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Biomedical subjects

M Colasanti

Publications and source records attributed to M Colasanti.

At least 37 records · Page 2Linked to original sources
PubMed1999

Stabilization of the T-state of human hemoglobin by proflavine, an antiseptic drug.

The effect of proflavine (3,6-diaminoacridine), an antiseptic drug, on the spectroscopic and oxygen binding properties of ferrous human adult hemoglobin (Hb) has been investigated. Upon binding of proflavine to the nitric oxide derivative of ferrous human adult hemoglobin (HbNO), the X-band EPR spectrum displays the characteristics which have been attributed to the T-state of the ligated tetramer. In parallel, oxygen affinity for the deoxygenated derivative of ferrous human adult Hb decreases in the presence of proflavine. The effect of proflavine on the spectroscopic and ligand binding properties of ferrous human adult Hb is reminiscent that of 2,3-D-glycerate bisphosphate, the physiological modulator of Hb action.

Allosteric Regulation↗
PubMed1999

S-nitrosylation of viral proteins: molecular bases for antiviral effect of nitric oxide.

Nitric oxide (NO) is considered an important signaling molecule implied in various different physiological processes, including nervous transmission, vascular regulation, and immune defence, as well as the pathogenesis of several diseases. NO reportedly also has an antiviral effect on several DNA and RNA virus families. The NO-mediated S-nitrosylation of viral and host (macro)molecules appears to be an intriguing general mechanism for the control of the virus life cycle. In this respect, NO is able to nitrosylate cysteine-containing enzymes (e.g., proteases, reverse transcriptase, and ribonucleotide reductase). Moreover, zinc-fingers and related domains present in enzymes (e.g., HIV-1-encoded integrase or herpes simplex virus type-1 heterotrimeric helicase-primase complex) or nucleocapsid proteins may be considered as NO targets. Also, NO may regulate both host (e.g., nuclear factor-kappaB) and viral-encoded (e.g., HIV-1 tat protein or Epstein-Barr virus Zta) transcriptional factors that are involved in virus replication. Finally, NO-mediated S-nitrosylation of cysteine-containing glycoproteins and hemagglutinin may also occur. Here, NO targets are summarised, and the molecular bases for the antiviral effect of NO are discussed.

Antiviral Agents↗
PubMed1999

Molecular bases for the anti-HIV-1 effect of NO. Commentary.

In infected human cells, nitric oxide (NO) has been shown to inhibit the replication of the human immunodeficiency virus-1 (HIV-1), the etiological agent of AIDS. Evidence suggests that NO may regulate HIV-1 replication by affecting the sulphydryl redox state. In this respect, it has been very recently demonstrated that NO-donors inactivate the HIV-1-encoded protease and reverse transcriptase in vitro. Further viral and host NO targets may be envisaged. Although no data are available on the anti-HIV-1 effect of NO in vivo, NO-releasing drugs, clinically used in the treatment of cardiovascular disorders, may represent a novel class of molecules for decreasing virus replication. Here, the possible molecular bases for the anti-HIV-1 effect of NO are discussed.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Expression of a NOS-III-like protein in human astroglial cell culture.

Evidence for the presence of a type-III nitric oxide synthase-like protein (NOS-III-like protein) in astroglial cells is reported. The mRNA of a NOS-III-like protein is constitutively expressed in human astrocytoma T67 cells, taken as an astroglial model. The nucleotide sequence of the PCR product (422 bp) shares more than 99% identity with the cDNA (from 1588 to 2009) of the human endothelial nitric oxide synthase (NOS-III). The molecular mass of the astroglial NOS-III-like protein is about 140 kDa, as observed for human NOS-III. Moreover, the astroglial NOS-III-like protein is constitutively tyrosine-phosphorylated and associated with caveolin-1. The astroglial NOS-III-like protein is apparently inactive, as reported for phosphorylated human NOS-III associated with caveolin-1.

Astrocytes↗
PubMed1998

Cysteine nitrosylation inactivates the HIV-1 protease.

Nitric oxide (NO) may modulate the catalytic activity of cysteine-containing enzymes. HIV-1 protease action is modulated by the redox equilibrium of Cys67 and Cys95 regulatory residues. In the present study, the inhibitory effect of NO, released by the NO-donor (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), on the aspartyl HIV-1 protease action is reported. HIV-1 protease inactivation via NO-mediated nitrosylation of Cys regulatory residue(s) may represent a possible mechanism for inhibition of HIV-1 replication.

Enzyme Activation↗
PubMed1998

Partial biochemical characterization of nitric oxide synthase in the caudal spinal cord of the teleost Oreochromis niloticus.

