We have measured the cosmic ray spectrum above 10(17.2) eV using the two air-fluorescence detectors of the High Resolution Fly's Eye observatory operating in monocular mode. We describe the detector, phototube, and atmospheric calibrations, as well as the analysis techniques for the two detectors. We fit the spectrum to a model consisting of galactic and extragalactic sources.

Journal Article↗

PubMed2003

An all-atom solution-equilibrated model for human extrinsic blood coagulation complex (sTF-VIIa-Xa): a protein-protein docking and molecular dynamics refinement study.

Tissue factor (TF)-bound factor (F)VIIa plays a critical role in activating FX, an event that rapidly results in blood coagulation. Despite recent advances in the structural information about soluble TF (sTF)-bound VIIa and Xa individually, the atomic details of the ternary complex are not known. As part of our long-term goal to provide a structural understanding of the extrinsic blood coagulation pathway, we built an all atom solution-equilibrated model of the human sTF-VIIa-Xa ternary complex using protein-protein docking and molecular dynamics (MD) simulations. The starting structural coordinates of sTF-VIIa and Xa were derived from dynamically equilibrated solution structures. Due to the flexible nature of the light-chain of the Xa molecule, a three-stage docking approach was employed in which SP (Arg195-Lys448)/EGF2 (Arg86-Arg139), EGF1 (Asp46-Thr85) and GLA (Ala1-Lys45) domains were docked in a sequential manner. The rigid-body docking approach of the FTDOCK method in conjunction with filtering based on biochemical knowledge from experimental site-specific mutagenesis studies provided the strategy. The best complex obtained from the docking experiments was further refined using MD simulations for 3 ns in explicit water. In addition to explaining most of the known experimental site-specific mutagenesis data pertaining to sTF-VIIa, our model also characterizes likely enzyme-binding exosites on FVIIa and Xa that may be involved in the ternary complex formation. According to the equilibrated model, the 140s loop of VIIa serves as the key recognition motif for complex formation. Stable interactions occur between the FVIIa 140s loop and the FXa -strand B2 region near the sodium-binding domain, the 160 s loop and the N-terminal activation loop regions. The helical-hydrophobic stack region that connects the GLA and EGF1 domains of VIIa and Xa appears to play a potential role in the membrane binding region of the ternary complex. The proposed model may serve as a reasonable structural basis for understanding the exosite-mediated substrate recognition of sTF-VIIa and to advance understanding of the TFPI-mediated regulatory pathway of the extrinsic blood coagulation cascade.

Binding Sites↗

PubMed2001

Four loops of the catalytic domain of factor viia mediate the effect of the first EGF-like domain substitution on factor viia catalytic activity.

The presence of tissue factor is essential for factor VIIa (FVIIa) to reach its full catalytic potential. The previous work in this laboratory demonstrated that substitution of the EGF1 domain of factor VIIa with that of factor IX (FVII((IXegf1))a) results in a substantial decrease in TF-binding affinity and catalytic activity. Supporting simulations of the solution structures of Ca(2+)-bound factor VIIa and FVII((IXegf1))a with tissue factor are provided. Mutants are generated, based on the simulation model, to study the effect of EGF1 substitution on catalytic activity. The simulations show larger Gla-EGF1 and EGF1-EGF2 inter-domain motions for FVII((IXegf1))a than for factor VIIa. The catalytic domain of the chimeric factor VIIa has been disturbed and several surface loops in the catalytic domain of FVII((IXegf1))a (Loop 170s (170-182), Loop 1 (185-188) and Loop 2 (221A-225)) manifest larger position fluctuations than wild-type. The position of Loop 140s (142-152) of FVII((IXegf1))a, near the N terminus insertion site of the catalytic domain, shifts relative to factor VIIa, resulting in a slight alteration of the active site. The results suggest that these four loops mediate the effect of the EGF1 domain substitution on the S1 site and catalytic residues. To test the model, we prepared mutations of these surface loops, including four FVII mutants, D186A, K188A, L144A and R147A, a FVII mutant with multiple mutations (MM3: L144A+R147A+D186A) and a FVII mutant with Loop 170s partially deleted, Loop 170s(del). The catalytic activities towards a small peptidyl substrate decreased 2.4, 4.5 and 9-fold for Loop 170s(del)a (a, activated), L144Aa and D186Aa, respectively, while MM3a lost almost all catalytic activity. The combined results of the simulations and mutants provide insight into the mechanism by which tissue factor enhances factor VIIa catalytic activity.

