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1.
Nutrients ; 15(2)2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36678216

RESUMO

Glucosylceramide (GlcCer) synthesis by the enzyme glucosylceramide synthase (GCS) occurs on the cytosolic leaflet of the Golgi and is the first important step for the synthesis of complex glycosphingolipids (GSLs) that takes place inside the lumen. Apart from serving as a precursor for glycosylation, newly synthesized GlcCer is also transported to the plasma membrane and secreted onto HDL in the circulation. The mechanism by which GlcCer is transported to HDL remains unclear. Recently, we showed that ATP-binding cassette transporter protein C10 (ABCC10) plays an important role in the synthesis and efflux of GlcCer in Huh-7 cells. In this study, we found that treatment of Huh-7 cells with an ABCC10 inhibitor, sorafenib, decreased the synthesis and efflux of GlcCer. However, treatment of cells with cepharanthine reduced only the efflux, but not synthesis, of GlcCer. These results indicate that ABCC10 may regulate the synthesis and efflux of GlcCer differentially in liver cells.


Assuntos
Glucosilceramidas , Complexo de Golgi , Transporte Biológico/fisiologia , Membrana Celular/metabolismo , Glicoesfingolipídeos/metabolismo , Complexo de Golgi/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo
2.
Nat Commun ; 14(1): 465, 2023 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-36709325

RESUMO

Oncogenic KRAS expression generates a metabolic dependency on aerobic glycolysis, known as the Warburg effect. We report an effect of increased glycolytic flux that feeds into glycosphingolipid biosynthesis and is directly linked to KRAS oncogenic function. High resolution imaging and genetic approaches show that a defined subset of outer leaflet glycosphingolipids, including GM3 and SM4, is required to maintain KRAS plasma membrane localization, with GM3 engaging in cross-bilayer coupling to maintain inner leaflet phosphatidylserine content. Thus, glycolysis is critical for KRAS plasma membrane localization and nanoscale spatial organization. Reciprocally oncogenic KRAS selectively upregulates cellular content of these same glycosphingolipids, whose depletion in turn abrogates KRAS oncogenesis in pancreatic cancer models. Our findings expand the role of the Warburg effect beyond ATP generation and biomass building to high-level regulation of KRAS function. The positive feedforward loop between oncogenic KRAS signaling and glycosphingolipid synthesis represents a vulnerability with therapeutic potential.


Assuntos
Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Membrana Celular/metabolismo , Transdução de Sinais , Glicólise , Glicoesfingolipídeos/metabolismo
3.
Methods Mol Biol ; 2613: 271-288, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36587085

RESUMO

Glucosylceramide synthase can be targeted by high affinity small molecular weight inhibitors for the study of glycosphingolipid metabolism and function or for the treatment of glycosphingolipid storage disorders, including Gaucher and Fabry disease. This work is exemplified by the discovery and development of eliglustat tartrate, the first stand-alone small chemical entity approved for the treatment of Gaucher disease type 1. The development of inhibitors of glucosylceramide synthase that have utility for either research or clinical purposes begins with a testing funnel for screening candidate inhibitors for activity against this enzyme and for activity in lowering the content of glucosylceramide in intact cells. Two common assays for glucosylceramide synthase, one enzyme based and another cell based, are the focus of this chapter.


Assuntos
Doença de Gaucher , Humanos , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/metabolismo , Glucosiltransferases/metabolismo , Glucosilceramidas , Glicoesfingolipídeos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico
4.
Adv Neurobiol ; 29: 281-304, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255679

