RESUMO
Ovarian cancer (OC) is the most lethal gynecologic cancer, mainly due to late diagnosis with widespread peritoneal spread at first presentation. We performed metabolomic analyses of ovarian and paired control tissues using capillary electrophoresis-mass spectrometry and liquid chromatography-mass spectrometry to understand its metabolomic dysregulation. Of the 130 quantified metabolites, 96 metabolites of glycometabolism, including glycolysis, tricarboxylic acid cycles, urea cycles, and one-carbon metabolites, showed significant differences between the samples. To evaluate the local and systemic metabolomic differences in OC, we also analyzed low or non-invasively available biofluids, including plasma, urine, and saliva collected from patients with OC and benign gynecological diseases. All biofluids and tissue samples showed consistently elevated concentrations of N1,N12-diacetylspermine compared to controls. Four metabolites, polyamines, and betaine, were significantly and consistently elevated in both plasma and tissue samples. These data indicate that plasma metabolic dysregulation, which the most reflected by those of OC tissues. Our metabolomic profiles contribute to our understanding of metabolomic abnormalities in OC and their effects on biofluids.
Assuntos
Metabolômica , Neoplasias Ovarianas , Humanos , Feminino , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Metabolômica/métodos , Pessoa de Meia-Idade , Metaboloma , Líquidos Corporais/metabolismo , Adulto , Saliva/metabolismo , Idoso , Poliaminas/metabolismo , Poliaminas/sangue , Cromatografia Líquida , Espectrometria de Massas , Eletroforese Capilar , Espermina/análogos & derivadosRESUMO
Oligoamines in cellular metabolism carry extremely diverse biological functions (i.e., regulating Ca2+-influx, neuronal nitric oxide synthase, membrane potential, Na+, K+-ATPase activity in synaptosomes, etc.). Furthermore, they also act as longevity agents and have a determinative role in autophagy, cell growth, proliferation, and death, while oligoamines dysregulation is a key in a variety of cancers. However, many of their mechanisms of actions have just begun to be understood. In addition to the numerous biosensing methods, only a very few simple small molecule-based tests are available for their selective but reversible tracking or fluorescent labeling. Motivated by this, we present herein a new fluorescent bis(acridino)-crown ether as a sensor molecule for biogenic oligoamines. The sensor molecule can selectively distinguish oligoamines from aliphatic mono- and diamino-analogues, while showing a reversible 1:2 (host:guest) complexation with a stepwise binding process accompanied by a turn-on fluorescence response. Both computational simulations on molecular docking and regression methods on titration experiments were carried out to reveal the oligoamine-recognition properties of the sensor molecule. The new fluorescent chemosensor molecule has a high potential for molecular-level functional studies on the oligoamine systems in cell processes (cellular uptake, transport, progression in cancers, etc.).
Assuntos
Éteres de Coroa , Simulação de Acoplamento Molecular , Espermina , Éteres de Coroa/química , Espermina/metabolismo , Espermina/química , Corantes Fluorescentes/química , Aminas/química , Aminas/metabolismo , Acridinas/químicaRESUMO
Systemic inflammation elicits sickness behaviors and fever by engaging a complex neuronal circuitry that begins in the preoptic area of the hypothalamus. Ectotherms such as teleost fish display sickness behaviors in response to infection or inflammation, seeking warmer temperatures to enhance survival via behavioral fever responses. To date, the hypothalamus is the only brain region implicated in sickness behaviors and behavioral fever in teleosts. Yet, the complexity of neurobehavioral manifestations underlying sickness responses in teleosts suggests engagement of higher processing areas of the brain. Using in vivo models of systemic inflammation in rainbow trout, we find canonical pyrogenic cytokine responses in the hypothalamus whereas in the telencephalon and the optic tectum il-1b and tnfa expression is decoupled from il-6 expression. Polyamine metabolism changes, characterized by accumulation of putrescine and decreases in spermine and spermidine, are recorded in the telencephalon but not hypothalamus upon systemic injection of bacteria. While systemic inflammation causes canonical behavioral fever in trout, blockade of bacterial polyamine metabolism prior to injection abrogates behavioral fever, polyamine responses, and telencephalic but not hypothalamic cytokine responses. Combined, our work identifies the telencephalon as a neuronal substrate for brain responses to systemic inflammation in teleosts and uncovers the role of polyamines as critical chemical mediators in sickness behaviors.
