Mobility of the SecA 2-helix-finger is not essential for polypeptide translocation via the SecYEG complex

The bacterial ATPase SecA and protein channel complex SecYEG form the core of an essential protein translocation machinery. The nature of the conformational changes induced by each stage of the hydrolytic cycle of ATP and how they are coupled to protein translocation are not well understood. The structure of the SecA–SecYEG complex revealed a 2-helix-finger (2HF) of SecA in an ideal position to contact the substrate protein and push it through the membrane. Surprisingly, immobilization of this finger at the edge of the protein channel had no effect on translocation, whereas its imposition inside the channel blocked transport. This analysis resolves the stoichiometry of the active complex, demonstrating that after the initiation process translocation requires only one copy each of SecA and SecYEG. The results also have important implications on the mechanism of energy transduction and the power stroke driving transport. Evidently, the 2HF is not a highly mobile transducing element of polypeptide translocation.     

GRIESBACHIAN AND DIENERIAN (EARLY TRIASSIC) AMMONOID FAUNAS FROM NORTHWESTERN GUANGXI AND SOUTHERN GUIZHOU (SOUTH CHINA)

Abstract: Intensive sampling of the Luolou (northwestern Guangxi) and the Daye (southern Guizhou) Formations in South China leads to the recognition of a regional Griesbachian and Dienerian ammonoid succession for this key palaeobiogeographical area. The new biostratigraphical sequence comprises the upper Griesbachian ‘Ophiceras beds’ and the lower Dienerian ‘Proptychites candidus beds’, which are separated from the uppermost Dienerian ‘Clypites beds’ by an unfossiliferous interval. These faunas contain some taxa with wide geographic distribution (e.g. Ambites, Pleurambites, Pleurogyronites, Proptychites candidus), thus facilitating correlation with faunal successions from other regions (i.e. British Columbia, Canadian Arctic, Himalayas and South Primorye). Two new genera (Jieshaniceras and Shangganites) and three new species (Anotoceras subtabulatus, Pleurambites radiatus and Shangganites shangganense) are described.     

Tuning in to multiple predators: conflicting demands for shell morphology in a freshwater snail

1. We examined the response to chemical cues from fish and crayfish, two predators with contrasting feeding modes, and their single and combined effect on shell morphology in the freshwater snail Radix balthica. 2. Snails were subjected to four treatments: tench (Tinca tinca), signal crayfish (Pacifastacus leniusculus), a combination of tench and signal crayfish and no predators (control). Shell shape, crushing resistance and shell thickness were quantified. We also analysed whether shape or shell thickness contributes most to crushing resistance. 3. Chemical cues from the fish induced a rounder shell shape in R. balthica, a thicker shell and a higher crushing resistance, whereas crayfish chemical cues had no effect on shell morphology, shell thickness or crushing resistance. Shell shape contributed more to crushing resistance than shell thickness. 4. The combined predator treatment showed an intermediate response between the fish and crayfish treatments. Shell roundness was reduced compared with the fish treatment, but the reduced crushing resistance that comes with a less rounded shell was compensated by an increased investment in extra shell material, exceeding that of the fish treatment. 5. Our study extends previous studies of multipredator effects on phenotypically plastic freshwater snails by showing that the snails are able to fine‐tune different elements of morphology to counter predator‐specific foraging modes.     

Role of glutamate-52 in the mechanism of L-lactate dehydrogenase from Bacillus stearothermophilus

A single residue of the NAD(H)-dependent lactate dehydrogenase (LDH) from Bacillus stearothermophilus has been changed in order to decrease substrate inhibition. The conserved aspartic acid residue at position 52 was replaced by glutamate using site-directed mutagenesis. The effect on substrate inhibition was measured. In the glutamate-52 mutant substrate inhibition is decreased twofold.     

Mutagenic exploration of the active site of lactate dehydrogenase from Plasmodium falciparum

Several site-directed mutations of residues around the active site of the lactate dehydrogenase from Plasmodium falciparum are described. These include changes to three highly, but not completely, conserved residues in the pocket of the active site and also three changes (including deletions) to the active site loop. Changes to residues in the active-site pocket resulted in little or no over-production of protein and no enzymic activity. Likewise, a five residue deletion from the active site loop gave no over-produced protein, while a two residue deletion and changes of residue type in this loop were tolerated. The results are discussed in the light of this protein being a suitable target for novel anti-malarials.     

