ER-27319, an acridone-related compound, inhibits release of antigen-induced allergic mediators from mast cells by selective inhibition of Fcɛ receptor I-mediated activation of Syk

Engagement of the mast cell high-affinity receptor for immunoglobulin E (IgE), FcɛRI, induces tyrosine phosphorylation of Syk, a non-receptor tyrosine kinase, that has been demonstrated as critical for degranulation. Herein we describe a synthetic compound, ER-27319, as a potent and selective inhibitor of antigen or anti-IgE-mediated degranulation of rodent and human mast cells. ER-27319 affected neither Lyn kinase activity nor the antigen-induced phosphorylation of the FcɛRI but did effectively inhibit the tyrosine phosphorylation of Syk and thus its activity. As a consequence, tyrosine phosphorylation of phospholipase C-γ1, generation of inositol phosphates, release of arachidonic acid, and secretion of histamine and tumor necrosis factor α were also inhibited. ER-27319 did not inhibit the anti-CD3-induced tyrosine phosphorylation of phospholipase C-γ1 in Jurkat T cells, demonstrating a specificity for Syk-induced signals. In contrast the tyrosine phosphorylation and activation of Syk, induced by in vitro incubation with the phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) of FcɛRI γ subunit or by antigen activation of RBL-2H3 cells, was specifically inhibited by ER-27319. However, when ER-27319 was added to immunoprecipitated Syk, derived from activated cells, no effect was seen on Syk activity. ER-27319 did not inhibit the tyrosine phosphorylation of Syk induced by activation in the presence of Igβ ITAM or the anti-IgM-induced phosphorylation of Syk in human peripheral B cells. Therefore, ER-27319 selectively interferes with the FcɛRI γ phospho-ITAM activation of Syk in vitro and in intact cells. These results confirm the importance of Syk in FcɛRI-mediated responses in mast cells and demonstrate the mast cell selectivity and therapeutic potential of ER-27319 in the treatment of allergic disease.

Purified hematopoietic stem cells without facilitating cells can repopulate fully allogeneic recipients across entire major histocompatibility complex transplantation barrier in mice

We report herein the successful long term engraftment of highly purified hematopoietic stem cells (HSCs) without any facilitating cells in fully allogeneic recipient mice across the entire major histocompatibility complex (MHC) transplantation barrier. This finding challenges the assumption that highly purified marrow HSCs alone cannot produce long-lived allogeneic bone marrow chimeras across the MHC barrier. In the present experiments, 1 × 105 HSCs from 5-fluorouracil (5-FU)-treated donors, without any facilitating cells, have been found to repopulate lethally irradiated fully allogeneic recipients. Low density, lineage-negative (CD4−, CD8−, B220−, Mac-1−, Gr-1−), CD71-negative, class I highly positive, FACS-sorted cells from 5-FU-treated C57BL/6 (B6) donor mice were transplanted into lethally irradiated BALB/c recipients. (BALB/c → BALB/c) → BALB/c T cell-depleted marrow cells used as compromised cells were also transplanted into the recipients to permit experiments to be pursued over a long period of time. Cells of donor origin in all recognized lineages of hematopoietic cells developed in these allogeneic chimeras. One thousand HSCs were sufficient to repopulate hemiallogeneic recipients, but 1 × 104 HSCs alone from 5-FU-treated donors failed to repopulate the fully allogeneic recipients. Transplantation of primary marrow stromal cells or bones of the donor strain into recipient, together with 1 × 104 HSCs, also failed to reconstitute fully allogeneic recipients. Suppression of resistance of recipients by thymectomy or injections of granulocyte colony-stimulating factor before stem cell transplantation enhanced the engraftment of allogeneic HSCs. Our experiments show that reconstitution of all lymphohematopoietic lineages across the entire MHC transplantation barriers may be achieved by transplanting allogeneic HSCs alone, without any facilitating cells, as long as a sufficient number of HSCs is transplanted.

