Biomedical subjects

M A Siani

Publications and source records attributed to M A Siani.

At least 19 recordsLinked to original sources

PubMed2000

Presentation of chemokine SDF-1 alpha by fibronectin mediates directed migration of T cells.

The role of chemokine-matrix interactions in integrin-dependent T-cell migration was examined to address the critical question of how chemokines provide directional information. The chemokine SDF-1 alpha binds fibronectin (Fn) with a low nanomolar K(d) (equilibrium dissociation constant). SDF-1 alpha presented by Fn induced directed migration. Spatial concentration gradients of chemokine were not required to maintain directed migration. Fn-presented chemokine induced the polarization of cells, including the redistribution of the SDF-1 alpha receptor, to the basal surface and leading edge of the cell. A new model for directed migration is proposed in which the co-presentation of an adhesive matrix and chemokine provides the necessary positional information independent of a soluble spatial gradient. (Blood. 2000;96:2682-2690)

Cell Adhesion↗

PubMed2000

An approach for obtaining perfect hybridization probes for unknown polyketide synthase genes: a search for the epothilone gene cluster.

An approach is described for obtaining 'perfect probes' for type I modular polyketide synthase (PKS) gene clusters that in turn enables the identification of all such gene clusters in a genome. The approach involves sequencing small fragments of a random genomic DNA library containing one or more modular PKS gene clusters, and identifying which fragments emanate from PKS genes. Knowing the approximate sizes of the genome and the target gene cluster, one can predict the the frequency that a PKS gene fragment will be present in the library sequenced. Computer simulations of the approach were applied to the known PKS and non-ribosomal peptide synthetase (NRPS) gene clusters in the Bacillus subtilus genome. The approach was then used to identify PKS gene fragments in a strain of Sorangium cellulosum that produces epothilone. In addition to identifying fragments of the epothilone gene cluster, we obtained 11 unique fragments from other PKS gene clusters; the results suggest that there may be six to eight PKS gene clusters in this organism. In addition, we identified four unique fragments of NRPS genes, demonstrating that the approach is also applicable for identification of these modular gene clusters.

Antineoplastic Agents↗

PubMed1999

Prevention of crescentic glomerulonephritis by immunoneutralization of the fractalkine receptor CX3CR1 rapid communication.

BACKGROUND: Fractalkine is a newly identified T-cell and monocyte/macrophage (Mphi) chemokine with a transmembrane domain and is a cell-surface protein on activated endothelium. It can mediate adhesion of cells expressing the fractalkine receptor CX3CR1. These unique features make fractalkine well suited for leukocyte recruitment in tissues with high blood flow as in the renal glomerulus. METHODS: Fractalkine expression in glomeruli and response of isolated glomerular inflammatory cells to fractalkine were studied in the Wistar-Kyoto (WKY) crescentic glomerulonephritis model. Antibody was used to confirm the proinflammatory role of fractalkine. RESULTS: Fractalkine was markedly induced in the endothelium of nephritic rat glomeruli, and inflammatory leukocytes infiltrating the glomeruli expressed increased levels of CX3CR1. Anti-CX3CR1 antibody treatment dramatically blocked leukocyte infiltration in the glomeruli, prevented crescent formation, and improved renal function. CONCLUSIONS: Fractalkine plays a central role in leukocyte trafficking at the endothelium in the high-flow glomerular circuit and, in turn, implicates CX3CR1 as a prime drug target for therapeutic intervention of endothelium-related inflammatory diseases.

Animals↗

PubMed1998

Accessibility of selenomethionine proteins by total chemical synthesis: structural studies of human herpesvirus-8 MIP-II.

The determination of high resolution three-dimensional structures by X-ray crystallography or nuclear magnetic resonance (NMR) is a time-consuming process. Here we describe an approach to circumvent the cloning and expression of a recombinant protein as well as screening for heavy atom derivatives. The selenomethionine-modified chemokine macrophage inflammatory protein-II (MIP-II) from human herpesvirus-8 has been produced by total chemical synthesis, crystallized, and characterized by NMR. The protein has a secondary structure typical of other chemokines and forms a monomer in solution. These results indicate that total chemical synthesis can be used to accelerate the determination of three-dimensional structures of new proteins identified in genome programs.

Amino Acid Sequence↗

PubMed1998

In vivo inhibition of CC and CX3C chemokine-induced leukocyte infiltration and attenuation of glomerulonephritis in Wistar-Kyoto (WKY) rats by vMIP-II.

