RESUMO
Cadaverine is an endogenous metabolite produced by the gut microbiome with various activity in physiological and pathological conditions. However, whether cadaverine regulates pain or itch remains unclear. In this study, we first found that cadaverine may bind to histamine 4 receptor (H4R) with higher docking energy score using molecular docking simulations, suggesting cadaverine may act as an endogenous ligand for H4R. We subsequently found intradermal injection of cadaverine into the nape or cheek of mice induces a dose-dependent scratching response in mice, which was suppressed by a selective H4R antagonist JNJ-7777120, transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine and PLC inhibitor U73122, but not H1R antagonist or TRPA1 antagonist or TRPV4 antagonist. Consistently, cadaverine-induced itch was abolished in Trpv1-/- but not Trpa1-/- mice. Pharmacological analysis indicated that mast cells and opioid receptors were also involved in cadaverine-induced itch in mice. scRNA-Seq data analysis showed that H4R and TRPV1 are mainly co-expressed on NP2, NP3 and PEP1 DRG neurons. Calcium imaging analysis showed that cadaverine perfusion enhanced calcium influx in the dissociated dorsal root ganglion (DRG) neurons, which was suppressed by JNJ-7777120 and capsazepine, as well as in the DRG neurons from Trpv1-/- mice. Patch-clamp recordings found that cadaverine perfusion significantly increased the excitability of small diameter DRG neurons, and JNJ-7777120 abolished this effect, indicating involvement of H4R. Together, these results provide evidences that cadaverine is a novel endogenous pruritogens, which activates H4R/TRPV1 signaling pathways in the primary sensory neurons.
Assuntos
Cadaverina , Gânglios Espinais , Camundongos Endogâmicos C57BL , Prurido , Canais de Cátion TRPV , Animais , Prurido/metabolismo , Prurido/induzido quimicamente , Canais de Cátion TRPV/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/efeitos dos fármacos , Masculino , Cadaverina/análogos & derivados , Cadaverina/farmacologia , Cadaverina/metabolismo , Camundongos , Camundongos Knockout , Humanos , Mastócitos/metabolismo , Mastócitos/efeitos dos fármacos , Canal de Cátion TRPA1/metabolismo , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Capsaicina/análogos & derivadosRESUMO
Cadaverine (Cad), which has an independent synthesis pathway compared to other polyamine (PA) types, contributes to the health of plants by regulating plant growth and development, abiotic stress tolerance and antioxidant defense mechanisms. In this work, experiments were carried out to understand the effects of exogenous Cad (10 µM) application under drought stress (%22 PEG 6000) and without stress on cell cycle, total protein content, endogenous PA levels, and biochemical enzyme activities in barley (Hordeum vulgare cv. Burakbey) considering the potential of Cad to stimulate the drought-related tolerance system. Cad application in a stress-free environment showed an effect almost like low-impact drought stress, causing changes in all parameters examined compared to samples grown in distilled water environment (Control). The results clearly show that Cad applied against the negative effects of drought stress on all parameters creates a drought resistance mechanism of the plant. Accordingly, Cad applied together with drought stress increased the density of cells in the cell cycle (G1-S and S-G2 phases) and the amount of endogenous (spermidine 10% and spermine 40%) PAs. In addition, while superoxide dismutase (SOD) (5%), (CAT) (55%) and ascorbate peroxidase (APX) (18%) enzyme levels increased, a stress response mechanism occurred due to the decrease in total protein content (20%) and malondialdehyde (MDA) (80%). As a result, exogenous application of 10 µM Cad showed that it reduced the negative effects of drought stress on endogenous PA amounts, cell division and biochemical activities in barley.
Assuntos
Hordeum , Poliaminas , Poliaminas/metabolismo , Hordeum/metabolismo , Cadaverina/farmacologia , Cadaverina/metabolismo , Plântula , Secas , Antioxidantes/metabolismo , Ciclo Celular , Divisão CelularRESUMO
Transglutaminase 2 (TGase 2) is a multifunctional protein which is involved in various physiological and pathophysiological processes. The latter also include its participation in the development and progression of malignant neoplasms, which are often accompanied by increased protein synthesis. In addition to the elucidation of the molecular functions of TGase 2 in tumor cells, knowledge of its concentration that is available for targeting by theranostic agents is a valuable information. Herein, we describe the application of a recently developed fluorescence anisotropy (FA)-based assay for the quantitative expression profiling of TGase 2 by means of transamidase-active enzyme in cell lysates. This assay is based on the incorporation of rhodamine B-isonipecotyl-cadaverine (R-I-Cad) into N,N-dimethylated casein (DMC), which results in an increase in the FA signal over time. It was shown that this reaction is not only catalyzed by TGase 2 but also by TGases 1, 3, and 6 and factor XIIIa using recombinant proteins. Therefore, control measurements in the presence of a selective irreversible TGase 2 inhibitor were mandatory to ascertain the specific contribution of TGase 2 to the overall FA rate. To validate the assay regarding the quality of quantification, spike/recovery and linearity of dilution experiments were performed. A total of 25 cancer and 5 noncancer cell lines were characterized with this assay method in terms of their activatable TGase 2 concentration (fmol/µg protein lysate) and the results were compared to protein synthesis data obtained by Western blotting. Moreover, complementary protein quantification methods using a biotinylated irreversible TGase 2 inhibitor as an activity-based probe and a commercially available ELISA were applied to selected cell lines to further validate the results obtained by the FA-based assay. Overall, the present study demonstrates that the FA-based assay using the substrate pair R-I-Cad and DMC represents a facile, homogenous and continuous method for quantifying TGase 2 activity in cell lysates.