In Vivo Observations of Myosin II Dynamics Support a Role in Rear RetractionV⃞

To investigate myosin II function in cell movement within a cell mass, we imaged green fluorescent protein-myosin heavy chain (GFP-MHC) cells moving within the tight mound of Dictyostelium discoideum. In the posterior cortex of cells undergoing rotational motion around the center of the mound, GFP-MHC cyclically formed a “C,” which converted to a spot as the cell retracted its rear. Consistent with an important role for myosin in rotation, cells failed to rotate when they lacked the myosin II heavy chain (MHC−) or when they contained predominantly monomeric myosin II (3xAsp). In cells lacking the myosin II regulatory light chain (RLC−), rotation was impaired and eventually ceased. These rotational defects reflect a mechanical problem in the 3xAsp and RLC− cells, because these mutants exhibited proper rotational guidance cues. MHC− cells exhibited disorganized and erratic rotational guidance cues, suggesting a requirement for the MHC in organizing these signals. However, the MHC− cells also exhibited mechanical defects in rotation, because they still moved aberrantly when seeded into wild-type mounds with proper rotational guidance cues. The mechanical defects in rotation may be mediated by the C-to-spot, because RLC− cells exhibited a defective C-to-spot, including a slower C-to-spot transition, consistent with this mutant’s slower rotational velocity.

The Balance of RanBP1 and RCC1 Is Critical for Nuclear Assembly and Nuclear Transport

Ran is a small GTPase that is essential for nuclear transport, mRNA processing, maintenance of structural integrity of nuclei, and cell cycle control. RanBP1 is a highly conserved Ran guanine nucleotide dissociation inhibitor. We sought to use Xenopus egg extracts for the development of an in vitro assay for RanBP1 activity in nuclear assembly, protein import, and DNA replication. Surprisingly, when we used anti-RanBP1 antibodies to immunodeplete RanBP1 from Xenopus egg extracts, we found that the extracts were also depleted of RCC1, Ran’s guanine nucleotide exchange factor, suggesting that these proteins form a stable complex. In contrast to previous observations using extracts that had been depleted of RCC1 only, extracts lacking both RanBP1 and RCC1 (codepleted extracts) did not exhibit defects in assays of nuclear assembly, nuclear transport, or DNA replication. Addition of either recombinant RanBP1 or RCC1 to codepleted extracts to restore only one of the depleted proteins caused abnormal nuclear assembly and inhibited nuclear transport and DNA replication in a manner that could be rescued by further addition of RCC1 or RanBP1, respectively. Exogenous mutant Ran proteins could partially rescue nuclear function in extracts without RanBP1 or without RCC1, in a manner that was correlated with their nucleotide binding state. These results suggest that little RanBP1 or RCC1 is required for nuclear assembly, nuclear import, or DNA replication in the absence of the other protein. The results further suggest that the balance of GTP- and GDP-Ran is critical for proper nuclear assembly and function in vitro.

Chimeric Cytokine Receptor Can Transduce Expansion Signals in Interleukin 6 Receptor α (IL-6Rα)-, IL-11Rα-, and gp130-low to -negative Primitive Hematopoietic Progenitors

We generated transgenic mice expressing chimeric receptors, which comprise extracellular domains of the human granulocyte–macrophage colony-stimulating factor (hGM-CSF) receptor and transmembrane and cytoplasmic domains of the mouse leukemia inhibitory factor receptor. In suspension cultures of lineage-negative (Lin−), 5-fluorouracil-resistant bone marrow cells of the transgenic mice, a combination of hGM-CSF and stem cell factor (SCF) induced exponential expansions of mixed colony-forming unit. The combination of hGM-CSF and SCF was effective on enriched, Lin−Sca-1+c-kit+ progenitors and increased either mixed colony-forming unit or cobblestone area–forming cells. In case of stimulation with hGM-CSF and SCF, interleukin-6 (IL-6) and SCF, or IL-11 and SCF, the most efficient expansion was achieved with hGM-CSF and SCF. When Lin−Sca-1+c-kit+CD34− further enriched progenitors were clone sorted and individually incubated in the presence of SCF, hGM-CSF stimulated a larger number of cells than did IL-6, IL-6 and soluble IL-6 receptor (IL-6R), or IL-11. These data suggest the presence of IL-6Rα-, IL-11Rα-, and gp130-low to -negative primitive hematopoietic progenitors. Such primitive progenitors are equipped with signal transduction molecules and can expand when these chimeric receptors are genetically introduced into the cells and stimulated with hGM-CSF in the presence of SCF.

Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells

Marrow stromal cells are adult stem cells from bone marrow that can differentiate into multiple nonhematopoietic cell lineages. Previous reports demonstrated that single-cell-derived colonies of marrow stromal cells contained two morphologically distinct cell types: spindle-shaped cells and large flat cells. Here we found that early colonies also contain a third kind of cell: very small round cells that rapidly self-renew. Samples enriched for the small cells had a greater potential for multipotential differentiation than samples enriched for the large cells. Also, the small cells expressed a series of surface epitopes and other proteins that potentially can be used to distinguish the small cells from the large cells. The results suggested it will be important to distinguish the major subpopulations of marrow stromal cells in defining their biology and their potential for cell and gene therapy.

