A mathematical study of non-equimolar ternary gas diffusion

In view of the increasing evidence that multicomponent diffusion effects could be significant in biological gas exchange systems, a non-equimolar film model of multicomponent diffusion was derived. “Osmotic” ternary diffusion was studied for the gas systems He−N₂−O₂, He−SF₆−O₂, and N₂−SF₆−O₂. Diffusional fluxes and concentration profiles were calculated under both the “square-root” and the “product” flux conditions. Results were also compared with those obtained using the equimolar flux condition. It was found that the greater the difference of the diffusibilities between the two active components in a system, the greater the osmotic fluxes, and also the more alinear the concentration profiles. These results support the suggestion that the “product” condition applies to molecular diffusion in free space, the “square-root” condition to molecular diffusion in pores, and the equimolar flux condition to closed diffusion systems.     

Unrestricted Diffusion of Exogenous and Endogenous PIP2 in Baby Hamster Kidney and Chinese Hamster Ovary Cell Plasmalemma

We used two approaches to characterize the lateral mobility of phosphatidylinositol 4,5-bisphosphate (PIP₂) in the plasmalemma of baby hamster kidney and Chinese hamster ovary fibroblasts. First, nitrobenzoxadiazole-labeled C6-phosphatidylcholine and C16-PIP₂ were incorporated into plasma membrane “lawns” (∼20 × 30 μm) from these cells and into the outer monolayer of intact cells. Diffusion coefficients determined by fluorescence recovery after photobleaching were similar for the two lipids and were higher in lawns, ∼0.3 μm²/s, than on the cell surface, ∼0.1 μm²/s. For membrane lawns, the fractional recoveries (75–90%) were close to those expected from the fraction of total membrane bleached, and labeling by the probes was several times greater than for intact cells. Second, we analyzed cells expressing M1 muscarinic receptors and green fluorescent protein fused with PIP₂-binding pleckstrin-homology domains, Tubby domains or diacylglycerol (DAG)-binding C1 domains. On-cell gigaseal patches were formed with pipette tips >5 μm in diameter. When the agonist carbachol (0.3 mm) was applied either within or outside of the pipette, lipid signals crossed the pipette barrier rapidly in both directions and membrane blebbing occurred on both membrane sides. Accurate simulations of lipid gradients required diffusion coefficients >1 μm²/s. Exogenous DAG also crossed the pipette barrier rapidly. In summary, we found no evidence for restricted diffusion of signaling lipids in these cells. The lower mobility and incorporation of phospholipid at the extracellular leaflet may reflect a more ordered and condensed extracellular monolayer, as expected from previous studies. An erratum to this article can be found at     

Conditional confined oscillatory dynamics of <Emphasis Type="Italic">Escherichia coli</Emphasis> strain K12-MG1655 in chemostat systems

A series of continuous- and sequencing-batch reactor experiments were performed to assess the growth dynamics of Escherichia coli strain K12-MG1655 in chemostat systems. Previous mathematical predictions and early experimental results had shown that confined oscillatory dynamics ensue in bioreactor populations, which relates to “group birth and death” events within the population. New results are reported here that generally verify the predictions of the model and show that confined oscillations occur under different initial conditions, but the characteristics of the oscillatory dynamics vary as a function of the hydraulic retention time (HRT). Bioreactors were operated at HRTs ranging from 2.7 to 35 h and, regardless of initial conditions or the imposition of transient operational instabilities, highly patterned oscillations developed when HRT was between ∼3 and 8 h. However, outside of this range, bioreactor populations tended to form biofilms on the reactor walls (although the majority of the cells remained suspended in the bulk solution) and stable oscillations were not seen in the bulk phase. This suggests that alternate operating “states” might exist in chemostat populations with biofilm formation and non-homogenous spatial growth influencing “system” dynamics at very low and high HRTs. Although the model accurately predicts a confined dynamic equilibrium for mid-range HRT operations, experimental data show that model predictions do not extend outside of this range, when an alternate stable-state seems to be attained.     

