Discovery and structures of the cyclotides: novel macrocyclic peptides from plants

Circular disulfide-rich polypeptides were unknown a decade ago but over recent years a large family of such molecules has been discovered, which we now refer to as the cyclotides. They are typically about 30 amino acids in size, contain an N- to C-cyclised backbone and incorporate three disulfide bonds arranged in a cystine knot motif. In this motif, an embedded ring in the structure formed by two disulfide bonds and their connecting backbone segments is penetrated by the third disulfide bond. The combination of this knotted and strongly braced structure with a circular backbone renders the cyclotides impervious to enzymatic breakdown and makes them exceptionally stable. This article describes the discovery of the cyclotides in plants from the Rubiaceae and Violaceae families, their chemical synthesis, folding, structural characterisation, and biosynthetic origin. The cyclotides have a diverse range of biological applications, ranging from uterotonic action, to anti-HIV and neurotensin antagonism. Certain plants from which they are derived have a history of uses in native medicine, with activity being observed after oral ingestion of a tea made from the plants. This suggests the possibility that the cyclotides may be orally bioavailable. They therefore have a range of potential applications as a stable peptide framework.

The influence of key chemical constituents in activated sludge on surface and flocculating properties

This paper examines the influence of the chemical constituents of activated sludge and extracted extracellular polymeric substances (EPS) on the surface properties, hydrophobicity, surface charge (SC) and flocculating ability (FA) of activated sludge floes. Activated sludge samples from 7 different full-scale wastewater treatment plants were examined. Protein and humic substances were found to be the dominant polymeric compounds in the activated sludges and the extracted EPS, and they significantly affected the FA and surface properties, hydrophobicity and SC, of the sludge floes. The polymeric compounds proteins, humic substances and carbohydrates in the sludge floes and the extracted EPS contributed to the negative SC, but correlated negatively to the hydrophobicity of sludge floes. The quantity of protein and carbohydrate within the sludge and the extracted EPS was correlated positively to the FA of the sludge floes, while increased amounts of humic substances resulted in lower FA. In contrast, increased amounts of total extracted EPS had a negative correlation to FA. The results reveal that the quality and quantity of the polymeric compounds within the sludge floes is more informative, with respect to understanding the mechanisms involved in flocculation, than if only the extracted EPS are considered. This is an important finding as it indicates that extracting EPS may be insufficient to characterise the EPS. This is due to the low extraction efficiency and difficulties involved in the separation of EPS from other organic compounds. Correlations were observed between the surface properties and FA of the sludge floes., This confirms that the surface properties of the, sludge flocs play an important role in the bioflocculation process but that also other interactions like polymer entanglement are important. (C) 2002 Elsevier Science Ltd. All rights reserved.

Chemical sterilization in in vitro propagation of Arundina bambusifolia Lindl. and Epidendrum ibaguense Kunth

There is a great demand for simpler and less costly laboratory techniques and for more accessible procedures for orchid breeders who do not have the necessary theoretical basis to use the traditional seed and clone production methods of orchids in vitro. The aim of this study was to assess the use of sodium hypochlorite (NaClO) as a decontaminant in the process of inoculating adult orchid explants of Arundina bambusifolia and Epidendrum ibaguenses. Solutions of NaClO (1.200, 2.400, 3.600, 4.800 and 6.000 mg L-1 - equivalent to 50, 100, 150, 200 and 250 mL L-1 of commercial bleach - CB) were sprayed on the explants (1.0 mL) and the culture medium (GB5), in the presence or absence of activated charcoal (2 g L-1). The explants used were nodal segments of field-grown adult plants. The procedures for inoculating the explants were conducted outside the laminar flow chamber (LFC), except for the control treatment (autoclaved medium and explant inoculation inside the LFC). The best results for fresh weight yield, height and number of shoots were obtained using NaClO in solution at 1.200 mg L-1 (equivalent to 50 mL L-1 commercial bleach) with activated charcoal in the culture medium. Fresh weight figures were 1.10 g/jar for Arundina bambusifolia and 0.16 g/jar for Epidendrum ibaguenses. Spraying the NaClO solutions controls the contamination of the culture medium already inoculated with the explants.

Sugarcane productivity correlated with physical-chemical attributes to create soil management zone

The socioeconomic importance of sugar cane in Brazil is unquestionable because it is the raw material for the production of ethanol and sugar. The accurate spatial intervention in the management of the crop, resulting zones of soil management, increases productivity as well as its agricultural yields. The spatial and Person's correlations between sugarcane attributes and physico-chemical attributes of a Typic Tropustalf were studied in the growing season of 2009, in Suzanápolis, State of São Paulo, Brazil (20°28'10'' S lat.; 50°49'20'' W long.), in order to obtain the one that best correlates with agricultural productivity. Thus, the geostatistical grid with 120 sampling points was installed to soil and data collection in a plot of 14.6 ha with second crop sugarcane. Due to their substantial and excellent linear and spatial correlations with the productivity of the sugarcane, the population of plants and the organic matter content of the soil, by evidencing substantial correlations, linear and spatial, with the productivity of sugarcane, were indicators of management zones strongly attached to such productivity.

