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Biomedical subjects

A Greco

Publications and source records attributed to A Greco.

At least 91 records · Page 5Linked to original sources
PubMed1995

A phase I study of etoposide phosphate administered as a daily 30-minute infusion for 5 days.

PURPOSE: To determine the maximum tolerated dose, toxicities, kinetics, and disposition of etoposide phosphate when administered as a daily 30-minute infusion for 5 days. PATIENTS AND METHODS: Twenty-eight patients were enrolled in this phase I dose-escalation trial. Cohorts of patients received etoposide phosphate in etoposide equivalent doses of 50, 75, 100 and 125 mg/m2 intravenously for 30 minutes each day for 5 days. Pharmacokinetic sampling of both blood and urine was performed and concentrations of etoposide and etoposide phosphate were determined on day 1 of study for each patient and on day 4 of study for three patients receiving the 100 mg/m2 dose. RESULTS: The dose-limiting toxicity was reversible myelosuppression as evidenced by leukopenia and neutropenia. Toxicities seen were comparable to those expected from etoposide administration. With this schedule, the 100 mg/m2 dose was the maximum tolerated dose. Nonhematologic toxicities were generally mild. Two patients had major responses and three others had minor responses. Pharmacokinetic analyses revealed rapid (< 15 minutes) extensive conversion of etoposide phosphate to etoposide. Peak plasma etoposide concentrations and etoposide areas under the curve were proportional to the dose of etoposide phosphate administered. Etoposide kinetics were similar to those expected after a comparable dose of etoposide. CONCLUSIONS: Etoposide phosphate is a water-soluble pro-drug of etoposide that is rapidly converted to etoposide in vivo with a toxicity profile similar to etoposide. Etoposide generated from etoposide phosphate exhibits linear kinetics over a dose range of 50 to 125 mg/m2. When administered as a daily 30-minute infusion for 5 days, the dose-limiting toxicity is myelosuppression and 100 mg/m2 daily is the maximum tolerated dose.

Adult↗
PubMed1995

Effects of nabumetone on prostanoid biosynthesis in humans.

BACKGROUND: The active metabolite of the anti-inflammatory drug nabumetone has been characterized as a selective inhibitor of the inducible prostaglandin H synthase (PGHS). The aim of this study was to investigate the rate of eicosanoid biosynthesis after oral dosing with nabumetone in nine healthy subjects. METHODS: We measured the urinary excretion of products of platelet (11-dehydro-thromboxane B2 [TXB2]) and renal (prostaglandin IF2 alpha [PGF2 alpha]) arachidonate metabolism as in vivo indexes of the constitutive PGHS-1 pathway. Moreover, the production of TXB2 during whole blood clotting was assessed as an index of the cyclooxygenase activity of platelet PGHS-1 ex vivo. RESULTS: At steady state, nabumetone (500 and 1000 mg daily for 7 days) was associated with statistically significant dose-dependent reduction in the urinary excretion of 11-dehydro-TXB2 and serum TXB2 levels by approximately 50% to 70%. However, the drug did not significantly affect the urinary excretion of PGF2 alpha. After discontinuation of nabumetone, urinary 11-dehydro-TXB2 excretion and whole blood TXB2 production returned to predrug levels with a similar timecourse that was consistent with the elimination half-life of its active metabolite. The daily administration of low-dose aspirin (40 mg), a selective inhibitor of platelet PGHS-1, caused a cumulative inhibition of urinary 11-dehydro-TXB2 and whole blood TXB2 production that recovered with a timecourse consistent with platelet turnover. CONCLUSIONS: Nabumetone does dose-dependently inhibit the cyclooxygenase activity of platelet PGHS-1 of healthy subjects both in vivo and ex vivo. Thus it is unlikely that its safety profile in patients may be related to selective inhibition of the inducible PGHS-2.

Administration, Oral↗
PubMed1995

Loss of function effect of RET mutations causing Hirschsprung disease.

We have introduced three Hirschsprung (HSCR) mutations localized in the tyrosine kinase domain of RET into the RET/PTC2 chimaeric oncogene which is capable of transforming NIH3T3 mouse fibroblasts and of differentiating pC12 rat pheochromocytoma cells. The three HSCR mutations abolished the biological activity of RET/PTC2 in both cell types and significantly decreased its tyrosine phosphorylation. By contrast, a rare polymorphism in exon 18 does not alter the transforming capability of RET/PTC2 or its tyrosine phosphorylation. These data suggest a loss of function effect of HSCR mutations which might act through a dominant negative mechanism. Our model system is therefore capable of discriminating between causative HSCR mutations and rare polymorphisms in the tyrosine kinase domain of RET.

