Biomedical subjects

Antonio Ferrante

Publications and source records attributed to Antonio Ferrante.

14 recordsLinked to original sources

PubMed2006

Drought, abscisic acid and transpiration rate effects on the regulation of PIP aquaporin gene expression and abundance in Phaseolus vulgaris plants.

BACKGROUND AND AIMS: Drought causes a decline of root hydraulic conductance, which aside from embolisms, is governed ultimately by aquaporins. Multiple factors probably regulate aquaporin expression, abundance and activity in leaf and root tissues during drought; among these are the leaf transpiration rate, leaf water status, abscisic acid (ABA) and soil water content. Here a study is made of how these factors could influence the response of aquaporin to drought. METHODS: Three plasma membrane intrinsic proteins (PIPs) or aquaporins were cloned from Phaseolus vulgaris plants and their expression was analysed after 4 d of water deprivation and also 1 d after re-watering. The effects of ABA and of methotrexate (MTX), an inhibitor of stomatal opening, on gene expression and protein abundance were also analysed. Protein abundance was examined using antibodies against PIP1 and PIP2 aquaporins. At the same time, root hydraulic conductance (L), transpiration rate, leaf water status and ABA tissue concentration were measured. KEY RESULTS: None of the treatments (drought, ABA or MTX) changed the leaf water status or tissue ABA concentration. The three treatments caused a decline in the transpiration rate and raised PVPIP2;1 gene expression and PIP1 protein abundance in the leaves. In the roots, only the drought treatment raised the expression of the three PIP genes examined, while at the same time diminishing PIP2 protein abundance and L. On the other hand, ABA raised both root PIP1 protein abundance and L. CONCLUSIONS: The rise of PvPIP2;1 gene expression and PIP1 protein abundance in the leaves of P. vulgaris plants subjected to drought was correlated with a decline in the transpiration rate. At the same time, the increase in the expression of the three PIP genes examined caused by drought and the decline of PIP2 protein abundance in the root tissues were not correlated with any of the parameters measured.

Abscisic Acid↗

PubMed2006

A novel beta-oxa polyunsaturated fatty acid downregulates the activation of the IkappaB kinase/nuclear factor kappaB pathway, inhibits expression of endothelial cell adhesion molecules, and depresses inflammation.

Several novel polyunsaturated fatty acids (PUFAs) that contain either an oxygen or sulfur atom in the beta-position were found to exhibit more selective antiinflammatory properties than their natural PUFA counterparts. One of these, beta-oxa-23:4n-6, unlike natural PUFAs, lacked ability to stimulate oxygen radical production in neutrophils but caused marked inhibition of agonist-induced upregulation of leukocyte adhesion to cultured human umbilical vein endothelial cells (HUVEC) and E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 expression. In addition, beta-oxa-23:4n-6 inhibited acute and chronic inflammatory responses in mice as well as the upregulation of adhesion molecule expression in arterial endothelium. This action of beta-oxa-23:4n-6 required a functional 12- but not 5-lipoxygenase or cyclooxygenases, consistent with its metabolism via the 12-lipoxygenase pathway. Whereas beta-oxa-23:4n-6 did not affect the activation of mitogen-activated protein kinases by tumor necrosis factor, activation of the IkappaB kinase/nuclear factor kappaB pathway was selectively inhibited. These novel PUFAs could form the basis for a potential new class of pharmaceuticals for treating inflammatory diseases, including atherosclerosis.

Animals↗

PubMed2006

Molecular approaches in the diagnosis of primary immunodeficiency diseases.

Over 120 inherited primary immunodeficiency diseases (PIDs) are known to exist. The genes responsible for many of these diseases have also been identified. Recent advances in diagnostic procedures have enabled these to be identified earlier and appropriately treated. While a number of approaches are available to identify mutations, direct sequencing remains the gold standard. This approach identifies the exact genetic change with substantial precision. We suggest that a sensitive and economical approach to mutation detection could be the direct sequencing of cDNA followed by the confirmatory sequencing of the corresponding exon. While screening techniques such as single-stranded conformation polymorphism (SSCP), heteroduplex analysis (HA), denaturing gradient gel electrophoresis (DGGE), and denaturing high-performance liquid chromatography (dHPLC) have proven useful, each has inherent advantages and disadvantages. We discuss these advantages and disadvantages and also discuss the potential of future sequencing technologies such as pyrosequencing, combinatorial sequencing-by-hybridization, multiplex polymerase colony (polony), and resequencing arrays as tools for future mutation detection. In addition we briefly discuss several high-throughput SNP detection technologies.