RESUMO

Gangliosides are sialylated glycosphingolipids (GSLs) with essential but enigmatic functions in brain activities and neural stem cell (NSC) maintenance. Our group has pioneered research on the importance of gangliosides for growth factor receptor signaling and epigenetic regulation of NSC activity and differentiation. The primary localization of gangliosides is on cell-surface microdomains and the drastic dose and composition changes during neural differentiation strongly suggest that they are not only important as biomarkers, but also are involved in modulating NSC fate determination. Ganglioside GD3 is the predominant species in NSCs and GD3-synthase knockout (GD3S-KO) revealed reduction of postnatal NSC pools with severe behavioral deficits. Exogenous administration of GD3 significantly restored the NSC pools and enhanced the stemness of NSCs with multipotency and self-renewal. Since morphological changes during neurogenesis require a huge amount of energy, mitochondrial functions are vital for neurogenesis. We discovered that a mitochondrial fission protein, the dynamin-related protein-1 (Drp1), as a novel GD3-binding protein, and GD3 regulates mitochondrial dynamics. Furthermore, we discovered that GM1 ganglioside promotes neuronal differentiation by an epigenetic regulatory mechanism. Nuclear GM1 binds with acetylated histones on the promoters of N-acetylgalactosaminyltransferase (GalNAcT; GM2 synthase) as well as on the NeuroD1 genes in differentiated neurons. In addition, epigenetic activation of the GalNAcT gene was detected as accompanied by an apparent induction of neuronal differentiation in NSCs responding to an exogenous supplement of GM1. GM1 is indeed localized in the nucleus where it can interact with transcriptionally active histones. Interestingly, GM1 could induce epigenetic activation of the tyrosine hydroxylase (TH) gene, with recruitment of nuclear receptor related 1 (Nurr1, also known as NR4A2), a dopaminergic neuron-associated transcription factor, to the TH promoter region. In this way, GM1 epigenetically regulates dopaminergic neuron specific gene expression. GM1 interacts with active chromatin via acetylated histones to recruit transcription factors at the nuclear periphery, resulting in changes in gene expression for neuronal differentiation. The significance is that multifunctional gangliosides modulate lipid microdomains to regulate functions of important molecules on multiple sites: the plasma membrane, mitochondrial membrane, and nuclear membrane. Versatile gangliosides could regulate functional neurons as well as sustain NSC functions via modulating protein and gene activities on ganglioside microdomains.


Assuntos
Gangliosídeo G(M1) , N-Acetilgalactosaminiltransferases , Humanos , Gangliosídeo G(M1)/metabolismo , Epigênese Genética , Histonas/genética , Histonas/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Gangliosídeos/genética , Gangliosídeos/metabolismo , Neurônios/metabolismo , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Glicoesfingolipídeos/metabolismo , Membranas Intracelulares/metabolismo , Biomarcadores/metabolismo , Cromatina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
EMBO J ; 42(2): e110553, 2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36504224

RESUMO

Epithelial-mesenchymal transition (EMT) is pivotal in the initiation and development of cancer cell metastasis. We observed that the abundance of glycosphingolipids (GSLs), especially ganglioside subtypes, decreased significantly during TGF-ß-induced EMT in NMuMG mouse mammary epithelial cells and A549 human lung adenocarcinoma cells. Transcriptional profiling showed that TGF-ß/SMAD response genes and EMT signatures were strongly enriched in NMuMG cells, along with depletion of UDP-glucose ceramide glucosyltransferase (UGCG), the enzyme that catalyzes the initial step in GSL biosynthesis. Consistent with this finding, genetic or pharmacological inhibition of UGCG promoted TGF-ß signaling and TGF-ß-induced EMT. UGCG inhibition promoted A549 cell migration, extravasation in the zebrafish xenograft model, and metastasis in mice. Mechanistically, GSLs inhibited TGF-ß signaling by promoting lipid raft localization of the TGF-ß type I receptor (TßRI) and by increasing TßRI ubiquitination and degradation. Importantly, we identified ST3GAL5-synthesized a-series gangliosides as the main GSL subtype involved in inhibition of TGF-ß signaling and TGF-ß-induced EMT in A549 cells. Notably, ST3GAL5 is weakly expressed in lung cancer tissues compared to adjacent nonmalignant tissues, and its expression correlates with good prognosis.


