Studies on the induction and expression of T cell-mediated immunity. VIII. Effector-target junctions and target cell membrane disruption during cytolysis.
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Animals↗
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Relationship of the light-induced proton uptake in bovine retinal outer segment fragments to triton-induced membrane disruption and to volume changes.
Light-induced proton uptake in bovine retinal outer segment (ROS) fragments was shown to be closely related to pH, salt concentration, membrane integrity, and perhaps secondarily to the volume of osmotic compartments. The principal findings were as follows: 1. As pH increased, both the discs and the plasmalemma swelled, and proton uptake markedly diminished. 2. As the discs were disrupted by increasing concentrations of Triton, proton uptake at slightly alkaline pH was supplanted by proton release. 3. Increasing the concentration of chloride salts caused increased H+ uptake roughly proportional to osmotic shrinkage of the ROS. Buffering by acetate prevented the measurement of proton uptake in the presence of acetate salts, although osmotic behavior of the ROS was similar to that observed in chloride salts. Although increasing the concentration of sucrose also resulted in osmotic shrinkage of the ROS, it was not accompanied by a systematic increase in the magnitude of proton uptake. 4. Light-induced H+ uptake was accompanied by small but reproducible changes in volume, probably of the discs. The magnitude and direction of these rapid volume changes were subject to influence by pH, solute, and other variables.
Animals↗
Ionophore A23187 disrupts membrane structure by modifying protein-lipid interactions.
The physiological function of membrane-boudn protein clearly depends on the nature of its neighbouring lipid. However, the question of how critical the protein is to the structure and function of membrane lipid has received much less attention. There is some evidence that lipid surrounding membrane protein, 'boundary lipid', may be more ordered than continuum lipid. Protein can also alter the enthalpy and temperature of pure lipid-phase transitions. However, controversy surrounds the question of whether membrane proteins determine long-range aspects of lipid structure or merly perturb their local environments. Here, we demonstrate that a lipophilic substance, the calcium ionophore, A23187 (Lilly), dramatically disorders the lipid structure of the membrane and that this phenomenon depends on the presence of proteins.
Animals↗
Antibacterial mechanisms and pathogen-dependent protective effects of the golden pompano LEAP2-derived peptide TroLEAP2-21.
Antimicrobial peptides (AMPs) are essential components of the innate immune system, with liver-expressed antimicrobial peptide 2 (LEAP2) playing a pivotal role in fish immunity. This study investigated the antimicrobial activity and mechanisms of TroLEAP2-21, a 21-amino-acid short peptide from golden pompano (Trachinotus ovatus), against Gram-positive (Lactococcus garvieae, Staphylococcus epidermidis) and Gram-negative (Vibrio alginolyticus, Vibrio harveyi) bacteria. The predicted three-dimensional structure and helical wheel projection of TroLEAP2-21 suggested typical AMP-like physicochemical features. troleap2 expression in the liver and intestine of T. ovatus was significantly upregulated post L. garvieae or V. harveyi infection, suggesting its potential involvement in antibacterial defense. In vitro, TroLEAP2-21 exhibited antibacterial activity against the tested bacterial strains, with membrane disruption, increased membrane permeability, cytoplasmic leakage, and membrane depolarization observed after peptide treatment. Gel retardation assays further indicated species-dependent association of TroLEAP2-21 with bacterial genomic DNA. In vivo, under the tested intraperitoneal injection conditions, TroLEAP2-21 was associated with improved survival and reduced tissue damage in V. harveyi-infected T. ovatus, whereas no significant survival benefit was observed against L. garvieae. Transcriptomic analysis at 48 h post-infection showed transcriptional changes in immune-related DEGs (rsad2, mx1/mx2, il-8) and enrichment of TLR and Jak-STAT signaling pathways at the transcriptional level in peptide-treated fish. FISH showed the tissue localization of tnf-α and nf-κb transcripts and revealed treatment-associated changes in fluorescence signals, and qRT-PCR of eight immune genes supported transcriptomic results in tissues at 48 h post-infection. Collectively, these findings characterize TroLEAP2-21 as a short LEAP2-derived peptide with antibacterial and immunomodulatory activities. Its comparative advantages over other LEAP2-related peptides and its practical application potential remain to be further investigated.
