The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1) sector catalyzes ATP synthesis, whereas the F(o) sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1) and F(o) sectors are highly conserved across prokaryotes and eukaryotes. Therefore, it was a surprise that genes encoding the a and b subunits as well as other components of the F(o) sector were undetectable in the sequenced genomes of a variety of apicomplexan parasites. While the parasitic existence of these organisms could explain the apparent incomplete nature of ATP synthase in Apicomplexa, genes for these essential components were absent even in Tetrahymena thermophila, a free-living ciliate belonging to a sister clade of Apicomplexa, which demonstrates robust oxidative phosphorylation. This observation raises the possibility that the entire clade of Alveolata may have invented novel means to operate ATP synthase complexes. To assess this remarkable possibility, we have carried out an investigation of the ATP synthase from T. thermophila. Blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed the ATP synthase to be present as a large complex. Structural study based on single particle electron microscopy analysis suggested the complex to be a <em>dimer</em> with several unique structures including an unusually large domain on the intermembrane side of the ATP synthase and novel domains flanking the c subunit rings. The two monomers were in a parallel configuration rather than the angled configuration previously observed in other organisms. Proteomic analyses of well-resolved ATP synthase complexes from <em>2</em>-<em>D</em> BN/BN-PAGE identified orthologs of seven canonical ATP synthase subunits, and at least 13 novel proteins that constitute subunits apparently limited to the ciliate lineage. A mitochondrially encoded protein, Ymf66, with predicted eight transmembrane domains could be a substitute for the subunit a of the F(o) sector. The absence of genes encoding orthologs of the novel subunits even in apicomplexans suggests that the Tetrahymena ATP synthase, despite core similarities, is a unique enzyme exhibiting dramatic differences compared to the conventional complexes found in metazoan, fungal, and plant mitochondria, as well as in prokaryotes. These findings have significant implications for the origins and evolution of a central player in bioenergetics.

OBJECTIVE

Plasminogen activator inhibitor-1 (PAI-1) production by adipose tissue is increased in obesity, and its circulating levels are high in type <em>2</em> diabetes. PAI-1 increases cardiovascular risk by favoring clot stability, interfering with vascular remodeling, or both. We investigated in obese diabetic persons whether an intensive lifestyle intervention for weight loss (ILI) would decrease PAI-1 levels independently of weight loss and whether PAI-1 reduction would be associated with changes in fibrinogen, an acute phase reactant, or fibrin fragment <em>D</em>-<em>dimer</em> (<em>D</em>-<em>dimer</em>), a marker of ambient coagulation balance.

RESULTS

We examined 1-year changes in PAI-1, <em>D</em>-<em>dimer</em>, and fibrinogen levels; adiposity; fitness; glucose; and lipid control with ILI in 1817 participants from Look AHEA<em>D</em>, a randomized trial investigating the effects of ILI, compared with usual care, on cardiovascular events in overweight or obese diabetic persons. Median PAI-1 levels decreased <em>2</em>9% with ILI and <em>2</em>.5% with usual care (P < 0.0001). Improvements in fitness, glucose control, and high-density lipoprotein cholesterol were associated with decreased PAI-1, independently of weight loss (P = 0.03 for fitness, P < 0.0001 for others). Fibrinogen and <em>D</em>-<em>dimer</em> remained unchanged.

CONCLUSIONS

Reductions in PAI-1 levels with ILI in obese diabetic individuals may reflect an improvement in adipose tissue health that could affect cardiovascular risk without changing fibrinogen or d-<em>dimer</em> levels. Clinical Trial Registration- URL: http://clinicaltrials.gov/ct<em>2</em>/show/NCT00017953. Unique identifier: NCT00017953.

An N-acetylneuraminic acid cytidylyltransferase (EC <em>2</em>.7.7.43) (CMP-NeuAc synthetase) was isolated from a Haemophilus ducreyi strain 35000 cell lysate and partially characterized. The enzyme catalyzes the reaction of CTP and NeuAc to form CMP-NeuAc, which is the nucleotide sugar donor used by sialyltransferases. Previous studies have shown that the outer membrane lipooligosaccharides of H. ducreyi contain terminal sialic acid attached to N-acetyllactosamine and that this modification is likely important to its pathogenesis. Therefore, to investigate the role of sialic acid in H. ducreyi pathogenesis, the gene encoding the CMP-NeuAc synthetase was cloned using degenerate oligonucleotide probes derived from NH<em>2</em>-terminal sequence data, and the nucleotide sequence was determined. The derived amino acid sequence of the CMP-NeuAc synthetase gene has homology to other CMP-NeuAc synthetases and to a lesser extent to CMP-<em>2</em>-keto-3-deoxy-<em>D</em>-manno-octulosonic acid synthetases. The gene was cloned into a T7 expression vector, the protein expressed in Escherichia coli, and purified to apparent homogeneity by anion exchange, Green 19 dye, and hydrophobic interaction chromatography. The final step yielded <em>2</em>0 mg of pure protein/liter of culture. The protein has a predicted molecular mass of <em>2</em>5440.6 <em>D</em>a, which was confirmed by electrospray mass spectrometry (Mexpt = <em>2</em>5439.9 +/- 1.4 <em>D</em>a). The enzyme appears to exist as a <em>dimer</em> by size exclusion chromatography. In contrast to other bacterial CMP-NeuAc synthetases, the H. ducreyi enzyme exhibited a different substrate specificity, being capable of also using N-glycolylneuraminic acid as a substrate.

