Showing papers by "Nagasaki International University published in 2012"

Dietary Crocin Inhibits Colitis and Colitis-Associated Colorectal Carcinogenesis in Male ICR Mice

Abstract: A natural carotenoid crocin is contained in saffron and gardenia flowers (crocuses and gardenias) and is used as a food colorant. This study reports the potential inhibitory effects of crocin against inflammation-associated mouse colon carcinogenesis and chemically induced colitis in male ICR mice. In the first experiment, dietary crocin significantly inhibited the development of colonic adenocarcinomas induced by azoxymethane (AOM) and dextran sodium sulfate (DSS) in mice by week 18. Crocin feeding also suppressed the proliferation and immunohistochemical expression of nuclear factor- (NF-) κB but increased the NF-E2-related factor 2 (Nrf2) expression, in adenocarcinoma cells. In the second experiment, dietary feeding with crocin for 4 weeks was able to inhibit DSS-induced colitis and decrease the mRNA expression of tumor necrosis factor α, interleukin- (IL-) 1β, IL-6, interferon γ, NF-κB, cyclooxygenase-2, and inducible nitric oxide synthase in the colorectal mucosa and increased the Nrf2 mRNA expression. Our results suggest that dietary crocin suppresses chemically induced colitis and colitis-related colon carcinogenesis in mice, at least partly by inhibiting inflammation and the mRNA expression of certain proinflammatory cytokines and inducible inflammatory enzymes. Therefore, crocin is a candidate for the prevention of colitis and inflammation-associated colon carcinogenesis.

Immunochemistry of pathogenic yeast, Candida species, focusing on mannan

Abstract: This review describes recent findings based on structural and immunochemical analyses of the cell wall mannan of Candida albicans, and other medically important Candida species Mannan has been shown to consist of α-1,2-, α-1,3-, α-1,6-, and β-1,2-linked mannopyranose units with few phosphate groups Each Candida species has a unique mannan structure biosynthesized by sequential collaboration between species-specific mannosyltransferases In particular, the β-1,2-linked mannose units have been shown to comprise a characteristic oligomannosyl side chain that is strongly antigenic For these pathogenic Candida species, cell-surface mannan was also found to participate in the adhesion to the epithelial cells, recognition by innate immune receptors and development of pathogenicity Therefore, clarification of the precise chemical structure of Candida mannan is indispensable for understanding the mechanism of pathogenicity, and for development of new antifungal drugs and immunotherapeutic procedures

Pharmacological Effects of Ginseng on Liver Functions and Diseases: A Minireview

Abstract: Ginseng, an ancient and famous medicinal herb in the Orient, has been used as a valuable tonic and for the treatment of various diseases including hepatic disorders. Ginseng saponins, commonly known as ginsenosides, are principal constituents and have believed to be responsible for multiple ginseng health benefits. There are more 40 ginsenosides isolated from ginseng. To date, treatment options for common liver diseases such as cirrhosis, fatty liver, and chronic hepatitis remain problematic. In this regard, ginseng extracts and individual ginsenosides have shown a wide array of beneficial role in the regulation of regular liver functions and the treatment of liver disorders of acute/chronic hepatotoxicity, hepatitis, hepatic fibrosis/cirrhosis, hepatocellular carcinoma, and so on in various pathways and mechanisms. In this paper, we first outline the pharmacological effects of ginseng and ginsenosides on the liver functions.

Involvement of NF-κB activation in the cisplatin resistance of human epidermoid carcinoma KCP-4 cells

