Biomedical subjects

G Ragnotti

Publications and source records attributed to G Ragnotti.

At least 37 records · Page 2Linked to original sources

PubMed1990

Liver genotoxic activity of an epoxide derivative of the hepatocarcinogenic beta-blocker DL-ZAMI 1305.

A single administration of the sex-dependent hepatocarcinogenic beta-blocker DL-1-(2-nitro-3-methyl-phenoxy)-3-tert-butylamino-propan-2-ol (DL-ZAMI 1305) induces dose-dependent liver DNA damage, as evaluated by alkaline sucrose gradient analysis, in female but not in male Fisher 344 rats. A single administration of the direct mutagenic epoxide-derivative of DL-ZAMI 1305 3-methyl-2-nitro-1-(2,3-epoxypropoxy)-benzene induces dose-dependent DNA damage in the liver of animals of both sexes. However, also in this case, the genotoxic activity of the compound appears to be significantly higher in female than in male rats. A DNA-damaging capacity similar in the two sexes is instead exerted by DL-ZAMI 1305-unrelated direct mutagens, like N-methyl-N-nitrosourea (MNU) and methyl-methanesulfonate (MMS). The data confirm the sex-dependent susceptibility of rat liver to the genotoxic activity of DL-ZAMI 1305-related molecules, also in the absence of an absolute requirement for a metabolic activation of the compound.

Animals↗

PubMed1990

Basic fibroblast growth factor in neuronal cultures of human fetal brain.

The presence of basic fibroblast growth factor (bFGF) was investigated in neuronal cells derived from 12 and 18 week-old human fetal brain cultures. To this purpose, the ability of bFGF to stimulate plasminogen activator (PA) production in fetal bovine aortic endothelial GM 7373 cells was used as an assay for this molecule in neuronal cell extracts. The identity of the PA-stimulating activity of neuronal cell extract with bFGF was confirmed by its high affinity for heparin and by its cross-reactivity with polyclonal antibodies to human placental bFGF. These antibodies recognized a Mr 18,000 cell-associated protein both in Western blot and in immuno-precipitation experiments. All the neurons showed bFGF immunoreactivity, as demonstrated by immunocytochemical staining, while nonneuronal cells were unstained. The data demonstrate for the first time that cultured human fetal brain neurons contain and synthesize bFGF.

Animals↗

PubMed1990

Characterization of a Mr 20,000 basic fibroblast growth factor-like protein secreted by normal and transformed fetal bovine aortic endothelial cells.

A mitogenic and plasminogen activator (PA)-inducing activity for endothelial cells has been identified in serum-free culture medium of normal AG 7680 and transformed tumorigenic GM 7373 fetal bovine aortic endothelial (FBAE) cells. The activity binds to heparin-Sepharose and it is quenched by polyclonal anti-human placental basic fibroblast growth factor (bFGF) antibodies. In the serum-free conditioned medium of FBAE cells, the anti-bFGF antiserum recognizes an immunorective Mr 20,000 molecule which co-purifies with the mitogenic and PA-inducing activity on a heparin-Sepharose column. The partially purified Mr 20,000 bFGF-like molecule competes with the typical Mr 18,000 125I-bFGF form for the binding to high-affinity bFGF receptors in intact GM 7373 cells. Immunoprecipitation of biosynthetically labeled GM 7373 cells with anti-bFGF antiserum confirms the presence of a Mr 20,000 bFGF-like molecule in the conditioned medium of these cells and identifies the typical Mr 16,000 and Mr 18,000 bFGF forms and two high-molecular-weight immunoreactive Mr 22,000 and Mr 25,000 bFGF forms in their cell extract. Immunoreactive Mr 20,000 bFGF is detectable also in the conditioned medium of transformed nontumorigenic FBAE GM 7372 cells and of adult bovine aortic endothelial cells, but not in the culture medium of nonendothelial cell types, including rat and mouse fibroblasts, human hepatoma, and human endometrial adenocarcinoma cells. The results indicate that bovine endothelial cells secrete a Mr 20,000 bFGF-like molecule which shares several biological, biochemical, and immunological characteristics with the typical cell-associated Mr 18,000 bFGF.

Animals↗

PubMed1990

Chemical structure and genotoxic activity of the hepatocarcinogenic beta-blocker DL-ZAMI 1305.

