Phenotype dependent differential effects of interleukin-1beta and amyloid-beta on viability and cholinergic phenotype of T17 neuroblastoma cells

Amyloid-beta accumulation in brains of Alzheimer's disease (AD) victims is accompanied by glial inflammatory reactions and preferential loss of cholinergic neurons. Therefore, the aim of this study was to find out whether proinflamatory cytokine interleukin 1beta (IL1beta) modifies effects of amyloid-beta (Abeta) on viability and cholinergic phenotype of septum derived T17 cholinergic neuroblastoma cells. In nondifferentiated T17 cells (NC) Abeta(25-35) (1 microg/ml) caused no changes in choline acetyltransferase (ChAT) activity, acetylcholine (ACh) release, subcellular distribution of acetyl-CoA, but doubled content of trypan blue positive cells. IL1beta (10 ng/ml) increased ACh release (125%) but did not change other parameters of NC. In the presence of Abeta IL1beta also increased ChAT activity (47%), ACh release (100%) but had no effect on acetyl-CoA distribution and cell viability. Differentiation with retinoic acid and dibutyryl cyclic AMP caused over two-fold increase of ChAT activity and ACh content, four-fold increase of ACh release and about 50% decrease of acetyl-CoA level in the mitochondria. In differentiated cells (DC), Abeta decreased ChAT activity (31%), ACh release (47%) and content of acetyl-CoA (80%) in cell cytoplasmic compartment, whereas IL1beta elevated ChAT activity (54%) and ACh release (32%). IL1beta totally reversed Abeta-evoked inhibition of ChAT activity and ACh release and restored control level of cytoplasmic acetyl-CoA but increased fraction of nonviable cells to 25%. Thus, IL1beta could compensate Abeta-evoked cholinergic deficits through the restoration of adequate expression of ChAT and provision of acetyl-CoA to cytoplasmic compartment in cholinergic neurons that survive under such pathologic conditions. These data indicate that IL1beta possess independent cholinotrophic and cholinotoxic activities that may modify Abeta effects on cholinergic neurons.     

Evidence of Nitrosative Damage in the Brain White Matter of Patients with Multiple Sclerosis

Nitric oxide (NO) has been implicated in the pathophysiology of both experimental autoimmune encephalomyelitis and multiple sclerosis (MS). NO-mediated protein damage in MS appears to be confined to large plaques where 3-nitrotyrosine has been detected. To determine whether nitrosative damage takes place beyond visible MS plaques, the occurrence of various NO-triggered protein modifications in normal-appearing white matter (NAWM) of eight MS brains was assessed and compared to that in white matter (WM) of four control brains. As determined by amino acid analysis and western blotting, no evidence of tyrosine nitration was found in the MS samples studied, suggesting that they did not contain appreciable amounts of plaque-derived material. The amino acid composition of total myelin proteins and proteolipid protein (PLP) was also unaltered in the diseased tissue, as was the fatty acid composition of PLP. In addition, we detected no changes in the number of protein free thiols suggesting that oxidation do not occur to any appreciable extent. However, the levels of nitrite in MS-NAWM were higher than those in control WM, while in the MS-gray matter (GM) the concentration of this ion was unaltered. Furthermore, five of the MS samples analyzed, and the same as those with high levels of glial fibrilary acidic protein, showed increased amounts of protein nitrosothiols as determined by the biotin switch method. S-nitrosation of GM proteins was again normal. There was no indication of N-nitrosation of tryptophan and N-terminal amino groups in both control and MS tissue. Overall, the data suggests that WM, but not GM, from MS brains is subjected to considerable nitrosative stress. This is the first report to present direct evidence of increased protein S-nitrosation and nitrite content in the brain parenchyma of MS patients.     

