Emanuele Persichetti

Università degli Studi di Perugia, Perugia, Umbria, Italy

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Publications (25)110.45 Total impact

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    ABSTRACT: Lysosomal dysfunction is thought to be a prominent feature in the pathogenetic events leading to Parkinson's disease (PD). This view is supported by the evidence that mutations in GBA gene, coding the lysosomal hydrolase β-glucocerebrosidase (GCase), are a common genetic risk factor for PD. Recently, GCase activity has been shown to be decreased in substantia nigra and in cerebrospinal fluid of patients diagnosed with PD or dementia with Lewy Bodies (DLB). Here we measured the activity of GCase and other endo-lysosomal enzymes in different brain regions (frontal cortex, caudate, hippocampus, substantia nigra, cerebellum) from PD (n = 26), DLB (n = 16) and age-matched control (n = 13) subjects, screened for GBA mutations. The relative changes in GCase gene expression in substantia nigra were also quantified by real-time PCR. The role of potential confounders (age, sex and post-mortem delay) was also determined. Substantia nigra showed a high activity level for almost all the lysosomal enzymes assessed. GCase activity was significantly decreased in the caudate (-23%) and substantia nigra (-12%) of the PD group; the same trend was observed in DLB. In both groups, a decrease in GCase mRNA was documented in substantia nigra. No other lysosomal hydrolase defects were determined. The high level of lysosomal enzymes activity observed in substantia nigra, together with the selective reduction of GCase in PD and DLB patients, further support the link between lysosomal dysfunction and PD pathogenesis, favoring the possible role of GCase as biomarker of synucleinopathy. Mapping the lysosomal enzyme activities across different brain areas can further contribute to the understanding of the role of lysosomal derangement in PD and other synucleinopathies.
    Molecular Neurodegeneration 03/2015; 10(1):15. DOI:10.1186/s13024-015-0010-2 · 5.29 Impact Factor
  • Movement Disorders 09/2014; 29(10). DOI:10.1002/mds.25976 · 5.68 Impact Factor
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    ABSTRACT: Measurements of the activities of lysosomal enzymes in cerebrospinal fluid have recently been proposed as putative biomarkers for Parkinson's disease and other synucleinopathies. To define the operating procedures useful for ensuring the reliability of these measurements, we analyzed several pre-analytical factors that may influence the activity of β-glucocerebrosidase, α-mannosidase, β-mannosidase, β-galactosidase, α-fucosidase, β-hexosaminidase, cathepsin D and cathepsin E in cerebrospinal fluid. Lysosomal enzyme activities were measured by well-established fluorimetric assays in a consecutive series of patients (n = 28) with different neurological conditions, including Parkinson's disease. The precision, pre-storage and storage conditions, and freeze/thaw cycles were evaluated. All of the assays showed within- and between-run variabilities below 10%. At -20°C, only cathepsin D was stable up to 40 weeks. At -80°C, the cathepsin D, cathepsin E, and β-mannosidase activities did not change significantly up to 40 weeks, while β-glucocerebrosidase activity was stable up to 32 weeks. The β-galactosidase and α-fucosidase activities significantly increased (+54.9±38.08% after 4 weeks and +88.94±36.19% after 16 weeks, respectively). Up to four freeze/thaw cycles did not significantly affect the activities of cathepsins D and E. The β-glucocerebrosidase activity showed a slight decrease (-14.6%) after two freeze/thaw cycles. The measurement of lysosomal enzyme activities in cerebrospinal fluid is reliable and reproducible if pre-analytical factors are accurately taken into consideration. Therefore, the analytical recommendations that ensue from this study may contribute to the establishment of actual values for the activities of cerebrospinal fluid lysosomal enzymes as putative biomarkers for Parkinson's disease and other neurodegenerative disorders.