Translocation of PKN from the cytosol to the nucleus induced by stresses.

Effects of environmental stresses on the subcellular localization of PKN were investigated in NIH 3T3, BALB/c 3T3, and Rat-1 cells. The immunofluorescence of PKN resided prominently in the cytoplasmic region in nonstressed cells. When these cells were treated at 42 degrees C, there was a time-dependent decrease of the immunofluorescence of PKN in the cytoplasmic region that correlated with an increase within the nucleus as observed by confocal microscope. After incubation at 37 degrees C following beat shock, the immunofluorescence of PKN returned to the perinuclear and cytoplasmic regions from the nucleus. The nuclear translocation of PKN by heat shock was supported by the biochemical subcellular fractionation and immunoblotting. The nuclear localization of PKN was also observed when the cells were exposed to other stresses such as sodium arsenite and serum starvation. These results raise the possibility that there is a pathway mediating stress signals from the cytosol to the nucleus through PKN.

Inhibition of NF-AT-dependent transcription by NF-kappa B: implications for differential gene expression in T helper cell subsets.

Activation of individual CD4+ T cells results in differential lymphokine expression: interleukin 2 (IL-2) is preferentially produced by T helper type 1 (TH1) cells, which are involved in cell-mediated immune responses, whereas IL-4 is synthesized by TH2 cells, which are essential for humoral immunity. The Ca(2+)-dependent factor NF-ATp plays a key role in the inducible transcription of both these lymphokine genes. However, while IL2 expression requires the contribution of Ca(2+)- and protein kinase C-dependent signals, we report that activation of human IL4 transcription through the Ca(2+)-dependent pathway is diminished by protein kinase C stimulation in Jurkat T cells. This phenomenon is due to mutually exclusive binding of NF-ATp and NF-kappa B to the P sequence, an element located 69 bp upstream of the IL4 transcription initiation site. Human IL4 promoter-mediated transcription is downregulated in Jurkat cells stimulated with the NF-kappa B-activating cytokine tumor necrosis factor alpha and suppressed in RelA-overexpressing cells. In contrast, protein kinase C stimulation or RelA overexpression does not affect the activity of a human IL4 promoter containing a mouse P sequence, which is a higher-affinity site for NF-ATp and a lower-affinity site for RelA. Thus, competition between two general transcriptional activators, RelA and NF-ATp, mediates the inhibitory effect of protein kinase C stimulation on IL4 expression and may contribute to differential gene expression in TH cells.

The only essential function of TFIIIA in yeast is the transcription of 5S rRNA genes.

We have developed a system to transcribe the yeast 5S rRNA gene in the absence of the transcription factor TFIIIA. A long transcript was synthesized both in vitro and in vivo from a hybrid gene in which the tRNA-like promoter sequence of the RPR1 gene was fused to the yeast 5S RNA gene. No internal initiation directed by the endogenous 5S rDNA promoter or any processing of the hybrid transcript was observed in vitro. Yeast cells devoid of transcription factor TFIIIA, which, therefore, could not synthesize any 5S rRNA from the endogenous chromosomal copies of 5S rDNA, could survive if they carried the hybrid RPR1-5S construct on a multicopy plasmid. In this case, the only source of 5S rRNA was the precursor RPR1-5S transcript that gave rise to two RNA species slightly larger than wild-type 5S rRNA. This establishes that the only essential function of TFIIIA is to promote the synthesis of 5S rRNA. However, cells devoid of TFIIIA and surviving with these two RNAs grew more slowly at 30 degrees C compared with wild-type cells and were thermosensitive at 37 degrees C.

Diffusion and formation of microtubule asters: physical processes versus biochemical regulation.

Microtubule asters forming the mitotic spindle are assembled around two centrosomes through the process of dynamic instability in which microtubules alternate between growing and shrinking states. By modifying the dynamics of this assembly process, cell cycle enzymes, such as cdc2 cyclin kinases, regulate length distributions in the asters. It is believed that the same enzymes control the number of assembled microtubules by changing the "nucleating activity" of the centrosomes. Here we show that assembly of microtubule asters may be strongly altered by effects connected with diffusion of tubulin monomers. Theoretical analysis of a simple model describing assembly of microtubule asters clearly shows the existence of a region surrounding the centrosome depleted in GTP tubulin. The number of assembled microtubules may in some cases be limited by this depletion effect rather than by the number of available nucleation sites on the centrosome.

