This study was performed to determine the accuracy of <em>D</em>-<em>Dimer</em> fibrin derivatives, thrombin-antithrombin III (TAT) complexes and prothrombin fragments 1 + <em>2</em> (F 1 + <em>2</em>) determinations for the diagnosis of deep vein thrombosis (<em>D</em>VT). One hundred and sixteen consecutive patients referred to the angiology unit of our hospital for a clinically suspected <em>D</em>VT were investigated. They were submitted to mercury strain gauge plethysmography and to ultrasonic duplex scanning examination; in cases of inconclusive results or of proximal <em>D</em>VT (n = 35), an ascending phlebography was performed. After these investigations were completed, the diagnosis of <em>D</em>VT was confirmed in 34 and excluded in 8<em>2</em>. One half of the patients were already under anticoagulant therapy at the time of investigation. The 3 biological markers were assayed using commercially available ELISA techniques and the <em>D</em>-<em>Dimer</em> was also assayed with a fast latex method. The normal distribution of these markers was established in 40 healthy blood donors. The most accurate assay for the diagnosis of <em>D</em>VT was the <em>D</em>-<em>Dimer</em> ELISA which had both a high sensitivity (94%) and a high negative predictive value (95%). The <em>D</em>-<em>Dimer</em> latex, TAT complexes and F 1 + <em>2</em> were far less sensitive and provided negative predictive values which ranged between 78 and 85%. In spite of positive and significant correlations between the levels of the 3 markers, their association did not improve their overall accuracy for detecting <em>D</em>VT. Therefore, with the exception of the <em>D</em>-<em>Dimer</em> ELISA, these markers were of little value for the diagnosis of <em>D</em>VT in this specific population.

The transcriptional activator RamA is involved in multidrug resistance (M<em>D</em>R) by increasing expression of the AcrAB-TolC RN<em>D</em>-type efflux system in several pathogenic Enterobacteriaceae. In Salmonella enterica serovar Typhimurium (S. Typhimurium), ramA expression is negatively regulated at the local level by RamR, a transcriptional repressor of the TetR family. We here studied the <em>D</em>NA-binding activity of the RamR repressor with the ramA promoter (P(ramA)). As determined by high-resolution footprinting, the <em>2</em>8-bp-long RamR binding site covers essential features of P(ramA), including the -10 conserved region, the transcriptional start site of ramA, and two 7-bp inverted repeats. Based on the RamR footprint and on electrophoretic mobility shift assays (EMSAs), we propose that RamR interacts with P(ramA) as a <em>dimer</em> of <em>dimers</em>, in a fashion that is structurally similar to the QacR-<em>D</em>NA binding model. Surface plasmon resonance (SPR) measurements indicated that RamR has a 3-fold-lower affinity (K(<em>D</em>) [equilibrium dissociation constant] = 191 nM) for the <em>2</em>-bp-deleted P(ramA) of an M<em>D</em>R S. Typhimurium clinical isolate than for the wild-type P(ramA) (K(<em>D</em>) = 66 nM). These results confirm the direct regulatory role of RamR in the repression of ramA transcription and precisely define how an alteration of its binding site can give rise to an M<em>D</em>R phenotype.

Ovarian cancer cells appear to be capable of both thrombin formation and induction of fibrin degradation which may be essential prerequisites for the development of deep vein thrombosis (<em>D</em>VT) as well as the spread of malignancy. To study further this coagulation-cancer interaction in 60 patients with untreated ovarian cancer of FIGO stage I-IV the incidence of <em>D</em>VT was recorded pre-operatively, post-operatively on day 1, 3, 5, 7, 10, before each of six cycles of Cisplatinum/ Epirubicin/Cyclophosphamide chemotherapy, during follow-up and in the post-operative period of second look surgery. In addition, blood coagulation tests results were determined prospectively. Two patients were excluded from these calculations due to previous <em>D</em>VT 5 to 6 weeks before the diagnosis of ovarian cancer but all patients were eligible for surgery and randomized to receive either daily low molecular weight heparin (LMWH) (n = <em>2</em>8) or unfractionated heparin (UFH) (n = 3<em>2</em>) for perioperative thrombosis prophylaxis until the 7th post-operative day. According to the FIGO stage, patients were equally distributed in the <em>2</em> heparin treatment groups. The predictive value of pre-operative coagulation test results, clinical parameters, and type of heparin used were tested in univariate and multivariate analysis for development of post-operative <em>D</em>VT and overall patients survival. Impedance plethysmography for <em>D</em>VT screening was used. The presence of <em>D</em>VT was then confirmed by phlebography. Only <em>D</em>-<em>dimer</em> and fibrinogen levels were correlated significantly with the FIGO stage while antithrombin, protein C, and plasminogen activator inhibitor activity were not. The incidence of <em>D</em>VT was 6.7% (4/60) up to the 7th and 8.3% (5/60) between the 8th and <em>2</em>9th post-operative day. <em>D</em>VT occurred in 10.6% (5/47) during chemotherapy. Pre-operative coagulation test results, the type of heparin used, and clinical parameters were not significant risk factors for post-operative <em>D</em>VT development in univariate analysis. The <em>D</em>-<em>dimer</em> and fibrinogen levels were significant risk factors for reduced overall survival in univariate analysis but only the FIGO stage was an independent predictor (in multivariate analysis). After a median follow up of <em>2</em>6.5 months (min. 8 months, max. 41 months), <em>2</em>1.4% of LMWH treated and 37.5% of UFH-treated patients died of cancer (p = 0.<em>2</em>6). Pre-operative test results were neither predictive for <em>D</em>VT nor the outcome of cancer but patients showed an improved though not statistically significant overall survival after LMWH treatment.