    PLoS ONE 07/2014; 9(7):e101453. DOI:10.1371/journal.pone.0101453 · 3.23 Impact Factor
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    ABSTRACT: Lysosomal enzymes are involved in macromolecules degradation; impairment of their functionality is the cause of lysosomal storage diseases. In recent years, lysosomal dysfunction has also been shown in neurodegenerative disorders such as Parkinson’s disease (PD). Mutations in the GBA1 gene, coding for the lysosomal beta-glucocerebrosidase, cause Gaucher’s disease. Interestingly, decreased beta-glucocerebrosidase activity has been described in substantia nigra of sporadic PD patients, further providing link between beta-glucocerebrosidase deficiency and PD. This is the first study investigating the expression of beta-hexosaminidase, alpha-fucosidase, beta-mannosidase, alpha-mannosidase, betagalactosidase, beta-glucocerebrosidase and cathepsin E in different areas of the human brain. Activity assays were performed on putamen, caudate, hippocampus, and frontal cortex from 13 PD patients and 11 control subjects and substantia nigra from 3 PD patients and 6 controls. Results revealed a 20% decrement of beta-glucocerebrosidase activity in substantia nigra of PD patients. Decreased activity (50%) of cathepsin E was also observed in caudate of PD patients. Interestingly, 21% and 60% increases in betahexosaminidase and alpha-mannosidase activities were found in caudate of PD patients. Lysosomal enzymes tested in this study showed different activities in different brain areas. These results may contribute to the understanding of the role of lysosomal enzymes in neurodegenerative disorders.
    European Biotechnology Congress 2014; 07/2014
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    ABSTRACT: To assess the discriminating power of multiple cerebrospinal fluid (CSF) biomarkers for Parkinson's disease (PD), we measured several proteins playing an important role in the disease pathogenesis. The activities of beta-glucocerebrosidase and other lysosomal enzymes, together with total and oligomeric alpha-synuclein, and total and phosphorylated tau, were thus assessed in CSF of 71 PD patients and compared to 45 neurological controls. Activities of beta-glucocerebrosidase, beta-mannosidase, beta-hexosaminidase, and beta-galactosidase were measured with established enzymatic assays, while alpha-synuclein and tau biomarkers were evaluated with immunoassays. A subset of PD patients (n=44) was also screened for mutations in the beta-glucocerebrosidase-encoding gene (GBA1). In the PD group, beta-glucocerebrosidase activity was reduced (P < 0.05) and patients at earlier stages showed lower enzymatic activity (P < 0.05); conversely, beta-hexosaminidase activity was significantly increased (P < 0.05). Eight PD patients (18%) presented GBA1 sequence variations; 3 of them were heterozygous for the N370S mutation. Levels of total alpha-synuclein were significantly reduced (P < 0.05) in PD, in contrast to increased levels of alpha-synuclein oligomers, with a higher oligomeric/total alpha-synuclein ratio in PD patients when compared with controls (P < 0.001). A combination of beta-glucocerebrosidase activity, oligomeric/total alpha-synuclein ratio, and age gave the best performance in discriminating PD from neurological controls (sensitivity 82%; specificity 71%, area under the receiver operating characteristic curve=0.87). These results demonstrate the possibility of detecting lysosomal dysfunction in CSF and further support the need to combine different biomarkers for improving the diagnostic accuracy of PD. (C) 2014 International Parkinson and Movement Disorder Society
    Movement Disorders 07/2014; 29(8). DOI:10.1002/mds.25772 · 5.63 Impact Factor
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    ABSTRACT: Free Man7-9GlcNAc2 are released during the biosynthesis pathway of N-linked glycans or from misfolded glycoproteins during the ERAD-process and are reduced to Man5GlcNAc in the cytosol. In this form free oligosaccharides can be transferred into the lysosomes to be completely degraded. Alpha-mannosidase (MAN2C1) is the enzyme responsible for the partial demannosylation occurring in the cytosol. It has been demonstrated that the inhibition of MAN2C1 expression induces accumulation of Man8-9GlcNAc oligosaccharides and apoptosis in vitro. We investigated the consequences caused by the lack of cytosolic alpha-mannosidase activity in vivo by the generation of Man2c1-deficient mice. Increased amounts of Man8-9GlcNAc oligosaccharides were recognized in all analyzed KO tissues. Histological analysis of CNS revealed neuronal and glial degeneration with formation of multiple vacuoles in deep neocortical layers and major telencephalic white matter tracts. Enterocytes of small intestine accumulate mannose-containing saccharides and glycogen particles in their apical cytoplasm as well as large clear vacuoles in retronuclear position. Liver tissue is characterized by groups of hepatocytes with increased contents of mannosyl compounds and glycogen, some of them undergoing degeneration by hydropic swelling. In addition, lectin screening showed the presence of mannose-containing saccharides in the epithelium of proximal kidney tubules, while scattered glomeruli appear collapsed or featured signs of fibrosis along Bowman 's capsule. Except for a moderate enrichment of mannosyl compounds and glycogen, heterozygous mice were normal, arguing against possible toxic effects of truncated Man2c1. These findings confirm the key role played by Man2c1 in the catabolism of free oligosaccharides.
