Biomedical subjects

W C Brown

Publications and source records attributed to W C Brown.

At least 91 records · Page 5Linked to original sources

PubMed1995

Feline immunodeficiency virus (FIV)-specific cytotoxic T lymphocytes from chronically infected cats are induced in vitro by retroviral vector-transduced feline T cells expressing the FIV capsid protein.

We have previously reported the presence of feline immunodeficiency virus (FIV)-specific, major histocompatibility complex (MHC)-restricted cytolytic T lymphocytes (CTL) in experimentally FIV-infected cats. However, the fine specificity of the CTL and the role of individual FIV proteins in inducing FIV-specific CTL responses remain unknown. In this study, we examined the in vitro induction and activity of FIV p24 capsid-specific CTL obtained from cats that had been experimentally infected with FIV Petaluma for 30 to 56 months. An amphotropic murine retroviral vector was used to generate transgenic primary feline T lymphoblasts that expressed the FIV capsid protein. When the autologous capsid-transduced T cells were used in vitro to stimulate CTL responses from peripheral blood mononuclear cells of chronically infected cats, MHC-restricted lysis of virus-infected target cells was observed. The majority of the CTL expressed CD8, and depletion of this population, but not CD4+ cells, effectively diminished the CTL activity. When the autologous capsid-transduced T cells were used as target cells, lysis by capsid-induced effectors was not observed. Analysis of capsid-transduced T cell clones revealed a variable and low level of capsid expression among the clones. This study demonstrates the potential for using retroviral vectors as a means of inducing CTL effector cells that will specifically kill lentivirus-infected cells during lentiviral infection.

Animals↗

PubMed1995

Expression and biological activities of bovine interleukin 4: effects of recombinant bovine interleukin 4 on T cell proliferation and B cell differentiation and proliferation in vitro.

Interleukin 4 (IL-4) is a pleotropic cytokine affecting a wide range of cell types in both the mouse and the human. These activities include regulation of the growth and differentiation of both T and B lymphocytes. The activities of IL-4 in nonprimate, nonmurine systems are not well established. Herein, we demonstrate in the bovine system that IL-4 upregulates production of IgM, IgG1, and IgE in the presence of a variety of costimulators including anti-IgM, Staphylococcus aureus cowan strain I, and pokeweed mitogen. IgE responses are potentiated by the addition of IL-2 to IL-4. Culture of bovine B lymphocytes with IL-4 in the absence of additional costimulators resulted in the increased surface expression of CD23 (low-affinity Fc epsilon RII), IgM, IL-2R, and MHC class II in a dose-dependent manner. IL-4 alone increased basal levels of proliferation of bulk peripheral blood mononuclear cells but in the presence of Con A inhibited proliferation. In contrast to the activities of IL-4 in the murine system, proliferation of TH1- and TH2-like clones was inhibited in a dose-dependent manner as assessed by antigen-or IL-2-driven in vitro proliferative responses. These observations are consistent with the role of IL-4 as a key player in regulation of both T and B cell responses.

Animals↗

PubMed1995

Ruptured ulnar artery pseudoaneurysm.

Ulnar artery aneurysms and pseudoaneurysms are rare lesions that usually occur distal to the wrist and cause symptoms as a result of embolization and not rupture. An elderly woman presented with acute rupture of an ulnar artery pseudoaneurysm proximal to the wrist, which caused severe neurologic compromise as a result of bleeding into Guyon's canal and the carpal tunnel. The patient had a remote wrist fracture resulting in a deformity that allowed the ulnar head to be in proximity to the ulnar artery. Rupture of the volar capsule with chronic abrasion of the artery eventually led to pseudoaneurysm formation and subsequent rupture. Emergency operative treatment with excision of the pseudoaneurysm, ulnar artery ligation, and repair of the volar capsule resulted in complete neurologic recovery without vascular compromise. This case is unique because of the proximal location of the pseudoaneurysm and the presentation with rupture. The anatomy, pathogenesis, and treatment options are discussed. Excision of the pseudoaneurysm with repair of the torn volar capsule is recommended to avoid recurrent problems. Vascular reconstruction is needed in cases where radial arterial flow is inadequate.

