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Featured researches published by A. Orlacchio.


Clinica Chimica Acta | 2000

β-Hexosaminidase, α-d-mannosidase, and β-mannosidase expression in serum from patients with carbohydrate-deficient glycoprotein syndrome type I

Tommaso Beccari; F Mancuso; Egidia Costanzi; C. Tassi; Rita Barone; Agata Fiumara; A Orlacchio; M.C Aisa; A. Orlacchio

The activity of beta-hexosaminidase, determined with 4-methylumbelliferyl-beta-N-acetylglucopyranoside substrate, and of beta-D-mannosidase was significantly higher in the serum of patients with carbohydrate-deficient glycoprotein (CDG) syndrome type IA (phosphomannomutase deficiency) than in controls. No significant differences were observed in the activity of beta-hexosaminidase, determined using 4-methylumbelliferyl-beta-N-acetylglucopyranoside-6-sulphate as substrate, and the activity of alpha-D-mannosidase. Using DEAE-cellulose chromatography, a greater amount of hexosaminidase B than hexosaminidase A was detected in CDG serum. In CDG serum, hexosaminidase A was eluted in a more basic position in the salt gradient. An isoenzyme of alpha-D-mannosidase and beta-D-mannosidase was identified in control and CDG sera. alpha-D-Mannosidase isoenzyme was eluted in a slightly more basic position in CDG serum than in control serum, whereas beta-D-mannosidase isoenzyme was eluted in the same position.


Journal of Inherited Metabolic Disease | 2003

Two novel mutations in the gene for human α-mannosidase that cause α-mannosidosis

Tommaso Beccari; Lucia Bibi; Roberta Ricci; Daniela Antuzzi; A. Burgalossi; Egidia Costanzi; A. Orlacchio

Summary: Mutation analysis performed on two Italian patients with α-mannosidosis allowed the identification of two new mutations, IVS20−2A>G and 322–323insA. The patients were both homozygous for these mutations. The first mutation causes skipping of exon 21, whereas the second causes a frameshift introducing a stop codon at position 160 of the amino acid sequence.


Clinica Chimica Acta | 1992

β-N-Acetylhexosaminidase in the urine, kidney and serum of bromobenzene-intoxicated mice

C. Tassi; Tommaso Beccari; A. Casini; A. Orlacchio

beta-N-Acetylhexosaminidase isoenzymes were separated from the kidney, serum and urine of normal mice and mice intoxicated with bromobenzene, using DEAE-cellulose chromatography. Both mouse serum and urine showed hexosaminidase profiles similar to the human counterparts with the presence of B (basic), I (intermediate) and A (acidic) isoenzymes. A notable feature was the presence of a high proportion of an intermediate form in mouse urine which is not always present in human urine. Hexosaminidase activity increased significantly in urine of mice intoxicated with bromobenzene. Its increase was time-dependent and due to kidney damage with a release in the urine of hexosaminidase A, I and, in higher proportion, B. No significant differences were observed in mouse kidney and serum profiles following intoxication with bromobenzene. The total activity of hexosaminidase, using 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside as substrate, did not increase in the serum of mice intoxicated with bromobenzene. Both hexosaminidase activity and the isoenzyme pattern in urine can be used as indicators of kidney damage by bromobenzene intoxication.


Mammalian Genome | 1998

Promoter characterization and structure of the gene encoding mouse lysosomal alpha-d-mannosidase

Sofia Stinchi; A. Orlacchio; Egidia Costanzi; John L. Stirling; Anna Rita Menghini; Aldo Orlacchio; Tommaso Beccari

Abstract. Mouse lysosomal α-d-mannosidase (EC 3.2.1.24) is an enzyme involved in the catabolism of N-linked glycoproteins. The gene is differentially expressed in mouse tissues, and the highest level of mRNA is found in the epididymis. The expression of mannosidase in the epididymis may be hormonally regulated, since its activity increases with age. To understand the factors affecting the expression of mouse mannosidase, we isolated and characterized the promoter and determined the exon-intron structure. The gene is about 15 kb, consists of 24 exons, and the 5′ flanking region contains GC-rich regions, TATA boxes, CAAT boxes, and putative binding sites for the transcription factors Sp1, AP2, and PEA3. PEA3 factor may participate in the transcriptional control of mannosidase expression in the mouse epididymis. In fact, it has been demonstrated that the PEA3 motif is spatially and temporally expressed within the mouse epididymis, and its accumulation is controlled by androgens and testicular factors. A 1279-bp fragment from the initiation codon had the strongest promoter activity, and three different transcription start sites were identified at positions -131, -149, and -174.


