Effect of cycloheximide on the process of "glucose-bleaching" in Chlorella protothecoides

The process of bleaching of Chlorella protothecoides inducedby the addition of glucose was strongly inhibited by cycloheximide,an inhibitor of protein synthesis, whereas it was suppressedonly weakly by chloramphenicol, puromycin and ethionine. Whencycloheximide was added simultaneously with glucose at the beginningof die bleaching experiment, no bleaching of algal cells occurredduring the subsequent incubation. When it was added after glucose,the bleaching of algal cells proceeded for a period of timeas actively as in the control, then gradually ceased. Cycloheximidewas found to suppress the uptake of glucose by algal cells,and to severely inhibit the assimilation of glucose into lipidswhen added at the beginning of the bleaching experiment. Theseinhibitory effects of cycloheximide are discussed in relationto the induction of "glucose-bleaching" in algal cells. (Received December 16, 1968; )     

Electron flow from hydrogen peroxide in photosystem II-catalyzed oxidation-reduction reactions of spinach chloroplast fragments

Oxidation-reduction reactions of photosystem II were investigatedin spinach chloroplast fragments. Chloroplast fragments treatedwith 8-hydroxyquinoline sulfate showed only a low activity forthe 2,6-dichlorophenolindophenol (DPIP) Hill reaction, as wasobserved in chloroplast fragments treated with a high-concentrationof Tris buffer. Hydrogen peroxide could donate electrons tophotoreaction center II in chloroplast fragments treated with8-hydroxyquinoline, high-concentration Tris, or ethylene glycol,but water could not serve as an electron donor in these preparations.Electrons from hydrogen peroxide were transferred to DPIP, ferricyanide,and p-benzoquinone viaphotosystem II. (Received May 12, 1971; )     

Reactions of photosystems I and II in spinach chloroplast fragments treated with ethylene glycol

Photochemical reactions of chloroplast fragments isolated fromspinach leaves were measured in the presence of ethylene glycolor were measured after washing with an ethylene glycol-containingmedium. 2,6-Dichlorophenolindophenol (DPIP) photoreduction,oxygen evolution and oxygen uptake (a photosystem I reaction)were investigated in ethylene glycol-treated chloroplast fragments.By washing with ethylene glycol, oxygen evolution was stronglyinhibited, but oxygen uptake was not much affected by ethyleneglycol washing. Chloroplast fragments in 50% ethylene glycolmaintained a high rate of DPIP photoreduction (85% of the controlactivity in an ethylene glycol-less medium). In 67% ethyleneglycol, DPIP photoreduction mediated by photosystem II was eliminatedand only a small rapid reduction mediated by photosystem I wasobserved. Chloroplast fragments inhibited by ethylene glycolphotoreduced DPIP in the presence of p-aminophenol added asan artificial electron donor to photosystem II. The restoredactivity of DPIP photoreduction was inhibited by 3-(3',4'- dichlorophenyl)-1,1-dimethylurea. (Received September 8, 1970; )     

DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES: V. DEGENERATION OF CHLOROPLASTS INDUCED BY DIFFERENT CARBON SOURCES, AND EFFECTS OF SOME ANTIMETABOLITES UPON THE PROCESS INDUCED BY GLUCOSE

1. The green cells of Chlorella protothecoides were bleached todifferent extents when incubated (in the dark) in the nitrogen-freemedia containing, besides basal mineral nutrients, glucose,fructose, galactose, glycerol or acetate. Glucose and fructosewere found to have the strongest bleaching effect. Additionof a nitrogen source (urea) caused a considerable reductionof the bleaching. It was assumed that from the different carbonsources a certain common intermediate(s) causing the bleachingis formed, and that in the presence of the nitrogen source thesubstance is removed by reacting with it.
2. Using glucose asbleach-inducing agent, the effects of someantimetabolites uponthe processes of bleaching, division andgrowth of green algalcells were investigated, and it was demonstratedthat the processof bleaching occurs without being accompaniedby growth anddivision of the algal cells.
3. It was found that during theprocess of bleaching no net increasesin RNA and protein tookplace.

