Type-I IFN signaling is required for the induction of antigen-specific CD8(+) T cell responses by adenovirus vector vaccine in the gut-mucosa

Microtubules in living cells are very important component for various cellular functions as well as to maintain the cell shape. Mechanical properties of microtubules play a vital role in their functions and structure. To understand the mechanical properties of microtubules in living cells, we developed an orthotropic-Pasternak model and investigated the vibrational behavior when microtubules are embedded in surrounding elastic medium. We considered microtubules as orthotropic elastic shell and its surrounding elastic matrix as Pasternak foundation. We found that due to mechanical coupling of microtubules with elastic medium, the flexural vibration is increased with the stiffening of elastic medium. We noticed that foundation modulus (H) and shear modulus (G) have more effect on radial vibrational mode as compared to longitudinal vibrational mode and torsional vibrational mode.     

Interaction of rifampicin with nonprotein thiols in mycobacterium smegmatis

In this study, the interaction of Rifampicin (RIF) with cellular glutathione (GSH) in Mycobacterium smegmatis has been investigated. Minimum inhibitory concentration of RIF for M. smegmatis was demonstrated to be 17 micrograms ml-1 medium. Three subinhibitory concentrations viz. 5, 10 and 15 micrograms RIF ml-1 medium were used to study its interaction with cellular non protein thiols (NPSH). Maximum depletion (57.8%) in NPSH levels [5, 5'-dithiobis (2-nitrobenzoic acid) assay] was observed on second day when the cells were grown in the presence of 15 micrograms RIF ml-1 medium. When the same samples were assayed for GSH levels (glyoxylase assay) the depletion of GSH levels by RIF was still observed, confirming the earlier findings. GSH depletion paralleled with growth inhibition and reached to normal level on 5th day of growth. Cellular depletion of GSH was also observed when 3 day grown cells of M. smegmatis were exposed to various concentrations of RIF (20, 40 and 60 micrograms ml-1 medium) for different time intervals. Maximum depletion of NPSH levels was observed when 3 day grown cultures were treated with 60 micrograms RIF ml-1 medium for a period of 6 h. The results of this study clearly demonstrate that RIF depletes cellular GSH levels regardless of the fact that the drug is included in the medium before inoculating it or after the cells have been grown for a period of three days. The depletion of cellular GSH levels by RIF in M. smegmatis may contribute towards its antituberculous activity.     

Change in energy metabolism of ascites cancer cells with a decrease in pH]

In Hank's balanced salt solution EL-4 ascites thymoma cells possessed endogenous respiration which was sufficient for the maintenance of their ATP level: pH decrease down to 6.0 had no effect either on endogenous respiration or the ATP level. Glucose had no influence on the respiration of EL-4 cells but inhibited that of Ehrlich ascites carcinoma (EAC) cells by 40% (Crabtree effect); respiration of the both cell lines was strongly (4-fold) inhibited after simultaneous addition of glucose, lactate and pH decrease. EL-4 cells had no endogenous glycolysis; EAC cells showed a low level of glycolysis only after pH decrease. Glucose addition led to activation of glycolysis (both inhibited 2-fold after a decrease of pH down to 6.0. The respiration inhibition at pH 7.3 and 6.0 caused no decrease of ATP depletion when glucose was present in the medium; this result may be due to suppression of ATP consumption. Incubation of EL-4 cells under respiration and glycolysis deficiency conditions resulted in a sharp ATP depletion; pH decrease delayed this depletion.     

Human cultured skin fibroblasts survive profound inherited ubiquinone depletion

Beside its role in electron transfer in the mitochondrial respiratory chain, ubiquinone is known to prevent lipid peroxidation and DNA damage by trapping cellular free radicals. Thanks to its antioxidant properties, ubiquinone may represent an important factor controlling both necrotic and apoptotic processes. We have investigated the consequences of a profound inherited ubiquinone depletion on cultured skin fibroblasts of a patient presenting with encephalomyopathy. Interestingly, cell respiration, mitochondrial oxidation of various substrates, and cell growth of ubiquinone-deficient fibroblasts were only partially decreased. Moreover, these cells did not apparently overproduce superoxide anions or lipoperoxides. Finally, apoptosis did not increase as compared to control, even after serum deprivation. These observations suggest that ubiquinone may not play a major role in the antioxidant defenses of cultured fibroblasts and that its role in controlling oxidative stress and apoptosis may greatly vary across cell types, especially as not all tissues were equally affected in the patient despite the widespread ubiquinone depletion in vivo.     

