A carbon monoxide-releasing molecule (CORM-3) uncouples mitochondrial respiration and modulates the production of reactive oxygen species

Carbon monoxide (CO), produced during the degradation of heme by the enzyme heme oxygenase, is an important signaling mediator in mammalian cells. Here we show that precise delivery of CO to isolated heart mitochondria using a water-soluble CO-releasing molecule (CORM-3) uncouples respiration. Addition of low-micromolar concentrations of CORM-3 (1–20 μM), but not an inactive compound that does not release CO, significantly increased mitochondrial oxygen consumption rate (State 2 respiration) in a concentration-dependent manner. In contrast, higher concentrations of CORM-3 (100 μM) suppressed ADP-dependent respiration through inhibition of cytochrome c oxidase. The uncoupling effect mediated by CORM-3 was inhibited in the presence of the CO scavenger myoglobin. Moreover, this effect was associated with a gradual decrease in membrane potential (ψ) over time and was partially reversed by malonate, an inhibitor of complex II activity. Similarly, inhibition of uncoupling proteins or blockade of adenine nucleotide transporter attenuated the effect of CORM-3 on both State 2 respiration and Δψ. Hydrogen peroxide (H₂O₂) produced by mitochondria respiring from complex I-linked substrates (pyruvate/malate) was increased by CORM-3. However, respiration initiated via complex II using succinate resulted in a fivefold increase in H₂O₂ production and this effect was significantly inhibited by CORM-3. These findings disclose a counterintuitive action of CORM-3 suggesting that CO at low levels acts as an important regulator of mitochondrial respiration.     

cDNA cloning and expression analysis of a putative alternative oxidase HsAOX1 from wild barley (Hordeum spontaneum)

A novel alternative oxidase (AOX1) gene, designated HsAOX1 (GenBank accession number JF440341) was cloned by RT-PCR from wild barley (Hordeum spontaneum). The full length of HsAOX1 is 1115 bp with an open reading frame of 987 bp, encoding a protein of 328 amino acids with molecular weight of 36.89 kDa and a theoretical isoelectric point of 6.81. As found in other plant AOX1 proteins, sequence alignment showed that HsAOX1 had conserved metal binding and hydrophobic ??-helix regions and had high homology to other AOX1 in plants. The expression analysis by semi-quantitative RT-PCR revealed that HsAOX1 was induced in response to cold stress, H₂O₂ treatment, SA, antimycin A and KCN. These results showed that HsAOX1 functions not only during inhibition of cytochrome electron transport but also during oxidative stresses, thus suggesting a role of HsAOX1 in preventing the generation of free radicals by the mitochondrial electron transport chain. The cloning and characterization of the HsAOX1 gene will be useful for further studies of biological roles of HsAOX1 in plants.     

Redox response of the endogenous calcineurin inhibitor Adapt 78

Adapt 78 (DSCR 1/calcipressin/MCIP 1) is a potent natural inhibitor of calcineurin, an important intracellular phosphatase that mediates many cellular responses to calcium. We previously reported two major cytosolic isoforms (1 and 4) of Adapt 78, and that isoform 4 is an oxidative and calcium stress-response protein. Using a higher cell culture density and new antibody, we again observed that both major isoforms localized to the cytosol, but a significant level of isoform 4 (but not isoform 1) was also detected in the nucleus where it was present in the non-soluble region and not associated with RNA. Exposure of cells to hydrogen peroxide led to the significant loss of isoform 4 from the nucleus with a moderate increase in cytosolic localization. The change in isoform 4 phosphorylation state in response to oxidative stress, characterized by a loss of the lesser (hypo) phosphorylated Adapt 78, was not due to accelerated degradation, although general Adapt 78 degradation was proteosome mediated. Finally, stimulation of Jurkat and primary T-lymphocyte signaling led to isoform 4 induction. This induction was BAPTA, diphenylene iodonium, and N-acetylcysteine inhibitable, and accompanied by induction of the classic immune response mediator and calcineurin-pathway-stimulated interleukin-2. These studies reveal new redox-related activities for Adapt 78 isoform 4, which may contribute to its known calcineurin-regulating and cytoprotective activities, and further suggest that Adapt 78 plays a role in basic T-cell response.     

