FLAVOPROTEIN IN CHLORELLA ELLIPSOIDEA CATALYZING TPNH-LINKED REDUCTION OF PLASTOCYANIN

1. An enzyme possessing a capacity of catalyzing reduction of thecopper protein, plastocyanin, with reduced pyridine nucleotides(TPNH-plastocyanin reductase) was isolated from Chlorella ellipsoidea.The procedures of purification and the properties of the purifiedenzyme are described.
2. From the results of chromatographicand enzymic tests, the prostheticgroup of the enzyme was identifiedas FAD. No evidence was obtainedto indicate the participationof metal ions in the reaction.
3. The enzyme utilizes both TPNHand DPNH as electron donors, butthe reaction is about 12 timesfaster with TPNH than with DPNH.
4. The enzyme, with TPNH aselectron donor, catalyzes the reductionof Chlorella plastocyanin,cytochrome c, 2,6-dichlorophenolindophenol and oxygen in adecreasing order of reaction rate.The reaction with oxygenas electron acceptor was found to bemuch more strongly acceleratedby the addition of higher concentrationsof flavins as comparedwith the reaction with other acceptors.FAD and FMN added tothe reaction mixture are not appreciablyreduced.
5. The propertiesof the enzyme are compared with those of alliedchloroplastenzymes reported by various investigators and thepossible roleof the new enzyme in photosynthesis is discussed.

(Received January 18, 1961; )     

ENDOGENOUS LEVELS OF INDOLE-3-ACETIC ACID IN SYNCHRONOUS CULTURES OF CHLORELLA PYRENOIDOSA12

Endogenous levels of indole-3-acetic acid were mesaured in synchronous cultures of Chlorella pyrenoidosa (TX-7-11-05). The cultures were synchronized by alternating light:dark periods of 15:9 hr at a temperature of 40 ± 1 C. After 2 synchronous cycles the cultures were exposed to a low light treatment of 350 ± 100 ft-c. The time to incipient cell division under these conditions was 6 hr and 15 min. Samples were taken at 3 sampling periods during the low light treatment period:low light 0 hr (LL0); low light 3 hr (LL3); and low light 6:15 hr (LL6:15). The algal extracts were analyzed by a fluorometric procedure which measured the indole-α-pyrone product formed by the action of the trifluoracetic acid-acetic anhydride reagent with IAA. The IAA levels increased gradually from the autospore stage (5.19 μg × 10^?4/mg dry wt) to the adolescent stage (7.13 μg × 10^?4/mg dry wt) and more rapidly when approaching the ripened adult stage (14.55 μg × 10^?4/mg dry wt). The mean percentage increase from autospore to adolescent was 36.9%, and from adolescent to ripened adult 104.6%. The total percentage increase from autospore to adult was 180.3%. Levels of IAA were 2 times higher just prior to division than in the autospore stage.     

Cd2+胁迫条件下椭圆小球藻的生理应答

研究了一株高重金属抗性的椭圆小球藻在Cd2+胁迫下的生理变化,结果显示,在10-240μmol/L浓度的Cd2+胁迫下,随着金属离子浓度提高,叶绿素的总量减少,光合放氧受到抑制.Cd2+离子浓度的提高,导致了氧自由基的大大增加,同时脯氨酸、SOD(超氧化物歧化酶)以及POD(过氧化物酶)水平均大大提高.结果提示这些与消除自由基有关的代谢产物的积累,有利于细胞减少胁迫造成的损伤.CAT(过氧化氢酶)对Cd2+离子是敏感的,其活性与胁迫浓度呈负相关.       

