Stephen F. Lincoln

Video image analysis of labile zinc in viable pancreatic islet cells using a specific fluorescent probe for zinc.

We used an intracellular zinc-specific fluorophore, Zinquin, in conjunction with fluorescence video image analysis, to reveal labile zinc in pancreatic islet cells, which concentrate this metal for use in synthesis, storage, and secretion of insulin. Zinquin vividly demonstrated zinc in the islet cell secretory granules, which formed a brightly labeled crescent in the cytoplasm between one side of the nucleus and the plasma membrane. Lower but still appreciable amounts of zinc were detected in the remaining cytoplasm, but there was little labeling in the nucleus. Fluorescence intensity varied among islet cells, suggesting differences in zinc content. Their average fluorescence intensity greatly surpassed that of the surrounding pancreatic acinar cells in frozen sections of pancreas and in all other types of cell studied, including lymphocytes, neutrophils, fibroblasts, and erythrocytes. Less labile zinc was detected in cells of the mouse insulinoma cell line NIT-1, regardless of whether they were maintained in long-term culture in the presence or absence of exogenous extracellular zinc. Exposure of islet or insulinoma cells to a high concentration of glucose or other secretagogue decreased the content of labile zinc. Zinquin should be a useful probe for revealing changes in zinc homeostasis in islet B-cells that may be important in their dysfunction and death during diabetes.

Modified cyclodextrins : scaffolds and templates for supramolecular chemistry

Cyclodextrin structure and complexation behaviour strategies for synthesis intramolecular complexation, chiral discrimination and photochemical devices cyclodextrin catalysts and enzyme mimics metallocyclodextrins multisite complexation of guests and linked cyclodextrins cyclodextrin rotaxanes and catenanes surface and interface behaviour.

Inclusion Complexes of the Cyclomalto-Oligosaccharides (Cyclodextrins)

Publisher Summary This chapter provides an overview of the inclusion complexes of cyclomalto-oligosaccharides (cyclodextrins). Inclusion complexes are chemical species consisting of two or more associated molecules in which one of the molecules—the “host”—forms or possesses a cavity into which it can admit a “guest” molecule, resulting in a stable association without formation of any covalent bonds. Secondary forces are alone responsible for the maintenance of the integrity of all inclusion complexes. One of the most important properties of the cyclodextrins is their ability to form complexes with a variety of organic and inorganic compounds. 1 H-Nuclear magnetic resonance (NMR) spectroscopy provided the first direct evidence of inclusion within the cyclodextrin cavity in solution. Using aromatic “guest” molecules, it was found that, on the addition of the guest, the resonances of the hydrogen atoms of the cyclodextrin situated on the inside of the cavity were shifted significantly upfield due to shielding by the aromatic guest. This chapter discusses determination of the structure of the cyclodextrins, and formation of the cyclodextrins from starch. It provides details about formation of inclusion complexes, detection of complex formation, and thermodynamics of complex formation. Kinetics of complex formation is also explained in the chapter.

Flux of intracellular labile zinc during apoptosis (gene-directed cell death) revealed by a specific chemical probe, Zinquin

BACKGROUND The transition metal Zn(II) is thought to regulate cell and tissue growth by enhancing mitosis (cell proliferation) and suppressing the counterbalancing process of apoptosis (gene-directed cell death). To investigate the role of Zn(II) further, we have used a UV-excitable Zn(II)-specific fluorophore, Zinquin. The ester group of Zinquin is hydrolyzed by living cells, ensuring its intracellular retention; this allows the visualization and measurement of free or loosely-bound (labile) intracellular Zn(II) by fluorescence video image analysis or fluorimetric spectroscopy. RESULTS Here we show that in cells undergoing early events of apoptosis, induced spontaneously or by diverse agents, there is a substantial increase in their Zinquin-detectable Zn(II). This increase occurred in the absence of exogenous Zn(II) and before changes in membrane permeability, consistent with a release of Zn(II) from intracellular stores or metalloproteins rather than enhanced uptake from the medium. We propose that there is a major redistribution of Zn(II) during the induction of apoptosis, which may influence or precipitate some of the later biochemical and morphological changes. CONCLUSIONS The phenomenon of Zn(II) mobilization, revealed by Zinquin, presents a new element in the process of apoptosis for investigation and may permit rapid and sensitive identification of apoptotic cells, particularly in those tissues where their frequency is low.

