Selectivity of Ingestion and Digestion in the Chrysomonad Flagellate Ochromonas malhamensis

Ochromonas malhamensis cells were allowed to feed on autoclaved Aerobacter aerogenes, biochemically inert polystyrene latex particles of comparable size, shape and density, or a combination of these to determine if this protozoan is capable of phagotrophic selectivity under defined experimental conditions. Electron microscopic examination of Gomoristained food vacuoles indicated that the type of particle ingested was determined entirely by the type available. Apparently the biochemical or physical configuration of the particle surface is not decisive in particle selection. Gomori acid-phosphatase reaction product, which indicates digestive enzyme activity, was present in all particle-containing vacuoles, suggesting that digestive activity in O. malhamensis is due to the presence of particulate material in the vacuole, but not necessarily to the nature of the particle.     

Cryopreservation of Ochromonas in Liquid Nitrogen with Reproducibility of Thiamin Assay

The first successful cryopreservation of Ochromonas danica and Ochromonas malhamensis is reported. The freezing method was consistently reproducible for the former, but not for the latter. Ochromonas danica cultures established from frozen material still could be used as test organisms for assay of thiamin. This is the first report of a protozoon retaining its assay property after being frozen to -196 C.     

Metabolism of 24-dihydrolanosterol in Ochromonas malhamensis and Chlorella ellipsoidea

24-Dihydrolanosterol-[2-³H] was converted to cholesterol in Chlorella ellipsoidea but ergost-5-enol, poriferasterol, clionasterol were not labelled. The absence of the necessary 24(25) double bond precursor eliminates the possibility of C₂₈ and C₂₉ sterol synthesis. However, it was confirmed that 24-dihydrolanosterol was metabolized by Ochromonas malhamensis to give cholesterol, brassicasterol, and poriferasterol.     

Identification of the Mitochondrial Genome in the Chrysophyte Alga Ochromonas danica

ABSTRACT. Analysis of total DNA isolated from the Chrysophyte alga Ochromonas danica revealed, in addition to nuclear DNA, two genomes present as numerous copies per cell. The larger genome (?120 kilobase pairs or kbp) is the plastid DNA, which is identified by its hybridization to plasmids containing sequences for the photosynthesis genes rbcL, psbA, and psbC. The smaller genome (40 kbp) is the mitochondrial genome as identified by its hybridization with plasmids containing gene sequences of plant cytochrome oxidase subunits I and II. Both the 120- and 40-kbp genomes contain genes for the small and large subunits of rDNA. The mitochondrial genome is linear with terminal inverted repeats of about 1.6 kbp. Two other morphologically similar species were examined, Ochromonas minuta and Poteriochromonas malhamensis. All three species have linear mitochondrial DNA of 40 kbp. Comparisons of endonuclease restriction-fragment patterns of the mitochondrial and chloroplast DNAs as well as those of their nuclear rDNA repeats failed to reveal any fragment shared by any two of the species. Likewise, no common fragment size was detected by hybridization with plasmids containing heterologous DNA or with total mitochondrial DNA of O. danica; these observations support the taxonomic assignment of these three organisms to different species. The Ochromonas mitochondrial genomes are the first identified in the chlorophyll a/c group of algae. Combining these results with electron microscopic observations of putative mitochondrial genomes reported for other chromophytes and published molecular studies of other algal groups suggests that all classes of eukaryote algae may have mitochondrial genomes < 100 kbp in size, more like other protistans than land plants.     

Metabolism of phenols by Ochromonas danica

Abstract This study investigated the catabolic potential of a eukaryotic alga to degrade one of the most common organic pollutants, phenol. The alga, Ochromonas danica (993/28), was selected for study after screening for its heterotrophic capabilities. The catabolic versatility of the alga was elucidated by incubating with a variety of phenolic compounds. The alga removed phenol, all the cresol isomers and 3,4-xylenol from its incubation media, with phenol being removed more rapidly than any of its methylated homologues. Consequently, the alga was found to have a greater specificity for phenol than for o - or p -cresols. This study shows that O. danica could catabolize phenol and its methylated homologues.     

Enantioselective Metabolism and Interference on Tryptophan Metabolism of Myclobutanil in Rat Hepatocytes

