<em>D</em>uring normal pregnancy the hemostatic balance changes in the direction of hypercoagulability, thus decreasing bleeding complications in connection with delivery. The most important initial factor for acute hemostasis at delivery is, however, uterine muscle contractions, which interrupt blood flow. Global tests such as Sonoclot signature, the Thromboelastogram, and a new method analyzing overall plasma hemostasis, all show changes representative of hypercoagulability during pregnancy. Increased endogenous thrombin generation, acquired activated protein C resistance, slightly decreased activated partial thromboplastin time (aPTT) and increased prothrombin complex level (PT) measured as international normalized ratio (INR) of less than 0.9 have been reported as well. In normal pregnancy, the platelet count is within normal range except during the third trimester when benign gestational thrombocytopenia, 80 to 150 x 10 9/L, can be observed. Platelet turnover is usually normal. Activation of platelets and release of beta-thromboglobulin and platelet factor 4 are reported. The bleeding time is unchanged during normal pregnancy. Most blood coagulation factors and fibrinogen increase during pregnancy. Factor (F) XI is the only blood coagulation factor that decreases. Blood coagulation inhibitors are mainly unchanged but the level of free protein S decreases markedly and the level of tissue factor pathway inhibitor increases. Thrombomodulin levels increase during pregnancy. Fibrinolytic capacity is diminished during pregnancy, mainly because of markedly increased levels of plasminogen activator inhibitor-1 (PAI-1) from endothelial cells and plasminogen activator inhibitor-<em>2</em> (PAI-<em>2</em>) from the placenta. Thrombin-activated fibrinolysis inhibitor is reported to be unaffected. The total hemostatic balance has been studied by analyses of prothrombin fragment 1+<em>2</em>, thrombin-antithrombin complex, fibrinopeptide A, soluble fibrin, <em>D</em>-<em>dimer</em>, and plasmin-antiplasmin complex. There is activation of blood coagulation and a simultaneous increase in fibrinolysis without signs of organ dysfunction during normal pregnancy. These changes increase as pregnancy progresses. <em>D</em>uring delivery, there is consumption of platelets and blood coagulation factors, including fibrinogen. Fibrinolysis improves and increases fast following childbirth and expulsion of the placenta, resulting in increased <em>D</em>-<em>dimer</em> levels. These changes are self-limiting at normal delivery. The hemostatic changes, noted during pregnancy, normalize after delivery within 4 to 6 weeks. Platelet count and free protein S, however, can be abnormal longer. Hemostasis should not be tested earlier than 3 months following delivery and after terminating lactation to rule out influences of pregnancy. PAI-1 and PAI-<em>2</em> levels decrease fast postpartum, but PAI <em>2</em> has been detected up to 8 weeks postpartum. alpha <em>2</em> -antiplasmin, urokinase, and kallikrein inhibitor levels have been reported to be increased 6 weeks postpartum.

Increasing evidence indicates the significance of platelet-derived growth factor receptor-β (β-P<em>D</em>GFR) signaling in prostate cancer (PCa). Accordingly, preclinical studies suggest the potential of β-P<em>D</em>GFR as a therapeutic target in metastatic PCa. However, a ligand responsible for β-P<em>D</em>GFR activation in PCa was unknown, and recent clinical trials with imatinib mesylate showed limited success due to normal tissue toxicity. Similarly, in spite of mounting evidence indicating the significance of matriptase in PCa, little is known about its substrates or molecular actions during PCa progression. Here, we identified P<em>D</em>GF-<em>D</em> as a ligand for β-P<em>D</em>GFR in PCa and discovered matriptase as its regulator. Matriptase activates P<em>D</em>GF-<em>D</em> by proteolytic removal of the CUB domain in a <em>2</em>-step process, creating a hemi<em>dimer</em>, followed by growth factor domain <em>dimer</em> (GF<em>D</em>-<em>D</em>) generation. Matriptase can deactivate P<em>D</em>GF-<em>D</em> by further proteolytic cleavage within the GF<em>D</em>, revealing its biphasic regulation. Importantly, P<em>D</em>GF-<em>D</em>/matriptase colocalization is accompanied with β-P<em>D</em>GFR phosphorylation in human PCa tissues. This study unveiled a novel signaling axis of matriptase/P<em>D</em>GF-<em>D</em>/β-P<em>D</em>GFR in PCa, providing new insights into functional interplay between serine protease and growth factor signaling networks.

By <em>2</em>1 March <em>2</em>0<em>2</em>0 infections related to the novel coronavirus SARS-CoV-<em>2</em> had affected people from 177 countries and caused 11,<em>2</em>5<em>2</em> reported deaths worldwide. Little is known about risk, presentation and outcomes of SARS-CoV-<em>2</em> (COVI<em>D</em>-19) infection in kidney transplantation recipients, who may be at high-risk due to long-term immunosuppression, comorbidity and residual chronic kidney disease. Whilst COVI<em>D</em>-19 is predominantly a respiratory disease, in severe cases it can cause kidney and multi-organ failure. It is unknown if immunocompromised hosts are at higher risk of more severe systemic disease. Therefore, we report on seven cases of COVI<em>D</em>-19 in kidney transplant recipients (median age 54 (range 45-69), three females, from a cohort of <em>2</em>08<em>2</em> managed transplant follow-up patients) over a six-week period in three south London hospitals. Two of seven patients presented within three months of transplantation. Overall, two were managed on an out-patient basis, but the remaining five required hospital admission, four in intensive care units. All patients displayed respiratory symptoms and fever. Other common clinical features included hypoxia, chest crepitation, lymphopenia and high C-reactive protein. Very high <em>D</em> <em>dimer</em>, ferritin and troponin levels occurred in severe cases and likely prognostic. Immunosuppression was modified in six of seven patients. Three patients with severe disease were diabetic. <em>D</em>uring a three week follow up one patient recovered, and one patient died. Thus, our findings suggest COVI<em>D</em>-19 infection in kidney transplant patients may be severe, requiring intensive care admission. The symptoms are predominantly respiratory and associated with fever. Most patients had their immunosuppression reduced and were treated with supportive therapy.

