Release of initiation factors from 48S complexes during ribosomal subunit joining and the link between establishment of codon-anticodon base-pairing and hydrolysis of eIF2-bound GTP

The 40S subunit in 48S complexes formed at the initiation codon of mRNA is bound to eukaryotic initiation factor (eIF) 3, eIF1, eIF1A, and an eIF2/GTP/Met-tRNAi(Met) ternary complex and can therefore not join a 60S subunit directly to form an 80S ribosome. We report that eIF5-induced hydrolysis of eIF2-bound GTP in 48S complexes led to release of eIF2-GDP but not eIF3 or eIF1. eIF5B did not influence factor release in the absence of 60S subunits. Therefore eIF3 and eIF1 dissociate from 40S subunits during, rather than before, the eIF5B-mediated subunit joining event. In the absence of eIF1, eIF5-stimulated hydrolysis of eIF2-bound GTP occurred at the same rate in 43S pre-initiation and 48S initiation complexes. GTP hydrolysis in 43S complexes assembled with eIF1 was much slower than in 43S or 48S complexes assembled without eIF1. Establishment of codon-anticodon base-pairing in 48S complexes relieved eIF1's inhibition. Thus, in addition to its role in initiation codon selection during 48S complex formation, eIF1 also participates in maintaining the fidelity of the initiation process at a later stage, hydrolysis of eIF2-bound GTP, by inhibiting premature GTP hydrolysis and by linking establishment of codon-anticodon base-pairing with GTP hydrolysis.     

Translation initiation factor eIF-5A, the hypusine-containing protein, is phosphorylated on serine and tyrosine and O-glycosylated in Trichomonas vaginalis

The eukaryotic translation factor eIF-5A is highly conserved throughout eukaryotes and undergoes an unusual polyamine-dependent post-translational modification called hypusination. Trichomonas vaginalis has two tveif-5a genes (tveif-5a1 and tveif-5a2), each encoding a 19-kDa protein. In this report, we describe the detection of two forms with different isoelectric points (5.2 and 5.5) that correspond to the precursor and mature TveIF-5A, respectively. In addition, we demonstrated that only the mature form of TveIF-5A is phosphorylated and glycosylated via two-dimensional gel electrophoresis-western blot (2DE-WB) assays using anti-phosphoserine and anti-phosphotyrosine antibodies and the SNA, ConA and MAA lectins. Interestingly, when the protozoa were grown in 1,4-diamino-2-butanone (DAB), an inhibitor of putrescine biosynthesis, and transferred to medium containing exogenous putrescine, a new spot with an isoelectric point of 5.3 was observed, presumably corresponding to a phosphorylated intermediate or deoxyhypusine form. Our data indicate that, in T. vaginalis, phosphorylations and glycosylations are necessary to obtain the mature TveIF-5A, and we confirm the identity of the precursor, intermediate and mature forms of TveIF-5A by mass spectrometry analysis.     

Immunization with a polyprotein vaccine consisting of the T-Cell antigens thiol-specific antioxidant, Leishmania major stress-inducible protein 1, and Leishmania elongation initiation factor protects against leishmaniasis

Development of an effective vaccine against Leishmania infection is a priority of tropical disease research. We have recently demonstrated protection against Leishmania major in the murine and nonhuman primate models with individual or combinations of purified leishmanial recombinant antigens delivered as plasmid DNA constructs or formulated with recombinant interleukin-12 (IL-12) as adjuvant. In the present study, we immunized BALB/c mice with a recombinant polyprotein comprising a tandem fusion of the leishmanial antigens thiol-specific antioxidant, L. major stress-inducible protein 1 (LmSTI1), and Leishmania elongation initiation factor (LeIF) delivered with adjuvants suitable for human use. Aspects of the safety, immunogenicity, and vaccine efficacy of formulations with each individual component, as well as the polyprotein referred to as Leish-111f, were assessed by using the L. major challenge model with BALB/c mice. No adverse reactions were observed when three subcutaneous injections of the Leish-111f polyprotein formulated with either MPL-squalene (SE) or Ribi 529-SE were given to BALB/c mice. A predominant Th1 immune response characterized by in vitro lymphocyte proliferation, gamma interferon production, and immunoglobulin G2A antibodies was observed with little, if any, IL-4. Moreover, Leish-111f formulated with MPL-SE conferred immunity to leishmaniasis for at least 3 months. These data demonstrate success at designing and developing a prophylactic leishmaniasis vaccine that proved effective in a preclinical model using multiple leishmanial antigens produced as a single protein delivered with a powerful Th1 adjuvant suitable for human use.     

