Inherited and acquired interactions between ACHE and PON1 polymorphisms modulate plasma acetylcholinesterase and paraoxonase activities

The 5.5 Mb chromosome 7q21-22 ACHE/PON1 locus harbours the ACHE gene encoding the acetylcholine hydrolyzing, organophosphate (OP)-inhibitable acetylcholinesterase protein and the paraoxonase gene PON1, yielding the OP-hydrolyzing PON1 enzyme which also displays arylesterase activity. In search of inherited and acquired ACHE-PON1 interactions we genotyped seven polymorphic sites and determined the hydrolytic activities of the corresponding plasma enzymes and of the AChE-homologous butyrylcholinesetrase (BChE) in 157 healthy Israelis. AChE, arylesterase, BChE and paraoxonase activities in plasma displayed 5.4-, 6.5-, 7.2- and 15.5-fold variability, respectively, with genotype-specific differences between carriers of distinct compound polymorphisms. AChE, BChE and arylesterase but not paraoxonase activity increased with age, depending on leucine at PON1 position 55. In contrast, carriers of PON1 M55 displayed decreased arylesterase activity independent of the - 108 promoter polymorphism. Predicted structural consequences of the PON1 L55M substitution demonstrated spatial shifts in adjacent residues. Molecular modelling showed substrate interactions with the enzyme variants, explaining the changes in substrate specificity induced by the Q192R substitution. Intriguingly, PON1, but not BChE or arylesterase, activities displayed inverse association with AChE activity. Our findings demonstrate that polymorphism(s) in the adjacent PON1 and ACHE genes affect each other's expression, predicting for carriers of biochemically debilitating ACHE/PON1 polymorphisms adverse genome-environment interactions.     

Limited left-right cell migration across the midline of the gastrulating avian embryo

During avian development the earliest phase in which the avian embryo expresses axial features of a left-right axis is at the primitive streak stage. Until the stage of definitive primitive streak (streak 4 H&H), the axis seems to possess morphological bilateral symmetry. Morphological asymmetry begins only during the next few hours of incubation, with development of overt morphological and molecular asymmetry within Hensen's node (stage 5 H&H). In this report, we present an experimental study aimed at following the pattern of cell movements during primitive streak formation and gastrulation of specific left-right regions from earlier stages of the avian embryo. To determine the origin of cells contributing to each side of the primitive streak, we applied the dye Lysinated-Rodamine-Dextran (LRD) to one half, either left or right, of the pre-streak blastoderm (stages X–XIII, EG&K). We tried to estimate the relative cell contribution to primitive streak formation, and to the three germ layers evolving during gastrulation in the context of the left-right axis. Moreover, we asked whether the midline serves as a border, that is, as a physiological barrier preventing cell passing during gastrulation. Our results demonstrate that on each side of the axis, either the right or the left, most of the cells originate from the same half of a pre-streak blastoderm, populate the same half of the PS and contribute to tissues largely confined to that particular side. However, along the primitive streak, a few cells were detected on the opposite side of the midline. Moreover, variation in the number of cells crossing the midline at specific regions along the primitive streak was found. Most crossing cells were located near the mid rostrocaudal extent of the primitive streak, from 25–85% of its length. At the posterior end of the primitive streak, fewer crossing cells were detected. At the anterior region of the PS, that is, within Hensen's node, cells do not cross the midline. These results suggest that differences occur in the process of ingression along the rostrocaudal extent of the PS. Dev. Genet. 23:175–184, 1998. © 1998 Wiley-Liss, Inc.     

Synthetic biology approach to reconstituting the ubiquitylation cascade in bacteria

Covalent modification of proteins with ubiquitin (Ub) is widely implicated in the control of protein function and fate. Over 100 deubiquitylating enzymes rapidly reverse this modification, posing challenges to the biochemical and biophysical characterization of ubiquitylated proteins. We circumvented this limitation with a synthetic biology approach of reconstructing the entire eukaryotic Ub cascade in bacteria. Co‐expression of affinity‐tagged substrates and Ub with E1, E2 and E3 enzymes allows efficient purification of ubiquitylated proteins in milligram quantity. Contrary to in‐vitro assays that lead to spurious modification of several lysine residues of Rpn10 (regulatory proteasomal non‐ATPase subunit), the reconstituted system faithfully recapitulates its monoubiquitylation on lysine 84 that is observed in vivo. Mass spectrometry revealed the ubiquitylation sites on the Mind bomb E3 ligase and the Ub receptors Rpn10 and Vps9. Förster resonance energy transfer (FRET) analyses of ubiquitylated Vps9 purified from bacteria revealed that although ubiquitylation occurs on the Vps9‐GEF domain, it does not affect the guanine nucleotide exchanging factor (GEF) activity in vitro. Finally, we demonstrated that ubiquitylated Vps9 assumes a closed structure, which blocks additional Ub binding. Characterization of several ubiquitylated proteins demonstrated the integrity, specificity and fidelity of the system, and revealed new biological findings.     

Delineating the Effect of Semantic Congruency on Episodic Memory: The Role of Integration and Relatedness

A fundamental challenge in the study of learning and memory is to understand the role of existing knowledge in the encoding and retrieval of new episodic information. The importance of prior knowledge in memory is demonstrated in the congruency effect—the robust finding wherein participants display better memory for items that are compatible, rather than incompatible, with their pre-existing semantic knowledge. Despite its robustness, the mechanism underlying this effect is not well understood. In four studies, we provide evidence that demonstrates the privileged explanatory power of the elaboration-integration account over alternative hypotheses. Furthermore, we question the implicit assumption that the congruency effect pertains to the truthfulness/sensibility of a subject-predicate proposition, and show that congruency is a function of semantic relatedness between item and context words.     

