RESUMO
Insig-1 and Insig-2 are endoplasmic reticulum (ER) proteins that inhibit lipid synthesis by blocking transport of sterol regulatory element-binding proteins (SREBP-1 and SREBP-2) from ER to Golgi. In the Golgi, SREBPs are processed proteolytically to release their transcription-activating domains, which enhance the synthesis of fatty acids, triglycerides, and cholesterol. Heretofore, the two Insigs have redundant functions, and there is no rationale for two isoforms. The current data identify a specific function for Insig-2. We show that eicosapentaenoic acid (EPA), a polyunsaturated fatty acid, inhibits fatty acid synthesis in human fibroblasts and rat hepatocytes by activating adenylate cyclase, which induces protein kinase A (PKA) to phosphorylate serine-106 in Insig-2. Phosphorylated Insig-2 inhibits the proteolytic processing of SREBP-1, thereby blocking fatty acid synthesis. Phosphorylated Insig-2 does not block the processing of SREBP-2, which activates cholesterol synthesis. Insig-1 lacks serine-106 and is not phosphorylated at this site. EPA inhibition of SREBP-1 processing was reduced by the replacement of serine-106 in Insig-2 with alanine or by treatment with KT5720, a PKA inhibitor. Inhibition did not occur in mutant human fibroblasts that possess Insig-1 but lack Insig-2. These data provide an Insig-2-specific mechanism for the long-known inhibition of fatty acid synthesis by polyunsaturated fatty acids.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico , Fibroblastos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Proteína de Ligação a Elemento Regulador de Esterol 1 , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Animais , Fosforilação , Ratos , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fibroblastos/metabolismo , Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos/metabolismo , Ácidos Graxos/biossíntese , Ácido Eicosapentaenoico/farmacologia , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Hepatócitos/metabolismoRESUMO
3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is the rate-limiting enzyme in cholesterol synthesis and target of cholesterol-lowering statin drugs. Accumulation of sterols in endoplasmic reticulum (ER) membranes accelerates degradation of HMGCR, slowing the synthesis of cholesterol. Degradation of HMGCR is inhibited by its binding to UBIAD1 (UbiA prenyltransferase domain-containing protein-1). This inhibition contributes to statin-induced accumulation of HMGCR, which limits their cholesterol-lowering effects. Here, we report cryo-electron microscopy structures of the HMGCR-UBIAD1 complex, which is maintained by interactions between transmembrane helix (TM) 7 of HMGCR and TMs 2-4 of UBIAD1. Disrupting this interface by mutagenesis prevents complex formation, enhancing HMGCR degradation. TMs 2-6 of HMGCR contain a 170-amino acid sterol sensing domain (SSD), which exists in two conformations-one of which is essential for degradation. Thus, our data supports a model that rearrangement of the TMs in the SSD permits recruitment of proteins that initate HMGCR degradation, a key reaction in the regulatory system that governs cholesterol synthesis.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , Colesterol/metabolismo , Microscopia Crioeletrônica , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Esteróis/metabolismoRESUMO
The polytopic, endoplasmic reticulum (ER) membrane protein 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase produces mevalonate, the key intermediate in the synthesis of cholesterol and many nonsterol isoprenoids including geranylgeranyl pyrophosphate (GGpp). Transcriptional, translational, and posttranslational feedback mechanisms converge on this reductase to ensure cells maintain a sufficient supply of essential nonsterol isoprenoids but avoid overaccumulation of cholesterol and other sterols. The focus of this review is mechanisms for the posttranslational regulation of HMG CoA reductase, which include sterol-accelerated ubiquitination and ER-associated degradation (ERAD) that is augmented by GGpp. We discuss how GGpp-induced ER-to-Golgi trafficking of the vitamin K2 synthetic enzyme UbiA prenyltransferase domain-containing protein-1 (UBIAD1) modulates HMG CoA reductase ERAD to balance the synthesis of sterol and nonsterol isoprenoids. We also summarize the characterization of genetically manipulated mice, which established that sterol-accelerated, UBIAD1-modulated ERAD plays a major role in regulation of HMG CoA reductase and cholesterol metabolism in vivo.
