Toll-like receptors in rheumatoid arthritis joint destruction mediated by two distinct pathways.
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Animals↗
Biomedical subjects
R Seibl
Publications and source records attributed to R Seibl.
[Classification and predictive quality of the "suicide intent" marker in parasuicides. A cluster analytic study].
The aim of the study was to investigate the discriminant and predictive quality of "suicide intent" (SIS) in relation to motives for deliberate self-harm (DSH). Cluster analysis was performed in a sample of 137 inpatients admitted to a psychiatric hospital after a DSH episode. Six different subgroups were isolated. The hypothesis was tested that the six groups would reveal distinct patterns of motives and different patterns of repeated DSH episodes during a 12-month observation period. Results indicated that one high-risk group for completed suicide was characterized by the highest death intention and remarkably low interpersonal motives. Two further groups were labeled as "moderate" risk groups. Two groups were found to be characterized by low suicide intent with prevailing patterns of distinct interpersonal oriented motives (manipulative vs appellative), whereas one group revealed an ambiguous motive structure reflecting death orientation and interpersonal motives. The DSH repetition rates were found to be significantly different between certain subgroups. The study results support the assumption that DSH patients represent heterogeneous populations with regard to suicide intent, motives, and the repetition of deliberate self-harm.
Adult↗
Human Toll-like receptor 2 mediates induction of the antimicrobial peptide human beta-defensin 2 in response to bacterial lipoprotein.
Recognition of pathogens by Drosophila Toll or human Toll-like receptors results in translocation of Dorsal or its human homologue NF-kappaB, respectively; in Drosophila, this is followed by the production of antimicrobial peptides serving as antimicrobial effector system of the innate immune response. We investigated whether human Toll-like receptors also mediate induction of the synthesis of antimicrobial peptides. We found that HEK293 cells transfected with Toll-like receptor 2, but not wild-type cells responded to stimulation with bacterial lipoprotein by production of human beta-defensin 2. Furthermore, the human lung epithelial cell line A549 was found to constitutively express Toll-like receptor 2 and to produce beta-defensin 2 in response to bacterial lipoprotein. This response was abrogated by blocking the signaling pathway activated through Toll-like receptors by transfecting the A549 cells with a dominant-negative form of IRAK-2. Thus, exposure of human cells to bacterial lipoprotein elicits production of the antimicrobial peptide beta-defensin 2 through Toll-like receptor 2.
Bacterial Proteins↗
[The consequences of a suicide for the bereaved - current knowledge, unresolved questions and tasks of future research].
UNLABELLED: Since the publication of Cain's landmark book Survivors of Suicide in 1972, the consequences of a suicide for the bereaved have been scientifically investigated. Mclntosh (1996) issued the most current bibliography of literature on the aftermath of suicide, including publications issued between 1986 and 1995. Mclntosh came to the conclusion that several unresolved issues still exist in this field of suicidology. OBJECTIVE: This paper aims at giving an overview on current knowledge of the aftermath of suicide. lt shall be investigated which questions and unresolved issues in this field still remain. This leads to a description of future tasks and challenges for researchers investigating the after effects of suicide. METHODS: At first, an overview on the most important aspects of current knowledge on the aftermath of suicide will be given. This will be done with reference to the unresolved issues and unanswered questions presented by McIntosh. How many of these issues and questions have come to a resolution after the publication of Mclntosh's bibliography shall be investigated. RESULTS: Upon examination of publications issued after 1995 it was found that several basic questions regarding the after effects of a suicide have not yet been answered in a satisfactory manner. To name a few, the term suicide survivor has not yet been clearly determined and reliable numbers as to how many people are bereaved by suicide do not exist. Moreover, a comprehensive theory regarding the consequences of a suicide for survivors is still lacking. CONCLUSIONS: Resolving these basic questions among a number of others can be seen as the main task of future research in this field. Additionally, it is pointed out that more affention should be given to the implementation of suicide postvention measures in order to alleviate the negative aftereffects of suicides.
Adaptation, Psychological↗
[Survivors of suicide victims searching for answers and ways out of the crisis].
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Adaptation, Psychological↗
Nonradioactive 3'-end-labeling of RNA molecules of different lengths by terminal deoxynucleotidyltransferase.
