Recognition of Hg 2+ ion through restricted imine isomerization: Crystallographic evidence and imaging in live cells

A newly synthesized imine-based receptor (L) showed remarkable specificity toward the Hg 2+ ion in aqueous media over other metal ions. Coordination of L to Hg 2+ induces a turn-on fluorescence response. This was explained based on the restricted imine isomerization along with PET on coordination to Hg 2+. X-ray structural evidence tends to favor a C-C bond rotation rather than C=N isomerization for adopting a favorable conformation in L for coordination to Hg 2+. This reagent could be used for imaging the accumulation of Hg 2+ ions in HeLa cells

Synthesis, photophysical, photochemical, DNA cleavage/binding and cytotoxic properties of pyrene oxime ester conjugates

A new series of (E)-pyrene oxime ester conjugates of carboxylic acids including amino acids were synthesized by coupling with an environment sensitive fluorophore 1-acetylpyrene. (E)-Pyrene oxime esters exhibited strong fluorescence properties and interestingly their fluorescence properties were found to be highly sensitive to the surrounding environment. Direct irradiation of the (E)-pyrene oxime esters by UV light (≥350 nm) resulted in both the photo-Beckmann rearrangement product and products resulting from N–O bond homolysis. Photoproduct formation and their distribution were found to be solvent dependent. Further, we also showed (E)-pyrene oxime esters intercalated into DNA efficiently and photo-cleaved DNA. Finally we also showed these oxime esters can permeate cells efficiently and may cause cytotoxicity upon irradiation of light

A novel encystation specific protein kinase regulates chitin synthesis in Entamoeba invadens

Phosphorylation is an important post-translational modification of proteins and is involved in the regulation of a variety of cellular events. The proteome of Entamoeba invadens, the reptilian counterpart of Entamoeba histolytica consists of an overwhelming number of putative protein kinases, and some may have a role to play in Entamoeba encystation. In this study, we have identified a novel protein kinase named as EiCSpk (Entamoeba invadens cyst specific protein kinase) which expressed almost exclusively during encystation. It is an active Protein kinase C with a characteristic substrate phosphorylation and auto-phosphorylation property. Gene silencing study has unveiled its role as a regulator of chitin synthesis through transcriptional activation of the chitin synthesis pathway genes along with glycogen phosphorylases that are involved in the influx of glucose from glycogen breakdown for chitin synthesis

Partial ligand-receptor engagement yields functional bias at the human complement receptor, C5aR1

The human complement component, C5a, binds two different seven transmembrane receptors termed C5aR1 and C5aR2. C5aR1 is a prototypical G protein-coupled receptor (GPCR) that couples to the Gαi sub-family of heterotrimeric G-proteins and β-arrestins (βarr) following C5a stimulation. Peptide fragments derived from the carboxyl-terminus of C5a can still interact with the receptor, albeit with lower affinity, and can act as agonists or antagonists. However, whether such fragments might display ligand bias at C5aR1 remains unexplored. Here, we compare C5a and a modified C-terminal fragment of C5a, C5apep, in terms of G protein coupling, βarr recruitment, endocytosis and ERK1/2 MAP kinase activation at the human C5aR1. We discover that C5apep acts as a full-agonist for Gαi-coupling as measured by cAMP response and ERK1/2 phosphorylation, but it displays partial agonism for βarr recruitment and receptor endocytosis. Interestingly, C5apep exhibits full-agonist efficacy with respect to inhibiting LPS induced IL-6 secretion in human macrophages, but its ability to induce human neutrophil migration is substantially lower compared to C5a although both these responses are sensitive to Pertussis toxin (PTX) treatment. Taken together, our data reveal that compared to C5a, C5apep exerts partial efficacy for βarr recruitment, receptor trafficking and neutrophil migration. Our findings therefore uncover functional bias at C5aR1, and also provide a framework that can potentially be extended to chemokine receptors which also typically interact with chemokines through a biphasic mechanism

Recognition of Hg²⁺ Ion through Restricted Imine Isomerization: Crystallographic Evidence and Imaging in Live Cells

A newly synthesized imine-based receptor (<b>L</b>) showed remarkable specificity toward the Hg²⁺ ion in aqueous media over other metal ions. Coordination of <b>L</b> to Hg²⁺ induces a <i>turn-on</i> fluorescence response. This was explained based on the restricted imine isomerization along with PET on coordination to Hg²⁺. X-ray structural evidence tends to favor a C–C bond rotation rather than CN isomerization for adopting a favorable conformation in <b>L</b> for coordination to Hg²⁺. This reagent could be used for imaging the accumulation of Hg²⁺ ions in HeLa cells

