The effect of intravenous adenosine diphosphate on the number of circulating platelets in experimental animals: inhibition by prostaglandin E1, dipyridamole, SH-869 and VK-774.

The number of circulating platelets was monitored in anaesthetized animals by a continuous flow technique, using a Technicon Autocounter. Intravenous infusions of adenosine diphosphate (ADP) produced transient, dose-dependent falls in circulating platelet numbers in rabbits, dogs, rats, pigs and squirrel monkeys. The rat was the most sensitive of the species investigated. In the rabbit, the effect of a submaximal dose of ADP was inhibited in a dose-dependent manner by intravenous infusions of prostaglandin E1 (PGE1), dipyridamole, and two derivatives of dipyridamole (SH-869 and VK-774). The dose-response curves for PGE1, SH-869 and VK-774 were approximately parallel, whereas that for dipyridamole was considerably less steep. PGE1 was the most potent inhibitor, but the duration of action was very short. Dipyridamole and SH-869 produced inhibition of long duration. The duration of action of VK-774 was intermediate. All inhibitors produced marked and often long-lasting hypotension. The fact no inhibition of ADP effects could be demonstrated with dibenzyline and hexamethonium, which also produced marked hypotension of long duration, indicated that inhibition of the ADP effect by the four antagonists studied was not due to changes in blood pressure.

Tyrosine-phosphorylated forms of Ig beta, CD22, TCR zeta and HOSS are major ligands for tandem SH2 domains of Syk.

The protein tyrosine kinase Syk plays an important role in signal transduction from the B cell antigen receptor and possibly also from the TCR. We have examined the binding specificity of Syk-derived SH2 domains in vitro and found that the tandem SH2 domains have two major ligands in activated Ramos B cells as well as in activated Jurkat T cells. The SH2-binding proteins in Ramos B cells were identified as the tyrosine-phosphorylated forms of the Ig alpha beta heterodimer and of CD22. Binding to the Ig alpha beta heterodimer seems to occur predominantly via Ig beta, indicating that the two receptor components might couple to distinct signaling pathways. In Jurkat T cells one of the SH2-binding proteins represents the tyrosine-phosphorylated TCR zeta chain. The identity of the second SH2 ligand, called HOSS, is not known. HOSS is discussed as a putative member of the receptor family characterized by the immunoreceptor tyrosine-based activation motif.

[Fracture movement and fracture healing in plaster case fixation (author's transl)]. / Die Relativität der Ruhigstellung im Gipsverband

The fixation of a fracture in a plaster cast, or in traction is not absolutely rigid. Experiments were carried out on adaver tibial fractures placed in long leg plaster casts. It was observed that the possible deformity ranged between 4-8 degrees in a well-padded cast and 2-5 degrees in a nonpadded cast. Further observation were conducted while hip osteotomies were compressed by external fixator. These osteotomies are further protected by hip spicas. It was demonstrated that increasing the length of the cast did not increase the degree of fixation. It is suggested that fracture healing in conservatively treated cases is optimal if movement of the fragments remains within the physiological elastic limits of the not fractured bone. Fracture healing can be disturbed not only by extensively denudation and soft tissue disturbance, but also by under-or overstressing the bone and by unphysiological immobilisation.

In vitro characterization of major ligands for Src homology 2 domains derived from protein tyrosine kinases, from the adaptor protein SHC and from GTPase-activating protein in Ramos B cells.