Aged↗

PubMed1995

Genetic and antigenic characterization of Babesia bovis merozoite spherical body protein Bb-1.

A Babesia bovis merozoite protein, Bb-1, was localized by immunoelectron microscopy to an apical organelle known as the spherical body. This unique structure appears to be analogous to dense granules of other apicomplexan protozoa. Similar to previously described dense granule proteins of Plasmodium spp., Bb-1 is secreted during or just after invasion of host erythrocytes and becomes associated with the cytoplasmic face of the infected cell. The amino terminal sequence of Bb-1 contains a predicted signal peptide and is similar to the amino terminus of another spherical body protein (BvVA1/225) which is also translocated to the erythrocyte membrane. Importantly, these two spherical body proteins are the major components of a protective fraction of B. bovis antigen. There is marked conservation of Bb-1 amino acid sequences and B-lymphocyte epitopes among geographic strains. However, a divergent Bb-1 allele (Bv80) in Australia strains encodes six regions of amino acid polymorphism, including a region of tetrapeptide repeats in the C-terminal half of the polypeptide. Two of the polymorphic regions map to previously defined Th1 epitopes on Bb-1.

Alleles↗

PubMed1995

Retroviral vector-transduced cells expressing the core polyprotein induce feline immunodeficiency virus-specific cytotoxic T-lymphocytes from infected cats.

The core polyprotein of feline immunodeficiency virus (FIV) was expressed in primary feline T-lymphocytes using a retroviral vector. These cells were used as antigen-presenting stimulator cells (APSC) for the in vitro induction of cytotoxic T-lymphocytes (CTL) from feline peripheral blood mononuclear cells (PBMC). CTL from 4 cats chronically infected with the Petaluma strain of FIV specifically lysed autologous FIV-infected targets in an MHC-restricted manner. The CD8 phenotype of more than 70% of the induced effector cells (97% for cells from one cat) was consistent with MHC class I-restricted cytotoxicity. In addition, it was possible to detect low levels of core polyprotein-specific lysis from effector cells of two of the FIV-infected cats. When observed, the level of lysis, measured as a percentage of specific 111In release, was lower for the transgenic gag-expressing targets than for FIV-infected targets. The difference in killing may reflect the low level of core CTL were not detected in either PBMC stimulated with cells transduced by a retroviral vector without the FIV gag sequence or PBMC from an uninfected cat stimulated with autologous transgenic APSC. The detection of FIV-specific CTL from infected cats following stimulation with transgenic APSC suggests a role for retroviral vectors in determining CTL specific for individual lentiviral proteins in protective immunity.

3T3 Cells↗

PubMed1995

Identification of candidate vaccine antigens of bovine hemoparasites Theileria parva and Babesia bovis by use of helper T cell clones.

