Franca Saccucci

Molecular cloning, chromosomal localization, tissue mRNA levels, bacterial expression, and enzymatic properties of human NMN adenylyltransferase.

A 1329-base pair clone isolated from a human placenta cDNA library contains a full-length 837-base pair coding region for a 31.9-kDa protein whose deduced primary structure exhibits high homology to consensus sequences found in other NMN adenylyltransferases. Northern blotting detected a major 3.1-kilobase mRNA transcript as well as a minor 4.1-kilobase transcript in all human tissues examined. In several cancer cell lines, lower levels of mRNA expression were clearly evident. The gene encoding the human enzyme was mapped to chromosome band 1p32–35. High efficiency bacterial expression yielded 1.5 mg of recombinant enzyme/liter of culture medium. The molecular and kinetic properties of recombinant human NMN adenylyltransferase provide new directions for investigating metabolic pathways involving this enzyme.

The human CD38 gene: polymorphism, CpG island, and linkage to the CD157 (BST-1) gene.

Abstract CD38 is a leukocyte activation antigen and ectoenzyme [NAD(P)+ glycohydrolase; EC 3.2.2.6] involved in numerous immune functions. The human CD38 gene is complex [eight exons, >80 kilobases (kb) long] located on Chromosome 4p15, and part of the eukaryotic NAD+ glycohydrolase/ADP-ribosyl cyclase gene family. Because of the increasing relevance of the CD38 molecule in the host immune response to infectious, tumoral, and metabolic diseases, we investigated the genetic variability and linkage of the human CD38 locus. We report that (1) the restriction endonuclease Pvu II identifies a bi-allelic polymorphism here defined as formed by the alleles CD38*A (12 kb) and CD38*B (9/2.5 kb); (2) their frequency in the healthy Italian Caucasian population is 14% and 86%, respectively; (3) the polymorphic Pvu II site is located at the 5′ end of the first intron of the CD38 gene; (4) in conjunction with the polymorphic site, we identified a 900 base pair CpG island associated with the CD38 gene, with two potential Sp1 binding sites; (5) the CpG island may play a role in the regulation of CD38 expression and is hypomethylated in various cell lines; (6) by pulsed-field gel electrophoresis we show that CD38 and its paralogue, the bone-marrow stromal cell antigen BST-1 (CD157), map to the same 800 kb Avi II fragment, indicating that the two human ecto-NADase genes are closely linked.

Studies on the life prolonging effect of food restriction: glutathione levels and glyoxalase enzymes in rat liver

Cytosolic and mitochondrial levels of glutathione (GSH) as well as the activities of glyoxalase I (GI) and glyoxalase II (GII), GSH-dependent enzymes involved in the detoxification of 2-ketoaldehydes, were investigated in the liver of ad libitum (AL) fed and food restricted (FR) rat during aging. Both cytosolic and mitochondrial GSH level was lower in old than in adult AL fed rats. Food restriction did not prevent this decrease, but its extent was attenuated considering the cytosolic GSH. As regards the mitochondrial GSH, its content was higher in adult FR animals than in the age-matched AL fed ones. Thus, the subsequent age-dependent decrease of GSH, occurring also in FR animals, resulted in a thiol concentration not different from that observed in young and adult AL fed animals. Considering the enzymatic activities, cytosolic GI decreased in old rats irrespective of diet, whereas GII activity remained constant in all the experimental groups. The higher glutathione content found in both cellular compartments of old FR rats as compared to the old AL fed ones, could help to explain the life prolonging effect of FR treatment. Moreover, the observation that the activity of glyoxalases was not influenced by food restriction does not necessarily mean that the cells of diet-conditioned animals are scarcely protected against the toxic effect of methylglyoxal. Indeed, the production of this compound should be lower in FR animals as compared to AL fed ones, due to the lower level serum glucose concentration during the life span of the former with respect to the latter group.

