From small molecules to polymeric probes: recent advancements of formaldehyde sensors.

Formaldehyde is a well-known industrial material regularly used in fishery, vegetable markets, and fruit shops for maintaining their freshness. But due to its carcinogenic nature and other toxic effects, it is very important to detect it in very low concentrations. In recent years, amine-containing fluorescent probes have gained significant attention for designing formaldehyde sensors. However, the major drawbacks of these small molecular probes are low sensitivity and long exposure time, which limits their real-life applications. In this regard, polymeric probes have gained significant attention to overcome the aforementioned problems. Several polymeric probes have been utilized as a coating material, nanoparticle, quartz crystal microbalance (QCM), etc., for the selective and sensitive detection of formaldehyde. The main objective of this review article is to comprehensively describe the recent advancements in formaldehyde sensors based on small molecules and polymers, and their successful applications in various fields, especially in situ formaldehyde sensing in biological systems.

Methemoglobin-induced signaling and chemokine responses in human alveolar epithelial cells.

Diffuse alveolar hemorrhage is characterized by the presence of red blood cells and free hemoglobin in the alveoli and complicates a number of serious medical and surgical lung conditions including the pulmonary vasculitides and acute respiratory distress syndrome. In this study we investigated the hypothesis that exposure of human alveolar epithelial cells to hemoglobin and its breakdown products regulates chemokine release via iron- and oxidant-mediated activation of the transcription factor NF-κB. Methemoglobin alone stimulated the release of IL-8 and MCP-1 from A549 cells via activation of the NF-κB pathway; additionally, IL-8 required ERK activation and MCP-1 required JNK activation. Neither antioxidants nor iron chelators and knockdown of ferritin heavy and light chains affected these responses, indicating that iron and reactive oxygen species are not involved in the response of alveolar epithelial cells to methemoglobin. Incubation of primary cultures of human alveolar type 2 cells with methemoglobin resulted in a similar pattern of chemokine release and signaling pathway activation. In summary, we have shown for the first time that methemoglobin induced chemokine release from human lung epithelial cells independent of iron- and redox-mediated signaling involving the activation of the NF-κB and MAPK pathways. Decompartmentalization of hemoglobin may be a significant proinflammatory stimulus in a variety of lung diseases.

The signaling protein CD38 is essential for early embryonic development.

CD38 is a multifunctional protein possessing ADP-ribosyl cyclase activity responsible for both the synthesis and the degradation of several Ca(2+)-mobilizing second messengers. Although a variety of functions have been ascribed to CD38, such as immune responses, insulin secretion, and social behavior in adults, nothing is known of its role during embryonic development when Ca(2+) signals feature prominently. Here, we report the identification and functional expression of CD38 from Xenopus laevis, a key model organism for the study of vertebrate development. We show that CD38 expression and endogenous ADP-ribosyl cyclase activity are developmentally regulated during cellular differentiation. Chemical or molecular inhibition of CD38 abolished ADP-ribosyl cyclase activity and disrupted elongation of the anterior-posterior axis and differentiation of skeletal muscle, culminating in embryonic death. Our data uncover a previously unknown role for CD38 as an essential regulator of embryonic development.

A single residue in a novel ADP-ribosyl cyclase controls production of the calcium-mobilizing messengers cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate.

Cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate are ubiquitous calcium-mobilizing messengers produced by the same family of multifunctional enzymes, the ADP-ribosyl cyclases. Not all ADP-ribosyl cyclases have been identified, and how production of different messengers is achieved is incompletely understood. Here, we report the cloning and characterization of a novel ADP-ribosyl cyclase (SpARC4) from the sea urchin, a key model organism for the study of calcium-signaling pathways. Like several other members of the ADP-ribosyl cyclase superfamily, SpARC4 is a glycoprotein targeted to the plasma membrane via a glycosylphosphatidylinositol anchor. However, unlike most other members, SpARC4 shows a remarkable preference for producing cyclic ADP-ribose over nicotinic acid adenine dinucleotide phosphate. Mutation of a single residue (tyrosine 142) within a noncanonical active site reversed this striking preference. Our data highlight further diversification of this unusual enzyme family, provide mechanistic insight into multifunctionality, and suggest that different ADP-ribosyl cyclases are fine-tuned to produce specific calcium-mobilizing messengers.

