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F H Sarkar

Publications and source records attributed to F H Sarkar.

At least 127 records · Page 7Linked to original sources
PubMed1984

Interferon receptor interaction. Internalization of interferon alpha 2 and modulation of its receptor on human cells.

Studies reported earlier [ Joshi et al. (1982) J. Biol. Chem. 257, 13884-13887] have indicated that human interferon-alpha 2 (HuIFN-alpha 2) binds to a specific macromolecular receptor on human cells as identified by cross-linking with bifunctional cross-linking reagents and analysis by polyacrylamide gel electrophoresis. We have carried out experiments to investigate the fate of the interferon-receptor complex on the cell surface under conditions which lead to cellular response. As analyzed by cross-linking and gel electrophoresis, the interferon-receptor complex, formed on incubation with 125I-IFN-alpha 2 at 4 degrees C, persisted at the cell surface for several hours at 4 degrees C; however, if the cells were switched to 37 degrees C, there was a rapid decline in the complex, apparently due to a loss of the interferon receptors from the cell surface. This was associated with an internalization of the 125I-interferon as indicated by the fact that, on incubation at 37 degrees C, an appreciable fraction of the cell-associated interferon (approximately equal to 50%) became resistant to trypsin digestion, or dissociation on incubation in growth medium or low-pH buffer. A large fraction of the trypsin-resistant (internalized) 125I-labeled material migrated as intact interferon in polyacrylamide gels, and it was immunoprecipitated by anti-(HuIFN-alpha)antibodies but not by anti-(HuIFN-beta)antibodies. The bulk of the internalized 125I-interferon was recovered in a particulate fraction and, on cross-linking with disuccinimidyl suberate, a 150000-Mr complex could be detected. The results suggest that interferon may be internalized as a complex with the receptor, which may account for the loss of the interferon-receptors on the cell surface. This modulation of the IFN-alpha/beta receptors was induced by HuIFN-alpha and HuIFN-beta but not by HuIFN-gamma. The recovery of the IFN-alpha/beta receptors, lost upon incubation with HuIFN-alpha, took several hours and required protein synthesis. The significance of the results is discussed.

Burkitt Lymphoma↗
PubMed1984

Receptors for human gamma interferon: binding and crosslinking of 125I-labeled recombinant human gamma interferon to receptors on WISH cells.

Purified recombinant human gamma interferon (HuIFN-gamma), labeled with 125I (125I-HuIFN-gamma), was used to study receptors for HuIFN-gamma on human WISH cells. 125I-HuIFN-gamma was bound to WISH cells, and this binding was displaced by unlabeled HuIFN-gamma but not by unlabeled recombinant HuIFN-alpha 2 or [Ser17]HuIFN-beta (HuIFN-beta with serine substituted for cysteine at position 17), indicating the presence of specific binding sites for HuIFN-gamma. The cell-bound 125I-HuIFN-gamma was crosslinked with disuccinimidyl suberate or ethylene glycol bis(succinimidyl succinate), which yielded a complex of Mr approximately 105,000 +/- 5000 as analyzed by NaDodSO4/PAGE. The formation of this complex was prevented by preincubation of cells with unlabeled HuIFN-gamma but not with HuIFN-alpha 2 or [Ser17]HuIFN-beta, indicating that HuIFN-gamma binds to a specific receptor molecule and that HuIFN-alpha 2 or HuIFN-beta do not interact with this receptor. Experiments were carried out with 125I-labeled recombinant [Ser17]HuIFN-beta (125I-[Ser17]HuIFN-beta) to verify this conclusion. Binding and crosslinking of 125I-[Ser17]HuIFN-beta to human WISH cells and Daudi cells yielded a complex of Mr approximately 150,000 similar to that obtained with 125I-HuIFN-alpha 2 as described earlier. The formation of this Mr 150,000 complex with 125I-[Ser17]HuIFN-beta was displaced by unlabeled [Ser17]HuIFN-beta and by HuIFN-alpha 2 but not by HuIFN-gamma, indicating that [Ser17]HuIFN-beta binds to the same receptor as does HuIFN-alpha 2, identified earlier, and that HuIFN-gamma does not compete with 125I-[Ser17]HuIFN-beta for this receptor. We conclude that HuIFN-gamma interacts with specific receptors that are distinctly different from the receptors recognized by HuIFN-alpha and HuIFN-beta.