The foundation of a light driven molecular muscle based on stilbene and α-cyclodextrin

The rotaxane 3(E,E) serves as the basis of a light driven molecular muscle, where reversible photoisomerisation of the stilbene units causes the cyclodextrins to move off and on the stilbene units, contracting and extending the distance between the blocking groups.

Substituent effects and chiral discrimination in the complexation of benzoic, 4-methylbenzoic and (RS)-2-phenylpropanoic acids and their conjugate bases by β-cyclodextrin and 6A-amino-6A-deoxy-β-cyclodextrin in aqueous solution: potentiometric titration and 1H nuclear magnetic resonance spectroscopic study

A potentiometric titration study in aqueous solution (l= 0.10 mol dm–3, KCl) of the complexation of benzoic, 4-methylbenzoic and (RS)–2-phenylpropanoic acids (HA) and their conjugate bases (A–) with β-cyclodextrin, βCD, and its substituted analogue, 6A-amino-6A-deoxy-β-cyclodextrin, βCDNH2, in which a primary hydroxy group is replaced by an amino group which may be protonated to produce a singly charged species, βCDNH+3, is reported. At 298.2 K the stability constants for the complexes have the values (in dm3 mol–1) shown in parentheses: benzoic acid ·βCD (K1HA= 590 ± 60); benzoate ·βCD (K1A= 60 ± 10); benzoic acid ·βCDNH+3(K2HA= 340 ± 30); benzoate ·βCDNH+3(K2A= 120 ± 20); benzoate ·βCDNH2(K3A= 50 ± 20); 4-methylbenzoic acid ·βCD (K1HA= 1680 ± 90); 4-methylbenzoate ·βCD (K1A= 110 ± 1); 4-methylbenzoic acid ·βCDNH+3(K2HA= 910 ± 20); 4-methylbenzoate ·βCDNH+3(K2A= 330 ± 20); and 4-methylbenzoate ·βCDNH2(K3A= 100 ± 20). These data indicate that for a given cyclodextrin the guest carboxylic acids form complexes of higher stability than do their conjugate base analogues, and that βCDNH+3 forms more stable complexes with the conjugate bases than do βCD and βCDNH2. These trends are also observed for the complexation of (RS)-2-phenylpropanoic acid and (RS)-2-phenylpropanoate where the complexes indicated are characterised by the stability constants (in dm3 mol–1) shown in parentheses: (RS)-2-phenylpropanoic acid ·βCD (K1RHA= 1090 ± 30, K1SHA= 1010 ± 40); (RS)-2-phenylpropanoate ·βCD (K1RA= 63 ± 8, K1SA= 52 ± 5); (RS)-2-phenylpropanoic acid ·βCDNH+3(K2RHA= 580 ± 20, K2SHA= 480 ± 10); (RS)-2-phenylpropanoate ·βCDNH+3(K2RA= 150 ± 8, K2SA= 110 ± 10); and (RS)-2-phenylpropanoate ·βCDNH2(K3RA= 36 ± 6, K3SA= 13 ± 7). These data also show that while K1RHA and K1SHA, and K1RA and K1SA are indistinguishable for (RS)-2-phenylpropanoic acid ·βCD and (RS)-2-phenylpropanoate ·βCD, chiral discrimination is indicated by K2RHA > K2SHA for (RS)-2-phenylpropanoic acid ·βCDNH+3, K2RA > K2SA for (RS)-2-phenylpropanoate ·βCDNH+3, and K3RA > K3SA for (RS)-2-phenylpropanoate ·βCDNH2. The 1H NMR spectra of the methyl groups of the enantiomers of (RS)-2-phenylpropanoic acid appear as two separate doublets, indicating chiral discrimination when complexed by βCD or βCDNH+3, but such chiral discrimination is not observed for (RS)-2-phenylpropanoate when complexed by βCDNH+3. The implications of these observations are discussed.