In the present study we describe a novel functional cell surface molecule, designated as Kp43, which is expressed among leukocytes by NK cells, TCR-gamma/<em>delta</em> + T lymphocytes, and some CD8+ CD56+TCR-alpha/beta + T cell clones. The Kp43 Ag is a 70-kDa disulfide-linked <em>dimer</em>, which migrates in SDS-PAGE under reducing conditions as a single 43-kDa band. Two-color immunofluorescence staining of fresh PBL revealed that only a fraction of CD16+, and of TCR-gamma/<em>delta</em> + T lymphocytes expressed the Ag. The analysis of TCR-alpha/beta + T cell clones showed that a small proportion (<em>2</em> out of <em>2</em>0) weakly expressed Kp43 together with the CD8 and CD56 molecules. By immunoperoxidase staining of different tissues the anti-Kp43, reactivity was detected exclusively in lymphoid organs, where a minority of scattered cells was stained, and in some liver sinusoidal cells. Essentially all NK cells acquired Kp43 when stimulated with a B lymphoblastoid cell line. By contrast, the pattern of distribution of Kp43 remained stable upon in vitro culture of T-gamma/<em>delta</em> lymphocytes, thus delineating two subsets according to its expression. In lymphokine-activated killer populations, obtained by culturing either PBL or NK cells with high concentration of IL-<em>2</em>, most CD16+ and CD56+ cells became Kp43+. The Kp43-specific mAb inhibited the IL-<em>2</em>-dependent proliferative response of cultured NK and TCR-gamma/<em>delta</em> + T cells without affecting their non-MHC-restricted cytotoxicity. The partial inhibitory effect, which was mediated as well by pepsin digested F(ab')<em>2</em> fragments, was lost upon reduction to Fab. The anti-Kp43 mAb did not interfere with the specific binding of IL-<em>2</em> to its surface receptors. Altogether the data point out that the Kp43 <em>dimer</em> is involved in the regulation of the IL-<em>2</em>-dependent proliferative response of NK cells and a subset of TCR-gamma/<em>delta</em> + T lymphocytes.

The interaction of lac operator <em>D</em>NA with lac repressor (LacI) is a classic example of a genetic regulatory switch. To dissect the role of stoichiometry, subunit association, and effects of <em>D</em>NA length in positioning this switch, we have determined binding isotherms for the interaction of LacI with a high affinity (Osym) operator on linearized plasmid (<em>2</em>500 bp) <em>D</em>NA over a wide range of macromolecular concentrations (10(-14) to 10(-8) M). Binding data were analyzed using a thermodynamic model involving four equilibria: dissociation of tetramers (T) into <em>dimers</em> (<em>D</em>), and binding of operator-containing plasmid <em>D</em>NA (O) to <em>dimers</em> and tetramers to form three distinct complexes, <em>D</em>O, TO, and TO<em>2</em>. Over the range of concentrations of repressor, operator, and salt (0.075 M K+ to 0.40 M K+) investigated, we find no evidence for any significant thermodynamic effect of LacI <em>dimers</em>. Instead, all isotherms can be interpreted in terms of just two equilibria, involving only T and the TO and TO<em>2</em> complexes. As a reference binding equilibrium, which we propose must approximate the <em>D</em>O binding interaction, we compare the plasmid Osym results with our extensive studies of the binding of a 40 bp Osym <em>D</em>NA fragment to LacI. On this basis, we obtain a lower bound on the LacI <em>dimer</em>-tetramer equilibrium constant and values of the equilibrium constants for formation of TO and TO<em>2</em> complexes. At a salt concentration of 0.40 M, the Osym plasmid binding data are consistent with a model with two independent and identical binding sites for operator per LacI tetramer, in which the binding to a site on the tetramer is only slightly more favorable than the reference binding interaction. Increasingly large deviations from the independent-site model are observed as the salt concentration is reduced; binding of a second operator to from TO<em>2</em> becomes strongly disfavored relative to formation of TO at low salt concentrations (0.075 to 0.1<em>2</em>5 M). In addition, binding of both the first and second plasmid operator <em>D</em>NA molecules to the tetramer becomes increasingly more favorable than the reference binding interaction as [K+] is reduced from 0.40 M to 0.1<em>2</em>5 M. At 0.075 M K+, however, the strength of binding of the second plasmid operator <em>D</em>NA to the LacI tetramer is dramatically reduced; this interaction is much less favorable than binding the first plasmid operator <em>D</em>NA, and becomes much less favorable than the reference binding interaction. We propose that these differences arise from changes in the nature of the TO and TO<em>2</em> complexes with decreasing salt concentration. At low salt concentration, we suggest the hypothesis that flanking non-operator sequences bind non-specifically (coulombically) by local wrapping, and that distant regions of non-operator <em>D</em>NA occupy the second operator-binding site by looping. We propose that wrapping stabilizes both 1:1 and <em>2</em>:1 complexes at low salt concentration, and that looping stabilizes the 1:1 complex but competitively destabilizes the <em>2</em>:1 TO<em>2</em> complex at low salt concentration. These effects must play a role in adjusting the stability and structure of the LacI-lac operator repression complex as the cytoplasmic [K+] varies in response to changes in extracellular osmolarity.

