Biomarker profiles in serum and saliva of experimental Sj?gren's syndrome: associations with specific autoimmune manifestations

Introduction  

Sj?gren's syndrome (SS) is a systemic autoimmune disease that mainly targets the exocrine glands. The aim of this study was to investigate the involvement of 87 proteins measured in serum and 75 proteins analyzed in saliva in spontaneous experimental SS. In addition, we intended to compute a model of the immunological situation representing the overt disease stage of SS.     

Allelic variation in HLA-B and HLA-C sequences and the evolution of the HLA-B alleles

Several new HLA-B (B8, B51, Bw62)- and HLA-C (Cw6, Cw7)-specific genes were isolated either as genomic cosmid or cDNA clones to study the diversity of HLA antigens. The allele specificities were identified by sequence analysis in comparison with published HLA-B and -C sequences, by transfection experiments, and Southern and northern blot analysis using oligonucleotide probes. Comparison of the classical HLA-A, -B, and -C sequences reveals that allele-specific substitutions seem to be rare events. HLA-B51 codes only for one allelespecific residue: arginine at position 81 located on the 1 helix, pointing toward the antigen binding site. HLA-B8 contains an acidic substitution in amino acid position 9 on the first central sheet which might affect antigen binding capacity, perhaps in combination with the rare replacement at position 67 (F) on the ul helix. HLA-B8 shows greatest homology to HLA-Bw42, -Bw41, -B7, and-Bw60 antigens, all of which lack the conserved restriction sites Pst I at position 180 and Sac I at position 131. Both sites associated with amino acid replacements seem to be genetic markers of an evolutionary split of the HLA-B alleles, which is also observed in the leader sequences. HLA-Cw7 shows 98% sequence identity to the JY328 gene. In general, the HLA-C alleles display lower levels of variability in the highly polymorphic regions of the 1 and 2 domains, and have more distinct patterns of locus-specific residues in the transmembrane and cytoplasmic domains. Thus we propose a more recent origin for the HLA-C locus.     

Purification and properties of the coenzyme A-linked acetaldehyde dehydrogenase of Acetobacterium woodii

Coenzyme A-linked acetaldehyde dehydrogenase (ACDH) of ethanol-grown cells of Acetobacterium woodii was purified to apparent homogeneity; a 28-fold purification was achieved with 13% yield. The enzyme proved to be oxygen-sensitive and was inactive in the absence of dithioerythritol. During the purification procedure addition of 1 mM MgCl₂ was necessary to maintain enzyme activity. Alcohol dehydrogenase (ADH) activity was separated from ACDH during anion exchange chromatography using DEAE Sephacel. A part of the ACDH activity coeluted with ADH, but both could be separately eluted from a Cibacron Blue 3GA-Agarose column, revealing the same subunit structure and activity band for ACDH as found before and, thus, indicating an aggregation of the enzyme. The remaining ADH activity could be separated by gel filtration. For the native ACDH a molecular mass of 255 kDa was determined by polyacrylamide gel electrophoresis and of 272 kDa by gel filtration using Superose 12. The enzyme subunit sizes were 28 kDa and 40 kDa, respectively, indicating a ₄₄ structure for the active form. The enzyme catalyzed the oxidation of several straight chain aldehydes although it was most active with acetaldehyde. NADH strongly inhibited oxidation of acetaldehyde whereas NADPH had no effect. The inhibition was noncompetitive.Non-standard abbrevations ACDH acetaldehyde dehydrogenase - ADH alcohol dehydrogenase - CHES 2-(N-cyclohexylamino)-ethanesulfonate - DTE dithioerythritol - KP-buffer 25 mM K-PO₄, pH 7.5, containing, 4 mM DTE - MES 2-(N-morpholino)-ethanesulfonate - TAPS N-Tris-(hydroxymethyl)-methyl-3-aminopropa-nesulfonate     

