RESUMO
The heat shock response is a highly conserved process essential for surviving environmental stress, including extremes of temperature. To investigate whether heat shock has an impact on intracellular Zn(2+) homeostasis, cells were subjected to heat shock, and subsequently the intracellular free zinc concentration was investigated. Sublethal heat shock induced a temperature-dependent and transient intracellular Zn(2+) release that was repeatable after 24 h. The free zinc was localized in round-shaped nuclear bodies identified as nucleoli. Metallothionein, the main cellular zinc storing protein, was found to be not functionally essential for this heat-shock-induced effect. No significant oxidative stress within the cells was detected after heat shock. Cold shock and subsequent rewarming did not result in disturbed intracellular zinc homeostasis. These results show that heat shock and cold shock differ with respect to intracellular Zn(2+) release. A role for zinc as signaling ion during fever is conceivable.
Assuntos
Temperatura Baixa , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Resposta ao Choque Térmico , Metalotioneína/metabolismo , Zinco/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Nucléolo Celular/metabolismo , Sobrevivência Celular , Células Cultivadas , Células Endoteliais/patologia , Fibroblastos/patologia , Homeostase , Metalotioneína/deficiência , Metalotioneína/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Astrocyte swelling is observed in different types of brain injury including hepatic encephalopathy (HE). This study investigates the role of astrocyte swelling on Zn2+ homeostasis in hypoosmotically treated astrocytes by using the Zn2+ indicators Newport-Green, Zinquin, and RhodZin-3. Hypoosmolarity (205 mosmol/L) led to a persistent increase of the intracellular "free" Zn2+ concentration [Zn2+](i) within 15 min, which was reversible after reinstitution of normoosmolarity (305 mosmol/L). The hypoosmotic [Zn2+](i) increase was abolished in the presence of the Zn2+ chelator TPEN, the NMDA receptor antagonists MK-801 and AP5, the antioxidant epigallocatechin gallate, and the nitric oxide synthase inhibitors L-NMMA and TRIM. Hypoosmolarity triggered nuclear accumulation of the metal response element-binding transcription factor MTF-1 and the specificity protein Sp1 and expression of the mRNAs encoding metallothionein and the Sp1-regulated peripheral-type benzodiazepine receptor (PBR). These effects were abolished by the Zn2+ chelator TPEN. The data suggest that astrocyte swelling affects gene expression by modulation of [Zn2+](i). Whereas Zn2+-dependent upregulation of metallothionein may help to counteract excessive astrocyte swelling and production of reactive oxygen and nitrogen oxide species, stimulation of PBR expression may augment HE development.
Assuntos
Astrócitos/citologia , Astrócitos/fisiologia , Tamanho Celular , Homeostase/fisiologia , Zinco/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Líquido Intracelular/metabolismo , Líquido Intracelular/fisiologia , Concentração Osmolar , Pressão Osmótica/fisiologia , Ratos , Ratos WistarRESUMO
Changes of the redox balance in cells alter the availability of intracellular free Zn(2+). Here, cells were exposed to ultraviolet (UV)-A, UV-B, or infrared (IR)-A light irradiation, and the intracellular free zinc pool was monitored. Under sublethal conditions only UV-A irradiation resulted in a transient cytoplasmic and nuclear increase of intracellular free Zn(2+). Likewise, tert-butyl hydroperoxide and singlet oxygen, but not H(2)O(2) or intracellular generation of O(2)(*-) by redox cyclers, mimicked the effects of UV-A irradiation, while disulfide stress by diamide only led to a transient cytoplasmic zinc release. These results show that only certain types of subtoxic cellular stress massively disturb the zinc homeostasis in cells.
Assuntos
Homeostase/efeitos da radiação , Raios Ultravioleta , Zinco/metabolismo , Animais , Linhagem Celular , Diamida/farmacologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Homeostase/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Camundongos , Oxirredução , Quinonas/metabolismo , Oxigênio Singlete/metabolismoRESUMO
Oxidative stress is one of the main causes of vascular disease. This study aims to investigate the antioxidant activity exerted by zinc in primary rat endothelial cells (EC). Using a 24-h treatment with hydrogen peroxide as a model for oxidative stress, we found that zinc supplementation protects from peroxide-induced cell death via increasing the transcription of the catalytic subunit (heavy chain) of glutamate-cysteine ligase (GCLC) and the concentrations of glutathione (GSH). Conversely, zinc depletion significantly decreased the expression of GCLC and the cellular GSH levels, resulting in an increased susceptibility of EC to oxidative stress. Using confocal microscopy and the RNA silencing technique, we found that zinc upregulates the expression of GCLC by activating the transcription factor Nrf2. Surprisingly, the intracellular zinc sensor, metal-responsive transcription factor-1, is not involved in the zinc-induced expression of GCLC. The present study shows that zinc controls the redox state of EC by regulating the de novo synthesis of GSH. This molecular mechanism may contribute to the elaboration of new nutritional and/or pharmaceutical approaches for protecting the endothelium against oxidative stress.
