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S M Liang

Publications and source records attributed to S M Liang.

17 recordsLinked to original sources
PubMed1992

Analysis of IL-2 functional structure by multiple cysteine substitutions.

IL-2 has three cysteine residues. The cysteines at positions 58 and 105 of active IL-2 form an intramolecular disulfide bond while that at position 125 remains as a free form. To evaluate the importance of correct disulfide bond, mutant proteins (muteins) that have triple and double substitutions of cysteines with alanines, namely A58/105/125 and A58/125, were made by polymerase chain reaction method respectively. Thymidine incorporation assay on CTLL-2 cells showed that although these two muteins were only 0.5-2.0% as potent as that of wild type IL-2, they were 50-200 fold more active than A58, a mutein that has substitution of cysteine at position 58 with alanine. Binding inhibition study showed that the relative affinity of muteins A58/125 and A58/105/125 for high affinity IL-2 receptors was 5-25 fold higher than that of A58. These results suggest that the dramatic decrease in the activity of mutein A58 may result from the formation of an incorrect disulfide bond between the cysteines at positions 105 and 125.

Binding, Competitive↗
PubMed1992

Effects of glutathione on the synthesis and turnover of interleukin-2 receptors.

Internalization of IL-2 is important for its biological activities. The internalization of IL-2 was regulated by the duration of glutathione (GSH) treatment in CTLL-2 and CT-4R cells. Flow cytometric studies showed that the level of surface IL-2 receptors was not increased by GSH treatment. Northern blot analysis showed that the mRNA of IL-2Rp55 and IL-2Rp70, the two major components of the high-affinity IL-2 receptors, was increased 6 hr after GSH treatment. The appearance rate of membrane IL-2 receptors in GSH-treated cells was faster than that of the untreated cells. GSH also shortened the half-life (from 5 to less than or equal to 3 hr) and thus increased the turnover of the surface high-affinity IL-2 receptors. These results suggest that although GSH does not affect the level of surface IL-2 receptors, GSH may regulate the internalization of IL-2 by enhancing the synthesis and turnover of surface IL-2 receptors.

Animals↗
PubMed1992

Dichotomy of glutathione regulation of the activation of resting and preactivated lymphocytes.

The present study has examined the effect of GSH on two lines of IL-2-dependent activated killer cells, LAK cells and alpha CD3-activated killer (CD3-AK) cells. We found that GSH added during first 24 hr decreased the generation of LAK and CD3-AK cells from resting lymphocytes, whereas after 48 hr of activation, the addition of GSH increased the killer cell activity. In addition, BSO, an inhibitor of GSH biosynthesis, decreased the proliferation and cytotoxic activities of activated killer cells, and the inhibitory effect was reversed by GSH. These results indicate that GSH downregulates the generation of LAK or CD3-AK cells from resting lymphocytes, but it upregulates the further differentiation of preactivated killer cells. The effect of GSH thus varied with the state of activation of the killer cells. Culturing CD3-AK cells in GSH did not change the distribution of T cell subsets, did not affect the cells' ability to produce lymphokine (IL-2), and did not induce suppressor cells. One striking change as revealed by flow cytometry analysis was that the levels of IL-2 receptor and TCR (alpha/beta)-CD3 were reduced by 80 and 30%, respectively, after 48 hr culturing in GSH. Determination of the mRNA of IL-2 receptor suggests that a post-transcriptional block existed. It appears that the negative effect of GSH on the function of surface IL-2 receptors or T cell receptors on resting lymphocytes severely affected the signal transduction through these receptors and thus abrogated or reduced LAK or CD3-AK cell response. In contrast, for preactivated killer cells, upregulation by intracellular GSH of IL-2 utilization is a dominant effect, thus allowing further differentiation of these killer cells. Our results indicate that the balance between the activation signal (IL-2 or alpha CD3) and the immunoregulatory signal (induced by GSH) may determine the outcome of the immune response.

Animals↗
PubMed1992

Regulation by glutathione of interleukin-4 activity on cytotoxic T cells.