Modulation of cellular mechanics during osteogenic differentiation of human mesenchymal stem cells

Recognition of the growing role of human mesenchymal stem cells (hMSC) in tissue engineering and regenerative medicine requires a thorough understanding of intracellular biochemical and biophysical processes that may direct the cell's commitment to a particular lineage. In this study, we characterized the distinct biomechanical properties of hMSCs, including the average Young's modulus determined by atomic force microscopy (3.2 +/- 1.4 kPa for hMSC vs. 1.7 +/- 1.0 kPa for fully differentiated osteoblasts), and the average membrane tether length measured with laser optical tweezers (10.6 +/- 1.1 microm for stem cells, and 4.0 +/- 1.1 microm for osteoblasts). These differences in cell elasticity and membrane mechanics result primarily from differential actin cytoskeleton organization in these two cell types, whereas microtubules did not appear to affect the cellular mechanics. The membrane-cytoskeleton linker proteins may contribute to a stronger interaction of the plasma membrane with F-actins and shorter membrane tether length in osteoblasts than in stem cells. Actin depolymerization or ATP depletion caused a two- to threefold increase in the membrane tether length in osteoblasts, but had essentially no effect on the stem-cell membrane tethers. Actin remodeling in the course of a 10-day osteogenic differentiation of hMSC mediates the temporally correlated dynamical changes in cell elasticity and membrane mechanics. For example, after a 10-day culture in osteogenic medium, hMSC mechanical characteristics were comparable to those of mature bone cells. Based on quantitative characterization of the actin cytoskeleton remodeling during osteodifferentiation, we postulate that the actin cytoskeleton plays a pivotal role in determining the hMSC mechanical properties and modulation of cellular mechanics at the early stage of stem-cell osteodifferentiation.     

Global analysis of endothelial cell line proliferation patterns based on nutrient-depletion models: implications for a standardization of cell proliferation assays

It is known that cell populations growing in different environmental conditions may exhibit different proliferation patterns. However, it is not clear if, despite the diversity of the so-observed patterns, inherent cellular growth characteristics of the population can nevertheless be determined. This study quantifies the proliferative behaviour of the permanent endothelial human cell line, Eahy926, and establishes to which extent the estimation of the cell proliferation rate depends on variations of the experimental protocols. Cell proliferation curves were obtained for cells cultured over 16 days and the influences of cell seeding densities, foetal bovine serum content and frequency of culture medium changes were investigated. Quantitative dynamic modelling was conducted to evaluate the kinetic characteristics of this cell population. We proposed successive models and retained a nutrient-depletion toxicity dependant model, which takes into account the progressive depletion of nutrients, as well as the increase of toxicity in the cell culture medium. This model is shown to provide a very good and robust prediction of the experimental proliferation curves, whatever are the considered frequency of culture medium changes and serum concentrations. Thus, the model enables an intrinsic quantification of the parameters driving in vitro EAhy926 proliferation, including proliferation, nutrient consumption and toxicity increase rates, rather independently of the experiments design. We therefore propose that such models could provide a basis for a standardized quantification of intrinsic cell proliferation kinetics.     

Inhibition of apoptosis in pulmonary endothelial cells by altered pH,mitochondrial function,and ATP supply

We investigated the effect of altered extracellular pH, mitochondrial function, and ATP content on development of apoptosis in human pulmonary artery endothelial cells after treatment with staurosporine (STS). STS produced a concentration- and time-dependent increase in caspase-3 activity in pH 7.4 medium that reached a peak at 6 h. The increase in caspase activity was associated with significant DNA fragmentation. Fluorescent imaging of treated monolayers in pH 7.4 medium with Hoechst-33342-propidium iodide demonstrated a large percentage of apoptotic cells ( approximately 40%) with no evidence of necrosis. Caspase activity, DNA fragmentation, and percentage of apoptotic cells were reduced after STS treatment in acidic media (pH 7.0 and 6.6). The Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM inhibited STS-induced apoptosis, whereas the rise in intracellular Ca2+concentration in STS-treated cells in pH 7.4 medium was reduced in pH 7.0 medium. These results suggest that one mechanism for inhibitory effects of acidosis may be a pH-induced alteration in Ca2+ signaling. Treatment with STS in the presence of oligomycin (10 microM), an inhibitor of the mitochondrial F(0)F(1)-ATPase, in glucose-free media abolished caspase activation and DNA fragmentation in association with severe ATP depletion ( approximately 2% of control cells). Imaging demonstrated a change in the mode of cell death from apoptosis to necrosis under these conditions. This change was linked to the level of ATP depletion, because STS treatment in the absence of glucose or the presence of oligomycin in media with glucose still leads to apoptosis in the presence of only moderate ATP depletion. These results demonstrate that pH, mitochondrial function, and ATP supply are important variables that regulate STS-induced apoptosis in human pulmonary artery endothelial cells.     

