Claudia Gargini | Università di Pisa (original) (raw)
Papers by Claudia Gargini
Molecules, Feb 10, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Frontiers in Neuroscience
Rhodopsin (RHO) mutations are responsible for 25–40% of the dominant cases of retinitis pigmentos... more Rhodopsin (RHO) mutations are responsible for 25–40% of the dominant cases of retinitis pigmentosa (RP) with different severity and progression rates. The Tvrm4 mice, heterozygous for an I307N dominant mutation of RHO, display a normal retinal phenotype when raised in ambient light conditions, but undergo photoreceptor degeneration when briefly exposed to strong white light. Here, The Tvrm4 mice is pre-treated with naringenin 100 mg/kg/die, quercetin 100 mg/kg/die, naringenin 50 + quercercetin 100 mg/kg/die or vehicle dimethyl sulfoxide (DMSO 0.025%) in the drinking water for 35 days. On the 30th day, retinal degeneration was induced by exposure for 1 min to the white light of 12,000 lux intensity, and the treatment was repeated for another 5 days. At the end of the protocol retinal functionality was tested by recording an electroretinogram (ERG). The retinal tissue was collected and was used for further analyses, including immunohistochemically, biochemical, and molecular biology a...
TMEM16A is associated with voltage-gated calcium channels in mouse retina and its function is dis... more TMEM16A is associated with voltage-gated calcium channels in mouse retina and its function is disrupted upon mutation of the auxiliary α2δ4 subunit
Nutrients, 2021
Retinal diseases can be induced by a variety of factors, including gene mutations, environmental ... more Retinal diseases can be induced by a variety of factors, including gene mutations, environmental stresses and dysmetabolic processes. The result is a progressive deterioration of visual function, which sometimes leads to blindness. Many treatments are under investigation, though results are still mostly unsatisfactory and restricted to specific pathologies, particularly in the case of gene therapy. The majority of treatments have been tested in animal models, but very few have progressed to human clinical trials. A relevant approach is to study the relation between the type of treatments and the degenerative characteristics of the animal model to better understand the effectiveness of each therapy. Here we compare the results obtained from different animal models treated with natural compounds (saffron and naringenin) to anticipate the potentiality of a single treatment in different pathologies.
Frontiers in Neuroscience, 2020
Tvrm4 mice, a model of autosomal dominant retinitis pigmentosa (RP), carry a mutation of Rhodopsi... more Tvrm4 mice, a model of autosomal dominant retinitis pigmentosa (RP), carry a mutation of Rhodopsin gene that can be activated by brief exposure to very intense light. Here, we test the possibility of an anatomical, metabolic, and functional recovery by delivering to degenerating Tvrm4 animals, Myriocin, an inhibitor of ceramide de novo synthesis previously shown to effectively slow down retinal degeneration in rd10 mutants (Strettoi et al., 2010; Piano et al., 2013). Different routes and durations of Myriocin administration were attempted by using either single intravitreal (i.v.) or long-term, repeated intraperitoneal (i.p.) injections. The retinal function of treated and control animals was tested by ERG recordings. Retinas from ERG-recorded animals were studied histologically to reveal the extent of photoreceptor death. A correlation was observed between Myriocin administration, lowering of retinal ceramides, and preservation of ERG responses in i.v. injected cases. Noticeably, the i.p. treatment with Myriocin decreased the extension of the retinal-degenerating area, preserved the ERG response, and correlated with decreased levels of biochemical indicators of retinal oxidative damage. The results obtained in this study confirm the efficacy of Myriocin in slowing down retinal degeneration in genetic models of RP independently of the underlying mutation responsible for the disease, likely targeting ceramide-dependent, downstream pathways. Alleviation of retinal oxidative stress upon Myriocin treatment suggests that this molecule, or yet unidentified metabolites, act on cellular detoxification systems supporting cell survival. Altogether, the pharmacological approach chosen here meets the necessary prerequisites for translation into human therapy to slow down RP.
Oxidative Medicine and Cellular Longevity, 2020
Sirtuin 1 (SIRT1) enzyme plays a pivotal role in the regulation of many physiological functions. ... more Sirtuin 1 (SIRT1) enzyme plays a pivotal role in the regulation of many physiological functions. In particular, it is implicated in ageing-related diseases, such as cardiac hypertrophy, myocardial infarct, and endothelial dysfunction; moreover, its expression decreases with age. Therefore, an effective strategy to extend the lifespan and improve cardiovascular function is the enhancement of the expression/activity of SIRT1 with exogenous agents. The Citrus flavonoid naringenin (NAR) presents structural similarity with the natural SIRT1 activator resveratrol. In this study, we demonstrate through in vitro assays that NAR significantly activates SIRT1 enzyme and shows antisenescence effects. The binding mode of NAR into SIRT1 was detailed investigated through in silico studies. Moreover, chronic administration (for six months) of NAR (100 mg/kg/day) to 6-month-old mice leads to an enhancement of SIRT1 expression and a marked reduction of reactive oxygen species production in myocardia...
