A light and electron microscopic study of the flagellate-to-ameba conversion in the MyxomyceteStemonitis pallida

Summary The flagellate-to-ameba conversion process of the MyxomyceteStemonitis pallida was investigated with Nomarski optics and electron microscopy. The flagellate has two flagella, a long and a short one. When the water film containing the flagellates becomes very thin, they retract their flagella, usually the short one first and then the long one. The short flagellum is retracted by only one method, in which the sheath membrane of the flagellum fuses with the cell membrane, consequently causing the axoneme to be absorbed into the cytoplasm. Retraction of the long flagellum can be divided into four types. In all cases, fusion of the sheath membrane and the cell membrane takes place. The retracted axoneme of the long flagellum sometimes beats convulsively for about 10 minutes after retraction, and after 10–15 minutes it became indistinguishable as it was detached from the blepharoplast.Analysis of thin sections shows that the retracted axonemes disintegrate in the following squence: B-tubules, A-tubules, spokes, central microtubules. In almost all cells the degradation begins immediately after retraction and is completed within 90 minutes. Only on rare occasions, structures which seem to have been derived from retracted axonemes are observed in the ameba about 90 minutes after conversion. The basal bodies and cytoplasmic microtubules are a little more stable than the retracted axonemes. Some basal bodies of the short flagellum, whose C-tubules are affected, are present in the amebae more than 90 minutes after conversion. Cytoplasmic microtubules decrease in number and become shorter in the amebae after about 24 hours, when newly formed regions filled with flocculent material appear.     

Characteristics of light-induced 515-nm absorbance change in spinach chloroplasts at lower temperatures I. Participation of structural changes of thylakoid membranes in light-induced 515-nm absorbance change

Light-induced absorbance change at 515 nm in spinach chloroplastswas studied in the temperature range from –2?C to 27?C.Lowering of temperature had no marked effect on the extentsof initial "light-on" spike and the steady-state change overthe temperature range examined, whereas the rate of recoveryof the 515-nm change was significantly reduced at lower temperatures.Above 15?C, recovery of the 515-nm change after continuous illuminationshowed a first-order kinetics. In contrast, the recovery wascomposed of a fast and a slow phases at lower temperatures. The fast phase of the recovery of the 515-nm change was acceleratedby carbonyl cyanide m-chlorophenylhydrazone, valinomycin plusK⁺ or sodium tetraphenylboron, while the slow phase was completelyeliminated in glutaraldehyde-fixed chloroplasts. Light-inducedchange in absorbance at 546 nm, an indicator of structural changesof membrane, showed almost the same dependency on temperatureas the slow phase of the recovery of the 515-nm change. Theseresults suggest that not only electric field formation acrossthe thylakoid membrane but also structural or conformationalchanges in the membrane participate in the 515-nm absorbancechange observed under steady illumination. (Received July 5, 1976; )     

Characteristics of light-induced 515-nm absorbance change in spinach chloroplasts at lower temperatures II. Relationship between 515-nm absorbance change and rapid H+ uptake in chloroplasts after a short flash illumination

The absorbance change at 515 nm induced by a short (7.6 µsec)light flash in spinach chloroplasts was studied at sub-roomtemperatures in relation to rapid H⁺ uptake into chloroplasts. Lowering of temperature caused a marked decrease in the rateof recovery of 515-nm absorbance change after a flash illumination.Initial rate of rapid H⁺ uptake, measured with absorbance changeof bromcresol purple (BCP), was also reduced at lower temperatures,in a parallel fashion. Half-recovery time of the absorbancechange at 515 nm and rise-time of the pH-indicating absorbanceincrease of BCP coincided well at each temperature studied.Values of the calculated activation energy for these two processeswere almost the same. The parallelism between the 515-nm absorbance change and therapid H⁺ uptake after a single flash illumination was also observedwhen the electric field decay and/or H⁺ translocation were acceleratedby ionophorous antibiotics, carbonylcyanide m-chlorophenylhydrazoneor phenazine methosulfate. From these results, it is suggestedthat the rapid H⁺ uptake into chloroplast is chemically coupledto electron transfer and at the same time diffusion- (or transport-)controlled. Membrane potential, reflected in the 515-nm absorbancechange is dissipated with the rapid H⁺ influx. A model for theelectron-transfer-coupled H⁺ translocation involving a plastosemiquinoneloop is presented. Dissipation of the illumination-formed inside-positivemembrane potential by the influx of H⁺ is explained by the model. (Received September 17, 1976; )     

