Influence of very low doses of mediators on fungal laccase activity - nonlinearity beyond imagination
© Malarczyk et al; licensee BioMed Central Ltd. 2009
Received: 20 April 2009
Accepted: 4 September 2009
Published: 4 September 2009
Laccase, an enzyme responsible for aerobic transformations of natural phenolics, in industrial applications requires the presence of low-molecular substances known as mediators, which accelerate oxidation processes. However, the use of mediators is limited by their toxicity and the high costs of exploitation. The activation of extracellular laccase in growing fungal culture with highly diluted mediators, ABTS and HBT is described. Two high laccase-producing fungal strains, Trametes versicolor and Cerrena unicolor, were used in this study as a source of enzyme. Selected dilutions of the mediators significantly increased the activity of extracellular laccase during 14 days of cultivation what was distinctly visible in PAGE technique and in colorimetric tests. The same mediator dilutions increased demethylation properties of laccase, which was demonstrated during incubation of enzyme with veratric acid. It was established that the activation effect was assigned to specific dilutions of mediators. Our dose-response dilution process smoothly passes into the range of action of homeopathic dilutions and is of interest for homeopaths.
Laccases (EC 220.127.116.11, p-diphenol: dioxygen oxidoreductases) are multi-copper proteins that use molecular oxygen to oxidize various phenolic and non-phenolic aromatic compounds by a radical-catalyzed reaction mechanism. Laccases can also recognize as glycoproteins since carbohydrates take part in the chemical stabilization of enzymatic molecules [1, 2]. Fungi produce laccases for degradation of polymeric phenolic compounds which are the main source of carbon. Molecule of laccase is too large for direct contact with the phenolics present in the inner part of such biopolymers as lignin or the lignocellulose complex, so the process of degradation runs very slowly and needs to cooperate with specific co-catalizators. They can enhance laccase activity by using low molecular compounds which accelerate the catalytic properties of enzyme by improving their affinity of specific radical forms to phenolic biopolymers . Fungal laccases, as well known biocatalysts, are readily used in many branches of industrial technology. Among them the depolymerization  and biobleaching of lignin , decolorization of artificial dyes in the textile industry [1, 6] or of natural pigments in food technology  could be mentioned. Laccases can also oxidize xenobiotic compounds  and remove of lipophilic compounds from paper pulp . Another important activity of laccase is demethylation of non-phenolic lignins [10, 11]. At the beginning of this process, non-phenolic lignin particles, rich in methoxylic compounds as veratrate or anisate, are degraded by laccase to phenolic compounds mainly via demethylation and hydroxylation and these processes are accelerated in combination with redox co-catalysts known as mediators [12, 13]. Many popular mediators of laccase are recognized also among the natural products of lignin and humus degradation and syringaldehyde, acetosyringone, vanillic, p-coumaric and ferulic acids can be mentioned [14, 15]. These natural mediators represent an alternative to synthetic mediators which are more efficient but unfortunately more toxic for environment and more expensive in exploitation.
Commonly used synthetic laccase mediators belong to phenolics. Most of them contain N-hydroxy-groups as 1-hydroxybenzotriazole (HBT), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), and N-hydroxy-acetanilide (NHA) or contain pirimidine origin such as violuric acid (VA), which can disturb the organization of nucleic acids [16, 17]. Also transition metal complexes, such as K4Mo(CN)8 or K4W(CN)8), were suggested as a new class of laccase mediators for pulp bleaching [18, 19]. For industrial purposes, both synthetic and metal-complex mediators must be used in large quantities, which is not very safe for the environment and is very costly. To diminish these difficulties, various methods have been tested and among them, electrochemical methods [20, 21] and production of laccase recombinants could be mentioned. In our earlier papers, the opportunity of using the low doses of laccase effectors during fungal cultivation was tested . The possibility to enhance laccase efficiency by the incubation with high dilutions of synthetic mediators is now presented. These experiments are based on our earlier results with changes of plant peroxidase activity in the presence of very low doses of selected phenolics [23, 24]. The oscillating character of the mentioned changes allowed us to indicate the dilutions of the tested aromatic substances which reveal a maximum and a minimum effect on enzymatic activity. These changes had been tested using spectrophotometrical and luminometrical methods . In the present study the technique of electrophoresis (PAGE) of native extracellular laccase from cultures of T. versicolor and C. unicolor was used to compare of isozymic patterns of laccase growing with or without low doses of mediators, ABTS and HBT.
