Sianette Kwee's research while affiliated with Aarhus University and other places

Publications (34)

This paper describes the effect of weak microwave fields on the amounts of heat-shock proteins in cell cultures at various temperatures. The field was generated by signal simulation of the Global System for Mobile communications (GSM) of 960 Mhz, used in portable phones. Transformed human epithelial amnion (AMA) cells, growing on glass coverslips, were exposed in a transverse electromagnetic (TEM) cell to a microwave field, generating a specific absorption rate (SAR) of 2.1 mW.kg−1 in the cells. Exposure temperatures were 35, 37, and 40 ± 0.1°C, respectively, and the exposure time was 20 min. The heat-shock proteins Hsp-70 and Hsp-27 were detected by immuno-fluorescence. Higher amounts of Hsp-70 were present in the cells exposed at 35 and 37°C than in the sham-exposed cells. These effects can be considered to be athermal, since the field strength was much lower than the safety standard for absence of heat generation by microwave fields. There was no significant response in the case of Hsp-27.
The number of reports on the effects induced by radiofrequency (RF) electromagnetic fields and microwave (MW) radiation in various cellular systems is still increasing. Until now no satisfactory mechanism has been proposed to explain the biological effects of these fields. One of the current theories is that heat generation by RF/MW is the cause, in spite of the fact that a great number of studies under isothermal conditions have reported significant cellular changes after exposure to RF/MW. Therefore, this study was undertaken to investigate which effect MW radiation from these fields in combination with a significant change of temperature could have on cell proliferation. The experiments were performed on the same cell line, and with the same exposure system as in a previous work [S. Kwee, P. Raskmark, Changes in cell proliferation due to environmental non-ionizing radiation: 2. Microwave radiation, Bioelectrochem. Bioenerg., 44 (1998), pp. 251-255]. The field was generated by signal simulation of the Global System for Mobile communications (GSM) of 960 MHz. Cell cultures, growing in microtiter plates, were exposed in a specially constructed chamber, a Transverse Electromagnetic (TEM) cell. The Specific Absorption Rate (SAR) value for each cell well was calculated for this exposure system. However, in this study the cells were exposed to the field at a higher or lower temperature than the temperature in the field-free incubator i.e., the temperature in the TEM cell was either 39 or 35 +/- 0.1 degrees C. The corresponding sham experiments were performed under exactly the same experimental conditions. The results showed that there was a significant change in cell proliferation in the exposed cells in comparison to the non-exposed (control) cells at both temperatures. On the other hand, no significant change in proliferation rate was found in the sham-exposed cells at both temperatures. This shows that biological effects due to RF/MW cannot be attributed only to a change of temperature. Since the RF/MW induced changes were of the same order of magnitude at both temperatures and also comparable to our previous results under isothermal conditions at 37 degrees C, cellular stress caused by electromagnetic fields could initiate the changes in cell cycle reaction rates. It is widely accepted that certain classes of heat-shock proteins are involved in these stress reactions.
In spite of numerous studies of the biological effects from exposure to weak electromagnetic(EM) fields, the issue of whether continuous exposure to these fields involves health risks, is still a subject for considerable debate. On cellular level effects on cell proliferation, enzyme activities, calcium transport, transcription and chromosome aberations have been reported. On the other hand there are several studies reporting negative results, where no effects of exposure to EM fields were found.
Due to the use of mobile telephones, there is an increased exposure of the environment to weak radiofrequency (RF) electromagnetic fields, emitted by these devices. This study was undertaken to investigate if the microwave radiation from these fields will have a similar effect on cell proliferation as weak electromagnetic (ELF) fields. The field was generated by signal simulation of the Global System for Mobile communications (GSM) of 960 MHz. Cell cultures, growing in microtiter plates, were exposed in a specially constructed chamber, a Transverse Electromagnetic (TEM) cell. The Specific Absorption Rate (SAR) values for each cell well were calculated for this exposure system. Experiments were performed on cell cultures of transformed human epithelial amnion cells (AMA), which were exposed to 960 MHz microwave fields at three different power levels and three different exposure times, respectively. It was found that cell growth in the exposed cells was decreased in comparison to that in the control and sham exposed cells. Cell proliferation during the period following exposure varied not only with the various SAR levels, but also with the length of exposure time. On the other hand, repeated periods of exposure did not seem to change the effects. There was a general linear correlation between power level and growth change. However, the exposure time required to obtain the maximum effect was not the same for the various power levels. It turned out that at low power level, a maximum effect was first reached after a longer exposure time than at higher power level. A similar phenomenon was registered in the studies on ELF electromagnetic fields. Here, it was found that there was a linear correlation between the length of exposure time to obtain maximum effect and field strength.
