Dibutyryl-cyclic AMP inhibits cholesterol esterification in J 774 monocyte-like cells

The effect of dibutyryl-cyclic AMP (dbcAMP) and theophylline was investigated on oleic acid incorporation into cholesteryl esters and triacylglycerols in the mouse monocyte-macrophage cell line J 774. 24h pretreatment of macrophages with dbcAMP decreased cholesteryl ester formation in a dose-dependent manner (about 4 fold reduction for dbcAMP 10(-4)M + theophylline 10(-3)M), while oleic acid incorporation into triacylglycerols was markedly (2 to 3 fold) enhanced. The catabolism of acetylated LDL was only slightly affected (about 15-20% reduction with dbcAMP 5 X 10(-4)M + theophylline 10(-3)M). Acyl Coenzyme A: cholesterol-O-acyl-transferase activity, measured in vitro on cell homogenates, was reduced in dbcAMP-treated cells, whereas diacylglycerol acyltransferase activity was increased. These results suggest that cyclic AMP can modulate cholesteryl ester and triacylglycerol formation in macrophages, and that these metabolisms are inversely regulated. Agents which increase cyclic AMP intracellular level could be of interest for reducing cholesteryl ester accumulation in macrophages.     

High threonine producer mutant ofNicotiana sylvestris (Spegg. and Comes)

Summary Mutagenesis and the subsequent selection of mesophyll diploid protoplasts ofNicotiana sylvestris on growth inhibitory concentrations of lysine plus threonine has led to the isolation of an LT-resistant mutant. Regeneration of this line (RLT 70) and analysis of its descendants demonstrated the dominant monogenic nuclear character of the resistance gene, further namedak-LT1. When the inhibition properties of aspartate kinase were examined in the homozygous mutant, lysine-sensitive activity could no longer be detected. In comparison, 70%–80% of the wild-type enzyme activity was usually inhibited by lysine, and the rest by threonine. Evidence for the existence of at least two AK isoenzymes was obtained by ion-exchange chromatography, where two peaks of activity could be detected: the first one to be eluted is lysine sensitive, and the second one threonine sensitive. One consequence of the altered regulation of AK in the mutant was the enhanced production of soluble threonine. Threonine accumulation was observed to occur throughout the life cycle of the mutant plant as well as in its different organs. In particular, leaves exhibited a 45-fold increment of soluble threonine, which corresponds to a 13-fold increase in total threonine: almost one-third of the total amino acids was free and proteinbound threonine. In RLT 70 seeds, 20% of the free amino acid pool was in the form of threonine (70-fold accumulation compared to the wild type), and total threonine content was increased five fold. As a general rule, the other amino acids were also more abundant in RLT 70 seeds, such that the total of amino acids present was between two to four times higher, but in contrast with the situation encountered in leaves, this was also due to a higher protein-bound amino acid content.     

Dependence on extracellular potassium of the positive inotropic response to St 587, a selective alpha-1 adrenoceptor agonist, in Zucker rat heart ventricle

The effects of St 587, a selective alpha-1 adrenoceptor agonist, were investigated in non obese and obese Zucker rat heart ventricles. In both groups, the numbers and affinity constants for alpha-1 adrenoceptors were found to be similar. At 4 or 10 mM [K]o, St 587 failed to increase the developed tension whereas at 14 mM [K]o, St 587 significantly increased it in both groups of rats. This effect was reversed by prazosin; St 587 also increased action potential duration at 14 mM [K]o. [K]o is thus important for the occurrence of the inotropic effect of St 587 in 12 week-old Zucker rats, either non obese or obese with reduced beta-adrenoceptor responsiveness. This suggests the participation of phosphoinositide metabolism in the mechanism of St 587 inotropic effect in the rat.     

