Reversible leukoencephalopathy associated with re-infusion of DMSO preserved stem cells.

We report a case of posterior reversible leuko- encephalopathy (PRL) following the infusion of dimethylsulfoxide (DMSO) cryopreserved autologous stem cells in the setting of myeloablative chemotherapy in a patient with recurrent Ewing's sarcoma. Magnetic resonance (MR) imaging revealed white matter changes which resolved over the next 2 months. Bone Marrow Transplantation (2000) 26, 797-800.

Direct photolinkage of GTP to the vaccinia virus mRNA (guanine-7-) methyltransferase GTP methyl acceptor site.

Direct UV photolinkage of [alpha-32P]GTP to the methyl acceptor site of the vaccinia virus (guanine-7-) methyltransferase was attempted in order to identify the GTP binding region of this enzyme. Low-efficiency photolinkage of GTP to the carboxyl terminal domain of the large subunit, D1R498-844, was achieved and shown to be specific by several criteria. The half-saturation value for GTP was determined to be 35 microM which is equivalent to the catalytic Km for the substrate. GTP photolinkage was shown to be inhibited by GpppA, a substrate for the methyltransferase reaction, better than GMepppA, the reaction product. The addition of MgCl2, known to prevent GTP from serving as a methyl group acceptor in this reaction, was found to eliminate GTP photolinkage. Finally, AdoHcy, a potent product inhibitor of AdoMet binding, failed to inhibit GTP photolinkage, demonstrating that GTP was not linked to the AdoMet binding site. Chemical cleavage of the GTP-labeled enzyme permitted the identification of multiple radioactive peptides, demonstrating the existence of multiple interaction sites in the carboxyl terminal domain of the D1R subunit. The addition of the small D12L subunit has been shown to activate the (guanine-7-) methyltransferase activity in D1R498-844 30-50-fold. The efficiency of GTP photolinkage to the isolated D1R498-844 domain, however, was found to be only marginally effected by the addition of the D12L subunit, demonstrating that this enhancement of mRNA (guanine-7-) methyltransferase activity mediated by D12L was not achieved by altering the strength of GTP binding.

The mRNA (guanine-7-)methyltransferase domain of the vaccinia virus mRNA capping enzyme. Expression in Escherichia coli and structural and kinetic comparison to the intact capping enzyme.

The mRNA (guanine-7-)methyltransferase active site of the heterodimeric vaccinia virus mRNA capping enzyme was previously localized to the carboxyl-terminal third of the large subunit, D1R, associated with the small subunit, D12L (Cong, P., and Shuman, S. (1992) J. Biol. Chem. 267, 16424-16429; Higman, M. A., Bourgeois, N., and Niles, E. G. (1992) J. Biol. Chem. 267, 16430-16437). A plasmid was constructed which directs the coexpression of the carboxyl terminus of the D1R subunit from amino acids 498 to 844 and the D12L subunit in Escherichia coli. The mRNA (guanine-7-)methyltransferase catalytic activity in the isolated domain was found to be kinetically equivalent to that present in the intact enzyme. Through mobility shift and ultraviolet photolinkage analyses, both domains were shown to bind RNA in a saturable fashion. RNA binding was localized predominantly to the large subunit, but a low level of linkage of RNA to D12L was also observed. A low, but reproducible, level of mRNA (guanine-7-)methyltransferase activity was detected in the isolated D1R498-844 subunit demonstrating that the active site resides solely within the large subunit of the capping enzyme. This activity is enhanced 30- to 50-fold by the association of the D12L subunit.

Location of the S-adenosyl-L-methionine binding region of the vaccinia virus mRNA (guanine-7-)methyltransferase.

The mRNA (guanine-7-)methyltransferase activity of the heterodimeric vaccinia virus mRNA capping enzyme was previously mapped to the carboxyl-terminal 396 amino acids of the large subunit, D1R. This activity is enhanced 30- to 50-fold by the association of the the small subunit, D12L (Higman, M. A., Christen, L. A., and Niles, E. G. (1994) J. Biol. Chem. 269, 14974-14981). Irradiation with ultraviolet light specifically photolinks S-[methyl-3H]S-adenosyl-L-methionine to the large subunit of both the intact capping enzyme and a methyltransferase subdomain, which consists of the D12L subunit associated with the carboxyl-terminal 346 amino acids of the D1R subunit. The extent of linkage was shown to be dependent on the length of incubation, intensity of ultraviolet light, and the concentration of both active enzyme and substrate. The covalent modification was inhibited by S-adenosyl-L-homocysteine, a known competitive inhibitor of the mRNA (guanine-7-)methyltransferase activity, demonstrating specific photolabeling of the active site. Sequence-specific chemical cleavage of the photolinked methyltransferase domain with mild acid or cyanogen bromide revealed linkage of S-adenosyl-L-methionine to two regions of the large subunit. Analysis of the products of cyanogen bromide and hydroxylamine cleavage mapped the photolinked fragments to amino acids 499 to 579 and 806 to 844 of the D1R subunit. Photolinkage of AdoMet to D1R498-844 was shown to be unaffected by the association of the small subunit.