Cd~(2 )胁迫条件下椭圆小球藻的生理应答

研究了一株高重金属抗性的椭圆小球藻在Cd2 胁迫下的生理变化 ,结果显示 ,在 10— 2 4 0 μmol/L浓度的Cd2 胁迫下 ,随着金属离子浓度提高 ,叶绿素的总量减少 ,光合放氧受到抑制。Cd2 离子浓度的提高 ,导致了氧自由基的大大增加 ,同时脯氨酸、SOD(超氧化物歧化酶 )以及POD(过氧化物酶 )水平均大大提高。结果提示这些与消除自由基有关的代谢产物的积累 ,有利于细胞减少胁迫造成的损伤。CAT(过氧化氢酶 )对Cd2 离子是敏感的 ,其活性与胁迫浓度呈负相关     

CHANGE OF FATTY ACID COMPOSITION OF CHLORELLA ELLIPSOIDEA DURING ITS CELL CYCLE

The lipids extracted from Chlorella cells at different developmentalstages were separated by chromatography on silicic acid into"nonpolar" (chloroform-eluate) and "polar" (methanol-eluate)lipid fractions. The lipids were also subjected to florisilchromatography to fractionate neutral glycerides and free fattyacids. Gas-liquid chromatographic analysis of these fractions has revealeda marked difference in their fatty acid compositions which werefound to undergo characteristic changes during the course ofalgal cell cycle. It was found that the fatty acids in the "nonpolar"lipid (fat) fraction are synthesized during the growth phasein the light and consumed during the process of cellular division. (Received September 24, 1966; )     

STUDIES ON THE METABOLISM OF UREA AND OTHER NITROGENOUS COMPOUNDS IN CHLORELLA ELLIPSOIDEA: III ASSIMILATION OF UREA

Assimilation of urea by Chlorella ellipsoidea was investigatedby using ¹⁴C-urea. It was revealed that urea carbon metabolizedby the N-starved cell was almost quantitatively recovered incarbon dioxide under dark-aerobic conditions. The rate of ureaconsumption was considerably influenced by the N-content ofalgal cells, practically no metabolism of urea being observedwith the cells containing more than 7 per cent N on a dry weightbasis. Similar results were also obtained when urea was replacedwith ammonia as nitrogen source. Based on the results obtained,a tentative scheme for the assimilation process of urea in Chlorellawas proposed. (Received February 7, 1960; )     

MODE OF NUCLEAR DIVISION IN SYNCHRONOUS CULTURES OF CHLORELLA: COMPARISON OF VARIOUS METHODS OF SYNCHRONIZATION

1. Chlorella ellipsoidea was grown synchronously using variouspossible techniques and the mode of nuclear division in eachcase was followed by staining the nuclei according to FEULGEN.
2. A satisfactory synchrony in respect to nuclear and cellulardivision was obtained by starting the culture from a homogeneouspopulation of young and small cells and by discontinuing theillumination at the stage which was called the L₃-stage. Thestarting young cells were invariably mononuclear and the L₃-cellswere either dinuclear or tetranuclear. When the L₃ were incubatedin the dark, they ripened further, and after passing througha tetranuclear stage (referred to as the L₄) divided into fourmononuclear daughter cells which have been called the D_n-cells.The most clear-cut and repetitive synchronous culture was obtainedwhen the culture (in the light) was started from the D_n-cellsand the illumination was discontinued at the L₃-stage untilthe fully ripened cells divided into four each of D_n-cells.
3. An apparently "synchronous" culture was also obtained by themethod of programmed light-and-dark regimen, in which a randomculture is subjected to a regular alternation of light and darkperiods of adequate durations. In this case, however, the cellsat different stages of culture showed irregular nuclear patterns,and the average "division number" of mother cells was not constant,being subject to change between 4.0 and 4.9.

(Received May 25, 1961; )     

SYNCHRONOUS CULTURE OF CHLORELLA: I. KINETIC ANALYSIS OF THE LIFE CYCLE OF CHLORELLA ELLIPSOIDEA AS AFFECTED BY CHANGES OF TEMPERATURE AND LIGHT INTENSITY

