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The present study is designed to find out if sesquiterpenes, α-humulene (HUM), valencene (VAL), β-caryphyllene-oxide (CAO) and trans-nerolidol (NER), are able to improve the antiproliferative effect of classical cytostatic drugs, 5-fluorouracil (FU) and oxaliplatin (1,2-diaminocyclohexaneoxalato-platinum, OxPt), in colon cancer cell lines Caco-2 and SW-620. In addition, the possible mechanisms of sesquiterpene action are studied. The results show significant ability of HUM and especially of CAO to enhance the anti-proliferative effects of FU and OxPt in cancer cell lines Caco-2 and SW-620. On the other hand, VAL and NER are ineffective. The action of CAO could be partly based on its ability to disrupt the mitochondrial membrane potential and to activate initiator caspases, but other mechanisms are probably also involved. Based on these results, CAO seems to have the potential for combination therapy of colon cancers and deserves further study.

Stepanka Beranova,1 Jan Stoklasa,2 Iva Dudova,1 Daniela Markova,3 Martina Kasparova,4 Jana Zemankova,5 Tomas Urbanek,6 Tomas Talasek,2 Pasi Luukka,7 Michal Hrdlicka1 1Department of Child Psychiatry, Charles University Second Faculty of Medicine and University Hospital Motol, Prague, 2Department of Applied Economics, Faculty of Arts, Palacky University, Olomouc, 3Department of Pediatrics and Adolescent Medicine, Charles University First Faculty of Medicine and General University Hospital, Prague, 4Department of Pediatrics, Charles University Second Faculty of Medicine and University Hospital Motol, Prague, 5Department of Pediatrics, Charles University Faculty of Medicine and University Hospital, Hradec Kralove, 6Institute of Psychology, Academy of Sciences, Brno, Czech Republic; 7Laboratory of Applied Mathematics, Lappeenranta University of Technology, Lappeenranta, Finland Objective: The objective of this study was to explore the potential of the Infant/Toddler Sensory Profile (ITSP) as a screening tool for autism spectrum disorders (ASD) in prematurely born children.Methods: Parents of 157 children with birth weights

Monocytes/macrophages are thought to play a fundamental role in the development of vascular lesions in atherosclerosis. In the present study, we evaluated circulating CD14+CD16+ monocytes, laboratory parameters of the risk of atherosclerosis, including serum cholesterol, homocysteine and C-reactive protein, urinary neopterin, serum a-tocopherol and retinol along with carotid intima-media thickness in patients with breast carcinoma. A significant correlation was observed between the absolute numbers of CD14+CD16+ monocytes, serum HDL cholesterol and triglyceride concentrations. In conclusion, present data extend the observation of an association between peripheral blood CD14+CD16+ monocyte counts and lipid metabolism to cancer patients. No correlation of CD14+CD16+ monocyte counts with urinary neopterin concentrations was observed.

Despite increased efficacy of regimens combining cytotoxic drugs and targeted agents, most patients with metastatic colorectal cancer will ultimately die of the disease. Only a limited number of drugs have reproducible activity in therapy of this tumor, and new active agents are urgently needed. Promising results in the therapy of metastatic colorectal cancer have been reported for patupilone, an epothilone analogue. Similarly to taxanes, the cytotoxic mechanism of epothilones involves the stabilization of microtubules. Increased serum or urinary concentrations of neopterin have been described in patients with tumors of different primary locations, including colorectal cancer. Neopterin concentrations were reported to increase during the administration of cytotoxic agents, including taxane-based chemotherapy. We have studied serum neopterin in patients with colorectal cancer before and during the therapy with patupilone. Urinary neopterin/creatinine concentrations were determined with highperformance liquid chromatography. Increased urinary neopterin concentrations were observed at baseline in the majority of the patients. In most patients, neopterin concentrations further increased during the therapy, and a significant increase of urinary neopterin was observed in patients with normal baseline neopterin concentrations. A trend of decreased survival was observed for patients with high initial neopterin concentration. In conclusion, urinary neopterin is increased in colorectal cancer patients presenting for second or higher line of treatment. An increase of urinary neopterin during patupilone therapy suggests an activation of immune response by this agent.