We have previously shown that cellular glutathione (GSH) regulates the T-cell proliferative activity of interleukin-2 (IL-2). Here, we examined whether and how GSH affects the activity of interleukin-4 (IL-4) on murine cytotoxic T cells. CT.4R, a T-cell line that is responsive to both IL-4 and IL-2, was used as a model. Although GSH alone had little effect on the thymidine incorporation of CT.4R cells, it enhanced the response of CT.4R to IL-4 and increased the level of thymidine incorporation up to more than 60-fold in a concentration-dependent manner. GSH affected the binding of IL-4 to cellular receptors. Scatchard plot analysis showed that GSH treatment did not change the dissociation constant significantly; however, it increased the receptor number from 1173 +/- 126 to 2112 +/- 492 molecules per cell. Internalization and degradation studies of IL-4 showed that the amount of IL-4 internalized and degraded in the GSH-treated cells was about twofold higher than those in the cells without GSH treatment. These results suggest that GSH regulates the binding, internalization, degradation and T-cell proliferative activity of IL-4; alteration of cellular GSH levels may thus affect the growth and replication of cytotoxic T cells through growth stimulating cytokines such as IL-2 and IL-4.

Animals↗
PubMed1991

Regulation by glutathione of the effect of lymphokines on differentiation of primary activated lymphocytes. Influence of glutathione on cytotoxic activity of CD3-AK-.

By activating murine lymphocytes with anti-CD3 antibodies for 1 to 2 days, we generated a subset of activated killer cells, namely CD3-AK-. CD3-AK- mediated the slow lysis (20-h 125I-UdR release assay) of allogeneic P815 but had little effect on syngeneic HFL/b cells. Addition of IL-2 (murine or human) or an IL-2 inducer such as PMA in the assay medium induced the cytolytic activity of CD3-AK- on HFL/b. The activating effect of murine IL-2 and PMA on CD3-AK- was decreased by anti-murine IL-2 mAb. Although anti-murine IL-4 mAb alone did not show any effect, it enhanced the inhibitory effect of anti-IL-2 mAb, suggesting that IL-2 and IL-4 may have a synergistic effect on the cytolytic activity of CD3-AK-. Incubation of CD3-AK- with L-buthionine-(SR)-sulfoximine (BSO), an inhibitor of de novo glutathione (GSH) synthesis, decreased cellular GSH levels and inhibited the cytolytic activity of CD3-AK-, in a concentration-dependent manner. This inhibitory effect of BSO was not primarily due to a general cytotoxic effect and was positively correlated with the requirement for IL-2 for the CD3-AK(-)-mediated killing of the target cells. Incubation of CD3-AK- with GSH or 2-ME, which increased the level of cellular GSH, reversed the inhibitory effect of BSO. These results suggest that cellular GSH may regulate the effect of lymphokine(s) such as IL-2 and thus affect the differentiation of activated primary cytotoxic lymphocytes.

Animals↗
PubMed1990

An anti-p24 monoclonal antibody shows cross-reactivity with multiple HIV-1 proteins.

We produced three murine monoclonal antibodies (mAbs) against the HIV-1 proteins. These three mAbs, namely CA-1, CA-2, CA-4, were IgG1 and all reacted with p24 on the HIV-1 Western blot. One of the mAbs, CA-4, also recognized p13, p21, p28, p29, p32, p39, p47, p55 on the Biotech/Du Pont HIV-1 Western blot strips and p21, p24, p28, p29, p39, p47, p55, p68, p80, p96; p110 on the Bio-Rad strips. CA-4 did not react with H-9 cell lysate nor with other retroviral antigens such as HTLV-1 or HIV-2 proteins. The binding of CA-4 to HIV-1 proteins was not blocked by deglycosylation. All three mAbs reacted with recombinant DNA derived capsid protein (p24) of HIV-1. These results suggest that many proteins in the HIV-1 Western blot contain antigenic epitope(s) similar to that of p24.

Animals↗
PubMed1989

Visualization of interleukin-2-like molecules in MPP(+)-lesioned rat brain.

The tissue distribution of interleukin-2 (IL-2) in normal and 1-methyl-4-phenyl-pyridinium (MPP+)-lesioned brains of rats was investigated. Intrastriatal administration of MPP+ caused visible damage in the vicinity of the injected region two weeks after injection. Autoradiography of the tissue section with anti-IL-2 antibodies plus trace amounts of radiolabeled IL-2 showed that the antibodies treatment elicited a selective radiolabeling of the brain tissues localized at the MPP(+)-lesioned region but not at normal cryo-sliced sections. Addition of radiolabeled IL-2 alone or normal rabbit immunoglobulins did not show any labeling effect. These autoradiographic imaging results suggest that there is an accumulation of cells bearing IL-2-like molecules at the MPP(+)-induced lesion sites.

1-Methyl-4-phenylpyridinium↗
PubMed1989

Glutathione regulates interleukin-2 activity on cytotoxic T-cells.

