A macrocyclic zinc(II) fluorophore as a detector of apoptosis.
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Our originally designed dansylamidoethylcyclen 4 as a biomimetic Zn(2+)-selective fluorophore has been demonstrated to be a good detector of the apoptosis (induced by an anticancer agent, etoposide, and H(2)O(2)) in cancer cells such as HeLa and HL60 cells. The macrocyclic Zn(2+) ligand 4 (mostly as a deprotonated form) is cell-permeable to show weak fluorescence (emission at 550 nm), which forms a strong fluorescent 1:1 Zn(2+) complex 5 (emission at 530 nm) when Zn(2+) is incorporated into the cells by a zinc(II) ionophore pyrithione. Thus formed, Zn(2+) complex 5 is cell-impermeable and remains intact over a few hours. When apoptosis in HeLa or HL60 cells is artificially induced, 4 selectively and strongly stains apoptotic cells only at early stages, which was verified by using the conventional apoptosis detection probe annexin V-Cy3. Detection of the apoptotic cells by 4 was perhaps due to significantly increased free Zn(2+) flux at early stages of apoptosis. Apoptotic detection by 4 has been compared with a presently available Zn(2+) fluorophore, Zinquin 1. We present that 4 has advantages in detection of apoptosis over annexin V-Cy3 and Zinquin 1. (+info)
Water-soluble quantum dots for multiphoton fluorescence imaging in vivo.
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The use of semiconductor nanocrystals (quantum dots) as fluorescent labels for multiphoton microscopy enables multicolor imaging in demanding biological environments such as living tissue. We characterized water-soluble cadmium selenide-zinc sulfide quantum dots for multiphoton imaging in live animals. These fluorescent probes have two-photon action cross sections as high as 47,000 Goeppert-Mayer units, by far the largest of any label used in multiphoton microscopy. We visualized quantum dots dynamically through the skin of living mice, in capillaries hundreds of micrometers deep. We found no evidence of blinking (fluorescence intermittency) in solution on nanosecond to millisecond time scales. (+info)
Viral assembly of oriented quantum dot nanowires.
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The highly organized structure of M13 bacteriophage was used as an evolved biological template for the nucleation and orientation of semiconductor nanowires. To create this organized template, peptides were selected by using a pIII phage display library for their ability to nucleate ZnS or CdS nanocrystals. The successful peptides were expressed as pVIII fusion proteins into the crystalline capsid of the virus. The engineered viruses were exposed to semiconductor precursor solutions, and the resultant nanocrystals that were templated along the viruses to form nanowires were extensively characterized by using high-resolution analytical electron microscopy and photoluminescence. ZnS nanocrystals were well crystallized on the viral capsid in a hexagonal wurtzite or a cubic zinc blende structure, depending on the peptide expressed on the viral capsid. Electron diffraction patterns showed single-crystal type behavior from a polynanocrystalline area of the nanowire formed, suggesting that the nanocrystals on the virus were preferentially oriented with their [001] perpendicular to the viral surface. Peptides that specifically directed CdS nanocrystal growth were also engineered into the viral capsid to create wurtzite CdS virus-based nanowires. Lastly, heterostructured nucleation was achieved with a dual-peptide virus engineered to express two distinct peptides within the same viral capsid. This work represents a genetically controlled biological synthesis route to a semiconductor nanoscale heterostructure. (+info)
Inhibitory effects of green tea catechins on the activity of human matrix metalloproteinase 7 (matrilysin).
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Inhibitory effects of green tea catechins and their derivatives on the matrilysin-catalyzed hydrolysis of a synthetic substrate, (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L- 2,3-diamino-propionyl]-L-Ala-L-Arg-NH(2) [MOCAc-PLGL(Dpa)AR], were examined. The 10 catechins examined were classified into three groups according to their inhibition potency. Catechins with a galloyl group at the 3 position, including a major component of green tea catechin, (-)-epigallo-3-catechin gallate [(-)-EGCG], were the most potent inhibitors and inhibited matrilysin in a non-competitive manner with K(i) values of 0.47-1.65 micro M. The inhibitory potency of (-)-EGCG was not influenced by the presence of an inhibitor, ZnCl(2), suggesting that the inhibitions of matrilysin by (-)-EGCG and by ZnCl(2) might be independent of each other. The inhibitory effects of green tea catechins suggest that a high intake of green tea might be effective for the prevention of tumor metastasis and invasion in which matrilysin is concerned. (+info)
The ERK pathway involves positive and negative regulations of HT-29 colorectal cancer cell growth by extracellular zinc.
