Diaryl-dialkyl-substituted pyrazoles: regioselective synthesis and binding affinity for the estrogen receptor

We have developed two novel series of tetrasubstituted pyrazoles, embodying 1,3-diaryl-4,5-dialkyl or 3,5-diaryl-1,4-dialkyl substitution patterns. The scope of a regioselective method, developed by us earlier, was expanded to allow the synthesis of the first series of these tetrasubstituted pyrazoles directly from alpha,beta-unsaturated ketones. The binding affinity of some of these pyrazoles for the estrogen receptor (ER) subtypes ERalpha and ERbeta is very high, and the overall affinity pattern suggests the importance of three phenol substituents for high affinity, ERalpha-selective binding.     

Genetic variability in mitochondrial and nuclear genes of Larus dominicanus (Charadriiformes,Laridae) from the Brazilian coast

Several phylogeographic studies of seabirds have documented low genetic diversity that has been attributed to bottleneck events or individual capacity for dispersal. Few studies have been done in seabirds on the Brazilian coast and all have shown low genetic differentiation on a wide geographic scale. The Kelp Gull is a common species with a wide distribution in the Southern Hemisphere. In this study, we used mitochondrial and nuclear markers to examine the genetic variability of Kelp Gull populations on the Brazilian coast and compared this variability with that of sub-Antarctic island populations of this species. Kelp Gulls showed extremely low genetic variability for mitochondrial markers (cytb and ATPase) and high diversity for a nuclear locus (intron 7 of the β-fibrinogen). The intraspecific evolutionary history of Kelp Gulls showed that the variability found in intron 7 of the β-fibrinogen gene was compatible with the variability expected under neutral evolution but suggested an increase in population size during the last 10,000 years. However, none of the markers revealed evidence of a bottleneck population. These findings indicate that the recent origin of Kelp Gulls is the main explanation for their nuclear diversity, although selective pressure on the mtDNA of this species cannot be discarded.     

Cancer/testis antigens expression and autologous serological response in a set of Brazilian non-Hodgkin’s lymphoma patients

Background

Based on their tumor-associated expression pattern, cancer/testis antigens (CTAs) are considered potential targets for cancer immunotherapy. We aim to evaluate the expression of CTAs in non-Hodgkin??s lymphoma (NHL) samples and the ability of these patients to elicit spontaneous humoral immune response against CTAs.

Methods

Expression of MAGE-A family, CT7/MAGE-C1, CT10/MAGE-C2, GAGE and NY-ESO-1 was analyzed by immunohistochemistry in a tissue microarray generated from 106 NHL archival cases. The humoral response against 19 CTAs was tested in 97 untreated NHL serum samples using ELISA technique.

Results

11.3?% of NHL tumor samples expressed at least 1 CTA. MAGE-A family (6.6?%), GAGE (5.7?%) and NY-ESO-1(4.7?%) were the most frequently expressed antigens. We found no statistically significant correlation between CTA positivity and clinical parameters such as NHL histological subtype, Ann Arbor stage, international prognostic index score, response to treatment and overall survival. Humoral response against at least 1 CTA was observed in 16.5?% of NHL serum samples. However, overall seroreactivity was low, and strong titers (>1:1000) were observed in only two diffuse large B-cell lymphomas patients against CT45.

Conclusion

Our findings are in agreement with most of published studies in this field to date and suggest an overall low expression of CTAs in NHL patients. However, as many new CTAs have been described recently and some of them are found to be highly expressed in NHL cell lines and tumor samples, further studies exploring the expression of different panels of CTAs are needed to evaluate their role as candidates for immunotherapy in NHL patients.     

Changes in membrane lipid composition cause alterations in epithelial cell–cell adhesion structures in renal papillary collecting duct cells

In epithelial tissues, adherens junctions (AJ) mediate cell–cell adhesion by using proteins called E-cadherins, which span the plasma membrane, contact E-cadherin on other cells and connect with the actin cytoskeleton inside the cell. Although AJ protein complexes are inserted in detergent-resistant membrane microdomains, the influence of membrane lipid composition in the preservation of AJ structures has not been extensively addressed. In the present work, we studied the contribution of membrane lipids to the preservation of renal epithelial cell–cell adhesion structures. We biochemically characterized the lipid composition of membranes containing AJ complexes. By using lipid membrane-affecting agents, we found that such agents induced the formation of new AJ protein-containing domains of different lipid composition. By using both biochemical approaches and fluorescence microscopy we demonstrated that the membrane phospholipid composition plays an essential role in the in vivo maintenance of AJ structures involved in cell–cell adhesion structures in renal papillary collecting duct cells.     

