Integrity of the P-site is probed during maturation of the 60S ribosomal subunit

Eukaryotic ribosomes are preassembled in the nucleus and mature in the cytoplasm. Release of the antiassociation factor Tif6 by the translocase-like guanosine triphosphatase Efl1 is a critical late maturation step. In this paper, we show that a loop of Rpl10 that embraces the P-site transfer ribonucleic acid was required for release of Tif6, 90 Å away. Mutations in this P-site loop blocked 60S maturation but were suppressed by mutations in Tif6 or Efl1. Molecular dynamics simulations of the mutant Efl1 proteins suggest that they promote a conformation change in Efl1 equivalent to changes that elongation factor G and eEF2 undergo during translocation. These results identify molecular signaling from the P-site to Tif6 via Efl1, suggesting that the integrity of the P-site is interrogated during maturation. We propose that Efl1 promotes a functional check of the integrity of the 60S subunit before its first round of translation.     

Nucleic acid solvation: from outside to insight

Nucleic acids are polyanionic molecules that were historically considered to be solely surrounded by a shell of water molecules and a neutralizing cloud of monovalent and divalent cations. In this respect, recent experimental and theoretical reports demonstrate that water molecules within complex nucleic acid structures can display very long residency times, and assist drug binding and catalytic reactions. Finally, anions can also bind to these polyanionic systems. Many of these recent insights are provided by state-of-the-art molecular dynamics simulations of nucleic acid systems, which will be described together with relevant methodological issues.     

Structure of the mammalian ribosomal pre-termination complex associated with eRF1?eRF3?GDPNP

Eukaryotic translation termination results from the complex functional interplay between two release factors, eRF1 and eRF3, in which GTP hydrolysis by eRF3 couples codon recognition with peptidyl-tRNA hydrolysis by eRF1. Here, we present a cryo-electron microscopy structure of pre-termination complexes associated with eRF1•eRF3•GDPNP at 9.7 -Å resolution, which corresponds to the initial pre-GTP hydrolysis stage of factor attachment and stop codon recognition. It reveals the ribosomal positions of eRFs and provides insights into the mechanisms of stop codon recognition and triggering of eRF3’s GTPase activity.     

Selection of Essential Medicines for Diabetes in Low and Middle Income Countries: A Survey of 32 National Essential Medicines Lists

Aim

Diabetes is a growing burden especially in low and middle income countries (LMICs). Inadequate access to diabetes care is of particular concern and selection of appropriate diabetes medicines on national essential medicines lists (NEMLs) is a first step in achieving adequate access. This selection was studied among LMICs and influences of various factors associated with selection decisions were assessed.

Methods

Countries were studied if they employed NEMLs for reimbursement or procurement purposes. Presence and number of essential diabetes medicines from different classes, both insulins and oral blood glucose lowering medicines, were surveyed and calculated. Data were also analyzed by country income level, geographic region, year of last update of the NEML and purpose of NEML employment. The effect of prevalence and burden of disease on the number of essential diabetes medicines was also studied. Non parametric tests and univariate linear regression analysis were used.

Results

Nearly all countries (n = 32) had chosen fast (97%) and intermediate acting insulin (93%), glibenclamide and metformin (100% both) as essential medicines. The median number of essential diabetes medicines was 6, equally divided between insulins and oral medicines. 20% of the countries had selected insulin analogues as essential medicines. Among all the studied factors, an increase in burden of diabetes and wealth of countries were associated with selection of higher numbers of essential diabetes medicines (p = 0.02 in both cases).

Conclusions

Nearly all the studied LMICs had included the minimum required medicines for diabetes management in their NEMLs. Selection can still be improved (e.g. exclusion of insulin analogues and replacement of glibenclamide by gliclazide). Nevertheless, the known suboptimal and inconsistent availability of essential diabetes medicines in LMICs cannot be explained by inadequate selection of essential medicines. Countries should therefore be encouraged to give precedence to implementation of NEMLs to make essential diabetes medicines more accessible.     