    Journal of Biological Chemistry 02/2014; 289(14). DOI:10.1074/jbc.M114.550509 · 4.57 Impact Factor
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    ABSTRACT: We proposed that group IIA secretory phospholipase A2 (GIIA) participates in neuritogenesis based on our observations that the enzyme migrates to growth cones and neurite tips when PC12 cells are induced to differentiate by nerve growth factor (NGF) (Ferrini et al., Neurochem Res 35:2168-2174, 2010). The involvement of other secretory PLA2 isoforms in neuronal development has been suggested by others but through different mechanisms. In the present study, we compared the subcellular distribution of GIIA and group X sPLA2 (GX) after stimulation of PC12 cells with NGF. We found that GIIA, but not GX, localized at the neuritic tips after treatment with NGF, as demonstrated by immunofluorescence analysis. We also found that NGF stimulated the expression and the activity of GIIA. In addition, NGF induced the expressed myc-tagged GIIA protein to migrate to neurite tips in its active form. We propose that GIIA expression, activity, and subcellular localization is regulated by NGF and that the enzyme may participate in neuritogenesis through intracellular mechanisms, most likely by facilitating the remodelling of glycerophospholipid molecular species by deacylation-reacylation reactions necessary for the incorporation of polyunsaturated fatty acids.
    Molecular Neurobiology 01/2014; 50(1). DOI:10.1007/s12035-013-8621-5 · 5.29 Impact Factor
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    ABSTRACT: Parkinson's disease (PD) is characterized neuropathologically by the cytoplasmic accumulation of misfolded α-synuclein in specific brain regions. The endolysosomal pathway appears to be involved in α-synuclein degradation and, thus, may be relevant to PD pathogenesis. This assumption is further strengthened by the association between PD and mutations in the gene encoding for the lysosomal hydrolase glucocerebrosidase. The objective of the present study was to determine whether endolysosomal enzyme activities in cerebrospinal fluid (CSF) differ between PD patients and healthy controls. Activity levels of 6 lysosomal enzymes (β-hexosaminidase, α-fucosidase, β-mannosidase, β-galactosidase, β-glucocerebrosidase, and cathepsin D) and 1 endosomal enzyme (cathepsin E) were measured in CSF from 58 patients with PD (Hoehn and Yahr stages 1-3) and 52 age-matched healthy controls. Enzyme activity levels were normalized against total protein levels. Normalized cathepsin E and β-galactosidase activity levels were significantly higher in PD patients compared with controls, whereas normalized α-fucosidase activity was reduced. Other endolysosomal enzyme activity levels, including β-glucocerebrosidase activity, did not differ significantly between PD patients and controls. A combination of normalized α-fucosidase and β-galactosidase discriminated best between PD patients and controls with sensitivity and specificity values of 63%. In conclusion, the activity of a number of endolysosomal enzymes is changed in CSF from PD patients compared with healthy controls, supporting the alleged role of the endolysosomal pathway in PD pathogenesis. The usefulness of CSF endolysosomal enzyme activity levels as PD biomarkers, either alone or in combination with other markers, remains to be established in future studies. © 2013 Movement Disorder Society.
