Biliverdin reductase A in the prevention of cellular senescence against oxidative stress

Biliverdin reductase A (BLVRA), an enzyme that converts biliverdin to bilirubin, has recently emerged as a key regulator of the cellular redox cycle. However, the role of BLVRA in the aging process remains unclear. To study the role of BLVRA in the aging process, we compared the stress responses of young and senescent human diploid fibroblasts (HDFs) to the reactive oxygen species (ROS) inducer, hydrogen peroxide (H2O2). H2O2 markedly induced BLVRA activity in young HDFs, but not in senescent HDFs. Additionally, depletion of BLVRA reduced the H2O2-dependent induction of heme oxygenase-1 (HO-1) in young HDFs, but not in senescent cells, suggesting an aging-dependent differential modulation of responses to oxidative stress. The role of BLVRA in the regulation of cellular senescence was confirmed when lentiviral RNAi- transfected stable primary HDFs with reduced BLVRA expression showed upregulation of the CDK inhibitor family members p16, p53, and p21, followed by cell cycle arrest in G0-G1 phase with high expression of senescence-associated β-galactosidase. Taken together, these data support the notion that BLVRA contributes significantly to modulation of the aging process by adjusting the cellular oxidative status.     

Two-photon fluorescence microscopy imaging of cellular oxidative stress using profluorescent nitroxides

A range of varying chromophore nitroxide free radicals and their nonradical methoxyamine analogues were synthesized and their linear photophysical properties examined. The presence of the proximate free radical masks the chromophore's usual fluorescence emission, and these species are described as profluorescent. Two nitroxides incorporating anthracene and fluorescein chromophores (compounds 7 and 19, respectively) exhibited two-photon absorption (2PA) cross sections of approximately 400 G.M. when excited at wavelengths greater than 800 nm. Both of these profluorescent nitroxides demonstrated low cytotoxicity toward Chinese hamster ovary (CHO) cells. Imaging colocalization experiments with the commercially available CellROX Deep Red oxidative stress monitor demonstrated good cellular uptake of the nitroxide probes. Sensitivity of the nitroxide probes to H(2)O(2)-induced damage was also demonstrated by both one- and two-photon fluorescence microscopy. These profluorescent nitroxide probes are potentially powerful tools for imaging oxidative stress in biological systems, and they essentially "light up" in the presence of certain species generated from oxidative stress. The high ratio of the fluorescence quantum yield between the profluorescent nitroxide species and their nonradical adducts provides the sensitivity required for measuring a range of cellular redox environments. Furthermore, their reasonable 2PA cross sections provide for the option of using two-photon fluorescence microscopy, which circumvents commonly encountered disadvantages associated with one-photon imaging such as photobleaching and poor tissue penetration.     

"Click" synthesis of small molecule-peptide conjugates for organelle-specific delivery and inhibition of lysosomal cysteine proteases

A click chemistry approach for the synthesis of small molecule inhibitor-peptide conjugates to achieve organelle-specific delivery has been developed. Biological testing showed that the inhibitor-Tat conjugate was successfully delivered to the lysosomes, leading to potent inhibition of lysosomal cysteine proteases in cultured cells.     

Analysis of the bacterial heat shock response to photodynamic therapy-mediated oxidative stress

Antimicrobial photodynamic therapy (PDT) has recently emerged as an effective modality for the selective destruction of bacteria and other pathogenic microorganisms. We investigated whether PDT induced protective responses such as heat shock proteins (HSPs) in bacteria. Using the photosensitizer Toluidine Blue O (TBO) at sublethal PDT conditions, a seven-fold increase in bacterial HSP GroEL and a three-fold increase in HSP DnaK were observed in Escherichia coli post PDT. Pretreatment with 50°C heat for 30 min reduced PDT killing in both E. coli and in Enterococcus faecalis, with the most pronounced inhibition occurring at 50 μm TBO with 5 J cm(-2) 635 nm light, where E. coli killing was reduced by 2 log(10) and E. faecalis killing was reduced by 4 log(10). Finally, inhibition of the highly conserved chaperone DnaK using a small molecule benzylidene lactam HSP inhibitor potentiated (but not significantly) the effect of PDT at a TBO concentration of 2.5 μm in E. faecalis; however, this effect was not observed in E. coli presumably because inhibitor could not gain access due to Gram-negative permeability barrier. Induction of HSPs may be a mechanism whereby bacteria could become resistant to PDT and warrants the need for further study in the application of dual PDT-HSP-inhibition therapies.     

