Critical cysteines in Akt1 regulate its activity and proteasomal degradation: implications for neurodegenerative diseases

Impaired Akt1 signaling is observed in neurodegenerative diseases, including Parkinson's disease (PD). In PD models oxidative modification of Akt1 leads to its dephosphorylation and consequent loss of its kinase activity. To explore the underlying mechanism we exposed Neuro2A cells to cadmium, a pan inhibitor of protein thiol disulfide oxidoreductases, including glutaredoxin 1 (Grx1), or downregulated Grx1, which led to dephosphorylation of Akt1, loss of its kinase activity, and also decreased Akt1 protein levels. Mutation of cysteines to serines at 296 and 310 in Akt1 did not affect its basal kinase activity but abolished cadmium- and Grx1 downregulation-induced reduction in Akt1 kinase activity, indicating their critical role in redox modulation of Akt1 function and turnover. Cadmium-induced decrease in phosphorylated Akt1 correlated with increased association of wild-type (WT) Akt1 with PP2A, which was absent in the C296-310S Akt1 mutant and was also abolished by N-acetylcysteine treatment. Further, increased proteasomal degradation of Akt1 by cadmium was not seen in the C296-310S Akt1 mutant, indicating that oxidation of cysteine residues facilitates degradation of WT Akt1. Moreover, preventing oxidative modification of Akt1 cysteines 296 and 310 by mutating them to serines increased the cell survival effects of Akt1. Thus, in neurodegenerative states such as PD, maintaining the thiol status of cysteines 296 and 310 in Akt1 would be critical for Akt1 kinase activity and for preventing its degradation by proteasomes. Preventing downregulation of Akt signaling not only has long-range consequences for cell survival but could also affect the multiple roles that Ala plays, including in the Akt-mTOR signaling cascade. (C) 2014 Elsevier Inc. All rights reserved.

Participação do L-NAME, da L-arginina e da N-acetilcisteína no enfisema pulmonar induzido por fumaça de cigarro em camundongos

O óxido nítrico (NO) constitui um dos mais importantes mediadores intra e extracelulares e tem sido descrita sua participação tanto em processos biológicos como patológicos. Nosso objetivo foi verificar se o aumento ou a diminuição do óxido nítrico apresenta um efeito benéfico na proteção do tecido pulmonar no enfisema pulmonar induzido por fumaça de cigarro em camundongos. Para tanto, utilizamos o L-NAME (inibidor do NO), a L-arginina (substrato para a formação do NO) e os comparamos com a N-acetilcisteína (utilizada no tratamento da DPOC). Foram utilizados 65 camundongos C57BL/6 machos. Cinquenta animais foram divididos em grupos controle, fumaça de cigarro (FC), fumaça de cigarro + L-NAME (FC+LN), fumaça de cigarro + L-arginina (FC+LA), fumaça de cigarro + N-acetilcisteína (FC+NAC) (n=10, por grupo). Durante sessenta dias 40 animais foram expostos a 12 cigarros comerciais por dia, 3 vezes ao dia. Os grupos controle e FC foram submetidos à gavagens orogástricas com o veículo. Os grupos FC+LN, FC+LA, FC+NAC receberam gavagens diárias de L-NAME (60 mg/kg), L-arginina (120 mg/kg) ou NAC (200 mg/kg) respectivamente. Quinze animais (n = 5, por grupo) foram expostos ao ar ambiente e tratados apenas com L-NAME, L-arginina e NAC. Realizamos a análise do perfil das células do lavado broncoalveolar após o sacrifício. O pulmão direito foi removido para as análises histológicas do alargamento dos espaços aéreos determinado pela medida do diâmetro alveolar médio (Lm) e da densidade de superfície (Sv) dos septos alveolares. Os pulmões esquerdos foram removidos e homogeneizados para a as análises da atividade enzimática (SOD, CAT e MPO) e do sistema glutationa (GSH/GSSG), para a análise dos valores de nitrito e da expressão de 4-HNE, MMP-12, NE, TIMP-1, TIMP-2. Nossos resultados apontam que o L-NAME tem uma ação voltada para a matriz extracelular (via protease-antiprotease), enquanto que a L-arginina possui uma ação voltada para os oxidantes, assim como a NAC. Porém a NAC atua aumentando os níveis de glutationa, o que interfere diretamente nos oxidantes (via oxidante-antioxidante), enquanto a L-arginina interfere aumentando o burden oxidativo concomitante a um aumento da velocidade de ação dos oxidantes o que aumenta as células inflamatórias, mas diminui seu tempo de ação permitindo uma maior proteção. Concluímos que tanto o favorecimento para a produção e liberação do NOatravés da administração da L-arginina quanto a inibição do NOpela utilização do L-NAME foi eficiente na proteção do pulmão, apesar de não terem alcançado um resultado tão bom quanto a NAC.

