The microtubule- and PP1-binding activities of Drosophila melanogaster Spc105 control the kinetics of SAC satisfaction

KNL1 is a large intrinsically disordered kinetochore (KT) protein that recruits spindle assembly checkpoint (SAC) components to mediate SAC signaling. The N-terminal region (NTR) of KNL1 possesses two activities that have been implicated in SAC silencing: microtubule (MT) binding and protein phosphatase 1 (PP1) recruitment. The NTR of Drosophila melanogaster KNL1 (Spc105) has never been shown to bind MTs or to recruit PP1. Furthermore, the phosphoregulatory mechanisms known to control SAC protein binding to KNL1 orthologues is absent in D. melanogaster. Here, these apparent discrepancies are resolved using in vitro and cell-based assays. A phosphoregulatory circuit that utilizes Aurora B kinase promotes SAC protein binding to the central disordered region of Spc105 while the NTR binds directly to MTs in vitro and recruits PP1-87B to KTs in vivo. Live-cell assays employing an optogenetic oligomerization tag and deletion/chimera mutants are used to define the interplay of MT and PP1 binding by Spc105 and the relative contributions of both activities to the kinetics of SAC satisfaction.     

Development, characterisation, and across-taxa utility of oil palm (Elaeis guineensis Jacq.) microsatellite markers.

The results of the development of oil palm (Elaeis guineensis Jacq.) microsatellite markers are given step by step, from the screening of libraries enriched in (GA)n, (GT)n, and (CCG)n simple-sequence repeats (SSRs) to the final characterisation of 21 SSR loci. Also published are primer sequences, estimates of allele size range, and expected heterozygosity in E. guineensis and in the closely related species E. oleifera, in which an optimal utility of the SSR markers was observed. Multivariate data analyses showed the ability of SSR markers to efficiently reveal the genetic-diversity structure of the genus Elaeis in accordance with known geographical origins and with measured genetic relationships based on previous molecular studies. High levels of allelic variability indicated that E. guineensis SSRs will be a powerful tool for genetic studies of the genus Elaeis, including variety identification and intra- or inter-specific genetic mapping. PCR amplification tests on a subset of 16 other palm species and allele-sequence data showed that E. guineensis SSRs are putative transferable markers across palm taxa. In addition, phenetic information based on SSR flanking region sequences makes E. guineensis SSR markers a potentially useful molecular resource for any researcher studying the phylogeny of palm taxa.     

Taenia solium metacestode immunodominant peptides recognized by IgG antibodies in cerebrospinal fluid and serum paired samples from patients with active and inactive neurocysticercosis

The aim of this study was to test if serological distinction between patients with active and inactive neurocysticercosis (NCC), could be accomplished by the recognition of immunodominant peptides in total saline antigenic extract of Taenia solium metacestodes by IgG antibody in cerebrospinal fluid (CSF) and serum paired samples. CSF and serum samples of 10 each, active NCC patients, inactive NCC, and individuals with other neurological disorders, were used to recognize the antigenic peptides by western blot (WB). In the active NCC the 28-32 and 39-42 kDa peptides were more frequently detected in CSF than in sera (p < 0.05). The 47-52, 64-68, and 70 kDa antigens showed high frequencies in both samples from patients with active NCC. All the CSF samples of inactive NCC and other neurological disorder (control) patients tested negative, while serum samples from these last two groups recognized mainly the 80, 86, 95, and 98 kDa bands. This finding eliminates the use of the high molecular weigh bands (>or= 80 kDa) for diagnosis of NCC. The final conclusions were that the difference between active and inactive NCC may be done with the detection of peptides only in the CSF samples and that the 47-52, 64-68, and 70 kDa bands may be included as specific markers for active NCC when detected in CSF samples by WB using total saline extract of T. solium metacestode.     

Use of the paired samples (cerebrospinal fluid and serum) in immunodiagnostic of active and inactive human neurocysticercosis

Paired samples of cerebrospinal fluid (CSF) and serum of 30 patients--10 with active, 10 with inactive neurocysticercosis (NCC), and 10 control subjects--were evaluated by enzyme-linked immunosorbent assay (ELISA) using two Taenia crassiceps metacestode extracts as antigen in order to detect IgG antibodies. In active NCC, high levels of IgG were detected (p < 0.05). The CSF samples showed 80% (CI 72-88) of reactivity in the saline extract (S) and 90% (CI 84-95) in sodium dodecyl sulphate (SDS) and the serum samples were reactive in 90% (CI 84-95) and 100% (CI 98-100) in the S and SDS antigenic extracts, respectively. The use of the paired samples of CSF and serum in active NCC showed equivalent results suggesting that the serum samples could be used as a screening in those patients whose CSF puncture is counter-indicated.     

A DNA vaccine encoding CCL4/MIP-1beta enhances myocarditis in experimental Trypanosoma cruzi infection in rats

Chagas' disease, caused by Trypanosoma cruzi, is a major cause of cardiovascular disease in Latin America. Exacerbated inflammation disproportional to parasite load characterizes chronic myocardial lesions in chagasic patients. Chemokines and their receptors are expected to account for the renewed inflammatory processes after the inoculation of the parasite, but their potential unique functions are far from being clear. Herein, we evaluated the effect of a DNA vaccine encoding CCL4/MIP-1beta, a CC-chemokine, in T. cruzi-elicited myocarditis in rats. Holtzman rats were given intramuscularly cardiotoxin and the CCL4/MIP-1beta DNA-containing plasmid (100microg) was delivered in this muscular site four times. Fourteen days after last immunization, animals were inoculated with a myotropical CL-Brener T. cruzi clone. Peak of parasitism was observed at day 15 after infection, preceding the peak of myocardial inflammation at day 20. Myocarditis was still intense at day 30, but the inflammatory infiltrates showed a more focal distribution. The expression of CCL2/MCP-1 and CCL4/MIP-1beta correlated closely with the kinetics of myocardial inflammation. The CCL4/MIP-1beta DNA vaccine induced an increase of the levels of the anti-CCL4/MIP-1beta observed in T. cruzi-infected animals. This was associated with an exacerbation of myocardial inflammation and fibrosis, although alterations in parasitemia and myocardial parasitism were not observed. Our data suggest that CCL4/MIP-1beta plays a role in preventing excessive inflammation and pathology rather than in controlling parasite replication.     

