BACKGROUND

Many inflammatory and haemostatic biomarkers show associations with acute ischaemic stroke outcome, but few studies compare a large range of markers.

METHODS

We assessed clinical status and 16 biomarkers within <em>2</em>4 h of onset in 180 consecutive acute ischaemic stroke patients.

RESULTS

A total of 94 patients had a poor outcome (dead or dependent at 30 days). C-reactive protein (CRP), IL-6, and fibrin <em>D</em>-<em>dimer</em> showed the strongest univariate associations with poor outcome >><em>2</em>-fold increase; p < 0.01). When all biomarkers were included with clinical variables in a multivariable model, only <em>D</em>-<em>dimer</em> (OR 1.54; 95% CI 1.09-<em>2</em>.17), CRP (OR 1.31; 95% CI 1.03-1.68) and Scandinavian Stroke Scale (OR 0.91; 95% CI 0.88-0.95) were associated with poor outcome.

CONCLUSIONS

D-dimer and CRP are independently associated with poor outcome in acute ischaemic stroke. More data is required to expand our understanding of these potential relationships with outcome.

15-Deoxy-<em>Delta</em> 1<em>2</em>,14-prostaglandin J<em>2</em> (15d-PGJ<em>2</em>), a cyclopentenone prostaglandin, displays a potent anti-inflammatory effect at micromolar concentrations >><em>2</em> microM) through direct inhibition of nuclear factor (NF)-kappa B activation. Here we show that at submicromolar concentrations (0.1-0.5 microM) 15d-PGJ<em>2</em> retains the ability to suppress the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in lipopolysaccharide (LPS)-activated murine J774 macrophages under the conditions of a prolonged incubation (>1<em>2</em> h). Western blot analysis revealed that the expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1), was induced and coincident with the anti-inflammatory action of 15d-PGJ<em>2</em>. Inhibition of HO-1 activity or scavenging carbon monoxide (CO), a byproduct derived from heme degradation, significantly attenuated the suppressive activity of 15d-PGJ<em>2</em>. Furthermore, LPS-induced NF-kappa B activation assessed by the inhibitory protein of NF-kappa B(I kappa B) degradation and p50 nuclear translocation was diminished in cells subjected to prolonged treatment with the low concentration of 15d-PGJ<em>2</em>. Treatment of cells with the protein synthesis inhibitor, cycloheximide, or the specific p38 MAP kinase inhibitor, SB<em>2</em>03580, blocked the induction of HO-1 and suppression of LPS-induced I kappa B degradation mediated by 15d-PGJ<em>2</em>. Likewise, HO inhibitor and CO scavenger were effective in abolishing the inhibitory effects of 15d-PGJ<em>2</em> on NF-kappa B activation induced by LPS. The functional role of CO was further demonstrated by the use of a CO releasing molecule, tricarbonyldichlororuthenium(II) <em>dimer</em>, which significantly suppressed LPS-induced nuclear translocation of p50 as assessed by confocal immunofluorescence. Collectively, these data suggest that even at submicromolar concentrations 15d-PGJ<em>2</em> can exert an anti-inflammatory effect in macrophages through a mechanism that involves the action of HO/CO.

Tubulin, the constituent protein of microtubules, is an alpha beta heterodimer; both alpha and beta exist in several isotypic forms whose functional significance is not precisely known. The antimitotic alkaloid colchicine binds to mammalian brain tubulin in a biphasic manner under pseudo-first-order conditions in the presence of a large excess of colchicine (Garland, <em>D</em>. L. (1978) Biochemistry 17, 4<em>2</em>66-4<em>2</em>7<em>2</em>). We have studied the kinetics of colchicine binding to purified beta-tubulin isotypes and find that each of the purified beta-tubulin isotypes binds colchicine in a monophasic manner. The apparent on-rate constants for the binding of colchicine to alpha beta II-, alpha beta III-, and alpha beta IV-tubulin <em>dimers</em> are respectively 13<em>2</em> +/- 5, 30 +/- <em>2</em>, and <em>2</em>36 +/- 7 M-1 s-1. When the isotypes are mixed, the kinetics become biphasic. Scatchard analysis revealed that the isotypes differ significantly in their affinity constants (Ka) for binding colchicine. The affinity constants are 0.<em>2</em>4 x 10(6), 0.1<em>2</em> x 10(6), and 3.31 x 10(6) M-1, respectively, for alpha beta II-, alpha beta III-, and alpha beta IV-tubulin <em>dimers</em>. Our results are in agreement with the hypothesis that the beta-subunit of tubulin plays a major role in the interaction of colchicine with tubulin. Our binding data raise the possibility that the tubulin isotypes might play important regulatory roles by interacting differently with other non-tubulin proteins in vivo, which in turn, may regulate microtubule-based functions in living cells.

We have investigated a number of mutations that alter the ability of the E. coli transcription factors CRP and FNR to activate transcription. In CRP, some mutations at position 159 (H159L, H159I and <em>delta</em> 159) prevent transcription activation at a number of naturally-occurring and semi-synthetic CRP-dependent promoters. We suggest that some feature of the surface-exposed turn around residue 159 is recognised by RNA polymerase during transcription activation at these promoters. Mutations at position 5<em>2</em> increase CRP activity and reverse the effects of H159L and <em>delta</em> 159, most likely by creating a new contact with RNA polymerase. However this new contact only gives increased expression when the CRP binding site is located 41 1/<em>2</em> base pairs upstream of the transcription start site and fails to reverse the effects of H159L and <em>delta</em> 159 at promoters where the CRP site is located further upstream. To explain our results we propose that the two surface-exposed turns around residues 5<em>2</em> and 159 contain elements that are potential RNA polymerase docking sites: in the CRP <em>dimer</em> these two active patches are located on adjacent faces of different subunits. FNR, a related transcription activator, contains amino acid sequences homologous to the CRP sequence around position 5<em>2</em>. Mutations in this zone (from residues 81-88 in FNR) reduce expression from an FNR-dependent promoter without stopping FNR binding to its target. This defines a patch on FNR, which is homologous to the CRP surface-exposed loop around position 5<em>2</em>, which is involved in transcription activation, most likely by contacting RNA polymerase.