Phosphorylase kinase, a regulatory enzyme of glycogenolysis in skeletal muscle, is a hexadecameric oligomer consisting of four copies each of a catalytic subunit (gamma) and three regulatory subunits (alpha, beta, and <em>delta</em>, the last being endogenous calmodulin). The enzyme is activated by a variety of effectors acting through its regulatory subunits. To probe the quaternary structure of nonactivated and activated forms of the kinase, we used the heterobifunctional, photoreactive cross-linker N-5-azido-<em>2</em>-nitrobenzoyloxysuccinimide. Mono-derivatization of the holoenzyme with the succinimidyl group, followed by photoactivation of the covalently attached azido group, resulted in intramolecular cross-linking to form two distinct hetero<em>dimer</em>s: a major (alphagamma) and a minor (beta<em>delta</em>) conjugate. Formation of both conjugates was significantly altered in activated conformations of the enzyme induced by phosphorylation, alkaline pH, and several allosteric activators (ADP, exogenous calmodulin/Ca<em>2</em>+, and Ca<em>2</em>+ alone). Of these activating mechanisms, all increased formation of alphagamma, except Ca<em>2</em>+ alone, which inhibited its formation. When cross-linking was carried out at alkaline pH or in the presence of ADP or exogenous calmodulin/Ca<em>2</em>+, the cross-linked enzyme remained activated following removal of the activators; however, cross-linking in the presence of Ca<em>2</em>+ resulted in sustained inhibition. The results indicate that perturbations in the subunit cross-linking forming the alphagamma <em>dimer</em> reflect the subsequent extent of sustained activation of the holoenzyme that is measured. The region cross-linked to the catalytic gamma subunit was confined to the C-terminal 1/6th of the alpha subunit, which contains known regulatory regions. These results suggest that activators of the phosphorylase kinase holoenzyme perturb interactions between the C-terminal region of the inhibitory alpha subunit and the catalytic gamma subunit, ultimately leading to activation of the latter.

OBJECTIVE

To investigate the effect of mechanical ventilation on alveolar fibrinolytic capacity.

METHODS

Randomized controlled animal study in 66 Sprague-Dawley rats.

METHODS

Test animals received intratracheal fibrinogen and thrombin instillations; six were killed immediately (fibrin controls), and the others were allocated to three ventilation groups (ventilation period: <em>2</em><em>2</em>5 min) differing in positive inspiratory pressure and positive end-expiratory pressure, respectively: group 1, 16 cmH<em>2</em>O and 5 cmH<em>2</em>O (n=17); group <em>2</em>, <em>2</em>6 cmH<em>2</em>O and 5 cmH<em>2</em>O (n=16); group 3, 35 cmH<em>2</em>O and of 5 cmH<em>2</em>O (n=17). Ten animals that had not been ventilated served as healthy controls.

RESULTS

After animals were killed, we measured D-dimers, plasminogen activator inhibitor (PAI) 1, and tumor necrosis factor alpha in the bronchoalveolar lavage fluid and calculated lung weight and pressure/volume (P/V) plots. The median D-dimer concentration (mg/l) decreased with increasing pressure amplitude (19<em>2</em> in group 1, IQR 119; 66 in group <em>2</em>, IQR 107; <em>2</em>9 in group 3, IQR 30) while median PAI-1 (U/ml) increased (undetectable in group 1; 0.55 in group <em>2</em>, IQR 4.55; 3.05 in group 3, IQR 4.85). PAI-1 level was correlated with increased lung weight per bodyweight (Spearman's rank correlation 0.708). Tumor necrosis factor alpha concentration was not correlated with PAI-1 level.

CONCLUSIONS

Alveolar fibrinolytic capacity is suppressed during mechanical ventilation with high pressure amplitudes due to local production of PAI-1.

In the Arc repressor <em>dimer</em>, the side chains of Ile37 and Val41 in alpha-helix B pack against each other and against the symmetry-related side chains of Ile37' and Val41' in alpha-helix B' to form part of the hydrophobic core and the <em>dimer</em> interface. Following combinatorial mutagenesis of these positions, only the wild-type combination of hydrophobic residues was recovered as a fully active protein, and only a few conservative replacements were recovered as stably folded or partially active proteins. Equilibrium and kinetic studies of the folding of purified mutants show that the <em>delta</em>-CH3 groups of Ile37 and Ile37' contribute approximately <em>2</em> kcal/mol of <em>dimer</em> to protein stability and are involved in interactions that are only partially formed in the transition state for protein folding. Alanine substitution at either position 37 or 41 results in proteins which differ from wild type in being monomeric at a concentration of 10 microM, having reduced secondary structure, having solvent-exposed tryptophans, and showing non-cooperative thermal and urea denaturation transitions. These mutants appear to exist in a physiologically denatured state that is similar in many ways to the molten globule state.

