Search Results (59)

The application of an ultrafiltration (UF) process with periodic membrane cleaning with the use of alkaline detergent solutions was proposed for the recovery of wash water from car wash effluent. In order to test the resistance of the membranes to the degradation caused by the cleaning solutions, a pilot plant study was carried out for almost two years. The installation included an industrial module with FP100 tubular membranes made of polyvinylidene fluoride (PVDF). The module was fed with synthetic effluent obtained by mixing foaming agents and hydrowax. To limit the fouling phenomenon, the membranes were cleaned cyclically with P3 Ultrasil 11 solution (pH = 11.7) or Insect solution (pH = 11.5). During plant shutdowns, the membrane module was maintained with a sodium metabisulphite solution. Changes in the permeate flux, turbidity, COD, and surfactant rejection were analysed during the study. Scanning electron microscopy (SEM), atomic force microscopy (AFM), differential scanning calorimetry (DSC), and Fourier-transform infrared spectroscopy (FTIR) analysis were used to determine the changes in the membrane structure. As a result of the repeated chemical cleaning, the pore size increased, resulting in a more than 50% increase in permeate flux. However, the quality of the recovered wash water did not deteriorate, as an additional separation layer was formed on the membrane surface due to the fouling phenomenon. Full article

Thread-based analytical devices are low-cost, portable, and easy to use, making them ideal for detecting various biomolecules like glucose and DNA with minimal sample requirements, while also offering environmental benefits through their biodegradability. This study explores the potential of zwitterionic poly(sulfobetaine methacrylate) brushes modified cotton thread (PSBMA@threads) as an innovative substitute for DNA solid-phase extraction. The PSBMA polymer brushes were synthesized on cotton threads via surface-initiated atom transfer radical polymerization (SI-ATRP). The usability of the PSBMA@threads for DNA extraction from cell lysates containing cell debris, proteins, and detergents was evaluated. Characterization using SEM, FTIR, and EDS confirmed the successful functionalization with PSBMA polymer brushes. The antifouling properties of PSBMA@threads, including resistance to non-specific protein adsorption and underwater oil repellency, were assessed. The results demonstrated selective DNA capture from protein and lipid-rich lysates. Optimized extraction parameters improved DNA yield, enabling efficient extraction from tumor cells, which successfully underwent PCR amplification. Comparative experiments with commercial silica membrane-based columns revealed that PSBMA@threads exhibited comparable DNA extraction capability. The PSBMA@threads maintained extraction capability after six months of ambient storage, highlighting its stability and cost-effectiveness for nucleic acid isolation in analytical applications. Full article

Rubber (cis-1,4-polyisoprene) is produced in cytosolic unilamellar vesicles called rubber particles (RPs), and the protein complex responsible for this synthesis, the rubber transferase (RTase), is embedded in, or tethered to, the membranes of these RPs. Solubilized enzyme activity is very difficult to achieve because the polymerization of highly hydrophilic substrates into hydrophobic polymers requires a polar/non-polar interface and a hydrophobic compartment. Using guayule (Parthenium argentatum) as a model rubber-producing species, we optimized methods to isolate RP unilamellear membranes and then a subset of membrane microdomains (detergent-resistant membranes) likely to contain protein complexes such as RTase. The phospholipid and sterol composition of these membranes and microdomains were analyzed using thin-layer chromatography (TLC) and liquid chromatography tandem mass spectroscopy (LC-MS/MS). Our data indicate that RP membranes consist predominantly of phosphatidic acid-containing membrane microdomains (DRMs or “lipid rafts”). Proteomic analyses of guayule RP membranes and membrane microdomains identified 80 putative membrane proteins covering 30 functional categories. From this population, we have tentatively identified several proteins in multiple functional domains associated with membrane microdomains which may be critical to RTase function. Definition of the mechanisms underlying rubber synthesis will provide targets for both metabolic engineering and breeding strategies designed to increase natural rubber production in latex-producing species. Full article