Participação do estresse oxidativo na lesão pulmonar induzida por lipopolissacarídeo: repercussões inflamatórias estruturais e funcionais

O objetivo do presente estudo foi investigar o envolvimento do estresse oxidativo na lesão pulmonar aguda (LPA) induzida por lipopolissacarídeo (LPS) e as repercussões inflamatórias, estruturais e funcionais, através de análises bioquímicas de estresse oxidativo, prova de função pulmonar, análise histológica e RT-PCR para citocinas e fatores de transcrição pró-inflamatórios. Na primeira etapa foram utilizados camundongos machos C57BL6 foram divididos em sete grupos: Grupo controle (CTR) (50 μL de solução fisiológica) administrados via intratraqueal [it], LPS 6 horas (10 μL de LPS) [it], LPS 12 horas (10 μL de LPS) [it], LPS 24 horas (10 μL de LPS) [i], LPS 48 horas (10 μL de LPS). Para verificar que as alterações observadas eram estresse oxidativo dependentes camundongos machos C57BL6 foram pré-tratados com N-acetilcisteína (NAC) 1 hora antes do estímulo com LPS e sacrifícados 24 horas depois do estímulo com LPS. Os animais foram divididos da seguinte forma: Grupo LPS 24 horas (10 μL) [it], grupo NAC 40 mg/kg (gavagem) + LPS (10 μL) [it] e grupo NAC 100 mg/kg (gavagem) + LPS (10 μL) [it]. O sistema antioxidante enzimático protegeu o pulmão do estresse oxidativo nas primeiras 12 horas. O estresse oxidativo foi caracterizado em 24 horas e em 48 horas observou-se falência do sistema antioxidante enzimático. Parâmetros de função pulmonar se mostraram alterados nos grupo estimulados com LPS principalmente no grupo LPS. A elastância (p<0,001), resistência de via aérea periférica (ΔP2) (p<0,001), resistência de via aérea central (ΔP1) (p<0,001) e resistência de via aérea total (ΔPtot) (p<0,001) se mostraram principlamente alteradas no grupo LPS 24 horas. O pré-tratamento com NAC impediu o aumento dos parâmetros de elastância (p<0,001), resistência de via aérea periférica (ΔP2) (p<0,001) resistência de via aérea central (ΔP1) (p<0,05) e resistência de via aérea total (ΔPtot) (p<0,001) comparado com o grupo LPS 24 horas. As alterações histológicas como espessamento de septo alveolar, influxo de células inflamatórias e hemorragia mostraram-se tempo dependentes. O pré-tratamento NAC impediu as alterações observadas nos grupo estimulados com LPS. Alterações inflamatórias foram observadas no grupo estimulado com LPS como IL-6 (p<0,001), iNOS (p<0,001), COX2 (p<0,05), TNF-α (p<0,001) e NFκB (p<0,001) quando comparados ao grupo controle. O pré-tratamento com NAC impediu o aumento desses parâmetros como IL-6 (p<0,001), iNOS (p<0,001), COX2 (p<0,05), TNF-α (p<0,05) e NFκB (p<0,001) quando comparados ao grupo LPS 24 horas. Nossos resultados sugerem que o estresse oxidativo desempenha um papel importante nas respostas inflamatórios, estruturais e funcionais no modelo de LPA induzido por LPS e que essas alterações são estresse oxidativo dependentes.

