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A Zilberstein

Publications and source records attributed to A Zilberstein.

At least 55 records · Page 3Linked to original sources
PubMed1990

Insulin-tumour interrelationship in EL4 lymphoma or thymoma-bearing mice. I. Alloxan-diabetic or non-diabetic mice.

A study has been carried out in which a comparison was made between EL4 lymphoma (assumed to be an insulin-producing secreting tumour) and thymoma (an insulin-dependent tumour). Tumour development and incidence, 3H-thymidine incorporation and insulin content in tumours, the host's food intake, blood insulin, glucose and cholesterol were determined in non-diabetic and alloxan-diabetic mice. Whereas no significant differences were observed between the diabetic and non-diabetic EL4 tumour-bearing mice, the diabetic, thymoma tumour-bearing mice showed reduced tumour growth and lower tumour incidence as compared with their non-diabetic counterparts. Insulin administration to diabetic tumour bearing mice, enhanced 3H-thymidine incorporation in the thymoma tumour cells only, and the insulin content of the EL4 tumours was found to be higher than that of the thymoma tumours. Rapid diabetes remission was observed in the diabetic, EL4 tumour-bearing mice as compared with the thymoma tumour-bearing mice.

Animals↗
PubMed1990

Tyrosine kinase activity is essential for the association of phospholipase C-gamma with the epidermal growth factor receptor.

Epidermal growth factor (EGF) treatment of NIH 3T3 cells transfected with wild-type EGF receptor induced tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma). The EGF receptor and PLC-gamma were found to be physically associated such that antibodies directed against PLC-gamma or the EGF receptor coimmunoprecipitated both proteins. The association between PLC-gamma and wild-type EGF receptor was dependent on the concentration of EGF, but EGF did not enhance the association between PLC-gamma and a kinase-negative mutant of the EGF receptor. Oligomerization of the EGF receptor was not sufficient to induce association of the EGF receptor with PLC-gamma, since the kinase-negative mutant receptor underwent normal dimerization in response to EGF yet did not associate with PLC-gamma. The form of PLC-gamma associated with the EGF receptor appeared to be primarily the non-tyrosine-phosphorylated form. It is concluded that the kinase activity of the EGF receptor is essential for association of PLC-gamma with the EGF receptor, possibly by stimulating receptor autophosphorylation.

Animals↗
PubMed1990

Characterization and cDNA cloning of phospholipase C-gamma, a major substrate for heparin-binding growth factor 1 (acidic fibroblast growth factor)-activated tyrosine kinase.

Heparin-binding growth factors (HBGFs) bind to high-affinity cell surface receptors which possess intrinsic tyrosine kinase activity. A Mr 150,000 protein phosphorylated on tyrosine in response to class 1 HBGF (HBGF-1) was purified and partially sequenced. On the basis of this sequence, cDNA clones were isolated from a human endothelial cell library and identified as encoding phospholipase C-gamma. Phosphorylation of phospholipase C-gamma in intact cells treated with HBGF-1 was directly demonstrated by using antiphospholipase C-gamma antibodies. Thus, HBGF-1 joins epidermal growth factor and platelet-derived growth factor, whose receptor activation leads to tyrosine phosphorylation and probable activation of phospholipase C-gamma.

Amino Acid Sequence↗
PubMed1990

Identification of autophosphorylation sites of HER2/neu.

HER2 or c-erbB-2 is a putative growth factor receptor with sequence homology to the epidermal growth factor receptor. It is the human homologue of the rat protooncogene neu and may have an important role in human malignancies such as breast and ovarian cancers. Like other growth factor receptors, HER2 has intrinsic protein tyrosine kinase activity and undergoes autophosphorylation. Recently, we have demonstrated that, similar to the epidermal growth factor receptor, all autophosphorylation sites of HER2 are localized in the carboxyl terminus of this protein. In the present study, immunopurified HER2 was allowed to autophosphorylate, and tryptic phosphopeptides were generated. After purification of these phosphopeptides by high performance liquid chromatography, microsequencing was performed. Utilizing this approach, two autophosphorylation sites were unequivocally identified at Y1023 and Y1248. The sequences of two other tyrosine phosphorylated tryptic peptides were determined, but the exact site of autophosphorylation could not be determined because multiple tyrosines were located on each peptide. However, each of these peptides contains tyrosines that correspond to major autophosphorylation sites of the epidermal growth factor receptor, suggesting that, in addition to Y1023 and Y1248, Y1139 and Y1222 also serve as autophosphorylation sites of HER2.