Assuntos
Inflamação , Oncorhynchus mykiss , Poliaminas , Telencéfalo , Animais , Telencéfalo/metabolismo , Poliaminas/metabolismo , Inflamação/metabolismo , Oncorhynchus mykiss/metabolismo , Oncorhynchus mykiss/imunologia , Neurônios/metabolismo , Hipotálamo/metabolismo , Espermina/metabolismo , Putrescina/metabolismo , Comportamento de Doença/fisiologia , Espermidina/metabolismoRESUMO
The only known pathway for biosynthesis of the polyamine norspermidine starts from aspartate ß-semialdehyde to form the diamine 1,3-diaminopropane, which is then converted to norspermidine via a carboxynorspermidine intermediate. This pathway is found primarily in the Vibrionales order of the γ-Proteobacteria. However, norspermidine is also found in other species of bacteria and archaea, and in diverse single-celled eukaryotes, chlorophyte algae and plants that do not encode the known norspermidine biosynthetic pathway. We reasoned that products of polyamine catabolism could be an alternative route to norspermidine production. 1,3-diaminopropane is formed from terminal catabolism of spermine and spermidine, and norspermidine can be formed from catabolism of thermospermine. We found that the single-celled chlorophyte alga Chlamydomonas reinhardtii thermospermine synthase (CrACL5) did not aminopropylate exogenously-derived 1,3-diaminopropane efficiently when expressed in Escherichia coli. In contrast, it completely converted all E. coli native spermidine to thermospermine. Co-expression in E. coli of the polyamine oxidase 5 from lycophyte plant Selaginella lepidophylla (SelPAO5), together with the CrACL5 thermospermine synthase, converted almost all thermospermine to norspermidine. Although CrACL5 was efficient at aminopropylating norspermidine to form tetraamine norspermine, SelPAO5 oxidizes norspermine back to norspermidine, with the balance of flux being inclined fully to norspermine oxidation. The steady-state polyamine content of E. coli co-expressing thermospermine synthase CrACL5 and polyamine oxidase SelPAO5 was an almost total replacement of spermidine by norspermidine. We have recapitulated a potential hybrid biosynthetic-catabolic pathway for norspermidine production in E. coli, which could explain norspermidine accumulation in species that do not encode the known aspartate ß-semialdehyde-dependent pathway.
Assuntos
Espermidina , Espermidina/metabolismo , Espermidina/análogos & derivados , Espermidina/biossíntese , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/genética , Vias Biossintéticas , Escherichia coli/metabolismo , Escherichia coli/genética , Espermina/metabolismo , Espermina/análogos & derivadosRESUMO
Acrolein is an environmental toxicant and is also generated by microbial metabolism in the intestinal tract. Aqueous acrolein rapidly dissipates from standard human cell culture media with nondetectable levels after 8 h, hindering cell-based studies to understand its biological impacts. Thus, we developed an extracellular acrolein biosynthesis system to continuously produce acrolein compatible with human cell culture conditions. The approach uses spermine as a precursor, amine oxidase found in fetal calf serum, and catalase to remove the hydrogen peroxide byproduct. We confirmed amine oxidase activity of calf serum using a colorimetric assay and further tested the requirement for catalase in the system to mitigate hydrogen peroxide-induced cytotoxicity. We calibrated responses of human colon cells to this enzymatic acrolein production system by comparing transcriptional responses, DNA adduct formation and cytotoxicity responses to either this system or pure acrolein exposures in a human colon cell line. Several genes related to oxidative stress including HMOX1, and the colorectal cancer-related gene SEMA4A were upregulated similarly between the enzymatic acrolein production system or pure acrolein. The acrolein-DNA adduct γ-OH-Acr-dG increased in a dose-dependent manner with spermine in the enzymatic acrolein production system, producing a maximum of 1065 adducts per 108 nucleosides when 400 µM spermine was used. This biosynthetic production method provides a relevant model for controlled acrolein exposure in cultured human cells and overcomes current limitations due to its physical properties and limited availability.
