Therapeutic Implications of Targeting Heat Shock Protein 70 by Immunization or Antibodies in Experimental Skin Inflammation.

Heat shock proteins (Hsp) are constitutive and stress-induced molecules which have been reported to impact innate and adaptive immune responses. Here, we evaluated the role of Hsp70 as a treatment target in the imiquimod-induced, psoriasis-like skin inflammation mouse model and related in vitro assays. We found that immunization of mice with Hsp70 resulted in decreased clinical and histological disease severity associated with expansion of T cells in favor of regulatory subtypes (CD4+FoxP3+/CD4+CD25+ cells). Similarly, anti-Hsp70 antibody treatment led to lowered disease activity associated with down-regulation of pro-inflammatory Th17 cells. A direct stimulating action of Hsp70 on regulatory T cells and its anti-proliferative effects on keratinocytes were confirmed in cell culture experiments. Our observations suggest that Hsp70 may be a promising therapeutic target in psoriasis and potentially other autoimmune dermatoses.

Biochemical and physiological aspects of chlorophyll breakdown / Biochemiczne i fizjologiczne aspekty rozkladu barwników chlorofilowych.

During leaf senescence and fruit ripening chlorophyll is broken down into nonfluorescent catabolites (NCCs). The chlorophyll degradation pathway includes a series of biochemical transformations ocurring sequentially in chloroplasts, cytosol and vacuoles. The path begins with enzymatic reduction of chlorophyll b to chlorophyll a. Next, the specific dechelatase and esterase remove the magnesium atom and the phytol chain resulting in the formation of pheophorbide a. In the next step, the porphyrin macroring is opened by pheophorbide a oxygenase and red catabolite reductase. The product of this transformation is an early fluorescent catabolite (pFCC), which after hydroxylation and species-specific modifications is imported into the vacuole. In acidic medium of the vacuole pFCC undergo isomerization to their respective colorless NCCs, which are final chlorophyll degradation products in higher plants. There are still no answers to a number of questions about the fate and significance of millions tons of chlorophyll catabolites released annually in the aquatic environment as a result of cellular senescence and death of phytoplankton. A few reports indicate that algae and cyanobacteria may metabolize their photosynthetic pigments in a similar way as higher plants do, however, the course of chlorophyll breakdown in these organisms has not been yet elucidated.

Exogenously applied hydrogen peroxide modifies the course of the Chlamydomonas reinhardtii cell cycle.

The interaction of NO and H2O2 in the regulation of plant development is well documented. We have recently shown that the content of NO and H2O2 changes in a characteristic way during the cell cycle of Chlamydomonas reinhardtii (Pokora et al., 2017), which implies participation of these molecules in the regulation of Chlamydomonas development. To verify this assumption, H2O2 was supplied at a concentration about 1.5 times higher than that determined in the control cells. Cells were synchronized by alternating the light/dark (10/14 h) regimen. H2O2 was added to zoospore suspensions, previously held in the dark, and cells growing for 3, 6, and 9 h in the light. The data indicate that, depending on the phase of the Chlamydomonas cell cycle, H2O2, via mild modification of redox homeostasis, may: a) accelerate or delay the duration of the cell cycle; b) increase the number of replication rounds occurring in one cell cycle; c) modify the biomass and cell volume of progeny cells and d) accelerate the liberation of daughter cells. This provides a tool to control the development of Chlamydomonas cell and thus offers the opportunity to obtain a population of cells with characteristics desired in biotechnology.

Mature Luffa Leaves (Luffa cylindrica L.) as a Tool for Gene Expression Analysis by Agroinfiltration.

We exploited the potential of cucurbits for ectopic gene expression. Agroinfiltration is a simple and commonly used method to obtain transient expression of foreign genes in plants. In contrast to in vitro transformation techniques, agroinfiltration can be used for genetic modification of mature plant tissues. Although the cucurbits are commonly used as model plants for molecular biology and biotechnology studies, to date there are no literature sources on the possibility of transient gene expression in mature cucurbit tissues. Our research has shown that mature leaves of Luffa cylindrica L. (luffa), in contrast to other cucurbit species, can be successfully transiently transformed with Agrobacterium tumefaciens. We efficiently transformed luffa leaves with a reporter gene encoding ß-glucuronidase (GUS). The GUS activity in transiently transformed leaf tissues was detected within 24 h after the infiltration with bacteria. Additionally, we have shown that the activity of a transiently expressed the GUS gene can be monitored directly in the EDTA-exudates collected from the cut petioles of the agroinfiltrated leaves. The results suggest that luffa leaves can be useful as a plant expression system for studies of physiological and biochemical processes in cucurbits.

