Evidence of ectokinase-mediated phosphorylation of osteopontin and bone sialoprotein by osteoblasts during bone formation in vitro.

Osteopontin (OPN) and bone sialoprotein (BSP) are phosphorylated glycoproteins that, together with osteonectin/secreted protein, acidic, rich in cysteine (SPARC) and osteocalcin, comprise the major non-collagen proteins of bone. Although phosphorylation of OPN and BSP, which is known to influence the biological properties of these proteins, has been shown to occur intracellularly, recent studies have demonstrated ectokinase activity in bone cell populations [Mikuni-Takagaki, Kakai, Satoyoshi, Kawano, Suzuki, Kawase and Saito (1995) J. Bone Miner. Res. 10, 231-241]. To determine whether OPN and BSP are phosphorylated by ectokinase activity we have used [gamma-32P]ATP and [gamma-32P]GTP as cell-impenetrable phosphate donors to analyse for ectokinase activity in osteoblastic UMR106.06 cells and fetal rat calvarial cells (FRCCs). By pulse-labelling confluent cells with radiolabelled nucleotides, the phosphorylation of endogenous and exogenously added OPN and BSP was demonstrated together with the labelling of a number of cell surface proteins. These phosphorylation reactions were inhibited by a cell-impermeable ectokinase inhibitor, K252b, and cell surface phosphorylation was also inhibited by exogenously added OPN and BSP substrates, indicating competition for the ectokinase enzyme. However, phosphorylation of OPN and BSP, both of which can mediate cell attachment through Arg-Gly-Asp (RGD) motifs, was not inhibited by an RGD peptide, suggesting that binding of OPN and BSP to cell surface integrins is not required. In similar experiments, ectokinase-mediated phosphorylation of OPN and BSP was demonstrated during mineralized tissue formation by FRCCs in vitro. These studies demonstrate that OPN and BSP secreted by bone cells are phosphorylated by a casein kinase II-like ectokinase present on the surface of osteoblastic cells.

Studies on the alignment of fibroblasts in uniform applied electrical fields.

Uniform electrical fields have been applied to human gingival fibroblasts by means of uniform ionic currents passed through a thin chamber. Cells were observed to align in fields between 0.1 and 1.5 V/mm but did not display directed motion toward the anode or the cathode of the chamber. Statistical analysis of directional data was used to distinguish threshold levels of orientation at low field intensities, to quantify the dependence of alignment on time and field intensity, and to analyze differences between alignment of cells treated with the Ca2+ transport modifiers A23187, verapamil, and lanthanum. Alignment occurred at a steady rate and was dependent in a saturating fashion on field strength. The Ca2+ ionophore A23187 had a significant inhibitory effect on cell alignment in applied electrical fields; however, the Ca2+ channel blockers lanthanum and verapamil did not have a significant effect on alignment.

Analysis of the diffusion theory of negative capacitance: the role of K+ and the unstirred layer thickness.

The diffusion theory of negative capacitance is extended to take into account potassium transport as well as proton or hydroxyl transport. It is shown that both the capacitance spectrum and the frequency at which the capacitance is zero can be used to experimentally test the theory. The effects of the fraction of potassium current, membrane conductance, NaCl concentration, and unstirred layer thickness on these two characteristics is investigated. Maximum negative capacitance can be obtained when the current flowing through the membrane is mainly carried by protons, the membrane conductance is high, the solution conductivity is low, and the unstirred layer thickness is large. The effect of a dominant hydroxyl transport in place of a proton transport is also discussed. We suggest simple experiments to test the theory on Characeaen plant cells.

Collagen remodelling in rat periodontal tissues: compensation for precursor reutilization confirms rapid turnover of collagen.

Measurement of collagen turnover is complicated by the reutilization of isotopic precursors used to label the collagen. In an earlier study a novel approach was used to circumvent the problems of precursor recycling and unusually short half-lives were determined for collagen in adult rat periodontal tissues (Sodek, 1977). To verify these results we have used an alternate procedure devised by Poole (1971) in which the decay profile for the radiolabelled protein is corrected in accordance with the decay of the radiolabelled precursor. In this manner real half-lives for mature, neutral salt-insoluble collagen were determined as 3 days in the molar periodontal ligament, 6 days in the continuously erupting incisor ligament and approximately 10 days in the lamina propria of the gingiva, compared to apparent half-lives for these tissues of 6, 12 and approximately 20 days, respectively. The values calculated for actual half-lives are, therefore, approximately two-fold faster than values determined without compensating for reutilization, a difference that is in agreement with other protein turnover studies in which the effects of precursor reutilization have been measured. Although the real half-lives determined in this study indicate turnover rates for the periodontal tissues that are slightly slower than reported previously, the relative differences between the tissues in the rates of collagen turnover are similar. Moreover, the study confirms the existence of a remarkably high rate of collagen remodelling in these tissues.