Assuntos
Acroleína , Humanos , Acroleína/metabolismo , Peróxido de Hidrogênio/metabolismo , Adutos de DNA/metabolismo , Catalase/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espermina/metabolismoRESUMO
In this paper the effects on the interaction of highly positively charged substitution-inert platinum polynuclear complexes (SI-PPCs) with negatively charged DNA and heparin are examined and compared by theoretical chemistry methods. Electrostatic and hydrogen bonding interactions contribute to the overall effects on the biomolecule. Root Mean Square (RMS) deviation, Solvent Accessible Surface, RMS fluctuation, and interaction analysis all confirm similar effects on both biomolecules, dictated predominantly by the total positive charge and total number of hydrogen bonds formed. Especially, changes in structural parameters suggesting condensation and reduction of available surface area will reduce or prevent normal protein recognition and may thus potentially inhibit biological mechanisms related to apoptosis (DNA) or reduced vascularization viability (HEP). Thermodynamic analyses supported these findings with favourable interaction energies. The comparison of DNA and heparin confirms the general intersectionality between the two biomolecules and confirms the intrinsic dual-nature function of this chemotype. The distinction between the two-limiting mode of actions (HS or DNA-centred) could reflect an intriguing balance between extracellular (GAG) and intracellular (DNA) binding and affinities. The results underline the need to fully understand GAG-small molecule interactions and their contribution to drug pharmacology and related therapeutic modalities. This report contributes to that understanding.
Assuntos
DNA , Simulação de Dinâmica Molecular , Espermidina , Espermina , Espermina/química , DNA/química , DNA/metabolismo , Espermidina/química , Espermidina/metabolismo , Heparina/química , Heparina/metabolismo , Termodinâmica , Ligação de Hidrogênio , Eletricidade EstáticaRESUMO
Polyamines are essential analytes due to their critical role in various biological processes and human health in general. Due to their role as regulators for cell growth and proliferation (putrescine and spermine), as neuroprotectors, gero-, and cardiovascular protectors (spermidine), and as bacterial growth indicators (cadaverine), rapid, simple, and cost-effective methods for polyamine detection in biofluids are in demand. The present study focuses on the development and investigation of self-assembled and fluorescent hostâ dye chemo-sensors based on sulfonated pillar[5]arene for the specific detection of polyamines. Binding studies, as well as stability and functionality assessments of the turn-on chemosensors for selective polyamine detection in saline and biologically relevant media, are shown. Furthermore, the practical applicability of the developed chemo-sensors is demonstrated in biofluids such as human urine and saliva.
Assuntos
Cadaverina , Calixarenos , Corantes Fluorescentes , Saliva , Espermidina , Espermina , Espermidina/análise , Espermidina/química , Humanos , Espermina/análise , Espermina/química , Cadaverina/análise , Corantes Fluorescentes/química , Calixarenos/química , Saliva/química , Espectrometria de Fluorescência/métodos , Compostos de Amônio Quaternário/química , Fluorescência , Solução Salina/químicaRESUMO
We studied the effects of some nitrogen-containing, heterocyclic, and cyclic compounds on the rate of oxidative deamination of polyamines and putrescine in tissues with a high proliferation rate. For this purpose, the specific activities of the main enzymes of polyamine oxidative degradation - spermine oxidase (SMO), polyamine oxidase (PAO), and diamine oxidase (DAO) were determined using a cell-free test system from regenerating rat liver. The compounds methyl 2-(5-formylfuran-2-yl)benzoate and 2,7-bis-[2-(diethylamino)ethoxy]-9H-fluoren-9-one (and in the form of dihydrochloride) showed mainly activating effect on oxidative degradation of putrescine, spermidine, and spermine, which indirectly indicates their antiproliferative effect. Nitrogen-free compounds inhibited this process, thus exhibiting potentially carcinogenic properties. Correlations were calculated for activity of DAO, PAO, and SMO with 5 topological indices: Wiener (W), Rouvray (R), Balaban (J) in the Trinaistich modification, detour (Ip), and electropy (Ie). The highest dependence was noted for DAO and the Balaban index (R=-0.55), for PAO and the detour index (R=0.78), and for SMO and the electropy index (R=0.53). The remaining dependencies showed insignificant correlation strength.
