Mieczysław Kuraś

Linker Histones Play a Role in Male Meiosis and the Development of Pollen Grains in Tobacco

To examine the function of linker histone variants, we produced transgenic tobacco plants in which major somatic histone variants H1A and H1B were present at ∼25% of their usual amounts in tobacco chromatin. The decrease in these major variants was accompanied by a compensatory increase in the four minor variants, namely, H1C to H1F. These minor variants are smaller and less highly charged than the major variants. This change offered a unique opportunity to examine the consequences to a plant of major remodeling of its chromatin set of linker histones. Plants with markedly altered proportions of H1 variants retained normal nucleosome spacing, but their chromosomes were less tightly packed than those of control plants. The transgenic plants grew normally but showed characteristic aberrations in flower development and were almost completely male sterile. These features correlated with changes in the temporal but not the spatial pattern of expression of developmental genes that could be linked to the abnormal flower phenotypes. Preceding these changes in flower morphology were strong aberrations in male gametogenesis. The earliest symptoms may have resulted from disturbances in correct pairing or segregation of homologous chromosomes during meiosis. No aberrations were observed during mitosis. We conclude that in plants, the physiological stoichiometry and distribution of linker histone variants are crucial for directing male meiosis and the subsequent development of functional pollen grains.

Anticancer activity of the Uncaria tomentosa (Willd.) DC. preparations with different oxindole alkaloid composition.

The activity of Uncaria tomentosa preparations on cancer cells was studied using in vitro and in vivo models. IC (50) values were calculated for preparations with different quantitative and qualitative oxindole alkaloid composition: B/W(37) --bark extracted in water at 37 °C, B/W(b)--bark extracted in boiling water, B/50E(37) --bark extracted in 50% ethanol at 37 °C, B/E(b)--bark extracted in boiling 96% ethanol, B/96E(37) --bark extracted in 96% ethanol at 37 °C and B/SRT--bark extracted in water and dichloromethane. Generally, the results obtained showed a high correlation between the total oxindole alkaloid content (from 0.43% to 50.40% d.m.) and the antiproliferative activity of the preparations (IC(50) from >1000 μg/ml to 23.57 μg/ml). B/96E(37) and B/SRT were the most cytotoxic preparations, whereas the lowest toxicity was observed for B/W(37). B/96E(37) were shown to be active against Lewis lung carcinoma (LL/2) [IC(50) =25.06 μg/ml], cervical carcinoma (KB) [IC(50) =35.69 μg/ml] and colon adenocarcinoma (SW707) [IC(50) =49.06 μg/ml]. B/SRT was especially effective in inhibiting proliferation of cervical carcinoma (KB) [IC(50) =23.57 μg/ml], breast carcinoma (MCF-7) [IC(50) =29.86 μg/ml] and lung carcinoma (A-549) [IC(50) =40.03 μg/ml]. Further animal studies on mice bearing Lewis lung carcinoma showed significant inhibition of tumor growth by B/W(37) administered for 21 days at daily doses of 5 and 0.5 mg (p=0.0009). There were no significant changes in the cell cycles of tumor cells with the exception of cell decrease at the G₂/M phase after the administration of B/96E(37) at a daily dose of 0.5 mg and the G(1)/G(0) cells cycle arrest demonstrated after the B/SRT therapy at a daily-dose of 0.05 mg. All tested preparations were non-toxic and well tolerated.

Antimitotic effect, G2/M accumulation, chromosomal and ultrastructure changes in meristematic cells of Allium cepa L. root tips treated with the extract from Rhodiola rosea roots

Abstract Rhodiola rosea is a Russian and Chinese medicinal plant used in medical practice as a stimulating, adaptogenic and antiarrhythmic agent. Many investigations have pointed to the anticancer and antimutagenic role of R. rosea. In spite of these therapeutic effects of R. rosea extract. little is known about the mechanism of its anticancer action, because few cytological investigations havebeen conducted on the mitotic activity, cellular cycle, structure, ultrastructure, and cytophysiology of both animal and plant cells treated with the extract. Our studies have shown that extract from R. rosea roots (containing rosavine and cinnamyl alcohol) cause the inhibition of a mitotic activity; however, cells resume their divisions after the postincubation period. During incubation in the extract, accumulation of G2/M cells was observed. Chromosomes inprophase and metaphase were shorter and thicker but the extract did not cause chromosomal aberrations or the formation of micronuclei. Ultrastructure changes after 24 h of incubation were connected with an increase in the electron density of ground cytoplasm, decrease in the number of mitochondria but with an increase of their surface. As early as after 6h postincubation, the number ofmitochondria doubled but their surface declined by over two times. After 24 h of incubation, the surface of plastids increased over twice in spite of their unchanged number. In postincubation (6h), their number doubled and the surface declined dramatically. The ultrastructure of the mitochondria was changed by the disappearance of cristae and a lowered electron density of the matrix, suggestingthat one of the mechanisms could be connected with changes in mitochondrial function.