Genotypic and Phenotypic Differences between Nodal and Extranodal Diffuse Large B-Cell Lymphomas

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous group of diseases that have diverse clinical, pathological, and biological features. Here, it is shown that primary nodal and extranodal DLBCLs differ genomically and phenotypically. Using conventional comparative genomic hybridization (CGH), the authors assessed the chromosomal aberrations in 18 nodal, 13 extranodal, and 5 mixed DLBCLs. The results demonstrate significantly distinct chromosomal aberrations exemplified by gains of chromosomal arms 1p, 7p, 12q24.21-12q24.31, and 22q and chromosome X and loss of chromosome 4, 6q, and 18q22.3-23 in extranodal compared with nodal DLBCLs. Nodal DLBCLs showed an increased tendency for 18q amplification and BCL2 protein overexpression compared with extranodal and mixed tumors. Using a panel of five antibodies against GCET1, MUM1, CD10, BCL6, and FOXP1 proteins to subclassify DLBCLs according to the recent Choi algorithm, the authors showed that the genomic profiles observed between the nodal and extranodal DLBCLs were not due to the different proportions of GCB vs ABC in the two groups. Further delineation of these genomic differences was illuminated by the use of high-resolution 21K BAC array CGH performed on 12 independent new cases of extranodal DLBCL. The authors demonstrated for the first time a novel genome and proteome-based signatures that may differentiate the two lymphoma types.     

High-dose atorvastatin causes a rapid sustained increase in human serum PCSK9 and disrupts its correlation with LDL cholesterol

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a key regulator of serum LDL-cholesterol (LDL-C) levels. PCSK9 is secreted by the liver into the plasma and binds the hepatic LDL receptor (LDLR), causing its subsequent degradation. We first demonstrated that a moderate dose of atorvastatin (40 mg) increases PCSK9 serum levels, suggesting why increasing statin doses may have diminished efficacy with regard to further LDL-C lowering. Since that initial observation, at least two other groups have reported statin-induced PCSK9 increases. To date, no analysis of the effect of high-dose atorvastatin (80 mg) on PCSK9 over time has been conducted. Therefore, we studied the time course of atorvastatin (80 mg) in human subjects. We measured PCSK9 and lipid levels during a 2-week lead-in baseline period and every 4 weeks thereafter for 16 weeks. We observed that atorvastatin (80 mg) caused a rapid 47% increase in serum PCSK9 at 4 weeks that was sustained throughout 16 weeks of dosing. Importantly, while PCSK9 levels were highly correlated with total cholesterol (TC), LDL-C, and triglyceride (TG) levels at baseline, atorvastatin (80 mg) completely abolished all of these correlations. Together, these results further suggest an explanation for why increasing doses of statins fail to achieve proportional LDL-C lowering.     

Expression in Escherichia coli and characterization of β-xylosidases GH39 and GH-43 from Bacillus halodurans C-125

To develop xylosidases as tools for the hydrolysis of wheat bran arabinoxylans, two β-xylosidases from Bacillus halodurans C-125 have been cloned and expressed in Escherichia coli. The recombinant (His)₆-tagged enzymes, designated as XylBH39 and XylBH43, were efficiently purified using Ni²⁺-affinity chromatography. Determination of native molecular masses indicated that XylBH43 is dimeric in solution, whereas a similar analysis of XylBH39 did not allow differentiation between the dimeric and trimeric states. Both enzymes had similar pH and temperature optima (pH 7.5 and 55 °C for XylBH39 and pH 8 and 60 °C for XylBH43) and were relatively stable over the pH range of 3.5–8.5. In contrast, XylBH39 was more thermostable. At 60 °C, XylBH39 and XylBH43 displayed approximate half-life values of 2.40 and 0.05 h, respectively. The comparison of the ratio k _cat/K _M revealed that XylBH43 hydrolyzed p-nitrophenyl-β-d-xyloside more efficiently (4.6-fold) than XylBH39. Similarly, while XylBH43 was 18-fold less active on p-nitrophenyl-α-l-arabinofuranoside, XylBH39 was essentially inactive on this substrate. Using either p-nitrophenyl-β-d-xyloside or xylotriose, XylBH39 performed transglycosylation, while xylobiose proved to be a poor substrate for both hydrolysis and transglycosylation. The use of XylBH39 and XylBH43 for the posttreatment of endoxylanase-generated wheat bran hydrolysates revealed that XylBH43 efficiently produced xylose monomers (385 μg/ml after 330 min incubation). Its activity was improved by the simultaneous deployment of an α-l-arabinofuranosidase. Together, these enzymes were able to release 521 μg/ml of xylose after 330 min. This constitutes an approximate yield improvement of 35%.     