Amino Acid Substitution↗

PubMed2001

Modeling human zymogen factor IX.

Modern theoretical techniques are employed to provide complete three dimensional structure for the zymogen and activated forms of human coagulation factors IX and IXa. These structures are fully calcium bound and equilibrated in an electrically neutral aqueous environment. The relationship of structure to mutational data is examined. We find that a substantial relative orientational change of the catalytic domain occurs on activation. Also, we find that the electrostatistically dipolar nature of the catalytic domain is substantially modified upon activation, with cleavage of the negatively charged activation peptide leaving behind a largely hydrophobic face in factor IXa. While the backbone atoms of the catalytic residues have little relative movement, nearby loops are found that do move. The presence or absence of these changes likely defines specificity.

Amino Acid Substitution↗

PubMed2000

Heparan sulfate biosynthesis: a theoretical study of the initial sulfation step by N-deacetylase/N-sulfotransferase.

Heparan sulfate N-deacetylase/N-sulfotransferase (NDST) catalyzes the deacetylation and sulfation of N-acetyl-D-glucosamine residues of heparan sulfate, a key step in its biosynthesis. Recent crystallographic and mutational studies have identified several potentially catalytic residues of the sulfotransferase domain of this enzyme (, J. Biol. Chem. 274:10673-10676). We have used the x-ray crystal structure of heparan sulfate N-sulfotransferase with 3'-phosphoadenosine 5'-phosphate to build a solution model with cofactor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and a model heparan sulfate ligand bound, and subsequently performed a 2-ns dynamics solution simulation. The simulation results confirm the importance of residues Glu(642), Lys(614), and Lys(833), with the possible involvement of Thr(617) and Thr(618), in binding PAPS. Additionally, Lys(676) is found in close proximity to the reaction site in our solvated structure. This study illustrates for the first time the possible involvement of water in the catalysis. Three water molecules were found in the binding site, where they are coordinated to PAPS, heparan sulfate, and the catalytic residues.

Amidohydrolases↗

PubMed2000

Modeling zymogen protein C.

A solution structure for the complete zymogen form of human coagulation protein C is modeled. The initial core structure is based on the x-ray crystallographic structure of the gamma-carboxyglutamic acid (Gla)-domainless activated form. The Gla domain (residues 1-48) is modeled from the x-ray crystal coordinates of the factor VII(a)/tissue factor complex and oriented with the epidermal growth factor-1 domain to yield an initial orientation consistent with the x-ray crystal structure of porcine factor IX(a). The missing C-terminal residues in the light chain (residues 147-157) and the activation peptide residues 158-169 were introduced using homology modeling so that the activation peptide residues directly interact with the residues in the calcium binding loop. Molecular dynamics simulations (Amber-particle-mesh-Ewald) are used to obtain the complete calcium-complexed solution structure. The individual domain structures of protein C in solution are largely unaffected by solvation, whereas the Gla-epidermal growth factor-1 orientation evolves to a form different from both factors VII(a) and IX(a). The solution structure of the zymogen protein C is compared with the crystal structures of the existing zymogen serine proteases: chymotrypsinogen, proproteinase, and prethrombin-2. Calculated electrostatic potential surfaces support the involvement of the serine protease calcium ion binding loop in providing a suitable electrostatic environment around the scissile bond for II(a)/thrombomodulin interaction.

1-Carboxyglutamic Acid↗

PubMed2000

Use of microsatellite DNA markers to investigate the level of genetic diversity and population genetic structure of coconut (Cocos nucifera L.).

