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R Prywes

Publications and source records attributed to R Prywes.

At least 55 records · Page 3Linked to original sources
PubMed1989

An AP1-binding site in the c-fos gene can mediate induction by epidermal growth factor and 12-O-tetradecanoyl phorbol-13-acetate.

We have demonstrated that two sequence elements in the c-fos promoter can mediate the response of the gene to epidermal growth factor and the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). The first is the previously described serum response element. The second is a sequence element highly homologous to the consensus binding site for the HeLa cell transcription factor AP1. Although recent reports have shown that fos protein binds to AP1-binding sites through an interaction with AP1 protein and have raised the speculation that fos protein may negatively regulate expression of the c-fos gene via this interaction, we found no role for the AP1 consensus homology in the downregulation of c-fos expression following induction by epidermal growth factor and TPA.

Binding Sites↗
PubMed1988

Phosphorylation of serum response factor, a factor that binds to the serum response element of the c-FOS enhancer.

Serum and growth factor regulation of c-FOS protooncogene transcription is mediated by the serum response element. A factor, serum response factor, binding to this element has already been identified. We demonstrate that serum response factor is phosphorylated in vivo on serine residues and that phosphatase treatment of this factor in vitro abolishes its DNA-binding activity. These results show phosphorylation of serum response factor to be required for its DNA-binding activity. The importance of serum response factor phosphorylation for the regulation of c-FOS expression is discussed.

Animals↗
PubMed1988

Release of a phorbol ester-induced mitogenic block by mutation at Thr-654 of the epidermal growth factor receptor.

The tumor promoter phorbol ester (TPA) modulates the binding affinity and the mitogenic capacity of the epidermal growth factor (EGF) receptor. Moreover, TPA-induced kinase C phosphorylation occurs mainly on Thr-654 of the EGF receptor, suggesting that the phosphorylation state of this residue regulates ligand-binding affinity and kinase activity of the EGF receptor. To examine the role of this residue, we prepared a Tyr-654 EGF receptor cDNA construct by in vitro site-directed mutagenesis. Like the wild-type receptor, the mutant receptor exhibited typical high- and low-affinity binding sites when expressed on the surface of NIH 3T3 cells. Moreover, TPA regulated the affinity of both wild-type and mutant receptors and stimulated receptor phosphorylation of serine and threonine residues other than Thr-654. The addition of TPA to NIH 3T3 cells expressing a wild-type human EGF receptor blocked the mitogenic capacity of EGF. However, this inhibition did not occur in cells expressing the Tyr-654 EGF receptor mutant. In the latter cells, EGF was able to stimulate DNA synthesis even in the presence of inhibitory concentrations of TPA. While phosphorylation of sites other than Thr-654 may regulate ligand-binding affinity, the phosphorylation of Thr-654 by kinase C appears to provide a negative control mechanism for EGF-induced mitogenesis in mouse NIH 3T3 fibroblasts.

Animals↗
PubMed1987

Purification of the c-fos enhancer-binding protein.

We have purified the c-fos enhancer-binding protein from HeLa cell nuclear extracts. The key purification steps involved chromatography on a nonspecific DNA affinity column, from which binding activity and other protein were eluted at low salt concentrations, followed by chromatography on a specific oligonucleotide affinity column, from which the enhancer binding activity was specifically eluted at high salt concentrations. The purified protein had a strong affinity for the c-fos enhancer dyad symmetry sequence, with an equilibrium dissociation constant of 3.3 x 10(-11) M. This affinity was at least 50,000-fold stronger than that found for nonspecific DNA sequences.

Chromatography↗
PubMed1987

c-fos sequence necessary for basal expression and induction by epidermal growth factor, 12-O-tetradecanoyl phorbol-13-acetate and the calcium ionophore.

We have investigated the sequence requirements for induction of the human c-fos gene by epidermal growth factor (EGF), 12-O-tetradecanoyl-13-acetate (TPA), and the calcium ionophore A23187 by transfecting c-fos promoter mutants into HeLa and A431 cells. Induction by both EGF and TPA in HeLa cells required the presence of the c-fos enhancer located at -317 to -298 relative to the mRNA cap site. A23187, however, did not induce expression of the transfected gene, even though it strongly induced expression of the endogenous gene, suggesting that it has different requirements for induction than do EGF and TPA. We have also investigated the role of promoter sequences downstream of the enhancer in general expression and induction of c-fos. A sequence between -97 and -76, which includes an 8-base-pair perfect direct repeat, was needed for efficient general expression but not for induction of the gene. A factor in nuclear extracts that bound specifically to this sequence was detected by a gel mobility shift assay. A 7-base-pair sequence, located between -63 and -57 relative to the mRNA cap site and previously shown to be important for general expression of mouse c-fos, was also important for general expression of the human gene. In addition, this element was important for inducibility by EGF and TPA, since induction was significantly reduced when internal deletion mutants that retained the enhancer but lacked the -63 to -57 sequence element were analyzed in transfecting assays.

