Biomedical subjects

Luca De Luca

Publications and source records attributed to Luca De Luca.

11 recordsLinked to original sources

PubMed2004

Quantum mechanical models of the resting state of the vanadium-dependent haloperoxidase.

Density functional theory has been used to investigate structural and electronic properties of complexes related to the resting form of the active site of vanadium haloperoxidase as a function of environment and protonation state. Results obtained by studying models of varying size and complexity highlight the influence of environment and protonation state on the structure and stability of the metal cofactor. The study shows that, in the trigonal bipyramidal active site, where one axial position is occupied by a key histidine, the trans position cannot contain a terminal oxo group. Further, a highly negatively charged vanadate unit is not stable. Protonation of at least one equatorial oxo ligand appears necessary to stabilize the metal cofactor. The study also indicates that, while at rest within the protein, the vanadate unit is most likely an anion with an axial hydroxide and an equatorial plane containing two oxos and a hydroxide. For the neutral, protonated state of the vanadate unit, there were two minima found. The first structure is characterized by an axial water with two oxo and one hydroxo group in the equatorial plane. The second structure contains an axial hydroxo group and an equatorial plane composed of one oxo and two hydroxo oxygen atoms. These two species are not significantly different in energy, indicating that either form may be important during the catalytic cycle. These data support the initial crystallographic assignment of an axially bound hydroxide, but an axial water is also a possibility. This study also shows that the protonation state of the vanadate ion is most likely greater than previously proposed.

Binding Sites↗

PubMed2004

Characterization of tissue specific expression of Notch-1 in human tissues.

Signaling through the Notch cell surface receptors is a highly conserved mechanism of cell fate specification. Notch signaling regulates proliferation, differentiation and cell death. In vertebrates, putative gene duplication has originated four Notch genes, Notch-1, -2, -3 and -4. They have been implicated in neurogenesis, hematopoiesis, T-cell development, vasculogenesis and brain cortical growth. We have investigated Notch-1 distribution in normal human tissues by immunohistochemistry and immunoblot. We detected widespread expression of Notch-1 cytoplasmatic staining, with different tissue distributions in the different organs examined. In particular, high expression of Notch-1 was detected in the intermediate suprabasal layers, but not in the dead cells at the extreme periphery of stratified epithelia. Moreover, a low/intermediate level of Notch-1 was observed in lymphocytes in several peripheral lymphoid tissues; in particular the germinal centers of lymph nodes showed the most abundant number of positive cells, which appeared to be centroblasts/immunoblasts based on nuclear morphology. Notch-1 participates in keratinocytes differentiation. We showed by Western blot analysis that Notch-1 level was clearly increased in HaCaT cells after Ca(++) addition and remained substantially elevated until late differentiation stages. These results suggest that Notch-1 may function in numerous cell types in processes beyond cell fate determination, such as neuronal plasticity, muscle hypertrophy, liver regeneration, and germinal center lymphopoiesis during the immune response.

Blotting, Western↗

PubMed2004

Evaluation of cyclin D1 expression and its subcellular distribution in mouse tissues.

Cyclin D1 is a key cell-cycle regulatory protein required for the cell to progress through G1 to S phase. We have shown by Western blot analysis that cyclin D1 has a wide distribution in adult mouse tissues, with its level of expression being tissue-dependent. Immunohistochemistry has also shown that cyclin D1 may be present in the cytoplasm, in the nucleus or in both these cell compartments: cytoplasmic staining was observed in both proliferating cells (e.g. kidney, intestine, stomach and salivary gland) and in the non-dividing cells (the mature neurons of adult brain), while nuclear staining was seen in the neurons of the embryonic nervous system. Immunoelectron microscopy results indicate that, in tissues where cyclin D1 is present in both compartments (e.g. intestinal enterocytes), it may move via nuclear pores from the nucleus to the cytoplasm, and vice versa. The findings as a whole suggest that cyclin D1 may play multiple roles within specific tissues, probably by interacting with different substrates, and that its transit between nuclear and cytoplasmic compartments may help maintain cell homeostasis.

Animals↗

PubMed2004

Pattern of expression of HtrA1 during mouse development.

The human HtrA family of proteases consists of four members: HtrA1, HtrA2, HtrA3, and HtrA4. In humans the four HtrA homologues appear to be involved in several important functions such as cell growth, apoptosis, and inflammatory reactions, and they control cell fate via regulated protein metabolism. In previous studies it was shown that the expression of HtrA1 was ubiquitous in normal adult human tissues. Here we examined the expression of HtrA1 protein and its corresponding mRNA during mouse embryogenesis using Northern blotting hybridization, RT-PCR, and immunohistochemical staining analyses. Our results indicate that HtrA1 is expressed in a variety of tissues in mouse embryos. Furthermore, this expression is regulated in a spatial and temporal manner. Relatively low levels of HtrA1 mRNA are detected in embryos at the beginning of organogenesis (E8), and the levels of expression increase during late organogenesis (E14-E19). Our results show that HtrA1 was expressed during embryonic development in specific areas where signaling by TGFbeta family proteins plays important regulatory roles. The expression of HtrA1, documented both at mRNA and protein levels by RT-PCR and immunohistochemistry in the developing nervous system, is consistent with a possible role of this protein both in dividing and postmitotic neurons, possibly via its documented inhibitory effects on TGFbeta proteins. An exhaustive knowledge of the different cell- and tissue-specific patterns of expression of HtrA1 in normal mouse embryos is essential for a critical evaluation of the exact role played by this protein during development.