Amnion↗
PubMed1984

Receptors for human alpha and beta interferon but not for gamma interferon are specified by human chromosome 21.

We examined the proposed role of human chromosome 21 in determining the cellular sensitivity to human alpha, beta, and gamma interferons (HuIFN-alpha, -beta, and -gamma) and the expression of the receptors for the HuIFNs with the use of mouse-human hybrid cells containing human chromosome 21. Hybrid cells (WA17) containing three copies of human chromosome 21 showed specific displaceable binding of 125I-labeled HuIFN-alpha 2 (125I-HuIFN-alpha 2), which was not observed with mouse parent (A9) cells. Crosslinking of 125I-HuIFN-alpha 2 bound to WA17 cells with disuccinimidyl suberate yielded a complex of Mr approximately equal to 150,000 similar to the 125I-HuIFN-alpha 2-receptor complex obtained with human cells as described earlier. Such a complex was not obtained with mouse parent (A9) cells or with hybrid cells containing certain other human chromosomes but not chromosome 21. Mice inoculated with mouse-human hybrid cells containing human chromosome 21 produce antibodies that block the antiviral action of HuIFN-alpha and -beta on human cells. Such antibodies could immunoprecipitate the 125I-HuIFN-alpha 2-receptor complex obtained from human cells but not free 125I-HuIFN-alpha 2, indicating that these antibodies were directed against the receptor. WA17 hybrid cells were highly sensitive to the antiviral action of HuIFN-alpha 2, -alpha (Le) and -beta but were completely insensitive to HuIFN-gamma. Furthermore, 125I-HuIFN-gamma showed specific binding to human WISH cells but not to WA17 hybrid cells or A9 mouse cells. The results indicate that the receptors for HuIFN-alpha and -beta but not for HuIFN-gamma are specified by human chromosome 21. Hybrid cells containing one, two, or three copies of human chromosome 21 were found to be increasingly sensitive to HuIFN-alpha 2, indicating that a chromosome 21-specified component (possibly the HuIFN-alpha receptor) may be a limiting factor in the cellular sensitivity to HuIFN-alpha.

Animals↗
PubMed1984

Receptors for human alpha interferon: are gangliosides involved?

Interferon (IFN) action on cells must begin with an interaction with cellular receptors. Binding and cross-linking experiments reported earlier with purified 125I-labeled recombinant human (Hu) IFN-alpha 2 have revealed that IFN-alpha 2 binds to a specific macromolecular receptor on human cells (Joshi et al., J. Biol. Chem. 257, 13884-13887, 1982). Based on indirect evidence such as neutralization of the antiviral action of IFN preparations by gangliosides and binding of IFNs to gangliosides coupled to solid supports, it has been suggested by various investigators that gangliosides may be a part of the IFN-alpha/beta receptors. Experiments presented here indicate that gangliosides could block the antiviral activity of HuIFN-beta, but not of HuIFN-alpha, although both species of IFN bound strongly to gangliosides coupled to poly-L-lysine-agarose. Furthermore, gangliosides did not inhibit the binding of 125I-labeled HuIFN-alpha 2 to specific receptors on human cells, and this binding was competed out by unlabeled HuIFN-alpha 2 and HuIFN-alpha(LE) which were preincubated with gangliosides. However, the capacity of HuIFN-beta to compete for the receptors was abolished by preincubation with gangliosides. These results were confirmed by cross-linking experiments to identify the IFN-receptor complex by gel electrophoresis. The results indicate that at least in the case of HuIFN-alpha species, the ganglioside binding is apparently not at the active site of the IFN molecules required for interaction with the receptors on the cell surface.

Binding Sites↗
PubMed1983

Characteristics of spontaneously produced and of virus-induced human LuKII cell interferons.

