Showing papers by "Torrey Pines Institute for Molecular Studies published in 1999"
TL;DR: Comparisons show that the new procedure, EPMR, can identify solutions using significantly less accurate or less complete search models than is possible with two existing molecular-replacement methods.
Abstract: A new procedure for molecular replacement is presented in which an efficient six-dimensional search is carried out using an evolutionary optimization algorithm. In this procedure, a population of initially random molecular-replacement solutions is iteratively optimized with respect to the correlation coefficient between observed and calculated structure factors. The sensitivity and reliability of the method is enhanced by uniform sampling of the rotational-search space and the use of continuously variable rotational and translational parameters. The process is several orders of magnitude faster than a systematic six-dimensional search, and comparisons show that it can identify solutions using significantly less accurate or less complete search models than is possible with two existing molecular-replacement methods. A program incorporating the method, EPMR, allows the rapid and highly automated solution of molecular-replacement problems involving single or multiple molecules in the asymmetric unit. EPMR has been used to solve a number of difficult molecular-replacement problems.
576 citations
TL;DR: By surveying the literature, this compilation can be used to make reasonable estimates for a wide range of organisms in the calculation of relative abundance and a phylogenetic analysis is used to offer insights into the evolution of both genome size and SSU rDNA copy number.
Abstract: > Abstract Determination of the relative abundance of a specific prokaryote in an environmental sample is of major interest in applied and environmental microbiology. Relative abundance can be calculated using knowledge of SSU rDNA copy number, amount of SSU rDNA in the sample, and a weighted average estimate of the genome sizes for organisms in the original sample. By surveying the literature, we provide estimates of genome size and SSU rDNA copy number for 303 and 101 prokaryotes, respectively. This compilation can be used to make reasonable estimates for a wide range of organisms in the calculation of relative abundance. A statistical analysis suggests that no correlation exists between genome size and SSU rDNA copy number. A phylogenetic analysis is used to offer insights into the evolution of both genome size and SSU rDNA copy number.http://link.springer-ny.com/link/service/journals/00248/bibs/38n2p93.html
332 citations
TL;DR: In this paper, the mean square potential associated with axisymmetric poloidal flows is given in terms of the linear response function and a model correlation function for the current source.
Abstract: As a result of turbulence and finite Larmor radius effects, random radial currents are present in a tokamak plasma and these drive sheared axisymmetric poloidal flows. We model these currents with a noise source with given statistical properties and calculate the linear kinetic response to this source. Without collisions, there is no long term damping of these flows; when collisions are included, poloidal flows are damped. The mean square potential associated with these flows is given in terms of the linear response function we calculate and a model correlation function for the current source. Without collisions, the mean square flow increases linearly with time, but with collisions, it reaches a steady state. In the long correlation time limit, the collisionless residual flows are important in determining the mean square flow.
246 citations
243 citations
TL;DR: The effect of polymer chemistry on adhesion, proliferation, and morphology of human articular cartilage (HAC) chondrocytes was evaluated on synthetic degradable polymer films and tissue culture polystyrene as a control and suggests that regardless of initial seeding density on these degradably polymer substrates, they will eventually populate the surfaces of all these polymers given sufficient space and time.
239 citations
TL;DR: A new method to effectively identify both microbial epitopes and candidate autoantigens is described and it is found that the antigen specificity of a single T-cell clone can be degenerate and yet the clone can preferentially recognize different peptides derived from the same organism, thus demonstrating that flexibility in T- cell recognition does not preclude specificity.
Abstract: Elucidating the cellular immune response to infectious agents is a prerequisite for understanding disease pathogenesis and designing effective vaccines. In the identification of microbial T-cell epitopes, the availability of purified or recombinant bacterial proteins has been a chief limiting factor. In chronic infectious diseases such as Lyme disease, immune-mediated damage may add to the effects of direct infection by means of molecular mimicry to tissue autoantigens. Here, we describe a new method to effectively identify both microbial epitopes and candidate autoantigens. The approach combines data acquisition by positional scanning peptide combinatorial libraries and biometric data analysis by generation of scoring matrices. In a patient with chronic neuroborreliosis, we show that this strategy leads to the identification of potentially relevant T-cell targets derived from both Borrelia burgdorferi and the host. We also found that the antigen specificity of a single T-cell clone can be degenerate and yet the clone can preferentially recognize different peptides derived from the same organism, thus demonstrating that flexibility in T-cell recognition does not preclude specificity. This approach has potential applications in the identification of ligands in infectious diseases, tumors and autoimmune diseases.
