RESUMO
Murine sickle cell disease (SCD) results in damage to multiple organs, likely mediated first by vasculopathy. While the mechanisms inducing vascular damage remain to be determined, nitric oxide bioavailability and sterile inflammation are both considered to play major roles in vasculopathy. Here, we investigate the effects of high mobility group box-1 (HMGB1), a pro-inflammatory damage-associated molecular pattern (DAMP) molecule on endothelial-dependent vasodilation and lung morphometrics, a structural index of damage in sickle (SS) mice. SS mice were treated with either phosphate-buffered saline (PBS), hE-HMGB1-BP, an hE dual-domain peptide that binds and removes HMGB1 from the circulation via the liver, 1-[4-(aminocarbonyl)-2-methylphenyl]-5-[4-(1H-imidazol-1-yl)phenyl]-1H-pyrrole-2-propanoic acid (N6022) or N-acetyl-lysyltyrosylcysteine amide (KYC) for three weeks. Human umbilical vein endothelial cells (HUVEC) were treated with recombinant HMGB1 (r-HMGB1), which increases S-nitrosoglutathione reductase (GSNOR) expression by â¼80%, demonstrating a direct effect of HMGB1 to increase GSNOR. Treatment of SS mice with hE-HMGB1-BP reduced plasma HMGB1 in SS mice to control levels and reduced GSNOR expression in facialis arteries isolated from SS mice by â¼20%. These changes were associated with improved endothelial-dependent vasodilation. Treatment of SS mice with N6022 also improved vasodilation in SS mice suggesting that targeting GSNOR also improves vasodilation. SCD decreased protein nitrosothiols (SNOs) and radial alveolar counts (RAC) and increased GSNOR expression and mean linear intercepts (MLI) in lungs from SS mice. The marked changes in pulmonary morphometrics and GSNOR expression throughout the lung parenchyma in SS mice were improved by treating with either hE-HMGB1-BP or KYC. These data demonstrate that murine SCD induces vasculopathy and chronic lung disease by an HMGB1- and GSNOR-dependent mechanism and suggest that HMGB1 and GSNOR might be effective therapeutic targets for reducing vasculopathy and chronic lung disease in humans with SCD.
Assuntos
Anemia Falciforme , Benzamidas , Proteína HMGB1 , Pneumopatias , Lesão Pulmonar , Pirróis , Doenças Vasculares , Humanos , Animais , Camundongos , Lesão Pulmonar/etiologia , Proteína HMGB1/genética , Células Endoteliais/metabolismo , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/genética , Inflamação , Doenças Vasculares/etiologiaRESUMO
Oxidative stress, inflammation, and endoplasmic reticulum (ER) stress sequentially occur in bronchopulmonary dysplasia (BPD), and all result in DNA damage. When DNA damage becomes irreparable, tumor suppressors increase, followed by apoptosis or senescence. Although cellular senescence contributes to wound healing, its persistence inhibits growth. Therefore, we hypothesized that cellular senescence contributes to BPD progression. Human autopsy lungs were obtained. Sprague-Dawley rat pups exposed to 95% oxygen between Postnatal Day 1 (P1) and P10 were used as the BPD phenotype. N-acetyl-lysyltyrosylcysteine-amide (KYC), tauroursodeoxycholic acid (TUDCA), and Foxo4 dri were administered intraperitoneally to mitigate myeloperoxidase oxidant generation, ER stress, and cellular senescence, respectively. Lungs were examined by histology, transcriptomics, and immunoblotting. Cellular senescence increased in rat and human BPD lungs, as evidenced by increased oxidative DNA damage, tumor suppressors, GL-13 stain, and inflammatory cytokines with decreased cell proliferation and lamin B expression. Cellular senescence-related transcripts in BPD rat lungs were enriched at P10 and P21. Single-cell RNA sequencing showed increased cellular senescence in several cell types, including type 2 alveolar cells. In addition, Foxo4-p53 binding increased in BPD rat lungs. Daily TUDCA or KYC, administered intraperitoneally, effectively decreased cellular senescence, improved alveolar complexity, and partially maintained the numbers of type 2 alveolar cells. Foxo4 dri administered at P4, P6, P8, and P10 led to outcomes similar to TUDCA and KYC. Our data suggest that cellular senescence plays an essential role in BPD after initial inducement by hyperoxia. Reducing myeloperoxidase toxic oxidant production, ER stress, and attenuating cellular senescence are potential therapeutic strategies for halting BPD progression.