A significant increase in cell growth was registered in human epithelial amnion (AMA) cells, when exposed to a sinusoidal 50 Hz, 50 μT electromagnetic field (S. Kwee and P. Raskmark, Bioelectrochem. Bioenerg., 36 (1995) 109). To study the effects of incoherent magnetic fields on the biological changes caused by the electromagnetic fields, varying levels of noise were superimposed on the above-mentioned field. No inhibition of the coherent magnetic field effects were seen at low noise magnetic field densities. However, when the noise reached a level of 70–80% of the magnetic field, a significant inhibition of the increased cell proliferation was registered. The imposed inhibition remained constant up to a noise level equal to the the magnetic field. No significant change in proliferation rate was seen when the cell culture was exposed to a noise field only.
To study the effect of extremely low frequency (ELF) magnetic fields on cell growth, human cells (AMA cells) and K14 skin fibroblasts cells, growing in monolayer culture, were exposed to a sinusoidal 50 Hz, 80 μT field. Exposure times varied from 15 to 90 min. Changes in cell proliferation rates were then studied during subsequent field-free incubation, for 24 h.The results showed that a 30 min exposure resulted in a much higher increase in proliferation rates in the AMA cells compared with non-exposed cells or cells exposed to electromagnetic fields for shorter or longer times. The magnitude of the increase also depended on the initial proliferation rate and confluency. The exposure to varying field densities showed that the greatest increase in proliferation occurred at 80 μT.
A comparative study of the electroporation of transformed human amnion (AMA) cells and their primary counterpart is presented. The differences in conditions for electroporation, membrane resealing and viability between these two cell types are reported, as well as the effects of the introduction of three different antibodies on cell proliferation and DNA replication.
A technique was developed to introduce monoclonal antibodies into cultured cells by electroporation, while retaining cell viability at the same time (Kwee et al., 1990). This enabled us to study cell growth and DNA synthesis in response to electroporation and the following uptake of specific antibodies.
Cultured cells grown in monolayers were permeabilized by applying low-voltage electric pulses. The permeabilized cells were able to incorporate extracellular nucleotides into their DNA, as well as various monoclonal antibodies as determined by indirect immunofluorescence. Complete reversibility and long term viability were retained. Both applied voltage and pulse duration were important parameters for this method and the results were different for different cell lines.
Cultured cells grown in monolayers were permeabilized by applying low-voltage electric pulses. The permeabilized cells were able to incorporate extracellular nucleotides into their DNA, as well as various monoclonal antibodies as determined by indirect immunofluorescence. Complete reversibility and long term viability were retained. Both applied voltage and pulse duration were important parameters for this method and the results were different for different cell lines.
Comprehensive, computerized databases of cellular protein information derived from the analysis of two-dimensional gels, together with recently developed techniques to microsequence proteins offer a new dimension to the study of genome organization and function. In particular, human protein databases provide an ideal framework in which to focus the human genome sequencing effort.
Databases of protein information from human embryonal lung fibroblasts (MRC-5) have been established using computer analyzed two-dimensional gel electrophoresis. One thousand four hundred and eighty-two cellular proteins (1060 with isoelectric focusing and 422 with nonequilibrium pH gradient electrophoresis, in the first dimension) ranging in molecular mass between 8 and 234 kDa were separated and numbered. Information entered in the database (in most cases for major proteins) includes: protein name, HeLa protein catalog number, mouse protein catalog number, proteins matched in transformed human epithelial amnion cells (AMA) and peripheral blood mononuclear cells (PBMC), transformation and/or proliferation sensitive proteins, synthesis in quiescent cells, cell cycle regulated proteins, mitochondrial and heat shock proteins, cytoskeletal proteins and proteins whose synthesis is affected by interferons. Additional information entered for a few transformation-sensitive proteins that have been selected for future studies includes levels of synthesis and amounts in fetal human tissues. A total of four hundred and seventy-six [35S]methionine labeled polypeptides (258 isoelectric focusing; 218, nonequilibrium pH gradient electrophoresis) secreted by MRC-5 fibroblasts were separated and recorded (J. E. Celis et al., Leukemia 1987, 1, 707-717). Information entered in this database includes molecular weight and transformation sensitive proteins. These databases, as well as those of epithelial and lymphoid cell proteins (J. E. Celis et al., Leukemia 1988, 9, 561-601), represent the initial stages of a systematic effort to establish comprehensive databases of human protein information. In the long run, these databases are expected to offer a useful framework in which to focus the human genome sequencing effort.