The regulation of the interaction between F-actin and muscle fructose 1,6-bisphosphatase

Interaction between rabbit muscle fructose 1,6-bisphosphatase (FBPase) and rabbit muscle F-actin results in heterologous complex formation [A. Gizak, D. Rakus, A. Dzugaj, Histol. Histopathol. 18 (2003) 135]. Calculated on the basis of co-sedimentation-binding experiments and ELISA assay-binding constant (Ka) revealed that FBPase binds to F-actin with Ka equal to 7.4 x 10(4) M(-1). The binding is down-regulated by ligands interacting with the FBPase active site (fructose 6-phosphate, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate) and with the FBPase allosteric inhibitory site (AMP). The binding and the kinetic data suggests that FBPase may bind F-actin using a bipartite motif which includes the amino acids residues involved in the binding of the substrate as well as of the allosteric inhibitor of the enzyme. The in situ co-localization experiment, in which FBPase was diffused into skinned muscle fibres pre-incubated with phalloidin (polymeric actin-interacting toxin), has shown that FBPase binds predominantly to the region of the Z-line.     

Fission yeast rad51 and dmc1, two efficient DNA recombinases forming helical nucleoprotein filaments

Homologous recombination is important for the repair of double-strand breaks during meiosis. Eukaryotic cells require two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, for meiotic recombination. To date, it is not clear, at the biochemical level, why two homologs of RecA are necessary during meiosis. To gain insight into this, we purified Schizosaccharomyces pombe Rad51 and Dmc1 to homogeneity. Purified Rad51 and Dmc1 form homo-oligomers, bind single-stranded DNA preferentially, and exhibit DNA-stimulated ATPase activity. Both Rad51 and Dmc1 promote the renaturation of complementary single-stranded DNA. Importantly, Rad51 and Dmc1 proteins catalyze ATP-dependent strand exchange reactions with homologous duplex DNA. Electron microscopy reveals that both S. pombe Rad51 and Dmc1 form nucleoprotein filaments. Rad51 formed helical nucleoprotein filaments on single-stranded DNA, whereas Dmc1 was found in two forms, as helical filaments and also as stacked rings. These results demonstrate that Rad51 and Dmc1 are both efficient recombinases in lower eukaryotes and reveal closer functional and structural similarities between the meiotic recombinase Dmc1 and Rad51. The DNA strand exchange activity of both Rad51 and Dmc1 is most likely critical for proper meiotic DNA double-strand break repair in lower eukaryotes.     

Identical triplets and twins developed from isolated blastomeres of 8- and 16-cell mouse embryos supported with tetraploid blastomeres

We studied the developmental potential of single blastomeres from early cleavage mouse embryos. Eight- and sixteen-cell diploid mouse embryos were disaggregated and single blastomeres from eight-cell embryos or pairs of sister blastomeres from sixteen-cell embryos were aggregated with 4, 5 or 6 tetraploid blastomeres from 4-cell embryos. Each diploid donor embryo gave eight sister aggregates, which later were manipulated together as one group (set). The aggregates were cultured in vitro until the blastocyst stage, when they were transferred (in sets) to the oviducts of pseudopregnant recipients. Eighteen live foetuses or pups were obtained from the transfer (11.0% of transferred blastocysts) and out of those, eleven developed into fertile adults (one triplet, one pair of twins and four singletons). In all surviving adults, pups and living foetuses, only diploid cells were detected in their organs and tissues as shown by analysis of coat pigmentation and distribution of glucose phosphate isomerase isoforms. In order to explain the observed high rate of mortality of transferred blastocysts, in an accompanying experiment, the diploid and tetraploid blastomeres were labelled with different fluorochromes and then aggregated. These experiments showed the diploid cells to be present not only in the inner cell mass (ICM) but also in the trophectoderm. The low number of diploid cells and the predominance of tetraploid cells in the ICM of chimaeric blastocysts might have been responsible for high postimplantation mortality of our experimental embryos.     

