Synthetic Compound Libraries Displayed on the Surface of Encoded Bacteriophage

AbstractWe describe a technology for attaching libraries of synthetic compounds to coat proteins of bacteriophage particles such that the identity of the chemical structure is encoded in the genome of the phage, analogous to peptides displayed on phage surfaces by conventional phage-display techniques. This format allows a library of synthetic compounds to be screened very efficiently as a single pool. Encoded phage serve as extremely robust reporters of the presence of each compound, providing exquisite sensitivity for identification of active compounds engaged in complex biological processes such as receptor-mediated endocytosis and transcytosis. To evaluate this approach, we constructed a library of 980 analogs of folic acid displayed on T7 phage, and demonstrated rapid identification of compounds that bind to folate receptor and direct endocytosis of associated phage particles into cells that express the targeted receptor

Human tribbles-1 controls proliferation and chemotaxis of smooth muscle cells via MAPK signaling pathways

Migration and proliferation of smooth muscle cells are key to a number of physiological and pathological processes, including wound healing and the narrowing of the vessel wall.Previous work has shown links between inflammatory stimuli and vascular smooth muscle cell proliferation and migration through mitogen activated protein kinase (MAPK) activation, though the molecular mechanisms of this process are poorly understood. Here we report that tribbles-1, a recently described modulator of MAPK activation controls vascular smooth muscle cell proliferation and chemotaxis via the Jun Kinase pathway. Our findings demonstrate that this regulation takes place via direct interactions between tribbles-1 and MKK4/SEK1, a Jun activator kinase. The activity of this kinase is dependent on tribbles-1 levels, whilst the activation and the expression of MKK4/SEK1 is not. In addition, tribbles-1 expression is elevated in human atherosclerotic arteries compared to non-atherosclerotic controls, suggesting that this protein may pay a role in disease in vivo. In summary, the data presented here suggest an important regulatory role for trb-1 in vascular smooth muscle cell biology

Interleukin-1 regulates multiple atherogenic mechanisms in response to fat feeding

Background: Atherosclerosis is an inflammatory process that develops in individuals with known risk factors that include hypertension and hyperlipidaemia, influenced by diet. However, the interplay between diet, inflammatory mechanisms and vascular risk factors requires further research. We hypothesised that interleukin-1 (IL-1) signaling in the vessel wall would raise arterial blood pressure and promote atheroma. Methodology/Principal Findings: Apoe(-/-) and Apoe(-/-)/IL-1R1(-/-) mice were fed high fat diets for 8 weeks, and their blood pressure and atherosclerosis development measured. Apoe(-/-)/IL-R1(-/-) mice had a reduced blood pressure and significantly less atheroma than Apoe(-/-) mice. Selective loss of IL-1 signaling in the vessel wall by bone marrow transplantation also reduced plaque burden (p<0.05). This was associated with an IL-1 mediated loss of endothelium-dependent relaxation and an increase in vessel wall Nox 4. Inhibition of IL-1 restored endothelium-dependent vasodilatation and reduced levels of arterial oxidative stress. Conclusions/Significance: The IL-1 cytokine system links atherogenic environmental stimuli with arterial inflammation, oxidative stress, increased blood pressure and atherosclerosis. This is the first demonstration that inhibition of a single cytokine can block the rise in blood pressure in response to an environmental stimulus. IL-1 inhibition may have profound beneficial effects on atherogenesis in man