Assuntos
Displasia Broncopulmonar , Hiperóxia , Ácido Tauroquenodesoxicólico , Recém-Nascido , Animais , Ratos , Humanos , Displasia Broncopulmonar/patologia , Hiperóxia/metabolismo , Ratos Sprague-Dawley , Pulmão/patologia , Senescência Celular , Peroxidase/metabolismo , Oxidantes , Animais Recém-Nascidos , Modelos Animais de DoençasRESUMO
An excessive blood level of homocysteine (HcySH) is associated with numerous cardiovascular and neurodegenerative disease conditions. It has been suggested that direct S-homocysteinylation, of proteins by HcySH, or N-homosteinylation by homocysteine thiolactone (HTL) could play a causative role in these maladies. In contrast, ascorbic acid (AA) plays a significant role in oxidative stress prevention. AA is oxidized to dehydroascorbic acid (DHA) and if not rapidly reduced back to AA may degrade to reactive carbonyl products. In the present work, DHA is shown to react with HTL to produce a spiro bicyclic ring containing a six-membered thiazinane-carboxylic acid moiety. This reaction product is likely formed by initial imine condensation and subsequent hemiaminal product followed by HTL ring opening and intramolecular nucleophilic attack of the resulting thiol anion to form the spiro product. The reaction product was determined to have an accurate mass of 291.0414 and a molecular composition C10H13NO7S containing five double bond equivalents. We structurally characterized the reaction product using a combination of accurate mass tandem mass spectrometry, 1D and 2D-nuclear magnetic resonance. We also demonstrated that formation of the reaction product prevented peptide and protein N-homocysteinylation by HTL using a model peptide and α-lactalbumin. Furthermore, the reaction product is formed in Jurkat cells when exposed to HTL and DHA.
Assuntos
Ácido Desidroascórbico , Doenças Neurodegenerativas , Humanos , Peptídeos , HomocisteínaRESUMO
Myeloperoxidase (MPO), oxidative stress (OS), and endoplasmic reticulum (ER) stress are increased in the lungs of rat pups raised in hyperoxia, an established model of bronchopulmonary dysplasia (BPD). However, the relationship between OS, MPO, and ER stress has not been examined in hyperoxia rat pups. We treated Sprague-Dawley rat pups with tunicamycin or hyperoxia to determine this relationship. ER stress was detected using immunofluorescence, transcriptomic, proteomic, and electron microscopic analyses. Immunofluorescence observed increased ER stress in the lungs of hyperoxic rat BPD and human BPD. Proteomic and morphometric studies showed that tunicamycin directly increased ER stress of rat lungs and decreased lung complexity with a BPD phenotype. Previously, we showed that hyperoxia initiates a cycle of destruction that we hypothesized starts from increasing OS through MPO accumulation and then increases ER stress to cause BPD. To inhibit ER stress, we used tauroursodeoxycholic acid (TUDCA), a molecular chaperone. To break the cycle of destruction and reduce OS and MPO, we used N-acetyl-lysyltyrosylcysteine amide (KYC). The fact that TUDCA improved lung complexity in tunicamycin- and hyperoxia-treated rat pups supports the idea that ER stress plays a causal role in BPD. Additional support comes from data showing TUDCA decreased lung myeloid cells and MPO levels in the lungs of tunicamycin- and hyperoxia-treated rat pups. These data link OS and MPO to ER stress in the mechanisms mediating BPD. KYC's inhibition of ER stress in the tunicamycin-treated rat pup's lung provides additional support for the idea that MPO-induced ER stress plays a causal role in the BPD phenotype. ER stress appears to expand our proposed cycle of destruction. Our results suggest ER stress evolves from OS and MPO to increase neonatal lung injury and impair growth and development. The encouraging effect of TUDCA indicates that this compound has the potential for treating BPD.
Assuntos
Displasia Broncopulmonar , Hiperóxia , Pneumonia , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Humanos , Recém-Nascido , Pulmão , Proteômica , Ratos , Ratos Sprague-Dawley , TunicamicinaRESUMO
Bronchopulmonary dysplasia (BPD) is caused primarily by oxidative stress and inflammation. To induce BPD, neonatal rat pups were raised in hyperoxic (>90% O2) environments from day one (P1) until day ten (P10) and treated with N-acetyl-lysyltyrosylcysteine amide (KYC). In vivo studies showed that KYC improved lung complexity, reduced myeloperoxidase (MPO) positive (+) myeloid cell counts, MPO protein, chlorotyrosine formation, increased endothelial cell CD31 expression, decreased 8-OH-dG and Cox-1/Cox-2, HMGB1, RAGE, TLR4, increased weight gain and improved survival in hyperoxic pups. EPR studies confirmed that MPO reaction mixtures oxidized KYC to a KYC thiyl radical. Adding recombinant HMGB1 to the MPO reaction mixture containing KYC resulted in KYC thiylation of HMGB1. In rat lung microvascular endothelial cell (RLMVEC) cultures, KYC thiylation of RLMVEC proteins was increased the most in RLMVEC cultures treated with MPO + H2O2, followed by H2O2, and then KYC alone. KYC treatment of hyperoxic pups decreased total HMGB1 in lung lysates, increased KYC thiylation of HMGB1, terminal HMGB1 thiol oxidation, decreased HMGB1 association with TLR4 and RAGE, and shifted HMGB1 in lung lysates from a non-acetylated to a lysyl-acetylated isoform, suggesting that KYC reduced lung cell death and that recruited immune cells had become the primary source of HMGB1 released into the hyperoxic lungs. MPO-dependent and independent KYC-thiylation of Keap1 were both increased in RLMVEC cultures. Treating hyperoxic pups with KYC increased KYC thiylation and S-glutathionylation of Keap1, and Nrf2 activation. These data suggest that KYC is a novel system pharmacological agent that exploits MPO to inhibit toxic oxidant production and is oxidized into a thiyl radical that inactivates HMGB1, activates Nrf2, and increases antioxidant enzyme expression to improve lung complexity and reduce BPD in hyperoxic rat pups.