Molecular evolution has evolved two metabolic routes for isoprenoid biosynthesis: the mevalonate and the <em>2</em>-C-methyl-<em>D</em>-erythritol-4-phosphate (MEP) pathway. The MEP pathway is used by most pathogenic bacteria and some parasitic protozoa (including the malaria parasite, Plasmodium falciparum) as well as by plants, but is not present in animals. The terminal reaction of the MEP pathway is catalyzed by (E)-4-hydroxy-3-methyl-but-<em>2</em>-enyl diphosphate (HMBPP) reductase (LytB), an enzyme that converts HMBPP into isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (<em>D</em>MAPP). Here, we present the structure of Aquifex aeolicus LytB, at 1.65 A resolution. The protein adopts a cloverleaf or trefoil-like structure with each monomer in the <em>dimer</em> containing three alpha/beta domains surrounding a central [Fe3S4] cluster ligated to Cys13, Cys96, and Cys193. Two highly conserved His (His 4<em>2</em> and His 1<em>2</em>4) and a totally conserved Glu (Glu1<em>2</em>6) are located in the same central site and are proposed to be involved in ligand binding and catalysis. Substrate access is proposed to occur from the front-side face of the protein, with the HMBPP diphosphate binding to the two His and the 4OH of HMBPP binding to the fourth iron thought to be present in activated clusters, while Glu1<em>2</em>6 provides the protons required for IPP/<em>D</em>MAPP formation.

A series of dideoxyribonucleoside methylphosphonate analogues, dNpN and dNpNp, which contain a nonionic 3'--5' methylphosphonyl internucleoside linkage were prepared. The two diastereoisomers, designated isomers 1 and <em>2</em>, of each <em>dimer</em> differ in configuration of the methylphosphonate group and were separated by column chromatography. The diastereoisomers of each <em>dimer</em> have different conformations in solution as shown by ultraviolet hypochromicity data and their circular dichroism spectra. For example, dApA isomer 1 is more highly stacked than isomer <em>2</em>, although both isomers are less stacked than the dinucleoside monophosphate, dApA. The circular dichroism spectrum of isomer 1 is very similar to that of dApA, while the C<em>D</em> spectrum of isomer <em>2</em> shows a loss of molecular ellipticity, [theta], at <em>2</em>70 nm and a greatly diminished [theta] at <em>2</em>50 nm. These results suggest that the stacked bases of dApA isomer 1 tend to orient in an oblique manner, while those in isomer <em>2</em> tend to orient in a parallel manner. This interpretation is verified by the 1H NMR study of these <em>dimers</em> (L. S. Kan, <em>D</em>. M. Cheng, P. S. Miller, J. Yano, and P. O. P. Ts'o, unpublished experiments). Both diastereoisomers of dAaA form <em>2</em>U:1A and <em>2</em>T:1A complexes with poly(U) and poly(dT), respectively. The higher Tm (Tm of poly(U)--isomer 1, 15.4 degrees C; Tm of poly(U)--isomer <em>2</em>, 19.8 degrees C; Tm of poly(dT)--isomer 1, 18.7 degrees C; Tm of poly(dT)--isomer <em>2</em>, 18.4 degrees C) values of these complexes vs. those of the corresponding dApA--polynucleotide complexes (Tm of poly(U)--dApA, 7.0 degrees C; Tm of poly(dT)--<em>D</em>ApA, 9.<em>2</em> degrees C) result from decreased charge repulsion between the nonionic <em>dimer</em> backbone and the negatively charged polymer backbone. The difference in conformations between dApA isomer 1 and dApA isomer <em>2</em> is reflected in the Tm of the isomer 1-poly(U) complex which is 4.4 degrees C lower than that of the isomer <em>2</em>-poly(U) complex. Since these nonionic oligonucleotide analogues are taken up by cells in culture, they show promise as molecular probes for the function and structure of nucleic acids inside living cells.

The composition of the P840-reaction center complex (RC), energy and electron transfer within the RC, as well as its topographical organization and interaction with other components in the membrane of green sulfur bacteria are presented, and compared to the FeS-type reaction centers of Photosystem I and of Heliobacteria. The core of the RC is homo<em>dimer</em>ic, since pscA is the only gene found in the genome of Chlorobium tepidum which resembles the genes psaA and -B for the hetero<em>dimer</em>ic core of Photosystem I. Functionally intact RC can be isolated from several species of green sulfur bacteria. It is generally composed of five subunits, PscA-<em>D</em> plus the BChl a-protein FMO. Functional cores, with PscA and PscB only, can be isolated from Prostecochloris aestuarii. The PscA-<em>dimer</em> binds P840, a special pair of BChl a-molecules, the primary electron acceptor A(0), which is a Chl a-derivative and FeS-center F(X). An equivalent to the electron acceptor A(1) in Photosystem I, which is tightly bound phylloquinone acting between A(0) and F(X), is not required for forward electron transfer in the RC of green sulfur bacteria. This difference is reflected by different rates of electron transfer between A(0) and F(X) in the two systems. The subunit PscB contains the two FeS-centers F(A) and F(B). STEM particle analysis suggests that the core of the RC with PscA and PscB resembles the PsaAB/PsaC-core of the P700-reaction center in Photosystem I. PscB may form a protrusion into the cytoplasmic space where reduction of ferredoxin occurs, with FMO trimers bound on both sides of this protrusion. Thus the subunit composition of the RC in vivo should be <em>2</em>(FMO)(3)(PscA)(<em>2</em>)PscB(PscC)(<em>2</em>)Psc<em>D</em>. Only 16 BChl a-, four Chl a-molecules and two carotenoids are bound to the RC-core, which is substantially less than its counterpart of Photosystem I, with 85 Chl a-molecules and <em>2</em><em>2</em> carotenoids. A total of 58 BChl a/RC are present in the membranes of green sulfur bacteria outside the chlorosomes, corresponding to two trimers of FMO (4<em>2</em> Bchl a) per RC (16 BChl a). The question whether the homo<em>dimer</em>ic RC is totally symmetric is still open. Furthermore, it is still unclear which cytochrome c is the physiological electron donor to P840(+). Also the way of NA<em>D</em>(+)-reduction is unknown, since a gene equivalent to ferredoxin-NA<em>D</em>P(+) reductase is not present in the genome.

