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- Hermetia illucens L. Frass in Promoting Soil Fertility in Farming SystemsPublication . Menino, Regina; Esteves, Catarina; Fareleira, Paula; Mano, Raquel; Antunes, Joana; Rehan, Iryna; Murta, Daniel; Moreira, Olga; Bioresources 4 Sustainability (GREEN-IT); MARE - Centro de Ciências do Mar e do Ambiente; DCEA - Departamento de Ciências e Engenharia do Ambiente; GeoBioTec - Geobiociências, Geoengenharias e Geotecnologias; Molecular Diversity Preservation International (MDPI)Following a pot trial with annual ryegrass (Lollium multiflorum Lam. (Pooideae: Poaceae)), where the effect of chemical fertilization was compared with organic fertilization with Black Soldier Fly larvae frass (BSFF), obtained by bio-digestion of cattle production effluents, and with mixed fertilization in proportions of 25%, 50%, and 75% of BSFF, the effect on crop production and soil fertility was tested in three soils of different textures, namely, sandy soil (Gleyic podzol), calcareous soil (Haplic calcisol), and clay soil (Haplic fluvisol). On top of the previous experimental device, a second year of testing was carried out with sowing of the same crop, but without any fertilizer input in all the residual soils for the different further modalities. With regard to the second sowing cycle production, the results are supportive of the expectation that fertilization with BSFF has a superior capacity for soil fertility resilience (assessed in terms of the ability to maintain or even increase soil production in the following year, in the absence of any fertilizer application) in all the soils tested in this experiment, with a significantly greater difference in the treatment corresponding to fertilization with only BSFF compared to the exclusively chemical treatment, in all the soils tested. Furthermore, BSFF, preferably as a mixed fertilizer (in a proportion until 75%), is shown to be a promising alternative for Gleyic podzol in the production of ryegrass as in the resilience and promotion of soil productivity. As far as more fertile soils are concerned (as in the case of Haplic calcisol and Haplic fluvisol), BSFF has not proved promising in terms of immediate crop production.
- Isotope effects on vapor phase 2nd virial coefficientsPublication . Van Hook, W. Alexander; Rebelo, Luis P. N.; Wolfsberg, Max; Bioresources 4 Sustainability (GREEN-IT); Instituto de Tecnologia Química e Biológica António Xavier (ITQB); Institute of Nuclear Chemistry and Technology; 1999Vapor phase 2nd virial coefficient isotope effects (VCIE's) are interpreted. A useful correlation is developed between -Δ(ℬ-bo)/(ℬ-bo) = (-VCIE) and the reference condensed phase reduced isotopic partition function ratio [ln(fc/fg)]*. ℬ is the second virial coefficient, bo = 2πσ3/3, σ is the Lennard-Jones size parameter, and Δ is an isotopic difference, light-heavy. [ln(fc/fg)]* can be obtained from vapor pressure isotope effects for T/TCRITICAL < 0.7. Also (-VCIE) = ln(fp/fg2), where ln(fp/fg2) is the reduced isotopic partition function ratio describing the equilibrium between monomers and interacting pairs. At temperatures well removed from crossovers in ln(fp/fg2) or [ln(fc/fg)]*, ln(fp/fg2) = (0.4±0.2) [ln(fc/fg)]*.
