Prp8 in a Reduced Spliceosome Lacks a Conserved Toggle that Correlates with Splicing Complexity across Diverse Taxa.

Conformational rearrangements are critical to regulating the assembly and activity of the spliceosome. The spliceosomal protein Prp8 undergoes multiple conformational changes during the course of spliceosome assembly, activation, and catalytic activity. Most of these rearrangements of Prp8 involve the disposition of the C-terminal Jab-MPN and RH domains with respect to the core of Prp8. Here we use x-ray structural analysis to show that a previously characterized and highly conserved ß-hairpin structure in the RH domain that acts as a toggle in the spliceosome is absent in Prp8 from the reduced spliceosome of the red alga Cyanidioschyzon merolae. Using comparative sequence analysis, we show that the presence or absence of this hairpin corresponds to the presence or absence of protein partners that interact with this hairpin as observed by x-ray and cryo-EM studies. The presence of the toggle correlates with increasing intron number suggesting a role in the regulation of splicing.

Microsporidian infection in a free-living marine nematode.

Microsporidia are unicellular fungi that are obligate endoparasites. Although nematodes are one of the most abundant and diverse animal groups, the only confirmed report of microsporidian infection was that of the "nematode killer" (Nematocida parisii). N. parisii was isolated from a wild Caenorhabditis sp. and causes an acute and lethal intestinal infection in a lab strain of Caenorhabditis elegans. We set out to characterize a microsporidian infection in a wild nematode to determine whether the infection pattern of N. parisii in the lab is typical of microsporidian infections in nematodes. We describe a novel microsporidian species named Sporanauta perivermis (marine spore of roundworms) and characterize its infection in its natural host, the free-living marine nematode Odontophora rectangula. S. perivermis is not closely related to N. parisii and differs strikingly in all aspects of infection. Examination by transmission electron microscopy (TEM) revealed that the infection was localized in the hypodermal and muscle tissues only and did not involve the intestines. Fluorescent in situ hybridization (FISH) confirmed infection in the muscle and hypodermis, and surprisingly, it also revealed that the parasite infects O. rectangula eggs, suggesting a vertical mode of transmission. Our observations highlight the importance of studying parasites in their natural hosts and indicate that not all nematode-infecting microsporidia are "nematode killers"; instead, microsporidiosis can be more versatile and chronic in the wild.

Morphogenesis during division and griseofulvin-induced changes of the microtubular cytoskeleton in the parasitic protist, Trichomonas vaginalis.

The behavior of microtubular structures during division was followed by immunofluorescence in Trichomonas vaginalis using an anti-alpha-tubulin monoclonal antibody together with nuclear staining by DAPI, allowing us to describe successive mitotic stages. In contrast to recent reports, we showed that: (1) the microtubular axostyle-pelta complex depolymerized during division, (2) the flagella were assembled during mitosis, and (3) the flagellar number was restored in each daughter kinetid before cytokinesis. Observation of griseofulvin-treated T. vaginalis cells revealed that the elongation of the mitotic spindle or paradesmosis was not the main motile force separating the daughter kinetids to opposite poles during division, suggesting the existence of other mechanisms and/or molecules involved in this morphogenetic event. Examination of treated cells re-incubated in fresh medium showed the nucleation of microtubules radiating from the perinuclear area, the origin of which is discussed. Finally, we confirm the effectiveness of griseofulvin against T. vaginalis and propose that this antifungal drug could be a promising antitrichomonal agent.

Alpha and beta subunits of pyruvate dehydrogenase E1 from the microsporidian Nosema locustae: mitochondrion-derived carbon metabolism in microsporidia.

Microsporidia are highly adapted eukaryotic intracellular parasites that infect a variety of animals. Microsporidia contain no recognisable mitochondrion, but recently have been shown to have evolved from fungi and to possess heat shock protein genes derived from mitochondria. These findings make it clear that microsporidian ancestors were mitochondrial, yet it remains unknown whether they still contain the organelle, and if so what its role in microsporidian metabolism might be. Here we have characterised genes encoding the alpha and beta subunits of pyruvate dehydrogenase complex E1 (PDH, EC 1.2.4.1) from the microsporidian Nosema locustae. All other amitochondriate eukaryotes studied to date have lost the PDH complex and replaced it with pyruvate:ferredoxin oxidoreductase (PFOR). Nevertheless, molecular phylogeny shows that these Nosema enzymes are most closely related to mitochondrial PDH from other eukaryotes, demonstrating that elements of mitochondrial metabolism have been retained in microsporidia, and that PDH has not been wholly lost. However, there is still no evidence for a mitochondrion in microsporidia, and neither PDH subunit is predicted to encode an amino terminal leader sequence that could function as a mitochondrion-targeting transit peptide, raising questions as to whether these proteins function in a relic organelle or in the cytosol. Moreover, it is also unclear whether these proteins remain part of the PDH complex, or whether they have been retained for another purpose. We propose that microsporidia may utilise a unique pyruvate decarboxylation pathway involving PDH, demonstrating once again the diversity of core metabolism in amitochondriate eukaryotes.

