RESUMO
Zero-grazing (ZG; the mechanical harvesting and feeding of fresh grass) is increasingly used in grass-based milk production systems alongside conventional grazing. It allows farmers to supply fresh grass from land parcels that are outside of the main grazing block during seasonal shortages and periods when climatic conditions limit animal grazing opportunities. The objective of this study was to establish an understanding of current ZG practices on Irish dairy farms, to capture farmer perceptions on the implementation of this management practice, and to identify farmer knowledge requirements on ZG. An online survey was distributed and completed by 130 dairy farmers who use or have used ZG. Zero-grazing was used alongside conventional grazing by 92% of respondents. These farms were particularly fragmented, with between 1 and 14 separate land blocks. Respondents felt ZG helped them overcome fragmentation, increase grass use, and extend grass feeding in spring and autumn. However, extra cost and time input associated with ZG were recognized as key challenges. The majority of respondents rated current technical information available on ZG in the Republic of Ireland as "poor" or "very poor," and knowledge deficits were identified in the areas of cost analysis, grass management and productivity, cow productivity, cow health and nutrition, and soil fertility.
Assuntos
Indústria de Laticínios , Lactação , Ração Animal/análise , Animais , Bovinos , Dieta , Fazendeiros , Fazendas , Feminino , Humanos , Irlanda , Leite , Percepção , Inquéritos e QuestionáriosRESUMO
Virtual fencing (VF) technology is gaining interest due to its potential to facilitate sustainable grazing management. It allows farmers to contain grazing livestock without physical fences, thereby reducing the time and labour associated with the implementation of conventional fences. From a conservation perspective, some sensitive areas within uplands should not be grazed during certain periods of the year, and VF provides an invisible and moveable fence line that can exclude livestock from these areas. However, there are also concerns associated with its use, including animal welfare impacts, cost-effectiveness, and public perception. The extent to which VF can contribute to make livestock systems more sustainable remains to be investigated. To address this gap, this study investigates the potential of VF to promote sustainable grazing management using the Efficiency, Substitution, and Redesign framework, which has been used for the first time in this context. The framework is particularly relevant in taking an active and normative approach to identify key aspects to focus on to help achieve sustainability. We consulted stakeholders including farmers, wildlife inspectors, veterinarians, policy officers, researchers, NGOs, farm advisors or certification managers, through focus groups (N = 4) and in-depth, semi-structured interviews (N = 5). Stakeholders have highlighted the potential of VF to provide new opportunities to increase the efficiency and sustainability of livestock grazing systems, enabling their redesign, and contributing to improved environmental and animal welfare outcomes, as well as higher financial and social performance. However, there are important aspects that remain to be addressed to achieve such redesign, including issues of reliability due to poor network signal, animals' ability to learn, biosecurity and safety issues related to the absence of physical fences, farm suitability and farmers' ability to use the systems effectively. This study highlights the need to ensure that the development and uptake of VF are mutually beneficial to farmers, animals, and the wider farming industry. This includes a highlight on the importance of participative approaches to involve key stakeholders to address concerns and maximise the potential of the technology.
RESUMO
Death effector domains (DEDs) are protein-protein interaction domains initially identified in proteins such as FADD, FLIP and caspase-8 involved in regulating apoptosis. Subsequently, these proteins have been shown to have important roles in regulating other forms of cell death, including necroptosis, and in regulating other important cellular processes, including autophagy and inflammation. Moreover, these proteins also have prominent roles in innate and adaptive immunity and during embryonic development. In this article, we review the various roles of DED-containing proteins and discuss recent developments in our understanding of DED complex formation and regulation. We also briefly discuss opportunities to therapeutically target DED complex formation in diseases such as cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Caspase 8/química , Proteína de Domínio de Morte Associada a Fas/química , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Caspase 8/genética , Caspase 8/metabolismo , Ensaios Clínicos como Assunto , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Humanos , Camundongos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de SinaisRESUMO
