Nutritional Quality, Techno-Functional Characteristics, and Safety of Biomass Powder and Protein Isolate Produced from Penicillium maximae.

This study investigated the suitability of Penicillium maximae biomass powder and protein isolate as a food product or food ingredient. The biomass powder is rich in proteins (34.8%) and insoluble fiber (36.2%) but poor in lipids (3.1%). Strong water hydration (8.3 g/g, 8.5 g/g) and oil holding (6.9 g/g, 16.3 g/g) capacity were observed in the biomass powder and protein isolate, respectively, besides 100% emulsion stability, indicating multiple applications in the food industry. No locomotor impairment was induced in Drosophila melanogaster flies after consuming extracts of P. maximae biomass powder. Furthermore, decreased production of reactive oxygen species and preservation of survival, viability, and fertility parameters were observed in the nematode Caenorhabditis elegans, which reinforces the potential of P. maximae biomass for human and animal consumption. Together, the results show the vast food applicability of P. maximae biomass and protein isolate as protein substitutes with several health and environmental benefits.

Cultivable fungi present in deep-sea sediments of Antarctica: taxonomy, diversity, and bioprospecting of bioactive compounds.

We accessed the culturable mycobiota present in marine sediments at different depths in Antarctica Ocean. Acremonium fusidioides, Penicillium allii-sativi, Penicillium chrysogenum, Penicillium palitans, Penicillium solitum, and Pseudogymnoascus verrucosus were identified. Penicillium allii-sativi was the dominant species. At least one isolate of each species was capable to present antifungal, trypanocidal, leishmanicidal, antimalarial, nematocidal, or herbicidal activities. Penicillium produced extracts with strong trypanocidal and antimalarial activities, and the extracts of P. solitum and P. chrysogenum demonstrated strong antimalarial activities. Acremonium fusidioides and P. verrucosus displayed strong selective herbicidal properties. The 1H NMR signals for extracts of A. fusidioides, P. chrysogenum, and P. solitum indicated the presence of highly functionalized secondary metabolites, which may be responsible for the biological activities detected. In the deep marine Antarctic sediments, we detected fungal assemblages in which the Penicillium species were found to be dominant and demonstrated capabilities to survive and/or colonise that poly-extreme habitat. Penicillium being a polyextremophile Antarctic species, exhibited strong biological activities and the presence of aromatic compounds in its extracts may indicate that they are wild ancient strains with high genetic and biochemical potentials that enable them to produce bioactive compounds which can be researched in further studies and used in the chemotherapy of neglected tropical diseases as well as in agriculture.

The eIF4E subunits of two distinct trypanosomatid eIF4F complexes are subjected to differential post-translational modifications associated to distinct growth phases in culture.

The eukaryotic eIF4F complex, the cap binding complex, functions during translation initiation through interactions mediated by its three subunits (eIF4E, eIF4G and eIF4A), other initiation factors and the ribosome. In trypanosomatids, various eIF4E and eIF4G homologues were identified, with two eIF4F-like complexes confirmed (EIF4E4/EIF4G3/EIF4AI and EIF4E3/EIF4G4/EIF4AI). Here, the expression pattern of these complexes was investigated during Leishmania amazonensis and Trypanosoma brucei growth. The two sets of eIF4E and eIF4G homologues were found represented by phosphorylated isoforms with multiple phosphorylation events targeting the two eIF4E homologues. Expression of these multiple isoforms was differentially affected by inhibitors of mRNA synthesis/processing and translation. Phosphorylated EIF4E4 was consistently associated with early/active growth phases in both organisms studied. In T. brucei phosphorylation of both EIF4E3 and 4, overexpressed as HA-tagged fusions, was partially mapped to their N-terminuses. Our results indicate that phosphorylation is associated with a further layer of complexity in translation initiation in trypanosomatids.

