Potentials and pitfalls of DNA array analysis of the endothelial stress response

Endothelial cells respond to inflammatory stimuli with complex genetic alterations that determine the immune response and the outcome of the inflammatory process. An additional layer of complexity is added by the different phenotypes and functional heterogeneity of endothelial cells in the various tissues. To understand these complex gene response patterns and the regulatory pathways involved, many investigators increasingly use DNA microarray analysis. There are, however, many potential pitfalls in the use of microarrays that can result in false data and erroneous conclusions. This review surveys the principles of DNA microarray technology and its applications in endothelial cell research. We also attempt to outline some of the caveats and standard criteria that have to be considered in order to realize the full potential of microarrays in inflammation research.     

Kinetic control of thiamin diphosphate activation in enzymes studied by proton-nitrogen correlated NMR spectroscopy

Proton-nitrogen correlated NMR studies were performed on thiamin diphosphate, which has been specifically labeled with (15)N at the 4'-amino group. After reconstitution of the labeled coenzyme with the apoenzymes of both wild-type pyruvate decarboxylase from Zymomonas mobilis and the E50Q variant, a high-field shift of the (15)N signal of approximately 4 ppm is observed at pH 5.9 when compared to that of the free coenzyme, indicating a higher electron density at the 4'-amino nitrogen in the enzyme-bound state. The pH dependence of the chemical shift of the (15)N signals in the (1)H-(15)N heteronuclear single-quantum coherence NMR spectra reveals typical titration curves for the free as well as the reconstituted coenzyme with nearly identical chemical shift end points. The midpoints of the transitions are at pH 5.3 and 5.0 for the free and enzyme-bound coenzyme, respectively. We conclude that the tremendous rate acceleration of C2-H deprotonation in ThDP enzymes is mainly the result of the enforced V conformation of the cofactor in the active site being perfectly suited to allowing intramolecular acid-base catalysis.     

Catalysis and binding of cyclophilin A with different HIV-1 capsid constructs

The prolyl isomerase cyclophilin A (CypA) is required for efficient HIV-1 replication and is incorporated into virions through a binding interaction at the Gly-Pro(222) bond located within the capsid domain of the HIV-1 Gag precursor polyprotein (Pr(gag)). It has recently been shown that CypA efficiently catalyzes the cis/trans isomerization of Gly-Pro(222) within the isolated N-terminal domain of capsid (CA(N)). To address the proposal that CypA interacts with Gly-Pro sequences in the C-terminal domain of a mature capsid, the interaction between CypA and the natively folded, full-length capsid protein (CA(FL)) has been investigated here using nuclear magnetic resonance spectroscopy. In addition, a fragment of the Pr(gag) protein encoding the full-matrix protein and the N-terminal domain of capsid (MA-CA(N)) has been used to probe the catalytic interaction between CypA and an immature form of the capsid. The results discussed herein strongly suggest that Gly-Pro(222) located within the N-terminal domain of the capsid is the preferential site for CypA binding and catalysis and that catalysis of Gly-Pro(222) is unaffected by maturational processing at the N-terminus of the capsid.     

Immune responses of breast cancer patients to mutated epidermal growth factor receptor (EGF-RvIII, Delta EGF-R, and de2-7 EGF-R)

Mutated epidermal growth factor receptor (EGF-RvIII, DeltaEGF-R, and de2-7 EGF-R) is the result of an 801-bp deletion within the extracellular domain of wild-type EGF-R and is expressed by breast carcinomas, but not by normal breast tissues. EGF-RvIII is expressed both on the surface and in the cytoplasm of tumor cells. Thus, EGF-RvIII is a potential tumor-specific target for both Abs and T cells. However, it is not known whether breast cancer patients can raise immune responses to EGF-RvIII expressed by their tumors. The demonstration of EGF-RvIII-specific immune responses in patients would suggest that immunization of patients with EGF-RvIII vaccines is feasible, because these vaccines may boost a pre-existing immune response. We have evaluated humoral and cellular immune responses to EGF-RvIII in 16 breast cancer patients and three healthy donors. Seven of 16 patients developed EGF-RvIII-specific Abs that bound to isolated EGF-RvIII protein or the protein expressed by EGF-RvIII-transfected mouse fibroblasts. The Abs that bound to EGF-RvIII did not bind to wild-type EGF-R, and anti-EGF-RvIII Abs were not found in the sera of healthy donors. Three patients had EGF-RvIII peptide-specific lymphoproliferative responses, and two of these patients also had humoral immune responses. Humoral and cellular immune responses correlated with EGF-RvIII expression by patients' tumors in most cases. These studies demonstrate that breast cancer patients specifically recognize EGF-RvIII with an overall immune response rate of 50%, suggesting that patients may benefit from vaccination against EGF-RvIII, boosting pre-existing immune responses.     

