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Jankowska K.I.,Rutgers University | Pagba C.V.,Rutgers University | Pagba C.V.,Center for Biophotonics | Piatnitski Chekler E.L.,Pfizer | And 2 more authors.
Journal of the American Chemical Society | Year: 2010

A water-soluble octacarboxyhemicarcerand was used as a shuttle to transport redox-active substrates across the aqueous medium and deliver them to the target protein. The results show that weak multivalent interactions and conformational flexibility can be exploited to reversibly bind complex supramolecular assemblies to biological molecules. Hydrophobic electron donors and acceptors were encapsulated within the hemicarcerand, and photoinduced electron transfer (ET) between the Zn-substituted cytochrome c (MW = 12.3 kD) and the host-guest complexes (MW = 2.2 kD) was used to probe the association between the negatively charged hemicarceplex and the positively charged protein. The behavior of the resulting ternary protein-hemicarcerand-guest assembly was investigated in two binding limits: (1) when Kencaps ≫ K assoc, the hemicarcerand transports the ligand to the protein while protecting it from the aqueous medium; and (2) when Kassoc > Kencaps, the hemicarcerand-protein complex is formed first, and the hemicarcerand acts as an artificial receptor site that intercepts ligands from solution and positions them close to the active site of the metalloenzyme. In both cases, ET mediated by the protein-bound hemicarcerand is much faster than that due to diffusional encounters with the respective free donor or acceptor in solution. The measured ET rates suggest that the dominant binding region of the host-guest complex on the surface of the protein is consistent with the docking area of the native redox partner of cytochrome c. The strong association with the protein is attributed to the flexible conformation and adaptable charge distribution of the hemicarcerand, which allow for surface-matching with the cytochrome. © 2010 American Chemical Society. Source


Millington O.R.,Center for Biophotonics | Millington O.R.,University of Strathclyde | Myburgh E.,University of Glasgow | Mottram J.C.,University of Glasgow | Alexander J.,University of Strathclyde
Experimental Parasitology | Year: 2010

An understanding of host-parasite interplay is essential for the development of therapeutics and vaccines. Immunoparasitologists have learned a great deal from 'conventional' in vitro and in vivo approaches, but recent developments in imaging technologies have provided us (immunologists and parasitologists) with the ability to ask new and exciting questions about the dynamic nature of the parasite-immune system interface. These studies are providing us with new insights into the mechanisms involved in the initiation of a Leishmania infection and the consequent induction and regulation of the immune response. Here, we review some of the recent developments and discuss how these observations can be further developed to understand the immunology of cutaneous Leishmania infection in vivo. © 2010 Elsevier Inc. Source


Awasthi S.,Center for Biophotonics | Izu L.T.,Biomedical Engineering | Mao Z.,Center for Biophotonics | Jian Z.,Biomedical Engineering | And 14 more authors.
Circulation Research | Year: 2016

Rationale: Cardiac myocyte contraction is caused by Ca2+ binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca2+ release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca2+ release, such as Ca2+ sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca2+-contraction coupling in live cardiac myocytes. Objective: To develop a method for imaging sarcomere level Ca2+-contraction coupling in healthy and disease model cardiac myocytes. Methods and Results: Freshly isolated cardiac myocytes were loaded with the Ca2+-indicator fluo-4. A confocal microscope equipped with a femtosecond-pulsed near-infrared laser was used to simultaneously excite second harmonic generation from A-bands of myofibrils and 2-photon fluorescence from fluo-4. Ca2+ signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca2+ sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca2+ activity and sarcomere strain were also imaged in paced cardiac myocytes under mechanical load, revealing spontaneous Ca2+ waves and correlated local contraction in pressure-overload-induced cardiomyopathy. Conclusions: Multimodal second harmonic generation 2-photon fluorescence microscopy enables the simultaneous observation of Ca2+ release and mechanical strain at the subsarcomere level in living cardiac myocytes. The method benefits from the label-free nature of second harmonic generation, which allows A-bands to be imaged independently of T-tubule morphology and simultaneously with Ca2+ indicators. Second harmonic generation 2-photon fluorescence imaging is widely applicable to the study of Ca2+-contraction coupling and mechanochemotransduction in both health and disease. © 2016 American Heart Association, Inc. Source

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