Bevacizumab, monoclonal antibody targeting vascular endothelial growth factor, is effective in different tumors, including colorectal carcinoma, non-small cell lung cancer, renal cell carcinoma and breast cancer. Increased serum or urinary concentrations of neopterin, an indicator of systemic immune response, have been described in patients with tumors of different primary locations, and further increase has been observed during anticancer therapy. An increase of urinary neopterin has been described after administration of cytokines, cytotoxic chemotherapy, or external beam radiation, but less is known about the effects of targeted agents on systemic immune response. We have studied serum homocysteine, C-reactive protein, α-tocopherol and retinol, and urinary neopterin in patients with metastatic breast cancer treated with bevacizumab, taxane and carboplatin. Homocysteine and C-reactive protein were determined immunochemically. α-tocopherol, retinol and urinary neopterin were determined by high performance liquid chromatography. Homocysteine, C-reactive protein and urinary neopterin decreased, while retinol and α-tocopherol increased during the therapy. In conclusion, the treatment of patients with metastatic breast cancer with bevacizumab, taxane and carboplatin resulted in the suppression of systemic inflammatory and immune response. The suppression of systemic inflammatory and immune response was associated with an increase in serum vitamin concentrations.

Increased serum or urinary concentrations of neopterin have been described in patients with tumors of different primary locations, but reports on neopterin in patients with esophageal carcinoma are scanty. We have studied serum neopterin, retinol and alpha-tocopherol in 45 patients with carcinoma of the esophagus or gastroesophageal junction. Serum neopterin was determined using radioimmunoassay. Retinol and alpha-tocopherol were determined by high-performance liquid chromatography. Serum neopterin in patients with carcinoma of the esophagus was significantly increased while retinol and alpha-tocopherol were significantly decreased compared to controls. No correlation of neopterin with hemoglobin or peripheral blood cell counts was observed. During chemoradiation, neopterin increased significantly, while retinol and alpha-tocopherol did not change. In conclusion, the present study demonstrates increased serum neopterin and decreased retinol and alpha-tocopherol concentrations in patients with esophageal carcinoma. Chemoradiation induced a further increase of serum neopterin concentrations.

Iva Dudova,1 Daniela Markova,2 Martina Kasparova,3 Jana Zemankova,4 Stepanka Beranova,1 Tomas Urbanek,5 Michal Hrdlicka1 1Department of Child Psychiatry, Charles University Second Faculty of Medicine and University Hospital Motol, Prague, Czech Republic; 2Department of Pediatrics and Adolescent Medicine, Charles University First Faculty of Medicine and General University Hospital, Prague, Czech Republic; 3Department of Pediatrics, Charles University Second Faculty of Medicine and University Hospital Motol, Prague, Czech Republic; 4Department of Pediatrics, Charles University Faculty of Medicine and University Hospital, Hradec Kralove, Czech Republic; 5Institute of Psychology, Academy of Sciences, Brno, Czech Republic Background: Preterm children seem to be at increased risk for autism spectrum disorders (ASD). Methods: Parents of 157 children with birth weights less than 1,500 g (age 2 years, corrected for prematurity; 88 boys, 69 girls) completed screening questionnaires. The screening battery included the Modified Checklist for Autism in Toddlers (M-CHAT), Communication and Symbolic Behavior Scales Developmental Profile Infant-Toddler Checklist (CSBS-DP-ITC), and the Infant/Toddler Sensory Profile (ITSP). Children with disabilities were excluded. All children who screened positive on any of the screening tools were subsequently assessed by clinical examination including the Autism Diagnostic Observation Schedule. Results: Fifty-six children (35.7%) screened positive on at least one of the parental screening questionnaires. Of the 56 children who tested positive, 33 participated in the detailed clinical follow-up assessment. A diagnosis of ASD was confirmed in 13 of the 33 children. The ASD prevalence was 9.7% of the sample. Analysis of children with and without an ASD diagnosis found significant differences relative to gestational age (26.9 weeks vs 28.3 weeks, P=0.033) and length of the stay in hospital (89.5 days vs 75.4 days, P=0.042). The screening tool with the most positive results was CSBS-DP-ITC (42 positive screens [PS]), followed by M-CHAT (28 PS), and ITSP (22 PS). Differences in the frequency of PS among the tests were significant (P=0.008). CSBS-DP-ITC had the highest sensitivity (0.846), followed by M-CHAT (0.692) and ITSP (0.462). Conclusion: Our results indicate a higher prevalence of autism in children with birth weights