O envolvimento do estresse oxidativo e nitrosativo em modelo de doença pulmonar obstrutiva crônica (DPOC) induzido por elastase

Esse estudo buscou investigar o papel do estresse oxidativo e nitrosativo no enfisema pulmonar induzido por elastase. Foram utilizados camundongos machos C57BL/6 submetidos a dois modelos de indução do enfisema por elastase pancreática suína (PPE): intratraqueal (i.t.) e intranasal (i.n.). No modelo intratraqueal a PPE foi instilada nas doses de 0,05 U ou 0,5 U/camundongo para avaliação temporal do enfisema 7, 14 e 21 dias após instilação de PPE. Em outra etapa, o papel da iNOS foi avaliado através da sua inibição farmacológica por aminoguanidina (AMG) 1% na água de beber ou pela sua exclusão genética em camundongos deficientes em iNOS que tiveram o enfisema induzido por 0,5 U PPE i.t. após 21 dias. No modelo intranasal a dose de PPE foi 3 U/camundongo para avaliação temporal do enfisema (1, 7, 14 e 21 dias após PPE). O papel do estresse oxidativo e nitrosativo foi avaliado com diferentes tratamentos antioxidantes na água de beber: tempol, apocinina+alopurinol, n-acetilcisteína, vitamina C+E, e aminoguanidina durante os 21 dias de indução do enfisema. Os grupos controles foram submetidos à instilação de salina. Lavado broncoalveolar, imunoensaios, análises bioquímicas de estresse oxidativo e ensaios morfométricos foram realizados nos pulmões dos animais. O enfisema foi histologicamente alcançado em 21 dias após 0,5 U PPE i.t., evidenciado pelo aumento do diâmetro alveolar médio Lm e da densidade de volume dos espaços alveolares - Vvair em comparação ao grupo controle. TNF-α foi aumentado em 7 e 14 dias após 0,5 U PPE comparados ao controle, concomitante com a redução de IL-10 nos mesmos períodos, comparados ao controle. O estresse oxidativo foi observado na fase inicial do enfisema, com aumento dos níveis de nitrito, TBARS e superóxido dismutase no grupo 7 dias após 0,5 U PPE (i.t.) quando comparados ao controle ao passo que no modelo intranasal as alterações típicas do estresse foram vistas no grupo 1 dia após 3 U de PPE. Atividade da glutationa peroxidase foi aumentada em todos os grupos PPE (i.t.). A exposição à 0,5 U PPE induziu o aumento da iNOS, eNOS e nitrotirosina, sendo revertido no grupo PPE+AMG. Os animais tratados com AMG 1% e os deficientes em iNOS tiveram o enfisema atenuado histologicamente, mantendo o Lm e o Vvair semelhantes ao grupo controle. Os grupos tratados com n-acetilcisteína e aminoguanidina no modelo i.n. tiveram redução do Lm quando comparados ao grupo PPE. Esses resultados sugerem que as vias de estresse oxidativo e nitrosativo são disparadas pela produção de óxido nítrico via iNOS no enfisema pulmonar. A modulação da iNOS parece uma estratégia promissora no estabelecimento do enfisema pulmonar

Efeitos do extrato de Chenopodium ambrosioides L. na cistite induzida pela ciclofosfamida

A cistite hemorrágica (CH) consiste em um processo inflamatório difuso de origem infecciosa ou não que resulta em um sangramento da mucosa vesical. As CH crônicas recorrentes induzidas pela ciclofosfamida (CYP) são um desafio na prática clínica pela alta morbidade e por vezes mortalidade dos pacientes. O tratamento da CH induzida pela ciclofosfamida consiste no uso de MESNA, disulfiram, N-acetil-cisteína, anti-inflamatório, oxigênio hiperbárico, hiper-hidratação e irrigação vesical, mas novas terapias têm sido investigadas, inclusive usando produtos naturais. A espécie vegetal Chenopodium ambrosioides L., conhecida popularmente como mastruz, mastruço e erva-de-Santa-Maria, tem sido relatada pela população como anti-inflamatório e analgésico. O presente estudo investigou os efeitos do extrato bruto hidroalcoólico de folhas de Chenopodium ambrosioides na CH induzida pela ciclofosfamida em ratos. Vinte e nove ratos receberam 150 mg/kg de CYP por via intraperitoneal (i.p.) para indução de CH e em seguida foram divididos em três grupos: controle negativo (CN), tratados com soro fisiológico a 0,9%; extrato bruto hidroalcoólico de Chenopodium ambrosioides (EBHCa), tratado com dose única de 50 mg/kg de extrato bruto hidroalcoólico de Chenopodium ambrosioides (EBH) e controle positivo (CP), tratados com dose única de 15 mg/kg de diclofenaco de potássio, todos por gavagem. Após 48 horas da indução da CH os animais foram sacrificados para retirada da bexiga, que foi preparada para análise histopatológica e imuno-histoquímica. O EBH foi capaz de diminuir o peso da bexiga e histologicamente a inflamação aguda e crônica da bexiga, a extensão do infiltrado inflamatório na parede vesical e a neoformação capilar do mesmo modo que o diclofenaco de potássio, quando comparados ao grupo CN. Observou-se ainda uma redução da expressão imuno-histoquímica de cicloxigenase-2 (COX-2) e do fator nuclear kappa B (NFB) na bexiga. No presente estudo o EBH das folhas de Chenopodium ambrosioides apresentou atividade anti-inflamatória, semelhante ao diclofenaco de potássio, no tratamento da CH induzida pela CYP.

