-
[show abstract]
[hide abstract]
ABSTRACT: The vasculature is the first organ to arise during development. Blood vessels assure metabolic homeostasis by supplying oxygen
and nutrients and removing waste products. Vasculature also mediates signals for correct organogenesis. Not surprisingly therefore,
vessels are critical for organ growth in the embryo and for repair of wounded tissue in the adult. In this chapter we will
focus in particular on molecular factors involved in differentiation and proliferation of arterial smooth muscle cells, endothelial
cells, and activated myofibroblasts. Moreover, we will provide a brief overview of the current experimental evidence about
the contribution of stem cells to arterial structure in pathophysiological conditions, with particular attention to injury-induced
arterial restenosis.
KeywordsArtery-Smooth muscle cells-Endothelial cells-Myofibroblasts-Stem cells-Restenosis-Differentiation-Cell cycle-TGF-β1-VEGF
12/2009: pages 203-228;
-
[show abstract]
[hide abstract]
ABSTRACT: Archaeological, anthropological and pathological data suggest that thirteen skeletons found in a house at the Pompeii archaeological site, dated to 79 A.D., belong to one family. To verify this and to identify the relationships between these individuals, we analyzed DNA extracted from bone specimens. Specifically, hypervariable segment 1 (HVS1) of the human mitochondrial DNA (mtDNA) control region was amplified in two overlapping polymerase chain reactions and the sequences were compared to the revised Cambridge Reference Sequence. As independent controls, other polymorphic sites in HVS1, HVS2 and in the coding region were analyzed. We also amplified some short tandem repeats of the thirteen specimens. This study revealed that six of the thirteen individuals are indeed closely related.
Annals of Human Genetics 08/2009; 73(Pt 4):429-37. · 2.57 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Restenosis is a pathophysiological phenomenon that can occur in patients submitted to revascularization procedures (bypass, endarterectomy, angioplasty), possibly resulting in new narrowing of injured vessels. Vascular restenosis remains a pressing clinical problem, despite the therapeutic strategies and devices developed so far. Stem cells hold a great potential for the regeneration of damaged tissues in cardiovascular diseases. Recent studies clearly indicated that different stem cell populations contribute to vascular remodeling after injury. Nevertheless, the exact role of vascular cell precursors in restenosis pathophysiology is not yet well defined, as heterogeneous and contrasting data are currently available. Mesenchymal stromal/stem cells (MSCs) are non-hematopoietic multi-potent stem-like cells able of differentiating into both mesenchymal and non-mesenchymal lineages. MSCs offer a series of advantages: a) they can be isolated from a small aspirate of bone marrow; b) extensively proliferate in vitro while preserving a normal karyotype and telomerase activity on several passages; c) express low immunogenicity and hence their use should not require a pharmacological immunosuppression. MSCs have an intrinsic ability to differentiate into functional cell types able to repair the diseased or injured tissue in which they are localised. For this reason, MSCs are currently under scrutiny for treatment of different cardiovascular diseases. Nevertheless, it has not yet been clearly determined whether MSCs can substantially contribute to a positive resolution of restenosis after vascular injury. This review will provide information about the mechanisms at the basis of vascular restenosis and the current knowledge of the role, positive or negative, played by MSCs in restenosis progression as highlighted by recent experimental protocols.
Current Vascular Pharmacology 08/2009; 7(3):381-93. · 2.90 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Random amplified polymorphic DNA (RAPD) is an efficient tool to detect DNA polymorphisms and to distinguish different clones
and cultivars. We have applied the RAPD technique to distinguish, starting from a single rice seed, among some Italian rice
cultivars from market and from National Institute for research on food and nutrition, Italy. We also estimated the genomic
similarity among the rice cultivars object of this study taking advantage of the amplification patterns. Despite the low genomic
variability among Italian rice cultivars, RAPD technique allowed us to distinguish among them. This indicates that RAPD can
be used routinely to verify the identity of Italian rice cultivars in order to prevent fraudulent commercial activity.
