RESUMO
Enzymatic production of D-mannose attracts increasing attention because of the health effects and commercial values of D-mannose. Several kinds of epimerases or isomerases have been used for enzymatic production of D-mannose from D-glucose or D-fructose. D-Mannose epimerase (MEase), belonging to N-acyl-D-glucosamine 2-epimerase superfamily enzymes, catalyzes the C-2 epimerization between D-glucose and D-mannose. In this study, a novel MEase was identified from Cytophagaceae bacterium SJW1-29. Sequence and structure alignments indicate that it is highly conserved with the reported R. slithyformis MEase with the known crystal structure. It was a metal-independent enzyme, with an optimal pH of 8.0 and an optimal temperature of 40⯰C. The specific activities on D-glucose and D-mannose were 2.90 and 2.96â¯U/mg, respectively. The Km, kcat, and kcat/Km on D-glucose were measured to be 194.9â¯mM, 2.72â¯s-1, and 0.014â¯mM-1 s-1, respectively. The purified enzyme produced 23.15â¯g/L of D-mannose from 100â¯g/L of D-glucose at pH 8.0 and 40 °C for 8â¯h, with a conversion rate of 23.15â¯%.
Assuntos
Carboidratos Epimerases , Glucose , Manose , Manose/metabolismo , Glucose/metabolismo , Especificidade por Substrato , Cinética , Carboidratos Epimerases/metabolismo , Carboidratos Epimerases/genética , Carboidratos Epimerases/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Concentração de Íons de Hidrogênio , Sequência de Aminoácidos , Clonagem Molecular , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Temperatura , Modelos Moleculares , Alinhamento de SequênciaRESUMO
A series of novel thio-derivatives of d-glucosamine has been synthesized using double inversion procedures at the C3 atom. New compounds were applied as ligands for the diethylzinc addition to benzaldehyde and the products of the addition were obtained with a low to good enantiomeric ratio. The direction and the level of the asymmetric induction were highly dependent on the type of protecting groups on the nitrogen and sulfur atoms.
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Benzaldeídos , Glucosamina , Benzaldeídos/química , Ligantes , Glucosamina/química , Glucosamina/análogos & derivados , Estereoisomerismo , Compostos Organometálicos/química , Compostos Organometálicos/síntese química , Estrutura MolecularRESUMO
A series of the first conjugates of N-acetyl-d-glucosamine with α-aminophosphonates was synthesized using the Kabachnik-Fields reaction, the Pudovik reaction, a copper(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC) and evaluated for the in vitro cytotoxicity against human cancer cell lines M - HeLa, HuTu-80, A549, PANC-1, MCF-7, T98G and normal lung fibroblast cells WI-38. The tested conjugates, with exception of compound 21b, considered as a lead compound, were either inactive against the used cancer cells or showed moderate cytotoxicity in the range of IC50 values 33-80 µM. The lead compound 21b, being non cytotoxic against normal human cells WI-38 (IC50 = 90 µM), demonstrated good activity (IC50 = 17 µM) against breast adenocarcinoma cells (MCF-7) which to be 1.5 times higher than the activity of the used reference anticancer drug tamoxifen (IC50 = 25.0 µM). A flexible receptor molecular docking simulation showed that the cytotoxicity of the synthesized conjugates of N-acetyl-d-glucosamine with α-aminophosphonates against breast adenocarcinoma MCF-7 cell line is due to their ability to inhibit EGFR kinase domain. In addition, it was found that conjugates 22a and 22b demonstrated antioxidant activity that was not typical for α-aminophosphonates.