外源物质对酵母拮抗菌生活力的影响

　　近年来，酵母拮抗菌在水果采后病害防治中展示了良好的应用前景。然而，在实际应用中,酵母拮抗菌在逆境条件下会因为发生凋亡或细胞损伤而引起生活力的下降，最终导致拮抗菌抑病能力降低。研究酵母拮抗菌生活力下降的规律，提高酵母拮抗菌的生产效率，减少剂型加工过程中的细胞损伤，增强其对逆境条件的耐受力是增加或稳定生防制剂防治效果的有效途径。本文主要研究酵母拮抗菌正常培养过程中生活力下降的规律，筛选剂型加工过程中对酵母拮抗菌具有保护作用的化学物质，并对酵母拮抗菌的培养条件进行了优化。主要研究结果如下： 　　1. 在正常培养过程中，酵母拮抗菌Rhodotorula glutinis和Cryptococcus laurentii中细胞染色质凝集或细胞膜破损的发生一般在6天以后。外源加入的N-乙酰半胱氨酸及硅酸钠等物质在超过一定浓度时会加速酵母菌的死亡。 　　2. 在不同的液体悬浮制剂中，对R. glutinis而言，使用磷酸缓冲液（PBS）悬浮时保护效果最好；而C. laurentii悬浮在NYDB培养基中或海藻糖、乳糖溶液中时的生活力最高。 　　3. 以10 %葡萄糖 + 5 %脱脂牛奶作保护剂，可以有效地保持酵母拮抗菌C. laurentii冻干制剂的生活力，配合使用的保护效果高于它们单独使用时的保护效果。添加1 mM N-乙酰半胱氨酸能更好地保持拮抗菌制剂在常温保存过程中的生活力，这可能与这种还原性物质缓解了细胞内活性氧的积累有关。 　　4. 不同酵母拮抗菌对不同碳、氮源的利用能力有明显差异。在9种不同的碳源和10种不同的氮源中，Pichia membranefaciens能够最有效利用的碳、氮源是葡萄糖、果糖和多价胨，而Candida guilliermondii的最佳碳源和氮源分别是果糖和肉蛋白胨。

Effects of tetrabrominated diphenyl ether and hexabromocyclododecanes in single and complex exposure to hepatoma HepG2 cells

This study was designed to determine cytotoxic effects of PBDE-47 and HBCDs individually or with a mixture of both compounds exposure to Hep G2 cells. The results showed PBDE-47 and HBCDs induced increase of nitric oxide synthase (NOS) activity, release of NO. dissipation of mitochondria membrane potential and cell apoptosis. Exposure to HBCDs induced ROS formation. Moreover, preincubation with PTIO (NO scavanger) and N-acetylcysteine (ROS scavanger) partially reversed cytotoxic effects of these compounds. The possible mechanism is that PBDE-47 and HBCDs could boost generation of NO and/or ROS, impact mitochondria, and result in start-ups of apoptosis program. Cells exposed to mixture of both compounds and each of them showed non-apoptotic rate significant difference, but the combination of them caused more adverse effects on cells. These results Suggest that PBDE-47 and HBCDs in single and complex exposure have the cytotoxic activity of anti-proliferation and induction of apoptosis in tumor cells in vitro. (C) 2008 Elsevier B.V. All rights reserved.