Abstract: cis-Diamminedichloroplatinum II (cisplatin) is one of the most potent antitumor agents for the treatment of various types of cancer. In spite of its therapeutic usefulness, the intrinsic resistance acquired under continuous treatment limits its benefit in cancer therapy. KCP-4, a cisplatin-resistant cell line, was derived from human epidermoid carcinoma KB-3-1 cells. Since the accumulation of cisplatin in KCP-4 cells is markedly reduced by the presence of an efflux pump, this pump is thought to be related to cisplatin resistance of the KCP-4 cells. However, given that KCP-4 cells are tremendously resistant to cisplatin compared with KB-3-1 cells, it is possible that another mechanism exists. The aim of this study was to investigate whether the activation of nuclear factor-kappa B (NF-κB) contributes to the cisplatin resistance of KCP-4 cells. We used the level of translocated NF-κB into the nucleus, determined by immunoblot analysis, as the indicator of NF-κB activation. The activation level of NF-κB was higher in KCP-4 cells than in KB-3-1 cells. KCP-4 cells were treated with a combination of cisplatin and curcumin, an inhibitor of NF-κB activation, and the cell viabilities were subsequently determined by the MTT assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. In the presence of 10 µmol/l curcumin, we found that the sensitivity of KCP-4 cells to 100 and 300 µmol/l cisplatin was augmented. Additionally, curcumin reduced the activation levels of NF-κB in KCP-4 cells, and suppressed the expression levels of Bcl-2, Bcl-xL and survivin, which are apoptosis-related proteins regulated by NF-κB. Our results suggest that the high cisplatin resistance of KCP-4 cells compared with KB-3-1 cells results from multiple mechanisms other than increased cisplatin efflux, including the activation of NF-κB.

Increases of CRF in the amygdala are responsible for reinstatement of methamphetamine-seeking behavior induced by footshock

Abstract: Recent evidence suggests the involvement of corticotropin-releasing factor (CRF) in drug abuse. Here, we evaluated whether CRF modulates the reinstatement of methamphetamine (METH)-seeking behavior induced by stress using a drug-self administration paradigm in rats. Rats were trained to lever-press for intravenous METH (0.02 mg/infusion) accompanied by light and tone (drug-associated cues) and then underwent extinction training (saline substituted for METH without cues). Under the extinction condition, the inhibitory effects of a CRF receptor antagonist on the stress-induced reinstatement of METH-seeking behavior were assessed. Anxiety-like behaviors during METH withdrawal in METH self-administered rats were also evaluated. The non-selective CRF receptor antagonist α-helical CRF(9-41) attenuated METH-seeking behavior induced by footshock stress. CRF levels both in the amygdala and in plasma were significantly increased on day 10 of withdrawal after METH self-administration. However, plasma corticosterone concentrations were unchanged during the withdrawal. In addition, METH-seeking behavior was not affected by an inhibitor of corticosterone synthesis, metyrapone. In the elevated plus maze test, METH self-administered rats showed a decrease in the duration of time spent in the open arms on day 10 of withdrawal. The increased CRF levels in the amygdala may, at least in part, contribute to the footshock-induced reinstatement of METH-seeking behavior and the increase in anxiety-like behavior. The present findings indicate that CRF receptor antagonists would be useful as a therapeutic agent for METH-dependence.

Crocin promotes non‐rapid eye movement sleep in mice

Abstract: Crocus sativus L. (saffron) has been traditionally used for the treatment of insomnia and other diseases of the nervous systems. Two carotenoid pigments, crocin and crocetin, are the major components responsible for the various pharmacological activities of C. sativus L. In this study, we examined the sleep-promoting activity of crocin and crocetin by monitoring the locomotor activity and electroencephalogram after administration of these components to mice. Crocin (30 and 100 mg/kg) increased the total time of non-rapid eye movement (non-REM) sleep by 60 and 170%, respectively, during a 4-h period from 20:00 to 24:00 after its intraperitoneal administration at a lights-off time of 20:00. Crocetin (100 mg/kg) also increased the total time of non-REM sleep by 50% after the administration. These compounds did not change the amount of REM sleep or show any adverse effects, such as rebound insomnia, after the induction of sleep.

Molecular Mechanisms Underlying Anti-Inflammatory Actions of 6-(Methylsulfinyl)hexyl Isothiocyanate Derived from Wasabi (Wasabia japonica)

Abstract: 6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC) is a major bioactive compound in wasabi (Wasabia japonica), which is a typical Japanese pungent spice. Recently, in vivo and in vitro studies demonstrated that 6-MSITC has several biological properties, including anti-inflammatory, antimicrobial, antiplatelet, and anticancer effects. We previously reported that 6-MSITC strongly suppresses cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and cytokines, which are important factors that mediate inflammatory processes. Moreover, molecular analysis demonstrated that 6-MSITC blocks the expressions of these factors by suppressing multiple signal transduction pathways to attenuate the activation of transcriptional factors. Structure-activity relationships of 6-MSITC and its analogues containing an isothiocyanate group revealed that methylsulfinyl group and the length of alkyl chain of 6-MSITC might be related to high inhibitory potency. In this paper, we review the anti-inflammatory properties of 6-MSITC and discuss potential molecular mechanisms focusing on inflammatory responses by macrophages.

