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J Mester

Publications and source records attributed to J Mester.

At least 91 records · Page 5Linked to original sources
PubMed1988

Resumption of cell cycle in BALB/c-3T3 fibroblasts arrested by polyamine depletion: relation with "competence" gene expression.

Serum deprivation arrests BALB/c-3T3 fibroblasts (clone A31) in G0 phase, where resumption of the cell division cycle can be induced by addition of serum or of specific growth factors in a defined sequence: PDGF (inducer of a state of "competence," characterized by the expression of a family of genes including c-myc), epidermal growth factor EGF and IGF1 (Leof et al., 1982, 1983). When exponentially growing A31 cells are placed for greater than or equal to 2 days in a medium containing the alpha-difluoromethylornithine (alpha DFMO), an irreversible inhibitor of ornithine decarboxylase, they become arrested in G1 phase as a consequence of polyamine depletion (Medrano et al., 1983). In the alpha DFMO-arrested cells, addition of putrescine (60 microM) in a culture medium containing 6% fetal calf serum (FCS), but not in serum-free medium, is sufficient to induce G1 progression and entry into S phase (as determined by 3H-thymidine incorporation). The level of "competence" mRNAs is high in alpha DFMO-arrested cells. After addition of putrescine in FCS-containing medium, these mRNAs continue to be present for at least 3 h. A large proportion of alpha DFMO-arrested cells can be induced to progress to S phase by insulin (1 microM, acting via IGF1 receptor) plus putrescine in a serum-free medium (greater than or equal to 50% of FCS effect). In this case, the levels of "competence" mRNAs become low or undetectable within 3 h, EGF (10 nM) plus insulin had only slightly greater effect than insulin alone on the progression of alpha DFMO-arrested cells. When the alpha DFMO-arrested cells are subsequently incubated during 3 days in a low-serum-containing medium (0.25% FCS), they do not replenish their supply of polyamines, and then continue to express the c-myc gene. The recruitment of the polyamine-depleted, serum-deprived cells into the cell division cycle does not require PDGF and can be induced by addition of EGF and insulin plus putrescine. These data indicate that alpha DFMO arrests majority of the cells at a point situated beyond the PDGF- and EGF-dependent portion of G1 phase. During the subsequent serum deprivation, the alpha DFMO-arrested cells remain "competent" (PDGF-independent), probably as a consequence of their continued expression of c-myc (and possibly other PDGF-inducible genes).

Animals↗
PubMed1988

Involvement of serum factor(s) adsorbed to the dish in the response of cycloheximide-pretreated BP-A31 cells to serum pulses.

The mitogenic effect of serum pulses, observed previously in quiescent BP-A31 cells, is an artifact due to adsorption of (unknown) serum mitogens to the culture dish; the continuous presence of growth factors is necessary for these cells to traverse the G1 phase. When pretreated with cycloheximide (CH) during the last 8-24 h of quiescence, the BP-A31 cells are particularly sensitive to the adsorbed serum mitogens, as well as to low concentrations of fetal calf serum (less than 0.25%) and to basic fibroblast growth factor. In contrast, the mitogenic activity of insulin was not influenced by CH pretreatment. The expression of the "competence genes" c-myc and JE was only transiently elevated in quiescent BP-A31 cells during CH exposure and did not correlate with the enhancement of the cells' sensitivity to mitogens.

Adsorption↗
PubMed1987

Transformation of the 8-9 S molybdate-stabilized estrogen receptor from low-affinity to high-affinity state without dissociation into subunits.

