RESUMO
Osteoarthritis belongs to the most common joint diseases in humans and animals and shows increased incidence in older patients. The bioactivities of collagen hydrolysates, sulfated glucosamine and a special fatty acid enriched dog-food were tested in a dog patient study of 52 dogs as potential therapeutic treatment options in early osteoarthritis. Biophysical, biochemical, cell biological and molecular modeling methods support that these well-defined substances may act as effective nutraceuticals. Importantly, the applied collagen hydrolysates as well as sulfated glucosamine residues from marine organisms were strongly supported by both an animal model and molecular modeling of intermolecular interactions. Molecular modeling of predicted interaction dynamics was evaluated for the receptor proteins MMP-3 and ADAMTS-5. These proteins play a prominent role in the maintenance of cartilage health as well as innate and adapted immunity. Nutraceutical data were generated in a veterinary clinical study focusing on mobility and agility. Specifically, key clinical parameter (MMP-3 and TIMP-1) were obtained from blood probes of German shepherd dogs with early osteoarthritis symptoms fed with collagen hydrolysates. Collagen hydrolysate, a chondroprotective food supplement was examined by high resolution NMR experiments. Molecular modeling simulations were used to further characterize the interaction potency of collagen fragments and glucosamines with protein receptor structures. Potential beneficial effects of collagen hydrolysates, sulfated glycans (i.e., sulfated glucosamine from crabs and mussels) and lipids, especially, eicosapentaenoic acid (extracted from fish oil) on biochemical and physiological processes are discussed here in the context of human and veterinary medicine.
Assuntos
Cartilagem Articular/efeitos dos fármacos , Colágeno/farmacologia , Dieta/veterinária , Suplementos Nutricionais , Doenças do Cão/dietoterapia , Osteoartrite/veterinária , Substâncias Protetoras/farmacologia , Animais , Organismos Aquáticos , Colágeno/química , Colágeno/uso terapêutico , Cães , Osteoartrite/dietoterapia , Substâncias Protetoras/química , Substâncias Protetoras/uso terapêuticoRESUMO
BACKGROUND: Neonatal thrombocytopenia is common in preterm and term neonates admitted to neonatal intensive care units. The etiology behind neonatal thrombocytopenia is complex. Inherited thrombocytopenia is rare and usually results from genetic mutations. CASE PRESENTATION: Here we report a case of twins with severe inherited thrombocytopenia presented in the neonatal period who were shown to be compound heterozygotes for 2 UDP-N-acetylglucosamine 2-epimerase (GNE) gene mutations, c.1351C > T and c.1330G > T, of which c.1330G > T is a novel mutation. CONCLUSION: These two GNE mutations may help in the diagnosis and management of thrombocytopenia diagnosed in neonates.
Assuntos
Heterozigoto , Complexos Multienzimáticos/genética , Mutação , Trombocitopenia Neonatal Aloimune/genética , Gêmeos/genética , Sequência de Bases , Feminino , Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Lactente , Unidades de Terapia Intensiva Neonatal , Modelos Moleculares , Complexos Multienzimáticos/deficiência , Estrutura Secundária de Proteína , Trombocitopenia Neonatal Aloimune/diagnóstico , Trombocitopenia Neonatal Aloimune/patologiaRESUMO
Chitin is the second most abundant polysaccharide in nature and linked to fungal infection and asthma. However, bona fide immune receptors directly binding chitin and signaling immune activation and inflammation have not been clearly identified because polymeric crude chitin with unknown purity and molecular composition has been used. By using defined chitin (N-acetyl-glucosamine) oligomers, we here identify six-subunit-long chitin chains as the smallest immunologically active motif and the innate immune receptor Toll-like receptor (TLR2) as a primary fungal chitin sensor on human and murine immune cells. Chitin oligomers directly bind TLR2 with nanomolar affinity, and this fungal TLR2 ligand shows overlapping and distinct signaling outcomes compared to known mycobacterial TLR2 ligands. Unexpectedly, chitin oligomers composed of five or less subunits are inactive, hinting to a size-dependent system of immuno-modulation that appears conserved in plants and humans. Since blocking of the chitin-TLR2 interaction effectively prevents chitin-mediated inflammation in vitro and in vivo, our study highlights the chitin-TLR2 interaction as a potential target for developing novel therapies in chitin-related pathologies and fungal disease.