Restoration of Dioxin-Induced Damage to Fetal Steroidogenesis and Gonadotropin Formation by Maternal Co-Treatment with α-Lipoic Acid

Abstract: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an endocrine disruptor, causes reproductive and developmental toxic effects in pups following maternal exposure in a number of animal models. Our previous studies have demonstrated that TCDD imprints sexual immaturity by suppressing the expression of fetal pituitary gonadotropins, the regulators of gonadal steroidogenesis. In the present study, we discovered that all TCDD-produced damage to fetal production of pituitary gonadotropins as well as testicular steroidogenesis can be repaired by co-treating pregnant rats with α-lipoic acid (LA), an obligate co-factor for intermediary metabolism including energy production. While LA also acts as an anti-oxidant, other anti-oxidants; i.e., ascorbic acid, butylated hydroxyanisole and edaravone, failed to exhibit any beneficial effects. Neither wasting syndrome nor CYP1A1 induction in the fetal brain caused through the activation of aryl hydrocarbon receptor (AhR) could be attenuated by LA. These lines of evidence suggest that oxidative stress makes only a minor contribution to the TCDD-induced disorder of fetal steroidogenesis, and LA has a restorative effect by targeting on mechanism(s) other than AhR activation. Following a metabolomic analysis, it was found that TCDD caused a more marked change in the hypothalamus, a pituitary regulator, than in the pituitary itself. Although the components of the tricarboxylic acid cycle and the ATP content of the fetal hypothalamus were significantly changed by TCDD, all these changes were again rectified by exogenous LA. We also provided evidence that the fetal hypothalamic content of endogenous LA is significantly reduced following maternal exposure to TCDD. Thus, the data obtained strongly suggest that TCDD reduces the expression of fetal pituitary gonadotropins to imprint sexual immaturity or disturb development by suppressing the level of LA, one of the key players serving energy production.

Characterization of Vitamin B12 Compounds in the Wild Edible Mushrooms Black Trumpet (Craterellus cornucopioides) and Golden Chanterelle (Cantharellus cibarius)

Abstract: This study determined the vitamin B₁₂ content of six wild edible mushrooms which are consumed by European vegetarians. Zero or trace levels (0.01-0.09 µg/100 g dry weight) of vitamin B₁₂ were determined in porcini mushrooms (Boletus spp.), parasol mushrooms (Macrolepiota procera), oyster mushrooms (Pleurotus ostreatus), and black morels (Morchella conica). By contrast, black trumpet (Craterellus cornucopioides) and golden chanterelle (Cantharellus cibarius) mushrooms contained considerable levels (1.09-2.65 µg/100 g dry weight) of vitamin B₁₂. To determine whether C. cornucopioides or C. cibarius contained vitamin B₁₂ or other corrinoid compounds that are inactive in humans, we purified a corrinoid compound using an immunoaffinity column and identified it as vitamin B₁₂ based on LC/ESI-MS/MS chromatograms.

Nuage proteins: their localization in subcellular structures of spermatogenic cells as revealed by immunoelectron microscopy.

Abstract: Chromatoid body (CB) was identified as granules stained by basic dye 130 years ago and called by various names. Electron microscopy revealed that the CB belonged to nuage (cloud in French) specific for germ cells. We described the localization of several proteins, including RNA helicases, in the nuage compartments classified into six types and in several spermatogenic cell-specific structures. All the proteins examined were detected in the nuage, including the CB with different staining intensities. Several proteins were localized to non-nuage structures, suggesting that these nuage proteins structures are related to nuage function.

Surface potential of methyl isobutyl carbinol adsorption layer at the air/water interface.

