Novel surface plasmon resonance biosensor that uses full-length Det7 phage tail protein for rapid and selective detection of Salmonella enterica serovar Typhimurium.

We report a novel surface plasmon resonance (SPR) biosensor that uses the full-length Det7 phage tail protein (Det7T) to rapidly and selectively detect Salmonella enterica serovar Typhimurium (S. Typhimurium). Det7T, which was obtained using recombinant protein expression and purification in Escherichia coli, demonstrated a size of ∼75 kDa upon SDS-PAGE and was homotrimeric in its native structure.

Rapid label-free detection of E. coli using a novel SPR biosensor containing a fragment of tail protein from phage lambda.

In efforts to speed up the assessment of microorganisms, researchers have sought to use bacteriophages as a biosensing tool, due to their host-specificity, wide abundance, and safety. However, the lytic cycle of the phage has limited its efficacy as a biosensor. Here, we cloned a fragment of tail protein J from phage lambda and characterized its binding with the host, E.

Comparative evaluation of an electrochemical bioreporter for detecting phenolic compounds.

In this study, we constructed an Escherichia coli-based electrochemical bioreporter (EB) harboring pLZCapR, which encodes the CapR regulatory protein (for phenol degradation) along with β-galactosidase, and examined its ability to detect phenolic compounds as compared with previously reported optical bioreporters (OBs) controlled by CapR and detected using a luminometer (OB-lum) or spectrophotometer (OB-spec). The recombinant E. coli bioreporter cells were immobilized in polyvinyl alcohol (PVA); p-aminophenyl-β-D-galactopyranoside (PAPG) was used as the enzymatic substrate; and electrochemical measurements were taken.

A recombinant Escherichia coli biosensor for detecting polycyclic aromatic hydrocarbons in gas and aqueous phases.

Recombinant microbial biosensors are known to be simple, cheap, and very efficient monitoring tools for detecting various environmental pollutants in the field. However, although various recombinant microbial biosensors have been developed for aqueous-phase samples, very few are applicable to the gas phase. Here, we report a recombinant Escherichia coli biosensor that can be used to monitor polycyclic aromatic hydrocarbons (PAHs) in both gas and aqueous phases by color development.

Urea, but not guanidinium, destabilizes proteins by forming hydrogen bonds to the peptide group.

The mechanism by which urea and guanidinium destabilize protein structure is controversial. We tested the possibility that these denaturants form hydrogen bonds with peptide groups by measuring their ability to block acid- and base-catalyzed peptide hydrogen exchange. The peptide hydrogen bonding found appears sufficient to explain the thermodynamic denaturing effect of urea.

Involvement of mitogen-activated protein kinase and NF-kappaB activation in Ca2+-induced IL-8 production in human mast cells.

Interleukin-8 (IL-8) is a potent proinflammatory chemokine that plays an important role in inflammation by activating and recruiting neutrophils, lymphocytes, and eosinophils. To demonstrate the effect of intracellular Ca(2+) on IL-8 production and related signaling, we stimulated human mast cell line HMC-1 with either calcium ionophore A23187 or thapsigargin. Increase of intracellular Ca(2+) resulted in inducing IL-8 gene expression and protein secretion, and addition of EGTA or BAPTA/AM before Ca(2+) stimulation inhibited the induction of IL-8 production.

Freeze-dried recombinant bacteria for on-site detection of phenolic compounds by color change.

We herein report the development of a recombinant bacterial biosensor for the rapid and easy detection of phenolic compounds in the field. A plasmid was designed to encode a beta-galactosidase reporter gene under the control of capR, an activator involved in phenolic compound degradation. The construct was transformed into Escherichia coli, and transformed cells were stored after being freeze-dried in the presence of sucrose.

In vitro binding of purified NahR regulatory protein with promoter Psal.

The NahR regulatory protein activates the naphthalene catabolic operon through binding to the Psal promoter in the presence of salicylate. Here, we investigated in vitro binding interaction between NahR and Psal using purified functional recombinant NahR. The T7-tagged NahR was shown to exist as a monomer in solution.

NahR: effects of replacements at Asn 169 and Arg 248 on promoter binding and inducer recognition.

NahR, a member of the LysR regulator family, is a positive transcriptional regulator for genes of the naphthalene degradation pathway in Pseudomonas sp. To study NahR binding properties, five single and six double mutants were made at residues 169 and/or 248, which are located in the central inducer recognition domain and the C-terminal multimerization domain of the protein, respectively. The effects of these mutations were examined by monitoring the expression of a firefly luciferase (luc) reporter gene under the control of NahR.

Construction and comparison of Escherichia coli whole-cell biosensors capable of detecting aromatic compounds.

The XylR regulatory protein is a transcription factor involved in the BTEX (benzene, toluene, ethylbenzene, and xylene) degradation pathway in Pseudomonas species. When XylR-dependent stimulation of transcription from a plasmid containing XylR and its cognate promoters Pr and Pu was monitored as firefly luciferase activities in Escherichia coli, a notably high level of basal activity was observed in the absence of inducers. To improve the response specificity of XylR in this system, two related but different promoters were tested for their activities; the XylS activator promoter Ps and the DmpR activator promoter Po.