Immunization of rhesus macaques with a polyvalent DNA prime/protein boost human immunodeficiency virus type 1 vaccine elicits protective antibody response against simian human immunodeficiency virus of R5 phenotype

The immunogenicity of a poylvalent HIV-1 vaccine comprised of Env antigens from primary R5 isolates was evaluated in rhesus macaques. DNA vaccines encoding four Env antigens from multiple HIV-1 subtypes and HIV-1 Gag antigen from a single subtype elicited a persistent level of binding antibodies to gp120 from multiple HIV-1 isolates that were markedly enhanced following boosting with homologous gp120 proteins in QS-21 adjuvant irrespective of the route of DNA immunization. These sera neutralized homologous and, to a lesser degree, heterologous HIV-1 isolates. Four of the six immunized animals were completely protected following rectal challenge with a SHIV encoding Env from HIV-1(Ba-L), whereas the virus load was reduced in the remaining animals compared to na?ve controls. Hence priming with DNA encoding Env antigens from multiple HIV-1 clades followed by boosting with homologous Env proteins elicits anti-HIV-1 immune responses capable of protecting macaques against mucosal transmission of R5 tropic SHIV isolate.     

Cloning and expression of a major allergen from Cupressus arizonica pollen, Cup a 3, a PR-5 protein expressed under polluted environment

BACKGROUND: This paper describes the cloning and expression of the Cupressus arizonica pollen protein Cup a 3. In addition, we present its modulation under polluted environmental conditions. Species of the Cupressaceae family are important because of their high sensitization prevalence. METHODS: Cup a 3 cloning is based on the sequence of the homologous protein Jun a 3. Cup a 3 was expressed with good yield in the methylotropic yeast Pichia pastoris. RESULTS: Recombinant Cup a 3 (rCup a 3) contains 199 amino acids, 10 potential phosphorylation sites and no glycosylation sites. By immunoblot 63% of cypress allergic patients had specific immunoglobulin E antibodies against rCup a 3 (n = 104). This major allergen is homologous to members of the pathogenesis-related proteins (PR-5 group) and contributes to the overall allergenicity of C. arizonica pollen. Our results show that the increased expression of Cup a 3 is dependent on the pollution in the area where the pollen has been collected, being higher under polluted conditions. CONCLUSIONS: Cup a 3 is a PR-5 protein derived from C. arizonica pollen. The expression of the protein under polluted conditions has a direct incidence on the pollen allergenicity, as has been demonstrated by skin tests and Radioallergosorbent test inhibition.     

Eosinophil leukocyte degranulation in response to serum-opsonized beads: C5a and platelet-activating factor enhance ECP release, with roles for protein kinases A and C

Background Eosinophils have a typical content of granule-bound, cytotoxic, cationic proteins which may. when released to the external milieu, play roles i n diseases such as asthma and parasitic infestation. Therefore, we have investigated possible mechanisms by which their release is regulated in eosinophils.
Methods The enzyme-linked immunosorhent assay (ELISA) was used to detect released eosinophil cationic protein (ECP). Release of ECP was induced by serum-opsonized, nonphagocytosable Sephadex beads (SOS). Results The complement fragment C5a and platelet-activating factor (PAF) were found to enhance ECP release in response to SOS in a dose-dependent fashion, and, contrary to previous reports, they were not found to act as secretagogues themselves on eosinophils in suspension. The role of protein kinase C (PKC) in eosinophil degranulation has been controversial. We found that ECP release induced by SOS was inhibited by the PKC inhibitors staurosporine and calphostin C. Activation of protein kinase A (PKA). by raising cAMP, also inhibited ECP release. Furthermore, pertussis toxin decreased ECP release on opsonized beads, indicating the involvement of pertussis-toxin-sensitive G proteins.
Conclusions C5a. and PAF enhance granule release, rather than acting as secretagogues themselves. PKC and PKA have opposing roles in the regulation of ECP release in response to SOS.     

