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1.
Proc Natl Acad Sci U S A ; 120(3): e2214750120, 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36623197

RESUMO

Nucleotide-binding leucine-rich repeat (NLR) receptor-mediated immunity includes rapid production of reactive oxygen species (ROS) and transcriptional reprogramming, which is controlled by transcription factors (TFs). Although some TFs have been reported to participate in NLR-mediated immune response, most TFs are transcriptional activators, and whether and how transcriptional repressors regulate NLR-mediated plant defenses remains largely unknown. Here, we show that the Alfin-like 7 (AL7) interacts with N NLR and functions as a transcriptional repressor. Knockdown and knockout of AL7 compromise N NLR-mediated resistance against tobacco mosaic virus, whereas AL7 overexpression enhances defense, indicating a positive regulatory role for AL7 in immunity. AL7 binds to the promoters of ROS scavenging genes to inhibit their transcription during immune responses. Mitogen-activated protein kinases (MAPKs), salicylic acid-induced protein kinase (SIPK), and wound-induced protein kinase (WIPK) directly interact with and phosphorylate AL7, which impairs the AL7-N interaction and enhances its DNA binding activity, which promotes ROS accumulation and enables immune activation. In addition to N, AL7 is also required for the function of other Toll interleukin 1 receptor/nucleotide-binding/leucine-rich repeats (TNLs) including Roq1 and RRS1-R/RPS4. Our findings reveal a hitherto unknown MAPK-AL7 module that negatively regulates ROS scavenging genes to promote NLR-mediated immunity.


Assuntos
Proteínas de Plantas , Fatores de Transcrição , Espécies Reativas de Oxigênio/metabolismo , Leucina/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Domínios Proteicos , Nucleotídeos/metabolismo , Imunidade Vegetal , Tabaco/metabolismo
2.
Phytomedicine ; 109: 154617, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36610140

RESUMO

BACKGROUND: Searching the targets of natural products is very important for drug discovery and elucidating the mechanism of drug action and disease. Honokiol (HK), as the major active component of Magnolia officinalis Rehder & E.H.Wilson, has been widely used in medicine and cosmetics. Among its bioactivities, its anti-inflammatory activity is particularly impressive. However, the target protein of HK in anti-inflammatory action and its regulatory mechanism are unclear. PURPOSE: Here, we identified the target protein and molecular mechanism of the anti- inflammatory action of HK. METHODS: First, an LPS-induced septic shock model and DSS-induced ulcerative colitis model were used to assess the anti-inflammatory efficacy of HK. Second, the drug affinity responsive target stability, proteomics analysis, thermal shift assays and cellular thermal shift assays were used to identify and validate the target of HK. Finally, western blot, ELISA, LDH immunofluorescence staining, shRNA and LC/MS for L-leucine analysis were performed to determine the mechanism of the anti-inflammatory action of HK. RESULTS: This study revealed that HK significantly alleviated LPS-induced septic shock and DSS-induced ulcerative colitis in vivo, suggesting that HK has significant anti-inflammatory activity. HK treatment dramatically reduced IL-1ß release and caspase-1 activation at different time points, showing that HK could inhibit both NLRP3 inflammasome priming and activation processes in cells. HK also suppressed adaptor apoptosis speck-like protein oligomerization. Mechanistically, SLC3A2 was identified as a direct target of HK in THP-1 cells. HK downregulated SLC3A2 expression by promoting its degradation via proteasome-mediated proteolysis. Further study demonstrated that HK triggered SLC3A2 to suppress NLRP3 inflammasome activation by significantly reducing the content of L-leucine transported into cells and lysosomes to block the mTORC1 pathway. CONCLUSIONS: Our work identified HK as a promising anti-inflammatory drug candidate through the SLC3A2/L-leucine/mTORC1/NLRP3 pathways.


Assuntos
Colite Ulcerativa , Choque Séptico , Humanos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/tratamento farmacológico , Lipopolissacarídeos , Leucina , Anti-Inflamatórios/farmacologia
3.
Mol Cell ; 83(1): 6-8, 2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36608671

RESUMO

The mechanistic target of rapamycin complex 1 (mTORC1) senses cellular leucine levels through the GATOR1/2-Rag axis. Jiang et al. show that the Ring domains of GATOR2 subunits maintain the integrity of the complex and promote ubiquitination and inhibition of GATOR1, thereby leading to mTORC1 activation.


