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Results for “cell deconvolution”

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PubMed1990

Estimation of in vivo reticuloendothelial system phagocytic activity in rats by direct blood clearance techniques and nuclear scintigraphy.

There has been considerable interest in the examination of reticuloendothelial system phagocytic blockade. In this study, the kinetics of phospholipid liposome-mediated and intraperitoneal silica-mediated phagocytic blockade were examined using five methods of analysis of in vivo 99mTc-labeled albumin clearance and reticuloendothelial cell uptake. Two direct blood sampling techniques revealed significant impairment in 99mTc-labeled albumin clearance after treatment with silica (P less than 0.05), while liposome treatment was not associated with such impairment. A method utilizing nuclear scintigraphy for the determination of blood clearance was incapable of detecting silica-mediated blockade but demonstrated significant impairment by liposomes at 2 hr (P less than 0.001), 6 hr (P less than 0.05), and 24 hr (P less than 0.001). Gamma camera imaging methods for determination of hepatic uptake demonstrated significant (P less than 0.05) and reversible impairment of 99mTc-labeled albumin uptake by liposomes. The most promising of these techniques utilizes deconvolutional analysis of liver region of interest time-activity curves to correct for continuously changing blood concentrations of tracer and for intracellular tracer processing and catabolism. Measurements of reticuloendothelial system phagocytic activity should include methods that take into account the observed discrepancies between blood clearance determinations and reticuloendothelial cell uptake.

Albumins↗

PubMed2006

Localization of antimicrobial peptides in normal and burned skin.

Severe burn causes immunosuppression, and the eschar remains a perfect culture medium for microbial growth. The resulting sepsis is a common complication of burns with a high mortality. The skin produces a number of molecules including antimicrobial peptides (AMPs) that act in the first line of host defense. Previous studies from our laboratory suggested decreased expression of human beta-defensin-2 (HBD-2) in burned wounds. Here, we have expanded our work by identifying HBD-1, HBD-2, HBD-3 and human neutrophil peptide (HNP) in normal and burn skin samples using fluorescence deconvolution microscopy. In normal skin, HBD-1 was localized to the perinuclear region of keratinocytes, while HBD-2 was seen primarily in the stratum basale of the epidermis. HBD-3 was found in dendritic cells of the stratum spinosum. HNP was distributed in a somewhat random pattern in the papillary dermis. In burned skin, in which the epidermis had been destroyed or disrupted, the presence of HBD-1 was localized to dermal glandular structures and hair shafts. HBD-2 was found in both the upper portions of the remaining keratin layers, and localized to lower, f-actin containing, acini-like structures, a pattern also evident with HBD-3. We conclude that although the upper layers of skin are destroyed and disrupted by burn, cells in the lower portions of the skin demonstrate an ability to synthesize most of the AMPs, thereby maintaining some barrier against infection. The results of these studies further contribute to an understanding of the role of AMPs in the pathophysiology of cutaneous burn and the possibility of using these sites for upregulation of AMP synthesis in the prevention of burn sepsis.

Burns↗

PubMed2006

Calcium-triggered exit of F-actin and IP(3) 3-kinase A from dendritic spines is rapid and reversible.

The structure of the actin cytoskeleton in dendritic spines is thought to underlie some forms of synaptic plasticity. We have used fixed and live-cell imaging in rat primary hippocampal cultures to characterize the synaptic dynamics of the F-actin binding protein inositol trisphosphate 3-kinase A (IP3K), which is localized in the spines of pyramidal neurons derived from the CA1 region. IP3K was intensely concentrated as puncta in spine heads when Ca(2+) influx was low, but rapidly and reversibly redistributed to a striated morphology in the main dendrite when Ca(2+) influx was high. Glutamate stimulated the exit of IP3K from spines within 10 s, and re-entry following blockage of Ca(2+) influx commenced within a minute; IP3K appeared to remain associated with F-actin throughout this process. Ca(2+)-triggered F-actin relocalization occurred in about 90% of the cells expressing IP3K endogenously, and was modulated by the synaptic activity of the cultures, suggesting that it is a physiological process. F-actin relocalization was blocked by cytochalasins, jasplakinolide and by the over-expression of actin fused to green fluorescent protein. We also used deconvolution microscopy to visualize the relationship between F-actin and endoplasmic reticulum inside dendritic spines, revealing a delicate microorganization of IP3K near the Ca(2+) stores. We conclude that Ca(2+) influx into the spines of CA1 pyramidal neurons triggers the rapid and reversible retraction of F-actin from the dendritic spine head. This process contributes to changes in spine F-actin shape and content during synaptic activity, and might also regulate spine IP3 signals.

