David I. Fisher

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A total of 17 Nudix hydrolases were tested for their ability to hydrolyze 5-phosphoribosyl 1-pyrophosphate (PRPP). All 11 enzymes that were active toward dinucleoside polyphosphates with 4 or more phosphate groups as substrates were also able to hydrolyze PRPP, whereas the 6 that could not and that have coenzyme A, NDP-sugars, or pyridine nucleotides as(More)
ATM (ataxia-telangiectasia mutated) is a phosphatidylinositol 3-kinase-related protein kinase (PIKK) best known for its role in DNA damage response. ATM also functions in oxidative stress response, insulin signaling, and neurogenesis. Our electron cryomicroscopy (cryo-EM) suggests that human ATM is in a dynamic equilibrium between closed and open dimers. In(More)
Nudix hydrolases form a protein family whose function is to hydrolyse intracellular nucleotides and so regulate their levels and eliminate potentially toxic derivatives. The genome of the radioresistant bacterium Deinococcus radiodurans encodes 25 nudix hydrolases, an unexpectedly large number. These may contribute to radioresistance by removing mutagenic(More)
Transient expression of heterologous proteins in mammalian systems is a powerful way to generate protein reagents quickly. However, it has historically suffered from poor yields in comparison to methods where the recombinant gene is stably integrated into the genome and high expressing clones isolated. Transient methods have been well described for(More)
The DR2356 nudix hydrolase gene from Deinococcus radiodurans has been cloned and the product expressed as an 18 kDa histidine-tagged protein. The enzyme hydrolysed adenosine and diadenosine polyphosphates, always generating ATP as one of the initial products. ATP and other (deoxy)nucleoside triphosphates were also substrates, yielding (d)NDP and Pi as(More)
This study investigated the use of large-scale transiently transfected cryopreserved cells for medium-throughput cellular screening. The data generated indicated that preprepared transiently transfected cryobanks can be used for cell-based assays and in fact can greatly enhance the consistency of data generated by cellular screens. In addition to this, a(More)
The pro-apoptotic Fhit tumor suppressor protein binds and hydrolyses diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) in vitro. We have measured the level of both these nucleotides in Fhit-positive HEK293 cells exposed to various apoptosis inducers. Cold shock, anti-Fas, cadmium ions and etoposide all increased the basal level of Ap4A(More)
The fatty acid synthase (FAS) enzyme in mammalian cells is a large multidomain protein responsible for de novo synthesis of fatty acids. The steps catalyzed by FAS involve the condensation of acetyl-CoA and malonyl-CoA moieties in the presence of NADPH until palmitate is formed. Inhibition of FAS causes an accumulation of intracellular malonyl-CoA, as this(More)
Certain recombinant proteins are deemed "difficult to express" in mammalian expression systems requiring significant cell and/or process engineering to abrogate expression bottlenecks. With increasing demand for the production of recombinant proteins in mammalian cells, low protein yields can have significant consequences for industrial processes. To(More)
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