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1.
Int J Syst Evol Microbiol ; 70(6): 3872-3877, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32511087

RESUMO

A Gram-stain-negative, aerobic, non-spore-forming, motile by single polar flagellum and ovoid or rod-shaped bacterial strain, designated JBTF-M18T, was isolated from tidal-flat sediment collected from the Yellow Sea, Republic of Korea. The neighbour-joining phylogenetic tree of 16S rRNA gene sequences showed that strain JBTF-M18T fell within the clade comprising the type strains of Shewanella species. Strain JBTF-M18T exhibited 16S rRNA gene sequence similarity values of 97.1-98.8 % to the type strains of S. loihica, S. aquimarina, S. waksmanii and S. marisflavi and of less than 96.9 % to the type strains of the other Shewanella species. The average nucleotide identity and digital DNA-DNA hybridization values between strain JBTF-M18T and the type strains of S. waksmanii and S. loihica were 72.0 and 89.5% and 18.9 and 38.1 %, respectively. DNA-DNA relatedness values between strain JBTF-M18T and the type strains of S. aquimarina and S. marisflavi were 14 and 19 %, respectively. The DNA G+C content of strain JBTF-M18T from genomic sequence data was 52.9 %. Strain JBTF-M18Tcontained MK-6 as the predominant menaquinone and Q-7 and Q-8 as the predominant ubiquinones. It had iso-C15 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and C16 : 0 as the major fatty acids. The major polar lipids of strain JBTF-M18T were phosphatidylethanolamine and phosphatidylglycerol. Distinguished phenotypic properties, along with the phylogenetic and genetic distinctiveness, revealed that strain JBTF-M18T is separated from recognized Shewanella species. On the basis of the data presented, strain JBTF-M18T is considered to represent a novel species of the genus Shewanella, for which the name Shewanella insulae sp. nov. is proposed. The type strain is JBTF-M18T (=KACC 19869T=NBRC 113583T).


Assuntos
Sedimentos Geológicos/microbiologia , Filogenia , Água do Mar/microbiologia , Shewanella/classificação , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Hibridização de Ácido Nucleico , Fosfatidiletanolaminas/química , Fosfatidilgliceróis/química , RNA Ribossômico 16S/genética , República da Coreia , Análise de Sequência de DNA , Shewanella/isolamento & purificação
2.
Int J Syst Evol Microbiol ; 70(6): 3801-3808, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32459166

RESUMO

Six Gram-stain-negative, catalase- and oxidase-positive, rod-shaped and motile strains (FT9WT, FT25W, FT26WT, FT109WT, FT134W and CY42WT) were isolated from subtropical streams in China. Comparisons based on 16S rRNA gene sequences showed that the six strains shared similarities of less than 98.1 % with other species within the family Oxalobacteraceae and formed two separately distinct clades in phylogenetic trees. The 16S rRNA gene sequence similarities between strains FT9WT and FT25W, and between strains FT109WT and FT134W were both 99.7 %. The genome sizes of strains FT9WT, FT25W, FT26WT, FT109WT, FT134W and CY42WT were 6.45, 6.45, 6.54, 6.43, 6.52 and 6.74 Mbp with G+C contents of 64.0, 64.0, 63.8, 63.2, 63.2 and 62.5 %, respectively. The calculated pairwise average nucleotide (ANI) values among the six strains and other related species were less than 93.9 %, except that the values were 99.9 % between strains FT9WT and FT25W, 98.2 % between strains FT109WT and FT134W, and 95.0 and 95.1 % between strain FT26WT and strains FT9WT and FT25W, respectively. However, strain FT26WT shared 16S rRNA gene sequence similarities of only 98.3 and 98.2 % with FT9WT and FT25W, respectively. The respiratory quinone of the six strains was determined to be Q-8. The major fatty acids were C16 : 1 ω7c, C16 : 0 and C12 : 0. The predominant polar lipids included phosphatidylethanolamine and phosphatidylglycerol. Considering the phenotypic, biochemical, genotypic and ANI data, strains FT9WT and FT25W, and FT109WT and FT134W may belong to the same species, respectively. Although the pairwise ANI values between strain FT26WT and each of strains FT9WT and FT25W were located in the transition region of species demarcation, the dissimilarities among them indicated that strain FT26WT could represent an independent novel species. The reconstructed phylogenomic tree based on a concatenation of 92 core genes showed that the six strains clustered closely with Duganella sacchari Sac-22T and Duganella radicis KCTC 22382T, and supported that these six strains belong to the genus Duganella. The names Duganella albus sp. nov. (type strain FT9WT=GDMCC 1.1637T=KACC 21313T), Duganella aquatilis sp. nov. (type strain FT26WT=GDMCC 1.1641T=KACC 21315T), Duganella pernnla sp. nov. (type strain FT109WT=GDMCC 1.1688T=KACC 21480T) and Duganella levis sp. nov. (type strain CY42WT=GDMCC 1.1673T=KACC 21465T) are proposed.


