Prevention of degradation of human polymorphonuclear leukocyte proteins by diisopropylfluorophosphate

Proteases can complicate the characterization of proteins from cells, especially human polymorphonuclear leukocytes (PMN), which contain abundant neutral proteases. We tested the ability of agents to inhibit proteolysis, with special reference to the subunit polypeptides of the contractile proteins actin, myosin, and actin-binding protein (ABP). Phenylmethylsulfonyl fluoride (PMSF), O-phenanthroline, EGTA, EDTA, N- ethylmaleimide, alone or in combinations, failed to prevent extensive proteolysis of the PMN proteins during solubilization of cells with dodecyl sulfate. These inhibitors and also alpha-1-antitrypsin and soybean trypsin inhibitor similarly could not prevent proteolysis during homogenization of cells in cold isosomolar sucrose. Treatment of PMN with greater than or equal to mM diisopropylfluorophosphate (DFP) prior to solubilization or homogenization markedly inhibited proteolysis. PMSF and DFP were equally effective in inhibiting proteolysis in PMN extracts, suggesting that the efficacy of DFP may result from its permeation of intact cells and granules before barriers are disrupted by detergents or homogenization. Treatment of PMN with DFP under conditions inhibiting proteolysis did not affect their rate of phagocytosis. We recommend the use of DFP in future studies correlating functions and protein structure of PMN.     

Improving care for patients with chronic heart failure in the community: the importance of a disease management program

STUDY OBJECTIVE: Utilizing a comparison group of patients with congestive heart failure (CHF) discharged to their primary care physicians, we sought to determine if disease management in a short-term, aggressive-intervention heart failure clinic (HFC) following hospital discharge is associated with improved outcomes. DESIGN: Chart review. SETTING: An integrated health-care center serving a tristate area. PATIENTS: Inclusion criteria were discharge from the hospital with a primary diagnosis of CHF, outpatient follow-up within the hospital system, and the presence of left ventricular systolic dysfunction as the basis for CHF. Patients were categorized into group 1 if they were referred to the HFC after hospital discharge, and into group 2 if follow-up care was provided by their primary care physician. MEASUREMENTS AND RESULTS: There were 38 patients in group 1 and 63 patients in group 2. There was a trend toward a shorter time to the first outpatient visit following discharge (11 days vs 15 days, p = 0.09), more outpatient visits within 90 days (10 visits vs 2 visits, p < 0.001), and more patient-initiated contacts (four contacts vs one contact, p = < 0.001) in group 1 compared to group 2, respectively. The combined hospital readmission and mortality rate at 90 days (10% vs 30%, p < 0.018) and 1 year (21% vs 43%, p < 0.02) was lower in group 1. There was a 77% relative risk reduction for 30-day hospital readmission in favor of group 1, and a statistically lower rate of readmissions at 90 days and 1 year. Utilization and maintenance of standardized CHF medications were significantly higher in patients who attended the HFC. CONCLUSIONS: A comprehensive disease management program for patients discharged with a diagnosis of CHF resulted in fewer rehospitalizations and improved event-free survival compared to patients followed up by their primary care physicians.     

Hematopoetic precursors respond to a unique B lymphocyte-derived factor in vivo

In this study we detected a factor that stimulates the proliferation of bone marrow-derived hematopoietic precursors in diffusion chambers implanted in mice. This factor, called diffusible colony-stimulating factor (D-CSF), was found in medium conditioned in the presence of spleen and peripheral blood cells from mice with B cell leukemia (BCL1). After the administration of D-CSF, the number of colonies formed in the plasma clot inside the chamber (CFU-DG) was increased, as were the number of hematopoietic precursors (CFU-MIX, CFU-S, CFU-C, and BFU-E) as judged by a subculture of diffusion chamber contents. Depletion of macrophages and T cells from the spleen cell suspension did not decrease the production of D-CSF, thereby indicating that it was derived from B cells. Neoplastic BCL1 cells appear to be the source because D-CSF could not be detected in medium conditioned with normal B cells. BCL1-conditioned medium (CM) did not enhance CFU-MIX, BFU-E, and CFU-C colony formation in vitro, which suggested that D-CSF is different from multi-CSF, EPA, or CSF. The addition of BCL1 CM to multi- CSF-, erythroid potentiating activity (EPA), and CSF (EL-4CM)- containing cultures had no effect on CFU-MIX, BFU-E, and CFU-C colony formation, thus indicating the absence of a synergistic or inhibitory activity. On the other hand, EL-4 CM, which stimulates CFU-MIX, BFU-E, and CFU-C in vitro, had no effect on CFU-DG in vivo. Biochemical characterization of BCL1 CM revealed that D-CSF is relatively heat stable and loses its bioactivity with protease treatments. It binds to lentil-lectin, according to gel-filtration chromatography has a relative molecular weight of approximately 43,000, and on reverse-phase high-performance liquid chromatography elutes with acetonitrile. These data also indicate that transformed B cells may serve as a source for hematopoietic regulators that act on hematopoietic precursors in vivo.     

