The nuclear factor kappaB (NF-kappaB) is involved in T cell activation and enhances HIV-1 gene expression. It is activated in response to numerous stimuli, including oxidative stress. Oxidative stress damages membrane lipids, proteins and nucleic acids. We have shown previously that oxidative <em>D</em>NA damage generated by photosensitization could trigger activation of NF-kappaB. We now show that a series of topoisomerase poisons (actinomycin <em>D</em>, camptothecin, daunomycin and etoposide) also activate NF-kappaB (NFKB1/RelA <em>dimer</em>) in ACH-<em>2</em> and CEM cells. This activation is inhibited by pyrrolidine dithiocarbamate. In ACH-<em>2</em> cells latently infected by HIV-1, camptothecin, daunomycin and etoposide are able to enhance virus production. Since topoisomerase poisons cause the formation of single- and double-strand breaks in <em>D</em>NA, these lesions might be capable of triggering NF-kappaB activation. Indeed, <em>D</em>NA damaging agents generating adducts (trans-platin and 4-nitroquinoline 1-oxide) and/or crosslinks in <em>D</em>NA (cisplatin and mitomycin C) do not or only weakly activate NF-kappaB in T cell lines.

OBJECTIVE

This study investigated the hypothesis that modern computed tomographic (CT) imaging is sufficient to exclude subarachnoid hemorrhage (SAH) in patients with severe headache.

METHODS

All 38,730 adult patients who presented to Hermann Hospital in Houston, Texas, during a 16-month period were prospectively screened to detect those with "the worst headache of my life." Two neuroradiologists blinded to the study hypothesis interpreted the CT scans. Patients with negative scans underwent comprehensive cerebrospinal fluid (CSF) analysis including cell count in first and last tubes, visual and spectrophotometric detection of xanthochromia, and CSF D-dimer assay.

RESULTS

A chief complaint of headache was elicited in 455 patients, and 107 of these had "worst headache" and were enrolled in the study. CT-confirmed SAH was found in 18 of the 107 (17%). Only 2 patients (2.5%, 95% confidence interval, .3% to 8.8%) had SAH detected by CSF analysis among those with negative CT imaging result. CSF spectrophotometric detection was the most sensitive test for blood. Three patients with less than 6 red blood cells in tube 1 had positive spectrophotometric results, but in all 3, tube 4 was negative on spectrophotometric analysis, suggesting a high false-positive rate.

CONCLUSIONS

Modern CT imaging is sufficient to exclude 97.5% of SAH in patients presenting to the ED with "worst headache" symptoms.

Nuclease A (NucA) is a nonspecific endonuclease from Anabaena sp. capable of degrading single- and double-stranded DNA and RNA in the presence of divalent metal ions. We have determined the structure of the <em>delta</em>(<em>2</em>-<em>2</em>4),D1<em>2</em>1A mutant of NucA in the presence of Zn<em>2</em>+ and Mn<em>2</em>+ (PDB code 1ZM8). The mutations were introduced to remove the N-terminal signal peptide and to reduce the activity of the nonspecific nuclease, thereby reducing its toxicity to the Escherichia coli expression system. NucA contains a betabeta alpha metal finger motif and a hydrated Mn<em>2</em>+ ion at the active site. Unexpectedly, NucA was found to contain additional metal binding sites approximately <em>2</em>6 A apart from the catalytic metal binding site. A structural comparison between NucA and the closest analog for which structural data exist, the Serratia nuclease, indicates several interesting differences. First, NucA is a monomer rather than a <em>dimer</em>. Second, there is an unexpected structural homology between the N-terminal segments despite a poorly conserved sequence, which in Serratia includes a cysteine bridge thought to play a regulatory role. In addition, although a sequence alignment had suggested that NucA lacks a proposed catalytic residue corresponding to Arg57 in Serratia, the structure determined here indicates that Arg93 in NucA is positioned to fulfill this role. Based on comparison with DNA-bound nuclease structures of the betabeta alpha metal finger nuclease family and available mutational data on NucA, we propose that His1<em>2</em>4 acts as a catalytic base, and Arg93 participates in the catalysis possibly through stabilization of the transition state.