Assuntos
Colesterol/biossíntese , Degradação Associada com o Retículo Endoplasmático/fisiologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Animais , Dimetilaliltranstransferase/metabolismo , Degradação Associada com o Retículo Endoplasmático/efeitos dos fármacos , Humanos , Hidroximetilglutaril-CoA Redutases/química , Hidroximetilglutaril-CoA Redutases/genética , Camundongos , Fosfatos de Poli-Isoprenil/metabolismo , Processamento de Proteína Pós-Traducional , Esteróis/metabolismo , Terpenos/metabolismo , Terpenos/farmacologia , UbiquitinaçãoRESUMO
Attributable mortality is an important metric that mirrors the public health effect of a potentially harmful infection by accounting not only for the effect of infection on mortality, but also for its prevalence within the target population. We did a systematic literature review to identify how attributable deaths were quantified and interpreted in core clinical infectious diseases journals between Jan 1, 2013, and April 6, 2020. Of the 1591 abstracts screened, 234 entered the primary analysis. Our summary of the epidemiological measures used in these articles reveals fundamental shortcomings in the conception of attributable mortality. Because of its importance as a basis for decision making on public health matters, it is essential to correctly quantify and report on attributable mortality. Our recommendation for quantification and reporting of attributable deaths aids clinical researchers in the correct statistical assessment of the burden of infections. Fictional as well as real data is used to illustrate these issues.
Assuntos
Doenças Transmissíveis/mortalidade , Interpretação Estatística de Dados , Humanos , Medição de Risco , Fatores de RiscoRESUMO
The autosomal dominant disorder Schnyder corneal dystrophy (SCD) is caused by mutations in UbiA prenyltransferase domain-containing protein-1 (UBIAD1), which uses geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K2 subtype menaquinone-4 (MK-4). SCD is characterized by opacification of the cornea, owing to aberrant build-up of cholesterol in the tissue. We previously discovered that sterols stimulate association of UBIAD1 with ER-localized HMG-CoA reductase, which catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids, including GGpp. Binding to UBIAD1 inhibits sterol-accelerated ER-associated degradation (ERAD) of reductase and permits continued synthesis of GGpp in cholesterol-replete cells. GGpp disrupts UBIAD1-reductase binding and thereby allows for maximal ERAD of reductase as well as ER-to-Golgi translocation of UBIAD1. SCD-associated UBIAD1 is refractory to GGpp-mediated dissociation from reductase and remains sequestered in the ER to inhibit ERAD. Here, we report development of a biochemical assay for UBIAD1-mediated synthesis of MK-4 in isolated membranes and intact cells. Using this assay, we compared enzymatic activity of WT UBIAD1 with that of SCD-associated variants. Our studies revealed that SCD-associated UBIAD1 exhibited reduced MK-4 synthetic activity, which may result from its reduced affinity for GGpp. Sequestration in the ER protects SCD-associated UBIAD1 from autophagy and allows intracellular accumulation of the mutant protein, which amplifies the inhibitory effect on reductase ERAD. These findings have important implications not only for the understanding of SCD etiology but also for the efficacy of cholesterol-lowering statin therapy, which becomes limited, in part, because of UBIAD1-mediated inhibition of reductase ERAD.
Assuntos
Autofagia/genética , Distrofias Hereditárias da Córnea/genética , Dimetilaliltranstransferase/genética , Dimetilaliltranstransferase/metabolismo , Variação Genética , Proteólise , Vitamina K 2/análogos & derivados , Linhagem Celular , Humanos , Espaço Intracelular/metabolismo , Transporte Proteico , Vitamina K 2/metabolismoRESUMO
This study aims to identify bioactive anticancer and anti-trypanosome secondary metabolites from the fermentation culture of Aspergillus flocculus endophyte assisted by modern metabolomics technologies. The endophyte was isolated from the stem of the medicinal plant Markhamia platycalyx and identified using phylogenetics. Principle component analysis was employed to screen for the optimum growth endophyte culturing conditions and revealing that the 30-days rice culture (RC-30d) provided the highest levels of the bioactive agents. To pinpoint for active chemicals in endophyte crude extracts and successive fractions, a new application of molecular interaction network is implemented to correlate the chemical and biological profiles of the anti-trypanosome active fractions to highlight the metabolites mediating for bioactivity prior to purification trials. Multivariate data analysis (MVDA), with the aid of dereplication studies, efficiently annotated the putatively active anticancer molecules. The small-scale RC-30d fungal culture was purified using high-throughput chromatographic techniques to yield compound 1, a novel polyketide molecule though inactive. Whereas, active fractions revealed from the bioactivity guided fractionation of medium scale RC-30d culture were further purified to yield 7 metabolites, 5 of which namely cis-4-hydroxymellein, 5-hydroxymellein, diorcinol, botryoisocoumarin A and mellein, inhibited the growth of chronic myelogenous leukemia cell line K562 at 30⯵M. 3-Hydroxymellein and diorcinol exhibited a respective inhibition of 56% and 97% to the sleeping sickness causing parasite Trypanosoma brucei brucei. More interestingly, the anti-trypanosomal activity of A. flocculus extract appeared to be mediated by the synergistic effect of the active steroidal compounds i.e. ergosterol peroxide, ergosterol and campesterol. The isolated structures were elucidated by using 1D, 2D NMR and HR-ESIMS.
