Effects of gamma radiation on the growth of Alternaria alternata and on the production of alternariol and alternariol monomethyl ether in sunflower seeds

The objective of the present study was to evaluate the effects of different gamma radiation doses on the growth of Alternaria alternata and on the production of toxins alternariol (AOH), and alternariol monomethyl ether (AME) in sunflower seed samples. After irradiation with 2, 5 and 7 kGy, the spore mass was resuspended in sterile distilled water and the suspension was inoculated into sunflower seeds. The number of colony-forming units per gram (CFU/g) was determined after culture on Dichloran Rose Bengal Chloramphenicol and Dichloran Chloramphenicol Malt Extract Agar. The presence of AOH and AME was investigated by liquid chromatography coupled to mass spectrometry. The radiation doses used resulted in a reduction of the number of A. alternata CFU/g and of AOH and AME levels when compared to the nonirradiated control group. Maximum reduction of the fungus (98.5%) and toxins (99.9%) was observed at a dose of 7 and 5 kGy, respectively. Under the present conditions, gamma radiation was found to be an alternative for the control of A. alternata and, consequently, of AOH and AME production in sunflower seeds. (C) 2009 Elsevier Ltd. All rights reserved.

Effects of nitrogen forms on the production of cyanobacterial toxin microcystin-LR by an isolated Microcystis aeruginosa

A cyanobacterial strain, which produced high content of microcystin-LR (MC-LR) but no rnicrocystin-RR (MC-RR), was isolated from the hypertrophic Dianchi Lake in China and identified as Microcystis aeruginosa DC-1. Effects of nitrogen containing chemicals and trace elements on the growth and the production of MC-LR by this strain were Studied. In the presence of bicine, compared with urea and ammonium, nitrate greatly promoted the growth and the production of MC-LR. However, leucine and arginine, which were the constitutional components in the molecular structure of MC-LR or RR, inhibited the production of MC-LR. Iron and silicon up to 10mg/L had little effects on the growth of M. aeruginosa DC-1, but the production of MC-LR was apparently enhanced. Under all conditions studied here, only MC-LR but no RR was detected within the cells of M. aeruginosa DC-1. Thus, chemical forms of nitrogen, rather than the usually concerned the total nitrogen, Lind trace elements played important roles in the production of MC toxins during cyanobacterial blooms.

Production of a broad specificity antibody for the development and validation of an optical SPR screening method for free and intracellular microcystins and nodularin in cyanobacteria cultures

A highly sensitive broad specificity monoclonal antibody was produced and characterised for microcystin detection through the development of a rapid surface plasmon resonance (SPR) optical biosensor based immunoassay. The antibody displayed the following cross-reactivity: MC-LR 100%; MC-RR 108%; MC-YR 68%; MC-LA 69%; MC-LW 71%; MC-LF 68%; and Nodularin 94%. Microcystin-LR was covalently attached to a CM5 chip and with the monoclonal antibody was employed in a competitive 4min injection assay to detect total microcystins in water samples below the WHO recommended limit (1µg/L). A 'total microcystin' level was determined by measuring free and intracellular concentrations in cyanobacterial culture samples as this toxin is an endotoxin. Glass bead beating was used to lyse the cells as a rapid extraction procedure. This method was validated according to European Commission Decision 96/23/EC criteria. The method was proven to measure intracellular microcystin levels, the main source of the toxin, which often goes undetected by other analytical procedures and is advantageous in that it can be used for the monitoring of blooms to provide an early warning of toxicity. It was shown to be repeatable and reproducible, with recoveries from spiked samples ranging from 74 to 123%, and had % CVs below 10% for intra-assay analysis and 15% for inter-assay analysis. The detection capability of the assay was calculated as 0.5ng/mL for extracellular toxins and 0.05ng/mL for intracellular microcystins. A comparison of the SPR method with LC-MS/MS was achieved by testing six Microcystis aeruginosa cultures and this study yielded a correlation R(2) value of 0.9989.