Protein N-myristoylation in Escherichia coli: reconstitution of a eukaryotic protein modification in bacteria.

Protein N-myristoylation refers to the covalent attachment of a myristoyl group (C14:0), via amide linkage, to the NH2-terminal glycine residue of certain cellular and viral proteins. Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes this cotranslational modification. We have developed a system for studying the substrate requirements and biological effects of protein N-myristoylation as well as NMT structure-activity relationships. Expression of the yeast NMT1 gene in Escherichia coli, a bacterium that has no endogenous NMT activity, results in production of the intact 53-kDa NMT polypeptide as well as a truncated polypeptide derived from proteolytic removal of its NH2-terminal 39 amino acids. Each E. coli-synthesized NMT species has fatty acid and peptide substrate specificities that are indistinguishable from those of NMT recovered from Saccharomyces cerevisiae, suggesting that the NH2-terminal domain of this enzyme is not required for its catalytic activity. By using a dual plasmid system, N-myristoylation of a mammalian protein was reconstituted in E. coli by simultaneous expression of the yeast NMT1 gene and a murine cDNA encoding the catalytic (C) subunit of cAMP-dependent protein kinase (PK-A). The fatty acid specificity of N-myristoylation was preserved in this system: [9,10(n)-3H]myristate but not [9,10(n)3H]palmitate was efficiently linked to Gly-1 of the C subunit. [13,14(n)-3H]10-Propoxydecanoic acid, a heteroatom-containing analog of myristic acid with reduced hydrophobicity but similar chain length, was an effective alternative substrate for NMT that also could be incorporated into the C subunit of PK-A. Such analogs have recently been shown to inhibit replication of certain retroviruses that depend upon linkage of a myristoyl group to their gag polyprotein precursors (e.g., the Pr55gag of human immunodeficiency virus type 1). A major advantage of the bacterial system over eukaryotic systems is the absence of endogenous NMT and substrates, providing a more straightforward way of preparing myristoylated, analog-substituted, and nonmyristoylated forms of a given protein for comparison of their structural and functional properties. The system should facilitate screening of enzyme inhibitors as well as alternative NMT fatty acid substrates for their ability to be incorporated into a specific target protein. Our experimental system may prove useful for recapitulating other eukaryotic protein modifications in E. coli so that structure-activity relationships of modifying enzymes and their substrates can be more readily assessed.     

National health accounts data from 1996 to 2010: a systematic review

Objective

To collect, compile and evaluate publicly available national health accounts (NHA) reports produced worldwide between 1996 and 2010.

Methods

We downloaded country-generated NHA reports from the World Health Organization global health expenditure database and the Organisation for Economic Co-operation and Development (OECD) StatExtract website. We also obtained reports from Abt Associates, through contacts in individual countries and through an online search. We compiled data in the four main types used in these reports: (i) financing source; (ii) financing agent; (iii) health function; and (iv) health provider. We combined and adjusted data to conform with OECD’s first edition of A system of health accounts manual, (2000).

Findings

We identified 872 NHA reports from 117 countries containing a total of 2936 matrices for the four data types. Most countries did not provide complete health expenditure data: only 252 of the 872 reports contained data in all four types. Thirty-eight countries reported an average not-specified-by-kind value greater than 20% for all data types and years. Some countries reported substantial year-on-year changes in both the level and composition of health expenditure that were probably produced by data-generation processes. All study data are publicly available at http://vizhub.healthdata.org/nha/.

Conclusion

Data from NHA reports on health expenditure are often incomplete and, in some cases, of questionable quality. Better data would help finance ministries allocate resources to health systems, assist health ministries in allocating capital within the health sector and enable researchers to make accurate comparisons between health systems.     

Images of a Healthy Worksite: A Group-Randomized Trial for Worksite Weight Gain Prevention With Employee Participation in Intervention Design

