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Nitrogen Transport, Transformation and Cycling through a Mountain lake, Bull Trout Lake, Idaho, USALockwood, Ryan Settle 01 May 2009 (has links)
The effects of a mountain lake on nitrogen dynamics in a sub-alpine watershed were examined via watershed monitoring, mesocosm experiments, microcosm experiments, and enzymatic assays during spring and summer of a single year. Our study addressed the questions: (1) How does hydrologic transport through the lake affect the net fluxes of dissolved nitrogen (N) species? (2) What are the net effects of the littoral zone biota on dissolved N fluxes? (3) What are the seston and benthic uptake rates of nitrate? (4) What is the magnitude of N retention in littoral zone sediments? (5) What role does microbial hydrolysis of amino-groups from organic matter play in the uptake of dissolved nitrogen, relative to rates of nitrate uptake? Our study found a net positive flux of total dissolved N and dissolved organic N (DON), and a net negative flux of nitrate through the lake. During snowmelt, when the majority of nutrients are transported in this watershed, DON was retained in the lake. Several experiments were run to more closely examine the mechanisms behind these observations. Experiments in 2.1 m3 mesocosms in June and July measured rates of DON flux from the littoral zone sediments into the water column that were similar to increments measured in the lake. 15N-nitrate mesocosm and microcosm tracer experiments quantified benthic and pelagic nitrate uptake and retention of that nitrate in the benthic sediments. Areal nitrate uptake was 65-times greater in the sediments than in the water column seston and the turnover rate (half life) of the newly input nitrate pool in the sediments was 33-64 days. Finally, the prevalence of DON relative to dissolved inorganic N (DIN) and high measured rates of enzymatic amino acid hydrolysis suggest the importance of DON as a source of N for this aquatic system.
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Charakterisierung der Eaf 1-Funktion für die Biogenese der Aminopeptidase 1 / Characterisation of the Eaf 1 function for the aminopeptidase 1 biogenesisBenkert, Tanja 03 July 2008 (has links)
No description available.
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Conception et synthèse d'inhibiteurs de l'Aminopeptidase membranaire N ([EC. 3.4.11.2], APN ou CD13) / Conception and synthesis of inhibitors of Aminopeptidase membranar N ([EC. 3.4.11.2], APN or CD13)Roux, Lionel 25 November 2010 (has links)
La lutte contre le cancer est l'un des défis majeurs du XXème siècle. Pour que les tumeurs puissent se développer dans l'organisme, elles ont besoin d'un apport en nutriment par le biais de vaisseaux sanguins pour se faire, elles vont avoir recours au processus angiogénique. Lors de ce processus, les cellules endothéliales qui tapissent la paroi des vaisseaux sanguins vont se multiplier et créer de nouveaux vaisseaux sanguins qui vont permettre la vascularisation des tumeurs. L'angiogenèse constitue donc aujourd'hui un axe de recherche pour la lutte contre la progression tumorale et donc contre le cancer. Lors de ce développement tumoral, une enzyme, l'aminopeptidase neutre APN est surexprimée sur les parois des cellules endothéliales. Différentes études ont été menées et montrent que l'inhibition de cette enzyme bloque la progression tumorale. Mon travail au sein de l'équipe du Pr Céline Tarnus consistait en la conception et la synthèse d'inhibiteurs de l'APN. Une relation structure activité de nos composés vis-à-vis de l'APN a tout d'abord été effectuée. Le développement de synthèse du composé le plus actif ont été faite, puis la synthèse d'inhibiteurs d'APN ayant pour objectif l'utilisation de la BNCT a été abordée. / The fight against the cancer is one of the most important struggles of this century. For the development of the tumors inside the body, they need to receive nutriments by the blood vessels and they use the angiogenic process. During this process, the endothelial cells being shown on the wall of the blood vessel will multiply and design new blood vessel, which will allow the tumor's vascularisation. Today, the angiogenesis is an axis of research for the fight against the cancer. During the tumoral development, the aminopeptidase N (APN) is overexpressed on the wall of endothelial cells. Various studies have shown that the inhibition of this enzyme stops the tumoral progression. My work in the Pr. Céline Tarnus Team consists in the conception and the synthesis of APN's inhibitors. In a first time, a structure activity relationship has been realized. Syntheses of a subnamolar compound have been developed, and then the synthesis of APN's inhibitors with the use of BNCT has been got onto.
