Selective inactivation of MAO-B by benzyl-dimethyl-silyl-methanamines in vitro

The alpha-silyl amines benzyl-dimethyl-silyl-methanamine and the p-fluoro and p-chloro derivatives are potent time-dependent inhibitors of rat brain MAO-B. The inhibition exhibits saturation kinetics, takes place in the enzyme active-site and is irreversible. The most potent inhibitor in the series is 4-fluorobenzyl-dimethyl-silyl-methanamine (KI = 11 microM, tau 1/2 = 2.3 min). Its selectivity for the B-form relative to the A-form of rat brain MAO is higher than 10(4). Benzyl-dimethyl-silyl-methanamines may represent a new family of anti-Parkinsonian agents.     

Insolubility of Cr2O3 in Bioaccessibility Tests Points to Requirement for a New Human Oral Reference Dose for Trivalent Chromium

Bioaccessibility measurements have the potential to improve the accuracy of risk assessments and reduce the potential costs of remediation when they reveal that the solubility of chemicals in a matrix (e.g., soil) differs markedly from that in the critical toxicity study (i.e., the key study from which a toxicological or toxicity reference value is derived). We aimed to apply this approach to a brownfield site contaminated with chromium, and found that the speciation was Cr^III, using a combination of alkaline digestion/diphenylcarbazide complexation and X-ray absorption near edge structure analysis. The bioaccessibility of Cr₂O₃, the compound on which a reference dose for Cr^III is based, was substantially lower (<0.1%) than that of the Cr^III in the soils, which was a maximum of 9%, giving relative bioaccessibility values of 13,000% in soil. This shows that the reference dose is based on essentially an insoluble compound, and thus we suggest that other compounds be considered for toxicity testing and derivation of reference dose. Two possibilities are CrCl₃·6H₂O and KCr(SO₄)₂·12H₂O, which have been used for derivation of ecological toxicity reference values and are soluble at a range of dosing levels in our bioaccessibility tests.     

A Non-Synonymous HMGA2 Variant Decreases Height in Shetland Ponies and Other Small Horses

The identification of quantitative trait loci (QTL) such as height and their underlying causative variants is still challenging and often requires large sample sizes. In humans hundreds of loci with small effects control the heritable portion of height variability. In domestic animals, typically only a few loci with comparatively large effects explain a major fraction of the heritability. We investigated height at withers in Shetland ponies and mapped a QTL to ECA 6 by genome-wide association (GWAS) using a small cohort of only 48 animals and the Illumina equine SNP70 BeadChip. Fine-mapping revealed a shared haplotype block of 793 kb in small Shetland ponies. The HMGA2 gene, known to be associated with height in horses and many other species, was located in the associated haplotype. After closing a gap in the equine reference genome we identified a non-synonymous variant in the first exon of HMGA2 in small Shetland ponies. The variant was predicted to affect the functionally important first AT-hook DNA binding domain of the HMGA2 protein (c.83G>A; p.G28E). We assessed the functional impact and found impaired DNA binding of a peptide with the mutant sequence in an electrophoretic mobility shift assay. This suggests that the HMGA2 variant also affects DNA binding in vivo and thus leads to reduced growth and a smaller stature in Shetland ponies. The identified HMGA2 variant also segregates in several other pony breeds but was not found in regular-sized horse breeds. We therefore conclude that we identified a quantitative trait nucleotide for height in horses.     

d-Aspartate in Human Brain

The presence of the biologically uncommon D-aspartic acid (D-aspartate) in human brain white matter has been previously reported. The earlier study has now been expanded to include D/L-aspartate ratios from 67 normal brains. The data show that the D-aspartate content increases rapidly from 1 year to approximately 35 years of age, levels off in middle age, and then appears to decrease somewhat. The D-aspartate content in gray matter remains at a consistently low level (half of that found in white matter) throughout the human life span. Within the limitations of current analytical methods, there was no detectable difference in D/L-aspartate ratios in white and gray matter of brains with Alzheimer's disease and several other pathologies when compared with brains of normal subjects. However, the presence of a significant D-aspartate level in white matter during the adult life span may lead to changes in protein configuration related to dysfunctions associated with the aging brain.     

Immobilization of microorganisms by adhesion: interplay of electrostatic and nonelectrostatic interactions

The adhesion of three microorganisms (Saccharomyces cerevisiae, Acetobacter aceti, and Moniliella pollinis) to different materials has been studied using various supports (glass, metals, plastics), some of which were treated by an Fe(III) solution. The surface properties of the cells were characterized by the zeta potential and an index of hydrophobicity; characterization of the supports involved surface chemical analysis (XPS) and contact angle measurements. Cell suspensions in pure water at a given pH were left to settle on plates; the latter were then rinsed and examined microscopically, Saccharomyces cerevisiae and A. aceti adhere to metals under certain pH conditions but do not adhere to any of the other materials tested unless it is previously treated by ferric ions; adhesion of these hydrophilic cells is essentially controlled by electrostatic interactions. Moniliella pollinis adhere spontaneously to glass and to polymeric materials, but its attachment is also influenced by cell-cell or cell-support electrostatic repulsions; near the cell isoelectric point, cell flocculation is competing with adhesion to a support.     