3T3 Cells↗
PubMed1995

Effects of the novel anti-inflammatory compounds, N-[2-(cyclohexyloxy)-4-nitrophenyl] methanesulphonamide (NS-398) and 5-methanesulphonamido-6-(2,4-difluorothio-phenyl)-1-inda none (L-745,337), on the cyclo-oxygenase activity of human blood prostaglandin endoperoxide synthases.

1. We have evaluated the selectivity of ketoprofen and two novel nonsteroidal anti-inflammatory drugs, N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulphonamide (NS-398) and 5-methanesulphonamido-6-(2,4-difluorothiophenyl)-1-indano ne (L-745,337), in inhibiting the cyclo-oxygenase activity of prostaglandin endoperoxide synthase-2 (PGHS-2) vs PGHS-1 in human blood monocytes and platelets, respectively. 2. Heparinized whole blood samples were drawn from healthy volunteers pretreated with aspirin, 300 mg 48 h before sampling, to suppress the activity of platelet PGHS-1 and incubated at 37 degrees C for 24 h with increasing concentrations of the test compounds in the presence of lipopolysaccharide (LPS, 10 micrograms ml-1). Immunoreactive PGE2 levels were measured in plasma by a specific radioimmunoassay as an index of the cyclo-oxygenase activity of LPS-induced monocyte PGHS-2. 3. The effects of the same inhibitors on platelet PGHS-1 activity were assessed by allowing whole blood samples, drawn from the same subjects in aspirin-free periods, to clot at 37 degrees C for 1 h in the presence of the compounds and measuring immunoreactive thromboxane B2 (TXB2) levels in serum by a specific radioimmunoassay. 4. Under these experimental conditions, ketoprofen enantioselectively inhibited the cyclo-oxygenase activity of both PGHS-1 and PGHS-2 with equal potency (IC50 ratio: approx. 0.5 for both enantiomers), while L-745,337 and NS-398 achieved selective inhibition of monocyte PGHS-2 (IC50 ratio: > 150). L-745,337 and NS-398 did not affect LPS-induced monocyte PGHS-2 biosynthesis to any detectable extent. 5. We conclude that L-745,337 and NS-398 are selective inhibitors of the cyclo-oxygenase activity of human monocyte PGHS-2. These compounds may provide adequate tools to test the contribution of this novel pathway of arachidonate metabolism to human inflammatory disease.

Adult↗
PubMed1995

The DNA rearrangement that generates the TRK-T3 oncogene involves a novel gene on chromosome 3 whose product has a potential coiled-coil domain.

Oncogenic rearrangements of the NTRK1 gene (also designated TRKA), encoding one of the receptors for the nerve growth factor, are frequently detected in thyroid carcinomas. Such rearrangements fuse the NTRK1 tyrosine kinase domain to 5'-end sequences belonging to different genes. In previously reported studies we have demonstrated that NTRK1 oncogenic activation involves two genes, TPM3 and TPR, both localized similarly to the receptor tyrosine kinase, on the q arm of chromosome 1. Here we report the characterization of a novel NTRK1-derived thyroid oncogene, named TRK-T3. A cDNA clone, capable of transforming activity, was isolated from a transformant cell line. Sequence analysis revealed that TRK-T3 contains 1,412 nucleotides of NTRK1 preceded by 598 nucleotides belonging to a novel gene that we have named TFG (TRK-fused gene). The TRK-T3 amino acid sequence displays, within the TFG region, a coiled-coil motif that could endow the oncoprotein with the capability to form complexes. The TRK-T3 oncogene encodes a 68-kDa cytoplasmic protein reacting with NTRK1-specific antibodies. By sedimentation gradient experiments the TRK-T3 oncoprotein was shown to form, in vivo, multimeric complexes, most likely trimers or tetramers. The TFG gene is ubiquitously expressed and is located on chromosome 3. The breakpoint producing the TRK-T3 oncogene occurs within exons of both the TFG gene and the NTRK1 gene and produces a chimeric exon that undergoes alternative splicing. Molecular analysis of the NTRK1 rearranged fragments indicated that the chromosomal rearrangement is reciprocal and balanced and involves loss of a few nucleotides of germ line sequences.

