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Biomedical subjects

G Taibi

Publications and source records attributed to G Taibi.

At least 19 recordsLinked to original sources
PubMed2002

Development and validation of a fast and sensitive chromatographic assay for all-trans-retinol and tocopherols in human serum and plasma using liquid-liquid extraction.

A sensitive HPLC assay for all-trans-retinol, alpha-tocopherol, and gamma-tocopherols in human serum and plasma is reported. Sample preparation is performed in one step and involves precipitation of proteins and extraction of lipids with two volumes of an ethanol-chloroform mixture (3:1, v/v) without I.S. addition. After removal of the precipitated protein, 20 microl aliquots of the supernatant (equivalent to 6.7 microl of serum or plasma) were injected into the HPLC system and analyzed using fluorometric detection. RP-HPLC was performed using a C(18) S3 ODS2 column with a methanol-water step gradient (97:3 to 100) at 1.0 ml/min. The quantification limit expressed as nanograms of analyte per milliliter of serum or plasma was approximately 30 ng for all-trans-retinol, 300 ng for alpha-tocopherol and 250 ng for gamma- and delta-tocopherol. The method was validated and applied to human serum and plasma from a total of 120 subjects. This procedure requires a small volume of serum or plasma and can therefore be a valuable tool for measuring low concentrations of these vitamins in preterm infants with sensitivity, precision and accuracy.

Chromatography, High Pressure Liquid↗
PubMed2001

Micellar electrokinetic chromatography of polyamines and monoacetylpolyamines.

A selective procedure for qualitative and quantitative analysis of ten polyamines by micellar electrokinetic chromatography (MEKC) was developed. Benzoylated polyamines and acetylpolyamines in micellar phase of SDS (10 mM) were separated at 25 degrees C by 20 mM borate buffer pH 8.5, containing 8% ethanol, with an applied voltage of 25 kV (5 microA) and then detected at 198 nm. The experimental factors and operational parameters were optimized by performing analysis at different surfactant concentrations, pH, voltage and temperature with and without ethanol. The repeatibility of migration times and peak heights is a peculiarity of the method here described.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2001

Xanthine oxidase catalyzes the synthesis of retinoic acid.

Milk xanthine oxidase (xanthine: oxygen oxidoreductase; XO; EC 1.1.3.22) was found to catalyze the conversion of retinaldehyde to retinoic acid. The ability of XO to synthesize all trans-retinoic acid efficiently was assessed by its turnover number of 31.56 min-1, determined at pH 7.0 with 1 nM XO and all trans-retinaldehyde varying between 0.05 to 2 microM. The determination of both retinoid and purine content in milk was also considered in order to correlate their concentrations with kinetic parameters of retinaldehyde oxidase activity. The velocity of the reaction was dependent on the isomeric form of the substrate, the all trans- and 9-cis-forms being the preferred substrates rather than 13-cis-retinaldehyde. The enzyme was able to oxidize retinaldehyde in the presence of oxygen with NAD or without NAD addition. In this latter condition the catalytic efficiency of the enzyme was higher. The synthesis of retinoic acid was inhibited 87% and 54% by 4 microM and 2 microM allopurinol respectively and inhibited 48% by 10 microM xanthine in enzyme assays performed at 2 microM all trans-retinaldehyde. The Ki value determined for xanthine as an inhibitor of retinaldehyde oxidase activity was 4 microM.

Animals↗
PubMed2000

Rapid and simultaneous high-performance liquid chromatography assay of polyamines and monoacetylpolyamines in biological specimens.

A rapid, resolutive and reproducible reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for polyamines and acetylpolyamines by adopting pre-column derivatization with benzoyl chloride. In a single run lasting less than 15 min ten polyamines were separated as well as traces of benzoic acid, methylbenzoate and benzoic anhydride. These contaminants, produced during the derivatization reaction, were almost all eliminated by washing steps envisaged in the same procedure. This simple and sensitive method can be applied to routine determination of polyamines in biological samples. A fine application of this procedure to the determination of endogenous content of polyamines in chick embryo retina was reported.

Biogenic Polyamines↗
PubMed1996

17 beta-hydroxysteroid oxidoreductase activity in intact cells significantly differs from classical enzymology analysis.