Assuntos
Antineoplásicos/isolamento & purificação , Aspergillus/química , Endófitos/química , Tripanossomicidas/isolamento & purificação , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Meios de Cultura , Fermentação , Humanos , Células K562 , Células PC-3 , Metabolismo Secundário , Tripanossomicidas/metabolismo , Tripanossomicidas/farmacologiaRESUMO
INTRODUCTION: Evidence-based guidelines or protocols regarding the perinatal management of babies born with gastroschisis are lacking. The aim of this work is to evaluate the different current treatment modalities for newborns with gastroschisis during the perinatal period in the German-speaking countries Germany, Austria, and Switzerland. These data could serve as a starting point for the development of a multicenter randomized controlled trial. MATERIALS AND METHODS: A questionnaire was developed with 30 questions divided into five sections: (1) prenatal diagnosis, (2) fetal therapy, (3) mode and timing of delivery, (4) operative management, and (5) postoperative management. All pediatric surgery institutions that treat newborns with gastroschisis were identified and asked to participate. Data were categorized by country and analyzed using descriptive statistics (frequency and percentage). RESULTS: The return rate of the questionnaire was 95% (89 hospitals). A standard procedure was identified regarding prenatal ultrasound monitoring, interdisciplinary team approach, planned delivery through cesarean section, postnatal coverage of the intestine with a silastic bag, first intervention within the first 6 hours after birth, attempt of primary abdominal wall closure, and perioperative antibiotic treatment. For many crucial parameters, management was not standardized. CONCLUSIONS: There is no gold standard in German-speaking countries on how to manage fetuses and babies with gastroschisis. Moreover, this report unveils some questionable elements of daily practice for which there is no evidence at all and which can jeopardize outcome and even prove fatal (fetal therapy, preterm delivery, lack of abdominal pressure monitoring). Prospective randomized-controlled multicenter studies are needed to set a standard.
Assuntos
Gastrosquise/cirurgia , Atresia Intestinal/cirurgia , Intestinos/cirurgia , Áustria , Criptorquidismo/complicações , Criptorquidismo/cirurgia , Parto Obstétrico/estatística & dados numéricos , Feminino , Terapias Fetais , Gastrosquise/complicações , Gastrosquise/diagnóstico , Alemanha , Idade Gestacional , Humanos , Recém-Nascido , Atresia Intestinal/complicações , Masculino , Cuidados Pós-Operatórios , Guias de Prática Clínica como Assunto , Gravidez , Diagnóstico Pré-Natal , Inquéritos e Questionários , SuíçaRESUMO
UBIAD1 (UbiA prenyltransferase domain-containing protein-1) utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize vitamin K2 We previously reported that sterols stimulate binding of UBIAD1 to endoplasmic reticulum (ER)-localized 3-hydroxy-3-methylglutaryl (HMG) CoA reductase. UBIAD1 binding inhibits sterol-accelerated, ER-associated degradation (ERAD) of reductase, one of several mechanisms for feedback control of this rate-limiting enzyme in the branched pathway that produces cholesterol and nonsterol isoprenoids such as GGpp. Accumulation of GGpp in ER membranes triggers release of UBIAD1 from reductase, permitting its maximal ERAD and ER-to-Golgi transport of UBIAD1. Mutant UBIAD1 variants associated with Schnyder corneal dystrophy (SCD), a human disorder characterized by corneal accumulation of cholesterol, resist GGpp-induced release from reductase and remain sequestered in the ER to block reductase ERAD. Using lines of genetically manipulated cells, we now examine consequences of UBIAD1 deficiency and SCD-associated UBIAD1 on reductase ERAD and cholesterol synthesis. Our results indicated that reductase becomes destabilized in the absence of UBIAD1, resulting in reduced cholesterol synthesis and intracellular accumulation. In contrast, an SCD-associated UBIAD1 variant inhibited reductase ERAD, thereby stabilizing the enzyme and contributing to enhanced synthesis and intracellular accumulation of cholesterol. Finally, we present evidence that GGpp-regulated, ER-to-Golgi transport enables UBIAD1 to modulate reductase ERAD such that synthesis of nonsterol isoprenoids is maintained in sterol-replete cells. These findings further establish UBIAD1 as a central player in the reductase ERAD pathway and regulation of isoprenoid synthesis. They also indicate that UBIAD1-mediated inhibition of reductase ERAD underlies cholesterol accumulation associated with SCD.
