ABSTRACT
The l-asparaginase (l-ASNase) enzyme catalyzes the conversion of the non-essential amino acid l-asparagine into l-aspartic acid and ammonia. Importantly, the l-ASNases are used as a key part of the treatment of acute lymphoblastic leukemia (ALL); however, despite their benefits, they trigger severe side effects because they have their origin in bacterial species (Escherichia coli and Erwinia chrysanthemi). Therefore, one way to solve these side effects is the use of l-ASNases with characteristics similar to those of bacterial types, but from different sources. In this sense, Cavia porcellus l-ASNase (CpA) of mammalian origin is a promising enzyme because it possesses similarities with bacterial species. In this work, the hydrolysis reaction for C. porcellus l-asparaginase was studied from an atomistic point of view. The QM/MM methodology was employed to describe the reaction, from which it was found that the conversion mechanism of l-asparagine into l-aspartic acid occurs in four steps. It was identified that the nucleophilic attack and release of the ammonia group is the rate-limiting step of the reaction. In this step, the nucleophile (Thr19) attacks the substrate (ASN) leading to the formation of a covalent intermediate and release of the leaving group (ammonia). The calculated energy barrier is 18.9 kcal mol-1, at the M06-2X+D3(0)/6-311+G(2d,2p)//CHARMM36 level of theory, which is in agreement with the kinetic data available in the literature, 15.9 kcal mol-1 (derived from the kcat value of 38.6 s-1). These catalytic aspects will hopefully pave the way toward enhanced forms of CpA. Finally, our work emphasizes that computational calculations may enhance the rational design of mutations to improve the catalytic properties of the CpA enzyme.
Subject(s)
Asparaginase , Asparagine , Animals , Guinea Pigs/metabolism , Ammonia/chemistry , Asparaginase/genetics , Asparaginase/metabolism , Asparaginase/therapeutic use , Asparagine/chemistry , Asparagine/genetics , Asparagine/metabolism , Aspartic Acid , Mammals/metabolism , MutationABSTRACT
This study evaluated the effect of the three inulin levels (0.1%, 0.2%, 0.4%) supplemented as a substitute for an antibiotic growth promoter (AGP, zinc bacitracin) and control in guinea pigs raised for human consumption. Fifty 14-day-old male guinea pigs were used. Productive parameters (weight gain, total dry matter intake, and feed conversion ratio (FCR)) and intestinal morphology of the duodenum, jejunum, and ileum at slaughter (70 days of age) were evaluated. An inverse relationship was observed between inulin levels and FCR (linear effect; P = 0.006). There was no statistically significant effect of the treatments on total dry matter intake and weight gain (P > 0.05). A linear effect of the inulin level on the villis length (VL), villis width (VW), and length/depth ratio (VL/DC) in the duodenum; VW in the jejunum; and VL in the ileum (P <0 .05) was reported. In conclusion, a linear effect of the increasing doses of inulin was found on the FCR and the morphological parameters of the duodenums integrity, and no differences in the effects of the inulin added to the diet and the treatment with AGP were found.(AU)
