<b>OBJECTIVEb>To study the molecular mechanism of GM(2) gangliosidosis.

<b>METHODSb>The skin fibroblasts from 4 patients with GM(2) gangliosidosis were subjected to culture. Enzyme activities assay, Western blot and immunocytochemical analysis were performed using the cultured fibroblasts.

<b>RESULTSb>The hexosaminidase (Hex) activities of 4 patients with GM(2) gangliosidosis were significantly decreased. The activities were 12% 3% 15% and 6% of control values, respectively. Western blot analysis indicated that the amount of Hex mature alpha- and beta- subunits (alpha m, beta m) was decreased in cells from patients 2 and 3, but only decreased alpha m was found in patient 1 and both alpha m and beta m were normal in cells from patient 4. Immunocytochemical analysis revealed the accumulated GM(2) ganglioside in cells from patients 1-4.

<b>CONCLUSIONb>The pathogenesis of GM(2) gangliosidosis was associated with deficiency of Hex alpha m and beta m and GM(2) activator caused by HEXA, HEXB and GM(2)A gene mutations.

Adult ; Blotting, Western ; Cells, Cultured ; Child, Preschool ; Female ; Gangliosidoses, GM2 ; enzymology ; pathology ; Hexosaminidase A ; Hexosaminidase B ; Humans ; Infant ; Male ; Protein Subunits ; metabolism ; beta-N-Acetylhexosaminidases ; metabolism

Tay-Sachs disease is an autosomal recessive, neurodegenerative disorder that results from excessive storage of the cell membrane glycolipid, and GM2 ganglioside within the lysosomes of cells. This disease is caused by deficiency of the isoenzyme beta-hexosaminidase A, produced in the endoplasmic reticulum. Patients with Tay-Sachs disease are characterized by normal motor development in the first few months of life, followed by progressive weakness and loss of motor skills beginning around 2 to 6 months of life. Neurodegeneration is relentless, with death occurring by the age of 4 or 5 years. Tay-Sachs disease could be diagnosed by hexosaminidase enzyme assay and DNA analysis of HEXA gene. However, specific treatment has not been developed. We report here on a case of Tay- Sachs disease in 18-month-old male who presented with delayed development and seizure. This patient showed hyperacusis and cherry red spot in macula on examination of the fundus. The hexosaminidase A activity was zero percent in the enzymatic assay and DNA analysis identified a mutation that glutamine is substituted by stop codon at position 390(Q390X). This patient is the first case of Tay-Sachs disease in Korea diagnosed by enzymatic assay and DNA analysis.
beta-N-Acetylhexosaminidases ; Cell Membrane ; Codon, Terminator ; DNA* ; Endoplasmic Reticulum ; Enzyme Assays* ; G(M2) Ganglioside ; Glutamine ; Hexosaminidase A ; Hexosaminidases ; Humans ; Hyperacusis ; Infant ; Korea* ; Lysosomes ; Male ; Motor Skills ; Neurodegenerative Diseases ; Prunus ; Seizures ; Tay-Sachs Disease*

GM2 gangliosidosis II(Sandhoff disease) is a lysosomal storage disease due to deficiency of beta-hexosaminidase activity, transmitted by mode of autosomal recessive. Clinical features are so variable, ranging from infantile onset resulting death before 4 years, to subacute or chronic forms with more slowly progressive neurologic condition. We experienced a case of GM2 gangliosidosis II in a 14 months old male who had developmental deterioration and seizures, so we report and review the related literatures.
beta-N-Acetylhexosaminidases ; Gangliosidoses, GM2* ; Hexosaminidases ; Humans ; Infant ; Lysosomal Storage Diseases ; Male ; Seizures

