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S Buxser

Publications and source records attributed to S Buxser.

12 recordsLinked to original sources
PubMed1992

Probing the structure-function relationship of nerve growth factor.

We compared the receptor binding, antigenicity, biological activation, and cell-mediated proteolytic degradation properties of mouse nerve growth factor (mNGF) and human NGF (hNGF). The affinity of hNGF toward human NGF-receptor is greater than that of mNGF, but the affinity of mNGF toward rat NGF-receptor is greater than that of hNGF. Thus, the specificity of the interaction between NGF and its receptor resides both on the NGF and on its receptor. Using a group of anti-NGF monoclonal antibodies that competitively inhibit the binding of NGF to receptor, sites differing between mNGF and hNGF were detected. Together, these results indicate that the sites on hNGF and mNGF, responsible for binding to NGF-receptor, are similar but not identical. In comparing the relative abilities of mNGF and hNGF to stimulate a biological response in PC12 cells, we observed that mNGF was better at stimulating neurite outgrowth than was hNGF, consistent with the differences observed for receptor binding affinity. However, the ED50 for biological activation is approximately 100-fold lower than the Kd for receptor occupancy, and, thus, the dose-response curve is not consistent with a simple activation proportional to receptor occupancy. The data are consistent with a model requiring a low-level threshold occupancy of NGF-receptor (Kd = 10(-9) M) in order to stimulate full biological activity. Finally, we observed the degradation of NGF by PC12 cells. We found that the NGF molecule is significantly degraded via a receptor-mediated uptake mechanism. Together, the data provide insight into regions of the NGF molecule involved in contacts with the receptor leading to formation of the NGF:NGF-receptor complex. Additionally, they establish the link between occupancy of receptor and biological activation and the requirement for receptor-mediated uptake in order to degrade NGF proteolytically in cultured PC12 cells.

Animals↗
PubMed1991

Single-step purification and biological activity of human nerve growth factor produced from insect cells.

Human nerve growth factor (NGF) was cloned and engineered for expression in a baculovirus-infected Spodoptera frugiperda (SF-9) insect cell system. Culture supernatants contained 2-3 mg/L of recombinant human NGF. The human NGF produced by this system was purified to apparent homogeneity with a single-step affinity chromatography procedure using a high-affinity monoclonal antibody originally raised against murine NGF. The purification procedure yielded 1-2 mg of pure, human NGF per liter of culture supernatant; i.e., approximately 60% recovery of the human NGF originally released into the culture medium. Although the gene transfected into the SF-9 cells coded for pro-NGF, the NGF recovered after purification was greater than 95% fully processed, mature protein. The KD for the affinity of the pure, recombinant human NGF for NGF receptor in PC12 membranes is 0.20 +/- 0.05 nM. Activation of neurite outgrowth in PC12 cells occurs with ED50 values of 85 +/- 20 pM and 9.6 +/- 1.5 pM for a 3-day primary response and a 1-day secondary response, respectively. The pure, recombinant human NGF also stimulates a significant increase in dopamine content of PC12 cells with an ED50 of 5.8 +/- 2.7 pM. These binding and biological activation properties are consistent with values observed using murine NGF purified from submaxillary glands.

Amino Acid Sequence↗
PubMed1990

Relationship among types of nerve growth factor receptors on PC12 cells.