Assuntos
Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases , Bioensaio , Cadaverina/farmacologia , Caseínas , Polarização de Fluorescência , Transglutaminases/metabolismoRESUMO
Some bacterial stress responses are involved in survival under antibiotic treatment and contribute to less susceptible microbial forms selection. Here, we tested the role of cadaverine, one of the biogenic polyamines considered as universal adaptogens, in the processes. The expression of ldcC and cadA genes, encoding cadaverine-producing lysine decarboxylase, increased in Escherichia coli cells exposed to ß-lactams and fluoroquinolones but not aminoglycosides. The transcriptional regulators RpoS and SoxS controlled the expression of ldcC and cadA, respectively, in response to antibiotics. Exogenous cadaverine had little effect on E. coli antibiotic susceptibility, whereas non-antibiotic-induced endogenous cadaverine contributed to its tolerance to ß-lactams, fluoroquinolones, and aminoglycosides. Antibiotic-induced cadaverine synthesis promoted bacterial survival under fluoroquinolone exposure, as well as could contribute to low-resistant bacterial forms development. Selection under the fluoroquinolone levofloxacin exposure toward bacteria with an increased ability to synthesize cadaverine and negative correlation between LdcC activity and fluoroquinolone susceptibility in the selected forms were demonstrated. The same correlation in a special group of low-level resistant clinical E. coli isolates was revealed. So, cadaverine biosynthesis appeared to be a significant player in decreased E. coli antibiotic susceptibility development.
Assuntos
Antibacterianos/farmacologia , Cadaverina/biossíntese , Carboxiliases/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Cadaverina/metabolismo , Cadaverina/farmacologia , Carboxiliases/metabolismo , Farmacorresistência Bacteriana , Tolerância a Medicamentos , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Fluoroquinolonas/farmacologia , Regulação Bacteriana da Expressão Gênica , Levofloxacino/farmacologia , Testes de Sensibilidade Microbiana , Mutação , beta-Lactamas/farmacologiaRESUMO
The liver is the primary target organ for perfluorooctane sulphonate (PFOS), a recently discovered persistent organic pollutant. However, the mechanisms mediating hepatotoxicity remain unclear. Herein, we explored the relationship between reactive oxygen species (ROS) and autophagy and apoptosis induced by PFOS in L-02 cells, which are incubated with different concentrations of PFOS (0, 50, 100, 150, 200, or 250 µmol/L) for 24 or 48 hrs at 37°C. The results indicated that PFOS exposure decreased cell activities, enhanced ROS levels in a concentration-dependent manner, decreased mitochondrial membrane potential (MMP), and induced autophagy and apoptosis. Compared with the control, 200 µmol/L PFOS increased ROS levels; enhanced the expression of Bax, cleaved-caspase-3, and LC3-II; induced autophagy; decreased MMP; and lowered Bcl-2, p62, and Bcl-2/Bax ratio. The antioxidant N-acetyl cysteine (NAC) protected MMP against PFOS-induced changes and diminished apoptosis and autophagy. Compared with 200 µmol/L PFOS treatment, NAC pretreatment reversed the increase in ROS, Bax, and cleaved-caspase-3 protein caused by PFOS, lowered the apoptosis rate increased by PFOS, and increased the levels of MMP and Bcl-2/Bax ratio decreased by PFOS. The autophagy inhibitor 3-methyladenine and chloroquine decreased apoptosis and cleaved-caspase-3 protein level and increased the Bcl-2/Bax ratio. In summary, our results suggest that ROS-triggered autophagy is involved in PFOS-induced apoptosis in L-02 cells.
