3,4-diaminopyridine. A potent new potassium channel blocker.

3,4-diaminopyridine has been found to act very potently in selectively blocking the potassium channels of squid axon membranes. The apparent dissociation constants for this action are estimated to be 5.8 micron and 0.7 micron for external and internal applications, respectively, the potency being about 50 times higher than that of 4-aminopyridine. The block depends upon the membrane potential, time, and stimulus frequency. 3,4-diaminopyridine shows great promise as a useful tool for the study of membrane ionic channels.     

On the activation-inactivation coupling in Shaker potassium channels.

The 'ball-and-chain' model suggests the existence of a negative site which may attract the positively charged inactivation ball to occlude the pore when the channel is in the open state. For Shaker K+ channels, we propose that the state-dependent negative site be tryptophan-435, which becomes negatively charged after receiving an electron from tyrosine-445. The kinetic scheme for the channel's activation-inactivation coupling as derived from the YW-gated model resembles a successful 'scheme 8' proposed by Zagotta and Aldrich. Our model suggests that the final rapid voltage-independent transition to the open state is due to the deprotonation of tyrosine-445.     

A new measure for upright stability

The control of balance is a primary objective in most human movements. In many cases, research or practice, it is essential to quantitatively know how good the balance is at a body posture or at every moment during a task. In this paper we suggest a new measure for postural upright stability which assigns a value to a body state based on the probability of avoiding a fall initiation from that state. The balance recovery problem is solved for a population sample using a strength database, and the probability of successfully maintaining the balance is found over the population and called the probability of recovery (PoR). It, therefore, describes an attribute of a body state: how possible the control of balance is, or how safe being at that state is. We also show the PoR calculated for a 3-link body model for all states on a plane, compare it to that found using a 2-link model, and compare it to a conventional metric: the margin of stability (MoS). It is shown, for example, that MoS may be very low at a state from which most of the people will be able to easily control their balance.     

Intracellular pH,intracellular free Ca,and junctional cell-cell coupling

Summary Intracellular pH (pH _i ) and intracellular Ca²⁺ ([Ca²⁺] _i ) were determined inChironomus salivary gland cells under various conditions of induced uncoupling. pH _i was measured with aThomas-type microelectrode, changes in [Ca²⁺] _i and their spatial distribution inside the cell were determined with the aid of intracellularly injected aequorin and an image intensifier-TV system, and cell-to-cell coupling was measured electrically. Treatments with NaCN (5mm), DNP (1.2mm), or ionophore A23187 (2m) caused fall in junctional conductance (uncoupling) that was correlated with [Ca²⁺] _i elevation, as was shown before (Rose & Loewenstein, 1976,J. Membrane Biol. 28:87) but not with changes in pH _i : during the uncoupling induced by CN, the pH _i (normally 7.5) decreased at most by 0.2 units; during the uncoupling induced by the ionophore, pH _i fell by 0.13 or rose by 0.3; and in any one of these three agents' uncouplings, the onset of uncoupling and recovery of coupling were out of phase with the changes in pH _i . Intracellular injection of Ca-citrate or Ca-EGTA solutions buffered to pH 7.2 or 7.5 produced uncoupling with little or no pH _i change when their free [Ca²⁺] _i was >10^–5 m. On the other hand, such a solution at pH 4, buffered to [Ca²⁺]<10^–6 m, lowered pH _i to 6.8 but produced no uncoupling. Thus, a decrease in pH _i is not necessary for uncoupling in any of these conditions. In fact, uncoupling ensued also during increase in pH _i : exposure to NH₄HCO₃ or withdrawal of propionate following exposure to a propionate-containing medium caused pH _i to rise to 8.74, accompanied by [Ca²⁺] _i elevation and uncoupling at pH _i >7.8.Cell acidification itself can cause elevation of [Ca²⁺] _i : injection (iontophoresis) of H⁺ invariably caused [Ca²⁺] _i elevation and uncoupling. These effects were produced also by an application of H⁺-transporting ionophore Nigericin at extracellular pH 6.5 which caused pH _i to fall to 6.8. Exposure to 100% CO₂ produced a fall in pH _i , associated in 10 out of 25 cases with [Ca²⁺] _i elevation and, invariably, with uncoupling. The absence of a demonstrable [Ca²⁺] _i elevation in a proportion of these trials is attributable to depression in Ca²⁺-measuring sensitivity; inin vivo tests, detection sensitivity for [Ca²⁺] _i by aequorin was found to be depressed by the CO₂ treatment. Upon CO₂ washout, pH _i and coupling recovered, but onset of recoupling set in at pH _i as low as 6.32–6.88, generally lower than at the pH _i at which uncoupling had set in. Exposure to 5% CO₂ lowered pH _i on the average by 0.3 and depressed coupling (in initially poorly coupled cells). After CO₂-washout, pH _i and coupling recovered. During the recovery phase [Ca²⁺] _i was elevated, an elevation associated with renewed uncoupling or decrease in rate of recoupling. The results are discussed in connection with possible regulatory mechanisms of junctional permeability.     