The env gene of Friend spleen focus-forming virus (SFFV) encodes a membrane glycoprotein (gp55) that is inefficiently (3 to 5%) processed from the rough endoplasmic reticulum to form a larger <em>dimer</em>ic plasma membrane derivative (gp55p). Moreover, the SFFV env glycoprotein associates with erythropoietin receptors (EpoR) to cause proliferation of infected erythroblasts [J.-P. Li, A. <em>D</em>. <em>D</em>'Andrea, H. F. Lodish, and <em>D</em>. Baltimore, Nature (London) 343:76<em>2</em>-764, 1990]. Interestingly, the mitogenic effect of SFFV is blocked in mice homozygous for the Fv-<em>2</em>r resistance gene, but mutant SFFVs can overcome this resistance. Recent evidence suggested that these mutants contain partial env deletions that truncate the membrane-proximal extracellular domain of the encoded glycoproteins (M. H. Majumdar, C.-L. Cho, M. T. Fox, K. L. Eckner, S. Kozak, <em>D</em>. Kabat, and R. W. Geib, J. Virol. 66:365<em>2</em>-3660, 199<em>2</em>). Mutant BB6, which encodes a gp4<em>2</em> glycoprotein that has a large deletion in this domain, causes erythroblastosis in <em>D</em>BA/<em>2</em> (Fv-<em>2</em>s) as well as in congenic <em>D</em><em>2</em>.R (Fv-<em>2</em>r) mice. Analogous to gp55, gp4<em>2</em> is processed inefficiently as a disulfide-bonded <em>dimer</em> to form cell surface gp4<em>2</em>p. Retroviral vectors with SFFV and BB6 env genes have no effect on interleukin 3-dependent BaF3 hematopoietic cells, but they cause growth factor independency of BaF3/EpoR cells, a derivative that contains recombinant EpoR. After binding 1<em>2</em>5I-Epo to surface EpoR on these factor-independent cells and adding the covalent cross-linking reagent disuccinimidyl suberate, complexes that had immunological properties and sizes demonstrating that they consisted of 1<em>2</em>5I-Epo-gp55p and 1<em>2</em>5I-Epo-gp4<em>2</em>p were isolated from cell lysates. Contrary to a previous report, SFFV or BB6 env glycoproteins did not promiscuously activate other members of the EpoR superfamily. Although the related env glycoproteins encoded by dualtropic murine leukemia viruses formed detectable complexes with EpoR, strong mitogenic signalling did not ensue. Our results indicate that the SFFV and BB6 env glycoproteins specifically activate EpoR; they help to define the glycoprotein properties important for its functions; and they strongly suggest that the Fv-<em>2</em> leukemia control gene encodes an EpoR-associated regulatory factor.

Because abnormalities in hemostatic factors may in part account for the risk of stroke and thromboembolism in atrial fibrillation, we measured plasma fibrinogen and fibrin <em>D</em>-<em>dimer</em> levels in 33 patients (18 men and 15 women, mean age 60.8 +/- 1.4 years [mean +/- SEM]) with paroxysmal atrial fibrillation (PAF) and 1<em>2</em> patients (3 men and 9 women, mean age 51.0 +/- 4.<em>2</em> years) with paroxysmal supraventricular tachycardia (PSVT). Levels of these markers were compared to levels in (1) patients with chronic atrial fibrillation; (<em>2</em>) hospital controls (age-matched [age +/- 5 years] and sex-matched patients in sinus rhythm with coronary artery disease and normal left ventricular function); and (3) healthy population controls in sinus rhythm. Patients with PAF had intermediate levels of median plasma fibrinogen and fibrin <em>D</em>-<em>dimer</em> when compared to patients with chronic atrial fibrillation and controls in sinus rhythm (both p < 0.001). There was no relation with atrial size or ventricular function on echocardiography. Patients with PSVT had plasma fibrinogen and fibrin <em>D</em>-<em>dimer</em> levels that were similar to the median levels of the population controls, suggesting that there was no excess in thrombogenesis. These findings are consistent with the hypothesis that atrial fibrillation is related to the increases in plasma fibrinogen and fibrin <em>D</em>-<em>dimer</em> levels. Patients with PAF have intermediate levels of these markers, a finding that is consistent with the intermediate risk of thromboembolism in such patients.

A sequence variation in the 3'-untranslated region of the prothrombin (PT) gene (<em>2</em>0<em>2</em>10 G->>A) was recently claimed to be associated with elevated plasma prothrombin levels and an increased risk for venous and arterial thrombosis. We examined the prevalence of the <em>2</em>0<em>2</em>10 A allele in the prothrombin gene in 400 healthy controls and in <em>2</em>63 patients with proven premature atherosclerotic disease. In addition, we measured prothrombin, prothrombin fragment 1 + <em>2</em>, thrombin-antithrombin (TAT) complex and <em>D</em>-<em>dimer</em> levels in plasma from carrier and non-carrier patients. The frequency of the variant allele was 1% in the control subjects and <em>2</em>.7% in the patient group, yielding a relative risk (RR) for the <em>2</em>0<em>2</em>10 A allele of <em>2</em>.7 (95% CI 0.8-9.4). All heterozygotes in the patient group were found to have had a myocardial infarction (MI), yielding a RR for MI of 4.<em>2</em> (95% CI 1.<em>2</em>-14.6). Plasma prothrombin levels in carriers (1<em>2</em>6+/-10) were higher than in non-carriers (103+/-1, P=0.0<em>2</em>). The levels of TAT complexes (16+/-9 v 6+/-1 microg/ml, P=0.0<em>2</em>) as well as of prothrombin fragment 1 + <em>2</em> (1.5+/-0.3 v 1.0+/-0.1 nmol/l, P=0.0<em>2</em>) were also elevated in carriers of the mutation. Our findings suggest that the <em>2</em>0<em>2</em>10 G->>A mutation in the prothrombin gene is a genetic risk factor for MI. In addition, our data provide evidence for an association of the mutation with excessive thrombin generation, which may contribute to the understanding of its role in venous and arterial disease.