As thrombin binding to the G protein-coupled proteinase activated receptor-1 (PAR-1) induces endothelial adhesivity to leukocytes through NF-kappaB activation and intercellular adhesion molecule-1 (ICAM-1) expression, we determined the signaling pathways mediating the response. Studies showed that the heterotrimeric G proteins, Galpha(q), and the Gbetagamma <em>dimer</em> were key determinants of the PAR-1 agonist peptide (TFLLRNPNDK)-induced NF-kappaB activation and ICAM-1 expression in endothelial cells. Cotransfection of RGS3T, a regulator of G-protein signaling that inhibits Galpha(q), or alpha-transducin (Galpha(t)), a scavenger of the Gbetagamma, markedly decreased NF-kappaB activity induced by PAR-1 activation. We determined the downstream signaling targets activated by Galpha(q) and Gbetagamma that mediate NF-kappaB activation. Expression of the kinase-defective protein kinase C (PKC)-<em>delta</em> mutant inhibited NF-kappaB activation induced by the constitutively active Galpha(q) mutant, but had no effect on NF-kappaB activity induced by Gbeta(1)gamma(<em>2</em>). In related experiments, NF-kappaB as well as ICAM-1 promoter activation induced by Gbeta(1)gamma(<em>2</em>) were inhibited by the expression of the dominant-negative mutant of 85-kDa regulatory subunit of PI 3-kinase; however, the expression of this mutant had no effect on the response induced by activated Galpha(q). Cotransfection of the catalytically inactive Akt mutant inhibited the NF-kappaB activation induced by the constitutively active PI 3-kinase mutant as well as that by the activated forms of Galpha(q) and PKC-<em>delta</em>. These results support a model in which ligation of PAR-1 induces NF-kappaB activation and ICAM-1 transcription by the engagement of parallel Galphaq/PKC-<em>delta</em> and Gbetagamma/PI3-kinase pathways that converge at Akt.

OBJECTIVE

To investigate the relationship between inflammatory [interleukin-6 (IL-6) and C-reactive protein (CRP)] and coagulation (D-dimer) biomarkers and cancer risk during HIV infection.

METHODS

A prospective cohort.

METHODS

HIV-infected patients on continuous antiretroviral therapy (ART) in the control arms of three randomized trials (N=5023) were included in an analysis of predictors of cancer (any type, infection-related or infection-unrelated). Hazard ratios for IL-6, CRP and <em>D</em>-<em>dimer</em> levels (log2-transformed) were calculated using Cox models stratified by trial and adjusted for demographics and C<em>D</em>4+ cell counts and adjusted also for all biomarkers simultaneously. To assess the possibility that biomarker levels were elevated at entry due to undiagnosed cancer, analyses were repeated excluding early cancer events (i.e. diagnosed during first 2 years of follow-up).

RESULTS

During approximately 24,000 person-years of follow-up (PYFU), 172 patients developed cancer (70 infection-related; 102 infection-unrelated). The risk of developing cancer was associated with higher levels (per doubling) of IL-6 (hazard ratio 1.38, P<0.001), CRP (hazard ratio 1.16, P=0.001) and D-dimer (hazard ratio 1.17, P=0.03). However, only IL-6 (hazard ratio 1.29, P=0.003) remained associated with cancer risk when all biomarkers were considered simultaneously. Results for infection-related and infection-unrelated cancers were similar to results for any cancer. Hazard ratios excluding 69 early cancer events were 1.31 (P=0.007), 1.14 (P=0.02) and 1.07 (P=0.49) for IL-6, CRP and D-dimer, respectively.

CONCLUSIONS

Activated inflammation and coagulation pathways are associated with increased cancer risk during HIV infection. This association was stronger for IL-6 and persisted after excluding early cancer. Trials of interventions may be warranted to assess whether cancer risk can be reduced by lowering IL-6 levels in HIV-positive individuals.

V(<em>D</em>)J recombination occurs at recombination signal sequences (RSSs) containing conserved heptamer and nonamer elements. RAG-1 and RAG-<em>2</em> initiate recombination by cleaving <em>D</em>NA between heptamers and antigen receptor coding segments. RAG-1 alone contacts the nonamer but interacts weakly, if at all, with the heptamer. RAG-<em>2</em> by itself has no <em>D</em>NA-binding activity but promotes heptamer occupancy in the presence of RAG-1; how RAG-<em>2</em> collaborates with RAG-1 has been poorly understood. Here we examine the composition of RAG-RSS complexes and the relative contributions of RAG-1 and RAG-<em>2</em> to heptamer binding. RAG-1 exists as a <em>dimer</em> in complexes with an isolated RSS bearing a 1<em>2</em>-bp spacer, regardless of whether RAG-<em>2</em> is present; only a single subunit of RAG-1, however, participates in nonamer binding. In contrast, multimeric RAG-<em>2</em> is not detectable by electrophoretic mobility shift assays in complexes containing both RAG proteins. <em>D</em>NA-protein photo-cross-linking demonstrates that heptamer contacts, while enhanced by RAG-<em>2</em>, are mediated primarily by RAG-1. RAG-<em>2</em> cross-linking, while less efficient than that of RAG-1, is detectable near the heptamer-coding junction. These observations provide evidence that RAG-<em>2</em> alters the conformation or orientation of RAG-1, thereby stabilizing interactions of RAG-1 with the heptamer, and suggest that both proteins interact with the RSS near the site of cleavage.

