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Proteins that oxidize extracellular substrates in Gram-positive bacteria are poorly understood. Ferrimicrobium acidiphilum is an actinobacterium that respires aerobically on extracellular ferrous ions at pH 1.5. In situ absorbance measurements were conducted on turbid suspensions of intact Fm. acidiphilum using an integrating cavity absorption meter designed for that purpose. Initial velocity kinetic studies monitored the appearance of product ferric ions in the presence of catalytic quantities of cells. Cell-catalyzed iron oxidation obeyed the Michaelis-Menten equation with K m and V max values of 71 μM and 0.29 fmol/min/cell, respectively. Limited-turnover kinetic studies were conducted with higher concentrations of cells to detect and monitor changes in the absorbance properties of cellular redox proteins when the cells were exposed to limited quantities of soluble reduced iron. A single a -type cytochrome with reduced absorbance peaks at 448 and 605 nm was the only redox-active chromophore that was visible as the cells respired aerobically on iron. The reduced cytochrome 605 exhibited mathematical and correlational properties that were consistent with the hypothesis that oxidation of the cytochrome constituted the rate-limiting step in the aerobic respiratory process, with a turnover number of 35 ± 2 s -1 Genomic and proteomic analyses showed that Fm. acidiphilum could and did express only two a -type heme copper terminal oxidases. Cytochrome 605 was associated with the terminal oxidase gene that is located between nucleotides 31,090 and 33,039, inclusive, in the annotated circular genome of this bacterium. IMPORTANCE The identities and functions of proteins involved in aerobic respiration on extracellular ferrous ions at acidic pH are poorly understood in the four phyla of Gram-positive eukaryotes and archaea where such activities occur. In situ absorbance measurements were conducted on Fm. acidiphilum as it respired on extracellular iron using an integrating cavity absorption meter that permitted accurate optical measurements in turbid suspensions of the intact bacterium under physiological conditions. The significance of these measurements is that they permitted a direct spectrophotometric examination of the extents and rates of biological electron transfer events in situ under noninvasive physiological conditions without disrupting the complexity of the live cellular environment. One thing is certain: one way to understand how a protein functions in an intact organism is to actually observe that protein as it functions in the intact organism. This paper provides an example of just such an observation., (Copyright © 2020 American Society for Microbiology.)

Metal-dependent lysine deacetylases (KDACs) are involved in regulation of numerous biological and disease processes through control of post-translational acetylation. Characterization of KDAC activity and substrate identification is complicated by inconsistent activity of prepared enzyme and a range of multi-step purifications. We describe a simplified protocol based on two-step affinity chromatography. The purification method is appropriate for use regardless of expression host, and we demonstrate purification of several representative members of the KDAC family as well as a selection of mutated variants. The purified proteins are highly active and consistent across preparations., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Absorbance spectra were collected on 12 different live microorganisms, representing six phyla, as they respired aerobically on soluble iron at pH 1.5. A novel integrating cavity absorption meter was employed that permitted accurate absorbance measurements in turbid suspensions that scattered light. Illumination of each microorganism yielded a characteristic spectrum of electrochemically reduced colored prosthetic groups. A total of six different patterns of reduced-minus-oxidized difference spectra were observed. Three different spectra were obtained with members of the Gram-negative eubacteria. Acidithiobacillus , representing Proteobacteria, yielded a spectrum in which cytochromes a and c and a blue copper protein were all prominent. Acidihalobacter , also representing the Proteobacteria, yielded a spectrum in which both cytochrome b and a long-wavelength cytochrome a were clearly visible. Two species of Leptospirillum , representing the Nitrospirae, both yielded spectra that were dominated by a cytochrome with a reduced peak at 579 nm. Sulfobacillus and Alicyclobacillus , representing the Gram-positive Firmicutes, both yielded spectra dominated by a -type cytochromes. Acidimicrobium and Ferrimicrobium , representing the Gram-positive Actinobacteria, also yielded spectra dominated by a -type cytochromes. Acidiplasma and Ferroplasma , representing the Euryarchaeota, both yielded spectra dominated by a ba 3 -type of cytochrome. Metallosphaera and Sulfolobus , representing the Crenarchaeota, both yielded spectra dominated by the same novel cytochrome as that observed in the Nitrospirae and a new, heretofore unrecognized redox-active prosthetic group with a reduced peak at around 485 nm. These observations are consistent with the hypothesis that individual acidophilic microorganisms that respire aerobically on iron utilize one of at least six different types of electron transfer pathways that are characterized by different redox-active prosthetic groups. In situ absorbance spectroscopy is shown to be a useful complement to existing means of investigating the details of energy conservation in intact microorganisms under physiological conditions.

Electron transfer reactions among three prominent colored proteins in intact cells of Acidithiobacillus ferrooxidans were monitored using an integrating cavity absorption meter that permitted the acquisition of accurate absorbance data in suspensions of cells that scattered light. The concentrations of proteins in the periplasmic space were estimated to be 350 and 25 mg/ml for rusticyanin and cytochrome c, respectively; cytochrome a was present as one molecule for every 91 nm(2) in the cytoplasmic membrane. All three proteins were rapidly reduced to the same relative extent when suspensions of live bacteria were mixed with different concentrations of ferrous ions at pH 1.5. The subsequent molecular oxygen-dependent oxidation of the multicenter respiratory chain occurred with a single macroscopic rate constant, regardless of the proteins' in vitro redox potentials or their putative positions in the aerobic iron respiratory chain. The crowded electron transport proteins in the periplasm of the organism constituted an electron conductive medium where the network of protein interactions functioned in a concerted fashion as a single ensemble with a standard reduction potential of 650 mV. The appearance of product ferric ions was correlated with the reduction levels of the periplasmic electron transfer proteins; the limiting first-order catalytic rate constant for aerobic respiration on iron was 7,400 s(-1). The ability to conduct direct spectrophotometric studies under noninvasive physiological conditions represents a new and powerful approach to examine the extent and rates of biological events in situ without disrupting the complexity of the live cellular environment., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Alcohol abuse has been associated with increased susceptibility to pulmonary infection. It is not fully defined how alcohol contributes to the host defense compromise. Here primary human airway epithelial cells were cultured at an air-liquid interface to form a differentiated and polarized epithelium. This unique culture model allowed us to closely mimic lung infection in the context of alcohol abuse by basolateral alcohol exposure and apical live bacterial challenge. Application of clinically relevant concentrations of alcohol for 24 hours did not significantly alter epithelial integrity or barrier function. When apically challenged with viable Klebsiella pneumoniae , the cultured epithelia had an enhanced tightness which was unaffected by alcohol. Further, alcohol enhanced apical bacterial growth, but not bacterial binding to the cells. The cultured epithelium in the absence of any treatment or stimulation had a base-level IL-6 and IL-8 secretion. Apical bacterial challenge significantly elevated the basolateral secretion of inflammatory cytokines including IL-2, IL-4, IL-6, IL-8, IFN-γ, GM-CSF, and TNF-α. However, alcohol suppressed the observed cytokine burst in response to infection. Addition of adenosine receptor agonists negated the suppression of IL-6 and TNF-α. Thus, acute alcohol alters the epithelial cytokine response to infection, which can be partially mitigated by adenosine receptor agonists.

Optimal microbicidal activity of human polymorphonuclear leukocytes (PMN) relies on the generation of toxic agents such as hypochlorous acid (HOCl) in phagosomes. HOCl formation requires H2O2 produced by the NADPH oxidase, myeloperoxidase derived from azurophilic granules, and chloride ion. Chloride transport from cytoplasm into phagosomes requires chloride channels which include cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. However, the phagosomal targeting of CFTR in PMN has not been defined. Using human peripheral blood PMN, we determined that 95-99% of lysosomal-associated membrane protein 1 (LAMP-1)-positive mature phagosomes were CFTR positive, as judged by immunostaining and flow cytometric analysis. To establish a model cell system to evaluate CFTR phagosomal recruitment, we stably expressed enhanced green fluorescent protein (EGFP) alone, EGFP-wt-CFTR and EGFP-DF508-CFTR fusion proteins in promyelocytic PLB-985 cells, respectively. After differentiation into neutrophil-like cells, CFTR presentation to phagosomes was examined. EGFP-wt-CFTR was observed to associate with phagosomes and colocalize with LAMP-1. Flow cytometric analysis of the isolated phagosomes indicated that such a phagosomal targeting was determined by the CFTR portion of the fusion protein. In contrast, significantly less EGFP-DF508-CFTR was found in phagosomes, indicating a defective targeting of the molecule to the organelle. Importantly, the CFTR corrector compound VRT-325 facilitated the recruitment of DF508-CFTR to phagosomes. These data demonstrate the possibility of pharmacologic correction of impaired recruitment of mutant CFTR, thereby providing a potential means to augment chloride supply to the phagosomes of PMN in patients with cystic fibrosis to enhance their microbicidal function., (Copyright © 2013 S. Karger AG, Basel.)