Calcimycin↗
PubMed1986

Inducible binding of a factor to the c-fos enhancer.

We have identified a factor in nuclear extracts that binds to the c-fos enhancer. Treatment of A431 cells with epidermal growth factor results in a rapid increase in the level of transcription and a concomitant increase in binding of the factor to the enhancer. Surprisingly, as transcription decreases rapidly, the enhancer-binding activity remains elevated. In addition, although HeLa cells exhibit no detectable transcription of the c-fos gene, they contain significant amounts of binding activity, comparable to those in induced A431 cells. These results suggest that regulation of c-fos transcription involves more than simply an increased level of a factor capable of binding the enhancer. Finally, transcription of c-fos in A431 cells is markedly induced by the tumor promoter TPA and the calcium ionophore A23187, yet neither induced an increased level of the enhancer-binding activity. These agents thus appear to activate c-fos transcription via a mechanism distinct from that used by epidermal growth factors.

Calcium↗
PubMed1986

Reconstitution of human epidermal growth factor receptors and its deletion mutants in cultured hamster cells.

DNA sequences encoding the human epidermal growth factor (EGF) receptor and various EGF-receptor deletion mutants were transfected into chinese hamster ovary (CHO) cells devoid of endogenous EGF receptors. A functional human EGF-receptor is expressed on the surface of heterologous CHO cells with the following properties: it exhibits typical high affinity (10%; Kd = 3 X 10(-10) M) and low affinity (90%; Kd = 3 X 10(-9) M) binding sites for 125I-EGF; it is expressed as a polypeptide of 170,000 molecular weight with intrinsic protein tyrosine kinase activity. EGF stimulates the kinase activity leading to self-phosphorylation and to phosphorylation of exogenous substrate; 125I-EGF is rapidly internalized into the CHO cells by receptor mediated endocytosis and; EGF stimulates DNA synthesis in the cells expressing the human EGF-receptor. Deletion of 63 amino acids from the C-terminal end of EGF-receptor, which removes two autophosphorylation sites, abolishes the high affinity state of the receptor. Nevertheless, this receptor mutant is able to undergo endocytosis and to respond mitogenically to EGF to a similar extent as the "wild type" receptor. Further deletions from the cytoplasmic domain give rise to low affinity endocytosis-defective receptor mutants. Finally, deletion of the transmembrane domain of the human receptor yields an EGF-receptor ligand binding domain which is secreted from the cells.

Animals↗
PubMed1986

Mutations in the cytoplasmic domain of EGF receptor affect EGF binding and receptor internalization.

Binding of epidermal growth factor (EGF) to its receptor results in a cascade of events that culminate in cell division. The receptor is present on the cell surface in two forms of high and low affinity binding for EGF. EGF binding activates the receptor's intracellular tyrosine kinase activity and subsequently causes the receptor to be rapidly internalized into the cell via clathrin-coated pits. We have cloned the EGF receptor cDNA into a retroviral expression vector and made mutations in vitro to investigate the function of different receptor domains. Deletion of cytoplasmic sequences abolishes high but not low affinity sites as well as impairing the ability of the protein to internalize into cells. Thus, cytoplasmic sequences must be involved in the regulation of high affinity sites and are required for EGF-induced receptor internalization. A four amino acid insertion mutation at residue 708 abolishes the protein-tyrosine kinase activity of the immunoprecipitated receptor. However, this receptor mutant exhibits both the high and low affinity states, internalizes efficiently and is able to cause cells to undergo DNA synthesis in response to EGF. Another four amino acid insertion mutation (residue 888) abolishes protein-tyrosine kinase activity, high affinity binding, internalization and mitogenic responsiveness. Finally, a chimaeric receptor composed of the extracellular EGF binding domain and the cytoplasmic v-abl kinase region transforms Rat-I cells. This chimaeric receptor possesses intrinsic protein tyrosine kinase activity which cannot be regulated by EGF. Moreover, EGF fails to induce the internalization of the chimaeric receptor.

Animals↗
PubMed1986

Large deletions in the cytoplasmic kinase domain of the epidermal growth factor receptor do not affect its laternal mobility.

