Seasonal metabolic changes in a year-round reproductively active subtropical tree-frog (Hypsiboas prasinus)

Although seasonal metabolic variation in ectothermic tetrapods has been investigated primarily in the context of species showing some level of metabolic depression during winter, but several species of anurans maintain their activity patterns throughout the year in tropical and subtropical areas. The tree-frog Hypsiboas prasinus occurs in the subtropical Atlantic Forest and remains reproductively active during winter, at temperatures below 10 degrees C. We compared males calling in summer and winter, and found that males of H. prasinus exhibit seasonal adjustments in metabolic and morphometric variables. Individuals calling during winter were larger and showed higher resting metabolic rates than those calling during summer. Calling rates were not affected by season. Winter animals showed lower liver and heart activity level of citrate synthase (CS), partially compensated by larger liver mass. Winter individuals also showed higher activity of pyruvate kinase (PK) and lower activity of CS in trunk muscles, and higher activity of CS in leg muscles. Winter metabolic adjustments seem to be achieved by both compensatory mechanisms to the lower environmental temperature and a seasonally oriented aerobic depression of several organs. The impact of seasonal metabolic changes on calling performance and the capacity of subtropical anurans for metabolic thermal acclimatization are also discussed.     

A New Recombinant BCG Vaccine Induces Specific Th17 and Th1 Effector Cells with Higher Protective Efficacy against Tuberculosis

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb) that is a major public health problem. The vaccine used for TB prevention is Mycobacterium bovis bacillus Calmette-Guérin (BCG), which provides variable efficacy in protecting against pulmonary TB among adults. Consequently, several groups have pursued the development of a new vaccine with a superior protective capacity to that of BCG. Here we constructed a new recombinant BCG (rBCG) vaccine expressing a fusion protein (CMX) composed of immune dominant epitopes from Ag85C, MPT51, and HspX and evaluated its immunogenicity and protection in a murine model of infection. The stability of the vaccine in vivo was maintained for up to 20 days post-vaccination. rBCG-CMX was efficiently phagocytized by peritoneal macrophages and induced nitric oxide (NO) production. Following mouse immunization, this vaccine induced a specific immune response in cells from lungs and spleen to the fusion protein and to each of the component recombinant proteins by themselves. Vaccinated mice presented higher amounts of Th1, Th17, and polyfunctional specific T cells. rBCG-CMX vaccination reduced the extension of lung lesions caused by challenge with Mtb as well as the lung bacterial load. In addition, when this vaccine was used in a prime-boost strategy together with rCMX, the lung bacterial load was lower than the result observed by BCG vaccination. This study describes the creation of a new promising vaccine for TB that we hope will be used in further studies to address its safety before proceeding to clinical trials.     

Blastocladiella emersonii expresses a centrin similar to Chlamydomonas reinhardtii isoform not found in late-diverging fungi

Centrins are members of the calcium-binding EF-hand protein superfamily which can be divided into two subfamilies, probably associated with different functions: one related to Chlamydomonas reinhardtii centrin, CrCenp, and the other, represented by Saccharomyces cerevisiae isoform, ScCdc31p. ESTs encoding the two isoforms (BeCen1 and BeCen3) from the chytridiomycete Blastocladiella emersonii were isolated, and expression of the CrCenp-type centrin, BeCen1, was analyzed throughout the fungus life cycle. Becen1 mRNA levels increase transiently during sporulation and protein levels present a similar pattern. Immunolocalization studies seem to localize BeCen1 at the basal body zone and in the cytoplasm surrounding the nuclear cap, a zoospore organelle.     

