Proteomic analyses of amniotic fluid: potential applications in health and diseases

Amniotic fluid (AF) is a potential source of biomarkers for many disorders which may occur during pregnancy. The purpose of this study was to evaluate the place of two-dimensional gel electrophoresis (2-DE) technologies to compare AF in both normal and pathological situations. Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE; Ettan DIGE) as well as two-dimensional gel electrophoresis and silver staining followed by image analysis were used. Differentially expressed proteins were identified by mass spectrometry. This approach was used to study electrophoregrams of normal AF obtained at 17 weeks of gestation and at term, as well as AF from fetuses presenting with congenital diaphragmatic hernia. Finally, the potential of two-dimensional electrophoresis was assessed by studying the protein profile of plasma containing AF proteins in a model of premature rupture of the membranes (PROM). Our results clearly show that two-dimensional electrophoresis technologies still have place for analyzing biological fluids such as AF.     

Glycosylation, transport, and complex formation of palmitoyl protein thioesterase 1 (PPT1) – distinct characteristics in neurons

Background

About 20 % of nemaline myopathies are thus far related to skeletal muscle alpha-actin. Seven actin mutants located in different parts of the actin molecule and linked to different forms of the disease were selected and expressed as EGFP-tagged constructs in differentiated C2C12 mytoubes. Results were compared with phenotypes in patient skeletal muscle fibres and with previous expression studies in fibroblasts and C2C12 myoblasts/myotubes.

Results

Whereas EGFP wt-actin nicely incorporated into endogenous stress fibres and sarcomeric structures, the mutants showed a range of phenotypes, which generally changed upon differentiation. Many mutants appeared delocalized in myoblasts but integrated into endogenous actin structures after 4–6 days of differentiation, demonstrating a poor correlation between the appearance in myotubes and the severity of the disease. However, for some mutants, integration into stress fibres induced aberrant structures in differentiated cells, like thickening or fragmentation of stress fibres. Other mutants almost failed to integrate but formed huge aggregates in the cytoplasm of myotubes. Those did not co-stain with alpha-actinin, a main component of nemaline bodies found in patient muscle. Interestingly, nuclear aggregates as formed by two of the mutants in myoblasts were found less frequently or not at all in differentiated cells.

Conclusion

Myotubes are a suitable system to study the capacity of a mutant to incorporate into actin structures or to form or induce pathological changes. Some of the phenotypes observed in undifferentiated myoblasts may only be in vitro effects. Other phenotypes, like aberrant stress fibres or rod formation may be more directly correlated with disease phenotypes. Some mutants did not induce any changes in the cellular actin system, indicating the importance of additional studies like functional assays to fully characterize the pathological impact of a mutant.     

Charged Amino Acids (R83, E567, D617, E625, R669, and K678) of CusA Are Required for Metal Ion Transport in the Cus Efflux System

Gram-negative bacteria expel various toxic chemicals via tripartite efflux pumps belonging to the resistance-nodulation-cell division superfamily. These pumps span both the inner and outer membranes of the cell. The three components of these tripartite systems are an inner-membrane, substrate-binding transporter (or pump); a periplasmic membrane fusion protein (or adaptor); and an outer-membrane-anchored channel. These three efflux proteins interact in the periplasmic space to form the three-part complexes. We previously presented the crystal structures of both the inner-membrane transporter CusA and membrane fusion protein CusB of the CusCBA tripartite efflux system from Escherichia coli. We also described the co-crystal structure of the CusBA adaptor-transporter, revealing that the trimeric CusA efflux pump assembles with six CusB protein molecules to form the complex CusB(6)-CusA(3). We here report three different conformers of the crystal structures of CusBA-Cu(I), suggesting a mechanism on how Cu(I) binding initiates a sequence of conformational transitions in the transport cycle. Genetic analysis and transport assays indicate that charged residues, in addition to the methionine pairs and clusters, are essential for extruding metal ions out of the cell.     