The lateral diffusion coefficients of various epidermal growth factor (EGF) receptor mutants with increasing deletions in their carboxy-terminal cytoplasmic domain were compared. A full size cDNA construct of human EGF receptor and different deletion constructs were expressed in monkey COS cells. The EGF receptor mutants expressed on the cell surface of the COS cells were labeled with rhodamine-EGF, and the lateral diffusion coefficients of the labeled receptors were determined by the fluorescence photo-bleaching recovery method. The lateral mobilities of three deletion mutants, including a mutant that has only nine amino acids in the cytoplasmic domain, are all similar (D approximately equal to 1.5 X 10(-10) cm2/s) to the lateral mobility of the "wild-type" receptor, which possess 542 cytoplasmic domain of EGF receptor, including its intrinsic protein kinase activity and phosphorylation state, are not required for the restriction of its lateral mobility.

Animals↗
PubMed1985

The minimum transforming region of v-abl is the segment encoding protein-tyrosine kinase.

Only 1.2 kilobases (kb) at the 5' end of the 3.9-kb v-abl sequence in Abelson murine leukemia virus is required for fibroblast transformation. A precise delineation of this minimum transforming region was made by using small 5' or 3' deletions. Insertions of four amino acids, generated by putting synthetic DNA linkers into various restriction enzyme cleavage sites, abolished transforming activity, indicating that much of the internal sequence of the minimum transforming region plays a critical role in the transformation process. This 5' 1.2 kb of v-abl encodes protein-tyrosine kinase activity when expressed in Escherichia coli. Each of the mutations which caused a loss of transformation activity also resulted in a loss of protein-tyrosine kinase activity when expressed in E. coli. The minimum transforming region of v-abl contains amino acid homology to other protein-tyrosine kinase oncogenes, and a comparison with these oncogenes is presented.

Abelson murine leukemia virus↗
PubMed1985

Protein stabilization explains the gag requirement for transformation of lymphoid cells by Abelson murine leukemia virus.

The single protein encoded by Abelson murine leukemia virus is a fusion of sequence from the retroviral gag genes with the v-abl sequence. Deletion of most of the gag region from the transforming protein results in a virus capable of transforming fibroblasts but no longer capable of transforming lymphoid cells. Smaller deletions in gag reveal that p15 gag sequences are responsible for this effect, whereas deletion of p12 sequences had no effect on lymphoid transformation. In transformed fibroblasts, p15-deleted and normal proteins had similar activities and subcellular localization. When the p15-deleted genome was introduced into previously transformed lymphoid lines, its protein product exhibited a marked instability. The tyrosine-specific autophosphorylation activity per cell was less than 1/20th that of the nondeleted protein. Although pulse-Ia-beling showed that the p15-deleted protein was synthesized efficiently, immunoblotting demonstrated that its steady-state level was less than 1/10th that of the nondeleted Abelson protein. The specific instability of the p15-deleted protein in lymphoid cells explains the requirement of these sequences for lymphoid but not fibroblast transformation.

Abelson murine leukemia virus↗
PubMed1983

Sequences of the A-MuLV protein needed for fibroblast and lymphoid cell transformation.

The role of various segments (gag or v-abl) of the Abelson murine leukemia virus (A-MuLV) genome in both lymphoid cell and fibroblast transformation was examined by deletion of areas from cloned, plasmid DNA representations of the genome. The deleted plasmids were tested by transfection into fibroblasts and by infection of bone marrow cells using virus stocks derived from the fibroblast transfectants. Deletion of gag coding sequence from the A-MuLV protein did not affect fibroblast transforming activity but abolished lymphoid transforming activity. The gag- A-MuLV genomes were very unstable in transformed fibroblasts leading to large secondary deletions in v-abl sequences. The gag- A-MuLV proteins also had lower autophosphorylation than their gag+ counterparts although cells transformed by gag- virus had a normal elevation of protein-linked phosphotyrosine. Systematic deletion of v-abl sequences showed that only 45,000 to the 130,000 molecular weight of v-abl sequence in the A-MuLV protein is needed for fibroblast transformation and, at most, slightly more is needed for lymphoid cell transformation.

Abelson murine leukemia virus↗
PubMed1983

Structure and function of the Abelson murine leukemia virus transforming gene.

The oncogene (v-abl) of A-MuLV is a fragment of a normal mouse gene. Only 1.2 kb on the 5'-end of the v-abl is sufficient to encode a tyrosine-specific protein kinase whose activity is important in transformation. This A-MuLV protein kinase can be active in E. coli and it phosphorylates bacterial proteins. The lack of discrimination in its selection for substrates is puzzling. Because of the demonstration of such promiscuity, the identification of physiologically-relevant phosphorylation events must rely on evidence of functional modification rather than physical modification of proteins.

Abelson murine leukemia virus↗
PubMed1980

Prevalence of hepatitis B markers (HBsAg and HBsAb) in women screened at time of delivery.

