Regeneration of nucleotides β-nicotinamide adenine soluble or immobilized enzyme coupled systems.

As bioconversões executadas com enzimas, que requerem NADP ou NADPH, são limitadas pela eficiência com que esta substância é mantida na forma particular requerida pelo catalisador ao longo de toda a reação, assim como pela sua recuperação no final do processo. Neste trabalho, foram caracterizadas as enzimas Glicose-6-fosfato desidrogenase (G6PDH) e Glutamato desidrogenase (GLUDH), além do estudo do reciclo NADP/NADPH em sistema constituído por essas enzimas. Determinou-se a temperatura (30ºC) e pH ótimo da G6PDH (pH 7,5), assim como a temperatura (40ºC) e o pH ótimo (pH 8,0) da GLUDH, além dos volumes e concentrações dos substratos utilizados na hidrólise enzimática. No caso da reação acoplada descontínua, a duração foi de 90min, às temperaturas de 30ºC e 40ºC, utilizando-se o cofator solúvel ou imobilizado. Em ambas as condições, mais de 85% das concentrações iniciais de G6P e NH4+, respectivamente, substratos da G6PDH e GLUDH, foram convertidas, sendo o reciclo NADP/NADPH mantido durante toda a reação. No caso da reação bienzimática em reator contínuo, não foi possível chegar ao estado estacionário da reação e a conversão dos subsratos G6P e NH4+ variaram de acordo com o período do teste.The Bioconversion performed with enzymes that require NADPH or NADP are limited by the efficiency with which this substance is maintained in the particular form required by the enzyme throughout the reaction, as well as for his recovery at the end of the process. The main aim of this work was to study the NADP/NADPH recycling through a bienzyme coupled reaction constituted by glucose-6-phosphate dehydrogenase (G6PDH) and glutamate dehydrogenase (GLUDH). The reaction was carried out in the discontinuous or continuous mode. The discontinuous process, which was carried out with soluble or immobilized NADP at 30°C or 40°C, had a total duration of 90min. Independently on the temperature used, around 85% of the initial concentration of glucose 6-phosphate (G6P) and ammonia were consumed, being the recycle of NADP/NADPH maintained throughout the reaction. In the continuous process, the addition of G6P and ammonia into the membrane reactor was made by turns of 2h. During the 15h-process the NADP/NADPH recycling was attained and the mean consumption yield of ammonia and G6P neared 30% and 60%, respectively

Regeneration of nucleotides β-nicotinamide adenine soluble or immobilized enzyme coupled systems.

As bioconversões executadas com enzimas, que requerem NADP ou NADPH, são limitadas pela eficiência com que esta substância é mantida na forma particular requerida pelo catalisador ao longo de toda a reação, assim como pela sua recuperação no final do processo. Neste trabalho, foram caracterizadas as enzimas Glicose-6-fosfato desidrogenase (G6PDH) e Glutamato desidrogenase (GLUDH), além do estudo do reciclo NADP/NADPH em sistema constituído por essas enzimas. Determinou-se a temperatura (30ºC) e pH ótimo da G6PDH (pH 7,5), assim como a temperatura (40ºC) e o pH ótimo (pH 8,0) da GLUDH, além dos volumes e concentrações dos substratos utilizados na hidrólise enzimática. No caso da reação acoplada descontínua, a duração foi de 90min, às temperaturas de 30ºC e 40ºC, utilizando-se o cofator solúvel ou imobilizado. Em ambas as condições, mais de 85% das concentrações iniciais de G6P e NH4+, respectivamente, substratos da G6PDH e GLUDH, foram convertidas, sendo o reciclo NADP/NADPH mantido durante toda a reação. No caso da reação bienzimática em reator contínuo, não foi possível chegar ao estado estacionário da reação e a conversão dos subsratos G6P e NH4+ variaram de acordo com o período do teste.The Bioconversion performed with enzymes that require NADPH or NADP are limited by the efficiency with which this substance is maintained in the particular form required by the enzyme throughout the reaction, as well as for his recovery at the end of the process. The main aim of this work was to study the NADP/NADPH recycling through a bienzyme coupled reaction constituted by glucose-6-phosphate dehydrogenase (G6PDH) and glutamate dehydrogenase (GLUDH). The reaction was carried out in the discontinuous or continuous mode. The discontinuous process, which was carried out with soluble or immobilized NADP at 30°C or 40°C, had a total duration of 90min. Independently on the temperature used, around 85% of the initial concentration of glucose 6-phosphate (G6P) and ammonia were consumed, being the recycle of NADP/NADPH maintained throughout the reaction. In the continuous process, the addition of G6P and ammonia into the membrane reactor was made by turns of 2h. During the 15h-process the NADP/NADPH recycling was attained and the mean consumption yield of ammonia and G6P neared 30% and 60%, respectively

Evaluation of the influence of voluntary physical exercise on neurogenesis and synaptic plasticity in offspring of conditioned PTEN deletion.