Chloride anion is critical for hypochlorous acid (HOCl) production and microbial killing in neutrophil phagosomes. However, the molecular mechanism by which this anion is transported to the organelle is poorly understood. In this report, membrane-enclosed and functionally active phagosomes were isolated from human neutrophils by using opsonized paramagnetic latex microspheres and a rapid magnetic separation method. The phagosomes recovered were highly enriched for specific protein markers associated with this organelle such as lysosomal-associated membrane protein-1, myeloperoxidase (MPO), lactoferrin, and NADPH oxidase. When FITC-dextran was included in the phagocytosis medium, the majority of the isolated phagosomes retained the fluorescent label after isolation, indicative of intact membrane structure. Flow cytometric measurement of acridine orange, a fluorescent pH indicator, in the purified phagosomes demonstrated that the organelle in its isolated state was capable of transporting protons to the phagosomal lumen via the vacuolar-type ATPase proton pump (V-ATPase). When NADPH was supplied, the isolated phagosomes constitutively oxidized dihydrorhodamine 123, indicating their ability to produce hydrogen peroxide. The preparations also showed a robust production of HOCl within the phagosomal lumen when assayed with the HOCl-specific fluorescent probe R19-S by flow cytometry. MPO-mediated iodination of the proteins covalently conjugated to the phagocytosed beads was quantitatively measured. Phagosomal uptake of iodide and protein iodination were significantly blocked by chloride channel inhibitors, including CFTRinh-172 and NPPB. Further experiments determined that the V-ATPase-driving proton flux into the isolated phagosomes required chloride cotransport, and the cAMP-activated CFTR chloride channel was a major contributor to the chloride transport. Taken together, the data suggest that the phagosomal preparation described herein retains ion transport properties, and multiple chloride channels including CFTR are responsible for chloride supply to neutrophil phagosomes., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Cystic fibrosis (CF) lung disease has a unique profile of pathogens predominated by Pseudomonas aeruginosa (PsA) and Staphylococcus aureus (SA). These microorganisms must overcome host immune defense to colonize the CF lungs. Polymorphonuclear neutrophils are a major component of the host defense against bacterial infection. A crucial microbicidal mechanism is the production of oxidants including hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) by neutrophils to achieve efficient bacterial killing. To determine to what degrees various CF pathogens resist the oxidants relative to non-CF pathogens, we compared the susceptibility of PsA, SA, Burkholderia cepacia (BC), Klebsiella pneumoniae (KP), and Escherichia coli (EC) to various concentrations of H2O2 or HOCl, in vitro. The comparative oxidant-resistant profiles were established. Oxidant-induced damage to ATP production and cell membrane integrity of the microbes were quantitatively assessed. Correlation of membrane permeability and ATP levels with bacterial viability was statistically evaluated., Results: PsA was relatively resistant to both H2O2 (LD50 = 1.5 mM) and HOCl (LD50 = 0.035 mM). SA was susceptible to H2O2 (LD50 = 0.1 mM) but resistant to HOCl (LD50 = 0.035 mM). Interestingly, KP was extremely resistant to high doses of H2O2 (LD50 = 2.5-5.0 mM) but was very sensitive to low doses of HOCl (LD50 = 0.015 mM). BC was intermediate to resist both oxidants: H2O2 (LD50 = 0.3-0.4 mM) and HOCl (LD50 = 0.025 mM). EC displayed the least resistance to H2O2 (LD50 = 0.2-0.3 mM) and HOCl (LD50 = 0.015 mM). The identified profile of H2O2-resistance was KP > PsA > BC > EC > SA and the profile of HOCl-resistance PsA > SA > BC > EC > KP. Moreover, both oxidants affected ATP production and membrane integrity of the cells. However, the effects varied among the tested organisms and, the oxidant-mediated damage correlated differentially with the bacterial viability., Conclusions: The order of HOCl-resistance identified herein best fits the clinical profile of CF infections. Even though oxidants are able to disrupt ATP production and cell membrane integrity, the degrees of damage vary among the organisms and correlate differentially with their viability.

Biosynthesis of hypochlorous acid, a potent antimicrobial oxidant, in phagosomes is one of the chief mechanisms employed by polymorphonuclear neutrophils to combat infections. This reaction, catalyzed by myeloperoxidase, requires chloride anion (Cl(-)) as a substrate. Thus, Cl(-) availability is a rate-limiting factor that affects neutrophil microbicidal function. Our previous research demonstrated that defective CFTR, a cAMP-activated chloride channel, present in cystic fibrosis (CF) patients leads to deficient chloride transport to neutrophil phagosomes and impaired bacterial killing. To confirm this finding, here we used RNA interference against this chloride channel to abate CFTR expression in the neutrophil-like cells derived from HL60 cells, a promyelocytic leukemia cell line, with dimethyl sulfoxide. The resultant CFTR deficiency in the phagocytes compromised their bactericidal capability, thereby recapitulating the phenotype seen in CF patient cells. The results provide further evidence suggesting that CFTR plays an important role in phagocytic host defense., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Alcohol abuse is associated with immunosuppressive and infectious sequelae. Particularly, alcoholics are more susceptible to pulmonary infections. In this report, gene transcriptional profiles of primary human airway epithelial cells exposed to varying doses of alcohol (0, 50, and 100 mM) were obtained. Comparison of gene transcription levels in 0 mM alcohol treatments with those in 50 mM alcohol treatments resulted in 2 genes being upregulated and 16 genes downregulated by at least 2-fold. Moreover, 0 mM and 100 mM alcohol exposure led to the upregulation of 14 genes and downregulation of 157 genes. Among the upregulated genes, glucocorticoid-induced leucine zipper (GILZ) responded to alcohol in a dose-dependent manner. Moreover, GILZ protein levels also correlated with this transcriptional pattern. Lentiviral expression of GILZ small interfering RNA in human airway epithelial cells diminished the alcohol-induced upregulation, confirming that GILZ is indeed an alcohol-responsive gene. Gene silencing of GILZ in A549 cells resulted in secretion of significantly higher amounts of inflammatory cytokines in response to IL-1beta stimulation. The GILZ-silenced cells were more resistant to alcohol-mediated suppression of cytokine secretion. Further data demonstrated that the glucocorticoid receptor is involved in the regulation of GILZ by alcohol. Because GILZ is a key glucocorticoid-responsive factor mediating the anti-inflammatory and immunosuppressive actions of steroids, we propose that similar signaling pathways may play a role in the anti-inflammatory and immunosuppressive effects of alcohol.

Chloride serves as a critical component of innate host defense against infection, providing the substrate for MPO-catalyzed production of HOCl in the phagosome of human neutrophils. Here, we used halide-specific fluorescent sensors covalently coupled to zymosan particles to investigate the kinetics of chloride and iodide transport in phagosomes of human neutrophils. Using the self-ratioable fluorescent probe specific for chloride anion, we measured chloride dynamics within phagosomes in response to extracellular chloride changes by quantitative fluorescence microscopy. Under the experimental conditions used, normal neutrophils showed rapid phagosomal chloride uptake with an initial influx rate of 0.31 +/- 0.04 mM/s (n=5). GlyH-101, a CFTR(inh), decreased the rate of uptake in a dose-dependent manner. Neutrophils isolated from CF patients showed a significantly slower rate of chloride uptake by phagosomes, having an initial influx rate of 0.043 +/- 0.012 mM/s (n=5). Interestingly, the steady-state level of chloride in CF phagosomes was approximately 26 mM, significantly lower than that of the control ( approximately 68 mM). As CFTR transports chloride as well as other halides, we conjugated an iodide-sensitive probe as an independent approach to confirm the results. The dynamics of iodide uptake by neutrophil phagosomes were monitored by flow cytometry. CFTR(inh)172 blocked 40-50% of the overall iodide uptake by phagosomes in normal neutrophils. In a parallel manner, the level of iodide uptake by CF phagosomes was only 20-30% of that of the control. Taken together, these results implicate CFTR in transporting halides into the phagosomal lumen.