OBJECTIVE

To determine the relationship between d-dimer (DD) and both proinflammatory and anti-inflammatory cytokine levels, and to confirm the association between DD status and outcomes in critically ill patients.

METHODS

Prospective observational study.

METHODS

Medical ICU (MICU) of a tertiary care, academic medical center.

METHODS

Individuals admitted to the MICU.

METHODS

Within 24 h of MICU admission, patients had DD status determined and interleukin (IL) levels (IL-6, IL-8, and IL-10) and tumor necrosis factor (TNF)-alpha measured. The strength of the DD level was also noted. Subjects were then monitored prospectively to determine mortality rate and the incidence of organ failure.

RESULTS

The study cohort included 79 patients (mean age, 65.2 years; 54.5% male patients). DD was present in 53.2% of subjects. The DD reaction was weak (1+) in 15 patients and strong (2+) in 27 patients. The TNF-alpha, IL-6, and IL-8 levels all increased in parallel with the increasing strength of the DD level. IL-10 levels did not differ based on DD status. Similarly, the severity of illness as measured by the APACHE (acute physiology and chronic health evaluation) II score was highest among those with higher DD levels: 24.7 +/- 6.2 for those with 2+ DD vs 17.2 +/- 3.1 and 11.5 +/- 2.7 for those with 1+ DD and no circulating DD, respectively (p < 0.001). For patients lacking DD, the mortality rate was 8.1%, compared to 13.3% and 55.6% for those with 1+ and 2+ DD levels, respectively (p < 0.001). No patient without DD had multisystem organ failure (MSOF) develop, while the incidence of MSOF also increased with increasing DD levels. As a screening test for mortality, the DD performed as well as the APACHE II system.

CONCLUSIONS

The coagulation system is active in critically ill patients, and DD levels correlate with activation of the proinflammatory cytokine cascade. The absence of a relationship between DD and anti-inflammatory cytokines (IL-10) suggests that the presence of DD may reflect the imbalance between proinflammatory and anti-inflammatory cytokines. DD identifies patients at increased risk for both MSOF and death.

BACKGROUND

Circulating levels of fibrinogen, fibrin D-dimer, C-reactive protein (CRP), tissue plasminogen activator antigen (t-PA) and von Willebrand factor are associated with incident coronary heart disease. We describe cross-sectional diurnal, seasonal, and blood-processing patterns for these variables, and assess whether they represent important sources of variability that should be taken into account in epidemiological studies or for additional risk prediction in individuals.

RESULTS

A total of 9377 men and women aged 45 years were visited in their homes and blood-sampled for fibrinogen, D-dimer, CRP, t-PA, and von Willebrand factor. These variables were examined in relation to the time of blood sample collection, day of the year, and delay in processing. All variables exhibited statistically significant diurnal sinusoidality (P < or = 0.02). Our models predicted a peak rise for fibrinogen and von Willebrand factor at midday, with overall diurnal variations of 3% and 10%, respectively, after adjustment for standard cardiovascular risk factors. D-dimer exhibited a peak at 14:00 hours, CRP at 15:00 hours, and t-PA at 10:00 hours with diurnal variations of 10%, 34%, and 55%, respectively, after full adjustment. All variables except CRP showed seasonal heterogeneity. Greater delays in processing blood samples were associated with higher levels of t-PA in particular. The proportion of variation attributed to the diurnal, seasonal, and processing effects was 2% for fibrinogen and von Willebrand factor; 9% for D-dimer, 1% for CRP, and 16% for t-PA.

CONCLUSIONS

Temporal variations are important sources of heterogeneity that may bias the analysis of epidemiological studies and coronary heart disease risk prediction in individuals. Sample-processing delay is particularly important for t-PA.

BACKGROUND

Patients with suspected deep vein thrombosis (DVT) of the lower extremities are usually investigated with ultrasonography either by the proximal veins (<em>2</em>-point ultrasonography) or the entire deep vein system (whole-leg ultrasonography). The latter approach is thought to be better based on its ability to detect isolated calf vein thrombosis; however, it requires skilled operators and is mainly available only during working hours. No randomized comparisons are yet available evaluating the relative values of these <em>2</em> strategies.

OBJECTIVE

To assess if the <em>2</em> diagnostic strategies are equivalent for the management of symptomatic outpatients with suspected DVT of the lower extremities.

METHODS

A prospective, randomized, multicenter study of consecutive symptomatic outpatients (n = <em>2</em>465) with a first episode of suspected DVT of the lower extremities who were randomized to undergo <em>2</em>-point or whole-leg ultrasonography. Data were taken from ultrasound laboratories of 14 Italian universities or civic hospitals between January 1, <em>2</em>003, and December <em>2</em>1, <em>2</em>006. Patients with normal ultrasound findings were followed up for 3 months, with study completion on March <em>2</em>0, <em>2</em>007.

METHODS

Objectively confirmed 3-month incidence of symptomatic venous thromboembolism in patients with an initially normal diagnostic workup.

RESULTS

Of <em>2</em>465 eligible patients, 345 met 1 or more exclusion criteria and <em>2</em><em>2</em> refused to participate; therefore, <em>2</em>098 patients were randomized to either <em>2</em>-point (n = 1045) or whole-leg (n = 1053) ultrasonography. Symptomatic venous thromboembolism occurred in 7 of 801 patients (incidence, 0.9%; 95% confidence interval [CI], 0.3%-1.8%) in the <em>2</em>-point strategy group and in 9 of 763 patients (incidence, 1.<em>2</em>%; 95% CI, 0.5%-<em>2</em>.<em>2</em>%) in the whole-leg strategy group. This met the established equivalence criterion (observed difference, 0.3%;95% CI, -1.4% to 0.8%).

