Radiolytic Mapping of Solvent-Contact Surfaces in Photosystem II of Higher Plants: EXPERIMENTAL IDENTIFICATION OF PUTATIVE WATER CHANNELS WITHIN THE PHOTOSYSTEM*

Photosystem II uses water as an enzymatic substrate. It has been hypothesized that this water is vectored to the active site for water oxidation via water channels that lead from the surface of the protein complex to the Mn₄O₅Ca metal cluster. The radiolysis of water by synchrotron radiation produces amino acid residue-modifying OH^• and is a powerful technique to identify regions of proteins that are in contact with water. In this study, we have used this technique to oxidatively modify buried amino acid residues in higher plant Photosystem II membranes. Fourier transform ion cyclotron resonance mass spectrometry was then used to identify these oxidized amino acid residues that were located in several core Photosystem II subunits (D1, D2, CP43, and CP47). While, as expected, the majority of the identified oxidized residues (≈75%) are located on the solvent-exposed surface of the complex, a number of buried residues on these proteins were also modified. These residues form groups which appear to lead from the surface of the complex to the Mn₄O₅Ca cluster. These residues may be in contact with putative water channels in the photosystem. These results are discussed within the context of a number of largely computational studies that have identified putative water channels in Photosystem II.     

A switch in the mechanism of communication between the two DNA-binding sites in the SfiI restriction endonuclease

While many Type II restriction enzymes are dimers with a single DNA-binding cleft between the subunits, SfiI is a tetramer of identical subunits. Two of its subunits (a dimeric unit) create one DNA-binding cleft, and the other two create a second cleft on the opposite side of the protein. The two clefts bind specific DNA cooperatively to give a complex of SfiI with two recognition sites. This complex is responsible for essentially all of the DNA-cleavage reactions by SfiI: virtually none is due to the complex with one site. The communication between the DNA-binding clefts was examined by disrupting one of the very few polar interactions in the otherwise hydrophobic interface between the dimeric units: a tyrosine hydroxyl was removed by mutation to phenylalanine. The mutant protein remained tetrameric in solution and could bind two DNA sites. But instead of being activated by binding two sites, like wild-type SfiI, it showed maximal activity when bound to a single site and had a lower activity when bound to two sites. This interaction across the dimer interface thus enforces in wild-type SfiI a cooperative transition between inactive and active states in both dimers, but without this interaction as in the mutant protein, a single dimer can undergo the transition to give a stable intermediate with one inactive dimer and one active dimer.     

A rapid reaction analysis of uracil DNA glycosylase indicates an active mechanism of base flipping

Uracil DNA glycosylase (UNG) is the primary enzyme for the removal of uracil from the genome of many organisms. A key question is how the enzyme is able to scan large quantities of DNA in search of aberrant uracil residues. Central to this is the mechanism by which it flips the target nucleotide out of the DNA helix and into the enzyme-active site. Both active and passive mechanisms have been proposed. Here, we report a rapid kinetic analysis using two fluorescent chromophores to temporally resolve DNA binding and base-flipping with DNA substrates of different sequences. This study demonstrates the importance of the protein–DNA interface in the search process and indicates an active mechanism by which UNG glycosylase searches for uracil residues.     

Pre-eclampsia and risk of cardiovascular disease and cancer in later life: systematic review and meta-analysis

Objective To quantify the risk of future cardiovascular diseases, cancer, and mortality after pre-eclampsia.Design Systematic review and meta-analysis.Data sources Embase and Medline without language restrictions, including papers published between 1960 and December 2006, and hand searching of reference lists of relevant articles and reviews for additional reports.Review methods Prospective and retrospective cohort studies were included, providing a dataset of 3 488 160 women, with 198 252 affected by pre-eclampsia (exposure group) and 29 495 episodes of cardiovascular disease and cancer (study outcomes).Results After pre-eclampsia women have an increased risk of vascular disease. The relative risks (95% confidence intervals) for hypertension were 3.70 (2.70 to 5.05) after 14.1 years weighted mean follow-up, for ischaemic heart disease 2.16 (1.86 to 2.52) after 11.7 years, for stroke 1.81 (1.45 to 2.27) after 10.4 years, and for venous thromboembolism 1.79 (1.37 to 2.33) after 4.7 years. No increase in risk of any cancer was found (0.96, 0.73 to 1.27), including breast cancer (1.04, 0.78 to 1.39) 17 years after pre-eclampsia. Overall mortality after pre-eclampsia was increased: 1.49 (1.05 to 2.14) after 14.5 years.Conclusions A history of pre-eclampsia should be considered when evaluating risk of cardiovascular disease in women. This association might reflect a common cause for pre-eclampsia and cardiovascular disease, or an effect of pre-eclampsia on disease development, or both. No association was found between pre-eclampsia and future cancer.     

