The rate and extent of disappearance of two <em>D</em>NA lesions (pyrimidine <em>dimers</em> and covalently bound acetylaminofluorene), both thought to be removed by the so-called wide-patch (approximately 100 nucleotides) repair process, were studied in a variety of cultured mammalian cells. With the exception of mouse cells, <em>dimers</em> were removed more rapidly and extensively than covalently bound acetylaminofluorene. In human cells, for example, about 50% of the <em>dimers</em> were excised from <em>D</em>NA in 1 hr while only <em>2</em>5-50% of the chemically induced lesions were excised from <em>D</em>NA after 48 hr. Surprisingly mouse cells, which remove few <em>dimers</em>, were about as competent as control human fibroblasts at removing acetylaminofluorene lesions; however, xeroderma pigmentosum cells (group <em>D</em>) removed fewer N-acetoxy-<em>2</em>-acetylaminofluorene-induced lesions than control human cells. Our data raise the possibility of separate repair processes for these two types of lesions and suggest that their expression may be under similar genetic control in human cells.

We combined fluorescence recovery after photobleaching (FRAP) beam-size analysis with biochemical assays to investigate the mechanisms of membrane recruitment and activation of phospholipase C-beta(<em>2</em>) (PLCbeta(<em>2</em>)) by G protein alpha(q) and betagamma <em>dimers</em>. We show that activation by alpha(q) and betagamma differ from activation by Rac<em>2</em> and from each other. Stimulation by alpha(q) enhanced the plasma membrane association of PLCbeta(<em>2</em>), but not of PLCbeta(<em>2</em>)<em>Delta</em>, which lacks the alpha(q)-interacting region. Although alpha(q) resembled Rac<em>2</em> in increasing the contribution of exchange to the FRAP of PLCbeta(<em>2</em>) and in enhancing its membrane association, the latter effect was weaker than with Rac<em>2</em>. Moreover, the membrane recruitment of PLCbeta(<em>2</em>) by alpha(q) occurred by enhancing PLCbeta(<em>2</em>) association with fast-diffusing (lipid-like) membrane components, whereas stimulation by Rac<em>2</em> led to interactions with slow diffusing membrane sites. On the other hand, activation by betagamma shifted the FRAP of PLCbeta(<em>2</em>) and PLCbeta(<em>2</em>)<em>Delta</em> to pure lateral diffusion 3- to 5-fold faster than lipids, suggesting surfing-like diffusion along the membrane. We propose that these different modes of PLCbeta(<em>2</em>) membrane recruitment may accommodate contrasting functional needs to hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdInsP(<em>2</em>)) in localized versus dispersed populations. PLCbeta(<em>2</em>) activation by Rac<em>2</em>, which leads to slow lateral diffusion and much faster exchange, recruits PLCbeta(<em>2</em>) to act locally on PtdInsP(<em>2</em>) at specific domains. Activation by alpha(q) leads to lipid-like diffusion of PLCbeta(<em>2</em>) accompanied by exchange, enabling the sampling of larger, yet limited, areas prior to dissociation. Finally, activation by betagamma recruits PLCbeta(<em>2</em>) to the membrane by transient interactions, leading to fast "surfing" diffusion along the membrane, sampling large regions for dispersed PtdInsP(<em>2</em>) populations.

We examined in vivo oral glucose tolerance tests and in vitro insulin binding, cellular response, and insulin-receptor structure of fibroblasts cultured from the skin of a patient with leprechaun syndrome and her parents. In response to oral glucose, the proband exhibited marked hyperinsulinism (maximum plasma insulin = 4,1<em>2</em>0 microU/ml), the father had mild hyperinsulinism (maximum plasma insulin = <em>2</em>40 microU/ml), and the mother was normal. [1<em>2</em>5I]insulin binding to monolayers of intact fibroblasts demonstrated complex kinetics that were interpreted using a two-receptor model. Normal high-affinity binding had an apparent KA of 1.6 X 10(10)/molar with 1,100 sites/cell. The proposed low-affinity state receptor had an apparent KA of 6.8 X 10(7)/molar with approximately 30,000 sites/cell. Insulin binding to the proband's cells had no high-affinity binding but had normal low-affinity binding. Cells from the mother had 60%, and cells from the father, <em>2</em>%, of control insulin binding to the high-affinity receptor, but normal, low-affinity site binding. Two different, insulin-stimulable responses were evaluated under experimental conditions identical with those used for insulin binding. Insulin stimulation of <em>2</em>-methylaminoisobutyric acid uptake occurred with half-maximal responses between <em>2</em>5 and 50 ng/ml insulin. This response was similar in cells from controls and the patient. By contrast, the uptake and phosphorylation of <em>2</em>-deoxy-<em>D</em>-glucose was stimulated at half-maximal insulin concentrations between 1 and 10 ng/ml in control cells but was not significantly increased in the proband's cells until 1,000 ng/ml concentrations of insulin were attained. In affinity crosslinking experiments, [1<em>2</em>5I]insulin was covalently bound to insulin receptors of fibroblast membranes using disuccinimidylsuberate. [1<em>2</em>5I]insulin specifically bound to 1<em>2</em>5,000 dalton monomeric subunits and <em>2</em>50,000 dalton <em>dimers</em>. In control cells, the ratio of monomer to <em>dimer</em> was approximately one, but significantly fewer <em>dimers</em> were crosslinked in insulin receptors from the patient's cells. We conclude that in this family two different recessive mutations impair high-affinity insulin-receptor binding and that the proband with leprechaunism is a compound heterozygote for these mutations. The two mutations produced structural changes in the receptor that altered subunit interactions and loss of high-affinity binding and cellular responsivity.

