The addition of Mn(II) to an early stationary-phase Deinococcus radiodurans RI culture could induce a new round of cell division (MnCD effect). The addition of Mn(II) could also stimulate the utilization of glucose and fructose in this bacterium. Class II fructose-1,6-bisphosphate aldolase (FBA) is an Mn-dependent key enzyme in pentose phosphate pathway. Therefore, in this research, we focused on the studies of the fba gene. Base on the gene sequence, FBA protein was composed of 306 amino acids, (M.W., 32.4 kDa¡F pI, 5.4). The expected PCR product size of the fba gene is 9.3 kbp. We had amplified the fba gene by using both Taq DNA polymerase and pfu turbo DNA polymerase. The sequence of the pfu turbo DNA polymerase products showed a higher homology with the fba gene than those of using Taq DNA polymerase. These amplified fba gene was cloned into three expression vectors, pGEX-4T-2, pQE30, and pET28a, and then further expressed in E. coli BL21(DE3)RIL and JM109. The recombinant GST-FBA protein could be overproduced in pTDA2/BL21(DE3)RIL. However, the expressed insoluble protein accumulated as inclusion bodies in the cells and exhibited no enzyme activity. After partial purification, and processing by thrombin protease cleavage, urea treatment, and the addition of Mn(II), this enzyme still showed no activity. The recombinant pEDA2/BL21(DE3)RIL strain cells grew in 18¢J and induced by 0.1mM IPTG could produced a soluble form His-Thrombin-T7-FBA protein which performed a 50X higher activities than those cells grew in 30¢J. This result indicated that decreasing the indicatioin temperature could improve the protein solubility and activity.

Abstract During surgery of the aortic arch and pediatric heart surgery, the blood flow to the brain has to be interrupted at times to allow a bloodless operation field and adequate conditions for surgical repair. During this no-flow period the brain is exposed to a high risk of ischaemic injury, as it will become irreversibly damaged after 5 minutes of circulatory arrest at 37°C. Additional time can be gained by cooling the patient with an extracorporeal heart-lung machine, as hypothermia reduces the cerebral metabolic rate and allows longer safe periods of circulatory standstill. This method of cerebral protection, called hypothermic circulatory arrest (HCA), is widely used in clinical practice. Thus the brain becomes susceptible to ischaemic injury after 30 minutes of HCA at 15°C. Lower temperatures than this are not practicable, however, as they require longer periods of cardiopulmonary bypass, which may further aggravate cerebral injury. To ensure a better outcome for patients undergoing these operations, additional ways of protecting the brain are required. The present work focuses on neuroprotective biochemical and fluid therapy methods for use during HCA, employing a surviving porcine model. Fructose-1,6-bisphosphate (FDP), a high-energy intermediate of glycolysis, was examined for potential neuroprotective properties in two cerebral injury settings associated with HCA. First, FDP was administered before and after a 75-minute period of HCA at a brain temperature of 18°C. This led to better survival, neurological recovery and brain histopathological findings and had supportive effects on brain metabolism (I). Second, a 25-minute period of HCA along with an iatrogenic embolic load produced by microsphere injection was used to generate a massive ischaemic injury to the brain. In this setting FDP did not affect the neurological outcome but had a clear supportive impact on cerebral metabolism (II). In addition, cerebral histopathological samples taken during the first study were analysed by electron microscopy, which revealed significant preservation of the ultrastructure in the FDP-treated animals (III). Hypertonic saline dextran (HSD) is a novel fluid therapy method which has been shown to enhance the outcome after hypovolaemic shock with or without head injury and is potentially very effective in reducing ischaemia-reperfusion injury. Its administration led to a decrease in intracranial pressure, improved brain metabolism, faster and better recovery and less histopathologically observable morphological damage (IV). The findings indicate that both FDP and HSD have significant neuroprotective properties and should be assessed in humans as well.

