No / Glutathione depletion is a consistent feature of the progression of mild to severe acute pancreatitis. In this study, we examined the temporal relationship between cysteine, homocysteine, and cysteinyl-glycine levels; total reduced erythrocyte glutathione; gamma-glutamyl transpeptidase activity; and disease severity. Initially, cysteine concentration was low, at levels similar to those of healthy controls. However, glutathione was reduced whilst cysteinyl glycine and gamma-glutamyl transpeptidase activity were increased in both mild and severe attacks. As the disease progressed, glutathione and cysteinyl glycine were further increased in mild attacks and cysteine levels correlated with homocysteine (r = 0.8, P < 0.001) and gamma-glutamyl transpeptidase activity (r = 0.75, P < 0.001). The progress of severe attacks was associated with glutathione depletion, reduced gamma-glutamyl transpeptidase activity, and increased cysteinyl glycine that correlated with glutathione depletion (r = 0.99, P = 0.01). These results show that glutathione depletion associated with severe acute pancreatitis occurs despite an adequate cysteine supply and could be attributed to heightened oxidative stress coupled to impaired downstream biosynthesis.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第25333号 / 農博第2599号 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 矢﨑 一史, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Except for tyrosine, the amino acid requirement of parenterally fed (PN) human neonates has not been derived. Methionine and cysteine are indispensable and dispensable sulphur amino acids respectively. Cysteine is synthesized from methionine. Cysteine is unstable in solution, and is left out or added in very small amounts to amino acid solutions. Methionine is added to compensate for the lack of cysteine, assuming that the neonate will convert methionine to cysteine to meet the body’s metabolic demand. Methionine is hepatotoxic and there is evidence that the neonate has limited ability for its conversion to cysteine. To determine the requirement of the neonate for methionine, PN-fed, stable, post-surgical neonates received graded intakes of methionine. The mean methionine requirement was estimated to be 49 mg.kg-1.day-1, which is 48 to 90% of the methionine content of current commercial amino acid solutions. Because cysteine is the rate limiting substrate for glutathione (GSH) synthesis and current methods of determining amino acid requirement measure requirement for protein synthesis, SAA requirements for maintenance of GSH status was deleniated in healthy adult males and in PN-fed human neonates. GSH kinetics was measured in healthy men receiving the mean methionine requirement and graded intakes of cysteine. GSH synthesis did not change with the addition of cysteine. Additionally, PN-fed post-surgical neonates recieved a methionine-adequate cysteine-free PN followed by cysteine supplemented PN for two 3-day periods and GSH kinetics measured on days 3 and 6. There was no change in GSH synthesis in response to cysteine supplementation. It is concluded that the PN-fed human neonate is capable of synthesizing enough cysteine from methionine not only for protein synthesis but for GSH synthesis. For both healthy men and stable post-surgical neonates, the requirement for GSH synthesis is met at the sulphur amino acid requirement derived using the indicator amino acid technique

Except for tyrosine, the amino acid requirement of parenterally fed (PN) human neonates has not been derived. Methionine and cysteine are indispensable and dispensable sulphur amino acids respectively. Cysteine is synthesized from methionine. Cysteine is unstable in solution, and is left out or added in very small amounts to amino acid solutions. Methionine is added to compensate for the lack of cysteine, assuming that the neonate will convert methionine to cysteine to meet the body’s metabolic demand. Methionine is hepatotoxic and there is evidence that the neonate has limited ability for its conversion to cysteine. To determine the requirement of the neonate for methionine, PN-fed, stable, post-surgical neonates received graded intakes of methionine. The mean methionine requirement was estimated to be 49 mg.kg-1.day-1, which is 48 to 90% of the methionine content of current commercial amino acid solutions. Because cysteine is the rate limiting substrate for glutathione (GSH) synthesis and current methods of determining amino acid requirement measure requirement for protein synthesis, SAA requirements for maintenance of GSH status was deleniated in healthy adult males and in PN-fed human