Amino Acid Sequence↗
PubMed1989

Tyrphostins inhibit epidermal growth factor (EGF)-receptor tyrosine kinase activity in living cells and EGF-stimulated cell proliferation.

Synthetic compounds called tyrphostins were examined for their effects on cells which are mitogenically responsive to epidermal growth factor (EGF). We studied in detail the effects of two tyrphostins on EGF binding, tyrosine phosphorylation in intact cells, EGF-receptor internalization, and mitogenesis. These compounds inhibited EGF-stimulated [3H]thymidine incorporation in a specific manner and the degree of selectivity varied. Both compounds inhibited EGF-stimulated receptor autophosphorylation and tyrosine phosphorylation of endogenous substrates in intact cells at doses that correlated with the IC50 for [3H] thymidine incorporation. These results are consistent with the notion that tyrosine phosphorylation is a crucial signal in transduction of the mitogenic message delivered by EGF. The compound RG50864 demonstrated specificity at inhibiting EGF-stimulated cell growth compared with stimulation with either platelet-derived growth factor or serum. For both compounds RG50864 and RG50810, long term exposure (16 h) of cells to tyrphostins was required for optimal inhibition because of the instability and slow action of these compounds. Tyrphostins did not alter cell surface display of EGF-receptor, EGF binding or EGF-induced internalization, degradation, and down-regulation of EGF receptors. These novel synthetic inhibitors, specific for EGF-receptor kinase, offer a new method to inhibit EGF-stimulated cell proliferation which may be useful in treating specific pathological conditions involving cellular proliferation, including different types of cancers.

Animals↗
PubMed1989

EGF induces tyrosine phosphorylation of phospholipase C-II: a potential mechanism for EGF receptor signaling.

Binding of EGF to cells expressing human EGF receptor stimulated rapid tyrosine phosphorylation of phospholipase C-II (PLC-II), as revealed by immunoblotting analysis with phosphotyrosine-specific antibodies. Tyrosine phosphorylation of PLC-II was stimulated by low physiological concentrations of EGF (1 nM), was quantitative, and was already maximal after a 30 sec incubation with 50 nM EGF at 37 degrees C. Interestingly, antibodies specific for PLC-II were able to coimmunoprecipitate the EGF receptor and antibodies against EGF receptor also coimmunoprecipitated PLC-II. According to this analysis, approximately 1% of EGF receptor molecules were associated with PLC-II molecules. The protein tyrosine kinase inhibitor tyrphostin RG50864, which blocks EGF-dependent cell proliferation, blocked EGF-induced tyrosine phosphorylation of PLC-II, its association with EGF receptor, and EGF-induced Ca2+ release. Hence, EGF-induced tyrosine phosphorylation of PLC-II may be a regulatory event linking the tyrosine kinase activity of EGF receptor to the PIP2 hydrolysis signaling pathway.

Antineoplastic Agents↗
PubMed1989

Phospholipase C-gamma, a substrate for PDGF receptor kinase, is not phosphorylated on tyrosine during the mitogenic response to CSF-1.