Chemokines play a central role in immune and inflammatory responses. It has been observed recently that certain viruses have evolved molecular piracy and mimicry mechanisms by encoding and synthesizing proteins that interfere with the normal host defense response. One such viral protein, vMIP-II, encoded by human herpesvirus 8, has been identified with in vitro antagonistic activities against CC and CXC chemokine receptors. We report here that vMIP-II has additional antagonistic activity against CX3CR1, the receptor for fractalkine. To investigate the potential therapeutic effect of this broad-spectrum chemokine antagonist, we studied the antiinflammatory activity of vMIP-II in a rat model of experimental glomerulonephritis induced by an antiglomerular basement membrane antibody. vMIP-II potently inhibited monocyte chemoattractant protein 1-, macrophage inflammatory protein 1beta-, RANTES (regulated on activation, normal T cell expressed and secreted)-, and fractalkine-induced chemotaxis of activated leukocytes isolated from nephritic glomeruli, significantly reduced leukocyte infiltration to the glomeruli, and markedly attenuated proteinuria. These results suggest that molecules encoded by some viruses may serve as useful templates for the development of antiinflammatory compounds.

Animals↗

PubMed1998

Crystal structure of chemically synthesized [N33A] stromal cell-derived factor 1alpha, a potent ligand for the HIV-1 "fusin" coreceptor.

Stromal cell-derived factor-1alpha (SDF-1alpha ) is a member of the chemokine superfamily and functions as a growth factor and chemoattractant through activation of CXCR4/LESTR/Fusin, a G protein-coupled receptor. This receptor also functions as a coreceptor for T-tropic syncytium-inducing strains of HIV-1. SDF-1alpha antagonizes infectivity of these strains by competing with gp120 for binding to the receptor. The crystal structure of a variant SDF-1alpha ([N33A]SDF-1alpha ) prepared by total chemical synthesis has been refined to 2.2-A resolution. Although SDF-1alpha adopts a typical chemokine beta-beta-beta-alpha topology, the packing of the alpha-helix against the beta-sheet is strikingly different. Comparison of SDF-1alpha with other chemokine structures confirms the hypothesis that SDF-1alpha may be either an ancestral protein from which all other chemokines evolved or the chemokine that is the least divergent from a primordial chemokine. The structure of SDF-1alpha reveals a positively charged surface ideal for binding to the negatively charged extracellular loops of the CXCR4 HIV-1 coreceptor. This ionic complementarity is likely to promote the interaction of the mobile N-terminal segment of SDF-1alpha with interhelical sites of the receptor, resulting in a biological response.

Binding Sites↗

PubMed1998

6-C-kine (SLC), a lymphocyte adhesion-triggering chemokine expressed by high endothelium, is an agonist for the MIP-3beta receptor CCR7.

The beta chemokine known as 6-C-kine, secondary lymphoid-tissue chemokine (SLC), TCA4, or Exodus-2 (herein referred to as 6CK/SLC) can trigger rapid integrin-dependent arrest of lymphocytes rolling under physiological shear and is highly expressed by high endothelial venules, specialized vessels involved in lymphocyte homing from the blood into lymph nodes and Peyer's patches. We show that 6CK/SLC is an agonist for the lymphocyte chemoattractant receptor, CCR7 (EBI-1, BLR-2), previously described as a receptor for the related beta chemokine MIP-3beta (ELC or Exodus-3). Moreover, 6CK/SLC and MIP-3beta attract the same major populations of circulating lymphocytes, including naive and memory T cells > B cells (but not natural killer cells); desensitization to MIP-3beta inhibits lymphocyte chemotaxis to 6CK/SLC but not to the alpha chemokine SDF-1 (stromal cell-derived factor); and 6CK/SLC competes for MIP-3beta binding to resting mouse lymphocytes. The findings suggest that the majority of circulating lymphocytes respond to 6CK/SLC and MIP-3beta in large part through their common receptor CCR7 and that these molecules may be important mediators of physiological lymphocyte recirculation in vivo.

Animals↗

PubMed1998

Chemokines and the arrest of lymphocytes rolling under flow conditions.

