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Poster at Open Repositories 2014, Helsinki, Finland, June 9-13, 2014

Sinningia allagophylla(Gesneriaceae): in vitro cultivation of a native plant of the Brazilian cerrado

We used axillary buds as initial explants for hormone interaction studies required for in vitro cultivation of S. allagophylla. Callus production was achieved on gelled Murashige & Skoog medium (MS) supplemented with indole-3-acetic acid (IAA= 0.1 and 0.5 mg.l­1 alone or combined with 6 benzylaminopurine) (BA= 0.01 and 0.1 mg.l-1). A hormone balance between IAA and BA that would encourage shoot bud development was not found. Nodal segments from axenic cultures grown in the presence of cytokinin (0.1 mg.1­1 of BA) without any auxin on MS medium with half-strength macronutrients were used as a standard explant source for subsequent experiments on optimum mineral culture media composition for S. allagophylla in vitro cultivation. We found that explants kept in vitro on gelled Gamborg et al. (B5) mineral composition culture medium showed better shoot and specially root growth than on MS medium. Comparisons of the ammonium and nitrate ratios of MS and B5 media indicate that B5 medium has a substantial reduced ammonium ion when compared to MS medium, as well as a lower total nitrogen level. The growth response pattern obtained in vitro may be evidence of the adaptation of this species to soils of poor mineral composition as found in the Brazilian cerrado, as well as an indication that nitrogen levels play a key role for S. allagophylla growth.

Fructan variation in the rhizophores of Vernonia herbacea (Vell.) Rusby, as influenced by temperature

The influence of climatic variations on fructan content in tropical regions is not well known. The present study deals with the effects of temperature on fructan contents in rhizophores of plants of Vernonia herbacea, a native species from the Brazilian cerrado vegetation. Intact plants and fragmented rhizophores were subjected to different temperatures under natural and controlled environmental conditions. Rhizophores of plants in pre-dormant stage (aerial parts showing some yellowish leaves) presented higher fructan content at 5oC than those kept at 25oC, whereas in dormant plants (aerial parts absent) temperature treatments did not affect fructan contents. Fragmented rhizophores obtained from dormant plants presented higher levels of fructo-polysaccharides at the end of the experiment than at the beginning of the treatment, regardless of the temperature they were stored, whereas fragments obtained from vegetative plants showed a decrease in fructan content under the same treatments. It was concluded that variations observed in fructan contents are related to the phenological state of the plants prior to the treatment rather than to extraneous temperatures they are subjected to during this stage.

Woody community dynamics in two fragments of "cerrado" stricto sensu over a seven-year period (1995-2002), MA, Brazil

This study was conducted in two fragments of "cerrado" stricto sensu in the Gerais de Balsas Colonization Project, located in southern Maranhão, Brazil. The objective was to evaluate the dynamics of the woody plant community, over seven years (1995-2002). Four transects of 160 × 20 m were monitored. All woody plants with a stem diameter > 3 cm, at 0.30 m above ground level, were recorded. In 1995, 983 and 1,177 stems were sampled in fragments 1 and 2, respectively; in 2002, 1057 and 1406 stems were sampled in the same fragments. In 1995, the Shannon diversity indices (H') were 3.07 and 3.33, in fragments 1 and 2, respectively, reaching their maximum value in 2002 of 3.11 and 3.35. The community of fragment 1 showed an increase of 7.5% in density and 4.4% in basal area between 1995 and 2002, while in fragment 2 there was an increment of 19.4% in density and 23.5% in basal area, over the same period. The annual increment in diameter was 0.13 cm year-1 and 0.17 cm year-1 in fragments 1 and 2, respectively. The mortality rate was 2.73% per year in fragment 1 and 4.88% per year in fragment 2, while the recruitment rate was 3.25% per year and 5.86% per year, respectively. The community presented high recruitment and mortality rates compared to the studies conducted in other sites, indicating a community that was highly dynamic in the period studied.

Population structure of Eschweilera coriacea (DC.) S. A. Mori in forest fragments in eastern Brazilian Amazonia

The population structure of a common canopy tree was examined in three sites to investigate the possible effects of forest fragmentation in eastern Amazonia. Evidence for the escape hypothesis of differential seed/seedling survival was evaluated. Two 1 ha plots were established at each site and all individuals of Eschweilera coriacea (DC.) S. A. Mori over 1 m tall were tagged, measured and mapped. Smaller individuals were recorded in the same way within subplots. Mature individuals were abundant at all sites with densities of 32-52 ha-1. The species exhibited substantial regeneration, although total population density varied fourfold among sites (1,256-4,805 individuals ha-1). Overall, juveniles were clumped while adults were randomly distributed. The difference between the dispersion pattern of adults and juveniles supported the escape hypothesis. However, no difference in population structure among sites could be related to the forest fragmentation.

