4-Epidoxycycline: an alternative to doxycycline to control gene expression in conditional mouse models.

Since the pioneering work by Gossen and Bujard in 1992 demonstrating the usefulness of the Escherichia coli derived tet resistance operon for regulating gene expression a large collection of doxycycline-controlled transgenic mice has been established. Gene switching in eukaryotic tissue culture cells or mice requires administration of tetracycline, anhydrotetracycline or doxycycline to efficiently inactivate the transactivator protein tTA (TET-OFF system) or alternatively to activate the reverse transactivator protein rtTA (TET-ON system). However, the antibiotic activity of doxycycline can create an imbalance of the intestinal flora, resulting in diarrhoea and in a smaller number of animals in colitis. Previous studies reported that 4-epidoxycycline (4-ED), a hepatic metabolite of doxycycline, does not function as an antibiotic in mice. This gave us the idea that 4-ED might be useful for controlling gene expression in mice without the unwanted antibiotic side effect. To study the applicability of 4-ED for control of gene expression we used cell lines expressing the oncogene HER2 under control of tTA (TET-OFF) as well as rtTA (TET-ON). 4-ED and doxycycline were similarly efficient in switching on or -off HER2 expression. In vivo we used a conditional mouse model that allows switching off HER2 in tumor tissue. We show that (i) doxycycline, 7.5mg/ml in drinking water (used as a positive control), (ii) 4-ED, 7.5mg/ml in drinking water, (iii) 4-ED, 10mg/kg body weight, s.c., and (iv) anhydrotetracycline, 10mg/kg, s.c. (used as a second positive control), were similarly efficient. Using mice with tumor volumes of 1.6cm(3) all four schedules led to a tumor remission of more than 95% within 7 days. In conclusion, 4-ED is similarly efficient as doxycycline to control gene expression in vitro and in mice. Since 4-ED lacks the antibiotic activity of doxycycline it may help to avoid adverse side effects and selection of resistant bacteria.

The role of UV-B radiation in aquatic and terrestrial ecosystems--an experimental and functional analysis of the evolution of UV-absorbing compounds.

