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Acetylcholine (ACh) promotes various cell migrations in vitro, but there are few investigations into this nonsynaptic role of ACh signaling in vivo. Here we investigate the function of a muscarinic receptor on an epithelial cell migration in Caenorhabditis elegans. We show that the migratory gonad leader cell, the linker cell (LC), uses an M1/M3/M5-like muscarinic ACh receptor GAR-3 to receive extrasynaptic ACh signaling from cholinergic neurons for its migration. Either the loss of the GAR-3 receptor in the LC or the inhibition of ACh release from cholinergic neurons resulted in migratory path defects. The overactivation of the GAR-3 muscarinic receptor caused the LC to reverse its orientation through its downstream effectors Gαq/egl-30, PLCβ/egl-8, and TRIO/unc-73. This reversal response only occurred in the fourth larval stage, which corresponds to the developmental time when the GAR-3::yellow fluorescent protein receptor in the membrane relocalizes from a uniform to an asymmetric distribution. These findings suggest a role for the GAR-3 muscarinic receptor in determining the direction of LC migration.

Background Aflatoxins (AFs) are carcinogenic mycotoxins. A simple, quick, and accurate method for the micro-analysis of AFs in foodstuffs, especially spices, is needed. Objective A sophisticated pretreatment method that combines solid-phase dispersive extraction (SPDE) and solid-phase fluorescence derivatization using immunoaffinity (IA) gel as the solid phase was developed to analyze AFs in spices simply, quickly, and sensitively by liquid chromatography with fluorescence detection. Method White and black pepper samples were extracted with a mixed solution of methanol/water (4:1) and then diluted with 7% aqueous solution of Triton-X. The solution was subjected to cleanup by SPDE using IA gel. Trifluoroacetic acid was added to the IA gel for on-site solid-phase fluorescence derivatization. Results Chromatograms containing well-separated peaks and few interference peaks from contaminants were obtained. The method detection limit of AFs in white and black pepper was 0.15–0.29 ng/g. Repeatability and intermediate precision were Conclusions The validity, reliability, practicality, and robustness of the developed method were verified. Highlights By using SPDE and solid-phase fluorescence derivatization in combination for AF analysis, fluorescence derivatization during cleanup was realized, leading to simplification of the pretreatment operation.

We have developed an easy and specific enzyme-linked immunoassay (ELISA) for the simultaneous determination of serum metallothinein-1 (MT-1) and 2 (MT-2) in both humans and experimental animals. A competitive ELISA was established using a specific polyclonal antibody against rat MT-2. The antibody used for this ELISA had exhibited the same cross-reactivity with MT in humans and experimental animals. The NH2 terminal peptide of MT containing acetylated methionine was shown to be the epitope of this antibody. The reactivity of this ELISA system with the liver, kidney and brain in MT1/2 knock-out mice was significantly low, but was normal in an MT-3 knock-out mouse. The lowest detection limit of this ELISA was 0.6ng/ml and the spiked MT-1was fully recovered from the plasma. We investigated the normal range of MT1/2 (25-75%tile) in 200 healthy human serum and found it to be 27-48ng/ml, and this was compared with the serum levels in various liver diseases. The serum MT1/2 levels in chronic hepatitis C (HCV) patients were significantly lower than healthy controls and also other liver diseases. In the chronic hepatitis cases, the MT1/I2 levels increased gradually, followed by the progression of the disease to liver cirrhosis and hepatocellular carcinoma. In particular, we found significantly elevated MT1/2 plasma levels in Wilson's disease patients, levels which were very similar to those in the Long-Evans Cinnamon (LEC) rat (model animal of Wilson's disease). Furthermore, a significantly elevated MT1/2 level was found in patients with Menkes disease, an inborn error of copper metabolism such as Wilson's disease.

A sample preparation method was developed for determination of quinolones in honey using immunoaffinity resin. For this purpose, an immunoaffinity resin for quinolones was prepared by coupling a quinolone-specific monoclonal antibody to agarose resin. Honey samples diluted with phosphate buffer were reacted with immunoaffinity resin. After the resin was washed, quinolones were eluted with glycine-HCl. Quinolones in the eluate were determined by HPLC with fluorescence detection. No interfering peak was found on the chromatograms of honey samples. The recoveries of quinolones from samples were over 70% at fortification levels of 20 ng/g (for norfloxacin, ciprofloxacin and enrofloxacin) and 10 ng/g (for danofloxacin). The quantification limits of quinolones were 2 ng/g. This sample preprocessing method using immunoaffinity resin was found to be effective and suitable for determining residual quinolones in honey.

