Jerry DiSalvo

CCR5, CXCR4, and CD4 Are Clustered and Closely Apposed on Microvilli of Human Macrophages and T Cells

ABSTRACT The chemokine receptors CCR5 and CXCR4 act synergistically with CD4 in an ordered multistep mechanism to allow the binding and entry of human immunodeficiency virus type 1 (HIV-1). The efficiency of such a coordinated mechanism depends on the spatial distribution of the participating molecules on the cell surface. Immunoelectron microscopy was performed to address the subcellular localization of the chemokine receptors and CD4 at high resolution. Cells were fixed, cryoprocessed, and frozen; 80-nm cryosections were double labeled with combinations of CCR5, CXCR4, and CD4 antibodies and then stained with immunogold. Surprisingly, CCR5, CXCR4, and CD4 were found predominantly on microvilli and appeared to form homogeneous microclusters in all cell types examined, including macrophages and T cells. Further, while mixed microclusters were not observed, homogeneous microclusters of CD4 and the chemokine receptors were frequently separated by distances less than the diameter of an HIV-1 virion. Such distributions are likely to facilitate cooperative interactions with HIV-1 during virus adsorption to and penetration of human leukocytes and have significant implications for development of therapeutically useful inhibitors of the entry process. Although the mechanism underlying clustering is not understood, clusters were observed in small trans-Golgi vesicles, implying that they were organized shortly after synthesis and well before insertion into the cellular membrane. Chemokine receptors normally act as sensors, detecting concentration gradients of their ligands and thus providing directional information for cellular migration during both normal homeostasis and inflammatory responses. Localization of these sensors on the microvilli should enable more precise monitoring of their environment, improving efficiency of the chemotactic process. Moreover, since selectins, some integrins, and actin are also located on or in the microvillus, this organelle has many of the major elements required for chemotaxis.

Angiogenic potential of perivascularly delivered aFGF in a porcine model of chronic myocardial ischemia

A number of heparin-binding growth factors, including basic (bFGF) and acidic (aFGF) fibroblast growth factors have been shown to promote angiogenesis in vivo. In this study, we employed a sustained-release polymer extravascular delivery system to evaluate the angiogenic efficacy of a novel form of genetically modified aFGF in the setting of chronic myocardial ischemia. Fifteen Yorkshire pigs subjected to Ameroid occluder placement on the left circumflex (LCX) artery were treated with perivascularly administered aFGF in ethylene vinyl acetate (EVAc) polymer (10 μg, n = 7) or EVAc alone (controls, n = 8). Seven to nine weeks later, after coronary angiography to document Ameroid-induced coronary occlusion, all animals underwent studies of coronary flow and global and regional left ventricular function. Microsphere-determined coronary flow in the Ameroid-compromised territory was significantly increased in aFGF-treated compared with control animals, and this improvement in perfusion was maintained during ventricular pacing. Left ventricular function studies demonstrated improved global and regional function in aFGF-treated animals. We conclude that local perivascular delivery of genetically modified aFGF results in significant improvement in myocardial flow and regional and global left ventricular function.A number of heparin-binding growth factors, including basic (bFGF) and acidic (aFGF) fibroblast growth factors have been shown to promote angiogenesis in vivo. In this study, we employed a sustained-release polymer extravascular delivery system to evaluate the angiogenic efficacy of a novel form of genetically modified aFGF in the setting of chronic myocardial ischemia. Fifteen Yorkshire pigs subjected to Ameroid occluder placement on the left circumflex (LCX) artery were treated with perivascularly administered aFGF in ethylene vinyl acetate (EVAc) polymer (10 micrograms, n = 7) or EVAc alone (controls, n = 8). Seven to nine weeks later, after coronary angiography to document Ameroid-induced coronary occlusion, all animals underwent studies of coronary flow and global and regional left ventricular function. Microsphere-determined coronary flow in the Ameroid-compromised territory was significantly increased in aFGF-treated compared with control animals, and this improvement in perfusion was maintained during ventricular pacing. Left ventricular function studies demonstrated improved global and regional function in aFGF-treated animals. We conclude that local perivascular delivery of genetically modified aFGF results in significant improvement in myocardial flow and regional and global left ventricular function.

