Anaerobic degradation of papermill sludge in a two-phase digester containing rumen microorganisms and colonized polyurethane foam

Summary The use of reticulated polyurethane foam as a support material for the immobilization of methanogenic associations and its application to the anaerobic treatment of fine particulate solid wastes was investigated. The colonization of polyurethane support particles in a continuous upflow reactor fed on a mixture of acetate, propionate and butyrate, was both rapid and dense. The combination of rumen microorganisms and colonized support particles in a two-phase digester resulted in an efficient anaerobic decomposition of papermill sludge.     

Catalytic domains of the LAR and CD45 protein tyrosine phosphatases from Escherichia coli expression systems: purification and characterization for specificity and mechanism.

The cytoplasmic domains of two human transmembrane protein tyrosine phosphatases (PTPases), LAR and CD45, have been expressed in Escherichia coli, purified to near-homogeneity, and compared for catalytic efficiency toward several phosphotyrosine-containing peptide substrates. A 615-residue LAR fragment (LAR-D1D2) containing both tandemly repeated PTPase domains shows almost identical specific activity and high catalytic efficiency as the 40-kDa single-domain LAR-D1 fragment, consistent with a single functional active site in the 70-kDa LAR-D1D2 enzyme. A 90-kDa fragment of the human leukocyte CD45 PTPase, containing two similar tandemly repeated PTPase domains, shows parallel specificity to LAR-D1 and LAR-D1D2 with a high kcat/Km value for a phosphotyrosyl undecapeptide. Sufficient purified LAR-D1 and LAR-D1D2 PTPases were available to demonstrate enzymatic exchange of 18O from 18O4 inorganic phosphate into H2(16)O at rates of approximately 1 x 10(-2) s-1. The oxygen-18 exchange probably proceeds via a phosphoenzyme intermediate. Brief incubation of all three PTPase fragments with a [32P]phosphotyrosyl peptide substrate prior to quench with SDS sample buffer and gel electrophoresis led to autoradiographic detection of 32P-labeled enzymes. Pulse/chase studies on the LAR 32P-enzyme showed turnover of the labeled phosphoryl group.     

Classical cardiovascular disease risk factors associate with vascular function and morphology in rheumatoid arthritis: a six-year prospective study

Introduction

Patients with rheumatoid arthritis (RA) are at an increased risk for cardiovascular disease (CVD). An early manifestation of CVD is endothelial dysfunction which can lead to functional and morphological vascular abnormalities. Classical CVD risk factors and inflammation are both implicated in causing endothelial dysfunction in RA. The objective of the present study was to examine the effect of baseline inflammation, cumulative inflammation, and classical CVD risk factors on the vasculature following a six-year follow-up period.

Methods

A total of 201 RA patients (155 females, median age (25th to 75th percentile): 61 years (53 to 67)) were examined at baseline (2006) for presence of classical CVD risk factors and determination of inflammation using C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). At follow-up (2012) patients underwent assessments of microvascular and macrovascular endothelium-dependent and endothelium-independent function, along with assessment of carotid atherosclerosis. The CRP and ESR were recorded from the baseline study visit to the follow-up visit for each patient to calculate cumulative inflammatory burden.

Results

Classical CVD risk factors, but not RA disease-related inflammation, predicted microvascular endothelium-dependent and endothelium-independent function, macrovascular endothelium-independent function and carotid atherosclerosis. These findings were similar in a sub-group of patients free from CVD, and not receiving non-steroidal anti-inflammatory drugs, cyclooxygenase 2 inhibitors or biologics. Cumulative inflammation was not associated with microvascular and macrovascular endothelial function, but a weak association was apparent between area under the curve for CRP and carotid atherosclerosis.

Conclusions

Classical CVD risk factors may be better long-term predictors of vascular function and morphology than systemic disease-related inflammation in patients with RA. Further studies are needed to confirm if assessments of vascular function and morphology are predictive of long-term CV outcomes in RA.     

Airway cellularity, lipid laden macrophages and microbiology of gastric juice and airways in children with reflux oesophagitis

Background and methods

Human metapneumovirus (hMPV) is a recently discovered respiratory virus associated with bronchiolitis, pneumonia, croup and exacerbations of asthma. Since respiratory viruses are frequently detected in patients with acute exacerbations of COPD (AE-COPD) it was our aim to investigate the frequency of hMPV detection in a prospective cohort of hospitalized patients with AE-COPD compared to patients with stable COPD and to smokers without by means of quantitative real-time RT-PCR.

Results

We analysed nasal lavage and induced sputum of 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. HMPV was detected in 3/130 (2.3%) AE-COPD patients with a mean of 6.5 × 10⁵ viral copies/ml in nasal lavage and 1.88 × 10⁵ viral copies/ml in induced sputum. It was not found in patients with stable COPD or smokers without COPD.

Conclusion

HMPV is only found in a very small number of patients with AE-COPD. However it should be considered as a further possible viral trigger of AE-COPD because asymptomatic carriage is unlikely.     

