Interleukin 6 in neutrophil cultures and mononuclear cells in blood serum of patients with Lyme disease

The aim of this study was to estimate the release of IL-6 by human neutrophils (PMN) and peripheral blood mononuclear cells (PBMC) in patients with Lyme disease confronted with the serum levels. The cells were isolated from whole blood of 25 patients and of 10 healthy donors and cultured in the presence of LPS. In the culture supernatants and serum the concentration of IL-6 with ELISA (BioSource) were measured. In patients we observed higher values of IL-6 released by unstimulated PMN and PBMC in compared with control. In contrast to control, we didn't observe increased the release of IL-6 by LPS-stimulated PMN and PBMC as compared to unstimulated cells. In the serum of patient we found increased the concentration of IL-6. The higher ability of PMN and PBMC from patients with Lyme disease to release of IL-6 and the lack response to additional stimulation indicate the activation of PMN and PBMC in vivo.     

Free radical scavengers can differentially modulate the genotoxicity of amsacrine in normal and cancer cells

Amsacrine is an acridine derivative drug applied in haematological malignancies. It targets topoisomerase II enhancing the formation of a cleavable DNA-enzyme complex and leading to DNA fragmentation in dividing cancer cells. Little is known about other modes of the interaction of amsacrine with DNA, by which it could affect also normal cells. Using the alkaline comet assay, we showed that amsacrine at concentrations from the range 0.01 to 10 microM induced DNA damage in normal human lymphocytes, human promyelocytic leukemia HL-60 cells lacking the p53 gene and murine pro-B lymphoid cells BaF3 expressing BCR/ABL oncogene measured as the increase in percentage tail DNA. The effect was dose-dependent. Treated cells were able to recover within a 120-min incubation. Amifostine at 14 mM decreased the level of DNA damage in normal lymphocytes, had no effect on the HL-60 cells and potentiated the DNA-damaging effect of the drug in BCR/ABL-transformed cells. Vitamin C at 10 and 50 microM diminished the extent of DNA damage in normal lymphocytes, but had no effect in cancer cells. Pre-treatment of the cells with the nitrone spin trap, N-tert-butyl-alpha-phenylnitrone or ebselen, which mimics glutathione peroxidase, reduced the extent of DNA damage evoked by amsacrine in all types of cells. The cells exposed to amsacrine and treated with endonuclease III and 3-methyladenine-DNA glycosylase II, the enzymes recognizing oxidized and alkylated bases, respectively, displayed greater extent of DNA damage than those not treated with these enzymes. The results obtained suggest that free radicals may be involved in the formation of DNA lesions induced by amsacrine. The drug can also methylate DNA bases. Our results indicate that the induction of secondary malignancies should be taken into account as diverse side effects of amsacrine. Amifostine may potentate DNA-damage effect of amsacrine in cancer cells and decrease this effect in normal cells and Vitamin C can be considered as a protective agent against DNA damage in normal cells.     

Paul-Bunnell antigen and a possible mechanism of formation of heterophile antibodies in patients with infectious mononucleosis

Sera of patients with infectious mononucleosis contain heterophile anti-Paul- Bunnell (PB) antibodies to erythrocytes of numerous mammalian species. Evidence is presented that the corresponding antigen of bovine erythrocytes is not, as previously described, a single molecule, but a series of glycoproteins with glycans terminated with N-glycolylneuraminic acid (Neu5Gc). The latter compound should be an important part of the PB epitope because, in agreement with the results of others, we found that desialylation of the PB antigen abolishes almost completely its activity. We examined three different preparations of GM3 ganglioside for their capacity to bind anti-PB and found that only GM3 from horse erythrocytes containing Neu5Gc exhibited a low although ELISA measurable PB activity. The other two GM3 preparations, from bovine milk and dog erythrocytes, containing N-acetylneuraminic acid (Neu5Ac) bound little if any anti-PB antibodies. This finding confirms a previous report that human erythrocyte Neu5Ac containing sialoglycoprotein with similar O-linked glycans as the PB-antigen of bovine erythrocytes exhibits only very low PB activity (Patarca & Fletcher, 1995, Crit Rev Oncogen., 6: 305). In conclusion, we present a hypothesis that anti-PB antibodies in patients with infectious mononucleosis are formed against infection-induced cell membrane glycoconjugates containing highly immunogenic Neu5Gc.     