A single administration of the sex-dependent hepatocarcinogenic beta-blocker DL-1-(2-nitro-3-methyl-phenoxy)3-tert-butylamino-propan-2-ol (DL-ZAMI 1305) induces liver DNA damage, as evaluated by alkaline sucrose gradient analysis, in female but not in male Fisher 344 rats. The shift of the methyl-group from the 3-position of the aromatic ring of DL-ZAMI 1305 to the 4- and 5-position causes the progressive decrease of the genotoxic activity of the molecule. No effect on the DNA-damaging capacity of DL-ZAMI 1305 is instead observed when the NO2 group of the aromatic ring is reduced to an NH2 group. Bis-demethylation of the side chain of DL-ZAMI 1305, or its modification to an alpha-hydroxicarboxylic acid or glycol, increases the DNA-damaging capacity of the molecule, which becomes genotoxic also for the liver of the male rat. Thus, modifications of the chemical structure of the aromatic ring or of the side chain of DL-ZAMI 1305 affect the genotoxic activity of the molecule both in male and female rat liver. The modifications of the side chain of DL-ZAMI 1305 investigated in the present work are likely to occur in vivo as a consequence of the hepatic metabolism of the molecule. Sex-dependent differences in the activity of the liver drug-metabolizing system might explain the different genotoxic and oncogenic activity of DL-ZAMI 1305 in the two sexes.

Adrenergic beta-Antagonists↗

PubMed1990

Synthesis of urokinase-type plasminogen activator and of type-1 plasminogen activator inhibitor in neuronal cultures of human fetal brain: stimulation by phorbol ester.

Human neuronal brain cultures established from 12- and 14-week-old fetuses synthesize and secrete urokinase-type plasminogen activator (uPA) and limited amounts of tissue-type plasminogen activator (tPA). These cells also produce and secrete the endothelial cell-type PA inhibitor (PAI-1), which forms sodium dodecyl sulfate-stable tPA/PAI-1 complexes in the culture medium. Immunocytochemistry shows a predominant localization of uPA, tPA, and PAI-1 in neuronal cells, with only a very weak positivity detectable in the few glial cells present in these cultures. The protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulates the synthesis of both uPA and PAI-1, resulting in a final increase in the plasmin-generating capacity of neuronal cell cultures. No significant effect is observed, however, when cells are treated with the TPA analogue 4 alpha-phorbol 12,13-didecanoate, which is inactive as a PKC inducer, or with the neurotrophic polypeptide basic fibroblast growth factor. These data represent the first characterization of the plasmin-generating system in human fetal brain neurons and suggest a role for PKC in the modulation of uPA and PAI-1 synthesis.

Brain↗

PubMed1990

Basic fibroblast growth factor in ovulatory cycle and postmenopausal human endometrium.

Biopsies of human endometrium were studied for the presence of basic fibroblast growth factor (bFGF). An immunoreactive Mr 18,000 bFGF-like molecule was detected at high levels both in ovulatory cycle and postmenopausal endometrium. This molecule was identified as bFGF on the basis of its molecular weight, its affinity for heparin, its capacity to induce plasminogen activator production and cell proliferation in endothelial GM 7373 cells, and its cross-reactivity with various anti-bFGF antibodies. The levels of endometrial bFGF do not change during the menstrual cycle but they increase significantly after menopause, as evaluated both by biological and immunological assays. Lower levels of an acidic FGF-like activity were also evident in ovulatory cycle endometrium but, at variance with bFGF, no significant increase of this activity was observed in postmenopausal endometrium. These data represent the first characterization of a polypeptide growth factor present in human endometrium.

Adult↗

PubMed1989

Characterization of a Mr 25,000 basic fibroblast growth factor form in adult, regenerating, and fetal rat liver.

A heparin-binding Mr 25,000 immunoreactive bFGF-like protein (ir-bFGF) is recognized in adult rat liver extract by affinity-purified polyclonal anti-human placental bFGF antibodies. Hepatic levels of this protein increase 4-fold in regenerating rat liver during the first 48 h after partial hepatectomy. Also, they appear to be higher in embryonic than in newborn or in adult rat liver. Mr 25,000 ir-bFGF from regenerating rat liver, partially purified by heparin-affinity chromatography, induces plasminogen activator activity and cell proliferation in transformed fetal bovine aortic endothelial GM 7373 cells and competes with Mr 18,000 [125I]bFGF for the binding to high affinity bFGF receptors. The data indicate the presence in rat liver of a high molecular weight form of bFGF whose expression is modulated during embryonic development and liver regeneration.

Aging↗

PubMed1989

Induction of plasminogen activator activity by phorbol ester in transformed fetal bovine aortic endothelial cells. Possible independence from protein kinase C.