Chemical treatment of papaya seeds aiming at long-term storage and control of damping off

Damping off is a nursery disease of great economic importance in papaya and seed treatment may be an effective measure to control. The aim of this work was to evaluate the quality of papaya seeds treated with fungicides and stored under two environmental and packaging conditions. Additionally, the efficiency of fungicide treatments in the control of damping-off caused by Rhizoctonia solani was evaluated. Papaya seeds were treated with the fungicides Captan, Tolylfluanid and the mixture Tolylfluanid + Captan (all commercial wettable powder formulations). Seeds of the control group were not treated. The seeds were stored for nine months in two conditions: packed in aluminum coated paper and kept at 7 ± 1ºC and in permeable kraft paper and kept in non-controlled environment. At the beginning of the storage and every three months the seed quality (germination and vigor tests), emergence rate index, height, dry mass and damping of plants in pre and post-emergence (in contaminated substrate and mycelia-free substrate) were analyzed. Both storage conditions as well as the fungicide treatments preserved the germination and seed vigor. In the infested substrate, seedling emergence was favored by fungicides, but in post-emergence, fungicides alone did not control the damping off caused by R. solani. Symptoms of damping off were not observed in the clean substrate. The results showed that the fungicide treatments may be used to pretreat papaya seed for long-term storage and commercialization.

Mathematical modelling of supercritical CO2 extraction of volatile oils from aromatic plants

The modelling of the experimental data of the extraction of the volatile oil from six aromatic plants (coriander, fennel, savoury, winter savoury, cotton lavender and thyme) was performed using five mathematical models, based on differential mass balances. In all cases the extraction was internal diffusion controlled and the internal mass transfer coefficienty (k(s)) have been found to change with pressure, temperature and particle size. For fennel, savoury and cotton lavender, the external mass transfer and the equilibrium phase also influenced the second extraction period, since k(s) changed with the tested flow rates. In general, the axial dispersion coefficient could be neglected for the conditions studied, since Peclet numbers were high. On the other hand, the solute-matrix interaction had to be considered in order to ensure a satisfactory description of the experimental data.

Mechanical performance of concrete made with aggregates from construction and demolition waste recycling plants

This research aims at analysing the mechanical performance of concrete with recycled aggregates (RA) from construction and demolition waste (CDW) from various locations in Portugal. First the characteristics of the various aggregates (natural and recycled) used in the production of concrete were thoroughly analysed. The composition of the RA was determined and several physical and chemical tests of the aggregates were performed. In order to evaluate the mechanical performance of concrete, compressive strength (in cubes and cylinders), splitting tensile strength, modulus of elasticity and abrasion resistance tests were performed. Concrete mixes with RA from CDW from several recycling plants were evaluated, in order to understand the influence that the RA's collection point, and consequently their composition, has on the characteristics of the mixes produced. The analysis of the mechanical performance allowed concluding that the use of RA worsens most of the properties tested, especially when fine RA are used. On the other hand, there was an increase in abrasion resistance when coarse RA were used. In global terms, the use of this type of aggregates, in limited contents, is viable from a mechanical viewpoint. (C) 2015 Elsevier Ltd. All rights reserved.

Study on the valorization routes of ashes from thermoelectric power plants working under mono-and co-combustion regimes

Thesis submitted to obtain the Doctoral degree in Energy and Bioenergy

Chemical composition and antifungal activity of essential oil from Eucalyptus smithii against dermatophytes

ABSTRACT INTRODUCTION: In this study, we evaluated the chemical composition of a commercial sample of essential oil from Eucalyptus smithii R.T. Baker and its antifungal activity against Microsporum canis ATCC 32903, Microsporum gypseum ATCC 14683, Trichophyton mentagrophytes ATCC 9533, T. mentagrophytes ATCC 11480, T. mentagrophytes ATCC 11481, and Trichophyton rubrum CCT 5507. METHODS: Morphological changes in these fungi after treatment with the oil were determined by scanning electron microscopy (SEM). The antifungal activity of the oil was determined on the basis of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values. RESULTS: The compound 1,8-cineole was found to be the predominant component (72.2%) of the essential oil. The MIC values of the oil ranged from 62.5μg·mL−1 to >1,000μg·mL−1, and the MFC values of the oil ranged from 125μg·mL−1 to >1,000μg·mL−1. SEM analysis showed physical damage and morphological alterations in the fungi exposed to this oil. CONCLUSIONS: We demonstrated the potential of Eucalyptus smithii essential oil as a natural therapeutic agent for the treatment of dermatophytosis.

