Microtubule nucleation at non-spindle pole body microtubule-organizing centers requires fission yeast centrosomin-related protein mod20p

BACKGROUND: Many types of differentiated eukaryotic cells display microtubule distributions consistent with nucleation from noncentrosomal intracellular microtubule organizing centers (MTOCs), although such structures remain poorly characterized. In fission yeast, two types of MTOCs exist in addition to the spindle pole body, the yeast centrosome equivalent. These are the equatorial MTOC, which nucleates microtubules from the cell division site at the end of mitosis, and interphase MTOCs, which nucleate microtubules from multiple sites near the cell nucleus during interphase. RESULTS: From an insertional mutagenesis screen we identified a novel gene, mod20+, which is required for microtubule nucleation from non-spindle pole body MTOCs in fission yeast. Mod20p is not required for intranuclear mitotic spindle assembly, although it is required for cytoplasmic astral microtubule growth during mitosis. Mod20p localizes to MTOCs throughout the cell cycle and is also dynamically distributed along microtubules themselves. We find that mod20p is required for the localization of components of the gamma-tubulin complex to non-spindle pole body MTOCs and physically interacts with the gamma-tubulin complex in vivo. Database searches reveal a family of eukaryotic proteins distantly related to mod20p; these are found in organisms ranging from fungi to mammals and include Drosophila centrosomin. CONCLUSIONS: Mod20p appears to act by recruiting components of the gamma-tubulin complex to non-spindle pole body MTOCs. The identification of mod20p-related proteins in higher eukaryotes suggests that this may represent a general mechanism for the organization of noncentrosomal MTOCs in eukaryotic cells.     

Multiple treatment comparisons in epilepsy monotherapy trials

Background

Previous trials of the RTS, S malaria candidate vaccine have shown that this vaccine is safe, tolerated and immunogenic. The development plan for this vaccine aims at administering it in the first year of life through the Expanded Program on Immunization (EPI). The objective was to evaluate the safety and reactogenicity of RTS, S/AS02D (0.5 ml dose), a pediatric formulation of GlaxoSmithKline Biologicals' current malaria candidate vaccine RTS, S/AS02A (0.25 ml dose). A 0.5 ml dose of AS02D is composed of the same active ingredients in the same quantities as in a 0.25 ml dose of AS02A and has been developed to be easily introduced into routine EPI practices.

Methods

We performed a phase I/IIb randomized double-blind bridging study in a malaria-endemic region of Mozambique, to compare the safety and immunogenicity of both candidate vaccines with the aim of replacing RTS, S/AS02A with RTS, S/AS02D as the candidate pediatric vaccine. 200 Mozambican children aged 3 to 5 years were randomized 1:1 to receive one of the 2 vaccines according to a 0, 1, 2 month schedule.

Results

Both vaccines were safe and had similar reactogenicity profiles. All subjects with paired pre and post-vaccination samples showed a vaccine response with respect to anti-circumsporozoite (CS) antibodies irrespective of initial anti-CS serostatus. Geometric mean titers (GMTs) were 191 EU/ml (95% CI 150–242) in recipients of RTS, S/AS02D compared to 180 EU/ml (95% CI 146–221) in recipients of RTS, S/AS02A. For the anti-hepatitis B surface antigen (HBsAg), all subjects were seroprotected at day 90, and the GMTs were 23978 mIU/ml (95% CI 17896–32127) in RTS, S/AS02D recipients and 17410 mIU/ml (95% CI 13322–22752) in RTS, S/AS02A recipients. There was a decrease in anti-CS GMTs between months 3 and 14 in both groups (191 vs 22 EU/mL in RTS, S/AS02D group and 180 vs 29 EU/mL in RTS, S/AS02A group).

Conclusion

Our data show that the RTS, S/AS02D is safe, well tolerated, and demonstrates non-inferiority (defined as upper limit of the 95% confidence interval of the anti-CS GMT ratio of RTS, S/AS02A to RTS, S/AS02D below 3.0) of the antibody responses to circumsporozoite and HBsAg induced by the RTS, S/AS02D as compared to the RTS, S/AS02A.     