Species richness at the guild level: effects of species pool and local environmental conditions on stream macroinvertebrate communities

1.?A fundamental question in ecology is which factors determine species richness. Here, we studied the relative importance of regional species pool and local environmental characteristics in determining local species richness (LSR). Typically, this question has been studied using whole communities or a certain taxonomic group, although including species with widely varying biological traits in the same analysis may hinder the detection of ecologically meaningful patterns. 2.?We studied the question above for whole stream macroinvertebrate community and within functional feeding guilds. We defined the local scale as a riffle site and the regional scale (i.e. representing the regional species pool) as a stream. Such intermediate-sized regional scale is rarely studied in this context. 3.?We sampled altogether 100 sites, ten riffles (local scale) in each of ten streams (regional scale). We used the local-regional richness regression plots to study the overall effect of regional species pool on LSR. Variation partitioning was used to determine the relative importance of regional species pool and local environmental conditions for species richness. 4.?The local-regional richness relationship was mainly linear, suggesting strong species pool effects. Only one guild showed some signs of curvilinearity. However, variation partitioning showed that local environmental characteristics accounted for a larger fraction of variance in LSR than regional species pool. Also, the relative importance of the fractions differed between the whole community and guilds, as well as among guilds. 5.?This study indicates that the importance of the local and regional processes may vary depending on feeding guild and trophic level. We conclude that both the size of the regional species pool and local habitat characteristics are important in determining LSR of stream macroinvertebrates. Our results are in agreement with recent large-scale studies conducted in highly different study systems and complement the previous findings by showing that the interplay of regional and local factors is also important at intermediate regional scales.     

Context dependency and metacommunity structuring in boreal headwater streams

We studied the relative importance of spatial and environmental factors as determinants of algal, bryophyte, and macroinvertebrate metacommunities in two boreal drainage basins differing in spatial extent. We used eigenfunction spatial analysis to model the spatial relationships among sites and distance‐based redundancy analysis to partition the variability in biotic communities between the spatial filters generated through spatial eigenfunction analysis and the environmental factors measured in the field. In the smaller study area, each metacommunity was structured mostly by environmental factors. This was evidenced by the fact that either the pure environmental effect was significant or environmental factors were strongly spatially structured. In the larger study area, only pure environmental effects were significant. These findings suggest that the environmental control prevails in boreal headwater streams. However, our findings also suggest that the specific details of the community‐environment and community–space relationships are dependent on the focal organism group and drainage basin.     

A potyvirus-based gene vector allows producing active human S-COMT and animal GFP, but not human sorcin, in vector-infected plants

Potato virus A (PVA), a potyvirus with a (+)ssRNA genome translated to a large polyprotein, was engineered and used as a gene vector for expression of heterologous proteins in plants. Foreign genes including jellyfish GFP (Aequorea victoria) encoding the green fluorescent protein (GFP, 27 kDa) and the genes of human origin (Homo sapiens) encoding a soluble resistance-related calcium-binding protein (sorcin, 22 kDa) and the catechol-O-methyltransferase (S-COMT; 25 kDa) were cloned between the cistrons for the viral replicase and coat protein (CP). The inserts caused no adverse effects on viral infectivity and virulence, and the inserted sequences remained intact in progeny viruses in the systemically infected leaves. The heterologous proteins were released from the viral polyprotein following cleavage by the main viral proteinase, NIa, at engineered proteolytic processing sites flanking the insert. Active GFP, as indicated by green fluorescence, and S-COMT with high levels of enzymatic activity were produced. In contrast, no sorcin was detected despite the expected equimolar amounts of the foreign and viral proteins being expressed as a polyprotein. These data reveal inherent differences between heterologous proteins in their suitability for production in plants.     

Microfabricated arrays of cylindrical wells facilitate single‐molecule enzymology of α‐chymotrypsin

Single‐molecule enzymology allows scientists to examine the distributions of kinetic rates among members of a population. We describe a simple method for the analysis of single‐molecule enzymatic kinetics and provide comparisons to ensemble‐averaged kinetics. To isolate our model enzyme, α‐chymotrypsin, into single molecules, we use an array of cylindrical poly(dimethylsiloxane) wells 2 μm in diameter and 1.35 μm in height. Inside the wells, a protease assay with a profluorescent substrate detects α‐chymotrypsin activity. We hold the concentration of α‐chymotrypsin at 0.39 nM in a given well with an enzyme‐to‐substrate ratio of 1:6,666 molecules. Fluorescence emitted by the substrate is proportional to enzyme activity and detectable by a charge‐coupled device. This method allows for the simultaneous real‐time characterization of hundreds of individual enzymes. We analyze single‐molecule kinetics by recording and observing their intensity trajectories over time. By testing our method with our current instruments, we confirm that our methodology is useful for the analysis of single enzymes for extracting static inhomogeneity. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009