Functional heterogeneity of cytokines and cytolytic effector molecules in human CD8+ T lymphocytes.

CD8(+) T cells use a number of effector mechanisms to protect the host against infection. We have studied human CD8(+) T cells specific for CMV pp65(495-503) epitope, or for staphylococcal enterotoxin B, for the expression patterns of five cytokines and cytolytic effector molecules before and after antigenic stimulation. Ex vivo, the cytolytic molecule granzyme B was detected in a majority of circulating CMV-specific CD8(+) T cells, whereas perforin was rarely expressed. Both were highly expressed after Ag-specific activation accompanied by CD45RO up-regulation. TNF-alpha, IFN gamma, and IL-2 were sequentially acquired on recognition of Ag, but surprisingly, only around half of the CMV-specific CD8(+) T cells responded to antigenic stimuli with production of any cytokine measured. A dominant population coexpressed TNF-alpha and IFN-gamma, and cells expressing TNF-alpha only, IFN-gamma only, or all three cytokines together also occurred at lower but clearly detectable frequencies. Interestingly, perforin expression and production of IFN-gamma and TNF-alpha in CD8(+) T cells responding to staphylococcal enterotoxin B appeared to be largely segregated, and no IL-2 was detected in perforin-positive cells. Together, these data indicate that human CD8(+) T cells can be functionally segregated in vivo and have implications for the understanding of human CD8(+) T cell differentiation and specialization and regulation of effector mechanisms.

Nuclear-encoded, plastid-targeted genes suggest a single common origin for apicomplexan and dinoflagellate plastids.

The phylum Apicomplexa encompasses a large number of intracellular protozoan parasites, including the causative agents of malaria (Plasmodium), toxoplasmosis (Toxoplasma), and many other human and animal diseases. Apicomplexa have recently been found to contain a relic, nonphotosynthetic plastid that has attracted considerable interest as a possible target for therapeutics. This plastid is known to have been acquired by secondary endosymbiosis, but when this occurred and from which type of alga it was acquired remain uncertain. Based on the molecular phylogeny of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes, we provide evidence that the apicomplexan plastid is homologous to plastids found in dinoflagellates-close relatives of apicomplexa that contain secondary plastids of red algal origin. Surprisingly, apicomplexan and dinoflagellate plastid-targeted GAPDH sequences were also found to be closely related to the plastid-targeted GAPDH genes of heterokonts and cryptomonads, two other groups that contain secondary plastids of red algal origin. These results address several outstanding issues: (1) apicomplexan and dinoflagellate plastids appear to be the result of a single endosymbiotic event which occurred relatively early in eukaryotic evolution, also giving rise to the plastids of heterokonts and perhaps cryptomonads; (2) apicomplexan plastids are derived from a red algal ancestor; and (3) the ancestral state of apicomplexan parasites was photosynthetic.

Tubulins in Trichomonas vaginalis: molecular characterization of alpha-tubulin genes, posttranslational modifications, and homology modeling of the tubulin dimer.

We have isolated and analysed an alpha-tubulin-encoding gene (atub1) in an early-diverging eukaryote, Trichomonas vaginalis. The complete atub1 open reading frame included 1.356 bp encoding a polypeptide of 452 amino-acyl residues. A second alpha-tubulin gene (atub2) was amplified by PCR using primers derived from consensus alpha-tubulin amino acid sequences. Both T. vaginalis alpha-tubulin sequences showed high identity to those described in other parabasalids (94.4%-97.3%), and exhibited a high degree of similarity to sequences from Metazoa (such as pig brain) and diplomonads (such as Giardia). Despite large evolutionary distances previously observed between trichomonads and mammals, the three-dimensional model of the T. vaginalis tubulin dimer was very similar to that of pig brain. Possible correlations between alpha-tubulin sequences and posttranslational modifications (PTMs) were examined. Our observations corroborated previous data obtained in T. vaginalis using specific anti-PTMs antibodies. As described in the related species Tritrichomonas mobilensis, microtubules are likely acetylated, non-tyrosinated, glutamylated, and non-glycylated in T. vaginalis. Evolutionary considerations concerning the time of appearance of these tubulin PTMs are also discussed since trichomonads are potentially one of the earliest diverging eukaryotic lineages.

