Acetyl-CoA Synthesizing Enzymes in Cholinergic Nerve Terminals

The activities of five enzymes involved in acetyl-CoA synthesis, pyruvate dehydrogenase complex, ATP citrate lyase, carnitine acetyltransferase, acetyl-CoA synthetase, and citrate synthase, were determined in normal nucleus interpeduncularis and nucleus interpeduncularis in which cholinergic terminals were removed following lesion of the habenulointerpeduncular tract. The activities of aspartate transaminase, fumarase, and GABA transaminase also were determined to compare the effect of lesion on other mitochondrial enzymes which are not linked to the biosynthesis of ACh. In normal nucleus interpeduncularis the activities of carnitine acetyltransferase and pyruvate dehydrogenase complex were higher than the activity of ChAT (choline acetyltransferase), whereas the activities of acetyl-CoA synthetase and citrate synthase were considerably lower than that of ChAT. The effect of the lesion separated the enzymes into two groups: the activities of pyruvate dehydrogenase complex, carnitine acetyltransferase, fumarase and aspartate transaminase decreased by 30--40%, whereas the activities of the other enzymes descreased 5--15%. ChAT activity was in all cases less than 15% of normal. It could be concluded that none of the acetyl-CoA synthesizing enzymes decreased to the degree that ChAT did. Only pyruvate dehydrogenase complex and carnitine acetyltransferase seem to be localized in cholinergic terminals to a significant degree. ATP citrate lyase as well as acetyl-CoA synthetase seem to have less significance in supporting acetyl-CoA formation in cholinergic nerve terminals.     

Quinacrine mustard fluorescence of a second Y chromosome in a Y-autosomal translocation

Summary Screening buccal smears from 97 prisoners by the quinacrine mustard technique revealed one XYY-individual and one Y-autosomal translocation of a second Y chromosome with a 46,XY, D-,t (?15q;Yq)+ karyotype. The translocation chromosome could be identified by its intense fluorescence of the short arm in all 75 metaphases examined.

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Zusammenfassung Untersuchungen von Abstrichen der Mundschleimhaut von 97 Gefängnisinsassen mit der Quinacrine-Mustard-Methode führten zur Aufdeckung eines XYY-Karyotyps und einer Y-autosomalen Translokation eines zweiten Y-Chromosoms mit einem Karyotyp von 46,XY,D-,t(?15q;Yq)+. Das Translokationschromosom konnte durch helle Fluorescenz des kurzen Armes in allen 75 Metaphasen identifiziert werden.

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This work was supported in part by grant number Pa 118/8 from the Deutsche Forschungsgemeinschaft and is part of a thesis by S. F.     

Dimerization of DNA methyltransferase 1 is mediated by its regulatory domain

DNA methylation is a major epigenetic modification and plays a crucial role in the regulation of gene expression. Within the family of DNA methyltransferases (Dnmts), Dnmt3a and 3b establish methylation marks during early development, while Dnmt1 maintains methylation patterns after DNA replication. The maintenance function of Dnmt1 is regulated by its large regulatory N‐terminal domain that interacts with other chromatin factors and is essential for the recognition of hemi‐methylated DNA. Gelfiltration analysis showed that purified Dnmt1 elutes at an apparent molecular weight corresponding to the size of a dimer. With protein interaction assays we could show that Dnmt1 interacts with itself through its N‐terminal regulatory domain. By deletion analysis and co‐immunoprecipitations we mapped the dimerization domain to the targeting sequence TS that is located in the center of the N‐terminal domain (amino acids 310–629) and was previously shown to mediate replication independent association with heterochromatin at chromocenters. Further mutational analyses suggested that the dimeric complex has a bipartite interaction interface and is formed in a head‐to‐head orientation. Dnmt1 dimer formation could facilitate the discrimination of hemi‐methylated target sites as has been found for other palindromic DNA sequence recognizing enzymes. These results assign an additional function to the TS domain and raise the interesting question how these functions are spatially and temporarily co‐ordinated. J. Cell. Biochem. 106: 521–528, 2009. © 2009 Wiley‐Liss, Inc.     

Comparative efficacy of two controlled-release gypsy moth mating disruption formulations

