Germ line mutations of the gene encoding the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) cause a hereditary cancer syndrome known as hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC-associated tumors harbor biallelic FH inactivation that results in the accumulation of the TCA cycle metabolite fumarate. Although it is known that fumarate accumulation can alter cellular signaling, if and how fumarate confers a growth advantage remain unclear. Here we show that fumarate accumulation confers a chronic proliferative signal by disrupting cellular iron signaling. Specifically, fumarate covalently modifies cysteine residues on iron regulatory protein 2 (IRP2), rendering it unable to repress ferritin mRNA translation. Simultaneously, fumarate increases ferritin gene transcription by activating the NRF2 (nuclear factor [erythroid-derived 2]-like 2) transcription factor. In turn, increased ferritin protein levels promote the expression of the promitotic transcription factor FOXM1 (Forkhead box protein M1). Consistently, clinical HLRCC tissues showed increased expression levels of both FOXM1 and its proliferation-associated target genes. This finding demonstrates how FH inactivation can endow cells with a growth advantage.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19607号 / 医博第4114号 / 新制||医||1015(附属図書館) / 32643 / 京都大学大学院医学研究科医学専攻 / (主査)教授 前川 平, 教授 中畑 龍俊, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The iron oxides are a group of materials with geological, biological, and technological importance. A thermodynamic understanding of these materials is important because it provides information about their relative stabilities, chemical reactivity, and transformations. This study provides the heat capacity of a nanocrystalline magnetite (Fe3O4) sample, bulk hematite (α-Fe2O3), nanocrystalline hematite, akaganéite (β-FeOOH), and lepidocrocite (γ-FeOOH) at temperatures as low as 0.5 K. These measurements were fit to theoretical functions at temperatures lower than 15 K, and the respective thermophysical properties of these materials are discussed. Also the molar entropies of bulk hematite and hydrous nanocrystalline hematite as well as hydrous akaganéite are given. Finally, a ferritin protein powder was prepared for heat capacity measurements by reconstituting the iron core in the presence of an imidazole buffer. This method allowed the introduction of almost 3000 iron atoms into each protein. Heat capacity measurements of apoferritin and the reconstituted ferritin sample are anticipated in the near future with plans to compare the heat capacity of the mineral core to that of other nanocrystalline iron oxides and oxyhydroxides.

Background - The first 1000 days of life (from conception to two years of age) is a critical period of nutritional vulnerability, affecting lifelong health. Iron deficiency (ID) and iron deficiency anaemia (IDA) are considered major public health problems that adversely affect development and growth, impair immunity, and increase morbidity and mortality in infants. ID and IDA in sub-Saharan Africa can be attributed to poor dietary, socioeconomic and disease conditions. One of the major obstacles in determining the prevalence of ID, using serum ferritin (SF) as marker of iron status, is that it not only reflects the amount of iron that is stored in the body, but also functions as an acute phase reactant that is raised in the presence of infection or inflammation. Aim - We conducted a re-analysis of the International Research on Infant Supplementation (IRIS) study’s baseline data to determine a more accurate estimation of the ID prevalence in apparently healthy four to thirteen-month-old infants from rural KwaZulu-Natal while accounting for the effect of chronic and acute inflammation on SF. Study design and methods - A cross-sectional analysis was performed on the baseline data (192 infants) of the IRIS study that was conducted in 2000. Infants’ haemoglobin (Hb), SF, C-reactive protein (CRP) and alpha-1 glycoprotein (AGP) concentrations were interpreted to determine the prevalence of ID. Literature of the past four years served as a guide to compare the ID prevalence obtained from four methods that account for the influence of inflammation on SF concentrations, to a reference method that does not take inflammation into consideration, and to what was reported in the original IRIS study. Weight and recumbent length measurements were converted to z-scores to interpret subjects’ anthropometric nutritional status. Results - A high prevalence of inflammation (52.6%) was present, with 11.5% of the subjects being in the incubation, 17.2% in the early convalescent, and 24% in the late convalescent phase of inflammation. SF was significantly associated with both CRP (ß = 0.200; P = 0.005) and AGP (ß = 0.223; P = 0.002) when adjusting for gender and age. The IRIS study reported an ID prevalence of 18.3%, whereas the results of this study ranged from 17.2 to 52.1%. We derived an IDA prevalence that ranged from 12 to 24.5% according to the different methods. The prevalence of stunting [length-for-age Z-score <-2SD] was 12.5%; while 25.1% of infants were overweight/obese [weight-for-length z-score >2SD]. Conclusion - A double burden of malnutrition was evident from the high prevalence of both overweight and ID, together with inflammation. The disconcertingly large variance in ID prevalence observed between the different methods that were employed highlights that iron supplementation interventions to treat anaemia must be based upon accurate estimates of IDA prevalence, otherwise they pose an increased risk of adverse effects to susceptible, iron-replete, but anaemic infants. Given the detrimental consequences of ID, it is imperative that governments, health care providers and parents must act to prevent or treat ID and IDA among vulnerable infants. / MSc (Dietetics), North-West University, Potchefstroom Campus, 2014

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No description available.