Upregulation of Metallothioneins After Exposure of Cultured Primary Astrocytes to Silver Nanoparticles

To test for the prolonged consequences of a short transient exposure of astrocytes to silver nanoparticles (AgNP), cultured primary astrocytes were incubated for 4 h in the presence of AgNP and the cell viability as well as various metabolic parameters were investigated during a subsequent incubation in AgNP-free medium. Acute exposure of astrocytes to AgNP led to a concentration-dependent increase in the specific cellular silver content to up to 46 nmol/mg protein, but did not compromise cell viability. During a subsequent incubation of the cells in AgNP-free medium, the cellular silver content of AgNP-treated astrocytes remained almost constant for up to 7 days. The cellular presence of AgNP did neither induce any delayed cell toxicity nor were alterations in cellular glucose consumption, lactate production or in the cellular ratio of glutathione to glutathione disulfide observed. However, Western blot analysis and immunocytochemical staining revealed that AgNP-treated astrocytes strongly upregulated the expression of metallothioneins. These results demonstrate that a prolonged presence of accumulated AgNP does not compromise the viability and the basal metabolism of cultured astrocytes and suggest that the upregulation of metallothioneins may help to prevent silver-mediated toxicity that could be induced by AgNP-derived silver ions.     

热带假丝酵母细胞内pH的测定及其与生长代谢活性的关系

应用荧光探针5(6)-双醋酸羧基荧光素 (Carboxyfluorescein diacetate) 测定了产长链二元酸热带假丝酵母 (Candida tropicalis) 细胞内pH (pHi) 值,确定了该探针载入C. tropicalis细胞的适宜条件。用摇瓶培养C. tropicalis细胞,考察了细胞外pH和生长碳源对pH_I的影响,实验结果表明：细胞外pH对pH_I略有影响,而生长碳源对pH_I的影响略为明显。利用5L发酵罐进一步研究了细胞生长代谢活性与pHi的关系,结果表明：细胞比生长速率、CO₂比生产速率和葡萄糖比消耗速率与pHi变化密切相关,pH_I的增加伴随着细胞生长活力的增加,反之亦然。在pH6.0条件下用葡萄糖和醋酸钠共作碳源培养C. tropicalis细胞时,测得的pH_I值维持在5.72～6.15范围内。     

Effects of methotrexate on the viscoelastic properties of single cells probed by atomic force microscopy

Methotrexate is a commonly used anti-cancer chemotherapy drug. Cellular mechanical properties are fundamental parameters that reflect the physiological state of a cell. However, so far the role of cellular mechanical properties in the actions of methotrexate is still unclear. In recent years, probing the behaviors of single cells with the use of atomic force microscopy (AFM) has contributed much to the field of cell biomechanics. In this work, with the use of AFM, the effects of methotrexate on the viscoelastic properties of four types of cells were quantitatively investigated. The inhibitory and cytotoxic effects of methotrexate on the proliferation of cells were observed by optical and fluorescence microscopy. AFM indenting was used to measure the changes of cellular viscoelastic properties (Young’s modulus and relaxation time) by using both conical tip and spherical tip, quantitatively showing that the stimulation of methotrexate resulted in a significant decrease of both cellular Young’s modulus and relaxation times. The morphological changes of cells induced by methotrexate were visualized by AFM imaging. The study improves our understanding of methotrexate action and offers a novel way to quantify drug actions at the single-cell level by measuring cellular viscoelastic properties, which may have potential impacts on developing label-free methods for drug evaluation.     

