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1.
Free Radic Biol Med ; 148: 170-181, 2020 02 20.
Article in English | MEDLINE | ID: mdl-31923584

ABSTRACT

The sperm tail outer dense fibres (ODFs) contribute passive structural role in sperm motility. The level of disulphide cross-linking of ODFs and their structural thickness determines flagellar bending curvature and motility. During epididymal maturation, proteins are internalized to modify ODF disulphide cross-linking and enable motility. Sperm thiol status is further altered during capacitation in female tract. This suggests that components in female reproductive tract acting on thiol/disulphides could be capable of modulating the tail stiffness to facilitate modulation of the sperm tail rigidity and waveform en route to fertilization. Understanding the biochemical properties and client proteins of ODFs in reproductive tract fluids will help bridge this gap. Using recombinant ODF2 (aka Testis Specific Antigen of 70 kDa) as bait, we identified client proteins in male and female reproductive fluids. A thiol-based interaction and internalization indicates sperm can harness reproductive tract fluids for proteins that interact with ODFs and likely modulate the tail stiffness en route to fertilization.


Subject(s)
Sperm Tail , Sulfhydryl Compounds , Female , Heat-Shock Proteins , Humans , Male , Sperm Motility , Spermatozoa , Testis
2.
Sci Rep ; 9(1): 1780, 2019 02 11.
Article in English | MEDLINE | ID: mdl-30741951

ABSTRACT

Cancer remains one of the biggest threats to human society. There are massive demands for compounds to selectively kill cancerous cells. Earlier studies have shown that bovine α -lactalbumin made lethal to tumor cells (BAMLET) becomes cytotoxic against cancer cells in complex with oleic acid {Hoque, M. et. al., PLoSOne 8, e68390 (2013)}. In our study, we obtained bovine α-lactalbumin complexed with lanthanum ion (La3+-B-α-LA) and determined its high resolution crystal structure. The natural calcium binding site of bovine α-lactalbumin is replaced by lanthanum. The La3+ complex formation by B-α-apo-LA was also supported by various biophysical methods. Interestingly, our complex, La3+-B-α-LA exhibits much greater anticancer activity against breast cancer cells as compared to the reported BAMLET-oleic acid complex. This study shows that La3+-B-α-LA complex is preferentially more toxic to MCF-7 cells as compared to KB (oral cancer) and HeLa (cervical) cells, while almost non-toxic to the healthy cells that we studied. Our data indicates that the cytotoxicity of La3+-B-α-LA against cancer cells is through apoptotic path way. The higher anticancer activity of La3+-B-α-LA is attributable to the requisite structural changes induced in the protein by La3+ binding as supported by the crystal structure of the complex.


Subject(s)
Apoproteins/pharmacology , Lactalbumin/pharmacology , Lanthanum/metabolism , Animals , Apoproteins/chemistry , Apoproteins/metabolism , Calcium/metabolism , Cattle , Cell Line, Tumor , Cell Survival/drug effects , Crystallography, X-Ray , Humans , Lactalbumin/chemistry , Lactalbumin/metabolism , Lanthanum/chemistry , Molecular Structure , Protein Binding
3.
Biochim Biophys Acta Gen Subj ; 1861(2): 365-374, 2017 Feb.
Article in English | MEDLINE | ID: mdl-27916677

ABSTRACT

BACKGROUND: Copper is an essential trace element required for the proper functioning of various enzymes present in the central nervous system. An imbalance in the copper homeostasis results in the pathology of various neurodegenerative disorders including Parkinson's Disease. Hence, residue specific interaction of Cu2+ to α-Syn along with the familial mutants H50Q and G51D needs to be studied in detail. METHODS: We investigated the residue specific mapping of Cu2+ binding sites and binding strength using solution-state NMR and ITC respectively. The aggregation kinetics, secondary structural changes, and morphology of the formed fibrils in the presence and absence of Cu2+ were studied using fluorescence, CD, and AFM respectively. RESULTS: Copper binding to α-Syn takes place at three different sites with a higher affinity for the region 48-53. While one of the sites got abolished in the case of H50Q, the mutant G51D showed a binding pattern similar to WT. The aggregation kinetics of these proteins in the presence of Cu2+ showed an enhanced rate of fibril formation with a pronounced effect for G51D. CONCLUSION: Cu2+ binding results in the destabilization of long-range tertiary interactions in α-Syn leading to the exposure of highly amyloidogenic NAC region which results in the increased rate of fibril formation. Although the residues 48-53 have a stronger affinity for Cu2+ in case of WT and G51D, the binding is not responsible for enhancing the rate of fibril formation in case of H50Q. GENERAL SIGNIFICANCE: These findings will help in the better understanding of Cu2+ catalyzed aggregation of synucleins.


