Acetylcholinesterase in Mouse Neuroblastoma NB2A Cells: Analysis of Production, Secretion, and Molecular Forms

The mouse neuroblastoma cell line NB2A produces cellular and secreted acetylcholinesterase (AChE). After incubation of the cells for 4 days the ratio between AChE secreted into the medium and AChE in the cells was 1:1. The cell-associated enzyme could be subdivided into soluble AChE (25%) and detergent-soluble AChE (75%). Both extracts contained predominantly monomeric AChE (4.6S) and minor amounts of tetrameric AChE (10.6S), whereas the secreted AChE in the culture supernatant contained only the tetrameric form. All forms were partially purified by affinity chromatography. It could be demonstrated that the secretory and the intracellular soluble tetramers were hydrophilic, whereas the detergent-soluble tetramer was an amphiphilic protein. On the other hand the soluble and the detergent-soluble monomeric forms were amphiphilic and their activity depended on the presence of detergent. By digestion with proteinase K amphiphilic monomeric and tetrameric AChE could be converted to a hydrophilic form that no longer required detergent for catalytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]diisopropylfluorophosphate-labelled AChE gave one band at 64 kilodaltons (kD) under reducing conditions and two additional bands at 120 kD and 140 kD under nonreducing conditions.     

Diurnal variations of amino acids and organic acids in xylem fluid from Lagerstroemia indica: an endogenous circadian rhythm

Diurnal variations in the concentrations of major organic compounds occurred in xylem fluid extracted from Lagerstroemia indica L. The concentration of amino acids and the N/C ratio was at a maximum and that of organic acids was at a minimum between 1230 and 2030 h. Since the concentrations of total organic nitrogen, total amino acids and most individual amino acids (but not organic acids or sugars) were also proportional to xylem tension two experiments were performed to discern whether variations in chemistry were a consequence of diurnal changes in moisture stress. In the first experiment, L. indica , exposed to variable levels of moisture stress during midday, manifested an increase in organic acids and a reduction in the N/C ratio. In the second experiment, chemical profiles of xylem fluid were collected and compared for plants exposed to a natural photoperiod, constant darkness or continuous light at noon and midnight. After 1 day amino acids increased in concentration during midday for all treatments; the variation was greatest (10-fold) for plants in constant darkness where xylem tension varied from 0.20 to 0.25 MPa. Only plants exposed to continuous light lost a diurnal rhythm after 3 days. Thus, the circadian rhythm was endogenous, terminated in continuous light and was not mediated by changes in moisture stress. Glutamine accounted for most of the diurnal variation in total amino acids, organic nitrogen and the N/C ratio in xylem fluid.     

Interactions of acetylcholine receptor and acetylcholinesterase with lipid monolayers

The interaction of acetylcholine receptor and acetylcholinesterase with lipid monolayers was followed by measuring changes in surface pressure.When injected into the subphase of a lipid monolayer, the proteins caused increases in surface pressure from 5 to 10 dynes/cm, indicating a penetration of protein into the monolayer. At pH values below the isoelectric point of the proteins the incorporation was improved. The same was observed when Ca²⁺ (2 mM) was added.The presence of the enzyme in the mixed film could be demonstrated by using diiso[³H]propyl fluorophosphate-labelled acetylcholinesterase as well as by measuring enzyme activity. Acetylcholine receptor was shown to be present in the mixed film by using a complex made of the receptor and α-[³H]neurotoxin.     

The membrane-anchoring systems of vertebrate acetylcholinesterase and variant surface glycoproteins of African trypanosomes share a common antigenic determinant

Amphiphilic detergent-soluble acetylcholinesterase (AChE) from Torpedo is converted to a hydrophilic form by digestion with phospholipase C from Trypanosoma brucei or from Bacillus cereus. This lipase digestion uncovers an immunological determinant which crossreacts with a complex carbohydrate structure present in the hydrophilic form of all variant surface glycoproteins (VSG) of T. brucei. This crossreacting determinant is also detected in human erythrocyte AChE after digestion with T. brucei lipase. From these results we conclude that the glycophospholipid anchors of protozoan VSG and of AChE of the two vertebrates share common structural features, suggesting that this novel type of membrane anchor has been conserved during evolution.     

