Calcium homeostasis and bone surface proteins, a postulated vital process for plasma calcium control

This report is a more in-depth explanation of a recently reported hypothesis for controlling the ionic calcium content of plasma and extracellular fluids (ECF). The hypothesis proposes a two-step process for returning calcium to the ECF against the established gradient continuously moving calcium from plasma to bone surfaces. The first step in this process is the predicted transfer of calcium directly from bone surfaces to the non-collagenous proteins, which are in contact with bone mineral. This calcium would be complexed to existing proteins and a portion would automatically become available for equilibration with ionic calcium in the ECF. The basis of the hypothesis is that the equilibration level helps to set the ionic calcium concentration of plasma. The gradient toward bone and the proposed two-step return occur in the ECF of bone and would be considered normal physiochemical processes. Thus, these processes are critical for mineral ion homeostasis in mammals. In this hypothesis, parathyroid hormone (PTH) is not required for the basic process. However, PTH works within the process to raise and set a precise plasma calcium concentration. The report to follow describes the process and discusses its relationship to normal and pathological conditions affecting human health.     

Energy Coupling Factor-Type ABC Transporters for Vitamin Uptake in Prokaryotes

Energy coupling factor (ECF) transporters are a subgroup of ATP-binding cassette (ABC) transporters involved in the uptake of vitamins and micronutrients in prokaryotes. In contrast to classical ABC importers, ECF transporters do not make use of water-soluble substrate binding proteins or domains but instead employ integral membrane proteins for substrate binding (named S-components). S-components form active translocation complexes with the ECF module, an assembly of two nucleotide-binding domains (NBDs, or EcfA) and a second transmembrane protein. In some cases, the ECF module is dedicated to a single S-component, but in many cases, the ECF module can interact with several different S-components that are unrelated in sequence and bind diverse substrates. The modular organization with exchangeable S-components on a single ECF module allows the transport of chemically different substrates via a common route. The recent determination of the crystal structures of the S-components that recognize thiamin and riboflavin has provided a first clue about the mechanism of S-component exchange. This review describes recent advances and the current views of the mechanism of transport by ECF transporters.     

Inhibition of the eosinophil chemotactic factor (ECF) release from human PMNs and rat mast cells by arachidonic acid analogs

An eosinophil chemotactic factor (ECF) can be released from human polymorphonuclear neutrophils (PMN) and rat mast cells by the calcium ionophore A23187, during phagocytosis, by arachidonic acid and melittin. It has been suggested that these stimuli lead to phospholipid turnover with the generation of arachidonic acid, which is subsequently transformed by a converting enzyme to ECF. Addition of polienoic (5,8,11-eicosatrienoic and 4,7,10,13-eicosatetraenoic acids) or poliynoic acids (5,8,11-eicosatriynoic and 4,7,10,13-eicosatetraynoic acids) induced a dose- and time-dependent inhibition of ECF release from the cells. Poliynoic acids are more potent inhibitors than polienoic acids. Among the former 4,7,10,13-eicosatetraynoic acid is the most effective substance.     

Difference in requirement of macrophage products for concanavalin A-induced production of eosinophil chemotactic factor and macrophage chemotactic factor from guinea pig lymphocytes in vitro

Differences between the conditions for an eosinophil chemotactic factor (ECF) and macrophage chemotactic factor (MCF) production by lymphoid cells of mesenteric lymph nodes and spleen were studied in guinea pigs. If lymphoid cells were washed less than 4 hr after concanavalin A (Con A) stimulation and were cultured for an additional 24 hr, they failed to produce ECF, whereas Con A stimulation for 1 hr before washing was sufficient to stimulate them to produce MCF. Subsequently, it was shown that heat-labile soluble factors (termed ECF-PF) with potentiating activity for ECF production are produced from macrophages by 5 micrograms/ml Con A activation. When ECF-PF were added to the cell culture with 5 micrograms/ml Con A, the lymphoid cells could produce ECF even when they were washed 2 hr after Con A stimulation and were cultured for an additional 24 hr, suggesting that ECF-PF plays a critical role in the early stage of ECF production. The lymphoid cells were also able to produce ECF even when they were cultured with ECF-PF and a suboptimal dose of Con A (1 microgram/ml) for ECF production. Protein synthesis seemed to be essential for ECF-PF production. The ECF-PF activity was associated with two separated molecular fractions with m.w. of about 50,000 to 70,000 and of 10,000 to 20,000. It is thus suggested that ECF is produced from T cells by Con A stimulation under conditions which differ, at least, from those for MCF in the requirement of ECF-PF.     