Mechanism of potentiation of a dysfunctional epilepsy-linked mutated GABA(A) receptor by a neurosteroid (3alpha, 21-dihydroxy-5alpha-pregnan-20-one): transient kinetic investigations.

The malfunction of a mutated GABA(A) receptor (alpha1beta2gamma2L(K289M)) in an inheritable form of epilepsy (GEFS+, generalized epilepsy with febrile seizures plus) in humans [Baulac, S., Huberfeld, G., Gourfinkel-An, I., Mitropoulou, G., Beranger, A., Prud'homme, J. F., Baulac, M., Brice, A., Bruzzone, R., and LeGuern, E. (2001) Nat. Genet. 28, 46-48] has been accounted for by a 5-fold decrease in the channel-opening equilibrium of the mutated receptor compared to the wild type [Ramakrishnan, L., and Hess, G. P. (2004) Biochemistry 43, 7534-7540]. Here we describe the mechanism by which the neurosteroid 3alpha, 21-dihydroxy-5alpha-pregnan-20-one (5alpha-THDOC) alleviates this malfunction of the mutated receptor transiently expressed in HEK293 cells. Two rapid reaction techniques, the cell-flow and the laser-pulse photolysis methods, were used in combination with whole-cell current recordings. 150-muM 5alpha-THDOC does not affect the rate constant for channel opening (k(op)) of approximately 250 s(-1) but does decrease the rate constant for channel closing (k(cl)) from 121 +/- 11 s(-1) to 56 +/- 21 s(-1). This results in an increase in the channel-opening equilibrium constant ((Phi(-1) = k(op)/k(cl)) by a factor of about 2, leading to about 50% alleviation of the malfunction of the inheritable mutated (alpha1beta2gamma2L(K289M)) GABA(A) receptor linked to GEFS+.

Pulmonary Arterial Hypertension: Iron Matters.

The interplay between iron and oxygen is longstanding and central to all aerobic life. Tight regulation of these interactions including homeostatic regulation of iron utilization ensures safe usage of this limited resource. However, when control is lost adverse events can ensue, which are known to contribute to an array of disease processes. Recently, associations between disrupted iron homeostasis and pulmonary artery hypertension (PAH) have been described with the suggestion that there is a contributory link with disease. This review provides a background for iron regulation in humans, describes PAH classifications, and discusses emerging literature, which suggests a role for disrupted iron homeostatic control in various sub-types of PAH, including a role for decompartmentalization of hemoglobin. Finally, the potential for therapeutic options to restore iron homeostatic balance in PAH are discussed.

The Hepcidin/Ferroportin axis modulates proliferation of pulmonary artery smooth muscle cells.

Studies were undertaken to examine any role for the hepcidin/ferroportin axis in proliferative responses of human pulmonary artery smooth muscle cells (hPASMCs). Entirely novel findings have demonstrated the presence of ferroportin in hPASMCs. Hepcidin treatment caused increased proliferation of these cells most likely by binding ferroportin resulting in internalisation and cellular iron retention. Cellular iron content increased with hepcidin treatment. Stabilisation of ferroportin expression and activity via intervention with the therapeutic monoclonal antibody LY2928057 reversed proliferation and cellular iron accumulation. Additionally, IL-6 treatment was found to enhance proliferation and iron accumulation in hPASMCs; intervention with LY2928057 prevented this response. IL-6 was also found to increase hepcidin transcription and release from hPASMCs suggesting a potential autocrine response. Hepcidin or IL-6 mediated iron accumulation contributes to proliferation in hPASMCs; ferroportin mediated cellular iron excretion limits proliferation. Haemoglobin also caused proliferation of hPASMCs; in other novel findings, CD163, the haemoglobin/haptoglobin receptor, was found on these cells and offers a means for cellular uptake of iron via haemoglobin. Il-6 was also found to modulate CD163 on these cells. These data contribute to a better understanding of how disrupted iron homeostasis may induce vascular remodelling, such as in pulmonary arterial hypertension.

ADP-ribosyl cyclases regulate early development of the sea urchin.

