Use of the frc gene as a molecular marker to characterize oxalate-oxidizing bacterial abundance and diversity structure in soil.

Oxalate catabolism, which can have both medical and environmental implications, is performed by phylogenetically diverse bacteria. The formyl-CoA-transferase gene was chosen as a molecular marker of the oxalotrophic function. Degenerated primers were deduced from an alignment of frc gene sequences available in databases. The specificity of primers was tested on a variety of frc-containing and frc-lacking bacteria. The frc-primers were then used to develop PCR-DGGE and real-time SybrGreen PCR assays in soils containing various amounts of oxalate. Some PCR products from pure cultures and from soil samples were cloned and sequenced. Data were used to generate a phylogenetic tree showing that environmental PCR products belonged to the target physiological group. The extent of diversity visualised on DGGE pattern was higher for soil samples containing carbonate resulting from oxalate catabolism. Moreover, the amount of frc gene copies in the investigated soils was detected in the range of 1.64x10(7) to 1.75x10(8)/g of dry soil under oxalogenic tree (representing 0.5 to 1.2% of total 16S rRNA gene copies), whereas the number of frc gene copies in the reference soil was 6.4x10(6) (or 0.2% of 16S rRNA gene copies). This indicates that oxalotrophic bacteria are numerous and widespread in soils and that a relationship exists between the presence of the oxalogenic trees Milicia excelsa and Afzelia africana and the relative abundance of oxalotrophic guilds in the total bacterial communities. This is obviously related to the accomplishment of the oxalate-carbonate pathway, which explains the alkalinization and calcium carbonate accumulation occurring below these trees in an otherwise acidic soil. The molecular tools developed in this study will allow in-depth understanding of the functional implication of these bacteria on carbonate accumulation as a way of atmospheric CO(2) sequestration.

Water-filtered infrared-A radiation (wIRA) is not implicated in cellular degeneration of human skin.

BACKGROUND: Excessive exposure to solar ultraviolet radiation is involved in the complex biologic process of cutaneous aging. Wavelengths in the ultraviolet-A and -B range (UV-A and UV-B) have been shown to be responsible for the induction of proteases, e. g. the collagenase matrix metalloproteinase 1 (MMP-1), which are related to cell aging. As devices emitting longer wavelengths are widely used in therapeutic and cosmetic interventions and as the induction of MMP-1 by water-filtered infrared-A (wIRA) had been discussed, it was of interest to assess effects of wIRA on the cellular and molecular level known to be possibly involved in cutaneous degeneration. OBJECTIVES: Investigation of the biological implications of widely used water-filtered infrared-A (wIRA) radiators for clinical use on human skin fibroblasts assessed by MMP-1 gene expression (MMP-1 messenger ribonucleic acid (mRNA) expression).Methods: Human skin fibroblasts were irradiated with approximately 88% wIRA (780-1400 nm) and 12% red light (RL, 665-780 nm) with 380 mW/cm(2) wIRA(+RL) (333 mW/cm(2) wIRA) on the one hand and for comparison with UV-A (330-400 nm, mainly UV-A1) and a small amount of blue light (BL, 400-450 nm) with 28 mW/cm(2) UV-A(+BL) on the other hand. Survival curves were established by colony forming ability after single exposures between 15 minutes and 8 hours to wIRA(+RL) (340-10880 J/cm(2) wIRA(+RL), 300-9600 J/cm(2) wIRA) or 15-45 minutes to UV-A(+BL) (25-75 J/cm(2) UV-A(+BL)). Both conventional Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and quantitative real-time RT-PCR techniques were used to determine the induction of MMP-1 mRNA at two physiologic temperatures for skin fibroblasts (30 degrees C and 37 degrees C) in single exposure regimens (15-60 minutes wIRA(+RL), 340-1360 J/cm(2) wIRA(+RL), 300-1200 J/cm(2) wIRA; 30 minutes UV-A(+BL), 50 J/cm(2) UV-A(+BL)) and in addition at 30 degrees C in a repeated exposure protocol (up to 10 times 15 minutes wIRA(+RL) with 340 J/cm(2) wIRA(+RL), 300 J/cm(2) wIRA at each time). RESULTS: Single exposure of cultured human dermal fibroblasts to UV-A(+BL) radiation yielded a very high increase in MMP-1 mRNA expression (11 +/-1 fold expression for RT-PCR and 76 +/-2 fold expression for real-time RT-PCR both at 30 degrees C, 75 +/-1 fold expression for real-time RT-PCR at 37 degrees C) and a dose-dependent decrease in cell survival. In contrast, wIRA(+RL) did not produce cell death and did not induce a systematic increase in MMP-1 mRNA expression (less than twofold expression, within the laboratory range of fluctuation) detectable with the sensitive methods applied. Additionally, repeated exposure of human skin fibroblasts to wIRA(+RL) did not induce MMP-1 mRNA expression systematically (less than twofold expression by up to 10 consecutive wIRA(+RL) exposures and analysis with real-time RT-PCR). CONCLUSIONS: wIRA(+RL) even at the investigated disproportionally high irradiances does not induce cell death or a systematic increase of MMP-1 mRNA expression, both of which can be easily induced by UV-A radiation. Furthermore, these results support previous findings of in vivo investigations on collagenase induction by UV-A but not wIRA and show that infrared-A with appropriate irradiances does not seem to be involved in MMP-1 mediated photoaging of the skin. As suggested by previously published studies wIRA could even be implicated in a protective manner.