Recently a dapF mutant of Escherichia coli lacking the diaminopimelate epimerase was found to have an unusual large LL-diaminopimelic acid (LL-<em>D</em>AP) pool as compared with that of meso-<em>D</em>AP (C. Richaud, W. Higgins, <em>D</em>. Mengin-Lecreulx, and P. Stragier, J. Bacteriol. 169:1454-1459, 1987). In this report, the consequences of high cellular LL-<em>D</em>AP/meso-<em>D</em>AP ratios on the structure and metabolism of peptidoglycan were investigated. For this purpose new efficient high-pressure liquid chromatography techniques for the separation of the <em>D</em>AP isomers were developed. Sacculi from dapF mutants contained a high proportion of LL-<em>D</em>AP that varied greatly with growth conditions. The same was observed with the two <em>D</em>AP-containing precursors, U<em>D</em>P-N-acetylmuramyl-tripeptide and U<em>D</em>P-N-acetylmuramyl-pentapeptide. The limiting steps for the incorporation of LL-<em>D</em>AP into peptidoglycan were found to be its addition to U<em>D</em>P-N-acetylmuramyl-L-alanyl-<em>D</em>-glutamate and the formation of the <em>D</em>-alanyl-<em>D</em>AP cross-bridges. The Km value of the <em>D</em>AP-adding enzyme for LL-<em>D</em>AP was 3.6 x 10(-<em>2</em>) M as compared with 1.1 x 10(-5) M for meso-<em>D</em>AP. When isolated sacculi were treated with Chalaropsis N-acetylmuramidase and the resulting soluble products were analyzed by high-pressure liquid chromatography, the proportion of the main peptidoglycan <em>dimer</em> was lower in the dapF mutant than in the parental strain. Moreover, the proportion of LL-<em>D</em>AP was higher in the main monomer than in the main <em>dimer</em>, where it was almost exclusively located in the donor unit. There are thus very few <em>D</em>-alanyl-LL-<em>D</em>AP cross-bridges, if any. We also observed that large amounts of LL-<em>D</em>AP and N-succinyl-LL-<em>D</em>AP were excreted in the growth medium by the dapF mutant.

Thrombosis and disseminated intravascular coagulation are common complications of cancer. Specific conditions associated with cancer such as stasis due to immobilization or blood flow obstruction, surgery, infections, endothelium damage due to chemotherapeutic agents and abnormalities of blood coagulation contribute to the hypercoagulable and thrombophilic state of cancer patients. This procoagulant state in cancer arises mostly from the capacity of tumor cells to express and release procoagulant activities (cancer procoagulant and tissue factor). <em>D</em>ecreased levels of inhibitors of coagulation, impaired fibrinolysis, the presence of antiphospholipid antibodies and an acquired activated protein C resistance contribute to the hypercoagulable state. The activation of coagulation is also implicated in tumor proliferation through interactions of coagulation with inflammation and increased tissue factor pathway inhibitor. Laboratory diagnosis of the thrombophilic state include (1) elevation of clotting factors, fibrinogen/fibrin degradation products, hyperfibrinogenemia and thrombocytosis and (<em>2</em>) elevation of specific markers of activation of coagulation: fibrinopeptide A, fragment 1 + <em>2</em>, thrombin-antithrombin complexes and <em>D</em>-<em>dimers</em>. However, none of the tests has any predictive value for the occurrence of thrombotic events in one individual patient. In patients with venous thromboembolism a noninvasive screening for occult cancer is able to detect a relatively high incidence of hidden cancer and the search for thrombophilia seems important in patients without known cancer.

The cytoplasmic domain of band 3 was released from spectrin-depleted, acetic acid-stripped erythrocyte membrane vesicles by mild chymotryptic digestion. After purification by ion exchange and gel filtration chromatography, the fragment preparation was found to be greater than 90% pure on polyacrylamide disc gels in the presence of 0.<em>2</em>% sodium dodecyl sulfate. The subunit Mr from the electrophoretic procedure was estimated at approximately 40,000. The isolated cytoplasmic fragment ws judged to be a <em>dimer</em>, since (i) the unmodified fragment and its disulfide-cross-linked (<em>dimer</em>ic) counterpart eluted in the same peak fraction from a Sephacryl S-<em>2</em>00 gel filtration column, and (ii) the sedimentation velocity molecular weight of the native fragment was calculated to be approximately 95,000. No evidence of either larger or smaller aggregates was obtained. The frictional ratio of the fragment was measured at 1.6, suggesting a highly elongated morphology. The circular dichroism spectrum of the fragment corresponded to approximately 37% alpha helix. Titration of the cytoplasmic fragment over the physiological pH range gave rise to a reversible <em>2</em>-fold increase in the intrinsic fluorescence quantum yield (lambda ex, <em>2</em>90 nm; lambda em, 335 nm) between pH 6 and 9. Computer analysis of the data yielded a temperature-dependent apparent pKa of 7.8 at 37 degrees C and 8.1 at <em>2</em>0 degrees C, both with Hill coefficients less than or equal to 1. Calorimetric experiments revealed a similar sensitivity to pH, where the denaturation temperature of the fragment titrated from 74 degrees C at pH 6 to 59 degrees C at pH 8.5, with an apparent pKa of 7.3 and a Hill coefficient less than 1. The enthalpies and widths at half-height of the transitions were also exquisitely sensitive to pH. The fluorescence and calorimetric data could all be described by the titration of a single ionizable group of apparent pKa of 7.8 at 37 degrees C and <em>delta</em> pKa/degrees C of -0.018. The ionization of this critical group is suggested to exert significant control over the structure/stability of the cytoplasmic domain of band 3.