Current vaccines for bovine hemoparasites utilize live attenuated organisms or virulent organisms administered concurrently with antiparasitic drugs. Although such vaccines can be effective, for most hemoparasites the mechanisms of acquired resistance to challenge infection with heterologous parasite isolates have not been clearly defined. Selection of potentially protective antigens has traditionally made use of antibodies to identify immunodominant proteins. However, numerous studies have indicated that induction of high antibody titers neither predicts the ability of an antigen to confer protective immunity nor correlates with protection. Because successful parasites have evolved antibody evasion tactics, alternative strategies to identify protective immunogens should be used. Through the elaboration of cytokines, T helper 1-(Th1)-like T cells and macrophages mediate protective immunity against many intracellular parasites, and therefore most likely play an important role in protective immunity against bovine hemoparasites. CD4+ T cell clones specific for soluble or membrane antigens of either Theileria parva schizonts or Babesia bovis merozoites were therefore employed to identify parasite antigens that elicit strong Th cell responses in vitro. Soluble cytosolic parasite antigen was fractionated by gel filtration, anion exchange chromatography or hydroxylapatite chromatography, or a combination thereof, and fractions were tested for the ability to induce proliferation of Th cell clones. This procedure enabled the identification of stimulatory fractions containing T. parva proteins of approximately 10 and 24 kDa. Antisera raised against the purified 24 kDa band reacted with a native schizont protein of approximately 30 kDa. Babesia bovis-specific Th cell clones tested against fractionated soluble Babesia bovis merozoite antigen revealed the presence of at least five distinct antigenic epitopes. Proteins separated by gel filtration revealed four patterns of reactivity, and proteins separated by anion exchange revealed two patterns of reactivity when selected T cell clones were assayed for stimulation by antigenic fractions. Studies using a continuous-flow electrophoresis apparatus have indicated the feasibility of identifying T cell-stimulatory proteins from parasite membranes as well as from the cytosolic fraction of B. bovis merozoites. The Th cell clones reactive with these different hemoparasites expressed either unrestricted or Th1 cytokine profiles, and were generally characterized by the production of high levels of IFN-gamma. A comprehensive study of T cell and macrophage responses to defined parasite antigens will help elucidate the reasons for vaccine failure or success, and provide clues to the mechanisms of acquired immunity that are needed for vaccine development.

Animals↗

PubMed1995

Interleukin-10 downregulates proliferation and expression of interleukin-2 receptor p55 chain and interferon-gamma, but not interleukin-2 or interleukin-4, by parasite-specific helper T cell clones obtained from cattle chronically infected with Babesia bovis or Fasciola hepatica.

Human recombinant interleukin-10 (IL-10) was previously shown to inhibit accessory cell (AC)-dependent proliferation of bovine parasite-specific T helper 1 (Th1), Th2, and Th0 cells in an IL-2-reversible manner (Brown, W.C., Woods, V.M., Chitko-McKown, C.G., Hash, S.M., and Rice-Ficht, A.C., 1994. Infect. Immun. 62, 4697-4708). The present study was therefore designed to determine whether the effect of IL-10 on T cell proliferation corresponded with downregulated expression of cytokines, or their receptors, important for T cell growth. The effects of IL-10 on cellular proliferation and expression of IL-2, IL-4, IL-2 receptor (IL-2R; p55), and IFN-gamma by Babesia bovis- or Fasciola hepatica-specific Th cell clones were simultaneously evaluated. As shown previously, IL-10 strongly inhibited proliferation of all types of Th cell clones, although this did not correspond with reduced expression of IL-2 or IL-4 mRNA or their products. In contrast, expression of IL-2R mRNA was consistently reduced in the IL-10-treated clones. These results indicate that IL-10 does not inhibit AC-dependent proliferation of bovine Th cells by downregulating T cell cytokines; rather, IL-10 may act by downregulating IL-2R p55 expression and subsequent signal transduction leading to decreased cellular proliferation. IFN-gamma production was also consistently downregulated in the presence of IL-10.

Animals↗

PubMed1995

Identification of Babesia bovis merozoite antigens separated by continuous-flow electrophoresis that stimulate proliferation of helper T-cell clones derived from B. bovis-immune cattle.