Relationship between exercise intervention and NO pathway in patients with heart failure with preserved ejection fraction

Objective: Elevated levels of arginine derivatives in the NO pathway, such as asymmetric dimethylarginine (ADMA), are related to disease severity and reduced exercise capacity in heart failure (HF). We investigated the influence of exercise intervention on these parameters and on L-arginine (L-Arg) and L-homoarginine (L-hArg) in HF with preserved ejection fraction (HFpEF) patients.

Material and methods: Sixty-two patients (65?±?6 years) were included in this analysis and randomized to supervised endurance/resistance training (ET) or to usual care (UC). EDTA-plasma was analysed for NO metabolites.

Results: There were baseline associations for adjusted values of maximum workload with ADMA (r=??0.322, p?=?0.028) and L-Arg/ADMA ratio (r?=?0.331, p?=?0.015), and for the 6-min walk test (6MWT) with ADMA (r=??0.314, p?=?0.024) and L-Arg/ADMA ratio (r?=?0.346, p?=?0.015). No significant differences between UC and ET changes of NO parameters were observed at 3-month follow-up. Higher L-hArg levels were associated with a greater improvement in peak oxygen uptake (peak O₂) at follow-up: 3.4?±?2.8 vs. 1.1?±?2.9?mL/min/kg (p?=?0.005).

Conclusions: Exercise intervention did not influence NO parameters in HFpEF patients, but L-hArg was related to change in peak O₂.     

High-sensitivity cardiac troponin T for diagnosis of NSTEMI in the elderly emergency department patient: a clinical cohort study

Purpose: The aim of this study is to evaluate the impact of age on the diagnostic performance of high-sensitivity troponin T (hsTnT) under routine conditions.

Materials and methods: Data of 4118 consecutive emergency department (ED) patients who underwent a routine TnT measurement between 11 October 2012 and 30 November 2013 were analysed. Diagnostic accuracy of hsTnT was compared in four age categories (<50, 50–64, 65–74, ≥75 years of age) for different cut-off values. Primary endpoint was a main hospital diagnosis of NSTEMI.

Results: The median age of the study population (n?=?4118) was 61 years (IQR: 45–75 years). NSTEMI was diagnosed in 3.3% (n?=?136) of all patients. There were significant differences in hsTnT concentrations between age-groups (p?p?=?0.297). 72.2% of all patients ≥75 years of age (583/808) without NSTEMI had hsTnT concentrations above the 99th percentile of a healthy reference population. Specificity at 14?ng/L was 93.6% (95% CI: 92.12–94.87) in patients below 50 years of age and 27.9% (95% CI: 24.78–31.08) in patients 75 years of age and older.

Conclusions: Patients’ age needs to be considered at least one influencing factor on hsTnT concentrations at admission and should be included in the clinical interpretation of hsTnT concentrations for further clinical workup beneath other influencing factors like comorbidities and symptom onset time. The implementation of age-specific cut-off values could be considered for single troponin testing at admission but is associated with an increased risk of underdiagnosis of NSTEMI.     

Comparative morphology and evolution of the cnidosac in Cladobranchia (Gastropoda: Heterobranchia: Nudibranchia)

Background

A number of shelled and shell-less gastropods are known to use multiple defensive mechanisms, including internally generated or externally obtained biochemically active compounds and structures. Within Nudipleura, nudibranchs within Cladobranchia possess such a special defense: the ability to sequester cnidarian nematocysts – small capsules that can inject venom into the tissues of other organisms. This ability is distributed across roughly 600 species within Cladobranchia, and many questions still remain in regard to the comparative morphology and evolution of the cnidosac – the structure that houses sequestered nematocysts (called kleptocnides). In this paper, we describe cnidosac morphology across the main groups of Cladobranchia in which it occurs, and place variation in its structure in a phylogenetic context to better understand the evolution of nematocyst sequestration.

Results

Overall, we find that the length, size and structure of the entrance to the cnidosac varies more than expected based on previous work, as does the structure of the exit, the musculature surrounding the cnidosac, and the position and orientation of the kleptocnides. The sequestration of nematocysts has originated at least twice within Cladobranchia based on the phylogeny presented here using 94 taxa and 409 genes.

Conclusions

The cnidosac is not homologous to cnidosac-like structures found in Hancockiidae. Additionally, the presence of a sac at the distal end of the digestive gland may have originated prior to the sequestration of nematocysts. This study provides a more complete picture of variation in, and evolution of, morphological characters associated with nematocyst sequestration in Cladobranchia.