Molecular cloning, gene expression, and tissue distribution of adiponectin and its receptors in the Japanese monkey, Macaca fuscata.

BACKGROUND: Adiponectin is an adipocyte-derived hormone that affects regulation of metabolic syndrome such as insulin resistance, type-2 diabetes, and obesity. It functions via seven transmembrane domain receptors [i.e.,adiponectin receptors 1 (AdipoR1) and 2 (AdipoR2)] that have been scarcely investigated in non-human primates. METHODS: Molecular cloning of cDNAs for adiponectin, AdipoR1, and AdipoR2 that included the whole protein-coding region in the Japanese monkey, Macaca fuscata, was carried out. Tissue-specific expression of respective genes was analyzed with Northern blot hybridization. RESULTS: The essential Cys36 and four lysine residues in adiponectin, and transmembrane-spanning domains in AdipoR1 and AdipoR2 appear well conserved. While adiponectin mRNA is expressed only in adipose tissues, AdipoR1 mRNA was found to be expressed in various tissues including the brain. CONCLUSIONS: These results significantly add to the understanding of the molecular basis of obesity-related adipokines and their receptors in nonhuman primates.

Effects of protein phosphatase inhibitors on the phosphorylation of spinal cord N-methyl-D-aspartate receptors following electroacupuncture stimulation in rats.

The present study investigated the role of inhibitor of protein phosphatases 1 and 2A on the modulation of the phosphorylation of the spinal N-methyl-D-aspartate receptor (NMDAR) NR1 and NR2B subunits following electroacupuncture (EA) stimulation in rats. Bilateral 2Hz EA stimulations with 1.0 mA were delivered at those acupoints corresponding to Zusanli and Sanyinjiao to men via needles for 30 min. EA analgesia was slightly reduced by the intrathecal injection of calyculin A during EA stimulation. At 60 min after the termination of EA stimulation, the levels of c-fos, serine phosphorylation of NR1 and NR2B by Western analysis had increased in the L(4-5) segments of the spinal cord after EA treatment. These expressions were enhanced by the intrathecal injection of calyculin A and immunohistochemical analyses confirmed the significant increase of these proteins. As for the regional reaction of NMDAR subunits, a mean integrated optical density of phosphorylated NR1 and NR2B subunits was potentiated by calyculin A injections in the superficial laminae and neck region and superficial laminae and nucleus proprius, respectively. It can be concluded that protein phosphatase may play an important role in EA analgesia by modulating the phosphorylation state of spinal NMDAR subunits.

Development of homogeneous enzyme immunoassay for the organophosphorus insecticide fenthion.

A rapid, convenient homogeneous competitive enzyme immunoassay for estimating the amount of fenthion is described. The assay utilizes glucose-6-phosphate dehydrogenase-hapten conjugates that are inhibited in solution by antibodies obtained from bovine serum albumin-hapten conjugates. In order to investigate the effects of bridging group recognition on the sensitivity of dose response characteristics, the bridging groups of varying alkyl chain length were attached at the phosphate position of fenthion. Among the antibodies used, the one obtained from the use of hapten (fenthion analog) with the same bridging group structure that was used in preparing the enzyme-fenthion conjugates showed maximum inhibition (up to 51.8%) in the absence of fenthion. In the presence of fenthion, the activity of the enzyme-hapten conjugate is regained in an amount proportional to the fenthion concentration. Under the optimized condition, the ED50 value for fenthion was 0.809 microg/ml. The assay developed in this study is a rapid effective screening method for fenthion prior to precise analysis.

Protective effects of Fructus sophorae extract on collagen-induced arthritis in BALB/c mice.