Actins↗

PubMed1980

Electron probe microanalysis of biological soft tissues: principle and technique.

Electron probe microanalysis is a method based upon X-ray spectrometry used for analyzing the chemical element content of very small amounts of material. The concentration of electrolytes in the microenvironment of cells, in cells, and in intracellular organelles can be measured. The main difficulties in using this method in biological soft tissue lie in sample preparation and in proper interpretation of the data. Best tissue preparation seems to be to quench the sample and to analyze it either freeze dried in thin or ultrathin sections, or frozen hydrated in thin sections or bulk samples. In all cases analysis should be performed using a cold stage and an ultra clean vacuum in order to minimize mass loss due to beam damage and mass gain due to contamination trapping. Interpretation of the data relies upon the knowledge of both the localization of the volume excited by the electron beam and the origin of the continuum and characteristic X-ray signals received by the X-ray spectrometer. This knowledge can be complicated by two facts: 1) when the electron beam is used in an analytical mode, viewing of the analyzed microarea can be lost, and 2) the X-ray signals received by the spectrometer can originate not only from the volume directly excited by the electron beam but from areas that can be far apart, excited by the electron beam tail, scattered electrons or secondary fluorescence, particularly when using energy dispersive spectrometers. Theoretical quantitation of the results is well developed. Practical quantitation could be complicated by the possibility of mass gain, mass loss, standard inhomogeneity, non-uniformity of sample thickness, possibility of shrinkage during freeze drying and, when using energy dispersive spectrometry, by the low signal over background for low atomic number elements (Na), the possibility of overlap of characteristic X-ray lines, and the use of complex and empirical methods for background stripping and peak deconvolution. All these difficulties can be overcome, making electron probe microanalysis one of the most powerful tools available to the biologist.

Animals↗

PubMed2004

Quantitative optical imaging of the pharmacokinetics of fluorescent-specific antibodies to tumor markers through tissuelike turbid media.

Fluorescent optical imaging of tumors deep within tissue depends on specific binding of antibodies to the tumors' surface markers. These fluorescent antibodies propagating in the vicinity of the tumor can be attached to and (or) diffused away from it. We illustrate application of a new tool, based on the random-walk theory in turbid media, for extracting the pharmacokinetics of these fluorescent antibodies by data deconvolution, excluding the effect of upper turbid tissue layers.

Animals↗

PubMed2003

In vitro-in vivo correlation in man of a topically applied local anesthetic agent using numerical convolution and deconvolution.

The aim of this study was to evaluate the relevance of the in vitro permeation method used at our laboratory in predicting in vivo dermal and transdermal absorption. Two different emulsions, a submicron oil-in-water (o/w) emulsion and a semisolid water-in-oil (w/o) emulsion, containing a model compound were investigated. The in vitro permeation rate of the compound from these emulsions was measured using static diffusion cells with human skin as membrane. The emulsions were allowed to remain in contact with the skin in the donor chamber for 15, 60, and 240 min. The study was monitored for 240 min and the steady state flux was calculated. The systemic concentration of the compound was measured in vivo as a function of time after dermal application to healthy volunteers with 15 and 60 min of application. A short-lasting i.v. infusion study in healthy volunteers was used to simulate the i.v. bolus dose. Numerical convolution was used to predict the in vivo plasma concentration of the compound while the in vivo absorption rate of the compound was estimated using numerical deconvolution. To establish correlation, the predicted in vivo flux was compared with the corresponding observed in vitro parameter after adjusting for the lag time. No major differences were seen in the systemic plasma levels between the two emulsions, which is in close agreement with the steady state flux measured in vitro. A linear correlation representing a point-to-point relationship was established for each of the investigated formulations and application times. The longer application time was predicted more accurately for both emulsions.

Administration, Cutaneous↗

PubMed2003

Identification of SNARE complex modulators that inhibit exocytosis from an alpha-helix-constrained combinatorial library.