Assuntos
Displasia Broncopulmonar , Hiperóxia , Amidas , Animais , Animais Recém-Nascidos , Humanos , Peróxido de Hidrogênio , Recém-Nascido , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Pulmão/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , RatosRESUMO
OBJECTIVE: Diabetes mellitus is a significant risk factor for peripheral artery disease. Diabetes mellitus induces chronic states of oxidative stress and vascular inflammation that increase neutrophil activation and release of myeloperoxidase. The goal of this study is to determine whether inhibiting myeloperoxidase reduces oxidative stress and neutrophil infiltration, increases vascularization, and improves blood flow in a diabetic murine model of hindlimb ischaemia. METHODS: Leptin receptor-deficient (db/db) mice were subjected to hindlimb ischaemia. Ischaemic mice were treated with N-acetyl-lysyltyrosylcysteine-amide (KYC) to inhibit myeloperoxidase. After ligating the femoral artery, effects of treatments were determined with respect to hindlimb blood flow, neutrophil infiltration, oxidative damage, and the capability of hindlimb extracellular matrix to support human endothelial cell proliferation and migration. RESULTS: KYC treatment improved hindlimb blood flow at 7 and 14 days in db/db mice; decreased the formation of advanced glycation end products, 4-hydroxynonenal, and 3-chlorotyrosine; reduced neutrophil infiltration into the hindlimbs; and improved the ability of hindlimb extracellular matrix from db/db mice to support endothelial cell proliferation and migration. CONCLUSION: These results demonstrate that inhibiting myeloperoxidase reduces oxidative stress in ischaemic hindlimbs of db/db mice, which improves blood flow and reduces neutrophil infiltration such that hindlimb extracellular matrix from db/db mice supports endothelial cell proliferation and migration.
Assuntos
Indutores da Angiogênese/farmacologia , Diabetes Mellitus/metabolismo , Inibidores Enzimáticos/farmacologia , Isquemia/tratamento farmacológico , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Oligopeptídeos/farmacologia , Peroxidase/antagonistas & inibidores , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus/genética , Diabetes Mellitus/fisiopatologia , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Membro Posterior , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Isquemia/enzimologia , Isquemia/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Estresse Oxidativo/efeitos dos fármacos , Peroxidase/metabolismo , Receptores para Leptina/deficiência , Receptores para Leptina/genética , Fluxo Sanguíneo Regional , Transdução de SinaisRESUMO
RATIONALE: Oxidative stress is an imbalance between reactive free radical oxygen species and antioxidant defenses. Its consequences can lead to numerous pathologies. Regulating oxidative stress is the complex interplay between antioxidant recycling and thiol-containing regulatory proteins. Understanding these regulatory mechanisms is important for preventing onset of oxidative stress. The aim of this study was to investigae S-thiol protein chemistry associated with oxidized vitamin C (dehydroascorbate, DHA), homocysteine (HcySH) and glutathione (GSH) using mass spectrometry. METHODS: Glutaredoxin-1 (Grx-1) was incubated with DHA, with and without GSH and HcySH. Disulfide formation was followed by electrospray ionization mass spectrometry (ESI-MS) of intact proteins and by LC/ESI-MS/MS of peptides from protein tryptic digestions. The mechanism of DHA-mediated S-thiolation was investigated using two synthetic peptides: AcFHACAAK and AcFHACE. Three proteins, i.e. human hemoglobin (HHb), recombinant peroxiredoxin 2 (Prdx2) and Grx-1, were S-homocysteinylated followed by S-transthiolyation with GSH and investigated by ESI-MS and ESI-MS/MS. RESULTS: ESI-MS analysis reveals that DHA mediates disulfide formation and S-thiolation by HcySH as well as GSH of Grx-1. LC/ESI-MS/MS analysis allows identification of Grx-1 S-thiolated cysteine adducts. The mechanism by which DHA mediates S-thiolation of heptapeptide AcFHACAAK is shown to be via initial formation of a thiohemiketal adduct. In addition, ESI-MS of intact proteins shows that GSH can S-transthiolate S-homocysteinylated Grx-1_ HHb and Prdx2. The GS-S-protein adducts over time dominate the ESI-MS spectrum profile. CONCLUSIONS: Mass spectrometry is a unique analytical technique for probing complex reaction mechanisms associated with oxidative stress. Using model proteins, ESI-MS reveals the mechanism of DHA-facilitated S-thiolation, which consists of thiohemiketal formation, disulfide formation or S-thiolation. Furthermore, protein S-thiolation by HcySH can be reversed by reversible GSH thiol exchange. The use of mass spectrometry with in vitro models of protein S-thiolation in oxidative stress may provide significant insight into possible mechanisms of action occurring in vivo.
