Reduced K-theory for Azumaya algebras

Abstract In the theory of central simple algebras, often we are dealing with abelian groups which arise from the kernel or co-kernel of functors which respect transfer maps (for example K-functors). Since a central simple algebra splits and the functors above are “trivial” in the split case, one can prove certain calculus on these functors. The common examples are kernel or co-kernel of the maps Ki(F)?Ki(D), where Ki are Quillen K-groups, D is a division algebra and F its center, or the homotopy fiber arising from the long exact sequence of above map, or the reduced Whitehead group SK1. In this note we introduce an abstract functor over the category of Azumaya algebras which covers all the functors mentioned above and prove the usual calculus for it. This, for example, immediately shows that K-theory of an Azumaya algebra over a local ring is “almost” the same as K-theory of the base ring. The main result is to prove that reduced K-theory of an Azumaya algebra over a Henselian ring coincides with reduced K-theory of its residue central simple algebra. The note ends with some calculation trying to determine the homotopy fibers mentioned above.

Synthesis, Kinetic Evaluation and Utilization of a Biotinylated Dipeptide Proline Diphenyl Phosphonate for the Disclosure of Dipeptidyl Peptidase IV-like Serine Proteases

In this study, we report on the synthesis, kinetic characterisation, and application of a novel biotinylated and active site-directed inactivator of dipeptidyl peptidase IV (DPP-IV). Thus, the dipeptide-derived proline diphenyl phosphonate NH(2)-Glu(biotinyl-PEG)-Pro(P)(OPh)(2) has been prepared by a combination of classical solution- and solid-phase methodologies and has been shown to be an irreversible inhibitor of porcine DPP-IV, exhibiting an over all second-order rate constant (k(i)/K(i)) for inhibition of 1.57 x 10(3) M(-1) min(-1). This value compares favourably with previously reported rates of inactivation of DPP-IV by dipeptides containing a P(1) proline diphenyl phosphonate grouping [B. Boduszek, J. Oleksyszyn, C.M. Kam, J. Selzler, R.E. Smith, J.C. Powers, Dipeptide phophonates as inhibitors of dipeptidyl peptidase IV, J. Med. Chem. 37 (1994) 3969-3976; B.F. Gilmore, J.F. Lynas, C.J. Scott, C. McGoohan, L. Martin, B. Walker, Dipeptide proline diphenyl phosphonates are potent, irreversible inhibitors of seprase (FAPalpha), Biochem, Biophys. Res. Commun. 346 (2006) 436-446.], thus demonstrating that the incorporation of the side-chain modified (N-biotinyl-3-(2-(2-(3-aminopropyloxy)-ethoxy)-ethoxy)-propyl) glutamic acid residue at the P(2) position is compatible with inhibitor efficacy. The utilisation of this probe for the detection of both purified dipeptidyl peptidase IV and the disclosure of a dipeptidyl peptidase IV-like activity from a clinical isolate of Porphyromonas gingivalis, using established electrophoretic and Western blotting techniques previously developed by our group, is also demonstrated.

Biotransformation of maximakinin, a bradykinin-related nonadecapeptide from toad venom, by mammalian kallikrein and salivary proteases

Maximakinin is an N-terminally extended bradykinin (DLPKINRKGPRPPGFSPFR) from the venom of a Chinese toad (Bombina maxima) that displays highly selective activity at mammalian arterial smooth muscle receptors. In this study, we report that incubation of maximakinin with either kallikrein or human saliva generates catabolites with enhanced bioactivity that retain the tissue selective effects of the parent molecule. In addition, we have observed that kallikrein rapidly cleaves the C-terminal arginyl residue of both maximakinin and bradykinin – a cleavage hitherto considered to be performed by a carboxypeptidase that facilitates selective bradykinin receptor targeting. Maximakinin has thus evolved as a `smart' defensive weapon in the toad with inherent resistance to the signal-terminating protease hardware in the potential predator. Thus, natural selection of amphibian skin peptides for antipredator defence, through interspecies delivery by an exogenous secretory mode, produces subtle structural stabilization modifications that can potentially provide new insights for the design of orally active and selectively targeted peptide therapeutics.

