Growth and Airborne Transmission of Cell-Sorted Life Cycle Stages of Pneumocystis carinii

Pneumocystis organisms are airborne opportunistic pathogens that cannot be continuously grown in culture. Consequently, the follow-up of Pneumocystis stage-to-stage differentiation, the sequence of their multiplication processes as well as formal identification of the transmitted form have remained elusive. The successful high-speed cell sorting of trophic and cystic forms is paving the way for the elucidation of the complex Pneumocystis life cycle. The growth of each sorted Pneumocystis stage population was followed up independently both in nude rats and in vitro. In addition, by setting up a novel nude rat model, we attempted to delineate which cystic and/or trophic forms can be naturally aerially transmitted from host to host. The results showed that in axenic culture, cystic forms can differentiate into trophic forms, whereas trophic forms are unable to evolve into cystic forms. In contrast, nude rats inoculated with pure trophic forms are able to produce cystic forms and vice versa. Transmission experiments indicated that 12 h of contact between seeder and recipient nude rats was sufficient for cystic forms to be aerially transmitted. In conclusion, trophic- to cystic-form transition is a key step in the proliferation of Pneumocystis microfungi because the cystic forms (but not the trophic forms) can be transmitted by aerial route from host to host.     

The preferred route for the degradation of silencing target RNAs in transgenic plants depends on pre-established silencing conditions

RNA silencing can be initiated upon dsRNA accumulation and results in homology-dependent degradation of target RNAs mediated by 21–23 nt small interfering RNAs (siRNAs). These small regulatory RNAs can direct RNA degradation via different routes such as the RdRP/Dicer- and the RNA-induced silencing complex (RISC)-catalysed pathways. The relative contribution of both pathways to degradation of target RNAs is not understood. To gain further insight in the process of target selection and degradation, we analysed production of siRNAs characteristic for Dicer-mediated RNA degradation during silencing of mRNAs and chimeric viral RNAs in protoplasts from plants of a transgenic tobacco silencing model line. We show that small RNA accumulation is limited to silencing target regions during steady-state mRNA silencing. For chimeric viral RNAs, siRNA production appears dependent on pre-established cellular silencing conditions. The observed siRNA accumulation profiles imply that silencing of viral target RNAs in pre-silenced protoplasts occurs mainly via a RISC-mediated pathway, guided by (pre-existing) siRNAs derived from cellular mRNAs. In cells that are not silenced at the time of infection, viral RNA degradation seems to involve Dicer action directly on the viral RNAs. This suggests that the silencing mechanism flexibly deploys different components of the RNA degradation machinery in function of the prevailing silencing status.     

Mapping functional associations in the entire genome of Drosophila melanogaster using fusion analysis

We have previously shown that the detection of gene fusion events can contribute towards the elucidation of functional associations of proteins within entire genomes. Here we have analysed the entire genome of Drosophila melanogaster using fusion analysis and two additional constraints that improve the reliability of the predictions, viz. low sequence similarity and low degree of paralogy of the component proteins involved in a fusion event. Imposing these constraints, the total number of unique component pairs is reduced from 18 654 to a mere 220 cases, which are expected to represent some of the most reliably detected functionally associated proteins. Using additional information from sequence databases, we have been able to detect pairs of functionally associated proteins with important functions in cellular and developmental pathways, such as spermatogenesis and programmed cell death.     

The Role of eIF1 in Translation Initiation Codon Selection in Caenorhabditis elegans