Algorithms↗

PubMed2005

Molecular action of 1,25-dihydroxyvitamin D3 and phorbol ester on the activation of the rat cytochrome P450C24 (CYP24) promoter: role of MAP kinase activities and identification of an important transcription factor binding site.

Although investigations of the transcriptional regulation of the rat cytochrome P450C24 [CYP24 (25-hydroxyvitamin D3 24-hydroxylase)] gene by 1,25D (1,25-dihydroxyvitamin D3) at either the genomic, or more recently at the non-genomic, level have provided insight into the mechanism of control of 1,25D levels, this regulation is still poorly characterized. Using HEK-293T cells (human embryonic kidney 293T cells), we reported that 1,25D induction of CYP24 requires JNK (c-Jun N-terminal kinase) but not the ERK1/2 (extracellular-signal-regulated kinase 1/2). The phenomenon of synergistic up-regulation of CYP24 expression by PMA and 1,25D is well known and was found to be protein kinase C-dependent. Whereas ERK1/2 was not activated by 1,25D alone, its activation by PMA was potentiated by 1,25D also. The importance of ERK1/2 for transcriptional synergy was demonstrated by transfection of a dominant-negative ERK1(K71R) mutant (where K71R stands for Lys71-->Arg), which resulted in a reduced level of synergy on a CYP24 promoter-luciferase construct. JNK was also shown to be required for synergy. We report, in the present study, the identification of a site located at -171/-163, about 30 bp upstream of the vitamin D response element-1 in the CYP24 proximal promoter. This sequence, 5'-TGTCGGTCA-3', is critical for 1,25D induction of CYP24 and is therefore termed the vitamin D stimulatory element. The vitamin D stimulatory element, a target for the JNK module, and an Ets-1 binding site were shown to be vital for synergy between PMA and 1,25D. This is the first report to identify the DNA binding sequences required for the synergy between PMA and 1,25D and a role for JNK on the CYP24 gene promoter.

Animals↗

PubMed2005

Novel role of lipoxygenases in the inflammatory response: promotion of TNF mRNA decay by 15-hydroperoxyeicosatetraenoic acid in a monocytic cell line.

The metabolism of arachidonic acid via the lipoxygenase and cyclooxygenase pathways generates metabolites that regulate the inflammatory response. Although products of lipoxygenase are classically proinflammatory, recently it has been demonstrated that lipoxins, 15-hydroperoxyeicosatetraenoic acid (15-HPETE) and 15-hydroxyeicosatetraenoic acid exhibit anti-inflammatory activity. We now demonstrate for the first time that 15-HPETE regulates the production of the proinflammatory cytokine TNF posttranscriptionally by promoting degradation of LPS-induced TNFmRNA in a human monocytic cell line, Mono Mac 6. 15-HPETE causes a significant increase in the rate of TNF but not G3PDHmRNA degradation in the presence of the transcription inhibitor, actinomycin D. The decay of TNFmRNA is accelerated 1.7-fold, and its half-life is decreased by 57%. In view of its chemical and physical properties, we propose that 15-HPETE may function by destabilizing TNFmRNA by interaction with a trans-activating protein bound to the AU-rich element of TNFmRNA.

Arachidonic Acid↗

PubMed2005

The immunomodulatory effects of novel beta-oxa, beta-thia, and gamma-thia polyunsaturated fatty acids on human T lymphocyte proliferation, cytokine production, and activation of protein kinase C and MAPKs.