Background - The first 1000 days of life (from conception to two years of age) is a critical period of nutritional vulnerability, affecting lifelong health. Iron deficiency (ID) and iron deficiency anaemia (IDA) are considered major public health problems that adversely affect development and growth, impair immunity, and increase morbidity and mortality in infants. ID and IDA in sub-Saharan Africa can be attributed to poor dietary, socioeconomic and disease conditions. One of the major obstacles in determining the prevalence of ID, using serum ferritin (SF) as marker of iron status, is that it not only reflects the amount of iron that is stored in the body, but also functions as an acute phase reactant that is raised in the presence of infection or inflammation. Aim - We conducted a re-analysis of the International Research on Infant Supplementation (IRIS) study’s baseline data to determine a more accurate estimation of the ID prevalence in apparently healthy four to thirteen-month-old infants from rural KwaZulu-Natal while accounting for the effect of chronic and acute inflammation on SF. Study design and methods - A cross-sectional analysis was performed on the baseline data (192 infants) of the IRIS study that was conducted in 2000. Infants’ haemoglobin (Hb), SF, C-reactive protein (CRP) and alpha-1 glycoprotein (AGP) concentrations were interpreted to determine the prevalence of ID. Literature of the past four years served as a guide to compare the ID prevalence obtained from four methods that account for the influence of inflammation on SF concentrations, to a reference method that does not take inflammation into consideration, and to what was reported in the original IRIS study. Weight and recumbent length measurements were converted to z-scores to interpret subjects’ anthropometric nutritional status. Results - A high prevalence of inflammation (52.6%) was present, with 11.5% of the subjects being in the incubation, 17.2% in the early convalescent, and 24% in the late convalescent phase of inflammation. SF was significantly associated with both CRP (ß = 0.200; P = 0.005) and AGP (ß = 0.223; P = 0.002) when adjusting for gender and age. The IRIS study reported an ID prevalence of 18.3%, whereas the results of this study ranged from 17.2 to 52.1%. We derived an IDA prevalence that ranged from 12 to 24.5% according to the different methods. The prevalence of stunting [length-for-age Z-score <-2SD] was 12.5%; while 25.1% of infants were overweight/obese [weight-for-length z-score >2SD]. Conclusion - A double burden of malnutrition was evident from the high prevalence of both overweight and ID, together with inflammation. The disconcertingly large variance in ID prevalence observed between the different methods that were employed highlights that iron supplementation interventions to treat anaemia must be based upon accurate estimates of IDA prevalence, otherwise they pose an increased risk of adverse effects to susceptible, iron-replete, but anaemic infants. Given the detrimental consequences of ID, it is imperative that governments, health care providers and parents must act to prevent or treat ID and IDA among vulnerable infants. / MSc (Dietetics), North-West University, Potchefstroom Campus, 2014

Female mosquitoes of the species, Aedes aegypti (yellow fever mosquito, Diptera), blood feed for oogenesis. Therefore, mosquitoes are exposed to high iron loads and possibly blood-borne pathogens. We are interested in studying iron metabolism in A. aegypti to find methods for controlling mosquito populations, and thereby reduce human exposure to these pathogens. First, we found that the expression of the Aedes ferritin light chain homologue (LCH) is up-regulated by blood feeding. Ferritin LCH and heavy chain homologue (HCH) genes are closely clustered together and both mRNA transcripts increase with iron and oxidative stress (H2O2 and hemin). Second, we show A. aegypti larval cells synthesize and secrete ferritin in response to iron. Cytoplasmic ferritin is maximal at low levels of iron, consists of a specific subunit composition and reflects cytoplasmic iron levels. Secreted ferritin increases in linear relationship to increasing iron dose and is composed of different subunits than cytoplasmic ferritin. HCH and LCH transcripts increase with increasing cytoplasmic iron suggesting transcriptional control of ferritin synthesis. We previously reported that the mosquito HCH mRNA has an iron responsive element (IRE), but LCH mRNA does not have a canonical IRE. We show that iron regulatory protein 1 (IRP1)/IRE binding activity declines in response to increasing cytoplasmic iron levels. These data would indicate that HCH synthesis is controlled at transcription and translation. Third, we report that A. aegypti larval cell cytoplasmic iron concentration does not change temporally with iron treatment. However, membrane iron levels increase with iron over time. Iron temporally up-regulates both HCH and LCH mRNA. Ferritin secretion increases with time in response to iron and reflects that most of the intracellular ferritin is found in the membrane fraction. Membrane ferritin has the same subunit composition as cytoplasmic ferritin. Finally, membrane ferritin is found in both non-iron and iron-treated cells. This suggests a mechanism to store iron from a blood meal in membrane ferritin. These results indicate Aedes ferritin could act as an antioxidant and holoferritin secretion is likely the mechanism whereby mosquito cells protect against iron overload and, thus reduce the intracellular potential for iron-mediated oxidative stress during blood feeding.

