Ulla Christensen

[28] Assay of coagulation proteases using peptide chromogenic and fluorogenic substrates

Publisher Summary Amino acid chromogenic and fluorogenic substrates have been used for many years for assaying proteases. The sensitivity of the assay procedures that employ these substrates and the convenience of spectrophotometric or fluorometric measurements has led to their widespread use. Most of the early amino acid chromogenic and fluorogenic substrates are highly selective for the primary specificity-determining (P1) amino acid; thus substrates such as benzoylarginine-p-nitroanilide, for assaying trypsin-like proteases, as well as aromatic amino acidp-nitrophenyl esters for chymotrypsin-like proteases, have been extensively investigated and employed for routine proteolytic enzyme assay. The recognition that both the selectivity of many proteases and their catalytic efficiency depend on interactions with subsite amino acids in the peptide substrate coupled with the availability of amino acid sequences around the cleavage sites in several zymogens of the coagulation and fibrinolytic systems has led to the synthesis and commercial availability of a variety of peptide chromogenic and fluorogenic substrates with much greater selectivity than the single amino acid chromogenic and fluorogenic substrates.

Income inequality, individual income, and mortality in Danish adults: analysis of pooled data from two cohort studies

Abstract Objective: To analyse the association between area income inequality and mortality after adjustment for individual income and other established risk factors. Design: Analysis of pooled data from two cohort studies. The relation between income inequality in small areas of residence (parishes) and individual mortality was examined with Cox proportional hazard analyses. Setting: Two population studies conducted in Copenhagen, Denmark. Participants: 13 710 women and 12 018 men followed for a mean of 12.8 years. Main outcome measure: All cause mortality. Results: Age standardised mortality was highest in the parishes with the least equal income distribution. After adjustment for individual risk factors, parish income inequality was not associated with mortality, whereas individual household income was. Thus, individuals in the highest income quarter had lower mortality than those in the lowest quarter (adjusted hazard ratio for men 0.51 (95% confidence interval 0.45 to 0.59) and for women 0.60 (0.54 to 0.68)). Conclusion: Area income inequality is not in itself associated with all cause mortality in this Danish population. Adjustment for individual risk factors makes the apparent effect disappear. This may be the result of Denmarks welfare system, based on a Nordic model. What is already known on this topic Several ecological studies have shown that higher levels of income inequality in countries, states, or smaller areas are associated with higher all cause mortality A few prospective studies from the United States have examined this after controlling for individual risk factors What this study adds Inequality in the distribution of income in parishes in Copenhagen is as high as inequality reported from metropolitan areas in the United States Area based income inequality did not affect all cause mortality after adjustment for individual income and other risk factors Denmarks welfare system (based on a Nordic model) may even out the effect of area inequality

Glucoamylase: structure/function relationships, and protein engineering.

Glucoamylases are inverting exo-acting starch hydrolases releasing beta-glucose from the non-reducing ends of starch and related substrates. The majority of glucoamylases are multidomain enzymes consisting of a catalytic domain connected to a starch-binding domain by an O-glycosylated linker region. Three-dimensional structures have been determined of free and inhibitor complexed glucoamylases from Aspergillus awamori var. X100, Aspergillus niger, and Saccharomycopsis fibuligera. The catalytic domain folds as a twisted (alpha/alpha)(6)-barrel with a central funnel-shaped active site, while the starch-binding domain folds as an antiparallel beta-barrel and has two binding sites for starch or beta-cyclodextrin. Certain glucoamylases are widely applied industrially in the manufacture of glucose and fructose syrups. For more than a decade mutational investigations of glucoamylase have addressed fundamental structure/function relationships in the binding and catalytic mechanisms. In parallel, issues of relevance for application have been pursued using protein engineering to improve the industrial properties. The present review focuses on recent findings on the catalytic site, mechanism of action, substrate recognition, the linker region, the multidomain architecture, the engineering of specificity and stability, and roles of individual substrate binding subsites.

The mediation proportion: a structural equation approach for estimating the proportion of exposure effect on outcome explained by an intermediate variable.

It is often of interest to assess how much of the effect of an exposure on a response is mediated through an intermediate variable. However, systematic approaches are lacking, other than assessment of a surrogate marker for the endpoint of a clinical trial. We review a measure of “proportion explained” in the context of observational epidemiologic studies. The measure has been much debated; we show how several of the drawbacks are alleviated when exposures, mediators, and responses are continuous and are embedded in a structural equation framework. These conditions also allow for consideration of several intermediate variables. Binary or categorical variables can be included directly through threshold models. We call this measure the mediation proportion, that is, the part of an exposure effect on outcome explained by a third, intermediate variable. Two examples illustrate the approach. The first example is a randomized clinical trial of the effects of interferon-α on visual acuity in patients with age-related macular degeneration. In this example, the exposure, mediator and response are all binary. The second example is a common problem in social epidemiology—to find the proportion of a social class effect on a health outcome that is mediated by psychologic variables. Both the mediator and the response are composed of several ordered categorical variables, with confounders present. Finally, we extend the example to more than one mediator.