We have clone<em>d</em> the M an<em>d</em> S genes of the restriction-mo<em>d</em>ification (R-M) system Ah<em>d</em>I an<em>d</em> have purifie<em>d</em> the resulting methyltransferase to homogeneity. M.Ah<em>d</em>I is foun<em>d</em> to form a 170 kDa tetrameric enzyme having a subunit stoichiometry M<em>2</em>S<em>2</em> (where the M an<em>d</em> S subunits are responsible for methylation an<em>d</em> DNA sequence specificity, respectively). Se<em>d</em>imentation equilibrium experiments show that the tetrameric enzyme <em>d</em>issociates to form a hetero<em>d</em>imer at low concentration, with K(<em>d</em>) approximately <em>2</em> microM. The intact (tetrameric) enzyme bin<em>d</em>s specifically to a 30 bp DNA <em>d</em>uplex containing the Ah<em>d</em>I recognition sequence GACN5GTC with high affinity (K(<em>d</em>) approximately 50 nM), but at low enzyme concentration the DNA bin<em>d</em>ing activity is governe<em>d</em> by the <em>d</em>issociation of the tetramer into <em>dimers</em>, lea<em>d</em>ing to a sigmoi<em>d</em>al DNA bin<em>d</em>ing curve. In contrast, only non-specific bin<em>d</em>ing is observe<em>d</em> if the <em>d</em>uplex lacks the recognition sequence. Methylation activity of the purifie<em>d</em> enzyme was assesse<em>d</em> by its ability to prevent restriction by the cognate en<em>d</em>onuclease. The subunit structure of the M.Ah<em>d</em>I methyltransferase resembles that of type I MTases, in contrast to the R.Ah<em>d</em>I en<em>d</em>onuclease which is typical of type II systems. Ah<em>d</em>I appears to be a novel R-M system with properties interme<em>d</em>iate between simple type II systems an<em>d</em> more complex type I systems, an<em>d</em> may represent an interme<em>d</em>iate in the evolution of R-M systems.

BACKGROUND

Growing evidence indicates that ambient air pollution is associated with exacerbation of chronic diseases like chronic pulmonary disease. A prospective panel study was conducted to investigate short-term changes of blood markers of inflammation and coagulation in response to daily changes in air pollution in Erfurt, Germany. 1<em>2</em> clinical visits were scheduled and blood parameters were measured in 38 male patients with chronic pulmonary disease during winter <em>2</em>001/<em>2</em>00<em>2</em>. Additive mixed models with random patient intercept were applied, adjusting for trend, weekday, and meteorological parameters. Hourly data on ultrafine particles (UFP, 0.01-0.1 mum), accumulation mode particles (ACP, 0.1-1.0 mum), PM10 (particulate matter <10 mum in diameter), elemental (EC) and organic carbon (OC), gaseous pollutants (nitrogen monoxide [NO], nitrogen dioxide [NO<em>2</em>], carbon monoxide [CO], and sulphur dioxide [SO<em>2</em>]) were collected at a central monitoring site and meteorological data were received from an official network. For each person and visit the individual <em>2</em>4-hour average of pollutants immediately preceding the blood withdrawal (lag 0) up to day 5 (lag1-4) and 5-day running means were calculated.

RESULTS

Increased levels of fibrinogen were observed for an increase in one interquartile range of UFP, PM10, EC, OC, CO, and NO revealing the strongest effect for lag 3. E-selectin increased in association with ACP and PM10 with a delay of one day. The ACP effect was also seen with the 5-day-mean. The pattern found for <em>D</em>-<em>dimer</em> was inconsistent. Prothrombin fragment 1+<em>2</em> decreased with lag 4 consistently for all particulate pollutants. Von Willebrand factor antigen (vWF) showed a consistent decrease in association with almost all air pollutants with all lags except for lag 0. No associations were found for C-reactive protein, soluble intercellular adhesion molecule 1, serum amyloid A and factor VII.

CONCLUSIONS

These results suggest that elevated concentrations of air pollution are associated with changes in some blood markers of inflammation and coagulation in patients with chronic pulmonary disease. The clinical implications of these findings need further investigation.

The electronic structure of the cation radical of the primary electron donor was investigated in genetically modified reaction centers of Rhodobacter sphaeroides. The site-directed mutations were designed to add or remove hydrogen bonds between the conjugated carbonyl groups of the primary donor, a bacteriochlorophyll <em>dimer</em>, and histidine residues of the protein and were introduced at the symmetry-related sites L168 His->>Phe, HF(L168), and M197 Phe->>His, FH(M197), near the <em>2</em>-acetyl groups of the <em>dimer</em> and at sites M160 Leu->>His, LH(M160), and L131 Leu->>His, LH(L131), in the vicinity of the 9-keto carbonyls of the <em>dimer</em>. The single mutants and a complete set of double mutants were studied using EPR, EN<em>D</em>OR, and TRIPLE resonance spectroscopy. The changes in the hydrogen bond situation of the primary donor were accompanied by changes in the <em>dimer</em> oxidation midpoint potential, ranging from 410 to 710 mV in the investigated mutants [Lin, X., Murchison, H. A., Nagarajan, V., Parson, W. W., Williams, J. C. & Allen, J. P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10<em>2</em>65-10<em>2</em>69]. It was found that the addition or removal of a hydrogen bond causes large shifts of the spin density between the two halves of the <em>dimer</em>. Measurements on double mutants showed that the unpaired electron can be gradually shifted from a localization on the L-half of the <em>dimer</em> to a localization on the M-half, depending on the hydrogen bond situation. As a control, the effects of the different hydrogen bonds on P.+ in the mutant HL(M<em>2</em>0<em>2</em>), which contains a BChlL-BPheM hetero<em>dimer</em> as the primary donor with localized spin on the BChl aL [Bylina, E. J., & Youvan, <em>D</em>. C. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7<em>2</em><em>2</em>6-7<em>2</em>30; Schenck, C. C., Gaul, <em>D</em>., Steffen M., Boxer S. G., Mc<em>D</em>owell L., Kirmaier C., & Holten <em>D</em>. (1990) in Reaction Centers of Photosynthetic Bacteria (Michel-Beyerle M. E., Ed.) pp <em>2</em><em>2</em>9-<em>2</em>38, Springer, Berlin] were studied. In this mutant only small local changes of the spin densities (< or = 10%) in the vicinity of the hydrogen bonds were observed. The effects of the introduced hydrogen bonds on the spin density distribution of the <em>dimer</em> in the mutants are discussed in terms of different orbital energies of the two BChl a moieties which are directly influenced by hydrogen bond formation. The observed changes of the spin density distribution for the double mutants are additive with respect to the single mutations.(ABSTRACT TRUNCATE<em>D</em> AT 400 WOR<em>D</em>S)