Assuntos
Dimetilaliltranstransferase/metabolismo , Hidroximetilglutaril-CoA Redutases/metabolismo , Esteróis/biossíntese , Terpenos/metabolismo , Células Cultivadas , Colesterol/metabolismo , Dimetilaliltranstransferase/genética , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático/efeitos dos fármacos , Degradação Associada com o Retículo Endoplasmático/fisiologia , Fibroblastos/metabolismo , Complexo de Golgi/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Ácido Mevalônico/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Esteróis/metabolismo , Vitamina K 2/metabolismoRESUMO
Endophytic fungi associated with medicinal plants are a potential source of novel chemistry and biology that may find applications as pharmaceutical and agrochemical drugs. In this study, a combination of metabolomics and bioactivity-guided approaches were employed to isolate secondary metabolites with cytotoxicity against cancer cells from an endophytic Aspergillus aculeatus. The endophyte was isolated from the Egyptian medicinal plant Terminalia laxiflora and identified using molecular biological methods. Metabolomics and dereplication studies were accomplished by utilizing the MZmine software coupled with the universal Dictionary of Natural Products database. Metabolic profiling, with aid of multivariate data analysis, was performed at different stages of the growth curve to choose the optimized method suitable for up-scaling. The optimized culture method yielded a crude extract abundant with biologically-active secondary metabolites. Crude extracts were fractionated using different high-throughput chromatographic techniques. Purified compounds were identified by HR-ESI-MS, 1D- and 2D-NMR. This study introduced a new method of dereplication utilizing both high-resolution mass spectrometry and NMR spectroscopy. The metabolites were putatively identified by applying a chemotaxonomic filter. We also present a short review on the diverse chemistry of terrestrial endophytic strains of Aspergillus, which has become a part of our dereplication work and this will be of wide interest to those working in this field.
Assuntos
Antibacterianos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Aspergillus/metabolismo , Bactérias/efeitos dos fármacos , Produtos Biológicos/farmacologia , Extratos Vegetais/farmacologia , Terminalia/química , Antibacterianos/química , Antibacterianos/metabolismo , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/metabolismo , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Metabolômica , Testes de Sensibilidade Microbiana , Estrutura Molecular , Extratos Vegetais/química , Extratos Vegetais/metabolismoRESUMO
UbiA prenyltransferase domain-containing protein-1 (UBIAD1) utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K2 subtype menaquinone-4. Previously, we found that sterols trigger binding of UBIAD1 to endoplasmic reticulum (ER)-localized HMG-CoA reductase, the rate-limiting enzyme in synthesis of cholesterol and nonsterol isoprenoids, including GGpp. This binding inhibits sterol-accelerated degradation of reductase, which contributes to feedback regulation of the enzyme. The addition to cells of geranylgeraniol (GGOH), which can become converted to GGpp, triggers release of UBIAD1 from reductase, allowing for its maximal degradation and permitting ER-to-Golgi transport of UBIAD1. Here, we further characterize geranylgeranyl-regulated transport of UBIAD1. Results of this characterization support a model in which UBIAD1 continuously cycles between the ER and medial-trans Golgi of isoprenoid-replete cells. Upon sensing a decline of GGpp in ER membranes, UBIAD1 becomes trapped in the organelle where it inhibits reductase degradation. Mutant forms of UBIAD1 associated with Schnyder corneal dystrophy (SCD), a human eye disease characterized by corneal accumulation of cholesterol, are sequestered in the ER and block reductase degradation. Collectively, these findings disclose a novel sensing mechanism that allows for stringent metabolic control of intracellular trafficking of UBIAD1, which directly modulates reductase degradation and becomes disrupted in SCD.
