Expression, purification, crystallization, and NMR studies of the helicase interaction domain of Escherichia coli DnaG primase

in Escherichia coli, the DnaG primase is the RNA polymerase that synthesizes RNA primers at replication forks. It is composed of three domains, a small N-terminal zinc-binding domain, a larger central domain responsible for RNA synthesis, and a C-terminal domain comprising residues 434-581 [DnaG(434-581)] that interact with the hexameric DnaB helicase. Presumably because of this interaction, it had not been possible previously to express the C-terminal domain in a stably transformed E coli strain. This problem was overcome by expression of DnaG(434-581) under control of tandem bacteriophage gimel-promoters, and the protein was purified in yields of 4-6 mg/L of culture and studied by NMR. A TOCSY spectrum of a 2 mM solution of the protein at pH 7.0, indicated that its structured core comprises residues 444-579. This was consistent with sequence conservation among most-closely related primases. Linewidths in a NOESY spectrum of a 0.5 mM sample in 10 mM phosphate, pH 6.05, 0.1 M NaCl, recorded at 36 degreesC, indicated the protein to be monomeric. Crystals of selenomethionine-substituted DnaG(434-581) obtained by the hanging-drop vapor-diffusion method were body-centered tetragonal, space group I4(1)22, with unit cell parameters a = b 142.2 Angstrom, c = 192.1 Angstrom, and diffracted beyond 2.7 Angstrom resolution with synchrotron radiation. (C) 2003 Elsevier Inc. All rights reserved.

Ten-year risk of first recurrent stroke and disability after first-ever stroke in the Perth Community Stroke Study

Background and Purpose-Limited information exists on the long-term prognosis after first-ever stroke. We aimed to determine the absolute frequency of first recurrent stroke and disability and the relative frequency of recurrent stroke over 10 years after first-ever stroke in Perth, Western Australia. Methods-For a 12-month period beginning February 1989, all individuals with suspected acute stroke or transient ischemic attack who lived in a geographically defined and representative region of Perth were registered prospectively. Patients with a definite first-ever stroke were followed up 10 years after the index event. Results-Over 10 years of follow-up, the cumulative risk of a first recurrent stroke was 43% (95% confidence interval [CI], 34 to 51). After the first year after first-ever stroke, the average annual risk of recurrent stroke was approximate to4%. Case fatality at 30 days after first recurrent stroke was 41%, which was significantly greater than the case fatality at 30 days after first-ever stroke (22%) (P=0.003). For 30-day survivors of first-ever stroke, the 10-year cumulative risk of death or new institutionalization was 79% (95% CI, 73 to 85) and of death or new disability was 87% (95% CI, 81 to 92). Conclusions-Over 10 years of follow-up, the risk of first recurrent stroke is 6 times greater than the risk of first-ever stroke in the general population of the same age and sex, almost one half of survivors remain disabled, and one seventh require institutional care. Effective strategies for prevention of stroke need to be implemented early, monitored frequently, and maintained long term after first-ever stroke.

Assessing cost-effectiveness - Mental health: introduction to the study and methods

Objective: The Assessing Cost-Effectiveness - Mental Health (ACE-MH) study aims to assess from a health sector perspective, whether there are options for change that could improve the effectiveness and efficiency of Australia's current mental health services by directing available resources toward 'best practice' cost-effective services. Method: The use of standardized evaluation methods addresses the reservations expressed by many economists about the simplistic use of League Tables based on economic studies confounded by differences in methods, context and setting. The cost-effectiveness ratio for each intervention is calculated using economic and epidemiological data. This includes systematic reviews and randomised controlled trials for efficacy, the Australian Surveys of Mental Health and Wellbeing for current practice and a combination of trials and longitudinal studies for adherence. The cost-effectiveness ratios are presented as cost (A$) per disability-adjusted life year (DALY) saved with a 95% uncertainty interval based on Monte Carlo simulation modelling. An assessment of interventions on 'second filter' criteria ('equity', 'strength of evidence', 'feasibility' and 'acceptability to stakeholders') allows broader concepts of 'benefit' to be taken into account, as well as factors that might influence policy judgements in addition to cost-effectiveness ratios. Conclusions: The main limitation of the study is in the translation of the effect size from trials into a change in the DALY disability weight, which required the use of newly developed methods. While comparisons within disorders are valid, comparisons across disorders should be made with caution. A series of articles is planned to present the results.

