Crystal structure of Leishmania tarentolae hypoxanthine-guanine phosphoribosyltransferase (HGPRT) with bound GMP.

O presente trabalho teve como objetivos a clonagem, expressão e purificação da proteína HGPRT de Leishimania tarentolae, para a caracterização e cristalização dessa enzima, a fim do seu estudo estrutural e funcional. O gene da HGPRT foi amplificado a partir de uma biblioteca genômica de Leishmania tarentolae da cepa UC Lambda ZAP Express BamHI-Sal3A I. Esse gene foi clonado no vetor de expressão pET29a(+) e usado na transformação da bactéria Escherichia coli BL21 (DE3). Esse sistema de expressão apresentou uma superexpressão da proteína recombinante, que foi purificada em cromatografia de forma iônica, com o uso da coluna de troca anônica POROS 20HQ. A proteína purificada foi utilizada para sua caracterização, como a determinação das constantes cinéticas (Km, Vmax e Kcat) para os diferentes substratos. A proteína foi encontrada como dímero em solução e o pI de aproximadamente 8,2. Além disso, essa proteína foi utilizada nos ensaios de cristalização pelo método de difusão de vapor em gotas suspensas. Os cristais obtidos foram utilizados nos testes da difração de raios-X sendo coletado um conjunto de dados no Laboratório Nacional de Luz Síncroton. Os dados de difração de raios-X foram processados com o uso do pacote de programa HKL. A resolução da estrutura cristalográfica foi obtida pelo método de substituição molecular através do programa AmoRe. Para o refinamento da estrutura foram utilizados os programas de refinamento automático CNS e REFMAC, e a manipulação na estação gráfica foi realizada através do programa O. A estrutura da HGPRT foi resolvida a 2,1 &#197 de resolução com os fatores de concordância Rwork e Rfree finais de 17,34 e 21,53%, respectivamente.The aims of this work were the cloning, expression and purification of the protein HGPRT from Leishimania tarentolae, for the characterization and crystallization of the enzyme for it structural and functional study. The full-length hgprt gene was isolated from a leishmania tarentolae UC strain Lambda ZAP Express BamHI-Sal3A I genomic library. This gene was cloned in the expression vector pET29a+, and used in the transformation of the bacteria Escherichia coli BL21(DE3). This expression system presented a super-expression of the recombinant protein, which was purified by ionic change chromatography, utilizing the anionic change column POROS 20HQ. The purified protein was utilized for its characterization, including its kinetics constants (Km, Vmax e Kcat) for different substrates. The protein was found to be a dimmer in solution with pI close to 8.2. Besides, this protein was used in the crystallization assays by the steam diffusion in suspended drop technique. The obtained crystals were utilized in the x-ray diffraction studies. The data collection was performed in the Laboratório Nacional de Luz Síncroton. The X-ray diffraction data were processed utilizing the package program HKL. The crystallographic structure resolution was obtained by the molecular replacement method from the AmoRe program. For the structure refinement, the automatic refinement programs CNS and REFMAC were used, and the graphic manipulation was made through the O program. The structure of the HGPRT was resolved with 2,1 &#197 of resolution and final agreement factors Rwork and Rfree of 17,34% and 21,53%, respectively

Crystal structure of Leishmania tarentolae hypoxanthine-guanine phosphoribosyltransferase (HGPRT) with bound GMP.

