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T K Hart

Publications and source records attributed to T K Hart.

At least 37 records · Page 2Linked to original sources
PubMed1996

Purification and characterization of fully functional human osteoclast precursors.

The identification and purification of human osteoclast precursors is essential to further our understanding of the mechanisms that control human osteoclast differentiation. Osteoclastoma tissue potentially provides a rich source of human osteoclast precursors, and in previous studies we have demonstrated the existence of a population of mononuclear cells within this tissue that is reactive with osteoclast-selective vitronectin receptor monoclonal antibodies. In this study, mononuclear cells expressing the vitronectin receptor, as defined by their ability to react with a murine monoclonal antibody to the beta 3 chain of the vitronectin receptor (87MEM1), were isolated from collagenase digests of osteoclastoma tissue using a fluorescence activated cell sorter. Based on their fluorescence signal and size, approximately 2-3% of the viable cells (typically 2 x 10(5)) were obtained and prepared for further phenotyping. The isolated cells demonstrated a number of phenotypic characteristics of osteoclasts: positive tartrate-resistant acid phosphatase (TRAP) activity, reactivity with human osteoclast-selective antibodies, expression of calcitonin receptors, cathepsin K (a novel osteoclast-selective cysteine proteinase) mRNA, and osteopontin mRNA and protein. These phenotypic characteristics were also detected in mononuclear cells within cryostat sections of the native osteoclastoma tissue as well as in resorption lacunae of sections of human bone. In contrast, isolated peripheral blood monocytes were negative for TRAP activity and osteopontin expression and, unlike the osteoclastoma-derived cells, demonstrated strong nonspecific esterase activity. Significantly, when the osteoclastoma-derived 87MEM1 positive cells were cocultured on whale dentine for 1-3 weeks with stromal cells, extensive resorption of the dentine surface was observed. This is the first demonstration of the purification of human osteoclast precursors. These cells provide an homogeneous cell population for studying cellular events that occur during human osteoclast differentiation.

Antibodies, Monoclonal↗
PubMed1996

An internalization signal in the simian immunodeficiency virus transmembrane protein cytoplasmic domain modulates expression of envelope glycoproteins on the cell surface.

A Tyr to Cys mutation at amino acid position 723 in the cytoplasmic domain of the simian immunodeficiency virus (SIV) transmembrane (TM) molecule has been shown to increase expression of envelope glycoproteins on the surface of infected cells. Here we show that Tyr-723 contributes to a sorting signal that directs the rapid endocytosis of viral glycoproteins from the plasma membrane via coated pits. On cells infected by SIVs with a Tyr at position 723, envelope glycoproteins were transiently expressed on the cell surface and then rapidly endocytosed. Similar findings were noted for envelope molecules expressed in the absence of other viral proteins. Immunoelectron microscopy demonstrated that these molecules were localized in patches on the cell surface and were frequently associated with coated pits. In contrast, envelope glycoproteins containing a Y723C mutation were diffusely distributed over the entire plasma membrane. To determine if an internalization signal was present in the SIV TM, chimeric molecules were constructed that contained the CD4 external and membrane spanning domains and a SIV TM cytoplasmic tail with a Tyr or other amino acids at SIV position 723. In Hela cells stably expressing these molecules, chimeras with a Tyr-723 were rapidly endocytosed, while chimeras containing other amino acids at position 723, including a Phe, were internalized at rates only slightly faster than a CD4 molecule that lacked a cytoplasmic domain. In addition, the biological effects of the internalization signal were evaluated in infectious viruses. A mutation that disrupted the signal and as a result, increased the level of viral envelope glycoprotein on infected cells, was associated with accelerated infection kinetics and increased cell fusion during viral replication. These results demonstrate that a Tyr-dependent motif in the SIV TM cytoplasmic domain can function as an internalization signal that can modulate expression of the viral envelope molecules on the cell surface and affect the biological properties of infectious viruses. The conservation of an analogous Tyr in all human and simian immunodeficiency viruses suggests that this signal may be present in other primate lentiviruses and could be important in the pathogenesis of these viruses in vivo.