Death receptor activation triggers recruitment of FADD, which via its death effector domain (DED) engages the DEDs of procaspase 8 and its inhibitor FLIP to form death-inducing signalling complexes (DISCs). The DEDs of FADD, FLIP and procaspase 8 interact with one another using two binding surfaces defined by α1/α4 and α2/α5 helices, respectively. Here we report that FLIP has preferential affinity for the α1/α4 surface of FADD, whereas procaspase 8 has preferential affinity for FADD's α2/α5 surface. These relative affinities contribute to FLIP being recruited to the DISC at comparable levels to procaspase 8 despite lower cellular expression. Additional studies, including assessment of DISC stoichiometry and functional assays, suggest that following death receptor recruitment, the FADD DED preferentially engages FLIP using its α1/α4 surface and procaspase 8 using its α2/α5 surface; these tripartite intermediates then interact via the α1/α4 surface of FLIP DED1 and the α2/α5 surface of procaspase 8 DED2.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Western Blotting , Cromatografia em Gel , Células HCT116 , Humanos , Imunoprecipitação , Ligação ProteicaRESUMO
Malignant pleural mesothelioma (MPM) is a highly pro-inflammatory malignancy that is rapidly fatal and increasing in incidence. Cytokine signaling within the pro-inflammatory tumor microenvironment makes a critical contribution to the development of MPM and its resistance to conventional chemotherapy approaches. SMAC mimetic compounds (SMCs) are a promising class of anticancer drug that are dependent on tumor necrosis factor alpha (TNFα) signaling for their activity. As circulating TNFα expression is significantly elevated in MPM patients, we examined the sensitivity of MPM cell line models to SMCs. Surprisingly, all MPM cell lines assessed were highly resistant to SMCs either alone or when incubated in the presence of clinically relevant levels of TNFα. Further analyses revealed that MPM cells were sensitized to SMC-induced apoptosis by siRNA-mediated downregulation of the caspase 8 inhibitor FLIP, an antiapoptotic protein overexpressed in several cancer types including MPM. We have previously reported that FLIP expression is potently downregulated in MPM cells in response to the histone deacetylase inhibitor (HDACi) Vorinostat (SAHA). In this study, we demonstrate that SAHA sensitizes MPM cells to SMCs in a manner dependent on its ability to downregulate FLIP. Although treatment with SMC in the presence of TNFα promoted interaction between caspase 8 and the necrosis-promoting RIPK1, the cell death induced by combined treatment with SAHA and SMC was apoptotic and mediated by caspase 8. These results indicate that FLIP is a major inhibitor of SMC-mediated apoptosis in MPM, but that this inhibition can be overcome by the HDACi SAHA.
Assuntos
Antineoplásicos/farmacologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Caspase 3/metabolismo , Caspase 8/metabolismo , Linhagem Celular Tumoral , Cisplatino/farmacologia , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Ativação Enzimática , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Mesotelioma , Mimetismo Molecular , Neoplasias Pleurais , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , VorinostatRESUMO
Non-small cell lung carcinoma remains by far the leading cause of cancer-related deaths worldwide. Overexpression of FLIP, which blocks the extrinsic apoptotic pathway by inhibiting caspase-8 activation, has been identified in various cancers. We investigated FLIP and procaspase-8 expression in NSCLC and the effect of HDAC inhibitors on FLIP expression, activation of caspase-8 and drug resistance in NSCLC and normal lung cell line models. Immunohistochemical analysis of cytoplasmic and nuclear FLIP and procaspase-8 protein expression was carried out using a novel digital pathology approach. Both FLIP and procaspase-8 were found to be significantly overexpressed in tumours, and importantly, high cytoplasmic expression of FLIP significantly correlated with shorter overall survival. Treatment with HDAC inhibitors targeting HDAC1-3 downregulated FLIP expression predominantly via post-transcriptional mechanisms, and this resulted in death receptor- and caspase-8-dependent apoptosis in NSCLC cells, but not normal lung cells. In addition, HDAC inhibitors synergized with TRAIL and cisplatin in NSCLC cells in a FLIP- and caspase-8-dependent manner. Thus, FLIP and procaspase-8 are overexpressed in NSCLC, and high cytoplasmic FLIP expression is indicative of poor prognosis. Targeting high FLIP expression using HDAC1-3 selective inhibitors such as entinostat to exploit high procaspase-8 expression in NSCLC has promising therapeutic potential, particularly when used in combination with TRAIL receptor-targeted agents.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Caspase 8/metabolismo , Western Blotting , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Caspase 8/genética , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Citometria de Fluxo , Humanos , Técnicas In Vitro , Estudos RetrospectivosRESUMO
FLIP is a potential anti-cancer therapeutic target that inhibits apoptosis by blocking caspase 8 activation by death receptors. We report a novel interaction between FLIP and the DNA repair protein Ku70 that regulates FLIP protein stability by inhibiting its polyubiquitination. Furthermore, we found that the histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) enhances the acetylation of Ku70, thereby disrupting the FLIP/Ku70 complex and triggering FLIP polyubiquitination and degradation by the proteasome. Using in vitro and in vivo colorectal cancer models, we further demonstrated that SAHA-induced apoptosis is dependant on FLIP downregulation and caspase 8 activation. In addition, an HDAC6-specific inhibitor Tubacin recapitulated the effects of SAHA, suggesting that HDAC6 is a key regulator of Ku70 acetylation and FLIP protein stability. Thus, HDAC inhibitors with anti-HDAC6 activity act as efficient post-transcriptional suppressors of FLIP expression and may, therefore, effectively act as 'FLIP inhibitors'.