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Acetilglucosamina , Antineoplásicos , Antioxidantes , Simulação de Acoplamento Molecular , Organofosfonatos , Humanos , Organofosfonatos/química , Organofosfonatos/farmacologia , Organofosfonatos/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Acetilglucosamina/química , Acetilglucosamina/farmacologia , Antioxidantes/farmacologia , Antioxidantes/síntese química , Antioxidantes/química , Ensaios de Seleção de Medicamentos Antitumorais , Relação Estrutura-Atividade , Linhagem Celular Tumoral , Estrutura Molecular , Proliferação de Células/efeitos dos fármacosRESUMO
We previously demonstrated that mice carrying natural mtDNA variants of the FVB/NJ strain (m.7778â¯G>T in the mt-Atp8 gene in mitochondrial complex V), namely C57BL/6â¯J-mtFVB/NJ (B6-mtFVB), exhibited (i) partial protection from experimental skin inflammatory diseases in an anti-murine type VII collagen antibody-induced skin inflammation model and psoriasiform dermatitis model; (ii) significantly altered metabolites, including short-chain fatty acids, according to targeted metabolomics of liver, skin and lymph node samples; and (iii) a differential composition of the gut microbiota according to bacterial 16â¯S rRNA gene sequencing of stool samples compared to wild-type C57BL/6â¯J (B6) mice. To further dissect these disease-contributing factors, we induced an experimental antibody-induced skin inflammatory disease in gnotobiotic mice. We performed shotgun metagenomic sequencing of caecum contents and untargeted metabolomics of liver, CD4+ T cell, and caecum content samples from conventional B6-mtFVB and B6 mice. We identified D-glucosamine as a candidate mediator that ameliorated disease severity in experimental antibody-induced skin inflammation by modulating immune cell function in T cells, neutrophils and macrophages. Because mice carrying mtDNA variants of the FVB/NJ strain show differential disease susceptibility to a wide range of experimental diseases, including diet-induced atherosclerosis in low-density lipoprotein receptor knockout mice and collagen antibody-induced arthritis in DBA/1â¯J mice, this experimental approach is valuable for identifying novel therapeutic options for skin inflammatory conditions and other chronic inflammatory diseases to which mice carrying specific mtDNA variants show differential susceptibility.
Assuntos
DNA Mitocondrial , Camundongos Endogâmicos C57BL , Animais , DNA Mitocondrial/genética , Microbioma Gastrointestinal , Camundongos , Pele/metabolismo , Pele/microbiologia , Pele/patologia , Dermatite/imunologia , Dermatite/microbiologia , Dermatite/genética , Dermatite/tratamento farmacológico , Dermatite/metabolismo , Inflamação/genética , Inflamação/imunologia , Modelos Animais de Doenças , Masculino , Vida Livre de Germes , Psoríase/tratamento farmacológico , Psoríase/imunologia , Psoríase/genética , Ceco/microbiologia , Doença Crônica , FemininoRESUMO
A new member of the family Flavobacteriaceae (termed Hal144T) was isolated from the marine breadcrumb sponge Halichondria panicea. Sponge material was collected in 2018 at Schilksee which is located in the Kiel Fjord (Baltic Sea, Germany). Phylogenetic analysis of the full-length Hal144T 16S rRNA gene sequence revealed similarities from 94.3 to 96.6% to the nearest type strains of the genus Maribacter. The phylogenetic tree of the 16S rRNA gene sequences depicted a cluster of strain Hal144T with its closest relatives Maribacter aestuarii GY20T (96.6%) and Maribacter thermophilus HT7-2T (96.3%). Genome phylogeny showed that Maribacter halichondriae Hal144T branched from a cluster consisting of Maribacter arenosus, Maribacter luteus, and Maribacter polysiphoniae. Genome comparisons of strain Maribacter halichondriae Hal144T with Maribacter sp. type strains exhibited average nucleotide identities in the range of 75-76% and digital DNA-DNA hybridisation values in the range of 13.1-13.4%. Compared to the next related type strains, strain Hal144T revealed unique genomic features such as phosphoenolpyruvate-dependent phosphotransferase system pathway, serine-glyoxylate cycle, lipid A 3-O-deacylase, 3-hexulose-6-phosphate synthase, enrichment of pseudogenes and of genes involved in cell wall and envelope biogenesis, indicating an adaptation to the host. Strain Hal144T was determined to be Gram-negative, mesophilic, strictly aerobic, flexirubin positive, resistant to aminoglycoside antibiotics, and able to utilize N-acetyl-ß-D-glucosamine. Optimal growth occurred at 25-30 °C, within a salinity range of 2-6% sea salt, and a pH range between 5 and 8. The major fatty acids identified were C17:0 3-OH, iso-C15:0, and iso-C15:1 G. The DNA G + C content of strain Hal144T was 41.4 mol%. Based on the polyphasic approach, strain Hal144T represents a novel species of the genus Maribacter, and we propose the name Maribacter halichondriae sp. nov. The type strain is Hal144T (= DSM 114563T = LMG 32744T).