Chloride anion is essential for myeloperoxidase (MPO) to produce hypochlorous acid (HOCl) in polymorphonuclear neutrophils (PMNs). To define whether chloride availability to PMNs affects their HOCl production and microbicidal capacity, we examined how extracellular chloride concentration affects killing of Pseudomonas aeruginosa (PsA) by normal neutrophils. PMN-mediated bacterial killing was strongly dependent on extracellular chloride concentration. Neutrophils in a chloride-deficient medium killed PsA poorly. However, as the chloride level was raised, the killing efficiency increased in a dose-dependent manner. By using specific inhibitors to selectively block NADPH oxidase, MPO, and cystic fibrosis transmembrane conductance regulator (CFTR) functions, neutrophil-mediated killing of PsA could be attributed to three distinct mechanisms: CFTR-dependent and oxidant-dependent; chloride-dependent but not CFTR- and oxidant-dependent; and independent of any of the tested factors. Therefore, chloride anion is involved in oxidant- and nonoxidant-mediated bacterial killing. We previously reported that neutrophils from CF patients are defective in chlorination of ingested bacteria, suggesting that the chloride channel defect might impair the MPO-hydrogen peroxide-chloride microbicidal function. Here, we compared the competence of killing PsA by neutrophils from normal donors and CF patients. The data demonstrate that the killing rate by CF neutrophils was significantly lower than that by normal neutrophils. CF neutrophils in a chloride-deficient environment had only one-third of the bactericidal capacity of normal neutrophils in a physiological chloride environment. These results suggest that CFTR-dependent chloride anion transport contributes significantly to killing PsA by normal neutrophils and when defective as in CF, may compromise the ability to clear PsA.

Tissue injury enhances homing and engraftment of mesenchymal stem cells (MSCs). However, the mechanisms by which MSCs sense the signals released by injured tissues and migrate toward injury sites have not been fully defined. In the current report, we investigated whether human MSCs express the N-formyl peptide receptor (FPR) and the formyl peptide receptor-like-1 (FPRL1). These receptors bind to N-formylated peptides by which phagocytes migrate to inflammatory sites and fibroblasts repopulate wounds to remodel the damaged tissues. Reverse-transcription polymerase chain reaction (PCR) demonstrated that MSCs express both FPR and FPRL1 at the transcriptional level. Flow cytometric analyses revealed expression of both receptors at the protein level. Fusion of the enhanced green fluorescence protein (eGFP) to the C terminus of each receptor showed localization to the cell surface. Moreover, MSCs responded to stimulation by N-formyl methionyl leucyl phenylalanine (fMLP), a prototypic N-formyl peptide, demonstrating rapid intracellular calcium mobilization that can be blocked by pertussis toxin or cyclosporin H. It is noteworthy that the fMLP-stimulated MSCs had an enhanced adhesion to extracellular matrix protein-coated surfaces. In addition, MSCs migrated toward gradients of increasing fMLP concentration, indicating that the receptors were functionally involved in positive chemotaxis to formylated peptides. Therefore, the N-formyl peptide receptors present in MSCs may play an important role in signaling stem cell adhesion, migration, and homing to injured and inflamed tissue for repair. Such a mechanism could potentially be exploited to direct the stem cells to target specific tissue sites, such as cystic fibrosis lungs, for therapy. Disclosure of potential conflicts of interest is found at the end of this article.

Production of hypochlorous acid (HOCl) in neutrophils, a critical oxidant involved in bacterial killing, requires chloride anions. Because the primary defect of cystic fibrosis (CF) is the loss of chloride transport function of the CF transmembrane conductance regulator (CFTR), we hypothesized that CF neutrophils may be deficient in chlorination of bacterial components due to a limited chloride supply to the phagolysosomal compartment. Multiple approaches, including RT-PCR, immunofluorescence staining, and immunoblotting, were used to demonstrate that CFTR is expressed in resting neutrophils at the mRNA and protein levels. Probing fractions of resting neutrophils isolated by Percoll gradient fractionation and free flow electrophoresis for CFTR revealed its presence exclusively in secretory vesicles. The CFTR chloride channel was also detected in phagolysosomes, a special organelle formed after phagocytosis. Interestingly, HL-60 cells, a human promyelocytic leukemia cell line, upregulated CFTR expresssion when induced to differentiate into neutrophils with DMSO, strongly suggesting its potential role in mature neutrophil function. Analyses by gas chromatography and mass spectrometry (GC-MS) revealed that neutrophils from CF patients had a defect in their ability to chlorinate bacterial proteins from Pseudomonas aeruginosa metabolically prelabeled with [(13)C]-l-tyrosine, unveiling defective intraphagolysosomal HOCl production. In contrast, both normal and CF neutrophils exhibited normal extracellular production of HOCl when stimulated with phorbol ester, indicating that CF neutrophils had the normal ability to produce this oxidant in the extracellular medium. This report provides evidence which suggests that CFTR channel expression in neutrophils and its dysfunction affect neutrophil chlorination of phagocytosed bacteria.

Human neutrophils synthesize hypochlorous acid, a nonradical oxidant, as one of the antimicrobial agents to kill phagocytosed pathogens within phagolysosomes. The production of HOCl is catalyzed by myeloperoxidase using chloride anions (Cl-). Even though various approaches have been documented to measure cytosolic Cl- concentrations, direct detection of phagolysosomal free Cl- in a real-time fashion is not available. Here we report the development of a new dual-labeled fluorescent probe by conjugation of the chloride-sensitive 6-methoxyquinoline and chloride-insensitive tetramethylrhodamine to the zymosan particles. On the surface of the zymosan particles, the chloride-sensitive fluorophore responded linearly to chloride concentrations when plotted according to the Stern-Volmer collisional quenching equation and the chloride-insensitive fluorophore served as an internal reference. After phagocytosis into neutrophils, the probe was able to monitor intraphagosomal chloride levels authentically. Human neutrophils in the medium containing 122 mM chloride had a phagolysosomal chloride level of 73.3 +/- 12.2 mM (N = 5). In contrast, the neutrophils in a chloride-free isoosmotic medium had a chloride level of 6.6 +/- 1.9 mM (N = 5) in the phagolysosomal compartment. These data clearly demonstrated that the extracellular chloride levels affect intraphagolysosomal chloride levels in human neutrophils. Moreover, when the extracellular chloride concentration was switched from high level (122 mM) to low level (0 mM) or vice versa, the probe demonstrated complete reversibility. The data indicate that the new zymosan-conjugated chloride probe described here can be used as an indicator for measurement of the free chloride level within the phagolysosomes of neutrophils and other phagocytic cell types.

Integration of a therapeutic gene into the host cell genome permits stable expression of the gene product in the target cells and its progeny. However, non-directional integration of any given gene can pose the risk of activating tumor genes or silencing tumor suppressor genes. Therefore, including a safety-control element into integrating vector systems is an important advance towards safer human gene therapy. Here, we report on a gene expression cassette that can be potentially exploited in integrating vector systems to eliminate post-therapeutic tumorigenesis. The Herpes simplex virus thymidine kinase (hsvTK) gene under the transcriptional control of the human telomere reverse transcriptase promoter (hTERTp) was incorporated into a self-inactivating HIV-based lentiviral vector. The hTERT promoter is silent in normal somatic cells and re-activated in tumor cells. Therefore, normal gene-corrected cells should not express hsvTK from the promoter. However, if some gene-corrected cells subsequently become tumorigenic and the hTERT promoter is re-activated, application of ganciclovir (GCV), a clinically used antiviral drug, will achieve selective deletion of the cancerous cells. Our experimental data indicated that the hTERTp-hsvTK cassette in the lentiviral vector was sufficient to differentiate between tumor cells and normal cells, thus eradicating tumor cells selectively in vitro and in vivo. These results proved the principle of using the element in integrating vectors for safer gene delivery., ((Cancer Sci 2005; 96: 607 -613).)