Assuntos
Oxalobacteraceae/classificação , Filogenia , Rios/microbiologia , Técnicas de Tipagem Bacteriana , Composição de Bases , China , DNA Bacteriano/genética , Ácidos Graxos/química , Hibridização de Ácido Nucleico , Oxalobacteraceae/isolamento & purificação , Fosfatidiletanolaminas/química , Fosfatidilgliceróis/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Ubiquinona/química
3.
PLoS Comput Biol ; 16(4): e1007818, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32298258

RESUMO

The activation process of G protein-coupled receptors (GPCRs) has been extensively studied, both experimentally and computationally. In particular, Molecular Dynamics (MD) simulations have proven useful in exploring GPCR conformational space. The typical behaviour of class A GPCRs, when subjected to unbiased MD simulations from their crystallized inactive state, is to fluctuate between inactive and intermediate(s) conformations, even with bound agonist. Fully active conformation(s) are rarely stabilized unless a G protein is also bound. Despite several crystal structures of the adenosine A2a receptor (A2aR) having been resolved in complex with co-crystallized agonists and Gs protein, its agonist-mediated activation process is still not completely understood. In order to thoroughly examine the conformational landscape of A2aR activation, we performed unbiased microsecond-length MD simulations in quadruplicate, starting from the inactive conformation either in apo or with bound agonists: endogenous adenosine or synthetic NECA, embedded in two homogeneous phospholipid membranes: 1,2-dioleoyl-sn-glycerol-3-phosphoglycerol (DOPG) or 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC). In DOPC with bound adenosine or NECA, we observe transition to an intermediate receptor conformation consistent with the known adenosine-bound crystal state. In apo state in DOPG, two different intermediate conformations are obtained. One is similar to that observed with bound adenosine in DOPC, while the other is closer to the active state but not yet fully active. Exclusively, in DOPG with bound adenosine or NECA, we reproducibly identify receptor conformations with fully active features, which are able to dock Gs protein. These different receptor conformations can be attributed to the action/absence of agonist and phospholipid-mediated allosteric effects on the intracellular side of the receptor.


Assuntos
Agonistas do Receptor A2 de Adenosina , Fosfolipídeos , Receptor A2A de Adenosina , Adenosina/química , Adenosina/metabolismo , Agonistas do Receptor A2 de Adenosina/química , Agonistas do Receptor A2 de Adenosina/metabolismo , Sítios de Ligação , Humanos , Simulação de Dinâmica Molecular , Fosfatidilcolinas , Fosfatidilgliceróis , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Conformação Proteica , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/metabolismo
4.
Nat Commun ; 11(1): 1455, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32193379

RESUMO

The lipopeptide daptomycin is used as an antibiotic to treat severe infections with gram-positive pathogens, such as methicillin resistant Staphylococcus aureus (MRSA) and drug-resistant enterococci. Its precise mechanism of action is incompletely understood, and a specific molecular target has not been identified. Here we show that Ca2+-daptomycin specifically interacts with undecaprenyl-coupled cell envelope precursors in the presence of the anionic phospholipid phosphatidylglycerol, forming a tripartite complex. We use microbiological and biochemical assays, in combination with fluorescence and optical sectioning microscopy of intact staphylococcal cells and model membrane systems. Binding primarily occurs at the staphylococcal septum and interrupts cell wall biosynthesis. This is followed by delocalisation of components of the peptidoglycan biosynthesis machinery and massive membrane rearrangements, which may account for the pleiotropic cellular events previously reported. The identification of carrier-bound cell wall precursors as specific targets explains the specificity of daptomycin for bacterial cells. Our work reconciles apparently inconsistent previous results, and supports a concise model for the mode of action of daptomycin.


Assuntos
Antibacterianos/farmacologia , Parede Celular/efeitos dos fármacos , Daptomicina/farmacologia , Lipídeos de Membrana/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Parede Celular/metabolismo , Humanos , Membranas Artificiais , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/fisiologia , Testes de Sensibilidade Microbiana , Fosfatidilgliceróis/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia
5.
Invest Ophthalmol Vis Sci ; 61(3): 29, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32186673