Respiration of tissue as a medium of heterogeneous permeability

Expressions are derived for the overall oxygen consumption and the O₂ penetration depth into a tissue section in terms of the basic parameters, of the system under steady-state conditions. The approach differs from many previous analyses in so far as the oxygen molecules are regarded as reaching their sites of chemical assimilation by diffusion through extracellular fluid followed by bulk diffusion into irregular cells of significantly lower permeability. This “two-phase” model would seem to be compatible with the major experimental features of steady-state respiration, and gives a ratio of cellular to extracellular diffusion coefficients of the same low order as that found for inert gases under transient conditions. The greater oxygen penetration predicted by this model is discussed in relation to the survival of ischemic tissue and is shown to be consistent with data for myocardial infarction.     

Membrane binding mechanisms of the PX domains of NADPH oxidase p40phox and p47phox

Phox (PX) domains are phosphoinositide (PI)-binding domains with broad PI specificity. Two cytosolic components of NADPH oxidase, p40(phox) and p47(phox), contain PX domains. The PX domain of p40(phox) specifically binds phosphatidylinositol 3-phosphate, whereas the PX domain of p47(phox) has two lipid binding sites, one specific for phosphatidylinositol 3,4-bisphosphate and the other with affinity for phosphatidic acid or phosphatidylserine. To delineate the mechanisms by which these PX domains interact with PI-containing membranes, we measured the membrane binding of these domains and respective mutants by surface plasmon resonance and monolayer techniques and also calculated the electrostatic potentials of the domains as a function of PI binding. Results indicate that membrane binding of both PX domains is initiated by nonspecific electrostatic interactions, which is followed by the membrane penetration of hydrophobic residues. The membrane penetration of the p40(phox) PX domain is induced by phosphatidylinositol 3-phosphate, whereas that of the p47(phox) PX domain is triggered by both phosphatidylinositol 3,4-bisphosphate and phosphatidic acid (or phosphatidylserine). Studies of enhanced green fluorescent protein-fused PX domains in HEK293 cells indicate that this specific membrane penetration is also important for subcellular localization of the two PX domains. Further studies on the full-length p40(phox) and p47(phox) proteins showed that an intramolecular interaction between the C-terminal Src homology 3 domain and the PX domain prevents the nonspecific monolayer penetration of p47(phox), whereas such an interaction is absent in p40(phox).     

The competition between transferrins labeled with59Fe,65Zn, and54Mn for the binding sites on lactating mouse mammary gland cells

Using a homologous competition of⁵⁴Mn-transferrin with Mntransferrin and⁶⁵Zn-transferrin with Zn-transferrin, it was found that on the plasma membrane of lactating mouse mammary gland cells there are receptor binding Mn-transferrin and Zn-transferrin. The heterologous competition between labeled and nonlabeled Fe-transferrin, Mn-transferrin and Zn-transferrin, as well as almost equal affinity constants of cellular receptors toward the three metals by competition of Fe-transferrin suggests that one and the same receptor accepts all three metals from the transferrin molecule. The cell receptors therefore possess a polymetal binding function. A model and a mechanism for regulation of the transport metal flow toward the mammary gland cell acting like “automated switching over” are proposed.     

Impermeant potential-sensitive oxonol dyes: II. The dependence of the absorption signal on the length of alkyl substituents attached to the dye

Summary We have measured the potential-dependent light absorption changes of 43 impermeant oxonol dyes with an oxidized cholesterol bilayer lipid membrane system. The size of the signal is strongly dependent on the chain length of alkyl groups attached to the chromophore. Dye molecules with intermediate chain lengths give the largest signals. To better understand the dependence of the absorbance signal on alkyl chain length, a simple equilibrium thermodynamic analysis has been derived. The analysis uses the free energy of dye binding to the membrane and the on-off model (E.B. George et al.,J. Membrane Biol.,103:245–253, 1988a) for the potential-sensing mechanism. In this model, a population of dye molecules in nonpolar membrane binding sites is in a potential-dependent equilibrium with a second population of dye that resides in an unstirred layer adjacent to the membrane. Dye in the unstirred layer is in a separate equilibrium with dye in the bulk bathing solution. The equilibrium binding theory predicts a sigmoidally shaped increase in signal with increasing alkyl chain length, even for very nonpolar dyes. We suggest that aggregation of the more hydrophobic dyes in the membrane bathing solution may be responsible for their low signals, which are not predicted by the theory.     