- Kinetic analysis of L1 homophilic interactionPublication . Gouveia, Ricardo M.; Gomes, Cláudio M.; Sousa, Marcos F. Q.; Alves, Paula M.; Costa, Júlia; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); ASBMB - American Society for Biochemistry and Molecular BiologyL1 is a cell adhesion molecule of the immunoglobulin (Ig) superfamily, critical for central nervous system development, and involved in several neuronal biological events. It is a type I membrane glycoprotein. The L1 ectodomain, composed of six Ig-like and five fibronectin (Fn) type-III domains, is involved in homophilic binding. Here, co-immunoprecipitation studies between recombinant truncated forms of human L1 expressed and purified from insect Spodoptera frugiperda Sf9 cells, and endogenous full-length L1 from human NT2N neurons, showed that the L1 ectodomain (L1/ECD) and L1/Ig1-4 interacted homophilically in trans, contrary to mutants L1/Ig1-3 and L1/Ig2-Fn5. All mutants were correctly folded as evaluated by combination of far-UV CD and fluorescence spectroscopy. Surface plasmon resonance analysis showed comparable dissociation constants of 116 ± 2 and 130 ± 6 nM for L1/ECD-L1/ECD and L1/ECD-L1/Ig1-4, respectively, whereas deletion mutants for Ig1 or Ig4 did not interact. Accordingly, in vivo, Sf9 cells stably expressing L1 were found to adhere only to L1/ECD- and L1/Ig1-4-coated surfaces. Furthermore, only these mutants bound to HEK293 cells overexpressing L1 at the cell surface. Enhancement of neurite outgrowth, which is the consequence of signaling events caused by L1 homophilic binding, was comparable between L1/ECD and L1/Ig1-4. Altogether, these results showed that domains Ig1 to Ig4 are necessary and sufficient for L1 homophilic binding in trans, and that the rest of the molecule does not contribute to the affinity under the conditions of the current study. Furthermore, they are compatible with a cooperative interaction between modules Ig1-Ig4 in a horseshoe conformation.
- Response to chitin in suspension-cultured Citrus aurantium cellsPublication . Gallão, Maria I.; Cortelazzo, Angelo L.; Fevereiro, Manoel P.S.; De Brito, Edy S.; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); Brazilian Journal of Plant Physiology / Sociedade Brasileira de Fisiologia VegetalMorphological changes and enzyme activity in suspension-cultured cells of Citrus aurantium were followed after chitin elicitation. Chitin oligomers presented a rapid effect with a maximum activity after 3 h followed by a gradual decrease to the original levels at 8 h of incubation. Cell walls presented a lignification process and the cytoplasmatic protein became less reactive to anionic stains. In the treated material a reduction in starch grain size, an increase in the number of autophagic vacuoles, deposition of secretion in the pericellular space and a defibrillation of the cell-wall polymers were observed. Chitin oligomers increased peroxidase and L-phenylalanine ammonia-lyase activities. Extracellular peroxidase activity increased from 0.20 U mL -1 after 1 h to 0.45 U mL-1 at 3 h followed by a gradual decrease up to 8 h. The peroxidase isoenzyme pattern revealed a predominance of basic isoenzymes in these cells.
- pH dependence of structural and functional properties of oxidized cytochrome c'' from Methylophilus methylotrophusPublication . Coletta, Massimo; Costa, Helena; De Sanctis, Giampiero; Neri, Francesca; Smulevich, Giulietta; Turner, David L.; Santos, Helena; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); ASBMB - American Society for Biochemistry and Molecular BiologyCytochrome c'' from Methylophilus methylotrophus is an unusual monoheme protein that undergoes a major redox-linked change in the heme arrangement: one of the two axial histidines bound to the iron in the oxidized form is detached upon reduction and a proton is taken up. The kinetics of reduction by sodium dithionite and the spectroscopic properties of the oxidized cytochrome c'' have been investigated over the pH range between 1.4 and 10.0. The rate of reduction displays proton-linked transitions of pK(a) ≃ 5.5 and 2.4, and a spectroscopic transition with a pK(a) ≃ 2.4 is also observed. The protein displays a complete reversibility after exposure to low pH, and both electronic absorption and resonance Raman spectroscopic properties suggest that the transition at lower pH brings about a drastic change in the heme coordination geometry. Circular dichroism spectra indicate that over the same proton-linked transition, the protein undergoes a marked decrease (~60%) of the α-helical content toward a random coil arrangement, which is re-covered upon increasing the ionic strength. The structural change at low pH is linked to a concerted two-proton transition, suggesting the detachment and protonation of axial histidine(s). Such kinetic and spectroscopic features along with the remarkable capacity of this protein to recover its native structure after exposure to extremely low pH values makes it a promising model for studying folding processes and stability in heme proteins.