Databases of protein information derived from the analysis of two-dimensional gels have been established from transformed human amnion cells (AMA) and peripheral blood mononuclear cells (PBMCs). A total of 1781 [35S]methionine-labeled AMA proteins (1274 IEF, 537 NEPHGE) and a total of 1311 proteins from PBMC (948 IEF, 363 NEPHGE) were resolved and recorded using computerized (PDQ-SCAN and PDQUEST softwares) two-dimensional gel electrophoresis. AMA and PBMC proteins (total, 454: 301 IEF, 153 NEPHGE) were matched both manually and by the computer. Information entered in the AMA database (in most cases for some major proteins) includes: molecular weight, protein name, HeLa protein catalogue number, mouse protein catalogue number, nuclear proteins, phosphorylated proteins, distribution of proteins in Triton X-100 supernatants and cytoskeletons, proliferation- and transformation-sensitive proteins, cell cycle-specific proteins, mitochondrial proteins, proteins matched in normal human embryonal lung MRC-5 fibroblasts and PBMC cells, heat shock proteins, proteins affected by interferons, cytoskeletal proteins, and the presence of antibody against protein in human sera. Additional information has been entered for the cell cycle-regulated and DNA replication protein cyclin (PCNA). Information entered in the PBMC database includes molecular weight and potential markers for sorted populations of lymphocyte subtypes. For those proteins that have been matched to AMA proteins, information contained in some entries may be transferred from the AMA database.
The process of the reduction of oxygen to water in the mitochondria has been widely studied in the course of time. It is generally accepted that oxygen is reduced by cytochrome oxidase, the terminal enzyme in the electron transport chain, in a single 4-electron 4-proton step. However, the discussion about the true mechanism of the process is still not closed. The electrochemical reduction of oxygen has also been studied extensively by direct methods1 as well as indirectly in the presence of mediators2–4. At the electrode the reduction of oxygen proceeds in two 2-electron steps. This process would imply hydrogen peroxide as the first reduction product, which is then reduced to water in the second step. In the presence of one specific porphyrin mediator, practically no hydrogen peroxide was found, which in this case could indicate that a direct reduction of oxygen to water has taken place5.
The oxidation of 6,7-dimethyltetrahydropterin (2-amino-6,7-dimethyl-5,6,7,8-tetrahydro-4-pteridone) was studied by electroanalytical techniques and by its reaction with the one-electron acceptors oxygen and ferricytochrome c. The results indicated that the oxidation process consisted of two one-electron steps, involving the transient generation of pterin radicals. It was also found that in the reaction between tetrahydropterin and oxygen, superoxide was formed and that oxygen was finally reduced to hydrogen peroxide.
The application of quinoxaline as a mediator in the indirect electrolysis of various hemoproteins is presented.Quinoxaline is reduced primarily to the radical anion, which can transfer its electron. Metalloproteins can serve as electron acceptors, in which case the metal ion is reduced. Hemoglobin, myoglobin, cytochrome c and cytochrome oxidase are reduced electrolytically in the presence of quinoxaline.In the presence of oxygen these proteins can be reduced partially by the electrogenerated superoxide anion radical without the aid of a mediator. The difference between the reduction by the superoxide and the quinoxaline radical is described.In the absence of an electron acceptor, in aqueous solutions, the quinoxaline radical anion is protonated to the neutral radical, which dimerizes to the tetrahydro-2,2′-biquinoxaline. This dimer is oxidized via unstable intermediates to 2,2′-biquinoxaline and/or is converted to the starting material during isolation procedures. At metal cathodes, such as mercury or lead, the dimer forms an insoluble metal complex, which can only be oxidized by a stronger oxidant, such as potassium ferricyanide.