Assuntos
Neoplasias Pulmonares , Fator de Crescimento Transformador beta , Humanos , Animais , Camundongos , Fator de Crescimento Transformador beta/metabolismo , Gangliosídeos , Transição Epitelial-Mesenquimal/genética , Peixe-Zebra/metabolismo , Neoplasias Pulmonares/metabolismo , Glicoesfingolipídeos , Catálise , Movimento Celular , Linhagem Celular Tumoral
6.
Methods Mol Biol ; 2613: 111-125, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36587075

RESUMO

Sphingolipids are ubiquitously expressed in eukaryotes and play various functional roles. The key characteristic of sphingolipids is their diversity of molecular species. Sphingomyelin (SM) and glycosphingolipids (GSLs) are the major components of sphingolipids in the plasma membrane, which are composed of ceramide and a polar head-group. SM is the most abundant sphingolipid species in mammalian cells, while GSLs have a wide variety of glycans as head groups. Various fatty acids in ceramide also contribute to the diversity of sphingolipid species. To analyze the cellular function of each sphingolipid species, precise gene manipulation is essential. Recent developments in genome editing technologies have facilitated complete gene disruption in cultured cells. This chapter describes protocols for the construction of various sphingolipid-related gene knockout HeLa cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system and for confirmation of changes in their lipid composition using radioisotopes and thin layer chromatography. This sphingolipid-remodeled cell panel is a useful tool for analyzing the cellular functions of sphingolipid species and as a reference for lipid analysis.


Assuntos
Edição de Genes , Esfingolipídeos , Animais , Humanos , Esfingolipídeos/metabolismo , Edição de Genes/métodos , Células HeLa , Ceramidas/metabolismo , Esfingomielinas/metabolismo , Glicoesfingolipídeos , Sistemas CRISPR-Cas , Mamíferos/metabolismo
7.
Methods Mol Biol ; 2613: 127-144, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36587076

RESUMO

This chapter describes the protocols for mass spectrometry (MS) applied to the structural characterization of neutral glycosphingolipids (GSLs) and the determination of neutral GSL contents in biological materials. The structural characterization is performed by thin layer chromatography-matrix assisted laser desorption ionization/mass spectrometry (TLC-MALDI/MS) and liquid chromatography-electrospray ionization/mass spectrometry (LC-ESI/MS) with reversed phase separation. The content determination is carried out by LC-ESI/MS with multiple reaction monitoring (MRM). These protocols provide clues for the functions of neutral GSLs at the level of a single GSL molecular species.


Assuntos
Glicoesfingolipídeos Neutros , Glicoesfingolipídeos Neutros/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas por Ionização por Electrospray , Cromatografia Líquida , Cromatografia em Camada Delgada/métodos , Glicoesfingolipídeos/química
8.
Methods Mol Biol ; 2613: 203-214, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36587081

RESUMO

Glycosphingolipids (GSLs) form GSL-enriched microdomains, together with sphingomyelin (SM), cholesterol, glycosylphosphatidylinositol (GPI)-anchored proteins, and membrane-associated signaling molecules. GSL-enriched microdomains mediate a variety of physiological functions, including innate immune responses. Innate immune responses are initialized by the binding of host pattern recognition receptors (PRRs) to pathogen-associated molecular patterns (PAMPs) expressed in microorganisms. This binding triggers phagocytosis and leads to the formation of a phagosome-containing microorganism and the subsequent lysosomal fusion with a phagosome. To detect the molecular interaction between GSL-enriched microdomains, sphingolipids, and signaling molecules from the uptake of the microorganism until the phagosome-containing microorganism fuses with lysosomes, biochemical and microscopic approaches are indispensable. Here, we describe the detailed methods for isolating phagosomes and observing the molecular interaction using a superresolution microscope. Our methodology provides a strategy for exploring the molecular interaction between the host and pathogen and for developing new treatment approaches.


