The anticonvulsant effect of citalopram on El mice,and the levels of tryptophan and tyrosine and their metabolites in the brain

Serotonin (5-HT) plays an important role in the seizures of El mice since the seizure threshold of El mice correlates with the 5-HT concentration in the central nervous system. In this study, the anticonvulsant effect of a 5-HT reuptake blocker, citalopram, was evaluated behaviorally and biochemically. El mouse convulsions were inhibited by oral administration of citalopram for 2 weeks. Citalopram increased tryptophan and tyrosine amounts, and decreased the 5-HT, 5-hydroxy-indoleacetic acid, kynurenine, and dopamine amounts in the brain. These findings show that citalopram depresses monoaminergic metabolism. Given the known convulsant effect of kynurenine, it is suggested that its decrease by citalopram may involve attenuation of El mice seizures.     

Photosynthetic Adaptation to High Temperature Associated with Thylakoid Membranes of Synechococcus PCC7002

Photosynthetic adaptation to high temperature was investigatedin intact cells and isolated thylakoid membranes of the cyanobacterium,Synechococcus PCC7002. In intact cells, the thermal stabilityof photosynthesis and photosystem 2-mediated electron transportfrom H₂O to 1,4-benzoquinone changed in concert with growthtemperature. The photosystem 2-mediated electron transport fromH₂O to phenyl-1,4-benzoquinone showed greater thermal stabilityin thylakoid membranes isolated from cells which had adaptedto high temperature than in those from non-adapted cells. Enhancedthermal stability was also observed in the thylakoid membranesin the transport of electrons from H₂O to 2,6-dichlorophenolindophenolbut not in the transport of electrons from diphenylcarbazideto 2,6-dichlorophenolindophenol. These observations suggestthat oxygen-evolving sites acquire enhanced thermal stability,and that factors which are responsible for thermal stabilityremain in isolated thylakoid membranes. (Received October 30, 1992; Accepted December 18, 1992)     

Functional analysis of the γ-glutamylcysteine synthetase of Escherichia coli B: effect of substitution of His-150 to Ala

A high expression system of the -glutamylcysteine synthetase gene (gshl) of Escherichia coli B was constructed, and rapid purification of GSH-I was performed. The active site of GSH-I was analysed by chemical modification, and Lys, Arg and His residues seemed to be involved in the active site of the enzyme. Among them, His residues were substituted to Ala by site-directed mutagenesis, and His-150 was found to be essential for the activity of GSH-I. Correspondence to: A. Kimura     

Cloning of the blasticidin S deaminase gene (BSD) from Aspergillus terreus and its use as a selectable marker for Schizosaccharomyces pombe and Pyricularia oryzae

Aspergillus terreus produces a unique enzyme, blasticidin S deaminase, which catalyzes the deamination of blasticidin S (BS), and in consequence confers high resistance to the antibiotic. A cDNA clone derived from the structural gene for BS deaminase (BSD) was isolated by transforming Escherichia coli with an Aspergillus cDNA expression library and directly selecting for the ability to grow in the presence of the antibiotic. The complete nucleotide sequene of BSD was determined and proved to contain an open reading frame of 393 bp, encoding a polypeptide of 130 amino acids. Comparison of its nulceotide sequence with that of bsr, the BS deaminase gene isolated from Bacillus cereus, indicated no homology and a large difference in codon usage. The activity of BSD expressed in E. coli was easily quantified by an assay based on spectrophotometric recording. The BSD gene was placed in a shuttle vector for Schizosaccharomyces pombe, downstream of the SV40 early region promoter, and this allowed direct selection with BS at high frequency, following transformation into the yeast. The BSD gene was also employed as a selectable marker for Pyricularia oryzae, which could not be transformed to BS resistance by bsr. These results promise that the BSD gene will be useful as a new dominant selectable marker for eukaryotes.     

Serum-free culture of rat keratinocytes

Summary Procedures for the serum-free culture of rat keratinocytes have been established. Basal cells prepared from epidermis of newborn rat were stored in liquid nitrogen and used for primary culture. Among the available media, MCDB 153, developed originally for human keratinocyte (HK) culture, was the best for the development of serum-free formulation. To grow rat keratinocytes, bovine serum albumin was arbitrarily substituted for the macromolecule supplements needed for HK culture, i.e. fetal bovine serum protein or bovine pituitary extract. Qualitative and quantitative adjustment of supplements was thereafter made to support rapid cell growth. Satisfactory cell growth was achieved in the optimized medium of MCDB 153 supplemented with growth factors and amino acids: insulin (10 μg/ml), hydrocortisone (0.1 μg/ml), epidermal growth factor (25 ng/ml), calcium chloride (0.2 mM), histidine (0.23 mM), isoleucine (0.05 mM), tryptophane (0.015 mM), threonine (1.25 mM), tyrosine (0.031 mM), alanine (4.08 mM), and albumin (2 mg/ml). This optimized culture system was superior to the original HK culture condition for rapid growth of rat keratinocytes. Under our condition, cells grew as a monolayer, becoming confluent, but without stratification, and were passaged 2 to 3 times without any changes in morphology. The serum-free formulation allows us to control more accurately the concentrations of biomolecules in the medium including lipids and hormones, and therefore will be suitable for the study focusing on lipid metabolism or hormonal regulation of rat keratinocytes.     

