Response to azacitidine in patients with chronic myelomonocytic leukemia according to overlap myelodysplastic/myeloproliferative neoplasms criteria.

BACKGROUND: Azacitidine (AZA) is approved for the treatment of high-risk chronic myelomonocytic leukemia (CMML) of myelodysplastic (MD) subtype. Data of response rates using the specific response criteria for this disease are scarce. The aim of this study was to evaluate the response to AZA in patients diagnosed with CMML from the Spanish Registry of Myelodysplastic Syndromes (MDS) applying the overlap myelodysplastic/myeloproliferative neoplasms (MDS/MPN) response criteria. METHODS: We retrospectively studied 91 patients with CMML treated with at least one cycle of AZA from the Spanish Registry of MDS. As it was a real-world study, the response rate was evaluated between cycle 4 and 6, applying the MDS/MPN response criteria FINDINGS: The overall response rate at cycle 4-6 was 58%. Almost half of the patients achieved transfusion independence and one quarter showed clinical benefit, regardless of the CMML French-American-British (FAB) and World Health Organization (WHO) subtypes and CMML Specific Prognosis Scoring (CPSS) risk groups. Toxicity was higher in the MD-CMML subtype. INTERPRETATION: In our series, most CMML patients achieved an overall response rate with AZA according to the overlap-MDS/MPN response criteria regardless of the CMML FAB and WHO subtypes and CPSS risk groups. Thus, AZA may also be a treatment option for patients with the myeloproliferative CMML subtype and those with a lower-risk CPSS, but symptomatic.

A sustainable approach by using microalgae to minimize the eutrophication process of Mar Menor lagoon.

The present study evaluates the removal capacity of microalgae photobioreactors of environmental pollutants present in wastewater from the dry riverbed El Albujón, as a way to minimize the eutrophication process of the Mar Menor. Particularly, the capacity of four autochthonous microalgae consortia collected from different locations of the salty lagoon to remove emerging contaminants (simazine, atrazine, terbuthylazine, adenosine and ibuprofen), nitrates, and phosphates, was evaluated. Among the four microalgae consortia, consortium 1 was the best in terms of biomass productivity (0.11 g L-1 d-1) and specific growth rate (0.14 d-1), providing 100% removal of emerging contaminants (simazine, atrazine, terbuthylazine, adenosine and ibuprofen), and a maximal reduction and consumption of macronutrients, especially nitrates and phosphates, reaching levels below 28 mg L-1, that is, a decrease of 89.90 and 99.70% of nitrates and phosphates, respectively. Therefore, this consortium (Monoraphidium sp., Desmodesmus subspicatus, Nannochloris sp.) could be selected as a green filter for successful large-scale applications. This study is the first one that combines the successful removal of herbicides, ibuprofen and adenosine as emerging contaminants, and nitrate removal.

Changes in the secretome of Vitis vinifera cv. Monastrell cell cultures treated with cyclodextrins and methyl jasmonate.

Elicitors induce defense responses that resemble those triggered by pathogen attack, including the synthesis of phytoalexins and pathogen-related proteins, which are accumulated in the extracellular space. In this work we analyze the changes in the secretome of Vitis vinifera cv. Monastrell cell cultures. This refers to the secreted proteome obtained from cell suspension cultures, in response to treatment with cyclodextrins and methyl jasmonate, separately or in combination using label-free quantitative approaches. Of the proteins found, thirty-three did not show significant differences in response to the different treatments carried out, indicating that these proteins were expressed in a constitutive way in both control and elicited grapevine cell cultures. These proteins included pathogenesis-related proteins 4 and 5, class III peroxidases, NtPRp-27, chitinases and class IV endochitinases, among others. Moreover, eleven proteins were differentially expressed in the presence of cyclodextrins and/or methyl jasmonate: three different peroxidases, two pathogenesis related protein 1, LysM domain-containing GPI-anchored protein 1, glycerophosphoryl diester phosphodiesterase, reticulin oxidase, heparanase, ß-1,3-glucanase and xyloglucan endotransglycosylase. Treatments with cyclodextrins reinforced the defensive arsenal and induced the accumulation of peroxidase V and xyloglucan endotransglycosylase. However, elicitation with methyl jasmonate decreased the levels of several proteins such as pathogenesis related protein 1, LysM domain-containing GPI-anchored protein 1, cationic peroxidase, and glycerophosphoryl diester phosphodiesterase, but increased the levels of new gene products such as heparanase, ß-1,3 glucanase, reticulin oxidase, and peroxidase IV, all of which could be used as potential biomarkers in the grapevine defense responses.

