It has been suggested for many decades that the essential and ubiquitous enzyme fructose 1,6-bisphosphate aldolase (FBA) could be a good drug target against bacteria and fungi, since lower organisms possess a metal-dependent (Class II) FBA, as opposed to higher organisms which possess a Schiff-base forming, metal-independent (Class I) FBA. The purpose of this doctoral project was to purify and study the inhibition of Class II FBA from pathogenic organisms. The capacity of various thiol compounds, as well as various derivatives of the metal-chelating compound dipicolinic acid, to inhibit the purified Class II FBAs from Mycobacterium tuberculosis, Pseudomonas aeruginosa, Bacillus cereus, and Magnaporthe grisea, was compared. The genes were subcloned in the Escherichia coli vector pT7-7 and the enzymes purified to near homogeneity, and characterized using a coupled assay. A small fed-batch fermentor was used to express the enzymes in E. coli, and yields of up to 2 grams of purified protein per liter of bacterial culture were obtained. The commercially available compound 2,3-dimercaptopropane sulfonate was found to be the most effective inhibitor against the aldolase from M. tuberculosis, with a second order binding rate constant of 500 +/- 4 M-1 s-1, which is three times and twenty times higher than the constants obtained with dipicolinic acid and EDTA, respectively. In an attempt to detect the enzyme dynamics during catalysis or inhibition, tryptophan residues were used as reporter groups and introduced by site-directed mutagenesis into the catalytic mobile loops and near the active site of the aldolases from M. tuberculosis, P. aeruginosa and B. cereus. The kinetic characterization of the mutants is described; as well as the effect of substrate binding on the steady-state and time-resolved fluorescence signals. Finally, the possibility of using the recombinant Class II FBP aldolases for industrial chemical synthesis was explored by measuring the enzymatic stability in organic solvents, at high temperatures and at different pH conditions. Surprisingly, the commercial Class I enzyme from rabbit muscle was more stable than the metalloenzymes in most conditions tested. The results presented in this thesis will be useful for the future design of Class II FBP aldolase inhibitors.

It has been suggested for many decades that the essential and ubiquitous enzyme fructose 1,6-bisphosphate aldolase (FBA) could be a good drug target against bacteria and fungi, since lower organisms possess a metal-dependent (Class II) FBA, as opposed to higher organisms which possess a Schiff-base forming, metal-independent (Class I) FBA. The purpose of this doctoral project was to purify and study the inhibition of Class II FBA from pathogenic organisms. The capacity of various thiol compounds, as well as various derivatives of the metal-chelating compound dipicolinic acid, to inhibit the purified Class II FBAs from Mycobacterium tuberculosis, Pseudomonas aeruginosa, Bacillus cereus, and Magnaporthe grisea, was compared. The genes were subcloned in the Escherichia coli vector pT7-7 and the enzymes purified to near homogeneity, and characterized using a coupled assay. A small fed-batch fermentor was used to express the enzymes in E. coli, and yields of up to 2 grams of purified protein per liter of bacterial culture were obtained. The commercially available compound 2,3-dimercaptopropane sulfonate was found to be the most effective inhibitor against the aldolase from M. tuberculosis, with a second order binding rate constant of 500 +/- 4 M-1 s-1, which is three times and twenty times higher than the constants obtained with dipicolinic acid and EDTA, respectively. In an attempt to detect the enzyme dynamics during catalysis or inhibition, tryptophan residues were used as reporter groups and introduced by site-directed mutagenesis into the catalytic mobile loops and near the active site of the aldolases from M. tuberculosis, P. aeruginosa and B. cereus. The kinetic characterization of the mutants is described; as well as the effect of substrate binding on the steady-state and time-resolved fluorescence signals. Finally, the possibility of using the recombinant Class II FBP aldolases for industrial chemical synthesis was explored by measuring the enzymatic stability in organic solvents, at high temperatures and at different pH conditions. Surprisingly, the commercial Class I enzyme from rabbit muscle was more stable than the metalloenzymes in most conditions tested. The results presented in this thesis will be useful for the future design of Class II FBP aldolase inhibitors.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1996. / Includes bibliographical references (leaves 185-196). / Microfiche. / xii, 196 leaves, bound ill. (some col.) 29 cm

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves [120]-134).

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19188号 / 工博第4065号 / 新制||工||1627(附属図書館) / 32180 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 跡見 晴幸, 教授 森 泰生, 教授 濵地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

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.