We have used eight pairs of simple sequence repeat (SSR) primers to analyse the genetic diversity in 130 individuals of coconut (Cocos nucifera L.) comprising 75 tall individuals and 55 dwarf individuals, representing 94 different coconut ecotypes throughout the world. A total of 51 alleles were detected, with an average of 6.4 alleles per locus. Fifty alleles were detected in tall coconuts (talls; mean alleles/locus 6.3) compared with only 26 (mean/locus 3.3) in dwarfs, and the average diversity value in talls (0.589) was also significantly higher than that in dwarfs (0.348). Using the eight SSRs we were able to uniquely discriminate 116 of the 130 individuals. A phenetic tree based on DAD (absolute distance) values clustered individuals into five groups, each mainly composed of either talls or dwarfs. These results provide evidence in support of previous hypotheses concerning the dissemination of coconut, as well as important new information for conservation and breeding purposes.

Cocos↗

PubMed2000

Expression of opioid receptors in osteoblast-like MG-63 cells, and effects of different opioid agonists on alkaline phosphatase and osteocalcin secretion by these cells.

We have previously shown that several stressful situations associated with tissue injury determine a decrease in serum osteocalcin concentration. Since reduced osteocalcin production is a marker of decreased osteoblastic activity, this finding could be related to the pathogenesis of osteoporosis secondary to some diseases. Endogenous opioids are involved in stress response. Proenkephalin-derived peptides have been shown to inhibit alkaline phosphatase activity, another marker of bone formation, in the murine cell line ROS-17/2.8. On the other hand, serum osteocalcin has been reported as being low in heroin abusers. We have therefore thought it of interest to study the presence of opioid receptors in the human osteoblast-like cell line MG-63, and to evaluate the effects of different opioid agonists on the secretion of alkaline phosphatase and osteocalcin by these cells. The presence of opioid receptors was studied by means of RT-PCR and immunohistochemistry. RT-PCR studies suggested the presence of specific mRNA for the three types of receptors, and immunohistochemistry clearly showed their occurrence. Osteocalcin synthesis was significantly inhibited by high concentrations of the mu agonists morphine and (D-Ala(2), N-MePhe(4),Gly(5)-ol)-enkephalin although no changes were seen with the delta agonist (D-Ala(2),D-leu(5))-enkephalin. Morphine-induced osteocalcin inhibition was abolished when osteoblastic cells were incubated simultaneously with naloxone, whereas it was potentiated when cells were preincubated with naloxone. None of the opioid agonists modified the secretion of alkaline phosphatase. In conclusion, human osteoblast-like cells MG-63 express the three types of opioid receptors. Endogenous opioids may be involved in the reduction of osteocalcin observed in stressful situations associated with tissue injury.

Alkaline Phosphatase↗

PubMed1999

New tricks for modelers from the crystallography toolkit: the particle mesh Ewald algorithm and its use in nucleic acid simulations.

Explore the source record for details and available documents.

Algorithms↗

PubMed1999

Probing the structural changes in the light chain of human coagulation factor VIIa due to tissue factor association.

The crystallographic structure of human coagulation factor VIIa/tissue factor complex bound with calcium ions was used to model the solution structure of the light chain of factor VIIa (residues 1-142) in the absence of tissue factor. The Amber force field in conjunction with the particle mesh Ewald summation method to accommodate long-range electrostatic interactions was used in the trajectory calculations. The estimated TF-free solution structure was then compared with the crystal structure of factor VIIa/tissue factor complex to estimate the restructuring of factor VIIa due to tissue factor binding. The solution structure of the light chain of factor VIIa in the absence of tissue factor is predicted to be an extended domain structure similar to that of the tissue factor-bound crystal. Removal of the EGF1-bound calcium ion is shown by simulation to lead to minor structural changes within the EGF1 domain, but also leads to substantial relative reorientation of the Gla and EGF1 domains.

Amino Acid Sequence↗

PubMed1999

Etidronate inhibits the production of IL-6 by osteoblast-like cells.