neonates. GSH kinetics was measured in healthy men receiving the mean methionine requirement and graded intakes of cysteine. GSH synthesis did not change with the addition of cysteine. Additionally, PN-fed post-surgical neonates recieved a methionine-adequate cysteine-free PN followed by cysteine supplemented PN for two 3-day periods and GSH kinetics measured on days 3 and 6. There was no change in GSH synthesis in response to cysteine supplementation. It is concluded that the PN-fed human neonate is capable of synthesizing enough cysteine from methionine not only for protein synthesis but for GSH synthesis. For both healthy men and stable post-surgical neonates, the requirement for GSH synthesis is met at the sulphur amino acid requirement derived using the indicator amino acid technique

La ligation chimique implique la liaison des molécules de manière covalente pour former un nouveau complexe ayant les propriétés combinées de ses composants individuels. Ainsi, les composés naturels ou synthétiques avec leurs activités individuelles peuvent être conjuguer pour créer des substances possédant des caractéristiques uniques. Un domaine d' intérêt particulier à ces procédures est le marquage de protéines. Afin de simplifier et d'accélérer la découverte de nouvelles réactions de ligation bioselectives, nous avons conçu un système de screening rapide pour attribuer de la sélectivité et de la réactivité d'un groupement fonctionnel vers une série de dérivés d'acides aminés traçable. Une fonction chimique à propriétés prometteuse – 3-arylpropiolonitrile (APN) – a été identifiée. Les études comparatives ont démontré que cette technique offrait une meilleure sélectivité et stabilité par rapport à la technologie classique basée sur l’utilisation du groupement maléimide. L’utilisation de l’APN permet d’obtenir des bioconjugués propres et résistants à la décomposition, ce qui est d’une importance cruciale pour les applications médicales. Étude structure-réactivité nous a permis d'optimiser ses propriétés et de préparer une série de sondes fonctionnelles, dont un a été utilisé pour tester la sélectivité d'APN sur les mélanges modèles de peptides. De plus, les APN ont été trouvés à posséder une sélectivité élevée vers sélénocystéine: un acide aminé rare mais très important présent dans de nombreux enzymes actives. Une série des APN a été testée pour son activité inhibitrice envers une enzyme contenant sélénocystéine – thiorédoxine réductase – et s'est révélé posséder des activités élevées Enfin, une approche combinatoire de type split and mix a été développée visant à identifier des séquences peptidiques possédant la réactivité élevée avec les réactifs biosélectifs déjà connus. / Chemical ligation involves the linking of molecules in covalent manner to form a novel complex having the combined properties of its individual components. Thus, natural or synthetic compounds with their individual activities can be chemically combined to create unique substances possessing carefully engineered characteristics. A field of especial interest in such ligation procedures is protein labeling.To accelerate the discovery of new bioselective ligation reactions, we designed a screening system for fast assigning of the selectivity and reactivity of a given functional group owards series of UVGtraceable amino acid derivatives. As a result of our screening a promising cysteineGselective scaffold–3Garylpropiolonitrile (APN)–was identified. Its remarkable selectivity, high reactivity and of both starting and addition products in aqueous and organic media represents an important advantage compared to methodologies classically used for cysteine tagging. StructureGreactivity study allowed us to optimise its properties and toprepare a series of funcional probes, one of which was used for!an accurate test of APN selectivity on model mixtures of peptides. Furthermore, APN were found to possess an elevated selectivity towards selenocysteine:ararebut very important amino!acid found in many active enzymes.A series of APN was tested for their inhibitory activity towards one of such selenocysteineGcontaining enzyme–thioredoxine reductase–and was found to possess promising activities, which however still must be!optimised.Lastly, a screening system devoted to the discovery of reagents reactivity towards a sequence of amino acid residue was elaborated and allowed us to determine presumable discrepancy in reactivity of APN depending on the amino acid residue neighbouring the cysteine moiety. Such difference in reactivity may represent an important advantage for bioconjugation, and is currently under further investigation.

