The structure of eukaryotic translation initiation factor-4E from wheat reveals a novel disulfide bond

Eukaryotic translation initiation factor-4E (eIF4E) recognizes and binds the m(7) guanosine nucleotide at the 5' end of eukaryotic messenger RNAs; this protein-RNA interaction is an essential step in the initiation of protein synthesis. The structure of eIF4E from wheat (Triticum aestivum) was investigated using a combination of x-ray crystallography and nuclear magnetic resonance (NMR) methods. The overall fold of the crystallized protein was similar to eIF4E from other species, with eight beta-strands, three alpha-helices, and three extended loops. Surprisingly, the wild-type protein did not crystallize with m(7)GTP in its binding site, despite the ligand being present in solution; conformational changes in the cap-binding loops created a large cavity at the usual cap-binding site. The eIF4E crystallized in a dimeric form with one of the cap-binding loops of one monomer inserted into the cavity of the other. The protein also contained an intramolecular disulfide bridge between two cysteines (Cys) that are conserved only in plants. A Cys-to-serine mutant of wheat eIF4E, which lacked the ability to form the disulfide, crystallized with m(7)GDP in its binding pocket, with a structure similar to that of the eIF4E-cap complex of other species. NMR spectroscopy was used to show that the Cys that form the disulfide in the crystal are reduced in solution but can be induced to form the disulfide under oxidizing conditions. The observation that the disulfide-forming Cys are conserved in plants raises the possibility that their oxidation state may have a role in regulating protein function. NMR provided evidence that in oxidized eIF4E, the loop that is open in the ligand-free crystal dimer is relatively flexible in solution. An NMR-based binding assay showed that the reduced wheat eIF4E, the oxidized form with the disulfide, and the Cys-to-serine mutant protein each bind m(7)GTP in a similar and labile manner, with dissociation rates in the range of 20 to 100 s(-1).     

Aberrant epigenetic and genetic marks are seen in myelodysplastic leukocytes and reveal Dock4 as a candidate pathogenic gene on chromosome 7q

Myelodysplastic syndromes (MDS) are characterized by abnormal and dysplastic maturation of all blood lineages. Even though epigenetic alterations have been seen in MDS marrow progenitors, very little is known about the molecular alterations in dysplastic peripheral blood cells. We analyzed the methylome of MDS leukocytes by the HELP assay and determined that it was globally distinct from age-matched controls and was characterized by numerous novel, aberrant hypermethylated marks that were located mainly outside of CpG islands and preferentially affected GTPase regulators and other cancer-related pathways. Additionally, array comparative genomic hybridization revealed that novel as well as previously characterized deletions and amplifications could also be visualized in peripheral blood leukocytes, thus potentially reducing the need for bone marrow samples for future studies. Using integrative analysis, potentially pathogenic genes silenced by genetic deletions and aberrant hypermethylation in different patients were identified. DOCK4, a GTPase regulator located in the commonly deleted 7q31 region, was identified by this unbiased approach. Significant hypermethylation and reduced expression of DOCK4 in MDS bone marrow stem cells was observed in two large independent datasets, providing further validation of our findings. Finally, DOCK4 knockdown in primary marrow CD34(+) stem cells led to decreased erythroid colony formation and increased apoptosis, thus recapitulating the bone marrow failure seen in MDS. These findings reveal widespread novel epigenetic alterations in myelodysplastic leukocytes and implicate DOCK4 as a pathogenic gene located on the 7q chromosomal region.     

Role of the p38 mitogen-activated protein kinase pathway in the generation of the effects of imatinib mesylate (STI571) in BCR-ABL-expressing cells