Cystic fibrosis (CF), the most prevalent, fatal genetic disorder in the Caucasian population, is caused by mutations of CF transmembrane conductance regulator (CFTR). The mutations of this chloride channel alter the transport of chloride and associated liquid and thereby impair lung defenses. Patients typically succumb to chronic bacterial infections and respiratory failure. Restoration of the abnormal CFTR function to CF airway epithelium is considered the most direct way to treat the disease. In this report, we explore the potential of adult stem cells from bone marrow, referred to as mesenchymal or marrow stromal stem cells (MSCs), to provide a therapy for CF. We found that MSCs possess the capacity of differentiating into airway epithelia. MSCs from CF patients are amenable to CFTR gene correction, and expression of CFTR does not influence the pluripotency of MSCs. Moreover, the CFTR-corrected MSCs from CF patients are able to contribute to apical Cl(-) secretion in response to cAMP agonist stimulation, suggesting the possibility of developing cell-based therapy for CF. The ex vivo coculture system established in this report offers an invaluable approach for selection of stem-cell populations that may have greater potency in lung differentiation.

We examined the effect of daily fat supplementation on intestinal gene expression and protein synthesis and plasma levels of apolipoprotein A-IV (apo A-IV). Rats were fasted overnight and then given intragastric bolus infusion of either saline or fat emulsion after 0, 1, 2, 4, 8, or 16 days of similar daily feedings. Four hours after the final saline or fat infusion, plasma and jejunal mucosa were harvested; plasma levels of apo A-IV, triglycerides, and leptin were measured, as well as mucosal apo A-IV mRNA levels and biosynthesis of apo A-IV protein. In response to fat, plasma apo A-IV showed an initial 40% increase compared with saline-injected control rats; with continued daily fat feeding, the plasma A-IV response showed rapid and progressive diminution such that by 4 days, plasma A-IV was not different between fat- and saline-fed groups. Jejunal mucosal apo A-IV synthesis and mRNA levels also showed time-dependent refractoriness to fat feeding. However, the kinetics of this effect were considerably slower than in the case of plasma, requiring 16 days for completion. There was no correlation between plasma leptin or triglyceride levels and intestinal apo A-IV synthesis or plasma apo A-IV. These results indicate rapid, fat-induced, posttranslational adapation of plasma apo A-IV levels and a slower, but similarly complete pretranslational adaptation of intestinal apo A-IV production, which are independent of plasma levels of leptin.

We examined the effect of ileal infusions of lipid emulsion on mRNA levels and biosynthesis of apolipoprotein A-IV (apo A-IV) in jejunal Thiry-Vella fistulas in rats. The rats were surgically prepared with jejunal Thiry-Vella fistulas; after recovery they were deprived of food, equipped with ileal infusion cannulas, then given 8 hr ileal infusions of fatty acid/monoglyceride emulsions. Mucosal synthesis and transcript levels of apo A-IV in the Thiry-Vella loop were then measured. Lipid infusion produced a two-fold stimulation in incorporation of 3H-leucine into apo A-IV-specific protein, but had no significant effect on apo A-IV mRNA levels. These results support the hypothesis that a lipid-elicited, distal gut-derived, systemic signal stimulates the production of apo A-IV by a post-transcriptional mechanism.

Neutrophil responses to alpha-N-formyl-L-Met-L-Leu-L-Phe (fMLF) are modulated by inhibitors of surface membrane neutral endopeptidase (NEP), such as phosphoramidon (PPAD). Because receptor recycling is presumably required for a sustained cellular response, the effect of PPAD on receptor reexpression was examined. After down-regulation of surface fMLF receptors by fMLF, PPAD blocked the normal reexpression of surface receptors in a manner that was related to the time of prior exposure to fMLF. Internalized fML[3H]F was hydrolyzed by NEP at a rate comparable to the rate of receptor reexpression at the cell surface, suggesting that ligand hydrolysis is rate limiting. To test this hypothesis, cells were incubated with fluorescein-labeled formyl-Met-Leu-Phe-Nle-Tyr-Lys at 15 degrees C. After binding was complete, but before internalization of receptor-ligand complexes, high-affinity antifluorescein antibody F(ab')2 fragments were added and the cells incubated at 37 degrees C for 60 min in the presence of PPAD. Under these conditions, the inhibitory effects of PPAD were largely reversed and nonimmune F(ab')2 fragments were without effect.

The regulation of prostacyclin (PGI2) synthesis by cultured human umbilical vein endothelium (HUVEC) was investigated. HUVEC monolayer generation of PGI2 was monitored by RIA of 6-keto PGF1 alpha and dose-dependent increases observed with human alpha- and gamma-thrombins, histamine, or arachidonate. Alpha thrombin (10 nM) produced levels of 6-keto PGF1 alpha approximating responses with 1 microM gamma-thrombin, 5 microM arachidonate, or 10 microM histamine. Diisopropyl phosphorofluoridate-inactivated alpha-thrombin did not stimulate PGI2 release, demonstrating that catalytic activity was required for thrombin-stimulated PGI2 release. Sodium fluoride (NaF), at concentrations known to activate guanine nucleotide regulatory proteins (G proteins), directly stimulated HUVEC PGI2 synthesis in a dose-dependent and time-dependent manner (20 mM NaF, 4.4 +/- 0.5-fold increase at 10 min, 11.9 +/- 1.5-fold increase at 30 min). Neither alpha-thrombin nor NaF-stimulated PGI2 release was dependent upon the availability of extracellular Ca++). The hypothesis that G proteins are involved in agonist-stimulated PGI2 synthesis was further supported by studies using digitonin-permeabilized HUVEC monolayers challenged with another G protein activator, guanosine 5'-0-3-thiotrisphosphate (GTP gamma S), which effected significant dose-dependent increases in PGI2 synthesis compared with control levels of 6-keto PGF1 alpha. In contrast, the G-protein inhibitor GDP beta S, (guanosine 5'-0-2-thiodiphosphate), attenuated alpha-thrombin-mediated prostaglandin generation. Treatment of HUVEC monolayers with pertussis toxin (1 microgram/ml) did not inhibit the PGI2 synthesis stimulated by either alpha-thrombin, NaF, or histamine but catalyzed the ADP ribosylation of a 40 kDa membrane protein which cross-reacted with antisera against a synthetic peptide corresponding to an amino acid sequence common to the alpha-subunit of other G-proteins. Preincubation of HUVEC microsomal membranes with alpha-thrombin diminished pertussis toxin-catalyzed ADP ribosylation in a time-dependent manner. These data suggest that thrombin stimulation of PGI2 synthesis by HUVEC monolayers requires the catalytically functional enzyme and further suggests that the thrombin-occupied receptor is coupled to phospholipase activities by a pertussis toxin-insensitive guanine nucleotide regulatory protein in human endothelial cell membranes.

We have compared the kinetics of the responses of neutrophils to the kinetics of ligand-receptor interaction and internalization, using as a model ligand the fluoresceinated hexapeptide N-CHO-Nle-Leu-Phe-Nle-Tyr-Lys-Fluorescein (Nle, norleucine). Cellular responses, ie, membrane depolarization, enzyme (elastase) secretion, and superoxide anion (O-2) generation, are all initiated within 10 sec of the exposure of cells to stimulus. In the cases of membrane depolarization and secretion (in cytochalasin B-treated cells), full responses are elicited by binding which occurs within 15 sec of peptide addition. Ligand binding and internalization have been analyzed over the same time frame with new spectroscopic techniques. The association of ligand and receptor is monitored using an antibody to fluorescein. The antibody to fluorescein specifically quenches the ligand which is in solution, but receptor-bound ligand is inaccessible to the antibody. The internalization of the receptor-bound ligand is monitored by the accessibility of the fluoresceinated peptide to quenching by an external pH change (7.4 leads to 4.0). Ligand which is either outside or on the cell surface is instantaneously quenched while intracellular peptide (or intracellular fluorescein derived from fluorescein diacetate) is only slowly quenched. No internalization is observed until 1 min after binding begins and internalization proceeds at a rate of up to 5,000 receptors/min/cell following a near optimal stimulatory ligand concentration (approximately 1 nM) while the occupied receptors are being cleared from the surface. A comparison of the kinetics of internalization and the cellular responses suggests that internalization of the ligand is too slow to be involved in the triggering of the cellular responses.