Assuntos
Quitina/química , Quitina/metabolismo , Fungos/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Receptor 2 Toll-Like/metabolismo , Animais , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Quitinases/metabolismo , Feminino , Humanos , Interações Hidrofóbicas e Hidrofílicas , Fatores Imunológicos/farmacologia , Ligantes , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células THP-1 , Receptor 1 Toll-Like/agonistas , Receptor 1 Toll-Like/metabolismo , Receptor 2 Toll-Like/química , Zimosan/metabolismoRESUMO
The genomes of several Acinetobacter species possess three distinct polysaccharide-producing operons [two poly-N-acetyl glucosamine (PNAG) and one K-locus]. Using a microfluidic device, an increased amount of polysaccharides and enhanced biofilm formation were observed following continuous exposure to H2O2 and removal of the H2O2-sensing key regulator, OxyR, in Acinetobacter oleivorans DR1 cells. Gene expression analysis revealed that genes located in PNAG1, but not those in PNAG2, were induced and that genes in the K-locus were expressed in the presence of H2O2. Interestingly, the expression of the K-locus gene was enhanced in the PNAG1 mutant and vice versa. The absence of either OxyR or PNAG1 resulted in enhanced biofilm formation, higher surface hydrophobicity, and increased motility, implying that K-locus-driven polysaccharide production in both the oxyR and PNAG1 deletion mutants may be related to these phenotypes. Both the oxyR and K-locus deletion mutants were more sensitive to H2O2 compared with the wildtype and PNAG1 mutant strains. Purified OxyR binds to the promoter regions of both polysaccharide operons with a higher affinity toward the K-locus promoter. Although oxidized OxyR could bind to both promoter regions, the addition of dithiothreitol further enhanced the binding efficiency of OxyR, suggesting that OxyR might function as a repressor for controlling these polysaccharide operons.
Assuntos
Acinetobacter/genética , Acinetobacter/fisiologia , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Polissacarídeos Bacterianos/biossíntese , Proteínas Repressoras/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Regiões Promotoras GenéticasRESUMO
The present study is focused on systematic process and kinetic investigation of hyaluronic acid (HA) production strategy unraveling the role of dissolved oxygen (DO) and N-acetyl glucosamine (GlcNAc) towards the enhancement of HA titer and its molecular weight. Maintaining excess DO levels (10-40% DO) through DO-stat control and the substitution of GlcNAc at a range (5-20 g/L) with glucose (Glc) critically influenced HA production. DO-stat control strategy yielded a promising HA titer (2.4 g/L) at 40% DO concentration. Controlling DO level at 20% (DO-stat) was observed to be optimum resulting in a significant HA production (2.1 g/L) and its molecular weight ranging 0.98-1.45 MDa with a consistent polydispersity index (PDI) (1.57-1.69). Substitution of GlcNAc with Glc at different proportions explicitly addressed the metabolic trade-off between HA titer and its molecular weight. GlcNAc substitution positively influenced the molecular weight of HA. The highest HA molecular weight (2.53 MDa) of two-fold increase compared with glucose as sole carbon substrate and narrower PDI (1.35 ± 0.18) was achieved for the 10:20 (Glc:GlcNAc) proportion. A novice attempt on modeling the uptake of dual substrates (Glc and GlcNAc) by Streptococcus zooepidemicus for HA production was successfully accomplished using double Andrew's growth model and the kinetic parameters were estimated reliably.
Assuntos
Acetilglucosamina/metabolismo , Ácido Hialurônico/biossíntese , Oxigênio/metabolismo , Streptococcus equi/crescimento & desenvolvimento , Streptococcus equi/metabolismo , Biomassa , Fermentação , Glucose/metabolismo , Cinética , Peso MolecularRESUMO
The microwave-assisted heating reaction of N-acetyl glucosamine (GlcNAc) in sulfolane is described. The reaction produces two major products that are assignable to 1,6-anhydro-2-acetamido-2-deoxy-ß-d-glucopyranose (AGPNAc) and 1,6-anhydro-2-acetamido-2-deoxy-ß-d-glucofuranose (AGFNAc). In order to reveal a general feature of the system, the 3, 5, and 10 min reactions were performed at 140, 160, 180, 200, and 220 °C to clarify the time course changes in the conversion of GlcNAc and the yields of the two produced 1,6-anhydrosugars. Temperature is a crucial factor that significantly affects the conversion of GlcNAc. The yields of AGPNAc and AGFNAc are also drastically changed depending on the reaction conditions. The 5-min reaction at 200 °C is shown to be the optimal condition to generate the 1,6-anhydrosugars with a high efficiency in which AGPNAc and AGFNAc are produced in the yields of 21% and 44%, respectively. Consequently, the microwave-assisted heating reaction of GlcNAc in sulfolane is shown to be a simple and promising pathway to generate 1,6-anhydrosugars consisting of amino monosaccharide backbones, which have high potentials as raw materials leading to biological oligosaccharides and biomimetic polysaccharides.
