Characterization of a monoclonal antibody specific for a novel primate cell surface marker with distinct biochemical properties on human erythroleukemia and myeloid cell lines.

A hybridoma, CSC-1, which secretes monoclonal antibody (MAb) specific for a cell surface molecule on African green monkey kidney cell line, BGMK, was isolated and characterized. The cell surface molecule recognized by CSC-1 is widely expressed on a variety of human cell lines. Among the hematopoietic cell lines examined, the CSC-1 marker seems to be preferentially expressed by lymphoid cell lines (e.

Mechanisms of metabotropic glutamate receptor desensitization: role in the patterning of effector enzyme activation.

Metabotropic glutamate receptors (mGluRs) constitute an unique subclass of G protein-coupled receptors (GPCRs). These receptors are activated by the excitatory amino acid glutamate and play an essential role in regulating neural development and plasticity. In the present review, we overview the current understanding regarding the molecular mechanisms involved in the desensitization and endocytosis of Group 1 mGluRs as well as the relative contribution of desensitization to the spatial-temporal patterning of glutamate receptor signaling.

Integrin alpha2beta1 on rat myeloma cells modulates interaction of alpha4beta1 integrin with vascular cell adhesion molecule-1 but not fibronectin.

It is well established that alpha2beta1 integrin functions as a receptor for collagen and laminin; whereas alpha4beta1 integrin binds fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In the present study, we showed that rat myeloma YB2/0 cells constitutively expressed alpha4beta1 but not alpha2beta1 integrin. Transfection of cDNA of mouse a2 integrin subunit resulted in the expression of heterologous alpha2beta1 integrin on YB2/0 cells (YBmalpha2).

Functional analysis of human hematopoietic repopulating cells mobilized with granulocyte colony-stimulating factor alone versus granulocyte colony-stimulating factor in combination with stem cell factor.

Using in vitro progenitor assays, serum-free in vitro cultures, and the nonobese diabetic/severe combined immune-deficient (NOD/SCID) ecotropic murine virus knockout xenotransplantation model to detect human SCID repopulating cells (SRCs) with multilineage reconstituting function, we have characterized and compared purified subpopulations harvested from the peripheral blood (PB) of patients receiving granulocyte colony-stimulating factor (G-CSF) alone or in combination with stem cell factor (SCF). Mobilized G-CSF plus SCF PB showed a 2-fold increase in total mononuclear cell content and a 5-fold increase in CD34-expressing cells depleted for lineage-marker expression (CD34(+)Lin(-)) as compared with patients treated with G-CSF alone. Functionally, G-CSF plus SCF-mobilized CD34(+)CD38(-)Lin(-) cells contained a 2-fold enhancement in progenitor frequency as compared with G-CSF-mobilized subsets.

On the influence of vessel planarity on local hemodynamics at the human carotid bifurcation.

The human carotid artery bifurcation is a complex, three-dimensional structure exhibiting non-planarity and both in- and out-of-plane curvature. The aim of this study was to determine the relative importance of vessel planarity, a potential geometric risk factor for atherogenesis, in determining the local hemodynamics. A combination of computational fluid dynamics and magnetic resonance imaging was used to reconstruct the subject-specific hemodynamics for three subjects.

Imaging inflammation: direct visualization of perivascular cuffing in EAE by magnetic resonance microscopy.

Purpose: To determine if the architectural features revealed by magnetic resonance microscopy (MRM) allow one to detect microscopic abnormalities associated with neuroinflammation in fixed brain sections from animals with experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis (MS).

Materials And Methods: Imaging was performed at the Center for In Vivo Microscopy (CIVM) using a 9.4-Tesla, 89-mm bore, superconducting magnet with actively shielded gradients capable of 850 mT/m.

Beta-arrestins regulate a Ral-GDS Ral effector pathway that mediates cytoskeletal reorganization.

beta-Arrestins are important in chemoattractant receptor-induced granule release, a process that may involve Ral-dependent regulation of the actin cytoskeleton. We have identified the Ral GDP dissociation stimulator (Ral-GDS) as a beta-arrestin-binding protein by yeast two-hybrid screening and co-immunoprecipitation from human polymorphonuclear neutrophilic leukocytes (PMNs). Under basal conditions, Ral-GDS is localized to the cytosol and remains inactive in a complex formed with beta-arrestins.

Phosphorylation-independent regulation of metabotropic glutamate receptor signaling by G protein-coupled receptor kinase 2.

The accepted paradigm for G protein-coupled receptor kinase (GRK)-mediated desensitization of G protein-coupled receptors involves GRK-mediated receptor phosphorylation followed by the binding of arrestin proteins. Although GRKs contribute to metabotropic glutamate receptor 1 (mGluR1) inactivation, beta-arrestins do not appear to be required for mGluR1 G protein uncoupling. Therefore, we investigated whether the phosphorylation of serine and threonine residues localized within the C terminus of mGluR1a is sufficient to allow GRK2-mediated attenuation of mGluR1a signaling.