Synthetic peptides patterned after the proteins involved in vesicle fusion [the so-called SNARE (soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins] are potent inhibitors of SNARE complex assembly and neuronal exocytosis. It is noteworthy that the identification of peptide sequences not related to the SNARE proteins has not been accomplished yet; this is due, in part, to the structural constraints and the specificity of the protein interactions that govern the formation of the SNARE complex. Here we have addressed this question and used a combinatorial approach to identify peptides that modulate the assembly of the SNARE core complex and inhibit neuronal exocytosis. An alpha-helix-constrained, mixture-based, 17-mer combinatorial peptide library composed of 137180 sequences was synthesized in a positional scanning format. Peptide mixtures were assayed for their ability to prevent the formation of the in vitro -reconstituted SDS-resistant SNARE core complex. Library deconvolution identified eight peptides that inhibited the assembly of the SNARE core complex. Notably, the most potent 17-mer peptide (acetyl-SAAEAFAKLYAEAFAKG-NH2) abolished both Ca2+-evoked catecholamine secretion from detergent-permeabilized chromaffin cells and L-glutamate release from intact hippocampal primary cultures. Collectively, these findings indicate that amino acid sequences that prevent SNARE complex formation are not restricted to those that mimic domains of SNARE proteins, thus expanding the diversity of molecules that target neuronal exocytosis. Because of the implication of neurosecretion in the aetiology of several human neurological disorders, these newly identified peptides may be considered hits for the development of novel anti-spasmodic drugs.

Amino Acid Sequence↗

PubMed1987

Quantitative renal blood flow in urinary tract obstruction.

Technetium-labeled red blood cell scintigraphy using a bolus injection was used to obtain a noninvasive quantitative measurement of renal blood flow. The method used is an adaptation of the one used for calculating cerebral blood flow which involves deconvolution of the first pass of the isotope through the kidneys. The relative renal blood flow of 18 normal patients and 18 patients with unilateral urinary tract obstruction was performed. All patients with obstruction, but one, were reexamined after surgery. There was a significant difference between the normal and partially obstructed patients (p less than 0.001) and between those with partial and complete obstruction (p less than 0.01). After surgery a statistically observable improvement in the relative renal blood flow was obtained.

Adolescent↗

PubMed1999

Cell membrane orientation visualized by polarized total internal reflection fluorescence.

In living cells, variations in membrane orientation occur both in easily imaged large-scale morphological features, and also in less visualizable submicroscopic regions of activity such as endocytosis, exocytosis, and cell surface ruffling. A fluorescence microscopic method is introduced here to visualize such regions. The method is based on fluorescence of an oriented membrane probe excited by a polarized evanescent field created by total internal reflection (TIR) illumination. The fluorescent carbocyanine dye diI-C(18)-(3) (diI) has previously been shown to embed in the lipid bilayer of cell membranes with its transition dipoles oriented nearly in the plane of the membrane. The membrane-embedded diI near the cell-substrate interface can be fluorescently excited by evanescent field light polarized either perpendicular or parallel to the plane of the substrate coverslip. The excitation efficiency from each polarization depends on the membrane orientation, and thus the ratio of the observed fluorescence excited by these two polarizations vividly shows regions of microscopic and submicroscopic curvature of the membrane, and also gives information regarding the fraction of unoriented diI in the membrane. Both a theoretical background and experimental verification of the technique is presented for samples of 1) oriented diI in model lipid bilayer membranes, erythrocytes, and macrophages; and 2) randomly oriented fluorophores in rhodamine-labeled serum albumin adsorbed to glass, in rhodamine dextran solution, and in rhodamine dextran-loaded macrophages. Sequential digital images of the polarized TIR fluorescence ratios show spatially-resolved time-course maps of membrane orientations on diI-labeled macrophages from which low visibility membrane structures can be identified and quantified. To sharpen and contrast-enhance the TIR images, we deconvoluted them with an experimentally measured point spread function. Image deconvolution is especially effective and fast in our application because fluorescence in TIR emanates from a single focal plane.

Adsorption↗

PubMed2026

Artificial Intelligence-Driven Multi-Omics Analysis Reveals Hydroxytyrosol Targeting of the TXNIP-NLRP3 Inflammasome Axis in Traumatic Brain Injury.

