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Biomedical subjects

C Emiliani

Publications and source records attributed to C Emiliani.

At least 19 recordsLinked to original sources
PubMed2005

A direct gene transfer strategy via brain internal capsule reverses the biochemical defect in Tay-Sachs disease.

Therapy for neurodegenerative lysosomal Tay-Sachs (TS) disease requires active hexosaminidase (Hex) A production in the central nervous system and an efficient therapeutic approach that can act faster than human disease progression. We combined the efficacy of a non-replicating Herpes simplex vector encoding for the Hex A alpha-subunit (HSV-T0alphaHex) and the anatomic structure of the brain internal capsule to distribute the missing enzyme optimally. With this gene transfer strategy, for the first time, we re-established the Hex A activity and totally removed the GM2 ganglioside storage in both injected and controlateral hemispheres, in the cerebellum and spinal cord of TS animal model in the span of one month's treatment. In our studies, no adverse effects were observed due to the viral vector, injection site or gene expression and on the basis of these results, we feel confident that the same approach could be applied to similar diseases involving an enzyme defect.

Animals↗
PubMed2003

Widespread distribution of beta-hexosaminidase activity in the brain of a Sandhoff mouse model after coinjection of adenoviral vector and mannitol.

Sandhoff disease is a severe inherited neurodegenerative disorder resulting from deficiency of the beta-subunit of hexosaminidases A and B, lysosomal hydrolases involved in the degradation of G(M2) ganglioside and related metabolites. Currently, there is no viable treatment for the disease. Here, we show that adenovirus-mediated transfer of the beta-subunit of beta-hexosaminidase restored Hex A and Hex B activity after infection of Sandhoff fibroblasts. Gene transfer following intracerebral injection in a murine model of Sandhoff disease resulted in near-normal level of enzymatic activity in the entire brain at the different doses tested. The addition of hyperosmotic concentrations of mannitol to the adenoviral vector resulted in an enhancement of vector diffusion in the injected hemisphere. Adenoviral-induced lesions were found in brains injected with a high dose of the vector, but were not detected in brains injected with 100-fold lower doses, even in the presence of mannitol. Our data underline the advantage of the adjunction of mannitol to low doses of the adenoviral vector, allowing a high and diffuse transduction efficiency without viral cytotoxicity.

Adenoviridae↗
PubMed2002

Muscle as a putative producer of acid alpha-glucosidase for glycogenosis type II gene therapy.

Glycogenosis type II (GSD II) is a lysosomal disorder affecting skeletal and cardiac muscle. In the infantile form of the disease, patients display cardiac impairment, which is fatal before 2 years of life. Patients with juvenile or adult forms can present diaphragm involvement leading to respiratory failure. The enzymatic defect in GSD II results from mutations in the acid alpha-glucosidase (GAA) gene, which encodes a 76 kDa protein involved in intralysosomal glycogen hydrolysis. We previously reported the use of an adenovirus vector expressing GAA (AdGAA) for the transduction of myoblasts and myotubes cultures from GSD II patients. Transduced cells secreted GAA in the medium, and GAA was internalized by receptor-mediated capture, allowing glycogen hydrolysis in untransduced cells. In this study, using a GSD II mouse model, we evaluated the feasibility of GSD II gene therapy using muscle as a secretary organ. Adenovirus vector encoding AdGAA was injected in the gastrocnemius of neonates. We detected a strong expression of GAA in the injected muscle, secretion into plasma, and uptake by peripheral skeletal muscle and the heart. Moreover, glycogen content was decreased in these tissues. Electron microscopy demonstrated the disappearance of destruction foci, normally present in untreated mice. We thus demonstrate for the first time that muscle can be considered as a safe and easily accessible organ for GSD II gene therapy.

Adenoviridae↗
PubMed2002

Restoration of the GM2 ganglioside metabolism in bone marrow-derived stromal cells from Tay-Sachs disease animal model.

The therapeutic potential of bone marrow-derived stromal cells for the therapy of Tay-Sachs disease is primarily related to the restoration of their own GM2 ganglioside storage. With this aim, we produced bone marrow-derived stromal cells from the adult Tay-Sachs animal model and transduced them with a retroviral vector encoding for the alpha-subunit of the lysosomal enzyme beta-hexosaminidase A (E.C. 3.2.1.52). Our results demonstrate that transduced Tay-Sachs bone marrow-derived stromal cells have beta-hexosaminidase A comparable to that of bone marrow-derived stromal cells from wild-type mice. Moreover, beta-hexosaminidase A in transduced Tay-Sachs bone marrow-derived stromal cells was able to hydrolyze the GM2 ganglioside in a feeding experiment, thus demonstrating the correction of the altered phenotype.

