Barnase-barstar high affinity interaction phenomenon as the base for the heterogenous bioluminescence pseudorabies virus' immunoassay

The effective new variant of "sandwich" bioluminescent enzyme immunoassay (BEIA) for the sensitive detection of glycoprotein B (gB) of pseudorabies virus (PrV) was presently developed. The high affinity interaction of barnase-barstar protein pair and photoprotein obelin as bioluminescent marker were for the first time successfully applied to BEIA development. Preliminary the two monoclonal antibodies, 11/5 and 34/2, were raised against gB for ELISA PrV detection. Presently we used the same immuno-"sandwich" principle for BEIA. To do this the two different bioconjugates were elaborated. Recombinant barnase was chemically conjugated with monoclonal anti-PrV's gB IgG, and also barstar was fused in frame to obelin. The characteristics of BEIA method have been compared to ELISA PrV detection. We have shown the proposed here gB-BEIA was 40-fold more sensitive as opposed to gB-ELISA test. The construction might have a broad promise in multiple potential immunological applications.     

Spectral analysis of Delayed Luminescence from human skin as a possible non-invasive diagnostic tool

In vivo measurements of Delayed Luminescence (DL), the low-level photo-induced emission which lasts for a longer time after switching off the excitation light, have been performed on human skin, with the aim to develop a technique for optical biopsy. Preliminary tests have been performed on healthy volunteers, measuring the time decays of the spectral components (λ_emiss = 400–800 nm) starting 10 μs after switching off the excitation (λ_exc = 337 nm). Significant differences in the decay trends of DL from different subjects were revealed and quite a good reproducibility for the same subject was observed. The modeling of experimental data has been examined in detail in order to get parameters, characterizing the theoretical fit, whose changes may be correlated with age differences and seasonal variations. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

Changes in photosynthetic electron transfer and state transitions in an herbicide-resistant D1 mutant from soybean cell cultures

Anomalies in photosynthetic activity of the soybean cell line STR7, carrying a single mutation (S268P) in the chloroplastic gene psbA that codes for the D1 protein of the photosystem II, have been examined using different spectroscopic techniques. Thermoluminescence emission experiments have shown important differences between STR7 mutant and wild type cells. The afterglow band induced by both white light flashes and far-red continuous illumination was downshifted by about 4 °C and the Q band was upshifted by 5 °C. High temperature thermoluminescence measurements suggested a higher level of lipid peroxidation in mutant thylakoid membranes. In addition, the reduction rate of P700⁺ was significantly accelerated in STR7 suggesting that the mutation led to an activation of the photosystem I cyclic electron flow. Modulated fluorescence measurements performed at room temperature as well as fluorescence emission spectra at 77 K revealed that the STR7 mutant is defective in state transitions. Here, we discuss the hypothesis that activation of the cyclic electron flow in STR7 cells may be a mechanism to compensate the reduced activity of photosystem II caused by the mutation. We also propose that the impaired state transitions in the STR7 cells may be due to alterations in thylakoid membrane properties induced by a low content of unsaturated lipids.     

Rare earth complexation with 1,3,5-trideoxy-1,3,5-tris(2-hydroxybenzyl)amino-cis-inositol

The protonation constants of 1,3,5-trideoxy-1,3,5-tris(2-hydroxyl-benzyl)amino-cis-inositol (thci) in I = 1 M (NaClO₄) were determined to be: pK_a1 5.96 ± 0.03, pK_a2 7.21 ± 0.01, pK_a3 8.32 ± 0.07, pK_a4 8.95 ± 0.06. The solvent extraction studies were consistent with the formation of the Ln(thci)³⁺ and complexes. The log of the stability constants (log β₁ and log β₂) at 25 °C in 1 M (NaClO₄) at pH 4 for formation of these complexes are reported. Laser luminescence measurements of the ⁷F₀-⁵D₀ transition of Eu(III) complexed by thci indicated two species. The shifts in the peaks relative to that of Eu(aq)³⁺ were comparable to the values reported for other complexes of Eu(III) with organic ligands, but the intensities were greater. Luminescence lifetime measurements of the fluorescence spectra indicated that the complex has 5 inner sphere water molecules bound to the Eu(III) cation at pH 6.71-8.52. This was consistent with bidentate chelation of Eu(III) with each thci molecule. gaussian view energy calculations indicated bonding for M(III) to the amino and hydroxyl groups of the cyclohexanetriol and (2-hydroxybenzyl)amino moieties in the Ln(thci)³⁺ complex.     