Two thousand women were screened at the time of delivery for two hepatitis B markers, surface antigen (HBsAg) and antibody (HBsAb), in order to determine prevalence rates, define high-risk groups and estimate the cost-efficiency of routine screening. It was found that 2.2% of the women were HBsAg positive and 32% of a 20% subsample were HBsAb positive. Jewish women of North African origin had the highest rates (HBsAg, 3.8%; HBsAb, 47%) and those of European origin had the lowest (1.2 and 14%, respectively). Bedouin women had intermediate rates (HBsAg, 1.1%; HBsAb, 28%). The issue of screening pregnant women for HBsAg is discussed in light of these results.

Adolescent↗
PubMed1977

The Jerusalem perinatal study: the first decade 1964--73.

This paper summarizes the main findings concerning Jewish births in the record-linked Jerusalem Perinatal Study. In the decade 1964--73 there were 63,638 births in which the birth weight was at least 1,000 g. The late fetal mortality rate was 9.1/1,000, and the neonatal and infant death rates were 10.1 and 15.5/1,000, respectively. The demographic characteristics of births changed over the decade, with a decrease in the proportion of high birth orders, of mothers with little education, of immigrants from Asia and North Africa, and of marriages within the same group of origin. Fertility fell, especially at the extremes of reproductive life. Illegitimacy was 1.2%. Year-by-year variations in mortality are discussed and the relationship of mortality to maternal age and education, birth order, social class, group of origin and birth weight are described. Frequencies of specific congenital malformations, infant and child admissions to hospital and various obstetric complications are also reported. Changes in obstetric interventions over the decade included an increasing proportion of induced labors, cesarean sections, forceps and vacuum deliveries, and interventions in the third stage of labor. The paper briefly indicates ways in which the data bank of the Jerusalem Perinatal Study is being exploited for a wide variety of health studies.

Adolescent↗
PubMed1977

Prevalence of elevated blood pressure in a village community with a high incidence of toxemia of pregnancy.

Blood pressure measurements were recorded in 95% of the men and 97% of the women aged 30 to 59 years in a small Israeli village populated by Jews of Kurdish origin and in which a high incidence of toxemia of pregnancy was previously observed. The prevalence of hypertension in the men, as well as in the women, was significantly higher in all age groups studied than in a sample of the general population. These findings support the hypothesis that toxemia of pregnancy is an expression of a preexisting hypertensive state rather than an etiological factor in the development of later hypertension in women.

Adult↗
PubMed1977

Maternal, perinatal and infant health in Bedouin and Jews in southern Israel.

A study has been made of 3,745 Bedouin and 9,422 Jewish babies born in 1972-73 to residents of the Beersheba district of southern Israel (the Negev). Newborn infants weighing less than 1 kg were excluded. Thirty-seven percent of the Bedouin babies were born at home; their mothers tended to be older and of higher parity than those choosing to deliver in hospital. Less than 6% of Bedouin mothers had been to school, compared with 90% of the Jews; 30% were aged under 20 or over 34 years, compared with 18% of the Jews, and 23% were having their seventh or later baby, compared with 12% of the Jews. Mean birth weight of babies born in hospital was about 200 g lower in Bedouin than in Jews, and 11.4% of Bedouin and 6.5% of Jewish infants weighed less than 2.5 kg. There was little variation in complications of labor between the 1,959 Bedouin and 8,877 Jewish women delivered in Beersheba's Soroka Medical Center. The cesarean section rate was 1.8% in Bedouin and 4.3% in Jews, while in 0.3% of Bedouin and 1.4% of Jews labor was induced. Monozygous twinning rates were similar in the two ethnic groups (4.8 and 4.5 sets/1,000 deliveries, respectively) but dizygous twinning was twice as common among the Bedouin as among the Jews (13.0 vs 6.0 sets/ 1,000). Male births accounted for 0.526 and 0.512 of the total in Bedouin and Jews, respectively. Perinatal mortality rates for hospital births were 31.1 and 18.3/1,000 in Bedouin and Jews, respectively. Infant deaths among Bedouin (31.0/1,000) were underreported; the rate was 16.8/1,000 for Jewish infants. Although rates of all specific causes of death were higher in Bedouin than in Jews, patterns of mortality in subgroups based on birth weight, sex, twinning and maternal age were quite similar in the two ethnic groups. There were six reported deaths from tetanus among Bedouin babies. For the cohort of babies born in 1972, admissions to the Soroka Medical Center pediatric wards were recorded in 366 (195.5/1,000) Bedouin and 787 (174.3/1,000) Jewish babies younger than the age of one year. Bedouin admission rates were higher than those of Jews for gastroenteritis (119.1 and 64.5/1,000 respectively), infectious and parasitic diseases (29.4 and 21.9), malnutrition (25.6 and 8.0) and external causes (10.1 and 4.4). Admission rates for bronchitis and pneumonia were, however, lower among Bedouin than Jews in the first six months of life.

Birth Order↗