Experiments were performed to examine how human granulocytes process the chemotactic peptide N-formyl-Met-Leu-Phe after stimulation by the same peptide. Purified human granulocytes were stimulated with 50 nM N-formyl-Met-Leu-[3H]Phe at 37 degrees C for various times, washed, lysed by N2 cavitation, and fractionated by isopycnic sucrose density gradient sedimentation. The major subcellular fractions identified were plasma membrane, Golgi, granules, endoplasmic reticulum, and mitochondria. After 1 min of stimulation, radioactivity was found only in the plasma membrane (sedimentable) and cytosol (soluble) fraction. At 5, 10, and 25 min, radioactivity also appeared in a sedimentable, low density fraction (25-28% sucrose) enriched in galactosyl transferase activity and containing Golgi structures. The accumulation in the sedimentable fractions was maximal after 5 min but continued to increase linearly in the cytosol fraction. Incorporation of radioactivity into cells or membrane and soluble fractions was 60 to 85% specific and was inhibited if incubation with N-formyl-Met-Leu-[3H]Phe was performed at 4 degrees C. 80-90% of the radiolabel in the plasma membrane or Golgi-containing fractions remained sedimentable despite freeze thawing or sonication. Solubilization of these fractions in Triton X-100 followed by Sepharose 4B column chromatography revealed that the radiolabel eluted in the void volume. Our results are consistent with internalization which proceeds by passage of an occupied receptor in a high affinity, supramolecular complex from the plasma membrane to the Golgi followed by accumulation of peptide in the cytosol.

Antifluorescein antibody molecules were used to interrupt the stimulation of neutrophils by a fluoresceinated chemotactic peptide. From the results we construct a semiquantitative relationship among ligand-receptor interaction, the time course of cell triggering and response, and aspects of cellular adaptation. The interaction of the antibody with the free fluoresceinated peptide is complete within a few seconds and the peptide-antibody complex neither stimulates the cells nor inhibits subsequent stimulation by unlabeled peptide. When antibody is added to a cell suspension that has been stimulated with the fluoresceinated peptide, we observe that: (i) the apparent membrane depolarization response monitored by a fluorescent dye can be inhibited only if antibody is added within 30 sec of stimulation; (ii) the superoxide response can be inhibited even if antibody is added more than 1 min after stimulation and decays with an intrinsic half-life of about 12 sec; (iii) responses to a second dose of nonfluoresceinated peptide are enhanced if the antibody is added within 2 min of stimulation by the fluoresceinated peptide. These results suggest that different neutrophil responses depend in individual ways on the time course and extent of ligand binding to its receptor. A comparison of these data with the time course of binding permits an estimate of the number of receptors involved in these responses.

Neutrophil homogenates contained a high affinity guanosine triphosphatase (GTPase) that was stimulatable (+27%) by the addition of 100 nM N-formyl chemotactic peptide (CHO-pep), but not by 1 microgram X ml-1 phorbolmyristate acetate (PMA). Kinetic analysis of the stimulation demonstrated an apparent lagtime of 14.3 +/- 6.9 s between the addition of CHO-pep and the optimal GTPase stimulation. The GTPase activity (but not CHO-pep-stimulated GTPase activity) was preserved in a highly purified plasma membrane fraction of the homogenate. From these observations we suggest that both a high affinity guanine nucleotide binding protein and GTPase are closely associated with the plasma membrane CHO-pep receptor. The possibility that GTPase activity may influence guanine nucleotide regulation of adenylate cyclase during CHO-pep stimulation of neutrophils is discussed.

Neutral endopeptidase (EC 3.4.24.11, NEP) is an integral membrane protein of human neutrophils. NEP is identical with the common acute lymphoblastic leukemia antigen (CALLA) of leukemic cells. The expression of NEP on the surface of neutrophils is down-regulated by endocytosis which can be induced by phorbol 12-myristate 13-acetate (PMA) at 37 degrees C. The activity of the enzyme on the surface of intact cells decreases by 76% within 5 min. The activity can be recovered, however, if the cells are lysed within 5 min of the endocytosis. After 30 min, only 32% of the NEP activity is present in the neutrophil lysates. The loss of activity is presumably due to proteolytic inactivation. Diacylglycerol and monoclonal antibody to CALLA/NEP also induce internalization of NEP. PMA induces endocytosis even at 4 degrees C, but NEP is not inactivated at that temperature. The disappearance of NEP activity after adding PMA was inhibited by various agents. Among the most active were the phospholipase inhibitor 4-bromophenacyl bromide and a combination of the serine protease and cathepsin inhibitors, diisopropylfluorophosphate and N-ethylmaleimide. The employment of fluorescent monoclonal antibody confirmed the down-regulation and internalization of NEP antigen on the neutrophils. Since NEP inactivates chemotactic peptides and thereby affects chemotaxis of neutrophils (Painter, R. G., Dukes, R., Sullivan, J., Carter, R., Erdös, E. G., and Johnson, A. R. (1988) J. Biol. Chem. 263, 9456-9461), the down-regulation of NEP activity on the cell membrane may modulate the function of these cells in inflammation.

Human granulocytes were disrupted by nitrogen cavitation and the lysates fractionated by sucrose density gradient centrifugation at 83000 X g for 20 min (rate zonal) or 3.5 h (isopycnic). The distribution of marker enzymes allowed the identification of the following subcellular components: plasma membrane, Golgi, endoplasmic reticulum, azurophil granules, specific granules, mitochondria and cytosol. Examination of the gradient fractions by electron microscopy confirmed the biochemical marker analysis. The protocol permitted isolation of vesicles highly enriched in either plasma membrane or Golgi (galactosyl transferase) activities. Absolute plasma membrane yields of 40-60% were achieved with a 20-70-fold increase in specific activity of surface marker over the cells. Plasma membrane sedimented to an average density of 1.14 g X cm-3. Galactosyl transferase activity was bimodal in distribution. The denser peak cosedimented with specific granules (p = 1.19). The lighter peak sedimented to unique position at an average density of 1.11, was enriched 18-fold over the low speed supernatant, and contained structures resembling Golgi. N-Formyl-Met-Leu-Phe binding and Mg2+-ATPase activities cosedimented with the plasma membrane as well as specific granule and/or high density galactosyl transferase fractions. These findings suggest that Mg2+-ATPase and N-formyl chemotactic peptide receptor activities may be localized in an internal pool of membranes as well as in the plasma membrane and that Golgi may have been a contaminant of previous granulocyte plasma membrane or specific granule preparations.

When intact platelets are incubated at 37 degrees C with Concanavalin A (ConA), the two major surface membrane proteins GPIIb and III become associated with the Triton-insoluble cytoskeleton. Preincubation of platelets with a variety of metabolic inhibitors, including cytochalasin B, 2-deoxy-D-glucose, and antimycin A or lidocaine, had no effect on the ability of ConA to produce this effect. These results suggested that the ConA-induced anchorage of GPIIb and III to the Triton-insoluble cytoskeleton is a passive process requiring clustering of GPIIb-III molecules but not requiring the metabolic energy of an intact cell. This was supported by experiments that showed that ConA binding to plasma membrane-rich fractions at 37 degrees C could induce association of GPIIb and III with a sedimentable actin-rich, Triton-insoluble membrane matrix. Similar results were obtained when membranes were first isolated from ConA-treated cells. Adding DNAse I, an actin depolymerizing agent, into the Triton extraction buffer inhibited the ConA-induced sedimentation of GPIIb-III and actin by 50% in the presence of Mg2+-ATP. Treatment of ConA-treated membranes with dimethyl-3,3'-dithiobispropiomidate, a bifunctional, reducible protein crosslinking agent, produced Triton-insoluble crosslinked species of discrete molecular weights. When these cross-linked species were analyzed by SDS-PAGE in the presence of beta-mercaptoethanol, they were found to be composed of a 180-200 K dalton protein, GPIIb, GPIII, and actin. Crosslinking of these components was equally effective after Triton treatment and indicated as well that the species crosslinked in the intact membrane was stable after Triton extraction. Addition of crosslinker to membranes not treated with ConA produced similar crosslinked species. Analysis of their composition on reduced gels revealed that the amounts of GPIIb and III were reduced greatly (less than 10% of the total input GPIIb and III) but that the 180-200 k dalton protein and actin content were similar to that seen with ConA-treated membranes. These results are consistent with the notion that ConA clusters mobile, unanchored molecules of GPIIb-III (approximately 90-95% of the total) around a small fraction of IIb-III that is associated with a submembranous cytoskeleton.

