Phenolic Compounds from Wild Plant and In Vitro Cultures of Ageratina pichichensis and Evaluation of Their Antioxidant Activity.

Ageratina pichichensis, is commonly used in traditional Mexican medicine. In vitro cultures were established from wild plant (WP) seeds, obtaining in vitro plant (IP), callus culture (CC), and cell suspension culture (CSC) with the objective to determine total phenol content (TPC) and flavonoids (TFC), as well as their antioxidant activity by DPPH, ABTS and TBARS assays, added to the compound's identification and quantification by HPLC, from methanol extracts obtained by sonication. CC showed significantly higher TPC and TFC than WP and IP, while CSC produced 2.0-2.7 times more TFC than WP, and IP produced only 14.16% TPC and 38.8% TFC compared with WP. There were identified compounds such as epicatechin (EPI), caffeic acid (CfA), and p-coumaric acid (pCA) in in vitro cultures that were not found in WP. The quantitative analysis shows gallic acid (GA) as the least abundant compound in samples, whereas CSC produced significantly more EPI and CfA than CC. Despite these results, in vitro cultures show lower antioxidant activity than WP, for DPPH and TBARS WP > CSC > CC > IP and ABTS WP > CSC = CC > IP. Overall, A. pichichensis WP and in vitro cultures produce phenolic compounds with antioxidant activity, especially CC and CSC, which are shown to be a biotechnological alternative for obtaining bioactive compounds.

Enhanced specialized metabolite, trichome density, and biosynthetic gene expression in Stevia rebaudiana (Bertoni) Bertoni plants inoculated with endophytic bacteria Enterobacter hormaechei.

Stevia rebaudiana (Bertoni) Bertoni is a plant of economic interest in the food and pharmaceutical industries due its steviol glycosides (SG), which are rich in metabolites that are 300 times sweeter than sucrose. In addition, S. rebaudiana plants contain phenolic compounds and flavonoids with antioxidant activity. Endophytic bacteria promote the growth and development and modulate the metabolism of the host plant. However, little is known regarding the role of endophytic bacteria in the growth; synthesis of SG, flavonoids and phenolic compounds; and the relationship between trichome development and specialized metabolites in S. rebaudiana, which was the subject of this study. The 12 bacteria tested did not increase the growth of S. rebaudiana plants; however, the content of SG increased with inoculation with the bacteria Enterobacter hormaechei H2A3 and E. hormaechei H5A2. The SG content in leaves paralleled an increase in the density of glandular, short, and large trichome. The image analysis of S. rebaudiana leaves showed the presence of SG, phenolic compounds, and flavonoids principally in glandular and short trichomes. The increase in the transcript levels of the KO, KAH, UGT74G1, and UGT76G1 genes was related to the SG concentration in plants of S. rebaudiana inoculated with E. hormaechei H2A3 and E. hormaechei H5A2. In conclusion, inoculation with the stimulating endophytes E. hormaechei H2A3 and E. hormaechei H5A2 increased SG synthesis, flavonoid content and flavonoid accumulation in the trichomes of S. rebaudiana plants.

An integrated biomarker approach: Non-monotonic responses to cadmium exposure in the suckermouth catfish Hypostomus plecostomus.

Environmental risk assessment in aquatic ecosystems typically uses biomarkers to detect interactions between potential hazards and biological systems. Next to knowing environmental contaminant levels in tissues and the environment, it is important to link to potentially deleterious effects at higher levels of biological organization such as biochemistry, physiology, and overall health status. In this laboratory study we assessed the toxicity of waterborne cadmium (Cd) over an exposure range of 0 - 100 µg l-1 for nine days to the loricariid suckermouth catfish Hypostomus plecostomus. We evaluated the integrated response of the fish at the biochemical to physiological level by means of a suite of tissue biomarkers of exposure and effects, including Cd concentrations in gills, liver metallothioneins (MT) and cholinesterase activity (ChE) in brain, before and after the inhibition of the alkaloid eserine, as well as whole-fish resting oxygen consumption rates and ingestion rate. Tissue biomarkers (MT and ChE) showed a non-monotonic relationship, with maximum/minimum responses at intermediate doses. i.e. 10 and 50 µg l-1, whereas biomarker responses of fish exposed at 100 µg l-1 more closely resembled biomarker responses seen at lower concentrations (< 10 µg l-1). Conversely, the oxygen consumption rate peaked at 100 µg l-1, suggesting a higher metabolic cost for higher metal exposure, with no significant correlation with fish body condition and food intake. Integrated Biomarker Response (IBR) values peaked at the intermediate exposure concentration of 50 µg l-1 Cd. The non-monotonic dose-response of the biochemical biomarkers of exposure, together with the higher metabolic rates of fish exposed to 50 - 100 µg l-1 of Cd and the non-significant effects on the more relevant physiological and histological variables suggests that H. plecostomus is capable of biochemically and physiologically regulating moderately high Cd concentrations, thus representing a suitable indicator organism to monitor metal pollution by Cd.