Purification and characterization of the α-glucosidase produced by thermophilic fungus <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> CBMAI 756

An α-glucosidase enzyme produced by the fungus Thermoascus aurantiacus CBMAI 756 was purified by ultra filtration, ammonium sulphate precipitation, and chromatography using Q Sepharose, Sephacryl S-200, and Superose 12 columns. The apparent molecular mass of the enzyme was 83 kDa as determined in gel electrophoresis. Maximum activity was observed at pH 4.5 at 70°C. Enzyme showed stability stable in the pH range of 3.0–9.0 and lost 40% of its initial activity at the temperatures of 40, 50, and 60°C. In the presence of ions Na⁺, Ba²⁺, Co²⁺, Ni²⁺, Mg²⁺, Mn²⁺, Al³⁺, Zn²⁺, Ca²⁺ this enzyme maintained 90–105% of its maximum activity and was inhibited by Cr³⁺, Ag⁺, and Hg²⁺. The enzyme showed a transglycosylation property, by the release of oligosaccharides after 3 h of incubation with maltose, and specificity for short maltooligosaccharides and α-PNPG. The K_m measured for the α-glucosidase was 0.07 μM, with a V_max of 318.0 μmol/min/mg.     

Knockdown of RBBP7 unveils a requirement of histone deacetylation for CPC function in mouse oocytes

During mouse oocyte maturation histones are deacetylated, and inhibiting this deacetylation leads to abnormal chromosome segregation and aneuploidy. RBBP7 is a component of several different complexes that contain histone deacetylases, and therefore could be implicated in histone deacetylation. We find that Rbbp7 is a dormant maternal mRNA that is recruited for translation during oocyte maturation to regulate the histone deacetylation. Importantly, we show that the maturation-associated decrease of histone acetylation is required for localization and function of the chromosomal passenger complex (CPC) during oocyte meiotic maturation. This finding can explain the phenotypes of oocytes where Rbbp7 is depleted by an siRNA/morpholino cocktail including severe chromosome misalignment, improper kinetochore–microtubule attachments, impaired SAC function, cytokinesis defects, and increased incidence of aneuploidy at metaphase II (Met II). These results implicate RBBP7 as a novel regulator of histone deacetylation during oocyte maturation and provide evidence that such deacetylation is required for proper chromosome segregation by regulating localized CPC function.     

Polymorphism of Microsatellite Markers in Papaya (<Emphasis Type="Italic">Carica papaya</Emphasis> L.)

A set of 81 new microsatellite markers for Carica papaya L. previously identified by data mining using freely available sequence information from Genbank were tested for polymorphism using 30 germplasm accessions from the Papaya Germplasm Bank (PGM) at Embrapa Mandioca e Fruticultura Tropical (CNPMF) and 18 landraces. The data were used to estimate pairwise genetic distances between the genotypes. A neighbor-joining based dendrogram was used to define clusters and infer possible genetic structuring of the collection. Most microsatellites were polymorphic (73%), with an observed number of alleles per locus ranging from one to eleven. The levels of observed and expected heterozygosity for 51 polymorphic loci varied from 0.00 to 0.85 and from 0.08 to 0.82, averaging 0.19 and 0.59, respectively. Forty-four percent of microsatellites showed polymorphism information content (PIC) higher than 0.50. The compound microsatellites seem to be more informative than dinucleotide and trinucleotide repeats in average alleles per locus and PIC. Among dinucleotides, AG/TC or GA/CT repeat motifs exhibited more informativeness than TA/AT, GT/CA and TG/AC repeat motifs. The neighbor-joining analysis based on shared allele distance could differentiate all the papaya accessions and landraces as well as differences in their genetic structure. This set of markers will be useful for examining parentage, inbreeding and population structure in papaya.     

Phenolic acid derivatives with potential anticancer properties--a structure-activity relationship study. Part 1: methyl, propyl and octyl esters of caffeic and gallic acids

The antiproliferative and cytotoxic properties of polyphenolic acid derivatives, structurally related with the natural models caffeic and gallic acids, have been tested in human cervix adenocarcinoma cells (HeLa). Simultaneous structural information was obtained for these compounds through theoretical ab initio methods. This study was conducted for the following esters: methyl caffeate (MC, 1), propyl caffeate (PC, 2), octyl caffeate (OC, 3), methyl gallate (MG, 4), propyl gallate (PG, 5) and octyl gallate (OG, 6). A significant growth-inhibition effect was assessed for some of these compounds, clearly dependent on their structural characteristics. Marked structure-activity relationships (SARs)--namely the number of hydroxyl ring substituents--were found to rule the biological effect of such systems.     