Circulating lymphocytes are recruited from the blood to the tissue by rolling along the endothelium until being stopped by a signaling event linked to the Gialpha subunit of a heterotrimeric GTP-binding protein; that event then triggers rapid integrin-dependent adhesion. Four chemokines are now shown to induce such adhesion to intercellular adhesion molecule-1 and to induce arrest of rolling cells within 1 second under flow conditions similar to those of blood. SDF-1 (also called PBSF), 6-C-kine (also called Exodus-2), and MIP-3beta (also called ELC or Exodus-3) induced adhesion of most circulating lymphocytes, including most CD4+ T cells; and MIP-3alpha (also called LARC or Exodus-1) triggered adhesion of memory, but not naïve, CD4+ T cells. Thus, chemokines can regulate the arrest of lymphocyte subsets under flowing conditions, which may allow them to control lymphocyte-endothelial cell recognition and lymphocyte recruitment in vivo.

Antigens, Surface↗

PubMed1998

Genetic subtype-independent inhibition of human immunodeficiency virus type 1 replication by CC and CXC chemokines.

We have studied the breadth and potency of the inhibitory actions of the CC chemokines macrophage inhibitory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES against macrophage-tropic (M-tropic) primary isolates of human immunodeficiency virus type 1 (HIV-1) and of the CXC chemokine stromal cell-derived factor 1alpha against T-cell-tropic (T-tropic) isolates, using mitogen-stimulated primary CD4+ T cells as targets. There was considerable interisolate variation in the sensitivity of HIV-1 to chemokine inhibition, which was especially pronounced for the CC chemokines and M-tropic strains. However, this variation was not obviously dependent on the genetic subtype (A through F) of the virus isolates. Peripheral blood mononuclear cell donor-dependent variation in chemokine inhibition potency was also observed. Among the CC chemokines, the rank order for potency (from most to least potent) was RANTES, MIP-1beta, MIP-1alpha. Some M-tropic isolates, unexpectedly, were much more sensitive to RANTES than to MIP-1beta, whereas other isolates showed sensitivities comparable to those of these two chemokines. Down-regulation of the CCR5 and CXCR4 receptors occurred in cells treated with the cognate chemokines and probably contributes to anti-HIV-1 activity. Thus, for CCR5, the rank order for down-regulation was also RANTES, MIP-1beta, MIP-1alpha.

Anti-HIV Agents↗

PubMed1997

Signal transduction due to HIV-1 envelope interactions with chemokine receptors CXCR4 or CCR5.

Infection with HIV-1 requires expression of CD4 and the chemokine receptors CXCR4 or CCR5 at the target cell surface. Engagement of these receptors by the HIV-1 envelope glycoprotein is essential for membrane fusion, but may additionally activate intracellular signaling pathways. In this study, we demonstrate that chemokines and HIV-1 envelope glycoproteins from both T-tropic and macrophage-tropic strains rapidly induce tyrosine phosphorylation of the protein tyrosine kinase Pyk2. The response requires CXCR4 and CCR5 to be accessible on the cell surface. The results presented here provide the first evidence for activation of an intracellular signaling event that can initiate multiple signaling pathways as a consequence of contact between HIV-1 and chemokine receptors.

CD4 Antigens↗

PubMed1997

Angiogenic and HIV-inhibitory functions of KSHV-encoded chemokines.

Unique among known human herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8) encodes chemokine-like proteins (vMIP-I and vMIP-II). vMIP-II was shown to block infection of human immunodeficiency virus-type 1 (HIV-1) on a CD4-positive cell line expressing CCR3 and to a lesser extent on one expressing CCR5, whereas both vMIP-I and vMIP-II partially inhibited HIV infection of peripheral blood mononuclear cells. Like eotaxin, vMIP-II activated and chemoattracted human eosinophils by way of CCR3. vMIP-I and vMIP-II, but not cellular MIP-1alpha or RANTES, were highly angiogenic in the chorioallantoic assay, suggesting a possible pathogenic role in Kaposi's sarcoma.

Animals↗

PubMed1997

Chemically synthesized SDF-1alpha analogue, N33A, is a potent chemotactic agent for CXCR4/Fusin/LESTR-expressing human leukocytes.

