IR and Raman studies of oil and seedcake extracts from natural and genetically modified flax seeds.

Flax plant of the third generation (F3) overexpressing key genes of flavonoid pathway cultivated in field in 2008 season was used as the plant material throughout this study. The biochemical properties of seed, oil and seedcake extracts from natural and transgenic flax plants were compared. Overproduction of flavonoids (kaempferol), phenolic acids (coumaric, ferulic/synapic) and lignan-secoisolariciresinol diglucoside (SDG) in oil and extracts from transgenic seeds has been revealed providing a valuable source of these compounds for biotechnological application. The changes in fatty acids composition and increase in their stability against oxidation along three plant generations were also detected. The analysis of oil and seedcake extracts was performed using Raman and IR spectroscopy. The wavenumbers and integral intensities of Raman and IR bands were used to identify the components of phenylpropanoid pathway in oil and seedcake extracts from control and transgenic flax seeds. The spectroscopic data were compared to those obtained from biochemical analysis.

The effect of Linola and W92/72 transgenic flax seeds on the rabbit caecal fermentation--in vitro study.

The effect of W92/72 transgenic flax seeds taken from a variety of Linola on the production of SCFA, ammonia and methane by bacteria inhabiting rabbit caecum was studied. The in vitro method was used where caecal contents from rabbits was incubated with W92/72 transgenic or Linola flax seeds, or without any additives (control samples). The total concentration of SCFA was higher in samples with the addition of flax seeds than in the control samples. The increase in concentrations of acetic, propionic and butyric acids was the highest in samples with Linola seeds added. A higher percentage of propionic and butyric acids was observed in the contents incubated with addition of flax seeds as compared to the control samples. This increase was the result of a percentage decrease in acetic acid. No differences were observed in the concentration of ammonia between fermented samples. Moreover, the addition of flax seeds resulted in slight decrease of pH in incubated samples. In gas samples, the methane level was higher in samples with flax seeds added, although the highest level was found in samples with transgenic seeds. In addition, gas pressure was significantly higher in samples with flax seeds added as compared to control samples, and this may indicate a higher intensity of microbiological fermentation processes. These studies suggest that neither Linola nor W92/72 flax seeds have any unfavorable effect on the caecal microflora activity of rabbits. A beneficial influence of flax seeds on the microbiological fermentation process in rabbit caecum was observed, based on an increase in percentage ratio of propionic acid in samples with flax seeds added.

Regeneration of flax ( Linum usitatissimum L.) plants from anther culture and somatic tissue with increased resistance to Fusarium oxysporum.

The aim of this study was to establish a protocol for the efficient production of flax plants of microspore origin. The results were compared to those obtained for plants regenerated from somatic explants from hypocotyls, cotyledons, leaves, stems and roots. All the plants obtained during the experiments were regenerated from callus that was grown for periods from a few weeks to a few months before the regeneration was achieved. Anther cultures were less effective in plant regeneration than somatic cell cultures. However, regenerants derived from anther cells showed valuable breeding features, including increased resistance to fungal wilt. The age of the donor plants and the season they grew in had a noticeable effect on their anther callusing and subsequent plant regeneration. Low temperature had a negative effect and dark pre-treatment a positive effect on callusing and plant regeneration. Different media were most effective for callus induction, shoot induction and rooting. For callus induction two carbon sources (2.5% sucrose and 2.5% glucose) were most effective; for shoots, only sucrose at lower concentration (2%) was effective. Rooting was most efficient in 1% sucrose and reduced (50%) mineral concentration in the medium. It was found that the length of in vitro cultivation significantly increases the ploidy and affects such features as regenerant morphological characteristics, petal colour, and resistance to Fusarium oxysporum-induced fungal wilt. The established plant regeneration system provides a basis for the creation of transgenic flax.

Transgenic 14-3-3 isoforms in plants: the metabolite profiling of repressed 14-3-3 protein synthesis in transgenic potato plants.

