Research advances in plant-made flavivirus antigens.

Outbreaks of flaviviruses such as dengue (DV), yellow fever (YFV), Japanese encephalitis (JEV), tick-borne encephalitis (TBEV) and West Nile (WNV) affect numerous countries around the world. The fast spread of these viruses is the result of increases in the human population, rapid urbanisation and globalisation. While vector control is an important preventive measure against vector-borne diseases, it has failed to prevent the spread of these diseases, particularly in developing countries where the implementation of control measures is intermittent. As antiviral drugs against flaviviruses are not yet available, vaccination remains the most important tool for prevention. Although human vaccines for YFV, TBEV and JEV are available, on-going vaccination efforts are insufficient to prevent infection. No vaccines against DENV and WNV are available. Research advances have provided important tools for flavivirus vaccine development, such as the use of plants as a recombinant antigen production platform. This review summarises the research efforts in this area and highlights why a plant system is considered a necessary alternative production platform for high-tech subunit vaccines.

Removal of azinphos methyl by alfalfa plants (Medicago sativa L.) in a soil-free system.

The objective of this study was to investigate the removal of azinphos methyl assisted by alfalfa plants, with special emphasis on the effects of this compound on some plant's physiological parameters. Hydroponic cultures of alfalfa (Medicago sativa L., var Romagnola) were employed as a model system. These cultures were exposed to a nutrient medium containing 10 mg/l of azinphos methyl. A first-order kinetic approach was used to describe the removal of azinphos methyl from the solution. After 20 days of culture, the initial amount of azinphos methyl was reduced to non-detectable levels in the presence of plants. In the absence of plants, 20% of azinphos methyl remained in the solution after 30 days of treatment. The half-life of the pesticide was reduced from 10.8 to 3.4 days in the presence of plants. The growth index of alfalfa plants exposed to azinphos methyl was negatively affected. Chlorophyll contents were reduced after 24 h of treatment and thereafter the levels were comparable to that of control plants. The peroxidase activity of alfalfa roots was not affected by the presence of azinphos methyl. In conclusion, alfalfa plants were able to survive when exposed to an effective concentration of 10 mg/l of azinphos methyl in the root zone, with some alterations on their physiological parameters.

Atividade antibacteriana em alguns extratos de vegetais do semi-árido brasileiro

Espécies nativas ou endêmicas do semi-árido brasileiro foram investigadas com o intuito de se descobrir novas drogas antimicrobianas. Os ensaios foram realizados contra cepas padrões de Staphylococcus aureus e Escherichia coli através do método de difusão em disco. Dos 137 extratos de vegetais avaliados, sete apresentaram atividade significativa contra o Staphylococcus aureus. Os extratos ativos foram preparados a partir de espécies pertencentes às famílias Leguminosae e Rutaceae e serão futuramente fracionados com o intuito de se chegar às moléculas ativas.

Species native or endemic of the Brazilian semi-arid were investigated with the intention of discovering new antibacterial drugs. The rehearsals were accomplished against standard strains of Staphylococcus aureus and Escherichia coli through the diffusion method in disk. Of the 137 extracts of appraised vegetables, seven presented significant activity against the Staphylococcus aureus. The active extracts were prepared starting from species belonging to the Leguminosae and Rutaceae families and they will be fractional hereafter with the intention of arriving to the active molecules.

Hairy roots of Brugmansia candida that grow without agitation: biotechnological implications.

Hairy roots of Brugmansia candida that grew without agitation were obtained. Kinetics of growth and production of the tropane alkaloids scopolamine and hyoscyamine, with and without agitation, were studied. The exponential growth rate was higher in the roots that were exposed to shaking (0.13 d(-1)) than in the nonagitated ones (0.09 d(-1)). The specific production and the levels per flask of both alkaloids were enhanced without shaking. The use of these roots in large-scale productions could be economically advantageous. It remains to be seen if the data obtained in shake flasks can be extrapolated to large-scale bioreactors.

