A robust microfluidic device for the synthesis and crystal growth of organometallic polymers with highly organized structures.

A simple and robust microfluidic device was developed to synthesize organometallic polymers with highly organized structures. The device is compatible with organic solvents. Reactants are loaded into pairs of reservoirs connected by a 15 cm long microchannel prefilled with solvents, thus allowing long-term counter diffusion for self-assembly of organometallic polymers. The process can be monitored, and the resulting crystalline polymers are harvested without damage. The device was used to synthesize three insoluble silver acetylides as single crystals of X-ray diffraction quality. Importantly, for the first time, the single-crystal structure of silver phenylacetylide was determined. The reported approach may have wide applications, such as crystallization of membrane proteins, synthesis and crystal growth of organic, inorganic, and polymeric coordination compounds, whose single crystals cannot be obtained using traditional methods.

Reversible mechanochromic luminescence at room temperature in cationic platinum(II) terpyridyl complexes.

Reversible mechanochromic luminescence in cationic platinum(II) terpyridyl complexes is described. The complexes [Pt(Nttpy)Cl]X2 (Nttpy = 4'-(p-nicotinamide-N-methylphenyl)-2,2':6',2″-terpyridine, X = PF6 (1), SbF6 (2), Cl (3), ClO4 (4), OTf (5), BF4 (6)) exhibit different colors under ambient light in the solid state, going from red to orange to yellow. All of these complexes are brightly luminescent at both room temperature and 77 K. Upon gentle grinding, the yellow complexes (4-6) turn orange and exhibit bright red luminescence. The red luminescence can be changed back to yellow by the addition of a few drops of acetonitrile to the sample. Crystallographic studies of the yellow and red forms of complex 5 suggest that the mechanochromic response is likely the result of a change in intermolecular Pt···Pt distances upon grinding.

Molecular cloning, functional characterization and phylogenetic analysis of B-cell activating factor in zebrafish (Danio rerio).

B-cell activating factor (BAFF), belonging to the TNF family, is a critical cytokine for B-cell survival, proliferation, maturation and differentiation. In the present study we cloned the cDNA of zebrafish (Danio rerio) BAFF (designated zBAFF) by reverse transcription-PCR (RT-PCR). The open reading frame (ORF) of zBAFF consists of 807 bases encoding a protein of 268 amino acids. The deduced amino acid sequence of its cDNA possessed the TNF family signature, a transmembrane domain, and three cysteine residues, which are the typical characteristics of TNF gene in mammals and birds. Phylogenetic analysis exhibits the highest identity score 67.6, 61.4 and 66.9% with the rainbow trout, tetraodon and salmon counterparts, respectively. The identity to avian and mammalian BAFFs ranges from 49.7 to 53.8%. Recombinant soluble zBAFF (zsBAFF) was fused with a small ubiquitin-related modifier gene (SUMO) to enhance the soluble expression level in Escherichia coli BL21 (DE3). The resulting fused protein SUMO-zsBAFF was highly expressed in BL21 (DE3) with a molecular weight of 38 kDa. The fusing protein was purified using metal chellate affinity chromatography (Ni-NTA) and cleaved by a SUMO-specific protease, then confirmed by SDS-PAGE and Western blotting analysis. In vitro, the MTT assay indicated that the purified zsBAFF as well as SUMO-zsBAFF proteins were able to promote spleen lymphocyte survival in a dose-dependent manner also to co-stimulate the proliferation of mammalian B-cells with anti-IgM. Thus, the fusion protein represents a readily obtainable source of biologically active zsBAFF that may prove useful in further studies on zebrafish BAFF and its receptors.

Molecular cloning and partial functional characterization of a proliferation inducing ligand (APRIL) in zebrafish (Danio rerio).

Here we describe the identification of a Danio rerio homologue of a proliferation-inducing ligand (APRIL) of the TNF family (designated zAPRIL). Sequence analysis showed that the open reading frame of zAPRIL consists of 600 bases encoding a protein of 199-amino acids. Recombinant soluble APRIL (zsAPRIL) was constructed consisting of fluorescence-enhanced green fluorescent protein (EGFP) and cloned into a pET28a vector. SDS-PAGE and western blotting analysis indicated a high-level expression of soluble EGFP/zsAPRIL protein in Escherichia coli BL21 (DE3). Observation by confocal microscopy demonstrated that EGFP/zsAPRIL could successfully bind to the surface receptors of zebrafish lymphocytes. In vitro survival analysis revealed that purified EGFP/zsAPRIL was able to promote the survival of zebrafish lymphocytes in a dose-dependent manner. The biological role of APRIL does not seem to be restricted to proliferation induction. Zebrafish may could served as a model organism for further study of APRIL.

Molecular cloning, bioinformatics analysis and functional characterization of B-cell activating factor in goat (Capra hircus).