Styphnolobium japonicum (L.) is utilized in Korean medicine for the treatment of various inflammatory diseases. The aim of the present study was to explore the effects of Fructus sophorae extract (FSE) isolated from the dried ripe fruit of S. japonicum (L.) on the development of type II collagen-induced arthritis (CIA) in BALB/c mice. The CIA mice were orally administered FSE or saline daily for 2 weeks. The incidence and severity of disease and the inflammatory response in the serum and the joint tissues were assessed. Macroscopic and histological investigation indicated that FSE protected against CIA development. FSE was associated with a significant reduction in the levels of total immunoglobulin G2a and proinflammatory cytokines and mediators in the serum. In addition, FSE suppressed the gene expression levels of proinflammatory cytokines and mediators, the mediator of osteoclastic bone remodeling, the receptor activator of nuclear factor κ-B ligand and matrix metalloproteinases in the joint tissues. The present results suggest that FSE may protect against inflammation and bone damage, and would be a valuable candidate for further investigation as a novel anti-arthritic agent.

Electroacupuncture inhibits inflammatory edema and hyperalgesia through regulation of cyclooxygenase synthesis in both peripheral and central nociceptive sites.

We investigated the anti-inflammatory effects of electroacupuncture (EA) on carrageenan-induced inflammatory model in association with peripheral and spinal COX-2 expression. EA with 2, 15 and 120 Hz, especially 2 Hz, had significant inhibitory effects on the developing of edema and hyperalgesia, which was measured in 30-min intervals after carrageenan injection. Therefore, we investigated whether the effect of 2 Hz EA on carrageenan-induced edema and hyperalgesia is associated with peripheral and spinal expression of inflammatory proteins. The expression of cyclooxygenase (COX)-1, COX-2, and inducible nitric oxide synthase (iNOS) was inhibited by 2 Hz EA in carrageenan-injected rat paws. Interestingly, we found that the mRNA of COX-1 and COX-2 expression in the spine was not induced by 2 Hz EA treatment after carrageenan-induced peripheral inflammation. In addition, synthesis of prostaglandin E(2) (PGE(2)) was partially inhibited by 2 Hz EA treatment in both peripheral and spinal nociceptive regions. In conclusion, EA treatment might be a useful therapy for mitigation of inflammatory edema and hyperalgesia through regulation of COX-2 expression in both peripheral and central nociceptive sites.

Ameliorative effects of Artemisia argyi Folium extract on 2,4­dinitrochlorobenzene­induced atopic dermatitis­like lesions in BALB/c mice.

Artemisia argyi Folium has been used to treat skin diseases, including eczema and dermatitis, in South Korean medicine. The present study investigated the curative effects of Artemisia argyi Folium extract (AAFE) on 2,4­dinitrochlorobenzene (DNCB)­induced atopic dermatitis (AD)­like skin lesions in a BALB/c mouse model. Briefly, the dorsal skin of the BALB/c mice was sensitized three times with DNCB, whereas the ears were challenged twice. Repeated treatment with DNCB induced AD­like lesions. The effects of AAFE on AD­like lesions were evaluated by clinical observation, histopathological analysis, immunohistochemistry and enzyme­linked immunosorbent assay. In addition, reverse transcription­polymerase chain reaction and western blotting were performed. Treatment with AAFE reduced AD­like lesions, as determined by clinical observation, histopathological analysis, and detection of the serum levels of histamine, immunoglobulin E and cytokines. With regards to its mechanism of action, AAFE inhibited the phosphorylation of Lck/yes­related novel tyrosine kinase (Lyn), spleen tyrosine kinase (Syk), mitogen­activated protein kinases (MAPKs), phosphoinositide 3­kinase (PI3K)/Akt and IκBα, which have essential roles in the production of various cytokines in lymph nodes. The suppressive activity of AAFE may be due to the inhibition of a series of immunopathological events, including the release of proinflammatory cytokines. The results of the present study strongly suggest that AAFE exerts an anti­AD effect by inhibiting the Lyn, Syk, MAPKs, PI3K/Akt and IκBα pathways. Therefore, AAFE may be considered an effective herbal remedy for the treatment of AD.

Pulse Wave Variation during the Menstrual Cycle in Women with Menstrual Pain.