Zur Problematik der Chromosomenautoradiographie

The distribution of silver grains on the chromosomes of group G was studied in 216 mitoses from five patients with Down's syndrome after incubation with tritated thymidine in order to test the replication pattern of these autosomes. The different labelling densities over the chromosomes were determined by comparative estimations of the blackened areas and subdivided into four degrees: very strongly labelled, strongly labelled, weakly labelled and unlabelled. The 56 combinations for five G chromosomes and four labelling densitives were divided into three groups: group A, with three strong and two weak labelling types, group B, with two strong and three weak labelling types, and group C, with the remaining combinations. Thereby 51 (23.6%) mitoses were counted in six combinations of group A, 31 (14.4%) in six combinations of group B, and 134 (62.0%) in 44 combinations of group C. In order to test the possibility of identifying the G chromosomes based on a different DNA replication pattern the findings were subjected to a statistical analysis. The expected values were calculated for the different combinations of labelling degrees and were compared with the observed values. This method being used, the question whether the extra chromosome is replicating late or early compared with the remaining G chromosomes could not be answered with sufficient certainty, because P_A=1, P_B=0 could not be assumed on our figures. We conclude from these findings that the termination phases for the chromosomes 21 as well as 22 lie close together and that apparently the autoradiographic representation of the replication pattern can be influenced by various factors. The influencing factors can only be understood in connection with the problems of quantitative autoradiography. They are subdivided into three groups and discussed in this way: A. Factors due to the physiology of the genetic material: 1. Duration of the DNA-synthesis and the G₂-period. 2. Chromosomal replication pattern. 3. The relation of ³H-Thymidin and unlabelled thymidine in the newly synthesized DNA. B. Factors due to the autoradiographic and cytologic method: 1. Statistics of the radioactive disintegrations. 2. -self-absorption. 3. Coincidence. 4. Effectivity. 5. Back-ground. 6. Fading. 7. Condensation of the chromosomes. 8. Cytologic preparation. 9. Thickness of the emulsion layer. 10. Duration of exposition and developping. C. Factors due to the method of evaluation: 1. Accuracy of counting. 2. Deficient analysis of the results. After considering these factors it seems clear that the autoradiographic technique is not capable to demonstrate subtle differencies of the DNA replication. Thus the autoradiographic identification of chromosomes remains questionable if the termination phases of DNA replication lie close together as this seems the case for the chromosomes 21 and 22.

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Studie im Rahmen der Assoziation Hämatologie EURATOM — GSF.     

Epinephrine potentiates calcium mobilization and activation of protein kinases in platelets stimulated by ADP through a mechanism unrelated to phospholipase C

ADP, added to suspensions of aspirinized 32P-prelabelled washed platelets, induced reversible platelet aggregation, the rapid elevation of cytosolic Ca2+ (maximum at 2 s), 20 kDa myosin light chain phosphorylation (maximum faster than 3 s), 40 kDa protein phosphorylation (maximum at 3-10 s) and phosphatidic acid formation (maximum at 30 s). Prior addition of epinephrine potentiated platelet aggregation, cytosolic Ca2(+)-elevation, 20 and 40 kDa protein phosphorylation evoked by ADP, but it did not enhance phosphatidic acid formation induced by ADP. The potentiating effect of epinephrine on aggregation, cytosolic Ca2(+)-increase and 20 and 40 kDa protein phosphorylation induced by ADP was also observed in the presence of EGTA. Ethylisopropylamiloride, an inhibitor of Na+/H(+)-exchange, did not affect the potentiation of ADP-induced platelet aggregation by epinephrine. We conclude that epinephrine primes platelets to increase Ca2(+)-influx and Ca2(+)-mobilization in response to ADP. The potentiation of cytosolic Ca2(+)-elevation by epinephrine leads to further stimulation of myosin light chain phosphorylation and protein kinase C activation and ultimately to enhanced platelet aggregation. These effects of epinephrine do not seem to take place at the level of phospholipase C.     

Isolation and biochemical characterization of fusicoccin-insensitive variant lines of Arabidopsis thaliana (L.) Heynh

Arabidopsis thaliana lines have been isolated that are insensitive to the fungal toxin fusicoccin (FC). Initial screening was done by selecting for plants that either grew well on high concentrations of FC or did not respond to FC by increases in H⁺-extrusion. All selected plants were tested, in several additional rounds of screening, for binding to microsomal proteins of a ³H-labeled radioligand of fusicoccin. A novel assay allowing for the direct selection of individual plants exhibiting reduced binding of FC was developed and used as screening procedure. Independent variant lines (43) with stably expressed, reduced binding of FC were isolated and subjected to a detailed characterization of their binding sites. The lines could be subdivided into several distinct classes with respect to these characteristics. In class-I lines, the data indicate a partial conversion of high-affinity binding sites to a low-affinity state. In class-II lines, the affinity of the binding site to FC is strongly reduced while the number of sites, as well as several other biochemical parameters, is completely unchanged, suggesting a specific alteration in the properties of the fusicoccin-binding protein. In class-III lines, the ligand-binding protein complex, while retaining its high affinity, is destabilized at supraoptimal concentrations of FC (such as those used for screening). In wild-type plants, only the high-affinity binding site was detected. Combined, these data prove that the high-affinity sites represent the plant's FC receptor.Abbreviations A_o binding site concentration - FC fusicoccin - FCBP fusicoccin-binding protein - FCol 9-nor-8-hydroxyfusicoccin - K_D dissociation constant of the FCBP-radioligand complex We are grateful to Iris Sandorf and Gudrun Henrichs for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany and by Fonds der Chemischen Industrie (literature provision).