S100B is a Ca(<em>2</em>+)-binding protein known to be a non-covalently associated <em>dimer</em>, S100B(beta beta), at high concentrations (0.<em>2</em>-3.0 mM) under reducing conditions. The solution structure of apo-S100B (beta beta) shows that the subunits associate in an antiparallel manner to form a tightly packed hydrophobic core at the <em>dimer</em> interface involving six of eight helices and the C-terminal loop (<em>D</em>rohat AC, Amburgey JC, Abildgaard F, Starich MR, Baldisseri <em>D</em>, Weber <em>D</em>J. 1996. Solution structure of rat apo-S100B (beta beta) as determined by NMR spectroscopy. Biochemistry 35:11577-11588). The C-terminal loop, however, is also known to participate in the binding of S100B to target proteins, so its participation in the <em>dimer</em> interface raises questions as to the physiological relevance of <em>dimer</em>ic S100B (beta beta). Therefore, we investigated the oligomerization state of S100B at low concentrations (1-10,000 nM) using large-zone analytical gel filtration chromatography with 35S-labeled S100B. We found that S100B exists >> 99%) as a non-covalently associated <em>dimer</em>, S100B (beta beta), at 1 nM subunit concentration (500 pM <em>dimer</em>) in the presence or absence of saturating levels of Ca<em>2</em>+, which implies a dissociation constant in the picomolar range or lower. These results demonstrate for the first time that in reducing environments and at physiological concentrations, S100B exists as <em>dimer</em>ic S100B (beta beta) in the presence or absence of Ca<em>2</em>+, and that the non-covalent <em>dimer</em> is most likely the form of S100B presented to target proteins.

Enzyme I of the bacterial phosphoenolpyruvate: glycose phosphotransferase system (PTS) exists in a monomer/<em>dimer</em> (M/<em>D</em>) equilibrium. These two forms are functionally different, and their interconversion may be a means of regulating the PTS. The M/<em>D</em> equilibrium was studied by fluorescence anisotropy of a pyrene derivative (Chauvin, F., Brand, L., and Roseman, S. (1994) J. Biol. Chem. <em>2</em>69, <em>2</em>0<em>2</em>63-<em>2</em>0<em>2</em>69). In this paper, the kinetics of the transition is investigated. The following apparent rate constants were found for the M/<em>D</em> transition of phospho-Enzyme I in the presence of Mg<em>2</em>+ and PEP at 6 degrees C: k*A = 3.4 x 10(3) M-1 s-1 and k*<em>D</em> = 1.04 x 10(3) s-1. The association rate is especially slow, <em>2</em>-3 orders of magnitude slower than the average <em>dimer</em>ization rate determined for other proteins. Furthermore, the rate of quaternary structure changes matches that of enzymatic activity changes, as well as that of tertiary structure changes (Chauvin, F., Toptygin, <em>D</em>., Roseman, S., and Brand, L. (199<em>2</em>) Biophys. Chem. 44, 163-173). Finally, the effect of two ligands is shown; PEP increases the relaxation rate by 3-fold at <em>2</em>3 degrees C, and Mg<em>2</em>+ addition causes a 4-fold increase in the relaxation rate.

The <em>D</em>cuS-<em>D</em>cuR system of Escherichia coli is a two-component sensor-regulator that controls gene expression in response to external C(4)-dicarboxylates and citrate. The <em>D</em>cuS protein is particularly interesting since it contains two PAS domains, namely a periplasmic C(4)-dicarboxylate-sensing PAS domain (PASp) and a cytosolic PAS domain (PASc) of uncertain function. For a study of the role of the PASc domain, three different fragments of <em>D</em>cuS were overproduced and examined: they were PASc-kinase, PASc, and kinase. The two kinase-domain-containing fragments were autophosphorylated by [gamma-(3<em>2</em>)P]ATP. The rate was not affected by fumarate or succinate, supporting the role of the PASp domain in C(4)-dicarboxylate sensing. Both of the phosphorylated <em>D</em>cuS constructs were able to rapidly pass their phosphoryl groups to <em>D</em>cuR, and after phosphorylation, <em>D</em>cuR dephosphorylated rapidly. No prosthetic group or significant quantity of metal was found associated with either of the PASc-containing proteins. The <em>D</em>NA-binding specificity of <em>D</em>cuR was studied by use of the pure protein. It was found to be converted from a monomer to a <em>dimer</em> upon acetylphosphate treatment, and native polyacrylamide gel electrophoresis suggested that it can oligomerize. <em>D</em>cuR specifically bound to the promoters of the three known <em>D</em>cuSR-regulated genes (dctA, dcuB, and frdA), with apparent K(<em>D</em>)s of 6 to 3<em>2</em> micro M for untreated <em>D</em>cuR and < or =1 to <em>2</em> microM for the acetylphosphate-treated form. The binding sites were located by <em>D</em>Nase I footprinting, allowing a putative <em>D</em>cuR-binding motif [tandemly repeated (T/A)(A/T)(T/C)(A/T)AA sequences] to be identified. The <em>D</em>cuR-binding sites of the dcuB, dctA, and frdA genes were located <em>2</em>7, 94, and 86 bp, respectively, upstream of the corresponding +1 sites, and a new promoter was identified for dcuB that responds to <em>D</em>cuR.

Passive Heymann nephritis with acute and severe proteinuria was produced in rats by a single injection of heterologous antibody against a purified glycoprotein which consisted of homologous subunits with a molecular weight of 108,000 (gp108). Gp108 was identified as one of the major antigens in rat renal tubular fraction (FX1A) on immunoblotting assay by using total proteins of FX1A and rabbit antiserum against FX1A. A band of gp330, which was identified as a pathogenic antigen of Heymann nephritis by Kerjaschki <em>D</em> and Farquhar MG (Proc Natl Acad Sci USA 79:5557, 198<em>2</em>) was detected as another band by Coomassie blue staining and immunoblotting. Autoantibodies in the sera of FX1A-injected (active Heymann nephritis) rats reacted to the band of gp330 but not to gp108. These results indicate that gp108 is a different glycoprotein from both gp330 and its degradation products. GP108 was subsequently purified to near homogeneity by extraction with Triton X-100, and then <em>D</em>EAE-cellulose and Bio-Gel A-1.5m column chromatographies. On gel permeation chromatography, the purified antigen showed a molecular weight of 310,000, suggesting that it consists of <em>dimer</em> or trimer of gp108. Rabbits immunized with gp108 produced an antibody which showed monospecific binding to gp108. The antibody stained with brush border of proximal renal tubules in addition to the capillary loops in rat glomeruli by indirect immunofluorescence. Injection of rabbit antiserum against gp108 in rats induced severe proteinuria within <em>2</em> days. On the <em>2</em>nd day after the injection, the glomeruli of the animals showed granular immune deposits along the capillary loops in addition to dominant staining of the brush border of the proximal tubules by immunofluorescence. These results indicate that gp108 is a pathogenic antigen in passive Heymann nephritis and that an antibody against gp108 has a nephritogenic and proteinuria-inducing activity.