Production of indole-3-acetic acid by Bacillus circulans E9 in a low-cost medium in a bioreactor.

Bacillus circulans E9 (now known as Niallia circulans) promotes plant growth-producing indole-3-acetic acid (IAA), showing potential for use as a biofertilizer. In this work, the use of a low-cost medium containing industrial substrates, soybean, pea flour, Solulys, Pharmamedia, yeast extract, and sodium chloride (NaCl), was evaluated as a substitute for microbiological Luria Broth (LB) medium for the growth of B. circulans E9 and the production of IAA. In Erlenmeyer flasks with pea fluor medium (PYM), the maximum production of IAA was 7.81 ± 0.16 µg mL-1, while in microbiological LB medium, it was 3.73 ± 0.15 µg mL-1. In addition, an oxygen transfer rate (OTR) of 1.04 kg O2 m-3 d-1 allowed the highest bacterial growth (19.3 ± 2.18 × 1010 CFU mL-1) and IAA production (10.7 µg mL-1). Consequently, the OTR value from the flask experiments was used to define the conditions for the operation of a 1 L stirred tank bioreactor. The growth and IAA production of B. circulans cultured in a bioreactor with PYM medium were higher (8 and 1.6 times, respectively) than those of bacteria cultured in Erlenmeyer flasks. IAA produced in a bioreactor by B. circulans was shown to induce the root system in Arabidopsis thaliana, similar to synthetic IAA. The results of this study demonstrate that PYM medium may be able to be used for the mass production of B. circulans E9 in bioreactors, increasing both bacterial growth and IAA production. This low-cost medium has the potential to be employed to grow other IAA-producing bacterial species.

Engineering Considerations to Produce Bioactive Compounds from Plant Cell Suspension Culture in Bioreactors.

The large-scale production of plant-derived secondary metabolites (PDSM) in bioreactors to meet the increasing demand for bioactive compounds for the treatment and prevention of degenerative diseases is nowadays considered an engineering challenge due to the large number of operational factors that need to be considered during their design and scale-up. The plant cell suspension culture (CSC) has presented numerous benefits over other technologies, such as the conventional whole-plant extraction, not only for avoiding the overexploitation of plant species, but also for achieving better yields and having excellent scaling-up attributes. The selection of the bioreactor configuration depends on intrinsic cell culture properties and engineering considerations related to the effect of operating conditions on thermodynamics, kinetics, and transport phenomena, which together are essential for accomplishing the large-scale production of PDSM. To this end, this review, firstly, provides a comprehensive appraisement of PDSM, essentially those with demonstrated importance and utilization in pharmaceutical industries. Then, special attention is given to PDSM obtained out of CSC. Finally, engineering aspects related to the bioreactor configuration for CSC stating the effect of the operating conditions on kinetics and transport phenomena and, hence, on the cell viability and production of PDSM are presented accordingly. The engineering analysis of the reviewed bioreactor configurations for CSC will pave the way for future research focused on their scaling up, to produce high value-added PDSM.

Photosynthetic performance and stevioside concentration are improved by the arbuscular mycorrhizal symbiosis in Stevia rebaudiana under different phosphate concentrations.

