Biomedical subjects

G Tesoriere

Publications and source records attributed to G Tesoriere.

At least 37 records · Page 2Linked to original sources

PubMed1997

Changes in protein composition during chick embryo retina development.

Two dimensional gel electrophoresis was employed to analyse the protein composition of chick embryo retina during "in ovo" development in order to individuate some components which are typical of different stages of growth. The study of protein composition of 7-day-old chick embryo retinas revealed the presence of about eighty different soluble components. In four of these proteins the staining intensity decreased during retina development. One of these components, of 35 KD with an isoelectric point of about 5.15, was partially purified by means of preparative isoelectric focusing. Other components, which were absent at day 7 appeared during the second week of development and were clearly visualised at day 16. These results were also compared with those obtained by means of 2DGE performed on liver and brain of chick embryo at day 7 and 16 of development.

Age Factors↗

PubMed1996

Role of insulin-like growth factors in autocrine growth of human retinoblastoma Y79 cells.

In this study, we have demonstrated that human retinoblastoma Y79 cells produce insulin-like growth factors (IGFs) type I and type II and release them into the medium. We have also ascertained, by means of competitive studies and cross-linking procedure, that Y79 cells contain the type-I IGF receptor (IGF-IR). Furthermore, surface-bound IGF-I is internalised by the receptor, then degraded to amino acids. Insulin, IGF-I and IGF-II caused down-regulation of IGF-IR; the effect is concentration and time dependent. Scatchard analysis demonstrated that incubation with insulin markedly decreased the binding capacity measured for IGF-I while the apparent Kd value calculated for IGF-I binding was not significantly modified. IGF-I, IGF-II and insulin induced tyrosine phosphorylation of IGF-IR. Tyrosine phosphorylation of this receptor with, however, a less strong signal, was detectable even in cells cultured in serum-free medium without the addition of any exogenous growth factor. Similar results have been found concerning the tyrosine phosphorylation of insulin receptor substrate-1 (IRS 1). Tyrosine phosphorylation of both IGF-IR and IRS 1, either under basal conditions or after stimulation with growth factors, was strongly inhibited when alpha-IR3, a monoclonal antibody to IGF-IR, was added to the culture. IGF-I was capable of inducing Y79 cell proliferation and its effect was entirely inhibited by the addition of alpha-IR3. This antibody also markedly reduced the proliferation of Y79 cells cultured in serum-free medium not supplemented with stimulatory factors. Our results indicate that IGF-I and IGF-IR mediate an autocrine growth mechanism in Y79 cells.

Binding, Competitive↗

PubMed1996

Insulin-like growth factors in chick embryo retina during development.

Evidence exists supporting an important role for insulin-like growth factors (IGFs) during fetal growth. In the present report we performed studies to define whether developing chick retina contains IGFs and whether IGFs play a role in the growth of this tissue. We have shown that both IGF-I and IGF-II are present in chick embryo retina throughout development (7th-18th day). The highest values, when expressed as ng/g of tissue, were found in the youngest retinas studied (7th-9th day) and at 16th-18th day of development. During whole development the content of IGF-II was about two to three times higher than that ascertained for IGF-I. The tissue also contains cell-surface binding for IGFs. However, the developmental pattern of IGF-I binding was quite different from that found for IGFs, showing the highest values during the second week of development. Competitive studies showed that this receptor has a high affinity for IGF-I, a lower affinity for IGF-II, and a very much lower affinity for insulin. Also anti-IGF-I receptor antibody (alpha IR3) inhibited 125I-labeled IGF-I binding to the receptor. Such results indicate the presence of type I IGF receptor in chick embryo retina. Affinity labeling experiments have confirmed this hypothesis. We have also shown that cultured retinal explants contain, synthesize and release into the medium appreciable amounts of IGFs. Both exogenous IGF-I and IGF-II added to the culture medium stimulated DNA synthesis of retinal explants. Evidence that the retinas produce IGFs and possess IGF-IR together with the growth-promoting effect of IGFs suggests that these factors play an important role as regulators of retinal growth.

Animals↗

PubMed1995

Chick embryo retina development in vitro: the effect of insulin.

