Phosphatases modulate transmission and serotonin facilitation at synapses: Studies with the inhibitor okadaic acid

We examined the role of phosphatases in synaptic transmission using the permeant phosphatase inhibitor okadaic acid (OA). In the crayfish neuromuscular junction (NMJ), postsynaptic effects including increases in input resistance occurred at doses greater than 5 μM OA. At lower doses (0.5–5 μM) the effects were solely presynaptic and transmitter release increased over three-fold despite small reductions in amplitude and duration of presynaptic action potentials. Potentiating effects of serotonin on transmitter release, Which depend on phosphorylation, were increased by OA. Frequency facilitation was reduced but its decay was not affected. In frog NMJs, OA increased spontaneous and evoked release two-fold through presynaptic mechanisms. An inactive analog of OA, OA tetra-acetate, had no effect on transmitter release at frog and crayfish NMJ. Therefore, phosphatases have a strong modulating influence on synaptic transmission.     

Multiple forms of O-methyltransferase involved in the microbial conversion of abietic acid into methyl abietate by Mycobacterium sp.

Six out of seven tested strains of mycobacteria transformed abietic acid to methyl abietate in shake culture. The conversion carried out by Mycobacterium sp. MB 3683 was induced by the substrate and stimulated by methionine. Fractionation of the cell extract of Mycobacterium sp. MB 3683 on DEAE cellulose, Ultrogel AcA 44 and MONO Q resulted in the separation of three distinct methyltransferase activities which could also esterify palmitic acid. The separated forms of the methyltransferase exhibited different activities towards these two substrates.     

Adenosine and Glutamate Modulate Each Other's Release from Rat Hippocampal Synaptosomes

Abstract: In rat hippocampal synaptosomes, adenosine decreased the K⁺ (15 mM) or the kainate (1 mM) evoked release of glutamate and aspartate. An even more pronounced effect was observed in the presence of the stable adenosine analogue, R-phenylisopropyladenosine. All these effects were reversed by the selective adenosine A₁ receptor antagonist 8-cyclo-pentyltheophylline. In the same synaptosomal preparation, K⁺ (30 mM) strongly stimulated the release of the preloaded [³H]adenosine in a partially Ca²⁺-dependent and tetrodotoxin (TTX)-sensitive manner. Moreover, in the same experimental conditions, both l -glutamate and l -aspartate enhanced the release of [³H]adenosine derivatives ([³H]ADD). The gluta-mate-evoked release was dose dependent and appeared to be Ca²⁺ independent and tetrodotoxin insensitive. This effect was not due to metabolism because even the nonmetabolizable isomers d -glutamate and d -aspartate were able to stimulate [³H]ADD release. In contrast, the specific glutamate agonists N-methyl-d -aspartate, kainate, and quisqualate failed to stimulate [³H]ADD release, suggesting that glutamate and aspartate effects were not mediated by known excitatory amino acid receptors. Moreover, NMDA was also ineffective in the absence of Mg²⁺ and l -glutamate-evoked release was not inhibited by adding the specific antagonists 2-amino-5-phosphonovaleric acid or 6–7-dinitroquinoxaline-2, 3-dione. The stimulatory effect did not appear specific for only excitatory amino acids, as γ-anunobutyric acid stimulated [³H]ADD release in a dose-related manner. These results suggest that, at least in synaptosomal preparations from rat hippocampus, adenosine and glutamate modulate each other's release. The exact mechanism of such interplay, although still, unknown, could help in the understanding of excitatory amino acid neurotoxicity.     

Septal and Hippocampal Glutamate Receptors Modulate the Output of Acetylcholine in Hippocampus: A Microdialysis Study

Abstract: In the present study, glutamate receptor agonists and antagonists were administered by retrograde microdialysis into either the medial septum/vertical limb of the diagonal band (MS/vDB), or hippocampus, and the output of acetylcholine (ACh) was measured in the hippocampus by using intracerebral microdialysis. Perfusion with N -methyl- d -aspartate (NMDA) and ( S )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) in the MS/vDB caused an increase in ACh output in the hippocampus. This increase was completely blocked by coadministration of their respective antagonists d (−)-2-amino-5-phosphonopentanoic acid ( d -AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Perfusion in the MS/vDB with kainic acid also caused an increase in ACh output, but coadministration of CNQX attenuated the increase only partially. Perfusion with d -AP5 or CNQX alone in the septal probe did not affect ACh output from the hippocampus. In contrast to the results of septal administration of NMDA and AMPA, local perfusion with the same drugs in the hippocampus caused a decrease in ACh output. Whereas the results of septal administration of drugs indicate that septal cholinergic neurons probably receive excitatory glutamatergic innervation, the decrease in ACh output caused by administration of NMDA and AMPA in the hippocampus is poorly understood.     