Molecular cloning of mRNA from toad granular gland secretion and lyophilized skin: identification of Bo8 - a novel prokineticin from Bombina orientalis

Prokineticins are small (8 kDa), biologically active secretory proteins whose primary structures have been highly conserved throughout the Animal Kingdom. Representatives have been identified in the defensive skin secretions of several amphibians reflecting the immense structural/functional diversity of polypeptides in such. Here we describe the identification of a prokineticin homolog (designated Bo8) from the skin secretion of the Oriental fire-bellied toad (Bombina orientalis). Full primary structural characterization was achieved using a combination of direct Edman microsequencing, mass spectrometry and cloning of encoding skin cDNA. The latter approach employed a recently described technique that we developed for the cloning of secretory peptide cDNAs from lyophilized skin secretion, and this was further extended to employ lyophilized skin as the starting material for cDNA library construction. The Bo8 precursor was found to consist of an open-reading frame of 96 amino acid residues consisting of a putative 19-residue signal peptide followed by a single 77-residue prokineticin (Mr = 7990 Da). Amino acid substitutions in skin prokineticins from the skin secretions of bombinid toads are confined to discrete sites affording the necessary information for structure/activity studies and analog design.

Kinestatin: a novel bradykinin B2 receptor antagonist peptide from the skin secretion of the Chinese toad, Bombina maxima

We have isolated a novel bradykinin B2-receptor antagonist peptide, kinestatin, from toad (Bombina maxima) defensive skin secretion. Mass spectroscopy established a molecular mass of 931.56 Da and a provisional structure: pGlu-Leu/Ile-Pro-Gly-Leu/Ile-Gly-Pro-Leu/Ile-Arg.amide. The unmodified sequence, -QIPGLGPLRG-, was located at the C-terminus of a 116-amino-acid residue open-reading frame following interrogation of a sequenced B. maxima skin cDNA library database. This confirmed the presence of appropriate primary structural attributes for the observed post-translational modifications present on the mature peptide and established residue 2 as Ile and residues 5/8 as Leu. Kinestatin represents a prototype novel peptide from amphibian skin.

Mutations affecting the Rossman fold of isoleucyl-tRNA synthetase are correlated with low-level mupirocin resistance in Staphylococcus aureus.

The isoleucyl-tRNA synthetase (ileS) gene was sequenced in toto from 9 and in part from 31 Staphylococcus aureus strains with various degrees of susceptibility to mupirocin. All strains for which the mupirocin MIC was greater than 8 µg/ml contained point mutations affecting the Rossman fold via Val-to-Phe changes at either residue 588 (V588F) or residue 631 (V631F). The importance of the V588F mutation was confirmed by an allele-specific PCR survey of 32 additional strains. Additional mutations of uncertain significance were found in residues clustered on the surface of the IleS protein.

Structure and kinetics of phosphonopyruvate hydrolase from Variovorax sp. Pal2: New insight into the divergence of catalysis within the PEP mutase/isocitrate lyase superfamily .