A simple and rapid assay method was developed for the detection of residual quinolones in contaminated milk. This assay uses specific anti-quinolones monoclonal antibody and can provide a simple, non-labour intensive method for on site detection.

Antibacterial reagents, especially quinolones, are widely used in animals and humans, and have caused serious problems to human health because of their residual contaminants in food. In order to screen for different kinds of newquinolones at the same time, a sensitive and specific enzyme-linked immunoassay (ELISA) has been developed. The anti-enrofloxacin monoclonal antibody was selected because of its ability to react with structurally related newquinolones in organic solvent. The antibody has 100% cross-reactivity with norfloxacin, ciprofloxacin and other newquinolones at 50% inhibition of control values IC50, but not with nitroflazone, sulphadimethoxine. The lowest detection limit of this ELISA was 0.7 ng/ml (ppb) when enrofloxacin was used as the calibrator. Eel extracts were spiked with enrofloxacin and the average recoveries at 10, 50, 100 ng/ml were 98, 102 and 91%, respectively. The proposed ELISA is a useful method for the practical microquantitation of various newquinolones in biologica...

SummaryMembrane proteins are the main gatekeepers of cellular state, especially in neurons, serving either to maintain homeostasis or instruct response to synaptic input or other external signals. Visualization of membrane protein localization and trafficking in live cells facilitates understanding the molecular basis of cellular dynamics. We describe here a method for specifically labeling the plasma membrane-localized fraction of heterologous membrane protein expression using channelrhodopsins as a case study. We show that the genetically encoded, covalent binding SpyTag and SpyCatcher pair from the Streptococcus pyogenes fibronectin-binding protein FbaB can selectively label membrane-localized proteins in living cells in culture and in vivo in Caenorhabditis elegans. The SpyTag/SpyCatcher covalent labeling method is highly specific, modular, and stable in living cells. We have used the binding pair to develop a channelrhodopsin membrane localization assay that is amenable to high-throughput screening for opsin discovery and engineering.

キノロン系抗菌剤は人や動物に対して治療を目的に幅広く使用されている．しかし，畜水産食品においてキノロン系抗菌剤の残留が数多く検出されていることから，薬物残留を評価するのに簡便且つ迅速に分析可能な市販ELISAキットによるスクリーニングが期待される．酵素免疫測定法（ELISA）法は多数の検体を一度に処理できるが，交差反応性に起因する同定能力等に課題を有する．そこで，ELISA法として開発されたNew Quinolone Kitの有用性を検証するために，高速液体クロマトグラフィー/蛍光検出法（HPLC/FL）を用いた高精度な機器分析法を構築し，比較検討した．HPLC/FLによる検出限界及び定量限界はエンロフロキサシンにおいて2 ng/g及び10 ng/gであった．エンロフロキサシン50 ng/gを添加したところ，回収率は107.8％ と良好な結果を得ることができた．ELISA法との相関性を検討するため，同一食肉を試料としてHPLC/FL及びELISA法をそれぞれ適用したところ，両者の値に相関性が認められた．HPLC/FLでは煩雑な前処理及び約120分の分析所要時間を必要とするため，ELISA法は1次スクリーニング法として有用であると考えられる．

Peroxisome proliferator-activated receptor gamma (PPARgamma) belongs to a superfamily of thyroid / steroid hormone receptors and regulates transcription of their target genes in a ligand-dependent manner. Recently, PPARgamma was reported to be expressed in several cell lines derived from breast, colon, stomach and lung cancers. Activation of PPARgamma by its ligand inhibits the growth of these tumor cells, suggesting that PPARgamma ligand is a potential anti-cancer agent in PPARgamma-expressing tumors. However, its expression in brain tumors has not been studied. We thus studied the expression in glioma samples with different pathological stages from 20 patients. It was demonstrated that 95% of the glioma tissue expressed PPARgamma mRNA. The results prompted us to study whether PPARgamma ligand affects the growth of cell lines derived from brain tumors. The receptor expression was studied in 9 cell lines either derived from malignant glioma or neuroblastoma. The expression was detected in a glioma cell line SK-MG-1 and in a neuroblastoma cell line NB-1. Addition of one of the PPARgamma ligands, troglitazone, induced growth inhibition in both cell lines. Further analyses revealed that this growth inhibition is caused by a PPARgamma-mediated induction of apoptosis. These results suggest that PPARgamma ligands could be a potential therapeutic agent for the treatment of the brain tumors expressing this receptor.