Abstract Interruption of cerebral blood flow during hypothermic circulatory arrest (HCA) predisposes neurons to glutamate excitotoxicity. Reperfusion is followed by leukocyte infiltration, which results in an inflammatory reaction in the brain tissue. In the first study, the presynaptic glutamate release inhibitor lamotrigine (L) and the leukocyte-depleting filter (LF) were studied to determine if their combination could mitigate brain injury after HCA (I). The aim of the second study was to evaluate the possible neuroprotective effect of a 14-hour period of mild (32°C) hypothermia after HCA (II). Recent experimental research has demonstrated the neuroprotective properties of erythropoietin (EPO) and fructose-1,6-bisphosphate (FDP), whose effects during and after HCA were evaluated in the third and the fourth studies (III, IV). A chronic porcine model was used. The animals were randomly assigned to the study groups as follows: 8 animals in the L+LF group, 8 in the L group, and 8 in the control group (I); 10 animals in the hypothermia group and 10 in the normothermia group (II); 10 animals in the EPO group and 10 in the control group (III), and 12 animals in the FDP group and 12 in the control group (IV). Monitoring of hemodynamics, metabolism, temperature, electroencephalogram (EEG), brain microdialysis, intracranial pressure (II-IV), and brain tissue oxygen (II-IV) was carried out. A daily behavioral assessment was performed until death or until elective sacrifice on the seventh postoperative day, after which the brain was prepared for a histopathologic examination. The results of these studies indicate that lamotrigine has a neuroprotective effect during HCA. This is observed in terms of EEG burst recovery, behavioral and histopathologic outcome, and brain microdialytic findings. The combined use of lamotrigine and leukocyte filtration may further improve survival. A 14-hour period of mild hypothermia after HCA is associated with a poor outcome. However, it may preserve its efficacy when used for no longer than 4 hours. Administration of EPO before HCA proved ineffective in reducing mortality or brain histopathologic injury. Findings from brain microdialysis, brain tissue oxygen tension, and neuronal apoptosis, however, suggest that the drug has neuroprotective properties. Administration of FDP before and after HCA is associated with better survival, behavioral outcome, and brain histopathologic scores. The metabolic and brain microdialytic findings also suggest that this drug has supportive effects on myocardial and brain metabolism.

Glucose 1,6-bisphosphate is an important small molecule involved in the regulation of glycolysis. Four enzymes synthesize this compound. One enzyme is known to degrade it, glucose 1,6-bisphosphatase. Other groups have produced work that indicates that there are two isoforms of this enzyme, one predominant in the brain and one in the muscle. This thesis contains the work performed in attempts to isolate and characterize the muscle isoform of glucose 1,6-bisphosphatase. While this enzyme was not isolated, much was learned about it and the results from this work may help in the future identification of this enzyme.

D-Frutose-1,6-difosfato (F1,6BP), um intermediário altamente energético da via glicolítica, é descrito na literatura como portador de diversas atividades farmacológicas, contudo seu mecanismo de ação ainda não fora esclarecido. Dessa forma, a possível atividade anti-inflamatória da F1,6BP sobre diferentes modelos de inflamação e sua via de ação em camundongos foram averiguadas com enfoque na inibição da produção de citocinas e na elevação dos níveis endógenos de adenosina. A hipernocicepção mecânica foi avaliada por método eletrônico de pressão crescente em patas de camundongos, o ensaio cinéticocolorimétrico de mieloperoxidase (MPO) foi utilizado na avaliação da migração leucocitária para o tecido subcutâneo plantar, e a performance motora dos animais foi determinada pelo teste rota-rod. A quantificação de citocinas foi realizada por ELISA, enquanto a quantificação de adenosina foi obtida em cromatografia líquida de alta