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Dietary zinc is an important trace element in the body and is related to both cell proliferation and growth arrest. A recent study found that extracellular zinc-sensing receptors trigger intracellular signal transduction in HT-29 human colorectal cancer cells. However, the signaling mechanism causing this growth regulation by extracellular zinc is not clearly understood. At 10- and 100-microM levels of ZnCl2 treatment, HT-29 cell growth and proliferation increased and decreased, respectively, in a minimally serum-starved medium (MSSM). A lack of significant increase in intracellular zinc levels after zinc treatment suggested that this differential growth regulation of HT-29 cells by extracellular zinc is acquired by receptor-mediated signal transduction. Moreover, this zinc-induced growth regulation was differentially affected by PD-98059, suggesting the involvement of the ERK pathway. Transient ERK activation and subsequent cyclin D1 induction were observed on adding 10 microM ZnCl2 in MSSM in the presence of cell proliferation. On the other hand, prolonged ERK activity was observed with a subsequent increase of cyclin D1 and p21(Cip/WAF1) on adding 100 microM ZnCl2 in MSSM, and this was associated with nonproliferation. Moreover, this ERK activation and cyclin D1 and p21(Cip/WAF1) induction were abolished by PD-98059 pretreatment. The differential regulations of cell growth, ERK activities, and cyclin D1 and p21(Cip/WAF1) inductions were also observed in serum-enriched medium containing higher zinc concentrations. Therefore, differential cell cycle regulator induction occurs by a common ERK pathway in the differential growth regulation of HT-29 cells by extracellular zinc. (+info)
Injury induced by chemical warfare agents: characterization and treatment of ocular tissues exposed to nitrogen mustard.
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PURPOSE: Mustard agents are highly toxic and abundant warfare chemicals, primarily affecting ocular tissues, with no specific treatment antidote. The purpose of the present study was to examine the efficacy of novel metallocomplexes, known to inhibit the formation of highly reactive free radicals, to reduce ocular injury induced by nitrogen mustard (NM). METHODS: One eye in each of 72 rabbits was exposed to 1% to 2% NM. Topical treatment with eye drops of a metallocomplex--either zinc- or gallium-desferrioxamine (Zn/DFO and Ga/DFO)--was compared with treatment with saline, zinc (chloride), or DFO alone. Examiners masked to the treatment groups assessed the extent of ocular injury and the response to treatment using clinical, histologic, and biochemical criteria. RESULTS: Exposure to NM followed by administration of carrier alone (saline) caused severe and long-lasting injury to ocular anterior segment structures. Treatment with either Zn/DFO or Ga/DFO yielded marked protection (52%-64%), including faster healing of corneal epithelial erosions, less scarring and neovascularization, decreased inflammation in the anterior chamber, better maintenance of intraocular pressure, and less severe changes in the iris and lens. These were also associated with better preservation of systemic antioxidant status. Zinc or DFO alone afforded lower levels of protection. No toxic effects of these complexes were observed. CONCLUSIONS: It is suggested that Zn/DFO or Ga/DFO, by virtue of their enhanced ability to infiltrate cells and inhibit transition metal-dependent formation of free radicals through the combined push-pull mechanism, be considered as a basis for treatment of mustard injuries. (+info)
Evidence for an intracellular ADP-ribosyl cyclase/NAD+-glycohydrolase in brain from CD38-deficient mice.
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Cyclic ADP-ribose, a metabolite of NAD+, is known to modulate intracellular calcium levels and signaling in various cell types, including neural cells. The enzymes responsible for producing cyclic ADP-ribose in the cytoplasm of mammalian cells remain unknown; however, two mammalian enzymes that are capable of producing cyclic ADP-ribose extracellularly have been identified, CD38 and CD157. The present study investigated whether an ADP-ribosyl cyclase/NAD+-glycohydrolase independent of CD38 is present in brain tissue. To address this question, NAD+ metabolizing activities were accurately examined in developing and adult Cd38-/- mouse brain protein extracts and cells. Low ADP-ribosyl cyclase and NAD+-glycohydrolase activities (in the range of pmol of product formed/mg of protein/min) were detected in Cd38-/- brain at all developmental stages studied. Both activities were found to be associated with cell membranes. The activities were significantly higher in Triton X-100-treated neural cells compared with intact cells, suggesting an intracellular location of the novel cyclase. The cyclase and glycohydrolase activities were optimal at pH 6.0 and were inhibited by zinc, properties which are distinct from those of CD157. Both activities were enhanced by guanosine 5'-O-(3-thiotriphosphate), a result suggesting that the novel enzyme may be regulated by a G protein-dependent mechanism. Altogether our results indicate the presence of an intracellular membrane-bound ADP-ribosyl cyclase/NAD+-glycohydrolase distinct from CD38 and from CD157 in mouse brain. This novel enzyme, which is more active in the developing brain than in the adult tissue, may play an important role in cyclic ADP-ribose-mediated calcium signaling during brain development as well as in adult tissue. (+info)
Insulinomimetic zinc(II) complexes with natural products: in vitro evaluation and blood glucose lowering effect in KK-Ay mice with type 2 diabetes mellitus.
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In vitro insulinomimetic activities of Zn(II) complexes with three natural products, betaine, L-lactic acid, and D-(-)-quinic acid (qui), were found in rat adipocytes treated with epinephrine in terms of the inhibition of free fatty acid release. Based on the results, the blood glucose lowering effect in KK-A(y) mice with type 2 diabetes mellitus was observed by daily i.p. injections of a monomeric zinc(II) complex, Zn(qui)(2), for 13 d. (+info)