Project description:Histones play a key role in chromatin remodeling and gene transcription. Further, free histones in the blood act as damage-associated molecules. Administration of histones to animals results in systemic inflammatory and toxic effects. Myelin basic protein is the principal constituent element of the myelin-proteolipid sheath of axons. Abzymes (antibodies with catalytic activities) are the original features of some autoimmune diseases. In this study, electrophoretically homogeneous IgGs against H1, H2A, H2B, H3, and H4 histones and myelin basic protein (MBP) were isolated from the blood sera of multiple sclerosis (MS) patients by several affinity chromatographies. Using MALDI mass spectrometry, the sites of H1 histone cleavage by IgGs against H1, H2A, H2B, H3, H4, and MBP were determined. It was shown that IgGs against H1 split H1 at 12 sites, while the number of cleavage sites by abzymes against other histones was lower: H2A (9), H2B (7), H3 (3), and H4 (3). The minimum rate of H1 hydrolysis was observed for antibodies against H3 and H4. A high rate of hydrolysis and the maximum number of H1 hydrolysis sites (17) were found for antibodies against MBP. Only a few sites of H1 hydrolysis by anti-H1 antibodies coincided with those for IgGs against H2A, H2B, H3, H4, and MBP. Thus, the polyreactivity of complexation and the enzymatic cross-activity of antibodies against H1, four other histones, and MBP have first been shown. Since histones act as damage molecules, abzymes against histones and MBP can play a negative role in the pathogenesis of MS and probably other different diseases as well.
Project description:The O antigen is an important cell wall antigen of gram-negative bacteria, and the genes responsible for its biosynthesis are located in a gene cluster. We have cloned and sequenced the DNA segment unique to the O-antigen gene cluster of Salmonella enterica group D3. This segment includes a novel O-antigen polymerase gene (wzyD3). The polymerase gives alpha(1-->6) linkages but has no detectable sequence similarity to that of group D2, which confers the same linkage. We find the remnant of a D3-like wzy gene in the O-antigen gene clusters of groups D1 and B and suggest that this is the original wzy gene of these O-antigen gene clusters.
Project description:Influenza A viruses are classified into 16 subtypes according to the serotypes of hemagglutinin (HA). It is generally thought that neutralizing antibodies (Abs) are not broadly cross-reactive among HA subtypes. We examined the repertoire of neutralizing Abs against influenza viruses in humans. B lymphocytes were collected from donors by apheresis, and Ab libraries were constructed by using phage-display technology. Anti-HA clones were isolated by screening with H3N2 viruses. Their binding activity was examined, and four kinds of Abs showing broad strain specificity were identified from one donor. Two of the Abs, F045-092 and F026-427, were extensively analyzed. They neutralized not only H3N2 but also H1N1, H2N2, and H5N1 viruses, although the activities were largely varied. Flow cytometry suggested that they have the ability to bind to HA and HA1 artificially expressed on the cell surface. They show hemagglutination inhibition activity and do not compete with C179, an Ab thought to bind to the stalk region. F045-092 competes with Abs that recognize sites A and B for binding to HA. Furthermore, the serine at residue 136 in site A could be a part of the epitope. Thus, it is likely that F045-092 and F026-427 bind to a conserved epitope in the head region formed by HA1. Interestingly, while the V(H)1-69 gene can encode MAbs against the HA stem that are group 1 specific, F045-092 and its relatives that recognize the head region also use V(H)1-69. The possible epitope recognized by these clones is discussed.
Project description:Neutrophils are able to neutralize pathogens by phagocytosis, by the release of antimicrobial components, as well as by the formation of neutrophil extracellular traps (NETs). The latter possibility is a DNA-meshwork mainly consisting of highly concentrated extracellular histones, which are not only toxic for pathogens, but also for endogenous cells triggering several diseases. To reduce the negative outcomes initiated by extracellular histones, different approaches like antibodies against histones, proteases, and the polysaccharide polysialic acid (polySia) were discussed. We examined whether each of the individual histones is a binding partner of polySia, and analyzed their respective cytotoxicity in the presence of this linear homopolymer. Interestingly, all of the histones (H1, H2A, H2B, H3, and H4) seem to interact with α2,8-linked sialic acids. However, we observed strong differences regarding the required chain length of polySia to bind histone H1, H2A, H2B, H3, and H4. Moreover, distinct degrees of polymerization were necessary to act as a cytoprotective agent in the presence of the individual histones. In sum, the outlined results described polySia-based strategies to bind and/or to reduce the cytotoxicity of individual histones using distinct polySia chain length settings.
Project description:Rotigotine acts as a dopamine receptor agonist with high affinity for the dopamine D2, D3, D4 and D5 receptors but with a low affinity for the dopamine D1 receptor. We have investigated this further in radioligand binding and functional studies and compared the profile of rotigotine with that of other drugs used in the treatment of Parkinson's disease (PD).The binding of rotigotine to human dopamine D1, D2, D3, D4 and D5 receptors was determined in radioligand binding studies using [(3)H]rotigotine and compared with that of standard antagonist radioligands. Functional interactions of rotigotine with human dopamine receptors was also determined.[(3)H]rotigotine can be used as an agonist radioligand to label all dopamine receptor subtypes and this can be important to derive agonist affinity estimates. Rotigotine maintains this high affinity in functional studies at all dopamine receptors especially D1, D2 and D3 receptors and, to a lesser extent, D4 and D5 receptors. Rotigotine, like apomorphine but unlike ropinirole and pramipexole, was a potent agonist at all dopamine receptors.Rotigotine is a high-potency agonist at human dopamine D1, D2 and D3 receptors with a lower potency at D4 and D5 receptors. These studies differentiate rotigotine from conventional dopamine D2 agonists, used in the treatment of PD, such as ropinirole and pramipexole which lack activity at the D1 and D5 receptors, but resembles that of apomorphine which has greater efficacy in PD than other dopamine agonists but has suboptimal pharmacokinetic properties.