Amino Acid Sequence↗
PubMed1995

Ultrastructural evidence of an interaction between Env and Gag proteins during assembly of HIV type 1.

Assembly and budding of retroviruses is believed to involve a complex interaction of envelope and capsid proteins at the host cell membrane. The nature of these interactions is, however, incompletely understood. Studies of the topography of the surface of HIV-1 have shown that the envelope glycoprotein projections (knobs) are arranged in a T = 7 levo rotational symmetry. Similarly, an icosahedral structure has been suggested for the p17 matrix of HIV-1. In an effort to investigate whether there is a structural interaction between these molecules, virions whose maturation was blocked by an inhibitor of HIV protease were studied using cytochemistry, morphometry, and 2D fast Fourier transform image enhancement. Analysis of the relationship between core morphology and the topographic distribution of envelope glycoprotein projections on HIV-1 provided structural evidence of an interaction between Env and Gag proteins. Furthermore, image enhancement revealed a periodic substructure in the Pr55gag plaque. Taken together, the data suggest an interaction between Pr55gag and the gp120-gp41 complex during assembly and budding of HIV-1. This interaction may, in part, contribute to determining the amount of Env glycoprotein that will be incorporated into a virion, and therefore play a role in the biology of HIV-1.

HIV Core Protein p24↗
PubMed1995

A single amino acid change in the cytoplasmic domain of the simian immunodeficiency virus transmembrane molecule increases envelope glycoprotein expression on infected cells.

We have described a virus termed CP-MAC, derived from the BK28 molecular clone of simian immunodeficiency virus, that was remarkable for its ability to infect Sup-T1 cells with rapid kinetics, cell fusion, and CD4 down-modulation (C. C. LaBranche, M. M. Sauter, B. S. Haggarty, P. J. Vance, J. Romano, T. K. Hart, P. J. Bugelski, and J. A. Hoxie, J. Virol. 68:5509-5522, 1994 [Erratum 68:7665-7667]). Compared with BK28, CP-MAC exhibited a number of changes in its envelope glycoproteins, including a highly stable association between the external (SU) and transmembrane (TM) molecules, a more rapid electrophoretic mobility of TM, and, of particular interest, a marked increase in the level of envelope protein expression on the surface of infected cells. These changes were shown to be associated with 11 coding mutations in the env gene (5 in SU and 6 in TM). In this report, we demonstrate that a single amino acid mutation of a Tyr to a Cys at position 723 (Y723C) in the TM cytoplasmic domain of CP-MAC is the principal determinant for the increased expression of envelope glycoproteins on the cell surface. When introduced into the env gene of BK28, the Y723C mutation produced up to a 25-fold increase in the levels of SU and TM on chronically infected cells, as determined by fluorescence-activated cell sorter analysis with monoclonal and polyclonal antibodies. A similar effect was observed when a Tyr-to-Cys change was introduced at the analogous position (amino acid 721) in the SIVmac239 molecular clone, which, unlike BK28 does not contain a premature stop codon in its TM cytoplasmic tail. Substituting other amino acids, including Ala, Ile, and Ser, at this position produced increases in surface envelope glycoproteins that were similar to that observed for the Cys substitution, while a Tyr-to-Phe mutation produced a smaller increase. These results could not be accounted for by differences in the kinetics or efficiency of envelope glycoprotein processing or by shedding of SU from infected cells. However, immunoelectron microscopy demonstrated that the Y723C mutation in BK28 produced a striking redistribution of cell surface envelope molecules from localized patches to a diffuse pattern that covered the entire plasma membrane. This finding suggests that mutation of a Tyr residue in the simian immunodeficiency virus TM cytoplasmic domain may disrupt a structural element that can modulate envelope glycoprotein expression on the surface of infected cells.