Stromal cell-derived factor (SDF) 1 is a potent chemoattractant for leukocytes through activation of the receptor CXCR4/Fusin/LESTR, which is a fusion co-factor for the entry of T lymphocytotropic human immunodeficiency virus type 1 (HIV-1). This CXCR4-mediated HIV-1 fusion can be inhibited by SDF-1. Because of its importance in the study of immunity and AIDS, large scale production of SDF-1 is desirable. In addition to recombinant technology, chemical synthesis provides means by which biologically active proteins can be produced not only in large quantity but also with a variety of designed modifications. In this study, we investigated the binding and function of an SDF-1alpha analogue, N33A, synthesized by a newly developed native chemical ligation approach. Radioiodinated N33A showed high affinity binding to human monocytes, T lymphocytes, as well as neutrophils, and competed equally well with native recombinant SDF-1alpha for binding sites on leukocytes. N33A also showed equally potent chemoattractant activity as native recombinant SDF-1alpha for human leukocytes. Further study with CXCR4/Fusin/LESTR transfected HEK 293 cells showed that N33A binds and induces directional migration of these cells in vitro. These results demonstrate that the chemically synthesized SDF-1alpha analogue, N33A, which can be produced rapidly in large quantity, possesses the same capacity as native SDF-1alpha to activate CXCR4-expressing cells and will provide a valuable agent for research on the host immune response and AIDS.

Binding, Competitive↗

PubMed1996

Receptor recognition and specificity of interleukin-8 is determined by residues that cluster near a surface-accessible hydrophobic pocket.

To determine the regions of interleukin-8 (IL-8) that allow high affinity and interleukin-8 receptor type 1 (IL8R1)-specific binding of chemokines, we produced chimeric proteins containing structural domains from IL-8, which binds to both IL8R1 and interleukin-8 receptor type 2 (IL8R2) with high affinity, and from GRO gamma, which does not bind to IL8R1 and binds to IL8R2 with reduced affinity. Receptor binding activity was tested by competition of 125I-IL-8 binding to recombinant IL8R1 and IL8R2 cell lines. Substitution into IL-8 of the GRO gamma sequences corresponding to either the amino-terminal loop (amino acids 1-18) or the first beta-sheet (amino acids 18-32) reduced binding to both IL8R1 and IL8R2. The third beta-sheet of IL-8 (amino acids 46-53) was required for binding to IL8R1 but not IL8R2. Exchanges of the second beta-sheet (amino acids 32-46) or the carboxyl-terminal alpha-helix (amino acids 53-72) had no significant effect. When IL-8 sequences were substituted into GRO gamma, a single domain containing the second beta-sheet of IL-8 (amino acids 18-32) was sufficient to confer high affinity binding for both IL8R1 and IL8R2. The amino-terminal loop (amino acids 1-18) and the third beta-sheet (amino acids 46-53) of IL-8 had little effect when substituted individually but showed increased binding to both receptors when substituted in combination. Individual amino acid substitutions were made at positions where IL-8 and GRO gamma sequences differ within the regions of residues 11-21 and 46-53. IL-8 mutations L49A or L49F selectively inhibited binding to IL8R1. Mutations Y13L and F21N enhanced binding to IL8R1 with little effect on IL8R2. A combined mutation Y13L/S14Q selectively decreased binding to IL8R2. Residues Tyr13, Ser14, Phe21, and Lys49 are clustered in and around a surface-accessible hydrophobic pocket on IL-8 that is physically distant from the previously identified ELR binding sequence. A homology model of GRO gamma, constructed from the known structure of IL-8 by refinement calculations, indicated that access to the hydrophobic pocket was effectively abolished in GRO gamma. These studies suggest that the surface hydrophobic pocket and/or adjacent residues participate in IL-8 receptor recognition for both IL8R1 and IL8R2 and that the hydrophobic pocket itself may be essential for IL8R1 binding. Thus this region contains a second site for IL-8 receptor recognition that, in combination with the Glu4-Leu5-Arg6 region, can modulate receptor binding affinity and IL8R1 specificity.

Amino Acid Sequence↗

PubMed1995

Measuring diversity: experimental design of combinatorial libraries for drug discovery.

Screening synthetic combinatorial libraries, such as mixtures of oligo(N-substituted)glycines, facilitates rapid drug lead discovery and optimization by vastly increasing the number of candidate molecules made and tested. Discovery efficiency and productivity can be further improved by using experimental design to maximize molecular diversity for a given library size or to bias the library with key features for a specific receptor. We describe new methods to quantify molecular diversity using descriptors that characterize lipophilicity, shape and branching, chemical functionality, and specific binding features. Experimental design methods select sets of side chains that are diverse in these properties, and "flower plots" allow the diversity to be graphically compared. We also quantify the overall diversity accessible to different families of combinatorial chemistry.