3T3 Cells↗
PubMed1995

[Spiral computed tomography with arteriography in the diagnosis of digestive system hemorrhages of obscure origin].

Some 5-20% of gastrointestinal (GI) bleeding cases are of obscure origin because bleeding is difficult to diagnose or locate with conventional imaging methods. The authors suggest arterial spiral CT (ASCT) of the abdomen as an original tool to study GI bleeding of obscure origin. This diagnostic method consists of two subsequent phases: first, after abdominal aorta catheterization, the catheter is positioned in the celiac artery; then, CT of the abdomen is performed before and after intra-arterial contrast agent injection. In 6 of 10 patients with GI bleeding of obscure origin, ASCT depicted small high-intensity areas in the intestinal lumen, which indicate a source of bleeding. The site of bleeding was studied only with topographic criteria according to scanning planes, and the morphological features of the intestinal segments were investigated. All patients were submitted to surgery: when ASCT identified a bleeding site, angiography was limited to the involved arterial district. Angiography alone could locate the source of bleeding in 5 patients; angiography and ASCT were in agreement in 4 patients. The combined techniques located the source of bleeding in 7/10 patients and the nature of bleeding was diagnosed in 3/10 patients (angiodysplasia). Even though their experience is limited, the authors believe that ASCT can be useful in locating GI bleeding of obscure origin, as well as in guiding subsequent selective angiography.

Adult↗
PubMed1994

Potentiometric determination of carbonic anhydrase activity in rabbit carotid bodies: comparison among normoxic, hyperoxic and hypoxic animals.

The catalytic activity of carbonic anhydrase (CA) contained in the glomus cells of mammalian carotid bodies has been determined in vitro by a potentiometric method. Experiments performed on whole rabbit carotid bodies have shown a very low variability, in terms of the overall CA activity, among organs belonging to different animals maintained in normoxic conditions. Repeated assays performed on each carotid body have shown a marked decrease of the overall CA activity after the first assay, thus suggesting the presence of at least two different forms of enzyme. Experiments performed on carotid bodies belonging to rabbits maintained in normal, hyperoxic and hypoxic conditions have shown that the overall CA activity follows the sequence: hypoxic > normoxic > hyperoxic, matching with the corresponding physiological activity of the carotid body.

Animals↗
PubMed1994

The DNA sequence coding for the 5' untranslated region of herpes simplex virus type 1 ICP22 mRNA mediates a high level of gene expression.

The sequence coding for the 5' untranslated region (UTR) of ICP22 mRNA of herpes simplex virus type 1 has been tested for its ability to regulate gene expression. This sequence was placed in frame with the chloramphenicol acetyltransferase (CAT) coding sequence and under the control of the simian virus 40 early promoter-enhancer. Under these conditions, the sequence coding for the 5'UTR led to an increase of about 13-fold in CAT activity, measured during transient expression. The use of mutants with progressive deletions within the sequence coding for the 5'UTR allowed localization of the sequence responsible for the enhancement of gene expression to the first exon of the ICP22 gene. Precise quantification of hybrid ICP22-CAT mRNA showed that the sequence coding for the 5'UTR induced an increase in the amounts of transcripts, which resulted in a parallel increase in CAT activity. This increase in the level of hybrid ICP22-CAT mRNA is not the result of an increase in mRNA stability, nor is it due to more efficient nucleo-cytoplasmic transport of the transcripts. Moreover, the distribution of hybrid mRNA in the different ribosomal populations indicates that the 5'UTR of ICP22 mRNA does not induce a preferential recruitment of the transcripts by the translational apparatus. Taken together, these results indicate that a cis-acting element located in the sequence coding for the 5'UTR of ICP22 mRNA can mediate a high level of gene expression independently of the viral promoter and of viral trans-acting factors.

Base Sequence↗
PubMed1994

Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases.

