Functional characterization of the Escherichia coli Fis-DNA binding sequence

The Escherichia coli protein Fis is remarkable for its ability to interact specifically with DNA sites of highly variable sequences. The mechanism of this sequence-flexible DNA recognition is not well understood. In a previous study, we examined the contributions of Fis residues to high-affinity binding at different DNA sequences using alanine-scanning mutagenesis and identified several key residues for Fis-DNA recognition. In this work, we investigated the contributions of the 15-bp core Fis binding sequence and its flanking regions to Fis-DNA interactions. Systematic base-pair replacements made in both half sites of a palindromic Fis binding sequence were examined for their effects on the relative Fis binding affinity. Missing contact assays were also used to examine the effects of base removal within the core binding site and its flanking regions on the Fis-DNA binding affinity. The results revealed that: (1) the − 7G and + 3Y bases in both DNA strands (relative to the central position of the core binding site) are major determinants for high-affinity binding; (2) the C⁵ methyl group of thymine, when present at the + 4 position, strongly hinders Fis binding; and (3) AT-rich sequences in the central and flanking DNA regions facilitate Fis-DNA interactions by altering the DNA structure and by increasing the local DNA flexibility. We infer that the degeneracy of specific Fis binding sites results from the numerous base-pair combinations that are possible at noncritical DNA positions (from − 6 to − 4, from − 2 to + 2, and from + 4 to + 6), with only moderate penalties on the binding affinity, the roughly similar contributions of − 3A or G and + 3T or C to the binding affinity, and the minimal requirement of three of the four critical base pairs to achieve considerably high binding affinities.     

Cytoplasmic and serum galectin-3 in diagnosis of thyroid malignancies

In order to address whether galectin-3 in the sera and fine needle aspirates serve as a diagnostic marker distinguishing between benign and malignant thyroid nodules, we developed an enzyme-linked immunosorbent assay. We quantified galectin-3 in fine needle aspirates from a series of 118 patients with thyroid nodules and serum galectin-3 from another series of 46 patients, which were compared with final histology after thyroidectomy. Relative galectin-3 value (ng/mg), defined as galectin-3 concentration (ng/ml) divided by total protein concentration (mg/ml) in fine needle aspirates, was significantly higher in papillary carcinoma than in the other thyroid entities. There was no significant difference in serum galectin-3 level among patients with thyroid nodules and healthy individuals. Accordingly, relative galectin-3 value allows a definitive diagnosis of papillary carcinoma independent of cellular morphology, whereas serum galectin-3 does not serve as a marker for papillary carcinoma.     

Immunogenic display of diverse peptides on virus-like particles of RNA phage MS2

The high level of immunogenicity of peptides displayed in dense repetitive arrays on virus-like particles makes recombinant VLPs promising vaccine carriers. Here, we describe a platform for vaccine development based on the VLPs of RNA bacteriophage MS2. It serves for the engineered display of specific peptide sequences, but will also allow the construction of random peptide libraries from which specific binding activities can be recovered by affinity selection. Peptides representing the V3 loop of HIV gp120 and the ECL2 loop of the HIV coreceptor, CCR5, were inserted into a surface loop of MS2 coat protein. Both insertions disrupted coat VLP assembly, apparently by interfering with protein folding, but these defects were suppressed efficiently by genetically fusing coat protein's two identical polypeptides into a single-chain dimer. The resulting VLPs displayed the V3 and ECL2 peptides on their surfaces where they showed the potent immunogenicity that is the hallmark of VLP-displayed antigens. Experiments with random-sequence peptide libraries show the single-chain dimer to be highly tolerant of six, eight and ten amino acid insertions. MS2 VLPs support the display of a wide diversity of peptides in a highly immunogenic format, and they encapsidate the mRNAs that direct their synthesis, thus establishing the genotype/phenotype linkage necessary for recovery of affinity-selected sequences. The single-chain MS2 VLP therefore unites in a single structural platform the selective power of phage display with the high immunogenicity of VLPs.     

dsRNA binding properties of RDE-4 and TRBP reflect their distinct roles in RNAi

Double-stranded RNA (dsRNA)-binding proteins facilitate Dicer functions in RNA interference. Caenorhabditis elegans RDE-4 facilitates cleavage of long dsRNA to small interfering RNA (siRNA), while human trans-activation response RNA-binding protein (TRBP) functions downstream to pass siRNA to the RNA-induced silencing complex. We show that these distinct in vivo roles are reflected in in vitro binding properties. RDE-4 preferentially binds long dsRNA, while TRBP binds siRNA with an affinity that is independent of dsRNA length. These properties are mechanistically based on the fact that RDE-4 binds cooperatively, via contributions from multiple domains, while TRBP binds noncooperatively. Our studies offer a paradigm for how dsRNA-binding proteins, which are not sequence specific, discern dsRNA length. Additionally, analyses of the ability of RDE-4 deletion constructs and RDE-4/TRBP chimeras to reconstitute Dicer activity suggest RDE-4 promotes activity using its dsRNA-binding motif 2 to bind dsRNA, its linker region to interact with Dicer, and its C-terminus for Dicer activation.     

