Improved high-performance liquid chromatography analysis of P-postlabeled 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-DNA adducts using in-line precolumn purification

An improved HPLC-based ³²P-postlabeling assay has been developed for the analysis of DNA modified with the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Postlabeled samples are loaded onto a C₁₈ precolumn and adducted bases are retained while excess radioactivity and unmodified DNA bases are eluted directly to waste through a switching valve. The use of this HPLC in-line precolumn purification (HIPP) technique allows entire postlabeled samples to be analyzed without prior removal of inorganic phosphate and unmodified DNA bases. The method has a sample to sample precision of 15% and accuracy of 20%, at adduct levels of 2 adducts/10⁷ bases and shows a linear relationship between signal and adduction levels from 1 adduct per 10⁴ to ≈ 2±1 adducts per 10⁹ bases. Individual postlabeled DNA samples can be analyzed by HPLC in less than 1 h, allowing high throughput. The use of calf-thymus DNA (CT-DNA), highly modified with PhIP, or DNA isolated from mice chronically fed a PhIP-modified diet shows two major PhIP-DNA adduct peaks and three additional minor adduct peaks when labeled under ATP-limiting conditions. Isolation of the HPLC purified peaks and analysis by thin layer chromatography (TLC) matches the five HPLC peaks to the spots typically seen by TLC, including N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (dG-C8-PhIP). Variations in digestion techniques indicate a potential resistance of the PhIP-DNA adducts to the standard enzymatic digestion methods. Attempts at adduct intensification by solid phase extraction, nuclease P1 enrichment or 1-butanol extraction decreased PhIP-DNA adduct peaks and introduced a large early eluting peak. Removal of the 3′-phosphate with nuclease P1 following the kinase labeling reaction simplifies the HPLC profile to one major peak (dG-C8-PhIP monophosphate) with several minor peaks. In addition to the high resolution provided by HPLC separation of the PhIP-DNA adducts, this method can be adjusted for analysis of other DNA adducts and is readily automated for high throughput.     

Respiratory and dissimilatory nitrate-reducing communities from an extreme saline alkaline soil of the former lake Texcoco (Mexico)

The diversity of the dissimilatory and respiratory nitrate-reducing communities was studied in two soils of the former lake Texcoco (Mexico). Genes encoding the membrane-bound nitrate reductase (narG) and the periplasmic nitrate reductase (napA) were used as functional markers. To investigate bacterial communities containing napA and narG in saline alkaline soils of the former lake Texcoco, libraries of the two sites were constructed (soil T3 with pH 11 and electrolytic conductivity in saturated extract (EC_SE) 160 dS m⁻¹ and soil T1 with pH 8.5 and EC_SE 0.8 dS m⁻¹). Phylogenetic analysis of napA sequences separated the clone families into two main groups: dependent or independent of NapB. Most of napA sequences from site T1 were grouped in the NapB-dependent clade, meanwhile most of the napA sequences from the extreme soil T3 were affiliated to the NapB-independent group. For both sites, partial narG sequences were associated with representatives of the Proteobacteria, Firmicutes and Actinobacteria phyla, but the proportions of the clones were different. Our results support the concept of a specific and complex nitrate-reducing community for each soil of the former lake Texcoco.     

Enhanced dissipation of polycyclic aromatic hydrocarbons in the rhizosphere of the Athel tamarisk (Tamarix aphylla L. Karst.) grown in saline-alkaline soils of the former lake Texcoco

Remediation of polycyclic aromatic hydrocarbons (PAHs) contaminated alkaline saline soil with phreatophyte or "water loving plants" was investigated by spiking soil from the former lake Texcoco with 100 mg phenanthrene (Phen) kg(-1) soil, 120 mg anthracene (Ant)kg(-1) soil and 45 mg benzo(a)pyrene (BaP) kg(-1) soil and vegetating it with Athel tamarisk (Tamarix aphylla L Karst.). The growth of the Athel tamarisk was not affected by the PAHs. In soil cultivated with Athel tamarisk, the leaching of PAHs to the 32-34 cm layer decreased 2-fold compared to the uncultivated soil. The BaP concentration decreased to 39% of the initial concentration at a distance smaller than 3 cm from the roots and to 45% at a distance larger than 3cm, but 59% remained in unvegetated soil after 240 days. Dissipation of Ant and Phen decreased with depth, but not BaP. The biodegradation of PAHs was affected by their chemical properties and increased in the presence of T. aphylla, but decreased with depth.     

Formation, derivatization and applications of bacterial cellulose

Acetobacter xylinum produces highly crystalline cellulose extracellulary using glucose as a carbon source. The polymer formed is free of other biogenic compounds, separable in a simple way and characterized by its high water-absorption capacity. Stepwise solvent exchange from water to unpolar solvents leads to a drastic decrease of the water content of the bacterial cellulose without decrease of the highly swollen and activated state. Heterogeneous as well as homogeneous derivatizations, e.g. carboxymethylation, silylation and acetylation, were performed on the wet or dried biopolymer. Furthermore, different methods for formation of hollow fibres during biosynthesis were investigated. Such tubes may have applications as biocompatible material in medicine.     