Assuntos
Complexos Multiproteicos , Serina-Treonina Quinases TOR , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Serina-Treonina Quinases TOR/genética , Complexos Multiproteicos/genética , Leucina , Lisossomos
4.
BMC Med Genomics ; 16(1): 10, 2023 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-36653841

RESUMO

BACKGROUND: Leucine-rich repeat sequence domains are known to mediate protein‒protein interactions. Recently, some studies showed that members of the leucine rich repeat containing (LRRC) protein superfamily may become new targets for the diagnosis and treatment of tumours. However, it is not known whether any of the LRRC superfamily genes is expressed in the stroma of ovarian cancer (OC) and is associated with prognosis. METHODS: The clinical data and transcriptional profiles of OC patients from the public databases TCGA (n = 427), GTEx (n = 88) and GEO (GSE40266 and GSE40595) were analysed by R software. A nomogram model was also generated through R. An online public database was used for auxiliary analysis of prognosis, immune infiltration and protein‒protein interaction (PPI) networks. Immunohistochemistry and qPCR were performed to determine the protein and mRNA levels of genes in high-grade serous ovarian cancer (HGSC) tissues of participants and the MRC-5 cell line induced by TGF-ß. RESULTS: LRRC15 and LRRC32 were identified as differentially expressed genes from the LRRC superfamily by GEO transcriptome analysis. PPI network analysis suggested that they were most enriched in TGF-ß signalling. The TCGA-GTEx analysis results showed that only LRRC15 was highly expressed in both cancer-associated fibroblasts (CAFs) and the tumour stroma of OC and was related to clinical prognosis. Based on this, we developed a nomogram model to predict the incidence of adverse outcomes in OC. Moreover, LRRC15 was positively correlated with CAF infiltration and negatively correlated with CD8 + T-cell infiltration. As a single indicator, LRRC15 had the highest accuracy (AUC = 0.920) in predicting the outcome of primary platinum resistance. CONCLUSIONS: The LRRC superfamily is related to the TGF-ß pathway in the microenvironment of OC. LRRC15, as a stromal biomarker, can predict the clinical prognosis of HGSC and promote the immunosuppressive microenvironment. LRRC15 may be a potential therapeutic target for reversing primary resistance in OC.


Assuntos
Neoplasias Ovarianas , Platina , Humanos , Feminino , Platina/metabolismo , Platina/uso terapêutico , Leucina/metabolismo , Leucina/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Prognóstico , Células Estromais/metabolismo , Células Estromais/patologia , Microambiente Tumoral , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo
6.
Biochem Biophys Res Commun ; 643: 88-95, 2023 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-36587526

RESUMO

Brahma (BRM) is one of the core ATPase subunits of SWI/SNF chromatin remodeling complex, and participates in various important cellular regulatory processes. However, the role of BRM in regulating gene expression of the mechanistic target of rapamycin (mTOR) still remains unknown. In this study, we explored the effects and the corresponding molecular mechanisms of BRM on Leucine (Leu)-stimulated mTOR activation in and proliferation of a mouse mammary epithelial cell (MEC) line (HC11 cell). Initially, we found that the abundance of BRM protein in mammary gland tissue during lactation was significantly higher than that during puberty and involution. BRM knockdown inhibited HC11 cell proliferation, mRNA expression of mTOR and subsequent protein phosphorylation, whereas BRM gene activation had the opposite effect. Leu affected the level of BRM protein and mTOR phospphorylation in a dose-dependent manner, and BRM knockdown totally blocked the stimulation of Leu on mTOR mRNA expression and protein phospphorylation. ChIP-PCR detected that BRM was bound to the -4368 âˆ¼ -4591 bp site of the mTOR promoter, and ChIP-qPCR further detected that Leu stimulated BRM to bind to this site. In conclusion, these data reveal that BRM is a positive regulator of HC11 cell proliferation and mediates Leu's stimulation on mTOR gene transcription and protein phosphorylation. Our data provide a new theoretical basis for the involvement of BRM in cell proliferation and regulation of the mTOR signaling pathway.