Assuntos
Flavobacteriaceae , Poríferos , Animais , Água do Mar , Fosfatidiletanolaminas/química , Filogenia , RNA Ribossômico 16S/genética , DNA Bacteriano/genética , Análise de Sequência de DNA , Técnicas de Tipagem Bacteriana , Vitamina K 2/química , Ácidos Graxos/químicaRESUMO
BACKGROUND: Breast cancer is a common cancer with high mortality rates. Early diagnosis is crucial for reducing the prognosis and mortality rates. Therefore, the development of alternative treatment options is necessary. OBJECTIVE: This study aimed to investigate the inhibitory effect of N-acetyl-D-glucosamine (D-GlcNAc) on breast cancer using a machine learning method. The findings were further confirmed through assays on breast cancer cell lines. METHODS: MCF-7 and 4T1 cell lines (ATCC) were cultured in the presence and absence of varying concentrations of D-GlcNAc (0.5 mM, 1 mM, 2 mM, and 4 mM) for 72 hours. A xenograft mouse model for breast cancer was established by injecting 4T1 cells into mammary glands. D-GlcNAc (2 mM) was administered intraperitoneally to mice daily for 28 days, and histopathological effects were evaluated at pre-tumoral and post-tumoral stages. RESULTS: Treatment with 2 mM and 4 mM D-GlcNAc significantly decreased cell proliferation rates in MCF-7 and 4T1 cell lines and increased Fas expression. The number of apoptotic cells was significantly higher than untreated cell cultures (p < 0.01 - p < 0.0001). D-GlcNAc administration also considerably reduced tumour size, mitosis, and angiogenesis in the post-treatment group compared to the control breast cancer group (p < 0.01 - p < 0.0001). Additionally, molecular docking/dynamic analysis revealed a high binding affinity of D-GlcNAc to the marker protein HER2, which is involved in tumour progression and cell signalling. CONCLUSION: Our study demonstrated the positive effect of D-GlcNAc administration on breast cancer cells, leading to increased apoptosis and Fas expression in the malignant phenotype. The binding affinity of D-GlcNAc to HER2 suggests a potential mechanism of action. These findings contribute to understanding D-GlcNAc as a potential anti-tumour agent for breast cancer treatment.
Assuntos
Neoplasias da Mama , Glucosamina , Camundongos , Humanos , Animais , Feminino , Acetilglucosamina/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Simulação de Acoplamento Molecular , Modelos Animais de DoençasRESUMO
N-acetyl-d-glucosamine (GlcNAc) is a commercially important amino sugar for its wide range of applications in pharmaceutical, food, cosmetics and biofuel industries. In nature, GlcNAc is polymerised into chitin biopolymer, which is one of the major constituents of fungal cell wall and outer shells of crustaceans. Sea food processing industries generate a large volume of chitin as biopolymeric waste. Because of its high abundance, chitinaceous shellfish wastes have been exploited as one of the major precursor substrates of GlcNAc production, both in chemical and enzymatic means. Nevertheless, the current process of GlcNAc extraction from shellfish wastes generates poor turnover and attracts environmental hazards. Moreover, GlcNAc isolated from shellfish could not be prescribed to certain groups of people because of the allergic nature of shell components. Therefore, an alternative route of GlcNAc production is advocated. With the advancement of metabolic construction and synthetic biology, microbial synthesis of GlcNAc is gaining much attention nowadays. Several new and cutting-edge technologies like substrate co-utilization strategy, promoter engineering, and CRISPR interference system were proposed in this fascinating area. The study would put forward the potential application of microbial engineering in the production of important pharmaceuticals. Very recently, autotrophic fermentation of GlcNAc synthesis has been proposed. The metabolic engineering approaches would offer great promise to mitigate the issues of low yield and high production cost, which are major challenges in microbial bio-processes industries. Further process optimization, optimising metabolic flux, and efficient recovery of GlcNAc from culture broth, should be investigated in order to achieve a high product titer. The current study presents a comprehensive review on microbe-based eco-friendly green methods that would pave the way towards the development of future research directions in this field for the designing of a cost-effective fermentation process on an industrial setup.