The homotetrameric protein transthyretin (TTR) must undergo rate-limiting dissociation to its constituent monomers in order to enable partial denaturation that allows the process of amyloidogenesis associated with human pathology to ensue. The TTR quaternary structure contains two distinct <em>dimer</em> interfaces, one of which creates the two binding sites for the natural ligand thyroxine. Tetramer dissociation could proceed through three distinct pathways; scission into <em>dimers</em> along either of the two unique quaternary interfaces followed by <em>dimer</em> dissociation represents two possibilities. Alternatively, the tetramer could lose monomers sequentially. To elucidate the TTR dissociation pathway, we employed two different TTR constructs, each featuring covalent attachment of proximal subunits. We demonstrate that tethering the A and B subunits of TTR with a disulfide bond (as well as the symmetrically disposed C and <em>D</em> subunits) allows urea-mediated dissociation of the resulting (TTR-S-S-TTR)(<em>2</em>) construct, affording (TTR-S-S-TTR)(1) retaining a stable 16-stranded beta-sheet structure that is equivalent to the <em>dimer</em> not possessing a thyroid binding site. In contrast, linking the A and C subunits employing a peptide tether (TTR-L-TTR)(<em>2</em>) affords a kinetically stable quaternary structure that does not dissociate or denature in urea. Both tethered constructs and wild-type TTR exhibit analogous stability based on guanidine hydrochloride denaturation curves. The latter denaturant can denature the tetramer, unlike urea, which can only denature monomeric TTR; hence urea requires dissociation to monomers to function. Under native conditions, the (TTR-S-S-TTR)(<em>2</em>) construct is able to dissociate and incorporate subunits from labeled WT TTR homotetramers at a rate equivalent to that exhibited by WT TTR. In contrast, the (TTR-L-TTR)(<em>2</em>) construct is unable to exchange any subunits, even after 180 h. All of the data presented herein and elsewhere demonstrate that the pathway of TTR tetramer dissociation occurs by scission of the tetramer along the crystallographic C(<em>2</em>) axis affording AB and C<em>D</em> <em>dimers</em> that rapidly dissociate into monomers. <em>D</em>etermination of the mechanism of dissociation provides an explanation for why small molecules that bind at the AB/C<em>D</em> <em>dimer</em>-<em>dimer</em> interface impose kinetic stabilization upon TTR and disease-associated variants thereof.

Both plant growth promoting Pseudomonas B10 and its yellow-green, fluorescent iron transfer agent (siderophore) pseudobactin enhance the growth of the potato and control certain phytopathogenic microorganisms. The structure of the little compound has been determined by single-crystal X-ray diffraction methods using counter data. The structure consisted of a linear hexapeptide, L-Lys-<em>D</em>-threo-beta-OH-Asp-L-Ala-<em>D</em>-allo-Thr-L-Ala-<em>D</em>-N <em>delta</em>-OH-Orn, in which the N <em>delta</em>-OH nitrogen of the ornithine was cyclized with the C-terminal carboxyl group, and the N epsilon-amino group of the lysine was linked via an amide bond to a fluorescent quinoline derivative. The iron-chelating groups consisted of a hydroxamate group derived from N <em>delta</em>-hydroxyornithine, an alpha-hydroxy acid derived from beta-hydroxyaspartic acid, and an o-dihydroxy aromatic group derived from the quinoline moiety. The combination of metal-chelating ligands and the alternating L- and <em>D</em>-amino acids was unusual. The little compound crystallized as a single coordination isomer with the lambda absolute configuration. The present study is the first structural determination of a fluorescent siderophore. In the crystal structure, ferric pseudobactin formed a <em>dimer</em>, which constituted the asymmetric unit. The asymmetric unit also contained <em>2</em>6 water molecules. The molecules in the <em>dimer</em> were related by a pseudo-<em>2</em>-fold symmetry axis. Red-brown crystals of ferric pseudobactin (C4<em>2</em>H57N1<em>2</em>O16Fe . 13H<em>2</em>O), obtained from pyridine-acetic acid buffer solution equilibrated with water, conformed to space group I<em>2</em> with a = <em>2</em>9.006 (<em>2</em>3) A, b = 14.511 (13) A, c = <em>2</em>8.791 (<em>2</em>1) A, and beta = 96.06 (5) degrees at -135 (<em>2</em>) degrees C. For eight molecules per unit cell, the calculated density was 1.38 g/cm3; the observed density was 1.40 g/cm3. The structure was refined by least-squares methods with anisotropic thermal parameters for all nonhydrogen atoms to a final R factor of 0.08 (8989 observed reflections).

Photoaffinity labeling methods have allowed a definition of the sites of interaction between Taxol and its cellular target, the microtubule, specifically beta-tubulin. Our previous studies have indicated that [(3)H]3'-(p-azidobenzamido)Taxol photolabels the N-terminal 31 amino acids of beta-tubulin (Rao, S., Krauss, N. E., Heerding, J. M., Swindell, C. S., Ringel, I., Orr, G. A., and Horwitz, S. B. (1994) J. Biol. Chem. <em>2</em>69, 313<em>2</em>-3134) and [(3)H]<em>2</em>-(m-azidobenzoyl)Taxol photolabels a peptide containing amino acid residues <em>2</em>17-<em>2</em>33 of beta-tubulin (Rao, S., Orr, G. A., Chaudhary, A. G., Kingston, <em>D</em>. G. I., and Horwitz, S. B. (1995) J. Biol. Chem. <em>2</em>70, <em>2</em>0<em>2</em>35-<em>2</em>0<em>2</em>38). The site of photoincorporation of a third photoaffinity analogue of Taxol, [(3)H]7-(benzoyldihydrocinnamoyl) Taxol, has been determined. This analogue stabilizes microtubules polymerized in the presence of GTP, but in contrast to Taxol, does not by itself enhance the polymerization of tubulin to its polymer form. CNBr digestion of [(3)H]7-(benzoyldihydrocinnamoyl)Taxol-labeled tubulin, with further arginine-specific cleavage by clostripain resulted in the isolation of a peptide containing amino acid residues <em>2</em>77-<em>2</em>93. Amino acid sequence analysis indicated that the photoaffinity analogue cross-links to Arg(<em>2</em>8<em>2</em>) in beta-tubulin. Advances made by electron crystallography in understanding the structure of the tubulin <em>dimer</em> have allowed us to visualize the three sites of photoincorporation by molecular modeling. There is good agreement between the binding site of Taxol in beta-tubulin as determined by photoaffinity labeling and electron crystallography.

BACKGROUND

Fatigue is commonly reported in patients receiving radiotherapy for breast conservation, but the underlying mechanisms remain unclear.

METHODS

Patients with early breast cancer participating in a prospective study of the impact of inflammatory processes on early and delayed breast morbidity were assessed for fatigue levels using the Functional Assessment of Cancer Therapy (FACT) fatigue subscale before and at intervals during and after radiotherapy. Blood for analysis of a variety of circulating cytokines, coagulation factors, peripheral blood indices and biochemical factors was collected at the same time points.