A new method to measure foot contact

A new method to measure foot contact is described. It consists of a pressure sensitive transducer attached to one end of a flexible silicone rubber tube. A reliable indicator of foot contact is obtained with the tube glued to the outer perimeter of the sole of a shoe.     

A new measure of cooperativity in protein-ligand binding

An allosteric binding system consisting of a single ligand and a nondissociating macromolecule having multiple binding sites can be represented by a binding polynomial. Various properties of the binding process can be obtained by analyzing the coefficients of the binding polynomial and such functions as the binding curve and the Hill plot. The Hill plot has an asymptote of unit slope at each end and the departure of the slope from unity at any point can be used to measure the effective interaction free energy at that point. Of particular interest in detecting and measuring cooperativity are extrema of the Hill slope and its value at the half-saturation point. If the binding polynomial is symmetric, then there is an extremum of the Hill slope at the half-saturation point. This value, the Hill coefficient, is a convenient measure of cooperativity. The purpose of this paper is to express the Hill coefficient for symmetric binding polynomials in terms of the roots of the polynomial and to give an interpretation of cooperativity in terms of the geometric pattern of the roots in the complex plane. This interpretation is then applied to the binding polynomials for the MWC (Monod-Wyman-Changeux) and KNF (Koshland-Nemethy-Filmer) models.     

A new measure of the robustness of biochemical networks

MOTIVATION: The robustness of a biochemical network is defined as the tolerance of variations in kinetic parameters with respect to the maintenance of steady state. Robustness also plays an important role in the fail-safe mechanism in the evolutionary process of biochemical networks. The purposes of this paper are to use the synergism and saturation system (S-system) representation to describe a biochemical network and to develop a robustness measure of a biochemical network subject to variations in kinetic parameters. Since most biochemical networks in nature operate close to the steady state, we consider only the robustness measurement of a biochemical network at the steady state. RESULTS: We show that the upper bound of the tolerated parameter variations is related to the system matrix of a biochemical network at the steady state. Using this upper bound, we can calculate the tolerance (robustness) of a biochemical network without testing many parametric perturbations. We find that a biochemical network with a large tolerance can also better attenuate the effects of variations in rate parameters and environments. Compensatory parameter variations and network redundancy are found to be important mechanisms for the robustness of biochemical networks. Finally, four biochemical networks, such as a cascaded biochemical network, the glycolytic-glycogenolytic pathway in a perfused rat liver, the tricarboxylic acid cycle in Dictyostelium discoideum and the cAMP oscillation network in bacterial chemotaxis, are used to illustrate the usefulness of the proposed robustness measure.     

Mechanically facilitated cell-cell electrofusion.