Evaluation of Antifungal Activity of Medicinal Plant Extracts Against Oral <Emphasis Type="Italic">Candida albicans</Emphasis> and Proteinases

Proteinases produced by Candida albicans are one kind of virulence factor expressed that contribute to adherence and invasion of host tissue. Proteinase inhibitor of human immunodeficiency virus in experimental candidiasis suggested reduction in fungal infection, and medicinal plants could be a source of alternative agent to prevent diseases. In this study, we investigated the production of proteinases by C. albicans from clinical isolates and the action of plant extracts against strains of C. albicans and its synthesized proteinases, comparing with antifungal fluconazole and amphotericin B and proteinase inhibitors pepstatin A, amprenavir, and ritonavir. The results reported here showed that these extracts have a certain kind of action and that the search for new antifungal agents could be found at the plants.     

The genomic ancestry of individuals from different geographical regions of Brazil is more uniform than expected

Based on pre-DNA racial/color methodology, clinical and pharmacological trials have traditionally considered the different geographical regions of Brazil as being very heterogeneous. We wished to ascertain how such diversity of regional color categories correlated with ancestry. Using a panel of 40 validated ancestry-informative insertion-deletion DNA polymorphisms we estimated individually the European, African and Amerindian ancestry components of 934 self-categorized White, Brown or Black Brazilians from the four most populous regions of the Country. We unraveled great ancestral diversity between and within the different regions. Especially, color categories in the northern part of Brazil diverged significantly in their ancestry proportions from their counterparts in the southern part of the Country, indicating that diverse regional semantics were being used in the self-classification as White, Brown or Black. To circumvent these regional subjective differences in color perception, we estimated the general ancestry proportions of each of the four regions in a form independent of color considerations. For that, we multiplied the proportions of a given ancestry in a given color category by the official census information about the proportion of that color category in the specific region, to arrive at a "total ancestry" estimate. Once such a calculation was performed, there emerged a much higher level of uniformity than previously expected. In all regions studied, the European ancestry was predominant, with proportions ranging from 60.6% in the Northeast to 77.7% in the South. We propose that the immigration of six million Europeans to Brazil in the 19th and 20th centuries--a phenomenon described and intended as the "whitening of Brazil"--is in large part responsible for dissipating previous ancestry dissimilarities that reflected region-specific population histories. These findings, of both clinical and sociological importance for Brazil, should also be relevant to other countries with ancestrally admixed populations.     

Quantification of Metabolic Rearrangements During Neural Stem Cells Differentiation into Astrocytes by Metabolic Flux Analysis

Proliferation and differentiation of neural stem cells (NSCs) have a crucial role to ensure neurogenesis and gliogenesis in the mammalian brain throughout life. As there is growing evidence for the significance of metabolism in regulating cell fate, knowledge on the metabolic programs in NSCs and how they evolve during differentiation into somatic cells may provide novel therapeutic approaches to address brain diseases. In this work, we applied a quantitative analysis to assess how the central carbon metabolism evolves upon differentiation of NSCs into astrocytes. Murine embryonic stem cell (mESC)-derived NSCs and astrocytes were incubated with labelled [1-¹³C]glucose and the label incorporation into intracellular metabolites was followed by GC-MS. The obtained ¹³C labelling patterns, together with uptake/secretion rates determined from supernatant analysis, were integrated into an isotopic non-stationary metabolic flux analysis (¹³C-MFA) model to estimate intracellular flux maps. Significant metabolic differences between NSCs and astrocytes were identified, with a general downregulation of central carbon metabolism during astrocytic differentiation. While glucose uptake was 1.7-fold higher in NSCs (on a per cell basis), a high lactate-secreting phenotype was common to both cell types. Furthermore, NSCs consumed glutamine from the medium; the highly active reductive carboxylation of alpha-ketoglutarate indicates that this was converted to citrate and used for biosynthetic purposes. In astrocytes, pyruvate entered the TCA cycle mostly through pyruvate carboxylase (81%). This pathway supported glutamine and citrate secretion, recapitulating well described metabolic features of these cells in vivo. Overall, this fluxomics study allowed us to quantify the metabolic rewiring accompanying astrocytic lineage specification from NSCs.