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Steroid receptor coactivator 1 (SRC-1) is one of the coactivators recruited by the nuclear receptors (NRs) when NRs are activated by steroid hormones, such as glucocorticoid. SRC-1 is abundant in hippocampus and hypothalamus and is also related to some major risk factors for depression, implicated by its reduced expression after stress and its effect on hypothalamus-pituitary-adrenal gland axis function. However, whether SRC-1 is involved in the formation of depression remains unclear. In this study, we firstly established chronic unpredictable stress (CUS) to induce depressive-like behaviors in mice and found that SRC-1 expression was reduced by CUS. A large number of studies have shown that neuroinflammation is associated with stress-induced depression and lipopolysaccharide (LPS) injection can lead to neuroinflammation and depressive-like behaviors in mice. Our result indicated that LPS treatment also decreased SRC-1 expression in mouse brain, implying the involvement of SRC-1 in the process of inflammation and depression. Next, we showed that the chronic unpredictable mild stress (CUMS) failed to elicit the depressive-like behaviors and dramatically promoted the expression of SRC-1 in brain of wild type mice. What's more, the SRC-1 knockout mice were more susceptible to CUMS to develop depressive-like behaviors and presented the changed expression of glucocorticoid receptor. However, SRC-1 deficiency did not affect the microglia activation induced by CUMS. Altogether, these results indicate a correlation between SRC-1 level and depressive-like behaviors, suggesting that SRC-1 might be involved in the development of depression induced by stress. [ABSTRACT FROM AUTHOR]

G protein-coupled receptor 50 (GPR50) belongs to the G protein-coupled receptor which is highly homologous with the sequence of melatonin receptor MT1 and MT2. GPR50 expression has previously been reported in many brain regions, like cortex, midbrain, pons, amygdala. But, the distribution of GPR50 in the hippocampus and cortex and the cell types expressing GPR50 is not yet clear. In this study, we examined the distribution of GPR50 in adult male mice by immunofluorescence. Our results showed that GPR50 was localized in the CA1-3 pyramidal cells and the granule cells of the dentate gyrus. GPR50 was also expressed in excitatory and inhibitory neurons. As inhibitory neurons also contain many types, we found that GPR50 was localized in some interneurons in which it was co-expressed with the calcium-binding proteins calbindin, calretinin, and parvalbumin. Besides, similar results were seen in the cortex. The widespread expression of GPR50 in the hippocampus and cortex suggests that GPR50 may be associated with synaptic plasticity and cognitive function. [ABSTRACT FROM AUTHOR]

A simple and rapid method of fluorescence polarization immunoassay (FPIA) for detection of Alternaria mycotoxin tenuazonic acid (TeA) was described. In this study, the tracers TeAH-FITC and TeAH-DTAF were synthesized from hapten 5-(sec-butyl)-3-(1-hydrazonoethyl)-4-hydroxy-1H-pyrrol-2(5H)-one (TeAH) and different fluoresceins isothiocyanate isomer I (FITC) or 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein hydrochloride (DTAF), respectively. The established FPIA showed an IC50 value of 3.0 μg/mL with a detection limit of 0.13 μg/mL for TeAH based on the tracer of TeAH-FITC, and with the same properties of 7.3 and 0.93 μg/mL based on TeAH-DTAF. The former gave better assay sensitivity than the latter, that the 2,4-dichloro-s-triazine group of TeAH-DTAF may affect the specific recognition of tracer and antibody. Models of the lowest energy conformation and the molecular electrostatic potential isosurface studies revealed that the -C=N-N- and -CHCH3(CH2CH3) groups were the characteristic chemical groups in anti-TeAH antibody-analyte recognition via molecular modeling, which could create a theoretical guide for hapten design of small molecules and provide the traditional theory with empirical proof. [ABSTRACT FROM AUTHOR]

In this paper, we report the development of a sensitive and specific monoclonal antibody-based immunodiagnostic method for the detection of iso-tenuazonic acid (ITeA), an Alternaria mycotoxin, in food samples. The ITeA was derivatized with hydrazine hydrate to produce the antigen (E)-3-(1-hydrazonoethyl)-4-hydroxy-5-isobutyl-1H-pyrrol-2(5H)-one (ITeAH) which was further reacted with glyoxalic acid to generate the hapten (E)-2-((Z)-(1-(4-hydroxy-5-isobutyl-2-oxo-2,5-dihydro-1H-pyrrol-3-yl)ethylidene) (ITeAHGA) which was used as an immunogen after conjugation to bovine serum albumin (BSA). A highly specific monoclonal antibody selectively binding to ITeAH was generated via the hybridoma technique and subsequently used to construct a heterologous indirect competitive enzyme-linked immunosorbent assay (icELISA) using ITeAH as the competitive antigen for the detection of ITeA with a limit of detection (LOD) of 0.5 ng/mL. Under the optimum conditions, the developed icELISA is highly sensitive (IC = 7.8 ng/mL) with recovery rates ranged from 82.3 to 109.8% for spiked food samples. The comparative analysis of results revealed a good correlation between the icELISA and the standard HPLC-MS/MS method, confirming the suitability of the developed icELISA for screening and detection of mycotoxin ITeA in food samples. [ABSTRACT FROM AUTHOR]

