Optimizing Human Synovial Fluid Preparation for Two-Dimensional Gel Electrophoresis

Background

Prenatal screening for Down Syndrome (DS) would benefit from an increased number of biomarkers to improve sensitivity and specificity. Improving sensitivity and specificity would decrease the need for potentially risky invasive diagnostic procedures.

Results

We have performed an in depth two-dimensional difference gel electrophoresis (2D DIGE) study to identify potential biomarkers. We have used maternal plasma samples obtained from first and second trimesters from mothers carrying DS affected fetuses compared with mothers carrying normal fetuses. Plasma samples were albumin/IgG depleted and expanded pH ranges of pH 4.5 - 5.5, pH 5.3 - 6.5 and pH 6 - 9 were used for two-dimensional gel electrophoresis (2DE). We found no differentially expressed proteins in the first trimester between the two groups. Significant up-regulation of ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4, complement proteins C1s subcomponent, C4-A, C5, and C9 and kininogen 1 were detected in the second trimester in maternal plasma samples where a DS affected fetus was being carried. However, ceruloplasmin could not be confirmed as being consistently up-regulated in DS affected pregnancies by Western blotting.

Conclusions

Despite the in depth 2DE approach used in this study the results underline the deficiencies of gel-based proteomics for detection of plasma biomarkers. Gel-free approaches may be more productive to increase the number of plasma biomarkers for DS for non-invasive prenatal screening and diagnosis.     

Mapping QTL for an Adaptive Trait: The Length of Caudal Fin in <Emphasis Type="Italic">Lates calcarifer</Emphasis>

The caudal fin represents a fundamental design feature of fishes and plays an important role in locomotor dynamics in fishes. The shape of caudal is an important parameter in traditional systematics. However, little is known about genes involved in the development of different forms of caudal fins. This study was conducted to identify and map quantitative trait loci (QTL) affecting the length of caudal fin and the ratio between tail length and standard body length in Asian seabass (Lates calcarifer). One F1 family containing 380 offspring was generated by crossing two unrelated individuals. One hundred and seventeen microsatellites almost evenly distributed along the whole genome were genotyped. Length of caudal fin at 90 days post-hatch was measured. QTL analysis detected six significant (genome-wide significant) and two suggestive (linkage-group-wide significant) QTL on seven linkage groups. The six significant QTL explained 5.5–16.6% of the phenotypic variance, suggesting these traits were controlled by multiple genes. Comparative genomics analysis identified several potential candidate genes for the length of caudal fin. The QTL for the length of caudal fin detected for the first time in marine fish may provide a starting point for the future identification of genes involved in the development of different forms of caudal fins in fishes.     

Decay of mRNAs targeted by RISC requires XRN1, the Ski complex, and the exosome

RNA interference (RNAi) is a conserved RNA silencing pathway that leads to sequence-specific mRNA decay in response to the presence of double-stranded RNA (dsRNA). Long dsRNA molecules are first processed by Dicer into 21-22-nucleotide small interfering RNAs (siRNAs). The siRNAs are incorporated into a multimeric RNA-induced silencing complex (RISC) that cleaves mRNAs at a site determined by complementarity with the siRNAs. Following this initial endonucleolytic cleavage, the mRNA is degraded by a mechanism that is not completely understood. We investigated the decay pathway of mRNAs targeted by RISC in Drosophila cells. We show that 5' mRNA fragments generated by RISC cleavage are rapidly degraded from their 3' ends by the exosome, whereas the 3' fragments are degraded from their 5' ends by XRN1. Exosome-mediated decay of the 5' fragments requires the Drosophila homologs of yeast Ski2p, Ski3p, and Ski8p, suggesting that their role as regulators of exosome activity is conserved. Our findings indicate that mRNAs targeted by siRNAs are degraded from the ends generated by RISC cleavage, without undergoing decapping or deadenylation.     

G148-GA3: a streptococcal virulence module with atypical thermodynamics of folding optimally binds human serum albumin at physiological temperatures

The third albumin binding domain of streptococcal protein G strain 148 (G148-GA3) belongs to a novel class of prokaryotic albumin binding modules that is thought to support virulence in several bacterial species. Here, we characterize G148-GA3 folding and albumin binding by using differential scanning calorimetry and isothermal titration calorimetry to obtain the most complete set of thermodynamic state functions for any member of this medically significant module. When buffered at pH 7.0 the 46-amino acid alpha-helical domain melts at 72 degrees C and exhibits marginal stability (15 kJ/mol) at 37 degrees C. G148-GA3 unfolding is characterized by small contributions to entropy from non-hydrophobic forces and a low DeltaCp (1.1 kJ/(deg mol)). Isothermal titration calorimetry reveals that the domain has evolved to optimally bind human serum albumin near 37 degrees C with a binding constant of 1.4 x 10 7 M(-1). Analysis of G148-GA3 thermodynamics suggests that the domain experiences atypically small per residue changes in structural dynamics and heat capacity while transiting between folded and unfolded states.     