Occurrence of cadaverine in hairy roots of Brugmansia candida.

The polyamine, cadaverine, was detected in transformed root cultures of Brugmansia candida (syn. Datura candida), a Solanaceae which produces the tropane alkaloids scopolamine and hyoscyamine. To the best of our knowledge, this is the first time that the existence of this uncommon polyamine has been detected in a Datura species. Cadaverine, however, could not be found in the whole plant. The occurrence of cadaverine in hairy roots could be a consequence of either the transformation or a response to stress. Also, cadaverine could be participating in other secondary pathways rather than to the tropane alkaloids. The common polyamines, putrescine, spermidine and spermine were also observed.

Peroxidase production in vitro by Armoracia lapathifolia (horseradish)-transformed root cultures: effect of elicitation on level and profile of isoenzymes.

Transformed roots of Armoracia lapathifolia (horseradish) were established by infection with Agrobacterium rhizogenes LBA 9402. They were used as a culture system in vitro for peroxidase production in vitro, to avoid many of the problems that affect the traditional production from field-grown species of Armoracia sp. The time course of growth of these cultures showed that total peroxidase attained maximum levels at the end of the exponential growth phase. At this stage of culture, elicitation assays were performed with AgNO3 and CuSO4 as abiotic elicitors and with fungal extracts of Verticillum sp., Monodyctis cataneae and Aspergillus niger as biotic elicitors. The best results were obtained with Verticillum sp., 24 h after elicitation, with an increase of approx. 100% in peroxidase activity. The isoenzyme pattern analysed by isoelectric focusing revealed predominantly basic and acidic isoenzymes in both plant roots and transformed root cultures. Elicited samples showed a similar isoenzyme pattern with a slight increase in basic isoenzymes.

Biotransformation of hydroquinone by hairy roots of Brugmansia candida and effect of sugars and free-radical scavengers.

Hairy roots of Brugmansia candida were used to bioconvert hydroquinone into arbutin. The highest bioconversion, with the lowest damage to the cells, was attained when concentrations of 20-40 mg/L hydroquinone were used. Sugars (sucrose, glucose, mannitol, and sorbitol) at concentrations of 30-120 g/L enhanced bioconversion, and, of these, sucrose was the most effective. Two different free-radical scavengers were also tested: sodium benzoate and gallic acid. The first one diminished biotransformation efficiency; gallic acid did not affect biotransformation at all. Preliminary permeabilization treatments tested failed to liberate arbutin into the medium, and provoked a total loss in cell viability.

Ritual and medicinal plants of the Ese'ejas of the Amazonian rainforest (Madre de Dios, Perú).

The Ese'eja is a hunter-fisher-gatherer tribe of Amerindians which occupies the south-eastern part of Perú. Their culture cannot be disassociated from religious beliefs. Disease can be caused by accident, distraction or indolence, or by evil powers. These evil powers come either from the direct action of a harmful shaman or by interactions with the Devil. A description of shamanic practices is given to elucidate the position of health practices in Ese'eja culture, which includes the use of medicinal and ritual plants. Aspects of ayahuasca (Banisteriopsis caapi (Spruce) Morton, Malpighiaceae) ritual in shaman initiation and in healing rituals are presented. Diagnosis and treatment include invocation to the ayahuasca spirit. Plants used as medicine or invoked for healing are presented.

Isolation and selection of biosurfactant-producing bacteria.

Haemolysis has been used as an initial selection criterion for the primary isolation of surfactant-producing bacteria. Only 37 of 492 strains of different origins had haemolytic activity. These 37 strains, together with 49 non-haemolytic ones chosen at random, were studied for surface activity. Only five strains, all of them haemolytic, tested positive. Haemolysis and biosurfactant-production are thus probably associated.

Design, formulation, and optimization of media.