Light-induced chemiluminescence of luminol in spinach chloroplast fragments: Reaction of O2- with electron transfer components

Chemiluminescence of luminol (CLL) was induced by illuminatedspinach chloroplast fragments. CLL was diminished by superoxidedismutase or under anaerobic conditions and increased by anautoxidizable electron acceptor, methyl viologen. The optimumpH for CLL was 10.0-10.5. Ferredoxin and cytochrome c reducing substance (CRS) did notaffect the intensity of CLL, but accelerated the dark decayin the absence of methyl viologen. In the presence of methylviologen, ferredoxin and CRS lowered the intensity and acceleratedthe dark decay. 3-(4-Chlorophenyl)-1,1-dimethylurea diminishedCLL. Carbonylcyanide m-chlorophenylhydrazone accelerated theinitial rate of CLL increase at low concentration and inhibitedit at high concentration. Half-decay time of CLL after the cessationof light was shortened by inhibiting electron transfer on theoxidizing side of photosystem II. We conclude that most of the CLL observed in illuminated chloroplastsis dependent on O₂^–. The results also suggest that O₂^–is reduced by reduced ferredoxin or CRS and oxidized on theoxidizing side of photosystem II. The half life of O₂^–in illuminated chloroplasts was estimated from the half-decaytime of CLL to be a few sec. ¹ Present address: Kyushu Dental College, Department of Biology,Kitakyushu 803, Japan. (Received May 30, 1977; )     

Discrimination and characterization of photosystem I-and photosystem II-induced 515-nm absorbance changes in chloroplasts II. Separate local fields in thylakoids indicated by differential responses of the 515-nm absorbance change

Flash-induced 515-nm and 475-nm absorbance changes in spinachchloroplasts were investigated in the presence of 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU). DCMU reduced the magnitude of the 515-nmabsorbance change by half and almost completely diminished theabsorbance change at 475-nm. The reduction of the 475-nm absorbancechange paralleled the inhibition of the photosystem II (PS II)light reaction. When chloroplasts were illuminated with red or far-red light,the ratio of A₅₁₅/A₄₇₅ changed depending on the photosystemactivated. Wide variations in the A₅₁₅/A₄₇₅ ratio observed insubchloroplast particle preparations were probably due to theenrichment and activation of one of the photosystems. We suggest that the photosynthetic pigments in the thylakoidmembrane are heterogeneously distributed, and chlorophyll bmolecules that may be responsible for the 475- nm absorbancechange are affected by the local field formed by the PS II lightreaction. On the other hand, an electric field due to the PSI reaction probably induced the absorbance change at 515-nm (Received February 24, 1978; )     

Sidedness of membrane structures in Rhodopseudomonas sphaeroides. Electrochemical titration of the spectrum changes of carotenoid in spheroplasts,spheroplast membrane vesicles and chromatophores

The shift of the carotenoid absorption spectrum induced by illumination and valinomycin-K⁺ addition was investigated in membrane structures with different characteristics and opposite sidednesses isolated from Rhodopseudomonas sphaeroides. Right-side-out membrane structures were prepared by isotonic lysozyme-EDTA treatment of the cells (spheroplasts) and by hypotonic treatment of spheroplasts (spheroplast membrane vesicles). Inside-out membrane structures (“chromatophores”) were obtained by treating spheroplast membrane vesicles by French press or sonication.The membrane structures with either sidedness showed the same light-induced change of the “red shift” type. However, the absorbance change by K⁺ addition in the presence of valinomycin in the right-side-out membrane structures were opposite to that in the inverted vesicles, “blue shift” in the former and “red shift” in the latter. The carotenoid absorbance change was linear to membrane potential, calculated from the concentration of KCl added, with a reference on the cytoplasmic side, through positive and negative ranges.     