Animals↗
PubMed2000

Evidence for the regulation of beta-N-acetylhexosaminidase expression during pregnancy in the rat.

It is believed that the lysosomal glycohydrolase beta-N-acetylhexosaminidase plays a part in several important processes of reproduction and it has been postulated that this enzyme is subject to hormonal regulation. During pregnancy, activity levels of the enzyme are strongly increased in both human and rat serum. However, little is known about the expression of this enzyme in the female reproductive apparatus and there is no evidence linking the production of hexosaminidase alpha- and beta-subunits to pregnancy. To clarify these aspects better, we examined the enzyme activity, isoenzyme subunit composition and distribution, as well as steady state levels of alpha- and beta-subunit mRNAs in the female reproductive organs and in other selected tissues of pregnant and non-pregnant rats. Among the female rat tissues tested, the ovary and kidney had the highest specific activity. Pregnancy modulated the hexosaminidase activity differently in the tissues examined. In pregnant rats, the activity decreased in the ovary but increased significantly in the uterus, liver and to a lesser extent in other tissues. The decreased hexosaminidase activity in the ovary from pregnant rats appeared to be accompanied by a disproportionately large decrease in beta-subunit mRNA abundance, whereas in the uterus and liver, an increased abundance of this transcript was detectable. The abundance of alpha-subunit mRNA was comparable in pregnant and control rat tissues. Hexosaminidase histochemical staining of tissue sections clearly demonstrates that the greatly increased activity of hexosaminidase in the uterus during pregnancy is largely due to the enzyme in the endometrium, and not to the uterus as a whole. The overall results provide evidence that, during pregnancy, a mechanism(s) of regulation of beta-N-acetylhexosaminidase expression is in operation, and that the enzyme is differentially regulated in rat tissues.

Animals↗
PubMed2000

Platelets release their lysosomal content in vivo in humans upon activation.

Platelets contain, besides alpha- and delta-granules, lysosomes which store glycohydrolases able to degrade glycoproteins, glycolipids and glycosaminoglycans. While several studies have shown that alpha- and delta-granule secretion takes place "in vivo" in humans upon platelet activation, no data are available on the "in vivo" release of lysosomes. We have studied the release of platelet lysosomal contents "in vivo" in healthy volunteers at a localized site of platelet activation by measuring markers of lysosomal secretion in the blood oozing from a skin wound inflicted for the measurement of the bleeding-time. The levels of beta-N-acetylhexosaminidase (Hex) were 13.1 +/- 0.85 mU/ml in bleeding-time blood and 10.2 +/- 0.66 mU/ml in plasma (p <0.001). Hex in serum was 16.4 +/- 0.72 mU/ml. The levels of beta-galactosidase were also higher in bleeding-time blood than in plasma (0.85 +/- 0.07 mU/ml vs 0.4 +/- 0.05 mU/ml, p <0.001). In bleeding-time blood collected at one minute intervals, Hex rose progressively consistent with ongoing platelet activation and flow-cytometry showed a progressive increase of the expression of LIMP and LAMP-2, two lysosomal associated proteins. In conclusion, our data demonstrate that platelet lysosomal glycohydrolases are released "in vivo" in humans upon platelet activation.

Bleeding Time↗
PubMed1998

Beta-N-acetylhexosaminidase in peripheral blood lymphocytes and monocytes in the different forms and stages of multiple sclerosis.

The activity of the acidic glycohydrolase beta-N-acetylhexosaminidase, an enzyme system normally participating in the stepwise degradation of glycoproteins, glycolipids, and proteoglycans, appears to be modulated in lymphocytes and monocytes from peripheral blood of patients affected by multiple sclerosis during different stages of the disease. In particular, a significant decrease in this enzyme activity, compared with healthy subjects, was observed in patients affected by the relapsing-remitting form both in a stable clinical status and during a relapse as well as in patients with the progressive form. The decrease in total intracellular hexosaminidase activity in lymphomonocytes of multiple sclerosis patients was accompanied by an enrichment of this activity associated with the plasma membrane fraction as demonstrated by experiments of subcellular fractionation. The analysis carried out using two synthetic substrates, 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide and its sulfate derivative, enables us to demonstrate that this accumulation is mainly due to isoenzymes with a betabeta structure, whereas lysosomal fractions confirmed the classical presence of both alphabeta and betabeta forms (hexosaminidases A and B, respectively). This was particularly evident in the plasma membrane fraction from mononuclear cells of patients with a clinical exacerbation of the disease. Considered together, these observations provide additional insight into the abnormality of peripheral blood immune cells in multiple sclerosis and may contribute to the understanding of the basic mechanisms underlying the pathological events resulting in the demyelinating process.