Assuntos
Acetilglucosamina/química , Calefação , Micro-Ondas , Oligossacarídeos/química , Tiofenos/química , Acetilação , Cromatografia Líquida de Alta Pressão , Estrutura MolecularRESUMO
Current treatments for hair follicle (HF) disruption are based on 5-α reductase inhibitors and prostaglandin modulators. Botanicals and nutraceutical compounds interfere with hair loss or stimulate its partial regrowth. Here, we used in vitro cocultures to investigate the activity of Serenoa repens ( SR) and N-acetyl glucosamine + milk proteins (NAG/Lac) on the paracrine interactions between human microvascular endothelial cells (HMVEC) and HF dermal papilla cells (FDPC). Both SR and NAG/Lac-induced endothelial tubulogenesis were enhanced by FDPC. SR promoted proliferation of both the cell types, while NAG/Lac was effective on endothelium. Vascular endothelial growth factor production, enhanced by SR, was further augmented by FDPC. In FDPC 5-α reductase-II and ß-catenin expressions were modified by SR and less by NAG/Lac, with no additional effect by HMVEC. SR and NAG/Lac prevented lipid peroxidation, whereas NAG/Lac was effective on interleukin 1ß production. Finally, SR and NAG/Lac differentially affected HMVEC permeability and tight junction proteins content. These data provide a mechanistic background for the potential use of these compounds as promoters of HF vascularization.
Assuntos
Acetilglucosamina/farmacologia , Indutores da Angiogênese/farmacologia , Células Endoteliais/efeitos dos fármacos , Folículo Piloso/efeitos dos fármacos , Proteínas do Leite/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Comunicação Parácrina/efeitos dos fármacos , Extratos Vegetais/farmacologia , Serenoa , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/metabolismo , Folículo Piloso/citologia , Folículo Piloso/metabolismo , Humanos , Interleucina-1beta/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Permeabilidade , Extratos Vegetais/isolamento & purificação , Serenoa/química , Transdução de Sinais , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Background: In the last few years, there has been increasing demand for aesthetic procedures to improve the effects of skin aging. Aim: To evaluate the anti-aging efficacy, tolerability and skin changes induced by the topical products containing hyaluronic acid, N-acetyl glucosamine and gamma-amino butyric acid through instrumental techniques, clinical and subjective evaluation. Patients/Method: Twenty female enrolled applied a day and night cream after applying a serum, once week applied a mask, for 2 months. A clinical assessment of smoothness, expression wrinkles, fine wrinkles and measurements of the parameters using Reveal Imager, X-Rite, Corneometer, Dermalab, Moisture Meter EpiD were taken at day 0, 15, 30 and 60 of study period. A final assessment questionnaire was submitted. Results: The products were accepted by all the volunteers. The hydration (Corneometer: T0 49.17 vs T60 61.11, average variation 24.28%) (Moisture Meter EpiD: T0 45.73 vs T60 61.10, average variation 33.60%), elasticity (Dermalab: T0 56.06 vs T60 62.78, average variation 11.98%) and lightening of the skin (X-Rite: T0 60.23 vs T60 63.36, average variation 5.26%) improved. All changes were statistically significant (p < 0.05). Conclusion: The efficacy of the topical products is confirmed by subjective, clinical and instrumental assessment. This should be a routine approach in dermatologic practice.