eficiência (CLAE) após validação laboratorial da metodologia segundo normas protocoladas pela ANVISA. O pré-tratamento de camundongos com F1,6BP reduziu a hipernocicepção induzida por injeção intraplantar de carragenina (45%), TNF-a (40%), IL-1B (46%), KC (33%), prostaglandina E2 (41%) e dopamina (55%). O tratamento com F1,6BP, no entanto, não alterou a produção de citocinas induzidas por carragenina (TNF-a, IL-1B e KC) e nem interferiu com a migração leucocitária para a região desafiada. Por outro lado, o efeito antinociceptivo da F1,6BP foi prevenido por tratamento sistêmico e local com antagonista de receptores A1 de adenosina (DPCPX) e o tratamento com adenosina apresentou efeitos similares os do tratamento com F1,6BP, sugerindo que o efeito da F1,6BP é mediado pela ação periférica da adenosina sobre receptores A1. Endossando isso, foi constatado por meio de cromatografia líquida de alta eficiência um aumento na concentração plasmática de adenosina em camundongos após administração de F1,6BP. Por conseguinte, a via de ação da F1,6BP parece ser dependente da produção de adenosina, mas não da modulação na produção de citocinas hipernociceptivas. A adenosina atua, então, perifericamente em receptores A1 inibindo a hipernocicepção, e a evidência farmacológica de que ela medeia a atividade da F1,6BP através dessa ação sobre receptores A1 foi corroborada por resultados in vivo que demonstram aumento dos níveis de adenosina decorrente da administração de F1,6BP. Em vista disso, esses resultados insinuam um possível potencial terapêutico da F1,6BP em reduzir a dor inflamatória. / D-Fructose-1,6-bisphosphate (F1,6BP), a high-energy glycolytic pathway intermediate, is reported to have a many pharmacologically effect, but its underlying mechanism of action in inflammation is incompletely understood. In this study, the aim was to examine the function of F1,6BP on cytokines and adenosine. Then, the possible F1,6BP anti-inflammatory activities in different models of inflammation and its mechanism of action in mice were addressed focusing inhibition of cytokine production or adenosine production enhancement. Mechanical hypernociception (decrease in the nociceptive threshold of animals) was evaluated by the electronic pressure meter test in mice, the myeloperoxidase (MPO) kineticcolorimetric assay was used to evaluate the leukocyte migration to the subcutaneous plantar tissue of mice hind paw, and mice motor performance were evaluated on the rota-rod test. The cytokines levels were measured by ELISA. Adenosine levels were determined by high performance liquid chromatography and this experimental procedure was validated according to ANVISA for this purpose. The pretreatment of mice with F1,6BP reduced the hypernociception induced by intraplantar injection of carrageenan (45%), TNF (40%), IL-1 (46%), KC (33%), prostaglandin E2 (41%) and dopamine (55%). However, FBP treatment did not alter carrageenin-induced cytokines (TNF-, IL-1 and KC) production or the leukocyte migration to the subcutaneous plantar tissue of mice hind paw. On the other hand, the antinociceptive effect of F1,6BP was prevented by systemic and local treatment with an adenosine A1 receptor antagonist (DPCPX) and adenosine treatment presented similar effect of F1,6BP, suggesting that F1,6BP effect is mediated by peripheral adenosine acting on A1 receptors. In agreement, the present work determined by high performance liquid chromatography that D-Fructose-1,6-bisphosphate administration increased the adenosine endogenous levels in the mice plasma. Therefore, F1,6BP mechanism of action seems dependent on adenosine production but not on modulation of hypernociceptive cytokine production. In turn, adenosine acts peripherally on A1 receptor inhibiting hypernociception. The pharmacological evidence that adenosine through activation of adenosine A1 receptor mediates the antinociceptive action of F1,6BP was supported by the finding that in vivo administration of F1,6BP increases the levels of adenosine in the blood samples. Thus, suggesting the treatment with F1,6BP as a possible therapeutic approach to reduce inflammatory pain.