The rate of dissociation of labeled estradiol from [3H] estradiol-8-9 S receptor complexes ([3H]E2-8-9 S ER) molybdate-stabilized was determined in the presence of either an excess of unlabeled hormone ("chase") or of charcoal/dextran suspension ("stripping"). Biphasic dissociation of the hormone was observed in both cases, but the fraction of the fast-dissociating component was dramatically reduced (5% instead of 60%) when stripping was used. As the dissociation patterns were independent of the degree of saturation of the receptor, the results do not favor the possibility of cooperative effects between binding sites in the 8-9 S ER. After pretreatment of cytosol by charcoal at 28 degrees C for 15 min, the dissociation studied by chase displayed only the slowly dissociating component (t1/2 approximately 65 min). This effect was dependent on temperature and influenced by the ligand bound to 8-9 S ER, being pronounced with estradiol (E2) and absent with [3H]4-hydroxytamoxifen. The slow-dissociating component obtained after charcoal treatment was reconverted to fast-dissociating state by adding dithiothreitol or by incubation with cytosol at 20 degrees C. The charcoal treatment did not change the sedimentation coefficient (approximately 9 S) and the Stokes radius (approximately 7 nm) of the [3H]E2-8-9 S ER, and the slow-dissociating form obtained did not bind to DNA-cellulose either in the presence or absence of molybdate ions. Thus there are likely small but functionally significant changes of structure in the 8-9 S ER which remain in a non-DNA-binding form, whereas the rate of estradiol dissociation is modified.

Animals↗
PubMed1987

Serum-induced G0/G1 transition in chemically transformed 3T3 cells. Independence of protein synthesis, stable "memory", and enhancement by cycloheximide pretreatment.

Quiescent, chemically transformed (benzo-alpha-pyrene) BALB/c 3T3 cells (BP A31) enter the cell division cycle when exposed to complete medium containing 10% fetal calf serum (FCS); the number of cells recruited is a function of the duration of serum exposure. The recruitment of cells by short (less than 4 h) serum pulses is not inhibited by simultaneous exposure to cycloheximide (CH), and therefore the initial commitment does not require protein synthesis. The cells enter S phase with a constant delay following the removal of CH, even if CH exposure has been continued for as long as 20 h after the end of the serum pulse. The cell recruitment by serum pulses was inhibited by 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), an inhibitor of cytoplasmic mRNA accumulation. These data suggest that serum exposure produces a stable 'memory' that is necessary and sufficient for the eventual progression through G1 to S phase that occurs when protein synthesis is resumed after the removal of CH; this 'memory' probably consists of mRNA species that are induced by serum and that are stable in the absence of protein synthesis. Unexpectedly, pretreatment of quiescent BP A31 cells with CH (8-24 h) dramatically increased the fraction of the total cell population that is recruited by a serum pulse of fixed duration.

Animals↗
PubMed1987

Activation of int-1 and int-2 mammary oncogenes in hormone-dependent and -independent mammary tumors of GR mice.

Mammary tumors in the GR mouse strain are caused by the expression of an endogenous provirus of the mouse mammary tumor virus (MMTV). The tumors progress from a hormone-dependent growth phase to autonomous, hormone-independent growth. We studied proviral insertion of MMTV at the int-1 and int-2 mammary oncogenes and the transcription of these genes during progression of a series of transplanted mammary tumors. During the hormone-dependent phase, 6 of 15 transplanted tumors were positive for proviral insertion at int-1 or int-2 or both. These tumors were oligoclonal with respect to the fraction of tumor cells with novel int-1 and int-2 restriction fragments and, apparently, consisted of different tumor cells with proviruses integrated at different oncogenes, including genes that are not yet known. In 10 tumors we detected expression of the int genes, indicating that most tumors contain minor populations of cells with int-1 or int-2 activations. On transplantation the tumors remained oligoclonal during the hormone-dependent phase. The hormone-independent variants of the tumors emerged as clonal outgrowths of cells with MMTV proviruses that could be traced back in the hormone-dependent tumors, but not always those of cells that were positive for insertions near int-1 or int-2. The maintenance of oligoclonality during the hormone-dependent phase suggests a growth-controlling effect of different populations of cells on each other. The clonal, hormone-independent tumors that arise later seem to be the result of mutations that are unrelated to int activation.

