Annamaria Mauro

Morphological and molecular tools in identifying the Mediterranean limpets Patella caerulea, Patella aspera and Patella rustica

Allozyme electrophoresis, a partial nucleotide sequence of a mitochondrial gene for cytochrome oxidase I (COI) and discriminant analysis of shell morphometric characters were used to study the relationships among the Sicilian marine gastropods of the Patella genus. Allozyme and mtDNA markers unequivocally distinguished the species and were very useful markers in correctly classifying the different species when morphological characters overlapped each other. Several allozyme loci and many nucleotide positions were diagnostic of species. In contrast, the discriminant analysis of simple morphometric shell characters failed to adequately discriminate the species, suggesting that environmental factors influence colouration and morphological patterns in the Patella species. Our results underline the importance of a genetic approach, as compared to a morphological approach, in discriminating the Mediterranean Patella species.

Immunohistochemical and transcriptional expression of matrix metalloproteinases in full-term human umbilical cord and Human Umbilical Vein Endothelial Cells

Matrix metalloproteinases (MMPs) are extracellular zinc-dependent endopeptidases involved in the degradation and remodelling of extracellular matrix in physiological and pathological processes. MMPs also have a role on cell proliferation, migration, differentiation, angiogenesis and apoptosis. Umbilical cord is a special organ subjected to many changes during pre-natal life and whose cells can maintain a certain degree of plasticity also in post-natal period; for example recently they have been used as a source of stem cells. In this work we investigated the expression of MMPs in human umbilical cord and Human Umbilical Vein Endothelial Cells (HUVEC) though immunohistochemistry, RT–PCR and gelatin zymography. MMP-2 protein is expressed in the amniotic epithelium of human umbilical cord and in few sub-epithelial fibroblasts, while MMP-3 and MMP-10 only in the umbilical epithelium. MMP-8, MMP-9 and MMP-13 immunoreactivity is localised in the epithelium and in Wharton’s jelly mesenchymal cells. Immunocytochemistry also revealed protein expression for MMP-2, 3, 8, 9 and 10 in cultured HUVEC. In agreement with immunohistochemical data, RT–PCR analysis performed on samples of whole umbilical cord confirmed the transcriptional expression for the genes encoding all the six matrix metalloproteinases investigated, while in HUVEC only the expression of MMP-2, 3, 9, 10 and 13 mRNAs was detected. Gelatin zymograpgy showed a clear MMP-2 and MMP-9 enzymatic activity in the conditioned medium of HUVEC at different culture passages, suggesting that HUVEC secrete gelatinases, that afterwards undergo extracellular activation, and this ability is not affected by passage number.

Expression of cyclooxygenase-1 and cyclooxygenase-2 in normal and pathological human oral mucosa.

Cyclooxigenase (COX) is the rate-limiting enzyme for the conversion of arachidonic acid (AA) to prostaglandins (PGs). Two isoforms of COX have been identified: COX-1 is constitutively expressed in many cells and is involved in cell homeostasis, angiogenesis and cell-cell signalling; COX-2 is not expressed in normal condition however it is strongly expressed in inflammation. The oral cavity is constantly exposed to physical and chemical trauma that could lead to mucosal reactions such as hyperplasia, dysplasia and cancer. Early diagnosis is the most important issue to address for a positive outcome of oral cancer; therefore it would be useful to identify molecular markers whose expression is associated with the various stages of oral cancer progression. Since COX enzyme has been involved, with different mechanisms, in the development and progression of malignancies we decided to investigate the expression and localization of COX-1 and COX-2 in normal human oral mucosa and three different pathologies (hyperplasia, dysplasia and carcinoma) by immunohistochemistry and RT-PCR. COX-1 mRNA and protein have been detected already in normal oral mucosa and their expression progressively increases from normal samples towards hyperplasia, dysplasia and finally carcinoma. On the contrary, COX-2 is not expressed in the normal tissue, starts to be expressed in hyperplasia, reaches the maximum activation in dysplasia and then starts to be downregulated in carcinoma.

Allozymic variation in Mediterranean hake Merluccius merluccius (Gadidae)

Abstract Four hundred and twenty individual hake from 10 sample sites in the Mediterranean Sea were analysed in order to study genetic variability and identify genetic stock structure. Twenty loci were identified, four of which were polymorphic at the 95% level: ADH*, PGI‐1*, PGI‐2* and SOD‐1*. Average observed and expected heterozygosity were 0.084 and 0.090, respectively. PG1–1* deviated from Hardy‐Weinberg expectations due to an excess of heterozygotes and F‐statistic analysis showed also a significant excess of heterozygosity at SOD‐1*. FST was not significant for each locus except for PGI‐2*, where a single sample from the Channel of Sicily (C5) showed a different pattern in allelic frequencies, probably caused by a stochastic event. The samples analyzed seemed to be homogeneous, but future analyses may be necessary using other complementary molecular markers.