Assuntos
Cromatina , Fatores de Transcrição , Feminino , Animais , Camundongos , Cromatina/metabolismo , Fatores de Transcrição/metabolismo , Leucina/farmacologia , Leucina/metabolismo , Maturidade Sexual , Células Epiteliais/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Transcrição Genética , RNA Mensageiro/metabolismo
7.
Cells ; 12(2)2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36672216

RESUMO

Autoimmune encephalitis (AE) associated with autoantibodies against leucine-rich glioma-inactivated protein-1 (LGI1) can present with faciobrachial dystonic seizures (FBDS) and/or limbic encephalitis (LE). The reasons for this heterogeneity in phenotypes are unclear. We performed autoantibody (abs) characterization per patient, two patients suffering from LE and two from FBDS, using isolated antibodies specified with single amino acid epitope mapping. Electrophysiological slice recordings were conducted alongside spine density measurements, postsynaptic Alpha-amino-3-hydoxy-5-methyl-4-isoaxole-proprionate-receptors (AMPA-R) and N-methyl-D-aspartate-receptors receptor (NMDA-R) cluster counting. These results were correlated with the symptoms of each patient. While LGI1 abs from LE patients mainly interacted with the Leucine-rich repeat section of LGI1, abs from both FBDS patients also recognized the Epitempin section as well. Six-hour incubation of mouse hippocampal slices with LE patients autoantibodies but not from the FBDS patients resulted in a significant decline in long-term potentiation (p = 0.0015) or short-term plasticity at CA3-CA1 neurons and in decreased hippocampal synaptic density. Cluster differentiation showed no decrease in postsynaptic AMPA-R and NMDA-R. LGI1 autoantibodies selected by phenotype show an almost distinct epitope pattern, elicit disparate functional effects on hippocampal neurons, and cause divergent effects on spine density. This data illuminates potential pathomechanisms for disease heterogeneity in LGI1 AE.


Assuntos
Encefalite , Encefalite Límbica , Animais , Camundongos , Peptídeos e Proteínas de Sinalização Intracelular , Leucina , N-Metilaspartato , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico , Autoanticorpos , Encefalite Límbica/complicações , Encefalite Límbica/diagnóstico , Convulsões/complicações , Fenótipo
8.
Biochim Biophys Acta Proteins Proteom ; 1871(2): 140886, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36496204

RESUMO

Pyridoxal-5'-phosphate-(PLP-) dependent D-amino acid transaminases (DAATs) catalyze stereoselective reversible transfer of the amino group between D-amino acids and keto acids. In vivo DAATs are commonly known to synthesize D-glutamate for cell wall peptidoglycans. Today DAATs meet increasing attention for application in the synthesis of D-amino acids, whereas little is known about the mechanism of substrate recognition and catalytic steps of the D-amino acids conversion by DAATs. In this work, the pre-steady-state kinetics of the half-reactions of DAAT from Haliscomenobacter hydrossis with D-glutamate, D-alanine, D-leucine, and D-phenylalanine was examined at two wavelengths, 416 and 330 nm, using a stopped-flow technique. Monophasic kinetics was observed with specific substrates D-glutamate and D-alanine, whereas half-reactions with D-leucine and D-phenylalanine exhibited biphasic kinetics. All half-reactions proceeded until the complete conversion of PLP due to the release of the pyridoxamine-5'-phosphate form of cofactor from the holoenzyme . Comparison of kinetic parameters of half-reactions and the overall transamination reactions for D-leucine, D-phenylalanine revealed the increase in the rates of deamination of these substrates in the overall reaction with α-ketoglutarate. In the overall transamination reaction, the catalytic turnover rates for D-leucine and D-phenylalanine increased by 260 and 60 times, correspondingly, comparing with the slowest step rate constants in the half-reactions. We suggested the activating effect by a specific substrate α-ketoglutarate in the overall transamination reaction. The study of half-reactions helped to quantify the specificity of DAAT from H. hydrossis for D-amino acids with different properties. The results obtained are the first detailed analysis of half-reactions catalyzed by DAAT.


