The tissue polarity gene nemo carries out multiple roles in patterning during Drosophila development

Drosophila nemo was first identified as a gene required for tissue polarity during ommatidial development. We have extended the analysis of nemo and found that it participates in multiple developmental processes. It is required during wing development for wing shape and vein patterning. We observe genetic interactions between nemo and mutations in the Notch, Wingless, Frizzled and Decapentaplegic pathways. Our data support the findings from other organisms that Nemo proteins act as negative regulators of Wingless signaling. nemo mutations cause polarity defects in the adult wing and overexpression of nemo leads to abdominal polarity defects. The expression of nemo during embryogenesis is dynamic and dsRNA inhibition and ectopic expression studies indicate that nemo is essential during embryogenesis.     

Phase separation rates of aqueous two-phase systems: correlation with system properties

The kinetics of phase separation in aqueous two-phase systems have been investigated as a function of the physical properties of the system. Two distinct situations for the settling velocities were found, one in which the light, organic-rich (PEG) phase is continuous and the other in which the heavier, salt-rich (phosphate) phase is continuous. The settling rate of a particular system is a crucial parameter for equipment design, and it was studied as a function of measured viscosity and density of each of the phases as well as the interfacial tension between the phases. Interfacial tension increases with increasing tie line length. A correlation that describes the rate of phase separation was investigated. This correlation, which is a function of the system parameters mentioned above, described the behavior of the system successfully. Different values of the parameters in the correlation were fitted for bottom-phase-continuous and top-phase-continuous systems. These parameters showed that density and viscosity play a role in the rate of separation in both top continuous- and bottom continuous-phase regions but are more dominant in the continuous top-phase region. The composition of the two-phase system was characterized by the tie line length. The rate of separation increased with increasing tie line length in both cases but at a faster rate when the bottom (less viscous) phase was the continuous phase. These results show that working in a continuous bottom-phase region is advantageous to ensure fast separation.     

The palaeoecology of the Upper Ndolanya Beds at Laetoli,Tanzania

The palaeoecology of the fauna from the Ndolanya Beds, Laetoli, Tanzania, has been analysed to reconstruct the environment of this 2.6 Ma site. Community profiles have been constructed in relation to three variables that carry ecological meaning: body weight, locomotor adaptations and feeding preferences. Comparing the similarities and differences in the structure of the fossil faunas with those of modern environments allows us to draw inferences about palaeoenvironmental conditions, and this is based on a comparative dataset of 15 modern environments (44 localities) covering a wide range of climatic and ecological conditions across Asia, Africa and Central America. In addition, 16 fossil sites in East and South Africa have been analysed in the same way, and both sets of comparative data have been used as the basis for comparison with an ecological diversity analysis of the fauna from the Upper Ndolanya Beds. The Ndolanya fauna is characterized by a predominance of medium to large sized terrestrial and herbivorous species.There is evidence of taphonomic bias that eliminated many of the smaller species. A comparison of multivariate analyses of 23 selected modern localities conducted both with and without the small species included, indicates that the loss of these species does not adversely influence the results of an ecological diversity analysis. The evidence suggests that at the time of the deposition of the Ndolanya Beds the Laetoli region was a semi-arid bushland. This is considerably drier and more open than is suggested for the earlier Laetolil Beds.     

Substrate recognition properties of oligopeptidase B from Salmonella enterica serovar Typhimurium