This paper reports on a model surface that is inert in biological fluids and that is important for studies in biointerfacial science. A self-assembled monolayer (SAM) terminated in the mannitol group was found to prevent the adsorption of proteins and the attachment of cells. Surface plasmon resonance spectroscopy showed that the mannitol-terminated SAM prevented the adsorption of several proteins and was indistinguishable from a SAM presenting tri(ethylene glycol)groups. In a second set of experiments, monolayers were patterned into circular regions of hexadecanethiolate with the surrounding area terminated in the mannitol group in order to evaluate the inert surface for patterning cell attachment. 3T3 fibroblasts, attached to the circular regions, proliferated to occupy these regions completely and remained patterned on these regions for 25 days. The use of oligo(ethylene glycol)-terminated SAMs, by contrast, showed a loss in fidelity of the pattern after one week in culture. The mannitol-terminated mon...

An easy and specific enzyme-linked immunoassay (ELISA) for the determination of metallothinein-3 (MT-3) in experimental animals for the research of heavy metal and chemical toxicity has not been reported yet. Therefore, we have developed a competitive ELISA, using a specific monoclonal antibody raised against human recombinant MT-3 (rMT-3). The epitope mapping of the antibody was conducted using mouse, rat, and human MT-3s and peptide fragments of human MT-3. MT-1/2, MT-3 knock-out (KO) mice and human brain and liver were used for the evaluation of the ELISA. A pretreatment method of the tissue homogenates was also examined. The antibody used for the ELISA had the same cross-reactivity with MT-3 in humans and experimental animals. The human MT- 3 NH(2) terminal peptide (Fr. 1-17) was the demonstrated epitope of this antibody. The reactivity of this ELISA in brain homogenate of MT-3 KO mouse was significantly low compared with the wild type and MT-1/2 KO mice. The lowest detection limit of the ELISA was 10 ng/ml and over 80% of the spiked rMT-3 was recovered in the brain homogenate. The assay linearity was intact with a 5-fold dilution in the brain homogenate. The inter- and intra-assay CV was 6.5%, respectively. An effective pretreatment procedure of the tissue homogenate was also established for this MT-3 ELISA. In conclusion, this competitive ELISA is an easy and specific method for measuring the brain MT-3 level in experimental animals.

Background An easy and specific enzyme-linked immunoassay (ELISA) for the determination of metallothinein-1 (MT-1) and 2 (MT-2) simultaneously in serum and other biological specimens in humans and experimental animals has not been developed yet. Methods We developed a competitive ELISA, a specific polyclonal antibody against rat MT-2. The epitope mapping of the antibody was conducted using MTs in mouse, rat, rabbit, human and the fragment peptides of human MT-2. MT1/2 and MT-3 knock-out mice and cadmium treated mice were used for the evaluation of the ELISA. Pretreatment method of serum was examined to deplete blocking factors for this assay. Results The antibody used for this ELISA had the same cross-reactivity with MT in humans and experimental animals. NH 2 terminal peptide of MT with acetylated methionine was proved to be the epitope of this antibody. The reactivity of this ELISA system with liver, kidney and brain in MT1/2 knock-out mice was significantly low, but was normal in MT-3 knock-out mouse. The lowest detection limit of this ELISA was 0.6 ng/ml and the added MT-1 was fully recovered from serum. The mean MT concentration in our preliminary study was 23 ± 4.6 ng/ml in human serum. Cadmium treatment to mice induced significantly higher amount of MT in serum, liver, kidney and spleen as reported previously by different established methods. Conclusion The proposed competitive ELISA is an easy and specific method for practical use, determining total MT-1 and -2 simultaneously in serum and other biological specimens of human and experimental animals.