Phosphonopyruvate (P-pyr) hydrolase (PPH), a member of the phosphoenolpyruvate (PEP) mutase/isocitrate lyase (PEPM/ICL) superfamily, hydrolyzes P-pyr and shares the highest sequence identity and functional similarity with PEPM. Recombinant PPH from Variovorax sp. Pal2 was expressed in Escherichia coli and purified to homogeneity. Analytical gel filtration indicated that the protein exists in solution predominantly as a tetramer. The PPH pH rate profile indicates maximal activity over a broad pH range.The steady-state kinetic constants determined for a rapid equilibrium ordered kinetic mechanism with Mg+2 binding first (Kd =140 ± 40 M), are kcat = 105 ± 2 s-1 and P-pyr Km = 5 ± 1 M. PEP (slow substrate kcat = 2 × 10-4 s-1), oxalate, and sulfopyruvate are competitive inhibitors with Ki values of 2.0 ± 0.1 mM, 17 ± 1 M, and 210 ± 10 M, respectively. Three PPH crystal structures have been determined, that of a ligand-free enzyme, the enzyme bound to Mg2+ and oxalate (inhibitor), and the enzyme bound to Mg2+ and P-pyr (substrate). The complex with the inhibitor was obtained by cocrystallization, whereas that with the substrate was obtained by briefly soaking crystals of the ligand-free enzyme with P-pyr prior to flash cooling. The PPH structure resembles that of the other members of the PEPM/ICL superfamily and is most similar to the functionally related enzyme, PEPM. Each monomer of the dimer of dimers exhibits an (/)8 barrel fold with the eighth helix swapped between two molecules of the dimer. Both P-pyr and oxalate are anchored to the active site by Mg2+. The loop capping the active site is disordered in all three structures, in contrast to PEPM, where the equivalent loop adopts an open or disordered conformation in the unbound state but sequesters the inhibitor from solvent in the bound state. Crystal packing may have favored the open conformation of PPH even when the enzyme was cocrystallized with the oxalate inhibitor. Structure alignment of PPH with other superfamily members revealed two pairs of invariant or conservatively replaced residues that anchor the flexible gating loop. The proposed PPH catalytic mechanism is analogous to that of PEPM but includes activation of a water nucleophile with the loop Thr118 residue.

Reactions of One-Electron-Oxidized Methionine with Oxygen: An ab initio study

A one-electron oxidation of a methionine residue is thought to be a key step in the neurotoxicity of the beta amyloid peptide of Alzheimer's disease. The chemistry of the radical cation of N-formylmethioninamide (11+) and two model systems, dimethyl sulfide (1+) and ethyl methyl sulfide (6+), in the presence of oxygen have been studied by B3LYP/6-31G(d) and CBS-RAD calculations. The stable form of 11+ has a three-electron bond between the sulfur radical cation and the carbonyl oxygen atom of the i - 1 residue. The radical cation may lose a proton from the methyl or methylene groups flanking the oxidized sulfur. Both 11+ and the resultant C-centered radicals may add oxygen to form peroxy radicals. The calculations indicate that unlike C-centered radicals the sulfur radical cation does not form a covalent bond to oxygen but rather forms a loose ion-induced dipole complex with an S-O separation of about 2.7 Å, and is bound by about 13 kJ mol-1 (on the basis of 1+ + O2). Direct intramolecular abstraction of an H atom from the C site is unlikely. It is endothermic by more than 20 kJ mol-1 and involves a high barrier (G = 79 kJ mol-1). The -to-S C-centered radicals will add oxygen to form peroxy radicals. The OH BDEs of the parent hydroperoxides are in the range of 352-355 kJ mol-1, similar to SH BDEs (360 kJ mol-1) and C-H BDEs (345-350 kJ mol-1). Thus, the peroxy radicals are oxidizing species comparable in strength to thiyl radicals and peptide backbone C-centered radicals. Each peroxy radical can abstract a hydrogen atom from the backbone C site of the Met residue to yield the corresponding C-centered radical/hydroperoxide in a weakly exothermic process with modest barriers in the range of 64-92 kJ mol-1.

Characterisation of two calmodulin-like proteins from the liver fluke, Fasciola hepatica.

Calmodulin is a calcium ion-sensing signalling protein found in eukaryotics. Two calmodulin-like gene sequences were identified in an EST library from adult liver flukes. One codes for a protein (FhCaM1) homologous to mammalian calmodulins (98% identity), whereas the other protein (FhCaM2) has only 41% identity. These genes were cloned into expression vectors and the recombinant proteins were expressed in Escherichia coli. Gel shift assays showed that both proteins bind to calcium, magnesium and zinc ions. Homology models have been built for both proteins. As expected, FhCaM1 has a highly similar structure to other calmodulins. Although FhCaM2 has a similar fold, its surface charge is higher than FhCaM1. One of the potential metal ion-binding sites has lower metal-ion co-ordination capability, while another has an adjacent lysine residue, both of which may decrease the metal-binding affinity. These differences may reflect a specialised role for FhCaM2 in the liver fluke.