Abstract: The surface potential (ΔV) and surface tension (γ) of MIBC (methyl isobutyl carbinol) were measured on the subphase of pure water and electrolyte solutions (NaCl at 0.02 and 2 M). In contrast to ionic surfactants, it was found that surface potential gradually increased with MIBC concentration. The ΔV curves were strongly influenced by the presence of NaCl. The available model in literature, in which surface potential is linearly proportional to surface excess, failed to describe the experimental data. Consequently, a new model, employing a partial charge of alcohol adsorption layer, was proposed. The new model predicted the experimental data consistently for MIBC in different NaCl solutions. However, the model required additional information for ionic impurity to predict adsorption in the absence of electrolyte. Such inclusion of impurities is, however, unnecessary for industrial applications. The modeling results successfully quantify the influence of electrolytes on surface potential of MIBC, which is critical for froth stability.

A Single Nucleotide Polymorphism within the Novel Sex-Linked Testis-Specific Retrotransposed PGAM4 Gene Influences Human Male Fertility

Abstract: Background The development of novel fertilization treatments, including in vitro fertilization and intracytoplasmic injection, has made pregnancy possible regardless of the level of activity of the spermatozoa; however, the etiology of male-factor infertility is poorly understood Multiple studies, primarily through the use of transgenic animals, have contributed to a list of candidate genes that may affect male infertility in humans We examined single nucleotide polymorphisms (SNPs) as a cause of male infertility in an analysis of spermatogenesis-specific genes Methods and Finding We carried out the prevalence of SNPs in the coding region of phosphoglycerate mutase 4 (PGAM4) on the X chromosome by the direct sequencing of PCR-amplified DNA from male patients Using RT-PCR and western blot analyses, we identified that PGAM4 is a functional retrogene that is expressed predominantly in the testes and is associated with male infertility PGAM4 is expressed in post-meiotic stages, including spermatids and spermatozoa in the testes, and the principal piece of the flagellum and acrosome in ejaculated spermatozoa A case-control study revealed that 45% of infertile patients carry the G75C polymorphism, which causes an amino acid substitution in the encoded protein Furthermore, an assay for enzymatic activity demonstrated that this polymorphism decreases the enzyme’s activity both in vitro and in vivo Conclusion These results suggest that PGAM4, an X-linked retrogene, is a fundamental gene in human male reproduction and may escape meiotic sex chromosome inactivation These findings provide fresh insight into elucidating the mechanisms of male infertility

Expression of DDX25 in nuage components of mammalian spermatogenic cells: immunofluorescence and immunoelectron microscopic study

Abstract: The localization of DDX25/GRTH and gonadotropin-stimulated RNA helicase was studied in the spermatogenic cells of rat, mouse, and guinea pig by immunofluorescence and immunoelectron microscopy (IEM). Immunofluorescence studies identified four kinds of granular staining: (1) fine particles observed in meiotic cells; (2) small granules associated with a mitochondrial marker, appearing in pachytene spermatocytes after stage V; (3) short strands lacking the mitochondrial marker in late spermatocytes; and, (4) large irregularly shaped granules in round spermatids. IEM identified DDX25 signals in nine compartments: (1) fine dense particles in the meiotic cells; (2) intermitochondrial cement; (3) loose aggregates of 70–90 nm particles; (4) chromatoid bodies; (5) late chromatoid bodies; (6) satellite bodies; (7) granulated bodies; (8) mitochondria-associated granules; and, (9) reticulated bodies. Compartments (1) to (6) were previously classified into nuage while (7) to (9) were classified as nuage components by the present study. The results suggest that DDX25 functions in these nine compartments.

Cytotoxicity and Glycan-Binding Properties of an 18 kDa Lectin Isolated from the Marine Sponge Halichondria okadai

Abstract: A divalent cation-independent lectin—HOL-18, with cytotoxic activity against leukemia cells, was purified from a demosponge, Halichondria okadai. HOL-18 is a 72 kDa tetrameric lectin that consists of four non-covalently bonded 18 kDa subunits. Hemagglutination activity of the lectin was strongly inhibited by chitotriose (GlcNAcβ1-4GlcNAcβ1-4GlcNAc), fetuin and mucins from porcine stomach and bovine submaxillary gland. Lectin activity was stable at pH 4–12 and temperatures lower than 60 °C. Frontal affinity chromatography with 16 types of pyridylaminated oligosaccharides indicated that the lectin had an affinity for N-linked complex-type and sphingolipid-type oligosaccharides with N-acetylated hexosamines and neuramic acid at the non-reducing termini. The lectin killed Jurkat leukemia T cells and K562 erythroleukemia cells in a dose- and carbohydrate-dependent manner.

