Angiogenin, a potent inducer of neovascularization, is the only angiogenic molecule known to exhibit ribonucleolytic activity. Its overall structure, as determined at 2.4 A, is similarto that of pancreatic ribonuclease A, but it differs markedly in several distinct areas, particularly the ribonucleolytic active center and the putative receptor binding site, both of which are critically involved in biological function. Most sri ly, the site that is spatially analogous to that for pyrimidine binding in ribonuclease A differs siificantly in conformation and is "obstructed" by glutamine-117. Movement of this and adjacent residues may be required for substrate binding to anginin and, hence, constitute a key part of its menism of action.Human angiogenin (Ang), a single-chain polypeptide (Mr 14,124) present in tumor cell conditioned medium and normal serum (1, 2), is a potent inducer of neovascularization (1). It binds specifically to endothelial cells in culture (3) and elicits second-messenger responses (4). It also binds heparin (38), can serve as a substratum for endothelial cell adhesion (5), and is translocated to the nucleus (39). Among angiogenic molecules, Ang is unique in that it is a ribonucleolytic enzyme (6) with an amino acid sequence 33% identical to that of bovine pancreatic ribonuclease (RNase) A (7). Moreover, although Ang has the same general catalytic properties as RNase A-it cleaves preferentially on the 3' side of pyrimidines and follows a transphosphorylation/hydrolysis mechanism-its activity differs markedly both in magnitude and in specificity (6,8).Efforts to delineate the structural basis for the characteristic enzymatic and biological activities of Ang have been guided in large part by the vast wealth of existing information on RNase A, much of it derived from x-ray crystallography (9,10 2915The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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The nematode Angiostrongylus vasorum is becoming more widely recorded globally, and is of increasing concern as a cause of disease in dogs. Apparent geographic spread is difficult to confirm due to a lack of standardized disease recording systems, increasing awareness among veterinary clinicians, and recent improvements in diagnostic technologies. This study examines the hypothesis that A. vasorum has spread in recent years by repeating the methods of a previous survey of the fox population. The hearts and lungs of 442 foxes from across Great Britain were collected and examined by dissection and flushing of the pulmonary circulation and microscopic inspection of tracheal scrapes. Sampling and parasite extraction methods were identical to an earlier survey in 2005 to ensure comparability. Prevalence of A. vasorum was 18·3% (exact binomial confidence bounds 14·9-22·3), compared with 7·3% previously (5·3-9·9, n = 546), and had increased significantly in most regions, e.g. 7·4% in the Northern UK (previously zero) and 50·8% in the south-east (previously 23·2%). Other nematodes identified were Crenosoma vulpis (prevalence 10·8%, CI 8·1-14·2) and Eucoleus aerophilus (31·6%, CI 27·3-36·2). These data support the proposal that A. vasorum has increased in prevalence and has spread geographically in Great Britain.

Seventy-four European hedgehogs (Erinaceus europaeus) that had died in wildlife rehabilitation centres were dissected and their parasite burdens documented. Overall parasite prevalence was 91%, and a total of six helminth species were isolated: five nematodes (Crenosoma striatum, Eucoleus aerophilus, Capillaria erinacei, Capillaria ovoreticulata and Capillaria spp.), one trematode (Brachylaemus erinacei) and one acanthocephalan (Oliganthorhynchus erinacei). The tick Ixodes hexagonus and flea Archeopsylla erinacei were also collected. The effect of parasite infection on body condition was assessed by correlation of burdens with the residuals of weight-skeletal length regression. Tick presence was positively related to body condition; for other parasites, no significant relationship was found. Faecal egg or larval count was closely correlated with adult parasite burden for C. striatum and Capillaria/Eucoleus spp., but not for other species. Coprological analysis should therefore be useful for in vivo studies of nematode parasite infection in hedgehogs. The epidemiology of parasites in hedgehogs and their possible role in recent population declines are discussed.