1. Using the technique of synchronous culture, investigationsweremade of the effects of temperature and light-intensityon cellularlife cycle of Chlorella ellipsoidea. Some improvementsin theculture technique for obtaining a good synchrony of algalgrowthwere described.
2. By following the changes of averagecell volume and cell numberoccurring during culturing, therates of the following processesof life cycle were determined:(i) "growth" (or the increasein cell mass) occurring from thestage of smaller cells (D_a)to the stage of ripened cell (L₃),(ii) "ripening" (or processofformation of "nuclear substances"as estimated from the averagenumber of daughter cells formedfrom single mother cell), and(iii) " maturing and division" which leads to the full maturationof mother cells (L-cells)and their division into separate daughtercells (D-cells).
3. "Growth"and "ripening" were found to be dependent in light,"maturingand division" light-independent. The time requiredfor "growth"and "ripening" (C) is dependent on temperaturebut independentof light intensity, the onset of "maturing anddivision" occurringat the same time (D) of culturing undervaried light intensities.The average cell volume at this stage(L₃),however, was foundto be markedly modified by light intensity;larger with highertemperatures (see Fig. 4).
4. Changes in incubation temperature(under the condition of saturatinglight intensities) were foundto affect the life cycle in thefollowing way: (i) The timeof onset of "maturing and division"(D), varies markedly withculturing temperature; earlier athigher temperatures, (ii)The average cell volume at this stagealso depends on temperature; smaller at higher temperatures.
5. The average number of daughtercells (n) emerging from singlemother cells, was found to beuninfluenced by culturing temperature;(4.0–4.1 underthe conditions of the present study). Itwas found that thedivision number n is remarkably varied bychanging the lightintensity in the "growth" and "ripening"phases; 2.0 at 1 kilolux,3.7 at 5 kilolux, 4.2 at saturatinglight intensities (10 and25 kilolux). This finding was explainedby assuming a light-dependentformation of "nuclear substances"during the "growth" and "ripening"phases, the quantity of thesubstances in the cell at L₃ stagedeterminig the division number.
6. The experimental data wereanalyzed reaction kinetically, therate constants and othercharacteristics of the reactions constitutingthe processesof life cycle were determined, and values forthe apparent activationenergy for each reaction were computed.The reactions were discussedwith special reference to theirrelationship with photosyntheticprocess was discussed.

(Received November 7, 1959; )     

PRODUCTION OF ANOMALOUS STRUCTURES IN QUERCUS RUBRA L. CALLUS CULTURES

Internodal stem segments of 2–4-month-old Quercus rubra L. seedlings were cultured on Murashige and Skoog agar medium containing NAA and BA. Structures, termed organoids, were initiated from callus on medium containing 5 mg/1 NAA and 0.1 mg/1 BA. Organoids consisted of parenchymatous cells with a distinct epidermis and a continuous vascular system. Growth occurred principally by cell division throughout the parenchymatous tissue resulting in a variety of morphological shapes. Although no organized shoot meristems were observed, normal roots were produced; such roots were connected to the vascular system of the organoid.     

STEROLS OF CHLORELLA. I. THE NATURALLY OCCURRING STEROLS OF CHLORELLA VULGARIS, C. ELLIPSOIDEA, AND C. SACCHAROPHILA

The characteristics of the sterols naturally occurring in threespecies of Chlorella were examined. The algae were grown heterotrophicallyon glucose. Sterols were extracted and isolated from the lipidfraction and were characterized by means of chemical and physicaltests. Chlorella vulgaris contained three sterols. Only the principalone, chondrillasterol, was identified. Chondrillasterol hasbeen isolated previously from the genus Scenedesmus. Chlorella ellipsoidea and Chlorella saccharophila were foundto contain sterols with ß-oriented alkyl groups atC-24 in contrast to the -oriented groups commonly found in higherplants. Poriferasterol was identified as the principal sterolof both algae. Clionasterol and 22-dihydrobrassicasterol wereidentified as the two secondary sterols present. None of thesesterols have previously been reported to occur in plants. Theisolation of 22-dihydrobrassicasterol has not been previouslyreported from any natural source. ¹Scientific Article A1153, Contribution No. 3623 of the Universityof Maryland Agricultural Experiment Station. ²This work has been supported in part by a grant from the NationalAeronautics and Space Administration.     

