RESUMEN

Peptides are remarkably interesting alternatives to several applications. In particular, antimicrobial sequences have raised major interest of the scientific community due to the resistance acquired by commonly used antibiotics. Amongst these, some dimeric peptides have shown very promising characteristics as strong biological activities and resistance against degradation by peptidases. However, despite such promising characteristics, a relatively small number of studies address dimeric peptides, mainly due to the synthesis-related obstacles in their production, whereas the well-implemented routines of solid phase peptide synthesis-which includes the possibility of automation-makes life significantly easier. Here, we present kinetic investigations of the dimerization of a cysteine-containing sequence to obtain the homodimeric antimicrobial peptide homotarsinin. Based on the structural and membrane interaction data already available for the dimer and its monomeric chain, we have proposed distinct dimerization protocols in selected environments, namely, aqueous buffer, TFE:H2O and micellar solutions. The experimental results were adjusted by a theoretical model. Both the kinetic profiles and the reaction yields are dependent on the reaction medium, clearly indicating that aggregation, peptide structure, and peptide-membrane interactions play major roles in the formation of the disulfide bond. Finally, the rationalization of the different aspects addressed here is expected to contribute to research and applications that demand the obtainment of dimeric peptides.

RESUMEN

Salicylate hydroxylase (NahG) has a single redox site in which FAD is reduced by NADH, the O2 is activated by the reduced flavin, and salicylate undergoes an oxidative decarboxylation by a C(4a)-hydroperoxyflavin intermediate to give catechol. We report experimental results that show the contribution of individual pieces of the FAD cofactor to the observed enzymatic activity for turnover of the whole cofactor. A comparison of the kinetic parameters and products for the NahG-catalyzed reactions of FMN and riboflavin cofactor fragments reveal that the adenosine monophosphate (AMP) and ribitol phosphate pieces of FAD act to anchor the flavin to the enzyme and to direct the partitioning of the C(4a)-hydroperoxyflavin reaction intermediate towards hydroxylation of salicylate. The addition of AMP or ribitol phosphate pieces to solutions of the truncated flavins results in a partial restoration of the enzymatic activity lost upon truncation of FAD, and the pieces direct the reaction of the C(4a)-hydroperoxyflavin intermediate towards hydroxylation of salicylate.

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The D37 and T100' side chains of orotidine 5'-monophosphate decarboxylase (OMPDC) interact with the C-3' and C-2' ribosyl hydroxyl groups, respectively, of the bound substrate. We compare the intra-subunit interactions of D37 with the inter-subunit interactions of T100' by determining the effects of the D37G, D37A, T100'G, and T100'A substitutions on the following: (a) kcat and kcat/Km values for the OMPDC-catalyzed decarboxylations of OMP and 5-fluoroorotidine 5'-monophosphate (FOMP) and (b) the stability of dimeric OMPDC relative to the monomer. The D37G and T100'A substitutions resulted in 2 kcal mol-1 increases in ΔG† for kcat/Km for the decarboxylation of OMP, while the D37A and T100'G substitutions resulted in larger 4 and 5 kcal mol-1 increases, respectively, in ΔG†. The D37G and T100'A substitutions both resulted in smaller 2 kcal mol-1 decreases in ΔG† for the decarboxylation of FOMP compared to that of OMP. These results show that the D37G and T100'A substitutions affect the barrier to the chemical decarboxylation step while the D37A and T100'G substitutions also affect the barrier to a slow, ligand-driven enzyme conformational change. Substrate binding induces the movement of an α-helix (G'98-S'106) toward the substrate C-2' ribosyl hydroxy bound at the main subunit. The T100'G substitution destabilizes the enzyme dimer by 3.5 kcal mol-1 compared to the monomer, which is consistent with the known destabilization of α-helices by the internal Gly side chains [Serrano, L., et al. (1992) Nature, 356, 453-455]. We propose that the T100'G substitution weakens the α-helical contacts at the dimer interface, which results in a decrease in the dimer stability and an increase in the barrier to the ligand-driven conformational change.

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RESUMEN

Herein, we present the light-induced synthesis and characterization of a La3+/spiropyran derivative complex (LaMC) and its application as a catalyst when incorporated into electrospun polycaprolactone (PCL) fibers. In addition to experimental methods, computational calculations were also essential to better understand the structure and electronic characteristics of LaMC. The LaMC complex was identified as a 10-coordinated structure with the La3+ ion coordinated by four oxygens from the phenolate and the carbonyl of the carboxyl acid group from both MC ligands and by six oxygens from three nitrate ligands. In addition, LaMC was capable of getting reversibly isomerized by UV or visible light cycling. All PCL fibers were successively obtained, and their morphologies, surface properties, and catalytic behavior were studied. Results showed that PCL/LaMC fibers were capable of catalyzing bis(2,4-dinitrophenyl)phosphate degradation efficiently. Complete hydrolysis was accomplished in only 1.5 days relative to the half-life time of 35 days for the uncatalyzed hydrolysis at pH 8.1 and 25 °C.