A long-term study in Merino sheep experimentally infected with Mycobacterium avium subsp paratuberculosis: clinical disease, faecal culture and immunological studies

Two longitudinal experiments involving Merino sheep challenged with either bovine or ovine strains of Mycobacterium avium subsp. paratuberculosis (Map) have been conducted over a period of 54 and 35 months, respectively. Blood samples for the interferon-gamma test, the absorbed ELISA and faecal samples for bacteriological culture were taken pre-challenge and monthly post-challenge. Infections were induced with either a bovine or ovine strain of Map in separate experiments with infections being more easily established, in terms of faecal bacterial shedding and clinical disease when the challenge inoculum was prepared from gut mucosal tissue than cultured bacteria. The patterns of response for shedding and clinical disease were similar. Cell-mediated immune responses were proportionally elevated by at least an order of magnitude in all sheep dosed with either a bovine or ovine strain of Map. Conversely, antibody responses were only elevated in a relatively small proportion of infected sheep. Neither of the clinically affected tissue challenged sheep developed an antibody response despite the presence of persistent shedding and the development and decline in cell-mediated immunity. The results indicated that for sheep the interferon-gamma test may be useful for determining if a flock has been exposed to ovine Johne's disease. (C) 2004 Elsevier B.V. All rights reserved.

The effects of predator odors in mammalian prey species: A review of field and laboratory studies

wPrey species show specific adaptations that allow recognition, avoidance and defense against predators. For many mammalian species this includes sensitivity towards predator-derived odors. The typical sources of such odors include predator skin and fur, urine, feces and anal gland secretions. Avoidance of predator odors has been observed in many mammalian prey species including rats, mice, voles, deer, rabbits, gophers, hedgehogs, possums and sheep. Field and laboratory studies show that predator odors have distinctive behavioral effects which include (1) inhibition of activity, (2) suppression of non-defensive behaviors such as foraging, feeding and grooming, and (3) shifts to habitats or secure locations where such odors are not present. The repellent effect of predator odors in the field may sometimes be of practical use in the protection of crops and natural resources, although not all attempts at this have been successful. The failure of some studies to obtain repellent effects with predator odors may relate to (1) mismatches between the predator odors and prey species employed, (2) strain and individual differences in sensitivity to predator odors, and (3) the use of predator odors that have low efficacy. In this regard, a small number of recent studies have suggested that skin and fur-derived predator odors may have a more profound lasting effect on prey species than those derived from urine or feces. Predator odors can have powerful effects on the endocrine system including a suppression of testosterone and increased levels of stress hormones such as corticosterone and ACTH. Inhibitory effects of predator odors on reproductive behavior have been demonstrated, and these are particularly prevalent in female rodent species. Pregnant female rodents exposed to predator odors may give birth to smaller litters while exposure to predator odors during early life can hinder normal development. Recent research is starting to uncover the neural circuitry activated by predator odors, leading to hypotheses about how such activation leads to observable effects on reproduction, foraging and feeding. (c) 2005 Elsevier Ltd. All rights reserved.

Achieving the millennium development goals for health - Cost effectiveness analysis of strategies for. maternal and neonatal health in developing countries