The purpose of this study is (1) to introduce a new approach for edge detection in orthopantograms (OPGs) and an improved automatic parameter selector for common edge detectors, (2) to present a comparison between our novel approach with common edge detectors and (3) to provide faster outputs without compromising quality. A new approach for edge detection based on statistical measures was introduced: (1) a set of N edge detection results is calculated from a given input image and a selected type of edge detector, (2) N correspondence maps are constructed from N edge detection results, (3) probabilities and average probabilities are computed, (4) an overall correspondence is evaluated for each correspondence map and (5) the correspondence map providing the best overall correspondence is taken as the result of edge detection procedure. A comparison with common edge detectors (the Roberts, Prewitt, Sobel, Laplacian of the Gaussian and Canny methods) with various parameter settings (304 combinations for each test image) was carried out. The methods were assessed objectively [edge mismatch error (EME), modified Hausdorff distance (MHD) and principal component analysis] and subjectively by experts in dentistry and based on time demands. The suitability of the new approach for edge detection in OPGs was confirmed by experts. The current conventional methods in edge detection in OPGs are inadequate (none of the tested methods reach an EME value or MHD value below 0.1). Our proposed approach for edge detection shows promising potential for its implementation in clinical dentistry. It enhances the accuracy of OPG interpretation and advances diagnosis and treatment planning.

Increased serum or urinary concentrations of neopterin have been described in patients with tumors of different primary locations, but reports on neopterin in patients with breast carcinoma are relatively innumerous. We have evaluated urinary neopterin in 456 patients with breast carcinoma. Urinary neopterin was determined using high-performance liquid chromatography. Neopterin in patients was increased only in a minority of patients with breast carcinoma. Increased urinary neopterin was associated with inferior overall survival. Prognostic significance of increased urinary neopterin was evident in patients with tumors expressing hormone receptors or/and human epidermal growth factor receptor (HER)-2, but not in patients with triple negative tumors. Among other parameters determined, C-reactive protein, hemoglobin, peripheral blood neutrophil count and platelet count were significant prognostic factors. On multivariate analysis, age, expression of hormone receptors, neutrophils, stage and hemoglobin concentration were independent prognostic indicators. In conclusion, serum neopterin is increased only in a minority of patients with breast carcinoma. Increased urinary neopterin was predictive of poor survival in univariate, but not multivariate analysis. Age, expression of hormone receptors, neutrophils, stage and hemoglobin concentration were independent prognostic indicators.