RESULTS

Fifty-two eligible patients were assessed. Twenty-one patients (43%) developed significant fatigue during radiotherapy, whereas <em>2</em>8 (54%) developed minimal or no fatigue. Fatigue appeared to plateau between week 4 of treatment and <em>2</em> weeks after treatment. The fatigue was beginning to settle by 6 weeks after treatment. Significant fatigue was predicted by a higher baseline fatigue score, red cell count, neutrophil count, and <em>D</em>-<em>dimer</em> level. Baseline fatigue correlated with higher body mass index, C-reactive protein, soluble thrombomodulin, tissue plasminogen activator, von Willebrand factor antigen, interleukin-6, ICAM-1, hemoglobin and red cell, monocyte, and neutrophil counts. There were also significant correlations between body mass index and tissue plasminogen activator, C-reactive protein, interleukin-6, ICAM-1, and red cell count. After these factors were controlled for, baseline fatigue was seen to be associated with higher body mass index, soluble thrombomodulin, tissue plasminogen activator, von Willebrand factor antigen, monocyte count, and neutrophil count. Multiple logistic regression procedures indicated that the most predictive factors for fatigue during radiotherapy were higher baseline fatigue level and higher baseline neutrophil and red cell counts.

CONCLUSIONS

This study has shown that significant fatigue is common in patients receiving breast irradiation and is precipitated during radiotherapy in some patients but not others. The factors shown to be associated with fatigue in this study will be helpful in shaping future studies.

Acanthamoeba capping protein increased the rate of actin polymerization from monomers with and without calcium. In the absence of calcium, capping protein also increased the critical concentration for polymerization. Various models were evaluated for their ability to predict the effect of capping protein on kinetic curves for actin polymerization under conditions where the critical concentration was not changed. Several models, which might explain the increased rate of polymerization from monomers, were tested. Two models which predicted the experimental data poorly were (1) capping protein was similar to an actin filament, bypassing nucleation, and (<em>2</em>) capping protein fragmented filaments. Three models in which capping protein accelerated, but did not bypass, nucleation predicted the data well. In the best one, capping protein resembled a nondissociable actin <em>dimer</em>. Several lines of evidence have supported the idea that capping protein blocks the barbed end of actin filaments, preventing the addition and loss of monomers [Cooper, J. A., Blum, J. <em>D</em>., & Pollard, T. <em>D</em>. (1984) J. Cell Biol. 99, <em>2</em>17-<em>2</em><em>2</em>5; Isenberg, G. A., Aebi, U., & Pollard, T. <em>D</em>. (1980) Nature (London) <em>2</em>88, 455-459]. This mechanism was also supported here by the effect of capping protein on the kinetics of actin polymerization which was nucleated by preformed actin filaments. Low capping protein concentrations slowed nucleated polymerization, presumably because capping protein blocked elongation at barbed ends of filaments. High capping protein concentrations accelerated nucleated polymerization because of capping protein's ability to interact with monomers and accelerate nucleation.

Thrombohemorrhagic complications are a major cause of morbidity and mortality in patients with essential thrombocythemia (ET) and polycythemia vera (PV). The pathogenesis of these complications is not completely clarified. Several studies have described abnormalities of red blood cells and platelets in these patients. However, no studies are available on changes in the polymorphonuclear leukocytes (PMNs), which can play an important role in the activation of the hemostatic system. In patients with ET (n = 37) and PV (n = 34), a series of PMN activation parameters (PMN membrane C<em>D</em>11b and leukocyte alkaline phosphatase [LAP] antigen expression, cellular elastase content, plasma elastase, and myeloperoxidase levels) was evaluated simultaneously with the levels of plasma markers of endothelial damage (thrombomodulin and von Willebrand factor antigen) and hypercoagulation (thrombin-antithrombin complex, prothrombin fragment 1 + <em>2</em>, and <em>D</em>-<em>dimer</em>). The results show the occurrence of PMN activation in both groups of patients compared with a control group of healthy subjects. An increase in C<em>D</em>11b and LAP expression by PMN membrane was observed, together with a significant increase in cellular elastase content, plasma elastase, and myeloperoxidase levels. In addition, patients had high plasma levels of endothelial and hypercoagulation markers compared with controls. For the first time, these data show that in ET and PV, <em>2</em> hematologic conditions that place patients at increased risk for thrombosis, an in vivo leukocyte activation occurs and is associated with laboratory signs of endothelium and coagulation system activation. (Blood. <em>2</em>000;96:4<em>2</em>61-4<em>2</em>66)

OBJECTIVE

Resistin induces insulin resistance in mice. In humans, recent data suggest that resistin functions as a proinflammatory cytokine. Here, we studied resistin up to <em>2</em> wks after admission in patients with septic shock and/or severe sepsis.

METHODS

Two prospective studies of patients with sepsis and in vitro studies of resistin interaction with monocytes.

METHODS

Intensive care unit at Karolinska University Hospital and Center for Infectious Medicine, Karolinska Institute, Huddinge, Sweden.

METHODS

Twenty-nine patients with severe sepsis and 66 with septic shock.

METHODS

None.

RESULTS

Ninety-five patients were studied, <em>2</em>5 of whom died within <em>2</em>8 days. Resistin and cytokine levels and routine biochemistry were measured at three to six defined time points during the first <em>2</em> wks after admission and were correlated to other cytokines, glucose levels, body mass index, Acute Physiology and Chronic Health Evaluation II, and Sepsis-related Organ Failure Assessment scores. Serum resistin was significantly elevated compared with healthy controls (p < .000001) and correlated with severity of disease as measured by Acute Physiology and Chronic Health Evaluation II and Sepsis-related Organ Failure Assessment scores, with an increasingly strong degree of correlation over time. Median levels were four- to eight-fold higher than controls and remained high up to <em>2</em> wks after admission to the intensive care unit. Levels correlated with interleukin-6, interleukin-8, interleukin-10, tumor necrosis factor-alpha, creatinine, D-dimer, and lactate, but not with p-glucose or body mass index. In vitro, resistin was released from monocytes after stimulation with either lipopolysaccharide or high mobility group box 1 protein. Recombinant resistin itself up-regulated intercellular adhesion molecule-1 on monocytes.

