Various effects of the photosystem II--inhibiting herbicides on 5-n-alkylresorcinol accumulation in rye seedlings.

The effect of three PSII-inhibiting herbicides, lenacil, linuron, and pyrazon, on the accumulation of 5-n-alkylresorcinols in rye seedlings (Secale cereale L.) grown under various light and thermal conditions was studied. All used chemicals increased resorcinolic lipid content in both green and etiolated plants grown at 29 °C. At 22 °C pyrazon and lenacil decreased the content of alkylresorcinols in plants kept in the darkness and increased their amount in the light-grown seedlings. In turn, level of resorcinolic lipids was decreased by linuron in both etiolated and green plants. At the lowest tested temperature lenacil enhanced production of alkylresorcinols only in etiolated rye seedlings, whereas the light-independent stimulatory action of pyrazon on alkylresorcinol accumulation in rye grown at 15 °C was observed. Additionally, only the latter did not exert a negative effect on rye seedling growth under any of tested conditions. Compared with respective controls, the herbicides used also markedly modified the qualitative pattern of resorcinolic homologs. Interestingly, the observed changes generally favored the enhanced antifungal activity of these compounds. Our study provides novel information on the influence of PSII inhibitors on alkylresorcinol metabolism in rye seedlings. The unquestionable achievement of this work is the observation that low dose of pyrazon mainly stimulated both growth and alkylresorcinol synthesis in rye seedlings, a non-target plant. Moreover, our experimental work showed unambiguously that the observed pyrazon-driven accumulation and homolog pattern modification of alkylresorcinols dramatically improved the resistance of winter rye to infections caused by Rhizoctonia cerealis.

Phenolic lipids affect the activity and conformation of acetylcholinesterase from Electrophorus electricus (Electric eel).

Phenolic lipids were isolated from rye grains, cashew nutshell liquid (CNSL) from Anacardium occidentale, and fruit bodies of Merrulius tremellosus, and their effects on the electric eel acetylcholinesterase activity and conformation were studied. The observed effect distinctly depended on the chemical structure of the phenolic lipids that were available for interaction with the enzyme. All of the tested compounds reduced the activity of acetylcholinesterase. The degree of inhibition varied, showing a correlation with changes in the conformation of the enzyme tested by the intrinsic fluorescence of the Trp residues of the protein.

Searching for new derivatives of neocryptolepine: synthesis, antiproliferative, antimicrobial and antifungal activities.

A series of novel amino acid and dipeptide derivatives of neocryptolepine were synthesized and tested for their antimicrobial, antifungal and antiproliferative activity in vitro against cancer cell lines (KB, A549, MCF-7, LoVo) and normal mice fibroblast cells (BALB/3T3). Biological evaluation revealed that almost all of the new compounds displayed high antiproliferative activity against the tested cells and moderate to potent antibacterial activities. Interestingly, these compounds were active against Candida albicans biofilms at doses significantly lower than those required against free-floating planktonic fungal cells. The most promising compounds are derivatives with glycine and L-proline as a substituent both at 2 and at 9 position of 5H-indolo[2,3-b]quinoline. In general, these new compounds (2a, 3a, 6a and 7a) showed the highest dual action against cancer lines and infectious pathogenic microbes in vitro.

Efficient human breast cancer xenograft regression after a single treatment with a novel liposomal formulation of epirubicin prepared using the EDTA ion gradient method.

Liposomes act as efficient drug carriers. Recently, epirubicin (EPI) formulation was developed using a novel EDTA ion gradient method for drug encapsulation. This formulation displayed very good stability and drug retention in vitro in a two-year long-term stability experiment. The cryo-TEM images show drug precipitate structures different than ones formed with ammonium sulfate method, which is usually used to encapsulate anthracyclines. Its pharmacokinetic properties and its efficacy in the human breast MDA-MB-231 cancer xenograft model were also determined. The liposomal EPI formulation is eliminated slowly with an AUC of 7.6487, while the free drug has an AUC of only 0.0097. The formulation also had a much higher overall antitumor efficacy than the free drug.

