High performance liquid chromatography analysis of a 4-anilinoquinazoline derivative (PD153035), a specific inhibitor of the epidermal growth factor receptor tyrosine kinase, in rat plasma

The quinazoline derivative, 4-N-(3'-bromo-phenyl)amino-6,7-dimethoxyquinazoline (PD153035), has recently been identified as a potential drug for the treatment of proliferative disease. Here, we report a sensitive high performance liquid chromatography (HPLC)-based quantitative detection method for measurement of PD153035 levels in rat plasma. Sample pretreatment involved a two-step extraction with chloroform. The analytes were separated on a column packed with OmniSpher C18 material and eluted with acetonitrile-0.1 M ammonium acetate, pH 7.2 (70:30, v/v). The column effluent was monitored by UV detection at 330 nm. A linear response was achieved over the concentration range 0.50-100.00 microM using multilevel calibration with an internal standard. The analytical method inter- and intra-run accuracy and precision were better than +/-15%. The lower limit of quantification was 0.50 microM. The method has been applied to study the preclinical pharmacokinetics of this compound in rats.     

Single domain antibodies derived from dromedary lymph node and peripheral blood lymphocytes sensing conformational variants of prostate-specific antigen

The importance of the lymphocyte source to generate hybridomas or to construct antibody gene libraries from which to identify potent monoclonal antibodies is understudied. However, the few comparative studies that exist seem to favor the lymph node tissue as a B-cell source. Here the peripheral blood and lymph node lymphocytes of a dromedary immunized with prostate-specific antigen (PSA) have been employed to clone two independent gene banks of the variable domains of heavy-chain antibodies (i.e. the VHHs). Several PSA-specific VHHs were retrieved after panning of these phage-displayed VHH libraries. Some of them were derived from the same B-cell lineage, possibly reflecting the restricted primary repertoire of heavy-chain antibodies. Other binders originated from different B-cell lineages and apparently converged toward a striking homologous amino acid sequence motif in their CDR3. This illustrates the strong somatic hypermutation and stringent antigen-driven selection ongoing in these animals. Although the various antigen binders exhibit a broad range of kinetic rate constants for their interaction with the PSA, leading to equilibrium constants from 70 pM to 100 nM, no significant difference existed between the binders from the two B-cell sources. The VHHs of both libraries were categorized in three groups based on nonoverlapping epitopes. Some of these VHHs could inhibit and others could enhance the proteolytic activity of the antigen. Remarkably, VHHs seem to sense or induce conformational changes on different PSA isoforms, a feature that might be exploited to study the PSA conformational flexibility and to discriminate the stages of prostate cancer.     

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis of oligosaccharides and oligosaccharide alditols obtained by hydrolysis of agaroses and carrageenans, two important types of red seaweed polysaccharides

MALDI-TOF mass spectrometry analyses of several oligosaccharides (aldoses) and oligosaccharide alditols derived from agaroses, kappa- and iota-carrageenans using different matrices (2,5-dihydroxybenzoic acid, nor-harmane, ferulic acid, and the ionic liquid matrices 2,5-dihydroxybenzoic acid-n-butylamine and ferulic acid-n-butylamine) were conducted. These carbohydrates were selected as model compounds to study the MALDI prompt and post-source decay (PSD) fragmentation processes of both families of oligosaccharides. Sulfated alditols showed in the negative-ion mode the molecular ion as [M−Na]⁻ together with the species yielded by their prompt fragmentation (mainly desulfation) while the sulfated oligosaccharides (aldoses) showed mainly glycosidic prompt fragmentation (glycosidic C-cleavages and desulfation). Non-sulfated aldoses and alditols, which could only be analyzed in positive-ion mode ([M+Na]⁺), did not suffer any prompt fragmentation. The former yielded cross-ring fragmentation in the PSD mode. Best results were obtained by using 2,5-dihydroxybenzoic acid and/or nor-harmane as matrices for all the compounds studied.     

Complete 1H and 13C NMR assignment of digeneaside, a low-molecular-mass carbohydrate produced by red seaweeds

Digeneaside (alpha-D-mannopyranosyl-(1-->2)-D-glycerate) was extracted from the red algae, Bostrychia binderii, and purified by adsorption and gel-filtration chromatography. HPLC and ESI-MS techniques were used to follow purification steps and characterize digeneaside. NMR spectroscopy experiments (1D 1H, 13C, DEPT and 2D HMQC, COSY and TOCSY) were used to fully assign the 1H and 13C spectra.     

