Teresa Lorenzi

Structure of nicotinamide mononucleotide adenylyltransferase: a key enzyme in NAD+ biosynthesis

BACKGROUND Nicotinamide adenine dinucleotide (NAD(+)) is an essential cofactor involved in fundamental processes in cell metabolism. The enzyme nicotinamide mononucleotide adenylyltransferase (NMN AT) plays a key role in NAD(+) biosynthesis, catalysing the condensation of nicotinamide mononucleotide and ATP, and yielding NAD(+) and pyrophosphate. Given its vital role in cell life, the enzyme represents a possible target for the development of new antibacterial agents. RESULTS The structure of NMN AT from Methanococcus jannaschii in complex with ATP has been solved by X-ray crystallography at 2.0 A resolution, using a combination of single isomorphous replacement and density modification techniques. The structure reveals a hexamer with 32 point group symmetry composed of alpha/beta topology subunits. The catalytic site is located in a deep cleft on the surface of each subunit, where one ATP molecule and one Mg(2+) are observed. A strictly conserved HXGH motif (in single-letter amino acid code) is involved in ATP binding and recognition. CONCLUSIONS The structure of NMN AT closely resembles that of phosphopantetheine adenylyltransferase. Remarkably, in spite of the fact that the two enzymes share the same fold and hexameric assembly, a striking difference in their quaternary structure is observed. Moreover, on the basis of structural similarity including the HXGH motif, we identify NMN AT as a novel member of the newly proposed superfamily of nucleotidyltransferase alpha/beta phosphodiesterases. Our structural data suggest that the catalytic mechanism does not rely on the direct involvement of any protein residues and is likely to be carried out through optimal positioning of substrates and transition-state stabilisation, as is proposed for other members of the nucleotidyltransferase alpha/beta phosphodiesterase superfamily.

Ghrelin: a metabolic signal affecting the reproductive system

Ghrelin, an acylated 28 amino acid gastric peptide, was isolated from the stomach as an endogenous ligand for growth hormone (GH) secretagogue receptor in 1999. Circulating ghrelin is mainly produced by specific cells in the stomachs oxyntic glands. Ghrelin potently stimulates GH release and food intake and exhibits diverse effects, including ones on glucose metabolism and on secretion and motility of the gastrointestinal tract. Besides these effects on food intake and energy homeostasis, ghrelin is also involved in controlling reproductive functions, and a role for it as a novel regulator of the hypothalamic-pituitary gonadal axis is clearly emerging. We review recent ghrelin research with emphasis on its roles in the reproductive axis.

Expression Patterns of Two Serine Protease HtrA1 Forms in Human Placentas Complicated by Preeclampsia with and without Intrauterine Growth Restriction

Preeclampsia (PE) and intrauterine growth restriction (IUGR) are pregnancy-specific disorders that have in common abnormal placental implantation, a marked proliferation of villous cytotrophoblastic cells and focal necrosis of the syncytiotrophoblast. Several studies show an ischemic placenta with a high-resistance vasculature, which cannot deliver an adequate blood supply to the feto-placental unit. The cause of PE is a matter of debate, but recently studies in mice suggest that the primary feto-placental lesions are sufficient to initiate the disease. HtrA1, a member of the family of HtrA proteins, is a secreted multidomain protein with serine protease activity. It is expressed in first and third trimester of gestation. In specimens from the first trimester of gestation, immunostaining for HtrA1 is generally found in both layers of villous trophoblast, syncytiotrophoblast and cytotrophoblast. Cytoplasm of extravillous trophoblast and extracellular matrix of cell islands and cell columns are labeled for HtrA1. Specimens from third trimester of gestation show a more intense positivity for HtrA1 in the syncytiotrophoblast than in cytotrophoblast. The extravillous trophoblast and the decidual cells, is positive for HtrA1. The purpose of this study is to investigate the expression pattern of HtrA1 in placentas from PE without IUGR (maternal PE) and with IUGR (fetal PE) by quantitative western blotting and immunohistochemistry. By quantitative western blotting analysis we observed a significant upregulation of approximately 30 kDa HtrA1 form in PE. Differently, we detected a significant total HtrA1 down-regulation in PE-IUGR. Moreover, immunostaining for HtrA1 was positive in the villous trophoblast, in the syncytial knots and irregularly in the fetal vessel walls in PE placentas while immunostaining for HtrA1was present particularly in the syncytial knots in PE-IUGR placentas. In conclusion, we suggest that the approximately 30 kDa HtrA1 form can be correlated to maternal PE while that the significant down-regulation of total HtrA1 can be correlated to placental PE. These HtrA1 alterations could be considered as possible markers to discriminate placental PE from maternal PE.

