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International Journal of Molecular... Apr 2024The thermo- and pain-sensitive Transient Receptor Potential Melastatin 3 and 8 (TRPM3 and TRPM8) ion channels are functionally associated in the lipid rafts of the...
The thermo- and pain-sensitive Transient Receptor Potential Melastatin 3 and 8 (TRPM3 and TRPM8) ion channels are functionally associated in the lipid rafts of the plasma membrane. We have already described that cholesterol and sphingomyelin depletion, or inhibition of sphingolipid biosynthesis decreased the TRPM8 but not the TRPM3 channel opening on cultured sensory neurons. We aimed to test the effects of lipid raft disruptors on channel activation on TRPM3- and TRPM8-expressing HEK293T cells in vitro, as well as their potential analgesic actions in TRPM3 and TRPM8 channel activation involving acute pain models in mice. CHO cell viability was examined after lipid raft disruptor treatments and their effects on channel activation on channel expressing HEK293T cells by measurement of cytoplasmic Ca concentration were monitored. The effects of treatments were investigated in Pregnenolone-Sulphate-CIM-0216-evoked and icilin-induced acute nocifensive pain models in mice. Cholesterol depletion decreased CHO cell viability. Sphingomyelinase and methyl-beta-cyclodextrin reduced the duration of icilin-evoked nocifensive behavior, while lipid raft disruptors did not inhibit the activity of recombinant TRPM3 and TRPM8. We conclude that depletion of sphingomyelin or cholesterol from rafts can modulate the function of native TRPM8 receptors. Furthermore, sphingolipid cleavage provided superiority over cholesterol depletion, and this method can open novel possibilities in the management of different pain conditions.
Topics: Animals; Sphingomyelin Phosphodiesterase; TRPM Cation Channels; Mice; Humans; CHO Cells; Cricetulus; beta-Cyclodextrins; HEK293 Cells; Disease Models, Animal; Membrane Microdomains; Pain; Cholesterol; Male; Analgesics; Pregnenolone; Cell Survival
PubMed: 38731855
DOI: 10.3390/ijms25094637 -
International Journal of... 2024This study investigated the raft-forming suspension of famotidine as an anti-reflux formulation to improve the oral bioavailability of narrow absorption window drugs by...
OBJECTIVE
This study investigated the raft-forming suspension of famotidine as an anti-reflux formulation to improve the oral bioavailability of narrow absorption window drugs by enhancing gastric residence time (GRT) and preventing gastro-esophageal reflux disease (GERD).
METHOD
Various combinations of raft-forming agents, such as Tragacanth gum (TG), guar gum (GG), and xanthan gum (XG), were evaluated alongside sodium alginate (SA) to develop an effective raft. Preformulation studies and preliminary screening were conducted to identify the most suitable raft-forming agent, and GG was chosen due to its mucilaginous properties. The formulation was optimized using a 32 full factorial design, with the quantities of GG and SA as independent factors and apparent viscosity and in-vitro drug release (%) as dependent factors. The in vivo floating behavior study was performed for optimized and stabilized formulation.
RESULTS
Among the tested batches, F6 was selected as the optimized formulation. It exhibited desirable characteristics such as adequate raft weight for extended floating in gastric fluid, improved apparent viscosity, and a significant percentage of drug release at 12 h. A mathematical model was applied to the in-vitro data to gain insights into the drug release mechanism of the formulation. The stability of the suspension was assessed under accelerated conditions, and it demonstrated satisfactory stability. The formulation remains floating in the Rabbit stomach for more than 12 h.
CONCLUSION
It concludes that the developed formulation has enhanced bioavailability in the combination of GG and SA. The floating layer of the raft prevents acid reflux, and the famotidine is retained for an extended period of time in the gastric region, preventing excess acid secretion. The developed formulations are effective for stomach ulcers and GERD, with the effect of reducing acid secretion by H2 receptor antagonists.
Topics: Famotidine; Animals; Drug Delivery Systems; Galactans; Drug Liberation; Alginates; Gastroesophageal Reflux; Biological Availability; Mannans; Plant Gums; Viscosity; Male; Rabbits; Gastric Mucosa; Polysaccharides, Bacterial; Drug Stability; Administration, Oral
PubMed: 38721971
DOI: 10.1177/03946320241249429 -
Frontiers in Immunology 2024Inhibitory natural killer (NK) cell receptors recognize MHC class I (MHC-I) in on target cells and suppress cytotoxicity. Some NK cell receptors recognize MHC-I in ,...