We analyzed the kinetics and thermodynamics of 125I-nerve growth factor (125I-NGF) binding to NGF-receptor on PC12 cells. We used conditions of pseudo-first order kinetics and techniques to quantitate internalized complexes, "slow" or high affinity binding complexes, and cell surface "fast" or low affinity complexes. Two possible models were examined: binding to two independent receptors at the cell surface (i.e. high and low affinity forms of NGF-receptor) and a model for consecutive formation of fast, low affinity binding followed by slow, high affinity binding or internalization. Our data are consistent with the consecutive model only. The rates of association and dissociation of NGF with slow, high affinity sites and internalized, acid wash-resistant sites are indistinguishable from each other. We also analyzed, in detail, the two assays primarily used to distinguish slow binding complexes from internalized complexes. Scatchard analysis of total binding and dissociation of pre-equilibrated 125I-NGF in the presence of unlabeled NGF at high concentration (cold wash). Neither of these assays shows any evidence that the slow, high affinity binding step is different from internalization of the 125I-NGF-receptor complex. Based on this analysis, there are only two detectable forms of NGF-receptor on PC12 cells: complexes on the surface of the cells with a binding affinity of 0.5 nM at 37 degrees C and complexes internalized by the cells. Furthermore, the data are consistent with a model in which NGF-receptor is internalized constitutively and independently of occupancy by NGF. We also examined the fate of internalized 125I-NGF. In the first 60 min after contact with PC12 cells, no degradation of 125I-NGF was observed. Moreover, a significant amount of 125I-NGF recirculates to the cell surface and is released as intact, Mr = 13,000 NGF. The cells were also stimulated by NGF in a primary neurite outgrowth assay with an ED50 of 2-16 pM under conditions of low initial cell numbers in a large extracellular volume of NGF-containing medium. Thus, low level occupancy of the cell surface receptors, Kd = 0.5 nM, for several days is sufficient to stimulate neurite outgrowth. This indicates the presence of spare NGF-receptors on the surface PC12 cells.

Adrenal Gland Neoplasms↗
PubMed1990

Bacterial proteins that mediate the association of a defined subset of T cell receptor:CD4 complexes with class II MHC.

We have examined the responses of cloned T cell lines and of normal T cells to staphylococcal enterotoxins A, B, and C1 (SEA, SEB, and SEC1). SEA, SEB, and SEC1 are all very potent mitogens for T cells in the presence of Ia+ APC. The minimal activating dose of all these SE varies from 1 to 100 ng/ml. As determined by mAb blocking of the responses of both normal T cells and cloned T cell lines, SEA required either the I-A or the I-E molecule on APC for stimulating T cells, whereas SEB required the I-E molecule predominantly over I-A molecule. The TCR:CD4 complex is also involved in the response to SE. The responses to SEB and SEC1 were inhibited by anti-V beta 8 antibody F23.1, whereas the response to SEA and to PHA was not affected by this antibody. Anti-CD4 effectively inhibited responses to all SE but not to PHA. The involvement of the TCR was also confirmed by flow microfluorimetry analysis of T cell blasts responding to SE and the responses of a panel of cloned T cell lines, both of which showed that V beta 8+ T cells preferentially responded to SEB, whereas V beta 8+ T cells failed to respond to SEA. By using fixed APC, it could be shown that processing is not required for the presentation of SE. Furthermore, pulsing experiments showed that SEB can bind to relevant sites on either B cells or T cells, whereas with conventional Ag only prepulsing of the APC has worked. In one case, SEB activates a cloned T cell line in the absence of APC, and this same clone also responds directly to anti-V beta 8 antibody. Thus, SEB appears to bring together V beta 8-expressing TCR with the I-E molecule, whereas SEA apparently has the same effect on TCR expressing different V beta with either the I-A or the I-E molecule, probably depending upon which TCR is bound. The close resemblance between T cell responses to SE and those to mixed-lymphocyte stimulating (Mls) locus suggests to us that a novel SE-like protein that binds both to class II MHC molecules on the APC surface and to V beta gene products on TCR could be the product of the Mls locus.

Animals↗
PubMed1990

Two-step purification of full-length nerve growth factor receptor and maintenance of receptor-specific binding activity.

A method for the purification of full-length nerve growth factor receptor (NGFRc) using membranes from three different cell lines was developed. We emphasized recovery of NGFRc that retained specific binding activity. Lipids were required to preserve binding activity during solubilization and throughout the purification procedure. Phosphatidylcholine was used for this purpose. Lectin affinity chromatography followed by high-resolution anion-exchange chromatography was used, and a 3000-fold increase in specific binding activity was obtained for NGFRc from human melanoma A875 membranes. Seven percent of the original binding activity was recovered as pure NGFRc. NGFRc binding activity eluted at 0.35 M NaCl in anion-exchange chromatography of solubilized A875, rat pheochromocytoma PC12, and human neuroblastoma MC-IXC membranes. Eight and three percent of the original binding activity were recovered as highly enriched NGFRc from membranes prepared from PC12 and MC-IXC cells, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of highly enriched, 125I-labeled NGFRc revealed several protein species. After chromatography, identification of proteins as NGFRc was verified both by immunoprecipitation using receptor-specific monoclonal antibodies and by covalent cross-linking to 125I-NGF using N-hydroxysuccinimidyl-4-azidobenzoate. Predominantly, NGFRc was recovered as a mixture of species of 80 and 160-180 kDa. Small amounts of larger species as well as smaller species were observed, consistent with minor amounts of receptor aggregation and proteolysis occurring during purification.