To characterize Babesia bovis merozoite antigens that stimulate anamnestic T helper (Th)-cell responses from B. bovis-immune cattle, B. bovis-specific Th-cell lines and clones, previously assigned to different antigenic groups (W. C. Brown, S. Zhao, A. C. Rice-Ficht, K. S. Logan, and V. M. Woods, Infect. Immun. 60:4364-4372, 1992), were tested in proliferation assays against fractionated merozoite antigens. The antigenic groups were determined by the patterns of response of Th clones to different parasite isolates and soluble or membrane forms of merozoite antigen. Soluble antigen fractionated by anion-exchange chromatography or gel filtration by using fast-performance liquid chromatography resolved two or three antigenic peaks, respectively. To enable fractionation of membrane-associated proteins and to resolve more precisely the proteins present in homogenized merozoites, a novel technique of continuous-flow electrophoresis was employed. Merozoite membranes or whole merozoites were homogenized and solubilized in sodium dodecyl sulfate-sample buffer, electrophoresed under reducing conditions on 15% or 10% acrylamide gels, eluted, and collected as fractions. Individual fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tested for the ability to stimulate Babesia-specific CD4+ T-cell lines and clones. CD4+ Th-cell lines from two cattle displayed differential patterns of reactivity and detected numerous peaks of antigenic activity, ranging from < 14 to 76 kDa. Th-cell clones previously categorized into different antigenic groups detected antigenic peaks unique for clones representative of a given group. Antigens of 29, 51 to 52, and 85 to 95 kDa (group I), 40 kDa (group III), 20 kDa (group IV), 58 to 60 kDa (group VI), and 38, 45, and 83 kDa (group VII) were identified in the stimulatory fractions. Immunization of rabbits with selected fractions produced a panel of antisera that reacted specifically on Western blots (immunoblots) with merozoite antigens of similar sizes, leading to the tentative identification of candidate antigens of B. bovis merozoites with molecular masses of 20, 40, 44, 51 to 52 or 95, and 58 to 60 kDa that stimulate proliferation of Th clones representative of five different antigenic groups. These antisera may be useful for isolating recombinant proteins that are immunogenic for Th cells of immune cattle and therefore potentially useful for vaccine development.

Animals↗

PubMed1994

Characterization of a cDNA encoding bovine interleukin 10: kinetics of expression in bovine lymphocytes.

A cDNA encoding bovine interleukin 10 (IL10) was cloned and sequenced using total cellular RNA derived from concanavalin A (ConA)-stimulated peripheral blood mononuclear cells (PBMC). A cDNA was produced with reverse transcriptase using oligo(dT) primers, and was amplified using primers chosen from consensus regions of the mouse and human IL10 genes. The nucleotide sequence derived from this cDNA shares 84, 79 and 78% homology with the human, mouse and rat cDNAs, respectively. The deduced amino-acid sequence shares an overall 77, 71 and 74% homology with the human, mouse and rat IL10 proteins, respectively. Northern blot analysis of the bovine IL10 mRNA reveals expression of a single 1.8-kb transcript, reaching maximal levels between 8 and 24 h, in ConA-stimulated peripheral T-cells, and weak expression in lipopolysaccharide-activated macrophages.

Amino Acid Sequence↗

PubMed1994

Functional and phenotypic characterization of WC1+ gamma/delta T cells isolated from Babesia bovis-stimulated T cell lines.

Functional studies with WC1+ gamma/delta T cell lines were performed to clarify the role of this subpopulation of gamma/delta T cells in the in vitro immune response to Babesia bovis. As CD4+ T cells decreased and gamma/delta T cells increased in B. bovis-stimulated T cell lines, antigen-specific proliferation declined to background levels. One irradiated gamma/delta T cell line inhibited proliferation of autologous Th1 cells, although unirradiated gamma/delta T cells either synergized with or had no effect on Th cell proliferation. gamma/delta T cells were not cytolytic for bovine alpha/beta T cells, but expressed natural killer (NK)-like cytotoxicity when assayed on xenogeneic NK-sensitive target cells. The gamma/delta T cells were IL-2 dependent and expressed IFN-gamma and TNF-alpha, but not TNF-beta, IL-2, or IL-4 mRNA. Together, these results raise the possibility that WC1+ gamma/delta T cells respond in vitro to autoantigens present on CD4+ T cells or to cytokines secreted by activated CD4+ T cells, resulting in modulation of the CD4+ T cell response and outgrowth of the gamma/delta T cells in parasite-stimulated lines.