The fundamental knowledge available about the microorganism, substrates, and process represent the basis on which a rational approach for the design and formulation of media for microbial processes can be attempted. In this respect, it is necessary to analyze critically the main objective to be optimized (yield, productivity, quality of final product, etc.). The first step of medium development is concerned with the decision to be taken about the adequate components to be used, followed by the calculation of their concentrations. After medium preparation and experimentation, the application of the most appropriate statistical optimization methodology will make it possible to attain the optimal medium. Medium design and formulation for plant cell processes are still carried out on an empirical basis owing to the lack of fundamental knowledge. However, some general guidelines can be given for growth and production media considering the experimental evidence available about the functions and influence of the medium components for promoting growth and product formation. Adequate manipulation of the carbon, nitrogen, and phosphorus sources and phytohormones and the inclusion of precursors and elicitors in the medium represent the best strategy for improving medium development for plant cell processes. The development of cost-effective medium supplies for mammalian cell culture production of proteins is a key element for a commercially successful process. Medium development is a task that requires experience, time, and resources in its solutions, which may be several. An optimal formulation is elusive because there will always be room for improvement, but through systematic, comprehensive work, practical combinations of nutrients, factors, and feeding schedules can be developed in a reasonable time. A properly developed formulation can bring the burden of the medium below 10% of the total cost of a mammalian cell process.

Content of thiophenes in transformed root cultures of argentinian species of tagetes.

The presence of thiophenes in four Argentinian species of TAGETES was studied. T. TERNIFLORA HBK and T. MINUTA L. seedlings contain 5-(4-hydroxy-1-butynyl)-2-2'-bithienyl (BBTOH); 5-(4-acetoxy-1-butynyl)-2,2'-bithienyl (BBTOAc), while T. CAMPANULATA Griseb and T. LAXA Cabrera seedlings also accumulated BBT and alpha-T. From the four TAGETES species tested only T. LAXA was able to produce transformed roots when infected with AGROBACTERIUM RHIZOGENES LBA 9402. Several clones of transformed roots were obtained in which the total thiophene content present showed considerable variations (277 to 1773 microg/g FW). The thiophene spectrum, however, was similar between different clones. In addition, the thiophene patterns in these transformed clones differed from that formed in the parent plants.

Effect of elicitation on the accumulation of solasodine by immobilized cells of Solanum eleagnifolium Cav.

The effect of a fungal elicitor obtained from Alternaria sp. on growth and solasodine production by free and alginate-entrapped cells of Solanum eleagnifolium Cav. was studied. Fourteen-day-old cultures were elicited with 1% FW/V autoclaved homogenates. The solasodine production increased from 0.9 to 1.5 mg g-1 DW (65%) in suspension cultures and from 0.75 to 1.4 mg g-1 DW (about 95%) in entrapped cells. The maximum accumulation was obtained after 72 h of elicitation. In order to induce alkaloid release from cells (suspension and entrapped cells), permeabilization with 10% dimethylsulfoxide (DMSO) for 30 min was used. In both cases (free and entrapped cells), about 50-60% of intracellular solasodine was released into the medium. The reuse of elicited and permeabilized entrapped cells was also carried out for three production cycles.

Tropane alkaloid production in transformed root cultures of brugmansia Candida.

Transformed root cultures of BRUGMANSIA CANDIDA were established by infection with AGROBACTERIUM RHIZOGENES LBA 9402. Several clones with different growth index and tropane alkaloid pattern and content were obtained and two were examined in depth. The alkaloid content and pattern changed during the time course. At 21 days of culture clone 1 revealed an alkaloid spectrum dominated by 3alpha-acetoxytropane (about 50% of the total alkaloid) and hyoscyamine (about 25%), with a ratio of hyoscyamine to scopolamine of 11.2. In clone 40 this ratio was 1.5 and the content of 3alpha-acetoxytropane was low (2%). The maximum concentrations of hyoscyamine were obtained at 3 weeks of culture, and were 700 and 500 microg/g FW in clone 1 and 40, respectively.

[Mutants with high butanol production from a strain of solventogenic Clostridium isolated from olive black-water]. / Mutantes con alta producción de butanol de una cepa de Clostridium solventogénica aislada de alpechín.