Assuntos
Ácido Desidroascórbico , Glutationa , Homocisteína , Espectrometria de Massas por Ionização por Electrospray/métodos , Compostos de Sulfidrila/análise , Ácido Desidroascórbico/análise , Ácido Desidroascórbico/química , Ácido Desidroascórbico/metabolismo , Glutationa/análise , Glutationa/química , Glutationa/metabolismo , Homocisteína/análise , Homocisteína/química , Homocisteína/metabolismo , Humanos , Estresse Oxidativo/fisiologia , Proteínas/análise , Proteínas/química , Proteínas/metabolismo , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Ascorbic acid (vitamin C) plays a significant role in the prevention of oxidative stress. In this process, ascorbate is oxidized to dehydroascorbate (DHA). We have investigated the impact of DHA on peptide/protein intramolecular disulfide formation as well as S-glutathionylation and S-homocysteinylation. S-glutathionylation of peptides/proteins is a reversible, potential regulatory mechanism in oxidative stress. Although the exact role of protein S-homocysteinylation is unknown, it has been proposed to be of importance in pathobiological processes such as onset of cardiovascular disease. Using an in vitro model system, we demonstrate that DHA causes disulfide bond formation within the active site of recombinant human glutaredoxin (Grx-1). DHA also facilities the formation of S-glutathionylation and S-homocysteinylation of a model peptide (AcFHACAAK) as well as Grx-1. We discuss the possible mechanisms of peptide/protein S-thiolation, which can occur either via thiol exchange or a thiohemiketal intermediate. A thiohemiketal DHA-peptide adduct was detected by mass spectrometry and its location on the peptide/protein cysteinyl thiol group was unambiguously confirmed by tandem mass spectrometry. This demonstrates that peptide/protein S-thiolation mediated by DHA is not limited to thiol exchange reactions but also takes place directly via the formation of a thiohemiketal peptide intermediate. Finally, we investigated a potential reducing role of glutathione (GSH) in the presence of S-homocysteinylated peptide/protein adducts. S-homocysteinylated AcFHACAAK, human hemoglobin α-chain and Grx-1 were incubated with GSH. Both peptide and proteins were reduced, and homocysteine replaced with GS-adducts by thiol exchange, as a function of time.
Assuntos
Ácido Desidroascórbico/química , Glutarredoxinas/química , Glutationa/química , Homocisteína/química , Peptídeos/química , Compostos de Sulfidrila/química , Antioxidantes/química , Domínio Catalítico , Cisteína/química , Dimerização , Dissulfetos/química , Hemoglobinas/química , Humanos , Oxirredução , Estresse OxidativoRESUMO
The eosinophilia-myalgia syndrome (EMS) outbreak that occurred in the USA and elsewhere in 1989 was caused by the ingestion of Showa Denko K.K. (SD) L-tryptophan (L-Trp). "Six compounds" detected in the L-Trp were reported as case-associated contaminants. Recently the final and most statistically significant contaminant, "Peak AAA" was structurally characterized. The "compound" was actually shown to be two structural isomers resulting from condensation reactions of L-Trp with fatty acids derived from the bacterial cell membrane. They were identified as the indole C-2 anteiso (AAA1-343) and linear (AAA2-343) aliphatic chain isomers. Based on those findings, we utilized a combination of on-line HPLC-electrospray ionization mass spectrometry (LC-MS), as well as both precursor and product ion tandem mass spectrometry (MS/MS) to facilitate identification of a homologous family of condensation products related to AAA1-343 and AAA2-343. We structurally characterized eight new AAA1-XXX/AAA2-XXX contaminants, where XXX represents the integer molecular ions of all the related homologs, differing by aliphatic chain length and isomer configuration. The contaminants were derived from the following fatty acids of the bacterial cell membrane, 5-methylheptanoic acid (anteiso-C8:0) for AAA1-315; n-octanoic acid (n-C8:0) for AAA2-315; 6-methyloctanoic acid (anteiso-C9:0) for AAA1-329; n-nonanoic acid (n-C9:0) for AAA2-329; 10-methyldodecanoic acid (anteiso-C13:0) for AAA1-385; n-tridecanoic acid (n-C13:0) for AAA2-385; 11-methyltridecanoic acid (anteiso-C14:0) for AAA1-399; and n-tetradecanoic acid (n-C14:0) for AAA2-399. The concentration levels for these contaminants were estimated to be 0.1-7.9⯵g / 500â¯mg of an individual SD L-Trp tablet or capsule The structural similarity of these homologs to case-related contaminants of Spanish Toxic Oil Syndrome (TOS) is discussed.
