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1.
Nat Commun ; 12(1): 5426, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34521824

RESUMO

Much hope in drug development comes from the discovery of positive allosteric modulators (PAM) that display target subtype selectivity and act by increasing agonist potency and efficacy. How such compounds can allosterically influence agonist action remains unclear. Metabotropic glutamate receptors (mGlu) are G protein-coupled receptors that represent promising targets for brain diseases, and for which PAMs acting in the transmembrane domain have been developed. Here, we explore the effect of a PAM on the structural dynamics of mGlu2 in optimized detergent micelles using single molecule FRET at submillisecond timescales. We show that glutamate only partially stabilizes the extracellular domains in the active state. Full activation is only observed in the presence of a PAM or the Gi protein. Our results provide important insights on the role of allosteric modulators in mGlu activation, by stabilizing the active state of a receptor that is otherwise rapidly oscillating between active and inactive states.


Assuntos
Ácido Glutâmico/farmacologia , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/química , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico , Aminoácidos/química , Aminoácidos/farmacologia , Compostos de Bifenilo/química , Compostos de Bifenilo/farmacologia , Compostos Bicíclicos com Pontes/química , Compostos Bicíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Domínio Catalítico , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ésteres do Colesterol/química , Ésteres do Colesterol/farmacologia , Diosgenina/análogos & derivados , Diosgenina/química , Diosgenina/farmacologia , Dissacarídeos/química , Dissacarídeos/farmacologia , Transferência Ressonante de Energia de Fluorescência , Expressão Gênica , Glucosídeos/química , Glucosídeos/farmacologia , Glicolipídeos/química , Glicolipídeos/farmacologia , Células HEK293 , Humanos , Indanos/química , Indanos/farmacologia , Micelas , Octoxinol/química , Octoxinol/farmacologia , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula , Xantenos/química , Xantenos/farmacologia
2.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209772

RESUMO

Due to the limited number of organ donors, 3D printing of organs is a promising technique. Tissue engineering is increasingly using xenogeneic material for this purpose. This study was aimed at assessing the safety of decellularized porcine pancreas, together with the analysis of the risk of an undesirable immune response. We tested eight variants of the decellularization process. We determined the following impacts: rinsing agents (PBS/NH3·H2O), temperature conditions (4 °C/24 °C), and the grinding method of native material (ground/cut). To assess the quality of the extracellular matrix after the completed decellularization process, analyses of the following were performed: DNA concentration, fat content, microscopic evaluation, proteolysis, material cytotoxicity, and most importantly, the Triton X-100 content. Our analyses showed that we obtained a product with an extremely low detergent content with negligible residual DNA content. The obtained results confirmed the performed histological and immuno-fluorescence staining. Moreover, the TEM microscopic analysis proved that the correct collagen structure was preserved after the decellularization process. Based on the obtained results, we chose the most favorable variant in terms of quality and biology. The method we chose is an effective and safe method that gives a chance for the development of transplant and regenerative medicine.


Assuntos
Matriz Extracelular/fisiologia , Pâncreas/ultraestrutura , Engenharia Tecidual/métodos , Tecidos Suporte , Animais , Bioimpressão/métodos , Células Cultivadas , Detergentes/química , Detergentes/farmacologia , Matriz Extracelular/química , Fibroblastos/citologia , Fibroblastos/fisiologia , Teste de Materiais , Camundongos , Octoxinol/química , Octoxinol/farmacologia , Pâncreas/citologia , Pós/química , Impressão Tridimensional , Proteômica , Controle de Qualidade , Suínos , Engenharia Tecidual/normas , Tecidos Suporte/química , Tecidos Suporte/normas
3.
Biochemistry (Mosc) ; 86(1): 44-58, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33705281

RESUMO

It is known that Triton X-100 (TX) reversibly inhibits activity of cytochrome c oxidase (CcO). The mechanism of inhibition is analyzed in this work. The action of TX is not directed to the reaction of CcO with cytochrome c, does not cause transition of the enzyme to the "slow" form, and is not associated with monomerization of the enzyme complex. TX completely suppresses oxygen reduction by CcO, but inhibition is prevented and partially reversed by dodecyl-ß-D-maltoside (DDM), a detergent used to maintain CcO in solution. A 1/1 stoichiometry competition is shown between DDM and TX for binding to CcO, with Ki = 0.3 mM and affinity of DDM for the enzyme of 1.2 mM. TX interaction with the oxidized enzyme induces spectral response with maximum at 421 nm and [TX]1/2 = 0.28 mM, presumably associated with heme a3. When CcO interacts with excess of H2O2 TX affects equilibrium of the oxygen intermediates of the catalytic center accelerating the FI-607 → FII-580 transition, inhibits generation of O2·- by the enzyme, and, to a lesser extent, suppresses the catalase partial activity. The observed effects can be explained by inhibition of the conversion of the intermediate FII-580 to the free oxidized state during the catalytic cycle. TX suppresses intraprotein electron transfer between hemes a and a3 during enzyme turnover. Partial peroxidase activity of CcO remains relatively resistant to TX under conditions that block oxidase reaction effectively. These features indicate an impairment of the K proton channel conductivity. We suggest that TX interacts with CcO at the Bile Acid Binding Site (BABS) that is located on the subunit I at the K-channel mouth and contacts with amphipathic regulators of CcO [Buhrow et al. (2013) Biochemistry, 52, 6995-7006]. Apparently, TX mimics the physiological ligand of BABS, whereas the DDM molecule mimics an endogenous phospholipid bound at the edge of BABS that controls effective affinity for the ligand.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Octoxinol/farmacologia , Animais , Bovinos , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Cinética , Ligantes , Mitocôndrias Cardíacas/enzimologia
4.
Int J Antimicrob Agents ; 57(3): 106283, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33503451

