Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H₂O₂ affair?

Class III peroxidases (Prxs) are plant enzymes capable of using H(2)O(2) to oxidize a range of plant secondary metabolites, notably phenolic compounds. These enzymes are localized in the cell wall or in the vacuole, which is a target for secondary metabolite accumulation, but very little is known about the function of vacuolar Prxs. Here, the physiological role of the main leaf vacuolar Prx of the medicinal plant Catharanthus roseus, CrPrx1, was further investigated namely by studying its capacity to oxidize co-localized phenolic substrates at the expense of H(2)O(2). LC-PAD-MS analysis of the phenols from isolated leaf vacuoles detected the presence of three caffeoylquinic acids and four flavonoids in this organelle. These phenols or similar compounds were shown to be good CrPrx1 substrates, and the CrPrx1-mediated oxidation of 5-O-caffeoylquinic acid was shown to form a co-operative regenerating cycle with ascorbic acid. Interestingly, more than 90% of total leaf Prx activity was localized in the vacuoles, associated to discrete spots of the tonoplast. Prx activity inside the vacuoles was estimated to be 1809 nkat ml(-1), which, together with the determined concentrations for the putative vacuolar phenolic substrates, indicate a very high H(2)O(2) scavenging capacity, up to 9 mM s(-1). Accordingly, high light conditions, known to increase H(2)O(2) production, induced both phenols and Prx levels. Therefore, it is proposed that the vacuolar couple Prx/secondary metabolites represent an important sink/buffer of H(2)O(2) in green plant cells.     

Pronounced hypoxia in models of murine and human leukemia: high efficacy of hypoxia-activated prodrug PR-104

Recent studies indicate that interactions between leukemia cells and the bone marrow (BM) microenvironment promote leukemia cell survival and confer resistance to anti-leukemic drugs. There is evidence that BM microenvironment contains hypoxic areas that confer survival advantage to hematopoietic cells. In the present study we investigated whether hypoxia in leukemic BM contributes to the protective role of the BM microenvironment. We observed a marked expansion of hypoxic BM areas in immunodeficient mice engrafted with acute lymphoblastic leukemia (ALL) cells. Consistent with this finding, we found that hypoxia promotes chemoresistance in various ALL derived cell lines. These findings suggest to employ hypoxia-activated prodrugs to eliminate leukemia cells within hypoxic niches. Using several xenograft models, we demonstrated that administration of the hypoxia-activated dinitrobenzamide mustard, PR-104 prolonged survival and decreased leukemia burden of immune-deficient mice injected with primary acute lymphoblastic leukemia cells. Together, these findings strongly suggest that targeting hypoxia in leukemic BM is feasible and may significantly improve leukemia therapy.     

Effects of the Commercial Flame Retardant Mixture DE-71 on Cytokine Production by Human Immune Cells

IntroductionAlthough production of polybrominated diphenyl ethers (PBDEs) is now banned, release from existing products will continue for many years. The PBDEs are assumed to be neurotoxic and toxic to endocrine organs at low concentrations. Their effect on the immune system has not been investigated thoroughly. We aimed to investigate the influence of DE-71 on cytokine production by peripheral blood mononuclear cells (PBMCs) stimulated with Escherichia Coli lipopolysaccharide (LPS) or phytohaemagglutinin-L (PHA-L).ResultsAt non-cytotoxic concentrations (0.01–10 μg/mL), DE-71 significantly enhanced secretion of IL-1β, IL-6, CXCL8, IL-10, and TNF-α (p<0.001–0.019; n = 6) from LPS-stimulated PBMCs. IFN-γ, TNF-α, IL-17A, and IL-17F (p = <0.001–0.043; n = 6) secretion were enhanced from PHA-L-stimulated PBMCs as well. Secretion of IL-1β, IL-2, IL-10, IL-8 and IL-6 was not significantly affected by DE-71.ConclusionsWe demonstrate an enhancing effect of DE-71 on cytokine production by normal human PBMCs stimulated with LPS or PHA-L ex vivo.     

Monoterpene biotransformation by <Emphasis Type="Italic">Colletotrichum</Emphasis> species

Objective

To investigate the biocatalytic potential of Colletotrichum acutatum and Colletotrichum nymphaeae for monoterpene biotransformation.

