Floristic similarities between lowland and montane areas of Atlantic Coastal Forest in Northeastern Brazil

Studies undertaken in the Atlantic Coastal Forest have revealed a notable floristic heterogeneity within this vegetation type in NE Brazil. However, there is still a great need for detailed comparisons of the floristic relationships between the various forest types found there. This work presents an analysis of the floristic similarity (at the species level) of these forest as revealed by floristic surveys, with the aim of better understanding and defining this vegetation. Using a binary matrix, grouping, ordering, and TWINSPAN analysis were performed on 742 tree/shrub species listed in 35 different plant surveys. These tree/shrub species were divided by these analysis into two large floristic groups – ombrophilous and semideciduous. The semideciduous group was formed, in general, by forest areas located at altitudes above 700 m (montane forests), and could itself be divided into two subgroups. The first subgroup was located more inland (Pernambuco), while the second subgroup was located nearer the coast (within the states of Pernambuco and Ceará). The ombrophilous group was quite heterogeneous, but could also be divided into two floristic subgroups: i) lowland forests (below 100 m a.s.l) in the states of Pernambuco, Paraíba, and in two areas of Bahia, as well as some montane forests (in Pernambuco, between 640 and 900 m a.s.l.); ii) lowlands forests in the states of Alagoas and Bahia. This latter subgroup is the largest and best defined, and has the highest degree of internal similarity. Nonetheless, it can be further subdivided into two smaller classes, one in Alagoas and the other in Bahia State. The results of this study demonstrate that the concept of the Atlantic Coastal Forest could also include montane ombrophilous forests in Pernambuco State, as these forests form a single floristic unit together with lowland forests in that state. This revised version was published online in June 2006 with corrections to the Cover Date.     

Insecticidal peptides from the theraposid spider Brachypelma albiceps: An NMR-based model of Ba2

Soluble venom and purified fractions of the theraposid spider Brachypelma albiceps were screened for insecticidal peptides based on toxicity to crickets. Two insecticidal peptides, named Ba1 and Ba2, were obtained after the soluble venom was separated by high performance liquid chromatography and cation exchange chromatography. The two insecticidal peptides contain 39 amino acid residues and three disulfide bonds, and based on their amino acid sequence, they are highly identical to the insecticidal peptides from the theraposid spiders Aphonopelma sp. from the USA and Haplopelma huwenum from China indicating a relationship among these genera. Although Ba1 and Ba2 were not able to modify currents in insect and vertebrate cloned voltage-gated sodium ion channels, they have noteworthy insecticidal activities compared to classical arachnid insecticidal toxins indicating that they might target unknown receptors in insect species. The most abundant insecticidal peptide Ba2 was submitted to NMR spectroscopy to determine its 3-D structure; a remarkable characteristic of Ba2 is a cluster of basic residues, which might be important for receptor recognition.     

Production and partial characterization of extracellular proteinases from <Emphasis Type="Italic">Streptomyces malaysiensis</Emphasis>, isolated from a Brazilian cerrado soil

Streptomyces malaysiensis AMT-3, isolated from a Brazilian cerrado soil, showed proteolytic activities detected by gelatin–sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum proteinase production was obtained when using 2.5% wheat bran and 0.1% yeast extract in the culture medium, after 5 days incubation at 30°C. The enzymatic complex degraded gelatin optimally at pH 7.0, and under these conditions eight proteolytic bands (four serine-proteinases and four metaloproteinases), ranging from 20 to 212 kDa, were detected on the culture supernatant filtrates. In addition, a 35-kDa proteinase was thermostable at 60°C for 120 min. These results point out to the applicability of gelatin zymograms in the characterization of crude enzymatic complexes. According to our results, this enzymatic complex could be used for biotechnological applications.     

Sugarcane bagasse enzymatic hydrolysis: rheological data as criteria for impeller selection

The aim of this work was to select an efficient impeller to be used in a stirred reactor for the enzymatic hydrolysis of sugar cane bagasse. All experiments utilized 100 g (dry weight)/l of steam-pretreated bagasse, which is utilized in Brazil for cattle feed. The process was studied with respect to the rheological behavior of the biomass hydrolysate and the enzymatic conversion of the bagasse polysaccharides. These parameters were applied to model the power required for an impeller to operate at pilot scale (100 l) using empirical correlations according to Nagata [16]. Hydrolysis experiments were carried out using a blend of cellulases, β-glucosidase, and xylanases produced in our laboratory by Trichoderma reesei RUT C30 and Aspergillus awamori. Hydrolyses were performed with an enzyme load of 10 FPU/g (dry weight) of bagasse over 36 h with periodic sampling for the measurement of viscosity and the concentration of glucose and reducing sugars. The mixture presented pseudoplastic behavior. This rheological model allowed for a performance comparison to be made between flat-blade disk (Rushton turbine) and pitched-blade (45°) impellers. The simulation showed that the pitched blade consumed tenfold less energy than the flat-blade disk turbine. The resulting sugar syrups contained 22 g/l of glucose, which corresponded to 45% cellulose conversion.     