Project description:Distinct clinical features of HBV infection have been associated with different viral genotype/subgenotype. HBV Genotype-D comprised of 10 subgenotypes, D1-D10, whose clinical implications still remain elusive. We investigated for the first-time, the virologic characteristics and cytopathic effects of four non-recombinant D-subgenotypes, D1/D2/D3/D5. Expressions of viral/host genes were evaluated in Huh7 cells transfected with full-length, linear-monomers of HBV/D-subgenotypes or pGL3-Basic vector carrying subgenotype-specific HBx. Intracellular HBV-DNA and pregenomic-RNA levels were high in D1/D2 than D3/D5. Expressions of PreC-mRNA and HBx were highest for D2 and D1 respectively, whereas PreS2/S-transcript was significantly reduced in D5. Increased apoptotic cell death and marked upregulation in caspase-3/Bax/TNF-R1/FasR/TRAIL-R1/ROS/MCP-1/IP-10/MIP-1β expression were noticed specifically in D2- and also in D3-transfected cells, while D5 resulted in over-expression of ER-stress-markers. D-subgenotype-transfected Huh7 cells were co-cultured with PBMC of healthy-donors or LX-2 cells and significant increase in pro-inflammatory cytokines in PBMC and fibrogenic-markers in LX-2 were noticed in presence of D2/D3. Further, Huh7 cells transfected with D1, in particular and also D5, displayed remarkable induction of EMT-markers and high proliferative/migratory abilities. Collectively, our results demonstrated that D2/D3 were more associated with hepatic apoptosis/inflammation/fibrosis and D1/D5 with increased risk of hepatocarcinogenesis and emphasize the need for determining HBV-subgenotype in clinical practice.
Project description:The major small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6, and U5 share a set of common proteins denoted B/B', D1, D2, D3, E, F, and G which play an important part in the biogenesis of the snRNPs. In addition, there is a link between the common proteins and autoimmunity; the three D proteins, together with B/B', are the major autoantigens for the so-called anti-Sm antibodies often produced by patients suffering from systemic lupus erythematosus. Here we describe the characterization of the human proteins D2 and D3 by cDNA cloning and immunological methods. D2 and D3 are encoded by distinct genes and are 118 and 126 amino acids in length, respectively. Both proteins prepared by in vitro translation exhibit Sm epitopes and can be precipitated by anti-Sm autoantibodies. They react differently with various patient sera, in a manner consistent with the reaction pattern on immunoblots of the D proteins isolated from HeLa cells. D1 and D2 synthesized in vitro form specific complexes, a result that is significant for the assembly pathway of the various core proteins into an snRNP's core ribonucleoprotein structure. The D3 protein is homologous to the human D1 protein, showing an overall amino acid sequence identity of 29%, including two regions with over 60% identity. D2 has less than 15% sequence identity with D1 and D3. A data bank search revealed a striking similarity (with more than 40% sequence identity) between human D3 and a Saccharomyces cerevisiae gene, previously published as the 5' flanking gene of yeast pep3 [Preston, R.A., Manolson, M., Becherer, K., Weidenhammer, E., Kirkpatrick, D., Wright, R. & Jones, E. (1991) Mol. Cell. Biol. 11, 5801-5812], suggesting that this gene encodes the yeast homologue of the human D3 protein.
Project description:Histones have a specific key role in the remodeling of chromatin and gene transcription. In the blood, free histones are damage-connected proteins. Myelin basic protein (MBP) is the major component of the myelin-proteolipid sheath of axons. Antibodies possessing enzymatic activities (abzymes, ABZs) are the specific features of several autoimmune pathologies. IgGs against five histones, MBP, and DNA were obtained from the sera of multiple sclerosis (MS) patients using several affinity chromatographies. The sites of H3 histone splitting by Abs against five individual histones, MBP, and DNA were revealed by MALDI mass spectrometry. It was shown that the number of H3 splitting sites by IgGs against five various histones is different (number of sites): H3 (11), H1 (14), H2A (11), H4 (17), MBP (22), and DNA (29). IgGs against five different histones hydrolyze H3 at different sites, and only a few them coincide. The main reason for the enzymatic cross-reactivity of Abs against H3 and four other histones, as well as MBP, might be the high level of these proteins' homology. The effective hydrolysis of the H3 histone at 29 sites with IgGs against DNA can be explained by the formation of chimeric abzymes against hybrid antigenic determinants formed by different histones and MBP at the junction of these protein sequences with DNA. The active centers of such abzymes contain structural elements of canonical DNases and proteases. Since free histones are pernicious proteins, antibodies-ABZs against five histones, MBP, and DNA could have a negative role in the pathogenesis of MS and probably other various autoimmune diseases.