ADP-ribosyl cyclases are multifunctional enzymes involved in the metabolism of nucleotide derivatives necessary for Ca2+ signalling such as cADPR and NAADP. Although Ca2+ signalling is a critical regulator of early development, little is known of the role of ADP-ribosyl cyclases during embryogenesis. Here we analyze the expression, activity and function of ADP-ribosyl cyclases in the embryo of the sea urchin - a key organism for study of both Ca2+ signalling and embryonic development. ADP-ribosyl cyclase isoforms (SpARC1-4) showed unique changes in expression during early development. These changes were associated with an increase in the ratio of cADPR:NAADP production. Over-expression of SpARC4 (a preferential cyclase) disrupted gastrulation. Our data highlight the importance of ADP-ribosyl cyclases during embryogenesis.

Galantamine reverses scopolamine-induced behavioral alterations in Dugesia tigrina.

In planaria (Dugesia tigrina), scopolamine, a nonselective muscarinic receptor antagonist, induced distinct behaviors of attenuated motility and C-like hyperactivity. Planarian locomotor velocity (pLMV) displayed a dose-dependent negative correlation with scopolamine concentrations from 0.001 to 1.0 mM, and a further increase in scopolamine concentration to 2.25 mM did not further decrease pLMV. Planarian hyperactivity counts was dose-dependently increased following pretreatment with scopolamine concentrations from 0.001 to 0.5 mM and then decreased for scopolamine concentrations ≥ 1 mM. Planarian learning and memory investigated using classical Pavlovian conditioning experiments demonstrated that scopolamine (1 mM) negatively influenced associative learning indicated by a significant decrease in % positive behaviors from 86 % (control) to 14 % (1 mM scopolamine) and similarly altered memory retention, which is indicated by a decrease in % positive behaviors from 69 % (control) to 27 % (1 mM scopolamine). Galantamine demonstrated a complex behavior in planarian motility experiments since co-application of low concentrations of galantamine (0.001 and 0.01 mM) protected planaria against 1 mM scopolamine-induced motility impairments; however, pLMV was significantly decreased when planaria were tested in the presence of 0.1 mM galantamine alone. Effects of co-treatment of scopolamine and galantamine on memory retention in planaria via classical Pavlovian conditioning experiments showed that galantamine (0.01 mM) partially reversed scopolamine (1 mM)-induced memory deficits in planaria as the % positive behaviors increased from 27 to 63 %. The results demonstrate, for the first time in planaria, scopolamine's effects in causing learning and memory impairments and galantamine's ability in reversing scopolamine-induced memory impairments.

Riluzole attenuates the effects of chemoconvulsants acting on glutamatergic and GABAergic neurotransmission in the planarian Dugesia tigrina.

Planarians, the non-parasitic flatworms, display dose-dependent, distinct (C-like and corkscrew-like) hyperkinesias upon exposure to 0.001-10 mM aqueous solutions of glutamatergic agonists (L-glutamate and N-methyl-D-aspartate (NMDA)) and 0.001-5 mM concentrations of the glutamate decarboxylase (GAD) inhibitor (semicarbazide). In the planarian seizure-like activity (PSLA) experiments the three chemoconvulsants displayed the following order of potency (EC50): L-glutamate (0.6mM)>NMDA (1.4 mM)>semicarbazide (4.5mM). Planarian hyperkinesias behavior counting experiments also revealed that riluzole (0.001 to 1mM), an anti-convulsive agent, displayed no significant behavioral activity by itself, but attenuated hyperkinesias elicited by the three chemoconvulsants targeting either glutamatergic or GABAergic neurotransmission with the following order of potency (IC50): NMDA (44.7 µM)>semicarbazide (88.3 µM)>L-glutamate (160 µM). Further, (+)-MK-801, a specific NMDA antagonist, alleviated 3mM NMDA (47%) or 3mM L-glutamate (27%) induced planarian hyperkinesias. The results provide pharmacological evidence for the presence of glutamatergic receptor-like and semicarbazide sensitive functional GAD enzyme-like proteins in planaria in addition to demonstrating, for the first time, the anti-convulsive effects of riluzole in an invertebrate model. High performance liquid chromatography coupled with fluorescence detection (HPLC-F) analysis performed on planarian extracts post no drug treatment (control) or treatment with 3mM semicarbazide, combination of 3mM semicarbazide and 0.1 mM riluzole, or 0.1 mM riluzole revealed that 3 mM semicarbazide induced 35% decrease in the GABA levels and a combination of 3mM semicarbazide and 0.1 mM riluzole induced 42% decrease in glutamate levels with respect to the control group.