OBJECTIVE

To determine whether patients with nonvalvular atrial fibrillation (NVAF) have prothrombotic changes compared with patients in sinus rhythm.

METHODS

Cross-sectional study. Hemostatic function compared in NVAF patients without prior embolic event (transient ischemic attack or embolic stroke) and control subjects without prior thrombotic stroke, and in NVAF patients with prior embolic event and control subjects with prior thrombotic stroke.

METHODS

Internal medicine outpatient group practice and anticoagulation clinic in <em>2</em> teaching hospitals.

METHODS

A total of 75 NVAF patients (50 without and <em>2</em>5 with prior embolic event) and 4<em>2</em> control patients (31 without and 11 with prior thrombotic stroke) recruited concurrently over 18 months during 1990-91.

METHODS

Platelet count, prothrombin time (PT), partial thromboplastin time (PTT), and plasma levels of hemoglobin, fibrinogen, von Willebrand factor antigen, factor VIII, fibrin D-dimer, antithrombin III, protein C, protein S, fibrinopeptide A and prothrombin fragment F1+<em>2</em>. All statistical analyses were performed after adjustments for age and sex.

RESULTS

The NVAF patients without a prior embolic event had significantly higher mean hemoglobin and fibrinogen levels (p < 0.001 and p = 0.05, respectively) than the control subjects without prior thrombotic stroke. The <em>2</em>9 NVAF patients not taking warfarin (none had had an embolic event) had significantly lower mean protein C and protein S levels (p = 0.01<em>2</em> and p < 0.001, respectively) and a significantly higher fibrinopeptide A level (p = 0.03, after exclusion of outliers) than the control subjects without prior stroke. The NVAF patients with a prior embolic event had alterations in the hemostatic variables similar to those seen in the control patients with a prior thrombotic stroke. The latter had significantly higher fibrinogen, von Willebrand factor antigen and factor VIII levels (p = 0.04, 0.00<em>2</em> and 0.00<em>2</em>, respectively) and significantly lower protein S levels (p = 0.0<em>2</em>) than the control subjects without prior stroke.

CONCLUSIONS

NVAF patients without a history of an embolic event show evidence of a prothrombotic state compared with patients in sinus rhythm who have not had a thrombotic stroke. NVAF patients with a history of an embolic event show evidence of a prothrombotic state similar to that of patients in sinus rhythm who have had a thrombotic stroke. Prospective studies are needed to determine whether these abnormalities predict higher risk of stroke in individual NVAF patients.

OBJECTIVE

The prevalence of thromboembolism might be higher than previously recognized in patients with atrial flutter (AFL) based on findings of transesophageal echocardiography (TEE). To evaluate the potential prothrombotic state in patients with AFL, TEE findings and hemostatic markers were compared among patient groups with AFL, normal sinus rhythm (NSR) and chronic nonvalvular atrial fibrillation (AF).

METHODS

Cross-sectional study at a university hospital.

METHODS

In <em>2</em>8 patients (mean age, 63 years) with AFL, 58 patients (mean age, 66 years) with AF, an<em>d</em> <em>2</em>7 patients (mean age, 61 years) with NSR who un<em>d</em>erwent TEE, plasma levels of markers for platelet activity (platelet factor 4 an<em>d</em> beta-thromboglobulin [beta-TG]), thrombotic status (thrombin-antithrombin III complex an<em>d</em> prothrombin fragments 1 an<em>d</em> <em>2</em>) an<em>d</em> fibrinolytic status (<em>d</em>-<em>dimer</em> an<em>d</em> plasmin-alpha(<em>2</em>)-plasmin inhibitor complex) were <em>d</em>etermine<em>d</em>.

RESULTS

Left atrial appen<em>d</em>age (LAA) bloo<em>d</em> flow velocity in patients with AFL was higher (p < 0.05) than that in patients with AF, but was lower (p < 0.05) than that in patients with NSR (AF, <em>2</em>5 +/- <em>2</em>; AFL, 44 +/- 4; NSR, 60 +/- 4 cm/s). Dense left atrial spontaneous echo contrast (SEC) was foun<em>d</em> in 4 patients (14%) with AFL an<em>d</em> 16 patients (<em>2</em>8%) with AF. There was no significant <em>d</em>ifference in plasma levels of hemostatic markers between the AFL group an<em>d</em> the NSR group. AFL patients with impaire<em>d</em> LAA function (LAA flow < 30cm/s, <em>d</em>ense SEC, or both), however, showe<em>d</em> higher level of <em>d</em>-<em>dimer</em> an<em>d</em> beta-TG than those without impaire<em>d</em> LAA function (<em>d</em>-<em>dimer</em>, 1.9 +/- 0.6 microg/mL vs 0.4 +/- 0.1 microg/mL; beta-TG, 73 +/- 17 ng/mL vs 33 +/- 5 ng/mL, p < 0.05).

CONCLUSIONS

Patients with AFL as a whole are not in the prothrombotic state as compared with those with AF. However, patients with AFL and impaired LAA function are at potentially high risk for thromboembolism and might require anticoagulation.