RESUMO

Purpose: In contact with the external environment, the cornea can easily be injured. Although corneal wounds generally heal rapidly, the pain and increased risk of infection associated with a damaged cornea, as well as the impaired healing observed in some individuals, emphasize the need for novel treatments to accelerate corneal healing. We previously demonstrated in epidermal keratinocytes that the glycerol channel aquaporin-3 (AQP3) interacts with phospholipase D2 (PLD2) to produce the signaling phospholipid phosphatidylglycerol (PG), which has been shown to accelerate skin wound healing in vivo. We hypothesized that the same signaling pathway might be operational in corneal epithelial cells. Methods: We used co-immunoprecipitation, immunohistochemistry, scratch wound healing assays in vitro, and corneal epithelial wound healing assays in vivo to determine the role of the AQP3/PLD2/PG signaling pathway in corneal epithelium. Results: AQP3 was present in human corneas in situ, and AQP3 and PLD2 were co-immunoprecipitated from corneal epithelial cell lysates. The two proteins could also be co-immunoprecipitated from insect cells simultaneously infected with AQP3- and PLD2-expressing baculoviruses, suggesting a likely direct interaction. A particular PG, dioleoylphosphatidylglycerol (DOPG), enhanced scratch wound healing of a corneal epithelial monolayer in vitro. DOPG also accelerated corneal epithelial wound healing in vivo, both in wild-type mice and in a mouse model exhibiting impaired corneal wound healing (AQP3 knockout mice). Conclusions: These results indicate the importance of the AQP3/PLD2/PG signaling pathway in corneal epithelial cells and suggest the possibility of developing DOPG as a pharmacologic therapy to enhance corneal wound healing in patients.


Assuntos
Epitélio Anterior/efeitos dos fármacos , Limbo da Córnea/efeitos dos fármacos , Fosfatidilgliceróis/farmacologia , Cicatrização/fisiologia , Animais , Aquaporina 3/metabolismo , Western Blotting , Movimento Celular , Proliferação de Células , Células Cultivadas , Epitélio Anterior/metabolismo , Humanos , Imunoprecipitação , Limbo da Córnea/metabolismo , Masculino , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Fosfolipase D/metabolismo , Células Sf9/metabolismo , Transdução de Sinais/fisiologia , Transfecção
6.
Mol Pharmacol ; 97(5): 324-335, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32173651

RESUMO

Skin serves not only as a protective barrier to microbial entry into the body but also as an immune organ. The outer layer, the epidermis, is composed predominantly of keratinocytes, which can be stimulated to produce proinflammatory mediators. Although some inflammation is useful to defend against infection, excessive or persistent inflammation can lead to the development of inflammatory skin diseases, such as psoriasis, a common skin disorder affecting approximately 2% of the US population. We have previously found that phosphatidylglycerol (PG) derived from soy can inhibit inflammation in a contact irritant ear edema mouse model. Here, we investigated the ability of soy PG to inhibit inflammatory mediator expression in response to activators of the pattern recognition receptors, toll-like receptor-2 (TLR2) and -4 (TLR4). We found that in epidermal keratinocytes, soy PG inhibited TLR2 and TLR4 activation and inflammatory mediator expression in response to a synthetic triacylated lipopeptide and lipopolysaccharide, respectively, as well as an endogenous danger-associated molecular pattern. However, at higher concentrations, soy PG alone enhanced the expression of some proinflammatory cytokines, suggesting a narrow therapeutic window for this lipid. Dioleoylphosphatidylglycerol (DOPG), but not dioleoylphosphatidylcholine, exerted a similar inhibitory effect, completely blocking keratinocyte inflammatory mediator expression induced by TLR2 and TLR4 activators as well as NFκB activation in a macrophage cell line (RAW264.7); however, DOPG was not itself proinflammatory even at high concentrations. Furthermore, DOPG had no effect on NFκB activation in response to a TLR7/8 agonist. Our results suggest that DOPG could be used to inhibit excessive skin inflammation. SIGNIFICANCE STATEMENT: Although inflammation is beneficial for clearing an infection, in some cases, the infection can be excessive and/or become chronic, thereby resulting in considerable tissue damage and pathological conditions. We show here that the phospholipid phosphatidylglycerol can inhibit the activation of toll-like receptors 2 and 4 of the innate immune system as well as the downstream inflammatory mediator expression in response to microbial component-mimicking agents in epidermal keratinocytes that form the physical barrier of the skin.


Assuntos
Mediadores da Inflamação/metabolismo , Queratinócitos/metabolismo , Padrões Moleculares Associados a Patógenos/farmacologia , Fosfatidilgliceróis/farmacologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Calgranulina B/farmacologia , Humanos , Imidazóis/farmacologia , Lipopeptídeos/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , NF-kappa B/metabolismo , Células RAW 264.7 , Receptores de Reconhecimento de Padrão/metabolismo , Proteínas Recombinantes/farmacologia , Soja/química
7.
Proc Natl Acad Sci U S A ; 117(6): 2938-2947, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31980523

