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At least 1,621 records · Page 90Linked to original sources
PubMed1999

Aiding clinical decisions with decision analysis.

As clinical decision making gets ever more complex, new analytical approaches are being developed to help. Decision analysis is used to structure complex decision problems in an uncertain environment by systematically linking decision choices with expected outcomes. Such models can include the probabilities of outcomes, patient preferences and costs. These models can help to advise about therapeutic avenues. This paper examines the nature of decision analysis, and explores the pitfalls that arise in interpreting the findings from published studies.

Decision Making↗
PubMed1998

Transgenically produced human antithrombin: structural and functional comparison to human plasma-derived antithrombin.

Recombinant human antithrombin (rhAT) produced in transgenic goat milk was purified to greater than 99%. The specific activity of the rhAT was identical to human plasma-derived AT (phAT) in an in vitro thrombin inhibition assay. However, rhAT had a fourfold higher affinity for heparin than phAT. The rhAT was analyzed and compared with phAT by reverse phase high-performance liquid chromatography, circular dichroism, fluorophore-assisted carbohydrate electrophoresis (FACE), amino acid sequence, and liquid chromatography/mass spectrography peptide mapping. Based on these analyses, rhAT was determined to be structurally identical to phAT except for differences in glycosylation. Oligomannose structures were found on the Asn 155 site of the transgenic protein, whereas only complex structures were observed on the plasma protein. RhAT contained a GalNAc for galactose substitution on some N-linked oligosaccharides, as well as a high degree of fucosylation. RhAT was less sialylated than phAT and contained both N-acetylneuraminic and N-glycolylneuraminic acid. We postulate that the increase in affinity for heparin found with rhAT resulted from the presence of oligomannose-type structures on the Asn 155 glycosylation site and differences in sialylation.

Animals↗
PubMed2003

Synthetic diacylglycerols (DAG) and DAG-lactones as activators of protein kinase C (PK-C).

The central role of protein kinase C (PK-C) in cellular signal transduction has established it as an important therapeutic target for cancer and other diseases. We have developed a series of 4,4-disubstituted-gamma-butyrolactones, which contain a constrained glycerol backbone (DAG-lactones) and behave as potent and selective activating ligands of PK-C with affinities that approach those of the structurally complex natural product agonists, such as the phorbol esters. This Account traces the design and construction of these molecules. Initially, we examined the consequences of reducing the entropic penalty associated with the transformation of a DAG into a DAG-lactone. Then, using molecular modeling to extend insights arising from the newly solved crystal structure of a C1 domain complexed with phorbol ester, we incorporated amino acid-specific branched hydrophobic chains to provide a new generation of DAG-lactones that have the capacity to bind to PK-C with low nanomolar affinity. Depending on the specific pattern of hydrophobic substitution, some DAG-lactones are able to induce selective translocation of individual PK-C isozymes to different cellular compartments, and since the specific nature of these hydrophobic interactions influences biological outcome, some of these compounds exhibit cell-specific antitumor activity. The ability to direct specific PK-C isozyme translocation with sets of structurally simple, yet highly potent molecules provides a powerful tool for engineering a plethora of molecules with novel biological functions.

Diglycerides↗
PubMed1994

The crystal structure of affinity-matured human growth hormone at 2 A resolution.

A variant of human growth hormone (hGH), in which 15 mutations were introduced with phage display mutagenesis to improve receptor binding affinity by 400-fold, yielded two related crystal forms diffracting to high resolution. The structure of this variant was determined in both crystal forms, one at 2.0 A resolution and one at 2.4 A resolution, using molecular replacement with wild-type hGH taken from the receptor complex structure as a search model. Crystallographic refinement of the 2 A structure gave an R-value R-value of 18.5% for data in the resolution range 8 to 2 A. The final model consists of residues 1 to 128 and 155 to 191, with three side-chains modeled in alternative conformations, together with 77 water molecules. Comparison of the structure with wild-type hGH shows that most of the secondary structural elements are unchanged. The exception is the first turn of the third helix in the four-helix bundle core, which is unraveled in the present variant. Analysis of the two related packing environments suggests that this change is caused by crystal packing forces. A large change in the orientation of a short segment of helix found in the connection between the first two core helices is interpreted as evidence for rigid-body variability of this helical segment. Analysis of the mutations in light of the structure of the wild-type hGH/receptor complex shows that six of the mutations are buried in the hormone, whereas the remaining nine involve residues that interact with the receptor in the complex.