Combination of the C1429T polymorphism in the G-protein beta-3 subunit gene and the A1330V polymorphism in the low-density lipoprotein receptor-related protein 5 gene is a risk factor for hypercholesterolaemia

The present study examined the relationship between genetic combinations of the C1429T polymorphism in the G-protein beta-3 subunit (GNB3) gene and the A1330V polymorphism in the low-density lipoprotein receptor-related protein 5 (LRP5) gene and the risk of hypercholesterolaemia in Japanese workers. The present study included observations from 1997 to 2002 in 927 males and 662 females who were not hypercholesterolaemic on entry. The endpoint was the development of hypercholesterolaemia, defined as a total cholesterol level ≥240 mg/dl. The odds ratios for the combination of polymorphisms were calculated using pooled logistic regression analyses that incorporated other potential factors into the model. The odds ratios in males and females with GNB3/1429TT and LRP5/1330VV or AV genotypes were 4.17 compared to males with the 1429CT or TT and 1330AA genotypes and 3.53 compared to females with the 1429CC, CT or TT and 1330AA genotypes. Assuming these effects were a mere addition of two independent effects, the odds ratios for both GNB3/1429TT and LRP5/1330VV or AV were estimated to be 3.27 for males and 1.42 for females. Therefore, the synergic effects were shown to be 1.28 times in males (not significant) and 2.49 times in females (P<0.05 by bootstrap method). These results provide clear evidence that the genetic combination has a synergic effect. This study indicates that the combination of GNB3/C1429T and LRP5/A1330V is a very useful marker for predicting the development of hypercholesterolaemia in the general Japanese population.     

IFN‐γ against the 38‐kDa antigen of Mycobacterium tuberculosis discriminates pulmonary tuberculosis from infection and infection from exposure: evidence from a study of human population in a high endemic setting

Mycobacterium tuberculosis (Mtb) 38‐kDa antigen is an immunogenic lipoprotein that induces strong T‐cell responses in experimental animals. However, there is limited information on the role of this antigen in human population. In this article, we present the dynamics of pro‐inflammatory (IFN‐γ and TNF‐α) and anti‐inflammatory cytokine (IL‐10) against the 38 kDa in cohorts of pulmonary TB (PTB) patients, household contacts (HHCs), and community controls (CCs) in a high endemic setting. Whole blood assay was used to determine the levels of cytokines in 149 patients, 149 HHCs, and 68 CCs at baseline, 6 months, and 12 months. At baseline, the level of IFN‐γ was significantly (p < 0.0001) higher in CCs and HHCs than in untreated patients. CCs had significantly (p < 0.05) higher level of IFN‐γ than HHCs. There was no significant difference between treated and untreated patients, and there was no significant change in HHCs over 12 months. At baseline, the levels of IL‐10 and TNF‐α were significantly (p < 0.0001) higher in patients than in HHCs and CCs. No significant change was observed between treated patients and untreated patients and HHCs over time. The study shows that IFN‐γ against the 38 kDa discriminates clinical TB from infection and infection from exposure, suggesting its potential for immune protection and diagnosis.     