Traumatic brain injury (TBI) induces secondary neuroinflammation driven by oxidative stress, inflammasome activation, and immune remodeling, yet specific mechanism-guided pharmacological interventions remain limited. This study established an artificial intelligence (AI)-integrated network pharmacology and multi-omics framework to evaluate whether hydroxytyrosol (HT), an olive-derived natural polyphenol, may regulate TBI-related neuroinflammatory targets centered on the TXNIP/NLRP3 inflammasome axis. Starting from the SMILES structure of HT, potential targets were predicted using PharmMapper, SwissTargetPrediction, and the Similarity Ensemble Approach and were standardized to UniProt identifiers. TBI-associated genes were integrated from GeneCards, DisGeNET, OMIM, and the Therapeutic Target Database. The overlapping target set was analyzed using STRING-based protein-protein interaction (PPI) networks, MCODE, CytoHubba, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. Public GEO transcriptomic datasets (GSE123831 and GSE104687) were used for cross-platform expression validation, differential expression analysis, and exploratory CIBERSORT-based immune infiltration estimation. Random forest (RF), multilayer perceptron (MLP), graph convolutional network (GCN), graph attention network (GAT), SHAP/LIME explainability analysis, LASSO inflammatory-risk scoring, and two-sample Mendelian randomization (MR) were further applied for target prioritization, immune phenotype mapping, and genetic association analysis. Seventy-three overlapping HT-TBI targets were identified. PPI and topology analyses prioritized TXNIP, NLRP3, CASP1, MAPK1, and TP53 as key hubs enriched in inflammasome activation, oxidative stress, apoptosis, and NOD-like receptor signaling. TXNIP, NLRP3, and CASP1 were consistently upregulated in both TBI transcriptomic datasets. LM22-based immune deconvolution suggested increased pro-inflammatory immune signatures and a positive TXNIP-M1 macrophage association (r = 0.63, p < 0.001), which should be interpreted as a transcriptome-derived hypothesis rather than validated murine immune-cell proportions. AI-based models consistently ranked TXNIP/NLRP3 as high-contribution features under internal validation, and removal of these targets reduced model performance. A five-gene inflammatory score achieved an internally evaluated AUC of 0.87, while two-sample MR supported positive genetic associations involving TXNIP expression, TBI risk, NLRP3 and IL-1β expression. Collectively, these findings prioritize the TXNIP/NLRP3/CASP1 module as a computationally supported candidate mechanism through which HT may influence oxidative stress-inflammasome-immune coupling in TBI. This study provides an interpretable drug-target-pathway-phenotype framework and identifies TXNIP, NLRP3, and CASP1 as priority nodes for future experimental validation.

Artificial Intelligence↗

PubMed2003

Identification of TNF-alpha inhibitors from a split-pool library based on a tyrosine-proline peptidomimetic scaffold.

The design and synthesis of a combinatorial library based on a 4-aryloxyproline scaffold with tyrosine as the aryl portion is described. The 1728 member library was prepared using the split-pool method to generate pools of compounds. Screening of the library components as mixtures followed by deconvolution led to the discovery of novel inhibitors of TNF-alpha induced apoptosis.

Apoptosis↗

PubMed1996

Structural organization of the major autolysin from Streptococcus pneumoniae.

LytA amidase is the best known bacterial autolysin. It breaks down the N-acetylmuramoyl-L-alanine bonds in the peptidoglycan backbone of Streptococcus pneumoniae and requires the presence of choline residues in the cell-wall teichoic acids for activity. Genetic experiments have supported the hypothesis that its 36-kDa chain has evolved by the fusion of two independent modules: the NH2-terminal module, responsible for the catalytic activity, and the COOH-terminal module, involved in the attachment to the cell wall. The structural organization of LytA amidase and of its isolated COOH-terminal module (C-LytA) and the variations induced by choline binding have been examined by differential scanning calorimetry and analytical ultracentrifugation. Deconvolution of calorimetric curves have revealed a folding of the polypeptide chain in several independent or quasi-independent cooperative domains. Elementary transitions in C-LytA are close but not identical to those assigned to the COOH-terminal module in the complete amidase, particularly in the absence of choline. These results indicate that the NH2-terminal region of the protein is important for attaining the native tertiary fold of the COOH terminus. Analytical ultracentrifugation studies have shown that LytA exhibits a monomer <--> dimer association equilibrium, through the COOH-terminal part of the molecule. Dimerization is regulated by choline interaction and involves the preferential binding of two molecules of choline per dimer. Sedimentation velocity experiments give frictional ratios of 1.1 for C-LytA monomer and 1.4 for C-LytA and LytA dimers; values that deviated from that of globular rigid particles. When considered together, present results give evidence that LytA amidase might be described as an elongated molecule consisting of at least four domains per subunit (two per module) designated here in as N1, N2, C1, and C2. Intersubunit cooperative interactions through the C2 domain in LytA dimer occur under all experimental conditions, while C-LytA requires the saturation of low affinity choline binding sites. The relevance of the structural features deduced here for LytA amidase is examined in connection with its biological function.