The selection of a proper AUG start codon requires the base-pairing interactions between the codon on the mRNA and the anticodon of the initiator tRNA. This selection process occurs in a pre-initiation complex that includes multiple translation initiation factors and the small ribosomal subunit. To study how these initiation factors are involved in start codon recognition in multicellular organisms, we isolated mutants that allow the expression of a GFP reporter containing a non-AUG start codon. Here we describe the characterization of mutations in eif-1, which encodes the Caenorhabditis elegans translation initiation factor 1 (eIF1). Two mutations were identified, both of which are substitutions of amino acid residues that are identical in all eukaryotic eIF1 proteins. These residues are located in a structural region where the amino acid residues affected by the Saccharomyces cerevisiae eIF1 mutations are also localized. Both C. elegans mutations are dominant in conferring a non-AUG translation initiation phenotype and lead to growth arrest defects in homozygous animals. By assaying reporter constructs that have base changes at the AUG start codon, these mutants are found to allow expression from most reporters that carry single base changes within the AUG codon. This trend of non-AUG mediated initiation was also observed previously for C. elegans eIF2β mutants, indicating that these two factors play a similar role. These results support that eIF1 functions in ensuring the fidelity of AUG start codon recognition in a multicellular organism.TRANSLATION initiation is thought to be one of the most complex cellular processes in eukaryotes. It involves at least 12 translation initiation factors (eIFs) comprising over 30 polypeptides (Pestova et al. 2007). These factors bring together an initiator methionyl tRNA (Met-tRNAi), the small ribosomal subunit, and a mRNA to form a 48S initiation complex. An important role performed by this complex is to select an AUG codon to initiate translation of the mRNA. Since the first AUG at the 5′ end of most mRNAs is selected as the start site, it is believed that the initiation complex scans for an AUG start codon as it moves from the 5′-capped end of the mRNA toward the 3′ end, as proposed in the ribosomal scanning model (Kozak 1978; Kozak 1989). The recognition of the AUG start codon is mediated by the anticodon of the Met-tRNAi, and the matching base-pairing interactions between the codon of the mRNA and the anticodon determine the site of initiation (Cigan et al. 1988). These base-pairing interactions are essential, but are likely not the only components required for accurately selecting the correct AUG start codon. Numerous initiation factors along with base-pairing interactions have been shown to aid in the AUG recognition process (Pestova et al. 2007).Translation initiation factors involved in start codon selection fidelity were first identified through genetic studies performed in the yeast Saccharomyces cerevisiae. Mutant strains with a modified His4 gene that had an AUU instead of an AUG at the native start site were selected for the ability to survive on media lacking histidine (Donahue et al. 1988; Castilho-Valavicius et al. 1990). These mutants were found to be able to produce the His4 protein by using a downstream inframe UUG codon (the third codon within the His4 coding region) as the translation start site. Further analyses determined that non-AUG initiation occurred mostly from a UUG codon and not significantly from other codons (Huang et al. 1997). These mutants defined five genetic loci and were named sui1-sui5 (suppressor of initiation codon) on the basis of their ability to initiate translation at a non-AUG codon.The sui1 suppressors were found to have missense mutations in eIF1. These missense mutations showed semidominant or codominant properties in non-AUG translation initiation while deletion of the eIF1 gene led to lethality in yeast (Yoon and Donahue 1992). eIF1 is a highly conserved protein with a size of approximately 12 kDa that plays a vital role in multiple translation initiation steps. eIF1 is incorporated into a multifactor complex that includes eIF1A, eIF3, and eIF5 and stimulates the recruiting of the ternary complex (consisting of eIF2 · GTP and the charged Met-tRNAi) to the small ribosomal subunit to form the 43S pre-initiation complex (Singh et al. 2004). eIF1 acts synergistically with eIF1A to promote continuous ribosomal scanning for AUG codons by stabilizing an open conformation that allows mRNA to pass through the complex (Maag et al. 2005; Cheung et al. 2007; Passmore et al. 2007). It also mediates the assembly of the ribosomal initiation complex at the AUG start codon (Pestova et al. 1998). eIF1 dissociates from the complex upon recognition of the AUG codon and this dissociation is necessary to trigger a series of conformational changes leading to the translation elongation phase (Algire et al. 2005). Consistent with these roles, sui1 mutations reduce the affinity of eIF1 for the ribosome and cause premature release of eIF1 at non-AUG codons (Cheung et al. 2007). Other sui mutations support the involvement of four additional genes in translation initiation fidelity in yeast. Mutations have been isolated in the heterotrimeric eIF2 as SUI2 (α-subunit) (Cigan et al. 1989), SUI3 (β-subunit) (Donahue et al. 1988), and SUI4 (γ-subunit) (Huang et al. 1997), and a mutation in eIF5 corresponds to the SUI5 mutant (Huang et al. 1997).However, the genetic studies that identified these translation fidelity mutants were conducted only in yeast. It is not known if there are similar mechanisms regulating translation initiation fidelity in multicellular organisms. To address this question, we designed a genetic system to isolate C. elegans mutants that have reduced fidelity in AUG start codon selection (Zhang and Maduzia 2010). Mutants were selected on the basis of their ability to express a GFP reporter that contains a GUG codon in place of its native translation start site. Here we report the characterization of two mutants that have mutations in eIF1. Unlike yeast sui1 mutants, which preferred the UUG codon, these mutants are capable of using a subset of non-AUG codons for translation initiation. Our results are consistent with eIF1 playing a role in the fidelity of AUG codon selection, perhaps by discriminating base-pairing interactions between the codon and anticodon during start-site selection.     