In plants, phosphorus (P) uptake occurs via arbuscular mycorrhizal (AM) symbiosis and through plant roots. The phosphate concentration is known to affect colonization by AM fungi, and the effect depends on the plant species. Stevia rebaudiana plants are valuable sources of sweetener compounds called steviol glycosides (SGs), and the principal components of SGs are stevioside and rebaudioside A. However, a detailed analysis describing the effect of the phosphate concentration on the colonization of AM fungi in the roots and the relationship of these factors to the accumulation of SGs and photochemical performance has not been performed; such an analysis was the aim of this study. The results indicated that low phosphate concentrations (20 and 200 µM KH2PO4) induced a high percentage of colonization by Rhizophagus irregularis in the roots of S. rebaudiana, while high phosphate concentrations (500 and 1,000 µM KH2PO4) reduced colonization. The morphology of the colonization structure is a typical Arum-type mycorrhiza, and a mycorrhiza-specific phosphate transporter was identified. Colonization with low phosphate concentrations improved plant growth, chlorophyll and carotenoid concentration, and photochemical performance. The transcription of the genes that encode kaurene oxidase and glucosyltransferase (UGT74G1) was upregulated in colonized plants at 200 µM KH2PO4, which was consistent with the observed patterns of stevioside accumulation. In contrast, at 200 µM KH2PO4, the transcription of UGT76G1 and the accumulation of rebaudioside A were higher in noncolonized plants than in colonized plants. These results indicate that a low phosphate concentration improves mycorrhizal colonization and modulates the stevioside and rebaudioside A concentration by regulating the transcription of the genes that encode kaurene oxidase and glucosyltransferases, which are involved in stevioside and rebaudioside A synthesis in S. rebaudiana.

Production of anti-inflammatory compounds in Sphaeralcea angustifolia cell suspension cultivated in stirred tank bioreactor.

Sphaeralcea angustifolia is a plant used for the treatment of inflammatory processes. Scopoletin, tomentin, and sphaeralcic acid were identified as the compounds with anti-inflammatory and immunomodulatory effects. Successful establishment of the cell culture in Erlenmeyer flasks has been reported previously. The aim of this study was to evaluate the ability of cells in suspension from S. angustifolia grown in a stirred tank bioreactor and demonstrate their capacity to produce bioactive compounds. Cells in suspension grown at 200 rpm reached a maximal cell biomass in dry weight at 19.11 g/L and produced 3.47 mg/g of sphaeralcic acid. The mixture of scopoletin and tomentin was only detected at the beginning of the culture (12.13 µg/g). Considering that the profile of dissolved oxygen during the cultures was lesser than 15%, it is possible that the low growth at 100 rpm could be due to oxygen limitations or to cell sedimentation. At 400 rpm, a negative effect on cell viability could be caused by the increase in the hydrodynamic stress, including the impeller tip, average shear rate, and Reynolds number. The sphaeralcic acid content in the cell suspension of S. angustifolia obtained in the bioreactor was two orders of magnitude greater than that reported for the culture grown in Erlenmeyer flasks.

Trends in biomarkers, biotic indices, and fish population size revealed contrasting long-term effects of recycled water on the ecological status of a Mediterranean river.

Recycled water is important for maintaining river flow in semi-arid regions. However, it has ecological risk, as suggested by comparison of habitat and white and red blood cell count in two wild fish species (Barbus meridionalis and Squalius laietanus) before and after an input of recycled water in Ripoll River (NE Spain) in 2009. Due to the lack of normal ranges for blood variables in wild fish, we surveyed seasonally the same river reaches in 2013 to test if blood alterations from 2009 compromised the viability of the fish populations. By examining other indicators of river health in baseline and polluted sites (fish abundance, mass-length relationships, and community indices in fish, diatoms and invertebrates), we tested for the superior utility of blood tests in biomonitoring. The comparison of water quality and scores of diatoms and invertebrate indices between polluted and reference sites showed that polluted sites improved from 2009 to 2013. The abundance of B. meridionalis also increased in polluted sites, but that of S. laietanus declined in 2013 compared to 2009. These results contrast with results of blood analyses in 2009, which suggested that B. meridionalis was more seriously affected by pollution than S. laietanus. The fish index did not reveal the risk of recycled water to fish health, whereas fish mass-length relationships suggested that S. laietanus individuals in 2013 had a better body condition in polluted than in reference sites. Given that the two fish species had opposite results in reference sites, and that the physical habitat was more suitable for B. meridionalis in polluted sites in 2013 than was for S. laietanus, trends in population size are not only explained by pollution. The role of phenology is suggested by peaks in blood disorders during the breeding season. However, more long-term studies combining indicators of river health at the individual and community scales are needed to fully assess the ecological risk of recycled water in this river. These studies will also help to develop blood tests as reliable health indicators in wild fish populations.