Assuntos
Suplementos Nutricionais/análise , Síndrome de Eosinofilia-Mialgia/induzido quimicamente , Ácidos Graxos/toxicidade , Contaminação de Alimentos , Indóis/toxicidade , Triptofano/análogos & derivados , Bacillus amyloliquefaciens/metabolismo , Caprilatos/análise , Caprilatos/química , Caprilatos/isolamento & purificação , Caprilatos/toxicidade , Centers for Disease Control and Prevention, U.S. , Cromatografia Líquida de Alta Pressão , Suplementos Nutricionais/efeitos adversos , Ácidos Graxos/análise , Ácidos Graxos/química , Ácidos Graxos/isolamento & purificação , Fermentação , Ácidos Heptanoicos/análise , Ácidos Heptanoicos/química , Ácidos Heptanoicos/isolamento & purificação , Ácidos Heptanoicos/toxicidade , Humanos , Indóis/análise , Indóis/química , Indóis/isolamento & purificação , Ácidos Láuricos/análise , Ácidos Láuricos/química , Ácidos Láuricos/isolamento & purificação , Ácidos Láuricos/toxicidade , Metilação , Estrutura Molecular , Miristatos/análise , Miristatos/química , Miristatos/isolamento & purificação , Miristatos/toxicidade , Espectrometria de Massas por Ionização por Electrospray , Estereoisomerismo , Triptofano/análise , Triptofano/química , Triptofano/isolamento & purificação , Estados UnidosRESUMO
The current paradigm of modern healthcare is a reactive response to patient symptoms, subsequent diagnosis and corresponding treatment of the specific disease(s). This approach is predicated on methodologies first espoused by the Cnidean School of Medicine approximately 2500years ago. More recently escalating healthcare costs and relatively poor disease treatment outcomes have fermented a rethink in how we carry out medical practices. This has led to the emergence of "P-Medicine" in the form of Personalized and Precision Medicine. The terms are used interchangeably, but in fact there are significant differences in the way they are implemented. The former relies on an "N-of-1" model whereas the latter uses a "1-in-N" model. Personalized Medicine is still in a fledgling and evolutionary phase and there has been much debate over its current status and future prospects. A confounding factor has been the sudden development of Precision Medicine, which has currently captured the imagination of policymakers responsible for modern healthcare systems. There is some confusion over the terms Personalized versus Precision Medicine. Here we attempt to define the key differences and working definitions of each P-Medicine approach, as well as a taxonomic relationship tree. Finally, we discuss the impact of Personalized and Precision Medicine on the practice of Critical Care Medicine (CCM). Practitioners of CCM have been participating in Personalized Medicine unknowingly as it takes the protocols of sepsis, mechanical ventilation, and daily awakening trials and applies it to each individual patient. However, the immediate next step for CCM should be an active development of Precision Medicine. This developmental process should break down the silos of modern medicine and create a multidisciplinary approach between clinicians and basic/translational scientists.