Disulfide bonds are neither required, present, nor compatible with full activity of human recombinant acidic fibroblast growth factor.

Human acidic fibroblast growth factor (aFGF) is a potent broad-spectrum mitogen that contains three Cys residues within its monomeric structure. We have found that site-directed mutants in which any one of these Cys residues is converted to serine remain highly active, although variably dependent on heparin, so none of the three possible intramolecular disulfide bonds that can be formed are required for mitogenic activity. Furthermore, a dispensable disulfide bond that might stabilize the active conformation is not present since all three Cys residues are accessible to chemical modification in recombinant as well as brain-derived aFGFs. Finally, formation of a disulfide bond between the two Cys residues conserved among all seven known members of the FGF family results in a virtually inactive product that can subsequently be reactivated by reduction. Thus, despite the extracellular function of aFGF, its Cys residues do not form intramolecular disulfide bonds in the active conformation.

Acidic Fibroblast Growth Factor Accelerates Dermal Wound Healing

Acidic fibroblast growth factor (aFGF) is a potent mitogen in vitro for many cells of ectodermal and mesodermal embryonic origin including skin-derived epidermal keratinocytes, dermal fibroblasts and vascular endothelial cells. Based on the mitogenic activity for these skin-derived cells, we tested the ability of topically applied aFGF to promote healing of full-thickness dermal wounds in healthy rodents. Low doses of aFGF can produce almost a two-fold maximum acceleration in the rate of closure of full-thickness dermal punch biopsy wounds in young healthy mice and rats. The mitogen also produces a 3 to 4 day acceleration in the time to complete closure in rats. Quantitative histomorphometric analysis of wound tissue shows that aFGF induces a marked stimulation of angiogenesis, granulation tissue formation and the growth of new epithelium, but does not promote dermal contraction. Application of aFGF to linear incisions in rat skin produces a transient increase in wound tensile strength accompanied by enhanced cellularity and deposition of collagen. Therefore, aFGF functions as a pharmacological agent that can accelerate dermal wound healing in rodents and could act therapeutically to promote dermal tissue repair in humans.

Discovery of structurally diverse natural product antagonists of chemokine receptor CXCR3

The chemokines (CXCL9, CXCL10 and CXCL11) and associated CXCR3 receptor are expressed during the inflammatory process from multiple sclerosis, atherosclerosis or organ transplantation resulting in the recruitment of lymphocytes leading to tissue damage. It is hypothesized that blocking of the ligand/CXCR3 receptor interaction has potential to provide opportunity for development of agents that would block tissue rejection. In this paper, four classes of natural product inhibitors (IC50 ranging 0.1–41 μM) have been described that block the CXCR3 receptor interaction of IP-10 ligand. These include a cyclic thiopeptide (duramycin), polyketide glycosides (roselipins), steroidal glycosides (hypoglausin A and dioscin) and a novel alkyl pyridinium alkaloid that were isolated by bioassay-guided fractionation of the organic extracts derived from actinomycete, fungal, plant and marine sources and discovered using 125 I IP-10/CXCR3 binding assay. Duramycin was the most potent with an IC50 of 0.1 μM. Roselipins 2A, 2B and 1A showed IC50 values of 14.6, 23.5, and 41 μM, respectively. Diosgenin glycosides dioscin, hypoglaucin A and kallstroemin D exhibited IC50 values of 2.1, 0.47 and 3 μM, respectively. A novel cyclic 3-alkyl pyridinium salt isolated from a sponge displayed a binding IC50 of 0.67 μ M.