Histochemical and biochemical studies of cholinesterases in the carotid labyrinth of amphibians

Acetylcholinesterase activity was detected in the carotid labyrinth of amphibians by both biochemical and histochemical methods. The histochemical tests showed enzyme activity in surfaces of afferent and efferent nerve terminals in contact with type I cells, but none within the type I cells. Acetylcholinesterase activity occurred on some, though not all, nerve fibers in the extra-cellular spaces. These fibers might be parasympathetic cholinergic fibers innervating blood vessels.     

Complex heterochrony underlies the evolution of Caenorhabditis elegans hermaphrodite sex allocation

Hermaphroditic organisms are key models in sex allocation research, yet the developmental processes by which hermaphrodite sex allocation can evolve remain largely unknown. Here we use experimental evolution of hermaphrodite‐male (androdioecious) Caenorhabditis elegans populations to quantify the developmental changes underlying adaptive shifts in hermaphrodite sex allocation. We show that the experimental evolution of increased early‐life self‐fertility occurred through modification of a suite of developmental traits: increased self‐sperm production, accelerated oogenesis and ovulation, and increased embryo retention. The experimental evolution of increased self‐sperm production delayed entry into oogenesis—as expected, given the sequentially coupled production of self‐spermatogenesis and oogenesis. Surprisingly, however, delayed oogenesis onset did not delay reproductive maturity, nor did it trade‐off with gamete or embryo size. Comparing developmental time dynamics of germline and soma indicates that the evolution of increased sperm production did not delay reproductive maturity due to a globally accelerated larval development during the period of self‐spermatogenesis. Overall, heterochrony in gametogenesis and soma can explain adaptive shifts in hermaphrodite sex allocation.     

Reaction norms of life history traits in response to zinc in Thlaspi caerulescens from metalliferous and nonmetalliferous sites

* We examined phenotypic plasticity of fitness components in response to zinc (Zn) in the Zn hyperaccumulator, Thlaspi caerulescens. * Two populations from Zn-enriched soils (M) and two populations from normal soils (NM) were grown in pots at three Zn concentrations (0, 1000 and 8000 mg kg(-1) Zn), for an entire life cycle. Growth, Zn accumulation and fitness components were assessed. * Based on vegetative growth, M and NM populations had similar Zn tolerance at 1000 mg kg(-1) Zn. However, reproductive output was markedly decreased in NM at 1000 and 8000 mg kg(-1) Zn. In M populations, Zn did not affect fitness. However, low Zn status enhanced reproductive output in year 1 compared with year 2 and decreased survival after the first flowering season. * M populations are able to achieve equal fitness across a broad range of Zn concentrations in soil by different combinations of fecundity and longevity. No cost of higher tolerance was demonstrated in M populations. Reproductive traits appeared to be a more sensitive indicator of tolerance than vegetative growth.     

Mixed human intra- and inter-subtype infections with the parasite Blastocystis sp

Because of their limitations, current subtyping methods likely underestimate mixed human intra- and inter-subtype infections with Blastocystis sp. leading to erroneous data in the context of epidemiological studies. We confirmed this hypothesis by the identification of several isolates belonging to three subtypes in a patient considered at high risk of mixed infection through her lifestyle in rural area and long history of travelling.