Reduced [3H]Cyclic AMP Binding in Postmortem Brain from Subjects with Bipolar Affective Disorder

Abstract: Findings of increased G_sα levels and forskolin-stimulated adenylyl cyclase activity in selective cerebral cortical postmortem brain regions in bipolar affective disorder (BD) implicate increased cyclic AMP (cAMP)-mediated signaling in this illness. Accumulating evidence suggests that intracellular levels of cAMP modulate the abundance and disposition of the regulatory subunits of cAMP-dependent protein kinase (cAMP-dPK). Thus, in the present study, we tested further whether hyperfunctional G_sα-linked cAMP signaling occurs in BD by determining [³H]cAMP binding, a measure of the levels of regulatory subunits of cAMP-dPK, in cytosolic and membrane fractions from discrete brain regions of postmortem BD brain. Specific [³H]cAMP (5 n M ) binding was determined in autopsied brain obtained from 10 patients with DSM-III-R diagnoses of BD compared with age- and postmortem delay-matched controls. [³H]cAMP binding was significantly reduced across all brain regions in cytosolic fractions of BD frontal (−22%), temporal (−23%), occipital (−22%) and parietal (−15%) cortex, cerebellum (−36%), and thalamus (−13%) compared with controls, but there were no differences in [³H]cAMP binding in the membrane fractions from these same regions. These results suggest that changes occur in the cAMP-dPK regulatory subunits in BD brain, possibly resulting from increased cAMP signaling. The possibility that antemortem lithium and/or other mood stabilizer treatment may contribute to the above changes, however, cannot be ruled out.     

N2 Fixation (C2H2-Reducing Activity) and Leghaemoglobin Content during Nitrate- and Water-Stress-Induced Senescence of Medicago sativa Root Nodules

Nitrate and water stress were used to induce senescence in rootnodules of alfalfa (Medicago sativa L. cv. Aragon). Nodule senescencewas assessed by determinations of the nitrogenase (C₂H₂-reducing)activity, and the leghaemoglobin (LHb) and total soluble proteincontents of the nodules. Nodules responded similarly to and water stress in many respects, but there was a significant difference.All parameters of nodule activity, expressed on the basis ofnodule dry weight (DW), consistently decreased following treatmentwith or during drought; there was a significant interaction (synergism) between the inhibitory effects of and water stress on nitrogenase activity, but sucheffects were merely additive in the case of LHb content or LHb/solubleprotein ratio. However, caused the selective decay of LHb with respect to other nodular soluble proteins,whereas the decrease of LHb during water stress was due to ageneral inhibition of protein synthesis and to an increasedproteolytic activity in the nodule cytosol rather than to aspecific proteolysis of LHb. Key words: Leghaemoglobin, Medicago saliva, nitrogen fixation, root nodule senescence, water stress     

ATP-Dependent Glutamate Uptake into Synaptic Vesicles from Cerebellar Mutant Mice

The ATP-dependent glutamate uptake system in synaptic vesicles prepared from mouse cerebellum was characterized, and the levels of glutamate uptake were investigated in the cerebellar mutant mice, staggerer and weaver, whose main defect is the loss of cerebellar granule cells, and the nervous mutant, whose main defect is the loss of Purkinje cells. The ATP-dependent glutamate uptake is stimulated by low concentrations of chloride, is insensitive to aspartate, and is inhibited by agents known to dissipate the electrochemical proton gradient. These properties are similar to those of the glutamate uptake system observed in the highly purified synaptic vesicles prepared from bovine cortex. The ATP-dependent glutamate uptake system is reduced by 68% in the staggerer and 57-67% in the weaver mutant; these reductions parallel the substantial loss of granule cells in those mutants. In contrast, the cerebellar levels of glutamate uptake are not altered significantly in the nervous mutant, which has lost Purkinje cells, but not granule cells. In view of evidence that granule cells are glutamatergic neurons and Purkinje cells are GABAergic neurons, these observations support the notion that the ATP-dependent glutamate uptake system is present in synaptic vesicles of glutamatergic neurons.     