Production of Trichophyton mentagrophytes antigens and their characterization in mice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Production of prostaglandins and leukotrienes by Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, the most prevalent deep mycosis in Latin America. Production of eicosanoids, including prostaglandins and leukotrienes, during fungal infections is theorized to play a critical role on fungal survival and/or growth as well as on host immune response modulation. Host cells are one source of these mediators; however another potential source may be the fungus itself. The purpose of our study was to assess whether P. brasiliensis strains with different degree of virulence (Pb18, Pb265, PbBT79, Pb192) produce both, prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)). Moreover, we asked if P. brasiliensis can use exogenous sources of arachidonic acid (AA), as well as metabolic pathways dependent on cyclooxygenase (COX) and lipoxygenase (5-LO) enzymes, for PGE(2) and LTB(4) production, respectively. Finally, a possible association between these eicosanoids and fungus viability was assessed. We demonstrated, using ELISA assays, that all P. brasiliensis strains, independently of their virulence, produce high PGE(2) and LTB(4) levels after a 4-hour culture, which were reduced after 8 hours. However, in both culture times, higher eicosanoids levels were detected when culture medium was supplemented with exogenous AA. Differently, treatment with indomethacin, a COX inhibitor, or MK886, a 5-LO inhibitor, induces a reduction on PGE(2) and LTB(4) levels, respectively, as well as in fungus viability. The data provide evidence that P. brasiliensis is able to metabolize either endogenous or exogenous AA by pathways that depend on COX and 5-LO enzymes for producing, respectively, PGE(2) and LTB(4) that are critical for its viability.

A Review on the Challenges for Increased Production of Castor

Castor (Ricinus communis L.) is one of the oldest cultivated crops, but currently it represents only 0.15% of the vegetable oil produced in the world. Castor oil is of continuing importance to the global specialty chemical industry because it is the only commercial source of a hydroxylated fatty acid. Castor also has tremendous future potential as an industrial oilseed crop because of its high seed oil content (more than 480 g kg(-1)), unique fatty acid composition (900 g kg(-1) of ricinoleic acid), potentially high oil yields (1250-2500 L ha(-1)), and ability to be grown under drought and saline conditions. The scientific literature on castor has been generated by a relatively small global community of researchers over the past century. Much of this work was published in dozens of languages in journals that are not easily accessible to the scientific community. This review was conducted to provide a compilation of the most relevant historic research information and define the tremendous future potential of castor. The article was prepared by a group of 22 scientists from 16 institutions and eight countries. Topics discussed in this review include: (i) germplasm, genetics, breeding, biotic stresses, genome sequencing, and biotechnology; (ii) agronomic production practices, diseases, and abiotic stresses; (iii) management and reduction of toxins for the use of castor meal as both an animal feed and an organic fertilizer; (iv) future industrial uses of castor including renewable fuels; (v) world production, consumption, and prices; and (vi) potential and challenges for increased castor production.

Effect of prostaglandins on the production of H2O2 and cytokines that modulate the fungicidal activity of human monocytes against Paracoccidioides brasiliensis

Human monocytes lack fungicidal activity against high virulent strains of Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis, even after IFN-γ activation. However, monocytes treated with indomethacin (INDO) or INDO plus IFN-γ effectively killed this fungus, suggesting an inhibitory role of prostaglandins in this process. Thus, the purpose of this work was to test if this regulatory effect of prostaglandin was associated with alterations on H2O2 production and/or on modulatory cytokines levels, such as TNF-α, IL-10, and IL-6. Peripheral blood monocytes obtained from 10 healthy donors were incubated for 18 hours in the presence or absence of IFN-γ, INDO, or IFN-γ plus INDO, and further challenged with a high virulent strain of P. brasiliensis (Pb18) for 4 hours. Then, the monocytes cultures were evaluated for H2O2 release and fungicidal activity calculated by counting the colony forming units after plating. Moreover, on supernatants of the same cultures, TNF-α, IL-10, IL-6, and PGE2 concentrations were evaluated by ELISA. Monocytes treated with INDO or INDO plus IFN-γ presented higher fungicidal activity associated with the release of higher levels of H2O2 and TNF-α, but lesser levels of PGE2, when compared to nontreated cells. However, the levels of IL-10 and IL-6 were similar between treated and nontreated cells. The results suggest that human monocytes when challenged with high virulent strains of P. brasiliensis produce prostaglandins that inhibit the fungicidal activity of these cells by reducing H2O2 and TNF-α levels.

