Priming effect as determined by adding 14C-glucose to modified controlled composting test

The development of new biodegradable packaging materials, especially biodegradable plastics, has created a need for biodegradability testing. The European standard for controlled composting test was used in this study for assessing if the addition of a test material results in excess CO₂ production in compost. This effect, designated as the priming effect, would give an erroneous result for biodegradation, which is based on CO₂ formation from the test material. Glucose was selected as a test substrate because it is the degradation product of starch and cellulose, which are major compounds of many packaging materials. Both ¹⁴C-glucose and non-labelled glucose was applied to nine compost samples of variable stability and agefrom two weeks to 1.5 years. CO₂ and ¹⁴CO₂ evolution were measured during the incubation. Biodegradation of glucose in unstable composts (age leq6 months) was negative and ¹⁴CO₂ evolution was poor, although the respective composts without glucose produced relatively high amounts of CO₂. It was concluded that a negative priming effect was observed in unstable composts, in which glucose remained mostly non-degraded and apparently inhibited the mineralization of native organic matter in the compost. In stable composts (age 6 months), biodegradation of glucose was high and approximately equal to ¹⁴C-glucose mineralization, i.e., the composts showed no priming effect. Young composts were unsuitable for controlled composting test due to lack of stability. It is important to ensure that the compost inoculum used for the test is sufficiently stable.     

Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin: Lipid composition and thermal phase behavior of the stratum corneum

The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present study was to compare the stratum corneum lipid content of ROC with the corresponding material from human skin. The lipid composition was determined by thin-layer chromatography (TLC) and mass-spectrometry, and the thermal phase transitions of stratum corneum were studied by differential scanning calorimetry (DSC). All major lipid classes of the stratum corneum were present in ROC in a similar ratio as found in human stratum corneum. Compared to human skin, the level of non-hydroxyacid-sphingosine ceramide (NS) was increased in ROC, while α-hydroxyacid-phytosphingosine ceramide (AP) and non-hydroxyacid-phytosphingosine ceramides (NP) were absent. Also some alterations in fatty acid profiles of ROC ceramides were noted, e.g., esterified ω-hydroxyacid-sphingosine contained increased levels of oleic acid instead of linoleic acid. The fraction of lipids covalently bound to corneocyte proteins was distinctly lower in ROC compared to human skin, in agreement with the results from DSC. ROC underwent a lipid lamellar order to disorder transition (T₂) at a slightly lower temperature (68 °C) than human skin (74 °C). These differences in stratum corneum lipid composition and the thermal phase transitions may explain the minor differences previously observed in drug permeation between ROC and human skin.     

Dopamine transport function is elevated in cocaine users

Dopaminergic transmission has been suggested to be a primary mechanism mediating reinforcement, withdrawal and craving associated with psychostimulant addiction. Pyscho-stimulants attenuate dopamine transporter (DAT) clearance efficiency, resulting in a net increase in synaptic dopamine levels. Re-uptake rate is determined by the number of functional DAT molecules at the membrane surface. Previous in vivo imaging studies in humans and in vitro studies in post-mortem human brain have demonstrated that chronic cocaine abuse results in a neuroadaptive increase in DAT-binding site density in the limbic striatum. Whether this increase in DAT availability represents an increase in the functional activity of the transporter is unknown. Here, we present evidence that DAT function is elevated by chronic cocaine abuse. The effect of increasing post-mortem interval on the functional viability of synaptosomes was modeled in the baboon brain. Baboon brains sampled under conditions similar to human brain autopsies yielded synaptosomal preparations that were viable up to 24 h post-mortem. Dopamine (DA) uptake was elevated twofold in the ventral striatum from cocaine users as compared to age-matched drug-free control subjects. The levels of [3H]DA uptake were not elevated in victims of excited cocaine delirium, who experienced paranoia and marked agitation prior to death. In keeping with the increase in DAT function, [3H]WIN 35,428 binding was increased in the cocaine users, but not in the victims of excited delirium. These results demonstrate that DA uptake function assayed in cryopreserved human brain synaptosomes is a suitable approach for testing hypotheses of the mechanisms underlying human brain disorders and for studying the actions of addictive drugs in man.     

Decreased anthocyanidin reductase expression strongly decreases silver birch (Betula pendula) growth and alters accumulation of phenolics

Phenolics, formed via a complex phenylpropanoid pathway, are important defensive agents in plants and are strongly affected by nitrogen (N) fertilization. Proanthocyanidins (PAs) are one possible endpoint of the phenylpropanoid pathway, and anthocyanidin reductase (ANR) represents a key enzyme in PA biosynthesis. In this study, the expression of silver birch (Betula pendula) anthocyanidin reductase BpANR was inhibited using the RNA interference (RNAi) method, in three consequent BpANR RNAi (ANRi birches) lines. The growth, the metabolites of the phenylpropanoid pathway, and the number of resin glands of the ANRi birches were studied when grown at two N levels. ANRi birches showed decreased growth and reduction in PA content, while the accumulation of total phenolics in both stems and leaves increased. Moreover, ANRi birches produced more resin glands than did wild‐type (WT) birches. The response of ANRi birches to N depletion varied compared with that of WT birches, and in particular, the concentrations of some phenolics in stems increased in WT birches and decreased in ANRi birches. Because the inhibition of PAs biosynthesis via ANR seriously affected birch growth and resulted in accumulation of the precursors, the native level of PAs in plant tissues is assumed to be the prerequisite for normal plant growth. This draws attention to the real plant developmental importance of PAs in plant tissues.     