Apparatus and methods were developed to enable mechanically facilitated cell-cell electrofusion to be performed. The apparatus and methods mechanically place cells in contact before fusion. The key component of this fusion system was a newly developed fusion chamber. The chamber was composed of two functionally identical electrodes that were housed in a multi-layer structure. The layers functioned as support for the electrodes. They also allowed adjustment of the distance between opposing electrode faces. The electrodes were constructed in a manner that allowed cells to be deposited, by vacuum, onto each face. Electrode faces were positioned at a predetermined distance from each other to mechanically force cell-cell contact between the deposited cells. Fusion was induced by delivering direct current pulses to the juxtaposed cells. Fusion products were detected and quantitated by flow cytometry. Details of the chamber design and a protocol for using the fusion chamber are given. Mechanically facilitated cell-cell electrofusion was demonstrated by using the chamber to produce fusion products from like fusion partners. The practical applicability of the chamber was demonstrated by fusing unlike cell types. Mechanically facilitated cell-cell electrofusion is not specific to the cells used in this study; the chamber can be adapted for use with other cell types.     

A new assay to measure RNA N-glycosidase activity

We have developed a convenient procedure to measure the activity of Ricin A-chain and other enzymes with RNA N-glycosidase activity. The method is based on the use of a tritiated oligoribonucleotide as a substrate. The enzymatic activity is directly determined by measuring the release of adenine from the substrate. This method should prove useful in the study of the molecular mechanism of action of Ricin A and other RNA N-glycosidases.     

A new dynamometric technique to measure and locate endocranial structures. Preliminary report

An apparatus for measuring and recording the resistance to penetration of endocranial tissue has been planned and built. The probe carrier, driven by a constant-speed electric motor, is fitted onto a stereotactic head frame which is used to guide the tool to the intended target. The displacement and resistance encountered when the tool penetrates intracranial structures are measured and recorded on an x-y recorder. Preliminary tests performed on calf brain specimens have documented that the apparatus can measure the different consistencies of normal cerebral tissue and suggest a new technique for morphological investigations based on the mechanical consistency of normal and pathological organic tissue. Moreover, the hypothesis of a plastic deformation in cerebral tissue has been confirmed, in that the same apparatus permits one to measure the displacement of tissues caused by the advancement of the surgical tool.     

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.     

Isolation and characterization of Chinese hamster cells defective in cell-cell coupling via gap junctions

Chinese hamster Wg3-h-o cells which were descended from DON cells have been mutagenized and selected for derivatives defective in metabolic cooperation via gap junctions (i.e., mec-). The selection protocol included four consecutive cycles of cocultivating mutagenized cells, deficient in hypoxanthine phosphoribosyltransferase (HPRT) and wild-type cells in the presence of thioguanine (cf Slack, C, Morgan, R H M & Hooper, M L, Exp cell res 117 (1978) 195-205) [8]. We carried out the last two selection cycles in the presence of 1 mM dibutyryl cyclic adenosine monophosphate (db-cAMP). The isolated Chinese hamster CI-4 cells which expressed the mec- phenotype most stringently showed the following characteristics: 1. In standard culture medium no cell-cell coupling was detected among CI-4 cells when assayed by injections of the fluorescent dye Lucifer yellow or by electrical measurements. Between 73 and 100% of the mec+ parental cells were coupled under these conditions. Up to 14% positive contacts were found between CI-4 cells and Chinese hamster Don cells (mec+). Confluent CI-4 cells grown in the presence of 1 mM db-cAMP showed 9% coupled cells. 2. No gap junction plaques were found on electron micrographs of freeze-fractured, confluent CI-4 cells. The mec+ parental cells showed small gap junction plaques (0.013% of the total cell surface analyzed). 3. CI-4 cells exhibited 16% positive contacts and the parental Wg3-h-o cells showed 92% positive contacts in autoradiographic measurements of metabolic cooperation with DON cells. On an extracellular matrix, prepared from normal embryonic fibroblasts, metabolic cooperation between CI-4 and DON cells was autoradiographically measured to be 68%. Other cells of spontaneous mec- phenotype (for example mouse L cells or human fibrosarcoma HT1080 cells) also appeared to exhibit increased metabolic cooperation when grown on an extracellular matrix and assayed by autoradiographic measurements. When tested by Lucifer yellow injections, however, only very few positive contacts were found for CI-4/DON cell pairs and no positive contacts were found among mouse L cells grown on an extracellular matrix. 4. The mec- defect in the genome of CI-4 cells was cured in somatic cell hybrids with mouse embryonic fibroblasts or with mouse embryonal carcinoma cells. The results of isozyme and karyotype studies of mec-, as well as mec+ somatic cell hybrids suggest that mouse chromosome 16 may be involved in complementation of the mec- defect.     

