Biomedical subjects

H J Ryser

Publications and source records attributed to H J Ryser.

At least 19 recordsLinked to original sources

PubMed1995

Characterization of fibroblasts with a unique defect in processing antigens with disulfide bonds.

A chinese hamster ovary (CHO) fibroblast, transfected with murine MHC class II genes, inefficiently stimulated CD4+ Th cells specific for OVA, hen egg lysozyme (HEL), and pork insulin which contain disulfide bonds. However, the fibroblasts elicited a T cell response to lambda repressor, which lacks disulfide bonds, and efficiently presented synthetic peptides. A somatic cell hybrid WALC, generated by fusing the hamster fibroblast with a murine L cell fibroblast, very efficiently processed OVA and HEL, suggesting that impaired processing was genetically complemented and was a recessive trait. The hamster fibroblasts were capable of processing two distinct denatured forms of OVA and carboxymethylated HEL, either as effectively or more efficiently than the B lymphoma cell. The CHO cells also displayed diminished disulfide reduction of an endocytosed [125I]tyramine linked to poly-(D-lysine) through a disulfide spacer compared with that of the cell hybrid, providing direct evidence for defective reductive cleavage by the CHO cells. Diminished aspartic acid-mediated proteolysis of Ag could not account for the phenotype, because cell lysates and separated organelles from the fibroblast possessed higher acidic aspartyl proteolytic activity than lysates and organelles from a B lymphoma cell. Thus, CHO cells exhibit a defect in processing Ag with disulfide bonds which is consistent with the impaired intracellular reduction of the disulfide bonds in endocytosed macromolecules.

Animals↗

PubMed1994

Inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfide interchange upon virus-receptor interaction.

The cell surface of mammalian cells is capable of reductively cleaving disulfide bonds of exogenous membrane-bound macromolecules (for instance, the interchain disulfide of diphtheria toxin), and inhibiting this process with membrane-impermeant sulfhydryl reagents prevents diphtheria toxin cytotoxicity. More recently it was found that the same membrane function can be inhibited by bacitracin, an inhibitor of protein disulfide-isomerase (PDI), and by monoclonal antibodies against PDI, suggesting that PDI catalyzes a thiol-disulfide interchange between its thiols and the disulfides of membrane-bound macromolecules. We provide evidence that the same reductive process plays a role in the penetration of membrane-bound human immunodeficiency virus (HIV) and show that HIV infection of human lymphoid cells is markedly inhibited by the membrane-impermeant sulfhydryl blocker 5,5'-dithiobis(2-nitrobenzoic acid), by bacitracin, and by anti-PDI antibodies. The results imply that HIV and its target cell engage in a thiol-disulfide interchange mediated by PDI and that the reduction of critical disulfides in viral envelope glycoproteins may be the initial event that triggers conformational changes required for HIV entry and cell infection. These findings suggest additional approaches to impede cell infection by HIV.

Amino Acid Sequence↗

PubMed1993

Inhibition of a reductive function of the plasma membrane by bacitracin and antibodies against protein disulfide-isomerase.

Evidence had been provided that a disulfide-linked [125I]iodotyramine/poly(D-lysine) conjugate was reductively cleaved when bound nonspecifically to the surface of Chinese hamster ovary (CHO) cells and that this cleavage was abolished by membrane-impermeant sulfhydryl blockers. The same blockers were subsequently found to inhibit the cytotoxicity of diphtheria toxin, a disulfide-linked heterodimer that binds to a specific surface receptor and must undergo chain separation to exert its cytotoxicity. This suggested that the disulfides of both macromolecules might be cleaved by a thiol-disulfide interchange reaction, possibly mediated by protein disulfide-isomerase (PDI, EC 5.3.4.1). We tested whether inhibitors of PDI--in particular, bacitracin and anti-PDI antibodies--might mimic the two effects of sulfhydryl blockers. Both bacitracin and anti-PDI antibodies were effective in inhibiting both reductive processes. This strongly suggests that the disulfide cleavage in the two membrane-bound macromolecules is mediated by PDI and that this enzyme, besides its known retention in the endoplasmic reticulum, must also be exposed at the plasma membrane. This paper points to other potentially important disulfide reductions that might be catalyzed by surface-associated PDI. It thereby broadens the known functions of an enzyme already known for its multifunctional properties.