Calorimetry, Differential Scanning↗

PubMedDate not supplied

Ultrastructural abnormalities in cultured exostosis chondrocytes.

Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by inappropriate chondrocyte proliferation and bone growth arising at the juxtaepiphyseal region of the long bones. HME is caused by mutations in the EXT 1 and EXT 2 genes, which have glycosyltransferase activity. These genes are responsible for synthesis of heparan sulfate (HS) chains, which are important signaling molecules in chondrocyte differentiation. HME chondrocytes in monolayer culture have been shown by transmission electron and deconvolution microscopy to contain enormous bundles of actin, cross-linked with muscle specific alpha-actinin. Here additional ultrastructural anomalies in HME chondrocytes are reported, including lobulated nuclei, shortened channels of rER, large numbers of cell processes and podosomes, nontypical junctions, elongated, bulbous-ended mitochondria, and reduced extracellular matrix. Microfilaments are present throughout the cytoplasm, compartmentalizing it, and isolating organelles. The excess microfilaments, attributed to increased cell adhesiveness, are likely to interfere with secretion and cytokinesis, and sterically hinder intracellular organelle differentiation. The observed surface modifications and cytoskeletal abnormalities are proposed to play a role in development of the mutant phenotype, via changes in cell adhesiveness and/or binding of signals to receptors, which results in loss of the unidirectionality of growth in the epiphyseal plate.

Cell Nucleus↗

PubMed2006

Effect of a simple versus a complex matrix on the polarity of cardiomyocytes in culture.

OBJECTIVE: The objective of this study was to observe the effects of cell culture on cellular polarity in cardiomyocytes as influenced by cytoskeletal proteins. METHODS: Cardiomyocytes from adult and neonatal rats were isolated and grown on 2 different extracellular matrices--laminin and a complex, fibroblast-derived extracellular matrix, cardiogel. The location of a number of proteins was visualized by means of fluorescence deconvolution microscopy, using specific fluorescent probes for alpha-adrenergic receptors, beta-adrenergic receptors, the sarcolemmal L-type calcium channel, and the sodium + potassium adenosine triphosphatase pump protein. Intracellular migration of these proteins during the first 4 days of culture was followed and microscopic stacked images were reconstructed. A fluorescein isothiocyanate-labeled probe for actin was used to ensure that cardiomyocytes were being examined, based on protein patterns. RESULTS: We examined 2 types of myocyte: freshly isolated neonates and cultured adult cardiomyocytes that undergo dedifferentiation. Initial, perinuclear clumping (endoplasmic reticulum/Golgi-associated) of the probes with an ensuing spread to the cytoplasm and periphery, accompanied by a better organization and more rapid response to biochemical stimuli, was seen on the complex matrix. CONCLUSIONS: A complex matrix overcomes cell polarity at a faster rate than myocytes cultured on a simple matrix, although both culture matrices were able to support cell growth and differentiation, and single-layer cultures are a good method by which structural and biochemical data can be obtained. The use of a native, complex matrix is preferable to employing a simple, single protein, although temporal aspects of cell growth must be considered regarding the particular aspect of the cell structure development/biochemical pathways that the researcher intends focusing on.

Journal Article↗

PubMed1998

Procedures for whole-mount immunohistochemistry and in situ hybridization of immature mammalian CNS.