The G-protein-regulated, inwardly rectifying K+ (GIRK) channels are critical for functions as diverse as heart rate modulation and neuronal post-synaptic inhibition. GIRK channels are distributed predominantly throughout the heart, brain, and pancreas. In recent years, GIRK channels have received a great deal of attention for their direct G-protein betagamma (Gbetagamma) regulation. Native cardiac IKACh is composed of GIRK1 and GIRK4 subunits (Krapivinsky, G., Gordon, E. A., Wickman, K. A., Velimirovic, B., Krapivinsky, L., and Clapham, <em>D</em>. E. (1995) Nature 374, 135-141). Here, we examine the quaternary structure of IKACh using a variety of complementary approaches. Complete cross-linking of purified atrial IKACh protein formed a single adduct with a total molecular weight that was most consistent with a tetramer. In addition, partial cross-linking of purified IKACh produced subsets of molecular weights consistent with monomers, <em>dimers</em>, trimers, and tetramers. Within the presumed protein <em>dimers</em>, GIRK1-GIRK1 and GIRK4-GIRK4 adducts were formed, indicating that the tetramer was composed of two GIRK1 and two GIRK4 subunits. This 1:1 GIRK1 to GIRK4 stoichiometry was confirmed by two independent means, including densitometry of both silver-stained and Western-blotted native atrial IKACh. Similar experimental results could potentially be obtained if GIRK1 and GIRK4 subunits assembled randomly as <em>2</em>:<em>2</em> and equally sized populations of 3:1 and 1:3 tetramers. We also show that GIRK subunits may form homotetramers in expression systems, although the evidence to date suggests that GIRK1 homotetramers are not functional. We conclude that the inwardly rectifying atrial K+ channel, IKACh, a prototypical GIRK channel, is a heterotetramer and is most likely composed of two GIRK1 subunits and two GIRK4 subunits.

The structure of <em>D</em>-xylose isomerase from Streptomyces rubiginosus has been determined at 4-A resolution using multiple isomorphous phasing techniques. The folding of the polypeptide chain has been established and consists of two structural domains. The larger domain consists of eight beta-strand alpha-helix (beta alpha) units arranged in a configuration similar to that found for triose phosphate isomerase, <em>2</em>-keto-3-deoxy-6-phosphogluconate aldolase, and pyruvate kinase. The smaller domain forms a loop away from the larger domain but overlapping the larger domain of another subunit so that a tightly bound <em>dimer</em> is formed. The tetramer then consists of two such <em>dimers</em>. The location of the active site in the enzyme has been tentatively identified from studies using a crystal grown from a solution containing the inhibitor xylitol.

BACKGROUND

The conversion of prothrombin to thrombin by factor Xa is the penultimate step in the blood clotting cascade. In vivo, where the conversion occurs primarily on activated platelets in association with factor Va and Ca<em>2</em>+ ions, meizothrombin is the major intermediate of the two step reaction. Meizothrombin rapidly loses the fragment 1 domain (F1) by autolysis to become meizothrombin des F1 (mzTBN-F1). The physiological properties of mzTBN-F1 differ dramatically from those of thrombin due to the presence of prothrombin fragment <em>2</em> (F<em>2</em>), which remains covalently attached to the activated thrombin domain in mzTBN-F1.

RESULTS

The crystal structure of mzTBN-F1 has been determined at 3.1 A resolution by molecular replacement, using only the thrombin domain, and refined to R and Rfree values of 0.<em>2</em>05 and 0.<em>2</em>4<em>2</em>, respectively. The protease active site was inhibited with D-Phe-Pro-Arg-chloromethylketone (PPACK) to reduce autolysis. The mobile linker chain connecting the so-called kringle and thrombin domains and the first two N-acetylglucosamine residues attached to the latter were seen in electron-density maps improved with the program SQUASH. Previously these regions had only been modeled.

CONCLUSIONS

The F<em>2</em> kringle domain in mzTBN-F1 is bound to the electropositive heparin-binding site on thrombin in an orientation that is systematically shifted and has significantly more interdomain contacts compared to a noncovalent complex of free F<em>2</em> and free thrombin. F<em>2</em> in mzTBN-F1 forms novel hydrogen bonds to the carbohydrate chain of thrombin and perhaps stabilizes a unique, rigid conformation of the gamma-autolysis loop through non-local effects. The F<em>2</em> linker chain, which does not interfere with the active site or fibrinogen-recognition site, is arranged so that the two sites cleaved by factor Xa are separated by 36 A. The two mzTBN-F1 molecules in the asymmetric unit share a tight 'dimer' contact in which the active site of one molecule is partially blocked by the F<em>2</em> kringle domain of its partner. This interaction suggests a new model for prothrombin organization.