Amino Acid Sequence↗
PubMed1995

Preclinical pharmacokinetic evaluation of the respiratory syncytial virus-specific reshaped human monoclonal antibody RSHZ19.

A preclinical evaluation of RSHZ19, a respiratory syncytial virus-specific reshaped human monoclonal antibody (IgG1 framework), has included pharmacokinetic studies in rats, adult cynomolgus macaques, and infant baboons after intravenous (iv), subcutaneous, or intramuscular (im) administration. After iv administration to rats and monkeys (1 mg/kg dose), a biphasic decline in plasma concentration was observed. The dominant terminal phase was characterized by an 11-day half-life in rats and a 21- to 24-day half-life in monkeys. Plasma clearances of 0.3 ml/hr/kg in the rat and 0.1-0.2 ml/hr/kg in the monkey were estimated. In the macaque, based on area under the curve, no evidence of significant nonlinearity in the pharmacokinetics was observed over a 200-fold dose range (1-200 mg/kg). In rat and monkey, absorption after extravascular administration was rapid relative to elimination (apparent half-lives < or = 24 hr), and bioavailability was high (> or = 82%). After iv or im administration to macaques (> or = 40 mg/kg), 1 of 3 animals in each group developed anti-RSHZ19 antibodies, and this resulted in rapid elimination of RSHZ19 from plasma. After the administration of a second im dose to macaques, no additional animals developed anti-RSHZ19 antibodies. Multiple-dose iv kinetics (5-day repeat dose) in infant baboons were modeled accurately by adult macaque data, suggesting that these species handled RSHZ19 similarly. The pharmacokinetic characteristics of RSHZ19 should support a convenient regimen for treatment or prophylaxis of human respiratory syncytial virus infection.

Animals↗
PubMed1994

HIV protease inhibitors: effects on viral maturation and physiologic function in macrophages.

Human immunodeficiency virus (HIV), the retrovirus believed to be the cause of acquired immunodeficiency syndrome (AIDS), infects a variety of CD4+ cells, including lymphocytes and cells of the monocyte/macrophage lineage. Encoded in the HIV genome are several precursor proteins that must undergo proteolytic cleavage to yield functional proteins. The gag precursor protein of HIV (p55) is cleaved by a virally encoded aspartate protease (HIV protease). Because cleavage of p55 is required for viral maturation and infectivity, inhibition of HIV protease is an attractive target for therapy designed to block the progression of HIV infection. Inhibitors of HIV protease from a variety of chemical classes have been synthesized and antiviral activity has been demonstrated in lymphocytes and cells from the monocyte/macrophage lineage. A few HIV protease inhibitors have progressed to the clinic and some have shown promise in early trials. There are, however, several important issues that will affect the development of a successful HIV protease inhibitor, including cell and tissue distribution and immunotoxicity. These issues are tissue distribution and immunotoxicity. These issues are discussed, with an emphasis on the complexities posed by cells of the monocyte/macrophage lineage.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Biological, molecular, and structural analysis of a cytopathic variant from a molecularly cloned simian immunodeficiency virus.

Some isolates of simian immunodeficiency virus (SIV) have been shown to infect Sup-T1 cells with slow kinetics and in the absence of cytopathic effects, including cell fusion or CD4 down-modulation (J. A. Hoxie, B. S. Haggarty, S. Bonser, J. Rackowski, H. Shan, and P. Kanki, J. Virol. 62:2557-2568, 1988). In the present study, we describe the isolation and characterization of a SIVmac variant, derived from the BK28 infectious molecular clone, that became highly cytopathic for Sup-T1 cells. This variant, termed CP-MAC, exhibited a number of differences from BK28, including (i) an altered tropism which largely restricted its host range to Sup-T1 cells, (ii) the ability to induce cell fusion and CD4 down-modulation, and (iii) a highly stable interaction of its external (SU) and transmembrane (TM) envelope glycoproteins. In addition, a marked increase in the level of surface envelope glycoproteins was observed both on CP-MAC-infected cells and on virions. The CP-MAC env gene was PCR amplified from infected cells, and sequence analysis identified five amino acid changes in SU and six in TM compared with BK28. The introduction of these changes into BK28 was shown to fully reconstitute the biological and morphological properties of CP-MAC. The limited number of mutations in CP-MAC should enable the molecular determinants to be more precisely defined and help to identify the underlying mechanisms responsible for the striking biological and structural alterations exhibited by this virus.

