A general method for studying the secretion of macromolecules by single cells. I. Detection of immunoglobulin-secreting cells.

A general method for detecting single cells secreting macromolecules has been developed and applied to the detection of mouse cells secreting antibodies. Secreted Ig molecules were precipitated in the immediate vicinity of an active cell with rabbit anti-mouse Ig antibody. Rapid removal of excess antibody not incorporated into immunoprecipitates was achieved with an electrophoresis technique. The immuno-precipitate surrounding the active cell was then stained with sheep anti-rabbit Ig antibody labeled with horseradish peroxidase. The use of enzyme markers has made the procedure much more convenient and rapid than previous precipitin assays for cell secretion that used radioiodine for detecting immunoprecipitates. Moreover, the availability of several enzyme markers makes possible the detection of cells secreting more than one molecular species. Experiments were also run in which cells producing antibodies specific for horseradish peroxidase (HRP) could be identified among the population of cells producing other immunoglobulins. Presumably, HRP-labeled antigens could be used to identify cells producing other specific antibodies. The generality of this procedure suggests that it may be useful for detecting single T-cells releasing regulatory molecules, since specific antisera are already available for several of these molecules.     

A biological interpretation of transient anomalous subdiffusion. I. Qualitative model

Anomalous subdiffusion has been reported for two-dimensional diffusion in the plasma membrane and three-dimensional diffusion in the nucleus and cytoplasm. If a particle diffuses in a suitable infinite hierarchy of binding sites, diffusion is well known to be anomalous at all times. But if the hierarchy is finite, diffusion is anomalous at short times and normal at long times. For a prescribed set of binding sites, Monte Carlo calculations yield the anomalous diffusion exponent and the average time over which diffusion is anomalous. If even a single binding site is present, there is a very short, almost artifactual, period of anomalous subdiffusion, but a hierarchy of binding sites extends the anomalous regime considerably. As is well known, an essential requirement for anomalous subdiffusion due to binding is that the diffusing particle cannot be in thermal equilibrium with the binding sites; an equilibrated particle diffuses normally at all times. Anomalous subdiffusion due to barriers, however, still occurs at thermal equilibrium, and anomalous subdiffusion due to a combination of binding sites and barriers is reduced but not eliminated on equilibration. This physical model is translated directly into a plausible biological model testable by single-particle tracking.     

Evidence for an initial fast nucleation process in the folding of human carbonic anhydrase I

Kinetic studies of the folding of carbonic anhydrase have indicated the occurrence of various conformational intermediates. Human carbonic anhydrase I contains a single cysteine residue, Cys-212, which in the native state is unavailable for alkylation. In the unfolded state, it can be specifically modified with iodoacetate. In this study the accessibility of Cys-212 in human carbonic anhydrase I to iodo[2-14C]acetate during the refolding process has been investigated. It is shown that Cys-212 is hidden to the alkylating agent as soon as the refolding is initiated. Since Cys-212 is located in the extensive beta-structure passing through the enzyme, it appears that the Cys-containing beta-strand is part of a rapidly formed nucleation center created during the folding process. This beta-strand (No. 7) together with its neighboring beta-strand (No. 6) constitute the most hydrophobic regions of the enzyme. Because hydrophobic contacts are considered to be important in predicting nucleation sites, these beta-strands probably partake in the formation of the nucleation center. These beta-strands are also partly involved in the bottom region of the active site cavity, indicating that this region is formed during the initial folding events. As a result of this study it was also observed that 2-mercaptoethanol is a potent inhibitor of the enzyme with a K1 = 26 microM at pH 8.0.     

A possible model for cell-cell recognition via surface macromolecules.

Alternative possibilities for the establishment of the proper cell distribution during embryogenesis are summarized at the beginning, followed by an assessment of the examples known so far where cell-cell recognition is known to be mediated via cell surface components. In the second part the species-specific recognition process which occurs during the sorting-out of dissociated sponge cells is analysed since it may serve as a possible model for cell-cell recognition in higher animals. Three possible mechanisms for the establishment of proper cell distribution are considered. These include, first, chemotaxis: secondly, guidance of cell or cell sheet movement by extracellular matrix or by surrounding cells and thirdly, random movement followed by recognition at the final point of destination. Recognition is necessary for both of the two latter processes, i.e. for cell guidance as well as for locking the cells into their final position after random movement. Two basically different recognition mechanisms should be distinguished from each other. On the one hand cells may recognize each other with the help of macromolecules situated in or just outside of the plasmamembrane which fit to each other like enzymes and substrates or antibodies and antigens. On the other hand, cells may exchange information by exchanging cytoplasmatic components via vesicles or gap junctions. The species-specific aggregation of dissociated sponge cells is considered to be a possible model for cell-cell recognition in higher animals. A proteoglycan-like intercellular macromolecule called aggregation factor seems to mediate recognition of a given species of cells in the reaggregation process of dissociated cells. The data available at the present time suggest that a monovalent surface macromolecule (baseplate) may mediate the recognition process probably by recognizing the carbohydrate side chains of the multivalent proteoglycan aggregation factor. A cell-free system was devised to mimic this aggregation process. Addition of aggregation factor to baseplate-coated sepharose beads of approximately the size of the original sponge cells has essentially the same characteristics as the cellular system. Macromolecule-coded surface information for the recognition between cells has not been established during the embryogenesis of higher animals and remains an interesting challenge.     

