Tumor necrosis factor-alpha inhibits myogenesis through redox-dependent and -independent pathways

Muscle wasting accompanies diseases that are associated with chronic elevated levels of circulating inflammatory cytokines and oxidative stress. We previously demonstrated that tumor necrosis factor-alpha (TNF-alpha) inhibits myogenic differentiation via the activation of nuclear factor-kappaB (NF-kappaB). The goal of the present study was to determine whether this process depends on the induction of oxidative stress. We demonstrate here that TNF-alpha causes a decrease in reduced glutathione (GSH) during myogenic differentiation of C(2)C(12) cells, which coincides with an elevated generation of reactive oxygen species. Supplementation of cellular GSH with N-acetyl-l-cysteine (NAC) did not reverse the inhibitory effects of TNF-alpha on troponin I promoter activation and only partially restored creatine kinase activity in TNF-alpha-treated cells. In contrast, the administration of NAC before treatment with TNF-alpha almost completely restored the formation of multinucleated myotubes. NAC decreased TNF-alpha-induced activation of NF-kappaB only marginally, indicating that the redox-sensitive component of the inhibition of myogenic differentiation by TNF-alpha occurred independently, or downstream of NF-kappaB. Our observations suggest that the inhibitory effects of TNF-alpha on myogenesis can be uncoupled in a redox-sensitive component affecting myotube formation and a redox independent component affecting myogenic protein expression.     

Risk factors of Coxiella burnetii (Q fever) seropositivity in veterinary medicine students

Background

Q fever is an occupational risk for veterinarians, however little is known about the risk for veterinary medicine students. This study aimed to assess the seroprevalence of Coxiella burnetii among veterinary medicine students and to identify associated risk factors.

Methods

A cross-sectional study with questionnaire and blood sample collection was performed among all veterinary medicine students studying in the Netherlands in 2006. Serum samples (n = 674), representative of all study years and study directions, were analyzed for C. burnetii IgG and IgM phase I and II antibodies with an immunofluorescence assay (IFA). Seropositivity was defined as IgG phase I and/or II titer of 1∶32 and above.

Results

Of the veterinary medicine students 126 (18.7%) had IgG antibodies against C. burnetii. Seropositivity associated risk factors identified were the study direction ‘farm animals’ (Odds Ratio (OR) 3.27 [95% CI 2.14–5.02]), advanced year of study (OR year 6: 2.31 [1.22–4.39] OR year 3–5 1.83 [1.07–3.10]) having had a zoonosis during the study (OR 1.74 [1.07–2.82]) and ever lived on a ruminant farm (OR 2.73 [1.59–4.67]). Stratified analysis revealed study direction ‘farm animals’ to be a study-related risk factor apart from ever living on a farm. In addition we identified a clear dose-response relation for the number of years lived on a farm with C. burnetii seropositivity.

Conclusions

C. burnetii seroprevalence is considerable among veterinary medicine students and study related risk factors were identified. This indicates Q fever as an occupational risk for veterinary medicine students.     

Efficient targeting of plant disease resistance loci using NBS profiling

The conserved sequences in the nucleotide-binding sites of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class of disease resistance (R) genes have been used for PCR-based R-gene isolation and subsequent development of molecular markers. Here we present a PCR-based approach (NBS profiling) that efficiently targets R genes and R-gene analogs (RGAs) and, at the same time, produces polymorphic markers in these genes. In NBS profiling, genomic DNA is digested with a restriction enzyme, and an NBS-specific (degenerate) primer is used in a PCR reaction towards an adapter linked to the resulting DNA fragments. The NBS profiling protocol generates a reproducible polymorphic multilocus marker profile on a sequencing gel that is highly enriched for R genes and RGAs. NBS profiling was successfully used in potato with several restriction enzymes, and several primers targeted to different conserved motifs in the NBS. Across primers and enzymes, the NBS profiles contained 50–90% fragments that were significantly similar to known R-gene and RGA sequences. The protocol was similarly successful in other crops (including tomato, barley, and lettuce) without modifications. NBS profiling can thus be used to produce markers tightly linked to R genes and R-gene clusters for genomic mapping and positional cloning and to mine for new alleles and new sources of disease resistance in available germplasm.Communicated by H.F. Linskens     

