Identification of protease and ADP-ribose 1''-monophosphatase activities associated with transmissible gastroenteritis virus non-structural protein 3.

The replicase polyproteins, pp1a and pp1ab, of porcine Transmissible gastroenteritis virus (TGEV) have been predicted to be cleaved by viral proteases into 16 non-structural proteins (nsp). Here, enzymic activities residing in the amino-proximal region of nsp3, the largest TGEV replicase processing product, were characterized. It was shown, by in vitro translation experiments and protein sequencing, that the papain-like protease 1, PL1pro, but not a mutant derivative containing a substitution of the presumed active-site nucleophile, Cys1093, cleaves the nsp2|nsp3 site at 879Gly|Gly880. By using an antiserum raised against the pp1a/pp1ab residues 526–713, the upstream processing product, nsp2, was identified as an 85 kDa protein in TGEV-infected cells. Furthermore, PL1pro was confirmed to be flanked at its C terminus by a domain (called X) that mediates ADP-ribose 1''-phosphatase activity. Expression and characterization of a range of bacterially expressed forms of this enzyme suggest that the active X domain comprises pp1a/pp1ab residues Asp1320–Ser1486.

Human coronavirus 229E nonstructural protein 13: characterization of duplex-unwinding, nucleoside triphosphatase, and RNA 5'-triphosphatase activities.

The human coronavirus 229E (HCoV-229E) replicase gene-encoded nonstructural protein 13 (nsp13) contains an N-terminal zinc-binding domain and a C-terminal superfamily 1 helicase domain. A histidine-tagged form of nsp13, which was expressed in insect cells and purified, is reported to unwind efficiently both partial-duplex RNA and DNA of up to several hundred base pairs. Characterization of the nsp13-associated nucleoside triphosphatase (NTPase) activities revealed that all natural ribonucleotides and nucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed most efficiently. Using the NTPase active site, HCoV-229E nsp13 also mediates RNA 5'-triphosphatase activity, which may be involved in the capping of viral RNAs.

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.

The human coronavirus 229E superfamily 1 helicase has RNA and DNA duplex-unwinding activities with 5'-to-3' polarity.

The human coronavirus 229E replicase gene encodes a protein, p66HEL, that contains a putative zinc finger structure linked to a putative superfamily (SF) 1 helicase. A histidine-tagged form of this protein, HEL, was expressed using baculovirus vectors in insect cells. The purified recombinant protein had in vitro ATPase activity that was strongly stimulated by poly(U), poly(dT), poly(C), and poly(dA), but not by poly(G). The recombinant protein also had both RNA and DNA duplex-unwinding activities with 5'-to-3' polarity. The DNA helicase activity of the enzyme preferentially unwound 5'-oligopyrimidine-tailed, partial-duplex substrates and required a tail length of at least 10 nucleotides for effective unwinding. The combined data suggest that the coronaviral SF1 helicase functionally differs from the previously characterized RNA virus SF2 helicases.

First report of a novel plant lysozyme with both antifungal and antibacterial activities.

A novel lysozyme exhibiting antifungal activity and with a molecular mass of 14.4 kDa in SDS–polyacrylamide gel electrophoresis was isolated from mung bean (Phaseolus mungo) seeds using a procedure that involved aqueous extraction, ammonium sulfate precipitation, ion exchange chromatography on CM-Sephadex, and high-performance liquid chromatography on POROS HS-20. Its N-terminal sequence was very different from that of hen egg white lysozyme. Its pI was estimated to be above 9.7. The specific activity of the lysozyme was 355 U/mg at pH 5.5 and 30 °C. The lysozyme exhibited a pH optimum at pH 5.5 and a temperature optimum at 55 °C. It is reported herein, for the first time, that a novel plant lysozyme exerted an antifungal action toward Fusarium oxysporum, Fusarium solani, Pythium aphanidermatum, Sclerotium rolfsii, and Botrytis cinerea, in addition to an antibacterial action against Staphylococcus aureus.

Size, albedo, and taxonomic type of potential spacecraft target Asteroid (10302) 1989 ML

The Amor-type near-Earth Asteroid (10302) 1989 ML has an “Earth-like” orbit (period 1.44 yr, eccentricity 0.14, inclination 4.4°), therefore the energy required to reach it from the Earth is relatively small making it a very attractive target for rendezvous missions. We have observed 1989 ML in the thermal infrared using the Spitzer Space Telescope, and compared these data with optical and near-infrared observations. The Spitzer results imply a diameter of 0.28±0.05 km and a geometric albedo of 0.37±0.15; together with the reflectance spectrum they are consistent with the relatively rare E classification.

Obesity in People with Learning Disabilities: the impact of nurse led health screenings and health promotion activities

Background. Obesity appears to be more common among people with intellectual disabilities, with few studies focusing on achieving weight reduction. Aim. Firstly, to follow up people identified as overweight and obese following special health screening clinics and to determine the actions taken. Secondly, to evaluate the impact of health promotion classes on participants' weight loss. Methods. A clinic led by two learning disbaility nurses was held for all people aged 10 years and over (n=464) who attended special services within the area of one Health and Social Services Trust in Northern Ireland. In a second study, the nurses organised health promotion classes for 20 people over a 6 - 8 week period. Findings. The health screen identified 64% of adults and 26% of 10 - 19 year olds as being overweight or obese. Moreover, those aged 40 - 49 years who were obese had significantly higher levels of blood pressure. However, information obtained from a follow up questionnaire sent after 3 months suggested that of the 122 people identified for wiehgt reduciton, action had been taken for only 34% of them and only three were reported to have lost weight. The health promotion classes, however, led to a significant reduction in weight and body mass index scores. Conclusion. Health screening per se has limited impact on reducing obesity levels in this client group. Rather, health personnel such as general practitioners, nurses and health promotion staff need to work in partnership with service staff, carers and people with intellectual disabiltieis to create more active lifestyles.

Neuromuscular adaptation during skill acquisition on a two degree-of-freedom target-acquisition task: Isometric torque production

In this study we attempted to identify the principles that govern the changes in neural control that occur during repeated performance of a multiarticular coordination task. Eight participants produced isometric flexion/extension and pronation/supination torques at the radiohumeral joint, either in isolation (e.g., flexion) or in combination (e.g., flexion - supination), to acquire targets presented by a visual display. A cursor superimposed on the display provided feedback of the applied torques. During pre- and postpractice tests, the participants acquired targets in eight directions located either 3.6 cm (20% maximal voluntary contraction [MVC]) or 7.2 cm (40% MVC) from a neutral cursor position. On each of five consecutive days of practice the participants acquired targets located 5.4 cm (30% MVC) from the neutral position. EMG was recorded from eight muscles contributing to torque production about the radiohumeral joint during the pre- and posttests. Target-acquisition time decreased significantly with practice in most target directions and at both target torque levels. These performance improvements were primarily associated with increases in the peak rate of torque development after practice. At a muscular level, these changes were brought about by increases in the rates of recruitment of all agonist muscles. The spatiotemporal organization of muscle synergies was not significantly altered after practice. The observed adaptations appear to lead to performances that are generalizable to actions that require both greater and smaller joint torques than that practiced, and may be successfully recalled after a substantial period without practice. These results suggest that tasks in which performance is improved by increasing the rate of muscle activation, and thus the rate of joint torque development, may benefit in terms of the extent to which acquired levels of performance are maintained over time.