Fibroblast-like synoviocytes from rheumatoid arthritis patients express functional IL-15 receptor complex: endogenous IL-15 in autocrine fashion enhances cell proliferation and expression of Bcl-x(L) and Bcl-2

The hallmarks of rheumatoid arthritis (RA) are leukocytic infiltration of the synovium and expansiveness of fibroblast-like synoviocytes (FLS). The abnormal proliferation of FLS and their resistance to apoptosis is mediated, at least in part, by present in RA joints proinflammatory cytokines and growth factors. Because IL-15 exerts properties of antiapoptotic and growth factors, and is produced by RA FLS, we hypothesized that IL-15 participates in RA FLS activation. To test this hypothesis, we first examined whether RA FLS express chains required for high affinity functional IL-15R. Indeed, RA FLS express IL-15Ralpha at mRNA and protein levels. Moreover, we confirmed the presence of IL-2Rbeta and common gamma-chains. Interestingly, TNF-alpha or IL-1beta triggered significant elevation of IL-15Ralpha chain at mRNA and protein levels. Next, we investigated the effects of exogenous or endogenous IL-15 on Bcl-2 and Bcl-x(L) expression, FLS proliferation, and apoptosis. Exogenous IL-15 enhanced RA FLS proliferation and increased the level of mRNA-encoding Bcl-x(L). To test the role of endogenous IL-15 in the activation of RA FLS, an IL-15 mutant/Fcgamma2a protein exerting properties of specific antagonist to the IL-15Ralpha chain was used. We found that blocking IL-15 biological activities using this protein substantially reduced endogenous expression of Bcl-2 and Bcl-x(L), and RA FLS proliferation that was reflected by increased apoptosis. Thus, we have demonstrated that a distinctive phenotype of RA FLS, i.e., persistent activation, proliferation, and resistance to apoptosis, is related to the autocrine activation of IL-15Rs by FLS-derived IL-15.     

Factors affecting the yield and properties of bacterial cellulose

Acetobacter xylinum E₂₅ has been applied in our studies in order to find optimal culture conditions for effective bacterial cellulose (BC) production. The strain displays significantly higher stability in BC production under stationary culture conditions. In contrast, intensive agitation and aeration appear to drastically reduce cellulose synthesis since such conditions induced formation of spontaneous cellulose nonproducing mutants (Cel−), which dominated in the culture. Mutation frequency strictly depends on the medium composition in agitated cultures. Enrichment of the standard SH and Yamanaka media with 1% ethanol significantly enhanced BC production in stationary cultures. Horizontal fermentors equipped with rotating discs or rollers were successfully applied in order to improve culture conditions. Relatively slow rotation velocity (4 rpm) and large surface area enabling effective cell attachment are optimal parameters for cellulose production. Physical properties of BC samples synthesized either in stationary cultures or in a horizontal fermentor revealed that cellulose from stationary cultures demonstrated a much higher value of Young's modulus, but a much lower value of water-holding capacity. Journal of Industrial Microbiology & Biotechnology (2002) 29, 189–195 doi:10.1038/sj.jim.7000303 Received 01 March 2002/ Accepted in revised form 18 July 2002     

Ex vivo apoptotic potential of peripheral blood mononuclear cells of the elderly human subject

Studies of immunosenescence have led to a detailed knowledge of immune system dysfunctions in the ageing human being. Apoptosis seems to be one of the process regulating an immune response after the antigenic stimulation. We examined whether commonly used methods of assessing apoptosis in the elderly human subject produce comparable results to young subjects. PBMC of young and elderly volunteers were isolated from the venous blood and cultured for 6 or 24 h with antigens of anti-influenza vaccine or PMA. The intensity of apoptosis was measured using the annexinV test, flow cytometric evaluation of DNA content (sub-G1 peak in DNA histograms), 'ladder' by DNA gel electrophoresis, and fluorescence microscope. Apoptosis in 6 h-lasting cultures of the elderly was more intense in annexinV test, while it was decreased assessing subG1 peak. Additionally, in the aged group, those changes were associated with cell cycle arrest. Our results suggest that the apoptosis after the stimulation with the vaccine antigens seems to be some kind of activation-induced cell death (AICD). Different patterns of apoptosis after stimulation may be associated with the cell cycle arrest of the PBMC in the elderly.     

Diabetes-induced changes of nitric oxide influence on the cardiovascular action of secretin

The modulation by condition of the lack or the excess of nitric oxide (NO) on cardiovascular action of secretin in diabetic rats was investigated. In vitro the isolated heart function and in vivo, the systolic (SBP), diastolic (DSP) blood pressure and heart rate (HR) were measured. Secretin evoked inotropic positive effect and increased coronary outflow (CO), in vivo did not increase systemic pressure and the highest dose of the peptide increased the heart rate. NO synthase inhibitor, N(G) nitro-L-arginine methyl ester (L-NAME) deeply increased the systemic pressure and in vitro decreased coronary outflow. L-arginine and sodium nitroprusside (SNP) did not influence the isolated heart function and in vivo decreased the systemic pressure. L-NAME preserved the inotropic positive effect of secretin and the increase of the coronary outflow. In vivo co-administration of L-NAME+secretin evoked hypotensive effect and abolished the increase of the heart rate after the highest dose of the peptide. L-arginine abolished inotropic positive effect of the peptide and the increase of coronary outflow. In vivo co-administration of these substances caused hypotension and attenuated the increase of the heart rate after the highest dose of secretin. Co-injection of SNP and secretin preserved the inotropic effect of secretin and abolished the increase of the coronary outflow. In vivo infusion of SNP+secretin evoked hypotension and similarly to L-arginine, SNP abolished tachycardia induced by the highest dose of secretin. Both the lack and the excess of nitric oxide changed the cardiovascular action of secretin in diabetic rats.     