    Movement Disorders 05/2013; 28(6). DOI:10.1002/mds.25495 · 5.68 Impact Factor
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    ABSTRACT: Clinical diagnosis of Parkinson disease (PD) is difficult in early stages of disease, with high risk of misdiagnosis. The long preclinical phase of PD provides the possibility for early therapeutic intervention once disease-modifying therapies have been developed, but lack of biomarkers for early diagnosis and monitoring of disease progression represents a major obstacle to achievement of this goal. Accordingly, research efforts aimed at identification of novel biomarkers have been increasing in the past 5 years. Cerebrospinal fluid (CSF) is an accessible source of brain-derived proteins, which mirror molecular changes that take place in the CNS. In this Review, we discuss evidence from numerous studies that have focused on identification of candidate CSF biomarkers for PD. Notably, molecular pathways related to α-synuclein, tau and β-amyloid peptides have received considerable attention. CSF levels of the protein DJ-1 are also of interest, although further investigation of this candidate marker is required. These studies support the usefulness of a combination of various CSF biomarkers of PD to increase diagnostic accuracy during early phases of the disease, and to differentiate PD from other neurodegenerative disorders.
    Nature Reviews Neurology 03/2013; 9(3):131-40. DOI:10.1038/nrneurol.2013.10 · 14.10 Impact Factor
  • Emanuele Persichetti · Alfredo De Michele · Michela Codini · Giovanna Traina
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    ABSTRACT: Objective The objective of this study has been to evaluate the performance of the overall antioxidant of Lactobacillus fermentum LF31 bacterium with prebiotic supplement in human colon cultured cells. Methods The antioxidant capability of Lactobacillus fermentum LF31 has been assayed in vitro on human colon adenocarcinoma HT-29 cell line using the Oxygen Radical Adsorbance Capacity (ORAC) method. Results The analysis has revealed that the interaction probiotic strain-cells supplemented with a prebiotic exerts a remarkable antioxidant capacity. Conclusion The Lactobacillus fermentum used in the present study exhibited significant in vitro antioxidant capacity, increasing the total antioxidant potential.
    Nutrition 01/2013; 30(7-8). DOI:10.1016/j.nut.2013.12.009 · 3.05 Impact Factor
  • S. Paciotti · E. Persichetti · M. Codini · T. Beccari
    Journal of Biotechnology 11/2012; 161:9. DOI:10.1016/j.jbiotec.2012.07.176 · 2.88 Impact Factor
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    ABSTRACT: We report the first newborn screening pilot study in an Italian region for four lysosomal disorders including Pompe disease, Gaucher disease, Fabry disease and mucopolysaccharidosis type 1. The screening has been performed using enzymatic assay on Dry Blood Spot on filter paper. A total of 3403 newborns were screened. One newborn showed a reduction of β-glucosidase activity in leucocytes. Molecular analysis revealed a status of compound heterozygous for the panethnic mutation N370S and for the sequence variation E388K, not yet correlated to Gaucher disease onset. The functional consequences of the E388K replacement on β-glucosidase activity were evaluated by in vitro expression, showing that the mutant protein retained 48% of wild type activity. Structural modeling predicted that the E388K replacement, localized to a surface of the enzyme, would change the local charges distribution which, in the native protein, displays an overwhelming presence of negative charges. However, the newborn, and a 4 year old sister showing the same genomic alterations, are currently asymptomatic. This pilot newborn screening for lysosomal diseases appears to be feasible and affordable to be extended to large populations. Moreover other lysosomal diseases for which a therapy is available or will be available, could be included in the screening.
    Clinica chimica acta; international journal of clinical chemistry 07/2012; 413(23-24):1827-31. DOI:10.1016/j.cca.2012.07.011 · 2.76 Impact Factor
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    ABSTRACT: Most lysosomal storage diseases are caused by defects in genes encoding for acidic hydrolases. Deficiency of an enzyme involved in the catabolic pathway of N-linked glycans leads to the accumulation of the respective substrate and consequently to the onset of a specific storage disorder. Di-N-acetylchitobiase and core specific α1-6mannosidase represent the only exception. In fact, to date no lysosomal disease has been correlated to the deficiency of these enzymes. We generated di-N-acetylchitobiase-deficient mice by gene targeting of the Ctbs gene in murine embryonic stem cells. Accumulation of Man2GlcNAc2 and Man3GlcNAc2 was evaluated in all analyzed tissues and the tetrasaccharide was detected in urines. Multilamellar inclusion bodies reminiscent of polar lipids were present in epithelia of a scattered subset of proximal tubules in the kidney. Less constantly, enlarged Kupffer cells were observed in liver, filled with phagocytic material resembling partly digested red blood cells. These findings confirm an important role for lysosomal di-N-acetylchitobiase in glycans degradation and suggest that its deficiency could be the cause of a not yet described lysosomal storage disease.