Assuntos
Acetilglucosamina , Glucosamina , Animais , Biotecnologia , Quitina/metabolismo , CrustáceosRESUMO
A chitinase (PbChi70) from Paenibacillus barengoltzii was engineered by directed evolution to enhance its hydrolysis efficiency towards powder chitin. Through two rounds of screening, a mutant (mPbChi70) with a maximum specific activity of 73.21 U/mg was obtained, which is by far the highest value ever reported. The mutant gene was further transformed into Aspergillus niger FBL-B (ΔglaA) which could secrete high level of endogenously ß-N-acetylglucosaminidase (GlcNAcase), thus a two-enzyme expression system was constructed. The highest chitinase activity of 61.33 U/mL with GlcNAcase activity of 353.1 U/mL was obtained in a 5-L fermentor by high-cell density fermentation. The chitin-degrading enzyme cocktail was used for the bioconversion of GlcNAc from powder chitin directly, and the highest conversion ratio reached high up to 71.9 % (w/w) with GlcNAc purity ≥95 % (w/w). This study may provide an excellent chitinase as well as a double enzyme cocktail system for efficient biological conversion of chitin materials.
Assuntos
Aspergillus , Quitina , Quitinases , Aspergillus niger/genética , Aspergillus niger/metabolismo , Glucosamina , Acetilglucosamina/metabolismo , Pós , Quitinases/genética , Quitinases/metabolismoRESUMO
In order to better utilize chitinolytic enzymes to produce high-value N-acetyl-D-glucosamine (GlcNAc) from chitinous waste, there is an urgent need to explore bi-functional chitinases with exceptional properties of temperature, pH and metal tolerance. In this study, we cloned and characterized a novel bi-functional cold-adaptive chitinase called CaChi18A from a newly isolated strain, Chitinilyticum aquatile CSC-1, in Bama longevity village of Guangxi Province, China. The activity of CaChi18A at 50 °C was 4.07 U/mg. However, it exhibited significant catalytic activity even at 5 °C. Its truncated variant CaChi18A_ΔChBDs, containing only catalytic domain, demonstrated significant activity levels, exceeding 40 %, over a temperature range of 5-60 °C and a pH range of 3 to 10. It was noteworthy that it displayed tolerance towards most metal ions at a final concentration of 0.1 mM, including Fe3+ and Cu2+ ions, retaining 122.52 ± 0.17 % and 116.42 ± 1.52 % activity, respectively. Additionally, it exhibited favorable tolerance towards organic solvents with the exception of formic acid. Interestedly, CaChi18A and CaChi18A_ΔChBDs had a low Km value towards colloidal chitin (CC), 0.94 mg mL-1 and 2.13 mg mL-1, respectively. Both enzymes exhibited chitobiosidase and N-acetyl-D-glucosaminidase activities, producing GlcNAc as the primary product when hydrolyzing CC. The high activities across a broader temperature and pH range, strong environmental adaptability, and hydrolytic properties of CaChi18A_ΔChBDs suggested that it could be a promising candidate for GlcNAc production.
Assuntos
Betaproteobacteria , Quitinases , Quitinases/química , China , Hexosaminidases , Quitina/química , ÍonsRESUMO
Microbiota are known to modulate the host response to influenza infection, but the mechanisms remain largely unknown. Gut metabolites are the key mediators through which gut microbes play anti-influenza effect. Transferring fecal metabolites from mice with high influenza resistance into antibiotic-treated recipient mice conferred resistance to influenza infections. By comparing the metabolites of different individuals with high or low influenza resistance, we identified and validated N-acetyl-D-glucosamine (GlcNAc) and adenosine showed strong positive correlations with influenza resistance and exerted anti-influenza effects in vivo or in vitro, respectively. Especially, GlcNAc mediated the anti-influenza effect by increasing the proportion and activity of NK cells. Several gut microbes, including Clostridium sp., Phocaeicola sartorii, and Akkermansia muciniphila, were positively correlated with influenza resistance, and can upregulate the level of GlcNAc in the mouse gut by exogenous supplementation. Subsequent studies confirmed that administering a combination of the three bacteria to mice via gavage resulted in similar modulation of NK cell responses as observed with GlcNAc. This study demonstrates that gut microbe-produced GlcNAc protects the host against influenza by regulating NK cells, facilitating the elucidation of the action mechanism of gut microbes mediating host influenza resistance.
