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Immunoinformatics is an emerging specialization of bioinformatics that focuses upon the structure, function and interactions of the molecules involved in immunity. Two major cell types, T-cells and B-cells, play significant roles in allergy, inflammation, infection and protective immunity. This review examines recently developed in silico tools and databases that can be used to identify, characterize or predict antigen epitopes recognized by T- and B-cells including the latest generation of B-cell epitope prediction tools that employ peptide-binding information derived from peptide phage display experiments. The application of these tools to facilitate drug development efforts is also discussed.

Tissue equivalents (TEs), formed by entrapping cells in a collagen gel, are an important model system for studying cell behavior. We have previously (Barocas and Tranquillo in J Biomech Eng 117:161-170, 1997a) developed an anisotropic biphasic theory of TE mechanics, which comprises five coupled partial differential equations describing interaction among cells and collagen fibers in the TE. The model equations, previously solved in one or two dimensions, were solved in three dimensions using an adaptive finite-element platform. The model was applied to three systems: a rectangular isometric cell traction assay, an otherwise- acellular gel containing two islands of cells, and an idealized tissue-engineered cardiac valve leaflet. In the first two cases, published experimental data were available for comparison, and the model results were consistent with the experimental observations. Fibers and cells aligned in the fixed direction in the isometric assay, and a region of strong fiber alignment arose between the two cell islands. For the valve problem, the alignment predicted by the model was generally similar to that observed experimentally, but an asymmetry in the experiment was not captured by the model.

Heart valve replacements composed of living tissue that can adapt, repair, and grow with a patient would provide a more clinically beneficial option than current inert replacements. Bioartificial valves were produced by entrapping human dermal fibroblasts within a fibrin gel. Using a mold design that presents appropriate mechanical constraints to the cell-induced fibrin gel compaction, gross fiber alignment (commissure-to-commissure alignment in the leaflets and circumferential alignment in the root) and the basic geometry of a native aortic valve were obtained. After static incubation on the mold in complete medium supplemented with transforming growth factor beta 1, insulin, and ascorbate, collagen fibers produced by the entrapped cells were found to coalign with the fibrin based on histological analyses. The resultant tensile mechanical properties were anisotropic. Ultimate tensile strength and tensile modulus of the leaflets in the commissural direction were 0.53 and 2.34 MPa, respectively. The constructs were capable of withstanding backpressure commensurate with porcine aortic valves in regurgitation tests (330 mmHg) and opened and closed under physiological pressure swings of 10 and 20 mmHg, respectively. These data support proof of principle of using cell-remodeled fibrin gel to produce tissue-engineered valve replacements.

Introduction: Treatment of osteoporosis with high-dose fluoride alone does not reduce fracture risk. We hypothesized that the antifracture efficacy of fluoride could be optimized by its use in low doses combined with an antiresorptive agent., Experimental Subjects: Subjects included 80 women with postmenopausal osteoporosis who had been taking estrogen for at least 1 yr., Methods: Subjects were randomized to receive monofluorophosphate (MFP) (fluoride content of 20 mg/d) or placebo over 4 yr in a double-blind trial., Results and Discussion: There were progressive increases in lumbar spine bone density over the duration of the study (MFP, 22%; placebo, 6%; P < 0.0001). In the trabecular bone of L3, these increases were even greater (MFP, 49%; placebo, 2.5%; P < 0.0001). In the proximal femur, there were smaller but significant treatment effects (P = 0.015). Total body scans and their subregions also showed significantly greater increases in the MFP group. Bone formation markers increased significantly in the MFP group at yr 1. Hyperosteoidosis was present in biopsies from five of seven MFP subjects, with osteomalacia in two of seven. The hazards ratio for vertebral fractures was 0.20 (95% confidence interval, 0.05-1.30), and the incidence rate ratio was 0.12 (95% confidence interval, 0.06-0.23; P < 0.01). The hazards ratio for nonvertebral fractures was 3.3 (95% confidence interval, 0.8-12.0)., Conclusions: We conclude that fluoride at 20 mg/d produces substantial increases in bone mineral density but still interferes with bone mineralization. This indicates that most previous studies with this ion have used toxic doses and that much lower doses should be assessed to find a safe dose window for the use of this powerful anabolic agent.

Objective: This pilot study tested the potential of puromycin (PUR) to inhibit protein synthesis and reduce oxygen utilization in a non-hibernating, whole animal preparation., Methods: After anesthesia and instrumentation, male rats received a single dose of PUR or 0.9% saline (control), followed 60 min later with [(35)S] methionine/cysteine radiolabeling. Thirty minutes after isotope injection, organ biopsies were taken for quantification of de novo protein synthesis. Arterial and central venous blood gases were obtained at baseline and 60 min after injection of PUR or 0.9% saline. Temperature, mean arterial pressure (MAP), and heart rate were recorded continuously., Results: Animals receiving PUR demonstrated significant reductions in protein synthesis in all organ systems sampled (p<0.05). The overall reduction averaged 67.8%. Central venous oxygen saturations (S(cv)O(2)) were higher in the PUR group than the controls at 60 min (90+/-2% versus 80+/-4%, p<0.05). The oxygen extraction ratio (O(2)ER) decreased from 16.1+/-1.7% to 6.8+/-1.2% in the PUR group (p<0.05) and increased from 12.5+/-3.2% to 16.0+/-4.2% in the controls (p=0.44). There was no difference in temperature, MAP, heart rate or blood gas variables, other than S(cv)O(2), at baseline or 60 min between groups., Conclusions: These results demonstrate that PUR is capable of reducing whole body protein synthesis significantly within a relatively short duration of time. This appears to decrease whole body oxygen utilization as evidenced by an increase in S(cv)O(2) and a decrease in O(2)ER. Protein synthesis inhibition may reduce metabolic demands and should be tested for its potential to improve outcomes where oxygen demands exceed oxygen delivery.

Electron paramagnetic resonance (EPR) spectroscopy reveals functional and structural similarities between the reaction centres of the chlorophyll d-binding photosystem I (PS I) and chlorophyll a-binding PS I. Continuous wave EPR spectrometry at 12K identifies iron-sulphur centres as terminal electron acceptors of chlorophyll d-binding PS I. A transient light-induced electron spin echo (ESE) signal indicates the presence of a quinone as the secondary electron acceptor (Q) between P(740)(+) and the iron-sulphur centres. The distance between P(740)(+) and Q(-) was estimated within point-dipole approximation as 25.23+/-0.05A, by the analysis of the electron spin echo envelope modulation.

The analysis of FDMR spectra, recorded at multiple emission wavelengths, by a global decomposition technique, has allowed us to characterise the triplet populations associated with Photosystem I and Photosystem II of thylakoids in the green alga Chlamydomonas reinhardtii. Three triplet populations are observed at fluorescence emissions characteristic of Photosystem II, and their zero field splitting parameters have been determined. These are similar to the zero field parameters for the three Photosystem II triplets previously reported for spinach thylakoids, suggesting that they have a widespread occurrence in nature. None of these triplets have the zero field splitting parameters characteristic of the Photosystem II recombination triplet observed only under reducing conditions. Because these triplets are generated under non-reducing redox conditions, when the recombination triplet is undetectable, it is suggested that they may be involved in the photoinhibition of Photosystem II. At emission wavelengths characteristic of Photosystem I, three triplet populations are observed, two of which are attributed to the P(700) recombination triplet frozen in two different conformations, based on the microwave-induced fluorescence emission spectra and the triplet minus singlet difference spectra. The third triplet population detected at Photosystem I emission wavelengths, which was previously unresolved, is proposed to originate from the antenna chlorophyll of the core or the unusually blue-shifted outer antenna complexes of this organism.