Adaptation of cyanobacteria to UV-B stress correlated with oxidative stress and oxidative damage

Cyanobacteria must cope with the negative effects of ultraviolet B (280-315 nm) (UV-B) stress caused by their obligatory light requirement for photosynthesis. The adaptation of the cyanobacterium Anabaena sp. to moderate UV-B radiation has been observed after 2 weeks of irradiation, as indicated by decreased oxidative stress, decreased damage, recovered photosynthetic efficiency and increased survival. Oxidative stress in the form of UV-B-induced production of reactive oxygen species was measured in vivo with the oxidative stress-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate. Photooxidative damage by UV-B radiation, including lipid peroxidation and DNA strand breakage, was determined by a modified method using thiobarbituric acid reactive substances and fluorometric analysis of DNA unwinding. Photosynthetic quantum yield was determined by pulse amplitude-modulated fluorometry. The results suggest that moderate UV-B radiation results in an evident oxidative stress, enhanced lipid peroxidation, increased DNA strand breaks, elevated chlorophyll bleaching as well as decreased photosynthetic efficiency and survival during the initial exposure. However, DNA strand breaks, photosynthetic parameters and chlorophyll bleaching returned to their unirradiated levels after 4-7 days of irradiation. Oxidative stress and lipid peroxidation appeared to respond later because decreases were observed after 7 days of radiation. The survival curve against irradiation time exhibited a close relationship with the changes in photosynthetic quantum yield and DNA damage, with little mortality after 4 days. Growth inhibition by UV-B radiation was observed during the first 7 days of radiation, whereas normal growth resumed even under UV-B stress thereafter. An efficient defense system was assumed to come into play to repair photosynthetic and DNA damage and induce the de novo synthesis of UV-sensitive proteins and lipids, allowing the organisms to adapt to UV-B stress successfully and survive as well as grow. No induction of mycosporine-like amino acids (MAA) was observed during the adaptation of Anabaena sp. to UV-B stress in our work. The adaptation of the cyanobacterium correlated with and could be caused by the oxidative stress and oxidative damage.     

A new fluorescent probe with ultralow background fluorescence for imaging of endogenous cellular selenol under oxidative stress

A new fluorescent probe (Rhod-Sec) for selenol detection with ultralow background fluorescence have been developed in this paper, which showed a 380-fold off-on fluorescence response, and can be applied to visualize the fluctuation of selenol in HepG2 cells through LPS-induced cells oxidation resistance.     

Site-specific oxidative cleavage of DNA by metallosalen-DNA conjugates

Ni-salen-DNA conjugates, prepared by template-directed synthesis, targeted oxidative adduct formation and strand scission at deoxyguanosine sites in complementary DNA strands of Watson-Crick duplexes.     

Peptide quinoline conjugates: a new class of RNA-binding molecules

[reaction: see text] A synthesis of 4,8-disubstituted 2-phenylquinoline amino acids is reported with the incorporation of one example into a peptide by solid-phase synthesis. The phenylquinoline-containing peptide binds an RNA target with nanomolar affinity (K(D) = 208 nM). The strategy can be used to prepare a variety of 2-substituted quinoline amino acids for alteration of affinity in intercalator peptides. Since quinolones represent an important class of antibacterials, these compounds may be useful in the discovery of new antibacterial agents.     

Synthesis, aggregation and cellular investigations of porphyrin-cobaltacarborane conjugates

A new series of porphyrin-cobaltacarborane conjugates (1-5) that contain four to sixteen carborane clusters per porphyrin macrocycle, were prepared in excellent yields (90-97 %) by means of a ring-opening reaction of the zwitterionic cobaltacarborane [3,3'-Co(8-C(4)H(8)O(2-)-1,2-C(2)B(9)H(10))(1',2'-C(2)B(9)H(11))]. The X-ray structure of one conjugate (3) is presented. The aggregation properties of these conjugates were investigated by using absorption and fluorescence spectrophotometry, and the stages of microcrystal formation were captured by using atomic force microscopy. All conjugates were found to aggregate in aqueous solutions, to form a broad dispersity of particle sizes. The cellular uptake, cytotoxicity, and preferential sites of subcellular localization of this series of conjugates were evaluated in human carcinoma HEp2 cells. The extent of conjugate cellular uptake depends on the number of cobaltacarborane units at the porphyrin periphery, their distribution, and the conjugate aggregation behavior. Conjugates 2 and 4, bearing either two adjacent or three 3,5-dicobaltacarboranephenyl groups, accumulated the most within HEp2 cells and are, therefore, the most promising boron neutron capture therapy agents. All conjugates showed very low dark- and photo-toxicity, probably due to their strong tendency for aggregation in aqueous solutions, and localized subcellularly within vesicles that correlated, to some extent, with the cell lysosomes.     