Cell Culture of Bursera linanoe in a Stirred Tank Bioreactor for Production of Linalool and Linalyl Acetate.

Bursera linanoe cell suspension cultures were initiated from callus grown in Murashige and Skoog medium supplemented with naphthalene acetic acid (3.0 mg L⁻¹) and 6-benzylaminopurine (0.5 mg L⁻¹). In flasks, B. linanoe cell cultures grew over a 9 day period, reaching a maximum biomass of 11.16 g DW L⁻¹. Throughout the growth phase, cell viability was constant at 60 - 70%. In contrast, B. linanoe cells growing in a bioreactor achieved a maximum biomass of 22.26 g DW L⁻¹ (after 7 days), and cell viability was constant at 75 - 85%. Production of linalool and linalyl acetate in the bioreactor (3.02 and 2.40 mg g⁻¹ DW, respectively) was significantly greater than that achieved from cells in flask cultures (1.05 and 0.97 mg g⁻¹ DW, respectively). B. linanoe cell suspension culture has potential as an alternative method for the production of essential oils.

A trait-based approach reveals the feeding selectivity of a small endangered Mediterranean fish.

Functional traits are growing in popularity in modern ecology, but feeding studies remain primarily rooted in a taxonomic-based perspective. However, consumers do not have any reason to select their prey using a taxonomic criterion, and prey assemblages are variable in space and time, which makes taxon-based studies assemblage-specific. To illustrate the benefits of the trait-based approach to assessing food choice, we studied the feeding ecology of the endangered freshwater fish Barbus meridionalis. We hypothesized that B. meridionalis is a selective predator which food choice depends on several prey morphological and behavioral traits, and thus, its top-down pressure may lead to changes in the functional composition of in-stream macroinvertebrate communities. Feeding selectivity was inferred by comparing taxonomic and functional composition (13 traits) between ingested and free-living potential prey using the Jacob's electivity index. Our results showed that the fish diet was influenced by 10 of the 13 traits tested. Barbus meridionalis preferred prey with a potential size of 5-10 mm, with a medium-high drift tendency, and that drift during daylight. Potential prey with no body flexibility, conical shape, concealment traits (presence of nets and/or cases, or patterned coloration), and high aggregation tendency had a low predation risk. Similarly, surface swimmers and interstitial taxa were low vulnerable to predation. Feeding selectivity altered the functional composition of the macroinvertebrate communities. Fish absence favored taxa with weak aggregation tendency, weak flexibility, and a relatively large size (10-20 mm of potential size). Besides, predatory invertebrates may increase in fish absence. In conclusion, our study shows that the incorporation of the trait-based approach in diet studies is a promising avenue to improve our mechanistic understanding of predator-prey interactions and to help predict the ecological outcomes of predator invasions and extinctions.

Ecological relevance of biomarkers in monitoring studies of macro-invertebrates and fish in Mediterranean rivers.

