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G Palumbo

Publications and source records attributed to G Palumbo.

At least 73 records · Page 4Linked to original sources
PubMed1996

Identification of differentially expressed mRNAs in normal and neoplastic (adenocarcinoma) human endometrium.

Individual mRNA species from normal and neoplastic endometrium obtained from the same patient were comparatively studied by exploiting the "differential display" methodology. The mRNAs were extracted from tissues, reverse transcribed, and amplified by PCR using appropriate primers. The cDNA electrophoretic bands which were frankly different on the basis of their quantitative expression were excised from the gels and reamplified. Each of these sequences was subsequently screened for the capacity to hybridize to RNA preparations from normal endometrium and endometrial adenocarcinoma samples, first from the original patient and then from a group of selected patients. Of many, only two sequences, thereafter named N5.5 and T16.2, respectively, successfully passed the two sieving tests and were chosen for further analysis. It appears that N5.5 recognizes an mRNA which is expressed more abundantly in normal than in neoplastic (adenocarcinoma) endometrium; in contrast, T16.2 seems to preferentially recognize an mRNA which is expressed in tumoral tissues. These two sequences have been cloned and sequenced; they do not show any identity or significant similarity to any other known sequence.

Adenocarcinoma↗
PubMed1996

Calcium-channel blockers inhibit human low-density lipoprotein oxidation by oxygen radicals.

Previous studies have shown that calcium channel blockers may reduce the development of experimental atherosclerosis, and that nifedipine may slow the progression of coronary atherosclerosis in humans. The mechanisms responsible for this antiatherogenic effect are still unclear. It has been recently proposed that oxygen free radicals can induce the oxidation of human low-density lipoproteins (LDL) and that oxidized LDL may be an atherogenic stimulus. Previous studies in other systems have shown that calcium channel blockers may effectively inhibit oxygen radical-induced lipid peroxidation in vitro. Thus, the aim of the present study was to investigate whether calcium channel blockers may also reduce LDL modifications induced by oxygen radicals. Isolated human LDL were exposed to oxygen radicals generated by CuSO4 (10 microM for 18 hours) after a 30 minute pre-incubation with different concentrations (1-100 microM) of nifedipine, diltiazem, and verapamil. Lipid peroxidation was measured from malonyldihaldehyde (MDA) production. Oxygen radical-induced damage on apolipoprotein-B100 was evaluated by acrylamide and agarose gel electrophoresis. Calcium channel blockers dose-dependently prevented oxidation of both the lipid and protein components of LDL. MDA formation was reduced in LDL pre-incubated with calcium antagonists before exposure to oxygen radicals (% MDA inhibition was 89.8 +/- 6.9 with 30 microM nifedipine, 68.6 +/- 4.9 with 30 microM verapamil, and 65.6 +/- 7.1 with 30 microM diltiazem; p < 0.01 vs. controls). Similarly, apolipoprotein-B100 integrity was preserved against oxygen radical attack in the presence of calcium antagonists. Thus, calcium channel blockers reduce the oxidation of human LDL in vitro. These data suggest that reduced formation of atherogenic oxidized LDL may be an additional mechanism for the antiatherosclerotic effects of calcium channel blockers in vivo.

Analysis of Variance↗
PubMed1996

Clinical and hormonal effects of gonadotropin-releasing hormone agonist plus an oral contraceptive in severely hirsute patients with polycystic ovary disease.

