Spatiotemporal laser inactivation of inositol 1,4,5-trisphosphate receptors using synthetic small-molecule probes

A malachite green-conjugated inositol 1,4,5-trisphosphate (MGIP(3)) induces specific inactivation of IP(3) receptor (IP(3)R) in tissue samples upon laser irradiation. To verify potential usefulness of the method for studies of cellular Ca(2+) signaling, we conducted laser inactivation at the single-cell level and show that IP(3)R was inactivated with extremely high spatiotemporal resolution. In the presence of MGIP(3), the Ca(2+) release function of IP(3)R in single B lymphoma cells decayed exponentially with increasing duration of laser irradiation with a time constant of 3.4 s. Moreover, by confining laser irradiation to a spatially distinct region of differentiated PC12 cells, subcellular inactivation of IP(3)R was attained, as revealed by a loss of local Ca(2+) signal. Such real-time inactivation of IP(3)R only within a subcellular region may provide a powerful method for investigating spatiotemporal dynamics of Ca(2+) signaling.     

Modification of intracellular Ca2+ dynamics by laser inactivation of inositol 1,4,5-trisphosphate receptor using membrane-permeant probes

A membrane-permeant malachite green-conjugated IP3 analog (MGIP3/PM) was synthesized as a probe for small molecule-based CALI (smCALI), and its effect on the Ca2+ signaling in intact DT40 chicken B cells was examined. In DT40 B cells treated with the smCALI probe, laser irradiation inhibited IP3-induced Ca2+ oscillations in response to B cell receptor stimulation, demonstrating that IP3R was acutely inactivated. We then applied smCALI to clarify the mechanism of capacitative Ca2+ entry (CCE), in which involvement of IP3R has been suggested. Despite the inactivation of IP3R by smCALI, thapsigargin-induced CCE remained unaffected, providing evidence that functional IP3R is not required for CCE in DT40 cells. These results demonstrate the potency of the smCALI technique for the study of the roles of IP3R in complex intracellular Ca2+ dynamics.     

Fluorescent bis-cyclen tweezer receptors for inositol (1,4,5)-trisphosphate

Herein, we report the development of two fluorescent sensors for inositol 1,4,5-trisphosphate (InsP₃), containing either two free cyclen or zinc(II)/cyclen groups preorganized into a binding cleft. Preorganization was achieved using a rigid acridine backbone, which was also exploited for fluorescence signal transduction. In addition, click chemistry was used to facilitate receptor synthesis and produce triazole moieties that add to structural rigidity and recognition. In binding studies, both receptors were found to bind InsP₃ with high affinity (K_a=0.5-1.0×10⁶ M⁻¹) in competitive media (1:1 methanol/water).     

Stochastic sensing of nanomolar inositol 1,4,5-trisphosphate with an engineered pore

The introduction of a ring of arginine residues near the constriction in the transmembrane beta barrel of the staphylococcal alpha-hemolysin heptamer yielded a pore that could be almost completely blocked by phosphate anions at pH 7.5. Block did not occur with other oxyanions, including nitrate, sulfate, perchlorate, and citrate. Based on this finding, additional pores were engineered with high affinities for important cell signaling molecules, such as the Ca(2+)-mobilizing second messenger inositol 1,4,5-trisphosphate (IP(3)), that contain phosphate groups. One of these engineered pores, P(RR-2), provides a ring of fourteen arginines that project into the lumen of the transmembrane barrel. Remarkably, P(RR-2) bound IP(3) with low nanomolar affinity while failing to bind another second messenger, adenosine 3', 5'-cyclic monophosphate (cAMP). The engineered alpha-hemolysin pores may be useful as components of stochastic sensors for cell signaling molecules.     

A kinetic Monte Carlo simulation study of inositol 1,4,5-trisphosphate receptor (IP3R) calcium release channel

Most of the previously theoretical studies about the stochastic nature of the IP3R calcium release channel gating use the chemical master equation (CME) approach. Because of the limitations of this approach we have used a stochastic simulation algorithm (SSA) presented by Gillespie. A single subunit of De Young-Keizer (DYK) model was simulated using Gillespie algorithm. The model has been considered in its complete form with eight states. We investigate the conditions which affect the open state of the model. Calcium concentrations were the subject of fluctuation in the previous works while in this study the population of the states is the subject of stochastic fluctuations. We found out that decreasing open probability is a function of Ca(2+) concentration in fast time domain, while in slow time domain it is a function of IP3 concentration. Studying the population of each state shows a time dependent reaction pattern in fast and medium time domains (10(-4) and 10(-3)s). In this pattern the state of X(010) has a determinative role in selecting the open state path. Also, intensity and frequency of fluctuations and Ca(2+) inhibitions have been studied. The results indicate that Gillespie algorithm can be a better choice for studying such systems, without using any approximation or elimination while having acceptable accuracy. In comparison with the chemical master equation, Gillespie algorithm is also provides a wide area for studying biological systems from other points of view.     