Recognition of Hg²⁺ Ion through Restricted Imine Isomerization: Crystallographic Evidence and Imaging in Live Cells

A newly synthesized imine-based receptor (<b>L</b>) showed remarkable specificity toward the Hg²⁺ ion in aqueous media over other metal ions. Coordination of <b>L</b> to Hg²⁺ induces a <i>turn-on</i> fluorescence response. This was explained based on the restricted imine isomerization along with PET on coordination to Hg²⁺. X-ray structural evidence tends to favor a C–C bond rotation rather than CN isomerization for adopting a favorable conformation in <b>L</b> for coordination to Hg²⁺. This reagent could be used for imaging the accumulation of Hg²⁺ ions in HeLa cells

Distinct phosphorylation sites in a prototypical GPCR differently orchestrate β-arrestin interaction, trafficking, and signaling

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a key determinant for their interaction with β-arrestins (βarrs) and subsequent functional responses. Therefore, it is important to decipher the contribution and interplay of different receptor phosphorylation sites in governing βarr interaction and functional outcomes. Here, we find that several phosphorylation sites in the human vasopressin receptor (V2R), positioned either individually or in clusters, differentially contribute to βarr recruitment, trafficking, and ERK1/2 activation. Even a single phosphorylation site in V2R, suitably positioned to cross-talk with a key residue in βarrs, has a decisive contribution in βarr recruitment, and its mutation results in strong G-protein bias. Molecular dynamics simulation provides mechanistic insights into the pivotal role of this key phosphorylation site in governing the stability of βarr interaction and regulating the interdomain rotation in βarrs. Our findings uncover important structural aspects to better understand the framework of GPCR-βarr interaction and biased signaling

Recognition of Hg²⁺ and Cr³⁺ in Physiological Conditions by a Rhodamine Derivative and Its Application as a Reagent for Cell-Imaging Studies

A new rhodamine-based receptor, derivatized with an additional fluorophore (quinoline), was synthesized for selective recognition of Hg²⁺ and Cr³⁺ in an acetonitrile/HEPES buffer medium of pH 7.3. This reagent could be used as a dual probe and allowed detection of these two ions by monitoring changes in absorption and the fluorescence spectral pattern. In both instances, the extent of the changes was significant enough to allow visual detection. More importantly, the receptor molecule could be used as an imaging reagent for detection of Hg²⁺ and Cr³⁺ uptake in live human cancer cells (MCF7) using laser confocal microscopic studies. Unlike Hg­(ClO₄)₂ or Hg­(NO₃)₂ salts, HgCl₂ or HgI₂ failed to induce any visually detectable change in color or fluorescence upon interaction with <b>L</b>_<b>1</b> under identical experimental conditions. Presumably, the higher covalent nature of Hg^II in HgCl₂ or HgI₂ accounts for its lower acidity and its inability to open up the spirolactam ring of the reagent <b>L</b>_<b>1</b>. The issue has been addressed on the basis of the single-crystal X-ray structures of <b>L</b>_<b>1</b>·HgX₂ (X^– = Cl^– or I^–) and results from other spectral studies

Recognition of Hg²⁺ and Cr³⁺ in Physiological Conditions by a Rhodamine Derivative and Its Application as a Reagent for Cell-Imaging Studies

A new rhodamine-based receptor, derivatized with an additional fluorophore (quinoline), was synthesized for selective recognition of Hg²⁺ and Cr³⁺ in an acetonitrile/HEPES buffer medium of pH 7.3. This reagent could be used as a dual probe and allowed detection of these two ions by monitoring changes in absorption and the fluorescence spectral pattern. In both instances, the extent of the changes was significant enough to allow visual detection. More importantly, the receptor molecule could be used as an imaging reagent for detection of Hg²⁺ and Cr³⁺ uptake in live human cancer cells (MCF7) using laser confocal microscopic studies. Unlike Hg­(ClO₄)₂ or Hg­(NO₃)₂ salts, HgCl₂ or HgI₂ failed to induce any visually detectable change in color or fluorescence upon interaction with <b>L</b>_<b>1</b> under identical experimental conditions. Presumably, the higher covalent nature of Hg^II in HgCl₂ or HgI₂ accounts for its lower acidity and its inability to open up the spirolactam ring of the reagent <b>L</b>_<b>1</b>. The issue has been addressed on the basis of the single-crystal X-ray structures of <b>L</b>_<b>1</b>·HgX₂ (X^– = Cl^– or I^–) and results from other spectral studies