14-3-3 proteins are abundant eukaryotic proteins that interact with many other proteins, thereby modulating their function and thus cell metabolism. The data from mRNA analysis confirm the developmental regulation of 14-3-3 isoform expression in potato plants. In order to test whether or not 14-3-3 protein expression affects plant phenotype and metabolism, transgenic potato plants either overexpressing Cucurbita pepo 14-3-3 or underexpressing endogenous 14-3-3 isoforms were analysed. An increase in tuber number and a decrease in tuber size in the overexpressed transformant was observed; the transgenic plants contain more chlorophyll than the control and they lose it more slowly than the control when transferred to the dark. The 14-3-3-repressed transgenic plants showed a decrease in tuber number and an increase in tuber size; an increase in the fresh weight of the transgenic tubers was also detected. The increased catecholamine level was accompanied by an increased ratio of soluble sugars to starch in overexpressed transformant. The opposite effect was detected in 14-3-3-repressed transgenic plants. All the repressed plants showed significant increases in nitrate reductase (NR) activity, suggesting that the regulation of NR occurs in vivo, and is not isoform-dependent. The increase in NR activity resulted in a significant decrease in nitrate level. The level of sucrose phosphate synthase activity was also significantly increased in all 14-3-3-underexpressed transgenes, and remarkably the increase in enzyme activity was accompanied by respective changes in sucrose levels in the tubers. The most intriguing finding was the significant (2-3-fold) increase in ethylene content in all the 14-3-3-repressed transgenic lines, which probably resulted from a methionine level increase. The substantial increase of ethylene level in the repressed forms might explain the significant shortening of the vegetation period of the analysed transgenic plants.

Quantitative and qualitative analysis of lipids in genetically modified potato tubers with varying rates of 14-3-3 protein synthesis.

In six transgenic lines of potatoes with the varying rates of 14-3-3 protein synthesis as well as in control cultivar Desiree the content and composition of the lipids extracted from the mature tubers from three years field trials (1998-2000) were analyzed. The transgenic lines J2 and J1 are both overexpressing gene encoding 14-3-3 protein. The J2 exhibited an overexpression of the protein 14-3-3 derived from pumpkin (Cucurbita pepo) cDNA and in J1 the 14-3-3 overexpression resulted from modifying of ADP-ribosylation factor synthesis. In the remaining lines, synthesis of the protein 14-3-3 was modified by the antisense technology. In tubers from 1998, the content of total lipids was within the range of 0.45-0.88% of tuber dry matter. The highest amount of fat was in tubers of line J2 (69% more than in the control). The content of lipids in tubers from subsequent years ranged from 0.36 to 0.63% of dry matter. Consistently the highest amount of fat was in tubers of line J2, however, the increase was very slight (8.6% more than in the control). The fractionation of lipids into polar and nonpolar fractions showed that all transgenic lines from field trials 1998 and 2000 contained more nonpolar lipids than the control (up to 270% in line J2). The percentage of nonpolar fractions in fats of tubers from all transgenes harvested in 1999 were similar, but they were higher than in tubers from the previous years, and they amounted to 44.4-49.1%. Chromatographic separation of methyl esters of fatty acids demonstrated that cis-alpha-linoleic acid was the main fatty acid present in potato tubers. This acid composed the biggest part of all lipids in G2 line. In the nonpolar fraction of lipids, palmitic acid followed by cis-alpha-linoleic acid showed the highest amounts.

Identification and quantification of catecholamines in potato plants (Solanum tuberosum) by GC-MS.

Dopamine, norepinephrine, and normetanephrine were identified by GC-MS in potato (Solanum tuberosum L.) plants, the latter was new for plants. The highest amount of catecholamines was found in leaves. A developmental stage dependent variation in potato leaf catecholamines accumulation was also observed with highest level in third leaves. Catecholamine contents decrease during cold storage of tubers to undetectable levels. Mechanical wounding of leaves led to a small increase in the level of catecholamines investigated.

Increase in lipid content in potato tubers modified by 14-3-3 gene overexpression.

