The eukaryotic translation initiation factor eIF4E recognizes the mRNA cap, a key step in translation initiation. Here we have characterized eIF4E from the human parasite Schistosoma mansoni. Schistosome mRNAs have either the typical monomethylguanosine (m 7 G) or a trimethylguanosine (m 2,2,7 G) cap derived from spliced leader trans-splicing. Quantitative fluorescence titration analyses demonstrated that schistosome eIF4E has similar binding specificity for both caps. We present the first crystal structure of an eIF4E with similar binding specificity for m 7 G and m 2,2,7 G caps. The eIF4E⅐m 7 GpppG structure demonstrates that the schistosome protein binds monomethyl cap in a manner similar to that of single specificity eIF4Es and exhibits a structure similar to other known eIF4Es. The structure suggests an alternate orientation of a conserved, key Glu-90 in the capbinding pocket that may contribute to dual binding specificity and a position for mRNA bound to eIF4E consistent with biochemical data. Comparison of NMR chemical shift perturbations in schistosome eIF4E on binding m 7 GpppG and m 2,2,7 GpppG identified key differences between the two complexes. Isothermal titration calorimetry demonstrated significant thermodynamics differences for the binding process with the two caps (m 7 G versus m 2,2,7 G). Overall the NMR and isothermal titration calorimetry data suggest the importance of intrinsic conformational flexibility in the schistosome eIF4E that enables binding to m 2,2,7 G cap. Eukaryotic initiation protein eIF4E2 is an essential translation factor that recognizes the mRNA cap (1-3). Recognition of the mRNA cap by eIF4E is the key and rate-limiting step in mRNA translation. The majority of translation in eukaryotic cells is cap-dependent; that is recruitment of mRNAs to the ribosome for translation is dependent on the interaction between eIF4E and the mRNA cap. eIF4E directly binds to the mRNA cap. However, for productive translation initiation to occur, eIF4E must interact with eIF4G. eIF4G acts as a bridge protein interacting with factors in the 40 S ribosomal subunit that facilitate ribosome recruitment to the mRNA. Increased expression of eIF4E is associated with a variety of cancers and cancer progression (4). Efforts in a number of laboratories are directed toward therapies against eIF4E in cancer, including the development of cap analogs (5-9).The mRNA cap in most eukaryotes is m 7 GpppN (where N is A, C, G, or U). The cap contains a 5Ј-5Ј triphosphate bridge with the first guanosine methylated at the N-7 position. However, spliced leader trans-splicing in metazoa adds a different cap to recipient mRNAs, a trimethylguanosine cap, m 2,2,7 GpppN (see Fig. 1A) (10 -14). trans-splicing is present in a variety of parasitic nematodes and flatworms, and these organisms remain a significant health problem in many parts of the world, infecting upward of 2 billion people (15-17). Translation of these trans-spliced mRNAs is thought to require eIF4E recognition of the m 2,2,7 G cap to facilitate ribosomal ...
Cap-dependent translation initiation in eukaryotes is a complex process involving many factors and serves as the primary mechanism for eukaryotic translation (37, 44). The first step in the initiation process, recruitment of the m 7 G (7-methylguanosine)-capped mRNA to the ribosome, is widely considered the rate-limiting step. It begins with recognition of and binding to the m 7 G cap at the 5Ј end of the mRNA by the eukaryotic translation initiation factor 4F (eIF4F) complex, which contains three proteins: eIF4E (a cap-binding protein), eIF4G (a scaffold protein with RNA binding sites), and eIF4A (an RNA helicase). eIF4G's interaction with eIF3, itself a multisubunit complex that interacts with the 40S ribosome, facilitates the actual recruitment of capped RNA to the ribosome. With the help of several other initiation factors, the small ribosomal subunit scans the mRNA from 5Ј to 3Ј until a translation initiation codon (AUG) in appropriate context is identified and an 80S ribosomal complex is formed, after which the first peptide bond is formed, thus ending the initiation process (37, 44). The AUG context can play an important role in the efficiency of translation initiation (23, 44). The length, structure, and presence of AUGs or open reading frames in the mRNA 5Ј untranslated region (UTR) can negatively affect cap-dependent translation and ribosomal scanning. In general, long and highly structured 5Ј UTRs, as well as upstream AUGs leading to short open reading frames, can impede ribosome scanning and lead to reduced translation (23, 44). In addition, 5Ј UTRs less than 10 nucleotides (nt) in length are thought to be too short to enable preinitiation complex assembly and scanning (24). Thus, several attributes of the mRNA 5Ј UTR are known to negatively affect translation initiation, whereas only the AUG context and the absence of negative elements are known to have a positive effect on translation initiation (44).Two of the important mRNA features associated with capdependent translation, the cap and the 5Ј UTR, are significantly altered by an RNA processing event known as spliced leader (SL) trans splicing (3,8,17,26,36,47). This takes place in members of a diverse group of eukaryotic organisms, including some protozoa, sponges, cnidarians, chaetognaths, flatworms, nematodes, rotifers, crustaceans, and tunicates (17,28,39,55,56). In SL trans splicing, a separately transcribed small exon (16 to 51 nucleotides [nt]) with its own cap gets added to the 5Ј end of pre-mRNAs. This produces mature mRNAs with a unique cap and a conserved sequence in the 5Ј UTR. In metazoa, the m 7 G cap is replaced with a trimethylguanosine (TMG) cap (m 2,2,7 GpppN) (27,30,46,49). In nematodes, ϳ70% of all mRNAs are trans spliced and therefore have a TMG cap and an SL (2). In general, eukaryotic eIF4E proteins do not effectively recognize the TMG cap (35). This raises the issues of how the translation machinery in trans-splicing