Increased Glucosinolate Production in Brassica oleracea var. italica Cell Cultures Due to Coronatine Activated Genes Involved in Glucosinolate Biosynthesis.

In this work, the effect of different elicitors and culture conditions on the production of glucosinolates in broccoli cell cultures was studied. The results showed that 0.5 µM coronatine was the best elicitor for increasing glucosinolate production (205-fold increase over untreated cells after 72 h of treatment). Furthermore, the expression levels of some genes related to the biosynthetic pathway of glucosinolates as well as three Myb transcription factors also have been studied. The highest glucosinolate levels found in coronatine-treated cells were closely correlated with the highest gene expression levels of Cyp79b2, Cyp83b1, St5a, Myb51, and Myb122 after 6 h of treatment. The data shown in this study provide new insight into the key metabolic steps involved in the biosynthesis of glucosinolates, which will be of use for future applications of metabolic engineering techniques in broccoli.

A new strategy to enhance the biosynthesis of trans-resveratrol by overexpressing stilbene synthase gene in elicited Vitis vinifera cell cultures.

In this work, transgenic lines of suspension cultured cells of Vitis vinifera cv. Monastrell containing the plasmid pMOG800-sts have been obtained. The cell growth of these transgenic cell lines decreased slightly as compared to non-transgenic suspension cultured cells, while cell viability was not affected. In addition, the elicitation with cyclodextrins and methyl jasmonate enhanced the production of trans-resveratrol, observing the highest levels of this compound in sts-expressing transgenic Vitis suspension cultured cells with the sts expression cassette in the forwards orientation. Moreover, the forwards 2 (F2) transgenic cell line produced the greater levels of trans-resveratrol in comparison with the non-transgenic cell line. In fact, when suspension cultured cells were treated with both elicitors, the accumulation of trans-resveratrol outside the cells in the F2 transgenic suspension cultured cells increased twice (1458 mg.L-1) as compared to non-transgenic cell lines (724 mg.L-1). In both cases, the levels of trans-resveratrol detected in the treatment with cyclodextrins and methyl jasmonate were greater than the sum of the individual treatments, and therefore we observed a synergistic effect in the presence of both elicitors. Moreover, the expression profile of sts gene in transgenic V. vinifera cell lines was similar to the expression profile detected for the endogenous sts gene in non-transgenic V. vinifera cell lines, being the expression levels greater in the treatment with methyl jasmonate and cyclodextrins, which was related to the high levels of trans-resveratrol found in the presence of both elicitors.

Bioactivity of Phytosterols and Their Production in Plant in Vitro Cultures.

Phytosterols are a kind of plant metabolite belonging to the triterpene family. These compounds are essential biomolecules for human health, and so they must be taken from foods. ß-Sitosterol, campesterol, and stigmasterol are the main phytosterols found in plants. Phytosterols have beneficial effects on human health since they are able to reduce plasma cholesterol levels and have antiinflammatory, antidiabetic, and anticancer activities. However, there are many difficulties in obtaining them, since the levels of these compounds produced from plant raw materials are low and their chemical synthesis is not economically profitable for commercial exploitation. A biotechnological alternative for their production is the use of plant cell and hairy root cultures. This review is focused on the biosynthesis of phytosterols and their function in both plants and humans as well as the different biotechnological strategies to increase phytosterol biosynthesis. Special attention is given to describing new methodologies based on the use of recombinant DNA technology to increase the levels of phytosterols.

Enhanced accumulation of phytosterols and phenolic compounds in cyclodextrin-elicited cell suspension culture of Daucus carota.