Assuntos
Técnicas Cosméticas , Face , Glucosamina/administração & dosagem , Ácido Hialurônico/administração & dosagem , Envelhecimento da Pele/efeitos dos fármacos , Ácido gama-Aminobutírico/administração & dosagem , Administração Cutânea , Adulto , Feminino , Humanos , Pessoa de Meia-Idade , Pomadas , Inquéritos e QuestionáriosRESUMO
N-acetyl-D-glucosamine (GlcNAc) is an important amino-monosaccharide with great potential for biotechnological applications. It has traditionally been produced by the chemical hydrolysis of chitin, despite certain industrial and environmental drawbacks, including acidic wastes, low yields and high costs. Therefore, enzymatic production has gained attention as a promising environmentally-friendly alternative to the chemical processes. In this study we demonstrate the GlcNAc bioproduction from colloidal α-chitin using an enzyme cocktail containing endochitinases and exochitinases (chitobiosidases and N-acetyl-glucosaminidases). The enzyme cocktail was extracted after fermentation in a bioreactor by Aeromonas caviae CHZ306, a chitinolytic marine bacterium with great potential for chitinase production. Hydrolysis parameters were studied in terms of temperature, pH, enzyme and substrate concentration, and reaction time, achieving over 90% GlcNAc yield within 6 h. The use of colloidal α-chitin as substrate showed a substantial improvement of GlcNAc yields, when compared with ß-chitin and α-chitin polymorphs. Such result is directly related to a significant decrease in crystallinity and viscosity from natural α-chitin, providing the chitinase with greater accessibility to the depolymerized chains. This study provides valuable information on the GlcNAc bioproduction from chitin using an enzymatic approach, addressing the key points for its production, including the enzyme cocktail composition and the substrate structures.
Assuntos
Acetilglucosamina/biossíntese , Aeromonas caviae/enzimologia , Quitina/metabolismo , Quitinases/metabolismo , Meios de Cultura/química , Concentração de Íons de Hidrogênio , Hidrólise , Espectroscopia de Ressonância Magnética , Peso Molecular , Temperatura , Viscosidade , Difração de Raios XRESUMO
BACKGROUND/AIMS: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential anti-cancer agent due to its selective toxicity. However, many human non-small cell lung cancer (NSCLC) cells are partially resistant to TRAIL, thereby limiting its clinical application. Therefore, there is a need for the development of novel adjuvant therapeutic agents to be used in combination with TRAIL. METHODS: In this study, the effect of N-acetyl-glucosamine (GlcNAc), a type of monosaccharide derived from chitosan, combined with TRAIL was evaluated in vitro and in vivo. Thirty NSCLC clinical samples were used to detect the expression of death receptor (DR) 4 and 5. After GlcNAc and TRAIL co-treatment, DR expression was determined by real-time PCR and western blotting. Cycloheximide was used to detect the protein half-life to further understand the correlation between GlcNAc and the metabolic rate of DR. Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to detect receptor clustering, and the localization of DR was visualized by immunofluorescence under a confocal microscope. Furthermore, a co-immunoprecipitation assay was performed to analyze the formation of death-inducing signaling complex (DISC). O-linked glycan expression levels were evaluated following DR5 overexpression and RNA interference mediated knockdown. RESULTS: We found that the clinical samples expressed higher levels of DR5 than DR4, and GlcNAc co-treatment improved the effect of TRAIL-induced apoptosis by activating DR5 accumulation and clustering, which in turn recruited the apoptosis-initiating protease caspase-8 to form DISC, and initiated apoptosis. Furthermore, GlcNAc promoted DR5 clustering by improving its O-glycosylation. CONCLUSION: These results uncovered the molecular mechanism by which GlcNAc sensitizes cancer cells to TRAIL-induced apoptosis, thereby highlighting a novel effective agent for TRAIL-mediated NSCLC-targeted therapy.
