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  • Author or Editor: Ernő Tyihák x
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The most current in vitro and in vivo results in the BioArena system and under greenhouse conditions provide a good opportunity for a fundamental renewal of biological detections and interactions in layer liquid chromatography. The adsorbent bed in a column liquid arrangement is not suitable for biological detection because the living cells do not grow there. Contrarily, the planar adsorbent layer enables the in situ biodetection of antimicrobials and the interactions among separated compounds, cells, and further various cofactors (molecules), making the study of mechanisms of action possible. The basic elements of the time- and dose-dependent quadruple immune response of plants to pathogens in relation to the function and reactions of formaldehyde and its reaction products (mainly endogenous ozone) were demonstrated. This finding opens a new horizon in the field of disease resistance in plants and perhaps in general in the biological world. These results give a good basis and possibility for studying and understanding the unique high-dilution phenomena as well, and at that time, they promise the elimination of century contradictions in this field.

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In the twenty-two years since a similar review in the first issue of this journal there has been much progress in forced-flow planar liquid chromatography (FFPLC). Innovative developments have resulted in extremely diverse new technical solutions. This review briefly summarizes techniques which are already in use as a result of this progress (overpressured-layer chromatography, OPLC, and rotation planar chromatography, RPC) and the future potential of other FFPLC techniques, for example electrochromatographic techniques and shear-driven chromatography (SDC). It seems that efforts in FFPLC provide real possibilities of efficient analytical and preparative separations of different complex mixtures exploiting, with increasing success, the objective advantages of planar layer chromatography. This review summarizes, first, the unique opportunities resulting from modern biological detection on the adsorbent layer (BioArena) and some recent results obtained. These results include — among others — the indirect detection of small key molecules (e.g. formaldehyde, HCHO, and ozone, O3) in chromatographic spots. Practical applications are mainly in-vivo investigations — already without chromatographic separations — with special emphasis on disease resistance and cell proliferation. It seems, however, that in the future BioArena-guided in-vivo investigation will enable characterization of biologically active substances.

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Direct bioautography performed with luminescence gene-tagged bacteria enables almost real-time detection of antimicrobial compounds in plant extracts. This method for the detection of chamomile ( Matricaria recutita ) components with antibacterial effect against Bacillus subtilis soil bacteria was more sensitive than commonly used bioautographic visualization by staining with a tetrazolium salt. Some compounds had a strong inhibiting effect only via the bioluminescence measurement. Extraction of antibacterial components of chamomile flowers was most effective with 50% ethanol; slightly lower efficiency was achieved with acetone and methanol, and hexane was least effective. The results were confirmed by using luminescent Pseudomonas syringae pv. maculicola plant pathogen bacteria.

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Singlet oxygen ( 1 O 2 )-catalyzed oxidation of water leads to a variety of oxidants (e.g. ozone, O 3 ) in host-parasite relationships as well. It can be detected indirectly in TLC or OPLC zones by use of the simple BioArena system and O 3 -eliminating molecules (e.g. d -limonene and indigo carmine) in the culture medium. It follows from these new findings that not only formaldehyde but also O 3 and related bioreactive compounds may play a crucial role in the mechanism of antibacterial activity of antibiotic-like compounds. The toxic potential of a molecule, however, originates from the ratio of the oxidants produced in the chromatographic spots.

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The present and more recent observations suggest that the ozone is an indispensable, endogenous molecule form, and so it can be detected and measured practically in all biological systems. There are already different indirect and direct methods for the detection and measurement of this small molecule. The endogenous formation of ozone in the biological world may open a totally new horizon, e.g., in the topics of disease resistance and cell proliferation. Special efforts and more and more efficient methods are needed for observing the endogenous reactions and functions of this very reactive key molecule.

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The accumulation of hydrogen peroxide (H 2 O 2 ) is an early and crucial step in paclitaxel-induced cancer cell death before commitment of the cells to apoptosis. In these intracellular events formaldehyde (HCHO) as endogenous, indispensable component may be present mainly as hydroxymethyl groups and so there is a possibility of its endogenous interaction with H 2 O 2 in which singlet oxygen ( 1 O 2 ) and excited HCHO (H*CHO) can be formed. 1 O 2 can interact with H 2 O molecules and in this interaction dihydrogen trioxide (H 2 O 3 ) is formed. The disproportion of this molecule — among others — results in ozone (O 3 ). It is supposed that this reaction series is also valid for the conditions in layer chromatographic spots after inoculation. Results with paclitaxel support this idea. Using BioArena as a complex bioautographic system the HCHO molecules could be captured with well-known endogenous HCHO capture molecules (l -arginine, glutathione) in the spots of paclitaxel on the TLC/OPLC adsorbent layer after inoculation. Capture of HCHO resulted in a dose-dependent decrease of the antibacterial activity of paclitaxel. The antibacterial activity of paclitaxel in the chromatographic spots can be increased dramatically by using Cu(II) ions as HCHO-mobilizing and carrier ions in the culture medium. The HCHO molecule can N -hydroxymethylate the C3’ amide of paclitaxel. By applying an O 3 scavenger (e.g. indigo carmine) this oxidant, as a key reaction product of HCHO, could be detected indirectly in chromatographic spots of paclitaxel. It seems that these small molecules — from HCHO to endogenous O 3 — may be crucial factors of the mechanism of antiproliferative action of the paclitaxel including killing of bystander cancer cells also. It seems that the basic molecule (paclitaxel) does not have a direct effect on the bacterial cells; its induction of the formation of H 2 O 2 molecules (and indirectly HCHO molecules) may, however, be resulting in this complicated process.

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Bioautography can be extended to a complex system called BioArena by linking different steps to it, for example dissolving a variety of compounds in the cell suspension to affect biological activity, measuring putative mediators of antibiosis, for example formaldehyde (HCHO) and hydrogen peroxide (H 2 O 2 ) in the inoculated layer, and performing densitometric and ex and in situ spectroscopic examination to follow the changes in the inhibition zones and active compounds (e.g. antibiotics and toxins). Possibilities of the basic elements of BioArena system are illustrated in this paper by results with aflatoxin B1 (AFB1). Target bacterial cells in the logarithmic growth phase were found to be the most sensitive for direct bioautography. Densitometric signals of bioautograms (negative densitometry) of 0.125–1 μg AFB1 spots showed logarithmic correlation with the amount of AFB1. The HCHO capturer L-arginine decreased whereas the HCHO generator-mobilizer Cu(II) ions increased the antibacterial-toxic effect of AFB1. The latter effect was also confirmed by negative densitometry. Besides higher levels of HCHO, a decrease of H 2 O 2 in the toxin spot was found. HCHO could also originate, among other sources, from demethylation of AFB1, which is apparent from the Fourier transform Raman spectra obtained in situ from the AFB1-containing spots. These results support the suggested role of HCHO and its reaction products with H 2 O 2 (e.g. singlet oxygen ( 1 O 2 ), ozone (O 3 )) in the antibacterial-toxic effect of AFB1.

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The shiitake mushroom is well known for its health-beneficial effects. In this study we examined its antimicrobial activity by use of bioautography. After bioactivity detection we identified one major antimicrobial compound by use of infrared spectroscopy and gas chromatography. It shares similarity with well-known fatty acids.

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