cDNA cloning and analysis of tissue-specific gene expression of rat urocortin II

The corticotropin-releasing hormone (CRH) family of neuropeptides includes CRH (a 41-amino acid hypothalamic peptide) and urocortin. Corticotropin-releasing factor (CRF), a peptide first isolated from mammalian, plays an important role in the regulation of the pituitary-adrenal axis, and in endocrine, autonomic, immune and behavioral responses to stress. In this study, we cloned rat urocortin II (UCN II) cDNA from rat mid-brain by RT-PCR. The rat UCN II clone contained an open reading frame (ORF) 109 amino acids which shared 90% and 63% homology with mouse and human homologues, respectively. The expression of UCN II mRNA is mainly distributed in bone marrow, ovary, uterus, hypophysis, adrenal gland, and skin. In this study, rat recombinant UCN was expressed in E. coli and purified in active form. Furthermore, purified recombinant UCN II protein specifically binds to CRF receptor 2 in rat ROS 17 / 2.8 and GH3 cells by flow-cytometry analysis. UCN II cDNA clone obtained in this study will be useful for further investigation on behavioral responses to stress in rats.     

Effects of lysine intake during late gestation and lactation on blood metabolites, hormones, milk composition and reproductive performance in primiparous and multiparous sows

Modern genotype primiparous and multiparous sows (Yorkshire x Landrace, n=48) were used to evaluate effects of dietary lysine intake during late gestation and lactation, and their interaction on reproductive performance. Sows were randomly allotted to two gestation lysine (G, 0.6% or 0.8% lysine) treatments based on parity in a 2 x 2 factorial arrangement, and each treatment had 12 replicates comprising 1 sow. Then all the sows were assigned to two lactation lysine (L, 1.0% or 1.3% lysine) treatments within parity and gestation treatments in a 2 x 2 x 2 factorial design, and each treatment comprised six replicates with 1 sow/replicate during lactation. Feeding higher lysine level during gestation increased sow body weight and backfat thickness (P=0.001) and body condition was better (P=0.001) in multiparous than that of primiparous sows. Both of the lysine levels during lactation and parity influenced sow body condition and reproductive performance (P<0.05). Higher lysine intake during lactation increased the concentrations of total solids (P=0.024), protein (P=0.001) and solids not-fat (P=0.042) in colostrum and total solids (P=0.001), protein (P=0.001), fat (P=0.001) and solids not-fat (P=0.005) in milk. Protein concentration of milk was greater (P=0.001) in multiparous sows than that of primiparous sows. Feeding of high lysine diets resulted in an increment of plasma urea N (P=0.010; P=0.047) and a decrease of creatinine (P=0.045; P=0.002) on the day of postfarrowing and weaning, respectively. Furthermore, as lysine intake increased, the secretions of insulin, FSH, and LH were increased (P<0.05) and multiparous sows showed higher (P<0.05) concentrations of FSH and LH pulses on the day of postfarrowing and weaning, respectively. These results indicated that higher lysine intake than that recommended by NRC [NRC, 1998. Nutrient Requirements of Swine, 10th ed. National Academy Press, 458 Washington, DC] could improve sow performance during late gestation and lactation. Furthermore primiparous sows need higher lysine intake than multiparous sows. Moreover, nutritional impacts on reproduction may be mediated in part through associated effects on circulating LH concentration.     

Carbon and nitrogen isotope composition of vegetation on King George Island,maritime Antarctic

We report abundance of ¹³C and ¹⁵N contents in terrestrial plants (mosses, lichens, liverworts, algae and grasses) from the area of Barton Peninsula (King George Island, maritime Antarctic). The investigated plants show a wide range of δ¹³C and δ¹⁵N values between −29.0 and −20.0‰ and between −15.3 and 22.8‰, respectively. The King George Island terrestrial plants show species specificity of both carbon and nitrogen isotope compositions, probably due to differences in plant physiology and biochemistry, related to their sources and in part to water availability. Carbon isotope compositions of Antarctic terrestrial plants are typical of the C₃ photosynthetic pathway. Lichens are characterized by the widest carbon isotope range, from −29.0 to −20.0‰. However, the average δ¹³C value of lichens is the highest (−23.6 ± 2.8‰) among King George Island plants, followed by grasses (−25.6 ± 1.7‰), mosses (−25.9 ± 1.6‰), liverworts (−26.3 ± 0.5‰) and algae (−26.3 ± 1.2‰), partly related to habitats controlled by water availability. The δ¹⁵N values of moss samples range widest (−9.0 to 22.8‰, with an average of 4.6 ± 6.6‰). Lichens are on the average most depleted in ¹⁵N (mean = −7.4 ± 6.4‰), whereas algae are most enriched in ¹⁵N (10.0 ± 3.3‰). The broad range of nitrogen isotope compositions suggest that the N source for these Antarctic terrestrial plants is spatially much variable, with the local presence of seabird colonies being particularly significant.     