OBJECTIVE: To evaluate the therapeutic effects of a GnRH agonist (GnRH-a), leuprolide acetate (LA) plus a pill containing ethinyl E2 plus cyproterone acetate (CPA) in a group of women with severe hirsutism who were unresponsive to oral contraceptive (OC) therapy. DESIGN: Twenty-four patients suffering from severe hirsutism secondary to polycystic ovary disease (PCOD) were treated for 12 months with 3.75 mg IM LA every 28 days in association with 0.035 mg/d ethinyl E2 plus 2 mg/d CPA (Diane; Schering, Berlin, Germany) for 21 d/mo. SETTING: Patients were recruited in the Institute of Obstetrics and Gynecology, St. Bambino Hospital, University of Catania, Catania, Italy. Hormonal assays were performed in the Hormone Laboratories of St. Bambino Hospital, University of Catania, Catania, Italy. MAIN OUTCOME MEASURES: Every 3 months the hirsutism score was evaluated. Mean serum concentrations of LH, FSH, E2, total and free T, androstenedione (A), sex hormone-binding globulin (SHBG), and DHEAS were determined. Every 6 months a vaginal ultrasound examination was performed. RESULTS: In all patients after 6 and 12 months of treatment with LA plus OC, the hirsutism score improved significantly. Serum levels of LH, FSH, E2, total and free T, A, and DHEAS decreased significantly, whereas SHBG showed a marked increase. A significant reduction in the ovarian size was observed. CONCLUSION: Gonadotropin-releasing hormone agonist, associated with a pill containing CPA, reduced the hirsutism in severely affected women with PCOD who are unresponsive to OC treatment alone.

Adult↗
PubMed1996

Targeted gene transfer in eucaryotic cells by dye-assisted laser optoporation.

The blue beam of an Argon laser (488 nm) has been focused on the cell membrane in the presence of phenol-red, an usual component of cell culture media, through a 100 x objective. At the site of the beam impact, due probably to local temperature changes, the cell membrane modifies its permeability. As a consequence of the hit, circular areas, whose radius may be apparently regulated by changing the irradiation time and/or the radiation intensity (energy), appear on the wall, last for a short time and fade spontaneously within 1-2 minutes. No evident sings of cell injury or hurt have been observed afterward. Plasmid DNA, purposely added to culture fluid, easily slips in the cytoplasm; utilizing such approach, thereafter indicated as "optoporation', we have successfully transfected two genes, namely beta-galactosidase and chloramphenicol-acetyl-transferase in murine NIH3T3 fibroblasts. Therefore optoporation represents an additional procedure for gene transfer with several advantages over already available methods: (1) it only takes advantage of the presence of phenol-red, a normal cell medium component, with no need of addition of extraneous substances; (2) it is a very mild treatment virtually suitable for any cell type and (3) it allows transfection of selected cells even in the presence of cells of different type (providing that they are morphologically distinguishable).

3T3 Cells↗
PubMed1996

Relationship between tumour necrosis factor alpha and sex steroid concentrations in the follicular fluid of women with immunological infertility.

Experimental evidence suggests a tight relationship between cytokines and the reproductive system. Tumour necrosis factor alpha (TNF alpha), a cytokine produced by activated macrophages and mesenchymal cells, seems to participate in the control of fertility. Therefore, the present study was undertaken to evaluate the concentrations of TNF alpha in the follicular fluid of female patients with immunological infertility, as well as the possible role of this cytokine in follicular development. Concentrations of TNF alpha, 17 beta-oestradiol, progesterone, androstenedione and testosterone were measured in the follicular fluid of patients with immunological infertility and patients with a tubal factor of infertility, who served as a control group. Patients with immunological infertility had significantly higher concentrations of TNF alpha in their follicular fluid compared to the control group. In contrast, oestradiol concentrations were significantly lower in the former group. The intrafollicular concentrations of the other steroids measured did not differ significantly between the two groups. The fertilization rate of ova from follicles included in the study was significantly lower in patients with immunological infertility compared to control subjects (19.1 and 57.1% respectively). In conclusion, this study shows that patients with infertility of immunological origin have increased follicular fluid concentrations of TNF alpha and lower oestradiol concentrations. We speculate that elevated TNF alpha concentrations in the human follicle may negatively influence both ovulation-and fertilization-related events.

Adult↗
PubMed1996

Human thyroglobulin reference material (CRM 457). 1st Part: Assessment of homogeneity, stability and immunoreactivity.