The action of Factor Xa on peptide p-nitroanilide substrates: substrate selectivity and examination of hydrolysis with different reaction conditions

Kinetic parameters for the action of bovine Factor Xa (EC 3.4.21.22) on 25 commercially available peptide p-nitroanilides have been determined. The selectivity constant, kc/Km, ranges from 1.5 X 10(1) to 2 X 10(6) M-1 X s-1 for the poorest and the best substates, respectively. The best substrates for Factor Xa were identified as those with arginine in the P1 position, and glycine in the P2 position. Quantitative distinction between lysine and arginine in the P1 position and other amino acids in the P2-P4 positions of the substrate is reported from the changes in the kinetic parameters for substrates differing in only a single amino acid in these positions. Effect of NaCl and CaCl2 concentrations and temperature on the action of Factor Xa on selected substrates have been assessed. Km values for Factor Xa hydrolysis of most substrates are greater than 100 microM. Solubility of the substrates consequently restricts measurements of reaction velocities to concentrations lower than desirable for optimally determining kc. Comparison of these kinetic parameters for Factor Xa with those of thrombin (Lottenberg, R., Hall, J.A., Blinder, M., Binder, E. and Jackson, C.M. (1983) Biochim. Biophys. Acta 742,539-557) for these same substrates indicates that the greater hydrolytic efficiency of thrombin is due primarily to lower Km values.

The longitudinal impact of partner coping in couples following 5 years of unsuccessful fertility treatments.

BACKGROUND Because there is a lack of longitudinal research examining the impact of partner coping in couples experiencing infertility, we know very little about the long-term nature of coping with infertility and how partner coping strategies impact personal, marital and social distress. METHODS Participants were Danish men and women about to start a cycle of assisted reproduction treatment who were followed for a 5 year period of unsuccessful treatments. Multilevel modeling using the actor-partner interdependence model was used to examine the couple as the unit of analysis. RESULTS Active and passive avoidance coping strategies were significantly related to increased personal, marital and social distress at the individual and partner level. Meaning-based coping strategies were related to decreases in a womans individual distress and her partners marital distress. CONCLUSIONS Partner coping strategies have a significant impact on the other member of the couple over time in men and women undergoing infertility treatments over a 5 year period. Physicians and mental health professionals can educate men and women regarding the ineffectiveness of avoidance coping strategies as well as the beneficial nature of finding new meaning and life goals while experiencing the stress of infertility.

Cynical hostility, socioeconomic position, health behaviors, and symptom load: a cross-sectional analysis in a Danish population-based study.

Objective: To analyze the cross-sectional association between cynical hostility and high symptom load in a Danish population-based study. Furthermore, the aim was to investigate to what extent health risk behaviors mediated this association. Methods: Data were based on a postal questionnaire in a Danish random sample of 3426 men and 3699 women aged 40 or 50 years. Cynical hostility was measured by the 8-item Cynical Distrust Scale. High symptom load was assessed by physiological and mental symptoms experienced within the last 4 weeks. Confounders were age and socioeconomic position, while potential mediators were alcohol consumption, smoking, physical activity, and BMI. Results: Higher cynical hostility was associated with self-reported symptom load. Health behaviors did not seem to mediate this effect. Socioeconomic position was a strong confounder for the effect on both health and health behaviors. After adjustment the effects of hostility on health remained with odds ratios of 2.1 (1.7–2.6) for women and 2.3 (1.8–2.8) for men. Conclusion: After adjustment for socioeconomic position, cynical hostility has an effect on self-reported high symptom load, and this effect is not mediated by health behaviors.

Predictors of return to work in employees sick-listed with mental health problems: findings from a longitudinal study

BACKGROUND Sickness absence due to mental health problems (MHPs) is increasing in several European countries. However, little is known about return to work (RTW) for employees with MHPs. This prospective study aimed to identify predictors for RTW in employees sick-listed with MHPs. METHODS Employees were recruited when applying for sickness benefit due to MHPs from the Municipality of Copenhagen (n = 644). Information about age, gender, occupation, self-reported RTW expectancy, self-reported reason for absence and prior absence with MHPs was retrieved from application forms for sickness benefit. Each participant was followed-up in the National Register for Social Transfer Payments for a maximum period of 52 weeks to estimate time to RTW. Hazard ratios for RTW with 95% confidence intervals were calculated using Cox proportional regression analyses. RESULTS Employees sick-listed with self-reported stress/burnout returned to work faster than those with self-reported depression (HR = 0.76), and other MHPs (HR = 0.56). A positive RTW expectancy of the sick-listed person (HR = 1.27) and no prior absence with MHPs (HR = 1.29) were associated with a shorter time to RTW. CONCLUSION Sickness absence due to self-reported stress/burnout, a positive RTW expectancy and no prior absence with MHPs predicted a shorter time to RTW among Danish employees sick listed with MHPs. Findings could help social insurance officers and other rehabilitation professionals to identify groups at high risk for prolonged absence.

C-terminal lysine residues of fibrinogen fragments essential for binding to plasminogen

C‐terminal lysine residue Fibrinogen Plasminogen Lysine binding site Fibrinogen fragment D Fibrinogen fragment E

Functional ability at age 75: is there an impact of physical inactivity from middle age to early old age?

The aim of this study is to analyze the impact of physical inactivity from middle age to early old age on functional ability at age 75. Physical activity is measured both as cumulated activity from age 50 to 60 to 70 and at three separate points in time.