Antigen receptors of B lymphocytes transmit their activation signal to the cell interior by associating with and activation of specific non-receptor tyrosine kinases. Most of these kinases as well as other cytoplasmic effectors contain at least one Src homology 2 (SH2) domain, known to bind tyrosine-phosphorylated proteins. We examined the binding specificity of SH2 domains from different signaling molecules in B cells and found that each of the SH2 domains tested bound distinct subsets of stimulation-dependent phosphoproteins in vitro. SH2 domains from Src-like tyrosine kinases bound predominantly to the HS1 phosphoprotein. The tandem SH2 domains of the ZAP-70 tyrosine kinase bound to phosphorylated Ig-beta but only weakly to Ig-alpha. Also the SHC-derived SH2 domain formed complexes with the tyrosine-phosphorylated Ig-alpha/beta heterodimer, while the C- and N-terminal SH2 domains of GTPase-activating protein displayed completely different binding preferences. These results suggest that cytoplasmic effector molecules can be recruited to the activated B cell receptor in an SH2-phosphotyrosine-mediated manner. The data also provide a possible explanation for the notion that Ig-alpha and Ig-beta might couple to different biochemical pathways.

All three IkappaB isoforms and most Rel family members are stably associated with the IkappaB kinase 1/2 complex.

Nuclear factor kappa B (NF-kappaB) is an important transcription factor for the genes of many pro-inflammatory proteins and is strongly activated by the cytokines interleukin-1 and tumor necrosis factor (TNF)alpha under various pathological conditions. In nonstimulated cells, NF-kappaB is present in the cytosol where it is complexed to its inhibitor IkappaB. Activation of NF-kappaB depends on the signal-induced phosphorylation of IkappaB by specific IkappaB kinases which initiates the inhibitor's conjugation to ubiquitin and subsequent degradation by the proteasome. We used both TNF-stimulated and okadaic-acid-stimulated HeLa cells to purify three biochemically distinct kinase activities targeting one or both of the two serines (S32 and S36) in IkappaBalpha which induce its rapid degradation upon cytokine stimulation. All three activities correspond to known IkappaB kinases: the mitogen-activated 90 kDa ribosomal S6 kinase (p90rsk1), the IkappaB kinase 1/2 complex (IKK1/2) and casein kinase II (CK II). However, we found that only one of the activities, namely the IKK1/2 complex, exists as a pre-assembled kinase-substrate complex in which the IKKs are directly or indirectly associated with several NF-kappaB-related and IkappaB-related proteins: RelA, RelB, cRel, p100, p105, Ikappa Balpha, Ikappa Bbeta and Ikappa Bepsilon. The existence of stable kinase-substrate complexes, the presence of all three known IkappaB isoforms in these complexes and our observation that the IKK complex is capable of phosphorylating Ikappa Balpha-, Ikappa Bbeta- and Ikappa Bepsilon-derived peptides at the respective degradation-relevant serines suggests that the IKK complex exerts a broad regulatory role for the activation of different NF-kappaB species. In contrast to previous studies, which locate CK II phosphorylation sites exclusively to the C-terminal PEST sequence of Ikappa Balpha, we observed efficient phosphorylation of serine 32 in Ikappa Balpha by the purified endogenous CK II complex. Therefore, both p90rsk1 and CK II have the same preference for phosphorylating only one of the two serines which are relevant for inducible degradation.

Serum amyloid A (apoSAA) expression is up-regulated in rheumatoid arthritis and induces transcription of matrix metalloproteinases.

The acute-phase reactant rabbit serum amyloid A 3 (SAA3) was identified as the major difference product in Ag-induced arthritis in the rabbit, a model resembling in many aspects the clinical characteristics of rheumatoid arthritis (RA) in humans. In Ag-induced arthritis, up-regulated SAA3 transcription in vivo was detected in cells infiltrating into the inflamed joint, in the area where pannus formation starts and, most notably, also in chondrocytes. The proinflammatory cytokine IL-1beta induced SAA3 transcription in primary rabbit chondrocytes in vitro. Furthermore, rSAA3 protein induced transcription of matrix metalloproteinases in rabbit chondrocytes in vitro. In the human experimental system, IL-1beta induced transcription of acute-phase SAA (A-SSA; encoded by SAA1/SAA2) in primary chondrocytes. Similar to the rabbit system, recombinant human A-SAA protein was able to induce matrix metalloproteinases' transcription in chondrocytes. Further, immunohistochemistry demonstrated that A-SAA was highly expressed in human RA synovium. A new finding of our study is that A-SSA expression was also detected in cartilage in osteoarthritis. Our data, together with previous findings of SAA expression in RA synovium, suggest that A-SAA may play a role in cartilage destruction in arthritis.

