Inhibitors of electron transport associated with photosystem II in chloroplasts

The modes of actions of six inhibitors on the electron transportsystem in the vicinity of system II in chloroplasts were studied. The first group, including piericidin A, ioxynil and broxynil,showed relatively simple modes of action on the Hill reaction,fluorescence of chlorophyll and the photobleaching of photosyntheticpigments, which are similar to the action of DCMU. As compared with inhibitors of the first group, the inhibitoryactions of salicylaldoxime, antimycin A and azide on the Hillreaction were more complicated in that they were influencedmore strongly by reaction conditions, i.e. duration of incubation,pH of the reaction mixture and illumination of chloroplasts.Inhibitors of the second group suppressed the rise in fluorescencein the induction period. However, this effect was not observedin the presence of DCMU or dithionite. Salicylaldoxime and azidewere effective in inducing photobleaching of photosyntheticpigments, whereas antimycin A inhibited the photobleaching inducedby ferricyanide or CCCP. Inhibition sites of the inhibitors in the first group are assumedto be similar to that of DCMU, whereas the inhibitors in thesecond group are effective in blocking electron transport onthe oxidizing side of system II between the primary electrondonor of system II and an intermediary electron carrier whichreceives electrons from artificial electron donors for systemII. (Received October 30, 1971; )     

Effects of growth hormone and nutrient on limb regeneration in hypophysectomized adult newts

The effect of hypophysectomy, growth hormone (GH) and an amino acid-glucose mixture on the regenerative ability of the hypophysectomized Triturus pyrrhogaster yielded the following results:

• 1 The survival time of hypophysectomized newts can be prolonged substantially by the sulfamide application.
• 2 Although the limb regeneration in the hypophysectomized newt is retarded as compared with that of the pituitary intact control, it finally completes morphogenetic process under such conditions of prolonged survival.
• 3 The injection of 100 μg of GH restored the speed of regeneration of pituitary-deprived limbs to almost a normal level.
• 4 Injections of the amino acid-glucose mixture also promoted the limb regeneration in hypophysectomized newts. However, initial delay in regeneration to the time of bud appearance was not restored by the nutrients.

     

Studies on chlorophyll fluorescence in chloroplasts II. Effect of ferricyanide on the induction of fluorescence in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea

1. The effect of preincubating spinach chloroplasts with ferricyanideon the time courses of chlorophyll- fluorescence in the presenceof 3-(3,4-dichlorophyl)-1,1-dimethylurea (DCMU) was studied.When DCMU was absent from the preincubation mixture, but wasadded just before the onset of excitation light, preincubationof chloroplasts with ferricyanide markedly affected the fluorescencekinetics. The rise-rate was lowered and consequently the areaabove the induction curve (S/Fv), which is proportional to thepool size of the electron acceptor(s) for photosystem 2, increased.The maximum increase in the S/Fv was attained after 3 min and10 min, respectively, of preincubation with 5?10^–4M and3?10^–5M ferricyanide.
2. When DCMU was present during preincubationwith ferricyanide,the effect of ferricyanide in increasingthe S/Fv, was completelyeliminated.
3. The effect of ferricyanidewas also suppressed by addition offerrocyanide to the preincubationmixture. The redox potentialof the ferri-ferrocyanide mixturewhich produced 50% suppressionof the ferricyanide effect wasabout 360 mV.
4. A similar dependency of the ferricyanide effecton the redoxpotential was observed in Tris-treated chloroplasts.However,the redox potential of cytochrome b-559 was markedlyloweredby Tris-treatment.
5. These results were explained byassuming the occurrence of asecondary electron acceptor, R,between the reaction centerof photosystem 2 and the DCMU-sensitivesite.

(Received February 27, 1973; )     

Studies on chlorophyll fluorescence in chloroplasts III. Effect of artificial electron donors for photosystexn 2 on reoxidation of the fluorescence quencher, Q, in spinach chloroplasts

1. Effects of various reducing reagents including dithionite,whichserve as artificial electron donors for photosystem 2,on therecovery of fluorescence induction in the presence of3-(3,4-dichlorophenyl)-l,l-dimethylurea(DCMU) during the darkincubation of preilluminated chloroplastswere investigated.
2. The dark recovery of fluorescence induction was not affectedby the addition of the p-phenylenediamine-ascorbate couple,the hydroquinone-ascorbate couple or manganese. Incubation ofchloroplasts with dithionite caused gradual suppression of thedark recovery.
3. Preillumination of chloroplasts caused partialinhibition ofthe recovery of fluorescence induction.
4. At lowintensities of excitation light, the fluorescence yieldincreasedvery slowly and continuously, and never reached asteady state.This continuous increase in fluorescence yieldunder weak lightwas due to photoinhibition of the dark recovery.A techniquewas devised to determine the steady state yieldof fluorescence,without the intervention of photoinhibition,at weak light intensities.The steady state yield of fluorescencein the presence of DCMUwas suppressed at lower excitation intensities.This drop inthe fluorescence yield was not altered by the presenceof addedreducing reagents but was suppressed after long preincubationof chloroplasts with dithionite.
5. The delayed fluorescencewith a decay time of seconds was affectedby dithionite butnot by other reductants.
6. Results are discussed in terms ofreoxidation of the reducedprimary electron acceptor, Q, bythe oxidized primary electrondonor for photosystem 2. A modelof the electron transport associatedwith photoreaction 2 isproposed to account for the experimentalresults obtained.