Assuntos
Aminoácidos , Transaminases , Transaminases/química , Ácido Glutâmico , Leucina , Ácidos Cetoglutáricos , Alanina , Fosfato de Piridoxal/química , Fenilalanina , Catálise , Fosfatos
9.
Pharmacol Res ; 187: 106604, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36503000

RESUMO

Fibrosis is a common pathological feature of organ diseases resulting from excessive production of extracellular matrix, which accounts for significant morbidity and mortality. However, there is currently no effective treatment targeting fibrogenesis. Recently, metabolic alterations are increasingly considered as essential factors underlying fibrogenesis, and especially research on metabolic regulation of amino acids is flourishing. Among them, branched-chain amino acids (BCAAs) are the most abundant essential amino acids, including leucine, isoleucine and valine, which play significant roles in the substance and energy metabolism and their regulation. Dysregulation of BCAAs metabolism has been proven to contribute to numerous diseases. In this review, we summarize the metabolic regulation of fibrosis and the changes in BCAAs metabolism secondary to fibrosis. We also review the effects and mechanisms of the BCAAs intervention, and its therapeutic targeting in hepatic, renal and cardiac fibrosis, with a focus on the fibrosis in liver and associated hepatocellular carcinoma.


Assuntos
Aminoácidos de Cadeia Ramificada , Isoleucina , Humanos , Aminoácidos de Cadeia Ramificada/metabolismo , Isoleucina/metabolismo , Valina , Leucina , Fibrose
10.
Nature ; 613(7942): 145-152, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36517600

RESUMO

Phytohormone signalling pathways have an important role in defence against pathogens mediated by cell-surface pattern recognition receptors and intracellular nucleotide-binding leucine-rich repeat class immune receptors1,2 (NLR). Pathogens have evolved counter-defence strategies to manipulate phytohormone signalling pathways to dampen immunity and promote virulence3. However, little is known about the surveillance of pathogen interference of phytohormone signalling by the plant innate immune system. The pepper (Capsicum chinense) NLR Tsw, which recognizes the effector nonstructural protein NSs encoded by tomato spotted wilt orthotospovirus (TSWV), contains an unusually large leucine-rich repeat (LRR) domain. Structural modelling predicts similarity between the LRR domain of Tsw and those of the jasmonic acid receptor COI1, the auxin receptor TIR1 and the strigolactone receptor partner MAX2. This suggested that NSs could directly target hormone receptor signalling to promote infection, and that Tsw has evolved a LRR resembling those of phytohormone receptors LRR to induce immunity. Here we show that NSs associates with COI1, TIR1 and MAX2 through a common repressor-TCP21-which interacts directly with these phytohormone receptors. NSs enhances the interaction of COI1, TIR1 or MAX2 with TCP21 and blocks the degradation of corresponding transcriptional repressors to disable phytohormone-mediated host immunity to the virus. Tsw also interacts directly with TCP21 and this interaction is enhanced by viral NSs. Downregulation of TCP21 compromised Tsw-mediated defence against TSWV. Together, our findings reveal that a pathogen effector targets TCP21 to inhibit phytohormone receptor function, promoting virulence, and a plant NLR protein has evolved to recognize this interference as a counter-virulence strategy, thereby activating immunity.


Assuntos
Capsicum , Doenças das Plantas , Reguladores de Crescimento de Plantas , Imunidade Vegetal , Proteínas de Plantas , Receptores de Reconhecimento de Padrão , Leucina , Doenças das Plantas/imunologia , Doenças das Plantas/virologia , Reguladores de Crescimento de Plantas/metabolismo , Imunidade Vegetal/imunologia , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Proteínas de Plantas/metabolismo , Receptores de Reconhecimento de Padrão/química , Receptores de Reconhecimento de Padrão/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Capsicum/imunologia , Capsicum/metabolismo , Capsicum/virologia , Virulência
11.
J Cereb Blood Flow Metab ; 43(1): 59-71, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36071616

