SUMMARYFoot-and-mouth disease virus (FMDV) A22 Iraq 24/64 adapted to grow in BHK monolayer cells induced antibodies which neutralized many isolates belonging to the A serotype. Plaque-purified virus isolated from this stock also induced broadly reactive antibodies, showing that this property is not due to the combined response to a mixture of variants in the original stock virus. However, viruses obtained by passage in suspension BHK cells of either the monolayer cell-adapted virus or a virus cloned from this stock resulted in the selection of virus which induced antibodies with highly specific neutralizing activity. In addition to their antigenic properties the monolayer and suspension cell-adapted viruses could be distinguished by plaque morphology, tendency to aggregate and ability to attach to BHK cells. Monoclonal antibodies (MAbs) induced with the plaque-purified monolayer-adapted virus had neutralizing activity almost as broad as polyclonal serum, showing that this property can be represented by a single epitope on the virus. These neutralizing MAbs recognize a trypsin-sensitive epitope on the virus. Surprisingly, sequence analysis of the structural protein-coding regions of the genomic RNAs of monolayer and suspension celladapted viruses showed no amino acid differences in VP1, the protein known to contain the major neutralization epitope in FMDV and to be the only protein susceptible to cleavage by trypsin in the virus particle. Although three coding differences were found in the capsid protein these were all located in VP2.

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Database searching and compound screening identified 1-benzyl-3-(3-dimethylaminopropyloxy)indazole (benzydamine, 3) as a potent activator of the nitric oxide receptor, soluble guanylate cyclase. A comprehensive structure-activity relationship study surrounding 3 clearly showed that the indazole C-3 dimethylaminopropyloxy substituent was critical for enzyme activity. However replacement of the indazole ring of 3 by appropriately substituted pyrazoles maintained enzyme activity. Compounds were evaluated for inhibition of platelet aggregation and showed a general lipophilicity requirement. Aryl-substituted pyrazoles 32, 34, and 43 demonstrated potent activation of soluble guanylate cyclase and potent inhibition of platelet aggregation. Pharmacokinetic studies in rats showed that compound 32 exhibits modest oral bioavailability (12%). Furthermore 32 has an excellent selectivity profile notably showing no significant inhibition of phosphodiesterases or nitric oxide synthases.

Background-Systemic hypotension as a consequence of vascular dysfunction is a well-recognized and important feature of critical illness. Although serine protease activation has been implicated as a cause of vascular dysfunction in systemic inflammation, the mechanism is unknown. Recently, a class of receptors with an entirely novel mechanism of action, protease-activated receptors (PARs), has been identified that would explain the link between protease activation and systemic hypotension. Our aim was to test the hypothesis that in vivo activation of protease-activated receptor 2 (PAR-2) in humans would mediate vasodilatation. Methods and Results-For these first-in-human studies, an activating peptide for the human PAR-2 receptor was synthesized and administered to healthy volunteers. Using both the dorsal hand vein technique and forearm plethysmography, we studied the effects of PAR-2 activation in human blood vessels and investigated the mechanism of vasodilation. Activation of PAR-2 receptors in vivo dilated human blood vessels in a dose-dependent manner, and the effects were reduced by inhibition of both nitric oxide and prostanoid synthesis Conclusions-These findings demonstrate that serine protease activity can cause human vasodilation and provide a possible explanation of why serine protease activation in critical illness is associated with vascular dysfunction.

Copper( 11) cleaves with moderate specificity peptides containing Ser-His or Thr-His sequences, at the Nterminal side of the hydroxyaminoacyl residue. The reaction is slow, and is first-order in peptide : Cu" complex, with a half-life of several hours at 62 "C in sodium bicarbonate buffer, pH 8. Cleavage of other histidine-containing peptides also occurs, at a rate around 10-100-fold less. EDTA completely quenches the cleavage. The reaction is stoichiometric in Cu" and is inhibited by amine-containing buffer components; Tris at 19 mM inhibits cleavage by 50%. The reaction has a complex pH-dependence, being very slow below pH 5 , and with rates increasing with pH from pH 7 to pH 9.5. Slower degradative side reactions do occur, with destruction of tyrosine residues, particularly in the presence of high concentrations of chloride ion, but the specific cleavage appears to be a hydrolysis, as determined by amino-acid analysis and mass spectrometry of the products. The cleavage is clearly different from the previously described oxidative degradation of proteins catalysed by copper ions. Cleavage of denatured IgG protein occurs with sufficient specificity to reveal distinct bands on SDS-polyacrylamide gel electrophoresis under reducing conditions. 0 Munksgaard 1996.