Whole-mount labeling techniques for staining in invertebrates or lower vertebrates cannot simply be applied to the mammalian central nervous system (CNS) because of its large size. Such techniques if possible would offer advantages over conventional methods based on sections since an immediate and 3-dimensional view of the stained components in a transparent CNS is provided. It thereby becomes possible to survey and count large number of cells and fibers in their natural relationships. The aim of our experiments is to follow developing and regenerating expression of proteins and mRNAs in the CNS of mouse embryos and newborn opossums (Monodelphis domestica). Accordingly, we have devised three techniques applicable to whole-mounts: (i) An effective immunohistochemical procedure. This comprises a peroxidase-antiperoxidase method (PAP-WM) based on protocols initially developed for Xenopus embryos and oocytes, including a variation to detect exogenously applied nucleotide analogs such as 5-bromo-2'-deoxyuridine (PAP[BrdU]-WM). For greater resolution we have introduced a novel gold-silver method (IGSS-WM). (ii) An in situ hybridization procedure (ISH[PAP]-WM) which combines PAP-WM with protocols described for Xenopus. (iii) A deconvolution (optical sectioning) procedure which improves resolution for bright-field microscopy. We show that reliable whole-mount staining can be obtained using isolated CNS aged up to mouse embryonic day 17 and newborn opossum up to 15 days. Examples are shown of preparations in which one can directly localize nerve cells containing neurotransmitters, cytoskeletal proteins, nucleotide analogs and growth factor messages.

Animals↗

PubMed2026

Multiregion profiling of genomic and transcriptional heterogeneity in head and neck squamous-cell carcinoma.

BACKGROUND: Intratumoral heterogeneity (ITH) is thought to contribute to tumour evolution and treatment resistance but its biological and clinical significance in localised head and neck squamous-cell carcinoma (HNSCC) remains incompletely understood. PATIENTS AND METHODS: In the prospective SCANDARE study, we analysed 87 patients with resectable HNSCC treated with upfront surgery. Two to five spatially distinct tumour regions per patient underwent pathological evaluation, targeted DNA sequencing, and bulk RNA sequencing. Genomic ITH (gITH) was quantified using clonal deconvolution and Shannon diversity indices, whereas transcriptional heterogeneity (tITH) was assessed using the intratumour expression distance metric. Associations between ITH, molecular features, tumour microenvironment composition, and clinical outcomes were explored using multivariable statistical models. RESULTS: Pathology-based spatial heterogeneity showed limited prognostic value. gITH was common, with 37% of tumours displaying regionally heterogeneous pathogenic variants, including spatially actionable alterations in 10% of patients. In an initial multivariable Cox model, higher gITH was associated with shorter disease-free survival. However, after Ridge-penalised modelling and bootstrap internal validation, the effect size was attenuated [corrected hazard ratio 1.42, 95% confidence interval (CI) 0.91-2.75]. The overall model retained moderate discriminative performance (optimism-corrected C-index 0.69, 95% CI 0.59-0.79). gITH was associated with tumour cellularity, reduced estimated endothelial cell infiltration, and alterations in KMT2C and PIK3CA. tITH differed according to human papillomavirus (HPV) status, with lower tITH in HPV-positive tumours, and was associated with distinct biological pathways and genomic alterations. Genomic and tITH were not correlated. CONCLUSIONS: This prospective multiregion study provides a comprehensive characterisation of genomic and tITH in localised HNSCC. Our findings highlight substantial spatial molecular diversity within primary tumours and suggest potential associations between heterogeneity, tumour biology, and clinical outcome that warrant validation in independent cohorts.

head and neck squamous-cell carcinoma (HNSCC)↗

PubMed1987

Influence of immune complexes on macrophage membrane fluidity: a nanosecond fluorescence anisotropy study.

Time-resolved fluorescence anisotropy (TRFA) and steady-state anisotropy measurements and fluorescence intensification microscopic observations were made on RAW264 macrophages labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) or 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Microscopic analysis revealed that the fluorescent probe DPH was found in association with plasma membranes and small vesicles. Macrophages treated with immune complexes could not be distinguished from untreated cells, indicating that the same membrane compartments were labeled. The probe TMA-DPH was exclusively localized to the plasma membrane. Steady-state anisotropy measurements indicated that in vitro culture conditions did not significantly affect membrane fluidity. TRFA measurements were conducted to determine the physical properties of macrophage membranes during immune recognition and endocytosis. Data were analyzed by iterative deconvolution to yield phi, the rotational correlation time, and r infinity, the limiting anisotropy. These parameters may be interpreted as the "fluidity" and order parameter of the membrane environment, respectively. Typical values for untreated macrophages were phi = 7.8 ns and r infinity = 0.12. Binding and endocytosis of immune complexes prepared in 4-fold antigen excess increase these values to phi = 22.1 ns and r infinity = 0.15. However, receptor-independent phagocytosis of latex beads decreases these values to phi = 2.2 ns and r infinity = 0.10. Addition of catalase before, but not after, immune complex incubation with cells diminishes the effect upon membrane structure, suggesting that H2O2 participates in fluidity changes. Pretreatment of macrophages with the membrane-impermeable sulfhydryl blocker p-(chloromercuri)benzenesulfonic acid also diminished these effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1998