Subject(s)
Amyloid/metabolism , Amyloidogenic Proteins/metabolism , Copper/metabolism , alpha-Synuclein/metabolism , Amyloidosis/metabolism , Amyloidosis/pathology , Binding Sites , Cell Line , Humans , Kinetics , Parkinson Disease/metabolism , Parkinson Disease/pathology , Protein Aggregation, Pathological/metabolism , Protein Aggregation, Pathological/pathology
4.
Chemistry ; 22(26): 8903-14, 2016 Jun 20.
Article in English | MEDLINE | ID: mdl-27167164

ABSTRACT

Anion recognition studies were performed with triazole-appended thiourea conjugates of calix[6]arene (i.e., compound (6) L) by absorption and (1) H NMR spectroscopy by using nineteen different anions. The composition of the species of recognition was derived from ESI mass spectrometry. The absorption spectra of compound (6) L showed a new band at λ=455 nm in the presence of F(-) due to a charge transfer from the anion to the thiourea moiety and the absorbance increases almost linearly in the concentration range 5 to 200 µm. This is associated with a strong visual color change of the solution. Other anions, such as H2 PO4 (-) and HSO4 (-) , exhibit a redshift of the λ=345 nm band and the spectral changes are associated with the formation of an isosbestic point at λ=343 nm. (1) H NMR studies further confirm the binding of F(-) efficiently to the thiourea group among the halides by shifting the thiourea proton signals downfield followed by their disappearance after the addition of more than one equivalent of F(-) . The other anions also showed interactions with compound (6) L, however, their binding strength follows the order F(-) >CO3 (2-) >H2 PO4 (-) ≈CH3 COO(-) >HSO4 (-) . The NMR spectral changes clearly revealed the anion-binding region of the arms in case of all these anions. The anion binding to compound (6) L indeed stabilizes a flattened-cone conformation as deduced based on the calix-aromatic proton signals and was further confirmed by VT (1) H NMR experiments. The stabilization of the flattened-cone conformation was further augmented by the interaction of the butyl moiety of the nBu4 N(+) counterion. The structural features of the anion-bound species were demonstrated by DFT computations and the resultant structures carried the features that were predicted based on the (1) H NMR spectroscopic measurements. In addition, SEM images showed a marigold flower-type morphology for compound (6) L and this has been transformed into a chain-like structure of connected spherical particles in the presence of F(-) . The anion-induced microstructural features are reflective of the binding strength, size, and shape of the anions. The binding strengths of the anions by compound (6) L were further compared with that of compound (4) L, a calix[4]arene analogue of compound (6) L, in order to address the role of the number of arms built on the calixarene platform based on absorption spectroscopy, (1) H NMR spectroscopy, and DFT computations and it was found that compound (6) L is a better receptor for F(-) , which extends its interactions from all the three arms.

