CHANGES IN CELL CHEMICAL COMPOSITION DURING THE LIFE CYCLE OF SCRIPPSIELLA TROCHOIDEA (DINOPHYCEAE)1

The cellular content of carbon, nitrogen, amino acids, polysaccharides, phosphorus and adenosine trtphosphate (ATP) was determined at several stages during the life cycle of the dinoflagellate Scrippsiella trochoidea (Stein) Loeblich. Carbon per cell decreased slightly between exponential and stationary phase growth in vegetative cells whereas nitrogen per cell did not change. Both of these cellular components increased markedly on encystment and then decreased to vegetative cell levels during dormancy and germination. C/N ratios increased gradually during cyst dormancy and activation, reflecting a more rapid decrease in N than in C pools, even though both decreased through time. Amino acid composition was relatively constant during the vegetative cell stages; glutamic acid was the dominant component. Arginine was notably higher in cysts than in vegetative cells but decreased significantly during germination, suggesting a role in nitrogen storage. The ratio of neutral ammo acids to total ammo acids (NAA/TAA) decreased as cysts were formed and then gradually increased during storage and germination. The ratio of basic ammo acids to total ammo acids (BAA/TAA) changed in the opposite direction of NAA/TAA, whereas the ratio of acidic acids to total amino adds (AAA/TAA) was generally invariant. Ammo acid pools were not static during the resting slate in the cysts: there was degradation or biosynthesis of certain, but not all, classes of these compounds. The monosacchande composition of cold and hot water extracted polysaccharides was quite different between cells and cysts. A high percentage of glucose in cysts suggests that the storage carbohydrate is probably in the form of glucan. Total cellular phosphorus was higher in all cyst stages than in vegetative cells. However, ATP-cell^?1 decreased as vegetative cells entered stationary phase and encysted, and continued to decrease in cysts during dark cold storage. ATP increased only as the cysts were activated at warm temperatures in the light and began to germinate. The above data demonstrate that dormancy and quiescence are not periods of inactive metabolism but instead are times when numerous biochemical transformations are occurring that permit prolonged survival in a resting state.     

PLD activation in Chinese hamster ovary (CHO) cells transfected with PGF2α receptor cDNA

Pertussis toxin-insensitive GTP-binding protein was observed to be involved in prostaglandin F2α(PGF2α)-induced phosphoinositide metabolism in Chinese hamster ovary (CHO) cells transfected with PGF2α receptor cDNA (CHO-PGF2α·R cells) (Ito, S. et al. Biochem. Biophys. Res. Commun. 200: 756, 1994). In the present study, we investigated PGF2α-induced PLD activation in CHO-PGF2α·R cells. PLD activation was examined by measuring the production of [³H]phosphatidylbutanol ([³H]PBut), a specific product of the PLD-catalyzed transphosphatidylation reaction. PGF2α-induced [³H]PBut formation was concentration-dependent with the maximal level obtained at 1 μM PGF2α. The maximal [³H]PBut formation was observed at 2 min after addition of PGF2α. Depletion of extracellular Ca²⁺ with EGTA suppressed PGF2α-induced PLD activation by 50%. PKC inhibitors Ro31–8425 and calphostin C inhibited PGF2α-induced [³H]PBut formation by 50%. PTK inhibitors genistein and herbimycin A failed to inhibit PGF2α-induced PLD activation. A combination of maximal effective concentrations of PGF2α (1 μM) and PMA (100 nM) enhanced PLD activation in an additive manner. Pretreatment of the cells with PMA for 2 h down-regulated PKCα and decreased PGF2α-induced PLD activation. These results suggest that PLD activation by PGF2α is mediated by both PKC-dependent and -independent pathways and that PKCα is involved in the former pathway.     

Effects of water content on the enantioselectivity of α-chymotrypsin catalysis in organic media

Summary The -chymotrypsin-catalyzed transesterification between N-trifluoroacetyl-DL-phenylalanine trifluoroethyl ester and 1-propanol was carried out in a variety of organic solvents. The addition of small quantities of water enhanced both the rate of reaction and enantioselectivity. A high enantioselectivity was achieved in ethyl acetate (E = 120), diethyl ether (86), or acetonitrile (60). The competing hydrolysis became significant at water content higher than 0.5% (w/w).     

