BACKGROUND:Newborn screening for lysosomal storage diseases (LSDs) has been gaining considerable interest owing to the availability of enzyme replacement therapies. We present a digital microfluidic platform to perform rapid, multiplexed enzymatic analysis of acid ␣-glucosidase (GAA) and acid ␣-galactosidase to screen for Pompe and Fabry disorders. The results were compared with those obtained using standard fluorometric methods.
Nucleotide-dependent unblocking of chain-terminated DNA by human immunodeficiency virus type 1 reverse transcriptase (RT) is enhanced by the presence of mutations associated with 3-azido-3-deoxythymidine (AZT) resistance. The increase in unblocking activity was greater for mutant combinations associated with higher levels of in vivo AZT resistance. The difference between mutant and wild-type activity was further enhanced by introduction of a methyl group into the nucleotide substrate and was decreased for a nonaromatic substrate, suggesting that -interactions between RT and an aromatic structure may be facilitated by these mutations.Many nucleoside analogues, including 3Ј-azido-3Ј-deoxythymidine (AZT), inhibit human immunodeficiency virus type 1 (HIV-1) replication. The phosphorylated forms of these compounds are incorporated during DNA synthesis by the HIV-1 reverse transcriptase (RT), resulting in chain termination and inhibition of viral replication (7,9,12,22,31,32). Mutations at codons 41, 67, 70, 210, 215, and 219 in the HIV-1 RT gene result in resistance of HIV-1 to AZT in cell culture assays (29). Substitutions of phenylalanine and tyrosine for threonine at position 215 (T215F and T215Y) are the predominant mutations observed in vivo and are considered the most important for the resistance phenotype (16,17,25).The inhibitory effect of incorporating a chain-terminating nucleotide analogue can be partially relieved by a reaction catalyzed by RT in which the terminating nucleotide is removed from the 3Ј end of a DNA chain by transfer to a nucleotide di-or triphosphate, producing an unblocked DNA chain and dinucleoside polyphosphate with the chain terminator linked to the nucleotide acceptor through a tri-or tetraphosphate chain (19,21). HIV-1 RT can also transfer the chain-terminating residue to pyrophosphate (PP i ), regenerating the triphosphate form of the chain terminator (1, 5, 10, 23). These observations have suggested that enhanced removal of 3Ј-azido-3Ј-deoxythymidine-5Ј-monophosphate (AZTMP) is a possible mechanism for AZT resistance, and an increase in the removal reaction has been reported for RT containing various AZT resistance mutations (1,2,18,19). The biochemical contribution of each of these mutations in the removal reaction remains unclear. Boyer et al. (2) have modeled the amino acid substitutions associated with AZT resistance, as well as ATP or PP i , into the three-dimensional structure of HIV-1 RT and concluded that several of these amino acid substitutions could affect the binding of ATP but are unlikely to affect binding of PP i .In this report we describe further investigation into the contributions made by specific mutations in HIV-1 RT to its removal activity and the effects of changes in the structure of the nucleotide substrate on the wild-type (WT) and mutant activities. The mutations associated with AZT resistance identify a region of HIV-1 RT that may interact with the nucleoside moiety of a transition intermediate to facilitate the formation of the dinucleoside tetraphospha...
Background: We used doxorubicin-based chemotherapy as a clinical model of oxidative assault in humans. Methods: The study recruited newly diagnosed breast cancer patients (n = 23). Urine samples were collected immediately before (T0) and at 1 hour (T1) and 24 hours (T24) after i.v. administration of treatment. Measurements included allantoin and the isoprostanes iPF(2α)-III, iPF(2α)-VI, and 8,12-iso-iPF(2α)-VI along with the prostaglandin 2,3-dinor-iPF(2α)-III, a metabolite of iPF(2α)-III. All biomarkers were quantified using liquid chromatography-tandem mass spectrometry.Results: In all subjects, the levels of the biomarkers increased at T1: allantoin by 22% (P = 0.06), iPF(2α)-III by 62% (P < 0.05), iPF(2α)-VI by 41% (P < 0.05), 8,12-iso-iPF(2α)-VI by 58% (P < 0.05), and 2,3-dinor-iPF(2α)-III by 52% (P < 0.05). At T24, the F2-isoprostanes returned to their baseline levels; the levels of allantoin continued to increase, although the T24-T0 difference was not statistically significant.Conclusions: These results indicate that urinary F2-isoprostanes are valid biomarkers and allantoin is a promising biomarker of oxidative status in humans.Impact: The levels of biomarkers change quickly in response to oxidative assault and can be used to monitor oxidative status in humans in response to treatments related either to generation of free radicals (chemotherapy and radiation therapy) or to antioxidants (inborn metabolic diseases and Down syndrome). Cancer
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