The dominant kanr marker gene plays an important role in gene disruption experiments in budding yeast, as this marker can be used in a variety of yeast strains lacking the conventional yeast markers. We have developed a loxP-kanMX-loxP gene disruption cassette, which combines the advantages of the heterologous kanr marker with those from the Cre-lox P recombination system. This disruption cassette integrates with high efficiency via homologous integration at the correct genomic locus (routinely 70%). Upon expression of the Cre recombinase the kanMX module is excised by an efficient recombination between the loxP sites, leaving behind a single loxP site at the chromosomal locus. This system allows repeated use of the kanr marker gene and will be of great advantage for the functional analysis of gene families.

Erythropoiesis is the process by which nucleated erythroid progenitors proliferate and differentiate to generate, every second, millions of nonnucleated red cells with their unique discoid shape and membrane material properties. Here we examined the time course of appearance of individual membrane protein components during murine erythropoiesis to throw new light on our understanding of the evolution of the unique features of the red cell membrane. We found that the accumulation of all of the major transmembrane and all skeletal proteins of the mature red blood cell, except actin, accrued progressively during terminal erythroid differentiation. At the same time, and in marked contrast, accumulation of various adhesion molecules decreased. In particular, the adhesion molecule, CD44 exhibited a progressive and dramatic decrease from proerythroblast to reticulocyte; this enabled us to devise a new strategy for distinguishing unambiguously between erythroblasts at successive developmental stages. These findings provide unique insights into the genesis of red cell membrane function during erythroblast differentiation and also offer a means of defining stage-specific defects in erythroid maturation in inherited and acquired red cell disorders and in bone marrow failure syndromes.E rythropoiesis is the process by which erythroid progenitors proliferate and differentiate into nonnucleated reticulocytes. Two distinct erythroid progenitors have been functionally defined in colony assays, namely, the early-stage burst-forming uniterythroid (BFU-E) and the later stage colony-forming uniterythroid (CFU-E) progenitor (1). The earliest morphologically recognizable erythroblast in hematopoietic tissues is the proerythroblast, which undergoes 3-4 mitoses to produce reticulocytes. Morphologically distinct populations of erythroblasts are produced by each successive mitosis, beginning with proerythroblasts and followed by basophilic, polychromatic, and orthochromatic erythroblasts. Finally, orthochromatic erythroblasts expel their nuclei to generate reticulocytes. This ordered differentiation process is accompanied by decreases in cell size, enhanced chromatin condensation, progressive hemoglobinization, and marked changes in membrane organization.During recent decades, detailed characterization of the protein composition and structural organization of the mature red cell membrane has led to insights into its function (2-6). A 2-dimensional spectrin-based skeletal network consisting of ␣-and ␤-spectrin, short actin filaments, protein 4.1R, ankyrin, protein 4.2, p55, adducin, dematin, tropomyosin, and tropomodulin has been shown to regulate membrane elasticity and stability. Mutations in some of these proteins result in loss of mechanical integrity and hemolytic anemia. The skeletal network is attached to the lipid bilayer through 2 major linkages (7). The first is through ankyrin, which itself forms part of a complex of band 3, glycophorin A, RhAG, CD47, and ICAM-4, while the second involves protein 4.1R, glycophorin C, and protei...

Steroid receptors activate and repress genes. An important class of genes that they repress is controlled by the transcription factor AP‐1. The activity of AP‐1 is inhibited by the receptor, a mechanism exploited for the therapy of various forms of pathological hyperproliferation in humans. We show here by point mutations in the DNA binding domain and by the choice of steroid ligands that repression of AP‐1 activity and transactivation functions of the glucocorticoid receptor (GR) are separable entities. While DNA binding and activation of glucocorticoid‐regulated promoters require GR dimerization, we present data that suggest that repression is a function of GR monomers.

Pluripotent hematopoietic stem cells have been studied extensively, but the events that occur during their differentiation remain largely uncharted. To develop a system that allows the differentiation of cultured multipotent progenitors by time-lapse fluorescence microscopy, myelomonocytic cells were labeled with green fluorescent protein (GFP) in vivo. This was achieved by knocking the enhanced GFP (EGFP) gene into the murine lysozyme M (lys) locus and using a targeting vector, which contains a neomycin resistant (neo) gene flanked by LoxP sites and “splinked” ends, to increase the frequency of homologous recombination. Analysis of the blood and bone marrow of thelys-EGFP mice revealed that most myelomonocytic cells, especially mature neutrophil granulocytes, were fluorescence-positive, while cells from other lineages were not. Removal of the neogene through breeding of the mice with the Cre-deleter strain led to an increased fluorescence intensity. Mice with an inactivation of both copies of the lys gene developed normally and were fertile.

The capsid domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein is a critical determinant of virus assembly, and is therefore a potential target for developing drugs for AIDS therapy. Recently, a 12-mer alpha-helical peptide (CAI) was reported to disrupt immature- and mature-like capsid particle assembly in vitro; however, it failed to inhibit HIV-1 in cell culture due to its inability to penetrate cells. The same group reported the X-ray crystal structure of CAI in complex with the C-terminal domain of capsid (C-CA) at a resolution of 1.7 A. Using this structural information, we have utilized a structure-based rational design approach to stabilize the alpha-helical structure of CAI and convert it to a cell-penetrating peptide (CPP). The modified peptide (NYAD-1) showed enhanced alpha-helicity. Experiments with laser scanning confocal microscopy indicated that NYAD-1 penetrated cells and colocalized with the Gag polyprotein during its trafficking to the plasma membrane where virus assembly takes place. NYAD-1 disrupted the assembly of both immature- and mature-like virus particles in cell-free and cell-based in vitro systems. NMR chemical shift perturbation analysis mapped the binding site of NYAD-1 to residues 169-191 of the C-terminal domain of HIV-1 capsid encompassing the hydrophobic cavity and the critical dimerization domain with an improved binding affinity over CAI. Furthermore, experimental data indicate that NYAD-1 most likely targets capsid at a post-entry stage. Most significantly, NYAD-1 inhibited a large panel of HIV-1 isolates in cell culture at low micromolar potency. Our study demonstrates how a structure-based rational design strategy can be used to convert a cell-impermeable peptide to a cell-permeable peptide that displays activity in cell-based assays without compromising its mechanism of action. This proof-of-concept cell-penetrating peptide may aid validation of capsid as an anti-HIV-1 drug target and may help in designing peptidomimetics and small molecule drugs targeted to this protein.