植物细胞培养生产抗癌药物研究进展

本文综述了国外通过植物细胞培养生产抗癌药物的进展情况。并着重介绍了长春碱、鬼臼毒素及紫杉醇的研究概况。     

VARIATION IN TOXIN PRODUCTION OF SYNCHRONIZED CULTURES OF CHRYSOCHROMULINA POLYLEPIS (PRYMNESIOPHYTA)

We studied spatial variability in giant kelp ( Macrocystis pyrifera ) forests at 84 sites along the west coast of North America in order to assess the impacts of the 1997–98 El Niño. Our sites spanned the geographic range of giant kelp in the Northern Hemisphere and were surveyed just before, immediately following, several months after, more than one year after, and nearly two years after the El Niño. Interspersion of sample units allowed us to compare the effects of this disturbance among spatial scales ranging from a few meters to more than a thousand kilometers. Variance components analyses revealed that El Niño shifted the relative importance of factors that regulate giant kelp communities from factors acting at the scale of a few meters (local control) to factors operating at hundreds of kilometers (regional control). Moreover, El Niño resulted in a near-to-complete loss of giant kelp populations throughout nearly two-thirds of the species' range. Evaluation of these effects along with oceanographic data (at the "appropriate" spatial scales), along with closer examination of giant kelp populations in the most severely impacted region (Baja) suggested that the among-region differences in giant kelp survival was due, at least in part, to El Niño-induced differences in ocean climate. Giant kelp recovery following El Niño was also scale-dependent, but driven by factors different from those of the disturbance. Here, we present results for several species of macroalgae in an attempt to relate the importance of El Niño to that of other processes in creating scale-dependent patterns of variability.     

BACTERIAL EFFECTS AND ALGAL BIOREMEDIATION BY CHLORELLA ELLIPSOIDEA GERNECK OF THE BERKELEY PIT LAKE SYSTEM

Mitman, G. G. Department of Biological Sciences, Montana Tech of The University of Montana, Butte, MT 59701 The Berkeley Pit Lake System is one of the largest contaminated sites in North America and is located near the headwaters of the largest superfund site in the U.S. It is filling at a rate of about 28.7 million liters per day with metal laden, acidic (pH 2.7) water. Chlorella ellipsoidea is one of the first autochthonous species of algae from the Berkeley Pit Lake System to be tested for its bioremediative potential. An experimental matrix was designed for this experiment using a completely randomized design (CRD). The matrix was set up with tissue culture flasks having the following treatments: Na2HPO4 at 0, 25, 50, 75, 100 mg/l, inoculated vs. no- inoculated with algae (187,500 algae/ml final), and filtered (0.2Fm) vs. non filtered pit water as variables, NaNO₃ amount was fixed at 53 mg/ml. Three replicates were made of each and the experiment lasted 60 days. The results of this experiment demonstrated significant remediation of most metals. These results will be presented. In addition, filtering seemed to negatively impact the bioremediative potential of the cultures. As a result, axenic cultures of Chlorella ellipsoidea will be tested in Berkeley pit water to determine if it is bacteria or filtration that caused these results.     

水杨酸对细胞培养生产紫杉烷的影响

研究了水杨酸对中国红豆杉细胞培养生产紫杉烷的影响。结果表明 ,适宜浓度的水杨酸对不同的紫杉烷的合成均有明显的促进作用     

唐古特山莨菪毛状根中东莨菪碱产生的研究

以唐古特山莨菪的无菌苗叶为外植体 ,通过发根农杆菌诱导获得了唐古特山莨菪的毛状根培养系统 ,应用PCR方法鉴定了转化毛状根 ,并应用薄层扫描方法对唐古特山莨菪毛状根生物碱进行了测定 ,结果表明 :在液体培养毛状根的培养基中东莨菪碱含量可达 10mg/L ,培养物中东莨菪碱的增加与莨菪碱的减少同步     

CARBON DIOXIDE PRODUCTION AND DURATION OF LIFE OF DROSOPHILA CULTURES

The total CO₂ produced by aseptic Drosophila cultures during the entire duration of life has been determined at 15°, 26°, and 30°C. in the dark and at 22–26°C. in the light. The total amount of CO₂ produced is not constant but is greater at 15° than at 26° or 30°, and is much greater in the light than in the dark. The total duration of life, therefore, is not determined by the time required to produce a limiting amount of CO₂.