Assuntos
Amina Oxidase (contendo Cobre) , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo CH-NH , Animais , Ratos , Oxirredução/efeitos dos fármacos , Desaminação , Amina Oxidase (contendo Cobre)/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Poliamina Oxidase , Putrescina/metabolismo , Putrescina/farmacologia , Compostos Heterocíclicos/farmacologia , Compostos Heterocíclicos/química , Sistema Livre de Células , Fígado/metabolismo , Fígado/efeitos dos fármacos , Poliaminas/metabolismo , Espermina/metabolismo , Espermina/farmacologia , Espermidina/metabolismo , Masculino , Nitrogênio/metabolismo , Ratos WistarRESUMO
BACKGROUND: Polyamines have been reported to be associated with neurological function, but the associations between polyamines and the prognosis of ischemic stroke remain unclear. We aimed to prospectively investigate whether elevated plasma polyamine levels are associated with adverse outcomes in patients with ischemic stroke. METHODS AND RESULTS: Plasma polyamine levels were measured at admission in 3570 patients with acute ischemic stroke, and clinical outcomes were assessed at 3 months after stroke onset. The primary outcome was a composite outcome of death and major disability (modified Rankin Scale score≥3), and secondary outcomes included the individual outcomes of death and major disability. During a 3-month follow-up period, 877 participants (25.1%) experienced the primary outcome. Increased putrescines were associated with a decreased risk of the primary outcome (the highest versus the lowest tertile: odds ratio, 0.72 [95% CI, 0.58-0.91]; P=0.005) and major disability (odds ratio, 0.59 [95% CI, 0.47-0.74]; P<0.001). Conversely, increased spermidines were associated with an increased risk of death (hazard ratio, 1.86 [95% CI, 1.10-3.14]; P=0.020), and increased spermines were associated with an increased risk of the primary outcome (odds ratio, 1.36 [95% CI, 1.08-1.71]; P=0.009) and major disability (odds ratio, 1.27 [95% CI, 1.01-1.59]; P=0.041). CONCLUSIONS: Among patients with ischemic stroke, high plasma putrescine levels were associated with a decreased risk of adverse outcomes, whereas high plasma spermidine and spermine levels were associated with an increased risk of adverse outcomes. Further studies are needed to investigate whether targeting these polyamines can improve the prognosis of patients with ischemic stroke. REGISTRATION: https://clinicaltrials.gov. Identifier: NCT01840072.
Assuntos
Biomarcadores , AVC Isquêmico , Poliaminas , Humanos , Masculino , Feminino , Idoso , Estudos Prospectivos , AVC Isquêmico/sangue , AVC Isquêmico/mortalidade , AVC Isquêmico/diagnóstico , Pessoa de Meia-Idade , Poliaminas/sangue , Prognóstico , Biomarcadores/sangue , Fatores de Tempo , Espermidina/sangue , Putrescina/sangue , Fatores de Risco , Avaliação da Deficiência , Espermina/sangue , Idoso de 80 Anos ou mais , Medição de RiscoRESUMO
Successful therapeutic delivery of siRNA with polymeric nanoparticles seems to be a promising but not vastly understood and complicated goal to achieve. Despite years of research, no polymer-based delivery system has been approved for clinical use. Polymers, as a delivery system, exhibit considerable complexity and variability, making their consistent production a challenging endeavor. However, a better understanding of the polymerization process of polymer excipients may improve the reproducibility and material quality for more efficient use in drug products. Here, we present a combination of Design of Experiment and Python-scripted data science to establish a prediction model, from which important parameters can be extracted that influence the synthesis results of polybeta-amino esters (PBAEs), a common type of polymer used preclinically for nucleic acid delivery. We synthesized a library of 27 polymers, each one at different temperatures with different reaction times and educt ratios using an orthogonal central composite (CCO-) design. This design allowed a detailed characterization of factor importance and interactions using a very limited number of experiments. We characterized the polymers by analyzing the resulting composition by 1H-NMR and the size distribution by GPC measurements. To further understand the complex mechanism of block polymerization in a one-pot synthesis, we developed a Python script that helps us to understand possible step-growth steps. We successfully developed and validated a predictive response surface and gathered a deeper understanding of the synthesis of polyspermine-based amphiphilic PBAEs.