European Food Research and Technology 03/2009; 228(6):875-881. · 1.57 Impact Factor
-
Amalia Forte,
Mauro Finicelli,
Pasquale De Luca,
Ina Nordström,
Francesco Onorati,
Cesare Quarto,
Pasquale Santè,
Attilio Renzulli,
Umberto Galderisi,
Liberato Berrino,
Marisa De Feo,
Per Hellstrand,
Francesco Rossi,
Maurizio Cotrufo, Antonino Cascino,
Marilena Cipollaro
[show abstract]
[hide abstract]
ABSTRACT: Vascular surgery aimed at stenosis removal induces local reactions often leading to restenosis. Although extensive analysis has been focused on pathways activated in injured arteries, little attention has been devoted to associated systemic vascular reactions. The aim of the present study was to analyse changes occurring in contralateral uninjured rat carotid arteries in the acute phase following unilateral injury. WKY (Wistar-Kyoto) rats were subjected to unilateral carotid arteriotomy. Contralateral uninjured carotid arteries were harvested from 4 h to 7 days after injury. Carotid arteries were also harvested from sham-operated rats and uninjured rats. Carotid morphology and morphometry were examined. Affymetrix microarrays were used for differential analysis of gene expression. A subset of data was validated by real-time RT-PCR (reverse transcription-PCR) and verified at the protein level by Western blotting. A total of 1011 genes were differentially regulated in contralateral uninjured carotid arteries from 4 h to 7 days after arteriotomy (P<0.0001; fold change, >or=2) and were classified into 19 gene ontology functional categories. To a lesser extent, mRNA variations also occurred in carotid arteries of sham-operated rats. Among the changes, up-regulation of members of the RAS (renin-angiotensin system) was detected, with possible implications for vasocompensative mechanisms induced by arteriotomy. In particular, a selective increase in the 69 kDa isoform of the N-domain of ACE (angiotensin-converting enzyme), and not the classical somatic 195 kDa isoform, was observed in contralateral uninjured carotid arteries, suggesting that this 69 kDa isoenzyme could influence local AngII (angiotensin II) production. In conclusion, systemic reactions to injury occur in the vasculature, with potential clinical relevance, and suggest that caution is needed in the choice of controls during experimental design in vivo.
Clinical Science 01/2009; 116(2):125-36. · 4.61 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Histone deacetylase inhibitors (HDACi) have received a great amount of attention for their antitumoral properties. Suberoyl anilide hydroxamic acid (SAHA) and MS-275 are among the more promising HDACi for cancer treatments. Although these HDACi compounds exert low toxicity on normal cells, the therapies based on these molecules can cause side effects that can greatly impair the functions of the bone marrow microenvironment. This is a complex system that contains several types of stem cells, such as mesenchymal stem cells (MSCs). We conducted comparative studies on the effects of SAHA and MS-275 on human MSCs in order to ascertain if these compounds can impair the physiology of MSCs. Both SAHA and MS-275 induced an arrest in the cell cycle along with the induction of apoptotic pathways as evidenced by flow cytometry, annexin assay, detection of activated caspase 9, and molecular analysis of Bax/Bcl-2 expression. The MS-275 treatment induced an increase of senescent cells, whereas in cells treated with SAHA, we detected a reduction of senescent cells compared to the control. We hypothesize that SAHA preferentially transactivates apoptotic genes, thereby inducing a great majority of the damaged cells to die by programmed cell death rather than senescence. Following the HDACi treatment, we observed a decrease in the expression of some genes that are involved in the regulation of stem cell properties. This suggests that SAHA and MS-275 could also be involved in the impairment of the stemness characteristics of MSCs. The phenomena that were induced by HDACi treatment were associated with an upregulation of several cyclin kinase inhibitors. By contrast, the p53-p21 pathway is apparently not involved in these processes.
