Ernő Tyihák

Formaldehyde promotes and inhibits the proliferation of cultured tumour and endothelial cells

Formaldehyde was applied in various doses (0.1–10.0 mm) to HT‐29 human colon carcinoma and HUV‐EC‐C human endothelial cell cultures. Cell number, apoptotic and mitotic index as well as proportion of cells in S‐phase was investigated by morphological methods and flow cytometry. Ten mM of formaldehyde caused high degree of cell damage and practically eradicated the cell cultures. One mM of formaldehyde enhanced apoptosis and reduced mitosis in both types of cell cultures, in a moderate manner. The low dose (0.1 mm) enhanced cell proliferation and decreased apoptotic activity of the cultured cells, the tumour cells appeared to be more sensitive. The possible role of this dose‐dependent effect of formaldehyde in various pathological conditions, such as carcinogenesis and atherogenesis is discussed with emphasis on the eventual interaction between formaldehyde and hydrogen peroxide.

Circular-Development with Overpressured Thin-Layer Chromatography

Abstract A novel type modification of circular thin-layer chromatography has been developed, in which the layer is tightly covered by a membrane, eliminating the vapour phase over the sorbent layer. The developing solvent is pumped through the apparatus.

Effect of formaldehyde on cell proliferation and death

Formaldehyde (HCHO) may reach living organisms as an exogenous agent or produced within cells. The so‐called formaldehydogenic compounds like S‐adenosyl‐l‐methionine, N‐hydroxymethyl‐l‐arginine, 1′‐methyl ascorbigen, methanol, E‐N‐trimethyl lysine and methylamine are special exogenous sources of HCHO. Endogenous HCHO can be formed from hydroxymethyl groups during enzymatic methylation and demethylation processes. HCHO, as a highly reactive compound, is considered to be involved in the induction of apoptosis, consequently in the pathogenesis of atherosclerosis and neurodegenerative processes. The biological action of HCHO is dose‐dependent. In vitro studies on tumour cell and endothelial cell cultures showed that HCHO in the concentration of 10.0 mM caused necrotic cell death, 1.0 mM resulted in enhanced apoptosis and reduced mitotic activity, while 0.5 and 0.1 mM enhanced cell proliferation and reduced apoptotic activity. Among formaldehydogenic compounds N‐hydroxymethyl‐l‐arginine, 1′‐methyl ascorbigen and the HCHO donor resveratrol may be considered as potential inhibitors of cell proliferation. Endogenous HCHO in plants apparently play a role in regulation of apoptosis and cell proliferation. The genotoxic and carcinogentic effects of HCHO is due to production of DNA—protein cross‐links. Low doses of HCHO, reducing apoptotic activity may also accumulate cells with such cross‐links. Experimental data point to the possible therapeutic use of methylated lysine residues and methylated arginine residues in the case of neoplasms.

Overpressured-Layer Chromatography

Abstract In the 1960s, the development of an ultramicro (UM) layer liquid chromatographic (LLC) separation chamber brought about a special combination of the two basic liquid chromatographic (LC) techniques, column LC and LLC. In this simple chamber the adsorbent layer of the chromatoplate was covered by a glass plate and/or plastic film during the separation process such that the end of the cover plate was not immersed in the mobile phase (eluent). Elimination of the vapor phase above the adsorbent layer in LLC was first achieved by use of this UM chamber. Further possibilities of this type of closed-layer chamber—for example, the use of a pumping system to increase and optimize the flow velocity through an optional development distance and the use of a special chromatographic plate—were subsequently realized by the development of an experimental pressurized UM (PUM) chamber. In this unique solution an external pressure is applied to the surface of the adsorbent layer by means of a cushion, forcing the mobile phase to flow (by overpressure) through the adsorbent layer as in HPLC. The PUM chamber is the basic instrument in so-called overpressured-layer chromatography (OPLC). The first commercially available OPLC instrument (Chrompres 10) was a completely off-line system. The second generation instrument (Chrompres 25) was suitable for both off-line and on-line separations. However, these conventional OPLC instruments and methodological solutions were suitable in general for of the advantages of OPLC versions over TLC and HPTLC in analytical and preparative separations. A new automated microprocessor-controlled separation system, new technology in the field of LLC, ensures rapid, efficient, and reproducible off-line and on-line isocratic and stepwise gradient separations. These technological solutions have opened new horizons in the field of LLC and exploit more attractively the advantages of layer liquid systems. This chapter summarizes the latest results obtained with conventional and modern OPLC versions. (The positive results of the BioArena system are demonstrated in Chapter 7 in this book.)