This clinical policy focuses on critical issues in the evaluation and management of patients with signs or symptoms of pulmonary embolism (PE). A ME<em>D</em>LINE search for clinical trials published from January 1995 through April <em>2</em>001 was performed using the key words "pulmonary embolus" with limits of "clinical investigations" and "clinical policies." Subcommittee members and expert peer reviewers also supplied articles with direct bearing on the policy. This policy focuses on <em>2</em> major areas of current interest and/or controversy: (1) diagnostic: utility of <em>D</em> -<em>dimer</em>, ventilation-perfusion scanning, and spiral computed tomography angiogram in the evaluation of PE; and (<em>2</em>) therapeutic: indications for fibrinolytic therapy. Recommendations for patient management are provided for each 1 of these topics based on strength of evidence (Level A, B, or C). Level A recommendations represent patient management principles that reflect a high degree of clinical certainty; Level B recommendations represent patient management principles that reflect moderate clinical certainty; and Level C recommendations represent other patient management strategies based on preliminary, inconclusive, or conflicting evidence, or based on panel consensus. This guideline is intended for physicians working in emergency departments or chest pain evaluation units.

HIV-1 reverse transcriptase (RT) has been an attractive target for the development of antiretroviral agents. Although this enzyme is bi-functional, having both <em>D</em>NA polymerase and ribonuclease H (RNH) activities, there is no clinically approved inhibitor of the RNH activity. Here, we characterize the structural basis and molecular interaction of an allosteric site inhibitor, BHMP07, with the wild-type (WT) RNH fragment. Solution NMR experiments for inhibitor titration on WT RNH showed relatively wide chemical shift perturbations, suggesting a long-range conformational effect on the inhibitor interaction. Comparisons of the inhibitor-induced NMR chemical shift changes of RNH with those of RNH <em>dimer</em>, in the presence and absence of Mg(<em>2</em>+) , were performed to determine and verify the interaction site. The NMR results, with assistance of molecular docking, indicate that BHMP07 preferentially binds to a site that is located between the RNH active site and the region encompassing helices B and <em>D</em> (the 'substrate-handle region'). The interaction site is consistent with the previous proposed site, identified using a chimeric RNH (p15-EC) [Gong et al. (<em>2</em>011) Chem Biol <em>D</em>rug <em>D</em>es 77, 39-47], but with slight differences that reflect the characteristics of the amino acid sequences in p15-EC compared to the WT RNH.

The quaternary structure of LacS, the lactose transporter of Streptococcus thermophilus, has been <em>d</em>etermine<em>d</em> for the <em>d</em>etergent-solubilize<em>d</em> an<em>d</em> the membrane-reconstitute<em>d</em> state of the protein. The quaternary structure of the n-<em>d</em>o<em>d</em>ecyl-beta-<em>d</em>-maltosi<em>d</em>e-solubilize<em>d</em> state was stu<em>d</em>ie<em>d</em> using a combination of se<em>d</em>imentation velocity an<em>d</em> equilibrium centrifugation analysis. From these measurements it followe<em>d</em> that the <em>d</em>etergent-solubilize<em>d</em> LacS un<em>d</em>ergoes reversible self-association with a monomer to <em>dimer</em> mo<em>d</em>e of association. The association constants were 5.4 +/- 3.6 an<em>d</em> 4.4 +/- 1.0 ml mg(-1) as <em>d</em>etermine<em>d</em> from the velocity an<em>d</em> equilibrium se<em>d</em>imentation measurements, respectively. The experiments <em>d</em>i<em>d</em> not in<em>d</em>icate significant changes in the shape of the protein-<em>d</em>etergent complex or the amount of <em>d</em>etergent boun<em>d</em> in going from the monomeric to <em>dimer</em>ic state of LacS. Importantly, a single Cys mutant of LacS is labele<em>d</em> by <em>2</em>-(4'-maleimi<em>d</em>ylanilino)naphthalene-6-sulfonic aci<em>d</em> in a substrate-<em>d</em>epen<em>d</em>ent manner, in<em>d</em>icating that the <em>d</em>etergent-solubilize<em>d</em> protein exhibits ligan<em>d</em> bin<em>d</em>ing activity. The quaternary structure of membrane-reconstitute<em>d</em> LacS was <em>d</em>etermine<em>d</em> by freeze-fracture electron microscopy analysis. Recent <em>d</em>evelopments in the analysis of freeze-fracture images (Eskan<em>d</em>ari, S. P., Wright, E. M., Freman, M., Starace, <em>D</em>. M., an<em>d</em> Zampighi, G. A. (1998) Proc. Natl. Aca<em>d</em>. Sci. U. S. A. 95, 11<em>2</em>35-11<em>2</em>40) allowe<em>d</em> us to <em>d</em>irectly correlate the cross-sectional area of the transmembrane segment to a <em>dimer</em>ic state of the functionally membrane-reconstitute<em>d</em> LacS protein. The cross-sectional area of the LacS protein was calibrate<em>d</em> using the membrane-reconstitute<em>d</em> transmembrane <em>d</em>omain of the mannitol transporter enzyme II, an intramembrane particle for which the cross-sectional area was obtaine<em>d</em> from maps of two-<em>d</em>imensional crystals. The consequences of the <em>d</em>etermine<em>d</em> quaternary structure for the transport function an<em>d</em> regulation of LacS are <em>d</em>iscusse<em>d</em>.

Properties of purified recombinant adenosine 3'-phosphate 5'-phosphosulfate (PAdoPS) reductase from Escherichia coli were investigated. The Michaelis constants for reduced thioredoxin and PAdoPS are <em>2</em>3 microM and 10 microM, respectively; the enzyme has a Vmax of 94-99 mumol min-1 mg-1 and a molecular activity/catalytically active <em>dimer</em> of 95 s-1. Adenosine 3',5'-bisphosphate (PAdoP) inhibits competitively (Ki 4 microM) with respect to PAdoPS; adenosine <em>2</em>',5'-bisphosphate and sulfite are not inhibitory. Alkylation by SH-group inhibitors irreversibly inactivates the enzyme. The structural gene (cysH) encodes for a small polypeptide with a single Cys residue located in a conserved cluster (KXECGI/LH) of amino acids. Involvement of the only Cys and of Tyr<em>2</em>09 in the reduction of PAdoPS to sulfite was investigated by site-specific mutagenesis: cysH was mutated by single-strand-overlay extension PCR; the mutated genes were cloned in pBTac1 and expressed in E. coli RL <em>2</em><em>2</em> (<em>delta</em> cysHIJ). Homogenous Cys<em>2</em>39Ser and Tyr<em>2</em>09Phe mutant PAdoPS reductases were investigated for altered catalytic properties. Mutation of the single Cys reduced Vmax by a factor of 4.5 x 10(3) (Vmax = 0.0<em>2</em>-0.013 mumol min-1 mg-1) with marginal effects on Km for PAdoPS (19 microM) and reduced thioredoxin (14 microM). Mutation of Tyr<em>2</em>09 drastically affected saturation with thioredoxin (Km 1.5 microM) and decreased Vmax (0.<em>2</em><em>2</em>-0.<em>2</em>5 mumol min-1 mg-1) in addition to a small increase in Km for PAdoPS (31 microM). Chromophores as prosthetic groups were absent from recombinant PAdoPS reductase. Difference absorption spectra between reduced and oxidized forms of wild-type and mutated proteins indicated that, in addition to Cys<em>2</em>39 and Tyr<em>2</em>09, an unidentified Trp (<em>delta</em> lambda max <em>2</em>9<em>2</em> nm) appears to be involved in the reduction. The data suggest a special ping-pong mechanism with PAdoPS reacting with the reduced enzyme isomer in a Theorell-Chance type mechanism.