CONCLUSIONS

The <em>2</em> diagnostic strategies are equivalent when used for the management of symptomatic outpatients with suspected DVT of the lower extremities.

BACKGROUND

clinicaltrials.gov Identifier: NCT00353093.

<em>D</em>iverse repertoires of antigen-receptor genes that result from combinatorial splicing of coding segments by V(<em>D</em>)J recombination are hallmarks of vertebrate immunity. The (RAG1-RAG<em>2</em>)<em>2</em> recombinase (RAG) recognizes recombination signal sequences (RSSs) containing a heptamer, a spacer of 1<em>2</em> or <em>2</em>3 base pairs, and a nonamer (1<em>2</em>-RSS or <em>2</em>3-RSS) and introduces precise breaks at RSS-coding segment junctions. RAG forms synaptic complexes only with one 1<em>2</em>-RSS and one <em>2</em>3-RSS, a dogma known as the 1<em>2</em>/<em>2</em>3 rule that governs the recombination fidelity. We report cryo-electron microscopy structures of synaptic RAG complexes at up to 3.4 Å resolution, which reveal a closed conformation with base flipping and base-specific recognition of RSSs. <em>D</em>istortion at RSS-coding segment junctions and base flipping in coding segments uncover the two-metal-ion catalytic mechanism. Induced asymmetry involving tilting of the nonamer-binding domain <em>dimer</em> of RAG1 upon binding of HMGB1-bent 1<em>2</em>-RSS or <em>2</em>3-RSS underlies the molecular mechanism for the 1<em>2</em>/<em>2</em>3 rule.

<strong class="sub-title"> Background: </strong> Vitamin <em>D</em> has an immunomodulatory role but the effect of therapeutic vitamin <em>D</em> supplementation in SARS-CoV-<em>2</em> infection is not known.

<strong class="sub-title"> Aim: </strong> Effect of high dose, oral cholecalciferol supplementation on SARS-CoV-<em>2</em> viral clearance.

Design: Randomised, placebo-controlled.

<strong class="sub-title"> Participants: </strong> Asymptomatic or mildly symptomatic SARS-CoV-<em>2</em> RNA positive vitamin <em>D</em> deficient (<em>2</em>5(OH)<em>D</em><<em>2</em>0 ng/ml) individuals.

<strong class="sub-title"> Intervention: </strong> Participants were randomised to receive daily 60 000 IU of cholecalciferol (oral nano-liquid droplets) for 7 days with therapeutic target <em>2</em>5(OH)<em>D</em>>50 ng/ml (intervention group) or placebo (control group). Patients requiring invasive ventilation or with significant comorbidities were excluded. <em>2</em>5(OH)<em>D</em> levels were assessed at day 7, and cholecalciferol supplementation was continued for those with <em>2</em>5(OH)<em>D</em> <50 ng/ml in the intervention arm. SARS-CoV-<em>2</em> RNA and inflammatory markers fibrinogen, <em>D</em>-dimer, procalcitonin and (CRP), ferritin were measured periodically.

<strong class="sub-title"> Outcome measure: </strong> Proportion of patients with SARS-CoV-<em>2</em> RNA negative before day-<em>2</em>1 and change in inflammatory markers.

<strong class="sub-title"> Results: </strong> Forty SARS-CoV-<em>2</em> RNA positive individuals were randomised to intervention (n=16) or control (n=<em>2</em>4) group. Baseline serum <em>2</em>5(OH)<em>D</em> was 8.6 (7.1 to 13.1) and 9.54 (8.1 to 1<em>2</em>.5) ng/ml (p=0.730), in the intervention and control group, respectively. 10 out of 16 patients could achieve <em>2</em>5(OH)<em>D</em>>50 ng/ml by day-7 and another two by day-14 [day-14 <em>2</em>5(OH)<em>D</em> levels 51.7 (48.9 to 59.5) ng/ml and 15.<em>2</em> (1<em>2</em>.7 to 19.5) ng/ml (p<0.001) in intervention and control group, respectively]. 10 (6<em>2</em>.5%) participants in the intervention group and 5 (<em>2</em>0.8%) participants in the control arm (p<0.018) became SARS-CoV-<em>2</em> RNA negative. Fibrinogen levels significantly decreased with cholecalciferol supplementation (intergroup difference 0.70 ng/ml; P=0.007) unlike other inflammatory biomarkers.

<strong class="sub-title"> Conclusion: </strong> Greater proportion of vitamin <em>D</em>-deficient individuals with SARS-CoV-<em>2</em> infection turned SARS-CoV-<em>2</em> RNA negative with a significant decrease in fibrinogen on high-dose cholecalciferol supplementation.

<strong class="sub-title"> Trial register number: </strong> <a href="http://clinicaltrials.gov/show/NCT04459<em>2</em>47" title="See in ClinicalTrials.gov">NCT04459<em>2</em>47</a>.

Keywords: Diabetes & endocrinology; Infectious diseases; Virology.

Cell surface-expressed receptors are often multichain complexes. One of these, the T cell receptor (TcR) alpha/beta-CD3 complex, is known to contain at least seven chains: the alpha and beta TcR chains plus the gamma, <em>delta</em>, epsilon and two zeta chains from the CD3 complex (alpha beta gamma <em>delta</em> epsilon zeta <em>2</em>). To gain insight into the structure of the complex we have used anti-peptide antisera specific for the individual subunits of the complex, and nonionic and ionic detergents to determine subunit interactions within the complex. Four closely associated pairs of chains could be identified: alpha beta, zeta <em>2</em>, gamma epsilon and <em>delta</em> epsilon. Interactions between the TcR alpha beta and either gamma epsilon or <em>delta</em> epsilon could be observed in the apparent absence of other CD3 chains. Furthermore, a hierarchy in the strength of the association between the TcR and the individual CD3 chains could be distinguished: TcR epsilon greater than TcR <em>delta</em> greater than TcR gamma. The zeta <em>2</em> <em>dimer</em> could only be detected in "intact" TcR-CD3 complexes shedding no light on possible interactions with either the TcR or CD3-gamma, <em>delta</em> and epsilon chains. Finally, cross-linking experiments suggest a close spatial relationship between the TcR alpha beta and both the CD3-gamma and CD3-epsilon chains. The results demonstrate that the methods used give valuable information on subunit interactions in a cell surface-expressed receptor complex and suggest a TcR-CD3 complex in which two epsilon chains are present, one linked to gamma and the other to <em>delta</em>. The data further indicate that gamma epsilon and <em>delta</em> epsilon complexes interact directly with the TcR chains. Based on the observations a model for the structure of the TcR-CD3 is presented and discussed.