Amino Acid Sequence↗
PubMedDate not supplied

Three-dimensional structure of a complex of E2020 with acetylcholinesterase from Torpedo californica.

The 3D structure of a complex of the anti-Alzheimer drug, E2020, also known as Aricept, with Torpedo californica acetylcholinesterase is reported. The X-ray structure, at 2.5 A resolution, shows that the elongated E2020 molecule spans the entire length of the active-site gorge of the enzyme. It thus interacts with both the 'anionic' subsite, at the bottom of the gorge, and with the peripheral anionic site, near its entrance, via aromatic stacking interactions with conserved aromatic residues. It does not interact directly with either the catalytic triad or with the 'oxyanion hole'. Although E2020 is a chiral molecule, and both the S and R enantiomers have similar affinity for the enzyme, only the R enantiomer is bound within the active-site gorge when the racemate is soaked into the crystal. The selectivity of E2020 for acetylcholinesterase, relative to butyrylcholinesterase, can be ascribed primarily to its interactions with Trp279 and Phe330, which are absent in the latter.

Acetylcholinesterase↗
PubMed1993

FK-506-binding protein: three-dimensional structure of the complex with the antagonist L-685,818.

L-685,818 differs only slightly in structure from the immunosuppressive drug FK-506, and both compounds bind with comparable affinity to the 12-kDa FK-506-binding protein (FKBP12), the major intracellular receptor for the drug. Despite these similarities, L-685,818 is a potent antagonist of both the immunosuppressive and toxic effects of the drug. Here, we present a structural analysis of this problem. Although FK-506 and L-685,818 differ greatly in pharmacology, we have found that the three-dimensional structures of their complexes with FKBP12 are essentially identical. Approximately half of each ligand is in contact with the receptor protein, and half is exposed to solvent; the exposed region includes the two sites where the compounds differ. These results indicate that the profound differences in the pharmacology of these two compounds are not caused by any difference in their interaction with FKBP12. Rather, these effects arise because relatively minor changes in the exposed part of a bound ligand have a strong effect on how FKBP12-ligand complexes interact with calcineurin, their putative intracellular target. In addition, FK-506 complexes with FKBP12 proteins from several species all inhibit mammalian calcineurin. Analysis of the three-dimensional structure of the complex with respect to residues conserved among these proteins suggests a small number of surface residues near the bound ligands that may play a critical role in interactions between the protein-drug complex and calcineurin.

Amino Acid Sequence↗
PubMed2003

Identification and characterization of ADAMTS-20 defines a novel subfamily of metalloproteinases-disintegrins with multiple thrombospondin-1 repeats and a unique GON domain.

We have cloned a mouse brain cDNA encoding a new protein of the ADAMTS family (a disintegrin and metalloproteinase domain, with thrombospondin type-1 repeats), which has been called ADAMTS-20. This protein shows a domain organization similar to that described for other ADAMTSs including signal sequence, propeptide, metalloproteinase domain, disintegrin domain, central TS-1 motif, cysteine-rich region, and C-terminal TS module. However, this last module is more complex than that of other ADAMTSs, being composed of a total of 14 repeats. The structural complexity of ADAMTS-20 is further increased by the presence of an additional domain 200 residues long and located immediately adjacent to the TS module. This domain has been tentatively called GON domain and can also be recognized in some ADAMTSs such as gon-1 from Caenorhabditis elegans and human and mouse ADAMTS-9. The presence of this domain is a hallmark of a novel subfamily of structurally and evolutionarily related ADAMTSs, called GON-ADAMTSs. Expression analysis demonstrated that ADAMTS-20 transcripts can be detected at low levels in several human and mouse tissues, especially in testis. This gene is also overexpressed in some human malignant tumors, including brain, colon, and breast carcinomas. Western blot analysis using polyclonal antibodies raised against recombinant ADAMTS-20 produced in Escherichia coli showed the presence of a 70-kDa band in mouse brain and testis extracts. This recombinant ADAMTS-20 hydrolyzed a synthetic peptide used for assaying matrix metalloproteinases. These data suggest that this novel enzyme may play a role in the tissue remodeling process occurring in both normal and pathological conditions.

ADAM Proteins↗
PubMed2006

Do structurally similar ligands bind in a similar fashion?

