Constitutive production of granulocyte/macrophage colony-stimulating factor by hypodense mononuclear eosinophils developed in vitro from hybrid eosinophil/basophil granulocytes.

We recently described the development in vitro of cells with granules characteristic of eosinophils and basophils (hybrid granulocytes) from normal human cord blood mononuclear cells cultured for 14 days with recombinant human (rh) interleukin (IL)-3, rhIL-5, and a soluble basement membrane, Matrigel. Hybrid granulocytes constitutively produced granulocyte/macrophage colony-stimulating factor (GM-CSF) and rapidly developed into eosinophils after the exogenous cytokines and Matrigel were removed. To characterize the developmental progression of hybrid granulocytes, cells were maintained for an additional 14 days in medium containing rhIL-3, rhIL-5, and Matrigel. After 28 days, 73% +/- 1% (mean +/- SEM; n = 6) of the nonadherent cells were mononuclear eosinophils, 13% +/- 3% were eosinophils with two or more nuclear lobes, 13% +/- 4% were hybrid granulocytes, and 0.2% +/- 0.1% were basophils. More than 90% of the mononuclear eosinophils were hypodense as determined by centrifugation through metrizamide gradients. After an additional 5 days of culture in medium without exogenous cytokines, 65% +/- 3% (n = 5) of the 28-day cells excluded trypan blue. In contrast, 2% +/- 1% of freshly isolated peripheral blood eosinophils survived 5 days of culture without exogenous cytokines (n = 5). Fifty percent conditioned medium from in vitro derived 28-day mononuclear eosinophils and 14-day hybrid granulocytes maintained the survival of 60% +/- 7% and 77% +/- 7%, respectively, of freshly isolated peripheral blood eosinophils for 72 h, compared with 20% +/- 8% survival in medium alone (n = 3). The eosinophil viability-sustaining activity of 50% mononuclear eosinophil-conditioned medium was neutralized with a GM-CSF antibody. A total of 88% of the 28-day cells exhibited immunochemical staining for GM-CSF. Thus, during eosinophilopoiesis, both hybrid eosinophil/basophil intermediates and immature mononuclear eosinophils exhibit autocrine regulation of viability due to constitutive production of GM-CSF.

Constitutive phosphorylation of I kappa B alpha by casein kinase II.

The NF-kappa B/Rel proteins are sequestered in the cytoplasm in association with the phosphorylated form of I kappa B alpha. Upon induction with a wide variety of agents, the activity of NF-kappa B/Rel proteins is preceded by the rapid degradation of I kappa B alpha protein. We report the identification and partial purification of a cellular kinase from unstimulated or stimulated murine cells, which specifically phosphorylates the C terminus of I kappa B alpha. There are several consensus sites for casein kinase II (CKII) in the C-terminal region of I kappa B alpha. Additionally, the activity of the cellular kinase is blocked by antibodies against the alpha subunit of CKII. No phosphorylation of the C-terminal region of I kappa B alpha can be detected if the five possible serine and threonine residues that can be phosphorylated by CKII are mutated to alanine. A two-dimensional tryptic phosphopeptide map of I kappa B alpha from unstimulated cells was identical to that obtained by in vitro phosphorylation of I kappa B alpha with the partially purified cellular kinase. We propose that constitutive phosphorylation of I kappa B alpha is carried out by CKII.

Constitutive and regulated membrane expression of aquaporin 1 and aquaporin 2 water channels in stably transfected LLC-PK1 epithelial cells.

The aquaporins (AQPs) are a family of homologous water-channel proteins that can be inserted into epithelial cell plasma membranes either constitutively (AQP1) or by regulated exocytosis following vasopressin stimulation (AQP2). LLC-PK1 porcine renal epithelial cells were stably transfected with cDNA encoding AQP2 (tagged with a C-terminal c-Myc epitope) or rat kidney AQP1 cDNA in an expression vector containing a cytomegalovirus promoter. Immunofluorescence staining revealed that AQP1 was mainly localized to the plasma membrane, whereas AQP2 was predominantly located on intracellular vesicles. After treatment with vasopressin or forskolin for 10 min, AQP2 was relocated to the plasma membrane, indicating that this relocation was induced by cAMP. The location of AQP1 did not change. The basal water permeability of AQP1-transfected cells was 2-fold greater than that of nontransfected cells, whereas the permeability of AQP2-transfected cells increased significantly only after vasopressin treatment. Endocytotic uptake of fluorescein isothiocyanate-coupled dextran was stimulated 6-fold by vasopressin in AQP2-transfected cells but was only slightly increased in wild-type or AQP1-transfected cells. This vasopressin-induced endocytosis was inhibited in low-K+ medium, which selectively affects clathrin-mediated endocytosis. These water channel-transfected cells represent an in vitro system that will allow the detailed dissection of mechanisms involved in the processing, targeting, and trafficking of proteins via constitutive versus regulated intracellular transport pathways.

