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J L Stirling - One of the best experts on this subject based on the ideXlab platform.

  • Evidence for the regulation of Beta-N-Acetylhexosaminidase expression during pregnancy in the rat.
    Biochimica et Biophysica Acta (BBA) - General Subjects, 2000
    Co-Authors: Brunella Tancini, J L Stirling, Carla Emiliani, Simona Mencarelli, Cristina Cavalieri, Aldo Orlacchio
    Abstract:

    Abstract It is believed that the lysosomal glycohydrolase β-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 α- and β-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 α- and β-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 β-subunit mRNA abundance, whereas in the uterus and liver, an increased abundance of this transcript was detectable. The abundance of α-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 β-N-acetylhexosaminidase expression is in operation, and that the enzyme is differentially regulated in rat tissues.

  • Distribution of active alpha- and beta-subunits of Beta-N-Acetylhexosaminidase as a function of leukaemic cell types.
    Biochimica et biophysica acta, 1997
    Co-Authors: S Martino, J L Stirling, C Emiliani, A Tabilio, F Falzetti, A Orlacchio
    Abstract:

    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.

  • Influence of cell differentiation and protein kinase C activation on sub-cellular distribution of Beta-N-Acetylhexosaminidases of HL 60 cells.
    Physiological chemistry and physics and medical NMR, 1995
    Co-Authors: Carla Emiliani, J L Stirling, Sabata Martino, Aldo Orlacchio
    Abstract:

    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.

  • Organization of the gene for the alpha-subunit of mouse Beta-N-Acetylhexosaminidase (HEXa).
    Biochemistry and molecular biology international, 1994
    Co-Authors: Bianconi S, J L Stirling, Tommaso Beccari, Sheardown S, Aldo Orlacchio
    Abstract:

    Genomic clones spanning the mouse HEXa gene were isolated and the exon-intron organization determined. The locations of all exon-intron junctions were identical to those of the human HEXa gene but the large intron 1 was shorter in the mouse (14Kb) than in the human gene (18Kb).

  • Blood testosterone levels are correlated with Beta-N-Acetylhexosaminidase activity in human caput epididymis.
    Biochemistry and molecular biology international, 1993
    Co-Authors: Alessandro Datti, J L Stirling, Carla Emiliani, Tommaso Beccari, Aldo Orlacchio
    Abstract:

    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.

K F Nielsen - One of the best experts on this subject based on the ideXlab platform.

  • Quantifying mold biomass on gypsum board: Comparison of ergosterol and Beta-N-Acetylhexosaminidase as mold biomass parameters
    Applied and Environmental Microbiology, 2003
    Co-Authors: M. Reeslev, M. Miller, K F Nielsen
    Abstract:

    Two mold species, Stachybotrys chartarum and Aspergillus versicolor, were inoculated onto agar overlaid with cellophane, allowing determination of a direct measurement of biomass density by weighing. Biomass density, ergosterol content, and Beta-N-Acetylhexosaminidase (3.2.1.52) activity were monitored from inoculation to stationary phase. Regression analysis showed a good linear correlation to biomass density for both ergosterol content and Beta-N-Acetylhexosaminidase activity. The same two mold species were inoculated onto wallpapered gypsum board, from which a direct biomass measurement was not possible. Growth was measured as an increase in ergosterol content and Beta-N-Acetylhexosaminidase activity. A good linear correlation was seen between ergosterol content and Beta-N-Acetylhexosaminidase activity. From the experiments performed on agar medium, conversion factors (CFs) for estimating biomass density from ergosterol content and Beta-N-Acetylhexosaminidase activity were determined. The CFs were used to estimate the biomass density of the molds grown on gypsum board. The biomass densities estimated from ergosterol content and Beta-N-Acetylhexosaminidase activity data gave similar results, showing significantly slower growth and lower stationary-phase biomass density on gypsum board than on agar.