Anacardic acid enhances the anticancer activity of liposomal mitoxantrone towards melanoma cell lines - in vitro studies.

THIS PAPER DESCRIBES A NOVEL FORMULATION OF ANTINEOPLASTIC DRUG: mitoxantrone loaded into liposomal carriers enriched with encapsulated anacardic acid in the liposomal bilayer using a vitamin C gradient. Anacardic acid is a potent epigenetic agent with anticancer activity. This is the first liposomal formulation to combine an actively encapsulated drug and anacardic acid. The liposomes were characterized in terms of basic parameters, such as size, zeta potential, optimal drug-to-lipid ratio, loading time and temperature, and stability at 4°C and in human plasma in vitro. The formulation was found to be stable, and the loading process was rapid and efficient (drug-to-lipid ratio of up to 0.3 with over 90% efficiency in 5 minutes). The cytotoxicity of these formulations was assessed using the human melanoma cell lines A375 and Hs294T and the normal human dermal fibroblast line. The results showed that anacardic acid and to a smaller extent vitamin C significantly increased the cytotoxicity of the drug towards melanoma compared to ammonium sulfate liposomes. On the other hand, vitamin C and anacardic acid both protected normal cells from damage caused by the drug. The formulation combining anacardic acid, vitamin C, and mitoxantrone showed promising results in terms of cytotoxicity and cytoprotection. Therefore, it has potential for anticancer treatment.

Vitamin C-driven epirubicin loading into liposomes.

The encapsulation of anticancer drugs in a liposome structure protects the drug during circulation and increases drug accumulation in the cancer tissue and antitumor activity while decreasing drug toxicity. This paper presents a new method of active drug loading based on a vitamin C pH/ion gradient. Formulations were characterized in terms of the following parameters: optimal external pH, time and drug-to-lipid ratio for the purpose of remote loading, and in vitro stability. In the case of the selected drug, epirubicin (EPI), its coencapsulation increases its anticancer activity through a possibly synergistic effect previously reported by other groups for a free nonencapsulated drug/vitamin C cocktail. The method also has another advantage over other remote-loading methods: it allows faster drug release through liposome destabilization at the tumor site, thanks to the very good solubility of the EPI vitamin C salt, as seen on cryogenic transmission electron microscopy images. This influences the drug-release process and increases the anticancer activity of the liposome formulation. The liposomes are characterized as stable, with very good pharmacokinetics (half-life 18.6 hours). The antitumor activity toward MCF-7 and 4T-1 breast cancer cells was higher in the case of EPI loaded via our gradient than via an ammonium sulfate gradient. Finally, the EPI liposomal formulation and the free drug were tested using the murine 4T-1 breast cancer model. The antitumor activity of the encapsulated drug was confirmed (tumor-growth inhibition over 40% from day 16 until the end of the experiment), and the free drug was shown to have no anticancer activity at the tested dose.

Membrane perturbations induced by new analogs of neocryptolepine.

Indoloquinoline alkaloids represent an important class of antimalarial, antibacterial and antiviral compounds. Indolo[2,3-b]quinolines are a family of DNA intercalators and inhibitors of topoisomerase II, synthetic analogs of neocryptolepine, an alkaloid traditionally used in African folk medicine. These cytotoxic substances are promising anticancer agents. Active representatives of indolo[2,3-b]quinolines affect model and natural membranes. The distinct structure and hydrophobicity of the compounds leads to marked differences in the disturbing effects on membrane organization and function. Our results also indicated a strong relationship between the presence of the chain and the Poct of the molecule as well as the capacity for incorporation into carboxyfluorescein-trapped liposomes in the 0.02-0.06 mM range. Moreover, a correlation between binding to neutral dimyristoylphosphatidylcholine (DMPC) or negative charged dimyristoylphosphatidylcholine:dimyristoylphosphatidylglycerol (DMPC:DMPG, 9:1 w/w) liposomes, as well as to erythrocyte ghosts and pKa, was also found. All the compounds cause hemolysis in isotonic conditions with concentration causing 50% hemolysis (HC50) in the 0.12-0.88 mM range. The concentration-dependent inhibitory effect of the tested agents on erythrocyte ghosts' acetylcholinesterase activity was also studied.