Human umbilical cord expresses several vasoactive peptides involved in the local regulation of vascular tone: protein and gene expression of Orphanin, Oxytocin, ANP, eNOS and iNOS

Full-term human umbilical cord contains three blood vessels: two arteries coiled around a vein and surrounded by Whartons jelly, a mucous tissue with few mesenchymal stromal cells and abundant extracellular matrix. Umbilical vessels lack innervations, thus endothelial cells must play a role in the control of blood flow. The aim of this study was to investigate in human umbilical cord the expression of five peptides that could be involved in the regulation of vascular tone: Orphanin FQ, Oxytocin, Atrial Natriuretic Peptide (ANP), endothelial Nitric Oxide Synthase (eNOS) and inducible Nitric Oxide Synthase (iNOS). The expression of these molecules in full-term human umbilical cord was investigated through immunohistochemistry and RT-PCR. Immunoreactivity for Orphanin FQ was detected in Whartons jelly, vessel musculature and endothelium; Oxytocin, ANP and eNOS were expressed by the umbilical epithelium, Whartons jelly and endothelium, whereas iNOS only by endothelial cells. RT-PCR analysis showed transcriptional expression of Oxytocin, ANP and eNOS mRNAs. The presence of Orphanin, Oxytocin, ANP, eNOS and iNOS proteins was identified in the human umbilical cord. mRNA expression for Oxytocin, ANP and eNOS suggest that these molecules are synthesized by umbilical cord cells themselves. The expression of these vasoactive molecules could be part of a general mechanism locally regulating vascular tone.

Expression of MMP-2 and MMP-9 in odontogenic myxoma in a child: report of a clinical case

BackgroundOdontogenic myxoma (OM) is a benign, locally invasive, non-metastasizing neoplasm of the jaw bones. Despite the benign nature of these lesions, there is a high rate of recurrence and the current recommended therapy, depending on the size and behaviour of the lesion, can vary from curettage with peripheral ostectomy, segmental resection up to radical resections for more aggressive lesions. OM is a rare tumour which occurs predominantly in the third decade of life and it is rare in children. Matrix metalloproteinases (MMPs) are a family of extracellular endopeptidases responsible for the degradation and remodelling of extracellular matrix, they are known to be involved in the progression and invasiveness of many types of tumour. MMPs have been studied in OM because of their well-known role in extracellular matrix degradation, tumour invasion and recurrence.Clinical case reportWe report a case of OM in a 6-year-old boy. A conservative excision was accomplished. The mass was excised without affecting the mandibular bone and the inferior alveolar nerve. Curettage and removal of the first right inferior molar were performed. After 6-month follow-up, no evidence of recurrence was found.Experimental dataWe investigated the expression of MMP-2 and MMP-9 in this case of OM in a child. RT-PCR showed the expression of both MMP-2 and MMP-9 mRNAs. Immunohistochemistry showed a weak MMP-2 protein expression while MMP-9 protein was not detected.ConclusionIn this case of OM in a child, we report lack of recurrence after excision associated with low MMP-2 protein expression and absence of MMP-9. We believe it is worthy to deeply investigate the relationship between MMPs expression and OM behaviour with the aim to use MMPs as prognostic and/or therapeutic markers in OM.

Expression of Gelatinases (MMP-2, MMP-9) and Cyclooxygenases (COX-1, COX-2) in Some Benign Salivary Gland Tumors

Salivary gland tumors, most of which are rare benign tumors, represent a histologically heterogenous group with the greatest diversity of morphological and cellular features. The aim of this study is to analyse the expression and possible interactions between gelatinases (MMP-2, MMP-9) and cyclooxygenases (COX-1, COX-2) in some benign salivary gland tumors. We investigated the expression of gelatinases and cyclooxigenases in control salivary gland, Pleomorphic adenoma and Warthins tumor through immunohistochemistry and Reverse Transcription – Polymerase Chain Reaction (PCR). We identified the expression of both classes of enzyme in normal samples and in the two types of pathological samples without any quantitative differences. From the present data no significant differences emerge in the expression of these enzymes among the different pathologies examined. Nevertheless, due to the small number of samples included in this study, general statements regarding correlation between the degree of severity of the tumoral pathology and the quantitative expression of these potential tumoral markers can not be made.

MMP-2, MMP-9, and iNOS expression in human dental pulp subjected to orthodontic traction.