Assuntos
Polímeros , Espermina , Polímeros/química , Polímeros/síntese química , Espermina/química , Nanopartículas/química , PolimerizaçãoRESUMO
Polyamines (PAs) including putrescine (PUT), spermidine (SPD) and spermine (SPM) are small, versatile molecules with two or more positively charged amino groups. Despite their importance for almost all forms of life, their specific roles in molecular and cellular biology remain partly unknown. The molecular structures of PAs suggest two presumable biological functions: (i) as potential buffer systems and (ii) as interactants with poly-negatively charged molecules like nucleic acids. The present report focuses on the question, whether the molecular structures of PAs are essential for such functions, or whether other simple molecules like small peptides with closely spaced positively charged side chains might be suitable as well. Consequently, we created titration curves for PUT, SPD, and SPM, as well as for oligolysines like tri-, tetra-, and penta-lysine. None of the molecules provided substantial buffering capacity at physiological intracellular pH values. Apparently, the most important mechanism for intracellular pH homeostasis in neurons is not a buffer system but is provided by the actions of the sodium-hydrogen and the bicarbonate-chloride antiporters. In a similar approach we investigated the interaction with DNA by following the extinction at 260 nm when titrating DNA with the above molecules. Again, PUT and tri-lysine were not able to interact with herring sperm DNA, while SPD and SPM were. Obviously, the presence of several positively charged groups on its own is not sufficient for the interaction with nucleic acids. Instead, the precise spacing of these groups is necessary for biological activity.
Assuntos
DNA , Peptídeos , Poliaminas , RNA , Concentração de Íons de Hidrogênio , DNA/química , DNA/metabolismo , Soluções Tampão , RNA/química , RNA/metabolismo , Poliaminas/química , Poliaminas/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Animais , Espermidina/química , Espermidina/metabolismo , Espermina/química , Espermina/metabolismo , Putrescina/química , Putrescina/metabolismoRESUMO
Introduction: The Hand, Foot and Mouth Disease (HFMD), caused by enterovirus 71 infection, is a global public health emergency. Severe HFMD poses a significant threat to the life and well-being of children. Numerous studies have indicated that the occurrence of severe HFMD is associated with cytokine storm. However, the precise molecular mechanism underlying cytokine storm development remains elusive, and there are currently no safe and effective treatments available for severe HFMD in children. Methods: In this study, we established a mouse model of severe HFMD to investigate the molecular mechanisms driving cytokine storm. We specifically analyzed metabolic disturbances, focusing on arginine/ornithine metabolism, and assessed the potential therapeutic effects of spermine, an ornithine metabolite. Results: Our results identified disturbances in arginine/ornithine metabolism as a pivotal factor driving cytokine storm onset in severe HFMD cases. Additionally, we discovered that spermine effectively mitigated the inflammatory injury phenotype observed in mice with severe HFMD. Discussion: In conclusion, our findings provide novel insights into the molecular mechanisms underlying severe HFMD from a metabolic perspective while offering a promising new strategy for its safe and effective treatment.
Assuntos
Arginina , Citocinas , Modelos Animais de Doenças , Doença de Mão, Pé e Boca , Ornitina , Animais , Doença de Mão, Pé e Boca/imunologia , Camundongos , Arginina/metabolismo , Humanos , Citocinas/metabolismo , Espermina/metabolismo , Feminino , Enterovirus Humano A/imunologia , Masculino , Camundongos Endogâmicos C57BL , Índice de Gravidade de DoençaRESUMO
Stimuli-responsive supramolecular chemotherapy, particularly in response to cancer biomarkers, has emerged as a promising strategy to overcome the limitations associated with traditional chemotherapy. Spermine (SPM) is known to be overexpressed in certain cancers. In this study, we introduced a novel supramolecular chemotherapy system triggered by SPM. The system featured pyridine salts of a diphenylanthracene derivative (PyEn) and a complementary water-soluble pillar[5]arene (WP5C5) with long alkyl chains. The diphenylanthracene unit of PyEn is effectively encapsulated within the long alkyl chains of WP5C5, resulting in a substantial reduction in the cytotoxicity of PyEn towards normal cells. The therapeutic effect of PyEn is selectively triggered intracellularly through SPM, leading to the endosomal release of PyEn and concurrent in situ cytotoxicity. This supramolecular chemotherapy system exhibits notable tumor inhibition against SPM-overexpressed cancers with reduced side effects on normal tissues. The supramolecular strategy for intracellular activation provides a novel tool with potential applications in chemotherapeutic interventions, offering enhanced selectivity and reduced cytotoxicity to normal cells.
