Phosphorus speciation in manure and manure-amended soils using XANES spectroscopy

Previous studies suggested an increase in the proportion of calcium phosphates (CaP) of the total phosphorus (P) pool in soils with a long-term poultry manure application history versus those with no or limited application histories. To understand and predict long-term P accumulation and release dynamics in these highly amended soils, it is important to understand what specific P species are being formed. We assessed forms of CaP formed in poultry manure and originally acidic soil in response to different lengths of mostly poultry manure applications using P K-edge X-ray absorption near-edge structure (XANES) spectroscopy. Phosphorus K-edge XANES spectra of poultry manure showed no evidences of crystalline P minerals but dominance of soluble CaP species and free and weakly bound phosphates (aqueous phosphate and phosphate adsorbed on soil minerals). Phosphate in an unamended neighboring forest soil (pH 4.3) was mainly associated with iron (Fe) compounds such as strengite and Fe-oxides. Soils with a short-term manure history contained both Fe-associated phosphates and soluble CaP species such as dibasic calcium phosphate (DCP) and amorphous calcium phosphate (ACP). Long-term manure application resulted in a dominance of CaP forms confirming our earlier results obtained with sequential extractions, and a transformation from soluble to more stable CaP species such as beta-tricalcium calcium phosphate (TCP). Even after long-term manure application (> 25 yr and total P in soil up to 13,307 mg kg(-1)), however, none of the manure-amended soils showed the presence of crystalline CaP. With a reduction or elimination of poultry manure application to naturally acidic soils, the pH of the soil is likely to decrease, thereby increasing the solubility of Ca-bonded inorganic P minerals. Maintaining a high pH is therefore an important strategy to minimize P leaching in these soils.     

Escherichia coli MutY protein has a guanine-DNA glycosylase that acts on 7,8-dihydro-8-oxoguanine:guanine mispair to prevent spontaneous G:C-->C:G transversions

Low rates of spontaneous G:C-->C:G transversions would be achieved not only by the correction of base mismatches during DNA replication but also by the prevention and removal of oxidative base damage in DNA. Escherichia coli must have several pathways to repair such mismatches and DNA modifications. In this study, we attempted to identify mutator loci leading to G:C-->C:G transversions in E.coli. The strain CC103 carrying a specific mutation in lacZ was mutagenized by random miniTn 10 insertion mutagenesis. In this strain, only the G:C-->C:G change can revert the glutamic acid at codon 461, which is essential for sufficient beta-galactosidase activity to allow growth on lactose. Mutator strains were detected as colonies with significantly increased rates of papillae formation on glucose minimal plates containing P-Gal and X-Gal. We screened approximately 40 000 colonies and selected several mutator strains. The strain GC39 showed the highest mutation rate to Lac+. The gene responsible for the mutator phenotypes, mut39 , was mapped at around 67 min on the E.coli chromosome. The sequencing of the miniTn 10 -flanking DNA region revealed that the mut39 was identical to the mutY gene of E.coli. The plasmid carrying the mutY + gene reduced spontaneous G:C-->T:A and G:C-->C:G mutations in both mutY and mut39 strains. Purified MutY protein bound to the oligonucleotides containing 7,8-dihydro-8-oxo-guanine (8-oxoG):G and 8-oxoG:A. Furthermore, we found that the MutY protein had a DNA glycosylase activity which removes unmodified guanine from the 8-oxoG:G mispair. These results demonstrate that the MutY protein prevents the generation of G:C-->C:G transversions by removing guanine from the 8-oxoG:G mispair in E.coli.     

Lowering of p27Kip1 levels by its antisense or by development of resistance to 1,25-dihydroxyvitamin D3 reverses the G1 block but not differentiation of HL60 cells

Cyclin-dependent kinase inhibitors are proteins with functions which appear to involve regulation of cell cycle traverse, and have been suggested to have a role in cell differentiation. However, there is as yet no rigorous proof that this is the case. We have addressed the participation of one of these inhibitors, p27Kip1, in the induction of differentiation and the subsequent G1 block induced in HL60 cells by 1,25-dihydroxyvitamin D3 (1,25D3). First, it was noted that sublines of HL60 cells able to grow rapidly in the presence of 1,25D3 have protein levels of p27Kip1 lower than the levels in cells subjected to 1,25D3-induced growth inhibition, but higher than in untreated parental cells. In contrast, there was no discernible relationship between the levels of p27Kip1 and the expression of differentiation markers. Further, HL60 cells treated with 1,25D3 and an oligonucleotide antisense, but not mismatched, to p27Kip1 showed an almost complete elimination of the 1,25D3-induced G1 block, but no decrease in the expression of differentiation markers. Similar results were obtained following transient transfection with an expression vector bearing the entire p27Kip1 coding sequence in the anti-sense orientation. This is the first direct demonstration that p27Kip1 plays a role in the 1,25D3-induced G1 arrest, and that partial reduction in its levels has no effect on the induction of differentiation in HL60 cells.