Involvement of L-Type-Like Amino Acid Transporters in S-Nitrosocysteine-Stimulated Noradrenaline Release in the Rat Hippocampus

Abstract: Nitrogen oxides, such as nitric oxide, have been shown to regulate neuronal functions, including neurotransmitter release. We investigated the effect of S-nitroso-l -cysteine (SNC) on noradrenaline (NA) release in the rat hippocampus in vivo and in vitro. SNC stimulated [³H]NA release from prelabeled hippocampal slices in a dose-dependent manner. SNC stimulated endogenous NA release within 30 min to almost five times the basal level in vivo (microdialysis in freely moving rats). In a Na⁺-containing Tyrode's buffer, SNC-stimulated [³H]NA release was inhibited 30% by the coaddition of l -leucine. In the Na⁺-free, choline-containing buffer, SNC-stimulated [³H]NA release, which was similar to that in the Na⁺-containing buffer, was inhibited markedly by l -leucine, l -alanine, l -methionine, l -phenylalanine, and l -tyrosine. The effects of the other amino acids examined were smaller or very limited. The effect of l -leucine was stronger than that of d -leucine. A specific inhibitor of the L-type amino acid transporter, 2-aminobicyclo[2.2.1]-heptane-2-carboxylate (BCH), inhibited the effects of SNC on [³H]NA release in the Na⁺-free buffer. Uptake of l -[³H]leucine into the slices in the Na⁺-free buffer was inhibited by SNC, BCH, and l -phenylalanine, but not by l -lysine. The effect of SNC on cyclic GMP accumulation was not inhibited by l -leucine, although SNC stimulated cyclic GMP accumulation at concentrations up to 25 µM, much less than the concentration that stimulates NA release. These findings suggest that SNC is incorporated into rat hippocampus via the L-type-like amino acid transporter, at least in Na⁺-free conditions, and that SNC stimulates NA release in vivo and in vitro in a cyclic GMP-independent manner.     

Establishment and characterization of a new,spontaneously immortalized,pancreatic ductal cell line from the Syrian golden hamster

Spontaneously immortal pancreatic cell lines are not available. By use of a defined culture medium, such a line (TAKA-1) was established from the Syrian golden hamster. Cytological, cytogenetic, molecular biological, enzymatic and receptor patterns as well as antigenicity were studied and were compared with those of the normal hamster pancreatic ductal cells in vivo. TAKA-1 cells grew exponentially in a monolayer on collagen gel in a defined medium but did not proliferate in soft agar. Ultrastructurally, the cells closely resembled the normal hamster pancreatic ductal cells. Similarities and dissimilarities were found between the normal ductal cells and TAKA-1 cells. Similarities included the presence of cytokeratin, carbonic anhydrase and some tumor-associated antigens. However, unlike the normal ductal cells, TAKA-1 cells expressed blood group A angigen and anti-vimentin, showed affinity to selected lectins, and an abnormality of chromosome 3, which is suggested to be associated with immortality. Moreover, unlike the hamster pancreatic ductal cancer cells but like the normal hamster pancreatic ductal cells, TAKA-1 cells did not have a c-Ki-ras mutation. EGF, TGF- and secretin, but not CCK or GRP, bound to the TAKA-1 cells. TAKA-1 cells produced TGF-, and their growth was stimulated by exogenous EGF in serum-free medium. This cell line presents a suitable model for biologic and pathologic study of the hamster pancreatic ductal cells in vitro.     

Characterization of Methanosarcina mazeii TMA isolated from a paddy field soil

We isolated a methanogenic strain, designated as strain TMA (=DSM 9195), from an enrichment culture inoculated with a Japanese paddy field soil. Strain TMA was Gram positive and strictly anaerobic. Cell shape was pseudosarcina-like, and cells were nonmotile. The strain was able to use methylamines, methanol, H₂–CO₂, and acetate as substrates for methanogenesis, but did not utilize formate. The optimum temperature and optimum pH were 30–37°C and 6.5–7.5 respectively. The G+C content of the DNA was 42.1 mol %. Strain TMA had DNA-DNA hybridization values of more than 80% with Methanosarcina mazeii S-6^T (T = type strain). On the basis of phenotypic and genotypic characteristics, we identified strain TMA as M. mazeii. This is the first methylotrophic methanogen isolated from a paddy field soil and identified to the species level.     