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Conception, synthèse et évaluation biologique d’inhibiteurs de PfA-M17, potentielle cible antipaludéenne / Design, synthesis and evaluation of PfA-M17 inhibitors, a potential antimalarial targetChaillou, Bérénice 30 October 2015 (has links)
Le paludisme, maladie infectieuse due à des parasites du genre Plasmodium, reste mortel et préoccupant dans les régions intertropicales d’Afrique, d’Asie et d’Amérique. Les phénomènes de résistance des parasites aux différentes générations de traitements représentent un problème majeur, auxquels s’ajoutent le coût des traitements et donc l’accès aux soins pour les populations les plus défavorisées. Il est ainsi urgent de trouver de nouvelles cibles et de développer de nouveaux agents antipaludiques agissant via des mécanismes originaux. La cible étudiée lors de ces travaux de thèse est la leucyl aminopeptidase bimétallique de P. falciparum, notée PfA-M17. Elle a récemment émergé comme potentielle cible antipaludéenne et jouerait un rôle essentiel à la croissance et à la survie du parasite. Elle serait impliquée dans la dernière étape du catabolisme de l’hémoglobine lors du cycle érythrocytaire du parasite. Ces travaux se sont concentrés sur la conception, la synthèse et l’évaluation d’inhibiteurs sélectifs de PfA-M17. En s’inspirant de travaux antérieurs menés au laboratoire et d’expériences de modélisation moléculaire, des analogues benzocycloheptanes trisubstitués ont été conçus pour inhiber sélectivement PfA-M17. Une voie de synthèse diastéréosélective partant de l’acide D-isoascorbique a été mise au point et a permis d’accéder à des analogues cycloheptanes et cyclohexanes trisubstitués. Cette voie de synthèse a aussi été étudiée afin d’obtenir des analogues benzocycloheptanes trisubstitués. / Malaria is an infectious disease due to Plasmodium parasites, still causing numerous deaths in intertropical areas of Africa, America and Asia. Existing treatments face problems of high cost and resistance, hence the need to discover new targets and to develop new compounds. This work focused on a recent antimalarial target, a bimetallic leucyl aminopeptidase, named PfA-M17. Inhibition of this aminopeptidase shows it is essential for parasite survival and growth. PfA-M17 may be involved in the last step of hemoglobin digestion during the intraerythrocytic cycle of the parasite. This study concerned the design, the synthesis and the evaluation of selective PfA-M17 inhibitors. Trisubstituted benzocycloheptane analogs were designed as PfA-M17 inhibitors, based on previous works performed in our team and based on molecular modelling. A diastereoselective pathway was developed from D-isoascorbic acid and gave trisubstituted cycloheptanes and cyclohexanes analogs. This pathway was also studied in order to get the trisubstituted benzocycloheptane scaffolds.
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Methanethiol and Cheddar Cheese FlavorDias, Benjamin 01 May 1999 (has links)
The use of slower acid-producing starter bacteria for the production of lower fat Cheddar cheese has lead to milder flavor Cheddar cheeses that lack intense Cheddar notes. The metabolism of methionine leads to the production of methanethiol, which is one of the desirable Cheddar cheese flavor compounds. The influence of NaCl and reduced pH was determined for aminopeptidase, lipase/ esterase, and methanethiol-producing capability in selected lactic acid bacteria and brevibacteria in simulated cheese-like conditions. The activity of each enzyme decreased with NaCl addition and pH reduction to approximate a Cheddar cheese environment (5% NaCl and pH 5.2).
The mechanism for methanethiol production by the starter and adjunct bacteria was also investigated. Different enzyme systems were found to be responsible for methanethiol production in starter lactococci, lactobacilli, and brevibacteria. In the lactococci, enzymes that acted primarily on cystathionine were responsible for methanethiol production from methionine. Lactobacilli also contained cystathionine-degrading enzymes, but these enzymes have properties different from the lactococcal enzymes. Brevibacterium linensBL2 lacked cystathionine-degrading enzymes, but was capable of the direct conversion of methionine to methanethiol.
L-Methionine γ-lyase from B. linens BL2 was purified to homogeneity, and was found to catalyze the α, γ elimination of methionine resulting in the production of methanethiol, α-ketobutyrate, and ammonia. Characterization of the pure enzyme demonstrated that it is pyridoxal phosphate dependent, which is active at salt and pH conditions existing in ripening Cheddar cheese. The addition of either B. linens BL2 or L-methionine γ-lyase to aseptic cheese curd slurries increased methanethiol and total volatile sulfur compound production.