Objective To determine the costs and benefits of interventions for maternal and newborn health to assess the appropriateness of current strategies and guide future plans to attain the millennium development goals. Design Cost effectiveness analysis. Setting Two regions classified by the World Health Organization according to their epidemiological grouping: Afr-E, those countries in sub-Saharan Africa with very high adult and high child mortality, and Sear-D, comprising countries in South East Asia with high adult and high child mortality. Data sources Effectiveness data from several sources, including trials, observational studies, and expert opinion. For resource inputs, quantifies came from WHO guidelines, literature, and expert opinion, and prices from the WHO choosing interventions that are cost effective database. Main outcome measures Cost per disability adjusted life year (DALY) averted in year 2000 international dollars. Results The most cost effective mix of interventions was similar in Afr-E and Sear-D. These were the community based newborn care package, followed by antenatal care (tetanus toxoid, screening for pre-eclampsia, screening and treatment of asymptomatic bacteriuria and syphilis); skilled attendance at birth, offering first level maternal and neonatal care around childbirth; and emergency obstetric and neonatal care around and after birth. Screening and treatment of maternal syphilis, community based management of neonatal pneumonia, and steroids given during the antenatal period were relatively less cost effective in Sear-D. Scaling up all of the included interventions to 95% coverage would halve neonatal and maternal deaths. Conclusion Preventive interventions at the community level for newborn babies and at the primary care level for mothers and newborn babies are extremely cost effective, but the millennium development goals for maternal and child health will not be achieved without universal access to clinical services as well.

Premovement activity of the pre-supplementary motor area and the readiness for action: Studies of time-resolved event-related functional MRI

The supplementary motor area (SMA) is thought to play in important role in the preparation and organisation of voluntary movement. It has long been known that cortical activity begins to increase up to 2 s prior to voluntary self-initiated movement. This increasing premovement activity measured in EEG is known as the Bereitschaftspotential or readiness potential. Modern functional brain imaging methods, using event-related and time-resolved functional MRI techniques, are beginning to reveal the role of the SMA, and in particular the more anterior pre-SMA, in premovement activity associated with the readiness for action. In this paper we review recent studies using event-related time-resolved fMRI methods to examine the time-course of activation changes within the SMA throughout the preparation, readiness and execution of action. These studies suggest that the preSMA plays a common role in encoding or representing actions prior to our own voluntary self-initiated movements, during motor imagery, and from the observation of others' actions. We suggest that the pre-SMA generates and encodes motor representations which are then maintained in readiness for action. (c) 2005 Elsevier B.V. All rights reserved.

A long-term study in Angora goats experimentally infected with Mycobacterium avium subsp paratuberculosis: Clinical disease, faecal culture and immunological studies

Two longitudinal experiments involving Angora goats challenged with either bovine or ovine strains of Mycobacterium avium subspecies paratuberculosis (Map) have been conducted over a period of 54 and 35 months, respectively. Blood samples for the interferon-gamma (IFN-gamma) test and the absorbed ELISA and faecal samples for bacteriological culture were taken pre-challenge and monthly post-challenge. Persistent shedding, IFN-gamma production, seroconversion and clinical disease occurred earlier with the bovine Map gut mucosal tissue challenge inoculum than with cultured bacteria. The IFN-gamma responses of the gut mucosal tissue and bacterial challenge groups were substantially and consistently higher than those of the control group. The in vivo and cultured cattle strains were much more pathogenic for goats than the sheep strains with persistent faecal shedding, seroconversion and clinical disease occurring in the majority of bovine Map challenged goats. With the ovine Map, 3 goats developed persistent antibody responses but only one of these goats developed persistent faecal shedding and clinical disease. However, there was no significant difference between the IFN-gamma responses of the tissue challenged, bacterial challenged and control groups. Compared with sheep, the ELISA appeared to have higher sensitivity and the IFN-gamma test lower specificity. (C) 2005 Elsevier B.V. All rights reserved.

Overweight and obesity increase the risk of coronary heart disease: A pooled analysis of 30 prospective studies

The importance of overweight as a risk factor for coronary heart disease (CHD) remains unsettled. We estimated the relative risk (RR) for CHD associated with underweight (body mass index, BMI < 20 kg/m2), overweight (25 – 30 kg/m2) and obesity (= 30 kg/m2), compared with normal weight (20 – 25 kg/m2) in a random effects meta-analysis of 30 prospective studies, including 389,239 healthy, predominantly Caucasian persons. We also explored sources of heterogeneity between studies and examined effects of systematic adjustment for confounding and intermediary variables. Pooled age-, sex- and smoking-adjusted RRs (95% confidence interval) for overweight, obesity and underweight compared with normal weight were 1.33 (1.24 – 1.43), 1.69 (1.44 – 1.99) and 1.01 (0.85 – 1.20), respectively. Stratified analyses showed that pooled RRs for BMI were higher for studies with longer follow-up (= vs. < 15 years) and younger populations (< vs. = 60 years). Additional adjustment for blood pressure, cholesterol levels and physical activity decreased the RR per 5 BMI units from 1.28 (1.21 – 1.34) to 1.16 (1.11 – 1.21). We conclude that overweight and obesity are associated with a substantially increased CHD risk in Caucasians, whereas underweight is not. Prevention and reduction of overweight and obesity, therefore, remain of importance for preventing CHD.

