小眼虫的核骨架研究

小眼虫细胞经轻度超声处理、选择性抽提后,利用DGD包埋-去包埋剂电镜技术及Westernblot分析技术对其核骨架、核纤层进行了研究.结果显示;细胞核内存在一个不被DNase所降解和热三氯醋酸所去除的纤维蛋白性网架结构;核的周围有一层明显的核纤层结构;Westernblot分析表明其核纤层有两种阳性蛋白成分,一为相当于高等真核细胞核纤层蛋白laminB的强阳性成分,另一为相当于laminA的弱阳性成分,但无相当于laminC的成分.本文认为小眼虫这种低等的单细胞真核生物已具有了核骨架、核纤层结构,其核纤层的蛋白组成应该代表了核纤层进化历程中早期的一个阶段.       

CYTOCHEMICAL LOCALIZATION OF ACID PHOSPHATASES IN EUGLENA GRACILIS

The localization of induced and constitutive acid phosphatase activity in Euglena was studied by light and electron microscopy, using two different cytochemical methods. Cells grown in high phosphate medium have constitutive acid phosphatase activity in three regions: in the Golgi complex, around the paramylum bodies, and in the peri-reservoir vesicles. Cells that have formed an induced acid phosphatase by exposure to a phosphate-deficient medium have, in addition to the constitutive activity localized exactly as in the uninduced cell, a strong activity in the pellicle. The induced activity is not uniformly distributed over the pellicle, but is localized at the notch of each pellicle complex, near a group of about four fibrils and near a characteristic vesicle of the endoplasmic reticulum. In the cytostome, where fission begins during division, there is an alternation of large and small pellicle complexes, both of which have induced phosphatase activity. A similar alternation is seen over the entire pellicle of dividing cells.     

光和暗条件培养的眼虫藻(Euglena gracilis)内RuBP羧化酶的免疫定位

应用免疫金标记技术证明,在眼虫藻和其它藻类中RuBP羧化酶主要分布在蛋白核部位,这与高等植物中RuBP羧化酶分布不同,在眼虫藻叶绿体间质中有少量RuBP羧化酶存在,这与高等植物中RuBP羧化酶的分布也有相似之处。 暗中培养的眼虫藻不能形成类囊体,无RuBP羧化酶,无光合能力,只能进行异养代谢。     

CORRELATED BIOCHEMICAL AND ULTRASTRUCTURAL CHANGES IN NITROGEN-STARVED EUGLENA GRACILIS1

Growth of Euglena gracilis Z Pringsheim under photoheterotrophic conditions in a nitrogen-deprived medium resulted in progressive loss of chloroplastic material until total bleaching of the cells occurred. Biochemical analysis and ultrastructural observation of the first stages of the starvation process demonstrated an early lag phase (from 0 to 9 h) in which cells increased in size, followed by a period of cell division, apparently supported by the mobilization of some chloroplastic proteins such as the photosynthetic CO₂-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. The degradation of the enzyme started after 9 h of starvation and was preceded by a transient concentration of this protein in pyrenoidal structures. Protein nitrogen and photosynthetic pigments as well as number of chloroplasts per cell decreased during proliferation through mere distribution among daughter cells. However, after 24 h, when cell division had almost ceased, there was a slow but steady decline of photosynthetic pigments. This was paralleled by observable ultrastructural changes including progressive loss of chloroplast structure and accumulation of paramylon granules and lipid globules in the cytoplasm. These findings reinforce the role of chloroplastic materials as a nitrogen source during starvation of E. gracilis in a carbon-rich medium. The excess of ribulose-1,5-bisphosphate carboxylase/oxygenase acts as a first reservoir that, once exhausted, is superseded by the generalized disassembly of the photosynthetic structures, if the adverse environment persists more than 24 h.     

