This article presents the first evidence that the DNA base analogue 1,3-diaza-2-oxophenoxazine, tCO, is highly fluorescent, both as free nucleoside and incorporated in an arbitrary DNA structure. tCO is thoroughly characterized with respect to its photophysical properties and structural performance in single- and double-stranded oligonucleotides. The lowest energy absorption band at 360 nm (ε = 9000 M−1 cm−1) is dominated by a single in-plane polarized electronic transition and the fluorescence, centred at 465 nm, has a quantum yield of 0.3. When incorporated into double-stranded DNA, tCO shows only minor variations in fluorescence intensity and lifetime with neighbouring bases, and the average quantum yield is 0.22. These features make tCO, on average, the brightest DNA-incorporated base analogue so far reported. Furthermore, it base pairs exclusively with guanine and causes minimal perturbations to the native structure of DNA. These properties make tCO a promising base analogue that is perfectly suited for e.g. photophysical studies of DNA interacting with macromolecules (proteins) or for determining size and shape of DNA tertiary structures using techniques such as fluorescence anisotropy and fluorescence resonance energy transfer (FRET).

Glutathione deficiency induced in newborn rats by giving buthionine sulfoximine, a selective inhibitor of y-glutamylcysteine synthetase, led to markedly decreased cerebral cortex glutathione levels and striking enlargement and degeneration ofthe mitochondria. These effects were prevented by giving glutathione monoethyl ester, which relieved the glutathione deficiency, but such effects were not prevented by giving glutathione, indicating that glutathione is not appreciably taken up by the cerebral cortex. Some of the oxygen used by mitochondria is known to be converted to hydrogen peroxide. We suggest that in glutathione deficiency, hydrogen peroxide accumulates and damages mitochondria. Glutathione, thus, has an essential function in mitochondria under normal physiological conditions. Observations on turnover and utilization of brain glutathione in newborn, preweaning, and adult rats show that (i) some glutathione turns over rapidly (t ., -30 min in adults, ==8 min in newborns), (ii) several pools of glutathione probably exist, and (iii) brain utilizes plasma glutathione, probably by y-glutamyl transpeptidase-initiated pathways that account for some, but not all, of the turnover; thus, there is recovery or transport of cysteine moieties. These studies provide an animal model for the human diseases involving glutathione deficiency and are relevant to oxidative phenomena that occur in the newborn.Studies in which glutathione (GSH) deficiency was induced in animals by administering L-buthionine (S,R)-sulfoximine (BSO) (1, 2), a transition-state inhibitor of y-glutamylcysteine synthetase (3,4), showed that GSH deficiency leads to myofiber degeneration in skeletal muscle (5), damage to type 2-cell lamellar bodies and capillary endothelial cells in the lung (6), and epithelial-cell damage to jejunum and colon (7) in adult mice, and to lens epithelial-cell degeneration and cataract formation in newborn mice (8, 9) and rats (9). These effects, which were invariably accompanied by markedly decreased mitochondrial GSH levels, were associated with mitochondrial swelling with vacuolization and rupture of cristae and mitochondrial membranes as seen by EM. The isolated mitochondria exhibited decreased citrate synthase activity.It should be emphasized that these effects occurred without application of stress (e.g., increased oxygen, drugs, radiation) and that they were completely prevented by administration of GSH monoesters (10-13). In the absence of evidence that BSO itself exerts a separate type of toxicity other than its effect on the enzyme that catalyzes the first step of GSH synthesis, it may be concluded that a major effect of GSH deficiency is mitochondrial damage. Although mitochondria have long been known to contain GSH, only recently was mitochondrial GSH found to originate from the cytosol and to be imported into mitochondria by a system that contains a high-affinity transporter (14,15). Not all oxygen used by mitochondria is reduced to water, but a significant fraction of it is converted, apparently throu...