A six-step mechanism is derived for the activation of kinesin K379 ATPase by microtubules. The data are fitted by the kinetic scheme [Formula see text] where T, <em>D</em>, and P refer to nucleotide triphosphate, nucleotide diphosphate, and inorganic phosphate, respectively; MtK refers to the complex of a K379 unit with the microtubule binding site. The initial binding and release steps, 1 and 6, are treated as rapid equilibria: k<em>2</em> = <em>2</em>00 s-1, k3 = 100 s-1, k5 = 35-40 s-1, maximum steady-state rate = <em>2</em>5 s-1 (50 mM NaCl, <em>2</em>0 degrees C). k<em>2</em> was obtained from the maximum rate of fluorescence enhancement with mant-ATP as substrate, k3 was obtained from the hydrolysis transient phase for ATP or mant-ATP, and k5 was obtained from the rate of decrease in fluorescence of mant-A<em>D</em>P in the reaction [Formula see text]. A large excess of ATP was present with the Mt to block rebinding of mant-A<em>D</em>P. The rate was measured as a function of microtubule concentration and extrapolated to give the maximum rate k5. The same method was used to obtain k5 for A<em>D</em>P by mixing K.A<em>D</em>P with microtubules plus excess mant-ATP. The enhancement of fluorescence for the binding of mant-ATP is followed by a decrease in fluorescence with a rate constant of 35-40 s-1. Since the decrease must occur after hydrolysis, it may be correlated with a step or steps leading to the low fluorescence MtK.<em>D</em> state. In the kinetic scheme, steps 4 and 5 both contribute to determining the maximum turnover rate. At higher ionic strengths or lower protein concentrations, the MtK complex is dissociated by ATP. The maximum rate is 1<em>2</em> +/- <em>2</em> s-1 in 50 mM NaCl; consequently, hydrolysis occurs before dissociation. The dissociation constant of MtK in the presence of A<em>D</em>P is twice as large as the dissociation constant in the presence of ATP and four times larger than the KM for microtubule activation. The proposed kinetic scheme, which treats the K379 units of a <em>dimer</em> as independent, provides a satisfactory description of the transient and steady-state properties of the system with the possible exception of results at very low substrate concentrations.

We cloned, expressed, and characterized a hemeprotein from <em>D</em>einococcus radiodurans (<em>D</em>. radiodurans NO synthase, deiNOS) whose sequence is 34% identical to the oxygenase domain of mammalian NO synthases (NOSoxys). deiNOS was <em>dimer</em>ic, bound substrate Arg and cofactor tetrahydrobiopterin, and had a normal heme environment, despite its missing N-terminal structures that in NOSoxy bind Zn(<em>2</em>+) and tetrahydrobiopterin and help form an active <em>dimer</em>. The deiNOS heme accepted electrons from a mammalian NOS reductase and generated NO at rates that met or exceeded NOSoxy. Activity required bound tetrahydrobiopterin or tetrahydrofolate and was linked to formation and disappearance of a typical heme-dioxy catalytic intermediate. Thus, bacterial NOS-like proteins are surprisingly similar to mammalian NOSs and broaden our perspective of NO biochemistry and function.

Postmenopausal hormone replacement therapy is associated with a reduction in the incidence of coronary heart disease. However, inconclusive results have been reported with respect to the risk of stroke, and recent studies consistently showed an increased risk of venous thromboembolism in postmenopausal women using oral estrogen. There are surprisingly few interventional studies to assess the true effects of estrogen-progestin regimens on blood coagulation and fibrinolysis, and the impact of the route of estrogen administration on hemostasis has not been well documented. Therefore, we investigated the effects of oral and transdermal estradiol/progesterone replacement therapy on hemostatic variables. Forty-five healthy postmenopausal women, aged 45 to 64 years, were assigned randomly to one of the three following groups: cyclic oral or transdermal estradiol, both combined with progesterone, or no hormonal treatment. Hemostatic variables were assayed at baseline and after a 6-month period. Pairwise differences in the mean change between the three groups were compared using nonparametric tests. Oral but not transdermal estradiol regimen significantly increased the mean value of prothrombin activation peptide (F1 + <em>2</em>) and decreased mean antithrombin activity compared with no treatment. <em>D</em>ifferences in fragment F1 + <em>2</em> levels between active treatments were significant. The oral estrogen group was associated with a significant decrease in both mean tissue-type plasminogen (t-PA) concentration and plasminogen activator inhibitor (PAI-1) activity and a significant rise in global fibrinolytic capacity (GFC) compared with the two other groups. A transdermal estrogen regimen had no significant effect on PAI-1, t-PA, and GFC levels. There were no significant changes in mean values of fibrinogen, factor VII, von Willebrand factor, protein C, fibrin <em>D</em>-<em>dimer</em>, and plasminogen between and within the three groups. We conclude that oral estrogen/progesterone replacement therapy may result in coagulation activation and increased fibrinolytic potential, whereas opposed transdermal estrogen appears without any substantial effects on hemostasis. Whereas these results may account for an increased risk of venous thromboembolism in users of oral postmenopausal estrogen, they emphasize the potential importance of the route of estrogen administration in prescribing hormone replacement therapy to postmenopausal women, especially to those at high risk of thrombotic disease.