A conserved tryptophan residue located between the A(1B) and F(X) redox centres on the PsaB side of the Photosystem I reaction centre has been mutated to a glycine in Chlamydomonas reinhardtii, thereby matching the conserved residue found in the equivalent position on the PsaA side. This mutant (PsaB:W669G) was studied using EPR spectroscopy with a view to understanding the molecular basis of the reported kinetic differences in forward electron transfer from the A(1A) and the A(1B) phyllo(semi)quinones. The kinetics of A(1)(-) reoxidation due to forward electron transfer or charge recombination were measured by electron spin echo spectroscopy at 265 K and 100 K, respectively. At 265 K, the reoxidation kinetics are considerably lengthened in the mutant in comparison to the wild-type. Under conditions in which F(X) is initially oxidised the kinetics of charge recombination at 100 K are found to be biphasic in the mutant while they are substantially monophasic in the wild-type. Pre-reduction of F(X) leads to biphasic kinetics in the wild-type, but does not alter the already biphasic kinetic properties of the PsaB:W669G mutant. Reduction of the [4Fe-4S] clusters F(A) and F(B) by illumination at 15 K is suppressed in the mutant. The results provide further support for the bi-directional model of electron transfer in Photosystem I of C. reinhardtii, and indicate that the replacement of the tryptophan residue with glycine mainly affects the redox properties of the PsaB bound phylloquinone A(1B).

A retrospective review of rotating-platform (n = 113) and fixed-bearing (n = 100) total knee arthroplasties at a minimum 2-year follow-up was performed. All patients in both groups in this study had osteoarthritis and underwent surgery by 1 of the 2 senior authors at 1 of 2 institutions. A cruciate-retaining total knee arthroplasty was implanted with patellar resurfacing in all cases. The only difference between the 2 groups was the design of the tibial bearing and its tibial tray. Through minimization of confounding variables relating to pathology, surgeon, institution, soft-tissue balancing, and type of prosthesis, this study isolates articular design as a variable. We could not demonstrate any significant difference in knee flexion after either fixed-bearing or rotating-platform cruciate-retaining total knee arthroplasty.

We report the first examination of exchangeable proton and MeOH interactions with the Mn catalytic cluster in photosystem II, under functional flash turnover conditions, using 2H ESEEM spectroscopy on the S2 and S0 multiline states. Deuterium-labeled water (D2O) and methyl d3-labeled methanol (DMeOH) are employed. It was discovered that a hyperfine resolved multiline S0 signal could be seen in the presence of D2O, the hyperfine structure of which depended on the presence or absence of methanol (MeOH). In the presence of DMeOH, significant dipolar coupling of the three methyl deuterons to the multiline centers in the S2 and S0 states was seen (S2, 0.65, 0.39(2) MHz; and S0, 0.60, 0.37(2) MHz). These are consistent with direct binding of the methoxy fragment to Mn. Assuming terminal Mn-OMe ligation, the couplings indicated a spin projection coefficient (rho) magnitude of approximately 2 for the ligating Mn in both the S2 and S0 states, with inferred Mn-O distances of approximately 1.9-2.0 A. In the presence of D2O, four classes of exchangeable deuterons were identified by ESEEM in S2 and S0. Three of these classes (1, 2, and 4) exhibited populations and coupling strengths that were essentially constant under various conditions of sample preparation, illumination turnover, and small alcohol addition. Class 3 could be modeled with constant coupling but a highly variable deuteron population (n3 approximately 0-10) depending in part on the preparation used. For all classes, the coupling parameters were very similar in S2 and S0. The favored interpretation is that the two strongest coupling classes (1 and 2) represent close binding of one water molecule to a single Mn which has an oxidation state of II in S0 and III in S2, and rho approximately 2 in both cases. This water is not displaced by MeOH, but either the water or MeOH is singly deprotonated upon MeOH binding. Class 4 represents approximately 2 water molecules which are not closely bound to Mn (Mn-deuteron distances of approximately 3.7-4.7 A). Class 3 probably represents protein matrix protons within approximately 4 A of the Mn in the cluster, which can be variably exchanged in different preparations.

The decay of the light-induced spin-correlated radical pair [P700+ A1-] and the associated electron spin echo envelope modulation (ESEEM) have been studied in either thylakoid membranes, cellular membranes, or purified photosystem I prepared from the wild-type strains of Synechocystis sp. PCC 6803, Chlamydomonas reinhardtii, and Spinaceae oleracea. The decay of the spin-correlated radical pair is described in the wild-type membrane by two exponential components with lifetimes of 2-4 and 16-25 micros. The proportions of the two components can be altered by preillumination of the membranes in the presence of reductant at temperatures lower than 220 K, which leads to the complete reduction of the iron-sulfur electron acceptors F(A), F(B), and F(X) and partial photoaccumulation of the reduced quinone electron acceptor A1A-. The "out-of-phase" (OOP) ESEEM attributed to the [P700+ A1-] radical pair has been investigated in the three species as a function of the preillumination treatment. Values of the dipolar (D) and the exchange (J) interactions were extracted by time-domain fitting of the OOP-ESEEM. The results obtained in the wild-type systems are compared with two site-directed mutants of C. reinhardtii [Santabarbara et al. (2005) Biochemistry 44, 2119-2128], in which the spin-polarized signal on either the PsaA- or PsaB-bound electron transfer pathway is suppressed so that the radical pair formed on each electron transfer branch could be monitored selectively. This comparison indicates that when all of the iron-sulfur centers are oxidized, only the echo modulation associated with the A branch [P700+ A1A-] radical pair is observed. The reduction of the iron-sulfur clusters and the quinone A1 by preillumination treatment induces a shift in the ESEEM frequency. In all of the systems investigated this observation can be interpreted in terms of different proportions of the signal associated with the [P700+ A1A-] and [P700+ A1B-] radical pairs, suggesting that bidirectionality of electron transfer in photosystem I is a common feature of all species rather than being confined to green algae.

Contractile stimuli can sensitize myosin to Ca2+ by activating RhoA kinase (ROK) and PKC that inhibit myosin light chain phosphatase (MLCP) activity. Relaxant stimuli, acting through PKA and PKG (cyclic nucleotide-dependent protein kinases), and pretreatment with contractile agents such as phenylephrine (PE), can desensitize myosin to Ca2+. It is unknown precisely how these stimuli cause Ca2+ desensitization. To test the hypothesis that PKA, PKG, and PE pretreatment signaling systems converge to cause relaxation by inhibition of ROK in intact, isolated tissues, we examined the effects of forskolin (FSK; PKA activation), 8-bromo-cGMP (8br-cGMP; PKG activation), and PE pretreatment on KCl-induced force maintenance in rabbit arteries, a response nearly completely dependent on ROK activation. PE pretreatment and agents activating PKA and PKG caused Ca2+ desensitization by inhibiting KCl-induced tonic force and MLC phosphorylation without inhibiting intracellular [Ca2+]. At pCa 5 in beta-escin-permeabilized muscle, FSK and 8b-cGMP accelerated the relaxation rate when tissues were returned to pCa 9, suggesting that both agents can elevate MLCP activity. However, a component of the Ca2+ desensitization attributed to PKG activation in intact tissues appeared to involve a MLC phosphorylation-independent component. Inhibition of KCl-induced tonic force by the ROK inhibitor, Y-27632, and by PE pretreatment, were synergistically potentiated by 8b-cGMP, but not FSK. FSK and PE pretreatment, but not 8b-cGMP, inhibited the KCl-induced increase in site-specific myosin phosphatase target protein-1 phosphorylation at Thr853. These data support the hypothesis that PKA and PE pretreatment converge on a common Ca2+-desensitization pathway, but that PKG can act by a mechanism different from that activated by PKA and PE pretreatment.