RESUMEN

Salicylate hydroxylase (NahG) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of salicylate into catechol in the naphthalene degradation pathway in Pseudomonas putida G7. We explored the mechanism of action of this enzyme in detail using a combination of structural and biophysical methods. NahG shares many structural and mechanistic features with other versatile flavin-dependent monooxygenases, with potential biocatalytic applications. The crystal structure at 2.0 Šresolution for the apo form of NahG adds a new snapshot preceding the FAD binding in flavin-dependent monooxygenases. The kcat/Km for the salicylate reaction catalyzed by the holo form is >105 M-1 s-1 at pH 8.5 and 25 °C. Hammett plots for Km and kcat using substituted salicylates indicate change in rate-limiting step. Electron-donating groups favor the hydroxylation of salicylate by a peroxyflavin to yield a Wheland-like intermediate, whereas the decarboxylation of this intermediate is faster for electron-withdrawing groups. The mechanism is supported by structural data and kinetic studies at different pHs. The salicylate carboxyl group lies near a hydrophobic region that aids decarboxylation. A conserved histidine residue is proposed to assist the reaction by general base/general acid catalysis.

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A detecção superprecoce de gestação é de grande relevância quando se trata do aumento da produtividade dos rebanhos, na tentativa de redução do tempo de serviço desses animais. Por tal relevância, objetivou-se utilizar a ultrassonografia (US) Doppler como ferramenta para predição de gestação, avaliando a irrigação do corpo lúteo (CL), vinte dias após IATF. Setenta e três vacas mestiças foram avaliadas 20 dias após IATF por US transretal. Para obtenção do diâmetro do CL (DCL) e sua área (ACL) utilizou-se o modo B, na mensuração da área de vascularização (VCL) e seu percentual no CL (VCL%) empregou-se o Doppler Colorido. As imagens foram classificadas com base na quantidade de pixels coloridos e sua distribuição no CL, em dois grupos: diagnóstico preditivo negativo de gestação (DPN) e preditivo positivo (DPP). No mesmo dia, a funcionalidade do CL foi determinada pela concentração plasmática de P4. O diagnóstico de gestação definitivo foi realizado por US 35 dias após as IATFs. Utilizou-se o teste t de Student (com P<0,05) na avaliação das variáveis. Grupo gestantes apresentou médias superiores (P<0,05) para todas as variáveis ao Grupo Não Gestantes. O diagnóstico preditivo demonstrou 83,33% de especificidade, sensibilidade de 100% e acurácia de 91,79%. Conclui-se que a US doppler do CL aos 20 dias pós IATF é um método confiável no diagnóstico precoce de gestação.(AU)

The super early gestation detection is of great relevance considering the increase of the herds' productivity, in an attempt to reduce the time of service of these animals. Due to this relevance, this study aimed to use Doppler ultrasonography as a tool for gestation prediction, evaluating the luteal body (CL) irrigation twenty days after FTAI. 73 crossbred cows were evaluated 20 days after FTAI through trans rectal US. In order to obtain the diameter of the CL (DCL) and its area (ACL), B mode was used. The Color Doppler was applied to measure the vascularization area (CLV) and its percentage in the CL (VCL %). The images were classified, based on the number of colored pixels and their distribution in CL, in two groups: negative predictive diagnosis of gestation (NPD) and positive predictive value (PPV). On the same day, LC functionality was determined based on P4 plasma concentration. The definitive gestation diagnosis was confirmed through US 35 days after the FTAIs. Student's T test (with P<0.05) was used to evaluate the variables. Pregnant group presented higher averages (P<0.05) for all variables compared to the Non-Pregnant Group. The predictive diagnosis showed 83.33% of specificity, 100% of sensitivity and 91.79% of accuracy. The conclusion is that the US Doppler at 20 days post FTAI is a reliable method for early gestation diagnosis.(AU)

Asunto(s)

