Distinct distribution of immunoreactive dynorphin and leucine enkephalin in various populations of isolated adrenal cromaffin cells

The distribution of immunoreactive-dynorphin (ir-Dyn) in isolated subpopulations of bovine adrenal chromaffin cells was examined and compared with that of adrenaline (A), noradrenaline (NA) and ir-Leucine-Enkephalin (ir-Leu-Enk). Using a stepwise bovine serum albumin (BSA) gradient, various populations of catecholamine-storing cells were separated and designated as cell layers I, II and III. Cell layer I contained more NA than A; cell layer II contained slightly more A than NA whereas cell layer III was highly enriched in A. The original cell preparation contained 2.9 times more ir-Leu-Enk than ir-Dyn (4.7 and 1.6 pmoles per 10(6) cells, respectively). After separation of the cells on BSA gradient, ir-Dyn was mainly detected in cell layer I (4.0 pmoles/10(6) cells) whereas ir-Leu-Enk was concentrated in cell layer III (8.3 pmoles/10(6) cells). Both peptides were secreted in response to acetylcholine (5 x 10(-5) M), but the amount secreted was in accordance with the cell content in each peptide. After subcellular fractionation of the adrenal medulla, the neuropeptides were found in close association with catecholamines in the secretory granules. These results indicate that bovine adrenal chromaffin cells can be isolated according to their specific content in A, NA and opioid peptides and are consistent with the hypothesis of distinct biosynthetic pathways for Dyn and the Enk.     

Identification of microtubule growth deceleration and its regulation by conserved and novel proteins

Microtubules (MTs) are cytoskeletal polymers that participate in diverse cellular functions, including cell division, intracellular trafficking, and templating of cilia and flagella. MTs undergo dynamic instability, alternating between growth and shortening via catastrophe and rescue events. The rates and frequencies of MT dynamic parameters appear to be characteristic for a given cell type. We recently reported that all MT dynamic parameters vary throughout differentiation of a smooth muscle cell type in intact Caenorhabditis elegans. Here we describe local differences in MT dynamics and a novel MT behavior: an abrupt change in growth rate (deceleration) of single MTs occurring in the cell periphery of these cells. MT deceleration occurs where there is a decrease in local soluble tubulin concentration at the cell periphery. This local regulation of tubulin concentration and MT deceleration are dependent on two novel homologues of human cylicin. These novel ORFs, which we name cylc-1 and -2, share sequence homology with stathmins and encode small, very basic proteins containing several KKD/E repeats. The TOG domain–containing protein ZYG-9^TOGp is responsible for the faster polymerization rate within the cell body. Thus we have defined two contributors to the molecular regulation for this novel MT behavior.     

The peroxisomal protein import machinery displays a preference for monomeric substrates

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and transported by the shuttling receptor PEX5 to the peroxisomal membrane docking/translocation machinery, where they are translocated into the organelle matrix. Under certain experimental conditions this protein import machinery has the remarkable capacity to accept already oligomerized proteins, a property that has heavily influenced current models on the mechanism of peroxisomal protein import. However, whether or not oligomeric proteins are really the best and most frequent clients of this machinery remain unclear. In this work, we present three lines of evidence suggesting that the peroxisomal import machinery displays a preference for monomeric proteins. First, in agreement with previous findings on catalase, we show that PEX5 binds newly synthesized (monomeric) acyl-CoA oxidase 1 (ACOX1) and urate oxidase (UOX), potently inhibiting their oligomerization. Second, in vitro import experiments suggest that monomeric ACOX1 and UOX are better peroxisomal import substrates than the corresponding oligomeric forms. Finally, we provide data strongly suggesting that although ACOX1 lacking a peroxisomal targeting signal can be imported into peroxisomes when co-expressed with ACOX1 containing its targeting signal, this import pathway is inefficient.     

Divergent selection along climatic gradients in a rare central European endemic species,Saxifraga sponhemica

Background and Aims The effects of habitat fragmentation on quantitative genetic variation in plant populations are still poorly known. Saxifraga sponhemica is a rare endemic of Central Europe with a disjunct distribution, and a stable and specialized habitat of treeless screes and cliffs. This study therefore used S. sponhemica as a model species to compare quantitative and molecular variation in order to explore (1) the relative importance of drift and selection in shaping the distribution of quantitative genetic variation along climatic gradients; (2) the relationship between plant fitness, quantitative genetic variation, molecular genetic variation and population size; and (3) the relationship between the differentiation of a trait among populations and its evolvability.Methods Genetic variation within and among 22 populations from the whole distribution area of S. sponhemica was studied using RAPD (random amplified polymorphic DNA) markers, and climatic variables were obtained for each site. Seeds were collected from each population and germinated, and seedlings were transplanted into a common garden for determination of variation in plant traits.Key Results In contrast to previous results from rare plant species, strong evidence was found for divergent selection. Most population trait means of S. sponhemica were significantly related to climate gradients, indicating adaptation. Quantitative genetic differentiation increased with geographical distance, even when neutral molecular divergence was controlled for, and Q_ST exceeded F_ST for some traits. The evolvability of traits was negatively correlated with the degree of differentiation among populations (Q_ST), i.e. traits under strong selection showed little genetic variation within populations. The evolutionary potential of a population was not related to its size, the performance of the population or its neutral genetic diversity. However, performance in the common garden was lower for plants from populations with reduced molecular genetic variation, suggesting inbreeding depression due to genetic erosion.Conclusions The findings suggest that studies of molecular and quantitative genetic variation may provide complementary insights important for the conservation of rare species. The strong differentiation of quantitative traits among populations shows that selection can be an important force for structuring variation in evolutionarily important traits even for rare endemic species restricted to very specific habitats.     