MECHANISTIC STUDIES OF SOME MACROCYCLIC COMPLEXES

Abstract A review of recent research carried out at Adelaide in three areas of macrocyche complex chemistry: (i) coronates, cryptates and related metal complexes: (ii) metal complexes of pendant arm macrocyclic ligands: and (iii) metallocyclodextrins is presented, and the relationship between complex stability, lability and structure is explored

Changes in distribution of labile zinc in mouse spermatozoa during maturation in the epididymis assessed by the fluorophore Zinquin

The Zn(II)-specific fluorophore Zinquin was used to determine the regional distribution of free or loosely-bound Zn(II) in mouse spermatozoa. Spermatozoa from the testes exhibited bright fluorescence over the entire head; those from the caput epididymides generally fluoresced more brightly in the post-acrosomal region; and spermatozoa from the caudae epididymides fluoresced less brightly, with foci of fluorescence over the sperm head which were lost after extraction with Triton X-100 and hence appeared to be membrane-associated. Treatment of cauda sperm with sodium dodecyl sulfate resulted in a bright uniform Zinquin fluorescence in the heads, similar to that observed in caput sperm, indicating that the two types of sperm have similar amounts of head Zn(II) but that the availability of Zn(II) for binding Zinquin is different. By contrast, the intensity of tail fluorescence was similar in spermatozoa from different regions of the male reproductive tract and was largely unaffected by Triton X-100 extraction, consistent with an intracellular location. Similar differences were observed between caput sperm and cauda sperm in the rat. It is concluded that visualization and measurement of free or loosely-bound Zn(II) in subcellular compartments of spermatozoa should facilitate investigation of the role of this metal in the development and function of spermatozoa and abnormalities that might accompany infertility and Zn(II) deficiency.

Cooperative binding and stabilization of the medicinal pigment curcumin by diamide linked γ-cyclodextrin dimers: a spectroscopic characterization

Diamide linked γ-cyclodextrin (γ-CD) dimers are used to capture curcumin and suppress its decomposition in water. In this study, succinamide and urea linked γ-CD dimers joined through the C6(A) carbon on each γ-CD are used. The γ-CD dimers, 66γCD(2)su and 66γCD(2)ur, show a remarkable ability to suppress the decomposition of curcumin and extend its half-life from less than 30 min to greater than 16 h. The 1:1 association of curcumin with 66γCD(2)su and 66γCD(2)ur has high stability constants of 8.7 × 10(6) M(-1) and 2.0 × 10(6) M(-1), respectively. In addition, 2D (1)H NOESY NMR results show specific hydrogen interactions in the association of curcumin with 66γCD(2)su and 66γCD(2)ur, consistent with the cooperative binding of curcumin by both γ-CD annuli of 66γCD(2)su and 66γCD(2)ur. The interactions between curcumin in the linked γ-CD dimers and surfactant micelles were studied using fluorescence spectroscopy. While linked γ-CD dimer-bound curcumin has a negligible fluorescence quantum yield, a significant increase in fluorescence intensity (Φ(fl) > 2%) in the presence of micelles suggests that curcumin is delivered to the micelle. The overall results indicate that the diamide linked γ-CD dimers are highly promising systems for curcumin delivery in vivo due to effective curcumin stabilization.

Use of a zinc fluorophore to measure labile pools of zinc in body fluids and cell-conditioned media

Here we describe a rapid and sensitive zinquin-based fluorometric assay that enables one to monitor levels of labile Zn(II) in body fluids, buffers, and cell-conditioned culture media as well as changes in these pools in disease. Labile pools of Zn(II) are free or loosely bound pools and more tightly bound but zinquin-accessible pools in contrast to the fixed pools of Zn(II) within metalloproteins. In human plasma, mean labile Zn(II) was 8.1 microM (SEM 0.53; n = 81) and constituted about 70% of the total plasma Zn(II) and >90% of human plasma albumin Zn(II). Plasma labile Zn(II) was significantly depleted after 7 days of Zn(II) deprivation in mice, despite only small changes in body weight. Labile Zn(II) concentrations were also measured in the induced sputum plugs, saliva, and urine of normal adults and were 1.30 microM (SEM 0.27; n = 73), 0.11 microM (SEM 0.11; n = 6), and 0.23 microM (SEM 0.08; n = 8), respectively. Urinary labile Zn(II) concentration was significantly increased in some patients with type II diabetes mellitus (overall mean was 0.90 microM, SEM 0.30; n = 12). The technique may be particularly useful in assessing extracellular Zn(II) levels in diseases associated with altered Zn(II) homeostasis, identifying those subjects most in need of Zn(II) supplementation, and defining the optimum concentrations of available Zn(II) in buffers and culture media.