Assuntos
Antracenos , Antineoplásicos , Calixarenos , Compostos de Amônio Quaternário , Solubilidade , Espermina , Água , Humanos , Antineoplásicos/química , Antineoplásicos/farmacologia , Calixarenos/química , Calixarenos/farmacologia , Água/química , Antracenos/química , Espermina/química , Espermina/farmacologia , Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/farmacologia , Animais , Ensaios de Seleção de Medicamentos Antitumorais , Tensoativos/química , Tensoativos/farmacologia , Tensoativos/síntese química , Sobrevivência Celular/efeitos dos fármacos , Camundongos , Estrutura Molecular , Proliferação de Células/efeitos dos fármacos , Tamanho da PartículaRESUMO
Biogenic polyamines are ubiquitous compounds. Dysregulation of their metabolism is associated with the development of various pathologies, including cancer, hyperproliferative diseases, and infections. The canonical pathway of polyamine catabolism includes acetylation of spermine and spermidine and subsequent acetylpolyamine oxidase (PAOX)-mediated oxidation of acetylpolyamines (back-conversion) or their direct efflux from the cell. PAOX is considered to catalyze a non-rate-limiting catabolic step. Here, we show that PAOX transcription levels are extremely low in various tumor- and non-tumor cell lines and, in most cases, do not change in response to altered polyamine metabolism. Its enzymatic activity is undetectable in the majority of cell lines except for neuroblastoma and low passage glioblastoma cell lines. Treatment of A549 cells with N1,N11-diethylnorspermine leads to PAOX induction, but its contribution to polyamine catabolism remains moderate. We also describe two alternative enzyme isoforms and show that isoform 4 has diminished oxidase activity and isoform 2 is inactive. PAOX overexpression correlates with the resistance of cancer cells to genotoxic antitumor drugs, indicating that PAOX may be a useful therapeutic target. Finally, PAOX is dispensable for the replication of various viruses. These data suggest that a decrease in polyamine levels is achieved predominantly by the secretion of acetylated spermine and spermidine rather than by back-conversion.
Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-NH , Poliaminas , Humanos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Poliaminas/metabolismo , Linhagem Celular Tumoral , Espermina/metabolismo , Espermina/análogos & derivados , Acetilação , Células A549RESUMO
Oral cancer accounts for 50%-70% of all cancer-related deaths in India and ranks sixth among the most frequent cancers globally. Roughly 90% of oral malignancies are histologically arise from squamous cells and are therefore called oral squamous cell carcinoma. Organic polycations known as biogenic polyamines, for example, putrescine (Put), spermidine (Spd), and spermine (Spm), are vital for cell proliferation, including gene expression control, regulation of endonuclease-mediated fragmentation of DNA, and DNA damage inhibition. Higher Spm and Spd levels have been identified as cancer biomarkers for detecting tumour development in various cancers. The current study utilises tannic acid, a polyphenolic compound, as a reducing and capping agent to fabricate AuNPs via a one-step microwave-assisted synthesis. The fabricated TA@AuNPs were utilised as a nanoprobe for colourimetric sensing of polyamines in PBS. When TA@AuNPs are added to the polyamine, the amine groups in polyamines interact with the phenolic groups of TA@AuNPs via hydrogen bonding or electrostatic interactions. These interactions cause the aggregation of TA@AuNPs, resulting in a red shift of the Surface Plasmon Resonance band of TA@AuNPs from 530 nm to 560 nm. The nanoprobe was found to be highly specific for Spm at low concentrations. TA@AuNPs were able to detect Spm successfully in artificial saliva samples. On recording the RGB values of the sensing process using a smartphone app, it was found that as the nanoparticles aggregated due to the presence of Spm, the intensity of theR-value decreased, indicating the aggregation of TA@AuNPs due to interaction with the polyamine.