Carbamazepine inhibits distinct chemoconvulsant-induced seizure-like activity in Dugesia tigrina.

Planaria, non-parasitic flatworms, were recently shown to be a simple yet sensitive model for investigating the pharmacology of convulsants and anticonvulsants. The present findings show that three distinct chemoconvulsants, (-)-nicotine, picrotoxin, and N-methyl-D-aspartate (NMDA), induce dose-dependent seizure-like paroxysms in the planarian Dugesia tigrina. Carbamazepine and oxcarbazepine, iminodibenzyl derivatives, exhibit anticonvulsive effects mediated mainly through the inactivation of voltage-gated sodium channels. Apart from these primary molecular targets, both carbamazepine and oxcarbazepine are known to activate γ-aminobutyric acid type A (GABA(A)) receptors and inhibit NMDA activated glutamate receptors and neuronal nicotinic acetylcholine receptors (nAChRs). The present study shows that in D. tigrina both carbamazepine and oxcarbazepine inhibit chemoconvulsant-induced seizure behaviors in a dose-dependent manner. Carbamazepine (100 µM) decreased by ~65% the cumulative mean planarian seizure-like activity (pSLA) observed in the presence of (-)-nicotine (10 µM), picrotoxin (5mM), or NMDA (3mM), whereas oxcarbazepine (1 µM) decreased by 45% the cumulative mean pSLA induced by (-)-nicotine (10µM). The results demonstrate, for the first time, the anti-seizure pharmacology of carbamazepine and oxcarbazepine in an invertebrate seizure model.

The endo-lysosomal system as an NAADP-sensitive acidic Ca(2+) store: role for the two-pore channels.

Accumulating evidence suggests that the endo-lysosomal system provides a substantial store of Ca(2+) that is tapped by the Ca(2+)-mobilizing messenger, NAADP. In this article, we review evidence that NAADP-mediated Ca(2+) release from this acidic Ca(2+) store proceeds through activation of the newly described two-pore channels (TPCs). We discuss recent advances in defining the sub-cellular targeting, topology and biophysics of TPCs. We also discuss physiological roles and the evolution of this ubiquitous ion channel family.

Molecular characterization of a novel cell surface ADP-ribosyl cyclase from the sea urchin.

The sea urchin is an extensively used model system for the study of calcium signalling by the messenger molecules NAADP and cyclic ADP-ribose. Both are synthesized by ADP-ribosyl cyclases but our molecular understanding of these enzymes in the sea urchin is limited. We have recently reported the cloning of an extended family of sea urchin ADP-ribosyl cyclases and shown that one of these enzymes (SpARC1) is active within the endoplasmic reticulum lumen. These studies suggest that production of messengers is compartmentalized. Here we characterize the properties of SpARC2. SpARC2 catalyzed both NAADP and cyclic ADP-ribose production. Unusually, the NAD surrogate, NGD was a poor substrate. In contrast to SpARC1, heterologously expressed SpARC2 localized to the plasma membrane via a glycosylphosphatidylinositol (GPI)-anchor. Transcripts for SpARC2 were readily detectable in sea urchin eggs and a majority of the endogenous membrane bound activity was found to be GPI-anchored. Our data reveal striking differences in the properties of sea urchin ADP-ribosyl cyclases and provide further evidence that messenger production may occur outside of the cytosol.

Picrotoxin inhibition mechanism of a gamma-aminobutyric acid A receptor investigated by a laser-pulse photolysis technique.