IMPACT is a developmentally regulated protein in neurons that opposes the eukaryotic initiation factor 2α kinase GCN2 in the modulation of neurite outgrowth.

The product of the mouse Imprinted and Ancient gene, IMPACT, is preferentially expressed in neurons. We have previously shown that IMPACT overexpression inhibits the activation of the protein kinase GCN2, which signals amino acid starvation. GCN2 phosphorylates the α-subunit of eukaryotic translation initiation factor 2 (eIF2α), resulting in inhibition of general protein synthesis but increased translation of specific messages, such as ATF4. GCN2 is also involved in the regulation of neuronal functions, controlling synaptic plasticity, memory, and feeding behavior. We show here that IMPACT abundance increases during differentiation of neurons and neuron-like N2a cells, whereas GCN2 displays lowered activation levels. Upon differentiation, IMPACT associates with translating ribosomes, enhances translation initiation, and down-regulates the expression of ATF4. We further show that endogenous IMPACT promotes neurite outgrowth whereas GCN2 is a strong inhibitor of spontaneous neuritogenesis. Together, these results uncover the participation of the GCN2-IMPACT module of translational regulation in a highly controlled step in the development of the nervous system.

Multiple RNAs from the mouse carboxypeptidase M locus: functional RNAs or transcription noise?

BACKGROUND: A major effort of the scientific community has been to obtain complete pictures of the genomes of many organisms. This has been accomplished mainly by annotation of structural and functional elements in the genome sequence, a process that has been centred in the gene concept and, as a consequence, biased toward protein coding sequences. Recently, the explosion of transcriptome data generated and the discovery of many functional non-protein coding RNAs have painted a more detailed and complex scenario for the genome. Here we analyzed the mouse carboxypeptidase M locus in this broader perspective in order to define the mouse CPM gene structure and evaluate the existence of other transcripts from the same genomic region. RESULTS: Bioinformatic analysis of nucleotide sequences that map to the mouse CPM locus suggests that, in addition to the mouse CPM mRNA, it expresses at least 33 different transcripts, many of which seem to be non-coding RNAs. We randomly chose to evaluate experimentally four of these extra transcripts. They are expressed in a tissue specific manner, indicating that they are not artefacts or transcriptional noise. Furthermore, one of these four extra transcripts shows expression patterns that differed considerably from the other ones and from the mouse CPM gene, suggesting that there may be more than one transcriptional unit in this locus. In addition, we have confirmed the mouse CPM gene RefSeq sequence by rapid amplification of cDNA ends (RACE) and directional cloning. CONCLUSION: This study supports the recent view that the majority of the genome is transcribed and that many of the resulting transcripts seem to be non-coding RNAs from introns of genes or from independent transcriptional units. Although some of the information on the transcriptome of many organisms may actually be artefacts or transcriptional noise, we argue that it can be experimentally evaluated and used to find and define biological functional elements on the genome. Furthermore, the transcription of other functional RNAs besides the protein coding RNA from a specific genomic locus imposes extra care when designing and interpreting experiments involving genetic manipulations or expression detection and quantification.

GCN2 activation and eIF2alpha phosphorylation in the maturation of mouse oocytes.

GCN2 is one of the four mammalian kinases that phosphorylate the alpha subunit of the translation initiation factor 2 (eIF2alpha) in a variety of stress situations, resulting in protein synthesis inhibition. GCN2 is involved in regulating metabolism, feeding behavior and memory in rodents. We show here that, relative to other cells, the beta isoform of the GCN2 transcript and the GCN2 protein are highly abundant in unfertilized mouse eggs. In addition, GCN2 in these cells is active, resulting in elevated levels of phosphorylated eIF2alpha. After fertilization, eIF2alpha phosphorylation decreases drastically. These results suggest that GCN2 mediated translational control may contribute to regulatory mechanisms operating during oocyte maturation.