The present study demonstrates that a NADPH/Ca2+-dependent nitric oxide synthase (NOS) activity is present in the soluble and in the particulate fractions of fish caudal spinal cord homogenates, both activities being inhibited by calmodulin inhibitors (W7 and/or TFP) and by the NOS inhibitor L-NAME. Moreover, Western blot analysis using either anti-NOS I or anti-NOS III antibodies shows that the soluble enzyme corresponds to the brain NOS (NOS-I) of mammals, whereas the particulate one is likely attributable to NOS I and/or NOS III (ecNOS) enzymes. To confirm the nitric oxide (NO) production by the caudal spinal cord homogenates, the NO-mediated conversion of oxyhemoglobin to methemoglobin was monitored spectroscopically. The present results are consistent with a constitutive, Ca2+-calmodulin-dependent, NO production by the caudal neurosecretory system.

Animals↗
PubMed1998

Aprotinin, the first competitive protein inhibitor of NOS activity.

Analogs of L-arginine represent the largest and potentially most useful class of NOS inhibitors. However, no competitive protein inhibitors of NOS activity are known so far. The effect of aprotinin (Kunitz inhibitor) on NOS activity is reported here, aprotinin being one of the most extensively studied globular proteins. Present data indicate that aprotinin, clinically used as a trypsin-like serine proteinase inhibitor, inhibits NOS-I and NOS-II with Ki values of 5.0 x 10(-5) M and 7.8 x 10(-5) M, respectively, at pH 7.5 and 37.0 degrees C, thereby representing the first competitive protein inhibitor of NOS activity. Therefore, the clinical use of aprotinin, as a drug, should be under careful control. In addition, aprotinin and aprotinin-like domains are present in a variety of organs, as well as in the Alzheimer's amyloid beta-protein precursor. Thus, the present findings open the way to novel mechanisms likely to be involved in the modulation of NOS activity, under physiological and pathological conditions.

Animals↗
PubMed1998

Effect of gabexate mesylate (FOY), a drug for serine proteinase-mediated diseases, on the nitric oxide pathway.

Considering the structural similarity between gabexate mesylate (FOY), a drug for serine proteinase-mediated diseases, and L-arginine, the effect of gabexate mesylate on the nitric oxide (NO) pathway has been investigated. Gabexate mesylate inhibits competitively constitutive and inducible NO synthase (cNOS and iNOS, respectively), with Ki values of 1.0 x 10(-4) M and 5.0 x 10(-3) M, respectively, at pH 7.4 and 37.0 degrees C. However, gabexate mesylate is not an NO precursor. Moreover, like other NOS inhibitors, gabexate mesylate increases iNOS mRNA expression in rat C6 glioma cells, as induced by E. coli lipopolysaccharide plus interferon-gamma. Finally, gabexate mesylate inhibits dose-dependently nitrite production (i.e. NO release) in rat C6 glioma cells, as induced by E. coli lipopolysaccharide plus interferon-gamma. Thus, this drug should be administered under careful control, since enzyme inhibition may occur also in vivo.

Animals↗
PubMed1998

Nitric oxide in invertebrates.

Nitric oxide (NO) is considered an important signaling molecule implied in different physiological processes, including nervous transmission, vascular regulation, immune defense, and in the pathogenesis of several diseases. The presence of NO is well demonstrated in all vertebrates. The recent data on the presence and roles of NO in the main invertebrate groups are reviewed here, showing the widespread diffusion of this signaling molecule throughout the animal kingdom, from higher invertebrates down to coelenterates and even to prokaryotic cells. In invertebrates, the main functional roles described for mammals have been demonstrated, whereas experimental evidence suggests the presence of new NOS isoforms different from those known for higher organisms. Noteworthy is the early appearance of NO throughout evolution and striking is the role played by the nitrergic pathway in the sensorial functions, from coelenterates up to mammals, mainly in olfactory-like systems. All literature data here reported suggest that future research on the biological roles of early signaling molecules in lower living forms could be important for the understanding of the nervous-system evolution.

Animals↗
PubMed1998

Cross-enzyme inhibition by gabexate mesylate: formulation and reactivity study.