RESUMEN

A detecção superprecoce de gestação é de grande relevância quando se trata do aumento da produtividade dos rebanhos, na tentativa de redução do tempo de serviço desses animais. Por tal relevância, objetivou-se utilizar a ultrassonografia (US) Doppler como ferramenta para predição de gestação, avaliando a irrigação do corpo lúteo (CL), vinte dias após IATF. Setenta e três vacas mestiças foram avaliadas 20 dias após IATF por US transretal. Para obtenção do diâmetro do CL (DCL) e sua área (ACL) utilizou-se o modo B, na mensuração da área de vascularização (VCL) e seu percentual no CL (VCL%) empregou-se o Doppler Colorido. As imagens foram classificadas com base na quantidade de pixels coloridos e sua distribuição no CL, em dois grupos: diagnóstico preditivo negativo de gestação (DPN) e preditivo positivo (DPP). No mesmo dia, a funcionalidade do CL foi determinada pela concentração plasmática de P4. O diagnóstico de gestação definitivo foi realizado por US 35 dias após as IATFs. Utilizou-se o teste t de Student (com P<0,05) na avaliação das variáveis. Grupo gestantes apresentou médias superiores (P<0,05) para todas as variáveis ao Grupo Não Gestantes. O diagnóstico preditivo demonstrou 83,33% de especificidade, sensibilidade de 100% e acurácia de 91,79%. Conclui-se que a US doppler do CL aos 20 dias pós IATF é um método confiável no diagnóstico precoce de gestação.(AU)

The super early gestation detection is of great relevance considering the increase of the herds' productivity, in an attempt to reduce the time of service of these animals. Due to this relevance, this study aimed to use Doppler ultrasonography as a tool for gestation prediction, evaluating the luteal body (CL) irrigation twenty days after FTAI. 73 crossbred cows were evaluated 20 days after FTAI through trans rectal US. In order to obtain the diameter of the CL (DCL) and its area (ACL), B mode was used. The Color Doppler was applied to measure the vascularization area (CLV) and its percentage in the CL (VCL %). The images were classified, based on the number of colored pixels and their distribution in CL, in two groups: negative predictive diagnosis of gestation (NPD) and positive predictive value (PPV). On the same day, LC functionality was determined based on P4 plasma concentration. The definitive gestation diagnosis was confirmed through US 35 days after the FTAIs. Student's T test (with P<0.05) was used to evaluate the variables. Pregnant group presented higher averages (P<0.05) for all variables compared to the Non-Pregnant Group. The predictive diagnosis showed 83.33% of specificity, 100% of sensitivity and 91.79% of accuracy. The conclusion is that the US Doppler at 20 days post FTAI is a reliable method for early gestation diagnosis.(AU)

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Objetivou-se avaliar o efeito de uma ou duas doses de prostaglandina F2α (PGF2α) associada ou não a gonadotrofina coriônica equina (eCG) sobre a dinâmica folicular, a função luteal pré-ovulatória, assim como as características morfofuncionais pós-ovulatórias do corpo lúteo (CL) em fêmeas mestiças cíclicas submetidas a um protocolo de inseminação artificial em tempo fixo (IATF). Para tanto, 29 vacas 3/4 Gir x Holandês multíparas foram submetidas ao exame de ultrassonografia (US) transretal e após a detecção do CL iniciou-se um protocolo de IATF em um dia denominado zero (D0), por meio da inserção do implante de progesterona (P4) associado à aplicação de 2,0mg de benzoato de estradiol. No D7 esses animais receberam 12,5mg de dinoprost trometamina. No D9 realizou a remoção dos dispositivos de P4 e aplicou 0,6mg de cipionato de estradiol. Nesse momento, as fêmeas foram subdivididas nos seguintes tratamentos: Grupo Controle (n=7), foi administrado 2,5mL de solução fisiológica; Grupo 2PGF (n=7), aplicou 12,5mg de dinoprost trometamina; Grupo eCG (n=7), administrou-se 300UI de eCG; Grupo 2PGF+eCG (n=8), realizou a aplicação de 300UI de eCG e 12,5mg de dinoprost trometamina. Para avaliar a dinâmica folicular foram realizados exames de US em modo B e power doppler (Mindray Z5, Shenzhen, China) a cada 12h do D7 até o momento da ovulação ou 96h após a remoção dos implantes de P4, mensurando-se o diâmetro folicular (DFOL), a área da parede folicular (AFOL) e a área de perfusão sanguínea da parede folicular (VFOL). Concomitante a cada exame, foram coletadas amostras de sangue sendo determinada a concentração sérica de P4 pré-ovulatória por meio da metodologia de quimioluminescência. No D24 foi realizada a US modo B e doppler analisando-se o diâmetro luteal (DCL), área luteal (ACL) e área de perfusão sanguínea do CL (VCL), assim como, foi coletada amostra de sangue para averiguar a concentração sérica de P4 pós-ovulatória.(AU)