Amino Acid Sequence↗
PubMed1993

Lack of effect of ammonium glycyrrhizinate on the morphology of ovine nasal mucosa in vitro.

Glycyrrhetinic acid derivatives are reported to be nasal absorption promoters (1). Effects of ammonium glycyrrhizinate (AMGZ) on the in vitro morphology of ovine nasal mucosa were therefore examined by light and electron microscopy. Nasal mucosa was stripped from the submucosa and mounted in Ussing chambers. Exposure of the apical surface to 2% ammonium glycyrrhizinate (24 mM) for 90 min caused no histopathological changes to the nasal epithelium. Epithelial integrity remained intact as evidenced by the continued presence of morphologically intact junctional complexes. No sloughing of the epithelial layer from the basement membrane was observed, and cilia and microvilli were not affected by treatment with AMGZ. The results indicate that short-term exposure in vitro to ammonium glycyrrhizinate caused no overt morphological damage to ovine nasal mucosa.

Animals↗
PubMed1993

Physicochemical dissociation of CD4-mediated syncytium formation and shedding of human immunodeficiency virus type 1 gp120.

The mechanism of CD4-mediated fusion via activated human immunodeficiency virus type 1 (HIV-1) gp41 and the biological significance of soluble CD4 (sCD4)-induced shedding of gp120 are poorly understood. The purpose of these investigations was to determine whether shedding of gp120 led to fusion activation or inactivation. BJAB cells (TF228.1.16) stably expressing HIV-1 envelope glycoproteins (the gp120-gp41 complex) were used to examine the effects of pH and temperature on sCD4-induced shedding of gp120 and on cell-to-cell fusion (syncytium formation) with CD4+ SupT1 cells. sCD4-induced shedding of gp120 was maximal at pH 4.5 to 5.5 and did not occur at pH 8.5. At physiologic pH, sCD4-induced shedding of gp120 occurred at 22, 37, and 40 degrees C but neither at 16 nor 4 degrees C. In contrast, syncytia formed at pH 8.5 (maximally at pH 7.5) but not at pH 4.5 to 5.5. At pH 7.5, syncytia formed at 37 and 40 degrees C but not at 22, 16, or 4 degrees C. Preincubation of cocultures of TF228.1.16 and SupT1 cells at 4, 16, or 22 degrees C before the shift to 37 degrees C resulted in similar, increased, or decreased syncytium formation, respectively, compared with the control. Furthermore, an activated intermediate of CD4-gp120-gp41 ternary complex may form at 16 degrees C; this intermediate rapidly executes fusion upon a shift to 37 degrees C but readily decays upon a shift to the shedding-permissive but fusion-nonpermissive temperature of 22 degrees C. These physicochemical data indicate that shedding of HIV-1 gp120 is not an integral step in the fusion cascade and that CD4 may inactivate the fusion complex in a process analogous to sCD4-induced shedding of gp120.

CD4 Antigens↗
PubMed1993

Morphometric analysis of envelope glycoprotein gp120 distribution on HIV-1 virions.