Lakes as nitrous oxide sources in the boreal landscape

Estimates of regional and global freshwater N₂O emissions have remained inaccurate due to scarce data and complexity of the multiple processes driving N₂O fluxes the focus predominantly being on summer time measurements from emission hot spots, agricultural streams. Here, we present four‐season data of N₂O concentrations in the water columns of randomly selected boreal lakes covering a large variation in latitude, lake type, area, depth, water chemistry, and land use cover. Nitrate was the key driver for N₂O dynamics, explaining as much as 78% of the variation of the seasonal mean N₂O concentrations across all lakes. Nitrate concentrations varied among seasons being highest in winter and lowest in summer. Of the surface water samples, 71% were oversaturated with N₂O relative to the atmosphere. Largest oversaturation was measured in winter and lowest in summer stressing the importance to include full year N₂O measurements in annual emission estimates. Including winter data resulted in fourfold annual N₂O emission estimates compared to summer only measurements. Nutrient‐rich calcareous and large humic lakes had the highest annual N₂O emissions. Our emission estimates for Finnish and boreal lakes are 0.6 and 29 Gg N₂O‐N/year, respectively. The global warming potential of N₂O from lakes cannot be neglected in the boreal landscape, being 35% of that of diffusive CH₄ emission in Finnish lakes.     

Catalytic activities of human liver cytochrome P-450 IIIA4 expressed in Saccharomyces cerevisiae

A human liver cytochrome P-450 (P-450) IIIA4 cDNA clone was inserted behind an alcohol dehydrogenase promoter in the plasmid vector pAAH5 and expressed in Saccharomyces cerevisiae (D12 and AH22 strains). A cytochrome P-450 with typical spectral properties was expressed at a level of approximately 8 x 10(5) molecules/cell in either strain of yeast. The expressed P-450 IIIA4 had the same apparent monomeric Mr as the corresponding protein in human liver microsomes (P-450NF) and could be isolated from yeast microsomes. Catalytic activity of the yeast microsomes toward putative P-450 IIIA4 substrates was seen in the reactions supported by cumene hydroperoxide but was often lower and variable when supported by the physiological donor NADPH. The catalytic activity of purified P-450 IIIA4 was also poor in some systems reconstituted with rabbit liver NADPH-P-450 reductase and best when both the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and a lipid extract (from liver or yeast microsomes) or L-alpha-1,2-dilauroyl-sn-glycero-3-phosphocholine were present. Under these conditions the expressed P-450 IIIA4 was an efficient catalyst for nifedipine oxidation, 6 beta-hydroxylation of testosterone and cortisol, 2-hydroxylation of 17 beta-estradiol and 17 alpha-ethynylestradiol, N-oxygenation and 3-hydroxylation of quinidine, 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate, erythromycin N-demethylation, the 10-hydroxylation of (R)-warfarin, the formation of 9,10-dehydrowarfarin from (S)-warfarin, and the activation of aflatoxins B1 and G1, sterigmatocystin, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (both + and - diastereomers), 3,4-dihydroxy-3,4-dihydrobenz[a]anthracene, 3,4-dihydroxy-3,4-dihydro-7, 12-dimethylbenz[a]anthracene, 9,10-dihydroxy-9,10-dihydrobenzo[b]fluoranthene, 6-aminochrysene, and tris(2,3-dibromopropyl) phosphate to products genotoxic in a Salmonella typhimurium TA1535/pSK1002 system where a chimeric umuC' 'lacZ plasmid is responsive to DNA alkylation. Reaction rates were stimulated by 7,8-benzoflavone and inhibited by rabbit anti-P-450 IIIA (anti-P-450NF), troleandomycin, gestodene, and cimetidine. Evidence was obtained that rates of reduction of ferric P-450 IIIA4 in yeast microsomes and the reconstituted systems are slow and at least partially responsible for the lower rates of catalysis seen in these systems (relative to liver microsomes). The results of these studies with a defined protein clearly demonstrate the ability of P-450 IIIA4 to catalyze regio- and stereoselective oxidations with a diverse group of substrates, and this enzyme appears to be one of the most versatile catalysts in the P-450 family.     

Inhibition of pancreatic lipase by mixed micelles of diethyl p-nitrophenyl phosphate and bile salts

Solubility and Sephadex filtration assays have shown that dissolved diethyl p-nitrophenyl phosphate can be included into bile salt micelles with a partition coefficient of 32 : 1. This inclusion is probably a prerequisite for the organophosphate to inhibit lipase. The essential role played by colipase confirms that the primary step in the inhibition is an interaction of lipase with bile salt containing micelles. Therefore, it appears that the requirements of lipase towards specific substrates and inhibitors are very similar. The inhibition rate strongly depends on the total bile salt concentration and on the micellar concentration of the organophosphate. This effect may be explained, at least qualitatively, by a competition between simple and mixed micelles for the binding of colipase and lipase.