- Escherichia coli di-iron YtfE protein is necessary for the repair of stress-damaged iron-sulfur clustersPublication . Justino, Marta C.; Almeida, Cláudia C.; Teixeira, Miguel; Saraiva, Lígia M; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); ASBMB - American Society for Biochemistry and Molecular BiologyDNA microarray experiments showed that the expression of the Escherichia coli ytfE gene is highly increased upon exposure to nitric oxide. We also reported that deletion of ytfE significantly alters the phenotype of E. coli, generating a strain with enhanced susceptibility to nitrosative stress and defective in the activity of several iron-sulfur-containing proteins. In this work, it is shown that the E. coli ytfE confers protection against oxidative stress. Furthermore, we found that the damage of the [4Fe-4S](2+) clusters of aconitase B and fumarase A caused by exposure to hydrogen peroxide and nitric oxide stress occurs at higher rates in the absence of ytfE. The ytfE null mutation also abolished the recovery of aconitase and fumarase activities, which is observed in wild type E. coli once the stress is scavenged. Notably, upon the addition of purified holo-YtfE protein to the mutant cell extracts, the enzymatic activities of fumarase and aconitase are fully recovered and at rates similar to the wild type strain. We concluded that YtfE is critical for the repair of iron-sulfur clusters damaged by oxidative and nitrosative stress conditions.
- Crystal structure of the human AAA+ protein RuvBL1Publication . Matias, Pedro M.; Gorynia, Sabine; Donner, Peter; Carrondo, Maria Arménia; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); ASBMB - American Society for Biochemistry and Molecular BiologyRuvBL1 is an evolutionarily highly conserved eukaryotic protein belonging to the AAA+-family of ATPases (ATPase associated with diverse cellular activities). It plays important roles in essential signaling pathways such as the c-Myc and Wnt pathways in chromatin remodeling, transcriptional and developmental regulation, and DNA repair and apoptosis. Herein we present the three-dimensional structure of the selenomethionine variant of human RuvBL1 refined using diffraction data to 2.2 Å of resolution. The crystal structure of the hexamer is formed of ADP-bound RuvBL1 monomers. The monomers contain three domains, of which the first and the third are involved in ATP binding and hydrolysis. Although it has been shown that ATPase activity of RuvBL1 is needed for several in vivo functions, we could only detect a marginal activity with the purified protein. Structural homology and DNA binding studies demonstrate that the second domain, which is unique among AAA+ proteins and not present in the bacterial homolog RuvB, is a novel DNA/RNA-binding domain. We were able to demonstrate that RuvBL1 interacted with single-stranded DNA/RNA and double-stranded DNA. The structure of the RuvBL1·ADP complex, combined with our biochemical results, suggest that although RuvBL1 has all the structural characteristics of a molecular motor, even of an ATP-driven helicase, one or more as yet undetermined cofactors are needed for its enzymatic activity.
- Pathway for the synthesis of mannosylglycerate in the hyperthermophilic archaeon Pyrococcus horikoshiiPublication . Empadinhas, Nuno; Marugg, Joey D.; Borges, Nuno; Santos, Helena; Da Costa, Milton S.; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); ASBMB - American Society for Biochemistry and Molecular BiologyThe biosynthetic pathway for the synthesis of the compatible solute α-mannosylglycerate in the hyperthermophilic archaeon Pyrococcus horikoshii is proposed based on the activities of purified recombinant mannosyl-3-phosphoglycerate (MPG) synthase and mannosyl-3-phosphoglycerate phosphatase. The former activity was purified from cell extracts, and the N-terminal sequence was used to identify the encoding gene in the completely sequenced P. horikoshii genome. This gene, designated PH0927, and a gene immediately downstream (PH0926) were cloned and overexpressed in Escherichia coli. The recombinant product of gene PH0927 catalyzed the synthesis of α-mannosyl-3-phosphoglycerate (MPG) from GDP-mannose and D-3-phosphoglycerate retaining the configuration about the anomeric carbon, whereas the recombinant gene product of PH0926 catalyzed the dephosphorylation of mannosyl-3-phosphoglycerate to yield the compatible solute α-mannosylglycerate. The MPG synthase and the MPG phosphatase were specific for these substrates. Two genes immediately downstream from mpgs and mpgp were identified as a putative bifunctional phosphomannose isomerase/mannose-1-phosphate-guanylyltransferase (PH0925) and as a putative phosphomannose mutase (PH0923). Genes PH0927, PH0926, PH0925, and PH0923 were contained in an operon-like structure, leading to the hypothesis that these genes were under the control of an unknown osmosensing mechanism that would lead to α-mannosylglycerate synthesis. Recombinant MPG synthase had a molecular mass of 45,208 Da, a temperature for optimal activity between 90 and 100 °C, and a pH optimum between 6.4 and 7.4; the recombinant MPG phosphatase had a molecular mass of 27,958 Da and optimum activity between 95 and 100 °C and between pH 5.2 and 6.4. This is the first report of the characterization of MPG synthase and MPG phosphatase and the elucidation of a pathway for the synthesis of mannosylglycerate in an archaeon.
