Overlapping functions of argonaute proteins in patterning and morphogenesis of Drosophila embryos

Argonaute proteins are essential components of the molecular machinery that drives RNA silencing. In Drosophila, different members of the Argonaute family of proteins have been assigned to distinct RNA silencing pathways. While Ago1 is required for microRNA function, Ago2 is a crucial component of the RNA-induced silencing complex in siRNA-triggered RNA interference. Drosophila Ago2 contains an unusual amino-terminus with two types of imperfect glutamine-rich repeats (GRRs) of unknown function. Here we show that the GRRs of Ago2 are essential for the normal function of the protein. Alleles with reduced numbers of GRRs cause specific disruptions in two morphogenetic processes associated with the midblastula transition: membrane growth and microtubule-based organelle transport. These defects do not appear to result from disruption of siRNA-dependent processes but rather suggest an interference of the mutant Ago2 proteins in an Ago1-dependent pathway. Using loss-of-function alleles, we further demonstrate that Ago1 and Ago2 act in a partially redundant manner to control the expression of the segment-polarity gene wingless in the early embryo. Our findings argue against a strict separation of Ago1 and Ago2 functions and suggest that these proteins act in concert to control key steps of the midblastula transition and of segmental patterning.     

The bspA locus of Lactobacillus fermentum BR11 encodes an L-cystine uptake system

BspA is a basic surface-exposed protein from Lactobacillus fermentum BR11. Sequence comparisons have shown that it is a member of family III of the solute binding proteins. It is 89% identical to the collagen binding protein, Cnb, from Lactobacillus reuteri. Compared with the database of Escherichia coli proteins, BspA is most similar to the L-cystine binding protein FliY. To investigate the function of BspA, mutants depleted for BspA were generated by homologous recombination with a temperature-sensitive plasmid. These mutants were significantly impaired in their abilities to take up L-cystine. Uptake rates of L-glutamine, L-histidine, and L-lysine, which are substrates for other binding proteins with similarity to BspA, were unaffected. Evidence was obtained that BspA is necessary for maximal resistance to oxidative stress. Specifically, inactivation of BspA causes defective growth in the presence of oxygen and sensitivity to paraquat. Measurements of sulfhydryl levels showed that incubation of L. fermentum BR11 with L-cystine resulted in increased levels of sulfhydryl groups both inside and outside the cell; however, this was not the case with a BspA mutant. The role of BspA as an extracellular matrix protein adhesin was also addressed. L. fermentum BR11 does not bind to immobilized type I collagen or laminin above background levels but does bind immobilized fibronectin. Inactivation of BspA did not significantly affect fibronectin binding; therefore, we have not found evidence to support the notion that BspA is an extracellular matrix protein binding adhesin. As BspA is most probably not a lipoprotein, this report provides evidence that gram-positive bacterial solute binding proteins do not necessarily have to be anchored to the cytoplasmic membrane to function in solute uptake.     

Immuno-Histochemical Analysis of Rod and Cone Reaction to RPE65 Deficiency in the Inferior and Superior Canine Retina

Mutations in the RPE65 gene are associated with autosomal recessive early onset severe retinal dystrophy. Morphological and functional studies indicate early and dramatic loss of rod photoreceptors and early loss of S-cone function, while L and M cones remain initially functional. The Swedish Briard dog is a naturally occurring animal model for this disease. Detailed information about rod and cone reaction to RPE65 deficiency in this model with regard to their location within the retina remains limited. The aim of this study was to analyze morphological parameters of cone and rod viability in young adult RPE65 deficient dogs in different parts of the retina in order to shed light on local disparities in this disease. In retinae of affected dogs, sprouting of rod bipolar cell dendrites and horizontal cell processes was dramatically increased in the inferior peripheral part of affected retinae, while central inferior and both superior parts did not display significantly increased sprouting. This observation was correlated with photoreceptor cell layer thickness. Interestingly, while L/M cone opsin expression was uniformly reduced both in the superior and inferior part of the retina, S-cone opsin expression loss was less severe in the inferior part of the retina. In summary, in retinae of young adult RPE65 deficient dogs, the degree of rod bipolar and horizontal cell sprouting as well as of S-cone opsin expression depends on the location. As the human retinal pigment epithelium (RPE) is pigmented similar to the RPE in the inferior part of the canine retina, and the kinetics of photoreceptor degeneration in humans seems to be similar to what has been observed in the inferior peripheral retina in dogs, this area should be studied in future gene therapy experiments in this model.     