Assuntos
Distrofias Hereditárias da Córnea/genética , Dimetilaliltranstransferase/metabolismo , Hidroximetilglutaril-CoA Redutases/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Distrofias Hereditárias da Córnea/metabolismo , Distrofias Hereditárias da Córnea/patologia , Dimetilaliltranstransferase/genética , Retículo Endoplasmático/enzimologia , Complexo de Golgi/enzimologia , Humanos , Metabolismo dos Lipídeos/genética , Transporte Proteico/genética , Proteólise , Terpenos/metabolismo , Vitamina K/biossíntese , Vitamina K/metabolismo , Vitamina K 2/análogos & derivados , Vitamina K 2/metabolismoRESUMO
Schnyder corneal dystrophy (SCD) is an autosomal dominant disorder in humans characterized by abnormal accumulation of cholesterol in the cornea. SCD-associated mutations have been identified in the gene encoding UBIAD1, a prenyltransferase that synthesizes vitamin K2. Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism. Geranylgeraniol inhibits binding of UBIAD1 to reductase, allowing its degradation and promoting transport of UBIAD1 from the ER to the Golgi. CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation. SCD-associated mutations in UBIAD1 block its displacement from reductase in the presence of geranylgeraniol, thereby preventing degradation of reductase. The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD.
Assuntos
Dimetilaliltranstransferase/metabolismo , Diterpenos/farmacologia , Degradação Associada com o Retículo Endoplasmático/efeitos dos fármacos , Hidroximetilglutaril-CoA Redutases/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Colesterol/biossíntese , Distrofias Hereditárias da Córnea/genética , Distrofias Hereditárias da Córnea/metabolismo , Dimetilaliltranstransferase/genética , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático/genética , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Immunoblotting , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , Esteróis/farmacologiaRESUMO
Eurycomanone and eurycomanol are two quassinoids from the roots of Eurycoma longifolia Jack. The aim of this study was to assess the bioactivity of these compounds in Jurkat and K562 human leukemia cell models compared to peripheral blood mononuclear cells from healthy donors. Both eurycomanone and eurycomanol inhibited Jurkat and K562 cell viability and proliferation without affecting healthy cells. Interestingly, eurycomanone inhibited NF-κB signaling through inhibition of IκBα phosphorylation and upstream mitogen activated protein kinase (MAPK) signaling, but not eurycomanol. In conclusion, both quassinoids present differential toxicity towards leukemia cells, and the presence of the α,ß-unsaturated ketone in eurycomanone could be prerequisite for the NF-κB inhibition.
