Purification and Characterization of Isocitrate Lyase from Ethanol-grown Euglena gracilis

Isocitrate lyase was purified to homogeneity from ethanol-grown Euglena gracilis. The specific activity was 0.26 μmol/min/mg protein. The molecular mass of the enzyme was calculated to be 380 kDa by gel filtration on a Superose 6 column. The subunit molecular mass of the enzyme was 116 kDa as determined by SDS-polyacrylamide gel electrophoresis. These results showed that the native form of this enzyme was a trimer composed of three identical subunits. The pH optimum for cleavage and condensation reactions was 6.5 and 7.0, respectively. The K_m values for isocitrate, glyoxylate and succinate were 3.8, 1.3 and 7.7 mM, respectively. Isocitrate lyase absolutely required Mg for enzymatic activity. This is the first report of the purification of isocitrate lyase to homogeneity from Euglena gracilis.     

Negative effects of tropospheric ozone on the feed value of rice straw are mitigated by an ozone tolerance QTL

High surface ozone concentrations are recognized as an emerging threat to food security in Asia. This study aimed at determining the effects of ozone on the nutritive quality of rice straw, a by‐product of rice grain production and a major feed resource for ruminant livestock. Further, the question was addressed whether negative effects of ozone can be mitigated through molecular breeding. Rice plants from three different genotypes were exposed to four different ozone treatments in fumigation chambers from transplanting to maturity. These genotypes were (i) IR64, one of the most wide spread indica varieties in the world, (ii) Nipponbare, a typical japonica variety, and (iii) SL41, an ozone tolerant breeding line that carried chromosomal inserts at the ozone tolerance QTL OzT9 in the genetic background of Nipponbare. The treatments consisted of (i) charcoal filtered air, (ii) simulated ambient ozone concentration, (iii) 2 × ambient ozone concentration, and (iv) 2.5 × ambient ozone concentration. The effects of ozone on the chemical composition of straw were clearly dependent of the ozone level, and were significant even at ambient ozone concentration. Increases in crude ash, lignin and phenolics concentration adversely affected the digestibility as demonstrated in incubation experiments simulating rumen digestion in vitro. Negative ozone effects included reductions in the rate and extent of gas production due to inhibition of microbial fermentation, reduced formation of short chain fatty acids (SCFA), and a decrease in the true organic matter digestibility. The ozone tolerant genotype SL41 was less responsive to ozone than its more susceptible recurrent parent Nipponbare in terms of lignin and phenolics formation, organic matter digestibility and SCFA production. These data demonstrate that the feed quality of rice straw is affected by ozone even at ambient concentration, and that these negative effects are mitigated by the ozone tolerance QTL OzT9.     

Airborne‐pollen pool and mating pattern in a hybrid zone between Pinus pumila and P. parviflora var. pentaphylla

The reproductive isolation barriers and the mating patterns among Pinus pumila, P. parviflora var. pentaphylla and their hybrids were examined by flowering phenology and genetic assays of three life stages: airborne‐pollen grains, adults and seeds, in a hybrid zone on Mount Apoi, Hokkaido, Japan. Chloroplast DNA composition of the airborne‐pollen was determined by single‐pollen polymerase chain reaction. Mating patterns were analysed by estimating the molecular hybrid index of the seed parent, their seed embryos and pollen parents. The observation of flowering phenology showed that the flowering of P. pumila precedes that of P. parviflora var. pentaphylla by about 6 to 10 days within the same altitudinal ranges. Although this prezygotic isolation barrier is effective, the genetic assay of airborne‐pollen showed that the two pine species, particularly P. pumila, still have chances to form F₁ hybrid seeds. Both parental species showed a strong assortative mating pattern; F₁ seeds were found in only 1.4% of seeds from P. pumila mother trees and not at all in P. parviflora var. pentaphylla. The assortative mating was concluded as the combined result of flowering time differentiation and cross‐incompatibility. In contrast to the parental species, hybrids were fertilized evenly by the two parental species and themselves. The breakdown of prezygotic barriers (intermediate flowering phenology) and cross‐incompatibility may account for the unselective mating. It is suggested that introgression is ongoing on Mount Apoi through backcrossing between hybrids and parental species, despite strong isolation barriers between the parental species.     