Structural studies on antibodies

Data from ¹H nuclear magnetic resonance studies on the Fv fragment of protein 315, a Dnp-binding BALB/c mouse lgA(λ2) myeloma protein, have been used to refine a predicted structure of the combining site of the protein. The Dnp-binding subsite in the modified structure is composed of the side chains of three aromatic amino acids Trp 93L, Tyr 34L and 34H. A fourth aromatic amino acid residue is close to the side chain-NH-CH 2 -group, this is Tyr 33 H - The antibody-hapten binding is a simple encounter process, which causes no extensive conformation change in the Fv fragment. A method for paramagnetic structural studies has been devised using Dnp derivatives with cllgophosphate side chains, which create a specific manganese binding site on the Fv fragment-hapten complex. The distances from the bound metal ion to the imidazole side chains of two of the three hlstldine residues of protein 315 have been determined. ¹H nuclear magnetic resonance has been used to study the histidine residues of the Fv fragment of protein 315. It has been shown that one of the three histidine residues (102H) is close to the combining site, but that this residue does not participate directly in binding haptens. 31 P nuclear magnetic resonance studies have shown the presence of a positively charged amino acid side chain near the entrance of the combining site of the Fv fragment. This residue has been identified as Arg 95L. The mode of binding of trini trophenyl derivatives to the Fv fragment has been studied by 1H nuclear magnetic resonance. It is concluded that these haptens, when bound to the Kv fragment, make contacts with the same amino acid side chains as Dnp derivatives.</p

Granulocyte Macrophage Colony-stimulating Factor mRNA and Protein A Potential Autocrine Role for Interleukin I in Epidermis Binds to Specific Receptors on Human Keratinocytes and Induces

Cultured human keratinocytes have been shown to produce IL-I alpha and beta mRNA and protein. IL-1 biological activity has been identified in normal human epidermis; in vitro, most biologically active IL-1 resides in a cell-associated compartment. The potential for autocrine effects of IL-1 on human keratinocytes was assessed by measurement of keratinocyte IL-1 receptors. Both high- and low-affinity cell surface receptors that bound recombinant (r) IL-1 alpha and beta with comparable affinities could be identified on cultured human keratinocytes, using III-labeled rIL-1. Chemical crosslinking experiments identified a cell surface molecule of roughly 72,500 M, that bound 1251I-labeled IL-1, similar to the molecular weight of previously described IL-1 receptors on fibroblasts, B cells, and T cells. To assess the biological consequences of keratinocyte IL-I binding, granulocyte-macrophage colonystimulating factor (GM-CSF) gene expression was measured. The addition of exogenous rIL-i alpha led to a dose-dependent increase in the accumulation of GM-CSF mRNA, as measured by a sensitive and specific Si nuclease assay. This increase in mRNA was reflected in a marked increase in GM-CSF biological activity as measured by proliferation of blast cells from chronic myelogenous leukemia patients. The biological activity was completely inhibitable by an antibody to human rGM-CSF. GM-CSF activates mature neutrophils and macrophages and appears to enhance the efficiency of Langerhans cell antigen presentation to T cells. Release of IL-i from injured or activated keratinocytes may lead to enhanced epidermal GM-CSF gene expression via an autocrine mechanism, thus enhancing local host defense

Structural studies on antibodies

Data from ¹H nuclear magnetic resonance studies on the Fv fragment of protein 315, a Dnp-binding BALB/c mouse lgA(λ2) myeloma protein, have been used to refine a predicted structure of the combining site of the protein. The Dnp-binding subsite in the modified structure is composed of the side chains of three aromatic amino acids Trp 93L, Tyr 34L and 34H. A fourth aromatic amino acid residue is close to the side chain-NH-CH 2 -group, this is Tyr 33 H - The antibody-hapten binding is a simple encounter process, which causes no extensive conformation change in the Fv fragment. A method for paramagnetic structural studies has been devised using Dnp derivatives with cllgophosphate side chains, which create a specific manganese binding site on the Fv fragment-hapten complex. The distances from the bound metal ion to the imidazole side chains of two of the three hlstldine residues of protein 315 have been determined. ¹H nuclear magnetic resonance has been used to study the histidine residues of the Fv fragment of protein 315. It has been shown that one of the three histidine residues (102H) is close to the combining site, but that this residue does not participate directly in binding haptens. 31 P nuclear magnetic resonance studies have shown the presence of a positively charged amino acid side chain near the entrance of the combining site of the Fv fragment. This residue has been identified as Arg 95L. The mode of binding of trini trophenyl derivatives to the Fv fragment has been studied by 1H nuclear magnetic resonance. It is concluded that these haptens, when bound to the Kv fragment, make contacts with the same amino acid side chains as Dnp derivatives.</p