Effect of charge density of cationic polyelectrolytes on complex formation with DNA

The interaction between DNA and ionen polymers, -[N⁺(CH₃)₂(CH₂)_mN⁺(CH₃)₂(CH₂)_n], with m-n of 3–3, 6–6, and 6–10 were examined in order to know how the binding behavior of cationic polymers with DNA depends on the charge density of polycation. The ionen polymer has no bulky side chain and the binding forces with DNA would be attributed mainly to electrostatic interaction. When 3–3 ionen polymers were added to DNA solution, precipitable complexes with the ratio of cationic residue to DNA phosphate (+/?) of 1/1 and the free DNA molecules were segregated, while 6–6 and 6–10 ionen polymers formed soluble complexes with DNA molecules up to (+/?) = 0.5. This suggests that 3–3 ionen polymers bind cooperatively with DNA while 6–6 and 6–10 ionen polymers bind noncooperatively. The cooperative binding of 3–3 ionen polymer and the noncooperative binding of 6–6 ionen polymer were also supported by the thermal melting and recooling profiles from the midpoint between first and second meltings. It was concluded that the charge density of DNA phosphate is a critical value determining whether the ionen polymers bind to DNA by a cooperative or by a noncooperative binding, since the distance between successive cationic charges of 3–3 ionen polymer is shorter than that between successive phosphate charges on DNA double helix and those of 6–6 and 6–10 ionen polymers are longer.     

Purification and partial characterization of squalene epoxidase from rat liver microsomes

Squalene epoxidase (EC 1.14.99.7, squalene 2,3-monooxygenase (epoxidizing) was purified to an apparent homogeneity from rat liver microsomes. The purification was carried out by solubilization of microsomes by Triton X-100, fractionation with ion exchangers, hydroxyapatite, Cibacron Blue Sepharose 4B, and chromatofocusing column chromatography. A total purification of 143-fold over the first DEAE-cellulose fraction was achieved. The purified enzyme gave a single major band on SDS-polyacrylamide gel electrophoresis and the Mr was estimated to be 51 000 as a single polypeptide chain. The enzyme showed no distinct absorption spectrum in the visible regions. The squalene epoxidase activity was reconstituted with the purified enzyme, NADPH-cytochrome P-450 reductase (EC 1.6.2.4), FAD, NADPH and molecular oxygen in the presence of Triton X-100. The apparent Michaelis constants for squalene and FAD were 13 microM and 5 microM, respectively. The Vmax was about 186 nmol per mg protein per 30 min for 2,3-oxidosqualene. The enzyme activity was not inhibited by potent inhibitors of cytochrome P-450. It is suggested that squalene epoxidase is distinct from cytochrome P-450 isozymes.     

Multiple Neurite Formation in Neuroblastoma Cell Lines by Griseolic Acid, a Potent Inhibitor of Cyclic Nucleotide Phosphodiesterases

The morphological change of several neuroblastoma cell lines induced by griseolic acid, a novel and potent inhibitor of cyclic nucleotide phosphodiesterase (PDE), was examined. In the cell lines tested, Neuro-2a (a murine neuroblastoma cell line) showed dose-dependent (1 microM-1 mM) neurite extension. Griseolic acid markedly increased the intracellular cyclic AMP level of Neuro-2a cells, suppressed DNA synthesis (82% at 1 mM), and induced multipolar (multiple-neurite-bearing)-type neuritogenesis. A similar type of neurite outgrowth was induced by 8-bromo-cyclic AMP, which also elevated the intracellular cyclic AMP concentration. In contrast, when Neuro-2a cells were treated with retinoic acid, neurite formation was of the monopolar (single-neurite-bearing) type. Papaverine and theophylline, which have been frequently used as PDE inhibitors, failed to induce these morphological changes up to 1 mM, probably owing to the lesser potency of these compounds as compared with griseolic acid on the inhibition of PDE. Retinoic acid, theophylline, and papaverine were ineffective at elevating the intracellular cyclic AMP level. These results suggest that multipolar-type cell shape change in Neuro-2a cells is correlated with the accumulation of intracellular cyclic AMP and that griseolic acid is a useful compound to induce neuroblastoma cells into terminal differentiation.     

Formation of functionally active chloroplasts is determined at a limited stage of leaf development in virescent mutants of rice

The ability to form functionally active chloroplasts is determined at a certain early stage of leaf development in three non-allelic temperature-sensitive virescent mutants of rice. Temperature-shift analysis, together with anatomical observations, indicates that the intrinsic developmental signals of the virescent genes are expressed at the stage immediately following the formation of basic leaf structure, but just before the onset of leaf elongation. These signals control the expression of chloroplast-encoded genes but do not affect the subsequent morphological development of the leaf or the photo-regulation of the expression of nuclear genes encoding chloroplast proteins.