This stu<em>d</em>y investigates the feasibility of using the enhance<em>d</em> cyan mutant of green fluorescent protein (ECFP) as a probe for two-photon fluorescence correlation spectroscopy (FCS). Molecular <em>d</em>ynamics an<em>d</em> other properties of ECFP an<em>d</em> an ECFP-tubulin fusion protein were investigate<em>d</em> in living Potorous tri<em>d</em>actylis (PTK<em>2</em>) cells. ECFP has high molecular brightness in the nucleus (eta=3.3 kcpsm) an<em>d</em> in the cytoplasm (3.<em>2</em> kcpsm) un<em>d</em>er our experimental con<em>d</em>itions. The <em>d</em>iffusion constants of ECFP were <em>d</em>etermine<em>d</em> to be <em>2</em>0+/-7 microm(<em>2</em>)/s in the nucleus an<em>d</em> <em>2</em>1+/-8 microm(<em>2</em>)/s in the cytoplasm. ECFP has stable molecular characteristics with negligible photobleaching an<em>d</em> photo<em>d</em>ynamic effects in our measurements. At the highest concentration of monomer ECFP (4<em>2</em>5 nM) the amount of <em>dimer</em> ECFP was estimate<em>d</em> to be negligible ( approximately 1.8 nM), consistent with our <em>d</em>ata analysis using a single species mo<em>d</em>el. ECFP-tubulin has a <em>d</em>iffusion constant of 6 microm(<em>2</em>)/s in the living cells. In a<em>d</em><em>d</em>ition, we <em>d</em>emonstrate that analysis of the molecular brightness can provi<em>d</em>e a new avenue for stu<em>d</em>ying the polymerization state of tubulin. We suggest that the tubulin in the vicinity of the nucleus exists primarily as a hetero<em>dimer</em> subunit while those in the area away from the nucleus (<em>d</em>)5 microm) are mostly oligomers. We conclu<em>d</em>e that ECFP is a useful genetic fluorescent probe for FCS stu<em>d</em>ies of various cellular processes when in fusion to other biomolecules of interest.

Diphtheria toxin repressor (DtxR) is a transition metal ion-activate<em>d</em> repressor in Corynebacterium <em>d</em>iphtheriae. DtxR is an iron sensor; metal-boun<em>d</em> DtxR represses transcription of genes <em>d</em>ownstream of the tox operator. Wil<em>d</em>-type DtxR [DtxR(wt)] an<em>d</em> several mutant forms were overexpresse<em>d</em> an<em>d</em> purifie<em>d</em> from Escherichia coli. DtxR was isolate<em>d</em> without boun<em>d</em> metal. Metal reconstitution gave a bin<em>d</em>ing stoichiometry of <em>2</em> per monomer for DtxR(wt) an<em>d</em> 1 per monomer for DtxR(H79A) an<em>d</em> DtxR(M10A). DNA bin<em>d</em>ing of DtxR(H79A) an<em>d</em> DtxR(M10A) in<em>d</em>icates that metal site <em>2</em> is essential for activity. Metal bin<em>d</em>ing lowers the <em>d</em>imerization K(<em>d</em>) of DtxR from low micromolar to 33 nM. Gel electrophoretic mobility-shift assays show that Fe(<em>2</em>+) an<em>d</em> not Fe(3+) activates DtxR for DNA bin<em>d</em>ing. This fin<em>d</em>ing suggests that gene regulation by DtxR may be sensitive not only to iron levels but also to re<em>d</em>ox state of the iron. Mutations in the tox operator sequence in<em>d</em>icate that DtxR <em>dimers</em> bin<em>d</em>ing to DNA may be highly cooperative.

OBJECTIVE

Rhamnogalacturonan-II (RG-II) is one of the pectin motifs foun<em>d</em> in the cell wall of all lan<em>d</em> plants. It contains sugars such as <em>2</em>-keto-3-<em>d</em>eoxy-<em>d</em>-lyxo-heptulosaric aci<em>d</em> (Dha) an<em>d</em> <em>2</em>-keto-3-<em>d</em>eoxy-<em>d</em>-manno-octulosonic aci<em>d</em> (K<em>d</em>o), an<em>d</em> within the wall RG-II is mostly foun<em>d</em> as a <em>dimer</em> via a borate <em>d</em>iester cross-link. To <em>d</em>ate, little is known regar<em>d</em>ing the biosynthesis of this motif. Here, after a brief review of our current knowle<em>d</em>ge on RG-II structure, biosynthesis an<em>d</em> function in plants, this stu<em>d</em>y explores the implications of the presence of a Golgi-localize<em>d</em> sialyltransferase-like <em>2</em> (SIA<em>2</em>) protein that is possibly involve<em>d</em> in the transfer of Dha or K<em>d</em>o in the RG-II of Arabi<em>d</em>opsis thaliana pollen tubes, a fast-growing cell type use<em>d</em> as a mo<em>d</em>el for the stu<em>d</em>y of cell elongation.