Assuntos
Ácidos Alcanossulfônicos/farmacologia , Apoptose , Autofagia , Embrião de Mamíferos/patologia , Fluorocarbonos/farmacologia , Fígado/embriologia , Fígado/patologia , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cadaverina/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Microambiente Celular/efeitos dos fármacos , Humanos , Fígado/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismoRESUMO
Polyamines are essential for biofilm formation in Escherichia coli, but it is still unclear which polyamines are primarily responsible for this phenomenon. To address this issue, we constructed a series of E. coli K-12 strains with mutations in genes required for the synthesis and metabolism of polyamines. Disruption of the spermidine synthase gene (speE) caused a severe defect in biofilm formation. This defect was rescued by the addition of spermidine to the medium but not by putrescine or cadaverine. A multidrug/spermidine efflux pump membrane subunit (MdtJ)-deficient strain was anticipated to accumulate more spermidine and result in enhanced biofilm formation compared to the MdtJ+ strain. However, the mdtJ mutation did not affect intracellular spermidine or biofilm concentrations. E. coli has the spermidine acetyltransferase (SpeG) and glutathionylspermidine synthetase/amidase (Gss) to metabolize intracellular spermidine. Under biofilm-forming conditions, not Gss but SpeG plays a major role in decreasing the too-high intracellular spermidine concentrations. Additionally, PotFGHI can function as a compensatory importer of spermidine when PotABCD is absent under biofilm-forming conditions. Last, we report here that, in addition to intracellular spermidine, the periplasmic binding protein (PotD) of the spermidine preferential ABC transporter is essential for stimulating biofilm formation.IMPORTANCE Previous reports have speculated on the effect of polyamines on bacterial biofilm formation. However, the regulation of biofilm formation by polyamines in Escherichia coli has not yet been assessed. The identification of polyamines that stimulate biofilm formation is important for developing novel therapies for biofilm-forming pathogens. This study sheds light on biofilm regulation in E. coli Our findings provide conclusive evidence that only spermidine can stimulate biofilm formation in E. coli cells, not putrescine or cadaverine. Last, ΔpotD inhibits biofilm formation even though the spermidine is synthesized inside the cells from putrescine. Since PotD is significant for biofilm formation and there is no ortholog of the PotABCD transporter in humans, PotD could be a target for the development of biofilm inhibitors.
Assuntos
Biofilmes/crescimento & desenvolvimento , Escherichia coli K12/fisiologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Periplásmicas de Ligação/metabolismo , Espermidina/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Acetiltransferases/metabolismo , Amida Sintases/metabolismo , Cadaverina/farmacologia , Meios de Cultura , Escherichia coli K12/efeitos dos fármacos , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Deleção de Genes , Proteínas de Membrana Transportadoras/genética , Mutação , Óperon , Proteínas Periplásmicas de Ligação/genética , Putrescina/farmacologia , Espermidina/farmacologia , Espermidina Sintase/genética , Espermidina Sintase/metabolismoRESUMO
The significant increase of periodontitis, chronic kidney disease (CKD), Alzheimer's disease and cancer can be attributed to an ageing population. Each disease produces a range of biomarkers that can be indicative of disease onset and progression. Biomarkers are defined as cellular (intra/extracellular components and whole cells), biochemical (metabolites, ions and toxins) or molecular (nucleic acids, proteins and lipids) alterations which are measurable in biological media such as human tissues, cells or fluids. An interesting group of biomarkers that merit further investigation are the polyamines. Polyamines are a group of molecules consisting of cadaverine, putrescine, spermine and spermidine and have been implicated in the development of a range of systemic diseases, in part due to their production in periodontitis. Cadaverine and putrescine within the periodontal environment have demonstrated cell signalling interfering abilities, by way of leukocyte migration disruption. The polyamines spermine and spermidine in tumour cells have been shown to inhibit cellular apoptosis, effectively prolonging tumorigenesis and continuation of cancer within the host. Polyamine degradation products such as acrolein have been shown to exacerbate renal damage in CKD patients. Thus, the use of such molecules has merit to be utilized in the early indication of such diseases in patients.