The structure of R-phycoerythrin (R-PE) from the red alga Griffithsia monilis was solved at 1.90-A resolution by molecular replacement, using the atomic coordinates of cyanobacterial phycocyanin from Fremyella diplosiphon as a model. The crystallographic R factor for the final model is 17.5% (Rfree <em>2</em><em>2</em>.7%) for reflections in the range 100-1.90 A. The model consists of an (alphabeta)<em>2</em> <em>dimer</em> with an internal noncrystallographic dyad and a fragment of the gamma-polypeptide. The alpha-polypeptide (164 amino acid residues) has two covalently bound phycoerythrobilins at positions alpha8<em>2</em> and alpha139. The beta-polypeptide (177 residues) has two phycoerythrobilins bound to residues beta8<em>2</em> and beta158 and one phycourobilin covalently attached to rings A and <em>D</em> at residues beta50 and beta61, respectively. The electron density of the gamma-polypeptide is mostly averaged out by threefold crystallographic symmetry, but a dipeptide (Gly-Tyr) and one single Tyr could be modeled. These two tyrosine residues of the gamma-polypeptide are in close proximity to the phycoerythrobilins at position beta8<em>2</em> of two symmetry-related beta-polypeptides and are related by the same noncrystallographic dyad as the (alphabeta)<em>2</em> <em>dimer</em>. Possible energy transfer pathways are discussed briefly.

The crystal structure of wheat germ agglutinin isolectin 1 (WGA1) complexed with a tryptic sialoglycopeptide fragment (T-5) from its erythrocyte receptor glycophorin A, which contains the O-linked tetrasaccharide NeuNAc-alpha <em>2</em>,3-Gal-beta 1,3-(alpha <em>2</em>,6-NeuNAc) Gal-NAc-alpha 1-O-Thr, has been determined by molecular replacement techniques and refined at <em>2</em>.0-A resolution (R = 18.1%). The structure reveals that association between WGA1 <em>dimers</em>, composed of two identical four-domain (A-<em>D</em>) monomers, and T-5 is asymmetric and involves sialic acid binding at three nonequivalent aromatic residue-rich sites. Two independent binding modes are observed. In the dominant ("major") binding mode, the two highest affinity sites are utilized to cross-link neighboring crystallographically related WGA1 <em>dimers</em>. The branched tetrasaccharide has an extended rigid conformation, and its terminal alpha <em>2</em>,6-NeuNAc and alpha <em>2</em>,3-NeuNAc residues occupy specificity sites in domains B1 (monomer 1) and C<em>2</em> (monomer <em>2</em>) on opposing <em>dimers</em>, respectively. This asymmetric selection of binding sites leads to infinite open-ended arrays of interlinked lectin molecules. In the subsidiary "minor" binding mode, only the terminal alpha <em>2</em>,6-NeuNAc, anchored to the aromatic residue-rich binding site in domain A<em>2</em>, is clearly visible. The remaining portion of T-5 is disordered. This structure presents the first evidence for NeuNAc binding in the aromatic residue-rich sites of domains A and C and suggests a preference of WGA for alpha <em>2</em>,6-linked NeuNAc. Moreover, the unusual asymmetric WGA1-tetrasaccharide association, involving domain binding sites that differ in their binding affinities for NeuNAc, offers explanations for the widely observed cooperative cell binding behavior of WGA.

BACKGROUND

The receptor for advanced glycation endproducts (RAGE) recognizes a variety of ligands that play an important role in the posttraumatic inflammatory response. However, whether soluble RAGE (sRAGE) is released early after trauma hemorrhage in humans and whether such a release is associated with the development of an inflammatory response and coagulopathy is not known and therefore constitutes the aim of this study.

METHODS

One hun<em>d</em>re<em>d</em> sixty-eight patients were stu<em>d</em>ie<em>d</em> as part of a prospective cohort stu<em>d</em>y of severe trauma patients a<em>d</em>mitte<em>d</em> to a single Level I Trauma center. Bloo<em>d</em> was <em>d</em>rawn within 10 minutes of arrival to the emergency <em>d</em>epartment before the a<em>d</em>ministration of any flui<em>d</em> resuscitation. sRAGE, tumor necrosis factor-alpha, interleukin-6, von Willebran<em>d</em> factor, angiopoietin-<em>2</em>, prothrombin time, prothrombin fragments 1 + <em>2</em>, soluble thrombomo<em>d</em>ulin, protein C, plasminogen activator inhibitor-1, an<em>d</em> <em>d</em>-<em>dimers</em> (fibrin <em>d</em>egra<em>d</em>ation pro<em>d</em>ucts) were measure<em>d</em> using stan<em>d</em>ar<em>d</em> techniques. Base <em>d</em>eficit was use<em>d</em> as a measure of tissue hypoperfusion. Measurements were compare<em>d</em> with outcome measures obtaine<em>d</em> from the electronic me<em>d</em>ical recor<em>d</em> an<em>d</em> trauma registry.

RESULTS

Plasma levels of sRAGE were increase<em>d</em> within 30 minutes after severe trauma in humans an<em>d</em> correlate<em>d</em> with the severity of injury, early posttraumatic coagulopathy an<em>d</em> hyperfibrinolysis, an<em>d</em> en<em>d</em>othelial cell activation (angiopoietin-1 an<em>d</em> complement). Furthermore, we foun<em>d</em> that there was a significant relationship between plasma levels of sRAGE an<em>d</em> the <em>d</em>evelopment of acute renal failure. This relationship was not quite significant for patients who <em>d</em>evelope<em>d</em> acute lung injury (p = 0.11), although patients with (<em>2</em>6 ventilator-free <em>d</em>ays ha<em>d</em> significantly higher plasma levels of sRAGE than those with>><em>2</em>6 ventilator-free <em>d</em>ays. Finally, there was no relationship between plasma levels of sRAGE an<em>d</em> mortality rate in trauma patients.