We have recently demonstrated that a novel n-3 long chain polyunsaturated fatty acid (PUFA) (beta-oxa 21:3n-3) was a more potent and more selective anti-inflammatory agent than n-3 PUFA. To gain further insights into this technology, we synthesized other novel PUFA consisting of beta-oxa, beta-thia, and gamma-thia compounds. All three types displayed anti-inflammatory activity. Each of the unsaturated beta-oxa fatty acids showed similar inhibition of PHA-PMA-induced T cell proliferation with a parallel inhibition of TNF-beta production. However, beta-oxa 25:6n-3 and beta-oxa 21:4n-3 displayed lower inhibitory action on IFN-gamma production. Surprisingly, beta-oxa 23:4n-6 and beta-oxa 21:3n-6 had marginal effect on IL-2 production. Thus, structural variation can generate selectivity for different immunological parameters. The beta-thia compounds 23:4n-6, 21:3n-6, and 21:3n-3 were highly effective in inhibiting all immunological responses. Of the two gamma-thia PUFA tested, gamma-thia 24:4n-6 was a strong inhibitor of all responses apart from IL-2, but gamma-thia 22:3n-6 had very little inhibitory effect. Two of the most active compounds, beta-thia 23:4n-6 and beta-thia 21:3n-6, were studied in more detail and shown to have an IC(50) of 1-2 muM under optimal conditions. Thus, these PUFA retain the immunosuppressive properties of the n-3 PUFAs, 20:5n-3 and 22:6n-3, but not the neutrophil-stimulating properties. Their action on T lymphocytes is independent of cyclooxygenase or lipoxygenase activity, and they act at a postreceptor-binding level by inhibiting the activation of protein kinase C and ERK1/ERK2 kinases.

Cell Proliferation↗

PubMed2004

Characterization of the MEK5-ERK5 module in human neutrophils and its relationship to ERK1/ERK2 in the chemotactic response.

The role of the extracellular signal-regulated kinase (ERK) 1 and ERK2 in the neutrophil chemotactic response remains to be identified since a previously used specific inhibitor of MEK1 and MEK2, PD98059, that was used to provide evidence for a role of ERK1 and ERK2 in regulating chemotaxis, has recently been reported to also inhibit MEK5. This issue is made more critical by our present finding that human neutrophils express mitogen-activated protein (MAP) kinase/ERK kinase (MEK)5 and ERK5 (Big MAP kinase), and that their activities were stimulated by the bacterial tripeptide, formyl methionyl-leucyl-phenylalanine (fMLP). Dose response studies demonstrated a bell-shaped profile of fMLP-stimulated MEK5 and ERK5 activation, but this was left-shifted when compared with the profile of fMLP-stimulated chemotaxis. Kinetics studies demonstrated increases in kinase activity within 2 min, peaking at 3-5 min, and MEK5 activation was more persistent than that of ERK5. There were some similarities as well as differences in the pattern of activation between fMLP-stimulated ERK1 and ERK2, and MEK5-ERK5 activation. The up-regulation of MEK5-ERK5 activities was dependent on phosphatidylinositol 3-kinase. Studies with the recently described specific MEK inhibitor, PD184352, at concentrations that inhibited ERK1 and ERK2 but not ERK5 activity demonstrate that the ERK1 and ERK2 modules were involved in regulating fMLP-stimulated chemotaxis and chemokinesis. Our data suggest that the MEK5-ERK5 module is likely to regulate neutrophil responses at very low chemoattractant concentrations whereas at higher concentrations, a shift to the ERK1/ERK2 and p38 modules is apparent.

Chemotaxis↗

PubMed2004

Detection and significance of the potentially pathogenic amoeboflagellate Naegleria italica in Australia.

Thermophilic amoeboflagellates in the genus Naegleria include both virulent and benign species. One of the less studied species, N. italica, has not been detected in the environment since the first reports from Italy in the 1980s; its virulence is known only from infection of laboratory mice. Two recent strains from recreational water in Western Australia (AWQC NG960, NG961) were tentatively identified as N. italica from the characteristic mobilities of seven isozymes. Sequences of the 5.8S rRNA gene and its flanking ITS aligned with a 380+bp length of the published sequence for N. italica with 98% identity. Differences from the type strain were confined to ITS2. Shorter alignments (<320 bp) were observed with other Naegleria species, corresponding to conserved regions of the 5.8S gene and ITS. Unlike the European type strain of N. italica, the Australian isolates failed to infect laboratory mice intranasally, confirming that infectivity of this species is variable and often lower than in N. fowleri.

Amebiasis↗

PubMed2004

Role of abscisic acid in perianth senescence of daffodil (Narcissus pseudonarcissus"Dutch Master").