The study aimed to evaluate the effect of one or two prostaglandin doses F2α (PGF2a) with or without equine chorionic gonadotropin (eCG) in the follicular dynamics, the preovulatory luteal function, as well as the structural and functional characteristics post-ovulatory of the corpus luteum (CL) in cyclic crossbred females subjected to a fixed time artificial insemination (FTAI) protocol. For this, 29 multiparous 3/4 Gyr x Holstein cows were subjected to transrectal ultrasound examination (US) and upon detection of CL initiated a FTAI protocol on day called zero (D0) by the insertion of progesterone implant (P4) associated with the application of 2.0mg estradiol benzoate. On D7, these animals received 12.5mg of dinoprost tromethamine. At D9 happened the removal of the P4 devices and was applied 0.6mg of estradiol cypionate. At that time, the females were divided into the following treatments: control group (n=7) - which received 2.5mL of saline solution, 2PGF group (n=7) - received 12.5mg of dinoprost tromethamine, eCG group (n=7) - was administered 300IU eCG and eCG+2PGF group (n=8) - which received 300 IU eCG and 12.5mg of dinoprost tromethamine. To assess follicular dynamics were performed US scans B-mode and power doppler (Mindray Z5, Shenzhen, China) each 12h on D7 until the time of ovulation or until 96h after removal of the P4 implants, considering the follicular diameter (DFOL), the area of the follicular wall (AFOL) and the blood perfusion area of the follicular wall (VFOL). Concomitant with each test, blood samples were collected to determine the serum concentration of P4 preovulatory by chemiluminescence methodology. In D24 had held US B-mode and doppler to analyse the luteal diameter (DCL), luteal area (ACL) and blood perfusion area CL (VCL). (AU)

Asunto(s)

RESUMEN

Objetivou-se avaliar o efeito de uma ou duas doses de prostaglandina F2α (PGF2α) associada ou não a gonadotrofina coriônica equina (eCG) sobre a dinâmica folicular, a função luteal pré-ovulatória, assim como as características morfofuncionais pós-ovulatórias do corpo lúteo (CL) em fêmeas mestiças cíclicas submetidas a um protocolo de inseminação artificial em tempo fixo (IATF). Para tanto, 29 vacas 3/4 Gir x Holandês multíparas foram submetidas ao exame de ultrassonografia (US) transretal e após a detecção do CL iniciou-se um protocolo de IATF em um dia denominado zero (D0), por meio da inserção do implante de progesterona (P4) associado à aplicação de 2,0mg de benzoato de estradiol. No D7 esses animais receberam 12,5mg de dinoprost trometamina. No D9 realizou a remoção dos dispositivos de P4 e aplicou 0,6mg de cipionato de estradiol. Nesse momento, as fêmeas foram subdivididas nos seguintes tratamentos: Grupo Controle (n=7), foi administrado 2,5mL de solução fisiológica; Grupo 2PGF (n=7), aplicou 12,5mg de dinoprost trometamina; Grupo eCG (n=7), administrou-se 300UI de eCG; Grupo 2PGF+eCG (n=8), realizou a aplicação de 300UI de eCG e 12,5mg de dinoprost trometamina. Para avaliar a dinâmica folicular foram realizados exames de US em modo B e power doppler (Mindray Z5, Shenzhen, China) a cada 12h do D7 até o momento da ovulação ou 96h após a remoção dos implantes de P4, mensurando-se o diâmetro folicular (DFOL), a área da parede folicular (AFOL) e a área de perfusão sanguínea da parede folicular (VFOL). Concomitante a cada exame, foram coletadas amostras de sangue sendo determinada a concentração sérica de P4 pré-ovulatória por meio da metodologia de quimioluminescência. No D24 foi realizada a US modo B e doppler analisando-se o diâmetro luteal (DCL), área luteal (ACL) e área de perfusão sanguínea do CL (VCL), assim como, foi coletada amostra de sangue para averiguar a concentração sérica de P4 pós-ovulatória. Os dados foram avaliados pelo Two-way ANOVA e análise de medidas repetidas considerando os efeitos do eCG, 2PGF e interação eCG*2PGF, P<0,05. Não houve diferença significativa entre os protocolos de sincronização para as variáveis DFOL, AFOL e VFOL ao longo do tempo da dinâmica folicular. Os grupos experimentais apresentaram uma concentração sérica de P4 pré-ovulatória semelhante em cada momento da avaliação. Não foi observada distinção da ACL e VCL entre os tratamentos hormonais, contudo o Grupo eCG demonstrou tendência (P=0,08) a apresentar maior DCL em relação ao Grupo 2PGF e 2PGF+eCG. Adicionalmente a estes achados, também foi constatado tendência (P=0,07) a maiores concentrações de progesterona no dia 24 do protocolo nos animais do Grupo eCG (11,00±3,32ng/mL) em relação ao Grupo 2PGF (6,37±1,31ng/mL), enquanto o Controle e 2PGF+eCG demonstraram resultados intermediários que se assemelham a ambos os grupos, com concentrações de 8,43±3,85 e 9,18±2,82ng/mL, respectivamente. As tentativas de ajustes no proestro foram incapazes de melhorar a qualidade folicular e minimizar a função luteal pré-ovulatória, assim como não incrementaram a morfologia do CL e a função luteal pós-ovulatória, sugerindo que em animais cíclicos mestiços protocolos de IATF com a utilização de uma única dose PGF2α e sem o suporte gonadotrófico da eCG parece promover adequada resposta folicular e luteal.(AU)