Alkylation of Histidine Residues of Bothrops jararacussu Venom Proteins and Isolated Phospholipases A(2): A Biotechnological Tool to Improve the Production of Antibodies

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

General characterization of Tityus fasciolatus scorpion venom. Molecular identification of toxins and localization of linear B-cell epitopes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A novel process for the production of castor oil by direct solvent extraction

This research was undertaken to: develop a process for the direct solvent extraction of castor oil seeds. A literature survey confirmed the desirability of establishing such a process with emphasis on the decortication, size, reduction, detoxification-deallergenization, and solvent·extraction operations. A novel process was developed for the dehulling of castor seeds which consists of pressurizing the beans and then suddenly releasing the pressure to vaccum. The degree of dehulling varied according to the pressure applied and the size of the beans. Some of the batches were difficult-to-hull, and this phenomenon was investigated using the scanning electron microscope and by thickness and compressive strength measurements. The other variables studied to lesser degrees included residence time, moisture, content, and temperature.The method was successfully extended to cocoa beans, and (with modifications) to peanuts. The possibility of continuous operation was looked into, and a mechanism was suggested to explain the method works. The work on toxins and allergens included an extensive literature survey on the properties of these substances and the methods developed for their deactivation Part of the work involved setting up an assay method for measuring their concentration in the beans and cake, but technical difficulties prevented the completion of this aspect of the project. An appraisal of the existing deactivation methods was made in the course of searching for new ones. A new method of reducing the size of oilseeds was introduced in this research; it involved freezing the beans in cardice and milling them in a coffee grinder, the method was found to be a quick, efficient, and reliable. An application of the freezing technique was successful in dehulling soybeans and de-skinning peanut kernels. The literature on the solvent extraction, of oilseeds, especially castor, was reviewed: The survey covered processes, equipment, solvents, and mechanism of leaching. three solvents were experimentally investigated: cyclohexane, ethanol, and acetone. Extraction with liquid ammonia and liquid butane was not effective under the conditions studied. Based on the results of the research a process has been suggested for the direct solvent extraction of castor seeds, the various sections of the process have analysed, and the factors affecting the economics of the process were discussed.

A 'deleterious' effect? : Australian legal education and the production of the legal identity

A body of critical legal scholarship argues that, by the time they have completed their studies, students who enter legal education holding social ideals and intending to use their legal education to achieve social change, have become cynical about the ability of the law to do so and no longer possess such ideals. This is explained by critical scholars to be the result of a process of ideological indoctrination, aimed at ensuring that graduates uphold the narrow and conservative interests of the legal profession and capitalist society, being exercised by law schools acting as adjuncts of the legal profession, and exercised upon the passive body of the law student. By using Foucault’s work on knowledge, power, and the subject to interrogate the assumptions upon which this narrative is based, this thesis intends to suggest a way of thinking differently to the approach taken by many critical legal scholars. It then uses an analytics of government (based on Foucault’s notion of ‘governmentality’) to consider the construction of the legal identity differently. It examines the ways in which the governance of the legal identity is rationalised, programmed, and implemented, in three Queensland law schools. It also looks at the way that five prescriptive texts to ‘surviving’ law school suggest students establish and practise a relation to themselves in order to construct their own legal identities. Overall, this analysis shows that governance is not simply conducted in the profession’s interests, but occurs due to a complex arrangement of different practices, which can lead to the construction of skilled legal professional identities as well as ethical lawyer-citizens that hold an interest in justice. The implications of such an analytics provide the basis for original ways of understanding legal education, and legal education scholarship.

Investigation of highly regulated promoters for the production of recombinant proteins in plants