Making the impact on research and society : nichesourcing of Uralic language material for the benefit of linguistic research and native-speakers

Presentation at the 12th Bibliotheca Baltica Symposium at Södertörn University Library

Multivariate analysis of pollen frequency of the native species Escallonia pulverulenta (Saxifragaceae) in Chilean honeys

The aim of this work was the identification of geographic zones suitable for the production of honeys in which pollen grains of Escallonia pulverulenta (Ruiz & Pav.) Pers. (Saxifragaceae) can be detected. The analysis of botanical origin of 240 honey samples produced between La Serena and Puerto Mont (the IV and X Administrative Regions of Chile), allowed the detection of pollen grains of E. pulverulenta in 46 Chilean honeys. The geographic distribution of the honeys studied is presented together with their affinities, through factor analysis and frequency tables. The study was based on the presence of E. pulverulenta pollen. Escallonia pulverulenta pollen percentages oscillated between 0.24% and 78.5%. Seventeen of the studied samples were designated as unifloral - i.e. samples showing more than 45% pollen of a determined plant species. Two of these corresponded to E. pulverulenta (corontillo, madroño or barraco) honeys. The remaining unifloral honeys correspond to 8 samples of Lotus uliginosus Schkuhr (birdsfoot trefoil), 2 samples of Aristotelia chilensis (Molina) Stuntz (maqui) and 1 sample of Escallonia rubra (Ruiz & Pav.) Pers. (siete camisas), Eucryphia cordifolia Cav. (ulmo or muemo), Weinmannia trichosperma Cav. (tineo), Rubus ulmifolius Schott (blackberry) and Brassica rapa L. (turnip). Honeys with different percentages of E. pulverulenta pollen - statistically analyzed through correspondence analysis - could be associated and assigned to one of three geographic types, defined on the basis of this analysis. The geographical type areas defined were the Northern Mediterranean Zone (samples from the IV Region), Central Mediterranean Zone (samples from the V to the VIII regions including two samples of unifloral Escallonia pulverulenta honey), and Southern Mediterranean Zone (samples from the IX Region).

Germination, initial growth, and biomass allocation in three native Cerrado species

The parameters of germination, initial growth, and biomass allocation of three native plant species of Cerrado (Copaifera langsdorffii, Dipteryx alata and Kielmeyera coriacea) were established. The species had germination percentages above 88% and average germination times longer than 139 hours. The average time for the opening of the first leaf pair was more than 538 hours for all three species. The average root length of C. langsdorffii and D. alata seedlings after 80 days of growth was around 40cm, four times larger than the average shoot length (<10cm), although the root and shoot biomasses were similar for both species. The average root length (>20cm) of K. coriacea seedlings was four times larger than the average shoot length (<5cm), and the root biomass was 243% greater than the shoot biomass. Increase in seedling biomass was sustained primarily by the cotyledons in C. langsdorffii and D. alata, which acted as reserve organs and showed progressive weight reductions. Increase in seedling biomass in K. coriacea was sustained primarily by photosynthesis, since the cotyledons showed no significant weight reduction, acting primarily as photosynthetic organs. The length of the root systems was at least four times larger than the length of the shoots parts in all three species. Higher investment in root length rather than in root biomass suggest that the initial growth of these species is primarily to ensure access to water resources, apparently putting off the function of the radicular system as storage organ.

Floristic composition and phytogeography of the tree component of Araucaria Forest fragments in southern Brazil

The present study examined the floristic composition of three fragments of Araucaria Forest (AF) in the Planalto Catarinense region of southern Brazil as well as the floristic contextualization of these areas in relation to other remnant AF sites. Three AF fragments at different altitudes were analyzed in the municipalities of Campos Novos, Lages, and Painel. Fifty 200 m² plots were examined in each fragment and all of the trees with CBH (circumference at breast height) > 15.7 cm were identified. In order to floristically contextualize the study fragments, comparisons were made with other remnant AF sites by way of dendrograms and NMDS (Non-metric multidimensional scaling). Environmental and spatial variables were plotted on the diagram produced by the NMDS to evaluate their influence on the floristic patterns encountered. The forest fragments studied demonstrated high floristic heterogeneity, indicating that AFs cannot be considered homogeneous formations and they could be classified into 3 phytogeographical categories: i) high altitude areas influenced by cloud cover/fog, including the Painel region; ii) areas of lesser altitude and greater mean annual temperatures situated in the Paraná River basin, and iii) areas situated in the Paraná and Upper-Uruguay river basins and the smaller basins draining directly into the southern Atlantic, near Campos Novos and Lages. The environmental variables most highly correlated with species substitutions among the sites were altitude, mean annual temperature, and the mean temperature of the most humid trimester.

