Uphill energy transfer in photosystem I from <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>. Time-resolved fluorescence measurements at 77 K

Energetic properties of chlorophylls in photosynthetic complexes are strongly modulated by their interaction with the protein matrix and by inter-pigment coupling. This spectral tuning is especially striking in photosystem I (PSI) complexes that contain low-energy chlorophylls emitting above 700 nm. Such low-energy chlorophylls have been observed in cyanobacterial PSI, algal and plant PSI–LHCI complexes, and individual light-harvesting complex I (LHCI) proteins. However, there has been no direct evidence of their presence in algal PSI core complexes lacking LHCI. In order to determine the lowest-energy states of chlorophylls and their dynamics in algal PSI antenna systems, we performed time-resolved fluorescence measurements at 77 K for PSI core and PSI–LHCI complexes isolated from the green alga Chlamydomonas reinhardtii. The pool of low-energy chlorophylls observed in PSI cores is generally smaller and less red-shifted than that observed in PSI–LHCI complexes. Excitation energy equilibration between bulk and low-energy chlorophylls in the PSI–LHCI complexes at 77 K leads to population of excited states that are less red-shifted (by ~?12 nm) than at room temperature. On the other hand, analysis of the detection wavelength dependence of the effective trapping time of bulk excitations in the PSI core at 77 K provided evidence for an energy threshold at ~?675 nm, above which trapping slows down. Based on these observations, we postulate that excitation energy transfer from bulk to low-energy chlorophylls and from bulk to reaction center chlorophylls are thermally activated uphill processes that likely occur via higher excitonic states of energy accepting chlorophylls.     

Energy dissipation mechanisms in the FCPb light-harvesting complex of the diatom Cyclotella meneghiniana

Transient absorption spectroscopy has been applied to investigate the energy dissipation mechanisms in the nonameric fucoxanthin-chlorophyll-a,c-binding protein FCPb of the centric diatom Cyclotella meneghiniana. FCPb complexes in their unquenched state were compared with those in two types of quenching environments, namely aggregation-induced quenching by detergent removal, and clustering via incorporation into liposomes. Applying global and target analysis, in combination with a fluorescence lifetime study and annihilation calculations, we were able to resolve two quenching channels in FCPb that involve chlorophyll-a pigments for FCPb exposed to both quenching environments. The fast quenching channel operates on a timescale of tens of picoseconds and exhibits similar spectral signatures as the unquenched state. The slower quenching channel operates on a timescale of tens to hundreds of picoseconds, depending on the degree of quenching, and is characterized by enhanced population of low-energy states between 680 and 710?nm. The results indicate that FCPb is, in principle, able to function as a dissipater of excess energy and can do this in vitro even more efficiently than the homologous FCPa complex, the sole complex involved in fast photoprotection in these organisms. This indicates that when a complex displays photoprotection-related spectral signatures in vitro it does not imply that the complex participates in photoprotection in vivo. We suggest that FCPa is favored over FCPb as the sole energy-regulating complex in diatoms because its composition can more easily establish the balance between light-harvesting and quenching required for efficient photoprotection.     

Thrombus Aspiration during Percutaneous coronary intervention in Acute non-ST-elevation myocardial infarction Study (TAPAS II)-Study design

Background and Objective. The Thrombus Aspiration during Percutaneous coronary intervention in Acute myocardial infarction Study (TAPAS) has shown that thrombus aspiration improves myocardial perfusion and clinical outcome compared with conventional primary percutaneous coronary intervention (PCI) in patients with ST-segment-elevation myocardial infarction. Impaired myocardial perfusion due to spontaneous or angioplasty-induced embolisation of atherothrombotic material also occurs in patients with non-ST-elevation myocardial infarction (NSTEMI). The aim of this study is to determine whether thrombus aspiration before stent implantation will result in improved myocardial perfusion in patients with NSTEMI compared with conventional PCI. Study design. The study is a single-centre, prospective, randomised trial with blinded evaluation of endpoints. The planned inclusion is 540 patients with acute NSTEMI who are candidates for urgent PCI. Patients are randomised to treatment with manual thrombus aspiration or to conventional PCI. The primary endpoint is the incidence of myocardial blush grade 3 after PCI. Secondary endpoints are coronary angiographic, histopathological, enzymatic, electrocardiographic and clinical outcomes including major adverse events at 30 days and one year. Implications. If thrombus aspiration leads to significant improvement of myocardial perfusion in patients with acute NSTEMI it may become part of the standard interventional approach. (Neth Heart J 2009;17:409–13.)     

Ion transport mechanisms linked to bicarbonate secretion in the esophageal submucosal glands

The esophageal submucosal glands (SMG) secrete HCO(3)(-) and mucus into the esophageal lumen, where they contribute to acid clearance and epithelial protection. This study characterized the ion transport mechanisms linked to HCO(3)(-) secretion in SMG. We localized ion transporters using immunofluorescence, and we examined their expression by RT-PCR and in situ hybridization. We measured HCO(3)(-) secretion by using pH stat and the isolated perfused esophagus. Using double labeling with Na(+)-K(+)-ATPase as a marker, we localized Na(+)-coupled bicarbonate transporter (NBCe1) and Cl(-)-HCO(3)(-) exchanger (SLC4A2/AE2) to the basolateral membrane of duct cells. Expression of cystic fibrosis transmembrane regulator channel (CFTR) was confirmed by immunofluorescence, RT-PCR, and in situ hybridization. We identified anion exchanger SLC26A6 at the ducts' luminal membrane and Na(+)-K(+)-2Cl(-) (NKCC1) at the basolateral membrane of mucous and duct cells. pH stat experiments showed that elevations in cAMP induced by forskolin or IBMX increased HCO(3)(-) secretion. Genistein, an activator of CFTR, which does not increase intracellular cAMP, also stimulated HCO(3)(-) secretion, whereas glibenclamide, a Cl(-) channel blocker, and bumetanide, a Na(+)-K(+)-2Cl(-) blocker, decreased it. CFTR(inh)-172, a specific CFTR channel blocker, inhibited basal HCO(3)(-) secretion as well as stimulation of HCO(3)(-) secretion by IBMX. This is the first report on the presence of CFTR channels in the esophagus. The role of CFTR in manifestations of esophageal disease in cystic fibrosis patients remains to be determined.     

