Regulation of Photosystem II core protein phosphorylation at the substrate level: Light induces exposure of the CP43 chlorophyll a protein complex to thylakoid protein kinase(s)

Light induces conformational changes in the CP43 chl-a-protein antenna complex in isolated PS II core-complexes exposing phosphorylation site(s) to PS II core-associated protein kinase(s), to added solubilized thylakoid protein kinase(s), as well as to tryptic cleavage. The substrate-activation effect is demonstrated by exposure of the PS II cores to light during the kinase assay as well as by preillumination of the PS II cores in which the endogenous kinase(s) has been inactivated by treatment with N-ethylmaleimid. In the latter case, phosphorylation was performed in darkness following addition of the solubilized protein kinase(s). The solubilized protein kinase(s) does not require light activation. The apparent molecular masses of the main protein kinase(s) associated with the PS II cores (about 31–35 kDa and 45 kDa) differ from that of the major protein kinase present in solubilized preparations obtained from spinach thylakoids (64 kDa). The light-induced exposure of CP43 increases with the light intensity in the range of 20–100 μmol photons m⁻² s⁻¹ as demonstrated by preillumination of N-ethylmaleimid treated cores followed by addition of the solubilized protein kinase(s) and performing the phosphorylation assay in darkness. This revised version was published online in June 2006 with corrections to the Cover Date.     

Inhibition of Photosystem II activity by saturating single turnover flashes in calcium-depleted and active Photosystem II

Inhibition of Photosystem II (PS II) activity induced by continuous light or by saturating single turnover flashes was investigated in Ca²⁺-depleted, Mn-depleted and active PS II enriched membrane fragments. While Ca²⁺- and Mn-depleted PS II were more damaged under continuous illumination, active PS II was more susceptible to flash-induced photoinhibition. The extent of photoinactivation as a function of the duration of the dark interval between the saturating single turnover flashes was investigated. The active centres showed the most photodamage when the time interval between the flashes was long enough (32 s) to allow for charge recombination between the S₂ or S₃ and Q_B ⁻ to occur. Illumination with groups of consecutive flashes (spacing between the flashes 0.1 s followed by 32 s dark interval) resulted in a binary oscillation of the loss of PS II-activity in active samples as has been shown previously (Keren N, Gong H, Ohad I (1995), J Biol Chem 270: 806–814). Ca²⁺- and Mn-depleted PS II did not show this effect. The data are explained by assuming that charge recombination in active PS II results in a back reaction that generates P₆₈₀ triplet and thence singlet oxygen, while in Ca²⁺- and Mn-depleted PS II charge recombination occurs through a different pathway, that does not involve triplet generation. This correlates with an up-shift of the midpoint potential of Q_A in samples lacking Ca²⁺ or Mn that, in term, is predicted to result in the triplet generating pathway becoming thermodynamically less favourable (G.N. Johnson, A.W. Rutherford, A. Krieger, 1995, Biochim. Biophys. Acta 1229, 201–207). The diminished susceptibility to flash-induced photoinhibition in Ca²⁺- and Mn-depleted PS II is attributed at least in part to this mechanism. This revised version was published online in June 2006 with corrections to the Cover Date.     

SYNTHESIS OF CELLULOSE BY ACETOBACTER XYLINUM : V. Ultrastructure of Polymer

Appearance of cellulose microfibrils in the medium of a suspension of cells of Acetobacter xylinum in buffered glucose solution was preceded by a stage during which the cellulose in the medium was amorphous within the available resolution. The size of the vertical axis of the microfibrils of the bacterial cellulose was found on the basis of measurement of shadow length to be only about 16 A. In good agreement with findings of earlier workers, the size of the lateral axis ("width") of the image of the metal-shadowed cellulose microfibrils was found to be 11 mµ. After correcting for a large part probably contributed by deposited metal in the observed width of the microfibrils, the real width is estimated roughly to be in the neighborhood of 3 mµ. To account for the occurrence of diverse morphological elements in the fields and for the fact that the cellulose fibrils are free entities rather than physical appendages of the cell, it is suggested that individual cellulose molecules are released at the cell surface and diffuse into the medium, wherein they finally enter into crystal-line patterns.     

Cold-acclimation protects photosystem II against freezing damage in the halotolerant alga Dunaliella salina

Cold-acclimation (CA) of the halotolerant alga Dunaliella was inhibited by light and by high salt. CA was associated with enhanced resistance to freezing in saline growth solutions, as manifested by protection of photosynthetic oxygen evolution and by reduced permeabilisation of the plasma membrane. Oxygen evolution activity in isolated chloroplasts was not affected by freezing, but was inhibited by high salt and the inhibition could be reversed or protected by glycerol. The activity of chloroplasts from cold-acclimated cells was more resistant to salt than of non-acclimated cells. Electron transport measurements in chloroplasts indicated that high salt inhibited PS-II, but not PS-I electron transport. High salt also inhibited PS-II thermoluminescence (TL) activity in chloroplasts. Similar inhibition of PS-II TL was observed by freezing intact cells in saline solutions. Chloroplasts from cold-acclimated cells had enhanced resistance to inhibition of PS-II electron transport and of PS-II TL by high salt. These results suggest that inhibition of oxygen evolution upon freezing Dunaliella cells may result from inactivation of PS-II due to massive influx of salt and loss of glycerol. The enhanced freeze-resistance of cold-acclimated cells to inhibition of oxygen evolution can be accounted for partly by protection of PS-II against high salt.     