Objectives. We assessed the effects of a worksite multiple-component intervention addressing diet and physical activity on employees’ mean body mass index (BMI) and the percentage of employees who were overweight or obese.Methods. This group-randomized trial (n = 3799) was conducted at 10 worksites in the northeastern United States. Worksites were paired and allocated into intervention and control conditions. Within- and between-groups changes in mean BMIs and in the percentage of overweight or obese employees were examined in a volunteer sample.Results. Within-group mean BMIs decreased by 0.54 kilograms per meter squared (P = .02) and 0.12 kilograms per meter squared (P = .73) at the intervention and control worksites, respectively, resulting in a difference in differences (DID) decrease of 0.42 kilograms per meter squared (P = .33). The within-group percentage of overweight or obese employees decreased by 3.7% (P = .07) at the intervention worksites and increased by 4.9% (P = .1) at the control worksites, resulting in a DID decline of 8.6% (P = .02).Conclusions. Our findings support a worksite population strategy that might eventually reduce the prevalence of overweight and obesity by minimizing environmental exposures to calorically dense foods and increasing exposures to opportunities for energy expenditure within worksite settings.Sixty-eight percent of adults residing in the United States are overweight or obese,1 and these conditions affect more than 1.4 billion adults worldwide.2 Traditional obesity control strategies, which have focused on changing diet and physical activity (PA) behaviors, provide significant individual benefits3 but are considered insufficient to reduce population disease burdens,4,5 for which broad, population-based approaches are needed.6 In addition to individual biology and behaviors, the physical, social, and cultural environment appears to contribute to the upward trend in population estimates of overweight and obesity7,8 by facilitating high-energy, low-nutrient diets and reducing the need to be physically active to perform activities of daily life.9Worksites are feasible self-contained environments with established communication systems in which interventions manipulating the food and PA environment and the social marketing of lifestyle changes can be implemented. Given that 58.4% of the US population aged 16 years or older is employed,10 worksite interventions have the potential to reach large number of adults11 and can foster the participation of employees in project development and sustainability.12–14 Moreover, participatory worksite interventions address workers’ needs, priorities, and interests and allow strategies to be adapted to the realities of individual sites.13 There is also a business case for weight control programs. In comparison with their nonobese counterparts, overweight and obese employees have higher absenteeism rates, have more work limitations, and are less productive.15–18With these issues in mind, the National Heart, Lung, and Blood Institute developed the Obesity Prevention in the Worksite initiative, a population-based approach to promoting behavioral change through environmental interventions that address prevention and control of weight gain.19 Prior to this initiative, worksite trials were either limited scope interventions, targeting a few aspects of the food or PA environment,9,20–23 or broader scope efforts simultaneously targeting risk factors for cardiovascular disease and cancer (e.g., smoking, diet).24–28 In addition, few studies addressed environmental influences related to excessive weight gain.Here we report the results of Images of a Healthy Worksite, one of the studies that is part of the Obesity Prevention in the Worksite initiative; this comprehensive nutrition and PA intervention was designed to promote healthy lifestyles and to stop the shift to the right of the population body mass index (BMI) curve. In this study, worksites were designated to receive an environmental intervention, and employees participated in intervention design. We hypothesized that mean BMIs among employees at the intervention worksites and the percentages of employees who were overweight or obese would not increase over a 2-year period or would increase less than at control worksites.     

The effect of influenza vaccination on IL2 production in healthy elderly: implications for current vaccination practices.

Age-related senescence of T-cell mediated responses is well recognized. This study was designed to determine how aging affects the T-cell mediated Interleukin 2 (IL2) response to influenza vaccination. A group of healthy elderly individuals were compared to a control group of healthy young adults for their response to the 1990 influenza vaccine. Cultures of peripheral blood mononuclear cells (PBMC) were prepared from venous blood samples taken prevaccination (pre) and 8 and 12 weeks post-vaccination (post). PBMC cultures stimulated with inactivated A/Shanghai/16/89 (contained in the 1990 vaccine) and A/Philippine/2/82 (not contained in the vaccine) were assayed for peak IL2 activity. We find that after influenza vaccination, there was an insignificant increase in IL2 activity when PBMC from the young control group were stimulated with A/Shanghai/16/89 (pre, 5.14 U/mL/10(6) PBMC; post, 6.64 U/mL/10(6) PBMC) but there was a significant increase in IL2 activity when stimulated with A/Phillippine/2/82 (pre, 1.5 U/mL/10(6) PBMC; post, 8.3 U/mL/10(6) PBMC). In similar cultures of PBMC from the elderly group, there was a significant increase in IL2 response to both A/Shanghai/16/89 (pre, 1.6 U/mL/10(6) PBMC; post, 3.5 U/mL/10(6) PBMC) and A/Philippine/2/82 (pre, 0.86 U/mL/10(6) PBMC; post, 8.3 U/mL/10(6) PBMC). Measurements of CD4+/CD8+ populations were not affected by vaccination and were not significantly different in the two groups. Subgroup analysis of the elderly group revealed that previous influenza vaccination in 1989 did not significantly affect IL2 levels measured in the present study. This study shows that in healthy elderly, influenza vaccination effectively restores IL2 activity to normal. There appears to be an age-related decrease in the duration of T-cell memory.(ABSTRACT TRUNCATED AT 250 WORDS)