Results

C. acutatum and C. nymphaeae used limonene, α-pinene, β-pinene, farnesene, citronellol, linalool, geraniol, perillyl alcohol, and carveol as sole carbon and energy sources. Both species biotransformed limonene and linalool, accumulating limonene-1,2-diol and linalool oxides, respectively. α-Pinene was only biotransformed by C. nymphaeae producing campholenic aldehyde, pinanone and verbenone. The biotransformation of limonene by C. nymphaeae yielded 3.34–4.01 g limonene-1,2-diol l^?1, depending on the substrate (R-(+)-limonene, S-(?)-limonene or citrus terpene (an agro-industrial by-product). This is among the highest concentrations already reported for this product.

Conclusions

This is the first report on the biotransformation of these terpenes by Colletotrichum spp. and the biotransformation of limonene to limonene-1,2-diol possibly involves enzymes similar to those found in Grosmannia clavigera.

     

Comparative proteomic analysis of Xanthomonas citri ssp. citri periplasmic proteins reveals changes in cellular envelope metabolism during in vitro pathogenicity induction

Citrus canker is a plant disease caused by Gram‐negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm‐enriched fraction was performed for XAC cells grown in pathogenicity‐inducing (XAM‐M) and pathogenicity‐non‐inducing (nutrient broth) media using two‐dimensional electrophoresis combined with liquid chromatography‐tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up‐regulated proteins related to cellular envelope metabolism included glucose‐1‐phosphate thymidylyltransferase, dTDP‐4‐dehydrorhamnose‐3,5‐epimerase and peptidyl‐prolyl cis–trans‐isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real‐time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up‐regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60‐kDa chaperonin and glyceraldehyde‐3‐phosphate dehydrogenase were identified, suggesting the presence of post‐translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence‐related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies.     

Glioprotective Effects of Lingonberry Extract Against Altered Cellular Viability,Acetylcholinesterase Activity,and Oxidative Stress in Lipopolysaccharide-Treated Astrocytes

Altered astrocytic function is a contributing factor to the development of neurological diseases and neurodegeneration. Berry fruits exert neuroprotective effects by modulating pathways involved in inflammation, neurotransmission, and oxidative stress. The aim of this study was to examine the effects of the lingonberry extract on cellular viability and oxidative stress in astrocytes exposed to lipopolysaccharide (LPS). In the reversal protocol, primary astrocytic cultures were first exposed to 1 µg/mL LPS for 3 h and subsequently treated with lingonberry extract (10, 30, 50, and 100 μg/mL) for 24 and 48 h. In the prevention protocol, exposure to the lingonberry extract was performed before treatment with LPS. In both reversal and prevention protocols, the lingonberry extracts, from 10 to 100 μg/mL, attenuated LPS-induced increase in reactive oxygen species (around 55 and 45%, respectively, P?<?0.01), nitrite levels (around 50 and 45%, respectively, P?<?0.05), and acetylcholinesterase activity (around 45 and 60%, respectively, P?<?0.05) in astrocytic cultures at 24 and 48 h. Also, in both reversal and prevention protocols, the lingonberry extract also prevented and reversed the LPS-induced decreased cellular viability (around 45 and 90%, respectively, P?<?0.05), thiol content (around 55 and 70%, respectively, P?<?0.05), and superoxide dismutase activity (around 50 and 145%, respectively, P?<?0.05), in astrocytes at both 24 and 48 h. Our findings suggested that the lingonberry extract exerted a glioprotective effect through an anti-oxidative mechanism against LPS-induced astrocytic damage.     

Effects of chronic allopurinol therapy on purine metabolism in Duchenne muscular dystrophy

Adenine, adenosine, inosine, hypoxanthine, xanthosine, xanthine, guanine and guanosine blood levels in 11 Duchenne muscular dystrophy patients treated with allopurinol, 10 untreated patients and 8 healthy controls, were determined by HPLC. Serum ADA, PNP and 5'-NT were also determined. Untreated patients showed lower adenine (p less than 0.001) and higher adenosine, xanthine, ADA and PNP levels (p less than 0.01) than controls. Treated patients had lower adenine and higher xanthine levels (p less than 0.001), but higher hypoxanthine, xanthosine and guanine levels (p less than 0.001), than controls, with normal ADA and PNP. The changes observed in ADA and PNP levels suggest an involvement of these enzymes in accelerated degradation of purines in Duchenne dystrophy.