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Biomedical subjects

M Gianni

Publications and source records attributed to M Gianni.

17 recordsLinked to original sources
PubMed2003

Design and synthesis of novel antihypertensive drugs.

AT1 antagonists constitute a new generation of drugs for the treatment of hypertension and are designed and synthesized to mimic the C-terminal segment of Angiotensin II (Ang II) and to block its binding action on AT1 receptor. For this reason, the conformational analysis of Ang II and its derivatives as well as the AT1 antagonists belonging to SARTANs class of molecules were studied. Such studies offer the possibility to reveal the stereoelectronic factors responsible for bioactivity of AT1 antagonists and to design and synthesize new analogues with better pharmacological and financial profiles. An example of a novel synthetic non-peptide molecule is given which mimics the His(6)-Pro(7)-Phe(8) part of Ang II and is based on the (S)-pyroglutamic acid.

Angiotensin II↗
PubMed2000

Retinoid-dependent growth inhibition, differentiation and apoptosis in acute promyelocytic leukemia cells. Expression and activation of caspases.

In the NB4 model of acute promyelocytic leukemia (APL), ATRA, 9-cis retinoic acid (9-cis RA), the pan-RAR and RARalpha-selective agonists, TTNPB and AM580, induce growth inhibition, granulocytic differentiation and apoptosis. By contrast, two RXR agonists, a RARbeta agonist and an anti-AP1 retinoid have very limited activity, ATRA- and AM580-dependent effects are completely inhibited by RAR antagonistic blockade, while 9-cis RA-induced cell-growth-inhibition and apoptosis are equally inhibited by RAR and RXR antagonists. ATRA, 9-cis RA and AM580 cause upregulation of the mRNAs coding for pro-caspase-1, -7, -8, and -9, which, however, results in increased synthesis of only pro-caspase-1 and -7 proteins. These phenomena are associated with activation of pro-caspase-6, -7 and -8, cytochrome c release from the mitochondria, inversion of Bcl-2/Bax ratio and degradation of PML-RARalpha. Caspase activation is fundamental for retinoid-induced apoptosis, which is suppressed by the caspase-inhibitor z-VAD.

Alitretinoin↗
PubMed1999

Retinoic acid induces proteasome-dependent degradation of retinoic acid receptor alpha (RARalpha) and oncogenic RARalpha fusion proteins.

Analyzing the pathways by which retinoic acid (RA) induces promyelocytic leukemia/retinoic acid receptor alpha (PML/RARalpha) catabolism in acute promyelocytic leukemia (APL), we found that, in addition to caspase-mediated PML/RARalpha cleavage, RA triggers degradation of both PML/RARalpha and RARalpha. Similarly, in non-APL cells, RA directly targeted RARalpha and RARalpha fusions to the proteasome degradation pathway. Activation of either RARalpha or RXRalpha by specific agonists induced degradation of both proteins. Conversely, a mutation in RARalpha that abolishes heterodimer formation and DNA binding, blocked both RARalpha and RXRalpha degradation. Mutations in the RARalpha DNA-binding domain or AF-2 transcriptional activation region also impaired RARalpha catabolism. Hence, our results link transcriptional activation to receptor catabolism and suggest that transcriptional up-regulation of nuclear receptors by their ligands may be a feedback mechanism allowing sustained target-gene activation.

Caspases↗
PubMed1999

Retinoic acid, but not arsenic trioxide, degrades the PLZF/RARalpha fusion protein, without inducing terminal differentiation or apoptosis, in a RA-therapy resistant t(11;17)(q23;q21) APL patient.

Primary blasts of a t(11;17)(q23;q21) acute promyelocytic leukaemia (APL) patient were analysed with respect to retinoic acid (RA) and arsenic trioxide (As2O3) sensitivity as well as PLZF/RARalpha status. Although RA induced partial monocytic differentiation ex vivo, but not in vivo, As203 failed to induce apoptosis in culture, contrasting with t(15;17) APL and arguing against the clinical use of As203 in t(11;17)(q23;q21) APL. Prior to cell culture, PLZF/RARalpha was found to exactly co-localize with PML onto PML nuclear bodies. However upon cell culture, it quickly shifted towards microspeckles, its localization found in transfection experiments. Arsenic trioxide, known to induce aggregation of PML nuclear bodies, left the microspeckled PLZF/RARalpha localization completely unaffected. RA treatment led to PLZF/RARalpha degradation. However, this complete PLZF/RARalpha degradation was not accompanied by differentiation or apoptosis, which could suggest a contribution of the reciprocal RARalpha/PLZF fusion product in leukaemogenesis or the existence of irreversible changes induced by the chimera.