Assuntos
Cuidados Críticos , Atenção à Saúde/normas , Medicina de Precisão , Cuidados Críticos/economia , Cuidados Críticos/tendências , Atenção à Saúde/economia , Custos de Cuidados de Saúde , Humanos , Medicina de Precisão/economia , Medicina de Precisão/tendências , Pesquisa Translacional BiomédicaRESUMO
The eosinophilia-myalgia syndrome (EMS) outbreak of 1989 that occurred in the USA and elsewhere was caused by the ingestion of l-Tryptophan (L-Trp) solely manufactured by the Japanese company Showa Denko K.K. (SD). Six compounds present in the SD L-Trp were reported to be case-associated contaminants. However, "one" of these compounds, Peak AAA has remained structurally uncharacterized, despite the fact that it was described as "the only statistically significant (p=0.0014) contaminant". Here, we employ on-line microcapillary-high performance liquid chromatography-electrospray ionization mass spectrometry (LC-MS), and tandem mass spectrometry (MS/MS) to determine that Peak AAA is in fact two structurally related isomers. Peak AAA1 and Peak AAA2 differed in LC retention times, and were determined by accurate mass-LC-MS to both have a protonated molecular ion (MH+) of mass 343.239Da (Da), corresponding to a molecular formula of C21H30N2O2, and possessing eight degrees of unsaturation (DoU) for the non-protonated molecule. By comparing the LC-MS and LC-MS-MS retention times and spectra with authentic synthetic standards, Peak AAA1 was identified as the intermolecular condensation product of L-Trp with anteiso 7-methylnonanoic acid, to afford (S)-2-amino-3-(2-((S,E)-7-methylnon-1-en-1-yl)-1H-indol-3-yl)propanoic acid. Peak AAA2 was determined to be a condensation product of L-Trp with decanoic acid, which produced (S)-2-amino-3-(2-((E)-dec-1-en-1-yl)-1H-indol-3-yl)propanoic acid.
Assuntos
Contaminação de Medicamentos , Síndrome de Eosinofilia-Mialgia/induzido quimicamente , Triptofano/análogos & derivados , Triptofano/química , Cromatografia Líquida de Alta Pressão , Suplementos Nutricionais , Humanos , Espectrometria de Massas , Espectrometria de Massas em Tandem , Triptofano/efeitos adversos , Triptofano/isolamento & purificaçãoRESUMO
Contraction of the actomyosin ring (AMR) provides the centripetal force that drives cytokinesis. In budding yeast (Saccharomyces cerevisiae), assembly and contraction of the AMR is coordinated with membrane deposition and septum formation at the bud neck. A central player in this process is Iqg1, which promotes recruitment of actin to the myosin ring and links AMR assembly with that of septum-forming components. We observed early actin recruitment in response to inhibition of cyclin-dependent kinase 1 (Cdk1) activity, and we find that the Cdk1-dependent phosphorylation state of Iqg1 is a determining factor in the timing of bud neck localization of both Iqg1 and actin, with both proteins accumulating prematurely in cells expressing nonphosphorylatable Iqg1 mutants. We also identified the primary septum regulator Hof1 as a binding partner of Iqg1, providing a regulatory link between the septation and contractile pathways that cooperate to complete cytokinesis.
Assuntos
Actomiosina/metabolismo , Proteína Quinase CDC28 de Saccharomyces cerevisiae/fisiologia , Citocinese , Saccharomyces cerevisiae/enzimologia , Proteínas Ativadoras de ras GTPase/metabolismo , Anáfase , Proteínas Associadas aos Microtúbulos/metabolismo , Cadeias Leves de Miosina/metabolismo , Fosforilação , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Transporte Proteico , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
We are all perplexed that current medical practice often appears maladroit in curing our individual illnesses or disease. However, as is often the case, a lack of understanding, tools and technologies are the root cause of such situations. Human individuality is an often-quoted term but, in the context of human biology, it is poorly understood. This is compounded when there is a need to consider the variability of human populations. In the case of the former, it is possible to quantify human complexity as determined by the 35,000 genes of the human genome, the 1-10 million proteins (including antibodies) and the 2000-3000 metabolites of the human metabolome. Human variability is much more difficult to assess, since many of the variables, such as the definition of race, are not even clearly agreed on. In order to accommodate human complexity, variability and its influence on health and disease, it is necessary to undertake a systematic approach. In the past decade, the emergence of analytical platforms and bioinformatics tools has led to the development of systems biology. Such an approach offers enormous potential in defining key pathways and networks involved in optimal human health, as well as disease onset, progression and treatment. The tools and technologies now available in systems biology analyses offer exciting opportunities to exploit the emerging areas of personalized medicine. In this article, we discuss the current status of human complexity, and how systems biology and personalized medicine can impact at the individual and population level.
RESUMO
Diet-induced obesity (DIO) leads to inflammatory activation of macrophages in white adipose tissue (WAT) and subsequently to insulin resistance. PPARgamma agonists are antidiabetic agents known to suppress inflammatory macrophage activation and to induce expression of the triacylglycerol (TG) synthesis enzyme acyl CoA: diacylglycerol acyltransferase 1 (DGAT1) in WAT and in adipocytes. Here, we investigated in mice the relationship between macrophage lipid storage capacity and DIO-associated inflammatory macrophage activation. Mice overexpressing DGAT1 in both macrophages and adipocytes (referred to herein as aP2-Dgat1 mice) were more prone to DIO but were protected against inflammatory macrophage activation, macrophage accumulation in WAT, systemic inflammation, and insulin resistance. To assess the contribution of macrophage DGAT1 expression to this phenotype, we transplanted wild-type mice with aP2-Dgat1 BM. These mice developed DIO similar to that of control mice but retained the protection from WAT inflammation and insulin resistance seen in aP2-Dgat1 mice. In isolated macrophages, Dgat1 mRNA levels correlated directly with TG storage capacity and inversely with inflammatory activation by saturated fatty acids (FAs). Moreover, PPARgamma agonists increased macrophage Dgat1 mRNA levels, and the protective effects of these agonists against FA-induced inflammatory macrophage activation were absent in macrophages isolated from Dgat1-null mice. Thus, increasing DGAT1 expression in murine macrophages increases their capacity for TG storage, protects against FA-induced inflammatory activation, and is sufficient to reduce the inflammatory and metabolic consequences of DIO.
