Detection of Papillomavirus DNA in Cutaneous Squamous Cell Carcinoma and Multiple Papillomas in Captive Reptiles.

Papillomaviruses (PVs) are small, non-enveloped DNA viruses that cause mucocutaneous tumours including squamous cell carcinoma (SCC) in man. In animals, evidence supports a causal role for PVs in the development of cutaneous and oral SCC in some species. In reptiles, three cases of papilloma or fibropapilloma have been associated with PV infection, but no association has been reported to date with SCC. Two cases of cutaneous epithelial tumours, multiple papillomas in a spiny-tailed lizard (Uromastyx acanthinura) and SCC in a Dumeril's boa (Acrantophis dumerili), were investigated by polymerase chain reaction. PV DNA was amplified from samples of both lesions. Typical microscopical features suggestive of PV infection (e.g. the presence of koilocytes) were observed in the lesions from the spiny-tailed lizard. This is the first report of an association between PV and SCC in reptiles. Further studies are needed to better clarify the role of PVs in these species and to characterize the PV strains involved.

An Acute Multispecies Episode of Sheep-Associated Malignant Catarrhal Fever in Captive Wild Animals in an Italian Zoo.

In July 2011, in a zoological garden in Rome, Italy, malignant catarrhal fever (MCF), a fatal, systemic disease of Artiodactyla, was suspected on the basis of neurological signs and gross lesions observed in a banteng, the first animal to die of this infection. An MCF type-specific PCR with subsequent sequencing of the PCR amplicon confirmed the aetiological agent as ovine herpesvirus-2 (OvHV-2). Biological samples were collected from the dead animals for gross, histological, bacteriological, virological and serological examinations. An epidemiological investigation was conducted to identify the source of the outbreak, as further deaths due to OvHV-2 still occurred after the removal of the acknowledged reservoirs, domestic sheep and goats. For this purpose, samples from other susceptible species and reservoir hosts were collected for virological and serological analysis. In conjunction, a retrospective sero-investigation was conducted on sera collected between 1999 and 2010 from some of the species involved in the present episode. In total, 11 animals belonging to four different species (banteng, Himalayan tahr, Nile lechwe and sika deer) died between July 2011 and October 2012. The severe gross and histological lesions were consistent with the disease, namely haemorrhages and congestion of several organs as well as lymphoid cell infiltrates and vasculitis of varying severity. The virological tests confirmed that all animals had died of sheep-associated MCF. The investigation indicated that the OvHV-2 infection could have been due to the arrival of sheep in the petting zoo, with cases commencing after first lambing and subsequent shedding of virus. This was also supported by the serological retrospective study that indicated limited previous MCF virus circulation. Further MCF cases that occurred even after the removal of the domestic sheep and goats were attributed to the mouflon. This episode confirms the importance of biosecurity measures in zoos, which house MCF susceptible species, especially those endangered.

Kinetic evidence for surface residues influencing the active site of Coprinus cinereus peroxidase: analysis of the pH dependence of G154E, P90H and P90H-G154E substrate entrance mutants.

Three mutants of Coprinus cinereus peroxidase (CIP) were made to mimic the substrate entrance histidine 82-glutamic acid 146 pair of the substrate channel in lignin peroxidase (LIP). Compound I formation of LIP has a low pH optimum around pH 3, while optimal formation of CIP compound I is obtained at pH 6-11. The mutants were glycine 154-->glutamic acid (G154E), proline 90-->histidine (P90H) and the double mutant P90H-G154E. All three showed kinetics of compound I formation similar to that of wt CIP between pH 3 and 9. However, the stability of compound I was strongly affected by these mutations. In wt CIP compound I is stable for approximately 30 min, while compound I of the mutants were stable for 5 s or less. The P90H and P90H-G154E mutants showed pK(a) values for the alkaline transition at least one pH unit lower than for wt CIP and the G154E mutant. We suggest that the changed electrostatic field results in destabilisation of the oxidised heme in compound I and II and that the P90H residue increases the electrostatic potential in the distal cavity thereby decreasing the pK(a) for the alkaline transition.

Effect of lysine residues on the deamidation reaction of asparagine side chains.

The effect of lysine residues on the deamidation reaction of the asparagine side chain has been studied on the peptide and on its lysine-acetylated derivative in a wide range of pH values. The amino acid sequence of these peptides is similar to the local sequence flanking the labile Asn-67 in RNAse A. The experimental data show that Lys influences both the deamidation rate and the relative yield of the two reaction products, i.e., the aspartic acid and beta-aspartic acid containing peptide. These effects are pH dependent and can be rationalized based on the mechanism previously proposed for the deamidation reaction via succinimide derivative.

Kinetic and thermodynamic control of the relative yield of the deamidation of asparagine and isomerization of aspartic acid residues.

Selective deamidation of Asn67 of RNase A to beta-Asp67 and Asp67 residues at neutral pH initially produces greater amounts of the beta-Asp derivative. As the reaction proceeds the relative concentration of [Asp67]-RNase A increases and, at equilibrium, becomes predominant. Such a discrepancy between the kinetic and thermodynamic control on reaction products is discussed in light of information from X-ray three-dimensional analysis and the lower thermodynamic stability of the beta-Asp derivative relative to the parent enzyme.

Formation of an RNase A derivative containing an aminosuccinyl residue in place of asparagine 67.

At acidic pH, Asp67 and beta-Asp67 (beta-Asp: isoaspartic acid residue) derivatives of RNase A, obtained by selective deamidation of the parent enzyme, spontaneously produces a new derivative containing an aminosuccinyl residue (Asu). The overall secondary structure of the protein chain does not change as a consequence of this substitution, while the catalytic activity on RNA is reduced to about 25%. The pH dependence of the first-order rate constants for the Asu formation has a bell-shaped profile, the maximum being close to the pK(a) of the aspartic acid side chains. Moreover, the values of the rate constants are of the same magnitude of those measured for Asp-containing peptides whose sequence mimics the Asu formation site of the enzyme. This feature indicates that Asp67 and beta-Asp67 residues in the deamidated RNase A derivatives are sited in a region flexible enough to permit the cyclization of the carboxylic side chain to succinimide ring. These results are discussed at the light on to the three-dimensional structure and the thermodynamic stability of the aspartic acid derivatives of RNase A.