The growth in refractive surgeries and corneal replacements has fueled interest in the development of a tissue-engineered cornea. This study characterizes the microstructure and biomechanical properties of film-based corneal stroma equivalents over time in culture. The increased collagen density in the films was hypothesized to result in improved mechanical properties both initially and over time. The microstructure of the film-based stromal equivalent was examined using atomic force microscopy and scanning electron microscopy; the mechanical properties, relaxed modulus, and ultimate tensile strength were quantified using uniaxial tensile testing. The dense, film-based stromal equivalent had a lamellae-like microstructure, which was notably different than the porous structure of sponges used previously. Seeded human corneal stromal fibroblasts remained on the surface of the film rather than migrating into the film and produced fibers of extracellular matrix with diameters of 35-75 nm. After an initial decrease during hydration, the relaxed modulus and ultimate tensile strength for fully hydrated collagen films were 0.4 +/- 0.2 MPa and 0.3 +/- 0.1 MPa, respectively. The mechanical properties of cell-seeded films mimicked those of control films. While further studies are needed to quantify the optical properties, the dense, lamellae-like structure of collagen films is a feasible scaffold for the development of tissue-engineered stroma.

N-terminal protein acetylation, catalyzed by N-terminal acetyltransferases (NATs) recognizing distinct N-terminal sequences, is gaining recognition as an essential regulator of normal cell function, but little is known of its role in vertebrate development. We previously cloned a novel gene, embryonic growth-associated protein (EGAP), the expression of which is associated with rapid vascular smooth muscle cell proliferation during development. We show herein EGAP is the mammalian/zebrafish homologue of yeast Mak10p, one subunit of the yeast NatC complex, and describe the cloning of its binding partners Mak3 and Mak31. The EGAP NAT forms a functional complex in mammalian cells, is evolutionarily conserved, and developmentally regulated. It is widely but not ubiquitously expressed during early zebrafish development but undetectable in later developmental stages. We demonstrate EGAP- and Mak3-deficient zebrafish fail to develop because of, in part, decreased cell proliferation, increased apoptosis, and poor blood vessel formation contributing to embryonic lethality. We examined the role of target of rapamycin (TOR), a highly conserved protein kinase controlling cell growth, as a physiological target of EGAP NAT acetylation. Compared with controls, TOR expression and signaling is significantly reduced in EGAP morphants. Pharmacological inhibition of TOR with rapamycin phenocopied the EGAP morpholino oligonucleotide-induced growth and vessel defects. Overexpression of constitutively active TOR rescued EGAP morphants, suggesting TOR is a direct or indirect endogenous substrate of the EGAP NAT complex. These data suggest the EGAP NAT complex is an essential regulatory enzyme controlling the function of a subset of proteins required for embryonic growth control and vessel development.

Promoters of many smooth muscle-specific genes (SM-genes) contain multiple CArG boxes, which represent a binding site for serum response factor (SRF). Transcriptional control through these regions involves interactions with SRF and specific coactivators such as myocardin. We have previously reported that suppression of SM-gene expression by platelet derived growth factor (PDGF) is associated with redistribution of SRF, leading to lower intra-nuclear levels, and a reduction in SRF transactivation. To further assess the role of SRF depletion on VSMC phenotype, the current study used RNA interference (RNAi). Two SRF-specific sequences constructed as hairpins were stably expressed in rat VSMC. Clones expressing SRF RNAi had no detectable SRF expression by immunoblotting, and showed diminished levels of SM alpha-actin protein and promoter activity. Unexpectedly, depletion of VSMC resulted in increased rates of proliferation and migration. Several genes whose expression is increased by PDGF stimulation, including c-Jun, were similarly induced in cells lacking SRF. Effects of SRF depletion were not attributable to altered PDGF receptor activity or alterations in activation of Akt. These data indicate that loss of SRF transactivation in VSMC, in this case through suppression via RNAi, induces biological responses similar to that seen with PDGF.

Photosystem I is a large macromolecular complex located in the thylakoid membranes of chloroplasts and in cyanobacteria that catalyses the light driven reduction of ferredoxin and oxidation of plastocyanin. Due to the very negative redox potential of the primary electron transfer cofactors accepting electrons, direct estimation by redox titration of the energetics of the system is hampered. However, the rates of electron transfer reactions are related to the thermodynamic properties of the system. Hence, several spectroscopic and biochemical techniques have been employed, in combination with the classical Marcus theory for electron transfer tunnelling, in order to access these parameters. Nevertheless, the values which have been presented are very variable. In particular, for the case of the tightly bound phylloquinone molecule A(1), the values of the redox potentials reported in the literature vary over a range of about 350 mV. Previous models of Photosystem I have assumed a unidirectional electron transfer model. In the present study, experimental evidence obtained by means of time resolved absorption, photovoltage, and electron paramagnetic resonance measurements are reviewed and analysed in terms of a bi-directional kinetic model for electron transfer reactions. This model takes into consideration the thermodynamic equilibrium between the iron-sulfur centre F(X) and the phylloquinone bound to either the PsaA (A(1A)) or the PsaB (A(1B)) subunit of the reaction centre and the equilibrium between the iron-sulfur centres F(A) and F(B). The experimentally determined decay lifetimes in the range of sub-picosecond to the microsecond time domains can be satisfactorily simulated, taking into consideration the edge-to-edge distances between redox cofactors and driving forces reported in the literature. The only exception to this general behaviour is the case of phylloquinone (A(1)) reoxidation. In order to describe the reported rates of the biphasic decay, of about 20 and 200 ns, associated with this electron transfer step, the redox potentials of the quinones are estimated to be almost isoenergetic with that of the iron sulfur centre F(X). A driving force in the range of 5 to 15 meV is estimated for these reactions, being slightly exergonic in the case of the A(1B) quinone and slightly endergonic, in the case of the A(1A) quinone. The simulation presented in this analysis not only describes the kinetic data obtained for the wild type samples at room temperature and is consistent with estimates of activation energy by the analysis of temperature dependence, but can also explain the effect of the mutations around the PsaB quinone binding pocket. A model of the overall energetics of the system is derived, which suggests that the only substantially irreversible electron transfer reactions are the reoxidation of A(0) on both electron transfer branches and the reduction of F(A) by F(X).

Ammonia and methanol both bind to the water oxidising complex of photosystem II during its turnover, possibly at sites where water binds during the normal water oxidation process. We have investigated the interaction between these two water analogues at the S2 state of the water oxidising cycle using electron magnetic resonance techniques. We find evidence that ammonia displaces methanol from its binding site.

In vascular smooth muscle cells (VSMC), platelet-derived growth factor (PDGF) suppresses expression of multiple smooth muscle contractile proteins, useful markers of differentiation. Conversely, hypertrophic agents induce expression of these genes. The goal of this study was to employ genomic approaches to identify classes of genes differentially regulated by PDGF and hypertrophic stimuli. Changes in gene expression were determined using Affymetrix RAE-230 GeneChips in rat aortic VSMC stimulated with PDGF. For comparison with a model hypertrophic stimulus, a microarray was performed with VSMC stably expressing constitutively active Galpha(16), which strongly induces smooth muscle marker expression. We identified 75 genes whose expression was increased by exposure to PDGF and decreased by expression of Galpha(16) and 97 genes whose expression was decreased by PDGF and increased by Galpha(16). These genes included many smooth muscle-specific proteins; several extracellular matrix, cytoskeletal, and chemotaxis-related proteins; cell signaling molecules; and transcription factors. Changes in gene expression for many of these were confirmed by PCR or immunoblotting. The contribution of signaling pathways activated by PDGF to the gene expression profile was examined in VSMC stably expressing gain-of-function H-Ras or myristoylated Akt. Among the genes that were confirmed to be differentially regulated were CCAAT/enhancer-binding protein delta, versican, and nexilin. All of these genes also had altered expression in injured aortas, consistent with a role for PDGF in the response of injured VSMC. These data indicate that genes that are differentially regulated by PDGF and hypertrophic stimuli may represent families of genes and potentially be biomarkers for vascular injury.