Animals↗
PubMed1985

Properties of the nerve growth factor receptor. Relationship between receptor structure and affinity.

Microsomal membranes from A875 human melanoma cells contain nerve growth factor receptors (NGF-receptors) which appear to belong to a single class with homogeneous binding properties, as determined by Scatchard plots. NGF-receptors in these membrane preparations are also uniformly highly sensitive to tryptic proteolysis, and 125I-NGF bound to NGF-receptor in these membranes is rapidly dissociated in the presence of a high concentration of unlabeled NGF. However, analysis of 125I-NGF dissociation kinetics indicated that two classes of NGF-receptor were present in these membranes. Thus, NGF-receptors can express either high or low affinity trypsin-sensitive states in addition to the high affinity trypsin resistant NGF-receptor state described previously (Buxser, S. E., Kelleher, D. J., Watson, L., Puma, P., and Johnson, G. L. (1983) J. Biol. Chem. 258, 3741-3749). The high affinity trypsin-sensitive and low affinity trypsin-sensitive states correlate with 200- and 90-kDa 125I-NGF X NGF-receptor complexes observed in photoaffinity cross-linking experiments. The absence of differences in peptide maps generated from the two sizes of NGF-receptor proteins together with structural and binding data strongly indicates that the 200-kDa NGF-receptor protein is a complex, probably a dimer, consisting of two 80-kDa NGF-receptor proteins associated with a single beta-NGF dimeric molecule. A model is proposed which relates structural states of NGF-receptors with specific receptor binding properties. The model provides an alternative explanation for binding phenomena previously attributed to negative cooperativity.

Animals↗
PubMed1982

Affinity labeling of a nerve growth factor receptor component on rat pheochromocytoma (PC12) cells.

Clonal PC12 rat pheochromocytoma cells were sequentially incubated with 125I-labeled nerve growth factor and the photoreactive bifunctional agent hydroxysuccinimidyl-p-azidobenzoate. This treatment effected the crosslinking of 125I nerve growth factor to a PC12 cell component that exhibits an apparent Mr = 148 000-158 000, and consists of a single polypeptide chain with internal disulfide bonds. The amount of label associated with this Mr = 148 000-158 000 species was proportional to the degree of occupancy of nerve growth factor receptors by 125I-labeled nerve growth factor. Affinity labeling of this species was inhibited by the presence of 0.2 microM unlabeled nerve growth factor during incubation of PC12 cells with 125I nerve growth factor. In membranes prepared from PC12 cells hydroxysuccinimidyl-p-azidobenzoate effected the crosslinking of 125I-labeled nerve growth factor to an Mr = 120 000-130 000 species but not to the Mr = 148 000-158 000 component observed in intact cells. The kinetics of 125I nerve growth factor affinity labeling of the Mr = 148 000-158 000 species closely paralleled the time-course of 125I nerve growth factor association to two kinetically distinct forms of nerve growth factor receptors in PC12 cells. The data indicate that the Mr = 148 000-158 000 species affinity-labeled by 125I nerve growth factor is the native form of a component associated with kinetically different nerve growth factor receptors in PC12 cells.

Adrenal Gland Neoplasms↗
PubMed1981

Staphylococcal enterotoxins fail to disrupt membrane integrity or synthetic functions of Henle 407 intestinal cells.

