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Detailed equilibrium binding studies were conducted on a monoclonal antibody directed against Pb(II) complexed with a protein conjugate of diethylenetriaminepentaacetic acid (DTPA). Binding curves obtained with DTPA and a cyclohexyl derivative of DTPA in the presence and absence of metal ions were consistent with the anticipated one-site homogeneous binding model. Binding curves obtained with aminobenzyl-DTPA or its complexes with Ca(II), Sr(II), and Ba(II) were highly sigmoidal, characterized by Hill coefficients of 2.3-6.5. Binding curves obtained with the Pb(II) and In(III) complexes of aminobenzyl-DTPA were hyperbolic, but in each case the apparent affinity of the antibody for the chelator-metal complex was higher in the presence of excess chelator than it was in the presence of excess metal ion. In the presence of excess chelator, the equilibrium dissociation constant for the binding of aminobenzyl-DTPA-Pb(II) to the antibody was 9.5 x 10(-)(10) M. Binding curves obtained with the Hg(II) and Cd(II) complexes of aminobenzyl-DTPA were biphasic, indicative of negative cooperativity. Further binding studies demonstrated that aminobenzyl-DTPA-Hg(II) opposed the binding of additional chelator-metal complexes to the antibody more strongly than did aminobenzyl-DTPA-Cd(II). The Fab fragment differed from the intact antibody only in that the apparent affinity of the Fab was generally lower for a given chelator-metal complex. These data are interpreted in terms of a model in which (i) aminobenzyl-DTPA and its complexes bind both to the antigen binding site and to multiple charged sites on the surface of the compact immunoglobulin; and (ii) the bound, highly charged ligands interact in a complicated fashion through the apolar core of the folded antibody.

Guggulipid is an extract of the guggul tree Commiphora mukul and has been widely used to treat hyperlipidemia in humans. The plant sterol guggulsterone (GS) is the active agent in this extract. Recent studies have shown that GS can act as an antagonist ligand for farnesoid X receptor (FXR) and decrease expression of bile acid-activated genes. Here we show that GS, although an FXR antagonist in coactivator association assays, enhances FXR agonist-induced transcription of bile salt export pump (BSEP), a major hepatic bile acid transporter. In HepG2 cells, in the presence of an FXR agonist such as chenodeoxycholate or GW4064, GS enhanced endogenous BSEP expression with a maximum induction of 400-500{\%} that induced by an FXR agonist alone. This enhancement was also readily observed in FXR-dependent BSEP promoter activation using a luciferase reporter construct. In addition, GS alone slightly increased BSEP promoter activation in the absence of an FXR agonist. Consistent with the results in HepG2, guggulipid treatment in Fisher rats increased BSEP mRNA. Interestingly, in these animals expression of the orphan nuclear receptor SHP (small heterodimer partner), a known FXR target, was also significantly increased, whereas expression of other FXR targets including cholesterol 7alpha-hydroxylase (Cyp 7a1), sterol 12alpha-hydroxylase (Cyp 8b1), and the intestinal bile acid-binding protein (I-BABP), remained unchanged. Thus, we propose that GS is a selective bile acid receptor modulator that regulates expression of a subset of FXR targets. Guggulipid treatment in rats lowered serum triglyceride and raised serum high density lipoprotein levels. Taken together, these data suggest that guggulsterone defines a novel class of FXR ligands characterized by antagonist activities in coactivator association assays but with the ability to enhance the action of agonists on BSEP expression in vivo.

Cellular immune responses play a critical role in the control of human immunodeficiency virus type 1 (HIV-1); however, the breadth of these responses at the single-epitope level has not been comprehensively assessed. We therefore screened peripheral blood mononuclear cells (PBMC) from 57 individuals at different stages of HIV-1 infection for virus-specific T-cell responses using a matrix of 504 overlapping peptides spanning all expressed HIV-1 proteins in a gamma interferon-enzyme-linked immunospot (Elispot) assay. HIV-1-specific T-cell responses were detectable in all study subjects, with a median of 14 individual epitopic regions targeted per person (range, 2 to 42), and all 14 HIV-1 protein subunits were recognized. HIV-1 p24-Gag and Nef contained the highest epitope density and were also the most frequently recognized HIV-1 proteins. The total magnitude of the HIV-1-specific response ranged from 280 to 25,860 spot-forming cells (SFC)/10(6) PBMC (median, 4,245) among all study participants. However, the number of epitopic regions targeted, the protein subunits recognized, and the total magnitude of HIV-1-specific responses varied significantly among the tested individuals, with the strongest and broadest responses detectable in individuals with untreated chronic HIV-1 infection. Neither the breadth nor the magnitude of the total HIV-1-specific CD8+-T-cell responses correlated with plasma viral load. We conclude that a peptide matrix-based Elispot assay allows for rapid, sensitive, specific, and efficient assessment of cellular immune responses directed against the entire expressed HIV-1 genome. These data also suggest that the impact of T-cell responses on control of viral replication cannot be explained by the mere quantification of the magnitude and breadth of the CD8+-T-cell response, even if a comprehensive pan-genome screening approach is applied.