Ultrastructure of neuroglial contacts in crayfish stretch receptor

In order to explore neuroglial relationships in a simple nervous system, we have studied the ultrastructure of the crayfish stretch receptor, which consists of only two mechanoreceptor neurons enwrapped by glial cells. The glial envelope comprises 10–30 glial layers separated by collagen sheets. The intercellular space between the neuronal and glial membranes is generally less than 10–15 nm in width. This facilitates diffusion between neurons and glia but restricts neuron communication with the environment. Microtubule bundles passing from the dendrites to the axon through the neuron body limit vesicular transport between the perikaryon and the neuronal membrane. Numerous invaginations into the neuron cytoplasm strengthen glia binding to the neuron and shorten the diffusion pathway between them. Double-membrane vesicles containing fragments of glial, but not neuronal cytoplasm, represent the captured tips of invaginations. Specific triads, viz., “flat submembrane cisterns - vesicles - mitochondria”, are presumably involved in the formation of the invaginations and double-membrane vesicles and in neuroglial exchange. The tubular lattice in the glial cytoplasm might transfer ions and metabolites between the glial layers. The integrity of the neuronal and glial membranes is impaired in some places. However, free neuroglial passage might be prevented or limited by the dense diffuse material accumulated in these regions. Thus, neuroglial exchange with cellular components might be mediated by transmembrane diffusion, especially in the invaginations and submembrane cisterns, by the formation of double-walled vesicles in which large glial masses are captured and by transfer through tubular lattices. This work was supported by RFBR (grants 05-04-48440 and 08-04-01322) and Minobrnauki RF (grant 2.1.1/6185).     

Transport of receptors

The axonal transport of neurotransmitter receptors is thought to be a common phenomenon in many neuronal systems. The “machinery” for receptor (protein) “assembly” is found in the cell bodies of neurons and the “manufacture” of receptors takes place there. These receptors are then “shipped” to their ultimate destinations by a transport process. This is an axonal transport mechanism in the case of presynaptic receptors. Some form of transport process may also exist to send receptors out into the dendritic arborizations of neurons, although the latter is more difficult to verify. Axonal transport has been demonstrated, in the peripheral nervous systems, for many different neurotransmitter receptors. In the central nervous system, the results are less clear, but indicate the presence of a transport mechanism for catecholamine, acetylcholine, and opiate sites. One important component then, in the development of receptors, is the transportation to terminal membrane sites where they are ultimately incorporated and available for interaction with neurotransmitters and drugs.     

Diffusion Models for the Squid Axon Schwann Cell Layer

The Schwann cell, basement membrane, and connective tissue layers that surround the squid giant axon and constitute barriers to diffusion, were modeled in a number of ways to analyze various experimental results. The experiments considered are (a) the time-course of the potassium concentration in the space between the Schwann cell and the axon membrane (from now on referred to as the F-H space) after an initial loading, (b) the time-course of sodium concentration in the F-H space after a sudden change in the sodium concentration in the external fluid; (c) the time-course of the concentration of tetrodotoxin (TTX) or saxitoxin (STX) in the F-H space after a sudden change in external concentration, including (or not) the effects of specific binding of TTX or STX to sites on the axon membrane and nonsaturable binding to sites in the F-H space or in the spaces (clefts) between Schwann cells; (d) the effects of the F-H space, clefts, and diffusion into the clefts from the outside (from now on referred to as convergence into the clefts) on the measured series resistance.

The analysis shows that (1) in no case is it necessary to include the effects of the convergence into the clefts from the outside; (2) in case a, the basement membrane, connective tissue layers, and the unstirred layer may be neglected, i.e., the clefts are rate limiting; (3) in case b the clefts may be neglected, i.e., the unstirred layer is rate limiting; (4) in most cases the clefts may be replaced by an equivalent thin diffusion barrier.