O presente trabalho teve como objetivos a clonagem, expressão e purificação da proteína HGPRT de Leishimania tarentolae, para a caracterização e cristalização dessa enzima, a fim do seu estudo estrutural e funcional. O gene da HGPRT foi amplificado a partir de uma biblioteca genômica de Leishmania tarentolae da cepa UC Lambda ZAP Express BamHI-Sal3A I. Esse gene foi clonado no vetor de expressão pET29a(+) e usado na transformação da bactéria Escherichia coli BL21 (DE3). Esse sistema de expressão apresentou uma superexpressão da proteína recombinante, que foi purificada em cromatografia de forma iônica, com o uso da coluna de troca anônica POROS 20HQ. A proteína purificada foi utilizada para sua caracterização, como a determinação das constantes cinéticas (Km, Vmax e Kcat) para os diferentes substratos. A proteína foi encontrada como dímero em solução e o pI de aproximadamente 8,2. Além disso, essa proteína foi utilizada nos ensaios de cristalização pelo método de difusão de vapor em gotas suspensas. Os cristais obtidos foram utilizados nos testes da difração de raios-X sendo coletado um conjunto de dados no Laboratório Nacional de Luz Síncroton. Os dados de difração de raios-X foram processados com o uso do pacote de programa HKL. A resolução da estrutura cristalográfica foi obtida pelo método de substituição molecular através do programa AmoRe. Para o refinamento da estrutura foram utilizados os programas de refinamento automático CNS e REFMAC, e a manipulação na estação gráfica foi realizada através do programa O. A estrutura da HGPRT foi resolvida a 2,1 &#197 de resolução com os fatores de concordância Rwork e Rfree finais de 17,34 e 21,53%, respectivamente.The aims of this work were the cloning, expression and purification of the protein HGPRT from Leishimania tarentolae, for the characterization and crystallization of the enzyme for it structural and functional study. The full-length hgprt gene was isolated from a leishmania tarentolae UC strain Lambda ZAP Express BamHI-Sal3A I genomic library. This gene was cloned in the expression vector pET29a+, and used in the transformation of the bacteria Escherichia coli BL21(DE3). This expression system presented a super-expression of the recombinant protein, which was purified by ionic change chromatography, utilizing the anionic change column POROS 20HQ. The purified protein was utilized for its characterization, including its kinetics constants (Km, Vmax e Kcat) for different substrates. The protein was found to be a dimmer in solution with pI close to 8.2. Besides, this protein was used in the crystallization assays by the steam diffusion in suspended drop technique. The obtained crystals were utilized in the x-ray diffraction studies. The data collection was performed in the Laboratório Nacional de Luz Síncroton. The X-ray diffraction data were processed utilizing the package program HKL. The crystallographic structure resolution was obtained by the molecular replacement method from the AmoRe program. For the structure refinement, the automatic refinement programs CNS and REFMAC were used, and the graphic manipulation was made through the O program. The structure of the HGPRT was resolved with 2,1 &#197 of resolution and final agreement factors Rwork and Rfree of 17,34% and 21,53%, respectively

In vitro maturation of bovine oocytes in medium supplemented with quercetin, and its effect on embryonic development

A quercetina é um flavonoide, amplamente encontrada em frutas, vegetais, grãos, flores, com elevada concentração no vinho tinto, e tem sido caracterizada funcionalmente pela atividade antioxidante. Para avaliação da maturação nuclear e do desenvolvimento embrionário bovino, os oócitos foram maturados por 22h na presença de quercetina (0,4, 2, 10 e 50µM), cisteamina (100µM) e na ausência dos antioxidantes. Os oócitos maturados foram corados com Hoechst para avaliação da maturação in vitro. Para avaliação do desenvolvimento embrionário, os oócitos foram fertilizados e cultivados in vitro, as taxas de desenvolvimento embrionário foram determinadas no sétimo dia de cultivo e o percentual de eclosão e o número de células dos embriões no oitavo dia. Os níveis de glutationa (GSH) dos oócitos foram mensurados por emissão de fluorescência com CMF₂HC. A porcentagem de maturação nuclear (±89%) não diferiu entre os grupos. O desenvolvimento embrionário variou entre os tratamentos, o percentual de blastocisto foi superior (P<0,05) nos grupos tratados com 0,4, 2, 10 e 50∝M de quercetina (56,9, 59,5, 53,6 e 49,6%, respectivamente) e com 100∝M de cisteamina (50,4%) em relação ao grupo controle (42,3%). Na comparação entre os dois antioxidantes, a quercetina (0,4 e 2µM) foi superior na produção de embriões (56,9 e 59,5%, respectivamente) em comparação com cisteamina (50,4%). As taxas de embriões eclodidos foram similares (P>0,05) entre os grupos (±63,0%). O número médio de células dos embriões também foi similar entre os grupos (±233). Os níveis intracelulares de GSH foram superiores nos oócitos maturados com cisteamina, mas similares entre os oócitos tratados com quercetina e o controle. A suplementação da maturação in vitro com antioxidantes melhora as taxas de blastocistos. A quercetina foi superior à cisteamina, que, por sua vez, foi superior ao controle. Mas os níveis de GSH foram superiores somente nos oócitos tratados com cisteamina.ABSTRACT: Quercetin is a flavonoid widely found in fruit, vegetables, grains and flowers, with a high concentration in red wine, and has been functionally characterized by its antioxidant activity. For assessment of nuclear maturation and bovine embryo, oocytes were matured for 22h in the presence of quercetin (0.4, 2, 10 and 50µM), cysteamine (100µM) and in the absence of antioxidants. The matured oocytes were stained with Hoechst to evaluate the in vitro maturation. To assess embryonic development, oocytes were fertilized and cultured in vitro and rates of embryo development were obtained in the seventh day of culture and the percentage of hatching and the number of cells on eighth day embryos. The levels of glutathione (GSH) of the oocytes were measured by fluorescence emission with CMF₂HC. The percentage of nuclear maturation (±89%) did not differ between groups. Embryonic development varied between treatments, the percentage of blastocyst was higher (P<0.05) in the groups treated with 0.4, 2, 10 and 50∝M of quercetin (56.9, 59.5, 53.6 and 49.6%, respectively) and 100 ∝M cysteamine (50.4%) compared to the control group (42.3%). Comparing the two antioxidants, quercetin (0.4 to 2µM) was superior in embryo production (56.9 and 59.5% respectively) compared with cysteamine (50.4%). The rates of hatched embryos were similar (P>0.05) between groups (±63.0%). The average number of embryo cells was also similar in both groups (±233). The intracellular GSH levels were higher in oocytes matured with cysteamine, but similar between the oocytes treated with quercetin and control. The supplementation of matured in vitro with antioxidants improves blastocyst rates. Quercetin was greater than cysteamine, which in turn was superior to the control. However, GSH levels were higher in oocytes treated only with cysteamine