The spin-correlated radical pair [P(700)(+)A(1)(-)] gives rise to a characteristic "out-of-phase" electron spin-echo signal. The electron spin-echo envelope modulation (ESEEM) of these signals has been studied in thylakoids prepared from the wild-type strain of Chlamydomonas reinhardtii and in two site-directed mutants, in which the methionine residue which acts as the axial ligand to the chlorin electron acceptor A(0) has been substituted with a histidine either on the PsaA (PsaA-M684H) or the PsaB (PsaB-M664H) reaction center subunits. The analysis of the time domain ESEEM provides information about the spin-spin interaction in the [P(700)(+)A(1)(-)] radical pair, and the values of the dipolar (D) and the exchange (J) interaction can be extracted. From the distance dependence of the dipolar coupling term, the distance between the unpaired electron spin density clouds of the primary donor P(700)(+) and the phyllosemiquinone A(1)(-) can be determined. The [P(700)(+)A(1)(-)] ESEEM spectrum obtained in wild-type thylakoids can be reconstructed using a linear combination of the spectra measured in the PsaA and PsaB A(0) mutants, demonstrating that electron transfer resulting in charge separation is occurring on both the PsaA and PsaB branches. The [P(700)(+)A(1B)(-)] distance in the point dipole approximation in the PsaA-M684H mutant is 24.27 +/- 0.02 A, and the [P(700)(+)A(1A)(-)] distance in the PsaB-M664H mutant is 25.43 +/- 0.01 A. An intermediate value of 25.01 +/- 0.02 A is obtained in the wild-type membranes which exhibit both spin-polarized pairs.

People of Indian origin develop type 2 diabetes at a relatively young age and low body mass index compared to people of other racial groups, possibly because of preferential accumulation of abdominal fat. We examined the relationship between abdominal fat and body mass index (BMI) in healthy premenopausal women of different racial groups, and compared abdominal fat measurements at different ages. Women of Chinese, European, Indian or Polynesian origin (50 of each, mean age 31) had dual energy x-ray absorptiometry of the lumbar spine, with estimation of the percentage fat in the abdominal area. Polynesian women had a significantly greater mean BMI (P< 0.001) than all other groups, and a higher abdominal fat than European and Chinese woman (P< 0.001). Although the Indian women had a similar mean BMI to the European and Chinese women, they had a significantly greater abdominal fat (P<0.001). When examined by age tertile, the Indian women had significantly more abdominal fat than Chinese or European women at all ages (P<0.001), with marked differences evident even in the youngest group (mean age 23 years). Compared to other racial groups, women of Indian origin preferentially accumulate abdominal fat--a trait that is well established in young adulthood.

Photosynthetic organisms harvest solar energy by absorbing light and ultimately transferring energy through a cascade of chemical reactions to power all cellular processes. Core components initiating this reaction cascade are the photosynthetic reaction centres Photosystem I and Photosystem II. Two recent publications on the structure of the reaction centres by Adam Ben-Shem et al. and Kristina Ferreira et al. represent a big step towards understanding the evolutionary development of the core energy conversion process and identifying the site of the water oxidation process, the source of atmospheric oxygen.

PAN, a yeast poly(A) nuclease, plays an important nuclear role in the posttranscriptional maturation of mRNA poly(A) tails. The activity of this enzyme is dependent on its Pan2p and Pan3p subunits, as well as the presence of poly(A)-binding protein (Pab1p). We have identified and characterized the associated network of factors controlling the maturation of mRNA poly(A) tails in yeast and defined its relevant protein-protein interactions. Pan3p, a positive regulator of PAN activity, interacts with Pab1p, thus providing substrate specificity for this nuclease. Pab1p also regulates poly(A) tail trimming by interacting with Pbp1p, a factor that appears to negatively regulate PAN. Pan3p and Pbp1p both interact with themselves and with the C terminus of Pab1p. However, the domains required for Pan3p and Pbp1p binding on Pab1p are distinct. Single amino acid changes that disrupt Pan3p interaction with Pab1p have been identified and define a binding pocket in helices 2 and 3 of Pab1p's carboxy terminus. The importance of these amino acids for Pab1p-Pan3p interaction, and poly(A) tail regulation, is underscored by experiments demonstrating that strains harboring substitutions in these residues accumulate mRNAs with long poly(A) tails in vivo.

The characteristic Mn hyperfine 'multiline' signal exhibited in the S2 state of the oxygen-evolving complex (OEC) complex of Photosystem II (PSII) has been shown to be heterogeneous in character. In this study, we have explored the effects that influence the proportions of the two forms of the S2 state multiline signal present in any sample. The narrow form of the signal is lost upon storage (weeks) at 77 K, whereas the broad form remains. In particular, we explore the roles of ethanol and methanol as well as effects of the second turnover of the enzyme on storage of the sample at 77 K. We find that in samples containing methanol, the narrow form may predominate upon the first flash, but the broad form predominates on the fifth flash and also in samples containing ethanol., (Copyright 2004 Elsevier B.V.)

In 1996, active surveillance in 5 Foodborne Diseases Active Surveillance Network (FoodNet) sites revealed up to a 9-fold difference in Escherichia coli O157:H7 (O157) infection incidence between sites. A matched case-control study of sporadic O157 cases was conducted in these sites from March 1996 through April 1997. Case subjects were patients with non-outbreak-related diarrheal illness who had O157 isolated from their stool samples. Control subjects were healthy persons matched by age and telephone number exchange. Overall, 196 case patients and 372 controls were enrolled. O157 infections were associated with farm exposure, cattle exposure, eating a pink hamburger (both at home and away from home), eating at a table-service restaurant, using immunosuppressive medication, and obtaining beef through a private slaughter arrangement. Variations in cattle exposures may explain a part of the regional variability of O157 infection incidence. O157 control measures should focus on reducing risks associated with eating undercooked hamburger, dining at table-service restaurants, and farm exposures.

This mini-review outlines the involvement of the tyrosine electron carriers, Y(D) and Y(Z), in the mechanism of electron transfer from water to P680. We discuss our data and put forward our ideas on the role of Y(D) and Y(Z).