The response of antioxidant systems in Nostoc sphaeroides against UV-B radiation and the protective effects of exogenous antioxidants

UV radiation is one of many harmful factors found in space that are detrimental to organisms on earth in space exploration. In the present work, we examined the role of antioxidant system in Nostoc sphaeroides Kutz (Cyanobacterium) and the effects of exogenously applied antioxidant molecules on its photosynthetic rate under UV-B radiation. It was found that UV-B radiation promoted the activity of antioxidant system to protect photosystem 11 (PSII) and exogenously applied antioxidant: sodium nitroprusside (SNP) and N-acetylcysteine (NAC) had an obvious protection on PSII activity under UV-B radiation. The activity of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7) and content of NIDA (malondialdehyde) and ASC (ascorbate) were improved by 0.5 mM and 1 mM SNP, but 0.1 mM SNP decreased the activity of antioxidant system. Addition of exogenous NAC decreased the activity of SOD, POD, CAT and the content MDA and ASC. In contrast, exogenously applied NAC increased GSH content. The results suggest that exogenous SNP and NAC may protect algae by different mechanisms: SNP may play double roles as both sources of reactive free radicals as well as ROS scavengers in mediating the protective role of PSII on algae under UV-B radiation. On the other hand, NAC functions as an antioxidant or precursor of glutathione, which could protect PSII directly from UV-B radiation. (c) 2007 COSPAR, Published by Elsevier Ltd. All rights reserved.

The involvement of the antioxidant system in protection of desert cyanobacterium Nostoc sp against UV-B radiation and the effects of exogenous antioxidants

In this study, we found that UV-B radiation decreased photosynthetic activity and boosted lipid peroxidation of desert Nostoc sp., and exogenous chemicals (ascorbate acid (ASC), N-acetylcysteine (NAC), and sodium nitroprusside (SNP)) had obvious protective effects on photosynthesis and membranes under UV-B radiation. High-concentration SNP boosted the activities of antioxidant enzymes, but low-concentration SNP reduced the activities of antioxidant enzymes. Both NAC and ASC treatments of cells decreased activities of antioxidant enzymes. The results suggested that those chemicals possibly had different mechanisms of protection of algae cells against UV-B radiation. SNP might play double roles as a signal molecule in the formation of algae cell protection of Photosystem 11 under UV-B radiation and as a (reactive oxygen species) scavenger, while NAC and ASC might function as antioxidant reagents or precursors of other antioxidant molecules, which could protect cells directly against ROS initiated by UV-B radiation. (c) 2006 Elsevier Inc. All rights reserved.

N-乙酰半胱氨酸对碳离子照射小鼠急性损伤的保护效应

探讨了N-乙酰半胱氨酸(N-acetylcysteine,NAC)对12C6+离子照射小鼠损伤的保护效应及可能的作用机制。预先给予昆明小鼠NAC(200mg/kg),后进行12C6+离子束4Gy的单次全身照射。照射后2h处死小鼠,取肝、肺组织,用化学法检测组织中超氧化物岐化酶(Superoxide dismutase,SOD)的活性,谷胱甘肽(Glutathione,GSH)含量及脂质过氧化产物丙二醛(Malondialdehyde,MDA)水平;用碱性单细胞凝胶电泳法检测DNA单链断裂;用流式细胞术检测组织细胞凋亡率。与照射对照组相比,提前给予NAC可极显著地减轻12C6+离子导致的肝、肺组织中DNA断裂(p<0.001)和细胞凋亡(p<0.001),并显著地提高组织中GSH的含量;NAC还明显地抑制了肺组织中碳离子辐照所致的MDA水平增高(p<0.01)并显著地诱导了组织中SOD活性的升高(p<0.01)。提示NAC可通过抵御组织内的氧化作用,合成、补充GSH的含量,阻止DNA链的断裂和细胞的凋亡,实现对碳离子辐照损伤的保护效应。