The surface of HIV-1, like that of other retroviruses, is studied with virally encoded glycoproteins which appear ultrastructurally as electron-dense spikes or knobs. The glycoprotein that forms the spike structure, gp120, is non-covalently bound to the transmembrane glycoprotein gp41. Mature HIV-1 virions do not have as many spikes as the genetically related retroviruses HIV-2 and SIV. gp120 is lost from HIV-1 during viral morphogenesis and after incubation of the virus with the soluble form of cellular receptor CD4. In this study we used ultrastructural cytochemistry and morphometry to quantitate the distribution of envelope glycoprotein spikes on budding and mature HIV-1 virions and to look for alternatives to the laborious and somewhat subjective spike-counting technique for envelope spike analysis on HIV-1. HIV-1, strain HTLV-IIIB, was examined after staining of envelope glycoproteins with either tannic acid, immunogold staining for gp120 (gp120-immunogold), or lectin-gold staining with concanavalin A for mannose residues (ConA-HRP-gold) and frequency distributions of spikes or gold particles per micron HIV-1 membrane generated. Envelope spikes were normally distributed on membranes of budding and mature HIV-1. However, the density of spikes per micron viral membrane on mature HIV-1 virions was approximately 50% of that observed on budding virions. ConA-HRP-gold and gp120-immunogold did not efficiently label budding virions. The shape of the frequency distribution for ConA-HRP-gold particles on mature virions was similar to that for envelope spikes and could be used to quantitate envelope glycoproteins on HIV-1. In addition, ConA-HRP-gold staining was able to detect the loss of envelope proteins after treatment of virus with soluble CD4. gp120-immunogold labeling was patchy and many virions were unlabeled. ConA-HRP-gold staining proved to be a rapid, reliable, and easily quantifiable method for estimation of envelope glycoprotein density on mature HIV-1. However, the loss of spike structures throughout the life cycle of HIV-1 can effectively be determined only by direct spike counting.

Concanavalin A↗
PubMed1992

Effect of a human immunodeficiency virus protease inhibitor on human monocyte function.

Human immunodeficiency virus (HIV) is the cause of acquired immunodeficiency syndrome (AIDS). Encoded by the HIV genome are several precursor proteins that undergo proteolytic cleavage to yield functional proteins. The env precursor protein is cleaved by a cellular protease. The gag precursor protein of HIV (p55), however, is cleaved by a virally encoded aspartate protease (HIV Protease). Cleavage of p55 is required for viral maturation and infectivity. There are also several host cell aspartate proteases that serve important homeostatic functions. Cathepsins D and E are lysosomal aspartate proteases which are believed to play an important role in macrophage function, and it has been suggested that inhibition of these enzymes by an HIV protease inhibitor may exacerbate immunosuppression in AIDS patients. We have studied the effect of SK&F 107461 (a hydroxyethylene dipeptide isostere inhibitor of HIV protease), on various host defense functions of human monocytes. Pepstatin A (an inhibitor of most aspartate proteases) and leupeptin (an inhibitor of serine and cysteine proteases) were included as controls. Although less potent than the prototypic aspartate protease inhibitor pepstatin, SK&F 107461 inhibited partially purified cathepsin D in vitro. However, in cell-based assays, SK&F 107461 had no effect on the degradation of hemoglobin, antigen processing of the protein antigen streptokinase, or secretion of 17-kD IL-1 beta by monocytes at concentrations which inhibit maturation of intracellular virus in HIV infected monocytes. Furthermore, SK&F 107461 had no effect on constitutive candidacidal activity. In contrast, leupeptin and pepstatin A partially inhibited accessory cell function of monocytes in the proliferative response to the recall antigen streptokinase. In addition, leupeptin partially inhibited degradation of hemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigen-Presenting Cells↗
PubMed1992

Strong association of simian immunodeficiency virus (SIVagm) envelope glycoprotein heterodimers: possible role in receptor-mediated activation.