The scope of the current work is to investigate whether structurally similar ligands bind in a similar fashion by exhaustively analyzing experimental data from the protein database (PDB). The complete PDB was searched for pairs of structurally similar ligands binding to the same biological target. The binding sites of the pairs of proteins complexing structurally similar ligands were found to differ in 83% of the cases. The most recurrent structural change among the pairs involves different water molecule architecture. Side-chain movements are observed in half of the pairs, whereas backbone movements rarely occurred. However, two structurally similar ligands generally confirm a high degree of structural conservation. That is, a majority of the ligand pairs occupy the same region in the binding sites, providing support for the use of shape matching in the drug design process. We allow ourselves to draw general conclusions because our data set consists of ligands with drug-like physicochemical properties complexed to a broad spectrum of different protein classes.

Crystallography, X-Ray↗
PubMed1999

Site-specific characterization of the N-linked glycans of murine prion protein by high-performance liquid chromatography/electrospray mass spectrometry and exoglycosidase digestions.

The murine prion protein PrP gene encodes a protein of 254 amino acids with two consensus sites for Asn-linked glycosylation at codons 180 and 196. A partial site-specific study of the N-linked glycans from hamster PrP has previously been carried out by mass spectrometry [Stahl, N., Baldwin, M. A., Teplow, D. B., Hood, L., Gibson, B. W., Burlingame, A. L., and Prusiner, S. B. (1993) Biochemistry 32, 1991-2002] and revealed that the glycosylation at Asn-181 (equivalent to mouse 180) is heterogeneous, comprising over 30 glycoforms. The identification of the glycosylated peptide spanning Asn-197 was not reported. Recent technical advances in electrospray mass spectrometry now provide the sensitivity to detect low femtomole quantities of glycopeptides with >5000 mass resolution and 30 ppm mass measurement [Medzihradszky, K. F., Besman, M. J., and Burlingame, A. L. (1998) Rapid Commun. Mass Spectrom. 12, 472-478]. This performance coupled with stepwise exoglycosidase digestion has been employed to establish the differential nature of the structural complexity (glycoforms) of the glycans at Asn-180 and Asn-196 from a single strain infected with the ME7 strain. Some sixty structures have been found characterized by neutral and sialylated bi-, tri-, and tetraantennary complex-type bearing outer-arm alpha(1-3)-fucosylation (the Lewisx and sialyl-Lewisx epitopes), core alpha(1,6) fucosylation, and the presence of terminal HexNAc residues. The Lewisx trisaccharide is the major nonreducing structure at Asn-180, and significant amounts of both Lewisx and sialyl Lewisx epitopes are observed at Asn-196. The abundance of the Lewisx and sialyl Lewisx epitopes on murine PrPSc may indicate a role for these structures in the normal function of PrPC or the pathophysiology of PrPSc.

Amino Acid Sequence↗
PubMed2003

Sperm ultrastructure and spermatogenesis in the lizard, Tropidurus itambere.

Spermatogenesis, with emphasis on spermiogenesis, is described for the lizard, Tropidurus itambere, using light microscopy, phase contrast and epifluorescence, as well as scanning and transmission electron microscopy. Cellular differentiation involves events of chromatin condensation, nuclear elongation and the formation of structural complexes, such as the acrosomal and axonemal ones. Other new characteristics, exclusive for this species, include various aspects of the subacrosomal granule, the insertion of the pro-acrosomal vesicle and the development of these structures to participate in the acrosomal complex. Radial projections occurjust above the nuclear shoulders, which have been recognized already from the beginning of cellular elongation. The development of the midpiece, the dense bodies, formation of the flagellum and elimination of residual cytoplasm result in the final characterization of the mature spermatozoon. Comparisons between Tropiduridae and other lizard families are made.

Acrosome↗
PubMed2025

Recurrent structural variation and recent turnover at the 17q21.31 locus in humans and great apes.