低剂量12C6+辐射对小鼠免疫系统损伤和抗氧化剂氮乙酰半胱氨酸的保护作用研究

实验目的：随着科技的发展，人类活动范围已经逐渐向外太空扩展，对于人类太空探索的最大威胁是太空中的各种粒子辐射。这些辐射包括太阳辐射（质子和电子）和银河辐射（质子占85%，氦离子占14%，重离子占1%）。众所周知，重离子与常规X和γ射线相比有较高的传能线密度（linear energy transfer, LET）和相对生物学效应（relative biological effectiveness, RBE），对机体组织和器官有较强的影响。放射治疗是肿瘤治疗的重要手段之一，由于肿瘤细胞的异质性，其对放、化疗的反应相差悬殊。本研究的目的是： 1评估辐射对健康机体产生的生物学风险； 2研究抗氧化剂氮乙酰半胱氨酸（NAC）对机体辐射损伤的保护作用 3不同肿瘤细胞辐射敏感性的差异。实验方法： 1 X射线或12C6+离子对小鼠进行不同剂量的全身辐射。NAC处理组小鼠在照射前1小时腹腔注射200mg/kg的NAC，对照组注射等体积的生理盐水。照射后不同时间点取样，利用流式细胞仪检测小鼠免疫细胞周期和凋亡情况，单细胞电泳检测淋巴细胞DNA损伤，MTT法（3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide）检测脾脏NK（natural killer，NK）细胞活性，微核法检测淋巴细胞染色体损伤情况，小鼠体内干扰素－γ（Interferon-γ，IFN-γ）由ELISA方法得到，小鼠血清中超氧化物岐化酶(Surperoxide dismutase SOD）由分光光度法测定，并观察胸腺和脾脏指数变化。 2 不同剂量X射线和12C6+离子辐射人肺腺癌细胞H1299和A549，用细胞克隆法检测照射后细胞存活曲线，流式细胞仪检测细胞周期和凋亡，Western-blot 检测A549 细胞P53蛋白表达。 结果： 1小鼠外周血淋巴细胞、胸腺细胞和脾脏淋巴细胞周期随着X射线照射剂量的增大而被阻滞在了G0/G1期，相同剂量的12C6+离子辐射时外周血淋巴细胞周期被阻滞在S期，分次连续X射线照射时，外周血淋巴细胞周期随着累积剂量的增加被阻滞在G2/M期；细胞凋亡比例随着照射剂量的增加而增加。小鼠血清中IFN-γ水平和脾脏中NK细胞活性在重离子照射剂量为0.05Gy时有显著增加，脾脏NK细胞活性随着照射剂量的增加而减弱。 2重离子照射后，小鼠淋巴细胞DNA和染色体的损伤随辐射剂量和照射后时间的延长而加剧。脾脏NK细胞活性在照射后各个时间点减弱，血清中IFN-γ水平和SOD酶活性随着重离子照射剂量的增加而降低。预防性给予NAC，12C6+离子辐射对淋巴细胞DNA和染色体所致损伤，胸腺细胞周期和凋亡，脾脏NK细胞活性，血清中IFN-γ的水平和SOD酶的活性的损伤与盐水组比较均有显著改善。 3 X射线照射对肺腺癌H1299细胞周期和凋亡率未产生明显影响，重离子照射后随着照射剂量的增加细胞周期被阻滞在G2/M期，细胞凋亡率也呈剂量依赖性；X射线和12C6+离子照射A549细胞后，细胞周期均被阻滞在G2/M期，凋亡率剂量依赖性增加。A549细胞P53蛋白的表达水平随着重离子照射剂量的增加而增加。结论： 1重离子辐射造成细胞DNA和染色体损伤随着照射剂量的增加和照射后时间的延长而增加，比X射线辐射损伤复杂和难以修复，产生这种现象的机理为辐射导致活性氧分子簇的产生，细胞因子和与细胞氧化反应有关的酶活性的变化，同时这种损伤对胸腺细胞周期、凋亡和胸腺、脾脏指数以及机体免疫系统都有影响；低剂量重离子辐射（0.05Gy）对小鼠机体的免疫力有刺激作用，机体免疫能力随着照射剂量增加和照射后时间的推移而减弱，不同的免疫器官对辐射的敏感性也不同； 2 200mg/kg 的NAC对辐射所致小鼠免疫系统损伤有很好的保护作用； 3 肺腺癌细胞H1299比同系A549具有较强的辐射敏感性，A549细胞凋亡的增加与P53蛋白表达水平升高有关

beta-arrestin-1 competitively inhibits insulin-induced ubiquitination and degradation of insulin receptor substrate 1.