Recognition of myo-inositol 1,4,5-trisphosphate using a fluorescent imidazolium receptor

The first example of an imidazolium-based fluorescent receptor for D-myo-inositol 1,4,5-trisphosphate (IP(3)) is reported in this study. The fluorescent receptor 1 bearing four imidazolium and pyrene groups displayed a large fluorescent quenching effect with myo-IP(3) compared to fluorescent changes with various IP series, pyrophosphate, and ATP.     

Development of inositol-based antagonists for the D-myo-inositol 1,4,5-trisphosphate receptor

The syntheses of four D-myo-inositol 1,4,5-trisphosphate (InsP(3)) derivatives, incorporating phosphate bioisosteres at the 5-position, are reported. The methyl phosphate ester and sulfate derivatives retain InsP(3) receptor (InsP(3)R) agonist activity; the compounds that possess a methylphosphonate or a carboxymethyl moiety are InsP(3)R antagonists.     

Novel real-time sensors to quantitatively assess in vivo inositol 1,4,5-trisphosphate production in intact cells

Real-time observation of messenger molecules in individual intact cells is essential for physiological studies of signaling mechanisms. We have developed a novel inositol 1,4,5-trisphosphate (IP(3)) sensor based on the pleckstrin homology (PH) domain from phospholipase C (PLC) delta. The environmentally sensitive fluorophore 6-bromoacetyl-2-dimethyl-aminonaphtalene was conjugated to the genetically introduced cysteine at the mouth of the IP(3) binding pocket for enhanced IP(3) selectivity and for rapid and direct visualization of intracellular IP(3) > or = 0.5 microM as fluorescence emission decreased. The probe, tagged with arginine-rich sequences for efficient translocation into various cell types, revealed a major contribution of Ca2+ influx to PLC-mediated IP(3) production that boosts Ca2+ release from endoplasmic reticulum. Thus, our IP(3) probe was extremely effective to quantitatively assess real-time physiological IP(3) production via those pathways formed only in the intact cellular configuration.     

Guanophostin A: Synthesis and evaluation of a high affinity agonist of the D-myo-inositol 1,4,5-trisphosphate receptor

Guanophostin A, the guanosine counterpart of the inositol 1,4,5-trisphosphate receptor agonist adenophostin A, has been synthesized and is the first synthetic adenophostin A-like analogue to be equipotent to its parent in stimulating intracellular Ca2+ release; its nucleotide moiety is proposed to interact with the receptor binding core by guanine base cation-pi stacking with Arg504 and hydrogen bonding with Glu505 and interaction of the ribosyl 2'-phosphate group with the helix-dipole of alpha6.     

A type 2 Ferrier rearrangement-based synthesis of d-myo-inositol 1,4,5-trisphosphate

The synthesis of d-myo-inositol 1,4,5-trisphosphate (InsP₃) from methyl α-d-glucopyranose, via a type 2 Ferrier rearrangement is reported. A key intermediate in this synthesis possesses orthogonal protecting groups at the 1-, 4- and 5-position, making it a versatile starting point for the synthesis of unnatural InsP₃ derivatives. Biological evaluation of the synthetic InsP₃ demonstrates that this compound evokes selective Ca²⁺ release via activation of InsP₃ receptors.     

Fluorinated analogs and tritiated enantiomers of inositol (1,3,4)-trisphosphate

The total syntheses of 2-fluoro- and 2,2-difluoro-2-deoxy analogs of DL-myo-Ins (1,3,4)P3 are described. Resolution of a key intermediate followed by borotritide reduction and phosphorylation provided both D- and L-[1-³H]-Ins(1,3,4)P₃ enantiomers with specific activities 15 Ci/mmol.     

Synthesis and biological action of novel 4-position-modified derivatives of D-myo-inositol 1,4,5-trisphosphate

The design of a range of 4-position-modified D-myo-inositol 1,4,5-trisphosphate derivatives is described. The enantioselective synthesis of these compounds is reported, along with initial biological analysis, which indicates that these compounds do not act as D-myo-inositol 1,4,5-trisphosphate receptor agonists or antagonists.     