Objective. This study is performed to obtain objective diagnostic indicators associated with menstrual pain using pulse wave analysis. Methods. Using a pulse diagnostic device, we measured the pulse waves of 541 women aged between 19 and 30 years, placed in either an experimental group with menstrual pain (n = 329) or a control group with little or no menstrual pain (n = 212). Measurements were taken during both the menstrual and nonmenstrual periods, and comparative analysis was performed. Results. During the nonmenstrual period, the experimental group showed a significantly higher value in the left radial artery for the radial augmentation index (RAI) (p = 0.050) but significantly lower values for pulse wave energy (p = 0.021) and time to first peak from baseline (T1) (p = 0.035) in the right radial artery. During the menstrual period, the experimental group showed significantly lower values in the left radial artery for cardiac diastole and pulse wave area during diastole and significantly higher values for pulse wave area during systole, ratio of systolic phase to the full heartbeat, and systolic-diastolic ratio. Conclusion. We obtained indicators of menstrual pain in women during the menstrual period, including prolonged systolic and shortened diastolic phases, increases in pulse wave energy and area of representative pulse wave, and increased blood vessel resistance.

Induction of apoptosis by Chan Su, a traditional Chinese medicine, in human bladder carcinoma T24 cells.

Chan Su is a traditional Chinese medicine prepared from the dried white secretion of the auricular and skin glands of toads, and has been used as an Oriental drug. However, little is known about the effect of Chan Su on the growth of human cancer cells. This study was undertaken to investigate the underlying mechanism of Chan Su-induced apoptosis in a human bladder carcinoma cell line, T24. The effects of this compound were also tested on cyclooxygenase (COX) activity. Treatment of T24 cells with Chan Su resulted in the inhibition of viability and induction of apoptosis in a concentration-dependent manner, which was proved by trypan blue counts, DAPI staining, agarose gel electrophoresis and flow cytometric analysis. Apoptosis of T24 cells by Chan Su was associated with a down-regulation of anti-apoptotic Bcl-2 and Bcl-X(S/L) expression and an up-regulation of pro-apoptotic Bax expression. Chan Su treatment induced the proteolytic activation of caspase-3 and caspase-9, and a concomitant degradation of poly(ADP-ribose)-polymerase and beta-catenin protein. Furthermore, Chan Su decreased the levels of COX-2 mRNA and protein expression without significant changes in the levels of COX-1, which was correlated with an inhibition in prostaglandin E(2) synthesis. Taken together, these findings partially provide novel insights into the possible molecular mechanisms of the anti-cancer activity of Chan Su.

Tetrandrine-induced cell cycle arrest and apoptosis in A549 human lung carcinoma cells.

Tetrandrine, isolated from the root of Stephania tetrandra, has been used in Chinese medicine for the treatment of silicosis and arthritis, and it also has anti-tumor/growth activities. However, the signaling pathways of tetrandrine-induced growth arrest and apoptosis in cancer cells remain unclear. We investigated the molecular mechanisms of tetrandrine-induced apoptosis and growth arrest in human lung carcinoma cells. Upon treatment with tetrandrine, a time-dependent inhibition of cell growth was observed and cells developed many of the hallmark features of apoptosis. Flow cytometry analysis confirmed that tetrandrine increased populations of both apoptotic sub-G1 and G1 phase. Tetrandrine-induced growth inhibition was associated with induction of Cdk inhibitor p21, inhibition of cyclin D1 and activation of caspase-3. Tetrandrine also affected the expression patterns of cytoskeletons including distribution of F-actin and expression level of microtubule. These results suggest that tetrandrine merits further investigation as a cell cycle blocker as well as a cancer chemopreventive agent.

Resveratrol inhibits cell proliferation and induces apoptosis of human breast carcinoma MCF-7 cells.