Development of a rapid screening test for veterinary sedatives and the beta-blocker carazolol in porcine kidney by ELISA

Sedatives and tranquillisers are frequently used to reduce stress during the transportation of food producing animals. The most widely used classes of sedatives include the butyrophenone azaperone, the phenothiazines acepromazine, propionylpromazine, chlorpromazine and the beta-blocker, carazolol. For regulatory control purposes, tolerances for azaperone and carazolol have been set by the European Union as 100 and 25 mug kg(-1), respectively. Furthermore, the use of the phenothiazines is prohibited and therefore has a zero tolerance. A method for the detection of residues of five tranquillisers and one beta-blocker using a single ELISA plate has been developed. Kidney samples (2.5 g) were extracted with dichloromethane and applied to a competitive enzyme immunoassay using three polyclonal antibodies raised in rabbits against azaperol, propionylpromazine and carazolol conjugates. In sample matrix, the azaperol antibody cross-reacted 28.0% with azaperone and the propionylpromazine antibody cross-reacted 24.9% with acepromazine and 11.7% with chlorpromazine. In the ELISA, the detection capabilities of the six sedatives, azaperol, azaperone, carazolol, acepromazine, chlorpromazine, and propionylpromazine are 5, 15, 5, 5, 20 and 5 mug kg(-1), respectively. The proposed method is a sensitive and rapid multi-residue technique that offers a cost effective alternative to current published procedures, without any concession on the ability to detect sedative misuse.

A complex zinc finger controls the enzymatic activities of nidovirus helicases.

Nidoviruses (Coronaviridae, Arteriviridae, and Roniviridae) encode a nonstructural protein, called nsp10 in arteriviruses and nsp13 in coronaviruses, that is comprised of a C-terminal superfamily 1 helicase domain and an N-terminal, putative zinc-binding domain (ZBD). Previously, mutations in the equine arteritis virus (EAV) nsp10 ZBD were shown to block arterivirus reproduction by disrupting RNA synthesis and possibly virion biogenesis. Here, we characterized the ATPase and helicase activities of bacterially expressed mutant forms of nsp10 and its human coronavirus 229E ortholog, nsp13, and correlated these in vitro activities with specific virus phenotypes. Replacement of conserved Cys or His residues with Ala proved to be more deleterious than Cys-for-His or His-for-Cys replacements. Furthermore, denaturation-renaturation experiments revealed that, during protein refolding, Zn2+ is essential for the rescue of the enzymatic activities of nidovirus helicases. Taken together, the data strongly support the zinc-binding function of the N-terminal domain of nidovirus helicases. nsp10 ATPase/helicase deficiency resulting from single-residue substitutions in the ZBD or deletion of the entire domain could not be complemented in trans by wild-type ZBD, suggesting a critical function of the ZBD in cis. Consistently, no viral RNA synthesis was detected after transfection of EAV full-length RNAs encoding ATPase/helicase-deficient nsp10 into susceptible cells. In contrast, diverse phenotypes were observed for mutants with enzymatically active nsp10, which in a number of cases correlated with the activities measured in vitro. Collectively, our data suggest that the ZBD is critically involved in nidovirus replication and transcription by modulating the enzymatic activities of the helicase domain and other, yet unknown, mechanisms.

Mutational analysis of the active centre of coronavirus 3C-like proteases.

Formation of the coronavirus replication-transcription complex involves the synthesis of large polyprotein precursors that are extensively processed by virus-encoded cysteine proteases. In this study, the coding sequence of the feline infectious peritonitis virus (FIPV) main protease, 3CL(pro), was determined. Comparative sequence analyses revealed that FIPV 3CL(pro) and other coronavirus main proteases are related most closely to the 3C-like proteases of potyviruses. The predicted active centre of the coronavirus enzymes has accepted unique replacements that were probed by extensive mutational analysis. The wild-type FIPV 3CL(pro) domain and 25 mutants were expressed in Escherichia coli and tested for proteolytic activity in a peptide-based assay. The data strongly suggest that, first, the FIPV 3CL(pro) catalytic system employs His(41) and Cys(144) as the principal catalytic residues. Second, the amino acids Tyr(160) and His(162), which are part of the conserved sequence signature Tyr(160)-Met(161)-His(162) and are believed to be involved in substrate recognition, were found to be indispensable for proteolytic activity. Third, replacements of Gly(83) and Asn(64), which were candidates to occupy the position spatially equivalent to that of the catalytic Asp residue of chymotrypsin-like proteases, resulted in proteolytically active proteins. Surprisingly, some of the Asn(64) mutants even exhibited strongly increased activities. Similar results were obtained for human coronavirus (HCoV) 3CL(pro) mutants in which the equivalent Asn residue (HCoV 3CL(pro) Asn(64)) was substituted. These data lead us to conclude that both the catalytic systems and substrate-binding pockets of coronavirus main proteases differ from those of other RNA virus 3C and 3C-like proteases.