This paper describes the assessment of the homogeneity and stability of a purified and lyophilized human thyroglobulin (Tg), and characterizes its immunoreactivity. The purified and lyophilized Tg is intended to be used as a primary reference material to establish calibration of working serum based reference material. The programme involved the participation of 15 European laboratories and one laboratory from the United States. The homogeneity of the content of the ampoules was considered acceptable (< 9%). The stability was tested by accelerated temperature degradation showing predicted annual relative losses of 0.01% at -70 degrees C and 1.04% at -20 degrees C. The immunoreactivity of the Tg material as measured in different laboratories varied mostly according to the method used rather than the laboratory. The interlaboratory variability showed that the two commercial methods used in several laboratories (kit 1 and 2) had an interlaboratory variation (CV) of 15.9% (N = 5) and 7.1% (N = 3), respectively, whereas the total interlaboratory CV was 64.3% (N = 18). The immunoreactive Tg had dilution curves parallel with other Tg calibrators (those of the methods). Dilution curves of the Tg material after storage at various temperatures and time were parallel in both RIA and IRMA. In conclusion, we have prepared a Tg reference material which in extensive studies in several participating laboratories has demonstrated a sufficient homogeneity and stability as well as dilution curves parallel to the calibrators of all the immunoassays tested in the study. This reference material is considered the first step towards decreasing the interlaboratory variability between Tg immunoassays.

Drug Stability↗
PubMed1996

Human thyroglobulin reference material (CRM 457). 2nd Part: Physicochemical characterization and certification.

This report describes the characterization of a purified human thyroglobulin (Tg) reference material, and details the procedures used in its certification. The purified Tg is intended to be used as a primary reference material to establish calibration of working serum based reference material for immunoassay procedures. The programme involved the participation of 15 European laboratories and one laboratory from the United States. The physicochemical characterization showed by polyacrylamide gel electrophoresis and immunoblotting that the purified Tg had for the major part the expected molecular size of 660 kDa with traces of lower molecular forms. The amino acid composition was close to that demonstrated for the cDNA and the content of iodine was in keeping with a moderately to highly iodinated Tg. The mass concentration in reference material RM 457 is certified to be (0.324 +/- 0.018) g/L on the basis of protein determined by the Lowry method and supported by nitrogen determination, absorbance measurement, and amino acid analysis. This reference material is considered the first step towards decreasing the interlaboratory variability between Tg methods of measurement.

Amino Acids↗
PubMed1995

Decreased phosphorylation of mutant insulin receptor by protein kinase C and protein kinase A.

We have recently reported that the Arg1152-->Gln insulin receptor mutation (QK single mutant) alters a conserved motif (RK motif) immediately next to the key tyrosine phosphorylation sites (Tyr1146, Tyr1150, Tyr1151) of the receptor and constitutively activates its kinase and metabolic signaling. To investigate further the function of the RK motif, we have expressed two additional mutant insulin receptors: a single mutant, in which the second basic residue in the RK motif (Lys1153) was substituted (RA mutant); and a double mutant, in which both the Arg and the Lys residues were replaced with noncharged amino acids (QA mutant). As compared with the transfected wild-type receptors (WT), both the single and the double mutant receptors were normally synthetized and transported to the plasma membrane and bound insulin normally. Whereas the double mutant receptor exhibited preserved insulin-dependent autophosphorylation, kinase activity, and 2-deoxyglucose uptake, all of these functions were grossly impaired in the two single mutant receptors. Two-dimensional analysis of tryptic phosphopeptides from receptor beta-subunits revealed that decreased autophosphorylation of the single mutant receptors mainly involved regulatory Tyr1150,1151 and carboxyl-terminal Tyr1316,1322. At variance with the insulin-stimulated, insulin-independent tyrosine kinase activity toward poly(Glu-Tyr) 4:1 was increased 3-fold in both the double and the single mutants. All mutant receptors induced a 2-fold increase in basal 2-deoxyglucose uptake in NIH-3T3 cells. Treatment of WT transfected cells with 12-O-tetradecanoyl-phorbol-13-acetate or 8-bromo-cAMP increased insulin receptor phosphorylation by 3-fold. No phosphorylation was observed in cells expressing the two single or the double mutant receptor. Consistently, purified preparations of PKC and PKA phosphorylated the WT but not the mutant receptors in vitro. A 17-amino acid synthetic peptide encoding the receptor sequence surrounding the RK motif inhibited phosphorylation of WT insulin receptors by both protein kinases A and C. A mutant peptide in which the RK sequence was replaced by QK (to mimic the mutation in the QK receptor) exhibited no inhibitory effect. Thus, the RK insulin receptor motif is required for insulin receptor phosphorylation by protein kinases C and A and may modulate insulin-independent receptor activity. The RK motif may also have an important structural role in allowing normal insulin regulation of the kinase.