Low temperature-induced modifications in cell ultrastructure and localization of phenolics in winter oilseed rape (Brassica napus L. var. oleifera L.) leaves

Acclimation of winter oilseed plants in the cold (i.e. at temperatures >0 degrees C) followed by short exposure to sub-lethal freezing temperatures resulted in pronounced ultrastructural changes of leaf epidermal and mesophyll cells. The following major changes were observed upon acclimation at 2 degrees C: increased thickness of cell walls; numerous invaginations of plasma membranes; the appearance of many large vesicles localized in the cytoplasm in close proximity to the central vacuole; the occurrence of abundant populations of microvesicles associated with the endoplasmic reticulum (ER) cisternae or located in the vicinity of dictyosomes; and the occurrence of paramural bodies and myelin-like structures. In addition, large phenolic deposits were observed in the vicinity of the plasma membrane and membrane-bound organelles such as chloroplasts, large vesicles or cytoplasm/tonoplast interfaces. Transient freezing (-5 degrees C for 18 h) of the cold-acclimated leaves led to reversible disorganization of the cytoplasm and to pronounced structural changes of the cellular organelles. Chloroplasts were swollen, with the stroma occupying one half of their volume and the thylakoid system being displaced to the other half. Large phenolic aggregates disappeared but distinct layers of phenolic deposits were associated with mitochondrial membranes and with chloroplast envelopes. In frost-thawed cells recovered at 2 degrees C for 24 h, dictyosomes and dictyosome- or ER-derived small vesicles reappeared in the ribosome-rich cytoplasm. Aberrations in the structure of chloroplasts and mitochondria were less pronounced. Few phenolic deposits were seen as small grains associated with chloroplast envelopes and vesicle membranes. These observations demonstrate that plants undergo different changes in cell ultrastructure depending on whether they are subjected to chilling or freezing temperatures. Results are discussed in relation to membrane recycling and the possible role of phenolics during the first and second stages of plant acclimation at low temperature.     

Assessment of RAPD markers for barley doubled haploid lines resistant and susceptible to Fusarium culmorum at seedling and adult plant growth stages

Thirty doubled haploid (DH) lines of barley derived from F(1) of a cross between the six-rowed cultivar Pomo and two-rowed cultivar Maresi were examined for susceptibility to Fusarium seedling blight (SB) and head blight (FHB), measured by mycotoxin (nivalenol) content of kernels. RAPD (random amplified polymorphic DNA) polymorphism was analysed by using 53 decamer primers. Amplification products (APs) were 200 bp up to 2000 bp in size on average 5.7 per primer and the total number of APs was 284, 51.06% of which were polymorphic. Only 32 APs differentiated the examined DH lines - 19 APs for nivalenol content of kernels and 13 for seedling resistance. DH lines segregated with continuous distribution of resistance to FHB and SB. At the seedling stage all DH lines exhibited lower susceptibility than parental cultivars, but in the adult stage only two lines (MP 2 and MP 7) appeared to be more resistant to FHB, i.e. accumulated in kernels a lower amount of mycotoxin than cultivars Maresi and Pomo.     

Organization of antibiotic amphotericin B in model lipid membranes. A mini review

Amphotericin B (AmB) is a polyene antibiotic frequently applied in the treatment of fungal infections. According to the general understanding, the mode of action of AmB is directly related to the molecular organization of the drug in the lipid environment, in particular to the formation of pore-like molecular aggregates. Electronic absorption and fluorescence techniques were applied to investigate formation of molecular aggregates of AmB in the lipid environment of liposomes and monomolecular layers formed at the argon-water interface. It appears that AmB dimers, stabilized by van der Waals interactions, are present in the membrane environment along with the aggregates formed by a greater number of molecules. Linear dichroism measurements reveal that AmB is distributed between two fractions of molecules, differently oriented with respect to the bilayer. Molecules in one fraction remain parallel to the plane of the membrane and molecules in the other one are perpendicular. Scanning Force Microscopy imaging of the surface topography of the monolayers formed with AmB in the presence of lipids reveals formation of pore-like structures characterized by the external diameter close to 17 A and the internal diameter close to 6 A. All the findings are discussed in terms of importance of the molecular organization of AmB in the pharmacological action, as well as of the toxic side effects of the drug.     

Ampicillin resistance in Listeria monocytogenes acquired as a result of transposon mutagenesis

We have used plasmid pLTV3, which carries transposon Tn917, to obtain a series of mutants of Listeria monocytogenes EGD showing varied degrees of resistance to ampicillin and other beta-lactam antibiotics, including imipenem. In this paper we focus on the characteristics of two strains in which decreased susceptibility to ampicillin is accompanied by changes in the structure of the cell wall murein and cell-wall related changes of phenotype.