Gelatin degradation assay reveals MMP-9 inhibitors and function of O-glycosylated domain

AIM: To establish a novel, sensitive and high-throughput gelatinolytic assay to define new inhibitors and compare domain deletion mutants of gelatinase B/matrix metalloproteinase (MMP)-9. METHODS: Fluorogenic Dye-quenched (DQ)TM-gelatin was used as a substrate and biochemical parameters (substrate and enzyme concentrations, DMSO solvent concentrations) were optimized to establish a highthroughput assay system. Various small-sized libraries (ChemDiv, InterBioScreen and ChemBridge) of hetero-cyclic, drug-like substances were tested and compared with prototypic inhibitors. RESULTS: First, we designed a test system with gelatin as a natural substrate. Second, the assay was validated by selecting a novel pyrimidine-2,4,6-trione (barbitu- rate) inhibitor. Third, and in line with present structural data on collagenolysis, it was found that deletion of the O-glycosylated region significantly decreased gelatinolytic activity (kcat/kM ± 40% less than full-length MMP-9). CONCLUSION: The DQTM-gelatin assay is useful in high-throughput drug screening and exosite targeting. We demonstrate that flexibility between the catalytic and hemopexin domain is functionally critical for gelatinolysis.     

Psychophysiological responses to stress after stress management training in patients with rheumatoid arthritis

Background

Stress management interventions may prove useful in preventing the detrimental effects of stress on health. This study assessed the effects of a stress management intervention on the psychophysiological response to stress in patients with rheumatoid arthritis (RA).

Methods

Seventy-four patients with RA, who were randomly assigned to either a control group or a group that received short-term stress management training, performed a standardized psychosocial stress task (Trier Social Stress Test; TSST) 1 week after the stress management training and at a 9-week follow-up. Psychological and physical functioning, and the acute psychophysiological response to the stress test were assessed.

Results

Patients in the intervention group showed significantly lower psychological distress levels of anxiety after the training than did the controls. While there were no between-group differences in stress-induced tension levels, and autonomic (α-amylase) or endocrine (cortisol) responses to the stress test 1 week after the intervention, levels of stress-induced tension and cortisol were significantly lower in the intervention group at the 9-week follow-up. Overall, the response to the intervention was particularly evident in a subgroup of patients with a psychological risk profile.

Conclusion

A relatively short stress management intervention can improve psychological functioning and influences the psychophysiological response to stress in patients with RA, particularly those psychologically at risk. These findings might help understand how stress can affect health and the role of individual differences in stress responsiveness.

Trial Registration

TrialRegister.nl NTR1193     

Cultivation of high-rate sulfate reducing sludge by pH-based electron donor dosage

A novel self-regulating bioreactor concept for sulfate reduction is proposed aiming for high biomass concentrations and treatment capacities. The system consists of a cell suspension of sulfate reducing bacteria in a continuous stirred tank reactor (30 degrees C) fed with a mixture of both electron donor and electron acceptor (formic acid and sulfuric acid, respectively), nutrients and phosphate buffer via a pH controller. The pH rise due to sulfate reduction is balanced with dosage of the sulfate reducing substrates as acids. The reactor concept was shown to be capable of full sulfate reduction without competition for the electron donor by methanogens and acetogens. Activity assays revealed that hardly any methanogenic activity on formate was left in the suspension by the end of the continuous run (130 days). In addition, the sulfidogenic activity with formate and H2/CO2 had increased, respectively, 3.9 and 11.6 times at the end of the experimental run. The evolution of the particle size distribution of the cell suspension over time indicated that newly grown cells have the tendency to attach together in flocs or to the existing agglomerates.     

Genetic and physiological architecture of early vigor in Aegilops tauschii, the D-genome donor of hexaploid wheat. A quantitative trait loci analysis

Plant growth can be studied at different organizational levels, varying from cell, leaf, and shoot to the whole plant. The early growth of seedlings is important for the plant's establishment and its eventual success. Wheat (Triticum aestivum, genome AABBDD) seedlings exhibit a low early growth rate or early vigor. The germplasm of wheat is limited. Wild relatives constitute a source of genetic variation. We explored the physiological and genetic relationships among a range of early vigor traits in Aegilops tauschii, the D-genome donor. A genetic map was constructed with amplified fragment-length polymorphism and simple sequence repeat markers, and quantitative trait loci (QTL) analysis was performed on the F(4) population of recombinant inbred lines derived from a cross between contrasting accessions. The genetic map consisted of 10 linkage groups, which were assigned to the seven chromosomes and covered 68% of the D genome. QTL analysis revealed 87 mapped QTLs (log of the odds >2.65) in clusters, 3.1 QTLs per trait, explaining 32% of the phenotypic variance. Chromosomes 1D, 4D, and 7D harbored QTLs for relative growth rate, biomass allocation, specific leaf area, leaf area ratio, and unit leaf rate. Chromosome 2D covered QTLs for rate and duration of leaf elongation, cell production rate, and cell length. Chromosome 5D harbored QTLs for the total leaf mass and area and growth rate of the number of leaves and tillers. The results show that several physiological correlations between growth traits have a genetic basis. Genetic links between traits are not absolute, opening perspectives for identification of favorable alleles in A. tauschii to improve early vigor in wheat.     