Plants used in traditional medicine of China and Brazil

Eventhough the rationale behind the use of medicinal plantes in Brazil and Chine is different, twenty four species are used in both countries. Scientific name, vulgar name and uses in both countries along with their chemical constituents are listed.

Antitumor activity of chemical modified natural compounds

Search of new activity substances starting from chemotherapeutic agents, continously appears in international literature. Perhaps this search has been done more frequently in the field of anti-tumor chemotherapy on account of the unsuccess in saving advanced stage patients. The new point in this matter during the last decade was computer aid in planning more rational drugs. In near future "the accessibility of supercomputers and emergence of computer net systems, willopen new avenues to rational drug design" (Portoghese, P. S. J. Med. Chem. 1989, 32, 1). Unknown pharmacological active compounds synthetized by plants can be found even without this eletronic devices, as tradicional medicine has pointed out in many contries, and give rise to a new drug. These compounds used as found in nature or after chemical modifications have produced successful experimental medicaments as FAA, "flavone acetic acid" with good results as inibitors of slow growing animal tumors currently in preclinical evaluation for human treatment. In this lecture some international contributions in the field of chemical modified compounds as antineoplasic drugs will be examined, particularly those done by Brazilian researches.

Some progress on the chemistry of natural bioactive terpenoids form Chinese medicinal plants

(1) Pseudolaric acids - Novel diterpenes, Pseudolaric acid A, B, C and D were isolated from Pseudolarix kaempferi Gorden (pinaceae). Their structures were assigned by spectroscopic data and chemical correlations. In the contineous studies, the absolute configurations, the conformations in the solutions, the framentation mechanisms of MS and assigments of all NMR spectral signals were also reported. They showed the antifungal and cytotoxic activities. (2) Daphnane diterpenes - In the further studies on the plants of Thymelaeaceae, besides 10 known diterpenes, 16 new daphnane diterpenes were isolated from Daphne genkwa, D. tangutica, D. giraldii, Wikstroemie chamaedaphne. They showed the antifertilities activities. (3) Tripterygium diterpenes 14 new diterpenes were isolated from Triperygium wilfordii, T. regeli and T. hypoglaucum. Some of them showed the antitumor activities. The CD spectra showed that A/B ring of all compoundshave trans configuration as same as tripdiolide and triptolide determined by X-ray diffraction (4) Pregnane glycosides from Marsdenia koi - Two new pregnane glycosides marsdenikoiside A and marsdenikoiside B which can terminate the early pregnancy were isolated from Marsdeia koi. Their structures were elucidated by hydrolysis and spectroscopic methods.

Activation of the heat shock response in plants by chlorophenols: transgenic Physcomitrella patens as a sensitive biosensor for organic pollutants.

The ability to detect early molecular responses to various chemicals is central to the understanding of biological impact of pollutants in a context of varying environmental cues. To monitor stress responses in a model plant, we used transgenic moss Physcomitrella patens expressing the beta-glucuronidase reporter (GUS) under the control of the stress-inducible promoter hsp17.3B. Following exposure to pollutants from the dye and paper industry, GUS activity was measured by monitoring a fluorescent product. Chlorophenols, heavy metals and sulphonated anthraquinones were found to specifically activate the hsp17.3B promoter (within hours) in correlation with long-term toxicity effects (within days). At mildly elevated physiological temperatures, the chemical activation of this promoter was strongly amplified, which considerably increased the sensitivity of the bioassay. Together with the activation of hsp17.3B promoter, chlorophenols induced endogenous chaperones that transiently protected a recombinant thermolabile luciferase (LUC) from severe heat denaturation. This sensitive bioassay provides an early warning molecular sensor to industrial pollutants under varying environments, in anticipation to long-term toxic effects in plants. Because of the strong cross-talk between abiotic and chemical stresses that we find, this P. patens line is more likely to serve as a direct toxicity bioassay for pollutants combined with environmental cues, than as an indicator of absolute toxicity thresholds for various pollutants. It is also a powerful tool to study the role of heat shock proteins (HSPs) in plants exposed to combined chemical and environmental stresses.

Recombinant " Physcomitrella patens " as a sensitive tool to study heat sensing and heat-shock signal transduction in plants