Changes in nitric oxide/hydrogen peroxide content and cell cycle progression: Study with synchronized cultures of green alga Chlamydomonas reinhardtii.

The present study aimed to evaluate the possible relationship between the changes in hydrogen peroxide (H2O2) and nitric oxide (NO) content and the course of growth and reproductive processes of the cell cycle of Chlamydomonas reinhardtii. The peak of H2O2 observed at the beginning of the cell cycle was found to originate from Fe-SOD and Mn-SODchl. activity and result from the alternation in the photosynthetic processes caused by the dark-to-light transition of daughter cells. A rapid increase in NO concentration, observed before the light-to-dark cell transition, originated from NR and NIR activity and was followed by a photosynthesis-independent, Mn-SODchl.-mediated increases in H2O2 production. This H2O2 peak overlapped the beginning of Chlamydomonas cell division, which was indicated by a profile of CYCs and CDKs characteristic of cells' passage through the G1/S and S/M checkpoints. Taken together, our results show that there is a clear relationship between the course of the Chlamydomonas cell cycle and typical changes in the H2O2/NO ratio, as well as changes in expression and activity of enzymes involved in generation and scavenging of these signaling molecules.

Effects of juglone and lawsone on oxidative stress in maize coleoptile cells treated with IAA.

Naphthoquinones are secondary metabolites widely distributed in nature and produced by bacteria, fungi and higher plants. Their biological activity may result from induction of oxidative stress, caused by redox cycling or direct interaction with cellular macromolecules, in which quinones act as electrophiles. The redox homeostasis is known as one of factors involved in auxin-mediated plant growth regulation. To date, however, little is known about the crosstalk between reactive oxygen species (ROS) produced by quinones and the plant growth hormone auxin (IAA). In this study, redox cycling properties of two naphthoquinones, juglone (5-hydroxy-1,4-naphthoquinone) and lawsone (2-hydroxy-1,4-naphthoquinone), were compared in experiments performed on maize coleoptile segments incubated with or without the addition of IAA. It was found that lawsone was much more effective than juglone in increasing both H2O2 production and the activity of antioxidative enzymes (SOD, POX and CAT) in coleoptile cells, regardless of the presence of IAA. An increase in the activity of Cu/Zn-SOD isoenzymes induced by both naphthoquinones suggests that juglone- and lawsone-generated H2O2 was primarily produced in the cytosolic and cell wall spaces. The cell potential to neutralize hydrogen peroxide, determined by POX and CAT activity, pointed to activity of catalase as the main enzymatic mechanism responsible for degradation of H2O2 Therefore, we assumed that generation of H2O2, induced more efficiently by LW than JG, was the major factor accounting for differences in the toxicity of naphthoquinones in maize coleoptiles. The role of auxin in the process appeared negligible. Moreover, the results suggested that oxidative stress imposed by JG and LW was one of mechanisms of allelopathic action of the studied quinones in plants.

Chlorophyll catabolites in conditioned media of green microalga Desmodesmus subspicatus.

Although the appearance of coloured chlorophyll degradation products of higher plants is well known, knowledge about such compounds produced and released particularly by planktonic algae is still limited. Colourless conditioned media (CM) obtained from autotrophic cultures of unicellular green alga Desmosdemus subspicatus turn red after acidification. The accumulation of red pigments in the medium and the growth rate of algae were inversely correlated. The red, crude solution isolated from CM by dialysis and ion exchange chromatography, and next purified by means of high-performance liquid chromatography, appeared to be a mixture of three compounds with characteristic UV/VIS absorption maxima near 330 and 505 nm. Electrospray ionization (ESI) mass spectrometry analysis revealed that the molecular mass of the most polar and most abundant compound was 637 Da and molecular masses of two other ones were 641 and 607 Da. Addition of 15 N isotope to the culture medium and subsequent mass spectrometry measurements revealed the occurrence of four nitrogen atoms per each molecule. The data suggest that red pigments isolated from algal-conditioned media are chlorophyll degradation compounds, the production of which depends on light intensity, and are released mainly during the stationary phase of growth.