12-O-tetradecanoyl phorbol 13-acetate (TPA) and 1,2-dioctanoyl-sn-glycerol (diC8) activate protein kinase C (PKC) in transformed fetal bovine aortic endothelial GM 7373 cells. Both molecules cause a similar increase in membrane-associated PKC activity and in the phosphorylation of a PKC-specific endogenous 87-kDa substrate in intact cells. Even though both TPA and diC8 exert a mitogenic activity in GM 7373 cells, only TPA induces also an increase in cell-associated plasminogen activator (PA) activity. Down-regulation of PKC which follows TPA-pretreatment completely abolishes the mitogenic activity of diC8 and the mitogenic and PA-inducing activity of TPA. However, both the PKC inhibitor H-7 and the down-regulation of PKC which follows a prolonged stimulation with diC8 do not abolish the PA-inducing activity of TPA. The PA-inducing activity of TPA is instead inhibited in cultures incubated in the presence of 1 mM EGTA or in a calcium-free medium. The data indicate that TPA and diC8 induce a different pattern of cellular activation in GM 7373 cells and that the PA-inducing activity of TPA might not be mediated by PKC.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1989

Basic fibroblast growth factor is released from endothelial extracellular matrix in a biologically active form.

Basic fibroblast growth factor (bFGF) binds to heparin-like molecules present in the extracellular matrix (ECM) of transformed fetal bovine aortic endothelial GM 7373 cells. Binding of bFGF to ECM can be competed by heparin or heparan sulfate, and ECM-bound bFGF can be released by treating the cells with heparinase or heparatinase. After binding to ECM, bFGF is slowly released into the medium in a biologically active form, as shown by its capacity to induce an increase of cell-associated plasminogen activator activity and cell proliferation. The increase is prevented upon removal of ECM-bound bFGF by a neutral 2 M NaCl wash. Soluble heparin and heparan sulfate reduce the amount of ECM-bound bFGF released into the medium, possibly competing with ECM polysaccharides for heparinase-like enzymes produced by endothelial cells, suggesting that these enzymes are involved in the mobilization of ECM-bound bFGF.

Animals↗

PubMed1989

Basic fibroblast growth factor: production, mitogenic response, and post-receptor signal transduction in cultured normal and transformed fetal bovine aortic endothelial cells.

Normal FBAE AG 7680 cells and chemically transformed FBAE GM 7373 cells were compared for their capacity to produce and to respond to bFGF. Normal FBAE cells showed higher levels of bFGF protein and of poly(A)+ bFGF mRNA than transformed GM 7373 cells, indicating that chemical transformation in FBAE cells is paralleled by a decrease of bFGF gene expression. Basic FGF induced cell proliferation in both normal and transformed FBAE cells. However, bFGF appeared to be much more potent in transformed than in normal cells. No differences in bFGF membrane receptors were observed between normal and transformed FBAE cells in terms of apparent molecular weight, number per cell, dissociation constant, and kinetic of downregulation. In respect to normal cells, however, transformed GM 7373 cells showed higher basal levels of PKC activity. This kinase is activated by bFGF and is involved in mediating the mitogenic activity of bFGF, as shown by the capacity of the PKC inhibitor H-7 to abolish the mitogenic activity of bFGF both in normal and transformed FBAE cells. Like bFGF, the PKC activators DAG and TPA exerted a stronger mitogenic activity in transformed than in normal FBAE cells. Thus, the different susceptibility of normal and transformed FBAE cells to bFGF appears to depend on differences in the post-receptor signal transduction mediated by PKC rather than on differences in bFGF receptors. The results indicate that chemical transformation causes significant modifications of bFGF physiology in FBAE cells. The relevance of these modifications to the genesis of tumors of vascular origin deserves further investigation.

Animals↗

PubMed1989

The mitogenic signaling pathway but not the plasminogen activator-inducing pathway of basic fibroblast growth factor is mediated through protein kinase C in fetal bovine aortic endothelial cells.

Basic fibroblast growth factor (bFGF) induces cell proliferation and plasminogen activator (PA) activity in transformed fetal bovine aortic endothelial (FBAE) GM 7373 cells. A similar response is observed after treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In these cells, bFGF and TPA cause activation of protein kinase C (PKC), as demonstrated by the induction of the phosphorylation of an 87-kD PKC substrate in intact cells and by the increase in membrane-associated PKC activity. Activation of PKC by bFGF or TPA is inhibited in cells made PKC-deficient by pretreatment with high concentrations of TPA. The mitogenic activity of bFGF or of TPA is completely inhibited in PKC-deficient cells or in naive cells treated with the PKC inhibitor H-7. However, these cells proliferate in response to serum, epidermal growth factor, and dibutyryl cyclic AMP. Similar results are obtained in normal FBAE AG 7680 cells. These data indicate that activation of PKC is responsible for the mitogenic activity of bFGF in FBAE cells. On the contrary, the PA-inducing activity of bFGF is unaffected by down-regulation of PKC or by treatment with the PKC inhibitor H-7 in both transformed GM 7373 and normal AG 7680 cells. bFGF induces a rapid 45Ca influx in naive and in PKC-deprived GM 7373 cells. In these cells, addition of EGTA to the incubation medium prevents both the 45Ca influx and the increase in PA activity induced by bFGF, without affecting its mitogenic activity. Even though the involvement of PKC in the increase of cell-associated PA activity induced by bFGF can not be completely dismissed, the present results suggest a role of calcium entry in the modulation of the PA-inducing activity of bFGF.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1988