Structures of wild-type and P28L/Y173F tryptophan synthase alpha-subunits from Escherichia coli.

The alpha-subunit of tryptophan synthase (alphaTS) catalyzes the cleavage of indole-3-glycerol phosphate to glyceraldehyde-3-phosphate and indole, which is used to yield the amino acid tryptophan in tryptophan biosynthesis. Here, we report the first crystal structures of wild-type and double-mutant P28L/Y173F alpha-subunit of tryptophan synthase from Escherichia coli at 2.8 and 1.

Crystallization and preliminary X-ray analysis of tryptophan synthase alpha-subunits from Escherichia coli.

Tryptophan synthase alpha-subunit (alphaTS) catalyzes the cleavage of indole-3-glycerolphosphate to glyceraldehyde-3-phosphate and indole, which is channelled to the active site of the associated beta-subunit (betaTS), where it reacts with serine to yield the amino acid tryptophan in tryptophan biosynthesis. The alphaTS from Escherichia coli is a 268 amino-acid protein with no disulfide bonds or prosthetic groups. Although the crystallization of the subunits from E.

A new variant activator involved in the degradation of phenolic compounds from a strain of Pseudomonas putida.

A new variant type of regulatory activator and relevant promoters (designated capR, Pr and Po) involved in the metabolism of phenolic compounds were cloned from Pseudomonas putida KCTC1452 by using PCR. The deduced amino acid sequence of CapR revealed a difference in nine amino acids from the effector binding domain of DmpR. To measure effector specificity, plasmids were constructed in such a way that the expression of luc gene for firefly luciferase or lacZ for beta-galactosidase as a reporter was under the control of capR.

Novel effects of On-Chung-Eum, the traditional plant medicine, on cytokine production in human mononuclear cells from Behçet's.

Plant medications have been used as treatment in various kinds of systemic inflammatory disorder such as Behçet's disease (BD). We investigated the roles of On-Chung-Eum (OCE), a traditional plant medicine, in cytokine regulation of BD. The effects of OCE on cytokine production from phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) of Behçet's patients and control subjects were measured by ELISA.

Protein hydrogen exchange mechanism: local fluctuations.

Experiments were done to study the dynamic structural motions that determine protein hydrogen exchange (HX) behavior. The replacement of a solvent-exposed lysine residue with glycine (Lys8Gly) in a helix of recombinant cytochrome c does not perturb the native structure, but it entropically potentiates main-chain flexibility and thus can promote local distortional motions and large-scale unfolding. The mutation accelerates amide hydrogen exchange of the mutated residue by about 50-fold, neighboring residues in the same helix by less, and residues elsewhere in the protein not at all, except for Leu98, which registers the change in global stability.

Fluorescence and folding properties of Tyr mutant tryptophan synthase alpha-subunits from Escherichia coli.

The fluorescence of tyrosine has been used to monitor a folding process of tryptophan synthase alpha-subunit from Escherichia coli, because this protein has 7 tyrosines, but not tryptophan. Here to assess the contribution of each Tyr to fluorescence properties of this protein during folding, mutant proteins in which Tyr was replaced with Phe were analyzed. The result shows that a change of Tyr fluorescence occurring during folding of this protein is contributed to approximately 40% each by Tyr(4) and Tyr(115), and to the remaining approximately 20% by Tyr(173) and Tyr(175).

Inhibition of cytokine production by the traditional oriental medicine, 'Gamcho-Sasim-Tang' in mitogen-stimulated peripheral blood mononuclear cells from Adamantiades-Behçet's patients.

Gamcho-Sasim-Tang (GS-Tang) is a traditional Chinese medication, which has been successfully used in Korea for the treatment of Adamantiades-Behçet's disease (ABD). We investigated the modulation effects of GS-Tang on cytokine production from phytohaemagglutinin-stimulated peripheral blood mononuclear cells of Behçet's patients. ABD is a systemic inflammatory disorder and might involve immune dysfunction.

Water-soluble chitosan inhibits the production of pro-inflammatory cytokine in human astrocytoma cells activated by amyloid beta peptide and interleukin-1beta.

A chronic inflammatory response associated with beta-amyloid (Abeta) and interleukin-1beta (IL-1beta) is responsible for the pathology of Alzheimer's disease (AD). Astrocytes are predominant neuroglial cells of the central nervous system and are actively involved in cytokine-mediated events in AD. To investigate the biological effect of water-soluble chitosan (WSC), we examined cytotoxicity, production of pro-inflammatory cytokines and inducible nitric-oxide synthase (iNOS) on human astrocytoma cell line CCF-STTG1 stimulated with IL-1beta and Abeta fragment 25-35 (Abeta[25-35]).