The study aimed to evaluate the effect of one or two prostaglandin doses F2α (PGF2a) with or without equine chorionic gonadotropin (eCG) in the follicular dynamics, the preovulatory luteal function, as well as the structural and functional characteristics post-ovulatory of the corpus luteum (CL) in cyclic crossbred females subjected to a fixed time artificial insemination (FTAI) protocol. For this, 29 multiparous 3/4 Gyr x Holstein cows were subjected to transrectal ultrasound examination (US) and upon detection of CL initiated a FTAI protocol on day called zero (D0) by the insertion of progesterone implant (P4) associated with the application of 2.0mg estradiol benzoate. On D7, these animals received 12.5mg of dinoprost tromethamine. At D9 happened the removal of the P4 devices and was applied 0.6mg of estradiol cypionate. At that time, the females were divided into the following treatments: control group (n=7) - which received 2.5mL of saline solution, 2PGF group (n=7) - received 12.5mg of dinoprost tromethamine, eCG group (n=7) - was administered 300IU eCG and eCG+2PGF group (n=8) - which received 300 IU eCG and 12.5mg of dinoprost tromethamine. To assess follicular dynamics were performed US scans B-mode and power doppler (Mindray Z5, Shenzhen, China) each 12h on D7 until the time of ovulation or until 96h after removal of the P4 implants, considering the follicular diameter (DFOL), the area of the follicular wall (AFOL) and the blood perfusion area of the follicular wall (VFOL). Concomitant with each test, blood samples were collected to determine the serum concentration of P4 preovulatory by chemiluminescence methodology. In D24 had held US B-mode and doppler to analyse the luteal diameter (DCL), luteal area (ACL) and blood perfusion area CL (VCL). Also, a blood sample was collected to determine the serum concentration of P4 post-ovulatory. All data was evaluated by Two-way ANOVA and repeated measures analysis considering the effects of eCG, 2PGF and eCG*2PGF, P<0.05. There was not significant difference between the synchronization protocols for DFOL, AFOL and VFOL variables over time of follicular dynamics. Experimental groups had a serum concentration of P4 preovulatory similar in every moment of evaluation. There wasn't distinction of ACL and VCL between hormone treatments. However, the eCG group showed a tendency (P=0.08) to present higher DCL compared to the 2PGF and 2PGF+eCG groups. In addition to these findings, there was also a tendency (P=0.07) to higher concentrations of P4 on D24 of the protocol in the animals of the eCG group (11.00±3.32ng/mL) compared to the 2PGF group (6,37±1.31ng/mL), meanwhile the Control and 2PGF+eCG showed intermediate results that resembled both groups, with concentrations of 8.43±3.85 and 9.18±2.82ng/mL, respectively. Attempts to adjust proestrus were unable to improve follicular quality and minimize preovulatory luteal function, nor did they increase CL morphology and post-ovulatory luteal function, suggesting that in cyclic animals, FTAI protocols using a single PGF2α dose and without the gonadotrophic support of eCG seems to promote adequate follicular and luteal responses.(AU)

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RESUMEN

The second enzyme of the naphthalene degradation pathway in Pseudomonas putida G7 is NahB, a dehydrogenase that converts cis-1,2-dihydroxy-1,2-dihydronaphthalene to 1,2-dihydroxynaphthalene. We report the cloning, optimization of expression, purification, kinetic studies and preliminary structural characterization of the recombinant NahB. The nahB gene was cloned into a T7 expression vector and the enzyme was overexpressed in Escherichia coli Rosetta (DE3) as an N-terminal hexa-histidine-tagged protein (6xHis-NahB). Using methods of enhancing protein stability in solution, we tested different expression, cell lysis, and purification protocols with and without ligand supplementation. The protein stability was evaluated by dynamic light scattering and circular dichroism spectroscopy assays. Best-derived protocols (expression at 18 °C, cell lysis with homogenizer, and three purification steps) were used to produce 20 mg of homogeneous 6xHis-NahB per liter of culture. The secondary and quaternary structures of 6xHis-NahB were assessed by circular dichroism and size-exclusion chromatography experiments, respectively. The enzyme was NAD+-dependent and active at pH 7.0 and 9.4 for the oxidation of the substrate. The Michaelis-Menten parameters determined at pH 7.0 and 25 °C for the substrate and cofactor, presented respective Km values of 6 and 350 µM, and a kcat value of 8.3 s-1. Furthermore, we identified conditions for the crystallization of 6xHis-NahB. X-ray diffraction data were collected from a single 6xHis-NahB crystal which diffracted to 2.21 Å. The crystal belongs to space group I222, with unit-cell parameters a = 63.62, b = 69.50, and c = 117.47 Å. The tertiary structure of 6xHis-NahB was determined using the molecular replacement method. Further structural refinement is currently underway.