Muscle activation and contraction: constitutive relations based directly on cross-bridge kinetics

This paper reviews recent work aimed at deriving tractable constitutive relations for skeletal muscle from biophysical cross-bridge theories. Discussion is focused on a model proposed previously by the first author (the Distribution-Moment Model), which emphasizes the important role of the moments of the actin-myosin bond-distribution function. The theory leads to a relatively simple third order state variable model for contraction dynamics in which the state variables are the three lowest order moments of the bond-distribution function; further, these three moments have simple macroscopic interpretations as muscle stiffness, force, and elastic energy. New results are presented on the formulation of a compatible model for excitation-contraction coupling, and this model requires the introduction of only one more state variable--the free calcium concentration.     

The epsilon subunit of Escherichia coli coupling factor 1 is required for its binding to the cytoplasmic membrane.

The coupling factor, F1-ATPase of Escherichia coli (ECF1) contains five different subunits, alpha, beta, gamma, delta, and epsilon. Properties of delta-deficient ECF1 have previously been described. F1-ATPase containing only the alpha, beta, and gamma subunits was prepared from E. coli by passage of delta-deficient ECF1 through an affinity column containing immobilized antibodies to the epsilon subunit. The delta, epsilon-deficient enzyme has normal ATPase activity but cannot bind to ECF1-depleted membrane vesicles. Both the delta and epsilon subunits are required for the binding of delta, epsilon-deficient ECF1 to membranes and the restoration of oxidative phosphorylation. Either delta or epsilon will bind to the deficient enzyme to form a four-subunit complex. Neither four-subunit enzyme binds to depleted membranes. The epsilon subunit, does, however, slightly improve the binding affinity between delta and delta-deficient enzyme suggesting a possible interaction between the two subunits. Neither subunit binds to trypsin-treated ECF1, which contains only the alpha and beta subunits. A role for gamma in the binding of epsilon to F1 is suggested. epsilon does not bind to ECF1-depleted membranes. Therefore, the in vitro reconstitution of depleted membranes requires an initial complex formation between epsilon and the rest of ECF1 prior to membrane attachment. Reconstitution experiments indicate that only one epsilon is required per functional ECF1 molecule.     

Siderophore-mediated cell signalling in Pseudomonas aeruginosa: divergent pathways regulate virulence factor production and siderophore receptor synthesis

Under iron-limiting conditions, Pseudomonas aeruginosa produces a siderophore called pyoverdine. Pyoverdine is secreted into the extracellular environment where it chelates iron, and the resulting ferri-pyoverdine complexes are transported back into the bacteria by a cell surface receptor protein FpvA. Pyoverdine also acts as a signalling molecule inducing the production of three secreted virulence factors. Binding of ferri-pyoverdine to FpvA transduces a signal to the periplasmic part of the membrane-spanning antisigma factor FpvR. The signal is transmitted to the cytoplasmic part of FpvR, which controls the activity of an extracytoplasmic family (ECF) sigma factor protein PvdS. This results in the production of the virulence factors pyoverdine, exotoxin A and PrpL endoprotease. Here, we show that a second divergent branch of this signalling pathway regulates the production of the FpvA protein. FpvR negatively regulates the activity of a second ECF sigma factor, FpvI, which is required for the synthesis of FpvA, and the presence of ferri-pyoverdine greatly increases the activity of FpvI so that production of FpvA is induced. To the best of our knowledge, this is the first example of a branched signalling system of this sort and the first example of an antisigma factor protein (FpvR) that directly regulates the activities of two different ECF sigma factor proteins (PvdS and FpvI).     