4-Chloromercuribenzenesulfonate↗

PubMed1991

Cell surface sulfhydryls are required for the cytotoxicity of diphtheria toxin but not of ricin in Chinese hamster ovary cells.

A previous study on cleavage of disulfide bonds in endocytosed model compounds had shown that an initial phase of cleavage was totally inhibited by membrane-impermeant sulfhydryl inhibitors and thus was mediated by cell surface sulfhydryls (Feener, E. P., Shen, W.-C., and Ryser, H. J.-P. (1990) J. Biol. Chem. 265, 18780-18785). This paper uses the same inhibitors (5,5'-dithiobis(2-nitrobenzoic acid) and p-chloromercuriphenylsulfonic acid) to examine the role of surface sulfhydryls in the cytotoxicity of diphtheria toxin (DT). Since the interchain disulfide of endocytosed DT must be cleaved prior to translocation of chain A from endosomes to cytoplasm, it was postulated that surface sulfhydryls might mediate the cleavage of that disulfide bond as well. Both sulfhydryl blockers did indeed markedly inhibit DT cytotoxicity. This effect was not due to inactivation of unbound DT, inhibition of receptor-mediated endocytosis, or impairment of acidification of endosomes. We conclude that cell surface sulfhydryls susceptible to blockage by 5,5'-dithiobis(2-nitro-benzoic acid) and p-chloromercuriphenylsulfonic acid are required for the cytotoxicity of DT and, most likely, for the reductive cleavage of DT's interchain disulfides. Ricin cytotoxicity was not decreased; this is consistent with the view that ricin reaches the cytoplasm from a late endocytic structure and with the finding that endocytosed disulfides are also cleaved in a cell fraction containing elements of the Golgi apparatus (Feener, E. P., Shen, W.-C., and Ryser, H. J.-P. (1990) J. Biol. Chem. 265, 18780-18785).

4-Chloromercuribenzenesulfonate↗

PubMed1991

Isolation of variants of Chinese hamster ovary cells with abnormally low levels of GSH: decreased ability to cleave endocytosed disulfide bonds.

Mutants of Chinese hamster ovary cells were selected for resistance to a 3 hour exposure to 4 microgram/ml N-methyl-N'-nitro-N-nitrosoguanidine and tested for glutathione (GSH) levels. Six of eight clones that survived the initial treatment had reduced GSH levels ranging from 26 to 61% of wild-type values. These eight cell lines were tested for their susceptibility to a drug conjugate in which methotrexate (MTX) is disulfide-linked to poly(D-lysine) (MTX-SS-PDL) to test their capacity to cleave the endocytosed disulfide bond and release free MTX from this otherwise undegradable drug conjugate. We had shown that wild-type cells were killed by approximately 1 x 10(-7) M MTX given as free drug, as MTX-poly(L-lysine) or as MTX-SS-PDL, but were not affected by MTX-poly(D-lysine). All six lines with abnormally low levels of GSH were resistant to MTX-SS-PDL. The variants with the lowest levels of GSH (MNR-5 and MNR-10) were tested further and showed near-normal sensitivity to MTX and MTX poly(L-lysine). As expected, both lines were hypersensitive to melphalan. They were, however, normally sensitive to diphtheria toxin and ricin, indicating that some cleavage of the interchain disulfides in these protein toxins occurs even when cellular GSH is abnormally low. The lesser GSH requirement for toxin activation may be due to their extraordinary potency.

Animals↗

PubMed1990

Cleavage of disulfide bonds in endocytosed macromolecules. A processing not associated with lysosomes or endosomes.

Whereas there is biological evidence that the reductive cleavage of disulfide bonds is critical for the activation of endocytosed macromolecules such as toxins, immunotoxins, and other drug carriers, virtually nothing is known about the specifics of this cleavage. To study this process, a model compound was synthesized in which a radioiodinated tyramine was linked through a disulfide bond to an undegradable carrier, poly(D-lysine), known to be efficiently endocytosed. Cultured Chinese hamster ovary cells were pulse-labeled with this probe, and the disulfide cleavage was measured as released acid-soluble radioactivity at different times of chase. Pulse-labeled cells were also subjected to subcellular fractionation to identify intracellular structures associated with disulfide cleavage. Cleavage began without lag, amounted to about approximately 7% of the initial cell-bound radioactivity in the first hour and continued for more than 6 h. It was abolished in the presence of N-ethylmaleimide. When sulfhydryl groups present at the cell surface were blocked with cell-impermeant sulfhydryl reagent, the initial phase of disulfide cleavage was inhibited, indicating that cleavage began at the cell surface. A long-lasting intracellular phase of disulfide cleavage began after about approximately 30 min of chase. Subcellular fractionation and kinetic analysis indicated that neither lysosomes nor endosomes were participating in that phase, leaving the Golgi apparatus as the most probable site of endocytic disulfide cleavage.