Daffodil (Narcissus pseudonarcissus L. 'Dutch Master') flowers detached at the base of their ovaries and held with their cut ends in 10-100 microM abscisic acid (ABA) senesced prematurely. Symptoms of the ABA treatment included water-soaking of the tepals and early collapse of the corona. No water-soaking was seen in tepals of flowers held in water. Instead, the tepals of these flowers dried. The ABA content increased in tepals of the potted flowers as they senesced. The rise in tepal ABA content coincided with the appearance of visual signs of senescence. When the flowers were cut and placed in water, a treatment that accelerated their senescence, the increase in ABA occurred earlier. Exogenously applied ABA enhanced the premature accumulation of senescence-associated transcripts in the tepals. Their ABA-mediated induction was not prevented when the flowers were pre-treated with 1-methylcyclopropene, an inhibitor of ethylene action, indicating that ABA induced the transcripts independently of ethylene. The transcripts accumulated in opened control flowers before the rise in endogenous ABA. Attempts to extend floral longevity by using putative inhibitors of ABA biosynthesis [tungstate, fluridone (applied as Sonar((R))) and 1,1-dimethyl-4-(phenylsulphonyl)semicarbazide (DPSS)] were unsuccessful. However, inclusion of 100 microM gibberellic acid (GA(3)) in the vase solution reduced the senescence-inducing effects of 50 microM ABA suggesting a possible mechanism for in-vivo control of senescence.

Journal Article↗

PubMed2003

Inhibition of neutrophil leukotriene B4 production by a novel synthetic N-3 polyunsaturated fatty acid analogue, beta-oxa 21:3n-3.

We recently reported the synthesis and anti-inflammatory properties of a novel long chain polyunsaturated fatty acid (PUFA) with an oxygen atom in the beta-position, beta-oxa-21:3 n-3 (Z,Z,Z)-(octadeca-9,12,15-trienyloxy) acetic acid). Our data, from studies aimed at elucidating the mechanism of its action, show that pretreatment of human neutrophils with the beta-oxa-PUFA substantially depresses the production of leukotriene B(4) (LTB(4)) in response to calcium ionophore, A23187, comparable to standard leukotriene inhibitors such as zileuton and nordihydroguaiaretic acid. Interestingly, the n-6 equivalent, beta-oxa 21:3 n-6, is also a strong inhibitor of LTB(4) production. In contrast, naturally occurring PUFA only slightly reduce, for eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids, or increase, for arachidonic acid (20:4n-6), the formation of LTB(4). The parent beta-oxa-21:3n-3 molecule, rather than its derivatives (methyl ester, saturated, monohydroperoxy, or monohydroxy forms), is exclusively responsible for attenuation of LTB(4) formation. beta-Oxa-21:3n-3 inhibits the conversion of [(3)H]20:4n-6 to [(3)H]5-hydroxyeicosatetraenoic acid and [(3)H]LTB(4) by neutrophils in the presence of calcium ionophore and also suppresses the activity of purified 5-lipoxygenase, but not cyclooxygenase 1 and 2. Beta-oxa-21:3n-3 is taken up by neutrophils and incorporated into phospholipids and neutral lipids. In the presence of calcium ionophore, the leukocytes convert a marginal amount of beta-oxa-21:3n-3 to a 16-monohydroxy-beta-oxa-21:3n-3 derivative. After administration to rodents by gavage or i.p. injection, beta-oxa-21:3n-3 is found to be incorporated into the lipids of various tissues. Thus, beta-oxa-21:3n-3 has the potential to be used in the treatment of inflammatory diseases, which are mediated by products of the lipoxygenase pathway.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2003

Unique effect of arachidonic acid on human neutrophil TNF receptor expression: up-regulation involving protein kinase C, extracellular signal-regulated kinase, and phospholipase A2.

Arachidonic acid (AA) regulates the function of many cell types, including neutrophils. Although much emphasis has been placed on agonist-induced down-regulation of TNFR, our data show that AA caused a rapid (10-20 min) and dose-dependent (0.5-30 micro M) increase in the surface expression of both classes of TNFR (TNFR1 and TNFR2) on human neutrophils. This increased TNFR expression correlated with an increase in TNF-induced superoxide production. In contrast, the omega3 fatty acids eicosapentaenoic acid, docosahexaenoic acid, and linolenic acid failed to stimulate TNFR expression. Although fMLP and LPS reduced the neutrophil expression of TNFR, when pretreated with AA, fMLP caused an increase in TNFR expression. Consistent with this result was the finding that AA prevented the fMLP-induced receptor release in neutrophil cultures. AA also caused an increase in TNFR expression in matured HL-60 cells (neutrophil-like cells), but a decrease in nonmatured cells and HUVEC. The AA effects were independent of the lipoxygenase and cyclooxygenase pathways, but dependent on protein kinase C, the extracellular signal-regulated kinases 1 and 2, and cytosolic phospholipase A(2). The data demonstrate a unique effect of AA in the inflammatory reaction, through its action on neutrophil TNFR expression, and suggest that AA may regulate the response of neutrophils to TNF by altering its receptor number.