An optimized model for rat liver perfusion studies

Conditions which influence the viability, integrity, and extraction efficiency of the isolated perfused rat liver were examined to establish optimal conditions for subsequent work in reperfusion injury studies including the choice of buffer, use of oncotic agents, hematocrit, perfusion flow rate, and pressure. Rat livers were perfused with MOPS-buffered Ringer solution with or without erythrocytes. Perfusates were collected and analyzed for blood gases, electrolytes, enzymes, radioactivity in MID studies, and lignocaine in extraction studies. Liver tissue was sampled for histological examinations, and wet:dry weight of the liver was also determined. MOPS-buffered Ringer solution was found to be superior to Krebs bicarbonate buffer, in terms of pH control and buffering capacity, especially during any prolonged period of liver perfusion. A pH of 7.2 is chosen for perfusion since this is the physiological pH of the portal blood. The presence of albumin was important as an oncotic agent, particularly when erythrocytes were used in the perfusate. Perfusion pressure, resistance, and vascular volume are how-dependent and the inclusion of erythrocytes in the perfusate substantially altered the flow characteristics for perfusion pressure and resistance but not vascular volume. Lignocaine extraction was relatively flow-independent. Perfusion injury as defined by enzyme release and tissue fine structure was closely related to the supply of O-2. The optimal conditions for liver perfusion depend upon an adequate supply of oxygen. This can be achieved by using either erythrocyte-free perfusate at a how rate greater than 6 ml/min/g liver or a 20% erythrocyte-containing perfusate at 2 ml/min/g. (C) 1996 Academic Press, Inc.

Mild myocardial infarction - A classification problem in epidemiologic studies

In studies assessing the trends in coronary events, such as the World Health Organization (WHO) MONICA Project (multinational MONItoring of trends and determinants of CArdiovascular disease), the main emphasis has been on coronary deaths and non-fatal definite myocardial infarctions (MI). It is, however, possible that the proportion of milder MIs may be increasing because of improvements in treatment and reductions in levels of risk factors. We used the MI register data of the WHO MONICA Project to investigate several definitions for mild non-fatal MIs that would be applicable in various settings and could be used to assess trends in milder coronary events. Of 38 populations participating in the WHO MONICA MI register study, more than half registered a sufficiently wide spectrum of events that it was possible to identify subsets of milder cases. The event rates and case fatality rates of MI are clearly dependent on the spectrum of non-fatal MIs, which are included. On clinical grounds we propose that the original MONICA category ''non-fatal possible MI'' could bt:divided into two groups: ''non fatal probable MI'' and ''prolonged chest pain.'' Non-fatal probable MIs are cases, which in addition to ''typical symptoms'' have electrocardiogram (EGG) or enzyme changes suggesting cardiac ischemia, but not severe enough to fulfil the criteria for non-fatal definite MI In more than half of the MONICA Collaborating Centers, the registration of MI covers these milder events reasonably well. Proportions of non-fatal probable MIs vary less between populations than do proportions of non fatal possible MIs. Also rates of non-fatal probable MI are somewhat more highly correlated with rates of fatal events and non-fatal definite MI. These findings support the validity of the category of non-fatal probable MI. In each center the increase in event rates and the decrease in case-fatality due to the inclusion of non-fatal probable MI was lar er for women than men. For the WHO MONICA Project and other epidemiological studies the proposed category of non-fatal probable MIs can be used for assessing trends in rates of milder MI. Copyright (C) 1997 Elsevier Science Inc.