In this work, suspension-cultured cells of Daucus carota were used to evaluate the effect of ß-cyclodextrins on the production of isoprenoid and phenolic compounds. The results showed that the phytosterols and phenolic compounds were accumulated in the extracellular medium (15100µgL(-1) and 477.46µgL(-1), respectively) in the presence of cyclodextrins. Unlike the phytosterol and phenolic compound content, ß-carotene (1138.03µgL(-1)), lutein (25949.54µgL(-1)) and α-tocopherol (8063.82µgL(-1)) chlorophyll a (1625.13µgL(-1)) and b (9.958 (9958.33µgL(-1)) were mainly accumulated inside the cells. Therefore, cyclodextrins were able to induce the cytosolic mevalonate pathway, increasing the biosynthesis of phytosterols and phenolic compounds, and accumulate them outside the cells. However, in the absence of these cyclic oligosaccharidic elicitors, carrot cells mainly accumulated carotenoids through the methylerythritol 4-phosphate pathway. Therefore, the use of cyclodextrins would allow the extracellular accumulation of both phytosterols and phenolic compounds by diverting the carbon flux towards the cytosolic mevalonate/phenylpropanoid pathway.

New strategies for the use of Linum usitatissimum cell factories for the production of bioactive compounds.

In this work, suspension-cultured cells of Linum usitatissimum L. were used to evaluate the effect of two types of cyclodextrins, ß-glucan and (Z)-3-hexenol separately or in combination on phytosterol and tocopherol production. Suspension-cultured cells of L. usitatissimum were able to produce high levels of phytosterols in the presence of 50 mM methylated-ß-cyclodextrins (1325.96 ± 107.06 µg g dry weight(-1)) separately or in combination with ß-glucan (1278.57 ± 190.10 µg g dry weight(-1)) or (Z)-3-hexenol (1507.88 ± 173.02 µg g dry weight(-1)), being cyclodextrins able to increase both the secretion and accumulation of phytosterols in the spent medium, whereas ß-glucan and (Z)-3-hexenol themselves only increased its intracellular accumulation. Moreover, the phytosterol values found in the presence of hydroxypropylated-ß-cyclodextrins were lower than those found in the presence of methylated-ß-cyclodextrins in all cases studied. However, the results showed that the presence of methylated-ß-cyclodextrins did not increase the tocopherols production and only an increase in tocopherol levels was observed when cells were elicited with 50 mM hydroxypropylated-ß-cyclodextrins in combination with ß-glucan (174 µg g dry weight(-1)) or (Z)-3-hexenol (257 µg g dry weight(-1)). Since the levels of tocopherol produced in the combined treatment were higher than the sum of the individual treatments, a synergistic effect between both elicitors was assumed. To sum up, flax cell cultures elicited with cyclodextrins alone or in combination with ß-glucan or (Z)-3-hexenol were able produce phytosterols and tocopherols, and therefore, these elicited suspension-cultured cells of L. usitatissimum can provide an alternative system, which is at the same time more sustainable, economical and ecological for their production.

A rational approach to improving the biotechnological production of taxanes in plant cell cultures of Taxus spp.

Taxol is a complex diterpene alkaloid scarcely produced in nature and with a high anticancer activity. Biotechnological systems for taxol production based on cell cultures of Taxus spp. have been developed, but the growing commercial demand for taxol and its precursors requires the optimization of these procedures. In order to increase the biotechnological production of taxol and related taxanes in Taxus spp. cell cultures, it is necessary not only to take an empirical approach that strives to optimize in-put factors (cell line selection, culture conditions, elicitation, up-scaling, etc.) and out-put factors (growth, production, yields, etc.), but also to carry out molecular biological studies. The latter can provide valuable insight into how the enhancement of taxane biosynthesis and accumulation affects metabolic profiles and gene expression in Taxus spp. cell cultures. Several rational approaches have focused on studying the transcriptomic profiles of key genes in the taxol biosynthetic pathway in Taxus spp. cell cultures treated with elicitors such as methyl jasmonate, coronatine and cyclodextrins in relation with the taxane pattern, production and excretion to the culture medium. These studies have provided new insights into the taxol biosynthetic pathway and its regulation. Additionally, identifying genes with low levels of expression even in the presence of elicitors, together with metabolomics studies, has shed light on the limiting steps in taxol biosynthesis and could help define suitable metabolic targets for engineering with the main aim of obtaining highly productive Taxus cultured cells. In this review, we have summarized the latest endeavors to enhance the molecular understanding of the action mechanism of elicitors in Taxus spp. cell cultures. Developments in the ongoing search for new and more effective elicitation treatments and the application of metabolic engineering to design new transgenic cell lines of Taxus with an improved capacity for taxane production are described.