In this paper we study the development of chick embryo retina cultured in vitro and the effects exerted by insulin. Retinas were removed from 7-day embryos and cultured in serum- and hormone-free medium for 7 additional days. Under these conditions retinal cells survived and underwent cholinergic differentiation, as previously ascertained by Hausman et al. (Dev. Brain Res., 1991, 59: 31-37). However, a great retardation of development was noted compared to uncultured control, 14-day retina. In fact both wet weight and DNA and protein content increased much slower than in ovo and the tubulin content decreased below even the starting value. In addition, although after 7 days in culture retinal cells were organized in identifiable layers, nevertheless the typical organization equivalent to 14-day in ovo retina was absent. The addition of insulin in the medium markedly increased the wet weight of cultured retinas, their protein content and the level of tubulin pools, particularly that of non-assembled fraction. Nevertheless insulin did not modify DNA synthesis and did not induce the increment of both neuron specific enolase and actin. Morphological observations show that insulin markedly increased the number and the thickening of the fiber layers. These results, together with the facts that retina synthesizes and secretes insulin and possesses specific insulin receptors suggest that insulin can have autocrine or paracrine regulatory functions in retinal development by exerting a general effect on retinal growth and a more specific one on tubulin production.

Animals↗

PubMed1994

High-performance liquid chromatographic method for the determination of insulin synthesis in biological systems.

This paper reports a two-step high-performance liquid chromatographic procedure which permits the study of the incorporation of [3H]leucine into insulin in biological systems. The first step of the procedure was size exclusion chromatography, performed on a GPC-100 column, which was eluted with 0.1 M KH2PO4-methanol (9:1, v/v). By this step the bulk of both protein and radioactivity was separated from tritiated insulin. The second step, which employs reversed-phase chromatography on an octadecylsilyl column, permits the separation of insulin from other contaminants by means of a linear gradient of acetonitrile. This simple and reproducible method was employed to test insulin synthesis in cultured human retinoblastoma Y79 cells.

Chromatography, Gel↗

PubMed1994

Synthesis of insulin and its effects in Y79 human retinoblastoma cells.

This paper demonstrates that Y79 human retinoblastoma cells contain immunoreactive insulin (IRI) and release it in the medium. Cells cultured either in suspension or in monolayer showed a similar content of IRI. Moreover, in both conditions, IRI concentration was higher in cells cultured in serum-supplemented medium rather than in serum-free medium. Retinoblastoma cells are capable of synthesizing insulin. This was demonstrated by incubating Y79 cells with [3H]leucine. The synthesized radioactive insulin was separated and assayed by means of a HPLC procedure described in this paper. Both cell growth and [3H]thymidine and [3H]uridine incorporation into acid-insoluble fraction was reduced (-75%) in Y79 cells cultured without serum with respect to those cultured in the presence of serum. The addition of insulin to the serum-free medium stimulated both cell division and DNA and RNA labeling, with values approaching those obtained with serum supplemented cultures. Insulin-like growth factor I exerted similar effects, but at a much lower concentration than insulin. We suggest that both insulin and IGF-I may represent mitogenic signals for these cells which might be mediated through insulin-like growth factor I receptors.

Cell Division↗

PubMed1994

Insulin synthesis in chick embryo retinas during development.

Retinas of chick embryos contain insulin and further, are capable of synthesizing it, as demonstrated by incubating retinas at different ages (7th-18th day) with [3H]leucine. The synthesized radioactive insulin was isolated and assayed by means of a HPLC procedure. The synthesis of insulin was found to be highest in the youngest retinas studied (day 7), afterwards it declined with age except for an increment found at 14-15 day. Explants of chick embryo retinas, cultured in vitro, rapidly degraded insulin. Nevertheless, the content of immunoreactive insulin in retinal explants diminished slowly with the age of culture, so that, after 8 days of incubation, it was about 60% of the content found in the retinas at the beginning of incubation. This was proof that cultured explants are capable of efficiently synthesizing insulin. The synthesized [3H]insulin was released from explants into the medium. This was evident also after 6-8 days in culture.

Animals↗

PubMed1994

Pattern of polyamines and related monoacetyl derivatives in chick embryo retina during development.

Polyamines and related monoacetyl derivatives were studied in chick embryo retina during development (6th-19th day). Putrescine, which is high in the first phase of retinogenesis, is necessary to sustain both tissue proliferation and via N-acetylputrescine, gamma-aminobutyric acid synthesis. A later increase in spermidine and particularly spermine may play a role in the last phase of development when the retina reaches maturation. The presence of N1-acetylspermidine already at the 8th day indicates that in chick embryo retina, putrescine synthesis can depend on two separate pathways. The first involves ornithine decarboxylase activity; the second, spermidine/spermine N1-acetyltransferase and probably polyamine oxidase that converts spermidine to putrescine via N1-acetylspermidine.

Animals↗

PubMed1992

A factor derived from chick embryo retina which inhibits DNA synthesis of retina itself.