This paper summarizes our most recent results of steroid enzyme studies on cultured breast and endometrial cancer cells. It deals mainly with estrogen 17 beta-hydroxysteroid oxidoreductase (17 beta HSOR) activity, which presides over estradiol (E2) and estrone (E1) interconversion, a major metabolic pathway of estrogens. Assessment of either the oxidative or reductive component of 17 beta HSOR was carried out on intact cells by means of an original approach based on reverse phase-high performance liquid chromatography and radioactive detection on line. This system allows the continuous monitoring of both precursor degradation and formation of several radiometabolites to assess rates and direction of steroid metabolism. Overall, hormone-responsive, estrogen receptor (ER)-positive cells, regardless of whether they were derived from breast (MCF7) or endometrial (Ishikawa) tumor tissues, showed a prevalence for reductive metabolism (E1-->E2), whilst oxidative pathways (E2-->E1) were largely dominant in non-responsive, ER-poor mammary (MDA-MB231) and endometrial (HEC-1A) cells. The above estimates of 17 beta HSOR activity were at variance with those obtained using the classical enzymology approach, not only in quantitative terms (being markedly lower using intact cell analysis), but also because the prevalent direction of estrogen metabolism was often reversed. Although striking methodological differences may well account for this discrepancy, intact cell analysis is undoubtedly more similar to the in vivo state than the artificial requirements of classical enzymology procedures.

17-Hydroxysteroid Dehydrogenases↗
PubMed1995

Polyamines and ripening of photoreceptor outer segments in chicken embryos.

Polyamines and their related monoacetyl derivatives were studied in rod outer segment (ROS) and cone outer segment (COS) of photoreceptor cells from chick embryo retina during eye development (7th-18th days). Putrescine was found to be necessary, in the second phase of retinogenesis, to sustain both ROS and COS differentiation and, after acetylation, gamma-aminobutyric acid synthesis. On the other hand, spermidine and even more spermine intervene in the third phase of development when photoreceptors mature. Moreover, the presence of N1-acetylspermidine already at the 7th day indicates that in the outer segment of photoreceptor cells too, as in the whole retina, putrescine synthesis comes about by two pathways. One pathway involves ornithine decarboxylase; the other, spermidine/spermine N1-acetyltransferase and FAD-dependent polyamine oxidase activities that convert spermidine to putrescine via N1-acetylspermidine. These different biosynthetic pathways are probably also decisive in permitting gamma-aminobutyric acid synthesis, which is very important in the ripening process of neural retina.

Acetyltransferases↗
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↗
PubMed1993

Simple high-performance liquid chromatographic assay for polyamines and their monoacetyl derivatives.

A rapid reversed-phase high-performance liquid chromatographic method, using pre-column derivatization with benzoyl chloride and ultraviolet detection at 254 nm, was developed for the simultaneous measurement of polyamines and their monoacetyl derivatives. Calibration curves were linear for concentrations from 1.25 to 25 nmol/ml. The method was employed to assay these compounds in chick embryo retina explants using organic solvent extraction and 1,7-diaminoheptane as an internal standard. This simple and sensitive method can be applied to routine determinations of these compounds in various biological samples.

Acetylation↗
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↗
PubMed1981

Kinetic properties of a nucleoside phosphotransferase of chick embryo.

1. A nonspecific nucleoside phosphotransferase (nucleotide : 3'-deoxynucleotide 5'-phosphotransferase, EC 2.7.1.77), purified from chick embryos, catalyzes the transfer of phosphate ester from a nucleotide donor to a nucleoside acceptor. 2. The enzyme exhibits sigmoidal kinetics with respect to nucleoside monophosphate donors, but with respect to nucleoside di- or triphosphate donors and nucleoside acceptors hyperbolic kinetics were obtained. 3. The nucleoside phosphotransferase of chick embryo is unstable to heat and is protected from inactivation by a large number of nucleosides. 4. Nucleoside di- and triphosphates lower both the concentration of nucleoside monophosphates required for half-maximal velocity and the kinetic order of reaction measured with these phosphate donors. On the contrary, nucleoside di- or triphosphate do not modify the kinetic parameters evaluated for nucleoside acceptors. 5. We suggest that the nucleoside phosphotransferase contains both substrate and regulatory sites. It seems that the free apoenzyme is converted, by means of cooperative interactions between regulatory sites, into an enzyme-nucleotide complex, which is particularly stable at 37 degrees C.

Animals↗