Cyclic sedimentation across a Middle Ordovician carbonate ramp (Duwibong Formation), Korea

Summary The Middle Ordovician Duwibong Formation (about 100 m thick), Korea, comprises various lithotypes deposited across a carbonate ramp. Their stacking patterns constitute several kinds of meter-scale, shallowing-upward carbonate cycles. Lithofacies associations are grouped into four depositional facies: deep- to mid-ramp, shoal-complex, lagoonal, and tidal-flat facies. These facies are composed of distinctive depositional cycles: deep subtidal, shallow subtidal, restricted marine, and peritidal cycles, respectively. The subtidal cycles are capped by subtidal lithofacies and indicate incomplete shallowing to the peritidal zone. The restricted marine and peritidal cycles are capped by tidal flat lithofacies and show evidence of subaerial exposure. These cycles were formed by higher frequency sea-level fluctuations with durations of 120 ky (fifth order), which were superimposed on the longer term sea-level events, and by sediment redistribution by storm-induced currents and waves. The stratigraphic succession of the Duwibong Formation represents a general regressive trend. The vertical facies change records the transition from a deep- to mid-ramp to shoal, to lagoon, into a peritidal zone. The depositional system of the Duwibong Formation was influenced by frequent storms, especially on the deep ramp to mid-ramp seaward of ooid shoals. The storm deposits comprise about 20% of the Duwibong sequence.     

trans-Sialidase catalyzed sialylation of beta-galactosyldisaccharide with an introduction of beta-galactosidase

Introduction of beta-galactosidase into a trans-sialidase reaction, i.e. sialic acid transfer reaction from a donor substrate (alpha2,3-sialyllactose) to an acceptor substrate (beta-galactosyldisaccharide), could improve the yield of desired sialylated trisaccharide by hydrolyzing lactose, a byproduct from the donor. When trans-sialidase reaction was performed with stoichiometric amounts (2 mM) of alpha2,3-sialyllactose and Galbeta(1,3)GlcNAc, the yield of NeuAcalpha(2,3)Galbeta(1,3)GlcNAc increased from 45% to 75% by the coupling of Escherichia coli beta-galactosidase. Furthermore, by changing the substrate ratio in the coupled reaction, i.e. two-fold excess of alpha2,3-sialyllactose to Galbeta(1,3)GlcNAc, above 95% of yield was achieved based on the amount of Galbeta(1,3)GlcNAc. However, two-fold excess of Galbeta(1,3)GlcNAc to alpha2,3-sialyllactose in this reaction was more desirable for the purification of NeuAcalpha(2,3)Galbeta(1,3)GlcNAc, since complete consumption of alpha2,3-sialyllactose was achieved. Efficiency of the coupled reaction was affected by the specificity of beta-galactosidase for acceptor substrate. When Galbeta(1,6)GlcNAc was used as the acceptor, E. coli beta-galactosidase hydrolyzed Galbeta(1,6)GlcNAc as well as lactose in the coupled reaction, resulting in a significant decrease in the yield of desired sialylated trisaccharide. The conversion yield of the sialylation of Galbeta(1,6)GlcNAc could be improved by employing Bacillus circulans beta-galactosidase.     

Cloning and expression of the beta-D-phosphogalactoside galactohydrolase gene of Lactobacillus casei in Escherichia coli K-12.

Lactose metabolism in Lactobacillus casei 64H is associated with the presence of plasmid pLZ64. This plasmid determines both phosphoenolpyruvate-dependent phosphotransferase uptake of lactose and beta-D-phosphogalactoside galactohydrolase. A shotgun clone bank of chimeric plasmids containing restriction enzyme digest fragments of pLZ64 DNA was constructed in Escherichia coli K-12. One clone contained the gene coding for beta-D-phosphogalactoside galactohydrolase on a 7.9-kilobase PstI fragment cloned into the vector pBR322 in E. coli strain chi 1849. The beta-D-phosphogalactoside galactohydrolase enzyme isolated from E. coli showed no difference from that isolated from L. casei, and specific activity of beta-D-phosphogalactoside galactohydrolase was stimulated 1.8-fold in E. coli by growth in media containing beta-galactosides. A restriction map of the recombinant plasmid was compiled, and with that information, a series of subclones was constructed. From an analysis of the proteins produced by minicells prepared from transformant E. coli cells containing each of the recombinant subclone plasmids, it was found that the gene for the 56-kilodalton beta-D-phosphogalactoside galactohydrolase was transcribed from an L. casei-derived promoter. The gene for a second protein product (43 kilodaltons) was transcribed in the opposite direction, presumably under the control of a promoter in pBR322. The relationship of this second product to the lactose metabolism genes of L. casei is at present unknown.     