De part ses propriétés physico-chimiques, le fer est un cofacteur de choix pour de nombreuses enzymes, impliquées dans de multiples processus biologiques, comme la photosynthèse ou la respiration. Cependant, sa capacité à gagner/perdre des électrons le rend très réactif, et potentiellement toxique. Son homéostasie doit donc être finement régulée. Chez A. thaliana, le gène de ferritine AtFER1 est régulé transcriptionnellement par le fer, et son expression est régulée par le rythme circadien. Au début de ce travail, aucun facteur de transcription impliqué dans cette régulation n'était identifié. Des cribles simple hybride en levure ont été réalisés, permettant l'identification de deux facteurs de transcription régulant le gène AtFER1: AtPHR1 et AtPIF7.PHR1 (Phosphate starvation Response1) et son homologue PHL1 (PHr1 Like 1) sont des facteurs de transcription impliqués dans la réponse à la carence en phosphate. Ils régulent directement le gène AtFER1, en se fixant sur l'élément 2 du promoteur d'AtFER1. Cette régulation ne fait pas intervenir l'IDRS et est indépendante du statut en fer des plantes. Par ailleurs, l'homéostasie du fer est affectée dans le double mutant phr1 phl1. Ces résultats montrent l'existence d'un lien moléculaire direct entre les homéostasies du fer et du phosphate.PIF7 (Phytochrome Interacting Factor 7) est un facteur de transcription de type bHLH impliqué dans la régulation circadienne des gènes DREB. Il se fixe probablement sur la G-box présente dans l'élément 5 du promoteur d'AtFer1. Dans un mutant perte de fonction pour le gène PIF7, l'amplitude des oscillations de l'expression du gène AtFer1 en cycles jour/nuit est augmentée. Un résultat similaire est obtenu lorsque l'élément 5 est muté dans des lignées trasngéniques exprimant le gène rapporteur LUC sous le contrôle du promoteur d'AtFer1. Ces résultats montrent que PIF7 est un répresseur de l'expression d'AtFer1. / Due to its redox properties, iron is a major cofactor for numerous proteins involved in many biological processes such as photosynthesis or respiration. Nevertheless, its ability to easily gain or lose electrons makes it highly reactive with oxygen and potentially toxic. Iron homeostasis has to be tightly regulated. In A. thaliana, AtFER1 ferritin gene is regulated at the transcriptional level by iron, and its expression is regulated by the circadian clock. Before this work, no transcription factor involved AtFer1 regulation has been identified. A yeast one hybrid was performed and allowed us to identify PHR1 and PIF7 as transcription factors involved in AtFER1 regulation. PHR1 (Phosphate starvation Response1) and its homolog PHL1 (PHr1 Like 1) are transcription factors involved in phosphate starvation response. They directly regulate AtFER1 expression and bind to the element 2 present in AtFER1 promoter. This regulation does not involve the IDRS sequence and is independent of iron status of plants. Moreover, iron homeostasis is affected in phr1phl1 double mutant. These results highlight a direct molecular link between iron and phosphate homeostasis.PIF7 (Phytochrome Interacting Factor 7) is a bHLH transcription factor involved in the circadian regulation of DREB genes. PIF7 probably interacts with the G-box found in element 5 in AtFER1 promoter region. In a pif7 knock-out mutant, amplitude of AtFer1 oscillations during light dark cycles are increased. Such a result was also obtained with transgenic lines expressing LUC reporter gene under the control of AtFER1 promoter region harboring a mutation in element 5. These results show that PIF7 is a repressor of AtFer1 expression.

Iron plays a potential role in oxidative stress-mediated injuries and pathologies e.g. rheumatoid arthritis (RA). Four decades ago it was suggested that iron may have a crucial role in the progression of inflammation in RA. Indeed, free radicals generated by iron can cause damage to lipids, proteins, carbohydrates, and DNA. It is this destructive process that is believed to occur in rheumatoid joints. However, none had differentiated between the role of iron in both acute and chronic phases of the disease and the origin of this 'labile' iron. Since RA cells are chronically exposed to oxidative stress, we have therefore chosen Jurkat cells to be our cell model. We used the parental (J16) cell line was used to mimic the acute phase of oxidative stress and the H2O2-resistant (HJ16) cells to mimic the chronic phase. By using hydrogen peroxide (H2O2) as the oxidising agent, we aim to study the role of iron in acute and chronic phase of oxidative stress and to know its origin. In the present study, we found that both antioxidants and H2O2-induced labile iron are modulated when cells are chronically exposed to H2O2. HJ16 cells contain higher total intracellular glutathione levels and glutathione peroxidase activity than J16 cells while the superoxide dismutase and catalase activity are similar. Haem oxygenase-1 (HO-1) was not detectable nor was it induced in these cell lines; HO-2 on the other hand was expressed but not induced. Although they had the same ‘basal’ LIP and L-Ft levels, J16 cells contain more than 7-fold higher H-Ft levels than in HJ16 cells. It was also found that H2O2-induced labile iron is directly correlated with necrotic cell death. These results are consistent with the conclusion that both antioxidant defence mechanism and labile iron status are modulated in cells chronically exposed to H2O2. We have also shown that the ‘basal’ and ‘H2O2-induced’ NFκB activation was higher in the HJ16 cells. We have also provided a link between labile iron release, lysosomal membrane damage and the ensuing necrotic cell death following H2O2 treatment.