Proceedings of the National Academy of Sciences, 2018
Significance Circadian rhythms are not simply a passive consequence of cyclic fluctuations in the... more Significance Circadian rhythms are not simply a passive consequence of cyclic fluctuations in the environment, but instead originate within the organism. Accumulating evidence indicates that disruption of circadian rhythms contributes to the development of many diseases. Here, we demonstrate that removal of the clock gene Bmal1 from the retina has multiple effects on rod and cone pathways. In the cone pathway, the primary effect is on cone viability, an effect accentuated in aging mice. In the rod pathway, we observed that retinal Bmal1 removal caused stunting of rod bipolar cell dendrites and thinning of the outer plexiform layer in both young and old mice. Hence, our data suggest that circadian clock dysfunction contributes to aberrant visual function during development and aging.
Molecular vision, 2017
Previous studies have shown that melatonin (MEL) signaling is involved in the modulation of photo... more Previous studies have shown that melatonin (MEL) signaling is involved in the modulation of photoreceptor viability during aging. Recent work by our laboratory suggested that MEL may protect cones by modulating the Fas/FasL-caspase-3 pathway. In this study, we first investigated the presence of MEL receptors (MTand MT) in 661W cells, then whether MEL can prevent HO-induced cell death, and last, through which pathway MEL confers protection. The mRNA and proteins of the MEL receptors were detected with quantitative PCR (q-PCR) and immunocytochemistry, respectively. To test the protective effect of MEL, 661W cells were treated with HOfor 2 h in the presence or absence of MEL, a MEL agonist, and an antagonist. To study the pathways involved in HO-mediated cell death, a Fas/FasL antagonist was used before the exposure to HO. Finally, Fas/FasL and caspase-3 mRNA was analyzed with q-PCR and immunocytochemistry in cells treated with HOand/or MEL. Cell viability was analyzed by using Trypan ...
Molecules, 2017
In this work, we reported the application and validation of an improved high-performance liquid c... more In this work, we reported the application and validation of an improved high-performance liquid chromatography method coupled with a fluorimetric detector (HPLC-FL) to screen the activity of two heterocyclic derivatives reported as serine palmitoyl transferase (SPT) inhibitors. The analytical conditions were optimized in terms of the derivatization procedure, chromatographic condition, extraction procedure, and method validation according to EMEA guidelines. Once fully optimized, the method was applied to assess the SPT-inhibitory activity of the above-mentioned derivatives and of the reference inhibitor myriocin. The obtained results, expressed as a percentage of residual SPT activity, were compared to those obtained with the reference radio immune assay (RIA). The good correlation between the two types of assay demonstrated that the improved HPLC-FL method is suitable for a preliminary and rapid screening of potential SPT-inhibitors.
Scientific Reports, 2017
Hallmarks of Retinitis Pigmentosa (RP), a family of genetic diseases, are a typical rod-cone-dege... more Hallmarks of Retinitis Pigmentosa (RP), a family of genetic diseases, are a typical rod-cone-degeneration with initial night blindness and loss of peripheral vision, followed by decreased daylight sight and progressive visual acuity loss up to legal blindness. Great heterogeneity in nature and function of mutated genes, variety of mutations for each of them, variability in phenotypic appearance and transmission modality contribute to make RP a still incurable disease. Translational research relies on appropriate animal models mimicking the genetic and phenotypic diversity of the human pathology. Here, we provide a systematic, morphological and functional analysis of RhoTvrm4/Rho+ rhodopsin mutant mice, originally described in 2010 and portraying several features of common forms of autosomal dominant RP caused by gain-of-function mutations. These mice undergo photoreceptor degeneration only when exposed briefly to strong, white light and allow controlled timing of induction of rod an...
