Inflammation-induced reactive nitrogen species cause proteasomal degradation of dimeric peroxiredoxin-1 in a mouse macrophage cell line

Peroxiredoxin 1 (PRDX1) is an antioxidant enzyme that, when secreted, can act as a proinflammatory signal. Here we studied the regulation of intracellular PRDX1 by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) in the RAW 264.7 mouse macrophage cell line. While LPS or IFN-γ alone did not affect PRDX1 protein levels, their combination led to an almost complete loss of the PRDX1 dimer. This was likely mediated by the increased production of nitric oxide (NO) as it was reversed by the NO synthase inhibitor L-N-methylarginine (L-NMMA), while a NO-releasing agent decreased PRDX1 levels. Inhibition of the proteasome with MG132 also prevented the loss of the PRDX1 dimer, suggesting that the decrease is due to a NO-activated proteasomal degradation pathway. By contrast with the decrease in protein levels, LPS increased PRDX1 mRNA and this effect was amplified by IFN-γ. Two other Nrf2 target genes, thioredoxin reductase (TXNRD1) and haem oxygenase (HMOX1), were also induced by LPS but IFN-γ did not increase their expression further. This study shows that inflammation differentially regulates PRDX1 at the levels of protein stability and gene expression, and that NO plays a key role in this mechanism.     

Calibration of the comet assay for the measurement of DNA damage in mammalian cells

We used X-rays from a linear accelerator and from a low energy therapeutic source to calibrate the single cell gel electrophoresis (comet assay), a widely used method to measure DNA damage. γ-Rays from ⁶⁰Co, with known efficiency in inducing DNA breakage, were used as reference. Human lymphocytes and one murine tumour cell line, F10-M3 cells, were irradiated under different experimental conditions. A similar relationship between radiation dose and induced DNA damage was obtained with γ- and X-rays. A calibration curve was constructed to convert the comet assay raw data into break frequency. The median levels of DNA breaks and oxidative damage in circulating lymphocytes from healthy volunteers were calculated to be 0.76 and 0.80 breaks/10⁹ Da, respectively, (0.50 and 0.52 breaks/10⁶ bp). The values of oxidative DNA damage were in the same order of magnitude as those found by others with HPLC methods.     

2- and 3-[(Aryl)(azolyl)methyl]indoles as Potential Non-steroidal Aromatase Inhibitors

The present study was designed to follow our pharmacomodulation work in the field of non-steroidal aromatase inhibitors. All target compounds 12a–h and 28a–h were tested in vitro for human placental aromatase inhibition, using testosterone or androstenedione as the substrate for the aromatase enzyme and the IC₅₀ and relative potency to aminoglutethimide data are included. A SAR study indicated that 3-[(4-fluorophenyl)(1H-imidazol-1-yl)methyl]-1-ethyl-2-methyl-1H-indole (28?g) was a highly potent and selective aromatase inhibitor with IC₅₀ value of 0.025?μM. 28?g was also a weak inhibitor of androstenedione synthesis.     

Subcellular localization of full-length human myeloid leukemia factor 1 (MLF1) is independent of 14-3-3 proteins

Myeloid leukemia factor 1 (MLF1) is associated with the development of leukemic diseases such as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, information on the physiological function of MLF1 is limited and mostly derived from studies identifying MLF1 interaction partners like CSN3, MLF1IP, MADM, Manp and the 14-3-3 proteins. The 14-3-3-binding site surrounding S34 is one of the only known functional features of the MLF1 sequence, along with one nuclear export sequence (NES) and two nuclear localization sequences (NLS). It was recently shown that the subcellular localization of mouse MLF1 is dependent on 14-3-3 proteins. Based on these findings, we investigated whether the subcellular localization of human MLF1 was also directly 14-3-3-dependent. Live cell imaging with GFP-fused human MLF1 was used to study the effects of mutations and deletions on its subcellular localization. Surprisingly, we found that the subcellular localization of full-length human MLF1 is 14-3-3-independent, and is probably regulated by other as-yet-unknown proteins.     

