New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures

A new family of high-affinity buffers and optical indicators for Ca2+ is rationally designed and synthesized. The parent compound is 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a relative of the well-known chelator EGTA [ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] in which methylene links between oxygen and nitrogen are replaced by benzene rings. BAPTA and its derivatives share the high (greater than 10(5)) selectivity for Ca2+ over Mg2+ of EGTA but are very much less affected by pH changes and are faster at taking up and releasing Ca2+. The affinity of the parent compound for Ca2+ (dissociation constant 1.1 x 10(-7) M in 0.1 M KCl) may be strengthened or weakened by electron-releasing or -withdrawing substituents on the aromatic rings. The Ca2+ and Mg2+ affinities may further be altered by replacing the ether oxygens by heterocyclic nitrogen atoms. The compounds described are fluorescent Ca2+ indicators absorbing in the ultraviolet region; the very large spectral shifts observed on binding Ca2+ fit the prediction that complexation should hinder the conjugation of the nitrogen lone-pair electrons with the aromatic rings. Derivatives with quinoline nuclei are notable for their high sensitivity of fluorescent quantum yield to the binding of Ca2+ but not of Mg2+. Preliminary biological tests have so far revealed little or no binding to membranes or toxic effects following intracellular microinjection.     

Modelling intraspecific resting egg shape variation in a freshwater fairy shrimp Tanymastix stagnalis (L., 1758) (Crustacea, Branchiopoda)

The shape of the resting eggs of a large branchiopod crustacean, the Anostraca Tanymastix stagnalis , is represented very accurately by analytical expressions. The occurrence of atypical shape of some T. stagnalis eggs may be viewed as a simple change of the analytical expression describing the usual egg shape. Their unusual shape may be explained by a higher embryo volume within an envelope of a given size. Biological implications are briefly discussed and hypothesized in an evolutionary point of view.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 90 , 55–60.     

Sequential activation and lethal hit measured by [Ca2+]i in individual cytolytic T cells and targets.

Changes in cytosolic free calcium ([Ca2+]i) have been continuously imaged during the interaction of the H-2Kb specific cytotoxic T cell lymphocyte (CTL) BM 3.3, with either the H-2Kb EL4.BU or the H-2Kk RDM4 cell lines. Activation of the CTLs by EL4.BU raises [Ca2+]i to several hundred nanomolar in the CTL. Frequently [Ca2+]i is preferentially elevated in the region of the CTL furthest from the site of target contact. These responses require external Ca2+ suggesting that they are generated by the plasma membrane and not internal stores. Inappropriate targets such as RDM4 evoke no changes in [Ca2+]i. Activation of the BM 3.3 CTL is followed by increases of [Ca2+]i to several micromolar or higher in the EL4.BU targets. This massive increase can be mimicked by direct application of cytolytic granules isolated from rat natural killer cells. The increase in plasma membrane permeability is ion-specific since external Mn2+ can also readily enter target cells that have been 'hit', as evidenced by the rapid selective quenching of fura-2 in those targets. The flood of Ca2+ into the target cell is followed by a leakage of the trapped fura-2. Since both processes continue after the CTL has disengaged, they provide a useful assay for the lethal hit. Furthermore, this technique can be used to follow complete cycles of CTL activation and lethal hit delivery, which under some circumstances can be as rapid as 6 min per cycle.     

Agonist-stimulated oscillations and cycling of intracellular free calcium in individual cultured muscle cells

Receptor regulation of [Ca2+]i was monitored in individual BC3H-1 muscle cells with intracellularly trapped fura-2 using digital imaging analysis techniques. Activation of alpha 1-adrenergic or H1-histaminergic receptors resulted in multiple bursts, or oscillations, of elevated [Ca2+]i with an average interval frequency of approximately 1.8 min-1. The duration of oscillatory behavior was generally more prolonged in response to phenylephrine than in response to histamine. Additionally, a larger fraction of the cells responded with [Ca2+]i oscillations to phenylephrine (approximately 90%) than to histamine (approximately 60%), although the majority of cells produced oscillations in response to both agonists. In most cells, the receptor-mediated [Ca2+]i oscillations continued for several minutes in the absence of extracellular Ca2+, although the amplitude of the individual peaks gradually decreased. The activation of [Ca2+]i oscillations by H1-receptors was more dependent upon extracellular Ca2+ than those elicited by alpha 1-receptors, reflecting the greater dependency of the histaminergic response on Ca2+ influx. Readdition of Ca2+ to the incubation buffer resulted in the resumption of the [Ca2+]i oscillations. These results indicate that considerable cycling of Ca2+ between the cytoplasm and the endoplasmic reticulum must occur. Receptor-mediated [Ca2+]i oscillations were much more prevalent in subconfluent cells than in confluent cells, possibly due to increased coupling of the cells at higher densities. The cells were capable of responding independently of one another, since sister cells displayed unique temporal responses immediately following cell division. Thus, the linkage of receptor occupancy to [Ca2+]i elevation is a functionally unique property for each individual cell and can be influenced by epigenetic factors.     

Ca2+ binding kinetics of fura-2 and azo-1 from temperature-jump relaxation measurements.