Mediterranean rivers are probably one of the most singular and endangered ecosystems worldwide due to the presence of many endemic species and a long history of anthropogenic impacts. Besides a conservation value per se, biodiversity is related to the services that ecosystems provide to society and the ability of these to cope with stressors, including climate change. Using macro-invertebrates and fish as sentinel organisms, this overview presents a synthesis of the state of the art in the application of biomarkers (stress and enzymatic responses, endocrine disruptors, trophic tracers, energy and bile metabolites, genotoxic indicators, histopathological and behavioural alterations, and genetic and cutting edge omic markers) to determine the causes and effects of anthropogenic stressors on the biodiversity of European Mediterranean rivers. We also discuss how a careful selection of sentinel species according to their ecological traits and the food-web structure of Mediterranean rivers could increase the ecological relevance of biomarker responses. Further, we provide suggestions to better harmonise ecological realism with experimental design in biomarker studies, including statistical analyses, which may also deliver a more comprehensible message to managers and policy makers. By keeping on the safe side the health status of populations of multiple-species in a community, we advocate to increase the resilience of fluvial ecosystems to face present and forecasted stressors. In conclusion, this review provides evidence that multi-biomarker approaches detect early signs of impairment in populations, and supports their incorporation in the standardised procedures of the Water Frame Work Directive to better appraise the status of European water bodies.

Production of dihydroxylated betalains and dopamine in cell suspension cultures of Celosia argentea var. plumosa.

Betalains are plant pigments of hydrophilic nature with demonstrated chemopreventive potential in cancer cell lines and animal models. Among the betalains, those containing an aromatic moiety with two free hydroxyl groups possess the strongest antioxidant and free radical scavenging activities. The betaxanthins dopaxanthin and miraxanthin V and the betacyanins betanidin and decarboxy-betanidin are the only natural betalains with catecholic substructures. These four pigments have been produced in cell cultures established from hypocotyls of the plant Celosia argentea. Two stable and differentially colored cell lines, yellow and red, were maintained on Murashige and Skoog medium supplemented with the plant growth regulators 6-benzylaminopurine (6.66 µM) and 2,4-dichlorophenoxyacetic acid (6.79 µM). Derived suspension cultures showed increased production of dihydroxylated betalains in the cells and secreted to the medium with a maximum reached after 8 days of culture. In addition, precursor molecules betalamic acid and dopamine, with content up to 42.08 mg/g dry weight, were also obtained. The joint presence of the bioactive betalains together with the production of dopamine and betalamic acid show the ability of cell cultures of C. argentea to become a stable source of valuable phytochemicals.

Metal concentration in water, sediment and four fish species from Lake Titicaca reveals a large-scale environmental concern.

Although intensive mining activity and urban sewage discharge are major sources of metal inputs to Lake Titicaca, the risk posed by metal pollution to wildlife and human populations has been poorly studied. In this study we compared the concentrations of Cu, Zn, Cd, Hg, Pb, Co, and Fe in water, sediment, and two tissues (liver and muscle) of four fish species (Odontesthes bonariensis, Orestias luteus, Orestias agassii, and Trichomycterus rivulatus) across important fishery areas in Lake Titicaca. The concentration of Pb in water at the discharge sites of the main rivers and of most elements, with the exception of Co and Fe, in all fish collected in this study exceeded the safety thresholds established by international legislation. The highest metal concentrations were observed in benthopelagic species, and liver tissue was identified as the main depository for all metals with the exception of mercury. The metal bioaccumulation pattern in fish was weakly related to the metal concentrations in the environment with the exception of Hg at the most polluted location, partly explained by the different metabolic role of essential and non-essential elements and the influence of other factors such as species' ecology and individual traits in the bioaccumulation of most metals. As metal pollution extended across the study area and high metal concentrations were detected in all four fish species, we urge the authorities to enforce legislation for water and fish consumption and to evaluate the effects of metal pollution on fish health.

Metal concentrations and pathological responses of wild native fish exposed to sewage discharge in a Mediterranean river.