Resveratrol, which is found in grapes and wine, has been reported to have a variety of important pharmacological effects including anti-inflammatory, anti-platelet, and anti-carcinogenetic properties. In this study, using the human breast cancer cell line MCF-7, we have analyzed a possible mechanism by which resveratrol could interfere with cell cycle control and induce cell death. Resveratrol treatment of MCF-7 cells resulted in a dose-dependent inhibition of the cell growth and the cells accumulated at the S phase transition of the cell cycle at low concentrations, but high concentrations do not induce S phase accumulation. The anti-proliferative effects of resveratrol were associated with a marked inhibition of cyclin D and cyclin-dependent kinase (Cdk) 4 proteins, and induction of p53 and Cdk inhibitor p21WAF1/CIP. Growth suppression by resveratrol was also due to apoptosis, as seen by the appearance of a sub-G1 fraction and chromatin condensation. In addition, the apoptotic process involves activation of caspase-9, a decrease of Bcl-2 as well as Bcl-XL levels, and an increase of Bax levels.

Development of a microtiter plate ELISA and a dipstick ELISA for the determination of the organophosphorus insecticide fenthion.

In previous studies, polyclonal antibodies against the organophosphorus insecticide fenthion were obtained and an indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed for this pesticide. In this study, using these antibodies and an enzyme tracer, direct competitive ELISAs for fenthion in microtiter plate and dipstick formats were developed. The microtiter plate ELISA showed an IC(50) value of 1.2 microg/L with a detection limit of 0.1 microg/L. The antibodies showed negligible cross-reactivity with other organophosphorus pesticides. The use of the dipstick format using Immunodyne as a support membrane allowed the quick visual detection of fenthion in concentrations >10 microg/L. The IC(50) value of the dipstick format using reflectance detection was 15 microg/L with a detection limit of 0.5 microg/L. The recoveries of fenthion from spiked vegetable samples using the two formats without any prior enrichment or cleanup steps were 87-116%.

Development of an enzyme-linked immunosorbent assay for the organophosphorus insecticide bromophos-ethyl.

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative detection of the organophosphorus insecticide bromophos-ethyl. Four bromophos-ethyl derivatives (haptens) were synthesized and were coupled to carrier proteins through the pesticide thiophosphate group to use as an immunogen or as a coating antigen. Rabbits were immunized with either one of two haptens coupled to bovine serum albumin for production of polyclonal antibodies, and the sera were screened against one of the haptens coupled to ovalbumin. Using the serum with highest titer, an antigen-coated ELISA was developed, which showed an IC(50) of 3.9 ng/mL with a detection limit of 0.3 ng/mL (20% inhibition). An antibody-coated ELISA using an enzyme tracer was also developed, which showed an IC(50) of 6.5 ng/mL with a detection limit of 1.0 ng/mL (20% inhibition). The antibodies showed negligible cross-reactivity with other organophosphorus pesticides except with the insecticides bromophos-methyl and chlorpyrifos in the antibody-coated assay only. Recoveries of bromophos-ethyl from fortified crop and water samples ranged from 82 to 128% and from 95 to 127%, respectively.

Oxidation of organophosphorus pesticides for the sensitive detection by a cholinesterase-based biosensor.

A potentiometric flow injection-type biosensor developed in our laboratory was used for the determination of organophosphorus pesticides (OPs). The principle of the biosensor is that the degree of inhibition of a sensor enzyme by an OP is dependent on the concentration of the pesticide. The sensor system consisted of a reactor with acetylcholinesterase (AChE) immobilized on a controlled pore glass and a detector with a tubular H(+)-selective membrane electrode. In order to examine the possibility of enhancing the sensitivity of the sensor by converting OPs to oxidized forms (stronger inhibitors), a comparison of the degree of enzyme inhibition by OPs at 10(-6) M before and after their oxidation was made. All of the ten pesticides tested exhibited greater inhibitory power toward the sensor enzyme following oxidation. All of the oxidized pesticides at 10(-6) M inhibited the sensor enzyme to a considerable degree, demonstrating the utility of the developed method for the class-specific determination of OPs. A calibration curve for diazinon, over the concentration range of 10(-11)-10(-4) M, was obtained. The lower detection limit was 2 x 10(-10) M. Treatment of the inhibited enzyme with pyridine-2-aldoxime restored the enzyme to near full activity, allowing repeated use of the sensor.

Combination of transcranial direct current stimulation and methylphenidate in subacute stroke.