RESUMO

A major determinant of ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) is the drug insensitive transpeptidase, PBP2a, encoded by mecA. Full expression of the resistance phenotype requires auxiliary factors. Two such factors, auxiliary factor A (auxA, SAUSA300_0980) and B (auxB, SAUSA300_1003), were identified in a screen against mutants with increased susceptibility to ß-lactams in the MRSA strain, JE2. auxA and auxB encode transmembrane proteins, with AuxA predicted to be a transporter. Inactivation of auxA or auxB enhanced ß-lactam susceptibility in community-, hospital- and livestock-associated MRSA strains without affecting PBP2a expression, peptidoglycan cross-linking or wall teichoic acid synthesis. Both mutants displayed increased susceptibility to inhibitors of lipoteichoic acid (LTA) synthesis and alanylation pathways and released LTA even in the absence of ß-lactams. The ß-lactam susceptibility of the aux mutants was suppressed by mutations inactivating gdpP, which was previously found to allow growth of mutants lacking the lipoteichoic synthase enzyme, LtaS. Using the Galleria mellonella infection model, enhanced survival of larvae inoculated with either auxA or auxB mutants was observed compared with the wild-type strain following treatment with amoxicillin. These results indicate that AuxA and AuxB are central for LTA stability and potential inhibitors can be tools to re-sensitize MRSA strains to ß-lactams and combat MRSA infections.


Assuntos
Antibacterianos/farmacologia , Lipopolissacarídeos/metabolismo , Proteínas de Membrana/metabolismo , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Proteínas de Ligação às Penicilinas/metabolismo , Ácidos Teicoicos/metabolismo , Amoxicilina/farmacologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cefoxitina/farmacologia , Parede Celular/metabolismo , DNA Bacteriano/genética , Farmacorresistência Bacteriana , Humanos , Larva/microbiologia , Proteínas de Membrana/genética , Meropeném/farmacologia , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Modelos Animais , Mariposas/microbiologia , Mutação , Octoxinol/farmacologia , Oxacilina/farmacologia , Peptidoglicano/metabolismo , Fenótipo , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Virulência , Resistência beta-Lactâmica , beta-Lactamas/farmacologia
5.
Lett Appl Microbiol ; 72(6): 669-676, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32955753

RESUMO

Acanthopanax (A.) henryi (Oliv.) Harms contain many bioactive compounds commonly used in traditional Chinese medicine. The objective of the present study was to investigate the antibacterial activity of the single constituent, Eleutheroside K (ETSK) isolated from the leaves of A. henryi (Oliv.) Harms, against methicillin-resistant Staphylococcus (S.) aureus (MRSA). Broth microdilution assay was used to measure the minimal inhibitory concentration (MIC) and the MIC values of ETSK against eight clinical S. aureus strains were all 50 µg ml-1 . At sub-inhibitory concentrations, a synergistic effect between oxacillin (OXA) and ETSK was confirmed using checkerboard dilution assay and time-kill curve analysis. The bacteriostatic effect became more pronounced when ETSK was used in combination with detergent (Triton X-100) or ATPase inhibitor (N, N'-dicyclohexylcarbodiimide). According to western blot analysis, the down-regulated expression of Penicillin-binding protein 2a (PBP2a) further validated that the bacterial activity was inhibited when treated with ETSK in a dose-dependent manner. Results based on our study verified that ETSK significantly suppressed MRSA infections and emphasized the potential application of ETSK as a novel anti-MRSA natural drug.


