Enzymatic activity of bone markers on Lithobates catesbeianus (Shaw, 1802) growth during the ossification process / Atividade enzimática de marcadores ósseos no crescimento de Lithobates catesbeianus (Shaw, 1802) durante o processo de ossificação

Abstract In order to better understand the ossification processes in anurans our study was carried out on tadpoles and adults of Lithobates catesbeianus. In this sense, we characterized the kinetic properties of alkaline phosphatase with p-nitrophenylphosphatase (pNPP) and pyrophosphate (PPi) and evaluated the activities of tartrate-resistant acid phosphatase and acid phosphatase. The enzyme extracts were obtained from tadpoles and adult femurs, which were divided into epiphysis and diaphysis. After homogenization, the samples were submitted to differential centrifugation to obtain cell membranes and, further, to phospholipase C (PIPLC) treatment, to remove membrane-bound proteins anchored by phosphatidylinositol. The average of specific activity for pNPP hydrolysis (at pH 10.5) by alkaline phosphatase released by phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus cereus among different bone regions at different animal ages was 1,142.57 U.mg-1, while for PPi hydrolysis (at pH 8.0), it was 1,433.82 U.mg-1. Among the compounds tested for enzymatic activity, the one that influenced the most was EDTA, with approximately 67% of inhibition for pNPPase activity and 77% for PPase activity. In the case of kinetic parameters, the enzyme showed a "Michaelian" behavior for pNPP and PPi hydrolysis. The Km value was around 0.6mM for pNPPase activity and ranged from 0.01 to 0.11mM for PPase activity, indicating that the enzyme has a higher affinity for this substrate. The study of pNPP and PPi hydrolysis by the enzyme revealed that the optimum pH of actuation for pNPP was 10.5, while for PPi, which is considered the true substrate of alkaline phosphatase, was 8.0, close to the physiological value. The results show that regardless of the ossification type that occurs, the same enzyme or isoenzymes act on the different bone regions and different life stages of anurans. The similarity of the results of studies with other vertebrates shows that anurans can be considered excellent animal models for the study of biological calcification.

Resumo Para melhor compreender o processo de ossificação em anuros, nosso estudo foi conduzido em girinos e adultos de Lithobates catesbeianus. Nesse sentido, as propriedades cinéticas da fosfatase alcalina com p-nitrofenilfosfato (pNPP) e pirofosfato (PPi) foram caracterizadas, e as atividades enzimáticas das fosfatases ácida e ácida tartarato resistente foram avaliadas. Os extratos enzimáticos foram obtidos de fêmur de girinos e adultos, divididos em epífise e diáfise. Após a homogeneização as amostras foram submetidas à centrifugação diferencial para obter membrana celular e, em seguida, ao tratamento com fosfolipase C (PIPLC), para remover as proteínas de membrana ancoradas por fosfatidilinositol. A média da atividade específica da fosfatase alcalina, liberada pela PIPLC de Bacillus cereus, para a hidrólise de pNPP (pH 10,5) nas diferentes regiões do fêmur e idades dos animais foi de 1.142,57 U.mg-1, enquanto para a hidrólise do PPi (pH 8,0) foi de 1.433,82 U.mg-1. Entre os compostos testados para a atividade enzimática, o de maior influência foi o EDTA, inibindo aproximadamente 67% e 77% das atividades de pNPPase e PPase, respectivamente. Quanto aos parâmetros cinéticos, a enzima apresentou comportamento Michaeliano para a hidrólise dos dois substratos. O valor de Km foi de 0,6 mM para a atividade de pNPPase e variou de 0,01 a 0,11 para a atividade de PPase, indicando uma maior afinidade por esse substrato. O estudo da hidrólise de pNPP e PPi revelou que o pH ótimo aparente de atuação foi de 10,5 para o pNPP e 8,0 para o PPi, próximo ao fisiológico, sendo que esse é considerado o substrato natural da fosfatase alcalina. Os resultados demonstram que, apesar do tipo de ossificação que ocorre, a mesma enzima ou isoenzimas, atuam nos diferentes locais do osso e estágios de vida dos anuros. A similaridade dos estudos com os realizados com outros vertebrados apontam que os anuros podem ser considerados excelentes modelos animais para o estudo da calcificação biológica.