BackgroundAngiostrongylus vasorum is a highly pathogenic metastrongylid nematode affecting dogs, which uses gastropod molluscs as intermediate hosts. The geographical distribution of the parasite appears to be heterogeneous or patchy and understanding of the factors underlying this heterogeneity is limited. In this study, we compared the species of gastropod present and the prevalence of A. vasorum along a rural–urban gradient in two cities in the south-west United Kingdom.MethodsThe study was conducted in Swansea in south Wales (a known endemic hotspot for A. vasorum) and Bristol in south-west England (where reported cases are rare). In each location, slugs were sampled from nine sites across three broad habitat types (urban, suburban and rural). A total of 180 slugs were collected in Swansea in autumn 2012 and 338 in Bristol in summer 2014. A 10 mg sample of foot tissue was tested for the presence of A. vasorum by amplification of the second internal transcribed spacer (ITS-2) using a previously validated real-time PCR assay.ResultsThere was a significant difference in the prevalence of A. vasorum in slugs between cities: 29.4 % in Swansea and 0.3 % in Bristol. In Swansea, prevalence was higher in suburban than in rural and urban areas. Comparing the sampled slug fauna, Arion rufus was found in greater numbers in Swansea than Bristol, and was commonly infected (prevalence 41 %). This, alongside the timing of slug collections in summer rather than autumn, could explain low infection prevalence in the Bristol sample. In the absence of Ar. rufus as a preferred host for A. vasorum, Ar. flagellus and Limacus maculatus appear to act as versatile hosts that are present in suburban and urban areas in Swansea (prevalence in Ar. flagellus 33 %; in L. maculatus 44 %) and in Bristol (prevalence in Ar. flagellus 0.9 %). These slug species might provide A. vasorum with an alternative vehicle to reach the final host, when the main host Ar. rufus is scarce or absent.ConclusionWe conclude that the composition of the slug fauna varies spatially, and that this could help explain patchiness in the prevalence of A. vasorum. A suburban peak was found in the prevalence of infection in intermediate hosts, perhaps explained by a higher density of competent intermediate and/or definitive hosts.

The three-dimensional structure of cat-muscle pyoruvate kinase has been refined at a resolution of 2.6 A. The details of the structure permit interpretation of the original heavy-atom studies and give insight into the importance of conserved residues in pyruvate kinases and the allosteric behaviour of the enzyme. There are a small number of essential residues which determine the relative orientations of domains and the precise nature of intersubunit contacts. Arginine residues are particularly important.

Recombinant yeast pyruvate kinase has been purified from a strain of Saccharomyces cerevisiae expressing the enzyme to very high levels. Expression was from a multicopy plasmid under the control of the yeast phosphoglycerate kinase promoter. The gene was expressed in the absence of the genomically encoded pyruvate kinase, using a strain of yeast in which the pyruvate kinase gene has been disrupted by the insertion of the yeast Ura3 gene. The purification procedure minimised proteolytic artefacts and enabled the covenient purification of 15 -20 mg enzyme from 1 1 culture. The purified enzyme was characterised by a high specific activity and by a lack of proteolytic degradation. Two active-site mutants of yeast pyruvate kinase have been produced, expressed and characterised in this system and preliminary results are described.Pyruvate kinase (ATP : pyruvate 2-0-phosphotransferase) is an enzyme of glycolysis that plays a major role in regulating the flux from fructose 1,6-bisphosphate (Fru(l,6)P2) to pyruvate. The enzyme is a homotetramer, with a subunit M , of 55 000 -60000, and catalyses the essentially irreversible reaction,Pyruvate kinase has been extensively studied from a wide range of organisms and much is known about its physical and catalytic properties. It exists as four isoenzymes in mammals designated M1, M2, L and R. The M1 isoenzyme is essentially non-regulated, whereas the remaining isoenzymes (as well as the enzymes from lower organisms) exhibit a sigmoidal kinetic profile with respect to phosphoenolpyruvate concentration, and are allosterically regulated by a large number of nonsubstrate molecules. All forms of the enzyme require both monovalent and bivalent cations for activity. The former is normally potassium whereas a number of bivalent cations will suffice. Two bivalent cations are required/active site, one primarily associated with the nucleotide and the other more closely enzyme associated (see Muirhead, 1987, for a review).The three-dimensional structure of cat muscle M1 isoenzyme has been solved to a resolution of 0.26 nm (Muirhead et al., 1986). Each subunit of cat muscle M1 pyruvate kinase consists of four domains, N, A, B and C. Domain A is an eight-stranded a/B barrel, typical of a number of enzymes (Farber and Petsko, 1990). The active site lies between do-1 Kt, Mg'+

A variant of bovine pancreatic ribonuclease A has been prepared with seven amino acid substitutions (Q55K, N62K, A64T, Y76K, S80R, E111G, N113K). These substitutions recreate in RNase A the basic surface found in bovine seminal RNase, a homologue of pancreatic RNase that diverged some 35 million years ago. Substitution of a portion of this basic surface (positions 55, 62, 64, 111 and 113) enhances the immunosuppressive activity of the RNase variant, activity found in native seminal RNase, while substitution of another portion (positions 76 and 80) attenuates the activity. Further, introduction of Gly at position 111 has been shown to increase the catalytic activity of RNase against double‐stranded RNA. The variant and the wild‐type (recombinant) protein were crystallized and their structures determined to a resolution of 2.0 Å. Each of the mutated amino acids is seen in the electron density map. The main change observed in the mutant structure compared with the wild‐type is the region encompassing residues 16–22, where the structure is more disordered. This loop is the region where the polypeptide chain of RNase A is cleaved by subtilisin to form RNase S, and undergoes conformational change to allow residues 1–20 of the RNase to swap between subunits in the covalent seminal RNase dimer.