Bicarbonate and CO₂ are essential substrates for carboxylation reactions in bacterial central metabolism. In Staphylococcus aureus, the bicarbonate transporter, MpsABC (membrane potential-generating system) is the only carbon concentrating system. An mpsABC deletion mutant can hardly grow in ambient air. In this study, we investigated the changes that occur in S. aureus when it suffers from CO₂/bicarbonate deficiency. Electron microscopy revealed that ΔmpsABC has a twofold thicker cell wall thickness compared to the parent strain. The mutant was also substantially inert to cell lysis induced by lysostaphin and the non-ionic surfactant Triton X-100. Mass spectrometry analysis of muropeptides revealed the incorporation of alanine into the pentaglycine interpeptide bridge, which explains the mutant’s lysostaphin resistance. Flow cytometry analysis of wall teichoic acid (WTA) glycosylation patterns revealed a significantly lower α-glycosylated and higher ß-glycosylated WTA, explaining the mutant’s increased resistance towards Triton X-100. Comparative transcriptome analysis showed altered gene expression profiles. Autolysin-encoding genes such as sceD, a lytic transglycosylase encoding gene, were upregulated, like in vancomycin-intermediate S. aureus mutants (VISA). Genes related to cell wall-anchored proteins, secreted proteins, transporters, and toxins were downregulated. Overall, we demonstrate that bicarbonate deficiency is a stress response that causes changes in cell wall composition and global gene expression resulting in increased resilience to cell wall lytic enzymes and detergents. Full article

Capitellacin is the β-hairpin membrane-active cationic antimicrobial peptide from the marine polychaeta Capitella teleta. Capitellacin exhibits antibacterial activity, including against drug-resistant strains. To gain insight into the mechanism of capitellacin action, we investigated the structure of the peptide in the membrane-mimicking environment of dodecylphosphocholine (DPC) micelles using high-resolution NMR spectroscopy. In DPC solution, two structural forms of capitellacin were observed: a monomeric β-hairpin was in equilibrium with a dimer formed by the antiparallel association of the N-terminal β-strands and stabilized by intermonomer hydrogen bonds and Van der Waals interactions. The thermodynamics of the enthalpy-driven dimerization process was studied by varying the temperature and molar ratios of the peptide to detergent. Cooling the peptide/detergent system promoted capitellacin dimerization. Paramagnetic relaxation enhancement induced by lipid-soluble 12-doxylstearate showed that monomeric and dimeric capitellacin interacted with the surface of the micelle and did not penetrate into the micelle interior, which is consistent with the “carpet” mode of membrane activity. An analysis of the known structures of β-hairpin AMP dimers showed that their dimerization in a membrane-like environment occurs through the association of polar or weakly hydrophobic surfaces. A comparative analysis of the physicochemical properties of β-hairpin AMPs revealed that dimer stability and hemolytic activity are positively correlated with surface hydrophobicity. An additional positive correlation was observed between hemolytic activity and AMP charge. The data obtained allowed for the provision of a more accurate description of the mechanism of the oligomerization of β-structural peptides in biological membranes. Full article

The purpose of this review is to succinctly examine the methodologies used in lipid raft research in the brain and to highlight the drawbacks of some investigative approaches. Lipid rafts are biochemically and biophysically different from the bulk membrane. A specific lipid environment within membrane domains provides a harbor for distinct raftophilic proteins, all of which in concert create a specialized platform orchestrating various cellular processes. Studying lipid rafts has proved to be arduous due to their elusive nature, mobility, and constant dynamic reorganization to meet the cellular needs. Studying neuronal lipid rafts is particularly cumbersome due to the immensely complex regional molecular architecture of the central nervous system. Biochemical fractionation, performed with or without detergents, is still the most widely used method to isolate lipid rafts. However, the differences in solubilization when various detergents are used has exposed a dire need to find more reliable methods to study particular rafts. Biochemical methods need to be complemented with other approaches such as live-cell microscopy, imaging mass spectrometry, and the development of specific non-invasive fluorescent probes to obtain a more complete image of raft dynamics and to study the spatio-temporal expression of rafts in live cells. Full article