     

Changes in the cell ultrastructure of the haloalkaliphilic endoevaporite cyanobacterium <Emphasis Type="Italic">‘Euhalothece natronophila’</Emphasis> during fossilization

The ultrastructure of the haloalkaliphilic endoevaporite cyanobacterium ‘Euhalothece natronophila’ Z-M001 from the soda Lake Magadi was investigated during the initial stages of fossilization in a model experimental system. The cyanobacterium was cultivated in concentrated carbonate solution supplemented with calcium chloride. It was revealed that the amorphous CaCO₃ formed under these conditions could interact with the cell wall during the first stages of ‘E. natronophila’ calcification. Evidence is presented that the surface layer of the ‘E. natronophila’ envelope, presumably containing polysaccharide and/or (glyco)protein components, can be involved in the adsorption and subsequent crystallization of CaCO₃ with the formation of a massive “shell” embedding the morphologically intact cells. It was established that the ultrastructure of the cell wall and the intrathylakoid space changed during CaCO₃ mineralization. During the later fossilization stages, cells covered by the calcium-containing “shell” were apparently mummified, and mostly retained their original shape. The encapsulation of cyanobacteria in the trona globule was characterized by a different pattern. It probably involved tight binding of the growing crystal to the glycocalyx components that are anchored in the outer membrane. This may result in its detachment from the underlying peptidoglycan layer. The peptidoglycan was retained, and the protoplasts were ultrastructurally similar to the intact ones. Cyanobacteria incorporated in large trona crystals underwent degradation, deformation, and destruction. This accounts for the fact that massive trona deposits of Lake Magadi lack cyanobacterial fossils that are abundant in calcium-containing strata.     

The effect of alkylhydroxybenzenes on the antigen-binding capacity of antibodies

The effect of the chemical analogues of microbial extracellular autoregulators belonging to alkylhydroxybenzenes (AHB), hexylresorcinol (HR), and methylresorcinol (MR), on the interactions between specific antibodies and the corresponding antigens was studied. Nonlinear dependency of the inhibition of binding of AHB-modified antibodies on the AHB chemical structures and concentrations was revealed by enzyme immunoassay. Hexylresorcinol was shown to decrease the antibody affinity and avidity indices and simultaneously increase the indices of nonspecific binding of AHB-modified antibodies to antigens, thereby promoting the formation of “false” antigen-antibody complexes. The nonspecificity of the influence of AHB on the antigenbinding capacity of antibodies is an important characteristic of these effects, which allows us to consider AHB as unique “superhaptenes”.     

Temporo-spatial distribution of matrix and microfilament components during odontoblast and ameloblast differentiation

Summary Several extracellular matrix components (procollagen type III, fibronectin, collagen type IV, laminin and nidogen) and microfilament constituents (actin, α-actinin and vinculin) were localized by indirect immunofluorescence microscopy in frozen sections of embryonic mouse molars. Nidogen was present at the epithelio-mesenchymal junction during polarization and initial steps of functional differentiation of odontoblasts. Nidogen disappeared at a stage where direct contacts between preameloblasts and predentin were required to allow the initiation of ameloblast polarization. Our observations concerning the distribution of procollagen type III and fibronectin during odontoblast differentiation add to current knowledge. Procollagen type III and fibronectin surrounding preodontoblasts accumulated at the apical part of polarizing and functional odontoblasts secreting “initial” predentin. Procollagen type III, but not fibronectin, disappeared in front of functional odontoblasts synthesizing “late” predentin and dentin. Fibronectin, present in “initial” predentin, was no longer detected in “late” predentin and dentin but was found between odontoblasts secreting “late” predentin and dentin. Actin, α-actinin and vinculin were concentrated in the peripheral cytoplasm of preameloblasts and accumulated at the apical and basal poles of functional ameloblasts. During differentiation of odontoblasts, the three proteins accumulated at the apical pole of these cells. Time and space correlations between matrix and microfilament modifications during odontoblast and ameloblast differentiation are documented. The possibility is discussed that there is transmembranous control of the cytoskeletal activities of odontoblasts and ameloblasts by the extracellular matrix.     