Although gastrointestinal toxicity is one of the most common side effects of anticancer therapy, the diagnosis and assessment of this toxicity still depend mostly on anamnestic data. Measurement of intestinal permeability is one of potential methods of non-invasive laboratory evaluation of gastrointestinal toxicity. The aim of the present study was to investigate intestinal permeability, vitamin A absorption, serum alpha-tocopherol, and urinary neopterin in patients with rectal carcinoma treated with chemoradiation. We have studied intestinal permeability, vitamin A absorption, serum alpha-tocopherol, and urinary neopterin in 17 patients with rectal carcinoma treated with chemoradiation. Urinary lactulose, mannitol, and xylose were measured by capillary gas chromatography, and serum alpha-tocopherol, retinol, retinyl esters, and urinary neopterin were determined by high-performance liquid chromatography. Lactulose/mannitol ratio was increased 5 and 6 weeks after the start of the treatment. Serum alpha-tocopherol was decreased significantly throughout the course of treatment, but no significant changes were observed in postprandial serum concentrations of retinyl esters or in the concentrations of urinary neopterin. A correlation was observed between baseline parameters of intestinal permeability and urinary neopterin. The measurement of intestinal permeability using the lactulose/mannitol test may represent a sensitive tool in the detection of changes associated with chemoradiation in patients with rectal carcinoma. The therapy is also associated with a decrease of alpha-tocopherol.

Cetuximab is a chimeric antibody registered for the therapy of advanced colorectal carcinoma. Cancer and anticancer therapy are associated with oxidative stress, and disorders of antioxidant balance may be involved in the toxicity associated with anticancer treatment. The aim of the present study was to investigate the changes of serum retinol, alpha-tocopherol and C-reactive protein during the first month of treatment with cetuximab and chemotherapy. Twenty-five consecutive patients with metastatic colorectal carcinoma treated with a combination of chemotherapy and cetuximab were included in the present study. Serum retinol and alpha-tocopherol were determined by high-performance liquid chromatography and serum C-reactive protein was determined using commercial kits. Significant correlation was observed between baseline concentrations of retinol and C-reactive protein (r(s)=-0.54, p<0.01). Median survival of patients who had baseline serum retinol below 1.25 µmol/L was 10 mo compared to 18 mo for patients who had serum retinol equal or above 1.25 µmol/L (p<0.05); median survival of patients who had serum C-reactive protein below 24 mg/L was significantly longer compared to patients with C-reactive protein levels equal or above 24 mg/L (18 vs. 7 mo, p<0.05), but no difference in survival was observed based on alpha-tocopherol levels. Twenty-two patients had evaluation of retinol, alpha-tocopherol and C-reactive protein at least once during the follow up. Serum concentration of alpha-tocopherol decreased significantly during the therapy, but retinol and C-reactive protein concentrations remained unchanged. In conclusion, a significant correlation was observed between serum retinol and C-reactive protein. Serum alpha-tocopherol decreased significantly during the first month of combination therapy with cetuximab. Low retinol and high C-reactive protein concentrations were predictive of poor prognosis in this patient population.

In an earlier study, we have observed an increase of alpha-tocopherol in breast cancer patients treated with third-generation aromatase inhibitors that was related to tamoxifen withdrawal. We report here the results of measurement of alpha-tocopherol in erythrocytes and alpha-tocopherol/cholesterol ratios in patients treated with letrozol. Alpha-tocopherol in lipoprotein fractions and erythrocytes was determined by high-performance liquid chromatography in 124 post-menopausal women with breast cancer treated with letrozol immediately before the start of treatment as well as 2 and 4 mo later. After a transient decrease after 2 mo of letrozol therapy, erythrocyte alpha-tocopherol concentrations returned to pre-treatment levels 4 mo after the start of treatment. Apart from lower cholesterol in patients pre-treated with tamoxifen, no significant differences were observed at baseline between patients previously treated with tamoxifen and patients who had no prior tamoxifen in any of the other parameters investigated, but the transient decrease of erythrocyte alpha-tocopherol was observed only in patients previously treated with tamoxifen. Alpha-tocopherol content of lipoprotein fractions was significantly increased 4 mo after the start of therapy, but this increase was evident mostly in patients not treated earlier with tamoxifen. In conclusion, only minor changes of alpha-tocopherol, including a transient decrease of alpha-tocopherol in erythrocyte membranes, and an increase of alpha-tocopherol in lipoprotein fractions were observed during the first 4 mo of letrozol therapy.

