Arf GAPs: Gatekeepers of vesicle generation

Arf GAP proteins are a versatile and diverse group of proteins. They control the activity of the GTP-binding proteins of the ARF family by inducing the hydrolysis of GTP that is bound to Arf proteins. The best-studied role of Arf GAPs is in intracellular traffic. In this review, we will focus mainly on the Arf GAPs that play a role in vesicle formation, Arf GAP1, Arf GAP2 and Arf GAP3 and their yeast homologues, Gcs1p and Glo3p. We discuss the roles of Arf GAPs as regulators and effectors for Arf GTP-binding proteins.     

A novel method for constructing pathogen-regulated small RNA cDNA library

Pathogen-responsive endogenous small non-coding RNAs regulate gene expression in relation to plant immune responses by serving as RNA silencing machinery. Decay caused by the bacterium, Erwinia carotovora subsp. carotovora (Ecc), often leads to soft rot disease in the plant Brassica campestris L. ssp. pekinensis (Bcp). To discover endogenous small RNA species in Bcp in response to Ecc infection, we developed a highly efficient approach for cloning pathogen-regulated small RNAs. A group of degenerate stem-loop reverse primers was designed to synthesize first single-stranded cDNA (sscDNA) and the sscDNA was then tailed with a poly(C) at its 3′ end to create a forward priming site. A novel cDNA/RNA subtractive hybridization was performed to capture Ecc-regulated small RNAs and this subsequently allowed construction of small RNA cDNA libraries for sequencing.     

Structure and properties of K141E mutant of small heat shock protein HSP22 (HspB8, H11) that is expressed in human neuromuscular disorders

Some properties of the K141E mutant of human HSP22 that is expressed in distal hereditary motor neuropathy were investigated. This mutation slightly decreased intrinsic fluorescence of HSP22 and induced changes in the far UV CD spectra that correlate with increase of disordered structure. Destabilized K141E mutant was more susceptible to trypsinolysis than the wild type protein. Mutation K141E did not significantly affect the hydrophobic properties measured by bis-ANS binding and did not affect the quaternary structure of HSP22. With insulin as a substrate the chaperone-like activity of K141E mutant and the wild type protein were similar. However with alcohol dehydrogenase and rhodanese the chaperone-like activity of K141E mutant was remarkably lower than the corresponding activity of the wild type protein. It is concluded that K141E mutation induces destabilization of HSP22 structure and probably by this means diminish the chaperone-like activity of HSP22 with certain protein substrates.     

Production in Escherichia coli of a recombinant C-terminal truncated precursor of surfactant protein B (rproSP-BΔc). Structure and interaction with lipid interfaces

SP-B, a protein absolutely required to maintain the lungs open after birth, is synthesized in the pneumocytes as a precursor containing C-terminal and N-terminal domains flanking the mature sequence. These flanking-domains are cleaved to produce mature SP-B, coupled with its assembly into pulmonary surfactant lipid-protein complexes. In the present work we have optimized over-expression in Escherichia coli and purification of rproSP-B_ΔC, a recombinant form of human proSP-B lacking the C-terminal flanking peptide, which is still competent to restore SP-B function in vivo. rProSP-B_ΔC has been solubilized, purified and refolded from bacterial inclusion bodies in amounts of about 4 mg per L of culture. Electrophoretic mobility, immunoreactivity, N-terminal sequencing and peptide fingerprinting all confirmed that the purified protein had the expected mass and sequence. Once refolded, the protein was soluble in aqueous buffers. Circular dichroism and fluorescence emission spectra of bacterial rproSP-B_ΔC indicated that the protein is properly folded, showing around 32% α-helix and a mainly hydrophobic environment of its tryptophan residues. Presence of zwitterionic or anionic phospholipids vesicles caused changes in the fluorescence emission properties of rproSP-B_ΔC that were indicative of lipid-protein interaction. The association of this SP-B precursor with membranes suggests an intrinsic amphipathic character of the protein, which spontaneously adsorbs at air-liquid interfaces either in the absence or in the presence of phospholipids. The analysis of the structure and properties of recombinant proSP-B_ΔC in surfactant-relevant environments will open new perspectives on the investigation of the mechanisms of lipid and protein assembly in surfactant complexes.     

A multivariate two-sample mean test for small sample size and missing data

We develop a new statistic for testing the equality of two multivariate mean vectors. A scaled chi-squared distribution is proposed as an approximating null distribution. Because the test statistic is based on componentwise statistics, it has the advantage over Hotelling's T2 test of being applicable to the case where the dimension of an observation exceeds the number of observations. An appealing feature of the new test is its ability to handle missing data by relying on only componentwise sample moments. Monte Carlo studies indicate good power compared to Hotelling's T2 and a recently proposed test by Srivastava (2004, Technical Report, University of Toronto). The test is applied to drug discovery data.     