Lipid membrane-induced optimization for ligand–receptor docking: recent tools and insights for the “membrane catalysis” model

Cells in living organisms are regulated by chemical and physical stimuli from their environment. Often, ligands interact with membrane receptors to trigger responses and Sargent and Schwyzer conceived a model to describe this process, “membrane catalysis”. There is a notion that the physical organization of membranes can control the response of cells by speeding up reactions. We revisit the “membrane catalysis” model in the light of recent technical, methodological and theoretical advances and how they can be exploited to highlight the details of membrane mediated ligand–receptor interactions. We examine the possible effects that ligand concentration causes in the membrane catalysis and focus our attention in techniques used to determine the partition constant. The hypothetical diffusional advantage associated with membrane catalysis is discussed and the applicability of existing models is assessed. The role of in-depth location and orientation of ligands is explored emphasizing the contribution of new analysis methods and spectroscopic techniques. Results suggest that membranes can optimize the interaction between ligands and receptors through several different effects but the relative contribution of each must be carefully investigated. We certainly hope that the conjugation of the methodological and technical advances here reported will revive the interest in the membrane catalysis model.     

A mechanism for indirect allosteric action of charged effectors

A mechanism for indirect allosteric action of charged effectors on substrate binding to a macromolecule is proposed. It is accounted for by electrostatic interaction among effectors in the solution, away from their receptors. The possibility of the mechanism proposed is tested in the allosteric action of univalent salt and 2,3-diphosphoglycerate on oxygen binding to hemoglobin. A model for electrostatic interaction between these two effectors in the solution and for their overall effect on oxygen binding is introduced. The 2,3-diphosphoglycerate binding constant to deoxygenated hemoglobin as a function of univalent salt concentration and the median ligand activity as a function of the concentration of univalent salt and 2,3-diphoshoglycerate are calculated and compared with experimental data. The obtained results indicate that electrostatic interaction in the solution may significantly contribute to indirect allosteric action of charged effectors. Partly presented at the “11th FEBS Meeting” in Copenhagen, August 1977     

FGF2 and dexamethasone increase the production of hyaluronan in two-dimensional culture of elastic cartilage-derived cells: in vitro analyses and in vivo cartilage formation

Elastic cartilage-derived cells cultured two-dimensionally with FGF2 and corticosteroid produce gel-type masses that become mature cartilage when injected into a subcutaneous pocket. This unique method has previously been clinically applied for treatments of nasal augmentation. However, the components of the gel-type mass and the mechanism of its synthesis remain unknown. Here, we have investigated the components of the gel-type mass produced by elastic cartilage-derived cells, and whether this gel-type mass can be produced by using other cell sources or other media. Human elastic cartilage-derived cells from auricular cartilage, hyaline cartilage-derived cells from articular cartilage, and mesenchymal stem cells from synovium were cultured in three media: “redifferentiation medium” containing FGF2 and dexamethasone; “chondrogenic medium” containing bone morphogenetic protein-2, transforming growth factor-β3, and dexamethasone specific for in vitro chondrogenesis of mesenchymal stem cells; control medium. The elastic cartilage-derived cells cultured in redifferentiation medium produced a gelatinous matrix positive for Alcian blue. During culture, the amount of chondroitin 4-sulfate, chondroitin 6-sulfate, and especially hyaluronan increased. However, the expression of RNAs for most chondrogenic genes did not increase. We also reproduced cartilage tissue formation by the injection of elastic cartilage-derived cells with the gelatinous mass into the subcutaneous space of the nude mouse. The synthesis of gelatinous matrix in vitro and the formation of cartilage tissue in vivo could be obtained only for the combination of elastic cartilage-derived cells with redifferentiation medium. This study was supported in part by grants from the “Japan Society for the Promotion of Science (19591752)” and “Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone in Tokyo Medical and Dental University” to Takeshi Muneta, and the “Japan Society for the Promotion of Science (18591657)” to Ichiro Sekiya.     