Assuntos
Acetilglucosamina/farmacologia , Apoptose/efeitos dos fármacos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/toxicidade , Células A549 , Acetilglucosamina/uso terapêutico , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Caspase 8/metabolismo , Linhagem Celular Tumoral , Glicosilação/efeitos dos fármacos , Humanos , Imunoprecipitação , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Microscopia Confocal , Poli(ADP-Ribose) Polimerases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/antagonistas & inibidores , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , Transplante Heterólogo , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Protein O-linked-ß-N-acetyl glucosamine (O-GlcNAc) is a post-translational modification to Ser/Thr residues that integrates energy supply with demand. Abnormal O-GlcNAc patterning is evident in several neurological disease states including epilepsy, Alzheimer's disease and autism spectrum disorder (ASD). A potential treatment option for these disorders includes the high-fat, low-carbohydrate, ketogenic diet (KD). The goal of this study was to determine whether the KD induces changes in O-GlcNAc in the BTBRT+tf/j (BTBR) mouse model of ASD. METHODS: Juvenile male (5weeks), age-matched C57 or BTBR mice consumed a chow diet (13% kcal fat) or KD (75% kcal fat) for 10-14days. Following these diets, brain (prefrontal cortex) and liver were examined for gene expression levels of key O-GlcNAc mediators, global and protein specific O-GlcNAc as well as indicators of energy status. RESULTS: The KD reduced global O-GlcNAc in the livers of all animals (p<0.05). Reductions were likely mediated by lower protein levels of O-GlcNAc transferase (OGT) and increased O-GlcNAcase (OGA) (p<0.05). In contrast, no differences in global O-GlcNAc were noted in the brain (p>0.05), yet OGT and OGA expression (mRNA) were elevated in both C57 and BTBR animals (p<0.05). CONCLUSIONS: The KD has tissue specific impacts on O-GlcNAc. Although levels of O-GlcNAc play an important role in neurodevelopment, levels of this modification in the juvenile mouse brain were stable with the KD despite large fluctuations in energy status. This suggests that it is unlikely that the KD exerts it therapeutic benefit in the BTBR model of ASD by O-GlcNAc related pathways.
Assuntos
Acetilglucosamina/metabolismo , Transtorno Autístico/metabolismo , Dieta Cetogênica , Proteínas do Tecido Nervoso/metabolismo , Córtex Pré-Frontal/metabolismo , Processamento de Proteína Pós-Traducional , Acetilglucosamina/genética , Animais , Transtorno Autístico/genética , Transtorno Autístico/patologia , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Córtex Pré-Frontal/patologiaRESUMO
OBJECTIVES: The effectiveness of various ligands against the protein structure of IcaA of the IcaABCD gene locus of Staphylococcus aureus were examined using the approach of structure based drug designing in reference with the protein's efficiency to form biofilms. RESULTS: Four compounds CID42738592, CID90468752, CID24277882, and CID6435208 were secluded from a database of 31,242 inhibitory ligands on the justification of the evaluated values falling under the four - tier structure based virtual screening. Under this principle value of least binding energy, human oral absorption and ADME properties were taken into consideration. Using the Glide module of Schrödinger, the above mentioned ligands showed an effective action against the protein IcaA which showed reduced activity as a glucosaminyl transferase. The complex of protein and ligand with best docking score was chosen for simulation studies. CONCLUSIONS: Structure based drug designing for the protein IcaA has given us potential leads as anti - biofilm agents. These screened out ligands might enable the development of new therapeutic strategies aimed at disrupting Staphylococcus aureus biofilms. The complex was showing stability towards the end of time for which it has been put for simulation. Thus molecule could be considered for making of biofilms.
Assuntos
Acetilglucosamina/metabolismo , Adesinas Bacterianas/metabolismo , Antibacterianos/metabolismo , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Adesinas Bacterianas/química , Antibacterianos/química , Avaliação Pré-Clínica de Medicamentos , Simulação de Acoplamento Molecular , Staphylococcus aureus/metabolismoRESUMO
A novel polymer-drug conjugate, polyethylene glycol-N-(acetyl)-glucosamine-doxorubicin (PEG-NAG-DOX) was evaluated in this study for its in vivo potential for treatment of tumours demonstrating improved efficacy and reduced toxicity. The proposed polymer-drug conjugate comprised of polyethylene glycol-maleimide (mPEG-MAL, 30000 Da) as a carrier, doxorubicin (DOX) as an anticancer drug and N-acetyl glucosamine (NAG) as a targeting moiety as well as penetration enhancer. Doxorubicin has a potent and promising anticancer activity; however, severe cardiotoxicity limits its application in cancer treatment. By modifying DOX in PEG-NAG-DOX prodrug conjugate, we aimed to eliminate this limitation. In vivo anticancer efficacy of the conjugate was evaluated using BDF mice-induced skin melanoma model by i.v. administration of DOX conjugates. Anticancer efficacy studies were done by comparing tumour volume, body weight, organ index and percent survival rate of the animals. Tumour suppression achieved by PEG-NAG-DOX at the cumulative dose of 7.5 mg/kg was two-fold better than that achieved by DOX solution. Also, the survival rate for PEG-NAG-DOX conjugate was >70% as compared to <50% survival rate for DOX solution. In addition, toxicity studies and histopathological studies revealed that while maintaining its cytotoxicity towards tumour cells, PEG-NAG-DOX conjugate showed no toxicities to major organs. Therefore, PEG-NAG-DOX conjugate can be suggested as a desirable candidate for targeted cancer therapy.