The effects of extremely low frequency electromagnetic fields (EMF) on intracellular Ca2+ mobilization and cellular function in RBL 2H3 cells were investigated. Exposure to EMF (60 Hz, 0.1 or 1 mT) for 4 or 16 h did not produce any cytotoxic effects in RBL 2H3 cells. Melittin, ionomycin and thapsigargin each dose-dependently increased the intracellular Ca2+ concentration. The increase of intracellular Ca2+ induced by these three agents was not affected by exposure to EMF (60 Hz, 1 mT) for 4 or 16 h in RBL 2H3 cells. To investigate the effect of EMF on exocytosis, we measured beta-hexosaminidase release in RBL 2H3 cells. Basal release of beta-hexosaminidase was 12.3+/-2.3% in RBL 2H3 cells. Exposure to EMF (60 Hz, 0.1 or 1 mT) for 4 or 16 h did not affect the basal or 1 microM melittin-induced beta-hexosaminidase release in RBL 2H3 cells. This study suggests that exposure to EMF (60 Hz, 0.1 or 1 mT), which is the limit of occupational exposure, has no influence on intracellular Ca2+ mobilization and cellular function in RBL 2H3 cells.
beta-N-Acetylhexosaminidases ; Electromagnetic Fields ; Exocytosis ; Ionomycin ; Melitten ; Occupational Exposure ; Thapsigargin

<b>OBJECTIVEb>To explore the clinical features and molecular mutation of HEXB gene in a case with juvenile Sandhoff disease.

<b>METHODb>We retrospectively reviewed the clinical, neuroimaging and biochemical findings in this Chinese child with juvenile Sandhoff disease. Hexosaminidase A and hexosaminidase A & B activities were measured in blood leukocytes by fluorometric assay. HEXB gene molecular analysis was performed by PCR and direct sequencing.

<b>RESULTb>The 9-year-old boy was admitted for psychomotor regression. He presented slowly progressive gait disorder and dysarthria during the last three years. Cranial MRI revealed a marked cerebellar atrophy with normal intensity in the thalamus and basal ganglia. Brain MRS showed normal in the thalamus and basal ganglia. Hexosaminidase A was 69.5 (mg·h) [normal controls 150-360 nmol/(mg·h)], hexosaminidase A & B activity was 119 nmol/(mg·h)[normal controls 600-3 500 nmol/(mg·h)], confirming the diagnosis of Sandhoff disease. The patient was a compound heterozygote for a novel deletion mutation c.1404delT (p. P468P fsX62) and a reported mutation c.1509-26G>A.

<b>CONCLUSIONb>The clinical features of juvenile Sandhoff disease include ataxia, dysarthria and cerebellar atrophy. The enzyme assay and molecular analysis of HEXB gene can confirm the diagnosis of Sandhoff disease. The novel mutation c.1404delT(p. P468P fsX62) is a disease-related mutation.

Brain ; diagnostic imaging ; pathology ; Cerebellar Ataxia ; diagnosis ; enzymology ; genetics ; Child ; DNA Mutational Analysis ; Heterozygote ; Hexosaminidase A ; blood ; metabolism ; Hexosaminidase B ; blood ; metabolism ; Humans ; Leukocytes ; enzymology ; Magnetic Resonance Imaging ; Male ; Mutation ; Radiography ; Retrospective Studies ; Sandhoff Disease ; diagnosis ; enzymology ; genetics ; beta-Hexosaminidase beta Chain ; genetics

In order to probe the biological function of O-GlcNAc and the pathogenesis of associated diseases, it is essential to prepare a potent and specific O-GlcNAcase (OGA) antibody. Based on protein sequence analysis, we found N terminal 1-350 amino acids of OGA (sOGA) has high antigenicity and hydrophilicity and then constructed it into plasmid pET28a vector. First, we optimized the expression of sOGA in Escherichia coli BL21(DE3) (0.05 mmol/L IPTG, 10 hours) and purified it with the Ni-NTA affinity chromatography and size exclusion chromatography respectively. SDS-PAGE verified the molecular weight (45 kDa) and the purity (>95%) of sOGA and the purified protein was subjected to immunize New Zealand rabbits. Finally, we obtained OGA polyclonal antibody by affinity purifying the antiserum with CNBr-activated Sepharose 4B beads. Western blotting and ELISA assay showed that this antibody could recognize three OGA isoforms with high specificity and the sensitivity was 0.11 ng/mL (the titer was 1:80 000). These results indicated the prepared polyclonal antibody of OGA can be used for the biological function study of OGA.
Animals ; Antibodies ; metabolism ; Antigen Presentation ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Immunization ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics ; beta-N-Acetylhexosaminidases ; biosynthesis ; genetics ; immunology