Assuntos
Ouro , Nanopartículas Metálicas , Neoplasias Bucais , Poliaminas , Smartphone , Espermina , Neoplasias Bucais/diagnóstico , Neoplasias Bucais/metabolismo , Humanos , Nanopartículas Metálicas/química , Poliaminas/química , Ouro/química , Espermina/química , Putrescina/análise , Espermidina/química , Taninos/química , Ressonância de Plasmônio de Superfície , Colorimetria/métodos , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/metabolismoRESUMO
Mitochondria play a crucial role in maintaining Ca2+ homeostasis in cells. Due to the critical regulatory role of the products of oxidative and non-oxidative metabolism of L-arginine, it is essential to clarify their effect on Ca2+ transport in smooth muscle mitochondria. Experiments were performed on the uterine myocytes of rats and isolated mitochondria. The possibility of NO synthesis by mitochondria was demonstrated by confocal microscopy and spectrofluorimetry methods using the NO-sensitive fluorescent probe DAF-FM and Mitotracker Orange CM-H2TMRos. It was shown that 50 µM L-arginine stimulates the energy-dependent accumulation of Ca2+ in mitochondria using the fluorescent probe Fluo-4 AM. A similar effect occurred when using nitric oxide donors 100 µM SNP, SNAP, and sodium nitrite (SN) directly. The stimulating effect was eliminated in the presence of the NO scavenger C-PTIO. Nitric oxide reduces the electrical potential in mitochondria without causing them to swell. The stimulatory effect of spermine on the accumulation of Ca2+ by mitochondria is attributed to the enhancement of NO synthesis, which was demonstrated with the use of C-PTIO, NO-synthase inhibitors (100 µM NA and L-NAME), as well as by direct monitoring of NO synthesis fluorescent probe DAF-FM. A conclusion was drawn about the potential regulatory effect of the product of the oxidative metabolism of L-arginine - NO on the transport of Ca2+ in the mitochondria of the myometrium, as well as the corresponding effect of the product of non-oxidative metabolism -spermine by increasing the synthesis of NO in these subcellular structures.
Assuntos
Arginina , Cálcio , Óxido Nítrico , Feminino , Animais , Arginina/metabolismo , Cálcio/metabolismo , Ratos , Óxido Nítrico/metabolismo , Oxirredução , Miométrio/metabolismo , Miométrio/efeitos dos fármacos , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/efeitos dos fármacos , Ratos Wistar , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Útero/metabolismo , Útero/efeitos dos fármacos , Espermina/metabolismo , Espermina/farmacologia , Doadores de Óxido Nítrico/farmacologia , Doadores de Óxido Nítrico/metabolismo , Músculo Liso/metabolismo , Músculo Liso/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacosRESUMO
After RNAi was first discovered over 20 years ago, siRNA-based therapeutics are finally becoming reality. However, the delivery of siRNA has remained a challenge. In our previous research, we found that spermine-based poly(ß-amino ester)s are very promising for siRNA delivery. However, the role of hydrophobic modification in siRNA delivery of spermine-based poly(ß-amino ester)s is not fully understood yet. In the current work, we synthesized spermine-based poly(ß-amino ester)s with different percentages of oleylamine side chains, named P(SpOABAE). The chemical structures of the polymers were characterized by 1H NMR. The polymers showed efficient siRNA encapsulation determined by SYBR Gold assays. The hydrodynamic diameters of the P(SpOABAE) polyplexes from charge ratio N/P 1 to 20 were 30-100 nm except for aggregation phenomena observed at N/P 3. Morphology of the polyplexes was visualized by atomic force microscopy, and cellular uptake was determined by flow cytometry in H1299 cells, where all the polyplexes showed significantly higher cellular uptake than hyperbranched polyethylenimine (25 kDa). The most hydrophobic P(SpOABAE) polyplexes were able to achieve more than 90% GFP knockdown in H1299/eGFP cells. The fact that gene silencing efficacy increased with hydrophobicity but cellular uptake was affected by both charge and hydrophobic interactions highlights the importance of endosomal escape. For pulmonary administration and improved storage stability, the polyplexes were spray-dried. Results confirmed the maintained siRNA activity after storage for 3 months at room temperature, indicating potential for dry powder inhalation.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , RNA Interferente Pequeno , Espermina , RNA Interferente Pequeno/química , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/farmacologia , Espermina/química , Humanos , Administração por Inalação , Polímeros/química , Pós/química , Linhagem Celular TumoralRESUMO
Prolonged activation of the type I interferon (IFN-I) pathway leads to autoimmune diseases such as systemic lupus erythematosus (SLE). Metabolic regulation of cytokine signaling is critical for cellular homeostasis. Through metabolomics analyses of IFN-ß-activated macrophages and an IFN-stimulated-response-element reporter screening, we identified spermine as a metabolite brake for Janus kinase (JAK) signaling. Spermine directly bound to the FERM and SH2 domains of JAK1 to impair JAK1-cytokine receptor interaction, thus broadly suppressing JAK1 phosphorylation triggered by cytokines IFN-I, IFN-II, interleukin (IL)-2, and IL-6. Peripheral blood mononuclear cells (PBMCs) from individuals with SLE showing decreased spermine concentrations exhibited enhanced IFN-I and lupus gene signatures. Spermine treatment attenuated autoimmune pathogenesis in SLE and psoriasis mice and reduced IFN-I signaling in monocytes from individuals with SLE. We synthesized a spermine derivative (spermine derivative 1 [SD1]) and showed that it had a potent immunosuppressive function. Our findings reveal spermine as a metabolic checkpoint for cellular homeostasis and a potential immunosuppressive molecule for controlling autoimmune disease.