Assuntos
Acetilglucosamina/toxicidade , Antineoplásicos/toxicidade , Doxorrubicina/toxicidade , Sistemas de Liberação de Medicamentos/métodos , Polietilenoglicóis/toxicidade , Polímeros/toxicidade , Acetilglucosamina/administração & dosagem , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Feminino , Humanos , Células MCF-7 , Camundongos , Neoplasias/tratamento farmacológico , Polietilenoglicóis/administração & dosagem , Polímeros/administração & dosagem , Pró-Fármacos/administração & dosagem , Pró-Fármacos/toxicidade , Ratos , Ratos Sprague-Dawley , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/fisiologiaRESUMO
N-Acetyl-D-glucosamine (GlcNAc) is a monosaccharide with great application potential in the food, cosmetic, pharmaceutical, and biomaterial areas. GlcNAc is currently produced by chemical hydrolysis of chitin, but the current processes are environmentally unfriendly, have low yield and high cost. This study demonstrates the potential to produce GlcNAc from α-chitin using chitinases of ten marine-derived Aeromonas isolates as a sustainable alternative to the current chemical process. The isolates were characterized as Aeromonas caviae by multilocus sequence analysis (MLSA) using six housekeeping genes (gltA, groL, gyrB, metG, ppsA, and recA), not presented the virulence genes verified (alt, act, ast, ahh1, aer, aerA, hlyA, ascV and ascFG), but showed hemolytic activity on blood agar. GlcNAc was produced at 37 °C, pH 5.0, 2% (w/v) colloidal chitin and crude chitinase extracts (0.5 U mL-1) by all the isolates with yields from 14 to 85% at 6 h, 17-89% at 12 h and 19-93% after 24 h. The highest yield of GlcNAc was observed by A. caviae CH129 (93%). This study demonstrates one of the most efficient chitin enzymatic hydrolysis procedures and A. caviae isolates with great potential for chitinases expression and GlcNAc production.
Assuntos
Acetilglucosamina/biossíntese , Aeromonas caviae/isolamento & purificação , Quitina/metabolismo , Quitinases/metabolismo , Aeromonas caviae/enzimologia , Aeromonas caviae/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Água do Mar/microbiologia , Zooplâncton/microbiologiaRESUMO
OBJECTIVE AND DESIGN: This study had investigated the anti-inflammatory activity of a seed lectin (LAL) isolated from Lonchocarpus araripensis. MATERIAL/METHODS: LAL was purified by affinity chromatography (chitin column) and ion exchange chromatography (DEAE-Sephacel). In vitro LAL was tested for hemagglutinating activity against rabbit erythrocytes. In vivo LAL was assessed for the anti-inflammatory activity via intravenous injection (i.v.) in Swiss mice (25-30 g; n = 6/group) in models of paw edema and peritonitis. STATISTICAL ANALYSIS: ANOVA (p < 0.05). RESULTS: LAL revealed two bands of 30 and 60 kDa (SDS-PAGE) and exhibited hemagglutinating activity. LAL (10 mg/kg) inhibited the paw edema (77%) and vascular permeability (26%) induced by carrageenan, and the paw edema induced by serotonin (80%), bradykinin (49%), sodium nitroprusside (83%), TNF-α (75%) and PGE2 (64%). LAL also inhibited the neutrophil migration induced by fMLP (70%) or carrageenan (69%). The intravital microscopy showed that LAL inhibited rolling (83%) and adhesion (70%) of leukocytes. LAL anti-inflammatory effect was reversed by its association with N-acetyl-glucosamine. The nine-daily treatment with LAL (10 mg/kg; i.v.) showed no toxicity. CONCLUSION: The novel N-acetyl-D-glucosamine-binding lectin isolated from L. araripensis seeds presents anti-inflammatory effect involving the lectin domain and the inhibition of 5-HT, BK, PGE2, NO, TNF-α and leukocyte rolling and adhesion.