Modulation of plumbagin production in Plumbago zeylanica using a single-chain variable fragment antibody against plumbagin

Abstract: A single-chain variable fragment antibody (scFv) against plumbagin (PL) accumulated the PL production in the hairy roots of Plumbago zeylanica. Recombinant Agrobacterium rhizogenes (ATCC 15834) containing an scFv gene against PL (PL-scFv) were obtained through triparental mating and transformed into P. zeylanica to induce PL-scFv protein in the hairy roots. Up to 40 μg recombinant PL-scFv were expressed per milligram of soluble protein in transgenic P. zeylanica hairy root cultures. The mean PL content obtained from transgenic hairy roots (12.24 μg/100 mg dry weight) exhibited 2.2 times higher than those obtained from wild-type (5.48 μg/100 mg dry weight). The high correlation between the PL-scFv expression level and PL content of the recombinant plants suggested that the PL biosynthesis pathway had been modulated by the expression of PL-scFv protein in the hairy roots of P. zeylanica.

Langmuir monolayer miscibility of perfluorocarboxylic acids with biomembrane constituents at the air-water interface.

Abstract: Over the past two or three decades, Langmuir monolayers comprising various fluorinated amphiphiles such as perfluorinated, partially fluorinated, and fluorinated-hydrogenated hybrid molecules at the air-water interface, have been investigated to deepen the understanding of their characteristic phenomena and to explore the mutual interaction of these amphiphiles with biomembrane constituents. Langmuir monolayers of these fluorinated amphiphiles are potentially applicable in the fields of materials, biological and clinical science, where they may be utilized in two-dimensional protein crystallization, microelectronics, and for surfactant replacement in the lung. This review article focuses on the miscibility between perfluorinated long-chain carboxylic acids (CF₃(CF₂)(n-1)COOH, or FnCOOH) and phospholipids or fatty acids in biomembranes upon lateral compression, and describes how the chain-length mismatch between the fluorinated and the hydrogenated tails relates to the interfacial phase behavior from the thermodynamic and the morphological aspects at the micrometer and nanometer levels.

Cardiovascular drift during low intensity exercise with leg blood flow restriction.

Abstract: Previous studies reported that aerobic-type exercise such as walking or cycling with blood flow restriction (BFR) has been shown to elicit increases in leg muscle hypertrophy and strength, as well as improved aerobic capacity. Although previous studies investigated cardiovascular responses during a relatively short duration of exercise (∼5 min), the effects of prolonged leg muscular BFR have remained unknown. The purpose of this study was to examine the cardiovascular effects of longer duration low intensity exercise combined with BFR. Eight men performed 30 min of exercise at 40% of a predetermined maximal oxygen uptake under both BFR and normal flow (CON) conditions. Cardiovascular parameters were measured at rest and every 10 min during exercise. The main findings were that 1) the SV and HR did not change significantly between 10 to 30 min of exercise in BFR and CON conditions, although BFR-induced reduction of SV and increased HR were found at 10 min exercise compared with normal flow, 2) blood pressure was increased at 10 min of exercise in BFR compared to the CON, however the blood pressure decreased gradually with BFR from 10 to 30 min of exercise, and 3) blood lactate and RPE increased gradually during exercise with BFR. In conclusion, our results suggest that the BFR-induced reduction of SV and increased HR within the first 10 min of exercise are representative of changes in these parameters.

Characterization of Magnetic Markers for Liquid-Phase Immunoassays Using Brownian Relaxation

Abstract: We characterized the magnetic markers used in biological immunoassays based on Brownian relaxation. Because the markers are composed of aggregated nanoparticles, i.e., magnetic nanoclusters, we first clarified their magnetic properties using AC susceptibility measurements, magnetization (M–H) curves, and magnetic relaxation properties. Analyzing the experimental results, we obtained the key parameters for the immunoassay, i.e., hydrodynamic diameter dh, magnetic moment mB, and anisotropy energy EB of the markers. Because these parameters were distributed in practical samples, we took their distribution into account in the analysis. Next, we showed the relationship between these parameters obtained from different samples. It was shown that mB increased approximately in proportion to dh. On the other hand, no clear correlation between mB and EB was obtained. These results were very different from those expected from single-domain nanoparticles and must be taken into account when magnetic markers are used in immunoassays based on Brownian relaxation.