Assuntos
Doença de Alzheimer/diagnóstico , Cadaverina/sangue , Neoplasias/diagnóstico , Periodontite/diagnóstico , Putrescina/sangue , Insuficiência Renal Crônica/diagnóstico , Espermidina/sangue , Espermina/sangue , Acroleína/sangue , Acroleína/farmacologia , Doença de Alzheimer/sangue , Apoptose/efeitos dos fármacos , Biomarcadores/sangue , Biotransformação , Cadaverina/farmacologia , Movimento Celular/efeitos dos fármacos , Humanos , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Neoplasias/sangue , Periodontite/metabolismo , Putrescina/farmacologia , Insuficiência Renal Crônica/sangue , Espermidina/farmacologia , Espermina/farmacologiaRESUMO
We recently reported that necroptosis contributed to compression-induced nucleus pulposus (NP) cells death. In the current study, we investigated the regulative effect of necroptosis inhibitor Necrostatin-1 on NP cells apoptosis and autophagy. Necrostatin-1, autophagy inhibitor 3-Methyladenine and apoptosis inhibitor Z-VAD-FMK were employed, and NP cells were exposed to 1.0 MPa compression for 0, 24 and 36 h. Necroptosis-associated molecules were measured by Western blot and RT-PCR. Autophagy and apoptosis levels were evaluated by Western blot and quantified by flow cytometry after monodansylcadaverine and Annexin V-FITC/propidium iodide staining, respectively. The cell viability and cell death were also examined. Furthermore, we measured mitochondrial membrane potential (MMP), mitochondrial permeability transition pore (MPTP) and indices of oxidative stress to assess mitochondrial dysfunction. The results established that Necrostatin-1 blocked NP cells autophagy, and 3-Methyladenine had little influence on NP cells necroptosis. The Necrostatin-1+3-Methyladenine treatment exerted almost the same role as Necrostatin-1 in reducing NP cells death. Necrostatin-1 restrained NP cells apoptosis, while Z-VAD-FMK enhanced NP cells necroptosis. The Necrostatin-1+Z-VAD-FMK treatment provided more prominent role in blocking NP cells death compared with Necrostatin-1, consistent with increased MMP, reduced opening of MPTP and oxidative stress. In summary, the synergistic utilization of Necrostatin-1 and Z-VAD-FMK is a very worthwhile solution in preventing compression-mediated NP cells death, which might be largely attributed to restored mitochondrial function.
Assuntos
Clorometilcetonas de Aminoácidos/farmacologia , Imidazóis/farmacologia , Indóis/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Núcleo Pulposo/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Cadaverina/análogos & derivados , Cadaverina/farmacologia , Morte Celular , Sobrevivência Celular , Força Compressiva , L-Lactato Desidrogenase/metabolismo , Mitocôndrias/metabolismo , Núcleo Pulposo/citologia , Pressão , Propídio/química , Ratos , Ratos Sprague-Dawley , Espécies Reativas de OxigênioRESUMO
A nutritional intervention, exclusive enteral nutrition (EEN) can induce remission in patients with pediatric Crohn's disease (CD). We characterized changes in the fecal microbiota and metabolome to identify the mechanism of EEN. Feces of 43 children were collected prior, during and after EEN. Microbiota and metabolites were analyzed by 16S rRNA gene amplicon sequencing and NMR. Selected metabolites were evaluated in relevant model systems. Microbiota and metabolome of patients with CD and controls were different at all time points. Amino acids, primary bile salts, trimethylamine and cadaverine were elevated in patients with CD. Microbiota and metabolome differed between responders and non-responders prior to EEN. EEN decreased microbiota diversity and reduced amino acids, trimethylamine and cadaverine towards control levels. Patients with CD had reduced microbial metabolism of bile acids that partially normalized during EEN. Trimethylamine and cadaverine inhibited intestinal cell growth. TMA and cadaverine inhibited LPS-stimulated TNF-alpha and IL-6 secretion by primary human monocytes. A diet rich in free amino acids worsened inflammation in the DSS model of intestinal inflammation. Trimethylamine, cadaverine, bile salts and amino acids could play a role in the mechanism by which EEN induces remission. Prior to EEN, microbiota and metabolome are different between responders and non-responders.