Reactivation of protein aggregates by mortalin and Tid1—the human mitochondrial Hsp70 chaperone system

The mitochondrial 70-kDa heat shock protein (mtHsp70), also known in humans as mortalin, is a central component of the mitochondrial protein import motor and plays a key role in the folding of matrix-localized mitochondrial proteins. MtHsp70 is assisted by a member of the 40-kDa heat shock protein co-chaperone family named Tid1 and a nucleotide exchange factor. Whereas, yeast mtHsp70 has been extensively studied in the context of protein import in the mitochondria, and the bacterial 70-kDa heat shock protein was recently shown to act as an ATP-fuelled unfolding enzyme capable of detoxifying stably misfolded polypeptides into harmless natively refolded proteins, little is known about the molecular functions of the human mortalin in protein homeostasis. Here, we developed novel and efficient purification protocols for mortalin and the two spliced versions of Tid1, Tid1-S, and Tid1-L and showed that mortalin can mediate the in vitro ATP-dependent reactivation of stable-preformed heat-denatured model aggregates, with the assistance of Mge1 and either Tid1-L or Tid1-S co-chaperones or yeast Mdj1. Thus, in addition of being a central component of the protein import machinery, human mortalin together with Tid1, may serve as a protein disaggregating machine which, for lack of Hsp100/ClpB disaggregating co-chaperones, may carry alone the scavenging of toxic protein aggregates in stressed, diseased, or aging human mitochondria.     

Thermoluminescence and flash-induced oxygen yield in herbicide resistant mutants of the D1 protein in Synechococcus PCC7942.

Several strains of Synechococcus PCC7942 carrying point mutations in the gene psbA were studied by thermoluminescence and polarographic measurement of flash-induced oxygen yield. The following results were obtained: (a) Replacement of Ser-264 in D1 by Ala (mutant Di1) or Gly (mutant G264) resulting in DCMU and atrazine resistance leads to a downshift of the thermoluminescence (TL) B-band peak temperature from 40 degrees C in wild-type thylakoids to about 30 degrees C. In dark adapted samples of both mutants the TL and oxygen yield pattern induced by a train of single turnover flashes were strongly damped indicative of a high miss factor. (b) In contrast to Ser-264 mutants, replacement of Phe-255 in D1 by Tyr (mutant Tyr5) induced strong resistance to atrazine but not to DCMU and did not affect the peak termperature of the B-band and the flash-induced TL and oxygen yield patterns. In this respect mutant Tyr5 resembles the wild type. (c) No significant differences have been found between strains with single site mutations in psbAI and normal psbAII/psbAIII genes, and strains with same mutations in psbAI but additional deletion of psbAII and psbAIII. Obviously in strains were psbAI is present, PS II complexes containing gene products of psbAII and psbAIII are not assembled in detectable amounts. (d) Strains with double mutations at positions 264 and 255 display a downshift of the B-band peak temperature. Their oscillatory patterns of B-band intensity and oxygen yield are highly damped. This behaviour is similar to strains D1 and G264 which are modified at position 264 only. We extend reports on additivity of mutation effects on herbicide binding to binding of QB. (e) Mutations at the QB site not only influence the binding of QB and herbicides but also change the thermoluminescence quantum yield and the lifetimes of the redox states S2 and S3 of the water oxidase. This finding might indicate long ranging effects on Photosystem II exerted by structural modifications of the QB site. From these data we conclude that Ser-264 is essential for binding of atrazine, DCMU and QB, whereas Phe-255 is involved in atrazine binding and its substitution by Tyr does not markedly affect QB or DCMU binding in Synechococcus PCC7942.     

Organizing the Carotid Revascularization Endarterectomy versus Stenting Trial (CREST): National Institutes of Health, Health Care Financing Administration, and industry funding

The Carotid Revascularization Endarterectomy versus Stenting Trial (CREST) is a prospective, randomized, multicenter clinical trial of carotid endarterectomy (CEA) versus carotid artery stenting (CAS) as prevention for stroke in patients with symptomatic stenosis greater than or equal to 50%. CREST is sponsored by the US National Institute of Neurological Disorders and Stroke (NINDS) of the US National Institutes of Health (NIH), with additional support by a device manufacturer, and will provide data to the US Food and Drug Administration (FDA) for evaluation of a stent device. Because of budget constraints for CREST, Health Care Financing Administration (HCFA) reimbursement for hospital costs incurred by CREST patients will be essential. The involvement of academic scientists, industry, and three separate government agencies (NIH, FDA, HCFA) has presented many challenges in conducting the trial. A review of the pathways followed to meet these challenges may be helpful to others seeking to facilitate sharing of the costs and burdens of conducting innovative clinical research.