Effect of Alkaloid-Free and Alkaloid-Rich preparations from Uncaria tomentosa bark on mitotic activity and chromosome morphology evaluated by Allium Test

ETHNOPHARMACOLOGICAL RELEVANCE Uncaria tomentosa (Willd.) DC. is the most popular Peruvian plant, used in folk medicine for different purposes. It contains thousands of active compounds with great content of alkaloids. AIM OF STUDY Two different fractions of Alkaloid-Rich and Alkaloid-Free were researched on chromosome morphology, mitotic activity and phases indexes. MATERIALS AND METHODS Cells of Allium Test (meristematic cells of root tips) were incubated up to 24h in different concentrations of Alkaloid-Free and Alkaloid-Rich fraction obtained from Uncaria tomentosa bark followed by 48 h of postincubation in water. The chromosome morphology was analyzed and the content of mitotic and phase indexes were done. Individual compounds, oxindole alkaloids, phenolic compounds and sugars were determined. RESULTS In Alkaloid-Rich and Alkaloid-Free fractions (different in chemical composition) we observed condensation and contraction of chromosomes (more in Alkaloid-Rich) with retardation and/or inhibition of mitoses and changed mitotic phases. Postincubation reversed results in the highest concentration which was lethal (in mostly Alkaloid-Rich fraction). CONCLUSIONS Our studies indicate that different action can depend on different groups of active compounds in a preparation either containing alkaloids or not. Other fraction analysis may be useful in the future.

Antimitotic effect of Selol and sodium selenate (IV) on Allium test cells

Selol is a mixture of selenitetriglycerides, obtained by esterification of pre-oxidated triglycerides from sunflower oil with selenous acid. Investigations have been carried out on the anticancer use of Selol and its antimitotic properties. The aim of our work was to compare the effect of two compounds (an organic and an inorganic one) containing selenium with oxidation number at +4: Selol and sodium selenate (IV), on plant cells of Allium test. Structural changes in cells, their mitotic activity and proportions of mitotic phases were subject to experimental analysis. Incubation in both selenium compounds led to changes in structure of chromosomes, consisting in their strong condensation and contraction (cc chromosomes), accompanied by gradual reduction or complete inhibition of cell division. High concentrations of Selol led to strong condensation of chromatin in interphase cells, while cells treated with high concentrations of sodium selenate (IV) displayed typical “pseudoapoptotic” bodies and totally fragmented nuclei. Both compounds caused inhibition of cell division; however, sodium selenate (IV) was more effective, while low concentrations of Selol stimulated division. Use of Selol resulted in changes in chromosome structure, condensation and contraction, which made it impossible to pass to further phases of cell division. The effect of sodium selenate (IV) consisted in raised prophase index, and high concentrations led to inhibition of divisions and arrest of cells at different stages of mitosis. The results obtained show markedly lower toxicity of Selol in comparison with sodium selenate, which makes it advisable to further investigate that compound as a potential safe anticancer drug.

Ultrastructural changes in onion (Allium cepa L.) root tip meristem cells treated with Selol and sodium selenate (IV)

Investigations on the antimitotic and anticancer effect of Selol have been carried out for more than a decade. In a previous work we analysed ultrastructural changes in onion root tip meristem cells (the Allium test), their division activity (the mitotic index), and proportion of division phases (the phase index). Our study revealed inhibition of cell divisions in response to both compounds; however, the effect of sodium selenate (IV) on cells was more toxic, involving frequent clastogenic aberrations. The aim of the present work was to compare the effect of both compounds (organic and inorganic), containing selenium with oxidation number at +4, i.e. Selol and sodium selenate (IV), on ultrastructure of plant cells of the Allium test, and their concentration in plant cells. Analysis of ultrastructural changes showed high toxicity of sodium selenate (IV), with degradation of cytoplasm, nucleus and cell organelles. Treatment with Selol did not cause such dramatic changes in ultrastructure. It resulted in gradual increase of heterochromatinization of cell nuclei and a change in their shape, correlated with increasing concentrations of Selol and incubation period. Additionally, a comparison of concentration of both compounds in Allium test cells showed that the organic form of selenium (Selol) penetrated them much more slowly than the inorganic one (sodium selenate (IV)). The results obtained indicate much lower toxicity of Selol than sodium selenate (IV), which supports the necessity of further investigations of this compound as a potential safe anticancer drug.

Perturbation in linker histone content has no effect on the cell cycle but affects the cell size of suspension grown tobacco BY-2 cells.

