SummaryThe bacterial tRNA Lys -specific anticodon nuclease is known as a phage T4 exclusion system. In the uninfected host cell anticodon nuclease is kept latent due to the association of its core protein PrrC with the DNA restriction-modification endonuclease EcoprrI. Stp, the T4-encoded peptide inhibitor of EcoprrI activates the latent enzyme. Previous in vitro work indicated that the activation by Stp is sensitive to DNase and requires added nucleotides. Biochemical and mutational data reported here suggest that Stp activates the latent holoenzyme when its EcoprrI component is tethered to a cognate DNA substrate. Moreover, the activation is driven by GTP hydrolysis, possibly mediated by the NTPase domain of PrrC. The data also reveal that Stp can be replaced as the activator of latent anticodon nuclease by certain pyrimidine nucleotides, the most potent of which is dTTP. The activation by dTTP likewise requires an EcoprrI DNA substrate and GTP hydrolysis but involves a different form of the latent holoenzyme/DNA complex. Moreover, whereas Stp relays its activating effect through EcoprrI, dTTP targets PrrC. The activation of the latent enzyme by a normal cell constituent hints that anticodon nuclease plays additional roles, other than warding off phage T4 infection.
The objective of the present study was to compare the mechanical, kinetic, and biochemical properties of fibrin clots produced using EVICEL Fibrin Sealant (Human) and TISSEEL Fibrin Sealant. The stiffness/elasticity and strength of fibrin clots formed with EVICEL and TISSEEL were assessed using applied mechanical force and thromboelastography (TEG). The factor XIII content of the fibrin clots was also evaluated. Mean Young modulus and tensile strength of the fibrin clots produced by EVICEL were significantly higher than those of clots produced by TISSEEL (P < 0.05 for both). The mean time to initial clot formation and mean time to the predefined level of clot formation were numerically shorter for EVICEL compared with TISSEEL. Furthermore, mean maximal amplitude of the clots formed with EVICEL was significantly greater than that for the clots formed with TISSEEL. Mean concentration of factor XIII for the EVICEL fibrinogen samples tested was 9 IU/ml compared with undetectable concentrations of factor XIII for the TISSEEL fibrinogen samples. Fibrin clots formed with EVICEL have a much higher resistance to stretching and tensile strength and are more capable of maintaining their structure against applied force than those formed with TISSEEL. EVICEL also allows more rapid development of fibrin clots than TISSEEL. This superior clot strength and resilience obtained with EVICEL relative to TISSEEL may be due in large part to the presence of factor XIII.
The bacterial tRNA Lys -specific PrrC-anticodon nuclease efficiently cleaved an anticodon stem-loop (ASL) oligoribonucleotide containing the natural modified bases, suggesting this region harbors the specificity determinants. Assays of ASL analogs indicated that the 6-threonylcarbamoyl adenosine modification (t 6 A37) enhances the reactivity. The side chain of the modified wobble base 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U34) has a weaker positive effect depending on the context of other modifications. The s 2 U34 modification apparently has none and the pseudouridine (⌿39) was inhibitory in most modification contexts. GC-rich but not IC-rich stems abolished the activity. Correlating the reported structural effects of the base modifications with their effects on anticodon nuclease activity suggests preference for substrates where the anticodon nucleotides assume a stacked A-RNA conformation and base pairing interactions in the stem are destabilized. Moreover, the proposal that PrrC residue Asp 287 contacts mnm 5 s 2 U34 was reinforced by the observations that the mammalian tRNA Lys-3 wobble base 5-methoxycarbonyl methyl-2-thiouridine (mcm 5 s 2 U) is inhibitory and that the D287H mutant favors tRNA Lys-3 over Escherichia coli tRNA Lys . The detection of this mutation and ability of PrrC to cleave the isolated ASL suggest that anticodon nuclease may be used to cleave tRNA Lys-3 primer molecules annealed to the genomic RNA template of the human immunodeficiency virus.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.