Different Glycosylation in Acetylcholinesterases from Mammalian Brain and Erythrocytes

Acetylcholinesterases (EC 3.1.1.7, AChE) have varying amounts of carbohydrates attached to the core protein. Sequence analysis of the known primary structures gives evidence for several asparagine-linked carbohydrates. From the differences in molecular mass determined on sodium dodecyl sulfate-polyacrylamide gel before and after deglycosylation with N-glycosidase F (EC 3.2.2.18), it is seen that dimeric AChE from red cell membranes is more heavily glycosylated than the tetrameric brain enzyme. Furthermore, dimeric and tetrameric forms of bovine AChE are more heavily glycosylated than the corresponding human enzymes. Monoclonal antibodies 2E6, 1H11, and 2G8 raised against detergent-soluble AChE from electric organs of Torpedo nacline timilei as well as Elec-39 raised against AChE from Electrophorus electricus cross-reacted with AChE from bovine and human brain but not with AChE from erythrocytes. Treatment of the enzyme with N-glycosidase F abolished binding of monoclonal antibodies, suggesting that the epitope, or part of it, consists of N-linked carbohydrates. Analysis of N-acetylglucosamine sugars revealed the presence of N-acetylglucosamine in all forms of cholinesterases investigated, giving evidence for N-linked glycosylation. On the other hand, N-acetylgalactosamine was not found in AChE from human and bovine brain or in butyrylcholinesterase (EC 3.1.1.8) from human serum, indicating that these forms of cholinesterase did not contain O-linked carbohydrates. Despite the notion that within one species, the different forms of AChE arise from one gene by different splicing, our present results show that dimeric erythrocyte and tetrameric brain AChE must undergo different postsynthetic modifications leading to differences in their glycosylation patterns.     

Active Sensing System with In Situ Adjustable Sensor Morphology

Background

Despite the widespread use of sensors in engineering systems like robots and automation systems, the common paradigm is to have fixed sensor morphology tailored to fulfill a specific application. On the other hand, robotic systems are expected to operate in ever more uncertain environments. In order to cope with the challenge, it is worthy of note that biological systems show the importance of suitable sensor morphology and active sensing capability to handle different kinds of sensing tasks with particular requirements.

Methodology

This paper presents a robotics active sensing system which is able to adjust its sensor morphology in situ in order to sense different physical quantities with desirable sensing characteristics. The approach taken is to use thermoplastic adhesive material, i.e. Hot Melt Adhesive (HMA). It will be shown that the thermoplastic and thermoadhesive nature of HMA enables the system to repeatedly fabricate, attach and detach mechanical structures with a variety of shape and size to the robot end effector for sensing purposes. Via active sensing capability, the robotic system utilizes the structure to physically probe an unknown target object with suitable motion and transduce the arising physical stimuli into information usable by a camera as its only built-in sensor.

Conclusions/Significance

The efficacy of the proposed system is verified based on two results. Firstly, it is confirmed that suitable sensor morphology and active sensing capability enables the system to sense different physical quantities, i.e. softness and temperature, with desirable sensing characteristics. Secondly, given tasks of discriminating two visually indistinguishable objects with respect to softness and temperature, it is confirmed that the proposed robotic system is able to autonomously accomplish them. The way the results motivate new research directions which focus on in situ adjustment of sensor morphology will also be discussed.     

Male- and female-specific variants of doublesex gene products have different roles to play towards regulation of Sex combs reduced expression and sex comb morphogenesis in Drosophila

Sexually dimorphic characters have two-fold complexities in pattern formation as they have to get input from both somatic sex determination as well as the positional determining regulators. Sex comb development in Drosophila requires functions of the somatic sex-determining gene doublesex and the homeotic gene Sex combs reduced. Attempts have not been made to decipher the role of dsx in imparting sexually dimorphic expression of SCR and the differential function of sex-specific variants of dsx products in sex comb development. Our results in this study indicate that male-like pattern of SCR expression is independent of dsx function, and dsx ^F must be responsible for bringing about dimorphism in SCR expression, whereas dsx ^M function is required with Scr for the morphogenesis of sex comb.     