RESUMO

The conformational changes required for activation and K+ conduction in inward-rectifier K+ (Kir) channels are still debated. These structural changes are brought about by lipid binding. It is unclear how this process relates to fast gating or if the intracellular and extracellular regions of the protein are coupled. Here, we examine the structural details of KirBac1.1 reconstituted into both POPC and an activating lipid mixture of 3:2 POPC:POPG (wt/wt). KirBac1.1 is a prokaryotic Kir channel that shares homology with human Kir channels. We establish that KirBac1.1 is in a constitutively active state in POPC:POPG bilayers through the use of real-time fluorescence quenching assays and Förster resonance energy transfer (FRET) distance measurements. Multidimensional solid-state NMR (SSNMR) spectroscopy experiments reveal two different conformers within the transmembrane regions of the protein in this activating lipid environment, which are distinct from the conformation of the channel in POPC bilayers. The differences between these three distinct channel states highlight conformational changes associated with an open activation gate and suggest a unique allosteric pathway that ties the selectivity filter to the activation gate through interactions between both transmembrane helices, the turret, selectivity filter loop, and the pore helix. We also identify specific residues involved in this conformational exchange that are highly conserved among human Kir channels.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Proteínas de Bactérias/genética , Domínio Catalítico , Transferência Ressonante de Energia de Fluorescência , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilgliceróis/química , Fosfatidilgliceróis/metabolismo , Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/genética , Conformação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína
8.
Sci Rep ; 10(1): 1385, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992800

RESUMO

CHF5633 is a novel synthetic clinical pulmonary surfactant preparation composed by two phospholipid species, dipalmitoyl phosphatidylcholine (DPPC) and palmitoyloleoyl phosphatidylglycerol (POPG), and synthetic analogues of the hydrophobic surfactant proteins SP-B and SP-C. In this study, the interfacial properties of CHF5633 in the absence and in the presence of inhibitory serum proteins have been assessed in comparison with a native surfactant purified from porcine lungs and with poractant alpha, a widely used clinical surfactant preparation. The study of the spreading properties of CHF5633 in a Wilhelmy balance, its ability to adsorb and accumulate at air-liquid interfaces as revealed by a multiwell fluorescence assay, and its dynamic behavior under breathing-like compression-expansion cycling in a Captive Bubble Surfactometer (CBS), all revealed that CHF5633 exhibits a good behavior to reduce and sustain surface tensions to values below 5 mN/m. CHF5633 shows somehow slower initial interfacial adsorption than native surfactant or poractant alpha, but a better resistance to inhibition by serum proteins than the animal-derived clinical surfactant, comparable to that of the full native surfactant complex. Interfacial CHF5633 films formed in a Langmuir-Blodgett balance coupled with epifluorescence microscopy revealed similar propensity to segregate condensed lipid domains under compression than films made by native porcine surfactant or poractant alpha. This ability of CHF5633 to segregate condensed lipid phases can be related with a marked thermotropic transition from ordered to disordered membrane phases as exhibited by differential scanning calorimetry (DSC) of CHF5633 suspensions, occurring at similar temperatures but with higher associated enthalpy than that shown by poractant alpha. The good interfacial behavior of CHF5633 tested under physiologically meaningful conditions in vitro and its higher resistance to inactivation by serum proteins, together with its standardized and well-defined composition, makes it a particularly useful therapeutic preparation to be applied in situations associated with lung inflammation and edema, alone or in combined strategies to exploit surfactant-facilitated drug delivery.


Assuntos
Proteínas Sanguíneas/química , Sistemas de Liberação de Medicamentos , Fragmentos de Peptídeos , Fosfatidilcolinas , Proteína B Associada a Surfactante Pulmonar , Proteína C Associada a Surfactante Pulmonar , Surfactantes Pulmonares , Animais , Produtos Biológicos/química , Humanos , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fosfatidilcolinas/antagonistas & inibidores , Fosfatidilcolinas/química , Fosfatidilgliceróis/química , Fosfolipídeos/química , Proteína B Associada a Surfactante Pulmonar/antagonistas & inibidores , Proteína B Associada a Surfactante Pulmonar/química , Proteína C Associada a Surfactante Pulmonar/antagonistas & inibidores , Proteína C Associada a Surfactante Pulmonar/química , Surfactantes Pulmonares/antagonistas & inibidores , Surfactantes Pulmonares/química , Relação Estrutura-Atividade , Tensão Superficial , Suínos
9.
Int J Syst Evol Microbiol ; 70(2): 1398-1403, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31860430

