CACP, encoding a secreted proteoglycan, is mutated in camptodactyly-arthropathy-coxa vara-pericarditis syndrome.

Altered growth and function of synoviocytes, the intimal cells which line joint cavities and tendon sheaths, occur in a number of skeletal diseases. Hyperplasia of synoviocytes is found in both rheumatoid arthritis and osteoarthritis, despite differences in the underlying aetiologies of the two disorders. We have studied the autosomal recessive disorder camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP; MIM 208250) to identify biological pathways that lead to synoviocyte hyperplasia, the principal pathological feature of this syndrome. Using a positional-candidate approach, we identified mutations in a gene (CACP) encoding a secreted proteoglycan as the cause of CACP. The CACP protein, which has previously been identified as both 'megakaryocyte stimulating factor precursor' and 'superficial zone protein', contains domains that have homology to somatomedin B, heparin-binding proteins, mucins and haemopexins. In addition to expression in joint synovium and cartilage, CACP is expressed in non-skeletal tissues including liver and pericardium. The similarity of CACP sequence to that of other protein families and the expression of CACP in non-skeletal tissues suggest it may have diverse biological activities.     

The effects of manganese and barium on the cardiac pacemaker current,i f,in rabbit sino-atrial node myocytes

Summary The isolation of ionic fluxes contributing to electric currents through cell membranes often requires block of other undesired components which can be achieved, among others, by divalent cations. Mn²⁺ and Ba²⁺ are often used, for example, to block Ca and K currents. Here we have investigated the effects of these two cations on the properties of the hyperpolarization-activated pacemaker current i_f, in rabbit sino-atrial node myocytes, as obtained by voltage clamp analysis. We find that 2 mM Mn²⁺ shifts the i_f activation curve by 3.2±0.3 mV towards more positive values. However, when 1 mM Ba²⁺ is also added, the positive shift is more than halved (1.3±0.2 mV). We find, too, that in the absence of blocking cations the ACh-induced i_f inhibition is slightly higher than in their presence. These results indicate that the alteration of i_f kinetic properties by Ba²⁺ plus Mn²⁺-containing solutions is minimal.     

Structure of pre-pro-von Willebrand factor and its expression in heterologous cells

Von Willebrand factor (vWF), a multifunctional haemostatic glycoprotein derived from endothelial cells and megakaryocytes, mediates platelet adhesion to injured subendothelium and binds coagulation factor VIII in the circulation. Native vWF is a disulphide-bonded homopolymer; the monomeric subunits, of apparent relative molecular mass (Mr) 220,000 (220K) are derived from an intracellular precursor estimated at 260-275K. Multimer assembly is preceded by the formation of dimers, linked near their C-termini, which then assemble into filamentous polymers. The importance of the removal of the large vWF pro-polypeptide during multimer assembly, and whether this or other stages of the complex post-translational processing require components specific to endothelial cells or megakaryocytes, is unknown. Here we report an analysis of the complete sequence of pre-pro-vWF and expression of the molecule in heterologous cells. The vWF precursor is composed of several repeated subdomains. When expressed in COS and CHO cells, it is cleaved and assembled into biologically active high relative molecular mass disulphide bonded multimers. This suggests that the information for assembly of this complex molecule resides largely within its primary structure.     

Steady-state kinetic studies with the polysulfonate U-9843, an HIV reverse transcriptase inhibitor

The tetramer of ethylenesulfonic acid (U-9843) is a potent inhibitor of HIV-1 RT^* and possesses excellent antiviral activity at nontoxic doses in HIV-1 infected lymphocytes grown in tissue culture. Kinetic studies of the HIV-1 RT-catalyzed RNA-directed DNA polymerase activity were carried out in order to determine if the inhibitor interacts with the template: primer or the deoxyribonucleotide triphosphate (dNTP) binding sites of the polymerase. Michaelis-Menten kinetics, which are based on the establishment of a rapid equilibrium between the enzyme and its substrates, proved inadequate for the analysis of the experimental data. The data were thus analyzed using steady-state Briggs-Haldane kinetics assuming that the template:primer binds to the enzyme first, followed by the binding of the dNTP and that the polymerase is a processive enzyme. Based on these assumptions, a velocity equation was derived which allows the calculation of all the specific forward and backward rate constants for the reactions occurring between the enzyme, its substrates and the inhibitor. The calculated rate constants are in agreement with this model and the results indicated that U-9843 acts as a noncompetitive inhibitor with respect to both the template:primer and dNTP binding sites. Hence, U-9843 exhibits the same binding affinity for the free enzyme as for the enzyme-substrate complexes and must inhibit the RT polymerase by interacting with a site distinct from the substrate binding sites. Thus, U-9843 appears to impair an event occurring after the formation of the enzyme-substrate complexes, which involves either an event leading up to the formation of the phosphoester bond, the formation of the ester bond itself or translocation of the enzyme relative to its template:primer following the formation of the ester bond.     

Role for microsomal Ca storage in mammalian neurones?

Alterations in the intracellular concentration of calcium ions [( Ca2+]i) are increasingly being found to be associated with regulatory functions in cells of all kinds. In muscle, an elevation of [Ca2+]i is the final link in excitation-contraction coupling while at nerve endings and in secretory cells, similar rises in [Ca2+]i are thought to mediate exocytosis. The discovery of calcium-activated ion channels indicated a role for intracellular calcium in the regulation of membrane excitability. Calcium transients associated with either intracellular release or the inward movement of Ca2+ across the membrane have been recorded in molluscan neurons and more recently in neurones of bullfrog sympathetic ganglia. Here, we report the first recordings of calcium transients in single mammalian neurones. In these experiments we have found that the methylxanthine, caffeine, causes the release of calcium from a labile intracellular store which can be refilled by Ca2+ entering the cell during action potentials.     

Plasmodium falciparum strain-specific antibody blocks binding of infected erythrocytes to amelanotic melanoma cells

An important feature of Plasmodium falciparum malaria which differentiates it from other human malarias is that erythrocytes infected with trophozoites and schizonts are not present in the peripheral blood but are sequestered along capillary and venular endothelium. Infected erythrocytes attach via parasite-induced ultrastructural modifications on the surface of the infected cells, called 'knobs'. This sequestration may be important for parasite survival because it prevents infected erythrocytes from circulating through the spleen where they could be eliminated. We have established an in vitro correlate of sequestration and used it to demonstrate that immune sera from repeatedly infected Aotus monkeys inhibit binding of infected erythrocytes to endothelial cells. We have investigated whether antiserum that blocks binding of one isolate of P. falciparum to target cells can block or reverse binding of other isolates. We report here that sera which block or reverse binding are strain-specific, indicating that the corresponding antigens on the surface of the infected erythrocytes are strain (isolate)-specific.