Phosphoenolpyruvic Acid, an Intermediary Metabolite of Glycolysis, as a Potential Cytoprotectant and Anti-oxidant in HeLa Cells

Abstract: This study examined the cytoprotective and anti-oxidative properties of phosphoenolpyruvic acid (PEP), a glycolysis metabolite with a high-energy phosphate group. PEP (0.1–10 mM) significantly attenuated the decrease in cell viability induced by hydrogen peroxide (H2O2) in HeLa cells in a dose-dependent manner. PEP also inhibited the decrease in calcein-acetomethoxy-stained cells and the increase in propidium iodide-stained cells that were induced by H2O2. The H2O2-stimulated increase in intracellular reactive oxygen species was significantly reduced by PEP. PEP also demonstrated scavenging potential against hydroxyl radicals, as assessed by the electron paramagnetic resonance method. In addition, PEP demonstrated scavenging potential against the 1,1-diphenyl-2-picrylhydrazyl radical, a representative artificial radical, although the potential is very weak. PEP (10 mM) slightly inhibited the decrease in cellular ATP content induced by H2O2, but did not show any effects at low doses (0.1, 1 mM). PEP (0.1–10 mM) also attenuated the cell injury but not the decrease in intracellular ATP content, induced by 2-deoxy-D-glucose, a glycolysis inhibitor. These results indicate that PEP exerts cytoprotective effects and has anti-oxidative potential, although the precise cytoprotective mechanisms are not fully elucidated. We suggest that PEP is a functional carbohydrate metabolite with cytoprotective and anti-oxidative activity, and is potentially useful as a therapeutic agent against diseases that involve the oxidative stress.

Preparation of a Single-Chain Variable Fragment and a Recombinant Antigen-Binding Fragment against the Anti-Malarial Drugs, Artemisinin and Artesunate, and Their Application in an ELISA

Abstract: Two different recombinant antibodies, a single-chain variable fragment (scFv) and an antigen-binding fragment (Fab), were prepared against artemisinin (AM) and artesunate (AS) and were developed for use in an enzyme-linked immunosorbent assay (ELISA). The recombinant antibodies, which were derived from a single monoclonal antibody against AM and AS (mAb 1C1) prepared by us, were expressed by Escherichia coli cells and their reactivity and specificity were characterized. As a result, to obtain sufficient signal in indirect ELISA, a much greater amount of a first antibody was needed in the use of scFv due to the differences of the secondary antibody and conformational stability. Therefore, we focused on the development of the recombinant Fab antibodies and applied it to indirect competitive ELISA. The specificity of the Fab was similar to that of mAb 1C1 in that it showed specific reactivity toward AM and AS only. The sensitivity of the icELISA (0.16 μg/mL to 40 μg/mL for AM and 8.0 ng/mL to 60 ng/mL for AS...

Eastern blotting analysis and isolation of two new dammarane-type saponins from American ginseng.

Abstract: Ginsenosides, the major active component of American ginseng, were analyzed using eastern blotting with anti-ginsenoside Rb(1) and Rg(1) monoclonal antibodies (MAbs). Immunoassay-guided fractionation of the methanol extract of American ginseng and column chromatography led to the isolation of two new minor dammarane-type saponins, named quinquenosides Ja (1) and Jb (2). Their structures were elucidated to be 6-O-[α-L-rhamnopyranosyl(1→2)-β-D-glucopyranosyl]-20-O-[β-D-glucopyranosyl(1→4)-β-D-glucopyranosyl]-3β,6α,12β,20β-tetrahydroxydammar-24-ene (1) and 3-O-[β-D-glucopyranosyl(1→2)-β-D-glucopyranosyl]-20-O-{[α-L-arabinofuranosyl(1→6)-β-D-glucopyranosyl](1→6)-β-D-glucopyranosyl}-3β,12β,20β-trihydroxydammar-24-ene (2) on the basis of chemical and spectroscopic methods.