Activated microglia (BV-2) facilitation of TNF-alpha-mediated motor neuron death in vitro.

We have studied the interactions between activated microglia and injured motor neurons using an immortalized murine microglial cell line (BV-2) stimulated with either lipopolysaccharide (LPS) (Escherichia coli) or supernatant from serum-deprived motor neurons (NSC-34 cell line). Both stimuli induced BV-2 activation. Although both BV-2 supernatants induced a subsequent increase in NO generation in otherwise healthy NSC-34 cells, only LPS-activated microglial supernatant induced NSC-34 cell death through a TNF-alpha-dependent pathway.

Image-based computational fluid dynamics modeling in realistic arterial geometries.

Local hemodynamics are an important factor in atherosclerosis, from the development of early lesions, to the assessment of stroke risk, to determining the ultimate fate of a mature plaque. Until recently, our understanding of arterial fluid dynamics and their relationship to atherosclerosis was limited by the use of idealized or averaged artery models. Recent advances in medical imaging, computerized image processing, and computational fluid dynamics (CFD) now make it possible to computationally reconstruct the time-varying, three-dimensional blood flow patterns in anatomically realistic models.

The C-terminal C1 cassette of the N -methyl-D-aspartate receptor 1 subunit contains a bi-partite nuclear localization sequence.

The N -methyl-D-aspartate receptor (NMDAR) is a multimeric transmembrane protein composed of at least two subunits. One subunit, NR1, is derived from a single gene and can be subdivided into three regions: the N-terminal extracellular domain, the transmembrane regions, and the C-terminal intracellular domain. The N-terminal domain is responsible for Mg2+ metal ion binding and channel activity, while the transmembrane domains are important for ion channel formation.

Clustering of a lipid-raft associated pool of ERM proteins at the immunological synapse upon T cell receptor or CD28 ligation.

Although ezrin is tyrosine phosphorylated following TCR ligation, its biological role in T cell activation is not known. Here, we shhow that ezrin clusters at the immunological synapse upon T cell stimulation. Clustering of ezrin can be triggered by TCR ligation, or, more efficiently, by CD28 ligation.

Inhibitors of nitric oxide synthase attenuate nerve growth factor-mediated increases in choline acetyltransferase expression in PC12 cells.

NGF can regulate nitric oxide synthase (NOS) expression and nitric oxide (NO) can modulate NGF-mediated neurotrophic responses. To investigate the role of NO in NGF-activated expression of cholinergic phenotype, PC12 cells were treated with either the nonselective NOS inhibitor L-NAME (N (omega)-nitro-L-arginine methylester) or the inducible NOS selective inhibitor MIU (s-methylisothiourea), and the effect on NGF-stimulated ChAT mRNA levels and ChAT specific activity was determined. NGF increased steady-state levels of mRNA and protein for both inducible and constitutive isozymes of NOS in PC12 cells, and led to enhanced NOS activity and NO production.

Overexpression of the signaling adapter FRS2 reconstitutes the cell cycle deficit of a nerve growth factor non-responsive TrkA receptor mutant.

We have characterized the cell cycle deficit of a novel TrkA receptor mutant (TrkAS3) that fails to support nerve growth factor (NGF)-dependent cell cycle arrest and neurite outgrowth. TrkAS3 receptors fail to support an NGF-dependent increase in the expression of cyclin D1 and the cell cycle inhibitor, p21(Waf1/Cip1), two important regulators of G(1) /S transition, and do not down-regulate expression of the G(2) /M phase marker, cdc2/cdk1, or the S phase marker, proliferating cell nuclear antigen. Moreover, NGF-activated TrkAS3 receptors do not down-regulate cyclin-dependent kinase 4 phosphorylation of the retinoblastoma protein, essential for G(1) arrest, in comparison to NGF-activated wild-type TrkA.

Human embryonic-derived hematopoietic repopulating cells require distinct factors to sustain in vivo repopulating function.

Objective: We have previously identified a novel circulating embryonic blood cell capable of pluripotent hematopoietic reconstitution, which may serve as a target for in utero stem cell therapy. Based on its unique biological properties and ontogenic origin, we aim to examine the ability to maintain and retrovirally transduce fetal blood (FB) reconstituting cells in ex vivo culture conditions previously optimized for pluripotent hematopoietic repopulating cells derived from later stages of human ontogeny.

Methods: FB cells were evaluated for proliferative potential, progenitor composition, and SCID-repopulating cell (SRC) capacity before and after 3 days of serum free (SF) ex vivo culture using the previously optimized growth factor conditions of SCF, Flt-3L, IL-3, IL-6, and G-CSF (GF Mix), in comparison to cultures using GF Mix + oncostatin M (OSM), or SCF + Flt-3L.

A precise radiographic technique for the measurement of dimensional changes in heart valve biomaterials following fixation.

Accurate tissue thickness measurements are difficult to acquire by present techniques. Error is introduced by tissue compression during measurements or by tissue processing prior to measurement. In the field of valve replacement, tissue dimensional changes from fixation prior to implantation may predispose implants to premature tissue failure and it becomes important to have an accurate method for comparing cusp dimensions pre- and post-fixation.