Assuntos
Antígenos Nucleares/metabolismo , Apoptose/efeitos dos fármacos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Proteínas de Ligação a DNA/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Acetilação , Sequência de Aminoácidos , Animais , Antígenos Nucleares/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/biossíntese , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Caspase 8/metabolismo , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Feminino , Células HCT116 , Células HT29 , Desacetilase 6 de Histona , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Autoantígeno Ku , Camundongos , Camundongos Endogâmicos BALB C , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transfecção , VorinostatRESUMO
We found that procaspase 8 was overexpressed in non-small-cell lung cancers (NSCLCs) compared with matched normal tissues. The caspase 8 inhibitor FLICE-inhibitory protein (FLIP) was also overexpressed in the majority of NSCLCs. Silencing FLIP induced caspase 8 activation and apoptosis in NSCLC cell lines, but not in normal lung cell lines. Apoptosis induced by FLIP silencing was mediated by the TRAIL death receptors DR4 and DR5, but was not dependent on ligation of the receptors by TRAIL. Furthermore, the apoptosis induced by FLIP silencing was dependent on the overexpression of procaspase 8 in NSCLC cells. Moreover, in NSCLC cells, but not in normal cells, FLIP silencing induced co-localization of DR5 and ceramide, and disruption of this co-localization abrogated apoptosis. FLIP silencing supra-additively increased TRAIL-induced apoptosis of NSCLC cells; however, normal lung cells were resistant to TRAIL, even when FLIP was silenced. Importantly, FLIP silencing sensitized NSCLC cells but not normal cells to chemotherapy in vitro, and silencing FLIP in vivo retarded NSCLC xenograft growth and enhanced the anti-tumour effects of cisplatin. Collectively, our results suggest that due to frequent procaspase 8 overexpression, NSCLCs may be particularly sensitive to FLIP-targeted therapies.
Assuntos
Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Caspase 8/metabolismo , Neoplasias Pulmonares/enzimologia , Animais , Antineoplásicos/farmacologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/fisiologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Caspase 8/genética , Caspase 8/fisiologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Precursores Enzimáticos/metabolismo , Feminino , Citometria de Fluxo , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Transplante HeterólogoRESUMO
Pro-inflammatory cytokines are implicated as the main mediators of beta-cell death during type 1 diabetes but the exact mechanisms remain unknown. This study examined the effects of interleukin-1beta (IL-1beta), interferon-gamma (IFNgamma) and tumour necrosis factor alpha (TNFalpha) on a rat insulinoma cell line (RIN-r) in order to identify the core mechanism of cytokine-induced beta-cell death. Treatment of cells with a combination of IL-1beta and IFNgamma (IL-1beta/IFNgamma)induced apoptotic cell death. TNFalpha neither induced beta-cell death nor did it potentiate the effects of IL-1beta, IFNgamma or IL-1beta/IFNgamma . The cytotoxic effect of IL-1beta/IFNgamma was associated with the expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide. Adenoviral-mediated expression of iNOS (AdiNOS) alone was sufficient to induce caspase activity and apoptosis. The broad range caspase inhibitor, Boc-D-fmk, blocked IL-1beta/IFNgamma -induced caspase activity, but not nitric oxide production nor cell death. However, pre-treatment with L-NIO, a NOS inhibitor, prevented nitric oxide production, caspase activity and reduced apoptosis. IL-1beta/IFNgamma -induced apoptosis was accompanied by loss of mitochondrial membrane potential, release of cytochrome c and cleavage of pro-caspase-9, -7 and -3. Transduction of cells with Ad-Bcl-X(L) blocked both iNOS and cytokine-mediated mitochondrial changes and subsequent apoptosis, downstream of nitric oxide. We conclude that cytokine-induced nitric oxide production is both essential and sufficient for caspase activation and beta-cell death, and have identified Bcl-X(L) as a potential target to combat beta-cell apoptosis.
Assuntos
Apoptose/fisiologia , Citocinas/farmacologia , Ilhotas Pancreáticas/metabolismo , Mitocôndrias/metabolismo , Proteína bcl-X/metabolismo , Adenoviridae/genética , Animais , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Insulinoma/patologia , Ilhotas Pancreáticas/citologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia de Fluorescência , Modelos Biológicos , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , RatosRESUMO
Tightly controlled proteolysis is a defining feature of apoptosis and caspases are critical in this regard. Significant roles for non-caspase proteases in cell death have been highlighted. Staurosporine causes a rapid induction of apoptosis in virtually all mammalian cell types. Numerous studies demonstrate that staurosporine can activate cell death under caspase-inhibiting circumstances. The aim of this study was to investigate the proteolytic mechanisms responsible for cell death under these conditions. To that end, we show that inhibitors of serine proteases can delay cell death in one such system. Furthermore, through profiling of proteolytic activation, we demonstrate, for the first time, that staurosporine activates a chymotrypsin-like serine protease-dependent cell death in HL-60 cells independently, but in parallel with the caspase controlled systems. Features of the serine protease-mediated system include cell shrinkage and apoptotic morphology, regulation of caspase-3, altered nuclear morphology, generation of an endonuclease and DNA degradation. We also demonstrate a staurosporine-induced activation of a putative 16 kDa chymotrypsin-like protein during apoptosis.