The Cellulase KORRIGAN Is Part of the Cellulose Synthase Complex

Plant growth and organ formation depend on the oriented deposition of load-bearing cellulose microfibrils in the cell wall. Cellulose is synthesized by a large relative molecular weight cellulose synthase complex (CSC), which comprises at least three distinct cellulose synthases. Cellulose synthesis in plants or bacteria also requires the activity of an endo-1,4-β-d-glucanase, the exact function of which in the synthesis process is not known. Here, we show, to our knowledge for the first time, that a leaky mutation in the Arabidopsis (Arabidopsis thaliana) membrane-bound endo-1,4-β-d-glucanase KORRIGAN1 (KOR1) not only caused reduced CSC movement in the plasma membrane but also a reduced cellulose synthesis inhibitor-induced accumulation of CSCs in intracellular compartments. This suggests a role for KOR1 both in the synthesis of cellulose microfibrils and in the intracellular trafficking of CSCs. Next, we used a multidisciplinary approach, including live cell imaging, gel filtration chromatography analysis, split ubiquitin assays in yeast (Saccharomyces cerevisiae NMY51), and bimolecular fluorescence complementation, to show that, in contrast to previous observations, KOR1 is an integral part of the primary cell wall CSC in the plasma membrane.Cellulose microfibrils are synthesized by a hexameric multiprotein complex at the plasma membrane called the cellulose synthase complex (CSC). Genetic analysis, expression data, and coimmunoprecipitation experiments have demonstrated that a functional CSC contains at least three different nonredundant cellulose synthase (CESA) isoforms (Höfte et al., 2007). CESA1, CESA3, and CESA6-like are involved in cellulose biosynthesis during primary cell wall deposition, whereas CESA4, CESA7, and CESA8 are essential for cellulose synthesis in the secondary cell wall (Taylor et al., 1999, 2000, 2003; Desprez et al., 2007; Persson et al., 2007). CSCs labeled by fluorescently tagged CESA proteins migrate in the plasma membrane along cortical microtubules (CMTs), propelled by the polymerization of the β-1,4-glucans (Paredez et al., 2006). Partial depolymerization of CMTs using oryzalin showed that the organized trajectories of CSCs depend on the presence of an intact CMT array. The CSC-microtubule interaction is mediated at least in part by a large protein, POMPOM2/CELLULOSE SYNTHASE INTERACTING1, that binds to both CESAs and microtubules (Lei et al., 2014). Interestingly, complete depolymerization of CMTs does not alter the velocity of the complexes, illustrating that CMTs are necessary for the guidance of CSCs but not for their movement (Paredez et al., 2006). The microtubule cytoskeleton also has a role in the secretion and internalization of CSCs (Crowell et al., 2009; Gutierrez et al., 2009)KORRIGAN1 (KOR1) is a membrane-bound endo-1,4-β-d-glucanase (EGase) that is also required for cellulose synthesis (Nicol et al., 1998). Enzymatic analysis of a recombinant and soluble form of the Brassica napus KOR1 homolog showed substrate specificity for low-substituted carboxymethyl cellulose and amorphous cellulose but no activity on crystalline cellulose, xyloglucans, or short cellulose oligomers (Mølhøj et al., 2001; Master et al., 2004). Fractionation of microsomes demonstrated that KOR1 is primarily present in plasma membrane fractions but also at low levels in a tonoplast-enriched fraction (Nicol et al., 1998). Similarly, the KOR1 ortholog from tomato (Solanum lycopersicum) was found in the plasma membrane and fractions enriched for the Golgi apparatus (Brummell et al., 1997). A GFP-KOR1 fusion protein expressed with the Cauliflower mosaic virus 35S promoter accumulated in the Golgi apparatus and post-Golgi compartments and the tonoplast (Robert et al., 2005). Surprisingly for an enzyme involved in cellulose synthesis, the protein could not be detected at the plasma membrane. Using this construct, it was also shown that KOR1 undergoes regulated intracellular cycling (Robert et al., 2005).Although numerous genetic studies indicate that KOR1 is required for cellulose synthesis in primary and secondary cell walls and during cell plate formation (Nicol et al., 1998; Peng et al., 2000; Zuo et al., 2000; Lane et al., 2001; Sato et al., 2001; Szyjanowicz et al., 2004), its precise role in the cellulose synthesis process remains unclear. It has been suggested that KOR1 might be a component of the CSC (Read and Bacic, 2002). However, until now there has been no experimental evidence for this in Arabidopsis (Arabidopsis thaliana), either with coprecipitation experiments or with localization studies (Szyjanowicz et al., 2004; Robert et al., 2005; Desprez et al., 2007). Numerous hypotheses have been proposed to explain the paradoxical role of KOR1 in cellulose synthesis (Robert et al., 2004). KOR1 might have a proofreading activity involved in hydrolyzing disordered amorphous cellulose to relieve stress generated during the assembly of glucan chains in cellulose microfibrils (Mølhøj et al., 2002). Alternatively, KOR1 may determine the length of individual cellulose chains, either during cellulose synthesis or once the microfibril has been incorporated in the wall. A third hypothesis is that KOR1 releases the cellulose microfibril from the CSC before the complex is internalized from the plasma membrane (Somerville, 2006). Studies in cotton (Gossypium hirsutum) fiber extracts identified sitosterol glucoside as a primer for the cellulose synthesis and suggested that KOR1 could be involved in their cleavage from the nascent glucan chain (Peng et al., 2002). However, this scenario is unlikely, since, at least for the bacterial CESA, which is homologous to plant CESAs, there is no evidence for the existence of lipid-linked precursors, as shown by the three-dimensional structure of an active complex (Morgan et al., 2013).In this study, we first confirmed previous observations (Paredez et al., 2008) that, in the leaky kor1-1 mutant, the velocity of the CSCs is reduced compared with that in a wild-type background but that, in addition, the mutation affects the ability of the cellulose synthesis inhibitor CGA325′615 (hereafter referred to as CGA) to induce the accumulation of GFP-CESA3 in a microtubule-associated compartment (MASC/small compartments carrying cellulose synthase complexes [SmaCCs]; Crowell et al., 2009; Gutierrez et al., 2009). This indicates that KOR1 plays a role both in the synthesis of cellulose and in the intracellular trafficking of the CSC. Using gel filtration approaches, we identified KOR1 in fractions of high molecular mass, suggesting that KOR1 is present in membranes as part of a protein complex. We next analyzed the dynamics of GFP-KOR1 expressed in the kor1-1 mutant background under the control of its endogenous promoter. GFP-KOR1 is found in discrete particles at the plasma membrane in the same cells as GFP-CESAs (Crowell et al., 2009). GFP-KOR1 plasma membrane particles migrate along linear trajectories with comparable velocities to those observed for GFP-CESAs. The organization of GFP-KOR1 at the plasma membrane also requires the presence of an intact microtubule array, suggesting that KOR1 and CESA trajectories in the plasma membrane are regulated in the same manner. GFP-KOR1 and mCherry-CESA1 partially colocalize in the plasma membrane, Golgi, and post-Golgi compartments. Finally, we provide evidence for direct interaction between KOR1 and primary cell wall CESA proteins using the membrane-based yeast (Saccharomyces cerevisiae NMY51) two-hybrid (MbYTH) system (Timmers et al., 2009) and bimolecular fluorescence complementation (BiFC). Our data support a new model in which KOR1 is an integral part of the CSC, where it plays a role not only in the synthesis of cellulose but also in the intracellular trafficking of the CSC.     