Influence of positive allosteric modulators on GABA(B) receptor coupling in rat brain: a scintillation proximity assay characterisation of G protein subtypes

Little is known concerning coupling of cerebral GABA_B receptors to G protein subtypes, and the influence of positive allosteric modulators (PAMs) has not been evaluated. These questions were addressed by an antibody-capture/scintillation proximity assay strategy. GABA concentration-dependently enhanced the magnitude of [³⁵S]GTPγS binding to Gαo and, less markedly, Gαi_1/3 in cortex, whereas Gq and Gs/olf were unaffected. ( R )-baclofen and SKF97581 likewise activated Gαo and Gαi_1/3, expressing their actions more potently than GABA. Similar findings were acquired in hippocampus and cerebellum, and the GABA_B antagonist, CGP55845A, abolished agonist-induced activation of Gαo and Gαi_1/3 in all structures. The PAMs, GS39783, CGP7930 and CGP13501, inactive alone, enhanced efficacy and potency of agonist-induced [³⁵S]GTPγS binding to Gαo in all regions, actions abolished by CGP55845A. In contrast, they did not modify efficacies at Gαi_1/3. Similarly, in human embryonic kidney cells expressing GABA_B(1a+2) or GABA_B(1b+2) receptors, allosteric modulators did not detectably enhance efficacy of GABA at Gαi_1/3, though they increased its potency. To summarise, GABA_B receptors coupled both to Gαo and to Gαi, but not Gq and Gs/olf, in rat brain. PAMs more markedly enhanced efficacy of coupling to Go versus Gi_1/3. It will be of interest to confirm these observations employing complementary techniques and to evaluate their potential therapeutic significance.     

Colorimetric assay for sensitive poly(styrene sulfonate) quantification in a template directed polyelectrolyte-assembling process

Template directed Layer-by-layer (LbL) technology recently moved into the center of scientific attention, particularly as a versatile tool for bioencapsulation purposes. Its major advantages can be found in the striking simplicity of tuning wall properties and the complete control over layer thickness and permeability. Yet, for the most commonly applied pair of polyelectrolytes, poly(allylamine) hydrochloride (PAH) and poly(styrene sulfonate) sodium salt (PSS), the mandatory control of the successful deposition on plane and colloidal surfaces is currently only attainable by means of sophisticated and expensive equipment. Here we describe an alternative quantification method based on a simple colorimetric assay using the Bradford reagent, a cost-effective commercially available dye, and standard laboratory technical devices. The binding of the dye to PSS causes a distinct shift of the absorption maximum from 465 to 680 nm, providing a method for spectral quantification of submicrogram amounts of dissolved PSS during LbL coating with significant accuracy and excellent reproducibility. The method was successfully employed to quantify accurate polyelectrolyte loadings on several particles that have a general importance as LbL templates. Thus, this method can be recommended as standard laboratory technique for control of LbL encapsulation and will considerably broaden the applicability of this promising technology in biotechnology.     

Protein A-based antibody immobilization onto polymeric microdevices for enhanced sensitivity of enzyme-linked immunosorbent assay

Highly efficient antibody immobilization is extremely crucial for the development of high-performance polymeric microdevices for enzyme-linked immunosorbent assay (ELISA). In this article, a site-selective tyrosinase (TR)-catalyzed protein A strategy for antibody immobilization was developed to enhance the sensitivity of ELISA in poly-(methyl methacrylate) (PMMA) microchannels for interferon-gamma (IFN-gamma) assay. To effectively immobilize the target antibodies, oxygen plasma was first used to activate the inert PMMA. This is followed by poly(ethyleneimine) (PEI) coating, an amine-containing functional polymer. For comparison, protein A was also immobilized through the commonly used amine-glutaraldehyde (GA) chemistry. Oxygen plasma treatment effectively increased the amount of PEI attachment and subsequent binding efficiency of the primary antibody. The antibody immobilized via TR-catalyzed protein A was able to provide much better specific antigen capture efficiency than GA chemistry due to the optimal spacing and orientation. Consequently, by using this new method, the detection signal and the signal-to-noise ratio of the ELISA immunoassay in microdevices were all significantly improved. In comparison to the standard assay carried out in the 96-well microtiter plate, the treated microchannels exhibited a broader detection range and a shorter detection time. And the detection limit was also decreased to 20 pg/mL, much lower than that obtained in other microdevices.