Interactions among Trypanosoma brucei RAD51 paralogues in DNA repair and antigenic variation

Homologous recombination in Trypanosoma brucei is used for moving variant surface glycoprotein (VSG) genes into expression sites during immune evasion by antigenic variation. A major route for such VSG switching is gene conversion reactions in which RAD51, a universally conserved recombinase, catalyses homology-directed strand exchange. In any eukaryote, RAD51-directed strand exchange in vivo is mediated by further factors, including RAD51-related proteins termed Rad51 paralogues. These appear to be ubiquitously conserved, although their detailed roles in recombination remain unclear. In T. brucei, four putative RAD51 paralogue genes have been identified by sequence homology. Here we show that all four RAD51 paralogues act in DNA repair, recombination and RAD51 subnuclear dynamics, though not equivalently, while mutation of only one RAD51 paralogue gene significantly impedes VSG switching. We also show that the T. brucei RAD51 paralogues interact, and that the complexes they form may explain the distinct phenotypes of the mutants as well as observed expression interdependency. Finally, we document the Rad51 paralogues that are encoded by a wide range of protists, demonstrating that the Rad51 paralogue repertoire in T. brucei is unusually large among microbial eukaryotes and that one member of the protein family corresponds with a key, conserved eukaryotic Rad51 paralogue.     

An essential Aurora-related kinase transiently associates with spindle pole bodies during Plasmodium falciparum erythrocytic schizogony

Aurora kinases compose a family of conserved Ser/Thr protein kinases playing essential roles in eukaryotic cell division. To date, Aurora homologues remain uncharacterized in the protozoan phylum Apicomplexa. In malaria parasites, the characterization of Aurora kinases may help understand the cell cycle control during erythrocytic schizogony where asynchronous nuclear divisions occur. In this study, we revisited the kinome of Plasmodium falciparum and identified three Aurora-related kinases, Pfark-1, -2, -3. Among these, Pfark-1 is highly conserved in malaria parasites and also appears to be conserved across Apicomplexa. By tagging the endogenous Pfark-1 gene with the green fluorescent protein (GFP) in live parasites, we show that the Pfark-1-GFP protein forms paired dots associated with only a subset of nuclei within individual schizonts. Immunofluorescence analysis using an anti-α-tubulin antibody strongly suggests a recruitment of Pfark-1 at duplicated spindle pole bodies at the entry of the M phase of the cell cycle. Unsuccessful attempts at disrupting the Pfark-1 gene with a knockout construct further indicate that Pfark-1 is required for parasite growth in red blood cells. Our study provides new insights into the cell cycle control of malaria parasites and reports the importance of Aurora kinases as potential targets for new antimalarials.     

Frequent detection of escape from cytotoxic T-lymphocyte recognition in perinatal human immunodeficiency virus (HIV) type 1 transmission: the ariel project for the prevention of transmission of HIV from mother to infant

Host immunologic factors, including human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL), are thought to contribute to the control of HIV type 1 (HIV-1) replication and thus delay disease progression in infected individuals. Host immunologic factors are also likely to influence perinatal transmission of HIV-1 from infected mother to infant. In this study, the potential role of CTL in modulating HIV-1 transmission from mother to infant was examined in 11 HIV-1-infected mothers, 3 of whom transmitted virus to their offspring. Frequencies of HIV-1-specific human leukocyte antigen class I-restricted CTL responses and viral epitope amino acid sequence variation were determined in the mothers and their infected infants. Maternal HIV-1-specific CTL clones were derived from each of the HIV-1-infected pregnant women. Amino acid substitutions within the targeted CTL epitopes were more frequently identified in transmitting mothers than in nontransmitting mothers, and immune escape from CTL recognition was detected in all three transmitting mothers but in only one of eight nontransmitting mothers. The majority of viral sequences obtained from the HIV-1-infected infant blood samples were susceptible to maternal CTL. These findings demonstrate that epitope amino acid sequence variation and escape from CTL recognition occur more frequently in mothers that transmit HIV-1 to their infants than in those who do not. However, the transmitted virus can be a CTL susceptible form, suggesting inadequate in vivo immune control.     