Standardization of immunophenotyping requires careful attention to reagents, sample handling, instrument setup, and data analysis, and is essential for successful cross-study and cross-center comparison of data. Experts developed five standardized, eight-color panels for identification of major immune cell subsets in peripheral blood. These were produced as pre-configured, lyophilized, reagents in 96-well plates. We present the results of a coordinated analysis of samples across nine laboratories using these panels with standardized operating procedures (SOPs). Manual gating was performed by each site and by a central site. Automated gating algorithms were developed and tested by the FlowCAP consortium. Centralized manual gating can reduce cross-center variability, and we sought to determine whether automated methods could streamline and standardize the analysis. Within-site variability was low in all experiments, but cross-site variability was lower when central analysis was performed in comparison with site-specific analysis. It was also lower for clearly defined cell subsets than those based on dim markers and for rare populations. Automated gating was able to match the performance of central manual analysis for all tested panels, exhibiting little to no bias and comparable variability. Standardized staining, data collection, and automated gating can increase power, reduce variability, and streamline analysis for immunophenotyping.

Immune thrombocytopenia (ITP) is an autoantibody-mediated bleeding disorder with both accelerated platelet destruction and impaired platelet production. We and others have described impaired regulatory CD4 ؉ CD25 hi T cells (Treg) numbers and/or suppressive function in ITP patients. Clinical trials using thrombopoietic agents to stimulate platelet production have shown favorable outcomes in ITP patients, but information on the immunologic responses of treated patients are lacking. We studied the immunologic profile of chronic ITP patients before (n ‫؍‬ 10) and during treatment with thrombopoietin receptor (TPO-R) agonists (n ‫؍‬ 9). Treg activity, as measured by suppression of proliferation of autologous CD4 ؉ CD25 ؊ cells, was improved in patients on treatment (P < .05), and the improvement correlated with reduction in interleukin-2-producing CD4 ؉ cells, consistent with dampening of immune responses. There was a concomitant increase in total circulating transforming growth factor-␤1 (TGF-␤1) levels (P ‫؍‬ .002) in patients on treatment, and the levels of TGF-␤1 correlated with the degree of improvement in platelet counts (r ‫؍‬ .8, P ‫؍‬ .0002). This suggests that platelets in patients on TPO-R treatment may play a role in improving Treg function, either directly or indirectly by enhanced release of TGF-␤1 as a result of greater platelet turnover. In conclusion, our findings suggest that thrombopoietic agents in patients with ITP have profound effects to restore immune tolerance. IntroductionImmune thrombocytopenia (ITP) is a bleeding disorder resulting from low platelet counts with an incidence of 2 and 12 per 100 000 adults and children, respectively, per year and a mortality rate of 1% to 3% per year in severely affected cases. 1,2 Autoreactive antibodies to platelet antigens, mainly the platelet glycoprotein IIb/IIIa complex, are considered responsible for accelerated destruction of platelets by the reticuloendothelial system and also reduced platelet production. 3 Whereas healthy persons harbor platelet-specific autoreactive T cells that are tolerized in the periphery, 4 patients with ITP possess activated platelet-autoreactive T cells with increasing cytokine imbalance toward interleukin-2 (IL-2) and interferon-␥, 5-9 especially in patients with chronic ITP with some also reporting higher levels of circulating proinflammatory cytokines tumor necrosis factor-␣ 10 and soluble CD40 ligand (sCD40L). 11 These data are consistent with loss of peripheral tolerance and an inflammatory phenotype in chronic ITP patients.CD4 ϩ regulatory T cells (Tregs) play a critical role in maintenance of peripheral tolerance by both directly and indirectly suppressing the activation and proliferation of many cell types, including T cells, B cells, dendritic cells, natural killer cells, and natural killer T cells in vivo and/or in vitro. 12 Because of their ability to control homeostasis and immunopathology, 13 the level of Tregs and their function are among the most informative criteria of a patient's immune status. Tregs are ...

Single cell PCR studies showed that hematopoietic stem cells (HSCs) express a variety of lineage-affiliated genes. However, it remains unclear whether these cells exhibiting "lineage priming" represent bona fide stem cells or a subpopulation earmarked for differentiation. Here we have used a Cre-Lox approach to follow the fate of cells expressing a lineage-affiliated marker. We crossed lysozyme Cre mice with yellow fluorescent protein (EYFP) reporter mice and found EYFP gene expression not only in myelomonocytic cells but also in a fraction of HSCs as well as B cells and T cells. Transplantation of EYFP+ HSCs into primary and secondary recipients generated mice in which all hematopoietic cells were EYFP+. In contrast, crosses between CD19 Cre and lck Cre mice with reporter mice showed no EYFP expression in HSCs or intermediate progenitors. Our results demonstrate that lysozyme expression does not mark myeloid commitment and that long-term repopulation potential is maintained in primed HSCs.