Time-dependent changes in antioxidative enzyme expression and photosynthetic activity of Chlamydomonas reinhardtii cells under acute exposure to cadmium and anthracene.

Heavy metals (HM) and polycyclic aromatic hydrocarbons (PAHs) are present in the freshwater environment at concentrations that can be hazardous to the biota. Among HMs and PAHs, cadmium (Cd) and anthracene (ANT) are the most prevalent and toxic ones. The response of Chlamydomonas cells to Cd and ANT at concentrations that markedly reduced the growth of algal population was investigated in this study. At such concentrations, both cadmium and anthracene were recognized as oxidative stress inducers, since high concentration of H2O2 in treated cultures was observed. Therefore, as a part of the "molecular phase" of the cell response to this stress, we examined the time-dependent expression of genes encoding the main antioxidative enzymes: superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), as well as the activity of these enzymes in cells, with special attention paid to chloroplastic and mitochondrial isoforms of SOD. To characterize the cell response at the "physiological level", we examined the photosynthetic activity of stressed cells via analysis of chlorophyll a fluorescence in vivo. In contrast to standard ecotoxicity studies in which the growth end-points are usually determined, herein we present time-dependent changes in algal cell response to Cd- and ANT-induced stress. The most significant effect(s) of the toxicants on photosynthetic activity was observed in the 6th hour, when strong depression of PI parameter value, an over 50 percent reduction of the active reaction center fraction (RC0) and a 3-fold increase in non-photochemical energy dissipation (DI0/RC) were noted. At the same time, the increase (up to 2.5-fold) in mRNA transcript of SOD and CAT genes, followed by the enhancement in the enzyme activity was observed. The high expression of the Msd 3 gene in treated Chlamydomonas cells probably complements the partial loss of chloroplast Fe-SOD and APX activity, while catalase and Mn-SOD 5 seem to be the major enzymes responsible for mitochondrion protection. The progressive increase in SOD and CAT activities seems to be involved in the recovery of photosynthesis within 12-24h after the application of the toxicants.

Adaptation strategies of two closely related Desmodesmus armatus (green alga) strains contained different amounts of cadmium: a study with light-induced synchronized cultures of algae.

During the Desmodesmus armatus cell cycle, 8-celled coenobia of 276-4d strain accumulated a much lower amounts of cadmium than unicells of B1-76 strain. Cadmium reduced growth and photosynthesis in the cells of strain B1-76, but not those of 276-4d strain. Cells of 276-4d strain revealed a higher activity of superoxide dismutase (SOD) isoforms, in particular the activity and protein content of Fe-SOD. Cu/Zn-SOD was earlier and much stronger induced by cadmium in 276-4d than in B1-76 strain, whereas Fe- and Mn-SOD activity and Fe-SOD synthesis were induced only in 276-4d strain. Cadmium did not affect the heat shock protein 70 synthesis in B1-76 strain, but significantly stimulated this process in 276-4d strain. The level of glutathione increased 30-fold during cell development of Cd-exposed 276-4d strain, while in B1-76 it increased about 12 timed. Matured cells of both strains exposed to cadmium produced comparable amounts of phytochelatins and other thiol peptides, but their production in young cells of B1-76 strain was much higher than in 276-4d strain. In conclusion, a complex of internal detoxification mechanisms appeared to be more efficient in cells of 276-4d strain than B1-76 one.

Induction time of Fe-SOD synthesis and activity determine different tolerance of two Desmodesmus (green algae) strains to chloridazon: a study with synchronized cultures.

