Requirement of B7-mediated costimulation in the induction of experimental autoimmune anterior uveitis.

PURPOSE: To study the role of costimulatory signaling through the CD28-B7 interaction in experimental autoimmune anterior uveitis (EAAU). METHODS: Naive Lewis rats were immunized with insoluble melanin-associated antigen (MAA) derived from bovine iris and ciliary body. CTLA4-Fc, a recombinant protein comprised of the extracellular domain of human CTLA4 bound to mouse IgG2a Fc, was used to block the CD28-B7 interaction. A mutant version (CTLA4-Fc-mutant) was used as a control. The effect of CTLA4-Fc on the in vivo induction of disease with MAA was studied. Subsequently, the mechanism by which CTLA4-Fc blocked the interaction of CD28 and B7 was investigated in vivo, using the adoptive transfer of T cells derived from CTLA4-Fc-treated rats, and in vitro, using the proliferative response and cytokine production of MAA-T cells in the presence of CTLA4-Fc. RESULTS: CTLA4-Fc markedly reduced the incidence and severity of EAAU in Lewis rats after sensitization with MAA. The adoptive transfer of sensitized T cells from CTLA4-Fc-treated donors did not induce EAAU in naive recipients. CTLA4-Fc inhibited the expansion of antigen-specific MAA-T cells and the production of TNF-alpha. CONCLUSIONS: The costimulatory signal delivered through CD28-B7 is required for the induction and pathogenesis of EAAU. In the absence of this signal, antigen-specific expansion of MAA reactive T cells as well as production of TNF-alpha is inhibited. Abrogation of this costimulatory signal may be an important therapeutic option for EAAU.

Acid beverage floc: protein-saponin interactions and an unstable emulsion model.

The occurrence of acid beverage floc (ABF) in acidified carbonated beverages has long been attributed to the presence of saponins. We have examined this assertion and have found evidence to suggest that traces of protein may also be a key factor, along with lipid material present in the floc. Turbidity levels of beet sugar protein (0.001 wt.%) and saponin (0.001 wt.%) solutions were examined over time using spectrophotometry. At neutral pH, no change in turbidity was observed in any combination (individually or mixed). Furthermore, acidified (pH 2) saponin and protein solutions, considered separately, also exhibited no change. However, a mixture of equal concentrations at pH 2 showed an initial increase in turbidity up to 2 h after mixing, followed by a decrease over the ensuing 12 h. Interfacial tension measurements also indicated interactions between the protein and saponin at pH 2. Photon correlation spectroscopy (PCS-Malvern Zetasizer 4) was used to quantitatively examine the particle size distributions and aggregation of a model, highly dilute dispersion, of bromohexadecane in 20 wt.% sucrose solution, prepared with a jet homogenisor. Beet sugar saponin (0.001 wt.%) and protein (0.001 wt.%) were added to the dispersion, and their emulsion-stabilising effects examined via oil droplet size measurement over time. At neutral pH, the size of oil droplets in the dispersion was unaffected by the addition of saponin or protein. At pH 2, the presence of saponin again caused no effect on droplet size. However, in acid conditions, protein appeared to destabilise the dispersion. The results indicate that the key to controlling the ABF problem may be the ratio of saponin to protein in the product, which may or may not stabilise dispersed lipid, depending on their interactions.

Potentiation of antitumor activity of irinotecan by chemically modified oligonucleotides.

Co-administration of synthetic chemically modified oligonucleotides with irinotecan, a selective topoisomerase I inhibitor, provided a significant enhancement in the antitumor activity of irinotecan. The enhancement of antitumor activity of irinotecan with co-administration of chemically modified oligonucleotides was observed in several tumor models--pancreatic cancer (Panc-1), colon cancer (HCT-116) and melanoma (A375). Inhibition of tumor growth in all three models required the co-administration of irinotecan and chemically modified oligonucleotides, but was independent of the nucleotide sequence of the oligonucleotides. The potentiation of antitumor activity was dependent on the dose of irinotecan and chemically modified oligonucleotides administered. The enhancement of antitumor activity of irinotecan was also observed by co-administration of a phosphorothioate oligonucleotide, however, to a lesser extent than did chemically modified oligonucleotides, suggesting that metabolic stability of the oligonucleotide contributes to the enhancement of antitumor activity seen with irinotecan. The co-administration of dextran sulfate sodium with irinotecan showed insignificant potentiation of antitumor activity of irinotecan, suggesting that the enhancement of antitumor activity of irinotecan observed was not a result of polyanionic characteristic of oligonucleotides. Co-administration of irinotecan and chemically modified oligonucleotides did not result in increased toxicity in the tumor models studied. Potentiation of antitumor activity of irinotecan observed with co-administration of oligonucleotides suggests that the oligonucleotides affect the pharmacokinetics and/or metabolism of irinotecan. The use of chemically modified oligonucleotides together with irinotecan may increase the therapeutic index of irinotecan in cancer patients and continued development of such agents should be considered.