PURIFICATION AND PROPERTIES OF THE MUCUS OF EUGLENA GRACILIS (EUGLENOPHYCEAE)1

Anionic groups were demonstrated in the mucus of Euglena gracilis Klebs var. bacillaris Cori by histochemical staining with alcian blue or diaminobenzidine tetrahydrochloride and were quantified during the growth cycle with an alcian blue dye-binding assay. Mucus in the culture increased during growth and became high when the culture entered the stationary phase. Cultures were grown under conditions which uniformly labeled all sulfur containing compounds with ³⁵S. A purification scheme was devised using 0.15 M NaCl and 0.10 M EDTA at pH 8 (Marmur's solution) to separate the mucus from the cells without cell breakage. The isolated purified mucus was fractionated with sodium dodecyl sulfate (SDS) at 100° C into soluble and insoluble components. The soluble fraction was separated by SDS polyacrylamide gel electrophoresis into 18 polypeptide bands ranging from 22 to 320 kdaltons that stained with Coomassie blue; 16 of these bands also stained for carbohydrates using periodic acid-Schiffs reagent, indicating their glycoprotein nature. On hydrolysis, the SDS soluble fraction yielded xylose, fucose, rhamnose, and hexose. The SDS insoluble fraction contained no ³⁵S label, and, therefore, presumably no protein or bound sulfate; this gelatinous material does not contain the same sugar residues as the glycoproteins in the SDS soluble fraction. Its staining properties with alcian blue and its resistance to hydrolysis suggested the presence of uronic acids. Comparison with other Euglena fractions showed that bands comigrating with the mucus glycoproteins were not detectable in the fractions containing the whole cells or the culture medium. Although the mucus of Euglena yielded appreciable sulfate during mild acid treatment, most if not all of this sulfate appears to have come from the oxidation of reduced sulfur rather than from the hydrolysis of covalently bound sulfate. An infrared spectrum of the mucus showed only minor peaks in the correct regions for the S-O linkage. Thus, the mucus of Euglena is composed of glycoproteins and polysaccharides which contain little or no ester sulfate.     

EUGLENA GRACILIS IN SYNCHRONOUS DIVISION I. DRY MASS AND VOLUME CHARACTERISTICS

Volume distributions and dry mass have been measured in synchronouspopulations of Euglena gracilis, grown in a salt medium at aconstant temperature. In keeping with the approximate doublingof cell number observed in each division burst, the averagedry mass and volume are doubled in each inter-division period. ¹ Present address: Department of Biology, Tokyo MetropolitanUniversity, Setagaya.ku, Tokyo. (Received February 17, 1961; )     

EFFECTS OF DARKNESS AND OF STREPTOMYCIN ON THE FINE STRUCTURE OF EUGLENA GRACILIS

Siegesmund , Kenneth A., Walter G. Rosen , and Stanley R. Gawlik . (Marquette (J., Milwaukee, Wis.) Effects of darkness and of streptomycin on the fine structure of Euglena gracilis. Amer. Jour. Bot. 49 (2) : 137–145. Illus. 1962.—Dark-grown Euglena gracilis cells, transferred from streptomycin (SM)-containing medium to SM-free medium 5 days before transfer to light, turn green normally, indicating that proplastids are unaffected by SM. SM-bleached cells, grown in light, contain numerous bodies composed of concentric lamellae (CL bodies). These differ from chloroplasts in that their lamellae lack electron-dense dots, are not coalesced, and they lack a 3-layered limiting membrane and pyrenoids. CL bodies are absent from dark-grown normal and dark-grown SM-bleached cells, as well as from light-grown normal cells. It is suggested that CL bodies result from a derangement of chloroplast synthesis caused by SM blockage of chlorophyll synthesis.     

THE ULTRASTRUCTURE OF THE PELLICLE COMPLEX OF EUGLENA GRACILIS

The pellicle complex of E. gracilis is composed of the cell membrane, the ridge and groove with the notch, four fibrils, and the subpellicular ER. The cell membrane is of unit membrane configuration and covers the outside of the cell, the cytostome, the gullet, and the reservoir. The notch of the pellicle complex has always a close topographic relationship to two particular fibrils, as well as the subpellicular ER. The gullet is that region between the reservoir and the cytostome which, in addition to longitudinal fibrils, is surrounded by a single row of circular fibrils. The circumference of the cytostome has twenty large pellicular ridges alternating with small pellicular ridges. Alternating tall and small pellicular ridges cover the entire cell during division.     