219 citations
TL;DR: Reversed phase-high performance chromatography (RP-HPLC) and surface plasmon resonance (SPR) are emerging techniques for the study of the dynamics of the interactions between cytolytic and antimicrobial peptides and lipid surfaces and immobilization of lipid moieties onto RP- HPLC sorbent allows the investigation of peptide conformational transition upon interaction with membrane surfaces.
196 citations
TL;DR: It is suggested that brain CRF may be substantially involved in the development of “anxiety-like” responses related to cocaine withdrawal and could be important for future drug dependence treatments.
Abstract: Rationale: Chronic cocaine abuse is associated with the development of anxiogenic states in humans. Corticotropin-releasing factor (CRF) is an endogenous neurotropic factor well known to modulate stress responses. It has been postulated that CRF is involved in the neurobiological mechanisms underlying the anxiety and/or stress responses associated with removal of cocaine after chronic administration. Objective: The present study investigated the role of endogenous CRF in mediating the “anxiety-like” effect 48 h after the cessation of saline or chronic cocaine treatment in rats, using the defensive burying paradigm and the elevated plus-maze. Methods: Rats received daily injections of cocaine (20 mg/kg IP, for 14 consecutive days) or vehicle. Forty-eight hours after the last injection, animals were tested in the plus-maze and then in the defensive burying paradigm. In a second experiment, intracerebroventricular (ICV) cannulae were implanted at the lateral ventricle. Animals were allowed a 1-week period for recovery before starting the chronic drug treatment. The defensive burying testing took place 48 h after cessation of the treatment. The CRF antagonist [DPhe12, Nle21,38, CαMe Leu37] r/h CRF(12–41), (also known as D-phe CRF(12–41)) (0.04, 0.2 and 1.0 μg/5 μl) was injected 5 min before the 15-min testing. Results: An “anxiogenic-like” effect following chronic cocaine treatment was demonstrated with the defensive burying paradigm, but not with the elevated plus-maze. This “anxiety-like” response was attenuated by ICV pretreatment with the CRF antagonist D-Phe CRF(12–41), with the highest dose of the CRF antagonist reversing the observed “anxiogenic-like” response. Conclusions: These data suggest that brain CRF may be substantially involved in the development of “anxiety-like” responses related to cocaine withdrawal and could be important for future drug dependence treatments.
189 citations
TL;DR: Evaluation of F1 hybrids and interspecific progenies, as well as the segregation of resistance in F2 and F3 lines of the IR64 × Gigante cross, provided results consistent with the presence of a single recessive resistance gene common to Tog5681 and Gigante.
Abstract: Three cultivars of Oryza sativa (IR64, Azucena, and Gigante) and four cultivars of O. glaberrima (Tog5681, Tog5673, CG14, and SG329) were evaluated for their resistance to two isolates of rice yellow mottle virus (RYMV) by enzyme-linked immunosorbent assay (ELISA) and symptomatology. Cultivars Tog5681 and Gigante were highly resistant, and no symptoms were observed when either virus isolate was inoculated at 10 or 20 days postgermination and assayed by ELISA at 7, 14, 22, 35, 50, or 64 days postinoculation. Azucena showed a partial resistance, whereas the other cultivars were susceptible. Symptom appearance was associated with increase in ELISA absorbance in the systemically infected leaves. The best discrimination among the cultivars occurred when the plants were inoculated at 10 days postgermination. Crosses were made between the highly resistant (Gigante and Tog5681) and the susceptible (IR64) cultivars to determine the genetic basis of resistance to RYMV. Evaluation of F1 hybrids and interspecific progenies, as well as the segregation of resistance in F2 and F3 lines of the IR64 × Gigante cross, provided results consistent with the presence of a single recessive resistance gene common to Tog5681 and Gigante.
