Purification of betulinic acid from Eugenia florida (Myrtaceae) by high-speed counter-current chromatography

A high yield of betulinic acid (up to 17% from the ethanolic extract) was found in the leaves of Eugenia florida collected in south-eastern Brazil, making this species a potential commercial source of the title compound. Extracts of E. florida were subjected to solvent partition, and rapid high-speed counter-current chromatography (HSCCC) was applied to the semi-crude extracts to afford betulinic acid in high purity. The mobile and stationary phases were derived from the two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (10:5:2.5:1). The developing solvent system (stationary and mobile phases) for optimum HSCCC separation was chosen by dissolving the fraction to be chromatographed in the proposed solvent mixture and determining the amount of betulinic acid in each phase by densitometric TLC. Purified betulinic acid was characterized by 13C-NMR, GC-MS and co-injection of its methyl ester with standards in GC-FID. The HSCCC technique is commonly employed to isolate triterpene glycosides, but is applied in this study to an aglycone.     

Syntheses, structures and electrochemical study of π-acetylene complexes of cobalt

The preparation and characterisation of the complexes [Co₂(CO)₄(PMe₃)₂][Co₂(CO)₆](Me₃SiC₂C₂SiMe₃) (4), [Co₂(CO)₄(dppm)][Co₂(CO)₆](Me₃SiC₂C₂H) (5), [Co₂(CO)₄(dppa)][Co₂(CO)₆](Me₃SiC₂C₂SiMe₃) (6), [Co₂(CO)₄(dppm)]₂[Co₂(CO)₆](Me₃SiC₂CCC₂C₂SiMe₃) (7) and [{SiMe₃(Co₂(CO)₄(dppm))C₂}₂(HCC)(1,3,5-C₆H₃)] (8) are described. An electrochemical study of the complexes 5-8 and of the related [Co₂(CO)₄(dppm)]₂(Me₃SiC₂(CC)₂C₂SiMe₃) (1), [Co₂(CO)₄(dppa)]₂(Me₃SiC₂C₂SiMe₃) (2) and [{SiMe₃(Co₂(CO)₄(dppm))C₂}(HCC)₂(1,3,5-C₆H₃)] (3) is presented by means of the cyclic and square-wave voltammetry techniques. Crystals of 8 suitable for single-crystal X-ray diffraction were grown and the molecular structure of this compound is discussed.     

Identification of a Site in Sar1 Involved in the Interaction with the Cytoplasmic Tail of Glycolipid Glycosyltransferases

Glycolipid glycosyltransferases (GGT) are transported from the endoplasmic reticulum (ER) to the Golgi, their site of residence, via COPII vesicles. An interaction of a (R/K)X(R/K) motif at their cytoplasmic tail (CT) with Sar1 is critical for the selective concentration in the transport vesicles. In this work using computational docking, we identify three putative binding pockets in Sar1 (sites A, B, and C) involved in the interaction with the (R/K)X(R/K) motif. Sar1 mutants with alanine replacement of amino acids in site A were tested in vitro and in cells. In vitro, mutant versions showed a reduced ability to bind immobilized peptides with the CT sequence of GalT2. In cells, Sar1 mutants (Sar1^D198A) specifically affect the exiting of GGT from the ER, resulting in an ER/Golgi concentration ratio favoring the ER. Neither the typical Golgi localization of GM130 nor the exiting and transport of the G protein of the vesicular stomatitis virus were affected. The protein kinase inhibitor H89 produced accumulation of Sec23, Sar1, and GalT2 at the ER exit sites; Sar1^D189A also accumulated at these sites, but in this case GalT2 remained disperse along ER membranes. The results indicate that amino acids in site A of Sar1 are involved in the interaction with the CT of GGT for concentration at ER exiting sites.     