We report the synthesis of a series of hydrophilic butadiyne-linked conjugated zinc porphyrin <em>dimers</em>, designed as photodynamic therapy (PDT) agents. These porphyrin <em>dimers</em> exhibit exceptionally high two-photon absorption cross sections (<em>delta</em>(max) approximately 8,000-17,000 GM) and red-shifted linear absorption spectra (lambda(max) approximately 700-800 nm) making them ideal candidates for one-photon and two-photon excited photodynamic therapy. Four polar triethyleneglycol substituents are positioned along the sides of each <em>dimer</em>, but, on their own, these TEG chains do not confer sufficient solubility in aqueous physiological media for reproducible delivery into live cells. Charged cationic (methylpyridinium and trimethylammonium) and anionic (sulfonate and carboxylate) substituents have been appended to the meso-positions of porphyrin <em>dimers</em> using three synthetic strategies: 1) Suzuki coupling, <em>2</em>) Sonogashira coupling, and 3) nucleophilic Senge arylation. Approaches 1 and 3 both allow attachment of aromatic substituents directly to the meso-positions of porphyrins. Approach <em>2</em> provides a route to hydrophilic porphyrin <em>dimers</em> with an ethyne link between the porphyrin and the polar aromatic substituent. The palladium-catalysed approaches 1 and <em>2</em> allow the synthesis of a broader range of meso-capped porphyrins, as many aryl halides are available. However the synthesis of the intermediate required for these routes necessitates a statistical reaction step, which decreases the overall yield. On the other hand, Senge-arylation provides highly regioselective nucleophilic aromatic substitution, and offers higher overall yield than the other routes. All these charged <em>dimers</em> exhibit good solubility in polar solvents (e.g. methanol) and aqueous solvent mixtures (aqueous DMSO or DMF).

The phiX174 external scaffolding protein <em>D</em> mediates the assembly of coat protein pentamers into procapsids. There are four external scaffolding subunits per coat protein. Organized as pairs of asymmetric <em>dimers</em>, the arrangement is unrelated to quasi-equivalence. The external scaffolding protein contains seven alpha-helices. The protein's core, alpha-helices <em>2</em> to 6, mediates the vast majority of intra- and interdimer contacts and is strongly conserved in all Microviridae (canonical members are phiX174, G4, and alpha3) external scaffolding proteins. On the other hand, the primary sequences of the first alpha-helices have diverged. The results of previous studies with alpha3/phiX174 chimeric external scaffolding proteins suggest that alpha-helix 1 may act as a substrate specificity domain, mediating the initial coat scaffolding protein recognition in a species-specific manner. However, the low sequence conservation between the two phages impeded genetic analyses. In an effort to elucidate a more mechanistic model, chimeric external scaffolding proteins were constructed between the more closely related phages G4 and phiX174. The results of biochemical analyses indicate that the chimeric external scaffolding protein inhibits two morphogenetic steps: the initiation of procapsid formation and <em>D</em>NA packaging. phiX174 mutants that can efficiently utilize the chimeric protein were isolated and characterized. The substitutions appear to suppress both morphogenetic defects and are located in threefold-related coat protein sequences that most likely form the pores in the viral procapsid. These results identify coat-external scaffolding domains needed to initiate procapsid formation and provide more evidence, albeit indirect, that the pores are the site of <em>D</em>NA entry during the packaging reaction.

The three-dimensional structure of the complex between methyl alpha-<em>D</em>-mannopyranoside and concanavalin A has been refined at <em>2</em>.0 A resolution. <em>D</em>iffraction data were recorded from a single crystal (space group P<em>2</em>(1)<em>2</em>(1)<em>2</em>(1), a = 1<em>2</em>3.7, b = 1<em>2</em>8.6, c = 67.<em>2</em> A) using synchrotron radiation at a wavelength of 1.488 A. The final model has good geometry and an R factor of 19.9% for 58 871 reflections (8<em>2</em>% complete), within the resolution limits of 8 to <em>2</em> A, with F>> 1.0sigma(F). The asymmetric unit contains four protein subunits arranged as a <em>dimer</em> of <em>dimers</em> with approximate <em>2</em><em>2</em><em>2</em> point symmetry. Each monomer binds one saccharide molecule. Each sugar is bound to the protein by hydrogen bonds and van der Waals contacts. Although the four subunits are not crystallographically equivalent, the protein-saccharide interactions are nearly identical in each of the four binding sites. The differences that do occur between the four sites are in the structure of the water network which surrounds each saccharide; these networks are involved in crystal packing. The structure of the complex is compared with a refined saccharide-free concanavalin A structure. The saccharide-free structure is composed of crystallographically identical subunits, again assembled as a <em>dimer</em> of <em>dimers</em>, but with exact <em>2</em><em>2</em><em>2</em> symmetry. In the saccharide complex the tetramer association is different in that the monomers tend to separate resulting in fewer intersubunit interactions. The average temperature factor of the mannoside complex is considerably higher than that of the saccharide-free protein. The binding site in the saccharide-free structure is occupied by three ordered water molecules and the side chain of Asp71 from a neighbouring molecule in the crystal. These occupy positions similar to those of the four saccharide hydroxyls which are hydrogen bonded to the site. Superposition of the saccharide-binding site from each structure shows that the major changes on binding involve expulsion of these ordered solvents and the reorientation of the side chain of Tyrl00. Overall the surface accessibility of the saccharide decreases from 370 to 100 A(<em>2</em>) when it binds to the protein. This work builds upon the earlier studies of <em>D</em>erewenda et al. [<em>D</em>erewenda, Yariv, Helliwell, Kalb (Gilboa), <em>D</em>odson, Papiz, Wan & Campbell (1989). EMBO J. 8, <em>2</em>198-<em>2</em>193] at <em>2</em>.9 A resolution, which was the first detailed study of lectin-saccharide interactions.

OBJECTIVE

In lung cancer, vascular endothelial growth factor (VEGF) is an important cytokine and is correlated with tumor vessel density, malignant pleural effusions, and coagulation-fibrinolysis factors in vitro. We investigated the correlation between serum VEGF level and stage progression in lung cancer to study the predicted value of VEGF level. We also studied whether coagulation-fibrinolysis factors and PaO(<em>2</em>) levels, which are also important factors for the prediction of the clinical course, are correlated with VEGF.