SUMMARY When exposed to heat stress, plants display a particular set of cellular and molecular responses, such as chaperones expression, which are highly conserved in all organisms. In chapter 1, I studied the ability of heat shock genes to become transiently and abundantly induced under various temperature regimes. To this aim, I designed a highly sensitive heat-shock dependent conditional gene expression system in the moss Physcomitrella patens, using the soybean heatinducible promoter (hsp17.3B). Heat-induced expression of various reporter genes was over three orders of magnitude, in tight correlation with the intensity and duration of the heat treatments. By performing repeated heating/cooling cycles, a massive accumulation of recombinant proteins was obtained. Interestingly, the hsp17.3B promoter was also activated by specific organic chemicals. Thus, in chapter 2, I took advantage of the extreme sensitivity of this promoter to small temperature variations to further address the role of various natural and organic chemicals and develop a plant based-bioassay that can serve as an early warning indicator of toxicity by pollutants and heavy metals. A screen of several organic pollutants from textile and paper industry showed that chlorophenols as well as sulfonated anthraquinones elicited a heat shock like response at noninducing temperatures. Their effects were synergistically amplified by mild elevated temperatures. In contrast to standard methods of pollutant detection, this plant-based biosensor allowed to monitor early stress-responses, in correlation with long-term toxic effect, and to attribute effective toxicity thresholds for pollutants, in a context of varying environmental cues. In chapter 3, I deepened the study of the primary mechanism by which plants sense mild temperature variations and trigger a cellular signal leading to the heat shock response. In addition to the above described heat-inducible reporter line, I generated a P. patens transgenic line to measure, in vivo, variations of cytosolic calcium during heat treatment, and another line to monitor the role of protein unfolding in heat-shock sensing and signalling. The heat shock signalling pathway was found to be triggered by the plasma membrane, where temperature up shift specifically induced the transient opening of a putative high afimity calcium channel. The calcium influx triggered a signalling cascade leading to the activation of the heat shock genes, independently on the presence of misfolded proteins in the cytoplasm. These results strongly suggest that changes in the fluidity of the plasma membrane are the primary trigger of the heatshocksignalling pathway in plants. The present thesis contributes to the understanding of the basic mechanism by which plants perceive and respond to heat and chemical stresses. This may contribute to developing appropriate better strategies to enhance plant productivity under the increasingly stressful environment of global warming. RÉSUME Les plantes exposées à des températures élevées déclenchent rapidement des réponses cellulaires qui conduisent à l'induction de gènes codant pour les heat shock proteins (HSPs). En fonction de la durée d'exposition et de la vitesse à laquelle la température augmente, les HSPs sont fortement et transitoirement induites. Dans le premier chapitre, cette caractéristique aété utilisée pour développer un système inductible d'expression de gènes dans la mousse Physcomitrella patens. En utilisant plusieurs gènes rapporteurs, j'ai montré que le promoteur du gène hsp17.3B du Soja est activé d'une manière. homogène dans tous les tissus de la mousse proportionnellement à l'intensité du heat shock physiologique appliqué. Un très fort taux de protéines recombinantes peut ainsi être produit en réalisant plusieurs cycles induction/recovery. De plus, ce promoteur peut également être activé par des composés organiques, tels que les composés anti-inflammatoires, ce qui constitue une bonne alternative à l'induction par la chaleur. Les HSPs sont induites pour remédier aux dommages cellulaires qui surviennent. Étant donné que le promoteur hsp17.3B est très sensible à des petites augmentations de température ainsi qu'à des composés chimiques, j'ai utilisé les lignées développées dans le chapitre 1 pour identifier des polluants qui déclenchent une réaction de défense impliquant les HSPs. Après un criblage de plusieurs composés, les chlorophénols et les antraquinones sulfonés ont été identifiés comme étant activateurs du promoteur de stress. La détection de leurs effets a été réalisée seulement après quelques heures d'exposition et corrèle parfaitement avec les effets toxiques détectés après de longues périodes d'exposition. Les produits identifiés montrent aussi un effet synergique avec la température, ce qui fait du biosensor développé dans ce chapitre un bon outil pour révéler les effets réels des polluants dans un environnement où les stress chimiques sont combinés aux stress abiotiques. Le troisième chapitre est consacré à l'étude des mécanismes précoces qui permettent aux plantes de percevoir la chaleur et ainsi de déclencher une cascade de signalisation spécifique qui aboutit à l'induction des gènes HSPs. J'ai généré deux nouvelles lignées afin de mesurer en temps réel les changements de concentrations du calcium cytosolique ainsi que l'état de dénaturation des protéines au cours du heat shock. Quand la fluidité de la membrane augmente après élévation de la température, elle semble induire l'ouverture d'un canal qui permet de faire entrer le calcium dans les cellules. Ce dernier initie une cascade de signalisation qui finit par activer la transcription des gènes HSPs indépendamment de la dénaturation de protéines cytoplasmiques. Les résultats présentés dans ce chapitre montrent que la perception de la chaleur se fait essentiellement au niveau de la membrane plasmique qui joue un rôle majeur dans la régulation des gènes HSPs. L'élucidation des mécanismes par lesquels les plantes perçoivent les signaux environnementaux est d'une grande utilité pour le développement de nouvelles stratégies afin d'améliorer la productivité des plantes soumises à des conditions extrêmes. La présente thèse contribue à décortiquer la voie de signalisation impliquée dans la réponse à la chaleur.