(Received March 11, 1965; )     

METABOLISM OF GLUCOSE IN THE PROCESS OF "GLUCOSE-BLEACHING" OF CHLORELLA PROTOTHECOIDES

1. As previously demonstrated, normal cells of Chlorella protothecoidesare bleached with degeneration of chloroplasts when they areincubated, under aerobic conditions—either in the lightor in darkness—, in a glucose-containing medium withoutadded nitrogen source ("glucose-bleaching"). It was found inthe present study that under the atmosphere of N₂, neither bleachingnor growth of algal cells occurs in the dark, while in the lighta significant growth of cells takes place with formation ofa certain amount of chlorophyll.
2. Studies on the effects ofvarious inhibitors (ammonium ion,DNP, CMU, -hydroxysulphonates,arsenate, cyanide, azide, andantimycin A) under different conditionsshowed that oxidativephosphorylation is a necessary processfor the occurrence ofthe glucosebleaching as well as the assimilationof glucose(cellular growth). Under light-anaerobic conditionsin the presenceof glucose, assimilation of glucose (cellulargrowth) takesplace being supported by photophosphorylation,but no bleachingoccurs.
3. When the algal cells in the courseof bleaching were transferredto the glucose-free mineral medium,the cell growth ceased immediatelybut the cell bleaching proceededfor several hours before itscessation. The respiratory activity,which was high in the glucose-containingmedium, became loweron transferring the algal cells into theglucose-free medium.The lowered level of respiration was maintained,for more than8 hr after the transfer of cells to the glucose-freemedium.
4. When the cells in the course of bleaching were placed underthe atmosphere of N₂, the cell bleaching ceased almost instantaneously.
5. Based on these observations and other inhibition experiments,it was inferred that a certain intermediate(s) produced by theaerobic respiration of glucose is closely associated with theoccurrence of cell bleaching, and that an O₂-requiring stepmay be involved in the process of chlorophyll degradation.

(Received September 9, 1965; )     

THE ROLE OF RESPIRATION AND PHOTOSYNTHESIS IN THE CHLOROPLAST REGENERATION IN THE "GLUCOSE-BLEACHED" CELLS OF CHLORELLA PROTOTHECOIDES

1. As previously demonstrated, entirely chlorophyll-less cellsof Chlorella protothecoides are obtained when the alga is grownin a medium rich in glucose and poor in nitrogen source (urea).These cells, which are referred to as "glucose-bleached" cells,have neither discernible chloroplast structures nor photosyntheticactivity. When the "glucose-bleached" cells are incubated, inthe light, in a nitrogen-enriched mineral medium without addedglucose, they turn green, after an induction period, with regenerationof chloroplasts and development of the capacity for performingnormal photosynthesis. In the present study, changes in respiratoryactivity of algal cells during the process of greening (chloroplastregeneration) were followed, and the effects of various inhibitorsof respiration and photosynthesis on the greening process wereexamined. 2. The glucose-bleached cells showed a very low activity ofrespiration, and the activity increased markedly during an earlyphase of chloroplast regeneration, showing, however, a decreaseduring the subsequent phase of greening. 3. Some antimetabolites which inhibited the cell respiration,were found to suppress also the greening of cells. 2,4-Dinitrophenoland azide, potent inhibitors of oxidative phosphorylation, acceleratedconsiderably both the respiration and greening of algal cells.CMU inhibited completely photosynthesis of the greening cells,but suppressed only slightly the greening process. 4. Based on these results it was concluded that the primaryrole of respiration in the chloroplast regeneration in the glucose-bleachedcells is to produce oxidized carbon compounds (and perhaps reducedforms of NAD and NADP) for various biosynthetic reactions. Itwas further suggested that ATP may be supplied for the chloroplastregeneration by a certain means different from the oxidativephosphorylation or photophosphorylation. The activities of photosyntheticphosphorylation and CO₂-fixation developing in the greeningcells do not appear to play any essential role in the chloroplastregeneration. (Received December 27, 1965; )     