Assuntos
Imunidade Inata , Esfingolipídeos , Fagocitose , Glicoesfingolipídeos/metabolismo , Fagossomos/metabolismo , Glicosilfosfatidilinositóis
9.
Methods Mol Biol ; 2613: 257-270, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36587084

RESUMO

Glycosphingolipids (GSLs) in the mammalian plasma membrane are essential for various biological events as they form glycolipid-rich membrane domains, such as lipid rafts. GSLs consist of a certain oligosaccharide head group and a ceramide tail with various lengths of acyl chains. The structure of the head group as well as the carbon number and degree of the unsaturation of the acyl chain are known to regulate the membrane distributions and interleaflet couplings of GSLs by altering physicochemical properties, such as dynamics, interactions, and cluster sizes. This chapter provides the detailed use of time-resolved fluorescence measurement for investigating the membrane properties of lactosylceramide (LacCer)-enriched domains in bilayer membranes. LacCer belongs to the neutral GSLs and is believed in forming a highly ordered phase in model membranes and biological membranes, while the details of the domain remain unclear. Here, we suggest using trans-parinaric acid (tPA) and tPA-LacCer fluorescent probes to reveal the dynamics and size of the GSL domains since they prefer to be distributed in the GSL-rich ordered phase. The fluorescence lifetime in the nanosecond timescale reveals the difference in the surrounding membrane environments, which relates to hydrocarbon chain ordering, membrane hydration, and submicrometer domain size. The fluorescence lifetime of these probes can thus provide important information on submicron- to nano-scale small GSL domains not only in model membranes but also in biological membranes.


Assuntos
Glicolipídeos , Lipossomos , Animais , Simulação de Dinâmica Molecular , Glicoesfingolipídeos/metabolismo , Membrana Celular/metabolismo , Lactosilceramidas , Mamíferos/metabolismo
10.
Methods Mol Biol ; 2613: 229-256, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36587083

RESUMO

Microbial glycosphingolipid (GSL)-degrading enzymes with unique specificity are useful tools for GSL research. On the other hand, some microbial glycolipids, not only GSLs but also steryl glucosides, are closely related to pathogenicity, and, thus, the metabolism of microbial glycolipids is attracting attention as a target for antibiotics. This chapter describes the assays and utilization of microbial enzymes useful for glycolipid research and those involved in pathogenicity or host immune reactions.


Assuntos
Glicolipídeos , Glicoesfingolipídeos , Glicolipídeos/metabolismo , Glicoesfingolipídeos/metabolismo , Fungos/metabolismo , Bactérias/metabolismo
11.
Methods Mol Biol ; 2613: 289-299, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36587086

RESUMO

Glycosphingolipids (GSLs) are glycolipids with ceramide and carbohydrate head groups that play an important role in numerous biological processes. Previously, we performed GSL-glycan analysis of various cell lines and virus-infected cells using a glycoblotting approach. Recently, we developed several methods for sialic acid linkage-specific chemical modification to distinguish sialylated glycan isomers by mass spectrometry. In this chapter, we describe a method for analyzing GSL-glycans in human serum/plasma using glycoblotting combined with aminolysis-SALSA (sialic acid linkage-specific alkylamidation) and lactone-driven ester-to-amide derivatization (LEAD)-SALSA for comprehensive and detailed structural glycomics.


Assuntos
Ácido N-Acetilneuramínico , Esfingolipídeos , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Glicoesfingolipídeos/metabolismo , Polissacarídeos/química
12.
Int J Mol Sci ; 23(23)2022 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-36499769

RESUMO

Diabetes contributes to about 30% morbidity and mortality world-wide and has tidal wave increases in several countries in Asia. Diabetes is a multi-factorial disease compounded by inflammation, dyslipidemia, atherosclerosis, and is sometimes accompanied with gains in body weight. Sphingolipid pathways that interplay in the enhancement of the pathology of this disease may be potential therapeutic targets. Thus, the application of advanced sphingolipidomics may help predict the progression of this disease and therapeutic outcomes in man. Pre-clinical studies using various experimental animal models of diabetes provide valuable information on the role of sphingolipid signaling networks in diabetes and the efficacy of drugs to determine the translatability of innovative discoveries to man. In this review, we discuss three major concepts regarding sphingolipids and diabetes. First, we discuss a possible involvement of a monosialodihexosylceramide (GM3) in insulin-insulin receptor interactions. Second, a potential role for ceramide (Cer) and lactosylceramide (LacCer) in apoptosis and mitochondrial dysfunction is proposed. Third, a larger role of LacCer in antioxidant status and inflammation is discussed. We also discuss how inhibitors of glycosphingolipid synthesis can ameliorate diabetes in experimental animal models.