METHODS

Forty-nine patients with lung cancer were investigated prospectively. VEGF levels of sera and malignant effusions, and plasma concentrations of coagulation-fibrinolysis factors were measured by enzyme-linked immunosorbent assay. We measured PaO(<em>2</em>) levels in all patients at rest.

RESULTS

Serum levels of VEGF were increased significantly according to stage progression. Additionally, plasma concentrations of D dimer, thrombin-antithrombin complex (TAT), and tissue plasminogen activator/plasminogen activator inhibitor type I complex were elevated significantly according to stage progression. The serum VEGF level had a significant positive correlation with the TAT and D dimer levels. Serum VEGF levels had a significant negative correlation with PaO(<em>2</em>) levels. The incidence of cerebral vascular disorder was significantly higher in the patients with systemic hypoxemia than in those without (p<0.05). Mean VEGF levels in malignant effusions in eight patients (five with pleural effusions, two with pericardial effusions, and one with both) were extremely high, especially in pericardial effusions ([mean +/- SD] pleural effusions, 531.9+/-<em>2</em>85.4 pg/mL; pericardial effusion, 3,071.6+/-81.3 pg/mL).

CONCLUSIONS

We predict that in lung cancer, VEGF production and the abnormality of the coagulation-fibrinolysis system differ depending on the stage of progression of disease. Serum VEGF levels would be affected by PaO(<em>2</em>) levels in lung cancer.

The molecular structure of ammonium <em>d</em>eoxycyti<em>d</em>ylyl-(3'-5')-<em>d</em>eoxyguanosine, crystallize<em>d</em> from aqueous acetone near pH 4, was <em>d</em>etermine<em>d</em> for X-ray <em>d</em>iffraction <em>d</em>ata. The crystals were tetragonal, space group P43<em>2</em>1<em>2</em> with a = b = 11.078 (1) A an<em>d</em> c = 45.8<em>2</em>6 (4) A. The structure was solve<em>d</em> by tangent expansion of phases base<em>d</em> on a <em>d</em>erive<em>d</em> phosphorus position an<em>d</em> refine<em>d</em> to R = 0.060 by full matrix least squares. Molecules relate<em>d</em> by a <em>2</em>-fol<em>d</em> symmetry axis are connecte<em>d</em> by hy<em>d</em>rogen bon<em>d</em>s between the bases an<em>d</em> form parallel right-han<em>d</em>e<em>d</em> <em>d</em>uplexes. Pairs of cytosines share a proton at N(3) an<em>d</em> are joine<em>d</em> by three hy<em>d</em>rogen bon<em>d</em>s: N(4)-H...O(<em>2</em>)...H-N(4), an<em>d</em> N(3)-H...N(3). Guanines are joine<em>d</em> by two hy<em>d</em>rogen bon<em>d</em>s: N(<em>2</em>)-H...N(3) an<em>d</em> N(3)...H-N(<em>2</em>). Base-stacking interactions within the <em>d</em>uplex are weak with the cytosine an<em>d</em> guanine ring planes incline<em>d</em> at <em>2</em>4 <em>d</em>egrees to each other in each monomer. Despite the unusual arrangement of the molecules, the sugar phosphate backbone has the g-g- conformation normally associate<em>d</em> with right-han<em>d</em>e<em>d</em> <em>d</em>ouble helical structures. Conformational parameters of the nucleosi<em>d</em>es are also typical with both sugars C(<em>2</em>')-en<em>d</em>o an<em>d</em> glycosi<em>d</em>ic torsion angles 55 <em>d</em>egrees for cyti<em>d</em>ine an<em>d</em> 94 <em>d</em>egrees for guanosine. The bon<em>d</em>ing geometry of the bases is influence<em>d</em> by hy<em>d</em>rogen bon<em>d</em>ing an<em>d</em> charge-transfer networks in the crystal lattice. The solvent molecules interact with the <em>dimer</em> in three fuse<em>d</em> circular hy<em>d</em>rogen bon<em>d</em>ing <em>d</em>omains with a single <em>d</em>isor<em>d</em>ere<em>d</em> ammonium cation per <em>d</em>(CpG) <em>dimer</em>. Parallels with the formation of self base pairs an<em>d</em> their implications in molecular biology are <em>d</em>iscusse<em>d</em>.

3-Deoxy-<em>d</em>-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway, was purifie<em>d</em> to electrophoretic homogeneity from tubers of Solanum tuberosum L. cv Superior. The enzyme is a <em>dimer</em> with a native molecular weight of 110,000. The enzyme appears to be hysteretic. The enzyme activity is stimulate<em>d</em> by Mn(<em>2</em>+) an<em>d</em> l-tryptophan. Chromatofocusing resolve<em>d</em> two forms of the enzyme with isoelectric points of 7.8 an<em>d</em> 8.4, respectively. The enzyme closely resembles an analogous activity previously isolate<em>d</em> from roots of Daucus carota (JA Suzich, JFD Dean, KM Herrmann 1985 Plant Physiol 79: 765-770).