Binding of oligoarginine to membrane lipids and heparan sulfate: structural and thermodynamic characterization of a cell-penetrating peptide

Cell-penetrating peptides (CPPs) comprise a group of arginine-rich oligopeptides that are able to deliver exogenous cargo into cells. A first step in the internalization of CPPs is their binding to the cell surface, a reaction likely to involve membrane phospholipids and/or heparan sulfate proteoglycans (HSPGs). The present work characterizes the interaction of R(9), one of the most efficient CPPs, with either heparan sulfate (HS) or lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG). Isothermal titration calorimetry shows that R(9) binds to HS with high affinity. Assuming that HS has n independent and equivalent binding sites for R(9), we find an association constant of 3.1 x 10(6) M(-1) at 28 degrees C. At this temperature, the reaction enthalpy is DeltaH(degrees)pep = - 5.5 kcal/mol and approximately 7 R(9) molecules bind per HS chain, which is equivalent to approximately 0.95 cationic/anionic charge ratio. Delta decreases in magnitude upon an increase in temperature, and the reaction becomes entropy-driven at higher temperatures (>or=37 degrees C). The positive heat-capacity change entailed by this reaction (DeltaC(degrees)P = +167 cal mol(-1) K(-1)) indicates the loss of polar residues on R(9)-HS binding, suggesting that hydrophobic forces play no major role on binding. Calorimetric analysis of the interaction of R(9) with POPC/POPG (75:25) vesicles reveals an association constant of 8.2 x 10(4) M(-1) at 28 degrees C. Using a surface partition equilibrium model to correct for electrostatic effects, we find an intrinsic partition constant of approximately 900 M(-1), a value that is also confirmed by electrophoretic mobility measurements. This corresponds to an electrostatic contribution of approximately 33% to the total free energy of binding. Deuterium nuclear magnetic resonance (NMR) shows no change in the headgroup conformation of POPC and POPG, suggesting that binding takes place at some distance from the plane of the polar groups. (31)P NMR indicates that the lipid bilayer remains intact upon R(9) binding. The fact that R(9) binds with greater affinity to HS than to anionic lipid vesicles makes the former molecule a more likely target in binding this CPP to the cell surface.     

Predictors of new atherosclerotic carotid plaque development in patients with rheumatoid arthritis: a longitudinal study

Introduction

Rheumatoid arthritis (RA) is associated with increased cardiovascular morbidity and mortality attributed to both classical risk factors and chronic inflammation. We assessed longitudinally the factors associated with new carotid plaques in nondiabetic RA patients and apparently healthy individuals.

Methods

In our present prospective observational study, carotid plaques were identified by ultrasonography at baseline and follow-up end, separated by an average of 3.6 ± 0.2 years, in 64 patients (mean age 59.2 ± 12.0 and disease duration at baseline 7.8 ± 6.2 years, 83% women, clinical and laboratory evaluation every 3 to 6 months). In a substudy, 35 of the patients were matched 1:1 for traditional cardiovascular risk factors with 'healthy' controls and were studied in parallel.

Results

New atherosclerotic plaques formed in 30% of patients (first plaque in 9%) who were significantly older than the remaining patients. Tobacco use, blood pressure, body mass index, average cumulative low-density lipoprotein, high-sensitivity C-reactive protein, erythrocyte sedimentation rate level, RA stage, functional class, disease duration and treatment modalities during follow-up did not differ significantly between subgroups after application of the Bonferroni correction. RA was in clinical remission, on average, for approximately 70% of the follow-up time and was not different between subgroups. Multivariate analysis including all the above parameters revealed that age (P = 0.006), smoking (P = 0.009) and duration of low-dose corticosteroid use (P = 0.016) associated independently with new plaque formation. RA patients displayed similar numbers of newly formed carotid plaques to the tightly matched for traditional cardiovascular risk factors 'healthy' controls, although more patients than controls had carotid plaques at baseline.

Conclusions

Formation of new atherosclerotic plaques in this small cohort of patients with well-controlled RA depended mainly on traditional cardiovascular risk factors and corticosteroid use, whereas an adverse effect of residual systemic inflammation was not readily detectable.     

The role of inflammation,the autonomic nervous system and classical cardiovascular disease risk factors on subendocardial viability ratio in patients with RA: a cross-sectional and longitudinal study

Introduction

Evidence indicates that rheumatoid arthritis (RA) patients have increased susceptibility to myocardial ischaemia that contributes to myocardial infarction. The subendocardial viability ratio (SEVR) can be measured using pulse wave analysis and reflects myocardial oxygen supply and demand. The objective of the present study was to examine specific predictors of SEVR in RA patients, with a specific focus on inflammation and classical cardiovascular disease (CVD) risk factors.

Methods

Two patient cohorts were included in the study; a primary cohort consisting of 220 RA patients and a validation cohort of 127 RA patients. All patients underwent assessment of SEVR using pulse wave analysis. Thirty-one patients from the primary cohort who were about to start anti-inflammatory treatment were prospectively examined for SEVR at pretreatment baseline and 2 weeks, 3 months and 1 year following treatment. Systemic markers of disease activity and classical CVD risk factors were assessed in all patients.