Mesenchymal stem cells (MSCs) have the capability for renewal and differentiation into various lineages of mesenchymal tissues. These features of MSCs attract a lot of attention from investigators in the context of cell-based therapies of several human diseases. Despite the fact that bone marrow represents the main available source of MSCs, the use of bone marrow-derived cells is not always acceptable due to the high degree of viral infection and the significant drop in cell number and proliferative/differentiation capacity with age. Thus, the search for possible alternative MSC sources remains to be validated. Umbilical cord blood is a rich source of hematopoietic stem/progenitor cells and does not contain mesenchymal progenitors. However, MSCs circulate in the blood of preterm fetuses and may be successfully isolated and expanded. Where these cells home at the end of gestation is not clear. In this investigation, we have made an attempt to isolate MSCs from the subendothelial layer of umbilical cord vein using two standard methodological approaches: the routine isolation of human umbilical vein endothelial cell protocol and culture of isolated cells under conditions appropriate for bone-marrow-derived MSCs. Our results suggest that cord vasculature contains a high number of MSC-like elements forming colonies of fibroblastoid cells that may be successfully expanded in culture. These MSC-like cells contain no endothelium- or leukocyte-specific antigens but express alpha-smooth muscle actin and several mesenchymal cell markers. Therefore, umbilical cord/placenta stroma could be regarded as an alternative source of MSCs for experimental and clinical needs.

We have previously examined the specificities of 28 commercially available compounds, reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases [Davies, Reddy, Caivano and Cohen (2000) Biochem. J. 351, 95-105]. In the present study, we have extended this analysis to a further 14 compounds. Of these, indirubin-3'-monoxime, SP 600125, KT 5823 and ML-9 were found to inhibit a number of protein kinases and conclusions drawn from their use in cell-based assays are likely to be erroneous. Kenpaullone, Alsterpaullone, Purvalanol, Roscovitine, pyrazolopyrimidine 1 (PP1), PP2 and ML-7 were more specific, but still inhibited two or more protein kinases with similar potency. Our results suggest that the combined use of Roscovitine and Kenpaullone may be useful for identifying substrates and physiological roles of cyclin-dependent protein kinases, whereas the combined use of Kenpaullone and LiCl may be useful for identifying substrates and physiological roles of glycogen synthase kinase 3. The combined use of SU 6656 and either PP1 or PP2 may be useful for identifying substrates of Src family members. Epigallocatechin 3-gallate, one of the main polyphenolic constituents of tea, inhibited two of the 28 protein kinases in the panel, dual-specificity, tyrosine-phosphorylated and regulated kinase 1A (DYRK1A; IC(50)=0.33 microM) and p38-regulated/activated kinase (PRAK; IC(50)=1.0 microM).

Killer immunoglobulin-like receptors (KIR) bind self-major histocompatibility complex class I molecules, allowing natural killer (NK) cells to recognize aberrant cells that have down-regulated class I. NK cells express variable numbers and combinations of highly homologous clonally restricted KIR genes, but uniformly express KIR2DL4. We show that NK clones express both 2DL4 alleles and either one or both alleles of the clonally restricted KIR 3DL1 and 3DL2 genes. Despite allele-independent expression, 3DL1 alleles differed in the core promoter by only one or two nucleotides. Allele-specific 3DL1 gene expression correlated with promoter and 5' gene DNA hypomethylation in NK cells in vitro and in vivo. The DNA methylase inhibitor, 5-aza-2'-deoxycytidine, induced KIR DNA hypomethylation and heterogeneous expression of multiple KIR genes. Thus, NK cells use DNA methylation to maintain clonally restricted expression of highly homologous KIR genes and alleles.

HOX genes, notably members of the HOXA cluster, and HOX cofactors have increasingly been linked to human leukemia. Intriguingly, HOXD13, a member of the HOXD cluster not normally expressed in hematopoietic cells, was recently identified as a partner of NUP98 in a t(2;11) translocation associated with t-AML/MDS. We have now tested directly the leukemogenic potential of the NUP98-HOXD13 t(2; 11) fusion gene in the murine hematopoietic model. NUP98-HOXD13 strongly promoted growth and impaired differentiation of early hematopoietic progenitor cells in vitro; this effect was dependent on the NUP98 portion and an intact HOXD13 homeodomain. Expression of the NUP98-HOXD13 fusion gene in vivo resulted in a partial impairment of lymphopoiesis but did not induce evident hematologic disease until late after transplantation (more than 5 months), when some mice developed a myeloproliferative-like disease. In contrast, mice transplanted with bone marrow (BM) cells cotransduced with NUP98-HOXD13 and the HOX cofactor Meis1 rapidly developed lethal and transplantable acute myeloid leukemia (AML), with a median disease onset of 75 days. In summary, this study demonstrates that NUP98-HOXD13 can be directly implicated in the molecular process leading to leukemic transformation, and it supports a model in which the transforming properties of NUP98-HOXD13 are mediated through HOX-dependent pathways. View Publication