O remodelamento ósseo é um processo metabólico, dentro do qual os osteoblastos e os osteoclastos, participam ativamente. Portanto, qualquer alteração neste equilíbrio, pode provocar uma modificação na densidade mineral do osso, situação observada em certas doenças osteolíticas como osteoporose, artrite reumatóide e periodontite. Nos últimos anos, há um crescente interesse em avaliar o papel da glicólise na proliferação, sobrevivência e diferenciação dos diferentes tipos celulares. Em particular, tem sido evidenciado o efeito regulador da frutose 1,6-bisfosfato (FBP), um intermediário da via glicolítica de alta energia. Considerando que ainda não existem dados na literatura que correlacionem a FBP com o funcionamento dos osteoclastos, este trabalho tem por finalidade avaliar seu papel na osteoclastogênese e reabsorção óssea in vitro. Para isso, pré-osteoclastos murinos derivados da medula óssea foram diferenciados em osteoclastos na presença de M-CSF, RANKL e duas concentrações da FBP (100 e 300 ?M). Os resultados obtidos amostram que a FBP inibe a diferenciação osteoclástica em uma relação dose-dependente, sem afetar a viabilidade celular. Observa-se também, que o tratamento com FBP diminui a expressão de genes marcadores como, Nfatc1, Trap e Catepsina K (p < 0.01) e das proteínas NFATc1 e catepsina K. Como também, promove uma redução na atividade reabsortiva dos osteoclastos depois de 96 h de cultura. O efeito inibidor da FBP não depende da atividade da piruvato quinase M2 (PKM2). Em conjunto, estes dados sugerem que a FBP é um metabolito regulador importante da osteoclastogênese, demonstrando ser um agente potencial para o tratamento de doenças osteolíticas. / Bone remodeling is a coordinated metabolic process, where the osteoblasts and osteoclasts participate actively. Therefore, any alteration in this balance may cause a change in the bone mineral density, a condition observed in certain bone loss-associated diseases such as osteoporosis, rheumatoid arthritis and periodontitis. Recently, there has been a growing interest in assessing the role of the glycolysis on the proliferation, survival, and differentiation of the different cell types. In particular, it has been demonstrated the protective effect of the Fructose 1,6-bisphosphate (FBP), a high-energy glycolytic intermediate. Considering that there is no evidence in the literature that associate FBP with the function of osteoclasts, this work aims to evaluate its role in osteoclastogenesis and bone resorption in vitro. To this end, murine bone marrow derived pre-osteoclasts were differentiated into osteoclasts in the presence of M-CSF, RANKL and two concentrations of FBP (100 and 300 ?M). The results showed that FBP inhibits the differentiation of osteoclasts in a dose dependent manner, without affecting the cell viability. It was also observed that the treatment with the FBP decreases the expression of marker genes such as Nfatc1, Trap and Cathepsin K (p < 0.01) and the NFATc1 and cathepsin K protein levels. As well, the treatment with FBP resulted in markedly fewer osteoclast activity after 96 h of culture. FBP osteoclast inhibitory effect does not involve Pyruvate Kinase M2 (PKM2) activity. Together, these data denote the important regulatory role of the FBP on osteoclastogenesis, proving to be a potential agent for the treatment of bone loss-associated diseases.