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Photoinduced electron transfer in donor-bridge-acceptor systems with zinc porphyrin (or its pyridine complex) as the donor and gold(III) porphyrin as the acceptor has been studied. The porphyrin moieties were covalently linked with geometrically similar bridging chromophores which vary only in electronic structure. Three of the bridges are fully conjugated pi-systems and in a fourth, the conjugation is broken. For systems with this bridge, the quenching rate of the singlet excited state of the donor was independent of solvent and corresponded to the rate of singlet energy transfer expected for a Förster mechanism. In contrast, systems with a pi-conjugated bridging chromophore show a solvent-dependent quenching rate that suggests electron transfer in the Marcus normal region. This is supported by picosecond transient absorption measurements, which showed formation of the zinc porphyrin radical cation only in systems with pi-conjugated bridging chromophores. On the basis of the Marcus and Rehm-Weller equations, an electronic coupling of 5-20 cm(-)(1) between the donor and acceptor is estimated for these systems. The largest coupling is found for the systems with the smallest energy gap between the donor and bridge singlet excited states. This is in good agreement with the coupling calculated with quantum mechanical methods, as is the prediction of an almost zero coupling in the systems with a nonconjugated bridging chromophore.

Glutathione (GSH) deficiency produced in mice by giving buthionine sulfoximine leads to severe degeneration of the epithelial cells of the jejunum and colon. This is prevented by giving GSH monoester (orally or i.p.) and also by giving GSH (orally, but not i.p.). The i.p. administration leads to high plasma levels of GSH but does not appreciably increase GSH levels in intestinal mucosa or pancreas. These and previous studies on lens, lung, lymphocytes, liver, heart, and skeletal muscle indicate that there is very little, if any, transport of intact GSH from plasma to these tissues. Cells can use extracellular GSH by a pathway involving its cleavage, uptake of products and intracellular GSH synthesis. Epithelial cells of the gastrointestinal tract may use this pathway and can also take up lumenal GSH (which arises partly from the bile) by a mechanism(s) that may involve transport of dipeptides or of GSH. It is suggested that biliary GSH normally functions in the protection of intestinal mucosa. Administration of GSH may be protective of the gastrointestinal epithelium and may also serve as a good source of cysteine moieties for intracellular GSH synthesis in the gastrointestinal tract and in other tissues. Administration of GSH delivery agents such as GSH esters is more effective than administration of GSH in increasing cellular and mitochondrial levels of GSH.Administration to experimental animals of L-buthionine-SR-sulfoximine (BSO), a transition-state inhibitor of yglutamylcysteine synthetase, leads to decreased synthesis of glutathione (GSH) in many tissues (1-4). Decreased cellular GSH levels and capacity for GSH synthesis sensitize cells to radiation and to certain drugs; these effects provide a potentially useful approach in cancer therapy (5-12). Administration of BSO offers several advantages over other methods of cellular GSH depletion, one of which is that it makes possible the selective decrease of cellular GSH for relatively long periods, thus facilitating study of the effects of GSH deficiency on cell structure and function. Administration of BSO to mice for 9-21 days, without application of stress, led to marked structural changes in skeletal muscle (13), lung (14), and epithelial cells ofnewborn lens (15); such effects were not found in heart (13) and liver (14). Mitochondrial damage is found in tissues affected by ; the mitochondrial levels of GSH in tissues markedly affected by GSH depletion (lung, muscle, lens) were -20%o of the untreated controls, whereas in heart and liver the mitochondrial GSH levels were >40% of the controls. Mitochondrial and other cellular damage was prevented by simultaneous administration of GSH monoester but not by administration of GSH. GSH monoesters can enter cells effectively and are converted to GSH intracellularly (16)(17)(18)(19). In contrast, GSH is poorly transported into cells.These studies were stimulated by the observation (13) that administration of BSO to 28-to 30-g mice for 9-21 days led to a marked decrease in weight gain and that simultaneous ...

We have studied singlet electronic energy transfer (EET) in two donor-bridge-acceptor series (D-B-A), in which the donor (zinc porphyrin or its pyridine complex) and the acceptor (free base porphyrin) were covalently connected by a geometrically well-defined bridging chromophore. We have investigated how the medium between a donor and an acceptor influences EET by separating the influence of the electronic structure of the bridging chromophore from other effects known to influence the energy transfer. The electronic structure of the bridging chromophore was varied by changing the central unit (bicyclo[2.2.2]octane, benzene, naphthalene, or anthracene) in the bridging chromophore. In all systems the excited state energy separation donor-bridge and bridge-acceptor is large enough to prevent stepwise singlet energy transfer. In addition, the systems were designed to minimize conjugation to preserve the identity of the separate chromophores (donor, bridge, acceptor). Compared with the rate constant expected from the Fo ¨rster theory, the bridging chromophore with bicyclo[2.2.2]octane as the central unit did not significantly enhance the energy transfer rate constant. However, the bridging chromophores with benzene and naphthalene as the central unit showed a moderate increase, whereas the bridging chromophore with anthracene as the central unit showed the largest increase in energy transfer rate constant. This increase is ascribed to a mediating effect of the bridging chromophore and it is proposed to be strongly correlated to the energy splitting between the singlet excited states of donor and bridging chromophores.