O objetivo do estudo foi avaliar o efeito da suplementação na dieta de cobaias com inulina, em níveis crescentes (0,1%, 0,2%, 0,4%) como substituto para um antibiótico promotor de crescimento (AGP, bacitracina de zinco) além do grupo controle (dieta padrão). Foram utilizados 50 porquinhos-da-índia machos com 14 dias de idade. Os parâmetros produtivos foram avaliados do desmame aos 70 dias de idade e os parâmetros morfológicos intestinais foram avaliados no duodeno, jejuno e íleo no momento do abate. Foi encontrado um efeito linear do nível de inulina sobre na taxa de conversão alimentar (FCR; P = 0,006), indicando que em níveis mais elevados de inulina o FCR diminui. Não houve diferença significativa entre os grupos quando avaliado o efeito dos diferentes tratamentos sobre o consumo de ração e ganho de peso corporal (P > 0,05). Um efeito linear do nível de inulina foi encontrado no comprimento das vilosidades (VL), na largura das vilosidades (VW) e na relação comprimento / profundidade (VL/DC) no duodeno, sobre a VW no jejuno; e no VL no íleo (P < 0,05). Em conclusão, um efeito linear do aumento do nível de inulina foi encontrado na taxa de conversão alimentar e nos parâmetros morfológicos da integridade do duodeno, além disso, não houve diferença entre a adição de inulina na dieta e o tratamento com um antibiótico promotor de crescimento.(AU)
Subject(s)
Animals , Male , Guinea Pigs , Guinea Pigs/anatomy & histology , Guinea Pigs/metabolism , Intestines/anatomy & histology , Inulin/administration & dosage , Inulin/adverse effectsABSTRACT
This study evaluated the effect of the three inulin levels (0.1%, 0.2%, 0.4%) supplemented as a substitute for an antibiotic growth promoter (AGP, zinc bacitracin) and control in guinea pigs raised for human consumption. Fifty 14-day-old male guinea pigs were used. Productive parameters (weight gain, total dry matter intake, and feed conversion ratio (FCR)) and intestinal morphology of the duodenum, jejunum, and ileum at slaughter (70 days of age) were evaluated. An inverse relationship was observed between inulin levels and FCR (linear effect; P = 0.006). There was no statistically significant effect of the treatments on total dry matter intake and weight gain (P > 0.05). A linear effect of the inulin level on the villis length (VL), villis width (VW), and length/depth ratio (VL/DC) in the duodenum; VW in the jejunum; and VL in the ileum (P <0 .05) was reported. In conclusion, a linear effect of the increasing doses of inulin was found on the FCR and the morphological parameters of the duodenums integrity, and no differences in the effects of the inulin added to the diet and the treatment with AGP were found.
O objetivo do estudo foi avaliar o efeito da suplementação na dieta de cobaias com inulina, em níveis crescentes (0,1%, 0,2%, 0,4%) como substituto para um antibiótico promotor de crescimento (AGP, bacitracina de zinco) além do grupo controle (dieta padrão). Foram utilizados 50 porquinhos-da-índia machos com 14 dias de idade. Os parâmetros produtivos foram avaliados do desmame aos 70 dias de idade e os parâmetros morfológicos intestinais foram avaliados no duodeno, jejuno e íleo no momento do abate. Foi encontrado um efeito linear do nível de inulina sobre na taxa de conversão alimentar (FCR; P = 0,006), indicando que em níveis mais elevados de inulina o FCR diminui. Não houve diferença significativa entre os grupos quando avaliado o efeito dos diferentes tratamentos sobre o consumo de ração e ganho de peso corporal (P > 0,05). Um efeito linear do nível de inulina foi encontrado no comprimento das vilosidades (VL), na largura das vilosidades (VW) e na relação comprimento / profundidade (VL/DC) no duodeno, sobre a VW no jejuno; e no VL no íleo (P < 0,05). Em conclusão, um efeito linear do aumento do nível de inulina foi encontrado na taxa de conversão alimentar e nos parâmetros morfológicos da integridade do duodeno, além disso, não houve diferença entre a adição de inulina na dieta e o tratamento com um antibiótico promotor de crescimento.