Enzymatic activity of bone markers on Lithobates catesbeianus (Shaw, 1802) growth during the ossification process

Abstract In order to better understand the ossification processes in anurans our study was carried out on tadpoles and adults of Lithobates catesbeianus. In this sense, we characterized the kinetic properties of alkaline phosphatase with p-nitrophenylphosphatase (pNPP) and pyrophosphate (PPi) and evaluated the activities of tartrate-resistant acid phosphatase and acid phosphatase. The enzyme extracts were obtained from tadpoles and adult femurs, which were divided into epiphysis and diaphysis. After homogenization, the samples were submitted to differential centrifugation to obtain cell membranes and, further, to phospholipase C (PIPLC) treatment, to remove membrane-bound proteins anchored by phosphatidylinositol. The average of specific activity for pNPP hydrolysis (at pH 10.5) by alkaline phosphatase released by phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus cereus among different bone regions at different animal ages was 1,142.57 U.mg-1, while for PPi hydrolysis (at pH 8.0), it was 1,433.82 U.mg-1. Among the compounds tested for enzymatic activity, the one that influenced the most was EDTA, with approximately 67% of inhibition for pNPPase activity and 77% for PPase activity. In the case of kinetic parameters, the enzyme showed a Michaelian behavior for pNPP and PPi hydrolysis. The Km value was around 0.6mM for pNPPase activity and ranged from 0.01 to 0.11mM for PPase activity, indicating that the enzyme has a higher affinity for this substrate. The study of pNPP and PPi hydrolysis by the enzyme revealed that the optimum pH of actuation for pNPP was 10.5, while for PPi, which is considered the true substrate of alkaline phosphatase, was 8.0, close to the physiological value. The results show that regardless of the ossification type that occurs, the same enzyme or isoenzymes act on the different bone regions and different life stages of anurans. The similarity of the results of studies with other vertebrates shows that anurans can be considered excellent animal models for the study of biological calcification.

Resumo Para melhor compreender o processo de ossificação em anuros, nosso estudo foi conduzido em girinos e adultos de Lithobates catesbeianus. Nesse sentido, as propriedades cinéticas da fosfatase alcalina com p-nitrofenilfosfato (pNPP) e pirofosfato (PPi) foram caracterizadas, e as atividades enzimáticas das fosfatases ácida e ácida tartarato resistente foram avaliadas. Os extratos enzimáticos foram obtidos de fêmur de girinos e adultos, divididos em epífise e diáfise. Após a homogeneização as amostras foram submetidas à centrifugação diferencial para obter membrana celular e, em seguida, ao tratamento com fosfolipase C (PIPLC), para remover as proteínas de membrana ancoradas por fosfatidilinositol. A média da atividade específica da fosfatase alcalina, liberada pela PIPLC de Bacillus cereus, para a hidrólise de pNPP (pH 10,5) nas diferentes regiões do fêmur e idades dos animais foi de 1.142,57 U.mg-1, enquanto para a hidrólise do PPi (pH 8,0) foi de 1.433,82 U.mg-1. Entre os compostos testados para a atividade enzimática, o de maior influência foi o EDTA, inibindo aproximadamente 67% e 77% das atividades de pNPPase e PPase, respectivamente. Quanto aos parâmetros cinéticos, a enzima apresentou comportamento Michaeliano para a hidrólise dos dois substratos. O valor de Km foi de 0,6 mM para a atividade de pNPPase e variou de 0,01 a 0,11 para a atividade de PPase, indicando uma maior afinidade por esse substrato. O estudo da hidrólise de pNPP e PPi revelou que o pH ótimo aparente de atuação foi de 10,5 para o pNPP e 8,0 para o PPi, próximo ao fisiológico, sendo que esse é considerado o substrato natural da fosfatase alcalina. Os resultados demonstram que, apesar do tipo de ossificação que ocorre, a mesma enzima ou isoenzimas, atuam nos diferentes locais do osso e estágios de vida dos anuros. A similaridade dos estudos com os realizados com outros vertebrados apontam que os anuros podem ser considerados excelentes modelos animais para o estudo da calcificação biológica.

Enzymatic activity of bone markers on Lithobates catesbeianus (Shaw, 1802) growth during the ossification process.

In order to better understand the ossification processes in anurans our study was carried out on tadpoles and adults of Lithobates catesbeianus. In this sense, we characterized the kinetic properties of alkaline phosphatase with p-nitrophenylphosphatase (pNPP) and pyrophosphate (PPi) and evaluated the activities of tartrate-resistant acid phosphatase and acid phosphatase. The enzyme extracts were obtained from tadpoles and adult femurs, which were divided into epiphysis and diaphysis. After homogenization, the samples were submitted to differential centrifugation to obtain cell membranes and, further, to phospholipase C (PIPLC) treatment, to remove membrane-bound proteins anchored by phosphatidylinositol. The average of specific activity for pNPP hydrolysis (at pH 10.5) by alkaline phosphatase released by phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus cereus among different bone regions at different animal ages was 1,142.57 U.mg-1, while for PPi hydrolysis (at pH 8.0), it was 1,433.82 U.mg-1. Among the compounds tested for enzymatic activity, the one that influenced the most was EDTA, with approximately 67% of inhibition for pNPPase activity and 77% for PPase activity. In the case of kinetic parameters, the enzyme showed a "Michaelian" behavior for pNPP and PPi hydrolysis. The Km value was around 0.6mM for pNPPase activity and ranged from 0.01 to 0.11mM for PPase activity, indicating that the enzyme has a higher affinity for this substrate. The study of pNPP and PPi hydrolysis by the enzyme revealed that the optimum pH of actuation for pNPP was 10.5, while for PPi, which is considered the true substrate of alkaline phosphatase, was 8.0, close to the physiological value. The results show that regardless of the ossification type that occurs, the same enzyme or isoenzymes act on the different bone regions and different life stages of anurans. The similarity of the results of studies with other vertebrates shows that anurans can be considered excellent animal models for the study of biological calcification.