In this study, we examined whether and how the cellular activity of interleukin-2 (IL-2) is affected by glutathione (GSH), an important tripeptide existing in most cells. Cell culture and thymidine incorporation assay showed that addition of GSH enhanced the effect of IL-2 on the proliferation and thymidine incorporation of IL-2-dependent cytotoxic T-cells such as CTLL-2 and CT-4R. Treatment of the cells with GSH resulted in a 2-fold increase in the amount of IL-2 bound to the cells and a rapid internalization of the bound IL-2. In addition, the degradation of IL-2 in the cells was enhanced by GSH treatment. These effects of GSH were accompanied by an increase in the intracellular GSH level. L-Buthionine-(S,R)-sulfoximine, an inhibitor of de novo GSH synthesis, blunted the increase of intracellular GSH level and modulated the effect of GSH on IL-2 activity. These results suggest that GSH regulates the binding, internalization, degradation, and T-cell proliferative activity of IL-2; alterations of cellular GSH concentration may thus affect the growth and replication of IL-2-sensitive cytotoxic T-cells.

Animals↗
PubMed1988

Biological characterization of human interleukin-2 mutant proteins. Structure-activity relationship studies.

Several human interleukin-2 (IL-2) mutant proteins have been produced previously by site-directed mutagenesis and found to have different capacities to induce T-cell proliferative activity. In this study, the abilities of these IL-2 mutant proteins to activate natural killer cells and to induce interferon-gamma production have been evaluated, and the binding of these proteins to IL-2 receptors analyzed. Natural killer cell activation and interferon-gamma induction assays showed that the relative activities of IL-2 mutant proteins were consistent with their relative activities in T-cell proliferation assay. Receptor-binding studies showed that the activities of most proteins correlated well with their respective affinities for high-affinity IL-2 receptors on CTLL-2 cells. Interestingly, although the mutant protein with deletion of cysteine 125 (des-Cys125) was biologically less active than the protein with substitution of alanine for cysteine 105 (Ala105), both proteins exhibited similar affinity. Des-Cys125, like IL-2 and Ala105, also caused down-regulation of high-affinity IL-2 receptors. Binding studies on MLA-144, a cell line expressing mainly intermediate-affinity IL-2 receptors (IL-2R beta), however, showed that des-Cys125 had much lower affinity than Ala105. These results suggest that binding of IL-2 and mutant proteins to the IL-2R beta component of the high-affinity receptor is essential for the induction of biological effects.

Animals↗
PubMed1986

Production and characterization of monoclonal antibodies against recombinant human tumor necrosis factor/cachectin.

Tumor necrosis factor is a monokine, which causes cytolysis of many transformed cells. In this study we have found that in addition to cytotoxicity recombinant Escherichia coli-derived human tumor necrosis factor, like cachectin, inhibited the lipoprotein lipase of 3T3-L1 preadipocytes. Both effects were inhibited by monoclonal anti-tumor necrosis factor antibodies. Monoclonal antibodies against recombinant human tumor necrosis factor were produced by fusing splenocytes of immune mice with P3X63Ag8 653 myeloma cells. The monoclonal antibodies, namely BG 2-4, were of IgG2a, IgG, and IgG2a subclasses. These monoclonal antibodies neutralized the cytotoxicity of natural and recombinant human tumor necrosis factor but not that of rabbit or mouse tumor necrosis factor. They also neutralized the cachectin activity of human tumor necrosis factor in the 3T3-L1 embryonic cell assay. These results indicate that the functional structure(s) of human tumor necrosis factor responsible for the cytotoxicity and cachectin activities are likely to be closely related.

Adipose Tissue↗
PubMed1986

Disulfide scrambling of interleukin-2: HPLC resolution of the three possible isomers.

Native interleukin-2 (IL-2) contains three cysteines; two exist in a disulfide bridge (Cys-58 and Cys-105) and the third Cys-125 is a free sulfhydryl. In the presence of 6 M guanidine hydrochloride at alkaline pH, IL-2 is converted into three isomers. Each isomer represents one of the three possible disulfide-linked forms that can be generated from three cysteines. These three isomers were resolved on a C4 reverse-phase HPLC system. The identity of each of the three forms was determined by carboxymethylation of the free cysteines in each isomer with [3H]iodoacetic acid followed by determination of the labelled cysteines by tryptic peptide mapping. Tryptic peptide mapping of the more predominant of the two scrambled peaks showed it to be the Cys-105-S-S-Cys-125 linked form of IL-2. A Ser-125 construction of IL-2, which lacks a free cysteine, did not scramble under these conditions. These experiments demonstrate the utility of reverse-phase HPLC in studies of protein folding and disulfide bond structure.

Amino Acid Sequence↗
PubMed1986

Studies of structure-activity relationships of human interleukin-2.