Mammalian Genome | 1997

Structural organization and expression of the gene for the mouse GM2 activator protein

C. Bertoni; M. G. Appolloni; John L. Stirling; Li Sc; Li Yt; A. Orlacchio; Tommaso Beccari

The GM2 activator protein is an essential component for the degradation of GM2 ganglioside by hexosaminidase A in vivo. Mutations in the human gene coding for the GM2 activator protein cause the AB variant of GM2-gangliosidosis, a condition that is clinically indistinguishable from Tay-Sachs disease. To understand better factors affecting the expression of the GM2 activator protein gene (Gm2a) in mouse tissues, we have determined its exon-intron organization and analyzed its promoter region.Gm2a is about 14 kb, has four exons, and the 5′ flanking region contains a CAAT box, Spl binding sites, AP-1, AP-2 sites, and a pair of IRE sites. A 1.2-kb fragment upstream from the initiation codon was shown to have promoter activity in NIH 3T3 cells. Similarities between the elements present in Gm2a and Hexa promoters might in part explain their similar expression patterns in mouse tissues. The different levels of GM2 activator protein mRNA in liver, kidney, brain, and testis are not owing to the use of different transcription start sites, because a single start site was found 50 bp upstream from the initiation codon in each these tissues. Northern blot analysis demonstrated variation in the GM2 activator protein mRNA expression during mouse development. Gm2a was mapped to Chromosome (Chr) 11, where it co-segregated with Csfgm.


Clinica Chimica Acta | 2000

beta-hexosaminidase, alpha-D-mannosidase, and beta-mannosidase expression in serum from patients with carbohydrate-deficient glycoprotein syndrome type I.

Tommaso Beccari; F Mancuso; Egidia Costanzi; C. Tassi; Rita Barone; Agata Fiumara; A. Orlacchio; M.C Aisa

The activity of beta-hexosaminidase, determined with 4-methylumbelliferyl-beta-N-acetylglucopyranoside substrate, and of beta-D-mannosidase was significantly higher in the serum of patients with carbohydrate-deficient glycoprotein (CDG) syndrome type IA (phosphomannomutase deficiency) than in controls. No significant differences were observed in the activity of beta-hexosaminidase, determined using 4-methylumbelliferyl-beta-N-acetylglucopyranoside-6-sulphate as substrate, and the activity of alpha-D-mannosidase. Using DEAE-cellulose chromatography, a greater amount of hexosaminidase B than hexosaminidase A was detected in CDG serum. In CDG serum, hexosaminidase A was eluted in a more basic position in the salt gradient. An isoenzyme of alpha-D-mannosidase and beta-D-mannosidase was identified in control and CDG sera. alpha-D-Mannosidase isoenzyme was eluted in a slightly more basic position in CDG serum than in control serum, whereas beta-D-mannosidase isoenzyme was eluted in the same position.


Clinica Chimica Acta | 1999

Elevated β-N-acetylhexosaminidase activity in focal dystonia fibroblasts

Egidia Costanzi; Tommaso Beccari; C. Tassi; A. Orlacchio; A. Rossi

Specific activities of beta-D-hexosaminidase, alpha-D-mannosidase, beta-D-galactosidase and beta-D-glucuronidase were determined in fibroblasts of patients with writers cramp and torticollis. These diseases show degenerative neurological disorders similar to those observed in lysosomal diseases. Hexosaminidase specific activities, determined using 4-methylumbelliferyl-beta-N-acetylglucopyranoside and 4-methylumbelliferyl-beta-N-acetylglucopyranoside-6-sulphate as substrates, were significantly higher in the fibroblasts of patients than in controls. No significant differences were observed in the specific activities of the other lysosomal enzymes. The increased hexosaminidase specific activities in torticollis and writers cramp may be additional markers for these diseases.


Clinica Chimica Acta | 1990

Identification of an intermediate form of β-N-acetylhexosaminidase in chorionic villi

M. Pieretti; Tommaso Beccari; Ermelando V. Cosmi; J.L. Stirling; A. Orlacchio

A minor form of beta-N-acetylhexosaminidase has been found in chorionic villi, in addition to the major forms A and B. This form does not hydrolyze the 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside-6-sulpha te substrate, is thermostable, has a higher mol mass (120,000) than A and B (100,000) and on analytical isoelectric focusing, it shows a microheterogeneity with values ranging between 6.3 and 7.0. For these characteristics, it resembles beta-N-Acetylhexosaminidase P from pregnancy serum, from which is chromatographically indistinguishable.


Biochemical Journal | 1987

Intermediate forms of human β-N-acetylhexosaminidase lack activity towards 4-methylumbelliferyl β-N-acetylglucosaminide 6-sulphate

Tommaso Beccari; Carla Emiliani; R Hosseini; A. Orlacchio; J L Stirling


Biochemical Journal | 1992

Cloning and sequence analysis of a cDNA encoding the alpha-subunit of mouse beta-N-acetylhexosaminidase and comparison with the human enzyme.

Tommaso Beccari; J. Hoade; A. Orlacchio; John L. Stirling

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C. Tassi

University of Perugia

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