Animals↗
PubMed1987

Comparison of some haemodynamic parameters determined by invasive methods as well as by the noninvasive radionuclide estimation of the left ventricular ejection fraction in conscious and anaesthetized dogs.

An attempt was made to elaborate an optimal method for noninvasive radionuclide determination of left ventricular ejection fraction in conscious and lightly anaesthetized dogs, as well as to assess the value of this procedure in estimating cardiac functional state. Repeated determinations in the same animals have shown that the 45 degree left anterior oblique position (LAO-45) for imaging is optimal, giving the most uniform values of the calculated left ventricular ejection fraction. Neither the latter, nor the haemodynamic parameters (LVSP, LVEDP and dP/dt) determined by invasive methods were changed by light pentobarbital anaesthesia (20 mg/kg i. v.). Results confirm the validity of noninvasive radionuclide determination of left ventricular ejection fraction for estimation of the functional state and of the pharmacological actions on the latter in both conscious and anaesthetized animals.

Angiocardiography↗
PubMed1986

A protein kinase copurified with chick oviduct progesterone receptor.

A magnesium-dependent protein kinase activity was copurified with both the molybdate-stabilized 8S form of the chick oviduct progesterone receptor (PR) and its B subunit. In each case, purification was performed by hormonal affinity chromatography followed by ion-exchange chromatography. The Km(app) values of the phosphorylation reaction for [gamma-32P]ATP and calf thymus histones were approximately 1.3 X 10(-5) M and approximately 1.6 X 10(-5) M, respectively, and only phosphorylated serine residues were found in protein substrates, including PR B subunit. Physicochemical parameters of the enzyme [pI approximately 5.3, Stokes radius approximately 7.2 nm, sedimentation coefficient (S20,w) approximately 5.6 S, and Mr approximately 200,000] were compared to those of purified forms of PR (B subunit, pI approximately 5.3, Stokes radius approximately 6.1 nm, and Mr approximately 110,000; 8S form, Stokes radius approximately 7.7 nm and Mr approximately 240,000). The results suggest that most of the protein kinase activity copurified with both oligomeric and monomeric forms of PR belongs to an enzyme distinct from currently known receptor components. Its physiological significance remains unknown.

Animals↗
PubMed1986

Detection and localization of ventricular preexcitation in Wolff-Parkinson-White syndrome (comparative study with body surface mapping).

21 patients with Wolff-Parkinson-White (WPW) syndrome were investigated through radionuclide imaging and body surface mapping. Ventricular preexcitation was localized by display of identical phase ventricular regions (phase display). In 79% of the cases radioisotope and body surface mapping methods have identical results for the site of preexcitation. In two patients of the control group (10 patients with Lown-Ganong-Levine (LGL) syndrome), false positive outside-of septum ventricular preexcitation was detected, i.e. an 80% specificity. It is concluded that phase display might be a suitable method in the diagnosis of WPW syndrome. The sensitivity of this method can be improved using several views.

Adult↗
PubMed1985

Transformation of chick oviduct progesterone receptor in vitro: effects of hormone, salt, heat, and adenosine triphosphate.