Animals↗

PubMed1989

In vivo antitumor effect of methotrexate conjugated to a monoclonal IgM antibody specific for stage-specific embryonic antigen-1, on MH-15 mouse teratocarcinoma.

Methotrexate (MTX) was coupled to an IgM monoclonal antibody specific for stage-specific embryonic antigen-1 (SSEA-1), and the resulting immunoconjugate (MTX-anti-SSEA-1) was used for in vivo drug targeting in mice bearing MH-15 teratocarcinoma. Immunoconjugates having an average of 65 mol MTX/mol antibody retained full antigen-binding capacity. Mice bearing well-established tumors (approx. 1 g) were treated i.v. using the immunoconjugate. MTX-anti-SSEA-1 at 15 mg/kg of drug had significant antitumor activity with no significant systemic toxicity. Neither an irrelevant isotype-matched conjugate, MTX-MOPC-104E, prepared from the MOPC 104E myeloma protein, nor free MTX injected alone or with either antibody had any significant antitumor effect. These results indicate that IgMs can be effective drug carriers for tumor targeting in spite of their high molecular mass, and that antigens that are selectively accessible in tumors, even though present in normal tissues, can be suitable targets for in vivo chemoimmunotherapy.

Antibodies, Monoclonal↗

PubMed1988

Methotrexate-poly(lysine) as a selective agent for mutants of Chinese hamster ovary cells defective in endocytosis.

Methotrexate (MTX) covalently linked to poly(L-lysine) [poly(Lys)] enters cells by endocytosis, is degraded in lysosomes and, upon liberation of small molecular methotrexate, is cytocidal to Chinese hamster cells in culture. This drug conjugate was used to select mutants resistant to MTX-poly(Lys), which were examined for defects in endocytosis. Two mutants resistant to MYX-poly(Lys) and sensitive to free MTX, MPL 3-4 and MPL 2-5, internalized the conjugate in normal fashion, but had a decreased ability to degrade it to small molecular drug. The magnitude of this defect in the two mutants correlated with their level of resistance. In addition, both mutants were cross resistant to diphtheria toxin and modeccin and hypersensitive to ricin. While MPL 3-4 internalized MTX-poly(Lys) and inulin normally, it showed decreased endocytosis via the mannose-6-phosphate receptor and decreased uptake of 125I-alpha-2 macroglobulin. Acidification of subcellular fractions was measured using the partitioning of acridine orange. In MPL 3-4, the ATP-driven acidification of the endosome-containing cell fractions was slightly decreased (80% of controls), while acidification of the heavy lysosome-containing fraction was normal. Complementation analysis using hybrids of MPL 3-4 x MPL 2-5 indicated that the mutations occurred at the same gene, but were expressed with different severity. This genotype is identical to that of the End 2 mutants described by Roff et al. (1986). Thus, surprisingly, mutants with identical genotypes were isolated independently by totally different selection procedures.

Animals↗

PubMed1988

Stable variant of LM fibroblast defective in fluid-phase but competent in receptor-mediated endocytosis.

The F-40 cell line, a stable variant of LM fibroblasts selected for its resistance to polyethylene glycol (PEG)-induced fusion (Roos and Davidson: Somatic Cell and Molecular Genetics 6:381-391, 1980), has a decreased capacity to internalize fluid-phase markers and nonspecifically surface-bound macromolecules. It is not defective in exocytosis since, after a short sucrose pulse, it releases the same fraction of ingested sucrose into the medium as does the parental line. F40 cells have a normal capacity to carry out receptor-mediated endocytosis, as tested with 125I-alpha-2 macroglobulin (alpha-2 MG) and 125I-transferrin (Tf), and to recycle Tf receptor to the cell surface. These data demonstrate that receptor-mediated and non-receptor mediated endocytosis are distinct processes that can be altered independently. Of the many membrane fusions occurring in the course of endocytosis, the only one that appears associated with the defect in cell fusion characteristic of F40 cells is the formation of primary endocytotic vesicles engaged in non-receptor-mediated internalizations.