AMPs↗
Melanoma development from subcellular fractions.
Amelanotic melanoma cells, RPMI-1846, were disrupted by sonic energy which caused cell membrane disruption, thereby allowing liberation of viable cellular organelles. These subcellular particles were injected into syngeneic Syrian and nonsyngeneic hamsters in six separate experiments to determine their possible growth potential. A sizable number of the injected hamsters subsequently died as a result of melanoma. In an attempt to be as certain as possible that no whole melanoma cells were injected with sonicated melanoma material, various techniques were used including collodian sections for debris analysis, phase microscopy and electron microscopy. Results of this study suggested that melanoma can be induced from particles within the melanoma cell which are liberated following cell membrane disruption. It appears that a mechanism exists for the transfer of oncogenic information that may not be dependent upon the presence of intact cells or require the participation of a viral agent.
Abdominal Muscles↗
Asymmetric membrane expansion and modification of active and passive cation permeability of human red cells by the fluorescent probe 1-anilino-8-napththalene sulfonate.
1. The membrane perturbations induced by the interaction of the fluorescent probe 1-anilino-8-naphthalene sulfonate (ANS) with human red blood cells were studied. 2. ANS below 0.5 mM inhibits partially (20% maximum) the ouabain-insensitive Na+ and K+ influx and efflux. Above 0.5 mM ANS increases both Na+ and K+ leak fluxes. The increased cation leaks are larger for Na+ than K+. 3. The (Na+ +K+)-ATPase and ouabain-sensitive Na+ and K+ fluxes are inhibited by ANS. Ouabain-insensitive, Mg2+-dependent ATPase activity of ghosts is stimulated by [ANS] less than 0.3 mM and inhibited by [ANS] greater than 0.3 mM. 4. ANS also inhibits the Na+-dependent, ouabain-insensitive K+ influx that is inhibited by ethacrynic acid and furosemide. 5. Red cells become crenated with [ANS] less than 1 mM and sphere at [ANS] greater than 1 mM. In the former conditions hypotonic hemolysis is decreased whereas the latter increase osmotic fragility. 6. It is suggested that ANS expands the membrane asymmetrically by binding preferentially to the external membrane surface. 7. It is concluded that ANS is a general inhibitor of ion transport, particularly of those processes thought to involve facilitated-diffusion mechanisms. The increased cation leaks observed at high ANS concentrations may be related to prehemolytic membrane disruption. 8. The membrane perturbations caused by ANS are compared to those caused by other reversible inhibitors of anion exchange in red blood cells. Their possible modes of action are discussed.
Adenosine Triphosphatases↗
Identification of polymorphonuclear leukocytes in cytologic samples for flow cytometry.
Inflammatory cells are commonly present in cytologic specimens obtained for flow cytometry, and may interfere with the analysis of epithelial cells. We have found that detergent (Triton X-100) pretreatment in the two-step acridine orange staining procedure disrupts granulocyte cell membranes to yield bare nuclei; bladder epithelial and squamous cells on the other hand are quite resistant to the detergent treatment. Being deprived of their cytoplasmic RNA, the granulocytes lose red fluorescence. Moreover, the shearing forces in the cytometer extend the multisegmented granulocyte nuclei and align them in the direction of flow. Thus, they present as elongated objects in the measuring system, giving a large DNA fluorescence pulsewidth (nuclear size). These two phenomena make it possible to identify granulocytes in the recorded data, where they are discernible from the mononucleated leukocytes and from epithelial cells. By data selection the granulocytes can be excluded, rendering epithelial cell populations more amenable to analysis. This method may make it unnecessary to remove physically leukocytes from the specimen before flow cytometry; it may also provide a way to analyze the morphology of granulocyte nuclei and to assess methods to manipulate their membrane stability. Full protection from membrane disruption is accomplished by alcohol fixation, and partial protection by 20-30% serum.
Cytological Techniques↗
Binding of viral glycoprotein mRNA to endoplasmic reticulum membranes is disrupted by puromycin.