Metal contamination is a considerable environmental problem because metals are persistent contaminants. Ion exchange is one of the most commonly used treatment options for trace metal removal. This research develops and evaluates a redox active modified ion exchange system that has the potential to reduce the ionic strength of ion exchange regeneration streams. Poly-L-cysteine (PLC) was selected as the redox active, adsorbing functional group on the surface of a reticulated vitreous carbon (RVC) electrode. PLC is an excellent soft acid metal chelator and is unique in that its thiol groups can form disulfide bonds with each other. The reduction of available thiols changes the metal binding capacity of the peptide since the thiol is the primary binding group. RVC provides a macroporous conductive monolithic resin to support the peptide. An experimental apparatus was designed to study the properties of this system and estimate performance. Distinct oxidized and reduced states of PLC on the surface of the RVC were confirmed by changes in metal binding characteristics. Adsorption edges showed a sharper pH dependence for the reduced electrode compared to the oxidized electrode from pH 3-7. Adsorption isotherms performed at pH 7 showed increased capacity for the reduced electrode. The change was reversible by chemical and electrical reduction. This difference was confirmed at the molecular level with Cd- EXAFS of oxidized and reduced electrodes. A greater degree of cadmium-sulfur coordination was observed on the reduced electrode and a greater cadmium-oxygen coordination was apparant on an oxidized electrode. A multidentate adsorption model was developed to model the pH dependent behavior of cadmium adsorption on the PLC-RVC surface. Nickel adsorption showed increased adsorption in the oxidized state. The most likely explanation is increased carboxylate complexation. The electronically switchable ion exchange system (ESIE) provides a framework for modifying traditional ion exchange processes. The system has 5 to 10 times less specifc capacity than current ion exchange systems, but uses solutions 10-100 times lower in ionic strength for regeneration. Further studies on the effect of ionic strength on adsorption and current usage are necessary to compare the cost of the ESIE process to traditional ion exchange. / text

Introduction. While opioid medications are commonly prescribed for management of neuropathic pain (NP), long-term use has been associated with increased risk for overdose, drug interactions and addiction. New strategies are necessary to better manage chronic pain, thereby reducing need for opioid medications and their associated adverse consequences. N-acetyl-L-cysteine (NAC), an over-the-counter supplement, has shown promise in the treatment of psychiatric and addictive disorders. In addition, NAC has shown promise for reducing physiological signs of NP in laboratory rat models, prompting this study. Purpose. The present study was an open-label clinical trial of NAC as an adjuvant to opioid treatment for poorly controlled, chronic NP. It examined whether 1200 mg NAC twice daily for 4 weeks was associated with: lower ratings of patient-reported pain; reductions in PRN opioid medication for breakthrough pain; and improvements in physical and mental health quality of life (QoL). The study also examined whether appraisal of pain impacts response to medication. Method. Participants were N=28 chronic NP patients who consented to study participation. This consisted of 2 baseline assessments, 4 weeks of NAC and 1 post-trial follow-up visit. The majority (N=17) dropped out or were excluded during baseline. Of the remaining participants, N = 11 started the study medication and N=10 completed the study, with daily recordings of pain severity ratings and use of PRN opioid medication. Small sample size limited analyses to qualitative case reviews and effect sizes. Results. Over 90% of participants receiving NAC completed the study. Case review found varied results. While 4 of 10 participants showed decrease in average pain ratings during NAC, estimated effect sizes for the whole sample were small, bordering on negligible (ω² from .003 to .027) as were those for PRN opioids (Partial Eta-Squared=.0003). Effect size for mental health QoL was medium (Cohen's d=.421). Conclusions. With N=10, findings must be interpreted with caution. Nonetheless, the study found some albeit small evidence supporting NAC for improving mental health QoL and pain ratings. Several participants reported improvements in pain and mental health domains while taking NAC. NAC was well tolerated with minimal side effects. Lessons from this study will inform design and implementation of future NAC studies.

TRPA1 is a thermosensitive ion channel from the family of TRP (transient receptor potential) receptors. In primary sensory neurons, TRPA1 is an important transducer of painful stimuli, where it contributes to detection of noxious, irritant and inflammatory compounds of endogenous and exogenous origin. The major activation mode of TRPA1 is covalent modification of N-terminal cysteines or lysines by electrophilic compounds. The potency of the electrophilic agonists is increased by voltage dependency of the TRPA1 channel, which contributes substantially during membrane depolarization. To date, the role of several cysteine residues in the N- terminus has been demonstrated. However, the functional role of six cysteines in the transmembrane domain is still unknown. The first part of the thesis focuses on the functional role of the transmembrane cysteines in the activation of human TRPA1 channel. Our results indicate that these sites do not mediate reactive-electrophile-induced activation but four of the six cysteines substantially contribute to voltage-dependent gating of the channel and two participate in calcium-dependent modulation of TRPA1. In the second part of this thesis we aim to explore the proximity of two specific charged residues, located in the linker between the fourth and the fifth...