Noninvasive transcranial direct current stimulation (tDCS) and methylphenidate (MP) are associated with motor recovery after stroke. Based on the potentially complementary mechanisms of these interventions, we examined whether there is an interactive effect between MP and tDCS. In this preliminary study, we randomized subacute stroke subjects to receive tDCS alone, MP alone or combination of tDCS and MP. A blinded rater measured safety, hand function, and cortical excitability before and after treatment. None of the treatments caused any major or severe adverse effects or induced significant differences in cortical excitability. Analysis of variance of gain score, as measured by Purdue pegboard test, showed a significant between-group difference (F(2,6)=12.167, p=0.008). Post hoc analysis showed that the combination treatment effected greater Purdue pegboard gain scores than tDCS alone (p=0.017) or MP alone (p=0.01). Our preliminary data with nine subjects shows an interesting dissociation between motor function improvement and lack of motor corticospinal plasticity changes as indexed by transcranial magnetic stimulation in subacute stroke subjects.

Importance of membrane selection in the development of immunochromatographic assays for low-molecular weight compounds.

This study was performed to demonstrate the importance of selecting an appropriate membrane when developing immunochromatographic assays (ICAs) for the sensitive detection of low-molecular weight compounds. Based on our findings, we propose a theoretical basis for selecting such a membrane. When eluting the sample solution for the competitive ICA using colloidal gold label for low-molecular analytes, the degree of binding inhibition is proportional to the collision frequency between the antibody-colloidal gold (Ab-CG) and analyte before Ab-CG binding to the capture antigen and a higher concentration of pesticides around the Ab-CG leads to a greater degree of inhibition. Therefore, we propose that the relative migration speed of the analyte and Ab-CG on the test strip is critically important for selecting a membrane in the development of sensitive competitive ICAs. We developed a novel method to estimate such a relative migration speed. We demonstrated the applicability of this proposal by using it to select an appropriate membrane for the development of an ICA of the pesticide diazinon.

Competitive immunochromatographic assay for the detection of the organophosphorus pesticide chlorpyrifos.

An immunochromatographic assay (ICA) based on competitive antigen-coated format using colloidal gold as the label was developed for the detection of the organophosphorus insecticide chlorpyrifos. The ICA test strip consisted of a membrane with a detection zone, a sample pad and an absorbent pad. The membrane was separately coated with chlorpyrifos Hapten-OVA conjugate (test line) and anti-mouse IgG (control line). Based on the fact that the competition is between the migrating analyte in the sample and the analyte hapten immobilized on the test strip for the binding sites of the antibody-colloidal gold (Ab-CG) conjugate migrating on the test strip, this study suggests that the relative migration speed between the two migrating substances is a critically important factor for the sensitive detection by competitive ICA. This criterion was utilized for the confirmation of appropriateness of a nitrocellulose (NC) membrane for chlorpyrifos ICA. The detection limit of the ICA for chlorpyrifos standard and chlorpyrifos spiked into agricultural samples were 10 and 50 ng mL(-1), respectively. The assay time for the ICA test was less than 10 min, suitable for rapid on-site testing of chlorpyrifos.

Monoclonal antibody-based enzyme-linked immunosorbent assays for the organophosphorus insecticide O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN).

This study aimed at developing competitive direct and indirect enzyme-linked immunosorbent assays (ELISAs) for the organophosphorus insecticide O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN) using a monoclonal antibody (mAb). Of the five EPN derivatives (haptens) prepared for use as an immunogen or as a competitor, two of them were used as the immunogen for the production of the mAbs. By using the antibody with the highest specificity and a coating antigen (hapten-OVA conjugate), a competitive indirect ELISA was developed, which showed an IC(50) of 2.9 ng/mL with a detection limit of 0.3 ng/mL. A competitive direct ELISA using a different antibody and an enzyme tracer was also developed, which showed an IC(50) of 0.6 ng/mL with a detection limit of 0.09 ng/mL. The mAbs in both assays showed negligible cross-reactivity with other organophosphorus pesticides. The recoveries of EPN from spiked samples determined by the developed ELISA ranged from 59 to 143%. Dilution of the samples improved the recovery. The assay performance of the present ELISAs based on the mAb was compared with that of the EPN ELISAs based on polyclonal antibodies (pAbs) that had been developed previously and was found to be better in dynamic response.