Human interleukin-2 (IL-2) has 3 cysteine residues; cysteines 58 and 105 form an intramolecular disulfide bridge, whereas cysteine 125 has a free sulfhydryl group. In this study, site-specific mutagenesis has been used to modify the cysteine residues of recombinant Escherichia coli-derived IL-2 (rIL-2) to evaluate the functional structure of IL-2. Substitution or deletion of cysteine 105 disrupted the disulfide bridge and yielded a mutant protein which was 8-10 times less active than wild type rIL-2. A similar modification at position 58, however, reduced the activity of rIL-2 by more than 250-fold. Although substitution of serine for cysteine 125 did not affect IL-2 activity, deletion of cysteine 125 or deletion of amino acids in the vicinity of this cysteine yielded mutant proteins with little, if any, activity. These results indicate that the protein structure in the vicinity of both positions 58 and 125 is more critical than that close to position 105. These findings may provide a clue to the understanding of the functional structure of human IL-2.

Amino Acids↗
PubMed1985

Characterization of human interleukin 2 derived from Escherichia coli.

Interleukin 2 isolated from Escherichia coli cells expressing the human interleukin gene has been characterized. The observed properties of the protein have been compared with those properties which can be deduced from the DNA sequence alone and the published properties of natural human interleukin 2. The purified E. coli-derived interleukin 2 is a monomeric protein of Mr 15 000 with a sedimentation velocity of 1.86S. The amino acid composition of the protein and isoelectric point (7.7) are consistent with that part of the translated DNA sequence of the gene corresponding to the mature protein. A single disulphide bridge was identified between Cys-58 and Cys-105. C.d. suggested that interleukin 2 is predominantly alpha-helical in secondary structure. The E. coli-derived protein differed from natural interleukin 2 in the presence of N-terminal methionine and also in the absence of a carbohydrate moiety. Removal of the coding region for the first three amino acids of the natural interleukin 2 protein sequence (Ala-Pro-Thr) by site-specific mutagenesis resulted in a protein with N-terminal serine. The possibility that the specificity of the E. coli ribosomal methionine aminopeptidase may not recognize the sequence NH2-Met-Xaa-Pro is discussed (where Xaa is any amino acid residue).

Amino Acids↗
PubMed1976

The measurement of ouabain binding and some related properties of digitonin-treated (Na+,K+)-ATPase.

1. Digitonin treated membrane preparations purified from dog kidney lose their (Na+,K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, but the K+-phosphatase and Na+-dependent ADP-ATP exchange activities survive and remain ouabain-sensitive. Because the enzyme preparations consist largely of pure (Na+,K+)-ATPase, these effects of digitonin must be intrinsic to the Na+ pump. 2. Concomitant with these enzymatic changes, digitonin treatment alters the sensitivity of the phosphatase and exchange activities to ouabain. 3. Attempts to measure ouabain binding by the usual centrifugation or filtration methods proved unsuccessful. A filtration method involving a double 0.01 mum filter and omitting water washes is necessary to demonstrate ouabain binding. Under these conditions, ouabain binding capacity appears to be unchanged in the presence of digitonin, but the apparent dissociation constant is doubled. 4. Ouabain binding is rendered more reversible by digitonin treatment, since washing filters with water removes a large fraction of bound ouabain without affecting the retention of exchange activity. 5. The double filter method traps essentially all of the ADP-ATP exchange activity on the filter. However, a large and somewhat variable proportion of the K+-phosphatase activity passes through the filter. Sodium dodecyl sulfate polyacrylamide gel analysis of the filtrate shows that a small amount of filtrable protein catalyzed this phosphatase activity at greatly increased turnover rates. Both subunits of the (Na+, K+)-ATPase are present in this latter protein fraction.

Adenosine Triphosphatases↗
PubMedDate not supplied

Mediation of mouse natural cytotoxic activity by tumour necrosis factor.

Natural cell-mediated cytotoxic activity in the mouse has been associated with two types of effector cells, the natural killer (NK) cell and the natural cytotoxic (NC) cell, which seem to differ with regard to their patterns of target selectivity, cell surface characteristics and susceptibility to regulatory factors. During studies on the mechanism of action of cytotoxic molecules, it became evident that WEHI-164, the prototype NC target cell, was highly susceptible to direct lysis by both human and mouse recombinant tumour necrosis factor (TNF). Here we show that NC, but not NK activity mediated by normal splenocytes, is abrogated by rabbit antibodies to recombinant and natural TNF, respectively. Thus, the cell-mediated activity defined as NC is due to release of TNF by normal spleen cells and does not represent a unique natural effector mechanism.

Animals↗