Assignment of lysosomal alpha-D-mannosidase to mouse chromosome 8.
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Animals↗
Biomedical subjects
T Beccari
Publications and source records attributed to T Beccari.
Disruption of murine Hexa gene leads to enzymatic deficiency and to neuronal lysosomal storage, similar to that observed in Tay-Sachs disease.
Tay-Sachs disease is an autosomal recessive lysosomal storage disease caused by beta-hexosaminidase A deficiency and leads to death in early childhood. The disease results from mutations in the HEXA gene, which codes for the alpha chain of beta-hexosaminidase. The castastrophic neurodegenerative progression of the disease is thought to be a consequence of massive neuronal accumulation of GM2 ganglioside and related glycolipids in the brain and nervous system of the patients. Fuller understanding of the pathogenesis and the development of therapeutic procedures have both suffered from the lack of an animal model. We have used gene targeting in embryonic stem (ES) cells to disrupt the mouse Hexa gene. Mice homozygous for the disrupted allele mimic several biochemical and histological features of human Tay-Sachs disease. Hexa-/- mice displayed a total deficiency of beta-hexosaminidase A activity, and membranous cytoplasmic inclusions typical of GM2 gangliosidoses were found in the cytoplasm of their neurons. However, while the number of storage neurons increased with age, it remained low compared with that found in human, and no apparent motor or behavioral disorders could be observed. This suggests that the presence of beta-hexosaminidase A is not an absolute requirement of ganglioside degradation in mice. These mice should help us to understand several aspects of the disease as well as the physiological functions of hexosaminidase in mice. They should also provide a valuable animal model in which to test new forms of therapy, and in particular gene delivery into the central nervous system.
Animals↗
Downregulation by cryptococcal polysaccharide of tumor necrosis factor alpha and interleukin-1 beta secretion from human monocytes.
The regulation by Cryptococcus neoformans encapsulation of interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) production by human monocytes was investigated. By using encapsulated and acapsular C. neoformans, we demonstrated that both strains induce cytokine production, although the acapsular strain was a better stimulator than the thinly encapsulated strain. The cytokine levels produced by cells stimulated by the two strains were lower and followed a different kinetic than those stimulated by lipopolysaccharide (LPS). Purified capsular polysaccharide inhibits TNF-alpha secretion induced by LPS or acapsular C. neoformans. In contrast, no regulator effect on IL-1 beta was observed when LPS was used. The secretory response of these cytokines follows different pathways of macrophage activation; in fact, complete inhibition of TNF-alpha does not affect IL-1 beta production and vice versa. These data indicate that purified capsular polysaccharide of C. neoformans could contribute to the in vivo progress of cryptococcosis by suppressing cytokine production of macrophages and suggest that a therapeutic approach to address the suppressive effect of cryptococal polysaccharide could be devised.
Adult↗
GM2 activator protein expression in mouse tissues.
The GM2 activator protein is an essential cofactor of hexosaminidase A in the degradation of GM2 ganglioside and it is responsible for the variant AB of GM2 gangliosidosis in man. In this study GM2 activator protein and its mRNA were determined in different mouse tissues. It was found that this protein is expressed mostly in the spleen and testis followed by brain and kidney which represent the main source in man. It is also interesting that in mouse testis there is a higher expression of the alpha subunit of hexosaminidase, thus suggesting a relationship between alpha subunit and GM2 activator protein. Furthermore the results indicate that the expression of GM2 activator protein is regulated, at least in part, at the transcriptional level.
Animals↗
Different expression of beta-N-acetylhexosaminidase in mouse tissues.
The expression of genes encoding for the alpha and beta-subunits of the lysosomal enzyme beta-N-acetylhexosaminidase was investigated in different mouse tissues. It was found, using fluorogenic substrates, that the amounts of alpha and beta subunits were not the same in different tissues: alpha-subunit was more abundant in the brain, beta-subunit in epididymis and brain. The different isoenzyme patterns and specific activities in mouse tissues are due to the differences in the amount of hexosaminidase subunits. The mRNA, evaluated by Northern blotting analyses, revealed a greater expression of alpha-subunit in the testis and of beta-subunit in the brain and epididymis. The results indicate, therefore, that gene expression and the amount of subunits are in good relationship for beta-subunit, whereas there is no correlation for alpha-subunit.
Animals↗
Organization of the gene for the alpha-subunit of mouse beta-N-acetylhexosaminidase (HEXa).
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).
Animals↗
Beta-N-acetylhexosaminidase in spinal muscular atrophy fibroblasts.
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Cells, Cultured↗
Cloning and sequence analysis of a cDNA clone coding for the mouse GM2 activator protein.
A cDNA (1.1 kb) containing the complete coding sequence for the mouse GM2 activator protein was isolated from a mouse macrophage library using a cDNA for the human protein as a probe. There was a single ATG located 12 bp from the 5' end of the cDNA clone followed by an open reading frame of 579 bp. Northern blot analysis of mouse macrophage RNA showed that there was a single band with a mobility corresponding to a size of 2.3 kb. We deduce from this that the mouse mRNA, in common with the mRNA for the human GM2 activator protein, has a long 3' untranslated sequence of approx. 1.7 kb. Alignment of the mouse and human deduced amino acid sequences showed 68% identity overall and 75% identity for the sequence on the C-terminal side of the first 31 residues, which in the human GM2 activator protein contains the signal peptide. Hydropathicity plots showed great similarity between the mouse and human sequences even in regions of low sequence similarity. There is a single N-glycosylation site in the mouse GM2 activator protein sequence (Asn151-Phe-Thr) which differs in its location from the single site reported in the human GM2 activator protein sequence (Asn63-Val-Thr).