CONCLUSIONS

This is the first study assessing systemic levels of resistin in patients with septic shock/severe sepsis. We show that resistin is a marker of severity of disease and possibly a mediator of the prolonged inflammatory state seen in infected critically ill patients. Further exploration of resistin as a therapeutic target and marker of disease is merited.

Protein kinase A (PKA), a central locus for cAMP signaling in the cell, is composed of regulatory (R) and catalytic (C) subunits. The C-subunits are maintained in an inactive state by binding to the R-subunit <em>dimer</em> in a tetrameric holoenzyme complex (R(<em>2</em>)C(<em>2</em>)). PKA is activated by cAMP binding to the R-subunits which induces a conformational change leading to release of the active C-subunit. Enzymatic activity of the C-subunit is thus regulated by cAMP via the R-subunit, which toggles between cAMP and C-subunit bound states. The R-subunit is composed of a <em>dimer</em>ization/docking (<em>D</em>/<em>D</em>) domain connected to two cAMP-binding domains (cAMP:A and cAMP:B). While crystal structures of the free C-subunit and cAMP-bound states of a deletion mutant of the R-subunit are known, there is no structure of the holoenzyme complex or of the cAMP-free state of the R-subunit. An important step in understanding the cAMP-dependent activation of PKA is to map the R-C interface and characterize the mutually exclusive interactions of the R-subunit with cAMP and C-subunit. Amide hydrogen/deuterium exchange mass spectrometry is a suitable method that has provided insights into the different states of the R-subunit in solution, thereby allowing mapping of the effects of cAMP and C-subunit on different regions of the R-subunit. Our study has localized interactions with the C-subunit to a small contiguous surface on the cAMP:A domain and the linker region. In addition, C-subunit binding causes increased amide hydrogen exchange within both cAMP-domains, suggesting that these regions become more flexible in the holoenzyme and are primed to bind cAMP. Furthermore, the difference in the protection patterns between RIalpha and the previously studied RIIbeta upon cAMP-binding suggests isoform-specific differences in cAMP-dependent regulation of PKA activity.

Isopentenyl pyrophosphate (IPP) is a common precursor for the synthesis of all isoprenoids, which have important functions in living organisms. IPP is produced by the mevalonate pathway in archaea, fungi, and animals. In contrast, IPP is synthesized by a mevalonate-independent pathway in most bacteria, algae, and plant plastids. 1-<em>D</em>eoxy-<em>D</em>-xylulose 5-phosphate synthase (<em>D</em>XS) catalyzes the first and the rate-limiting step of the mevalonate-independent pathway and is an attractive target for the development of novel antibiotics, antimalarials, and herbicides. We report here the first structural information on <em>D</em>XS, from Escherichia coli and <em>D</em>einococcus radiodurans, in complex with the coenzyme thiamine pyrophosphate (TPP). The structure contains three domains (I, II, and III), each of which bears homology to the equivalent domains in transketolase and the E1 subunit of pyruvate dehydrogenase. However, <em>D</em>XS has a novel arrangement of these domains as compared with the other enzymes, such that the active site of <em>D</em>XS is located at the interface of domains I and II in the same monomer, whereas that of transketolase is located at the interface of the <em>dimer</em>. The coenzyme TPP is mostly buried in the complex, but the C-<em>2</em> atom of its thiazolium ring is exposed to a pocket that is the substrate-binding site. The structures identify residues that may have important roles in catalysis, which have been confirmed by our mutagenesis studies.

OBJECTIVE

To quantify changes in variables of inflammation, coagulation, and fibrinolysis in blunt trauma patients with lower extremity fractures who underwent different types of surgical procedures.

METHODS

Prospective, cohort study.

METHODS

Level I university trauma center.

METHODS

We allocated 83 blunt trauma patients in stable condition and <em>2</em><em>2</em> patients eligible for elective hip replacement to four treatment groups.

METHODS

In 34 multiply traumatized patients with femoral fracture (group PTFF) and in <em>2</em>8 patients with an isolated femoral fracture (group IFF), primary unreamed intramedullary nailing for stabilization of the femoral shaft fracture was performed. In <em>2</em><em>2</em> patients, an elective uncemented total hip arthroplasty (group THA) was inserted for osteoarthritis, and in <em>2</em>1 control patients, an isolated ankle fracture (group AF) was acutely stabilized.

RESULTS

From serially sampled central venous blood, the perioperative concentrations of interleukin (IL)-6, of tumor necrosis factor-alpha, of prothrombin fragments 1 + <em>2</em>, and of <em>D</em>-<em>dimer</em> cross-linked fibrin degradation products were evaluated. Intramedullary instrumentation for an isolated femur fracture caused a significant perioperative increase in the concentrations of IL-6 (preoperative IL-6, 5<em>2</em> +/- 1<em>2</em> pg/mL; IL-6 30 mins postinsertion, 78 +/- 14 pg/mL; p = .0<em>2</em>). This increase was comparable with group THA (preoperative IL-6, 46 +/- 16 pg/mL; IL-6 30 mins postinsertion, 67 +/- 11 pg/mL; p = .03). A positive correlation occurred between both groups (r = .83, p < .0004). Multiple trauma patients demonstrated significantly (p = .000<em>2</em>) higher IL-6 concentrations than all other groups throughout the study period and showed a significant increase after femoral nailing (preoperative IL-6, 570 +/- <em>2</em>1 pg/mL; IL-6 30 mins postinsertion, 690 +/- <em>2</em>4 pg/mL; p = .003), whereas no perioperative change was seen in group AF. The highest IL-6 increases were associated with a longer ventilation time (group PTFF) and a longer period of positive fluid balances (groups PTFF, IFF, THA). The coagulatory variables demonstrated similar perioperative increases in groups IFF and THA, but not in groups PTFF and AF. The IL-6 concentrations and the prothrombin fragments 1 + <em>2</em> concentrations correlated between groups THA and IFF at 30 mins and at 1 hr after surgery (r<em>2</em> = .64, p < .0<em>2</em>). In all patients the clinical variables were stable perioperatively.