Assuntos
Diacilglicerol O-Aciltransferase/biossíntese , Dieta/efeitos adversos , Resistência à Insulina , Macrófagos/enzimologia , Obesidade/enzimologia , Triglicerídeos/metabolismo , Adipócitos/enzimologia , Tecido Adiposo Branco/enzimologia , Animais , Diacilglicerol O-Aciltransferase/genética , Ácidos Graxos , Regulação Enzimológica da Expressão Gênica/genética , Inflamação/genética , Inflamação/mortalidade , Camundongos , Camundongos Knockout , Obesidade/etiologia , Obesidade/genética , PPAR gama/agonistas , PPAR gama/genética , PPAR gama/metabolismo , Triglicerídeos/genéticaRESUMO
The myelin sheath allows axons to conduct action potentials rapidly in the vertebrate nervous system. Axonal signals activate expression of specific transcription factors, including Oct6 and Krox20, that initiate myelination in Schwann cells. Elevation of cyclic adenosine monophosphate (cAMP) can mimic axonal contact in vitro, but the mechanisms that regulate cAMP levels in vivo are unknown. Using mutational analysis in zebrafish, we found that the G protein-coupled receptor Gpr126 is required autonomously in Schwann cells for myelination. In gpr126 mutants, Schwann cells failed to express oct6 and krox20 and were arrested at the promyelinating stage. Elevation of cAMP in gpr126 mutants, but not krox20 mutants, could restore myelination. We propose that Gpr126 drives the differentiation of promyelinating Schwann cells by elevating cAMP levels, thereby triggering Oct6 expression and myelination.
Assuntos
Bainha de Mielina/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Células de Schwann/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Axônios/fisiologia , Axônios/ultraestrutura , Diferenciação Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Sistema da Linha Lateral/inervação , Dados de Sequência Molecular , Mutação , Proteína Básica da Mielina/metabolismo , Neuregulina-1/metabolismo , Fator 6 de Transcrição de Octâmero/genética , Fator 6 de Transcrição de Octâmero/metabolismo , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Receptores Acoplados a Proteínas G/genética , Células de Schwann/citologia , Transdução de Sinais , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genéticaRESUMO
The kinesin motor protein Kif1b has previously been implicated in the axonal transport of mitochondria and synaptic vesicles. More recently, KIF1B has been associated with susceptibility to multiple sclerosis (MS). Here we show that Kif1b is required for the localization of mbp (myelin basic protein) mRNA to processes of myelinating oligodendrocytes in zebrafish. We observe the ectopic appearance of myelin-like membrane in kif1b mutants, coincident with the ectopic localization of myelin proteins in kif1b mutant oligodendrocyte cell bodies. These observations suggest that oligodendrocytes localize certain mRNA molecules, namely those encoding small basic proteins such as MBP, to prevent aberrant effects of these proteins elsewhere in the cell. We also find that Kif1b is required for outgrowth of some of the longest axons in the peripheral and central nervous systems. Our data demonstrate previously unknown functions of kif1b in vivo and provide insights into its possible roles in MS.
Assuntos
Axônios/metabolismo , Cinesinas/metabolismo , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Humanos , Cinesinas/genética , Dados de Sequência Molecular , Esclerose Múltipla , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
PURPOSE: We present work that demonstrates that cisplatin reacts rapidly with dimethyl sulfoxide (DMSO) in solution and identify the structure and reactivity of the resulting compound. METHODS: Electrospray ionization-mass spectrometry (ESI-MS) and NMR were used to identify the chemical structure of compounds formed when DMSO reacts with cisplatin. We studied the reactivity of the identified compound with DNA. In vitro toxicity studies in neurons and cancer cells and in vivo toxicity studies in rats were used to determine both the cancer chemotherapeutic and toxic effects of the identified compound. RESULTS: Cisplatin binds rapidly with DMSO to form a DMSO adduct. The resulting compound has reduced ability to bind to double-stranded DNA both in vitro and in cells. This compound has reduced toxicity for cancer cells and neurons in vitro. In vivo nephrotoxicity studies show that the adducted compound has different nephrotoxicity and elimination characteristics than cisplatin. CONCLUSIONS: From this work, we conclude that dissolving cisplatin in DMSO results in formation of an adducted compound with different therapeutic and biological characteristics. Furthermore, future studies which propose using DMSO in combination with cisplatin for chemotherapeutic treatment in patients must be reconsidered. Due to the rapidity and nature of the reaction, DMSO and cisplatin should not be combined for patient treatment.