Background: Targeted agents present with a new spectrum of side-effects, including toxicities that negatively impact the risk of atherosclerosis. The aim of the present study was to evaluate the effect of the combination of targeted therapy and chemotherapy on serum homocysteine and other laboratory parameters of cardiovascular risk in patients with metastatic colorectal carcinoma., Patients and Methods: Thirty-one patients with metastatic colorectal carcinoma treated with the combination of bevacizumab, oxaliplatin, 5-fluorouracil and leucovorin were studied before and during the therapy., Results: Serum homocysteine decreased significantly throughout the course of treatment. Total cholesterol and low-density lipoprotein cholesterol also decreased significantly during the first month of therapy. In contrast, serum retinol significantly increased during the second and third months of treatment. A significant increase in glycosylated hemoglobin was also observed. After an initial rise, serum C-reactive protein (CRP) and carcinoembryonic antigen (CEA) were significantly lower compared to baseline throughout the course of treatment. Serum ferritin increased throughout most of the course of treatment. A significant correlation was observed between CRP and high-density lipoprotein cholesterol, retinol, ferritin, and CEA. CEA correlated with hemoglobin, retinol, and ferritin. Retinol correlated significantly with hemoglobin., Conclusion: Tumor control, reflected in lower CEA, resulted in suppression of the acute phase response and generally in favorable effects on laboratory parameters indicative of risk factors of atherosclerosis, including lower homocysteine concentrations, and lower total and LDL cholesterol.

A simple and rapid HPLC method requiring small volumes (250 microL) of human serum after C18 SPE sample preparation was developed using monolithic technology for simultaneous determination of all-trans-retinoic acid, 13-cis-retinoic acid, retinol, gamma- and alpha-tocopherol. The monolithic column, Chromolith Performance RP-18e (100x4.6 mm), was operated at ambient temperature. The mobile phase consisted of a mixture of acetonitrile (ACN) and 1% ammonium acetate in water (AMC) at pH 7.0. The mobile phase started at 98:2 (v/v) ACN/AMC (column pre-treatment) at a flow rate of 2 mL/min, then changed to 95:5 (v/v) ACN/AMC for 4 min at a flow rate of 1.5 mL/min and a further 3 min at a flow rate of 3.2 mL/min. Detection and identification were performed using a photodiode array detector. Retinol, 13-cis- and all-trans-retinoic acid were monitored at 325 nm. Both alpha- and gamma-tocopherol were detected at 295 nm. The total analysis time was 7.2 min. Tocol (synthesized tocopherol, not occurring in humans) was used as internal standard. The method was linear in the range of 0.125-10.00 micromol/L for all-trans-retinoic acid, 0.125-5.00 micromol/L for 13-cis-retinoic acid, 0.25-10.00 micromol/L for retinol, 0.5-50.00 micromol/L for gamma-tocopherol, and 0.5-50.00 micromol/L for alpha-tocopherol. The present method may be useful for monitoring of retinoids and tocopherols in clinical studies.

Rheological conditions basically influence tissue perfusion, oxygen and nutrient supply, tissue regeneration and in its consequence the course of pathological processes, especially in microcirculation and partially even in macrocirculation. Haemorheotherapy has shown to be successful in several indications (critical disorders in microcirculation) when other methods of therapy have failed. In this study we describe the changes of effectivity indicators in haemorheopheresis treatment and their clinical importance in cases of eye microcirculation, statoacoustic apparatus and disorders of the lower extremities. We treated with haemorheopheresis (separator Cobe.Spetra + Evaflux filter) 24 patients (16 patients with age related macular degeneration, 5 with critical peripheral arterial foot disease, and 3 with acute hearing loss). After the procedures alpha2-macroglobulin decreases by about 58%, fibrinogen by about 69%, IgM by about 61%, LDL-cholesterol by about 77%, apolipoprotein B by about 76% and lipoprotein(a) by about 63%. It corresponds with a decrease in blood and plasma viscosity and clinical improvement in the observed patients, i.e. visual improvement, acceleration of tissue defect healing and improvement in hearing. We noticed 7.1% clinically insignificant side-effects. The method of haemorheopheresis was safe. It led to improvement in patients' clinical condition in the above mentioned indications when other methods of treatment failed.