METHODS

Two heterozygous mutant lines of arabi<em>d</em>opsis (sia<em>2</em>-1+/- an<em>d</em> qrt1 × sia<em>2</em>-<em>2</em>+/-) were investigate<em>d</em>. sia<em>2</em>-<em>2</em>+/- was in a quartet1 backgroun<em>d</em> an<em>d</em> the inserte<em>d</em> T-DNA containe<em>d</em> the reporter gene β-glucuroni<em>d</em>ase (GUS) un<em>d</em>er the pollen-specific promoter LAT5<em>2</em>. Pollen germination an<em>d</em> pollen tube phenotype an<em>d</em> growth were analyse<em>d</em> both in vitro an<em>d</em> in vivo by microscopy.

RESULTS

Self-pollination of heterozygous lines pro<em>d</em>uce<em>d</em> no homozygous plants in the progeny, which may suggest that the mutation coul<em>d</em> be lethal. Heterozygous mutants <em>d</em>isplaye<em>d</em> a much lower germination rate overall an<em>d</em> exhibite<em>d</em> a substantial <em>d</em>elay in germination (<em>2</em>0 h of <em>d</em>elay to reach 30 % of pollen grain germination compare<em>d</em> with the wil<em>d</em> type). In both lines, mutant pollen grains that were able to pro<em>d</em>uce a tube ha<em>d</em> tubes that were either bursting, abnormal (swollen or <em>d</em>ichotomous branching tip) or much shorter compare<em>d</em> with wil<em>d</em>-type pollen tubes. In vivo, mutant pollen tubes were restricte<em>d</em> to the style, whereas the wil<em>d</em>-type pollen tubes were <em>d</em>etecte<em>d</em> at the base of the ovary.

CONCLUSIONS

This stu<em>d</em>y highlights that the mutation in arabi<em>d</em>opsis SIA<em>2</em> enco<em>d</em>ing a sialyltransferase-like protein that may transfer Dha or K<em>d</em>o on the RG-II motif has a <em>d</em>ramatic effect on the stability of the pollen tube cell wall.

Enolase (<em>2</em>-phospho-<em>D</em>-glycerate hydrolase; EC 4.<em>2</em>.1.11) from the hyperthermophilic bacterium Thermotoga maritima was purified to homogeneity. The N-terminal <em>2</em>5 amino acids of the enzyme reveal a high degree of similarity to enolases from other sources. As shown by sedimentation analysis and gel-permeation chromatography, the enzyme is a 345-k<em>D</em>a homoctamer with a subunit molecular mass of 48 +/- 5 k<em>D</em>a. Electron microscopy and image processing yield ring-shaped particles with a diameter of 17 nm and fourfold symmetry. Averaging of the aligned particles proves the enzyme to be a tetramer of <em>dimers</em>. The enzyme requires divalent cations in the activity assay, Mg<em>2</em>+ being most effective. The optimum temperature for catalysis is 90 degrees C, the temperature dependence yields a nonlinear Arrhenius profile with limiting activation energies of 75 kJ mol-1 and 43 kJ mol-1 at temperatures below and above 45 degrees C. The pH optimum of the enzyme lies between 7 and 8. The apparent Km values for <em>2</em>-phospho-<em>D</em>-glycerate and Mg<em>2</em>+ at 75 degrees C are 0.07 mM and 0.03 mM; with increasing temperature, they are decreased by factors <em>2</em> and 30, respectively. Fluoride and phosphate cause competitive inhibition with a Ki of 0.14 mM. The enzyme shows high intrinsic thermal stability, with a thermal transition at 90 and 94 degrees C in the absence and in the presence of Mg<em>2</em>+.

Human proliferating cell nuclear antigen (PCNA) was overexpressed in Escherichia coli as a soluble protein. Recombinant PCNA was purified to homogeneity by phosphocellulose, Q-Sepharose, Sephacryl S-<em>2</em>00, and hydroxylapatite chromatography. Approximately <em>2</em>0 mg of PCNA was isolated from E. coli cells derived from <em>2</em> L of culture. Characterization of the recombinant protein showed that it was functionally active and that its properties were similar to those of purified human placental PCNA. Recombinant PCNA stimulated human DNA polymerase <em>delta</em> activity at least <em>2</em>5-fold with poly(dA)/oligo-(dT) as the template. Recombinant PCNA eluted with a M(r) = 10<em>2</em>,000 and a Stokes radius of 37 Angstrum by high-performance gel-permeation chromatography. The sedimentation coefficient determined by glycerol gradient ultracentrifugation was 6.3 S. The molecular weight calculated from the Stokes radius and S value was 96,800. The behavior of PCNA was entirely consistent with its being a trimeric protein. Analytical ultracentrifugation and gel filtration revealed the existence of a <em>dimer</em>ic species at low dilution. Cross-linking experiments revealed the presence of PCNA <em>dimers</em> which predominated, as well as a trimeric species. These studies provide biophysical evidence that PCNA is an oligomeric protein which behaves as a trimeric species at high protein concentrations but dissociates to a <em>dimer</em> at low protein concentrations.

Type VII procollagen has been characterized as a product of epithelial cell lines. As secreted, it contains a large triple-helical domain terminated by a multi-globular-domained carboxyl terminus (NC-1), and a smaller amino-terminal globule (NC-<em>2</em>). The triple helix and the NC-1 domain have previously been identified in anchoring fibril-containing tissues by biochemical and immunochemical means, leading to the conclusion that type VII collagen is a major component of anchoring fibrils. In order to better characterize the tissue form of type VII collagen, we have produced a panel of monoclonal antibodies which recognize the NC-1 domain. Peptide mapping of these epitopes indicate that they are independent and span approximately 1<em>2</em>5,000 k<em>D</em>a of the total 150,000 k<em>D</em>a of each alpha chain contained in NC-1. All these antibodies elicit immunofluorescent staining of the basement membrane zone in tissues. Type VII collagen has been extracted from tissues. As previously reported, it is smaller than type VII procollagen, (Woodley, <em>D</em>. T., Burgeson, R. E., Lunstrum, G. P., Bruckner-Tuderman, L., and Briggaman, R. A., submitted for publication), and we now find that it predominantly occurs as a <em>dimer</em>. Following clostridial collagenase digestion, intact NC-1 has been recognized, indicating that the difference in apparent Mr between the tissue form of the molecule and type VII procollagen results from modification of the amino terminus. The size of the amino-terminal globule has been determined to be between approximately 96 and 10<em>2</em> k<em>D</em>a. Rotary shadowing analyses of extracted molecules indicate that <em>dimer</em>ic molecules contain the NC-1 domain, but are missing intact NC-<em>2</em>. We propose that the tissue form monomer, Mr = 960,000, be referred to as "type VII collagen." These studies strongly suggest that anchoring fibrils contain <em>dimer</em>ic molecules with intact NC-1 domains. The data also support the previous suggestion that the NC-<em>2</em> domain is involved in the formation of disulfide bond-stabilized type VII collagen <em>dimers</em>, and is subsequently removed by physiological proteolytic processing.

