Phylogeographical pattern of Euplotes nobilii,a protist ciliate with a bipolar biogeographical distribution

Nuclear (18S and ITS) and mitochondrial (16S) ribosomal RNA gene sequences were determined from genetically distinct wild‐type strains of Antarctic (nine strains), Fuegian (four strains), Greenland (nine strains) and Svalbard (three strains) populations of the marine ciliate, Euplotes nobilii, and analysed for their nucleotide polymorphisms. A close genetic homogeneity was found within and between the Antarctic and Fuegian populations, while more significant levels of genetic differentiation were detected within and between the two Arctic populations, as well as between these populations and the Antarctic/Fuegian ones. The phylogeographical pattern that was derived from these data indicates that gene flow is not limited among Arctic populations; it equally connects the Arctic and Antarctic populations either directly, or through the Fuegian population. This indication reinforces previous evidence from laboratory assays of mating interactions between some of the strains analysed in this work that Southern and Northern polar populations of E. nobilii belong to a unique, panmictic population that substantially share the same gene pool.     

Metabolic fingerprinting reveals differences between northern and southern strains of the cryptic diatom Chaetoceros socialis

Morphology and molecular phylogeny constitute the structural elements of diatom taxonomy. These approaches do not, however, give information on the functioning of taxa. Additional methods to serve a more integrated and wide-ranging taxonomy have therefore been called for. Metabolic fingerprinting is one approach used within the field of metabolomics, often applied in classification of samples. Here we apply metabolic fingerprinting in a taxonomic study of a cryptic diatom species. Strains of the cosmopolitan diatom Chaetoceros socialis from two geographical areas; the north-east Atlantic and Arctic and the Gulf of Naples, were cultivated at three different temperatures; 2.5, 8 and 13°C. The strains from the two different geographical areas exhibited different growth rates as well as different photosynthetic efficiencies. Algal extracts, collected at the end of the growth experiments, were analysed by Ultra-Performance Liquid Chromatography High Resolution Mass Spectrometry. The two groups of strains were separated by principal component analysis of their metabolic fingerprints. Analysis of the data revealed both qualitative and quantitative differences in metabolite markers. These phenotypic differences reinforce differences also found for morphology, phylogenetic markers and growth rates, and point at different adaptive characteristics in organisms living under different temperature regimes.     

Gene delivery mediated by cationic liposomes: from biophysical aspects to enhancement of transfection

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. However, the relationship between the features of the lipid-DNA complexes (`lipoplexes') and their mode of interaction with cells, the efficiency of gene transfer and gene expression remain to be clarified. To gain insights into these aspects, the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3- (trimethylammonium) propane (DOTAP) and its mixture with phosphatidylethanolamine (PE)), and their complexes with DNA at different (+/-) charge ratios were determined. A lipid mixing assay was used to assess the interaction of liposomes and lipoplexes with monocytic leukaemia cells. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. However, very limited transfection of these cells was achieved using the above complexes. It is possible that the topology of the cationic liposome-DNA complexes does not allow the entry of DNA into cells through a fusion process at the plasma membrane. In an attempt to enhance transfection mediated by lipoplexes composed of DOTAP and its equimolar mixture with dioleoylphosphatidylethanolamine (DOPE) two different strategies were explored: (i) association of a targeting ligand (transferrin) to the complexes to promote their internalization, presumably by receptor-mediated endocytosis; and (ii) association of synthetic fusogenic peptides (GALA or the influenza haemagglutinin Nterminal peptide HA-2) to the complexes to promote endosomal destabilization and release of the genetic material into the cytoplasm. These strategies were effective in enhancing transfection in a large variety of cells, including epithelial and lymphoid cell lines, as well as human macrophages, especially with the use of optimized lipid/ DNA (+/-) charge ratios. Besides leading to high levels of transfection, the ternary complexes of cationic liposomes, DNA, and protein or peptide, have the advantages of being active in the presence of serum and being non-toxic. Moreover, such ternary complexes present a net negative charge and, thus, are likely to alleviate the problems associated with the use of highly positively charged complexes in vivo, such as avid complexation with serum proteins. Overall, the results indicate that these complexes, and their future derivatives, may constitute viable alternatives to viral vectors for gene delivery in vivo.     

Falcipain-2 inhibition by suramin and suramin analogues

Falcipain-2 is a cysteine protease of the malaria parasite Plasmodium falciparum that plays a key role in the hydrolysis of hemoglobin, a process that is required by intraerythrocytic parasites to obtain amino acids. In this work we show that the polysulfonated napthylurea suramin is capable of binding to falcipain-2, inhibiting its catalytic activity at nanomolar concentrations against both synthetic substrates and the natural substrate hemoglobin. Kinetic measurements suggest that the inhibition occurs through an noncompetitive allosteric mechanism, eliciting substrate inhibition. Smaller suramin analogues and those with substituted methyl groups also showed inhibition within the nanomolar range. Our results identify the suramin family as a potential starting point for the design of falcipain-2 inhibitor antimalarials that act through a novel inhibition mechanism.     

Trypsin inhibitors in xoconostle seeds (Opuntia joconostle Weber.)