3T3 Cells↗
PubMed1995

The Ste locus, a component of the parasitic cry-Ste system of Drosophila melanogaster, encodes a protein that forms crystals in primary spermatocytes and mimics properties of the beta subunit of casein kinase 2.

Males of Drosophila melanogaster lacking the Y chromosome-linked crystal locus show multiple meiotic alterations including chromosome disorganization and prominent crystal formation in primary spermatocytes. These alterations are due to the derepression of the X chromosome-linked Stellate sequences. To understand how the derepression of the Stellate elements gives rise to these abnormalities, we have expressed the protein encoded by the Stellate sequences in bacteria and produced an antibody against the fusion protein. Immunostaining of crystal- testes has clearly shown that the Stellate protein is a major component of the crystals. Moreover, in vitro experiments have shown that this protein can interact with the catalytic alpha subunit of casein kinase 2 enzyme, altering its activity.

Amino Acid Sequence↗
PubMed1995

Oxidative structural modifications of low density lipoprotein in homozygous familial hypercholesterolemia.

Patients with homozygous familial hypercholesterolemia (FH), as a result of the increased levels and prolonged residence time of low density lipoprotein (LDL) in plasma, have a strong tendency toward accumulation of LDL-cholesterol in the arterial wall, causing premature atherosclerosis. This phenomenon may enhance per se the physiological degradation of both protein and lipid component of LDL, which be more susceptible to oxidative damage induced by oxygen radicals. It is well known that LDL may undergo oxidative modification before being taken up by macrophages which are then transformed into foam cells. It has been suggested that platelet-activating factor (PAF) may play an important role in atherogenesis and PAF catabolism is known to be mediated by serum acetylhydrolase, an enzyme that is normally associated with LDL. Thus, the present study was designed to investigate the structural properties of LDL, including acetylhydrolase activity, in homozygous FH as compared to normolipidemic subjects before and after xanthine/xanthine oxidase-mediated oxidation. We studied 8 homozygous FH patients matched with 8 normolipidemic volunteers. Lipids of LDL fraction were extracted and verified by thin layer chromatography (TLC) analysis. Fatty acids were methylated and injected into a gas chromatograph/mass spectrometer. Vitamin E in LDL was determined by high performance liquid chromatography (HPLC). As an index of susceptibility of LDL to oxidative modifications, the formation of lipid-conjugated dienes was continuously monitored at 234 nm. Lipid peroxidation was also evaluated from the amount of both lipid peroxides (LPO) and malonyldialdehyde (MDA) content. Apolipoprotein (apo) B-100 on LDL was carried on polyacrylamide and agarose gel electrophoresis. In the homozygous FH patients, the relative content of cholesteryl ester was slightly increased. Interestingly, the relative amount of arachidonic acid (20:4) was constantly increased in each lipid fraction in homozygous FH patients. The amount of vitamin E was not significantly different in the patient group from that in the control group. However, LDL from patients carried lower levels of vitamin E (nmol/mg LDL) than controls (2.7 +/- 0.4 vs. 2.9 +/- 0.3 P = NS). The results shows that lag time (min) was decreased (82 +/- 19 vs. 111 +/- 21; P < 0.05) and the maximal rate of diene production and total diene production was increased in homozygous FH patients. Mean levels of MDA were similar in both groups before oxidation, but levels after initiation of oxidation were significantly higher in the patient group. In contrast, mean levels of LPO were already higher in patients before oxidation (58 vs. 27 nmol/mg of protein; P < 0.05), and after initiation of oxidation were also significantly higher at each time points. When oxidized LDL was run on a polyacrylamide gel, an extensive apo B-100 fragmentation replaced by lower molecular mass fragments ranging from 45,000 to 205,000 m.wt., was observed only in LDL from homozygotes. Relative LDL agarose gel mobility shows that LDL from patients migrated higher than LDL of controls. Finally acetylhydrolase activity associated with LDL in patients was significantly reduced as compared to controls. Thus, in homozygous FH patients, LDL appeared more susceptible to oxidation in vitro; the indices for LDL oxidizability were all significantly different from those of controls. This phenomenon might be due to prolonged residence time of LDL in these patients, as suggested from high basal LPO levels and lower vitamin E levels carried by LDL. This hypothesis may explain together with the high content of arachidonic acid, the enhanced susceptibility of LDL from homozygous FH patients to oxidative damage.