Animals↗

PubMed1994

Use of helper T cells to identify potential vaccine antigens of Babesia bovis.

Babesia bovis is an economically important hemoprotozoon parasite o f cattle that is prevalent in many, tropical and subtropical regions o f the world. Effective vaccines against this tick-transmitted parasite consist o f live organisms attenuated by passage through splenectomized calves. However, the nature o f acquired resistance to challenge infection with heterologous isolates of this parasite has not been clearly defined. Unsuccessful attempts to select protective antigens have relied upon the use of antibodies to identify immunodominant proteins. In this review, Wendy Brown and Allison Rice-Ficht discuss the limitations of this approach and the rationale behind using helper T cells to select potential vaccine antigens.

Journal Article↗

PubMed1994

Interleukin-10 is expressed by bovine type 1 helper, type 2 helper, and unrestricted parasite-specific T-cell clones and inhibits proliferation of all three subsets in an accessory-cell-dependent manner.

Murine interleukin-10 (IL-10) is produced by type 2 helper (Th2) cells and selectively inhibits cytokine synthesis by type 1 helper (Th1) cells, whereas human IL-10 is produced by and inhibits proliferation and cytokine synthesis by both Th1 and Th2 subsets. This study reports that bovine IL-10 mRNA is expressed by Th0, Th1, and Th2 clones of bovine T cells specific for either Babesia bovis or Fasciola hepatica but not by two CD8+ T-cell clones. The antigen-induced proliferative responses of all three subsets of CD4+ cells were inhibited by human IL-10, and low levels (10 U/ml) of exogenous human IL-2 restored the suppressed response. However, proliferation of one Th1 clone was never inhibited but was enhanced by IL-10. Human IL-10 also inhibited the expression of gamma interferon and IL-4 mRNA in Th0 clones. In the absence of accessory cells (AC), the responses of Th clones to concanavalin A or IL-2 were not inhibited by IL-10, whereas antigen-specific responses of Th1 and Th2 cells were reduced when IL-10-pretreated macrophages were used as AC. Together, our results with bovine T cells support the concept that IL-10 primarily affects AC function and does not directly inhibit CD4+ T cells and demonstrate that the immunoregulatory effects of IL-10 are not selectively directed at Th1 populations, as they are in mice.

Animals↗

PubMed1994

CD4+ T-cell clones obtained from cattle chronically infected with Fasciola hepatica and specific for adult worm antigen express both unrestricted and Th2 cytokine profiles.

The well-established importance of helper T (Th)-cell subsets in immunity and immunoregulation of many experimental helminth infections prompted a detailed study of the cellular immune response against Fasciola hepatica in the natural bovine host. T-cell lines established from two cattle infected with F. hepatica were characterized for the expression of T-cell surface markers and proliferative responses against F. hepatica adult worm antigen. Parasite-specific T-cell lines contained a mixture of CD4+, CD8+, and gamma/delta T-cell-receptor-bearing T cells. However, cell lines containing either fewer than 10% CD8+ T cells or depleted of gamma/delta T cells proliferated vigorously against F. hepatica antigen, indicating that these T-cell subsets are not required for proliferative responses in vitro. Seventeen F. hepatica-specific CD4+ Th-cell clones were examined for cytokine expression following concanavalin A stimulation. Biological assays to measure interleukin-2 (IL-2) or IL-4, gamma interferon (IFN-gamma), and tumor necrosis factor and Northern (RNA) blot analysis to verify the expression of IL-2, IL-4, and IFN-gamma revealed that the Th-cell clones expressed a spectrum of cytokine profiles. Several Th-cell clones were identified as Th2 cells by the strong expression of IL-4 but little or no IL-2 or IFN-gamma mRNA. The majority of Th-cell clones were classified as Th0 cells by the expression of either all three cytokines or combinations of IL-2 and IL-4 or IL-4 and IFN-gamma. No Th1-cell clones were obtained. All of the Th-cell clones expressed a typical memory cell surface phenotype, characterized as CD45Rlow, and all expressed the lymph node homing receptor (L selectin). These results are the first to describe cytokine responses of F. hepatica-specific T cells obtained from infected cattle and extend our previous analysis of Th0 and Th1 cells from cattle immune to Babesia bovis (W. C. Brown, V. M. Woods, D. A. E. Dobbelaere, and K. S. Logan, Infect. Immun. 61:3273-3281, 1993) to include F. hepatica-specific Th2 cells.