M. Reeslev - One of the best experts on this subject based on the ideXlab platform.

  • Quantifying mold biomass on gypsum board: Comparison of ergosterol and Beta-N-Acetylhexosaminidase as mold biomass parameters
    Applied and Environmental Microbiology, 2003
    Co-Authors: M. Reeslev, M. Miller, K F Nielsen
    Abstract:

    Two mold species, Stachybotrys chartarum and Aspergillus versicolor, were inoculated onto agar overlaid with cellophane, allowing determination of a direct measurement of biomass density by weighing. Biomass density, ergosterol content, and Beta-N-Acetylhexosaminidase (3.2.1.52) activity were monitored from inoculation to stationary phase. Regression analysis showed a good linear correlation to biomass density for both ergosterol content and Beta-N-Acetylhexosaminidase activity. The same two mold species were inoculated onto wallpapered gypsum board, from which a direct biomass measurement was not possible. Growth was measured as an increase in ergosterol content and Beta-N-Acetylhexosaminidase activity. A good linear correlation was seen between ergosterol content and Beta-N-Acetylhexosaminidase activity. From the experiments performed on agar medium, conversion factors (CFs) for estimating biomass density from ergosterol content and Beta-N-Acetylhexosaminidase activity were determined. The CFs were used to estimate the biomass density of the molds grown on gypsum board. The biomass densities estimated from ergosterol content and Beta-N-Acetylhexosaminidase activity data gave similar results, showing significantly slower growth and lower stationary-phase biomass density on gypsum board than on agar.

Tommaso Beccari - One of the best experts on this subject based on the ideXlab platform.

  • CONSTITUTIVE EXPRESSION OF BETA -N-ACETYLHEXOSAMINIDASE IN A MICROGLIAL CELL LINE: TRANSCRIPTIONAL MODULATION BY LIPOPOLYSACCHARIDE AND SERUM FACTORS
    Journal of Neuroscience Research, 1997
    Co-Authors: Tommaso Beccari, Antonio Orlacchio, Egidia Costanzi, Maria Grazia Appolloni, Assunta Laurenzi, Virginia Bocchini
    Abstract:

    We investigated the expression of the alpha- and beta-subunits of the lysosomal enzyme Beta-N-Acetylhexosaminidase in the BV-2 microglial cell line under different culture conditions. Beta-N-Acetylhexosaminidase from BV-2 microglia cells was separated into its constituent isoenzymes on diethylaminoethyl (DEAE) cellulose, and its activity was monitored with 4-methylumbelliferyl-beta-N-acetylglucosamine and 4-methylumbelliferyl-beta-N-acetylglucosamine-6-sulphate substrates. Forms corresponding to the mouse isoenzymes A and B were present in the cells incubated in serum-supplemented medium as well as in serum-free medium. Lipopolysaccharide, a well-known activator of microglia in vitro, added to the BV-2 cells in serum-supplemented medium induced a decrease in the specific enzymatic activity determined with the 4-methylumbelliferyl-beta-N-acetylglucosamine substrate. Lipopolysaccharide had no effect on hexosaminidase isoenzyme pattern of BV-2 cells in serum-supplemented medium. The level of alpha-subunit mRNA was increased and the level of beta-subunit mRNA was decreased in BV-2 cells incubated in serum-supplemented medium plus lipopolysaccharide. In the cells incubated in a serum-free medium no significant changes in the hexosaminidase-specific activities towards the above substrates were observed. Interestingly, increased expression of alpha- and beta-subunit mRNA was evident in comparison with cultures in serum-supplemented medium. The present results suggest that the BV-2 cell line may be a useful tool to study the possible role of microglia in the metabolism of brain glycolipids.