Chimeric tumor necrosis factor receptors with constitutive signaling activity.

Many hormone and cytokine receptors are crosslinked by their specific ligands, and multimerization is an essential step leading to the generation of a signal. In the case of the tumor necrosis factor (TNF) receptors (TNF-Rs), antibody-induced crosslinking is sufficient to trigger a cytolytic effect. However, the quaternary structural requirements for signaling--i.e., the formation of dimers, trimers, or higher-order multimers--have remained obscure. Moreover, it has not been clear whether the 55-kDa or 75-kDa TNF-R is responsible for initiation of cytolysis. We reasoned that an obligate receptor dimer, targeted to the plasma membrane, might continuously signal the presence of TNF despite the actual absence of the ligand. Such a molecule, inserted into an appropriate vector, could be used to project receptor-specific "TNF-like" activity to specific cells and tissues in vivo. Accordingly, we constructed sequences encoding chimeric receptors in which the extracellular domain of the mouse erythropoietin receptor (Epo-R) was fused to the "stem," transmembrane domain, and cytoplasmic domain of the two mouse TNF-Rs. Thus, the Epo-R group was used to drive dimerization of the TNF-R cytoplasmic domain. These chimeric proteins were well expressed in a variety of cell lines and bound erythropoietin at the cell surface. Both the 55-kDa and the 75-kDa Epo/TNF-R chimeras exerted a constitutive cytotoxic effect detected by cotransfection or clonogenic assay. Thus, despite the lack of structural homology between the cytoplasmic domains of the two TNF-Rs, a similar signaling endpoint was observed. Moreover, dimerization (rather than trimerization or higher-order multimerization) was sufficient for elicitation of a biological response.

CT-2576, an inhibitor of phospholipid signaling, suppresses constitutive and induced expression of human immunodeficiency virus.

Viruses such as human immunodeficiency virus (HIV) require cellular activation for expression. Cellular activation in lymphoid cells is associated with augmented accumulation of certain phosphatidic acid (PA) species derived from the hydrolysis of glycan phosphatidylinositol (GPI). This suggests that activation of a phospholipid pathway may play a role in initiation of viral replication. To test this hypothesis, we examined the effect of tat gene expression on the production of cellular PA species, as the Tat protein is essential for HIV expression and has been implicated in activating the expression of multiple host cellular genes. Expression of tat increased the expression of PA. We then tested whether synthetic inhibitors of PA metabolism would inhibit activation of the HIV long terminal repeat by Tat and tumor necrosis factor alpha (TNF-alpha). CT-2576 suppressed both PA generation induced by Tat and HIV long terminal repeat-directed gene expression in response to Tat or TNF-alpha at a posttranscriptional step. CT-2576 also inhibited constitutive as well as TNF-alpha- and interleukin 6-induced expression of HIV p24 antigen in chronically infected U1 cells and in peripheral blood lymphocytes acutely infected with a clinical isolate of HIV. Pharmacological inhibition of synthesis of selected PA species may therefore provide a therapeutic approach to suppression of HIV replication.

Constitutive activation of phototransduction by K296E opsin is not a cause of photoreceptor degeneration.

The missense mutation Lys-296-->Glu (K296E) in the rhodopsin gene produces an opsin with no chromophore binding site and therefore is not activated by light. Nevertheless, the mutant opsin constitutively activates transducin in vitro and causes photoreceptor degeneration in vivo, possibly by continuously activating the phototransduction cascade, analogous to constant exposure to environmental light. We studied the K296E mutation in eight lines of transgenic mice. Each line developed photoreceptor degeneration with the rate of degeneration increasing monotonically as the ratio of mutant:wild-type opsin mRNA increased. At no time in the course of degeneration was there endogenous light adaptation in the retina as measured by the electroretinogram. The mutant opsin was found to be invariably phosphorylated and stably bound to arrestin. Light-independent activation of transducin was demonstrated only after the removal of arrestin and dephosphorylation of K296E opsin. Thus, K296E opsin in vivo does not activate the phototransduction cascade because it is shut off by photoreceptor inactivation mechanisms. Our data show that the K296E mutation does not cause photoreceptor degeneration by continuous activation of phototransduction.

