Biomedical subjects

Egidia Costanzi

Publications and source records attributed to Egidia Costanzi.

4 recordsLinked to original sources

PubMed2006

Cloning and expression of mouse cytosolic alpha-mannosidase (Man2c1).

It is known that the neutral/cytosolic alpha-mannosidase (Man2c1) which can cleave alpha 1,2-, alpha 1,3-, and alpha 1,6-linked mannose residues, is stimulated by cobalt and is inhibited by furanose analogues swainsonine (SW) and 1,4-dideoxy-1,4-imino-d-mannitol (DIM). The enzyme is involved in the degradation of oligomannosides derived from dolichol intermediates and the degradation of newly synthesized glycoproteins. An immunological relationship has been demonstrated between the rat endoplasmic reticulum alpha-mannosidase and the cytosolic alpha-mannosidase. In fact antibodies raised against the soluble alpha-mannosidase recognized the membrane form of the ER alpha-mannosidase. A cDNA encoding the mouse cytosolic alpha-mannosidase was obtained by RZPD (Deutsches Ressourcenzentrum fur Genomforschung GmbH), Berlin, Germany. Comparison of the mouse genomic sequence with the cDNA sequence revealed the presence of 25 introns within the cytosolic alpha-mannosidase gene. The gene spans 11.5 kb from the major transcription initiation site to the RNA cleavage site. The transcription initiation site of mouse cytosolic alpha-mannosidase was mapped to 170 bases upstream of the ATG codon using 5' RACE. Northern blotting analysis revealed expression of a major transcript of 3.8 kb in all tissues examined. COS cells transfected with the cDNA showed a 20-fold increase in cytosolic alpha-mannosidase activity. This enzyme activity was stimulated by cobalt and inhibited by DIM and EDTA. Furthermore we demonstrated that the expressed enzyme was active towards the radiolabeled substrate Man9GlcNAc1 giving the final product Man5GlcNAc1 through the formation of Man8GlcNAc1 isomer C as intermediate.

3' Untranslated Regions↗

PubMed2005

Expression and purification of a human, soluble Arylsulfatase A for Metachromatic Leukodystrophy enzyme replacement therapy.

The production of active Arylsulfatase A is a key step in the development of enzyme replacement therapy for Metachromatic Leukodystrophy. To obtain large amounts of purified Arylsulfatase A for therapeutic use, we combined a retroviral expression system with a versatile and rapid purification protocol that can easily and reliably be adapted to high-throughput applications. The purification method consists of an initial ion-exchange DEAE-cellulose chromatography step followed by immuno-affinity purification using a polyclonal antibody against a 29-mer peptide of the Arylsulfatase A sequence. Immuno-adsorbed protein was eluted with a combination of acidic pH and an optimal concentration of the 29-mer peptide. This protocol reproducibly yielded approximately 100 microg of >99% pure human Arylsulfatase A, corresponding to 152 mU of enzyme activity, per liter of culture medium with properties similar to those of human non-recombinant protein.

Animals↗

PubMed2003

Mouse sulphamidase gene: characterization of the promoter region of the gene and expression in mouse tissues.

Sulphamidase is a lysosomal enzyme necessary for the degradation of heparan sulphate. The deficiency of this hydrolase causes a disorder known as mucopolysaccharidosis type IIIA, characterized by a profound neurological deterioration. Human and mouse exon-intron structures were reported without any characterization of their promoter regions [DNA Res. 3 (1996) 269; Mamm. Genome 11 (2000) 436]. The promoter region was isolated and characterized to understand the factors affecting the expression of mouse sulphamidase. The 5'-flanking region was shown to contain a GC-rich region and putative binding sites for the transcription factors SRY, MZF1 and Nkx-2.5 with no TATA or CAAT boxes present. The 5' region had promoter activity to drive luciferase gene expression in transfected COS cells. The transcription initiation site of mouse sulphamidase was mapped to a single adenine residue 355 bases upstream of ATG codon. Northern blot analysis revealed differential expression of a major transcript of 4.5 kb in all tissues examined. Finally, the 3'-untranslated region of the mouse sulphamidase gene was isolated and found to be longer than the region identified in the human gene.

3' Flanking Region↗

PubMed2003

Cathepsin B in osteoblasts.

Active cathepsin B has been found in cell extract and medium of human osteoblast-like cells and MG-63 cells. The released form is stable at neutral and alkaline pH and, in both cell types, intracellular and extracellular cathepsin B activities are increased by interleukin-1 beta (IL-1beta) and parathyroid hormone (PTH). To evaluate the physiological role of cathepsin B in osteoblasts, we investigated the production and secretion of this enzyme in normal human synovial fibroblasts and modulation by IL-1beta and PTH. Lactate secretion concurrent with release of cathepsin B and comparable responses in osteoblasts were also examined. Our data show that synovial fibroblasts respond differently to treatment with the two agents, suggesting a cell-specific regulation of cathepsin B and possible involvement in osteoblast physiology. Cathepsin B involvement was then evaluated in the activation of plasminogen activator (PA) in MG-63 cells using two specific inhibitors of cathepsin B, CA074 and CA074-Me, in constitutive conditions and after treatment with IL-1beta. As results of PA activity obtained in the presence of IL-beta were in contrast with previous reports, we examined the activities of PA, pro-PA activated with trypsin, and plasmin in cell extract and media of MG-63 cells after 24-h treatment with IL-1beta. Results show that in normal conditions and in the presence of IL-1beta, cathepsin B is involved in the activation of PA. Moreover, IL-1beta stimulates PA, pro-PA activated by trypsin, and plasmin activity in medium, whereas in cell extract it stimulates pro-PA activated by trypsin and plasmin activity. IL-1beta has no effect on cell extract-associated PA.

Cathepsin B↗