Antimicrobial activity of fractions and compounds from Calophyllumbrasiliense (Clusiaceae/Guttiferae)

Calophyllum brasiliense (Clusiaceae/Guttiferae) is a native Brazilian medicinal plant traditionally used against several diseases, including infectious pathologies. Crude methanolic extracts (CME) and two fractions, denoted non-polar (soluble in chloroform) and polar (nonsoluble in chloroform), were prepared from different parts of the plant (roots, stems, leaves, flowers and fruits) and studied. The following compounds were isolated and tested against pathogenic bacteria and yeasts by determination of the minimal inhibitory concentration (MIC): brasiliensic acid (1), gallic acid (2), epicatechin (3), protocatechuic acid (4), friedelin (5) and 1,5-dihydroxyxanthone (6). The results indicated that all the parts of the plant exhibited antimicrobial activity against Gram-positive bacteria, which are selectively inhibited by components of C. brasiliense. No activity was observed against Gram-negative bacteria and yeasts tested. Regarding the isolated compounds, substance 4 showed antimicrobial activity against all the tested microorganisms, whereas compound 6 exhibited antimicrobial activity only against Gram-positive bacteria. The results from the current study confirm and justify the popular use of this plant to treat infectious processes.     

Synthesis and biological evaluation of new non-imidazole H3-receptor antagonists of the 2-aminobenzimidazole series

A novel series of non-imidazole H(3)-receptor antagonists was developed, by chemical modification of a potent lead H(3)-antagonist composed by an imidazole ring connected through an alkyl spacer to a 2-aminobenzimidazole moiety (e.g., 2-[[3-[4(5)-imidazolyl]propyl]amino]benzimidazole), previously reported by our research group. We investigated whether the removal of the imidazole ring could allow retaining high affinity for the H(3)-receptor, thanks to the interactions undertaken by the 2-aminobenzimidazole moiety at the binding site. The imidazole ring of the lead was replaced by a basic piperidine or by a lipophilic p-chlorophenoxy substituent, modulating the spacer length from three to eight methylene groups; moreover, the substituents were moved to the 5(6) position of the benzimidazole nucleus. Within both the 2-alkylaminobenzimidazole series and the 5(6)-alkoxy-2-aminobenzimidazole one, the greatest H(3)-receptor affinity was obtained for the piperidine-substituted compounds, while the presence of the p-chlorophenoxy group resulted in a drop in affinity. The optimal chain length was different in the two series. Even if the new compounds did not reach the high receptor affinity shown by the imidazole-containing lead compound, it was possible to get good H(3)-antagonist potencies with 2-aminobenzimidazoles having a tertiary amino group at appropriate distance.     

TLR9-independent activation of B lymphocytes by bacterial DNA

The intracellular Toll-like receptor 9 (TLR9) is unique in its ability to recognize single-stranded DNA unmethylated at CpG motifs. Work from this laboratory showed that plasmid DNA is spontaneously internalized in B lymphocytes. This event is followed by the upregulation of costimulatory molecules and the acquisition of antigen presenting function by these cells. However, it is not known whether this phenomenon depends on TLR9. Because of the relevant role played by DNA-based drugs in immunotherapy and vaccination, and the central role of TLR9 signaling by CpG motifs, we decided to investigate whether signaling through TLR9 is a prerequisite for spontaneous transgenesis of lymphocytes. Here we found that transgene expression and upregulation of CD40 and CD86 costimulatory molecules was not inhibited by chloroquine treatment. Spontaneous transgenesis also occurred in B lymphocytes from TLR9-/- mice, and the injection of TLR9-/- transgenic B lymphocytes in C57Bl/6 mice induced both CD4 and CD8 T cell responses comparable to those induced by wild-type B lymphocytes. Collectively, these results suggest that plasmid DNA activates mammalian B lymphocytes through a TLR9 independent pathway.     

Pattern of expression of HtrA1 during mouse development.

The human HtrA family of proteases consists of four members: HtrA1, HtrA2, HtrA3, and HtrA4. In humans the four HtrA homologues appear to be involved in several important functions such as cell growth, apoptosis, and inflammatory reactions, and they control cell fate via regulated protein metabolism. In previous studies it was shown that the expression of HtrA1 was ubiquitous in normal adult human tissues. Here we examined the expression of HtrA1 protein and its corresponding mRNA during mouse embryogenesis using Northern blotting hybridization, RT-PCR, and immunohistochemical staining analyses. Our results indicate that HtrA1 is expressed in a variety of tissues in mouse embryos. Furthermore, this expression is regulated in a spatial and temporal manner. Relatively low levels of HtrA1 mRNA are detected in embryos at the beginning of organogenesis (E8), and the levels of expression increase during late organogenesis (E14-E19). Our results show that HtrA1 was expressed during embryonic development in specific areas where signaling by TGFbeta family proteins plays important regulatory roles. The expression of HtrA1, documented both at mRNA and protein levels by RT-PCR and immunohistochemistry in the developing nervous system, is consistent with a possible role of this protein both in dividing and postmitotic neurons, possibly via its documented inhibitory effects on TGFbeta proteins. An exhaustive knowledge of the different cell- and tissue-specific patterns of expression of HtrA1 in normal mouse embryos is essential for a critical evaluation of the exact role played by this protein during development.     