April 4, 1968: Death, Difference, and Dialogue

Robert Kennedy's announcement of the assassination of Martin Luther King, Jr., in an Indianapolis urban community that did not revolt in riots on April 4, 1968, provides one significant example in which feelings, energy, and bodily risk resonate alongside the articulated message. The relentless focus on Kennedy's spoken words, in historical biographies and other critical research, presents a problem of isolated effect because the power really comes from elements outside the speech act. Thus, this project embraces the complexities of rhetorical effectivity, which involves such things as the unique situational context, all participants (both Kennedy and his audience) of the speech act, aesthetic argument, and the ethical implications. This version of the story embraces the many voices of the participants through first hand interviews and new oral history reports. Using evidence provided from actual participants in the 1968 Indianapolis event, this project reflects critically upon the world disclosure of the event as it emerges from those remembrances. Phenomenology provides one answer to the constitutive dilemma of rhetorical effectivity that stems from a lack of a framework that gets at questions of ethics, aesthetics, feelings, energy, etc. Thus, this work takes a pedagogical shift away from discourse (verbal/written) as the primary place to render judgments about the effects of communication interaction. With a turn to explore extra-sensory reasoning, by way of the physical, emotional, and numinous, a multi-dimensional look at public address is delivered. The rhetorician will be interested in new ways of assessing effects. The communication ethicist will appreciate the work as concepts like answerability, emotional-volitional tone, and care for the other, come to life via application and consideration of Kennedy's appearance. For argumentation scholars, the interest comes forth in a re-thinking of how we do argumentation. And the critical cultural scholar will find this story ripe with opportunities to uncover the politics of representation, racialized discourse, privilege, power, ideological hegemony, and reconciliation. Through an approach of multiple layers this real-life tale will expose the power of the presence among audience and speaker, emotive argument, as well as the magical turn of fate which all contributes the possibility of a dialogic rhetoric.

E. Committee to frame rules, directions, and statutes of the Boylston Professorship of Rhetoric and Oratory, 1804

This subseries consists of a paper notebook containing a handwritten draft of the report presented to the Harvard Corporation on April 30, 1804 by the Committee to frame Rules, Directions, and Statutes of the Boylston Professorship of Rhetoric and Oratory. The handwritten report provides a numbered list of rules related to the Boylston Professorship and is dated April 16, 1804. The report is followed by a certification signed May 1, 1804 from President Joseph Willard that he was unable to attend the meeting of the Corporation to discuss the professorship.

Draft of the Rules, Directions, and Statutes of the Boylston Professorship of Rhetoric and Oratory in Harvard College, [ca. 1804]

This original draft was probably written by Eliphalet Pearson (1752-1826) as a member of the committee charged with the task of establishing the rules, directions, and statutes for the Boylston Professorship by the Harvard Corporation and the Board of Overseers. This draft is heavily edited and contains many cross outs through the text.

E. Committee to frame rules, directions, and statutes of the Boylston Professorship of Rhetoric and Oratory, 1804

This subseries consists of a paper notebook containing a handwritten draft of the report presented to the Harvard Corporation on April 30, 1804 by the Committee to frame Rules, Directions, and Statutes of the Boylston Professorship of Rhetoric and Oratory. The handwritten report provides a numbered list of rules related to the Boylston Professorship and is dated April 16, 1804. The report is followed by a certification signed May 1, 1804 from President Joseph Willard that he was unable to attend the meeting of the Corporation to discuss the professorship.

Common places, new places. The labour law rhetoric of the crisis. WP C.S.D.L.E. "Massimo D'Antona" .INT - 92/2012

'Common places', as argued in this paper, may at times fulfil a persuasive function. This is the case of messages enshrined in Europe 2020. In the aftermath of an unprecedented economic and financial crisis they may sound like common places. European institutions have given precedence to measures on financial and budgetary stability, thus marginalising social and employment policies. The only promising developments, the 'new places' in labour law, must be searched in the new synergies among employment and cohesion policies. National and subnational levels of decison-making should be favoured in order to attain the delivery of new partnership agreements, supported by European structural funds.

Federalist rhetoric or political tactics? The what, where, who, when and why of Juncker’s call for a common European army. EPC Commentary, 12 March 2015

In an interview with the German newspaper Welt am Sonntag on 8 March 2015, the President of the European Commission, Jean-Claude Juncker, strongly advocated a common European army to consolidate peace in Europe, react to security threats both within the continent and in its neighbourhood, defend European values vis-à-vis aggressive powers, notably Russia, and create economies of scale by consolidating demand for military equipment.