Lipid mediators in marine diatoms

Aquatic Ecology - Diatoms are eukaryotic microalgae representing one of the major groups in the marine phytoplankton, accounting for up to 40% of annual productivity at sea. They are widely...     

Crystal structure at 1.5-A resolution of Pyrus pyrifolia pistil ribonuclease responsible for gametophytic self-incompatibility

The crystal structure of the Pyrus pyrifolia pistil ribonuclease (S(3)-RNase) responsible for gametophytic self-incompatibility was determined at 1.5-A resolution. It consists of eight helices and seven beta-strands, and its folding topology is typical of RNase T(2) family enzymes. Based on a structural comparison of S(3)-RNase with RNase Rh, a fungal RNase T(2) family enzyme, the active site residues of S(3)-RNase assigned were His(33) and His(88) as catalysts and Glu(84) and Lys(87) as stabilizers of an intermediate in the transition state. Moreover, amino acid residues that constitute substrate binding sites of the two RNases could be superimposed geometrically. A hypervariable (HV) region that has an S-allele-specific sequence comprises a long loop and short alpha-helix. This region is far from the active site cleft, exposed on the molecule's surface, and positively charged. Four positively selected (PS) regions, in which the number of nonsynonymous substitutions exceeds that of synonymous ones, are located on either side of the active site cleft, and accessible to solvent. These structural features suggest that the HV or PS regions may interact with a pollen S-gene product(s) to recognize self and non-self pollen.     

Evidence for the association of ultraviolet-C and H(2)O(2)-induced apoptosis with acid sphingomyelinase activation

We measured the temperature dependence of oxygen evolution in thylakoids from tobacco using mass spectrometry and high resolution polarography. We determined the initial S-state distribution and the efficiency of the transition between these states including the probability of the O(2) yield through a fast mode. We observed discontinuous changes of the parameters at the temperatures 11 degrees C, 15 degrees C and 21 degrees C. Due to the mass spectroscopy data we think that the irregularity observed at 11 degrees C is due to conformational changes within the water catalytic site. We show that the different contributions of the slow and fast modes of oxygen evolution and of the water molecule exchange are correlated and that their behavior can be explained in terms of the H(2)O accessibility to the water splitting enzyme.     

Effects of nitric oxide on matrix metalloproteinase-2 production by rheumatoid synovial cells

Nitric oxide (NO) is a multifunctional messenger molecule generated from L-arginine by a family of enzymes, including nitric oxide synthase (NOS). This study was performed to examine whether NO modulates the production of matrix metalloproteinases (MMPs), which degrade all components of extracellular matrix (ECM), in rheumatoid synovial cells. We investigated the effects of exogenously generated NO by a NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), on the MMPs production by rheumatoid synovial cells. Culture media conditioned by SNAP-treated synovial cells were examined by gelatin zymography and immunoblot analysis. Incubation of synovial cells with SNAP resulted in gelatinase A production in a dose-dependent fashion. Furthermore, RT-PCR analysis demonstrated that MMP-2 mRNA expression was induced in SNAP-treated synovial cells. In contrast, SNAP did not influence the production of TIMP-1 and TIMP-2, which preferentially inhibit MMP-2, by rheumatoid synovial cells. Our data indicate that NO could modulate MMP production by rheumatoid synovial cells and therefore contribute to ECM degradation of articular components in RA.     

Chemo-enzymatic synthesis and structure-activity study of artificially N-glycosylated eel calcitonin derivatives with a complex type oligosaccharide

Starting from N-glycosylated eel calcitonin derivatives that contain an N-acetyl-D-glucosamine residue specifically at the 3rd, 14th, 20th or 26th amino acid residue, corresponding glycopeptides with a complex-type oligosaccharide attached to the respective amino acid residue were synthesized by means of a transglycosylation reaction catalyzed by an endo-beta-N-acetylglucosaminidase from Mucor hiemalis . The use of a recombinant enzyme and an excess of a glycosyl donor led to a yield in excess of 60%. Calcitonin derivatives containing truncated oligosaccharides were also prepared via digestion of the complex-type N-glycan with exoglycosidases. Using these N-glycosylated calcitonin derivatives, the effect of carbohydrate structure and glycosylation site on the three-dimensional structure and the biological activity of the peptide were studied. The conformation of the peptide backbone did not change irrespective of the carbohydrate structure or the glycosylation site. However, hypocalcemic activity, calcitonin-receptor binding activity and the biodistribution of the derivatives were affected by the glycosylation and were dependent on both the carbohydrate structure and the glycosylation site. Although the larger oligosaccharides tended to hinder receptor binding, the biodistribution altered by N-glycosylation appeared to enhance the hypocalcemic activity in some cases, and the magnitude of the effect was dependent on the site of glycosylation.     