Antineoplastic Agents↗
PubMed1997

Stat1 is induced and activated by all-trans retinoic acid in acute promyelocytic leukemia cells.

Treatment of freshly isolated acute promyelocytic leukemia (APL) cells and the myelogenous leukemia cell lines, NB4, HL-60, and U937, with all-trans retinoic acid (ATRA) results in a remarkable elevation in the amounts of Stat1 alpha and Stat2 proteins. Stat1 alpha protein levels are augmented by ATRA as a consequence of elevated amounts of the corresponding transcripts. The retinoid increases the levels of nuclear complexes that are capable of binding to interferon (IFN)-regulated consensus sequences and contain Stat1 and/or Stat2 proteins, and causes a rapid and long-lasting elevation in Stat1 alpha tyrosine phosphorylation. Transient transfection experiments show that ATRA enhances the transactivating properties of Stat1 alpha observed on an appropriate reporter gene, in the presence of the RAR alpha retinoic acid receptor, but not in the presence of the PML-RAR protein. Treatment of NB4 cells with ATRA is associated with a remarkable upregulation of the two IFN-responsive genes IFN-responsive factor 1 and 2'-5' oligoadenylate synthetase, as well as with an augmentation in the levels of IFN alpha secretion. Our data show that ATRA is capable of modulating the amounts and the state of activation of some of the components of the IFN intracellular signaling pathways. They also suggest that the retinoid can bypass IFN/IFN-receptor interactions and induce the expression of IFN-regulated genes.

2',5'-Oligoadenylate Synthetase↗
PubMed1996

Leukocyte alkaline phosphatase a specific marker for the post-mitotic neutrophilic granulocyte: regulation in acute promyelocytic leukemia.

Leukocyte alkaline phosphatase (LAP) is the product of the gene coding for the liver/bone/kidney-type alkaline phosphatase. In the normal hematopoietic system, the only cell type expressing LAP in basal conditions is the post-mitotic neutrophilic granulocyte. Thus LAP represents a specific and restrictive marker for the terminal maturation of the neutrophilic granulocyte. The study of the factors and the molecular mechanisms responsible for the expression of LAP in cells undergoing granulocytic maturation may shed light on this complex biological process. Acute promyelocytic leukemia (APL) represents a unique biological model in which it is possible to investigate neutrophilic differentiation. APL blasts undergo rapid and irreversible maturation towards cells morphologically and biochemically resembling normal mature granulocytes upon in vivo and in vitro challenge with all-trans retinoic acid (ATRA). In this cellular context, we studied the endogenous factors involved in the expression of LAP. The phosphatase is not synthesized in undifferentiated APL blasts and it is expressed only upon treatment with combinations between ATRA and a second cyto-differentiating signal. The second signal may be given by G-CSF, cAMP analogs, IL-6 and to a lesser extent by IL-1 beta. The molecular mechanisms underlying the induction of LAP by combinations of ATRA and G-CSF or cAMP analogs were studied in detail and are the object of this review.

Alkaline Phosphatase↗
PubMed1995

Tyrosine kinases but not cAMP-dependent protein kinase mediate the induction of leukocyte alkaline phosphatase by granulocyte-colony-stimulating factor and retinoic acid in acute promyelocytic leukemia cells.

Leukocyte alkaline phosphatase (LAP) is synergistically induced by the combination of all-trans retinoic acid (ATRA) and granulocyte-colony-stimulating factor (G-CSF) in acute promyelocytic leukemia (APL) cells (Gianni' M. et al., Blood 83: 1909-1921, 1994). The role of cAMP and tyrosine kinases in the induction of LAP was investigated. In the APL cell line NB4, adenosine-3': 5'-monophosphothioate, cyclic, Rp isomer, a reversible inhibitor of cAMP-dependent protein kinase (PKA), has no effect on the induction of LAP enzymatic activity and mRNA triggered by ATRA+G-CSF, in conditions where this compound completely blocks the upregulation of LAP transcript caused by the combination of the PKA agonist, dibutyryl-cAMP (db-cAMP), and ATRA. Challenge of NB4 cells with G-CSF, dbcAMP and ATRA causes a much higher induction of LAP relative to that observed in the presence of ATRA+G-CSF or ATRA+dbcAMP. Treatment of NB4 with ATRA and G-CSF results in increases in the tyrosine phosphorylation of several proteins. In the presence of the cytokine and the retinoid, tyrosine kinase inhibitors decrease LAP enzymatic activity and mRNA.