Dose-dependent effects of UVB-induced skin carcinogenesis in hairless p53 knockout mice

Exposure to (solar) UVB radiation gives rise to mutations in the p53 tumor suppressor gene that appear to contribute to the earliest steps in the molecular cascade towards human and murine skin cancer. To examine in more detail the role of p53, we studied UVB-induced carcinogenesis in hairless p53 knock-out mice. The early onset of lymphomas as well as early wasting of mice interfered with the development of skin tumors in p53 null-mice. The induction of skin tumors in the hairless p53+/- mice was accomplished by daily exposure to two different UV-doses of approximately 450 J/m2 and 900 J/m2 from F40 lamps corresponding to a fraction of about 0.4 and 0.8 of the minimal edemal dose. Marked differences in skin carcinogenesis were observed between the p53+/- mice and their wild type littermates. Firstly, at 900 J/m2, tumors developed significantly faster in the heterozygotes than in wild types, whereas at 450 J/m2 there was hardly any difference, suggesting that only at higher damage levels loss of one functional p53 allele is important. Secondly, a large portion (25%) of skin tumors in the heterozygotes were of a more malignant, poorly differentiated variety of squamous cell carcinomas, i.e. spindle cell carcinomas, a tumor type that was rarely observed in daily UV exposed wild type hairless mice. Thirdly, the p53 mutation spectrum in skin tumors in heterozygotes is quite different from that in wild types. Together these results support the notion that a point mutation in the p53 gene impacts skin carcinogenesis quite differently than allelic loss: the former is generally selected for in early stages of skin tumors in wild type mice, whereas the latter enhances tumor development only at high exposure levels (where apoptosis becomes more prevalent) and appears to increase progression (to a higher grade of malignancy) of skin tumors.     

Cytokine production of stimulated whole blood cultures in rheumatoid arthritis patients receiving short-term infliximab therapy

Patients with rheumatoid arthritis (RA) treated with anti-tumor necrosis factor (TNF) strategies have an increased susceptibility to infections, especially those caused by intracellular pathogens. In this study we assessed the cytokine production capacity in patients with RA and we further investigated whether anti-TNF therapy modulates the production of pro-inflammatory cytokines involved in the resistance against infections. Whole blood cultures from 10 RA patients and 10 healthy controls were stimulated with heat-killed Candida albicans, Salmonella typhimurium, Staphyloccocus aureus, Aspergillus fumigatus or Mycobacterium tuberculosis and production of interleukin (IL)-1beta, IL-6, IL-10, interferon (IFN)-gamma and TNF-alpha was measured. Before anti-TNF therapy, whole blood cultures from RA patients released significantly less IFN-gamma than healthy controls after stimulation with all tested microorganisms. Short-term anti-TNF therapy did not have an inhibitory effect on the release of the cytokines tested. We conclude that cells of patients with RA have a strongly reduced production capacity of IFN-gamma after bacterial challenge. Although short-term therapy with anti-TNF agents did not further decrease the release of other proinflammatory cytokines, the combination of defective IFN-gamma production in basal conditions and TNF neutralization during anti-TNF therapy is likely to be responsible for the higher susceptibility to infections in patients with RA.     

DAS28: a useful instrument to monitor infliximab treatment in patients with rheumatoid arthritis

The Disease Activity Score using 28 joint counts (DAS28) has been developed in a cohort of patients with rheumatoid arthritis in which only conventional anti-rheumatic treatments were used. It has extensively been validated to monitor disease activity in daily clinical practice as well as in clinical trials. The study of Vander Cruyssen and colleagues showed that the DAS28 correlated best with the decisions of rheumatologists to increase the infliximab dose because of insufficient response. This result once more confirms the validity of the DAS28 to monitor disease activity in patients with rheumatoid arthritis and to titrate treatment with biologicals.     