Photoactive Photosystem I Particles with a Molar Ratio of Chlorophyll a to P700 of 9

Lyophilized photosystem I particles from spinach were treatedwith diethyl ether that contained various organic solvents withdifferent dielectric constants. More pigments were extractedas the dielectric constant of the solvent added to ether increased.The reaction-center chlorophylldimer, P700, was more resistantto extraction than the rest of the chlorophyll. Particles thatcontained only 6 chlorophylls in addition to P700 and the primaryelectron acceptor (A₀), in a single reaction-center unit, wereprepared by extraction with a mixture of ether and acetaldehyde.A distinct shoulder at 695 nm due to P700 or at 686 nm due toP700⁺ was observed in the absorption spectra of the reducedor oxidized particles, respectively, even at room temperature.No secondary acceptor phylloquinone remained in the particles.Stable charge separation was restored upon the addition of 2-amino-anthraquinone,even though the particles had the lowest molar ratio of chlorophyllto P700 of any reported particles. (Received February 20, 1995; Accepted May 8, 1995)     

The Expression of Two Splice Variants of Metabotropic Glutamate Receptor Subtype 5 in the Rat Brain and Neuronal Cells During Development

Abstract: We previously reported that a variant with extra amino acid residues exists in the metabotropic glutamate receptor subtype 5 (mGluR5). Either of the two isoforms, named mGluR5b and mGluR5a for the isoforms with and without the inserted sequence, respectively, generated Ca²⁺-activated Cl⁻ current when expressed in Xenopus oocytes. We herein report that these two isoforms are produced by the alternative splicing of the exon skipping type. When examined during the course of postnatal development, the major mGluR5 isotype mRNA was observed to switch from mGluR5a to mGluR5b in the rat hippocampus and the cerebral cortex. We also investigated two cell lines that could be differentiated into neuron-like cells in vitro. Whereas the mGluR5b mRNA was hardly detectable in either undifferentiated or differentiated NG108-15 cells, the relative amounts of the two variant mRNAs changed after the induction of differentiation in the P19 cells. An extracellular application of trans - d,l -1-amino-1,3-cyclopentanedicarboxylate on the neuron-like P19 cells induced intracellular Ca²⁺ mobilization, thus suggesting that the cells could express functional mGluR(s) coupled to phospholipase C and other components that could mediate the signal transduction pathway. This cell line may thus provide a model system for studying both mGluR5 expression and other mGluR-induced phenomena at the molecular level.     

Nitrogen-flow in the rotifer Brachionus rotundiformis and its significance in mass cultures

The nitrogen budget in the rotifer Brachionus rotundiformis wasmeasured by the stable-isotope technique. The budget was estimatedusing the difference in the turnover time between egestion andexcretion. The rotifer was fed on the algae Nannochloropsiswhich was labeled with ¹⁵N as a tracer. The turnover time ofegestion and excretion were 20 min and 2.5 hours, respectively. Where77% of the ingested nitrogen was egested, and of the assimilated23%, 18% were devoted to growth and 5% to excretion.As for the unassimilated nitrogen egested as faeces, it recycled tothe rotifer through bacteriovory. When the algae provided as foodwere almost fully consumed, bacteriovory became dominant. Thethreshold occurred when the concentration of algae in the culture wasbetween 1.5 and 0.5 million cells of Nannochloropsis per ml. Ina chemostat operated with un-limited food condition, bacterialnitrogen corresponding to 20% of algal feeding, was consumed by therotifer.In a semi-continuous mass culture where food condition was limited,bacteriovory was more effective in supporting the rotiferreproduction. It contributed to the extremely high nitrogen recoveryfrom the provided foods (algae and oil-yeast) to the harvestedrotifers. The rapid and large nitrogen outflow from rotifersaccelerated the propagation of edible bacteria and can explain thestrange paradox observed in the culture; daily supply of foods didnot cover the sum of growth and excretion.It is not too exaggerated to state that the rotifer mass culture issupported by bacteria. The future strategy for maintenance of masscultures should consider this aspect.     

A mutation at proline-115 in the A-factor receptor protein of Streptomyces griseus abolishes DNA-binding ability but not ligand-binding ability.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) and its specific receptor protein (ArpA) are required for streptomycin production and aerial mycelium formation in Streptomyces griseus. A mutant strain HO1 that produced streptomycin and formed aerial mycelium and spores was derived from an A-factor-deficient mutant, S. griseus HH1. The phenotypes of mutant HO1 were found to result from a single amino acid replacement of ArpA; the proline residue at position 115 in the wild-type ArpA was replaced by serine, yielding mutant ArpA (P115S). The mutant ArpA (P115S) was still able to form a homodimer and possessed A-factor-binding ability but lost the ability to bind DNA. The properties of P115S suggest that ArpA consists of two independently functional domains, one for A-factor binding and one for DNA binding, and that proline-115 plays an important role in DNA binding. This is in agreement with the idea that A-factor binding to the COOH-terminal domain of ArpA causes a subtle conformational change of the distal NH2-terminal DNA-binding domain, resulting in dissociation of ArpA from DNA.