The requirements of the Water Framework Directive suggest the need for further research to test and develop sensitive tools that will allow freshwater managers to detect impacts on fish communities. Diagnostic refinement often encompasses the use of lethal diagnostic tools that are incompatible with the conservation of native ichthyofauna. Here we determine the metal concentration and the pathological response of Squalius laietanus exposed to sewage discharges in the Ripoll river (north-eastern Spain), and compare these findings with our previous studies on Barbus meridionalis using lethal and non-lethal diagnostic tools. Metals concentrations (Zn, Cu, Pb, Hg, Fe, Cd and Ni) were determined in liver and muscle. A complete blood cell profile (haematocrit, haemoglobin, differential leukocyte count, erythrocytic nuclear abnormalities, erythrocytes in division and the development stage of erythrocytes) was used as a non-lethal diagnostic tool to determine early warning signs of disease in these two fish species. As the reference range for these haematological variables is lacking, liver histology, calculation of body condition (CF) and organosomatic indices (HSI and GSI) were employed to support the findings of the blood analyses. Compared to our previous results on B. meridionalis, S. laietanus appeared to have fewer pathological responses than B. meridionalis under the environmental conditions measured and the fish size range examined in this study. Both species showed a similar bioaccumulation pattern, but B. meridionalis stored high Hg and Cu concentrations in muscle and liver, respectively. Hg, Cu and Pb concentrations in fish tissues exceeded the thresholds of European and Spanish legislation. Our findings pinpoint the potential suitability of the blood variables determined in the health diagnoses of these species. Further research will be necessary to establish the natural variability of these and other haematological variables to convert haematology into a sensitive and useful non-lethal diagnostic tool in wild fish populations.

Metal bioaccumulation in the Mediterranean barbel (Barbus meridionalis) in a Mediterranean river receiving effluents from urban and industrial wastewater treatment plants.

Although sewage treatment plants (STPs) play a crucial role in maintaining the water quality and flow of Mediterranean rivers, particularly during drought periods, few studies have addressed their impact on aquatic fauna. Here we analyzed the role of STPs as a source of metals in the Ripoll River, a heavily urbanized and industrialized watercourse with a long history of anthropogenic disturbance. For this purpose, we measured iron, mercury, cadmium, zinc, lead, nickel and copper accumulation in the liver and muscle of the Mediterranean barbel, Barbus meridionalis and also the concentrations of these metals in the river water. Industrial and urban sewage treatment plants are source of metals in Ripoll River but the former mainly increases Zn and Ni values. Significant differences in metal bioaccumulation between reference and polluted sites were detected. Nevertheless, there was only a significant positive relationship between bioaccumulation of Cu and Hg, and their concentration in water. In addition, the lead concentration in fish was not clearly associated with the presence of STPs. On the basis of morphometric parameters, the hepato-somatic index was the only one denoting significant differences between polluted and references sites. Given that fish are key elements in food webs, recreational fishing is practice in this area and that river water is used for agricultural purposes, we recommend long-term studies to analyze the impact of metal pollution in this river.

Changes in non-specific biomarkers in the Mediterranean barbel (Barbus meridionalis) exposed to sewage effluents in a Mediterranean stream (Catalonia, NE Spain).

The effluents from sewage treatment plants strongly influence the water quality and flow of Mediterranean streams. These effluents play a crucial role in maintaining the aquatic communities of these ecosystems, particularly in the absence of natural flow resulting from climate constraints or intensive water use. To detect the ecological effects of these effluents, we used non-lethal biomarkers in Barbus meridionalis and traditional assessment protocols in the Ripoll River (NE of Spain). Our results demonstrate the utility of haematological parameters. In comparison with fish at reference sites, we detected a decrease in haematocrit and haemoglobin, neutrophilia, lymphopenia, monocytosis, a rise in the nucleo-cytoplasmatic ratio of erythrocytes and an increase in the frequency of abnormal, immature and senescent erythrocytes. Many haematological parameters correlated significantly with the environmental parameters measured. In addition to these haematological alterations, histopathological examination also revealed damage in fish livers but no impact was detected by the regional index of biotic integrity using fish as bioindicators. To the best of our knowledge, this study is the first to report this battery of haematological parameters as biomarkers on a freshwater fish in a Mediterranean stream. In a region in which more than 50% of native fish species are classified as endangered or vulnerable, there is an urgent need to find biomarkers that do not harm the animals.

Transformed cell suspension culture of Galphimia glauca producing sedative nor-friedelanes.