RESUMO

A Gram-stain-positive, rod-shaped (0.3-0.4×1.2-2.0 µm), strictly aerobic and beige-pigmented bacterium, designated B3227T, was isolated from the sediment of a sea cucumber culture pond in Rongcheng, China (122.2° E 36.9° N). Its biochemical characteristics analysis revealed that the cells of this bacterium were catalase-positive and oxidase-negative. Cell growth occurred at 15-45 °C (optimum, 37 °C), pH 6.5-9.0 (pH 7.5-8.0) and in the presence of 0.0-22.0 % (w/v) NaCl (6.0-9.0 % NaCl). Phylogenetic analysis based on 16S rRNA gene sequencing indicated that strain B3227T exhibited similarities of 95.7, 95.5, 95.5 and 95.3 % to the type strains of Filobacillus milensis, Piscibacillus salipiscarius, Halalkalibacillus halophilus and Piscibacillus halophilus, respectively, and the results of physiological analyses revealed that strain B3227T was most similar to the genus Halalkalibacillus. The cells were endospore-forming and comprised an A1-γ-meso-diaminopimelic acid-type peptidoglycan. The respiratory quinone of strain B3227T was MK-7, and the dominant fatty acids were anteiso-C15 : 0 and anteiso-C17 : 0. The major polar lipids were diphosphatidylglycerol and phosphatidylethanolamine. The genomic DNA G+C content was 38.7 mol%. The average nucleotide identity values between strain B3227T and H. halophilus JCM 14192T (ANIb 69.5%, ANIm 84.2 %) and F. milensis JCM 12288T (ANIb 70.1 %, ANIm 84.1 %) were below the cut-off level (95-96  %) for species delineation. The results of kegg analysis revealed that strain B3227T could biosynthesize shikimate acid, a base compound for the formulation of the swine flu drug. Based on its morphological and physiological properties, as well as phylogenetic distinctiveness, strain B3227T should be placed into the genus Halalkalibacillus as a representative of a new species, for which the name Halalkalibacillus sediminis sp. nov. is proposed. The type strain is B3227T (=KCTC 33093T=MCCC 1H00193T).


Assuntos
Bacillaceae/classificação , Sedimentos Geológicos/microbiologia , Filogenia , Pepinos-do-Mar/microbiologia , Água do Mar/microbiologia , Animais , Bacillaceae/isolamento & purificação , Técnicas de Tipagem Bacteriana , Composição de Bases , China , DNA Bacteriano/genética , Ácido Diaminopimélico/química , Ácidos Graxos/química , Peptidoglicano/química , Fosfatidiletanolaminas/química , Fosfatidilgliceróis/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Vitamina K 2/análogos & derivados , Vitamina K 2/química
10.
PLoS One ; 14(12): e0226072, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31800629

RESUMO

Pulmonary surfactant preparations extracted from natural sources have been used to treat millions of newborn babies with respiratory distress syndrome (RDS) and can possibly also be used to treat other lung diseases. Due to costly production and limited supply of animal-derived surfactants, synthetic alternatives are attractive. The water insolubility and aggregation-prone nature of the proteins present in animal-derived surfactant preparations have complicated development of artificial surfactant. A non-aggregating analog of lung surfactant protein C, SP-C33Leu is used in synthetic surfactant and we recently described an efficient method to produce rSP-C33Leu in bacteria. Here rSP-C33Leu obtained by salt precipitation of bacterial extracts was purified by two-step liquid gel chromatography and analyzed using mass spectrometry and RP-HPLC, showing that it is void of modifications and adducts. Premature New Zealand White rabbit fetuses instilled with 200mg/kg of 2% of rSP-C33Leu in phospholipids and ventilated with a positive end expiratory pressure showed increased tidal volumes and lung gas volumes compared to animals treated with phospholipids only. This shows that rSP-C33Leu can be purified from bacterial lipids and that rSP-C33Leu surfactant is active against experimental RDS.


Assuntos
Lipopeptídeos/química , Pulmão/efeitos dos fármacos , Surfactantes Pulmonares/farmacologia , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Cromatografia Líquida , Feminino , Lipopeptídeos/genética , Lipopeptídeos/metabolismo , Pulmão/fisiologia , Espectrometria de Massas , Fosfatidilgliceróis/química , Fosfolipídeos/farmacologia , Respiração com Pressão Positiva , Gravidez , Surfactantes Pulmonares/química , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Volume de Ventilação Pulmonar/efeitos dos fármacos
11.
Chem Commun (Camb) ; 55(100): 15141-15144, 2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31789329

RESUMO

The terahertz dynamics of water nanodroplets confined in phospholipid reverse micelles have been developed as a probe to explore the interactions of metal ions with lipid membrane interfaces. The terahertz absorption coefficients of reverse micelles vary greatly with different Cu2+ concentrations and with different headgroups. The results proved that the metal ion effects on water dynamics near a membrane surface can be detected via terahertz spectroscopy, thus providing a novel tool for investigating ion-lipid interactions.


Assuntos
Micelas , Fosfolipídeos/química , Cobre/química , Íons/química , Nanoestruturas/química , Fosfatidilgliceróis/química , Espectroscopia Terahertz
12.
J Chromatogr Sci ; 58(1): 53-59, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31867607

RESUMO

A high-performance liquid chromatography method with evaporative light-scattering detection (ELSD) was performed for simultaneous determination of dipalmitoyl phosphatidylglycerol (DPPG), dierucoyl phosphatidylcholine (DEPC) and cholesterol in propofol liposome by the pretreatment of alkaline hydrolysis (temperature, concentration of KOH anhydrous ethanol solution and reaction time were 90°C, 1 mol · L-1 and 10 min, respectively). The analysis was carried out on an Agilent TC-C18 column (4.6 mm × 250 mm, 5 µm) with isocratic elution of methanol and 0.1% acetic acid aqueous solution (95:5, v/v) at a flow rate of 1.0 mL · min-1. The column temperature was 30°C. The drift tube temperature of the ELSD system was set at 30°C, and the pressure of carrier gas was 350 KPa. The regression equation revealed a good linear relationship (r = 0.9990-0.9993) during the test ranges. The RSD of stability and repeatability (n = 6) was found to be less than 1.96 and 1.46%, respectively. The average recoveries ranged from 97.90 to 101.00%. The proposed method was validated and showed good precision, stability, repeatability and recovery, which indicated that the method could be readily utilized as a quality evaluation method for the determination of DPPG, DEPC and cholesterol in propofol liposome.


