Characteristics of the Photosynthetic Metabolism of Carbon in Deuterated Chlorella ellipsoidea C-27

The photosynthetic metabolism of carbon in fully deuteratedcells of Chlorella ellipsoidea C-27 (D-Chlorella), obtainedby culture in medium prepared with 100 mol% D₂O, was characterizedby examining the activities of several enzymes and the levelsof metabolic regulators in a comparison with those of ordinarycells (H-Chlorella). The cellular content of starch in D-Chlorellawas more than twice that in H-Chlorella, whereas those of sucroseand glucose were significantly lower in D-Chlorella. Deuterationof Chlorella caused marked alterations in the activities ofenzymes involved in starch metabolism. There was a significantdecrease in the activity of phosphorylase, a catabolic enzyme,and a significant increase in the activity of starch synthase,an anabolic enzyme. These alterations are probably responsiblefor the increase in the amount of starch in cells. By contrast,no marked changes were observed in the activities of enzymesand the levels of metabolic inhibitors that are involved inthe synthesis of sucrose. It seems likely, therefore, that thedecrease in the amount of sucrose in D-Chlorella was causedmainly by a deficiency in sources of carbon in the cytoplasm,as a consequence of an increase in levels of starch in chloroplasts. (Received May 13, 1992; Accepted December 1, 1992)     

Detection and first characterization of a cell-wall lytic activity in Chlorella ellipsoidea C-27

Cell wall lytic activity was detected in Chlorella ellipsoidea Gernick IAM C-27 using the [¹⁴C]-labeled cell wall as substrate. The highest activity was obtained at pH 8. and the solubilized product was a polysaccharide of high molecular weight. The lytic activity appeared to be a protease and did not hydrolyse glycosidic bonds of the cell wall polysaccharides. The activity probably solubilizes the cell wall by cleaving the peptide bonds that interconnect the polysaccharides of the cell wall.     

Growth Delay and Intracellular Changes in Chlorella ellipsoidea C-27 as a Result of Deuteration

The effects of deuterium (D) on Chlorella ellipsoidea C-27 wereinvestigated. Cells grown in a medium prepared with deuteriumoxide (D₂O) showed pronounced delays in cell growth and division;the length of a cell cycle in medium with 100 mol% D₂O was morethan 5 times longer than that in medium prepared in H₂O Thedelay caused by D₂O was not overcome by either indoleaceticacid or kinetin. The biological and ultrastractural characteristicsof deuterated .Chlorella (D-Chlorella) cells were examined.The responses of D-Chlorella to cell wall-digesting enzymesdid not differ from those of normal (H-Chlorella) cells. D-Chlorellacells were enlarged, and cellular components, such as proteins,nucleic acids, lipids and ATP, were present in larger quantitiesthan those in H-cells. The chloroplast of D-Chlorella was enlarged,but the levels of component photosynthetic pigments were significantlyreduced. By contrast, mitochondria of D-Chlorella were smallerthan those of H-cells. These changes in levels of cellular componentsand in the sizes of organelles seem to be unique to deuteration. (Received May 13, 1992; Accepted July 28, 1992)     

Purification and properties of a lytic enzyme from the cell wall of Chlorella ellipsoidea C-87

Cell wall lytic activity was detected in the culture medium and cell wall of 1AM Chlorella ellipsoidea C-87. The enzymes of both fractions had their highest activity at pH 5. The lytic activity bound to the cell wall consisted of a polysaccharide releasing enzyme, an exo-type enzyme releasing disaccharide, and glucosidase; but only the polysaccharide releasing enzyme was solubilized by lithium chloride. A polysaccharide releasing enzyme with a molecular weight around 40 kDa was isolated from the culture medium. Hemicellulose is degraded by the polysaccharide releasing enzyme, and the rigid wall by the exo-type enzyme.     