Soluble forms of a human cell-surface molecule expressed on T lymphocytes (CD4) neutralize diverse strains of both human (HIV) and simian (SIV) immunodeficiency viruses through the induction of envelope shedding and direct competition with cellular CD4 for virus binding. However, we have previously shown that sCD4 enhances infection of simian immunodeficiency viruses from African green monkeys (SIVagm) and have theorized that this enhancement is due to the induction of conformational changes leading to viral fusion (receptor-mediated activation). In this report, we compared the relative association of the envelope glycoproteins of SIVagm with HIV type 1 (HIV-1) in order to determine if a more stable association of SIVagm envelope glycoproteins might account for the differential effects of sCD4 on the infectious process. Monospecific antisera to each of the SIVagm glycoproteins were generated and used to detect stable heterodimers by radioimmunoprecipitation. Standard solubilization buffers containing both ionic and nonionic detergents or saturating concentrations of sCD4 failed to disrupt SIVagm gp120 interactions with the transmembrane protein, gp36, whereas HIV-1 heterodimers were easily dissociated. Higher concentrations of SDS (1%) were necessary to disrupt the SIVagm envelope complexes demonstrating the existence of strong noncovalent interactions between these membrane glycoproteins. In addition, morphometric analysis by electron microscopy revealed that the linear density of SIVagm spikes was stable and resisted shedding when virus was incubated with sCD4 whereas a significant decrease in linear spike density was noted for HIV-1. Based on our original hypothesis, the strong association of SIVagm glycoprotein spikes during soluble receptor binding may allow for highly stable conformational intermediates important for viral fusion, while neutralization of HIV-1 by sCD4 results from less stable envelope associations.

Animals↗
PubMed1992

Human immunodeficiency virus type 1 protease inhibitors irreversibly block infectivity of purified virions from chronically infected cells.

Synthetic peptide analog inhibitors of human immunodeficiency virus type 1 (HIV-1) protease were used to study the effects of inhibition of polyprotein processing on the assembly, structure, and infectivity of virions released from a T-cell line chronically infected with HIV-1. Inhibition of proteolytic processing of both Pr55gag and Pr160gag-pol was observed in purified virions from infected T cells after treatment. Protease inhibition was evident by the accumulation of precursors and processing intermediates of Pr55gag and by corresponding decreases in mature protein products. Electron microscopy revealed that the majority of the virion particles released from inhibitor-treated cells after a 24-h treatment had an immature or aberrant capsid morphology. This morphological change correlated with the inhibition of polyprotein processing and a loss of infectivity. The infectivity of virion particles purified from these chronically infected cell cultures was assessed following treatment with the inhibitor for 1 to 3 days. Virions purified from cultures treated with inhibitor for 1 or 2 days demonstrated a 95- to 100-fold reduction in virus titers, and treatment for 3 days resulted in complete loss of detectable infectivity. The fact that virions from treated cultures were unable to establish infection over the 7- to 10-day incubation period in the titration experiments strongly suggests that particles produced by inhibitor-treated cells were unable to reactivate to an infectious form when they were purified away from exogenous protease inhibitor. Thus, a block of HIV-1 protease processing of viral polyproteins by specific inhibitors results in a potent antiviral effect characterized by the production of noninfectious virions with altered protein structures and immature morphologies.

Blotting, Western↗
PubMed1992

Myeloma-like cast nephropathy caused by human recombinant soluble CD4 (sCD4) in monkeys.

CD4 is the receptor for human immunodeficiency virus (HIV) on lymphocytes and macrophages. Soluble CD4 (sCD4), a recombinant truncated form of CD4, has been shown to inhibit HIV-1 in vitro and is being tested as a therapy for AIDS. Preclinical studies in cynomolgus monkeys revealed a protein cast nephropathy after four daily intravenous doses of 100 mg/kg/day. Renal lesions were not found in monkeys that received 10 mg/kg/day. The renal lesions consisted of proteinaceous tubular casts associated with multinucleate giant cells and neutrophils located in the tubules of the distal nephron. The affected tubules were surrounded by an interstitial mixed inflammatory cell infiltrate. By electron microscopy, the casts were composed of moderately electron dense, paracrystalline material. Immunostaining demonstrated that the casts contained sCD4-derived material and Tamm-Horsfall protein. Moreover, biochemical analysis of urine showed that a portion of sCD4 was excreted as intact protein. Because infection with HIV-1 can be associated with clinically significant nephropathy, these data suggest that renal function should be closely monitored in patients receiving soluble forms of CD4.

