Saltatory propagation of Ca2+ waves by Ca2+ sparks.

Punctate releases of Ca2+, called Ca2+ sparks, originate at the regular array of t-tubules in cardiac myocytes and skeletal muscle. During Ca2+ overload sparks serve as sites for the initiation and propagation of Ca2+ waves in myocytes. Computer simulations of spark-mediated waves are performed with model release sites that reproduce the adaptive Ca2+ release observed for the ryanodine receptor. The speed of these waves is proportional to the diffusion constant of Ca2+, D, rather than D, as is true for reaction-diffusion equations in a continuous excitable medium. A simplified "fire-diffuse-fire" model that mimics the properties of Ca2+-induced Ca2+ release (CICR) from isolated sites is used to explain this saltatory mode of wave propagation. Saltatory and continuous wave propagation can be differentiated by the temperature and Ca2+ buffer dependence of wave speed.     

Model of beta -cell mitochondrial calcium handling and electrical activity. I.Cytoplasmic variables

We continue our development of a kineticmodel of bursting electrical activity in the pancreatic -cell( J. Keizer and G. Magnus. Biophys. J. 56: 229-242,1989), including the influence of Ca²⁺ handling by themitochondria. Our minimal model of mitochondrial Ca²⁺handling [G. Magnus and J. Keizer. Am. J. Physiol. 273 (Cell Physiol. 42): C717-C733, 1997] is expanded toinclude the D-glucose dependence of the rate of productionof mitochondrial reducing equivalents. The Ca²⁺ dependenceof the mitochondrial dehydrogenases, which is also included in themodel, plays only a small role in the simulations, since thedehydrogenases appear to be maximally activated when D-glucose concentrations are sufficient to producebursting. A previous model of ionic currents in the plasma membrane isupdated using a recent experimental characterization of the dependence of the conductance of the ATP-sensitive K⁺(K_ATP) current on adenine nucleotides. The resultingwhole cell model is complex, involving 12 dynamic variables that coupleCa²⁺ handling in the cytoplasm and the mitochondria withelectrical activity in the plasma and inner mitochondrial membranes.Simulations with the whole cell model give rise to bursting electricalactivity similar to that seen in pancreatic islets and clusters ofpancreatic -cells. The full D-glucose dose response ofelectrical activity is obtained if the cytosolic rate of ATP hydrolysisis a sigmoidal function of glucose. The simulations give the correctshape, period, and phase of the associated oscillations in cytosolicCa²⁺, predict that the conductance of the K_ATPcurrent oscillates out of phase with electrical activity [as recentlyobserved in ob/ob mice (O. Larsson, H. Kindmark, R. Bränstrom, B. Fredholm, and P.-O. Berggren. Proc. Natl. Acad.Sci. USA 93: 5161-5165, 1996)], and make other novelpredictions. In this model, bursting results because Ca²⁺uptake into mitochondria during the active phase reduces the mitochondrial inner membrane potential, reducing the rate of production of ATP, which in turn activates the K_ATP current andrepolarizes the plasma membrane.

     

Maintenance of immune tolerance depends on normal tissue homeostasis

Ags expressed at immune privileged sites and other peripheral tissues are able to induce T cell tolerance. In this study, we analyzed whether tolerance toward an intraocular tumor expressing a highly immunogenic CTL epitope is maintained, broken, or reverted into immunity in the event the anatomical integrity of the eye is lost. Inoculation of tumor cells into the anterior chamber of the eye of naive B6 mice leads to progressive intraocular tumor growth, an abortive form of CTL activation in the tumor-draining submandibular lymph node, and systemic tolerance as evidenced by the inability of these mice to reject an otherwise benign tumor cell inoculum. Loss of anatomical integrity of the eye as a consequence of phthisis resulted in loss of systemic tolerance and the emergence of effective antitumor immunity against an otherwise lethal tumor challenge. Phthisis was accompanied by dendritic cell maturation and preceded the induction of systemic tumor-specific CTL immunity. Our data show that normal tissue homeostasis and anatomical integrity is required for the maintenance of ocular tolerance and prevention of CTL-mediated immunity. These data also indicate that tissue injury in the absence of viral or microbial infection can act as a switch for the induction of CTL immunity.     