CONCLUSIONS

The results of this study demonstrate that the release of sRAGE in the bloodstream of trauma patients requires severe injury and is associated with coagulation abnormalities and endothelial cell and complement activation.

We describe 5 children with severe SARS-CoV-<em>2</em> infection, hemodynamic instability and suspected acute abdomen. This form of the disease has not been previously documented. Four of the cases were confirmed SARS-CoV-<em>2</em> infection and 1 probable. All of them were previously healthy and needed a pediatric critical care unit admission. The respiratory symptoms were not dominant or were absent. Also, fever was observed. Laboratory testing revealed lymphopenia and high levels of C-reactive protein and procalcitonin with <em>D</em>-<em>dimer</em>, ferritin and interleukin-6 usually elevated. Respiratory support and inotropic support were almost always necessary. In all of them, deterioration occurred on the day of admission.

We have examined the interaction of the Escherichia coli trp aporepressor with its ligand, L-tryptophan, using both equilibrium dialysis and flow dialysis methods. Results obtained by the two procedures were equivalent and indicate that the trp aporepressor binds L-tryptophan with an equilibrium dissociation constant (Kd) of 40 microM at <em>2</em>5 degrees C under standard binding assay conditions (10 mM potassium phosphate, pH 7.4, 0.<em>2</em> M potassium chloride, 0.1 mM EDTA, 5% glycerol). Molecular sizing of the purified trp aporepressor shows that in the absence of ligand the regulatory protein exists as a <em>dimer</em>ic species with greater than 99% purity and an apparent molecular weight of 30,000. Under the storage and assay conditions used, the <em>dimer</em> appears quite stable, and essentially no monomer or higher multimeric species are detected. Analysis of binding data by Scatchard and direct linear plot methods shows two identical and independent ligand-binding sites/native trp aporepressor <em>dimer</em>. When examined as a function of temperature, L-tryptophan binding by trp aporepressor varied over 7-fold (Kd = <em>2</em>8 microM at 6.5 degrees C to Kd = <em>2</em>17 microM at 40 degrees C). At the optimal growth temperature for E. coli (37 degrees C), the dissociation constant was 160 microM for the ligand, L-tryptophan. From the relationship between temperature and L-tryptophan binding by trp aporepressor, the apparent enthalpy change <em>delta</em> H = -10.6 +/- 0.6 kcal mol-1 and the apparent entropy change <em>delta</em> S = -17 +/- <em>2</em> cal degree-1 mol-1 were determined.

The anticancer antibiotic chromomycin A3 (Chro) is a <em>D</em>NA minor groove bin<em>d</em>ing <em>d</em>rug belonging to the aureolic family. Chro likely exerts its activity by interfering with replication an<em>d</em> transcription. Chro forms a <em>dimer</em>, me<em>d</em>iate<em>d</em> by a <em>d</em>ivalent metal ion, which bin<em>d</em>s to G/C-rich <em>D</em>NA. Herein we report the first crystal structure of Chro boun<em>d</em> to <em>d</em>(TTG GCCAA)<em>2</em> <em>D</em>NA <em>d</em>uplex solve<em>d</em> by multiwavelength anomalous <em>d</em>iffraction (MA<em>D</em>) base<em>d</em> on the chelate<em>d</em> Co3+ ion. The structure of the Mg<em>2</em>+ complex was subsequently refine<em>d</em> at <em>2</em>.15 A resolution, which reveale<em>d</em> two complexes of metal-coor<em>d</em>inate<em>d</em> <em>dimers</em> of Chro boun<em>d</em> to the octamer <em>D</em>NA <em>d</em>uplex in the asymmetric unit. The metal ion is octahe<em>d</em>rally coor<em>d</em>inate<em>d</em> to the O1 an<em>d</em> O9 oxygen atoms of the chromophore (CPH), an<em>d</em> two water molecules act as the fifth an<em>d</em> sixth ligan<em>d</em>s. The two coor<em>d</em>inate<em>d</em> water molecules are hy<em>d</em>rogen bon<em>d</em>e<em>d</em> to O<em>2</em> atoms of C5 an<em>d</em> C13 bases. The Chro <em>dimer</em> bin<em>d</em>s at an<em>d</em> significantly wi<em>d</em>ens the minor groove of the GGCC sequence. The long axis of each chromophore lies along an<em>d</em> stacks over the sugar-phosphate backbone with the two attache<em>d</em> sacchari<em>d</em>e moieties (rings A/B an<em>d</em> C/<em>D</em>/E) wrapping across the minor groove. <em>D</em>NA is kinke<em>d</em> by 30 <em>d</em>egrees an<em>d</em> 36 <em>d</em>egrees in the two complexes, respectively. Six G-specific hy<em>d</em>rogen bon<em>d</em>s between Chro an<em>d</em> <em>D</em>NA provi<em>d</em>e the GGCC sequence specificity. Interestingly, <em>D</em>NA in concert with Chro appears to act as an effective template to catalyze the <em>d</em>eamination of Co(NH3)6(3+), as shown by circular <em>d</em>ichroism an<em>d</em> crystal structure <em>d</em>ata. Our results present useful structural information for <em>d</em>esigning new anticancer <em>d</em>rug <em>d</em>erivatives in the future.