Binding of dodecyl octaethyleneglycol monoether (C12E8), purified Triton X-100 and dodecyldimethylamino oxide (DDAO) to various integral membrane proteins was studied by chromatographic procedures. Binding capacity decreased in the following order: bacteriorhodopsin ~ mammalian rhodopsin > photochemical reaction center > sarcoplasmic reticulum Ca2+-ATPase. The detergents were bound in different amounts to the proteins (DDAO > polyoxyethylene glycol surfactants). Appreciable binding of C12E8, as well as of DDAO to Ca2+-ATPase was observed far below the critical micelle concentration, consistent with the interaction of the membrane protein with non-micellar detergent.
An electrochemical synthesis of isomer-free 6-methylpterin from folic acid has been developed. Folic acid is reduced to 7,8-dihydrofolic acid via 5,8-dihydrofolic acid. Under acidic conditions the C(9)N(10) bond in 7,8-dihydrofolic acid is reduced electrochemically, resulting in 6-methyl-7,8-dihydropterin and p-aminobenzoylglutamic acid. 6-Methylpterin can be obtained by oxidation of the corresponding dihydropterin. 7,8-Dihydrofolic acid is stable under anaerobic conditions between pH 4 and 9. Under strongly acidic conditions hydrolysis of the C(9)N(10) bond takes place and 6-hydroxymethyl-7,8-dihydropterin and p-aminobenzoylglutamic acid are obtained. Under aerobic conditions oxidative cleavage of the C(9)N(10) bond results in various pterin derivatives.Some 6-azomethinpterins were synthesized. Voltammetric studies of these compounds show that the reduction of the pterin nucleus follows the same general pathway described for the reduction of pterins. Reduction of the C(9)N(10) double bond proceeds through a reversible two-electron step, followed by a transformation, resulting in saturation of the bond.
Binding of dodecyloctaethyleneglycol monoether (C12E3) and purified Triton X-100 to various integral membrane proteins was studied by chromatographic procedures. Binding capacity decreased in the following order: bovine rhodopsin greater than photochemical reaction center greater than sarcoplasmic reticulum Ca2+-ATPase. The detergents were bound in different amounts to the proteins and less than corresponding to the aggregation number of the pure micelles. Appreciable binding of C12E8 to Ca2+-ATPase was observed far below the critical micelle concentration, consistent with interaction of the membrane protein with non-micellar detergent. Model calculations indicate that the detergents cannot combine with the membrane proteins, forming an oblate ring similar to that of pure detergent micelles, such as has been previously proposed for e.g. cytochrome b5 [Robinson and Tanford (1975) Biochemistry, 14, 365-378]. Other arrangements (prolate and monolayer rings), in which all detergent molecules are in contact with the protein, are considered as alternatives for covering the hydrophobic surface of the membrane protein with a continuous layer of detergent.
Tetrahydrofolic acid (H4PteGlu) functions as a cofactor for a large group of enzyme-catalyzed reactions. Due to the stereoselective nature of these reactions only one diastereomer can be utilized. In the reduction of L-folic acid to tetrahydrofolic acid a second asymmetric centre is created (at C-6) yielding 2 diastereomers. Tetrahydrofolic acid can be prepared electrochemically from L-folic acid [1]. In analogy to chemical and catalytic reductions equal amounts of l, L-H4PteGlu and d,L-H4PteGlu could be expected from the electrochemical reductions. Since folic acid is strongly adsorbed to a mercury electrode, a change of the chiral environment at the electrode might induce stereospecificity. To this purpose L-folic acid was reduced in the presence of small amounts of optically active compounds such as different proteins and alkaloids. At the same time the effects of a change in electrode shape and material were studied. Preliminary results showed that enantiomeric excesses up to 20 % could be obtained as determined by polarimetry and enzyme activity.
An electrochemical synthesis of 5,6,7,8-tetrahydrofolic acid (H4PteGlu) from folic acid (PteGlu) has been developed. In the first step folic acid is reduced, via 5,8-dihydrofolic acid (5,8-H2PteGlu), to 7,8-H2PteGlu. Reduction of the protonated 7,8-H2PteGlu cleaves C(9)-N(10), but reduction of the unprotonated compound at a more negative potential leads to H4PteGlu in high yield. The reduction is made at relatively low buffer concentration which permits having a neutral pH in the bulk of the solution, but a high pH at the electrode surface due to the electrogenerated base.Besides the preparation of H4PteGlu, the reduction of 5,10-methylidyne-H4PteGlu and 5,10-methylene-H4PteGlu was studied.