Visual Neuroscience, 2017
Rod-cone gap junctions mediate the so-called “secondary rod pathway”, one of three routes that co... more Rod-cone gap junctions mediate the so-called “secondary rod pathway”, one of three routes that convey rod photoreceptor signals across the retina. Connexin 36 (Cx36) is expressed at these gap junctions, but an unidentified connexin protein also seems to be expressed. Cx36 knockout mice have been used extensively in the quest to dissect the roles in vision of all three pathways, with the assumption, never directly tested, that rod-cone electrical coupling is abolished by deletion of this connexin isoform. We previously showed that when wild type mouse cones couple to rods, their apparent dynamic range is extended toward lower light intensities, with the appearance of large responses to dim flashes (up to several mV) originating in rods. Here we recorded from the cones of Cx36del[LacZ]/del[LacZ] mice and found that dim flashes of the same intensity evoked at most small sub-millivolt responses. Moreover, these residual responses originated in the cones themselves, since: (i) their spec...
Journal of Medicinal Chemistry, 2017
The gasotransmitter hydrogen sulfide (H2S) is an important tuner of the cardiovascular homeostasi... more The gasotransmitter hydrogen sulfide (H2S) is an important tuner of the cardiovascular homeostasis and its deficiency is etiologically associated with a number of cardiovascular diseases. Therefore, the research of original moieties able to release H2S represents a timely issue for drug discovery. In this work, we developed a collection of iminothioethers (ITEs), exhibiting H2S-releasing properties and producing vasorelaxing effects on rat aortic rings. Derivatives 4 and 11, selected as representative of slow and fast rate H2Sdonors respectively, produced a complete recovery of the basal coronary flow, reverting the AngII-induced effects in isolated rat hearts. In addition, studies on human aortic smooth muscle cells (HASMCs) demonstrated membrane hyperpolarizing effects, well related with intracellular generation of H2S. Taken together, the results obtained support ITEs 4 and 11 as new pharmacological tools, as well as effective and innovative H2S-donors for cardiovascular drug discovery. INTRODUCTION: Hydrogen sulfide (H2S) is presently recognized as a fundamental mediator, which controls the homeostasis of many biological systems in the mammalian body. 1,2 This gasotransmitter is biosynthetized by specific enzymes, such as cystathionine-beta-synthase (CBS), cystathioninegamma-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST) starting from the aminoacid L-Cysteine. Among its numerous roles, H2S is a key regulator of the cardiovascular system, where it is mainly produced by CSE. H2S acts as a vasodilator 3 through several mechanisms of action often involving the modulation of ion channels or phosphodiesterase (PDE) in vascular smooth muscle. 4-8 The deletion of CSE gene in experimental animals is associated with a significant reduction of endogenous H2S in blood, and in vascular and myocardial tissues; such a reduction leads to the impairment of endothelium-mediated vasorelaxation and increase in blood pressure. 9 These data clearly indicate that vascular H2S is a key factor in the regulation of blood pressure and the defective production of endogenous H2S is likely to be one of the most important etiopathogenetic factor in several forms of hypertension. 10 The roles played by endogenous H2S in the regulation of the cardiovascular homeostasis pave the way to appealing therapeutic purposes, based on effective and rational pharmacological modulation of the H2S pathway. 11 Indeed, the administration of exogenous H2S has been proven to exert significant anti-hypertensive effects in several experimental models of hypertension, 12,13 indicating that "druggable" H2S-releasing agents can actually be viewed as promising tools to obtain novel cardiovascular drugs. 14,15 The poor posological control and the high probability of toxic effects strongly exclude the use of gaseous H2S. Some sulfide salts, such as sodium hydrogen sulfide (NaHS) and calcium sulfide (CaS) 16 are H2S-generating agents widely used for experimental purposes, but the rapid formation of H2S (due to the protonation of hydrosulfide and sulfide anions, respectively, at physiological pH) seems to be poorly appropriate for clinical uses. Ideal H2S-donor drugs should produce H2S with relatively slow and constant rates. Accordingly, the search of novel H2S-releasing chemical moieties suitable for the development of clinically effective H2S-donors is strongly required. An interesting H2S-donor feature has been early recognized in natural organosulfur derivatives, such as the polysulfides of Alliaceae (for example, diallyl disulfide 1 (DADS), Chart 1). 17 More recently, H2S-releasing properties have been recognized also in another important class of natural sulfur compounds: the isothiocyanates typical of Brassicaceae. 18 Synthetic H2S-releasing agents are also known; among them, the 4methoxyphenyl(morpholino)phosphinodithioatemorpholinium salt 2 (GYY4137, Chart 1) is one of the most widely used in pharmacological studies. 19 As well, H2S-releasing dithiolethiones and thioamides (TAs) are largely used, especially for the synthesis of multitarget drugs. 20-22 Satisfactory H2S-releasing features of aminothiol and aryl isothiocyanate derivatives have been also reported. 23,24 Very recently, original examples of "smart" H2S-donors, able to generate the gasotransmitter based on specific mechanisms of release, which may be useful in specific biological targets, have been described. Among these, molecules exhibiting esterase-mediated production of H2S 25 , pHcontrolled mechanisms 26 or initial release of intermediates such as carbonyl sulfide 27 have been reported. All the target compounds 3-15 were finally purified by flash chromatography, when necessary (see Experimental section). Scheme 2. Synthesis of imithioether 9-11 and thioamide 15 derivatives.