Endometriotic cyst fluid induces reactive oxygen species (ROS) in human immortalized epithelial cells derived from ovarian endometrioma

Objectives: Endometriotic cyst fluid (ECF) contains a large amount of reactive oxygen species (ROS), and endometriotic cysts are exposed to strong oxidative stress, which may cause malignant transformation. In this study, ROS production by ECF was clinically analysed.

Methods: Human immortalized epithelial cells derived from ovarian endometrioma (EMosis-CC/TERT 1) were treated with ECF. In addition, ROS production in EMosis-CC/TERT 1 was measured, and its clinical significance was analysed.

Results: A total of 38 ECF samples were obtained from patients diagnosed with endometriotic cysts. In EMosis-CC/TERT1, significantly higher levels of ROS were induced by ECF than by the vehicle control and ferric nitrilotriacetate. There were no significant differences in ROS production by laterality and preoperative serum CA125 values. There were several patients whose cyst sizes were approximately 5?cm and had relatively high ROS production. Production of ROS by ECF was relatively higher in patients older than 40 years of age than in those younger than 40.

Discussion: Our study revealed that ROS are highly produced by ECF in EMosis-CC/TERT1 cells; therefore, exposure to ECF induced strong oxidative stress. Development of a therapeutic strategy to reduce ROS production might be useful for preventing malignant transformation of endometriotic cysts.     

A novel key–lock mechanism for inactivating amino acid neurotransmitters during transit across extracellular space

There are two kinds of neurotransmissions that occur in brain. One is neuron to neuron at synapses, and the other is neuron to glia via extracellular fluid (ECF), both of which are important for maintenance of proper neuronal functioning. For neuron to neuron communications, several potent amino acid neurotransmitters are used within the confines of synaptic space. However, their presence at elevated concentrations in extra-synaptic space could be detrimental to well organized neuronal functioning. The significance of the synthesis and release of N-acetylaspartylglutamate (NAAG) by neurons has long been a puzzle since glutamate (Glu) itself is the “key” that can interact with all Glu receptors on membranes of all cells. Nonetheless, neurons synthesize this acetylated dipeptide, which cannot be catabolized by neurons, and release it to ECF where its specific physiological target is the Glu metabotropic receptor 3 on the surface of astrocytes. Since Glu is excitotoxic at elevated concentrations, it is proposed that formation and release of NAAG by neurons allows large quantities of Glu to be transported in ECF without the risk of injurious excitotoxic effects. The metabolic mechanism used by neurons is a key–lock system to detoxify Glu during its intercellular transit. This is accomplished by first synthesizing N-acetylaspartate (NAA), and then joining this molecule via a peptide bond to Glu. In this paper, a hypothesis is presented that neurons synthesize a variety of relatively nontoxic peptides and peptide derivatives, including NAA, NAAG, homocarnosine (γ-aminobutyrylhistidine) and carnosine (β-alanylhistidine) from potent excitatory and inhibitory amino acids for the purpose of releasing them to ECF to function as cell-specific neuron-to-glia neurotransmitters.     

Production models for nonlinear stochastic age-structured fisheries

The overriding feature of stock-recruitment data for most fisheries is the amount of variability involved. Previous production models have assumed either an underlying linear stock-recruitment relationship [11] or an equilibrium condition [23]. Here a production model is derived for an age-structured fishery exhibiting nonlinear stochastic recruitment under nonequilibrium conditions. In the first section deterministic age-structured production models are reviewed, and in the next section corresponding random variable models are presented. Equations for the first and second order moments for each age class, for the stock, and for the yield are then derived using two approaches. The first approach assumes that third and order higher moments associated with the noise can be neglected (thus extending the “small noise” approach in [23]). The second approach assumes that the distributions associated with the random variables can be characterized by a particular two parameter distribution. This latter approximation can be applied to systems with “large noise,” and precision will not be lost for situations where the exact form of the distribution, associated with the stock-recruitment data, is unknown. Equations are derived for the solution under equilibrium recruitment and constant harvesting conditions. Detailed expressions are also obtained for the case where the random variables are assumed to satisfy a gamma distribution.     