In an attempt to increase methanethiol production and Cheddar cheese flavor in reduced-fat Cheddar cheese, B. linens BL2 was added as a starter adjunct to 60% reduced-fat cheese. Sensory evaluation of the cheese indicated that B. linens BL2 improved the flavor of 60% reduced-fat Cheddar cheese. This suggests that the addition of B. linens BL2 is an alternative to the addition of lactic acid bacteria to improve Cheddar cheese flavor via the metabolism of methionine.
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Design and Synthesis of Angiotensin IV Peptidomimetics Targeting the Insulin-Regulated Aminopeptidase (IRAP)Andersson, Hanna January 2010 (has links)
Peptidomimetics derived from the bioactive hexapeptide angiotensin IV (Ang IV, Val1-Tyr2-Ile3-His4-Pro5-Phe6) have been designed and synthesized. These peptidomimetics are aimed at inhibiting the insulin-regulated amino peptidase (IRAP), also known as the AT4 receptor. This membrane-bound zinc-metallopeptidase is currently under investigation regarding its potential as a target for cognitive enhancers. The work presented herein was based on stepwise replacement of the amino acid residues in Ang IV by natural and unnatural amino acids, non-peptidic building blocks, and also on the introduction of conformational constraints. Initially, we focused on the introduction of secondary structure mimetics and backbone mimetics. The C-terminal tripeptide His-Pro-Phe was successfully replaced by a γ-turn mimetic scaffold, 2-(aminomethyl)phenylacetic acid (AMPA), which was coupled via an amide bond to the carboxyl terminus of Val-Tyr-Ile. Substitution of Val-Tyr-Ile, Val-Tyr, Tyr-Ile and Tyr, respectively, by 4-hydroxydiphenylmethane scaffolds comprising a 1,3,5-substituted benzene ring as a central moiety unfortunately rendered peptidomimetics that were less potent than Ang IV. The subsequent approach involved the introduction of conformational constraints into Val-Tyr-Ile-AMPA by replacing Val and Ile by amino acid residues appropriate for disulfide cyclization or ring-closing metathesis. Chemically diverse structures encompassing an N-terminal 13- or 14-membered macrocyclic tripeptide and a C-terminal non-peptidic moiety were developed. Tyr2 and AMPA were modified to acquire further knowledge about the structure-activity relationships and, in addition, to improve the metabolic stability and reduce the polarity. Several of the compounds displayed a high capacity to inhibit IRAP and exhibited Ki values in the low nanomolar range. Hence, the new compounds were more than ten times more potent than the parent peptide Ang IV. Enhanced selectivity over the closely related aminopeptidase N (AP-N) was achieved, as well as improved stability against proteolysis by metallopeptidases present in the assays. However, additional investigations are required to elucidate the bioactive conformation(s) of the relatively flexible N-terminal macrocycles. The compounds presented in this thesis have provided important information on structure-activity relationships regarding the interaction of Ang IV-related pseudopeptides and peptidomimetics with IRAP. The best compounds in the series constitute important starting points for further discovery of Ang IV peptidomimetics suitable as tools in the investigation of IRAP and other potential targets for Ang IV. The literature presents strong support for the hypothesis that drug-like IRAP inhibitors would serve as a new type of future cognitive enhancers with potential use in the treatment of cognitive disorders, e.g. Alzheimer’s disease.
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Peptidase N, A Major Aminopeptidase Belonging To The M1 Family : Biochemical And Functional ImplicationsAnujith Kumar, K V 12 1900 (has links)
Intracellular protein degradation is required for maintaining the cellular proteome and regulating cellular processes. This pathway involves proximal ATP-dependent proteases that unfold and translocate proteins targeted for degradation into catalytic chambers. The large peptides produced are further cleaved by ATP independent endopeptidases, aminopeptidases and carboxypeptidases to release free amino acids. Lon and Clp are the key ATP-dependent proteases in prokaryotes and 26S proteasomes in eukayotes. In general, enzymes involved in the distal processing of peptides are ATP-independent, display greater redundancy and their orthologs are present in most organisms. The aim of the present study was to generate biochemical and functional insights on the ATP-independent enzyme, Peptidase N (PepN), which belongs to the M1 family. Previous studies in our laboratory identified Escherichia Coli PepN, to harbor both amino and endopeptidase activitities. In addition, it is responsible for the cleavage of majority of aminopeptidase substrates in E. Coli and is known to be involved in Sodium salicylate(NaSal)-induced stress.
The present study consists of four parts.