Assuntos
Antibacterianos/farmacologia , Eleutherococcus/metabolismo , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Oxacilina/farmacologia , Extratos Vegetais/farmacologia , Dicicloexilcarbodi-Imida/farmacologia , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Eleutherococcus/química , Resistência a Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Octoxinol/farmacologia , Proteínas de Ligação às Penicilinas/biossíntese , Folhas de Planta/química
6.
J Biosci Bioeng ; 131(3): 234-240, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33189544

RESUMO

Trypsin is a serine protease with important applications such as protein sequencing and tissue dissociation. Preserving protein structure and its activity during freeze-thawing and prolonging its shelf life is one of the most interesting tasks in biochemistry. In the present study, trypsin cryoprotection was achieved by altering buffer composition. Sodium phosphate buffer at pH 8.0 led to pH shift-induced destabilization of trypsin and formation of a molten globule, followed by significant activity loss (about 70%). Potassium phosphate and ammonium bicarbonate buffers at pH 8.0 were used with up to 90% activity recovery rate after 7 freeze-thaw cycles. The addition of non-ionic surfactants Tween 20 and Tween 80 led to up to 99% activity recovery rate. Amide I region changes, corresponding to specific secondary structures in the Fourier transform infrared (FTIR) spectrum, were modest in the case of Tween 20 and Tween 80. On the other hand, the addition of Triton X-100 led to the destabilization of α-helicoidal segments of trypsin structure after 7 freeze-thaw cycles but also increased protein substrate availability.


Assuntos
Congelamento , Tensoativos/farmacologia , Tripsina/metabolismo , Octoxinol/farmacologia , Fosfatos/farmacologia , Compostos de Potássio/farmacologia , Tensoativos/química
7.
Int J Biol Macromol ; 167: 736-745, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33278448

RESUMO

Identification of functionalities responsible for prevention of fibrillation in proteins is important to design effective drugs in addressing neurodegenerative diseases. We have used nonionic surfactant triton X-100 (TX-100) and antithyroid drug methimazole (MMI) to understand mechanistic aspects of action of these molecules having different functionalities on hen egg-white lysozyme at different stages of fibrillation. After establishing the nucleation, elongation and maturation stages of fibrillation of protein at 57 °C, energetics of interactions with these molecules have been determined by using isothermal titration calorimetry. Differential scanning calorimetry has permitted assessment of thermal stability of the protein at these stages, with or without these molecular entities. The enthalpies of interaction of TX-100 and MMI with protein fibrils suggest importance of hydrogen bonding and polar interactions in their effectiveness towards prevention of fibrils. TX-100, in spite of several polar centres, is unable to prevent fibrillation, rather it promotes. MMI is able to establish polar interactions with interacting strands of the protein and disintegrate fibrils. A rigorous comparison with inhibitors reported in literature highlights importance -OH and >CO functionalities in fibrillation prevention. Even though MMI has hydrogen bonding centres, its efficiency as inhibitor falls after the inhibited lysozyme fibrils further interact and form amorphous aggregates.


Assuntos
Amiloide/química , Fenômenos Químicos , Metimazol/farmacologia , Muramidase/química , Octoxinol/farmacologia , Agregados Proteicos/efeitos dos fármacos , Amiloide/ultraestrutura , Varredura Diferencial de Calorimetria , Ligação de Hidrogênio , Cinética , Metimazol/química , Modelos Biológicos , Octoxinol/química , Dobramento de Proteína , Termodinâmica
8.
Bull Exp Biol Med ; 169(4): 600-604, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32910398

RESUMO

We compared the capability of human fibroblasts to populate porous polycaprolactone (PCL) scaffolds modified during fabrication with surface-active agents Triton Ð¥-100 (type 1 scaffold) and polyvinylpyrrolidone (type 2 scaffold). The mean fiber diameter in both scaffolds was almost the same: 3.90±2.19 and 2.46±2.15 µ, respectively. Type 1 scaffold had higher surface density and hydrophilicity, when type 2 scaffold was 1.6 times thicker. The cells were seeded on the scaffolds by the dynamic seeding technique and then cultured in Petri dishes with nutrient medium in a humid atmosphere. During 3-day culturing, no cell release from the matrix was noted. DAPI staining proved the presence of cells in both scaffolds. However, in type 1 scaffold the cells populated the whole thickness, while in type 2 scaffold, the cells were present only in the superficial layer. These findings suggest that PCL scaffolds modified with Triton Ð¥-100 or polyvinylpyrrolidone are not cytotoxic, but the structure of the scaffold treated with Triton Ð¥-100 is more favorable for population with cells.


Assuntos
Proliferação de Células/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Octoxinol/farmacologia , Poliésteres/farmacologia , Povidona/farmacologia , Tecidos Suporte , Materiais Biocompatíveis , Técnicas Eletroquímicas , Fibroblastos/citologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Octoxinol/química , Poliésteres/química , Porosidade , Povidona/química , Cultura Primária de Células , Pele/citologia , Pele/efeitos dos fármacos , Tensoativos/química , Tensoativos/farmacologia , Engenharia Tecidual
9.
Biotechnol Prog ; 36(6): e3036, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32533632