Changes in the Carbonic Anhydrase Activity and the Rate of Photosynthetic O2 Evolution during the Cell Cycle of Chlorella ellipsoidea C-27

Rhythmical changes in carbonic anhydrase activity(CA) and inphotosynthesis were observed during the cell cycle of Chlorellaellipsoidea C-27 synchronized at various concentrations of dissolvedCO₂ (dCO₂ with a regime of 16 h of light and 8 h of darkness.At a constant low concentration of dCO₂ (11 {diaeresis}M), intracellularCA activity showed obvious fluctuations with a peak at 8 h afterthe initiation of illumination, while extracellular CA activity,located on the cell surface, showed only minor fluctuationsalthough the activity was as high as the maximum activity ofintracellular CA. In contrast, obvious changes in the activitiesof intra- and extracellular CA activities were not observedat a high concentration of dCO₂ (520 {diaeresis}M). The ratioof photosynthetic activity at limiting versus saturating concentrationsof dCO₂, which is indicative of the affinity of cells for CO₂,showed clear rhythmical changes during the cell cycle and theratio was higher in low-CO₂ cells than in high-CO₂ cells. Thechanges in the ratio seemed to reflect the changes in CA activity. When the cells that had been synchronized under high CO₂ conditionswere transferred to low CO₂ conditions at any given stage inthe cell cycle, CA activity was induced in every case but thecapacity for induction of CA was greater in young cells thanin mature cells. This result suggests that the capacity of cellsto induce CA over the course of the cell cycle is closely relatedto endogenous aging of the cell. (Received August 29, 1988; Accepted December 28, 1988)     

Enzymatic Inactivation of Extracellular Carbonic Anhydrase and its Effect on K1/2 (CO2) for Photosynthesis in Chlorella ellipsoidea C-27

The ratio of the extracellular to the intracellular activityof carbonic anhydrase (CA) in cells of Chlorella ellipsoideaC-27, adapted to low levels of CO₂ for 24 h (low-CO₂ cells),was about one to one. Treatment of intact cells with PronaseP inactivated about one-half of the extracellular CA activitywithout affecting photosynthetic activity. The CA activity incell homogenates and in cell-wall ghosts liberated during celldivision was completely inactivated by the same treatment. Pretreatmentwith Glycosidase mix, Chitosanase and Macerozyme enhanced theinactivation of the CA activity in intact cells. These resultssuggest that extracellular CA is evenly distributed throughoutthe whole cell-wall region. The apparent K_1/2 for dissolved inorganic carbon (DIC) in low-CO₂cells doubled when extracellular CA was inactivated by treatmentwith Pronase P, but the K_1/2 obtained was still one-half ofthat in high-CO₂ cells. Photosynthetic ¹⁴CO₂-fixation in low-CO₂cells was enhanced by acetazolamide, whereas H¹⁴CO₃^–-fixationwas suppressed. The results suggest that CO₂ is a dominant substrateutilized by cells and that HCO₃^– is utilized after conversionto CO₂. The present results show that both intracellular andextracellular CA contribute to the increase in affinity forDIC during photosynthesis in low-CO₂ cells of Chlorella ellipsoideaC-27. (Received May 7, 1990; Accepted July 18, 1990)     

Preparation and Regeneration of Chalara paradoxa Protoplasts

A lignan mixture from sesame salad oil containing episesamin and sesamin as major components was fed to rats. Lignans at the dietary level of approximately 0.2% tended to decrease plasma and liver cholesterol levels with an accompanying increase in the fecal excretion of neutral steroids, particularly when the dietary fat source was evening primrose oil containing γ-linolenic acid. There was a decreasing trend in the specific activity of Δ5-desaturase in liver microsomes whereas that of Δ6-desaturase tended to increase, in particular in rats fed with safflower oil. The proportion of dihomo-γ-linolenate increased in response to the reduction of Δ5-desaturation activity, and that of docosapentaenoate (n-6) decreased in liver phosphatidylcholine in both groups of rats, suggesting that lignans interfered with various steps of linoleate metabolism. However, the production by the aorta of prostacyclin and by platelets of thromboxane A₂ was not influenced by lignans. Thus, episesamin and/or sesamin functioned as a regulator of cholesterol and linoleate metabolism in rats.     