First, intracellular proteolysis plays an important role for virulence in pathogens. Therefore, it becomes important to study the biochemical properties and roles of enzymes involved in protein degradation. In this direction, a study was initiated to characterize the biochemical properties of Peptidase N from Salmonella enterica serovar Typhimurium(S. typhimurium). To study the contribution of PepN to the overall cystosolic protein degradation in S.typhimurium, a targeted deletion in pepN was generated. Cystosolic lysates of S. typhimurium wild type(WT) and ΔpepN strains were examined for their ability to cleave a panel of aminopeptidase and endopeptidase substrates. The ΔpepN strain displayed greatly reduced cleavage of nine out of a total of thirteen exopeptidase substrates, demonstrating a significant contribution of PepN to cytosolic aminopeptidase activity. S. typhimurium PepN also cleaved the endopeptidase substrate Suc-LLVY-AMC, similar to E. Coli PepN. To understand the physiological role of PepN, WT and ΔpepN were subjected to different stress conditions. During nutritional downshift in combination with high temperature stress, the growth of ΔpepN was significantly reduced compared to WT. Importantly, the PepN overexpressing strains grew better than WT, demonstrating an enhanced ability to overcome this stress combination. The above study clearly underscores the importance of PepN, to play distinct roles during stress. The significance of this study lies in understanding the biochemical and functional properties of a M1 family member from a pathogenic organism.
Second, peptidases belonging to the M1 family are widely distributed with orthologs found across different kingdoms. The key amino acids in the catalytic domain are conserved in this family. However, amino acids present in the C-termini are variable and the three available crystal structures of M1 family members display distint differences in organization of this domain. To investigate the functional role of C-termini, progressive deletions were generated in PepN from E.Coli and Tricorn interacting factor F2 from Thermoplasma acidophilum(F2). Catalytic activity was partially reduced inPepN lacking four aa from C-terminus (PepNΔC4) whereas it is greatly reduced in F2 lacking ten amino acids from C-terminus(F2ΔC10) or eleven amino acids from PepN (PepNΔC11). To understand the mechanistic reasons involved, biochemical and biophysical studies were performed on purified WT and C-termini deleted proteins. Increased binding to 8-amino- 1- naphthalene sulphonic acid (ANS) was observed for all C-termini deleted proteins revealing greater numbers of surface exposed hydrophobic amino acids. Further, trypsin sensitivity studies demonstrated that mutant proteins were more sensitive compared to WT. Notably, expression of PepNΔC4, but not PepNΔC11, in E ColiΔpepN increased its ability to resist nutritional and high temperature stress, demonstrating a physiological role for the C-terminus. Together, these studies reveal involvement of distal amino acids in the C-termini of two distant M1 family members in repressing the exposure of apolar residues and enhancing enzyme function.
Third, the crystal structure of E. coliPepN displayed the presence of Zn2+. To study the role of metal cofactor, apo-PepN was isolated by chelating the holoenzyme with 1,10-phenanthroline. Among different metals tested, only Zn2+ rescued the greatly reduced catalytic activity of the apo-PepN. Further confirmatory studies were performed using pepN mutants in the conserved GXMEN and HEXXH motifs. No major structural differences were observed in purified mutants(E264A, H297A, and E298A) using circular dichroism (CD) and intrinsic fluorescence studies; however, they lacked catalytic activity. These studies clearly demonstrate that Zn2+ was essential for catalysis but not for the overall structural integrity of PepN. Estimation of the Zn2+ content by atomic absorption spectrometry demonstrated that the WT contained one molecule of zinc per molecule of enzyme. Similar results were obtained in purified proteins of E264A and E298A. residues involved in catalysis. However the Zn2+ amount was greatly reduced in H297A, which is involved in Zn2+ binding. Further, the in vivo role of metal cofactor and catalyis were studied during two established stress conditions. Over expression of the mutants, unlike WT, was unable to rescue the growth of ΔpepN during nutritional down shift and high temperature stress. These results demonstrate that E264, H297 and E298 were required for PepN function during nutritional downshift and high temperature stress.
However during NaSal-induced stress condition, overexpression of WT or mutants reduced growth of ΔpepN, demonstrating that PepN function was independent of catalytic activity or metal cofactor. Further studies identified the YL motif, which is conserved in all members of the M1 family, to play a role during NaSal-induced stress. Over expression of Y185F or L186Q did not modulate catalytic activity although growth reduction of ΔpepN in the presence of NaSal was compromised. To understand the mechanisms by which the YL motif plays a role during this condition, Y185F and L186Q mutant proteins were purified. In vitro, both mutant proteins were found to aggregate at a lower temperature and their catalytic activities were more sensitive to temperature, compared to WT. Steady state analysis of WT, Y185F and L186Q were performed to study the modulation of PepN amount during stress conditions. Steady state amounts of Y185F and L186Q mutant proteins were greatly decreased compared to WT, during NaSal-induced stress. Most likely, the lowered amounts of Y185F and L186Q mutant proteins contribute to growth advantage during NaSal-induced stress. Thus, the YL motif in E. Coli PepN reduces protein aggregation and enhances the structural integrity of PepN during selective stress conditions in vivo. In summary, this study clearly identifies metal cofactor and peptidase-dependent and –independent motifs to play distinct functional roles in PepN.