Animals↗
PubMed1991

Binding of soluble CD4 proteins to human immunodeficiency virus type 1 and infected cells induces release of envelope glycoprotein gp120.

Human immunodeficiency virus (HIV) infects cells after binding of the viral envelope glycoprotein gp120 to the cell surface recognition marker CD4. gp120 is noncovalently associated with the HIV transmembrane envelope glycoprotein gp41, and this complex is believed responsible for the initial stages of HIV infection and cytopathic events in infected cells. Soluble constructs of CD4 that contain the gp120 binding site inhibit HIV infection in vitro. This is believed to occur by competitive inhibition of viral binding to cellular CD4. Here we suggest an alternative mechanism of viral inhibition by soluble CD4 proteins. We demonstrate biochemically and morphologically that following binding, the soluble CD4 proteins sT4, V1V2,DT, and V1[106] (amino acids 1-369, 1-183, and -2 to 106 of mature CD4) induced the release of gp120 from HIV-1 and HIV-1-infected cells. gp120 release was concentration-, time-, and temperature-dependent. The reaction was biphasic at 37 degrees C and did not take place at 4 degrees C, indicating that binding of soluble CD4 was not sufficient to release gp120. The appearance of free gp120 in the medium after incubation with sT4 correlated with a decrease in envelope glycoprotein spikes on virions and exposure of a previously cryptic epitope near the amino terminus of gp41 on virions and infected cells. The concentration of soluble CD4 proteins needed to induce the release of gp120 from virally infected cells also correlated with those required to inhibit HIV-mediated syncytium formation. These results suggest that soluble CD4 constructs may inactivate HIV by inducing the release of gp120. We propose that HIV envelope-mediated fusion is initiated following rearrangement and/or dissociation of gp120 from the gp120-gp41 complex upon binding to cellular CD4, thus exposing the fusion domain of gp41.

Blotting, Western↗
PubMed1991

Quantitative immunocytochemical staining for recombinant tissue-type plasminogen activator in transfected Chinese hamster ovary cells.

Tissue-type plasminogen activator (tPA) is a serine protease which cleaves plasminogen to its active form, plasmin. tPA plays a physiologic role in hemostasis, wound healing, and embryogenesis. Therapeutically, recombinant human tPA is used as a thrombolytic in myocardial infarction. Although production of therapeutic quantities of tPA in Chinese hamster ovary (CHO) cells transfected with the human gene for tPA is practical, production costs remain high. One important factor which determines the ultimate cost of tPA (or any other recombinant protein expressed in mammalian cells) is its production level on a per cell basis. We have used postembedding immunocytochemical staining with colloidal gold to study the subcellular localization of tPA in CHO cells expressing recombinant tPA (rCHO) in an effort to understand the factor(s) which might limit secretion. Staining for tPA was evaluated visually and by morphometric analysis and was specific and reproducible. Serially passaged rCHO showed no significant change in staining density over 31 serial passages. Staining density was greatest over dilated cisternae of the rough endoplasmic reticulum and nuclear envelope. Golgi stacks and large acid phosphatase-positive vacuoles (probably lysosomes) were also heavily stained. Staining of lysosomal vacuoles suggested that rCHO might be degrading nascent tPA. Incubation of rCHO with 125I-tPA showed that the cells were not internalizing tPA from the media. These results suggest that rCHO fail to secrete a portion of the tPA they synthesize and that it is degraded in lysosomes. This observation may have important implications on the choice of expression systems for efficient production of large quantities of recombinant proteins.

Animals↗