Arachidonic Acid↗

PubMed2003

Selective deficiency in protein kinase C isoenzyme expression and inadequacy in mitogen-activated protein kinase activation in cord blood T cells.

The biochemical basis for the reduced lymphokine production by neonatal T cells compared with adult T cells remains poorly defined. Previous studies have raised the possibility that neonatal T cells could be deficient in their ability to transmit signals via protein kinase (PK) C. We now report that while PKC-dependent activation of the mitogen-activated protein (MAP) kinases, c-Jun N-terminal protein kinase and the extracellular signal-regulated protein kinase (ERK)1/ERK2, was deficient in cord blood T cells compared with adult blood T cells, marked activation of the MAP kinases in cord blood T cells was achieved via PKC-independent means. Consistent with a deficiency in the signalling capability of PKC, cord blood T cells were selectively deficient in the expression of PKC beta I, epsilon, theta and zeta. Stimulation of cord blood T cells resulted in a time-dependent increase in PKC expression, with increases detectable by 4 h. This was accompanied by an enhancement in MAP kinase activation via PKC-dependent means. These novel data suggest that an inadequacy in PKC-MAP kinase signalling may be responsible, at least in part, for the phenotype of cord blood T cells.

Enzyme Activation↗

PubMed2002

Role of MAP kinases in the 1,25-dihydroxyvitamin D3-induced transactivation of the rat cytochrome P450C24 (CYP24) promoter. Specific functions for ERK1/ERK2 and ERK5.

The current study investigated the action of 1,25-dihydroxyvitamin D(3) (1,25D) at the genomic and signal transduction levels to induce rat cytochrome P450C24 (CYP24) gene expression. A rat CYP24 promoter containing two vitamin D response elements and an Ets-1 binding site was used to characterize the mechanism of actions for the 1,25D secosteroid hormone. The Ets-1 binding site was determined to function cooperatively with the most proximal vitamin D response element in a hormone-dependent fashion. Evidence was obtained for distinct roles of ERK1/ERK2 and ERK5 in the 1,25D-inductive actions. Specifically, 1,25D stimulated the activities of ERK1/ERK2 and ERK5 in a Ras-dependent manner. Promoter induction was inhibited by mitogen-activated protein (MAP) kinase inhibitors (PD98059 and U0126) and a dominant-negative Ras mutant (Ras17N). Induction of CYP24 by 1,25D was also inhibited by overexpression of dominant-negative mutants of ERK1 and MEK5 (ERK1K71R and MEK5(A)). The p38 and JNK MAP kinases were not required for the action of 1,25D. 9-cis retinoid X receptor alpha (RXR alpha) interacted with ERK2 but not ERK5 in intact cells, whereas Ets-1 interacted preferentially with ERK5. Increased phosphorylation of RXR alpha and Ets-1 was detected in response to 1,25D. Activated ERK2 and ERK5 specifically phosphorylated RXR alpha and Ets-1, respectively. Mutagenesis of Ets-1 (T38A) reduced CYP24 promoter activity to levels observed with the dominant-negative MEK5(A) and inhibited ERK5-directed phosphorylation. Mutated RXR alpha (S260A) inhibited 1,25D-induced CYP24 promoter activity and abolished phosphorylation by activated ERK2. The 1,25D-inductive action through ERK5 involved Ets-1 phosphorylation at threonine 38, whereas hormone stimulation of ERK1/ERK2 required RXR alpha phosphorylation on serine 260. The ERK1/ERK2 and ERK5 modules provide a novel mechanism for linking the rapid signal transduction and slower transcription actions of 1,25D to induce CYP24 gene expression.

Animals↗

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Humans↗