N-terminal guanidinylation of TIPP (Tyr-Tic-Phe-Phe) peptides results in major changes of the opioid activity profile

Derivatives of peptides of the TIPP (Tyr-Tic-Phe-Phe; Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) family containing a guanidino (Guan) function in place of the N-terminal amino group were synthesized in an effort to improve their blood–brain barrier permeability. Unexpectedly, N-terminal amidination significantly altered the in vitro opioid activity profiles. Guan-analogues of TIPP-related δ opioid antagonists showed δ partial agonist or mixed δ partial agonist/μ partial agonist activity. Guanidinylation of the mixed μ agonist/δ antagonists H-Dmt-Tic-Phe-Phe-NH₂ (DIPP-NH₂) and H-Dmt-TicΨ[CH₂NH]Phe-Phe-NH₂ (DIPP-NH₂[Ψ]) converted them to mixed μ agonist/δ agonists. A docking study revealed distinct positioning of DIPP-NH₂ and Guan-DIPP-NH₂ in the δ receptor binding site. Lys³-analogues of DIPP-NH₂ and DIPP-NH₂[Ψ] (guanidinylated or non-guanidinylated) turned out to be mixed μ/κ agonists with δ antagonist-, δ partial agonist- or δ full agonist activity. Compounds with some of the observed mixed opioid activity profiles have therapeutic potential as analgesics with reduced side effects or for treatment of cocaine addiction.     

Aldehyde oxidase cross-reacting material in the Aldox n,cin, mal,and lxd mutants of Drosophila melanogaster

Rocket immunoelectrophoresis was used to estimate aldehyde oxidase cross-reacting material (AO-CRM) in larval hemolymph and adult fly extracts in mutants with reduced AO enzymatic activity. Hemolymph of larvae homozygous for Aldox ⁿ, which is a mutation of the presumed structural gene for AO, contains 30% of the wild-type CRM. The demonstration of AO-CRM in Aldox ⁿ larval hemolymph is surprising since this genotype has been reported to lack CRM. By contrast, adult Aldox ⁿ flies lack detectable CRM. The other AO-deficient mutants that were examined are cin, mal, and lxd; each has appreciable levels of CRM in both larval hemolymph and adult extracts. Detection of CRM in these mutants helps to clarify conflicting reports in the literature.This research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to L.W.B.     

Effect of stocking rate on soil-atmosphere CH4 flux during spring freeze-thaw cycles in a northern desert steppe, China

BACKGROUND: Methane (CH(4)) uptake by steppe soils is affected by a range of specific factors and is a complex process. Increased stocking rate promotes steppe degradation, with unclear consequences for gas exchanges. To assess the effects of grazing management on CH(4) uptake in desert steppes, we investigated soil-atmosphere CH(4) exchange during the winter-spring transition period. METHODOLOGY/MAIN FINDING: The experiment was conducted at twelve grazing plots denoting four treatments defined along a grazing gradient with three replications: non-grazing (0 sheep/ha, NG), light grazing (0.75 sheep/ha, LG), moderate grazing (1.50 sheep/ha, MG) and heavy grazing (2.25 sheep/ha, HG). Using an automatic cavity ring-down spectrophotometer, we measured CH(4) fluxes from March 1 to April 29 in 2010 and March 2 to April 27 in 2011. According to the status of soil freeze-thaw cycles (positive and negative soil temperatures occurred in alternation), the experiment was divided into periods I and II. Results indicate that mean CH(4) uptake in period I (7.51 μg CH(4)-C m(-2) h(-1)) was significantly lower than uptake in period II (83.07 μg CH(4)-C m(-2) h(-1)). Averaged over 2 years, CH(4) fluxes during the freeze-thaw period were -84.76 μg CH(4)-C m(-2) h(-1) (NG), -88.76 μg CH(4)-C m(-2) h(-1) (LG), -64.77 μg CH(4)-C m(-2) h(-1) (MG) and -28.80 μg CH(4)-C m(-2) h(-1) (HG). CONCLUSIONS/SIGNIFICANCE: CH(4) uptake activity is affected by freeze-thaw cycles and stocking rates. CH(4) uptake is correlated with the moisture content and temperature of soil. MG and HG decreases CH(4) uptake while LG exerts a considerable positive impact on CH(4) uptake during spring freeze-thaw cycles in the northern desert steppe in China.     