The potential cytotoxic activity of purified staphylococcal enterotoxins for mammalian cells was evaluated. The effects of staphylococcal enterotoxins A (SEA) and B (SEB) on cell membrane integrity as measured by leakage of labeled cytoplasmic constituents ([3H]uridine), amino acid transport (lysine and aminoisobutyric acid), and macromolecular synthesis (protein, ribonucleic acid, and deoxyribonucleic acid) was evaluated for a human intestinal epithelial cell (Henle 407). No evidence of cytotoxicity by any of these criteria could be detected for cell monolayers incubated with SEA for periods of between 30 min and 24 h. Purified staphylococcal hemolysins (alpha- and delta-toxins) were shown to exert cytotoxicity by the leakage and amino acid uptake assays. In efforts to detect synergistic effects between enterotoxin and the staphylococcal cytotoxins, membrane functions were evaluated after sequential or combined treatment with enterotoxin and alpha-toxin or with enterotoxin and delta-toxin. In no instance could a contribution to cytotoxicity by the staphylococcal enterotoxin be detected. That the assays were sufficiently sensitive to detect synergistic effects was shown by the greater than additive effects achieved with a combination of alpha- and delta-toxins. The data, contrary to previous reports, showed that staphylococcal enterotoxins did not behave as bacterial cytotoxins.

Animals↗
PubMed1981

Specific receptor binding of staphylococcal enterotoxins by murine splenic lymphocytes.

We describe a reliable assay to measure the specific binding of 125I-labeled staphylococcal enterotoxin A (SEA) by murine spleen cells. Toxin binding by lymphocytes was specific in that it was inhibited by unlabeled SEA but not by unrelated proteins. The biological activity of SEA (T-lymphocyte mitogenesis) correlated with toxin binding to splenic lymphocytes. In the presence of high concentrations of [125I]SEA, specific binding increased rapidly and approached saturation after 2 h. Toxin binding was sensitive to temperature and pH and was directly proportional to the concentration of spleen cells in the incubation mixture. We estimated that there was a single class of toxin-binding sites, which had an apparent equilibrium dissociation constant (Kd) of 8 x 10(-7) M and numbered 3,600 sites per cell. SEA and the antigenically distinct compounds staphylococcal enterotoxins B and E in excess competitively inhibited binding of [125I]SEA to mouse spleen cells. Our data suggest a common class of binding sites for the three staphylococcal enterotoxins.

Animals↗
PubMedDate not supplied

Staphylococcal enterotoxin B stimulation of BALB/c lymphocyte mitogenesis and potential relationship to the Mls response.

Staphylococcal enterotoxin B (SEB) is a T cell mitogen with properties different from the plant lectin mitogens. We examined the stimulation of mitogenesis induced by SEB in BALB/c mouse spleen cells and its relationship to major histocompatibility complex (MHC) and related cell surface proteins. Based on the ability of specific monoclonal antibodies to block mitogenesis, SEB stimulation appears to be more dependent on interaction with I-E than with I-A class II MHC molecules. Additionally, anti-L3T4, and possibly other antibodies specific for proteins related to the T cell receptor complex, were inhibitory. When A20 cells were treated with SEB and used to stimulate BALB/c spleen cells which were not otherwise exposed to SEB, the treated A20 cells were capable of stimulating mitogenesis of the BALB/c spleen cells. The data support the hypothesis that SEB stimulation is mediated primarily by interactions with class II MHC proteins and possibly proteins in the T cell receptor complex. We also observed that the presence of SEB in DBA/2 (Mlsa)-stimulated BALB/c (Mlsb) spleen cell cultures enhanced the BALB/c mitogenesis three-fold over the sum of the SEB-plus Mls-stimulated mitogenesis. These results suggest that SEB may be a useful tool for further exploration of the Mls response.

Animals↗
PubMedDate not supplied

An external stimulus that mimics Mls locus responses.

The response to a novel set of T cell mitogens has been analysed and compared to the response to Mls locus differences. These polyclonal T cell activators, staphylococcal enterotoxins A and B, stimulate T cells in a way that requires an antigen-presenting cell bearing class II MHC products and involves the CD4:T cell receptor complex. However, the specificity of MHC recognition by the T cell receptor is lost in this response. Thus, these mitogens produce a response with characteristics similar to that induced by Mls locus differences. These mitogens can be used to analyse the immunobiology of this response, and may help in understanding and identifying the Mls locus product as well.

Animals↗