Previous studies showed that the glycoprotein (G) of vesicular stomatitis virus is synthesized in association with the endoplasmic reticulum (ER) membrane and that all G mRNA co-fractionates with ER membrane. Here, we show that treatment of infected cells with puromycin results in dissociation of G mRNA, and presumably the associated ribosomes, from the ER membrane. Even it extracts from treated cells are kept at low ionic strength (0.01 M KCl), over 80% of G mRNA is found unattached to membranes. There is no evidence for direct interaction of GmRNA with membranes; rather, the linkage apparently is mediated by the nascent G polypeptide.
Animals↗
Reinnervation of original synaptic sites on muscle fiber basement membrane after disruption of the muscle cells.
Regenerating axons form new synapses precisely at sites of original synapses in denervated skeletal muscle. To determine what role the muscle cell plays in this phenomenon, we studied reinnervation of frog muscle at intervals after crushing the nerve and damaging the muscle fibers. Damaged muscle fibers degenerate and are phagocytized, but their basement membrane persists and acts as a scaffold for regenerating muscle cells. Specializations of the basement membrane serve to mark original synaptic sites after nerve and muscle have degenerated. Regenerating axons enter the region of damage and form functional synapses with regenerating myofibers. The new nerve terminals are found almost exclusively at the original synaptic sites, demonstrating that the integrity of the original postsynaptic cell is not necessary for topographically precise reinnervation of denervated muscle.
Animals↗
TamL is a Key Player of the Outer Membrane Homeostasis in Bacteroidota.
In Proteobacteria, the outer membrane protein TamA and the inner membrane-anchored protein TamB form the Translocation and Assembly Module (TAM) complex, which facilitates the transport of autotransporters, virulence factors, and likely lipids across the two membranes. In Bacteroidota, TamA is replaced by TamL, a TamA-like lipoprotein with a lipid modification at its N-terminus that likely anchors it to the outer membrane. This structural difference suggests that TamL may have a distinct function compared to TamA. However, the role of TAM in bacterial phyla other than Proteobacteria remains unexplored. Our study aimed to elucidate the function of TamL in Flavobacterium johnsoniae, an environmental Bacteroidota. Unlike its homologs in Proteobacteria, we found that TamL and TamB are essential in F. johnsoniae. Through genetic, phenotypic, proteomic, and lipidomic analyses, we show that TamL depletion severely compromises outer membrane integrity, as evidenced by reduced cell viability, altered cell shape, increased susceptibility to membrane-disrupting agents, and elevated levels of outer membrane lipoproteins. Notably, we did not observe an overall decrease in the levels of β-barrel outer membrane proteins, nor substantial alterations in outer membrane lipid composition. By pull-down assays, we found TamL co-purifying with TamB in F. johnsoniae, suggesting an interaction. Furthermore, we found that while TamL and TamB monocistronic genes are conserved among Bacteroidota, only some species encode multiple TamL, TamB and TamA proteins. To our knowledge, this study is the first to provide functional insights into a TAM subunit beyond Proteobacteria.
Bacterial Outer Membrane Proteins↗
Fine structure of epithelial cells of Lieberkühn's crypts in feline panleukopenia.
Electron microscopic observation was carried out on epithelial cells of Lieberkühn's crypts of cats naturally affected with feline panleukopenia. The most important change was the replication of feline panleukopenia. The most important change was the replication of feline panleukopenia virus in the nucleus with associated alterations in the lining epithelial cells of the crypts. In these cells in the early stage of infection, virus particles 20 nm in average diameter were found either singly or in small regularly arrayed clusters everywhere in the markedly swollen nucleus. In the course of infection, the nucleus of infected cells became rather atrophic with a marked margination of chromatin granules. Its major portion was occupied with masses of fine fibrillar substance. It was a "viral matrix area" in which appeared a large compact aggregate of virus particles showing a crystalline array. At the same time, the outer membrane of the nuclear envelope partially extended and disrupted. Membranous elements related to it in the cytoplasm were regularly distributed almost always with particles indistinguishable from the virus particles in the nucleus. From these results it was suggested that the major portion of the infected nucleus, or the site of viral replication, might correspond to the amphophilic intranuclear inclusion body revealed by light microscopy.
Animals↗
Identification of a Nonribosomal Peptide Analog With Activity Against Multiple Gram-Positive Bacteria via a Synthetic Bioinformatic Natural Product Discovery Approach.