Submaximal stimulation of porcine endothelial cells causes focal Ca2+ elevation beneath the cell membrane.

1. Endothelial cell activation is correlated with increased cytosolic Ca2+ concentration, often monitored with cytoplasmic Ca2+ dyes, such as fura-2 and Calcium Green-1. We tested the hypothesis that during weak stimulation of porcine coronary artery endothelial cells, focal, subplasmalemmal Ca2+ elevations occur which are controlled by cell membrane Na(+)-Ca2+ exchange near mitochondrial membrane and superficial endoplasmic reticulum (SER). 2. Bulk Ca2+ concentration ([Ca2+]b) was monitored using fura-2 or Calcium Green-1 and subplasmalemmal Ca2+ concentration ([Ca2+]sp) was determined with FFP-18. The distribution of the SER network was estimated using laser scanning and deconvolution microscopy. 3. Sodium fluoride (10 mmol l-1) and submaximal concentrations of bradykinin (Bk; 1 nmol l-1) stimulated Ca2+ entry with no increase in [Ca2+]b. Although inositol 1,4,5-trisphosphate formation and intracellular Ca2+ release in response to both stimuli were similar, Ca2+ entry in response to NaF exceeded that in response to 1 nmol l-1 BK by fourfold, suggesting additional effects of NaF on Ca+ entry pathways but stimulation via intracellular Ca2+ release. 4. Prevention of Na(+)-Ca2+ exchange activity by decreasing extracellular Na+ unmasked intracellular Ca2+ release in response to NaF and 1 nmol l-1 Bk, indicated by an increase in [Ca2+]b. Thereby, NaF depleted Bk-releasable Ca2+ pools, while mitochondrial Ca2+ content (released with FCCP or oligomycin) and the amount of Ca2+ stored within the cells (released with ionomycin) was increased compared with cells treated with NaF under normal Na+ conditions. The NaF-initiated increase in [Ca2+]b and depletion of Bk-releasable Ca2+ pool(s) in the low-Na+ condition was diminished by 25 mumol l-1 ryanodine, indicating the involvement of Ca(2+)-induced Ca2+ release (CICR). 5. In simultaneous recordings of [Ca2+]sp (with FFP-18) and [Ca2+]b (with Calcium Green-1), 1 nmol l-1 Bk or 10 mmol l-1 NaF yielded focal [Ca2+] elevation in the subplasmalemmal region with no increase in the perinuclear area. 6. Treatment with 10 mumol-1 nocodazole caused the SER to collapse and unmasked Ca2+ release in response to 1 nmol l-1 Bk and 10 mmol l-1 NaF, similar to low-Na+ conditions, while the effect of thapsigargin was not changed. 7. These data show that in endothelial cells, focal, subplasmalemmal Ca2+ elevations in response to small or slow IP3 formation occur due to vectorial Ca2+ release from the SER towards the plasmalemma followed by Ca2+ extrusion by Na(+)-Ca2+ exchange. While these local Ca2+ elevations are not detectable with Ca2+ dyes for the determination of [Ca2+]b, prevention of Ca2+ extrusion or SER disruption yields increases in [Ca2+]b partially due to CICR. 8. All of the data support our hypothesis that in weakly stimulated endothelial cells, intracellular Ca2+ release and [Ca2+] elevation are limited to the subplasmalemmal region. We propose that the SER co-operates with associated parts of the plasma membrane to control Ca2+ homeostasis, Ca2+ distribution and Ca2+ entry. The existence of such a subplasmalemmal Ca2+ control unit (SCCU) needs to be considered in discussions of Ca2+ signalling, especially when cytoplasmic Ca2+ dyes, such as fura-2 or Calcium Green-1, are used.

Animals↗