The aim of our study was to characterize a model of human prostaglandin endoperoxide synthase-2 (PGHS-2) expression allowing the assessment of pharmacological inhibition in vitro and ex vivo. Heparinized human whole blood samples were incubated with lipopolysaccharide (LPS, 0.1-50 micrograms/ml) for 0 to 24 hr at 37 degrees C. The contribution of platelet PGHS-1 was suppressed by either pretreating the subjects with aspirin (300 mg 48 hr before sampling) or adding aspirin (10 micrograms/ml) in vitro at time 0. PGE2 was measured by radioimmunoassay. LPS induced expression of cyclooxygenase activity in a time- and concentration-dependent fashion. After 24 hr at 10 micrograms/ml LPS, PGE2 production averaged 12.1 +/- 6.2 ng/ml (mean +/- S.D., n = 7). Cyclooxygenase activity increased in parallel with the mass of a monocyte protein doublet analyzed by Western blot using antibodies directed against the carboxyl-terminal portion of human PGHS-2. Dexamethasone (2 microM) inhibited LPS-induced PGE2 production by 96 +/- 4% (mean +/- S.D., n = 3). Four different inhibitors were tested in vitro on the cyclooxygenase activity of LPS-induced monocyte PGH-2 and thrombin-stimulated platelet PGHS-1. IC50 values (microM) for inhibition of PGHS-1 and PGHS-2 were: indomethacin, 0.70 +/- 0.20 vs 0.36 +/- 0.10 (P < .05); S-indobufen, 0.64 +/- 0.22 vs. 14.9 +/- 8 (P < .05), R-indobufen, 38 +/- 18 vs. 230 +/- 68 (P < .01), 6-methoxy-2-naphthyl acetic acid (the active metabolite of nabumetone), 278 +/- 96 vs. 187 +/- 96.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

The oncogenic versions of the Ret and Trk tyrosine kinases bind Shc and Grb2 adaptor proteins.

Proto-TRK and proto-RET genes encode receptor type tyrosine kinases. Oncogenic rearrangements of both proto-oncogenes have been detected with a significant frequency in human papillary thyroid carcinomas. Chimeric Ret and Trk oncoproteins, encoded by different rearrangements of proto-TRK and proto-RET genes, display a constitutive phosphorylation on tyrosine. Moreover, it has been shown that phosphorylated tyrosine receptors, activated by their ligands, form multiprotein complexes responsible for transducing mitogenic or differentiation signals. We have therefore begun to analyse in this study the signal transduction pathways triggered by different Ret and Trk oncoproteins. We have shown that the SH2 domain of the adaptor protein Shc coimmunoprecipitates with all the Ret and Trk oncoproteins as well as with NGF-activated proto-Trk receptor. Tyrosine phosphorylation of Trk proteins both normal and oncogenic is necessary for their binding to Shc. In addition, in cells containing either Ret or Trk oncoproteins, Shc proteins are constitutively phosphorylated on tyrosine and bound to Grb2. Only in in vitro experiments were Ret and Trk oncoproteins shown to bind the SH2 region of Grb2. Finally, when proto-Trk product is stimulated by NGF, Shc phosphorylation and association with Grb2 are induced. In conclusion, we have shown that Ret and Trk oncoproteins can form multiprotein complexes, however, the functional meaning of the described interactions has to be elucidated.

3T3 Cells↗
PubMed1993

Precise quantitation of chloramphenicol acetyl transferase reporter mRNA by competitive polymerase chain reaction.

A strategy, based on competitive polymerase chain reaction (PCR) after reverse transcription, was developed to quantitate mRNA of the E. coli chloramphenicol acetyl transferase (CAT) gene. Precise quantitation of this rare mRNA is achieved by co-amplification of a constant amount of the complementary DNA (cDNA) with successive dilutions of a competitive DNA template of known concentration and with the use of the same primers. The unique EcoRI site located in the CAT coding sequence has been abolished in the competitive DNA. The two oligonucleotide primers used are both located in the CAT coding sequence at equal distance from the EcoRI site. After amplification, the PCR products from the cDNA to be quantified and from the competitor DNA are discriminated by EcoRI digestion, followed by separation of the resulting fragments by agarose gel electrophoresis. DNA amplified from the target cDNA is the only DNA digested by EcoRI into two fragments of identical size co-migrating in an agarose gel. After ethidium bromide staining, comparison of the intensity of fluorescence of the resulting bands in a competition series permits us to estimate precisely the amount of CAT cDNA and therefore of the mRNA to be quantified.

Base Sequence↗