O gene da PTEN (Phosphatase and tensin homolog on chromosome ten) foi primeiramente estudado como um gene supressor de tumor. No entanto, devido as características abrangentes desta fosfatase que podem influenciar a proliferação, sobrevivência e migração celular, a PTEN também parece estar envolvida nos fenômenos da neurogênese e plasticidade sináptica. Estudos sugerem que não somente fatores genéticos, mas também ambientais podem influenciar o processo de neurogênese. Entender os mecanismos pelos quais isso ocorre pode trazer benefícios no tratamento ou até mesmo na prevenção de doenças relacionadas ao sistema nervoso central (SNC). Como exemplo, o exercício físico tem demonstrado exercer efeitos benéficos em processos associados ao envelhecimento e a doenças neurodegenerativas. A prática de exercício físico durante a gestação também pode favorecer a neurogênese e a plasticidade sináptica na prole, e esses efeitos benéficos podem perdurar até a idade adulta desses animais. Portanto, este projeto teve como escopo avaliar os efeitos do exercício físico voluntário na neurogênese e na plasticidade sináptica em animais com a deleção parcial do gene da PTEN. Foram observadas alterações positivas quanto ao aprendizado da prole com a deleção condicionada da PTEN durante o aprendizado da tarefa do Labirinto Aquático de Morris, além do aumento de marcadores moleculares relacionados ao fenômeno da plasticidade sináptica da subunidade NR1 do receptor glutamatérgico nesses animais e o aumento da do fator neurotrófico derivado do cérebro. De forma geral o exercício físico materno foi capaz de aumentar alguns marcadores relacionados ao fenômeno da plasticidade sináptica, envolvido diretamente na formação de memória e da capacidade de aprendizado. Portanto, os resultados sugerem que o exercício físico materno, desde que respeitada as condições do cuidado materno durante o puerpério e a amamentação, pode ser benéfico para a prole wild type e com a deleção parcial da PTEN.PTEN was first studied as a tumor suppressor gene. However, due to the comprehensive characteristics of this phosphatase that can influence cell proliferation, survival and migration, PTEN may be also involved in the neurogenesis and synaptic plasticity. Studies suggest that not only genetic but also environmental factors can influence the process of neurogenesis. Understanding the mechanisms by which this occurs can bring benefits in the treatment or even the prevention of diseases related to the central nervous system (CNS). As an example, physical exercise has been shown to exert beneficial effects in processes associated with aging and neurodegenerative diseases. The practice of physical exercise during pregnancy seem also favor neurogenesis and synaptic plasticity in the offspring, and these beneficial effects may last until the adulthood of these animals. Therefore, the purpose of this project was to evaluate the effects of voluntary physical exercise on neurogenesis and synaptic plasticity in animals with partial deletion of PTEN gene. Positive alterations were observed regarding offspring learning with the conditional PTEN deletion during the learning of the Morris water maze task, as well as the increase of the subunit N-Methyl-D- Aspartate receptor 1 (NR1) in these animals and an increase in brain derived neurotrophic factor levels. In general, maternal physical exercise was able to increase some markers related to the phenomenon of synaptic plasticity, directly involved in the formation of memory and learning capacity. Therefore, the results suggest that maternal physical exercise, while respecting the conditions of maternal care during the puerperium and breastfeeding, may be beneficial to the wild type offspring and with the partial deletion of PTEN

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Abstract: Aging is a multifactorial process associated with an increased susceptibility to neurodegenerative disorders which can be related to chronic inflammation. Chronic inflammation, however, can be characterized by the persistent elevated glucocorticoid (GCs) levels, activation of the proinflammatory transcription factor NF-кB, as well as an increase in cytokines. Interestingly, both NF-кB and cytokines can be even modulated by Glycogen Synthase Kinase 3 beta (GSK-3β) activity, which is a key protein that can intermediate inflammation and metabolism, once it has a critical role in AKT signaling pathway, and can also intermediate WNT/β-CATENIN signaling pathway. The aim of this study was to verify agerelated changes in inflammatory status, as well as in the AKT and WNT signaling pathways. Results showed an age-related increase in neuroinflammation as indicated by NF-кB activation, TNF-α and GCs increased levels, a decrease in AKT activation and an increase in GSK-3β activity in both 12-and 24-month old animals. Aging also seems to induce a progressive decrease in canonical WNT/β-CATENIN signaling pathway once there is a decrease in DVL-2 levels and in the transcription of Axin2 gene. Little is known about the DVL-2 regulation as well as its roles in WNT signaling pathway, but for the first time it was suggested that DVL-2 expression can be changed along aging