112 citations
TL;DR: The definition of epitopes for human B and T cells is fundamental for the understanding of the immune response mechanism and its role in the prevention and cause of human disease and the design of diagnostics and synthetic vaccines is applied.
108 citations
TL;DR: The consensus of computational results suggests a set close to 300 genes, which will be evaluated by engineering of small bacterial genomes, sufficient for cellular life.
TL;DR: Comparison of the IRF-Smad alignment to the known three-dimensional structure of human tumor suppressor Smad4 suggests that a conserved loop, equivalent to Loop 3 in Smad 4, is a determinant of protein-protein interaction in IRFs.
Abstract: Interferon regulatory factors (IRFs) regulate the transcription of both interferon-inducible genes and interferons themselves. Along with the N-terminal, DNA-binding, winged-helix domain, most IRFs contain the C-terminal domains that are shown to be related to the C-terminal domains in the proteins of the Smad family that mediate transcription activation in the transforming growth factor response pathway. Comparison of the IRF-Smad alignment to the known three-dimensional structure of human tumor suppressor Smad4 suggests that a conserved loop, equivalent to Loop 3 in Smad 4, is a determinant of protein-protein interaction in IRFs.
TL;DR: In non-B cell lines, like the murine erythroleukemia cell line (MEL), the most distal IgH constant region gene, C alpha, replicates early in S; other heavy chain constant region genes, joining and diversity segments, and the most proximal Vh gene replicate successively later in S in a 3' to 5' direction proportional to their distance from C alpha.
Journal Article•
TL;DR: It is demonstrated that many of these deduced ligands are not only effective immunogens in vivo, but are capable of inducing T cell responses to the original native ligands used to generate the clones.
Abstract: Recent studies have demonstrated the utility of synthetic combinatorial libraries for the rapid identification of peptide ligands that stimulate clonotypic populations of T cells. Here we screen a decapeptide combinatorial library arranged in a positional scanning format with two different clonotypic populations of CD4+ T cells to identify peptide epitopes that stimulate proliferative responses by these T cells in vitro. An extensive collection of mimic peptide sequences was synthesized and used to explore the fine specificity of TCR/peptide/MHC interactions. We also demonstrate that many of these deduced ligands are not only effective immunogens in vivo, but are capable of inducing T cell responses to the original native ligands used to generate the clones. These results have significant implications for considerations of T cell specificity and the design of peptide vaccines for infectious disease and cancer using clinically relevant T cell clones of unknown specificity.
TL;DR: Northern hybridization and RT-PCR demonstrated that the expression level of MAGED1 in different normal adult tissues is comparable to that in testis and fetal liver, suggesting that the biology of the MAGE-family genes is more complex than previously thought.
TL;DR: This chapter describes a method for the in vitro generation of a 3‐dimensional cartilage matrix from articular chondrocytes seeded onto a bioresorbable polymeric scaffold, containing the major cartilage constituents: sulfated proteoglycan, collagen type II, and water.
Abstract: This chapter describes a method for the in vitro generation of a 3-dimensional cartilage matrix from articular chondrocytes seeded onto a bioresorbable polymeric scaffold. This particular growth system was chosen for the subject of this chapter owing to the relative simplicity of the methods required and the ease with which the necessary materials can be obtained. The tissue produced using this protocol is a cellular, metabolically active hyaline-like matrix, containing the major cartilage constituents: sulfated proteoglycan, collagen type II, and water. It serves as a useful in vitro tool for studying the influence of various mechanical and chemical factors on cartilage metabolism, as well as providing an implantable material for in vivo cartilage repair studies.
TL;DR: The synthesis of polyamines is a rapidly developing area of vital importance to biomedical science as mentioned in this paper, and N-alkylation followed by N-terminal acylation and the complete reduction of carbonyl amide bonds enables the preparation by parallel solid phase synthesis of a wide range of N 1,N 5,1,4-tetrasubstituted-1,5-diamino-3-azapentane derivatives.