Structural role for Tyr-104 in Escherichia coli isopentenyl-diphosphate isomerase: site-directed mutagenesis, enzymology, and protein crystallography

Isopentenyl-diphosphate (IPP):dimethylallyl diphosphate isomerase is a key enzyme in the biosynthesis of isoprenoids. The mechanism of the isomerization reaction involves protonation of the unactivated carbon-carbon double bond in the substrate, but identity of the acidic moiety providing the proton is still not clear. Multiple sequence alignments and geometrical features observed in crystal structures of complexes with IPP isomerase suggest that Tyr-104 could play an important role during catalysis. A series of mutants was constructed by directed mutagenesis and characterized by enzymology. Crystallographic and thermal denaturation data for Y104A and Y104F mutants were obtained. Those data demonstrate the importance of residue Tyr-104 for proper folding of Escherichia coli type I IPP isomerase.     

Association of plasma sRAGE,but not esRAGE with lung function impairment in COPD

Rationale

Plasma soluble Receptor for Advanced Glycation End Product (sRAGE) is considered as a biomarker in COPD. The contribution of endogenous sRAGE (esRAGE) to the pool of plasma sRAGE and the implication of both markers in COPD pathogenesis is however not clear yet. The aim of the current study was therefore to measure plasma levels of esRAGE comparative to total sRAGE in patients with COPD and a control group. Further, we established the relations of esRAGE and total sRAGE with disease specific characteristics such as lung function and D_LCO, and with different circulating AGEs.

Methods

Plasma levels of esRAGE and sRAGE were measured in an 88 patients with COPD and in 55 healthy controls. FEV₁ (%predicted) and FEV₁/VC (%) were measured in both groups; D_LCO (%predicted) was measured in patients only. In this study population we previously reported that the AGE N^ϵ-(carboxymethyl) lysine (CML) was decreased, N^ϵ-(carboxyethyl) lysine (CEL) increased and pentosidine was not different in plasma of COPD patients compared to controls.

Results

Plasma esRAGE (COPD: 533.9 ± 412.4, Controls: 848.7 ± 690.3 pg/ml; p = 0.000) was decreased in COPD compared to controls. No significant correlations were observed between plasma esRAGE levels and lung function parameters or plasma AGEs. A positive correlation was present between esRAGE and total sRAGE levels in the circulation. Confirming previous findings, total sRAGE (COPD: 512.6 ± 403.8, Controls: 1834 ± 804.2 pg/ml; p < 0.001) was lower in patients compared to controls and was positively correlated FEV₁ (r = 0.235, p = 0.032), FEV₁/VC (r = 0.218, p = 0.047), and D_LCO (r = 0.308, p = 0.006). sRAGE furthermore did show a significant positive association with CML (r = 0.321, p = 0.003).

Conclusion

Although plasma esRAGE is decreased in COPD patients compared to controls, only total sRAGE showed a significant and independent association with FEV₁, FEV₁/VC and D_LCO, indicating that total sRAGE but not esRAGE may serve as marker of COPD disease state and severity.     

Distribution of the human intracellular serpin protease inhibitor 8 in human tissues.

Ovalbumin-like serine protease inhibitors are mainly localized intracellularly and their in vivo functions are largely unknown. To elucidate their physiological role(s), we studied the expression of one of these inhibitors, protease inhibitor 8 (PI-8), in normal human tissues by immunohistochemistry using a PI-8-specific monoclonal antibody. PI-8 was strongly expressed in the nuclei of squamous epithelium of mouth, pharynx, esophagus, and epidermis, and by the epithelial layer of skin appendages, particularly by more differentiated epithelial cells. PI-8 was also expressed by monocytes and by neuroendocrine cells in the pituitary gland, pancreas, and digestive tract. Monocytes showed nuclear and cytoplasmic localization of PI-8, whereas neuroendocrine cells showed only cytoplasmic staining. In vitro nuclear localization of PI-8 was confirmed by confocal analysis using serpin-transfected HeLa cells. Furthermore, mutation of the P(1) residue did not affect the subcellular distribution pattern of PI-8, indicating that its nuclear localization is independent of the interaction with its target protease. We conclude that PI-8 has a unique distribution pattern in human tissues compared to the distribution patterns of other intracellular serpins. Additional studies must be performed to elucidate its physiological role.