Structure-activity studies on position 14 of human alpha-calcitonin gene-related peptide

A structure-activity study was performed to examine the role of position 14 of human alpha-calcitonin gene-related peptide (h-alpha-CGRP) in activating the CGRP receptor. Interestingly, position 14 of h-alpha-CGRP contains a glycyl residue and is part of an alpha-helix spanning residues 8-18. Analogues [Ala(14)]-h-alpha-CGRP, [Aib(14)]-h-alpha-CGRP, [Asp(14)]-h-alpha-CGRP, [Asn(14)]-h-alpha-CGRP, and [Pro(14)]-h-alpha-CGRP were synthesized by solid phase peptide methodology and purified by RP-HPLC. Secondary structure was measured by circular dichroism spectroscopy. Agonist activities were determined as the analogues' ability to stimulate amylase secretion from guinea pig pancreatic acini and to relax precontracted porcine coronary arteries. Analogues [Ala(1)4]-h-alpha-CGRP, [Aib(14)]-h-alpha-CGRP, [Asp(14)]-h-alpha-CGRP, and [Asn(14)]-h-alpha-CGRP, all containing residues with a high helical propensity in position 14, were potent full agonists compared to h-alpha-CGRP in both tissues. Interestingly, replacement of Gly(14) of h-alpha-CGRP with these residues did not substantially increase the helical content of these analogues. [Pro(14)]-h-alpha-CGRP, predictably, has significantly lower helical content and is a 20-fold less potent agonist on coronary artery, known to contain CGRP-1 receptor subtypes, and an antagonist on pancreatic acini, known to contain CGRP-2 receptor subtypes. In conclusion, the residue in position 14 plays a structural role in stabilizing the alpha-helix spanning residues 8-18. The alpha-helix is crucial for maintaining highly potent agonist effects of h-alpha-CGRP at CGRP receptors. The wide variety of functional groups that can be tolerated in position 14 with no substantial modification of agonist effects suggests the residue in this position is not in contact with the CGRP receptor. [Pro(14)]-h-alpha-CGRP may be a useful pharmacological tool to distinguish between CGRP-1 and CGRP-2 receptor subtypes.

Cloning of the (Thr6)-phyllokinin precursor from Phyllomedusa sauvagei skin confirms a non-consensus tyrosine 0-sulfation motif.

Nine bradykinin-related peptides were identified in Phyllomedusa sauvagei skin secretion using QTOF MS/MS fragmentation sequencing. The major peptides were (Thr6)-bradykinin, (Hyp3, Thr6)-bradykinin, (Thr6)-phyllokinin and (Hyp3, Thr6)-phyllokinin. The phyllokinins occurred in both sulfated and non-sulfated forms. All (Thr6)-substituted bradykinins/phyllokinins could be generated from a common precursor by differential post-translational processing and modification. The open-reading frame of the cloned precursor cDNA consisted of 62 amino acid residues with a single bradykinin/phyllokinin coding sequence located at the C-terminus. Structural features included a Glu-Arg processing site at the N-terminus of the bradykinin/phyllokinin domain and the absence of an acidic amino acid residue adjacent to the C-terminal Tyr residue in the phyllokinins. However, the neutral amino acid residue (Ile) at position -1 and the basic amino acid residue (Arg) at position -2 from the Tyr residue, constitute a sulfation motif previously identified only in a protochordean.