Assuntos
Autoimunidade , Citocinas , Lúpus Eritematoso Sistêmico , Transdução de Sinais , Espermina , Animais , Espermina/metabolismo , Espermina/farmacologia , Humanos , Transdução de Sinais/efeitos dos fármacos , Camundongos , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Citocinas/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Janus Quinase 1/metabolismo , Fosforilação , Interferon Tipo I/metabolismo , Interferon Tipo I/imunologia , Psoríase/imunologia , Psoríase/metabolismo , Camundongos Endogâmicos C57BL , Janus Quinases/metabolismo , Feminino , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismoRESUMO
Chronic inflammation, oxidative stress, and airway remodelling represent the principal pathophysiological features of chronic respiratory disorders. Inflammation stimuli like lipopolysaccharide (LPS) activate macrophages and dendritic cells, with concomitant M1 polarization and release of pro-inflammatory cytokines. Chronic inflammation and oxidative stress lead to airway remodelling causing irreversible functional and structural alterations of the lungs. Airway remodelling is multifactorial, however, the hormone transforming growth factor-ß (TGF-ß) is one of the main contributors to fibrotic changes. The signalling pathways mediating inflammation and remodelling rely both on the transcription factor nuclear factor-κB (NFκB), underlying the potential of NFκB inhibition as a therapeutic strategy for chronic respiratory disorders. In this study, we encapsulated an NFκB-inhibiting decoy oligodeoxynucleotide (ODN) in spermine-functionalized acetalated dextran (SpAcDex) nanoparticles and tested the in vitro anti-inflammatory and anti-remodelling activity of this formulation. We show that NF-κB ODN nanoparticles counteract inflammation by reversing LPS-induced expression of the activation marker CD40 in myeloid cells and counteracts remodelling features by reversing the TGF-ß-induced expression of collagen I and α-smooth muscle actin in human dermal fibroblast. In summary, our study highlights the great potential of inhibiting NFκB via decoy ODN as a therapeutic strategy tackling multiple pathophysiological features underlying chronic respiratory conditions.
Assuntos
Anti-Inflamatórios , Lipopolissacarídeos , NF-kappa B , Nanopartículas , Oligodesoxirribonucleotídeos , Espermina , Oligodesoxirribonucleotídeos/farmacologia , Oligodesoxirribonucleotídeos/química , Humanos , Nanopartículas/química , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/química , NF-kappa B/metabolismo , Espermina/farmacologia , Espermina/química , Lipopolissacarídeos/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose/tratamento farmacológicoRESUMO
Molecular carriers are necessary for the controlled release of drugs and genes to achieve the desired therapeutic outcomes. DNA hydrogels can be a promising candidate in this application with their distinctive sequence-dependent programmability, which allows precise encapsulation of specific cargo molecules and stimuli-responsive release of them at the target. However, DNA hydrogels are inherently susceptible to the degradation of nucleases, making them vulnerable in a physiological environment. To be an effective molecular carrier, DNA hydrogels should be able to protect encapsulated cargo molecules until they reach the target and release them once they are reached. Here, we develop a simple way of controlling the enzyme resistance of DNA hydrogels for cargo protection and release by using cation-mediated condensation and expansion. We found that DNA hydrogels condensed by spermine are highly resistant to enzymatic degradation. They become degradable again if expanded back to their original, uncondensed state by sodium ions interfering with the interaction between spermine and DNA. These controllable condensation, expansion, and degradation of DNA hydrogels pave the way for the development of DNA hydrogels as an effective molecular carrier.