Quiescent mouse NIH3T3 cells expressing a transduced human c-fms gene encoding the receptor for colony stimulating factor-1 (CSF-1) were stimulated with mitogenic concentrations of platelet-derived growth factor (PDGF) or CSF-1. Immunoprecipitated phospholipase C-gamma (PLC-gamma) was phosphorylated on tyrosine and calcium was mobilized following treatment of intact cells with PDGF. In contrast, only trace amounts of phosphotyrosine were incorporated into PLC-gamma and no intracellular calcium signal was detected after CSF-1 stimulation. Similarly, CSF-1 treatment did not stimulate phosphorylation of PLC-gamma on tyrosine in a CSF-1-dependent. SV40-immortalized mouse macrophage cell line that expresses high levels of the CSF-1 receptor. In fibroblasts, antiserum to PLC-gamma co-precipitated a fraction of the tyrosine phosphorylated form of the PDGF receptor (PDGF-R) after ligand stimulation, implying that phosphorylated PDGF-R and PLC-gamma were associated in a stable complex. Pre-treatment of cells with orthovanadate also led to tyrosine phosphorylation of PLC-gamma which was significantly enhanced by PDGF, but not by CSF-1. Thus, although the PDGF and CSF-1 receptors are structurally related and appear to be derived from a single ancestor gene, only PDGF-induced mitogenesis in fibroblasts correlated with tyrosine phosphorylation of PLC-gamma.

Animals↗
PubMed1988

Growth inhibition of human breast carcinoma and leukemia/lymphoma cell lines by recombinant interferon-beta 2.

Recombinant human interferon-beta 2 was produced in Escherichia coli by direct expression of cDNA encoding the mature protein sequence. At concentrations that stimulate DNA synthesis and growth in B-cell hybridomas and plasmacytomas, the cytokine was found to exert a strong inhibition on the growth of a number of carcinoma and leukemia/lymphoma cell lines. This antigrowth effect was observed in clonogenic assays and by measurements of cell number and thymidine incorporation in growing cultures. The effect was blocked by antibodies to a synthetic peptide from the N terminus of the molecule. Normal diploid fibroblasts were inhibited at concentrations higher than those needed for breast carcinoma cells.

Breast Neoplasms↗
PubMed1988

Interferon-beta 2 (BSF-2) mRNA is expressed in human monocytes.

We previously have reported the presence of interferon-beta 2 (IFN-beta 2) mRNA in PHA-stimulated human peripheral blood leukocytes (PBL), as well as in nonstimulated cells, although at a lower level. The IFN-beta 2 cloned from a leukocyte library appeared to be similar to that of the fibroblast IFN-beta 2 gene first described in fibroblasts. To assess the nature of the cell population in which the synthesis of IFN-beta 2 takes place, PBL were fractionated in adherent and nonadherent cells. The antiviral activity of the culture supernatants of adherent cells was characterized as the IFN-beta type by neutralization with polyclonal antibodies raised against purified fibroblast IFN-beta 2. IFN-beta 2 mRNA was observed in enriched monocyte populations and accumulated very rapidly, peaking at 2.5 h. RNA extracted from these cultures encoded in a reticulocyte lysate a protein immunoprecipitated by the anti-IFN-beta 2 antiserum. In addition, IFN-beta 2 secreted in monocyte supernatants also was immunoprecipitated by the specific antiserum and was able to compete with the fibroblast IFN-beta 2, suggesting a strong similarity between the fibroblast and monocyte proteins.

Cell Adhesion↗
PubMed1987

A quantitative approach to sequence comparisons of nitrogenase MoFe protein alpha- and beta-subunits including the newly sequenced nifK gene from Klebsiella pneumoniae.

The nucleotide sequence was determined for part of the Klebsiella pneumoniae nif gene cluster containing the 3' end of the nifD gene and the entire length of the nifK gene (encoding the alpha- and beta-subunits of the nitrogenase MoFe protein respectively), as well as the putative start of the nifY gene, a gene of as yet unknown function. A broad-based comparison of a number of MoFe protein alpha-subunits, beta-subunits and alpha-versus beta-subunits was carried out by the use of a computer program that simultaneously aligns three protein sequences according to the mutation data matrix of Dayhoff. A new kind of quantitative statistical measure of the similarity between the aligned sequences was obtained by calculating and plotting standardized similarity scores for overlapping segments along the aligned proteins. This calculation determines if a test sequence is similar to the consensus sequence of two other proteins that are known to be related to each other. The different beta-subunits compared were found to be significantly similar along most of their sequence, with the exception of two relatively short regions centred around residues 225 and 300, which contain insertions/deletions. The overall pattern of similarity between different alpha-subunits exhibits resemblance to the overall pattern of similarity between different beta-subunits, including regions of low similarity centred around residues 225 and 340. Comparison of alpha-subunits with beta-subunits showed that a region of significant similarity between the two types of subunits was located approximately between residues 120 and 180 in both subunits, but other parts of the proteins were only marginally similar. These results provide insights into likely tertiary structural features of the MoFe protein subunits.