In vitro anti-Neisseria gonorrhoeae activity of Terminalia macroptera leaves

We used the agar dilution method to evaluate the antibacterial effect of Terminalia macroptera leaf (Tml) extract against nine reference and clinical Neisseria gonorrhoeae strains, including penicillin- and tetracycline-resistant and -susceptible strains. Tml possesses anti-N. gonorrhoeae activity against all of the strains and the minimum inhibitory concentrations (MIC) were between 100 and 200 microg ml(-1). We then used a liquid-liquid partition method to divide the Tml extract into five fractions and determined the anti-N. gonorrhoeae activity of each of the fractions. All of the fractions showed antibacterial activity. The most active one was identified as the diethyl ether fraction and had MIC values of between 25 and 50 microg ml(-1) against all of the strains.     

Genetic diversity and population structure in the Brazilian Cattleya labiata (Orchidaceae) using RAPD and ISSR markers

Brazilian orchids are currently threatened with extinction due to habitat loss and, because of their high ornamental value, intense collecting pressure. Genetic diversity can play a key role in the survival of endangered orchid species. Here we provide the first data on genetic diversity and structure of wild populations in the genus Cattleya, in particular C.?labiata, using random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) markers. We studied 130 individuals, 117 belonging to Cattleya?labiata and 13 from 10 other species in the same genus. Data generated from 12 ISSR and 12 RAPD primers were used to determine genetic variability via a model-based Bayesian procedure (Structure) and molecular variance analysis. In addition, Shannon index, genetic diversity and Jaccard coefficients were also estimated. The marker data indicated that C. labiata has a high level of polymorphism, and five reconstructed populations were identified by Structure. The unweighted pair group method with arithmetic mean dendrogram did not group the samples by origin, which was also confirmed by Bayesian analysis, demonstrating the complex genetic structure of C.?labiata. Other Cattleya species showed no relationship with any C.?labiata sample. This genetic characterization of Cattleya from northeast Brazil contributes to knowledge of the genetic structure of the species and can be used to define strategies for conservation and breeding programmes.     

The impact of highly active antiretroviral therapy on the survival of vertically HIV-infected children and adolescents in Belo Horizonte, Brazil

The use of highly active antiretroviral therapy (HAART) for human immunodeficiency virus (HIV)-infected patients has reduced the number of acquired immune deficiency syndrome-related deaths worldwide. This study assessed the impact of HAART on the survival and death rates of vertically HIV-infected children and adolescents in Belo Horizonte, Brazil. Data were obtained from a historic cohort of vertically HIV-infected children and adolescents aged zero-19 years old who were admitted from March 1989-December 2004 and were followed until June 2006. Patients who used HAART were included if they were treated for at least 12 weeks. Of 359 patients, 320 patients met the inclusion criteria. The overall mortality rate was 9.7% [31/320; 95% confidence interval (CI): 6.0-13%]. The median survival for the non-HAART and HAART groups was 31.5 and 55.9 months, respectively (log rank = 22.11, p < 0.0001). In the multivariate analysis, the statistically significant variables were HAART and the weight-for-age Z score < -2, with HAART constituting a protective factor [relative risk (RR): 0.13; CI 95%: 0.05-0.33] and malnutrition constituting a risk factor (RR: 3.44; CI 95%: 1.60-7.40) for death. The incidence of death was 5.1/100 person-years in the non-HAART group and 0.8/100 person-years in the HAART group (p < 0.0001).     

Synthesis and characterization of (±)-13-hydroxy-3,11-diaza steroids

An efficient strategy for introducing a nitrogen atom in positions 3 and 11 of the steroidal skeleton, which are key positions for biological purposes, is described. This procedure involves an intramolecular Diels-Alder cycloaddition of o-quinodimethanes which are generated from a 3-azabicyclo[4.2.0]octa-1,3,5-trien-7-one. The characteristic (1)H and (13)C NMR spectroscopic features of the synthesized compounds are reported.