Animals↗

PubMed1987

Marked synergism of dimethylnitrosamine carcinogenesis in rats exposed to cadmium.

The combined carcinogenic effect of cadmium and dimethylnitrosamine (DMN) was examined in male Wistar rats, to test whether the previously observed synergism in mutagenicity between cadmium and 2 N-nitroso compounds would be paralleled by a synergism in carcinogenicity. In experiment 1, 50 five-month old rats received 18 mg DMN/kg i.p. followed by cadmium i.m. in two injections totaling either 1.5 or 3.0 mg Cd2+/kg. In experiment 2, 30 weanling rats received five i.m. doses totaling 6 mg Cd2+/kg followed by 18 mg DMN/kg. After 52 weeks, the incidence of renal tubular neoplasms was significantly increased above additivity in both experiments (P = 0.0005 to 0.017). Experiment 1 also showed a synergistic increase in the incidence of neoplastic (P = 0.024) and preneoplastic (P less than 0.01) microscopic liver lesions, of tumors of organs other than liver and kidney (P less than 0.01), of malignant versus benign tumors (P = 0.038), and of multiple versus single tumors (P = 0.0019). In experiment 2, the incidence of DMN-induced hepatocellular adenomas was significantly lower than additivity, suggesting an antagonistic, protective effect of cadmium pretreatment. The overall incidence of tumors of any type was 17.5 versus 67.7% in control (one agent or none) versus test animals. Malignant tumors included carcinomas, sarcomas, and lymphomas, involving nine different sites. Cadmium appears to enhance the initiation of carcinogenesis induced by DMN. Both cadmium and nitrosamines are known environmental contaminants present in air, food, water supplies, and tobacco smoke.

Animals↗

PubMed1987

Mechanisms of synergism in the mutagenicity of cadmium and N-methyl-N-nitrosourea in Salmonella typhimurium: the effect of pH.

Cadmium enhances the mutagenic effect of N-methyl-N-nitrosourea (MNU) in a synergistic manner in Salmonella typhimurium. In the range of doses that yield synergistic effects, it is by itself highly cytotoxic and only weakly mutagenic. A decrease in pH from 7 to 6 markedly decreases cadmium toxicity, causing a 4-fold increase in the surviving fraction. The same shift in pH markedly increases the dose-dependent mutagenesis of MNU, whether MNU is acting alone or in combination with cadmium and increases the synergism at lower doses of cadmium. Thus, the synergism appears to depend on the mutagenicity of MNU and not on the cytotoxicity of Cd. The combined mutagenic effect of Cd and MNU is comparable in tester strain TA1535 and TA100, which contains the error-prone (SOS) repair-enhancing pKM101 plasmid. Thus the synergistic effect of cadmium is not enhanced by the induction of SOS processing. The hydrolysis of MNU is higher at pH 7 than at pH 6 but cadmium has no effect on the rate of MNU hydrolysis at either pH, and therefore does not influence the concentration of the active electrophile of MNU. Cadmium does not enhance the mutagenesis of ethylnitrosourea, the ethylating analogue of MNU, indicating that the synergism is specific for methylated DNA lesions. These data suggest that cadmium acts either directly by modifying the nature or indirectly by inhibiting the repair of methylation damage.

Cadmium↗

PubMed1986

Altered endocytosis in a mutant of LM fibroblasts defective in cell-cell fusion.