Genetic polymorphism in Taenia solium metacestodes from different Brazilian geographic areas

The aim of the present study is to investigate genetic polymorphisms in Taenia solium metacestodes from different Brazilian geographical areas and to relate them to antibody recognition in serum samples of neurocysticercosis (NC) patients. Metacestodes were obtained from the Distrito Federal (DF), Bahia, Minas Gerais (MG) and S?o Paulo (SP) regions of Brazil. Samples of human sera from 49 individuals with NC, 68 individuals with other helminthiasis and 40 healthy volunteers were analysed (157 individuals in total). Antigens were prepared and used in enzyme-linked immunosorbent assay and western blotting assays to detect specific immunoglobulin G antibodies. Genetic distances between metacestode populations were analysed using random amplified polymorphic DNA (RAPD) analysis. Our results show that there was a higher frequency of reactivity in the DF region in the sera from NC patients (p < 0.05), while discrimination between active and inactive NC was seen only in extracts from the MG and SP regions (p < 0.05). Using RAPD, the sample from the DF region presented a greater increase compared to the other regions. A relationship between genetic polymorphisms among T. solium metacestodes from different areas in Brazil and the differences in antibody detection in patients with NC were established.     

Synthesis of methoxylated goniothalamin, aza-goniothalamin and γ-pyrones and their in vitro evaluation against human cancer cells

The present work describes the preparation of three novel series of compounds based on the structure of goniothalamin, a natural styryl lactone which has been found to display cytotoxic and antiproliferative activities against a variety of cancer cell lines. A focused library of 29 novel goniothalamin analogues was prepared and evaluated against seven human cancer cell lines. While the γ-pyrones and the aza-goniothalamin analogues were less potent than the lead compound, 2,4-dimethoxy analogue 88 has shown to be more potent in vitro than goniothalamin against all cancer cell lines evaluated. Furthermore, it was more potent than doxorubicin against NCI-ADR/RES, OVCAR-03 and HT-29 while being less toxic to human keratinocytes (HaCat). The 3,5-dimethoxy analogue 90 and 2,4,5-trimethoxy analogue 92 also displayed promising antiproliferative activity when compared to goniothalamin (1). These results provide new elements for the design and synthesis of novel representatives of this family of natural compounds.     

Anatomical and physiological changes of in vitro-propagated Vriesea imperialis (Bromeliaceae) in the function of sucrose and ventilated containers

Abstract

Under in vitro culture conditions, plants may present physiological and anatomical disorders, which can interfere negatively after ex vitro transfer. The aim of this investigation was to analyze the impacts of natural ventilation and sucrose supply on the anatomy and physiology of Vriesea imperialis. Plants previously grown in vitro were transferred to culture medium containing 0, 15, 30 or 45?g L^?1 sucrose. Three different culture container sealing systems were tested: lids with a green filter (81.35 gas exchanges per day), yellow filter (13.09 gas exchanges per day) or lids with a yellow filter covered with three layers of transparent polyvinylchloride (PVC) film (blocking fluent gas exchange). Sucrose concentrations influenced thickness, lignin and suberin deposition of exodermis cell wall. The modifications verified in leaves, such as higher density of stomata and trichome scales, showed that sucrose can induce osmotic stress in the plants. Photomixotrophic conditions, using containers with intermediate rate of gas exchange (yellow filter) and with 15–30?g L^?1 sucrose, produced an improvement in the growth traits and did not induce anatomical and physiological disturbances.     

Community composition has greater impact on the functioning of marine phytoplankton communities than ocean acidification

Ecosystem functioning is simultaneously affected by changes in community composition and environmental change such as increasing atmospheric carbon dioxide (CO₂) and subsequent ocean acidification. However, it largely remains uncertain how the effects of these factors compare to each other. Addressing this question, we experimentally tested the hypothesis that initial community composition and elevated CO₂ are equally important to the regulation of phytoplankton biomass. We full‐factorially exposed three compositionally different marine phytoplankton communities to two different CO₂ levels and examined the effects and relative importance (ω²) of the two factors and their interaction on phytoplankton biomass at bloom peak. The results showed that initial community composition had a significantly greater impact than elevated CO₂ on phytoplankton biomass, which varied largely among communities. We suggest that the different initial ratios between cyanobacteria, diatoms, and dinoflagellates might be the key for the varying competitive and thus functional outcome among communities. Furthermore, the results showed that depending on initial community composition elevated CO₂ selected for larger sized diatoms, which led to increased total phytoplankton biomass. This study highlights the relevance of initial community composition, which strongly drives the functional outcome, when assessing impacts of climate change on ecosystem functioning. In particular, the increase in phytoplankton biomass driven by the gain of larger sized diatoms in response to elevated CO₂ potentially has strong implications for nutrient cycling and carbon export in future oceans.