QTL analysis and QTL-based prediction of flowering phenology in recombinant inbred lines of barley

Combining ecophysiological modelling and genetic mapping has increasingly received attention from researchers who wish to predict complex plant or crop traits under diverse environmental conditions. The potential for using this combined approach to predict flowering time of individual genotypes in a recombinant inbred line (RIL) population of spring barley (Hordeum vulgare L.) was examined. An ecophysiological phenology model predicts preflowering duration as affected by temperature and photoperiod, based on the following four input traits: f(o) (the minimum number of days to flowering at the optimum temperature and photoperiod), theta1 and theta2 (the development stages for the start and the end of the photoperiod-sensitive phase, respectively), and delta (the photoperiod sensitivity). The model-input trait values were obtained from a photoperiod-controlled greenhouse experiment. Assuming additivity of QTL effects, a multiple QTL model was fitted for the model-input traits using composite interval mapping. Four to seven QTL were identified for each trait. Each trait had at least one QTL specific to that trait alone. Other QTL were shared by two or all traits. Values of the model-input traits predicted for the RILs from the QTL model were fed back into the ecophysiological model. This QTL-based ecophysiological model was subsequently used to predict preflowering duration (d) for eight field trial environments. The model accounted for 72% of the observed variation among 94 RILs and 94% of the variation among the two parents across the eight environments, when observations in different environments were pooled. However, due to the low percentage (34-41%) of phenotypic variation accounted for by the identified QTL for three model-input traits (theta1, theta2 and delta), the QTL-based model accounted for somewhat less variation among the RILs than the model using original phenotypic input trait values. Nevertheless, days to flowering as predicted from the QTL-based ecophysiological model were highly correlated with days to flowering as predicted from QTL-models per environment for days to flowering per se. The ecophysiological phenology model was thus capable of extrapolating (QTL) information from one environment to another.     

In vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine

We report on chloroquine, a 4-amino-quinoline, as an effective inhibitor of the replication of the severe acute respiratory syndrome coronavirus (SARS-CoV) in vitro. Chloroquine is a clinically approved drug effective against malaria. We tested chloroquine phosphate for its antiviral potential against SARS-CoV-induced cytopathicity in Vero E6 cell culture. Results indicate that the IC50 of chloroquine for antiviral activity (8.8 +/- 1.2 microM) was significantly lower than its cytostatic activity; CC50 (261.3 +/- 14.5 microM), yielding a selectivity index of 30. The IC50 of chloroquine for inhibition of SARS-CoV in vitro approximates the plasma concentrations of chloroquine reached during treatment of acute malaria. Addition of chloroquine to infected cultures could be delayed for up to 5h postinfection, without an important drop in antiviral activity. Chloroquine, an old antimalarial drug, may be considered for immediate use in the prevention and treatment of SARS-CoV infections.     

Structural and thermodynamic basis for the binding of TMC114, a next-generation human immunodeficiency virus type 1 protease inhibitor

TMC114, a newly designed human immunodeficiency virus type 1 (HIV-1) protease inhibitor, is extremely potent against both wild-type (wt) and multidrug-resistant (MDR) viruses in vitro as well as in vivo. Although chemically similar to amprenavir (APV), the potency of TMC114 is substantially greater. To examine the basis for this potency, we solved crystal structures of TMC114 complexed with wt HIV-1 protease and TMC114 and APV complexed with an MDR (L63P, V82T, and I84V) protease variant. In addition, we determined the corresponding binding thermodynamics by isothermal titration calorimetry. TMC114 binds approximately 2 orders of magnitude more tightly to the wt enzyme (K(d) = 4.5 x 10(-12) M) than APV (K(d) = 3.9 x 10(-10) M). Our X-ray data (resolution ranging from 2.2 to 1.2 A) reveal strong interactions between the bis-tetrahydrofuranyl urethane moiety of TMC114 and main-chain atoms of D29 and D30. These interactions appear largely responsible for TMC114's very favorable binding enthalpy to the wt protease (-12.1 kcal/mol). However, TMC114 binding to the MDR HIV-1 protease is reduced by a factor of 13.3, whereas the APV binding constant is reduced only by a factor of 5.1. However, even with the reduction in binding affinity to the MDR HIV protease, TMC114 still binds with an affinity that is more than 1.5 orders of magnitude tighter than the first-generation inhibitors. Both APV and TMC114 fit predominantly within the substrate envelope, a property that may be associated with decreased susceptibility to drug-resistant mutations relative to that of first-generation inhibitors. Overall, TMC114's potency against MDR viruses is likely a combination of its extremely high affinity and close fit within the substrate envelope.