RESUMO

Triton X-100 detergent treatment is a robust enveloped virus inactivation unit operation included in biopharmaceutical manufacturing processes. However, the European Commission officially placed Triton X-100 on the Annex XIV authorization list in 2017 because a degradation product of Triton X-100, 4-(1,1,3,3-tetramethylbutyl) phenol (also known as 4-tert-octylphenol), is considered to have harmful endocrine disrupting activities. As a result, the use of Triton X-100 in the European Economic Area (EEA) would not be allowed unless an ECHA issued authorization was granted after the sunset date of January 4, 2021. This has prompted biopharmaceutical manufacturers to search for novel, environment-friendly alternative detergents for enveloped virus inactivation. In this study, we report the identification of such a novel detergent, Simulsol SL 11W. Simulsol SL 11W is an undecyl glycoside surfactant produced from glucose and C11 fatty alcohol. We report here that Simulsol SL 11W was able to effectively inactive enveloped viruses, such as xenotropic murine leukemia virus (XMuLV) and pseudorabies virus (PRV). By using XMuLV as a representative enveloped virus, the influence of various parameters on the effectiveness of virus inactivation was evaluated. Virus inactivation by Simulsol SL 11W was effective across different clarified bioreactor harvests at broad concentrations, pH, and temperature ranges. Simulsol SL 11W concentration, temperature of inactivation, and treatment time were identified as critical process parameters for virus inactivation. Removal of Simulsol SL 11W was readily achieved by Protein A chromatography and product quality was not affected by detergent treatment. Taken together, these results have shown the potential of Simulsol SL 11W as a desirable alternative to Triton X-100 for enveloped virus inactivation that could be readily implemented into biopharmaceutical manufacturing processes.


Assuntos
Produtos Biológicos/química , Detergentes/química , Disruptores Endócrinos/efeitos adversos , Inativação de Vírus/efeitos dos fármacos , Animais , Produtos Biológicos/síntese química , Produtos Biológicos/farmacologia , Detergentes/síntese química , Disruptores Endócrinos/farmacologia , Humanos , Camundongos , Octoxinol/efeitos adversos , Octoxinol/farmacologia , Fenóis/efeitos adversos
10.
Transfus Clin Biol ; 27(3): 172-178, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32340867

RESUMO

OBJECTIVES: The use of leukoreduction filters has been highly increased in Iranian Blood Transfusion Centers within the last decade to provide sufficient leukoreduced blood products from healthy repeated donors for alloimmunized or sensitive recipients. Leucoflex LCR5, the dominant brand which procured by the Iranian Blood Transfusion Organization, is the most updated generation of the filters used around the world. MATERIAL AND METHODS: In this study, we recovered trapped leukocytes from these filters using different buffer solutions and optimized elution method. The count of recovered cells assessed by cell counter, and cell viability was detected using trypan blue staining. The percent of leukocyte subpopulations was evaluated using a panel of monoclonal antibodies and flow cytometric analysis. RESULTS: It illustrated that a buffer solution consistent with PBS in pH 7.2 containing 2mM EDTA and 4% (w/w) Dextran 40 was the best buffer for LCR5 filter backflushing. The white cell counted as 4.56×108 Granulocytes, 3.34×108 Lymphocytes, and 0.64×108 Monocytes according to analysis with auto hemoanalysis and flow cytometric methods. CONCLUSION: The study guides and assists blood management systems in arranging a national blood profile database for future cell therapy strategies. Also, the recovered cells could be of significance in stem cell research, cellular interaction studies as well as novel molecular developments in drug discovery.


Assuntos
Separação Celular/instrumentação , Procedimentos de Redução de Leucócitos/instrumentação , Leucócitos , Tampões (Química) , Separação Celular/métodos , Sobrevivência Celular , Ácido Edético/farmacologia , Desenho de Equipamento , Citometria de Fluxo , Humanos , Contagem de Leucócitos , Leucócitos/citologia , Octoxinol/farmacologia , Temperatura
11.
Carbohydr Res ; 490: 107962, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32169671

RESUMO

Three large (2084-, 984-, and 2104-amino acids) endo-α-N-acetylgalactosaminidase candidate genes from the commensal human gut bacterium Tyzzerella nexilis were successfully cloned and subsequently expressed in Escherichia coli. Activity tests of the purified proteins revealed that two of the candidate genes (Tn0153 and Tn2105) were able to hydrolyze the disaccharide unit from Galß1-3GalNAc-α-pNP. The biochemical characterization revealed optimum pH conditions of 4.0 for both enzymes and temperature optima of 50 °C. The addition of 2-mercaptoethanol, Triton X-100 and urea had only minor effects on the activity of the enzymes, and the addition of imidazole and sodium dodecyl sulfate led to a significant reduction of the enzymes' activities. A mutational study identified and confirmed the role of the catalytically significant amino acids. The present study describes the first functional characterization of members of the GH101 family from this human gut symbiont.