灵芝原生质体的分离及其转化

对灵芝（赤芝）进行了原生质体的形成和再生实验,研究了渗透压稳定剂、菌龄、酶解条件对原生质体分离的影响,以及渗透压稳定剂、培养基对原生质体现生的影响,另外还尝试用电穿孔在灵芝原生质体中进行报告基因（report gene）β-gal的转化,结果β-gal在菌丝体中有表达。     

Purification, Kinetic and Regulatory Properties of Phosphofructokinase from Chiorella pyrenoidosa

Phosphofructokinase was purified 585-fold from Chlorella pyrenoidosaby using a combination of ammonium sulphate fractionation, filtrationthrough Sepharose 4B and chromatography on DEAE-Sephacel. Enzymestability was maintained by the presence of 50 mM P_i at pH 6.6.The optimum pH for activity was 7.7. Concentrations of substratesrequired to achieve half maximal velocity in the standard assaywere 9 µM (ATP) and 0.2 mM (fructose-6-P). ATP above 0.5mM was inhibitory. Enzyme activity was inhibited by high concentrations(10–100 mM) of P_i but lower concentrations (1–5mM) were effective in relieving the influence of other inhibitorssuch as P enolpyruvate. Inhibition by P-enolpyruvate was greaterat lower pH and with less P_i in reaction mixtures: 50% inhibitioncould be attained with 0.1 mM P-enolpyruvate. Fructose-2,6-bisphosphate,which was shown to be present in Chlorella, had no effect onthe phosphofructokinase. Chlorella appeared to contain onlyone form of phosphofructokinase, possibly in the chloroplast.No pyrophosphate :D-fructose-6-P 1-phospho transferase activitycould be detected. (Received February 20, 1984; Accepted December 5, 1984)     

Preparation and Regeneration of Mycelial Protoplasts of Alternaria eichhomiae

Preparation and regeneration of mycelial protoplasts from Alternaria eichhorniae were examined. A commercially available muralytic enzyme, Novozym 234, was used for isolation of protoplasts. The mycelial age and the pH of the stabilized buffer affected the formation of protoplasts. The maximum production of protoplasts (3,9 × 10⁸/g fresh weight mycelia) was obtained from 24-h-old mycelia digested with Novozym 234 (20 mg/ml) in a stabilized buffer of pH 6.4 and incubated in the dark at 30°C on a rotary shaker (90 r.p.m.) for 6 h. Morphological characteristics of the protoplasts varied and depended on the age of the mycelia used in protoplast production. Moreover, mycelial age had a highly significant influence (P = 0.0001) on the frequency of protoplast regeneration.     

提高酵母菌原生质体制备与再生的方法研究

研究了提高酵母菌原生质体制备与再生率的技术路线:选用酵母菌对数生长中期,约培养9～12h(A600为0.5～1.0)的细胞,采用1.5%蜗牛酶和1.0%纤维素酶的混合酶液水解去除细胞壁,经特定条件,可获得98%的原生质体制备率,通过分离到含有0.01mol/LCaCl2和15%蔗糖的YEPDS再生培养基上培养,再生率可达95%以上。     

Preparation of Metabolically Active Protoplasts and Particle Preparations from the Blue-Green Alga, Phormidium luridum

A method is described for the preparation of metabolically active protoplasts from the blue-green alga, Phormidium luridum. The isolated protoplasts are stable and are capable of endogenous respiration and photoassimilation of carbon dioxide at rates not appreciably different from the untreated filaments. The protoplasts can be ruptured by dilution of the stabilizing osmoticum and, therefore, they provide a convenient starting point for the production of cell-free preparations within a biochemical reaction mixture of interest.     

Protoplasts from Aspergillus nidulans.