Testes em condições para o controle de Dysmicoccus texensis (Tinsley) (Hemiptera, Pseudococcidae) em cafeeiro com nematóides entomopatogênicos do gênero Heterorhabditis(Rhabditida, Heterorhabditidae) / Tests for the control of coffee root mealybug Dysmicoccus texensis (Tinsley) (Hemiptera, Pseudococcidae) with Heterorhabditis (Rhabditida, Heterorhabditidae)

Os nematóides entomopatogênicos (NEPs) apresentam potencial para o controle biológico de pragas e têm sido usados com sucesso, em vários países, no controle de pragas de solo e de ambientes crípticos, como a cochonilha-da-raiz-do-cafeeiro Dysmicoccus texensis (Tinsley). Testes de laboratório demonstram que estes agentes apresentam alta virulência sobre este inseto, no entanto, são necessários testes que avaliem a eficiência dos NEPs em condições de casa-de-vegetação e campo, sendo este o objetivo do presente trabalho. O experimento em condição de casa-de-vegetação para o controle da cochonilha foi realizado em vasos infestados, usando dois isolados do nematóide e dois métodos de aplicação (cadáver infectado e suspensão aquosa), conduzido em delineamento inteiramente casualisado com cinco repetições. O experimento em condições de campo foi conduzido em blocos casualisados (6 blocos), para avaliar a eficiência de dois isolados heterorhabditídeos no controle da cochonilha-da-raiz-do-cafeeiro. Os resultados mostraram que, em casa-de-vegetação, o método de suspensão aquosa apresentou melhores resultados para os dois isolados, sendo que JPM3 aplicado em suspensão aquosa foi o melhor tratamento, apresentando eficiência de controle de 70 por cento. No experimento de campo, apenas o tratamento com o inseticida Actara 250 WG (thiamethoxam), usado como padrão de comparação, e JPM3, aplicado em suspensão aquosa, diferiram da testemunha, apresentando 81 e 65 por cento de eficiência de controle, respectivamente.

Entomopathogenic nematodes (EPNs) have potential for biological pest control and have been successfully used in several countries in soil and cryptic pests control, as for example the coffee root mealybug Dysmicoccus texensis (Tinsley). Laboratory tests demonstrated that these agents are highly virulent to the insect, but semi-field and field tests are needed to determine their efficiency. Greenhouse tests were made in infested pots with two isolates and two application methods dead insect bodies and aqueous suspension in a complete randomized design with five replicates. Field tests were made in randomized plots (six plots) to evaluate six isolates of Heterorhabditis on coffee root for mealybug control. Greenhouse results demonstrate that aqueous suspension was more efficient for the two isolates, with 70 percent control efficiency for JPM3. In field experiments, treatments with aqueous suspensions of insecticide Actara 250 WG (thiamethoxam), used for comparison, and JPM3 were the only ones statistically different from control, with 81 and 65 percent control efficiency, respectively.

Novel membrane-bound eIF2alpha kinase in the flagellar pocket of Trypanosoma brucei.

Translational control mediated by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2alpha) is central to stress-induced programs of gene expression. Trypanosomatids, important human pathogens, display differentiation processes elicited by contact with the distinct physiological milieu found in their insect vectors and mammalian hosts, likely representing stress situations. Trypanosoma brucei, the agent of African trypanosomiasis, encodes three potential eIF2alpha kinases (TbeIF2K1 to -K3). We show here that TbeIF2K2 is a transmembrane glycoprotein expressed both in procyclic and in bloodstream forms. The catalytic domain of TbeIF2K2 phosphorylates yeast and mammalian eIF2alpha at Ser51. It also phosphorylates the highly unusual form of eIF2alpha found in trypanosomatids specifically at residue Thr169 that corresponds to Ser51 in other eukaryotes. T. brucei eIF2alpha, however, is not a substrate for GCN2 or PKR in vitro. The putative regulatory domain of TbeIF2K2 does not share any sequence similarity with known eIF2alpha kinases. In both procyclic and bloodstream forms TbeIF2K2 is mainly localized in the membrane of the flagellar pocket, an organelle that is the exclusive site of exo- and endocytosis in these parasites. It can also be detected in endocytic compartments but not in lysosomes, suggesting that it is recycled between endosomes and the flagellar pocket. TbeIF2K2 location suggests a relevance in sensing protein or nutrient transport in T. brucei, an organism that relies heavily on posttranscriptional regulatory mechanisms to control gene expression in different environmental conditions. This is the first membrane-associated eIF2alpha kinase described in unicellular eukaryotes.