Assuntos
Acetilglucosamina/farmacologia , Anti-Inflamatórios/farmacologia , Fabaceae/química , Inflamação/prevenção & controle , Lectinas/farmacologia , Animais , Permeabilidade Capilar/efeitos dos fármacos , Edema/induzido quimicamente , Edema/prevenção & controle , Eritrócitos/efeitos dos fármacos , Hemaglutinação/efeitos dos fármacos , Técnicas In Vitro , Inflamação/patologia , Masculino , Camundongos , Peritonite/induzido quimicamente , Peritonite/prevenção & controle , Coelhos , Sementes/químicaRESUMO
The psychrotolerant Pseudoalteromonas issachenkonii PAMC 22718 was isolated for its high exo-acting chitinase activity in the Kara Sea, Arctic. An exo-acting chitinase (W-Chi22718) was homogeneously purified from the culture supernatant of PAMC 22718, the molecular weight of which was estimated to be approximately 112 kDa. Due to its ß-N-acetylglucosaminidase activity, W-Chi22718 was able to produce N-acetyl-D-glucosamine (GlcNAc) monomers from chitin oligosaccharide substrates. W-Chi22718 displayed chitinase activity from 0 to 37°C (optimal temperature of 30°C) and maintained activity from pH 6.0 to 9.0 (optimal pH of 7.6). W-Chi22718 exhibited a relative activity of 13 and 35% of maximal activity at 0 and 10°C, respectively, which is comparable to the activities of previously characterized, cold-adapted bacterial chitinases. W-Chi22718 activity was enhanced by K+, Ca2+, and Fe2+, but completely inhibited by Cu2+ and SDS. We found that W-Chi22718 can produce much more (GlcNAcs) from colloidal chitin, working together with previously characterized cold-active endochitinase W-Chi21702. Genome sequencing revealed that the corresponding gene (chi22718_IV) was 2,856 bp encoding a 951 amino acid protein with a calculated molecular weight of approximately 102 kDa.
Assuntos
Acetilglucosamina/metabolismo , Acetilglucosaminidase/metabolismo , Pseudoalteromonas/enzimologia , Quitina/metabolismo , Quitinases/metabolismo , Hidrólise , Microbiologia Industrial , Cinética , Pseudoalteromonas/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
Predation plays a major role in energy and nutrient flow in the biological food chain. Plant carnivory has attracted much interest since Darwin's time, but many fundamental properties of the carnivorous lifestyle are largely unexplored. In particular, the chain of events leading from prey perception to its digestive utilization remains to be elucidated. One of the first steps after the capture of animal prey, i.e. the enzymatic breakup of the insects' chitin-based shell, is reflected by considerable chitinase activity in the secreted digestive fluid in the carnivorous plant Venus flytrap. This study addresses the molecular nature, function, and regulation of the underlying enzyme, VF chitinase-I. Using mass spectrometry based de novo sequencing, VF chitinase-I was identified in the secreted fluid. As anticipated for one of the most prominent proteins in the flytrap's "green stomach" during prey digestion, transcription of VF chitinase-I is restricted to glands and enhanced by secretion-inducing stimuli. In their natural habitat, Venus flytrap is exposed to high temperatures. We expressed and purified recombinant VF chitinase-I and show that the enzyme exhibits the hallmark properties expected from an enzyme active in the hot and acidic digestive fluid of Dionaea muscipula. Structural modeling revealed a relative compact globular form of VF chitinase-I, which might contribute to its overall stability and resistance to proteolysis. These peculiar characteristics could well serve industrial purposes, especially because of the ability to hydrolyze both soluble and crystalline chitin substrates including the commercially important cleavage of α-chitin.