We have refine<em>d</em> the initial <em>d</em>ocking mo<em>d</em>el of the Mg(II)-co-or<em>d</em>inate<em>d</em> chromomycin-<em>d</em>(A<em>2</em>G<em>2</em>C<em>2</em>T<em>2</em>) complex (<em>2</em> <em>d</em>rug equivalents per <em>d</em>uplex) by a complete relaxation matrix analysis simulation of the two-<em>d</em>imensional nuclear Overhauser effect (NOESY) spectrum of the complex in <em>2</em>H<em>2</em>O solution. This relaxation matrix refine<em>d</em> structure of the complex exhibits the following characteristics. (1) We observe an unwoun<em>d</em> an<em>d</em> elongate<em>d</em> <em>d</em>uplex that exhibits characteristics <em>d</em>istinct from the A an<em>d</em> B-<em>D</em>NA family of helices at the central (G-G-C-C).(G-G-C-C) chromomycin <em>dimer</em> bin<em>d</em>ing an<em>d</em> flanking sites. On the other han<em>d</em> sugar puckers, glycosi<em>d</em>ic torsion angles, <em>d</em>isplacement of the base-pairs from the helix axis an<em>d</em> the minor groove wi<em>d</em>th for this central tetranucleoti<em>d</em>e segment all fall within the A-family of helical parameters. (<em>2</em>) The chromomycin monomers are aligne<em>d</em> in a hea<em>d</em>-to-tail orientation in the Mg(II)-co-or<em>d</em>inate<em>d</em> <em>dimer</em> in the complex. The chromophores are aligne<em>d</em> with a slight tilt relative to each other an<em>d</em> make an angle of 75 <em>d</em>egrees between their planes. The C-<em>D</em>-E trisacchari<em>d</em>e segments from in<em>d</em>ivi<em>d</em>ual monomers a<em>d</em>opt an exten<em>d</em>e<em>d</em> conformation that projects in opposite <em>d</em>irections in the <em>dimer</em>. The <em>d</em>ivalent metal cation is co-or<em>d</em>inate<em>d</em> to the O(1) carbonyl an<em>d</em> O(9) enolate atoms of the chromophores an<em>d</em> aligns them such that the O(9)-Mg-O(9) angle is 170 <em>d</em>egrees while all other O-Mg-O angles are in the 95(+/- 15)<em>d</em>egrees range. (3) The sequence specificity of the chromomycin <em>dimer</em> for the wi<em>d</em>ene<em>d</em> an<em>d</em> shallower (G3-G4-C5-C6).(G3-G4-C5-C6) minor groove bin<em>d</em>ing site is associate<em>d</em> with intermolecular hy<em>d</em>rogen bon<em>d</em>s forme<em>d</em> between the OH group at C(8) of the chromophore an<em>d</em> the minor groove NH<em>2</em> group at position <em>2</em> an<em>d</em> N(3) groups of G4 an<em>d</em> between the O(1) oxygen of the E-sugar an<em>d</em> the minor groove NH<em>2</em> group at position <em>2</em> of G3 in the complex. (4) A<em>d</em><em>d</em>itional intermolecular interactions are primarily van <em>d</em>er Waals contacts between anomeric an<em>d</em> a<em>d</em>jacent CH<em>2</em> protons on each sugar in the C-<em>D</em>-E trisacchari<em>d</em>e segments of the chromomycin <em>dimer</em> an<em>d</em> the minor groove surface of the <em>D</em>NA. These results provi<em>d</em>e insights into the in<em>d</em>uce<em>d</em> conformational transitions require<em>d</em> to generate a complementary match between the <em>d</em>rug <em>dimer</em> an<em>d</em> its <em>D</em>NA bin<em>d</em>ing site on complex formation.

The equilibrium unfolding transition of class pi glutathione S-transferase, a homo<em>dimer</em>ic protein, from porcine lung was monitored by spectroscopic methods (fluorescence emission and ultraviolet absorption), and by enzyme activity changes. Solvent (guanidine hydrochloride and urea)-induced denaturation is well described by a two-state model involving significant populations of only the folded <em>dimer</em> and unfolded monomer. Neither a folded, active monomeric form nor stable unfolding intermediates were detected. The conformational stability, <em>delta</em> Gu (H<em>2</em>O), of the native <em>dimer</em> was estimated to be about <em>2</em>5.3 +/- <em>2</em> kcal/mol at <em>2</em>0 degrees C and pH6.5.

Here we report the crystal structures of human hematopoietic prostaglandin (PG) <em>D</em> synthase bound to glutathione (GSH) and Ca<em>2</em>+ or Mg<em>2</em>+. Using GSH as a cofactor, prostaglandin <em>D</em> synthase catalyzes the isomerization of PGH<em>2</em> to PG<em>D</em><em>2</em>, a mediator for allergy response. The enzyme is a homo<em>dimer</em>, and Ca<em>2</em>+ or Mg<em>2</em>+ increases its activity to approximately 150% of the basal level, with half maximum effective concentrations of 400 microM for Ca<em>2</em>+ and 50 microM for Mg<em>2</em>+. In the Mg<em>2</em>+-bound form, the ion is octahedrally coordinated by six water molecules at the <em>dimer</em> interface. The water molecules are surrounded by pairs of Asp93, Asp96 and Asp97 from each subunit. Ca(<em>2</em>+) is coordinated by five water molecules and an Asp96 from one subunit. The Asp96 residue in the Ca<em>2</em>+-bound form makes hydrogen bonds with two guanidium nitrogen atoms of Arg14 in the GSH-binding pocket. Mg<em>2</em>+ alters the coordinating water structure and reduces one hydrogen bond between Asp96 and Arg14, thereby changing the interaction between Arg14 and GSH. This effect explains a four-fold reduction in the K(m) of the enzyme for GSH. The structure provides insights into how Ca<em>2</em>+ or Mg<em>2</em>+ binding activates human hematopoietic PG<em>D</em> synthase.

The aggregation of <em>delta</em> 5,7,9(11),<em>2</em><em>2</em>-ergostatetraen-3 beta-ol (dehydroergosterol or DHE), a fluorescent analog of cholesterol, was studied by photophysical techniques. It was concluded that the aqueous dispersions of DHE consist of strongly fluorescent microcrystals, and no evidence for self-quenching in micellar-type aggregates was found. The organization of DHE in model systems of membranes (phospholipid vesicles) is strongly dependent on the vesicle type. In small unilamellar vesicles, no evidence for aggregation is obtained, and the fluorescence anisotropy is rationalized on the basis of a random distribution of fluorophores. On the contrary, in large unilamellar vesicles (LUVs), a steeper concentration depolarization was observed. To explain this, a model that takes into account transbilayer <em>dimer</em> formation was derived. This was further confirmed from observation of excitonic absorption bands of <em>2</em><em>2</em>-(N-7-nitrobenz-<em>2</em>-oxa-1,3-diazol-4-yl-amino)-<em>2</em>3,<em>2</em>4-bisnor- 5-cholen-3 beta-ol (NBD-cholesterol) in LUV, which disappear upon sonication. It is concluded that, in agreement with recent works, sterol aggregation is a very efficient process in large vesicles (and probably in natural membranes), even at very low concentrations (approximately 5 mol%).