Translation elongation factor 1A is a component of the tombusvirus replicase complex and affects the stability of the p33 replication co-factor

Host RNA-binding proteins are likely to play multiple, integral roles during replication of plus-strand RNA viruses. To identify host proteins that bind to viral RNAs, we took a global approach based on the yeast proteome microarray, which contains 4080 purified yeast proteins. The biotin-labeled RNA probes included two distantly related RNA viruses, namely Tomato bushy stunt virus (TBSV) and Brome mosaic virus (BMV). Altogether, we have identified 57 yeast proteins that bound to TBSV RNA and/or BMV RNA. Among the identified host proteins, eleven bound to TBSV RNA and seven bound to BMV RNA with high selectivity, whereas the remaining 39 host proteins bound to both viral RNAs. The interaction between the TBSV replicon RNA and five of the identified host proteins was confirmed via gel-mobility shift and co-purification experiments from yeast. Over-expression of the host proteins in yeast, a model host for TBSV, revealed 4 host proteins that enhanced TBSV replication as well as 14 proteins that inhibited replication. Detailed analysis of one of the identified yeast proteins binding to TBSV RNA, namely translation elongation factor eEF1A, revealed that it is present in the highly purified tombusvirus replicase complex. We also demonstrate binding of eEF1A to the p33 replication protein and a known cis-acting element at the 3′ end of TBSV RNA. Using a functional mutant of eEF1A, we provide evidence on the involvement of eEF1A in TBSV replication.     

Prevalence of hepatitis C virus genotype 1a in Japan and correlation of mutations in the NS5A region and single-nucleotide polymorphism of interleukin-28B with the response to combination therapy with pegylated-interferon-alpha 2b and ribavirin

Hepatitis C virus (HCV) genotype 1a is rare in Japanese patients and the clinical characteristics of this genotype remain unclear. The interferon (IFN) sensitivity‐determining region (ISDR) and single‐nucleotide polymorphisms (SNPs) of interleukin‐28B (IL28B) among patients with HCV genotype 1b are associated with IFN response, but associations among patients with genotype 1a are largely unknown. This study investigated the clinical characteristics of genotype 1a and examined whether genomic heterogeneity of the ISDR and SNPs of IL28B among patients with HCV genotype 1a affects response to combination therapy with pegylated‐IFN‐α2b and ribavirin. Subjects comprised 977 patients infected with HCV genotype 1, including 574 men and 412 women (mean age, 55.2 ± 10.6 years). HCV was genotyped by direct sequencing of the 5′‐untranslated region and/or core regions and confirmed by direct sequencing of the NS5A region. HCV genotypes 1a (n = 32) and 1b (n = 945) were detected. Twenty‐three (71.9%) of the 32 patients with genotype 1a were patients with hemophilia who had received imported clotting factors. Prevalence of genotype 1a after excluding patients with hemophilia was thus 0.9%. Of the 23 patients with genotype 1a who completed IFN therapy, 11 (47.8%) were defined as achieving sustained virological response. Factors related to sustained virological response by univariate analysis were IL28B and ISDR. In conclusion, HCV genotype 1a is rare in Japan. The presence of IL28B genotype TT, and more than two mutations, in the ISDR are associated with a good response to IFN therapy in patients with HCV genotype 1a. J. Med. Virol. 84:438–444, 2012. © 2011 Wiley Periodicals, Inc.     

NS5A resistance leading to failure of 24-week therapy with sofosbuvir/ledipasvir and ribavirin for the treatment of hepatitis C genotype 1a infection in a HIV-1 co-infected patient

Herein we report a previously undescribed case of treatment-emergent non-structural protein 5A (NS5A) resistance mutations, Q30H and Y93C, leading to a failure of 24-week course of sofosbuvir/ledipasvir + ribavirin therapy for the treatment of hepatitis C virus (HCV) genotype 1a in interferon-experienced, human immunodeficiency virus type 1 (HIV-1) co-infected patient with cirrhosis.     