Two species of white rot fungi with a high index of extracellular-laccase production, Trametes versicolor (L, ex Fr.) Pil and Cerrena unicolor (Bull. Ex Fr.) Murr, [26–28] originated from the culture collection of the Department of Biochemistry of Maria Curie-Sklodowska University in Lublin were used in the study.
Preparation of mediator dilutions
The substances affecting the activation of fungal laccase were chosen from among mediators well-known from the literature. These were ABTS and HBT, the both at an initial concentration of 1 mol/l. Each mediator was subjected to dynamic dilution in 75% ethanol by successive transfer to new portion of the diluent at 1:100 ratios. The Eppendorf tubes, every fulfilled with 1,98 ml of 75% ethanol, were shaken with 10 vertical strokes after serial transfer of 0,02 ml of mediator dilution according to homeopathic manner [23–25]. The dilutions were numbered in accordance with the number of transfers so that n = 1 corresponded to the one-hundredth dilution of the stock solution, n = 2 was a one-hundredth dilution of solution n = 1, etc. The transfers were usually repeated 10 times to obtain the final solution with n = 10 at a concentration of 100-10 mol/l. In the same manner the successive dilutions of 75% ethanol were prepared for control purpose. For comparative studies, dilution series which required 31 successive transfers (iterations) were prepared for ABTS.
Stock cultures grew stationary in liquid medium according to earlier report . In these conditions fungal mycelium is growing in the shape of flat comb. The common used technique with transfer of mycelia disks from stock cultures  were used for production of parallel cultures for comparison an action of serial dilutions. Mycelial discs were punched out with a 7 mm diameter cork borer after 10 days of cultivation from the stock culture mycelium under sterile conditions and every 3 discs were placed in flasks containing 10 ml of fresh medium. Starting from the second day of cultivation, each culture was supplemented every two days with 200 μl of serial dilution of ABTS and HBT, prepared as described in the above manner. The control cultures were lead with 200 μl of serial dilutions of 75% ethanol, puted in the same days as dilutions of mediators for dividing the effect of ethanol and mediators . To average the results, three cultures were performed for each dilution. The cultures were grown in stationary mood for fourteen days. After that time, the media were separated and the activity of extracellular laccase was measured.
Laccase activity assay
Laccase activity was measured spectrophotometrically using syringaldazine (2.5 μM) as the substrate in 0.1 M citrate-phosphate buffer, pH 5.2 according to the method . The activity was expressed as nkatals/l, using molar absorption coefficients of 65,000 M-1 cm-1 at 525 nm.
Non-denaturing polyacrylamide gel electrophoresis
PAGE technique with 12% poliacrylamide slabs without SDS was used to compare the native laccase isozyme patterns. For electrophoretic test the 5 ml volume of every medium after the end of cultivation was used according to . The media samples were demineralized on the column with Sephadex G25, concentrated by ultrafiltration using Microcon Centrifugal Filter Units, 3000 NMWL (Millipore) and lyophylised. After dissolution, 25 μl of every sample were deposited on 9-well slab, and the routine electrophoresis was run. After finishing of experiment the slabs were immediately specifically stained for laccase activity and enzymatic bands were visualized by the rapid color reaction with guaiacol in 0.1 M citrate-phosphate buffer at 25°C and pH 4.8 .
Veratrate demethylation assay
Laccase from T. versicolor was purified in routine manner according to .
1. Changes in laccase activity in cultures of T. versicolor and C. unicolorin the presence of low doses of mediators
Fungal strains were grown for 14 days with the addition of the mediators, ABTS and HBT, diluted serially in 75% ethanol as described in Methods. Each culture was treated every two days with a new portion of 200 μl of a given dilution. It can be mentioned that although after 14 days of culture the mycelium growth was similar for all trials, including the control cultures, but the activity of extracellular laccase was different, depending on the type of mediator and the degree of its dilution. The curves of laccase activities had always sine shape, which were characteristic for the type of mediator and kind of fungi.