    Biochimica et Biophysica Acta 03/2012; 1822(7):1137-46. DOI:10.1016/j.bbadis.2012.03.005 · 4.66 Impact Factor
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    Etiology and Pathophysiology of Parkinson's Disease, 10/2011; , ISBN: 978-953-307-462-7
  • Current Opinion in Biotechnology 09/2011; 22. DOI:10.1016/j.copbio.2011.05.025 · 8.04 Impact Factor
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    ABSTRACT: Deficiency in human lysosomal α-mannosidase (MAN2B1) results in α-mannosidosis, a lysosomal storage disorder; patients present a wide range of neurological, immunological, and skeletal symptoms caused by a multisystemic accumulation of mannose-containing oligosaccharides. Here, we describe the expression of recombinant MAN2B1 both transiently in Nicotiana benthamiana leaves and in the leaves and seeds of stably transformed N. tabacum plants. After purification from tobacco leaves, the recombinant enzyme was found to be N-glycosylated and localized in vacuolar compartments. In the fresh leaves of tobacco transformants, MAN2B1 was measured at 10,200 units/kg, and the purified enzyme from these leaves had a specific activity of 32-45 U/mg. Furthermore, tobacco-produced MAN2B1 was biochemically similar to the enzyme purified from human tissues, and it was internalized and processed by α-mannosidosis fibroblast cells. These results strongly indicate that plants can be considered a promising expression system for the production of recombinant MAN2B1 for use in enzyme replacement therapy.
    Plant Biotechnology Journal 06/2011; 9(9):1061-73. DOI:10.1111/j.1467-7652.2011.00630.x · 5.68 Impact Factor
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    ABSTRACT: Mucolipidosis type III (MLIII) is an autosomal recessive disorder affecting lysosomal hydrolase trafficking. In a study of 10 patients from seven families with a clinical phenotype and enzymatic diagnosis of MLIII, six novel GNPTG gene mutations were identified. These included missense (p.T286M) and nonsense (p.W111X) mutations and a transition in the obligate AG-dinucleotide of the intron 8 acceptor splice site (c.610-2A>G). Three microdeletions were also identified, two of which (c.611delG and c.640_667del28) were located within the coding region whereas one (c.609+28_610-16del) was located entirely within intron 8. RT-PCR analysis of the c.610-2A>G transition demonstrated that the change altered splicing, leading to the production of two distinct aberrantly spliced forms, viz. the skipping of exon 9 (p.G204_K247del) or the retention of introns 8 and 9 (p.G204VfsX28). RT-PCR analysis, performed on a patient homozygous for the intronic deletion (c.609+28_610-16del), failed to detect any GNPTG RNA transcripts. To determine whether c.609+28_610-16del allele-derived transcripts were subject to nonsense-mediated mRNA decay (NMD), patient fibroblasts were incubated with the protein synthesis inhibitor anisomycin. An RT-PCR fragment retaining 43 bp of intron 8 was consistently detected suggesting that the 33-bp genomic deletion had elicited NMD. Quantitative real-time PCR and GNPTG western blot analysis confirmed that the homozygous microdeletion p.G204VfsX17 had elicited NMD resulting in failure to synthesize GNPTG protein. Analysis of the sequences surrounding the microdeletion breakpoints revealed either intrinsic repetitivity of the deleted region or short direct repeats adjacent to the breakpoint junctions. This is consistent with these repeats having mediated the microdeletions via replication slippage and supports the view that the mutational spectrum of the GNPTG gene is strongly influenced by the properties of the local DNA sequence environment.
    Human Mutation 06/2009; 30(6):978-84. DOI:10.1002/humu.20959 · 5.05 Impact Factor
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    ABSTRACT: The autophagy-lysosomal degradation pathway plays a role in the onset and progression of neurodegenerative diseases. Clinical and genetic studies indicate that mutations of beta-glucocerebrosidase represent genetic risk factors for synucleinopathies, including Parkinson's Disease (PD) and Dementia with Lewy Bodies (DLB). We recently found a decreased activity of lysosomal hydrolases, namely beta-glucocerebrosidase, in cerebrospinal fluid of PD patients. We have thus measured the activity of these enzymes - alpha-mannosidase (EC, beta-mannosidase (EC, beta-glucocerebrosidase (EC, beta-galactosidase (EC and beta-hexosaminidase (EC - in cerebrospinal fluid of patients suffering from DLB, Alzheimer's Disease (AD), Fronto-Temporal Dementia (FTD) and controls. Alpha-mannosidase activity showed a marked decrease across all the pathological groups as compared to controls. Conversely, beta-glucocerebrosidase activity was selectively reduced in DLB, further suggesting that this enzyme might specifically be impaired in synucleinopathies.