Subject(s)
Male , Animals , Guinea Pigs , Guinea Pigs/anatomy & histology , Guinea Pigs/metabolism , Intestines/anatomy & histology , Inulin/administration & dosage , Inulin/adverse effectsABSTRACT
Para a metabolização de hormônios esteroides sexuais é essencial a participação de enzimas esteroidogênicas. A enzima citocromo P450c17 é responsável pela produção de andrógenos e a enzima citocromo P450 aromatase é responsável pela produção de estrógenos, sendo que, ambas as enzimas necessitam formar um complexo com uma enzima parceira, denominada NADPH citocromo P450 redutase, para realizar a metabolização destes hormônios essenciais para a diferenciação sexual. Objetivou-se imunolocalisar as três enzimas acima citadas no tecido vaginal de fêmeas de roedores Galea spixii. Para tanto, o experimento foi desenvolvido utilizando técnicas de citologia esfoliativa vaginal para definição das fases do ciclo estral, técnicas de microscopia de Luz, Eletrônica de Varredura e testes de imunohistoquímica para detecção das enzimas: citocromo P450c17, citocromo P450 aromatase, e NADPH citocromo P450 redutase. Constatou-se no presente estudo que o ciclo estral das fêmeas de preás silvestres do semiárido apresenta ciclo estral com duração de 15,85 ± 1,4 dias, com a formação e ruptura da membrana de oclusão vaginal durante cada ciclo estral. A vagina apresenta um clitóris hipertrofiado com o óstio uretral que se abre no topo do mesmo, e ausência de um vestíbulo vaginal. O epitélio da mucosa vaginal sofre modificações proliferativas, se espessando e se adelgando de acordo com a respectiva fase do ciclo estral. As enzimas citocromo P450 aromatase e NADPH redutase estão imunolocalizadas durante todo o ciclo estral no epitélio e tecido conjuntivo da vagina de Galea spixii. Por outro lado, a enzima P450c17 está imunolocalizada no epitélio vaginal durante todas as fases do ciclo estral, porém não está presente no estro. Com estes dados pode-se sugerir que ocorre uma metabolização local de hormônios estrógenos e andrógenos no tecido vaginal o que podem estar relacionado com a proliferação celular, variação na vascularização e inervação e ruptura da membrana de oclusão vaginal
To the metabolism of sex steroid hormones is essential the participation of esteroidogenic enzymes. The enzyme cytochrome P450c17 is responsible for androgen production; and the enzyme aromatase cytochrome P450 is responsible for estrogen production, however, both enzymes require forming a complex with an enzyme partner called NADPH cytochrome P450 reductase to perform the metabolism of these hormones, which are essential for sexual differentiation. This study aimed to immunolocalization of the three enzymes above mentioned in the vaginal tissue from female rodents Galea spixii. Therefore, the experiment was carried out using vaginal cytology techniques for definition of the estrous cycle phases, techniques for light microscopy, scanning electron and immunohistochemical tests for the detection of enzymes: cytochrome P450c17, cytochrome P450 aromatase, and NADPH cytochrome P450 reductase. It was found that the estrus cycle of Galea spixii female lasts 15.85 ± 1.4 days, with the formation and rupture of vaginal closure membrane during each oestrous cycle. The vagina has a hypertrophied clitoris with the urethral orifice that opens on top of it, and it is observed the absence of a vaginal vestibule. The vaginal mucosal epithelium undergoes proliferative changes, with thickening and thinning according to the respective estrous cycle phase. The enzymes cytochrome P450 aromatase and NADPH reductase are immunolocated throughout the oestrous cycle in the epithelium and connective tissue of the vagina from Galea spixii. On the other hand, the enzyme cytochrome P450c17 is immune located in the vaginal epithelium during all stages of the estrous cycle, except at estrous. With these data may be suggested that there is a local estrogens and androgens metabolism in vaginal tissue which may be associated with cell proliferation, vascularization and innervation variation besides the formation and rupture of the vaginal closure membrane
Subject(s)
Female , Animals , Guinea Pigs/metabolism , Gonadal Steroid Hormones/biosynthesis , Androgens/metabolism , Estrogens/metabolismABSTRACT
Primary Na+ transport has been essentially attributed to Na+/K+ pump. However, there are functional and biochemical evidences that suggest the existence of a K+-independent, ouabain-insensitive Na+ pump, associated to a Na+-ATPase with similar characteristics, located at basolateral plasma membrane of epithelial cells. Herein, membrane protein complex associated with this Na+-ATPase was identified. Basolateral membranes from guinea-pig enterocytes were solubilized with polyoxyethylene-9-lauryl ether and Na+-ATPase was purified by concanavalin A affinity and ion exchange chromatographies. Purified enzyme preserves its native biochemical characteristics: Mg2+ dependence, specific Na+ stimulation, K+ independence, ouabain insensitivity and inhibition by furosemide (IC50: 0.5 mM) and vanadate (IC50: 9.1 µM). IgY antibodies against purified Na+-ATPase did not recognize Na+/K+-ATPase and vice versa. Analysis of purified Na+-ATPase by SDS-PAGE and 2D-electrophoresis showed that is constituted by two subunits: 90 (α) and 50 (ß) kDa. Tandem mass spectrometry of α-subunit identified three peptides, also present in most Na+/K+-ATPase isoforms, which were used to design primers for cloning both ATPases by PCR from guinea-pig intestinal epithelial cells. A cDNA fragment of 1148 bp (atna) was cloned, in addition to Na+/K+-ATPase α1-isoform cDNA (1283 bp). In MDCK cells, which constitutively express Na+-ATPase, silencing of atna mRNA specifically suppressed Na+-ATPase α-subunit and ouabain-insensitive Na+-ATPase activity, demonstrating that atna transcript is linked to this enzyme. Guinea-pig atna mRNA sequence (2787 bp) was completed using RLM-RACE. It encodes a protein of 811 amino acids (88.9 kDa) with the nine structural motifs of P-type ATPases. It has 64% identity and 72% homology with guinea-pig Na+/K+-ATPase α1-isoform. These structural and biochemical evidences identify the K+-independent, ouabain-insensitive Na+-ATPase as a unique P-type ATPase.