- The role of the hybrid cluster protein in oxidative stress defensePublication . Almeida, Cláudia C.; Romão, Célia V.; Lindley, Peter F.; Teixeira, Miguel; Saraiva, Lígia M.; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); ASBMB - American Society for Biochemistry and Molecular BiologyHybrid cluster proteins (HCP) contain two types of Fe/S clusters, namely a [4Fe-4S]2+/1+ or [2Fe-2S]2+/1+ cluster and a novel type of hybrid cluster, [4Fe-2S-2O], in the as-isolated state. Although first isolated from anaerobic sulfate-reducing bacteria, the analysis of the genomic sequences reveals that genes encoding putative hybrid cluster proteins are present in a wide range of organisms, aerobic, anaerobic, or facultative, from the Bacteria, Archaea, and Eukarya domains. Despite a detailed spectroscopic and structural characterization, the precise physiological function of these proteins remained unknown. The present work shows that the transcription of the Escherichia coli hcp gene is induced by hydrogen peroxide, and this induction is regulated by the redox-sensitive transcriptional activator, OxyR. The E. coli hcp mutant strain exhibits higher sensitivity to hydrogen peroxide, a behavior that reverts to the wild type phenotype once a plasmid carrying the hcp gene is reintroduced. Furthermore, the purified HCPs from E. coli and Desulfovibrio desulfuricans ATCC 27774 show an alternative enzymatic activity, which under physiological conditions exhibited Km values for hydrogen peroxide (∼0.3 mM) within the range of other peroxidases. Altogether, the results reveal that HCP is involved in oxidative stress protection.
- Characterization of the murMN operon involved in the synthesis of branched peptidoglycan peptides in Streptococcus pneumoniaePublication . Filipe, Sérgio R.; Pinho, Mariana G.; Tomasz, Alexander; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); ASBMB - American Society for Biochemistry and Molecular BiologyThe murMN operon, recently identified in the genome of Streptococcus pneumoniae, encodes for enzymes involved in the synthesis of branched structured muropeptides in the pneumococcal peptidoglycan; inactivation of murMN causes production of a peptidoglycan composed exclusively of linear muropeptides and a virtually complete loss of resistance in penicillin-resistant strains (Filipe, S. R., and Tomasz, A. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4891-4896). The experiments described in this paper follow up these observations. Primer extension analysis was used to identify the putative promoter region of the murMN operon in penicillin-susceptible and -resistant strains. Selective inactivation of the murN gene in the penicillin-resistant strain Pen6 caused production of an unusual peptidoglycan that contained only single amino acid residues in the muropeptide branches, indicating that the product of murN was involved with the addition of the second amino acid and the product of murM was involved with the addition of the first amino acid (alanine or serine) to the peptidoglycan cross-bridge. Allelic replacement of the mosaic murM gene of strain Pen6 with murM of the penicillin-susceptible laboratory strain caused enrichment of the peptidoglycan in linear muropeptides. The findings suggest that the genetic determinant primarily controlling the synthesis of branched muropeptides in the pneumococcal peptidoglycan is murM.