The influence of the multi-basic cleavage site of the H5 hemagglutinin on the attenuation, immunogenicity and efficacy of a live attenuated influenza A H5N1 cold-adapted vaccine virus

A recombinant live attenuated influenza virus ΔH5N1 vaccine with a modified hemagglutinin (HA) and intact neuraminidase genes from A/Vietnam/1203/04 (H5N1) and six remaining genome segments from A/Ann Arbor/6/60 (H2N2) cold-adapted (AA ca) virus was previously shown to be attenuated in chickens, mice and ferrets. Evaluation of the recombinant H5N1 viruses in mice indicated that three independent factors contributed to the attenuation of the ΔH5N1 vaccine: the attenuating mutations specified by the AA ca loci had the greatest influence, followed by the deletion of the H5 HA multi-basic cleavage site (MBS), and the constellation effects of the AA genes acting in concert with the H5N1 glycoproteins. Restoring the MBS in the H5 HA of the vaccine virus improved its immunogenicity and efficacy, likely as a consequence of increased virus replication, indicating that removal of the MBS had a deleterious effect on the immunogenicity and efficacy of the ΔH5N1 vaccine in mice.     

Human C5a and C5a analogs as probes of the neutrophil C5a receptor

Quantitative assessment of the human neutrophil chemotactic response and immunologic evidence demonstrate that human C5a and/or its physiological equivalent, C5a_{des Arg'} serves as the predominant chemotactic factor in complement activated serum. Both C5a and its des Arg74 derivative are capable of promoting directed cellular migration as well as lysosomal enzyme release. When neutrophil chemotaxis is assessed by an ‘under agarose methodology’, in the absence of extraneous human serum proteins. C5a is found to be some 10- to 30-fold more active than C5a_{des Arg} Quantitatively similar results are obtained when each factor is assessed for its ability to promote secretion of azurophilic granular enzymes from cytochalasin B-treated neutrophils. The C5a structural analog C5a-(1–69), lacking five residues of the C-terminal portion of the parent molecule, and a synthetic pentapeptide l-methionyl-l-glutaminyl-l-leucylglycyl-l-arginine, which mimics the C-terminal linear sequence of C5a (Chenoweth et al., 1979a), are found to be devoid of biological activity when examined either-alone or in combination. These findings imply that the C-terminal portions of C5a contribute importantly to modulating ligand-receptor interactions on neutrophils. Based on these observations, a conceptual model of the interaction of human C5a with the neutrophil C5a receptor is proposed. We hypothesize that C5a possesses an internal ‘recognition’ site in addition to the ‘activation’ site contained in the C-terminal region of the molecule.     

Characterization of human variable domain antibody fragments against the U1 RNA-associated A protein,selected from a synthetic and a patient-derived combinatorial V gene library

This is the first study describing recombinant human antibody fragments directed to the U1 RNA-associated A protein (U1A). Three anti-U1A antibody fragments (Fab) were isolated from a semi-synthetic human Fab library and one anti-U1A single-chain variable fragment (scFv) was isolated from a library which was derived from the IgG-positive splenic lymphocytes of an autoimmune patient. Competition studies with autoantibodies against the U1 small nuclear ribonucleoprotein (snRNP) particle from patients with systemic lupus erythematosus (SLE) and SLE-overlap syndromes revealed that U1A binding of these antibody fragments can be inhibited by about 40% of the patient sera. All antibody fragments recognized the native U1 snRNP in immunoprecipitation assays. Two of three Fab clones as well as the scFv clone derived from the repertoire of an autoimmune patient use the same heavy chain germ-line gene DP-65. Epitope mapping revealed that these three clones appear to recognize an identical epitope domain present on the C-terminal RNP motif of the U1A protein. The DP-65 heavy chain gene is used in less than 1% of the B cells in healthy individuals, while three out of four anti-U1A antibody fragments use this gene. This points to a restricted V_H gene usage in the case of U1A, suggesting that the DP-65 heavy chain has a natural shape complementarity to the U1A protein.     