Nonribosomal peptide (NRP) antibiotics exhibit potent biological activities and are broadly used in clinical therapy. Because most microorganisms are difficult to culture and many antibiotic biosynthetic genes are silent, traditional activity tracking approaches face major limitations in the discovery of novel NRPs. Here, based on a synthetic bioinformatic natural product (syn-BNP) discovery approach that integrates bioinformatics and chemical synthesis, a novel nonribosomal peptide synthetase (NRPS) gene cluster from the genome of Rhodococcus erythropolis D-1 was mined. A putative NRP scaffold synthesized by the NRPS encoded by this cluster was predicted. Through chemical synthesis and four rounds of structure-activity relationship (SAR) studies, 37 NRP analogs were ultimately generated. Among these analogs, ZURJC28 shows activity against multiple Gram-positive bacteria, including two drug-resistant strains. Mechanistic studies and metabolomics analyses revealed that ZURJC28 exerts membrane-disruptive activity associated with interaction with phosphatidylglycerol (PG)-enriched Gram-positive membranes, leading to membrane damage and widespread metabolic dysregulation. ZURJC28 also shows low cytotoxicity and low hemolytic activity, suggesting its preliminary in vitro safety profile.
Gram-Positive Bacteria↗
Electron spin resonance evidence for vertical asymmetry in animal cell membranes.
Two electron spin resonance (ESR) spin labels were used to monitor the physical state of bacterial and animal cell membranes: 5N10, a nitroxide derivative of decane, and 12NS-GA, a glucosamine derivative of 12-nitroxide stearic acid. Spectra were recorded at 1 degrees C intervals from approximately 5 to 45 degrees C. Arrhenius plots of log hH/hP vs. 1/K were obtained by measuring the amplitudes of the hydrocarbon and water signals, hH and hP, respectively. Two discontinuities in the Arrhenius plot (at characteristic temperatures t1 and th) were observed with bacterial cell membranes independent of the spin label employed. Analysis of sealed animal cell membrane samples revealed four characteristic temperatures when the hydrophobic spin lable 5N10 was used, but only two when the amphiphilic spin label 12NS-GA was used. The specific set of characteristic temperatures revealed with 12NS-GA depended on whether the membrane preparation was inside out (ISO) or right side out (RSO). Analysis of Newcastle disease virus, a source of RSO plasma membrane derived from host, revealed two characteristic temperatures at approximately 14 and 33 degrees C. Analysis of phagosomes, a source of ISO plasma membrane derived from LM cells, revealed two characteristic temperatures at approximately 23 and 38 degrees C. When unsealed or disrupted membrane preparations were spin labeled with 12NS-GA, both sets (RSO and ISO) of characteristic temperatures were revealed. The results indicate that the inner and outer monolayers of animal cell membranes are physically distinct and that the glycosylated spin label, 12NS-GA, is apparently restricted in its ability to flip across the membrane bilayer. In this study, characteristic temperatures were pinpointed by computer analysis of the ESR spectral data.
Cell Membrane↗
Inactivation and reactivation of mitochondrial respiration by charged detergents.
Respiration of submitochondrial preparations can be inhibited by the cationic detergent cetyl trimethyl ammonium bromide and the anionic detergent sodium dodecyl sulfate in the range of 0.3-2 mumol of detergent per mg of mitochondrial membrane protein depending on the substrate and detergent used. This inhibition can be rapidly reversed by neutralizing a given detergent by the detergent of the opposite charge. At higher levels of the inhibiting detergent, no such reactivation was observed. Spin labeling assays of membrane structure were used to correlate structural effects with the loss and recovery of respiratory functions. Because the detergents progressively disrupt membrane structure, mitochondrial were cross-linked with bifunctional imidoesters to an extent that osmotic properties and detergent lysis were gone, but respiration remained. Such fixed respiring mitochondria also show inhibition reactivation phenomena.
Binding, Competitive↗
The influence of propranolol on the thyrotropin receptor.