Stem cells and development 09/2008; 18(4):573-81. · 4.15 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Restenosis following vascular injury remains a pressing clinical problem. Mesenchymal stem cells (MSCs) promise as a main actor of cell-based therapeutic strategies. The possible therapeutic role of MSCs in vascular stenosis in vivo has been poorly investigated so far. We tested the effectiveness of allogenic bone marrow-derived MSCs in reduction of stenosis in a model of rat carotid arteriotomy. MSCs were expanded in vitro retaining their proliferative and differentiation potentiality. MSCs were able to differentiate into adipocyte and osteocyte mesenchymal lineage cells, retained specific antigens CD73, CD90, and CD105, expressed smooth muscle alpha-actin, were mainly in proliferative phase of cell cycle and showed limited senescence. WKY rats were submitted to carotid arteriotomy and to venous administration with 5 x 10(6) MSCs. MSCs in vivo homed in injured carotids since 3 days after arteriotomy but not in contralateral uninjured carotids. Lumen area in MSC-treated carotids was 36% greater than in control arteries (P = 0.016) and inward remodeling was limited in MSC-treated carotids (P = 0.030) 30 days after arteriotomy. MSC treatment affected the expression level of inflammation-related genes, inducing a decrease of IL-1beta and Mcp-1 and an increase of TGF-beta in injured carotids at 3 and 7 days after arteriotomy (P < 0.05). Taken together, these results indicate that allogenic MSC administration limits stenosis in injured rat carotids and plays a local immunomodulatory action.
Journal of Cellular Physiology 08/2008; 217(3):789-99. · 3.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Hypertension has been traditionally considered a risk factor for restenosis following carotid arteriotomy. Genetic and morphological response to carotid arteriotomy in normotensive Wystar-Kyoto (WKY), spontaneously hypertensive (SHR), and Milan hypertensive (MHS) rats were analyzed.
C-myc, angiotensin II receptor-1 (AT1), angiotensin II receptor-2 (AT2), endothelin-1 receptor A (ET(A)), endothelin-1 receptor B (ET(B)), Bcl-2 family-members (Bcl-2/Bax, Bcl-X(L/S)) were analyzed in surgically injured as well as uninjured carotids of WKY and hypertensive strains (HS). Thirty-day histology and morphometry were accomplished on injured and uninjured carotids.
C-myc mRNA is activated earlier and/or to a greater extent in hypertensive strains than in WKY. AT1 mRNA increases in WKY after injury, while it decreases in SHR and MHS. AT2 shows the opposite, decreasing in WKY and increasing in hypertensive strains. ET(A) mRNA decreases in all strains although with different timing and levels, associated with a replacement by ET(B) mRNA. Bcl-2/Bax ratio gradually decreases in WKY, while it shows only a transient decrease in SHR and MHS 4 h after the injury. Negative remodeling is observed in all injured carotids, although neointima was detected in WKY only. Thirty days following arteriotomy, morphometry demonstrated a significant decrease of luminal area, with consistent gain in the medial area in WKY, whereas hypertensive strains showed significant increase of the luminal area, consistent with a contemporary decrease of the medial area.
Vaso-relaxant AT2 and ET(B) induced limited vasoconstriction in HS. Less apoptosis in hypertensive rats reduced cell proliferation, contrasting c-myc. These responses favorably modulated media/lumen area ratio following arteriotomy in HS.