Terminal deoxynucleotidyltransferase is well known for its ability to add nucleotides to the 3'-ends of DNA; here we demonstrate a corresponding activity with RNA as acceptor molecule. Using DIG- or biotin-dUTP as substrate, a tailing reaction is used for nonradioactive 3'-end-labeling of RNA oligonucleotides, isolated RNA molecules such as tRNA from E. coli, and MS2 RNA as well as in vitro transcripts. The tail length and associated sensitivity of detection can be varied using DIG-ddUTP, DIG-dUTP, or a nucleotide mixture of DIG-dUTP and dATP.
Base Sequence↗
The digoxigenin (DIG) system for non-radioactive labelling and detection of nucleic acids--an overview.
The use of non-radioactive nucleic acid probes has increased dramatically over the last years. The convenience of not having to deal with radioactive isotopes combined with the stability and economy of the non-radioactive systems has led to applications in various techniques. One of the most successful labelling and detection systems is based on the hapten digoxigenin. Here, the different methods for labelling nucleic acids with digoxigenin as well as the various possibilities for detection are described. Some typical applications illustrate the utility of the DIG system.
Animals↗
A new method for measuring reverse transcriptase activity by ELISA.
A new and sensitive assay of reverse transcriptase (RT) activity of retroviruses measures the incorporation of digoxigenin-labelled dUTP in newly synthesized DNA instead of radioactively labelled (3H- or 32P-)dTTP. To avoid difficulties associated with separation of non-incorporated nucleotides from the newly synthesized DNA, biotin-labelled dUTP is added to the reaction mixture in very low concentrations. After reverse transcription, the newly synthesized, doubly labelled DNA is immobilized on streptavidin-coated ELISA wells and evaluated photometrically by binding of peroxidase-conjugated anti-digoxigenin-antibodies (sheep) and subsequent colour development with 2,2'-azino-di[3-ethylbenzthiazolin-sulfonate(6)] (ABTSR) as substrate. For better standardization, it is suggested that RT activity is given in units (one unit of RT is the amount of enzyme incorporating one nanomole of labelled dNTP in 10 min at 37 degrees C into an acid precipitable DNA) rather than in cpm (counts per minute). The method is specific and easy to perform.
Avian Myeloblastosis Virus↗
Nonradioactive in situ hybridization with digoxigenin labeled DNA probes.
Nonradioactive in situ hybridization techniques are becoming increasingly important tools for rapid analysis of the topological organization of DNA and RNA sequences within cells. Prerequisite for further advances with these techniques are multiple labeling and detection systems for different probes. Here we summarize our results with a recently developed labeling and detection system. The DNA probe for in situ hybridization is modified with digoxigenin-labeled deoxyuridine-triphosphate. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Labeled DNA probes were hybridized in situ to chromosome preparations. The hybridization signal was detected using digoxigenin-specific antibodies covalently coupled to enzyme markers (alkaline phosphatase or peroxidase) or to fluorescent dyes. Color reactions catalyzed by the enzymes resulted in precipitates located on the chromosomes at the site of probe hybridization. This was verified by hybridizing DNA probes of known chromosomal origin. The signals were analyzed by bright field, reflection contrast and fluorescence microscopy. The results indicate that the new technique gives strong signals and can also be used in combination with other systems (e.g., biotin) to detect differently labeled DNA probes on the same metaphase plate.
Animals↗
Nonradioactive labeling of oligonucleotides in vitro with the hapten digoxigenin by tailing with terminal transferase.
A procedure for the nonradioactive labeling of oligonucleotides with the hapten digoxigenin (DIG) has been developed. The label is introduced by enzymatic tailing of the 3'-end. Two different modified nucleotides were applied. DIG-dUTP allows the synthesis of hapten-modified oligonucleotides with longer tails containing several DIG molecules, whereas the novel compound DIG-ddUTP leads to the addition of only a single DIG hapten. The efficiency of the labeling reactions with respect to variation of the different parameters was analyzed and data on application of labeled oligonucleotide probes to different blot formats are shown.
Biotin↗
Non-radioactive labeling and detection of nucleic acids. I. A novel DNA labeling and detection system based on digoxigenin: anti-digoxigenin ELISA principle (digoxigenin system).