DIGE analysis of proteome changes accompanying large resveratrol production by grapevine (Vitis vinifera cv. Gamay) cell cultures in response to methyl-ß-cyclodextrin and methyl jasmonate elicitors.

We had previously shown that Vitis vinifera cv. Gamay cell suspension accumulates extracellularly large amounts of the phytoalexin trans-resveratrol (tR) in response to elicitation with methylated cyclodextrins (MBCD), which can be triplicated when the elicitor is combined with methyl jasmonate (MeJA). In parallel, new pathogenesis-related proteins accumulated in the apoplast-like extracellular space. The aim of this study was to investigate changes in the grapevine cell proteome potentially related to tR accumulation in response to the above elicitors. The DIGE technique was used to detect statistically significant changes in the cell's proteome. A total number of 1031 unique spots were detected, 67 of which were de-regulated upon elicitation. Sixty-four spots were successfully identified by nLC-MS/MS database search analysis. The tR biosynthetic pathway enzymes were up-regulated by MBCD alone or combined with MeJA, but not by treatment with MeJA alone, in agreement with tR accumulation pattern. Seven spots contained stilbene synthase encoded by four different isogenes. Likewise, four glutathione-S-transferases, potentially involved in tR trafficking within the cell and across membranes, were up-regulated in the same fashion as stilbene synthases. The relation of other de-regulated proteins with other effects caused by elicitors on grapevine cells, namely defense response and cell growth inhibition, is discussed.

New method to enhance ajmalicine production in Catharanthus roseus cell cultures based on the use of cyclodextrins.

The joint use of cyclodextrins and methyljasmonate, when accompanied by a short exposure to UV, enhanced extracellular ajmalicine accumulation to 1040 ± 26.6 mg/l in suspension cultured cells of Catharanthus roseus. The success of this strategy is due to the use of cyclodextrins, which not only induce ajmalicine biosynthesis but also promote adduct formation. This removes ajmalicine from the medium, reduces feedback inhibition and ajmalicine degradation, and allows its accumulation in the culture medium at elevated concentrations.

Changes of defense proteins in the extracellular proteome of grapevine (Vitis vinifera cv. Gamay) cell cultures in response to elicitors.

In plant cells, elicitors induce defense responses that resemble those triggered by pathogen attack, such as the synthesis of phytoalexins and pathogen-related proteins which accumulate in the extracellular space. In the search for the particular proteins involved in defense responses, we investigated the changes in the extracellular proteome of a grapevine (Vitis vinifera cv. Gamay) cell suspension in response to elicitation with methylated cyclodextrins (MBCD) and methyl jasmonate (MeJA). Twenty-five of the 39 spots differentially expressed in 2-D gels were identified and found to be encoded by 10 different genes: three secretory peroxidases, chitinase-III, beta-1,3-glucanase, thaumatin-like, SGNH plant lipase-like, NtPR27-like, xyloglucan endotransglycosylase and subtilisin-like protease. Most of them belong to the pathogenesis-related type proteins. A new class III secretory basic peroxidase and chitinase III were strongly induced in cultures treated with MBCD alone or combined with MeJA, while cultures treated with MeJA alone displayed a general repression of most of the extracellular proteins. Some of the proteins induced in grapevine cell cultures by MBCD are induced in other species by activators of systemic acquired resistance (SAR), a form of plant immunity. Collectively, the results suggest that treatment with MBCD resembles the effect of SAR induction agents in cell cultures.

Reproducibilidad de las mediciones de radiografías axiales de rótula a 30° / Reproducibility of measurements of axial patellar views at 30°

Introducciónpara conseguir un diagnóstico de las posibles alteraciones femoropatelares se han definido distintos tipos de exploraciones complementarias y gran cantidad de parámetros radiográficos; uno de los más utilizados es la proyección radiográfica axial de rótula a 30°. Objetivovalorar la reproducibilidad de las mediciones patelares en la radiografía axial de rótula a 30°. Material y métodoen 23 pacientes se realizaron 2 radiografías axiales de la misma rótula, en ángulo fijo de 30° y con la misma técnica, con un intervalo entre ambas de 5min. Se utilizaron como mediciones radiográficas los 3 parámetros más usados para medir las alteraciones en la alineación patelofemoral: ángulo patelofemoral lateral de Laurin, desviación lateral de Sasaki y Yagi y el ángulo de congruencia de Merchant. Un mismo observador realizó estas mediciones en todas las radiografías. Se realizó un análisis estadístico de los resultados comparando cada medición en las 2 radiografías y se analizó el coeficiente de correlación intraclase. Resultadosencontramos una concordancia muy buena con un coeficiente de correlación intraclase para los tres ángulos analizados >0,90. Conclusionesla técnica usada tiene un alto grado de reproducibilidad