Phosphorylation of a membrane curvature-sensing motif switches function of the HOPS subunit Vps41 in membrane tethering

Tethering factors are organelle-specific multisubunit protein complexes that identify, along with Rab guanosine triphosphatases, transport vesicles and trigger their SNARE-mediated fusion of specific transport vesicles with the target membranes. Little is known about how tethering factors discriminate between different trafficking pathways, which may converge at the same organelle. In this paper, we describe a phosphorylation-based switch mechanism, which allows the homotypic vacuole fusion protein sorting effector subunit Vps41 to operate in two distinct fusion events, namely endosome-vacuole and AP-3 vesicle-vacuole fusion. Vps41 contains an amphipathic lipid-packing sensor (ALPS) motif, which recognizes highly curved membranes. At endosomes, this motif is inserted into the lipid bilayer and masks the binding motif for the δ subunit of the AP-3 complex, Apl5, without affecting the Vps41 function in endosome-vacuole fusion. At the much less curved vacuole, the ALPS motif becomes available for phosphorylation by the resident casein kinase Yck3. As a result, the Apl5-binding site is exposed and allows AP-3 vesicles to bind to Vps41, followed by specific fusion with the vacuolar membrane. This multifunctional tethering factor thus discriminates between trafficking routes by switching from a curvature-sensing to a coat recognition mode upon phosphorylation.     

Selectivity of neuronal adaptation does not match response selectivity: a single-cell study of the FMRI adaptation paradigm

fMRI-based adaptation paradigms (fMR-A) have been used to infer neuronal stimulus selectivities in humans. Inferring neuronal selectivities from fMR-A, however, requires an understanding of the relationship between the stimulus selectivity of neuronal adaptation and responses. We studied this relationship by recording single cells in macaque inferior temporal (IT) cortex, an area that shows fMRI adaptation. Repetition of identical object images reduced the responsiveness of single IT neurons. Presentation of an image to which the neuron was unresponsive did not alter the response to a subsequent image that activated the neuron. Successive presentation of two different images to which the neuron responded similarly produced adaptation, but less so than the repeated presentation of an image. The neuronal adaptation at the single-cell level showed a greater degree of stimulus selectivity than the responses. This complicates the interpretation of fMR-A paradigms when inferring neuronal selectivity.     

Cooperative regulation of the activity of factor Xa within prothrombinase by discrete amino acid regions from factor Va heavy chain

The prothrombinase complex catalyzes the activation of prothrombin to alpha-thrombin. We have repetitively shown that amino acid region (695)DYDY(698) from the COOH terminus of the heavy chain of factor Va regulates the rate of cleavage of prothrombin at Arg(271) by prothrombinase. We have also recently demonstrated that amino acid region (334)DY(335) is required for the optimal activity of prothrombinase. To assess the effect of these six amino acid residues on cofactor activity, we created recombinant factor Va molecules combining mutations at amino acid regions 334-335 and 695-698 as follows: factor V(3K) ((334)DY(335) --> KF and (695)DYDY(698) --> KFKF), factor V(KF/4A) ((334)DY(335) --> KF and (695)DYDY(698) --> AAAA), and factor V(6A) ((334)DY(335) --> AA and (695)DYDY(698) --> AAAA). The recombinant factor V molecules were expressed and purified to homogeneity. Factor Va(3K), factor Va(K4/4A), and factor Va(6A) had reduced affinity for factor Xa, when compared to the affinity of the wild-type molecule (factor Va(Wt)) for the enzyme. Prothrombinase assembled with saturating concentrations of factor Va(3K) had a 6-fold reduced second-order rate constant for prothrombin activation compared to the value obtained with prothrombinase assembled with factor Va(Wt), while prothrombinase assembled with saturating concentrations of factor Va(KF/4A) and factor Va(6A) had approximately 1.5-fold reduced second-order rate constants. Overall, the data demonstrate that amino acid region 334-335 together with amino acid region 695-698 from factor Va heavy chain are part of a cooperative mechanism within prothrombinase regulating cleavage and activation of prothrombin by factor Xa.     

Genbank accession #: AF111812

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