    Neurobiology of Disease 04/2009; 34(3):484-6. DOI:10.1016/j.nbd.2009.03.002 · 5.20 Impact Factor
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    ABSTRACT: Beta-mannosidosis (OMIM # 248510) is an autosomal-recessive lysosomal storage disorder caused by deficiency of the lysosomal enzyme beta-mannosidase (MANBA, E.C. The disorder has been reported in goat, cattle and man. The human disorder is rare and only 20 cases in 16 families have been reported. We have sequenced the exons and exon-intron borders in a European patient with infantile onset of beta-mannosidosis. The patient was compound heterozygous for a silent mutation (c.375A>G) in exon 3 causing alternative splicing, and a missense mutation (c.1513T>C, p.Ser505Pro) in exon 12. The alternative splicing event deleted four nucleotides from the transcript and was predicted to result in premature termination of translation. In order to evaluate the consequence of the missense mutation, we inserted the human beta-mannosidase gene into an expression vector, performed site-directed mutagenesis and expressed the normal and mutant enzyme in COS-7 cells. We also included the previously reported beta-mannosidosis-associated missense mutations c.544C>T (p.Arg182Trp) and c.1175G>A (p.Gly392Glu), which were found in patients presenting a milder phenotype. Cells transfected with the wild-type construct showed a 33-fold increase in beta-mannosidase activity compared to mock-transfected cells, whereas cells transfected with the mutant constructs showed no detectable increase in activity. We propose that the milder phenotype described in some beta-mannosidosis patients with missense mutations in the MANBA gene is not due to residual beta-mannosidase activity, but rather caused by epigenetic and/or environmental factors.
    Molecular Genetics and Metabolism 08/2008; 94(4):476-80. DOI:10.1016/j.ymgme.2008.04.010 · 2.83 Impact Factor
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    ABSTRACT: The lysosomal enzyme di-N-acetylchitobiase hydrolyzes N-acetylglucosamine from the reducing-end of the N,N' diacetylchitobiose core of N-linked-oligosaccharides. The presence of chitobiase in the tissues of different species is probably responsible for differences in the structure of oligosaccharides accumulated in the lysosomal storage disease beta-mannosidosis. The disease has so far been described in humans, cats, cattle and goats. Low chitobiase activity has been observed in the tissues of ruminants and it has been hypothesized that in cattle this low level of expression is due to evolutionary changes in the promoter region. A cDNA encoding the mouse chitobiase has been isolated, sequenced and its identity confirmed by expression in COS-7 cells. Comparison of the mouse genomic sequence with the cDNA sequence revealed the presence of seven exons within the chitobiase gene. The gene spans about 15 kb and a single transcription initiation site was determined by 5'RACE. Chitobiase is differentially and ubiquitously expressed in mouse tissues as demonstrated by qRT-PCR analysis. Chitobiase is differentially expressed at lower levels in bovine tissues. In two bovine tissues (heart and muscle) mRNA was not detectable. Mouse and bovine promoters have been isolated and sequenced and their activities compared. The activity of the bovine promoter is very low and might explain the low activity of chitobiase observed in cattle.
    Gene 07/2008; 416(1-2):85-91. DOI:10.1016/j.gene.2008.03.009 · 2.08 Impact Factor

Publication Stats

215 Citations
110.45 Total Impact Points


  • 2006–2015
    • Università degli Studi di Perugia
      • • Department of Economics and Food Sciences
      • • Department of Internal Medicine
      • • Department of Experimental Medicine and Biochemical Sciences
      Perugia, Umbria, Italy
  • 2008
    • University Hospital of North Norway
      • Department of Medical Genetics
      Tromsø, Troms, Norway