Isolation, growth and identification of colony-forming cells with erythroid, myeloid, dendritic cell and NK-cell potential from human fetal liver

The study of hematopoietic stem cells (HSCs) and the process by which they differentiate into committed progenitors has been hampered by the lack of in vitro clonal assays that can support erythroid, myeloid and lymphoid differentiation. We describe a method for the isolation from human fetal liver of highly purified candidate HSCs and progenitors based on the phenotypes CD38⁻CD34⁺⁺ and CD38⁺CD34⁺⁺, respectively. We also describe a method for the growth of colony-forming cells (CFCs) from these cell populations, under defined culture conditions, that supports the differentiation of erythroid, CD14/CD15⁺ myeloid, CD1a⁺ dendritic cell and CD56⁺ NK cell lineages. Flow cytometric analyses of individual colonies demonstrate that CFCs with erythroid, myeloid and lymphoid potential are distributed among both the CD38⁻ and CD38⁺ populations of CD34⁺⁺ progenitors. Published: June 11, 2002.     

Aequorin-based measurements of intracellular Ca2+-signatures in plant cells

Due to the involvement of calcium as a main second messenger in the plant signaling pathway, increasing interest has been focused on the calcium signatures supposed to be involved in the patterning of the specific response associated to a given stimulus. In order to follow these signatures we described here the practical approach to use the non-invasive method based on the aequorin technology. Besides reviewing the advantages and disadvantages of this method we report on results showing the usefulness of aequorin to study the calcium response to biotic (elicitors) and abiotic stimuli (osmotic shocks) in various compartments of plant cells such as cytosol and nucleus. Published: December 9, 2002     

Cell-based expression cloning for identification of polypeptides that hypersensitize mammalian cells to mitotic arrest

Microtubule inhibitors such as Vinblastine and Paclitaxel are chemotherapy agents that activate the mitotic spindle checkpoint, arresting cells in mitosis and leading to cell death. The pathways that connect mitotic arrest to cell death are not well characterized. We developed a mammalian cell-based cDNA cloning method to isolate proteins and protein fragments whose expression inhibits colony formation in the presence of microtubule inhibitors. Understanding how these proteins impact cellular responses to microtubule drugs will lead to better understanding of the biochemical pathways connecting mitotic arrest and cell death in mammalian cells and may provide novel targets that can enhance microtubule inhibitor-mediated chemotherapy.     

A fluorescence microscopy method for quantifying the detection of prostaglandin endoperoxide H synthase-1 and CD-41 in MEG-01 cells

In platelets, PGHS-1-dependant formation of thromboxane A₂ is an important modulator of platelet function and a target for pharmacological inhibition of platelet function by aspirin. Since platelets are anucleated cells, we have used the immortalized human megakaryoblastic cell line MEG-01, which can be induced to differentiate into platelet-like structures upon addition of TPA as a model system to study PGHS-1 gene expression. Using a specific antibody to PGHS-1 we have developed a technique using immunofluorescence microscopy and analysis of multiple digital images to monitor PGHS-1 protein expression as MEG-01 cells were induced to differentiate by a single addition of TPA (1.6 × 10⁻⁸ M) over a period of 8 days. The method represents a rapid and economical alternative to flow cytometry. Using this technique we observed that TPA induced adherence of MEG-01 cells, and only the non-adherent TPA-stimulated cells demonstrated compromised viability. The differentiation of MEG-01 cells was evaluated by the expression of the platelet-specific cell surface antigen, CD-41. The latter was expressed in MEG-01 cells at the later stages of differentiation. We demonstrated a good correlation between PGHS-1 expression and the overall level of cellular differentiation of MEG-01 cells. Furthermore, PGHS-1 protein expression, which shows a consistent increase over the entire course of differentiation can be used as an additional and better index by which to monitor megakaryocyte differentiation. Published: December 12, 2001     

T cell integrin overexpression as a model of murine autoimmunity

Integrin adhesion molecules have important adhesion and signaling functions. They also play a central role in the pathogenesis of many autoimmune diseases. Over the past few years we have described a T cell adoptive transfer model to investigate the role of T cell integrin adhesion molecules in the development of autoimmunity. This report summarizes the methods we used in establishing this murine model. By treating murine CD4+ T cells with DNA hypomethylating agents and by transfection we were able to test thein vitro effects of integrin overexpression on T cell autoreactive proliferation, cytotoxicity, adhesion and trafficking. Furthermore, we showed that the ability to inducein vivo autoimmunity may be unique to the integrin lymphocyte function associated antigen-1 (LFA-1). Published: October 24, 2003     

Expression and purification of recombinant vesicular glutamate transporter VGLUT1 using PC12 cells and High Five insect cells

In synaptic vesicles, the estimated concentration of the excitatory amino acid glutamate is 100–150 mM. It was recently discovered that VGLUT1, previously characterized as an inorganic phosphate transporter (BNPI) with 9–11 predicted transmembrane spanning domains, is capable of transporting glutamate. The expression and His-tag based purification of recombinant VGLUT1 from PC12 cells and High Five insect cells is described. Significantly better virus and protein expression was obtained using High Five rather than Sf9 insect cells. PC12 cell expressed VGLUT1 is functional but not the Baculovirus expressed protein. The lack of functionality of the Baculovirus expressed VGLUT1 is discussed. The data indicate that VGLUT1 readily oligomerizes/dimerizes. The data are discussed in the context of developing this system further in order to reconstitute vesicular glutamate uptake in vitro using lipid-detergent vesicles. Published: June 7, 2004.     