Apolipoprotein B-100↗
PubMed1995

Simultaneous determination of 5-aminosalicylic acid, acetyl-5-aminosalicylic acid and 2,5-dihydroxybenzoic acid in endoscopic intestinal biopsy samples in humans by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method for the simultaneous determination of 5-aminosalicylic acid (5-ASA), acetyl-5-aminosalicylic acid (Ac-5-ASA) and 2,5-dihydroxybenzoic acid (5-HSA) in human endoscopic intestinal biopsy with electrochemical detection has been developed and validated. A liquid-liquid extraction procedure was used to isolate these drugs from the biological material prior to analysis. The compounds were separated on an Erbasil S reversed-phase column using methanol-critic acid-sodium hydrogenphosphate-heptane-sulfonic acid-disodium ethylenediaminetetraacetate (pH 3) as mobile phase. The method was linear from 1.0 to 300 ng ml-1 for 5-ASA, from 10 to 1000 ng ml-1 for Ac-5ASA and from 0.1 to 10 ng m-1 for 5-HSA. The limit of detection for 5-ASA and for Ac-5-ASA was 1 ng ml-1 and that for 5-HSA was 0.1 ng ml-1. This procedure is suitable for pharmacological and clinical studies of 5-ASA.

Aminosalicylic Acids↗
PubMed1995

Clinical and endocrine effects of finasteride, a 5 alpha-reductase inhibitor, in women with idiopathic hirsutism.

OBJECTIVE: To evaluate the effects of long-term administration of finasteride on hirsutism score, basal gonadotropin, and androgen secretion in women with idiopathic hirsutism. DESIGN: Randomized single-blinded study. PATIENTS: Eighteen patients with moderate-severe hirsutism were recruited for the study. INTERVENTIONS: Nine hirsute patients received 7.5 mg/d oral finasteride for a period of 9 months whereas the other nine were treated with placebo. Hirsutism score, serum basal gonadotropin, androgens, estrogen, and sex hormone-binding globulin (SHBG) levels were evaluated in all patients before treatment and every 3 months during treatment. RESULTS: After 6 and 9 months of treatment, the hirsutism score improved significantly in the patients receiving finasteride, whereas no significant modifications were observed in patients treated with placebo. The side effects observed were headache and depression of modest entity during the 1st month of treatments, whereas libido did not change. Serum levels of LH, FSH, androstenedione, unbound T, DHEAS, E2, 17 alpha-hydroxyprogesterone, and SHBG did not change during therapy. Hirsute patients treated with finasteride exhibited a marked decrease of dihydrotestosterone and a significant increase of T serum levels from the 3rd and 6th months of treatment, respectively. CONCLUSION: Finasteride decreased the hirsutism score of patients affected by idiopathic hirsutism with few side effects during treatment. No modification of libido was observed.

5-alpha Reductase Inhibitors↗
PubMed1995

Simultaneous determination of rufloxacin and theophylline by high-performance liquid chromatography in human plasma.