Journal of Surgical Research 01/2008; 143(2):300-10. · 2.25 Impact Factor
-
Amalia Forte,
Mauro Finicelli,
Pasquale De Luca,
Cesare Quarto,
Francesco Onorati,
Pasquale Santè,
Attilio Renzulli,
Umberto Galderisi,
Liberato Berrino,
Marisa De Feo,
Francesco Rossi,
Maurizio Cotrufo, Antonino Cascino,
Marilena Cipollaro
[show abstract]
[hide abstract]
ABSTRACT: Vascular injury aimed at stenosis removal induces local reactions often leading to restenosis. The aim of this study was a concerted transcriptomic-proteomics analysis of molecular variations in a model of rat carotid arteriotomy, to dissect the molecular pathways triggered by vascular surgical injury and to identify new potential anti-restenosis targets. RNA and proteins extracted from inbred WKY rat carotids harvested 4hrs, 48hrs and 7 days after arteriotomy were analyzed by Affymetrix rat microarrays and by bidimensional electrophoresis followed by liquid chromatography and tandem mass spectrometry, using as reference the RNA and the proteins extracted from uninjured rat carotids. Results were classified according to their biological function, and the most significant KEGG pathways were identified. A total of 1163 mRNAs were differentially regulated in arteriotomy-injured carotids 4hrs, 48hrs and 7 days after injury (p<0.0001, fold-change >/=2), while 48 spots exhibited significant changes after carotid arteriotomy (p<0.05, fold-change >/=2). Among them, 16 spots were successfully identified and resulted to correspond to a set of 19 proteins. mRNAs were mainly involved in signal transduction, oxidative stress/inflammation and remodeling, including many new potential targets for limitation of surgically-induced (re)stenosis (e.g. Arginase I, Kruppel like factors). Proteome analysis confirmed and extended the microrarray data, revealing time-dependent post-translational modifications of Hsp27, haptoglobin and contrapsin-like protease inhibitor 6, and the differential expression of proteins mainly involved in contractility. Transcriptomic and proteomic methods revealed functional categories with different preferences, related to the experimental sensitivity and to mechanisms of regulation. The comparative analysis revealed correlation between transcriptional and translational expression for 47% of identified proteins. Exceptions from this correlation confirm the complementarities of these approaches.
Journal of Cellular and Molecular Medicine 12/2007; · 4.13 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Self-renewal, proliferation and differentiation properties of stem cells are controlled by key transcription factors. However, their activity is modulated by chromatin remodeling factors that operate at the highest hierarchical level. Studies on these factors can be especially important to dissect molecular pathways governing the biology of stem cells. SWI/SNF complexes are adenosine triphosphate (ATP)-dependent chromatin remodeling enzymes that have been shown to be required for cell cycle control, apoptosis and cell differentiation in several biological systems. The aim of our research was to investigate the role of these complexes in the biology of mesenchymal stem cells (MSCs). To this end, in MSCs we caused a forced expression of the ATPase subunit of SWI/SNF (Brg1 - also known as Smarca4) by adenoviral transduction. Forced Brg1 expression induced a significant cell cycle arrest of MSCs in culture. This was associated with a huge increase in apoptosis that reached a peak 3 days after transduction. In addition, we observed signs of senescence in cells having ectopic Brg1 expression. At the molecular level these phenomena were associated with activation of Rb- and p53-related pathways. Inhibition of either p53 or Rb with E1A mutated proteins allowed us to hypothesize that both Rb and p53 are indispensable for Brg1-induced senescence, whereas only p53 seems to play a role in triggering programmed cell death. We also looked at the effects of forced Brg1 expression on canonical MSC differentiation in adipocytes, chondrocytes and osteocytes. Brg1 did not induce cell differentiation per se; however, this protein could contribute, at least in part, to the adipocyte differentiation process. In conclusion, our results suggest that whereas some ATP-dependent chromatin remodeling factors, such as ISWI complexes, promote stem cell self-renewal and conservation of an uncommitted state, others cause an escape from 'stemness' and induction of differentiation along with senescence and cell death phenomena.