Phenotypic and Genotypic Features of a Salmonella Heidelberg Strain Isolated in Broilers in Brazil and Their Possible Association to Antibiotics and Short-Chain Organic Acids Resistance and Susceptibility

Salmonella enterica serovar Heidelberg is a human pathogen also found in broilers. A strain (UFPR1) has been associated with field reports of resistance to short-chain organic acids (SCOA) in broilers in the South of Brazil, but was susceptible to a Bacillus subtilis-based probiotic added in feed in a related study. This work aimed to (i) report clinical symptoms caused by SH UFPR1 in broilers, (ii) study its susceptibility to some antibiotics in vitro, and (iii) SCOA in vivo; and (iv) relate these phenotypic observations with its genome characteristics. Two in vivo trials used 1-day-old chicks housed for 21 days in 8 sterilized isolated negative pressure rooms with 4 battery cages of 12 birds each. Birds were challenged or not with 107 CFU/bird of SH UFPR1 orally and exposed or not to SCOA in a 2 × 2 factorial design. Zootechnical parameters were unaffected (P &gt; 0.05), no clinical signs were observed, and few cecal and hepatic histologic and immune-related alterations were seen, in birds challenged with SH. Formic and propionic acids added together in drinking water, fumaric and benzoic acid in feed (Trial 1), and coated calcium butyrate in feed (Trial 2) did not reduce the SH isolation frequencies seen in cecum and liver in broilers after SH challenge (P &gt; 0.05). SH UFPR1 was susceptible to amikacin, amoxicillin + clavulanate, ceftiofur, cephalexin, doxycycline and oxytetracycline; and mildly susceptible to ampicillin + sulbactam, cephalothin, ciprofloxacin, enrofloxacin, and gentamycin in an in vitro minimum inhibitory concentration model using Mueller–Hinton agar. The whole genome of SH UFPR1 was sequenced and consisted of a circular chromosome, spanning 4,760,321 bp with 52.18% of GC-content encoding 84 tRNA, 22 rRNA, and 4,427 protein-coding genes. The comparison between SH UFPR1 genome and a multidrug-resistant SL476 strain revealed 11 missing genomic fragments and 5 insertions related to bgt, bgr, and rpoS genes. The deleted genes codify proteins associated with cell cycle regulation, virulence, drug resistance, cellular adhesion, and salt efflux which collectively reveal key aspects of the evolution and adaptation of SH strains such as organic acids resistance and antibiotic sensitivity and provide information relevant to the control of SH in poultry

Effect of Feeding Bacillus subtilis Spores to Broilers Challenged with Salmonella enterica serovar Heidelberg Brazilian Strain UFPR1 on Performance, Immune Response, and Gut Health