Phosphorylation of the alpha subunit of translation initiation factor-2 by PKR mediates protein synthesis inhibition in the mouse brain during status epilepticus.

In response to different cellular stresses, a family of protein kinases phosphorylates eIF2alpha (alpha subunit of eukaryotic initiation factor-2), contributing to regulation of both general and genespecific translation proposed to alleviate cellular injury or alternatively induce apoptosis. Recently, we reported eIF2alpha(P) (phosphorylated eIF2alpha) in the brain during SE (status epilepticus) induced by pilocarpine in mice, an animal model of TLE (temporal lobe epilepsy) [Carnevalli, Pereira, Longo, Jaqueta, Avedissian, Mello and Castilho (2004) Neurosci. Lett. 357, 191-194]. We show in the present study that one eIF2alpha kinase family member, PKR (double-stranded-RNA-dependent protein kinase), is activated in the cortex and hippocampus at 30 min of SE, reflecting the levels of eIF2alpha(P) in these areas. In PKR-deficient animals subjected to SE, eIF2alpha phosphorylation was clearly evident coincident with activation of a secondary eIF2alpha kinase, PEK/PERK (pancreatic eIF2alpha kinase/RNA-dependent-protein-kinase-like endoplasmic reticulum kinase), denoting a compensatory mechanism between the two kinases. The extent of eIF2alpha phosphorylation correlated with the inhibition of protein synthesis in the brain, as determined from polysome profiles. We also found that C57BL/6 mice, which enter SE upon pilocarpine administration but are more resistant to seizure-induced neuronal degeneration, showed very low levels of eIF2alpha(P) and no inhibition of protein synthesis during SE. These results taken together suggest that PKR-mediated phosphorylation of eIF2alpha contributes to inhibition of protein synthesis in the brain during SE and that sustained high levels of eIF2alpha phosphorylation may facilitate ensuing cell death in the most affected areas of the brain in TLE.

Gir2 is an intrinsically unstructured protein that is present in Saccharomyces cerevisiae as a group of heterogeneously electrophoretic migrating forms.

Gir2 is a highly acidic cytoplasmic protein of Saccharomyces cerevisiae of unknown function that shows an anomalous migration on SDS-PAGE. Based on its large Stokes radius and thermostability, we have previously suggested that Gir2 lacks extensive secondary structure. Here we report that Gir2 is extremely sensitive to proteolysis when compared to glutathione-S-transferase, a highly structured protein, further indicating its unfolded nature. Prediction based on the FoldIndex program also indicates that Gir2 is a disordered protein. Using truncated forms of Gir2 we show that the N-terminal half of this protein, with its high content of acidic amino acid residues, is responsible for the anomalous electrophoretic behavior of Gir2. Because all these features are hallmarks of intrinsically unstructured proteins (IUP), we propose that Gir2 is another representative of the IUP group of proteins. Additionally, we describe that the endogenous yeast Gir2 shows heterogeneous electrophoretic mobility, which is not due to proteolytic cleavage.