A requirement for cholesterol for phorbol ester-induced adhesion of a human monocyte-like cell line

The human monocyte/macrophage-like cell line U937, which is a cholesterol auxotroph, is nonadherent. However, it becomes adherent after treatment with phorbol 12-myristate 13-acetate (phorbol ester). We investigated the effects of cellular cholesterol depletion and repletion on the effectiveness of phorbol ester to induce adhesion to substratum. Almost 70% of cellular cholesterol is depleted by incubation of the cells for 24 hrs in the growth medium in which delipidated fetal calf serum is substituted for fetal calf serum without affecting viability or the rate of growth. The use of delipidated fetal calf serum inhibited phorbol ester-induced adhesion by 40%. If the cells were preincubated in the medium containing delipidated fetal calf serum 6 hrs prior to addition of phorbol ester, adhesion was inhibited by 90%. Addition of cholesterol to the medium containing delipidated fetal calf serum, which replenishes cellular cholesterol, restored the ability of phorbol ester to induce adhesion to levels seen in cells cultured in the medium containing fetal calf serum. Epicholesterol was not as effective as cholesterol in supporting adhesion. Cholesterol depletion did not inhibit phorbol ester stimulation of superoxide anion production. These observations indicate a function for cholesterol in phorbol ester-induced adhesion that is independent of phorbol ester-induced superoxide anion production. It is proposed that cholesterol is required for synthesis and/or proper orientation and distribution, in the plasma membrane, of macromolecule(s) that mediate phorbol ester-induced adhesion.     

The morphology of the denuded epidermal basal cell layer of the hairless mouse after different preparation methods. A scanning and transmission electron microscopical study

The denuded basal cell layer of the hairless mouse epidermis is described in the present scanning (SEM) and transmission electron microscopical (TEM) study. The suprabasal layers were removed mechanically after trypsinization or by extracellular calcium depletion. Trypsinization before removal of the suprabasal cells caused the basal cells to shrink. Characteristic surface plication and hemi-desmosomal attachment to the basement membrane were generally preserved. SEM revealed partly maintained intercellular bridging, whereas by TEM such contacts were absent because half desmosomes were internalized. Total calcium depletion induced more serious damage to the basal cell surface, which was smooth with apparent perforations. However, cell bridges, and occasional desmosomes were present. The cell interior demonstrated important cellular injury. If the calcium deprived explants were allowed to recover in calcium-containing medium, the cells acquired an activated "regenerative" morphology, without junctions, similar to that observed in wound healing. Epidermal non-keratinocytes were seen only after trypsinization. Control experiments revealed that they adapted poorly to organ culture conditions. By TEM, we observed several interesting aspects of the differences, between dark and clear basal keratinocytes. This was unexpected because fixation studies had shown, that with the present fixation method, typical dark and clear cells do not occur in untreated epidermis. We believe that membrane injury through mechanical stripping of partly adhering epidermal layers induced "clear cells", whereby the neighboring cells appeared darker. This provides additional evidence as to the origin of the two sub-populations, dark and clear basal cells. The clear cells may be injured cells, caused by cell damage, and not by processes of cellular differentiation. The results of the present investigation supports the view that basal keratinocytes have a polygonal shape with numerous free surface extensions and they are anchored to the basement membrane with "foot pads". Our study also shows that SEM of the epidermal basal layer might be feasible. Various artifacts, however, must be considered, depending on the denudation method used. We prefer trypsinization to calcium depletion because it is less time-consuming and results in a cell morphology which in TEM is comparable to that of basal cells in untreated whole epidermis. Extra-cellular calcium depletion, however, might be useful as a method to prepare single cell suspensions for flow cytometry. Restoration of a normal calcium concentration after stripping, provides an opportunity to mimic wound healing in situ, as an alternative t     

Variations in tumor cell growth rates and metabolism with oxygen concentration, glucose concentration, and extracellular pH.