Adult↗
PubMed1997

Distribution of active alpha- and beta-subunits of beta-N-acetylhexosaminidase as a function of leukaemic cell types.

beta-N-Acetylhexosaminidase isoenzymes, and the distribution of the alpha- and beta-subunits forming the enzyme in a representative series of fresh leukaemic cells and in established leukaemic cell lines, were obtained by using a combination of DEAE-cellulose chromatography and assay with the fluorogenic substrates 4-methylumbelliferyl-beta-N-acetylglucosaminide hydrolyzed by both alpha and beta subunits, and 4-methylumbelliferyl-beta-N-acetylglucosaminide-6-SO4 hydrolyzed only by hexosaminidase isoenzymes containing alpha-subunits. The presence of hexosaminidase S (the alpha alpha dimer), was found in all the leukaemic cell populations we surveyed, but not in normal human cells. The presence of this isoenzyme can therefore be considered as an additional marker of leukaemic cells. A prevalence of hexosaminidase A and A-like intermediate forms (alpha beta structure), characterize leukaemic cells of myeloid origin, whereas greater amounts of hexosaminidase B and B-like intermediate forms (beta beta structure), were consistent attributes of leukaemic cells of lymphoid origin. An over-expression of beta-subunits in blasts might be related to their undifferentiated status. These changes in the isoenzymes of hexosaminidase may prove informative about a variety of changes in the biology of leukaemic cells that could range from chromosomal alterations to changes in the proteolytic processing and glycosylation.

Hexosaminidase A↗
PubMed1996

Purification and properties of human urinary beta-D-mannosidase.

Two forms of the lysosomal enzyme beta-mannosidase were identified and purified from human urine. The purification strategy employed allowed sufficient quantities of both forms to be obtained for subunit analysis and for further characterizations. The two beta-mannosidases were identified as beta-mannosidase B and A, in order of their elution from an ion-exchange column. In all samples examined, the A form was predominant, and the B/A ratio was consistently 0.14. The two forms displayed the same optimum pH (i.e., 4.3) and both were retained by a Concanavalin-A Sepharose column, but showed different isoelectric points, molecular masses and subunit compositions. Native- and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of pure beta-mannosidases B and A suggest that active protein B (160 kDa) consists of three subunits, one 75 kDa and two 49 kDa subunits. Protein A is smaller and appears to be composed of three subunits of 75 kDa, 49 kDa and 37 kDa. Two forms of beta-mannosidase, exhibiting a chromatographic behaviour comparable to the urinary forms, were also detected in human kidney. Nevertheless, in this tissue their relative distribution was different, the B/A ratio being 19.

Chromatography, Agarose↗
PubMed1995

Beta-N-acetylhexosaminidases A and S have similar sub-cellular distributions in HL-60 cells.

In HL-60 cells the most abundant isoenzymes of beta-N-acetyl-hexosaminidase are A (alpha beta) and S (alpha alpha). Sub-cellular fractionation of HL-60 cells by differential centrifugation showed that both A and S forms were present in the lysosomal and post-lysosomal (soluble) fractions in approximately equal abundance. Ion-exchange chromatography showed that a fraction enriched with plasma membranes had the A form, and a form of beta-N-acetylhexosaminidase less acidic than A, but there was no S. Analysis of the alpha-subunits of beta-N-acetylhexosaminidases A and S using Western blotting and immuno-detection with antisera raised to synthetic peptides showed that mature alpha-subunits were present in both A and S isolated from the lysosomal fraction. This observation establishes that the alpha alpha-dimer of beta-N-acetyl-hexosaminidase (S) can be transported to lysosomes in HL-60 cells whereas there is good evidence that this does not take place in fibroblasts. HL-60 cells were not stimulated to secrete lysosomal enzymes by incubating them with NH4Cl and, unlike fibroblasts, are unlikely to use mannose-6-phosphate mediated transport of beta-N-acetylhexosaminidases to lysosomes. Comparison of the sequence of the beta-N-acetylhexosaminidase alpha-subunit with a 43 amino acid sequence of cathepsin D, though to function in the mannose-6-phosphate independent targeting of this enzyme to lysosomes, showed alignment in a region towards the C-terminus in which 21% of the residues were identical with the interposition of a one amino acid gap.

Amino Acid Sequence↗
PubMed1995

Partial sequence of the purified protein confirms the identity of cDNA coding for human lysosomal alpha-mannosidase B.