IL-6 is a resorbing cytokine synthesized by osteoblasts and monocytes that has been implicated in the pathogenesis of osteoporosis. Bisphosphonates are well-known antiresorptive drugs, the antiosteoclastic effect of which has been recently suggested to be brought about at least in part through osteoblasts. Based on these facts, we have studied the effect of etidronate on the production of IL-6 by two tumoral cell lines of human osteoblastic phenotype (MG63 and SaOs cells), and by peripheral blood mononuclear cells (PBMC). For comparison, another antiresorptive drug, estradiol, was included in the study. MG63 cells were stimulated with LPS and IL-1 beta, SaOs cells with LPS, IL-1 beta and PMA, and PBMC with LPS and PMA. Etidronate was tested at 10(-7), 10(-6), 10(-5), and 10(-4) M, and 17beta-estradiol was tested at 10(-10), 10(-9), 10(-8), and 10(-7) M. IL-6 was determined in supernatants by an ELISA. No significant effect of either etidronate or estradiol on IL-6 secretion by LPS or PMA-stimulated PBMC was found. However, in osteoblastic-like cells, an inhibition of IL-6 production by etidronate in LPS-stimulated cultures was found. At the highest concentrations tested, IL-6 production values were 58 +/- 9% and 53 +/- 8% of those at base line for MG63 and SaOs cells, respectively. Estradiol did not modify IL-6 secretion under any condition. In conclusion, our study supports the contention that the antiresorptive effect of bisphosphonates may be due in part to a decrease in IL-6 production by osteoblasts.

Cells, Cultured↗

PubMed1999

Identification and characterization of microsatellite loci in coconut (Cocos nucifera L.) and the analysis of coconut populations in Sri Lanka.

Explore the source record for details and available documents.

Cocos↗

PubMed1998

Trans-cis isomerization of proline 22 in bovine prothrombin fragment 1: a surprising result of structural characterization.

The calcium ion-mediated interaction of bovine prothrombin (BF1) with negatively charged phospholipid membranes is assumed to be largely via the Gla domain of BF1 with the fold of the Gla domain essential for binding. It has been reported that Pro22 undergoes classical trans to cis isomerization in the presence of calcium ions with the cis conformation of Pro22 of BF1 responsible for membrane binding [Evans, T. C., Jr., and Nelsestuen, G. L. (1996) Biochemistry 35, 8210-8215]. However, Pro22 was found to be in the trans conformation in the crystal structure of BF1. In the present work, we have used molecular dynamics simulations to investigate the relative importance of the two conformations of Pro22 to the structural and dynamical properties of BF1. The initial trans conformation of Pro22 in BF1 was slowly converted to cis-Pro22 using constrained dynamics. The second-generation AMBER force field in conjunction with the particle mesh Ewald method to accommodate long-range interaction was employed in the trajectory calculations. Comparison of the BF1(trans-Pro22) and BF1(cis-Pro22) equilibrated structures reveals surprisingly that the overall structural changes associated with the trans-cis isomerization is minimal and only minor modifications to the hydrogen bond network and the network of N-terminus Ala1 take place. The calculated electrostatic potential energy surfaces of the two protein structures also appear to be very similar, indicating the near equality of the local interaction site environments in the protein prior to lipid binding.

1-Carboxyglutamic Acid↗

PubMed1998

Engineering of betabellin-15D: a 64 residue beta sheet protein that forms long narrow multimeric fibrils.

The betabellin target structure is a beta-sandwich protein consisting of two 32 residue beta-sheets packed against one another by interaction of their hydrophobic faces. The 32 residue chain of betabellin-15S (HSLTAKIpkLTFSIAphTYTCAV pkYTAKVSH, where p=DPro, k=DLys, and h=DHis) did not fold in water at pH 6.5. Air oxidation of betabellin-15S provided betabellin-15D, the 64 residue disulfide bridged two-chain molecule, which also remained unfolded in water at pH 6.5. By circular dichroic spectropolarimetry, the extent of beta structure observed for betabellin-15D increased with the pH and ionic strength of the solution and the betabellin-15D concentration. By electron microscopy, in 5.0 mM MOPS and 0.25 M NaCl at pH 6.9, betabellin-15D formed long narrow multimeric fibrils. A molecular model was constructed to show that the dimensions of these betabellin-15D fibrils are consistent with a single row of beta-sandwich molecules joined by multiple intersheet H-bonds.

Amino Acid Sequence↗