Multiple equilibrium states in a micro-vascular network

We use a simple model of micro-vascular blood flow to explore conditions that give rise to multiple equilibrium states in a three-node micro-vascular network. The model accounts for two primary rheological effects: the Fåhræus-Lindqvist effect, which describes the apparent viscosity of blood in a vessel, and the plasma skimming effect, which governs the separation of red blood cells at diverging nodes. We show that multiple equilibrium states are possible, and we use our analytical and computational tools to design an experiment for validation.     

Multiple kinetic states for the flagellar motor switch.

By means of a computerized video processing system, the flagellar motors of Escherichia coli were shown to have multiple kinetic states for each rotational direction. High-resolution analysis of flagellar motors revealed new kinetic states both in wild-type cells and in a strain deleted of other signal-transducing genes to which CheY had been introduced. This strain, RP1091, retained residual kinase activity that could phosphorylate CheY, complicating the biochemical identification of certain kinetic states. The behavioral effect of CheY on single flagellar motors was ultrasensitive, with an apparent Hill coefficient of 5.5 +/- 1.9 (SD) and a half-maximal effect at 10.1 +/- 0.5 (SD) microM CheY. Based on the CheY concentration dependence, a two-state model is clearly excluded, even for the simpler system of CheY-induced rotational reversals in the deletion strain. The data are best described by a four-state model, with two clockwise and two counterclockwise states.     

Qualitative analysis of a model generating long potential waves in Ba-treated nerve cells—I. Reduced systems

This study is related to a model describing the behavior of barium-treatedAplysia neurons generating regular burst-plateau patterns. The model is represented by an autonomous dynamical system, defined inR ⁴ and depending on a small parameter. This paper is restricted to the qualitative study of three “reduced systems” deduced from the “complete system”. Part of the study is performed with the use of the qualitative theory of singular perturbations. The predicted behaviors are compared with experimental results.     

Evolution of macromolecules: I. Dual coding systems

The evolution of proteins determined by two independently mutable coding mechanisms (e.g., one in which nucleic acids operate with unit coding ratio) has been analyzed kinetically in two ways. The first presumes a system of mutating and reproducing proteins; the dependent variables are the numbers of the various kinds of proteins—wild types, and mutants obtained by mutations in one or another of the two coding mechanisms. The second approach deals with kinds rather than numbers of proteins; the reproductive element in the evolving system is dealt with by assuming a specific rate of extinction for members of each protein class, due to the occurrence of lethal mutations in the proteins themselves or in other proteins in the organisms that carry them. If the two kinds of mutants occur at different rates, it is shown in both treatments that time will not necessarily extinguish the initial advantage of one of them—that is, the notion that the slower class will eventually occur often enough to produce a random distribution of the two classes after long periods of evolution is not in general true. The effects of mutation rate, reproduction rate, and extinction rate on the distribution of the various protein classes are analyzed. Contribution No. 666 from the Division of Basic Health Sciences.     

Stochastic analysis of a nonlinear model for selection of biological macromolecules

A stochastic analysis of a nonlinear selection model is presented. The model, based on Eigen and Schuster's theory of selection and evolution of biological macromolecules, considers the effects of fluctuations on the individual concentrations of macromolecules as well as the total population numbers in constrained systems. Our analysis shows that one of the models most often treated deterministically (referred to as constant organization in the literature) becomes unstable when fluctuations in the total population number are considered. An alternative model which apparently has built in self-regulating properties is analyzed and proves to be stable except for some special cases of degeneracy.     

On one-dimensional nucleation and growth of “living” polymers I. Homogeneous nucleation

The kinetics of one-dimensional polymerization are described in terms of successive monomer additions. During homogeneous nucleation, the concentration c_n of critical nuclei is proportional to the nth power of monomer concentration c₁ and, initially, to the (n?1)th power of time.     

Matrix macromolecules in hard tissues control the nucleation and hierarchical assembly of hydroxyapatite

Biogenic minerals found in teeth and bones are synthesized by precise cell-mediated mechanisms. They have superior mechanical properties due to their complex architecture. Control over biomineral properties can be accomplished by regulation of particle size, shape, crystal orientation, and polymorphic structure. In many organisms, biogenic minerals are assembled using a transient amorphous mineral phase. Here we report that organic constituents of bones and teeth, namely type I collagen and dentin matrix protein 1 (DMP1), are effective crystal modulators. They control nucleation of calcium phosphate polymorphs and the assembly of hierarchically ordered crystalline composite material. Both full-length recombinant DMP1 and post-translationally modified native DMP1 were able to nucleate hydroxyapatite (HAP) in the presence of type I collagen. However, the N-terminal domain of DMP1 (amino acid residues 1-334) inhibited HAP formation and stabilized the amorphous phase that was formed. During the nucleation and growth process, the initially formed metastable amorphous calcium phosphate phase transformed into thermodynamically stable crystalline hydroxyapatite in a precisely controlled manner. The organic matrix-mediated controlled transformation of amorphous calcium phosphate into crystalline HAP was confirmed by x-ray diffraction, selected area electron diffraction pattern, Raman spectroscopy, and elemental analysis. The mechanical properties of the protein-mediated HAP crystals were also determined as they reflect the material structure. Such understanding of biomolecule controls on biomineralization promises new insights into the controlled synthesis of crystalline structures.