Assuntos
Microbioma Gastrointestinal , Influenza Humana , Microbiota , Camundongos , Animais , Humanos , Células Matadoras Naturais , Fezes/microbiologiaRESUMO
The ability of cartilage to regenerate and repair is limited. N-acetyl- d-glucosamine (GlcNAc) is a nutritional supplement commonly used to activate chondrocytes. To prolong the duration of action of GlcNAc and improve its curative effect after cartilage injury, a GlcNAc thermosensitive hydrogel is prepared based on Pluronic F127 (PF127). The physicochemical properties results indicate that this hydrogel is injectable and retards the release of GlcNAc. Further, the therapeutic benefits of GlcNAc hydrogel are detected through intra-articular injection in rat specimens with cartilage injury. Behavioral experiments results indicate that the rats treated with GlcNAc hydrogel had longer step lengths, smaller foot angles and slower fall times. Compared with the sham group, the expression of Sox9 was 1.5 times and the level of collagen II was 2.4 times in the hydrogel treated group. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining result confirmed that the GlcNAc hydrogel reduce apoptosis by about 50%. Our results of immunohistochemical staining, Western blotting assays and enzyme activity detection all suggested that GlcNAc hydrogel reduce the expression of cleaved-caspase3 and caspase8 (Compared to the sham group, the protein contents were reduced by about 50% in the GlcNAc hydrogel group). We also found that GlcNAc hydrogel activates autophagy through ERK signal pathway. The results of Western blotting indicated that GlcNAc hydrogel increase the levels of LC3B and Becline1 (hydrogel group & sham group, LC3B: 1.56 ± 0.07 & 1.00 ± 0.14; Becline1: 1.98 ± 0.07 & 1.00 ± 0.13). Whereas, the content of P62 reduced after GlcNAc hydrogel treatment, the relative level in sham group and hydrogel group are 1.00 ± 0.02 and 0.73 ± 0.06. Our results revealed that the number of P-ERK positive cells in the hydrogel group (57.36 ± 3.56%) was higher when compared with the sham (24.82 ± 2.72%). And, the ratio of P-ERK and ERK was higher than that in the sham group (1.48 ± 0.07 & 1.00 ± 0.08). The GlcNAc thermosensitive hydrogel is a promising and sustainable drug delivery system for intra-articular injection in the treatment of cartilage injury.
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Chitosans are partially acetylated polymers of glucosamine, structurally characterized by their degree of polymerization as well as their fraction and pattern of acetylation. These parameters strongly influence the physico-chemical properties and biological activities of chitosans, but structure-function relationships are only poorly understood. As an example, we here investigated the influence of acetylation on chitosan-copper complexation using density functional theory. We investigated the electronic structures of completely deacetylated and partially acetylated chitosan oligomers and their copper-bound complexes. Frontier molecular orbital theory revealed bonding orbitals for electrophiles and antibonding orbitals for nucleophiles in fully deacetylated glucosamine oligomers, while partially acetylated oligomers displayed bonding orbitals for both electrophiles and nucleophiles. Our calculations showed that the presence of an acetylated subunit in a chitosan oligomer affects the structural and the electronic properties of the oligomer by generating new intramolecular interactions with the free amino group of neighboring deacetylated subunits, thereby influencing its polarity. Furthermore, the band gap energy calculated from the fully and partially deacetylated oligomers indicates that the mobility of electrons in partially acetylated chitosan oligomers is higher than in fully deacetylated oligomers. In addition, fully deacetylated oligomers form more stable complexes with higher bond dissociation energies with copper than partially acetylated ones. Interestingly, in partially acetylated oligomers, the strength of copper binding was found to be dependent on the pattern of acetylation. Our study provides first insight into the influence of patterns of acetylation on the electronic and ion binding properties of chitosans. Depending on the intended application, the obtained results can serve as a guide for the selection of the optimal chitosan for a specific purpose.
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NAcetylDneuraminic acid (Neu5Ac) is the crucial compound for the chemical synthesis of antiflu medicine Zanamivir. Chemoenzymatic synthesis of Neu5Ac involves N-acetyl-D-glucosamine 2-epimerase (AGE)-catalyzed epimerization of N-acetyl-D-glucosamine (GlcNAc) to N-acetyl-D-mannosamine (ManNAc), and aldolase-catalyzed condensation between ManNAc and pyruvate. Host optimization plays an important role in the whole-cell biotransformation of value-added compounds. In this study, via single-plasmid biotransformation system, we showed that the AGE gene BT0453, cloned from human gut microorganism Bacteroides thetaiotaomicron VPI-5482, showed the highest biotransformation yield among the AGE genes tested; and there is no clear Neu5Ac yield difference between the BT0453 coupled with one aldolase coding nanA gene and two nanA genes. Next, Escherichia coli chromosomal genes involved in substrate degradation, product exportation and pH change were deleted via recombineering and CRISPR/Cas9. With the final E. coli BL21(DE3) ΔnanA Δnag ΔpoxB as host, a significant 16.5% yield improvement was obtained. Furthermore, precursor (pyruvate) feeding resulted in 3.2% yield improvement, reaching 66.8% molar biotransformation. The result highlights the importance of host optimization, and set the stage for further metabolic engineering of whole-cell biotransformation of Neu5Ac.