Quantitative analysis of ligand-occupied receptor interactions with elements of the cytoskeleton and with intracellular compartments requires a sensitive and simple method of identifying the receptor-ligand complex in living cells. Toward this goal, we have prepared a photoactivatable arylazide derivative of the chemotactic peptide N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys, which can be radiolabeled to high specific activity with 125I. This derivative was biologically active as judged by its ability to elicit superoxide anion production by human PMNL at nanomolar concentrations (ED50 approximately 0.7 nM). When incubated at 0 degree C with whole PMNL, radioactive ligand became specifically and saturably associated with a 60-70,000-dalton species (as assessed by SDS-PAGE) after exposure to UV light. Addition of 10-100-fold excess of unlabeled parent or unlabeled azidopeptide derivative completely blocked uptake into this species. Approximately 20-40% of the available surface receptor-binding sites were covalently labeled under these conditions. Subcellular fractionation of the labeled cells on sucrose gradients after homogenization showed that the labeled species was primarily associated with plasma membrane-rich fractions. The labeled receptor could be completely solubilized with Triton X-100 in a form which eluted as a single species with a Stoke's radius of less than 50 A on Sepharose 4B columns. In addition, the solubilized receptor-ligand complex bound specifically to wheat germ agglutinin, indicating that it is probably a glycoprotein. The ability to label the receptor in living PMNL with a high efficiency should facilitate the study of receptor dynamics and receptor physiochemical properties in this system.

The intracellular distribution of F-actin and myosin has been examined in mouse peritoneal macrophages by immunofluorescence microscopy. In resting, adherent cells, F-actin was distributed in a fine networklike pattern throughout the cytoplasm. Myosin, in contrast, was distributed in a punctate pattern. After treatment with cytochalasin B (CB), both proteins showed a coarse punctate pattern consistent with a condensation of protein around specific foci. After CB-pretreated cells were exposed to opsonized zymosan particles, immunofluorescent staining for F-actin and myosin showed an increased staining under particle binding sites. Transmission electron microscope (TEM) examination of whole-cell mounts of such preparations revealed a dense zone of filaments beneath the relatively electron-translucent zymosan particles. At sites where particles had detached during processing, these filament-rich areas were more clearly delineated. At such sites dense arrays of filaments that appeared more or less randomly oriented were apparent. The filaments could be decorated with heavy meromyosin, suggesting that they were composed, in part, of F-actin and were therefore identical to the structures giving rise to the immunofluorescence patterns. After viewing CB-treated preparations by whole-mount TEM, we examined the cells by scanning electron microscopy (SEM). Direct SEM comparison of the filament-rich zones seen by TEM showed that these structures resulted from the formation of short lamellipodial protrusions below the site of particle binding. Electron micrographs of thin-sectioned material established that these lamellipodial protrusions were densely packed with microfilaments that were in part associated with the cytoplasmic surface of the plasma membrane. The formation of particle-associated lamellipodia did not appear to represent merely a slower rate of ingestion in the presence of CB, because they formed within minutes of particle contact with the cell membrane and were not followed by particle ingestion even after a 1-h or longer incubation. Furthermore, their formation required cellular energy. These results suggest that cytochalasin B blocks phagocytosis of large particles by affecting the distances over which any putative actomyosin-mediated forces are generated.

We have developed a continuous spectroscopic method to analyze the kinetics of elastase secretion by human neutrophils. We have used the elastase-specific substrate methylsuccinylalanylalanylprolylvalylmethylcoumarin amide (Castillo, M. J., Nakajima, K., Zimmerman, J., and Powers, J. C. (1979) Anal. Biochem. 99, 53-64), which liberates the fluorophore, aminomethylcoumarin, when cleaved by elastase. We find that secretion of elastase in cytochalasin B-treated cells is initiated within approximately 5 s of exposure of the cells to the fluoresceinated chemotactic peptide stimulus, N-formyl-norleucylleucylphenylalanylnorleucyltyrosyllysine-fluorescein, and that secretion is completed within 30 s. The kinetics of this response is only slightly dependent on the concentration of the stimulus. Up to 100% (approximately 0.5 pg/neutrophil) of the elastase can be released in a dose-dependent manner by stimulated cells. We have also used this fluoresceinated stimulus and an antibody to fluorescein (Sklar, L. A., Oades, Z. G., Jesaitis, A. J., Painter, R. G., and Cochrane, C. G. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 7540-7544) to probe the temporal relationship between the binding of stimulus to the receptors and the cellular response. We find that the entire response is elicited by the binding which occurs within the first 15 s after the addition of stimulus, regardless of the dose. We estimate that an occupancy of no more than 20% of the cellular receptors for the stimulus is required to evoke the optimal response.

Previous studies have indicated that the receptor for N-formylated peptides present on human neutrophils can exist in several ligand-dissociation states at least one of which is sensitive to guanine nucleotides. Human neutrophil membranes rich in cell surface enzyme markers have been isolated from cells pretreated at 37 degrees C with 5 nM fluoresceinated chemotactic peptide (N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein; Fl-peptide) or a buffer control and analyzed for receptor-ligand dissociation states using a previously published fluorescence assay for estimating ligand binding and dissociation rates (Sklar, L. A., et al. 1984. J. Biol. Chem. 259:5661-5669). Fractionation of crude microsomes derived from homogenates of unstimulated cells by ultracentrifugation on linear D2O gradients yielded two plasma membrane-rich fractions termed fast and slow microsomes. Analysis of Fl-peptide dissociation rates from receptor present in fast membrane fractions of unstimulated cells yielded data that could be best fit by assuming that the receptor exists in three distinct ligand-dissociation states. The intermediate ligand-dissociation state (state B) accounted for 47% of the total and was converted to the fastest ligand-dissociation state (state A) by incubation of membranes with GTP or GTP-gamma-S. The remainder of the receptor (17%) present in unstimulated membranes was in a state from which ligand was virtually nondissociable (state C). This form of the receptor was insensitive to GTP-gamma-S. When cells were stimulated with Fl-peptide, most of the receptor present in slow and fast membranes was of the state C type. In contrast to unstimulated cells, slow membranes derived from cells exposed to Fl-peptide contained the majority of the recoverable receptor indicating that receptor was transferred to a physically isolatable membrane domain after ligand binding to the intact cell. The ligand-induced formation of state C in both fast and slow microsome fractions was inhibited by treatment of cells with dihydrocytochalasin B. However, the drug had no effect on translocation of the receptor to slow membranes. Pertussis toxin treatment of intact cells had no effect on ligand-induced formation of state C in either fraction even though other cellular responses were inhibited. Both slow and fast membranes contained a 41-kD G protein as assayed by immunoblot analysis. The data suggest that ligand induces a segregation of receptor-ligand complexes into a membrane domain in which the receptor is functionally uncoupled from the 41-kD neutrophil G protein.(ABSTRACT TRUNCATED AT 400 WORDS)

The distribution and transport of thrombospondin (TSP), fibrinogen (Fbg), fibronectin (Fn), and Factor VIII-related antigen (VIII:RAg) in resting and thrombin-stimulated platelets was investigated by immunofluorescence microscopy. In resting intact cells, little surface staining was seen for these proteins. In permeable resting cells, punctate staining similar to that reported for platelet factor 4 was observed. Double-label immunofluorescence staining for Fbg and either beta-thromboglobulin (beta TG), TSP, or Fn demonstrated co-localization, indicating their presence in the same intracellular structures. VIII:RAg showed general co-localization; however, the staining was finer, suggesting a possible differential intragranular localization. Thrombin stimulation induced the appearance of larger (approximately 0.5 mu) immunofluorescent masses of these proteins. In thrombin-stimulated cells, co-localization of all proteins in these masses was observed by double label immunofluorescence. Thus, TSP, Fbg, Fn, and beta TG are localized in the same structure in resting cells. Thrombin stimulates formation of common larger masses of these proteins prior to their release, suggesting that they reach the cell surface through a common intermediate.