Folic acid (FA) is an important B vitamin naturally found in foods and also used in dietary supplements and food fortification. To monitor FA content is important to guarantee food quality and also to prevent its abuse in dietary supplements. In this work, we developed a simple and robust lateral flow immunochromatographic assay (ICA) for the routine screening of FA in food samples. FA was conjugated to carrier protein as immunogen and used to produce specific polyclonal antibody (pAb). The pAb was conjugated with gold nanoparticle and used to develop a lateral flow ICA. After the careful optimization of ICA conditions, the assay showed an IC of 51.8 ng mL and linear range from 23.4 to 114.5 ng mL for FA in assay buffer, with the assist of a DY6510 strip reader within 8 min. Using milk powder as a matrix model, after a 15-fold dilution to eliminate matrix effect, the ICA showed intra-assay recoveries from 94.2 to 107.0% with coefficient of variance (CV) below 15% and inter-assay recoveries from 91.4% to 111.3% with CV below 18%. Good agreement was obtained between the results of ICA and ciELISA and standard HPLC. It is an ideal screening tool for the fast monitoring of FA in a simple, fast, and low-cost manner. [ABSTRACT FROM AUTHOR]

Scorpion venom has been used in the Orient to treat central nervous system diseases for many years, and the protein/peptide toxins in Buthus martensii Karsch (BmK) venom are believed to be the effective components. Scorpion venom heat-resistant peptide (SVHRP) is an active component of the scorpion venom extracted from BmK. In a previous study, we found that SVHRP could inhibit the formation of a glial scar, which is characterized by enhanced glial fibrillary acidic protein (GFAP) expression, in the epileptic hippocampus. However, the cellular and molecular mechanisms underlying this process remain to be clarified. The results of the present study indicate that endogenous GFAP expression in primary rat astrocytes was attenuated by SVHRP. We further demonstrate that the suppression of GFAP was primarily mediated by inhibiting both c-Jun expression and its binding with AP-1 DNA binding site and other factors at the GFAP promoter. These results support that SVHRP contributes to reducing GFAP at least in part by decreasing the activity of the transcription factor AP-1. In conclusion, the effects of SVHRP on astrocytes with respect to the c-Jun/AP-1 signaling pathway in vitro provide a practical basis for studying astrocyte activation and inhibition and a scientific basis for further studies of traditional medicine. [ABSTRACT FROM AUTHOR]

A polyclonal antibody (PcAb)-based enzyme-linked immunosorbent assay (ELISA) for the determination of benzoic acid (BA) was developed. For this, 4-aminobenzoic acid was conjugated to keyhole limpet hemocyanin (KLH) by diazotization and used as an immunogen to produce a PcAb against BA. Two spectrophotometric formats, indirect and direct competitive ELISA (ci- and cd-ELISA, respectively), were developed, and the effects of heterologous coatings and assay conditions on assay performance were examined. Under optimal conditions, comparative sensitivities were obtained for a heterologous ci-ELISA and homologous cd-ELISA. The cd-ELISA showed a detection limit (LOD) of 1.25, 2.5 and 2.5 μg mL, linear range of 5.0-530.0, 10.0-1,060.0 and 10.0-1,060.0 μg mL, in carbonated drink, sausage and jelly, respectively. The assay time was 55 min (simple sum of incubations without time for the necessary intermediate manipulations). This assay exhibited good specificity for BA and its salt, sodium benzoate, and no cross-reactivity with structurally related food additives. Averaged recoveries from BA-spiked carbonated drink, sausage and jelly using this proposed dc-ELSIA ranged from 85.8 to 104.6 %. In addition, good correlation was observed between results from cd-ELISA and standard HPLC analyses ( R = 0.9484). Overall, these results indicated that this cd-ELISA could be used effectively for simple, rapid and low-cost screening of BA abuse in food samples. [ABSTRACT FROM AUTHOR]

The expression efficiency was improved for the recombinant single-chain variable fragment (scFv) against clenbuterol (CBL) obtained from mouse and expressed in the methylotrophic yeast Pichia pastoris GS115, by redesigning and synthesizing the DNA sequence encoding for CBL-scFv based on the codon bias of P. pastoris. The codons enco4ding 124 amino acids were optimized, in which a total of 156 nucleotides were changed, and the G+C ratio was simultaneously decreased from 53 to 47.2 %. Under the optimized expression conditions, the yield of the recombinant CBL-scFv (41 kDa) antibodies was 0.223 g L in shake culture. Compared to the non-optimized control, the expression level of the optimized recombinant CBL-scFv based on preferred codons in P. pastoris demonstrated a 2.35-fold higher yield. Furthermore, the recombinant CBL-scFv was purified by Ni-NTA column chromatography, and the purity was 95 %. The purified CBL-scFv showed good CBL recognition by a competitive indirect enzyme-linked immunoassay. The average concentration required for 50 % inhibition of binding and the limit of detection for the assay were 5.82 and 0.77 ng mL, respectively. [ABSTRACT FROM AUTHOR]

A series of haptens were rationally designed for producing monoclonal antibodies specific for EC and a simple fluorescence immunoassay platform was developed for the sensitive determination of EC based on alkaline phosphatase (ALP)-triggered Cu+ quenching of CdSe quantum dots (QDs). It was noted that Cd as a fluorescence substrate in CdSe QDs can be selectively substituted by Cu+ that resulted in a more significant fluorescence quenching in comparison with Cu2+. Meanwhile, because ALP catalyzed ascorbic acid phosphate and then assisted the transformation of Cu2+ to Cu+, the change in fluorescence intensity was found to be proportional to ALP concentration. After simple magnetic separation, the sensitivity and linear range of the established assay were improved approximately 53-fold and an order of magnitude, respectively, when compared with the conventional ELISA. The proposed platform was able to both amplify the signal and eliminate matrix interferences, making it a promising to determine EC as well as other contaminants in complex food matrix in a highly sensitive and simple manner. [ABSTRACT FROM AUTHOR]