Effect of Cobalamin Deficiency on the Biosynthesis of Phosphatidylcholine in Euglena gracilis

Euglena gracilis requires cobalamin (Cbl) as an essential growth factor. Phosphatidylcholine (PC) synthesis was greatly reduced by Cbl deficiency. Rapid cell division occurred after Cbl was replenished, and PC was actively synthesized during the cell divisions. When the deficient cells were given methionine (a precursor for the choline moiety), active synthesis of PC occurred even without the Cbl supplement, although cell division was not induced. As methionine synthase in Euglena requires methylcobalamin as a coenzyme, decrease in methionine synthesis may account for reduced PC synthesis under Cbl-deficient conditions. Phosphatidyleth-anolamine and phosphatidylserine synthesis were also suppressed, commensurate with decrease of PC synthesis, under Cbl deficiency, even though Cbl is not thought to participate in their synthesis. In contrast, a lot of triglyceride and wax ester accumulated in Cbl-deficient cells. Moreover, Cbl depletion altered fatty acid composition, notably due to increased proportion of odd-numbered fatty acids     

Mono-ADP-Ribosylation of Arginine Residue of Euglena gracilis Z in Synchronous Culture

ABSTRACT. In Euglena gracilis Z, a considerably high activity of mono-ADP-ribosyltransferase occurred and change of it was accompanied by a cell cycle induced by a light-dark cycle. The enzyme activity was strongly inhibited by L-arginine and supported in the presence of poly-L-arginine as a substrate, indicating that ADP-ribosylated amino acid is an arginine residue. Arginine: mono-ADP-ribosyltransferase activity was found in the chloroplasts, mitochondria, microsomes and cytosol as judged from marker enzyme activities and the activity in each organelle fluctuated with the cell cycle.     

Subcellular Localization of the GABA-Shunt Enzymes in Euglena gracilis Strain Z

SYNOPSIS. Glutamate decarboxylase, γ-aminobutyrate-α-ketoglutarate aminotransferase and NAD-linked and NADP-linked succinic semialdehyde dehydrogenase, all constituting the GABA (γ-aminobutyrate)-shunt pathway of glutamate metabolism are localized in the mitochondrial matrix in a streptomycin-bleached mutant of Euglena gracilis strain Z. Glutamate dehydrogenase, requiring NADP as the cofactor, was distributed in the cytoplasm. An improved version of the controlled digestion method for preparing Euglena mitochondria, which involves use of trypsin and a trypsin inhibitor and removal of broken cells before mechanical disruption of cells, is also described.     

DEVELOPMENT OF VAGINAL EPITHELIUM SHOWING IRREVERSIBLE PROLIFERATION AND CORNIFICATION IN NEONATALLY ESTROGENIZED MICE: AN ELECTRON MICROSCOPE STUDY *

Irreversible proliferation and cornification of the mouse vaginal epithelium were induced by 10 daily injections of 20μg estradiol-17β starting on the day of birth. Development of the irreversible vaginal epithelium during the period of estrogen injections in early postnatal life was observed under light and electron microscopes. Small electron-dense cells (A-cells) in clusters were present in the columnar vaginal epithelium of newborn mice. A-cells were proliferated by 2 daily estrogen injections. At the sites of A-cell clumps, large electron-dense cells (B-cells) characterized by long winding cytoplasmic processes appeared in mice given 3 daily injections, forming nodules which then fused together to form a layer under the columnar epithelium after 4 daily injections. In mice given 7 daily injections, the primary epithelium was shed by the superficial cornification of the newly formed layer. The B-cell membrane bore fewer desmosomes than in the basal and intermediary cells of the vaginal epithelium of ovariectomized ‘normal’ adult mice after 5 daily injections of 100μg estradiol-17β. Hyperplastic epithelial downgrowths in old ovariectomized mice given neonatal estrogen injections contained another type of cells with reduced density which formed much fewer processes and only a few desmosomes (C-cells).