Tumors and multicellular tumor spheroids can develop gradients in oxygen concentration, glucose concentration, and extracellular pH as they grow. In order to calculate these gradients and assess their impact on tumor growth, it is necessary to quantify the effect of these variables on tumor cell metabolism and growth. In this work, the oxygen consumption rates, glucose consumption rates, and growth rates of EMT6/Ro mouse mammary tumor cells were measured at a variety of oxygen concentrations, glucose concentrations, and extracellular pH levels. At an extracellular pH of 7.25, the oxygen consumption rate of EMT6/Ro cells increased by nearly a factor of 2 as the glucose concentration was decreased from 5.5 mM to 0.4 mM. This effect of glucose concentration on oxygen consumption rate, however, was slight at an extracellular pH of 6.95 and disappeared completely at an extracellular pH of 6.60. The glucose consumption rate of EMT6/Ro cells increased by roughly 40% when the oxygen concentration was reduced from 0.21 mM to 0.023 mM and decreased by roughly 60% when the extracellular pH was decreased from 7.25 to 6.95. The growth rate of EMT6/Ro cells decreased with decreasing oxygen concentration and extracellular pH; however, severe conditions were required to stop cell growth (0.0082 mM oxygen and an extracellular pH of 6.60). Empirical correlations were developed from these data to express EMT6/Ro cell growth rates, oxygen consumption rates, and glucose consumption rates, as functions of oxygen concentration, glucose concentration, and extracellular pH. These empirical correlations make it possible to mathematically model the gradients in oxygen concentration, glucose concentration, and extracellular pH in EMT6/Ro multicellular spheroids by solution of the diffusion/reaction equations. Computations such as these, along with oxygen and pH microelectrode measurements in EMT6/Ro multicellular spheroids, indicated that nutrient concentration and pH levels in the inner regions of spheroids were low enough to cause significant changes in nutrient consumption rates and cell growth rates. However, pH and oxygen concentrations measured or calculated in EMT6/Ro spheroids where quiescent cells have been observed were not low enough to cause the cessation of cell growth, indicating that the observed quiescence must have been due to factors other than acidic pH, oxygen depletion, or glucose depletion.     

Ezrin regulates skin fibroblast size/mechanical properties and YAP-dependent proliferation

Ezrin acts as a dynamic linkage between plasma membrane and cytoskeleton, and thus involved in many fundamental cellular functions. Yet, its potential role in human skin is virtually unknown. Here we investigate the role of Ezrin in primary skin fibroblasts, the major cells responsible extracellular matrix (ECM) production. We report that Ezrin play an important role in the maintenance of skin fibroblast size/mechanical properties and proliferation. siRNA-mediated Ezrin knockdown decreased fibroblast size and mechanical properties, and thus impaired the nuclear translocation of YAP, a protein commonly response to cell size and mechanical force. Functionally, depletion of Ezrin significantly inhibited YAP target gene expression and fibroblast proliferation. Conversely, restoration of YAP nuclear translocation by overexpression of constitutively active YAP reversed YAP target genes expression and rescued proliferation in Ezrin knockdown cells. These data reveal a novel role for Ezrin in maintenance of fibroblast size/mechanical force and regulating YAP-mediated proliferation.     

Effects of blocking integrin β1 and N-cadherin cellular interactions on mechanical properties of vascular smooth muscle cells

Experimental measurements of cellular mechanical properties have shown large variability in whole-cell mechanical properties between cells from a single population. This heterogeneity has been observed in many cell populations and with several measurement techniques but the sources are not yet fully understood. Cell mechanical properties are directly related to the composition and organization of the cytoskeleton, which is physically coupled to neighboring cells through adherens junctions and to underlying matrix through focal adhesion complexes. This high level of heterogeneity may be attributed to varying cellular interactions throughout the sample. We tested the effect of cell-cell and cell-matrix interactions on the mechanical properties of vascular smooth muscle cells (VSMCs) in culture by using antibodies to block N-cadherin and integrin β1 interactions. VSMCs were cultured on substrates of varying stiffness with and without tension. Under each of these conditions, cellular mechanical properties were characterized by performing atomic force microscopy (AFM) and cellular structure was analyzed through immunofluorescence imaging. As expected, VSMC mechanical properties were greatly affected by the underlying culture substrate and applied tension. Interestingly, the cell-to-cell variation in mechanical properties within each sample decreased significantly in the antibody conditions. Thus, the cells grown with blocking antibodies were more homogeneous in their mechanical properties on both glass and soft substrates. This suggests that diversified adhesion binding between cells and the ECM is responsible for a significant amount of mechanical heterogeneity that is observed in 2D cell culture studies.     