Assuntos
Eurycoma/química , Inflamação/tratamento farmacológico , Leucemia/tratamento farmacológico , Extratos Vegetais/administração & dosagem , Quassinas/administração & dosagem , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Inflamação/metabolismo , Células Jurkat , Leucemia/metabolismo , Leucemia/patologia , Leucócitos Mononucleares/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Extratos Vegetais/química , Quassinas/química , Transdução de Sinais/efeitos dos fármacosRESUMO
Here we describe the synthesis and biological activity of novel amino-pyrazolopyridines with anti-NF-κB and pro-apoptotic potential. α-Methylene ketones were used as a starting point for synthesis of amino-pyrazolopyridine 3. The alkylidene malononitriles 1 were obtained by the Knoevenagel reaction of ketones with malononitriles. Vilsmeier-Haack reaction allowed direct access to 2-chloro-3-cyanopyridines 2. Those products, by refluxing with hydrazine hydrate, allowed cyclization to amino-pyrazolopyridines 3a-g, which were not previously described in the literature. Bioactivity results indicated that amino-pyrazolopyridines 3a, 3b and 3g induced apoptotic cell death in K562 cancer cells with an IC50 of 36.5 ± 3.9 µM, 27.6 ± 4.5 µM and 35.0 ± 2.3 µM, respectively, after 72 h. In addition, compounds 3a, 3b and 3g exerted NF-κB inhibition activity with an IC50 of 4.7 ± 1.6 µM, 6.9 ± 1.9 µM and 39.8 ± 3.9 µM, respectively, after 8 h in K562 cells activated with TNFα. Compounds 3b and 3g showed interesting differential toxicity as viability of peripheral blood mononuclear cells (PBMCs) from healthy donors remained largely unaffected by this treatment.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Pirazóis/síntese química , Pirazóis/farmacologia , Piridinas/síntese química , Piridinas/farmacologia , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Técnicas de Química Sintética , Humanos , Concentração Inibidora 50 , Células K562 , Cetonas/química , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Pirazóis/química , Piridinas/química , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
OBJECTIVE: To evaluate whether surgical complications after kidney transplantation correlate with surgeon's experience and whether individual surgeons' complication rates improve during their learning process. STUDY DESIGN: Retrospective analysis: A generalized linear mixed-effects model was used to identify risk factors for surgical complications. Plots of cumulative sums of complications were used to evaluate the individual surgeons' performance. SETTING: Single-center experience of a teaching hospital in Switzerland. PARTICIPANTS: Consecutive kidney transplant recipients operated from 1962 until 2003. RESULTS: A total of 1496 kidney transplants were analyzed; 73% were from deceased donors and 27% from living donors. At least 1 surgical complication occurred in 352 patients (24%). Male gender (odds ratio [OR] = 1.35, 95% CI: 1.04-1.74), donor's age (OR = 1.14, 95% CI: 1.06-1.24 per decade increment), and third or fourth vs. first or second transplant in a recipient (OR = 2.90, 95% CI: 1.02-8.24) were significantly associated with surgical complications. The surgeon's transplant experience was not found to be associated with surgical complications. Even surgeons with an experience of less than 10 kidney transplants did not have higher complication rates, 30-day mortality, or 1-year graft survival. Individual surgeons' complication rates analyzed by cumulative sum plots did not improve with increasing experience. CONCLUSIONS: We present the largest single-center study on surgical complications after kidney transplantation, with unique data on the surgeon's experience for every single procedure. We found no evidence for a learning curve during training for kidney transplantation. We conclude that carefully selected experienced general and vascular surgeons can achieve good results in kidney transplantation after a relatively short training period.
Assuntos
Transplante de Rim/efeitos adversos , Transplante de Rim/educação , Curva de Aprendizado , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
(R)-(+)-Goniothalamin (GTN), a styryl-lactone isolated from the medicinal plant Goniothalamus macrophyllus, exhibits pharmacological activities including cytotoxic and anti-inflammatory effects. In this study, GTN modulated TNF-α induced NF-κB activation. GTN concentrations up to 20 µM showed low cytotoxic effects in K562 chronic myelogenous leukemia and in Jurkat T cells. Importantly, at these concentrations, no cytotoxicity was observed in healthy peripheral blood mononuclear cells. Our results confirmed that GTN inhibited tumor necrosis factor-α (TNF-α)-induced NF-κB activation in Jurkat and K562 leukemia cells at concentrations as low as 5 µM as shown by reporter gene assays and western blots. Moreover, GTN down-regulated translocation of the p50/p65 heterodimer to the nucleus, prevented binding of NF-κB to its DNA response element and reduced TNF-α-activated interleukin-8 (IL-8) expression. In conclusion, GTN inhibits TNF-α-induced NF-κB activation at non-apoptogenic concentrations in different leukemia cell models without presenting toxicity towards healthy blood cells underlining the anti-leukemic potential of this natural compound.