Introduction: Different types of studies and radiographic parameters have been established to diagnose potential patellofemoral alterations; one of the most commonly used of these is the axial patellar view at 30°. PurposeTo assess the reproducibility of patellar measurements on axial patella X-rays at 30°. Materials and methodsIn 23 patients we performed 2 axial X-rays of the same patella, at a fixed 30° angle; both films were obtained with the same technique, at an interval of 5min between them. The 3 most frequently used parameters were used to measure alterations in patellofemoral alignment: Laurin lateral patellofemoral angle, Sasaki and Yagi lateral deviation and Merchant congruence angle. These measurements were taken by one same observer in all radiographs. A statistical analysis was made of the results, comparing each measurement on the 2 radiographs and analyzing the intraclass correlation coefficient. ResultsWe found very good concordance levels with an intraclass correlation coefficient for the three angles studied greater than 0.90. ConclusionsThe technique described boasts a high degree of reproducibility (AU)

Class III peroxidases in plant defence reactions.

When plants are attacked by pathogens, they defend themselves with an arsenal of defence mechanisms, both passive and active. The active defence responses, which require de novo protein synthesis, are regulated through a complex and interconnected network of signalling pathways that mainly involve three molecules, salicylic acid (SA), jasmonic acid (JA), and ethylene (ET), and which results in the synthesis of pathogenesis-related (PR) proteins. Microbe or elicitor-induced signal transduction pathways lead to (i) the reinforcement of cell walls and lignification, (ii) the production of antimicrobial metabolites (phytoalexins), and (iii) the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Among the proteins induced during the host plant defence, class III plant peroxidases (EC 1.11.1.7; hydrogen donor: H(2)O(2) oxidoreductase, Prxs) are well known. They belong to a large multigene family, and participate in a broad range of physiological processes, such as lignin and suberin formation, cross-linking of cell wall components, and synthesis of phytoalexins, or participate in the metabolism of ROS and RNS, both switching on the hypersensitive response (HR), a form of programmed host cell death at the infection site associated with limited pathogen development. The present review focuses on these plant defence reactions in which Prxs are directly or indirectly involved, and ends with the signalling pathways, which regulate Prx gene expression during plant defence. How they are integrated within the complex network of defence responses of any host plant cell will be the cornerstone of future research.

Mitigation of lethal effects of Karlodinium veneficum and K. armiger on Sparus aurata: changes in haematocrit and plasma osmolality.

Between January and April 2000, several experiments were performed during a Karlodinium spp. proliferation in Alfacs Bay (Ebro delta, NW Mediterranean) to determine the effects of these dinoflagellates on sea bream Sparus aurata cultivated in the area. Moribund fish showed an increase in plasma osmolality together with a decrease in the haematocrit percentage compared to control fish. The efficacy of copper sulphate, hydrogen peroxide, potassium permanganate and formalin against Karlodinium spp. was also tested. None of these treatments had mitigation effects when applied in the presence of fish; on the contrary, lethal effects appeared at lower Karlodinium spp. densities compared to fish control groups. When a lytic agent, such as copper sulphate, was used as a water pre-treatment, in the absence of fish, Karlodinium spp. toxicity was significantly reduced. Protocols for water pre-treatments were studied as a potential tool for combating Karlodinium spp. in fish farms.

Characterization of the last step of lignin biosynthesis in Zinnia elegans suspension cell cultures.

The last step of lignin biosynthesis in Zinnia elegans suspension cell cultures (SCCs) catalyzed by peroxidase (ZePrx) has been characterized. The k(3) values shown by ZePrx for the three monolignols revealed that sinapyl alcohol was the best substrate, and were proportional to their oxido/reduction potentials, signifying that these reactions are driven exclusively by redox thermodynamic forces. Feeding experiments demonstrate that cell wall lignification in SCCs is controlled by the rate of supply of H(2)O(2). The results also showed that sites for monolignol beta-O-4 cross-coupling in cell walls may be saturated, suggesting that the growth of the lineal lignin macromolecule is not infinite.