Molecular analysis of the aspartate kinase-homoserine dehydrogenase gene from Arabidopsis thaliana

The gene encoding Arabidopsis thaliana aspartate kinase (ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) was isolated from genomic DNA libraries using the carrot ak-hsdh gene as the hybridizing probe. Two genomic libraries from different A. thaliana races were screened independently with the ak probe and the hsdh probe. Nucleotide sequences of the A. thaliana overlapping clones were determined and encompassed 2 kb upstream of the coding region and 300 bp downstream. The corresponding cDNA was isolated from a cDNA library made from poly(A)⁺-mRNA extracted from cell suspension cultures. Sequence comparison between the Arabidopsis gene product and an AK-HSDH bifunctional enzyme from carrot and from the Escherichia coli thrA and metL genes shows 80%, 37.5% and 31.4% amino acid sequence identity, respectively. The A. thaliana ak-hsdh gene is proposed to be the plant thrA homologue coding for the AK isozyme feedback inhibited by threonine. The gene is present in A. thaliana in single copy and functional as evidenced by hybridization analyses.The apoprotein-coding region is interrupted by 15 introns ranging from 78 to 134 bp. An upstream chloroplast-targeting sequence with low sequence similarity with the carrot transit peptide was identified. A signal sequence is proposed starting from a functional ATG initiation codon to the first exon of the apoprotein. Two additional introns were identified: one in the 5 non-coding leader sequence and the other in the putative chloroplast targeting sequence. 5 sequence analysis revealed the presence of several possible promoter elements as well as conserved regulatory motifs. Among these, an Opaque2 and a yeast GCN4-like recognition element might be relevant for such a gene coding for an enzyme limiting the carbon-flux entry to the biosynthesis of several essential amino acids. 3 sequence analysis showed the occurrence of two polyadenylation signals upstream of the polyadenylation site.This work is the first report of the molecular cloning of a plant ak-hsdh genomic sequence. It describes a promoter element that may bring new insights to the regulation of the biosynthesis of the aspartate family of amino acids.Abbreviations AK aspartate kinase - HSDH homoserine dehydrogenase - ID intermediate domain - Tp transit peptide     

A multivariate diagnosis approach applied to celery

Celery (Apium graveolens L. var Dulce) is a high value crop affected at different growth stages by a variety of nutrient disorders. Each nutrient concentration can be corrected for its dependence on concentrations of other nutrients by recognizing plant composition as a closed system whose components add up to one. New variables z _i are computed as logratioed values of individual nutrients, where each nutrient concentration is corrected for the geometric mean of all nutrient concentrations. The z _i are used together with principal component analysis (PCA) to relate celery composition to yield, deficiency symptoms and quality parameters. A survey of commercial celery fields suggested that (1) celery growth is most often limited by P and N deficiencies associated with Fe toxicity; (2) K uptake is most likely to become limiting when the crop reaches 15 cm in height; (3) blackheart incidence can be traced to low levels of K and Mg in external petioles, and (4) cracked stem incidence is related to low B when the crop is 30 cm in height.     

Studies on the mechanism of NADPH oxidation by the granule fraction isolated from human resting polymorphonuclear blood cells

Various factor affecting NADPH-oxidation by resting human leucocyte granules (LG) at acid pH, have been investigated.It was found that:

1) oxidation of NADPH by LG was increasingly inhibited by increased cyanide concentrations in the medium and was abolished by 4 mM cyanide.

2) with or without cyanide in the incubation medium, LG omitted, Mn⁺⁺, in the presence of NADPH induced superoxide anion (O¯₂) production, as evidenced by oxygen consumption and H₂O₂ production, which were abolished (in the absence of cyanide) by cytochrome C (a potent O¯₂ scavenger).

3) Both NADPH oxidation in the presence of 2 mM cyanide (cyanide-resistant) and in its absence (cyanide-sensitive) by LG occured only in the presence of Mn⁺⁺, and both were inhibited by superoxide dismutase.

4) Cyanide-resistant NADPH oxidation by LG generated H₂O₂, was inhibited by H₂O₂ and was not modified by «active catalase. The ratio of cyanide-resistant NADPH oxidation/O₂ uptake was 1 up to 1.25 mM NADPH, and increased above this concentration.

5) Cyanide-sensitive NADPH oxidation was inhibited by catalase and increased upon addition of H₂O₂. The ratio of cyanide-sensitive NADPH oxidation/O₂ uptake was 2.

It was concluded that after initiation by O¯₂, produced independently of LG, two sequential types of LG dependent NADPH oxidations occur. First, an O¯₂-dependent protein mediated NADPH oxidation (cyanide-resistant) which generates H₂O₂ and O¯₂ occurs. Second, NADPH peroxidation (cyanide-sensitive) which utilizes H₂O₂ takes place.     