Generation of mucosal cytotoxic T cells against soluble protein by tissue-specific environmental and costimulatory signals.

We compared peripheral and mucosal primary CD8 T cell responses to inflammatory and noninflammatory forms of antigen in a T cell-adoptive transfer system. Immunization with the soluble antigen, ovalbumin (ova), administered i.p. or orally without adjuvant, activated nonmucosal CD8 T cells but did not induce cytotoxic activity. However, after activation, the transferred cells entered the intestinal mucosa and became potent antigen-specific killers. Thus, exogenous intact soluble protein entered the major histocompatibility complex class I antigen presentation pathway and induced mucosal cytotoxic T lymphocytes. Moreover, distinct costimulatory requirements for activation of peripheral versus mucosal T cells were noted in that the CD28 ligand, B7-1, was critical for activated mucosal T cell generation but not for activation of peripheral CD8 T cells. The costimulator, B7-2, was required for optimum activation of both populations. Infection with a new recombinant vesicular stomatitis virus encoding ovalbumin induced lytic activity in mucosal as well as peripheral sites, demonstrating an adjuvant effect of inflammatory mediators produced during virus infection. Generation of antiviral cytotoxic T lymphocytes was also costimulation-dependent. The results indicated that induction of peripheral tolerance via antigen administration may not extend to mucosal sites because of distinct costimulatory and inflammatory signals in the mucosa.

The biogenesis of the MHC class II compartment in human I-cell disease B lymphoblasts.

The localization and intracellular transport of major histocompatibility complex (MHC) class II molecules nd lysosomal hydrolases were studied in I-Cell Disease (ICD) B lymphoblasts, which possess a mannose 6-phosphate (Man-6-P)-independent targeting pathway for lysosomal enzymes. In the trans-Golgi network (TGN), MHC class II-invariant chain complexes colocalized with the lysosomal hydrolase cathepsin D in buds and vesicles that lacked markers of clathrin-coated vesicle-mediated transport. These vesicles fused with the endocytic pathway leading to the formation of "early" MHC class II-rich compartments (MIICs). Similar structures were observed in the TGN of normal beta lymphoblasts although they were less abundant. Metabolic labeling and subcellular fractionation experiments indicated that newly synthesized cathepsin D and MHC class II-invariant chain complexes enter a non-clathrin-coated vesicular structure after their passage through the TGN and segregation from the secretory pathway. These vesicles were also devoid of the cation-dependent mannose 6-phosphate (Man-6-P) receptor, a marker of early and late endosomes. These findings suggest that in ICD B lymphoblasts the majority of MHC class II molecules are transported directly from the TGN to "early" MIICs and that acid hydrolases cam be incorporated into MIICs simultaneously by a Man-6-P-independant process.

Differential effects of CTLA-4 substitutions on the binding of human CD80 (B7-1) and CD86 (B7-2).

CTLA-4 expressed on activated T cells binds to CD80 (B7-1) and CD86 (B7-2) molecules present on APC with high avidity and appears to deliver a negative regulatory signal to the T cell. We have investigated the kinetics of CTLA-4 binding to CD80 and CD86, together with the effects of selected CTLA-4 mutations on binding activity. The dissociation constants (Kd) for binding of CTLA-4-Ig to CD80 and CD86 transfectants were 8.1 and 6.7 nM, respectively. Surface plasmon resonance was used to determine kinetic parameters of CTLA-4-Ig binding to CD80-Ig and CD86-Ig fusion proteins and revealed enhanced association (ka) and dissociation (kd) rate constants for CD86-Ig compared with CD80-Ig. Furthermore, CD80-Ig and CD86-Ig fusion molecules demonstrated variable abilities to cross-compete for binding to several modified forms of CTLA-4-Ig. Differential binding of CD80 and CD86 to CTLA-4 was further revealed by analysis of 10 discrete CTLA-4 mutants. Five single amino acid substitutions within the CTLA-4 MYPPPY domain exerted modest effects on CD80 binding, but each of these substitutions completely abrogated CD86 binding. In addition, substitutions just N-terminal of the MYPPPY region, and within the CDR1-like region of CTLA-4, eliminated both CD80 and CD86 binding. Hence, CD80 and CD86 bind with different association/dissociation kinetics to similar, but distinct, sites on CTLA-4.