Salmonellosis is a poultry industry and public health concern worldwide. Recently, Salmonella enterica serovar Heidelberg (SH) has been reported in broilers in Brazil. The effect of feeding a blend of three strains of Bacillus subtilis (PRO) was studied in broilers orally challenged (107 CFU/chick) or not with a SH isolated in south of Brazil (UFPR1 strain). Twelve male Cobb 500 broilers per pen were randomly assigned to six treatments in a 3 × 2 factorial experiment where PRO was added at 0, 250, or 500 g/ton of broiler feed and fed to either SH-challenged (SH Control, SH + PRO 250, and SH + PRO 500) or non-challenged birds (Control, PRO 250, and PRO 500). Broiler performance, histologic alterations in intestinal morphology, Salmonella quantification and immune cells counts in liver (macrophages, T CD4+ and T CD8+) were analyzed. Changes in the intestinal microbiota of broilers were also studied by metagenomics for Control, SH Control, SH + PRO 250, and SH + PRO 500 only. Feeding PRO at 250 or 500 g/ton reduced SH counts and incidence in liver and cecum at 21 days of age. It was observed that PRO groups increased the macrophage mobilization to the liver in SH-challenged birds (P &lt; 0.05) but reduced these cells in the liver of non-challenged birds, showing an interesting immune cell dynamics effect. PRO at 250 g/ton did not affect gut histology, but improved animal performance (P &lt; 0.05) while PRO at 500/ton did not affect animal performance but increased histologic alteration related to activation of the defense response in the ileum in SH challenged birds compared to control birds (P &lt; 0.05). SH + PRO 500 group presented a more diverse cecal microbiota (Shannon–Wiener index; P &lt; 0.05) compared to Control and SH Control groups; while SH + PRO 250 had greater ileal richness (JackkNife index) compared to Control (P &lt; 0.05). PRO was effective in reducing Salmonella colonization in liver and cecum when fed at 250 or 500 g/ton to broilers inoculated with SH strain UFPR1. PRO promotes positive alterations in performance (at 250 g/ton), immune modulatory effect in the gastrointestinal tract, SH reduction, and intestinal microbiota modulation

Heat treatment at 65°C induces the formation of recombinant <i>Tc</i>CYS4 dimers.

<p><b>A</b>, native gel of <i>Tc</i>CYS4 incubated at temperatures ranging between 45 and 80°C for 10 minutes. <b>B</b>, percentages of dimers and monomers of <i>Tc</i>CYS4 obtained by densitometry of the gels of A in triplicate using the software Image2D Platinum 7.0. <b>C</b>, size exclusion chromatography of <i>Tc</i>CYS4 after heat treatment. The retention volumes for the standards are indicated as Al, BSA with 66.5 kDa; Ov, ovalbumin with 45 kDa; Ch, Chymotrypsinogen A with 25 kDa; AR, ribonuclease A with 13.8 kDa. The size expected for monomers, dimers ande trimers for His-tagged protein is approximately 24, 48 and 72 kDa, respectively. <b>D</b>, Spectral profiles by circular dichroism at the wavelengths from 190 to 250 nm of the <i>Tc</i>CYS4 treated at 20°C (solid line), 65°C (dashed line), and 90°C (dotted line) showing that the protein undergoes minor alterations at 65°C, and, when analyzed at 90°C, it undergoes loss of structure when compared with the protein at 20°C.</p

The<i>Tc</i>CYS4 dimers are stable after storage at 8°C.

<p>A, residual activity of papain in reactions containing aliquots of the protein treated at 0, 0.4, 0.5, 1, 2, 2, 4, 5, and 10 minutes at 65°C. The bars correspond to the standard deviation of the mean (n = 4). <b>B</b>, Percentage of residual activity of papain in reactions with <i>Tc</i>CYS4 treated at 65°C and stored at 8°C (Control) over 20 days. The bars correspond to the standard deviations of the mean (n = 3). <b>C</b>, percentage of residual activity of papain in reactions with <i>Tc</i>CYS4 treated at 65°C, 80°C, and 65°C for 10 minutes and then treated at 80°C for 10 minutes, showing that the <i>Tc</i>CYS4 dimers were reconverted to monomers, when treated at 80°C. The activity of <i>Tc</i>CYS4-His-tag (histidine tailed) was similar to that of the protein <i>Tc</i>CYS4 (without histidine tail), demonstrating that the histidine tag does not interfere with the protein’s activity. The bars correspond to the standard deviation of the mean (n = 4). Means followed by the same capital letters, for the same inhibitor,do not differ among themselves by the Scott-Knott test at 5% probability. Means followed by the same lowercase letter, for the same treatment, do not differ among themselves by t test at 5% probability.</p

Unfolding and refolding of TcCYS4 expressed in percentage of structue.

<p>Unfolding of <i>Tc</i>CYS4 by heating from 26 to 92°C previously incubated at 8°C (empty circle) and 65°C (full circle), and refolding of this protein from 92 to 26°C previously incubated at 8°C (empty square) and 65°C (full square), showing that the protein pretreated at 65°C re-coils, similarly to protein pretreated at 8°C. Three spectra were performed to each treatment.”</p

Variation of acidity in infected cacao leaves.

<p><b>A</b>, image of the cocoa leaves with the same level of expansion and at each stage of development of the disease.<b>B</b>, Level of titratable acidity (TA) in healthy leaves and three developmental stages of witches’ broom disease. The bars correspond to the standard deviation of the mean (n = 5). Means followed by the same letters do not differ by the Scott-Knott test at 5% probability.</p