3,3'-<em>D</em>iindolylmethane is a <em>dimer</em> of indole-3-carbinol formed both in vivo and in vitro. In this study, human cancer cells MCF-7 (with wild-type p53), T47-<em>D</em> (mutant p53), and Saos-<em>2</em> (deficient in p53 gene), were used to examine the anticancer activities of 3,3'-diindolylmethane. The dose-dependent growth inhibitory effect was found in all these cell lines. Exposure of the cells to 50 microM solution of 3,3'-diindolylmethane for 48 h, apoptosis (programmed cell death) was evidenced by the characteristic morphology of cell nuclei under fluorescence microscope and the <em>D</em>NA "ladder" in agarose gel electrophoresis. The percentage of apoptotic cells in each cell line was found to be 1<em>2</em>% for MCF-7, 14% for T47<em>D</em> and 13% for Saos<em>2</em> cells. Exposure of MCF-7 cells to 100 microM 3,3'-diindolylmethane for <em>2</em>4 h, 19% of apoptotic cells were detected by flow cytometry analysis. The lowest dose required for induction of apoptosis in MCF-7 cells was found to be 10 microM after 7<em>2</em> h incubation. Western blot showed that wild-type p53 protein was unchanged after MCF-7 cells had been exposed to 50 microM 3,3'-diindolylmethane for 8 h. This study provides evidences that 3,3'-diindolylmethane induces apoptosis in human cancer cells and that the induction of apoptosis is independent of p53 pathway.

The main pathogenic process underlying dialysis-related amyloidosis is the accumulation of β-<em>2</em>-microglobulin (β<em>2</em>m) as amyloid fibrils in the musculoskeletal system, and some evidence suggests that Cu(II) may play a role in β<em>2</em>m amyloid formation. Cu(II)-induced β<em>2</em>m fibril formation is preceded by the formation of discrete, oligomeric intermediates, including <em>dimers</em>, tetramers, and hexamers. In this work, we use selective covalent labeling reactions combined with mass spectrometry to investigate the amino acids responsible for mediating tetramer formation in wild-type β<em>2</em>m. By comparing the labeling patterns of the monomer, <em>dimer</em>, and tetramer, we find evidence that the tetramer interface is formed by the interaction of <em>D</em> strands from one <em>dimer</em> unit and G strands from another <em>dimer</em> unit. These covalent labeling data along with molecular dynamics calculations allow the construction of a tetramer model that indicates how the protein might proceed to form even higher-order oligomers.

Twenty two patients with exacerbation of inflammatory bowel disease (19 with Crohn's disease, 3 with ulcerative colitis) and thrombocytosis were tested for possible activation of the coagulation and platelet system. Fifteen patients had abnormal platelet function i.e. unphysiologically high sensitivity in vitro towards A<em>D</em>P <em>2</em> mumol/l aggregation induction. In 81.8% of the patients we found enhanced fibrinogen concentrations. In <em>2</em><em>2</em>.7% of the patients thrombin-antithrombin III values exceeded the upper limit of the reference range, and in 68.<em>2</em>% of the patients the <em>D</em>-<em>Dimer</em> concentration exceeded the upper reference limit as a result of reactive fibrinolysis. The altered platelet count and function, and the increased levels of fibrinogen and thrombin-antithrombin III with reactive fibrinolysis activation indicate the presence of prethrombotic factors in patients with exacerbation of inflammatory bowel disease. The presence of enhanced fibrinolysis in these patients might have consequences for the therapeutic treatment.

The OpuA system of Bacillus subtilis is a member of the substrate-binding-protein-dependent ABC transporter superfamily and serves for the uptake of the compatible solute glycine betaine under hyperosmotic growth conditions. Here, we have characterized the nucleotide-binding protein (OpuAA) of the B.subtilis OpuA transporter in vitro. OpuAA was overexpressed heterologously in Escherichia coli as a hexahistidine tag fusion protein and purified to homogeneity by affinity and size exclusion chromatography (SEC). <em>D</em>ynamic monomer/<em>dimer</em> equilibrium was observed for OpuAA, and the K(<em>D</em>) value was determined to be 6 microM. Under high ionic strength assay conditions, the monomer/<em>dimer</em> interconversion was diminished, which enabled separation of both species by SEC and separate analysis of both monomeric and <em>dimer</em>ic OpuAA. In the presence of 1 M NaCl, monomeric OpuAA showed a basal ATPase activity (K(M)=0.45 mM; k(<em>2</em>)=<em>2</em>.3 min(-1)), whereas <em>dimer</em>ic OpuAA showed little ATPase activity under this condition. The addition of nucleotides influenced the monomer/<em>dimer</em> ratio of OpuAA, demonstrating different oligomeric states during its catalytic cycle. The monomer was the preferred species under post-hydrolysis conditions (e.g. A<em>D</em>P/Mg(<em>2</em>+)), whereas the <em>dimer</em> dominated the nucleotide-free and ATP-bound states. The affinity and stoichiometry of monomeric or <em>dimer</em>ic OpuAA/ATP complexes were determined by means of the fluorescent ATP-analog TNP-ATP. One molecule of TNP-ATP was bound in the monomeric state and two TNP-ATP molecules were detected in the <em>dimer</em>ic state of OpuAA. Binding of TNP-A<em>D</em>P/Mg(<em>2</em>+) to <em>dimer</em>ic OpuAA induced a conformational change that led to the decay of the <em>dimer</em>. On the basis of our data, we propose a model that couples changes in the oligomeric state of OpuAA with ATP hydrolysis.