Extracellular laccase activity (Δ max - min) in media of 3 series of Trametes versicolor cultures and 3 series of Cerrena unicolor cultures growing 14 days in presence of dilutions of ABTS and HBT with extreme action on laccase activity (in katals/l).
6500 ± 150
1550 ± 92
4.2 ± 1.6
950 ± 65
275 ± 45
3.4 ± 1.4
6200 ± 120
1410 ± 79
4.4 ± 1.51
1220 ± 83
370 ± 72
3.3 ± 1.15
5900 ± 180
1311 ± 103
4.5 ± 1.74
980 ± 75
280 ± 55
3.5 ± 1.36
The electrophoretic patterns, prepared in the same manner for all variants, confirmed changes in the intensity of enzymatically active bands parallel to sine curve of enzymatic activity visible after specific guaiacol staining for laccase activity. A particular distinctive change occurred in cultures activated with ABTS, where all seven laccase isozymes underwent a significant intensification (Figures 3 and 4). The next experiments were lead only with ABTS dilutions in cultures of T. versicolor for the higher results then with C. unicolor cultures and HBT.
With series of ABTS dilutions extended to 31 iterations the next uncommon observations in changes of laccase activity were obtained. After crossing the value of Avogadro number (dilution with n = 12) the differences in activity were visible in agreement with sine curve shape. It was also confirmed by electrophoretic patterns. The amplitude of maximal and minimal values showed the declining character (Figure 5)
2. The influence of selected mediator dilutions on the demethylation activity of purified laccase
In the next part of studies on demethylation two extreme dilutions for the both mediators were chosen based on the results of experiments with PAGE (Figures 1, 2, 3, and 4), and incubation with pure laccase and veratrate was lead as above mentioned. The dilutions with maximal activity in changing the isozymic patterns were also very active in the demethylation processes.
These results confirmed that isozymic patterns, demethylation activities and activation/deactivation processes were influenced differently by mediators of different dilutions n, shaped like sine curve.
The presented results of activation or inhibition of extracellular laccase in laboratory cultures under the influence of diluted synthetic mediators ABTS and HBT are in accordance with the action of the law of hormesis. This law describes the opposing biological effect of diluted effectors and concerns regulation of the speed of biological response to a given stimulus based on the phenomenon of feedback and allosteric properties of complex protein systems . During gradual dilution, the intensity of a biological effect oscillates in a sinusoidal manner in accordance with a characteristic hormetic curve, which changes dynamically in time. The universality of the oscillatory influence of high dilutions on processes taking place in human and animal organisms has been pointed out in a growing number of studies, for instance those cataloged in the years 2001-2008 by Calabrese [37, 38].
In the experiments on fungal laccase, studies of the influence of the individual mediator dilutions in the order in which they had been prepared made it possible to distinguish those which activated laccase from those which inhibited it, and the use of two fungal species additionally showed that each of them had an individual pattern of the sine curve. This is an important observation, from which it follows that the results obtained in the present study cannot be put directly into practice without constructing appropriate hormetic curves in the conditions of a given laboratory. Also, there are yet other species of industrial fungi that need to be studied in which selective use of highly diluted mediators may strengthen the ability to produce laccase. The studies presented here show that among the molar dilutions, a clearly activating effect was exerted by those of the order of picomoles, corresponding to the 100-6 mol/l concentration of the mediator. Use of picogram, instead of gram amounts of a mediator in industry may lead to a significant reduction of industrial-scale use of mediators without lowering the effects of their action, which is promising not only on account of the general reduction of production costs but also because of a decreased pollution of the environment. It can also be mentioned that diluted mediators may act on fungal cultures without additional sterilization procedures if high percent ethanol is used in preparing the dilutions. These dependencies allow applying experimentally selected dilutions of fungal laccase mediators for intensification of oxidation processes during transformation of laccase substrates such as phenols and methoxyphenols.