Assuntos
Aldeído Liases , Escherichia coli , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Aldeído Liases/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Ácido Pirúvico/metabolismo , Biotransformação , Ácido N-Acetilneuramínico/metabolismoRESUMO
N-acetylneuraminic acid (Neu5Ac) possesses the ability to promote mental health and enhance immunity and is widely used in both medicine and food fields as a supplement. Enzymatic production of Neu5Ac using N-acetyl-D-glucosamine (GlcNAc) as substrate was significant. However, the high-cost GlcNAc limited its development. In this study, an in vitro multi-enzyme catalysis was built to produce Neu5Ac using affordable chitin as substrate. Firstly, exochitinase SmChiA from Serratia proteamaculans and N-acetylglucosaminosidase CmNAGase from Chitinolyticbacter meiyuanensis SYBC-H1 were screened and combined to produce GlcNAc, effectively. Then, the chitinase was cascaded with N-acetylglucosamine-2-epimerase (AGE) and N-neuraminic acid aldolase (NanA) to produce Neu5Ac; the optimal conditions of the multi-enzyme catalysis system were 37°C and pH 8.5, the ratio of AGE to NanA (1:4) and addition of pyruvate (70 mM), respectively. Finally, 9.2 g/L Neu5Ac could be obtained from 20 g/L chitin within 24 h along with two supplementations with pyruvate. This work will lay a good foundation for the production of Neu5Ac from cheap chitin resources.
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The therapeutics available for cancer treatment have the major hurdle of site-specific delivery of anti-cancer drugs to the tumor site and non-target specific side effects. The standard therapy for ovarian cancer still poses numerous pitfalls due to the irrational use of drugs affecting healthy cells. As an appealing approach, nanomedicine could revamp the therapeutic profile of anti-cancer agents. Owing to the low manufacturing cost, increased biocompatibility, and modifiable surface properties, lipid-based nanocarriers, particularly solid lipid nanoparticles (SLN), have remarkable drug delivery properties in cancer treatment. Given the extra-ordinary benefits, we developed anti-neoplastic (paclitaxel) drug-loaded SLN (PTX-SLN) and functionalized with N-acetyl-d-glucosamine (GLcNAc) (GLcNAc-PTX-SLN) to reduce the rate of proliferation, growth, and metastasis of ovarian cancer cells over-expressing GLUT1 transporters. The particles presented considerable size and distribution while demonstrating haemocompatibility. Using GLcNAc modified form of SLNs, confocal microscopy, MTT assay, and flow cytometry study demonstrated higher cellular uptake and significant cytotoxic effect. Also, molecular docking results established excellent binding affinity between GLcNAc and GLUT1, complimenting the feasibility of the therapeutic approach in targeted cancer therapy. Following the compendium of target-specific drug delivery by SLN, our results demonstrated a significant response for ovarian cancer therapy.