EARLY HISTOGENESIS OF TRANSPLANTED MOUSE MAMMARY GLANDS III. PARENCHYMAL-STROMAL INTERACTIONS *

Mammary isografts dissociated in gland-free fat pads in 24 hr but regenerated single glands. Isografts in the anterior chamber of the eye did not dissociate but regenerated only after collagenase treatment and retransplantation to gland-free fat pads.     

Acetate metabolism in the process of "acetate-bleaching" of Chlorella protothecoides

Green cells of Chlorella protothecoides when incubated in amedium containing acetate but no nitrogen source, have beenshown to be bleached as strongly as in glucose-induced bleaching.Using U-¹⁴C-acetate as tracer, the acetate metabolism of algalcells during the process of acetate-induced bleaching was investigated.Changes in algal cell activities for respiration and assimilationof added ¹⁴C-acetate were followed during bleaching processesin "acetate-adapted" and "non-adapted" green cells. As in glucose-inducedbleaching of algal cells, algal cell activity for incorporating¹⁴C into lipids showed the most characteristic change, suggestingthat lipogenesis is causally related to the occurrence of bleachingin algal cells. (Received March 5, 1969; )     

Detection of recombinant haplotypes in wild mice (Mus musculus) provides new insights into the origin of Japanese mice

Japanese house mice (Mus musculus molossinus) are thought to be a hybrid lineage derived from two prehistoric immigrants, the subspecies M. m. musculus of northern Eurasia and M. m. castaneus of South Asia. Mice of the western European subspecies M. m. domesticus have been detected in Japanese ports and airports only. We examined haplotype structuring of a 200 kb stretch on chromosome 8 for 59 mice from throughout Eurasia, determining short segments (≈ 370–600 bp) of eight nuclear genes (Fanca, Spire2, Tcf25, Mc1r, Tubb3, Def8, Afg3l1 and Dbndd1) which are intermittently arranged in this order. Where possible we identified the subspecies origin for individual gene alleles and then designated haplotypes for concatenated alleles. We recovered 11 haplotypes among 19 Japanese mice examined, identified either as ‘intact’ haplotypes derived from the subspecies musculus (57.9%), domesticus (7.9%), and castaneus (2.6%), or as ‘recombinant’ haplotypes (31.6%). We also detected recombinant haplotypes unique to Sakhalin. The complex nature of the recombinant haplotypes suggests ancient introduction of all three subspecies components into the peripheral part of Eurasia or complicated genomic admixture before the movement from source areas. ‘Intact’domesticus and castaneus haplotypes in other Japanese wild mice imply ongoing stowaway introductions. The method has general utility for assessing the history of genetic admixture and for disclosing ongoing genetic contamination.     

STUDIES ON A CAROTENE-PROTEIN COMPLEX ISOLATED FROM GREEN LEAVES

1. Two new methods of preparing a carotene-protein complex fromchloroplasts were described, in addition to the one previouslyreported by the present authors.
2. Light-scattering measurementswere performed and from the dissymmetryratio of the scattering,the particle size of the complex wascalculated. The mean radiuscorresponding to the weightaverageparticle weight was foundto be 56 mµ. This result wasdiscussed with referenceto the previously reported values obtainedfrom sedimentationmeasurements and electron micrographic determinations.
3. Thedepolarization of scattered light was measured. Large valuesobtained for the degree of depolarization of the incident lightrevealed a high degree of anisotropy in the particle of thecarotene-protein complex.

¹Present address: Johnson Foundation, University of Pennsylvania,Philadelphia, Pa., U.S.A. (Received July 8, 1960; )