Cross-species Proteomics Reveals Specific Modulation of Signaling in Cancer and Stromal Cells by Phosphoinositide 3-kinase (PI3K) Inhibitors

The tumor microenvironment plays key roles in cancer biology, but its impact on the regulation of signaling pathway activity in cancer cells has not been systemically investigated. We designed an analytical strategy that allows differential analysis of signaling between cancer and stromal cells present in tumor xenografts. We used this approach to investigate how in vivo growth conditions and PI3K inhibitors regulate pathway activities in both cancer and stromal cell populations. We found that, despite inducing more modest changes in protein expression, in vivo growing conditions extensively rewired protein kinase networks in cancer cells. As a result, different sets of phosphorylation sites were modulated by PI3K inhibitors in cancer cells growing in tumors relative to when these cells were in culture. The p110δ PI3K-selective compound CAL-101 (Idelalisib) did not inhibit markers of PI3K activity in cancer or stromal cells; however, unexpectedly, it induced phosphorylation on SQ motifs in both subpopulations of tumor cells in vivo but not in vitro. Thus, the interaction between cancer cells and the stroma modulated the ability of PI3K inhibitors to induce the activation of apoptosis in solid tumors. Our study provides proof-of-principle of a proteomics workflow for measuring signaling specifically in cancer and stromal cells and for investigating how cancer biochemistry is modulated in vivo.Solid tumors contain a heterogeneous population of cells. Transformed epithelial cells recruit different types of somatic cells to the tumor microenvironment where they influence varying aspects of cancer biology. The role of heterotypic communication between normal stromal cells and transformed cancer cells is well established (1, 2). Different somatic cell types, including fibroblasts, epithelial cells, and cells of the immune system—all of which are found in tumors—promote cancer cell development by means of gap-junction intercellular communication, direct cell-to-cell contacts, and by the release of growth factors, enzymes, and cytokines that act on neighboring malignant cells (3–6).The tumor microenvironment determines the ability of cancer cells to survive in specific organs and their ability to proliferate and metastasize (7–9). Growth factors released from tumor-associated stromal cells also influence how cancer cells respond to drug administration (10). Therefore, the advancement of targeted cancer therapies requires an understanding of how the tumor microenvironment modulates the biochemistry of transformed cancer cells. In addition, targeting the tumor stroma is emerging as an intriguing concept for the development of anti-cancer therapies (11). It is therefore important to investigate specific effects of compounds in clinical development on stromal cells in addition to those exerted toward malignant cancer cells (12).Here we investigated the effects that changes in growing conditions from a two-dimensional cell culture to an in vivo three-dimensional tumor environment had in modulating protein and phosphoprotein expression in human cancer cells. For this, we used mass spectrometry (MS) to specifically measure cancer and stromal proteomes and phosphoproteomes within mouse tumor xenografts.We also investigated the effects that the pharmacological inhibitors of PI3K, namely GDC-0941 or CAL-101, would have on the phosphoproteomes of stromal cells relative to cancer cells in solid tumors. GDC-0941 is an inhibitor with specificity for class I PI3Ks, whereas CAL-101 specificity is restricted to the p110δ isoform of PI3K (13, 14), which in untransformed tissues is mainly found in leukocytes (15). The PI3K signaling pathway is often deregulated in different cancer types (16), including colorectal cancer (17), and both compounds used in this study are in different stages of clinical development (18–20). PI3K signaling has also been implicated in mediating the effects that the microenvironment has on cancer cells (21).We found that in vivo growth conditions had profound effects on phosphoprotein expression, which was reflected on the phosphorylation sites modulated by PI3K inhibitors in vivo relative to in vitro and in their ability to induce apoptotic markers across these two cell culture conditions.     

PfeIK1, a eukaryotic initiation factor 2α kinase of the human malaria parasite Plasmodium falciparum, regulates stress-response to amino-acid starvation

Background

Plasmodium falciparum -parasitized red blood cells (RBCs) are equipped with protective antioxidant enzymes and heat shock proteins (HSPs). The latter are only considered to protect against thermal stress. Important issues are poorly explored: first, it is insufficiently known how both systems are expressed in relation to the parasite developmental stage; secondly, it is unknown whether P. falciparum HSPs are redox-responsive, in view of redox sensitivity of HSP in eukaryotic cells; thirdly, it is poorly known how the antioxidant defense machinery would respond to increased oxidative stress or inhibited antioxidant defense. Those issues are interesting as several antimalarials increase the oxidative stress or block antioxidant defense in the parasitized RBC. In addition, numerous inhibitors of HSPs are currently developed for cancer therapy and might be tested as anti-malarials. Thus, the joint disruption of the parasite antioxidant enzymes/HSP system would interfere with parasite growth and open new perspectives for anti-malaria therapy.

