Differential Effects of Hypoxia on Ligand Binding Properties of Nicotinic and Muscarinic Acetylcholine Receptors on Cultured Bovine Adrenal Chromaffin Cells

Abstract: Hypoxia is known to disturb neuronal signal transmission at the synapse. Presynaptically, hypoxia is reported to suppress the release of neurotransmitters, but its postsynaptic effects, especially on the function of neurotransmitter receptors, have not yet been elucidated. To clarify the postsynaptic effects, we used cultured bovine adrenal chromaffin cells as a model of postsynaptic neurons and examined specific binding of l -[³H]nicotine (an agonist for nicotinic acetylcholine receptors: nAChRs) and ²²Na⁺ flux under control and hypoxic conditions. Experiments were performed in media preequilibrated with a gas mixture of either 21% O₂/79% N₂ (control) or 100% N₂ (hypoxia). Scatchard analysis of the specific binding to the cells revealed that the K_D under hypoxic conditions was twice as large as that under control conditions, whereas the B _max was unchanged. When the specific [³H]nicotine binding was kinetically analyzed, the association constant ( k ₁) but not the dissociation constant ( k ₋₁) was decreased to 40% of the control value by hypoxia. When the binding assay was performed using the membrane fraction, these changes were not observed. Nicotine-evoked ²²Na⁺ flux into the cells was suppressed by hypoxia. In contrast, specific [³H]quinuclidinyl benzilate binding to the intact cells was unaffected by hypoxia. These results demonstrate that hypoxia specifically suppresses the function of nAChRs (and hence, neuronal signal transmission through nAChRs), primarily by acting intracellularly.     

Characterization of a gene cluster for glycogen biosynthesis and a heterotetrameric ADP-glucose pyrophosphorylase from Bacillus stearothermophilus.

A chromosomal region of Bacillus stearothermophilus TRBE14 which contains genes for glycogen synthesis was cloned and sequenced. This region includes five open reading frames (glgBCDAP). It has already been demonstrated that glgB encodes branching enzyme (EC 2.4.1.18 [H. Takata et al., Appl. Environ. Microbiol. 60:3096-3104, 1994]). The putative GlgC (387 amino acids [aa]) and GlgD (343 aa) proteins are homologous to bacterial ADP-glucose pyrophosphorylase (AGP [EC 2.7.7.27]): the sequences share 42 to 70% and 20 to 30% identities with AGP, respectively. Purification of GlgC and GlgD indicated that AGP is an alpha2beta2-type heterotetrameric enzyme consisting of these two proteins. AGP did not seem to be an allosteric enzyme, although the activities of most bacterial AGPs are known to be allosterically controlled. GlgC protein had AGP activity without GlgD protein, but its activity was lower than that of the heterotetrameric enzyme. The GlgA (485 aa) and GlgP (798 aa) proteins were shown to be glycogen synthase (EC 2.4.1.21) and glycogen phosphorylase (EC 2.4.1.1), respectively. We constructed plasmids harboring these five genes (glgBCDAP) and assayed glycogen production by a strain carrying each of the derivative plasmids on which the genes were mutated one by one. Glycogen metabolism in B. stearothermophilus is discussed on the basis of these results.     

Transient Ischemia Differentially Increases Tyrosine Phosphorylation of NMDA Receptor Subunits 2A and 2B

Abstract: Activation of the N -methyl- d -aspartate (NMDA) receptor has been implicated in the events leading to ischemia-induced neuronal cell death. Recent studies have indicated that the properties of the NMDA receptor channel may be regulated by tyrosine phosphorylation. We have therefore examined the effects of transient cerebral ischemia on the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B in different regions of the rat brain. Transient (15 min) global ischemia was produced by the four-vessel occlusion procedure. The tyrosine phosphorylation of NR2A and NR2B subunits was examined by immunoprecipitation with anti-tyrosine phosphate antibodies followed by immunoblotting with antibodies specific for NR2A or NR2B, and by immunoprecipitation with subunit-specific antibodies followed by immunoblotting with anti-phosphotyrosine antibodies. Transient ischemia followed by reperfusion induced large (23–29-fold relative to sham-operated controls), rapid (within 15 min of reperfusion), and sustained (for at least 24 h) increases in the tyrosine phosphorylation of NR2A and smaller increases in that of NR2B in the hippocampus. Ischemia-induced tyrosine phosphorylation of NR2 subunits in the hippocampus was higher than that of cortical and striatal NR2 subunits. The enhanced tyrosine phosphorylation of NR2A or NR2B may contribute to alterations in NMDA receptor function or in signaling pathways in the postischemic brain and may be related to pathogenic events leading to neuronal death.     

A new alkaline serine protease from alkalophilic Bacillus sp.: Cloning,sequencing, and characterization of an intracellular protease

To obtain a new serine protease from alkalophilic Bacillus sp. NKS-21, shotgun cloning was carried out. As a result, a new protease gene was obtained. It encoded an intracellular serine protease (ISP-1) in which there was no signal sequence. The molecular weight was 34,624. The protease showed about 50% homology with those of intracellular serine proteases (ISP-1) from Bacillus subtilis, B. polymyxa, and alkalophilic Bacillus sp. No. 221. The amino acid residues that form the catalytic triad, Ser, His and Asp, were completely conserved in comparison with subtilisins (the extracellular proteases from Bacillus). The cloned intracellular protease was expressed in Escherichia coli, and its purification and characterization were carried out. The enzyme showed stability under alkaline condition at pH 10 and tolerance to surfactants. The cloned ISP-1 digested well nucleoproteins, clupein and salmin, for the substrates.The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases with the accession number D37921.     