OBJECTIVE To test the hypothesis that some metalloproteinases (MMP-2, MMP-9) and inducible nitric oxide synthetase (iNOS) enzymes in dental pulp samples do not vary when subjected to orthodontic treatment. MATERIALS AND METHODS Human dental pulps were taken from male and female patients (N=10; age 10-14 years). A straight wire technique was used with nickel-titanium or steel archwires. The increase of pressure applied on teeth was gradual. Five patients were subjected to premolar extractions after 14 months of treatment and one after 24 months. Samples were Bouin-fixed, paraffin-embedded, and afterwards processed for immunohistochemistry using anti-MMP-2, anti-MMP-9, and anti-iNOS antibodies. RESULTS A reduction of MMP-2, MMP-9, and iNOS expression occurred in treated samples. This became more evident with increased treatment time. CONCLUSION The hypothesis is rejected. The reduction of expression of those proteins revealed a time-dependent relationship.

Expression of matrix metalloproteinases in full-term human umbilical cord and Human Umbilical Vein Endothelial Cells.

background Matrix metalloproteinases (MMPs) are extracellular zinc-dependent endopeptidases involved in breakdown and remodelling of extracellular matrix in physiological and pathological processes. MMPs have also a role on cell proliferation, migration, differentiation, angiogenesis and apoptosis. Umbilical cord is a special organ subjected to many changes during pre-natal life and whose cells can maintain a certain degree of plasticity also in post-natal period; for example recently they have been used as a source of stem cells. For these reasons we believe it is interesting to investigate the expression of some remodelling enzymes in this tissue. Human Umbilical Vein Endothelial cells (HUVEC) have shown to be endowed with an high degree of plasticity; they have the potentiality to differentiate into different cell types and can be considered one of the territories of umbilical cord more dynamic and involved in remodelling/renewal phenomena, for this reason we decided to extend our research investigating the expression of MMPs also in these cells. Aim In this study we investigated by immunohistochemistry and RT-PCR the expression of the gelatinases MMP-2 and MMP-9, the collagenases MMP-8 and MMP-13 and the stromelysin MMP-3 and MMP-10 in the whole human umbilical cord and in HUVEC. Results MMP-2 protein is expressed in the amniotic epithelium of human umbilical cord and in few sub-epithelial fibroblasts, while MMP-3 and MMP-10 only in the umbilical epithelium. MMP-8, MMP-9 and MMP-13 immunoreactivity is localised in the epithelium and in Wharton’s jelly mesenchymal cells. Immunohistochemistry also revealed protein expression for MMP-2, 3, 8, 9 and 10 in cultured Human Umbilical cord Vein Endothelial Cells. In agreement with immunohistochemical data, RT-PCR analysis performed on samples of whole umbilical cord confirmed the transcriptional expression for the genes encoding all the six matrix metalloproteinases investigated, while in HUVEC cells only the expression of MMP-2, 3, 9, 10 and 13 mRNAs was detected.

Expression of remodelling enzymes (MMP2, MMP9) in samples of salivary gland’s tumors

Introduction Salivary gland tumors are morphologically and clinically diverse group of neoplasms and the most common tumor is the pleomorphic adenoma. Objective The aim of our work is to examine the expression of MMPs in tumors of salivary gland and to investigate the relationship between the expression and the biological behaviour of the tumor. Material and methods In this paper we investigate two normal samples of salivary gland and twelve samples of tumors’s salivary gland. These pathological samples are so distributed pleomorphic adenoma, five Whartin’s tumor, two carcinoma, one myepithelioma and one lymphadenoma. All samples arise from parotid gland except one of pleomorphic adenoma which arises from submandibular gland.We evidentiated the localization of MMP2 and MMP9 through IHC and gene expression through RT-PCR analysis. Results In control samples we identified an expression of MMP2 and MMP9 by IHC, these data are confirmed by RT-PCR analysis. In lymphadenoma we identified gene expression of MMP2 and MMP9 these data are confirmed by IHC. In myepithelioma we identified either the gene expression of MMP2 and MMP9 or in the protein’s expression of these two enzymes by IHC. In cases of pleomorphic adenoma we identified the expression of MMP2 and MMP9 by IHC, these data are confirmed by RT-PCR analysis. In cases of Warthin’s tumors we identified the expression of all two MMPs by IHC and by RT-PCR analysis. In cases of carcinoma we identified the expression of MMP2 and MMP9 by IHC, these data are confirmed by RT-PCR analysis. Conclusions In our cases examined we found that the mechanisms of remodelling about MMPs are present either in all physiological or in pathological tissues. Infact there is not clear difference between the groups we investigated, so that we conclude that there is not difference about the remodelling by MMP2 and MMP9 in samples investigated, so we are studying an other MMP, that is MMP8, to investigate if the behaviour of MMP8 will be similar.