Mapping the ribonucleolytic active site of eosinophil-derived neurotoxin (EDN). High resolution crystal structures of EDN complexes with adenylic nucleotide inhibitors

Eosinophil-derived neurotoxin (EDN), a basic ribonuclease found in the large specific granules of eosinophils, belongs to the pancreatic RNase A family. Although its physiological function is still unclear, it has been shown that EDN is a neurotoxin capable of inducing the Gordon phenomenon in rabbits. EDN is also a potent helminthotoxin and can mediate antiviral activity of eosinophils against isolated virions of the respiratory syncytial virus. EDN is a catalytically efficient RNase sharing similar substrate specificity with pancreatic RNase A with its ribonucleolytic activity being absolutely essential for its neurotoxic, helminthotoxic, and antiviral activities. The crystal structure of recombinant human EDN in the unliganded form has been determined previously (Mosimann, S. C., Newton, D. L., Youle, R. J., and James, M. N. G. (1996) J. Mol. Biol. 260, 540-552). We have now determined high resolution (1.8 A) crystal structures for EDN in complex with adenosine-3',5'-diphosphate (3',5'-ADP), adenosine-2',5'-di-phosphate (2',5'-ADP), adenosine-5'-diphosphate (5'-ADP) as well as for a native structure in the presence of sulfate refined at 1.6 A. The inhibition constant of these mononucleotides for EDN has been determined. The structures present the first detailed picture of differences between EDN and RNase A in substrate recognition at the ribonucleolytic active site. They also provide a starting point for the design of tight-binding inhibitors, which may be used to restrain the RNase activity of EDN.     

An investigation of the activity of recombinant rat skeletal muscle cytosolic sialidase

Rat cytosolic sialidase is expressed at elevated levels in skeletal muscle and is believed to play a role in the myogenic differentiation of muscle cells. Here, we observed varying levels of enhancement of sialidase activity in the presence a range of divalent cations. In particular, a significant enhancement of activity was observed in the presence of Ca2+. Conversely, inhibition of the sialidase activity was found when the enzyme was incubated in the presence of Cu2+, EDTA, and a range of carbohydrate-based inhibitors. Finally, an investigation of the enzymatic hydrolysis of a synthetic substrate, 4-methylumbelliferyl N-acetyl-alpha-D-neuraminide, by 1H NMR spectroscopy revealed that the reaction catalysed by rat skeletal muscle cytosolic sialidase proceeds with overall retention of anomeric configuration. This result further supports the notion that all sialidases appear to be retaining enzymes.     

Results of the Ontario Survey on the Prevalence and Control of Hypertension

Background

Available information on the prevalence and management of hypertension in the Canadian population dates back to 1986–1992 and probably does not reflect the current status of this major risk factor for cardiovascular disease. We sought to evaluate the current prevalence and management of hypertension among adults in the province of Ontario.

Methods

Potential respondents from randomly selected dwellings within target neighbourhoods in 16 municipalities were contacted at their homes to request participation in the study. For potential respondents who agreed to participate, blood pressure was measured with an automated device. Estimation weights were used to obtain representative estimates of population parameters. Responses were weighted to the total adult population in Ontario of 7 996 653.

Results

From 6436 eligible dwellings, contact was made with 4559 potential participants, of whom 2992 agreed to participate. Blood pressure measurements were obtained for 2551 of these respondents (age 20–79 years). Hypertension, defined as systolic blood pressure of 140 mm Hg or more, diastolic blood pressure of 90 mm Hg or more, or treatment with an antihypertensive medication, was identified in 21.3% of the population overall (23.8% of men and 19.0% of women). Prevalence increased with age, from 3.4% among participants 20–39 years of age to 51.6% among those 60–79 years of age. Hypertension was more common among black people and people of South Asian background than among white people; hypertension was also associated with higher body mass index. Among participants with hypertension, 65.7% were undergoing treatment with control of hypertension, 14.7% were undergoing treatment but the hypertension was not controlled, and 19.5% were not receiving any treatment (including 13.7% who were unaware of their hypertension). The extent of control of hypertension did not differ significantly by age, sex, ethnic background or comorbidities.