Assuntos
Doenças Cardiovasculares , Diabetes Mellitus , Animais , Glicoesfingolipídeos/metabolismo , Doenças Cardiovasculares/prevenção & controle , Esfingolipídeos/metabolismo , Lactosilceramidas/metabolismo , Estresse Oxidativo , Inflamação , Modelos Animais
13.
Nat Commun ; 13(1): 7868, 2022 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-36550101

RESUMO

Patients with multiple myeloma, an incurable malignancy of plasma cells, frequently develop osteolytic bone lesions that severely impact quality of life and clinical outcomes. Eliglustat, a U.S. Food and Drug Administration-approved glucosylceramide synthase inhibitor, reduced osteoclast-driven bone loss in preclinical in vivo models of myeloma. In combination with zoledronic acid, a bisphosphonate that treats myeloma bone disease, eliglustat provided further protection from bone loss. Autophagic degradation of TRAF3, a key step for osteoclast differentiation, was inhibited by eliglustat as evidenced by TRAF3 lysosomal and cytoplasmic accumulation. Eliglustat blocked autophagy by altering glycosphingolipid composition whilst restoration of missing glycosphingolipids rescued autophagy markers and TRAF3 degradation thus restoring osteoclastogenesis in bone marrow cells from myeloma patients. This work delineates both the mechanism by which glucosylceramide synthase inhibition prevents autophagic degradation of TRAF3 to reduce osteoclastogenesis as well as highlighting the clinical translational potential of eliglustat for the treatment of myeloma bone disease.


Assuntos
Doenças Ósseas , Mieloma Múltiplo , Humanos , Mieloma Múltiplo/patologia , Fator 3 Associado a Receptor de TNF/metabolismo , Qualidade de Vida , Osteoclastos/metabolismo , Doenças Ósseas/tratamento farmacológico , Doenças Ósseas/metabolismo , Autofagia , Glicoesfingolipídeos/metabolismo
14.
Anal Chem ; 94(46): 16042-16049, 2022 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-36367338

RESUMO

Interactions between glycan-binding proteins (GBPs) and glycosphingolipids (GSLs) are involved in numerous physiological and pathophysiological processes. Many model membrane systems are available for studying GBP-GSL interactions, but a systematic investigation has not been carried out on how the nature of the model membrane affects binding. In this work, we use electrospray ionization mass spectrometry (ESI-MS), both direct and competitive assays, to measure the binding of cholera toxin B subunit homopentamer (CTB5) to GM1 ganglioside in liposomes, bilayer islands [styrene maleic acid lipid particles (SMALPs), nanodiscs (NDs), and picodiscs (PDs)], and micelles. We find that direct ESI-MS analysis of CTB5 binding to GM1 is unreliable due to non-uniform response factors, incomplete extraction of bound GM1 in the gas phase, and nonspecific CTB5-GM1 interactions. Conversely, indirect proxy ligand ESI-MS measurements show that the intrinsic (per binding site) association constants of CTB5 for PDs, NDs, and SMALPs are similar and comparable to the affinity of soluble GM1 pentasaccharide (GM1os). The observed affinity decreases with increasing GM1 content due to molecular crowding stemming from GM1 clustering. Unlike the smaller model membranes, the observed affinity of CTB5 toward GM1 liposomes is ∼10-fold weaker than GM1os and relatively insensitive to the GM1 content. GM1 glycomicelles exhibit the lowest affinity, ∼35-fold weaker than GM1os. Together, the results highlight experimental design considerations for quantitative GBP-GSL binding studies involving multisubunit GBPs and factors to consider when comparing results obtained with different membrane systems. Notably, they suggest that bilayer islands with a low percentage of GSL, wherein clustering is minimized, are ideal for assessing intrinsic strength of GBP-GSL interactions in a membrane environment, while binding to liposomes, which is sub-optimal due to extensive clustering, may be more representative of authentic cellular environments.