A poxvirus-encoded uracil DNA glycosylase is essential for virus viability.

Infection of cultured mammalian cells with the Leporipoxvirus Shope fibroma virus (SFV) causes the induction of a novel uracil DNA glycosylase activity in the cytoplasms of the infected cells. The induction of this activity, early in infection, correlates with the early expression of the SFV BamHI D6R open reading frame which possesses significant protein sequence similarity to eukaryotic and prokaryotic uracil DNA glycosylases. The SFV BamHI D6R open reading frame and the homologous HindIII D4R open reading frame from the Orthopoxvirus vaccinia virus were cloned under the regulation of a phage T7 promoter and expressed in Escherichia coli as insoluble high-molecular-weight aggregates. During electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, the E. coli-expressed proteins migrate with an apparent molecular mass of 25 kDa. The insoluble protein aggregate generated by expression in E. coli was solubilized in urea and, following a subsequent refolding step, displayed the ability to excise uracil residues from double-stranded plasmid DNA substrates, with the subsequent formation of apyrimidinic sites. The viral enzyme, like all other characterized uracil DNA glycosylases, is active in the presence of high concentrations of EDTA, is substrate inhibited by uracil, and does not display any endonuclease activity. Attempts to inactivate the HindIII D4R gene of vaccinia virus by targeted insertion of a dominant xanthine-guanine phosphoribosyltransferase selection marker or direct insertion of a frame-shifted oligonucleotide were uniformly unsuccessful demonstrating that, unlike the uracil DNA glycosylase described for herpesviruses, the poxvirus enzyme is essential for virus viability.

Phenotypic characterization of three temperature-sensitive mutations in the vaccinia virus early gene transcription initiation factor.

Vaccinia virus gene D6R encodes the small subunit of the virion early gene transcription initiation factor. Three temperature-sensitive mutations have been mapped to this gene. The biochemical phenotype exhibited by each mutation was examined. All mutants displayed altered viral protein synthesis in pulse-labelling analyses at both the permissive and non-permissive temperatures. The onset of early protein synthesis was delayed, and the rate of early protein synthesis was reduced in each case. Furthermore the shut-off of both host and early protein synthesis was delayed. In pulse-chase experiments, the stability of the D6R protein in E93- or C46-infected cells was shown to be reduced at 40 degrees C relative to that at 31 degrees C. Early mRNA was quantified in cells at 2 h post-infection and shown to be reduced substantially. The ability of each mutant virus to support transcription in vitro was examined at both temperatures and, of the three mutants, only S4 transcription was shown to exhibit reversible temperature sensitivity.

The vaccinia virus mRNA (guanine-N7-)-methyltransferase requires both subunits of the mRNA capping enzyme for activity.

Plasmid vectors capable of expressing the large and small subunits of the vaccinia virus mRNA capping enzyme were constructed and used to transform Escherichia coli. Conditions for the induction of the dimeric enzyme or the individual subunits in a soluble form were identified, and the capping enzyme was purified to near homogeneity. Proteolysis of the capping enzyme in bacteria yields a 60-kDa product shown previously to possess the mRNA triphosphatase and guanyltransferase activities (Shuman, S. (1990) J. Biol. Chem. 265, 11960-11966) was isolated and shown by amino acid sequence analysis to be derived from the NH2 terminus of D1R. The individual subunits lacked methyltransferase activity when assayed alone. However, mixing the D1R and D12L subunits permitted reconstitution of the methyltransferase activity, and this appearance in activity accompanied the association of the subunits. In contrast, mixing the D12L subunit with the D1R-60K proteolytic fragment failed to yield methyltransferase activity or result in a physical association of the two proteins. These results demonstrate that the methyltransferase active site requires the presence of the D12L subunit with the carboxyl-terminal portion of the D1R subunit. Furthermore, since the mRNA triphosphatase and guanyltransferase active sites reside in the NH2-terminal domain of the D1R subunit, and the methyltransferase activity is found in the carboxyl-terminal portion of this subunit and D12L, there must be at least two separate active sites in this enzyme.