Assuntos
Células Endoteliais/metabolismo , Glutamato-Cisteína Ligase/biossíntese , Glutationa/biossíntese , Peróxido de Hidrogênio/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Zinco/fisiologia , Animais , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Endotélio Vascular/metabolismo , Estresse Oxidativo , Ratos , Fatores de Transcrição/metabolismo , Sulfato de Zinco/farmacologia , Fator MTF-1 de TranscriçãoRESUMO
Aberrant production of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathogenesis of endothelial dysfunction and vascular disease. Mechanisms responsible for the fine-tuning of iNOS activity in inflammation are still not fully understood. Zinc is an important structural element of NOS enzymes and is known to inhibit its catalytical activity. In this study we aimed to investigate the effects of zinc on iNOS activity and expression in endothelial cells. We found that zinc down-regulated the expression of iNOS (mRNA+protein) and decreased cytokine-mediated activation of the iNOS promoter. Zinc-mediated regulation of iNOS expression was due to inhibition of NF-κB transactivation activity, as determined by a decrease in both NF-κB-driven luciferase reporter activity and expression of NF-κB target genes, including cyclooxygenase 2 and IL-1ß. However, zinc did not affect NF-κB translocation into the nucleus, as assessed by Western blot analysis of nuclear and cytoplasmic fractions. Taken together our results demonstrate that zinc limits iNOS-derived high output NO production in endothelial cells by inhibiting NF-κB-dependent iNOS expression, pointing to a role of zinc as a regulator of iNOS activity in inflammation.
Assuntos
Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/biossíntese , Zinco/farmacologia , Animais , Aorta/citologia , Células Endoteliais/citologia , Células Endoteliais/enzimologia , RatosRESUMO
INTRODUCTION: Core competencies have progressively gained importance in medical education. In other contexts, especially personnel selection and development, assessment centers (ACs) are used to assess competencies, but there is only a limited number of studies on competency-based ACs in medical education. To the best of our knowledge, the present study provides the first data on the criterion-related validity of a competency-based AC in medical education. METHODS: We developed an AC tailored to measure core competencies relevant to medical education (social-ethical, communicative, self, and teaching) and tested its validity in n=30 first-year medical students using 3- to 4-year follow-up measures such as (a) objective structured clinical examinations (OSCE) on basic clinical skills (n=26), (b) OSCE on communication skills (n=21), and (c) peer feedback (n=18). The AC contained three elements: interview, group discussion, and role play. Additionally, a self-report questionnaire was provided as a basis for the interview. RESULTS: Baseline AC average score and teaching competency correlated moderately with the communication OSCE average score (r=0.41, p=0.03, and r=0.38, p=0.04, respectively). Social-ethical competency in the AC showed a very strong convergent association with the communication OSCE average score (r=0.60, p<0.01). The AC total score also showed a moderate correlation with the overall peer feedback score provided in Year 4 (r=0.38, p=0.06). In addition, communicative competency correlated strongly with the overall peer feedback (r=0.50, p=0.02). We found predominantly low and insignificant correlations between the AC and the OSCE on basic clinical skills (r=-0.33 to 0.30, all p's>0.05). CONCLUSION: The results showed that competency-based ACs can be used at a very early stage of medical training to successfully predict future performance in core competencies.
Assuntos
Competência Clínica , Educação Baseada em Competências/organização & administração , Educação Médica/organização & administração , Avaliação Educacional , Competência Clínica/normas , Currículo , Feminino , Seguimentos , Alemanha , Humanos , Masculino , Pesquisa Qualitativa , Inquéritos e QuestionáriosRESUMO
Despite zinc ions being redox inert in biologic systems, zinc-finger structures act as redox-sensitive molecular switches controlling several crucial cellular processes. Oxidative or nitrosative stress, via modification of zinc finger cysteine thiols, leads to a release of Zn(2+) from these structures, causing not only a loss of zinc-finger function but also an increase of cytoplasmic or nuclear free Zn(2+) that may, in turn, stimulate and interfere with cellular signaling cascades. A signaling cascade stimulated by exposure of cells to zinc ions or to stressful stimuli that are reported to cause an intracellular release of zinc ions involves phosphoinositide 3'-kinases and the Ser/Thr protein kinase Akt, resulting in an inactivation of transcriptional regulators of the FoxO family. Possible modes of action of zinc ions to stimulate this signaling cascade and consequences of stimulation are discussed. Moreover, we present an overview on human diseases or disorders characterized by an intracellular Zn(2+) dyshomeostasis.