CONCLUSIONS

Major surgery of the lower extremity causes changes to the inflammatory, fibrinolytic, and coagulatory cascades in patients with stable cardiopulmonary function. The inflammatory response induced by femoral nailing is biochemically comparable to that induced by uncemented total hip arthroplasty. In multiple trauma patients, increases, which occurred in addition to those induced by the initial trauma, were measured. Definitive primary femoral stabilization by intramedullary nailing imposes an additional burden to the patient with blunt trauma. A careful preoperative investigation is required to evaluate whether primary definitive stabilization can be performed safely.

Two polypepti<em>d</em>es of Mr approximately <em>2</em>9,000 an<em>d</em> <em>2</em>7,000 have been i<em>d</em>entifie<em>d</em> in human erythrocyte membranes that cross-react specifically with affinity purifie<em>d</em> antibo<em>d</em>ies to chicken gizzar<em>d</em> tropomyosin. The cross-reacting polypepti<em>d</em>es are quantitatively retaine<em>d</em> on the membrane after cell lysis if millimolar concentrations of magnesium are inclu<em>d</em>e<em>d</em> in the lysis an<em>d</em> wash buffers, in<em>d</em>icating that they are membrane-boun<em>d</em> proteins un<em>d</em>er physiological con<em>d</em>itions. Milligram quantities of these immunoreactive polypepti<em>d</em>es have been purifie<em>d</em> to greater than 95% purity from a low salt extract of membranes by DEAE-chromatography, precipitation at pH 4.4, an<em>d</em> heating to 85 <em>d</em>egrees C to <em>d</em>enature contaminants. Physical similarities of the erythrocyte protein to other tropomyosins inclu<em>d</em>e (a) amino aci<em>d</em> composition (b) anomalous migration of the Mr approximately <em>2</em>9,000 an<em>d</em> <em>2</em>7,000 polypepti<em>d</em>es on so<em>d</em>ium <em>d</em>o<em>d</em>ecyl sulfate-gels in the presence of 6 M urea to apparent Mr approximately 43,000 an<em>d</em> 38,000, respectively (c) arrangement of chains as <em>dimers</em> of Mr approximately 60,000 base<em>d</em> on cross-linking stu<em>d</em>ies an<em>d</em> calculation of molecular weight from hy<em>d</em>ro<em>d</em>ynamic values (Rs = 5.9 nm, se<em>d</em>imentation coefficient = <em>2</em>.5 S; partial specific volume = 0.7<em>2</em> cm3/g) an<em>d</em> (<em>d</em>) highly asymmetric shape, base<em>d</em> on a frictional ratio of <em>2</em>.07. Bin<em>d</em>ing of erythrocyte tropomyosin to muscle F-actin saturates at one tropomyosin molecule (Mr approximately 60,000) to 6-7 actin monomers an<em>d</em> is highly cooperative with a Hill coefficient of about <em>2</em>.8, similar to muscle tropomyosins. Bin<em>d</em>ing also exhibits a high <em>d</em>egree of cooperativity as a function of the magnesium concentration with a transition between no bin<em>d</em>ing an<em>d</em> complete bin<em>d</em>ing between 1 an<em>d</em> <em>2</em> mM MgCl<em>2</em>. Increasing the magnesium concentration from <em>2</em> to 10 mM increases the apparent affinity of tropomyosin for actin from approximately <em>2</em>.6 X 10(6) M-1 to approximately <em>2</em>.7 X 10(7) M-1 without effect on the Hill coefficient. The tropomyosin polypepti<em>d</em>es comprise about 1% of the erythrocyte membrane protein an<em>d</em> are present in a ratio of one Mr approximately 60,000 tropomyosin molecule to 7-8 actin monomers, an amount almost sufficient to coat all of the F-actin on the membrane. These <em>d</em>ata are consistent with the association of two tropomyosin molecules with each of the short actin filaments (1<em>2</em>-17 monomers long) thought to exist in the erythrocyte membrane cytoskeleton. The erythrocyte tropomyosin coul<em>d</em> function to mechanically stabilize these actin filaments as well as play a role in regulating the interaction of spectrin with actin.(ABSTRACT TRUNCATED AT 400 WORDS)

The structural protein Gag is the only viral product required for retrovirus assembly. Purified Gag proteins or fragments of Gag are able in vitro to spontaneously form particles resembling immature virions, but this process requires nucleic acid, as well as the nucleocapsid domain of Gag. To examine the role of nucleic acid in the assembly in vitro, we used a purified, slightly truncated version of the Rous sarcoma virus Gag protein, <em>Delta</em> MBD <em>Delta</em> PR, and DNA oligonucleotides composed of the simple repeating sequence GT. Apparent binding constants were determined for oligonucleotides of different lengths, and from these values the binding site size of the protein on the DNA was calculated. The ability of the oligonucleotides to promote assembly in vitro was assessed with a quantitative assay based on electron microscopy. We found that excess zinc or magnesium ion inhibited the formation of virus-like particles without interfering with protein-DNA binding, implying that interaction with nucleic acid is necessary but not sufficient for assembly in vitro. The binding site size of the <em>Delta</em> MBD <em>Delta</em> PR protein, purified in the presence of EDTA to remove zinc ions at the two cysteine-histidine motifs, was estimated to be 11 nucleotides (nt). This value decreased to 8 nt when the protein was purified in the presence of low concentrations of zinc ions. The minimum length of DNA oligonucleotide that promoted efficient assembly in vitro was <em>2</em><em>2</em> nt for the zinc-free form of the protein and 16 nt for the zinc-bound form. To account for this striking 1:<em>2</em> ratio between binding site size and oligonucleotide length requirement, we propose a model in which the role of nucleic acid in assembly is to promote formation of a species of Gag <em>dimer</em>, which itself is a critical intermediate in the polymerizaton of Gag to form the protein shell of the immature virion.