Assuntos
Gangliosídeo G(M1) , Glicoesfingolipídeos , Glicoesfingolipídeos/química , Gangliosídeo G(M1)/química , Lipossomos , Proteínas/química , Toxina da Cólera/química , Espectrometria de Massas por Ionização por Electrospray/métodos
15.
Int J Mol Sci ; 23(22)2022 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-36430817

RESUMO

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2)-induced disease (COVID-19) and Gaucher disease (GD) exhibit upregulation of complement 5a (C5a) and its C5aR1 receptor, and excess synthesis of glycosphingolipids that lead to increased infiltration and activation of innate and adaptive immune cells, resulting in massive generation of pro-inflammatory cytokines, chemokines and growth factors. This C5a-C5aR1-glycosphingolipid pathway- induced pro-inflammatory environment causes the tissue damage in COVID-19 and GD. Strikingly, pharmaceutically targeting the C5a-C5aR1 axis or the glycosphingolipid synthesis pathway led to a reduction in glycosphingolipid synthesis and innate and adaptive immune inflammation, and protection from the tissue destruction in both COVID-19 and GD. These results reveal a common involvement of the complement and glycosphingolipid systems driving immune inflammation and tissue damage in COVID-19 and GD, respectively. It is therefore expected that combined targeting of the complement and sphingolipid pathways could ameliorate the tissue destruction, organ failure, and death in patients at high-risk of developing severe cases of COVID-19.


Assuntos
Doença de Gaucher , Humanos , Doença de Gaucher/tratamento farmacológico , Esfingolipídeos , SARS-CoV-2 , Proteínas do Sistema Complemento , Complemento C5a/metabolismo , Inflamação , Glicoesfingolipídeos
16.
Molecules ; 27(22)2022 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-36431892

RESUMO

Due to the high propensity of drug resistance in Acinetobacter baumannii, the number of currently available therapeutic drugs has become very limited. Thus, it becomes incredibly important to prepare an effective vaccine formulation capable of eliciting an effective immune response against A. baumannii. In this study, we prepared a liposomal vaccine formulation bearing glycosphingolipids (GSLs) from Sphingomonas paucimobilis and loaded with the whole cell antigen (WCAgs-GSLs-liposomes) of A. baumannii. The immune-stimulating potential and prophylactic efficacy of WCAgs-GSLs-liposomes were compared with those of WCAgs-liposomes (without GSLs) or free WCAgs in both immunocompetent and immunodeficient mice. The efficacy of vaccine formulations was determined by analyzing antibody titer, cytokine levels, and survival studies in the immunized mice. The findings revealed that vaccination with WCAgs-GSLs-liposomes stimulated a greater secretion of antibodies and cytokines, higher lymphocyte proliferation, and increased expression of the co-stimulatory molecules. Anti-sera from WCAgs-GSLs-liposomes-immunized mice remarkably reduced the biofilm formation by A. baumannii. Most importantly, WCAgs-GSLs-liposomes-vaccinated mice demonstrated a higher defiance against the pathogen, as compared to the immunizations with WCAgs-liposomes (without GSLs) or free WCAgs. Immunocompetent mice immunized with WCAgs-GSLs-liposomes showed a 100% survival rate, while those immunized with WCAgs-liposomes exhibited a 60% survival rate. The protective effect of WCAgs-GSLs-liposomes was also found to be higher in immunocompromised mice, as the immunized mice showed a 50% survival rate, which was greater than the 20% survival rate of those immunized with WCAgs-liposomes. The survival data was also supported by the findings of bacterial load and histological analysis that substantiated the greatest prophylactic potential of the WCAgs-GSLs-liposomes. These findings recommend that WCAgs-GSLs-liposomes may be reckoned as a prospective vaccine to protect the persons against A. baumannii infection.