Factors affecting tissue nutrient concentrations in aCarex meadow

Summary The influence of community and edaphic variables on tissue nutrient concentration was assessed for seven species on aCarex wetland in southern Quebec, Canada.Potassium and sodium tissue levels were considerably higher and Ca and Mg 35% lower than in a deciduous forest. Macronutrient concentrations decreased in the order K>N>Ca>Mg>Na>P. Micronutrient concentrations (Fe>Mn>Zn>Cu) ranged from 0.038–0.005 mg/g. This was 2–3 times less abundant than in an adjacentScirpus wetland. Inter-species coefficient of variation in N, P and K was low (14%) compared to variation in Ca, Mg, and the micronutrients (35%).Principal components analysis of interrelations between tissue elements indicated a clear distinction between N, P, K, Cu, Mn, and Zn levels and ash, Ca, Mg, Na, and Fe levels on the first component. This difference related closely to water depth and fire incidence. The coincidence of burning with water depth and the period of maximum snowmelt and runoff in the Spring suggested the loss of N, P, K, Cu, Mn and Zn by volotilization, runoff, or leaching.Stem density was the most important parameter influencing tissue N, P, and K concentrations whereas soil nitrogen levels were important in ash, Ca, and Mg concentrations. Water depth was the most important variable in the case of Cu, Fe, Mn, Na and Zn levels. Typha angustifolia had the highest level of total nutrients in green tissue,Carex lanuginosa the lowest. Principal components analysis indicated soil nitrogen, water depth, and soil potassium levels, in that order, were the three most important variables influencing the patterns of tissue element variation among species.Potassium and sodium levels in 1-year old litter were 11% and 0.4% compared to concentrations in green tissue. Iron and manganese, both subject to oxidation and adsorption to litter at the soil surface, were distinctly higher (2247% and 199%) in litter than green tissue. Concentrations of these and other elements in litter were consistent with results reported in literature and indicated litter was especially active as a site of cation exchange in the system.     

Altered plasma membrane H(+)-ATPase from the Dio-9-resistant pma1-2 mutant of Schizosaccharomyces pombe.

The pma1-2 mutation affecting the plasma membrane H(+)-ATPase of Schizosaccharomyces pombe has been selected for resistance to the antibiotic Dio-9. In membrane fractions purified from glucose-starved cells, the mutant ATPase activity is reduced by 96%, is insensitive to inhibition by vanadate and has a pH profile displaced in the acidic pH range when compared to the wild type. The maximum velocity of the H(+)-ATPase activity of plasma membranes from glucose-activated pma1-2 cells is activated 20-fold. This is in striking contrast with the wild-type ATPase activity, the maximal velocity of which is not affected by glucose. However, similar to the wild-type enzyme, glucose activation of the pma1-2 mutant H(+)-ATPase reduces the Km for MgATP 9-2 mM and shifts the optimal pH from 4.8 to 6.0-6.5. The pma1-2 mutation modifies Lys250 to a threonine, which is highly conserved in fungal and plant H(+)-ATPases. These results, compared to those reported for mutations of neighbour residues in yeast or mammalian P-type ATPases, suggest that Lys250 could play a significant role, not only in phosphate binding and/or in the E1P-E2P conformational isomerisation, but also in glucose activation of the H(+)-ATPase.     

Response to host age of the egg parasitoid Anaphes n.sp. (Hymenoptera: Mymaridae)

The mymarid Anaphes n.sp. was observed to parasitize the eggs of the carrot weevil Listronotus oregonensis (LeConte) up to 138 h old at 23±1°C and 18L:6D. Adult emergence of the parasitoid was observed in 85%‐97.5% of the weevil eggs 42–138 h old and host embryogenesis was interrupted in nearly 100% of these eggs. In host eggs of 18 h old, parasitism was very high but not all parasitoids completed their development and their subsequent emergence was generally retarded. An.sp. could parasitize host eggs 162 h old, but such an attack rarely retarded the normal development of the host. The parasitoid was unable to complete its development in sterile carrot weevil eggs.