Assuntos
Clonagem Molecular/métodos , Clostridiales/fisiologia , Trato Gastrointestinal/microbiologia , alfa-N-Acetilgalactosaminidase/genética , alfa-N-Acetilgalactosaminidase/metabolismo , Proteínas de Bactérias , Clostridiales/enzimologia , Dissacarídeos/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Humanos , Hidrólise , Mercaptoetanol/farmacologia , Mutação , Octoxinol/farmacologia , Especificidade por Substrato , Simbiose , Ureia/farmacologia
12.
Int J Mol Sci ; 21(3)2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32019072

RESUMO

Shiraia mycelial culture is a promising biotechnological alternative for the production of hypocrellin A (HA), a new photosensitizer for anticancer photodynamic therapy (PDT). The extractive fermentation of intracellular HA in the nonionic surfactant Triton X-100 (TX100) aqueous solution was studied in the present work. The addition of 25 g/L TX100 at 36 h of the fermentation not only enhanced HA exudation to the broth by 15.6-fold, but stimulated HA content in mycelia by 5.1-fold, leading to the higher production 206.2 mg/L, a 5.4-fold of the control on day 9. After the induced cell membrane permeabilization by TX100 addition, a rapid generation of nitric oxide (NO) and hydrogen peroxide (H2O2) was observed. The increase of NO level was suppressed by the scavenger vitamin C (VC) of reactive oxygen species (ROS), whereas the induced H2O2 production could not be prevented by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), suggesting that NO production may occur downstream of ROS in the extractive fermentation. Both NO and H2O2 were proved to be involved in the expressions of HA biosynthetic genes (Mono, PKS and Omef) and HA production. NO was found to be able to up-regulate the expression of transporter genes (MFS and ABC) for HA exudation. Our results indicated the integrated role of NO and ROS in the extractive fermentation and provided a practical biotechnological process for HA production.


Assuntos
Ascomicetos/química , Peróxido de Hidrogênio/metabolismo , Óxido Nítrico/metabolismo , Octoxinol/farmacologia , Perileno/análogos & derivados , Fármacos Fotossensibilizantes/metabolismo , Quinonas/metabolismo , Biotecnologia , Membrana Celular/metabolismo , Fermentação , Micélio/química , Perileno/metabolismo , Fenol , Fotoquimioterapia
13.
Int J Mol Sci ; 21(2)2020 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-31936522

RESUMO

ß-N-Acetylhexosaminidases are glycoside hydrolases (GHs) acting on N-acetylated carbohydrates and glycoproteins with the release of N-acetylhexosamines. Members of the family GH20 have been reported to catalyze the transfer of N-acetylglucosamine (GlcNAc) to an acceptor, i.e., the reverse of hydrolysis, thus representing an alternative to chemical oligosaccharide synthesis. Two putative GH20 ß-N-acetylhexosaminidases, PhNah20A and PhNah20B, encoded by the marine bacterium Paraglaciecola hydrolytica S66T, are distantly related to previously characterized enzymes. Remarkably, PhNah20A was located by phylogenetic analysis outside clusters of other studied ß-N-acetylhexosaminidases, in a unique position between bacterial and eukaryotic enzymes. We successfully produced recombinant PhNah20A showing optimum activity at pH 6.0 and 50 °C, hydrolysis of GlcNAc ß-1,4 and ß-1,3 linkages in chitobiose (GlcNAc)2 and GlcNAc-1,3-ß-Gal-1,4-ß-Glc (LNT2), a human milk oligosaccharide core structure. The kinetic parameters of PhNah20A for p-nitrophenyl-GlcNAc and p-nitrophenyl-GalNAc were highly similar: kcat/KM being 341 and 344 mM-1 s-1, respectively. PhNah20A was unstable in dilute solution, but retained full activity in the presence of 0.5% bovine serum albumin (BSA). PhNah20A catalyzed the formation of LNT2, the non-reducing trisaccharide ß-Gal-1,4-ß-Glc-1,1-ß-GlcNAc, and in low amounts the ß-1,2- or ß-1,3-linked trisaccharide ß-Gal-1,4(ß-GlcNAc)-1,x-Glc by a transglycosylation of lactose using 2-methyl-(1,2-dideoxy-α-d-glucopyrano)-oxazoline (NAG-oxazoline) as the donor. PhNah20A is the first characterized member of a distinct subgroup within GH20 ß-N-acetylhexosaminidases.