Modulation of plasminogen activator activity in human endometrial adenocarcinoma cells by basic fibroblast growth factor and transforming growth factor beta.

Human endometrial adenocarcinoma HEC-1-A and HEC-1-B cells produce and secrete the urokinase-type plasminogen activator and trace amounts of the tissue-type plasminogen activator. The two clones, which originate from the same tumor, possess high affinity binding sites for basic fibroblast growth factor (bFGF) and they respond to the addition of human recombinant bFGF with an increase of the synthesis and secretion of urokinase-type and tissue-type plasminogen activators and with an increase in cell proliferation. Transforming growth factor beta, (TGF beta), when added alone or 6 h before bFGF, induces an increase of plasminogen activator synthesis in both cell lines and stimulates the production of the endothelial cell-type plasminogen activator inhibitor in HEC-1-A cells, but not in HEC-1-B cells. Moreover, TGF beta inhibits basal proliferation of both cell lines and suppresses the mitogenic activity of bFGF. We have previously demonstrated that HEC-1-A and HEC-1-B cells produce significant amounts of bFGF. On the basis of the well-established coordinate modulation of solid tumor growth and plasminogen activator production, our data suggest that bFGF may contribute, in an autocrine fashion, to the development of endometrial carcinomas. Moreover, endometrial tumor cells appear also to be a target for TGF beta. Our results demonstrate also that significant qualitative and quantitative differences in the response to growth factors exist in clones derived from the same tumor, and support the view that the properties of in vivo tumors cannot be extrapolated from results obtained with a single isolate of tumor cells.

Adenocarcinoma↗

PubMed1988

Purification of basic fibroblast growth factor from rat brain: identification of a Mr 22,000 immunoreactive form.

A 18,000-dalton protein which stimulates plasminogen activator (PA) activity in endothelial GM 7373 cells has been purified from rat brain by using heparin affinity chromatography and ion-exchange chromatography. The purified molecule stimulates PA activity in a dose-dependent manner between 1 and 30 ng/ml. It also stimulates proliferation of GM 7373 cells and DNA synthesis in NIH 3T3 cells in a similar concentration range. The molecule has been identified as a bFGF-like molecule on the basis of its biological activity, its affinity for heparin-Sepharose, and its cross-reactivity with anti-human bFGF antibodies. In the final preparation of the rat brain bFGF, trace amounts (less than 5%) of a contaminant were detectable. This contaminant has a molecular weight of 22,000 and cross reacts with several anti-human placental bFGF antibodies. On the basis of its affinity for heparin-Sepharose and its immunological characteristics, this protein appears to be an high molecular weight form of bFGF.

Animals↗

PubMed1988

High molecular weight immunoreactive basic fibroblast growth factor-like proteins in rat pituitary and brain.

Four proteins immunologically related to basic fibroblast growth factor (bFGF) have been detected by Western blot analysis in the extract from rat anterior pituitary. Their apparent molecular weights are 29, 27, 18, and 14 kDa, respectively. A similar immunoreactive pattern has been observed in the rat tumor pituitary GH3 cell line. In the extracts from rat neurohypophysis, hypothalamus, hippocampus, striatum, olfactory tubercles, cerebellum, and cortex only the 29 kDa form is detectable in a significant amount.

Animals↗

PubMed1987

Critical role of gonadal hormones on the genotoxic activity of the hepatocarcinogen DL-ZAMI 1305.

The DNA damaging capacity of the sex-dependent hepatocarcinogen beta-blocker DL-1-(2-nitro-3-methyl-phenoxy)-3-tert-butylamino-propan-2-ol (DL-ZAMI 1305) was evaluated in different sex hormonal conditions. A single injection of DL-ZAMI 1305 causes DNA damage in the liver of the female but not the male Wistar rat. When the hormonal environment of the female rat is converted to 'male type' by ovariectomy and 1 week of treatment with testosterone, DNA damage by DL-ZAMI 1305 is completely abolished. On the contrary, in male rats orchiectomy coupled to 17 beta-estradiol administration increases the amount of hepatic DNA damage by DL-ZAMI 1305 to values similar to those observed in intact female rats. DL-ZAMI 1305 induces hepatic DNA damage also when administered to female Sprague-Dawley and Fisher 344 female rats. It is uneffective instead on the male rats of these strains. Moreover, in the female Fisher 344 rat phenobarbital pretreatment reduces the DNA damaging capacity of DL-ZAMI 1305. Our data indicate that the genotoxic activity of DL-ZAMI 1305 depends on the sex-hormonal status of the animal and that this is possibly due to a modulation of the microsomal mixed function oxidase system by sex hormones.