Fluobodies against Bioactive Natural Products and their Application in Fluorescence-Linked Immunosorbent Assay

Abstract: An enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody (MAb), Fab antibody, and single-chain variable fragment (scFv) antibody has become one of the most promising analytical methods owing to its rapidity, sensitivity, and reliability. Recently, a chimera of green fluorescent protein (GFP) with a scFv antibody, named fluobody, was proposed as a probe for an alternative immunosorbent assay; i.e., fluorescence-linked immunosorbent assay (FLISA). In this FLISA, an even more sensitive, simple, and rapid immunoassay can be performed by detecting the highly sensitive fluorophore of GFP that is genetically and directly fused to the scFv antibody. In addition, the time- and cost-consuming secondary antibody reaction and the following enzyme-substrate reaction, necessary for conventional ELISA, can be avoided, making it possible to complete the assay more rapidly. Focusing on naturally occurring bioactive products, fluobody recognizing 1,4-naphthoquinone, plumbagin and triterpenoid saponin, ginsenosides were successfully expressed in Escherichia coli (E. coli) and applied to FLISA. The construction, the expression, and the potential use of fluobody in quantitative/qualitative analysis of bioactive natural products are reviewed in this article.

Identification and recombinant analysis of botrocetin-2, a snake venom cofactor for von Willebrand factor-induced platelet agglutination.

Abstract: Botrocetin is a heterodimer snake venom protein that induces von Willebrand factor (VWF)- and platelet glycoprotein Ib (GPIb)-dependent platelet agglutination in vitro. We have cloned cDNAs for a botrocetin-2 from a cDNA library of the venom gland of Bothrops jararaca having a high similarity with botrocetin subunits. Recombinant botrocetin-2, expressed in 293T cells, showed cofactor activity comparable to natural botrocetin. In a single subunit expression experiment, a dimer of the β subunit was obtained, and it showed reduced, but apparent, platelet agglutination activity. Ala scanning mutagenesis showed that substitutions at Asp62, Asp70, Arg115, or Lys117 in the β subunit reduced platelet agglutination activity. The 3D homology modeling of botrocetin-2 complexed with the VWF A1 domain and GPIbα indicated that Asp62, Arg115, and Lys117 of the β subunit are located near Arg218 and Asp222 of GPIbα, respectively, and that Aspβ70 is in proximity to Gln1391 of the A1 domain. Our results indicate that these charged amino acid residues in the β subunit have a preferential role in the activity of botrocetin-2. Since it has been time-consuming and difficult to obtain homogeneous botrocetin from natural venom, recombinant botrocetin-2 has potential benefits for clinical and basic investigations into hemostasis and thrombosis as a standard reagent.

Localization of BRUNOL2 in Rat Spermatogenic Cells as Revealed by Immunofluorescence and Immunoelectron Microscopic Techniques

Abstract: Distribution and localization of a RNA-binding protein, BRUNOL2 in rat spermatogenic cells were studied by dot blotting of cell fractions, immunofluorescence (IF), and immunoelectron microscopy (IEM) BRUNOL2 distributed in nuclear (23%), mitochondrial (19%), microsomal (15%), and cytosol fractions (43%) BRUNOL2 was detected in all spermatogenic cells In the cytoplasm and nucleoplasm of the spermatogonia, spermatocytes and spermatids, both diffuse and granular staining patterns were observed Many cytoplasmic granules were stained also for DDX4 and DDX25 Large granules in the cytoplasm of elongated spermatids were stained for BRUNOL2 but not for the nuage proteins IEM showed that gold signals for BRUNOL2 were concentrated in nuage components including loose aggregates of small particles, chromatoid body (CB), intermitochondrial cement (IMC), and satellite body (SB) In addition, many non-nuage structures such as ER-attached small granules, less dense material surrounding connecting piece of flagellum, reticulated body, mitochondria-associated granules (MAG), granulated body, ribosome aggregate, and manchette, were stained for BRUNOL2 with different staining intensities In the nucleus, gold signals were concentrated in heterochromatin area and nucleolus The results suggest that BRUNOL2 is one of the nuage proteins and also associated with the other non-nuage structures, suggesting multiple functions of this protein