Background: At distinct times during embryonic development and after vascular injury, smooth muscle cells (SMCs) exhibit a highly proliferative, serum-independent growth phenotype. The aim of the present study was to evaluate the functional role of S6 ribosomal protein (S6RP) and upstream positive and negative regulators in the control of SMC serum-independent growth., Methods and Results: We previously reported increased expression of S6RP mRNA was associated with this unique growth phenotype. Using immunohistochemistry and Western blot analysis, we report high levels of total and phospho-S6RP and increased levels of Akt and p70S6K phosphorylation, upstream positive regulators of S6RP, in rat embryonic aortas and adult balloon-injured carotid arteries compared with quiescent adult aortas and uninjured carotid arteries. Western blot analysis demonstrated that cultured embryonic and neointimal SMCs that exhibited serum-independent growth capabilities expressed high levels of S6RP and constitutively active Akt, mTOR, and p70S6K. Pharmacological and molecular inhibition of phosphatidylinositol 3-kinase (PI3K) signaling pathways, using PI3K inhibitors, rapamycin, or dominant-negative Akt adenovirus, suppressed embryonic and neointimal SMC serum-independent growth. Finally, decreased activity of PTEN, an endogenous negative regulator of PI3K signaling, was associated with high in vivo SMC growth rates, and morpholino-mediated loss of endogenous PTEN induced a serum-independent growth phenotype in cultured serum-dependent SMCs., Conclusions: The possibility exists that cells that exhibit a distinct embryonic-like growth phenotype different from traditional SMCs are major contributors to intimal thickening. Growth of SMCs that exhibit this phenotype is dependent on constitutive Akt and mTOR/p70S6K signaling and is actively inhibited through the timed acquisition of the endogenously produced growth suppressor PTEN.

We were interested in the elucidation of the interaction between the heparan sulfate proteoglycan, perlecan, and PTEN in the regulation of vascular smooth muscle cell (SMC) growth. We verified serum-stimulated DNA synthesis, and Akt and FAK phosphorylation were significantly reduced in SMCs overexpressing wild-type PTEN. Our previous studies showed perlecan is a potent inhibitor of serum-stimulated SMC growth. We report in the present study, compared with SMCs plated on fibronectin, serum-stimulated SMCs plated on perlecan exhibited increased PTEN activity, decreased FAK and Akt activities, and high levels of p27, consistent with SMC growth arrest. Adenoviral-mediated overexpression of constitutively active Akt reversed perlecan-induced SMC growth arrest while morpholino antisense-mediated loss of endogenous PTEN resulted in increased growth and phosphorylation of FAK and Akt of SMCs on perlecan. Immunohistochemical and Western analyses of balloon-injured rat carotid artery tissues showed a transient increase in phosphoPTEN (inactive) after injury, correlating to high rates of neointimal cell replication; phosphoPTEN was largely limited to actively replicating SMCs. Similarly, in the developing rat aorta, we found increased PTEN activity associated with increased perlecan deposition and decreased SMC replication rates. However, significantly decreased PTEN activity was detected in aortas of perlecan-deficient mouse embryos, consistent with SMC hyperplasia observed in these animals, compared with E17.5 heterozygous controls that produce abundant amounts of perlecan at this developmental time point. Our data show PTEN is a potent endogenously produced inhibitor of SMC growth and increased PTEN activity mediates perlecan-induced suppression of SMC proliferation.

The interaction of water with the water oxidizing Mn complex of photosystem II has been investigated using electron spin-echo envelope modulation spectroscopy in the presence of H(2)(17)O. The spectra show interaction of the (17)O with the preparation in the S(2) state induced by 200 K illumination. The modulation is observed only in the center of the multiline spectrum. The inferred hyperfine coupling terms are compatible with water (not hydroxyl) oxygen bound to a particular quasi-axial Mn(III) center in a coupled Mn cluster.

Acute wounds normally heal in a very orderly and efficient manner characterized by four distinct, but overlapping phases: hemostasis, inflammation, proliferation and remodeling. Specific biological markers characterize healing of acute wounds. Likewise, unique biologic markers also characterize pathologic responses resulting in fibrosis and chronic non-healing ulcers. This review describes the major biological processes associated with both normal and pathologic healing. The normal healing response begins the moment the tissue is injured. As the blood components spill into the site of injury, the platelets come into contact with exposed collagen and other elements of the extracellular matrix. This contact triggers the platelets to release clotting factors as well as essential growth factors and cytokines such as platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-beta). Following hemostasis, the neutrophils then enter the wound site and begin the critical task of phagocytosis to remove foreign materials, bacteria and damaged tissue. As part of this inflammatory phase, the macrophages appear and continue the process of phagocytosis as well as releasing more PDGF and TGF beta. Once the wound site is cleaned out, fibroblasts migrate in to begin the proliferative phase and deposit new extracellular matrix. The new collagen matrix then becomes cross-linked and organized during the final remodeling phase. In order for this efficient and highly controlled repair process to take place, there are numerous cell-signaling events that are required. In pathologic conditions such as non-healing pressure ulcers, this efficient and orderly process is lost and the ulcers are locked into a state of chronic inflammation characterized by abundant neutrophil infiltration with associated reactive oxygen species and destructive enzymes. Healing proceeds only after the inflammation is controlled. On the opposite end of the spectrum, fibrosis is characterized by excessive matrix deposition and reduced remodeling. Often fibrotic lesions are associated with increased densities of mast cells. By understanding the functional relationships of these biological processes of normal compared to abnormal wound healing, hopefully new strategies can be designed to treat the pathological conditions.

Objectives: This study was conducted to investigate the occurrence of antimicrobial resistance among Salmonella Weltevreden isolates from different sources in South-East Asia (Indonesia, Laos, Malaysia, Taiwan, Thailand, Vietnam), Australia, Denmark, New Zealand and the USA., Methods: A total of 503 isolates were examined for susceptibility to antimicrobial agents, and resistant isolates were examined for the presence of selected resistance genes by PCR., Results: Only 48 (9.5%) of the isolates were resistant to one or more of the antimicrobial agents tested. A low frequency of resistance was found towards ampicillin (1.8%), chloramphenicol (1.6%), florphenicol (0.4%), nalidixic acid (1.6%), neomycin (0.6%), streptomycin (4.4%), sulfamethoxazole (4.2%), tetracycline (4.0%) and trimethoprim (1.4%), whereas all isolates were susceptible to co-amoxiclav, ceftiofur, ciprofloxacin, colistin and gentamicin. All nine ampicillin-resistant isolates contained a sequence similar to the blaTEM-1b gene, one of the eight chloramphenicol-resistant isolates a sequence similar to the catA1 gene, all three neomycin-resistant isolates a sequence similar to the aphA-2 gene, 16 (73%) of the 22 streptomycin-resistant isolates a sequence similar to the aadA gene, the remaining six (27%) a sequence similar to the strA gene, and all 21 sulfamethoxazole-resistant isolates a sequence similar to the sul2 gene. Thirteen (65%) of the 20 tetracycline-resistant isolates contained the tet(A) gene, four (20%) the tet(B) gene, and one (5%) the tet(C) gene., Conclusions: This study showed a low frequency of resistance among Salmonella Weltevreden isolated from humans and other reservoirs in South-East Asia and elsewhere. There was no major difference in the occurrence of resistance between source or geographical origin.

The attainment of optimal peak bone mass during adolescence is important in the primary prevention of osteoporosis. Exercise may contribute to skeletal development and bone density during growth, although competitive exercise is suggested to have an adverse effect. This study assesses the effect of moderate exercise on the bone density of adolescent females. Additionally, other factors which significantly influence attainment of peak bone mass were identified. This was a cross-sectional study of 42 adolescent females, classified as runners (n=15) or non-runners (n=27). Nutrient intake, energy expenditure, menstrual history and pubertal stage were recorded. Bone age, skinfold thickness, body composition and bone mineral density (BMD) of total body, lumbar spine and proximal femur were measured. Statistical analyses used Student's t-test, Pearson correlation and multiple regression analyses. Runners had lower fat mass and higher lean mass, with a trend to higher BMD in all sites measured. There were no significant differences in menstrual cycle regularity, age at menarche or number who had attained menarche. Pubic hair development was similar in both groups. Breast development was delayed in runners, although this may have been a function of lower fat mass in this group. When subjects were categorized according to menarchal status, postmenarchal girls were significantly taller and heavier, with higher fat mass and significantly higher total body and lumbar spine BMD. There was no significant relationship between BMD at any site and dietary nutrient intake. Multiple regression analyses, using BMD as the dependent variable, identified running status, pubertal stage, fat mass and lean mass as significant determinants. When BMD/height was used, significant determinants in total body BMD were fat mass, pubertal stage and running status, while in the lumbar spine, only the latter two variables remained significant. In conclusion, body composition, physical activity and sexual maturity were identified as significant determinants of bone density during adolescence. Runners had significantly lower body fat than non-runners, but this did not interfere with hormonal cyclicity, and caused no detriment to their bone density. The results of this study are reassuring, since they indicate that sub-elite competitive athletics has no detrimental effect on bone mass accrual in adolescent females.