Myclobutanil, (RS)‐2‐(4‐chlorophenyl)‐2‐(1H‐1, 2, 4‐triazol‐1‐ylmethyl) hexanenitrile is a widely used triazole fungicide. In this study, enantioselective metabolism and cytotoxicity were investigated in rat hepatocytes by chiral HPLC‐MS/MS and the methyl tetrazolium (MTT) assay, respectively. Furthermore, tryptophan metabolism disturbance in rat hepatocytes after myclobutanil exposure was also evaluated by target metabolomics method. The half‐life (t1/2) of (+)‐myclobutanil was 10.66 h, whereas that for (?)‐myclobutanil was 15.07 h. Such results indicated that the metabolic process of myclobutanil in rat hepatocytes was enantioselective with an enrichment of (?)‐myclobutanil. For the cytotoxicity research, the calculated EC₅₀ (12h) values for rac‐myclobutanil, (+)‐ and (?)‐myclobutanil were 123.65, 150.65 and 152.60 µM, respectively. The results of tryptophan metabolites profiling showed that the levels of kynurenine (KYN) and XA were both up‐regulated compared to the control, suggesting the activation effect of the KYN pathway by myclobutanil and its enantiomers which may provide an important insight into its toxicity mechanism. The data presented here could be useful for the environmental hazard assessment of myclobutanil. Chirality 27:643–649, 2015. © 2015 Wiley Periodicals, Inc.     

Distribution of Folates in Ochromonas malhamensis

SYNOPSIS. Ochromonas malhamensis contains folate derivatives active for Streptococcus faecalis, Pediococcus cerevisiae and Lactobacillus casei. These activities increase about 6-, 4- and 3-fold, respectively, on treatment with chicken liver conjugase, establishing the presence of polyglutamyl folates in the organism. The nature of the folate derivatives as well as their functional groups has been ascertained by chromatography on DEAE cellulose columns and assay of the activities of the eluted fractions, before and after the conjugase treatment, using differential microbiologic assay technic. The folate complex has been resolved into 7 fractions corresponding to N¹⁰-formyltetrahydrofolic acid, N⁵-formyltetrahydrofolic acid, unsubstituted tetrahydrofolic acid, and 4 polyglutamyl folates. The implications of these findings are indicated.     

Biosynthesis of Deoxyribonucleotides in Ochromonas malhamensis

SYNOPSIS. Uniformly ¹⁴C-labeled pyrimidine ribonucleosides and orotic-6-¹⁴C acid were fed to growing cultures of Ochromonas malhamensis and the radioactivity appearing in RNA and DNA was determined. The carbon skeletons of uridine and cytidine were incorporated intact into all of the pyrimidine nucleotides from RNA and DNA. Incorporation of radioactivity into the purine nucleotides was negligible. The evidence supports the conclusion that deoxyribonucleotide biosynthesis in this organism proceeds via a pathway involving the direct reduction of the corresponding ribonucleosides or ribonucleotides. An important role for a trans-N-deoxyribosylase in deoxyribonucleotide biosynthesis here appears to be ruled out.     

Competition of Ethionine and Methionine for Aminoacyl-tRNA Formation in an In Vitro System from Ochromonas danica*

Aminoacyl-tRNA synthetase and tRNA were isolated from the chrysomonad Ochromonas danica. The mutual effect of methionine and ethionine, and the effect of other amino acids on methionyl- and ethionyl-tRNA formation, were tested in an in vitro system. The tRNA^Met had a similar accepting capacity for either methionine or ethionine. Ethionine and methionine, but none of the other amino acids tested, competed for the same aminoacyl-tRNA synthetase. The K_m of methionine was 0.88 × 10^–5 M, and that of ethionine 5 × 10^–4 M. Ethionine inhibited methionine binding; K_i 3.4 × 10^–4 M. The respective values in a similar system isolated from E. coli were 2.2 × 10^–5, 1.95 × 10^–3, and 1.95 × 10^–3.     

Subcellular Distribution of Enzymes in Ochromonas malhamensis*

SYNOPSIS. The activity and distribution of 7 enzymes in Ochromonas malhamensis were studied. Subcellular organelles were separated by centrifugation at 648,000 g min to precipitate the larger particles; the resulting supernatant was centrifuged at 5,560,000 g min to separate the microsomal fraction from the supernatant. Sixty-four percent of the cytochrome oxidase (1.9.3.1 ferrocytochrome c:oxygen oxidoreductase, 81% of the catalase (1.11.1.6 hydrogen-peroxide: hydrogen-peroxide oxidoreductase) and 70% of the urate oxidase (1.7.3.3 urate:oxygen oxidoreductase) activity was associated with the larger particles, altho only 20% of the total protein was found in this fraction. Three acid hydrolases, cathepsin (3.4.4.9 cathepsin C, acid phosphatase (3.1.3.2 orthophosphoric monoesterphosphohydrolase) and acid ribonuclease (2.7.7.17 ribonucleate nucleotido-2′-transferase) were found mostly in the supernate (50-60%, yet their latency and their similar subcellular distribution indicated the presence of lysosomes. After 2.5 hr centrifugation in a sucrose density gradient (ρ= 1.08–1.25, the acid hydrolases showed a broad distribution which differed greatly from cytochrome oxidase associated with mitochondria. Catalase, which could not be separated from cytochrome oxidase by centrifuging on this gradient, had a different distribution after centrifugation on a kinetic gradient. Urate oxidase had a similar distribution to catalase and both these enzymes were latent, indicating the presence of peroxisomes.