We have examined the linkage between pH and monovalent salt concentration (NaCl and NaF) on the equilibrium binding of the Escherichia coli SSB protein to single stranded poly(U) in its (SSB)65 binding mode. In this mode, single-stranded nucleic acid interacts with all four subunits of the SSB tetramer covering approximately 65 nucleotides and nearest-neighbor cooperative interactions can form between DNA bound SSB tetramers, although protein clusters are limited to <em>dimers</em> of tetramers (octamers). The intrinsic association equilibrium constant, K(obs), and the "limited" cooperativity parameter, omega T/O, have been determined from titrations that monitor the quenching of the SSB tryptophan fluorescence upon binding poly(U). The cooperativity parameter, omega T/O, is independent of salt concentration and type and increases only slightly with increasing pH. However, K(obs) decreases with increasing salt concentration due to a net release of ions accompanying complex formation. This net ion release has contributions from cation release from the nucleic acid as well as differential binding of both cations and anions to the protein. The dependence of K(obs) on [NaF] is independent of pH with <em>delta</em> logK(obs)/<em>delta</em> log[NaF] = -4.5(+/- 0.5). However, there is a strong linkage between the effects of [NaCl] and pH, such that (<em>delta</em> logK(obs)/<em>delta</em> log[NaCl]) ranges from -1<em>2</em>.0(+/- 0.8) at pH 5.5, to -6.0(+/- 0.5) at pH 9.0 (at <em>2</em>5 degrees C). Thus Cl- release increases with decreasing pH due to a linkage between chloride binding and protonation of the protein, whereas there is essentially no release of F-. The linkages of ion concentration and pH on K(obs) can be described in terms of: (1) cation release from the polynucleotide; (<em>2</em>) release of Cl- from sites on the SSB tetramer that require protonation to bind Cl-; (3) binding of cations to sites on the SSB tetramer which require deprotonation for cation binding, and (4) required binding of two-to-three protons by the SSB tetramer in order to form the SSB-poly(U) complex. Thus, the influence of salt concentration on protein-nucleic acid equilibria can be quite complex with contributions from differential ion binding to both the protein and the nucleic acid; however, these can be resolved by examining the linked effects of pH and salt concentration on these interactions.

UV irradiation of purified mengovirus resulted in a very rapid inactivation of the infectivity of the virions (<em>D</em>(37) [37% survival dose] = 700 ergs/mm(<em>2</em>)) which correlated in time with the formation of uracil <em>dimers</em> in the viral RNA. <em>D</em>uring the first <em>2</em> min of irradiation, an average of 1.7 uracil <em>dimers</em> were formed per PFU of virus inactivated. Hemagglutination activity of the virions began to decrease only after a lag period of about 5 min and at a much lower rate (<em>D</em>(37) = 84,000 ergs/mm(<em>2</em>)). This decrease coincided in time with the appearance of altered proteins in the capsid and a structural change in the capsid. Although 10- to <em>2</em>0-min irradiated virions appeared intact in the electron microscope and sedimented at 150S in sucrose density gradients, the RNA of the virions became accessible to RNase and extractable by low concentrations of sodium dodecyl sulfate, and the virions broke down upon equilibrium centrifugation in CsCl gradients. <em>D</em>uring longer periods of irradiation (30 to 60 min), a progressively greater proportion of the virions were converted to 14S protein particles and 80S ribonucleoprotein particles composed of intact viral RNA and about 30% of the capsid proteins, alpha, beta, and gamma. Empty capsids were not detectable at any time during 60 min of irradiation, by which time disruption of the virions was complete. Irradiation of complete virions also resulted in an increased sedimentation rate of the viral RNA and in the covalent linkage to the viral RNA of about 1% of the total capsid protein in the form of heterogeneous low-molecular-weight polypeptides. The two observations seem to be causally related, since irradiation of isolated viral RNA did not result in an increase in sedimentation rate of the RNA, even though uracil <em>dimer</em> formation in viral RNA occurred at about the same rate and to the same extent whether intact virions or viral RNA were irradiated.

It has been shown that type <em>2</em> diabetes (<em>D</em>M) is associated with enhanced thrombin generation and formation of denser fibrin clots of reduced lysability. We sought to investigate the impact of diabetes duration versus glycaemia control on fibrin clot phenotype and its determinants in type <em>2</em> diabetic patients. In 156 consecutive Caucasian patients with type <em>2</em> diabetes we investigated ex vivo thrombin generation, fibrinolytic proteins, along with plasma fibrin clot permeation (Ks), compaction, turbidity, and efficiency of tissue plasminogen activator (t-PA)-mediated fibrinolysis. Patients with longer diabetes duration (>5 years, median; n=68) had higher peak thrombin generation (+16.3%, p<0.001), plasminogen activator inhibitor-1 (PAI-1) antigen (+14.8%, p=0.001), t-PA antigen (+13.9%, p=0.00<em>2</em>) compared with those with duration ≤5 years (n=88). No such differences were observed between patients with inadequate glycaemic control, defined as glycated haemoglobin (HbA1C) >6.5% (48 mmol/mol) (n=77), versus those with HbA1C≤6.5% (n=79). Fibrinogen, thrombin-activatable fibrinolysis inhibitor antigen, plasminogen and soluble thrombomodulin were unaffected by disease duration or glycaemia control. Lower clot permeability, longer clot lysis, and higher maximum <em>D</em>-<em>dimer</em> levels released from clots (all p<0.05 after adjustment for fibrinogen, age, body mass index, insulin, acetylsalicylic acid treatment, and HbA1c or diabetes duration) were also observed in patients with diabetes duration >5 years and those with HbA1C>6.5%. We conclude that prolonged duration of type <em>2</em> diabetes is associated with increased thrombin formation, hypofibrinolysis, and prothrombotic fibrin clot phenotype. The impact of disease duration on coagulation is different and stronger than that observed during inadequate glycaemia control.

BACKGROUND

Interleukin 6 (IL-6), high-sensitivity C-reactive protein (hsCRP), and D-dimer levels are linked to adverse outcomes in human immunodeficiency virus (HIV) infection, but the strength of their associations with different clinical end points warrants investigation.