Voltage-Dependent K-Channel in Protoplasmic Droplets of Chara corallina: A Single Channel Patch Clamp Study.

Passive transport of potassium through the plasma membrane of a protoplasmic droplet isolated from large internodal cells of Chara corallina Klein ex Willd., em, R.D.W. has been investigated using the patchclamp technique. When the membrane is hyperpolarized the conductance of a single K(+)-channel is of the order of magnitude of 100 picoSiemens and is reduced by tetraethylammonium chloride. Its open time is voltage dependent. This voltage-dependent K(+)-channel displays rectifying properties. The channel density is about 0.1 channel per square micrometer of membrane. When the membrane is depolarized the conductance of a single channel is of the order of magnitude of 30 picoSiemens and is insensitive to tetraethylammonium chloride. These results suggest that K(+)-channels are incorporated in the plasma membrane during membranogenesis of a protoplasmic droplet. They constitute further evidence for the existence of voltage-dependent K(+)-channels in plant cells.

Correlation of collagenolytic enzymes and inhibitors in gingival crevicular fluid with clinical and microscopic changes in experimental periodontitis in the dog.

The quadrants of the jaw in 2 beagle dogs had various forms of periodontal disease induced by ligatures placed around second, third and fourth premolars in one quadrant and, 2.5 months later, around the same teeth in a second quadrant; gingivitis was allowed to develop in a third quadrant after 4 months; the fourth quadrant served as a healthy control. Crevicular fluid flow, plaque index, gingival index, attachment levels and recession were determined at intervals and collagenolytic activity measured in the fluid. The dogs were killed after 5 months and sections of each site prepared for histomorphometry. Clinically-inflamed and degenerating sites had significantly higher collagenolytic activities (p less than 0.001), lower collagenase inhibitor activities and greater fluid flow than control sites which showed abundant inhibitor activity and minimal active enzyme. Periodontitis sites had higher active enzyme, compared to latent enzyme activities, whereas latent collagenase was predominant in control and gingivitis sites. The collagenolytic activities in periodontitis sites fluctuated with time, suggesting a cyclical pattern. Active enzyme activities correlated strongly with gingival crevicular fluid flow and attachment loss. Periodontitis sites had much more inflammatory-cell infiltration than control and gingivitis sites (p less than 0.001). Thus periodontal disease may be monitored by examination of crevicular fluid collagenolytic enzymes, inhibitors and fluid flow, and these criteria may prove more meaningful than current clinical criteria.

Apparent Bicarbonate Uptake and Possible Plasmalemma Proton Efflux in Chara corallina.

It is shown that the apparent uptake of bicarbonate by cells of Chara corallina could be the result of a proton efflux coupled to extracellular production of CO(2) from bicarbonate, with CO(2) being taken up by the cell. The theoretical results presented here show that the influx of CO(2) across the plasmalemma can be much greater than previously thought, if there is a large efflux of protons across the plasmalemma, and that, if this occurs, there would be a much steeper gradient of pH near the cell surface than previously thought possible.

Plasmalemma Voltage Noise in Chara corallina.

Voltage noise analysis is applied to plasmalemma ion transport in Chara corallina. There is a component in the noise power spectrum that is probably associated with current fluctuations within passive transport channels, and another component that may be associated either with fluctuations in the number of open channels, or with active transport. The data allow the calculation of time constants that may be attributable to molecular level events in these transport processes.

Water Flow in Beta vulgaris Storage Tissue.

The relative magnitudes of the hydraulic resistances, water capacities, and water potential equilibration time constants for the single cell, for the apoplast, and for the symplast in higher plant tissue are assessed. Swelling of beetroot (Beta vulgaris, var. ;Detroit Red') storage tissue sections in pure water is measured using a displacement transducer. This method of measurement avoids the difficulty of solute diffusion in the apoplast. Theoretical analysis of the experimental results shows that the main path of water flow into the tissue is the apoplast rather than the symplast, that the main resistance to water flow into the cells is usually the cell membrane rather than the apoplast, but that in some cases the apoplast resistance and water capacity can contribute significantly to the water potential equilibration time constant of the tissue.

Role of Concentration-dependent Unloading in Mathematical Models of Münch Transport.