Assuntos
Antineoplásicos , Nanopartículas , Neoplasias Ovarianas , Humanos , Feminino , Portadores de Fármacos/química , Transportador de Glucose Tipo 1 , Simulação de Acoplamento Molecular , Linhagem Celular Tumoral , Paclitaxel , Neoplasias Ovarianas/tratamento farmacológico , Nanopartículas/química , Proteínas de Membrana TransportadorasRESUMO
The establishment of defense reactions to protect plants against pathogens requires the recognition of invasion patterns (IPs), mainly detected by plasma membrane-bound pattern recognition receptors (PRRs). Some IPs, also termed elicitors, are used in several biocontrol products that are gradually being developed to reduce the use of chemicals in agriculture. Chitin, the major component of fungal cell walls, as well as its deacetylated derivative, chitosan, are two elicitors known to activate plant defense responses. However, recognition of chitooligosaccharides (COS) in Vitis vinifera is still poorly understood, hampering the improvement and generalization of protection tools for this important crop. In contrast, COS perception in the model plant Arabidopsis thaliana is well described and mainly relies on a tripartite complex formed by the cell surface lysin motif receptor-like kinases (LysM-RLKs) AtLYK1/CERK1, AtLYK4 and AtLYK5, the latter having the strongest affinity for COS. In grapevine, COS perception has for the moment only been demonstrated to rely on two PRRs VvLYK1-1 and VvLYK1-2. Here, we investigated additional players by overexpressing in Arabidopsis the two putative AtLYK5 orthologs from grapevine, VvLYK5-1 and VvLYK5-2. Expression of VvLYK5-1 in the atlyk4/5 double mutant background restored COS sensitivity, such as chitin-induced MAPK activation, defense gene expression, callose deposition and conferred non-host resistance to grapevine downy mildew (Erysiphe necator). Protein-protein interaction studies conducted in planta revealed a chitin oligomer-triggered interaction between VvLYK5-1 and VvLYK1-1. Interestingly, our results also indicate that VvLYK5-1 mediates the perception of chitin but not chitosan oligomers showing a part of its specificity.
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Ultrashort cationic lipopeptides (USCLs) are promising antimicrobial agents that may be used to combat pathogens such as bacteria and fungi. USCLs consist of a few basic amino acid residues and at least one lipid moiety, usually a fatty acid chain. Generally, USCLs are potent antimicrobials but their major shortcoming is a relatively high cytotoxicity and hemolytic activity. Glycopeptide antibiotics (e.g. vancomycin) are essential in combating bacterial infections and are popular in medicinal practice. However, literature concerning the effect of glycosylation of peptides on their antimicrobial activity is rather scarce. For the first time, this study highlights the effect of USCLs glycosylation on in vitro biological activity. The aim of this study was to evaluate the impact of glycosylation of a series of USCLs on antimicrobial activity, cytotoxicity and hemolytic activity. Straight-chain fatty acids (C14, C16, C18) were attached to the N-terminal amino group of tripeptides-SRR-NH2, RSR-NH2 and RRS-NH2. Two groups of the lipopeptides were synthetized, the first with unmodified L-serine (USCLs) and the other with L-serine O-glycosylated by N-acetyl-ß-d-glucosamine to produce new class of glycosylated ultrashort cationic lipopeptide (gUSCLs). Both USCLs and gUSCLs were tested against planktonic and biofilm cultures of ESKAPE strains (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) and Candida glabrata, and hemolytic activity on human erythrocytes and cytotoxicity against the HaCaT cell line was examined. Generally, USCLs and gUSCLs proved to be active against all the tested strains. The highest activity displayed was by lipopeptides containing the C18 fatty acid. Antimicrobial, hemolytic and cytotoxic activities were mainly correlated with amino acid sequence (position of serine/glycosylated serine) and hydrophobicity of molecule and were found to be highly strain-dependent. In general, glycosylation did not guarantee an increased antimicrobial activity or a decreased hemolytic and cytotoxic activities. However, in some cases, gUSCLs proved to be superior to their USCLs analogs. The most pronounced differences were found for peptides with C18 fatty acid and serine at the first and second position against both planktonic cells and biofilm of C. glabrata, as well as the second and third position against S. aureus. It is noteworthy that gUSCLs were also more active against biofilm than were USCLs.
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Anti-Infecciosos , Lipopeptídeos , Humanos , Lipopeptídeos/farmacologia , Lipopeptídeos/química , Glicosilação , Staphylococcus aureus , Testes de Sensibilidade Microbiana , Anti-Infecciosos/farmacologia , Anti-Infecciosos/química , Antibacterianos/farmacologia , Antibacterianos/química , Ácidos Graxos/química , SerinaRESUMO
Achieving the early diagnosis of breast cancer, through ultrasensitive detection of tumor marker miRNA-155, is a significant challenge. Therefore, an ultrasensitive hairpin electrochemical biosensor based on graphite-like phase carbon nitride composite was proposed. In this paper, poly(D-glucosamine) (PDG) was used as a stabilizer and reducing agent to prepare gold nanoparticles at room temperature, and then a graphite-like phase with a two-dimensional lamellar structure carbon nitride was further combined with it to obtain the poly(D-glucosamine)/gold nanoparticles/graphite-like phase carbon nitride nanocomposite (PDG/AuNPs/g-C3N4), in order to achieve the goal of signal amplification. The specific hairpin capture probe (HP) that recognized and bound miRNA-155 was then grafted. The hairpin biosensor showed a linear range of 0.1 fM-1 pM with a detection limit of 0.05 fM using differential pulse voltammetry (DPV) electrochemical analysis. Furthermore, the excellent performance hairpin electrochemical biosensor had been applied to the detection of miRNA-155 in human serum samples with good recovery.