Monoclonal antibody modification of the ATPase activity of Escherichia coli F1 ATPase

Monoclonal antibodies (mAbs) have been made against each of the five subunits of ECF1 (alpha, beta, gamma, delta, and epsilon), and these have been used in topology studies and for examination of the role of individual subunits in the functioning of the enzyme. All of the mAbs obtained reacted with ECF1, while several failed to react with ECF1F0, including three mAbs against the gamma subunit (gamma II, gamma III, and gamma IV), one mAb against delta, and two mAbs against epsilon (epsilon I and epsilon II). These topology data are consistent with the gamma, delta, and epsilon subunits being located at the interface between the F1 and F0 parts of the complex. Two forms of ECF1 were used to study the effects of mAbs on the ATPase activity of the enzyme: ECF1 with the epsilon subunit tightly bound and acting to inhibit activity and ECF1* in which the delta and epsilon subunits had been removed by organic solvent treatment. ECF1* had an ATPase activity under standard conditions of 93 mumol of ATP hydrolyzed min-1 mg-1, cf. an activity of 7.5 units mg-1 for our standard ECF1 preparation and 64 units mg-1 for enzyme in which the epsilon subunit had been removed by trypsin treatment. The protease digestion of ECF1* reduced activity to 64 units mg-1 in a complicated process involving an inhibition of activity by cleavage of the alpha subunit, activation by cleavage of gamma, and inhibition with cleavage of the beta subunit. mAbs to the gamma subunit, gamma II and gamma III, activated ECF1 by 4.4- and 2.4-fold, respectively, by changing the affinity of the enzyme for the epsilon subunit, as evidenced by density gradient centrifugation experiments. The gamma-subunit mAbs did not alter the ATPase activity of ECF1*- or trypsin-treated enzyme. The alpha-subunit mAb (alpha I) activated ECF1 by a factor of 2.5-fold and ECF1F0 by 1.3-fold, but inhibited the ATPase activity of ECF1* by 30%.     

Quantitative determination of extracellular glutamine concentration in rat brain, and its elevation in vivo by system A transport inhibitor, alpha-(methylamino)isobutyrate

The basal concentration of glutamine in the extracellular fluid, [GLN(ECF)], was determined to be 385 +/- 16 microm in the cortico-striatal region of awake rats. This in vivo concentration was determined by measuring glutamine concentrations in dialysates collected at several flow rates (0.2-4 microL/min), and extrapolating to the concentration at zero flow-rate. Dialysate glutamine concentrations in the somatosensory cortex, hippocampus and thalamus showed no statistically significant difference. In these brain regions, [GLN(ECF)] was elevated 1.5- to 1.8-fold upon perfusion of 50-250 mmalpha-(methylamino)isobutyrate (MeAIB), a competitive inhibitor of glutamine uptake by system A amino acid transporter. The results show, for the first time, that MeAIB causes elevation of brain GLN(ECF)in vivo. The MeAIB-induced elevation of [GLN(ECF)] provides additional support for the current view that system A GLN transporter (Gln T/SAT 1) is the major pathway for the uptake of GLN(ECF) by neurons, while GLN release from glia is mainly mediated by a system N transporter (SN1) which is not inhibitable by MeAIB. The steady-state GLN(ECF) concentration and the effectiveness of MeAIB in inhibiting neuronal GLN uptake in vivo, reported in this study, will be useful, when combined with the known in vitro kinetic properties of the GLN transporters, for study of GLN transport in the intact brain.     

