorsal root ganglion (DRG) neurons and are an important target for the proinflammatory mediator prostaglandin E2, which augments these currents. Prostaglandin D2 (PGD2) is released in the tissue together with prostaglandin E2, and it was reported to be antiinflammatory, but its effect on primary afferent neurons is unclear. In the present study we localised Gs‐protein‐coupled DP1 and Gi‐protein‐coupled DP2 receptors in DRG neurons, and we assessed the effect of PGD2 on TTX‐R Na+ currents in patch‐clamp recordings from small‐ to medium‐sized cultured DRG neurons from adult rats. DP1 and DP2 receptor‐like immunoreactivity was localised in the vast majority of DRG neurons. In all neurons, PGD2 shifted conductance to more hyperpolarised potentials, depending on an action at Nav1.9 channels. In about one third of the neurons, PGD2 additionally influenced Nav1.8 channels by facilitating conductance and by increasing maximal current amplitudes. Selective DP1 receptor activation increased the amplitude of TTX‐R Na+ currents of most neurons, but this effect was counteracted by DP2 receptor activation, which by itself had no effect. In the current‐clamp mode, PGD2 lowered the threshold for elicitation of an action potential and increased the number of action potentials per stimulus, an effect mainly depending on DP1 receptor activation. Thus, the net effect of PGD2 on DRG neurons is pronociceptive, although the magnitude of the TTX‐R Na+ currents depends on the balance of DP1 and DP2 receptor activation. Prostaglandin D2 regulates conductance and current amplitudes of tetrodotoxin‐resistant sodium channels on dorsal root ganglion neurons by a balanced action at DP1 and DP2 receptor subtypes.

Optimization of heterocyclic 4-hydroxystyryl derivatives for histological localization of endogenous and immunobound peroxidase activity

Assisted by the development of light excitation and measuring techniques and the commercial availability of highly sensitive equipment, luminescent labels are sensitive detection tools for life sciences research. By contrast to a wide variety of well established chromogenic techniques, fluorescent labels for detecting peroxidase (PO) have been confined to only a few substrates. We describe here novel fluorescent substrates of PO derived from heterocyclic 4-hydroxy styrenes as useful tools for detecting endogenous and exogenous targets in fixed cells and tissues. Excellent localization, high staining sensitivity, outstanding photostability, and exceptionally low background staining were achieved by optimizing the substrate through chemical synthesis. Structure/staining behavior relationships are discussed. By contrast to tyramine-fluorochrome conjugates employed in the catalyzed reporter deposition (CARD) technique, reporting and anchoring functions are no longer separated. Consequently, enzymatic cross-linking of the substrate yields an altered fluorochrome with different properties. Spectral properties and anchoring capability are interdependent and influenced by environmental effects and pH. We screened overall staining capability of 4-hydroxy styryl derivatives using an iterative semi-empirical approach, and ascertained optimal substitution patterns for high PO staining specificity and high fluorescence response. Reliable staining performance was achieved with alkyl chains of short or medium length at the positively charged nitrogen, whereas introducing polar groups often impaired the staining specificity of PO. Catalytic cross-linking of heterocyclic 4-hydroxy-styryl derivatives is a promising approach for permanent fluorescent staining of PO in fixed cells and tissues, and complements the CARD technique. Histochemical and immunohistochemical applications are presented using conventional and confocal fluorescence microscopes with different excitation sources. Spectral properties of selected stains are discussed. Novel stains also are of potential interest as “reactive-tracers” for living cells under multi-photon laser excitation conditions, because they exhibit pronounced nonlinear optical properties.

[The altered membrane of the erythrocyte. I. Ultrahistochemical and biocellular investigations for the detection of activated acetylcholinesterase (ACHE) and demasking of IgG receptor sites (author's transl)].

Zusammenfassung Es wurde versucht, verschiedene Zustandsformen der Erythrocytenmembran im Hinblick auf den Grad ihrer Desintegration naher zu charakterisieren. Als Kriterien wurden die Bindungsfahigkeit gegenuber autologem bzw. allogenem IgG, der Aktivierungsgrad der am intakten Erythrocytenplasmalemm inhibierten membransessilen Acetylcholinesterase und die Mernbranvesiculation herangezogen. Die Befunde belegen, das offenbar mit dem Schweregrad der Membrangefugestorung die IgG-Bindung anwachst und die Acetylcholinesterase zunehmend aktiviert wird. Das Enzym kann dann spektrophotometrisch und ultrahistochemisch erfast werden. Die Mikrovesiculation wird als Ausdruck tiefgreifender Membrangefugestorungen auigefast. Am konservierten Erythrocyten wird sie als ein lokaler Membran-Remodellierungsprozes angesehen, Starkere Erythrocytenschadigung, wie nach mechanischem Stress, Warmesohock und Inkubation in Harnstoff, fuhrt zu vergleichsweise hoheren Vesiculationsraten, teilweise zu Zellfragmentation. An stark spektrinverarmten Erythrocytenschatten tritt Mikrovesiculation spontan ein und fuhrt zum volligen mikrovesicularen Zerfall der Schattenmembran. Das membranassoziierte autologe IgG wird mit immunologischen und ultrahistochemischen Methoden nachgewiesen. Seine Bedeutung als homoostatisch wirksames Immunsignal fur die Elimination von in vivo oder in vitro gealterten Erythrocyten, Erythrocytenschatten und -vesiceln durch das Reticulo-Histiocytare-System wird anhand von Modellversuchen aufgezeigt. Molekulare Mechanismen zur IgG-Receptordemaskierung und A.cetylcholinesteraseaktivierung in der alterierten Erythrocytenmembran werden diskutiert.

The rate of lateral diffusion of phospholipids in erythrocyte microvesicles.

31P-NMR spectra of phospholipids in membranes of erythrocyte microvesicles isolated from outdated blood units were recorded in the temperature range 5 to 55 degrees C. Within that range the lineshape is strongly influenced by an increasing rate of lateral diffusion of phospholipids. At 36 degrees C a diffusion constant, D, of (2 +/- 1) X 10(-12) m2/s was obtained. The diffusion rate is by a factor of 3 to 10 greater than in erythrocyte membranes measured by the photobleaching technique and is comparable with values obtained for several lipid model membranes. The differences in lateral diffusion rates are probably connected with the depletion of microvesicle membranes in membrane proteins.

Topo-optical investigations of human erythrocyte glycocalyx conformational changes induced by dextran

Cell surface properties are involved in the aggregation process of red blood cells. Using the topo-optical toluidine blue reaction, conformational changes of the glycocalyx (main component glycophorin A) were found when red blood cells were incubated and fixed in the presence of dextran. Relative differences in optical path as a measure of red blood cell membrane anisotropy decreased in relation to dextran concentration during fixation. These conformational changes could not be detected by electrophoretic measurements. When incubating, fixing and staining red blood cells in the presence of dextran, anisotropy decreased only at low dextran concentrations and increased at rising dextran concentrations. This biphasic course of differences in optical path seems to be due to different effects of dextran superimposing upon each other: a disturbing influence on the spatial order of sialic acid carrying oligosaccharide side chains due to H-bond interaction, and an increase in the size of dye aggregates and suppression of the thermal motion of macromolecules at higher dextran concentrations.