Antigen-dependent clonal expansion of a trace population of antigen-specific CD4+ T cells in vivo is dependent on CD28 costimulation and inhibited by CTLA-4.

The importance of CD28 costimulation to a primary T cell response in vivo was assessed in an adoptive transfer system where a small population of peptide-specific CD4+ TCR transgenic T cells can be physically tracked. Ag-dependent clonal expansion of the transgenic T cells in draining lymph nodes was blocked by cyclosporin A and required a CD28 signal that was completely inhibited by CTLA-4-Ig or a combination of anti-B7-1 and anti-B7-2 mAbs, but not by either Ab alone. In vivo treatment with the combination of anti-B7-1 and anti-B7-2 mAbs also blocked conversion of the Ag-specific T cells to the activated phenotype. In contrast, anti-CTLA-4 Fab greatly enhanced the in vivo clonal expansion of the Ag-specific T cells. These results suggest that Ag-driven proliferation and phenotype conversion of naive CD4+ T cells is dependent on CD28-derived signals and is inhibited by CTLA-4.

Delivery of nascent MHC class II-invariant chain complexes to lysosomal compartments and proteolysis of invariant chain by cysteine proteases precedes peptide binding in B-lymphoblastoid cells.

The intracellular trafficking, proteolysis, and dissociation of invariant chain (li) associated with nascent class II molecules was examined in B-lymphoblastoid cells. Metabolic labeling and Percoll gradient centrifugation was used to assess the kinetics of delivery and processing of class II-li complexes within the endocytic pathway. Catabolism of class II-li complexes rapidly followed their delivery from post-Golgi compartments to dense lysosome-like compartments distinct from early and late endosomes. Direct peptide binding assays revealed that class II molecules associated with even small N-terminal fragments of li failed to bind peptide. Cysteine protease inhibitors alone blocked li proteolysis/dissociation and accumulation of class II-li biosynthetic intermediates within lysosome-containing compartments. Active-site labeling of cysteine proteases in B cells was used to identify cysteine proteases capable of mediating li proteolysis within endosomal compartments. Our results indicate rapid, possibly direct, transport of nascent class II-li complexes from the Golgi/trans-Golgi network to dense lysosomal compartments wherein cysteine protease(s), likely including cathepsin B, mediate complete removal of li. Inhibition of cysteine protease activity results in the accumulation of incompletely processed class II-li complexes, which lack peptide binding ability, within lysosomal compartments.

High-power mode-locked hybrid pulse source using two-section laser diodes.

We describe a mode-locked hybrid pulse source with a two-section laser diode to obtain short mode-locked pulses (23 ps) with an average power of 7.8 mW, a high peak power of 137 mW, and a repetition rate of 2.51 GHz. The hybrid laser incorporates a two-section laser and an optical fiber cavity with an integrated Bragg reflector. The Bragg reflector controls the operating wavelength to subnanometer precision and also confines the bandwidth of the pulses so as to keep the time-bandwidth product below 1.

Endocytosis and lysosomal delivery of tissue plasminogen activator-inhibitor 1 complexes in Hep G2 cells.

Receptor-mediated endocytosis of tissue-type plasminogen activator (t-PA)-plasminogen activator inhibitor type 1 (PAI-1) complexes results in their clearance by Hep G2 cells. After complexes are internalized, the t-PA component is degraded. However, neither the locus of intracellular catabolism nor the fate of PAI-1 has been elucidated. To characterize these aspects of t-PA-PAI-1 catabolism, the subcellular distribution of a prebound cohort of ligand molecules was delineated after internalization at 37 degrees C. 125I-t-PA.PAI-1 and t-PA.125I-PAI-1 were compared in separate experiments. After ligand uptake, intracellular vesicles were separated on density gradients. Internalized 125I-t-PA.PAI-1 concentrated initially in endosomes. After 20 minutes of uptake, the complex began to appear in lysosomes. Subsequently, low molecular weight labeled ligand fragments were detected in culture media. A panel of lysosomotropic agents, including primaquine, chloroquine, ammonium chloride, and a combination of leupeptin and pepstatin A, inhibited degradation. When t-PA.125I-PAI-1 rather than 125I-t-PA.PAI-1 was internalized, strikingly different results were observed. Although the kinetics of internalization and the intracellular itinerary were indistinguishable for the differently labeled complexes, the 125I-PAI-1 component of t-PA.125I-PAI-1 resisted rapid degradation. After a rapid loss of t-PA, the 125I-PAI-1 moiety persisted in lysosomes for up to 180 minutes. Thus, internalized t-PA.PAI-1 is targeted to lysosomes in which PAI-1 is relatively more stable than t-PA.