Two distinct cell sources of H2O2 in the lignifying Zinnia elegans cell culture system.

The use of transdifferentiating Zinnia elegans mesophyll cells has proved useful in investigations of the process of xylem differentiation from cambial derivatives. Cultured mesophyll cells can be induced by external stimuli to proceed through temporally controlled developmental programs which conclude in the formation of single-cell-derived dead vascular tracheids and parenchyma-like elements. However, there is a gap in our knowledge concerning the role played by reactive oxygen species (O(2) (-) and H(2)O(2)) in the development of these vascular elements. In this study, we show by the following four independent and highly selective methods that transdifferentiating Z. elegans mesophyll cells are capable of producing reactive oxygen species: the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay, which monitors O(2) (-) production, and the xylenol orange, 2,7-dichlorofluorescein diacetate, and CeCl(3) assays, which monitor H(2)O(2) production and localization. The joint use of these biochemical (XTT and xylenol orange) assays and cytochemical (2,7-dichlorofluorescein diacetate and CeCl(3)) probes revealed that transdifferentiating Z. elegans mesophyll cells do not show an oxidative burst but live in a strongly oxidative state during the entire culture period. In this state, H(2)O(2) is produced by both tracheary and parenchyma-like elements, the nonlignifying parenchyma-like cells acting quantitatively as the main source. The existence of these two sources of H(2)O(2) in this in vitro cell culture system may be especially relevant during the later stages of tracheary cell wall lignification, in which lignifying tracheary elements become hollow. In the case of differentiating tracheary elements, H(2)O(2) was located in the same place and at the same time as the onset of tracheary element lignification, i.e., at the primary cell wall during secondary thickening, supporting the view that the H(2)O(2) produced by this in vitro culture system is destined for use during lignin biosynthesis.

[Do our patients know the economic cost of medication of the chronic type? Are they interested?]. / Conocen nuestros pacientes el coste económico de la medicación que toman de forma crónica? Les interesa?

OBJECTIVES: To identify how much patients know about the cost of the long-term medication they take and what importance they give to its cost. DESIGN: Descriptive study with a questionnaire to people collecting their prescriptions for long-term treatment. SETTING: Patients of working age and pensioners of our health centre who take medication for chronic illnesses. PARTICIPANTS: 100 patients of working age and 100 pensioners who take medication for chronic illnesses. MEASUREMENTS: They were asked how much they thought each drug they picked up in CLT prescriptions cost. The figure given was compared with the real price. An opinion questionnaire, with 5 questions on the economic aspects of medication, was conducted. RESULTS: Patients of working age knew the price of 64% of their prescriptions with under 25% error. Pensioners only knew the cost of 27% of their prescriptions with under 25% error. 50% of patients of working age were very interested in the cost of what their doctor prescribed, whereas only 30.4% of pensioners were. CONCLUSIONS: Patients of working age know quite accurately how much the drugs they are prescribed for long-term treatment cost, but pensioners know a lot less. Patients believed that doctors were sufficiently sensitive to the cost of prescriptions.

Effect of fosetyl-A1 on peroxidase from grapevine (Vitis vinifera) cells.

Grapevine (Vitis vinifera cv. Monastrell) suspension cell cultures were treated with fosetyl-A1, a widely used systemic fungicide for grapevine diseases caused by oomycetes, and examined at the electron microscope level for peroxidase cytochemistry. The results showed that treatment with fosetyl-A1 provokes an activation of both vacuolar sap and tonoplast-located peroxidase, already described as due to the basic peroxidase isoenzyme, B5, which was previously characterized as a constitutive marker of disease resistance against Plasmopara viticola in axillary bud cultures of Vitis spp. This activation of peroxidase isoenzyme B5, as seen at the electron microscope level, was confirmed by cytophotometric methods, but is in contrast with the unchanged enzyme level determined by biochemical methods. These results suggest a metabolic activation of peroxidase isoenzyme B5 as a consequence of fosetyl-A1 treatment, probably due to an acidification of the vacuole. This response was accompanied by the appearance of myelin-like structures inside the cytoplasm and osmiophylic-bodies inside the mitochondria. However, the latter structural changes cannot easily be related to the above described specific peroxidase response.