Hydrogen bonding, solvent exchange, and coupled proton and electron transfer in the oxidation and reduction of redox-active tyrosine Y(Z) in Mn-depleted core complexes of photosystem II

The redox-active tyrosines, Y(Z) and Y(D), of Photosystem II are oxidized by P680+ to the neutral tyrosyl radical. This oxidation thus involves the transfer of the phenolic proton as well as an electron. It has recently been proposed that tyrosine Y(Z) might replace the lost proton by abstraction of a hydrogen atom or a proton from a water molecule bound to the manganese cluster, thereby increasing the driving force for water oxidation. To compare and contrast with the intact system, we examine here, in a simplified Mn-depleted PSII core complex, isolated from a site-directed mutant of Synechocystis PCC 6803 lacking Y(D), the role of proton transfer in the oxidation and reduction of Y(Z). We show how the oxidation and reduction rates for Y(Z), the deuterium isotope effect on these rates, and the Y(Z)* - Y(Z) difference spectra all depend on pH (from 5.5 to 9.5). This simplified system allows examination of electron-transfer processes over a broader range of pH than is possible with the intact system and with more tractable rates. The kinetic isotope effect for the oxidation of P680+ by Y(Z) is maximal at pH 7.0 (3.64). It decreases to lower pH as charge recombination, which shows no deuterium isotope, starts to become competitive with Y(Z) oxidation. To higher pH, Y(Z) becomes increasingly deprotonated to form the tyrosinate, the oxidation of which at pH 9.5 becomes extremely rapid (1260 ms(-1)) and no longer limited by proton transfer. These observations point to a mechanism for the oxidation of Y(Z) in which the tyrosinate is the species from which the electron occurs even at lower pH. The kinetics of oxidation of Y(Z) show elements of rate limitation by both proton and electron transfer, with the former dominating at low pH and the latter at high pH. The proton-transfer limitation of Y(Z) oxidation at low pH is best explained by a gated mechanism in which Y(Z) and the acceptor of the phenolic proton need to form an electron/proton-transfer competent complex in competition with other hydrogen-bonding interactions that each have with neighboring residues. In contrast, the reduction of Y(Z)* appears not to be limited by proton transfer between pH 5.5 and 9.5. We also compare, in Mn-depleted Synechocystis PSII core complexes, Y(Z) and Y(D) with respect to solvent accessibility by detection of the deuterium isotope effect for Y(Z) oxidation and by 2H ESEEM measurement of hydrogen-bond exchange. Upon incubation of H2O-prepared PSII core complexes in D2O, the phenolic proton of Y(Z) is exchanged for a deuterium in less than 2 min as opposed to a t(1/2) of about 9 h for Y(D). In addition, we show that Y(D)* is coordinated by two hydrogen bonds. Y(Z)* shows more disordered hydrogen bonding, reflecting inhomogeneity at the site. With 2H ESEEM modulation comparable to that of Y(D)*, Y(Z)* would appear to be coordinated by two hydrogen bonds in a significant fraction of the centers.     

Etiopathogenesis of ankylosing spondylitis and reactive arthritis

In ankylosing spondylitis and reactive arthritis, an interplay of microbe and major histocompatibility complex initiates a sequence of events resulting in chronic inflammation. With the use of molecular probes as direct evidence and immune response patterns as indirect evidence, a strong case has been made for a central role of local microbial antigen in reactive arthritis. Cofactors such as gender, persistent gut inflammation, and antibiotic treatment may contribute to this process. Studies of transgenic rats and of familial spondylitis implicate B27 itself as the critical host variable. The results of recent studies point to intimate B27-bacteria interrelationships. HLA-B27 and proteins from enteric bacteria are structurally related, in a manner that may affect T cell response to enteric pathogens. B27 also may directly affect host-microbe interactions by modulating the invasive potential of these bacteria into target cells. Studies are in progress to apply the predictions of these in vitro systems to the in vivo situations of these diseases. The insights of research in the spondyloarthropathies may find broad applications in the rheumatic diseases.     

Computerized microassay of keratinocyte cell-plastic attachment and proliferation for assessing net stimulatory, inhibitory and toxic effects of compounds on nonimmortalized cell lines

Testing of pharmacological agents that affect growth of epidermal keratinocytes (EK) requires a standardized assay. We have developed an assay measuring net effects of stimulatory (e.g. growth factors), inhibitory (e.g. methotrexate) or toxic (e.g. Triton X-100) compounds. The amount of crystal violet staining viable EK attached to the wells of standard 96-well microplates is measured in situ using an ELISA plate reader. Optical density readings are directly converted into cell counts by computer software. Counts obtained by this method strongly correlate with the results obtained using the [3H]thymidine uptake assay and direct cell counts. The assay standardizes measurements of nonimmortalized EK lines with different innate proliferative properties and allows accurate quantitation of EK numbers in the range of 2,500-500,000 EK/well.     

