Human ribosomal protein L13a is dispensable for canonical ribosome function but indispensable for efficient rRNA methylation

Previously, we demonstrated that treatment of monocytic cells with IFN-gamma causes release of ribosomal protein L13a from the 60S ribosome and subsequent translational silencing of Ceruloplasmin (Cp) mRNA. Here, evidence using cultured cells demonstrates that Cp mRNA silencing is dependent on L13a and that L13a-deficient ribosomes are competent for global translational activity. Human monocytic U937 cells were stably transfected with two different shRNA sequences for L13a and clonally selected for more than 98% abrogation of total L13a expression. Metabolic labeling of these cells showed rescue of Cp translation from the IFN-gamma mediated translational silencing activity. Depletion of L13a caused significant reduction of methylation of ribosomal RNA and of cap-independent translation mediated by Internal Ribosome Entry Site (IRES) elements derived from p27, p53, and SNAT2 mRNAs. However, no significant differences in the ribosomal RNA processing, polysome formation, global translational activity, translational fidelity, and cell proliferation were observed between L13a-deficient and wild-type control cells. These results support the notion that ribosome can serve as a depot for releasable translation-regulatory factors unrelated to its basal polypeptide synthetic function. Unlike mammalian cells, the L13a homolog in yeast is indispensable for growth. Thus, L13a may have evolved from an essential ribosomal protein in lower eukaryotes to having a role as a dispensable extra-ribosomal function in higher eukaryotes.     

Roles of the negatively charged N-terminal extension of Saccharomyces cerevisiae ribosomal protein S5 revealed by characterization of a yeast strain containing human ribosomal protein S5

Ribosomal protein (rp) S5 belongs to a family of ribosomal proteins that includes bacterial rpS7. rpS5 forms part of the exit (E) site on the 40S ribosomal subunit and is essential for yeast viability. Human rpS5 is 67% identical and 79% similar to Saccharomyces cerevisiae rpS5 but lacks a negatively charged (pI approximately 3.27) 21 amino acid long N-terminal extension that is present in fungi. Here we report that replacement of yeast rpS5 with its human homolog yielded a viable yeast strain with a 20%-25% decrease in growth rate. This replacement also resulted in a moderate increase in the heavy polyribosomal components in the mutant strain, suggesting either translation elongation or termination defects, and in a reduction in the polyribosomal association of the elongation factors eEF3 and eEF1A. In addition, the mutant strain was characterized by moderate increases in +1 and -1 programmed frameshifting and hyperaccurate recognition of the UAA stop codon. The activities of the cricket paralysis virus (CrPV) IRES and two mammalian cellular IRESs (CAT-1 and SNAT-2) were also increased in the mutant strain. Consistently, the rpS5 replacement led to enhanced direct interaction between the CrPV IRES and the mutant yeast ribosomes. Taken together, these data indicate that rpS5 plays an important role in maintaining the accuracy of translation in eukaryotes and suggest that the negatively charged N-terminal extension of yeast rpS5 might affect the ribosomal recruitment of specific mRNAs.     

Pathogenic Potential,Genetic Diversity,and Population Structure of Escherichia coli Strains Isolated from a Forest-Dominated Watershed (Comox Lake) in British Columbia,Canada