The avian sarcoma/leukemia virus protease (PR), purified from avian myeloblastosis virus has a native molecular mass of <em>2</em>6 k<em>D</em>a, suggesting a <em>dimer</em> structure. The enzymatic activity of PR has been characterized using synthetic peptide substrates. PR is most active at pH 5.5, 35 degrees C and <em>2</em>-3 M NaCl. Under these conditions PR cleaves decapeptides which are resistant in low ionic strength. This high, nonphysiological, salt concentration also increases the proteolytic activity of a cellular aspartic protease, pepsin. PR and pepsin show additional similarities: they both cleave a synthetic decapeptide at the same Tyr-Pro bond in low and high salt, while the cleavage site preferences of human renin and cathepsin-<em>D</em> in this substrate are altered at high salt concentrations. In addition, iodination of the tyrosine residue in this decapeptide causes an increase in the rates of hydrolysis by both PR and pepsin. However, Km values are too high to be estimated accurately for PR using Tyr-Pro and Tyr(I)-Pro decapeptides as substrates. Comparison of the digestion products of two additional decapeptides, altered in a single amino acid residue, shows that PR cleaves at fewer sites than all three cellular enzymes. Furthermore pepstatin, a strong inhibitor of pepsin, renin, and cathepsin-<em>D</em> has little effect on PR.

Pseudomonas putida uses L-phenylalanine as the sole nitrogen source for growth by converting L-phenylalanine to L-tyrosine, which acts as a donor of the amino group. This metabolic step requires the products of the phhA and phhB genes, which form an operon. Expression of the phhA promoter is mediated by the phhR gene product in the presence of L-phenylalanine or L-tyrosine. The PhhR protein belongs to the NtrC family of enhancers. In contrast with most members of this family of regulators, transcription from the promoter of the phhAB operon (P(phhA)) is mediated by RNA polymerase with sigma(70) rather than with sigma(54). The PhhR regulator binds two similar but non-identical upstream PhhR motifs (5'-TGTAAAATTATCGTTACG-3' and 5'-ACAAAAACTGTGTTTCCG-3') that are located 39 and 97 nucleotides upstream of the proposed -35 hexamer for RNA polymerase, respectively. These motifs are called PhhR proximal and PhhR distal binding motifs because of their position with respect to the RNA polymerase binding site. Affinity of PhhR for its target sequences was determined by isothermal titration calorimetry and was found to be around 30 nM for the proximal site and <em>2</em> microM for the distal site, and the binding stoichiometry is of a <em>dimer</em> per binding site. Both target sequences are sine qua non requirements for transcription, since inactivation of either of them resulted in no transcription from the phhA promoter. An IHF binding site overlaps the proximal PhhR proximal motif, which is recognized by IHF with a K(<em>D</em>) of around 1.<em>2</em> microM. IHF may consequently compete with PhhR for binding and indeed inhibits PhhR-dependent phhAB operon expression.

BACKGROUND

To test the hypothesis that tissue factor release, thrombin activation, fibrin formation, and fibrinolysis after an isolated head injury are equal to those in patients without head injury, as well as to investigate the precise time course of the coagulation and fibrinolytic abnormalities after head injury, we performed prospective and retrospective studies.

RESULTS

In the prospective study, 5 patients with isolated head injury and 11 trauma patients without head injury took part in this study. Tissue factor antigen concentration, prothrombin fragment F1+<em>2</em>, thrombin antithrombin complex, fibrinopeptide A, and fibrin degradation products (<em>D</em>-<em>dimer</em>) were measured on the day of admission, and days 1, <em>2</em>, 3, and 4 after admission. The levels of all five hemostatic molecular markers were markedly elevated on the day of admission, and then gradually decreased to day 4. The levels and the time course of these hemostatic markers in patients with isolated head injury were not different from those in the control patients. The same incidence of disseminated intravascular coagulation between the two groups was also observed. In the retrospective study, the records of fibrinopeptide Bbeta15-4<em>2</em>, plasmin antiplasmin complex, plasminogen activator inhibitor-1 antigen concentration (PAI-1 antigen), and PAI-1 activity in 76 trauma patients were reviewed. On the basis of the exclusion criteria, 9 patients with isolated head injury and 30 control patients were selected for the study group. Fibrinopeptide Bbeta15-4<em>2</em> and plasmin antiplasmin complex markedly elevated on the day of admission, then decreased on day 1, and tended to increase to day 5. Markedly elevated PAI-1 antigen and PAI-1 activity on the day of admission significantly decreased on day 1 and recovered to the normal values on day 5. The changes of these molecular markers in patients with isolated head injury were equal to those in the control patients.

CONCLUSIONS

We systematically elucidated the time course of coagulation and fibrinolysis after isolated head injury. We further demonstrated that changes in coagulofibrinolytic and antifibrinolytic systems in patients with isolated head injury are not different from those in patients without head injury.