Accurate determination of chemical composition of urinary calculi by spiral computerized tomography

PURPOSE: Choice of efficacious clinical management of symptomatic renal calculi can be facilitated by ascertaining the precise chemical composition of the calculus. Spiral computerized tomography (CT) is becoming a frequently used radiographic examination to establish the diagnosis and severity of calculus disease. Our objective for this study was to determine the precision of spiral CT in identifying the chemical composition of 6 different types of urinary calculi with region of interest measurements using spiral CT. MATERIALS AND METHODS: A total of 102 chemically pure stones were separated into 6 groups. The stones along with phantoms containing butter (fat) and jello (water) were mounted vertically in the scanner gantry. Then 1 mm. thickness scanning was performed with a high speed scanner at the 2 energy levels of 80 and 120 kV. The determination of the chemical composition was performed using the absolute CT value measured at 120 kV. and the dual kilovolt CT values measured at 80 and 120 kV. Hounsfield unit at 80 kV.-Hounsfield unit at 120 kV.). RESULTS: The absolute CT value measured at 120 kV. was able to identify precisely the chemical composition of uric acid, struvite and calcium oxalate stones. It was imprecise in differentiating calcium oxalate from brushite stone and struvite from cystine stone. However, dual kilovolt CT value was able to differentiate these latter stones with statistical significance (p < 0.03). Uric acid stones were easily differentiated from all other stones using the absolute CT value. CONCLUSIONS: This study demonstrates that the chemical composition of urinary calculi can be accurately determined by CT scanning in an in vitro setting.     

Transfer of human adenine deaminase gene into murine hematopoietic stem cells: sequential study of spleen colony-forming units from bone marrow of living mice and the requirement of the microenvironment

PURPOSE: To explore relationships between pregnant women's childhood experiences with discipline and 1) prenatal plans for disciplining own child and 2) later maternal-infant interactions DESIGN: Prospective correlation study. SETTING: Prenatal clinics and home visits PARTICIPANTS: Low-income pregnant women (N = 205) with a mean educational level of 12 years MAIN OUTCOME MEASURES: Adapted Ways of Handling Irritating Behaviors (Mother and You) Scales, NCAST Teaching Scale, Home Observation for Measurement of the Environment Scale RESULTS: Correlations between childhood experiences with discipline and 1) plans for later discipline strategies with own child and 2) infant components of maternal-infant interactions, including clarity of cues and responsiveness to the mother CONCLUSIONS: Prenatal assessment for childhood experiences with discipline may uncover factors associated with a break in the trajectory for optimal maternal-child interactions described by Barnard's theoretical framework.     

News: Rapid, large-scale solid waste composting focus of company research

Technology.     

Clinical relevance of calreticulin in systemic lupus erythematosus

Calreticulin is an abundant intracellular protein which is proposed to have numerous biological functions. However, there is increasing evidence to suggest that calreticulin plays a multifunctional role as an autoantigen present in patients with systemic lupus erythematosus. In this review we detail some of the recent evidence which indicate that calreticulin may play a supportive role in the formation of the autoantigen complex-Ro/SS-A. In addition, several proposed mechanisms of release and surface expression of calreticulin are described in relation to SLE mediated responses to the autoantigen. In particular, the generation of autoantibodies to specific regions of the protein and the ability of calreticulin to interfere with complement mediated inflammatory processes.     

A high efficiency technique for the generation of transgenic sugar beets from stomatal guard cells

An optimized protocol has been developed for the efficient and rapid genetic modification of sugar beet (Beta vulgaris L.). A polyethylene glycol-mediated DNA transformation technique could be applied to protoplast populations enriched specifically for a single totipotent cell type derived from stomatal guard cells, to achieve high transformation frequencies. Bialaphos resistance, conferred by the pat gene, produced a highly efficient selection system. The majority of plants were obtained within 8 to 9 weeks and were appropriate for plant breeding purposes. All were resistant to glufosinate-ammonium-based herbicides. Detailed genomic characterization has verified transgene integration, and progeny analysis showed Mendelian inheritance.