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Descoberta de Drogas , Leucemia/tratamento farmacológico , NF-kappa B/metabolismo , Pironas/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Anti-Inflamatórios não Esteroides/efeitos adversos , Anti-Inflamatórios não Esteroides/isolamento & purificação , Antineoplásicos Fitogênicos/efeitos adversos , Antineoplásicos Fitogênicos/isolamento & purificação , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Genes Reporter/efeitos dos fármacos , Goniothalamus/química , Humanos , Interleucina-8/antagonistas & inibidores , Interleucina-8/metabolismo , Células Jurkat , Células K562 , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Malásia , NF-kappa B/agonistas , NF-kappa B/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Raízes de Plantas/química , Transporte Proteico/efeitos dos fármacos , Pironas/efeitos adversos , Pironas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Elementos de Resposta/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Metastases destroy the function of infested organs and are the main reason of cancer-related mortality. Heteronemin, a natural product derived from a marine sponge, was tested in vitro regarding its properties to prevent tumour cell intravasation through the lymph-endothelial barrier. In three-dimensional (3D) cell cultures consisting of MCF-7 breast cancer cell spheroids that were placed on lymph-endothelial cell (LEC) monolayers, tumour cell spheroids induce "circular chemorepellent-induced defects" (CCIDs) in the LEC monolayer; 12(S)-Hydroxyeicosatetraenoic acid (12(S)-HETE) and NF-κB activity are major factors inducing CCIDs, which are entry gates for tumour emboli intravasating the vasculature. This 3D co-culture is a validated model for the investigation of intravasation mechanisms and of drugs preventing CCID formation and hence lymph node metastasis. Furthermore, Western blot analyses, NF-κB reporter, EROD, SELE, 12(S)-HETE, and adhesion assays were performed to investigate the properties of heteronemin. Five micromolar heteronemin inhibited the directional movement of LECs and, therefore, the formation of CCIDs, which were induced by MCF-7 spheroids. Furthermore, heteronemin reduced the adhesion of MCF-7 cells to LECs and suppressed 12(S)-HETE-induced expression of the EMT marker paxillin, which is a regulator of directional cell migration. The activity of CYP1A1, which contributed to CCID formation, was also inhibited by heteronemin. Hence, heteronemin inhibits important mechanisms contributing to tumour intravasation in vitro and should be tested in vivo.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Células Endoteliais/efeitos dos fármacos , Metástase Linfática/prevenção & controle , Terpenos/farmacologia , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Western Blotting , Neoplasias da Mama/patologia , Movimento Celular , Técnicas de Cocultura , Citocromo P-450 CYP1A1/metabolismo , Células Endoteliais/metabolismo , Feminino , Humanos , Células MCF-7 , NF-kappa B/metabolismo , Paxilina/metabolismoRESUMO
Cancer continues to be a major public health problem despite the efforts that have been made in the search for novel drugs and treatments. The current sources sought for the discovery of new molecules are plants, animals and minerals. During the past decade, the search for anticancer agents of marine origin to fight chemo-resistance has increased greatly. Each year, several novel anticancer molecules are isolated from marine organisms and represent a renewed hope for cancer therapy. The study of structure-function relationships has allowed synthesis of analogues with increased efficacy and less toxicity. In this report, we aim to review 42 compounds of marine origin and their derivatives that were published in 2011 as promising anticancer compounds.
Assuntos
Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Organismos Aquáticos/química , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/classificação , Produtos Biológicos/classificação , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The two LIM domain-containing proteins from plants (LIMs) typically exhibit a dual cytoplasmic-nuclear distribution, suggesting that, in addition to their previously described roles in actin cytoskeleton organization, they participate in nuclear processes. Using a south-western blot-based screen aimed at identifying factors that bind to plant histone gene promoters, we isolated a positive clone containing the tobacco LIM protein WLIM2 (NtWLIM2) cDNA. Using both green fluorescent protein (GFP) fusion- and immunology-based strategies, we provide clear evidence that NtWLIM2 localizes to the actin cytoskeleton, the nucleus, and the nucleolus. Interestingly, the disruption of the actin cytoskeleton by latrunculin B significantly increases NtWLIM2 nuclear fraction, pinpointing a possible novel cytoskeletal-nuclear crosstalk. Biochemical and electron microscopy experiments reveal the ability of NtWLIM2 to directly bind to actin filaments and to crosslink the latter into thick actin bundles. Electrophoretic mobility shift assays show that NtWLIM2 specifically binds to the conserved octameric cis-elements (Oct) of the Arabidopsis histone H4A748 gene promoter and that this binding largely relies on both LIM domains. Importantly, reporter-based experiments conducted in Arabidopsis and tobacco protoplasts confirm the ability of NtWLIM2 to bind to and activate the H4A748 gene promoter in live cells. Expression studies indicate the constitutive presence of NtWLIM2 mRNA and NtWLIM2 protein during tobacco BY-2 cell proliferation and cell cycle progression, suggesting a role of NtWLIM2 in the activation of basal histone gene expression. Interestingly, both live cell and in vitro data support NtWLIM2 di/oligomerization. We propose that NtWLIM2 functions as an actin-stabilizing protein, which, upon cytoskeleton remodeling, shuttles to the nucleus in order to modify gene expression.