Quantification of HIV-1-specific T-cell responses at the mucosal cervicovaginal surface.

OBJECTIVE: To characterize HIV-1 specific cellular immune responses at mucosal surfaces using a rapid, sensitive enzyme-linked immuno-spot (ELISPOT) technique. DESIGN: Cervicovaginal mononuclear cells obtained from cytobrush and cervicovaginal lavage were assessed for production of interferon-gamma (IFN-gamma) in response to stimulation by HIV-1 antigens. HIV-1 specific responses were compared in a cross-sectional study of two HIV-1-positive patient groups: women not currently on antiretroviral therapy with peripheral CD4 cell counts > 250 x 10(6)/l (n = 12); and women on highly active antiretroviral therapy (HAART) (n = 9). METHODS: Mononuclear cells from peripheral blood or cervicovaginal specimens were assessed in an ELISPOT assay for responses to HIV-1 antigens expressed by recombinant vaccinia viruses. This assay detects primarily CD8 T cells and shows good correlation with MHC class I tetramer staining of cytotoxic T lymphocytes. RESULTS: HIV-1 specific IFN-gamma spot-forming cells were detected in cervicovaginal samples of one out of nine women (11%) on HAART and five out of 12 women (42%) not currently on HAART. In peripheral blood mononuclear cells, HIV-1 specific IFN-gamma spot-forming cells were significantly more numerous in women not currently on HAART than in women on HAART (P = 0.009). In most cases, antigens recognized by mucosal T cells were also recognized by PBMC; however, there were exceptions. CONCLUSIONS: HIV-1-specific antigen-reactive T cells may be detected in routine, noninvasive gynecological specimens. The results suggest that cervicovaginal HIV-1-specific T cells may be less numerous in individuals on HAART than in those not on HAART, as shown previously for HIV-1-specific cytotoxic T lymphocytes in the peripheral blood.

Evolutionary relationship between translation initiation factor eIF-2gamma and selenocysteine-specific elongation factor SELB: change of function in translation factors.

Eubacterial and eukaryotic translation initiation systems have very little in common, and therefore the evolutionary events that gave rise to these two disparate systems are difficult to ascertain. One common feature is the presence of initiation, elongation, and release factors belonging to a large GTPase superfamily. One of these initiation factors, the gamma subunit of initiation factor 2 (eIF-2gamma), is found only in eukaryotes and archaebacteria. We have sequenced eIF-2gamma gene fragments from representative diplomonads, parabasalia, and microsporidia and used these new sequences together with new archaebacterial homologues to examine the phylogenetic position of eIF-2gamma within the GTPase superfamily. The archaebacterial and eukaryotic eIF-2gamma proteins are found to be very closely related, and are in turn related to SELB, the selenocysteine-specific elongation factor from eubacteria. The overall topology of the GTPase tree further suggests that the eIF-2gamma/SELB group may represent an ancient subfamily of GTPases that diverged prior to the last common ancestor of extant life.

U2 and U6 snRNA genes in the microsporidian Nosema locustae: evidence for a functional spliceosome.

The removal of introns from pre-messenger RNA is mediated by the spliceosome, a large complex composed of many proteins and five small nuclear RNAs (snRNAs). Of the snRNAs, the U6 and U2 snRNAs are the most conserved in sequence, as they interact extensively with each other and also with the intron, in several base pairings that are necessary for splicing. We have isolated and sequenced the genes encoding both U6 and U2 snRNAs from the intracellularly parasitic microsporidian Nosema locustae . Both genes are expressed. Both RNAs can be folded into secondary structures typical of other known U6 and U2 snRNAs. In addition, the N.locustae U6 and U2 snRNAs have the potential to base pair in the functional intermolecular interactions that have been characterized by extensive analyses in yeast and mammalian systems. These results indicate that the N.locustae U6 and U2 snRNAs may be functional components of an active spliceosome, even though introns have not yet been found in microsporidian genes.

Selfish DNA: the best defense is a good offense.

The recent discovery of novel biochemical activities of intron-encoded endonucleases emphasizes the selfish nature of mobile genetic elements.