Recently, transgenic potato plants were created with overexpression of the 14-3-3 protein derived from Cucurbita pepo. Detailed analysis of those plants suggested that the function of the isolated 14-3-3 isoform is in the control of carbohydrate and lipid metabolism in the plants. 14-3-3 protein overexpression gave rise to an increase in soluble sugar and catecholamine contents in both leaves and tubers. It is proposed that 14-3-3 protein affects carbohydrate metabolism in potato plants via regulation of catecholamine synthesis. Furthermore, genetically modified potato tubers with 14-3-3 protein overexpression showed changes in lipid content and composition. The transgenic potato tubers contained 69% more total fat compared to the wild-type plant. Separation of tuber lipids into polar and nonpolar fractions revealed that the transgenic potato tubers contained almost 3 times more nonpolar lipids than the control. Analysis of fatty acid composition, conducted by the means of gas chromatography, showed that linoleic acid was the main fatty acid present in the tubers of both modified and control potato plants. In the nonpolar fraction of the fat of the transgenic tubers the unsaturated fatty acids exhibited a higher participation in the sum of all fatty acids.

A novel apoptosis-like pathway, independent of mitochondria and caspases, induced by curcumin in human lymphoblastoid T (Jurkat) cells.

We have shown previously [E. Sikora, A. Bielak-Zmijewska, K. Piwocka, J. Skierski, and E. Radziszewska (1997) Biochem. Pharmacol. 54, 899-907] that curcumin prevents formation of oligonucleosomal DNA fragmentation in rat thymocytes and human leukemic T lymphocytes (Jurkat cells) induced to undergo apoptosis. In this paper we show that 50 microM curcumin by itself induces cell death in Jurkat cells, but its symptoms differ from those observed after a short ultraviolet (uv) irradiation. Ultraviolet-irradiated Jurkat cells displayed typical symptoms of apoptosis: morphological changes, internucleosomal and high-molecular-weight DNA fragmentation, formation of sub-G1 fractions in DNA content frequency histograms, and dissipation of the mitochondrial transmembrane electric potential (Delta psi). In contrast, curcumin-treated Jurkat cells exhibited DNA splitting into high-, but not low-, molecular-weight fragments. These cells retained their high mitochondrial Delta psi, and the content of Ca2+ in endoplasmic reticulum stores remained at the level typical for untreated cells. The frequency of opening of the mitochondrial permeability transition pores in curcumin-treated cells was decreased compared to the controls, whereas uv irradiation made these pores completely open. Curcumin did not produce any change in the activity of caspase-3, whereas uv irradiation considerably activated this protease. The morphology of curcumin-treated cells displayed chromatin condensation, which was insensitive to the caspase inhibitor z-VAD-fmk, but no formation of typical apoptotic bodies, as was the case after uv irradiation. In contrast to uv-irradiated cells, curcumin-treated Jurkat cells considerably increased the level of Bcl-2. It is concluded that the programmed cell death induced by curcumin in Jurkat cells differs from "classical" by the lack of mitochondrial depolarization and of the involvement of caspases.

Brain spectrin (fodrin) interacts with phospholipids as revealed by intrinsic fluorescence quenching and monolayer experiments.

We demonstrate that phospholipid vesicles affect the intrinsic fluorescence of isolated brain spectrin. In the present studies we tested the effects of vesicles prepared from phosphatidylcholine (PtdCho) alone, in addition to vesicles containing PtdCho mixed with other phospholipids [phosphatidylethanolamine (PtdEtn) and phosphatidylserine] as well as from total lipid mixture extracted from brain membrane. The largest effect was observed with PtdEtn/PtdCho (3:2 molar ratio) vesicles; the effect was markedly smaller when vesicles were prepared from egg yolk PtdCho alone. Brain spectrin injected into a subphase induced a substantial increase in the surface pressure of monolayers prepared from phospholipids. Results obtained with this technique indicated that the largest effect is again observed with monolayers prepared from a PtdEtn/PtdCho mixture. The greatest effect was observed when the monolayer contained 50-60% PtdEtn in a PtdEtn/PtdCho mixture. This interaction occurred at salt and pH optima close to physiological conditions (0.15 M NaCl, pH7.5). Experiments with isolated spectrin subunits indicated that the effect of the beta subunit on the monolayer surface pressure resembled that measured with the whole molecule. Similarly to erythrocyte spectrin-membrane interactions, brain spectrin interactions with PtdEtn/PtdCho monolayer were competitively inhibited by isolated erythrocyte ankyrin. This also suggests that the major phospholipid-binding site is located in the beta subunit and indicates the possible physiological significance of this interaction.