BACKGROUND

Studies of fibrinolysis/coagulation status in women with polycystic ovary syndrome (PCOS) are contradictory.

OBJECTIVE

The aim of the study was to investigate whether women with PCOS have disturbed circulating levels of fibrinolysis/coagulation markers and, if so, whether the disturbances are related to hemodynamics, metabolic variables, sex steroids, SHBG, lipids, and inflammatory variables in women with PCOS. DESIGN/MAIN OUTCOME MEASURES: Anthropometric variables, hemodynamics, circulating hemostatic and inflammatory markers, and serum lipid profile were measured in women with untreated PCOS (n = 74) and controls (n = 31).

RESULTS

After adjustments for age and body mass index (BMI), circulating plasminogen activator inhibitor 1 (PAI-1) activity and fibrinogen levels were higher in women with PCOS than controls; lipid profile, blood pressure, and levels of <em>D</em>-<em>dimer</em>, von Willebrand factor, factor VIII, tissue plasminogen activator, and inflammatory markers were comparable in the two groups. In multiple linear regression analyses including women with PCOS, low SHBG and high insulin predicted high PAI-1 activity (R(<em>2</em>) = 0.5<em>2</em>6; P < 0.001); elevated high-sensitivity C-reactive protein and soluble E-selectin in combination with heart rate predicted high fibrinogen (R(<em>2</em>) = 0.333; P < 0.001). <em>D</em>ifferences in PAI-1 activity were not significant after adjustments for age, BMI, SHBG, and insulin.

CONCLUSIONS

PCOS is characterized by a prothrombotic state, as reflected by increased PAI-1 activity and fibrinogen, without signs of dyslipidemia or a proinflammatory state. Low SHBG and high insulin may partly explain the BMI-independent difference in PAI-1 activity between women with PCOS and controls. High-sensitivity C-reactive protein and E-selectin may be involved in regulating fibrinogen in PCOS.

The RAG1 and RAG<em>2</em> proteins together constitute the nuclease that initiates the assembly of immunoglobulin and T cell receptor genes in a reaction known as V(<em>D</em>)J recombination. RAG1 plays a central role in recognition of the recombination signal sequence (RSS) by the RAG1/<em>2</em> complex. To investigate the parameters governing the RAG1-RSS interaction, the murine core RAG1 protein (amino acids 377-1008) fused to a short Strep tag has been purified to homogeneity from bacteria. The Strep-RAG1 (StrRAG1) protein exists as a <em>dimer</em> at a wide range of protein concentrations (<em>2</em>5-500 nM) in the absence of <em>D</em>NA and binds with reasonably high affinity and specificity (apparent K(<em>D</em>) = 41 nM) to the RSS. Both electrophoretic mobility shift assays and polarization anisotropy experiments indicate that only a single StrRAG1-<em>D</em>NA species exists in solution. Anisotropy decay measured by frequency domain spectroscopy suggests that the complex contains a <em>dimer</em> of StrRAG1 bound to a single <em>D</em>NA molecule. Using measurements of protein intrinsic fluorescence and circular dichroism, we demonstrate that StrRAG1 undergoes a major conformational change upon binding the RSS. Steady-state fluorescence and acrylamide quenching studies reveal that this conformational change is associated with a repositioning of intrinsic protein fluorophores from a hydrophobic to a solvent-exposed environment. RSS-induced conformational changes of StrRAG1 may influence the interaction of RAG1 with RAG<em>2</em> and synaptic complex formation.

The pharmacokinetics an<em>d</em> quality of planar an<em>d</em> SPECT brain imaging of two 99mTc-labele<em>d</em> brain perfusion agents, <em>d</em>,l-hexamethyl propylene amine oxime (HMPAO) an<em>d</em> ethyl cysteinate <em>dimer</em> (ECD), were compare<em>d</em> in seven healthy, normal subjects. Both ra<em>d</em>iopharmaceuticals showe<em>d</em> rapi<em>d</em> brain uptake an<em>d</em> ha<em>d</em> a net brain washout of less than 5% <em>d</em>uring the first <em>2</em>0 min after <em>d</em>rug a<em>d</em>ministration. However, <em>d</em>uring the same time perio<em>d</em>, 99mTc-ECD images of the hea<em>d</em> showe<em>d</em> significantly less backgroun<em>d</em> facial uptake an<em>d</em> retention when compare<em>d</em> to 99mTc-HMPAO images. The brain-to-backgroun<em>d</em> contrast ratio of 99mTc-ECD (brain/neck) continue<em>d</em> to improve over time an<em>d</em> by 5 hr posta<em>d</em>ministration was 17 to 1 versus <em>2</em> to 1 for 99mTc-HMPAO. SPECT brain images of both agents show gray/white matter ratios that were unchange<em>d</em> over time an<em>d</em> an intracerebral <em>d</em>istribution consistent with bloo<em>d</em> flow. A blin<em>d</em> rea<em>d</em> of these SPECT images also shows 99mTc-ECD to pro<em>d</em>uce images that were "easier to interpret" with less extracerebral activity as compare<em>d</em> to 99mTc-HMPAO. Repeat, whole-bo<em>d</em>y planar spot imaging suggests that 99mTc-ECD was cleare<em>d</em> more rapi<em>d</em>ly from the bo<em>d</em>y than was 99mTc-HMPAO.