A novel highly sensitive non-radioactive DNA labeling and detection system based on the ELISA principle has been developed. DNA is modified with the cardenolide-hapten digoxigenin by enzymatic incorporation of digoxigenin-labeled deoxyuridine-triphosphate with Klenow enzyme. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Following hybridization of membrane-bound target-DNA with a digoxigenin-labeled probe, the hybrids are detected by an ELISA reaction using digoxigenin-specific antibodies covalently coupled to the marker enzyme alkaline phosphatase [(Dig):CIAP]. This binding of antibody: marker enzyme-conjugate is followed by an enzyme-catalysed coupled redox reaction with the colour substrates 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium salt (NBT) giving rise to a deep-blue coloured, water-insoluble precipitate directly adhering to the membrane. The digoxigenin system allows the detection of 0.1 pg homologous DNA within 16 h in dot- and Southern-blots on nitrocellulose or nylon membranes avoiding any significant background even after a prolonged period of color development. Due to its high sensitivity and specificity, the new system is appropriate for detection of single-copy genes in genomic blots as well as for Northern, slot, colony, plaque and in situ hybridizations.
Animals↗
Non-radioactive labeling and detection of nucleic acids. II. Optimization of the digoxigenin system.
The random-primed DNA labeling technique was modified for the incorporation of digoxigenin into DNA as basic component of the digoxigenin-based non-radioactive DNA labeling and detection system. Digoxigenin molecules act as reporter groups for highly sensitive DNA detection by a digoxigenin-specific antibody:alkaline phosphatase-conjugate-catalysed color reaction. The parameters affecting the individual reaction steps of the digoxigenin labeling, hybridization and detection reactions were optimized to maximal sensitivity and specificity.
Blotting, Northern↗
Non-radioactive labeling and detection of nucleic acids. III. Applications of the digoxigenin system.
The digoxigenin-based non-radioactive DNA labeling and detection system was applied in various hybridization protocols using digoxigenin-labeled probes obtained by enzymatic incorporation of Dig-[11]-dUTP. In genomic blots single-copy genes (human tissue-type plasminogen activator, constant part of immunoglobulin kappa light chain) can be detected with only 0.5 to 5 micrograms human DNA depending on the type of probe and the length of the hybridizing region. Due to its high sensitivity and specificity, the digoxigenin system is also appropriate for colony-, plaque-, and in situ hybridizations with metaphase chromosome spreads and fixed cells. Especially in the latter applications it is of great advantage, that with the digoxigenin system any significant background or unspecific side reactions with biological materials are avoided.
Blotting, Southern↗
Identification of proteins encoded by Epstein-Barr virus trans-activator genes.
Specific antisera were generated to characterize Epstein-Barr virus proteins reported to have trans-activating properties. Open reading frame BRLF1 was found to be expressed in two modifications in vivo, with molecular sizes ranging from 94 to 98 kilodaltons (kDa) depending on the cell line, whereas only one protein (Raji cells, 96 kDa) was detected by in vitro translation. Open reading frame BZLF1 encoded polypeptides of 38 and 35 kDa and additional smaller forms. A BZLF1-encoded 30-kDa protein could be detected under conditions in which expression was restricted to immediate early genes. Nuclear localization could be detected under conditions in which expression was restricted to immediate early genes. Nuclear localization could be shown for the proteins derived from reading frames BZLF1 and BMLF1. BMLF1 expression gave a heterogeneous protein pattern, with molecular sizes between 45 and 70 kDa, including a predominant 60-kDa protein detected in different B-cell lines.
Antibodies, Viral↗
Polymorphic proteins encoded within BZLF1 of defective and standard Epstein-Barr viruses disrupt latency.
These experiments identify an Epstein-Barr virus-encoded gene product, called ZEBRA (BamHI fragment Z Epstein-Barr replication activator) protein, which activates a switch between the latent and replicative life cycle of the virus. Our previous work had shown that the 2.7-kilobase-pair WZhet piece of rearranged Epstein-Barr virus DNA from a defective virus activated replication when introduced into cells with a latent genome, but it was not clear whether a protein product was required for the phenomenon. We now use deletional, site-directed, and chimeric mutagenesis, together with gene transfer, to show that a 43-kilodalton protein, encoded in the BZLF1 open reading frame of het DNA, is responsible for this process. The rearrangement in defective DNA does not contribute to the structural gene for the protein. Similar proteins with variable electrophoretic mobility (37 to 39 kilodaltons) were encoded by BamHI Z fragments from standard, nondefective Epstein-Barr virus genomes. Plasmids expressing the ZEBRA proteins from B95-8 and HR-1 viruses were less efficient at activating replication in D98/HR-1 cells than those which contained the ZEBRA gene from the defective virus. It is not yet known whether these functional differences are due to variations in expression of the plasmids or to intrinsic differences in the activity of these polymorphic polypeptides.