Identification and partial characterization by chemical cross-linking of a binding protein for tissue-type plasminogen activator (t-PA) on rat hepatoma cells. A plasminogen activator inhibitor type 1-independent t-PA receptor.

Plasma tissue-type plasminogen activator (t-PA) is cleared rapidly in vivo by the liver. Previous studies with the human hepatoma cell line HepG2 have identified a clearance system for t-PA modulated by plasminogen activator inhibitor type 1 (PAI-1). In the present study, a rat hepatoma cell line MH1C1 is shown to contain a PAI-1-independent t-PA clearance system. At 4 degrees C, binding of 125I-t-PA to MH1C1 cells was rapid, specific, and saturable. Scatchard analysis of the binding data yielded a mean estimate of 105,000 high affinity binding sites per cell (Kd = 4.1 nM). When the bound ligand was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the majority (about 90%) of the specific binding was in the form of uncomplexed 125I-t-PA. This is in contrast to HepG2 cells in which specific binding was mainly in the form of a sodium dodecyl sulfate-stable 125I-t-PA.PAI-1 complex. When availability of matrix-associated PAI-1 was blocked by preincubation with anti-PAI-1 antibody or removed by elastase treatment, specific 125I-t-PA binding to MH1C1 cells was unaffected, whereas most of the specific 125I-t-PA binding to HepG2 cells was abolished. Furthermore, when the active site of t-PA was inactivated with diisopropyl fluorophosphate, the diisopropyl fluorophosphate-t-PA specifically competed for binding of 125I-t-PA to MH1C1 cells, but failed to block specific 125I-t-PA binding to HepG2 cells. At 37 degrees C, PAI-1-independent t-PA binding to MH1C1 cells was followed by ligand uptake and degradation with kinetics similar to that seen in HepG2 cells. Chemical cross-linking of t-PA to MH1C1 cells revealed a specific t-PA binding protein with a molecular mass of about 500,000 daltons. Ligand-receptor complexes generated by chemical cross-linking were immunoprecipitable by anti-t-PA antibody but not by anti-PAI-1 antibody, further supporting the finding that binding of t-PA to MH1C1 cells is PAI-1-independent.

Binding of plasminogen activator inhibitor type-1 to extracellular matrix of Hep G2 cells. Evidence that the binding protein is vitronectin.

Catabolism of plasminogen activators by Hep G2 cells is mediated by a specific receptor which recognizes complexes of these serine proteases with their physiological inhibitor, plasminogen activator inhibitor type-1 (PAI-1). This catabolic process is initiated by interaction of exogenous plasminogen activators with bioactive PAI-1, which is secreted and localizes in an active form to the extracellular matrix (ECM) of Hep G2 cells. We now report that vitronectin (VN) mediates the specific binding of PAI-1 to the ECM of these cells. Purified bovine or human VN competes for specific binding of PAI-1 to Hep G2 ECM, and ligand blotting reveals specific binding of PAI-1 to ECM-associated VN. Hep G2 cells secrete both VN and PAI-1, and pulse-chase studies strongly suggest that these proteins associate only following secretion. Although Hep G2 cell-derived VN does not significantly bind to ECM in vitro, 30-40% of endogenous PAI-1 binds to the ECM, even in the presence of human serum, suggesting that ECM-associated VN is entirely derived from bovine serum. PAI-1 was localized by indirect immunofluorescence to ECM beneath cells and at cell margins, whereas VN exhibited a uniform distribution throughout the growth substratum. VN associated with the ECM may confer retention and bioactivity to PAI-1, potentially facilitating both pericellular regulation of plasmin generation and the rapid hepatic clearance of plasminogen activators.

Identification of determinants involved in binding of tissue-type plasminogen activator-plasminogen activator inhibitor type 1 complexes to HepG2 cells.