Characterization and modelling of the hydrophobic domain of a sunflower oleosin

The oleosins are a group of hydrophobic proteins present on the surface of oil bodies in seeds, where they are thought to prevent coalescence. They contain a central hydrophobic domain of 68-74 residues that is thought to form a loop into the triacylglycerol matrix of the oil body, but the conformation adopted by this sequence is uncertain. We have therefore expressed an oleosin cDNA from sunflower (Helianthus annuus L.) in Escherichia coli as a fusion with maltose-binding protein (MBP) and isolated a peptide corresponding to the hydrophobic domain by sequential digestion with factor Xa (to remove the MBP) followed by trypsin and Staphylococcus V8 protease to remove the N- and C-terminal domains of the oleosin. Circular dichroism spectroscopy of the peptide in two solvent systems chosen to mimic the environment within the oil body (trifluoroethanol and SDS) demonstrated high proportions of alpha-helical structure, with no beta-sheet. A model was therefore developed in which the domain forms an alpha-helical hairpin structure, the two helices being separated by a turn region. We consider that this model is consistent with our current knowledge of oleosin structure and properties.     

Development, chromosomal composition, and cell allocation of bovine cloned blastocyst derived from chemically assisted enucleation and cultured in conditioned media

Treatment of in vitro matured bovine oocytes with colcemid results in a membrane protrusion that contains maternal chromosomes, which can be easily removed by aspiration. Four experiments were designed to evaluate the overall and temporal effects of conditioned medium (CM) by bovine cumulus cells on development of nuclear transfer (NT) bovine embryos and to examine the chromosomal composition and allocation of inner cell mass (ICM) and trophectoderm (TE) of the subsequent blastocysts. The nuclear transfer embryos were cultured in various CR1aa media conditioned by preculture with bovine cumulus cells. Development to the blastocyst stage in BSA-containing CM (BCM) and serum-containing CM (SCM) were similar to co-culture group (24-30%). The 24 hr-conditioned BCM yielded higher blastocyst development than 48 and 72 hr-conditioned BCM. Temporary exposure of embryos to BCM and SCM followed by CR1aa was also studied. Morula and blastocyst development were not different among the groups cultured in BCM for 72, 96, and 168 hr, but were significantly higher (P < 0.01) than groups exposed to BCM for 24 and 48 hr, respectively. Blastocyst development in SCM for 24 hr (29%), 96 hr (25%), and 168 hr (27%) were much higher (P < 0.05) than those in SCM for 48 hr (12%) and 72 hr (10%). The analyses of chromosomal composition of the resulting blastocysts indicate approximately 80% of the blastocysts cultured in CR1aa with co-culture or groups initially exposed to BCM for 24 hr followed by culture in CR1aa were diploid. However, the incidence of diploidy were only 36-60% in SCM-cultured groups and groups cultured in BCM beyond 48 hr. Conditioned media did not affect the allocation of ICM and TE in the blastocyst. No difference was found in the ratio of inner cell mass to total cells in co-culture, BCM or SCM groups (0.424, 0.441, and 0.473, respectively). In conclusion, bovine cumulus cell-CM and CR1aa with co-culture supported comparable development and blastocyst ICM:total cell ratio of bovine NT embryos. However, CM affected the blastocyst chromosomal composition and induced higher mixploidy.     

Production of Infectious Dengue Virus in Aedes aegypti Is Dependent on the Ubiquitin Proteasome Pathway

Dengue virus (DENV) relies on host factors to complete its life cycle in its mosquito host for subsequent transmission to humans. DENV first establishes infection in the midgut of Aedes aegypti and spreads to various mosquito organs for lifelong infection. Curiously, studies have shown that infectious DENV titers peak and decrease thereafter in the midgut despite relatively stable viral genome levels. However, the mechanisms that regulate this decoupling of infectious virion production from viral RNA replication have never been determined. We show here that the ubiquitin proteasome pathway (UPP) plays an important role in regulating infectious DENV production. Using RNA interference studies, we show in vivo that knockdown of selected UPP components reduced infectious virus production without altering viral RNA replication in the midgut. Furthermore, this decoupling effect could also be observed after RNAi knockdown in the head/thorax of the mosquito, which otherwise showed direct correlation between infectious DENV titer and viral RNA levels. The dependence on the UPP for successful DENV production is further reinforced by the observed up-regulation of key UPP molecules upon DENV infection that overcome the relatively low expression of these genes after a blood meal. Collectively, our findings indicate an important role for the UPP in regulating DENV production in the mosquito vector.