Alkaline Phosphatase↗
PubMed1993

Effects of synthetic retinoids and retinoic acid isomers on the expression of alkaline phosphatase in F9 teratocarcinoma cells.

Expression of ALP in F9 teratocarcinoma cells is induced by all-trans retinoic acid (ATRA) (Gianni' et al., Biochem. J. 274: 673-678, 1991). The specific ligand for retinoic acid related receptors (RXRs), 9-cis retinoic acid (9-cis RA), and three synthetic analogs binding to the alpha, beta and gamma forms of the retinoic acid receptors (RARs), AM580, CD2019, and CD437, were used to study their effects on alkaline phosphatase (ALP) enzymatic activity and mRNA levels. At concentrations close to the Kd for their respective receptors, 9-cis RA, AM580 (the RAR alpha agonist) and CD437 (the RAR gamma agonist) clearly upregulate the expression of the ALP gene, whereas the effect of CD2019 (the RAR beta agonist) is very modest. A specific inhibitor of the RAR alpha, Ro 41-5253, completely blocks the induction of ALP triggered by AM580, while it has minor effects on the upregulation caused by ATRA, 9-cis RA, CD437 and CD2019. The induction of ALP observed with the various retinoids is inhibited by the contemporaneous treatment with dibutyryl cAMP. The levels of the RAR alpha and gamma transcripts are unaltered, while RAR beta mRNAs are induced by ATRA, AM580, CD437 and to a lower extent by 9-cis RA and CD2019.

Alkaline Phosphatase↗
PubMed1992

Inhibition of melanogenesis by BMY-28565, a novel compound depressing tyrosinase activity in B16 melanoma cells.

The mechanism of a novel melanin synthesis inhibitor, BMY-28565, was studied using mouse B16 melanoma cells. This compound was active in depressing the intracellular accumulation of melanin with an IC50 of 5 microM. At dose levels causing no cytotoxicity, the melanolytic effect of this compound was correlated strongly with depression of the enzymatic activity of tyrosinase (monophenol oxygenase, EC 1.14.18.1), the key enzyme in the melanin synthesis pathway. Transcription of the tyrosinase gene was not inhibited by BMY-28565, as determined by RNA blotting analysis. BMY-28565 and three other active derivatives of this compound caused increased glycosylation of proteins in B16 melanoma cells, as assessed by radioactive mannose incorporation. It is, thus, suggested that the mechanism of inhibition of tyrosinase might be related to modifications of the sugar moiety of this enzyme or of a protein(s) that is essential for the expression of its enzymatic activity.

Animals↗
PubMed1991

Characterization of a second promoter for the mouse liver/bone/kidney-type alkaline phosphatase gene: cell and tissue specific expression.

The second leader exon and the relative promoter of the mouse liver/bone/kidney-type alkaline phosphatase gene were identified and characterized. The transcription initiation site was determined by S1 mapping analysis. The differential expression of the two alternatively spliced transcripts was assessed in cell lines of different origin and in various tissues by polymerase chain reaction and RNase mapping analysis. The first promoter is active in embryo derived cells, whereas the second promoter is silent in basal conditions but it is activated by dibutyryl cAMP in fibroblastic cells. In the whole animal, the transcript driven by the first promoter is found in most tissues albeit at different levels, while the one driven by the second promoter is specifically expressed at high levels only in the heart.

Alkaline Phosphatase↗
PubMed1991

Retinoic acid induces liver/bone/kidney-type alkaline phosphatase gene expression in F9 teratocarcinoma cells.

All-trans retinoic acid (RA) induces alkaline phosphatase (ALP) activity by 3-8-fold in murine F9 teratocarcinoma cells, in parallel with their differentiation towards primitive endoderm. The elevation of ALP activity is associated with increases in the amounts of liver/bone/kidney-type ALP protein and the respective transcript. These effects of RA are due to activation of ALP gene transcription rather than to an increase in the half-life of the mRNA. Induction of ALP mRNA does not require de novo protein synthesis, since it is not blocked by treatment with cycloheximide. Dibutyryl cyclic AMP, which is known to induce further differentiation of F9 cells from the primitive to the parietal endoderm, blocks the induction of ALP mRNA by RA.

Alkaline Phosphatase↗
PubMed1988

Expression of hepatitis B surface antigen in human cells by a recombinant BK virus DNA vector.