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.     

Analysis of secondary structure and self‐assembly of amelogenin by variable temperature circular dichroism and isothermal titration calorimetry

Amelogenin is a proline‐rich enamel matrix protein known to play an important role in the oriented growth of enamel crystals. Amelogenin self‐assembles to form nanospheres and higher order structures mediated by hydrophobic interactions. This study aims to obtain a better insight into the relationship between primary–secondary structure and self‐assembly of amelogenin by applying computational and biophysical methods. Variable temperature circular dichroism studies indicated that under physiological pH recombinant full‐length porcine amelogenin contains unordered structures in equilibrium with polyproline type II (PPII) structure, the latter being more populated at lower temperatures. Increasing the concentration of rP172 resulted in the promotion of folding to an ordered β‐structured assembly. Isothermal titration calorimetry dilution studies revealed that at all temperatures, self‐assembly is entropically driven due to the hydrophobic effect and the molar heat of assembly (ΔH_A) decreases with temperature. Using a computational approach, a profile of domains in the amino acid sequence that have a high propensity to assemble and to have PPII structures has been identified. We conclude that the assembly properties of amelogenin are due to complementarity between the hydrophobic and PPII helix prone regions. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Down-regulation of FoxO-dependent c-FLIP expression mediates TRAIL-induced apoptosis in activated hepatic stellate cells

Activated hepatic stellate cells which contribute to liver fibrosis have represented an important target for antifibrotic therapy. In this study, we found that TRAIL inhibited PI3K/Akt-dependent FoxO phosphorylation and relocated FoxO proteins into the nucleus from the cytosol in activated human hepatic stellate LX-2 cells. The accumulated FoxO proteins in the nucleus led to down-regulation of c-FLIP_L/S expression, resulting in the activation of apoptosis-related signaling molecules including the activation of caspase-8, -3, and Bid, as well as mitochondrial cytochrome c release. These results were supported by showing that siRNA-mediated knockdown of FoxO led to restoration of c-FLIP_L/S expression and resistance to TRAIL-induced apoptosis after treatment of LX-2 cells with TRAIL. Furthermore, c-FLIP_L/S-transfected LX-2 cells showed the decreased sensitivity to TRAIL-induced apoptosis. Collectively, our data suggest that sequential activation of FoxO proteins under conditions of suppressed PI3K/Akt signaling by TRAIL can down-regulate c-FLIP_L/S, consequently promoting TRAIL-induced apoptosis in LX-2 cells. Therefore, the present study suggests TRAIL may be an effective strategy for antifibrotic therapy in liver fibrosis.     

Design and synthesis of 4-aryl-4-oxobutanoic acid amides as calpain inhibitors

The involvement of μ-calpain in neurological disorders, such as stroke and Alzheimer’s disease has attracted considerable interest in the use of calpain inhibitors as therapeutic agents. 4-Aryl-4-oxobutanoic acid amide derivatives 4 were designed as acyclic variants of μ-calpain inhibitory chromone and quinolinone derivatives. Of the compounds synthesized, 4c-2, which possesses a 2-methoxymethoxy group at the phenyl ring and a primary amide at the warhead region most potently inhibited μ-calpain (IC₅₀ = 0.34 μM). Our findings suggest that the 4-aryl-4-oxobutanoic acid amide derivatives should be considered as a new family of μ-calpain inhibitors.     

Synthesis and antifungal activity of 1H-pyrrolo[3,2-g]quinoline-4,9-diones and 4,9-dioxo-4,9-dihydro-1H-benzo[f]indoles

1H-Pyrrolo[3,2-g]quinoline-4,9-diones and 4,9-dioxo-4,9-dihydro-1H-benzo[f]indoles were synthesized and tested for in vitro antifungal activity against fungi. Among them tested, many compounds showed good antifungal activity. The results suggest that 1H-pyrrolo[3,2-g]quinoline-4,9-diones and 4,9-dioxo-4,9-dihydro-1H-benzo[f]indoles would be potent antifungal agents.