Human lysosomal alpha-mannosidase has been purified by a simple and rapid method in sufficient quantities for the analysis of its subunit composition and partial protein sequencing. Analysis of the N-terminal residues of the 30 kDa polypeptide has enabled us to confirm the identity of the recently cloned cDNA that was tentatively identified as that of lysosomal alpha-mannosidase [Nebes and Schmidt (1994) Biochem. Biophys. Res. Commun. 200, 239-245] and to locate the position of this polypeptide within the total deduced amino acid sequence. This finding will therefore provide a firm foundation for the characterization of alpha-mannosidosis mutations.

Amino Acid Sequence↗
PubMed1995

Platelet glycohydrolase activities: characterization and release.

Granules containing acid hydrolases have been detected in human platelets but have not been thoroughly characterized. We have studied the activity and characteristics of glycohydrolases present in normal human platelets, evaluated their release upon stimulation with thrombin, and assessed the contribution of platelet - released lysosomal contents to the glycohydrolase activity present in normal serum. Platelets contained a remarkable glycohydrolase activity with a prevalence of beta - N-acetylhexosaminidase. All glycohydrolases were released to some extent upon stimulation with thrombin and contributed to the glycohydrolase activity found in human serum. alpha-Mannosidase and alpha-galactosidase were partially inactivated after release by a mechanism as yet undefined. In addition, thrombin stimulation affects the intraplatelet isoenzyme pattern of beta-N-acetylhexosaminidase by producing the appearance of a new form.

Blood Platelets↗
PubMed1995

Influence of cell differentiation and protein kinase C activation on sub-cellular distribution of beta-N-acetylhexosaminidases of HL 60 cells.

There have been several accounts regarding the alterations of the lysosomal enzyme beta-N-acetylhexosaminidase in human leukaemic cells. In addition to Hex A (alpha beta) and Hex B (beta beta) forms, leukaemic cells contain a third isoenzyme displaying many characteristics in common with Hex S, the alpha alpha dimer representing the residual activity in patients with Sandhoff's disease. In the human leukaemic cell line HL 60, A (alpha beta) and S (alpha alpha) are the most abundant forms. Sub-cellular fractionation of HL 60 cells showed that both A and S forms were present in the lysosomal and post-lysosomal fractions, however, a proportion of activity was found to be associated with the plasma membrane. The phorbol ester 12-O-tetra-decanoylphorbol-13-acetate (TPA) exerts complex effects on the physiology of HL 60 cells, leading to cell differentiation along the macrophage pathway and including activation of Protein Kinase C (PKC). In order to assess the extent to which cell differentiation and PKC activation plays a role in modulating the expression of hexosaminidase during cell differentiation, we treated HL 60 cells with TPA and in parallel with the more specific activator of PKC, 1-oleoyl-2-acetyl diglycerol (OAG) which does not cause cell differentiation. We observed that 24 h exposure of HL 60 cells to TPA or OAG produced significant modification of the hexosaminidase isoenzyme pattern of HL 60 cells. The most remarkable effect was seen in both cases in the plasma membrane fraction. Taken together, our results suggest a correlation between hexosaminidase expression and kinase(s) activation.

Cell Differentiation↗
PubMed1993

Extinction and viruses.

The micropaleontological record of deep-sea sediments is demonstrably continuous through time and, therefore, it can be used to study in detail the evolutionary process. Several taxa, including Coccolithophorida, planktic Foraminifera, Radiolaria, and diatoms, are abundantly represented. Some species, apparently evolved in the marginal marine environment, appear suddenly in the record while other species grade into different ones at rates that range from 100,000 years to millions of years. The rates are usually different for different morphological characters within the same lineage. Every few million years a mass extinction occurs--the disappearance of a number of taxa apparently caused by an environmental upset of some kind. These events are used by stratigraphers to subdivide geologic time. Interspersed between mass extinctions are the background extinctions, the individual extinctions of well-established species while the sympatric species exhibit no sign of stress. It is hypothesized that background extinctions are caused by host-specific viral action. It is further hypothesized that background extinctions are a fundamental component of the process of evolution.

Animals↗
PubMed1993

Blood testosterone levels are correlated with beta-N-acetylhexosaminidase activity in human caput epididymis.

beta-N-acetylhexosaminidase exhibits a relatively high activity in human epididymis. Its isoenzymatic profile and immunological properties led us to conclude that the increased activity was not due to the expression of an isoform unrelated to the HEX A and B present in other human tissues. Measurement of enzyme levels in the three distinct epididymal regions revealed that in any given sample, activity was higher in the caput than in the corpus or cauda. Moreover, we found a striking correlation between beta-HEX activity in the caput region and concentrations of blood testosterone, suggesting a possible involvement of the hormone in modulating enzyme expression. Since the caput epididymis plays a role in the maturation of sperm cells, our data may be an indication that beta-HEX activity in the caput has a physiological relevance in human epididymis functions.

Chromatography, Ion Exchange↗