Assuntos
Bactérias/classificação , Doença de Crohn/terapia , Nutrição Enteral/métodos , Microbioma Gastrointestinal/efeitos dos fármacos , Metabolômica/métodos , Adolescente , Aminoácidos/análise , Bactérias/genética , Biodiversidade , Cadaverina/análise , Cadaverina/farmacologia , Estudos de Casos e Controles , Criança , Doença de Crohn/imunologia , Nutrição Enteral/efeitos adversos , Fezes/microbiologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Interleucina-6/metabolismo , Lipopolissacarídeos/efeitos adversos , Masculino , Metilaminas/análise , Metilaminas/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Estudos Prospectivos , RNA Ribossômico 16S/genética , Resultado do TratamentoRESUMO
A tight junction (TJ) makes a physical barrier in the epidermal cells of skin. Ultraviolet (UV) light may disrupt the TJ barrier, but the mechanism has not been well clarified. Weak UVB (5 mJ/cm2) caused mislocalization of claudin-1 (CLDN1), a component of the TJ strand, and disruption of TJ barrier in human keratinocyte-derived HaCaT cells. The UVB-induced mislocalization of CLDN1 was inhibited by monodansylcadaverine (MDC), a clathrin-dependent endocytosis inhibitor, suggesting that UVB enhances the internalization of CLDN1. Transepidermal electrical resistance and paracellular flux of lucifer yellow, a fluorescent hydrophilic marker, were rescued by MDC. UVB changed neither the total nor phosphorylation levels of CLDN1, but it increased both mono-ubiquitination and tyrosine nitration levels of CLDN1. Fluorescence measurements revealed that UVB increased intracellular free Ca2+, nitric oxide (NO), and peroxynitrite contents, which were inhibited by Opsin2 (OPN2) siRNA, suggesting that OPN2 functions as a UVB sensor. The effects of UVB were inhibited by an antagonist of transient receptor potential type vanilloid 1 (TRPV1) and Ca2+ chelator. Both NO donor and peroxynitrite donor induced the mislocalization of CLDN1 and disruption of TJ barrier, which were rescued by a NO synthase (NOS) inhibitor and a peroxynitrite scavenger. Weak UVB irradiation induced the disruption of TJ barrier mediated by mislocalization of CLDN1 in HaCaT cells. The OPN2/TRPV1/NOS signaling pathway may be a novel target for preventing destruction of the TJ barrier by UVB irradiation.
Assuntos
Claudina-1/metabolismo , Queratinócitos/metabolismo , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/biossíntese , Transdução de Sinais/efeitos da radiação , Raios Ultravioleta , Cadaverina/análogos & derivados , Cadaverina/farmacologia , Sobrevivência Celular/efeitos da radiação , Endocitose/efeitos dos fármacos , Células HaCaT , Humanos , Óxido Nítrico Sintase/metabolismo , Fosforilação/efeitos da radiação , Transdução de Sinais/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , Junções Íntimas/metabolismo , Junções Íntimas/efeitos da radiação , Ubiquitinação/efeitos da radiaçãoRESUMO
Polyamines are small amines that accumulate during stress and contribute to disease resistance through as yet unknown signaling pathways. Using a comprehensive RNA-sequencing analysis, we show that early transcriptional responses triggered by each of the most abundant polyamines (putrescine, spermidine, spermine, thermospermine and cadaverine) exhibit specific quantitative differences, suggesting that polyamines (rather than downstream metabolites) elicit defense responses. Signaling by putrescine, which accumulates in response to bacteria that trigger effector triggered immunity (ETI) and systemic acquired resistance (SAR), is largely dependent on the accumulation of hydrogen peroxide, and is partly dependent on salicylic acid (SA), the expression of ENHANCED DISEASE SUSCEPTIBILITY (EDS1) and NONEXPRESSOR of PR GENES1 (NPR1). Putrescine elicits local SA accumulation as well as local and systemic transcriptional reprogramming that overlaps with SAR. Loss-of-function mutations in arginine decarboxylase 2 (ADC2), which is required for putrescine synthesis and copper amine oxidase (CuAO), which is involved in putrescine oxidation, compromise basal defenses, as well as putrescine and pathogen-triggered systemic resistance. These findings confirm that putrescine elicits ROS-dependent SA pathways in the activation of plant defenses.
Assuntos
Arabidopsis/efeitos dos fármacos , Reguladores de Crescimento de Plantas/metabolismo , Putrescina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Ácido Salicílico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Arabidopsis/metabolismo , Cadaverina/farmacologia , Perfilação da Expressão Gênica , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Espermidina/farmacologia , Espermina/análogos & derivados , Espermina/farmacologiaRESUMO
Plant responses to salinity have been extensively studied over the last decades. Despite the vast accumulated knowledge, the ways Arabidopsis lateral roots (LR) cope with lethal salinity has not been fully resolved. Here we compared the primary root (PR) and the LR responses during events leading to lethal salinity (NaCl 200 mM) in Arabidopsis. We found that the PR and young LR responded differently to lethal salinity: While the PR died, emerging and young LR's remained strikingly viable. Moreover, "age acquired salt tolerance" (AAST) was observed in the PR. During the 2 days after germination (DAG) the PR was highly sensitive, but at 8 DAG there was a significant increase in the PR cell survival. Nevertheless, the young LR exhibited an opposite pattern and completely lost its salinity tolerance, as it elongated beyond 400 µm. Examination of several cell death signatures investigated in the young LR showed no signs of an active programmed cell death (PCD) during lethal salinity. However, Autophagic PCD (A-PCD) but not apoptosis-like PCD (AL-PCD) was found to be activated in the PR during the high salinity conditions. We further found that salinity induced NADPH oxidase activated ROS, which were more highly distributed in the young LR compared to the PR, is required for the improved viability of the LR during lethal salinity conditions. Our data demonstrated a position-dependent resistance of Arabidopsis young LR to high salinity. This response can lead to identification of novel salt stress coping mechanisms needed by agriculture during the soil salinization challenge.