Photosystem I with its full antenna complement (PSI-LHCI) has been prepared by mild detergent solubilization with octyl beta-<em>D</em>-glucopyranoside from maize thylakoids. A preliminary polypeptide analysis is presented. At room temperature, the steady-state fluorescence derives from an almost perfectly thermalized state, as demonstrated by a Stepanov analysis, in which about 90% of the excited states are associated with the red chlorophyll spectral forms absorbing above 700 nm. Equilibration is temperature-sensitive and is lost at T < <em>2</em>00 K. A careful analysis of fluorescence between 75 and <em>2</em>80 K clearly demonstrates the presence of at least three red chlorophyll spectral forms with emission maxima at 7<em>2</em>0, 730, and 74<em>2</em> nm, the absorption origin bands of which have been calculated at 714, 7<em>2</em>5, and 738 nm. On the basis of a minor deviation from thermal equilibration around 695 nm, it is suggested that at least 3-4 antenna chlorophylls, with an average absorption near 695 nm, are strongly coupled to P700. Thermodynamic analysis of absorption and fluorescence spectra indicates that the equilibrium, absorption-weighted excited state population of the P700 <em>dimer</em> is around 0.013 assuming that the low-energy exciton state possesses all the oscillator strength. The average free energy for excitation transfer from antenna to P700 is thus calculated to be -0.<em>2</em>6 kT at room temperature. This indicates that P700 is almost isoenergetic with its antenna at room temperature when the red forms are taken fully into account. From the calculated excited state population of P700, we estimate that the primary charge separation rate in PSI is 1-<em>2</em> ps-1.

OBJECTIVE

First, to examine and explain the relationship between religious service attendance and plasma Interleukin-6 (IL-6) levels, and second, to examine the relationship between religious attendance and other immune-system regulators and inflammatory substances.

METHODS

During the thir<em>d</em> in-person interview (199<em>2</em>) of the Establishment of Populations for Epi<em>d</em>emiologic Stu<em>d</em>ies of the El<em>d</em>erly (EPESE) project, Duke site, 1718 subjects age sixty-five or over ha<em>d</em> bloo<em>d</em> <em>d</em>rawn for analysis of immune regulators an<em>d</em> inflammatory factors, inclu<em>d</em>ing IL-6 measurements. IL-6 was examine<em>d</em> both as a continuous variable an<em>d</em> at a cutoff of 5 pg/ml. Information on atten<em>d</em>ance at religious services was available from the 199<em>2</em> interview an<em>d</em> two prior interviews (1986 an<em>d</em> 1989).

RESULTS

Religious atten<em>d</em>ance was inversely relate<em>d</em> to high IL-6 levels >> 5 pg/ml), but not to IL-6 measure<em>d</em> as a continuous variable. Bivariate analyses reveale<em>d</em> that high religious atten<em>d</em>ance in 1989 pre<em>d</em>icte<em>d</em> a lower proportion of subjects with high IL-6 in 199<em>2</em> (beta-.10, p = .01) High religious atten<em>d</em>ance in 199<em>2</em> also pre<em>d</em>icte<em>d</em> a lower proportion of subjects with high IL-6 levels in 199<em>2</em> (beta-.14, p = .0005). When age, sex, race, e<em>d</em>ucation, chronic illnesses, an<em>d</em> physical functioning were controlle<em>d</em>, 1989 religious atten<em>d</em>ance weakene<em>d</em> as a pre<em>d</em>ictor of high IL-6 (beta-.07, p = .10), but 199<em>2</em> religious atten<em>d</em>ance retaine<em>d</em> its effect (beta-.10, p = .0<em>2</em>). When religious atten<em>d</em>ers were compare<em>d</em> to non- atten<em>d</em>ers, they were only about one-half as likely to have IL-6 levels greater than 5 ng/ml (OR 0.58, 95% CI 0.40-0.84, p < .005). Religious atten<em>d</em>ance was also relate<em>d</em> to lower levels of the immune-inflammatory markers alpha-<em>2</em> globulin, fibrin <em>d</em>-<em>dimers</em>, polymorphonuclear leukocytes, an<em>d</em> lymphocytes. While controlling for covariates weakene<em>d</em> most of these relationships, a<em>d</em>justing analyses for <em>d</em>epression an<em>d</em> negative life events ha<em>d</em> little effect.

CONCLUSIONS

There is a weak relationship between religious attendance and high IL-6 levels that could not be explained by other covariates, depression, or negative life events. This finding provides some support for the hypothesis that older adults who frequently attend religious services have healthier immune systems, although mechanism of effect remains unknown.

OBJECTIVE

There are conflicting data regarding relationships of systemic biomarkers of inflammation, hemostasis, and homocysteine with diabetic retinopathy. We examined these relationships in the Multi-Ethnic Study of Atherosclerosis.

METHODS

A total of 9<em>2</em>1 participants with <em>d</em>iabetes were inclu<em>d</em>e<em>d</em>. Diabetic retinopathy was gra<em>d</em>e<em>d</em> from retinal photographs. We <em>d</em>efine<em>d</em> two outcomes: any <em>d</em>iabetic retinopathy an<em>d</em> vision-threatening <em>d</em>iabetic retinopathy (severe nonproliferative <em>d</em>iabetic retinopathy or worse). Systemic markers analyze<em>d</em> were C-reactive protein, homocysteine, fibrinogen, plasmin-alpha(<em>2</em>)-antiplasmin complex (PAP), interleukin-6, <em>d</em>-<em>dimer</em>, factor VIII, serum creatinine, an<em>d</em> urinary albumin-to-creatinine (UAC) ratio.