Frontiers in Pharmacology, 2017
Background and Purpose: Incidence of cardiovascular disorders increases with age, because of a dr... more Background and Purpose: Incidence of cardiovascular disorders increases with age, because of a dramatic fall of endogenous self-defense mechanisms and increased vulnerability of myocardium. Conversely, the effectiveness of many cardioprotective drugs is blunted in hearts of 1 year old rat. The Citrus flavanone naringenin (NAR) was reported to promote cardioprotective effects against ischemia/reperfusion (I/R) injury, through the activation of mitochondrial large conductance calcium-activated potassium channel (mitoBK). These effects were observed in young adult rats, but no data are available about the possible cardioprotective effects of NAR in aged animals. Experimental Approach: This study aimed at evaluating the potential cardioprotective effects of NAR against I/R damage in 1 year old rats, and the possible involvement of mitoBK. Key Results: Naringenin protected the hearts of 1 year old rats in both ex vivo and in vivo I/R protocols. Noteworthy, these effects were antagonized by paxilline, a selective BK-blocker. The cardioprotective effects of NAR were also observed in senescent H9c2 cardiomyoblasts. In isolated mitochondria from hearts of 1 year old, NAR exhibited the typical profile of a mitoBK opener. Finally, Western Blot analysis confirmed a significant (albeit reduced) presence of BK-forming alpha and beta subunits, both in cardiac tissue of 1 year old rats and in senescent H9c2 cells. Conclusion and Implications: This is the first work reporting cardioprotective effects of NAR in 1 year old rats. Although further studies are needed to better understand the whole pathway involved in the NAR-mediated cardioprotection, these preliminary data represent a promising perspective for a rational nutraceutical use of NAR in aging.
Scientific Reports, 2016
Retinitis pigmentosa (RP) comprises a group of inherited pathologies characterized by progressive... more Retinitis pigmentosa (RP) comprises a group of inherited pathologies characterized by progressive photoreceptor degeneration. In rodent models of RP, expression of defective genes and retinal degeneration usually manifest during the first weeks of postnatal life, making it difficult to distinguish consequences of primary genetic defects from abnormalities in retinal development. Moreover, mouse eyes are small and not always adequate to test pharmacological and surgical treatments. An inducible paradigm of retinal degeneration potentially extensible to large animals is therefore desirable. Starting from the serendipitous observation that intraocular injections of a Rho GTPase activator, the bacterial toxin Cytotoxic Necrotizing Factor 1 (CNF1), lead to retinal degeneration, we implemented an inducible model recapitulating most of the key features of Retinitis Pigmentosa. The model also unmasks an intrinsic vulnerability of photoreceptors to the mechanism of CNF1 action, indicating st...
British journal of pharmacology, Nov 22, 2016
Hydrogen sulfide (H2 S) modulates many pathophysiological processes, including inflammation and a... more Hydrogen sulfide (H2 S) modulates many pathophysiological processes, including inflammation and allergic reactions, in which mast cells act as major effector cells. IgE receptor (FcεRI) cross-linking leads to an increase in intracellular calcium ([Ca(+2) ]i ), a critical step in mast cell degranulation. The aim of this study was to investigate the role of H2 S in [Ca(+2) ]i -dependent mast cell activation. We investigated the effects of H2 S, either endogenously produced or released by the slow H2 S donor 4-carboxy-phenyl isothiocyanate (PhNCS-COOH), on antigenic- and non-antigenic degranulation of native murine mast cells (BMMC), human (HMC-1) and rat (RBL-2H3) mast cell lines. We measured the release of specific mast cell degranulation markers (β-hexosaminidase and renin), as well as changes in [Ca(+2) ]i and phosphorylation of proteins downstream of FcεRI activation. Endogenously produced H2 S inhibited antigen-induced degranulation in RBL-2H3. Similarly, H2 S released by PhNCS-C...
Investigative Ophthalmology Visual Science, 2009
Frontiers in Cellular Neuroscience, 2016
Science Signaling, 2013
Melatonin stimulates a heteromeric G protein–coupled receptor to modulate the eye’s response to l... more Melatonin stimulates a heteromeric G protein–coupled receptor to modulate the eye’s response to light flashes at night.