Mutants of LM fibroblasts selected for their decreased ability to undergo polyethylene glycol-induced cell-to-cell fusion (F40 subline) were examined for possible alterations of their ability to carry out endocytosis. Both fluid-phase endocytosis of inulin and horseradish peroxidase and nonreceptor mediated adsorptive endocytosis of poly(L-lysine) were reduced to 60% of control values. Comparable results were obtained when the uptake of poly(L-lysine) was measured as internalization of surface-bound label in label-free medium or following continuous exposure. Accelerated breakdown of internalized label was ruled out as a cause for decreased label accumulation. Accelerated exocytosis is an unlikely cause, and it is suggested that the decreased uptake is due to a decrease in the constitutive membrane vesiculation process that leads to the formation of endocytotic vesicles. The capacity of F40 cells to degrade internalized horseradish peroxidase and poly(L-lysine) was not impaired, nor was their susceptibility to the cytotoxic action of methotrexate-poly(L-lysine). This drug conjugate must be degraded inside cells and release small molecular methotrexate in order to be cytocidal. These data suggest that only the first step of nonspecific endocytosis is impaired, while the subsequent steps that require fusion of endosomes to lysosomes proceed normally. Since the formation of primary endosomes requires membrane fusion through the external aspect of the plasma membrane and in that respect resembles cell-cell fusion, we propose the hypothesis that the observed decrease in endocytosis is related to the decreased ability of F40 cells to fuse with each other, and reflects a decreased efficiency of fusion processes at the external face of the plasma membrane.

Animals↗

PubMed1986

Tumor location and drug targeting using a monoclonal antibody (anti-SSEA-1) and antigen-binding fragments.

Both murine and heterotransplanted human nonseminomatous germ-cell tumors have been successfully located by external scintigraphy using radioiodinated anti-SSEA-1, a monoclonal IgM, and its pepsin-derived antigen-binding fragment, F(ab')2 mu. Antibody localization in the tumor is mainly due to antigenic specificity, rather than to nonspecific trapping, and depends strongly on the amount of time after injection. The antibody has been used for drug targeting in vitro and in vivo.

Animals↗

PubMed1986

Targeting, internalization, and cytotoxicity of methotrexate-monoclonal anti-stage-specific embryonic antigen-1 antibody conjugates in cultured F-9 teratocarcinoma cells.

Methotrexate (MTX) conjugates of a monoclonal antibody, anti-SSEA-1, containing an average of 45 mol MTX/mol of immunoglobulin M, were prepared by a carbodiimide coupling reaction. Binding experiments indicate that conjugation does not decrease the affinity of the antibody for its antigen. The conjugate strongly inhibits the growth of SSEA-1-bearing F-9 teratocarcinoma cells, with 50% inhibitory dose of 4.5 nM MTX, which makes it more active than free MTX (50% inhibitory dose of 15 nM). The drug-free antibody is not cytotoxic to F-9 cells at the concentrations used. The high efficacy of the conjugated drug may be due in part to the fact that anti-SSEA-1 antibody is an immunoglobulin M. MTX conjugated to nonspecific immunoglobulin M has little inhibitory effect (50% inhibitory dose of 150 nM). When acting on SSEA-1 negative cells, the two conjugates have only a small but identical effect. Thiamine pyrophosphate, an inhibitor of MTX transport, can prevent the cytotoxicity of the free MTX but not that of the anti-SSEA-1 conjugate. Leupeptin, an inhibitor of lysosomal protease, can partially protect F-9 cells against the antibody conjugate but not against free MTX. These results indicate that the MTX antibody conjugate binds specifically to F-9 cells, and is internalized and intracellularly degraded to release a small molecular active drug. Pretreatments of F-9 cells for 1 h with unlabeled antibody inhibits the subsequent uptake of identical concentration of labeled conjugate. The rate of internalization, however, regains almost normal values within 4 h, indicating a rapid reappearance of free antigenic sites at the cell surface.

Animals↗

PubMed1985

Disulfide spacer between methotrexate and poly(D-lysine). A probe for exploring the reductive process in endocytosis.

Poly(D-lysine) is taken up avidly by cultured cells through adsorptive endocytosis and can serve as a carrier to increase cellular uptake of other molecules. While direct conjugation of methotrexate to poly(D-lysine) yields a conjugate devoid of cytotoxic effects because poly(D-lysine) is not digested in lysosomes, the indirect conjugation using a triglycine spacer or a disulfide spacer strongly inhibits the growth of both the wild type and the methotrexate transport-defective Chinese hamster ovary cells. Cell treatment with 3 mM NH4Cl or 50 micrograms/ml leupeptin prevents the effect of conjugate with the triglycine spacer, but not of conjugate with the disulfide spacer. On the other hand, preincubation with 2-mercaptoethanol abolishes the effect of the drug-disulfide conjugate in the methotrexate transport-defective mutant, but not the effect of the drug-triglycine conjugate. The disulfide conjugate shows an identical cytotoxic effect in alpha-minimal essential medium and RPMI 1640 media, even though cells grown in the latter have only half the glutathione content as cells grown in the former medium. We conclude that the reductive process through which methotrexate is released from the disulfide spacer (a) occurs inside cells and not at the cell surface, (b) requires neither acid pH nor lysosomal enzymes, and (c) is not mediated by a glutathione-disulfide exchange reaction requiring high glutathione concentrations. Although the cellular compartment in which this reductive process occurs is not yet identified, there are reasons to assume that it is prelysosomal.