Cells of two Desmodesmus armatus strains (276-4a and 276-4d) grown asynchronously in batch cultures after 24-h treatment revealed different tolerance to chloridazon (photosynthetic herbicide) applied at a concentration of 3.45 mg L(-1). To find time- and cell cycle-dependent biochemical reasons leading to such a difference, a population of young autospores of both strains synchronized by a light/dark (14/10) regime were exposed to chloridazon at the initiation of the light period. Chloridazon reduced the growth and number of divisions of cell strain 276-4d. In consequence, at the end of the dark phase the number of released autospores was reduced by 50% compared with the control. In contrast, the growth and reproductive processes of cell strains 276-4a was unaffected. Moreover, chloridazon treatment speeded up cell development, as a result of which the release of autospores took this process observed in the control cells over. There is a relationship between photosynthetic activity response to chloridazon and time-dependent changes in Fe-SOD content and activity. The energy trapped in the reaction centre (RC) was similar in both strains, but the amount of energy absorbed by RCs was twice as high in strain 276-4d as in 276-4a. In consequence, non-photochemical energy dissipation occurring in the cells of 276-4d strain far exceed the value obtained for 276-4a strain. The control cells of both strains differed significantly in the content of FSD 1 and FSD 2 proteins, whereas the differences in Fe-SOD isoforms activities were slight. The 8-fold increase in SOD content in CHD treated cells of strain 276-4a was associated with the transience of photosynthetic efficiency impairment. In CHD treated cells of strain 276-4d, neither activity of Fe-SOD nor FSDs protein content was instantly affected. Different response of developing cells of two Desmodesmus strains to CHD is influenced by the inherent features of cells; the short time required to induce stress adaptive mechanism involving chloroplastic Fe-SOD activity and biosynthesis in the cells of CHD tolerant strain 276-4a seems to play the key role, being an overriding on the high, but not induced in response to stress, FSD protein level and activity in cells of strain 276-4d.

Phytotoxicity and antioxidative enzymes of green microalga (Desmodesmus subspicatus) and duckweed (Lemna minor) exposed to herbicides MCPA, chloridazon and their mixtures.

In this study, we evaluate the toxicity of MCPA (auxin-like growth inhibitor), chloridazon (CHD) (PSII-inhibitor) and their mixtures to floating plants and planktonic algae. Toxicity of MCPA (4-chloro-2-methylphenoxyacetic acid) and CHD (5-amino-4-chloro-2-phenyl-3(2H)-pyridazinone) was first assessed in two growth inhibition tests with Lemna minor (ISO/DIS 20079) and Desmodesmus subspicatus (ISO 8692). Next, herbicide mixtures at concentrations corresponding to the EC values were used to assess their interactive effects, and the biomarkers were: for duckweed fresh weight, frond area, chlorophyll content and number of fronds, and for algae cell count and cell volume. The 3d EC10 and EC50 values using cell counts of D. subspicatus were 142.7 and 529.1 mg/L for MCPA and 1.7 and 5.1 mg/L for CHD. The 7d EC10 and EC50 values using frond number of L. minor amounted to 0.8 and 5.4 mg/L for MCPA and 0.7 and 10.4 mg/L for CHD. Higher sensitivity of reproductive (number of cells/fronds) than growth processes (cell volume/frond area) to herbicides applied individually and in mixtures was especially pronounced in the responses of Desmodesmus. Herbicide interactions were assessed by the two-way ANOVA and Abbott's formula. Generally, an antagonistic interaction with Lemna was revealed by MCPA and chloridazon, whereas additive effect of both herbicides was observed for Desmodesmus. A significant stimulation of SOD and APX activity by binary mixtures was noted in algal cells mainly after 24 and 48 hours of exposure. The extremely high stimulation of the activity of both enzymes was induced by the combination EC10CHD + EC50MCPA (48 h). Presumably due to oxidative stress, the treatment with CHD at concentration EC50 after 72 h was lethal for algae grown in aerated cultures, in contrast to standardized test conditions. Taking into account the consequences of risk assessment for herbicide mixtures we can state that a relatively low toxicity, as well as the lack of significant synergy between MCPA and CHD to non-target plants appears to be the most important result.

The mechanism of anthracene interaction with photosynthetic apparatus: a study using intact cells, thylakoid membranes and PS II complexes isolated from Chlamydomonas reinhardtii.

Intact cells of Chlamydomonas reinhardtii as well as isolated thylakoid membranes and photosystem II complexes were used to examine a possible mechanism of anthracene (ANT) interaction with the photosynthetic apparatus. Since ANT concentrations above 1 mM were required to significantly inhibit the rate of oxygen evolution in PS II membrane fragments it may indicate that the toxicant did not directly interact with this photosystem. On the other hand, stimulation of oxygen uptake by ANT-treated thylakoids suggested that ANT could either act as an artificial electron acceptor in the photosynthetic electron transport chain or function as an uncoupler. Electron transfer from excited chlorophyll to ANT is impossible due to the very low reduction potential of ANT and therefore we propose that toxic concentrations of ANT increase the thylakoid membrane permeability and thereby function as an uncoupler, enhancing electron transport in vitro. Hence, its unspecific interference with photosynthetic membranes in vitro suggests that the inhibitory effect observed on intact cell photosynthesis is caused by uncoupling of phosphorylation.

