Purification and partial characterization of human neutrophil elastase inhibitors from the marine snail Cenchritis muricatus (Mollusca)

Human neutrophil elastase inhibition was detected in a crude extract of the marine snail Cenchritis muricatus (Gastropoda, Mollusca). This inhibitory activity remained after heating this extract at 60 °C for 30 min. From this extract, three human neutrophil elastase inhibitors (designated CmPI–I, CmPI–II and CmPI–III) were purified by affinity and reversed-phase chromatographies. Homogeneity of CmPI–I and CmPI–II was confirmed, while CmPI–III showed a single peak in reversed-phase chromatography, but heterogeneity in SDS-PAGE with preliminary molecular masses in the range of 18.4 to 22.0 kDa. In contrast, MALDI-TOF mass spectrometry of CmPI–I and CmPI–II showed that these inhibitors are molecules of low molecular mass, 5576 and 5469 Da, respectively. N-terminal amino acid sequences of CmPI–I (6 amino acids) and CmPI–II (20 amino acids) were determined. Homology to Kazal-type protease inhibitors was preliminarily detected for CmPI–II. Both inhibitors, CmPI–I and CmPI–II are able to inhibit human neutrophil elastase strongly, with equilibrium dissociation constant (K_i) values of 54.2 and 1.6 nM, respectively. In addition, trypsin and pancreatic elastase were also inhibited, but not plasma kallikrein or thrombin. CmPI–I and CmPI–II are the first human neutrophil elastase inhibitors described in a mollusk.     

Bioprospecting of Saprobe Fungi from the Semi‐Arid North‐East of Brazil for the Control of Anthracnose on Sorghum

Anthracnose, caused by the hemiotrophic fungus Colletotrichum sublineolum, is one of the most important diseases affecting sorghum production worldwide. The main goal of this study was to select saprobe fungi from the semi‐arid north‐east of Brazil that could increase sorghum resistance to anthracnose and investigate this increased resistance at both physiological and biochemical levels. Plants were sprayed with Curvularia inaequalis, Gonytrichum macroladum, Memnoniella levispora, Pithomyces chartarum, Periconia hispidula, Phaeoisaria clematidia, Dictyochaeta heteroderae, Sarcopodium circinatum, Periconia byssoides, Moorella speciosa, Stachybotrys chartarum, Pseudobotrytis terrestres, Memnoniella echinata, Stachybotrys globosa and Gonytrichum clamydosporium 24 h before inoculation with C. sublineolum. Plants sprayed with water served as the control treatment. The area under the anthracnose progress curve was significantly reduced in comparison with the control treatment only for plants sprayed with C. inaequalis. Therefore, C. inaequalis was selected for physiological and biochemical evaluations. The peroxidases, chitinases and β‐1,3‐glucanases activities were significantly higher for plants sprayed with C. inaequalis and inoculated with C. sublineolum than for plants not sprayed with C. inaequalis and inoculated with C. sublineolum. There was no apparent decrease in the values of net carbon assimilation rate, stomatal conductance to water vapour or transpiration rate for plants sprayed with C. inaequalis and infected by C. sublineolum in comparison with plants not sprayed with C. inaequalis and infected by C. sublineolum. In conclusion, sorghum resistance against C. sublineolum infection was greatly potentiated by C. inaequalis without any apparent change in the photosynthetic capacity of the infected plants.     

Heterozygous Reelin Mutations Cause Autosomal-Dominant Lateral Temporal Epilepsy

Autosomal-dominant lateral temporal epilepsy (ADLTE) is a genetic epilepsy syndrome clinically characterized by focal seizures with prominent auditory symptoms. ADLTE is genetically heterogeneous, and mutations in LGI1 account for fewer than 50% of affected families. Here, we report the identification of causal mutations in reelin (RELN) in seven ADLTE-affected families without LGI1 mutations. We initially investigated 13 ADLTE-affected families by performing SNP-array linkage analysis and whole-exome sequencing and identified three heterozygous missense mutations co-segregating with the syndrome. Subsequent analysis of 15 small ADLTE-affected families revealed four additional missense mutations. 3D modeling predicted that all mutations have structural effects on protein-domain folding. Overall, RELN mutations occurred in 7/40 (17.5%) ADLTE-affected families. RELN encodes a secreted protein, Reelin, which has important functions in both the developing and adult brain and is also found in the blood serum. We show that ADLTE-related mutations significantly decrease serum levels of Reelin, suggesting an inhibitory effect of mutations on protein secretion. We also show that Reelin and LGI1 co-localize in a subset of rat brain neurons, supporting an involvement of both proteins in a common molecular pathway underlying ADLTE. Homozygous RELN mutations are known to cause lissencephaly with cerebellar hypoplasia. Our findings extend the spectrum of neurological disorders associated with RELN mutations and establish a link between RELN and LGI1, which play key regulatory roles in both the developing and adult brain.     

Real-time PCR in HIV/Trypanosoma cruzi coinfection with and without Chagas disease reactivation: association with HIV viral load and CD4 level

Background

Reactivation of chronic Chagas disease, which occurs in approximately 20% of patients coinfected with HIV/Trypanosoma cruzi (T. cruzi), is commonly characterized by severe meningoencephalitis and myocarditis. The use of quantitative molecular tests to monitor Chagas disease reactivation was analyzed.