We analysed and compared the functioning of UV-B screening pigments in plants from marine, fresh water and terrestrial ecosystems, along the evolutionary line of cyanobacteria, unicellular algae, primitive multicellular algae, charophycean algae, lichens, mosses and higher plants, including amphibious macrophytes. Lichens were also included in the study. We were interested in the following key aspects: (a) does the water column function effectively as an 'external UV-B filter'?; (b) do aquatic plants need less 'internal UV-B screening' than terrestrial plants?; (c) what role does UV screening play in protecting the various plant groups from UV-B damage, such as the formation of thymine dimers?; and (d) since early land 'plants' (such as the predecessors of present-day cyanobacteria, lichens and mosses) experienced higher UV-B fluxes than higher plants, which evolved later, are primitive aquatic and land organisms (cyanobacteria, algae, lichens, mosses) better adapted to present-day levels of UV-B than higher plants? Furthermore, polychromatic action spectra for the induction of UV screening pigments of aquatic organisms have been determined. This is relevant for translating 'physical' radiation measurements of solar UV-B into 'biological' and 'ecological' effects. From the action spectra, radiation amplification factors (RAFs) have been calculated. These action spectra allow us to determine any mitigating or antagonistic effects in the ecosystems and therefore qualify the damage prediction for the ecosystems under study. We summarize and discuss the main results based on three years of research of four European research groups. The central theme of the work was the investigation of the effectiveness of the various screening compounds from the different species studied in order to gain some perspective of the evolutionary adaptations from lower to higher plant forms. The induction of mycosporine-like amino acids (MAAs) was studied in the marine dinoflagellate Gyrodinium dorsum, the green algal species Prasiola stipitata and in the cyanobacterium Anabaena sp. While visible (400-700 nm) and long wavelength UV-A (315-400 nm) showed only a slight effect, MAAs were effectively induced by UV-B (280-315 nm). The growth of the lower land organisms studied, i.e. the lichens Cladina portentosa, Cladina foliacaea and Cladonia arbuscula, and the club moss Lycopodiumannotinum, was not significantly reduced when grown under elevated UV-B radiation (simulating 15% ozone depletion). The growth in length of the moss Tortula ruralis was reduced under elevated UV-B. Of the aquatic plants investigated the charophytes Chara aspera showed decreased longitudinal growth under elevated UV-B. In the 'aquatic higher plants' studied, Ceratophyllum demersum, Batrachium trichophyllum and Potamogeton alpinus, there was no such depressed growth with enhanced UV-B. In Chara aspera, neither MAAs nor flavonoids could be detected. Of the terrestrial higher plants studied, Fagopyrum esculentum, Deschampsia antarctica, Vicia faba, Calamagrostis epigejos and Carex arenaria, the growth of the first species was depressed with enhanced UV-B, in the second species length growth was decreased, but the shoot number was increased, and in the latter two species of a dune grassland there was no reduced growth with enhanced UV-B. In the dune grassland species studied outdoors, at least five different flavonoids appeared in shoot tissue. Some of the flavonoids in the monocot species, which were identified and quantified with HPLC, included orientin, luteolin, tricin and apigenin. A greenhouse study with Vicia faba showed that two flavonoids (aglycones) respond particularly to enhanced UV-B. Of these, quercetin is UV-B inducible and mainly located in epidermal cells, while kaempferol occurs constitutively. In addition to its UV-screening function, quercetin may also act as an antioxidant. Polychromatic action spectra were determined for induction of the UV-absorbing pigments in three photosynthetic organisms, representing very different taxonomic groups and different habitats. In ultraviolet photobiology, action spectra mainly serve two purposes: (1) identification of the molecular species involved in light absorption; and (2) calculation of radiation amplification factors for assessing the effect of ozone depletion. Radiation amplification factors (RAFs) were calculated from the action spectra. In a somewhat simplified way, RAF can be defined as the percent increase of radiation damage for a 1% depletion of the ozone layer. Central European summer conditions were used in the calculations, but it has been shown that RAF values are not critically dependent on latitude or season. If only the ultraviolet spectral region is considered, the RAF values obtained are 0.7 for the green alga Prasiola stipitata, 0.4 for the dinoflagellate Gyrodinium dorsum, and 1.0 for the cyanobacterium Anabaena sp. In the case of P. stipitata, however, the effect of visible light (PAR, photosynthetically active radiation, 400-700 nm) is sufficient to lower the RAF to about 0.4, while the PAR effect for G. dorsum is negligible. RAFs for some damage processes, such as for DNA damage (RAF=2.1 if protective effects or photorepair are not considered [1]), are higher than those above. Our interpretation of this is that if the ozone layer is depleted, increased damaging radiation could overrule increased synthesis of protective pigments. In addition to investigating the functional effectiveness of the different screening compounds, direct UV effects on a number of key processes were also studied in order to gain further insight into the ability of the organisms to withstand enhanced UV-B radiation. To this end, the temperature-dependent repair of cyclobutane dimers (CPD) and (6-4) photoproducts induced by enhanced UV-B was studied in Nicotiana tabacum, and the UV-B induction of CPD was studied in the lichen Cladonia arbuscula. Also, photosynthesis and motility were monitored and the response related to the potential function of the screening compounds of the specific organism.

Hybrid liver support system in a short term application on hepatectomized pigs.

A short term application of a hybrid liver support system in circuits with continuous plasma-separation was investigated in a model of hepatectomized pigs under general anesthesia. Primary pig hepatocytes were immobilized in a bioreactor with three independent capillary systems. An immune barrier is achieved by avoiding the direct contact of blood cells with the hepatocytes by a plasmaseparation step and by an outflow filtration within the reactor. In three groups (hepatectomized pigs and system with- or without hepatocytes as well as untreated pigs with system without hepatocytes), the short term metabolism of the reactors was positively demonstrated by investigating ammonia detoxification, phenylalanine- and lactate metabolism. Limitations of the presented model are discussed.

[Immunological diagnosis of cutaneous lymphomas. II. Membrane receptor for Helix pomatia lectin on lymphocytes: a new immunohistological marker for T-lymphocytes and Sezary cells]. / Immunologische Diagnostik kutaner Lymphome. II. Membranrezeptor für Helix Pomatia Lectin auf Lymphozyten: Ein neuer immunhistologischer Marker für T-Lymphozyten und Sezary-Zellen.

Lymphocytes carrying the receptor for helix pomatia lectin on their cytomembranes can be detected in lymphatic tissue and cutaneous T-cell lymphoma by means of peroxidase-labelled helix pomatia lectin. Cells positive for the HPL-receptor are identical with the population of T-lymphocytes, including their pathological variants, i.e. lymphoma cells and Sezary's cells.