Influence of hormones and drugs on glutathione-S-transferase levels in primary culture of adult rat hepatocytes

GST activities against 1-Chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) were measured in isolated and cultured adult rat hepatocytes. Within 24 h in culture, both GST activities decreased to about 70% and either stabilized at this level (CDNB) or recovered (DCNB) to the initial level. Use of hyaluronidase in addition to collagenase during the isolation of the cells strongly reduced both activities and its stimulation by various drugs for up to 168 h. The hormones insulin, glucagon, triiodothyronine, estradiol, testosterone, and progesterone did not affect GST activity, while dexamethasone showed some interference. In the presence of dexamethasone the activity against CDNB was mainly stimulated by the combination of methylcholanthrene (MC) and phenobarbital (PB) to about 260% within 168 h. The activity against DCNB was stimulated predominantly by MC alone reaching 170% after 168 h. Quantification of the GST subunits Ya, Yb₁ and Yp by an ELISA technique revealed a strong decrease of Ya, a transient increase of Yb₁ after 24 h followed by a moderate decrease, and a stable low level of the transformation marker Yp during cultivation. The level of Ya was markedly induced by PB, particularly in combination with MC. The level of Yb₁ was equally induced by MC or PB with no synergistic effect. Yp was not affected by these drugs. None of the hormones affected the level of these GST subunits. These results indicate that the physiological type of regulation of the GSTs is maintained during primary culture and no signs of dedifferentiation or transformation are observed. Furthermore, they demonstrate that the interaction of drugs and hormones and their inducing potential can be efficiently studied in the cultured hepatocytes.Abbreviations ABTS 2,2-Azino-bis(3-ethylbenzthiazoline-6-sulfonate) - CDNB I-Chloro-2,4-dinitrobenzene - DCNB 1,2-dichloro-4-nitrobenzene; DEX, dexamethasone - DMSO dimethylsulfoxide - GST glutathione Stransferase - MC methylcholanthrene - N, NIC nicotinamide - -NF -naphthoflavone - PB phenobarbital - PBS phosphate buffered saline     

Conditions promoting stability of solventogenesis or culture degeneration in continuous fermentations of Clostridium acetobutylicum

Summary The stability of solvent production by Clostridium acetobutylicum has been studied in continuous single-stage and two-stage fermentations. At low dilution rates, metabolic oscillations resulting from product inhibition have been observed especially in the case of fermentations controlled by product accumulation. A second type of instability also observed in product-controlled fermentations, but not in fermentations controlled by nitrogen limitation, was a long-term metabolic drift towards acid production. This acid drift has been shown to be identical to the phenomenon of culture degeneration occurring upon subculturing in batch fermentation. In addition, it was found that acid drift could be reversed by decreases in pH, temperature and dilution rate, by growth limitation in nitrogen-deficient conditions and by the addition of butyric and acetic acids. The existence of two distinct mechanisms, a short-term control (shift) and a long-term control (drift), both triggered by the same physiological conditions, is proposed in the regulation of acid and solvent production.     

Calcium-dependent protease of the cyanobacterium Anabaena: molecular cloning and expression of the gene in Escherichia coli, sequencing and site-directed mutagenesis

Summary It has been suggested that a calcium-dependent intracellular protease of the cyanobacterium, Anabaena sp., participates in the differentiation of heterocysts, cells that are specialized for fixation of N₂. Clones of the structural gene (designated prcA) for this protease from Anabaena variabilis strain ATCC 29413 and Anabaena sp. strain PCC 7120 were identified via their expression in Escherichia coli. The prcA gene from A. variabilis was sequenced. The genes of both strains, mutated by insertion of a drug resistance cassette, were returned to these same strains of Anabaena on suicide plasmids. The method of sacB-mediated positive selection for double recombinants was used to achieve replacement of the wild-type prcA genes by the mutated forms. The resulting mutants, which lacked Ca²⁺-dependent protease activity, were not impaired in heterocyst formation and grew on N₂ as sole nitrogen source.     

Relative Rates of Nitric Oxide and Nitrous Oxide Production by Nitrifiers, Denitrifiers, and Nitrate Respirers

Biogenic emissions of nitric and nitrous oxides have important impacts on the photochemistry and chemistry of the atmosphere. Although biogenic production appears to be the overwhelming source of N₂O, the magnitude of the biogenic emission of NO is very uncertain. In soils, possible sources of NO and N₂O include nitrification by autotrophic and heterotrophic nitrifiers, denitrification by nitrifiers and denitrifiers, nitrate respiration by fermenters, and chemodenitrification. The availability of oxygen determines to a large extent the relative activities of these various groups of organisms. To better understand this influence, we investigated the effect of the partial pressure of oxygen (pO₂) on the production of NO and N₂O by a wide variety of common soil nitrifying, denitrifying, and nitrate-respiring bacteria under laboratory conditions. The production of NO per cell was highest by autotrophic nitrifiers and was independent of pO₂ in the range tested (0.5 to 10%), whereas N₂O production was inversely proportional to pO₂. Nitrous oxide production was highest in the denitrifier Pseudomonas fluorescens, but only under anaerobic conditions. The molar ratio of NO/N₂O produced was usually greater than unity for nitrifiers and much less than unity for denitrifiers. Chemodenitrification was the major source of both the NO and N₂O produced by the nitrate respirer Serratia marcescens. Chemodenitrification was also a possible source of NO and N₂O in nitrifier cultures but only when high concentrations of nitrite had accumulated or were added to the medium. Although most of the denitrifiers produced NO and N₂O only under anaerobic conditions, chemostat cultures of Alcaligenes faecalis continued to emit these gases even when the cultures were sparged with air. Based upon these results, we predict that aerobic soils are primary sources of NO and that N₂O is produced only when there is sufficient soil moisture to provide the anaerobic microsites necessary for denitrification by either denitrifiers or nitrifiers.