The electrochemical reduction of the disulfide bonds in α-chymotrypsin and trypsin is described. After reduction a separation of the respective chains is attempted by gel-filtration. It is shown that biological activity is dependent on the number of disulfide bonds cleaved.
Direct electrolysis of proteins in neutral solutions has been met with limited success, but an electrochemical reduction of disulfide bonds in proteins may be obtained in the presence of a pteridone, which has been previously employed as a mediator in other systems. In certain cases the addition of guanidine hydrochloride made possible a more complete reduction of the disulfide bonds.
Some Macromolecular compounds have been reduced or oxidized electrolytically by means of two mediator systems, both derived from 2-aminopteridone-4. As substrates NAD+ (reduction to NADH), NADH (oxidation to NAD+, cytochrome c (reduction), hemoglobin (reduction to ferrooxyhemoglobin or to ferrohemoglobin), and insulin (reduction of two or three disulphide bonds) were used; the mediator systems were of the types; aminopteridone/5,8-dihydroaminopteridone and 6,7-dihydroaminopteridone/5,6,7,8-tetrahydroaminopteridone. The indirect electrolytic reaction by means of these mediator systems offers, compared to the direct electrolysis of the substrates, several advantages such as rapid reaction and high enzymatic activity of the products.
Some substituted 4(3H)-pteridones have been investigated by classical polarography, cyclic voltammetry, and controlled potential electrolysis; based on these results, the following reaction path for substituted 2-amino-4(3H)-pteridones seems likely. In neutral and slightly alkaline solution the first step is a reversible two-electron reduction to the unreducible 5,8-dihydro derivative, which tautomerizes into a reducible 7,8-dihydropteridone at a rate depending on the substituents. At a more negative potential the 7,8-dihydro derivative is reduced in a two-electron reaction to the unreducible 5,6,7,8-tetrahydropteridone. This compound can be oxidized in a reversible reaction to a 6,7-dihydropteridone, a “quinonoid” form, which tautomerizes into the 7,8-dihydropteridone. The data from cyclic voltammetry favour the formulation of the “quinonoid” form as the 6,7-dihydropteridone, an “o-quinonoid” structure.
In acidic and neutral solution 4-dimethylamino, 4-amino, 4-mercapot, 4-methylrnercapto-derivatives of quinazolines are reduced in a 2 e step to their respective 3,4-dihydro derivatives. This is followed by elimination of the substituent group, resulting in a generation of the 3,4 double bond and formation of quinazoline itself. Quinazoline is then reduced further to the 3,4-dihydroquinazoline, since the half-wave potential of quinazoline is less negative then those of its 4-substituted derivatives. In alkaline solution there is a second wave which corresponds to the further reduction of the 3,4-dihydroquinazoline to the 1,2,3,4-tetrahydro-compound. Adenine and 6-methylaminopurine show only one wave in acidic solution, catalyzing hydrogen evolution. Macro-scale electrolysis proved that the same mechanism is operating here as in the case of the quinazolines. First a 2 e reduction to the 1,6-dihydroadenine, followed by deamination, yielding purine itself, which is reduced further to the 1,2,3,6-tetrahydropurine at the same potential. Purine itself is reduced in two 2 e steps to its tetrahydro derivative at potentials less negative than that of adenine. 1-Methyladenine shows a similar behaviour and yields 1-methyl-1,2,3,6-tetrahydropurine on reduction and also here ammonia is eliminated in the second step.
Durch elektrolytische Reduktion der Nitroverbindungen (I) bei kontrolliertem Kathodenpotential zu den Benzamidrazonen (II), anschließenden Ringschluß und Ammoniakabspaltung unter Bildung der Dihydrotriazine (III) und Oxidation entstehen die Benzotriazine (IV) (85-96% Ausbeute).

Citations

Top co-authors (39)

Henning Lund
  • Aarhus University
Julio Celis
  • Danish Cancer Society
Borbala Gesser
  • Aarhus University
Henrik Vendelbo Nielsen
  • Aarhus University Hospital
Jesper Vuust Møller
  • Aarhus University
Svetlozar Velizarov
  • Universidade NOVA de Lisboa
Peder Madsen
  • Aarhus University

Affiliations

Aarhus University
Department
  • Department of Medical Biochemistry

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