Journal of Cell Science 09/2007; 120(Pt 16):2904-11. · 6.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Mechanisms governing commitment and differentiation of the cells of the nervous system begin to be elucidated: how extrinsic and intrinsic components are related remains poorly understood. To investigate this issue, we overexpressed genes of the retinoblastoma (Rb) family RB and RB2/p130, which play an important role during nerve cell maturation, in rat neural stem cells (NSCs). Immunostaining of neurons, astrocytes and oligodendrocytes in cultures overexpressing pRb or pRb2/p130 revealed that these genes affect lineage specification of differentiating NSCs. We observed modifications in percentage of differentiated cells indicating a shift towards the phenotype induced by culture conditions. Results were confirmed by detection of the expression levels of differentiation markers by RT-PCR. Analysis of BrdU incorporation and detection of an early marker of apoptosis suggest that the effect of pRb and pRb2/p130 overexpression is not dependent on the inhibition of cell proliferation, nor does it rely on the regulation of cell survival. Our findings suggest that Rb family genes are involved in fate determination of the cells of the nervous system. However, their role seems subsidiary to that of the extrinsic signals that promote lineage specification and appear to be mediated by a direct effect on the acquisition of a specific phenotype.
Molecular and Cellular Neuroscience 04/2007; 34(3):299-309. · 3.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Mesenchymal stem cells (MSCs) promise as a main actor of cell-based therapeutic strategies, due to their intrinsic ability to differentiate along different mesenchymal cell lineages, able to repair the diseased or injured tissue in which they are localized. The application of MSCs in therapies requires an in depth knowledge of their biology and of the molecular mechanisms leading to MSC multilineage differentiation. The knockdown of target genes through small interfering RNA (siRNA) carried by adenoviruses (Ad) represents a valid tool for the study of the role of specific molecules in cell biology. Unfortunately, MSCs are poorly transfected by conventional Ad serotype 5 (Ad5) vectors. We set up a method to obtain a very efficient transduction of rat MSCs with low doses of unmodified Ad5, carrying the siRNA targeted against the mRNA coding for Rb2/p130 (Ad-siRNA-Rb2), which plays a fundamental role in cell differentiation. This method allowed a 95% transduction rate of Ad-siRNA in MSC, along with a siRNA-mediated 85% decrease of Rb2/p130 mRNA and a 70% decrease of Rb2/p130 protein 48 h after transduction with 50 multiplicities of infection (MOIs) of Ad5. The effect on Rb2/p130 protein persisted 15 days after transduction. Finally, Ad-siRNA did not compromise the viability of transduced MSCs neither induced any cell cycle modification. The effective Ad-siRNA-Rb2 we constructed, together with the efficient method of delivery in MSCs we set up, will allow an in depth analysis of the role of Rb2/p130 in MSC biology and multilineage differentiation.
Journal of Cellular Biochemistry 03/2007; 100(2):293-302. · 2.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: c-myc is the main proto-oncogene responsible for restenosis in cardiovascular surgery. The aim of our study was to evaluate the effects of c-myc antisense (AS) phosphorothioate oligodeoxynucleotides (ODNs) in the remodelling process induced by surgical carotid arteriotomy on an experimental rat model.
Fifty-five rats with carotid stenosis and apoptosis induced by arteriotomy were submitted to gene expression analysis 4 h after surgery, to TUNEL assay 48 h after surgery and to histological analysis 30 days later.
AS ODNs induced a 60% decrease in target c-myc mRNA in injured carotid arteries compared to control sense and scrambled ODN-treated carotid arteries (P < 0.05). Histological evaluation revealed that stenosis stimulated by arteriotomy was mainly due to adventitial constrictive remodelling rather than to neointimal hyperplasia, observed only in a limited number of samples. Morphometric analysis showed that lumen area in c-myc AS ODN-treated carotid arteries was 35% greater than in control arteries (P < 0.05), whereas the media/lumen area ratio showed a 63% reduction in AS ODN-treated carotid arteries in comparison to control arteries (P < 0.05). Surgical injury affected the expression of apoptosis-related genes Bcl-2, Bax, Bcl-xL and Bcl-xS, inducing a mean 3.5-fold decrease in the Bcl-2/ Bax ratio and a 9-fold decrease in the Bcl-xL/S ratio 4 h after injury as compared with uninjured carotid arteries. TUNEL assay experiments revealed increased apoptosis in AS ODN-treated carotid arteries in comparison to control carotid arteries.
c-myc AS ODNs reduce the negative remodelling induced by arteriotomy. The imbalance between proliferative stimulus represented by surgery and the c-myc mRNA decrease induced greater apoptosis in AS ODN-treated carotid arteries without further affecting mRNA levels of Bcl-2, Bax, Bcl-xL and Bcl-xS genes.
