In Cellulo Examination of a Beta-Alpha Hybrid Construct of Beta-Hexosaminidase A Subunits,Reported to Interact with the GM2 Activator Protein and Hydrolyze GM2 Ganglioside

The hydrolysis in lysosomes of GM2 ganglioside to GM3 ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.C. # 3.2.1.52 (encoded by the HEXA and HEXB genes, respectively), and the GM2-activator protein (GM2AP, encoded by the GM2A gene). Mutations in any one of these genes can result in one of three neurodegenerative diseases collectively known as GM2 gangliosidosis (HEXA, Tay-Sachs disease, MIM # 272800; HEXB, Sandhoff disease, MIM # 268800; and GM2A, AB-variant form, MIM # 272750). Elements of both of the hexosaminidase A subunits are needed to productively interact with the GM2 ganglioside-GM2AP complex in the lysosome. Some of these elements have been predicted from the crystal structures of hexosaminidase and the activator. Recently a hybrid of the two subunits has been constructed and reported to be capable of forming homodimers that can perform this reaction in vivo, which could greatly simplify vector-mediated gene transfer approaches for Tay-Sachs or Sandhoff diseases. A cDNA encoding a hybrid hexosaminidase subunit capable of dimerizing and hydrolyzing GM2 ganglioside could be incorporated into a single vector, whereas packaging both subunits of hexosaminidase A into vectors, such as adeno-associated virus, would be impractical due to size constraints. In this report we examine the previously published hybrid construct (H1) and a new more extensive hybrid (H2), with our documented in cellulo (live cell- based) assay utilizing a fluorescent GM2 ganglioside derivative. Unfortunately when Tay-Sachs cells were transfected with either the H1 or H2 hybrid construct and then were fed the GM2 derivative, no significant increase in its turnover was detected. In vitro assays with the isolated H1 or H2 homodimers confirmed that neither was capable of human GM2AP-dependent hydrolysis of GM2 ganglioside.     

Changes in Compositional Properties during Fruit Development and On-Tree Ripening of Two Common Apricot (Prunus armeniaca L.) Cultivars

The objective of the present study was to investigate the variations in some major primary (sugars and organic acids) and secondary (phenolics, β-carotene) metabolite contents during fruit development and ripening in two important apricot cultivars (Hacıhaliloğlu and Kabaaşı). The changes in the compositional properties of two apricot cultivars were monitored during fruit development with one-week intervals from 56 to 119 days after blossom. During fruit development, the contents of organic acids and phenolics decreased whereas that of sucrose and sorbitol increased. p-Coumaric acid was the only phenolic compound which increased in concentration during fruit development regardless of the cultivar. The content of the other phenolic compounds decreased in a cultivar-dependent manner. The β-carotene content of the cultivars showed distinct patterns of change such that 3 fold increase in β-carotene content of Kabaaşı cultivar was observed whereas the β-carotene content of the Hacıhaliloğlu cultivar did not show any significant change during fruit development.