Determination of the rate and yield of B-side quinone reduction in Rhodobacter capsulatus reaction centers

In the native purple bacterial reaction center (RC), light-driven charge separation utilizes only the A-side cofactors, with the symmetry related B-side inactive. The process is initiated by electron transfer from the excited primary donor (P*) to the A-side bacteriopheophytin (P* --> P+ H(A)-) in approximately 3 ps. This is followed by electron transfer to the A-side quinone (P+ H(A)- --> P+ Q(A)-) in approximately 200 ps, with an overall quantum yield of approximately 100%. Using nanosecond flash photolysis and RCs from the Rhodobacter capsulatus F(L181)Y/Y(M208)F/L(M212)H mutant (designated YFH), we have probed the decay pathways of the analogous B-side state P+ H(B)-. The rate of the P+ H(B)- --> ground-state charge-recombination process is found to be (3.0 +/- 0.8 ns)(-1), which is much faster than the analogous (10-20 ns)(-1) rate of P+ H(A)- --> ground state. The rate of P+ H(B)- --> P+ Q(B)- electron transfer is determined to be (3.9 +/- 0.9 ns)(-1), which is about a factor of 20 slower than the analogous A-side process P+ H(A)- --> P+ Q(A)-. The yield of P+ H(B)- --> P+ Q(B)- electron-transfer calculated from these rate constants is 44%. This value, when combined with the known 30% yield of P+ H(B)- from P in YFH RCs, gives an overall yield of 13% for B-side charge separation P* --> P+ H(B)- --> P+ Q(B)- in this mutant. We determine essentially the same value (15%) by comparing the P-bleaching amplitude at approximately 1 ms in YFH and wild-type RCs.     

Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.      

The sensitivity of Afromontane tarns in the Maloti-Drakensberg region of South Africa and Lesotho to acidic deposition

Despite their remoteness from sources of atmospheric pollutant emissions, the Afromontane tarns in the Maloti-Drakensberg region are perfect candidates to study the negative effects of acidifying atmospheric pollution, because mountain lakes are widely recognised as sentinel ecosystems, unimpacted by direct human disturbance within their catchments. Thirty-four tarns were sampled in the Maloti-Drakensberg region and most were found to be extremely sensitive to acidic deposition, as indicated by their low acid neutralising capacity. There are very few studies of freshwater critical loads for any region within South Africa. The steady-state water chemistry model (SSWC) was adapted and used to determine critical loads, whereas exceedance was estimated relative to modelled regional deposition data, in order to understand the risk of harmful effects to aquatic ecosystems. Seventy-six percent of sampled sites across the Maloti-Drakensberg would exceed critical loads even at the lowest modelled deposition levels, but there are no current measured deposition data for the region. The sensitivity of the Maloti-Drakensberg tarns needs to be considered in future policy formulation regarding acceptable levels of acidifying atmospheric pollution from South Africa’s energy sector and indicates the need for assessing aquatic ecosystem impacts in other regions of South Africa.     

Influence of shape-memory stent grafts on local aortic compliance

The effect of repair techniques on the biomechanics of the aorta is poorly understood, resulting in significant levels of postoperative complications for patients worldwide. This study presents a computational analysis of the influence of Nitinol-based devices on the biomechanical performance of a healthy patient-specific human aorta. Simulations reveal that Nitinol stent-grafts stretch the artery wall so that collagen is stretched to a straightened high-stiffness configuration. The high-compliance regime (HCR) associated with low diastolic lumen pressure is eliminated, and the artery operates in a low-compliance regime (LCR) throughout the entire cardiac cycle. The slope of the lumen pressure–area curve for the LCR post-implantation is almost identical to that of the native vessel during systole. This negligible change from the native LCR slope occurs because the stent-graft increases its diameter from the crimped configuration during deployment so that it reaches a low-stiffness unloading plateau. The effective radial stiffness of the implant along this unloading plateau is negligible compared to the stiffness of the artery wall. Provided the Nitinol device unloads sufficiently during deployment to the unloading plateau, the degree of oversizing has a negligible effect on the pressure–area response of the vessel, as each device exerts approximately the same radial force, the slope of which is negligible compared to the LCR slope of the native artery. We show that 10% oversizing based on the observed diastolic diameter in the mid descending thoracic aorta results in a complete loss of contact between the device and the wall during systole, which could lead to an endoleak and stent migration. 20% oversizing reaches the Dacron enforced area limit (DEAL) during the pulse pressure and results in an effective zero-compliance in the later portion of systole.

     

Correlating ultrafast function with structure in single crystals of the photosynthetic reaction center

Femtosecond transient absorbance spectroscopy was applied to the study of primary electron transfer in single reaction center crystals from Rhodobacter sphaeroides. Polarized transient absorption spectra of individual crystals are shown to correlate with polarized ground-state absorption spectra and to track cofactor transition moment directions calculated from the crystallographic structure. Electron transfer from the bacteriochlorophyll dimer to the bacteriopheophytin acceptor was found to be multiphasic in crystals and approximately 2-fold slower than in solution. This work demonstrates the ability to resolve ultrafast photosynthetic function in single crystals and allows ultrafast function to be directly correlated with structure.     

Non-contiguous finished genome sequence of Staphylococcus capitis CR01 (pulsetype NRCS-A)

Staphylococcus capitis is a coagulase-negative staphylococcus (CoNS) commonly found in the human microflora. Recently, a clonal population of Staphylococcus capitis (denominated NRCS-A) was found to be a major cause of late-onset sepsis (LOS) in several neonatal intensive care units in France. Here, we report the complete genome sequence and annotation of the prototype Staphylococcus capitis NCRS-A strain CR01. The 2,504,472 bp long genome (1 chromosome and no plasmids) exhibits a G+C content of 32.81%, and contains 2,468 protein-coding and 59 tRNA genes and 4 rRNA genes.     

Protamine sulfate protects exogenous DNA against nuclease degradation but is unable to improve the efficiency of bovine sperm mediated transgenesis

The present study investigated whether protamine sulfate can be used to improve the efficiency of bovine sperm mediated transgenesis (SMT) by protecting the plasmid pCX-EGFP against nuclease activity. A high proportion (31%) of bovine spermatozoa transfected with the plasmid pCX-EGFP maintain their motility. Using an in vitro assay, protamine sulfate protected the plasmid against degradation by DNase I. However, upon transfecting spermatozoa, the plasmid remained intact regardless of whether it was complexed to protamine sulfate. When in vitro fertilisation (IVF) was undertaken using transfected sperm, 14.6 and 10.2% of blastocysts derived from pCX-EGFP only and pCX-EGFP-protamine transfected sperm, respectively, were PCR positive for the plasmid. In conclusion, using spermatozoa transfected with either pCX-EGFP or pCX-EGFP-protamine complexes, produced PCR positive blastocysts after SMT. However, the use of protamine sulfate does not improve the efficiency of SMT suggesting that factors other than nuclease activity could be limiting.