The encapsulation of idarubicin within liposomes using the novel EDTA ion gradient method ensures improved drug retention in vitro and in vivo.

The purpose of this study was to design a new stable liposomal formulation for the anticancer drug idarubicin. Idarubicin is a relatively hydrophobic member of the anthracycline family. It exhibits pronounced bilayer interactions leading to rapid in vivo drug release from liposomes. This rapid drug leakage is due to the presence of cholesterol and charged lipids in the liposomal bilayer. Therefore, a novel method of remote drug loading was developed to prevent rapid drug release from PEGylated cholesterol-containing liposomes. This method uses EDTA disodium or diammonium salt as an agent to form low solubility complexes between the drug and EDTA molecules inside the liposomes, thus yielding improved drug retention. The efficiency of idarubicin encapsulation is close to 98% at a drug to lipid molar ratio of 1:5. An in vitro long-term storage experiment confirmed the high stability of the liposomes. The in vivo studies also showed the superiority of the new idarubicin formulation over the recently used remote loading methods. The plasma level of idarubicin was much higher when EDTA liposomes were used. The presented results fully demonstrate the superiority of the proposed method of idarubicin encapsulation over existing methods. The method offers the possibility of encapsulating not only all the anthracyclines, but also other weakly amphiphilic bases within the liposomes.

Cycloate, an inhibitor of fatty acid elongase, modulates the metabolism of very-long-side-chain alkylresorcinols in rye seedlings.

BACKGROUND: Cycloate inhibits the biosynthesis of very-long-chain fatty acids, the essential constituents of plant waxes and suberin. Fatty acids also serve as precursors of aliphatic carbon chains in resorcinolic lipids, which play a fundamental role in the plant defence system against fungal pathogens. In this study, the effect of cycloate on the biosynthesis of 5-n-alkylresorcinols in rye seedlings (Secale cereale L.) grown under various light and thermal conditions was examined. RESULTS: The content of alkylresorcinols biosynthesised in rye was generally increased by the herbicide in both green and etiolated plants. The presence of cycloate also affected patterns of alkylresorcinol homologues in plants grown at 15 and 22 degrees C; very-long-side-chain compounds were less abundant, whereas both short-chain saturated and unsaturated homologues were generally accumulated. No cycloate-related effects caused by homologue pattern modifications were observed at elevated temperature. CONCLUSION: This study extends present understanding of the mode of action of thiocarbamate herbicides. Cycloate markedly affected the biosynthesis of very-long-side-chain resorcinolic lipids in rye seedlings, confirming the existence of parallels in both fatty acid and alkylresorcinol biosynthetic pathways. The observed cycloate-driven accumulation of 5-n-alkylresorcinols may improve the resistance of cereals to infections caused by microbial pathogens.

Disturb or stabilize? A molecular dynamics study of the effects of resorcinolic lipids on phospholipid bilayers.

Resorcinolic lipids, or resorcinols, are commonly found in plant membranes. They consist of a substituted benzene ring forming the hydrophilic lipid head, attached to an alkyl chain forming the hydrophobic tail. Experimental results show alternative effects of resorcinols on lipid membranes. Depending on whether they are added to lipid solutions before or after the formation of the liposomes, they either stabilize or destabilize these liposomes. Here we use atomistic molecular dynamics simulations to elucidate the molecular nature of this dual effect. Systems composed of either one of three resorcinol homologs, differing in the alkyl tail length, interacting with dimyristoylphosphatidylcholine lipid bilayers were studied. It is shown that resorcinols preincorporated into bilayers induce order within the lipid acyl chains, decrease the hydration of the lipid headgroups, and make the bilayers less permeable to water. In contrast, simulations in which the resorcinols are incorporated from the aqueous solution into a preformed phospholipid bilayer induce local disruption, leading to either transient pore formation or even complete rupture of the membrane. In line with the experimental data, our simulations thus demonstrate that resorcinols can either disturb or stabilize the membrane structure, and offer a detailed view of the underlying molecular mechanism.

