Biocontrol evaluation of extracts and a major component,clusianone, from Clusia fluminensis Planch. & Triana against Aedes aegypti

Studies evaluated the effects of hexanic extracts from the fruits and flowers ofClusia fluminensis and the main component of the flower extract, a purified benzophenone (clusianone), against Aedes aegypti. The treatment of larvae with the crude fruit or flower extracts from C. fluminensis did not affect the survival ofAe. aegypti (50 mg/L), however, the flower extracts significantly delayed development of Ae. aegypti. In contrast, the clusianone (50 mg/L) isolate from the flower extract, representing 54.85% of this sample composition, showed a highly significant inhibition of survival, killing 93.3% of the larvae and completely blocking development of Ae. aegypti. The results showed, for the first time, high activity of clusianone against Ae. aegypti that both killed and inhibited mosquito development. Therefore, clusianone has potential for development as a biopesticide for controlling insect vectors of tropical diseases. Future work will elucidate the mode of action of clusianone isolated from C. fluminensis.     

A novel mutation in the mitochondrial tRNAAla gene (m.5636T>C) in a patient with progressive external ophthalmoplegia

We report a heteroplasmic novel mutation m.5636T>C in the mt-tRNA^Ala in a patient with bilateral ptosis and ophthalmoparesis in whom a muscle biopsy showed cytochrome c oxdidase (COX) negative and ragged red fibers. Using laser capture microdissection we have isolated COX negative fibers and COX positive fibers from the muscle of the patient and determined that the mutation load was clearly increased in COX negative muscle fibers. Additionally, the mutated m.5636T nucleotide is conserved in all the mammal and non-mammal species analyzed and might be structurally relevant as it is located in a position involved in the formation of tertiary structure of canonical mitochondrial tRNAs.     

Moderate caloric restriction during gestation in rats alters adipose tissue sympathetic innervation and later adiposity in offspring

Maternal prenatal undernutrition predisposes offspring to higher adiposity in adulthood. Mechanisms involved in these programming effects, apart from those described in central nervous system development, have not been established. Here we aimed to evaluate whether moderate caloric restriction during early pregnancy in rats affects white adipose tissue (WAT) sympathetic innervation in the offspring, and its relationship with adiposity development. For this purpose, inguinal and retroperitoneal WAT (iWAT and rpWAT, respectively) were analyzed in male and female offspring of control and 20% caloric-restricted (from 1-12 d of pregnancy) (CR) dams. Body weight (BW), the weight, DNA-content, morphological features and the immunoreactive tyrosine hydroxylase and Neuropeptide Y area (TH+ and NPY+ respectively, performed by immunohistochemistry) of both fat depots, were studied at 25 d and 6 m of age, the latter after 2 m exposure to high fat diet. At 6 m of life, CR males but not females, exhibited greater BW, and greater weight and total DNA-content in iWAT, without changes in adipocytes size, suggesting the development of hyperplasia in this depot. However, in rpWAT, CR males but not females, showed larger adipocyte diameter, with no changes in DNA-content, suggesting the development of hypertrophy. These parameters were not different between control and CR animals at the age of 25 d. In iWAT, both at 25 d and 6 m, CR males but not females, showed lower TH(+) and NPY(+), suggesting lower sympathetic innervation in CR males compared to control males. In rpWAT, at 6 m but not at 25 d, CR males but not females, showed lower TH(+) and NPY(+). Thus, the effects of caloric restriction during gestation on later adiposity and on the differences in the adult phenotype between internal and subcutaneous fat depots in the male offspring may be associated in part with specific alterations in sympathetic innervation, which may impact on WAT architecture.     

Marked mitochondrial DNA depletion associated with a novel SUCLG1 gene mutation resulting in lethal neonatal acidosis,multi-organ failure,and interrupted aortic arch

The aim of this study was to identify the causative genetic lesion in two apparently unrelated newborns having lethal lactic acidosis, multi-organ failure and congenital malformations including interrupted aortic arch, who exhibited mild methylmalonic aciduria, combined mitochondrial respiratory chain deficiency, and marked muscle mitochondrial DNA depletion. A novel mutation in the SUCLG1 gene was identified. Phenotype severity in Succinate-CoA ligase dysfunction appears to be more correlated to the muscle mtDNA content than to the tissue distribution of the heterodimer subunits. Prominent impairment of mitochondrial respiratory chain may result in deep ravages in developmental tissues leading to multiple organ failure and malformations.     