Genotoxicity assessment of Copaiba oil and its fractions in Swiss mice

Copaiba oil-resin, extracted from the trunk of Copaifera, and traditionally used in folk medicine in the treatment of various disorders, has been shown to be an effective antiinflamatory, antitumor, antitetanus, antiseptic and anti-blenorrhagea agent. As, there are few studies evaluating its genotoxicity, this aspect of the commercial oil-resin, and its volatile and resinous fractions, were evaluated in mice by comet assay and micronucleus (MN) test. A single dose of oil resin, volatile or resin fractions (500; 1,000 or 2,000 mg/kg b.w.) was administered by gavage. The chemical compositions of Copaiba oil resin and its fractions was analyzed by gas chromatography. According to comet assaying, treatment with either one did not increase DNA damage, and as to MN testing, there was no alteration in the incidence of micronucleated polychromatic erythrocytes. Chromatographic analysis of the oil-resin itself revealed sesquiterpenes, diterpenic carboxylic acid methyl esters and high levels of β-caryophyllene. Thus, it can be assumed that the oil resin and volatile and resinous fractions from the commercial product are not genotoxic or mutagenic.     

Helminthic therapy: improving mucosal barrier function

The epidemiology of autoimmune diseases and helminth infections led to suggestions that helminths could improve inflammatory conditions, which was then tested using animal models. This has translated to clinical investigations aimed at the safe and controlled reintroduction of helminthic exposure to patients suffering from autoimmune diseases (so-called 'helminthic therapy') in an effort to mitigate the inflammatory response. In this review, we summarize the results of recent clinical trials of helminthic therapy, with particular attention to mechanisms of action. Whereas previous reviews have emphasized immune regulatory mechanisms activated by helminths, we propose that enhancement of mucosal barrier function may have an equally important role in improving conditions of inflammatory bowel diseases.     

Pharmacological blockade of mGlu2/3 metabotropic glutamate receptors reduces cell proliferation in cultured human glioma cells

Glial cell proliferation in culture is under the control of metabotropic glutamate (mGlu) receptors. We have examined whether this control extends to human glioma cells. Primary cultures were prepared from surgically removed human glioblastomas. RT-PCR combined with western blot analysis showed that most of the cultures (eight out of 11) expressed group-II mGlu receptors. In two selected cultures (MZC-12 and FCN-9), the mGlu2/3 receptor antagonist, LY341495, slowed cell proliferation when applied to the growth medium from the second day after plating. This effect was reversible because linear cell growth was restored after washing out the drug. LY341495 reduced glioma cell proliferation at concentrations lower than 100 nm, which are considered as selective for mGlu2/3 receptors. In addition, its action was mimicked by the putative mGlu2/3 receptor antagonist (2S)-alpha-ethylglutamate. The anti-proliferative effect of LY341495 was confirmed by measuring [methyl-3H]-thymidine incorporation in cultures arrested in G0 phase of the cell cycle and then stimulated to proliferate by the addition of 10% fetal calf serum or 100 ng/mL of epidermal growth factor (EGF). In cultures treated with EGF, LY341495 was also able to reduce the stimulation of the mitogen-activated protein kinase (MAPK) pathway, as well as the induction of cyclin D1. Both effects, as well as decreased [methyl-3H]-thymidine incorporation, were partially reduced by co-addition of the potent mGlu2/3 receptor agonist, LY379268. We conclude that activation of group-II mGlu receptors supports the growth of human glioma cells in culture and that antagonists of these receptors should be tested for their ability to reduce tumour growth in vivo.     

Endotheliochorial Placentation in a Pangolin,Manis tetradactyla

The placenta of a pangolin, Manis tetradactyla, was examined grossly and histologically. The placenta was arranged in longitudinal bands of 2–3 mm width. Microscopically there was a deep labyrinth and an underlying layer of distended endometrial glands. A narrow junctional zone was present containing syncytiotrophoblast. Thoughout the labyrinth cytotrophoblast and syncytiotrophoblast were observed in contact with maternal capillaries. The placentation was considered to be endotheliochorial in type.     

Elastic moduli of excised constricted rat lungs

When airways constrict, the surrounding parenchyma undergoesstretch and distortion. Because of the mechanical interdependence between airways and parenchyma, the material properties of the parenchyma are important factors that modulate the degree ofbronchoconstriction. The purpose of this study was to investigate theeffect of changes in transpulmonary pressure (Ptp) and inducedconstriction on parenchymal bulk (k)and shear (µ) moduli. In excised rat lungs, pressure was measured atthe airway opening, and pressure-volume curves were obtained byimposing step decreases in volume with a calibrated syringe from totallung inflation. Calculation was made ofk during small-volume oscillations (1 Hz). Absolute lung volume at 0 cmH₂O Ptp was obtained bysaline displacement. To calculate µ, a lung-indentation test wasperformed. The lung surface was deformed with a cylindrical punch(diameter = 0.45 cm) in 0.25-mm increments, and the force required toeffect this displacement was measured by a weight balance. Measurementsof k and µ were obtained at 4 and 10 cmH₂O Ptp, and again at 4 cmH₂O Ptp, after delivery ofmethacholine aerosol (100 mg/ml) into the trachea. Values ofk and µ in rat lungs were similar tothose reported in other species. In addition, k and µ were dependent on Ptp. Afterinduced constriction, k and µ increased significantly. That k and µ can increase after induced constriction has important implicationsvis a vis the factors modulating airway narrowing.