Autophagy regulates cytoplasmic remodeling during cell reprogramming in a zebrafish model of muscle regeneration

Cell identity involves both selective gene activity and specialization of cytoplasmic architecture and protein machinery. Similarly, reprogramming differentiated cells requires both genetic program alterations and remodeling of the cellular architecture. While changes in genetic and epigenetic programs have been well documented in dedifferentiating cells, the pathways responsible for remodeling the cellular architecture and eliminating specialized protein complexes are not as well understood. Here, we utilize a zebrafish model of adult muscle regeneration to study cytoplasmic remodeling during cell dedifferentiation. We describe activation of autophagy early in the regenerative response to muscle injury, while blocking autophagy using chloroquine or Atg5 and Becn1 knockdown reduced the rate of regeneration with accumulation of sarcomeric and nuclear debris. We further identify Casp3/caspase 3 as a candidate mediator of cellular reprogramming and Fgf signaling as an important activator of autophagy in dedifferentiating myocytes. We conclude that autophagy plays a critical role in cell reprogramming by regulating cytoplasmic remodeling, facilitating the transition to a less differentiated cell identity.     

Subcellular localization of flavonoid synthesizing enzymes in Pisum,Phaseolus, Brassica and Spinacia cultivars

Functionally-intact chloroplasts were obtained from 11-day-old pea (Pisum sativum cv Midfreezer) seedlings. Enzyme-distribution studies with ribulose bisphosphate carboxylase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase showed that ca 2.1% of the total tissue chloroplasts were present in the chloroplast preparation. The rate of intactness of chloroplast preparations was 34–82%. SAM:caffeic acid methyltransferase, flavanone synthase, UDPG:flavonoid-3-O-glucosyltransferase and SAM:quercetin methyltransferase activities were measured in the homogenate, supernatant and chloroplast lysate fractions. Significant activities of the above four enzymes could only be detected in the homogenate and supernatant fractions. Similar experiments with 11-day-old seedlings of green beans (Phaseolus vulgaris cv Early Gallatin), red cabbage (Brassica oleracea cv Red Danish) and 6-week-old plants of spinach (Spinacia oleracea cv Bloomsdale) showed a similar distribution of the flavonoid synthesizing enzymes. We conclude that under the reported conditions chloroplasts are not involved in flavonoid biosynthesis.     

Herbicidal effects under field conditions of Ailanthus altissima bark extract,which contains ailanthone

Extracts of Ailanthus altissima stem bark were evaluated for herbicidal effects under field conditions in two outdoor trials. Previous investigations had shown A. altissima bark, extracted with methanol, yielded a strongly phytotoxic extract that contained ailanthone as one of the major herbicidal compounds. The first field trial investigated the level of activity and selectivity of the extract. A. altissima bark extract was sprayed post-emergence onto 17 species of weeds and crops at rates of 366, 177, 93, 47, 23, and 0 kg ha^–1. These application rates provided herbicidal activity equivalent to 4.5, 2.2, 1.1, 0.6, 0.3, and 0.0 kg of pure ailanthone per hectare, based on the results of a laboratory bioassay of extract and pure ailanthone. Strong herbicidal effects were observed within several days. Even the lowest rate caused mortality and injury in excess of 50% for nine of the 17 species, and a significant reduction in shoot biomass for 13 species. The second field trial tested the ability of bark extract to control weeds under field conditions with horticultural crops (bush bean, cauliflower, sweet corn, tomato). A. altissima bark extract was sprayed post-emergence at rates of 99, 50, 26, 13, and 0 kg ha^–1, providing herbicidal activity equivalent to 1.1, 0.6, 0.3, 0.14, and 0.0 kg of pure ailanthone per hectare. Extract treatment provided partial weed control (greatest reduction in weed biomass was 40%), but also caused serious crop injury. Bush bean was the only crop that showed a significant increase in shoot biomass and fruit yield, compared to the non-weeded control. None of the crops, regardless of application rate, showed a level of shoot biomass or fruit yield comparable to the hand-weeded control. The herbicidal effects of A. altissima bark extract declined within the first few weeks after application, supporting previous evidence that ailanthone is rapidly degraded under field conditions.     