Consequences of the Lack of TNFR1 in Ouabain Response in the Hippocampus of C57BL/6J Mice

Ouabain is a cardiac glycoside that has a protective effect against neuroinflammation at low doses through Na+/K+-ATPase signaling and that can activate tumor necrosis factor (TNF) in the brain. TNF plays an essential role in neuroinflammation and regulates glutamate receptors by acting on two different receptors (tumor necrosis factor receptor 1 [TNFR1] and TNFR2) that have distinct functions and expression. The activation of constitutively and ubiquitously expressed TNFR1 leads to the expression of pro-inflammatory cytokines. Thus, this study aimed to elucidate the effects of ouabain in a TNFR1 knockout (KO) mouse model. Interestingly, the hippocampus of TNFR1 KO mice showed a basal increase in both TNFR2 membrane expression and brain-derived neurotrophic factor (BDNF) release, suggesting a compensatory mechanism. Moreover, ouabain activated TNF-α-converting enzyme/a disintegrin and metalloprotease 17 (TACE/ADAM17), decreased N-methyl-D-aspartate (NMDA) receptor subunit 2A (NR2A) expression, and induced anxiety-like behavior in both genotype animals, independent of the presence of TNFR1. However, ouabain induced an increase in interleukin (IL)-1β in the hippocampus, a decrease in IL-6 in serum, and an increase in NMDA receptor subunit 1 (NR1) only in wild-type (WT) mice, indicating that TNFR1 or TNFR2 expression may be important for some effects of ouabain. Collectively, our results indicate a connection between ouabain signaling and TNFR1, with the effect of ouabain partially dependent on TNFR1

Ouabain Reverts CUS-Induced Disruption of the HPA Axis and Avoids Long-Term Spatial Memory Deficits

Ouabain (OUA) is a cardiotonic steroid that modulates Na+, K+ -ATPase activity. OUA has been identified as an endogenous substance that is present in human plasma, and it has been shown to be associated with the response to acute stress in both animals and humans. Chronic stress is a major aggravating factor in psychiatric disorders, including depression and anxiety. The present work investigates the effects of the intermittent administration of OUA (1.8 μg/kg) during the chronic unpredictable stress (CUS) protocol in a rat’s central nervous system (CNS). The results suggest that the intermittent OUA treatment reversed CUS-induced HPA axis hyperactivity through a reduction in (i) glucocorticoids levels, (ii) CRH-CRHR1 expression, and by decreasing neuroinflammation with a reduction in iNOS activity, without interfering with the expression of antioxidant enzymes. These changes in both the hypothalamus and hippocampus may reflect in the rapid extinction of aversive memory. The present data demonstrate the ability of OUA to modulate the HPA axis, as well as to revert CUS-induced long-term spatial memory deficits

Altered KLOTHO and NF-κB-TNF-α Signaling Are Correlated with Nephrectomy-Induced Cognitive Impairment in Rats.

Renal insufficiency can have a negative impact on cognitive function. Neuroinflammation and changes in klotho levels associate with chronic kidney disease (CKD) and may play a role in the development of cognitive impairment (CI). The present study evaluates the correlation of cognitive deficits with neuroinflammation and soluble KLOTHO in the cerebral spinal fluid (CSF) and brain tissue of nephrectomized rats (Nx), with 5/6 renal mass ablation. Nx and sham Munich Wistar rats were tested over 4 months for locomotor activity, as well as inhibitory avoidance or novel object recognition, which started 30 days after the surgery. EMSA for Nuclear factor-κB and MILLIPLEXMAP or ELISA kit were used to evaluate cytokines, glucocorticoid and KLOTHO levels. Nx animals that showed a loss in aversive-related memory and attention were included in the CI group (Nx-CI) (n=14) and compared to animals with intact learning (Nx-M n=12 and Sham n=20 groups). CSF and tissue samples were collected 24 hours after the last behavioral test. The results show that the Nx-groups have increased NF-κB binding activity and tumor necrosis factor-alpha (TNF-α) levels in the hippocampus and frontal cortex, with these changes more pronounced in the Nx-CI group frontal cortex. In addition, the Nx-CI group showed significantly increased CSF glucocorticoid levels and TNF-α /IL-10 ratio compared to the Sham group. Klotho levels were decreased in Nx-CI frontal cortex but not in hippocampus, when compared to Nx-M and Sham groups. Overall, these results suggest that neuroinflammation mediated by frontal cortex NF-κB, TNF-α and KLOTHO signaling may contribute to Nx-induced CI in rats