The 17q21.31 locus in humans harbors several complex structural haplotypes including a ~970kb inversion. Different inversion haplotypes have been associated with susceptibility to microdeletions causing Koolen-de Vries syndrome and variation in fecundity and recombination rates. Here, using 210 haplotype-resolved human genome assemblies and pangenome graph-based approaches we characterize 11 distinct structural haplotypes, several of which have not been previously described. Extending our analyses to a set of haplotype-resolved great-ape genomes, we characterize the structure of an independent inversion in chimpanzees which extends an additional 650kb, encompasses 5 additional genes, and is ~2 million years younger than the human inversion. We further determine that gorillas exhibit an independent duplication of the KANSL1 gene which may predispose them to Koolen-de Vries syndrome causing microdeletions. Using short read sequencing data we characterize 17q21.31 haplotype diversity worldwide in ~5174 individuals from 107 populations finding increased frequencies of KANSL1 duplication-containing haplotypes in both European and South Asian populations as well as 8 double recombination events between inverted and non-inverted haplotypes ranging in size from 20-180kb. Finally, using 626 ancient Eurasian human genomes we show the frequency of haplotypes containing KANSL1 duplications has increased ~6-fold over the past 12 thousand years in Europe. Together, our results highlight the dynamics, complexity, and recurrent, independent evolution of a medically relevant locus across humans and great apes.

Journal Article↗
PubMed1998

Evolutionary classification of homeodomains.

PURPOSE AND METHODS: We performed multiple comparisons between available amino acid (aa) sequences of homeodomain(HOM)-containing proteins from a wide spectrum of animals to create an evolutionary classification of the proteins. RESULTS: Based on results of statistical and special computational analyses of over 500 homeodomain aa sequences (HOMs) a novel system of concepts describing complex structural correlations between homologous proteins is proposed. This system includes such notions as differentiated isofunctionality of aa, chemotype, stereotype, local functional motifs, gradual conservativeness of aa positions, and group-specific domain patterns, as well as major categories of the evolutionary classification of HOMs (Division, Type, Branch, Class, Family, Series, Variety, Sort). Using this approach, a complete structural systematics of HOMs belonging to proteomes of eukaryotic animals is proposed. CONCLUSIONS: The proposed structural classification of HOMs is in full agreement with the bulk of experimental data revealing complex functional similarities and differences among HOMs in terms of their expression patterns in developing embryos. It turn, this classification can provide answers regarding homology among homeodomains when experimental data are conflicting.

Amino Acid Sequence↗
PubMed1996

Generation of polyketide libraries via combinatorial biosynthesis.

Polyketides are a family of structurally complex natural products that include a number of important pharmaceuticals. Motivated by the value of these natural products, there has been much research focused on developing guidelines for engineering polyketide synthases (PKSs) to generate novel polyketides. Recent studies have provided interesting insights into the enzymatic specificity of the polyketide synthesis pathway, and have demonstrated that various PKSs can be genetically manipulated to synthesize 'unnatural' polyketide natural products. In this article, we discuss the synthesis of polyketides and polyketide libraries by combinatorial biosynthesis.

Anti-Bacterial Agents↗
PubMed2004

Theoretical analysis of the three-dimensional structure of tetrathiolato iron complexes.

The three-dimensional structures of a number of [M(SR)(4)](n-) complexes, where M is a 3d transition metal and R is an alkyl or aryl group, have been analyzed using density functional theory (DFT). Special attention is paid to the Fe(II)/Fe(III) mimics of rubredoxin. The Fe(II) model complex [Fe(SCH(3))(4)](2-) has an equilibrium conformation with D2d symmetry. The DFT energy has been decomposed into contributions for ligand-ligand and metal-ligand interactions. The latter contribution is analyzed with the angular overlap model (AOM) and constitutes the dominant stereospecific interaction in the Fe(II) complex. The sulfur lone-pair electrons exert anisotropic pi interactions on the 3d(6) shell of Fe(II), which are controlled by the torsion angles, omega(i), for the rotations of the S(i)-C(beta) bonds around the Fe-S(i) axes. In contrast, the pi interactions acting on the high-spin 3d(5) shell of Fe(III) are isotropic. As a consequence, the stereochemistry of the Fe(III) complexes is determined by the Coulomb repulsions between the ligands and has S(4) symmetry. The electrostatic repulsions between the lone pairs of the sulfurs are an essential component of the ligand-ligand interaction. The lone-pair repulsions distort the 90 degree angle SFeS' angles (delta + delta(t)) and give rise to a correlation between delta and omega, which is confirmed by crystallographic data. Both the Fe(II) and Fe(III) complexes exhibit structural bistability due to the presence of low-lying equilibrium conformations with S(4) symmetry in which the complex can be trapped by the crystalline host.