The important substances of secondary metabolism include flavonoids and isoflavonoids. The Trifolium pratense L. (Fabaceae) suspension culture's yield of these metabolites is low, thus an attempt was made to increase the production by elicitation. The endogenous signal substances of the plants' defensive responses include jasmonic acid that also functions as a biotic elicitor in the case of exogenous application. In the experiment the authors monitored the impact of 4 different concentrations of jasmonic acid on the Trifolium pratense L. (variety DO-8 and variety DO-9) suspension culture's yield. The culture was cultivated in Gamborg medium to which 2 mg.l(-1) of 2,4-dichlorophenoxyacetic acid and 2 mg.l(-1) of 6-benzylaminopurine were added. The maximum increase in the production of flavonoids was achieved, when compared with the control samples, with both varieties of the 500 micromol concentration (DO-9, 6-hour application by 140%; DO-8, 24-hour application by 65%). The production of isoflavonoids (genistin, daidzein, genistein, and formononetine) was best stimulated in both varieties by the 50 micromol concentration (in the case of DO-9 variety, the 48-hour application increased the content of genistin by up to 845%).

Scopoletin is a coumarin possessing many interesting biological effects, e.g., spasmolytic, anti-inflammatory, antimutagenic, antioxidant, antifungal, apoptosis-inducing, antiproliferative, acetylcholinesterase-inhibitory, and hypouricemic activities. Plant tissue cultures represent a promising alternative source of valuable plant-derived substances. A number of physical and chemical factors influence the cell growth and secondary metabolite biosynthesis in plant tissue cultures. The mechanism of their action is not completely understood. Besides other factors, plant growth regulators and light conditions play an important role. Effects of four auxins (2,4-dichlorophenoxyacetic acid, 2,4-D, alpha-naphthaleneacetic acid, NAA, beta-indoleacetic acid, IAA or beta-indolebutyric acid, IBA) at four concentrations (0.2, 2, 10 or 20 mg/l) on the culture growth and accumulation of scopoletin in the medium were tested in Angelica archangelica cell suspension cultures cultured under continuous light or in the dark. The highest culture growth was achieved with 2 mg/l 2,4-D, and 10 mg/l IAA. The best scopoletin levels were obtained with 0.2 mg/l 2,4-D, 2 mg/l 2,4-D, 10 mg/l NAA, and 20 mg/l IAA. The effects of light conditions were less marked than those of auxins and their concentrations in influencing both the cell growth and scopoletin accumulation in Angelica archangelica cell suspension cultures. The changes brought about by auxins were modified by light conditions.

Important substances of secondary metabolism include flavonoids and isoflavonoids. The Trifolium pratense L. (Fabacae) suspension culture is characterized by low production of these metabolites and therefore we tried to influence the production output with elicitation. From their origin point of view, the elicitors are divided into two groups - biotic and abiotic. The latter group includes, for instance, the salts of heavy metals. Our work was aimed at observing the effect of the copper sulphate abiotic elicitor on the production of the Trifolium pratense L. suspension culture (variety DO-8 and variety DO-9) that was cultivated in Gamborg media supplemented with 2 mg l(-1) of 2,4-dichlorophenoxyacetic acid and 2 mg l(-1) of 6-benzylaminopurine. The maximum increase in the flavonoid production took place, when compared with the test check, during the 168-hour application of the 100 micromol concentration. The DO-8 variety isoflavonoids production was stimulated namely during the 48-hour application of the I micromol concentration; the best elicitation effect of the DO-9 variety was achieved with the 168-hour application of the 10 micromol concentration.