Optimization of gene delivery methods in <Emphasis Type="Italic">Xenopus laevis</Emphasis> kidney (A6) and Chinese hamster ovary (CHO) cell lines for heterologous expression of <Emphasis Type="Italic">Xenopus</Emphasis> inner ear genes

The Xenopus inner ear provides a useful model for studies of hearing and balance because it shares features with the mammalian inner ear, and because amphibians are capable of regenerating damaged mechanosensory hair cells. The structure and function of many proteins necessary for inner ear function have yet to be elucidated and require methods for analysis. To this end, we seek to characterize Xenopus inner ear genes outside of the animal model through heterologous expression in cell lines. As part of this effort, we aimed to optimize physical (electroporation), chemical (lipid-mediated; Lipofectamine? 2000, Metafectene® Pro), and biological (viral-mediated; BacMam virus Cellular Lights? Tubulin-RFP) gene delivery methods in amphibian (Xenopus; A6) cells and mammalian (Chinese hamster ovary (CHO)) cells. We successfully introduced the commercially available pEGFP-N3, pmCherry-N1, pEYFP-Tubulin, and Cellular Lights? Tubulin-RFP fluorescent constructs to cells and evaluated their transfection or transduction efficiencies using the three gene delivery methods. In addition, we analyzed the transfection efficiency of a novel construct synthesized in our laboratory by cloning the Xenopus inner ear calcium-activated potassium channel β1 subunit, then subcloning the subunit into the pmCherry-N1 vector. Every gene delivery method was significantly more effective in CHO cells. Although results for the A6 cell line were not statistically significant, both cell lines illustrate a trend towards more efficient gene delivery using viral-mediated methods; however the cost of viral transduction is also much higher. Our findings demonstrate the need to improve gene delivery methods for amphibian cells and underscore the necessity for a greater understanding of amphibian cell biology.     

Nanomolar ouabain elicits apoptosis through a direct action on HeLa cell mitochondria

The steroid Na⁺/K⁺ ATPase (NKA) blocker ouabain has been shown to exhibit pro-apoptotic effects in various cell systems; however, the mechanism involved in those effects is unclear. Here, we have demonstrated that incubation of HeLa cells during 24 h with nanomolar concentrations of ouabain or digoxin causes apoptotic death of 30–50% of the cells. Ouabain caused the activation of caspases-3/7 and -9; however, caspase-8 was unaffected. The fact that compound Z-LEHD-FMK reduced both apoptosis and caspase-9 activation elicited by ouabain, suggest a mitochondrially-mediated pathway. This was strengthened by the fact that ouabain caused ATP depletion and the release of mitochondrial cytochrome c into the cytosol. Furthermore, upon ouabain treatment mitochondrial disruption and redistribution into the cytosol were observed. A mitochondrial site of action for ouabain was further corroborated by tight co-localisation of fluorescent ouabain with mitochondria. Finally, in ouabain-treated cells the histamine-elicited elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_c) suggests an additional effect on the endoplasmic reticulum (ER) leading to Ca²⁺ store depletion. We conclude that fluorescent ouabain is taken up and tightly co-localises with mitochondria of HeLa cells. This indicates that apoptosis may be triggered by a direct action of ouabain on mitochondria.     

In vitro and in vivo antimalarial activity and cytotoxicity of extracts,fractions and a substance isolated from the Amazonian plant Tachia grandiflora (Gentianaceae)

Tachia sp. are used as antimalarials in the Amazon Region and in vivo antimalarial activity of a Tachia sp. has been previously reported. Tachia grandiflora Maguire and Weaver is an Amazonian antimalarial plant and herein its cytotoxicity and antimalarial activity were investigated. Spectral analysis of the tetraoxygenated xanthone decussatin and the iridoid aglyone amplexine isolated, respectively, from the chloroform fractions of root methanol and leaf ethanol extracts was performed. In vitro inhibition of the growth of Plasmodium falciparum Welch was evaluated using optical microscopy on blood smears. Crude extracts of leaves and roots were inactive in vitro. However, chloroform fractions of the root and leaf extracts [half-maximal inhibitory concentration (IC50) = 10.5 and 35.8 µg/mL, respectively] and amplexine (IC50= 7.1 µg/mL) were active in vitro. Extracts and fractions were not toxic to type MRC-5 human fibroblasts (IC50> 50 µg/mL). Water extracts of the roots of T. grandiflora administered by mouth were the most active extracts in the Peters 4-day suppression test in Plasmodium berghei-infected mice. At 500 mg/kg/day, these extracts exhibited 45-59% inhibition five to seven days after infection. T. grandiflora infusions, fractions and isolated substance have potential as antimalarials.     

Cross-Talk Between Body Iron Stores and Diabetes: Iron Stores are Associated with Activity and Microsatellite Polymorphism of the Heme Oxygenase and Type 2 Diabetes

To assess the relationship between the length of (GT) _n repeats in HO-1 gene promoter and heme oxygenase (HO) enzymatic activity in mononuclear cells with iron (Fe) stores in type 2 diabetic mellitus (DM2) patients and metabolic syndrome (MS) subjects, we studied 163 patients with DM2, 185 with MS, and 120 controls subjects. We evaluated iron status (hemoglobin and serum Fe, ferritin, and transferrin receptor), and we determined the length of (GT) _n repeats in HO-1 gene promoter by capillary electrophoresis and HO enzymatic activity in mononuclear cells and assessed the relationship between these results and Fe stores. Only 1/163, 6/185, and 7/120 had iron deficiency anemia in DM2 patients, MS subjects, and controls, respectively. No iron overload (ferritin >200 μg/L) was detected in all the subjects studied. DM2 patients had higher iron deposits, total body iron, and heme oxygenase activity (a suggestion of high oxidative stress condition) than MS subjects and controls. In DM2, we found a positive association between serum iron and HO activity. There were no difference in allelic frequency between the three groups; however, among DM2 and MS patients, the frequency of short/medium (SM) genotype of (GT) _n repetition was increased and medium/medium (MM) genotype of (GT) _n repetition was lower than controls. These results imply that DM2 patients and individuals with MS carrying SM repeats might have higher susceptibility to develop diabetes consequences. This increased susceptibility could be Fe-mediated oxidative stress.