It has been erroneously claimed (Goeschl et al. [1976] Plant Physiol. 58: 556-562) that concentration-dependent unloading is required for a correct mathematical solution for Münch phloem transport. Although it may prove to be physiologically correct, concentration-dependent unloading is not a mathematical necessity. Furthermore, its use in a mathematical model may not be desirable, because there is an infinite family of solutions for any given unloading distribution, irrespective of whether concentration-dependent unloading is assumed. Some illustrative numerical results are presented.

Plasmalemma transport of OH- in Chara corallina: dynamics of activation and deactivation.

The light-mediated, time-dependent rise in the pH value at the center of an alkaline band was analyzed using the methods of numerical analysis. From this analysis an expression of the time-dependent build-up of OH- efflux was obtained for these bands. This information can now be employed to determine whether the light-activated transport of OH- and HCO3- influences the electrical properties of the plasmalemma. The dark-induced deactivation of OH- transport was also characterized, revealing a transition from efflux to a transient influx phase during deactivation. Numerical analysis of the steady-state OH- diffusion pattern, established along the surface of an alkaline band, revealed that the OH- efflux width was wider than previously envisaged. It was also found the OH- sink regions exist on either side of the efflux zone. These, and other characteristics revealed by the numerical analysis, enabled us to extend the OH- transport model proposed by Lucas (J. Exp. Bot. 1975, 26:347).

Time-dependent Behavior of a Mathematical Model for Munch Translocation: Application to Recovery from Cold Inhibition.

The time evolution of a Munch pressure-flow translocation system is calculated using a numerical computer method. Results are obtained for the time course of the system variables following application of a large resistance in the translocation path, intended to simulate a cold block. The resistance factor required to produce translocation inhibition indicates that even moderate inhibition is primarily due to sieve plate pore block-age, rather than to the viscosity increase. The calculated time for recovery from cold inhibition and the shape of the translocation recovery curve agree with experimental results. The time for translocation recovery and the level of velocity recovery depend on the rate of sucrose unloading in the sink; on the sucrose concentration in the sieve tube; on the position, length, and resistance factor of the cold block; and on the hydraulic conductivities.

A simpler iterative steady state solution of münch pressure-flow systems applied to long and short translocation paths.

A simple steady state iterative solution of Münch pressure-flow in unbranched sieve tubes containing only water and sucrose is derived. The iterative equations can be solved on a programmable desk calculator. Solutions are presented for steady state transport with specific mass transfer rates up to 1.5 x 10(-5) mole second(-1) centimeters(-2) (= 18.5 grams hour(-1) centimeters(-2)) over distances in excess of 50 meters. The calculations clearly indicate that a Münch pressure-flow system can operate over long distances provided (a) the sieve tube is surrounded by a semipermeable membrane; (b) sugars are actively loaded in one region and unloaded at another; (c) the sieve pores are unblocked so that the sieve tube hydraulic conductivity is high (around 4 centimeters(2) second(-1) bar(-1)); (d) the sugar concentration is kept high (around one molar in the source region); and (e) the average sap velocity is kept low (around 20-50 centimeters hour(-1)). The dimensions of sieve cells in several species of plants are reviewed and sieve tube hydraulic conductivities are calculated; the values range from 0.2 to 20 centimeters(2) second(-1) bar(-1). For long distance pressure-flow to occur, the hydraulic conductivity of the sieve cell membranes must be about 5 x 10(-7) centimeters second(-1) bar(-1) or greater.

A Mathematical Treatment of Munch's Pressure-Flow Hypothesis of Phloem Translocation.

The steady state solutions of two mathematical models are used to evaluate Münch's pressure-flow hypothesis of phloem translocation. The models assume a continuous active loading and unloading of translocate but differ in the site of loading and unloading and the route of water to the sieve tube. The dimensions of the translocation system taken are the average observed values for sugar beet and are intended to simulate translocation from a mature source leaf to an expanding sink leaf. The volume flow rate of solution along the sieve tube, water flow rate into the sieve tube, hydrostatic pressure, and concentration of sucrose in the sieve tube are obtained from a numerical computer solution of the models. The mass transfer rate, velocity of translocation, and osmotic and hydrostatic pressures are consistent with empirical findings. Owing to the resistance to water flow offered by the lateral membranes, the hydrostatic pressure generated by the osmotic pressure can be considerably less than would be predicted by the solute concentration. These models suggest that translocation at observed rates and velocities can be driven by a water potential difference between the sieve tube and surrounding tissue and are consistent with the pressure-flow hypothesis of translocation.