We have used pulsed electron paramagnetic resonance (EPR) measurements of the electron spin polarised (ESP) signals arising from the geminate radical pair P700(z.rad;+)/A(1)(z.rad;-) to detect electron transfer on both the PsaA and PsaB branches of redox cofactors in the photosystem I (PSI) reaction centre of Chlamydomonas reinhardtii. We have also used electron nuclear double resonance (ENDOR) spectroscopy to monitor the electronic structure of the bound phyllosemiquinones on both the PsaA and PsaB polypeptides. Both these spectroscopic assays have been used to analyse the effects of site-directed mutations to the axial ligands of the primary chlorophyll electron acceptor(s) A(0) and the conserved tryptophan in the PsaB phylloquinone (A(1)) binding pocket. Substitution of histidine for the axial ligand methionine on the PsaA branch (PsaA-M684H) blocks electron transfer to the PsaA-branch phylloquinone, and blocks photoaccumulation of the PsaA-branch phyllosemiquinone. However, this does not prevent photoautotrophic growth, indicating that electron transfer via the PsaB branch must take place and is alone sufficient to support growth. The corresponding substitution on the PsaB branch (PsaB-M664H) blocks kinetic electron transfer to the PsaB phylloquinone at 100 K, but does not block the photoaccumulation of the phyllosemiquinone. This transformant is unable to grow photoautotrophically although PsaA-branch electron transfer to and from the phyllosemiquinone is functional, indicating that the B branch of electron transfer may be essential for photoautotrophic growth. Mutation of the conserved tryptophan PsaB-W673 to leucine affects the electronic structure of the PsaB phyllosemiquinone, and also prevents photoautotrophic growth.

Previous work in many laboratories has established that hydroxylamine reduces the S(1) state of the water oxidizing complex (WOC) in one-electron steps. Significant levels of what can now be defined as the S(-1)* state are achieved by specific (concentration and incubation length) hydroxylamine treatments. This state has already been studied by electron paramagnetic resonance spectrometry (EPR), and unusual EPR signals were noted (for example, see Sivaraja, M., and Dismukes, G. C. (1988) Biochemistry 27, 3467-3475). We have now reinvestigated these initial experiments and confirmed many of the original observations. We then utilized more recent EPR markers for the S(0) and S(1) states to further explore the S(-1)* state. The broad radical "split" type EPR signal, produced by 200 K illumination of samples prepared to give a high yield of the S(-1)* state, is shown to most likely reflect a trapped intermediate state between S(-1)* and S(0)*, since samples where this signal is present can be warmed in the dark to produce S(0)*. The threshold for advancement from S(-1)* to S(0)* is near 200 K, as the yield of broad radical decreases and S(0)* multiline EPR signal increases with length of 200 K illumination. Advancement of S(0)* to S(1) is limited at 200 K, but S(1) can be restored by 273 K illumination. Illumination of these hydroxylamine-treated samples at temperatures below 77 K gives a second broad radical EPR signal. The line shape, decay, and other properties of this new radical signal suggest that it may arise from an interaction in the S(-2)* or lower S states, which are probably present in low yield in these samples. Illumination below 20 K of S(0)* state samples containing methanol, and therefore exhibiting the S(0) multiline signal, gives rise to a third broad radical with distinctive line shape. The characteristics of the three broad radicals are similar to those found from interactions between Y(Z)(*) and other S states. The evidence is presented that they do represent intermediate states in S state turnover. Further work is now needed to identify these radicals.

We previously reported that fully assembled basement membranes are nonpermissive to smooth muscle cell (SMC) replication and that perlecan (PN), a basement membrane heparan sulfate proteoglycan, is a dominant effector of this response. We report here that SMC adhesion to basement membranes, and perlecan in particular, up-regulate the expression of focal adhesion kinase-related nonkinase (FRNK), a SMC-specific endogenous inhibitor of FAK, which subsequently suppresses FAK-mediated, ERK1/2-dependent growth signals. Up-regulation of FRNK by perlecan is actively and continuously regulated. Relative to the matrix proteins studied, the effects are unique to perlecan, because plating of SMCs on several other basement membrane proteins is associated with low levels of FRNK and corresponding high levels of FAK and ERK1/2 phosphorylation and SMC growth. Perlecan supports SMC adhesion, although there is reduced cell spreading compared with fibronectin (FN), laminin (LN), or collagen type IV (IV). Despite the reduction in cell spreading, we report that perlecan-induced up-regulation of FRNK is independent of cell shape changes. Growth inhibition by perlecan was rescued by overexpressing a constitutively active FAK construct, but overexpressing kinase-inactivated mutant FAK or FRNK attenuated fibronectin-stimulated growth. These data indicate that perlecan functions as an endogenously produced inhibitor of SMC growth at least in part through the active regulation of FRNK expression. FRNK, in turn, may control SMC growth by downregulating FAK-dependent signaling events.

The use of antimicrobial growth promoters in Danish food animal production was discontinued in 1998. Contrary to concerns that pathogen load would increase; we found a significant decrease in Salmonella in broilers, swine, pork, and chicken meat and no change in the prevalence of Campylobacter in broilers.

Objective: Vascular smooth muscle cells (SMCs), activated by growth factors after arterial injury, migrate and proliferate to expand the intima of the blood vessel. During intimal expansion, proliferation is suppressed and an increasingly large proportion of the neointimal mass is composed of newly synthesized extracellular matrix (ECM). We sough to determine whether the ECM heparan sulfate proteoglycan (HSPG) perlecan, which inhibits SMC proliferation in vitro, also accumulates and limits SMC proliferation during neointimal expansion., Methods and Results: Perlecan expression and accumulation were analyzed by immunohistochemistry and in situ hybridization during neointima formation after balloon catheter injury to the rat carotid artery. Perlecan expression was low in uninjured vessels and up to 7 days after injury, during maximal SMC proliferation. By 14 days after injury, perlecan was dramatically increased, and immunostaining remained heavy throughout the advanced lesion, 35 to 42 days after injury. Finally, explants of intimal tissue from 35- to 42-day neointimal lesions were digested with glycosaminoglycanases to determine whether endogenous HSPGs inhibit intimal SMC proliferation. SMCs within HS-depleted, but not chondroitinase ABC-treated or mock-incubated, explants were found to proliferate in response to platelet-derived growth factor BB., Conclusions: HSPGs, such as perlecan, may inhibit the proliferative response of SMCs after vascular injury.