EPR characterization of half-integer-spin iron molecule-based magnets

We report single crystal high-frequency electron paramagnetic resonance studies of two recently discovered half-integer Fe₇ complexes: [Fe₇O₃(O₂CBu^t)₉(mda)₃(H₂O)₃] (1) and [Fe₇O₄(O₂CPh)₁₁(dmem)₂] (2). The obtained spectra confirm spin S = 5/2 ground states for both complexes. On the basis of detailed frequency and field orientation dependent studies, we find that complex 1 is a single-molecule magnet (uniaxial zero-field-splitting parameter, D < 0) while complex 2 is not (D > 0). The EPR linewidth for complex 1 is considerably narrower in comparison to spectra obtained for other single-molecule magnets, suggesting the possibility of reduced decoherence from nuclear spins (⁵⁶Fe has no nuclear moment).     

Vibrational spectroscopy as a probe of molecule-based devices

This critical review discusses the applicability of vibrational spectroscopic techniques, specifically Raman and mid-infrared, to the study of molecule-based electronics through a series of examples. We focus on a number of devices currently of interest, such as solar cells, organic light emitting diodes, molecular junctions, switches and transistors. Infrared and Raman spectroscopic techniques and their variations, the main focus of this article, can be used to investigate properties such as crystallinity, multiphasic distributions in three dimensions, as well as lifetimes, structures and energetics of excited-states on ultrashort to very long timescales (210 references).     

Simple molecule-based fluorescent sensors for vapor detection of TNT

1,4-Diarylpentiptycenes (1a-e) were synthesized from 1,4-dichloro- or 1,4-difluoro-2,5-diarylbenzene derivatives by double base-promoted dehydrohalogenation to give corresponding arynes, which in the presence of anthracene undergo cycloaddition providing 1,4-diarylpentiptycenes in moderate overall yields. The resulting 1,4-diarylpentiptycenes show fluorescence modulated by the 1,4-aryl residues. The fluorescence is quenched in the presence of vapors of nitroaromatic compounds suggesting potential application in sensing of explosives.     

Impact of molecule-based magnetic materials: A critical outlook

A critical outlook of the field of molecular magnetic materials is presented. This article is inspired by an international symposium devoted to the “Design, Characterization and Modelling of Molecule-Based Magnetic Materials (DCM4-II)” that took place at Strasbourg (France), from May 28th to June 1st, within the E-MRS 2007 Spring Meeting (Symposium R) organized by the European Materials Research Society in collaboration with the European Science Foundation. A series of papers linked to this symposium are published in this issue and in the previous issue (Volume 11, Issue 4) of Solid State Sciences.     

Nonlinear magneto-optical effects and photomagnetism of electrochemically synthesized molecule-based magnets

This article describes novel optical functionalities such as photomagnetic effects and magnetization-induced second harmonic generation (MSHG) in several cyano-bridged metal assemblies. Single crystal- and film-types of a cyano-bridged Cu–Mo bimetallic assembly, , were electrochemically prepared. When this compound was irradiated with light, spontaneous magnetization with a Curie temperature (T _C) of 23 K was observed. Electrochemically prepared Fe^II[Cr^III(CN)₆]_2/3·5H₂O thin film, which was a ferromagnet with T _C=21 K, showed photoreduced magnetization. This photomagnetism is due to the change of ferromagnetic coupling between Fe^II and Cr^III. MSHG was observed in Cs^ICo^II[Cr^III(CN)₆]·0.5H₂O. This -type Prussian blue analog-based magnet is proven to be a piezoelectric ferromagnet, i.e., condensed matter with both piezoelectric and ferromagnetism. This MSHG is due to the coupling between a piezoelectric structure of and ferromagnetism with a T _C of 46 K.

Effect of uniaxial strain on a molecule-based ferrimagnet with crystal chirality

Bimetallic ferrimagnet [Cr(CN)₆][Mn(R)-pnH(H₂O)]H₂O with crystallographical chirality of space group P2₁2₁2₁ exhibits a giant third-order harmonic susceptibility at around 38 K. This anomaly appears as precursory magnetic phenomenon of the magnetic ordering. In order to investigate the effect of lattice distortion on this giant nonlinear magnetic response (NLMR) and finally elucidate the correlation between the structural chirality and the giant NLMR, we performed the ac susceptibility measurement for the single crystal in the situation of bringing about lattice distortion along the direction parallel to the a-axis. This giant NLMR was drastically suppressed even by slight strain, whereas the traces could be confirmed in the region up to 3.7 kbar. We conclude that the appearance of the giant NLMR requires crystal chirality well-regulated over the crystal.