Imatinib mesylate (STI571), a specific inhibitor of the BCR-ABL tyrosine kinase, exhibits potent antileukemic effects in vitro and in vivo. Despite the well established role of STI571 in the treatment of chronic myelogenous leukemia, the precise mechanisms by which inhibition of BCR-ABL tyrosine kinase activity results in generation of antileukemic responses remain unknown. In the present study we provide evidence that treatment of CML-derived BCR-ABL-expressing leukemia cells with STI571 results in activation of the p38 mitogen-activated protein (MAP) kinase signaling pathway. Our data indicate that STI571 induces phosphorylation of the p38 and activation of its kinase domain, in KT-1 cells and other BCR-ABL-expressing cell lines. We also identify the kinases MAP kinase-activated protein kinase-2 and Msk1 as two downstream effectors of p38, activated during inhibition of BCR-ABL activity by STI571. Importantly, pharmacological inhibition of p38 reverses the growth inhibitory effects of STI571 on primary leukemic colony-forming unit granulocyte/macrophage progenitors from patients with CML. Altogether, our data establish that activation of the p38 MAP kinase signaling cascade plays an important role in the generation of the effects of STI571 on BCR-ABL-expressing cells. They also suggest that, in addition to activation of mitogenic pathways, BCR-ABL promotes leukemogenesis by suppressing the function of growth inhibitory signaling cascades.     

Generation of Functional CLL-Specific Cord Blood CTL Using CD40-Ligated CLL APC

Though remissions have been observed following allo-HSCT for the treatment of CLL, many CLL patients are ineligible for transplant due to the lack of HLA-compatible donors. The use of umbilical cord blood (UCB) permits transplantation of many patients who lack HLA-compatible donors due to reduced requirements for stringent HLA matching between graft and recipient; however, disease relapse remains a concern with this modality. The generation of CLL-specific CTL from UCB T-cells, primed and expanded against the leukemic clone, might enhance the GVL effect and improve outcomes with UCB transplantation. Here we report the generation of functional, CLL-specific CTL using CD40-ligated CLL cells to prime partially-HLA matched UCB T-cells. Functionality and specificity were demonstrated by immune synapse assay, IFN-γ ELISpot, multi-parametric intracellular cytokine flow cytometry, and ⁵¹Cr release assay. The use of patient-specific, non-CLL controls demonstrated the generation of both alloantigen and CLL-specific responses. Subsequently, we developed a clinically-applicable procedure permitting separation of alloreactive CTL from leukemia-specific CTL. Leukemia-specific CTL were able to mediate in vivo killing of CLL in humanized mice without concurrent or subsequent development of xenoGVHD. Our results demonstrate that generation of CLL-specific effectors from UCB is feasible and practical, and the results support further exploration of this strategy as a treatment modality for CLL.     

Establishing bioequivalence of racemic venlafaxine formulations using stereoselective assay method: Is it necessary?

A bioequivalence study for venlafaxine generic formulation was conducted as an open label, balanced, randomized, two‐way crossover, single‐dose study. In this study, a comparison of various pharmacokinetic parameters of venlafaxine hydrochloride 150 mg modified release capsules of Ranbaxy and EFEXOR®‐XR 150 mg capsules of Wyeth, in healthy, adult, male, human subjects under fasting condition was performed to conclude bioequivalence. Venlafaxine and its major active metabolite O‐desmethylvenlafaxine (ODV) are racemates. The “(S)‐(+)” and “(R)‐(−)” enantiomers of venlafaxine and ODV are established as being active. Hence, subject samples were analyzed using nonstereoselective and stereoselective assay methods. Both (S)‐(+) and (R)‐(−) enantiomers of venlafaxine and ODV showed similar absorption and disposition. The 90% confidence intervals for venlafaxine, (R)‐(−)‐venlafaxine as well as (S)‐(+)‐venlafaxine were within acceptance range concluding bioequivalence. The results obtained by stereoselective assay were comparable to the nonstereoselective analysis, as sum of concentrations of (S)‐(+)‐ and (R)‐(−)‐enantiomers of venlafaxine and ODV. The mean (S)‐(+)/(R)‐(−) ratios of the enantiomers of venlafaxine and ODV at various time points were consistent in the study subjects. Therefore, the estimation of venlafaxine and ODV using nonstereoselective assay method is effective in distinguishing formulation differences (if any) in bioequivalence studies in a cost‐effective manner. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Activation of the p70 S6 kinase and phosphorylation of the 4E-BP1 repressor of mRNA translation by type I interferons