Specificity of amphiphilic anionic peptides for fusion of phospholipid vesicles.

We have synthesized five amphiphilic anionic peptides derived from E5 peptide [Murata, M., Takahashi, S., Kagiwada, S., Suzuki, A., Ohnishi, S. 1992. Biochemistry 31:1986-1992. E5NN and E5CC are duplications of the N-terminal and the C-terminal halves of E5, respectively, and E5CN is an inversion of the N- and the C-terminal halves. E5P contains a Pro residue in the center of E5 and E8 has 8 Glu residues and 9 Leu residues. We studied fusion of dioleoylphosphatidylcholine (DOPC) large unilamellar vesicles assayed by fluorescent probes. The peptides formed alpha-helical structure with different degrees; E5NN, E5CN, and E8 with high helical content and E5CC and E5P with low helical content. These peptides bound to DOPC vesicles at acidic pH in proportion to the helical content of peptide. The peptides caused leakage of DOPC vesicles which increased with decreasing pH. The leakage was also proportional to the helicity of peptide. Highly helical peptides E5NN, E5CN, and E8 caused hemolysis at acidic pH but not at neutral pH. The fusion activity was also dependent on the helicity of peptides. In fusion induced by an equimolar mixture of E5 analogues and K5 at neutral pH, E8, E5NN, and E5CN were most active but E5CC did not cause fusion. In fusion induced by E5-analogue peptides alone, E5CN was active at acidic pH but not at neutral pH. Other peptides did not cause fusion. Amphiphilic peptides also appear to require other factors to cause fusion.     

Establishment of a human anaplastic thyroid cancer cell line secreting granulocyte colony-stimulating factor in response to cytokines

Summary A human anaplastic thyroid cancer cell line K-119, derived from a 77-yr-old woman who had developed marked neutrophilia and underwent surgery for anaplastic thyroid cancer, has been established. The spindlelike and polygonal cells in shape are stably proliferating since the beginning of its culture 2 yr ago. The cells grow rapidly and the population doubling time is 26 h. The chromosomes show many abnormalities and many marker chromosomes have been observed. Heterotransplantation of the cells into nude mice has resulted in the formation of tumors that are histologically interpreted as anaplastic cancer. The most noteworthy characteristics of the cell line are the many Ki-67-positive cells (86.3%) and that the cell line spontaneously secretes granulocyte colony-stimulating factor (G-CSF) and releases increased amounts of G-CSF in response to the stimulation of tumor necrosis factor, interleukin 1α, and interleukin 1β. The conditioned medium obtained from K-119 cells contains an autocrine factor stimulating the proliferation of themselves.     

Induction of antibacterial protein synthesis by soluble peptidoglycan in isolated fat body from larvae of the silkworm, Bombyx mori

Synthesis and secretion of bactericidal protein (cecropin) and lysozyme were induced by soluble peptidoglycan fragments (SPG) from Escherichia coli in a culture of fat body from Bombyx mori larvae. The rate of the secretion by fat body increased as a function of SPG concentration added to the culture medium. The induction of bactericidal activity was specific for peptidoglycan of a particular structure. Thus, SPG from Micrococcus luteus was 500-times less potent than E. coli SPG, and various glucans and peptides structurally related to peptidoglycan were all ineffective as elicitor. These results support the hypothesis that bacteria invading the haemocoel have to be partially degraded to generate peptidoglycan fragments as a signal molecule, which subsequently acts on a receptor on fat body cells and induces antibacterial protein synthesis.     

Vegetative growth and productivity ofEichhornia azurea with special emphasis on leaf dynamics

The lifeform and the biological production of pure stands ofEichhornia azurea Kunth in three lakes in tropical Brazil were studied. The lifeform ofE. azurea is termed ‘semi-emergent’, because the plant has well developed trailing stems just under the water, and the aerial lamina emerges with the thick petiole. The density of shoot apices was 9.9, 17.2 and 17.1 m⁻² in Lake Dom Helvecio, Lake Jacaré and Lake Carioca, respectively. The mean daily increment of the apical shoot biomass was between 1.8 and 4.8 g m⁻² day⁻¹. The mean leaf life-span in Lake Dom Helvecio, Lake Jacaré and Lake Carioca was estimated to be 78, 49 and 64 days in the wet season and 73, 70 and 73 days in the dry season, respectively. The stem life-span was estimated to be about 28 months. Starch content in the current years' stem ranged from 24 to 118 mg g⁻¹ dry matter with fluctuations, the amplitude of which decreased with age. The differences for most of the growth parameters, such as density of shoot apices, daily increment of biomass and leaf life-span, between dry and wet season are smaller than those among the three lakes. Both the decrease in daily dry matter production and the increase in leaf life-span occurred in order from Lake Dom Helvecio to Lake Jacaré and Lake Carioca. The low productivity ofE. azurea is considered to be related to a low leaf area index, a long time interval for the emergence of new leaves, long leaf life-span and a low capacity for branching.