Chick embryo retinas contain a peptide factor that inhibits DNA synthesis in explants of chick embryo retina. The inhibitory factor, obtained by acid/ethanol extraction from 15-day-old chick embryo retinas, was partially purified by affinity chromatography on heparin-sepharose CL-6B and gel filtration on Sephadex G-100. The inhibitor reduced DNA synthesis with maximal effects observed in retinal explants from 7 to 8-day-old chick embryos. The inhibitory effect became apparent after 10 h of incubation and reached the maximum levels after 16 h. DNA-inhibiting activity was heat and acid-stable and was destroyed by trypsin and alkaline treatments. The inhibitory effect was observed in retinal explants incubated in a medium free from L-glutamine, and the addition of this compound to the medium reduced the inhibitory effect in a concentration-dependent manner.

Animals↗

PubMed1992

Identification of insulin in chick embryo retina during development and its inhibitory effect on DNA synthesis.

Incubation of chick embryo retinal explants with insulin resulted in a pronounced inhibition of thymidine uptake and incorporation into trichloroacetic acid-insoluble fraction. The inhibitory effect was highest with explants from embryos at day 7 and day 8, and thereafter it declined markedly with the age of embryos until day 11. A time-course study of the effect revealed that the inhibition occurred after a lag time; both thymidine uptake and incorporation were not altered significantly after 2-6 h of incubation with insulin, but began to decrease thereafter, reaching the maximum after 16 h. The effect was also dose dependent. After 16 h of incubation, the maximal inhibition (65%) was found with 10(-8) M insulin. Insulin caused similar effects also on thymidine kinase activity. All these effects were obtained by using minimal essential medium without glutamine. The addition of glutamine to the medium reduced the inhibitory effect of insulin. Retinas of chick embryos contain immunoreactive insulin. Retinal immunoreactive insulin was at the highest level (1.12 ng/mg of protein) in the youngest retinas studied (day 6), then it declined with age, reaching the lowest value (0.58 ng/mg of protein) at day 14. This value did not vary significantly during the third week of development. A potential biological role of insulin in retinal development is discussed.

Animals↗

PubMed1989

Inhibition of DNA synthesis in chick embryo retinas, in vitro, by a factor from fetal bovine serum.

Fetal bovine serum inhibited deoxyribonucleic acid (DNA) synthesis in chick embryo retina explants. The inhibitory activity was precipitated from fetal bovine serum by 45% saturated ammonium sulfate and isolated by means of Sephadex G-100 and Bio-Gel P-60 columns as a peak with an apparent molecular weight of 7000 Da. DNA-inhibiting activity was heat- and acid-stable and was destroyed by dithiothreitol and alkaline treatment. The purified factor inhibited similarly both DNA synthesis and thymidine kinase activity; 50% inhibitory effect was found with 160 ng, 17 h after the addition into the incubation medium.

Animals↗

PubMed1989

Biochemical aspects of chick embryo retina development: the effects of glucocorticoids.

In chick embryo retina during development, DNA synthesis and the activities of DNA polymerase, thymidine kinase, thymidylate synthetase, and ornithine decarboxylase (ODC) declined in parallel from day 7 to 12. The administration in ovo of hydrocortisone reduced significantly, particularly at 8-10 days of incubation, both DNA synthesis and the four enzyme activities tested. The effect was dose dependent, reaching the maximum with 50-100 nmol of hydrocortisone, 8-16 h after treatment. The highest inhibition was found for ODC activity (70%), followed by thymidine kinase activity (62%) and DNA synthesis (45%), whereas activities of DNA polymerase and thymidylate synthetase were reduced only by 30%. The inhibitory effect was exerted by all the glucocorticoids tested, with dexamethasone and hydrocortisone being the most efficacious. The results support the view that glucocorticoids reduce the proliferative events in chick embryo retina, particularly at 8-10 days of embryonic life.

Animals↗

PubMed1988

Morphological and biochemical effects of glucocorticoids in chick embryo hepatocytes during development.

The administration in ovo of hydrocortisone-21-phosphate caused, in chick embryo liver, a reduction of the number of hepatocytes which can be isolated from 1 mg dry weight of liver and a marked increase of their size. Moreover, the treatment diminished the incorporation of thymidine into acid-insoluble fraction in these cells whilst it augmented the content of protein, RNA, DNA and the level of thymidine kinase/cell. These effects were highest at 8-10 days, then declined with the age, disappearing after 18th day of incubation. Similar effects were obtained by injecting other glucocorticoids or ACTH. Combined treatment with metopirone abolished the effects found with ACTH, but did not modify the action of hydrocortisone. These findings suggest that glucocorticoids interfere with the proliferative cycle of hepatocytes by inhibiting the mitotic phase and favouring the production of abnormally large cells.

Adrenocorticotropic Hormone↗

PubMed1987

Influence of hydrocortisone on chick embryo retina development.