RESULTS

Prevalence of <em>d</em>iabetic retinopathy was 33.<em>2</em>% an<em>d</em> vision-threatening <em>d</em>iabetic retinopathy 7.1%. After a<em>d</em>justing for establishe<em>d</em> risk factors (<em>d</em>iabetes <em>d</em>uration, A1C, systolic bloo<em>d</em> pressure, waist-to-hip ratio, an<em>d</em> use of <em>d</em>iabetes me<em>d</em>ications), fibrinogen (o<em>d</em><em>d</em>s ratio 1.14 [95% CI 1.01-1.3<em>2</em>], P = 0.05) an<em>d</em> PAP (1.<em>2</em>5 [1.05-1.50], P = 0.01) were associate<em>d</em> with any <em>d</em>iabetic retinopathy, while PAP (1.54 [1.13-<em>2</em>.11], P = 0.007) an<em>d</em> homocysteine (1.57 [1.16-<em>2</em>.11], P = 0.003) were associate<em>d</em> with vision-threatening <em>d</em>iabetic retinopathy. Only PAP remaine<em>d</em> significant after a<em>d</em><em>d</em>itional a<em>d</em>justment for serum creatinine an<em>d</em> UAC ratio. Area un<em>d</em>er receiver-operator characteristic curve (AUROC) for <em>d</em>iabetic retinopathy was constructe<em>d</em> for establishe<em>d</em> an<em>d</em> novel risk factors. Establishe<em>d</em> risk factors accounte<em>d</em> for a 39.<em>2</em>% increase of the AUROC, whereas novel markers (fibrinogen, PAP, homocysteine, serum creatinine, an<em>d</em> UAC ratio) only accounte<em>d</em> for an a<em>d</em><em>d</em>itional <em>2</em>.<em>2</em>%.

CONCLUSIONS

There were few associations of novel markers of inflammation, hemostasis, and homocysteine with diabetic retinopathy after controlling for established risk factors. These data suggest that there is limited clinical use of these biomarkers for prediction of diabetic retinopathy.

BACKGROUND

Disseminated intravascular coagulation (DIC) with an antifibrinolytic phenotype is characterized by microvascular thrombosis leading to poor outcome at the late-stage of trauma. To test the hypothesis that DIC with a fibrinolytic phenotype at an early stage of trauma also contributes to a poor outcome due to severe bleeding, we conducted a retrospective, cohort study.

METHODS

The subjects included 314 consecutive severe trauma patients. A systematic review of medical records of the patients was conducted to provide the base line characteristics and DIC-related variables. The data of these variables were obtained at 4 time points within <em>2</em>4 hr after arrival to the emergency department (ED); Time Point 1, immediately after arrival to the ED to 4 hr after arrival; Time Point <em>2</em>, 4 to 8 hr after arrival; Time Point 3, 8 to 16 hr after arrival; Time Point 4, 16 to <em>2</em>4 hr after arrival.

RESULTS

Nonsurvivors (87.3%, 48/55) met the Japanese Association for Acute Medicine (JAAM) DIC criteria showing lower fibrinogen levels, a prolonged prothrombin time, and higher fibrin/fibrinogen degradation products (FDP) and D-dimer levels in comparison to those of the <em>2</em>89 survivors. The FDP/D-dimer ratio and lactate level were significantly higher in the nonsurvivors than those of the survivors. Lower fibrinogen levels and higher FDP/D-dimer ratio suggest fibrinogenolysis in DIC of the nonsurvivors. Furthermore a stepwise logistic regression analysis showed that the JAAM DIC score, levels of fibrinogen, FDP and lactate at Time Point 1 are independent predictors of death. Low levels of fibrinogen and high FDP but not D-dimer predict massive bleeding at an early stage of trauma. The optimal cutoff points for the prediction of death and massive bleeding were fibrinogen (1.90, 1.90 g/L) and FDP (35.<em>2</em>, 68.7 mg/L), respectively.

CONCLUSIONS

DIC with a fibrinolytic phenotype modified through fibrinogenolysis at an early phase of trauma contributes to poor prognosis due to massive bleeding. Tissue hypoperfusion may be involved in the pathogenesis of this type of DIC.

Brain-derived neurotrophic factor (B<em>D</em>NF) has potential for the treatment of human neurodegenerative diseases. However, the general lack of success of neurotrophic factors in clinical trials has led to the suggestion that low molecular weight neurotrophic drugs may be better agents for therapeutic use. Here we describe small, <em>dimer</em>ic peptides designed to mimic a pair of solvent-exposed loops important for the binding and activation of the B<em>D</em>NF receptor, trkB. The monomer components that make up the <em>dimers</em> were based on a monocyclic monomeric peptide mimic of a single loop of B<em>D</em>NF (loop <em>2</em>) that we had previously shown to be an inhibitor of B<em>D</em>NF-mediated neuronal survival (O'Leary, P. <em>D</em>., and Hughes, R. A. (1998) J. Neurochem. 70, 171<em>2</em>-17<em>2</em>1). Bicyclic <em>dimer</em>ic peptides behaved as partial agonists with respect to B<em>D</em>NF, promoting the survival of embryonic chick sensory neurons in culture. We reasoned that the potency and/or efficacy of these compounds might be improved by reducing the conformational flexibility about their <em>dimer</em>izing linker. Thus, we designed a highly conformationally constrained tricyclic <em>dimer</em>ic peptide and synthesized it using an efficient, quasi-one-pot approach. Although still a partial B<em>D</em>NF-like agonist, the tricyclic <em>dimer</em> was particularly potent in promoting neuronal survival in vitro (EC50 11 pm). The peptides described here, which are greatly reduced in size compared with the parent protein, could serve as useful lead compounds for the development of true neurotrophic drugs and indicate that the structure-based design approach could be used to obtain potent mimetics of other growth factors that <em>dimer</em>ize their receptors.

OBJECTIVE

To investigate the relationship between preoperative plasma D-dimer levels and extent of tumor involvement in operable breast cancer patients.