Platelet lipid rafts are critical membrane domains for adhesion, aggregation, and clot retraction. Lipid rafts are isolated as a detergent-resistant membrane fraction via sucrose density gradient centrifugation. The platelet detergent-resistant membrane shifted to a higher density on the sucrose density gradient upon thrombin stimulation. The shift peaked at 1 min and returned to the control level at 60 min. During this time, platelets underwent clot retraction and spreading on a fibronectin-coated glass strip. Thrombin induced the transient tyrosine phosphorylation of several proteins in the detergent-resistant membrane raft fraction and the transient translocation of fibrin and myosin to the detergent-resistant membrane raft fraction. The level of phosphatidylserine (36:1) was increased and the level of phosphatidylserine (38:4) was decreased in the detergent-resistant membrane raft fraction via the thrombin stimulation. Furthermore, Glanzmann’s thrombasthenia integrin αIIbβ3-deficient platelets underwent no detergent-resistant membrane shift to a higher density upon thrombin stimulation. As the phosphorylation of the myosin regulatory light chain on Ser19 was at a high level in Glanzmann’s thrombasthenia resting platelets, thrombin caused no further phosphorylation of the myosin regulatory light chain on Ser19 or clot retraction. These observations suggest that the fibrin–integrin αIIbβ3–myosin axis and compositional change of phosphatidylserine species may be required for the platelet detergent-resistant membrane shift to a higher density upon stimulation with thrombin. Full article

Outer membrane proteins (Omps) of Gram-negative bacteria represent porins involved in a wide range of virulence- and pathogenesis-related cellular processes, including transport, adhesion, penetration, and the colonization of host tissues. Most outer membrane porins share a specific spatial structure called the β-barrel that provides their structural integrity within the membrane lipid bilayer. Recent data suggest that outer membrane proteins from several bacterial species are able to adopt the amyloid state alternative to their β-barrel structure. Amyloids are protein fibrils with a specific spatial structure called the cross-β that gives them an unusual resistance to different physicochemical influences. Various bacterial amyloids are known to be involved in host-pathogen and host-symbiont interactions and contribute to colonization of host tissues. Such an ability of outer membrane porins to adopt amyloid state might represent an important mechanism of bacterial virulence. In this work, we investigated the amyloid properties of the OmpC and OmpF porins from two species belonging to Enterobacteriaceae family, Escherichia coli, and Salmonella enterica. We demonstrated that OmpC and OmpF of E. coli and S. enterica form toxic fibrillar aggregates in vitro. These aggregates exhibit birefringence upon binding Congo Red dye and show characteristic reflections under X-ray diffraction. Thus, we confirmed amyloid properties for OmpC of E. coli and demonstrated bona fide amyloid properties for three novel proteins: OmpC of S. enterica and OmpF of E. coli and S. enterica in vitro. All four studied porins were shown to form amyloid fibrils at the surface of E. coli cells in the curli-dependent amyloid generator system. Moreover, we found that overexpression of recombinant OmpC and OmpF in the E. coli BL21 strain leads to the formation of detergent- and protease-resistant amyloid-like aggregates and enhances the birefringence of bacterial cultures stained with Congo Red. We also detected detergent- and protease-resistant aggregates comprising OmpC and OmpF in S. enterica culture. These data are important in the context of understanding the structural dualism of Omps and its relation to pathogenesis. Full article

The cell–cell contact between HIV-1-infected and uninfected cells forms a virological synapse (VS) to allow for efficient HIV-1 transmission. Not only are HIV-1 components polarized and accumulate at cell–cell interfaces, but viral receptors and lipid raft markers are also. To better understand the nature of the HIV-1 VS, detergent-resistant membrane (DRM) fractions were isolated from an infected–uninfected cell coculture and compared to those from non-coculture samples using 2D fluorescence difference gel electrophoresis. Mass spectrometry revealed that ATP-related enzymes (ATP synthase subunit and vacuolar-type proton ATPase), protein translation factors (eukaryotic initiation factor 4A and mitochondrial elongation factor Tu), protein quality-control-related factors (protein disulfide isomerase A3 and 26S protease regulatory subunit), charged multivesicular body protein 4B, and vimentin were recruited to the VS. Membrane flotation centrifugation of the DRM fractions and confocal microscopy confirmed these findings. We further explored how vimentin contributes to the HIV-1 VS and found that vimentin supports HIV-1 transmission through the recruitment of CD4 to the cell–cell interface. Since many of the molecules identified in this study have previously been suggested to be involved in HIV-1 infection, we suggest that a 2D difference gel analysis of DRM-associated proteins may reveal the molecules that play crucial roles in HIV-1 cell–cell transmission. Full article