The Type I IFN receptor-generated signals required for initiation of mRNA translation and, ultimately, induction of protein products that mediate IFN responses, remain unknown. We have previously shown that IFNalpha and IFNbeta induce phosphorylation of insulin receptor substrate proteins and downstream engagement of the phosphatidylinositol (PI) 3'-kinase pathway. In the present study we provide evidence for the existence of a Type I IFN-dependent signaling cascade activated downstream of PI 3'-kinase, involving p70 S6 kinase. Our data demonstrate that p70 S6K is rapidly phosphorylated on threonine 421 and serine 424 and is activated during treatment of cells with IFNalpha or IFNbeta. Such activation of p70 S6K is blocked by pharmacological inhibitors of the PI 3'-kinase or the FKBP 12-rapamycin-associated protein/mammalian target of rapamycin (FRAP/mTOR). Consistent with this, the Type I IFN-dependent phosphorylation/activation of p70 S6K is defective in embryonic fibroblasts from mice with targeted disruption of the p85alpha and p85beta subunits of the PI 3'-kinase (p85alpha-/-beta-/-). Treatment of sensitive cell lines with IFNalpha or IFNbeta also results in phosphorylation/inactivation of the 4E-BP-1 repressor of mRNA translation. Such 4E-BP1 phosphorylation is also PI3'-kinase-dependent and rapamycin-sensitive, indicating that the Type I IFN-inducible activation of PI3'-kinase and FRAP/mTOR results in dissociation of 4E-BP1 from the eukaryotic initiation factor-4E (eIF4E) complex. Altogether, our data establish that the Type I IFN receptor-activated PI 3'-kinase pathway mediates activation of the p70 S6 kinase and inactivation of 4E-BP1, to regulate mRNA translation and induction of Type I IFN responses.     

The crystal structure of the N-terminal region of the alpha subunit of translation initiation factor 2 (eIF2alpha) from Saccharomyces cerevisiae provides a view of the loop containing serine 51, the target of the eIF2alpha-specific kinases

The alpha subunit of translation initiation factor 2 (eIF2alpha) is the target of specific kinases that can phosphorylate a conserved serine residue as part of a mechanism for regulating protein expression at the translational level in eukaryotes. The structure of the 20 kDa N-terminal region of eIF2alpha from Saccharomyces cerevisiae was determined by X-ray crystallography at 2.5A resolution. In most respects, the structure is similar to that of the recently solved human eIF2alpha; the rather elongated protein contains a five-stranded antiparallel beta-barrel in its N-terminal region, followed by an almost entirely helical domain. The S.cerevisiae eIF2alpha lacks a disulfide bridge that is present in the homologous protein in humans and some of the other higher eukaryotes. Interestingly, a conserved loop consisting of residues 51-65 and containing serine 51, the putative phosphorylation site, is visible in the electron density maps of the S.cerevisiae eIF2alpha; most of this functionally important loop was not observed in the crystal structure of the human protein. This loop is relatively exposed to solvent, and contains two short 3(10) helices in addition to some extended structure. Serine 51 is located at the C-terminal end of one of the 3(10) helices and near several conserved positively charged residues. The side-chain of serine 51 is sufficiently exposed so that its phosphorylation would not necessitate a substantial change in the protein structure. The structures and relative positions of residues that have been implicated in kinase binding and in the interaction with guanine nucleotide exchange factor (eIF2B) are described.     

Overcoming the barriers to umbilical cord blood transplantation

Umbilical cord blood (UCB) transplantation (UCBT) has seen a marked increase in utilization in recent years, especially in the pediatric population; however, graft failure, delayed engraftment and profound delay in immune reconstitution leads to significant morbidity and mortality in adults. The lack of cells available for post-transplant therapies, such as donor lymphocyte infusions, has also been considered a disadvantage. To overcome the cell–dose barrier, the combination of two UCB units is becoming commonplace in adolescent and adult populations, and is currently being studied in pediatrics as well. In some studies, the use of two UCB units appears to have a positive impact on outcomes; however, engraftment is still suboptimal. A possible additional way to improve outcome and extend applicability of UCBT is via ex vivo expansion. Studies to develop optimal expansion conditions are still in the exploratory phase; however, recent studies suggest expanded UCB is safe and can improve outcomes. The ability to transplant across HLA disparities, rapid procurement time and decreased graft-versus-host disease (GvHD) seen with UCBT makes it a promising stem cell source and, while barriers exist, consistent progress is being made to overcome them.