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.     

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.     

Construction of shuttle vectors derived fromAcetobacter xylinum for cellulose-producing bacteriumAcetobacter xylinum

Summary Shuttle vector pUF106 was constructed by ligation ofAcetobacter xylinum plasmid pFF6 toEscherichia coli plasmid pUC18. It had unique restriction sites suitable for insertion of a foreign DNA fragment and conferred ampicillin resistance to a host. pUF106 transformed cellulose-producingA. xylinum ATCC10245 as well asE. coli JM109.     

A possible clinical application of multicytokine-producing cytotoxic mononuclear cell (MCCM) therapy

When peripheral blood mononuclear cells (PBMC) were incubated with a streptococcal preparation, OK-432, for 24 h, PBMC acquired cytolytic activity against cultured and fresh human tumor cells. Such PBMC were called OK-432-activated mononuclear cells (OK-MC). OK-MC produce several kinds of cytokines such as interferon (IFN), IFN, and tumor growth inhibitory factor (TGIF) bothin vitro andin vivo. OK-MC-produced cytokines also inhibited the growth of cultured and fresh human tumor cells. The growth inhibition was examined by human tumor clonogenic assay using a double-layer agar technique. The results indicate that two pathways of anti-tumor activity are induced in OK-MC, i.e., cell-mediated and cytokine-mediated.     

Actin cytoskeleton of resting bovine platelets

Actin filaments in resting discoid bovine platelets were examined by fluorescence and electron microscopy. Rhodamine-phalloidin staining patterns showed a characteristic wheel-like structure which consisted of a central small circle connected by several radial spokes to a large peripheral circle. This wheel-like structure was composed of actin filaments forming a characteristic arrowhead structure with heavy meromyosin from muscle. Actin filaments were densely arrayed in parallel with a marginal microtubule band and radiated out from the center to the periphery. Platelets treated with colchicine lost their marginal microtubule band but retained their wheel-like structure and normal discoid form. Cytochalasin B disrupted the wheel-like structure but not the marginal microtubule band or the normal discoid form. After simultaneous treatment with both cytochalasin B and colchicine, platelets lost their discoid shape. These results suggest that actin filaments and microtubules both play important roles in the maintenance of the discoid shape of resting bovine platelets.     

Evaluation of chemical pretreatment for enzymatic solubilization of rice straw

Summary Rice straw was treated with NaOH, peracetic acid (PA), and sodium chlorite (NaClO₂). Quantitative changes in the composition of the treated straw, crystallinity of the treated straw and extracted cellulose, and susceptibility of the treated straw to Trichoderma reesei cellulase were studied. The alkali treatment resulted in a remarkable decrease in hemicellulose as well as lignin. Consequently, the recovery of residual straw after NaOH treatment was lowest among the three chemical reagents evaluated. The treatment with PA or NaCIO₂ resulted in a slight loss in hemicellulose and cellulose in the straw. The three chemical treatments caused little or no breakdown of the crystalline structure of cellulose in the straw. The treated straw was solubilized with the culture filtrate of T. reesei. The degree of enzymatic solubilization relative to the amount of residual straw was 69% after treatment with 0.25 N NaOH, 42% after treatment with 20% PA, and 50% after treatments with NaClO₂ (twice). The degree of enzymatic solubilization relative to the amount of the untreated straw, however, was 30% after treatment with 0.25 N NaOH, 32% after treatment with 20% PA, and 37% after treatments with NaClO₂ (twice).