Halophytes represent important models for studying the key mechanisms of salt tolerance. One approach to the development of new knowledge of salt tolerance is to study the properties of detergent-resistant membranes (DRMs). In this work, the lipid profiles of DRMs of chloroplasts and mitochondria of euhalophyte Salicornia perennans Willd, before and after their exposure to shock concentrations of NaCl, have been investigated. We found that DRMs of chloroplasts are enriched in cerebrosides (CERs) and that sterols (STs) dominate the mass of mitochondrial DRMs. Also, it has been proven that (i) the impact of salinity provokes obvious growth in the content of CERs in DRMs of chloroplasts; (ii) the content of STs in DRMs of chloroplasts does not change under the influence of NaCl; (iii) salinity also causes some elevation in the content of monounsaturated and saturated fatty acids (FAs). Considering the fact that DRMs represent integral parts of both chloroplast and mitochondrial membranes, the authors have come to the conclusion that the cells of euhalophyte S. perennans, under the impact of salinity, presumes the choice (by the cell) of some specific composition of lipids and FAs in the membrane. This may be considered as a specific protection reaction of the plant cell against salinity. Full article

We evaluated the effects of detergents based on sodium dodecyl sulfoxide (SDS) on the functional parameters of collared peccary frozen–thawed sperm. Semen aliquots from ten individuals were diluted in a Tris–egg yolk–glycerol extender alone or with 0.5% Equex STM^® paste or SDS (at 0.1%, 0.3% or 0.5% (v/v) concentration). Samples were fast frozen in liquid nitrogen with a post-thaw evaluation of motility, membrane functionality and integrity, mitochondrial activity, sperm binding ability and thermal resistance. The treatments without SDS (41.8 ± 3.5%) and those containing Equex (41.8 ± 4.4%) or 0.1% SDS (41.2 ± 5.5%) provided greater sperm motility (p < 0.05) than those containing SDS 0.3% (30.5 ± 4.7%) and 0.5% (31.2 ± 6.3%). Immediately after thawing, only treatments containing 0.1% SDS effectively preserved sperm straightness (STR) when compared to the negative control. All treatments preserved the amplitude of lateral head (ALH) and straightness (STR) during a thermal resistance test (p > 0.05), but SDS 0.5% impaired the membrane functionality and mitochondrial activity after thawing (p < 0.05). All treatments provided a similar recovery of sperm binding ability after thawing (p < 0.05). Our results showed that the addition of 0.1% SDS to the Tris–yolk–glycerol extender optimized the freeze–thaw recovery of peccary semen. Full article

Cationic antimicrobial peptides (CAMPs) offer a promising strategy to counteract bacterial resistance, mostly due to their membrane-targeting activity. W-BP100 is a potent broad-spectrum cecropin-melittin CAMP bearing a single N-terminal Trp, which was previously found to improve its antibacterial activity. W-BP100 has high affinity toward anionic membranes, inducing membrane saturation at low peptide-to-lipid (P/L) ratios and membrane permeabilization, with the unique property of promoting the aggregation of anionic vesicles only at specific P/L ratios. Herein, we aimed to investigate this unusual behavior of W-BP100 by studying its aggregation and fusion properties with negatively-charged large (LUVs) or giant (GUVs) unilamellar vesicles using biophysical tools. Circular dichroism (CD) showed that W-BP100 adopted an α-helical conformation in anionic LUVs, neutralizing its surface charge at the aggregation P/L ratio. Its fusion activity, assessed by Förster resonance energy transfer (FRET) using steady-state fluorescence spectroscopy, occurred mainly at the membrane saturation/aggregation P/L ratio. Confocal microscopy studies confirmed that W-BP100 displays aggregation and detergent-like effects at a critical P/L ratio, above which it induces the formation of new lipid aggregates. Our data suggest that W-BP100 promotes the aggregation and fusion of anionic vesicles at specific P/L ratios, being able to reshape the morphology of GUVs into new lipid structures. Full article