Escherichia coli isolates (n = 658) obtained from drinking water intakes of Comox Lake (2011 to 2013) were screened for the following virulence genes (VGs): stx₁ and stx₂ (Shiga toxin-producing E. coli [STEC]), eae and the adherence factor (EAF) gene (enteropathogenic E. coli [EPEC]), heat-stable (ST) enterotoxin (variants STh and STp) and heat-labile enterotoxin (LT) genes (enterotoxigenic E. coli [ETEC]), and ipaH (enteroinvasive E. coli [EIEC]). The only genes detected were eae and stx₂, which were carried by 37.69% (n = 248) of the isolates. Only eae was harbored by 26.74% (n = 176) of the isolates, representing potential atypical EPEC strains, while only stx₂ was detected in 10.33% (n = 68) of the isolates, indicating potential STEC strains. Moreover, four isolates were positive for both the stx₂ and eae genes, representing potential EHEC strains. The prevalence of VGs (eae or stx₂) was significantly (P < 0.0001) higher in the fall season, and multiple genes (eae plus stx₂) were detected only in fall. Repetitive element palindromic PCR (rep-PCR) fingerprint analysis of 658 E. coli isolates identified 335 unique fingerprints, with an overall Shannon diversity (H′) index of 3.653. Diversity varied among seasons over the years, with relatively higher diversity during fall. Multivariate analysis of variance (MANOVA) revealed that the majority of the fingerprints showed a tendency to cluster according to year, season, and month. Taken together, the results indicated that the diversity and population structure of E. coli fluctuate on a temporal scale, reflecting the presence of diverse host sources and their behavior over time in the watershed. Furthermore, the occurrence of potentially pathogenic E. coli strains in the drinking water intakes highlights the risk to human health associated with direct and indirect consumption of untreated surface water.     

Potentiating effects of MPL on DSPC bearing cationic liposomes promote recombinant GP63 vaccine efficacy: high immunogenicity and protection

Background

Vaccines that activate strong specific Th1-predominant immune responses are critically needed for many intracellular pathogens, including Leishmania. The requirement for sustained and efficient vaccination against leishmaniasis is to formulate the best combination of immunopotentiating adjuvant with the stable antigen (Ag) delivery system. The aim of the present study is to evaluate the effectiveness of an immunomodulator on liposomal Ag through subcutaneous (s.c.) route of immunization, and its usefulness during prime/boost against visceral leishmaniasis (VL) in BALB/c mice.

Methodology/Principal Findings

Towards this goal, we formulated recombinant GP63 (rGP63)-based vaccines either with monophosphoryl lipid A-trehalose dicorynomycolate (MPL-TDM) or entrapped within cationic liposomes or both. Combinatorial administration of liposomes with MPL-TDM during prime confers activation of dendritic cells, and induces an early robust T cell response. To investigate whether the combined formulation is required for optimum immune response during boost as well, we chose to evaluate the vaccine efficacy in mice primed with combined adjuvant system followed by boosting with either rGP63 alone, in association with MPL-TDM, liposomes or both. We provide evidences that the presence of either liposomal rGP63 or combined formulations during boost is necessary for effective Th1 immune responses (IFN-γ, IL-12, NO) before challenge infection. However, boosting with MPL-TDM in conjugation with liposomal rGP63 resulted in a greater number of IFN-γ producing effector T cells, significantly higher levels of splenocyte proliferation, and Th1 responses compared to mice boosted with liposomal rGP63, after virulent Leishmania donovani (L. donovani) challenge. Moreover, combined formulations offered superior protection against intracellular amastigote replication in macrophages in vitro, and hepatic and splenic parasite load in vivo.

Conclusion

Our results define the immunopotentiating effect of MPL-TDM on protein Ag encapsulated in a controlled release system against experimental VL.     

Synthesis and pharmacological activities of some mononuclear Ru(II) complexes

A series of mononuclear Ru(II) complexes of the type [Ru(M)2(U)]2+, where M = 2,2'-bipyridine/1,10-phenanthroline and U = tpl (Ru1), 4-Cl-tpl (Ru2), 4-CH3-tpl (Ru3), 4-CH3O-tpl (Ru4), and 4-NO2-tpl (Ru5), -pai (Ru6), where tpl = thiopicolinanilide and pai = 2-phenyl-azo-imidazole, have been prepared and characterized by IR, UV-Vis, 1H NMR, 13C-NMR, FAB-Mass spectrophotometer, and elemental analysis. The complexes display metal-ligand charge transfer (MLCT) transitions in the visible region. The title complexes were subjected to in vivo anticancer activity tests against a transplantable murine tumor cell line, Ehrlich's ascitic carcinoma (EAC) and in vitro antibacterial activity against Gram positive and Gram negative microorganisms. Ru1-Ru6 were found to increase the life span of the tumor hosts by 19-52%, and decreased tumor volume and viable ascitic cell count. The results of the present study clearly demonstrated the tumor inhibitory activity of the ruthenium chelates against transplantable murine tumor cell line. The treatment with ruthenium complexes could be secondary to tumor regression or due to the action of the compounds itself. The significant antibacterial activity was observed for Ru1-Ru4 against microorganisms like Vibrio cholera 865, Staphylococcus aureus 6571, and Shigella flexneri as compared to that of standard drug chloramphenical. Ru5 showed moderate activity against S. aureus 8530. However, all the complexes fail to show significant antibacterial activity against V. cholera 14033 and Shigella sonnai.     