OBSERVATIONS ON THE ACID PHOSPHATASES OF EUGLENA GRACILIS

When a bleached strain of Euglena is maintained in a medium containing very low con centrations of phosphate, the acid phosphatase activity increases. The increase in acid phosphatase activity is prevented by Actinomycin D and by p-fluorophenylalanine (PFA), indicating that the increased activity is due to de novo synthesis of acid phosphatase. When phosphate is replenished, the acid phosphatase activity decreases to the level characteristic of uninduced cells before there is any appreciable cell division. When cell division resumes in the presence of PFA, the level of acid phosphatase activity remains approximately constant. This indicates that there are two different phosphatases: a constitutive enzyme, whose synthesis is insensitive to the presence of PFA, and an induced enzyme, whose synthesis is sensitive to PFA. These enzymes are not equally sensitive to changes in pH and in fluoride concentration, thus permitting them to be assayed individually in whole toluene-treated cells. Induced cells also acquire the ability to remove phosphate from the medium very rapidly.     

ROLE OF GOLGI APPARATUS IN MUCILAGE PRODUCTION AND CYST FORMATION IN EUGLENA GRACILIS (EUGLENOPHYCEAE)1

Dark grown cells of Euglena gracilis Klebs (strain Z Pringsheim) encyst when placed in minus nitrogen media for 48–72 h in the dark. The number of cisternae per dictyosome decreases from 10–20 to 6–12 during encystment. Cisternae dilate and fill with mucilage within 12–18 h after induction. The material is secreted into the reservoir and deposited onto the cell surface. The encysting cells rotate as they develop resulting in the deposition of a thick mucilaginous layer over the cell surface. The secretion product has been identified as polysaccharide with the periodic acid-silver methenamine reaction. Mucilage has not been observed in the endoplasmic reticulum adjacent to the pellicle. The product present in the dictyosornes and on the cell surface react identically to the silver reagent.     

LE PLASTIDOME CHEZ EUGLENA GRACILIS Z1

Chloroplast morphology changes cyclically during each cell generation of synchronous photoheterotrophic cultures of Euglena gracilis Z; these cycles persist until the transition to stationary phase. Structural modifications, development of pyrenoids, and increase in chloroplast volume accompany the cessation of cell division. Chloroplasts of stationary phase heterotrophic cells resemble those of exponential phase autotrophic cells, suggesting a relationship between chloroplast morphology and available carbon.     

TREHALOSE SYNTHESIS IN EUGLENA GRACILIS (EUGLENOPHYCEAE) OCCURS THROUGH AN ENZYME COMPLEX1

The aim of this study was to isolate and characterize a trehalose‐synthesizing enzyme from Euglena gracilis Klebs. After purification by anion exchange chromatography, gel filtration, isoelectric focusing, and native electrophoresis, trehalose‐6‐phosphate synthase (TPS, EC 2.4.1.15) and trehalose‐6‐phosphate phosphatase (TPP, EC 3.1.3.12) activities could not be separated. Consequently, a TPS/TPP enzyme complex of about 250 kDa was suggested as responsible for trehalose synthesis in E. gracilis. The TPS activity was shown to be highly specific for glucose‐6‐P, and UDP‐Glc was the preferred glucose donor, but GDP‐Glc and CDP‐Glc could also act as TPS substrates. The TPP activity was highly specific for trehalose‐6‐P. In vitro phosphorylation assays revealed rapid decreases in TPS and TPP activities. These changes corresponded to variations in the elution profile of gel filtration chromatography after the phosphorylation treatment. Taken together, these results suggest that the proposed TPS/TPP complex might be regulated through a protein phosphorylation/dephosphorylation‐mediated mechanism that could affect the association state of the complex. Such a regulatory mechanism might lead to a rapid change in trehalose synthesis in response to variations in environmental conditions.     

ISOLATION AND CHARACTERIZATION OF PARAMYLON SYNTHASE FROM EUGLENA GRACILIS (EUGLENOPHYCEAE)1,

The aim of this study was to isolate and characterize the paramylon synthesizing enzyme from Euglena gracilis Klebs. A method for enzyme solubilization with high synthase activity using the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate is presented. Fractionated purification showed that the main enzyme activity was associated with the paramylon granula fraction, isolated from heterotrophically grown cells of E. gracilis. Further purification by sucrose density centrifugation resulted in a large enzyme complex with an apparent molar mass of 670 kDa (native). The complex remained active throughout the isolation procedures and produced beta-1,3-glucan in vitro. Two polypeptides of 37 and 54 kDa could be identified by photoaffinity labeling with [³²P]-UDP-glucose as substrate after SDS-PAGE.