Assuntos
Arabidopsis/fisiologia , Raízes de Plantas/fisiologia , Tolerância ao Sal , Apoptose/efeitos dos fármacos , Arabidopsis/efeitos dos fármacos , Autofagia , Cadaverina/análogos & derivados , Cadaverina/farmacologia , Morte Celular , Endossomos/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Germinação , Proteínas de Fluorescência Verde/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Transmissão , NADPH Oxidases/metabolismo , Raízes de Plantas/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Salinidade , Sais , Plântula/metabolismo , Cloreto de Sódio/farmacologiaRESUMO
Chikungunya virus (CHIKV) is a re-emerging arbovirus known to cause chronic myalgia and arthralgia with high morbidity. CHIKV is now considered endemic in many countries across Asia and Africa. In this study, the susceptibility of various human, mammalian and mosquito cell lines to CHIKV infection was evaluated. CHIKV infection was found to be cell-type dependent and virus strain-specific. Furthermore, SJCRH30 (human rhabdomyosarcoma cell line) was showed to be highly permissive to CHIKV infection, with maximum production of infectious virions observed at 12 h.p.i. Pre-infection treatment of SJCRH30 with various inhibitors of endocytosis, including monodansylcadaverine (receptor-mediated endocytic inhibitor), dynasore (clathrin-mediated endocytic inhibitor), as well as filipin (caveolin-mediated endocytosis inhibitor), resulted in minimal inhibition of CHIKV infection. In contrast, dose-dependent inhibition of CHIKV infection was observed with the treatment of macropinocytosis inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Furthermore, siRNA-mediated knockdown of sortin nexin 9 (SNX9) a protein involved in macropinosome formation, also resulted in a significant dose-dependent reduction in viral titre. By performing a virus entry assay, CHIKV particles were also observed to colocalize with FITC-dextran, a macropinosome marker. This study shows for the first time, that the infectious entry of CHIKV into human muscle cells is mediated by macropinocytosis. Together, the data from this study may pave the way for the development of specific inhibitors that target the entry process of CHIKV into cells.
Assuntos
Febre de Chikungunya/virologia , Vírus Chikungunya/fisiologia , Músculos/virologia , Pinocitose/fisiologia , Internalização do Vírus , Amilorida/análogos & derivados , Amilorida/farmacologia , Animais , Cadaverina/análogos & derivados , Cadaverina/farmacologia , Linhagem Celular , Sobrevivência Celular , Vírus Chikungunya/crescimento & desenvolvimento , Clatrina/antagonistas & inibidores , Endocitose/efeitos dos fármacos , Filipina/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Hidrazonas/farmacologia , Cinética , Pinocitose/efeitos dos fármacos , Pinocitose/genética , RNA Interferente Pequeno , Rabdomiossarcoma , Nexinas de Classificação/genética , Carga Viral , Ensaio de Placa ViralRESUMO
Despite evidence linking the human microbiome to health and disease, how the microbiota affects human physiology remains largely unknown. Microbiota-encoded metabolites are expected to play an integral role in human health. Therefore, assigning function to these metabolites is critical to understanding these complex interactions and developing microbiota-inspired therapies. Here, we use large-scale functional screening of molecules produced by individual members of a simplified human microbiota to identify bacterial metabolites that agonize G-protein-coupled receptors (GPCRs). Multiple metabolites, including phenylpropanoic acid, cadaverine, 9-10-methylenehexadecanoic acid, and 12-methyltetradecanoic acid, were found to interact with GPCRs associated with diverse functions within the nervous and immune systems, among others. Collectively, these metabolite-receptor pairs indicate that diverse aspects of human health are potentially modulated by structurally simple metabolites arising from primary bacterial metabolism.