Using mass spectrometry to explore the neglected glycan moieties of the antiophidic proteins DM43 and DM64

The resistance of the opossum Didelphis aurita to Bothrops snake venoms is attributed to the opossum's antihemorrhagic (DM43) and antimyotoxic (DM64) acidic serum glycoproteins. The aim of this study was to characterize the N-glycosylation sites of these antiophidic proteins and to determine whether their glycans influence the biological activity measured by in vitro assays. Our experimental pipeline included the sequential enzymatic digestion of the inhibitors with two different proteinases (trypsin and endoproteinase Asp-N) and eventually with trypsin, peptide-N-glycosidase F (PNGase F) and endoproteinase Asp-N, used in that order. All of the peptide and protein samples were analyzed by MALDI-TOF/TOF MS. The results experimentally confirmed the putative N-glycosylation sites of DM43 (Asn23, Asn156, Asn160, and Asn175) and DM64 (Asn46, Asn179, Asn183, and Asn379). Following treatments with specific glycosidases, complex-type oligosaccharides containing galactose and sialic acid could be assigned to both proteins. The removal of these monosaccharide units by exoglycosidase digestion did not measurably affect the inhibitory activity. In contrast, partially deglycosylated DM43 treated with PNGase F under nondenaturing conditions was half as effective as native DM43. In conclusion, we have demonstrated that the contribution of the carbohydrate portion of these potentially therapeutic molecules, for their mechanism of action, should not be overlooked.     

Proteomic analysis of a multi-resistant clinical Escherichia coli isolate of unknown genomic background

Horizontal transfer of gene clusters occurs in Escherichia coli (E. coli), which could lead to evolution of new pathovars and improve survival fitness. However, this genetic event results in genomic plasticity which is a hindrance for proteomic characterization of strains with unknown genetic backgrounds. To characterize such isolate with many specific genetic variations we used the recently in-house designed MSMSpdbb software which merges protein databases from several sources of E. coli including type strains and other commensal and pathogenic isolates. We selected a multidrug resistant clinical isolate in order to check the capacity of our approach to identify selected protein markers. From the 1596 identified proteins, we found important virulence factors such as IutA, OmpA, TraT and selected enzymes conferring antibiotic resistance, such as CTX-M-15 (Extended-Spectrum Beta Lactamase - ESBL) and AAC(6′)-Ib-cr (to aminoglycoside + fluoroquinolone). In addition, we compared the protein identifications with E. coli gene annotation and found that 27% of the proteins identified in the present study corresponded to the pan-genome of E. coli species and are only present in a subset of strains. This demonstrates the ability of our approach to characterize the proteome of bacterial strains with complex genomic plasticity even without its genomic information.     

α-Gal immunization positively impacts Trypanosoma cruzi colonization of heart tissue in a mouse model

Chagas disease, caused by the parasite Trypanosoma cruzi, is considered endemic in more than 20 countries but lacks both an approved vaccine and limited treatment for its chronic stage. Chronic infection is most harmful to human health because of long-term parasitic infection of the heart. Here we show that immunization with a virus-like particle vaccine displaying a high density of the immunogenic α-Gal trisaccharide (Qβ-αGal) induced several beneficial effects concerning acute and chronic T. cruzi infection in α1,3-galactosyltransferase knockout mice. Approximately 60% of these animals were protected from initial infection with high parasite loads. Vaccinated animals also produced high anti-αGal IgG antibody titers, improved IFN-γ and IL-12 cytokine production, and controlled parasitemia in the acute phase at 8 days post-infection (dpi) for the Y strain and 22 dpi for the Colombian strain. In the chronic stage of infection (36 and 190 dpi, respectively), all of the vaccinated group survived, showing significantly decreased heart inflammation and clearance of amastigote nests from the heart tissue.     

Genotoxic evaluation of extracts from Aplysina fulva, a Brazilian marine sponge

A range of biologically active secondary metabolites with pharmacological application has been reported to occur in marine sponges. The present study was undertaken to provide a set of data on the safety of a hydro-alcoholic extract (ALE) and an aqueous fraction (AQE) from Aplysina fulva Pallas, 1766 (Aplysinidae, Verongida, Porifera). Salmonella typhimurium strains TA97, TA98, TA100 and TA102, Escherichia coli strains PQ65, OG40, OG100, PQ35 and PQ37 and Balb/c 3T3 mouse fibroblasts were used to detect induction of DNA lesions by ALE and AQE. Assays used for these analyses were a bacterial (reverse) mutation assay (Ames test), the SOS-chromotest and the comet assay. Both extracts presented identical infrared 2-oxazolidone spectra. ALE treatment induced a higher frequency of type-4 comets, indicative of increasing DNA migration, in the alkaline comet assay. ALE also induced a weak genotoxic effect, as expressed by the induction factor (IF) values in the test with E. coli strain PQ35 (IF=1.5) and by cytotoxic effects in strains PQ35, PQ65 and PQ37. Positive SOS induction (IF=1.7) was detected in strain PQ37 treated with diluted AQE. No genotoxic effects were observed in strains PQ35, PQ65, OG40 and OG 100 after treatment with AQE dilutions. Using the bacterial (reverse) mutation test and survival assays with or without S9 mix, after 60min of pre-incubation, we observed for strain TA97 treated with ALE a weak mutagenic response (MI=2.2), while cytotoxic effects were seen for strains TA98, TA100 and TA102. AQE did not show mutagenic activity in any of the strains tested, but a weak cytotoxic effect was noted in strain TA102. Our data suggest that both ALE and AQE from A. fulva induce DNA breaks leading to cytotoxicity and mutagenicity under the conditions used.     

Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant major surface protein 5 (rMSP5) and initial body (IB) antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2%) and specificities (100% for rMSP5 and 93.8% for IB ELISA) which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA) to 15% (IB ELISA) of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1% by rMSP5 ELISA and 79.7% by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.