METHODS

Participants receiving standard of care in 2 HIV trials with measured biomarker levels were followed to ascertain all-cause death, non-AIDS-related death, AIDS, cardiovascular disease (CVD), and non-AIDS-defining malignancies. Hazard ratios (HRs) and 95% confidence intervals (CIs) of each end point for quartiles and log2-transformed IL-6, hsCRP, and D-dimer levels were calculated using Cox models. Marginal models modelling multiple events tested for equal effects of biomarker levels on different end points.

RESULTS

Among 4304 participants, there were 157 all-cause deaths, 117 non-AIDS-related deaths, 101 AIDS cases, 121 CVD cases, and 99 non-AIDS-defining malignancies. IL-6 was more strongly associated with most end points, compared with hsCRP. IL-6 appeared to be a stronger predictor than D-dimer for CVD and non-AIDS-defining malignancies, but 95% CIs overlapped. Independent associations of IL-6 were stronger for non-AIDS-related death (HR, 1.71; 95% CI, 1.43-2.04) and all-cause death (HR, 1.56; 95% CI, 1.33-1.84) and similar for CVD (HR, 1.35; 95% CI, 1.12-1.62) and non-AIDS-defining malignancies (HR, 1.30; 95% CI, 1.06-1.61). There was heterogeneity of IL-6 (P < .001) but not hsCRP (P = .15) or D-dimer (P = .20) as a predictor for different end points.

CONCLUSIONS

IL-6 is a stronger predictor of fatal events than of CVD and non-AIDS-defining malignancies. Adjuvant antiinflammatory and antithrombotic therapies should be tested in HIV-infected individuals.

Photochemical reaction of a plant blue-light photoreceptor, Arabidopsis phototropin 1-LOV (light-oxygen-voltage sensing) domain <em>2</em>, was studied with a view to the diffusion coefficients (<em>D</em>) using the pulsed-laser-induced transient grating method. Although the reaction dynamics completes at a rate of several microseconds as long as it is monitored by the absorption change, the diffusion coefficient was found to be time-dependent in a time range of submilliseconds to seconds. The observed signal can be analyzed by the two-state model, which includes the <em>D</em>-value decrease from <em>D</em> of the reactant (9.8 +/- 0.4) x 10(-11) m<em>2</em>/s to <em>D</em> of the product (8.0 +/- 0.4) x 10(-11) m<em>2</em>/s. The <em>D</em>-value of the reactant implies that the dominant form in the ground state of phototropin 1 LOV<em>2</em> is the monomeric form in a concentration range of 50-<em>2</em>00 microM. According to the Stokes-Einstein relationship, the <em>D</em>-change can be explained by a volume increase of 1.8 times. Furthermore, the rate of the <em>D</em>-change was roughly proportional to the concentration of the sample. These two observations indicate that the LOV<em>2</em> domain transiently forms a <em>dimer</em> upon photoexcitation. When the sample concentration is increased (>180 microM), a new signal component appears within a few milliseconds. This signal represents a <em>D</em> increase from 8.0 x 10(-11) m<em>2</em>/s to 9.8 x 10(-11) m<em>2</em>/s with a time constant of 300 micros. The completely opposite <em>D</em>-change from that observed in a lower concentration, as well as the concentration dependence, implies that a <em>dimer</em> is formed in the ground state in a higher concentration range, even though the fraction of the <em>dimer</em> is still minor in this range. This <em>dimer</em> is photodissociated, with a time constant of 300 micros. This research clearly shows that the time-resolved diffusion measurement is a very powerful tool for detecting spectrally silent association/dissociation processes during chemical reactions. The photoreaction of the LOV<em>2</em> domain is discussed.

Beta-<em>2</em>-microglobulin (beta<em>2</em>m) self-associates into fibrillar amyloid deposits in the musculoskeletal system of patients undergoing hemodialysis treatment. Previous studies have shown that stoichiometric amounts of Cu(II) at near physiological conditions can cause beta<em>2</em>m to organize into native-like <em>dimers</em> prior to forming amyloid fibrils. Here, we report the results from selective covalent labeling reactions combined with mass spectrometry that provide insight into the amino acid residues that mediate <em>dimer</em> formation in the wild-type protein. Using three complementary covalent labeling reagents, we find that the <em>dimer</em> interface is formed by the antiparallel stacking of ABE<em>D</em> beta-sheets from two beta<em>2</em>m monomers. In addition, our data clearly indicate that a <em>dimer</em> interface involving the interactions of <em>D</em>-<em>D</em> strands from separate protein units as seen in the recent crystal structures of two mutant beta<em>2</em>m oligomers is unlikely.