[Transformations of erythrocytes shape and its regulation]. / Zmiany ksztaltu erytrocytów i czynniki je wywolujace.

Erythrocytes can occur in many different shapes. Most of them are pathological and can be involved in diseases such a hemolytic anemia's and sickle cell anemia. Only three kinds of red blood cells are no pathological. Echinocytes, stomatocytes and discocytes can occure in blood stream of healthy organism. The echinocyte-dyscocyte-stomatocyte transformation protects red blood cells from lysis caused by echinocytogenic agents (hypertonic saline, basic pH, vanadate, anionic amphiphiles, ATP depletion etc.) or stomatocytogenic agents (hypotonic saline, acidic pH, cationic amphiphiles etc.). Mechanisms of these transformations can be classified in three group: redistribution of bilayer's lipid, modification Donnan's equilibrium and interaction of band 3 protein with different type of external factors.

Membrane perturbing properties of natural phenolic and resorcinolic lipids.

The effects induced by natural phenolic and resorcinolic lipids on membrane permeability were investigated. All of the compounds tested perturbed the phospholipid bilayer and stabilized erythrocytes against hypoosmotically induced hemolysis. Dipalmitoylphosphatidylcholine liposomes with two preincorporated fluorescent dyes (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatrien p-toluenesulfonate (TMA-DPH) and N-(-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoetanolamine triethylammonium salt (NBD-PE)) were used to determine the effects of tested compounds on the core and surface of the bilayer. Resorcinolic lipids from rye and cardol increased the polarization of TMA-DPH fluorescence more than that of NBD-PE, but anacardic acid, methylocardol, and alkylphenol increased NBD-PE dye fluorescence.

Alkylresorcinols in selected Polish rye and wheat cereals and whole-grain cereal products.

The alkylresorcinol content and homologue composition in selected Polish rye and wheat cultivars and selected whole-grain cereal products were determined in this study. Cereal grains and whole-grain cereal products were extracted with acetone, whereas bread types were extracted with hot 1-propanol. The average alkylresorcinol content in tested rye (approximately 1100 mg/kg DM) and wheat (approximately 800 mg/kg DM) grains harvested in Poland was within the range previously reported in Swedish and Finnish samples. The total alkylresorcinol content in tested cereal products available on the Polish market varied from very low levels in barley grain-based foods up to 3000 mg/kg DM in wheat bran. The total alkylresorcinol content in 14 bread samples extracted with hot 1-propanol varied from approximately 100 mg/kg DM in whole bread made with honey up to approximately 650 mg/kg DM in whole-rye bread. Calculated ratios of C17:0 to C21:0 homologues, a useful parameter previously used to distinguish between rye and wheat cereals and their derived products, was about 1.2-1.4 in rye products, about 0.2 in wheat products, and varied between 0.2 and 0.6 in cereal-derived products containing a mixture of whole rye and/or wheat. The data set obtained were subsequently compared using cluster and principal component analysis, which allowed the tested cereal products to be classified into two major groups consisting of whole-rye or whole-wheat products, respectively. On the basis of that approach, mixed cereal products containing rye and wheat bran or whole rye and wheat flour were grouped between those two well-defined clusters. Our work not only provides a detailed examination of alkylresorcinols in selected Polish rye and wheat cultivars and selected whole-grain cereal products, but also demonstrates that this type of analysis accompanied by the use of proper statistical algorithms offers an objective way to evaluate the quality of whole-grain rye and/or wheat and their derived products.

Liposomal formulation of DIMIQ, potential antitumor indolo[2,3-b]quinoline agent and its cytotoxicity on hepatoma Morris 5123 cells.