Animals↗

PubMed1984

Colorimetric determination of microgram quantities of polylysine by trypan blue precipitation.

A simple and sensitive method for the determination of polylysine in solution is described. Polylysine is quantitatively precipitated with trypan blue. The absorbance of unbound dye in the supernatant is inversely proportional to the concentration of this polyamino acid. The precipitation is identical for all sizes of polylysine of molecular weight 13,000 or higher, and is prevented by the addition of either polyanions or serum. The measurable range of polylysine hydrobromide is between 1 and 10 micrograms/ml, which is about 10-fold lower than that by the published methyl orange precipitation method.

Azo Compounds↗

PubMed1984

Binding sites and endocytosis of heparin and polylysine are changed when the two molecules are given as a complex to Chinese hamster ovary cells.

This paper characterizes the complex formed in vitro between polylysine and heparin in the presence of heparin excess, and investigates the interaction of this complex with the surface of Chinese hamster ovary cells. It examines the kinetics of surface binding and cellular uptake of the complex and shows that both processes can be distinguished from those of free heparin and free polylysine. The view that these three ligands bind to different surface sites is further supported by the fact that their interaction with cells is influenced differently by cell detachment with trypsin, detachment with EGTA or exposure to acid pH. Membrane transport of the complex is a saturable process suggestive of receptor-mediated endocytosis. It is, however, less effective than would be expected on the basis of the binding kinetics. Only 40% of the complex bound at 0 degrees C is internalized during a 2 h reincubation period at 37 degrees C, suggesting some degree of uncoupling between binding and endocytosis. These data confirm prior results obtained with methotrexate-polylysine conjugates. We had shown that the addition of heparin to a medium containing a methotrexate-polylysine conjugate leads unexpectedly to a marked cellular uptake of drug conjugate, which is capable of killing cells that are otherwise resistant to free methotrexate (Shen, W.-C. and Ryser, H.J.-P. (1981) Proc. Natl. Acad. Sci. USA 78, 7589-7593). The polylysine X heparin complex is therefore of interest as a potential carrier for intracellular drug delivery through endocytosis.

Animals↗

PubMed1984

Mutagenicity of cadmium in Salmonella typhimurium and its synergism with two nitrosamines.

Cadmium chloride (CdCl2) at concentrations of 0.5 mM was significantly mutagenic in Salmonella typhimurium tester strains and reverted histidine auxotrophy due either to missense (TA1975 and TA1535) or to frameshift (TA1537) mutations. It also induced forward mutations to 8-azaguanine resistance in each strain, but failed to increase mutation frequencies in strain TA100. More importantly, CdCl2 increased the mutagenicity of two common nitrosamines in synergistic fashion, at a level up to 30-fold greater than expected from simple additivity. The mutation frequency induced by N-methyl-N'-nitro-N-nitrosoguanidine was increased about 10-fold in the presence of 0.5 mM CdCl2. This synergism was seen both in the induction of 8-azaguanine resistance and the reversion of histidine auxotrophy and was observed in the repair-proficient strain TA1975 as well as its repair-defective (uvrB-) derived strain TA1535. The synergism was dependent upon Cd concentration and was much reduced at 0.25 mM CdCl2. The strongest synergism was observed in the reversion of histidine auxotrophy in TA1975 by 180 microM methylnitrosourea and 0.5 mM CdCl2. In contrast to mutagenicity, there was no evidence for synergism in the toxicity of CdCl2. These data suggest that cadmium might interfere with the repair of both spontaneous and nitrosamine-induced mutations. They also raise the possibility that cadmium and nitrosamines may have synergistic effects as environmental carcinogens.

Cadmium↗