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RESUMEN

The salicylaldehyde dehydrogenase (NahF) catalyzes the oxidation of salicylaldehyde to salicylate using NAD(+) as a cofactor, the last reaction of the upper degradation pathway of naphthalene in Pseudomonas putida G7. The naphthalene is an abundant and toxic compound in oil and has been used as a model for bioremediation studies. The steady-state kinetic parameters for oxidation of aliphatic or aromatic aldehydes catalyzed by 6xHis-NahF are presented. The 6xHis-NahF catalyzes the oxidation of aromatic aldehydes with large kcat/Km values close to 10(6) M(-1) s(-1). The active site of NahF is highly hydrophobic, and the enzyme shows higher specificity for less polar substrates than for polar substrates, e.g., acetaldehyde. The enzyme shows α/ß folding with three well-defined domains: the oligomerization domain, which is responsible for the interlacement between the two monomers; the Rossmann-like fold domain, essential for nucleotide binding; and the catalytic domain. A salicylaldehyde molecule was observed in a deep pocket in the crystal structure of NahF where the catalytic C284 and E250 are present. Moreover, the residues G150, R157, W96, F99, F274, F279, and Y446 were thought to be important for catalysis and specificity for aromatic aldehydes. Understanding the molecular features responsible for NahF activity allows for comparisons with other aldehyde dehydrogenases and, together with structural information, provides the information needed for future mutational studies aimed to enhance its stability and specificity and further its use in biotechnological processes.

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Phosphoimidazole-containing compounds are versatile players in biological and chemical processes. We explore catalytic and mechanistic criteria for the efficient formation of cyclic aryl phosphoimidazoles in aqueous solution, viewed as a template reaction for the in situ synthesis of related compounds. To provide a detailed analysis for this reaction a series of o-(2'-imidazolyl)naphthyl (4-nitrophenyl) phosphate isomers were examined to provide a basis for analysis of both mechanism and the influence of structural factors affecting the nucleophilic attack of the imidazolyl group on the phosphorus center of the substrate. Formation of the cyclic aryl phosphoimidazoles was probed by NMR and ESI-MS techniques. Kinetic experiments show that cyclization is faster under alkaline conditions, with an effective molarity up to 2900 M for the imidazolyl group, ruling out competition from external nucleophiles. Heavy atom isotope effect and computational studies show that the reaction occurs through a SN2(P)-type mechanism involving a pentacoordinated phosphorus TS, with apical positions occupied by the incoming imidazolyl nucleophile and the p-nitrophenolate leaving group. The P-O bond to the leaving group is about 50-60% broken in the transition state.

RESUMEN

Hybrid quantum mechanical/effective fragment potential (QM/EFP) calculations, in conjunction with the quantum theory of atoms in molecules (QTAIM) and energy decomposition analysis (EDA), were employed to investigate the reaction mechanism and stereo-electronic effects along the alkaline hydrolysis of the monoethyl phosphate dianion (MEP) and the diethylphosphate monoanion (DEP). Reactions proceed through a synchronous bimolecular ANDN mechanism for MEP and a stepwise (AN + DN) mechanism for DEP, with the formation of a phosphorane intermediate, having an overall reaction free energy and barrier of 11.5 and 43.0 kcal mol(-1), respectively. In addition, ab initio molecular dynamics simulations were performed to investigate the stability of the phosphorane pentacoordinate intermediate observed in the reaction of the phosphate diester. The phosphorane intermediate has a lifetime of ∼1 ps after which it decomposes into the corresponding alcohol and phosphate monoester dianion. Electrostatics governs the interaction between the nucleophile and the phosphate ester. However, some degree of covalence in the interaction starts to appear at distances shorter than 2.45 Šfor MEP and 2.63 Šfor DEP. For the monoester, the electrostatic repulsive terms are the dominant contributions for the formation of the transition state. On the other hand, for the phosphate diester, the formation of the P-OH bond is dominated by associative terms of electrostatic nature.