O remodelamento ósseo é um processo metabólico, dentro do qual os osteoblastos e os osteoclastos, participam ativamente. Portanto, qualquer alteração neste equilíbrio, pode provocar uma modificação na densidade mineral do osso, situação observada em certas doenças osteolíticas como osteoporose, artrite reumatóide e periodontite. Nos últimos anos, há um crescente interesse em avaliar o papel da glicólise na proliferação, sobrevivência e diferenciação dos diferentes tipos celulares. Em particular, tem sido evidenciado o efeito regulador da frutose 1,6-bisfosfato (FBP), um intermediário da via glicolítica de alta energia. Considerando que ainda não existem dados na literatura que correlacionem a FBP com o funcionamento dos osteoclastos, este trabalho tem por finalidade avaliar seu papel na osteoclastogênese e reabsorção óssea in vitro. Para isso, pré-osteoclastos murinos derivados da medula óssea foram diferenciados em osteoclastos na presença de M-CSF, RANKL e duas concentrações da FBP (100 e 300 ?M). Os resultados obtidos amostram que a FBP inibe a diferenciação osteoclástica em uma relação dose-dependente, sem afetar a viabilidade celular. Observa-se também, que o tratamento com FBP diminui a expressão de genes marcadores como, Nfatc1, Trap e Catepsina K (p < 0.01) e das proteínas NFATc1 e catepsina K. Como também, promove uma redução na atividade reabsortiva dos osteoclastos depois de 96 h de cultura. O efeito inibidor da FBP não depende da atividade da piruvato quinase M2 (PKM2). Em conjunto, estes dados sugerem que a FBP é um metabolito regulador importante da osteoclastogênese, demonstrando ser um agente potencial para o tratamento de doenças osteolíticas. / Bone remodeling is a coordinated metabolic process, where the osteoblasts and osteoclasts participate actively. Therefore, any alteration in this balance may cause a change in the bone mineral density, a condition observed in certain bone loss-associated diseases such as osteoporosis, rheumatoid arthritis and periodontitis. Recently, there has been a growing interest in assessing the role of the glycolysis on the proliferation, survival, and differentiation of the different cell types. In particular, it has been demonstrated the protective effect of the Fructose 1,6-bisphosphate (FBP), a high-energy glycolytic intermediate. Considering that there is no evidence in the literature that associate FBP with the function of osteoclasts, this work aims to evaluate its role in osteoclastogenesis and bone resorption in vitro. To this end, murine bone marrow derived pre-osteoclasts were differentiated into osteoclasts in the presence of M-CSF, RANKL and two concentrations of FBP (100 and 300 ?M). The results showed that FBP inhibits the differentiation of osteoclasts in a dose dependent manner, without affecting the cell viability. It was also observed that the treatment with the FBP decreases the expression of marker genes such as Nfatc1, Trap and Cathepsin K (p < 0.01) and the NFATc1 and cathepsin K protein levels. As well, the treatment with FBP resulted in markedly fewer osteoclast activity after 96 h of culture. FBP osteoclast inhibitory effect does not involve Pyruvate Kinase M2 (PKM2) activity. Together, these data denote the important regulatory role of the FBP on osteoclastogenesis, proving to be a potential agent for the treatment of bone loss-associated diseases.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2006. / Fructose 2,6-bisphosphate (Fru 2,6-P2) is an important regulatory molecule in plant carbohydrate metabolism. There were three main objectives in this study. Firstly, to determine whether the recombinant rat 6-phosphofructo 2-kinase (6PF2K, EC 2.7.1.105) and fructose 2,6-bisphosphatase (FBPase2, EC 3.1.3.11) enzymes, which catalyse the synthesis and degradation of Fru 2,6-P2 respectively, showed any catalytic activity as fusion proteins. Secondly, to alter the levels of Fru 2,6-P2 in sugarcane, an important agricultural crop due to its ability to store large quantities of sucrose, by expressing the recombinant genes. Thirdly, to investigate whether sugar metabolism in photosynthetic- (leaves) and non-photosynthetic tissue (internodes) were subsequently influenced. Activity tests performed on the bacterially expressed glutathione-S-transferase (GST) fusion 6PF2K and FBPase2 enzymes showed that they were catalytically active. In addition antibodies were raised against the bacterially expressed proteins. Methods for extracting and measuring Fru 2,6-P2 from sugarcane tissues had to be optimised because it is known that the extraction efficiencies of Fru 2,6-P2 could vary significantly between different plant species and also within tissues from the same species. A chloroform/methanol extraction method was established that provided Fru 2,6-P2 recoveries of 93% and 85% from sugarcane leaves and internodes respectively. Diurnal changes in the levels of Fru 2,6-P2, sucrose and starch were measured and the results suggested a role for Fru 2,6-P2 in photosynthetic sucrose metabolism and in the partitioning of carbon between sucrose and starch in sugarcane leaves. Transgenic sugarcane plants expressing either a recombinant rat FBPase2 (ODe lines) or 6PF2K (OCe lines) were generated. The ODe lines contained decreased leaf Fru 2,6-P2 levels but increased internodal Fru 2,6-P2 levels compared to the control plants. Higher leaf sucrose and reducing sugars (glucose and fructose) were measured in the transgenic plants than the control plants. The transgenic lines contained decreased internodal sucrose and increased reducing sugars compared to the control plants. Opposite trends were observed for Fru 2,6-P2 and sucrose when leaves, internodes 3+4 or internodes 7+8 of the different plant lines were compared. In contrast, no consistent trends between Fru 2,6-P2 and sucrose were evident in the OCe transgenic lines.

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.