The adhesion of cells is mediated by the binding of several cell-surface receptors to ligands found in the extracellular matrix. These receptors often have overlapping specificities for the peptide ligands, making it difficult to understand the roles for discrete receptors in cell adhesion, migration, and differentiation as well as to direct the selective adhesion of cell types in tissue-engineering applications. To overcome these limitations, we developed a strategy to rewire the receptor-ligand interactions between a cell and substrate to ensure that adhesion is mediated by a single receptor with unique specificity. The strategy combines a genetic approach to engineer the cell surface with a chimeric integrin receptor having a unique ligand binding domain with a surface chemistry approach to prepare substrates that present ligands that are bound by the new binding domain. We show that Chinese hamster ovary cells that are engineered with a chimeric beta1 integrin adhere, signal, and even migrate on a synthetic matrix.

The adhesion of mammalian cells is mediated by the binding of cell-surface integrin receptors to peptide ligands from the extracellular matrix and the clustering of these receptors into focal adhesion complexes. This paper examines the effect of one mechanistic variable, ligand affinity, on the assembly of focal adhesions (FAs) in order to gain mechanistic insight into this process. This study uses self-assembled monolayers of alkanethiolates on gold as a substrate to present either a linear or cyclic Arg-Gly-Asp peptide at identical densities. Inhibition assays showed that the immobilized cyclic RGD is a higher affinity ligand than linear RGD. 3T3 Swiss fibroblasts attached to substrates presenting the cyclic peptide at twice the rate they attached to substrates presenting the linear peptide. Quantitation of focal adhesions revealed that cells on cyclic RGD had twice the number of FAs as did cells on linear RGD and that these focal adhesions were on average smaller. These findings show that affinity affects the assembly of integrins into focal adhesions and support a model based on competing rates of nucleation and growth of FAs to explain the change in distribution of FAs with ligand affinity. This study is important because it provides a model system that is well-suited for biophysical studies of integrin-mediated cell adhesion and reveals insight into one mechanism utilized by cells to perceive environmental changes.

The gene encoding Toxoplasma gondii P24 has been reported previously. To determine the function of P24 against immune systems in the near future, we prepared recombinant P24 antigens using Escherichia coli, insect cells infected with recombinant baculovirus and mammalian cells infected with recombinant vaccinia virus. The P24 antigens derived from E. coli, insect cells and mammalian cells were detected with mouse immune sera against P24 or T. gondii homogenates by Western blot analysis; these corresponded to the authentic P24 and secreted into the supernatants of the insect and mammalian cell cultures. These proteins were not effected by tunicamycin treatment in cultured cells, indicating that recombinant P24 did not contain N-linked sugars. Recombinant P24 was separated by two-dimensional electrophoresis and analyzed by Western blotting. From these results, P24 was acidic protein and had identical isoelectric point with the authentic P24.

The accessory olfactory bulb (AOB) is a primary center of the vomeronasal system. In the dog, the position and morphology of the AOB remained vague for a long time. Recently, the morphological characteristics of the dog AOB were demonstrated by means of lectin-histochemical, histological, and immunohistochemical staining, although the distribution of each kind of neuron, especially granule cells, remains controversial in the dog AOB. In the present study, we examined the distribution of neuronal elements in the dog AOB by means of immunohistochemical and enzyme-histochemical staining. Horizontal paraffin or frozen sections of the dog AOB were immunostained with antisera against protein gene product 9.5 (PGP 9.5), brain nitric oxide synthase (NOS), glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH), substance P (SP), and vasoactive intestinal polypeptide (VIP) by avidin-biotin peroxidase complex method. In addition, frozen sections were stained enzyme-histochemically for NADPH-diaphorase. In the dog AOB, vomeronasal nerve fibers, glomeruli, and mitral/tufted cells were PGP 9.5-immunopositive. Mitral/tufted cells were observed in the glomerular layer (GL) and the neuronal cell layer (NCL). In the NCL, a small number of NOS-, GAD-, and SP-immunopositive and NADPH-diaphorase positive granule cells were observed. In the GL, GAD-, TH-, and VIP-immunopositive periglomerular cells were observed. In the GL and the NCL, TH-, and VIP-immunopositive short axon cells were also observed. In addition to these neurons, TH- and SP-immunopositive afferent fibers were observed in the GL and the NCL. We could distinctly demonstrate the distribution of neuronal elements in the dog AOB. Since only a small number of granule cells were present in the dog AOB, the dog AOB did not display such a well-developed GCL as observed in the other mammals.