Application of a colorimetric assay to identify putative ribofuranosylaminobenzene 5′-phosphate synthase genes expressed with activity in<Emphasis Type="Italic">Escherichia coli</Emphasis>

Tetrahydromethanopterin (H₄MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5′-phosphate synthase (RFAP synthase). Given the importance of RFAP synthase in H₄MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H4MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies inEscherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase fromArchaeoglobus fulgidus was produced inE. coli and purified to homogeneity. The production of active RFAP synthase fromMethanothermobacter thermautotrophicus was achieved by coexpression of the geneMTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase. Florida Agricultural Experiment Station Journal Series no. R-09353 Published: March 4, 2003     

An improved method for constructing and selectively silanizing double-barreled,neutral liquid-carrier,ion-selective microelectrodes

We describe an improved, efficient and reliable method for the vapour-phase silanization of multi-barreled, ion-selective microelectrodes of which the silanized barrel(s) are to be filled with neutral liquid ion-exchanger (LIX). The technique employs a metal manifold to exclusively and simultaneously deliver dimethyldichlorosilane to only the ion-selective barrels of several multi-barreled microelectrodes. Compared to previously published methods the technique requires fewer procedural steps, less handling of individual microelectrodes, improved reproducibility of silanization of the selected microelectrode barrels and employs standard borosilicate tubing rather than the less-conventional theta-type glass. The electrodes remain stable for up to 3 weeks after the silanization procedure. The efficacy of a double-barreled electrode containing a proton ionophore in the ion-selective barrel is demonstrated in situ in the leaf apoplasm of pea (Pisum) and sunflower (Helianthus). Individual leaves were penetrated to depth of ∼150 μm through the abaxial surface. Microelectrode readings remained stable after multiple impalements without the need for a stabilizing PVC matrix.     

Force and compliance measurements on living cells using atomic force microscopy (AFM)

We describe the use of atomic force microscopy (AFM) in studies of cell adhesion and cell compliance. Our studies use the interaction between leukocyte function associated antigen-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1) as a model system. The forces required to unbind a single LFA-1/ICAM-1 bond were measured at different loading rates. This data was used to determine the dynamic strength of the LFA-1/ICAM-1 complex and characterize the activation potential that this complex overcomes during its breakage. Force measurements acquired at the multiple- bond level provided insight about the mechanism of cell adhesion. In addition, the AFM was used as a microindenter to determine the mechanical properties of cells. The applications of these methods are described using data from a previous study. Published: January 15, 2004     

Measuring solution viscosity and its effect on enzyme activity

In proteins, some processes require conformational changes involving structural domain diffusion. Among these processes are protein folding, unfolding and enzyme catalysis. During catalysis some enzymes undergo large conformational changes as they progress through the catalytic cycle. According to Kramers theory, solvent viscosity results in friction against proteins in solution, and this should result in decreased motion, inhibiting catalysis in motile enzymes. Solution viscosity was increased by adding increasing concentrations of glycerol, sucrose and trehalose, resulting in a decrease in the reaction rate of the H⁺-ATPase from the plasma membrane ofKluyveromyces lactis. A direct correlation was found between viscosity (η) and the inhibition of the maximum rate of catalysis (V _max). The protocol used to measure viscosity by means of a falling ball type viscometer is described, together with the determination of enzyme kinetics and the application of Kramers’ equation to evaluate the effect of viscosity on the rate of ATP hydrolysis by the H⁺-ATPase. Published: May 1, 2003     

Inactivation of sortilin (a novel lysosomal sorting receptor) by dominant negative competition and RNA interference

To assess the role of sortilin in the sorting and trafficking of sphingolipid activator proteins (SAPs) the function of sortilin was abolished by a dominant-negative mutant and by the use of RNAi. Mutant sortilin lacking the carboxyl-terminal region that contains the sorting signal abolished the trafficking of SAPs to the lysosomes. Both sortilin and SAPs were retained in the Golgi apparatus. The use of chemically synthesized siRNA effectively blocked the trafficking of SAPs to the lysosomes as well. Additionally, we created a stable COS-7 cell line transfected with the pSilencer 3.1 H1 neo vector containing a selected siRNA template oligonucleotide (small hairpin interference RNA) where the levels of sortilin were greatly suppressed. The elimination of sortilin by this method will permit to determine whether or not sortilin is involved in a general mechanism of lysosomal sorting that involves the trafficking of various soluble lysosomal proteins other than SAPs.     

Assessment of different protocols for the isolation and purification of gut associated lymphoid cells from the gilthead seabream (<Emphasis Type="Italic">Sparus aurata</Emphasis> L.)

Teleost gut associated lymphoid tissue (GALT) consists of leucocyte populations located both intraepithelially and in the lamina propria with no structural organization. The present study aims to assess different protocols for the isolation of GALT cells from an important fish species in the Mediterranean aquaculture, the gilthead seabream. Mechanical, chemical and enzymatic treatments were assayed. Nylon wool columns and continuous density gradients were used for further separation of cell subpopulations. Light microscopy and flow cytometry showed that the highest density band (HD) consisted of a homogeneous lymphocytic population, whereas the intermediate density band (ID) corresponded to epithelial and secretory cells and some lymphocytes. Respiratory burst activity of total cell suspensions revealed very low numbers of potential phagocytic cells, reflecting results from light microscopy and reports in other teleost species. The present data set up the basis for future functional characterization of GALT in seabream.