Complexes between tissue-type plasminogen activator (t-PA) and its rapidly acting inhibitor plasminogen activator inhibitor type 1 (PAI-1) are bound, internalized, and degraded by HepG2 cells. The mechanism involves endocytosis mediated by a specific high-affinity receptor. However, the particular domains of the complex that are recognized by the receptor have not been elucidated. To identify the determinants involved in ligand binding to the receptor, several variants of t-PA were assessed for their ability to form complexes with PAI-1 and thereby to inhibit specific cellular binding of complexes between structurally unmodified 125I-t-PA and PAI-1. Catalytically active variants lacking selected structural domains form complexes with PAI-1 and inhibit 125I-t-PA.PAI-1 binding to HepG2 cells. In addition, several forms of the plasminogen activator urokinase (u-PA), which shares partial structural homology with t-PA, were evaluated as competitors of cellular binding. The catalytically active two-chain forms of u-PA, but not the inactive proenzyme single-chain form, complex with PAI-1 and inhibit specific binding of 125I-t-PA.PAI-1, suggesting that the serine protease domain, rather than other domains, may confer the determinants required for cellular binding. However, a mutant t-PA with markedly reduced catalytic activity, resulting from replacement of the active site serine with threonine, not only forms complexes with PAI-1 but also inhibits specific cellular binding of unmodified 125I-t-PA.PAI-1. These data indicate that specific binding of t-PA.PAI-1 to HepG2 cells does not require a serine-containing catalytic site in the protease domain. To determine whether binding of the complex is mediated through other components of t-PA or through structural elements of PAI-1, both t-PA and PAI-1 were examined separately for capacity to bind directly to HepG2 cells. To exclude potential interactions with components of the extracellular matrix which contains binding sites for PAI-1, ligand binding to HepG2 cells in suspension was assessed. Although neither t-PA nor PAI-1 alone binds specifically to HepG2 cells, the preformed t-PA.PAI-1 complexes do. These findings suggest that specific binding of t-PA.PAI-1 requires elements of the PAI-1 moiety and/or parts of the protease domain of t-PA.

Design of 1.3-microm GaInAsP surface-emitting lasers for high-bandwidth operation.

Important design considerations for high-speed GaInAsP 1.3-microm surface-emitting lasers are described. Modified rate equations for surface-emitting lasers and a small-signal analysis are used to calculate the frequency response versus the mirror reflectivity. For the structure analyzed, the results predict optimum reflectivities of 98% for highfrequency operation and maximum quantum efficiency. Strong gain-saturation effects are predicted owing to the high photon densities that occur in these devices. As the mirror thickness increases, the frequency response of the laser reduces greatly. The effect of heating is found to be important and is included in the analysis.

Interactions between tissue-type plasminogen activator and extracellular matrix-associated plasminogen activator inhibitor type 1 in the human hepatoma cell line HepG2.

Hepatic parenchymal cells contribute to the clearance of circulating tissue-type plasminogen activator (t-PA) in vivo. The hepatocyte extracellular matrix is interposed between the endothelial-lined sinusoids and the parenchymal cell surface and thus may influence t-PA clearance. To test this hypothesis, the well differentiated human hepatoma cell line HepG2 was used to characterize the role of extracellular matrix in t-PA clearance in vitro. Previous studies with these cells demonstrated their capacity for specific catabolism of t-PA in a system modulated by plasminogen activator inhibitor type 1 (PAI-1). In the present study the extracellular matrix growth substratum of HepG2 cells is shown to contain active PAI-1. PAI-1 is distributed in a punctuate pattern throughout the substratum. Components of the substratum confer stability to active PAI-1 for intervals of at least 24 h. Exposing substratum to 125I-t-PA leads rapidly to the formation and release of a sodium dodecyl sulfate-stable 95-kDa 125I-t-PA.PAI-1 complex. In comparison, cell monolayers have the additional capacity for specific binding of the complex. However, PAI-1 is not detected at the surface of HepG2 cells in suspension, suggesting that 125I-t-PA.PAI-1 complexes form in substratum and subsequently bind to cells. Specific binding of performed 125I-t-PA.PAI-1, but not 125I-t-PA, was demonstrated for HepG2 cells in suspension. These results suggest that components of extracellular matrix participate in the clearance of t-PA by hepatocytes.

Catabolism of tissue-type plasminogen activator by the human hepatoma cell line Hep G2. Modulation by plasminogen activator inhibitor type 1.