Important similarities are reported between human smooth muscle actomyosin and the human erythrocyte spectrin complex, primarily components 1, 2, and 5 (Fairbanks G., Steck, T.L., and Wallach, D.F.H. (1971), Biochemistry 10, 2606). The actin-like protein, component 5, is identical with human uterine actin in its ability to form 50-70-A filaments to stimulate myosin ATPase activity, and to bind rabbit heavy meromyoson specit heavy meromyosin specifically. Antibodies to human smooth muscle myosin(uterine) were prepared which were monospecific. A weak but specific cross-reaction of these antisera with components 1 and/or 2 (spectrin) was characterized and at least 25% of the antimyosin antibodies showed a low affinity reaction iwth spectrin. Antibodies generated against a soluble complex of spectrin components 1 and 2 reacted only with component 1 and did not cross-react with myosin. In addition to these structural similarities between smooth muscle actomyosin and the spectrin complex, we have found that spectrin is involved in ATP-dependent erythrocyte shape changes (Sheetz, M.P., Painter, R.G., AND Singer, S.J. (1976B), Cold Spring Harbor Symp. Cell Motility (in press) and, therefore, the spectrin complex is also a mechanochemical protein system.

Parallel cytometric and fluorimetric analyses of the interaction of a fluoresceinated N-formyl hexapeptide (N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein, Nle = norleucine) with its receptors on human neutrophils are presented. The cytometric analyses take advantage of the ability of the fluorescence flow cytometer to discriminate free and receptor-bound ligand in a homogeneous real-time assay. The spectrofluorometric analysis relies on a high affinity antibody to fluorescein to discriminate free and bound ligand. We find that the number of receptors for formyl peptides on the surface of a resting cell is 53,000 +/- 13,000 (Kd approximately 0.6 +/- 0.2 nM). We use commercially available cytometric standards to calibrate the cytometer and we obtain similar values for the number of receptors. The temperature dependence of the kinetics of ligand-receptor interactions have been examined. The association rate constant varies from approximately 3 X 10(8) M-1 min-1 at 4 degrees C to approximately 10(9) M-1 min-1 at 37 degrees C (delta H approximately 8 kcal/mol). While ligand internalization is blocked at 4 degrees C, at 37 degrees C internalization proceeds at an initial rate of approximately 24% of the occupied receptors/min following a latency period of approximately 20-30 s. Intermediate rates and longer latency periods are found at 15 and 25 degrees C. Dissociation of the ligand is heterogeneous and depends both on the length of time of association and the temperature. After short periods of association, the ligand dissociates with t1/2 approximately 1-5 min. After longer periods (30 min at 15 degrees C or 100 min at 4 degrees C), but while the ligand-receptor complex remains on the cell surface, t1/2 increases to greater than 30 min. It appears that the ligand-receptor complex undergoes an alteration in affinity, with a time course at elevated temperatures, which parallels or lags behind the time course of the participation of the occupied receptors in cell activation.

Spectrin, a protein complex which is peripherally attached to the cytoplasmic surface of the human erythrocyte membrane, cannot be detected (by complement fixation with anti-spectrin antibodies) in homogenates of several different human non-muscle cells studied. On the other hand, a protein antigenically identical or similar to human smooth muscle myosin was detected (by complement fixation with antibodies to uterine smooth muscle myosin) in these cells. In the case of human fibroblast line WI38, this smooth muscle myosin like component was shown (by ferritin-antibody experiments in electron microscopy) to be at least partly associated with cytoplasmic surface of the plasma membrane of the cell. It is proposed that the spectrin complex of the erythrocyte membrane and the smooth muscle myosin-like component of the fibroblast membrane play similar roles in regulating the translational mobilities of integral proteins in their respective membranes.

Platelets are cells which develop adhesive properties following stimulation. Since fibronectin (fn) mediates adhesive properties of several cells, we sought evidence for platelet associated fn. Lysates of suspensions of washed human platelets containing less than or equal to 50 ng soluble fn/10(9) cells contained 2.85 micrograms fn antigen per 10(9) cells. The platelet fn antigen competition curve showed a similar slope to the curve for purified plasma fn suggesting antigenic identity. Immunofluorescent staining for fn was minimal in intact cells suggesting that the majority of fn antigen is intracellular. In permeable platelets, fluorescent staining for fn was seen in a punctate distribution suggesting a granule localization. Stimulation of platelet secretion by thrombin released platelet fn antigen. Suramin, a drug which inhibits platelet secretion, inhibited fn release. The apparent secretion of platelet fn, taken with the immunofluorescent data, support the localization of a portion of platelet fn antigen in a storage granule.

The response of neutrophils to N-formyl peptides is mediated via a specific 50,000- to 60,000-dalton (Mr) receptor. Real-time kinetic analysis indicated that most of the cellular responses elicited by this ligand began within 5-10 s of addition to the cells at 37 C. Of three possible biochemical changes measured that could serve as transducers or second messengers, two, i.e., increases in cyclic AMP (cAMP) and intracellular free Ca2+, occurred within 5 s of stimulus addition. In contrast, internalization of the ligand by cells showed a latency time of 20-30 s, which indicates that it probably plays no role in triggering later responses. Using pulse binding techniques that allow the level of a given response to be measured as a function of the measured level of surface receptor occupancy, we found that O2- production required up to 30% receptor occupancy to elicit 50% of maximal response. In contrast, secretion, cAMP changes, Ca2+ changes, and membrane potential changes required less than 5% occupancy. Within 5 s, occupied receptors were converted at the cell surface to a slowly dissociating form. The receptors, exhibiting apparent higher affinity, were transiently associated with the cell cytoskeleton as defined by their conversion to a Triton X-100-insoluble form. Internalized receptor-ligand complexes were transported, in large part, to the Golgi apparatus. Further analyses of the mechanism of stimulation of leukocytes have been performed with monoclonal antibodies directed against the neutrophil surface. Data with these antibodies, which are not directed to the N-formyl peptide receptor, reveal that some modulated the N-formyl peptide-mediated responses and other antibodies initiated responses of the cells.

When N-formyl chemotactic peptides bind to granulocyte receptors at 37 degrees C they rapidly form a high-affinity ligand-receptor complex whose coisolation with cytoskeletal residues of Triton X-100-extracted cells is under cellular control [Jesaitis et al: J Cell Biol 98:1378, 1984]. Experiments were performed to investigate the significance of this coisolation. When the granulocytes were preincubated with dihydrocytochalasin B (dhCB) for 10 min at 37 degrees C and then stimulated with 50 nM N-formyl-Met-Leu-[3H]Phe, the rate of uptake of the radioligand by the cells was inhibited. Colocalization of the retained peptide with the Triton X-100 fraction of these cells was also reduced relative to this fraction of the untreated cells. This inhibition was apparent before the onset of FMLP endocytosis. The inhibition was 50% effective at 0.25 microgram dhCB/ml. Maximal inhibition (80-90%) occurred at doses of dhCB greater than 1 microgram/ml. The 90% retention of two plasma membrane markers by the cytoskeleton was marginally affected. These results support the hypothesis that coisolation of the high-affinity receptor-peptide complexes with granulocyte cytoskeletons occurs because of specific association of the complexes with the cytoskeleton at the cell surface. In addition, since these events precede internalization, they suggest that formation of the association between the ligand-receptor complex and cytoskeleton may be necessary for ligand-receptor endocytosis. Experiments were also performed to evaluate other functional consequences of cytoskeletal disruption on chemotactic peptide-stimulated functions. f-Met-Leu-Phe stimulation of O2- production was potentiated due to a prolongation of and an increase in the rate of O2- production. This potentiation had the same dose dependency as the inhibition of receptor modulation. The possible relationship of these various functions is discussed.

