Yael Cohen

Cytokine-inducible SH2 protein (CIS3) and JAK2 binding protein (JAB) abolish prolactin receptor-mediated STAT5 signaling

The ability of five members of the cytokine‐inducible SH2 protein family (CIS1–4) and JAK2 binding (JAB) protein to affect prolactin receptor (PRLR)‐mediated activity was tested in human 293 embryonic kidney fibroblasts transiently transfected with rat PRLR, five concentrations of CIS/JAB Myc‐tagged cDNAs and a STAT5‐responsive reporter gene encoding luciferase. The protein expressions of CIS1, CIS2, CIS3 and JAB were comparable, whereas the level of CIS4 was slightly lower. PRLR‐mediated luciferase activity was abolished in a dose‐dependent manner in cells transfected with cDNA of CIS3 or JAB, even at concentrations below the level of protein detection by anti‐Myc antibody. In contrast, CIS1, CIS2 and CIS4 had little or no effect, despite similar levels of expression. CIS1 expression in postpartum mouse mammary glands was high and changed little in the course of 3 days. CIS2 and CIS3 expression was also high and increased further, whereas JAB expression was very low. These results hint that at least in mammary gland CIS3 is likely the main physiological negative regulator of the PRLR‐mediated JAK2/STAT5 pathway.

Modified monolayer electrodes for electrochemical and piezoelectric analysis of substrate-receptor interactions: novel immunosensor electrodes

Abstract Monolayer-modified Au-electrodes were used to analyze electrochemically host-guest binding interactions of biomaterials. Two configurations to sense the binding of an antibody and a lectin to the complementary substrate monolayer are addressed. In one configuration, a fluorescein monolayer was assembled on an Au-electrode and binding of the complementary anti-fluorescein antibody Flc-Ab was followed by the examination of electrode insulation by the antibody towards a solubilized redox probe, Fe(CN) 6 3− /Fe(CN) 6 4− . The extent of electrode insulation is controlled by the Flc-Ab concentration in the sample and the electrode responds amperometrically to Flc-Ab concentrations as low as 0.7 μI. The second configuration applies a redox-modified protein to analyze competitively the protein itself. An Au-electrode was modified by an α- d -mannopyranose monolayer, and a bipyridinium-modified concanavalin A was used to analyze concanavalin A (Con. A). Competitive binding of the redox-modified Con. A and the analyzed Con. A to the monolayer-modified electrode occurred, and the amperometric response was inversely proportional to the Con. A concentration. Quartz crystals coated with Au-electrodes were applied for the piezoelectric QCM analyses of Flc-Ab and Con. A. The crystal electrodes are modified with a fluorescein antigen monolayer. The Flc-Ab was sensed by the changes in the crystal frequencies as a result of the antibody association to the electrode. Flc-Ab at a concentration as low as 5 ng ml −1 was detected. The series of monosaccharides α- d -mannopyranose, β- d -glucose or α- d -glucose was assembled onto the Au-electrodes of the quartz crystals and used as a sensing interface for concanavalin A. The α- d -mannopyranose monolayer revealed high affinity for the binding of Con. A, whereas the β- d -glucose monolayer showed lower affinity for the protein, and the α- d -glucose monolayer lacked association to Con. A. The monolayer-modified quartz crystal electrodes revealed specificity for the respective complementary proteins.

Evidence of rainbow trout prolactin interaction with its receptor through unstable homodimerisation

This study aims to characterise Prolactin receptor (PRLR) in rainbow trout for which no information is available despite the availability of Salmonid PRL preparations. By screening a freshwater rainbow trout intestine cDNA library with a probe corresponding to the extracellular domain (ECD) of tilapia PRLR, we have cloned a 2.5 kb insert coding for the PRLR. The mature protein of 614 amino acid residues is similar to PRLR isolated in tilapia and also the long form of mammalian PRLR. Analysis of PRLR gene expression in osmoregulatory organs revealed the presence of a unique transcript, thus confirming the involvement of this hormone in the control of osmoregulation in this fish species. By using surface plasmon resonance (SPR) technology, kinetic measurement of interaction between trout PRL and its receptor ECD was studied. This approach allowed us to demonstrate the formation of a transient, unstable homodimeric complex. This unstability could explain the inability to perform binding experiments using homologous PRL. In contrast, heterologous lactogenic ligands were able to interact through a more stable complex. Whether these characteristics of PRL-receptor interaction in rainbow trout are different to what occurs in tilapia where a homologous radioreceptor assay was developed would require further studies.