Assuntos
Técnicas Biossensoriais , Neoplasias da Mama , Grafite , Nanopartículas Metálicas , MicroRNAs , Nanocompostos , Biomarcadores Tumorais , Técnicas Biossensoriais/métodos , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Técnicas Eletroquímicas/métodos , Feminino , Glucosamina , Ouro/química , Grafite/química , Humanos , Limite de Detecção , Nanopartículas Metálicas/química , Nanocompostos/química , Nitrilas , Compostos de Nitrogênio , Substâncias RedutorasRESUMO
Sugar phosphates are emerging as potential therapeutic candidates for certain diseases. However, their high polarity makes them poorly absorbed by the body and their penetration inside the cell is even more difficult without a proper transporter. Amino sugar phosphates (n-amino-n-deoxy-sugars, carbohydrates in which a hydroxyl group has been replaced with an amine group), such as N-acetyl-D-mannosamine (ManNac)-6-phosphate have shown potential as a treatment for a muscular disease called GNE myopathy caused by a deficiency in the production of sialic acid. However, its high polarity leads to poor absorption and consequent high dosage in humans, causing unwanted side effects. Herein, we describe the application of phosphoramidate prodrug chemistry to 1,3,4-O-acetylated N-acetylmannosamine (Ac3ManNAc) to deliver ManNAc-6-phosphate (ManNAc-6-P), a critical intermediate in sialic acid biosynthesis. Sialic acid deficiency is a hallmark of GNE myopathy, a rare congenital disorder of glycosylation (CDG), caused by mutations in the gene "GNE," that limit the production of ManNAc-6-P. Synthetic methods were developed to provide a library of Ac3ManNAc-6-phosphoramidates that were evaluated in a series of studies for their potential as a treatment for GNE myopathy. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 2-Acetamido-1,3,4-tri-O-acetyl-2-deoxy-D-mannopyranose. Basic Protocol 2: Preparation of 3-acetamido-6-((((((S)-1-ethoxy-4-methyl-1-oxo-pentan-2-yl) amino) (phenoxy)phosphoryl) oxy) methyl) tetrahydro-2H-pyran-2,4,5-triyl triacetate (5). Support Protocol: Preparation of ethyl (chloro(phenoxy)phosphoryl)-l-leucinate.
Assuntos
Pró-Fármacos , Miopatias Distais , Humanos , Manose , Ácido N-Acetilneuramínico , Fosfatos , Pró-Fármacos/uso terapêuticoRESUMO
Similar to canine inflammatory enteropathy, inflammatory bowel disease (IBD) is a chronic idiopathic condition characterized by remission periods and recurrent flares in which diarrhea, visceral pain, rectal bleeding/bloody stools, and weight loss are the main clinical symptoms. Intestinal barrier function alterations often persist in the remission phase of the disease without ongoing inflammatory processes. However, current therapies include mainly anti-inflammatory compounds that fail to promote functional symptoms-free disease remission, urging new drug discoveries to handle patients during this step of the disease. ALIAmides (ALIA, autacoid local injury antagonism) are bioactive fatty acid amides that recently gained attention because of their involvement in the control of inflammatory response, prompting the use of these molecules as plausible therapeutic strategies in the treatment of several chronic inflammatory conditions. N-palmitoyl-D-glucosamine (PGA), an under-researched ALIAmide, resulted in being safe and effective in preclinical models of inflammation and pain, suggesting its potential engagement in the treatment of IBD. In our study, we demonstrated that micronized PGA significantly and dose-dependently reduces colitis severity, improves intestinal mucosa integrity by increasing the tight junction proteins expression, and downregulates the TLR-4/NLRP3/iNOS pathway via PPAR-α receptors signaling in DNBS-treated mice. The possibility of clinically exploiting micronized PGA as support for the treatment and prevention of inflammation-related changes in IBD patients would represent an innovative, effective, and safe strategy.