EDI3 links choline metabolism to integrin expression,cell adhesion and spreading

Endometrial carcinoma differential 3 (EDI3) was the first member of the glycerophosphodiesterase (GDE) protein family shown to be associated with cancer. Our initial work demonstrated that endometrial and ovarian cancer patients with primary tumors overexpressing EDI3 had a higher risk of developing metastasis and decreased survival. Further analysis indicated that EDI3 cleaves glycerophosphocholine to choline and glycerol-3-phosphate, increases the levels of active PKC, and enhances the migratory activity of tumor cells. Despite these initial findings, EDI3 remained mainly uncharacterized. Therefore, to obtain an overview of processes in which EDI3 may be involved, gene array analysis was performed using MCF-7 breast cancer cells after EDI3 knockdown compared with a non-targeting control siRNA. Several biological motifs were altered, including an enrichment of genes involved in integrin-mediated signaling. More specifically, silencing of EDI3 in MCF-7 and OVCAR-3 cells was associated with reduced expression of the key receptor subunit integrin β1, leading to decreased cell attachment and spreading accompanied by delayed formation of cell protrusions. To confirm these results, we stably overexpressed EDI3 in MCF-7 cells which led to elevated integrin β1 expression associated with enhanced cell attachment and spreading - two processes critical for metastasis. In conclusion, our data provide further insight into the role of EDI3 during cancer progression.     

Choline Uptake by the Neuroblastoma X Glioma Hybrid, NG108-15

The neuroblastoma X glioma hybrid clone NG108-15 is able to release acetylcholine upon depolarization and form cholinergic neuromuscular synapses in culture. Normal functioning of cholinergic synapses is thought to be dependent on the ability of a neuron to take up extracellular choline, since neurons are unable to synthesize choline de novo. For these two reasons it became important to characterize the choline uptake system of NG108-15 cells. The uptake system appears to bear little if any resemblance to the Na⁺-dependent high-affinity choline uptake system normally associated with cholinergic neurons. Although the cells appear to possess both high- and low- affinity choline uptake systems, neither system is dependent on Na⁺ and uptake actually is increased about 60% by the substitution of sucrose for NaCl. Acetylcholine synthesis also is not dependent on Na⁺, since sucrose, substituted for NaCl, also stimulates acetylcholine synthesis. Changes in the concentrations of the other ions in the uptake medium have little effect on uptake, with the exception that elevated Ca²⁺ or Mg²⁺ reverses the stimulation of choline uptake produced by substitution of sucrose for NaCl. Choline uptake is inhibited by hemicholinium-3, but only at high concentrations of the drug (IC₅₀= 30–80 μm ). The metabolic poisons cyanide and iodoacetate inhibit uptake by only 30-40%. Growth of the cells in N⁶,O^2′ dibutyryladenosine-3′,5′-cyclic monoposphate, which promotes functional and morphological differentiation of the cells, decreased slightly the total amount of choline taken up but had no additional effect on the uptake system. Thus, it appears that NG108-15 cells are capable of forming functional cholinergic synapses with muscle cells even though the neuroblastoma does not possess the high-affinity choline uptake system normally associated with cholinergic neurons.     

A radiometric microassay for choline acetyltransferase. Some observations on the spinal cord of the chicken embryo

This paper describes cation-exchange methods for separating acetyl[³H] coenzyme A from [acetyl-³H]choline. Blanks for the routine method were approximately 0.05% of the substrate radioactivity; product recoveries were approximately 97%. The cation-exchange method was moreefficient than the standard methods using either anion-exchange chromatography or periodide precipitation. The cation-exchange method was also morespecific than either of the other two standard methods for estimating choline acetyltransferase (ChAT) activity. ChAT activity was detected in the chicken lumbar spinal cord on embryonic day (E) 2 1/4 with the cation-exchange method. This developmental stage is about 6 hours before the final mitosis of any neuroblast in the ventral horn. Total ChAT activity per lumbar spinal cord increased more than 10,000-fold between E 3 and E 18. Changes in ChAT activity in the lumbar spinal cord following limb-bud extirpation appeared to mirror (with a phase lag) the changes in the number of motoneurons in the lateral motor column.     