[Immunologic diagnosis of cutaneous lymphomas. I. A new immunoperoxidase technic for the demonstration of cell- and tissue-bound antigens in formalin-fixed paraffin-embedded tissues using protein A-peroxidase as a universal second antibody for mono- and polyclonal antibodies]. / Immunologische Diagnostik kutaner Lymphome. I. Eine neue Immunperoxidase-Technik zur Darstellung gewebs- und zellgebundener Antigene in formalinfixiertem Paraffin-eingebetteten Gewebe, mit Protein-A-Peroxidase als universeller "Second Antibody" für mono- und polyclonale Antiköper.

Immunoperoxidase techniques, applied to formalin-fixed paraffin embedded tissues, provide a simultaneous histopathological and immunological diagnosis. Application of protein-A-peroxidase constitutes a new technical variant: The technique is relatively simple, decreases non-specific background staining, and binds mono- and polyclonal antibodies from different mammalian species.

[Preliminary experiences in the treatment of onychomycosis with ketoconazole]. / Erste Erfahrungen bei der Behandlung der Onychomykjose mit Ketoconazol.

17 patients with proven onychomycosis were treated with Ketoconazole (200 mg daily) over 6 months. In 15, a distinct improvement was seen, 2 were slightly improved. No therapeutic failures were to be seen. Few patients complained about side effects; biochemical parameters were neither consistently changed, nor caused discontinuing of treatment.

In situ-differentiation of lymphocytes in fixed tissues with peroxidase-labeled Helix pomatia lectin: revelation of the Helix pomatia lectin receptor on cell surfaces. Application in lymph node tissues and cutaneous lymphomas.

A receptor for Helix pomatia lectin (HPL) occurs on 71.5% of peripheral blood lymphocytes. With double incubation techniques we could show that the population of HPL-positive lymphocytes is by far (greater than 90%) identical with the population of T lymphocytes, as determined by "classical" markers (E rosetting) and newly developed T cell markers like OKT3 or anti-human Lyt3 monoclonal antibodies. In formaldehyde-fixed lymph node sections we could demonstrate the homing area of HPL-positive lymphocytes, the paracortical regions. Conventional histological counterstaining was readily possible. We have employed HPL-peroxidase for differentiation of lymphocytes in benign and malignant infiltrates in human skin. In reactive cutaneous infiltrates, as well as in cutaneous lymphomas, HPL-peroxidase staining allows a clear differentiation of the involved lymphatic cells, and may serve as a tumor marker in certain T lymphomas, where we could demonstrate the lymphoma cells to carry the HPL-receptor.

[Acral bullae following PUVA-therapy (author's transl)]. / Akrale Blasenbildung unter PUVA-Therapie.

Six patients with mycosis fungoides, psoriasis or generalized granuloma anulare developed acral bullae showing the clinical and histological picture of bullous pemphigoid during systemically applied PUVA-therapy. Immunohistological investigations (Protein A-Per-oxidase, Immunofluorescence) produced no proof of bullous pemphigoid.

Eritadenine: a new tool for investigation of the adenosine P site in plasma membranes of rat fat cells.

Eritadenine was investigated for its effects on adenylate cyclase activity of rat fat cell plasma membranes on cyclic AMP accumulation and lipolysis in isolated rat fat cells. In rat fat cell plasma membranes, a 50% inhibition of noradrenaline (100 mumol/l) stimulated adenylate cyclase was obtained at 11.6 mumol/l eritadenine or 9.0 mumol/l adenosine. NaF (3 mmol/l) stimulated adenylate cyclase was inhibited at concentrations of eritadenine (IC25 5.8 mumol/l) lower than those of adenosine (IC25 51.9 mumol/l). Eritadenine ethyl ester (100 mumol/l) was almost ineffective on adenylate cyclase. The inhibitory effect of eritadenine was resistant to adenosine deaminase. Isolated rat fat cells eritadenine (100 mumol/l) was completely ineffective to block noradrenaline-stimulated cyclic AMP accumulation or lipolysis stimulated by theophylline or adenosine deaminase. It is suggested that eritadenine is an effector of the adenosine P site of fat cell plasma membranes.

[Improvement of the IgM-FTA-ABS Test--Short Communication (author's transl)]. / Verbesserung der Aussagefähigkeit des IgM-FTA-ABS-Testes.