A very effective lytic enzyme system for massive micro/macro-scale production of protoplasts from the filamentous fungus Aspergillus nidulans is described. A striking coincidence was observed between maximal lytic activity towards Aspergillus mycelium and the presece of both chitinase and alpha-(1 leads to 3)-glucanase activities. The release of protoplasts was greatly enhanced by preincubating the mycelium with 2-deoxy-D-glucose. Furthermore, protoplast formation was influenced by fungal age, culture conditions, pH of incubation and the osmotic stabilizer used. From 40 mg of fresh mycelium, grown for 14--16 h on 1% glucose in a low phosphate-citrate medium, preincubated with 2-deoxy-D-glucose for 45 min, and then incubated with the lytic enzyme mixture at pH 6.5 in the presence of 0.3--0.4 M (NH4) SO4, 2.5 x 10(8) stable protoplasts were produced within 3 h of incubation at 30 degrees C. Comparable results were obtained with 40--50 g of mycelium. At low osmotic stabilizer concentrations a peculiar type of regeneration was observed in the presence of the lytic enzyme system; within 12 h of incubation aberrant hyphal structure emerged from the large vacuolated protoplasts.     

Formation of Protoplasts from Resting Spores

Coat-stripped spores suspended in hypertonic solutions and supplied with two essential cations can be converted into viable protoplasts by lysozyme digestion of both cortex and germ cell wall. Calcium ions are necessary to prevent membrane rupture, and magnesium ions are necessary for changes indicative of hydration of the core, particularily the nuclear mass. Since remnant spore coat covered such protoplasts of Bacillus subtilis and the germ cell wall of B. cereus spores is not lysozyme digestible, coatless spores of B. megaterium KM were more useful for these studies. Lysozyme digestion in cation-free environment produced a peculiar semi-refractile spore core free of a cortex but prone to rapid hydration and lytic changes on the addition of cations. Strontium could replace Ca(2+) but Mn(2+) could not replace Mg(2+) in these digestions. When added to the spores, dipicolinic acid and other chelates appeared to compete with the membrane for the calcium needed for stabilization during lysozyme conversion to protoplasts. It is argued that calcium could function to stabilize the inner membrane anionic groups over the anhydrous dipicolinic acid-containing core of resting spores.     

传统中药漆姑草（Sagina japonica）中的C-27甾体成分

利用Diaion HP 20及硅胶柱层析进行化合物的分离,从乙酸乙酯萃取部位分离得到了4个化合物,借助多种光谱技术进行结构鉴定分别鉴定为(25R)-螺甾-5-烯-1β,3β-二醇1-O{O-α-L-鼠李吡喃糖苷-(1→2)-O-[β-D-木糖吡喃糖苷-(1→3)]1β-D-岩藻吡喃糖苷}(ophiopogonin D,1),(25R)-ruscogenin 1-O-[2-O-(乙酰基)-α-L-鼠李吡喃糖苷-(1→2)][β-D-木糖吡喃糖苷-(1→3)]-β-D-岩藻吡喃糖苷(2),(25R)-rascogenin 1-O-[3-0-(乙酰基)-α-L-鼠李吡喃糖苷-(1→2)][β-D-木糖吡喃糖苷-(1→3)]-β-D-岩藻吡喃糖苷(3),蜕皮甾酮(4).所有化合物均为首次从该植物中分得.     

地衣芽孢杆菌原生质体的制备、再生及转化研究

目的：提高地衣芽孢杆菌原生质体的产量和形成率,为进一步提高原生质体转化率打下基础。方法：通过酶解法对地衣芽孢杆菌工业生产菌株Bacillus licheniformis303原生质体的制备及再生条件进行了研究。考察了菌体生长状态、溶菌酶浓度、处理时间、渗透压稳定剂和再生培养基等因素对地衣芽孢杆菌原生质体的制备及再生的影响。结果：对数生长后期的菌体,以SMMP作渗透压稳定剂,溶菌酶浓度为100mg/mL,37℃下酶解30min,原生质体生成量可达8×109个/mL;再生培养基选用含1mol/L琥珀酸钠的DM3时,再生率最高可达17%。在此条件下,采用PEG法将游离型质粒pHY-P43-secQ转化宿主菌B.lichenifor-mis303,转化率可达10~15 CFU/μg DNA。