D-Frutose-1,6-difosfato (F1,6BP), um intermediário altamente energético da via glicolítica, é descrito na literatura como portador de diversas atividades farmacológicas, contudo seu mecanismo de ação ainda não fora esclarecido. Dessa forma, a possível atividade anti-inflamatória da F1,6BP sobre diferentes modelos de inflamação e sua via de ação em camundongos foram averiguadas com enfoque na inibição da produção de citocinas e na elevação dos níveis endógenos de adenosina. A hipernocicepção mecânica foi avaliada por método eletrônico de pressão crescente em patas de camundongos, o ensaio cinéticocolorimétrico de mieloperoxidase (MPO) foi utilizado na avaliação da migração leucocitária para o tecido subcutâneo plantar, e a performance motora dos animais foi determinada pelo teste rota-rod. A quantificação de citocinas foi realizada por ELISA, enquanto a quantificação de adenosina foi obtida em cromatografia líquida de alta eficiência (CLAE) após validação laboratorial da metodologia segundo normas protocoladas pela ANVISA. O pré-tratamento de camundongos com F1,6BP reduziu a hipernocicepção induzida por injeção intraplantar de carragenina (45%), TNF-a (40%), IL-1B (46%), KC (33%), prostaglandina E2 (41%) e dopamina (55%). O tratamento com F1,6BP, no entanto, não alterou a produção de citocinas induzidas por carragenina (TNF-a, IL-1B e KC) e nem interferiu com a migração leucocitária para a região desafiada. Por outro lado, o efeito antinociceptivo da F1,6BP foi prevenido por tratamento sistêmico e local com antagonista de receptores A1 de adenosina (DPCPX) e o tratamento com adenosina apresentou efeitos similares os do tratamento com F1,6BP, sugerindo que o efeito da F1,6BP é mediado pela ação periférica da adenosina sobre receptores A1. Endossando isso, foi constatado por meio de cromatografia líquida de alta eficiência um aumento na concentração plasmática de adenosina em camundongos após administração de F1,6BP. Por conseguinte, a via de ação da F1,6BP parece ser dependente da produção de adenosina, mas não da modulação na produção de citocinas hipernociceptivas. A adenosina atua, então, perifericamente em receptores A1 inibindo a hipernocicepção, e a evidência farmacológica de que ela medeia a atividade da F1,6BP através dessa ação sobre receptores A1 foi corroborada por resultados in vivo que demonstram aumento dos níveis de adenosina decorrente da administração de F1,6BP. Em vista disso, esses resultados insinuam um possível potencial terapêutico da F1,6BP em reduzir a dor inflamatória. / D-Fructose-1,6-bisphosphate (F1,6BP), a high-energy glycolytic pathway intermediate, is reported to have a many pharmacologically effect, but its underlying mechanism of action in inflammation is incompletely understood. In this study, the aim was to examine the function of F1,6BP on cytokines and adenosine. Then, the possible F1,6BP anti-inflammatory activities in different models of inflammation and its mechanism of action in mice were addressed focusing inhibition of cytokine production or adenosine production enhancement. Mechanical hypernociception (decrease in the nociceptive threshold of animals) was evaluated by the electronic pressure meter test in mice, the myeloperoxidase (MPO) kineticcolorimetric assay was used to evaluate the leukocyte migration to the subcutaneous plantar tissue of mice hind paw, and mice motor performance were evaluated on the rota-rod test. The cytokines levels were measured by ELISA. Adenosine levels were determined by high performance liquid chromatography and this experimental procedure was validated according to ANVISA for this purpose. The pretreatment of mice with F1,6BP reduced the hypernociception induced by intraplantar injection of carrageenan (45%), TNF (40%), IL-1 (46%), KC (33%), prostaglandin E2 (41%) and dopamine (55%). However, FBP treatment did not alter carrageenin-induced cytokines (TNF-, IL-1 and KC) production or the leukocyte migration to the subcutaneous plantar tissue of mice hind paw. On the other hand, the antinociceptive effect of F1,6BP was prevented by systemic and local treatment with an adenosine A1 receptor antagonist (DPCPX) and adenosine treatment presented similar effect of F1,6BP, suggesting that F1,6BP effect is mediated by peripheral adenosine acting on A1 receptors. In agreement, the present work determined by high performance liquid chromatography that D-Fructose-1,6-bisphosphate administration increased the adenosine endogenous levels in the mice plasma. Therefore, F1,6BP mechanism of action seems dependent on adenosine production but not on modulation of hypernociceptive cytokine production. In turn, adenosine acts peripherally on A1 receptor inhibiting hypernociception. The pharmacological evidence that adenosine through activation of adenosine A1 receptor mediates the antinociceptive action of F1,6BP was supported by the finding that in vivo administration of F1,6BP increases the levels of adenosine in the blood samples. Thus, suggesting the treatment with F1,6BP as a possible therapeutic approach to reduce inflammatory pain.