Suppression of glial glutamine release to the extracellular fluid studied in vivo by NMR and microdialysis in hyperammonemic rat brain

Release of glial glutamine (GLN) to the extracellular fluid (ECF), mainly mediated by the bidirectional system N transporter SN1, was studied in vivo in hyperammonemic rat brain, using (15)N-nuclear magnetic resonance (NMR) to monitor intracellular [5-(15)N]GLN and microdialysis/gradient (1)H-(15)N heteronuclear single-quantum correlation NMR to analyse extracellular [5-(15)N]GLN. GLN(ECF) was elevated to 2.4 +/- 0.2 mm after 4.5 h of intravenous ammonium acetate infusion. The [GLN(i)]/[GLN(ECF)] ratio (i = intracellular) was 9.6 +/- 0.9, compared with 17-20 in normal brain. GLN(ECF) then decreased substantially at t = 4.9 +/- 0.1 h. Comparison of the time-courses of intra- and extra-cellular [5-(15)N]GLN strongly suggested that the observed decrease reflects partial suppression of glial GLN release to ECF. Suppression also followed elevation of GLN(ECF) to 1.9 mM, resulting in a [GLN](i)/[GLN(ECF)] ratio of 8.4, upon perfusion of alpha-(methylamino)isobutyrate which inhibits neuronal uptake of GLN(ECF) mediated by sodium-coupled amino acid transporter (SAT). The results provide first evidence for bidirectional operation of SN1 in vivo, and clarify the effect of transmembrane GLN gradient on glial GLN release at physiological Na(+) gradient. Implications of the results for SN1 as an additional regulatory site in the glutamine/glutamate cycle and utility of this approach for examining the role of GLN in an experimental model of fulminant hepatic failure are discussed.     

Structure-function relationships of domains of the delta subunit in Escherichia coli adenosine triphosphatase.

The topology of the and subunit of the Escherichia coli adenosinetriphosphatase (ECF1) has been explored by proteinase digestion and chemical labeling methods. The delta subunit of ECF1 could be cleaved selectively by reaction of the enzyme complex with very low amounts of trypsin (1:5000, w/w). Cleavage of the delta subunit occurred serially from the C-terminus. The N-terminal fragments of the delta subunit remained bound to the core ECF1 complex through sucrose gradient centrifugation, indicating that part of the binding of this subunit involves the N-terminal segment. ECF1, in which around 20 amino acids had been removed from the C-terminus of delta, still bound to ECF0 but DCCD sensitivity of the ATPase activity was lost. When ECF1 was reacted with N-ethyl[14C]maleimide ([14C]NEM) in the native state, only one of the two Cys residues on the delta subunit was modified. This residue, Cys-140, was also labeled in ECF1F0. Cys-140 was shown to be involved in the disulfide bridge between alpha and delta subunits that is generated when ECF1 is treated with CuCl2. Thus, the C-terminal part of the delta subunit around Cys-140 can interact with the core ECF1 complex. These results suggest a model for the delta subunit in which the central part of polypeptide is a part of the stalk, with both N- and C-termini associated with ECF1.     

Brain stem extracellular fluid pH and respiratory drive during hypoxia in newborn pigs

In response to moderate hypoxia many newborn animals are capable of increasing ventilation only transiently. To examine the hypothesis that changes in brain stem extracellular fluid (ECF) pH explain this transient ventilatory response, we measured brain stem ECF pH and respiratory drive during hypoxia in newborn pigs. The animals were anesthetized with alpha-chloralose-urethan, paralyzed, vagotomized, and mechanically ventilated with a servo-controlled ventilator to regulate end-tidal CO2. Hypoxic ventilation for 6 min was achieved by changing inspired gas from 100% to 10-15% O2. Respiration, measured as integrated phrenic nerve activity, showed a range of responses. In 13 trials increased phrenic activity early in the hypoxic period was sustained or further augmented for the duration of the period. In contrast, in eight other trials phrenic activity increased and then declined. Regardless of the respiratory response, ECF pH (measured with a flat-surface electrode) increased slightly (0.009 +/- 0.002 U) during the first 2.5 min of hypoxia and then declined 0.061 +/- 0.017 U by the 6th min. This acidotic shift in ECF pH is inconsistent with the hypothesis that an alkalotic shift causes the nonsustained respiratory response of newborn pigs.