Subject(s)
Enterocytes/enzymology , Guinea Pigs/genetics , Sodium-Potassium-Exchanging ATPase/genetics , Sodium-Potassium-Exchanging ATPase/isolation & purification , Amino Acid Sequence , Animals , Base Sequence , Biocatalysis/drug effects , Cell Line , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , Dose-Response Relationship, Drug , Furosemide/pharmacology , Gene Expression Regulation, Enzymologic , Guinea Pigs/metabolism , Immunoblotting , Isoenzymes/genetics , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Membrane Proteins/genetics , Membrane Proteins/isolation & purification , Membrane Proteins/metabolism , Molecular Sequence Data , Ouabain/pharmacology , Potassium/pharmacology , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Sodium Potassium Chloride Symporter Inhibitors/pharmacology , Sodium-Potassium-Exchanging ATPase/metabolism , Vanadates/pharmacologyABSTRACT
El cobayo (Cavia porcellus) es un roedor perteneciente al Orden Rodentia y a la Familia Caviidae, utilizado como animal de laboratorio y de consumo humano. Los parámetros cuantitativos del riñón entregan importante información de su morfofunción dada su labor en la homeostasis del organismo. El objetivo de este estudio fue describir el riñón de cobayo (Cavia porcellus), analizando las características estereológicas para futuros estudios experimentales. Se utilizaron 5 cobayos machos, obtenidos del Bioterio de la Universidad de La Frontera, Temuco, Chile. El riñón de cobayo pesó 3,2 g, aproximadamente. El riñón posee 140.298 glomérulos en total, Nv de 458 mm³, Vv de 7,89% y Sv de 3,58 mm²/ mm³. El volumen glomerular del riñón fue de 1,73 x 10(4)mm³ y el diámetro glomerular de 90 jm. Factores como especie, edad, peso corporal, peso y volumen renal, son importantes a considerar, ya que diferencian los resultados en investigaciones morfofuncionales.
The guinea pig, (Cavia porcellus) is a rodent pertaining to the Rodentia group and the Caviidae family, used as a laboratory animal and for human consumption. Quantitative parameters of the kidney provides important information of its morphofunction, given its labor in the organism's homeostasis. The aim or this study was to describe the kidney of the guinea pig (Cavia porcellus), analyzing the stereological characteristics for future experimental studies. Five male guinea pigs (Cavia porcellus) obtained from the Biotery of the Universidad de la Frontera, Temuco, Chile, were used. The kidney of the guinea pig weighed approximately 3.2g. The kidney has 140,298 total glomerulus, Nv of 458 mm³, Vv of 7.89% and Svof 3.58mm²/mm³. The glomerular volume of the kidney was of 1.73 x 10(4)mm³ and a glomerular diameter of 90 urn. Factors such as species, age, body weight and renal volume, are important to consider, as they differentiate the results in the morphofunctional investigations.