Our studies of equilibrium solubilization of crystals of unconjugated bilirubin (UCB) in buffered aqueous NaCl (1988. J. Lipid Res. <em>2</em>9: 335-348) suggested that the two carboxylic pKa values were 6.8 and 9.3 and the solubility of UCB diacid was 0.1 microM. These data, however, were not ideal, due to possible effects of crystal size, metastability, 96-h incubation times with formation of polar derivatives, impurities in the bilirubin, and imprecision of analyses at low concentrations of UCB ([UCB]). In the present study, designed to determine the pKa values and self-association of UCB, these problems were minimized by solvent partition of UCB from solution in CHCl3 into buffered aqueous NaCl. There was no crystal phase. Equilibrium was attained rapidly (10 min); UCB and CHCl3 were highly purified; and accurate diazo assay of low [UCB] in the aqueous phase, [Bw], was achieved by concentrating the UCB through back-extraction into a small volume of CHCl3. By determining effects on partition rations of varying the [UCB] in the CHCl3 phase, [Bc], we could assess also the self-association of UCB species in the aqueous phase. Partition ratios (P = Bw/Bc) did not differ between initial and repeat extractions, indicating insignificant concentrations of polar UCB derivatives. Similar P ratios were obtained when equilibrium was approached from a supersaturated aqueous phase. At <em>2</em>1-<em>2</em>5 degrees C, mu = 0.15, the data (n = 76) fit the equation: log P = log Po + log[1 + 10(pH-A) + 10(<em>2</em>pH-B) + Bc.10(4pH-<em>D</em>)]; the bracketed terms reflect P for H<em>2</em>Bo (diacid), HB- (monoanion), B= (dianion), and (B=)<em>2</em> <em>dimer</em>, respectively. Computer-fitted values for constants (+/- S<em>D</em>) were: Po = P for H<em>2</em>Bo = 5.79 x 10(-5); A = pK1 = 8.1<em>2</em> +/- 0.<em>2</em>3; B = pK1 + pK<em>2</em> = 16.56 +/- 0.10; pK<em>2</em> = 8.44 +/- 0.33; <em>D</em> = pk<em>2</em><em>2</em> + <em>2</em>(pK1 + pK<em>2</em>) -log(<em>2</em>Po) = 37.64 +/- 0.07, and k<em>2</em><em>2</em> = 0.<em>2</em>6 microM-1 [formation constant of (B=)<em>2</em> <em>dimer</em>]. In ancillary studies, multiple cycles of direct dissolution of UCB crystals revealed a progressive decrease in aqueous solubility of UCB as fine crystals were removed; this effect was minimal in CHCl3. Unlike in water, moreover, varied UCB crystal forms had similar solubilities in CHCl3, with [Bc] = 1.14 mM at saturation. As determined from [Bc]sat.Po, the aqueous solubility of H<em>2</em>Bo was 66 nM.(ABSTRACT TRUNCATE<em>D</em> AT 400 WOR<em>D</em>S)

Low molecular weight compoun<em>d</em>s were isolate<em>d</em> by high-performance liqui<em>d</em> chromatography from the maggot or haemolymph extracts of Lucilia sericata (Meigen) (Diptera: Calliphori<em>d</em>ae). Using gas chromatography-mass spectrometry analysis, three compoun<em>d</em>s were obtaine<em>d</em>: p-hy<em>d</em>roxybenzoic aci<em>d</em> (molecular weight 138 Da), p-hy<em>d</em>roxyphenylacetic aci<em>d</em> (molecular weight 15<em>2</em> Da) an<em>d</em> octahy<em>d</em>ro-<em>d</em>ipyrrolo[1,<em>2</em>-a;1',<em>2</em>'-<em>d</em>] pyrazine-5,10-<em>d</em>ione (molecular weight 194 Da), also known as the cyclic <em>dimer</em> of proline (or proline <em>d</em>iketopiperazine or cyclo[Pro,Pro]). All three molecules reveale<em>d</em> antibacterial activity when teste<em>d</em> against Micrococcus luteus an<em>d</em>/or Pseu<em>d</em>omonas aeruginosa, an<em>d</em> the effect was even more pronounce<em>d</em> when these molecules were teste<em>d</em> in combination an<em>d</em> cause<em>d</em> lysis of these bacteria.

NhaA, the Na(+)/H(+) antiporter of Escherichia coli, exists in the native membrane as a homo<em>dimer</em> of which two monomers have been suggested to be attached by a beta-hairpin at the periplasmic side of the membrane. Constructing a mutant deleted of the beta-hairpin, NhaA/<em>Delta</em>(Pro(45)-Asn(58)), revealed that in contrast to the <em>dimer</em>ic mobility of native NhaA, the mutant has the mobility of a monomer in a blue native gel. Intermolecular cross-linking that monitors <em>dimers</em> showed that the mutant exists only as monomers in the native membrane, proteoliposomes, and when purified in beta-dodecyl maltoside micelles. Furthermore, pull-down experiments revealed that, whereas as expected for a <em>dimer</em>, hemagglutinin-tagged wild-type NhaA co-purified with His-tagged NhaA on a Ni(<em>2</em>+)-NTA affinity column, a similar version of the mutant did not. Remarkably, under routine stress conditions (0.1 m LiCl, pH 7 or 0.6 m NaCl, pH 8.3), the monomeric form of NhaA is fully functional. It conferred salt resistance to NhaA- and NhaB-deleted cells, and whether in isolated membrane vesicles or reconstituted into proteoliposomes exhibited Na(+)/H(+) antiporter activity and pH regulation very similar to wild-type <em>dimers</em>. Remarkably, under extreme stress conditions (0.1 m LiCl or 0.7 m NaCl at pH 8.5), the <em>dimer</em>ic native NhaA was much more efficient than the monomeric mutant in conferring extreme stress resistance.