Most eukaryotic mRNAs are subject to considerable post-transcriptional modification, including capping, splicing, and polyadenylation. The process of polyadenylation adds a 3' poly(A) tail and provides the mRNA with a binding site for a major class of regulatory factors, the poly(A)-binding proteins (PABPs). These highly conserved polypeptides are found only in eukaryotes; single-celled eukaryotes each have a single PABP, whereas humans have five and Arabidopis has eight. They typically bind poly(A) using one or more RNA-recognition motifs, globular domains common to numerous other eukaryotic RNA-binding proteins. Although they lack catalytic activity, PABPs have several roles in mediating gene expression. Nuclear PABPs are necessary for the synthesis of the poly(A) tail, regulating its ultimate length and stimulating maturation of the mRNA. Association with PABP is also a requirement for some mRNAs to be exported from the nucleus. In the cytoplasm, PABPs facilitate the formation of the 'closed loop' structure of the messenger ribonucleoprotein particle that is crucial for additional PABP activities that promote translation initiation and termination, recycling of ribosomes, and stability of the mRNA. Collectively, these sequential nuclear and cytoplasmic contributions comprise a cycle in which PABPs and the poly(A) tail first create and then eliminate a network of cis- acting interactions that control mRNA function.

Intra-subunit interactions in the environment of the iron-sulfur cluster F(X) in Photosystem I (PS I) of Synechocystis sp. PCC 6803 were studied by site-directed and second site suppressor mutations. In subunit PsaB, the cysteine ligand (C565) of F(X) and a conserved aspartate (D566) adjacent to C565 were modified. The resulting mutants D566E, C556S/D566E, C556H/D566E and C565H/D566E did not assemble PS I in the thylakoids of the cyanobacterium. Yet, this is the first report of cells of the second site-suppressor mutant (D566E/L416P) and of second site-directed mutant (C565S/D566E) in PsaB that could grow autotrophically in light and were found to assemble a stable functional PS I containing all three iron-sulfur centers, F(X) and F(A/B). The newly resolved structure of PS I (PDB 1JB0) was used to interpret the functional interactions among the amino acid residues. It is suggested that the stability of F(X) is supported by a salt bridge formed between D566, which is adjacent to the cysteine ligand C565 of the iron-sulfur cluster located on loop hi, and R703 located at the start of loop jk. Hydrogen bond between R703 and D571 at the start of loop hi further stabilizes the arginine. Lengthening of the side by 1.2 A chain in mutation D566E caused destabilization of F(X). The extended side-chain was compensated for by the Fe-O, which is 0.3 A shorter than the Fe-S bond resulting in stabilization of the F(X) in the double mutations C565S/D566E. The suppressor mutation D566E/L416P allowed greater freedom for the salt bridge E566-R703, thus relieving the pressure introduced by the D566E replacement and enabling the formation of F(X). F(X) and R703 are therefore stabilized through short- and long-range interactions of the inter-helical loops between h-i, j-k and f-g, respectively.

Photoaccumulation of membrane preparations of Chlamydomonas reinhardtii at pH 8 and 220 K reduces the primary and secondary electron acceptors in the Photosystem I (PSI) reaction centre, and produces a maximum of two spins per P700(z.rad;+). Proton electron nuclear double resonance (ENDOR) spectra demonstrate that the phyllosemiquinone produced is that attributed to the PsaA branch of electron transfer. Photoaccumulation at pH 10 and 220 K produces a maximum of four spins per P700(z.rad;+), and proton ENDOR spectra indicate that a second phyllosemiquinone is being photoaccumulated, with markedly different proton hyperfine couplings (hfcs). This phyllosemiquinone is unaffected by mutation of PsaAW693, confirming that it does not arise from the PsaA branch of electron transfer, and we therefore attribute it to the PsaB phyllosemiquinone.

Objective: To prospectively assess bone density and the factors determining the rate of bone loss over a 10-year period of postmenopausal life., Design: Prospective, observational study., Methods: One hundred and four normal White postmenopausal women, baseline mean age 59 years (range 47-71 years) completed the study (mean duration of follow-up 10.2 years, range 9.4-10.6 years). None had diseases or were taking medications affecting bone metabolism at entry to the study. Information was collected on medical, fracture and smoking history, alcohol use, dietary calcium intake and physical activity. Body composition and bone density were measured by dual-energy X-ray absorptiometry at baseline and at 10 years. Biochemical, haematological and hormonal analyses were performed., Results: Twenty-four percent of the women started hormone replacement therapy (HRT) during the study period; most of these remained on therapy at follow-up. The mean duration of therapy was 6.6 years (range 2.8-10.4 years). The use of HRT was associated with significant gains in bone density (total body + 3.0%, trochanter + 4.2%, Ward's triangle + 4.4%, spine + 10.5%) and a significant reduction in vertebral fracture risk [standardized risk ratio compared with non-HRT users 0.42 (confidence interval 0.18-0.83)]. HRT use was not associated with greater weight gain than that occurring in other members of the cohort. The baseline and follow-up bone densities in the non-HRT users were highly correlated (0.82 < or = r < or = 0.91, P < or = 0.0001) and baseline bone density accounted for the majority of the variance in the 10-year results. Multivariate analyses showed that the independent correlates of rate of change of bone density were weight and fat mass (both baseline values and changes during follow-up), time after menopause, sex hormone concentrations, urinary calcium loss, PTH levels and haemoglobin concentration (which may reflect nutrition and health)., Conclusions: Bone density is highly predictable over an extended period of time in normal postmenopausal women. Maintenance of body weight and good health reduce bone loss. HRT is effective for treating osteoporosis, with improvement in bone density and reduction in vertebral fractures. Good compliance with HRT long-term is achievable. These findings are relevant to deciding the frequency of bone density measurement, and in advising women regarding prevention and treatment of postmenopausal bone loss.

The 25-kDa Family 4 uracil-DNA glycosylase (UDG) from Pyrobaculum aerophilum has been expressed and purified in large quantities for structural analysis. In the process we observed it to be colored and subsequently found that it contained iron. Here we demonstrate that P. aerophilum UDG has an iron-sulfur center with the EPR characteristics typical of a 4Fe4S high potential iron protein. Interestingly, it does not share any sequence similarity with the classic iron-sulfur proteins, although four cysteines (which are strongly conserved in the thermophilic members of Family 4 UDGs) may represent the metal coordinating residues. The conservation of these residues in other members of the family suggest that 4Fe4S clusters are a common feature. Although 4Fe4S clusters have been observed previously in Nth/MutY DNA repair enzymes, this is the first observation of such a feature in the UDG structural superfamily. Similar to the Nth/MutY enzymes, the Family 4 UDG centers probably play a structural rather than a catalytic role.

Purpose: To determine the effect of supplementation with calcium citrate on circulating lipid concentrations in normal older women., Subjects and Methods: As part of a study of the effects of calcium supplementation on fractures, we randomly assigned 223 postmenopausal women (mean [+/- SD] age, 72 +/- 4 years), who were not receiving therapy for hyperlipidemia or osteoporosis, to receive calcium (1 g/d, n = 111) or placebo (n = 112) for 1 year. Fasting serum lipid concentrations, including high-density lipoprotein (HDL) cholesterol and low-density lipoprotein (LDL) cholesterol, were obtained at baseline, and at 2, 6, and 12 months., Results: After 12 months, HDL cholesterol levels and the HDL cholesterol to LDL cholesterol ratio had increased more in the calcium group than in the placebo group (mean between-group differences in change from baseline: for HDL cholesterol, 0.09 mmol/L (95% confidence interval [CI]: 0.02 to 0.17; P = 0.01); for HDL/LDL cholesterol ratio, 0.05 (95% CI: 0.02 to 0.08; P = 0.001). This was largely due to a 7% increase in HDL cholesterol levels in the calcium group, with a nonsignificant 6% decline in LDL cholesterol levels. There was no significant treatment effect on triglyceride level (P = 0.48)., Conclusion: Calcium citrate supplementation causes beneficial changes in circulating lipids in postmenopausal women. This suggests that a reappraisal of the indications for calcium supplementation is necessary, and that its cost effectiveness may have been underestimated.

