Oligogalacturonides inhibit the induction of late but not of early auxin-responsive genes in tobacco

Oligogalacturonides (OGs) released from the plant cell wall regulate several defense responses, as well as various aspects of plant growth and development. In these latter effects, OGs exhibit auxin-antagonist activity. To shed light on the mechanism by which OGs antagonise auxin, we analysed the ability of these oligosaccharides to inhibit the activity of four auxin-up-regulated promoters [pGm-GH3 of soybean (Glycine max L. Merr.), pNt114 of tobacco (Nicotiana tabacum L.), and prolB and prolD of Agrobacterium rhizogenes] driving the expression of the beta-glucuronidase reporter gene (GUS) in transgenic tobacco seedlings. Our results indicate that OGs at submicromolar concentrations inhibit the activation by auxin of pNt114, prolB and prolD, but not that of pGm-GH3. Comparative analysis of the kinetics of activation of the four promoters in response to the hormone shows that, while pGm-GH3 is rapidly activated, the other three promoters exhibit a delayed activation, with a lag of at least 4 h before the appearance of GUS activity. The lack of effect of the OGs on early auxin-responsive genes was confirmed by RNA gel blot analysis of the tobacco genes Nt-GH3 and Nt-iaa2.3/2.5. Our results suggest that the auxin-antagonist action of OGs affects the expression of late but not of early auxin-responsive genes.     

SNAP-25 and synaptotagmin 1 function in Ca2+-dependent reversible docking of granules to the plasma membrane

In neuroendocrine cells, Ca²⁺ triggers fusion of granules with the plasma membrane and functions at earlier steps by increasing the size of the readily releasable pool of vesicles. The effect of Ca²⁺ at early steps of secretion may be due to the recruitment at the plasma membrane of granules localized in the cytoplasm. To study the mechanism of granule docking, a new in vitro assay is designed using membrane fractions from mouse pituitary AtT-20 cells. By using this assay, it is found that granule docking to the plasma membrane is controlled by Ca²⁺ concentrations in the micromolar range, is reversible and requires intact SNAP-25, but not VAMP-2. In the docking assay, addition of Ca²⁺ induces the formation of a SNAP-25-Synaptotagmin 1 complex. The cytosolic domain C2AB of Synaptotagmin 1 and anti-Synaptotagmin 1 antibodies block granule docking. These results show that Ca²⁺ modulates dynamic docking of granules to the plasma membrane and that this process is due to a Ca²⁺-dependent interaction between SNAP-25 and Synaptotagmin 1 .     

Computer-based design of an HLA-haplotype and HIV-clade independent cytotoxic T-lymphocyte assay for monitoring HIV-specific immunity

BACKGROUND: Human immunodeficiency virus (HIV)- specific CD8-positive cytotoxic T-lymphocytes (CTL) play a key role in controlling HIV infection. Monitoring CTL response could be clinically relevant during structured therapy interruption (STI), HIV exposure, and vaccine trials. However, HLA patients' restriction and HIV variability limited the development of a CTL assay with broad specificity. MATERIALS AND METHODS: We designed an HLA-class I/HIV-1 clade independent assay for assessing HIV- specific CTL by using a computer-assisted selection ofthe CTL epitopes. Twenty-eight 15-mers were selected by peptide-binding motifs analysis using different databases (HIV-Immunology Database, SYFPEITHI, BIMAS). Altogether they putatively bind to more than 90% of HLA haplotypes in different populations, with an overall HIV-1 variability below 9%. The peptide pool was used as an antigen in an intracellular cytokine staining (ICS) assay for quantifying HIV-specific CTL response. RESULTS: The test can be performed using both fresh and cryopreserved peripheral blood mononuclear cells (PBMC), whereas GAG protein as antigen works only on fresh PBMC. A significantly higher CTL response with respect to HIV-negative controls was detected in all HIV-1 infected subjects of two groups of patients with different ethnicities (Caucasians and Africans) and coming from areas with different HIV-1 clade prevalences (clade B and A/G, respectively). In Caucasian patients, after month of STI, the number of HIV-1 specific CTL (2,896 +/- 2,780 IFN-gamma specific CD8 cells/ml) was significantly higher than that found at enrolment (2,125 +/- 4,426 IFN-gamma specific CD8 cells/ml, p< 0.05). CONCLUSIONS: These data indicate that this CTL assay is broadly specific and could represent a useful clinical tool for HIV immunodiagnostic independent of HLA-haplotype and HIV-clade variabilities.     

