Specific alterations of U1-C protein or U1 small nuclear RNA can eliminate the requirement of Prp28p, an essential DEAD box splicing factor

While some members of the ubiquitous DExD/H box family of proteins have RNA helicase activity in vitro, their roles in vivo remain virtually unknown. Here, we show that the function of an otherwise essential DEAD box protein, Prp28p, can be bypassed by mutations that alter either the protein U1-C or the U1 small nuclear RNA. Further analysis suggests that the conserved L13 residue in the U1-C protein makes specific contact to stabilize the U1 snRNA/5' splice site duplex in the prespliceosome, and that Prp28p functions to counteract the stabilizing effect of the U1-C protein, thereby promoting the dissociation of the U1 small nuclear ribonucleoprotein particle from the 5' splice site. Thus, in addition to unwinding RNA, the DExD/H box proteins may affect RNA-RNA rearrangements by antagonizing specific RNA-stabilizing proteins.     

Multiple functions for the invariant AGC triad of U6 snRNA

The invariant AGC triad of U6 snRNA plays an essential, unknown role in splicing. The triad has been implicated in base-pairing with residues in U2, U4, and U6. Through a genetic analysis in S. cerevisiae, we found that most AGC mutants are suppressed both by restoring pairing with U2, supporting the significance of U2/U6 helix Ib, and by destabilizing U2 stem I, indicating that this stem regulates helix Ib formation. Intriguingly, one of the helix Ib base pairs is required specifically for exon ligation, raising the possibility that the entirety of helix Ib is required only for exon ligation. We also found that U4 mutations that reduce complementarity in U4 stem I enhance U2-mediated suppression of an AGC mutant, suggesting that U4 stem I competes with the AGC-containing U4/U6 stem I. Implicating an additional, essential function for the triad, three triad mutants are refractory to suppression--even by simultaneous restoration of pairing with U2, U4, and U6. An absolute requirement for a purine at the central position of the triad parallels an equivalent requirement in a catalytically important AGC triad in group II introns, consistent with a role for the AGC triad of U6 in catalysis.     

Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, reduction of NAD(+) to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD(+). At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial 'internal' NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol-acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate-oxaloacetate shuttle, a malate-aspartate shuttle and a malate-pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate-citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments.     

Anaerobic Naphthalene Degradation by Microbial Pure Cultures under Nitrate-Reducing Conditions

Pure bacterial cultures were isolated from a highly enriched denitrifying consortium previously shown to anaerobically biodegrade naphthalene. The isolates were screened for the ability to grow anaerobically in liquid culture with naphthalene as the sole source of carbon and energy in the presence of nitrate. Three naphthalene-degrading pure cultures were obtained, designated NAP-3-1, NAP-3-2, and NAP-4. Isolate NAP-3-1 tested positive for denitrification using a standard denitrification assay. Neither isolate NAP-3-2 nor isolate NAP-4 produced gas in the assay, but both consumed nitrate and NAP-4 produced significant amounts of nitrite. Isolates NAP-4 and NAP-3-1 transformed 70 to 90% of added naphthalene, and the transformation was nitrate dependent. No significant removal of naphthalene occurred under nitrate-limited conditions or in cell-free controls. Both cultures exhibited partial mineralization of naphthalene, representing 7 to 20% of the initial added ¹⁴C-labeled naphthalene. After 57 days of incubation, the largest fraction of the radiolabel in both cultures was recovered in the cell mass (30 to 50%), with minor amounts recovered as unknown soluble metabolites. Nitrate consumption, along with the results from the ¹⁴C radiolabel study, are consistent with the oxidation of naphthalene coupled to denitrification for NAP-3-1 and nitrate reduction to nitrite for NAP-4. Phylogenetic analyses based on 16S ribosomal DNA sequences of NAP-3-1 showed that it was closely related to Pseudomonas stutzeri and that NAP-4 was closely related to Vibrio pelagius. This is the first report we know of that demonstrates nitrate-dependent anaerobic degradation and mineralization of naphthalene by pure cultures.     

Genome-Wide Association Study of CSF Levels of 59 Alzheimer's Disease Candidate Proteins: Significant Associations with Proteins Involved in Amyloid Processing and Inflammation