The Mexican species GALPHIMIA GLAUCA (Cav.) Kuntze (Malphigiaceae) synthesises a family of sedative and anxiolytic nor-secofriedelanes, designated as galphimines. These active principles accumulate at low concentration in the aerial parts of plants from wild populations. Transformed calluses and cell suspension cultures of this species were established in order to induce a greater production of nor-friedelanes. The cell suspension line GgBa was selected and grown over a period of two years of continuous subculturing in MS nutrient medium in the absence of growth regulators. PCR and Southern blot analyses were employed in order to confirm that the ROL A gene had been integrated into the plant genome. Batch cultures of the GgBa cell line were grown over a 32-day period and first-order growth kinetics was observed, reaching a specific growth rate (micro) of 0.13 d (-1). The production of glaucacetalin A ( 10), a triterpenoid related to the known galphimines, was quantified in the nutrient medium by HPLC. The transformed cell suspension culture GgBa also synthesised a novel nor-friedelane, given the name glaucacetalin D ( 13). High-resolution spectroscopic and spectrometric techniques were employed to elucidate the structure of 13. This triterpene has never been observed in wild plant tissues or in other IN VITRO cultures. Maslinic acid ( 14) was identified in cell biomasses. The triterpene production of the cell line GgBa was as follows: glaucacetalin A, 2.7 mg/L; glaucacetalin D, 2.9 mg/L and maslinic acid, 2.4 mg/g dry weight. The sedative activity of compounds 10 and 13 was demonstrated in ICR mice by using the sodium pentobarbital-induced hypnosis model. No cytotoxicity of 10 and 13 was exhibited against KB, MCF-7 and HF6 human cancer cell lines.

Las proteínas arabinogalactanos en cultivos de células vegetales / Arabinogalactan proteins in plant cell cultures / As proteínas arabinogalactanos em cultivos de células vegetais

Las proteínas arabinogalactanos (AGPs) son macromoléculas que se encuentran prácticamente en todos los órganos de las plantas, siendo asociadas con varios aspectos del crecimiento y desarrollo vegetal. Estas moléculas se caracterizan bioquímicamente por contener carbohidratos y proteínas en relación 9:1. El carbohidrato está compuesto principalmente por arabinogalactanos tipo II; mientras que la parte proteica está organizada en dominios que definen a las AGPs como clásicas o no clásicas. Las primeras se caracterizan además por presentar una secuencia C-terminal que predice la incorporación de un grupo glicosilfosfatidilinositol (GFI), que permite su unión a la membrana plasmática. En cultivos de células vegetales se reportan varias especies que liberan AGPs al medio de cultivo. Se presenta una revisión de las características bioquímicas de las AGPs liberadas al medio y de las propuestas sobre los mecanismos bioquímicos y celulares por los cuales las AGPs participan en la diferenciación y crecimiento de las células vegetales. Los cultivos de células liberan al medio de cultivo AGPs clásicas y no clásicas, y se propone que podrían provenir de la membrana plasmática o la pared celular. Las AGPs intervienen en el control del crecimiento celular, además de estar relacionadas con la embriogénesis somática y la organogénesis, procesos de diferenciación celular importantes en los sistemas de micropropagación de plantas. El mecanismo bioquímico por el cual las AGPs participan en el crecimiento celular y la diferenciación implica que éstas, o los productos de su degradación, quizás actúen como moléculas de señalización.

The arabinogalactan proteins (AGPs) are macromolecules found in practically all plant organs, being associated with several aspects of the plant growth and development. These molecules contain carbohydrates and proteins in a 9:1 relation. The carbohydrate moiety is composed mainly of type II arabinogalactans, whereas the protein has particular amino acid domains that allow classifying the AGPs into two groups, classical and non-classical. In addition, the former are characterized by a C-terminal tail that predicts the incorporation of a glycosylphosphatidylinositol group (GPI) that allows the attachment of the AGPs to the plasma membrane. Plant cell cultures of several species release AGPs into the culture medium. The biochemical characteristics of the AGPs released into the medium, and the proposed biochemical and cellular mechanisms by which AGPs participate in plant cell differentiation and growth are reviewed. The plant cells release classical as well as non-classical AGPs into the culture medium. The origin of these AGPs could likely be the plasma membrane or the cell wall. They are involved in the control of cellular growth and differentiation processes, aspects that have fundamental importance in the induction of somatic embryogenesis and organogenesis, key steps in plant micropropagation programs. The biochemical mechanism by which the AGPs participate in cell growth and differentiation implies that the AGPs or their degradation products participate like signal molecules.