  • Hexosaminidase in Trichinella spiralis is a single protein with alpha- and beta-subunits catalytic activities.
    Cellular and molecular biology (Noisy-le-Grand France), 1997
    Co-Authors: Egidia Costanzi, Tommaso Beccari, Antonio Orlacchio, M. A. Della Fazia, Giuseppe Servillo, C. Tassi, F. Bruschi
    Abstract:

    Beta-N-Acetylhexosaminidase is expressed as a single protein in Trichinella spiralis and has catalytic properties similar to the alpha- and beta-subunits of human and mouse isoenzymes A and B. It can hydrolyze the artificial substrates, 4-methylumbelliferyl-beta-D-glucosamine and 4-methylumbelliferyl-beta-D-glucosamine-6-sulphate which are respectively hydrolyzed by the beta- and alpha-subunits. The enzyme is thermostable, has a basic isoelectric point, and thus is similar to the B isoenzyme. Northern blotting experiments indicate that the enzyme is encoded by a single gene. Hexosaminidase from Trichinella spiralis shows that the substrate specificities of alpha- and beta-subunits precede the duplication of their genes.

  • Organization of the gene for the alpha-subunit of mouse Beta-N-Acetylhexosaminidase (HEXa).
    Biochemistry and molecular biology international, 1994
    Co-Authors: Bianconi S, J L Stirling, Tommaso Beccari, Sheardown S, Aldo Orlacchio
    Abstract:

    Genomic clones spanning the mouse HEXa gene were isolated and the exon-intron organization determined. The locations of all exon-intron junctions were identical to those of the human HEXa gene but the large intron 1 was shorter in the mouse (14Kb) than in the human gene (18Kb).

  • Blood testosterone levels are correlated with Beta-N-Acetylhexosaminidase activity in human caput epididymis.
    Biochemistry and molecular biology international, 1993
    Co-Authors: Alessandro Datti, J L Stirling, Carla Emiliani, Tommaso Beccari, Aldo Orlacchio
    Abstract:

    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.

  • Cloning and sequence analysis of a cDNA encoding the α-subunit of mouse β-N-acetylhexosaminidase and comparison with the human enzyme
    Biochemical Journal, 1992
    Co-Authors: Tommaso Beccari, Antonio Orlacchio, J Hoade, J L Stirling
    Abstract:

    cDNAs encoding the mouse Beta-N-Acetylhexosaminidase alpha-subunit were isolated from a mouse testis library. The longest of these (1.7 kb) was sequenced and showed 83% similarity with the human alpha-subunit cDNA sequence. The 5' end of the coding sequence was obtained from a genomic DNA clone. Alignment of the human and mouse sequences showed that all three putative N-glycosylation sites are conserved, but that the mouse alpha-subunit has an additional site towards the C-terminus. All eight cysteines in the human sequence are conserved in the mouse. There are an additional two cysteines in the mouse alpha-subunit signal peptide. All amino acids affected in Tay-Sachs-disease mutations are conserved in the mouse.

M. Miller - One of the best experts on this subject based on the ideXlab platform.

  • Quantifying mold biomass on gypsum board: Comparison of ergosterol and Beta-N-Acetylhexosaminidase as mold biomass parameters
    Applied and Environmental Microbiology, 2003
    Co-Authors: M. Reeslev, M. Miller, K F Nielsen
    Abstract:

    Two mold species, Stachybotrys chartarum and Aspergillus versicolor, were inoculated onto agar overlaid with cellophane, allowing determination of a direct measurement of biomass density by weighing. Biomass density, ergosterol content, and Beta-N-Acetylhexosaminidase (3.2.1.52) activity were monitored from inoculation to stationary phase. Regression analysis showed a good linear correlation to biomass density for both ergosterol content and Beta-N-Acetylhexosaminidase activity. The same two mold species were inoculated onto wallpapered gypsum board, from which a direct biomass measurement was not possible. Growth was measured as an increase in ergosterol content and Beta-N-Acetylhexosaminidase activity. A good linear correlation was seen between ergosterol content and Beta-N-Acetylhexosaminidase activity. From the experiments performed on agar medium, conversion factors (CFs) for estimating biomass density from ergosterol content and Beta-N-Acetylhexosaminidase activity were determined. The CFs were used to estimate the biomass density of the molds grown on gypsum board. The biomass densities estimated from ergosterol content and Beta-N-Acetylhexosaminidase activity data gave similar results, showing significantly slower growth and lower stationary-phase biomass density on gypsum board than on agar.