Rapid generation of knockdown transgenic mice by silencing lentiviral vectors

Lentiviral vectors are potent gene delivery vehicles that enable stable expression of transgenes in both dividing and postmitotic cells, including preimplantation embryos. We have developed lentiviral vectors carrying silencing cassettes consisting of an RNA polymerase III promoter expressing short hairpin RNAs. Transgenic mice can be generated rapidly by transduction of early embryos with lentiviral silencing vectors, resulting in mice with downregulated target genes. We describe two alternative early embryo transduction protocols (removal of zona pellucida and subzonal microinjection). These methodologies offer the possibility of large-scale generation of knockdown transgenic mice for functional genomic studies and enable the production of transgenic mice in 7 weeks.     

Pre-steady-state decoding of the Bicoid morphogen gradient

Morphogen gradients are established by the localized production and subsequent diffusion of signaling molecules. It is generally assumed that cell fates are induced only after morphogen profiles have reached their steady state. Yet, patterning processes during early development occur rapidly, and tissue patterning may precede the convergence of the gradient to its steady state. Here we consider the implications of pre-steady-state decoding of the Bicoid morphogen gradient for patterning of the anterior–posterior axis of the Drosophila embryo. Quantitative analysis of the shift in the expression domains of several Bicoid targets (gap genes) upon alteration of bcd dosage, as well as a temporal analysis of a reporter for Bicoid activity, suggest that a transient decoding mechanism is employed in this setting. We show that decoding the pre-steady-state morphogen profile can reduce patterning errors caused by fluctuations in the rate of morphogen production. This can explain the surprisingly small shifts in gap and pair-rule gene expression domains observed in response to alterations in bcd dosage.     

Assessing diversity and biogeography of aerobic anoxygenic phototrophic bacteria in surface waters of the Atlantic and Pacific Oceans using the Global Ocean Sampling expedition metagenomes

Aerobic anoxygenic photosynthetic bacteria (AAnP) were recently proposed to be significant contributors to global oceanic carbon and energy cycles. However, AAnP abundance, spatial distribution, diversity and potential ecological importance remain poorly understood. Here we present metagenomic data from the Global Ocean Sampling expedition indicating that AAnP diversity and abundance vary in different oceanic regions. Furthermore, we show for the first time that the composition of AAnP assemblages change between different oceanic regions, with specific bacterial assemblages adapted to open ocean or coastal areas respectively. Our results support the notion that marine AAnP populations are complex and dynamic, and compose an important fraction of bacterioplankton assemblages in certain oceanic areas.     

ACETOACETATE AND d-(-)-BETA-HYDROXYBUTYRATE AS PRECURSORS FOR STEROL SYNTHESIS BY CALF OLIGODENDROCYTES IN SUSPENSION CULTURE: EXTRAMITOCHONDRIAL PATHWAY FOR ACETOACETATE METABOLISM

Abstract— Oligodendroglia prepared from calf cerebral white matter were incubated with [3^-14C]aceto-acetate (AcAc), d -[3^-14C](-)-beta-hydroxybutyrate (OHbut) or ³H₂O. Addition to the medium of the ATP-rate oxaloacetate lyase inhibitor (-)-hydroxycitrate diminished sterol labelling from OHbut and ³H₂O, but caused 4-fold stimulation of sterol labelling from AcAc. The discrepancy between results with the two ketone bodies indicates the existence in oligodendroglia of an extrarnitochondrial pathway for conversion of AcAc but not OHbut to acetyl CoA. Acetoacetyl CoA synthetase, the first enzyme in this pathway, was more than 20-fold enriched in oligodendroglia over whole brain.     

Nutrient study for the transition from earthen sedimentation ponds to ones lined with pvc in integrated mariculture systems, what needs to be done?

Stable diatom populations in earthen ponds for fishpond effluent treatment supported fast rates of bivalve growth in integrated mariculture systems. However, when these ponds were lined with PVC plastic sheets to prevent seepage, the populations of benthic diatoms dwindled, and did not support, as before, a commercially acceptable rate of growth by oysters and clams. Experiments were undertaken to understand the problem and restore the diatom productivity of such ponds. Clones of Amphora luciae, A,cftenerrima, Cylindrotheca closterium, Navicula cf lineola, N.cf lenzii, N. salinicola, N. cf viminoides, and Nitzschia laevis were isolated in axenic culture from an earthen sedimentation pond. Their N, P, Si, and trace element requirements for growth in fully defined media, and in media formulated with mariculture effluent, were studied in axenic batch culture. In fully defined batch culture tests, most of the isolates achieved their highest density in media with 32 M P, 0.7 mM N, 20 M Fe, 10 nM–20 M Mn, 10–20 M Zn and Co, and 17.5 M Si. Enrichment by trace elements and Si stimulated the populations of these diatoms even in media based on nutrient-enriched mariculture effluents. However, in large flow-through agnotobiotic mesocosms (700 L), only Si enrichment was needed. Si concentration >100 M was required to promote the sustained blooms of diatoms in full-sized and commercial PVC-lined fishpond effluent treatment ponds (300 m², 1 m depth). Except for Si, the requirements of the diatoms for micronutrients were apparently fully satisfied by the fishpond effluents (uneaten food and fish-waste). A molar ratio of 1:1 between Si and N is necessary to sustained dense diatom populations in the pond water. It is therefore recommended to enrich plastic lined mariculture effluent treatment/sedimentation ponds with Si, if the goal is to raise bivalves as a secondary crop.