Animals↗

PubMed1994

Mechanical stretch increases proto-oncogene expression and phosphoinositide turnover in vascular smooth muscle cells.

OBJECTIVE: To determine whether changes in haemodynamic load, simulated in vitro by mechanically stretching cultured vascular smooth muscle cells, could be transduced into biochemical signals similar to those produced by growth factors. DESIGN: A system was developed which was capable of stretching cultured vascular smooth muscle cells from 0 to 20%. The effect of stretching quiescent vascular smooth muscle cells on both c-fos messenger RNA (mRNA) expression and release of total inositol phosphates was determined over a time interval of 0-360 min. METHODS: Rat mesenteric artery vascular smooth muscle cells were grown using standard cell culture methods. Induction of the proto-oncogene, c-fos, was determined by Northern blotting. Phosphoinositide breakdown was assessed by measuring [3H]-inositol phosphates released from prelabelled cells. RESULTS: A 20% fixed stretch resulted in a rapid induction of c-fos mRNA which reached maximal levels by 15 min. The amount of c-fos mRNA detected was dependent on the degree of stretch, with maximum induction obtained for 15 and 20% stretch. The effects of mechanical stretch were also assessed on phosphoinositide turnover by measuring [3H]-inositol phosphates released from prelabelled cells. A 20% fixed stretch of vascular smooth muscle cells for 20 min resulted in a 3.2-fold increase in total [3H]-inositol phosphates released compared with unstretched cells. CONCLUSIONS: Our results show that mechanical stretch increases proto-oncogene expression and phosphoinositide turnover in vascular smooth muscle cells in vitro. These observations suggest that mechanical stretch and growth factors share common signal transduction pathways which may be important in the development of vascular hypertrophy.

Animals↗

PubMed1993

Interaction of proliferating cell nuclear antigen with yeast DNA polymerase delta.

Previously, we described the purification of the catalytic core subunit of yeast DNA polymerase delta from Escherichia coli carrying the yeast POL3 gene cloned in a vector that allowed efficient expression. The basic catalytic properties of the single subunit enzyme are virtually identical to the heterodimeric core enzyme purified from yeast cells (Brown, W. C., Duncan, J. A., and Campbell, J. L. (1993) J. Biol. Chem. 268, 982-990). In this work, we investigate the ability of yeast proliferating cell nuclear antigen (PCNA) to stimulate processive synthesis by the bacterially produced, single-subunit DNA polymerase delta. Yeast PCNA was found to stimulate the full-length single-subunit yeast DNA polymerase delta and to increase its processivity. A truncated version of DNA polymerase delta, from which the NH2-terminal 220 amino acids had been removed, was prepared and similarly investigated. While the catalytic properties of the truncated protein were nearly identical to those of the full-length enzyme, neither the extent of synthesis nor processivity was increased in the presence of PCNA. Thus, we conclude that the single-subunit DNA polymerase can associate productively with PCNA in the absence of other proteins and that the NH2-terminal domain of the catalytic subunit must be intact for this interaction.

Animals↗

PubMed1993

A new cloning vector and expression strategy for genes encoding proteins toxic to Escherichia coli.