RESUMO

During sleep, reduced brain energy demands provide an opportunity for biosynthetic processes like protein synthesis. Sleep is required for some forms of memory consolidation which requires de novo protein synthesis. We measured regional cerebral protein synthesis rates (rCPS) in human subjects to ascertain how rCPS is affected during sleep. Subjects underwent three consecutive L-[1-11C]leucine PET scans with simultaneous polysomnography: 1. rested awake, 2. sleep-deprived awake, 3. sleep. Measured rCPS were similar across the three conditions. Variations in sleep stage times during sleep scans were used to estimate rCPS in sleep stages under the assumption that measured rCPS is the weighted sum of rCPS in each stage, with weights reflecting time and availability of [11C]leucine in that stage. During sleep scans, subjects spent most of the time in N2, N3, and awake and very little time in N1 and REM; rCPS in N1 and REM could not be reliably estimated. When stages N1 and N2 were combined [N1,N2], estimates of rCPS were more robust. In selective regions, estimated rCPS were statistically significantly higher (30-39%) in [N1,N2] compared with N3; estimated rCPS in N3 were similar to values measured in sleep-deprived awake scans. Results indicate increased rates of protein synthesis linked to [N1,N2] sleep.


Assuntos
Sujeitos da Pesquisa , Sono , Humanos , Leucina , Encéfalo/diagnóstico por imagem , Tomografia por Emissão de Pósitrons
12.
Oncol Rep ; 49(1)2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36453240

RESUMO

Microcystin­leucine arginine (MC­LR) is an environmental toxin produced by cyanobacteria and is considered to be a potent carcinogen. However, to the best of our knowledge, the effect of MC­LR on colorectal cancer (CRC) cell proliferation has never been studied. The aim of the present study was to investigate the effect of MC­LR on CRC cell proliferation and the underlying mechanisms. Firstly, a Cell Counting Kit­8 (CCK­8) assay was conducted to determine cell viability at different concentrations, and 50 nM MC­LR was chosen for further study. Subsequently, a longer CCK­8 assay and a cell colony formation assay showed that MC­LR promoted SW620 and HT29 cell proliferation. Furthermore, western blotting analysis showed that MC­LR significantly upregulated protein expression of PI3K, p­Akt (Ser473), p­GSK3ß (Ser9), ß­catenin, c­myc and cyclin D1, suggesting that MC­LR activated the PI3K/Akt and Wnt/ß­catenin pathways in SW620 and HT29 cells. Finally, the pathway inhibitors LY294002 and ICG001 were used to validate the role of the PI3K/Akt and Wnt/ß­catenin pathways in MC­LR­accelerated cell proliferation. The results revealed that MC­LR activated Wnt/ß­catenin through the PI3K/Akt pathway to promote cell proliferation. Taken together, these data showed that MC­LR promoted CRC cell proliferation by activating the PI3K/Akt/Wnt/ß­catenin pathway. The present study provided a novel insight into the toxicological mechanism of MC­LR.


Assuntos
Neoplasias Colorretais , beta Catenina , Humanos , Leucina/farmacologia , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Microcistinas/toxicidade , Arginina , Sincalida , Proliferação de Células , Receptores Proteína Tirosina Quinases
13.
J Chromatogr A ; 1688: 463728, 2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36566571