Human herpesvirus 8 interleukin-6 (vIL-6) displays <em>2</em>5% amino acid identity with human IL-6 (hIL-6) and shares an overall four-helix-bundle structure and gp130-mediated STAT/mitogen-activated protein kinase signaling with its cellular counterpart. However, vIL-6 is distinct in that it can signal through gp130 alone, in the absence of the nonsignaling gp80 alpha-subunit of the IL-6 receptor. To investigate the structural requirements for gp80 independence of vIL-6, a series of expression vectors encoding vIL-6/hIL-6 chimeric and site-mutated IL-6 proteins was generated. The replacement of hIL-6 residues with three vIL-6-specific tryptophans implicated in gp80 independence from crystallographic studies or the A and C helices containing these residues did not confer gp80 independence to hIL-6. The N- and C-terminal regions of vIL-6 could be substituted with hIL-6 sequences with the retention of gp80-independent signaling, but substitutions of other regions of vIL-6 (helix A, A/B loop, helix B, helix C, and proximal half of helix <em>D</em>) with equivalent sequences of hIL-6 abolished gp80 independence. Interestingly, the B helix of vIL-6 was absolutely required for gp80 independence, despite the fact that this region contains no receptor-binding residues. Point mutational analysis of helix C, which contains residues involved in physical and functional interactions with gp130 domains <em>2</em> and 3 (cytokine-binding homology region), identified a variant, VI1<em>2</em>0EE, that was able to signal and dimerize gp130 only in the presence of gp80. gp80 was also found to stabilize gp130:g130 <em>dimers</em> induced by a distal <em>D</em> helix variant of vIL-6 that was nonetheless able to signal independently of gp80. Together, our data reveal the crucial importance of overall vIL-6 structure and conformation for gp80-independent signaling and provide functional and physical evidence of the stabilization of vIL-6-induced gp130 signaling complexes by gp80.

The intrinsic tryptophan fluorescence of bacteriophage T4-co<em>d</em>e<em>d</em> gene 3<em>2</em>-protein is foun<em>d</em> to be partially quenche<em>d</em> on bin<em>d</em>ing a variety of mono-, oligo-, an<em>d</em> polynucleoti<em>d</em>es. This phenomenon is exploite<em>d</em> to partially "map" the nucleic aci<em>d</em> bin<em>d</em>ing site of the protein. The intrinsic fluorescence spectrum of the protein peaks at about 347 nm, compare<em>d</em> to 359 nm for the fully solvate<em>d</em> mo<em>d</em>el fluorophore, N-acetyl-L-tryptophanami<em>d</em>e. Nucleoti<em>d</em>e bin<em>d</em>ing, or collisional quenching by io<em>d</em>i<em>d</em>e ion, re<em>d</em>uces the intensity of the fluorescence, with little or no peak shift. Small ligan<em>d</em>s, ranging in size from ribose- an<em>d</em> <em>d</em>eoxyribose-phosphate to tetranucleoti<em>d</em>es, quench the fluorescence by <em>2</em> to 6%; larger ligan<em>d</em>s quench from <em>2</em>0 to 35% of the intrinsic protein fluorescence. Io<em>d</em>i<em>d</em>e quenching experiments subjecte<em>d</em> to Stern-Vollmer analysis suggest that the bin<em>d</em>ing of short nucleoti<em>d</em>e-containing ligan<em>d</em>s brings about a conformational change in the protein, fully exposing a tryptophan si<em>d</em>e chain to the solvent environment. The fluorescence of this tryptophan is fully quenche<em>d</em> by the bin<em>d</em>ing of <em>d</em>(Ap)<em>2</em>, but is largely unaffecte<em>d</em> by the bin<em>d</em>ing of <em>d</em>(ApA) or <em>d</em>(pA)<em>2</em>, in<em>d</em>icating both that this (tryptophan) "reporter" resi<em>d</em>ue is locate<em>d</em> in the nucleic aci<em>d</em> bin<em>d</em>ing site an<em>d</em> that bin<em>d</em>ing is polar, i.e. polynucleoti<em>d</em>e chains of only one orientation are complexe<em>d</em>. Long oligonucleoti<em>d</em>es fully quench the fluorescence of this bin<em>d</em>ing site tryptophan. At high salt concentration (<em>2</em> M NaCl), gene 3<em>2</em>-protein forms self-limite<em>d</em> <em>dimers</em> (Carroll, R.B., Neet, K.E., an<em>d</em> Gol<em>d</em>thwait, D.A. (197<em>2</em>) Proc. Natl, Aca<em>d</em>. Sci. U.S.A. 69, <em>2</em>741-<em>2</em>744; (1975) J. Mol. Biol. 91, <em>2</em>75-<em>2</em>91). These <em>dimers</em>, in either high salt or in low salt after cross-linking, fail to bin<em>d</em> nucleoti<em>d</em>es, suggesting that <em>dimer</em> formation partially occlu<em>d</em>es the nucleic aci<em>d</em> bin<em>d</em>ing site an<em>d</em> thus that these <em>dimers</em> are probably not involve<em>d</em> as interme<em>d</em>iates in cooperative protein bin<em>d</em>ing to the DNA. On the other han<em>d</em>, <em>dimer</em>ization apparently results in a conformational change which fully exposes the "reporter" tryptophan to io<em>d</em>i<em>d</em>e quenching. These results are use<em>d</em> to formulate a mo<em>d</em>el of some of the nucleic aci<em>d</em>-protein an<em>d</em> protein-protein interactions involve<em>d</em> in the cooperative bin<em>d</em>ing of gene 3<em>2</em>-protein to single-stran<em>d</em>e<em>d</em> DNA.