NAD glycohydrolase activity in dog cardiac tissue.

1. Dog heart tissue suspension hydrolyzes NAD, NADP and NMN, and releases nicotinamide stoichiometrically. 2. Maximum activity was observed at 50 degrees C and the activation energy was 10 kcal/mol. 3. Optimum pH range was 6.2-7.6. 4. Compounds with adenine-ribose moiety increased the enzymatic activity. 5. Nicotinamide released during incubation produced reaction nonlinearity. 6. Km for NAD and NADP were about the same; Vmax was higher for NAD. Similar findings have been reported for rabbit heart. 7. Dog enzyme appears to be more sensitive than the rabbit enzyme to noncompetitive inhibitors. 8. Pyrophosphatase activity was not detected in dog heart in contrast to rabbit and rat heart preparations.     

Changes in the bacterial populations of the highly alkaline saline soil of the former lake Texcoco (Mexico) following flooding

Flooding an extreme alkaline-saline soil decreased alkalinity and salinity, which will change the bacterial populations. Bacterial 16S rDNA libraries were generated of three soils with different electrolytic conductivity (EC), i.e. soil with EC 1.7 dS m⁻¹ and pH 7.80 (LOW soil), with EC 56 dS m⁻¹ and pH 10.11 (MEDIUM soil) and with EC 159 dS m⁻¹ and pH 10.02 (HIGH soil), using universal bacterial oligonucleotide primers, and 463 clone 16S rDNA sequences were analyzed phylogenetically. Library proportions and clone identification of the phyla Proteobacteria, Actinobacteria, Acidobacteria, Cyanobacteria, Bacteroidetes, Firmicutes and Cloroflexi showed that the bacterial communities were different. Species and genera of the Rhizobiales, Rhodobacterales and Xanthomonadales orders of the α- and γ-subdivision of Proteobacteria were found at the three sites. Species and genera of the Rhodospirillales, Sphingobacteriales, Clostridiales, Oscillatoriales and Caldilineales were found only in the HIGH soil, Sphingomonadales, Burkholderiales and Pseudomonadales in the MEDIUM soil, Myxococcales in the LOW soil, and Actinomycetales in the MEDIUM and LOW soils. It was found that the largest diversity at the order and species level was found in the MEDIUM soil as bacteria of both the HIGH and LOW soils were found in it.     

Denitrification in extreme alkaline saline soils of the former lake Texcoco

Plant and Soil - Extreme alkalinity and salinity affects different processes involved in the N cycle, such as mineralization and nitrification, so it might affect the denitrification process and...     

Changes in methane oxidation activity and methanotrophic community composition in saline alkaline soils

The soil of the former Lake Texcoco is a saline alkaline environment where anthropogenic drainage in some areas has reduced salt content and pH. Potential methane (CH₄) consumption rates were measured in three soils of the former Lake Texcoco with different electrolytic conductivity (EC) and pH, i.e. Tex-S1 a >18 years drained soil (EC 0.7 dS m^?1, pH 8.5), Tex-S2 drained for ~10 years (EC 9.0 dS m^?1, pH 10.3) and the undrained Tex-S3 (EC 84.8 dS m^?1, pH 10.3). An arable soil from Alcholoya (EC 0.7 dS m^?1, pH 6.7), located nearby Lake Texcoco was used as control. Methane oxidation in the soil Tex-S1 (lowest EC and pH) was similar to that in the arable soil from Alcholoya (32.5 and 34.7 mg CH₄ kg^?1 dry soil day^?1, respectively). Meanwhile, in soils Tex-S2 and Tex-S3, the potential CH₄ oxidation rates were only 15.0 and 12.8 mg CH₄ kg^?1 dry soil day^?1, respectively. Differences in CH₄ oxidation were also related to changes in the methane-oxidizing communities in these soils. Sequence analysis of pmoA gene showed that soils differed in the identity and number of methanotrophic phylotypes. The Alcholoya soil and Tex-S1 contained phylotypes grouped within the upland soil cluster gamma and the Jasper Ridge, California JR-2 clade. In soil Tex-S3, a phylotype related to Methylomicrobium alcaliphilum was detected.     

Noninvasive characterization of human stratum corneum of undiseased skin of patients with atopic dermatitis and psoriasis as studied by Fourier transform Raman spectroscopy

Etiopathogenetic regulatory disorders of epidermal metabolism and the subsequent changes in the molecular pattern of the stratum corneum play an important role in the clinical differentiation of particular dermatoses (e.g., psoriasis, atopic dermatitis). In this study we present in vitro Fourier transform Raman spectra of the stratum corneum from healthy skin, as well as from clinically undiseased skin of the right heel of atopic and psoriatic volunteers. Differences in the averaged spectra were detected, particularly in the spectral ranges of 1112-1142 (lipid band), 1185-1220, and 1394-1429 cm(-1). By using the first derivative of the averaged spectra and/or a statistical evaluation of the spectroscopic data it was possible to distinguish the skin types examined.