Distribution of glycosylphosphatidylinositol-specific phospholipase D mRNA in bovine tissue sections

It has been reported that mammalian serum, and to a lower extent mammalian liver, brain, pancreas, udder, and milk, contain glycosylphosphatidylinositolspecific phospholipase D activity. However, the sites of synthesis have not been determined. In order to study in which cells(s) of the organism synthesis of glycosylphosphatidylinositol-specific phospholipase D takes place, we undertook a systematic screening of 12 different bovine tissues. In situ hybridization experiments with a specific anti-sense RNA probe, derived from a bovine liver cDNA, revealed that glycosylphosphatidylinositol-specific phospholipase D mRNA is present in mast cells of the adrenal gland, lung, and liver. On the other hand, our specific probe detected no mRNA in bovine pancreas, brain, and udder, although enzyme activity has been reported in these tissues. Northern blot analysis of total bovine liver RNA demonstrated two distinct glycosylphosphatidylinositol-specific phospholipse D mRNAs of approximately 3.3 kb and 4 kb length suggesting that two forms of the enzyme may exist.     

Two splice variants of protein kinase B gamma have different regulatory capacity depending on the presence or absence of the regulatory phosphorylation site serine 472 in the carboxyl-terminal hydrophobic domain

We have reported previously the cloning and characterization of human and mouse protein kinase B gamma (PKB gamma), the third member of the PKB family of second messenger-regulated serine/threonine kinases (Brodbeck, D., Cron, P., and Hemmings, B. A. (1999) J. Biol. Chem. 274, 9133--9136). Here we report the isolation of human and mouse PKB gamma 1, a splice variant lacking the second regulatory phosphorylation site Ser-472 in the hydrophobic C-terminal domain. Expression of PKB gamma 1 is low compared with PKB gamma, and it is regulated in different human tissues. We show that PKB gamma and PKB gamma 1 differ in their response to stimulation by insulin, pervanadate, peroxide, or okadaic acid. Activation of PKB gamma 1 requires phosphorylation at a single regulatory site Thr-305. Interestingly, this site is phosphorylated to a higher extent in PKB gamma compared with PKB gamma 1 upon maximal stimulation by pervanadate, and this is reflected in the respective specific kinase activities. Furthermore, upon insulin stimulation of transfected cells, PKB gamma 1 translocates to the plasma membrane to a lesser extent than PKB gamma. Taken together, these results suggest that phosphorylation of the hydrophobic motif at the extreme C terminus of PKB gamma may facilitate translocation of the kinase to the membrane and/or its phosphorylation on the activation loop site by phosphoinositide-dependent protein kinase-1.     

Anti-mouse GPI-PLD antisera highlight structural differences between murine and bovine GPI-PLDs

Despite its well characterised biochemistry, the physiological role of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is unknown. Most of the previous studies investigating the distribution of GPI-PLD have focused on the human and bovine forms of the enzyme. Studies on mouse GPI-PLD are rare, partly due to the lack of a specific anti-mouse GPI-PLD antibody, but also due to the apparent low reactivity of existing antibodies to rodent GPI-PLDs. Here we describe the isolation of a mouse liver cDNA, the construction and expression of a recombinant enzyme and the generation of an affinity-purified rabbit anti-mouse GPI-PLD antiserum. The antibody shows good reactivity to partially purified murine and purified bovine GPI-PLD. In contrast, a rat anti-bovine GPI-PLD antibody shows no reactivity with the mouse enzyme and the two antibodies recognise different proteolytic fragments of the bovine enzyme. Comparison between the rodent, bovine and human enzymes indicates that small changes in the amino acid sequence of a short peptide in the mouse and bovine GPI-PLDs may contribute to the different reactivities of the two antisera. We discuss the implications of these results and stress the importance of antibody selection while investigating GPI-PLD in the mouse.