Effects of salt, heat, and ATP on the rate of sedimentation of chick oviduct progesterone receptor (PR) were examined under various conditions. Cytosol [3H]PR complex (PRc) sedimented as an 8S molecule in 10-35% glycerol or 5-20% sucrose gradients. Incubation of the oviduct cytosol containing [3H]PRc at either 23 or 0 C with 0.3 M KCl or 5-10 mM ATP for 1-4 h resulted in the appearance of a slower migrating form with a sedimentation rate of approximately 4S and a complete and concomitant disappearance of the 8S PR form. This transformation of the receptor was inhibited by molybdate and paralleled an increase in the affinity of the cytosol PRc toward DNA-cellulose, ATP-Sepharose, and isolated nuclei. The 8S to 4S transformation of PR could be achieved with the unliganded receptor. The effect of the hormone on the rate of the transformation of PR was examined. A gradual transformation of the 8S PR occurred with increasing time of incubation at 23 or 0 C with KCl or with 10 mM ATP. The ATP-induced transformation of the 8S form was complete by 2-4 h in both the presence and absence of progesterone. The transformation of PR by salt was complete by 1-2 h of incubation of the cytosol with 0.3 M KCl at 0 C and was slightly accelerated in the presence of the steroid. However, when the cytosol PR was thermally transformed by incubation at 23 C, the appearance of the 4S PR form was significantly accelerated in the presence of the hormone. While the addition of 10 mM ATP to the incubation mixture enhanced the rate of transformation of PRc by heat and salt, lower nucleotide concentrations (0.1-2 mM) inhibited the thermal conversion of the 8S PR (in both its liganded and unliganded forms) to the 4S form. In addition, other nucleoside triphosphates (CTP, GTP, and UTP) were also effective in inducing the 8S to 4S transformation of the unoccupied and the steroid-bound PR. The transforming effects of heat, salt, and ATP were cumulative, and a complete 8S to 4S transformation could be achieved within 5 min when all three were applied simultaneously. We conclude that ATP and other nucleoside triphosphates are effective modifiers of the process of transformation of the chick oviduct PR in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1984

Antibodies against highly purified B-subunit of the chick oviduct progesterone receptor.

A rabbit was immunized with the highly purified B-subunit (110kDa) (20 to 50 micrograms per injection) of the chick oviduct progesterone receptor (PR). Specific antibodies (IgG-RB) were observed 2 weeks after the first booster injection and high antibody titers in the serum were found after the second and third booster injections (with Kdeq of interaction integral of 2 nM). IgG-RB were tested by immunoprecipitation, immunoblotting, density gradient ultracentrifugation and protein A-sepharose assay methods. They recognized not only the B-subunit but also the A-subunit (79K), the nuclear PR, the mero-receptor (proteolytic cleavage product) and the "non-activated" molybdate-stabilized "8S" PR. However, IgG-RB did not interact with the 90K non hormone-binding component of this 8S-PR. IgG-RB did not affect the binding of the hormone to PR, whether incubated with the receptor before or after labelling with tritiated progesterone. They did not cross-react with glucocorticosteroid receptor of the chick oviduct. Weak interaction was observed with estrogen receptor of the chick oviduct and with KC1 activated "4S" forms of the rabbit and human uterus PR.

Animals↗
PubMed1984

Purification by affinity chromatography and immunological characterization of a 110kDa component of the chick oviduct progesterone receptor.

A 110kDa component of the chick oviduct progesterone receptor (PR) has been purified to homogeneity according to electrophoretic criteria and specific activity (assuming one progestagen-binding site/110kDa). The procedure involved affinity chromatography of 0.3 M-KCl-prepared cytosol, followed by DEAE-Sephacel chromatography (elution at 0.2 M-KCl). The final yield was about 12% in terms of binding activity. Properties of the 110kDa component indicate that it is identical with the 'B' subunit described previously [Stokes radius approximately 6.1 nm; sedimentation coefficient, (S20, w) approximately 4S; frictional ratio approximately 1.77]. It reacted with the IgG-G3 polyclonal antibody, but not with BF4 monoclonal antibody raised against the 8S molybdate-stabilized chick oviduct PR and reacting with its 90kDa component. Another progesterone-binding component, corresponding to the 'A' subunit, also previously described, was eluted from the DEAE-Sephacel column at approximately 0.08 M-KCl, and contained a peptide of molecular mass approx. 75-80kDa, which had S20, w approximately 4S in a sucrose gradient. This component was also recognized by IgG-G3, but not by BF4; it was very unstable in terms of hormone-binding activity.

Animals↗
PubMed1984

Effects of calcium on the chick oviduct progesterone receptor.