The construction of the first stable human cell lines that express and secrete authentic hepatitis B virus surface antigen (HBsAg), using a BK virus (BKV) episomal plasmid vector, is described. The amount of HBsAg produced by BKV vectors (up to 600 ng/10(7) cells) was comparable to other eukaryotic vector systems. The level of HBsAg expression remained the same regardless of the orientation of the HBsAg gene, substitution of the HBsAg gene promoter with the mouse metallothionein I gene promoter or the tissue origin of the human cell lines used to establish stable cellular transformants. Northern blot analysis also indicated synthesis of normal HBsAg transcripts. Surprisingly, however, the vectors were maintained at far lower than expected copy number (one to five copies/cell). Reasons for this are discussed.

BK Virus↗
PubMed1986

Isolation of genes preferentially expressed during Bacillis subtilis spore outgrowth.

From the Charon 4A library of Ferrari et al (J. Bacteriol. 146:430-432, 1981) we isolated three genes involved in Bacillus subtilis spore outgrowth by screening the library by hybridization with labeled RNA from outgrowing spores in the presence of an excess of unlabeled vegetative RNA. Hybridization competition experiments with purified clones showed that the clones contained sequences that were transcribed only during spore outgrowth or preferentially during spore outgrowth. Fragments of the cloned DNAs were subcloned in plasmid pJH101, and by using plasmid integration and PBS1 transduction the chromosomal loci were mapped. The three loci which we mapped are outG and outH, which are located between polC and dnaA, and outI, which is located near pycA. Using the cloned DNAs and derived plasmids in dot hybridization experiments with RNA extracted from cells at different developmental stages, we defined for two clones a region that is transcribed only during the outgrowth phase.

Bacillus subtilis↗
PubMedDate not supplied

Anaplastic lymphoma kinase (ALK) is not expressed in Hodgkin's disease: results with ALK-11 antibody in 327 untreated patients.

The t(2;5)(p23;q35) or other rare chromosomal abnormalities involving 2p23 upregulate the ALK gene, which is not expressed in normal lymphocytes. Thus, detection of ALK protein is presumptive evidence of these 2p23 abnormalities. The t(2;5) and ALK immunoreactivity are common in anaplastic large cell lymphoma of T/null-cell lineage. However, a small subset of cases of Hodgkin's disease (HD) have been reported to either carry the t(2;5) or express ALK. In this study, we have immunohistochemically evaluated 327 cases of HD with the ALK-11 antibody. ALK-11 is a well characterized polyclonal antibody raised against an intracellular portion of the ALK protein. We detected ALK-11 immunoreactivity in 8 (2.4%) cases of HD. We further studied these positive cases with ALK-1 monoclonal antibody, which reacts with an intracellular portion of ALK, similar to ALK-11. All 8 ALK-11 positive cases were negative for ALK-1. These results indicate that rare cases of HD may react with ALK-11 antibody, similar to previous reports by others using different polyclonal anti-ALK antibodies. However, the absence of ALK-1 expression in these HD cases suggests that ALK protein is not truly present and that polyclonal anti-ALK antibodies may rarely yield non-specific cross reactivity. These results further support the use of anti-ALK antibodies in the differential diagnosis of HD from ALCL.

Adolescent↗
PubMedDate not supplied

Flow cytometry to estimate circulating hematopoietic progenitors for autologous transplantation: comparative analysis of different CD34 monoclonal antibodies.

We report that the 2 fluorescein (FITC) conjugated CD34 monoclonal antibodies available to date, namely FITC-8G12 and FITC-QBEND10, exhibit different capabilities of detecting circulating hematopoietic progenitors (CHP) as cells expressing the CD34 antigen (CD34+) by direct immunofluorescence flow cytometry. Mean fluorescence intensity conferred by FITC-QBEND10 to CD34+ CHP is 2.8-4.3 times lower than that conferred by FITC-8G12. By indirect immunofluorescence, native unconjugated QBEND10 and 8G12 antibodies detect CD34+ CHP in a comparable manner, thus indicating that the decrease in QBEND10 affinity is due to FITC-conjugation. Utilization of FITC-QBEND10 instead of FICT-8G12 to estimate infrequent (usually less than or equal to 5%) CD34+ CHP for clinical decision-making in autologous transplantation of CHP exposes clinicians to the risk of either overestimating (false positive) or underestimating (false negative) these cells in peripheral blood. Recently published guidelines for large-scale collection of CHP for autologous transplantation in cancer patients were based on data obtained with FITC-8G12. The same guidelines cannot be considered valid if FITC-QBEND10 is employed. We recommend FITC-8G12 as the optimal reagent to date for standardizing results of autologous CHP transplantation in cancer therapy.

Antibodies, Monoclonal↗