Patent•
05 Mar 1999TL;DR: In this article, the authors describe a prodrugs of formula (I), their uses, their intermediates, and their method of manufacture, where the prodrug is attached to a carbon, oxygen, or nitrogen atom.
Abstract: Prodrugs of formula (I), their uses, their intermediates, and their method of manufacture are described, wherein V is selected from the group consisting of -H, aralkyl, alicyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, 1-alkenyl, 1-alkynyl, and -R9; or together V and Z are connected via 3-5 atoms to form a cyclic group, optionally containing 1 heteroatom, substituted with hydroxy, acyloxy, alkoxycarbonyloxy, or aryloxycarbonyloxy attached to a carbon atom that is three atoms from an oxygen attached to the phosphorus; or together V and Z are connected via 3-5 atoms to form a cyclic group, optionally containing 1 heteroatom, that is fused to an aryl group at the beta and gamma position to the oxygen attached to the phosphorus; or together V and W are connected via 3 carbon atoms to form an optionally substituted cyclic group containing 6 carbon atoms and substituted with one substituent selected from the group consisting of hydroxy, acyloxy, alkoxycarbonyloxy, alkylthiocarbonyloxy, and aryloxycarbonyloxy, attached to a carbon atom that is three atoms from an oxygen attached to the phosphorus; W and W' are independently selected from the group consisting of -H, alkyl, aralkyl, alicyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, 1-alkenyl, 1-alkynyl, and -R9; Z is selected from the group consisting of -CHR2OH, -CHR?2OC(O)R3, -CHR2OC(S)R3, -CHR2OC(S)OR3, -CHR2OC(O)SR3, -CHR2OCO?2R?3, -OR2, -SR2, -CHR2N?3, -CH2aryl, -CH(aryl)OH, -CH(CH=CR22)OH, -CH(C CR?2)OH, -R2, -NR2?2, -OCOR3, -OCO?2?R?3, -SCOR3?, -SCO?2R?3, -NHCOR2, -NHCO?2?R?3, -CH?2NHaryl, -(CH2)p-OR2, and -(CH?2?)p-SR?2; R2? is an R3 or -H; R3 is selected from the group consisting of alkyl, aryl, aralkyl, and alicyclic; and R9 is selected from the group consisting of alkyl, aralkyl, and alicyclic; p is an integer from 2 to 3; with the provisos that a) V, Z, W, and W' are not all -H; and b) when z is -R2, then at least one of V and W is not -H, or -R9; and M is selected from the group that attached to PO?3?2-, P2O63-, or P?3O9?4- is biologically active in vivo, and that is attached to the phosphorus in formula (I) via a carbon, oxygen, or nitrogen atom; and pharmaceutically acceptable prodrugs and salts thereof.
TL;DR: In this article, the authors presented the results of molecular docking simulations with HIV-1 protease for the sb203386 and skf107457 inhibitors by Monte Carlo simulated annealing.
Abstract: We present the results of molecular docking simulations with HIV-1 protease for the sb203386 and skf107457 inhibitors by Monte Carlo simulated annealing. A simplified piecewise linear energy function, the standard AMBER force field, and the AMBER force field with solvation and a soft-core smoothing component are employed in simulations with a single-protein conformation to determine the relationship between docking simulations with a simple energy function and more realistic force fields. The temperature-dependent binding free energy profiles of the inhibitors interacting with a single protein conformation provide a detailed picture of relative thermodynamic stability and a distribution of ligand binding modes in agreement with experimental crystallographic data. Using the simplified piecewise linear energy function, we also performed Monte Carlo docking simulations with an ensemble of protein conformations employing preferential biased sampling of low-energy protein conformations, and the results are analyzed in connection with the free energy profiles. Q 1999 John Wiley & Sons, Inc. Int J Quant Chem 72: 73)84, 1999
TL;DR: These studies illustrate the ability to synthesize tissue‐engineered cartilage under convective‐flow conditions for potential human tissue repair.