Identification of the functional expression of adenosine A<Subscript>3</Subscript> receptor in pancreas using transgenic mice expressing jellyfish apoaequorin

To investigate the functional expression of adenosine A₃ receptor (A3AR) in mammalian living tissues, we generated an apoaequorin-transgenic mouse that expresses jellyfish apoaequorin throughout its body. The expression of apoaequorin under the control of a strong CAG promoter was detected in various tissues, including the abdominal skin, adipose, ear, brain, esophagus, heart, inferior vena cava vessel, kidney, lens, liver, lung, pancreas, skeletal muscle, spleen, tail, testis, and thymus. The transgene was mapped to the C1–2 region of chromosome 16 by Fluorescence in situ hybridization analysis. Among these transgenic mouse tissues, we succeeded in detecting elevated responses of intracellular Ca²⁺ as a light emission of aequorin induced by the A3AR agonist in the pancreas, brain, and testis, the last two of which are known to be main tissues abundantly expressing A3AR. The A3AR agonist led to the phosphorylation of both extracellular signal-regulated kinase 1/2 and protein kinase B in mouse pancreas, and all the intracellular responses via A3AR were antagonized by the A3AR-specific antagonist. In addition, the mRNA expression of A3AR and the A3AR-induced intracellular responses were also found in the rat pancreatic acinar cell line AR42J. These results suggest that pancreas is one of the main tissues functionally expressing A3AR in mammalians in vivo, and that the present approach using transgenic mice that express apoaequorin throughout their bodies will facilitate the functional analysis of proteins of interest. Kazuya Yamano and Katsuhiro Mori contributed equally to this work     

Expression, purification and characterization of a photoprotein, clytin, from Clytia gregarium

A novel histidine-tagged secretion vector in Escherichia coli was constructed and large amounts of highly pure clytin, a calcium-binding photoprotein, was prepared. The histidine-tagged apoclytin expressed into the periplasmic space in E. coli was purified by nickel chelate affinity chromatography. Recombinant clytin was regenerated from apoclytin by incubation with coelenterazine in the presence of dithiothreitol and then purified by anion-exchange chromatography and hydrophobic chromatography. The yield of recombinant clytin was 20mg from 2L of cultured cells with purity greater than 95%. Luminescence properties of recombinant clytin were identical to that of native clytin (phialidin). The Ca(2+) sensitivity of recombinant clytin is lower than that of aequorin and clytin is suited for measuring higher concentration of Ca(2+). Semi-synthetic clytins were also prepared with coelenterazine analogues, and the initial intensity, luminescence capacity and half decay time were characterized.     

Circularly polarized luminescence of Eu(III) complexes with chiral 1,1′-bi-2-naphtol-derived bisphosphate ligands

The interest for lanthanide circularly polarized luminescence (CPL) has been quickly growing for 10 years. However, very few of these studies have involved correlation between the dissymmetry factor (g_lum) and the chemical modifications in a series of chiral ligands. Four polymeric compounds of Eu(III) were prepared by using a series of binaphtyl derivatives for which the size of the π system as well as the number of stereogenic elements (i.e., the binaphtyl moiety) are modulated. The resulting {[Eu(hfac)₃((S)/(R)-L^x)]}_n (x = 1 and 3) and {[Eu(hfac)₃((S,S,S)/(R,R,R)-L^x)]}_n (x = 2 and 4) have been characterized by powder X-ray diffraction by comparison with the X-ray structures on single crystal of the Dy(III) analogs. In solution, the structure of the complexes is deeply modified and becomes monomeric. The nature of the ligand induces change in the shape of the CPL spectra in CH₂Cl₂ solution. Furthermore, a large |g_lum| = 0.12 of the magnetic-dipole transition for the [Eu(hfac)₃((S,S,S)/(R,R,R)-L²)] complex involving the ligand with three stereogenic elements and an extended ?? system has been measured. This report also shows CPL measurements in solid state for the series of {[Eu(hfac)₃((S)/(R)-L^x)]}_n (x = 1 and 3) and {[Eu(hfac)₃((S,S,S)/(R,R,R)-L^x)]}_n (x = 2 and 4) polymers.     

Effect of concentrating and exposing the bioluminescent bacteria to the non‐luminescent allo‐bacterial extracellular products on their luminescence

Bioluminescence is a biochemical process occurring in many organisms. Bacterial bioluminescence has been investigated extensively that lead to many applications of such knowledge. Quorum sensing in the bioluminescent bacteria is a chemical signal process to recognize the strength of its own population to start luminescence in harmony. There is a mechanism in these bacteria to also recognize inter‐species strength. When there is a higher number of these bacteria, the possibility and frequency of cell–cell physical contact will be high. In this study, the physical proximity was artificially enhanced between cells and the effect on luminescence in the concentrated cells in the normal culture medium and in the presence of other non‐bacterial cell‐free supernatants was investigated. The role of such physical contact in the quorum sensing in the bioluminescence is not known. Increase in the luminescence of V. fischeri when concentrated shows that the presence of physical proximity facilitates the quorum sensing for their bioluminescence. Copyright © 2009 John Wiley & Sons, Ltd.     

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.