Methods

Stage-dependent mRNA expression of ten representative P. falciparum antioxidant enzymes and hsp 60/70–2/70–3/75/90 was studied by quantitative real-time RT-PCR in parasites growing in normal RBCs, in RBCs oxidatively-stressed by moderate H2O2 generation and in G6PD-deficient RBCs. Protein expression of antioxidant enzymes was assayed by Western blotting. The pentosephosphate-pathway flux was measured in isolated parasites after Sendai-virus lysis of RBC membrane.

Results

In parasites growing in normal RBCs, mRNA expression of antioxidant enzymes and HSPs displayed co-ordinated stage-dependent modulation, being low at ring, highest at early trophozoite and again very low at schizont stage. Additional exogenous oxidative stress or growth in antioxidant blunted G6PD-deficient RBCs indicated remarkable flexibility of both systems, manifested by enhanced, co-ordinated mRNA expression of antioxidant enzymes and HSPs. Protein expression of antioxidant enzymes was also increased in oxidatively-stressed trophozoites.

Conclusion

Results indicated that mRNA expression of parasite antioxidant enzymes and HSPs was co-ordinated and stage-dependent. Secondly, both systems were redox-responsive and showed remarkably increased and co-ordinated expression in oxidatively-stressed parasites and in parasites growing in antioxidant blunted G6PD-deficient RBCs. Lastly, as important anti-malarials either increase oxidant stress or impair antioxidant defense, results may encourage the inclusion of anti-HSP molecules in anti-malarial combined drugs.     

Olfactory function in Australian aboriginal children and chronic otitis media

Chronic suppurative otitis media (CSOM), a severe form of middle ear infection, affects most Australian Aboriginal children with up to 50% in some communities suffering hearing loss as a consequence. To date, there is no information on whether repeated exposure to the pathogens that characterize CSOM and that are present in the upper respiratory airway affect olfactory function. Accordingly, this study aimed to determine whether 1) there was a high prevalence of olfactory loss in Aboriginal children and 2) hearing loss is a predictor of olfactory loss. Two hundred and sixty one 9- to 12-year-old Aboriginal children from 16 rural communities reported to have high prevalences of CSOM and hearing loss were assessed for olfactory loss using a 16-odor identification test and hearing loss. One child was found to be anosmic, 4 were slightly hyposmic, and 42 had hearing loss. No relationship was found between olfactory loss and hearing loss. The test-retest reliability of the 16-odor identification test was 0.98. It was concluded that CSOM does not appear to affect olfactory function in the long term and that hearing loss in Aboriginal children is not a predictor of olfactory loss.     

Twenty-five dyneins in Tetrahymena: A re-examination of the multidynein hypothesis

Dyneins are responsible for essential movements in eukaryotic cells. The motor activity of each dynein complex resides in its complement of heavy chains. In the present study, we examined 136 heavy chain sequences from the completed genomes of 11 diverse model organisms, including examples from Viridiplantae, Excavata, Chromalveolata, and Metazoa. In many cases, we discovered dynein heavy chains previously not identified. For example, Tetrahymena expresses a total of 25 DYH genes rather than the previously identified 14. The Tetrahymena DYH genes are nonaxonemal DYH1 and DYH2; axonemal outer arm alpha, beta, and gamma; axonemal two-headed inner arm 1alpha and 1beta; and 18 single-headed inner arm heavy chains. The heavy chains divide into nine classes; six of these are highly conserved in sequence and number of isoforms in a given organism. The other three are single-headed inner arm dyneins, whose numbers vary significantly in different organisms. These findings lead to two conclusions. One, the last common ancestor of all eukaryotes expressed nine different dynein heavy chains. Two, subsequent to the divergences leading to different organisms, additional dynein heavy chains emerged. These newer dyneins are not well conserved across species and the variation may reflect different motility requirements in different organisms. Together, these results suggest that each of the nine classes of dyneins is functionally distinct, but members within some of the classes are not specialized. An understanding of the relationships among the various dynein heavy chains is important when deducing functions across species.