A new model for the mechanism of stimulus-secretion coupling

By combining ultrastructural techniques with a biochemical approach to study the mechanism of mast cell stimulus-secretion coupling and by using purified secretory granules to confirm those early biochemical events which originate from within the secretory granule, a new model for the mechanism of secretory granule exocytosis has emerged. This model not only provides the mechanism by which an activated granule can achieve fusion with the plasma membrane, but it also provides the rationale for the linking of the various early biochemical events to the process of granule activation and thus to exocytosis. Although we still do not understand how the 'activating signal', which results from the stimulation of cell surface receptors, can be conveyed to the granule to cause its activation, we are certain that this 'signal' must cause an influx of water into the matrix of the target granule. This influx of water is what initiates the granule activation process. The major intragranular events which are triggered by this water influx include: (i) de novo membrane assembly; (ii) protein proteolysis; (iii) release of arachidonic acid from matrix-bound phospholipid by phospholipase A2; (iv) initiation of the arachidonic acid cascade and the synthesis of eicosanoids; (v) rapid phospholipid turnover; and (vi) the discharge of matrix materials into the cytoplasm of the activated cell via the fusion of de novo generated vesicles with the perigranular membrane. The ejection of some matrix contents which may include histamine, Ca2+, calmodulin, protease, the products of the arachidonic acid cascade and the products of phospholipid turnover into the cytosole, may serve to turn on the various metabolic machineries needed to initiate a cellular recovery phase.     

A new method of coupling proteins to insoluble polymers

The A chain isolated from biologically active human thrombin is thirteen residues shorter from the amino terminus than the A chain isolated from bovine thrombin. Automated Edman degradation of the reduced, alkylated A chain permitted the direct identification of 34 out of the 36 residues and established all tryptic overlaps. The NH₂-terminal residue is threonine. This residue is homologous to threonine-14 of the A chain of bovine thrombin. Human thrombin A chain has one half cystine linking it to the larger B chain. Methionine, histidine, valine and tryptophan are not present. There are nine acid residues (Glu, Asp) but none of their respective amides.     

A new dissimilarity measure and a new optimality criterion in phytosociological classification

A new dissimilarity measure, Uppsala dissimilarity, is proposed. It is a Manhattan-type measure in between the Canberra and Gower measures, based on the differences between scores in relevés compared, but it also takes both the sums of scores and the difference between maximum and minimum score into account. The measure is considered realistic for phytosociological material.A new optimality criterion has been developed after unsatisfactory results had been obtained with the DOL criterion (Popma et al. 1983) which was developed previously by our group. Problems with DOL were especially met when the criterion was applied to the distribution of only one species over the cluster array obtained. The new criterion takes both internal cluster homogeneity and between-cluster dissimilarity into account. Between-cluster dissimilarity is calculated for all other clusters and not only for the nearest neighbour, as in DOL. The new criterion has both an unweighted form: SOM, and a form with weighting for cluster size: SWOM.This new criterion was successfully applied to the evaluation of the sharpness of distribution of individual species over cluster arrays, under the name of SIM: species indication measure and SWIM, species weighted indication measure.The measures were applied to some test data. Differences between the unweighted and weighted forms were found which could not be easily interpreted.Some remarks are made on the coherence of d-SAHN and h-SAHN approaches in agglomerative clustering within the new strategy proposed.Abbreviations DOL = Detection of Optimal Level - S(W)IM = Species (Weighted) Indication Measure - S(W)OM = Standardized (Weighted) Optimality Measure - UD = Uppsala Dissimilarity measure - WPGMA = Weighted Pair-Group Method Average linking clustering - SAHN = Sequential Agglomerative Hierarchical Non-overlapping clustering