Amino Acid Sequence↗
PubMed1987

Nitrogenase MoFe protein subunits from Klebsiella pneumoniae expressed in foreign hosts. Characteristics and interactions.

The expression of selected nitrogen fixation (nif) genes from Klebsiella pneumoniae in foreign hosts provides an approach to determine the pathway, minimal genetic requirements, and host dependence of nitrogenase assembly. In this study, we investigated the assembly of the alpha 2 beta 2 MoFe protein, responsible for substrate binding and reduction, by introducing nifD and nifK (encoding respectively, the alpha and beta subunits) into Escherichia coli and the yeast Saccharomyces cerevisiae. In E. coli, both genes were expressed from the nifHDKY operon; in yeast, the genes, separately fused to the yeast ADH1 promoter, were introduced on two different plasmids. Denaturing immunoblot analyses demonstrated the presence of significant amounts of NifD and NifK in both hosts. In E. coli, the level or perhaps modification of NifD depended on the growth medium of the bacteria. Nondenaturing, anaerobic immunoblot assays revealed in E. coli, nif-specific antigens of lower electrophoretic mobility than Kp1, which may represent assembly intermediates. In yeast, no putative assembled products were evident, and the predominant antigens corresponded to the monomeric forms of the polypeptides. These results indicate that, unlike NifH, the Fe protein subunit (Berman, J., Gershoni, J. M., and Zamir, A. (1985) J. Biol. Chem. 260, 5240-5243), NifD and NifK are insufficient for the assembly of an electrophoretically Kp1-like structure. Homodimerization of nifK and probably of nifD primary gene products does not appear to occur spontaneously and hence is unlikely to represent the initial step in the assembly. The difference between the two hosts suggests that the cellular environment or mode of expression could affect the interaction between the two subunits.

Escherichia coli↗
PubMed1986

Structure and expression of cDNA and genes for human interferon-beta-2, a distinct species inducible by growth-stimulatory cytokines.

Induced human fibroblasts produce several mRNAs encoding interferon (IFN) activity. We previously cloned cDNA for a 1.3-kb RNA designated IFN-beta 2 and distinct from the 0.9-kb IFN-beta 1 mRNA. In vitro transcription--translation mapping of the full-length IFN-beta 2 cDNA sequence, shows that it encodes a 23.7-kd protein of 212 amino acids. This cDNA, fused to the SV40 early gene promoter, was transfected and amplified in Chinese hamster ovary cells and clones were obtained which constitutively produce human interferon activity. Two IFN-beta 2 genomic clones were isolated and their expression in hamster and mouse cells also produces biologically active rIFN-beta 2. Specific immunoassays show that IFN-beta 2 secreted by DNA-transformed rodent cells is a processed 21-kd protein, whose activity is cross-neutralized by antibodies to human IFN-beta 1 but not to IFN-alpha or gamma. The immunoassay also demonstrates the induction of IFN-beta 2 secretion by fibroblasts in response to growth-regulatory cytokines, such as interleukin-1 and tumor necrosis factor. The function of this IFN-beta 2 as an autoregulatory inhibitor of cell growth is discussed.

Animals↗
PubMed1986

Human chromosome 7 carries the beta 2 interferon gene.

A cDNA clone (pAE20-4) corresponding to the 1.3-kilobase human beta 2 interferon mRNA was used as a probe in blot-hybridization experiments of DNA from a panel of human-rodent somatic cell hybrids containing overlapping subsets of human chromosomes. The DNA hybridization experiments showed that the human beta 2 interferon gene is located on human chromosome 7. This assignment is consistent with previous experimental data in which the expression of the translationally active 1.3-kilobase beta 2 interferon mRNA was assayed in various somatic cell hybrids. Blot-hybridization experiments using DNA from different human cell strains and cell lines reveal distinct EcoRI restriction fragment length polymorphisms of the human beta 2 interferon gene.