beta-arrestin-1 is an adaptor protein that mediates agonist-dependent internalization and desensitization of G-protein-coupled receptors (GPCRs) and also participates in the process of heterologous desensitization between receptor tyrosine kinases and GPCR signaling. In the present study, we determined whether beta-arrestin-1 is involved in insulin-induced insulin receptor substrate 1 (IRS-1) degradation. Overexpression of wild-type (WT) beta-arrestin-1 attenuated insulin-induced degradation of IRS-1, leading to increased insulin signaling downstream of IRS-1. When endogenous beta-arrestin-1 was knocked down by transfection of beta-arrestin-1 small interfering RNA, insulin-induced IRS-1 degradation was enhanced. Insulin stimulated the association of IRS-1 and Mdm2, an E3 ubiquitin ligase, and this association was inhibited to overexpression of WT beta-arrestin-1, which led by decreased ubiquitin content of IRS-1, suggesting that both beta-arrestin-1 and IRS-1 competitively bind to Mdm2. In summary, we have found the following: (i) beta-arrestin-1 can alter insulin signaling by inhibiting insulin-induced proteasomal degradation of IRS-1; (ii) beta-arrestin-1 decreases the rate of ubiquitination of IRS-1 by competitively binding to endogenous Mdm2, an E3 ligase that can ubiquitinate IRS-1; (iii) dephosphorylation of S412 on beta-arrestin and the amino terminus of beta-arrestin-1 are required for this effect of beta-arrestin on IRS-1 degradation; and (iv) inhibition of beta-arrestin-1 leads to enhanced IRS-1 degradation and accentuated cellular insulin resistance.

Impacts of Mountaintop Removal Coal Mining on the Mud River, West Virginia: Selenium Accumulation, Trophic Transfer, and Toxicity in Fish

Selenium (Se) is a micronutrient necessary for the function of a variety of important enzymes; Se also exhibits a narrow range in concentrations between essentiality and toxicity. Oviparous vertebrates such as birds and fish are especially sensitive to Se toxicity, which causes reproductive impairment and defects in embryo development. Selenium occurs naturally in the Earth's crust, but it can be mobilized by a variety of anthropogenic activities, including agricultural practices, coal burning, and mining.

Mountaintop removal/valley fill (MTR/VF) coal mining is a form of surface mining found throughout central Appalachia in the United States that involves blasting off the tops of mountains to access underlying coal seams. Spoil rock from the mountain is placed into adjacent valleys, forming valley fills, which bury stream headwaters and negatively impact surface water quality. This research focused on the biological impacts of Se leached from MTR/VF coal mining operations located around the Mud River, West Virginia.

In order to assess the status of Se in a lotic (flowing) system such as the Mud River, surface water, insects, and fish samples including creek chub (Semotilus atromaculatus) and green sunfish (Lepomis cyanellus) were collected from a mining impacted site as well as from a reference site not impacted by mining. Analysis of samples from the mined site showed increased conductivity and Se in the surface waters compared to the reference site in addition to increased concentrations of Se in insects and fish. Histological analysis of mined site fish gills showed a lack of normal parasites, suggesting parasite populations may be disrupted due to poor water quality. X-ray absorption near edge spectroscopy techniques were used to determine the speciation of Se in insect and creek chub samples. Insects contained approximately 40-50% inorganic Se (selenate and selenite) and 50-60% organic Se (Se-methionine and Se-cystine) while fish tissues contained lower proportions of inorganic Se than insects, instead having higher proportions of organic Se in the forms of methyl-Se-cysteine, Se-cystine, and Se-methionine.

Otoliths, calcified inner ear structures, were also collected from Mud River creek chubs and green sunfish and analyzed for Se content using laser ablation inductively couple mass spectrometry (LA-ICP-MS). Significant differences were found between the two species of fish, based on the concentrations of otolith Se. Green sunfish otoliths from all sites contained background or low concentrations of otolith Se (< 1 µg/g) that were not significantly different between mined and unmined sites. In contrast creek chub otoliths from the historically mined site contained much higher (≥ 5 µg/g, up to approximately 68 µg/g) concentrations of Se than for the same species in the unmined site or for the green sunfish. Otolith Se concentrations were related to muscle Se concentrations for creek chubs (R2 = 0.54, p = 0.0002 for the last 20% of the otolith Se versus muscle Se) while no relationship was observed for green sunfish.