Expression of basic fibroblast growth factor, its receptors and syndecans in bladder cancer

Basic Fibroblast Growth Factor (bFGF) is a heparin-binding cationic protein involved in a variety of pathological conditions including angiogenesis and solid tumour growth. The basic Fibroblast Growth Factor Receptor (FGFR) family comprises at least 4 high affinity tyrosine kinase receptors that require syndecans for their function. Mounting evidence indicates that syndecans, that bind both bFGF and their FGFRs, will act as stimulators, whereas syndecans that only bind bFGF will act as inhibitors of signaling by sequestering the growth factor. Recent findings have highlighted the importance of syndecans in urological cancers. The aim of this study is to investigate the expression of bFGF, its receptors (R1 and R2) and syndecans (1-4) in invasive urothelial carcinoma and normal-looking urothelium by Western blotting, RT-PCR, and immunohistochemistry analyses. Interestingly, bFGF, FGFR1 and FGFR2 protein levels statistically increased in bladder cancer tissues. mRNA of FGFR1 and syndecans (1-4), showed a statistically significant increase while an mRNA increase in the other molecules analysed was not significant. bFGF, its receptors and syndecan immunostaining were mainly present in the urothelium both in normal-looking tissues and urothelial neoplastic cells. In conclusion, our data report that the bFGF, FGFR and syndecan expressions are altered in bladder tumours.

HtrA1 in human urothelial bladder cancer: A secreted protein and a potential novel biomarker

Our aim was to analyze the expression of the serine protease HtrA1 in human bladder tissue and urine in order to point out its possible association with the presence of urothelial bladder cancer. Bladder tissue and urine specimens from cancer patients with different tumor grades and stages (n = 68) and from individuals with cystitis (n = 16) were collected along with biopsy specimens and urine from healthy individuals (n = 68). For the first time, we demonstrated by immunohistochemistry that HtrA1 protein is produced by bladder urothelium in both physiological and inflammatory conditions, whereas it is not detectable in urothelial cancer cells regardless of tumor grade and stage. A different HtrA1 expression between normal‐looking and neoplastic bladder tissue, despite similar HtrA1 mRNA levels, was also found by western blotting, which disclosed the presence of two forms of HtrA1, a native form of ∼50 kDa and an autocatalytic form of ∼38 kDa. Our investigations documented the presence of the two forms of HtrA1 also in urine. The ∼38 kDa form was significantly down‐regulated in neoplastic tissue, whereas significantly higher amounts of both HtrA1 forms were found in urine from cancer patients compared with both healthy subjects and patients with cystitis. Our findings suggest that HtrA1 is a downexpressed molecule since an early stage of bladder urothelial carcinoma development and that urinary HtrA1 protein may be considered, if successfully validated, as an early and highly sensitive and specific biomarker for this neoplasia (the sensitivity and specificity of HtrA1 are 92.65% and 95.59%, respectively).

Effects of enamel matrix derivative on vascular endothelial growth factor expression and microvessel density in gingival tissues of periodontal pocket: a comparative study.

BACKGROUND Vascular endothelial growth factor (VEGF) stimulates proliferation and migration of endothelial cells, and correlates with inflammatory resolution and periodontal tissue healing. Enamel matrix derivative (EMD) seems to stimulate soft tissue healing. Our aim was to assess if topical EMD application in an instrumented periodontal pocket could affect angiogenesis at the gingival level. METHODS A total of 56 periodontal sites in 28 patients were treated with a single session of comprehensive scaling and root planing under local anesthesia after recording the clinical attachment level (CAL). EMD gel in the test site or only the vehicle propylene glycol alginate in aqueous solution in the control site of the same mouth was applied onto the root surfaces and into the pocket and left in place for 3 minutes. After 48 hours, gingival biopsies were collected for histologic and immunohistochemical analysis for VEGF and CD34 (for microvessel density [MVD] count) antibodies. Statistical comparisons were performed by analysis of variance test. RESULTS Endothelial VEGF expression and MVD were statistically different in the test site compared to the control site. VEGF expression and MVD of the control site were not correlated with CAL, whereas the test site showed high correlations among CAL and endothelial VEGF or MVD. CONCLUSIONS EMD induces proliferation and viability and angiogenesis of human microvascular cells. Recent clinical and histologic studies found EMD to be useful as an adjunct to scaling and root planing in single-rooted teeth. Our findings may help to understand the mechanisms involved in soft tissue healing, through the ability of EMD to increase angiogenesis at periodontal pockets.

Role of Electron Microscopy in the Diagnosis of Cadasil Syndrome: A Study of 32 Patients

Background and Purpose Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by NOTCH3 gene mutations that result in vascular smooth muscle cell (VSMC) degeneration. Its distinctive feature by electron microscopy (EM) is granular osmiophilic material (GOM) detected in VSMC indentations and/or the extracellular space close to VSMCs. Reports of the sensitivity of EM in detecting GOM in biopsies from CADASIL patients are contradictory. We present data from 32 patients clinically suspected to have CADASIL and discuss the role of EM in its diagnosis in this retrospective study. Methods Skin, skeletal muscle, kidney and pericardial biopsies were examined by EM; the NOTCH3 gene was screened for mutations. Skin and muscle biopsies from 12 patients without neurological symptoms served as controls. Results and Discussion All GOM-positive patients exhibited NOTCH3 mutations and vice versa. This study i) confirms that EM is highly specific and sensitive for CADASIL diagnosis; ii) extends our knowledge of GOM distribution in tissues where it has never been described, e.g. pericardium; iii) documents a novel NOTCH3 mutation in exon 3; and iv) shows that EM analysis is critical to highlight the need for comprehensive NOTCH3 analysis. Our findings also confirm the genetic heterogeneity of CADASIL in a small Italian subpopulation and emphasize the difficulties in designing algorithms for molecular diagnosis.