In vitro effects of arachidonic acid and of inhibitors of its metabolism on the dog thyroid gland

Incubation of dog thyroid tissue with arachidonic acid (10 to 200 μM) led to the following events:

- low conversion to prostaglandins E₂ and F_2α: 0.07% and 0.02% per hour and 100 mg tissue, respectively

- inhibition of the stimulatory effect of low concentrations of TSH on thyroid secretion: the secretory effect of supra-maximal concentrations of TSH and of dB-cAMP was unaffected

- inhibition of the cyclic AMP accumulation induced by TSH: this effect was inhibited neither by indomethacin nor by ETYA; cyclic AMP accumulation in response to cholera toxin or PGE₁ was unaffected

- no effect on cyclic GMP level

- stimulation of thyroid proteins iodination.

ETYA, but not indomethacin, depressed the iodination of thyroid proteins in resting and stimulated tissue. These data show that arachidonic acid-or a metabolite-can modulate thyroid responsiveness to TSH and suggest that lipoxygenase-products of arachidonic acid metabolism could be involved in thyroid proteins iodination.     

Legal constraints and opportunities for biochar: a case analysis of EU law

This article addresses biochar from a legal point of view. It analyses different policies and regulations from a European (Flemish) point of view and provides a first and general insight in what potential legal constraints the development of a biochar industry might face and what opportunities lie ahead. This is due to the fact that biochar is a recent product and a lot of scientific uncertainty still exists regarding the consequences of its application. From the analysis it appears a multitude of policies and legislative measures influence the development of the biochar industry. Hence, it is important that all these policies and legislative measures are analyzed in an appropriate manner. Moreover, considerable lobbying, negotiating and cooperation between different disciplines (legal, scientific, economical, etc.) will be required so as to develop a feasible and safe biochar framework.     

Enhanced leishmanicidal activity of cryptopeptide chimeras from the active N1 domain of bovine lactoferrin

Two antimicrobial cryptopeptides from the N1 domain of bovine lactoferrin, lactoferricin (LFcin17–30) and lactoferrampin (LFampin265–284), together with a hybrid version (LFchimera), were tested against the protozoan parasite Leishmania. All peptides were leishmanicidal against Leishmania donovani promastigotes, and LFchimera showed a significantly higher activity over its two composing moieties. Besides, it was the only peptide active on Leishmania pifanoi axenic amastigotes, already showing activity below 10?μM. To investigate their leishmanicidal mechanism, promastigote membrane permeabilization was assessed by decrease of free ATP levels in living parasites, entrance of the vital dye SYTOX Green (MW?=?600?Da) and confocal and transmission electron microscopy. The peptides induced plasma membrane permeabilization and bioenergetic collapse of the parasites. To further clarify the structural traits underlying the increased leishmanicidal activity of LFchimera, the activity of several analogues was assessed. Results revealed that the high activity of these hybrid peptides seems to be related to the order and sequence orientation of the two cryptopeptide moieties, rather than to their particular linkage through an additional lysine, as in the initial LFchimera. The incorporation of both antimicrobial cryptopeptide motifs into a single linear sequence facilitates chemical synthesis and should help in the potential clinical application of these optimized analogues.     

Alterations in the Fatty Acid Composition of Rat Brain Cells (Neurons, Astrocytes, and Oligodendrocytes) and of Subcellular Fractions (Myelin and Synaptosomes) Induced by a Diet Devoid of n-3 Fatty Acids

Abstract: Rats were fed through four generations with a semisynthetic diet containing 1.0% sunflower oil (6.7 mg/ g n-6 fatty acids, 0.04 mg/g n-3 fatty acids). Ten days before mating, half of the animals received a diet in which sunflower was replaced by soya oil (6.6 mg/g n-6 fatty acids, 0.8 mg/g n-3 fatty acids) and analyses were performed on their pups. Fatty acid analysis in isolated cellular and subcellular material from sunflower-fed animals showed that the total amount of unsaturated fatty acids was not reduced in any cellular or subcellular fraction (except in 60-day-old rat neurons). All material from animals fed with sunflower oil showed an important reduction in the docosahexaenoic acid content, compensated (except in 60-day-old rat neurons) by an increase in the n-6 fatty acids (mainly C22:5 n-6). When comparing 60-day-old animals fed with soya oil or sunflower oil, the n-3/n-6 fatty acid ratio was reduced 16-fold in oligodendrocytes, 12-fold in myelin, twofold in neurons, sixfold in synaptosomes, and threefold in astrocytes. No trienes were detected. Saturated and monounsaturated fatty acids were hardly affected. This study provides data on the fatty acid composition of isolated brain cells.