Assuntos
Artrópodes/fisiologia , Quitinases/metabolismo , Digestão , Droseraceae/enzimologia , Cadeia Alimentar , Sequência de Aminoácidos , Animais , Quitina/metabolismo , Quitinases/química , Quitinases/genética , Clonagem Molecular , Droseraceae/genética , Modelos Moleculares , Dados de Sequência Molecular , Pichia , Estrutura Secundária de ProteínaRESUMO
Protein glycosylation with O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification of serine/threonine residues in nucleocytoplasmic proteins. O-GlcNAc has been shown to play a role in many different cellular processes and O-GlcNAcylation is often found at sites that are also known to be phosphorylated. Unlike phosphorylation, O-GlcNAc levels are regulated by only two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (O-GlcNAcase or OGA). So far, no obvious consensus sequence has been found for sites of O-GlcNAcylation. Additionally, O-GlcNAcase recognizes and cleaves all O-GlcNAcylated proteins, independent of their sequence. In this work, we generate and analyze five models of O-GlcNAcylated peptides in complex with a bacterial OGA. Each of the five glycopeptides bind to OGA in a similar fashion, with OGA-peptide interactions primarily, but not exclusively, involving the peptide backbone atoms, thus explaining the lack of sensitivity to peptide sequence. Nonetheless, differences in peptide sequences, particularly at the -1 to -4 positions, lead to variations in predicted affinity, consistent with observed experimental variations in enzyme kinetics. The potential exists, therefore, to employ the present analysis to guide the development glycopeptide-specific inhibitors, or conversely, the conversion of OGA into a reagent that could target specific O-GlcNAcylated peptide sequences.
Assuntos
Proteínas de Bactérias/química , Bacteroides/enzimologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Análise de Sequência de Proteína , beta-N-Acetil-Hexosaminidases/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Bacteroides/genética , Inibidores Enzimáticos/química , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , beta-N-Acetil-Hexosaminidases/genéticaRESUMO
N-acetyl glucosamine (NAG) is a natural amino sugar found in various human tissues with previously described anti-inflammatory effects. Various chemical modifications of NAG have been made to promote its biomedical applications. In this study, we synthesized two bi-deoxygenated NAG, BNAG1 and BNAG2 and investigated their anti-inflammatory properties, using an in vivo and in vitro inflammation mouse model induced by lipopolysaccharide (LPS). Among the parent molecule NAG, BNAG1 and BNAG2, BNAG1 showed the highest inhibition against serum levels of IL-6 and TNF α and the leukocyte migration to lungs and peritoneal cavity in LPS challenged mice, as well as IL-6 and TNF α production in LPS-stimulated primary peritoneal macrophages. BNAG2 displayed an anti-inflammatory effect which was comparable to NAG. These findings implied potential application of these novel NAG derivatives, especially BNAG1, in treatment of certain inflammation-related diseases.
Assuntos
Acetilglucosamina , Anti-Inflamatórios , Lipopolissacarídeos , Macrófagos Peritoneais , Fator de Necrose Tumoral alfa , Animais , Acetilglucosamina/farmacologia , Camundongos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/síntese química , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-6/metabolismo , Interleucina-6/sangue , Inflamação/tratamento farmacológico , Masculino , Modelos Animais de DoençasRESUMO
Intestinal alkaline phosphatases (IAPs) are involved in the cleavage of phosphate prodrugs to liberate the drug for absorption in the intestine. To facilitate in vitro characterization of phosphate prodrugs, we have cloned, expressed, purified and characterized IAPs from rat and cynomolgus monkey (rIAP and cIAP respectively) which are important pre-clinical species for drug metabolism studies. The recombinant rat and monkey enzymes expressed in Sf9 insect cells (IAP-Ic) were found to be glycosylated and active. Expression of rat IAP in Escherichia coli (rIAP-Ec) led to ~200-fold loss of activity that was partially recovered by the addition of external Zn(2+) and Mg(2+) ions. Crystal structures of rIAP-Ec and rIAP-Ic were determined and they provide rationale for the discrepancy in enzyme activities. Rat IAP-Ic retains its activity in presence of both Zn(2+) and Mg(2+) whereas activity of most other alkaline phosphatases (APs) including the cIAP was strongly inhibited by excess Zn(2+). Based on our crystal structure, we hypothesized the residue Q317 in rIAP, present within 7 Å of the Mg(2+) at M3, to be important for this difference in activity. The Q317H rIAP and H317Q cIAP mutants showed reversal in effect of Zn(2+), corroborating the hypothesis. Further analysis of the two structures indicated a close linkage between glycosylation and crown domain stability. A triple mutant of rIAP, where all the three putative N-linked glycosylation sites were mutated showed thermal instability and reduced activity.