With immobilized Protein A from Staph. aureus, treponema-specific IgG can be removed from patient's serum by affinity chromatography. The IgM-FTA-ABS-test can be improved decisively by eliminating competition at the antigen of treponema-specific IgM and IgG and thus false negative results.

Immuno-electron microscopical features of in vivo antibody binding during pemphigus blister formation. Revelation with a new technique: peroxidase-labeled protein A.

Pemphigus autoantibodies, bound in the epidermis during different stages of acantholysis, were demonstrated with a new technique for immuno-electron microscopy. Peroxidase-labeled Protein A was used as a new and specific tracer for tissue-bound antibodies of the IgG-type. Advantages were: (1) A small molecular weight of the tracer, (2) a rapid tissue penetration, and (3) shortened incubation times, thus better preserved tissue fine structures. Unspecific adsorption in tissues and on cells was found to be comparatively low.

Immuno-electron microscopical investigations with a new tracer: peroxidase-labeled protein A: application for detection of pemphigus and bullous pemphigoid antibodies.

Peroxidase-labeled Protein A, stable immunoenzyme tracer of high reactivity and comparatively low molecular weight, has been applied in immuno-electron microscopy for detection of bound IgG-type pemphigus and bullous pemphigoid antibodies. Comparing Protein A-peroxidase with peroxidase-labeled immunoglobulins, we obtained similar morphological results in corresponding incubation techniques, but lower nonspecific adsorption of Protein A-peroxidase complexes in tissues. The Protein A-peroxidase molecules showed good tissue penetration abilities. Our rapid one-step incubation procedure led to enhanced preservation of tissue fine structures, without the need of prior tissue fixation. It seems that Protein A-peroxidase is able to replace peroxidase-labeled anti-IgG for immuno-electron microscopical purposes.

A new immunoenzyme tracer for localization of antibodies in immunohistology: peroxidase-labeled protein A.

Protein A from Staphylococcus aureus, characterized by high affinity binding properties for IgG-type antibodies, was labeled with peroxidase to form a stable immuno-histological tracer molecule of comparatively low molecular weight. It has been used for demonstration of antibodies against tissue antigens, in direct and indirect techniques, and the findings were consistent with those in routine immunofluorescence and in staining with peroxidase-coupled anti-IgG. In comparison, the lowest unspecific tissue adsorption and staining was obtained with Protein A-peroxidase in buffer containing glucose and mannose. The immunohistological preparations were mounted and stored for documentation without apparent loss of staining.

A new immunoenzyme tracer for immunohistology: peroxidase-labeled protein A. Its application for determination of antinuclear antibodies.

Protein A from Staphylococcus aureus, a cell wall protein with high affinity binding properties for IgG-type antibodies, has been labeled with peroxidase to form a stable immunohistological tracer molecule of relatively low molecular weight. It has been used for demonstration and titration of antinuclear antibodies in SLE sera on mouse liver sections in an indirect technique. The findings were consistent with those obtained by immunofluorescence and by staining with peroxidase-coupled anti-IgG. In contrast to immunofluorescence, the stained sections could be mounted and stored for documentation. In comparison, unspecific tissue adsorption and staining could be minimized by addition of glucose, galactose, and mannose as well as bovine serum albumine to the buffer containing Protein A-peroxidase.

Specific binding of 3H-adenosine to rat brain membranes.

The binding of 3H-adenosine to rat brain membranes was studied by a microcentrifugation technique. Specific binding of 3H-adenosine was rapid, reversible, saturable and dependent on pH and temperature. Scatchard plots of equilibrium binding data were nonlinear suggesting the existence of two different binding sites for adenosine. The dissociation constants (Kd) were 1.7 muM and 13.6 muM and the maximal number of binding sites (Bmax) 31 and 165 pmol adenosine bound per mg of membrane protein. Ten adenosine derivatives were studied for their ability to compete with 3H-adenosine binding. The phosphorylated adenosine compounds 5'-AMP, cyclic AMP and ATP were most potent in displacing 3H-adenosine from its binding sites and the IC50-values ranged from 11--25 muM. N6-Phenylisopropyladenosine produced only partial inhibition (30%) of 3H-adenosine binding and no stereospecific difference between the (-)- and (+)isomer was observed. Several methylxanthines known as adenosine antagonists competed for the 3H-adenosine binding sites parallel with their pharmacological potency. The results offer a first approach for the study of adenosine binding sites in brain membranes.