Assuntos
Antineoplásicos/química , Antineoplásicos/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/química , Cisplatino/toxicidade , Adutos de DNA/química , Adutos de DNA/toxicidade , Dimetil Sulfóxido/química , Dimetil Sulfóxido/toxicidade , Síndromes Neurotóxicas/patologia , Animais , Células Cultivadas , DNA/química , DNA/efeitos dos fármacos , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Feminino , Humanos , Leucemia L1210/metabolismo , Espectrometria de Massas , Neurônios/efeitos dos fármacos , Células PC12 , Ratos , Ratos Sprague-Dawley , Soluções , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Although theoretical systems analysis has been available for over half a century, the recent advent of omic high-throughput analytical platforms along with the integration of individual tools and technologies has given rise to the field of modern systems biology. Coupled with information technology, bioinformatics, knowledge management and powerful mathematical models, systems biology has opened up new vistas in our understanding of complex biological systems. Currently there are two distinct approaches that include the inductively driven computational systems biology (bottom-up approach) and the deductive data-driven top-down analysis. Such approaches offer enormous potential in the elucidation of disease as well as defining key pathways and networks involved in optimal human health and nutrition. The tools and technologies now available in systems biology analyses offer exciting opportunities to develop the emerging areas of personalized medicine and individual nutritional profiling.
Assuntos
Pesquisa Biomédica/métodos , Saúde , Ciências da Nutrição , Promoção da Saúde , Humanos , Modelos Teóricos , Fenômenos Fisiológicos da NutriçãoRESUMO
Mutations in Kif1-binding protein/KIAA1279 (KBP) cause the devastating neurological disorder Goldberg-Shprintzen syndrome (GSS) in humans. The cellular function of KBP and the basis of the symptoms of GSS, however, remain unclear. Here, we report the identification and characterization of a zebrafish kbp mutant. We show that kbp is required for axonal outgrowth and maintenance. In vivo time-lapse analysis of neuronal development shows that the speed of early axonal outgrowth is reduced in both the peripheral and central nervous systems in kbp mutants. Ultrastructural studies reveal that kbp mutants have disruption to axonal microtubules during outgrowth. These results together suggest that kbp is an important regulator of the microtubule dynamics that drive the forward propulsion of axons. At later stages, we observe that many affected axons degenerate. Ultrastructural analyses at these stages demonstrate mislocalization of axonal mitochondria and a reduction in axonal number in the peripheral, central and enteric nervous systems. We propose that kbp is an important regulator of axonal development and that axonal cytoskeletal defects underlie the nervous system defects in GSS.
Assuntos
Anormalidades Múltiplas/metabolismo , Anormalidades Múltiplas/patologia , Axônios/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Axônios/ultraestrutura , Padronização Corporal , Proteínas de Transporte/genética , Citoesqueleto/ultraestrutura , Sistema Nervoso Entérico/embriologia , Sistema Nervoso Entérico/metabolismo , Sistema Nervoso Entérico/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Mutação/genética , Bainha de Mielina/ultraestrutura , Vesículas Sinápticas/metabolismo , Síndrome , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genéticaRESUMO
The advent of on-line multidimensional liquid chromatography-mass spectrometry has significantly impacted proteomic analyses of complex biological fluids such as plasma. However, there is general agreement that additional advances to enhance the peak capacity of such platforms are required to enhance the accuracy and coverage of proteome maps of such fluids. Here, we describe the combination of strong-cation-exchange and reversed-phase liquid chromatographies with ion mobility and mass spectrometry as a means of characterizing the complex mixture of proteins associated with the human plasma proteome. The increase in separation capacity associated with inclusion of the ion mobility separation leads to generation of one of the most extensive proteome maps to date. The map is generated by analyzing plasma samples of five healthy humans; we report a preliminary identification of 9087 proteins from 37,842 unique peptide assignments. An analysis of expected false-positive rates leads to a high-confidence identification of 2928 proteins. The results are catalogued in a fashion that includes positions and intensities of assigned features observed in the datasets as well as pertinent identification information such as protein accession number, mass, and homology score/confidence indicators. Comparisons of the assigned features reported here with other datasets shows substantial agreement with respect to the first several hundred entries; there is far less agreement associated with detection of lower abundance components.