ABSTRACT
Flavonoid and limonoid glycosides influence taste properties as well as marketability of Citrus fruit and products, particularly grapefruit. In this work, nine grapefruit putative natural product glucosyltransferases (PGTs) were resolved by either using degenerate primers against the semiconserved PSPG box motif, SMART-RACE RT-PCR, and primer walking to full-length coding regions; screening a directionally cloned young grapefruit leaf EST library; designing primers against sequences from other Citrus species; or identifying PGTs from Citrus contigs in the harvEST database. The PGT proteins associated with the identified full-length coding regions were recombinantly expressed in Escherichia coli and/or Pichia pastoris and then tested for activity with a suite of substrates including flavonoid, simple phenolic, coumarin, and/or limonoid compounds. A number of these compounds were eliminated from the predicted and/or potential substrate pool for the identified PGTs. Enzyme activity was detected in some instances with quercetin and catechol glucosyltransferase activities having been identified.
Subject(s)
Citrus paradisi/enzymology , Glucosyltransferases/analysis , Glucosyltransferases/genetics , Recombinant Proteins/genetics , Amino Acid Sequence , Coumarins/metabolism , Escherichia coli/metabolism , Flavonoids/metabolism , Gene Expression , Genes, Plant/genetics , Limonins/metabolism , Molecular Sequence Data , Phenols/metabolism , Phylogeny , Pichia/metabolism , Seeds/enzymology , Sequence Alignment , Substrate SpecificityABSTRACT
Athletic injuries of the foot and lower extremity are commonly treated with custom foot orthoses. These devices usually provide immediate relief of the athlete's pain and dysfunction. Occasionally, however, they do not help, or even increase the patient's discomfort. We discuss a method of using in-shoe pressure-measurement systems to analyze the athletic patient's foot and lower-extremity function before and after treatment with custom foot orthoses, with a focus on sagittal plane biomechanics. Case histories are presented of athletes whose gait pathologies were identified and treated successfully using an in-shoe pressure-measurement system.