Zinc uptake into MCF-10A cells is inhibited by cholesterol depletion

The mechanism for cellular Zn uptake was investigated by depleting cell cholesterol levels, a treatment that disrupts lipid rafts/caveolae-dependent processes and inhibits coated-pit budding. Incubation of MCF-10A human breast epithelial cells with hydroxypropyl-beta-cyclodextrin significantly lowered cell cholesterol levels and significantly inhibited cellular zinc uptake measured at 10 min, but had no effect on 2-deoxyglucose uptake. Replacing potassium for sodium in the uptake buffer significantly stimulated Zn uptake by 20%. The effects of potassium depletion and chlorpromazine on Zn uptake were investigated to determine the contribution of coated-pit endocytosis. Potassium depletion following hypotonic shock significantly inhibited Zn uptake into MCF-10A cells approximately 15%. Chlorpromazine at 20 microg/ml inhibited uptake approximately 30%. The data support the hypothesis that Zn uptake into MCF-10A cells involves lipid rafts/caveolae. The relatively mild effects of potassium depletion and chlorpromazine suggest that a small portion of Zn uptake may require coated pit endocytosis.     

Influence of Culture Medium pH on Growth, Aggregation, and Biofilm Formation of Xylella fastidiosa

The major feature of Xylella fastidiosa growing in its hosts, as well as in culture media, is its cellular aggregation and biofilm formation, leading to partial obstruction of the xylem causing water stress in the plant. We report that growth, aggregation, and biofilm formation of X. fastidiosa are influenced by the medium pH. We have verified that X. fastidiosa cell aggregation is reversibly inhibited by decreasing the medium pH from 6.6 to 6.4. Biofilm formation on glass walls was affected as well, and a concomitant decrease in cell multiplication was observed below pH 6.4. The manipulation of culture medium pH can be used as a tool for the cloning of X. fastidiosa strains isolated from plant hosts, because different strains can inhabit the same plant. Also, X. fastidiosa mutants produced by gene manipulation can be isolated from cell aggregates containing transformed and untransformed cells.     

Impact of baculoviral transduction of fluorescent actin on cellular forces

Live staining of actin brings valuable information in the field of mechanobiology. Gene transfer of GFP-actin has been reported to disturb cell rheological properties while gene transfer of fluorescent actin binding proteins was not. However the influence of gene transfer on cellular forces in adhered cells has never been investigated. This would provide a more complete picture of mechanical disorders induced by actin live staining for mechanobiology studies. Indeed, most of these techniques were shown to alter cell morphology. Change in cell morphology may in itself be sufficient to perturb cellular forces. Here we focus on quantifying the alterations of cellular stresses that result from baculoviral transduction of GFP-actin in MDCK cell line. We report that GFP-actin transduction increases the proportion of cells with large intracellular or surface stresses, especially in epithelia with low cell density. We show that the enhancement of the mechanical stresses is accompanied by small perturbations of cell shape, but not by a significant change in cell size. We thus conclude that this live staining method enhances the cellular forces but only brings subtle shape alterations.     

Single integrin molecule adhesion forces in intact cells measured by atomic force microscopy.

Cross-talk between cells and the extracellular matrix is critically influenced by the mechanical properties of cell surface receptor-ligand interactions; these interactions are especially well defined and regulated in cells capable of dynamically modifying their matrix environment. In this study, attention was focused on osteoclasts, which are absolutely dependent on integrin extracellular matrix receptors in order to degrade bone; other bone cells, osteoblasts, were used for comparison. Integrin binding forces were measured in intact cells by atomic force microscopy (AFM) for several RGD-containing (Arg-Gly-Asp) ligands and ranged from 32 to 97 picoNewtons (pN); they were found to be cell and amino acid sequence specific, saturatable and sensitive to the pH and divalent cation composition of the cellular culture medium. In contrast to short linear RGD hexapeptides, larger peptides and proteins containing the RGD sequence, such as osteopontin (a major non-collagenous bone protein) and echistatin (a high affinity RGD sequence containing antagonist snake venom protein), showed different binding affinities. This demonstrates that the context of the RGD sequence within a protein has considerable influence upon the final binding force for receptor interaction. These data also demonstrate that AFM, as a methodological approach, can be adapted to cell biology studies wherever cell-matrix interactions play a critical role, and, moreover, may have applicability to the analysis of receptor-ligand interactions in cell membranes in general.