Plants have been identified as promising expression systems for the commercial production of recombinant proteins. Plant-based protein production or “biofarming” offers a number of advantages over traditional expression systems in terms of scale of production, the capacity for post-translation processing, providing a product free of contaminants and cost effectiveness. A number of pharmaceutically important and commercially valuable proteins, such as antibodies, biopharmaceuticals and industrial enzymes are currently being produced in plant expression systems. However, several challenges still remain to improve recombinant protein yield with no ill effect on the host plant. The ability for transgenic plants to produce foreign proteins at commercially viable levels can be directly related to the level and cell specificity of the selected promoter driving the transgene. The accumulation of recombinant proteins may be controlled by a tissue-specific, developmentally-regulated or chemically-inducible promoter such that expression of recombinant proteins can be spatially- or temporally- controlled. The strict control of gene expression is particularly useful for proteins that are considered toxic and whose expression is likely to have a detrimental effect on plant growth. To date, the most commonly used promoter in plant biotechnology is the cauliflower mosaic virus (CaMV) 35S promoter which is used to drive strong, constitutive transgene expression in most organs of transgenic plants. Of particular interest to researchers in the Centre for Tropical Crops and Biocommodities at QUT are tissue-specific promoters for the accumulation of foreign proteins in the roots, seeds and fruit of various plant species, including tobacco, banana and sugarcane. Therefore this Masters project aimed to isolate and characterise root- and seed-specific promoters for the control of genes encoding recombinant proteins in plant-based expression systems. Additionally, the effects of matching cognate terminators with their respective gene promoters were assessed. The Arabidopsis root promoters ARSK1 and EIR1 were selected from the literature based on their reported limited root expression profiles. Both promoters were analysed using the PlantCARE database to identify putative motifs or cis-acting elements that may be associated with this activity. A number of motifs were identified in the ARSK1 promoter region including, WUN (wound-inducible), MBS (MYB binding site), Skn-1, and a RY core element (seed-specific) and in the EIR1 promoter region including, Skn-1 (seed-specific), Box-W1 (fungal elicitor), Aux-RR core (auxin response) and ABRE (ABA response). However, no previously reported root-specific cis-acting elements were observed in either promoter region. To confirm root specificity, both promoters, and truncated versions, were fused to the GUS reporter gene and the expression cassette introduced into Arabidopsis via Agrobacterium-mediated transformation. Despite the reported tissue-specific nature of these promoters, both upstream regulatory regions directed constitutive GUS expression in all transgenic plants. Further, similar levels of GUS expression from the ARSK1 promoter were directed by the control CaMV 35S promoter. The truncated version of the EIR1 promoter (1.2 Kb) showed some differences in the level of GUS expression compared to the 2.2 Kb promoter. Therefore, this suggests an enhancer element is contained in the 2.2 Kb upstream region that increases transgene expression. The Arabidopsis seed-specific genes ATS1 and ATS3 were selected from the literature based on their seed-specific expression profiles and gene expression confirmed in this study as seed-specific by RT-PCR analysis. The selected promoter regions were analysed using the PlantCARE database in order to identify any putative cis elements. The seed-specific motifs GCN4 and Skn-1 were identified in both promoter regions that are associated with elevated expression levels in the endosperm. Additionaly, the seed-specific RY element and the ABRE were located in the ATS1 promoter. Both promoters were fused to the GUS reporter gene and used to transform Arabidopsis plants. GUS expression from the putative promoters was consitutive in all transgenic Arabidopsis tissue tested. Importantly, the positive control FAE1 seed-specific promoter also directed constitutive GUS expression throughout transgenic Arabidopsis plants. The constitutive nature seen in all of the promoters used in this study was not anticipated. While variations in promoter activity can be caused by a number of influencing factors, the variation in promoter activity observed here would imply a major contributing factor common to all plant expression cassettes tested. All promoter constructs generated in this study were based on the binary vector pCAMBIA2300. This vector contains the plant selection gene (NPTII) under the transcriptional control of the duplicated CaMV 35S promoter. This CaMV 35S promoter contains two enhancer domains that confer strong, constitutive expression of the selection gene and is located immediately upstream of the promoter-GUS fusion. During the course of this project, Yoo et al. (2005) reported that transgene expression is significantly affected when the expression cassette is located on the same T-DNA as the 35S enhancer. It was concluded, the trans-acting effects of the enhancer activate and control transgene expression causing irregular expression patterns. This phenomenon seems the most plausible reason for the constitutive expression profiles observed with the root- and seed-specific promoters assessed in this study. The expression from some promoters can be influenced by their cognate terminator sequences. Therefore, the Arabidopsis ARSK1, EIR1, ATS1 and ATS3 terminator sequences were isolated and incorporated into expression cassettes containing the GUS reporter gene under the control of their cognate promoters. Again, unrestricted GUS activity was displayed throughout transgenic plants transformed with these reporter gene fusions. As previously discussed constitutive GUS expression was most likely due to the trans-acting effect of the upstream CaMV 35S promoter in the selection cassette located on the same T-DNA. The results obtained in this study make it impossible to assess the influence matching terminators with their cognate promoters have on transgene expression profiles. The obvious future direction of research continuing from this study would be to transform pBIN-based promoter-GUS fusions (ie. constructs containing no CaMV 35S promoter driving the plant selection gene) into Arabidopsis in order to determine the true tissue specificity of these promoters and evaluate the effects of their cognate 3’ terminator sequences. Further, promoter truncations based around the cis-elements identified here may assist in determining whether these motifs are in fact involved in the overall activity of the promoter.