Overpressured layer chromatography: From the pressurized ultramicro chamber to BioArena system

The pressurized ultramicro (UM) chamber as a closed adsorbent layer chamber enables the use of a special chromatoplate and a pump to increase and optimize the mobile phase flow velocity through an optional development distance in an adsorbent layer. This chamber is the basic instrument of overpressured-layer chromatography (OPLC), which is a separation technique that combines the advantages of conventional TLC/HPTLC with those of HPLC. The versions of OPLC instrument, the character and achievement of off-line and on-line OPLC systems in analytical and preparative use are described. The development of BioArena as a complex bioautographic system means an exploitation of the unique advantages of planar-layer system for detection, isolation and identification of new antimicrobials, antineoplastics, biopesticides and other biologically active substances as well as for studying fundamental biochemical reactions and mechanisms.

Possible role of formaldehyde in the apoptotic and mitotic effect of 1-methyl-ascorbigen

PC-3 human prostate carcinoma cells were treated with 100 ug/ml 1-methyl-ascorbigen (Me-Asc). This treatment resulted in a significant decrease in tumor cell number in parallel with an increase in apoptotic cells. The formaldehyde (HCHO) level in the culture medium was also increased. Dimedone (Di), a known capture molecule forming formaldemethone with HCHO, applied simultaneously with Me-Asc in 10 ug/ml doses diminished the apoptosis-inducing effect of Me-Asc. The possible role of in situ generated HCHO in the induction of apoptosis is discussed.

Separation and detection of aflatoxins using overpressured-layer chromatography and bioautography

It has been established, by use of bioautographic detection with the phytopathogenic bacteria Pseudomonas savastanoi pv. phaseolicola, that the common aflatoxins have antimicrobial activity after OPLC separation. Our preliminary results suggest this antimicrobial activity originates from the activity of formaldehyde formed by the bacterial cells and/or from the methoxy groups of aflatoxin molecules.

Comparison of thin-layer chromatography and overpressured layer chromatographic techniques for the separation of ascorbigen and 1'-methylascorbigen

Abstract Simple and efficient methods are described for the separation of biologically active ascorbigen and 1′-methylascorbigen by normal-phase TLC, conventional as well as new personal overpressured layer chromatographic (OPLC) systems using the same eluent. Results obtained with these techniques were compared on the basis of the separation parameters with special emphasis on the presentation of the advantages of the automated personal OPLC system. For the characterization of the personal OPLC system dependence of average plate height and resolution on the external pressure, front velocity and development distance was examined on TLC and HPTLC sorbent layers using ascorbigen and 1′-methylascorbigen as model compounds.

Micro-preparative OPLC — rapid isolation by transfusion and infusion-transfusion processes

A new OPLC procedure, infusion—transfusion OPLC, has been developed and compared with conventional transfusion OPLC. Spot and/or band deformation caused by the total wetness front (which results from pore filling) was reduced, as was the bubble effect in on-line detection. Both techniques were used for rapid micro-preparative OPLC isolation on analytical adsorbent layers. In-situ clean-up and separation were used to isolate trigonelline from Leuzea extract. Modeling of loading capacity for isolation of ascorbigen was accomplished by fully off-line OPLC. Under optimized conditions ascorbigen of high purity was isolated from cabbage extract by transfusion and infusion—transfusion OPLC.

The influence of L-ascorbic acid on the antibacterial-toxic activity of aflatoxins on adsorbent layer.

Aims:  To substantiate the role of formaldehyde (HCHO) and its reaction products in the mechanism of the antibacterial‐toxic effect of aflatoxins B1 (AFB1), B2, G1 and G2.