Phosphorus in the sediment of the Loosdrecht lakes and its implications for lake restoration perspectives

In 1984 the external phosphorus load of the shallow eutrophic Loosdrecht lakes was reduced from 3.3 to 1.0 mg m^–2 d^–1. The effect of phosphorus release from the sediment on lake restoration was investigated. Diffusive release under aerobic conditions (20 °C) decreased from 1 mg m^–2 d^–1 in 1984 to 0.3 mg m^–2 d^–1 in 1990. The generation of inorganic phosphorus due to mineralization during summer equals 3 mg m^–2 d^–1, which is much higher than the measured rate of diffusive release. Despite that, the phosphorus release is hardly stimulated by anaerobic conditions, which indicates that only a small amount of phosphorus is adsorbed by ferric iron in the top sediment layer. This apparent discrepancy is probably caused by the uptake of inorganic phosphorus uptake during resuspension and the loss of inorganic phosphorus with downward seepage.The estimated removal of phosphorus due to downward seepage of 0.8 mg m^–2 d^–1 agrees well with the average phosphorus retention in the lake. This indicates that sediment burial and diagenesis are unimportant mechanisms for withdrawing phosphorus from the nutrient cycle.Between 1982 and 1991 the total phosphorus content of the upper 2 cm of the sediment decreased from 0.94 to 0.60 g kg^–1 DW. At present, about 20% of total phosphorus in this layer is potentially bioavailable, but largely incorporated in easily degradable organic matter. This pool is much smaller in deeper layers. Based on the estimated and measured rates and pool sizes, the annual average phosphorus cycle in the lakes was modelled to evaluate the effects of various restoration measures. The main predictions of the model are: 1) further reduction of the external load may cause a gradual decrease of the total phosphorus concentration in the lake water; 2) dredging and iron addition, without reduction of the external load, may give a rapid improvement followed by a slow return to the present situation; and 3) reduction of the external load, combined with a cut off of downward seepage will not improve the water quality.     

Two roles of Ca2+ in agonist stimulated Ca2+ oscillations.

We propose a mechanism for agonist-stimulated Ca2+ oscillations that involves two roles for cytosolic Ca2+: (a) inhibition of inositol-1,4,5-trisphosphate (IP3) stimulated Ca2+ release from the endoplasmic reticulum (ER) and (b) stimulation of the production of IP3 through its action on phospholipase C (PLC), via a Gq protein related mechanism. Relying on quantitative experiments by Parker, I., and I. Ivorra (1990. Proc. Natl. Acad. Sci. USA. 87:260-264) on the inhibition of Ca2+ release from the ER using caged-IP3, we develop a kinetic model of inhibition that allows us to simulate closely their experiments. The model assumes that the ER IP3 receptor is a tetramer of independent subunits that can bind both Ca2+ and IP3. Upon incorporation of the action of Ca2+ on PLC that leads to production of IP3, we observe in-phase-oscillations of Ca2+ and IP3 at intermediate values of agonist stimulation. The oscillations occur on a time scale of 10-20 s, which is comparable to the time scale for inhibition in Xenopus oocytes. Analysis of the mechanism shows that Ca(2+)-inhibition of IP3-stimulated Ca2+ release from the ER is an essential step in the mechanism. We also find that the effect of Ca2+ on PLC can lead to an indirect increase of cytosolic Ca2+, superficially resembling "Ca(2+)-induced Ca(2+)-release." The mechanism that we propose appears to be consistent with recent experiments on REF52 cells by Harootunian, A. T., J. P. Y. Kao, S. Paranjape, and R. Y. Tsien. (1991. Science [Wash. DC]. 251:75-78.) and we propose additional experiments to help test its underlying assumptions.     