Assuntos
Alteromonadaceae/enzimologia , Organismos Aquáticos/enzimologia , beta-N-Acetil-Hexosaminidases/biossíntese , Alteromonadaceae/genética , Organismos Aquáticos/genética , Biocatálise/efeitos dos fármacos , Estabilidade Enzimática , Genoma Bacteriano , Glicosilação , Concentração de Íons de Hidrogênio , Cinética , Octoxinol/farmacologia , Filogenia , Domínios Proteicos , Soroalbumina Bovina/farmacologia , Cloreto de Sódio/farmacologia , Especificidade por Substrato/efeitos dos fármacos , Temperatura , Fatores de Tempo , beta-N-Acetil-Hexosaminidases/química
14.
Mol Hum Reprod ; 26(3): 167-178, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-31980817

RESUMO

Uterus tissue engineering may dismantle limitations in current uterus transplantation protocols. A uterine biomaterial populated with patient-derived cells could potentially serve as a graft to circumvent complicated surgery of live donors, immunosuppressive medication and rejection episodes. Repeated uterine bioengineering studies on rodents have shown promising results using decellularised scaffolds to restore fertility in a partially impaired uterus and now mandate experiments on larger and more human-like animal models. The aim of the presented studies was therefore to establish adequate protocols for scaffold generation and prepare for future in vivo sheep uterus bioengineering experiments. Three decellularisation protocols were developed using vascular perfusion through the uterine artery of whole sheep uteri obtained from slaughterhouse material. Decellularisation solutions used were based on 0.5% sodium dodecyl sulphate (Protocol 1) or 2% sodium deoxycholate (Protocol 2) or with a sequential perfusion of 2% sodium deoxycholate and 1% Triton X-100 (Protocol 3). The scaffolds were examined by histology, extracellular matrix quantification, evaluation of mechanical properties and the ability to support foetal sheep stem cells after recellularisation. We showed that a sheep uterus can successfully be decellularised while maintaining a high integrity of the extracellular components. Uteri perfused with sodium deoxycholate (Protocol 2) were the most favourable treatment in our study based on quantifications. However, all scaffolds supported stem cells for 2 weeks in vitro and showed no cytotoxicity signs. Cells continued to express markers for proliferation and maintained their undifferentiated phenotype. Hence, this study reports three valuable decellularisation protocols for future in vivo sheep uterus bioengineering experiments.


Assuntos
Derme Acelular , Engenharia Tecidual/métodos , Útero/citologia , Animais , Ácido Desoxicólico/farmacologia , Matriz Extracelular/ultraestrutura , Feminino , Células HEK293 , Células-Tronco Hematopoéticas/citologia , Humanos , Modelos Anatômicos , Octoxinol/farmacologia , Preservação de Órgãos , Perfusão , Ovinos , Dodecilsulfato de Sódio/farmacologia , Soluções/toxicidade , Artéria Uterina , Útero/irrigação sanguínea
15.
Colloids Surf B Biointerfaces ; 187: 110602, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31761521

RESUMO

OBJECTIVE: This study was conducted to investigate the wetting behavior of different surfactant solutions on the leaf surfaces of apple during the fruit formation stage. METHODS: Five surfactants, including C12E5, Tween-20, Triton X-100, DTAB, and SDS were evaluated in this study. The contact angle, surface tension, adhesion tension, work of adhesion, and solid-liquid interface tension of droplets on the leaf surface were determined by the drop method. RESULTS: The results showed that the nonionic surfactants C12E5 and Triton X-100 had better wetting effects than other surfactants. Moreover, when the concentration of C12E5 and Triton X-100 was 1 × 10-3 mol/L, the leaves reached a completely wet state. Toxicity measurement showed that the incubation rate of Carposina niponensis eggs decreased gradually with increasing content of C12E5 or Triton X-100. Additionally, field efficacy analysis showed that adding C12E5 or Triton X-100 significantly improved the beta-cyfluthrin 3% water emulsion (EW) against C. niponensis. CONCLUSIONS: These results indicate that the surfactants C12E5 and Triton X-100 can significantly improve pesticide application, which will be helpful for reducing pesticide use and developing new pesticides.


Assuntos
Malus/anatomia & histologia , Folhas de Planta/anatomia & histologia , Tensoativos/farmacologia , Árvores/anatomia & histologia , Adesividade , Animais , Lepidópteros/fisiologia , Malus/efeitos dos fármacos , Nitrilas/toxicidade , Octoxinol/farmacologia , Óvulo/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Piretrinas/toxicidade , Soluções , Tensão Superficial/efeitos dos fármacos , Árvores/efeitos dos fármacos , Molhabilidade
16.
J Hazard Mater ; 385: 121616, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31780289