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Optimisation of strain rate imaging for application to stress echocardiography

We sought to improve the feasibility of strain rate imaging (SRI) during dobutamine stress echocardiography (DSE) in 56 subjects at low risk of coronary disease. The impact of several SRI changes during acquisition were studied, including: (1) changing from fundamental to harmonic imaging; (2) parallel beam-forming; (3) alteration of spatial resolution and (4) narrow sector acquisition. We assessed SR signal quality, a quantitative measure of signal noise and measurements of SRI. Of 1462 segments evaluated, 6% were uninterpretable at rest and 8% at peak stress. Signal quality was optimised by increasing temporal (p = 0.01) and spatial resolution (p<0.0001 vs. baseline imaging) at rest and peak. Increasing spatial resolution also minimised signal noise (p<0.0001). Inter-observer variability of time to peak SR and peak SR were less with high temporal and spatial resolution. SRI quality can be improved with harmonic imaging and higher temporal resolution but optimisation of spatial resolution is critical. (C) 2004 World Federation for Ultrasound in Medicine Biology.

Potent cyclic antagonists of the complement C5a receptor on human polymorphonulcear leukocytes. Relationships between structures and activity

Human C5a is a plasma protein with potent chemoattractant and pro-inflammatory properties, and its overexpression correlates with severity of inflammatory diseases. C5a binds to its G protein-coupled receptor (C5aR) on polymorphonuclear leukocytes (PMNLs) through a high-affinity helical bundle and a low-affinity C terminus, the latter being solely responsible for receptor activation. Potent and selective C5a antagonists are predicted to be effective anti-inflammatory drugs, but no pharmacophore for small molecule antagonists has yet been developed, and it would significantly aid drug design. We have hypothesized that a turn conformation is important for activity of the C terminus of C5a and herein report small cyclic peptides that are stable turn mimics with potent antagonism at C5aR on human PMNLs. A comparison of solution structures for the C terminus of C5a, small acyclic peptide ligands, and cyclic antagonists supports the importance of a turn for receptor binding. Competition between a cyclic antagonist and either C5a or an acyclic agonist for C5aR on PMNLs supports a common or overlapping binding site on the C5aR. Structure-activity relationships for 60 cyclic analogs were evaluated by competitive radioligand binding with C5a (affinity) and myeloperoxidase release (antagonist potency) from human PMNLs, with 20 compounds having high antagonist potencies (IC50, 20 nM(-1) muM). Computer modeling comparisons reveal that potent antagonists share a common cyclic backbone shape, with affinity-determining side chains of defined volume projecting from the cyclic scaffold. These results define a new pharmacophore for C5a antagonist development and advance our understanding of ligand recognition and receptor activation of this G protein-coupled receptor.

Genetic basis of human testicular germ cell cancer: insights from the fruitfly and mouse

The prevalence of tumours of the germ line is increasing in the male population. This complex disease has a complex aetiology. We examine the contribution of genetic mutations to the development of germ line tumours in this review. In particular, we concentrate on fly and mouse experimental systems in order to demonstrate that mutations in some conserved genes cause pathologies typical of certain human germ cell tumours, whereas other mutations elicit phenotypes that are unique to the experimental model. Despite these experimental systems being imperfect, we show that they are useful models of human testicular germ cell tumourigenesis.

A wideband array antenna with beam-steering capability using real valued weights

This article presents an array antenna with beam-steering capability in azimuth over a wide frequency band using real-valued weighting coefficients that can be realized in practice by amplifiers or attenuators. The described beamforming scheme relies on a 2D (instead of 1D) array structure in order to make sure that there are enough degrees of freedom to realize a given radiation pattern in both the angular and frequency domains. In the presented approach, weights are determined using an inverse discrete Fourier transform (IDFT) technique by neglecting the mutual coupling between array elements. Because of the presence of mutual coupling, the actual array produces a radiation pattern with increased side-lobe levels. In order to counter this effect, the design aims to realize the initial radiation pattern with a lower side-lobe level. This strategy is demonstrated in the design example of 4 X 4 element array. (C) 2005 Wiley Periodicals. Inc.