Glutathione, an essential cellular antioxidant required for mitochondrial function, is not synthesized by mitochondria but is imported from the cytosol. Rat liver mitochondria have a multicomponent system that underlies the remarkable ability of mitochondria to take up and retain glutathione. At external glutathione levels of <1 mM, glutathione is transported into the mitochondrial anatrix by a high-affinity component (K., -'60 FM-; V ,K, 0.5 nmol/min per mg of protein), which is saturated at levels of 1-2 mM and stimulated by ATP. Another component has lower affinity (Km, -5.4 mM; V,,,,, -5.9 nmol/min per mg of protein) and is stimulated by ATP and ADP. Both components are inhibited by carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), glutamate, and ophthalmic acid. Increase of extramitochondrial glutathione promotes uptake and exchange; the intermembranous space seems to function as a recovery zone that promotes efficient recycling of matrix glutathione. The findings are in accord with in vivo data showing that (i) rapid exchange occurs between mitochondrial and cytosolic glutathione, (ii) lowering of cytosolic glutathione levels (produced by administration of buthionine siilfoximine) decreases export of glutathione from mitochondria to cytosol, and (iii) administration of glutathione esters increases glutathione levels in mitochondria more than those in the cytosol.A small but significant fraction of the oxygen utilized by mitochondria is converted to hydrogen peroxide (1). Much has been written about the toxicity of hydrogen peroxide, superoxide anion, and other reactive oxygen compounds and also about the antioxidant defenses that seem to protect cells against oxygen toxicity (see, for example, refs. 2-5). The suggested "primary defenses" (5) include the activities of such enzymes as superoxide dismutase, catalase, and glutathione (GSH) peroxidases and also smaller molecules such as ascorbate, a-tocopherol, GSH, B-carotene, and uric acid. Superoxide dismutase converts superoxide anion to hydrogen peroxide, which is destroyed in mitochondria (which lack catalase) by GSH peroxidase. GSH is involved in the reduction of dehydroascorbate to ascorbate and also in the maintenance of a-tocopherol in the reduced state. Thus, it appears that GSH plays a crucial role as a cellular antioxidant.In the course of studies in this laboratory on the functions of GSH, we induced GSH deficiency in vivo in mice and rats by administration of buthionine sulfoximine (BSO), a selective and irreversible transition-state inhibitor of y-glutamylcysteine synthetase (4, 6-9), the enzyme that catalyzes the first step of GSH synthesis. GSH deficiency leads to marked structural damage in several tissues, including skeletal muscle (10), lung (11), lens epithelia of newborns (12), and epithelia of the jejunum and colon (13). Cellular damage in each instance is characterized by severe mitochondrial degeneration and very low levels of mitochondrial GSH. Administration of GSH monoester (14-17) eliminated the BSO-induced GSH de...

For many years, neutrophil gelatinase-associated lipocalin (NGAL) has been considered the most promising biomarker of acute kidney injury (AKI). Commercial assays and point-of-care instruments, now available in many hospitals, allow rapid NGAL measurements intended to guide the clinician in the management of patients with or at risk of AKI. However, these assays likely measure a mixture of different NGAL forms originating from different tissues. Systemic inflammation, commonly seen in critically ill patients, and several comorbidities contribute to the release of NGAL from haematopoietic and non-haematopoietic cells. The unpredictable release and complex nature of the molecule and the inability to specifically measure NGAL released by tubular cells have hampered its use a specific marker of AKI in heterogeneous critically ill populations. In this review, we describe the nature and cellular sources of NGAL, its biological role and diagnostic ability in AKI and the increasing concerns surrounding its diagnostic and clinical value.