Journal of Cardiovascular Medicine 02/2006; 7(1):61-7. · 1.51 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Analysis of gene expression profiles in patients or in animal models affected by cardiovascular diseases may provide insight into therapeutic strategies. In this study, 3 rat strains, Wistar Kyoto (WKY), spontaneously hypertensive rats (SHR) and the Milan hypertensive rat strain (MHS), have been investigated to assess the influence of genetic background and/or of hypertension on gene expression in arteriotomy-injured carotid arteries (CAs). Expression profiles of genes, c-myc, AT1, AT2, ETA, ETB, Bcl-2, Bax and Bcl-X, were determined by reverse transcription-polymerase chain reaction (RT-PCR) in the acute phase, from 1 to 48 h, following CA arteriotomy. WKY, SHR and MHS show significant differences in gene expression profiles after CA arteriotomy. c-Myc mRNA is activated earlier and/or to a greater extent in hypertensive strains than in WKY (p<0.05). AT1 mRNA increases in WKY after injury, while it decreases in both SHR and MHS (p<0.05). AT2 shows the opposite behaviour, decreasing in WKY and increasing in hypertensive strains (p<0.05). ETA mRNA decreases in all strains although with different timing and levels, associated with a replacement by ETB mRNA (p<0.05). Bcl-2/Bax ratio gradually decreases in WKY, while it decreases only transiently in SHR and MHS 4 h after injury (p<0.05). Overall data indicate that therapeutic strategies for stenosis prevention should carefully consider the gene expression profile after injury, the genetic background, the kind of vascular trauma and the diseases affecting the animal model or the patient.
International Journal of Molecular Medicine 12/2005; 16(6):1057-64. · 1.98 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In recent years several reports have claimed to demonstrate trans-differentiation, namely that stem cells have been derived from a given tissue and have differentiated into phenotypes characteristic of different tissues following transplantation or in vitro treatment. For example, the mesenchymal stem cells, also referred to as marrow stromal stem cells (MSCs), present in bone marrow, have been induced to differentiate into neurons. We decided to investigate this phenomenon more in depth by a molecular and morphological follow-up. We analyzed the biochemical pathways that are currently induced to trigger neuron-like commitment and maturation of MSCs. Our studies suggest that: (i) the increase in cAMP, induced to differentiate MSCs, activates the classical PKA pathway and not through the exchange protein directly activated by cAMP (EPAC), a guanine nucleotide exchange factor for the small GTPase Rap1 and Rap2; (ii) MEK-ERK signaling could contribute to neural commitment and differentiation; (iii) CaM KII activity seems dispensable for neuron differentiation. On the contrary, its inhibition could contribute to rescuing differentiating cells from death. Our research also indicates that the currently used in vitro differentiation protocols, while they allow the early steps of neural differentiation to take place, are not able to further sustain this process.