Abstract: The development of tissue engineered cartilage is emerging as a potential treatment for the repair of cartilage defects. By seeding chondrocytes onto poly-glycolic acid (PGA) biodegradable scaffolds within a convective-flow bioreactor, the synthesis of tissue-engineered articular cartilage has been recently demonstrated. The ability to cultivate and manipulate this cell-polymer construct to possess specific dimensions, as well as biochemical and biomechanical properties is critical for potential application as an in vivo therapy of damaged articular surfaces. Bioreactor design requirements for stages from research to development to commercialization are discussed. Advantages and limitations to various bioreactor designs are critiqued. These studies illustrate the ability to synthesize tissue-engineered cartilage under convective-flow conditions for potential human tissue repair.
TL;DR: The results call into question a long accepted theory regarding the interaction of chymotrypsin family serine proteases with substrates and suggest that the canonical interactions observed between these enzymes and standard inhibitors may represent nonproductive rather than productive, substrate-like interactions.
TL;DR: It is shown how evolutionary ligand selection for a receptor active site can produce well‐optimized ligand–protein systems such as MTX–DHFR complex with the thermodynamically stable native structure and a direct transition mechanism of binding from unbound conformations to the unique native structure.
Abstract: The thermodynamic and kinetic aspects of molecular recognition for the methotrexate (MTX)-dihydrofolate reductase (DHFR) ligand-protein system are investigated by the binding energy landscape approach. The impact of 'hot' and 'cold' errors in ligand mutations on the thermodynamic stability of the native MTX-DHFR complex is analyzed, and relationships between the molecular recognition mechanism and the degree of ligand optimization are discussed. The nature and relative stability of intermediates and thermodynamic phases on the ligand-protein association pathway are studied, providing new insights into connections between protein folding and molecular recognition mechanisms, and cooperativity of ligand-protein binding. The results of kinetic docking simulations are rationalized based on the thermodynamic properties determined from equilibrium simulations and the shape of the underlying binding energy landscape. We show how evolutionary ligand selection for a receptor active site can produce well-optimized ligand-protein systems such as MTX-DHFR complex with the thermodynamically stable native structure and a direct transition mechanism of binding from unbound conformations to the unique native structure.
TL;DR: The results suggest the existence of a functional epitope essential for heterodimerization between Pip and PU.1 and possibly, in general, between interferon regulatory factor family members and their partners.
Abstract: The PU.1 interaction partner (Pip) is a member of the interferon regulatory factor family that regulates gene expression through heterodimerization with the ETS transcription factor PU.1. Binding of Pip alone to DNA is weak, and usually it is recruited by phosphorylated PU.1 to form a strong ternary complex with specific DNA sequences. An approach combining sequence homology analysis, secondary structure predictions, and a precise mutational strategy has been used to determine critical residues within the Pip heterodimerization domain that contribute to ternary complex formation. We have delimited the Pip interaction domain to residues 245–422 by using deletion analysis. Site-directed mutagenesis of conserved polar amino acids within two predicted α-helices contained in this region, and which are highly conserved in the IRF family, confirmed the importance of these residues for Pip–PU.1 interaction with DNA as well as for trans-activation activity. Our results suggest the existence of a functional epitope essential for heterodimerization between Pip and PU.1 and possibly, in general, between interferon regulatory factor family members and their partners.
TL;DR: Challenges remain in the application of gene therapy techniques to skin substitutes, both the control of transgene expression and in the selection of suitable genes to transfect, and a challenge that may be diminishing is the importance of acute rejection of allogeneic tissue-engineered skin substitutes.
Abstract: The development of tissue engineered skin substitutes has reached a mature stage; major remaining questions relate to areas of greatest potential utility. Research is mainly concerned with extending indications of existing products, including treatment of the full range of acute, chronic, surgical and cosmetic wounds, and in other applications such as soft tissue augmentation and gene delivery. Among the few advances in the composition of skin substitutes is the inclusion of endothelial cells to provide a pathway for vascularisation by the host system.