Contrasting Patterns of Genetic Differentiation among Blackcaps (Sylvia atricapilla) with Divergent Migratory Orientations in Europe

Migratory divides are thought to facilitate behavioral, ecological, and genetic divergence among populations with different migratory routes. However, it is currently contentious how much genetic divergence is needed to maintain distinct migratory behavior across migratory divides. Here we investigate patterns of neutral genetic differentiation among Blackcap (Sylvia atricapilla) populations with different migratory strategies across Europe. We compare the level of genetic divergence of populations migrating to southwestern (SW) or southeastern (SE) wintering areas with birds wintering in the British Isles following a recently established northwesterly (NW) migration route. The migratory divide between SW and SE wintering areas can be interpreted as a result of a re-colonization process after the last glaciation. Thus we predicted greater levels of genetic differentiation among the SW/SE populations. However, a lack of genetic differentiation was found between SW and SE populations, suggesting that interbreeding likely occurs among Blackcaps with different migratory orientations across a large area; therefore the SW/SE migratory divide can be seen as diffuse, broad band and is, at best, a weak isolating barrier. Conversely, weak, albeit significant genetic differentiation was evident between NW and SW migrants breeding sympatrically in southern Germany, suggesting a stronger isolating mechanism may be acting in this population. Populations located within/near the SW/SE contact zone were the least genetically divergent from NW migrants, confirming NW migrants likely originated from within the contact zone. Significant isolation-by-distance was found among eastern Blackcap populations (i.e. SE migrants), but not among western populations (i.e. NW and SW migrants), revealing different patterns of genetic divergence among Blackcap populations in Europe. We discuss possible explanations for the genetic structure of European Blackcaps and how gene flow influences the persistence of divergent migratory behaviors.     

207-nm UV Light - A Promising Tool for Safe Low-Cost Reduction of Surgical Site Infections. I: In Vitro Studies

Background

0.5% to 10% of clean surgeries result in surgical-site infections, and attempts to reduce this rate have had limited success. Germicidal UV lamps, with a broad wavelength spectrum from 200 to 400 nm are an effective bactericidal option against drug-resistant and drug-sensitive bacteria, but represent a health hazard to patient and staff. By contrast, because of its limited penetration, ∼200 nm far-UVC light is predicted to be effective in killing bacteria, but without the human health hazards to skin and eyes associated with conventional germicidal UV exposure.

Aims

The aim of this work was to test the biophysically-based hypothesis that ∼200 nm UV light is significantly cytotoxic to bacteria, but minimally cytotoxic or mutagenic to human cells either isolated or within tissues.

Methods

A Kr-Br excimer lamp was used, which produces 207-nm UV light, with a filter to remove higher-wavelength components. Comparisons were made with results from a conventional broad spectrum 254-nm UV germicidal lamp. First, cell inactivation vs. UV fluence data were generated for methicillin-resistant S. aureus (MRSA) bacteria and also for normal human fibroblasts. Second, yields of the main UV-associated pre-mutagenic DNA lesions (cyclobutane pyrimidine dimers and 6-4 photoproducts) were measured, for both UV radiations incident on 3-D human skin tissue.

Results

We found that 207-nm UV light kills MRSA efficiently but, unlike conventional germicidal UV lamps, produces little cell killing in human cells. In a 3-D human skin model, 207-nm UV light produced almost no pre-mutagenic UV-associated DNA lesions, in contrast to significant yields induced by a conventional germicidal UV lamp.

Conclusions

As predicted based on biophysical considerations, 207-nm light kills bacteria efficiently but does not appear to be significantly cytotoxic or mutagenic to human cells. Used appropriately, 207-nm light may have the potential for safely and inexpensively reducing surgical-site infection rates, including those of drug-resistant origin.     

Microarray Analysis of Microbiota of Gingival Lesions in Noma Patients

Noma (cancrum oris) is a gangrenous disease of unknown etiology affecting the maxillo-facial region of young children in extremely limited resource countries. In an attempt to better understand the microbiological events occurring during this disease, we used phylogenetic and low-density microarrays targeting the 16S rRNA gene to characterize the gingival flora of acute noma and acute necrotizing gingivitis (ANG) lesions, and compared them to healthy control subjects of the same geographical and social background. Our observations raise doubts about Fusobacterium necrophorum, a previously suspected causative agent of noma, as this species was not associated with noma lesions. Various oral pathogens were more abundant in noma lesions, notably Atopobium spp., Prevotella intermedia, Peptostreptococcus spp., Streptococcus pyogenes and Streptococcus anginosus. On the other hand, pathogens associated with periodontal diseases such as Aggregatibacter actinomycetemcomitans, Capnocytophaga spp., Porphyromonas spp. and Fusobacteriales were more abundant in healthy controls. Importantly, the overall loss of bacterial diversity observed in noma samples as well as its homology to that of ANG microbiota supports the hypothesis that ANG might be the immediate step preceding noma.