The process of incubation of high-molecular-weight lignin polymers with laccase applied in the paper industry is long-lasting and its supplementation with diluted effectors may have a practical use in shortening the contact of wood with a fungus. In accordance with the results shown in Fig. 6, an activating concentration of ABTS which corresponds to dilution n = 6 is within the range of 100-6 mol/l and is thus a picogram quantity. Even repeated supplementation with such a strongly-diluted effector will remain safe for the environment and will considerably decrease the costs of applying these useful laccase-activating compounds.
It seems that this remains true for both native laccase from in vitro cultures and for the pure enzyme, after its isolation and purification. PAGE data indicate that the effect of the diluted effectors reaches as far as the molecular level of cells, increasing the processes of isozyme production in the fungal cell. A well-visible strengthening of the bands of laccase activity, corresponding to the process of intensification of laccase induction by the most active dilutions, dependent on the type of mediator and kind of fungal strain, was observed during our study. We also noted a simultaneous augmentation of the pool of laccase secreted extracellularly from cells to the culture medium.
Although the double action of biologically active substances, dependent on the degree of dilution, has been confirmed in numerous experiments, this phenomenon has not received a complete explanation within the accepted scientific canons. Currently, more and more studies point to the possibility of explaining the biological effect of low doses by means of physical and chemical instruments [39–43]. Probably, in the future it will be possible to use those instruments to explain the phenomenon, described in this work, of reaction continued for dilutions greater than the Avogadro number. This phenomenon was already signaled earlier in the 1980s by French researchers  for human and animal organisms and has now also been demonstrated for plant material . Also similar results with fungal material or pure enzymes were presented earlier by us, and in all those experiments the conformity and repeatability of the results were very high. All these data seem to constitute a prelude to recognizing hormesis, so far considered in molecular categories, to be an initial stage of the dose-response dilution process which smoothly passes into the range of action of homeopathic dilutions greater than the Avogadro number. Although the phenomenon has not yet been explained by contemporary science (cf. ), the growing number of common observations enforces the need to acknowledge the facts and the possibility of their practical implementation just as unfamiliarity with the scientific basis of fermentation did not prevent the Ancients from consuming large amounts of wine. The physico-chemical character of the discussed phenomenon seems important for the biology and biotechnology of fungal laccase because of its practical consequences in reducing the toxic effect of industrially applied laccase mediators on the environment.
During cultivation of two fungal strains, Trametes versicolor and Cerrena unicolor, in the presence of high diluted laccase mediators (ABTS, HBT), changes in the production and activity of extracellular laccase in the culture medium were observed. The profile of the activity had the character of a sine curve with distinct maxima and minima. These changes were also noticed in the enzymatic patterns after specific visualization of laccase activity on PAGE gel, and the correlation between maximum activities on the sine curve and the abundance of laccase-active protein bands was very distinct. The selected dilutions of the mediators caused similar changes in the demethylation abilities of pure laccase isolated from T. versicolor. All these results strongly bear out the new features of diluted substances against selected enzymes tested in in vitro and in vivo conditions. The biphasic role of high diluted low molecular effectors on the laccase system of fungal cells is best understood as an instance of hormesis, in which every substance after dilution can act in vivo either as an inhibitor or as an activator of a biological process dependent on the rate of dilution. As for laccase applications, the possibility of radically diminishing the amount of its mediators in industrial processes with simultaneous augmentation of its production during fungal cultivation is very real. For this reason, the mentioned results are very interesting both from the economic point of view and from the perspective of environmental protection against chemical pollution.
The presented study should be treated mainly as a report on the newly discovered ability of selected dilutions of commonly used mediators to change the activity of laccase. Practical application of these results in industrial processes will require from factories adaptation of this knowledge to their own experimental conditions.
This work was supported by the Polish Scientific Project BS/UMCS.