A new brain metalloendopeptidase which degrades the Alzheimer ß-amyloid 1-40 peptide producing soluble fragments without neurotoxic effects

A new metalloendopeptidase was purified to apparent homogeneity from a homogenate of normal human brain using successive steps of chromatography on DEAE-Trisacryl, hydroxylapatite and Sephacryl S-200. The purified enzyme cleaved the Gly33-Leu34 bond of the 25-35 neurotoxic sequence of the Alzheimer ß-amyloid 1-40 peptide producing soluble fragments without neurotoxic effects. This enzyme activity was only inhibited by divalent cation chelators such as EDTA, EGTA and o-phenanthroline (1 mM) and was insensitive to phosphoramidon and captopril (1 µM concentration), specific inhibitors of neutral endopeptidase (EC 3.4.24.11) and angiotensin-converting enzyme (EC 3.4.15.1), respectively. The high affinity of this human brain endopeptidase for ß-amyloid 1-40 peptide (Km = 5 µM) suggests that it may play a physiological role in the degradation of this substance produced by normal cellular metabolism. It may also be hypothesized that the abnormal accumulation of the amyloid ß-protein in Alzheimer's disease may be initiated by a defect or an inactivation of this enzyme.

pH titration of native and unfolded ß-trypsin: evaluation of the D D G0 titration and the carboxyl pK values

The stabilizing free energy of ß-trypsin was determined by hydrogen ion titration. In the pH range from 3.0 to 7.0, the change in free energy difference for the stabilization of the native protein relative to the unfolded one (D D G0 titration) was 9.51 ± 0.06 kcal/mol. An isoelectric point of 10.0 was determined, allowing us to calculate the Tanford and Kirkwood electrostatic factor w. This factor presented a nonlinear behavior and indicated more than one type of titratable carboxyl groups in ß-trypsin. In fact, one class of carboxyl group with a pK = 3.91 ± 0.01 and another one with a pK = 4.63 ± 0.03 were also found by hydrogen ion titration of the protein in the folded state

Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing

Reactivity of anti-thyroid antibodies to thyroglobulin tryptic fragments: comparison of autoimmune and non-autoimmune thyroid diseases

Studies concerning the antigenicity of thyroglobulin fragments allow the characterization of the epitopes but do not consider the role of heavier antigenic fragments that could result in vivo from the action of endoproteases. Here we assess the relative importance of the fragments obtained from thyroglobulin by limited proteolysis with trypsin and compare by immunoblotting their reactivity to serum from patients with autoimmune (Graves' disease and Hashimoto's thyroiditis) and non-autoimmune (subacute thyroiditis) disease. The results showed no difference in frequency of recognition of any peptide by sera from patients with autoimmune thyroiditis. In contrast, sera from patients with subacute thyroiditis reacted more frequently with a peptide of 80 kDa. These results suggest the presence of antibody subpopulations directed at fragments produced in vivo by enzymatic cleavage of thyroglobulin. This fragment and antibodies to it may represent markers for subacute thyroiditis.

Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity

The genome of Mycobacterium tuberculosis H37Rv contains three contiguous genes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginosa phospholipase C (PLC) genes. Expression of mycobacterial PLC-a and PLC-b in E. coli and M. smegmatis has been reported, whereas expression of the native proteins in M. tuberculosis H37Rv has not been demonstrated. The objective of the present study was to demonstrate that native PLC-a is expressed in M. tuberculosis H37Rv. Sera from mice immunized with recombinant PLC-a expressed in E. coli were used in immunoblots to evaluate PLC-a expression. The immune serum recognized a 49-kDa protein in immunoblots against M. tuberculosis extracts. No bands were visible in M. tuberculosis culture supernatants or extracts from M. avium, M. bovis and M. smegmatis. A 550-bp DNA fragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of ß-galactosidase activity. ß-Galactosidase activity was detected in M. smegmatis transformed with recombinant pJEM12 grown in vitro and inside macrophages. The putative promoter was active both in vitro and in vivo, suggesting that expression is constitutive. In conclusion, expression of non-secreted native PLC-a was demonstrated in M. tuberculosis.

Screening of antibacterial extracts from plants native to the Brazilian Amazon Rain Forest and Atlantic Forest

More than 20% of the world's biodiversity is located in Brazilian forests and only a few plant extracts have been evaluated for potential antibacterial activity. In the present study, 705 organic and aqueous extracts of plants obtained from different Amazon Rain Forest and Atlantic Forest plants were screened for antibacterial activity at 100 µg/ml, using a microdilution broth assay against Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Escherichia coli. One extract, VO581, was active against S. aureus (minimum inhibitory concentration (MIC) = 140 µg/ml and minimal bactericidal concentration (MBC) = 160 µg/ml, organic extract obtained from stems) and two extracts were active against E. faecalis, SM053 (MIC = 80 µg/ml and MBC = 90 µg/ml, organic extract obtained from aerial parts), and MY841 (MIC = 30 µg/ml and MBC = 50 µg/ml, organic extract obtained from stems). The most active fractions are being fractionated to identify their active substances. Higher concentrations of other extracts are currently being evaluated against the same microorganisms.