Cell Line↗
Expression of the Epstein-Barr virus 138-kDa early protein in Escherichia coli for the use as antigen in diagnostic tests.
We have attempted to produce the 138-kDa early protein (ep 138) of Epstein-Barr virus (EBV) in Escherichia coli. This protein was found, by immunoprecipitation, to be a clinically relevant antigen, especially for the determination of the IgA-titer in patients with nasopharyngeal carcinoma (NPC). Since the expression of the entire ep 138 coding region was unsuccessful, we synthesized only the antigenic parts of this protein. Potential antigenic sites were predicted from the amino acid sequence by combining values for hydrophilicity with calculated estimates of the secondary structure. The two predicted fragments were found to be antigenic, but only one of them was stably expressed in E. coli as a non-fusion protein. This stable protein fragment was, in turn, able to stabilize the second antigenic fragment forming an autologous fusion protein, consisting exclusively of EBV-derived sequences. The resulting product reacts particularly well with IgA antibodies of NPC patients indicating its diagnostic value for NPC.
Antigens, Viral↗
Strain-specific transcription and translation of the BamHI Z area of Epstein-Barr Virus.
The expression of the 1,800-base-pair BamHI Z region of Epstein-Barr virus DNA was analyzed by hybrid-selected translation with several DNA subclones and RNA from different cell lines. Furthermore, large segments of the three reading frames extending in this area were expressed as fusion proteins into Escherichia coli. The fusion proteins were partially purified and used to immunize rabbits. These sera were used to confirm our mapping assignments and to identify the respective posttranslationally modified proteins in in vivo labeling experiments. The reading frame BRLF1 (the first reading frame starting in the BamHI R fragment in leftward orientation) encoded a 93- to 96-kilodalton (kDa) protein depending on the cell line. The molecular weight of in vivo-labeled proteins was increased relative to that of in vitro-translated proteins, indicating that a posttranslational modification had occurred. The BZLF1 reading frame encoded a 35-kDa protein. It was posttranslationally cleaved from a 38-kDa precursor in induced B95-8 and induced Raji cells and from a 40-kDa precursor in induced P3HR1 cells. In Raji cells superinfected with virus derived from P3HR1 cells, the protein seemed to be expressed both from endogenous Raji genomes and from infecting genomes. The transcripts for the 93- to 96-kDa and the 35-kDa protein overlapped partially. The serum against the expressed third reading frame BZLF2 specifically precipitated a 140-kDa protein. This reading frame contains only 650 nucleotides, and therefore further coding sequences were presumably spliced to BZLF2. The latter is deleted in the Raji cell line; therefore, the observed 140kDa protein in superinfected Raji cells was expressed from infecting P3HR1 genomes.
Chromosome Mapping↗
Mapping of Epstein-Barr virus proteins on the genome by translation of hybrid-selected RNA from induced P3HR1 cells and induced Raji cells.
RNA was isolated from induced P3HR1 cells which synthesize Epstein-Barr virus (EBV) particles and therefore a full set of early and late antigens and from induced Raji cells which synthesize only early EBV proteins and hybridized to cloned EBV-DNA fragments spanning the entire genome. Bound mRNA was eluted and translated in vitro with rabbit reticulocyte lysate. The translation products were analyzed on SDS-polyacrylamide gels either directly or after immunoprecipitation with human sera. Most proteins could be mapped to short defined regions of the EBV genome using short restriction fragments and overlapping sheared fragments and there is evidence of splicing for some mRNA species. The synthesis of five early proteins can be seen only with hybrid-selected RNA from induced Raji cells. These mRNAs seem to be enriched in the cells restricted to early antigen synthesis.
Cell Line↗