Cerebrospinal fluid (CSF) 42 amino acid species of amyloid beta (Aβ42) and tau levels are strongly correlated with the presence of Alzheimer''s disease (AD) neuropathology including amyloid plaques and neurodegeneration and have been successfully used as endophenotypes for genetic studies of AD. Additional CSF analytes may also serve as useful endophenotypes that capture other aspects of AD pathophysiology. Here we have conducted a genome-wide association study of CSF levels of 59 AD-related analytes. All analytes were measured using the Rules Based Medicine Human DiscoveryMAP Panel, which includes analytes relevant to several disease-related processes. Data from two independently collected and measured datasets, the Knight Alzheimer''s Disease Research Center (ADRC) and Alzheimer''s Disease Neuroimaging Initiative (ADNI), were analyzed separately, and combined results were obtained using meta-analysis. We identified genetic associations with CSF levels of 5 proteins (Angiotensin-converting enzyme (ACE), Chemokine (C-C motif) ligand 2 (CCL2), Chemokine (C-C motif) ligand 4 (CCL4), Interleukin 6 receptor (IL6R) and Matrix metalloproteinase-3 (MMP3)) with study-wide significant p-values (p<1.46×10⁻¹⁰) and significant, consistent evidence for association in both the Knight ADRC and the ADNI samples. These proteins are involved in amyloid processing and pro-inflammatory signaling. SNPs associated with ACE, IL6R and MMP3 protein levels are located within the coding regions of the corresponding structural gene. The SNPs associated with CSF levels of CCL4 and CCL2 are located in known chemokine binding proteins. The genetic associations reported here are novel and suggest mechanisms for genetic control of CSF and plasma levels of these disease-related proteins. Significant SNPs in ACE and MMP3 also showed association with AD risk. Our findings suggest that these proteins/pathways may be valuable therapeutic targets for AD. Robust associations in cognitively normal individuals suggest that these SNPs also influence regulation of these proteins more generally and may therefore be relevant to other diseases.     

Vital role for Plasmodium berghei Kinesin8B in axoneme assembly during male gamete formation and mosquito transmission

Sexual development is an essential phase in the Plasmodium life cycle, where male gametogenesis is an unusual and extraordinarily rapid process. It produces 8 haploid motile microgametes, from a microgametocyte within 15 minutes. Its unique achievement lies in linking the assembly of 8 axonemes in the cytoplasm to the three rounds of intranuclear genome replication, forming motile microgametes, which are expelled in a process called exflagellation. Surprisingly little is known about the actors involved in these processes. We are interested in kinesins, molecular motors that could play potential roles in male gametogenesis. We have undertaken a functional characterization in Plasmodium berghei of kinesin‐8B (PbKIN8B) expressed specifically in male gametocytes and gametes. By generating Pbkin8B‐gfp parasites, we show that PbKIN8B is specifically expressed during male gametogenesis and is associated with the axoneme. We created a ΔPbkin8B knockout cell line and analysed the consequences of the absence of PbKIN8B on male gametogenesis. We show that the ability to produce sexually differentiated gametocytes is not affected in ΔPbkin8B parasites and that the 3 rounds of genome replication occur normally. Nevertheless, the development to free motile microgametes is halted and the life cycle is interrupted in vivo. Ultrastructural analysis revealed that intranuclear mitoses are unaffected whereas cytoplasmic microtubules, although assembled in doublets and elongated, fail to assemble in the normal axonemal ‘9+2' structure and become motile. Absence of a functional axoneme prevented microgamete assembly and release from the microgametocyte, severely reducing infection of the mosquito vector. This is the first functional study of a kinesin involved in male gametogenesis. These results reveal a previously unknown role for PbKIN8B in male gametogenesis, providing new insights into Plasmodium flagellar formation.     

Drought and Root Herbivory Interact to Alter the Response of Above-Ground Parasitoids to Aphid Infested Plants and Associated Plant Volatile Signals

Multitrophic interactions are likely to be altered by climate change but there is little empirical evidence relating the responses of herbivores and parasitoids to abiotic factors. Here we investigated the effects of drought on an above/below-ground system comprising a generalist and a specialist aphid species (foliar herbivores), their parasitoids, and a dipteran species (root herbivore).We tested the hypotheses that: (1) high levels of drought stress and below-ground herbivory interact to reduce the performance of parasitoids developing in aphids; (2) drought stress and root herbivory change the profile of volatile organic chemicals (VOCs) emitted by the host plant; (3) parasitoids avoid ovipositing in aphids feeding on plants under drought stress and root herbivory. We examined the effect of drought, with and without root herbivory, on the olfactory response of parasitoids (preference), plant volatile emissions, parasitism success (performance), and the effect of drought on root herbivory. Under drought, percentage parasitism of aphids was reduced by about 40–55% compared with well watered plants. There was a significant interaction between drought and root herbivory on the efficacy of the two parasitoid species, drought stress partially reversing the negative effect of root herbivory on percent parasitism. In the absence of drought, root herbivory significantly reduced the performance (e.g. fecundity) of both parasitoid species developing in foliar herbivores. Plant emissions of VOCs were reduced by drought and root herbivores, and in olfactometer experiments parasitoids preferred the odour from well-watered plants compared with other treatments. The present work demonstrates that drought stress can change the outcome of interactions between herbivores feeding above- and below-ground and their parasitoids, mediated by changes in the chemical signals from plants to parasitoids. This provides a new insight into how the structure of terrestrial communities may be affected by drought.