The chick oviduct cytosol progesterone receptor can be transformed to a small form (Rs = 21A, S20,w:2.9) denoted "mero-receptor" by incubation in the presence of Ca2+ [8]. In the molybdate-free cytosol all the progestin binding components could be completely transformed to mero-form by 1 h treatment with 100 mM Ca2+ at 0 degrees C. If EDTA was secondarily added, the ligand was rapidly released. If molybdate (20 mM) containing cytosol was incubated with Ca2+, no radioactivity was found in the meroposition on the Agarose A 0.5 m column, but the bound steroid sedimented at 2.9 S in sucrose gradients containing Ca2+ (and no molybdate). When 20 nM molybdate was added to cytosol containing receptor activated by 0.3 M KCl, complete mero-transformation by Ca2+ was obtained also by the gel filtration criterion, indicating that molybdate does not inhibit the mero-transforming factor. Ligand-free progesterone receptor could also be completely converted to mero-form by endogenous cytosolic transforming factor and calcium. The transforming factor was completely inactivated, when cytosol was run through Agarose A 0.5 m gel. Mero-transformation was found to be irreversible. The purified progesterone receptor subunit 110 K (B) was partially converted to smaller forms by calcium alone (100 mM, 0 degrees C, 1 h) whereas addition of a small amount of cytosol allowed complete conversion to mero-form.

Animals↗
PubMed1984

Chick oviduct progesterone receptor: structure, immunology, function.

Analysis of the purified chick oviduct progesterone receptor using biochemical and immunological approaches indicates that while the 'activated' receptor ('4S') is a mixture of two progestin-binding polypeptides, 'A' (Mr approximately 79 kDa) and 'B' (Mr approximately 110 kDa), the non-activated receptor ('8S') is a population of complexes containing a hormone-binding polypeptide (A or B, but probably not both) bound to a non-hormone-binding protein (Mr approximately 90 kDa). Two molecules of the 90 kDa protein appear to be present in each '8S' receptor molecule. The 90 kDa protein is also associated with the non-activated forms of receptors of other steroid hormones in the chick. Molybdate stabilizes the non-activated receptors, probably by forming weak coordination bonds with radicals provided by the subunits of the '8S' structure. Activation implies separation of the subunits, without a change in their primary structure, and does not require intervention of any protein other than those present in the '8S' receptor form. The presence of ligand at the binding site accelerates the activation process but, in vitro, is not necessary for it to occur. Unlike the non-activated form, activated receptors bind to the cell nuclei. However, histological studies with anti-progesterone receptor antibodies indicate that in the non-hormone-exposed tissue the (non-activated) receptors could be localized in the nuclei.

Animals↗
PubMed1984

The effect of exposure to charcoal and ion-exchange chromatography on the dissociation rate of estrogen from the nuclear estrogen receptor of hen oviduct.

The method of initiating dissociation of 3H-estradiol from the nuclear estrogen receptor of hen oviduct was found to have a profound effect on the dissociation rate. Likewise, prior exposure to charcoal or partial purification by ion-exchange chromatography had an effect on the dissociation rate. When the reaction was initiated by isotopic dilution with the addition of 1 microM unlabeled estradiol, dissociation of the complexes was rapid (t 1/2 approximately 3 min). When the reaction was initiated by the addition of charcoal to adsorb free steroid, the dissociation of the complexes proceeded slowly (t 1/2 approximately 30 min). Partial purification of the receptors by DEAE-Sephacel chromatography or 15 min exposure to charcoal at 0 degree C prior to initiation of the dissociation reaction by isotopic dilution produced a form of the receptor that exhibited an intermediate dissociation rate (t 1/2 approximately 10 min). The partially purified receptor that exhibited an intermediate dissociation rate was reconverted to the rapidly dissociating form in a reconstitution experiment. These data raise the possibility of a nuclear substance that regulates the rates of estrogen dissociation.

Animals↗