Plant tissue cultures represent a promising source of substances of natural origin. The main problem of their use is a low production of the majority of secondary metabolites. One of the methods of increasing the production of these substances is elicitation, because the biosynthesis of many secondary metabolites in plant cells is part of the defensive reaction against biological or abiotic stress influences. The paper tested vanadium compounds as potential elicitors of the production of coumarins: sodium vanadate (0.2; 1; 10; 100 and 1000 microM/l of medium) and vanadyl sulfate (10; 20; 50; 100; 200 and 500 microM/l of medium). The toxicity of these substances for the culture was simultaneously monitored by means of the evaluation of the effects on growth (characterized by fresh and dry weights of biomass at the end of two-week cultivation). The cultures were grown both in the light and dark. The growth of the cultures was not influenced by sodium vanadate at concentrations of 0.2 to 100 microM. A vanadate concentration of 1000 microM acted already toxically (in comparison with the control culture, the fresh weight was decreased by 28 % and the dry weight by 41% when cultivated in the light; the fresh weight by 69% and the dry weight by 66% when cultivated in the dark). Vanadyl sulfate in concentrations of 10 to 50 microM did not affect the growth of the culture, at higher concentrations it decreased it gradually: a concentration of 500 microM acted already toxically, again more markedly when cultivated in the light (in comparison with the control culture, the fresh weight was decreased by 27% and dry weight by 38% when cultivated in the light; the fresh weight by 65% and the dry weight by 61% when cultivated in the dark). The production of coumarins was stimulated by sodium vanadate in a concentration of 0.2 and 1 microM when cultivated in the light. The content of coumarins increased in comparison with the control culture mainly in the medium by 46% and by 25% at vanadate concentrations of 0.2 and 1 microM, respectively. Vanadyl sulfate did not increase the production of coumarins.

The red cloverl seems to be a prospective source of flavonoids and isoflavonoids with phytoestrogenic activity. Young seedlings of four different varieties of Trifolium pratense L. were employed to derive callus and suspension cultures. The optimal medium for cultivation is Gamborg's cultivating medium with an addition of 2 mg x l(-1) of 2,4-dichlorphenoxyacetic acid and 2 mg x l(-1) of 6-benzylaminopurine. On the basis of growth and production characteristics, subcultivating intervals of 29 to 43 days in callus culture and 16 to 23 days in suspension culture were determined. TLC and HPLC revealed that the explant culture of Trifolium pratense L. contains the isoflavonoid formononetin.

The plant cell reacts to an increased concentration of metals in the environment by various mechanisms. They include an increase in the formation of heat-shock proteins, metallothioneins, phytochelatins, amino acids (cysteine, histidine), organic acids (citric, malic), or secondary metabolites. The latter mechanism is being investigated for its possible use in explant cultures for the stimulation of secondary metabolism, which is the source of substances of pharmaceutical importance. The study tested manganese (II) (0, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, and 50 mM in the medium), cobalt (II), and nickel (II) ions (0, 0.1, 0.5, 1, 5, 10, 50, 100, 200, and 500 microM in the medium) as potential elicitors of coumarin production. At the same time, toxicity of these metals for the culture was examined by evaluating their effect on growth (characterized by fresh and dry weight of biomass at the end of a two-week cultivation). Cultures were cultivated in the dark and in the light. It has been found that the growth of cultures is not influenced by manganese in concentrations ranging from 0 to 2 mM, then it slightly decreases, at a concentration of 50 mM it is lower by 20 % when cultivated in the dark and by 30 % when cultivated in the light in comparison with the control. Cobalt in concentrations of 0 to 50 microM does not significantly influence the growth of the culture, higher concentrations decrease the biomass yields, more markedly when cultivated in the light (at 500 microM Co by 60 %, in the dark only by 30 % in comparison with the controls). Nickel in concentrations of 0.1 to 200 microM does not influence growth, and in a concentration of 500 microM decreases it by approximately 30 % in comparison with the control both in the light and dark. Production of coumarins was not stimulated by any metal in comparison with the control cultures, only the removal of manganese from the medium in the culture cultivated in the dark increased production by about 15 % versus the control.