The present study was to investigate anti-oxidative and anti-inflammatory activity of Perillae semen in RBL-2H3 basophilic leukemia cells. Inhibitory effect of Perillae semen onto free radical generation was determined by measuring DPPH and hydroxyl radical scavenging activities in vitro. Anti-inflammatory actions of Perillae semen extracts (100, 250, 500 microgram/mL) were assessed by testing their effects on the degranulation of mast cells. For this, beta-hexosaminidase released from RBL-2H3 cells was used and proinflammatory cytokines were measured by an ELISA kit. Our results indicated that Perillae semen water extracts effectively inhibited free radical generation. At the concentration of 500 microgram/mL of water extract, the degranulation of RBL-2H3 cells were inhibited by 42.1%. The IgE-antigen complex increased the accumulation of IL-4 and TNF-alpha secretion in RBL-2H3 cells and treatments with 250 and 500 microgram/mL of Perillae semen extracts suppressed the IgE induced secretion of IL-4 and TNF-alpha protein by 20.5, 26.9% and 14.5, 16.5% respectively. We observed that Perillae semen water extract reduced beta-hexosaminidase, IL-4, and TNF-alpha secretion in RBL-2H3 cells. These results provide that Perillae semen may be beneficial in the treatment of allergic inflammatory disease.
Basophils ; beta-N-Acetylhexosaminidases ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; Hydroxyl Radical ; Immunoglobulin E ; Interleukin-4 ; Leukemia ; Mast Cells ; Perilla ; Semen ; Tumor Necrosis Factor-alpha ; Water

Siglecs are sialic acid binding Ig-like lectins, subset of the immunoglobulin superfamily. They are characterized by a homologous N-terminal V-set Ig-like domain and C2 set Ig-like domains. N-terminal domains have sialic acid binding activity. In humans, 11 Siglecs have been described sialoadhesin(Siglec-1), CD22(Siglec-2), CD33(Siglec-3), MAG(Siglec-4), more recently described CD33-related Siglecs(Siglec 5-11). Siglecs express most signal via immunoreceptor tyrosine-based inhibition motif(ITIM) cytoplasmic domains. The cytoplasmic tails of all Siglecs except sialoadhesin have one or more tyrosine residues within potential signaling motifs. Inhibitory function of other Siglecs such as Siglec-7 or Siglec-9 was shown in RBL-2H3 cells. Co-crosslinking of Siglec-7 or Siglec-9 and Fc epsilon R1 substantially reduced the serotonin release of RBL-7 and RBL-9 cells. Siglec-8 is expressed on human eosinophils, mast cells and basophils. Siglec-8 has two tyrosine motifs, a proximal motif and a distal motif. They have some inhibitory functions in immune system. We have observed that Siglec-8 is able to inhibit the IgE receptor-mediated beta-hexosaminidase release of RBL-2H3 cells following co-crosslinking. Co-crosslinking of Siglec-8 and Fc epsilon R1 reduced the hexosaminidase release of RBL-2H3 cells. These results show that Siglec-8 is as potent as Siglec-7 and Siglec-9 in delivering inhibitory signals to RBL-2H3 cells. Siglec-8 should be a new member of the inhibitory receptor superfamily and the membrane-proximal ITIM is essential for the inhibitory function of Siglec-8 molecules. Although these molecules present specific marker for the allergic cell types, more work is needed to understand the signaling mechanism and the role in various disease processes.
Basophils ; beta-N-Acetylhexosaminidases ; Cytoplasm ; Eosinophils ; Hexosaminidases ; Humans ; Immune System ; Immunoglobulin E ; Immunoglobulins ; Inflammation* ; Lectins ; Mast Cells ; N-Acetylneuraminic Acid ; Serotonin ; Sialic Acid Binding Ig-like Lectin 1 ; Sialic Acid Binding Immunoglobulin-like Lectins* ; Tyrosine