Treatment of chick embryos in ovo with hydrocortisone-21-phosphate (a single dose of 150 micrograms) caused a marked reduction of retinal thymidine kinase activity 24 h later. The inhibitory effect was highest (65-70%) in 8-10-day-old embryos and declined with age, disappearing after day 15. It was accompanied by a reduction in thickness of the retinal layers. Adrenocorticotropic hormone (ACTH) treatment (10 micrograms daily for 2 days) also produced an age-dependent inhibitory effect on retinal thymidine kinase, whereas treatment with a single dose of 200 micrograms of metopirone, a compound that prevents the 11 beta-hydroxylation of steroid molecules in the adrenal glands, impeded the decrease in thymidine kinase activity that normally occurs in chick embryo retina after day 9 of development. In addition, metopirone prevented the inhibition exerted by ACTH on thymidine kinase activity but had no effect on the action of hydrocortisone.

Adrenocorticotropic Hormone↗

PubMed1986

Uridine enhances the cytotoxic effect of D-glucosamine in rat C6 glioma cells.

This paper studies the influence of uridine on the effects exerted by D-glucosamine in rat C6 glioma cells. 2 mM uridine increased markedly both the cytotoxic effect of the aminosugar and the inhibition of thymidine incorporation into acid-insoluble fraction. Furthermore the complete resumption of the capacity to incorporate either 3H-thymidine or 3H-mannose which was observed after the removal of the aminosugar, was impeded when the cells were treated contemporaneously with D-glucosamine and uridine. An exposure for 4 hr to 20 mM glucosamine alone enhanced about 15-fold the cellular pool of UDP-N-acetylhexosamines; the addition of 2 mM uridine intensified the expansion of this pool, which became about 35-fold the control value. The findings suggest a connection between the accumulation of UDP-N-acetylhexosamines in the cells and the appearance of D-glucosamine cytotoxicity.

Animals↗

PubMed1985

Nucleoside phosphotransferase in animal tissues. Tissue distribution and kinetic properties.

Amphibian, avian and mammal tissues contain a nucleoside phosphotransferase clearly different from those previously described in vegetables and bacteria. Whatever the animal source, the enzyme showed many similar characteristics as far as substrate specificity, dependence upon Mg2+, instability at 37 degrees C, and the protecting effect of nucleotides were concerned. Moreover, when submitted to gel filtration, the enzyme behaved in all cases as a dissociable high molecular weight protein, whose degree of association was controlled by nucleotides. In amphibian and avian tissues multiple forms of the enzyme seem to be present which differ for the substrate concentration at half-maximal velocity (S0.5); the concentration of nucleotide effector which affords half-maximal protection at 37 degrees C (P0.5); and the Hill coefficient for monophosphate donor. Within each single species, the higher the interaction coefficient was, the lower S0.5 and P0.5 values were. In mammalian tissues one form of nucleoside phosphotransferase seems to prevail where cooperative interactions are almost absent and whose S0.5 as well as P0.5 values do not vary significantly from one tissue to another.

Animals↗

PubMed1984

The inhibitory effect of D-glucosamine on thymidine kinase in chick embryo retinas and HeLa cells.

D-Glucosamine markedly inhibits thymidine incorporation into the TCA-insoluble fraction and thymidine kinase activity in HeLa cells. Both the inhibitory effects are also observed in isolated retinas of chick embryos. In this case the inhibitory effects are age-dependent and the magnitude of the responses decreases with embryonic development. In addition the time of exposure to D-glucosamine which is necessary to reveal the inhibitory effect on thymidine kinase increases with the age of the embryos.

Age Factors↗

PubMed1984

The purification and properties of nucleoside phosphotransferase from mucosa of chicken intestine.

Nucleoside phosphotransferase (nucleotide: 3'-deoxynucleoside 5'-phosphotransferase, EC 2.7.1.77) has been purified from chicken intestine mucosa to apparent homogeneity. The enzyme is represented by a multisubunit protein at different degrees of association. It can dissociate into a component with a marked fall in catalytic activity. The associated forms are similar to the enzyme previously purified from chick embryo as regards: substrate specificity both with respect to nucleoside monophosphate donors and to deoxyribonucleoside acceptors; sigmoidicity in the rate curve with a variable phosphate donor; instability to heat, dilution and lowering of pH; the activating and protecting effect of nucleotides, particularly the diphosphate forms. The dissociated form displays lower Vmax and higher S0.5 than the associated ones; and the Hill constants are always about 1. With this form, nucleotides show only a modest activating effect and do not protect. Mg2+, Mn2+ or Co2+ are required for catalytic activity, whereas the protective effect of nucleotides is independent of divalent metals. Inorganic phosphate stabilizes associated forms of the enzyme, but inhibits its activity by competing with nucleotide effectors. The enzyme behaves also as a phosphohydrolase, particularly with respect to deoxyribonucleoside monophosphates; deoxyuridine and deoxythymidine inhibit hydrolytic activity.

Alcohols↗