The gamma-aminobutyric acid(A) (GABA(A)) receptor, a major inhibitory neurotransmitter receptor, belongs to a family of membrane-bound proteins that regulate signal transmission between approximately 10(12) cells of the nervous system. It plays a major role in many neurological disorders, including epilepsy. It is the target of many pharmacological agents, including the convulsant picrotoxin. Here, we present the mechanism of inhibition by picrotoxin of the rat alpha1beta2gamma2L GABA(A) receptor investigated using rapid kinetic techniques in combination with whole-cell current recordings. The following new results were obtained by using transient kinetic techniques, the cell-flow method and the laser-pulse photolysis (LaPP) technique with a microsecond to millisecond time resolution. (i) The apparent dissociation constant of picrotoxin for the open-channel form of the receptor was approximately 5 times higher than that of the closed-channel form. (ii) Picrotoxin increased the channel-closing rate constant (k(cl)) approximately 4-fold, while the rate constant for channel opening (k(op)) remained essentially unaffected. (iii) The mechanism indicates that picrotoxin binds to an allosteric site of the receptor with higher affinity for the closed-channel form than for the open-channel form and thereby inhibits the receptor by decreasing 4-fold its channel-opening equilibrium constant [Phi(I)(-)(1) = k(op(I))/k(cl(I))]. (iv) The mechanism further indicates that compounds that bind with equal affinity to the picrotoxin-binding site on the open-channel form of the receptor and the closed-channel form will not affect the channel-opening equilibrium and can, therefore, displace picrotoxin and prevent inhibition of the GABA(A) receptor by picrotoxin. Such compounds may be therapeutically useful in counteracting the effects of compounds and diseases that unfavorably affect the channel-opening equilibrium of the receptor channel.

On the mechanism of a mutated and abnormally functioning gamma-aminobutyric acid (A) receptor linked to epilepsy.

A recent report indicates that a lysine-to-methionine mutation (K289M) in the gamma2 subunit of a human gamma-aminobutyric acid neurotransmitter receptor, the GABA(A) receptor, is linked to generalized epilepsy with febrile seizures [Baulac et al. (2001) Nat. Genet. 28, 46-48]. This mutation caused a decreased current response to GABA [Baulac et al. (2001) Nat. Genet. 28, 46-48]. Here we determine changes that occur in the mechanism of opening and closing of transmembrane channels formed by the GABA(A) receptor as a result of this mutation. The K289M mutation was introduced into the gamma2L subunit of the rat GABA(A) receptor, and the mutated subunit was coexpressed with the alpha1 and beta2 subunits in HEK293 cells. Transient kinetic techniques suitable for investigating reactions on cell surfaces with a microsecond-to-millisecond time resolution [Hess, G. P., and Grewer, C. (1998) Methods Enzymol. 291, 443-473] were used. They allow one to determine not only the channel-opening probability and rates of receptor desensitization but also the opening and closing rates of the mutated GABA(A) receptor channel. The channel-opening equilibrium constant of the mutated receptor was found to be 5-fold lower than that of the wild type. We calculated that this decrease in the channel-opening equilibrium accounts for the dysfunction of the mutated receptor. We discuss how a knowledge of the mechanism of the mutated receptor indicates an approach for alleviating this dysfunction.

Toward the development of new photolabile protecting groups that can rapidly release bioactive compounds upon photolysis with visible light.

The synthesis and characterization of a new photolabile protecting group (caging group) for carboxylic acids, the 2-(dimethylamino)-5-nitrophenyl (DANP) group, is described. This compound has a major absorption band in the visible wavelength region with a maximum near 400 nm (epsilon400 = 9077 M(-1) cm(-1) at pH 7.4 and 21 degrees C). The caging group is attached through an ester linkage to the carboxyl functionality of beta-alanine, which activates the inhibitory glycine receptor in the mammalian central nervous system. Such caged compounds play an important role in transient kinetic investigations of fast cellular processes. Upon photolysis of DANP-caged beta-alanine, the caging group is released within 5 micros. Quantum yields of 0.03 and 0.002 were obtained in the UV region (308 and 360 nm) and the visible region (450 nm), respectively. Laser-pulse photolysis experiments, using 337 or 360 nm light, were performed with the caged compound equilibrated with HEK 293 cells transiently transfected with cDNA encoding the alpha1 homomeric, wild-type glycine receptor. The experiments demonstrated that neither DANP-caged beta-alanine nor its byproducts inhibit or activate the glycine receptors on the cell surface. Under physiological conditions, the DANP-caged beta-alanine is water-soluble and stable and can be used for transient kinetic measurements.