5.
Chemistry ; 22(26): 8980-9, 2016 Jun 20.
Article in English | MEDLINE | ID: mdl-27218408

ABSTRACT

A phenylenediamine-capped conjugate of calix[4]arene (Lamino ) was synthesized by reducing its precursor, Limino , with sodium borohydride in methanol. The Lamino sample binds to anions due to the more flexible and bent conformation of the capped aminophenolic binding core, compared to the precursor Limino . The Lamino sample showed selectivity towards H2 PO4 (-) by exhibiting a ratiometric increase in emission by about 11-fold with a detection limit of (1.2±0.2) µm ((116±20) ppb) over 15 anions studied, including other phosphates, such as P2 O7 (4-) , adenosine monophosphate (AMP(2-) ), adenosine diphosphate (ADP(2-) ), and adenosine triphosphate (ATP(2-) ). The Lamino sample shows an increase in the absorbance at λ=315 nm in the presence of H2 PO4 (-) , CO3 (2-) , HCO3 (-) , CH3 CO2 (-) , and F(-) . The (1) H NMR spectroscopic titration of Lamino with H2 PO4 (-) , F(-) , and CH3 CO2 (-) showed major changes in the phenylene-capped and salicyl moieties, and thereby, confirming the aminophenolic region as the binding core. However, the binding strength of these anions followed the trend H2 PO4 (-) >F(-) ≫CH3 CO2 (-) >HSO4 (-) . The heat changes observed by isothermal titration calorimetry support this trend. The Lamino sample showed reversible sensing towards H2 PO4 (-) and F(-) in the presence of Mg(2+) and Ca(2+) , respectively. NOESY studies of Lamino , in comparison with its anionic complexes, revealed that major conformational changes occurred in the capping region to facilitate the binding of anion. ESI-MS and the Job's method revealed 1:1 stoichiometry between Lamino and H2 PO4 (-) or F(-) . In the SEM micrographs of Lamino , the spherical particles are converted into spherical aggregates and further form large agglomerates and even branched sheets in the presence of anions, depending upon their binding strength.


Subject(s)
Calixarenes/chemistry , Phenols/chemistry , Phenylenediamines/chemistry , Adenosine Diphosphate/chemistry , Adenosine Monophosphate/chemistry , Adenosine Triphosphate/chemistry , Anions/chemistry , Borohydrides/chemistry , Calixarenes/chemical synthesis , Calorimetry , Magnetic Resonance Spectroscopy , Methanol/chemistry , Microscopy, Electron, Scanning , Phenols/chemical synthesis , Phosphates/chemistry , Spectrometry, Fluorescence , Spectrometry, Mass, Electrospray Ionization
6.
Anal Chem ; 87(24): 12396-403, 2015 Dec 15.
Article in English | MEDLINE | ID: mdl-26581844

ABSTRACT

Dansyl-derivatized, triazole-linked, glucopyranosyl conjugates, (5F)LOH, (2F)LOH, (1F)LOH, and (0F)LOH were synthesized and characterized. While the (5F)LOH acts as a molecular probe for CN(-), (2F)LOH, (1F)LOH, and (0F)LOH acts as control molecules. The reactivity of CN(-) toward (5F)LOH has been elicited through the changes observed in NMR, ESI MS, emission, and absorption spectroscopy. The conjugate (5F)LOH releases a fluorescent product upon reaction by CN(-) in aqueous acetonitrile medium by exhibiting an ∼125-fold fluorescence enhancement even in the presence of other anions. Fluorescence switch-on behavior has been clearly demonstrated on the basis of the nucleophilic substitution reaction of CN(-) on (5F)LOH. A minimum detection limit of (2.3 ± 0.3) × 10(-7) M (6 ± 1 ppb) was shown by (5F)LOH for CN(-) in solution. All the other anions studied showed no change in the fluorescence emission. The utility of (5F)LOH has been demonstrated by showing its reactivity toward CN(-) on a thin layer of silica gel as well as on Whatman No. 1 cellulose filter paper strips. The role of glucose moiety and the penta-fluorobenzenesulfonyl reactive center present in (5F)LOH in the selectivity of CN(-) over other anions has been demonstrated by fluorescence, absorption and thermodynamics study. Similar studies carried out with the control molecules showed no selectivity for CN(-). The mechanistic aspects of the reactivity of CN(-) toward (5F)LOH were supported by DFT computational study.