As proteínas arabinogalactanos (AGPs) são macromoléculas que se encontram praticamente em todos os órgãos das plantas, sendo associadas com vários aspectos do crescimento e desenvolvimento vegetal. Estas moléculas se caracterizam bioquímicamente por conter carboidratos e proteínas em relação 9:1. O carboidrato está composto principalmente por arabinogalactanos tipo II; enquanto que a parte protéica está organizada em domínios que definem as AGPs como clássicas ou não clássicas. As primeiras se caracterizam, além disso, por apresentar uma sequência C-terminal que prediz a incorporação de um grupo glicosilfosfatidilinositol (GFI), que permite sua união à membrana plasmática. Em cultivos de células vegetais se relatam várias espécies que liberam AGPs ao meio de cultivo. Apresenta-se uma revisão das características bioquímicas das AGPs liberadas ao meio e das propostas sobre os mecanismos bioquímicos e celulares pelos quais as AGPs participam na diferenciação e crescimento das células vegetais. Os cultivos de células liberam ao meio de cultivo AGPs clássicas e não clássicas, e se propõe que poderiam provir da membrana plasmática ou da parede celular. As AGPs intervêm no controle do crescimento celular, além de estar relacionadas com a embriogênese somática e a organogênese, processos de diferenciação celular importantes nos sistemas de micropropagação de plantas. O mecanismo bioquímico pelo qual as AGPs participam no crescimento celular e a diferenciação, implica que estas, ou os produtos de sua degradação, talvez atuem como moléculas de sinalização.

La agregación celular en la producción de metabolitos secundarios en cultivos vegetales in vitro / Celular aggregation in secondary metabolite production in in vitro plant cell cultures

El cultivo de células vegetales es una alternativa biotecnológica para la producción de metabolitos secundarios. Sin embargo, la productividad de los sistemas in vitro es menor a la obtenida de plantas. En esta revisión se ilustra la diferenciación y compartamentalización celular como eventos necesarios para la síntesis de metabolitos secundarios en las plantas. Se discute la inducción de la agregación celular en los cultivos in vitro como una de las estrategias para favorecer la acumulación de estos compuestos químicos. Este efecto positivo podría ser explicado como consecuencia de la formación de estructuras morfogénicas y/o por una condición de estrés por limitaciones de oxígeno al interior de los agregados. Finalmente, se muestra que la combinación de la agregación con otras estrategias tales como la selección de líneas celulares, la elicitación y la adición de precursores constituye una alternativa para desarrollar bioprocesos a partir de células vegetales in vitro para la producción de compuestos químicos de alto valor agregado.

Hydrodynamic stress induces monoterpenoid oxindole alkaloid accumulation by Uncaria tomentosa (Willd) D. C. cell suspension cultures via oxidative burst.

Uncaria tomentosa cell suspension cultures were grown in a 2-L stirred tank bioreactor operating at a shear rate gamma(.)(avg)=86 s(-1). The cultures showed an early monophasic oxidative burst measured as H2O2 production (2.15 micromol H2O2 g(-1) dw). This response was followed by a transient production of monoterpenoid oxindole alkaloids (178 +/- 40 microg L(-1) at 24 h). At the stationary phase (144 h), the increase of the shear rate gamma(.)(avg) up to 150 s(-1) and/or oxygen tension up to 85% generated H2O2, restoring oxindole alkaloid production. U. tomentosa cells cultured in Erlenmeyer flasks also exhibited the monophasic oxidative burst but the H2O2 production was 16-fold lower and the alkaloids were not detected. These cells exposed to H2O2 generated in situ produced oxindole alkaloids reaching a maximum of 234 +/- 40 microg L(-1). A positive correlation was observed between the oxindole alkaloid production and the endogenous H2O2 level. On the other hand, addition of 1 microM diphenyleneiodonium (NAD(P)H oxidase inhibitor) or 10 microM sodium azide (peroxidases inhibitor) reduced both H2O2 production and oxindole alkaloids build up, suggesting that these enzymes might play a role in the oxidative burst induced by the hydrodynamic stress.