A novel artificial substrate for cell culture: Effects of substrate flexibility/malleability on cell growth and morphology

Summary Gels of glyoxyl agarose (GA) are evaluated as a novel flexible substrate for cell culture with physical properties comparable to extracellular matrix (ECM) gels. We show here that cells adhere well to pure GA gels; in addition, specific interactions involving matrix receptors can be studied when individual matrix molecules are bound to the gel covalently. When cells are grown on such substrates, morphology is comparable to that observed on “natural” matrix gels (reconstituted gels of collagen type I or of Matrigel): rather than being flattened as in monolayer cultures on tissue culture plastic the cells assume a rounded morphology and tend to form tissue-like aggregates. The effects of the artificial matrix gels are discussed in the context of previous publications on cell interactions with the extracellular matrix, suggesting that in addition to specific recognition of matrix molecules the physical properties of ECM by themselves can be decisive for cell differentiation. We conclude that gels of glycoxyl agarose a) provide a useful model to mimic the physical properties of matrix gels without the presence of specific adhesion factors; b) may be useful as a general, non-specific ECM allowing cells to be cultured in vitro under conditions favorable for differentiation; and c) allow to design a variety of “synthetic” ECM models composed of a chemically defined gel matrix, which can be supplemented with covalently bound molecules to be recognized by cell surface receptors.     

Two new motile phototrophic consortia: “Chlorochromatium lunatum” and “Pelochromatium selenoides”

Two new phototrophic consortia, “Chlorochromatium lunatum” and “Pelochromatium selenoides”, were observed and collected in the hypolimnion of several dimictic lakes in Wisconsin and Michigan (USA). The two consortia had the same morphology but different pigment composition. The cells of the photosynthetic components of the consortia were half-moon-shaped. This morphology was used to differentiate them from the previously described motile phototrophic consortia “Chlorochromatium aggregatum” and “Pelochromatium roseum”. These phototrophic cells did not resemble any described unicellular green sulfur bacteria. The predominant pigments detected were bacteriochlorophyll d and chlorobactene for the green-colored “Clc. lunatum”, and bacteriochlorophyll e and isorenieratene for the brown-colored “Plc. selenoides”. Their pigment compositions and the presence of chlorosomes attached to the inner face of the cytoplasmic membrane in both kinds of photosynthetic cells confirmed this new half-moon-shaped morphotype as a green sulfur bacterium. Both consortia were found thriving in lakes with low concentrations of sulfide (< 60 μM), below the layers of “Clc. aggregatum” and “Plc. roseum”. The green consortia were observed in lakes where the oxic-anoxic interface was located at shallow depths (2–7 m), while the brown consortia were found at greater depths (8–16 m). The two newly described consortia were never detected together at the same depth in any lake. Received: 30 April 1997 / Accepted: 17 January 1998     

Interaction of auxin-binding protein 1 with maize coleoptile plasma membranes in vitro

In a search for membrane “docking proteins” interacting with Zea mays auxin-binding protein (ABP1) the binding of purified ABP1 to maize coleoptile plasma-membrane vesicles was investigated. Concentration-dependent, saturable binding of ABP1 to the membrane vesicles was observed in binding assays using 10⁻⁸–10⁻⁶␣M ABP1. Biotinylated ABP1 was displaced from the membrane binding sites by competition with unlabeled ABP1, demonstrating specific binding. The association step proved to be pH-dependent with maximum binding at pH 5.0 or lower. Auxins did not influence the ABP1 binding to plasma-membrane vesicles, but ABP1 associated with plasma-membrane vesicles was still able to specifically bind [³H]naphthalene-1-acetic acid. The rather stable interaction of ABP1 with plasma-membrane vesicles was only affected by strong alkaline buffers or detergents. The binding capacity was calculated to be in the range of 0.2 pmol ABP1 per g coleoptile fresh weight. Received: 29 April 1996 / Accepted: 20 September 1996