Medicago sativa - Sinorhizobium meliloti Symbiosis Promotes the Bioaccumulation of Zinc in Nodulated Roots

In this study we investigated effects of Zn supply on germination, growth, inorganic solutes (Zn, Ca, Fe, and Mg) partitioning and nodulation of Medicago sativa This plant was cultivated with and without Zn (2 mM). Treatments were plants without (control) and with Zn tolerant strain (S532), Zn intolerant strain (S112) and 2 mM urea nitrogen fertilisation. Results showed that M. sativa germinates at rates of 50% at 2 mM Zn. For plants given nitrogen fertilisation, Zn increased plant biomass production. When grown with symbionts, Zn supply had no effect on nodulation. Moreover, plants with S112 showed a decrease of shoot and roots biomasses. However, in symbiosis with S532, an increase of roots biomass was observed. Plants in symbiosis with S. meliloti accumulated more Zn in their roots than nitrogen fertilised plants. Zn supply results in an increase of Ca concentration in roots of fertilised nitrogen plants. However, under Zn supply, Fe concentration decreased in roots and increased in nodules of plants with S112. Zn supply showed contrasting effects on Mg concentrations for plants with nitrogen fertilisation (increase) and plants with S112 (decrease). The capacity of M. sativa to accumulate Zn in their nodulated roots encouraged its use in phytostabilisation processes.     

Telomerase Inhibition Decreases Alpha-Fetoprotein Expression and Secretion by Hepatocellular Carcinoma Cell Lines: In Vitro and In Vivo Study

Alpha-fetoprotein (AFP) is a diagnostic marker for hepatocellular carcinoma (HCC). A direct relationship between poor prognosis and the concentration of serum AFP has been observed. Telomerase, an enzyme that stabilizes the telomere length, is expressed by 90% of HCC. The aim of this study was to investigate the effect of telomerase inhibition on AFP secretion and the involvement of the PI3K/Akt/mTOR signaling pathway. Proliferation and viability tests were performed using tetrazolium salt. Apoptosis was determined through the Annexin V assay using flow cytometry. The concentrations of AFP were measured using ELISA kits. The AFP mRNA expression was evaluated using RT-PCR, and cell migration was evaluated using a Boyden chamber assay. The in vivo effect of costunolide on AFP production was tested in NSG mice. Telomerase inhibition by costunolide and BIBR 1532 at 5 and 10 μM decreased AFP mRNA expression and protein secretion by HepG2/C3A cells. The same pattern was obtained with cells treated with hTERT siRNA. This treatment exhibited no apoptotic effect. The AFP mRNA expression and protein secretion by PLC/PRF/5 was decreased after treatment with BIBR1532 at 10 μM. In contrast, no effect was obtained for PLC/PRF/5 cells treated with costunolide at 5 or 10 μM. Inhibition of the PI3K/Akt/mTOR signaling pathway decreased the AFP concentration. In contrast, the MAPK/ERK pathway appeared to not be involved in HepG2/C3A cells, whereas ERK inhibition decreased the AFP concentration in PLC/PRF/5 cells. Modulation of the AFP concentration was also obtained after the inhibition or activation of PKC. Costunolide (30 mg/kg) significantly decreased the AFP serum concentration of NSG mice bearing HepG2/C3A cells. Both the inhibition of telomerase and the inhibition of the PI3K/Akt/mTOR signaling pathway decreased the AFP production of HepG2/C3A and PLC/PRF/5 cells, suggesting a relationship between telomerase and AFP expression through the PI3K/Akt/mTOR pathway