FKBP12.6 Activates RyR1: Investigating the Amino Acid Residues Critical for Channel Modulation

We have previously shown that FKBP12 associates with RyR2 in cardiac muscle and that it modulates RyR2 function differently to FKBP12.6. We now investigate how these proteins affect the single-channel behavior of RyR1 derived from rabbit skeletal muscle. Our results show that FKBP12.6 activates and FKBP12 inhibits RyR1. It is likely that both proteins compete for the same binding sites on RyR1 because channels that are preactivated by FKBP12.6 cannot be subsequently inhibited by FKBP12. We produced a mutant FKBP12 molecule (FKBP12_{E31Q/D32N/W59F}) where the residues Glu³¹, Asp³², and Trp⁵⁹ were converted to the corresponding residues in FKBP12.6. With respect to the functional regulation of RyR1 and RyR2, the FKBP12_{E31Q/D32N/W59F} mutant lost all ability to behave like FKBP12 and instead behaved like FKBP12.6. FKBP12_{E31Q/D32N/W59F} activated RyR1 but was not capable of activating RyR2. In conclusion, FKBP12.6 activates RyR1, whereas FKBP12 activates RyR2 and this selective activator phenotype is determined within the amino acid residues Glu³¹, Asp³², and Trp⁵⁹ in FKBP12 and Gln³¹, Asn³², and Phe⁵⁹ in FKBP12.6. The opposing but different effects of FKBP12 and FKBP12.6 on RyR1 and RyR2 channel gating provide scope for diversity of regulation in different tissues.     

FKBP12.6 Activates RyR1: Investigating the Amino Acid Residues Critical for Channel Modulation

We have previously shown that FKBP12 associates with RyR2 in cardiac muscle and that it modulates RyR2 function differently to FKBP12.6. We now investigate how these proteins affect the single-channel behavior of RyR1 derived from rabbit skeletal muscle. Our results show that FKBP12.6 activates and FKBP12 inhibits RyR1. It is likely that both proteins compete for the same binding sites on RyR1 because channels that are preactivated by FKBP12.6 cannot be subsequently inhibited by FKBP12. We produced a mutant FKBP12 molecule (FKBP12_{E31Q/D32N/W59F}) where the residues Glu³¹, Asp³², and Trp⁵⁹ were converted to the corresponding residues in FKBP12.6. With respect to the functional regulation of RyR1 and RyR2, the FKBP12_{E31Q/D32N/W59F} mutant lost all ability to behave like FKBP12 and instead behaved like FKBP12.6. FKBP12_{E31Q/D32N/W59F} activated RyR1 but was not capable of activating RyR2. In conclusion, FKBP12.6 activates RyR1, whereas FKBP12 activates RyR2 and this selective activator phenotype is determined within the amino acid residues Glu³¹, Asp³², and Trp⁵⁹ in FKBP12 and Gln³¹, Asn³², and Phe⁵⁹ in FKBP12.6. The opposing but different effects of FKBP12 and FKBP12.6 on RyR1 and RyR2 channel gating provide scope for diversity of regulation in different tissues.     

Activation of thiazide-sensitive co-transport by angiotensin II in the cyp1a1-Ren2 hypertensive rat

Transgenic rats with inducible expression of the mouse Ren2 gene were used to elucidate mechanisms leading to the development of hypertension and renal injury. Ren2 transgene activation was induced by administration of a naturally occurring aryl hydrocarbon, indole-3-carbinol (100 mg/kg/day by gastric gavage). Blood pressure and renal parameters were recorded in both conscious and anesthetized (butabarbital sodium; 120 mg/kg IP) rats at selected time-points during the development of hypertension. Hypertension was evident by the second day of treatment, being preceded by reduced renal sodium excretion due to activation of the thiazide-sensitive sodium-chloride co-transporter. Renal injury was evident after the first day of transgene induction, being initially limited to the pre-glomerular vasculature. Mircoalbuminuria and tubuloinsterstitial injury developed once hypertension was established. Chronic treatment with either hydrochlorothiazide or an AT1 receptor antagonist normalized sodium reabsorption, significantly blunted hypertension and prevented renal injury. Urinary aldosterone excretion was increased ≈ 20 fold, but chronic mineralocorticoid receptor antagonism with spironolactone neither restored natriuretic capacity nor prevented hypertension. Spironolactone nevertheless ameliorated vascular damage and prevented albuminuria. This study finds activation of sodium-chloride co-transport to be a key mechanism in angiotensin II-dependent hypertension. Furthermore, renal vascular injury in this setting reflects both barotrauma and pressure-independent pathways associated with direct detrimental effects of angiotensin II and aldosterone.     

Response of blanket bog vegetation to drain‐blocking

Question: Does blocking of moorland drains increase bog vegetation on blanket peat? Location: Two sites with blocked drains and two with unblocked drains on Forsinard Flows National Nature Reserve, Sutherland, UK. Methods: Vegetation cover was recorded from 70 locations, with 12 sampling points at different distances (0.5‐14.5 m) from moorland drains in each location. Gradients in the cover of species indicative of wet and dry conditions, as well as bog recovery and degradation in relation to distance from drain, were compared from a sample of drains at two sites with blocked drains and two with unblocked drains. Results: There was evidence for drain‐blocking having a negative effect on vegetation indicative of drier conditions and bog degradation. One of the blocked sites had the lowest values of these indices near to the drain and increasing at greater distances perpendicular from the drain. The two unblocked sites, and the other blocked site, had a contrasting pattern of highest values of these indices close to the drain declining with distance. Cover of species indicative of bog recovery was greater where the drains had been blocked for the longest time. Conclusions: In some cases drain‐blocking can improve the ecological functioning of blanket bogs by increasing cover of healthy bog vegetation. Further studies into the causes of such variability in restoring vegetation through drain‐blocking are needed to aid targeting of peatland restoration projects to areas or methods most likely to be effective.