Synthesis and cellular uptake of cell delivering PNA-peptide conjugates

The synthesis and cellular uptake of fluorescently labelled PNA-peptide conjugates is described; Dde/Mmt protected PNA monomers, fully orthogonal to Fmoc chemistry, were used to develop a flexible strategy to give Peptide Nucleic Acids conjugated to tri- and hepta-arginine and the short basic Tat(48-57) peptide as examples of cellular penetrating peptides, thereby allowing efficient cellular delivery of PNA into cells.     

Rapid uptake of oxidized ascorbate induces loss of cellular glutathione and oxidative stress in liver slices

The observation that ascorbate known to retain pro-oxidant properties induces cell death in a number of immortal cell lines, led us to examine its mechanism and whether it is involved in oxidative stress injury in such asocorbate-enriched tissue cells as hepatocytes. In rat liver homogenates, higher concentrations (1 and 3 mM) of ascorbate suppressed lipid peroxide productions but lower concentrations (0.1 and 0.3 mM) did not. In contrast to the homogenate, ascorbate increased lipid peroxide production in liver slices in a concentration dependant manner. Iso-ascorbate, the epimer of ascorbate did not cause an increase the oxidative stress in liver slices. This differential effect between homogenates and liver slices implies that cellular integrity is required for ascorbate to induce oxidative stress. Wortmannin, an inhibitor of the GLUT (glucose transporter) thought to transport dehydroascorbate into cells, inhibited [(14)C]-ascorbate uptake and suppressed oxidative stress in liver slices. Wortmannin suppressed that [(14)C]-ascorbate uptake by GLUT following oxidation to [(14)C]dehydroascorbate. Taken together, these observations support our hypothesis that ascorbate is oxidized to dehydroascorbate by molecular oxygen in solution (i.e., plasma and culture medium) which is then carried into hepatocytes (via a GLUT) where it is reduced back to ascorbate causing oxidative stress.     

Self-assembled thermosensitive luminescent nanoparticles with peptide-Au conjugates for cellular imaging and drug delivery

A facile and efficient strategy has been developed to fabricate a multifunctional,theranostic anticancer drug delivery platform featuring active targeting,controlled drug release and fluorescence imaging for real-time control of delivery.To this end,thermo sensitive poly(N-isopropyl acrylamide)(PNIPAM)nanospheres are decorated with peptide-Au cluster conjugates as a smart nanomedicine platform.A sophisticated trifunctional peptide is designed to release the anticancer drug doxorubicin(DOX),target cells and reduce Au^3+ions to form luminescent Au cluste rs.Importantly,the peptide-Au cluster moieties are attached to the PNIPAM nanospheres via amide bonds rather than noncovalent interactions,significantly improving their stability in biological medium and drug release efficiency.The in vitro experiments showed that DOX was released in an efficient and controlled manner under physiological conditions.     

Rearrangement regulated cysteine fluorescent probe for cellular oxidative stress evaluation induced by copper(II)

Cysteine (Cys) plays an important role in regulating cellular redox balance. But due to the constant changes in the concentration of Cys in organisms, fast response sensors are urgent required for practical application. In this work, a fluorescent probe with a fast response was developed by linking coumarin derivatives containing α,β-unsaturated ketones to NBD. The PET effect made the system non-fluorescent. When the probe reacted with Cys, the bond between the coumarin derivative and the NBD was cut off, meanwhile a rapid rearrangement and reactive site passivation occurred. Then two fluorophores with the same emission peak are released, among them, strong fluorescence signal of NBD dominated. Thus, although the similar reaction occurred for Hcy, the rate of NBD derivative rearrangement was slow, in a short time, fluorescence signal was still weak. As for GSH, cleavage could occur, but no rearrange within the NBD molecule due to GSH with large volume. Because of strong fluorescent emission, this probe was successfully used in biological imaging about cell and zebrafish. More importantly, the probe was successfully used to evaluate the oxidative stress caused by copper(II) in living cells. This fluorescence strategy and application will provide a new way of studying intracellular oxidative stress processes and damage.     