Fourth, the crystal structures of known M1 family members have shown that the catalytic domain and mechanism of action are similar. To identify novel residues that may modulate the catalytic activity of PepN, multiple sequence alignment of important M1 family members were performed. The alignment identified a subset of M1 family members, including PepN, containing an aspargine residue which is present two amino acids before glycine in the GAMEN motif. A closer investigation of thecrystal structure of PepN revealed an interaction between N259(Catalytic domain) with Q821 (C-terminal domain). To understand the functional role of this interaction, site-specific mutants were generated: N259D, Q821E and a double mutant, N259D & Q821E. Spectroscopic studies did not reveal any significant differences with respect to global structure or protein stability between purified WT and mutant enzymes. Also, binding to substrates by mutant enzymes was not affected as judged by Km values. However, the Kcat of PepN containing N259D or Q821E was enhanced with respect to both aminopeptidase and endopeptidase substrates. On the other hand, there was significant decrease in the catalytic activity of the double mutant. Modeling studies demonstrate that the N259-Q821 interaction is located in the vicinity of residues important for catalysis in PepN and specific alterations in this interaction may affect the compactness of the catalytic domain. In summary, this study provides a functional role for the N259-Q821 interaction in modulating the catalytic activity of PepN.
Mammalian orthologs of M1 family members play important roles in different physiological processes, e.g. angiogenesis, blood pressure, inflammation, MHC class I antigen presentation etc. PepN is a well characterized M1 family member of microbial origin. The present study on E. Coli PepN provides new knowledge on the roles of: a) distal C-terminal amino acids in repressing exposed hydrophobic amino acids; b) the conserved YL motif during NaSal-induced stress condition; c) the N259 and Q821 interaction in modulating enzymatic activity. The implications of these results on other members of the M1 family are discussed.
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Analysis of the Role of Cytosolic Aminopeptidases in the Generation of MHC-Class I Presented Peptides: a DissertationTowne, Charles Fenton 27 February 2006 (has links)
To detect viral infections and tumors, CD8 T lymphocytes monitor cells for the presence of antigenic peptides bound to MHC class I molecules. The majority of MHC class I-presented peptides are generated from the cleavage of cellular and viral proteins by the ubiquitin-proteasome pathway. Many of the oligopeptides produced by this process are too long to stably bind to MHC class I molecules and require further trimming for presentation. Cytosolic aminopeptidases such as leucine aminopeptidase (LAP), which is IFN-inducible, Bleomycin Hydrolase (BH), and puromycin-sensitive aminopeptidase (PSA) can trim precursor peptides to mature epitopes and have been thought to play an important role in antigen presentation. To examine the role of these aminopeptidases in generating MHC class I peptides in vivo, we generated mice deficient in LAP or PSA, as well as cell lines deficient in LAP, PSA, or BH. LAP mutant mice and cells are viable and grow normally, whereas PSA mutant mice are smaller than their wild-type and heterozygote littermates, are subfertile as adults, and are subviable as embryos.
The trimming of peptides in LAP-deficient cells is not reduced under basal conditions or after stimulation with IFN. Similarly, there is no reduction in presentation of peptides from precursor or full length antigen constructs or in the overall supply of peptides from cellular proteins to MHC class I molecules, even after stimulation with IFN. After viral infection, LAP-deficient mice generate normal CTL responses to seven epitopes from three different viruses. Similarly, PSA deficient mice and BH deficient mice generate normal CTL responses to viral epitopes. These data demonstrate that LAP, BH, and PSA are not essential enzymes for generating most MHC class I-presented peptides and reveal redundancy in the function of cellular aminopeptidases in most cell types.
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Perfusão do fígado de rato com triton x-100: remoção e caracterização de uma aminopeptidase cinino-conversora e de uma arilamidaseTermignoni, Carlos January 1980 (has links)
Resumo não disponível
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Perfusão do fígado de rato com triton x-100: remoção e caracterização de uma aminopeptidase cinino-conversora e de uma arilamidaseTermignoni, Carlos January 1980 (has links)
Resumo não disponível
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