Sheldon M. Schuster

ADVERTISEMENT RETURN TO ISSUEPREVCommunicationNEXTDesigning a Novel Molecular Beacon for Surface-Immobilized DNA Hybridization StudiesXiaohong Fang, Xiaojing Liu, Sheldon Schuster, and Weihong TanView Author Information Department of Chemistry and UF Brain Institute University of Florida, Gainesville, Florida 32601 Cite this: J. Am. Chem. Soc. 1999, 121, 12, 2921–2922Publication Date (Web):March 11, 1999Publication History Received29 October 1998Published online11 March 1999Published inissue 1 March 1999https://pubs.acs.org/doi/10.1021/ja9837809https://doi.org/10.1021/ja9837809rapid-communicationACS PublicationsCopyright © 1999 American Chemical SocietyRequest reuse permissionsArticle Views2526Altmetric-Citations210LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose SUBJECTS:Fluorescence,Genetics,Hybridization,Imaging probes,Molecules Get e-Alerts

Asparagine synthetase B catalyzes the assembly of asparagine from aspartate, Mg(2+)ATP, and glutamine. Here, we describe the three-dimensional structure of the enzyme from Escherichia colidetermined and refined to 2.0 A resolution. Protein employed for this study was that of a site-directed mutant protein, Cys1Ala. Large crystals were grown in the presence of both glutamine and AMP. Each subunit of the dimeric protein folds into two distinct domains. The N-terminal region contains two layers of antiparallel beta-sheet with each layer containing six strands. Wedged between these layers of sheet is the active site responsible for the hydrolysis of glutamine. Key side chains employed for positioning the glutamine substrate within the binding pocket include Arg 49, Asn 74, Glu 76, and Asp 98. The C-terminal domain, responsible for the binding of both Mg(2+)ATP and aspartate, is dominated by a five-stranded parallel beta-sheet flanked on either side by alpha-helices. The AMP moiety is anchored to the protein via hydrogen bonds with O(gamma) of Ser 346 and the backbone carbonyl and amide groups of Val 272, Leu 232, and Gly 347. As observed for other amidotransferases, the two active sites are connected by a tunnel lined primarily with backbone atoms and hydrophobic and nonpolar amino acid residues. Strikingly, the three-dimensional architecture of the N-terminal domain of asparagine synthetase B is similar to that observed for glutamine phosphoribosylpyrophosphate amidotransferase while the molecular motif of the C-domain is reminiscent to that observed for GMP synthetase.

Through clever design of an oligonucleotide probe, the molecular beacon (MB), protein – DNA interactions can be studied and proteins quantified. The principle involves a design in which the fluorophore (F) of the MB is quenched by intramolecular interaction with a quenching group (Q) in close proximity. This quenching is relieved by the interaction of the molecular beacon with a single-stranded DNA binding protein (SSB). As a result, fluorescence is restored and can be monitored. This approach could be developed into a powerful method for directly monitoring protein production in living cells and organs.

Summary We examined the effectiveness of various anti‐tumour agents to natural killer (NK)‐cell tumour cell lines and samples, which are generally resistant to chemotherapy, using flow cytometric terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end‐labelling (TUNEL) assay. Although NK‐YS and NK‐92 were highly resistant to various anti‐tumour agents, l ‐asparaginase induced apoptosis in these two NK‐cell lines. NK‐cell leukaemia/lymphoma and acute lymphoblastic leukaemia (ALL) samples were selectively sensitive to l ‐asparaginase and to doxorubicin (DXR) respectively. Samples of chronic NK lymphocytosis, an NK‐cell disorder with an indolent clinical course, were resistant to both drugs. Our study clearly separated two major categories of NK‐cell disorders and ALL according to the sensitivity to DXR and l ‐asparaginase. We examined asparagine synthetase levels by real‐time quantitative polymerase chain reaction (RQ‐PCR) and immunostaining in these samples. At least in nasal‐type NK‐cell lymphoma, there was a good correlation among asparagine synthetase expression, in vitro sensitivity and clinical response to l ‐asparaginase. In aggressive NK‐cell leukaemia, although asparagine synthetase expression was high at both mRNA and protein levels, l ‐asparaginase induced considerable apoptosis. Furthermore, samples of each disease entity occupied a distinct area in two‐dimensional plotting with asparagine synthetase mRNA level (RQ‐PCR) and in vitro l ‐asparaginase sensitivity (TUNEL assay). We confirmed rather specific anti‐tumour activity of l ‐asparaginase against NK‐cell tumours in vitro , which provides an experimental background to the clinical use of l ‐asparaginase for NK‐cell tumours.