Assuntos
Bactérias/metabolismo , Interações entre Hospedeiro e Microrganismos/imunologia , Interações entre Hospedeiro e Microrganismos/fisiologia , Microbiota/imunologia , Microbiota/fisiologia , Receptores Acoplados a Proteínas G/agonistas , Proteínas Angiogênicas/agonistas , Animais , Cadaverina/metabolismo , Cadaverina/farmacologia , Ácidos Graxos/metabolismo , Ácidos Graxos/farmacologia , Fermentação , Vida Livre de Germes , Agonistas dos Receptores Histamínicos , Humanos , Sistema Imunitário , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Propionatos/metabolismo , Propionatos/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos/efeitos dos fármacos , Receptores de Neurotransmissores/agonistasRESUMO
Recent studies showed that changes to the gut microbiome alters the microbiome-derived metabolome, potentially promoting carcinogenesis in organs that are distal to the gut. In this study, we assessed the relationship between breast cancer and cadaverine biosynthesis. Cadaverine treatment of Balb/c female mice (500 nmol/kg p.o. q.d.) grafted with 4T1 breast cancer cells ameliorated the disease (lower mass and infiltration of the primary tumor, fewer metastases, and lower grade tumors). Cadaverine treatment of breast cancer cell lines corresponding to its serum reference range (100-800 nM) reverted endothelial-to-mesenchymal transition, inhibited cellular movement and invasion, moreover, rendered cells less stem cell-like through reducing mitochondrial oxidation. Trace amino acid receptors (TAARs), namely, TAAR1, TAAR8 and TAAR9 were instrumental in provoking the cadaverine-evoked effects. Early stage breast cancer patients, versus control women, had reduced abundance of the CadA and LdcC genes in fecal DNA, both responsible for bacterial cadaverine production. Moreover, we found low protein expression of E. coli LdcC in the feces of stage 1 breast cancer patients. In addition, higher expression of lysine decarboxylase resulted in a prolonged survival among early-stage breast cancer patients. Taken together, cadaverine production seems to be a regulator of early breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Cadaverina/farmacologia , Microbiota , Receptores de Aminoácido/metabolismo , Neoplasias da Mama/etiologia , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Transição Epitelial-Mesenquimal , Feminino , Humanos , Estimativa de Kaplan-Meier , Modelos BiológicosRESUMO
A whole-cell (cadaverine-producing strain, Escherichia coli AST3) immobilization method was developed for improving catalytic activity and cadaverine tolerance during cadaverine production. Cell-immobilized beads were prepared by polyvinyl alcohol (PVA) and sodium alginate (SA) based on their advantages in biocatalyst activity recovery and mechanical strength. The following optimal immobilization conditions were established using response surface methodology: 3.62% SA, 4.71% PVA, 4.21% CaCl2, calcification, 12 h, and freezing for 16 h at - 80 °C, with a cell concentration of 0.3% (g dry cell weight (DCW) per 100 mL) of immobilized beads. After a 2-h bioconversion, the immobilized beads maintained 85% of their original biocatalyst activity, which was 1.8-fold higher than that of free cells. Furthermore, the effects of cell protectants on immobilized biocatalyst activity were examined by fed-batch bioconversion experiments. The results showed that the addition of polyvinylpyrrolidone (PVP) into the immobilized matrix effectively protected biocatalyst activity, with 95% of the relative activity remaining after the 2-h bioconversion. The performance of PVA-SA-PVP-immobilized E. coli AST3 showed continuous production of cadaverine, with an average cadaverine yield of 29 ± 1 g gDCW-1 h-1 after 12 h, suggesting that this method is capable of industrial scale cadaverine production.
Assuntos
Cadaverina/metabolismo , Cadaverina/farmacologia , Citoproteção/efeitos dos fármacos , Alginatos/metabolismo , Cadaverina/biossíntese , Catálise , Álcool de Polivinil/metabolismoRESUMO
AIMS: Accumulating evidence supports that cerebrospinal fluid (CSF) in the subarachnoid space (SAS) could reenter the brain parenchyma via the glymphatic influx. The present study was designed to characterize the detailed pathway of subarachnoid CSF influx by using a novel CSF tracer. MAIN METHODS: Fluorescently conjugated cadaverine (A488-ca), for the first time, was employed to investigate CSF movement in the brain. Following intracisternal infusion of CSF tracers, mice brain was sliced and prepared for fluorescence imaging. Some brain sections were immunostained in order to observe tracer distribution and cellular uptake. KEY FINDINGS: A488-ca moved into the brain parenchyma rapidly, and the influx was time and region dependent. A488-ca entered the mice brain more readily and spread more widely than another commonly used CSF tracer-fluorescently conjugated ovalbumin (OA-45). Furthermore, A488-ca could enter the brain parenchyma either along the paravascular space or across the pial surface. Suppression of glymphatic transport by administration with acetazolamide strikingly reduced the influx of A488-ca. More importantly, relative to OA-45 largely remained in the extracellular space, A488-ca exhibited obvious cellular uptake by astrocytes surrounding the blood vessels and neurons in the cerebral cortex. SIGNIFICANCE: Subarachnoid CSF could flow into the brain parenchyma via the glymphatic influx, in which the transcellular pathway was faithfully traced by intracisternal infusion with fluorescently conjugated cadaverine. These observations extend our comprehension on the glymphatic influx pathway.