Animals↗
PubMed1986

Efficient constitutive production of human IFN-gamma in Chinese hamster ovary cells.

A human genomic DNA segment of 5.6 kb containing the entire gene for immune interferon-gamma was fused through its 5'-untranslated region to the corresponding region of the simian virus 40 (SV40) T-antigen gene. The SV40 early promoter used contained a modified transcriptional enhancer element with a 93-bp repeat. Supercoiled plasmid DNA was used to transfect Chinese hamster ovary (CHO) cells, the selectable marker being a SV40-dihydrofolate gene construct. Constitutive expression of the IFN-gamma gene in primary transformants was high, especially if a Harvey murine sarcoma virus long terminal repeat (LTR) was present in addition to the SV40 promoter. After gene amplification by methotrexate selection, CHO-gamma cell lines were obtained that produce 1.5-2 million units of IFN-gamma per million cells and per day (200,000 molecules per cell per minute). Metabolic labeling showed that over 90% of the protein secreted by such cells is human IFN-gamma. A one-step immuno-affinity chromatography on monoclonal antibodies yielded pure IFN-gamma with 1-2 X 10(8) units/mg protein. Like IFN-gamma from human white blood cells, the IFN-gamma from CHO-gamma cells is a mixture of two glycoproteins of 26,000 and 20,000 daltons with traces of the unglycosylated 17,000-dalton polypeptide. Large-scale cultures in 1% serum routinely yield over 600,000 units of human IFN-gamma/ml culture per day.

Animals↗
PubMed1985

Expression of a nitrogen-fixation gene encoding a nitrogenase subunit in yeast.

Biological nitrogen fixation is catalyzed by nitrogenase, an enzyme complex exclusive to prokaryotes. We used the yeast Saccharomyces cerevisiae to study the synthesis, and subsequently the assembly, of nitrogenase components in a eukaryote. Here, the Klebsiella pneumoniae nifH gene, encoding the subunit of the Fe protein (Kp2) component of nitrogenase, was expressed in S. cerevisiae from the yeast ADHI promoter. The nifH gene product, detected in yeast by immunoblot analysis with anti-Kp2 antibodies, exhibited the same electrophoretic mobility in SDS-polyacrylamide gels as that of the Kp2 subunit synthesized in K. pneumoniae. Estimates of Kp2 antigen and assays of beta-galactosidase activity specified by nifH'-'lacZ fusions showed that the level of nifH product was similar in anaerobically and aerobically grown yeast, but varied with different transforming plasmids and in various haploid and diploid yeast strains. A cistron located downstream to nifH in a transcript resembling the polycistronic mRNA of the nifHDKY operon in K. pneumoniae is not translated in yeast.

Gene Expression Regulation↗
PubMed1983

Promoter mutations that allow nifA-independent expression of the nitrogen fixation nifHDKY operon.

The nifHDKY operon of Klebsiella pneumoniae encodes for structural polypeptides of nitrogenase and requires the nifA gene product for transcription. Mutations that allow transcription of the nifHDKY operon in absence of the nifA gene product were characterized in plasmids containing the regulatory region of nifHDKY and nifH fused in phase to lacZ. beta-Galactosidase activity served as a measure for nifH expression. Most mutations were located in the nif regulatory region and included insertion sequence 2 (IS2) insertions, a sequence duplication, and a base substitution. In Escherichia coli, beta-galactosidase activity expressed from the mutant plasmids in the absence of nifA was 6-30% of the nifA-activated, parental level. Expression from most mutant plasmids was further increased by nifA. In K. pneumoniae, IS2-containing plasmids expressed low levels of beta-galactosidase and responded poorly, if at all, to activation by nifA, whereas expression from other mutant types was similar to that observed in E. coli. Nucleotide sequence analysis of two mutants indicated that sequences within 41 base pairs upstream to the nifH coding sequence were involved in nif-specific regulation. The results suggest that an inverted repeat element in this region, which could theoretically form a cruciform structure in the DNA, is involved in the transcriptional control of the nifHDKY operon.

Bacterial Proteins↗