Drug Design↗

PubMed1994

Discovery of nanomolar ligands for 7-transmembrane G-protein-coupled receptors from a diverse N-(substituted)glycine peptoid library.

Screening a diverse, combinatorial library of ca. 5000 synthetic dimer and trimer N-(substituted)glycine "peptides" yielded novel, high-affinity ligands for 7-transmembrane G-protein-coupled receptors. The peptoid library was efficiently assembled using readily available chemical building blocks. The choice of side chains was biased to resemble known ligands to 7-transmembrane G-protein-coupled receptors. All peptides were screened in solution-phase, competitive radioligand-binding assays. Peptoid trimer CHIR 2279 binds to the alpha 1-adrenergic receptor with a Ki of 5 nM, and trimer CHIR 4531 binds to the mu-opiate receptor with a Ki of 6 nM. This represents the first example of the discovery of high-affinity receptor ligands from a combinatorial library of non-natural chemical entities.

Amino Acid Sequence↗

PubMed1992

The interdependence of protein surface topography and bound water molecules revealed by surface accessibility and fractal density measures.

To characterize water binding to proteins, which is fundamental to protein folding, stability and activity, the relationships of 10,837 bound water positions to protein surface shape and residue type were analyzed in 56 high-resolution crystallographic structures. Fractal atomic density and accessibility algorithms provided an objective characterization of deep grooves in solvent-accessible protein surfaces. These deep grooves consistently had approximately the diameter of one water molecule, suggesting that deep grooves are formed by the interactions between protein atoms and bound water molecules. Protein surface topography dominates the chemistry and extent of water binding. Protein surface area within grooves bound three times as many water molecules as non-groove surface; grooves accounted for one-quarter of the total surface area yet bound half the water molecules. Moreover, only within grooves did bound water molecules discriminate between different side-chains. In grooves, main-chain surface was as hydrated as that of the most hydrophilic side-chains, Asp and Glu, whereas outside grooves all main and side-chains bound water to a similar, and much decreased, extent. This identification of the interdependence of protein surface shape and hydration has general implications for modelling and prediction of protein surface shape, recognition, local folding and solvent binding.

Algorithms↗

PubMed1992

Identification of highest-affinity ligands by affinity selection from equimolar peptide mixtures generated by robotic synthesis.

A fully automated peptide synthesizer has been constructed that is capable of the simultaneous synthesis of up to 36 individual peptides and the synthesis of equimolar peptide mixtures. The instrument consists of an array of reaction vessels, a series of solenoid valves to control liquid flow, and a Zymark robot to deliver solvents and reagents; all components are computer controlled and coordinated. Equimolar peptide mixtures are obtained by algorithms that automate the mixing and distribution of peptide-resin particles. This technology was used to synthesize a library of 361 peptides, generated by randomizing two critical binding residues of a 10-mer epitope known to bind an anti-human immuno-deficiency virus gp120 monoclonal antibody. Each critical residue was substituted with 19 amino acids consisting of all the natural amino acids except cysteine. The library was synthesized as 19 pools, each containing 19 peptides. Each pool was screened in a solution-phase competition ELISA assay. The 12 most inhibitory peptides in the library were isolated by a rapid affinity-selection method and were identified by mass spectrometry and amino acid analysis. The binding properties of these 12 selected peptides were verified by synthesis and assay of the individual peptides. The two critical residues investigated were found to contribute independently to antibody binding.

Amino Acid Sequence↗

PubMed1992

Design, construction and application of a fully automated equimolar peptide mixture synthesizer.

A fully automated peptide synthesizer has been constructed that is capable of the synthesis of equimolar peptide mixtures and the simultaneous synthesis of 36 individual peptides. The synthesizer was constructed from a workstation of our own design utilizing a Zymark robot arm. A Macintosh II computer coordinates the movements of the robotic arm, the switching of over 40 solenoid valves and the monitoring of sensors in the workstation. The robot hands are used to deliver solvents from pressurized spigot lines and to pipet amino acid solutions from reservoirs to an array of reaction vessels. Liquid dispensing, reagent mixing and solvent removal are controlled from a multifunction I/O board in the computer. The design features of the synthesizer are presented, as well as the characterization of multiple individual peptides, a simple mixture of 19 components, and a complex mixture of 15,625 components.

Amino Acid Sequence↗