Butanol high producing mutants of a solventogenic Clostridium sp. capable of degrading olive black water, were selected according to ethanol or butanol resistance after treatment with N-methyl-N'-nitro-N-nitrosoguanidine. Mutants were quickly screened from isolated colonies and then characterized in standard culture conditions.

Mutantes con alta producción de butanol de una cepa de Clostridium solventogénica aislada de alpechín / [Mutants with high butanol production from a strain of solventogenic Clostridium isolated from olive black-water]

Butanol high producing mutants of a solventogenic Clostridium sp. capable of degrading olive black water, were selected according to ethanol or butanol resistance after treatment with N-methyl-N’-nitro-N-nitrosoguanidine. Mutants were quickly screened from isolated colonies and then characterized in standard culture conditions.

Mutantes con alta producción de butanol de una cepa de Clostridium solventogénica aislada de alpechín. / [Mutants with high butanol production from a strain of solventogenic Clostridium isolated from olive black-water]

Butanol high producing mutants of a solventogenic Clostridium sp. capable of degrading olive black water, were selected according to ethanol or butanol resistance after treatment with N-methyl-N-nitro-N-nitrosoguanidine. Mutants were quickly screened from isolated colonies and then characterized in standard culture conditions.

The influence of auxins, light and cell differentiation on solasodine production bySolanum eleagnifolium Cav. calli.

The effect of auxins, light and cellular production ofSolanum eleagnifolium Cav. calli were studied. 2,4-dichlorophenoxyacetic acid (4.5 µM) was the plant growth regulator used for calli initiation and this produced the highest solasodine concentration. The solasodine concentration in darkness was significantly lower than that achieved under a photoperiod of 16 h. Differentiated tissue obtained by adequate hormonal balance (several ratios of 3-indolebutyric acid to 6-benzylaminopurine) produced higher yields of solasodine than non-differentiated tissue. 3-indolebutyric acid (2.5 µM) and 6-benzylaminopurine (8.8 µM) increased the productivity of solasodine by 100%.

Production of solasodine by calli from different parts of Solanum eleganifolium Cav. plants.

Callus tissues from different explants (hypocotyl, cotyledon, root, leaf and fruit) of Solanum eleagnifolium Cav. were cultured on a modified Murashige-Skoog medium, with 1 mg.1(-1) 2,4-D as the sole growth regulator. The presence of the alkaloid solasodine was determined by spectrophotometric and TLC methods. Its concentration ranged from 1.00 to 2.15 mg.g(-1) DW. The calli from different explants showed a direct association between the solasodine production and their growth, although they have a different production rate. It was also observed that about the seventh week of culture the metabolite concentration decreased in all cases.

[Biomass production and biological purification of distillation slops in a 2-step process]. / Producción de biomasa y depuración biológica de vinaza de destilería en un proceso de dos etapas.

In order to lower the chemical demand (COD) of slops from cane molasses alcohol a treatment of two steps which allows the production of single cell protein of Candida utilis and Paecilomyces variotii has been performed. Its use reduces the treatment cost. In the first step the slops without sterilization supplemented with ammonium sulphate (5 g.l-1) and dipotassium phosphate (0.5 g.l-1) was inoculated with C. utilis and P. variotii. The yield was 24 and 18 g.l-1 of dry biomass and COD reduction of 36 and 75% respectively. In the second step, the remainder effluents were treated with Aspergillus niger. The final COD reduction attained was 93 and 92% respectively.

Influencia de la velocidad de crecimiento sobre la formacion de toxinas en microorganismos del genero Clostridium. / Influence of growth velocity on the toxin production in microorganisms of the genus Clostridium

Se analizo la relacion entre velocidad de crecimiento y formacion de toxina en cultivos de Clostridium botulinum tipo A y Clostridium perfringens tipo D obtenidos con distintos medios y sistemas de cultivo Los resultados obtenidos indican una relacion inversa entre velocidad de crecimiento y actividad toxigenica especifica