Subject(s)
Male , Adult , Animals , Guinea Pigs , Guinea Pigs/anatomy & histology , Guinea Pigs/physiology , Guinea Pigs/metabolism , Kidney/anatomy & histology , Kidney/physiology , Kidney/ultrastructure , Zona Glomerulosa/anatomy & histology , Zona Glomerulosa/physiology , Zona Glomerulosa/ultrastructure , Photogrammetry/methods , Photogrammetry/veterinary , Homeostasis/physiology , Body Weights and Measures/methods , Body Weights and Measures/veterinaryABSTRACT
This study aimed to assess the body weight and food and water intake of alendronate-treated Wistar rats. Thirty rats were divided in 3 groups of 10. Group A (control group) received saline, and Groups B and C received alendronate 4mg and alendronate 0.033mg, respectively through gavage. Body weight and food and water intake were measured daily for 10 days. From the first to the last day, the mean body weight ranged from 188.2g (SD 7.3) to 183.2g (SD 5.7) in Group A, from 183.0g (SD 7.7) to 177.5g (SD 8.2) in Group B, and from 188.9g (SD 17.1) to 184.5 (SD 16.4) in Group C. Mean food intake ranged from 15.0g (SD 1.8) to 17.5g (SD 1.1)in Group A, from 14.0g (SD 2.2) to 15.4g (SD 2.6) in Group B, and from 23.3g (SD 2.0) to 14.9g (SD 2.6) in Group C. Mean water intake ranged from 17.0ml (SD 6.5) to 20.6ml (SD 5.6) in Group A, from 20.8ml (SD1.3) to 22.1ml (SD 3.6) in Group B, and from 16.0ml (SD 5.7) to 19.3ml (SD 2.7) in Group C. Alendronate caused significant differences in body weight (the higher the dose the lower the weight) and water intake (the control group consumed less water). No difference regarding food intake was found.
Subject(s)
Animals , Female , Rats , Body Weight , Alendronate/adverse effects , Body Weight Changes , Guinea Pigs/metabolismABSTRACT
Gonadotropin-releasing hormone (GnRH) is a decapeptide widely known for its role in regulating vertebrate reproduction by serving as a signal from the hypothalamus to pituitary gonadotropes. The first form of GnRH to be identified was isolated from mammals (mGnRH) and the same form has been reported for all mammals studied, which includes marsupials and placental mammals. Later, another variant, chicken GnRH-II (cGnRH-II) was shown to be expressed together with mGnRH in the brains of all jawed vertebrates, including mammals such as rats, monkeys and humans. Our objective was to characterize a third form of GnRH that was isolated previously as mRNA from guinea pigs (gpGnRH), but has not been reported for any other mammal to date. Furthermore, the gonadotropic activity of gpGnRH has not been fully characterized. Our results, using chromatographical and immunological methods, show for the first time that gpGnRH is expressed together with mGnRH in some rodents (wild guinea pig and capybara), but not in others (mouse and hamster). Also, the gonadotropic activity of gpGnRH and mGnRH was tested in two different rat cell culture systems. Although there have been reports that the salmon(s) form of GnRH is present in mammals, we did not detect sGnRH in capybara, wild guinea pigs, hamsters, rats or mice. Taken together with previous reports, the present results support the idea that the expression of multiple GnRH variants in a single species is a common pattern in most vertebrate groups.
Subject(s)
Brain/metabolism , Gonadotropin-Releasing Hormone/physiology , Guinea Pigs/metabolism , Rodentia/metabolism , Animals , Cells, Cultured , Chromatography, High Pressure Liquid , Female , Follicle Stimulating Hormone/metabolism , Gonadotropin-Releasing Hormone/metabolism , Gonadotropin-Releasing Hormone/pharmacology , Luteinizing Hormone/metabolism , Male , Peptide Fragments/pharmacology , Pituitary Gland, Anterior/cytology , Pituitary Gland, Anterior/metabolism , RadioimmunoassayABSTRACT
Pancreatic sections from diverse tetrapods, including various species of caviomorph rodents, were immunohistochemically investigated using two antisera which reacted with the N- and C-terminal portions of the glucagon molecule. While the antiserum against the N-terminal portion stained alpha cells in all the species studied, the antiserum against the C-portion failed to stain alpha cells in two caviomorphs of the Caviidae family (guinea pig and cuis) and in one of the Octodontidae family (degu). The observations in guinea pig and degu were expected, since their glucagons differ from those of many other tetrapods in the C-terminal portion of the molecule. In this paper, the cuis was added to these two species. It is noteworthy that among the caviomorphs studied herein (nine species), immunohistochemical differences were detected only in the three above-mentioned species and did not involve higher taxa, thus suggesting that these modifications are relatively recent in the evolution of this group of rodents.