Activating protein-1 family of transcription factors in the human placenta complicated by preeclampsia with and without fetal growth restriction

Preeclampsia (PE) is a serious disorder of human pregnancy, it is often associated with fetal growth restriction (FGR) which is a failure of the fetus to reach its own growth potential. Activator protein-1 (AP-1) is a family of transcription factors inducible in response to a variety of extracellular stimuli and functions. AP-1 plays a complex role in the regulation of different fundamental cellular processes, including cell proliferation, survival, death and transformation. We investigate the expression pattern of AP-1 transcription factors in normal placentas during gestation and in placentas from PE without and with FGR using semiquantitative RT-PCR and immunohistochemistry techniques. The most interesting data concern the alterations of protein expression patterns of c-fos, Jun D and c-jun in normal gestation as well as in PE and PE-FGR pathologies. In addition, alterations but not significant changes are detected in mRNA expressions for these transcription factors. We strongly suggest that c-fos is implicated in regulating invasiveness mechanism of extravillous trophoblast in normal gestation as well as in PE placentas. In addition, we suggest that the opposite modulation of Jun D and c-jun in PE and PE-FGR supports the recent hypothesis that PE and PE-FGR could be considered two pathologies with different origin (maternal and placental) each of which has a different molecular pattern of expression.

Possible role of RKIP in cytotrophoblast migration: immunohistochemical and in vitro studies

Raf kinase inhibitor protein (RKIP) regulates growth and differentiation signaling of mitogen‐activated protein kinases (MAPK), GRK2 and NF‐kappaB pathways each of which regulates cytotrophoblast differentiation and normal placental development. We show here that RKIP is expressed in human normal and preeclampic placentas as detected by immunostaining. RKIP was detected in villous cytotrophoblast in normal placenta and switched to syncytiotrophoblast in pre‐eclampsia (PE)‐complicated pregnancies. RKIP was also localized in extravillous cytotrophoblast of cell islands and cell columns both in normal and in PE placentas, although staining was less uniform in the latter specimens. In order to test RKIP involvement in cytotrophoblast function, we performed in vitro studies on HTR‐8/SVneo cells, a first trimester cytotrophoblast cell line. We show that the RKIP inhibitor locostatin reduces ERK phosphorylation and impairs HTR‐8/SV neo cells motility in wound closure experiments. We also document the presence of GRK2 mRNA, the reduction of phosphorylated RKIP expression by locostatin and the induction of PAI mRNA expression in HTR‐8/SV neo cells, suggesting the involvement of GRK2 and NF‐kappaB pathways in these cells. In conclusion, our work provides evidence that RKIP is a novel factor expressed in cytotrophoblast cells where it likely regulates cell migration. J. Cell. Physiol. 227: 1821–1828, 2012.

Constitutive expression of ciliary neurotrophic factor in mouse hypothalamus

Ciliary neurotrophic factor (CNTF) is a potent survival molecule for a large number of neuronal and glial cells in culture; its expression in glial cells is strongly upregulated after a variety of nerve tissue injuries. Exogenously administered CNTF produces an anorectic effect via activation of hypothalamic neurons and stimulates neurogenesis in mouse hypothalamus. To determine whether CNTF is produced endogenously in the hypothalamus, we sought cellular sources and examined their distribution in adult mouse hypothalamus by immunohistochemistry. CNTF immunoreactivity (IR) was predominantly detected in the ependymal layer throughout the rostrocaudal extension of the third ventricle, where numerous ependymocytes and tanycytes exhibited specific staining. Some astrocytes in the grey matter of the anterior hypothalamus and in the median eminence of the hypothalamic tuberal region were also positive. Stimulation of cells bearing CNTF receptor α (CNTFRα) induces specific activation of the signal transducer and activator of transcription 3 (STAT3) signalling system. Treatment with recombinant CNTF and detection of the nuclear expression of phospho‐STAT3 (P‐STAT3) showed that CNTF‐producing ependymal cells and tanycytes were intermingled with, or very close to, P‐STAT3‐positive, CNTFRα‐bearing cells. A fraction of CNTF‐producing ependymal cells and tanycytes and some median eminence astrocytes also exhibited P‐STAT3 IR. Thus, in normal adult mice the ependyma of the third ventricle is both a source of and a target for CNTF, which may play hitherto unknown roles in hypothalamic function in physiological conditions.