Selection of low-molecular-mass trypsin and chymotrypsin inhibitors based on the binding loop of CMTI-III using combinatorial chemistry methods

Using a combinatorial chemistry approach, a decapaptide library containing the N-terminal fragment of trypsin inhibitor CMTI-III was synthesized by the solid-phase method. The peptide library was screened for trypsin and chymotrypsin inhibitory activity applying the iterative method in solution. Two decapeptides were selected and resynthesized for each enzyme. The association equilibrium constants ((1.1+/-0.2)x10(8) and (7.3+/-1.6)x10(7)) determined for peptides with trypsin inhibitory activity indicate that they are 3-4-fold less active than the CMTI inhibitors. On the other hand, they are significantly more effective as compared with the starting sequence. Two peptides selected as chymotrypsin inhibitors displayed about 10 times higher activity (1.7+/-0.4)x10(7) and (1.1+/-0.2)x10(7), respectively) than those monosubstituted in position P(1) of the CMTI-III analogue. Considering low molecular weight of peptides selected and the lack of conformational constraints in their structures, the results are promising. They are good templates as starting sequences for further selection of small, peptidomimetic proteinase inhibitors.     

PPARdelta activation induces COX-2 gene expression and cell proliferation in human hepatocellular carcinoma cells

Cyclooxygenase-2 (COX-2) has been suggested to be associated with carcinogenesis. Recently, many studies have shown increased expression of COX-2 in a variety of human malignancies, including hepatocellular carcinoma (HCC). Therefore, it becomes important to know more about what determines COX-2 expression. In this work, we have studied the effect of PPARdelta activation on COX-2 expression using a selective agonist (GW501516) in human hepatocellular carcinoma (HepG2) cells. Activation of PPARdelta resulted in increased COX-2 mRNA and protein expression. The mechanism behind the induction seems to be increased activity of the proximal promoter of the COX-2 gene, spanning nucleotides -327 to +59. The increased COX-2 protein expression and promoter activity induced by the GW501516 was also confirmed in the monocytic cell line THP-1. Induced levels of COX-2 have previously been associated with resistance to apoptosis and increased cell proliferation in many cell types. In HepG2 cells, we observed a dose-dependent increase in cell number by GW501516 treatment for 72h. The levels of PCNA, used as an indicator of cell division were induced, and the cell survival promoting complex p65 (NF-kappaB) was phosphorylated under GW501516 treatment. We conclude that PPARdelta activation in HepG2 cells results in induced COX-2 expression and increased cellular proliferation. These results may suggest that PPARdelta plays an important role in the development of HCC by modulating expression of COX-2.     

Paramecium decaurelia and Paramecium dodecaurelia from the P. aurelia spp. complex in Japan

The presence of Paramecium decaurelia (three strains) and Paramecium dodecaurelia (two strains) were recorded in Japan, for the first time in this country and outside the USA.     

Unravelling cell wall formation in the woody dicot stem

Populus is presented as a model system for the study of wood formation (xylogenesis). The formation of wood (secondary xylem) is an ordered developmental process involving cell division, cell expansion, secondary wall deposition, lignification and programmed cell death. Because wood is formed in a variable environment and subject to developmental control, xylem cells are produced that differ in size, shape, cell wall structure, texture and composition. Hormones mediate some of the variability observed and control the process of xylogenesis. High-resolution analysis of auxin distribution across cambial region tissues, combined with the analysis of transgenic plants with modified auxin distribution, suggests that auxin provides positional information for the exit of cells from the meristem and probably also for the duration of cell expansion. Poplar sequencing projects have provided access to genes involved in cell wall formation. Genes involved in the biosynthesis of the carbohydrate skeleton of the cell wall are briefly reviewed. Most progress has been made in characterizing pectin methyl esterases that modify pectins in the cambial region. Specific expression patterns have also been found for expansins, xyloglucan endotransglycosylases and cellulose synthases, pointing to their role in wood cell wall formation and modification. Finally, by studying transgenic plants modified in various steps of the monolignol biosynthetic pathway and by localizing the expression of various enzymes, new insight into the lignin biosynthesis in planta has been gained.     