The cytotoxic and antitumor activity of DIMIQ (5,11-dimethyl-5H-indolo[2,3-b]quinoline), synthetic analog of neocryptolepine, makes this compound a potential antitumor agent. An attempt to obtain liposomal form of DIMIQ.HCl was undertaken in the present study. Standard experimental conditions were chosen and information on the physicochemical parameters of the liposome dispersion containing studied indoloquinoline agent was collected. The effective and efficient encapsulation of DIMIQ.HCl (66.6%) in conventional liposomes (FAT-MLV, DMPC:DMPG 7:3 w/w at pH 7.0), uniformity of the size of liposomal vesicles, and high stability at pH 6.5 were demonstrated. Hemolysis of sheep erythrocytes induced by free form of DIMIQ.HCl was dramatically decreased after addition of liposome-entrapped DIMIQ.HCl. Treatment of hepatoma Morris 5123 cells for 24 hr with different concentrations of both free and its liposomal formulation of DIMIQ.HCl resulted in significant changes in cell morphology accompanied by reduction of cell viability.

Inhibitory effect of some natural and semisynthetic phenolic lipids upon acetylcholinesterase activity.

The effect of phenolic lipids isolated from rye grains and cashew nut shell liquid (CNSL) from Anacardium occidentale and their semisynthetic derivatives on erythrocyte ghost's acetylcholinesterase activity was studied. It has been shown that all tested compounds decreased the enzymatic activity of acetylcholinesterase. This effect depends on the type of studied compounds. Three of them completely inhibit acetylcholinesterase activity at the micromolar concentration.

Effect of norflurazon on resorcinolic lipid metabolism in rye seedlings.

Norflurazon is a selective pyridazinone herbicide excessively employed in the control of many annual grasses and broad-leaved weeds. This chemical causes plant bleaching due to the inhibition of the carotenoid pigment biogenesis as well as induces irreparable changes to chloroplasts, which are considered the organelles where the biosynthesis of resorcinolic lipids takes place. Resorcinolic lipids, a group of phenolic compounds, constitute not only an essential part of the plant antifungal defense system, but also are an important component of the human cereal diet. The aim of this study was to investigate the effect of norflurazon on the biosynthesis of resorcinolic lipids in 5-day-old rye plants (Secale cereale L.) that were grown at three different temperatures under light or dark conditions. At all tested temperatures, norflurazon decreased the fresh biomass of light-grown rye seedlings and increased the weight of plants grown in darkness. Compared with respective controls, this herbicide caused an increase in total content of alkylresorcinols in both green and etiolated plants with the exception of dark-grown norflurazon-treated rye at 29 degrees C. The general level of saturated homologues was markedly decreased by norflurazon in all etiolated plants and in light-grown seedlings at 15 degrees C. Independent of thermal and light conditions, in all norflurazon-treated samples two alkylresorcinol derivatives predominated: 1,3-dihydroxy-5-n-heptadecylbenzene and 1,3-dihydroxy-5-n-nonadecylbenzene. Thus, our results suggest that norflurazon affected the metabolism of alkylresorcinols in rye seedlings and its action was dependent on external stimuli.

Does isoprene protect plant membranes from thermal shock? A molecular dynamics study.

The question of why plants release isoprene when heat stressed has been continuously debated for more than half a century. In this work we use molecular dynamics simulation techniques to directly investigate the interaction between isoprene and a model phospholipid membrane in atomic detail. It is found that isoprene partitions preferentially in the center of the membrane and in a dose dependent manner enhances the order within the membrane without significantly changing the dynamical properties of the system. At a concentration of 20 mol% isoprene (16 isoprene molecules per 64 lipid molecules) the effect of the addition of isoprene on the membrane order is equivalent to a reduction in temperature of 10 K, rising to a reduction of 30 K at 43 mol% isoprene. The significance of the work is that it provides for the first time direct evidence that isoprene stabilizes lipid membranes and reduces the likelihood of a phospholipid membrane undergoing a heat induced phase transition. Furthermore it provides a clear mechanistic picture as to why plants specifically utilize isoprene for this purpose.