RESUMO

Fabricating functional electrospun nanofiber coating for highly selective extraction of microcystin-LR (MC-LR) was of significant importance for water-safety monitoring. Herein, a novel MOF@aptamer functionalized nanofabric was presented via a facile and reliable strategy integrating polydopamine (PDA) mediation and thiol-ene chemistry and applied for specific recognition of the MC-LR model analyte. Using polydopamine (PDA) as the mediating layer, vinyl-UiO-66 MOF was grown in situ, followed by post-synthetic modification (PSM) of Zr4+ with vinyl phosphate and rapid UV-initiated click reaction of aptamers. Uniform deposition of Zr-based MOF (vinyl-UiO-66) on the nanofibers was directly produced, and the tedious co-electrospinning process was abandoned to prevent the aggregation and encapsulation of MOF. Via an efficient "thiol-ene" chemistry, massive thiol-terminated aptamers were grafted on MOF within one step under friendly conditions, rather than the time-consuming nanoparticle adsorption or unfriendly covalent chemical reactions. As a result, the robust MOF@aptamer-coated nano-fabrics were obtained, and a highly selective performance towards MC-LR was illustrated with a limit of detection (LOD) at 0.002 ng/mL, good precision (CV<8.3%), good repeatability (2.2∼6.0%) when coupled with LC-MS. Almost 1∼2 orders of magnitude higher detection sensitivity was exhibited than that of the common non-specific SPE/SPME fiber reported so far. Applied to water samples, the good matrix-resistance ability, and acceptable recovery yields were achieved with high specificity. This strategy might provide a rapid and friendly protocol to efficiently fabricate MOF@aptamer functionalized nano-fabrics through electrospinning and interfacial "thiol-ene" chemistry for highly-selective microextraction.


Assuntos
Aptâmeros de Nucleotídeos , Estruturas Metalorgânicas , Compostos Organometálicos , Arginina , Leucina , Água , Compostos de Sulfidrila
14.
Food Funct ; 14(1): 133-147, 2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36524418

RESUMO

The study investigated the effects of dietary leucine (Leu) and fish oil (FO) on skeletal myofiber type transformations in pigs and their potential interactions. The results showed that Leu increased the content of Leu, upregulated myocyte enhancer factor-2C (MEF2C) and activated the CaMKII-AMPK/SIRT1-PGC-1α pathway in the longissimus dorsi (LD) muscle. FO increased adiponectin and fatty acid beta-oxidation of LD muscle. Activation of the adiponectin signaling pathway by FO further enhanced the CaMKII pathway and upregulated the expression of MEF2C. Moreover, we found that Leu increased cyclic AMP and caffeine, and FO increased linoleic acid and glutamine in muscle metabolites, which may be the cause of myofiber conversion. In conclusion, this study demonstrated that dietary Leu and FO co-regulated the transformation from glycolytic to oxidative skeletal myofiber type. It is hypothesized that there is an interaction between amino acids and polyunsaturated fatty acids, possibly via the CaMKII signaling pathway to upregulate MEF2 and mitochondrial biogenesis.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Óleos de Peixe , Animais , Suínos , Leucina/farmacologia , Leucina/metabolismo , Óleos de Peixe/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/farmacologia , Adiponectina/metabolismo , Músculo Esquelético/metabolismo , Transdução de Sinais
15.
Int J Pharm ; 631: 122550, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36577481

RESUMO

N-acetylcysteine (NAC) has both antioxidant and immunomodulatory activities and has been used as adjuvant therapy in several viral infections. Recently, NAC attracted attention for its possible role in reducing the affinity of the spike protein receptor binding domain to angiotensin-converting enzyme (ACE2) receptors. Since only NAC solutions are available for inhalation, the purpose of the work was to develop a NAC dry powder for inhalation using mannitol or leucine as excipient. The powder was successfully produced using co-spray-drying with leucine. ATR-FTIR analyses evidenced spectral variations ascribed to the formation of specific interactions between NAC and leucine. This effect on the NAC environment was not evident for NAC-mannitol powders, but mannitol was in a different polymorphic form compared to the supplied material. Both the feedstock concentration and the leucine content have an impact on the powder aerodynamic features. In particular, to maximize the respirable fraction, it is preferable to produce the powder starting from a 0.5 % w/v feedstock solution using 33 to 50 % w/w leucine content. The NAC-leucine powder was stable for ten months maintaining NAC content of 50 % (w/w) and about 200 µg of NAC was able to deposit on a transwell insert, useful for future in vitro studies.