Effects of Toxoplasma gondii and Toxocara canis Antigens on WEHI-164 Fibrosarcoma Growth in a Mouse Model

Cancer is the main cause of death in developed countries. However, in underdeveloped countries infections and parasitic diseases are the main causes of death. There are raising scientific evidences indicating that parasitic infections induce antitumor activity against certain types of cancers. In this study, the effects of Toxoplasma gondii and Toxocara canis egg antigens in comparison with Bacillus Calmette Guerin (BCG) (known to have anticancer distinctive) on WEHI-164 fibosarcoma transplanted to BALB/c mice was investigated. Groups of 6 male BALB/c mice injected with T. gondii antigen, BCG, or T. canis egg antigen as case groups and alum alone as control groups. All mice were then challenged with WEHI-164 fibrosarcoma cells. The mice were examined for growth of the solid tumor and the tumor sizes were measured every other day up to 4 wk. The mean tumor area in T. gondii, BCG, or alum alone injected mice in 4 different days of measurements was 25 mm², 23 mm², and 186 mm² respectively. Also the mean tumor area in T. canis injected mice in 4 different days was 25.5 mm² compared to the control group (alum treated) which was 155 mm². T. gondii parasites and T. canis egg antigens induced inhibition of the tumor growth in the fibrosarcoma mouse model. We need further study to clarify the mechanisms of anti-cancer effects.     

Bauhinia bauhinioides cruzipain inhibitor reduces endothelial proliferation and induces an increase of the intracellular Ca2+ concentration

Proteinase inhibitors, isolated from different types of Bauhinia, have an effect on apoptosis, angiogenesis and inflammation. The Bauhinia bauhinioides cruzipain inhibitor (BbCI) is a Kunitz-type inhibitor and inactivates the cysteine proteinases cruzipain and cruzain from Trypanosoma cruzi. Cruzipain and tissue kallikrein have similar biochemical properties, e.g. the proteolytic cleavage of the kininogen precursor of lys-bradykinin. Tissue kallikrein stimulation in endothelial cells causes migration and capillary tube formation. The aim of this study was to examine whether the antiproliferative effect of BbCI is dependent on changes of the intracellular calcium concentration and membrane hyperpolarization. Endothelial cells were isolated from human umbilical cord veins (HUVEC). For proliferation experiments, HUVEC were incubated with BbCI (10–100 μmol/L) for 48 h. The proliferation was detected by cell counting with a Neubauer chamber. The effect of BbCI (10–100 μM) on the membrane potential was measured with the fluorescence dye DiBAC₄(3) and the effect on [Ca⁺²] _i with the fluorescence probe Fluo-3 AM. The change of the fluorescence intensity was determined with a GENios plate reader (Tecan). The experiments showed that BbCI (10–100 μmol/L) reduces the endothelial cell proliferation significantly in a concentration-dependent manner with a maximum effect at 100 μmol/L (35.1?±?1.8% as compared to control (p?≤?0.05; n?=?45)). As compared to the control, the addition of BbCI (100 μmol/L) caused a significant increase of systolic Ca²⁺ of 28.4?±?5.0% after 30 min incubation. HUVEC treatment with BbCI (100 μmol/L) showed a weak but significant decrease of the membrane potential of 9.5?±?0.9% as compared to control (p?≤?0.05; n?=?80). BbCI influenced significantly the endothelial proliferation, the intracellular Ca²⁺ concentration and the membrane potential.