HLA-G and pregnancy adverse outcomes

There is growing evidence that pregnancy complications such as preeclampsia, recurrent pregnancy loss (RPL), IUGR, and premature birth could be associated with abnormal immunologic interactions at the fetal-maternal interface. The restricted expression of HLA-G to the subpopulation of trophoblast cells which invade the uterus has generated much interest. The alternative splicing of HLA-G primary transcript, gives origin to seven isoforms, including both membrane-bound forms (HLA-G1, G2, G3, G4) and soluble forms (sHLA-G: sHLA-G5, G6, G7). sHLA-G consists predominantly of sHLA-G1 after its shedding by metalloproteinases, and secreted sHLA-G5 representing the quantitatively dominating and full-length isoforms. HLA-G expression and HLA-G genetic variations in both the mother and the embryo/fetus may be important for pregnancy outcome. It is also intuitively apparent that a gene with putative immunosuppressive and immunotolerant potential might be functional in both the mother and the embryo/fetus/placenta. Reduced or aberrant HLA-G expression seems to be associated with certain complications of pregnancy, among which preeclampsia and possibly the risk of miscarriage, and that this may be further linked to HLA-G polymorphisms. Most of the studies aimed at assessing the role of HLA-G in pregnant diseases have considered only the maternal genotype and ignored the contribution of the fetus. In this regard, the mother, placenta and the fetus form a synthesis. Therefore, studies on placental diseases should address HLA-G expression and genetic variations also to the fetus/placenta.

Identification and Characterization of Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma Cell Lines

Background/Aims: Head and neck squamous cell carcinoma (HNSCC) ranks sixth worldwide for tumor-related mortality. A subpopulation of tumor cells, termed cancer stem cells (CSCs), has the ability to support cancer growth. Therefore, profiling CSC-enriched populations could be a reliable tool to study cancer biology. Methods: We performed phenotypic characterization of 7 HNSCC cell lines and evaluated the presence of CSCs. CSCs from Hep-2 cell line and HNSCC primary cultures were enriched through sphere formation and sphere-forming cells have been characterized both in vitro and in vivo. In addition, we investigated the expression levels of Nicotinamide N-methyltransferase (NNMT), an enzyme overexpressed in several malignancies. Results: CSC markers were markedly expressed in Hep-2 cell line, which was found to be highly tumorigenic. CSC-enriched populations displayed increased expression of CSC markers and a strong capability to form tumors in vivo. We also found an overexpression of CSC markers in tumor formed by CSC-enriched populations. Interestingly, NNMT levels were significantly higher in CSC-enriched populations compared with parental cells. Conclusion: Our study provides an useful procedure for CSC identification and enrichment in HNSCC. Moreover, results obtained seem to suggest that CSCs may represent a promising target for an anticancer therapy.

The involvement of inflammatory cytokines in the pathogenesis of recurrent miscarriage.

OBJECTIVE To investigate the inflammatory cytokine expression pattern in trophoblastic tissue from women with unexplained recurrent miscarriage (RM). STUDY DESIGN Trophoblasts were obtained during uterine evacuation from 11 women with RM and from 20 healthy pregnant women undergoing elective termination of pregnancy, who served as controls. The array was performed using GEArray Q Series Human Inflammatory Cytokines & Receptors Gene Array HS-015 membranes. Data were confirmed by quantitative real-time PCR. The Mann-Whitney U test was performed for statistical analysis. RESULTS Microarray analysis identified three genes that were differentially expressed between RM patients and controls. We observed significant downregulation of Transforming Growth Factor beta 3 (TGF-β3) and Interleukin 25 (IL-25) (5-fold reduction and 2.5-fold reduction, respectively) and significant upregulation of CD-25, also known as Interleukin 2 receptor alpha (IL-2RA) (7-fold increase) in women with RM compared with controls. The median ΔC(t) of TGF-β3 was 8.2 (interquartile range, 7.67-8.9) in RM patients vs. 5.85 (interquartile range, 5.3-6.09) in controls; the median ΔC(t) of IL-25 was 5.18 (interquartile range, 4.46-5.76) in RM patients vs. 3.85 (interquartile range, 3.6-4.51) in controls, and the median ΔC(t) of CD-25 was 9.62 (interquartile range, 7.81-12.42) in RM patients vs. 12.44 (interquartile range, 11.02-13.86) in controls. DISCUSSION Our results suggest that the immunological and inflammatory regulation mechanisms of the placental environment play a key role in recurrent miscarriage. The observed trophoblast cytokine expression pattern at the maternal-fetal interface confirms the immunotrophic theory, as demonstrated by a switch from a T-helper-1 (Th1) profile to a T-helper-2 (Th2) profile in women who experience recurrent miscarriages.