A radioterapia é um dos principais tratamentos para pacientes com câncer de cabeça e pescoço e a cárie relacionada à radioterapia é um de seus efeitos colaterais, apresentando-se com alta taxa de ocorrência. Além disso, falhas precoces em restaurações realizadas em dentes de pacientes irradiados em cabeça e pescoço também são observadas. Como a degradação enzimática do colágeno ocorre principalmente através da atividade das metaloproteinases de matriz e das cisteínacatepsinas, o objetivo deste estudo foi avaliar a atividade enzimática da dentina hígida e restaurada de dentes submetidos à radioterapia in vivo e in vitro. Os dentes irradiados in vivo foram extraídos de pacientes submetidos à radioterapia com uma dose cumulativa que variou de 40 a 70 Gy. As extrações foram feitas de 3 a 12 meses após a RT devido a doenças periodontais. Para os dentes irradiados in vitro, as amostras foram submersas em água destilada com uma irradiação total e única de 70 Gy. O estudo foi dividido em 2 fases independentes: Fase 1: Dentina Não-Restaurada (avaliação de amostras não irradiadas, dentes submetidos à radioterapia in vitro e in situ). Fase 2: Dentina Restaurada (avaliação de amostras não irradiadas e dentes submetidos à radioterapia in vitro) com 3 adesivos. Para o ensaio de zimografia (fase 1), os grupos irradiados in vitro, in vivo e não irradiados foram divididos em dois subgrupos: 1) mineralizado; 2) desmineralizado com ácido fosfórico10%. As proteínas dentinárias foram extraídas e submetidas à análise zimográfica de acordo com Mazzoni et al., 2007. Para a zimografia in situ (fase 2), os espécimes foram divididos em 6 grupos, de acordo com a forma de irradiação (não irradiada e irradiada in vitro) e o sistema adesivo testado (Adper Single Bond, 3M ESPE, ClearFil SE Bond, Kuraray ou Scotchbond Universal, 3M ESPE). Uma gelatina conjugada com fluoresceína autoextinguível foi usada como substrato para as proteases endógenas. A atividade enzimática gelatinolítica foi observada em microscópio confocal (Zeiss LSM 780-NLO, Carl Zeiss Microscopy GmbH). Para a análise da microscopia eletrônica de varredura, amostras restauradas e hígidas foram submetidas a técnica de pré-imunomarcação usando anticorpo monoclonal primário anti-CT-K e anti-CT-B, e anticorpo secundário conjugado com nano-partículas de ouro de 15nm. Um aumento na atividade gelatinolítica pós radioterapia para ambos os substratos (dentina restaurada e hígida) pôde ser observada. Houve uma maior expressão das formas ativas das MMP-2 e MMP-9 pós radioterapia para ambas as formas de radioterapia em dentina não restaurada. Nenhuma diferença na imuno-marcação para CT-K e CT-B entre os grupos irradiados e não irradiados foi observada. Adesivos autocondicionantes apresentaram uma imuno-marcação mais fraca para CT-K quando comparado ao adesivo de condicionamento total. Com isso, pode-se concluir que a radiação ionizante foi capaz de influenciar a atividade enzimática das proteínas endógenas da dentina restaurada e não restaurada. Palavras-chave: Radioterapia, metaloproteinases de matriz, MMP, cisteinocatepsinas, CT, Cárie relacionada à radiação. / Radiotherapy is one of the main treatments for head and neck cancer patients. Radiation-related caries and early restorations failures are side-effects with high rate of recurrence. As enzymatic degradation of collagen occurs mainly through the activity of matrix metalloproteinases (MMPs) and cysteine-cathepsins (CTs), the objective of this study was to evaluate the influence of in vivo and in vitro radiotherapy on endogenous proteases of the restored and non-restored dentin. In vivo irradiated teeth were extracted from patients who underwent clinical radiation protocols with a cumulative dose of radiation that ranged from 40 to 70 Gy. Extractions were performed 3 to 12 months after radiotherapy conclusion due to periodontal reasons. For the in vitro irradiated teeth, samples were submerged in distilled water with a total and single irradiation dose of 70 Gy. For gelatin zymography assay, irradiated in vivo, in vitro and non-irradiated groups were divided in two subgroups: 1) mineralized or 2) demineralized with 10% phosphoric acid. Dentin proteins were extracted and submitted to zymographic analysis in accordance to Mazzoni et al., 2007. For in situ zymography, specimens were divided into 6 groups, according to its irradiation form (non-irradiated and irradiated in vitro) and the adhesive system tested (Adper Single Bond, 3M ESPE, ClearFil SE Bond, Kuraray or Scotchbond Universal, 3M ESPE) using a self-quenched fluorescein-conjugated gelatin as the endogenous proteases substrate. The endogenous gelatinolytic enzyme activity was assessed by confocal laser-scanning microscope (Zeiss LSM 780-NLO, Carl Zeiss Microscopy GmbH). For SEM analysis of the HL, restored specimens were submitted to a pre-embedding immunolabeling technique using primary monoclonal antibody anti-CT-K and anti-CTB and a secondary antibody conjugated with 15nm gold nanoparticles. Radiotherapy groups presented increased gelatinolytic activity on both restored and non-restored dentin. MMP-2 and MMP-9 active form presented higher expression on both irradiated groups for non-restored dentin. Labeling for CT-K and CT-B did not differ from irradiated to non-irradiated groups. SE adhesives presenter weaker labeling for CT-K when compared to the E&R adhesive. Herewith, ionizing radiation may be able to influence the enzymatic activity of the endogenous proteins of restored and unrestored dentin