Molecules, Feb 10, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Frontiers in Neuroscience
Rhodopsin (RHO) mutations are responsible for 25–40% of the dominant cases of retinitis pigmentos... more Rhodopsin (RHO) mutations are responsible for 25–40% of the dominant cases of retinitis pigmentosa (RP) with different severity and progression rates. The Tvrm4 mice, heterozygous for an I307N dominant mutation of RHO, display a normal retinal phenotype when raised in ambient light conditions, but undergo photoreceptor degeneration when briefly exposed to strong white light. Here, The Tvrm4 mice is pre-treated with naringenin 100 mg/kg/die, quercetin 100 mg/kg/die, naringenin 50 + quercercetin 100 mg/kg/die or vehicle dimethyl sulfoxide (DMSO 0.025%) in the drinking water for 35 days. On the 30th day, retinal degeneration was induced by exposure for 1 min to the white light of 12,000 lux intensity, and the treatment was repeated for another 5 days. At the end of the protocol retinal functionality was tested by recording an electroretinogram (ERG). The retinal tissue was collected and was used for further analyses, including immunohistochemically, biochemical, and molecular biology a...
TMEM16A is associated with voltage-gated calcium channels in mouse retina and its function is dis... more TMEM16A is associated with voltage-gated calcium channels in mouse retina and its function is disrupted upon mutation of the auxiliary α2δ4 subunit
Nutrients, 2021
Retinal diseases can be induced by a variety of factors, including gene mutations, environmental ... more Retinal diseases can be induced by a variety of factors, including gene mutations, environmental stresses and dysmetabolic processes. The result is a progressive deterioration of visual function, which sometimes leads to blindness. Many treatments are under investigation, though results are still mostly unsatisfactory and restricted to specific pathologies, particularly in the case of gene therapy. The majority of treatments have been tested in animal models, but very few have progressed to human clinical trials. A relevant approach is to study the relation between the type of treatments and the degenerative characteristics of the animal model to better understand the effectiveness of each therapy. Here we compare the results obtained from different animal models treated with natural compounds (saffron and naringenin) to anticipate the potentiality of a single treatment in different pathologies.
Frontiers in Neuroscience, 2020
Tvrm4 mice, a model of autosomal dominant retinitis pigmentosa (RP), carry a mutation of Rhodopsi... more Tvrm4 mice, a model of autosomal dominant retinitis pigmentosa (RP), carry a mutation of Rhodopsin gene that can be activated by brief exposure to very intense light. Here, we test the possibility of an anatomical, metabolic, and functional recovery by delivering to degenerating Tvrm4 animals, Myriocin, an inhibitor of ceramide de novo synthesis previously shown to effectively slow down retinal degeneration in rd10 mutants (Strettoi et al., 2010; Piano et al., 2013). Different routes and durations of Myriocin administration were attempted by using either single intravitreal (i.v.) or long-term, repeated intraperitoneal (i.p.) injections. The retinal function of treated and control animals was tested by ERG recordings. Retinas from ERG-recorded animals were studied histologically to reveal the extent of photoreceptor death. A correlation was observed between Myriocin administration, lowering of retinal ceramides, and preservation of ERG responses in i.v. injected cases. Noticeably, the i.p. treatment with Myriocin decreased the extension of the retinal-degenerating area, preserved the ERG response, and correlated with decreased levels of biochemical indicators of retinal oxidative damage. The results obtained in this study confirm the efficacy of Myriocin in slowing down retinal degeneration in genetic models of RP independently of the underlying mutation responsible for the disease, likely targeting ceramide-dependent, downstream pathways. Alleviation of retinal oxidative stress upon Myriocin treatment suggests that this molecule, or yet unidentified metabolites, act on cellular detoxification systems supporting cell survival. Altogether, the pharmacological approach chosen here meets the necessary prerequisites for translation into human therapy to slow down RP.
Oxidative Medicine and Cellular Longevity, 2020
Sirtuin 1 (SIRT1) enzyme plays a pivotal role in the regulation of many physiological functions. ... more Sirtuin 1 (SIRT1) enzyme plays a pivotal role in the regulation of many physiological functions. In particular, it is implicated in ageing-related diseases, such as cardiac hypertrophy, myocardial infarct, and endothelial dysfunction; moreover, its expression decreases with age. Therefore, an effective strategy to extend the lifespan and improve cardiovascular function is the enhancement of the expression/activity of SIRT1 with exogenous agents. The Citrus flavonoid naringenin (NAR) presents structural similarity with the natural SIRT1 activator resveratrol. In this study, we demonstrate through in vitro assays that NAR significantly activates SIRT1 enzyme and shows antisenescence effects. The binding mode of NAR into SIRT1 was detailed investigated through in silico studies. Moreover, chronic administration (for six months) of NAR (100 mg/kg/day) to 6-month-old mice leads to an enhancement of SIRT1 expression and a marked reduction of reactive oxygen species production in myocardia...