Over-expression and characterization of the recombinant small heat shock protein from Pyrococcus furiosus

The gene encoding a small heat shock protein (sHSP) from Pyrococcus furiosus was redesigned and chemically synthesized by using bacteria-preferred codons. The gene product was over-expressed in Escherichia coli BL21(DE)₃ and purified to homogeneity. In the presence of this protein, the activities of Taq DNA polymerase, DNA restriction endonuclease HindIII and lysozyme were protected at elevated temperature, and also, thermal aggregation of lysozyme was prevented by this purified recombinant sHSP.Huayou Chen, Zhongmei Chu, Contributed equally to this work     

Structural and functional analysis of the coupling subunit F in solution and topological arrangement of the stalk domains of the methanogenic A1AO ATP synthase

The first low-resolution shape of subunit F of the A₁A_O ATP synthase from the archaeon Methanosarcina mazei Gö1 in solution was determined by small angle X-ray scattering. Independent to the concentration used, the protein is monomeric and has an elongated shape, divided in a main globular part with a length of about 4.5 nm, and a hook-like domain of about 3.0 nm in length. The subunit-subunit interaction of subunit F inside the A₁A_O ATP synthase in the presence of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide EDC was studied as a function of nucleotide binding, demonstrating movements of subunits F relative to the nucleotide-binding subunit B. Furthermore, in the intact A₁A_O complex, crosslinking of subunits D-E, A-H and A-B-D was obtained and the peptides, involved, were analyzed by MALDI-TOF mass spectrometry. Based on these data the surface of contact of B-F could be mapped in the high-resolution structure of subunit B of the A₁A_O ATP synthase.     

SK3 Trafficking in Hippocampal Cells: The Role of Different Molecular Domains

The regulative steps that control trafficking of ion channels are fundamental determinants of their qualitative and quantitative expression on the cell membrane. In this work the trafficking of the small conductance calcium-activated potassium channel, SK3 was studied in neurons in order to identify relevant molecular domains involved in this process. Hippocampal cell cultures were transfected with fusion proteins of green fluorescent protein (GFP) and different SK3 subunit truncations. The differential distribution of the mutants was analyzed by confocal microscopy and compared to the localization of the control fusion protein with full length SK3. The transport of chimeric proteins was quantified from fluorescence images by developing a morphometric analytical method. We found that the full length SK3 was distributed in cell body, axon and dendrites, whereas the deleted forms GFPΔ578–736 (deletion of the entire C-terminal domain), GFPΔCaMBD (deletion of the calmodulin-binding site) and GFPΔN (deletion of the N-terminal domain) were not transported into cell processes but accumulated in the cell body. The GFPΔ640–736 (deletion of the distal C-terminal domain) showed a distribution similar to control. The quantification and statistical analysis confirmed the differences in distribution across the three groups. In conclusion, the current work provides evidence for a fundamental role of the N-terminal domain and the calmodulin binding domain in SK3 trafficking in neurons.     

Induction of CML28-specific cytotoxic T cell responses using co-transfected dendritic cells with CML28 DNA vaccine and SOCS1 small interfering RNA expression vector

CML28 is an attractive target for antigen-specific immunotherapy. SOCS1 represents an inhibitory control mechanism for DC antigen presentation and the magnitude of adaptive immunity. In this study, we evaluated the potential for inducing CML28-specific cytotoxic T lymphocytes (CTL) responses by dendritic cells (DCs)-based vaccination. We constructed a CML28 DNA vaccine and a SOCS1 siRNA vector and then cotransfect monocyte-derived DCs. Flow cytometry analysis showed gene silencing of SOCS1 resulted in higher expressions of costimulative moleculars in DCs. Mixed lymphocyte reaction (MLR) indicated downregulation of SOCS1 stronger capability to stimulate proliferation of responder cell in DCs. The CTL assay revealed transfected DCs effectively induced autologous CML28-specific CTL responses and the lytic activities induced by SOCS1-silenced DCs were significantly higher compared with those induced by SOCS1-expressing DCs. These results in our study indicates gene silencing of SOCS1 remarkably enhanced the cytotoxicity efficiency of CML28 DNA vaccine in DCs.     

Semiautomated computer-assisted image analysis to quantify 3,3'-diaminobenzidine tetrahydrochloride-immunostained small tissues

This work aimed to develop a technique to measure stained areas in images from sample tissue sections, namely when the structure of interest does not fill the entire image field of the microscope. We propose a semiautomated computer-assisted image analysis (SACAIA) method in which brightfield color images of 3,3'-diaminobenzidene tetrahydrochloride (DAB)-stained antigens are converted to their blue component and boundaries are delineated to extract the object of interest. The number of pixels of a defined color (elicited by DAB) is counted and used to measure the stained area relative to the total area of the tissue under study. The percentages of area stained with adenosine A(1) receptor were 40.76+/-2.08 and 42.44+/-2.26% for manual analysis and SACAIA, respectively (P=0.582). A strong linear correlation of A(1) receptor quantification was found (r=0.98, P<0.001, and 95% CI=0.97 to 0.99 for manual method; r=0.99, P<0.001, and 95% CI=0.98 to 0.99 for SACAIA method). The extent to which misclassification affected staining quantification was evaluated by Bland-Altman analysis, indicating that this method can be applied accurately to quantify the immunohistochemical staining area (occupied by a specific antigen) in small sample tissues that do not fill the entire image field of the microscope.