Rearrangement regulated cysteine fluorescent probe for cellular oxidative stress evaluation induced by copper(II)

Cysteine (Cys) plays an important role in regulating cellular redox balance. But due to the constant changes in the concentration of Cys in organisms, fast response sensors are urgent required for practical application. In this work, a fluorescent probe with a fast response was developed by linking coumarin derivatives containing α,β-unsaturated ketones to NBD. The PET effect made the system non-fluorescent. When the probe reacted with Cys, the bond between the coumarin derivative and the NBD was cut off, meanwhile a rapid rearrangement and reactive site passivation occurred. Then two fluorophores with the same emission peak are released, among them, strong fluorescence signal of NBD dominated. Thus, although the similar reaction occurred for Hcy, the rate of NBD derivative rearrangement was slow, in a short time, fluorescence signal was still weak. As for GSH, cleavage could occur, but no rearrange within the NBD molecule due to GSH with large volume. Because of strong fluorescent emission, this probe was successfully used in biological imaging about cell and zebrafish. More importantly, the probe was successfully used to evaluate the oxidative stress caused by copper(II) in living cells. This fluorescence strategy and application will provide a new way of studying intracellular oxidative stress processes and damage.     

Discovery and identification of potential biomarkers for alcohol‐induced oxidative stress based on cellular metabolomics

Biomarkers involved in alcohol‐induced oxidative stress play an important role in alcoholic liver disease prevention and diagnosis. Alcohol‐induced oxidative stress in human liver L‐02 cells was used to discover the potential biomarkers. Metabolites from L‐02 cells induced by alcohol were measured by high‐performance liquid chromatography and mass spectrometry. Fourteen metabolites that allowed discrimination between control and model groups were discovered by multivariate statistical data analysis (i.e. principal components analysis, orthogonal partial least‐squares discriminate analysis). Based on the retention time, UV spectrum and LC–MS findings of the samples and compared with the authentic standards, eight biomarkers involved in alcohol‐induced oxidative stress, namely, malic acid, oxidized glutathione, γ‐glutamyl‐cysteinyl‐glycine, adenosine triphosphate, phenylalanine, adenosine monophosphate, nitrotyrosine and tryptophan, were identified. These biomarkers offered important targets for disease diagnosis and other researches.     

Deconvolution of pulse trains with the L0 penalty

The output of many instruments can be modeled as a convolution of an impulse response and a series of sharp spikes. Deconvolution considers the inverse problem: estimate the input spike train from an observed (noisy) output signal. We approach this task as a linear inverse problem, solved using penalized regression. We propose the use of an L(0) penalty and compare it with the more common L(2) and L(1) penalties. In all cases a simple and iterative weighted regression procedure can be used. The model is extended with a smooth component to handle drifting baselines. Application to three different data sets shows excellent results.     

Peptide heterocycle conjugates: a diverted edman degradation protocol for the synthesis of N-terminal 2-iminohydantoins

[reaction: see text] A modified Edman degradation procedure provides an effective means of introducing a heterocycle at the N-terminus of an alpha-amino acid amide or peptide. Reaction of a peptide with an isothiocyanate, followed by dehydrothiolative trapping of the intermediate thiourea, by intramolecular cyclization of the weakly nucleophilic adjacent amide nitrogen, generates an iminohydantoin. A solution-phase parallel synthesis of iminohydantoins and a polymer-supported synthesis of dipeptide- and tripeptide-derived iminohydantoins were also achieved.     

Releasable luciferin-transporter conjugates: tools for the real-time analysis of cellular uptake and release

The design, synthesis, and evaluation of conjugates of arginine-rich transporters and luciferin are described that release luciferin only after entry into cells that are stably transfected with luciferase. Each molecule of free luciferin that is released after entry generates a photon that can be measured allowing for real-time quantification of uptake and release in cells. The process provides a method to assay uptake and release of free luciferin as a function of variations in the releasable linker and in the transporter.     

Spectroscopic study of bituminous oxidative stress

Bitumen, as each organic substance, is a product which alters over time. Indeed, roads deteriorate under the effect of several phenomena. A number of studies have been undertaken to increase the quality of road's coating, mostly by adding polymer to bitumen. This work was based on the study, by electron paramagnetic resonance (EPR), FTIR and Synchronous UV fluorescence, of different base and modified bitumens after different treatments used to simulate the ageing (gamma irradiation, thermal treatment). Our purpose was to compare and correlate the results obtained by different techniques to improve the knowledge of bitumen's reactivity and evolution submitted to ageing phenomena.