Proceedings of the National Academy of Sciences, 2018
Significance Circadian rhythms are not simply a passive consequence of cyclic fluctuations in the... more Significance Circadian rhythms are not simply a passive consequence of cyclic fluctuations in the environment, but instead originate within the organism. Accumulating evidence indicates that disruption of circadian rhythms contributes to the development of many diseases. Here, we demonstrate that removal of the clock gene Bmal1 from the retina has multiple effects on rod and cone pathways. In the cone pathway, the primary effect is on cone viability, an effect accentuated in aging mice. In the rod pathway, we observed that retinal Bmal1 removal caused stunting of rod bipolar cell dendrites and thinning of the outer plexiform layer in both young and old mice. Hence, our data suggest that circadian clock dysfunction contributes to aberrant visual function during development and aging.
Molecular vision, 2017
Previous studies have shown that melatonin (MEL) signaling is involved in the modulation of photo... more Previous studies have shown that melatonin (MEL) signaling is involved in the modulation of photoreceptor viability during aging. Recent work by our laboratory suggested that MEL may protect cones by modulating the Fas/FasL-caspase-3 pathway. In this study, we first investigated the presence of MEL receptors (MTand MT) in 661W cells, then whether MEL can prevent HO-induced cell death, and last, through which pathway MEL confers protection. The mRNA and proteins of the MEL receptors were detected with quantitative PCR (q-PCR) and immunocytochemistry, respectively. To test the protective effect of MEL, 661W cells were treated with HOfor 2 h in the presence or absence of MEL, a MEL agonist, and an antagonist. To study the pathways involved in HO-mediated cell death, a Fas/FasL antagonist was used before the exposure to HO. Finally, Fas/FasL and caspase-3 mRNA was analyzed with q-PCR and immunocytochemistry in cells treated with HOand/or MEL. Cell viability was analyzed by using Trypan ...
Molecules, 2017
In this work, we reported the application and validation of an improved high-performance liquid c... more In this work, we reported the application and validation of an improved high-performance liquid chromatography method coupled with a fluorimetric detector (HPLC-FL) to screen the activity of two heterocyclic derivatives reported as serine palmitoyl transferase (SPT) inhibitors. The analytical conditions were optimized in terms of the derivatization procedure, chromatographic condition, extraction procedure, and method validation according to EMEA guidelines. Once fully optimized, the method was applied to assess the SPT-inhibitory activity of the above-mentioned derivatives and of the reference inhibitor myriocin. The obtained results, expressed as a percentage of residual SPT activity, were compared to those obtained with the reference radio immune assay (RIA). The good correlation between the two types of assay demonstrated that the improved HPLC-FL method is suitable for a preliminary and rapid screening of potential SPT-inhibitors.
Scientific Reports, 2017
Hallmarks of Retinitis Pigmentosa (RP), a family of genetic diseases, are a typical rod-cone-dege... more Hallmarks of Retinitis Pigmentosa (RP), a family of genetic diseases, are a typical rod-cone-degeneration with initial night blindness and loss of peripheral vision, followed by decreased daylight sight and progressive visual acuity loss up to legal blindness. Great heterogeneity in nature and function of mutated genes, variety of mutations for each of them, variability in phenotypic appearance and transmission modality contribute to make RP a still incurable disease. Translational research relies on appropriate animal models mimicking the genetic and phenotypic diversity of the human pathology. Here, we provide a systematic, morphological and functional analysis of RhoTvrm4/Rho+ rhodopsin mutant mice, originally described in 2010 and portraying several features of common forms of autosomal dominant RP caused by gain-of-function mutations. These mice undergo photoreceptor degeneration only when exposed briefly to strong, white light and allow controlled timing of induction of rod an...