Animals↗

PubMed2003

Frequent loss of hMLH1 by promoter hypermethylation leads to microsatellite instability in adenomatous polyps of patients with a single first-degree member affected by colon cancer.

The first-degree relatives of patients affected by colorectal cancer, who do not belong to familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer families, have a doubled risk of developing tumors of the large intestine. We have previously demonstrated that subjects with a single first-degree relative (SFDR) with colon cancer have a doubled risk for developing colorectal adenomas, and in these cases, polyps recur more frequently. The mechanism underlying this predisposition has not been clarified. In this study, we evaluated the frequency of microsatellite instability (MSI) using the five markers suggested by the National Cancer Institute workshop, target gene mutations, hMLH1 and hMSH2 expression, and hMLH1 promoter hypermethylation in the adenomas of patients with and without a SFDR affected by colon cancer. Seventy polyps were obtained from 70 patients: 27 with a single FDR with colon cancer and 43 without such a history. Of the 70 polyps, 12 were MSI-H (17.1%), 20 were MSI-L (28.6%), and 30 were microsatellite stable (42.9%). Of the 27 patients with positive family history, 8 polyps (29.6%) were MSI-H compared with those with negative history in which 4 polyps (9.3%) were MSI-H (P < 0.02). Of the 12 MSI-H polyps, all of the polyps obtained from patients with positive family history had loss of hMLH1 immunostaining versus one with negative family history (P < 0.02). Of the MSI-H polyps, 2 had a somatic frameshift mutation of the MBD4 gene, 1 of MSH6, 1 of BAX, and 2 of transforming growth factor betaRII. Furthermore, 6 of 8 polyps from patients with positive family history with MSI-H and loss of MLH1 had hypermethylation of the MLH1promoter versus none of the MSI-H with negative family history (P < 0.02). All 6 polyps of the 27 from SFDR positive subjects, with hMLH1 promoter hypermethylation loss of hMLH1 and MSI, were located in the right colon (P < 0.02). Hypermethylation of the promoter of hMLH1, consequent loss of hMLH1 expression, and MSI are at the basis of approximately 25% of adenomatous polyps developed in subjects with a SFDR affected by colorectal cancer.

Adaptor Proteins, Signal Transducing↗

PubMed2003

p300/cAMP-response-element-binding-protein ('CREB')-binding protein (CBP) modulates co-operation between myocyte enhancer factor 2A (MEF2A) and thyroid hormone receptor-retinoid X receptor.

Thyroid hormone receptors (TRs) and members of the myocyte enhancer factor 2 (MEF2) family are involved in the regulation of muscle-specific gene expression during myogenesis. Physical interaction between these two factors is required to synergistically activate gene transcription. p300/cAMP-response-element-binding-protein ('CREB')-binding protein (CBP) interacting with transcription factors is able to increase their activity on target gene promoters. We investigated the role of p300 in regulating the TR-MEF2A complex. To this end, we mapped the regions of these proteins involved in physical interactions and we evaluated the expression of a chloramphenicol acetyltransferase (CAT) reporter gene in U2OS cells under control of the alpha-myosin heavy chain promoter containing the thyroid hormone response element (TRE). Our results suggested a role of p300/CBP in mediating the transactivation effects of the TR-retenoid X receptor (RxR)-MEF2A complex. Our findings showed that the same C-terminal portion of p300 binds the N-terminal domains of both TR and MEF2A, and our in vivo studies demonstrated that TR, MEF2A and p300 form a ternary complex. Moreover, by the use of CAT assays, we demonstrated that adenovirus E1A inhibits activation of transcription by TR-RxR-MEF2A-p300 but not by TR-RxR-MEF2A. Our data suggested that p300 can bind and modulate the activity of TR-RxR-MEF2A at TRE. In addition, it is speculated that p300 might modulate the activity of the TR-RxR-MEF2A complex by recruiting a hypothetical endogenous inhibitor which may act like adenovirus E1A.

Animals↗

PubMed2003

Role of endogenous endothelin-1 in ethanol-induced gastric mucosal damage in humans.