Assuntos
Acinetobacter baumannii , Pneumonia , Camundongos , Animais , Lipossomos , Glicoesfingolipídeos
17.
Int J Mol Sci ; 23(19)2022 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-36232754

RESUMO

Due to the role of cancer stem cells (CSCs) in tumor resistance and glycosphingolipid (GSL) involvement in tumor pathogenesis, we investigated the effect of a newly synthesized compound (3-amino-N-(3-chloro-2-methylphenyl)-5-oxo-5,6,7,8-tetrahydrothieno[2,3-b]quinoline-2-carboxamide 1 on the percentage of CSCs and the expression of six GSLs on CSCs and non-CSCs on breast cancer cell lines (MDA-MB-231 and MCF-7). We also investigated the effect of 1 on the metabolic profile of these cell lines. The MTT assay was used for cytotoxicity determination. Apoptosis and expression of GSLs were assessed by flow cytometry. A GC-MS-coupled system was used for the separation and identification of metabolites. Compound 1 was cytotoxic for both cell lines, and the majority of cells died by treatment-induced apoptosis. The percentage of CSCs was significantly lower in the MDA-MB-231 cell line. Treatment with 1 caused a decrease of CSC IV6Neu5Ac-nLc4Cer+ MDA-MB-231 cells. In the MCF-7 cell line, the percentage of GalNAc-GM1b+ CSCs was increased, while the expression of Gg3Cer was decreased in both CSC and non-CSC. Twenty-one metabolites were identified by metabolic profiling. The major impact of the treatment was in glycolysis/gluconeogenesis, pyruvate and inositol metabolism. Compound 1 exhibited higher potency in MBA-MB-231 cells, and it deserves further examination.


Assuntos
Antineoplásicos , Neoplasias da Mama , Quinolinas , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Glucose/metabolismo , Glicoesfingolipídeos/metabolismo , Humanos , Inositol/farmacologia , Células-Tronco Neoplásicas/metabolismo , Piridinas/metabolismo , Piridinas/farmacologia , Piruvatos/metabolismo , Quinolinas/farmacologia
18.
Int J Syst Evol Microbiol ; 72(10)2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36268862

RESUMO

A Gram-stain-negative, aerobic, motile, rod-shaped bacterium, designated CMS5P-6T, was isolated from a surface-sterilized bark of Aegiceras corniculatum collected from Guangxi Zhuang Autonomous Region, PR China, and investigated by a polyphasic approach to determine its taxonomic position. Strain CMS5P-6T was found to grow optimally with 0-1 % (w/v) NaCl, at 30 °C and pH 6.0-7.0. Substrate mycelia and aerial mycelia were not formed, and no diffusible pigments were observed on the media tested. Phylogenetic analysis showed that strain CMS5P-6T showed high 16S rRNA gene sequence similarity of 96.7 % to Hephaestia caeni DSM 25527T and Sphingomonas colocasiea CC-MHH0539T. The average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity values between strain CMS5P-6T and H. caeni DSM 25527T were 78.0, 21.7 and 70.8 %, respectively. The average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity values between strain CMS5P-6T and S. colocasiea JCM 31229T were 74.0, 19.9 and 61.4 %, respectively. Phylogenomic analyses based on genome sequences showed that strain CMS5P-6T and H. caeni DSM 25527T formed a distinct cluster within the family Sphingomonadaceae and far away from S. colocasiea JCM 31229T. The DNA G+C content of strain CMS5P-6T was determined to be 65.6 mol%. The cell-wall peptidoglycan was found to contain meso-diaminopimelic acid as the diagnostic diamino acid and ubiquinone Q-10 was identified as the respiratory lipoquinone. The polar lipids were found to comprise diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, sphingoglycolipid and two unidentified aminolipids, and the major fatty acids were identified as C18 : 1 ω7c, C19 : 0 cycloω8c and C16 : 0. On the basis of phylogenetic, genomic, chemotaxonomic and phenotypic data, strain CMS5P-6T can be concluded to represent a novel species of the genus Hephaestia, for which the name Hephaestia mangrovi sp. nov. is proposed. The type strain is CMS5P-6T (=JCM 33125T=CGMCC 1.13868T).