The Escherichia coli gamma complex serves as a clamp loader, catalyzing ATP-dependent assembly of beta protein clamps onto primed DNA templates during DNA replication. These ring-shaped clamps tether DNA polymerase III holoenzyme to the template, facilitating rapid and processive DNA synthesis. This report focuses on the role of ATP binding and hydrolysis catalyzed by the gamma complex during clamp loading. We show that the energy from ATP binding to gamma complex powers several initial events in the clamp loading pathway. The gamma complex (gamma<em>2</em> <em>delta</em> <em>delta</em>'chi psi) binds two ATP molecules (one per gamma subunit in the complex) with high affinity (Kd = 1-<em>2</em>. 5 x 10(-6) M) or two adenosine 5'-O-(3-thiotriphosphate)(ATPgammaS) molecules with slightly lower affinity (Kd = 5-6.5 x 10(-6) M). Experiments performed prior to the first ATP turnover (kcat = 4 x 10(-3) s-1 at 4 degreesC), or in the presence of ATPgammaS (kcat = 1 x 10(-4) s-1 at 37 degreesC), demonstrate that upon interaction with ATP the gamma complex undergoes a change in conformation. This ATP-bound gamma complex binds beta and opens the ring at the <em>dimer</em> interface. Still prior to ATP hydrolysis, the composite of gamma complex and the open beta ring binds with high affinity to primer-template DNA. Thus ATP binding powers all the steps in the clamp loading pathway leading up to the assembly of a gamma complex. open beta ring.DNA intermediate, setting the stage for ring closing and turnover of the clamp loader, steps that may be linked to subsequent hydrolysis of ATP.

Cancer patients are at increase<em>d</em> risk of <em>d</em>eep vein thrombosis an<em>d</em> pulmonary embolism. The inci<em>d</em>ence among <em>d</em>ifferent groups of cancer patients varies consi<em>d</em>erably <em>d</em>epen<em>d</em>ing on clinical factors, the most important being tumor entity an<em>d</em> stage. Biomarkers have been specifically investigate<em>d</em> for their capacity of pre<em>d</em>icting venous thromboembolism (VTE) <em>d</em>uring the course of <em>d</em>isease. Parameters of bloo<em>d</em> count analysis (elevate<em>d</em> leukocyte an<em>d</em> platelet count an<em>d</em> <em>d</em>ecrease<em>d</em> hemoglobin) have turne<em>d</em> out to be useful in risk pre<em>d</em>iction. Associations between elevate<em>d</em> levels an<em>d</em> future VTE have been foun<em>d</em> for <em>d</em>-<em>dimer</em>, prothrombin fragment 1+<em>2</em>, an<em>d</em> soluble P-selectin an<em>d</em> also for clotting factor VIII an<em>d</em> the thrombin generation potential. The results for tissue factor-bearing microparticles are heterogeneous: an association with occurrence of VTE in pancreatic cancer might be present, whereas in other cancer entities, such as glioblastoma, colorectal, or gastric carcinoma, this coul<em>d</em> not be confirme<em>d</em>. Risk assessment mo<em>d</em>els were <em>d</em>evelope<em>d</em> that inclu<em>d</em>e clinical an<em>d</em> laboratory markers. In the high-risk categories, patient groups with up to a>><em>2</em>0% VTE rate within 6 months can be i<em>d</em>entifie<em>d</em>. A further improvement in risk stratification woul<em>d</em> allow better i<em>d</em>entification of patients for primary VTE prevention using in<em>d</em>irect or novel <em>d</em>irect anticoagulants.

OBJECTIVE

To determine whether chronic atrial fibrillation is associated with abnormalities in plasma fibrinogen, von Willebrand factor (vWF) (a marker of endothelial disturbance), or fibrin D- dimer (a measure of fibrin turnover); and if so, whether such levels are related to haemodynamic disturbance (enlarged left atrium, poor left ventricular function) or existing treatment with warfarin or aspirin. To investigate the effects of introducing warfarin in patients with atrial fibrillation on fibrinogen and D- dimer levels.

METHODS

Cross sectional population sample controlled study and longitudinal study of patients undergoing anticoagulation.

METHODS

District general hospital.

METHODS

87 patients (44 men and 43 women of mean (SEM) age 63.0 (1.0)) with chronic atrial fibrillation. At the time of the study, 37 were taking no antithrombotic medication (group 1), 31 were taking warfarin (including two on warfarin and aspirin) (group 2) and 19 were taking aspirin alone (group 3). They were compared with 158 population controls from a random population sample (the second Glasgow monitoring trends and determinants in cardiovascular disease study). As part of clinical treatment warfarin was introduced in 20 patients with chronic atrial fibrillation (14 men and six women of mean (SEM) (range) age 63.9 (2.35 (32-74) years).

RESULTS

Plasma fibrinogen remained significantly increased in patients of group 1 (no antithrombotic medication) compared with that of the population controls (median difference 1.23 g/l; 95% confidence interval (CI) 0.88 to 1.62, P < 0.0001). There was also a significant increase in plasma D-dimer levels (median difference 77 ng/ml; 95% CI 38 to 122, P < 0.01) and vWF (median difference 63 IU/dl; 95% CI 38 to 89, P < 0.0001). There was no significant difference in plasma fibrinogen (median difference 0.14 g/l; 95% CI -0.44 to 0.77, P = 0.65) or vWF (median difference 3.5 IU/dl; 95% CI - 41 to 41, P = not significant in patients of group 2 (warfarin treatment) compared with that of patients in group 1. Levels of D-dimer were significantly lower in group 2 (median difference 90 ng/ml, 95% CI 39 to 150, P < 0.0001) than in group 1. There were no significant differences in plasma fibrinogen (median difference 0.08 g/l; 95% CI - 0.52 to 0.77, P = 0.73), D-dimer (median difference - 34 ng/ml; 95% CI - 114 to 21.0, P = 0.25), or vWF (median difference 2%; 95% CI - 35 to 41, P = not significant) levels between patients of groups 1 and 3. There were no significant correlations between the coagulation indices and left atrial volume or ventricular function. There was a significant positive correlation between plasma fibrin D-dimer and vWF levels in patients of groups 1 and 3 (r = 0.52, P < 0.001). There was a significant reduction in median plasma fibrin D-dimer levels at 2 months after the introduction of warfarin (181 ng/ml v 80 ng/ml, P < 0.001), but no effect on plasma fibrinogen.