Localization and Functionality of the Inflammasome in Neutrophils

Neutrophils represent the major fraction of circulating immune cells and are rapidly recruited to sites of infection and inflammation. The inflammasome is a multiprotein complex that regulates the generation of IL-1 family proteins. The precise subcellular localization and functionality of the inflammasome in human neutrophils are poorly defined. Here we demonstrate that highly purified human neutrophils express key components of the NOD-like receptor family, pyrin domain containing 3 (NLRP3), and absent in melanoma 2 (AIM2) inflammasomes, particularly apoptosis-associated speck-like protein containing a CARD (ASC), AIM2, and caspase-1. Subcellular fractionation and microscopic analyses further showed that inflammasome components were localized in the cytoplasm and also noncanonically in secretory vesicle and tertiary granule compartments. Whereas IL-1β and IL-18 were expressed at the mRNA level and released as protein, highly purified neutrophils neither expressed nor released IL-1α at baseline or upon stimulation. Upon inflammasome activation, highly purified neutrophils released substantially lower levels of IL-1β protein compared with partially purified neutrophils. Serine proteases and caspases were differentially involved in IL-1β release, depending on the stimulus. Spontaneous activation of the NLRP3 inflammasome in neutrophils in vivo affected IL-1β, but not IL-18 release. In summary, these studies show that human neutrophils express key components of the inflammasome machinery in distinct intracellular compartments and release IL-1β and IL-18, but not IL-1α or IL-33 protein. Targeting the neutrophil inflammasome may represent a future therapeutic strategy to modulate neutrophilic inflammatory diseases, such as cystic fibrosis, rheumatoid arthritis, or sepsis.     

DNA damage in the kidney tissue cells of the fish Rhamdia quelen after trophic contamination with aluminum sulfate

Even though aluminum is the third most common element present in the earth''s crust, information regarding its toxicity remains scarce. It is known that in certain cases, aluminum is neurotoxic, but its effect in other tissues is unknown. The aim of this work was to analyze the genotoxic potential of aluminum sulfate in kidney tissue of the fish Rhamdia quelen after trophic contamination for 60 days. Sixty four fish were subdivided into the following groups: negative control, 5 mg, 50 mg and 500 mg of aluminum sulfate per kg of fish. Samples of the posterior kidney were taken and prepared to obtain mitotic metaphase, as well as the comet assay. The three types of chromosomal abnormalities (CA) found were categorized as chromatid breaks, decondensation of telomeric region, and early separation of sister chromatids. The tests for CA showed that the 5 mg/kg and 50 mg/kg doses of aluminum sulfate had genotoxic potential. Under these treatments, early separation of the sister chromatids was observed more frequently and decondensation of the telomeric region tended to increase in frequency. We suggest that structural changes in the proteins involved in DNA compaction may have led to the decondensation of the telomeric region, making the DNA susceptible to breaks. Moreover, early separation of the sister chromatids may have occurred due to changes in the mobility of chromosomes or proteins that keep the sister chromatids together. The comet assay confirmed the genotoxicity of aluminum sulfate in the kidney tissue of Rhamdia quelen at the three doses of exposure.     

DNA vaccines against dengue virus type 2 based on truncate envelope protein or its domain III

Two DNA vaccines were constructed encoding the ectodomain (domains I, II and III) of the DENV2 envelope protein (pE1D2) or only its domain III (pE2D2), fused to the human tissue plasminogen activator signal peptide (t-PA). The expression and secretion of recombinant proteins was confirmed in vitro in BHK cells transfected with the two plasmids, detected by immunofluorescence or immunoprecipitation of metabolically labeled gene products, using polyclonal and monoclonal antibodies against DENV2. Besides, results reveal that the ectodomain of the E protein can be efficiently expressed in vivo, in a mammalian system, without the prM protein that is hypothesized to act as a chaperonin during dengue infection. Balb/c mice were immunized with the DNA vaccines and challenged with a lethal dose of DENV2. All pE1D2-vaccinated mice survived challenge, while 45% of animals immunized with the pE2D2 died after infection. Furthermore, only 10% of pE1D2-immunized mice presented some clinical signs of infection after challenge, whereas most of animals inoculated with the pE2D2 showed effects of the disease with high morbidity degrees. Levels of neutralizing antibodies were significantly higher in pE1D2-vaccinated mice than in pE2D2-immunized animals, also suggesting that the pE1D2 vaccine was more protective than the pE2D2.     

Sugarcane (Saccharum X officinarum): A Reference Study for the Regulation of Genetically Modified Cultivars in Brazil

Global interest in sugarcane has increased significantly in recent years due to its economic impact on sustainable energy production. Sugarcane breeding and better agronomic practices have contributed to a huge increase in sugarcane yield in the last 30?years. Additional increases in sugarcane yield are expected to result from the use of biotechnology tools in the near future. Genetically modified (GM) sugarcane that incorporates genes to increase resistance to biotic and abiotic stresses could play a major role in achieving this goal. However, to bring GM sugarcane to the market, it is necessary to follow a regulatory process that will evaluate the environmental and health impacts of this crop. The regulatory review process is usually accomplished through a comparison of the biology and composition of the GM cultivar and a non-GM counterpart. This review intends to provide information on non-GM sugarcane biology, genetics, breeding, agronomic management, processing, products and byproducts, as well as the current technologies used to develop GM sugarcane, with the aim of assisting regulators in the decision-making process regarding the commercial release of GM sugarcane cultivars.     