- Alcalde M: Laccase: biological functions, molecular structure and industrial applications. Industrial Enzymes: structure, function and applications. Edited by: Polaina J, MacCabe AP. 2007, New York: Springer, 459-474.Google Scholar
- Nishizawa Y, Nakabayashi K, Shinagawa E: Purification and characterization of laccase from white rot fungus Trametes sanguinea M85-2. J Ferment Bioeng. 1995, 80 (1): 91-93. 10.1016/0922-338X(95)98183-L.View ArticleGoogle Scholar
- Li K, Xu F, Eriksson KEL: Comparison of fungal laccases and redox mediators in oxidation of a nonphenolic lignin model compound. Appl Environ Microbiol. 1999, 65 (6): 2654-2660.Google Scholar
- Moldes D, Díaz M, Tzanov T, Vidal T: Comparative study of the efficiency of synthetic and natural mediators in laccase-assisted bleaching of eucalyptus kraft pulp. Bioresource Technol. 2008, 99 (17): 7959-7965. 10.1016/j.biortech.2008.04.002.View ArticleGoogle Scholar
- Knutson K, Ragauskas A: Laccase-mediator biobleaching applied to a direct yellow dyed paper. Biotechnol Prog. 2004, 20 (6): 1893-1896. 10.1021/bp049833+.View ArticleGoogle Scholar
- Machado KMG, Matheus DR, Bononi VLR: Ligninolytic enzymes production and Remazol brilliant blue R decolorization by tropical brazilian basidiomycetes fungi. Brazil J Microbiol. 2005, 36 (3): 211-216. [http://www.scielo.br/pdf/bjm/v36n3/arq08.pdf]View ArticleGoogle Scholar
- Kunamneni A, Camarero S, García-Burgos C, Plou FJ, Ballesteros A, Alcalde M: Engineering and Applications of fungal laccases for organic synthesis. Microbiol Cell Fact. 2008, 7: 1-32. 10.1186/1475-2859-7-32.View ArticleGoogle Scholar
- Rodríguez E, Nuero O, Guillén F, Martínez AT, Martínez MJ: Degradation of phenolic and non-phenolic aromatic pollutants by four Pleurotus species: the role of laccase and versatile peroxidase. Soil Biol Biochem. 2004, 36 (6): 909-916. 10.1016/j.soilbio.2004.02.005.View ArticleGoogle Scholar
- Gutiérrez A, Rencoret J, Ibarra D, Molina S, Camarero S, Romero J, Del Río JC, Martínez AT: Removal of lipophilic extractives from paper pulp by laccase and lignin-derived phenols as natural mediators. Environ Sci Technol. 2007, 41 (11): 4124-4129. 10.1021/es062723+.View ArticleADSGoogle Scholar
- Srebotnik E, Hammel KE: Degradation of nonphenolic lignin by the laccase/1-hydroxybenzotriazole system. J Biotechnol. 2000, 81 (2-3): 179-188. 10.1016/S0168-1656(00)00303-5.View ArticleGoogle Scholar
- Bourbonnais R, Paice MG: Oxidation of non-phenolic substrates. An expanded role of laccase in lignin biodegradation. FEBS Lett. 1990, 267: 99-102. 10.1016/0014-5793(90)80298-W.View ArticleGoogle Scholar
- Crestini C, Argyropoulos DS: The early oxidative biodegradation steps of residual kraft lignin models with laccase. Bioorgan Med Chem. 1998, 6 (11): 2161-2169. 10.1016/S0968-0896(98)00173-4.View ArticleGoogle Scholar
- Kawai S, Nakagawa M, Ohashi H: Degradation mechanisms of a nonphenolic β-O-4 lignin model dimer by Trametes versicolor laccase in the presence of 1-hydroxybenzotriazole. Enzyme Microb Technol. 2002, 30 (4): 482-489. 10.1016/S0141-0229(01)00523-3.View ArticleGoogle Scholar
- Johannes Ch, Majcherczyk A: Natural mediators in the oxidation of polycyclic aromatic hydrocarbons by laccase mediator systems. Appl Environ Microbiol. 2000, 66 (2): 524-528. 10.1128/AEM.66.2.524-528.2000.View ArticleGoogle Scholar