Animals↗

PubMed1986

Further studies on the tumor-initiating activity of the beta-blocker DL-ZAMI 1305.

The purpose of this study was to evaluate the initiating activity of the hepatocarcinogen beta-blocker DL-1-(2-nitro-3-methyl-phenoxy)-3-tert-butylamino-propan-2-ol (DL-ZAMI 1305) by the initiation-promotion protocol of Pereira. Female Wistar rats were given a single dose 150 mg/kg of body weight of DL-ZAMI 1305 by gavage 24 hours before or 24 hours after partial hepatectomy. One week later rats were given phenobarbital (0.05%) in the diet for a period of 7 weeks. DL-ZAMI 1305-treatment resulted in the appearance of gamma-glutamyltranspeptidase foci and of other preneoplastic lesions in all animals. Preneoplastic lesions were also present in a fraction of DL-ZAMI 1305-treated animals not subjected to partial hepatectomy, whether given or not phenobarbital. Results obtained in a separate experiment demonstrated that DL-ZAMI 1305-treatment inhibits cell proliferation and induces DNA damage in the regenerating rat liver. The results of this study clearly demonstrated that the beta-blocker DL-ZAMI 1305 is an initiating carcinogen for the liver of female Wistar rats.

Adrenergic beta-Antagonists↗

PubMed1985

Influence on cell-cell communication (dye-transfer) of the oncogenic beta-blocker DL-ZAMI 1305: possible relation to tumor promotion.

The effect of the oncogenic beta-blocker DL-1-(2-nitro-3-methyl-phenoxy)-3-tert-butyl-amino-propan-2-ol (DL-ZAMI 1305) on intercellular communication between cultured cells was studied. Intercellular communication of Chinese hamster V79 cells was measured by the dye-transfer method in which the spread of intracellularly microinjected fluorescent probe, Lucifer Yellow CH, through gap-junctions was used as an index of intercellular communication. When V79 cells are cultured with non-toxic doses (1-60 micrograms/ml) of DL-ZAMI 1305, a significant inhibition of dye-transfer is observed after 4 h. The inhibition is dose-related and greater than 90% inhibition is seen at the dose of 50 micrograms/ml. When DL-ZAMI 1305 is added at 0 and 24 h of experiment, its inhibitory effect is maintained for at least 48 h at high doses (50-60 micrograms/ml), whereas for lower doses of DL-ZAMI 1305, some recovery is seen after 24 h incubation. These results are suggestive of a possible tumor-promoting activity of DL-ZAMI 1305; in vivo studies on this carcinogen are in progress.

Adrenergic beta-Antagonists↗

PubMed1985

Inhibition of DNA and RNA synthesis in rat liver nuclei by oncogenic and non-oncogenic beta-blockers.

The beta-blocker DL-1-(2-nitro-3-methylphenoxy)-3-tert-butylaminopropan-2-ol (ZAMI 1305), hepatocarcinogenic to the female rat, and the non-oncogenic beta-blockers DL-1-(2-nitro-5-methylphenoxy)-3-tert-butylaminopropan-2-ol (ZAMI 1327), DL-propranolol, and DL-atenolol were studied for their capacity to interfere with hepatic DNA and RNA synthesis. These moieties inhibit DNA and RNA synthesis, in a dose-dependent fashion, when added in vitro to nuclei isolated from the liver of male or female rats. The inhibition is due to a decrease of the initial rate of synthesis and of the total amount of labeled precursor incorporated into the growing chains. When administered in vivo both the oncogenic ZAMI 1305 and its non-oncogenic isomer ZAMI 1327 inhibit hepatic DNA and RNA synthesis in female rats, as evaluated by the determination of nucleic acids synthesis in liver nuclei isolated from female rats 5 and 15 min after the injection of the drug. No influence on hepatic DNA and RNA synthesis is observed when the molecules are administered to male rats. The in vivo administration of DL-propranolol causes an increase of hepatic DNA and RNA synthesis in male rats, while it is uneffective in female rats.

Adrenergic beta-Antagonists↗