The association between obesity and academic performance in youth: a systematic review.

Previous studies have found that obesity could influence academic performance. The aim of this study was to systematically review the scientific evidence on the association between obesity and academic performance in school children. A systematic review of English articles was undertaken by using databases PubMed/Medline, ERIC, LILACS, SciELO and Web of Science. Cross-sectional and longitudinal studies examining the association between obesity and academic performance in children and adolescents, published between January 1990 and December 2016, were included. Risk of bias was assessed by using Strengthening the Reporting of Observational Studies in Epidemiology. Thirty-four studies (23 cross-sectional and 11 longitudinal) matched all inclusion criteria and were included. Seven studies were classified as low risk of bias, 23 as medium risk and four as high risk. After controlling for covariates such as socio-economic status, parental education and physical activity, the association between obesity and academic performance becomes uncertain for most of the studies (55.9%). Therefore, at present, there is insufficient evidence to support a direct link between obesity and poor academic performance in school age children. In order to clarify this issue, we need more longitudinal studies with adequate sample sizes and that control for potential confounders.

Physical fitness and academic performance in youth: A systematic review.

Physical fitness (PF) is a construct of health- and skill-related attributes which have been associated with academic performance (AP) in youth. This study aimed to review the scientific evidence on the association among components of PF and AP in children and adolescents. A systematic review of articles using databases PubMed/Medline, ERIC, LILACS, SciELO, and Web of Science was undertaken. Cross-sectional and longitudinal studies examining the association between at least one component of PF and AP in children and adolescents, published between 1990 and June 2016, were included. Independent extraction of articles was carried out by the two authors using predefined data fields. From a total of 45 studies included, 25 report a positive association between components of PF with AP and 20 describe a single association between cardiorespiratory fitness (CRF) and AP. According to the Strengthening the Reporting of Observational Studies in Epidemiology guidelines: 12 were classified as low, 32 as medium risk, and 1 as high risk of bias. Thirty-one studies reported a positive association between AP and CRF, six studies with muscular strength, three studies with flexibility, and seven studies reported a positive association between clustered of PF components and AP. The magnitude of the associations is weak to moderate (ß = 0.10-0.42 and odds = 1.01-4.14). There is strong evidence for a positive association between CRF and cluster of PF with AP in cross-sectional studies; and evidence from longitudinal studies for a positive association between cluster of PF and AP; the relationship between muscular strength and flexibility with AP remains uncertain.

Utilização do filme de quitosana na reparação de tendão em coelhos / Use of the film of chitosan in the repair of tendons in rabbits

Com este trabalho objetivou-se avaliar o processo de cicatrização do tendão em coelhos, utilizando-se no grupo tratamento o filme de quitosana, por meio de uma análise clínico-cirúrgica e histológica. Foram utilizados 12 coelhos adultos, separados em grupo controle (GC) e grupo tratamento (GT), nos quais se realizou uma secção parcial do tendão gastrocnêmio de ambos os membros pélvicos. A avaliação clínica baseou-se na presença de reação inflamatória, infecção, dor e deiscência da sutura. Para a avaliação histológica, foi realizado um estudo comparativo do processo cicatricial por meio do tipo de células, da quantidade de tecido conjuntivo e da organização das fibras colágenas entre os grupos e os momentos. Nas feridas cirúrgicas, não foram observadas secreção, dor ou deiscência. Na histologia comparativa entre os grupos, o GC apresentou melhor processo cicatricial em relação ao GT, aos 60 dias. Aos 90 dias, no GT a cicatrização já esboça recuperação do tendão, com reorganização da celularidade e das fibras colágenas no tecido conjuntivo denso modelado. Concluiu-se que a quitosana estimula rápido crescimento celular, mas de forma desorganizada, e que a cicatrização completa só ocorre após 90 dias da sua implantação no tecido...