RESUMEN

Historical perspective has revealed the many aspects of Portugal's interest in the sea, evident in a series of initiatives and entities throughout the twentieth century. From the beginning of the century until the 1974 Revolution, the genesis of organizations devoted to the scientific study of the sea is analyzed, observing their specific missions in the context of the formulation of science policy, and more specifically "ocean policies." The Portuguese valued knowledge of the sea due to their maritime vocation, coastal life and geographic position. Traversing different historical and political contexts and development cycles, the assumptions and political implications that accentuate the strategic dimension of science policy, visible in the geopolitical affirmation of oceanography, are studied.

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RESUMEN

A perspectiva histórica revelou o interesse português pelo mar numa série de iniciativas e entidades ao longo do século XX. Desde o início do século à Revolução de 1974, estuda-se a génese de organismos vocacionados para a investigação científica do mar, observando suas missões específicas no contexto da formulação de políticas científicas, concretamente na definição de “políticas do mar”. A vocação marítima portuguesa, a realidade costeira e a posição geográfica estimulam a valorização do conhecimento sobre o mar. Percorrendo diferentes conjunturas histórico-políticas e ciclos de desenvolvimento, captam-se pressupostos e implicações políticas que acentuam a dimensão estratégica da política científica, visível na afirmação geopolítica das questões do mar agregadas sob a oceanografia.

Historical perspective has revealed the many aspects of Portugal’s interest in the sea, evident in a series of initiatives and entities throughout the twentieth century. From the beginning of the century until the 1974 Revolution, the genesis of organizations devoted to the scientific study of the sea is analyzed, observing their specific missions in the context of the formulation of science policy, and more specifically “ocean policies.” The Portuguese valued knowledge of the sea due to their maritime vocation, coastal life and geographic position. Traversing different historical and political contexts and development cycles, the assumptions and political implications that accentuate the strategic dimension of science policy, visible in the geopolitical affirmation of oceanography, are studied.

Asunto(s)

RESUMEN

This paper aims to evaluate the influence of milking time on the ingestive behavior of crossbred cows in the production system with suckling calves. The experiment lasted 48 days. Sixteen crossbred cows with different lineages were submitted to the treatments and divided into two groups that alternated these treatments. Treatments were: traditional (5:30 a.m.) and alternative schedule (8:00 a.m.). The cows were evaluated by visual observation and recording of time spent grazing, ruminating and other activities. This observation was conducted every 5 minutes, 24 hours a day, to determine the time spent with feeding, rumination and other activities. That time was not affected (p > 0.05) by treatments. There were significant effects (p 0.05) for the number of rumination periods, being lower for the 5:30 a.m. milking. The number of bites per minute differed (p 0.05), being fewer for the alternative milking time. The implementation schedule of the alternative milking did not influence feeding behavior, rumination and other activities. However, it affected the number of rumination periods and bites per minute.

Objetivou-se avaliar a influência do horário de ordenha no comportamento ingestivo de vacas mestiças leiteiras no sistema de produção com o bezerro em pé. O experimento teve duração de 48 dias, no qual foram utilizadas 16 vacas leiteiras mestiças, com diferentes composições genéticas, em ensaio rotativo, com avaliação por período de 12 dias. Os tratamentos avaliados foram horário de ordenha tradicional (5h 30 min.) e alternativo (8h). As vacas foram submetidas à observação visual para avaliação do comportamento ingestivo. Os animais foram avaliados a cada 5 min. durante 24h, para determinação do tempo despendido em alimentação, ruminação e outras atividades. Os tempos gastos com estes itens não foram influenciados (p > 0,05) pelos tratamentos. Houve efeito significativo (p 0,05) para o número de períodos de ruminação, que foi menor na ordenha das 5h 30 min. O número de bocados por minuto diferiu (p 0,05) e foi menor no horário alternativo. A realização da ordenha no horário alternativo não influenciou os tempos de alimentação, ruminação e outras atividades. Todavia, influenciou os números de períodos de ruminação e de bocado por minuto.

RESUMEN

Objetivou-se avaliar a influência do horário de ordenha no comportamento ingestivo de vacas mestiças leiteiras no sistema de produção com o bezerro em pé. O experimento teve duração de 48 dias, no qual foram utilizadas 16 vacas leiteiras mestiças, com diferentes composições genéticas, em ensaio rotativo, com avaliação por período de 12 dias. Os tratamentos avaliados foram horário de ordenha tradicional (5h 30 min.) e alternativo (8h). As vacas foram submetidas à observação visual para avaliação do comportamento ingestivo. Os animais foram avaliados a cada 5 min. durante 24h, para determinação do tempo despendido em alimentação, ruminação e outras atividades. Os tempos gastos com estes itens não foram influenciados (p > 0,05) pelos tratamentos. Houve efeito significativo (p < 0,05) para o número de períodos de ruminação, que foi menor na ordenha das 5h 30 min. O número de bocados por minuto diferiu (p < 0,05) e foi menor no horário alternativo. A realização da ordenha no horário alternativo não influenciou os tempos de alimentação, ruminação e outras atividades. Todavia, influenciou os números de períodos de ruminação e de bocado por minuto.