The effects of aerial applications of the gypsy moth sex pheromone, disparlure, on mating disruption and suppression of growth of populations of the gypsy moth, Lymantria dispar (L.), were investigated. Two formulations of disparlure, plastic laminate flakes applied in a single application and polymethacrylate beads applied in two applications, were compared in two separate tests conducted in 1993 and 1994. The beads were applied in two applications spaced 2 weeks apart because preliminary tests had indicated that they released pheromone too rapidly to maintain adequate emission rates throughout the period of male flight. In 1993, the flakes were applied at a rate of 50 g a.i./ha, and the beads were applied at a rate of 15 g a.i./ha for each application. In 1994, the flakes were applied at a rate of 75 g a.i./ha and the beads were applied at rates of 32.5 and 42.5 g a.i./ha for the two applications. Beads with larger average particle size were used in 1994 to prolong disparlure release. The treatments applied in 1993 resulted in >97% reduction in mating and >82% suppression of population growth in the following year. Because of a 1995 collapse of gypsy moth populations in the vicinity of the tests, reliable population growth data were not available for the treatments applied in 1994, but significant mating disruption did occur under both treatments. Based on measurements of residual disparlure after field aging, the flakes released 32 and 48% of their disparlure content during the 6 weeks of male moth flight in 1993 and 1994, respectively. The smaller beads used in 1993 released 75% of their disparlure content, and the larger beads used in 1994 released 52% of their disparlure content, during the 6 weeks of male flight. The biological efficacy data suggest that the bead and flake formulations, as applied in these tests, have similar effects on gypsy moth mating disruption and subsequent population growth. Based on the observed release rates from both 1993 and 1994, a single application of the beads would provide emission rates equal to or greater than those provided by the flakes when applied at an equal dose.     

Pulsed extremely low-frequency magnetic fields stimulate microvesicle release from human monocytic leukaemia cells

Microvesicles are released from cell surfaces constitutively during early apoptosis or upon activation with various stimuli including sublytic membrane attack complex (MAC). This study shows that an alternating current, pulsed, extremely low-frequency electromagnetic field (0.3 μT at 10 Hz, 6 V AC) induced transient plasma membrane damage that allowed calcium influx. This in turn caused a release of stimulated microvesicles (sMV). When extracellular calcium was chelated with EGTA, sMV biogenesis initiated by ELFMF was markedly reduced and the reduction was less than when the stimulation was the deposition of sublytic MAC. This suggested that pulsed ELFMF resulted in transcellular membrane pores causing organelles to leak additional calcium into the cytoplasm (which EGTA would not chelate) which itself can lead to sMV release.     

BDNF and its pro-peptide are stored in presynaptic dense core vesicles in brain neurons

Although brain-derived neurotrophic factor (BDNF) regulates numerous and complex biological processes including memory retention, its extremely low levels in the mature central nervous system have greatly complicated attempts to reliably localize it. Using rigorous specificity controls, we found that antibodies reacting either with BDNF or its pro-peptide both stained large dense core vesicles in excitatory presynaptic terminals of the adult mouse hippocampus. Both moieties were ~10-fold more abundant than pro-BDNF. The lack of postsynaptic localization was confirmed in Bassoon mutants, a seizure-prone mouse line exhibiting markedly elevated levels of BDNF. These findings challenge previous conclusions based on work with cultured neurons, which suggested activity-dependent dendritic synthesis and release of BDNF. They instead provide an ultrastructural basis for an anterograde mode of action of BDNF, contrasting with the long-established retrograde model derived from experiments with nerve growth factor in the peripheral nervous system.     

Analysis of centriole elimination during C. elegans oogenesis

Centrosomes are the principal microtubule organizing centers (MTOCs) of animal cells and comprise a pair of centrioles surrounded by pericentriolar material (PCM). Centriole number must be carefully regulated, notably to ensure bipolar spindle formation and thus faithful chromosome segregation. In the germ line of most metazoan species, centrioles are maintained during spermatogenesis, but eliminated during oogenesis. Such differential behavior ensures that the appropriate number of centrioles is present in the newly fertilized zygote. Despite being a fundamental feature of sexual reproduction in metazoans, the mechanisms governing centriole elimination during oogenesis are poorly understood. Here, we investigate this question in C. elegans. Using antibodies directed against centriolar components and serial-section electron microscopy, we establish that centrioles are eliminated during the diplotene stage of the meiotic cell cycle. Moreover, we show that centriole elimination is delayed upon depletion of the helicase CGH-1. We also find that somatic cells make a minor contribution to this process, and demonstrate that the germ cell karyotype is important for timely centriole elimination. These findings set the stage for a mechanistic dissection of centriole elimination in a metazoan organism.     

Plant peroxisomes: biogenesis and function

Peroxisomes are eukaryotic organelles that are highly dynamic both in morphology and metabolism. Plant peroxisomes are involved in numerous processes, including primary and secondary metabolism, development, and responses to abiotic and biotic stresses. Considerable progress has been made in the identification of factors involved in peroxisomal biogenesis, revealing mechanisms that are both shared with and diverged from non-plant systems. Furthermore, recent advances have begun to reveal an unexpectedly large plant peroxisomal proteome and have increased our understanding of metabolic pathways in peroxisomes. Coordination of the biosynthesis, import, biochemical activity, and degradation of peroxisomal proteins allows for highly dynamic responses of peroxisomal metabolism to meet the needs of a plant. Knowledge gained from plant peroxisomal research will be instrumental to fully understanding the organelle's dynamic behavior and defining peroxisomal metabolic networks, thus allowing the development of molecular strategies for rational engineering of plant metabolism, biomass production, stress tolerance, and pathogen defense.