Biological and conformational evaluation of angiotensin II lactam bridge containing analogues

Angiotensin II (AII) is the active octapeptide product of the renin enzymatic cascade, which is responsible for sustaining blood pressure. In an attempt to establish the AII-receptor-bound conformation of this octapeptide, we designed conformationally constrained analogues by scanning the entire AII sequence with an i-(i+2) and i-(i+3) lactam bridge consisting of an Asp-(Xaa)(n)-Lys scaffold. Most analogues presented low agonistic activity when compared to AII in the different bioassays tested. The exceptions are cyclo(0-1a) [Asp(0), endo-(Lys(1a))]-AII (1) and [Asp(0), endo-(Lys(1a))]-AII (2), both of which showed activity similar to AII. Based on peptide 1 and the analogue cyclo(3-5)[Sar(1), Asp(3), Lys(5)]-AII characterized by Matsoukas et al., we analyzed the agonistic and antagonistic activities, respectively, through a new monocyclic peptide series synthesized by using the following combinations of residues as bridgehead elements for the lactam bond formation: D- or L-Asp combined with D- or L-Lys or L-Glu combined with L-Orn. Six analogues showed an approximately 20% increase in biological activity when compared with peptide (1) and were equipotent to AII. In contrast, six analogues presented antagonistic activity. These results suggest that the position of the lactam bridge is more important than the bridge length or chirality for recognition of and binding to the angiotensin II AT1-receptor.     

Towards a framework for the study of the neural correlates of aesthetic preference

Aiming to provide a tentative framework for the study of the neural correlates of aesthetic preference, we review three recent neuroimaging studies carried out with the purpose of locating brain activity associated with decisions about the beauty of visual stimuli (Cela-Conde et al., 2004; Kawabata and Zeki, 2004; Vartanian and Goel, 2004). We find that the results of the three studies are not in line with previous neuropsychological data. Moreover, there are no coincidences among their results. However, when they are mapped on to Chatterjee's (2003) neuropsychological model of aesthetic preference it becomes clear that neuroimaging data are not contradictory, but complementary, and their interpretation is enriched. The results of these studies suggest that affective processes have an important role in aesthetic preference, and that they are integrated with cognitive processes to reach a decision regarding the beauty of visual stimuli. Future studies must aim to clarify whether certain methodological procedures are better suited to study any of the particular cognitive operations involved in aesthetic preference, and ascertain the extent to which the proposed framework is compatible with the aesthetic appreciation of musical stimuli.     

Early Transplantation of Bone Marrow Mononuclear Cells Promotes Neuroprotection and Modulation of Inflammation After Status Epilepticus in Mice by Paracrine Mechanisms

Status epilepticus (SE) is a severe clinical manifestation of epilepsy associated with intense neuronal loss and inflammation, two key factors involved in the pathophysiology of temporal lobe epilepsy. Bone marrow mononuclear cells (BMMC) attenuated the consequences of pilocarpine-induced SE, including neuronal loss, in addition to frequency and duration of seizures. Here we investigated the effects of BMMC transplanted early after the onset of SE in mice, as well as the involvement of soluble factors produced by BMMC in the effects of the cell therapy. Mice were injected with pilocarpine for SE induction and randomized into three groups: transplanted intravenously with 1 × 10⁷ BMMC isolated from GFP transgenic mice, injected with BMMC lysate, and saline-treated controls. Cell tracking, neuronal counting in hippocampal subfields and cytokine analysis in the serum and brain were performed. BMMC were found in the brain 4 h following transplantation and their numbers progressively decreased until 24 h following transplantation. A reduction in hippocampal neuronal loss after SE was found in mice treated with live BMMC and BMMC lysate when compared to saline-treated, SE-induced mice. Moreover, the expression of inflammatory cytokines IL-1β, TNF-α, IL-6 was decreased after injection of live BMMC and to a lesser extent, of BMMC lysate, when compared to SE-induced controls. In contrast, IL-10 expression was increased. Analysis of markers for microglia activation demonstrated a reduction of the expression of genes related to type 1-activation. BMMC transplantation promotes neuroprotection and mediates anti-inflammatory effects following SE in mice, possibly through the secretion of soluble factors.