A new xanthene‐based two‐photon fluorescent probe for the imaging of 1,4‐dithiothreitol (DTT) in living cells

1,4‐Dithiothreitol (DTT) has wide applications in cell biology and biochemistry. Development of effective methods for monitoring DTT in biological systems is important for the safe handling and study of toxicity to humans. Herein, we describe a two‐photon fluorescence probe (Rh‐DTT) to detect DTT in living systems for the first time. Rh‐DTT showed high selectivity and sensitivity to DTT. Rh‐DTT can be successfully used for the two‐photon imaging of DTT in living cells, and also can detect DTT in living tissues and mice.     

The chlorophyll fluorescence quenching and changes of absorbance in pea chloroplasts

Chlorophyll (Chl) fluorescence quenching parameters were measured in dark-adapted pea leaves and chloroplasts with the purpose to find the conditions of high and low non-photochemical quenching, that would be stable during a prolonged irradiation. A PAM fluorometer was used for measuring induction curves in the range of actinic radiation of 3-35 W m-2, with an ordinary value of about 15 W m-2. The effects of various mediators, i.e., ascorbate, methyl viologen (MV), dithiothreitol (DTT) and nigericin, on the quenching process were tested. Simultaneously, the absorbance was measured during a 15-20 min period of irradiation and after the actinic radiation was turned off, i.e., in the recovery period. The pH values of chloroplast suspensions were 5.5, 6.5 and 8.0, the largest non-photochemical quenching was observed at pH of 6.5. The irradiation of chloroplasts led to an absorption decrease within the entire photosynthetically active range, attaining saturation when the fluorescence reached Fs level, and to an absorption increase during the recovery period. Absorbance changes at the maximum of red band were 10-20 %. A decrease in Chl concentration (10 %) after irradiation was found only at pH of 5.5, when the recovery time was the longest, i.e., about 60 min. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phycobilisome from Anabaena Variabilis Kütz. and its Model Conjugates

The model conjugates phycocyanin-allophycocyanin (C-PC-APC) and phycoerythrocyanin-phycocyanin-allophycocyanin (PEC-C-PC-APC) were synthesized by using a heterobifunctional coupling reagent N-succinimidyl-3-(2-pyridyldithio)propionate. The rod-core complex (αβ)₆ ^PCL_RC ²⁷(αβ)₃ ^APCL_C ^8.9 and phycobilisomes were separated from Anabaena variabilis. Energy transfer features for the conjugates and the complexes were compared. The absorption and fluorescence emission spectra indicated that the linker-peptides mediate interaction of phycobiliproteins and prompt energy transfer. The energy transfer in the conjugates was detected by fluorescence emission spectra and confirmed by the addition of dithiothreitol. The conjugates may be used as models for studying the energy transfer mechanism in phycobilisomes. This revised version was published online in September 2006 with corrections to the Cover Date.     

Recombinant expression and characterization of Lucilia cuprina CYP6G3: Activity and binding properties toward multiple pesticides

The Australian sheep blowfly, Lucilia cuprina, is a primary cause of sheep flystrike and a major agricultural pest. Cytochrome P450 enzymes have been implicated in the resistance of L. cuprina to several classes of insecticides. In particular, CYP6G3 is a L. cuprina homologue of Drosophila melanogaster CYP6G1, a P450 known to confer multi-pesticide resistance. To investigate the basis of resistance, a bicistronic Escherichia coli expression system was developed to co-express active L. cuprina CYP6G3 and house fly (Musca domestica) P450 reductase. Recombinant CYP6G3 showed activity towards the high-throughput screening substrates, 7-ethoxycoumarin and p-nitroanisole, but not towards p-nitrophenol, coumarin, 7-benzyloxyresorufin, or seven different luciferin derivatives (P450-Glo™ substrates). The addition of house fly cytochrome b₅ enhanced the k_cat for p-nitroanisole dealkylation approximately two fold (17.8 ± 0.5 vs 9.6 ± 0.2 min⁻¹) with little effect on K_M (13 ± 1 vs 10 ± 1 μM). Inhibition studies and difference spectroscopy revealed that the organochlorine compounds, DDT and endosulfan, and the organophosphate pesticides, malathion and chlorfenvinphos, bind to the active site of CYP6G3. All four pesticides showed type I binding spectra with spectral dissociation constants in the micromolar range suggesting that they may be substrates of CYP6G3. While no significant inhibition was seen with the organophosphate, diazinon, or the neonicotinoid, imidacloprid, diazinon showed weak binding in spectral assays, with a Kd value of 23 ± 3 μM CYP6G3 metabolised diazinon to the diazoxon and hydroxydiazinon metabolites and imidacloprid to the 5-hydroxy and olefin metabolites, consistent with a proposed role of CYP6G enzymes in metabolism of phosphorothioate and neonicotinoid insecticides in other species.     