Folding, stability and polymerization properties of FtsZ chimeras with inserted tubulin loops involved in the interaction with the cytosolic chaperonin CCT and in microtubule formation

To attain its native conformation, the cytoskeletal protein tubulin needs the concourse of several molecular chaperones, among others the cytosolic chaperonin CCT. It has been previously described that denatured tubulin interacts with CCT in a quasi-folded conformation using several loops located throughout its sequence. These loops are also involved in microtubule formation and are absent in its prokaryote homologue FtsZ, which in vitro folds by itself and does not interact with CCT. Several FtsZ/tubulin chimeric proteins were generated by inserting consecutively one, two or three of the CCT-binding domains of tubulin into the corresponding sequence of FtsZ from Methanococccus jannaschii. The insertion of any of the CCT-binding loops generates in the FtsZ/tubulin chimeras the ability to interact with CCT. The accumulation of CCT-binding loops induces in the FtsZ/tubulin chimeras unfolding and refolding properties that are more similar to tubulin than to its prokaryote counterpart. Finally, the insertion of some of these loops generates in the FtsZ/tubulin chimeras more complex polymeric structures than those found for FtsZ. These results reinforce the notion that CCT has coevolved with tubulin to deal with the folding problems encountered by the eukaryotic protein with the appearance of the new sequences involved in microtubule formation.     

Changes in lignocellulolytic enzyme activites in six <Emphasis Type="Italic">Pleurotus</Emphasis> spp. strains cultivated on coffee pulp in confrontation with <Emphasis Type="Italic">Trichoderma</Emphasis> spp.

Summary The antagonistic effect of six Pleurotus spp. strains was studied in confrontation with three strains of Trichoderma spp. Pleurotus strains were cultivated on sterile coffee pulp, with and without a Trichoderma inoculant. Laccase, Mn peroxidase and endoglucanase activities were determined during incubation. Laccase production was also studied by PAGE analysis to detect enzymatic isoforms. Results show that the presence of Trichoderma induced a significant increase in oxidase production by the Pleurotus strains. Nevertheless, Trichoderma was not observed to induce laccase isoforms.     

Macromolecular accessibility of fluorescent taxoids bound at a paclitaxel binding site in the microtubule surface

The macromolecular accessibility of the paclitaxel binding site in microtubules has been investigated using a fluorescent taxoid and antibodies against fluorescein, which cannot diffuse into the microtubule lumen. The formation of a specific ternary complex of microtubules, Hexaflutax (7-O-{N-[6-(fluorescein-4'-carboxamido)-n-hexanoyl]-l-alanyl}paclitaxel) and 4-4-20 IgG (a monoclonal antibody against fluorescein) has been observed by means of sedimentation and electron microscopy methods. The kinetics of binding of the antibody to microtubule-bound Hexaflutax has been measured. The quenching of the observed fluorescence is fast (k+ 2.26 +/- 0.25 x 10(6) m(-1) s(-1) at 37 degrees C), indicating that the fluorescein groups of Hexaflutax are exposed to the outer solvent. The velocity of the reaction is linearly dependent on the antibody concentration, indicating that a bimolecular reaction is being observed. Another fluorescent taxoid (Flutax-2) bound to microtubules has also been shown to be rapidly accessible to polyclonal antibodies directed against fluorescein. A reduced rate of Hexaflutax quenching by the antibody is observed in microtubule-associated proteins containing microtubules or in native cellular cytoskeletons. It can be concluded that the fluorescent taxoids bind to an outer site on the microtubules that is shared with paclitaxel. Paclitaxel would be internalized in a further step of binding to reach the known luminal site, this step being blocked in the case of the fluorescent taxoids. Because the fluorescent ligands are able to induce microtubule assembly, binding to the outer site should be enough to induce assembly by a preferential binding mechanism.     

Structural dissection of a highly knotted peptide reveals minimal motif with antimicrobial activity

The increasing occurrence of bacterial resistance to antibiotics is driving a renewed interest on antimicrobial peptides, in the hope that understanding the structural features responsible for their activity will provide leads into new anti-infective drug candidates. Most chemical studies in this field have focused on linear peptides of various eukaryotic origins, rather than on structures with complex folding patterns found also in nature. We have undertaken the structural dissection of a highly knotted, cysteine-rich plant thionin, with the aim of defining a minimal, synthetically accessible, structure that preserves the bioactive properties of the parent peptide. Using efficient strategies for directed disulfide bond formation, we have prepared a substantially simplified (45% size reduction) version with undiminished antimicrobial activity against a representative panel of pathogens. Analysis by circular dichroism shows that the downsized peptide preserves the central double alpha-helix of the parent form as an essential bioactive motif. Membrane permeability and surface plasmon resonance studies confirm that the mechanism of action remains unchanged.