- Wells A, Teria M, Eve T: Green oxidations with laccase-mediator systems. Biochem Soc Trans. 2006, 34 (2): 304-308. 10.1042/BST20060304.View ArticleGoogle Scholar
- Mickel M, Kim H-C, Hampp N: Origin of the mediator losses in electrochemical delignification processes: Primary and secondary reactions of violuric acid and N, N'-dimethylvioluric acid radicals with lignin model compounds. Green Chem. 2003, 5: 8-14. 10.1039/b208812f.View ArticleGoogle Scholar
- d'Acunzo F, Galli C, Masci B: Oxidation of phenols by laccase and laccase-mediator systems: Solubility and steric issues. Eur J Biochem. 2002, 269: 5330-5335. 10.1046/j.1432-1033.2002.03256.x.View ArticleGoogle Scholar
- Korpainen T: Capital effectiveness in the paper industry. TAPPSA. 2002Google Scholar
- Bourbonnais R, Rochefort D, Paice MG, Renaud S, Leech D: Transition metal complexes: A new class of laccase mediators for pulp bleaching. Tappi J. 2000, 83 (10): 68-76.Google Scholar
- Hill CL, Prosser-McCartha CM: Homogeneous catalysis by transition metal oxygen anion clusters. Coord Chem Rev. 1995, 143: 407-455. 10.1016/0010-8545(95)01141-B.View ArticleGoogle Scholar
- Padtberg C, Kim H-C, Mickel M, Bartling S, Hampp N: Electrochemical delignification of softwood pulp with violuric acid. Tappi J. 2001, 84: 68-77.Google Scholar
- Claus H, Faber G, König H: Redox-mediated decolorization of synthetic dyes by fungal laccases. Appl Microbiol Biotechnol. 2002, 59 (6): 672-678. 10.1007/s00253-002-1047-z.View ArticleGoogle Scholar
- Malarczyk E, Jarosz-Wilkołazka A, Kochmańska-Rdest J: Effects of low doses of guaiacol and ethanol on enzymatic activity of fungal cultures. Nonlinear Biol Toxicol Med. 2003, 1 (2): 176-184.View ArticleGoogle Scholar
- Malarczyk E, Kochmańska-Rdest J, Paździoch-Czochra M: Effect of low and very low doses of simple phenolics on plant peroxidase activity. Nonlinear Biol Toxicol Med. 2004, 2 (2): 129-141. 10.1080/15401420490464466.View ArticleGoogle Scholar
- Malarczyk E: Kinetic changes in the activity of HRperoxidase induced by very low doses of phenol. Int J High Dilut Res. 2008, 7 (23): 3-11. [http://www.feg.unesp.br/~ojs/index.php/ijhdr/article/view/37/349]Google Scholar
- Hatakka A: Lignin-modifying enzymes from selected white-rot fungi production and role in lignin degradation. FEMS Microbiol Rev. 1994, 13: 125-135. 10.1111/j.1574-6976.1994.tb00039.x.View ArticleGoogle Scholar
- Janusz G, Rogalski J, Szczodrak J: Increased production of laccase by Cerrena unicolor in submerged liquid cultures. World J Microbiol Biotechnol. 2007, 23: 1459-1464. 10.1007/s11274-007-9390-y.View ArticleGoogle Scholar
- De Carvalho MEA, Monteiro MC, Sant'Anna GL: Laccase from Trametes versicolor. Appl Biochem Biotechnol. 1999, 77-79: 723-728. 10.1385/ABAB:79:1-3:723.View ArticleGoogle Scholar
- Ishikawa NK, Kasuya MCM, Vanetti MCD: Antibacterial activity of Lentinula edodes grown in liquid medium. Brazil J Microbiol. 2001, 32: 206-210. [http://www.scielo.br/pdf/bjm/v32n3/7738.pdf]View ArticleGoogle Scholar
- Lee I-Y, Jung K-H, Lee C-H, Park Y-H: Enhanced production of laccase in Trametes versicolor by the addition of ethanol. Biotechnol Lett. 1999, 21: 965-968. 10.1023/A:1005646812381.View ArticleGoogle Scholar
- Leonowicz A, Grzywnowicz K: Quantitative estimation of laccase forms in some white-rot fungi using syringaldazine as a substrate. Enzyme Microb Technol. 1981, 3: 55-62. 10.1016/0141-0229(81)90036-3.View ArticleGoogle Scholar