Assuntos
Primulaceae , Sphingomonadaceae , RNA Ribossômico 16S/genética , Composição de Bases , Peptidoglicano/química , Ácido Diaminopimélico/química , Filogenia , Fosfatidiletanolaminas , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Ubiquinona/química , Cloreto de Sódio , Cardiolipinas , Ácidos Graxos/química , Análise de Sequência de DNA , Fosfolipídeos/química , China , Primulaceae/microbiologia , Sphingomonadaceae/genética , Fosfatidilcolinas , Nucleotídeos , Glicoesfingolipídeos
19.
Int J Syst Evol Microbiol ; 72(10)2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36260506

RESUMO

A Gram-negative, aerobic, rod-shaped bacterium, designated DM2-R-LB4T was isolated from Cannabis sativa L. 'Cheungsam' in Andong, Republic of Korea. The strain DM2-R-LB4T grew at temperatures of 15-45 °C (optimum, 30-37 °C), pH of 5.5-9 (optimum, 8.0), and 0-2 % (w/v) NaCl concentration (optimum, 0%). Phylogenetic analyses based on the 16S rRNA gene sequences revealed that strain DM2-R-LB4T is related to species of the genus Sphingomonas, and shared 97.8 and 97.5% similarity to Sphingomonas kyenggiensis KCTC 42244T and Sphingomonas leidyi DSM 4733T, respectively. The DNA G+C content was 67.9 mol% and genome analysis of the strain DM2-R-LB4T revealed that the genome size was 4 386 171 bp and contained 4 009 predicted protein-coding genes. The average nucleotide identity (ANI) values between strain DM2-R-LB4T and S. kyenggiensis KCTC 42244T, and S. leidyi DSM 4733T was 76.8 and 76.7 %, respectively, while the values of digital DNA-DNA hybridization (dDDH) were 20.7 and 20.6 %, respectively. C14 : 0 2-OH, C16 : 0, and summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c) were the major fatty acids (>10 %) in the strain DM2-R-LB4T. The polar lipids comprised diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC), sphingoglycolipid (SGL), glycolipid (GL), phospholipid (PL), and two unidentified polar lipids (L1 and L2). Ubiquinone-10 (Q-10) was the only respiratory quinone. The polyamine pattern was found to contain homospermidine, putrescine, and spermidine. The results of phylogenetic anlayses, polyphasic studies, revealed that strain DM2-R-LB4T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas cannabina sp. nov., is proposed. The type strain is DM2-R-LB4T (=KCTC 92075T = GDMCC 1.3018T).


Assuntos
Cannabis , Sphingomonas , RNA Ribossômico 16S/genética , Filogenia , Cannabis/genética , Fosfatidiletanolaminas , Composição de Bases , Ubiquinona/química , Espermidina/química , Microbiologia do Solo , Cloreto de Sódio , Putrescina , Cardiolipinas , DNA Bacteriano/genética , Técnicas de Tipagem Bacteriana , Ácidos Graxos/química , Análise de Sequência de DNA , Fosfolipídeos/química , Glicolipídeos/química , Fosfatidilcolinas , Glicoesfingolipídeos/análise , Nucleotídeos
20.
Cell Rep ; 41(3): 111510, 2022 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-36261008

RESUMO

Septin GTPases polymerize into higher-ordered structures as a part of the cytoskeleton and are involved in interactions of the host with a wide spectrum of pathogens. Many pathogens foster an ambiguous relationship with septins. They exploit septins for uptake, but septins also prevent their intracellular replication and target them for autophagy. We demonstrate that septins are involved in a defense mechanism against the pathogen Pseudomonas aeruginosa, which enters cells via a lipid zippering mechanism relying on interaction of the lectin LecA with the glycosphingolipid Gb3 on the host membrane. LecA-dependent invagination of the plasma membrane triggers septin recruitment to the site of bacterial attachment. We also find a septin-dependent reinforcement of cortical actin at attachment sites. Atomic force microscopy reveals formation of a septin-dependent rigid barrier below the membrane, preventing bacterial penetration. Our data suggest that septin barriers represent a cellular defense against bacteria inducing membrane curvature for invasion.


Assuntos
Pseudomonas aeruginosa , Septinas , Animais , Septinas/metabolismo , Pseudomonas aeruginosa/metabolismo , Actinas/metabolismo , Glicoesfingolipídeos/metabolismo , Lectinas/metabolismo , Mamíferos/metabolismo
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