Interpretation

In Ontario, the overall prevalence of hypertension is high in the older population but appears not to have increased in recent decades. Hypertension management has improved markedly among all age groups and for both sexes.Hypertension is a major risk factor for premature cardiovascular morbidity and mortality. Epidemiologic studies have indicated that, for people 40–69 years of age, each increase of 20 mm Hg in usual systolic blood pressure is associated with a doubling of mortality rates for stroke and ischemic heart disease.¹ In 2002 the World Health Organization estimated that at least 50% of cases of cardiovascular disease and at least 75% of strokes were caused by elevated blood pressure.² At the same time, randomized clinical trials have demonstrated that these risks can be markedly reduced by antihypertensive drug therapy. The substantial burden of suffering associated with hypertension, combined with the availability of feasible and accurate means of detection and a clear benefit from treatment have led to worldwide recommendations for hypertension screening and management.Currently available Canadian data on the prevalence and management of hypertension are based on Canadian Heart Health Surveys that took place between 1986 and 1992. At that time, 22% of adult Canadians had hypertension, but only 16% of these had their blood pressure treated and controlled.³ These data are clearly out of date, and there has been no way of assessing the impact of changes in lifestyle, including higher rates of obesity and a sedentary lifestyle, on the prevalence of hypertension or determining whether hypertension management has improved with the widespread use of newer classes of antihypertensive drugs such as calcium antagonists and blockers of the renin–angiotensin system.We sought to evaluate the current prevalence and management of hypertension among adults in the province of Ontario.     

Multiresistant Gram-negative bacteria: the role of high-risk clones in the dissemination of antibiotic resistance

Multilocus sequence typing reveals that many bacterial species have a clonal structure and that some clones are widespread. This underlying phylogeny was not revealed by pulsed-field gel electrophoresis, a method better suited to short-term outbreak investigation. Some global clones are multiresistant and it is easy to assume that these have disseminated from single foci. Such conclusions need caution, however, unless there is a clear epidemiological trail, as with KPC carbapenemase-positive Klebsiella pneumoniae ST258 from Greece to northwest Europe. Elsewhere, established clones may have repeatedly and independently acquired resistance. Thus, the global ST131 Escherichia coli clone most often has CTX-M-15 extended-spectrum β-lactamase (ESBL), but also occurs without ESBLs and as a host of many other ESBL types. We explore this interaction of clone and resistance for E. coli, K. pneumoniae, Acinetobacter baumannii- a species where three global lineages dominate--and Pseudomonas aeruginosa, which shows clonal diversity, but includes the relatively 'tight' serotype O12/Burst Group 4 cluster that has proved adept at acquiring resistances--from PSE-1 to VIM-1 β-lactamases--for over 20 years. In summary, 'high-risk clones' play a major role in the spread of resistance, with the risk lying in their tenacity--deriving from poorly understood survival traits--and a flexible ability to accumulate and switch resistance, rather than to constant resistance batteries.     

Host-parasite interface of the urinary bladder-inhabiting nematode Trichosomoides crassicauda: changes induced in the urothelium of infected rats

The urinary bladders of rats infected with Trichosomoides crassicauda were studied by light, scanning and transmission electron microscopy. The bladder epithelium of infected rats showed a diffuse, mild, flat hyperplasia, four to six cells thick. The anterior parts of adult female worms were embedded in tunnels within the hyperplastic epithelium with the posterior portions of the parasites lying free in the bladder lumen. The hyperplastic epithelial cells forming the inner layer of the tunnel wall, adjacent to the parasite, showed degenerative changes. These cells contained single or multiple pycnotic nuclei. Their cytoplasm was fibro-granular in appearance, with few distinct organelles, and the luminal surfaces of the cells were not limited by plasma membranes. Small numbers of granular cells, similar in appearance to mast cells, were seen in, and possibly crossing, the epithelium. There was no infection-related increase in the number or type of inflammatory cells in the lamina propria. Questions of interest emerging from this study relate to the nutrition of an adult nematode occupying an intra-epithelial location, and the absence of a significant chronic inflammatory response to the mature worm. It is suggested that the avascular hyperplastic epithelium of the bladder is an immunologically 'protected site' for the mature female T. crassicauda.