Assuntos
Acetilcisteína , Manitol , Pós/química , Leucina/química , Administração por Inalação , Aerossóis/química , Manitol/química , Tamanho da Partícula , Inaladores de Pó Seco
16.
Life Sci ; 314: 121327, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36584912

RESUMO

AIMS: This study investigated whether l-glutamine (Gln) and/or l-leucine (Leu) administration could attenuate muscle atrophy in a mouse model of cecal ligation and puncture (CLP)-induced sepsis. MATERIALS AND METHODS: Septic mice were given a daily intraperitoneal injection of Gln, Leu, or Gln plus Leu, and mice were sacrificed on either day 1 or 4 after CLP. Blood and muscles were collected for analysis of amino acid contents and markers related to protein degradation, muscle regeneration, and protein synthesis. KEY FINDINGS: Leu treatment alone increased both muscle mass and total muscle protein content on day 4 after CLP. Gln administration reduced muscular Gln contents on day 1 and enhanced plasma Gln levels on day 4. Higher plasma branched-chain amino acid (BCAA) abundances and lower muscular BCAA levels were observed in Leu-treated mice on day 4. Gln and Leu individually suppressed muscle expressions of the E3 ubiquitin ligase genes, Trim63 and Fbxo32, on day 4 after CLP. As to muscle expressions of myogenic genes, both Gln and Leu upregulated Myog expression on day 1, but Leu alone enhanced Myf5 gene expression, whereas Gln plus Leu increased MyoD and Myog expression levels on day 4. Akt/mammalian target of rapamycin (mTOR) signaling was only activated by Gln and Leu when individually administered. SIGNIFICANCE: Gln and/or Leu administration reduces sepsis-induced muscle degradation and promotes myogenic gene expressions. Leu treatment alone had more-pronounced effects on maintaining muscle mass during sepsis. A combination of Gln and Leu failed to show synergistic effects on alleviating sepsis-induced muscle atrophy.


Assuntos
Glutamina , Sepse , Camundongos , Animais , Glutamina/farmacologia , Glutamina/metabolismo , Leucina/farmacologia , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/etiologia , Atrofia Muscular/prevenção & controle , Aminoácidos de Cadeia Ramificada/metabolismo , Músculo Esquelético/metabolismo , Sepse/complicações , Sepse/tratamento farmacológico , Sepse/metabolismo , Mamíferos/metabolismo
17.
BMC Plant Biol ; 22(1): 567, 2022 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-36471245

RESUMO

BACKGROUND: Downy mildew of foxtail millet, which is caused by the biotrophic oomycete Sclerospora graminicola (Sacc.) Schroeter, is one of the most disruptive diseases. The foxtail millet-S. graminicola interaction is largely unexplored. Transcriptome sequencing technology can help to reveal the interaction mechanism between foxtail millet and its pathogens. RESULTS: Transmission electron microscopy observations of leaves infected with S. graminicola showed that the structures of organelles in the host cells gradually became deformed and damaged, or even disappeared from the 3- to 7-leaf stages. However, organelles in the leaves of resistant variety were rarely damaged. Moreover, the activities of seven cell wall degrading enzymes in resistant and susceptible varieties were also quite different after pathogen induction and most of enzymes activities were significantly higher in the susceptible variety JG21 than in the resistant variety G1 at all stages. Subsequently, we compared the transcriptional profiles between the G1 and JG21 in response to S. graminicola infection at 3-, 5-, and 7-leaf stages using RNA-Seq technology. A total of 473 and 1433 differentially expressed genes (DEGs) were identified in the resistant and susceptible varieties, respectively. The pathway analysis of the DEGs showed that the highly enriched categories were related to glutathione metabolism, plant hormone signalling, phenylalanine metabolism, and cutin, suberin and wax biosynthesis. Some defence-related genes were also revealed in the DEGs, including leucine-rich protein kinase, Ser/Thr protein kinase, peroxidase, cell wall degrading enzymes, laccases and auxin response genes. Our results also confirmed the linkage of transcriptomic data with qRT-PCR data. In particular, LRR protein kinase encoded by Seita.8G131800, Ser/Thr protein kinase encoded by Seita.2G024900 and Seita. 2G024800, which have played an essential resistant role during the infection by S. graminicola. CONCLUSIONS: Transcriptome sequencing revealed that host resistance to S. graminicola was likely due to the activation of defence-related genes, such as leucine-rich protein kinase and Ser/Thr protein kinase. Our study identified pathways and genes that contribute to the understanding of the interaction between foxtail millet and S. graminicola at the transcriptomic level. The results will help us better understand the resistance mechanism of foxtail millet against S. graminicola.