Subject(s)
Benzyl Compounds/chemistry , Cellulose/chemistry , Cyanides/analysis , Electrons , Glycosides/chemistry , HEPES/chemistry , Paper , Silica Gel/chemistry , Buffers , Models, Molecular , Molecular Structure
7.
Anal Chem ; 87(18): 9344-51, 2015 Sep 15.
Article in English | MEDLINE | ID: mdl-26301897

ABSTRACT

A phenylene diimine capped conjugate of 1,3-calix[4]arene (L) was synthesized and characterized, and its Mg(2+) complex has been isolated and characterized. The chemo sensing ensemble of Mg(2+) bound L provides distinguishable features of response toward phosphates, viz., HPO4(2-), P2O7(4-), and AMP(2-) (Set A) and H2PO4(-), ATP(2-), and ADP(2-) (Set B). While the Set A shows the formation of ternary complex, the Set B does not exhibit any intermediate complex, but both show the release of Mg(2+) and L at different equivalents. The structures of {L + Mg(2+)} and its phosphate bound ternary complexes have been established by computational calculations, and the corresponding results agree well with the experimental ones. The microscopy studies show an aggregation-disaggregation phenomenon in the presence of different equivalents of phosphates in both of the sets. Using the fluorescence data, an INHIBIT logic gate has been built.


Subject(s)
Calixarenes/chemistry , Magnesium/chemistry , Microscopy , Models, Molecular , Organometallic Compounds/chemistry , Phenols/chemistry , Phosphates/analysis , Phosphates/chemistry , Molecular Conformation , Spectrometry, Fluorescence
8.
ACS Appl Mater Interfaces ; 7(21): 11555-66, 2015 Jun 03.
Article in English | MEDLINE | ID: mdl-25946024

ABSTRACT

Gels are interesting soft materials owing to their functional properties leading to potential applications. This paper deals with the synthesis of monocholesteryl derivatized calix[4]arene (G) and its instantaneous gelation at a minimum gelator concentration of 0.6% in 1:1 v/v THF/acetonitrile. The gel shows remarkable thermoreversibility by exhibiting Tgel→sol at ∼48 °C and is demonstrated for several cycles. The gel shows an organized network of nanobundles, while that of the sol shows spherical nanoaggregates in microscopy. A bundle with ∼12 nm diameter possessing hydrophobic pockets in itself is obtained from computationally modeled gel, and hence the gel is suitable for storage and release applications. The guest-entrapped gels exhibit the same microstructures as that observed with simple gels, while fluorescence spectra and molecular mechanics suggests that the drug molecules occupy the hydrophobic pockets. All the entrapped drug molecules are released into water, suggesting a complete recovery of the trapped species. The reusability of the gel for the storage and release of the drug into water is demonstrated for four consecutive cycles, and hence the gel formed from G acts as a functional material that finds application in drug delivery.


Subject(s)
Antineoplastic Agents/chemistry , Calixarenes/chemistry , Coloring Agents/chemistry , Gels/chemistry , Nanocapsules/chemistry , Phenols/chemistry , Antineoplastic Agents/administration & dosage , Coloring Agents/administration & dosage , Curcumin/administration & dosage , Curcumin/chemistry , Diffusion , Doxorubicin/administration & dosage , Doxorubicin/chemistry , Materials Testing , Nanocapsules/ultrastructure , Temperature , Tocopherols/administration & dosage , Tocopherols/chemistry
9.
Anal Chem ; 87(9): 4988-95, 2015.
Article in English | MEDLINE | ID: mdl-25867699