Psychological treatment of essential hypertension: A controlled comparison of meditation and meditation plus biofeedback

Twenty-one patients with essential hypertension were randomly allocated to eight 1-hour sessions of meditation training, meditation plus biofeedback-aided relaxation, or a no-treatment control group. Statistically significant falls in systolic and diastolic blood pressure occurred after both training programs, although overall reductions in blood pressure were not significantly greater in either program than in the control group. Meditation plus biofeedback-aided relaxation produced falls in diastolic blood pressure earlier in the training program than did meditation alone. All patients practiced meditation regularly between training sessions: The amount of practice did not correlate with the amount of blood pressure reduction after training. On questionnaire measures of psychological symptoms and personality, sex differences emerged, with females showing significant abnormalities in hostility scores and males showing significantly raised levels of somatopsychic symptoms. In females, outward-directed hostility fell significantly and assertiveness increased after training, but in males, somatopsychic symptoms were unchanged.The author is grateful for financial support from the research fund of St. George's Hospital, London, where this project was conducted, and from the Society for Psychosomatic Research. He is indebted to Dr. Chandra H. Patel for invaluable help in conducting the meditation and biofeedback training.     

Identification of a Bis-molybdopterin Intermediate in Molybdenum Cofactor Biosynthesis in Escherichia coli

The molybdenum cofactor is an important cofactor, and its biosynthesis is essential for many organisms, including humans. Its basic form comprises a single molybdopterin (MPT) unit, which binds a molybdenum ion bearing three oxygen ligands via a dithiolene function, thus forming Mo-MPT. In bacteria, this form is modified to form the bis-MPT guanine dinucleotide cofactor with two MPT units coordinated at one molybdenum atom, which additionally contains GMPs bound to the terminal phosphate group of the MPTs (bis-MGD). The MobA protein catalyzes the nucleotide addition to MPT, but the mechanism of the biosynthesis of the bis-MGD cofactor has remained enigmatic. We have established an in vitro system for studying bis-MGD assembly using purified compounds. Quantification of the MPT/molybdenum and molybdenum/phosphorus ratios, time-dependent assays for MPT and MGD detection, and determination of the numbers and lengths of Mo–S and Mo–O bonds by X-ray absorption spectroscopy enabled identification of a novel bis-Mo-MPT intermediate on MobA prior to nucleotide attachment. The addition of Mg-GTP to MobA loaded with bis-Mo-MPT resulted in formation and release of the final bis-MGD product. This cofactor was fully functional and reconstituted the catalytic activity of apo-TMAO reductase (TorA). We propose a reaction sequence for bis-MGD formation, which involves 1) the formation of bis-Mo-MPT, 2) the addition of two GMP units to form bis-MGD on MobA, and 3) the release and transfer of the mature cofactor to the target protein TorA, in a reaction that is supported by the specific chaperone TorD, resulting in an active molybdoenzyme.     

Single Vector System for Efficient N-myristoylation of Recombinant Proteins in E. coli

Background

N-myristoylation is a crucial covalent modification of numerous eukaryotic and viral proteins that is catalyzed by N-myristoyltransferase (NMT). Prokaryotes are lacking endogeneous NMT activity. Recombinant production of N-myristoylated proteins in E. coli cells can be achieved by coexpression of heterologous NMT with the target protein. In the past, dual plasmid systems were used for this purpose.

Methodology/Principal Findings

Here we describe a single vector system for efficient coexpression of substrate and enzyme suitable for production of co- or posttranslationally modified proteins. The approach was validated using the HIV-1 Nef protein as an example. A simple and efficient protocol for production of highly pure and completely N-myristoylated Nef is presented. The yield is about 20 mg myristoylated Nef per liter growth medium.

Conclusions/Significance

The single vector strategy allows diverse modifications of target proteins recombinantly coexpressed in E. coli with heterologous enzymes. The method is generally applicable and provides large amounts of quantitatively processed target protein that are sufficient for comprehensive biophysical and structural studies.     

Movement patterns of the bush cricket Platycleis albopunctata in different types of habitat: matrix is not always matrix

Abstract.  1. Inter-patch movement is usually assumed to be homogeneous across a landscape. As the intervening area between suitable patches is usually richly textured, it cannot be assumed to be uniform in real landscapes.
2. In an experimental mark-and-resight study, the movement behaviour of the bush cricket Platycleis albopunctata in four habitat types as well as at the border between two of these habitat types was observed.
3. Analysis of recapture data indicated differences in mortality risk (or emigration rates) between habitat types.
4. When released at the border between suitable habitat and a crop field, P. albopunctata did not show a consistent preference for the suitable habitat. This suggests that the crop field is at least temporarily attractive for P. albopunctata .
5. Movement in suitable habitat was not always different from movement in the matrix, and movement between different types of matrix also differed.
6. The results indicate that the movement behaviour of P. albopunctata is influenced not only by suitability for breeding but also by structural resistance and other factors (e.g. food availability or habitat-specific mortality risk).