Predicting urinary stone composition using X-ray coherent scatter: a novel technique with potential clinical applications.

Purpose: Coherent scatter properties depend on the molecular structure of the scattering medium and measured scatter patterns are often characteristic of a chemical species. We explored the usefulness of coherent scatter analysis as a basis for identifying urinary calculus composition.

Materials And Methods: A laboratory system for collecting coherent scatter signals from biological specimens was developed.

An in vitro system for Doppler ultrasound flow studies in the stenosed carotid artery bifurcation.

To investigate the correlation between disease severity and Doppler spectral measurements in the carotid artery bifurcation, a unique in vitro system has been developed that mimics the human vasculature with respect to both anatomy and flow perfusion. Agar-based carotid phantoms are perfused with a blood-mimicking fluid using a computer-controlled pump and realistic pulsatile flow waveform. A three-axis translational stage allows the lumen to be interrogated with a 0.

Influence of hypoxia on wavelength dependence of differential pathlength and near-infrared quantification.

Continuous wave near-infrared spectroscopy (NIRS) measurements of cardiac correlated changes in attenuation in the adult human head were computed using a Fourier analysis technique that eliminates the positive error bias associated with the magnitude of the Fourier coefficient. These attenuation changes were used to determine wavelength dependence of differential pathlength, DP(lambda), at four stages during progressive hypoxia (21, 17, 13 and 9% FIO2) in normal volunteers. The effects of incorporating DP(lambda) into NIRS algorithms to compute relative concentration changes and absolute concentration of oxyhaemoglobin and deoxyhaemoglobin are discussed.

Cardiomyopathy in congenital complete lipodystrophy.

Molecular genetic studies have pointed to a relationship between congenital lipodystrophy syndromes and some cardiac disorders. For instance, mutations in LMNA cause either lipodystrophy or cardiomyopathy, indicating that different mutations in the same gene can produce these clinical syndromes. The present authors describe a 10-year-old female with Berardinelli-Seip congenital complete lipodystrophy (MIM 606158) caused by homozygosity for a frameshift mutation in BSCL2.

Regulation of autoimmune disease by natural killer T cells.

Natural killer T (NKT) cells express phenotypic characteristics shared by conventional natural killer cells and T cells, and reside in several primary and secondary lymphoid as well as nonlymphoid organs. Although these cells possess important effector functions in immunity against cancer and microbial pathogens, their immunoregulatory function has received much recent attention. There is convincing evidence to suggest a regulatory role for these cells in the control of susceptibility to autoimmune disease.

Surface cytotoxic T lymphocyte-associated antigen 4 partitions within lipid rafts and relocates to the immunological synapse under conditions of inhibition of T cell activation.

T cell activation through the T cell receptor (TCR) involves partitioning of receptors into discrete membrane compartments known as lipid rafts, and the formation of an immunological synapse (IS) between the T cell and antigen-presenting cell (APC). Compartmentalization of negative regulators of T cell activation such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is unknown. Recent crystal structures of B7-ligated CTLA-4 suggest that it may form lattices within the IS which could explain the mechanism of action of this molecule.

Role of donor MHC class III genes in the destruction of transplanted islets in NOD mice.

MHC class III genes are important in immune regulation and inflammation, and the gene products of this region are well conserved between species. Their role in diabetes is, however, unknown. We used islets from NOD mice that lacked expression of both MHC class I and class II molecules to test the effect of class III differences on the injury of transplanted NOD islets.

Regulatory natural killer T cells protect against spontaneous and recurrent type 1 diabetes.

Autoimmune diseases, especially type 1 diabetes (T1D), may be caused by dysregulation of the immune system, which leads to hyporesponsiveness of regulatory T helper 2 (Th2) cells and promotion of autoimmune Th1 cells. Natural killer T (NKT) cells, which comprise a minor subpopulation of T cells, play a critical role in immunoregulation as a result of a rapid burst of IL-4 and IFN-gamma secretion. These cells are functionally and numerically deficient in individuals at risk of T1D, as well as in nonobese diabetic (NOD) mice.

ShcB and ShcC activation by the Trk family of receptor tyrosine kinases.

Activation of the neurotrophin Trk receptors is a key process in the survival and development of the nervous system. The signaling adapters ShcB and ShcC, but not ShcA, are thought to be the primary Shc adaptor proteins in neurons as both are highly expressed in both the developing and adult nervous system. Although a previous study suggested that ShcB and ShcC do not strongly interact with the Trk receptors (1), we find that ShcB and ShcC bind the Trk receptors in a phosphotyrosine-dependent manner via their N-terminal phosphotyrosine binding domain at Tyr(499) (TrkA) and Tyr(515) (TrkB), they are tyrosine-phosphorylated in response to neurotrophin stimulation, and they enhance the activation of mitogen-activated protein kinase in Trk-expressing cells.