Investigations on the effects of rape oil quality,choline and methionine concentration in diets for laying hens on the trimethylamine content of the eggs,on trimethylamine metabolism and on laying performance

Abstract

An experiment was conducted to study the effects of graded levels of choline addition (0, 500, 1000 and 4000 mg/kg diet) in laying hen diets prepared either with degummed or refined rape oil on the performance, sensory properties and trimethylamine (TMA) contents of the eggs. Furthermore, the diets containing no supplemented choline or 4000 mg choline/kg diet were tested with adequate or inadequate methionine supply (4.2 vs. 2.8 g methionine/kg diet). TMA metabolism and N-balance were measured for the latter diet types, but only with the diets containing refined rape oil. Therefore, a total of 12 and 4 diets were tested in the feeding (n = 60) and balance study (n = 9). Laying performance (23 – 75 weeks of age) was not significantly influenced by increasing choline additions with the exception of feed-to-egg mass ratio which decreased significantly linearly (p _linear = 0.003). However, a significant interaction between choline addition and laying month was detected which was caused by a depression of performance of the unsupplemented control group occurring from the sixth laying month. The most obvious effect of an inadequate methionine supply was a temporary drop in performance between the third and sixth laying months. The mean TMA-concentration in pooled egg yolks [μg/g] increased with dietary choline concentration [mg/kg] in an exponentially related fashion (y = 1.14 + 4E^?10 ? x^2.71, r² = 0.962) and suggested only a minor influence of total dietary choline on TMA content up to approximately 2000 mg choline/kg. Individual TMA-concentrations varied greatly from 0.4 – 1.5 μg/g, from 2.2 – 34 μg/g and from 18.4 – 75 μg/g for eggs with a normal, aberrant and heavily aberrant odour, respectively. It is concluded that a total choline concentration of at least approximately 1500 mg/kg is necessary to maintain a maximal laying performance. An inadequate methionine supply cannot be compensated by an increased addition of choline. Neither degummed nor refined rape oil influenced the TMA content of eggs.     

Biomolecules at phase boundaries

Experimental and theoretical studies of biomolecules at water surfaces and metal surfaces are presented. We studied lecithin molecules (monolayers) and phospholipase A₂ at a water surface with molecular dynamics (MD) simulations. Results were compared with those obtained for a pure water surface. We also studied amino acids at a TiO₂ surface with thermal desorption spectroscopy in the presence and absence of water.     

Measurement of choline acetyltransferase with [14C]acetate by a cycling procedure

A multiple enzyme and multisubstrate cycling system is described for the radiometric determination of cholineacetyltransferase (ChAT) activity in crude tissue homogenates. The methods employs [¹⁴C]acetate coupled with the enzymes acetate kinase (AK) and phosphotransacetylase (PTA) for the generation of [¹⁴C]acetyl CoA. By recycling it was possible to avoid product inhibition of ChAT by CoA, ATP was maintained constant by rephosphorylation of ADP. Kinetics of the individual enzyme reactions were studied and the parameters obtained were used to select appropriate conditions to maintain linearity of varying amounts ChAT activity over a sixty minute time course. The sensitivity of the method is limited only by the specific activity of commercially available isotope labeled acetate.Special issue dedicated to Dr. O. H. Lowry.     