Translocation of nucleoside analogs across the plasma membrane in hematologic malignancies

Nucleoside analogs are currently used in the treatment of various hematologic malignancies due to their ability to induce apoptosis of lymphoid cells. For nucleoside-derived drugs to exert their action, they must enter cells via nucleoside transporters from two gene families, SLC28 and SLC29 (CNT and ENT, respectively). Once inside the cell, these drugs must be phosphorylated to their active forms. In contrast, some members of the ATP-binding cassette (ABC) protein family have been identified as responsible for the efflux of the phosphorylated forms of these nucleoside-derived drugs. Here, we review the main nucleoside analogs used in hematologic malignancies and focus especially on those that are currently used in chronic lymphocytic leukemia (CLL). Moreover, we discuss the pharmacological profile of the nucleoside transporters, which determines the bioavailability of and cell sensitivity to these nucleoside-derived drugs. We also discuss the expression of nucleoside transporters and their activities in CLL as well as the possibility of modulating these transporter activities as a means of modulating intracellular drug availability and, consequently, responsiveness to therapy.     

Functional proteomic insights in B-cell chronic lymphocytic leukemia

Introduction: B cell chronic lymphocytic leukemia (B-CLL) is a hematological malignancy considered as the most common leukemia in the Western world. The understanding of B cell differentiation is crucial for the diagnosis, prognosis, and treatment of the disease.

Areas covered: In this review, B-cell ontogeny and its relation with the CLL development, in combination with the proteomic approaches which could provide a deep characterization of the disease through the characterization of the cellular signaling pathways involved in the pathological cells is described.

Expert commentary: Although conventional strategies (genome sequencing, morphology assays, and immunophenotyping by flow cytometry and/or immunochemistry) have allowed the establishment of the disease stage based on different parameters, it is still necessary to utilize novel approaches (e.g., proteomics) that have the potential to simultaneously analyze thousands of molecules to improve understanding of CLL.     

Mitochondrial DNA of pre‐last glacial maximum red deer from NW Spain suggests a more complex phylogeographical history for the species

The major climatic oscillations that characterized the Quaternary had a great influence on the evolution and distribution of several species. During cold periods, the distribution of temperate‐adapted species became fragmented with many surviving in southern refugia (Iberian, Italian, and Balkan Peninsulas). Red deer was one of the species that contracted its original range to southern refugia. Currently, two main lineages have been described for the species: western and eastern. We have analyzed fossils pre‐dating the last glacial maximum (LGM) from Liñares cave (NW Spain) that belongs to the peripheral range of the western clade, and fossils from the Danish Holocene belonging to the central part of the same clade. Phylogenetic analyses place our samples in the western clade. However, some specimens from Liñares represent an early split in the tree along with other pre‐LGM western samples from previous studies. Despite low bootstrap values in the Bayesian phylogenies, haplotype networks connect these foreign haplotypes to the eastern clade. We suggest a mixed phylogeographical model to explain this pattern with range expansions from the east during the expansion phase after the cold periods in marine isotope stage 3. We find slight isolation by distance in post‐LGM populations that could be a consequence of the recolonization from southern refugia after the LGM.     

Stability and kinetic behavior of immobilized laccase from Myceliophthora thermophila in the presence of the ionic liquid 1‐ethyl‐3‐methylimidazolium ethylsulfate

The use of ionic liquids (ILs) as reaction media for enzymatic reactions has increased their potential because they can improve enzyme activity and stability. Kinetic and stability properties of immobilized commercial laccase from Myceliophthora thermophila in the water‐soluble IL 1‐ethyl‐3‐methylimidazolium ethylsulfate ([emim][EtSO₄]) have been studied and compared with free laccase. Laccase immobilization was carried out by covalent binding on glyoxyl–agarose beads. The immobilization yield was 100%, and the activity was totally recovered. The Michaelis‐Menten model fitted well to the kinetic data of enzymatic oxidation of a model substrate in the presence of the IL [emim][EtSO₄]. When concentration of the IL was augmented, the values of V_max for free and immobilized laccases showed an increase and slight decrease, respectively. The laccase–glyoxyl–agarose derivative improved the laccase stability in comparison with the free laccase regarding the enzymatic inactivation in [emim][EtSO₄]. The stability of both free and immobilized laccase was slightly affected by small amounts of IL (<50%). A high concentration of the IL (75%) produced a large inactivation of free laccase. However, immobilization prevented deactivation beyond 50%. Free and immobilized laccase showed a first‐order thermal inactivation profile between 55 and 70°C in the presence of the IL [emim][EtSO₄]. Finally, thermal stability was scarcely affected by the presence of the IL. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:790–796, 2014     

Adenovirus-mediated hepatic overexpression of scavenger receptor class B type I accelerates chylomicron metabolism in C57BL/6J mice