Beryllium binding at neutral pH: the importance of the Be-O-Be motif

Beryllium speciation at physiological conditions is critical to understanding chronic beryllium disease (CBD). The MHC-class II receptor alleles that have been linked to CBD have more than six carboxylates in a short 20 amino acid segment of the binding pocket and it has been suggested that beryllium may bind within the MHC-class II receptor via the carboxylates. Previous reports also show that citric acid binds beryllium significantly stronger than similar carboxylate ligands such as tartaric acid and is one of the few ligands that can compete with hydrolysis to solubilize beryllium across the entire pH range at molar concentrations. We have characterized the binding of Be to citric acid and shown using a combination of NMR, mass spectrometry and ligand competition studies that Be2L and Be4L2 species dominate. A Be-O-Be linkage with the bridging oxygen coming from the aliphatic alcohol is critical to the stability of the complex. We show through competition experiments that the most stable Be-O-Be arrangement has one Be in a five-member ring and the other Be in a six-member ring. The unusual deprotonation of an aliphatic alcohol (pK(a) = 18) at neutral pH has significant ramifications on the potential interactions of Be with biological ligands such as carbohydrates and Ser and Thr residues.     

Structure and inter-domain interactions of domain II from the blood-stage malarial protein, apical membrane antigen 1

The malarial surface antigen apical membrane antigen (AMA1), from Plasmodium falciparum, is a leading candidate for inclusion in a vaccine against malaria. AMA1 is synthesised by mature blood-stages of the parasite and is located initially in the apical organelles of the merozoite. Prior to merozoite invasion of host erythrocytes, it is processed into a 66 kDa type 1 integral membrane protein on the merozoite surface. The pattern of disulphide bonds in AMA1 has been the basis for separation of the ectodomain into three domains, with three, two and three disulphide bonds, respectively. We have determined the solution structure of a 16kDa construct corresponding to the putative second domain of AMA1. While circular dichroism and hydrodynamic data were consistent with a folded structure for domain II, its NMR spectra were characterised by broad lines and significant peak overlap, more typical of a molten globule. Consistent with this, domain II bound the fluorescent dye 8-anilino-1-naphthalene sulphonate (ANS). We have nonetheless determined a structure, which defines the secondary structure elements and global fold. The two disulphide bonds link the N and C-terminal regions of the molecule, which come together to form a four-stranded beta-sheet linked to a short helix. A long loop linking the N and C-terminal regions contains four other alpha-helices, the locations of which are not fixed relative to the beta-sheet core, even though they are well-defined locally. Very recently this region of domain II has been shown to contain the epitope recognised by the invasion-inhibitory antibody 4G2, even though it does not contain any of the polymorphisms that are regarded as having arisen in response to the pressure of immune recognition.     

Pulmonary aspiration in preschool children with cystic fibrosis

Pulmonary aspiration of gastric refluxate (PAGR) has been demonstrated in association with pulmonary inflammation in school aged children with Cystic Fibrosis (CF). We sought to determine if similar findings were present in preschool children. Pepsin was measured in Broncho-alveolar lavage (BAL) fluid collected from clinically stable preschool children with CF and controls. Elevated pepsin levels were found in a subgroup of children with CF, but this was not found to be associated with pulmonary infection, pulmonary inflammation or respiratory or gastrointestinal symptoms.     

Sensing and refilling calcium stores in an excitable cell.

Inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ mobilization leads to depletion of the endoplasmic reticulum (ER) and an increase in Ca2+ entry. We show here for the gonadotroph, an excitable endocrine cell, that sensing of ER Ca2+ content can occur without the Ca2+ release-activated Ca2+ current (Icrac), but rather through the coupling of IP3-induced Ca2+ oscillations to plasma membrane voltage spikes that gate Ca2+ entry. Thus we demonstrate that capacitative Ca2+ entry is accomplished through Ca(2+)-controlled Ca2+ entry. We develop a comprehensive model, with parameter values constrained by available experimental data, to simulate the spatiotemporal behavior of agonist-induced Ca2+ signals in both the cytosol and ER lumen of gonadotrophs. The model combines two previously developed models, one for ER-mediated Ca2+ oscillations and another for plasma membrane potential-driven Ca2+ oscillations. Simulations show agreement with existing experimental records of store content, cytosolic Ca2+ concentration ([Ca2+]i), and electrical activity, and make a variety of new, experimentally testable predictions. In particular, computations with the model suggest that [Ca2+]i in the vicinity of the plasma membrane acts as a messenger for ER content via Ca(2+)-activated K+ channels and Ca2+ pumps in the plasma membrane. We conclude that, in excitable cells that do not express Icrac, [Ca2+]i profiles provide a sensitive mechanism for regulating net calcium flux through the plasma membrane during both store depletion and refilling.