Assuntos
Actinas/metabolismo , Histonas/genética , Nicotiana/genética , Nicotiana/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Transcrição Gênica , Citoesqueleto de Actina/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Clonagem Molecular , DNA Complementar/genética , Genes Reporter/genética , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Protoplastos/metabolismo , Nicotiana/citologiaRESUMO
Traditional pharmacopeia is strongly involved in the continuous search for the well being of African populations. The World Health Organization (WHO) estimates that 80% of the population of developing countries relies on traditional medicine for their primary care needs. Medicinal plants are the major resource of this folk medicine where several species are used for the treatment of diseases with an inflammatory and/or infectious component as it is the case of old wounds, skin diseases and malfunctions affecting internal organs such as liver, lung, prostate and kidney. Many of these pathologies described by practitioners of traditional medicine have similarities with certain cancers, but the lack of training of many of these healers does not allow them to establish a link with cancer. However, ethnobotanical and ethnopharmacological surveys conducted by several researchers allowed to identify plants of interest for cancer treatment. Most scientific investigations on these plants demonstrated an anti-inflammatory or antioxidant effect, and sometimes, antiproliferative and cytotoxic activities against cancer cells were reported as well. The emergence of resistance to cancer chemotherapy has forced researchers to turn to natural products of plant and marine origin. In the West African sub-region, research on natural anti-cancer molecules is still in its infancy stage because of very limited financial resources and the scarcity of adequate technical facilities. However, several plants were investigated for their anticancer properties through north-south or south-south partnerships. In this review, we will review the role of West African traditional pharmacopeia in cancer treatment as well as medicinal plants with anti-cancer properties.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Medicinas Tradicionais Africanas , Neoplasias/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Plantas Medicinais/química , África Ocidental , Alcaloides/isolamento & purificação , Alcaloides/farmacologia , Alcaloides/uso terapêutico , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Atenção à Saúde , Países em Desenvolvimento , Humanos , Fitoterapia , Extratos Vegetais/farmacologia , Polifenóis/isolamento & purificação , Polifenóis/farmacologia , Polifenóis/uso terapêutico , Esteroides/isolamento & purificação , Esteroides/farmacologia , Esteroides/uso terapêutico , Terpenos/isolamento & purificação , Terpenos/farmacologia , Terpenos/uso terapêuticoRESUMO
Garlic-derived organosulfur compounds including diallyl polysulfides are well known for various health-beneficial properties and recent reports even point to a potential role of diallyl polysulfides as chemopreventive and therapeutic agents in cancer treatment due to their selective antiproliferative effects. In this respect, diallyl tri- and tetrasulfide are reported as strong inducers of an early mitotic arrest and subsequent apoptosis, but the underlying molecular mechanisms and the link between these two events are not yet fully elucidated. Our data revealed that diallyl tetrasulfide acts independently of reactive oxygen species and tubulin represents one of its major cellular targets. Tubulin depolymerization prevents the formation of normal spindle microtubules, thereby leading to G2/M arrest. Here, we provide evidence that c-jun N-terminal kinase, which is activated early in response to diallyl tetrasulfide treatment, mediates multisite phosphorylation and subsequent proteolysis of the anti-apoptotic protein B-cell lymphoma 2. As the latter event occurs concomitantly with the onset of apoptosis and the chemical c-jun N-terminal kinase inhibitor SP600125 not only prevented B-cell lymphoma 2 phosphorylation and proteolysis but also apoptosis following diallyl tetrasulfide treatment, we suggest that these c-jun N-terminal kinase-mediated modulations of B-cell lymphoma 2 represent the missing link connecting early microtubule inactivation to the induction of apoptosis.