Gabexate mesylate (GM; commercialized under the brand name FOY) is a nonantigenic synthetic inhibitor of plasmatic and pancreatic serine proteinases that is used therapeutically in the treatment of pancreatitis and disseminated intravascular coagulation and as a regional anticoagulant for hemodialysis. The inhibitory effect of GM on nitric oxide synthase as well as serine proteinases and swine kidney copper amine oxidase, all acting on cationic substrates, has been investigated. On the basis of the available X-ray crystal structures of the enzymes considered, the possible binding mode(s) of GM has(have) been analyzed. The enzyme cross-inhibition by GM suggests that the use of this drug should be under careful control. With the aim to improve the scarce plasma stability of GM, the positively charged drug has been complexed to the surface of preformed anionic liposomes. The liposome-complexed GM half-life increases about five-fold, indicating the protective effect of liposomes on GM degradation. Moreover, the GM complexation with liposomes does not alter its inhibitory activity on NOS-I and porcine pancreatic trypsin.

Drug Carriers↗
PubMed1998

The effect of nitric oxide on cytokine-induced release of PGE2 by human cultured astroglial cells.

1. The role of the L-arginine-nitric oxide (NO) pathway on the formation of prostaglandin E2 (PGE2) by human cultured astroglial cells incubated with interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) was investigated. 2. Incubation of T 67 astroglial cell line with IL-beta (10 ng ml(-1)) and TNF-alpha (500 u ml(-1)) produced a significant (P<0.05) increase of both nitrite (the breakdown product of NO), cyclic GMP and PGE2 levels in cell supernatants. N omega-nitro-L-arginine methyl ester (L-NAME; 20-300 microM), an inhibitor of NO synthase (NOS), inhibited the increase of cyclic GMP and nitrite levels found in supernatants of cytokine-treated astroglial cells and reduced the release of PGE2. The latter effect showed that the enhanced arachidonic acid (AA) metabolism subsequent to stimulation of astroglial cells with IL-1beta and TNF-alpha was, at least in part, induced by NO. This occurred also when sodium nitroprusside (SNP; 120 microM), an NO donor, was incubated with astroglial cells, an effect antagonized by oxyhaemoglobin (OxyHb; 10 microM). 3. The inhibition elicited by L-NAME on PGE2-release by cytokine-treated astroglial cells was reversed by adding AA (40 microM), showing that the effect of NO on cytokine-dependent PGE2 release occurred at the cyclo-oxygenase (COX) level. Furthermore, the release of PGE2 in cytokine-treated astroglial cells was inhibited by indomethacin (10 microM), a COX inhibitor as well as by preincubating cells with dexamethasone (20 microM), an inhibitor of inducible enzymes, showing that the inducible isoform of COX (COX-2) was involved. 4. On the other hand, pretreating astroglial cells with methylene blue (MB; 10 microM), an inhibitor of NO biological activity acting at the guanylate cyclase level, failed to affect PGE2 release in cytokine-treated astroglial cells, leading to the conclusion that cyclic GMP changes related to NO formation are not involved in the generation of AA metabolites. 5. The present experiments demonstrated that the release of PGE2 by astroglial cells pretreated with IL-1beta and TNF-alpha is due to enhanced COX-2 activity via activation of the L-arginine-NO pathway, and this may be relevant to the understanding of the pathophysiological mechanisms underlying neuroimmune disorders.

Astrocytes↗
PubMed1998

Cys25-nitrosylation inactivates papain.

Nitric oxide (NO) may modulate the catalytic activity of cysteine proteases. In the present study, the inhibitory effect of NO, released by the NO-donors (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide and nitroprusside, on papain action is reported. Papain inactivation via NO-mediated nitrosylation of the Cys25 catalytic residue represents a molecular model for cysteine protease inhibition.

Cysteine↗
PubMed1997

Nitric oxide synthase in the caudal neurosecretory system of the teleost Oreochromis niloticus.

This study provides evidence that, within the caudal neurosecretory system of the teleost Oreochromis niloticus, neurons express nitric oxide synthase (NOS)-like molecules. The presence of NOS-like molecules was demonstrated by means of NADPH-diaphorase (NADPHd) staining and NOS immunohistochemistry. In the caudal spinal cord, NOS-positive neurosecretory cell bodies and neurosecretory fibers were observed. In addition, NOS-positive structures were found in the urophysis which correspond to neurosecretory axon terminals. Cellular co-localization of NOS and ovine corticotropin-releasing factor (oCRF) immunoreactivities confirmed that the NOS-positive structures belong to the caudal neurosecretory system. The present results suggest that NO may participate in the caudal neuroendocrine function.

Animals↗
PubMed1997

Bacterial lipopolysaccharide plus interferon-gamma elicit a very fast inhibition of a Ca2+-dependent nitric-oxide synthase activity in human astrocytoma cells.