Methodology

Polymerase chain reaction (PCR) of kDNA sequences, competitive (C-) PCR and real-time quantitative (q) PCR were compared with blood cultures and xenodiagnosis in samples from 91 patients (57 patients with chronic Chagas disease and 34 with HIV/T. cruzi coinfection), of whom 5 had reactivation of Chagas disease and 29 did not.

Principal Findings

qRT-PCR showed significant differences between groups; the highest parasitemia was observed in patients infected with HIV/T. cruzi with Chagas disease reactivation (median 1428.90 T. cruzi/mL), followed by patients with HIV/T. cruzi infection without reactivation (median 1.57 T. cruzi/mL) and patients with Chagas disease without HIV (median 0.00 T. cruzi/mL). Spearman''s correlation coefficient showed that xenodiagnosis was correlated with blood culture, C-PCR and qRT-PCR. A stronger Spearman correlation index was found between C-PCR and qRT-PCR, the number of parasites and the HIV viral load, expressed as the number of CD4⁺ cells or the CD4⁺/CD8⁺ ratio.

Conclusions

qRT-PCR distinguished the groups of HIV/T. cruzi coinfected patients with and without reactivation. Therefore, this new method of qRT-PCR is proposed as a tool for prospective studies to analyze the importance of parasitemia (persistent and/or increased) as a criterion for recommending pre-emptive therapy in patients with chronic Chagas disease with HIV infection or immunosuppression. As seen in this study, an increase in HIV viral load and decreases in the number of CD4⁺ cells/mm³ and the CD4⁺/CD8⁺ ratio were identified as cofactors for increased parasitemia that can be used to target the introduction of early, pre-emptive therapy.     

Occurrence of co-infection by Leishmania (Leishmania) chagasi and Trypanosoma (Trypanozoon) evansi in a dog in the state of Mato Grosso do Sul, Brazil

A natural case of co-infection by Leishmania and Trypanosoma is reported in a dog (Canis familiaris) in south- western state of Mato Grosso do Sul, Brazil. Both amastigote and trypomastigote forms were observed after Giemsa staining of cytological preparations of the dog's bone marrow aspirate. No parasite was detected using medium culture inoculation of the sample. DNA obtained from the bone marrow aspirate sample and from the blood buffy coat was submitted to polymerase chain reaction (PCR) with a set of rDNA-based primers S4/S12. The nucleotide sequence of the PCR product was identical to that of Trypanosoma (Trypanozoon) evansi. The S4/S12 PCR was then used as template in a nested-PCR using a specific Leishmania set S17/S18 as primers, to explain the amastigote forms. The nucleotide sequence of the new PCR product was identical to that of Leishmania (Leishmania) chagasi. This case, as far as we know, is the first report of a dog co-infected with these parasites, suggesting that besides L. (L.) chagasi, the natural transmission of T. (T.) evansi occurs in the area under study.     

Evaluation of methoprene effect on Aedes aegypti (Diptera: Culicidae) development in laboratory conditions

Several Brazilian Aedes aegypti populations are resistant to the larvicidae temephos. Methoprene, that inhibits adult emergence, is one of the alternatives envisaged by the Brazilian Dengue Control Program (PNCD). However, at Brazil vector infestation rates are measured through larvae indexes and it has been claimed that methoprene use in the field could face operational problems. In order to define a standardized protocol, methoprene effect was evaluated in laboratory conditions after continuous exposure of larvae (Rockefeller strain) to a methoprene formulation available to the PNCD. Methoprene-derived mortality occurs mainly at the pupa stage and pupa development is inversely proportional to methoprene concentration. Number and viability of eggs laid by treated and control females are equivalent. A methoprene dose-dependent delay in the development was noted; however, strong correlations were found for total mortality or adult emergence inhibition if data obtained when all control mosquitoes have emerged are compared to data obtained when methoprene-treated groups finish development. The cumulative record of total methoprene-induced mortality at the time control adults emerge is proposed for routine evaluation of field populations. Mortality of all specimens, but not of larva, could account for adult emergence inhibition, confirming the inadequacy of larvae indexes to evaluate methoprene effect.     

The effects of intraspinal L-NOARG or SIN-1 on the control by descending pathways of incisional pain in rats

The modulation by spinal nitric oxide (NO) of descending pathways travelling through the dorsal lateral funiculus (DLF) is a mechanism proposed for the antinociceptive effects of drugs that changes the NO metabolism. In this study we confirm that a surgical incision in the mid-plantar hind paw of rats reduces the threshold to mechanical stimulation with von Frey filaments. The incisional pain was further increased in rats with ipsilateral DLF lesion. Intrathecal L-NOARG (50-300 microg), or SIN-1 (0.1-5.0 microg) reduced, while SIN-1 (10 and 20 microg) intensified the incisional pain in rats with sham or effective lesion of the DLF. Stimulation of the dorsal raphe (DRN) or anterior pretectal (APtN) nuclei with stepwise increased electrical currents (7, 14, 21, 28 and 35 microA r.m.s.) produced a current-related reduction of the incisional pain. These nuclei activate pain inhibitory pathways that descend to the spinal cord mainly through the DLF. Intrathecal SIN-1 (5 microg) reduced, SIN-1 (20 microg) decreased and L-NOARG (150 microg) did not change the EC50 for the DRN or APtN stimulation-induced reduction of incisional pain. We conclude that the antinociceptive effects of L-NOARG or low doses of SIN-1 are independent on the activity of descending pain control pathways travelling via the DLF, but the antinociceptive effect of stimulating electrically the DRN or APtN can be summated to the effect of low dose of SIN-1 or overcome by the high dose of SIN-1.     

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.