OBJECTIVE: To detect potential mutations of HEXB gene in an infant with Sandhoff disease (SD). METHODS: Genomic DNA was extracted from peripheral blood sample of the infant. All coding exons (exons 1 to 14) and splicing sites of the HEXB gene were subjected to PCR amplification and direct sequencing.PubMed Protein BLAST system was employed to analyze cross-species conservation of the mutant amino acid. PubMed BLAST CD-search was performed to identify functional domains destroyed by thecandidate mutations. Impact of the mutations was analyzed with software including PolyPhen-2, Mutation Taster and SIFT. Whole-exome sequencing was carried out to identify additional mutations. RESULTS: The infant was found to carry compound heterozygous mutations c.1652G>A(p.Cys551Tyr) and c.1389C>G (p.Tyr463*) of the HEXB gene. The c.1389C>G (p.Tyr463*) mutation may lead to destruction of two functional domains in β subunit of the Hex protein. The c.1652G>A(p.Cys551Tyr) mutation, unreported previously,was predicted to be probably damaging by Bioinformatic analysis. CONCLUSION Compound heterozygous mutations c.1652G>A(p.Cys551Tyr) and c.1389C>G (p.Tyr463*) in the HEXB gene probably underlie the disease in this patient.
DNA Mutational Analysis ; Exons ; Heterozygote ; Humans ; Infant ; Mutation ; Polymerase Chain Reaction ; Sandhoff Disease ; genetics ; beta-Hexosaminidase beta Chain ; genetics

BACKGROUND: IgE is closely related to the development of allergies. However, the poor relationship between the specific IgE level and the severity of allergic diseases suggests the possibility of functionally different IgE isoforms. With this in mind, rat basophilic leukemia (RBL)-2H3 activation was analyzed with each type of rat IgE for two parameters, exocytosis and IL-4 mRNA production. RBL-2H3 has been well documented in the rat mucosal mast cell line. METHODS: RBL-2H3 cells sensitized with each kind of rat IgE was activated by cross-linking FcRI with B5 (monoclonal anti-rat IgE mouse IgG antibodies). The RBL-2H3 exocytosis was measured by analyzing the beta-hexosaminidase level, and the level of IL-4 mRNA synthesis was analyzed using semi- quantitative RT-PCR. Rat IgE, which was produced by a parasite infection (REP), was prepared using either Paragonimus westermani metacercariae (REP-PW) or Anisakis simplex third stage larvae (REP-AS). A rat IgE prototype of IR162 was prepared by a peritoneal injection of immunocytoma. RESULTS: The level of exocytosis showed a linear relationship with the rat IgE concentration when REP-PW or REP-AS was applied. However, it exhibited a biphasic response with IR162. In addition, the time course of heating at 56oC illustrated the similarity between REP-PW and REP-AS, which differed from that of IR162. In contrast, the level of IL-4 mRNA synthesis in the RBL-2H3 cells with IR162 was comparable to that of either REP-PW or REP-AS. CONCLUSION: These results suggest that functionally different rat IgE isoforms exists in RBL-2H3 exocytosis.
Animals ; Anisakis ; Basophils ; beta-N-Acetylhexosaminidases ; Exocytosis* ; Heating ; Hot Temperature ; Hypersensitivity ; Immunoglobulin E* ; Immunoglobulin G ; Interleukin-4 ; Larva ; Leukemia ; Mast Cells ; Metacercariae ; Mice ; Paragonimus westermani ; Parasites ; Protein Isoforms ; Rats* ; RNA, Messenger