GM$ sb2$ ganglioside hydrolysis requires the formation of a ternary complex consisting of substrate, enzyme ($ beta$-N-acetylhexosaminidase A = Hex A), and the GM$ sb2$ activator protein. In order to study the interaction between Hex A and GM$ sb2$ activator, the human GM$ sb2$ activator cDNA was cloned into the p-FLAG vector. The fusion protein (FLAG-AP) was expressed in E. coli, and purified to homogeneity using immunoaffinity chromatography. / A retardation assay was designed using the immunoaffinity column to detect transient interactions between FLAG-AP and Hex A. Hex A and Hex S are retarded by the column, but not Hex B or unrelated proteins. Hex A retardation is absolutely dependent upon the presence of immobilized FLAG-AP, but does not require the presence of GM$ sb2$ ganglioside. Interaction of GM$ sb2$ activator and Hex A does not involve the enzyme's active site, but does appear to depend upon hydrophobic interactions between the two proteins.

No description available.

No description available.

This thesis describes a search for DNA polymorphisms at the HEXA locus as well as the characterization of three Tay-Sachs disease (TSD) mutations in the Chinese population. / No polymorphisms were detected in the HEXA gene by three different methods: (1) Southern blotting; (2) PCR amplification and restriction enzyme digestion of intronic sequences; (3) single strand conformational polymorphism (SSCP) analysis of introns. The apparent deficiency of accessible polymorphisms is a handicap in studying the origin, distribution, and frequency of mutant HEXA alleles in human populations. / I have characterized five of six infantile TSD alleles segregating in three unrelated Chinese families in which there is no known consanguinity. Two of the mutations described are novel, the third is a transition previously reported in an Italian patient (Nakano et al, 1988). / The two novel mutations occur in homozygous form in the affected individuals investigated. They are: (1) an insertion of an A at nucleotide 547 (Family 1) and (2) a T1453C transition (Family 2). (Abstract shortened by UMI.)

Thesis (Ph.D.)--Tufts University, 2001. / Adviser: Andrew McClellan. Submitted to the Dept. of Interdisciplinary Studies. Includes bibliographical references (leaves 492-531). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Thesis (Ph. D.)--John Hopkins University, 1911. / Vita. "Chapter I. Various views on the meter of Hans Sachs. Chapter II. The development of the Knittelvers up to the time of Hans Sachs." "The two remaining chapters of this treatise will appear in 'Hesperai, Schriften zur germanischen philologie, nr. 2 ... together with these two first."

No description available.

Note:

Note:

Tay-Sachs disease and its related disorders (GM2 gangliosidoses) are neurodegenerative diseases caused by the excessive accumulation of ganglioside GM2, an otherwise non-toxic plasma membrane component, in the lysosomes of cells of the central nervous system. The accumulation of ganglioside GM2 is the result of a defect in the gene encoding the α-subunit of β-hexosaminidase A (Hex A), an acid hydrolase responsible for the metabolism of gangloside GM2 in the lysosome. Though a debilitating disease in humans, Tay-Sachs model mice (𝘏𝘦𝘹𝘢-/-) escape symptoms by the action of lysosomal sialidase, which is expressed in mice at levels sufficient to metabolize ganglioside GM2 and effectively "bypass" Hex A deficiency. In an attempt to understand why a lysosomal sialidase-mediated bypass of Hex A deficiency is not observed in humans, we cloned ~ 2.9 kb of the human lysosomal sialdiase promoter and began characterization of the regulatory machinery that determines its activity. The transcription factor CDP (CCAAT -Displacement Protein) and truncations thereof were found to have a clear and consistent effect on promoter activity 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰, with the truncation CDP⁸³¹⁻¹⁵⁰⁵ resulting in a near 50-fold increase in activity. Adenovirus-mediated gene transfer of CDP⁸³¹⁻¹⁵⁰⁵ into CRB/TSD cells, a human Tay-Sachs neuroglia cell line, resulted in elevated lysosomal sialidase activity and a decrease in ganglioside GM2 stores. These results suggest that the regulatory machinery responsible for lysosomal sialidase expression may be manipulated in such a way as to "activate" a sialidase-mediated bypass of Hex A deficiency in human Tay-Sachs cells. Thus, induction of lysosomal sialidase may have a potential therapeutic benefit in human Tay-Sachs disease and other Hex A-deficient GM2 gangliosidoses. / Thesis / Master of Science (MS)