Structure of a new 2-deoxy-2-[(R)-3-hydroxybutyramido]-D-glucose-containing O-specific polysaccharide from the lipopolysaccharide of Citrobacter gillenii PCM 1542

The O-specific polysaccharide of Citrobacter gillenii PCM 1542 from serotype O-12a,12 b is composed of one residue each of D-glucose, D-GlcNAc, 2-deoxy-2-[(R)-3-hydroxybutyramido]-D-glucose (D-GlcNAcyl) and two GalNAc residues. On the basis of sugar and methylation analyses of the intact and Smith degraded polysaccharides, along with 1D and 2D 1H and 13C NMR spectroscopy, the following structure of the branched pentasaccharide repeating unit of the O-specific polysaccharide was established:This structure differs significantly from that of the O-specific polysaccharide of C. gillenii PCM 1544 from the same serotype O-12a,12 b, which has been established earlier (Kübler-Kielz.shtsls;b, J. et al. Carbohydr. Res. 2001, 331, 331-336). Serological studies confirmed that the two O-antigens are not related and suggested that strains PCM 1542 and 1544 should be classified into different O-serogroups.     

Phenol utilisation by fungi isolated from activated sludge

The paper presents the efficiency of phenol removal (concentrations from 500 to 2000 mg/l) by fungi isolated from activated sludge purifying wastewater with high phenol concentration. Five fungal strains were isolated and identified. All isolated strains appeared to be Moniliales from the class of Fungi Imperfecti (Candida sp., Monosporium sp., Trichosporon sp.) Stationary cultures of the individual strains and their mixtures were maintained in Czapek medium containing phenol in concentration from 500 to 2000 mg/l. All isolated strains (except one) were capable of utilising phenol up to a concentration of 1500 mg/l. Depending on investigated strain, phenol in concentration of 500 mg/l was decomposed during 4-25 days, 750 mg/l during 4-14 days. After 20 days, a phenol decline of 1000 mg/l was observed. After 16 days, the phenol decline was 1500 mg/l. Higher phenol concentrations (1500 mg/l) were utilised only by a mixture of two strains. The investigated fungal strains showed good efficiency of phenol removal from high phenol concentration in wastewater and they may be proposed for use in the process of purifying wastewater of this type.     

Integrative nuclear FGFR1 signaling (INFS) pathway mediates activation of the tyrosine hydroxylase gene by angiotensin II,depolarization and protein kinase C

The integrative nuclear FGFR1 signaling (INFS) pathway functions in association with cellular growth, differentiation, and regulation of gene expression, and is activated by diverse extracellular signals. Here we show that stimulation of angiotensin II (AII) receptors, depolarization, or activation protein kinase C (PKC) or adenylate cyclase all lead to nuclear accumulation of fibroblast growth factor 2 (FGF-2) and FGFR1, association of FGFR1 with splicing factor-rich domains, and activation of the tyrosine hydroxylase (TH) gene promoter in bovine adrenal medullary cells (BAMC). The up-regulation of endogenous TH protein or a transfected TH promoter-luciferase construct by AII, veratridine, or PMA (but not by forskolin) is abolished by transfection with a dominant negative FGFR1TK-mutant which localizes to the nucleus and plasma membrane, but not by extracellularly acting FGFR1 antagonists suramin and inositolhexakisphosphate (IP6). Mechanism of TH gene activation by FGF-2 and FGFR1 was further investigated in BAMC and human TE671 cultures. TH promoter was activated by co-transfected HMW FGF-2 (which is exclusively nuclear) but not by cytoplasmic FGF-1 or extracellular FGFs. Promoter transactivation by HMWFGF-2 was accompanied by an up-regulation of FGFR1 specifically in the cell nucleus and was prevented FGFR1(TK-) but not by IP6 or suramin. The TH promoter was also transactivated by co-transfected wild-type FGFR1, which localizes to both to the nucleus and the plasma membrane, and by an exclusively nuclear, soluble FGFR1(SP-/NLS) mutant with an inserted nuclear localization signal. Activation of the TH promoter by nuclear FGFR1 and FGF-2 was mediated through the cAMP-responsive element (CRE) and was associated with induction of CREB- and CBP/P-300-containing CRE complexes. We propose a new model for gene regulation in which nuclear FGFR1 acts as a mediator of CRE transactivation by AII, cell depolarization, and PKC.