Journal of Cellular Biochemistry 04/2005; 94(4):645-55. · 2.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Marrow stromal cells (MSCs) are stem-like cells having a striking somatic plasticity. In fact, besides differentiating into mesenchymal lineages (bone, cartilage, and fat), they are capable of differentiating into neurons and astrocytes in vitro and in vivo. The RB and RB2/P130 genes, belonging to the retinoblastoma gene family, play a key role in neurogenesis, and for this reason, we investigated their role in neural commitment and differentiation of MSCs. In MSCs that were either uncommitted or committed toward neural differentiation, we ectopically expressed RB and RB2/P130 genes and analyzed their role in regulating the cell cycle, apoptosis and differentiation. In uncommitted MSCs, the activity of RB and RB2/P130 appeared limited to negatively regulating cell cycle progression, having no role in apoptosis and differentiation (toward either mesenchymal or neural lineages). On the other hand, in MSCs committed toward the neural phenotype, both RB and RB2/P130 reduced cell proliferation rate and affected the apoptotic process. RB protected differentiating cells from programmed cell death. On the contrary, RB2/P130 increased the percentage of cells in apoptosis. All of these activities were accomplished mainly in an HDAC-independent way. The retinoblastoma genes also influenced differentiation in neural committed MSCs. RB2/P130 contributes mainly to the induction of generic neural properties, while RB triggers cholinergic differentiation. These differentiating activities are HDAC-dependent. Our research shows that there is a critical temporal requirement for the RB genes during neuronal differentiation of MSCs: they are not required for cell commitment but play a role in the maturation process. For the above reasons, RB and RB2/P130 may have a role in neural differentiation but not in neural determination.
Journal of Cellular Physiology 09/2004; 200(2):201-12. · 3.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: DNA extracted from the skeletons of five equids discovered in a Pompeii stable and of a horse found in Herculaneum was investigated. Amino acid racemization level was consistent with the presence of DNA. Post-mortem base modifications were excluded by sequencing a 146 bp fragment of the 16S rRNA mitochondrial gene. Sequencing of a 370 bp fragment of mitochondrial (mt)DNA control region allowed the construction of a phylogenetic tree that, along with sequencing of nuclear genes (epsilon globin, gamma interferon, and p53) fragments, gave us the possibility to address some questions puzzling archaeologists. What animals-donkeys, horses, or crossbreeds-were they? And, given they had been evidently assigned to one specific job, were they all akin or were they animals with different mitochondrial haplotypes? The conclusions provided by molecular analysis show that the Pompeii remains are those of horses and mules. Furthermore one of the equids (CAV5) seems to belong to a haplotype, which is either not yet documented in the GenBank or has since disappeared. As its characteristics closely recall those of donkeys, which is the out group chosen to construct the tree, that appears to have evolved within the Equidae family much earlier than horses, this assumption seems to be nearer the truth.
Journal of Cellular Physiology 06/2004; 199(2):200-5. · 3.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We used rats with a sciatic nerve chronic constrictive injury (CCI) and combined behavioural, molecular and morphological approaches to assess the involvement of mGlu5 receptors in neuropathic pain-associated hyperalgesia and spinal cord neuron apoptosis. Mechanical and thermal hyperalgesia developed 2-3 days after surgery. Morphological changes in the ipsilateral L4-L5 lamina II consisted of: (i) cell loss (38 +/- 5%), (ii) increased TUNEL-positive profiles, (iii) decreased SP-immunoreactive primary afferents, and (iv) reactive gliosis. Molecular expression data suggested a bi-phasic response of bcl-2 family genes in CCI. An early (2-3 days post-CCI) E2F1- and p53-independent apoptosis appeared in the spinal cord as the pro-apoptotic bax gene increased (320 +/- 19%), followed by an increased expression of the anti-apoptotic bcl-2 and bcl-xL genes (60 +/- 11% and 110 +/- 15%, respectively) 7 days from CCI. The selective mGlu5 receptor antagonist, MPEP (2 mg/kg i.p. twice daily), prevented the development of thermal hyperalgesia and transiently reduced mechanical hyperalgesia. Despite the MPEP treatment, which normalised bax/bcl-2 and bcl-xL/bcl-xS ratios at all times post-CCI, mechanical hyperalgesia reappeared by 7 days after CCI. Similarly, MPEP was cytoprotective at 3, but not 7 days post-CCI. This study shows that: (a) spinal cord neuron loss may be triggered by a p53- and E2F1-independent apoptosis in lamina II with the participation of glutamate mGlu5 receptors, (b) these receptors seem to be involved transiently, as their blockade was no longer protective by 7 days CCI, and (c) this delayed cell death occurred in the absence of Bax activation, suggesting the involvement of an alternative death pathway.