Time- and dose-dependent induction of HSP70 in Lemna minor exposed to different environmental stressors.

The objective of this study was to examine the influence of different stressors, including cadmium (heavy metal), anthracene (polycyclic aromatic hydrocarbon-PAH) and chloridazon (herbicide), on population growth and biosynthesis of cytoplasmic HSP70 in Lemna minor (duckweed) in short (4 h)- and long (7 days)-term tests. A heat shock response was confirmed in Lemna exposed to high temperature: 35, 37.5, 40, or 42.5°C in short-term (4 h) treatments. The chemicals tested stimulated the biosynthesis of the cytoplasmic HSP70 protein in a concentration-dependent way (0.5-5 µM), higher in fronds exposed to lower doses of stressors. Additionally, production of HSP70 was greater after 4 h of incubation than after 7 days. The results suggest that HSP70 could be applied as a non-specific and sensitive detector of stress induced by different chemicals at concentrations below those that produce the type of response observed in classical cytotoxicity tests, such as growth inhibition.

Toxicity of cadmium, anthracene, and their mixture to Desmodesmus subspicatus estimated by algal growth-inhibition ISO standard test.

Cells of Desmodesmus subspicatus 86.81 were used to examine the toxicity of cadmium chloride (CdCl(2)) and anthracene (ANT) applied individually and in combination. The experiments were performed according to standardized ISO (International Organization for Standardization) 8692 protocol (2004). Parameters measured were the number of cells and chlorophyll a fluorescence parameters. E(r)C(10) and E(r)C(50) values (growth rate [r] inhibition by 10% and 50%, respectively) for single toxicants were determined separately. The effect of mixtures of the substances (Cd + ANT) at concentrations corresponding to E(r)C(10) (E(r)C(10) + E(r)C(10)) and E(r)C(50) (E(r)C(50) + E(r)C(50)) values was characterized. The toxicity of individual chemicals after a 72-h exposure was as follows: ANT (E(r)C(10) = 0.06; E(r)C(50) = 0.26 mg l(-1)) and CdCl(2) (E(r)C(10) = 0.12; E(r)C(50) = 0.30 mg l(-1)). The combination Cd + ANT decreased the population growth rate more strongly than the substances applied individually. Cadmium at a concentration corresponding to E(r)C(10) slightly influenced the parameters of chlorophyll a fluorescence as measured by the OJIP test (O, J, I, and P are the different steps of fluorescence induction curve), whereas the influence of ANT was not statistically significant. In Cd + ANT-treated samples, the photosynthetic "vitality" (PI), the maximum quantum yield of primary photochemistry (φ(Po)), and the fraction of active PS II reaction centre (RC) decreased, but the values of ABS/RC, TR(0)/RC, and DI(0)/RC increased. The type of interaction between Cd and ANT depended on the concentration of chemicals used. When the substances were applied at concentrations of E(r)C(10), synergistic effects were observed, whereas the mixture of chemicals used at an E(r)C(50) concentration showed an antagonistic interaction.

The combined effect of anthracene and cadmium on photosynthetic activity of three Desmodesmus (Chlorophyta) species.

Individual toxicity of heavy metals (HM) and polycyclic aromatic hydrocarbons (PAH) to plants living in water bodies is well-documented. In view of frequent joint occurrence of these compounds in the environment, plants are subjected to damage from their combined action. Cadmium and anthracene can generate production of reactive oxygen species (ROS). We have recently detected elevated activity of Fe- and Mn-SOD isoforms, indicating chloroplast and mitochondrion as the main sites of combined toxicity of HM and PAH. In the present paper, short-term (1-24 h) experiments on the mechanism of combined toxicity of anthracene and cadmium to the photosynthesis of three Desmodesmus species are reported. Inhibition, stimulation or no effect on the oxygen evolution was observed following the treatment with the contaminants when applied either separately or jointly. The response pattern was both strongly species- and time-dependent. In contrast, the photosynthetic activity of cells, expressed by chlorophyll fluorescence parameters, was substantially unaffected, since no effect or, in several cases, a slight stimulation of PS II quantum efficiency (Phi PS II) were noted. A characteristic relationship between the SOD activity and the qN values was observed. The treatment of Desmodesmus cells with anthracene or cadmium had either no effect or slightly enhanced either the SOD activity or the qN value, whereas the mixture of the contaminants resulted in a multifold increase in both the SOD activity and the qN values. The results suggest that chloroplasts of algae are well protected against the combined action of the two contaminants the toxicity of which should be attributed to nucleocytoplasmic compartments and reproductive processes of the cell cycle.