RESUMO

The efficient bioremediation of estrogen contamination in complex environments is of great concern. Here the strain Stenotrophomonas maltophilia SJTH1 was found with great and stable estrogen-degradation efficiency even under stress environments. The strain could utilize 17ß-estradiol (E2) as a carbon source and degrade 90% of 10 mg/L E2 in a week; estrone (E1) was the first degrading intermediate of E2. Notably, diverse pH conditions (3.0-11.0) and supplements of 4% salinity, 6.25 mg/L of heavy metal (Cd2+ or Cu2+), or 1 CMC of surfactant (Tween 80/ Triton X-100) had little effect on its cell growth and estrogen degradation. The addition of low concentrations of copper and Tween 80 even promoted its E2 degradation. Bioaugmentation of strain SJTH1 into solid clay soil achieved over 80% removal of E2 contamination (10 mg/kg) within two weeks. Further, the whole genome sequence of S. maltophilia SJTH1 was obtained, and a series of potential genes participating in stress-tolerance and estrogen-degradation were predicted. Four dehydrogenases similar to 17ß-hydroxysteroid dehydrogenases (17ß-HSDs) were found to be induced by E2, and the four heterogenous-expressed enzymes could oxidize E2 into E1 efficiently. This work could promote bioremediation appliance potential with microorganisms and biodegradation mechanism study of estrogens in complex real environments.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Estradiol Desidrogenases/isolamento & purificação , Estradiol/metabolismo , Stenotrophomonas maltophilia/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biodegradação Ambiental , Estradiol Desidrogenases/química , Estradiol Desidrogenases/genética , Cinética , Octoxinol/farmacologia , Oxirredução , Polissorbatos/farmacologia , Alinhamento de Sequência , Stenotrophomonas maltophilia/efeitos dos fármacos , Stenotrophomonas maltophilia/enzimologia , Stenotrophomonas maltophilia/genética , Tensoativos/farmacologia
17.
Elife ; 82019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31829935

RESUMO

We develop magnetic resonance (MR) methods for real-time measurement of tissue microstructure and membrane permeability of live and fixed excised neonatal mouse spinal cords. Diffusion and exchange MR measurements are performed using the strong static gradient produced by a single-sided permanent magnet. Using tissue delipidation methods, we show that water diffusion is restricted solely by lipid membranes. Most of the diffusion signal can be assigned to water in tissue which is far from membranes. The remaining 25% can be assigned to water restricted on length scales of roughly a micron or less, near or within membrane structures at the cellular, organelle, and vesicle levels. Diffusion exchange spectroscopy measures water exchanging between membrane structures and free environments at 100 s-1.


Assuntos
Membrana Celular/ultraestrutura , Imagem de Difusão por Ressonância Magnética/métodos , Membranas Intracelulares/ultraestrutura , Espectroscopia de Ressonância Magnética/métodos , Medula Espinal/ultraestrutura , Potenciais de Ação , Animais , Animais Recém-Nascidos , Anisotropia , Células do Corno Anterior/fisiologia , Água Corporal , Detergentes/farmacologia , Deutério , Difusão , Imagem de Difusão por Ressonância Magnética/instrumentação , Desenho de Equipamento , Espectroscopia de Ressonância Magnética/instrumentação , Lipídeos de Membrana/química , Camundongos , Movimento (Física) , Octoxinol/farmacologia , Medula Espinal/efeitos dos fármacos
18.
J Nutr Sci ; 8: e32, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31595188

RESUMO

Melatonin-rich and 1,8-cineole-rich extracts have been successfully obtained from yellow mustard (YM) and small cardamom (SC) seeds, respectively, employing green technology of supercritical CO2 (SC-CO2) extraction. Chemical profiling confirmed the presence of melatonin and 1,8-cineole and co-extractants in the respective extracts. Electron paramagnetic resonance spectroscopy attested strong antioxidant activities of the extracts foregoing pan-assay interference compounds involved in spectroscopic analysis. These extracts also exhibited synergistic efficacies greater than unity confirming antioxidant synergy among the co-extracted bioactives therein. To ascertain hypocholesterolaemic efficacies, these extracts were co-administered orally with Triton X (at the pre-optimised dose of 175 mg/kg body weight (BW)) to Wistar albino rats at doses of 550, 175 and 55 mg/kg BW. Serum total cholesterol levels in the rats were monitored on days 3, 7, 15 and 21. On day 21, total cholesterol level reduced appreciably by 49·44 % in rats treated with YM seed extract and by 48·95 % in rats treated with SC seed extract, comparable with atorvastatin-administered rats (51·09 %). Either extract demonstrated inhibitory effects on hepatic 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase activity. A molecular docking exercise identified specific compounds in the extracts which possessed binding affinities comparable with therapeutically used HMG-CoA reductase inhibitors. In silico and in vivo studies concertedly concluded that the consortium of bioactive components in the extracts cannot be considered as invalid metabolic panaceas and therefore these 'green' extracts could be safely subjected to clinical studies as preventive biotherapeutics for hypercholesterolaemia. These extracts could be consumed per se as hypocholesterolaemic supplements or could be ingredients of new spice-based therapeutic foods.