Seed proteins recovered after heating a seed extract from Opuntia joconostle Weber (xoconostle, an acid cactus pear) were screened for different biochemical activities, detecting only trypsin-like inhibitory activity. Two trypsin-like inhibitor forms from seeds were separated by RP-HPLC and partially sequenced and characterized as an enriched mixture. They were evaluated for inhibition on several serine proteinases, but only trypsin-like inhibition was detected by the inhibitor extract. The two isolated forms, OjTI 1 and OjTI 2 showed low molecular weights of 4.26 and 4.17 kDa as determined by mass spectrometry. An enriched inhibitory fraction showed a high thermal stability by retaining the activity after heating the sample for 1 h at 90 °C, as well as after heating for at 120 °C under 1 kg/cm² for 15 min at different pH values. Partial sequence of the two forms was determined by mass spectrometry indicating that they were similar and after alignments analysis they showed the highest similarity with the trypsin inhibitor from O. streptacantha and to a lesser extent to other trypsin inhibitors of the MEROPS database families. The inhibitory spectrum was evaluated against several digestive enzymes from pests and beneficial insects from several taxonomic orders.     

Trends of karyotypical evolution in the pearl cichlid,Geophagus brasiliensis,from southern Brazil

Chromosomal rearrangements such as inversions can facilitate speciation even in the presence of gene flow. The present study aims to analyze the karyotypic variation in six populations of Geophagus brasiliensis from southern Brazil. All specimens showed 2n = 48 chromosomes, but three karyotypes were found to have one, two or three pairs of submetacentric chromosomes. Although G. brasiliensis did not exhibit variation in the diploid number, it presented a wide interpopulational variation mainly regarding the karyotype formula and specific chromosomal markers. Differences in the location of the major and minor rDNA loci were observed among the populations. Moreover, different patterns were observed in the distribution of the constitutive heterochromatin, presenting intra- and interpopulational variation. This supports the hypothesis that this taxon represents a complex species or that cryptic species are included in this group, indicating a possibleprocess of sympatric speciation. By potentially restricting gene flow between heterokaryotypes, the segregating chromosome rearrangements we describe for G. brasiliensis may play a role in diversification in this species complex.     

Fish oil reverses the altered glucose transporter,phosphorylation, insulin receptor substrate-1 protein level and lipid contents in the skeletal muscle of sucrose-rich diet fed rats

The role and underlying mechanisms by which n?3 polyunsaturated fatty acids (PUFA) prevent/reverse SRD-induced insulin resistance (IR) in the muscle are not completely understood. Therefore, we examined: triglyceride, diacylglycerol, PKCθ, Glut-4, enzymatic hexokinase activity, IRS-1 protein mass level, and fatty acid composition of muscle phospholipids. Rats were fed a SRD during 6 months. Thereafter, half the animals continued with SRD up to 8 months; the other half was fed a SRD in which CO (8% wt/wt) was replaced by FO (7%+1% CO) for 2 months. Results were compared with those obtained in rats fed a control diet (CD). In SRD-fed rats, FO oil normalized/improved lipid storage and PKCθ protein mass level. Effects of insulin were comparable with those of CD-fed rats. FO reversed impaired glucose phosphorylation, IRS-1, and, under insulin stimulation, Glut-4 protein mass level. FO normalized insulin resistance and increased n?3 PUFAs in muscle phospholipids.     

Microplates as a microreactor platform for microalgae research

High‐throughput platforms for microalgae screening are not yet commercially available. In this study, the feasibility of 96‐well microplates was analyzed for microalgae research. Equivalence among wells, as culture microreactors, was investigated in controlled high CO₂ conditions. Specific growth rates of two microalgae species, Scenedesmus sp. UTEX1589 and an environmental isolate, were significantly higher in border wells than in internal positions. Furthermore, growth rate gradients analyzed as contours throughout the platform were observed for Scenedesmus sp. However, the output variable exhibited high precision associated with a low coefficient of variation (CV), between 6.8 and 7.8%. In a demonstrative experiment to determine the effect of culture media dilution on six microalgae species, treatments were randomized in the central subset of a microplate. Results were consistent and statistically sound (CV 9.4–12.9%), and showed that microalgae species could grow with no detrimental effect in 50% (v/v) dilution of the culture medium. Provided border wells exclusion and a randomized design, 96‐well microplates are a practical and statistical robust platform for microalgae research. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:638–644, 2013     

Distribution patterns of Dikarya in arid and semiarid soils of Baja California,Mexico

Approximately four-fifths of the land area of Baja California (BC) in Mexico are occupied by arid and semiarid soils, the mycobiota of which is virtually uncharacterized. In the first culture-independent study of the mycobiota of BC, we collected soil from five different locations in the region and constructed a Dikarya-specific gene library for the ITS region of nuclear ribosomal DNA. Clones were analyzed by RFLP, were sequenced for phylogenetic analyses, and diversity and similarity indices were calculated. The ascomycete Penicillium dipodomyicola was the most frequent fungus found in soil at the most arid location studied, and the basidiomycete Coprinellus radians was the most frequent at the location receiving the highest rainfall. Other frequent members of the soil mycobiota were identified as Alternaria spp., Ceratobasidium sp., Coniozyma leucospermi, Nematoctonus robustus, Penicillium griseofulvum, Tulostoma kotlabae and uncultured members of the Dikarya. Several sequences were identified as those of uncultured fungi, one of which was previously reported from other hot deserts. Arid soils and the transitional zones between arid and semiarid soils had the most similar fungal diversity, with the former soils having a community from which basidiomycetes were absent, and the soil receiving the highest precipitation having a community dominated by basidiomycetes.