Probing the carotenoid content of intact Cyclotella cells by resonance Raman spectroscopy

In this study, we demonstrate the selective in vivo detection of diadinoxanthin (DD) and diatoxanthin (DT) in intact Cyclotella cells using resonance Raman spectroscopy. In these cells, we were able to assess both the content of DD and DT carotenoids relative to chlorophyll and their conformation. In addition, the sensitivity and selectivity of the technique allow us to discriminate between different pools of DD on a structural basis, and to follow their fate as a function of the illumination conditions. We report that the additional DD observed when cells are grown in high-light conditions adopts a more twisted conformation than the lower levels of DD present when the cells are grown in low-light (LL) conditions. Thus, we conclude that this pool of DD is more tightly bound to a protein-binding site, which must differ from the one occupied by the DD present in LL conditions.     

Mutations that modify or exclude binding of the QA ubiquinone and carotenoid in the reaction center from Rhodobacter sphaeroides

Three single-site mutations have been introduced at positions close to the Q_A ubiquinone in the reaction centre from Rhodobacter sphaeroides. Two of these mutations, Ala M260 to Trp and Ala M248 to Trp, result in a reaction centre that does not support photosynthetic growth of the bacterium, and in which electron transfer to the Q_A ubiquinone is abolished. In the reaction centre with an Ala to Trp mutation at the M260 residue, electron transfer from the primary donor to the acceptor bacteriopheophytin is not affected by the mutation, but electron transfer from the acceptor bacteriopheophytin to Q_A is not observed. The most likely basis for these effects is that the mutation produces a structural change that excludes binding of the Q_A ubiquinone. A third mutation, Leu M215 to Trp, produces a reaction centre that has an impaired capacity for supporting photosynthetic growth. The mutation changes the nature of ubiquinone binding at the Q_A site, and renders the site sensitive to quinone site inhibitors such as o- phenanthroline. Adopting a similar approach, in which a small residue located close to a cofactor is changed to a more bulky residue, we show that the reaction centre can be rendered carotenoid-less by the mutation Gly M71 to Leu.     

Apoprotein heterogeneity increases spectral disorder and a step-wise modification of the B850 fluorescence peak position

It has already been established that the quaternary structure of the main light-harvesting complex (LH2) from the photosynthetic bacterium Rhodopseudomonas palustris is a nonameric ‘ring’ of PucAB heterodimers and under low-light culturing conditions an increased diversity of PucB synthesis occurs. In this work, single molecule fluorescence emission studies show that different classes of LH2 ‘rings’ are present in “low-light” adapted cells and that an unknown chaperon process creates multiple sub-types of ‘rings’ with more conformational sub-states and configurations. This increase in spectral disorder significantly augments the cross-section for photon absorption and subsequent energy flow to the reaction centre trap when photon availability is a limiting factor. This work highlights yet another variant used by phototrophs to gather energy for cellular development.     

Microbial Communities Inhabiting a Rare Earth Element Enriched Birnessite-Type Manganese Deposit in the Ytterby Mine,Sweden

The dominant initial phase formed during microbially mediated manganese oxidation is a poorly crystalline birnessite-type phyllomanganate. The occurrence of manganese deposits containing this mineral is of interest for increased understanding of microbial involvement in the manganese cycle. A culture independent molecular approach is used as a first step to investigate the role of microorganisms in forming rare earth element enriched birnessite-type manganese oxides, associated with water bearing rock fractures in a tunnel of the Ytterby mine, Sweden. 16S rRNA gene results show that the chemotrophic bacterial communities are diverse and include a high percentage of uncultured unclassified bacteria while archaeal diversity is low with Thaumarchaeota almost exclusively dominating the population. Ytterby clones are frequently most similar to clones isolated from subsurface environments, low temperature milieus and/or settings rich in metals. Overall, bacteria are dominant compared to archaea. Both bacterial and archaeal abundances are up to four orders of magnitude higher in manganese samples than in fracture water. Potential players in the manganese cycling are mainly found within the ferromanganese genera Hyphomicrobium and Pedomicrobium, and a group of Bacteroidetes sequences that cluster within an uncultured novel genus most closely related to the Terrimonas. This study strongly suggest that the production of the YBS deposit is microbially mediated.     

Dynamic structure of human serum transferrin from transient electric birefringence experiments

In order to investigate the secondary, tertiary, and dynamic structure of the iron-free (apo) and iron-saturated (holo) forms of human serum transferrin and its amino (N)-terminal lobe at the physiologically relevant pHs 7.4 and 5.0, we have combined ultraviolet circular dichroism (CD) spectroscopy with transient-electric birefringence (TEB) measurements. No significant changes are found in the protein's secondary structure under the different conditions studied. The tertiary structure as monitored by near-UV CD is affected by iron binding, but does not change upon decrease in pH. In contrast, TEB results indicate dramatic changes in the dynamic structure of transferrin both upon binding of iron and decrease of pH. In apotransferrin freedom of movement is found for the lobes with respect to each other, and for the domains within the lobes. The interlobal flexibility is considerably enhanced at the lower pH. Holotransferrin is found to behave as a rigid molecule. © 1995 Wiley-Liss, Inc.