This work aimed to evaluate the process of tendon healing in rabbits, using the treatment group in chitosan film through a clinical, surgical and histological analysis. In the experiment, 12 adult rabbits were used, divided into control group (CG) and treatment group (TG), which were held in the partial section of the gastrocnemius tendon of both limbs. Clinical assessment was based on the presence of inflammation, infection, pain and suture dehiscence. For histological evaluation a comparative study of the healing process through the cell type, amount of connective tissue and collagen fiber organization was performed between groups and times. In surgical wounds no secretion, pain or dehiscence were observed. In the comparative histology between groups, the CG showed better healing compared to TG, at 60 days. On day 90 the TG already outlines tendon recovery with reorganization of cellularity and collagen fibers in the dense connective tissue modeled. It was concluded that chitosan stimulates faster cell growth, but it is haphazardly, and that complete healing occurs after 90 days of their implantation in tissue...

Inflammation and structural changes of splenic lymphoid tissue in visceral leishmaniasis: a study on naturally infected dogs.

The aim of this study was to identify splenic immuno-inflammatory patterns associated with natural infection by Leishmania chagasi. Spleen samples were obtained from 72 stray dogs from an endemic area of visceral leishmaniasis. The animals were grouped into four categories as follows: (i) potentially resistant to visceral leishmaniasis, with a positive leishmanin skin test result, and negative splenic culture for Leishmania parasites (ii) potentially susceptible to visceral leishmaniasis, with a negative leishmanin skin test and positive splenic culture for Leishmania (iii) infected with undefined susceptibility status, with a positive leishmanin skin test and positive splenic culture for Leishmania, and (iv) noninfected, with a negative leishmanin skin test, negative splenic culture for Leishmania, and negative serology for anti-Leishmania antibodies. Histopathological analyses showed that there was a higher frequency of perisplenitis (18/25, P < 0.0001), granuloma (7/25, P = 0.0102), structural disorganization (14/25, P < 0.0001), and atrophy of the lymphoid follicles (20/25, P = 0.0036) and of the marginal zone (15/25, P = 0.0025) in the potentially susceptible group than in the other groups. The data presented here show changes in the white pulp of the spleen that are associated with naturally acquired visceral leishmaniasis.

Adsorption of inulinases in ion-exchange columns.

The use of adsorption columns packed with ion-exchange resins for recovering, concentrating and purifying proteins is now widespread. The present work consists of a study on the dynamic behavior of adsorption columns that uses two kinds of adsorbents: a cationic and an anionic resin. A frontal analysis of the columns was performed with experimental data obtained from Fructozyme, a mixture of inulinase enzymes. The parameters of a Langmuir type of isotherm and adsorption kinetics were obtained from experimental tests in a batch system. A numerical technique based on orthogonal collocation and a fourth-order Runge-Kutta method was coupled with a nonlinear optimization method to predict the coefficients of the rate equations, which are fundamental for scale-up purposes.

Brazilian bioethanol program.

Brazil is the largest producer of bioethanol, and sugarcane is the main raw material. Bioethanol is produced by both batch and continuous processes, and in some cases, flocculating yeast is used. This article analyzes the Brazilian Ethanol Program. For the 1996-1997 harvest, Brazil produced 14.16 billion L of ethanol and 13.8 million metric t of sugar, from 286 million metric t of sugarcane. These products were produced by 328 industries in activity, with 101 autonomous ethanol plants producing only ethanol, and 227 sugar mills producing sugar and ethanol. The sugar-ethanol market reaches about 7.5 billion US$/yr, accounting for direct and indirect revenues.

Recovery of cellulase by HPMC-salt precipitation: analysis by statistical experimental design.

Production of industrial enzymes including cellulases requires minimum cost with the downstream processing. The objective of this work was to analyze the precipitation of cellulases by ammonium sulfate in the presence of hydroxypropyl(methylcellulose) as a co-precipitant through the use of statistical experimental design. The model generated with the experimental results showed that high protein recovery can be achieved at high levels of temperature, aging times, and rate of salt-solution addition, and at a low mixing level. The results also allowed the observation that activity recovery was improved at high levels of temperature, rate of salt addition and mixing level, and a low level of aging time.

Expanded-bed adsorption utilizing ion-exchange resin to purify extracellular beta-galactosidase.

The application of expanded-bed ion-exchange resins allows the elimination of intermediary particulate separation steps like filtration or centrifugation prior to adsorption steps in enzyme-purification processes from crude fermentation broths. This work is concerned with the experimental evaluation data of a process related to the adsorption of an extracellular p-galactosidase from the fungi Scopulariopsis. The protein recovery in the ion-exchange resin Accell Plus QMA was accomplished using a continuous-monitoring method. The direct adsorption step was followed by a elution step with concentrated NaCl solutions aiming to improve the enzyme-specific activity. Experimental data for fixed and expanded bed were compared.