Visual Neuroscience, 2017
Rod-cone gap junctions mediate the so-called “secondary rod pathway”, one of three routes that co... more Rod-cone gap junctions mediate the so-called “secondary rod pathway”, one of three routes that convey rod photoreceptor signals across the retina. Connexin 36 (Cx36) is expressed at these gap junctions, but an unidentified connexin protein also seems to be expressed. Cx36 knockout mice have been used extensively in the quest to dissect the roles in vision of all three pathways, with the assumption, never directly tested, that rod-cone electrical coupling is abolished by deletion of this connexin isoform. We previously showed that when wild type mouse cones couple to rods, their apparent dynamic range is extended toward lower light intensities, with the appearance of large responses to dim flashes (up to several mV) originating in rods. Here we recorded from the cones of Cx36del[LacZ]/del[LacZ] mice and found that dim flashes of the same intensity evoked at most small sub-millivolt responses. Moreover, these residual responses originated in the cones themselves, since: (i) their spec...
Journal of Medicinal Chemistry, 2017
The gasotransmitter hydrogen sulfide (H2S) is an important tuner of the cardiovascular homeostasi... more The gasotransmitter hydrogen sulfide (H2S) is an important tuner of the cardiovascular homeostasis and its deficiency is etiologically associated with a number of cardiovascular diseases. Therefore, the research of original moieties able to release H2S represents a timely issue for drug discovery. In this work, we developed a collection of iminothioethers (ITEs), exhibiting H2S-releasing properties and producing vasorelaxing effects on rat aortic rings. Derivatives 4 and 11, selected as representative of slow and fast rate H2Sdonors respectively, produced a complete recovery of the basal coronary flow, reverting the AngII-induced effects in isolated rat hearts. In addition, studies on human aortic smooth muscle cells (HASMCs) demonstrated membrane hyperpolarizing effects, well related with intracellular generation of H2S. Taken together, the results obtained support ITEs 4 and 11 as new pharmacological tools, as well as effective and innovative H2S-donors for cardiovascular drug discovery. INTRODUCTION: Hydrogen sulfide (H2S) is presently recognized as a fundamental mediator, which controls the homeostasis of many biological systems in the mammalian body. 1,2 This gasotransmitter is biosynthetized by specific enzymes, such as cystathionine-beta-synthase (CBS), cystathioninegamma-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST) starting from the aminoacid L-Cysteine. Among its numerous roles, H2S is a key regulator of the cardiovascular system, where it is mainly produced by CSE. H2S acts as a vasodilator 3 through several mechanisms of action often involving the modulation of ion channels or phosphodiesterase (PDE) in vascular smooth muscle. 4-8 The deletion of CSE gene in experimental animals is associated with a significant reduction of endogenous H2S in blood, and in vascular and myocardial tissues; such a reduction leads to the impairment of endothelium-mediated vasorelaxation and increase in blood pressure. 9 These data clearly indicate that vascular H2S is a key factor in the regulation of blood pressure and the defective production of endogenous H2S is likely to be one of the most important etiopathogenetic factor in several forms of hypertension. 10 The roles played by endogenous H2S in the regulation of the cardiovascular homeostasis pave the way to appealing therapeutic purposes, based on effective and rational pharmacological modulation of the H2S pathway. 11 Indeed, the administration of exogenous H2S has been proven to exert significant anti-hypertensive effects in several experimental models of hypertension, 12,13 indicating that "druggable" H2S-releasing agents can actually be viewed as promising tools to obtain novel cardiovascular drugs. 14,15 The poor posological control and the high probability of toxic effects strongly exclude the use of gaseous H2S. Some sulfide salts, such as sodium hydrogen sulfide (NaHS) and calcium sulfide (CaS) 16 are H2S-generating agents widely used for experimental purposes, but the rapid formation of H2S (due to the protonation of hydrosulfide and sulfide anions, respectively, at physiological pH) seems to be poorly appropriate for clinical uses. Ideal H2S-donor drugs should produce H2S with relatively slow and constant rates. Accordingly, the search of novel H2S-releasing chemical moieties suitable for the development of clinically effective H2S-donors is strongly required. An interesting H2S-donor feature has been early recognized in natural organosulfur derivatives, such as the polysulfides of Alliaceae (for example, diallyl disulfide 1 (DADS), Chart 1). 17 More recently, H2S-releasing properties have been recognized also in another important class of natural sulfur compounds: the isothiocyanates typical of Brassicaceae. 18 Synthetic H2S-releasing agents are also known; among them, the 4methoxyphenyl(morpholino)phosphinodithioatemorpholinium salt 2 (GYY4137, Chart 1) is one of the most widely used in pharmacological studies. 19 As well, H2S-releasing dithiolethiones and thioamides (TAs) are largely used, especially for the synthesis of multitarget drugs. 20-22 Satisfactory H2S-releasing features of aminothiol and aryl isothiocyanate derivatives have been also reported. 23,24 Very recently, original examples of "smart" H2S-donors, able to generate the gasotransmitter based on specific mechanisms of release, which may be useful in specific biological targets, have been described. Among these, molecules exhibiting esterase-mediated production of H2S 25 , pHcontrolled mechanisms 26 or initial release of intermediates such as carbonyl sulfide 27 have been reported. All the target compounds 3-15 were finally purified by flash chromatography, when necessary (see Experimental section). Scheme 2. Synthesis of imithioether 9-11 and thioamide 15 derivatives.