Bacterial cell wall-expressed protein A triggers supraclonal B-cell responses upon in vivo infection with Staphylococcus aureus

We have previously shown that staphylococcal protein A (SpA) anchored to the cell wall of Staphylococcus aureus acts as a virulence factor in septic arthritis. Apart from the ability of SpA to interact with Fcgamma, it also binds to Fab-regions with immunoglobulin heavy chains encoded by the V(H) clan III gene family. The objective of the present study was to investigate whether in vivo expression of SpA by staphylococci induces V(H)III-dependent supraclonal B-cell responses, and whether such responses may affect the ability of the host to produce anti-staphylococcal antibodies. Upon primary infection of mice, a SpA-expressing staphylococcal strain gave rise to significantly higher serum levels of V(H)III-encoded antibodies specific for SpA devoid of Fcgamma-binding ability (MSpA) than an isogeneic spa deletion mutant strain. The V(H)III-dependence of MSpA-specific antibody responses was affected by the size of the staphylococcal inoculum, and differed for IgM and IgG isotypes. Mice that had recovered from a prior mild infection from a SpA-expressing strain were protected against infection-induced weight loss upon reinfection. Although no lasting MSpA-specific IgG was induced by previous mild infection, these protected mice possessed IgG specific for clumping factor A, a conventional staphylococcal protein antigen. Our findings demonstrate that the expression of a B-cell superantigen during staphylococcal infection causes supraclonal changes to the immune system. Notably, while superantigen-triggered B-cell responses do not favor the development of SpA-specific memory B-cells, such responses do not interfere with the development of antibodies specific for a staphylococcal protein antigen associated with protective immunity.     

Parallel solid-phase synthesis and characterization of new sulfonamide and carboxamide proline derivatives as potential CNS agents

A solid-phase synthesis of the 64-member library of novel sulfonamide and carboxamide proline derivatives, focused on the 5-HT7 receptor antagonist SB-258741, was described. The final compounds were obtained in good yields and high purity upon cleavage from SynPhase Lanterns, functionalized by a BAL linker. The library representatives were screened for 5-HT7, 5-HT1A and D2 receptors to explore the impact of a tertiary amine moiety, the length of an alkylene spacer and the aryl fragment on the receptor affinity. The preliminary biological results provided data for further investigation aimed at a search for 5-HT7 receptor agents, and permitted the identification of several compounds with significant 5-HT1A receptor affinity.     

Major structural proteins of type 1 and type 3 Klebsiella fimbriae are effective protein carriers and immunogens in conjugates as revealed from their immunochemical characterization

Fimbriae are filamentous structures present on the cell surface of many bacteria, including genus Klebsiella. The use of fimbriae as protein carriers in conjugates may allow to formulate effective multivalent vaccines and suitable diagnostics. However, the evidences have been reported that fimbriae may enhance the inflammatory response. This prompted us to examine the degree of cytokine induction by the type 1 and type 3 Klebsiella fimbriae and their conjugates. Fimbriae were assessed as carrier proteins for Escherichia coli K12 endotoxin core oligosaccharide. MALDI-MS revealed the molecular mass of fimbrial monomer major protein, which was 15,847 Da for type 1 and 18,574 Da for type 3 fimbriae of Klebsiella. These two types of fimbriae were moderate inductors of IL-6 and interferon and almost inactive with regard to the stimulation of TNF when tested in human whole blood assay. Coupling of fimbriae with E. coli K12 core oligosaccharide gave immunogenic conjugates with respect to a saccharide ligand and protein carrier, although only 10% of the pilin monomers possessed the attached oligosaccharide. Rabbit antiserum reacted with a broad spectrum of lipopolysaccharides, as measured by ELISA and immunoblotting assays. The antibodies against glycoconjugates were bactericidal for the wild, S-type bacteria of some species. Regarding the induction of cytokines by conjugates only the TNF level was noticeably elevated. These results prompt for the practical use of fimbriae, as effective protein carriers for conjugates to obtain broad-spectrum antisera for diagnostic applications.