A sensitive, specific and rapid liquid chromatographic procedure to monitor rufloxacin and theophylline selectively in human plasma has been developed and validated. Plasma samples were extracted with a mixture of dichloromethane-diethyl ether. The organic layer was evaporated to dryness, reconstituted with mobile phase, and the analytes were separated and quantified by LC on an octadecylsilane column with acetonitrile-0.1 mol dm-3 phosphate buffer (pH3) as mobile phase and UV detection at 280 nm. The procedure was linear from 0.05 to 5.0 micrograms cm-3, with a detection limit of 0.03 micrograms cm-3 for rufloxacin, and from 0.5 to 30 micrograms cm-3, with a detection limit of 0.02 micrograms cm-3 for theophylline. The accuracy of the method was defined by relative errors of 4.2, 5.0 and 1.6% for human plasma containing 0.1, 1.0 and 5.0 micrograms cm-3 of rufloxacin and 2.4, 2.0 and 2.7% at the concentrations of 1.0, 5.0 and 20 micrograms cm-3 for theophylline. The corresponding relative standard deviations were 3.4, 2.8 and 1.7% for rufloxacin and 2.1, 3.7 and 3.8% for theophylline at the same concentrations, respectively. The assay has been successfully applied to pharmacology, toxicology and pharmacokinetics studies in humans.

Anti-Infective Agents↗
PubMed1995

Levels of flurithromycin in female genital tissue.

The levels of flurithromycin in gynecological tissue in 20 female patients were studied after preoperative administration. The tissue flurithromycin levels obtained were comparable to those obtained in serum at 3 and 4 h but were frequently higher than those in serum at 6 and 12 h. Flurithromycin reached the highest concentrations in ovary at 4 h and in endometrium at 6 h.

Anti-Bacterial Agents↗
PubMed1995

Ph positive acute lymphoblastic leukemia in adults: molecular and clinical studies.

Fifty-six patients with ALL were investigated for bcr involvement by PCR. Breakpoints were found in 15 patients (26.8%). There were no differences in clinical and hematologic features or the percentages of complete response (CR) between the Ph+ and Ph- cases. The duration of CR was 6 and 8 months, respectively. In 7/9 Ph1 relapsed ALL we observed increased expression of myeloid markers and 2/9 showed a switch of cytotype (Ly-->My). In none of the 13 Ph- relapsed ALL patients did we observe these findings. 7/15 of Ph+ cases expressed P190 and mRNA ela2 and 8/15 patients showed P210, with mRNA b3a2 in 5 and b2a2 in 3, respectively. The percentage of CR was 57% in the P190+ and 87% in the P210+ group. Investigation of more Ph1+ ALL cases treated with a uniform protocol should be performed in the future in order to determine whether any such biological and clinical differences exist.

Adolescent↗
PubMed1994

Internalization of the constitutively active arginine 1152-->glutamine insulin receptor occurs independently of insulin at an accelerated rate.

Signals controlling the insulin receptor endocytotic pathway have been investigated using the R1152Q insulin receptor mutant (M). This mutant receptor exhibits high levels of insulin-independent kinase activity, impaired autophosphorylation, and lack of an insulin stimulatory effect on both auto- and substrate phosphorylation. NIH-3T3 fibroblasts expressing M receptors displayed a 2.5-fold higher 125I-insulin internalization rate than wild type (WT) but lacked insulin-induced receptor internalization and down-regulation. Cell surface recycling of internalized receptors also occurred at a higher rate in M cells and was unaffected by insulin. Cell preincubation with 35 mM Tris, which inhibits the insulin receptor degradative route, elicited no effect on M receptor recycling but inhibited that of WT by 40%. In contrast, the energy depleter 2,4-dinitrophenol, which inhibits normal insulin receptor retroendocytosis, impaired M receptor recycling 4-fold more effectively than that of WT. The release of internalized intact 125I-insulin was 6-fold greater in M than in WT fibroblasts and was almost completely inhibited by dinitrophenol, whereas insulin degradation by M cells was 4-fold decreased as compared with WT. Thus, internalization and recycling of the constitutively active Gln1152 receptor kinase occur in the absence of autophosphorylation. However, tyrosine phosphorylation appears to be required for proper sorting of endocytosed insulin receptors.

2,4-Dinitrophenol↗