TL;DR: An efficient method for the solid-phase synthesis of 1,3,4-trisubstituted-2-imidazolidones and 1,2,2- trisubstantiales from reduced N-acylated dipeptide with borane in THF is described.
Abstract: An efficient method for the solid-phase synthesis of 1,3,4-trisubstituted-2-imidazolidones and 1,3,4-trisubstituted-2-imidazolidinethiones from reduced N-acylated dipeptides is described. The complete reduction of the amide bonds of an N-acylated dipeptide with borane in THF results in two secondary and one tertiary amine. Reaction of the resulting resin-bound polyamine 4 with carbonyldiimidazole or thiocarbonyldiimidazole affords, respectively, cyclic ureas 6 and cyclic thioureas 7 in high yield and purity. Details on the selection of the building blocks and characterization of the controls for the libraries' synthesis will be presented. These procedures have also been used to generate parallel arrays and mixture-based combinatorial libraries of cyclic ureas and thioureas.
TL;DR: In this article, the solid phase synthesis of 1,4-benzothiazepin-5-one derivatives, resulting from the reaction of resin-bound protected cysteine with 2-fluoro-5nitro-benzoic acid followed by a reductive alkylation and an intra molecular cyclization, is described.
TL;DR: The use of combinatorial libraries in opioid receptor assays is reviewed and new opioid compounds identified from peptidomimetic libraries, such as peptoids and alkylated dipeptides, and those identified from acyclic and heterocyclic libraries are reviewed.
Abstract: Here we review the use of combinatorial libraries in opioid receptor assays. Following a brief description of the history of the combinatorial field, methods for the generation of synthetic libraries and the deconvolution of mixture-based libraries are presented. Case studies involving opioid assays used to demonstrate the viability of combinatorial libraries are described. The identification of new opioid peptides from combinatorial libraries is reviewed. The peptides found are composed of L-amino acids, D-amino acids, or L-, D-, and unnatural amino acids, and range from tetrapeptides to decapeptides. Likewise, new opioid compounds identified from peptidomimetic libraries, such as peptoids and alkylated dipeptides, and those identified from acyclic (e.g., polyamine, urea) and heterocyclic (e.g., bicyclic guanidine) libraries, are reviewed.
TL;DR: A physical map of the entire mouse Chr 16/human Chr 22 region of conserved synteny has been constructed to provide a substrate for gene discovery, genomic sequencing, and animal model development.
Abstract: Proximal mouse Chromosome (Chr) 16 shows conserved synteny with human Chrs 16, 8, 22, and 3 The mouse Chr 16/human Chr 22 conserved synteny region includes the DiGeorge/Velocardiofacial syndrome region of human Chr 22q112 A physical map of the entire mouse Chr 16/human Chr 22 region of conserved synteny has been constructed to provide a substrate for gene discovery, genomic sequencing, and animal model development A YAC contig was constructed that extends ca 54 Mb from a region of conserved synteny with human Chr 8 at Prkdc through the region conserved with human Chr 3 at DVL3 Sixty-one markers including 37 genes are mapped with average marker spacing of 90 kb Physical distance was determined across the 26-Mb region from D16Mit74 to Hira with YAC fragmentation The central region from D16Jhu28 to Igl-C1 was converted into BAC and PAC clones, further refining the physical map and providing sequence-ready template The gene content and borders of three blocks of conserved linkage between human Chr 22q112 mouse Chr 16 are refined
TL;DR: In this paper, the determination of sample size for such studies when the number of tests to perform is given, or when the sample size is given is discussed, and simple examples to illustrate some of the problems that arise.
Abstract: With discovery of an increasing number of candidate genes that may affect inter-individual variability in response to drugs, the design of drug trials that incorporate their study has become relevant. We discuss the determination of sample size for such studies when the number of tests to perform is given, or, alternatively, the number of tests to perform when the sample size is given. In many cases, a uniformly most powerful test does not exist and normal approximations are not sufficiently accurate to determine sample size. We discuss briefly various tests of interest and we give simple examples to illustrate some of the problems that arise.