Patogenicidade de Steinernema glaseri e S. carpocapsae (Nematoda: Rhabdita) contra o cascudinho, Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) / Pathogenicity of Steinernema glaseri and S. carpocapsae (Nematoda: Rhabdita) against the lesswer mealworm, Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae)

A importância econômica do cascudinho, Alphitobius diaperinus (Panzer), para a avicultura e a ausência de medidas de controle eficientes e seguras têm levado à busca de alternativas no controle microbiano. Este trabalho foi desenvolvido com o objetivo de avaliar a patogenicidade de nematóides entomopatogênicos da família Steinernematidae contra larvas e adultos do inseto. Verificou-se que as larvas foram mais suscetíveis que os adultos e que dois isolados de S. carpocapsae foram os mais patogênicos.

The lesser mealworm, Alphitobius diaperinus (Panzer), is an important pest in poultry production systems. Due to the lack of effective and safe tactics to control this insect the interest on developing alternative control tactics such as microbial control has increased. The pathogenicity of entomopathogenic nematodes (Steinernematidae) was evaluated in laboratory conditions, against larvae and adults of the lesser mealworm. Adults were less susceptible than larvae and two strains of S. carpocapsae were the most pathogenic.

Ocorrência de Metarhizium anisopliae (Metsch. ) Sorok. em adultos de cascudinho (Alphitobius diaperinus) (Panzer) (Coleoptera: Tenebrionidae) em aviários comerciais em Cascavel, PR / Natural occurrence of Metarhizium anisopliae (Metsch. ) Sorok. on adults of the lesser mealworm (Alphitobius diaperinus) (Panzer) (Coleoptera: Tenebrionidae) in poultry houses in Cascavel, PR, Brazil

Alphitobius diaperinus (Panzer) é uma das pragas mais importantes que atacam criações comerciais de frango de corte em todo o mundo. Os insetos são hospedeiros de microrganismos patogênicos às aves, provocam ferimentos no trato digestivo das mesmas e afetam a conversão alimentar. Embora seja praticado, o controle dos insetos com inseticidas químicos é insatisfatório, na maioria das vezes, e oferece risco às aves. O controle biológico destaca-se como alternativa viável, uma vez que os entomopatógenos são inócuos a animais homeotérmicos, não oferecendo risco de contaminação às aves e ao produtor. A presença de entomopatógenos no ambiente contribui para o controle natural dos insetos-praga e, neste sentido, o presente trabalho teve como objetivo registrar a ocorrência natural de Metarhizium anisopliae sobre adultos de A. diaperinus, em aviários comerciais no município de Cascavel, PR.

Alphitobius diaperinus (Panzer), the lesser mealworm, is the most important pest in large-scale poultry production and is capable of harboring several types of poultry pathogens. Chickens feed readily on the beetles in infested litter and this sometimes causes nutritional problems and the weight gain may be affected. Chemical insecticides are usually applied, however beetle infestation are very difficult to control using these products. Besides, this control method can also offer risks to chicken and man. Microbial control may be a promising strategy to control A. diaperinus in poultry houses and natural occurrence of entomopathogenic fungi is very important to control the lesser mealworm in poultry houses. The objective of this work was to register the occurrence of Metarhizium anisopliae on adults of the lesser mealworm in commercial poultry houses in Brazil.

Biophysical characterization of Gir2, a highly acidic protein of Saccharomyces cerevisiae with anomalous electrophoretic behavior.

Gir2 is an uncharacterized protein of Saccharomyces cerevisiae, containing a RWD/GI domain. In this work, we report the biophysical characterization of Gir2. His-tagged Gir2, expressed and purified from Escherichia coli, showed an abnormally slow migration on SDS-PAGE. The yeast expressed protein behaves similarly. Using mass spectrometry and peptide mass fingerprinting we demonstrated that the protein has the expected molecular mass (34kDa). EDC modification of carboxylate groups reverted the anomalous migration on SDS-PAGE. Size exclusion chromatography showed that Gir2 has a Stokes radius larger than expected. Gir2 is thermostable and lacks extensive structure, as determined by CD analysis. Based on these findings, we suggest that Gir2 is a representative of the growing group of "natively unfolded" proteins.