Results

The SEVR (mean ± standard deviation) for RA in the primary cohort was 148 ± 27 and in the validation cohort was 142 ± 25. Regression analyses revealed that all parameters of RA disease activity were associated with SEVR, along with gender, blood pressure and heart rate. These findings were the same in the validation cohort. Analysis of longitudinal data showed that C-reactive protein (P < 0.001), erythrocyte sedimentation rate (P < 0.005), Disease Activity Score in 28 joints (P < 0.001), mean blood pressure (P < 0.005) and augmentation index (P < 0.001) were significantly reduced after commencing anti-TNFα treatment. Increasing C-reactive protein was found to be associated with a reduction in SEVR (P = 0.02) and an increase in augmentation index (P = 0.001).

Conclusion

The present findings reveal that the SEVR is associated with markers of disease activity as well as highly prevalent classical CVD risk factors in RA, such as high blood pressure and diabetes. Further prospective studies are required to determine whether the SEVR predicts future cardiac events in RA.     

The role of inflammation and cardiovascular disease risk on microvascular and macrovascular endothelial function in patients with rheumatoid arthritis: a cross-sectional and longitudinal study

Introduction

Rheumatoid arthritis (RA) is associated with an increased risk for cardiovascular disease (CVD), and it has been postulated that RA disease-related inflammation contributes to endothelial dysfunction. The aim of the present work was to examine predictors (RA-related and CVD risk factors) and anti-tumor necrosis factor-alpha (anti-TNF-α) treatment effects on endothelial function in different vascular beds.

Methods

Microvascular endothelial function (laser Doppler imaging with iontophoresis of acetylcholine and sodium-nitroprusside), and macrovascular endothelial function (flow-mediated dilatation and glyceryl-trinitrate-mediated dilatation) were analyzed in parallel with disease activity. Individual CVD risk factors and global CVD risk were assessed cross-sectionally in 99 unselected RA patients and longitudinally (baseline, 2 weeks, and 3 months) in 23 RA patients commencing anti-TNF-α therapy.

Results

In this cross-sectional study, regression analyses revealed that markers of RA disease-related inflammation were not associated with microvascular or macrovascular endothelium-dependent function (P > 0.05); global CVD risk inversely correlated with microvascular endothelium-dependent function (P < 0.01) and with macrovascular endothelium-independent function (P < 0.01). In the longitudinal study, only microvascular endothelium-dependent function showed an improvement after 2 weeks of anti-TNF-α treatment when compared with baseline (437% ± 247% versus 319% ± 217%; P = 0.001), but no association was evident between change in endothelial function and change in inflammatory markers.

Conclusions

Classical CVD risk may influence endothelial function more than disease-related markers of inflammation in RA. Classical CVD risk factors and anti-TNF-α medication have different effects on microvascular and macrovascular endothelial function, suggesting that combined CVD-prevention approaches may be necessary. Prospective studies examining whether assessments of vascular function are predictive of long-term CV outcomes in RA are required.     

Structural and thermodynamic aspects of the interaction between heparan sulfate and analogues of melittin

Melittin is an amphipathic cationic peptide derived from honeybee venom with well-known cytolytic and antimicrobial properties. When coupled to cationic polymers or lipid molecules, it forms conjugates with high transfection efficiency and low toxicity with promising applications in gene therapy. A first step in the internalization of melittin and its conjugates is their binding to the cell surface, a reaction likely to involve heparan sulfate proteoglycans (HSPG). In the present work, we characterize the binding equilibrium of heparan sulfate (HS) with two melittin analogues, [Cys(1)]melittin (mel-SH) and retro-inverso [Cys(1)]melittin (ri-mel-SH). The terminal cysteine found in these peptides replaces the N-terminal glycine present in native melittin and allows covalent binding to other molecules. Isothermal titration calorimetry (ITC) reveals a high affinity of each melittin analogue to HS. Association constants of 4.7 x 10(4) and 3.5 x 10(5) M(-)(1) are found at physiological ionic strength and 15 degrees C for ri-mel-SH and mel-SH, respectively. The reaction enthalpy measured under these conditions is DeltaH(degrees)pep= 4.2 kcal/mol for ri-mel-SH and DeltaH(degrees)pep= 1.1 kcal/mol for mel-SH. The peptide-to-HS stoichiometry is approximately 20 for ri-mel-SH and approximately 14 for mel-SH under the same conditions. Temperature dependence studies using ri-mel-SH (mel-SH) show that DeltaH(degrees)pep decreases in magnitude upon increase in temperature, which results in a molar heat capacity of DeltaH(degrees)pep= -322 cal mol(-)(1) K(-)(1) (-45 cal mol(-)(1) K(-)(1)). Such a negative heat capacity change is not expected for a purely electrostatic interaction and indicates that hydrophobic and other interactions are also involved in the binding equilibrium. Salt dependence studies of the binding constants confirm that nonelectrostatic forces are an important component of the HS-melittin interaction. Binding to HS induces conformational changes in both peptides, with ri-mel-SH showing a 6-fold increase of the alpha-helix content when incubated with HS under saturation conditions.