Caveolin-1 is a cholesterol-binding scaffold protein, which is localized in detergent-resistant membrane (DRM) rafts and interacts with components of signal transduction systems, including visual cascade. Among these components are neuronal calcium sensors (NCSs), some of which are redox-sensitive proteins that respond to calcium signals by modulating the activity of multiple intracellular targets. Here, we report that the formation of the caveolin-1 complex with recoverin, a photoreceptor NCS serving as the membrane-binding regulator of rhodopsin kinase (GRK1), is a redox-dependent process. Biochemical and biophysical in vitro experiments revealed a two-fold decreased affinity of recoverin to caveolin-1 mutant Y14E mimicking its oxidative stress-induced phosphorylation of the scaffold protein. At the same time, wild-type caveolin-1 demonstrated a 5–10-fold increased affinity to disulfide dimer of recoverin (dRec) or its thiol oxidation mimicking the C39D mutant. The formation of dRec in vitro was not affected by caveolin-1 but was significantly potentiated by zinc, the well-known mediator of redox homeostasis. In the MDCK cell model, oxidative stress indeed triggered Y14 phosphorylation of caveolin-1 and disulfide dimerization of recoverin. Notably, oxidative conditions promoted the accumulation of phosphorylated caveolin-1 in the plasma membrane and the recruitment of recoverin to the same sites. Co-localization of these proteins was preserved upon depletion of intracellular calcium, i.e., under conditions reducing membrane affinity of recoverin but favoring its interaction with caveolin-1. Taken together, these data suggest redox regulation of the signaling complex between recoverin and caveolin-1. During oxidative stress, the high-affinity interaction of thiol-oxidized recoverin with caveolin-1/DRMs may disturb the light-induced translocation of the former within photoreceptors and affect rhodopsin desensitization. Full article

The necessity to include plants as a component of a Bioregenerative Life Support System leads to investigations to optimize plant growth facilities as well as a better understanding of the plant cell membrane and its numerous activities in the signaling, transport, and sensing of gravity, drought, and other stressors. The cell membrane participates in numerous processes, including endo- and exocytosis and cell division, and is involved in the response to external stimuli. Variable but stabilized microdomains form in membranes that include specific lipids and proteins that became known as (detergent-resistant) membrane microdomains, or lipid rafts with various subclassifications. The composition, especially the sterol-dependent recruitment of specific proteins affects endo- and exo-membrane domains as well as plasmodesmata. The enhanced saturated fatty acid content in lipid rafts after clinorotation suggests increased rigidity and reduced membrane permeability as a primary response to abiotic and mechanical stress. These results can also be obtained with lipid-sensitive stains. The linkage of the CM to the cytoskeleton via rafts is part of the complex interactions between lipid microdomains, mechanosensitive ion channels, and the organization of the cytoskeleton. These intricately linked structures and functions provide multiple future research directions to elucidate the role of lipid rafts in physiological processes. Full article

Caveolin-1 (Cav-1) is the basic component of caveolae, a specialized form of lipid raft that plays an essential role in endocytic viral entry. However, the evidence of direct involvement of caveolae and Cav-1 in avian reovirus (ARV) entry remains insufficient. In this study, the membrane lipid rafts were isolated as detergent-resistant microdomains (DRMs) by sucrose gradient centrifugation, and the capsid protein σB of ARV was found to associate with Cav-1 in DRMs fractions. Additionally, the interaction between ARV σB protein and Cav-1 was demonstrated by immunofluorescence co-localization and co-immunoprecipitation assays. Furthermore, we found that the internalization of ARV is sensitive to caveolae and dynamin inhibitors, while it is insensitive to clathrin inhibitors. In conclusion, these results indicate that the ARV σB protein interacts with Cav-1 during dynamin-dependent caveolae-mediated endocytosis for the entry of ARV. Full article