A sensitive enzyme immunoassay for measuring cotinine in passive smokers.

BACKGROUND: Both active smoking and passive exposure to tobacco smoke are major risk factors for cardiovascular, pulmonary, and oncological diseases. The serum level of cotinine, a major proximate metabolite of nicotine, reflects active or passive exposure to tobacco smoke. However, currently available enzyme-linked immunosorbent assays (ELISAs) for cotinine have limited sensitivity, and a high-throughput quantification of the severity of passive exposure to tobacco smoke has not been possible thus far. METHODS: We generated a phage display of combinatorial antibody library, from which we selected a recombinant antibody against cotinine, developed a sensitive ELISA using this antibody, and evaluated the method in a clinical setting and an animal model. RESULTS: The limits of detection and the lower limit of quantification were 31pg/mL and 1ng/mL cotinine, respectively. The intra- and inter-assay precisions based on three quality control samples were 3.8-13.5% and 14.0-15.0%, respectively. No significant interference from nicotine, trans-3'-hydroxy cotinine, tobacco alkaloids, or other serum components was found. When we applied our ELISA to serum samples from 36 volunteers, the serum cotinine levels were clustered into two groups, which exactly corresponded to their smoking behavior and this ELISA yielded reproducible and accurate results, which were comparable to those of LC/MS in a split assay. In animal studies, we were able to distinguish between rats injected with a nicotine dose equivalent to that of passive exposure to tobacco and rats without exposure. CONCLUSION: The competitive ELISA described here is useful for the detection and quantification of the severity of risk of passive smoking.

Development of ELISAs for the class-specific determination of organophosphorus pesticides.

Enzyme-linked immunosorbent assays (ELISAs) for the class-specific determination of organophosphorus (OP) pesticides were developed from monoclonal antibodies raised against haptens with the functional group common to OP pesticides. To develop antigen-coated, indirect, competitive ELISAs, four haptens with different spacer arm structures were used to prepare antibodies, while eight haptens were tested for use as coating antigens. A total of 32 ELISAs were developed with one selected as the most suitable one based on average IC(50) and % CV values. The chosen ELISA showed class-selective response to O,O-diethyl phosphorothioate and phosphorodithioate OP pesticides with negligible cross-reactivity to other types of pesticides. Average IC(50) and % CV values of this ELISA for the 12 OP pesticides were 89 ng/mL and 96%, respectively. Compared to ELISAs previously developed with the same objective, the current ELISA demonstrates better sensitivity based on much lower mean IC(50) values in addition to improved class-selective determination based on considerably lower % CV values as well as precise discrimination against other types of pesticides.

Development of enzyme-linked immunosorbent assays for the organophosphorus insecticide EPN.

Competitive enzyme-linked immunosorbent assays (ELISAs) in indirect and direct format were developed for the quantitative detection of the organophosphorus insecticide EPN. Five EPN derivatives (haptens) were synthesized and coupled to carrier proteins to use as an immunogen or as a competitor. Rabbits were immunized with two of the five haptens coupled to KLH for production of polyclonal antibodies, and the sera were screened against one of the haptens coupled to ovalbumin (OVA). Using the serum with the highest specificity and a coating antigen (hapten-OVA conjugate), an indirect (antigen-coated) ELISA was developed, which showed an IC(50) of 5.6 ng/mL with a detection limit of 0.2 ng/mL (20% inhibition). A direct (antibody-coated) ELISA using an enzyme tracer (hapten-enzyme conjugate) was also developed, which showed an IC(50) of 8.4 ng/mL with a detection limit of 0.9 ng/mL (20% inhibition). The antibodies showed negligible cross-reactivity with other organophosphorus pesticides except with the insecticide parathion-ethyl only in the direct ELISA. The recoveries of EPN from spiked samples determined by the indirect ELISAs were between 37 and 164%.