METHODS

A total of 140 preoperative plasma specimens were obtained from women scheduled to undergo diagnostic breast biopsies. Ninety-five patients in the initial group went on to undergo axillary lymph node dissection. Of the 140 patients from whom plasma samples were obtained, 102 were subsequently diagnosed with invasive breast carcinoma, nine were subsequently diagnosed with ductal carcinoma-in-situ, and 20 were subsequently diagnosed with benign breast disease. Plasma D-dimer levels were quantitated using a commercially available immunoassay kit (DIMERTEST; American Diagnostica, Greenwich, CT). The relationships between plasma D-dimer and other prognostic variables (tumor size, estrogen receptor, progesterone receptor, nuclear grade, histologic grade, lymphovascular invasion, and clinical stage grouping) were then examined using univariate and multivariate linear and logistic regression analyses.

RESULTS

Median plasma D-dimer levels were significantly higher in patients with invasive carcinoma than those patients with either benign breast disease or carcinoma-in-situ (P =.0001). A significant relationship existed between the presence of elevated D-dimer >> 100 ng/mL) and involved axillary lymph nodes (chi(2) test; P =.001). Elevated D-dimer levels predicted positive lymph node involvement in both univariate regression (P =.0035) and multivariate linear regression (P =.012) models. In addition, elevated D-dimer levels predicted the presence of lymphovascular invasion in univariate logistic regression (P =. 0025) and multivariate logistic regression analysis (P =.0053). Quantitative D-dimer levels were highly correlated with clinical stage grouping (analysis of variance test; P =.002).

CONCLUSIONS

Plasma D-dimer levels were markers of lymphovascular invasion, clinical stage, and lymph node involvement in operable breast cancer. This correlation suggests that detectable fibrin degradation, as measured by plasma D-dimer, is a clinically important marker for lymphovascular invasion and early tumor metastasis in operable breast cancer.

A ground-state <em>dimer</em> (denoted <em>D</em>(I)) exhibiting a strong absorption maximum at 477 nm (epsilon = 97 000 M(-1)cm(-1)) can form between adjacent BO<em>D</em>IPY groups attached to mutant forms of the protein, plasminogen activator inhibitor type 1 (PAI-1). No fluorescence from excited <em>D</em>(I) was detected. A locally high concentration of BO<em>D</em>IPY groups was also achieved by doping lipid phases (micelles, vesicles) with BO<em>D</em>IPY-labeled lipids. In addition to an absorption band located at about 480 nm, a new weak absorption band is also observed at ca. 570 nm. Both bands are ascribed to the formation of BO<em>D</em>IPY <em>dimers</em> of different conformation (<em>D</em>(I) and <em>D</em>(II)). Contrary to <em>D</em>(I) in PAI-1, the <em>D</em>(II) aggregates absorbing at 570 nm are emitting light observed as a broad band centered at about 630 nm. The integrated absorption band of <em>D</em>(I) is about twice that of the monomer, which is compatible with exciton coupling within a <em>dimer</em>. The Förster radius of electronic energy transfer between a BO<em>D</em>IPY excited monomer and the ground-state <em>dimer</em> (<em>D</em>(I)()) is 57 +/- <em>2</em> A. A simple model of exciton coupling suggests that in <em>D</em>(I) two BO<em>D</em>IPY groups are stacked on top of each other in a sandwich-like configuration with parallel electronic transition dipoles. For <em>D</em>(II) the model suggests that the S(0) ->> S(1) transition dipoles are colinear. An explanation for the previously reported (J. Am. Chem. Soc. 1994, 116, 7801) exceptional light spectroscopic properties of BO<em>D</em>IPY is also presented. These are ascribed to the extraordinary electric properties of the BO<em>D</em>IPY chromophore. First, changes of the permanent electric dipole moment (<em>Delta</em>(mu) approximately -0.05 <em>D</em>) and polarizability (-<em>2</em>6 x 10(-40) C m(<em>2</em>) V(-1)) between the ground and the first excited states are small. Second, the S(0) <->> S(1) electronic transition dipole moments are perpendicular to <em>Delta</em>(mu).

<strong class="sub-title">Background:</strong> Coronavirus disease-<em>2</em>019 (COVI<em>D</em>-19), a respiratory disease has been associated with ischemic complications, coagulation disorders, and an endotheliitis.

Objectives: To explore endothelial damage and activation-related biomarkers in COVID-19 patients with criteria of hospitalization for referral to intensive care unit (ICU) and/or respiratory worsening.

Methods: Analysis of endothelial and angiogenic soluble markers in plasma from patients at admission.

<strong class="sub-title">Results:</strong> Study enrolled 40 consecutive COVI<em>D</em>-19 patients admitted to emergency department that fulfilled criteria for hospitalization. Half of them were admitted in conventional wards without any ICU transfer during hospitalization; whereas the <em>2</em>0 others were directly transferred to ICU. Patients transferred in ICU were more likely to have lymphopenia, decreased SpO<em>2</em> and increased <em>D</em>-<em>dimer</em>, CRP and creatinine levels. In those patients, soluble E-selectin and angiopoietin-<em>2</em> were significantly increased (p value at 0.009 and 0.003, respectively). Increase in SELE gene expression (gene coding for E-selectin protein) was confirmed in an independent cohort of 3<em>2</em> patients using a whole blood gene expression profile analysis. In plasma, we found a strong association between angiopoetin-<em>2</em> and CRP, creatinine and <em>D</em>-<em>dimers</em> (with p value at 0.001, 0.001 and 0.003, respectively). ROC curve analysis identified an Angiopoietin-<em>2</em> cut-off of 5000 pg/mL as the best predictor for ICU outcome (Se = 80.1%, Sp = 70%, PPV = 7<em>2</em>.7%, NPV = 77%), further confirmed in multivariate analysis after adjustment for creatinine, CRP or <em>D</em>-<em>dimers</em>.

<strong class="sub-title">Conclusion:</strong> Angiopoietin-<em>2</em> is a relevant predictive factor for ICU direct admission in COVI<em>D</em>-19 patients. This result showing an endothelial activation reinforces the hypothesis of a COVI<em>D</em>-19-associated microvascular dysfunction.

<strong class="sub-title">Keywords:</strong> Angiogenesis; Angiopoietin-<em>2</em>; Biomarker; COVI<em>D</em>-19; E-selectin; Endothelial.