Elicitation-produced stress activates the defensive reactions of the plant or plant explant, which result, among others, in a change in the transcription of the genes coding the enzymes influencing biosynthesis of secondary metabolites. The present paper investigated the effect of ions of heavy metals in concentrations od 1; 10, and 100 microM on the production of anthracene derivatives by the explant culture of Rheum palmatum L. cultivated on Murashige-Skoog medium with an addition of 10 mg.l(-1) of alpha-naphthylacetic acid. The periods of application of elicitation were 6; 24; 48, and 168 hours. It follows from the results that the applied abiotic elicitation positively influenced the production mainly in suspension culture. The maximal increase in the content of anthracene derivatives versus the control by 66% was observed after 48-hour action of 10 microM concentration of cadmium chloride. Aluminium chloride produced the largest increase in the production after 6-hour application of a 100 microM concentration, when in comparison with the control culture the production was stimulated by 60%.

Elicitation is based on signal (elicitor)-induced expression of genes, which subsequently results in increased synthesis of secondary metabolities in plants and in vitro cultures. Elicitors do not usually influence gene activity directly but by means of transmitters of the signal, which include primarily calcium ions. The paper examined the effect of four concentrations of calcium ions on the production of anthracene derivatives by means of the callus and suspension cultures of Rheum palmatum L. (Polygonaceae), which was elicited by an abiotic elicitor--a 20 microM solution of lead dichloride. The culture was cultivated on a Murashige-Skoog medium with an addition of 10 mg.l-1 of alpha-naphthylacetic acid. It follows from the results that the maximal content of anthracene derivatives, determined by the photometric determination according to PhBs 4, was demonstrated in the suspension culture after 24-hr administration of the elicitor and a 10 mM calcium chloride solution. In comparison with the control and elicited groups, the production was stimulated by 44% and 17%, respectively. After an addition of calcium ions to the elicited callus culture, no positive influence on the production was observed.

The paper examined the effect of selected auxins (2,4-dichlorophenoxyacetic acid, alpha-naphthalene-acetic acid, beta-indoleacetic acid, beta-indoleburytic acid; each in four concentrations--0.2, 2, 10, and 20 mg/l) on the production of coumarins in the suspension culture of Angelica archangelica L. cultinated in the dark and under permanent lighting(3500 lux). The effect of the light regimen is, in comparison with auxins, less marked--the content of coumarins is mostly comparable both under permanent lighting and in the dark. The highest coumarin content was achieved with the use of alpha-naphthalene-acetic acid in a concentration of 0.2 mg/l with cultivation in the dark.

The process of elicitation makes use of the capacity of plants and plant cells cultivated in vitro to react to various stress stimuli by a number of protective reactions leading to increased accumulation of secondary metabolites. The endogenic signal substances of plant protective reactions include jasminic acid, which in the case of exogenous application also acts as an elicitor. The paper examined the effect of four concentrations of jasminic acid on the production of anthracene derivatives by a three-year-old tissue culture, which was derived from the roots of a two-year-old intact plant Rheum palmatum L. (Polygonaceae). The culture was cultivated on Murashige-Skoog medium with an addition of 10 mg.l-1 of alpha-naphthylacetic acid. The results show that the optimal effect of jasminic acid on the production of callus culture was manifested after a 12-hour application of the strongest concentration of 5 mM, and on the production of suspension culture after a 48-hour application of a concentration of 0.05 mM, when the photometric determination according to PhBs 4 demonstrated the maximal content of anthracene derivatives (1.26%) and the production was stimulated by 109% in comparison with the control.