Artificial lipi<em>d</em> membranes are wi<em>d</em>ely use<em>d</em> as a mo<em>d</em>el system to stu<em>d</em>y single ion channel activity using electrophysiological techniques. In this stu<em>d</em>y, we characterize the properties of the artificial bilayer system with respect to its <em>d</em>ynamics of lipi<em>d</em> phase separation using single-molecule fluorescence fluctuation an<em>d</em> electrophysiological techniques. We <em>d</em>etermine<em>d</em> the rotational motions of fluorescently labele<em>d</em> lipi<em>d</em>s on the nanosecon<em>d</em> timescale using confocal time-resolve<em>d</em> anisotropy to probe the microscopic viscosity of the membrane. Simultaneously, long-range mobility was investigate<em>d</em> by the lateral <em>d</em>iffusion of the lipi<em>d</em>s using fluorescence correlation spectroscopy. <em>D</em>epen<em>d</em>ing on the solvent use<em>d</em> for membrane preparation, lateral <em>d</em>iffusion coefficients in the range <em>D</em>(lat) = 10-<em>2</em>5 mum(<em>2</em>)/s an<em>d</em> rotational <em>d</em>iffusion coefficients ranging from <em>D</em>(rot) = <em>2</em>.8 - 1.4 x 10(7) s(-1) were measure<em>d</em> in pure liqui<em>d</em>-<em>d</em>isor<em>d</em>ere<em>d</em> (L(<em>d</em>)) membranes. In ternary mixtures containing saturate<em>d</em> an<em>d</em> unsaturate<em>d</em> phospholipi<em>d</em>s an<em>d</em> cholesterol, liqui<em>d</em>-or<em>d</em>ere<em>d</em> (L(o)) <em>d</em>omains segregate<em>d</em> from the L(<em>d</em>) phase at <em>2</em>3 <em>d</em>egrees C. The lateral mobility of lipi<em>d</em>s in L(o) <em>d</em>omains was aroun<em>d</em> eightfol<em>d</em> lower compare<em>d</em> to those in the L(<em>d</em>) phase, whereas the rotational mobility <em>d</em>ecrease<em>d</em> by a factor of 1.5. Burst-integrate<em>d</em> stea<em>d</em>y-state anisotropy histograms, as well as anisotropy imaging, were use<em>d</em> to visualize the rotational mobility of lipi<em>d</em> probes in phase-separate<em>d</em> bilayers. These experiments an<em>d</em> fluorescence correlation spectroscopy measurements at <em>d</em>ifferent focal <em>d</em>iameters in<em>d</em>icate<em>d</em> a heterogeneous microenvironment in the L(o) phase. Finally, we <em>d</em>emonstrate the potential of the optoelectro setup to stu<em>d</em>y the influence of lipi<em>d</em> <em>d</em>omains on the electrophysiological properties of ion channels. We foun<em>d</em> that the electrophysiological activity of gramici<em>d</em>in A (gA), a well-characterize<em>d</em> ion-channel-forming pepti<em>d</em>e, was relate<em>d</em> to lipi<em>d</em>-<em>d</em>omain partitioning. <em>D</em>uring liqui<em>d</em>-liqui<em>d</em> phase separation, gA was largely exclu<em>d</em>e<em>d</em> from L(o) <em>d</em>omains. Simultaneously, the number of electrically active gA <em>dimers</em> increase<em>d</em> <em>d</em>ue to the increase<em>d</em> surface <em>d</em>ensity of gA in the L(<em>d</em>) phase.

The NA<em>D</em>(+)-dependent <em>D</em>-lactate dehydrogenase was purified to apparent homogeneity from Lactobacillus bulgaricus and its complete amino acid sequence determined. Two gaps in the polypeptide chain (10 residues) were filled by the deduced amino acid sequence of the polymerase chain reaction amplified <em>D</em>-lactate dehydrogenase gene sequence. The enzyme is a <em>dimer</em> of identical subunits (specific activity <em>2</em>800 +/- 100 units/min at <em>2</em>5 degrees C). Each subunit contains 33<em>2</em> amino acid residues; the calculated subunit M(r) being 36,831. Isoelectric focusing showed at least four protein bands between pH 4.0 and 4.7; the subunit M(r) of each subform is 36,000. The pH dependence of the kinetic parameters, Km, Vm, and kcat/Km, suggested an enzymic residue with a pKa value of about 7 to be involved in substrate binding as well as in the catalytic mechanism. Treatment of the enzyme with group-specific reagents <em>2</em>,3-butanedione, diethylpyrocarbonate, tetranitromethane, or N-bromosuccinimide resulted in complete loss of enzyme activity. In each case, inactivation followed pseudo first-order kinetics. Inclusion of pyruvate and/or NA<em>D</em>H reduced the inactivation rates manyfold, indicating the presence of arginine, histidine, tyrosine, and tryptophan residues at or near the active site. Spectral properties of chemically modified enzymes and analysis of kinetics of inactivation showed that the loss of enzyme activity was due to modification of a single arginine, histidine, tryptophan, or tyrosine residue. Peptide mapping in conjunction with peptide purification and amino acid sequence determination showed that Arg-<em>2</em>35, His-303, Tyr-101, and Trp-19 were the sites of chemical modification. Arg-<em>2</em>35 and His-303 are involved in the binding of <em>2</em>-oxo acid substrate whereas other residues are involved in binding of the cofactor.

Hemostatic changes might contribute to the increased risk of cardiovascular and cerebrovascular events in patients with obstructive sleep apnea (OSA). We investigated the effect of a short-term isocapnic hypoxic challenge on coagulation activation markers thrombin/antithrombin III complexes (TAT) and <em>D</em>-<em>dimer</em> in OSA. Thirty-two OSA patients (mean age 48 +/- 11 years) inhaled a gas mixture containing 10% O(<em>2</em>) and 90% N(<em>2</em>) and further adjusted to yield pulse oximetry saturation of 80-85% for 5 minutes. Plasma levels of TAT and <em>D</em>-<em>dimer</em> were measured immediately before and immediately after the hypoxic challenge. The hypoxic challenge provoked a significant increase in TAT (p < 0.001) and in <em>D</em>-<em>dimer</em> (p = 0.037). Mean nocturnal oxygen saturation from the sleep recordings correlated with <em>D</em>-<em>dimer</em> increase (r = -0.37, p = 0.041). Also, OSA patients with a history of hypertensive parents had greater <em>D</em>-<em>dimer</em> increase in response to hypoxia than patients having normotensive parents (p = 0.035). Parental hypertension independently explained 15% of the variance in <em>D</em>-<em>dimer</em> increase after hypoxia (p = 0.035). Oxygen desaturation during sleep may predispose OSA patients, in particular those with a parental history of hypertension, to a hypercoagulable state providing one explanation for the increased risk of atherothrombotic events in this population.