Purification and properties of pyruvate kinase from Thermoplasma acidophilum

Thermoplasma acidophilum is a thermoacidophilic archaebacterium occupying a paradoxical place in phylogenetic trees (phenotypically it is a thermoacidophile but phylogenetically it classifies with the methanogens). To better understand its phylogeny, the pyruvate kinase from this organism is being investigated as a molecular marker. The enzyme has been purified and has a native M(r) of 250,000. It consists of four, apparently identical subunits each of M(r) 60,000. No remarkable kinetic differences have been found between this thermophilic enzyme and its mesophilic counterparts other than its greater thermostability. Its amino acid composition has been determined and some partial sequencing has been done.     

Activation of the pro-migratory bone morphogenetic protein receptor 1B gene in human MDA-MB-468 triple-negative breast cancer cells that over-express CYP2J2

Secondary metastases are the leading cause of mortality in patients with breast cancer. Cytochrome P450 (CYP) 2J2 (CYP2J2) is upregulated in many human tumors and generates epoxyeicosanoids from arachidonic acid that promote tumorigenesis and metastasis, but at present there is little information on the genes that mediate these actions. In this study MDA-MB-468 breast cancer cells were stably transfected with CYP2J2 (MDA-2J2 cells) and Affymetrix microarray profiling was undertaken. We identified 182 genes that were differentially expressed in MDA-2J2 cells relative to control (MDA-CTL) cells (log[fold of control] ≥2). From gene ontology pathway analysis bone morphogenetic protein (BMP) receptor 1B (BMPR1B) emerged as an important upregulated gene in MDA-2J2 cells. Addition of the BMPR1B ligand BMP2 stimulated the migration of MDA-2J2 cells, but not MDA-CTL cells, from 3D-matrigel droplets. Migration of MDA-2J2 cells was prevented by the BMPR antagonist dorsomorphin. These findings indicate that over-expression of CYP2J2 in MDA-MB-468-derived breast cancer cells activates BMPR1B expression that may contribute to increased migration. Targeting BMPR1B may be a novel approach to inhibit the metastatic activity of breast cancers that contain high levels of CYP2J2.     

WFS1 and non-syndromic low-frequency sensorineural hearing loss: A novel mutation in a Portuguese case

Low-frequency sensorineural hearing loss (LFSNHL) is an unusual type of HL in which frequencies at 2000 Hz and below are predominantly affected. Most of the families with LFSNHL carry missense mutations in WFS1 gene, coding for wolframin.     

Effect of externally added carnitine on the synthesis of acetylcholine in rat cerebral cortex cells

Acetylcholine synthesis from radiolabelled glucose was monitored in cerebral cortex cells isolated from brains of suckling and adult rats. Acetylcholine synthesis was found much higher in suckling animals, both in the absence and presence of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) inhibitor, paraoxon. Together with choline (20 μM), carnitine was found to stimulate acetylcholine synthesis in a synergistic way in cortex cells from adult rats (18%). Choline, however, was incapable of reversing an inhibitory effect exerted by carnitine on acetylcholine synthesis in cortex cells from suckling animals. Distribution of carnitine derivatives was found significantly different in the cells from young and old animals, the content of acetylcarnitine decreased with age with a corresponding increase of free carnitine. The observed differences in carnitine effect on acetylcholine synthesis suggested that high acetylcarnitine in cells capable of β-oxidation might be correlated with the lower level of acetylcholine synthesis.     

The neutralization of acidic coal mine lakes by additions of natural organic matter: a mesocosm test

Cylindrical polyethylene enclosures 3 m in length and 1 m in diameter reaching from the surface to the bottom were constructed in an acid (pH=3.1) lake on a coal surface mine in southern Illinois. Wheat straw was added to the enclosures to test the effects of dissimilatory sulfate reduction on water chemistry. Added straw increased sulfide concentrations, raised pH to 6.5, reduced O₂ and increased acid neutralizing capacity of the enclosed water columns when compared with a control enclosure and with the open lake. Generation of acid neutralizing capacity exceeded the standing stock of sulfide indicating that sulfide was removed either by precipitation of FeS or outgassing of H₂S. The pH and acid neutralizing capacity within the enclosures eventually returned to the level of the surrounding lake because of water exchange around the enclosure walls. Our results show that additions of organic matter to acid surface mine lakes result in the generation of acid neutralizing capacity.     

A novel potent metal-binding NDM-1 inhibitor was identified by fragment virtual,SPR and NMR screening

NDM-1 can hydrolyze nearly all available β-lactam antibiotics, including carbapenems. NDM-1 producing bacterial strains are worldwide threats. It is still very challenging to find a potent NDM-1 inhibitor for clinical use. In our study, we used a metal-binding pharmacophore (MBP) enriched virtual fragment library to screen NDM-1 hits. SPR screening helped to verify the MBP virtual hits and identified a new NDM-1 binder and weak inhibitor A1. A solution NMR study of ¹⁵N-labeled NDM-1 showed that A1 disturbed all three residues coordinating the second zinc ion (Zn2) in the active pocket of NDM-1. The perturbation only happened in the presence of zinc ion, indicating that A1 bound to Zn2. Based on the scaffold of A1, we designed and synthesized a series of NDM-1 inhibitors. Several compounds showed synergistic antibacterial activity with meropenem against NDM-1 producing K. pneumoniae.