Histone H1, a key structural element of eukaryotic chromosomes can be perturbed in plants in vivo by overexpression or by a change in the proportion of native H1 variants (Prymakowska-Bosak M., Przewloka M., Iwkiewicz J., Egierszdorff S., Kuras M., Chaubert N., Gigot C., Spiker S., Jerzmanowski A., Histone H1 overexpressed to high level in tobacco affects certain developmental programs but has limited effect on basal cellular functions, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 10250-10255; Prymakowska-Bosak M., Slusarczyk J., Przewloka M., Kuras M., Lichota J., Kilianczyk B., Jerzmanowski A., Linker Histones Play a Role in Male Meiosis and the Development of Pollen Grains in Tobacco, Plant Cell 11 (1999) 2317-2330). In order to analyze the possible causes of the specific phenotypic changes observed in whole plants we employed a simpler system of tobacco BY-2 cell line. We show that the BY-2 cells engineered to overexpress a major variant of Arabidopsis H1 or with the level of native major variants of H1 decreased by antisense strategy maintain the normal ability to grow and the normal length of the cell cycle. In the cells with perturbed H1 histones no change was observed in the organization of mitotic spindle or actin filaments of the cytoskeleton. The only visible phenotypic change occurred in cells overexpressing H1 that showed an increased frequency of cells with unusually large size. This phenotype was correlated with subtle but reproducible changes in the organization of cortical microtubules.

Characterisation of Solanum lycopersicoides Dun. root primordia culture with plant recovery

A liquid meristematic root primordia culture (RPC) of Solanum lycopersicoides Dun. based on persistent rhizogenesis in a modified Murashige and Skoog (1962) medium supplemented with NAA (15 mg·l−1) or 2,4-D (1 mg·l−1) was described. The meristematic clumps (2–3 mm in diameter) originating from NAA supplemented medium were capable of regenerating plants through the callus stage (up to 70 %). Efficient direct plant regeneration (up to 21 %) was possible from numerous single globular-shaped root primordia (RP) structures liberated from the parental aggregates in 2,4-D supplemented proliferation medium without NH4NO3 and with a 2.5 fold increase in KNO3. The RP converted into plantlets (artificial seedlings) on solid or liquid media without growth growth regulators through the unipolar followed by the mace-shaped bipolar structure stages. The use of apical shoot bud, root apices or root segments as a primary explants brought about RPC induction and plant regeneration. The plants derived from 2 years old culture were phenotypically identical to their parental S. lycopersicoides plants and possessed the same ploidy.

Mitotic activity of a new phytotherapeutic product with a trade name of “Ganomix”

Abstract Water extracts of Uncaria tomentosa (Vilcacora) bark and Ganoderma lucidum (Reishi) spores that are rich in biologically active compounds, are increasingly widely used in prevention and treatment of many serious medical conditions. They are considered an important treatment option, complementary to phytotherapeutics and synthetic medicines. A new formulation with a trade name of Ganomix was developed based on these two components. The aim of this study was to determine mitotic activity of Ganomix and to compare the activity of Ganomix with the activity of its individual components. The biological activity was assessed using the following plant tests: Allium test (Levan test) and Phytotoxkit biotest. In addition, the chemical composition of selected components was analyzed. Water extracts of Vilcacora bark and Reishi spores, as well as their combination in Ganomix, exhibited varying biological activities. The Vilcacora bark extracts had inhibitory effects at the great majority of concentrations tested, while Reishi spore extracts were mainly stimulatory at the same range of concentrations. However, Ganomix extract showed an intermediate effect compared with Vilcacora bark and Reishi spore extracts. The promising results of these investigations give hope for the effective use of Ganomix in prevention and therapy of different sicknesses.

Ultrastructural changes in the mycelium of Hericium erinaceum (Bull.; Fr.) Pers. under selenium-induced oxidative stress

BACKGROUND In this study we examined the influence of various forms of selenium (organic and inorganic) on the vivacity of Hericium erinaceum mycelium and structural changes and ultrastructure occurring during its development in submerged culture. RESULTS The mycelium was grown on sodium selenite (Na₂SeO₃), Selol (with 20 and 50 g kg⁻¹ Se, respectively) and a mixture of Na₂SeO₃ and Selol. Samples of the mycelium were collected on day 3 and day 24 of the incubation and viewed under an electron microscope. Selol at concentration 20 g kg⁻¹ did not cause any damage to the cell ultrastructure, but it contributed to the thickening of the cell wall, which implied an influence on polysaccharide production. In the other cases, degradation changes appeared in the protoplasm and the thickness of the cell wall did not increase. CONCLUSION The nature of the effect exerted by various sources of selenium in the culture medium on the formation of polysaccharides probably results from the differences in their chemical composition and differences in the toxicity of these compounds towards the cells, but is also connected with the decomposition of the wall surrounding degraded fungal cells.