Algorithms↗
PubMed1999

Electronic Structure of Metallacyclophosphazene and Metallacyclothiazene Complexes.

The electronic structure of metallacyclotriphosphazene complexes with several substituents at the phosphorus atoms and metallacyclothiazene complexes is explored for a variety of transition metal elements using density functional theory methods. Accordingly the metallacyclophosphazenes possess a large HOMO-LUMO energy separation while the metallacyclothiazenes bear stronger open-shell character. In addition our calculations predict the existence of experimentally so far unknown dimetallacyclophosphazenes. All structures show to be highly dynamical. The double bond character of the transition metal nitrogen bond is much less pronounced than in nitrido or imido complexes. For the ring compounds vibrational spectra are reported and compared with experimental data.

Journal Article↗
PubMed2025

A small cationic probe for accurate, punctate discovery of RNA tertiary structure.

RNA molecules fold into intricate three-dimensional tertiary structures that are central to their biological functions. Yet reliably discovering new motifs that form true tertiary interactions remains a major challenge. Here we show that RNA tertiary folding occasionally generates electronegative motifs that react selectively with the small, positively-charged probe trimethyloxonium (TMO). Sites with enhanced reactivity to TMO, compared with the neutral reagent dimethyl sulfate (DMS), are indicative of tertiary structure and define T-sites. These positions share a structural signature in which a reactive nucleobase is adjacent to non-bridging phosphate oxygens, creating localized regions of negative charge. T-sites consistently map to the cores of higher-order structural interactions and functional centers across diverse RNAs, including distinct states in conformational ensembles. In the 10,723-nt dengue virus genome, three strong T-sites were detected, each within a complex structure required for viral replication. Cation-based covalent chemistry enables high-confidence discovery and analysis of functional RNA tertiary motifs across long and complex RNAs, opening new opportunities for transcriptome-wide structural analysis.

RNA electrostatics↗
PubMed2004

Determination of binding constants of cyclodextrin inclusion complexes with amino acids and dipeptides by potentiometric titration.

Cyclodextrins are well known for their ability to separate enantiomers of drugs, natural products, and other chiral substances using HPLC, GC, or CE. The resolution of the enantiomers is due to the formation of diastereomeric complexes between the cyclodextrin and the pairs of enantiomers. The aim of this study was to determine the binding constants of the complexes between alpha- and beta-cyclodextrin and the enantiomers of a series of aliphatic and aromatic amino acids, and dipeptides, using a potentiometric titration method. The results of this method are compared to other methods, and correlated to findings in cyclodextrin-modified capillary electrophoresis and possible complex structures. Potentiometric titration was found to be an appropriate tool to determine the binding constants of cyclodextrin inclusion complexes.

Amino Acids↗
PubMed2006

Fatty-acid-binding protein from the flight muscle of Locusta migratoria: evolutionary variations in fatty acid binding.

Intracellular lipid-binding proteins have evolved from a common ancestral gene with the appearance of mitochondrial oxidation, to guarantee, for example, transport of fatty acids through the aqueous cytosol to their site of utilization. The mammalian forms of these lipid carriers are structurally well-characterized and have been categorized, on the basis of sequence similarities and several typical ligand-binding features, into four subfamilies. Only a single complex structure of an invertebrate fatty-acid-binding protein (FABP) has been reported to date, which reveals a unique ligand-binding arrangement yet unknown in vertebrate FABPs. In the present study, the structure of a second invertebrate FABP (locust muscle) complexed with a fatty acid has been determined on the basis of intermolecular NOE connectivities between the protein and the uniformly (13)C-enriched oleate ligand. The resulting ligand conformation, although resembling the closely related mammalian heart- and adipocyte-type FABPs, is characterized by certain binding features that differ significantly from the typical hairpin-turn ligand shapes of the latter forms. This is primarily due to an alanine-to-leucine substitution in locust FABPs that produces a steric hindrance for ligand binding. A comparison with an FABP from tobacco hornworm larvae furthermore demonstrates that certain amino acid substitutions that appear to be specific for invertebrates decidedly influence the binding arrangement inside the protein cavity. Hence, as a result of these evolutionary variations, invertebrate FABPs may display a much greater diversity in intracellular lipid binding than observed for the mammalian transport proteins, thus possibly providing new insights for the design of modified lipid carriers.

Amino Acid Sequence↗