B-cell activating factor of the TNF family (BAFF) induces B cell survival, proliferation, immunoglobulin secretion and has a role in enhancing immune responses. In the present study, we amplified the cDNA of goat (Capra hircus) BAFF (designated gBAFF) from spleen by reverse transcription-PCR (RT-PCR). The open reading frame (ORF) of gBAFF covers 843 bp encoding 280 amino acids, with a 152-aa mature peptide. Sequence comparison indicated that the amino acid of gBAFF possessed the TNF signature, a transmembrane domain, a putative furin protease cleavage site and three cysteine residues. The predicted three-dimensional (3D) structure of the soluble part of gBAFF (gsBAFF) analyzed by "comparative protein modelling" revealed that it was very similar to its counterparts. Real-time quantitative PCR (qPCR) analysis indicated that gBAFF mRNA was predominantly expressed in goat lymphoid tissue spleen. Recombinant gsBAFF was fused with a small ubiquitin-related modifier (SUMO) gene to enhance the soluble expression level in Escherichia coli BL21 (DE3). The resulting fused protein SUMO-gsBAFF was efficiently expressed and purified using metal chelate affinity chromatography (Ni-NTA), then confirmed by SDS-PAGE and Western blotting analysis. In vitro, the MTT assays and flow cytometric analysis indicated that SUMO-gsBAFF as well as gsBAFF could promote the survival/proliferation of goat splenic B cells or mouse splenic B cells. Therefore, BAFF may be a potential immunologic factor for enhancing immunological efficacy in goat.

Molecular structure, expression analysis and functional characterization of APRIL (TNFSF 13) in goat (Capra hircus).

A proliferation-inducing ligand (APRIL) is an important member of the tumor necrosis factor (TNF) superfamily. In the present study, a novel cDNA was isolated from the spleen of goat by RT-PCR and designated as goat APRIL (gAPRIL). The open reading frame (ORF) of this cDNA covered 753bp, encoding a protein of 250 amino acids. Sequence comparison showed that gAPRIL contains a predicted transmembrane domain, a putative furin protease cleavage site, and two cysteine residues, which are the typical characteristics of TNF gene in mammals. The predicted three dimensional (3D) structure of soluble part of the gAPRIL (gsAPRIL) monomer analyzed by comparative protein modeling revealed that it is very similar to its counterparts. Real-time PCR analysis revealed that gAPRIL was constitutively expressed in various tissues. Recombinant gsAPRIL fused with NusA tag was efficiently produced in Escherichia coli BL21 (DE3) and then analyzed by the SDS-PAGE as well as western blot. Laser scanning confocal microscopy analysis showed gsAPRIL could bind to its receptors. In vitro, the MTT and flow cytometric methods revealed that purified gsAPRIL protein was not only able to promote survival/proliferation of goat splenocytes, but also able to stimulate survival/proliferation of mouse B cells. These results indicated that gAPRIL plays an important role in survival/proliferation of goat splenocytes and provided a basis for investigating its potential to be used as an immunoadjuvant for enhancing vaccine efficacy and as an immunotherapeutic in goats.

Expression and purification of moricin CM4 and human ß-defensins 4 in Escherichia coli using a new technology.

Different strategies have been developed to produce small antimicrobial peptides using recombinant techniques. Here we report a new technology of biosynthesis of moricin CM4 and human ß-defensins 4 (HßD4) in the Escherichia coli. The CM4 and HßD4 gene were cloned into a vector containing the tags elastin-like peptide (ELP) and intein to construct the expression vector pET-EI-CM4 and pET-EI-HßD4. All the peptides, expressed as soluble fusions, were isolated from the protein debris by the method called inverse transition cycling (ITC) rather than traditional immobilized metal affinity chromatography (IMAC) and separated from the fusion leader by self-cleavage. Fully reduced peptides that were purified exhibited expected antimicrobial activity. The approach described here is a low-cost, convenient and potential way for generating small antimicrobial peptide.

Molecular structure, expression, cell and tissue distribution, immune evolution and cell proliferation of the gene encoding bovine (Bos taurus) TNFSF13 (APRIL).

A novel bovine cDNA has been isolated by EST assembly and subsequently confirmed by using RT-PCR and designated bovine A Proliferation-Inducing Ligand belonging to TNF family (bAPRIL). The open reading frame (ORF) of this cDNA covers 753 bp, encoding 250 amino acids. The functional soluble part of bAPRIL (bsAPRIL) shows 97% and 92% identity with its pig and human counterparts, respectively, at the level of the primary protein structure. The bAPRIL genomic sequence consists of six exons and five introns, is approximately 1.8 kb in size, and maps to bovine chromosome 19q. Real-time quantitative PCR (qPCR) analysis revealed that bAPRIL is predominantly expressed in bovine lymphoid tissues spleen. The predicted three-dimensional (3D) structure of the bsAPRIL monomer analyzed by "comparative protein modelling" revealed that it is very similar to its mouse counterpart. The bsAPRIL and EGFP/bsAPRIL were efficiently expression in Escherichia coli BL21 (DE3) and its expression was confirmed by SDS-PAGE and Western blotting analysis. After purification, the EGFP/bsAPRIL fusion protein obtained similar fluorescence spectrum to those of EGFP. Laser scanning confocal microscopy analysis showed EGFP/bsAPRIL could bind to its receptor. In vitro, bsAPRIL could promote the proliferation of bovine or mouse splenic B cells together with/without SAC or anti-mouse IgM. Furthermore, compared to mouse soluble APRIL, the bovine soluble APRIL has the similar proliferation to mouse B cell. Those findings indicated that bsAPRIL plays an important role in proliferation of bovine B cells and has functional cross-reactivity among cow and other mammalians. Therefore, APRIL may be a potential immunologic factor for enhancing immunological efficacy in animals.