High-level expression of the Listeria monocytogenes listeriolysin O in Escherichia coli and preliminary characterization of the purified protein

Listeriolysin O (LLO) is a cholesterol-binding sulfhydryl-activated hemolysin encoded by Listeria monocytogenes hlyA gene. After analyzing the nucleotide coding sequence of this gene from the ATCC 9525 L. monocytogenes strain, we cloned it in a pET vector for expression in Escherichia coli. Thanks to the optimization of the induction protocol, we achieved a high-level LLO synthesis (about 10% of total cell proteins) in hemolytically active form. The expressed hemolysin was then purified to homogeneity, as revealed by SDS-PAGE and Western blot analysis, by a hydroxyapatite adsorption chromatography, followed by an SP Sepharose ion-exchange chromatography. The recombinant protein showed the same properties determined for LLO purified from L. monocytogenes cultures and the characteristics of the sulfhydryl-activated toxins such as inactivation by oxidation and by reaction with cholesterol. By a combination of the pET expression system and the simple purification method, we obtained a significant amount of toxin (4.5 mg/litre cell culture) in a hemolytically active form (1.25 x 10(6)HU/mg protein). This procedure can solve the problem of LLO isolation from L. monocytogenes cultures, which is a difficult task, mainly owing to the low levels of toxin released in the culture media. The recombinant hemolysin, purified in sufficient quantities, could be very useful for structural studies and for diagnostic and pharmaceutical applications.     

The impact of different antioxidant agents alone or in combination on reactive oxygen species,antioxidant enzymes and cytokines in a series of advanced cancer patients at different sites: correlation with disease progression

In the present study we tested the ability of different antioxidant agents, used alone or in combination, to reduce the reactive oxygen species (ROS) levels and to increase the glutathione peroxidase (GPx) activity. Moreover, we tested the ability of such antioxidant agents to reduce the serum levels of proinflammatory cytokines IL-6 and TNFalpha. Fifty-six advanced stage cancer patients with tumors at different sites were included in the study: they were mainly stage III (12.5%) and stage IV (82.1%). The study was divided into two phases. In the 1st phase 28 patients were divided into five groups and a single different antioxidant agent was administered to each group. The selected antioxidant agents were: alpha lipoic acid or carboxycysteine-lysine salt, amifostine, reduced glutathione, vitamin A plus vitamin E plus Vitamin C. In the 2nd phase of the study 28 patients were divided into five groups and a combination of two different antioxidant agents was administered to each group. The antioxidant treatment was administered for 10 consecutive days. The patients were studied at baseline and after antioxidant treatment. Our results show that all single antioxidants tested were effective in reducing the ROS levels and three of them in increasing GPx activity, too. Among the combinations of antioxidant agents, three were effective in reducing ROS, while three were effective in increasing GPx activity (arm 4 was effective in both instances). Comprehensively, the "antioxidant treatment" was found to be effective both on ROS levels and GPx activity. Moreover, the antioxidant treatment was able to reduce serum levels of IL-6 and TNFalpha. Furthermore, a correlation was shown between the Eastern Cooperative Oncology Group Performance Status of patients and blood levels of ROS, GPx activity, serum levels of proinflammatory cytokines.     

Antioxidant activity of oligomeric acylphloroglucinols from Myrtus communis L

The use of myrtle (Myrtus communis L.) as a culinary spice and as a flavoring agent for alcoholic beverages is widespread in the Mediterranean area, and especially in Sardinia. Myrtle contains unique oligomeric non-prenylated acylphloroglucinols, whose antioxidant activity was investigated in various systems. Both semimyrtucommulone (1) and myrtucommulone A (2) showed powerful antioxidant properties, protecting linoleic acid against free radical attack in simple in vitro systems, inhibiting its autoxidation and its FeCl3- and EDTA-mediated oxidation. While both compounds lacked pro-oxidant activity, semimyrtucommulone was more powerful than myrtucommulone A, and was further evaluated in rat liver homogenates for activity against lipid peroxidation induced by ferric-nitrilotriacetate, and in cell cultures for cytotoxicity and the inhibition of TBH- or FeCl3-induced oxidation. The results of these studies established semimyrtucommulone as a novel dietary antioxidant lead.