Additional experiments using biofilms grown in the Mud River showed increased Se in mined site biofilms compared to the reference site. When we fed fathead minnows (Pimephales promelas) on these biofilms in the laboratory they accumulated higher concentrations of Se in liver and ovary tissues compared to fathead minnows fed on reference site biofilms. No differences in Se accumulation were found in muscle from either treatment group. Biofilms were also centrifuged and separated into filamentous green algae and the remaining diatom fraction. The majority of Se was found in the diatom fraction with only about 1/3rd of total biofilm Se concentration present in the filamentous green algae fraction

Finally, zebrafish (Danio rerio) embryos were exposed to aqueous Se in the form of selenate, selenite, and L-selenomethionine in an attempt to determine if oxidative stress plays a role in selenium embryo toxicity. Selenate and selenite exposure did not induce embryo deformities (lordosis and craniofacial malformation). L-selenomethionine, however, induced significantly higher deformity rates at 100 µg/L compared to controls. Antioxidant rescue of L-selenomethionime induced deformities was attempted in embryos using N-acetylcysteine (NAC). Pretreatment with NAC significantly reduced deformities in the zebrafish embryos secondarily treated with L-selenomethionine, suggesting that oxidative stress may play a role in Se toxicity. Selenite exposure also induced a 6.6-fold increase in glutathione-S-transferase pi class 2 gene expression, which is involved in xenobiotic transformation. No changes in gene expression were observed for selenate or L-selenomethionine-exposed embryos.

The findings in this dissertation contribute to the understanding of how Se bioaccumulates in a lotic system and is transferred through a simulated foodweb in addition to further exploring oxidative stress as a potential mechanism for Se-induced embryo toxicity. Future studies should continue to pursue the role of oxidative stress and other mechanisms in Se toxicity and the biotransformation of Se in aquatic ecosystems.

Mechanisms of modified LDL-induced pericyte loss and retinal injury in diabetic retinopathy

Aims/hypothesis: In previous studies we have shown that extravasated, modified LDL is associated with pericyte loss, an early feature of diabetic retinopathy (DR). Here we sought to determine detailed mechanisms of this LDLinduced pericyte loss.

Methods: Human retinal capillary pericytes (HRCP) were exposed to ‘highly-oxidised glycated’ LDL (HOG-LDL) (a model of extravasated and modified LDL) and to 4-hydroxynonenal or 7-ketocholesterol (components of oxidised LDL), or to native LDL for 1 to 24 h with or without 1 h of pretreatment with inhibitors of the following: (1) the scavenger receptor (polyinosinic acid); (2) oxidative stress (N-acetyl cysteine); (3) endoplasmic reticulum (ER) stress (4-phenyl butyric acid); and (4) mitochondrial dysfunction (cyclosporin A). Oxidative stress, ER stress, mitochondrial dysfunction, apoptosis and autophagy were assessed using techniques including western blotting, immunofluorescence, RT-PCR, flow cytometry and TUNEL assay. To assess the relevance of the results in vivo, immunohistochemistry was used to detect the ER stress chaperon, 78 kDa glucose-regulated protein, and the ER sensor, activating transcription factor 6, in retinas from a mouse model of DR that mimics exposure of the retina to elevated glucose and elevated LDL levels, and in retinas from human participants with and without diabetes and DR.

Results: Compared with native LDL, HOG-LDL activated oxidative and ER stress in HRCP, resulting in mitochondrial dysfunction, apoptosis and autophagy. In a mouse model of diabetes and hyperlipidaemia (vs mouse models of either condition alone), retinal ER stress was enhanced. ER stress was also enhanced in diabetic human retina and correlated with the severity of DR.

Conclusions/interpretation: Cell culture, animal, and human data suggest that oxidative stress and ER stress are induced by modified LDL, and are implicated in pericyte loss in DR.

The role of lipid peroxidation products and oxidative stress in activation of the canonical wingless-type MMTV integration site (WNT) pathway in a rat model of diabetic retinopathy

Our recent studies suggest that activation of the wingless-type MMTV integration site (WNT) pathway plays pathogenic roles in diabetic retinopathy and age-related macular degeneration. Here we investigated the causative role of oxidative stress in retinal WNT pathway activation in an experimental model of diabetes.