Effects of various chemical agents and early ethylene production on floral senescence of Hibiscus syriacus L.

To understand the factors that induce floral senescence in Hibiscus syriacus L., we have investigated the effects of various chemical agents on flower senescence at two different flowering stages, before and after full bloom, as well as the relationship between flower longevity and endogenous ethylene production before full bloom. Treatments with ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), and ethephon enhanced floral senescence, while aminoethoxyvinylglycine (AVG) promoted flower longevity regardless of treatment timing. Although ethanol slightly extended flower longevity, abscisic acid (ABA), nitric oxide, boric acid and sucrose, which have been reported to affect flower longevity or senescence, had no effect on H. syriacus floral senescence. The polyamine spermine (SPM), methylglyoxal-bis(guanylhydrazone) (MGBG), an inhibitor of SPM biosynthesis, and cycloheximide (CHI) accelerated flower senescence when applied before full bloom, but had no effect when applied after full bloom. SPM, MGBG and CHI treatments resulted in enhanced ethylene production during flower opening, and the promotion of flower senescence is mediated by ethylene production prior to full bloom. Furthermore, endogenous ethylene, spontaneously produced before blooming, was closely associated with floral senescence. These results suggest that ethylene production during flower opening plays a key role in determining the timing of Hibiscus flower senescence.     

Aberrant expression of retinol-binding protein, osteopontin and fibroblast growth factor 7 in the porcine uterine endometrium of pregnant recipients carrying embryos produced by somatic cell nuclear transfer

The technique of somatic cell nuclear transfer (NT) is a useful tool to produce cloned animals for various purposes, but the efficiency to generate cloned animals using this technique is still very low. To improve the low efficiency in production of cloned pigs it is critical to understand the reprogramming process during development of cloned embryos, but it is also essential to understand the uterine function interacting with the transferred cloned embryos during implantation and placentation. Thus, to understand the uterine responsiveness to NT cloned embryos during pregnancy, we investigated expression of retinol-binding protein (RBP), osteopontin (OPN) and fibroblast growth factor 7 (FGF7), which play important roles in implantation and/or maintenance of pregnancy as a transport protein, an extracellular matrix protein and a growth factor, respectively, in the uterine endometrium in pigs. The uterine tissue samples were obtained by C-section from pigs with NT cloned normal (NT-normal) embryos and NT cloned abnormal (NT-abnormal) embryos and pigs with non-NT (Non-NT) embryos at term. Immunoblot analysis showed that expression of RBP and FGF7 decreased in the uterine endometrium of recipient gilts carrying NT embryos than in the endometrium of gilts carrying Non-NT embryos. Levels of OPN protein of 70 and 45kDa were not different in between the uterine endometrium of gilts carrying Non-NT and NT-normal embryos, but in the uterine endometrium of gilts carrying NT-abnormal embryos 70 and 45kDa OPN proteins increased compared to those in the endometrium of gilts carrying Non-NT embryos. Immunohistochemistry results showed that RBP expression was lower in the endometrial glandular epithelial cells, while OPN expression was higher in the endometrial luminal epithelial cells of the uterus of gilts carrying NT embryos than in the uterus of gilts carrying Non-NT embryos. Results of this study showed that maternal uterine genes were aberrantly expressed in the uterine endometrium of gilts carrying NT cloned embryos in varying degrees depending on the normality of the developing embryos. These results indicate that abnormal maternal-fetal interactions of the uterus carrying the developing NT cloned embryos may cause problems in development of cloned embryos.     

Quercetin‐induced upregulation of human GCLC gene is mediated by cis‐regulatory element for early growth response protein‐1 (EGR1) in INS‐1 beta‐cells

The catalytic subunit of γ‐glutamylcysteine ligase (GCLC) catalyses the rate‐limiting step in the de novo synthesis of glutathione (GSH), which is involved in maintaining intracellular redox balance. GSH is especially important for antioxidant defense system since beta‐cells show intrinsically low expression of antioxidant enzymes. In the present study, we investigated the regulatory mechanisms by which quercetin, a flavonoid, induces the expression of the GCLC gene in rat pancreatic beta‐cell line INS‐1. Promoter study found that the proximal GC‐rich region (from ?90 to ?34) of the GCLC promoter contained the quercetin‐responsive cis‐element(s). The quercetin‐responsive region contains consensus DNA binding site for early growth response 1 (EGR1) at ‐67 (5′‐CGCCTCCGC‐3′) which overlaps with a putative Sp1 binding site. Electrophoretic mobility shift assay showed that an oligonucleotide containing the EGR1 site was bound to nuclear factors EGR1, Sp1, and Sp3. In the promoter analysis, mutation of EGR1 site significantly reduced the quercetin response, whereas mutation of Sp1 site decreased only the basal activity of the GCLC promoter. Additionally, the transient overexpression of EGR1 significantly increased basal activity of the GCLC promoter. Finally, we showed that quercetin potently induced both EGR1 mRNA and its protein levels without affecting the expression of Sp1 and Sp3 proteins. Therefore, we concluded that EGR1 was bound to GC‐rich region of the GCLC gene promoter, which was prerequisite for the transactivation of the GCLC gene by quercetin. J. Cell. Biochem. 108: 1346–1355, 2009. © 2009 Wiley‐Liss, Inc.     