Fibronectins (fn) are adhesive glycoproteins which bind to collagen and to fibrin and appear to be important in cellular adhesion to other cells or surfaces. Fn-related antigen is present in human platelets, suggesting a possible role for fn in the adhesive properties of platelets. We have studied the localization of fn in resting and thrombin-stimulated platelets by immunofluorescence and quantitative binding of radiolabeled antibody. In resting fixed platelets, variable light surface staining for fn was observed. When these cells were made permeable to antibody with detergent, staining for fn was markedly enhanced and was present in a punctate distribution, suggesting intracellular localization. Stimulation with thrombin, which is associated with increased platelet adhesiveness, resulted in increased staining for fn antigen on intact platelets. These stimulated cells did not leak 51Cr nor did they stain for F-actin, thus documenting that the increased fn staining was not due to loss of plasma membrane integrity. The thrombin-induced increase in accessible platelet fn antigen was confirmed by quantitative antibody binding studies in which thrombin-stimulated platelets specifically bound 15 times as much radiolabeled F(ab')2 anti-fn as did resting cells. Thus, thrombin stimulation results in increased expression of fn antigen on the platelet surface. Here it may participate in interactions with fibrin, connective tissue, or other cells.

Triton X-100-insoluble residues, or skeletons, of plasma membrane-rich vesicles obtained from unstimulated human platelets were isolated by high speed centrifugation. About 10-15% of the total surface iodinatable glycoproteins IIb and III (GPIIb and GPIII, respectively) co-isolated with the insoluble fraction. After sonication and centrifugation the solubilized material was further purified by affinity chromatography on Lens culinaris lectin-Sepharose. SDS PAGE analysis of this material revealed the presence of at least three major proteins, which were shown to be GPIIb, GPIII, and membrane actin, as judged by their electrophoretic properties and on the basis of immunological criteria. Antibodies directed against platelet surface glycoproteins and antibodies directed against rabbit actin were able to immunoprecipitate all three proteins, which indicates that they were noncovalently associated with one another. Gel filtration of the Lens lectin-purified Triton-insoluble complex on Ultrogel AcA 22 showed that greater than 85% of the total surface GPIIb and III was associated with an actin-rich peak that eluted in the void volume. In contrast, the form of GPIIb-III present in the Triton-soluble membrane fraction behaved as monomeric species when chromatographed under identical conditions. Finally, the GPIIb-III membrane actin complex bound with high efficiency to rabbit f-actin in vitro in a Ca++-independent manner, whereas the monomeric forms found in the Triton-soluble fraction did not bind to actin. These results indicate that two forms of GPIIb and III exist: one that binds directly to endogenous membrane actin and one that does not.

When human granulocytes were exposed to 50 nM N-formyl-Met-Leu-[3H]Phe at 37 degrees C they rapidly formed ligand-receptor complexes that dissociated 50-100 times more slowly than those on cells initially exposed to the peptide at 4 degrees C. These complexes of apparent higher affinity were stable after detergent solubilization of the cells with Triton X-100. The complexes co-isolated with the detergent insoluble cytoskeletal residues and were free of the cytosolic and Golgi markers, lactate dehydrogenase and galactosyl transferase, respectively. After 5 s of exposure to f-Met-Leu-Phe, 2,000-3,000 molecules of ligand per cell were trapped in such complexes. Continued exposure resulted in capture of a maximum of 14,000 molecules per cell by 5 min. Exposure at 15 degrees C, a temperature at which endocytosis of the receptor is prevented, resulted in complex formation at a linear rate for at least 20 min to levels twice those measured at 37 degrees C. At 4 degrees C, complex formation was approximately 10% of the maximum amount formed at 37 degrees C. Pulse-chase experiments revealed that the complex was in transient association with the cytoskeleton with a half life ranging between 30 s to 4 min depending on the length of the original incubation. Electron microscopic autoradiography indicated that after 1 min of incubation at 37 degrees C, the majority of the specific autoradiographic grains were localized to the outer circumference of the cellular cytoskeleton. After 4 min of incubation, the grains were less frequent at the cytoskeleton periphery but still threefold enriched over a random cellular distribution. We conclude that a metabolically controlled modulation of the state of the N-formyl chemotactic peptide receptor occurs in the plasma membrane which may be the result of transient association of ligand-receptor complex and the cell cytoskeleton.

We have measured the association of platelet surface membrane proteins with Triton X-100 (Triton)-insoluble residues in platelets surface labeled with 125I. In both concanavalin A (Con A)-stimulated and resting platelets, this fraction is composed largely of polypeptides with apparent molecular weights of 45,000, 200,000, and 250,000 which comigrate with authentic actin, myosin heavy chain, and actin binding protein, respectively, as judged by PAGE in SDS. Less than 10% of the two major 125I-labeled surface glycoproteins, GPiib and GPIII, were associated with the Triton residue in resting platelets. Within 45 s after Con A addition, 80-95% of these two glycoproteins became associated with the Triton residue and the amount of sedimentable actin doubled. No cosedimentation of GPIIb and III with the cytoskeletal protein-containing Triton residue was seen when Con A was added to a Triton extract of resting cells, indicating that the sedimentation of GPIIb and III seen in Con A-stimulated platelets was not due to precipitation of the glycoproteins by Con A after detergent lysis. Treatment of Triton extracts of Con A-stimulated platelets with DNase I (deoxyribonucleate 5'-oligonucleotidido-hydrolase [EC 3.1.4.5]) inhibited the sedimentation of actin and the two surface glycoproteins in a dose-dependent manner. This inhibition of cosedimentation was not due to an effect of DNase I on Con A-glycoprotein interactions since these two glycoproteins could be quantitatively recovered by Con A-Sepharose affinity absorption in the presence of DNase I. When the Con A bound to the Triton residue was localized ultrastructurally, it was associated with cell-sized structures containing filamentous material. In intact cells, there was simultaneous immunofluorescent coredistribution of surface-bound Con A and myosin under conditions which induced a redistribution of platelet myosin. These data suggest that Con A can, in the intact platelet, induce physical interactions between certain surface glycoproteins and the internal cytoskeleton.

When exposed to the N-formylated chemoattractant peptides, neutrophils undergo a transient ruffling followed by a polarization that involves a redistribution of F-actin (Fechheimer, M., and S. H. Zigmond, 1983, Cell Motil., 3:349-361). The cells also undergo a biphasic right angle light scatter response whose first phase is maximal 10-15 s after exposure to the stimulus, and whose second phase is longer in duration and maximal only after 1 min or more (Yuli, I., and R. Snyderman, 1984, J. Clin. Invest. 73:1408-1417). We now report that the first phase is accompanied by a transient polymerization of actin (monitored by cytometric analysis of phallacidin staining according to the method of Howard, T. H., and W. H. Meyer, 1984, J. Cell Biol., 98:1265-1271) and the second phase is accompanied by a more sustained polymerization of actin. Based on correlated measurements of ligand binding (Sklar, L. A., D. A. Finney, Z. G. Oades, A. J. Jesaitis, R. G. Painter, and C. G. Cochrane, 1984, J. Biol. Chem., 259:5661-5669) and intracellular Ca++ elevation (under conditions where we use the fluorescent Ca++ chelator Quin 2 to modulate intracellular Ca++ levels), we conclude that this first phase requires less than 100 receptors/cell (out of 50,000) and does not require the release of intracellular stores of Ca++. In contrast, the sustained polymerization requires both the occupancy of thousands of receptors (an estimated 10% of the receptors per minute) and may be somewhat sensitive to the availability of intracellular Ca++. When ligand binding is interrupted, F-actin rapidly depolymerizes with a half-time of no greater than approximately 15 s, and the transient light scatter response decays toward its initial value in parallel. Partial disaggregation of the cells follows the recovery of these responses. Based on these observations, we suggest that transient actin polymerization and transient cell ruffling give rise to transient aggregation as long as degranulation is limited.