We report the detection of a "split" electron paramagnetic resonance (EPR) signal during illumination of dark-adapted (S(1) state) oxygen-evolving photosystem II (PSII) membranes at <20 K. The characteristics of this signal indicate that it arises from an interaction between an organic radical and the Mn cluster of PSII. The broad radical signal decays in the dark following illumination either by back-reaction with Qa*- or by forward electron transfer from the Mn cluster. The forward electron transfer (either from illumination at 11 K followed by incubation in the dark at 77 K or by illumination at 77 K) results in the formation of a multiline signal similar to, but distinct from, other well-characterized multiline forms found in the S0 and S2 states. The relative yield of the "S1 split signal", which we provisionally assign to S1X*, where X could be YZ* or Car*+, and that of the 77 K multiline signal indicate a relationship between the two states. An approximate quantitation of the yield of these signals indicates that up to 40-50% of PSII centers can form the S1 split signal. Ethanol addition removes the ability to observe the S1 split signal, but the multiline signal is still formed at 77 K. The multiline forms with <700 nm light and is not affected by near-infrared (IR) light, showing that we are detecting electron transfer in centers not responsive to IR illumination. The results provide important new information about the mechanism of electron abstraction from the water oxidizing complex (WOC).

Transcriptional activation of the thiostrepton-inducible promoter, ptipA, in Streptomyces lividans is mediated by TipAL. This transcriptional activator belongs to the MerR/SoxR family that characteristically binds an operator sequence located between the -10 and -35 hexamers normally occupied by RNA polymerase. As for the Escherichia coli merT promoter, the ptipA hexamers are separated by a long 19 bp spacer and hence a topological transition of the DNA is likely to be a requisite for alignment with RNA polymerase. Growth conditions that could facilitate this conformational change were investigated using transcriptional fusions of ptipA with reporter genes. Adjustment of growth medium osmolarity led to increased and prolonged TipAL-dependent expression, both with and without the inducer, thiostrepton. These effects correlated with increases in negative DNA supercoiling. Moreover, an inability to induce the promoter with thiostrepton in strain TK64 was corrected by increasing the concentration of osmolyte, compensating for an apparent reduced level of negative DNA supercoiling in the strain. Prolonging the time of activation of tipA in the wild-type by manipulating growth conditions revealed that mycelial autolysis could be induced by thiostrepton in 4-d-old cultures.

Background: Perimenopausal and postmenopausal fractures are well-recognized, strong, independent predictors of subsequent fractures. However, it is unknown whether premenopausal fractures are predictive of postmenopausal fractures., Objective: To determine whether self-reported fractures sustained before the age of 50 years are associated with fractures after this age., Subjects and Methods: Cross-sectional study of 1284 women (mean +/- SD age, 73 +/- 4 years) who were 10 or more years' postmenopausal and who were recruited from electoral rolls in Auckland, New Zealand. Detailed information on their fracture, medical, menstrual, alcohol, and smoking histories was obtained using a standardized questionnaire., Results: Nine percent of the women reported fractures before the age of 20 years; 7%, between the ages of 20 and 50 years; and 29%,after the age of 50 years. Fractures sustained between the ages of 20 and 50 years were associated with a 74% increase in the risk of fractures after the age of 50 years (odds ratio, 1.74; 95% confidence interval, 1.12-2.70), while fractures occurring before the age of 20 years were not (odds ratio, 1.01; confidence interval, 0.66-1.56). Multivariate analysis showed that after bone density, age, maternal history of hip fractures, age at menopause, weight, history of hormone replacement therapy, and smoking and alcohol histories were adjusted for, a history of fractures between the ages of 20 and 50 years remained a significant independent predictor of risk of fractures after the age of 50 years (risk ratio, 1.83; confidence interval, 1.12-2.76)., Conclusions: Any fracture (unrelated to motor vehicle accidents) sustained between the ages of 20 and 50 years is associated with increased risk of fractures after the age of 50 years. Therefore, this is an important clinical risk factor that points to the need for bone density measurement, consideration of lifestyle modification, and antiosteoporosis therapies in these women.

Kinetic analysis using pulsed electron paramagnetic resonance (EPR) of photosynthetic electron transfer in the photosystem I reaction centres of Synechocystis 6803, in wild-type Chlamydomonas reinhardtii, and in site directed mutants of the phylloquinone binding sites in C. reinhardtii, indicates that electron transfer from the reaction centre primary electron donor, P700, to the iron-sulphur centres, Fe-S(X/A/B), can occur through either the PsaA or PsaB side phylloquinone. At low temperature reaction centres are frozen in states which allow electron transfer on one side of the reaction centre only. A fraction always donates electrons to the PsaA side quinone, the remainder to the PsaB side.

To investigate the environment of the phylloquinone secondary electron acceptor A(1) within the photosystem I reaction center, we have carried out site-directed mutagenesis of two tryptophan residues (W693 and W702) in the PsaA subunit of Chlamydomonas reinhardtii. One of these conserved tryptophans (W693) is predicted to be close to the phylloquinone and has been implicated in the interaction of A(1) with an aromatic residue through pi--pi stacking. We find that replacement of W702 with either histidine or leucine has no effect on the electronic structure of A(1)(*-) or on forward electron transfer from A(1)(*-) to the iron--sulfur center F(x). In contrast, the same mutations of W693 alter the electronic structure of the photoaccumulated A(1)(*-) and slow forward electron transfer as measured by the decay of the electron spin-polarized signal arising from the P700(*+)/A(1)(*-) radical pair. These results provide support for the hypothesis that W693 has a role in poising the redox potential of A(1)/A(1)(*-) so it can reduce F(x), and they indirectly provide evidence for electron transfer along the PsaA-side branch of cofactors in PSI.

This mini-review outlines the current theories on the mechanism of electron transfer from water to P680, the location and structure of the water oxidising complex and the role of the manganese cluster. We discuss how our data fit in with current theories and put forward our ideas on the location and mechanism of water oxidation.

Objective: To examine the factors influencing the levels of sex hormone-binding globulin (SHBG) in normal postmenopausal women by assessing the relationship between SHBG and measured anthropometric, metabolic and hormonal variables., Design: Cross-sectional, observational study., Subjects and Methods: Seventy normal postmenopausal women aged 47-71 years (mean 58 years), participated in the study. Information was collected on medical, reproductive and smoking history, alcohol use, dietary intake and physical activity. Body composition measurements using dual-energy absorptiometry, and analyses of biochemical and hormonal indices were performed., Results: Bivariate correlation coefficients indicated that SHBG was inversely related to body weight (r = - 0.44), fat mass (r = - 0.35), and abdominal obesity (r = - 0.42). It was also inversely related to the glucose and insulin levels during an oral glucose tolerance test (- 0.24 < r < - 0.40), serum oestradiol (r = - 0.26), and physical activity (r = - 0.24). Multiple regression analysis indicated that significant independent correlates of SHBG concentration were fat mass, physical activity, alcohol intake, serum oestradiol, and insulin-like growth factor-1, all having a negative impact on SHBG., Conclusions: From these observed associations, it is concluded that maintenance of body weight, moderate alcohol consumption, and physical activity will tend to reduce SHBG concentrations in postmenopausal women, thereby increasing the levels of free oestradiol. This mechanism could mediate the beneficial effects of these factors in preventing the development of osteoporosis and cardiovascular disease.