[5-n-alkylresorcinols of whole grain cereals and whole grain cereal products as biomarkers of healthy food]. / 5-n-alkilorezorcynole ziaren zbóz i pelnoziarnistych produktów spozywczych jako biomarkery zdrowej zywnosci.

Epidemiological studies suggest that consumption of whole grain cereals and whole grain cereal products have many benefical health effects, including reducing risk of diabetes, obesity, coronary heart diseases, stroke and even some cancers. Precise knowledge protective compounds present in cereal grains can be achieved only when specific biomarkers (biological marker, indicator), that could provide estimation of grain cereals absorption and intake, are established and determined. 5-n-alkylresorcinols (main fraction of phenolic compounds in cereals), because of their specific occurrence only in bran fraction, obtained in refining of milling fractions process, could be a very good candidate to play the role of biomarker of whole grain intake. They are absorbed by animals and humans, present in human plasma and as metabolites in urine. Because composition of saturated homologues of 5-n-alkylresorcinols is different in rye and wheat grains, they could be used as an indicator of the intake of the specific type of cereals and whole grain cereal products.

A semisynthetic 5-n-alkylresorcinol derivative and its effect upon biomembrane properties.

MSAR (1-sulfate-3-myristoyl-5-pentadecylbenzene) is a semisynthetic derivative of 5-n-pentadecylresorcinol (C15:0). MSAR exhibits hemolytic activity against sheep erythrocytes with a EH50 value of (35 +/- 1.7) microM. At low concentrations MSAR also exhibits the ability to protect cells against their hypoosmotic lysis. This protective effect is significant as, at 0.1 microM of MSAR, the extent of osmotically induced cell lysis is reduced by approx. 20%. It was demonstrated that the 9-anthroyloxystearic acid signal was most intensively quenched by MSAR molecules, suggesting a relatively deep location of these molecules within the lipid bilayer. MSAR causes an increase of the fluorescence of the membrane potential sensitive probe. This indicates an alteration of the surface charge and a decrease of the local pH value at the membrane surface. At low bilayer content (1-4 mol%) this compound causes a significant increase of the phospholipid bilayer fluidity (both under and above the main phase transition temperature) of dipalmitoylphosphatidylcholine (DPPC) liposomes. At this low content MSAR slightly decreases the main phase transition temperature (T(c)) value. The effects induced in the phospholipid bilayer by higher contents of MSAR molecules (5-10 mol%) make it impossible to determine the T(c) value and to evaluate changes of the membrane fluidity by using pyrene-labeled lipid. MSAR also causes a decrease of the activity of membrane-bound enzymes - red blood cell acetylcholinesterase (AChE) and phospholipase A2 (PLA2). MSAR decreases the AChE activity by 40% at 100 microM. The presence of MSAR in the liposomal membrane induces a complete abolishment of the lag time of the PLA2 activity, indicating that these molecules induce the formation of packing defects in the bilayer which may result from imperfect mixing of phospholipids.

A simply and sensitive fluorometric method for determination of gentamicin in liposomal suspensions.

A new method for measuring gentamicin in liposomes fluorometrically is described. The assay is based on the reaction between the amino groups in the gentamicin molecule and o-phthaldialdehyde (OPA), under basic pH conditions; the product's fluorescence can be read directly on a simple fluorimeter. The effects of several factors (time of reaction, volume of the OPA reagent, and product stability) were investigated. The standard curve was linear in the concentration range of 0.5-4.0microg, showing an excellent determination coefficient of r(2)=0.99. Additionally, the influence of different liposomal lipids on gentamicin determination was tested. Liposomal lipids containing no free amino groups (PC, Chol, DOTAP) have no influence on the reaction when present in the reaction mixture. In contrast, amino groups containing lipid (SA) showed intense method interference. Therefore, a method of lipid extraction was adapted to remove undesired lipids. The described method was successfully utilised during 2 years of liposomal gentamicin experiments.