Inhibitory natural killer (NK) cell receptors recognize MHC class I (MHC-I) in on target cells and suppress cytotoxicity. Some NK cell receptors recognize MHC-I in , but the role of this interaction is uncertain. Ly49Q, an atypical Ly49 receptor expressed in non-NK cells, binds MHC-I in and mediates chemotaxis of neutrophils and type I interferon production by plasmacytoid dendritic cells. We identified a lipid-binding motif in the juxtamembrane region of Ly49Q and found that Ly49Q organized functional membrane domains comprising sphingolipids via sulfatide binding. Ly49Q recruited actin-remodeling molecules to an immunoreceptor tyrosine-based inhibitory motif, which enabled the sphingolipid-enriched membrane domain to mediate complicated actin remodeling at the lamellipodia and phagosome membranes during phagocytosis. Thus, Ly49Q facilitates integrative regulation of proteins and lipid species to construct a cell type-specific membrane platform. Other Ly49 members possess lipid binding motifs; therefore, membrane platform organization may be a primary role of some NK cell receptors.
Topics: Animals; Humans; Sphingolipids; Killer Cells, Natural; Phagocytosis; Phagocytes; NK Cell Lectin-Like Receptor Subfamily A; Cell Membrane; Protein Binding
PubMed: 38720899
DOI: 10.3389/fimmu.2024.1401294 -
BioRxiv : the Preprint Server For... Apr 2024Cholesterol- and sphingolipid-enriched domains called lipid rafts are hypothesized to selectively coordinate protein complex assembly within the plasma membrane to...
Cholesterol- and sphingolipid-enriched domains called lipid rafts are hypothesized to selectively coordinate protein complex assembly within the plasma membrane to regulate cellular functions. Desmosomes are mechanically resilient adhesive junctions that associate with lipid raft membrane domains, yet the mechanisms directing raft association of the desmosomal proteins, particularly the transmembrane desmosomal cadherins, are poorly understood. We identified the desmoglein-1 (DSG1) transmembrane domain (TMD) as a key determinant of desmoglein lipid raft association and designed a panel of DSG1 variants to assess the contribution of TMD physicochemical properties (length, bulkiness, and palmitoylation) to DSG1 lipid raft association. Sucrose gradient fractionations revealed that TMD length and bulkiness, but not palmitoylation, govern DSG1 lipid raft association. Further, DSG1 raft association determines plakoglobin recruitment to raft domains. Super-resolution imaging and functional assays uncovered a strong relationship between the efficiency of DSG1 lipid raft association and the formation of morphologically and functionally robust desmosomes. Lipid raft association regulated both desmosome assembly dynamics and DSG1 cell surface stability, indicating that DSG1 lipid raft association is required for both desmosome formation and maintenance. These studies identify the biophysical properties of desmoglein transmembrane domains as key determinants of lipid raft association and desmosome adhesive function.
PubMed: 38712246
DOI: 10.1101/2024.04.24.590936 -
Advanced Materials (Deerfield Beach,... May 2024A significant amount of research has been conducted in carbon dioxide (CO) capture, particularly over the past decade, and continues to evolve. This review presents the... (Review)
Review
A significant amount of research has been conducted in carbon dioxide (CO) capture, particularly over the past decade, and continues to evolve. This review presents the most recent advancements in synthetic methodologies and CO capture capabilities of diverse polymer-based substances, which includes the amine-based polymers, porous organic polymers, and polymeric membranes, covering publications in the last 5 years (2019-2024). It aims to assist researchers with new insights and approaches to develop innovative polymer-based materials with improved capturing CO capacity, efficiency, sustainability, and cost-effective, thereby addressing the current obstacles in carbon capture and storage to sooner meeting the net-zero CO emission target.
PubMed: 38709571
DOI: 10.1002/adma.202403324 -
RSC Advances Apr 2024Stimuli-responsive membranes play an important role in the fields of biomedicine, food and chemical industries, and environmental applications, including separation of...