Modification of the apoptotic-like effects of MBP protein overexpression in E. coli by fusion with 14-3-3 derived polypeptides.

Overexpression of even non-toxic proteins in bacteria causes a starvation-like response: the arrest of bacterial proliferation and apoptotic-like suicidal cell death. We have shown here that, as in the cells of higher organisms, these effects are accompanied by DNA degradation. The fusion with the bacterial MBP of a polypeptide, belonging to the 14-3-3 family and normally expressed in pumpkin (C. pepo), modifies the apoptotic-like effects of overexpression of this protein in E. coli. Fusion of the full length 14-3-3 protein with the MBP considerably slows down the DNA degradation caused by overexpression of the unmodified MBP. Overexpression of the construct containing a truncated version of the 14-3-3 polypeptide causes immediate arrest of bacterial growth and rapid degradation of the chromosomal DNA. This result suggests that the DNA degradation in bacteria is an active process which can be modified to some extent by an endogenous protein.

Identification of the proteins responsible for SAR DNA binding in nuclear matrix of Cucurbita pepo.

The nuclear matrices from White bush (Cucurbita pepo var. patisonina) cell nuclei have been isolated using three methods: I, standard procedure involving extraction of cell nuclei with 2 M NaCl and 1% Triton X-100; II, the same with pre-treatment of cell nuclei with 0.5 mM CuSO4 (stabilisation step); and III, method with extraction by lithium diiodosalicylate (LIS), and compared the polypeptide pattern. The isolated matrices specifically bind SAR DNA derived from human beta-interferon gene in the exogenous SAR binding assay and in the gel mobility shift assay. Using IgG against the 32 kDa endonuclease we have found in the DNA-protein blot assay that this protein is one of the proteins binding SAR DNA. We have identified three proteins with molecular mass of 65 kDa, 60 kDa and 32 kDa which are responsible for SAR DNA binding in the gel mobility shift assay experiments.

Immunoglobulins anti-endonuclease 32 kDa from Cucurbita pepo var patissonina affect process of DNA synthesis.

Immunoglobulins anti-endonuclease 32 kDa inhibit DNA synthesis. We observed that low concentrations of IgGs (about 50 micrograms IgG per 1 x 10(6) cell nuclei) temporary inhibit DNA synthesis. This inhibition concerns only the synthesis of DNA bound to the nuclear matrix (associated with isolated nuclear matrix). Preincubation of cell nuclei of White bush with IgG generates longer DNA fragments than in controls. Involvement of the 32 kDa endonuclease or an endonuclease-65 kDa protein complex from the nuclear matrix in replication or structural organisation of replication is considered.

Expression analysis of a Cucurbita cDNA encoding endonuclease.

The nuclear matrices of plant cell nuclei display intrinsic nuclease activity which consists in nicking supercoiled DNA. A cDNA encoding a 32 kDa endonuclease has been cloned and sequenced. The nucleotide and deduced amino-acid sequences show high homology to known 14-3-3 protein sequences from other sources. The amino-acid sequence shows agreement with consensus sequences for potential phosphorylation by protein kinase A and C and for calcium, lipid and membrane-binding sites. The nucleotide-binding site is also present within the conserved part of the sequence. By Northern blot analysis, the differential expression of the corresponding mRNA was detected; it was the strongest in sink tissues. The endonuclease activity found on DNA-polyacrylamide gel electrophoresis coincided with mRNA content and was the highest in tuber.

Modification of the lipid component modulates nuclear matrix nucleolytic activity.