ABSTRACT

Lower rim amide linked 8-amino quinoline and 8-amino naphthalene moiety 1,3,5-triderivatives of calix[6]arene L1 and L2 have been synthesized and characterized. While the L1 acts as a receptor molecule, the L2 acts as a control molecule. The complexation between L1 and Cu(2+) or Zn(2+) was delineated by the absorption and electrospray ionization (ESI) MS spectra. The binding ability of these molecules toward biologically important metal ions was studied by fluorescence and absorption spectroscopy. The derivative L1 detects Zn(2+) by bringing ratiometric change in the fluorescence signals at 390 and 490 nm, but in the case of Cu(2+), it is only the fluorescence quenching of 390 nm band that is observed, while no new band is observed at 390 nm. The stoichiometry of both the complexes is 1:1 and was confirmed in both the cases by measuring the ESI mass spectra. The isotopic peak pattern observed in the ESI MS confirmed the presence of Zn(2+) or Cu(2+) present in the corresponding complex formed with L1. Among these two ions, the Cu(2+) exhibits higher sensitivity. The density-functional theory (DFT) studies revealed the conformational changes in the arms and also revealed the coordination features in the case of the metal complexes. The arm conformational changes upon Zn(2+) binding were supported by nuclear Overhauser effect spectrometry (NOESY) studies. The stronger binding of Cu(2+) over that of Zn(2+) observed from the absorption study was further supported by the complexational energies computed from the computational data. While the L1 exhibited spherical particles, upon complexation with Cu(2+), it exhibits chain like morphological features in scanning electron microscopy (SEM) but only small aggregates in the case of Zn(2+). Thus, even the microscopy data can differentiate the complex formed between L1 and Cu(2+) from that formed with Zn(2+).


Subject(s)
Calixarenes/chemistry , Copper/analysis , Phenols/chemistry , Quinolines/chemistry , Zinc/analysis , Binding Sites , Ions/analysis , Macromolecular Substances/chemical synthesis , Macromolecular Substances/chemistry , Models, Molecular , Molecular Structure , Particle Size , Quantum Theory , Spectrometry, Fluorescence , Spectrometry, Mass, Electrospray Ionization , Surface Properties
10.
J Org Chem ; 79(21): 10477-86, 2014 Nov 07.
Article in English | MEDLINE | ID: mdl-25310270

ABSTRACT

Herein we report the synthesis and characterization of 7-oxanorbornadiene (OND)-appended 1,3,5-tris conjugate of calix[6]arene (L2). L2 has been shown to exhibit selective reactivity toward cysteine (Cys) over homocysteine (Hcy) and glutathione (GSH) under stoichiometric conditions. The selectivity of L2 is attributed to the steric crowding of three Diels-Alder centers possessing OND units present on the calix[6]arene platform, while a control molecular system possessing only one such unit without the calix[6]arene platform (L1) does not show any selectivity toward Cys. While L2 exhibited spherical particles, its reactivity with Cys resulted in flowerlike morphological features, as revealed by scanning electron microscopy. However, the reaction with GSH did not result in any such morphological features, a result that is in agreement with that observed from fluorescence studies in solution. L2 has been shown to react with Cys present in HeLa and Jurkat E6 cells by fluorescence microscopy.


Subject(s)
Calixarenes/chemistry , Calixarenes/chemical synthesis , Cysteine/chemistry , Glutathione/chemistry , Homocysteine/chemistry , Norbornanes/chemistry , Norbornanes/chemical synthesis , Phenols/chemistry , Phenols/chemical synthesis , Cycloaddition Reaction , HeLa Cells , Humans , Magnetic Resonance Spectroscopy , Microscopy/methods , Microscopy, Fluorescence/methods
11.
Chemistry ; 20(44): 14378-86, 2014 Oct 27.
Article in English | MEDLINE | ID: mdl-25225120

ABSTRACT

A triazolium-anthracenyl calix[4]arene conjugate (L) was synthesized by methylating the precursor triazole derivative and then characterized. The potential of the cationic L to differentiate nucleoside triphosphates (NTPs) from their mono- and diphosphates was demonstrated. Due to its unique combination of arms with the calix-platform, a fluorescence enhancement was observed for L with all the NTPs, whereas there is no report with such enhancement being exhibited in case of all the NTPs. This has been supported by the aggregation of L observed from microscopy. Selectivity of L towards NTPs over other phosphates was a result of specific weak interactions, namely, ion-ion, hydrogen bonding and π⋅⋅⋅π, present in the 1:2 complex of L and NTPs (based on ESI MS), which were absent in their congener-phosphates as delineated by NMR and computational studies. Thus, L stands as a unique receptor for NTPs.


Subject(s)
Calixarenes/chemistry , Organophosphates/chemistry , Triazoles/chemistry , Triazoles/chemical synthesis , Calixarenes/chemical synthesis , Magnetic Resonance Spectroscopy , Models, Molecular , Organophosphates/chemical synthesis
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