Several human lymphoblastoid cell lines produced significant levels of interferon (IFN) activity in the absence of any IFN induces. We have partially purified spontaneous IFN (SpIFN) from LuKII cells and compared it with the IFN activity produced by the same cells following Sendai virus induction. Virus-induced LuKII IFN reached maximum titers of 1000-6000 reference units/ml at 12 to 15 houts post-induction and produced a heterogeneous electrophoretic profile with major (18,500 dalton) and minor (23,500 dalton) species. Spontaneous LuKII IFN was produced very slowly over several days and reached maximum titers of 100-1000 reference units/ml. Crude SpIFN (25-100 ref. units/ml) was purified to 1-6 X 10(5) ref. units/ml with a 53-80 percent overall recovery, and it consistently migrated as a homogeneous 20,000 dalton band upon SDS gel electrophoresis. Although spontaneously produced and virus-induced lymphoblastoid IFNs differed in their ability to be neutralized by anti-mouse IFN antiserum (18), both types of IFN were only 1 percent cross-reactive on heterologous mouse L cells. We conclude that IFN definitely can be produced by many human lymphoblastoid cell lines in the absence of inducers, and that LuKII SpIFN has different characteristics than the IFNs produced by the same cell line following viral induction. Since it is now known that there are many IFN-alpha genes (3, 10), it seems likely that these cell lines may provide a system for studying the selective expression of one or more of these genes.

Animals↗
PubMed1982

Inhibition of interferon secretion in human fibroblast by vinblastine.

Vinblastine treatment of superinduced human fibroblast (FS-4) cultures strongly inhibited interferon (IFN) secretion and resulted in intracellular accumulation of IFN activity. The cell-associated IFN migrated slower than secreted IFN in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating a larger size. The results suggest the presence of a possible precursor form of mature HuIFN-beta with biological activity.

Cell Line↗
PubMed1982

Pharmacokinetic comparison of leukocyte and Escherichia coli-derived human interferon type alpha.

Human alpha interferons derived from leukocytes (HuIFN-alpha-Le) and from an Escherichia coli clone (HuIFN-alpha 1) were compared for their relative abilities to distribute systemically in mice following intramuscular or intraperitoneal injections, and for their rate of clearance from the blood. HuIFN alpha 1, which is about 30-50 tines more active on bovine cells than on human cells, was as efficient as HuIFN-alpha-Le at entering the bloodstream following injections by either route. However, HuIFN-alpha-Le was still present in the circulation at about 30% of peak serum IFN levels at 8 h post-inoculation and was present at about 3-5% at 24 h, whereas HuIFN-alpha 1 was present at only about 3% of peak IFN serum levels at 8 h and was undetectable (less than 1%) at 24 h. These data indicate the importance of evaluating the pharmacokinetic properties of each of the molecular forms of interferons obtained from genetically enginered organisms, as each molecular species of interferon may have importantly distinct pharmacologic properties.

Animals↗
PubMed1980

Comparisons of several biological and physicochemical properties of human leukocyte interferons produced my human leukocytes and by E. coli.

Human interferon (IFN) prepared from virus-induced human leukocyte suspensions (leukocyte-derived interferon) was compared to the IFN extracted from Escherichia coli harboring a human interferon-alpha cDNA hybrid plasmid (Hif-SN35-AH-L6). E coli-derived IFN was 20 to 50 times more active than leukocyte-derived IFN on heterologous bovine, feline, murine and guinea pig cells, relative to the activity on human cells. After partial purification by affinity chromatography on an anti-human lymphoblastoid IFN antibody column, the IFN was analyzed by SDS-polyacrylamide gel electrophoresis. While leukocyte-derived IFN gave a heterogeneous pattern with major peaks of activity of 24000 and 19000 daltons, E. coli-derived IFN gave a heterogeneous peak of activity at about 17-18000 daltons. The leading edge of leukocyte-derived IFN in SDS-polyacrylamide gels was significantly more active on bovine cells than on human cells and coincided in mobility with E. coli-derived IFN, which was also much more active on bone than on human cells. After reduction with mercaptoethanol in SDS, the E. coli-derived IFN lost no activity, whereas the leukocyte-derived IFN lost about 90% of its activity. After reduction, E. coli-derived IFN migrated in SDS-polyacrylamide gels as a single peak at 24000 daltons, as did the residual activity of reduced leukocyte-derived interferon. Out data suggest that the interferon produced by the E. coli harboring the clone Hif-SN35-AH-L6 is analogous in size and cross-species activity to one of the molecular species of leukocyte-derived interferon.

Chromatography, Affinity↗