- Jaszek M, Grzywnowicz K, Malarczyk E, Leonowicz A: Enhanced extracellular laccase activity as a part of the response system of white rot fungi: Trametes versicolor and Abortiporus biennis to paraquat-caused oxidative stress conditions. Pestic Biochem Physiol. 2006, 85: 147-154. 10.1016/j.pestbp.2006.01.002.View ArticleGoogle Scholar
- Xiao YZ, Wu J, Hong YZ, Wang YP, Chen Q, Hang J, Shi YY: Selective induction, purification and characterization of a laccase isozyme from the basidiomycete Trametes sp. AH28-2. Mycologia. 2004, 96 (1): 26-35. 10.2307/3761984.View ArticleGoogle Scholar
- Jarosz-Wilkolazka A, Janusz G, Ruzgas T, Gorton L, Malarczyk E, Leonowicz A: Development of a laccase-modified electrode for amperometric detection of mono- and diphenols. The influence of enzyme storage method. Anal Lett. 2004, 37 (8): 1497-1513. 10.1081/AL-120037583.View ArticleGoogle Scholar
- Pazdzioch-Czochra M, Malarczyk E, Sielewisiuk J: Relationship of demethylation processes to veratric acid concentration and cell density in cultures of Rhodococcus erythropolis. Cell Biol Int. 2003, 27: 325-336. 10.1016/S1065-6995(02)00282-2.View ArticleGoogle Scholar
- Tuomela M: Degradation of lignin and other 14C-labelled compounds in compost and soil with an emphasis on white-rot fungi. 2002, Academic dissertation in microbiology, Faculty of Agriculture and Forestry of the University of Helsinki, [http://ethesis.helsinki.fi/julkaisut/maa/skemi/vk/tuomela/degradat.pdf]Google Scholar
- Calabrese EJ: Overcompensation stimulation: A mechanism for hormetic effects. Crit Rev Toxicol. 2001, 31: 425-470. 10.1080/20014091111749.View ArticleGoogle Scholar
- Calabrese EJ: Hormesis and medicine. Br J Clin Pharmacol. 2008, 66: 594-617.Google Scholar
- Stebbing AR: Hormesis-the stimulation of growth by low levels of inhibitors. Sci Total Environ. 1982, 22 (3): 213-34. 10.1016/0048-9697(82)90066-3.View ArticleGoogle Scholar
- Lenger K: Homeopathic potencies identified by a new magnetic resonance method. Subtle Energies & Energy Medicine. 2006, 15 (3): 225-243.Google Scholar
- Lenger K, Bajpai RP, Drexel M: Delayed luminescence of sugar globule with high homeopathic potencies. Homeopathy. 2008, 97 (3): 134-40. 10.1016/j.homp.2008.05.003.View ArticleGoogle Scholar
- Elia V, Niccoli M: Thermodynamics of extremely diluted aqueous solutions. An NY Acad Sci. 1999, 879: 241-248. 10.1111/j.1749-6632.1999.tb10426.x.View ArticleADSGoogle Scholar
- Elia V, Niccoli M: New physico-chemical properties of extremely diluted aqueous solutions. A calorimetric and conductivity study at 25°C. J Therm Anal Calorimet. 2004, 75: 815-836. 10.1023/B:JTAN.0000027178.11665.8f.View ArticleGoogle Scholar
- Belon P, Cumps J, Ennis M, Mannaioni PF, Roberfroid M, Sainte-Laudy J, Wiegant FA: Histamine dilutions modulate basophil activation. Inflamm Res. 2004, 53 (5): 181-8. 10.1007/s00011-003-1242-0. Epub 2004 Apr 21.View ArticleGoogle Scholar
- Tyihak E, Steiner U, Schönbeck F: Time- and dose-dependent double immune response of plants to pathogens. Modern Fungicide and Antifungal Compounds III, 13th Intern Reinhardsbrunn Symp, May 14th-18th 2001, Friedrichroda, Germany (AgroConcept GmbH Bonn). 2002, 187-196.Google Scholar
- Klonowski W: From conformons to human brains: an informal overview of nonlinear dynamics and its applications in biomedicine. Nonlinear Biomed Phys. 2007, 1: 5-8. 10.1186/1753-4631-1-5.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.