Crystal structure of pyridoxine 4-oxidase from Mesorhizobium loti

Pyridoxine 4-oxidase (PNOX) from Mesorhizobium loti is a monomeric glucose–methanol–choline (GMC) oxidoreductase family enzyme, catalyzes FAD-dependent oxidation of pyridoxine (PN) into pyridoxal, and is the first enzyme in pathway I for the degradation of PN. The tertiary structures of PNOX with a C-terminal His₆-tag and PNOX–pyridoxamine (PM) complex were determined at 2.2 Å and at 2.1 Å resolutions, respectively. The overall structure consisted of FAD-binding and substrate-binding domains. In the active site, His460, His462, and Pro504 were located on the re-face of the isoalloxazine ring of FAD. PM binds to the active site through several hydrogen bonds. The side chains of His462 and His460 are located at 2.7 and 3.1 Å from the N4′ atom of PM. The activities of His460Ala and His462Ala mutant PNOXs were very low, and 460Ala/His462Ala double mutant PNOX exhibited no activity. His462 may act as a general base for the abstraction of a proton from the 4′-hydroxyl of PN. His460 may play a role in the binding and positioning of PN. The C4′ atom in PM is located at 3.2 Å, and the hydride ion from the C4′ atom may be transferred to the N5 atom of the isoalloxazine ring. The comparison of active site residues in GMC oxidoreductase shows that Pro504 in PNOX corresponds to Asn or His of the conserved His–Asn or His–His pair in other GMC oxidoreductases. The function of the novel proline residue was discussed.     

Studies on the characterization of rat prostate androgen receptors

Summary Choline used as the sole carbon or carbon and nitrogen source induces in Pseudomonas aeruginosa an active transport system. The induction of the choline uptake is repressed by succinate independently of the presence of ammonium ion in the culture medium. The repression mediated by succinate was insensitive to cyclic AMP. Substitution for dibutyryl-cyclic AMP was without effect. Choline metabolites that also support the growth of Pseudomonas aeruginosa were poor inducer agents of the choline transport. Kinetic evidence and the employment of choline metabolites as effectors indicated that the choline uptake system of this bacterium is formed by at least two components: one of high affinity (Km=3 µM) and another of low affinity (Km=400 µM). Contrary to what occurs in the synaptosome system, the high affinity form for the choline uptake was not dependent on Na⁺ ions and is not inhibited by hemicholinium-3. Since Pseudomonas aeruginosa can utilize choline as the sole carbon and nitrogen source, the induction of the choline transport with two components in this bacterium may be related to its own strategy to survive and grow in an adverse environment.     

Some properties of frog vestibular choline acetyltransferase and acetylcholinesterase

The amount and some properties of choline acetyltransferase (ChAT) and of acetylcholinesterase (AchE) were investigated in the frog vestibule. Enzyme activities were found to be of the same order of magnitude as in frog nervous tissue and various properties of vestibular ChAT (dependence on pH, chloride and Triton X-100 activation, phosphate sensitivity) and AchE (inhibition by eserine but not by Tetraisopropylpyrophosphoramide) were also similar as those of the homologous central nervous system enzymes. Although the precise localization of ChAT and AchE is not yet certain the efferent neurotransmitter in the vertebrate vestibular sensory periphery is believed to be acetylcholine and thus the enzymes responsible for its synthesis and degradation may participate in regulating inner ear function.     

Decreased Serum Choline Concentrations in Humans after Surgery,Childbirth, and Traumatic Head Injury

The serum levels of choline decreased by approximately 50% in patients having a surgery under general as well as epidural anesthesia. The decrease is lasts for two days after surgery. Intravenous administration of succinylcholine, either by a single bolus injection or by a slow continuous infusion, increased the serum choline levels several folds during surgery. In these patients, a significant decrease in the serum choline levels was observed one and two days after surgery. In 16 pregnant women at the term, serum choline levels were higher than the value observed in 19 non-pregnant women. The serum choline levels decreased by about 40% or 60% after having a child-birth either by vaginal delivery or caesarean section, respectively. Serum choline levels in blood obtained from 9 patients with traumatic head injury were significantly lower than the observed levels in blood samples obtained from healthy volunteers. These observations show that serum choline levels increase during pregnancy and decrease during stressful situations in humans.