Assuntos
Oomicetos , Pennisetum , Setaria (Planta) , Setaria (Planta)/genética , Setaria (Planta)/metabolismo , Leucina/genética , Pennisetum/genética , Oomicetos/fisiologia , Perfilação da Expressão Gênica , Proteínas Quinases/genética , Transcriptoma
18.
Org Lett ; 24(49): 9049-9053, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36475781

RESUMO

A newly devised route to the Pfizer drug nirmatrelvir is reported that reduces the overall sequence to a 1-pot process and relies on a commercially available, green coupling reagent, T3P. The overall yield of the targeted material, isolated as its MTBE solvate, is 64%.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Leucina , Antivirais/farmacologia
19.
Cells ; 11(23)2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36497142

RESUMO

Recent studies have suggested that mouse cathelicidin-related antimicrobial peptide (CRAMP) and its human homologue leucine leucine-37 (LL-37) play critical roles in innate immune responses. Here, we studied the role of mouse CRAMP in bacterial endotoxin lipopolysaccharide (LPS)-induced neuroinflammation. CRAMP peptide treatment significantly inhibited LPS-mediated inflammatory activation of glial cells in culture. In the animal model of LPS-induced neuroinflammation, CRAMP expression was highly induced in multiple cell types, such as astrocytes, microglia, and neurons. Injection of exogenous CRAMP peptide significantly inhibited inflammatory cytokine expression and the reactivity of glial cells in the mouse brain following intraperitoneal or intracerebroventricular LPS administration. Altogether, results of the study suggest that CRAMP plays an important part in containment of LPS-induced neuroinflammatory responses, and that CRAMP can be exploited for the development of targeted therapies for neuroinflammatory conditions associated with bacterial infection.


Assuntos
Peptídeos Catiônicos Antimicrobianos , Microglia , Animais , Camundongos , Humanos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Leucina , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo
20.
Front Immunol ; 13: 1028994, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36569927

RESUMO

Introduction: Osteoarthritis (OA) is a degenerative disease of the joints mainly affecting older individuals. Since the etiology behind the progression of OA is not well understood, several associated consequences, such as synovial joint stiffness and its progression due to joint fibrosis, are still poorly understood. Although a lot of developments have been achieved in the diagnosis and management of OA, synovial fibrosis remains one of the major challenging consequences. The present study was therefore focused on understanding the mechanism of synovial fibrosis, which may further contribute to improving symptomatic treatments, leading to overall improvements in the treatment outcomes of patients with OA. Methods: We used advanced proteomic techniques including isobaric tag for relative and absolute quantitation and sequential window acquisition of all theoretical mass spectra for the identification of differentially expressed proteins in the plasma samples of patients with OA. An in silico study was carried out to evaluate the association of the identified proteins with their biological processes related to fibrosis and remodeling of the extracellular matrix (ECM). The most significantly upregulated protein was then validated by Western blot and enzyme-linked immunosorbent assay. The target protein was then further investigated for its role in inflammation and joint fibrosis using an in vitro study model. Results: Leucine-rich alpha-2 glycoprotein (LRG1) was found to be the most highly differentially expressed upregulated (9.4-fold) protein in the plasma samples of patients with OA compared to healthy controls. The knockdown of LRG1 followed by in vitro studies revealed that this protein promotes the secretion of the ECM in synovial cells and actively plays a role in wound healing and cell migration. The knockdown of LRG1 further confirmed the reduction of the inflammatory- and fibrosis-related markers in primary cells. Conclusion: LRG1 was identified as a highly significant upregulated protein in the plasma samples of patients with OA. It was found to be associated with increased fibrosis and cell migration, leading to enhanced inflammation and joint stiffness in OA pathogenesis.


Assuntos
Osteoartrite do Joelho , Humanos , Osteoartrite do Joelho/metabolismo , Leucina/metabolismo , Regulação para Cima , Proteômica , Articulação do Joelho/patologia , Inflamação/metabolismo , Fibrose , Glicoproteínas/metabolismo
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