Amino Acid Sequence↗
Glial fibrillary acidic protein and its encoding mRNA exhibit mosaic expression in a glioblastoma multiform cell line of clonal origin.
The expression of two astroglial differentiation markers, vimentin and glial fibrillary acidic protein, was investigated in a previously established human glioma cell line of clonal origin (GL15). Vimentin immunolabelling was homogeneously expressed in all cells. Glial fibrillary acidic protein and its encoding message, investigated by immunocytochemistry and in situ hybridization, showed a mosaic-like expression. Only 30% of the cell population expressed glial fibrillary acidic protein and its mRNA. Western and Northern blots performed for both markers confirmed the presence of both proteins and messages, and their level was correlated with the observed antigenic and molecular probe labelling. The overall antigenic pattern suggests that GL-15 cells do not belong to the O-2A progenitor cell lineage and may arise from a clonal expansion of astrocyte precursors.
Biomarkers↗
Inhibition of candidacidal activity of polymorphonuclear cells by alveolar macrophage-derived factor from lung cancer patients.
Culture supernatants of alveolar macrophages (AM) from lung cancer patients are able to inhibit the candidacidal activity of polymorphonuclear cells (PMN) in vitro. This phenomenon is ascribed to a factor secreted in the culture medium by unstimulated AM from tumor-bearing patients, but not from normal subjects. The inhibitor does not apparently affect the phagocytic activity of PMN, but the superoxide release during phagocytosis is significantly impaired when cells are pretreated with supernatants containing the factor. The secretion of the inhibitor seems to be restricted to the pulmonary compartment of lung cancer patients, since culture supernatants of peripheral blood monocytes (PBM) from the same subjects are not capable of depressing the candidacidal activity of PMN. The AM-derived factor is not inactivated after exposure to heat (60 degrees C) and when supernatants are analyzed by HPLC, the inhibitory activity is recovered in the fractions corresponding to a low molecular weight (800 D). In conclusion, AM from lung cancer patients are able to produce a factor capable of inhibiting the antimicrobial activity of PMN. This could account, at least in part, for the enhanced susceptibility to local infections observed in lung cancer patients.
Biological Factors↗
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↗
Cloning and sequence analysis of a cDNA encoding the alpha-subunit of mouse beta-N-acetylhexosaminidase and comparison with the human enzyme.
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.
Amino Acid Sequence↗
Beta-N-acetylhexosaminidase in the urine, kidney and serum of bromobenzene-intoxicated mice.
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.
Animals↗
Calcium ionophore A-23187 inhibits the secretion of beta-hexosaminidase from the GG2EE mouse macrophage cell line.
Secretion of the lysosomal enzyme beta-N-acetylhexosaminidase is inhibited by calcium ionophore A-23187 in the GG2EE macrophage cell line. Such inhibition is time and dose dependent. Calcium ionophore A-23187 treatment causes a change in the pattern of hexosaminidase isoenzymes detectable in the cell extract, as assessed by DEAE-cellulose chromatography. In particular, control cells show two hexosaminidase isoenzymes corresponding to hexosaminidase A and B, whereas cells treated with calcium ionophore A-23187 express a third isoenzyme form with properties similar to hexosaminidase S.
Animals↗
beta-N-acetylhexosaminidases from Serratia marcescens.
Two forms of beta-N-acetylhexosaminidase from Serratia marcescens with an optimum pH of 5.0 and 6.5, respectively, to 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside were separated by DEAE-cellulose chromatography and Sephacryl S-200 chromatography. On the basis of their molecular weights, thermal stability, substrate specificity and isoelectric points, the form with an acidic pH optimum resembled hexosaminidase B, whereas the form with a neutral pH optimum resembled hexosaminidase C. Lectin binding studies showed that the acidic form does not bind to concanavalin-A-Sepharose, Tetragonolobus purpurea-agarose, wheat germ-agglutinin-Sepharose or Ricinus communis-agglutinin-agarose, whereas the neutral form binds to the last two lectin columns.
Hexosaminidases↗
Analysis of the patterns of expression of mRNAs for the alpha- and beta-subunits of the lysosomal enzyme beta-N-acetylhexosaminidase in mouse epididymis and testis.
mRNA for the alpha- and beta-subunits of mouse beta-N-acetylhexosaminidase were detected in tissue sections of epididymis and testis using 3H-labelled antisense RNA probes by hybridization in situ. There were clear differences in the intensity of the signals given by the two tissues with alpha-subunit mRNA being more abundant in testis than epididymis, while the opposite was so with beta-subunit mRNA. Differences in the isoenzyme patterns of the two tissues are likely to be due, at least in part, to differences in transcription by the Hexa and Hexb genes.
Animals↗
beta-Hexosaminidase expression in chick embryo fibroblasts in vitro.
1. Two forms of beta-hexosaminidase, similar to hexosaminidase A and hexosaminidase C, were separated by DEAE-cellulose chromatography in chick embryo skin fibroblasts in vitro. 2. beta-Hexosaminidase specific activity increases during development in cultured chick embryo skin fibroblasts in vitro. 3. Concanavalin-A treatment determines the increase of the neutral form, hexosaminidase C, during development. 4. Concanavalin-A reduces the specific activity of beta-hexosaminidase during development.
Animals↗
Amphotericin B stimulates secretion of beta-hexosaminidase from mouse adherent spleen cells.
Secretion of the lysosomal enzyme hexosaminidase is induced by amphotericin B in mouse spleen adherent cells that show a significant increase in their candidacidal activity. The stimulation of beta-hexosaminidase is both time and dose dependent. Amphotericin B treatment did not change hexosaminidase expression that is represented mainly by "A-type" hexosaminidase in macrophages, on the basis of its biochemical properties.
Acetylglucosamine↗