The function of scavenger receptor class B type I (SR-BI) in mediating the selective uptake of HDL cholesteryl esters is well established. In SR-BI-deficient mice, we recently observed a delayed postprandial triglyceride (TG) response, suggesting an additional role for SR-BI in facilitating chylomicron (CM) metabolism. Here, we assessed the effect of adenovirus-mediated hepatic overexpression of SR-BI (Ad.SR-BI) in C57BL/6J mice on serum lipids and CM metabolism. Infection of 5 x 10(8) plaque-forming units per mouse of Ad.SR-BI significantly decreases serum cholesterol (>90%), phospholipids (>90%), and TG levels (50%), accompanied by a 41.4% reduction (P < 0.01) in apolipoprotein B-100 levels. The postprandial TG response is 2-fold lower in mice treated with Ad.SR-BI compared with control mice (area under the curve = 31.4 +/- 2.4 versus 17.7 +/- 3.2; P < 0.05). Hepatic mRNA expression levels of genes known to be involved in serum cholesterol and TG clearance are unchanged and thus could not account for the decreased plasma TG levels and the change in postprandial response. We conclude that overexpression of SR-BI accelerates CM metabolism, possibly by mediating the initial capture of CM remnants by the liver, whereby the subsequent internalization can be exerted by additional receptor systems such as the LDL receptor (LDLr) and LDLr-related protein 1.     

Proteomic analysis of reaper 5' untranslated region-interacting factors isolated by tobramycin affinity-selection reveals a role for La antigen in reaper mRNA translation

Translational control is a key step in gene expression regulation during apoptosis. To understand the mechanisms of mRNA translation of a pro-apoptotic gene, reaper (rpr), we adapted the tobramycin-aptamer technique described by Hartmuth et al. (Proc. Natl. Acad. Sci. USA 2002, 99, 16719-16724) for the analysis of proteins interacting with rpr 5' untranslated region (UTR). We assembled ribonucleoprotein complexes in vitro using translation extracts derived from Drosophila embryos and purified the RNA-protein complexes for mas spectrometry analysis. We identified the proteins bound to the 5' UTR of rpr. One of them, the La antigen, was validated by RNA-crosslinking experiments using recombinant protein and by the translation efficiency of reporter mRNAs in Drosophila cells after RNAinterference experiments. Our data provide evidence of the involvement of La antigen in the translation of rpr and set a protocol for purification of tagged-RNA-protein complexes from cytoplasmic extracts.     

Root gravitropism requires lateral root cap and epidermal cells for transport and response to a mobile auxin signal

Re-orientation of Arabidopsis seedlings induces a rapid, asymmetric release of the growth regulator auxin from gravity-sensing columella cells at the root apex. The resulting lateral auxin gradient is hypothesized to drive differential cell expansion in elongation-zone tissues. We mapped those root tissues that function to transport or respond to auxin during a gravitropic response. Targeted expression of the auxin influx facilitator AUX1 demonstrated that root gravitropism requires auxin to be transported via the lateral root cap to all elongating epidermal cells. A three-dimensional model of the root elongation zone predicted that AUX1 causes the majority of auxin to accumulate in the epidermis. Selectively disrupting the auxin responsiveness of expanding epidermal cells by expressing a mutant form of the AUX/IAA17 protein, axr3-1, abolished root gravitropism. We conclude that gravitropic curvature in Arabidopsis roots is primarily driven by the differential expansion of epidermal cells in response to an influx-carrier-dependent auxin gradient.     

Assessment of micronucleus frequency in normal oral mucosa of patients exposed to carcinogens

OBJECTIVE: To assess the presence of micronuclei in exfoliated oral mucosal cells collected from 3 anatomic sites in patients exposed to tobacco and alcohol. STUDY DESIGN: Smears were prepared with normal oral mucosal cells obtained from the lower lip, tongue border and floor of the mouth of 21 controls, 28 tobacco users and 19 tobacco/alcohol users. Slides were stained with Feulgen stain for quantification of micronucleated cells, karyorrhexis and "broken eggs." RESULTS: The groups were similar in terms of the mean number of micronucleated cells and cells undergoing karyorrhexis. In the comparison of anatomic sites, the mean number of cells undergoing karyorrhexis was higher on the lower lip than on the tongue border or floor of the mouth (all groups). A significantly higher number of broken eggs was observed in the control group when compared to the tobacco and tobacco/alcohol groups at all anatomic sites. CONCLUSION: The higher number of broken eggs in patients not exposed to tobacco and/or alcohol suggests that this nuclear alteration may be associated with DNA repair or a healthy mucosa. A trend toward an increased number of micronucleated cells was observed for tobacco and/or alcohol users at all anatomic sites.