Frontiers in Pharmacology, 2017
Background and Purpose: Incidence of cardiovascular disorders increases with age, because of a dr... more Background and Purpose: Incidence of cardiovascular disorders increases with age, because of a dramatic fall of endogenous self-defense mechanisms and increased vulnerability of myocardium. Conversely, the effectiveness of many cardioprotective drugs is blunted in hearts of 1 year old rat. The Citrus flavanone naringenin (NAR) was reported to promote cardioprotective effects against ischemia/reperfusion (I/R) injury, through the activation of mitochondrial large conductance calcium-activated potassium channel (mitoBK). These effects were observed in young adult rats, but no data are available about the possible cardioprotective effects of NAR in aged animals. Experimental Approach: This study aimed at evaluating the potential cardioprotective effects of NAR against I/R damage in 1 year old rats, and the possible involvement of mitoBK. Key Results: Naringenin protected the hearts of 1 year old rats in both ex vivo and in vivo I/R protocols. Noteworthy, these effects were antagonized by paxilline, a selective BK-blocker. The cardioprotective effects of NAR were also observed in senescent H9c2 cardiomyoblasts. In isolated mitochondria from hearts of 1 year old, NAR exhibited the typical profile of a mitoBK opener. Finally, Western Blot analysis confirmed a significant (albeit reduced) presence of BK-forming alpha and beta subunits, both in cardiac tissue of 1 year old rats and in senescent H9c2 cells. Conclusion and Implications: This is the first work reporting cardioprotective effects of NAR in 1 year old rats. Although further studies are needed to better understand the whole pathway involved in the NAR-mediated cardioprotection, these preliminary data represent a promising perspective for a rational nutraceutical use of NAR in aging.
Scientific Reports, 2016
Retinitis pigmentosa (RP) comprises a group of inherited pathologies characterized by progressive... more Retinitis pigmentosa (RP) comprises a group of inherited pathologies characterized by progressive photoreceptor degeneration. In rodent models of RP, expression of defective genes and retinal degeneration usually manifest during the first weeks of postnatal life, making it difficult to distinguish consequences of primary genetic defects from abnormalities in retinal development. Moreover, mouse eyes are small and not always adequate to test pharmacological and surgical treatments. An inducible paradigm of retinal degeneration potentially extensible to large animals is therefore desirable. Starting from the serendipitous observation that intraocular injections of a Rho GTPase activator, the bacterial toxin Cytotoxic Necrotizing Factor 1 (CNF1), lead to retinal degeneration, we implemented an inducible model recapitulating most of the key features of Retinitis Pigmentosa. The model also unmasks an intrinsic vulnerability of photoreceptors to the mechanism of CNF1 action, indicating st...
British journal of pharmacology, Nov 22, 2016
Hydrogen sulfide (H2 S) modulates many pathophysiological processes, including inflammation and a... more Hydrogen sulfide (H2 S) modulates many pathophysiological processes, including inflammation and allergic reactions, in which mast cells act as major effector cells. IgE receptor (FcεRI) cross-linking leads to an increase in intracellular calcium ([Ca(+2) ]i ), a critical step in mast cell degranulation. The aim of this study was to investigate the role of H2 S in [Ca(+2) ]i -dependent mast cell activation. We investigated the effects of H2 S, either endogenously produced or released by the slow H2 S donor 4-carboxy-phenyl isothiocyanate (PhNCS-COOH), on antigenic- and non-antigenic degranulation of native murine mast cells (BMMC), human (HMC-1) and rat (RBL-2H3) mast cell lines. We measured the release of specific mast cell degranulation markers (β-hexosaminidase and renin), as well as changes in [Ca(+2) ]i and phosphorylation of proteins downstream of FcεRI activation. Endogenously produced H2 S inhibited antigen-induced degranulation in RBL-2H3. Similarly, H2 S released by PhNCS-C...
Investigative Ophthalmology Visual Science, 2009
Frontiers in Cellular Neuroscience, 2016
Science Signaling, 2013
Melatonin stimulates a heteromeric G protein–coupled receptor to modulate the eye’s response to l... more Melatonin stimulates a heteromeric G protein–coupled receptor to modulate the eye’s response to light flashes at night.