This paper aims to evaluate the influence of milking time on the ingestive behavior of crossbred cows in the production system with suckling calves. The experiment lasted 48 days. Sixteen crossbred cows with different lineages were submitted to the treatments and divided into two groups that alternated these treatments. Treatments were: traditional (5:30 a.m.) and alternative schedule (8:00 a.m.). The cows were evaluated by visual observation and recording of time spent grazing, ruminating and other activities. This observation was conducted every 5 minutes, 24 hours a day, to determine the time spent with feeding, rumination and other activities. That time was not affected (p > 0.05) by treatments. There were significant effects (p < 0.05) for the number of rumination periods, being lower for the 5:30 a.m. milking. The number of bites per minute differed (p < 0.05), being fewer for the alternative milking time. The implementation schedule of the alternative milking did not influence feeding behavior, rumination and other activities. However, it affected the number of rumination periods and bites per minute.

Asunto(s)

RESUMEN

This paper aims to evaluate the influence of milking time on the ingestive behavior of crossbred cows in the production system with suckling calves. The experiment lasted 48 days. Sixteen crossbred cows with different lineages were submitted to the treatments and divided into two groups that alternated these treatments. Treatments were: traditional (5:30 a.m.) and alternative schedule (8:00 a.m.). The cows were evaluated by visual observation and recording of time spent grazing, ruminating and other activities. This observation was conducted every 5 minutes, 24 hours a day, to determine the time spent with feeding, rumination and other activities. That time was not affected (p > 0.05) by treatments. There were significant effects (p 0.05) for the number of rumination periods, being lower for the 5:30 a.m. milking. The number of bites per minute differed (p 0.05), being fewer for the alternative milking time. The implementation schedule of the alternative milking did not influence feeding behavior, rumination and other activities. However, it affected the number of rumination periods and bites per minute.

Objetivou-se avaliar a influência do horário de ordenha no comportamento ingestivo de vacas mestiças leiteiras no sistema de produção com o bezerro em pé. O experimento teve duração de 48 dias, no qual foram utilizadas 16 vacas leiteiras mestiças, com diferentes composições genéticas, em ensaio rotativo, com avaliação por período de 12 dias. Os tratamentos avaliados foram horário de ordenha tradicional (5h 30 min.) e alternativo (8h). As vacas foram submetidas à observação visual para avaliação do comportamento ingestivo. Os animais foram avaliados a cada 5 min. durante 24h, para determinação do tempo despendido em alimentação, ruminação e outras atividades. Os tempos gastos com estes itens não foram influenciados (p > 0,05) pelos tratamentos. Houve efeito significativo (p 0,05) para o número de períodos de ruminação, que foi menor na ordenha das 5h 30 min. O número de bocados por minuto diferiu (p 0,05) e foi menor no horário alternativo. A realização da ordenha no horário alternativo não influenciou os tempos de alimentação, ruminação e outras atividades. Todavia, influenciou os números de períodos de ruminação e de bocado por minuto. 

RESUMEN

Two imidazole groups act together to catalyze the hydrolysis of the phosphodiester bis(2-(1-methyl-1H-imidazolyl)phenyl) phosphate (BMIPP). A full investigation involving searching computational and electrospray ionization (ESI-MS-/MS) and ultra mass spectrometry (LTQ-FT) experiments made possible a choice between two kinetically equivalent mechanisms. The preferred pathway, involving intramolecular nucleophilic catalysis by imidazole, assisted by intramolecular general acid catalysis by the imidazolium group, offers the first simple model for the mechanism used by the extensive phospholipase D superfamily.

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RESUMEN

8-Quinolyl phosphate (8QP) in the presence of the trivalent lanthanide ions (Ln = La, Sm, Eu, Tb, and Er) forms a [Ln x 8QP]+ complex where the lanthanide ion catalyzes hydrolysis of 8QP. In reactions with Tb3+ or Er3+, there is evidence of limited intervention by a second lanthanide ion. Rate constants are increased by more than 10(7)-fold, and kinetic data and B3LYP/ECP calculations indicate that the effects are largely driven by leaving group and metaphosphate ion stabilization. The lanthanides favor a single-step D(N)A(N) mechanism with a dissociative transition state, with limited nucleophilic assistance, consistent with the low hydroxide ion dependence and the small kinetic effect of Ln3+ radii.

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