Gastric microcirculatory disturbances are involved in the ethanol-induced gastric mucosal damage. In this study in humans we evaluated the time course of plasma and gastric mucosal endothelin-1 (ET-1) concentrations after intragastric ethanol administration; furthermore we determined the correlation among changes in gastric tissue endothelin-1 and microscopic and gross gastric hemorrhagic damage. ET-1 concentrations in plasma and gastric mucosa were measured by radioimmunoassay. The endoscopic appearance of the gastric mucosa was evaluated and scored on a scale of 0-5, and gastric biopsies for histological evaluation were obtained from the antral and the corpus mucosa just before and 30 min after 40% ethanol administration in seven healthy volunteers. Plasma ET-1 concentration increased as soon as 20 min after ethanol administration, reached a significant peak at 30 min (P < 0.01), and returned to near basal level within 120 min. Gastric mucosal ET-1 concentration significantly increased 30 min after ethanol administration in both the body (P < 0.05) and the antrum (P < 0.05) of the stomach; however the ET-1 increase was significantly higher in the body. Moreover, data obtained 30 min after ethanol administration showed a significant correlation between gastric mucosal ET-1 levels and gross hemorrhagic damage (r = 0.84). A significant correlation was also observed between antral gastric mucosal ET-1 and microscopic lesions (r = 0.70). We conclude that 40% ethanol, given orally, stimulates the release of gastric mucosal endothelin-1 and causes a rapid and time-dependent increase of ET-1 plasma level in humans. The increased plasma and gastric tissue endothelin-1 concentration may play a role in ethanol-induced gastric hemorrhagic injury in humans.

Adult↗

PubMed2002

Endothelin-B (ET(B)) receptor distribution in tissues of the lizard Podarcis sicula.

Although the structural and pharmacological properties of endothelin (ET) receptors have been studied, little is known concerning their physiological significance, even if each subtype is supposed to have a distinct physiological action. Thus, to further elucidate the physiological function of this receptor, we examined the presence and distribution of the endothelin-B receptor (ET(B)) subtype in tissues of the lizard Podarcis sicula, using immunoblotting and immunohistochemistry. Immunoblotting indicated that, although the ET(B) receptor appears to be ubiquitous, it is present at different levels in the tissues examined. Furthermore, immunohistochemistry showed that this receptor is very abundant in endothelial cells of all tissues, suggesting that there is an ET(B)-mediated autocrine system of endothelin, which plays an important role in the regulation of endothelial cell function. On the other hand, the presence of ET(B) immunoreactivity also in endocrine systems such as adrenal and thyroid glands suggests an involvement also in the paracrine system of these organs.

Adrenal Glands↗

PubMedDate not supplied

Apelin expression in normal human tissues.

Apelin is an endogenous ligand of the human orphan receptor APJ (orphan G protein-coupled receptor). This peptide is produced through processing from the C-terminal portion in the pre-proprotein consisting of 77 amino acid residues and exists in multiple molecular forms. Although the main physiological functions of apelin have not been clarified yet, it has been demonstrated that apelin partially suppresses cytokine production from mouse spleen and, specifically, induces the promotion of extracellular acidification and inhibition of cAMP production in Chinese hamster ovary cells. Moreover, it is involved in the regulation of blood pressure and blood flow. In this study we have analyzed, by immunohistochemistry, apelin distribution in several human tissues, demonstrating that apelin has a widespread pattern of expression. These results seem to confirm that apelin functions widely in various tissues interacting with its specific receptor APJ.

Apelin↗

PubMedDate not supplied

Immunohistochemical study of apelin, the natural ligand of receptor APJ, in a case of AIDS-related cachexia.

Apelin, a peptide first isolated from bovine stomach extracts, was discovered as an endogenous ligand for the APJ receptor. APJ has been shown to be a co-receptor for human and simian immunodeficiency virus (HIV and SIV). Apelin specifically inhibited the entry of primary T-tropic and dualtropic HIV-1 isolated from different clones expressing antiviral CD4 and APJ. On the basis of these results, we decided to compare the apelin expression level between normal and AIDS-infected tissues by immunohistochemistry. We found that apelin expression was less intense in AIDS-infected tissues compared to normal tissues, in particular in the pancreas, kidney, adrenal glands and lymphoid organs. These results suggest an involvement of this peptide in immunodeficiency and in the immune response to AIDS.

Adult↗

PubMedDate not supplied

Expression of surface protein receptors in lung cancer.

Cell adhesion is a basic count in inter- and intracellular communication and plays an important role in tumor progression. In this study, the expression of E-selectin, ICAM-1 and VCAM-1 were evaluated by means of immunohistochemistry in a group of 153 lung cancer specimens. E-selectin immunoreactivity was localized mostly on endothelial cell venules and capillaries with an average staining intensity of 75% of cells in the NSCLC, while in SCLC the intensity of the staining was 69%. The staining pattern for ICAM-1 reached an average intensity of 57%, in both NSCLC and SCLC. Finally, VCAM-1 immunoreactivity was detected only in NSCLC with an average intensity of 12% on endothelial cell venules and capillaries. This study provides a contribution towards the understanding of the basic mechanisms of cell adhesion in lung cancer progression.

Adenocarcinoma↗