Preparation and expression of biologically active prolactin and growth hormone receptors and suppressor of cytokine signaling proteins 1, 2, 3, and 6 tagged with cyan and yellow fluorescent proteins

To prepare reagents for a study of the interactions of prolactin (PRL) and growth hormone (GH) receptors (Rs) with suppressor of cytokine signaling (SOCS) proteins in living cells by fluorescence resonance energy transfer methodology, the respective proteins were tagged with cyan (CFP) or yellow (YFP) fluorescent protein. Constructs encoding ovine (o)PRLR-YFP, oPRLR-CFP, oGHR-YFP, and oGHR-CFP tagged downstream of the receptor DNA were prepared in the plasmid pcDNA plasmid and tested for biological activity in HEK 293T cells transiently cotransfected with those constructs and the reporter gene encoding luciferase. All four constructs were biologically active and as potent as their untagged counterparts. Cells transfected with those proteins exhibited fluorescence in the cytoplasm and the membrane. Constructs encoding DNA tagged with YFP or CFP upstream of SOCS1, SOCS2, SOCS3, and SOCS6 were prepared in pECFP-C1 and pEYFP-C1 plasmids. The biological activities of SOCS1 and SOCS3 tagged at their amino termini were assayed by their ability to inhibit placental lactogen (PL)- or GH-induced activation of JAK2/STAT5-mediated luciferase transcription in HEK 293T cells; the activity of SOCS2 was assayed by its ability to abolish SOCS1-induced inhibition. The tagged proteins exhibited biological activity that was equal to or even more potent than their untagged counterparts. The biological activities of CFP-SOCS2 and YFP-SOCS2 were also assayed using GST-GHR binding assay. Their interaction with the cytosolic domain of GHR was equivalent to their respective untagged counterparts. The biological activity of the construct encoding SOCS6 was not tested because of lack of a suitable assay. Cells transfected with eight of these tagged constructs expressed the fluorescent proteins in both the nucleus and cytosol; the tagged SOCS2 was localized mostly in the latter compartment.

Recombinant human CIS2 (SOCS2) protein: subcloning, expression, purification, and characterization.

The 1x myc-tagged cDNA encoding for human CIS2 protein was subcloned into a pET-29a+ vector in order to express and produce a recombinant S-peptide tagged and 1x myc-tagged protein in Escherichia coli BL21(DE3). The constitutively expressed protein was isolated from inclusion bodies by a simple solubilization-renaturation procedure and purified by anion-exchange chromatography on Q-Sepharose. The recombinant form was found to be pure and monomeric as judged by both SDS-PAGE and gel-filtration chromatography and its biological activity was proven by its ability to bind to the tyrosine-phosphorylated cytosolic fragment of human growth hormone receptor fused to glutathione-S-transferase. Recombinant CIS2 was compared by biochemical, immunological, and molecular methods to the CIS2 protein expressed in eukaryotic cells. This report describes the first substantial production of biologically active recombinant human CIS2.

A new view of grue

SummaryProfessor Goodman first presented his “new riddle of induction” in 19461 but it was mainly the more elaborated version published in hisFact, Fiction and Forecast in 1955 that has captured the attention of philosophers. Since then, numerous attempts to solve his “paradox of grue” appeared in press; none of them, however, proved to be wholly satisfactory.In this paper I want to present a solution to this 30-years old puzzle. In the first section I shall try to show that my solution, which is based on aGedankenexperiment, is immune to the objections leveled against previous attempts. In light of this solution I shall re-examine (in the second part of the paper) the status of the paradox and show that in order to preserve the meaningfulness of the paradox some type of Platonic framework for the theory of meaning should have to be assumed. In the last section I shall discuss a proposed solution to the paradox using counterfactual claims. I will show that despite the similarity between counterfactuals and thought-experiments, the counterfactual approach does not lead to a satisfactory solution.