Regulation of neuropeptide expression in the brain by neurotrophins

Neurotrophins, which are structurally related to nerve growth factor, have been shown to promote survival of various neurons. Recently, we found a novel activity of a neurotrophin in the brain: Brain-derived neurotrophic factor (BDNF) enhances expression of various neuropeptides. The neuropeptide differentiation activity was then compared among neurotrophins both in vivo and in vitro. In cultured neocortical neurons, BDNF and neurotrophin-5 (NT-5) remarkably increased levels of neuropeptide Y and somatostatin, and neurotrophin-3 (NT-3) also increased these peptides but required higher concentrations. At elevating substance P, however, NT-3 was as potent as BDNF. In contrast, NGF had negligible or no effect. Neurotrophins administered into neonatal brain exhibited slightly different potencies for increasing these neuropeptides: The most marked increase in neuropeptide Y levels was obtained in the neocortex by NT-5, whereas in the striatum and hippocampus by BDNF, although all three neurotrophins increased somatostatin similarly in all the brain regions examined. Overall spatial patterns of the neuropeptide induction were similar among the neurotrophins. Neurons in adult rat brain can also react with the neurotrophins and alter neuropeptide expression in a slightly different fashion. Excitatory neuronal activity and hormones are known to change expression of neurotrophins. Therefore, neurotrophins, neuronal activity, and hormones influence each other and all regulate neurotransmitter/peptide expression in developing and mature brain. Physiological implication of the neurotransmitter/peptide differentiation activities is also discussed.     

Bsr-REMI: An improved method for gene tagging using a new vector inDictyostelium

Using a plasmid pBsr2 which carries a blasticidin S-resistant gene, we have improved the method of REMI (restriction enzyme-mediated integration) provided for insertional mutagenesis inDictyostelium discoideum (bsr-REMI). To confirm usefulness of thebsr-REMI, transformation efficiency, copy number of integrated DNA, and randomness of integration into genome were examined.     

Chronic ethanol administration decreases fatty acyl-CoA desaturase activities in rat liver microsomes

The Δ⁹-desaturase system in liver microsome from rats treated chronically with ethanol was studied. Stearoyl-CoA desaturase activity decreased by 80% and palmitoyl-CoA desaturase activity was not detectable in microsomes from ethanol-fed rats, while activities of electron transport components such as NADH-cytochrome c and NADH-ferricyanide reductases remained unchanged. However, chronic ethanol administration resulted in an adaptive induction of the activity of NADPH-cytochrome c reductase and the contents of cytochrome b₅ and P-450. The activity of the terminal component (cyanide-sensitive factor; CSF) of the desaturase system was greatly depressed by ethanol treatment. The NADH/NAD ratio in microsomes of ethanol-fed rats increased over 2-fold. These results suggest that, during chronic ethanol ingestion, decreased activities of Δ⁹-desaturases are due mainly to a decreased content of the terminal component of the desaturase system.     

Successive spontaneous abortions including one with whole-arm translocation between chromosomes 2

Summary A family with five induced and seven spontaneous abortions and no live births is described. Four of the seven spontaneous abortuses were available for cytogenetic examination and three were successfully karyotyped. Their karyotypes were 46,XX; 46,XX/46,XX,t(2;2)(2p2p;2q2q); and 46,XY. The karyotypes of the parents were normal. The origin of the 2p/2p and 2p/2p translocation in one of the abortuses was assigned to an interhomologous whole-arm translocation in an early mitotic division in a conceptus with a 46,XX karyotype.