Swiprosin‐1 is expressed in mast cells and up‐regulated through the protein kinase CβI/η pathway

Swiprosin‐1 exhibits the highest expression in CD8⁺ T cells and immature B cells and has been thought to play a role in lymphocyte physiology. Here we report that swiprosin‐1 is also expressed in mast cells and up‐regulated in both in vitro cultured mast cells by phorbol ester and in vivo model tissues of passive cutaneous anaphylaxis and atopic dermatitis. Targeted inhibition of the specific protein kinase C (PKC) isotypes by siRNA revealed that PKC‐βI/η are involved in the expression of swiprosin‐1 in the human mast cell line HMC‐1. In contrast, down‐regulation of swiprosin‐1 by A23187 or ionomycin suggests that calcium‐signaling plays a negative role. The ectopic expression of swiprosin‐1 augmented PMA/A23187‐induced NF‐κB promoter activity, and resulted in increased expression of cytokines. Moreover, knock‐down of swiprosin‐1 attenuated PMA/A23187‐induced cytokine expression. Collectively, these results suggest that swiprosin‐1 is a PKC‐βI/η‐inducible gene and it modulates mast cell activation through NF‐κB‐dependent pathway. J. Cell. Biochem. 108: 705–715, 2009. © 2009 Wiley‐Liss, Inc.     

Proteomic analysis of differentially expressed proteins in human cholangiocarcinoma cells treated with Clonorchis sinensis excretory–secretory products

Severe Clonorchis sinensis infection is a significant risk factor for malignant changes in bile ducts and surrounding liver tissues occurring as a result of direct contact with C. sinensis worms and their excretory–secretory products (ESP). However, the intrinsic molecular mechanisms involved in these processes remain obscure. To determine the effects of C. sinensis infection on protein expression in host bile duct epithelium, we examined proteomic profile changes in the human cholangiocarcinoma cell line (HuCCT1) treated with ESP at 24 h. Using a combination of 2‐DE, quantitative image and MALDI‐TOF MS analysis, we identified 83 proteins that were translationally modulated in response to ESP, among which 49 were up‐regulated and 34 down‐regulated. These proteins were classified under various biological categories, including metabolism, cell structure and architecture, proteolysis, protein modification, transport, signal transduction, and reactive oxygen species (ROS) detoxification. In particular, ESP induced the expression of redox‐regulating proteins, including peroxiredoxins (Prdx 2, 3, and 6) and thioredoxin 1 (Trx 1), possibly via intracellular ROS generation. Application of the proteomic approach to identify ESP response proteins should be a prerequisite before further investigation to clarify the molecular pathways and mechanisms involved in C. sinensis infection of host cells. J. Cell. Biochem. 108: 1376–1388, 2009. © 2009 Wiley‐Liss, Inc.     

All‐atom chain‐building by optimizing MODELLER energy function using conformational space annealing

We have investigated the effect of rigorous optimization of the MODELLER energy function for possible improvement in protein all‐atom chain‐building. For this we applied the global optimization method called conformational space annealing (CSA) to the standard MODELLER procedure to achieve better energy optimization than what MODELLER provides. The method, which we call MODELLERCSA , is tested on two benchmark sets. The first is the 298 proteins taken from the HOMSTRAD multiple alignment set. By simply optimizing the MODELLER energy function, we observe significant improvement in side‐chain modeling, where MODELLERCSA provides about 10.7% (14.5%) improvement for χ₁ (χ₁ + χ₂) accuracy compared to the standard MODELLER modeling. The improvement of backbone accuracy by MODELLERCSA is shown to be less prominent, and a similar improvement can be achieved by simply generating many standard MODELLER models and selecting lowest energy models. However, the level of side‐chain modeling accuracy by MODELLERCSA could not be matched either by extensive MODELLER strategies, side‐chain remodeling by SCWRL3, or copying unmutated rotamers. The identical procedure was successfully applied to 100 CASP7 template base modeling domains during the prediction season in a blind fashion, and the results are included here for comparison. From this study, we observe a good correlation between the MODELLER energy and the side‐chain accuracy. Our findings indicate that, when a good alignment between a target protein and its templates is provided, thorough optimization of the MODELLER energy function leads to accurate all‐atom models. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Multiple isoforms of beta-TrCP display differential activities in the regulation of Wnt signaling