CONCLUSIONS

Increased median plasma fibrinogen and vWF levels were found in patients with chronic atrial fibrillation. Plasma D-dimer levels were also increased in patients with chronic atrial fibrillation not receiving warfarin, suggesting increased intravascular thrombogenesis in such patients. Introduction of warfarin normalised circulating fibrin D- dimer levels, suggesting that warfarin treatment was effective in preventing excessive fibrin turnover, consistent with the antithrombotic effects of warfarin. These results suggest three possible thrombotic markers to assess patients with atrial fibrillation who are at high risk of thrombogenesis; D-dimer also merits assessment as a measure of reduction in thrombotic risk in patients receiving warfarin.

The products of recombination-activating genes RAG1 and RAG<em>2</em> mediate the assembly of antigen receptor genes during lymphocyte development in a process known as V(<em>D</em>)J recombination. Lack of structural information for the RAG proteins has hindered mechanistic studies of this reaction. We report here the crystal structure of an essential <em>D</em>NA binding domain of the RAG1 catalytic core bound to its nonamer <em>D</em>NA recognition motif. The RAG1 nonamer binding domain (NB<em>D</em>) forms a tightly interwoven <em>dimer</em> that binds and synapses two nonamer elements, with each NB<em>D</em> making contact with both <em>D</em>NA molecules. Biochemical and biophysical experiments confirm that the two nonamers are in close proximity in the RAG1/<em>2</em>-<em>D</em>NA synaptic complex and demonstrate the functional importance of the protein-<em>D</em>NA contacts revealed in the structure. These findings reveal a previously unsuspected function for the NB<em>D</em> in <em>D</em>NA synapsis and have implications for the regulation of <em>D</em>NA binding and cleavage by RAG1 and RAG<em>2</em>.

BACKGROUND

Activation of coagulation and fibrinolysis play a role in the pathophysiology of experimental arthritis.

OBJECTIVE

To determine the extent of activation of the coagulation and fibrinolytic pathways in different joint diseases in humans and to ascertain the factors that may influence fibrin deposition within the joint.

METHODS

Plasma from normal subjects (controls, n= <em>2</em>1) an<em>d</em> plasma an<em>d</em> synovial flui<em>d</em> samples from patients with rheumatoi<em>d</em> arthritis (RA; n = 64), osteoarthritis (OA; n = <em>2</em>9), spon<em>d</em>yloarthropathy (SpA; n = <em>2</em><em>2</em>) an<em>d</em> crystal arthritis (CA; n = <em>2</em>5) were analyze<em>d</em> for the levels of TF (tissue factor) an<em>d</em> tissue factor pathway inhibitor (TFPI) activities, thrombin-antithrombin III (TAT) complexes, an<em>d</em> F1 + <em>2</em> (thrombin fragment), fibrin <em>d</em>-<em>dimer</em> an<em>d</em> thrombin-activate<em>d</em> fibrinolysis inhibitor (TAFI) antigenic levels. The measurements were analyze<em>d</em> by pairwise correlation with each other as well as with stan<em>d</em>ar<em>d</em> parameters of inflammation [C-reactive protein (CRP), joint leukocyte count]. Inter-group comparisons were performe<em>d</em> to look for <em>d</em>isease-specific <em>d</em>ifferences.

RESULTS

Compare<em>d</em> with healthy controls, patients with joint <em>d</em>iseases ha<em>d</em> higher levels of TAT, F1 + <em>2</em> an<em>d</em> <em>d</em>-<em>dimer</em>s in their plasma. In the synovial flui<em>d</em>, TF activity, TAT, <em>d</em>-<em>dimer</em>s, an<em>d</em> TAFI were significantly higher in inflammatory arthriti<em>d</em>es than in OA. The levels were highest in RA patients. In the plasma, TF activity was correlate<em>d</em> with TAT an<em>d</em> <em>d</em>-<em>dimer</em> levels with CRP, TFPI, an<em>d</em> TAT. In the synovial flui<em>d</em>, TF activity correlate<em>d</em> with plasma CRP levels, synovial flui<em>d</em> leukocyte count, an<em>d</em> synovial TAT an<em>d</em> TAFI levels. In a<em>d</em><em>d</em>ition, synovial <em>d</em>-<em>dimer</em>s correlate<em>d</em> with CRP, an<em>d</em> synovial TAFI levels were correlate<em>d</em> with synovial F1 + <em>2</em> an<em>d</em> TAT.

CONCLUSIONS

Activation of the coagulation and fibrinolytic cascades in the joint and in the circulation is evident in both inflammatory and degenerative joint diseases. Within the joint, inflammatory mechanisms leading to TF-mediated activation of the coagulation pathway and subsequent fibrin deposition is the most likely explanation for the observed findings. In the plasma, the link between inflammation (CRP increase) and TF activation is weak, and a non-TF-mediated mechanism of coagulation activation could explain these findings. RA is characterized by significantly higher levels of TAT in the synovial fluid and plasma than other arthritides. Although fibrinolytic activity is linked to inflammation, the increased amounts of TAFI in the joint, particularly in RA, may explain why fibrin formation is so prominent in this condition compared with other joint diseases.

The ability to excise (repair) UV-induced pyrimidine <em>dimers</em> in Escherichia coli is not related to its ability to remove N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced O6-methylguanine (O6-MeG) from <em>D</em>NA. It was therefore surprising that certain xeroderma pigmentosum cell lines, deficient in <em>dimer</em> excision, were also unable to remove O6-MeG. We find that removal of O6-MeG occurs rapidly with a half life of less than 1 h. Two cell types can be distinguished: mex+, which remove O6-MeG residues produced by incubation with 0.5 microgram ml-1 MNNG, and mex- cells, which are unable to remove the adduct. Xeroderma pigmentosum-derived lymphoblastoid lines of complementation groups A, C or <em>D</em> may be either mex+ or mex-. The biochemical mechanism for the removal of O6-MeG in human cells is distinct from the excision of adducts produced by compounds such as N-acetoxy-N-<em>2</em>-acetylaminofluorene (AAAF) or by UV irradiation but it is not clear whether the distinction between mex+ and mex- lines is genetic or epigenetic.