Optimization of a medium for the production of 1,2-epoxytetradecane by Nocardia corallina B-276

Summary A medium for the production of 1,2-epoxytetradecane from 1-tetradecene by Nocardia corallina B-276 was optimized. The activity of cells producing 1,2-epoxytetradecane increased when cell growth was suppressed by limiting nitrogen or potassium ion in the medium. Mg²⁺ was found to be essential for the production of 1,2-epoxide. Cell mass was increased, without reducing production of 1,2-epoxide, by increasing the concentration of yeast extract and limiting the concentration of potassium ion. The concentration of 1,2-epoxytetradecane reached 80 g/l in 6 days after optimization and the yield of 1,2-epoxytetradecane was 65 mol% based on consumed 1-tetradecene. Long chain 1,2-epoxyalkanes containing 13–17 carbon atoms also were produced under these conditions.     

Identification of glycosphingolipid-glycoprotein hybrids in gastric epithelium

A glycolipid fraction, obtained from dog gastric epithelium by detergent solubilization, was found to contain the glycoconjugates with features common to both glycosphingolipids and glycoproteins. Three such compounds have been purified to homogeneity and their composition and structural characteristics determined. The purified glycosphingolipid-glycoprotein hybrids had a molecular weight in the range of 14,000 and contained 88.2 - 91.1% carbohydrate, 3.1 - 4.5% protein, 2.0 - 2.4% sphingosine, and 1.1 - 1.9% fatty acids. The oxidation of the olefinic bond of sphingosine followed by beta-elimination reaction led to the release from each compound of glucose and mannose containing oligosaccharides. Deglycosylation with trifluoromethanesulfonic acid exposed the core regions of the hybrid molecules which were found to consist of sphingosine, glucose, N-acetylglucosamine and amino acids. The data suggest that glycosphingolipid-glycoprotein hybrid molecules are held together by amide linkages between the sphingosine and aspartic or glutamic acid.     

Changes in Organelle Movement in the Nuclear Region during the Cell Cycle of Adiantum Protonema

Movements of organelles in the nuclear region as the cell cycleprogresses in single-celled protonemata of Adiantum capillus-veneriswere examined by digital image processing techniques and microscopyof particle movement. Organelles in the nuclear region werenot very crowded and moving directionally along the longitudinalaxis of the filamentous cell in the G₁ and S phases. They beganto gather and accumulate in the nuclear region in early G₂ phase,after which directional movement changed to undirectional Brownianmotion-like movement in late G₂ phase. Movement of organelleslocated on the lateral surface of the nucleus slowed after premitoticpositioning of nucleus and lasted until the nucleolus disappeared.Movement of organelles in the cytoplasm surrounding the nucleoplasmresumed just after the nucleolus disappeared, whereas organelleslocated in the outer regions of the apical and basal surfacesof the nucleus moved rapidly during prophase but did not moveduring metaphase, movement being resumed after chromosome separation.Thus, organelle movement in the nuclear region showed temporaland spatial change during the cell cycle. (Received August 24, 1983; Accepted December 28, 1983)     

Termination of early pregnancy by ONO-802 suppositories (16,16-dimethyl-trans-Δ-PGE1 methyl ester)

ONO-802 was used in the form of vaginal suppositories for the termination of early pregnancy in 63 healthy volunteers. Fifty-four (86%) of the 63 cases had complete abortions and remaining 9 (14%) had incomplete abortions.One (1.6%) of the 63 cases complained of nausea and vomiting, and 3 (4.8%) complained of headaches. No other side effects were observed.These results suggest that ONO-802 is acceptable in the form of vaginal suppositories for the termination of early pregnancy.     

Morphine-like analgesic actions of enkephalin-like peptides containing the Tyr-Arg unit

The analgesic actions of some synthetically prepared peptides having the Tyr-D-Arg unit at the N terminal portion of met- and leu-enkephalin were measured by the intra-cisternal injection method in mice. Among them, Tyr-D-Arg-Gly-Phe (DR-4) induced the most potent naloxone-reversible analgesia and was also effective by s.c. injection. DR-4 showed the good affinity to mu-receptor, and the resistance to the enzymatic degradation.     

Increase in arginine and citrulline production by 6-azauracil-resistant mutants of Bacillus subtilis.

In the arginine producer AHr-5, an L-arginine hydroxamate-resistant mutant of Bacillus subtilis, accumulation of N8-acetyl-L-ornithine increased as the level of L-arginine accumulation increased in the medium containing L-glutamic acid. Ornithine carbamoyltransferase of this strain was genetically derepressed. These results suggested that carbamoylphosphate might be deficient in vivo. With the intention to increase endogenous carbamoylphosphate, pyrimidine analogs inhibiting growth were selected and the mutants resistant to these compounds were derived from the AHr-5 mutant. Of the resistant mutants derived, the 6-azauracil-resistant mutant AAr-9 produced 28 mg of L-arginine per ml, which corresponded to more than twofold the amount produced by the parent strain. Derivation of an arginine-requiring mutant from the double-resistant mutant AAr-9 provides a new advantageous method for the production of L-citrulline. The increase in arginine and citrulline production is discussed.