Fas-, caspase 8-, and caspase 3-dependent signaling regulates the activity of the aminophospholipid translocase and phosphatidylserine externalization in human erythrocytes

Apoptosis and erythrocyte senescence share the common feature of exposure of phosphatidylserine (PS) in the outer leaflet of the cells. Western analysis showed that mature red cells contain Fas, FasL, Fas-associated death domain (FADD), caspase 8, and caspase 3. Circulating, aged cells showed colocalization of Fas with the raft marker proteins Galpha(s) and CD59; the existence of Fas-associated FasL, FADD and caspase 8; and caspase 8 and caspase 3 activity. Aged red cells had significantly lower aminophospholipid translocase activity and higher levels of PS externalization in comparison with young cells. In support of our contention that caspases play a functional role in the mature red cell, the oxidatively stressed red cell recapitulated apoptotic events, including translocation of Fas into rafts, formation of a Fas-associated complex, and activation of caspases 8 and 3. These events were independent of calpain but dependent on reactive oxygen species (ROS) as evident from the effects of the ROS scavenger N-acetylcysteine. Caspase activation was associated with loss of aminophospholipid translocase activity and with PS externalization. ROS was not generated by treatment of cells with t-butyl hydroperoxide at 10 degrees C, and Fas did not translocate into rafts. Concomitantly, neither formation of a Fas-associated signaling complex nor caspase activation could be observed, supporting the view that translocation of Fas into rafts was the trigger for the chain of events leading to caspase 3 activation. Our data demonstrate for the first time the novel involvement of Fas/caspase 8/caspase 3-dependent signaling in an enucleated cell leading to PS externalization, a central feature of erythrophagocytosis and erythrocyte biology.     

Molecular modeling and NMR studies of benzyl substituted mannosyl trisaccharide binding to two mannose‐specific lectins: Allium sativam agglutinin I and Concanavalin A

The interaction of trimannoside, α?benzyl 3, 6‐di‐O‐(α‐D ‐mannopyranosyl)‐α‐D ‐mannopyranoside, 1 with ASAI (Allium sativam agglutinin I, garlic lectin) was studied to reveal the conformational preferences of this ligand in bound‐state and detailed binding mode at atomic level. The binding phenomenon was then compared with another well‐known mannose‐binding lectin, ConA (Concanavalin A). Structural studies of the ligand in free state were done using NMR spectroscopy and Molecular Dynamics simulations. It is found that the substituted‐trimannoside can undergo conformational transitions in solution, with one major and one minor conformation per glycosidic linkage (α 1→3 and α 1→6). On the other hand in the bound‐state only one of the two major conformations was significantly populated. The role of phenyl ring in the binding process was explored. An extended binding site was observed for the trimannoside in ASAI utilizing the aromatic substituent, which is not seen in ConA. Binding data from difference absorption spectroscopy supported this fact that the binding of benzyl‐substituted ligand is tighter with ASAI than ConA. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 952–967, 2010.     

Limitation of glucose oxidase method of glucose estimation in jaundiced neonates

The most widely used method for estimation of plasma glucose is that adopted by Trinder's using glucose oxidase-peroxidase (GOD-POD) system. This method gives much lower blood glucose values with blood samples of neonatal jaundice (plasma bilirubin level > 10 mg/dL) of age 10 +/- 5 daysthan with samples of neonates of the same age group without jaundice or older children suffering from other diseases like acute respiratory distress, septicemia.