Assuntos
Colesterol/análise , Cromatografia Líquida de Alta Pressão/métodos , Lipossomos/química , Fosfatidilcolinas/análise , Fosfatidilgliceróis/análise , Propofol/química , Hidrólise , Reprodutibilidade dos Testes , Temperatura
13.
Elife ; 82019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31724949

RESUMO

Pentameric ligand-gated ion channels (pLGICs) are essential determinants of synaptic transmission, and are modulated by specific lipids including anionic phospholipids. The exact modulatory effect of anionic phospholipids in pLGICs and the mechanism of this effect are not well understood. Using native mass spectrometry, coarse-grained molecular dynamics simulations and functional assays, we show that the anionic phospholipid, 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG), preferentially binds to and stabilizes the pLGIC, Erwinia ligand-gated ion channel (ELIC), and decreases ELIC desensitization. Mutations of five arginines located in the interfacial regions of the transmembrane domain (TMD) reduce POPG binding, and a subset of these mutations increase ELIC desensitization. In contrast, a mutation that decreases ELIC desensitization, increases POPG binding. The results support a mechanism by which POPG stabilizes the open state of ELIC relative to the desensitized state by direct binding at specific sites.


Assuntos
Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Fosfatidilgliceróis/metabolismo , Regulação Alostérica , Análise Mutacional de DNA , Canais Iônicos de Abertura Ativada por Ligante/química , Canais Iônicos de Abertura Ativada por Ligante/genética , Espectrometria de Massas , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Transmissão Sináptica
14.
Commun Biol ; 2: 402, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31701030

RESUMO

Human ß-defensins (hBD) play central roles in antimicrobial activities against various microorganisms and in immune-regulation. These peptides perturb phospholipid membranes for function, but it is not well understood how defensins approach, insert and finally disrupt membranes on the molecular level. Here we show that hBD-3 analogs interact with lipid bilayers through a conserved surface that is formed by two adjacent loops in the solution structure. By integrating a collection of 13C, 1H and 31P solid-state NMR methods with long-term molecular dynamic simulations, we reveal that membrane-binding rigidifies the peptide, enhances structural polymorphism, and promotes ß-strand conformation. The peptide colocalizes with negatively charged lipids, confines the headgroup motion, and deforms membrane into smaller, ellipsoidal vesicles. This study designates the residue-specific, membrane-bound topology of hBD-3 analogs, serves as the basis for further elucidating the function-relevant structure and dynamics of other defensins, and facilitates the development of defensin-mimetic antibiotics, antifungals, and anti-inflammatories.


Assuntos
beta-Defensinas/química , Sequência de Aminoácidos , Sítios de Ligação , Isótopos de Carbono/química , Humanos , Hidrogênio/química , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Isótopos de Nitrogênio/química , Ressonância Magnética Nuclear Biomolecular/métodos , Fosfatidilgliceróis/química , Ligação Proteica , Conformação Proteica em Folha beta , Estabilidade Proteica , beta-Defensinas/genética
15.
Phys Rev E ; 100(3-1): 032404, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31640048

RESUMO

We have studied the kinetics of the interaction between antimicrobial peptide nisin and Langmuir monolayers of phospholipids DPPC and DPPG at the air-water interface using the surface manometry technique. The charge on the nisin and the lipid molecules is controlled by varying the pH of the subphase, and the interactions between them are studied by measuring the surface pressure of the lipid monolayer as a function of time after injecting the nisin in the subphase. A model based on the diffusion of particles under the influence of a constant force is developed to obtain an analytical expression for surface pressure as a function of time. The expression was found to fit well with the experimental data. The average hydrodynamic radius and the translational diffusion constant of the nisin molecules are calculated from the fit parameters for the different subphase pH solutions.


Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Nisina/química , Fosfatidilgliceróis/química , Ar , Difusão , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Conformação Molecular , Pressão , Ligação Proteica , Água/química
16.
Anal Chim Acta ; 1084: 60-70, 2019 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-31519235

RESUMO

Bis(monoacylglycero)phosphate (BMP) and phosphatidylglycerol (PG) are structural isomeric phospholipids with very different properties and biological functions. Due to their isomeric nature, it has thus far been challenging to simultaneously quantify BMP and PG lipids in tissue samples by mass spectrometry. Therefore, we have developed a sensitive LC-MS/MS based approach with prior methylation derivatization that is able to handle large batches of samples. Using this high throughput platform, a simulated MS/MS database was established for confident lipid assignment. In this work, we have simultaneously identified and quantified BMP and PG lipid molecules in different body tissues of rats and mice. We report for the first time a quantitative molecular atlas of BMP and PG lipids for 14 different tissues and organs in Wistar rats, NMRI and CD1 mice. Organ- and species-specificity was analyzed and compared for both lipid molecule classes. A total of 34 BMP and 10 PG molecules were quantified, with PG concentrations being generally much higher across tissues than BMP, but BMP lipids showing a much higher molecular diversity between animal organs. The large diversity of the BMP lipids with regard to their abundance and molecular composition suggests distinct biological function(s) of the individual BMP molecules in different tissues and organs of body. Particularly high tissue levels of BMP were seen in spleen, lung, liver, kidney and small intestines, i.e. tissues that are known for their high abundance and/or activity level of lysosomes late and endosomes. Elevated BMP levels in brain tissue of APP/PSEN transgenic compared to age matched wild-type mice were also observed using this platform. This analytical methodology presented a high throughput LC-based approach incorporating simulated MS/MS database to identify and quantify BMP lipids as well as PG molecules.


Assuntos
Lisofosfolipídeos/análise , Lipídeos de Membrana/química , Monoglicerídeos/análise , Fosfatidilgliceróis/análise , Animais , Cromatografia Líquida , Masculino , Lipídeos de Membrana/isolamento & purificação , Metilação , Camundongos , Camundongos Endogâmicos , Ratos , Ratos Wistar , Espectrometria de Massas em Tandem
17.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1864(12): 158522, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31487556

RESUMO

Organisms use various adaptive strategies against phosphate stress, including lipid remodeling. Here, the response of major membrane lipids to phosphate stress was analyzed in Synechococcus sp. PCC 7942. Unlike plants and eukaryotic microalgae, no significant increases in neutral lipids were found, whereas glycolipids content increased to as high as 6.13% (of dry cell weight, DCW) and phospholipids decreased to 0.34% (of DCW) after 16 days of cultivation without phosphate. Glycolipids accumulation were mainly attributed to the significant increase of digalactosyldiacylglycerol (DGDG) by 50% and sulfoquinovosyldiaclglycerol (SQDG) by 90%, both of which acted as complementary lipids for phosphatidylglycerol (PG) in the cyanobacterial membrane. Also, a notable increase in content (by 48%) of C18 fatty acids (especially C18:1) was observed in all glycolipids at the expense of C12 and C14 (72%). These changes may contribute to membrane fluidity and photosynthetic activity for basic cell metabolism and phosphate stress adaptation. Lipidomic analyses showed the reduction of PG 18:1/16: 0 (by 52%) with the increase of DGDG 18:1/16:0 (133%) and SQDG 18:1/16:0 (245%), strongly suggesting a direct conversion of PG to DGDG and SQDG. Moreover, the decreasing amount of monogalactosyldiacylglycerol (MGDG) 16:1/16:0 (22%) was consistent with the increase of free fatty acids (125%) on day 2 of phosphate absence, which suggested that MGDG is more likely to provide a pool of fatty acids for de novo synthesis of glycolipids. This study provides valuable insight into cyanobacteria adaptation strategies to phosphate stress by membrane lipid remodeling and unveils the underlying acyl chain fluxes into glycolipids.


Assuntos
Glicolipídeos/metabolismo , Lipídeos de Membrana/metabolismo , Fosfatos/metabolismo , Synechococcus/metabolismo , Galactolipídeos/metabolismo , Lipidômica , Fosfatidilgliceróis/metabolismo
18.
J Microbiol ; 57(11): 953-958, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31463785

RESUMO

A strictly aerobic, motile, endospore-forming, rod-shaped bacterium, designated HS21T, was isolated from rhizospheric soil of the Korean fir tree (Abies koreana) from Halla mountain on Jeju island, Korea. Growth of strain HS21T was observed at pH 6.0-8.0 (optimum: pH 7.0), 0-2% (w/v) NaCl and 4-30°C (optimum: 25°C). A comparative analysis of 16S rRNA gene sequences showed that strain HS21T was most closely related to Cohnella luojiensis HY-22RT (97.6%), followed by C. lupini RLAHU4BT (97.4%) and C. collisoli NKM-5T (97.2%). The genome of strain HS21T comprised a circular chromosome of 7,059,027 bp with 44.8% G + C content. The DNA-DNA relatedness values between strain HS21T and C. luojiensis HY-22RT and C. lupini RLAHU4BT were 18.1% and 13.8%, respectively. The major cellular fatty acids (> 5%) of the isolate were anteiso-C15:0, iso-C16:0, C16:0, and iso-C15:0. The polar lipids present were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, lysylphosphatidylglycerol, and three unidentified aminophospholipids. Based on its phenotypic, phylogenetic, genomic, and chemotaxonomic properties, strain HS21T represents a novel species of the genus Cohnella, for which the name Cohnella abietis sp. nov. is proposed. The type strain is HS21T (= KCTC 43028T = CCTCC AB 2019010T).