We investigate the performance of contemporary semilocal an<em>d</em> hybri<em>d</em> <em>d</em>ensity functionals for bon<em>d</em> energetics, structures, <em>d</em>ipole moments, an<em>d</em> harmonic frequencies of 3<em>d</em> transition-metal (TM) compoun<em>d</em>s by comparison with gas-phase experiments. Special attention is given to the nonempirical metageneralize<em>d</em> gra<em>d</em>ient approximation (meta-GGA) of Tao, Per<em>d</em>ew, Staroverov, an<em>d</em> Scuseria (TPSS) [Phys. Rev. Lett. 91, 146401 (<em>2</em>003)], which has been implemente<em>d</em> in TURBOMOLE for the present work. Tren<em>d</em>s an<em>d</em> error patterns for classes of homologous compoun<em>d</em>s are analyze<em>d</em>, inclu<em>d</em>ing <em>dimers</em>, monohy<em>d</em>ri<em>d</em>es, mononitri<em>d</em>es, monoxi<em>d</em>es, monofluori<em>d</em>es, polyatomic oxi<em>d</em>es an<em>d</em> halogeni<em>d</em>es, carbonyls, an<em>d</em> complexes with organic pi ligan<em>d</em>s such as benzene an<em>d</em> cyclopenta<em>d</em>ienyl. Weakly boun<em>d</em> systems such as Ca(<em>2</em>), Mn(<em>2</em>), an<em>d</em> Zn(<em>2</em>) are <em>d</em>iscusse<em>d</em>. We propose a reference set of reaction energies for benchmark purposes. Our all-electron results with qua<em>d</em>ruple zeta valence basis sets vali<em>d</em>ate semilocal <em>d</em>ensity-functional theory as the workhorse of computational TM chemistry. Typical errors in bon<em>d</em> energies are substantially larger than in (organic) main group chemistry, however. The Becke-Per<em>d</em>ew'86 [Phys. Rev. A 38, 3098 (1988); Phys. Rev. B 33, 88<em>2</em><em>2</em> (1986)] GGA an<em>d</em> the TPSS meta-GGA have the best price/performance ratio, while the TPSS hybri<em>d</em> functional achieves a slightly lower mean absolute error in bon<em>d</em> energies. The popular Becke three-parameter hybri<em>d</em> B3LYP un<em>d</em>erbin<em>d</em>s significantly an<em>d</em> ten<em>d</em>s to overestimate bon<em>d</em> <em>d</em>istances; we give a possible explanation for this. We further show that hybri<em>d</em> mixing <em>d</em>oes not re<em>d</em>uce the wi<em>d</em>th of the error <em>d</em>istribution on our reference set. The error of a functional for the s-<em>d</em> transfer energy of a TM atom <em>d</em>oes not pre<em>d</em>ict its error for TM bon<em>d</em> energies an<em>d</em> bon<em>d</em> lengths. For semilocal functionals, self-interaction error in one- an<em>d</em> three-electron bon<em>d</em>s appears to be a major source of error in TM reaction energies. Nevertheless, TPSS pre<em>d</em>icts the correct groun<em>d</em>-state symmetry in the vast majority of cases an<em>d</em> rarely fails qualitatively. This further confirms TPSS as a general purpose functional that works throughout the perio<em>d</em>ic table. We also give workstation timing comparisons for the 645-atom protein crambin.

Type I restriction-modification (R-M) systems encode multisubunit/multidomain enzymes. Two genes (M and S) are required to form the methyltransferase (MTase) that methylates a specific base within the recognition sequence and protects <em>D</em>NA from cleavage by the endonuclease. The <em>D</em>NA methyltransferase M.AhdI is a 170 k<em>D</em>a tetramer with the stoichiometry M(<em>2</em>)S(<em>2</em>) and has properties typical of a type I MTase. The M.AhdI enzyme has been prepared with deuterated S subunits, to allow contrast variation using small-angle neutron scattering (SANS) methods. The SANS data were collected in a number of (1)H:(<em>2</em>)H solvent contrasts to allow matching of one or other of the subunits in the multisubunit enzyme. The radius of gyration (R(g)) and maximum dimensions (<em>D</em>(max)) of the M subunits in situ in the multisubunit enzyme (50 A and 190 A, respectively) are close of those of the entire MTase (51 A and 190 A). In contrast, the S subunits in situ have experimentally determined values of R(g)=35 A and <em>D</em>(max)=110 A, indicating their more central location in the enzyme. Ab initio reconstruction methods yield a low-resolution structural model of the shape and subunit organization of M.AhdI, in which the Z-shaped structure of the S subunit <em>dimer</em> can be discerned. In contrast, the M subunits form a much more elongated and extended structure. The core of the MTase comprises the two S subunits and the globular regions of the two M subunits, with the extended portion of the M subunits most probably forming highly mobile regions at the outer extremities, which collapse around the <em>D</em>NA when the MTase binds.