The F-box proteins beta-TrCP1 and 2 (beta-transducin repeat containing protein) have 2 and 3 isoforms, respectively, due to alternative splicing of exons encoding the N-terminal region. We identified an extra exon in between the previously known exons 1 and 2 of beta-TrCP1 and beta-TrCP2. Interestingly, sequence analysis suggested that many more isoforms are produced than previously identified, via the alternative splicing of all possible combination of exons II to V of beta-TrCP1 and exons II to IV of beta-TrCP2. Different mouse tissues show specific expression patterns of the isoforms, and the level of expression of the isoform that has been used in most published papers was very low. Yeast two-hybrid assays show that beta-TrCP1 isoforms containing exon III, which are the most highly expressed isoforms in most tissues, do not interact with Skp1. Indirect immunofluorescence analysis of transiently expressed beta-TrCP1 isoforms suggests that the presence of exon III causes beta-TrCP1 to localize in nuclei. Consistent with the above findings, isoforms including exon III showed a reduced ability to block ectopic embryonic axes induced via injection of Wnt8 or beta-catenin in Xenopus embryos. Overall, our data suggest that isoforms of beta-TrCPs generated by alternative splicing may have different biological roles.     

Kaempferol from the leaves of Apocynum venetum possesses anxiolytic activities in the elevated plus maze test in mice

The present work evaluated the anxiolytic activity of an aqueous extract of Apocynum venetum L. (Apocynaceae) and bioguided its fractionation using the elevated plus maze (EPM) in mice as a model of anxiety. A single treatment of AV extract markedly increased the percentage time spent on the open arms of the EPM in two distinct concentration ranges of 22.5–30 and 100–125 mg/kg p.o., respectively, indicating a putative anxiolytic-like activity. Fractions showing anxiolytic effects in concentrations equal to 30 or 125 mg/kg of whole extract were antagonized using the benzodiazepine antagonist flumazenil (3 mg/kg i.p.) or the 5-HT_1A receptor antagonist WAY-100635 (0.5 mg/kg i.p.). All active fractions in a concentration equal to 125 mg/kg were effectively blocked by the benzodiazepine antagonist flumazenil, while the anxiolytic activities of fractions in the lower dose equivalent to 30 mg/kg of whole extract were inhibited by the 5-HT_1A receptor antagonist WAY-100635. Through further separation of AV fractions it was possible to isolate and characterize the flavonol kaempferol which showed an anxiolytic-like activity in concentrations from 0.02 to 1.0 mg/kg p.o. The anxiolytic activity of kaempferol was partially antagonized by concomitant administration of flumazenil, but not by WAY-100635. In conclusion, our study clearly demonstrates that AV extract possesses anxiolytic-like activity and that at least one of its flavonoids, kaempferol, can elicit the same kind of neuropharmacological activity.     

Molasses and microbial inoculants improve fermentability and silage quality of cotton waste-based spent mushroom substrate

A small-silo study was conducted to develop an effective ensiling storage method for the use of cotton waste-based spent mushroom substrate (SMS) as an animal feed. The SMS was ensiled with 5% molasses (DM basis), 0.5% (v/w) lactic acid bacteria (LAB, Lactobacillus plantarum) inoculant or 0.5% (v/w) yeast (Saccharomyces cerevisiae) inoculant. The treatments included 100% SMS (control), 95% SMS+5% molasses (T1), 95% SMS+5% molasses+0.5% LAB (T2) and 95% SMS+5% molasses+5% LAB+0.5% yeast (T3). The treatments were ensiled for 10. Change in chemical compositions was little (P>0.05) according to the ensiling process and treatments. Compared with those before ensiling, 100% SMS (control) after ensiling showed unstable fermentative properties with high pH (5.2) and little lactic acid production. Compared with the ensiled control, treatments (T1, T2 and T3) resulted in decreased pH, 18-20 times higher concentrations of lactic acid, and greater populations of total bacteria (P<0.07), LAB and yeast (P<0.07). The addition of 5% molasses, 0.5% LAB and 0.5% yeast (T3) to the SMS resulted in the lowest pH (4.25) and the greatest microbial populations. Treatment T3 was selected for a large scale silo study which was ensiled for 10, 20 and 30 d. As in the small-silo study, the T3 treatment showed favorable fermentative and microbial parameters, compared with the control, by decreasing pH and increasing lactic acid concentrations, LAB and yeast populations. The minimum ensiling period was 20 d, when pH was reasonably low and LAB and yeast populations were greatest. In conclusion, molasses and microbial inoculation improved silage quality of SMS.