Stimuli-responsive membranes play an important role in the fields of biomedicine, food and chemical industries, and environmental applications, including separation of water-oil emulsions. In this study, we present a method to fabricate pH-sensitive membranes using UV-initiated RAFT graft copolymerization of styrene (ST) and acrylic acid (AA) on poly(ethylene terephthalate) (PET) track-etched membranes (TeMs). The optimization of polymerization conditions led to successful grafting of polystyrene (PS) and poly(acrylic acid) (PAA) onto PET TeMs, resulting in membranes with stable hydrophobicity and pH change responsiveness. The membranes show a contact angle of 65° in basic environments (pH 9) and 97° in acidic environments (pH 2). The membranes were characterized by atomic force microscopy (AFM), scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX), thermogravimetric analyses (TGA), Fourier transform infrared spectroscopy (FTIR), contact angle (CA) methods. The PET TeMs--PS--PAA exhibited good performance in separating water-oil emulsions with a high efficiency of more than 90% and flux for direct chloroform-water 2500 L m h and reverse emulsions of benzene-water 1700 L m h. This method of preparing stimuli-responsive membranes with controlled wettability and responsiveness to environmental pH provides versatility in their use in separating two types of emulsions: direct and reverse.
PubMed: 38694549
DOI: 10.1039/d4ra02117g -
Biophysical Journal Apr 2024Lateral lipid heterogeneity (i.e., raft formation) in biomembranes plays a functional role in living cells. Three-component mixtures of low- and high-melting lipids plus...
Lateral lipid heterogeneity (i.e., raft formation) in biomembranes plays a functional role in living cells. Three-component mixtures of low- and high-melting lipids plus cholesterol offer a simplified experimental model for raft domains in which a liquid-disordered (Ld) phase coexists with a liquid-ordered (Lo) phase. Using such models, we recently showed that cryogenic electron microscopy (cryo-EM) can detect phase separation in lipid vesicles based on differences in bilayer thickness. However, the considerable noise within cryo-EM data poses a significant challenge for accurately determining the membrane phase state at high spatial resolution. To this end, we have developed an image-processing pipeline that utilizes machine learning (ML) to predict the bilayer phase in projection images of lipid vesicles. Importantly, the ML method exploits differences in both the thickness and molecular density of Lo compared to Ld, which leads to improved phase identification. To assess accuracy, we used artificial images of phase-separated lipid vesicles generated from all-atom molecular dynamics simulations of Lo and Ld phases. Synthetic ground-truth data sets mimicking a series of compositions along a tieline of Ld + Lo coexistence were created and then analyzed with various ML models. For all tieline compositions, we find that the ML approach can correctly identify the bilayer phase with >90% accuracy, thus providing a means to isolate the intensity profiles of coexisting Ld and Lo phases, as well as accurately determine domain-size distributions, number of domains, and phase-area fractions. The method described here provides a framework for characterizing nanoscopic lateral heterogeneities in membranes and paves the way for a more detailed understanding of raft properties in biological contexts.
PubMed: 38689500
DOI: 10.1016/j.bpj.2024.04.029 -
Nihon Yakurigaku Zasshi. Folia... Jul 2024The primary cilium, an antenna-like structure of cell membrane, detects various signals and regulates cellular functions such as proliferation and differentiation. The... (Review)
Review
The primary cilium, an antenna-like structure of cell membrane, detects various signals and regulates cellular functions such as proliferation and differentiation. The impairment of primary cilium is associated with the etiologies of diseases including cancer, obesity, and congenital anomalies. In this review, novel functions of trichoplein, a suppressor of ciliogenesis, on the regulation of adipose progenitors and fibro-adipogenic progenitors are focused. Trichoplein-knockout mice show resistance to high-fat diet-induced obesity and accelerated regeneration after skeletal muscle injury. The primary cilia of adipose progenitors from trichoplein-knockout mice are elongated, leading to the inhibitions of the accumulation of lipid raft to the base of primary cilia and the phosphorylation of AKT. The primary cilia of fibro-adipogenic progenitors from trichoplein-knockout mice are also elongated, causing the increased expression of IL-13 through IL-33 receptor signaling. These mechanisms are involved in the resistance to diet-induced obesity and improved regeneration. These findings suggest that targeting the primary cilia of specific cells may be a novel therapeutic approach through modulating cellular functions.
Topics: Animals; Cilia; Humans; Stem Cells; Adipogenesis; Adipose Tissue; Mice; Cell Differentiation; Signal Transduction; Obesity; Tumor Suppressor Proteins
PubMed: 38684399
DOI: 10.1254/fpj.23108 -
The Journal of Physical Chemistry. B May 2024G protein-coupled receptors (GPCRs) are a major gateway to cellular signaling, which respond to ligands binding at extracellular sites through allosteric conformational...