Rapid identification of phosphopeptide ligands for SH2 domains. Screening of peptide libraries by fluorescence-activated bead sorting.

A method for the identification of high-affinity ligands to SH2 domains by fluorescence-activated bead sorting (FABS) was established. Recombinant SH2 domains, expressed as glutathione S-transferase (GST) fusion proteins, were incubated with a phosphotyrosine (Y*)-containing peptide library. 6.4 x 10(5) individual peptides of nine amino acids in length (EPX6Y*X19X7X19X7X6) were each displayed on beads. Phosphopeptide interaction of a given SH2 domain was monitored by binding of fluorescein isothiocyanate-labeled antibodies directed against GST. High-fluorescence beads were isolated by flow cytometric sorting. Subsequent pool sequencing of the selected beads revealed a distinct pattern of phosphotyrosine-containing motifs for each individual SH2 domain: the SH2 domain of the adapter protein Grb2 predominantly selected beads with the sequence Y*ENDP, whereas the C-terminal SH2 domain of the tyrosine kinase Syk selected Y*EELD, each motif representing the most frequently found residues C-terminal to the phosphotyrosine. For deconvolution studies, soluble phosphopeptides comprising variations of the Grb2 motifs were resynthesized and analyzed by surface plasmon resonance.

The kinetics of association and phosphorylation of IkappaB isoforms by IkappaB kinase 2 correlate with their cellular regulation in human endothelial cells.

Activation of the transcription factor NF-kappaB depends on the specific dual phosphorylation of its inhibitor protein IkappaB by the homologous cytokine-inducible IkappaB kinases 1 and 2 (IKK1/2). Various IkappaB isoforms exist: IkappaBalpha, IkappaBbeta1/2 (two alternative splice variants), and IkappaBepsilon. However, the individual relevance and the specific regulation of these isoforms is not well-understood. We have studied the direct interaction of recombinant IkappaBalpha, IkappaBbeta1, IkappaBbeta2, and IkappaBepsilon with the recombinant homodimeric IKK2. Fluorescence-based active site titration revealed that each IKK2 dimer contains two binding sites for IkappaB. By using surface plasmon resonance analysis, we found that all IkappaB proteins interact with the IKK2 dimer following a noncooperative binding mechanism. Further, the four IkappaB proteins bind to the kinase with equilibrium dissociation constants (KD) in the range of 50-300 nM; the association rate constants for all IkappaB isoforms with IKK2 were between 6.0 x 10(3) and 22.5 x 10(3) M-1 s-1, and the dissociation rate constants were between 1.25 x 10(-3) and 1.75 x 10(-3) s-1. This high-affinity binding suggests that the previously observed preassociation of all analyzed IkappaB proteins with the biochemically purified 700 kDa IkappaB kinase (IKK) complex is based on a direct enzyme-substrate association between the various IkappaB isoforms and the IKK proteins. The apparent catalytic efficiencies (kcat/KM) of IKK2 for IkappaBalpha, IkappaBbeta1, IkappaBbeta2, and IkappaBepsilon were 22 x 10(3), 10 x 10(3), 5.4 x 10(3), and 8.5 x 10(3) s-1 M-1, respectively, with KM values ranging between 1.7 x 10(-6) and 3.2 x 10(-6) M and kcat values ranging between 1.5 x 10(-2) and 3.7 x 10(-2) s-1. The relative affinities and catalytic efficiencies of IKK2 for the IkappaB isoforms were also reflected by the kinetics observed for the TNF-induced, phosphorylation-dependent degradation of the alpha, beta1, beta2, and epsilon isoforms of IkappaB in human umbilical vein endothelial cells. Therefore, differential regulation of the IkappaB isoforms in some cell types is not a direct result of the IKK activity, but appears to be due to parallel events.