Assuntos
Abies/microbiologia , Bacillales/classificação , Bacillales/isolamento & purificação , Filogenia , Rizosfera , Microbiologia do Solo , Bacillales/genética , Bacillales/fisiologia , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Lipídeos/química , Lisina/química , Fosfatidilgliceróis/química , Fosfolipídeos/química , RNA Ribossômico 16S/genética , República da Coreia , Nódulos Radiculares de Plantas/microbiologia , Análise de Sequência de DNA , Solo , Sequenciamento Completo do Genoma
19.
Eur J Pharm Biopharm ; 143: 70-79, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31446045

RESUMO

Controlled drug delivery to the lungs is promising with plentiful advantages over current rapid release products. However, alveolar macrophage clearance has severely hindered the application of inhaled controlled release preparations. The objective of our study was to explore the feasibility to decorate poly(lactide-co-glycolide) (PLGA) microparticles with endogenous phospholipids found in the deep lungs, thus, to regulate the interplay with alveolar macrophages. The influence of the phospholipid amount and type on macrophage uptake of PLGA microparticles was investigated systemically under both in vitro (RAW264.7 and NR8383) and in vivo conditions. The uptake rate (k) by macrophages, in vivo elimination rate from the bronchoalveolar lavage fluid (k') and elimination rate from the whole lung (k″) were used as parameters for evaluation. Our data showed that a modification with dipalmitoyl phosphatidylcholine (DPPC) enhanced the macrophage phagocytosis significantly over the unmodified counterparts. Thereafter, using the same modification ratio, remarkable enhancement of macrophage uptake was found in the presence of different types of other phospholipids, especially with distearoyl phosphatidylethanolamine (DSPE). When replaced by a poly(ethylene glycol)-conjugated version of DSPE the uptake of the modified PLGA microparticles was reduced by ~200%. Meanwhile, the drug content in the lung tissue was improved by 3-fold (area under the curve value). Finally, it was possible to establish a correlation between in vitro phagocytosis and in vivo lung elimination rate for the investigated formulations. Overall, our study demonstrated that phospholipids play an important role in modulating the clearance of microparticle-based drug delivery vehicles, which gives a meaningful insight into the development of prolonged drug release system for inhalation.


Assuntos
Macrófagos Alveolares/metabolismo , Fosfolipídeos/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , 1,2-Dipalmitoilfosfatidilcolina/química , Administração por Inalação , Animais , Linhagem Celular , Preparações de Ação Retardada/química , Sistemas de Liberação de Medicamentos/métodos , Pulmão/metabolismo , Camundongos , Fagocitose/efeitos dos fármacos , Fosfatidilgliceróis/química , Polietilenoglicóis/química , Células RAW 264.7
20.
Int J Pharm ; 569: 118603, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31401296

RESUMO

Conventional quantitation of drug content in the liposome formulation involves the breakdown of bulk liposomes, which ignores details on the distribution of the active pharmaceutical ingredient (API) and excipients in liposomes of different sizes. The objective of this study is to develop an analytical method which can separate the liposomes into different sizes and obtain information of the drug and excipient distribution in the different sized liposomes. We developed an asymmetric flow field-flow fractionation (AF4) method for size-based separation of AmBisome, an amphotericin B liposomal formulation, and a high-performance liquid chromatography ultraviolet-visible and charged aerosol detection (HPLC-UV-CAD) method for simultaneous quantitation of the API (Amphotericin B) and the lipid excipients [1,2-Distearoyl-sn-glycero-3-phosphoglycerol (DSPG), hydrogenated soy phosphatidylcholine (HSPC), and cholesterol]. The measured drug content in the bulk liposome formulation was consistent with the drug product labeling. Liposomes were separated using AF4 into eleven size fractions and the liposomes particles sizes of each fraction were measured with nanoparticle tracking analysis. The drug to total lipid ratios in fractionated liposomes increased from 0.1 to 0.45 when the liposome size increased from 75 nm to 124 nm, while the lipid composition remained constant throughout the fractioned size range (cholesterol:DSPG, 0.7 and HSPC:DSPG, 0.3). These study results suggest that, for liposomal formulations of Amphotericin B in liposomes, the drug to lipid ratio increases with the size of the liposomes. This new analytical method provided a more in-depth characterization of liposomes, i.e., determining drug and excipient distributions in different sizes of liposomes, in a more efficient manner with more specific size-based composition information.


Assuntos
Anfotericina B/química , Excipientes/química , Colesterol/química , Nanopartículas/química , Tamanho da Partícula , Fosfatidilcolinas/química , Fosfatidilgliceróis/química
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