Dans cette thèse, l’auto-assemblage en molécules colloïdales de virus en forme de filament, les bactériophages M13, est étudié. Comme première approche, l’affinité de la streptavidine pour la biotine ou un Strep-tag est utilisée et quantitativement comparée. Pour ce faire, des virus modifiés génétiquement, M13-AS, présentant des Strep-tag et des virus M13C7C chimiquement bioconjugués par de la biotine ont réagi via leur extrémité proximale avec des nanoparticules fonctionnalisées par de la streptavidine. Il en résulte la formation de molécules colloïdales en étoile, dont la valence ou nombre de virus par structure, peut être simplement contrôlée par l’excès molaire initial. Cependant, la stabilité de ces molécules colloïdales est limitée par la libération progressive et la dégradation de la streptavidine. Nous avons alors développé une seconde approche basée sur l’affinité soufre-métal, qui s’est avérée à la fois pratique expérimentalement et fiable. Grâce aux groupements disulfures présents sur les cystéines de la protéine P3, des nanoparticules métalliques peuvent se lier à l’extrémité des virus. Le caractère générique de cette méthode est vérifié en faisant varier la nature du métal des nanoparticules ainsi que la souche des virus, dont la sauvage. La valence des structures formées est déterminée en fonction de plusieurs paramètres, dont l’excès molaire initial, la taille des nanoparticules et la force ionique. Un modèle rendant compte des résultats expérimentaux a été élaboré, dont les principales variables sont la surface des nanoparticules et le diamètre effectif électrostatique des virus. Cette approche est étendue à la réalisation de diblocs colloïdaux hétéro bifonctionnels, utilisant les virus comme briques constitutives. Comme preuve de concept, des diblocs bicolores à base de virus sont obtenus par auto-assemblage et leur dynamique est étudiée à l’échelle du bloc élémentaire en microscopie optique de fluorescence. Ainsi, nous avons montré dans cette thèse la réalisation par auto-assemblage d’une nouvelle génération de molécules colloïdales, dont l’auto-organisation peut conduire à la formation de superstructures hiérarchiques hybrides de complexité croissante, potentiellement utiles en sciences des matériaux. / In this thesis, the self-assembly of rod-like viral particles, specifically the M13 bacteriophages, into colloidal molecules is studied. As the first method, the affinity of streptavidin to biotin or Strep-tag is used and quantitatively compared. In this case, both biologically engineered M13-AS displaying Strep-tags and chemically biotinylated M13C7C viruses have reacted with streptavidin activated nanoparticles via their functionalized proximal ends. This results in star-like colloidal molecules, whose valency – or number of viruses par structure – can be solely controlled by tuning the initial molar excess. However, the stability of these colloidal molecules is limited by streptavidin release and degradation. Thus, we develop the second method based on the sulfur—metal interactions, which is more convenient and reliable. Thanks to the exposed disulfide groups located at p3 proteins, metallic nanoparticles are able to bind to proximal ends of the M13 virus. The generic feature of this method is verified by using different metals and two virus strains including wt-M13. Afterwards, the control of the valency is explored by varying the initial molar excess, the nanoparticle size and the ionic strength. A quantitative model is built correspondingly, using the surface area of Au nanobead and the effective electrostatic diameter of the virus as variables, which accounts for the assembly of colloidal molecules with desired valencies. This method is further applied to assemble heterobifunctional diblocks by using filamentous viruses as building units. As a proof-of-concept experiment, bicolored diblocks are produced and tracked by each block simultaneously. Overall, we demonstrate the synthesis of a new generation of hybrid colloidal molecules, whose self-organization could serve as a promising means to create novel hierarchical biologic/inorganic superstructures that may find applications in materials science.