Proteomic analysis reveals rattlesnake venom modulation of proteins associated with cardiac tissue damage in mouse hearts

Snake envenomation is a common but neglected disease that affects millions of people around the world annually. Among venomous snake species in Brazil, the tropical rattlesnake (Crotalus durissus terrificus) accounts for the highest number of fatal envenomations and is responsible for the second highest number of bites. Snake venoms are complex secretions which, upon injection, trigger diverse physiological effects that can cause significant injury or death. The components of C. d. terrificus venom exhibit neurotoxic, myotoxic, hemotoxic, nephrotoxic, and cardiotoxic properties which present clinically as alteration of central nervous system function, motor paralysis, seizures, eyelid ptosis, ophthalmoplesia, blurred vision, coagulation disorders, rhabdomyolysis, myoglobinuria, and cardiorespiratory arrest. In this study, we focused on proteomic characterization of the cardiotoxic effects of C. d. terrificus venom in mouse models. We injected venom at half the lethal dose (LD50) into the gastrocnemius muscle. Mouse hearts were removed at set time points after venom injection (1 h, 6 h, 12 h, or 24 h) and subjected to trypsin digestion prior to high-resolution mass spectrometry. We analyzed the proteomic profiles of >1300 proteins and observed that several proteins showed noteworthy changes in their quantitative profiles, likely reflecting the toxic activity of venom components. Among the affected proteins were several associated with cellular deregulation and tissue damage. Changes in heart protein abundance offer insights into how they may work synergistically upon envenomation. Venom of the tropical rattlesnake (Crotalus durissus terririficus) is known to be neurotoxic, myotoxic, nephrotoxic and cardiotoxic. Although there are several studies describing the biochemical effects of this venom, no work has yet described its proteomic effects in the cardiac tissue of mice. In this work, we describe the changes in several mouse cardiac proteins upon venom treatment. Our data shed new light on the clinical outcome of the envenomation by C. d. terrificus, as well as candidate proteins that could be investigated in efforts to improve current treatment approaches or in the development of novel therapeutic interventions in order to reduce mortality and morbidity resulting from envenomation. [Display omitted] •C. d. terrificus rattlesnake venom treatment of mice showed changes in cardiac protein profile•C. d. terrificus venom modulated proteins involved in biological and cellular process - C. d. terrificus venom modulated proteins related to molecular function and phenotypic profiles•C. d. terrificus venom modulated Gene Ontology categories related to cardiac cardiac tissue damage•Network analysis showed disturbances of proteins related to cardiac tissue damag

Changes in TNF-α in hippocampus and frontal cortex at 121 days in sham and Nx (Nx-M and Nx-CI) groups.

<p>Values are the mean ± S.E.M. (n = 21). *p<0.05 versus sham group and ** versus Nx-M group—one-way ANOVA followed by Newman-Keuls test. TNF-α Frontal cortex: F(2,14) = 39.43, p<0.001; TNF-α hippocampus: F(2,14) = 9.24, p<0.05.</p

Histological kidney analysis in sham and Nx groups at 121 days after the surgical procedure.

<p>(A,B). The photomicrographs are representative samples of glomerular sclerosis observed in sham and Nx kidneys that were stained with periodic acid-Schiff (SPADES). The results showed a significant increase in the number of glomeruli with sclerosis (yellow arrow) in the Nx group (B) (n = 8) when compared to the sham group (A) (n = 5). (C,D) The photomicrographs are representative samples of cortical interstitial area expansion observed in Nx (D) (yellow arrow) when compared to sham (C) that were stained with SPADES. The analysis showed a significant increase in cortical interstitial area in the Nx group (n = 8) when compared to the sham group (n = 5). (E) The graphical representation of the percent of glomerular sclerosis (A,B) and (F) of cortical interstitial area (C,D), respectively. *p<0.05 versus sham—Student's t test.</p