Cofactor regulation of C5a chemotactic activity in physiological fluids. Requirement for the vitamin D binding protein, thrombospondin-1 and its receptors

Factors in physiological fluids that regulate the chemotactic activity of complement activation peptides C5a and C5a des Arg are not well understood. The vitamin D binding protein (DBP) has been shown to significantly enhance chemotaxis to C5a/C5a des Arg. More recently, platelet-derived thrombospondin-1 (TSP-1) has been shown to facilitate the augmentation of C5a-induced chemotaxis by DBP. The objective of this study was to better characterize these chemotactic cofactors and investigate the role that cell surface TSP-1 receptors CD36 and CD47 may play in this process. The chemotactic activity in C-activated normal serum, citrated plasma, DBP-depleted serum or C5 depleted serum was determined for both normal human neutrophils and U937 cell line transfected with the C5a receptor (U937–C5aR). In addition, levels of C5a des Arg, DBP and TSP-1 in these fluids were measured by RIA or ELISA. Results show that there is a clear hierarchy with C5a being the essential primary signal (DBP or TSP-1 will not function in the absence of C5a), DBP the necessary cofactor and TSP-1 a dependent tertiary factor, since it cannot function to enhance chemotaxis to C5a without DBP. Measurement of the C5a-induced intracellular calcium flux confirmed the same hierarchy observed with chemotaxis. Moreover, analysis of bronchoalveolar lavage fluid (BALF) from patients with the adult respiratory distress syndrome (ARDS) demonstrated that C5a-dependent chemotactic activity is significantly decreased after anti-DBP treatment. Finally, results show that TSP-1 utilizes cell surface receptors CD36 and CD47 to augment chemotaxis, but DBP does not bind to TSP-1, CD36 or CD47. The results clearly demonstrate that C5a/C5a des Arg needs both DBP and TSP-1 for maximal chemotactic activity and suggest that the regulation of C5a chemotactic activity in physiological fluids is more complex than previously thought.     

Apoptosis-inducing factor: a matter of neuron life and death

The mitochondrial flavoprotein apoptosis-inducing factor (AIF) is the main mediator of caspase-independent apoptosis-like programmed cell death. Upon pathological permeabilization of the outer mitochondrial membrane, AIF is translocated to the nucleus, where it participates in chromatin condensation and is associated to large-scale DNA fragmentation. Heavy down-regulation of AIF expression in mutant mice or reduced AIF expression achieved with small interfering RNA (siRNA) provides neuroprotection against acute neurodegenerative insults. Paradoxically, in addition to its pro-apoptotic function, AIF likely plays an anti-apoptotic role by regulating the production of reactive oxygen species (ROS) via its putative oxidoreductase and peroxide scavenging activities. In this review, we discuss accumulating evidence linking AIF to both acute and chronic neurodegenerative processes by emphasising mechanisms underlying the dual roles apparently played by AIF in these processes.     

Eating ourselves to death (and despair): The contribution of adiposity and inflammation to depression

Obesity and related metabolic conditions are of epidemic proportions in most of the world, affecting both adults and children. The accumulation of lipids in the body in the form of white adipose tissue in the abdomen is now known to activate innate immune mechanisms. Lipid accumulation causes adipocytes to directly secrete the cytokines interleukin (IL) 6 and tumor necrosis factor α (TNFα), but also monocyte chemoattractant protein 1 (MCP-1), which results in the accumulation of leukocytes in fat tissue. This sets up a chronic inflammatory state which is known to mediate the association between obesity and conditions such as cardiovascular disease, type 2 diabetes, and cancer. There is also a substantial literature linking inflammation with risk for depression. This includes the observations that: (1) people with inflammatory diseases such as multiple sclerosis, cardiovascular disease, and psoriasis have elevated rates of depression; (2) many people administered inflammatory cytokines such as interferon α develop depression that is indistinguishable from depression in non-medically ill populations; (3) a significant proportion of depressed persons show upregulation of inflammatory factors such as IL-6, C-reactive protein, and TNFα; (4) inflammatory cytokines can interact with virtually every pathophysiologic domain relevant to depression, including neurotransmitter metabolism, neuroendocrine function, and synaptic plasticity. While many factors may contribute to the association between inflammatory mediators and depression, we hypothesize that increased adiposity may be one causal pathway. Mediational analysis suggests a bi-directional association between adiposity and depression, with inflammation possibly playing an intermediary role.