Assuntos
Dedos de Zinco , Zinco/metabolismo , Humanos , Luz , Oxirredução , Transdução de SinaisRESUMO
Silver compounds used as topical antimicrobial agents are known to exert toxic effects on skin cells. The aim of this study was to investigate whether the toxicity of silver ions, in analogy to other transition metal ions, depends on pro-oxidant effects. We treated human skin fibroblasts with concentrations of AgNO(3) not affecting cell proliferation, mitochondrial activity, or cell viability and found that Ag(+) strongly increases the production of reactive oxygen species, including superoxide anion radicals. These effects correspond to a strong decrease in intracellular reduced glutathione and to an increased susceptibility to H(2)O(2)-induced cell death. In addition, AgNO(3) down-regulates the expression of antioxidant genes such as the transcription factor Nrf2 and its target gene glutamate-cysteine ligase catalytic subunit. Furthermore Ag(+) induces a transient intracellular zinc release and increases the mRNA and protein expression of the zinc-binding protein metallothionein by activating the metal-responsive transcription factor 1, as verified by RNA interference. In conclusion, we show for the first time that Ag(+) induces oxidative stress and affects intracellular zinc homeostasis in human skin fibroblasts. The understanding of the mechanism involved in silver toxicity might contribute to new strategies for managing the therapy of skin infections.
Assuntos
Anti-Infecciosos Locais/farmacologia , Fibroblastos/metabolismo , Estresse Oxidativo , Nitrato de Prata/farmacologia , Zinco/metabolismo , Adulto , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Regulação da Expressão Gênica , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa/metabolismo , Humanos , Infecções/tratamento farmacológico , Íons , Metalotioneína/biossíntese , Pessoa de Meia-Idade , Fator 2 Relacionado a NF-E2/biossíntese , Fator 2 Relacionado a NF-E2/genética , RNA Interferente Pequeno/genética , Pele/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator MTF-1 de TranscriçãoRESUMO
Impaired cutaneous nitric oxide (NO) production is associated with diminished antioxidative capacity in skin cells, hindered wound healing, unbalanced inflammatory reactions, and disturbed immunological responses. Use of topically applied NO donors might represent an auspicious new therapeutic approach in the field of dermatology. But what is the appropriate NO-generating compound or system? In this issue, Mowbray et al. describe a new chemical inert NO donor that per se produces little inflammation in the skin.
Assuntos
Doadores de Óxido Nítrico/uso terapêutico , Óxido Nítrico/metabolismo , Pele/metabolismo , Cicatrização/efeitos dos fármacos , Humanos , Cicatrização/fisiologiaRESUMO
Various stressful conditions like oxidative or nitrosative stress, heavy metal load or thiol-modifying compounds have been shown to disturb the intracellular zinc homeostasis leading to increasing concentrations of free zinc within the cytoplasm or nuclei of cells. However, much less is known about the consequences of a disturbed intracellular Zn2+ homeostasis under these conditions. Current knowledge is reviewed here.
Assuntos
Zinco/metabolismo , Animais , Homeostase , Humanos , Metais Pesados/toxicidade , Camundongos , Óxido Nítrico/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismoRESUMO
The impact of nitric oxide (NO) synthesized after activation by proinflammatory cytokines and/or bacterial products by an inducible NO synthase (iNOS) is still contradictory. Expression of iNOS in inflammatory reactions is often found predominantly in cells of epithelial origin, and in these cases NO may serve as a protective agent limiting pathogen spreading, downregulating local inflammatory reactions by inducing production of Th2-like responses in a classical feedback circle, or limiting tissue damage during stress conditions. However, an abundant amount of data on chronic human disorders with predominant proinflammatory Th1-like reactions points to a destructive role of iNOS activity calling for a specific inhibition. Various methods to inhibit iNOS have been established to elucidate a protective versus a destructive role of NO during various stresses. In this review, we focus on antisense (AS)-mediated gene knock-down as a relatively new method to inhibit NO production and summarize the techniques applied and their successes. At least in theory, it provides a specific, rapid, and potentially high-throughput method for inhibiting gene expression and function. We here discuss the opportunities of iNOS-directed AS-ODN, and extensively deal with limitations and experimental problems.
Assuntos
Regulação Enzimológica da Expressão Gênica , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Animais , Humanos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II , Oligonucleotídeos Antissenso/genética , TransfecçãoRESUMO
Zn2+ is critical for the functional and structural integrity of cells and contributes to a number of important processes including gene expression. It has been shown that NO exogenously applied via NO donors resulting in nitrosative stress leads to cytoplasmic Zn2+ release from the zinc storing protein metallothionein (MT) and probably other proteins that complex Zn2+ via cysteine thiols. We show here that, in cytokine-activated murine aortic endothelial cells, NO derived from the inducible NO synthase (iNOS) induces a transient nuclear release of Zn2+. This nuclear Zn2+ release depends on the presence of MT as shown by the lack of this effect in activated endothelial cells from MT-deficient mice and temporally correlates with nuclear MT translocation. Data also show that NO is an essential but not sufficient signal for MT-mediated Zn2+ trafficking from the cytoplasm into the nucleus. In addition, we found that, endogenously via iNOS, synthesized NO increases the constitutive mRNA expression of both MT-1 and MT-2 genes and that nitrosative stress exogenously applied via an NO donor increases constitutive MT mRNA expression via intracellular Zn2+ release. In conclusion, we here provide evidence for a signaling mechanism based on iNOS-derived NO through the regulation of intracellular Zn2+ trafficking and homeostasis.