The Ca2+-binding kinetics of fura-2 and azo-1 were studied using temperature-jump relaxation methods. In 140 mM KCl at 20 degrees C, the association and dissociation rate constants for fura-2 were 6.02 x 10(8) M-1s-1 and 96.7 s-1, respectively. The fura-2 kinetics were insensitive to pH over the range 7.4 to 8.4. Azo-1 was studied in 140 mM KCl, at pH 7.4, at 10 degrees and 20 degrees C. At 10 degrees C, azo-1 exhibited association and dissociation rate constants of 1.43 x 10(8) M-1s-1 and 777.9 s-1, respectively; while at 20 degrees C, the corresponding values were 3.99 x 10(8) M-1s-1 and 1,177 s-1. The kinetic results demonstrate that fura-2 and azo-1 are well suited to monitoring rapid changes in intracellular [Ca2+].     

Effect of Temperature on the Distribution of Membrane Particles in Streptococcus faecalis as Seen by the Freeze-Fracture Technique

When cells of Streptococcus faecalis ATCC 9790 were incubated at temperatures above 10 C before being frozen for freeze-fracture, a random distribution of particles was observed on the outer fracture face of the freeze-cleaved cell membrane. However, when cells were incubated below 10 C before freezing, particleless patches were seen on this membrane surface. The size of the patches produced on chilling could be increased by centrifugation or by storing the chilled cells overnight at about 3 C. Patch formation appeared readily reversible, since the medium and large patches that formed on chilling could not be observed in cells warmed for 10 s at 25 C. However, during the transition from the patch to patchless state, smaller patches not seen in the chilled cells were observed. This suggested that the smaller patches might have been intermediate forms produced by the fragmentation of larger patches on warming.     

Biodegradation of low-molecular-weight halogenated hydrocarbons by methanotrophic bacteria

Low-molecular-weight halogenated hydrocarbons are susceptible to degradation by anaerobic and aerobic bacteria. The methanotrophic bacterium Methylosinus trichosporium 0B3b degrades trichloroethylene more rapidly than other bacteria examined to date. Expression of soluble methane monooxygenase (MMO) is correlated with high rates of biodegradation. An analysis of 16 S rRNA sequences of 11 ribosomal RNAs from type I, type II and type X methanotrophs and methanol-utilizing bacteria have revealed four clusters of phylogenetically related methylotrophs. This information may be useful for the identification and enumeration of methylotrophs in bioreactors and other environments during remediation of contaminated waters.     

Fluorescence measurements of cytosolic free Na concentration, influx and efflux in gastric cells.

Regulation of cytosolic free Na (Nai) was measured in isolated rabbit gastric glands with the use of a recently developed fluorescent indicator for sodium, SBFI. Intracellular loading of the indicator was achieved by incubation with an acetoxymethyl ester of the dye. Digital imaging of fluorescence was used to monitor Nai in both acid-secreting parietal cells and enzyme-secreting chief cells within intact glands. In situ calibration of Nai with ionophores indicated that SBFI fluorescence (345/385 nm excitation ratio) could resolve 2 mM changes in Nai and was relatively insensitive to changes in K or pH. Measurements on intact glands showed that basal Nai was 8.5 +/- 2.2 mM in parietal cells and 9.2 +/- 3 mM in chief cells. Estimates of Na influx and efflux were made by measuring rates of Nai change after inactivation or reactivation of the Na/K ATPase in a rapid perfusion system. Na/K ATPase inhibition resulting from the removal of extracellular K (Ko) caused Nai to increase at 3.2 +/- 1.5 mM/min and 3.5 +/- 2.7 mM/min in parietal and chief cells, respectively. Na buffering was found to be negligible. Addition of 5 mM Ko and removal of extracellular Na (Nao) caused Nai to decrease rapidly toward 0 mM Na. By subtracting passive Na efflux under these conditions (the rate at which Nai decreased in Na-free solution containing ouabain), an activation curve (dNai/Nai) for the Na/K ATPase was calculated. The pump demonstrated the greatest sensitivity between 5 and 20 mM Nai. At 37 degrees C the pump rate was less than 3 mM/min at 5 mM Nai and 26 mM/min at 25 mM Nai, indicating that the pump has a great ability to respond to changes in Nai in this range. Carbachol, which stimulates secretion from both cell types, was found to stimulate Na influx in both cell types, but did not have detectable effects on Na efflux. dbcAMP+IBMX, potent stimulants of acid secretion, had no effect on Na metabolism.     

Fluctuations in membrane current driven by intracellular calcium in cardiac Purkinje fibers

Spontaneous oscillatory fluctuations in membrane potential are often observed in heart cells, but their basis remains controversial. Such activity is enhanced in cardiac Purkinje fibers by exposure to digitalis or K-free solutions. Under these conditions, we find that voltage noise is generated by current fluctuations that persist when membrane potential is voltage clamped. Power spectra of current signals are not made up of single time-constant components, as expected from gating of independent channels, but are dominated by resonant characteristics between 0.5 and 2 HZ. Our evidence suggests that the periodicity arises from oscillatory variations in intracellular free Ca that control ion movements across the surface membrane. The current fluctuations are strongly cross-correlated with oscillatory fluctuations in contractile force, and are inhibited by removing extracellular Ca or exposure to D600. Chelating intracellular Ca with injected EGTA also abolishes the current fluctuations. The oscillatory mechanism may involve cycles of Ca (or Sr) movement between sarcoplasmic reticulum and myoplasm, as previously suggested for skinned cardiac preparations. Our experiments in intact cells indicate that changes in surface membrane potential can modulate cytoplasmic Ca oscillations in frequency and perhaps amplitude as well. A two-way interaction between surface membrane potential and intracellular Ca stores may be a common feature of heart, neuron, and other cell types.