It was shown that lipid composition of plant nuclear matrix depends on procedure of its isolation. The matrix isolated with the use of lithium diiodosalicylate (LiS) differs in its lipid composition from the preparation isolated with the use of nonionic detergent (Triton X-100). It was also shown that the nucleolytic activity of the matrix is related to its lipid component. Matrix depleted in lipids loses half of its nucleolytic activity which is recovered after supplementation with previously extracted lipids. The extent of recovery of the nucleolytic activity is also dependent on the presence of residual DNA in matrix preparation. The recoveries of nucleolytic activities were higher in matrices not depleted in their DNA content.

Modulation of some nuclear matrix enzymatic activities by free fatty acids.

It was shown that two of main enzymatic activities of plant nucleus and nuclear matrix, namely RNA-polymerasic and DNA-nucleolytic are susceptible to modulation with free fatty acids. The effects observed were dependent to both fatty acid length and degree of unsaturation. In nuclei a stimulation of nuclease activity was observed whereas in matrices short chain fatty acids inhibited the studied activity. The effect of fatty acids on RNA-polymerase was also different in nuclei and matrices. In nuclei all fatty acids studied inhibited polymerasic activity whereas in matrices short chain fatty acids stimulated this activity by up to 80% and the long chain fatty acids inhibited by up over 70%. The overall alteration of studied activities in nuclei and matrices by unsaturated fatty acids was similar. Nucleolytic activity was stronger inhibited and polymerasic activity was stimulated when the effects of linoleic and linolenic acids were studied. The results suggest possible importance of lipid component in nuclear matrix biological function.

Interaction of the Pisum sativum nuclear matrix proteins with SAR DNA.

We have isolated the nuclear matrices from Pisum sativum cell nuclei using three methods: i. standard procedure involving extraction of cell nuclei with 2 M NaCl and 1% Triton X-100; ii. the same with pretreatment of cell nuclei with 0.5 mM CuSO4 (stabilisation step); and iii. method including lithium diiodosalicylate extraction. We compared the polypeptide pattern and residual DNA content of the nuclear matrices isolated. The nuclear matrices displayed a specific endonuclease activity which was due to the presence of a 32 kDa protein. The isolated nuclear matrices bound specifically the scaffold-attached (SAR) DNA derived from human beta interferon gene, in the exogenous SAR binding assay. Using the DNA-protein binding blot assay we demonstrated the presence of two nuclear matrix proteins of 66 kDa and 62 kDa which bound specifically SAR DNA.

Interaction of erythrocyte spectrin with some nonbilayer phospholipids.

Bovine erythrocyte spectrin was found to interact with lysophosphatidylcholine and lysophospatidylserine what was detected by small changes of the intrinsic fluorescence of spectrin. Lysophosphatidylethanolamine in contrast to its diacyl, natural counterpart did not affect the intrinsic fluorescence of spectrin at all. Dioleoylphosphatidylethanolamine induced distinct changes in the intrinsic fluorescence from these induced by natural phosphatidylethanolamine suspensions. Our data may indicate an importance of the presence of both fatty acyl chains in phosphatidylethanolamine molecule and perhaps, its bilayer structure for the interaction of this phospholipid aggregates with spectrin.

[DNA metabolism in lymphocytes of patients with lupus nephropathy]. / Metabolizm DNA w limfocytach chorych na nefropatie toczniowa.

DNA metabolism in lymphocytes was evaluated in patients suffering from the nephrotic syndrome decompensation, basing on measurement of endonucleases (DNases) activity in these cells. The examination involved 17 patients, aged 27.7 +/- 7.11 with clinical and biochemical active disease, all with the nephrotic syndrome decompensation and erythrocytes in urine. A significant rise in the enzyme activity was observed in T and B lymphocytes (p < 0.001) in all the patients, with a distinct 3-fold increase in enzyme activity in B lymphocytes, when compared with the other cell populations. To elucidate the nature of the observed changes in DNases activity in systemic lupus erythematosus, nucleus proteins of the cells were separated electrophoretically in acrylamide gradient with immobilized DNA. Degradation processes in nucleic acids were considerably more efficient than the DNA synthesis in B lymphocytes of these cells. A smaller increase in the enzyme activity in T cells than in B lymphocytes results exclusively from more intense catabolism of nucleic acids not parallel to the intensified DNA synthesis in these cells.