Akt-mediated phosphorylation of the G protein-coupled receptor EDG-1 is required for endothelial cell chemotaxis

The role of the protein kinase Akt in cell migration is incompletely understood. Here we show that sphingosine-1-phosphate (S1P)-induced endothelial cell migration requires the Akt-mediated phosphorylation of the G protein-coupled receptor (GPCR) EDG-1. Activated Akt binds to EDG-1 and phosphorylates the third intracellular loop at the T(236) residue. Transactivation of EDG-1 by Akt is not required for G(i)-dependent signaling but is indispensable for Rac activation, cortical actin assembly, and chemotaxis. Indeed, T236AEDG-1 mutant sequestered Akt and acted as a dominant-negative GPCR to inhibit S1P-induced Rac activation, chemotaxis, and angiogenesis. Transactivation of GPCRs by Akt may constitute a specificity switch to integrate rapid G protein-dependent signals into long-term cellular phenomena such as cell migration.     

The Arabidopsis LOS5/ABA3 Locus Encodes a Molybdenum Cofactor Sulfurase and Modulates Cold Stress– and Osmotic Stress–Responsive Gene Expression

To understand low temperature and osmotic stress signaling in plants, we isolated and characterized two allelic Arabidopsis mutants, los5-1 and los5-2, which are impaired in gene induction by cold and osmotic stresses. Expression of RD29A-LUC (the firefly luciferase reporter gene under the control of the stress-responsive RD29A promoter) in response to cold and salt/drought is reduced in the los5 mutants, but the response to abscisic acid (ABA) remains unaltered. RNA gel blot analysis indicates that the los5 mutation reduces the induction of several stress-responsive genes by cold and severely diminishes or even completely blocks the induction of RD29A, COR15, COR47, RD22, and P5CS by osmotic stresses. los5 mutant plants are compromised in their tolerance to freezing, salt, or drought stress. los5 plants are ABA deficient, as indicated by increased transpirational water loss and reduced accumulation of ABA under drought stress in the mutant. A comparison with another ABA-deficient mutant, aba1, reveals that the impaired low-temperature gene regulation is specific to the los5 mutation. Genetic tests suggest that los5 is allelic to aba3. Map-based cloning reveals that LOS5/ABA3 encodes a molybdenum cofactor (MoCo) sulfurase. MoCo sulfurase catalyzes the generation of the sulfurylated form of MoCo, a cofactor required by aldehyde oxidase that functions in the last step of ABA biosynthesis in plants. The LOS5/ABA3 gene is expressed ubiquitously in different plant parts, and the expression level increases in response to drought, salt, or ABA treatment. Our results show that LOS5/ABA3 is a key regulator of ABA biosynthesis, stress-responsive gene expression, and stress tolerance.     

Accumulation of Polyhydroxyalkanoic Acid Containing Large Amounts of Unsaturated Monomers in Pseudomonas fluorescens BM07 Utilizing Saccharides and Its Inhibition by 2-Bromooctanoic Acid

A psychrotrophic bacterium, Pseudomonas fluorescens BM07, which is able to accumulate polyhydroxyalkanoic acid (PHA) containing large amounts of 3-hydroxy-cis-5-dodecenoate unit up to 35 mol% in the cell from unrelated substrates such as fructose, succinate, etc., was isolated from an activated sludge in a municipal wastewater treatment plant. When it was grown on heptanoic acid (C₇) to hexadecanoic acid (C₁₆) as the sole carbon source, the monomer compositional characteristics of the synthesized PHA were similar to those observed in other fluorescent pseudomonads belonging to rRNA homology group I. However, growth on stearic acid (C₁₈) led to no PHA accumulation, but instead free stearic acid was stored in the cell. The existence of the linkage between fatty acid de novo synthesis and PHA synthesis was confirmed by using inhibitors such as acrylic acid and two other compounds, 2-bromooctanoic acid and 4-pentenoic acid, which are known to inhibit β-oxidation enzymes in animal cells. Acrylic acid completely inhibited PHA synthesis at a concentration of 4 mM in 40 mM octanoate-grown cells, but no inhibition of PHA synthesis occurred in 70 mM fructose-grown cells in the presence of 1 to 5 mM acrylic acid. 2-Bromooctanoic acid and 4-pentenoic acid were found to much inhibit PHA synthesis much more strongly in fructose-grown cells than in octanoate-grown cells over concentrations ranging from 1 to 5 mM. However, 2-bromooctanoic acid and 4-pentenoic acid did not inhibit cell growth at all in the fructose media. Especially, with the cells grown on fructose, 2-bromooctanoic acid exhibited a steep rise in the percent PHA synthesis inhibition over a small range of concentrations below 100 μM, a finding indicative of a very specific inhibition, whereas 4-pentenoic acid showed a broad, featureless concentration dependence, suggesting a rather nonspecific inhibition. The apparent inhibition constant K_i (the concentration for 50% inhibition of PHA synthesis) for 2-bromooctanoic acid was determined to be 60 μM, assuming a single-site binding of the inhibitor at a specific inhibition site. Thus, it seems likely that a coenzyme A thioester derivative of 2-bromooctanoic acid specifically inhibits an enzyme linking the two pathways, fatty acid de novo synthesis and PHA synthesis. We suggest that 2-bromooctanoic acid can substitute for the far more expensive (2,000 times) and cell-growth-inhibiting PHA synthesis inhibitor, cerulenin.