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N McLellan

Publications and source records attributed to N McLellan.

5 recordsLinked to original sources
PubMed2002

Nonradioactive detection of retroviral-associated RNase H activity in a microplate-based, high-throughput format.

None of the available antiretroviral drugs that are currently used in the clinic to treat infection with HIV-1 is directed against the RNase H active site of the reverse transcriptase. Here we developed a nonradioactive, 96-well plate assay designed to be used for high-throughput screening of compounds capable of inhibiting the RNase H activity of HIV-1 reverse transcriptase. We employed a tRNA as substrate that was labeled with digoxygenin-modified reporter residues. The labeled tRNA was prehybridized with a DNA oligonucleotide that contained a single biotinylated residue at its 5'-terminus to ensure its attachment to streptavidin-coated microplates. The uncleaved, immobilized DNA/tRNA substrate was detected through the use of established ELISA protocols. Incubation with purified HIV-1 reverse transcriptase initiated RNase H degradation and caused a signal reduction to negligible background levels. In contrast, the signal intensity remained unaffected when using an RNase H deficient mutant enzyme. The assay was validated using the hydrazone derivative BBNH that was previously shown to inhibit RNase H degradation below concentrations of 10 microM.

Bacteriological Techniques↗
PubMed2000

Analysis of efficiency and fidelity of HIV-1 (+)-strand DNA synthesis reveals a novel rate-limiting step during retroviral reverse transcription.

We have analyzed the efficiency and accuracy of polymerization at several different stages during the initiation of human immunodeficiency virus type 1 (HIV-1) (+)-strand DNA synthesis. This reaction is of particular interest, as it involves the recruitment by reverse transcriptase of an RNA primer that serves as substrate for both the polymerase and RNase H activities of the enzyme. We found that the correct incorporation of the first two nucleotides was severely compromised and that formation of mismatches was completely absent at this stage of initiation. Although the fidelity of incorporations decreased concomitantly with ensuing polymerization, the elongation of mispaired primers was literally blocked. Instead, mispaired primer strands initiated a switch from active synthesis of DNA to premature RNase H-mediated primer removal. These findings suggest the existence of a fragile equilibrium between these two enzymatic activities that is shifted toward RNase H cleavage once the polymerization process is aggravated. Our data show that the initiation of HIV-1 (+)-strand DNA synthesis differs significantly from reactions involving other primer/template combinations, including tRNA-primed (-)-strand DNA synthesis.

Base Pair Mismatch↗
PubMed1989

Are severe acute retinopathy of prematurity and severe periventricular leucomalacia both ischaemic insults?

Over a period of 20 months six preterm infants have been seen who developed severe acute retinopathy of prematurity (ROP) and who also had ultrasound evidence of extensive cerebral parenchymal changes compatible with severe periventricular leucomalacia. Only one of these infants had a birthweight of less than 1000 g, and their gestational ages ranged from 27 to 30 weeks. The association between these two important complications of preterm birth has led us to postulate that an episode of hypoperfusion of the cerebral circulation sufficient to result in cerebral ischaemia could also reduce an already compromised ocular blood flow and further exacerbate retinal ischaemia, thereby increasing the severity of ROP.

Brain Ischemia↗
PubMed1983

Microcomputer organization of parenteral nutrition in the neonate.

The use of a microcomputer is described to simplify the organization of neonatal parenteral feeding and extend the flexibility of the technique, providing a hard copy prescription and regime analysis for each patient. Its use can save medical and pharmacy time and conserve expensive reagents. The programme is readily adaptable to different feeding policies and the system is simple, works well in practice and is cheap to run.

Amino Acids↗