Assuntos
Dióxido de Carbono/química , Colesterol/sangue , Suplementos Nutricionais , Elettaria/química , Mostardeira/química , Sementes/química , Especiarias/análise , Animais , Anticolesterolemiantes/análise , Anticolesterolemiantes/farmacologia , Antioxidantes/análise , Antioxidantes/farmacologia , Cromatografia com Fluido Supercrítico , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Inibidores de Hidroximetilglutaril-CoA Redutases/análise , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Hipercolesterolemia/tratamento farmacológico , Masculino , Simulação de Acoplamento Molecular , Octoxinol/análise , Octoxinol/farmacologia , Extratos Vegetais/análise , Extratos Vegetais/farmacologia , Ratos , Ratos Wistar , Testes de Toxicidade Aguda
19.
Int J Biochem Cell Biol ; 116: 105612, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31546020

RESUMO

BACKGROUND/AIMS: Epigallocatechin-3-gallate (EGCG), a major catechin found in green tea, plays an important anti-tumor role and is involved in various other biological processes, such as, neuroprotection by prevention of aggregation of misfolded proteins generated because of genetic defects. Surfactant protein A2 mutations (G231V and F198S) have been identified to be associated with pulmonary fibrosis and lung cancer, and these mutations cause protein aggregation, instability as well as secretion deficiency. The present study focused on investigating the inhibitory effects of EGCG on aggregation of mutant SP-A2 and elucidating the potential mechanisms underlying this action. METHODS: Wild-type and mutant SP-A2 were transiently expressed in CHO-K1 cells. The aggregated and soluble proteins were separated into NP-40-insoluble and NP-40-soluble fractions. Protein stability was validated by chymotrypsin limited proteolysis assay. Western blot and RT-PCR were used to determine the protein and mRNA expression level, respectively. RESULTS: Mutant SP-A2 alone or wild-type SP-A2 co-expressed with G231V formed NP-40-insoluble aggregates in CHO-K1 cells. EGCG significantly suppressed this aggregation and alleviated mutant SP-A2 accumulation in the ER. When combined with 4-PBA, EGCG treatment completely blocked mutant SP-A2 aggregate formation. Though secretion of mutant protein was not affected, EGCG facilitated protein instability in both wild-type and mutant protein. Importantly, MG132, a proteasome inhibitor, reversed EGCG-induced aggregate reduction. CONCLUSIONS: EGCG inhibits aggregation of misfolded SP-A2 via induction of protein instability and activation of proteasomal pathway for aggregate degradation.


Assuntos
Catequina/análogos & derivados , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Agregados Proteicos/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteína A Associada a Surfactante Pulmonar/química , Animais , Butilaminas/farmacologia , Células CHO , Catequina/farmacologia , Cricetulus , Inibidores de Cisteína Proteinase/farmacologia , Detergentes/farmacologia , Expressão Gênica , Leupeptinas/farmacologia , Mutação , Octoxinol/farmacologia , Estabilidade Proteica , Fibrose Pulmonar/metabolismo , Proteína A Associada a Surfactante Pulmonar/genética , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidade
20.
Influenza Other Respir Viruses ; 13(5): 504-516, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31411006

RESUMO

BACKGROUND: Formulation of neuraminidase (NA) within influenza vaccines is gaining importance in light of recent human studies. The enzyme-linked lectin assay (ELLA) is considered a reliable assay to evaluate human anti-NA antibodies. OBJECTIVES: To overcome interference by hemagglutinin (HA)-specific antibodies and detect neuraminidase inhibitory (NI) antibodies only, two different sources of antigen have been studied in ELLA: reassortant viruses with a mismatched avian origin-HA or Triton X-100 (Tx)-treated wild-type viruses. Pseudotypes or pseudovirus (PV), characterized by a lentivirus core bearing human influenza NA and avian influenza HA, were investigated as an alternative source of antigen and compared to HA-mismatched and Tx-treated viruses, since represent a safer product to be handled. METHODS: Two independent panels of sera were analyzed by ELLA to evaluate the anti-NA response against N1 (A/California/07/2009 (H1N1pdm)) and N2 (A/Hong Kong/4801/2014 (H3N2)). The NA inhibition (NI) antibody titers measured as either the 50% end point or 50% inhibitory concentration (IC50 ) were compared for every source of antigen. RESULTS: The ELLA assay performed well with all three sources of antigen. NI titers measured using each antigen type correlated well when reported either as end point titers or as the IC50 . CONCLUSIONS: This study suggests that HA-mismatched whole virus, Triton-treated wild-type virus or PV can be used to measure NI antibody titers of human sera, but further comparability/validation assays should be performed to assess statistical differences. The data support the use of PV as an attractive alternative source of antigen and justify further investigation to improve stability of this antigen source.


Assuntos
Antígenos Virais/imunologia , Ensaios Enzimáticos/normas , Vírus da Influenza A/imunologia , Lectinas/química , Neuraminidase/imunologia , Octoxinol/farmacologia , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Aves/virologia , Ensaios Enzimáticos/métodos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza A/efeitos dos fármacos , Vacinas contra Influenza/imunologia , Influenza Aviária/virologia , Influenza Humana/virologia , Lentivirus/genética , Lentivirus/imunologia , Neuraminidase/antagonistas & inibidores , Vírus Reordenados/genética , Vírus Reordenados/imunologia
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