Characterization of specific binding of [125I]L-762,459, a selective α1A-adrenoceptor radioligand to rat and human tissues

L-762,459 ((+/-)1-(3-¿[5-carbamoyl-2-2-[(4-hydroxy-3-iodobenzimidoyl)-amino] -ethoxy-methy¿-6-methyl-4-(4-nitropheny)-1,4-dihydropyridine -3-carbonyl]-amino¿-propyl)-4-phenyl-1-piperidine-4-carboxylic acid methyl ester), an analog of a series of dihydropyridines previously reported to be selective alpha1A-adrenoceptor subtype antagonists was found to have alpha1A-adrenoceptor subtype selectivity (Ki (nM), la = 1.3, lb = 240, Id = 280). Specific [125I]L-762,459 binding was detected in rat cerebral cortex, hippocampus, vas deferens, kidney, heart and prostate tissues known to contain the alpha1A-adrenoceptor subtype, but not in tissues known to contain alpha1B-adrenoceptor (spleen, liver) and alpha1D-adrenoceptor (aorta). Scatchard analysis of [125I]L-762,459 binding in rat cerebral cortex and prostate indicated a single binding site with a Kd of 0.7 nM and Bmax of 11 (cerebral cortex) and 1 (prostate) pmole/g tissue. Specific and saturable [125I]L-762,459 binding was also found in human cerebral cortex, liver, prostate and vas deferens (Kd = 0.2-0.4 nM, Bmax = 0.4-4 pmole/g tissue). The specific binding in rat and human tissues was competed by non-selective alpha1-adrenoceptor compounds (Ki values in nM: prazosin (0.14-1.2), terazosin (1.8-5.9) and phentolamine (2.4-11)) and selective alpha1A-adrenoceptor compounds [Ki values in nM: (+) niguldipine (0.04-1.2) and SNAP 5399 ((+/-)-2-((2-aminoethyl)oxy)methyl-5-carboxamido-6-ethyl-4-(4-nitropheny l)-3-N-(3-(4,4-diphenylpiperidin-1-yl)propyl)carboxamido-1,4-dihyd ropyridine hydrate (0.5-4.8)]. The results were consistent with the selective binding of [125I]L-762,459 to the alpha1A-adrenoceptor. The specific labeling of the alpha1A-adrenoceptor subtype by [125I]L-762,459 may make it a useful tool to localize the distribution of the alpha1A-adrenoceptor.

Heterocyclic acetamide and benzamide derivatives as potent and selective β3-adrenergic receptor agonists with improved rodent pharmacokinetic profiles

A series of amide derived beta(3)-adrenergic receptor (AR) agonists is described. The discovery and optimization of several series of compounds derived from 1, is used to lay the SAR foundation for second generation beta(3)-AR agonists for the treatment of overactive bladder.

High‐performance liquid chromatography fraction marker‐timer controller

A fraction marker‐timer controller has been designed that partially automates peak collection and provides event marking during high‐performance liquid chromatography of partially purified samples having complex chromatograms. The device is activated via a pushbutton switch when a peak of interest elutes. The apparatus places an event mark on the detector’s chart recording, provides a time delay to account for detector‐to‐fraction‐collector dead volume, and then advances a fraction collector to capture the peak in a test tube. If more than one peak elutes in the dead volume window, up to three independent time delays may be triggered.

Structure, Homologies and Activities of Acidic Fibroblast Growth Factor

Fibroblast growth factors (FGFS, reviewed by Thomas and Gimenez-Gallego, 1986) are mitogens for a wide variety of cells in culture including not only fibroblasts but also vascular endothelial cells, myoblasts, chondrocytes, osteoblasts and glial cells. In vivo, FGFS have been shown to promote blood vessel growth. Acidic and basic forms of FGFS have been identified, purified and characterized which, although structurally related to each other, are unique gene products. Alternative names for these molecules have been proposed based on their source (retina-derived growth factors, eye-derived growth factors, brain-derived growth factors), target cells (endothelial cell growth factor, astroglial growth factors, prostatropins) and ability to bind to heparin (heparin-binding growth factors). The amino acid sequence, homologies and activities of acidic FGF (aFGF) are reported.

Primary structure and mitogenic and angiogenic activities of brain-derived acidic fibroblast growth factor

Pure brain-derived acidic fibroblast growth factor (aFGF) is a protein that is mitogenic for a variety of types of cells in culture, including vascular endothelial cells, and is angiogenic in vivo. The complete amino acid sequence of the 140-residue bovine aFGF has been determined and recognized to be homologous to basic fibroblast growth factor and both interleukin-1α and-1β. These four growth factors define a new class of homologous proteins that presumably diverged from a common ancestor.