Morphology agrees with molecular data: phylogenetic affinities of Euptychiina butterflies (Nymphalidae: Satyrinae)

Euptychiina is the most species‐rich subtribe of Neotropical Satyrinae, with over 450 known species in 47 genera (14 monotypic). Here, we use morphological characters to examine the phylogenetic relationships within Euptychiina. Taxonomic sampling included 105 species representing the majority of the genera, as well as five outgroups. A total of 103 characters were obtained: 45 from wing pattern, 48 from genitalia and 10 from wing venation. The data matrix was analysed using maximum parsimony under both equal and extended implied weights. Euptychiina was recovered as monophyletic with ten monophyletic genera, contrasting previous DNA sequence‐based phylogenies that did not recover the monophyly of the group. In agreement with sequence‐based hypotheses, however, three main clades were recognized: the ‘Megisto clade’ with six monophyletic and three polyphyletic genera, the ‘Taygetis clade’ with nine genera of which three were monophyletic, and the ‘Pareuptyhia clade’ with four monophyletic and two polyphyletic genera. This is the first morphology‐based phylogenetic hypothesis for Euptychiina and the results will be used to complement molecular data in a combined analysis and to provide critical synapomorphies for clades and genera in this taxonomically confused group.     

Plant phenylacetaldehyde synthase is a bifunctional homotetrameric enzyme that catalyzes phenylalanine decarboxylation and oxidation

We have isolated and characterized Petunia hybrida cv. Mitchell phenylacetaldehyde synthase (PAAS), which catalyzes the formation of phenylacetaldehyde, a constituent of floral scent. PAAS is a cytosolic homotetrameric enzyme that belongs to group II pyridoxal 5'-phosphate-dependent amino-acid decarboxylases and shares extensive amino acid identity (approximately 65%) with plant L-tyrosine/3,4-dihydroxy-L-phenylalanine and L-tryptophan decarboxylases. It displays a strict specificity for phenylalanine with an apparent Km of 1.2 mM. PAAS is a bifunctional enzyme that catalyzes the unprecedented efficient coupling of phenylalanine decarboxylation to oxidation, generating phenylacetaldehyde, CO2, ammonia, and hydrogen peroxide in stoichiometric amounts.     

Phenyltriazolinones as potent factor Xa inhibitors

We have discovered that phenyltriazolinone is a novel and potent P₁ moiety for coagulation factor Xa. X-ray structures of the inhibitors with a phenyltriazolinone in the P₁ position revealed that the side chain of Asp189 has reoriented resulting in a novel S₁ binding pocket which is larger in size to accommodate the phenyltriazolinone P₁ substrate.     

Carbon and nitrogen content based estimation of the fat content of animal carcasses in various species

The objective of this study was to explore whether the C and N content can be used to estimate the fat content of animal carcasses. Considering the mean C and N contents of body fat and body protein, the fat content (EE) [%] can be predicted from C and N values [%] according to the generally valid equation EE=1.3038·C – 4.237·N. The application of this equation to estimate the total fat content of all animal carcasses results in significant differences in fat content between predicted and measured values. Therefore, we derived specific equations for rats, pigs, cattle, sheep, broilers and mice to predict the fat content by dual linear regression analysis (y=EE [% DM], x₁=C [% DM], x₂=N [% DM]) based on measured fat, C, and N contents of animal body samples. The specific equations for different animals showed residual standard deviations of 1.55, 1.63, 1.12, 1.35, 1.85 and 0.92% fat for rats, pigs, cattle, sheep, broilers and mice, respectively.