Here, we describe a modification of a plasmid, pT7-7 [Tabor and Richardson, Proc. Natl. Acad. Sci. USA 262 (1985) 1074-1078], that allows expression of inserted genes from the phage T7 RNA polymerase promoter. The modification is designed to suppress readthrough transcription from cryptic promoters and start points on the plasmid, in order to reduce expression in the absence of T7 RNA polymerase and thus improve the vector for use in the expression of highly toxic gene products. This vector (pT7SC) was used to stably clone the POL3 gene (encoding DNA polymerase delta) of Saccharomyces cerevisiae, which destabilizes all other cloning and expression vectors tested. Previously described expression strategies proved ineffective in overexpressing the POL3 gene. A new strategy was developed which relies on induction by infection with mutant T7 phage. This system efficiently overproduced the POL3 gene product.

Bacteriophage T7↗

PubMed1993

Purification and characterization of the Saccharomyces cerevisiae DNA polymerase delta overproduced in Escherichia coli.

In order to further define the enzymatic properties of yeast DNA polymerase delta, the Saccharomyces cerevisiae POL3 gene, whose expression is highly toxic to bacteria in most cloning vectors, was cloned into a new T7 expression vector (W. C. Brown and J. L. Campbell, submitted for publication) which allowed efficient overexpression in bacteria. Fifteen mg of polymerase were obtained from 3 g of cells. Since the protein is produced in insoluble form, to obtain active polymerase, inclusion bodies were solubilized with urea. DNA polymerase delta (124 kDa) was purified in the presence of urea and then renatured by dialysis against buffers containing decreasing concentrations of urea. Optimal protein concentration for refolding was 5 micrograms/ml. By several criteria the enzyme obtained is comparable with that from yeast: specific activity, electrophoretic mobility, template preference, sensitivity to inhibitors, and processivity. The electrophoretic mobility suggests that, unlike DNA polymerase alpha, polymerase delta is not posttranslationally modified in yeast. Polyclonal antibody was raised against the full-length DNA polymerase delta from bacteria and shown to cross-react with the protein purified from yeast on protein blots. The renatured protein also exhibits an exonucleolytic activity. Further examination of this nuclease determined it to be a 3' to 5' exonuclease with the characteristics of a proofreading activity. The presence of this nuclease in the highly purified bacterial polymerase provides biochemical confirmation of earlier genetic evidence (Simon, M., Giot, L., and Faye, G. (1991) EMBO J. 10, 2165-2170) that suggested that DNA polymerase delta's core catalytic subunit contains an intrinsic 3' to 5' exonuclease.

Aphidicolin↗

PubMed1993

Nucleated cells response to protein electroinsertion.

Application of an electrical pulse field at a strength slightly below the value required for electroporation to a suspension of red blood cells in the presence of membrane xenoproteins leads to the insertion of those proteins in the erythrocyte plasma membrane. This observation is extended to nucleated cells. In the presence of glycophorin A, application of such pulses leads to the insertion of 10(4)-10(5) molecules of glycophorin A per cell in CEM-CM3, Hela S3, and bovine CD8+ T cells. Electroinserted glycophorin A is detected by flow cytometry using anti-glycophorin monoclonal antibodies. The survival of the cells subjected to electroinsertion was 55% for CEM-CM3 cells, 69% for Hela S3 cells, and 65% for CD8+ T cells. Cells cultured after electroinsertion lost the electroinserted glycophorin A, with two different rates, by a temperature and cell type-dependent mechanism. During the first 2 h after electroinsertion, the CD8+ T cells lost 12.5% of the inserted glycophorin A per h, the CEM-CM3 cells lost 7.7% per h, whereas the Hela S3 cells lost only 0.8% of the inserted protein per h. After 2 h, the rate increased substantially, to 41.7% per h for the CD8+ T cells, 13.5% for the CEM-CM3 cells, and 8.9% for the Hela S3 cells. Cytochalasin D efficiently inhibited the disappearance of electroinserted glycophorin A during the first 2 h after electroinsertion only.

Animals↗