Purified <em>D</em>rosophila lebanonensis alcohol dehydrogenase (Adh) revealed one enzymically active zone in starch gel electrophoresis at pH 8.5. This zone was located on the cathode side of the origin. Incubation of <em>D</em>. lebanonensis Adh with NA<em>D</em>+ and acetone altered the electrophoretic pattern to more anodal migrating zones. <em>D</em>. lebanonensis Adh has an Mr of 56,000, a subunit of Mr of <em>2</em>8 000 and is a <em>dimer</em> with two active sites per enzyme molecule. This agrees with a polypeptide chain of <em>2</em>47 residues. Metal analysis by plasma emission spectroscopy indicated that this insect alcohol dehydrogenase is not a metalloenzyme. In studies of the substrate specificity and stereospecificity, <em>D</em>. lebanonensis Adh was more active with secondary than with primary alcohols. Both alkyl groups in the secondary alcohols interacted hydrophobically with the alcohol binding region of the active site. The catalytic centre activity for propan-<em>2</em>-ol was 7.4 s-1 and the maximum velocity of most secondary alcohols was approximately the same and indicative of rate-limiting enzyme-coenzyme dissociation. For primary alcohols the maximum velocity varied and was much lower than for secondary alcohols. The catalytic centre activity for ethanol was <em>2</em>.4 s-1. With [<em>2</em>H6]ethanol a primary kinetic <em>2</em>H isotope effect of <em>2</em>.8 indicated that the interconversion of the ternary complexes was rate-limiting. Pyrazole was an ethanol-competitive inhibitor of the enzyme. The difference spectra of the enzyme-NA<em>D</em>+-pyrazole complex gave an absorption peak at 305 nm with epsilon 305 14.5 X 10(3) M-1 X cm-1. Concentrations and amounts of active enzyme can thus be determined. A kinetic rate assay to determine the concentration of enzyme active sites is also presented. This has been developed from active site concentrations established by titration at 305 nm of the enzyme and pyrazole with NA<em>D</em>+. In contrast with the amino acid composition, which indicated that <em>D</em>. lebanonensis Adh and the <em>D</em>. melanogaster alleloenzymes were not closely related, the enzymological studies showed that their active sites were similar although differing markedly from those of zinc alcohol dehydrogenases.

BACKGROUND

Currently, serum biomarkers, which are sufficiently sensitive and specific for early detection and risk classification of gastric adenocarcinoma do not exist. Therefore, this study identified a panel of serum biomarkers for the diagnosis of gastric adenocarcinoma.

METHODS

A <em>2</em>9-plex array platform with <em>2</em>9 biomarkers, consisting of 11 proteins discovered through proteomics and 18 previously known to be cancer-associated, was constructed. A test/training set consisting of 1<em>2</em>0 gastric adenocarcinoma and 1<em>2</em>0 control samples were examined. After 13 proteins were selected as candidate biomarkers, multivariate classification analyses were used to identify algorithms for diagnostic biomarker combinations. These algorithms were independently validated using a set of 95 gastric adenocarcinoma and 51 control samples.

RESULTS

Epidermal growth factor receptor (EGFR), pro-apolipoprotein A1 (proApoA1), apolipoprotein A1, transthyretin (TTR), regulated upon activation, normally T-expressed and presumably secreted (RANTES), <em>D</em>-<em>dimer</em>, vitronectin (VN), interleukin-6, α-<em>2</em> macroglobulin, C-reactive protein and plasminogen activator inhibitor-1 were selected as classifiers in the two algorithms. These algorithms differentiated between the majority of gastric adenocarcinoma and control serum samples in the training/test set with high accuracy (>88%). These algorithms also accurately classified in the validation set (>85%).

CONCLUSIONS

Two panels of combinatorial biomarkers, including EGFR, TTR, RANTES, and VN, are developed, which are less invasive method for the diagnosis of gastric adenocarcinoma. They could supplement clinical gastroscopic evaluation of symptomatic patients to enhance diagnostic accuracy.

Cocaine abuse is a worl<em>d</em>-wi<em>d</em>e public health an<em>d</em> social problem without a US Foo<em>d</em> an<em>d</em> Drug A<em>d</em>ministration-approve<em>d</em> me<em>d</em>ication. An i<em>d</em>eal anticocaine me<em>d</em>ication woul<em>d</em> accelerate cocaine metabolism, pro<em>d</em>ucing biologically inactive metabolites by a<em>d</em>ministration of an efficient cocaine-specific exogenous enzyme. Our recent stu<em>d</em>ies have le<em>d</em> to the <em>d</em>iscovery of the <em>d</em>esirable, highly efficient cocaine hy<em>d</em>rolases (CocHs) that can efficiently <em>d</em>etoxify an<em>d</em> inactivate cocaine without affecting normal functions of the CNS. Preclinical an<em>d</em> clinical <em>d</em>ata have <em>d</em>emonstrate<em>d</em> that these CocHs are safe for use in humans an<em>d</em> are effective for accelerating cocaine metabolism. However, the actual therapeutic use of a CocH in cocaine a<em>d</em><em>d</em>iction treatment is limite<em>d</em> by its short biological half-life (e.g., 8 h or shorter in rats). Here we <em>d</em>emonstrate a novel CocH form, a catalytic antibo<em>d</em>y analog, which is a fragment crystallizable (Fc)-fuse<em>d</em> CocH <em>dimer</em> (CocH-Fc) constructe<em>d</em> by using CocH to replace the Fab region of human IgG1. The CocH-Fc not only has a high catalytic efficiency against cocaine but also, like an antibo<em>d</em>y, has a consi<em>d</em>erably longer biological half-life (e.g., ∼107 h in rats). A single <em>d</em>ose of CocH-Fc was able to accelerate cocaine metabolism in rats even after <em>2</em>0 <em>d</em> an<em>d</em> thus block cocaine-in<em>d</em>uce<em>d</em> hyperactivity an<em>d</em> toxicity for a long perio<em>d</em>. Given the general observation that the biological half-life of a protein <em>d</em>rug is significantly longer in humans than in ro<em>d</em>ents, the CocH-Fc reporte<em>d</em> in this stu<em>d</em>y coul<em>d</em> allow <em>d</em>osing once every <em>2</em>-4 wk, or longer, for treatment of cocaine a<em>d</em><em>d</em>iction in humans.