Characterization and phylogenetic epitope mapping of CD38 ADPR cyclase in the cynomolgus macaque

BackgroundThe CD38 transmembrane glycoprotein is an ADP-ribosyl cyclase that moonlights as a receptor in cells of the immune system. Both functions are independently implicated in numerous areas related to human health. This study originated from an inherent interest in studying CD38 in the cynomolgus monkey (Macaca fascicularis), a species closely related to humans that also represents a cogent animal model for the biomedical analysis of CD38.ResultsA cDNA was isolated from cynomolgus macaque peripheral blood leukocytes and is predicted to encode a type II membrane protein of 301 amino acids with 92% identity to human CD38. Both RT-PCR-mediated cDNA cloning and genomic DNA PCR surveying were possible with heterologous human CD38 primers, demonstrating the striking conservation of CD38 in these primates. Transfection of the cDNA coincided with: (i) surface expression of cynomolgus macaque CD38 by immunofluorescence; (ii) detection of ~42 and 84 kDa proteins by Western blot and (iii) the appearance of ecto-enzymatic activity. Monoclonal antibodies were raised against the cynomolgus CD38 ectodomain and were either species-specific or cross-reactive with human CD38, in which case they were directed against a common disulfide-requiring conformational epitope that was mapped to the C-terminal disulfide loop.ConclusionThis multi-faceted characterization of CD38 from cynomolgus macaque demonstrates its high genetic and biochemical similarities with human CD38 while the immunological comparison adds new insights into the dominant epitopes of the primate CD38 ectodomain. These results open new prospects for the biomedical and pharmacological investigations of this receptor-enzyme.

Specific interaction of cytosolic and mitochondrial glyoxalase II with acidic phospholipids in form of liposomes results in the inhibition of the cytosolic enzyme only.

Kinetics of cytosolic recombinant human glyoxalase II and bovine liver mitochondrial glyoxalase II were studied in the presence of liposomes made of different phospholipids (PLs). Neutral PLs such as egg phosphatidylcholine or dipalmitoylphosphatidylcholine did not affect the enzymatic activity of either enzymatic form. Liposomes made of dioleoyl phosphatidic acid or cardiolipin or phosphatidylserine also did not affect the enzymatic activity of mitochondrial glyoxalase II. Conversely, these negatively charged PLs exerted noncompetitive inhibition on cytosolic glyoxalase II only, dioleoyl phosphatidic acid and bovine brain phosphatidylserine exerting the highest and lowest inhibition, respectively. Binding studies, carried out by using a resonant mirror biosensor, revealed that liposomes made of negatively charged PLs interact specifically with both enzymatic forms of glyoxalase II, whereas interactions were not detected with neutral PLs. Once bound on glyoxalase II, negatively charged liposomes could not be removed by 3 M NaCl, suggesting that interactions between glyoxalase II and negatively charged PLs, besides ionic, may be also hydrophobic.

Thrombomodulin is silenced in malignant mesothelioma by a poly(ADP-ribose)polymerase-1-mediated epigenetic mechanism

Malignant mesothelioma (MM) is often complicated by thromboembolic episodes, with thrombomodulin (TM) playing a critical role in the anticoagulant process. Heterogeneous expression of TM has been observed in cancer, and low or no TM expression in cancer cells is associated with poor prognosis. In this study, we analyzed TM expression in biopsies of MM patients and compared them with normal mesothelial tissue. The role of DNA methylation-associated gene silencing in TM expression was investigated. To evaluate poly(ADP-ribose) polymerase-1 (PARP1) as responsible for gene promoter epigenetic modifications, nonmalignant mesothelial cells (Met-5A) and MM cells (H28) were silenced for PARP1 and the DNA methylation/acetylation-associated TM expression evaluated. A correlation between low TM expression and high level of TM promoter methylation was found in MM biopsies. Low expression of TM was restored in MM cells by their treatment with 5-aza-2′-deoxycytidine and, to a lesser extent, with trichostatin, whereas the epigenetic agents did not affect TM expression in Met-5A cells. Silencing of PARP1 resulted in a strong down-regulation of TM expression in Met-5A cells, while restoring TM expression in H28 cells. PARP1 silencing induced TM promoter methylation in Met-5A cells and demethylation in MM cells, and this was paralleled by corresponding changes in the DNA methyltransferase activity. We propose that methylation of the TM promoter is responsible for silencing of TM expression in MM tissue, a process that is regulated by PARP1.

Unexplained fetal loss: the fetal side of thrombophilia

Carrier status of the fetus for factor V polymorphism or double homozygosity for mutant alleles of the PAI-1 4 G/4 G and MTHFR T677 T polymorphisms must be considered risk factors for intrauterine fetal death. The clinical implications of these data need to be addressed in a prospective study to confirm our preliminary data and to answer the question of whether or not double homozygous individuals should be treated with low molecular-weight heparin and/or low-dose aspirin.