(Received February 27, 1973; )     

Activity of a two-domain antifreeze protein is not dependent on linker sequence

The reported NMR structure of RD3, a naturally occurring two-domain antifreeze protein, suggests that the two nearly identical domains are oriented to allow simultaneous binding of their active regions to the ice surface. It is implied that the nine residues linking the two domains play a role in this alignment, but this has not been established. We have designed and expressed a modified form of RD3 that replaces the nine-residue linker with a generic sequence of one serine and eight glycine residues to test the importance of the linker amino acid sequence. The modified linker is shown to have significantly different characteristics compared to the original linker. Heteronuclear nuclear Overhauser effect experiments show that the new linker residues have more mobility than the linker residues in the native protein. Further, NMR data show that the folding of the C-terminal domain is somewhat perturbed by the altered linker. Finally, distributions of residual dipolar couplings indicate that the two domains tumble and move independently of each other. Nevertheless, the thermal hysteresis activity of the modified protein is indistinguishable from that of native RD3, proving that increased activity of the two-domain antifreeze protein is not dependent on structure of the linker.     

Brain-type creatine kinase has a crucial role in osteoclast-mediated bone resorption

Osteoclasts differentiate from precursor cells of the monocyte-macrophage lineage and subsequently become activated to be competent for bone resorption through programs primarily governed by receptor activator of nuclear factor-kappaB ligand in cooperation with macrophage colony-stimulating factor. Proteins prominently expressed at late phases of osteoclastogenesis and with a supportive role in osteoclast function are potential therapeutic targets for bone-remodeling disorders. In this study, we used a proteomics approach to show that abundance of the brain-type cytoplasmic creatine kinase (Ckb) is greatly increased during osteoclastogenesis. Decreasing Ckb abundance by RNA interference or blocking its enzymatic activity with a pharmacological inhibitor, cyclocreatine, suppressed the bone-resorbing activity of osteoclasts grown in vitro via combined effects on actin ring formation, RhoA GTPase activity and vacuolar ATPase function. Activities of osteoclasts derived from Ckb-/- mice were similarly affected. In vivo studies showed that Ckb-/- mice were better protected against bone loss induced by ovariectomy, lipopolysaccharide challenge or interleukin-1 treatment than wild-type controls. Furthermore, administration of cyclocreatine or adenoviruses harboring Ckb small hairpin RNA attenuated bone loss in rat and mouse models. Our findings establish an important role for Ckb in the bone-resorbing function of osteoclasts and underscore its potential as a new molecular target for antiresorptive drug development.     

Phytoplankton blooms under dim and cold conditions in freshwater lakes of East Antarctica

The seasonal variations of limnological (water temperature, light availability, turbidity, and chlorophyll a concentration) parameters were recorded continuously from January 2004 to February 2005 at two freshwater lakes: Oyako-ike and Hotoke-ike, Sôya Coast, East Antarctica. Water was in a liquid phase throughout the year, with temperatures ranging from 0 to 10°C. The maximum photosynthetically active radiation in Lake Oyako-ike was 23.16 mol m^?2 day^?1 (at 3.8 m) and Hotoke-ike was 53.01 mol m^?2 day^?1 (at 2.2 m) in summer, and chlorophyll a concentration ranged from ca. 0.5 to 2.5 μg L^?1 (Oyako-ike) and from ca. 0.1 to 0.8 μg L^?1 (Hotoke-ike) during the study period. Increase in chlorophyll a fluorescence occurred under dim-light conditions when the lakes were covered with ice in spring and autumn, but the signals were minimum in ice-free summer in both the lakes. During spring and summer, as a result of decreasing snow cover, the chlorophyll a concentration similarly decreased when PAR was relatively high, following periods of heavy winds. The autumnal and spring increase occurred under different PAR levels (ca. 20-fold and 90-fold stronger, respectively, in autumn in both the lakes). Differences in the autumn and spring increases suggest that the spring algal community is more shade-adapted than the autumn algal community. Antarctic phytoplankton appears especially adapted to low-light levels and inhibited by strong light regimes.