Anticoagulation therapy is administered to patients to prevent or stop thrombin generation in vivo. Although plasma tests of in vivo thrombin generation have been available for more than 2 decades, they are not routinely used in clinical trials or practice to monitor anticoagulation therapy. We observed a fall in one such marker, the D-dimer antigen, in patients receiving anticoagulation therapy. We therefore conducted a systematic review of the medical literature to document the change in serum biomarkers of thrombin generation following the initiation of anticoagulation therapy. Using a defined search strategy, we screened PubMed and Embase citations and identified full-length articles published in English. Eighteen articles containing serial changes in 1 of 3 markers of thrombin generation (D-dimer antigen, thrombin-antithrombin complexes, and prothrombin fragment 1þ2 antigen levels) in the 14 days following the initiation of anticoagulation were identified. Even though the assays used varied considerably, each of the 3 markers of thrombin generation declined in the initial period of anticoagulation therapy, with changes evident as early as 1 day after beginning therapy. These observations provide a rationale for further exploration of these markers as measures of the adequacy of anticoagulation using classic as well as novel anticoagulants. Particular patient groups that would benefit from additional means of monitoring anticoagulation therapy are discussed.
Macronuclear chromosomes of spirotrichous ciliates are mainly "nanochromosomes" containing only a single gene. We identified a two-gene chromosome in the spirotrich Sterkiella histriomuscorum (formerly Oxytricha trifallax) which, unlike other characterized two-gene molecules, contains reading frames oriented tail to tail. These are homologs of ribosomal protein L29 (RPL29) and cyclophilin. We found that both genes are transcribed, with their polyadenylation sites on opposite strands separated by only 135 bp. Furthermore, both genes in S. histriomuscorum are present only on one macronuclear chromosome and do not occur alone or linked to other genes. The corresponding micronuclear locus is fragmented into three nonscrambled gene segments (MDSs), separated by two noncoding segments (IESs). We also found that these two genes are linked on a macronuclear chromosome, similarly arranged tail to tail, in the three spirotrichs Stylonychia lemnae, Uroleptus sp., and Holosticha sp.. In addition, single-gene macronuclear chromosomes containing only the RPL29 gene were detected in the earlier diverged Holosticha and Uroleptus. These observations suggest a possible evolutionary trend towards loss of chromosomal breakage between these two genes. This study is the first to examine gene linkage in the macronucleus of several spirotrichs and may provide insight into the evolution of multi-gene macronuclear chromosomes and chromosomal fragmentation in spirotrichs.
Objectives: We used SRA testing for samples positive in a HIT ELISA assay for the past 5 years. In recent years we have used an IgG-specific HIT ELISA, rather than a polytypic assay. We analyzed the incidence of SRA positivity in samples that were abnormal by either ELISA assay and computed the costs/benefits of our reflex testing policy. Methods: A query of our laboratory database identified 75 SRA tests performed during a 20-month period on patients with an elevated IgG HIT ELISA OD. We divided the samples into low (0.4-1), intermediate (1-2) and high (>2) subgroups based upon the OD obtained. We then calculated the 4 Ts clinical score for the 51 patients with available clinical data. Finally, we reviewed the clinical charts to identify patients who had direct thrombin inhibitor (DTI) therapy changed to heparin as a result of SRA testing. Results: Using a polytypic HIT ELISA, the percentage of patients in the low, intermediate, and high OD groups with positive SRA tests were 7%, 21% and 66%. Use of the IgG-specific HIT ELISA revealed that 5%, 33%, 68% of samples, respectively, were positive by SRA testing. 4 Ts scores were statistically different between high and low IgG HIT ELISA OD (P < .0001) patients, between positive and negative SRA patients (P = .0035) but did not discriminate SRA results on intermediate and high HIT ELISA OD patients. Eight patients had DTIs discontinued after a negative SRA, and were switched to a less-expensive anticoagulant during the study period (>55 patient days). Utilizing average wholesale acquisition cost for DTIs, we estimate that the reflex SRA testing saved over $40,000 in DTI costs during a 20-month period. Further, we estimate that at least an additional $40,000 in DTI costs were avoided during six subsequent hospitalizations. SRA testing laboratory cost during the study period was approximately $25,000. Conclusions: Our results suggest that the OD of IgG-specific HIT ELISAs should be reported to clinicians, because there are vast differences in likelihoods of having a subsequent positive SRA based on the OD value. A positive IgG HIT ELISA result should not be considered diagnostic of HIT. A policy of SRA reflex testing for IgG positive HIT ELISAs results in cost savings.
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