Intact anthracene inhibits photosynthesis in algal cells: a fluorescence induction study on Chlamydomonas reinhardtii cw92 strain.

Short-term (24h) experiments were performed to examine the effect of anthracene (ANT) on Chlamydomonas reinhardtii cw92 grown in a batch culture system aerated with 2.5% CO(2). At concentrations ranging from 0.7 to 5.6 microM, ANT inhibited the growth of population in a concentration-dependent manner and EC(50) calculated amounted to 1.6 microM. At concentrations from 0.7 to 4.2 microM ANT stimulated respiration and inhibited the intensity of photosynthesis but did not affect chlorophyll content in the cells. ANT influenced chlorophyll a fluorescence parameters, measured by OJIP test (O, J, I and P are the different steps of fluorescence induction curve). ANT diminished the performance index (PI), the yield of primary photochemistry (phi(Po)), the yield of electron transport (phi(Epsilonomicron), the efficiency of moving the electron beyond Qa(-) (Psi(0)) and the fraction of active oxygen evolving complexes (OEC). The fraction of active PS II reaction centres in the treated samples dramatically dropped. The most pronounced changes in ANT-treated cells were observed in the stimulation of energy dissipation parameter (DI(0)/RC). The only OJIP parameter that was not influenced by ANT was energy absorption by photosynthetic antennae (ABS). The results lead to a conclusion that the inhibition of photosynthesis may be a consequence of unspecific ANT-membrane interaction, resulting from hydrophobic character of this hydrocarbon.

Toxic effects of anthraquinone and phenanthrenequinone upon Scenedesmus strains (green algae) at low and elevated concentration of CO2.

Short-term (24h) experiments were performed to examine the effect of anthraquinone (ANTQ) and phenanthrenequinone (PHEQ) on two Scenedesmus armatus strains (B1-76 and 276-4d) grown in a batch culture system aerated with CO2 at a low (0.1%) or elevated (2%) concentration. ANTQ at concentrations within the range of 0.156-1.250 mg dm-3 inhibited the growth of B1-76 population in a concentration-dependent manner, and calculated EC50 for low-CO2 cells was 0.56 mg dm-3. The toxic effect of ANTQ on this strain was more pronounced in high-CO2 cells, where not only growth but also photosynthesis, respiration and SOD activity were significantly inhibited. In contrast, except for SOD activity, no ANTQ effects on strain 276-4d were found. PHEQ at concentrations within the range of 0.063-0.125 mg dm-3 inhibited the growth of B1-76 population in a concentration-dependent manner. The value of EC50 for low-CO2 B1-76 cells was 0.10 mg dm-3. PHEQ inhibited the growth of both strains regardless of CO2 concentration. In B1-76 cells affected by PHEQ, inhibition of photosynthesis was independent of the CO2 level, whereas the SOD activity was much higher in cultures aerated with 2% than with 0.1% CO2. Higher toxicity of PHEQ to strain 276-4d grown at 2% CO2 was accompanied by strong inhibition of photosynthesis, while in low-CO2 cells this process was slightly stimulated. The SOD activity in both low- and high-CO2 cells of strain 276-4d treated with PHEQ was 2-3 times higher compared with the controls. The pattern of SOD isoforms (PAGE analysis) obtained from cells exposed to ANTQ or PHEQ did not change compared with the controls, but the location of the SOD isoforms bands on gel was affected by the concentration of CO2. The results suggest that the strain-specific toxicity of ANTQ and PHEQ may result from oxidative stress. In addition, carbon dioxide appears to play an important role in the toxicity of quinones to algae.