Previous results indicate that induction of inducible nitric-oxide synthase (iNOS) expression may be kept suppressed by the endogenous NO level as produced by a constitutive NOS (cNOS) enzyme. In cell types possessing both cNOS and iNOS, this may represent an evident paradox. Here, we report that lipopolysaccharide and interferon-gamma, which are able to strongly induce iNOS in astrocytoma cells, can rapidly inhibit the NO production generated by the constitutive NOS isoform, thus obtaining the best conditions for iNOS induction and resolving the apparent paradox. In fact, a 30-min treatment of T67 cells with the combination of lipopolysaccharide plus interferon-gamma (MIX) strongly inhibits the cNOS activity, as determined by measuring [3H]citrulline production. In addition, the effect of MIX is also observed by measuring nitrite, the stable breakdown product of NO: a 30-min pretreatment of T67 cells with MIX is able to reduce significantly the N-methyl-D-aspartate-induced nitrite production. Finally, using reverse transcriptase-polymerase chain reaction, we have observed that a 30-min treatment of T67 cells with MIX does not affect expression of mRNA coding for the neuronal NOS-I isoform. These results suggest the novel concept of a possible role of a cNOS isoform in astrocytes as a control function on iNOS induction.

Astrocytes↗
PubMed1997

Nitric oxide involvement in Hydra vulgaris very primitive olfactory-like system.

Hydra feeding response is a very primitive olfactory-like behavior present in a multicellular organism. We investigated the role of nitric oxide (NO) in the induction and control of hydra feeding response. Under basal conditions, hydra specimens produce detectable amounts of nitrite (NO2-), the breakdown product of NO. When hydra were incubated with reduced glutathione (GSH), the typical activator of feeding response, an increase of basal NO production was observed. This effect was inhibited by glutamic or alpha-aminoadipic acids, two GSH antagonists, which block GSH-induced feeding response, and by the NO synthase (NOS) inhibitor L-NAME. Moreover, we found that hydra possess a calcium-dependent (but calmodulin-independent) NOS isoform. By using exogenous NO donors and NOS inhibitors, we demonstrated that NO stimulus can participate both in triggering tentacular movements and in recruiting neighbor tentacles during hydra feeding response. By using dbt2-cGMP, an analog to cGMP, we observed that the NO effect was independent of cGMP pathway. Our results strongly implicate NO involvement in hydra very primitive feeding behavior, thus confirming its preservation throughout evolution.

Animals↗
PubMed1995

Human ramified microglial cells produce nitric oxide upon Escherichia coli lipopolysaccharide and tumor necrosis factor alpha stimulation.

This study shows that human ramified microglial cells derived from fetal brain primary cultures, are able to produce nitric oxide (NO). In fact, stimulation with Escherichia coli lipopolysaccharide (LPS) (1 microgram ml-1) or tumor necrosis factor alpha (TNF alpha) (500 U ml-1) enhances nitrite release in cell supernatants, as determined by the Griess reaction. A synergistic effect is achieved following treatment with LPS plus TNF alpha, this effect being inhibited by pretreating cells with NOS inhibitor N omega-nitro-L-arginine methyl ester (L-NAME). Using reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot analysis, we also found that LPS/TNF alpha produce an increase of inducible NO synthase (iNOS) mRNA expression.

Base Sequence↗
PubMed1995

Induction of nitric oxide synthase mRNA expression. Suppression by exogenous nitric oxide.

The reactive nitrogen species, nitric oxide (NO), plays an important role in the pathogenesis of neurodegenerative diseases. The suppression of NO production may be fundamental for survival of neurons. Here, we report that pretreatment of human ramified microglial cells with nearly physiological levels of exogenous NO prevents lipopolysaccharide (LPS)/tumor necrosis factor alpha (TNF alpha)-inducible NO synthesis, because by affecting NF-kappa B activation it inhibits inducible Ca(2+)-independent NO synthase isoform (iNOS) mRNA expression. Using reverse transcriptase polymerase chain reaction, we have found that both NO donor sodium nitroprusside (SNP) and authentic NO solution are able to inhibit LPS/TNF alpha-inducible iNOS gene expression; this effect was reversed by reduced hemoglobin, a trapping agent for NO. The early presence of SNP during LPS/TNF alpha induction is essential for inhibition of iNOS mRNA expression. Furthermore, SNP is capable of inhibiting LPS/TNF alpha-inducible nitrite release, as determined by Griess reaction. Finally, using electrophoretic mobility shift assay, we have shown that SNP inhibits LPS/TNF alpha-elicited NF-kappa B activation. This suggests that inhibition of iNOS gene expression by exogenous NO may be ascribed to a decreased NF-kappa B availability.

Base Sequence↗