Assuntos
Cadaverina/farmacologia , Líquido Cefalorraquidiano/metabolismo , Linfa/fisiologia , Espaço Subaracnóideo/metabolismo , Acetazolamida/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Cadaverina/administração & dosagem , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Líquido Cefalorraquidiano/efeitos dos fármacos , Cisterna Magna , Diuréticos/farmacologia , Corantes Fluorescentes , Injeções , Masculino , Camundongos , Camundongos Endogâmicos ICR , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Pia-Máter/metabolismo , Espaço Subaracnóideo/efeitos dos fármacosRESUMO
Root growth architecture is a major determinant of agricultural productivity and plant fitness in natural ecosystems. Here we describe the methods used in a Quantitative Trait Loci (QTL) study that allowed the identification of ORGANIC CATION TRANSPORTER 1 (OCT1) as a determinant of root growth response to cadaverine treatment in Arabidopsis thaliana. This protocol screens natural accessions to characterize the variation in root growth response to the naturally occurring polyamine cadaverine, then uses recombination mapping to identify loci that are responsible for the variation existing between two accessions with contrasting phenotypes.
Assuntos
Arabidopsis/efeitos dos fármacos , Arabidopsis/fisiologia , Cadaverina/farmacologia , Fenótipo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/fisiologia , Locos de Características Quantitativas , Alelos , Mapeamento Cromossômico , Regulação da Expressão Gênica de Plantas , Genótipo , Mutação , Plântula/genéticaRESUMO
OBJECTIVES: Indium compounds are used in manufacturing displays of mobile phones and televisions. However, these materials cause interstitial pneumonia in exposed workers. Animal experiments demonstrated that indium compounds caused lung cancer. Chronic inflammation is considered to play a role in lung carcinogenesis and fibrosis induced by particulate matters. 8-Nitroguanine (8-nitroG) is a mutagenic DNA lesion formed during inflammation and may participate in carcinogenesis. To clarify the mechanism of carcinogenesis, we examined 8-nitroG formation in indium-exposed cultured cells. METHODS: We treated RAW 264.7 mouse macrophages with indium oxide (In2O3) nanoparticles (primary diameter: 30-50 nm), and performed fluorescent immunocytochemistry to detect 8-nitroG. The extent of 8-nitroG formation was evaluated by quantitative image analysis. We measured the amount of nitric oxide (NO) in the culture supernatant of In2O3-treated cells by the Griess method. We also examined the effects of inhibitors of inducible NO synthase (iNOS) and endocytosis on In2O3-induced 8-nitroG formation. RESULTS: In2O3 significantly increased the intensity of 8-nitroG formation in RAW 264.7 cells in a dose-dependent manner. In2O3-induced 8-nitroG formation was observed at 2 h and further increased at 4 h, and the amount of NO released from In2O3-exposed cells was significantly increased at 2-4 h compared with the control. 8-NitroG formation was suppressed by 1400W (an iNOS inhibitor), methyl-ß-cyclodextrin and monodansylcadaverine (inhibitors of caveolae- and clathrin-mediated endocytosis, respectively). CONCLUSIONS: These results suggest that endocytosis and NO generation participate in indium-induced 8-nitroG formation. NO released from indium-exposed inflammatory cells may induce DNA damage in adjacent lung epithelial cells and contribute to carcinogenesis.
Assuntos
Dano ao DNA/efeitos dos fármacos , Guanina/análogos & derivados , Índio/farmacologia , Macrófagos/efeitos dos fármacos , Amidinas/farmacologia , Animais , Benzilaminas/farmacologia , Cadaverina/análogos & derivados , Cadaverina/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Guanina/biossíntese , Imuno-Histoquímica , Camundongos , Nanopartículas , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Tamanho da Partícula , beta-Ciclodextrinas/farmacologiaRESUMO
The insulin-like growth factor-1 receptor (IGF1R) mediates the biological actions of IGF1 and IGF2. The IGF1R is involved in both physiological and pathological activities and is usually overexpressed in most types of cancer. In addition to its classical mechanism of action, recent evidence has shown a nuclear presence of IGF1R, associated with novel genomic/transcriptional types of activities. The present study was aimed at evaluating the hypothesis that nuclear IGF1R localization is not restricted to cancer cells and might constitute a novel physiologically relevant regulatory mechanism. Our data shows that nuclear translocation takes place in a wide array of cells, including normal diploid fibroblasts. In addition, we provide evidence for a synergistic effect of a nuclear translocation blocker along with selective IGF1R inhibitors in terms of decreasing cell proliferation. Given the important role of the IGF1R in mitogenesis, the present results may be of translational relevance in cancer research. In conclusion, results are consistent with the concept that nuclear IGF1R fulfills important physiological and pathological roles.