Catalytic activity of tissue-type plasminogen activator (t-PA) in plasma is regulated in part by formation of complexes with specific inhibitors as well as by hepatic clearance. Potential interaction of these two regulatory mechanisms was examined in the human hepatoma cell line Hep G2. These cells secrete plasminogen activator inhibitor type-1 (PAI-1) and initiate catabolism of exogenous t-PA by receptor-mediated endocytosis. Specific binding of 125I-t-PA to cells at 4 degrees C results in dose-dependent formation of a 95-kDa species recognized by monospecific anti-PAI-1 and anti-t-PA antibodies and stable in the presence of low (0.2%) concentrations of sodium dodecyl sulfate (SDS). Specific binding of 125I-t-PA and formation of the 95-kDa SDS-stable species are inhibited in a concentration-dependent manner following preincubation of cells with anti-PAI-1 antibodies. High and low molecular weight forms of urokinase plasminogen activator (u-PA) capable of forming specific complexes with PAI-1 complete for 125I-t-PA binding sites. However, the proenzyme form of u-PA (scu-PA), incapable of forming complexes with PAI-1, does not compete for 125I-t-PA binding sites. The role of the serine protease active site of t-PA in mediating both interaction with PAI-1 and specific binding was examined using 125I-t-PA that had been functionally inactivated with D-phenylalanyl-L-propyl-L-arginyl-chloromethyl ketone (PPACK). 125I-t-PA-PPACK, despite a 6-fold lower affinity than active 125I-t-PA, exhibited specific binding to cells without detectable formation of SDS-stable complexes with PAI-1. Both surface-bound 125I-t-PA and 125I-t-PA-PPACK are internalized and degraded by cells at 37 degrees C. 125I-t-PA is internalized as a stable complex with PAI-1, whereas 125I-t-PA-PPACK is internalized with similar kinetics but without the presence of an SDS-stable complex. Thus, PAI-1 appears capable of modulating t-PA catabolism in the human hepatocyte.

Extracellular and cellular proteins of rat cells with O-glycosidically linked fucose.

Fucose-labelled proteins were examined for the release of low-Mr O-linked fucose substituents after mild alkaline-borohydride treatment. A component tentatively identified as glucosylfucitol (DS) and an apparently higher-Mr component (TS), which also contained fucitol, were observed to be released over a broad molecular-size range of proteins. Approx. 90% of the DS-releasing proteins were in the particulate fraction, whereas only approx. 66% of the TS-releasing proteins were in that fraction. In addition to cell-associated proteins, a substantial proportion of DS-containing proteins were shed into the medium. For example, after 96 h of labelling there was 6-fold more of these components in the growth medium than were cell-associated. Moreover, the incorporation of labelled fucose into both the DS and TS appeared to be cell-population-density-dependent. Despite the apparent wide distribution of these novel fucose substituents in cellular proteins, it seems reasonable to suggest that they have not been routinely observed largely because each represents less than 0.5% of the fucose bound to protein.

Metabolism of a transformation-sensitive amino acid fucoside in normal and simian virus 40-transformed human embryonic lung cells.

We have demonstrated previously that there is a marked decrease in the level of labeled fucose incorporated into an amino acid fucoside, i.e., fucosyl-N-acetylglucosaminylasparagine (FL4c) in SV40-transformed human embryonic lung cells (SV40-WI-38) as compared to WI-38 cells (Morton, P.A., Klinger, M. M., and Steiner, S. Cancer Res., 42: 3022-3027, 1982). In the current study, we have observed that the reduction in labeled fucose incorporated into FL4c in the SV40-WI-38 cells is paralleled by reduced chemical quantity of that component. [3H]-Fucose pulse/chase and long-term fucose labeling/chase studies, in some instances in the presence of tunicamycin, have revealed that FL4c is a relatively stable end produce of N-linked-type glycoprotein metabolism. The relative metabolic stability argues against breakdown of FL4c as the basis for the markedly reduced level in the SV40-WI-38 cells. However, the transformed cells manifested an almost 3-fold higher level of alpha-L-fucosidase activity when p-nitrophenyl-alpha-fucoside was used as substrate. These results raise the possibility that the parent glycoprotein of FL4c might be more rapidly catabolized in the SV40-WI-38 cells than in the WI-38 cells. Whatever the biochemical basis for the decrease in level of FL4c in transformed human cells, it would seem to underlie a difference between normal and transformed cells in membrane glycoprotein catabolism.