Oligopeptidase B (OpdB) is a serine peptidase broadly distributed among unicellular eukaryotes, gram-negative bacteria, and spirochetes which has emerged as an important virulence factor and potential therapeutic target in infectious diseases. We report here the cloning and expression of the opdB homologue from Salmonella enterica serovar Typhimurium and demonstrate that it exhibits amidolytic activity exclusively against substrates with basic residues in P(1). While similar to its eukaryotic homologues in terms of substrate specificity, Salmonella OpdB differs significantly in catalytic power and inhibition and activation properties. In addition to oligopeptide substrates, restricted proteolysis of histone proteins was observed, although no cleavage was seen at or near residues that had been posttranslationally modified or at defined secondary structures. This supports the idea that the catalytic site of OpdB may be accessible only to unstructured oligopeptides, similar to the closely related prolyl oligopeptidase (POP). Salmonella OpdB was employed as a model enzyme to define determinants of substrate specificity that distinguish OpdB from POP, which hydrolyzes substrates exclusively at proline residues. Using site-directed mutagenesis, nine acidic residues that are conserved in OpdBs but absent from POPs were converted to their corresponding residues in POP. In this manner, we identified a pair of glutamic acid residues, Glu(576) and Glu(578), that define P(1) specificity and direct OpdB cleavage C terminal to basic residues. We have also identified a second pair of residues, Asp(460) and Asp(462), that may be involved in defining P(2) specificity and thus direct preferential cleavage by OpdB after pairs of basic residues.     

Kinetic model of BiP- and PDI-mediated protein folding and assembly

A mechanism for heavy chain binding protein (BiP)- and protein disulfide isomerase (PDI)- mediated protein folding and assembly has been proposed. It considers BiP chaperoning action and PDI catalytic activity. A kinetic model has been developed based on the proposed mechanism. The model was used for quantifying the influence of polypeptide concentration and ratio, and the effect of BiP and PDI concentration on the kinetics of folding and assembly. An optimum value for polypeptide concentration that minimizes assembly times was found, and different kinetic behaviors were identified for polypeptide concentrations higher or lower than the optimum. Pulse-chase experiments and the dependence of assembly time on unassembled polypeptides ratio predicted by the model are similar to those found during in vitro and in vivo folding and assembly of antibodies and human chorionic gonadotropin (hCG), as well as bovine pancreatic trypsin inhibitor (BPTI) folding. The model also explains the increase in folding and assembly rates during overexpression of BiP and PDI.     

Characterization of Dir: a putative potassium inward rectifying channel in Drosophila

Potassium channels vary in their function and regulation, yet they maintain a number of important features - they are involved in the control of potassium flow, cell volume, cell membrane resting potential, cell excitability and hormone release. The potassium (K(+)) inward rectifier (Kir) superfamily of channels are potassium selective channels, that are sensitive to the concentration of K(+) ions. They are termed inward rectifiers since they allow a much greater K(+) influx than efflux. There are at least seven subfamilies of Kir channels, grouped according to sequence and functional similarities (Curr. Opin. Neurobiol. 5 (1995) 268; Annu. Rev. Physiol. 59 (1997) 171). While numerous Kir channels have been discovered in a variety of organisms, Drosophila inward rectifier (Dir) is the first putative inward rectifier to be studied in Drosophila. In fact, there are only three genes (including Dir) encoding putative inward rectifiers in the Drosophila genome. Though there are other known potassium channels in Drosophila such as ether-a-go-go and shaker, most are voltage-gated channels. As an important first step in characterizing Kir channels in Drosophila, we initiated studies on Dir.     

Adiabatic compressibility and intrinsic viscosity studies on peptide aggregates

Density and sound velocity measurements and ¹H NMR investigations were carried out in aqueous solution at various temperatures for determining the adiabatic compressibility () and hydration of the tetrapeptide, TFA. Tyr-Gly-Phe-Ala-Obz I. The present investigation showed changes in the temperature coefficient of adiabatic compressibility at 40 °C. ¹H NMR studies indicated the inverse temperature transition in the concentration range studied.     

To 5D and beyond: quantitative fluorescence microscopy in the postgenomic era

Digital fluorescence microscopy is now a standard technology for assaying molecular localisation in cells and tissues. The choice of laser scanning (LSM) and wide-field microscopes (WFM) largely depends on the type of sample, with LSMs performing best on thick samples and WFMs performing best on thin ones. These systems are increasingly used to collect large multidimensional datasets. We propose a unified image structure that considers space, time, and fluorescence wavelength as integral parts of the image. Moreover, the application of fluorescence imaging to large-scale screening means that large datasets are now routinely acquired. We propose that analysis of these data requires querying tools based on relational databases and describe one such system.