Neuropharmacology 04/2004; 46(4):468-79. · 4.81 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: DNA extracted from the skeletons of five equids discovered in a Pompeii stable and of a horse found in Herculaneum was investigated. Amino acid racemization level was consistent with the presence of DNA. Post-mortem base modifications were excluded by sequencing a 146 bp fragment of the 16S rRNA mitochondrial gene. Sequencing of a 370 bp fragment of mitochondrial (mt)DNA control region allowed the construction of a phylogenetic tree that, along with sequencing of nuclear genes (epsilon globin, gamma interferon, and p53) fragments, gave us the possibility to address some questions puzzling archaeologists. What animals—donkeys, horses, or crossbreeds—were they? And, given they had been evidently assigned to one specific job, were they all akin or were they animals with different mitochondrial haplotypes? The conclusions provided by molecular analysis show that the Pompeii remains are those of horses and mules. Furthermore one of the equids (CAV5) seems to belong to a haplotype, which is either not yet documented in the GenBank or has since disappeared. As its characteristics closely recall those of donkeys, which is the out group chosen to construct the tree, that appears to have evolved within the Equidae family much earlier than horses, this assumption seems to be nearer the truth. J. Cell. Physiol. 199: 200–205, 2004© 2003 Wiley-Liss, Inc.
Journal of Cellular Physiology 01/2004; 199(2):200 - 205. · 3.87 Impact Factor
-
Amalia Forte,
Salvatore Esposito,
Marisa De Feo,
Umberto Galderisi,
Cesare Quarto,
Ferdinando Esposito,
Attilio Renzulli,
Liberato Berrino,
Marilena Cipollaro,
Lucio Agozzino,
Maurizio Cotrufo,
Francesco Rossi, Antonino Cascino
[show abstract]
[hide abstract]
ABSTRACT: Milan hypertensive rats (MHS) are characterised by an increase in renal sodium reabsorption mainly related to adducin mutations. Interest in this model relies on the genetic link between adducin polymorphisms and primary hypertension, observed also in a subset of patients.
To investigate the molecular and morphological events involved in carotid stenosis and triggered by surgery in MHS model.
Stenosis was induced through arteriotomy. At different times after injury, the expression profiles of various gene families were investigated by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), while histological techniques were used to follow morphometric and morphological changes. Apoptotic nuclei were revealed by terminal deoxynucleotidyl transferase-dUTP nick end labelling (TUNEL).
mRNAs coding for transcription factors c-jun, c-fos and c-myc were rapidly induced by injury. Analysis of apoptosis-related genes revealed a decrease of the Bcl-2/Bax ratio 4 h after vascular trauma (P<0.05), followed by a recovery of antiapoptotic factors 24 and 48 h later. ET(A) and receptor mRNAs decreased after the injury and were replaced by ET(B) and AT2 mRNAs. Both ET(A) and AT1 turned to basal level 48 h after injury. Expression profiles of chatepsins B and D were also determined. A marked neoadventitia led to maximal 60+/-9% lumen reduction (P<0.05) 30 days after surgery. Media substitution by fibrotic and granulomatous tissue was also evident. Maximal 47+/-2% apoptotic nuclei were detected 48 h after the injury (P<0.05).
The injury applied to MHS carotids induces negative remodelling and a limited apoptotic reaction. These findings could arise from the balancing among proliferative factors, apoptosis-related molecules and relaxant anti-proliferative receptors, all stimulated by the injury.
Cardiovascular Research 12/2003; 60(3):654-63. · 6.06 Impact Factor