Polyclonal T-cell activation protocol stimulates preferential expansion of EBV-specific T-cell clones in vitro

Purpose Allogeneic bone marrow transplantation (AlloBMT) can be curative for patients with leukemia. The most important anti-leukemic effect may be mediated by the T-cells contained within the graft; however, the allogeneic T-cells may also give rise to graft-vs-host disease (GVHD). One way to control GVHD might be to transduce the donor T-cells with a drug-inducible suicide gene. If a retrovirus vector is to be used for this transduction, activation of the T-cells is required for integration of the transgene to occur. The activation protocol should ensure expansion of a broad repertoire of donor T-cells. Notably, T-cells specific for herpes virus family antigens are important for adoptive immunoprotection.Methods To define optimal activation conditions for retrovirus-mediated suicide gene transduction of donor T-cells, we examined the repertoire of CD8+ T-cells in general, and Epstein-Barr virus (EBV) specific T-cells in particular, following two different activation and expansion procedures.Results We found that repeated CD3/CD28 stimulation resulted in a high level of activation-induced T-cell death, affecting in vivo expanded clones, some of which were specific for EBV, in particular. In contrast, initial CD3/CD28 activation followed by proliferation in interleukin-2 lead to expansion of EBV-specific clones over and above the expansion observed for CD8+ T-cells in general.Conclusion These results should impact on protocols for ex vivo activation of T-cells prior to suicide gene transduction.     

Role of follicular dendritic cells in the early HIV-1 infection: in vitro model without specific antibody

About 90% of HIV-1 RNA in the lymph nodes is reported to localize in follicular dendritic cellsnetwork (FDC-NW) as early as several days after infection and as much as that in the late stage. But the mechanism remains to be fully understood. To elucidate the role of follicular dendritic cells (FDC) in the early stage of HIV-1 infection, FDC-like cell strains (FDCLC) were established and they were characterized in the co-culture system with T cells for their effect on HIV-1 trapping and replication in p24 immunoassay, immunohistochemistry as well as confocal and electronmicroscopy. Established FDCLC were positive for CNA-42, S-100alpha and intercellular desmosome-like junctions. L-SIGN and DC-SIGN were also detected in FDCLC. Alu-HIV-1 PCR analysis showed no HIV-1 integration in FDCLC. FDCLC trapped HIV-1 and transferred them to uninfected MOLT-4 T cells (MOLT-4) efficiently in the absence of specific antibody. FDCLC also accelerated HIV-1 replication in HIV-1-pre-exposed MOLT-4. These unique FDCLC effects were explained, at least partly, by the fact that FDCLC up-regulated CD4 expression in MOLT-4 and helped T cells escape from apoptosis in the co-culture. These data suggest that FDC/FDCLC engage not only in trapping but also in active expansion of HIV-1 in the absence of specific antibody.     

Rapid and sensitive NMR method for osmolyte determination

We propose a rapid and sensitive method for osmolyte determination, based on one-dimensional and two-dimensional 1H NMR spectroscopy applied directly on culture of haloalkalophilic Halomonas pantelleriensis and acidothermophilic archaeon Sulfolobus solfataricus, without any extraction procedure. The osmoprotectants hydroxyectoine, ectoine, glutamate, glycine-betaine and treahalose can easily be quantified by integrating the peak areas with respect to an internal standard, and the concentrations evaluated with this method are in excellent agreement with the values previously reported. Furthermore, trace amount of osmoprotectants, often undetectable after extraction procedures, can also be evaluated.     

Catheter-mediated delivery of adenoviral vectors expressing beta-adrenergic receptor kinase C-terminus inhibits intimal hyperplasia and luminal stenosis in rabbit iliac arteries

BACKGROUND: Previous studies have shown that incubation of balloon-injured rat carotid arteries with adenoviral vectors encoding the carboxyl terminus of the beta-adrenergic receptor kinase (Ad2/betaARKct) for 30 min reduces neointima formation. However, it is unclear whether this beneficial effect of betaARKct could be achieved using a catheter-based vector delivery system and whether the observed inhibition of neointima formation translated into a reduction of vessel stenosis. METHODS: In this study, Ad2/betaARKct was infused into the balloon-injured site of rabbit iliac arteries using a porous infusion catheter over 2 min. Twenty-eight days after gene transfer, angiographic and histological assessments were performed. RESULTS: Angiographic and histological assessments indicate significant (p < 0.05) inhibition of iliac artery neointima formation and lumen stenosis by Ad2/betaARKct. Our studies demonstrate that an inhibitory effect of Ad2/betaARKct on neointima formation is achievable using a catheter-based vector delivery system and that the inhibition of neointima formation translates into a gain in the vessel minimal luminal diameter. The extent of inhibition (35%) was comparable to that observed with adenoviral-mediated expression of thymidine kinase plus ganciclovir treatment, a cytotoxic gene therapy approach for restenosis. CONCLUSIONS: These results suggest that adenoviral-mediated gene transfer of betaARKct is a clinically viable cytostatic gene therapy strategy for the treatment of restenosis.