Although the antigen-binding pocket of all antibodies consists of VL + VH <em>dimers</em> (where VL and VH represent immunoglobulin light and heavy chain regions, respectively), subgroups of their VH largely determine their antigen-binding specificities. This VH subgroup dependence automatically relegates subsidiary roles to VL as a whole and to the complementarity-determining region 3 (C<em>D</em>R-3) of VH encoded by independent diversity (<em>D</em>) and joining (J) coding segments in determining antigen-binding specificities of individual antibodies. As a sequel to our previous paper, which emphasized the role conserved residues in C<em>D</em>R-1 and C<em>D</em>R-<em>2</em> of VH play in general shaping of the primordial antigen-binding cavity, here we propose that the three short clusters of amino acid sequences in C<em>D</em>R-1 and C<em>D</em>R-<em>2</em> that are placed in the immediate vicinity of the tryptophan loop primarily determine subgroup-dependent antigen preference of individual VH, therefore, antibodies. The three clusters are the 31st to 35th positions of C<em>D</em>R-1 and the 50th to 5<em>2</em>nd and 58th to 60th positions of C<em>D</em>R-<em>2</em>. Of those, the 3<em>2</em>nd, 34th, 51st, and 59th positions tend to be occupied by tyrosine, methionine, isoleucine, and tyrosine, respectively. Nevertheless, free amino acid substitutions at the remaining seven sites can generate <em>2</em>0(7) or 1.<em>2</em>8 X 10(9) varieties of amino acid sequence combinations. Some of these astronomically numerous sequence combinations no doubt contribute to the maintenance of the vast repertoire of antigen-combining diversity, which might be as large as 10(7), whereas others serve to vary binding affinities toward the same antigen. Ironically, but not surprisingly, a single nonconservative amino acid substitution at one of these sites often suffices to change the antigen preference of VH from one to another, whereas more substitutions affecting two or more clusters are apparently required to change the binding affinity toward the same antigen. In the case of mouse anti-p-azophenylarsonate antibodies, the principle of VH subgroup dependence is violated, their VH belonging to either subgroup 1 or 3. It appears that the mouse genome lacks anti-p-azophenylarsonate germ line VH, residues of C<em>D</em>R-3 derived from one particular JH coding segment coming to rescue to cope with this unnatural man-made antigen.

Rheumatoid arthritis is a complex inflammatory disease of unknown cause. Although various laboratory and clinical measurements are useful in managing these patients, there is a need for better tests to quantitatively assess disease activity. The purpose of this study was to investigate the association of certain immune and inflammation (I-I) parameters with four traditional disease severity measures and a functional measure in rheumatoid arthritis patients. A single set of patient blood samples was analyzed, and four traditional disease severity measures and patient functional statuses were determined from 64 consecutive outpatients with rheumatoid arthritis. Plasma tumor necrosis factor-alpha (TNF), soluble interleukin-<em>2</em> receptor (sIL-<em>2</em>R), sC<em>D</em>4 and sC<em>D</em>8 (and the sC<em>D</em>4/sC<em>D</em>8 ratio), neopterin, and fibrin <em>D</em>-<em>dimer</em> were analyzed in relationship to Westergren erythrocyte sedimentation rate (ESR), physician assessment of disease activity, joint pain count, grip strength, and Arthritis Impact Measurement Scale (AIMS) scores. Rheumatoid arthritis patients had higher mean levels of all I-I measures (except sC<em>D</em>4) compared to healthy subjects. Initial significant correlations between TNF, sIL-<em>2</em>R, and <em>D</em>-<em>dimer</em> and several disease severity and functional measures were detected. When we controlled for the covariates age, gender, race, and medications, regression analyses indicated that, as a group, the I-I measures were significantly related to grip strength, physician disease severity rating, ESR, and total joint pain. When the predictive values of the I-I measures were tested controlling for the covariates and ESR, <em>D</em>-<em>dimer</em> was independently and significantly associated with variability in grip strength, physician disease severity, and AIMS physical disability, while TNF was associated with a significant amount of variability in total joint pain.(ABSTRACT TRUNCATE<em>D</em> AT <em>2</em>50 WOR<em>D</em>S)

BACKGROUND

The incidence of deep vein thrombosis and pulmonary embolism following laparoscopic surgery is unknown and studies on alterations of hemostasis after laparoscopy are inconclusive.

METHODS

In this study we prospectively evaluated changes in prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen (Fg), antithrombin III (ATIII), prothrombin fragment F 1 + <em>2</em>, beta-thromboglobulin (betaTG) and <em>D</em>-<em>dimer</em> (<em>D</em>-<em>D</em>), preoperatively and <em>2</em>4 h after laparoscopic surgery in 16 patients.

RESULTS

Comparing pre- and postoperative values, no statistical differences were observed in aPTT, F1 + <em>2</em>, and ATIII measurements. Postoperative PT values increased slightly (p approximately 0.05) after surgery. Conversely, Fg, betaTG, and <em>D</em>-<em>D</em> values were statistically higher in the <em>2</em>4-h evaluation (p = 0.008, 0.01, and 0.045, respectively).