Oligomerization of H-pyrophosphatase and its structural and functional consequences

The H⁺-pyrophosphatase (H⁺-PPase) consists of a single polypeptide, containing 16 or 17 transmembrane domains. To determine the higher order oligomeric state of Streptomyces coelicolor H⁺-PPase, we constructed a series of cysteine substitution mutants and expressed them in Escherichia coli. Firstly, we analyzed the formation of disulfide bonds, promoted by copper, in mutants with single cysteine substitutions. 28 of 39 mutants formed disulfide bonds, including S545C, a substitution at the periplasmic side. The formation of intermolecular disulfide bonds suppressed the enzyme activity of several, where the substituted residues were located in the cytosol. Creating disulfide links in the cytosol may interfere with the enzyme's catalytic function. Secondly, we prepared double mutants by introducing second cysteine substitutions into the S545C mutant. These double-cysteine mutants produced cross-linked complexes, estimated to be at least tetramers and possibly hexamers. Thirdly, we co-expressed epitope-tagged, wild type, and inactive mutant H⁺-PPases in E. coli and confirmed the formation of oligomers by co-purifying one subunit using the epitope tag used to label the other. The enzyme activity of these oligomers was markedly suppressed. We propose that H⁺-PPase is present as an oligomer made up of at least two or three sets of dimers.     

Prosurvival Bcl-2 proteins stabilize pancreatic mitochondria and protect against necrosis in experimental pancreatitis

Acinar cells in pancreatitis die through apoptosis and necrosis, the roles of which are different. The severity of experimental pancreatitis correlates directly with the extent of necrosis and inversely, with apoptosis. Apoptosis is mediated by the release of cytochrome c into the cytosol followed by caspase activation, whereas necrosis is associated with the mitochondrial membrane potential (ΔΨm) loss leading to ATP depletion. Here, we investigate the role of Bcl-2 proteins in apoptosis and necrosis in pancreatitis. We found up-regulation of prosurvival Bcl-2 proteins in pancreas in various experimental models of acute pancreatitis, most pronounced for Bcl-xL. This up-regulation translated into increased levels of Bcl-xL and Bcl-2 in pancreatic mitochondria. Bcl-xL/Bcl-2 inhibitors induced ΔΨm loss and cytochrome c release in isolated mitochondria. Corroborating the results on mitochondria, Bcl-xL/Bcl-2 inhibitors induced ΔΨm loss, ATP depletion and necrosis in pancreatic acinar cells, both untreated and hyperstimulated with CCK-8 (in vitro pancreatitis model). Together Bcl-xL/Bcl-2 inhibitors and CCK induced more necrosis than either treatment alone. Bcl-xL/Bcl-2 inhibitors also stimulated cytochrome c release in acinar cells leading to caspase-3 activation and apoptosis. However, different from their effect on pronecrotic signals, the stimulation by Bcl-xL/Bcl-2 inhibitors of apoptotic responses was less in CCK-treated than control cells. Therefore, Bcl-xL/Bcl-2 inhibitors potentiated CCK-induced necrosis but not apoptosis. Correspondingly, transfection with Bcl-xL siRNA stimulated necrosis but not apoptosis in the in vitro pancreatitis model. Further, in animal models of pancreatitis Bcl-xL up-regulation inversely correlated with necrosis, but not apoptosis. Results indicate that Bcl-xL and Bcl-2 protect acinar cells from necrosis in pancreatitis by stabilizing mitochondria against death signals. We conclude that Bcl-xL/Bcl-2 inhibition would aggravate acute pancreatitis, whereas Bcl-xL/Bcl-2 up-regulation presents a strategy to prevent or attenuate necrosis in pancreatitis.     

Reduction of gene expression by a hairpin-loop structured oligodeoxynucleotide: Alternative to siRNA and antisense

Background

We previously described the inhibition of HIV-1 replication by a 54-mer hairpin-loop structured oligodeoxynucleotide (ODN) A, which binds the polypurine tract (PPT) on HIV-1 RNA. ODN A was shown to lead to reduced viral RNA in virions or early during infection.

Methods and results

Here we demonstrated that ODN A was able to cause hydrolysis of viral RNA not only by retroviral RT-associated RNase H but also cellular RNase H1 and RNase H2 in vitro. Furthermore, ODN A reduced gene expression in a dose-dependent manner in a cell-based reporter assay where a PPT sequence was inserted in the 5′ untranslated region of the reporter gene. The efficacy of ODN A was higher than that of its siRNA and antisense counterparts. By knocking down cellular RNases H, we showed that RNase H1 contributed to the gene silencing by ODN A but the possibility of a partial contribution of RNase H-independent mechanisms could not be ruled out.

General significance

Our findings highlight the potential application of hairpin-loop structured ODNs for reduction of gene expression in mammalian cells and underscore the possibility of using ODN A to trigger the hydrolysis of HIV RNA in infected cells by cellular RNases H.