Propranolol, a beta adrenergic blocking drug, is known to inhibit the thyrotropin (TSH) stimulation of adenosine-3',5'-monophosphate (cyclic AMP production in thyroid membranes but the mechansim of this inhibitory action is known. We have therefore investigated the influence of propranolol on the binding of 125I-labelled TSH to human thyroid membranes. Both d- and l-propranolol were found to enhance the binding of 125I-labelled TSH to thyroid membranes. The amount of label bound increased from about 30% in the absence of propranolol to about 60% in the presence of 3.3 x 10(-3)M propranolol. Scatchard analysis of the binding data indicated that propranolol increased the association constant of the thyrotropin-thyrotropin receptor interaction. Practolol, lithium carbonate, methimazole, and somatostatin had no effect on thyrotropin binding. This effect of propranolol appeared to be due to a direct reversible action of propranolol on the thyroid membranes and could be attributed to the membrane-disrupting properties of the drug rather than its beta-blocking activity. The increased TSH receptor occupancy which resulted from the increased association constant of the TSH-thyroid membrane interaction corresponded with a decrease in TSH-stimulated cyclic AMP formation. These data could indicate that propranolol reduced the efficiency of the receptor-adenylyl cyclase coupling system.
Cell Membrane↗
Transport by the lactose permease of Escherichia coli as the basis of lactose killing.
Lactose killing is a peculiar phenomenon in which 80 to 98% of the Escherichia coli cells taken from a lactose-limited chemostat die when plated on standard lactose minimal media. This unique form of suicide is caused by the action of the lactose permease. Since uptake of either lactose or galactose by the lactose permease caused death, the action of rapid transport across the membrane must be the cause of the phenomenon. Alternative causes of lactose killing, such as accumulation of toxic metabolic intermediates or action of the beta-galactosidase, have been eliminated. It is proposed that rapid uptake of sugars by the lactose permease disrupts membrane function, perhaps causing collapse of the membrane potential.
Biological Transport↗
Proline transport carrier-defective mutants of Escherichia coli K-12: properties and mapping.
A series of mutants of Escherichia coli K-12 requiring a high concentration of L-proline for growth were isolated from a proline auxotroph strain, JE2133. Genetic studies of the mutants, PT19, PT21, and PT22, showed that all the mutations (proT) were point mutations, and these were mapped at 82 min on the E. coli genetic map. Intact cells and cytoplasmic membrane vesicles of these mutants were specifically defective in L-proline transport activity. Strain PT21 had no detectable activity of the L-proline transport carrier at all, and strains PT19 and PT22 had only 1/35 and 1/70, respectively, of the transport activity of the parental strain. The mutants were also shown to have a defect in proline-binding function of the carrier by measuring specific binding of proline to sonically disrupted membranes. These results indicate that the gene proT determines the function of proline carrier in the cytoplasmic membrane.
Bacterial Proteins↗
Unlocking antifungal mechanisms of natural 3-(oxazole-5-yl) indole compound derived from Streptomyces syringium against plant gray mold caused by Botrytis cinerea.
BACKGROUND: Plant fungal diseases cause significant agricultural losses, and Streptomyces-derived antifungal compounds offer a promising biocontrol strategy. This study aimed to isolate and characterize bioactive metabolites from Streptomyces syringium LZ036 and evaluate their activity and mechanism of action against Botrytis cinerea. RESULTS: A strain LZ036 with broad-spectrum antifungal activity was identified as Streptomyces syringium. The 3-(oxazole-5-yl) indole compound NL3 isolated from this strain exhibited potent broad-spectrum antifungal activity, especially against Botrytis cinerea. Compound NL3 inhibited fungal growth and development by inducing severe oxidative damage and membrane disruption. And it could trigger jasmonic acid (JA)-dependent induced systemic resistance (ISR) in plants. Transcriptomic analysis of compound NL3-treated Botrytis cinerea revealed genome-wide transcriptional alterations, including disruption of energy metabolism and mitochondrial function. Key genes related to mitogen-activated protein kinase (MAPK) signaling pathway down-regulated significantly, among which the catalytic S_TKc domain of Bcste7 exhibited a predicted interaction with compound NL3 through hydrophobic interactions and hydrogen bonding. CONCLUSION: The Streptomyces syringium-derived compound NL3 shows high potential as a green fungicide, acting through multiple mechanisms. These findings advance the development of Streptomyces-based antifungal agents. © 2026 Society of Chemical Industry.
3‐(oxazole‐5‐yl) indole compo↗