CONCLUSIONS

These data suggest that laparoscopic surgery induces activation of coagulation and fibrinolytic pathways and, additionaly, betaTG elevation, which has never been reported and might account for postoperative platelet activation and a greater risk of thrombogenicity. Therefore, routine thromboembolic prophylaxis in patients undergoing laparoscopic surgery is recommended.

OBJECTIVE

To evaluate if elevated D-dimer level is specific for venous malformations (VMs) and thus useful for differential diagnosis, which can be problematic even in specialized interdisciplinary centers. Localized intravascular coagulopathy, characterized by elevated D-dimer levels, has been observed in approximately 40% of patients with VMs.

METHODS

Prospective convenience sample accrued from 2 interdisciplinary sites.

METHODS

Two interdisciplinary centers for vascular anomalies in Brussels, Belgium, and Caen, France

METHODS

The study population comprised 280 patients with clinical data, Doppler ultrasonograms (for 251 patients), and coagulation parameter measurements. Main Outcome Measure Measurement of D-dimer levels.

RESULTS

A VM was diagnosed in 195 of 280 patients (69.6%), and 83 of them had elevated D-dimer levels; the sensitivity of D-dimer dosage was 42.6% (95% confidence interval, 35.6%-49.5%). Among the 85 patients without VM, D-dimer levels were elevated only in 3 patients; the specificity of the dosage was 96.5% (95% confidence interval, 92.5%-100%).

CONCLUSIONS

Elevated D-dimer level is highly specific for VMs (pure, combined, or syndromic), and therefore this easy and inexpensive biomarker test should become part of the clinical evaluation of vascular anomalies. It can detect hidden VMs and help differentiate glomuvenous malformation (normal D-dimer levels) from other multifocal venous lesions. Elevated D-dimer level also differentiates a VM from a lymphatic malformation. Moreover, slow-flow Klippel-Trenaunay syndrome (capillaro-lymphatico-venous malformation with limb hypertrophy) can be distinguished from fast-flow Parkes Weber syndrome (capillary malformation with underlying multiple microfistulas and limb hypertrophy). For these reasons, D-dimer level measurement is a useful complementary tool for diagnosing vascular anomalies in everyday practice.

Microtubules are highly dynamic structures, composed of alpha/beta-tubulin hetero<em>dimer</em>s. Biosynthesis of the functional <em>dimer</em> involves the participation of several chaperones, termed cofactors A-E, that act on folding intermediates downstream of the cytosolic chaperonin CCT (1, <em>2</em>). We show that cofactor <em>D</em> is also a centrosomal protein and that overexpression of either the full-length protein or either of two centrosome localization domains leads to the loss of anchoring of the gamma-tubulin ring complex and of nucleation of microtubule growth at centrosomes. In contrast, depletion of cofactor <em>D</em> by short interfering RNA results in mitotic spindle defects. Because none of these changes in cofactor <em>D</em> activity produced a change in the levels of alpha-or beta-tubulin, we conclude that these newly discovered functions for cofactor <em>D</em> are distinct from its previously described role in tubulin folding. Thus, we describe a new role for cofactor <em>D</em> at centrosomes that is important to its function in polymerization of tubulin and organization of the mitotic spindle.

OBJECTIVE

To determine the involvement of coagulation in bleeding and poor outcome in patients with severe leptospirosis.

METHODS

In a prospective study, parameters of the coagulation system were measured on admission and during follow-up in 5<em>2</em> consecutive patients with severe leptospirosis.

RESULTS

All patients showed coagulation disorders, such as prolonged prothrombin time (PT) and activated partial thromboplastin time, marked procoagulant activity [thrombin-antithrombin (TAT) complexes, prothrombin fragment 1+<em>2</em>, <em>D</em>-<em>dimer</em>], reduced levels of anticoagulant markers (protein C, antithrombin) and increased (anti-) fibrinolytic activity [plasmin-antiplasmin (PAP) complexes, plasminogen activator inhibitor-1]. These disorders were more pronounced in patients who died eventually. PT prolongation was associated with mortality (OR 1.4, 95% CI: 1.0-1.8, P = 0.04). Bleeding occurred in 31 subjects (60%). Of these, <em>2</em>4 had mild bleeding and seven had severe haemorrhages. Thrombocytopenia (platelets </=100 x 10(9)/l) was significantly associated with clinical bleeding (OR 4.6, 95% CI: 1.3-16). A subanalysis of patients with and without severe bleeding revealed a more pronounced imbalance of the coagulation system in patients with severe bleeding, as reflected by a significant association with PT (OR 1.4, 95% CI: 1.0-1.8, P = 0.05) and the TAT/PAP ratio (OR 1.3, 95% CI: 1.0-1.6, P = 0.05), which is an indicator of the balance between coagulation and fibrinolysis. Overt disseminated intravascular coagulation (<em>D</em>IC) was found in 10 (<em>2</em><em>2</em>%) of the 46 patients for whom the score could be calculated. There was no significant association between <em>D</em>IC scores, bleeding diathesis or poor outcome.

CONCLUSIONS

The coagulation system was strongly activated in patients with leptospirosis. This was more pronounced in the deceased and in patients with severe bleeding than in than the survivors and in those without severe bleeding.