HIV-1 replication in simian cells is restricted at an early postentry step because of the presence of an inhibitory cellular factor. This block reduces the usefulness of HIV-1-based lentiviral vectors in primate animal models. Here, we demonstrate that substitution of the cyclophilin A (CyPA) binding region in the capsid of an HIV-1-based lentiviral vector (LV) with that of the macrophage tropic HIV-1 Ba-L resulted in a vector that was resistant to the inhibitory effect and efficiently transduced simian cells. Notably, the chimeric gag LV efficiently transduced primary simian hematopoietic progenitor cells, a critical cellular target in gene therapy. The alterations in the CyPA binding region did not affect CyPA incorporation; however, transduction by the gag chimeric LV seemed to be relatively insensitive to cyclosporin A, indicating that it does not require CyPA for early postentry steps. In dual infection experiments, the gag chimeric LV failed to remove the block to transduction of the WT LV, suggesting that the gag chimeric LV did not saturate the inhibitory simian cellular factor. These data suggest that the CyPA binding region of capsid contains a viral determinant involved in the postentry restriction of HIV-1-based lentiviral vectors. Overall, the findings demonstrate that the host range of HIV-1-based LV can be altered by modifications in the packaging construct. View Publication

Embryonic stem cells are pluripotent cells derived from the inner cell mass of mouse blastocysts that have been shown to differentiate spontaneously into cell types representing all three germ layers. This study shows that ES cells were induced to differentiate in vitro into mineralized osteoblasts under the influence of ascorbic acid, beta-glycerophosphate and 1alpha,25-OH vitamin D3. The activity of alkaline phosphatase, an early osteoblast marker, was found to be increased around day 12 of culture. Mineralized cells were clearly identified by histochemical staining, which detects mineralized calcium. The major noncollagenous component of bone matrix, osteocalcin, was localized to the mineralized cells by immunofluorescence. The expression of bone-specific genes was analyzed by real-time quantitative PCR. Osteocalcin and bone sialoprotein (BSP) were identified as early as in the fourth week of embryonic stem cell culture, both being characteristic for late stages of osteoblastic differentiation, indicating that at this time of culture the identified cells represent mature" osteoblasts. The osteoblast-specific transcription factor Cbfa1 was induced a few days earlier. The expression of osteopontin and osteonectin�

Here it is shown that the phenotype of adult mice lacking the first enhancer (DNA hypersensitive site I) and the distal promoter of the GATA-1 gene (neo Delta HS or GATA-1(low) mutants) reveals defects in mast cell development. These include the presence of morphologically abnormal alcian blue(+) mast cells and apoptotic metachromatic(-) mast cell precursors in connective tissues and peritoneal lavage and numerous (60-70% of all the progenitors) unique" trilineage cells committed to erythroid� View Publication

It is largely unknown how hematopoietic progenitors are positioned within specialized niches of the bone marrow microenvironment during development. Chemokines such as CXCL12, previously called stromal cell-derived factor 1, are known to activate cell integrins of circulating leukocytes resulting in transient adhesion before extravasation into tissues. However, this short-term effect does not explain the mechanism by which progenitor cells are retained for prolonged periods in the bone marrow. Here we show that in human bone marrow CXCL12 triggers a sustained adhesion response specifically in progenitor (pro- and pre-) B cells. This sustained adhesion diminishes during B cell maturation in the bone marrow and, strikingly, is absent in circulating mature B cells, which exhibit only transient CXCL12-induced adhesion. The duration of adhesion is tightly correlated with CXCL12-induced activation of focal adhesion kinase (FAK), a known molecule involved in integrin-mediated signaling. Sustained adhesion of progenitor B cells is associated with prolonged FAK activation, whereas transient adhesion in circulating B cells is associated with short-lived FAK activation. Moreover, sustained and transient adhesion responses are differentially affected by pharmacological inhibitors of protein kinase C and phosphatidylinositol 3-kinase. These results provide a developmental cell stage-specific mechanism by which chemokines orchestrate hematopoiesis through sustained rather than transient activation of adhesion and cell survival pathways.

Caspase-directed apoptosis usually fragments cells, releasing nonfunctional, prothrombogenic, membrane-bound apoptotic bodies marked for rapid engulfment by macrophages. Blood platelets are functional anucleate cells generated by specialized fragmentation of their progenitors, megakaryocytes (MKs), but committed to a constitutive caspase-independent death. Constitutive formation of the proplatelet-bearing MK was recently reported to be caspase-dependent, apparently involving mitochondrial release of cytochrome c, a known pro-apoptogenic factor. We extend those studies and report that activation of caspases in MKs, either constitutively or after Fas ligation, yields platelets that are functionally responsive and evade immediate phagocytic clearance, and retain mitochondrial transmembrane potential until constitutive platelet death ensues. Furthermore, the exclusion from the platelet progeny of caspase-9 present in the progenitor accounts for failure of mitochondrial release of cytochrome c to activate caspase-3 during platelet death. Thus, progenitor cell death by apoptosis can result in birth of multiple functional anucleate daughter cells.