Subject(s)
Glucagon/analysis , Glucagon/immunology , Guinea Pigs/metabolism , Immune Sera/immunology , Rodentia/metabolism , Amino Acid Sequence , Animals , Biological Evolution , Bufonidae , Ducks , Glucagon/chemistry , Immunohistochemistry , Molecular Sequence Data , Pancreas/chemistry , Pancreas/cytology , Pancreas/metabolism , Rats , SnakesABSTRACT
O desenvolvimento pós-natal do pâncreas da cobaia (Cavia porcellus) foi estudado por métodos estereológicos ao microscópio de luz, no período de 2 a 140 dias de idade. As modificaçöes morfométricas detectadas no estudo dos dados numéricos foram acompanhadas por uma análise morfológica qualitativa. A massa pancreática exibiu um aumento acentuado de 805 por cento, no período de 2 a 140 dias de vida pós-natal. Este crescimento pode ser expresso pela equaçäo linear Y = 519,2 + 14,2x (coeficiente de correlaçäo r= 0,95) e o tempo de duplicaçäo calculado por essa equaçäo foi de 38,6 dias. Este crescimento de massa pancreática ocorreu devido ao aumento de volume em todos os compartimentos morfológicos, notadamente o dos ácinos e o das outras estruturas (estroma). A relativa estabilidade da densidade de volume dos vários compartimentos mostrou que durante este crescimento as relaçöes volumétricas säo mantidas. O volume do compartimento dos ácinos aumentou 756 por cento, no período total analisado. Este crescimento pode ser representado pela equaçäo Y = 204,1 + 4,7x (r= 0,92) e o tempo de duplicaçäo calculado para o período de 2 a 140 dias foi de 45,6 dias. Este aumento de volume do compartimento dos ácinos ocorreu por dois mecanismos de crescimento: a atividade proliferativa e o aumento de volume celular. O estudo da evoluçäo do número absoluto de células acinosas mostrou um aumento de 361 por cento, no período de 2 a 140 dias. A equaçäo obtida pela análise de regressäo para exprimir esse aumento foi Y = 110,1 + 1,6x (r=0,94) e o tempo de duplicaçäo calculado foi de 70,8 dias. Por outro lado, o volume celular médio das células acinosas aumentou 210 por cento, no mesmo período. A equaçäo linear obtida foi Y = 755,3 + 6,4x (r=0,86) e o tempo de duplicaçäo foi de 120,0 dias. Os dados mostraram que no período total de 2 a 140 dias, o aumento de volume do compartimento dos ácinos ocorreu com um predomínio de atividade proliferativa sobre aumento de volume celular
Subject(s)
Animals , Male , Infant, Newborn , Infant , Guinea Pigs , Pancreas/growth & development , Pancreas/metabolism , Guinea Pigs/growth & development , Guinea Pigs/metabolism , Pancreas/ultrastructure , Pathology, OralABSTRACT
A tetraiodinated derivative of bovine insulin, prepared at pH 1 with stable iodine, was unable to cause signs of hypoglycemia in doses up to 2.4 micrograms/g in fasting mice, when native insulin caused 100% mortality. In neutral and acidic solutions, in absence of chaotropic agents, it behaved as the monomer, and could be separated from less iodinated, active species, that appeared as dimers, by conventional gel filtration. To generate antibodies in guinea-pigs, the tetraiodinated insulin was injected in doses three times higher than native insulin, without any harm to recipient animals. The induced antiserum was compared with antiserum generated by conventional methods in radioimmunoassay (RIA) of native insulin, and parallel curves were obtained.