G protein-coupled receptors (GPCRs) are a major gateway to cellular signaling, which respond to ligands binding at extracellular sites through allosteric conformational changes that modulate their interactions with G proteins and arrestins at intracellular sites. High-resolution structures in different ligand states, together with spectroscopic studies and molecular dynamics simulations, have revealed a rich conformational landscape of GPCRs. However, their supramolecular structure and spatiotemporal distribution is also thought to play a significant role in receptor activation and signaling bias within the native cell membrane environment. Here, we applied single-molecule fluorescence techniques, including single-particle tracking, single-molecule photobleaching, and fluorescence correlation spectroscopy, to characterize the diffusion and oligomerization behavior of the muscarinic M receptor (MR) in live cells. Control samples included the monomeric protein CD86 and fixed cells, and experiments performed in the presence of different orthosteric MR ligands and of several compounds known to change the fluidity and organization of the lipid bilayer. M receptors exhibit Brownian diffusion characterized by three diffusion constants: (∼0.01 μm/s), (∼0.04 μm/s), and (∼0.14 μm/s), whose populations were found to be modulated by both orthosteric ligands and membrane disruptors. The lipid raft disruptor C6 ceramide led to significant changes for CD86, while the diffusion of MR remained unchanged, indicating that M receptors do not partition in lipid rafts. The extent of receptor oligomerization was found to be promoted by increasing the level of expression and the binding of orthosteric ligands; in particular, the agonist carbachol elicited a large increase in the fraction of MR oligomers. This study provides new insights into the balance between conformational and environmental factors that define the movement and oligomerization states of GPCRs in live cells under close-to-native conditions.
Topics: Ligands; Receptor, Muscarinic M1; Diffusion; Humans; Cell Membrane; Protein Multimerization; Animals; Spectrometry, Fluorescence; Molecular Dynamics Simulation; Lipid Bilayers
PubMed: 38683784
DOI: 10.1021/acs.jpcb.4c01035 -
Biomedicines Apr 2024The entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into human embryonic kidney (HEK293T) cells has been shown to be a cholesterol-rich, lipid...
The entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into human embryonic kidney (HEK293T) cells has been shown to be a cholesterol-rich, lipid raft-dependent process. In this study, we investigated if the presence of a cholesterol uptake receptor Niemann-pick type c1-like1 (NPC1L1) impacts SARS-CoV-2 cell entry. Initially, we utilized reporter-based pseudovirus cell entry assays and a spike (S) glycoprotein-mediated cell-to-cell fusion assay. Using Chinese hamster ovary (CHO-K1) cells, which lack endogenous receptors for SARS-CoV-2 entry, our data showed that the co-expression of NPC1L1 together with the ACE2 receptor synergistically increased SARS-CoV-2 pseudovirus entry even more than the cells expressing ACE-2 receptor alone. Similar results were also found with the HEK293T cells endogenously expressing the ACE2 receptor. Co-cultures of effector cells expressing S glycoprotein together with target cells co-expressing ACE-2 receptor with NPC1L1 significantly promoted quantitative cell-to-cell fusion, including syncytia formation. Finally, we substantiated that an elevated expression of NPC1L1 enhanced entry, whereas the depletion of NPC1L1 resulted in a diminished SARS-CoV-2 entry in HEK293T-ACE2 cells using authentic SARS-CoV-2 virus in contrast to their respective control cells. Collectively, these findings underscore the pivotal role of NPC1L1 in facilitating the cellular entry of SARS-CoV-2. Importance: Niemann-Pick type C1-like1 (NPC1L1) is an endosomal membrane protein that regulates intracellular cholesterol trafficking. This protein has been demonstrated to play a crucial role in the life cycle of several clinically important viruses. Although SARS-CoV-2 exploits cholesterol-rich lipid rafts as part of its viral entry process, the role of NPC1L1 in SARS-CoV-2 entry remains unclear. Our research represents the first-ever demonstration of NPC1L1's involvement in facilitating SARS-CoV-2 entry. The observed role of NPC1L1 in human kidney cells is not only highly intriguing but also quite relevant. This relevance stems from the fact that NPC1L1 exhibits high expression levels in several organs, including the kidneys, and the fact that kidney damages are reported during severe cases of SARS-CoV-2. These findings may help us understand the new functions and mechanisms of NPC1L1 and could contribute to the identification of new antiviral targets.
PubMed: 38672177
DOI: 10.3390/biomedicines12040821