Isolation of the protein tyrosine phosphatase 1B inhibitory metabolite from the marine-derived fungus Cosmospora sp. SF-5060

In the course of bioassay-guided study on the EtOAc extract of a culture broth of the marine-derived fungus Cosmospora sp. SF-5060, aquastatin A (1) was isolated as a protein tyrosine phosphatase 1B (PTP1B) inhibitory component produced by the fungus. The compound was isolated by various chromatographic methods, and the structure was determined mainly by analysis of NMR spectroscopic data. Compound 1 exhibited potent inhibitory activity against PTP1B with IC₅₀ value of 0.19 μM, and the kinetic analyses of PTP1B inhibition by compound 1 suggested that the compound is inhibiting PTP1B activity in a competitive manner. Aquastatin A (1) also showed modest but selective inhibitory activity toward PTP1B over other protein tyrosine phosphatases, such as TCPTP, SHP-2, LAR, and CD45. In addition, the result of hydrolyzing aquastatin A (1) suggested that the dihydroxypentadecyl benzoic acid moiety in the molecule is responsible for the inhibitory activity.     

Quantifying and Understanding Voltage Losses Due to Nonradiative Recombination in Bulk Heterojunction Organic Solar Cells with Low Energetic Offsets

Open‐circuit voltage (V_OC) losses in organic photovoltaics (OPVs) inhibit devices from reaching V_OC values comparable to the bandgap of the donor–acceptor blend. Specifically, nonradiative recombination losses (?V_nr) are much greater in OPVs than in silicon or perovskite solar cells, yet the origins of this are not fully understood. To understand what makes a system have high or low loss, an investigation of the nonradiative recombination losses in a total of nine blend systems is carried out. An apparent relationship is observed between the relative domain purity of six blends and the degree of nonradiative recombination loss, where films exhibiting relatively less pure domains show lower ?V_nr than films with higher domain purity. Additionally, it is shown that when paired with a fullerene acceptor, polymer donors which have bulky backbone units to inhibit close π–π stacking exhibit lower nonradiative recombination losses than in blends where the polymer can pack more closely. This work reports a strategy that ensures ?V_nr can be measured accurately and reports key observations on the relationship between ?V_nr and properties of the donor/acceptor interface.     

Pituitary Adenylate Cyclase-activating Polypeptide (PACAP) Targets Down Syndrome Candidate Region 1 (DSCR1/RCAN1) to control Neuronal Differentiation

Pituitary adenylate cyclase-activating peptide (PACAP) is a neurotrophic peptide involved in a wide range of nervous functions, including development, differentiation, and survival, and various aspects of learning and memory. Here we report that PACAP induces the expression of regulator of calcineurin 1 (RCAN1, also known as DSCR1), which is abnormally expressed in the brains of Down syndrome patients. Increased RCAN1 expression is accompanied by activation of the PKA-cAMP response element-binding protein pathways. EMSA and ChIP analyses demonstrate the presence of a functional cAMP response element in the RCAN1 promoter. Moreover, we show that PACAP-dependent neuronal differentiation is significantly disturbed by improper RCAN1 expression. Our data provide the first evidence of RCAN1, a Down syndrome-related gene, as a novel target for control of the neurotrophic function of PACAP.     

Purification and characterization of nitric oxide synthase from Staphylococcus aureus

We previously reported the presence of nitric oxide synthase (NOS) in Staphylococcus aureus ATCC6538P whose activity was induced by methanol. In the present study, the methanol-induced NOS was purified 900-fold from S. aureus by means of Mono Q ion exchange column, 2',5'-ADP-agarose affinity column, and Superdex 200HR gel permeation column chromatography. The purified bacterial NOS showed two protein bands with 67 and 64 kDa molecular mass on SDS-PAGE. However, the molecular mass of the NOS was 135 kDa on Superdex 200HR gel permeation column chromatography, indicating that the native enzyme exists as a heterodimer. This bacterial NOS had K(m) value of 13.4x10(-6) M for L-arginine and V(max) of 35.3 nmol min(-1) mg(-1) protein. In addition, reduced nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, flavin mononucleotide, tetrahydrobiopterin, calmodulin and Ca(2+) were required as cofactors in the conversion of L-arginine to L-citrulline, and NOS inhibitors selectively inhibited the activity of the purified NOS.     

Inhibitory effect of chlorinated guaiacols on methanogenic activity of anaerobic digester sludge

Of four chlorinated guaiacols, tetrachloroguaiacol at 62 M inhibited acetate methanogenesis, the strongest decreasing activity by 50%. 4,5,6-Trichloroguaiacol, 4,5-dichloroguaiacol, and 4-chloroguaiacol showed 50% inhibition at 0.13, 0.32, and 1.50 mM, respectively. Degradation test results of volatile fatty acids (acetic, propionic, and butyric acid) by anaerobic digester sludge (stored 5 weeks) indicated that syntrophic butyrate degraders of this sludge were more sensitive to tetrachloroguaiacol than acetoclastic methanogens and syntrophic propionate degraders.     

Generation of fusion genes carrying drug resistance, green fluorescent protein, and herpes simplex virus thymidine kinase genes in a single cistron

We generated new fusion genes carrying positive- and negative-selection markers, and a reporter gene in a single reading frame. The new genes were constructed by sequentially linking the coding sequences of drug-resistance genes (hygro, or puro), a green fluorescence protein (GFP) gene (gfp), and the thymidine kinase gene (tk). The new synthetic genes (hygro/gfp/tk and puro/ gfp/tk) were inserted into retroviral vectors to test their usefulness as selective markers and reporters. The genes were functional in a positive selection in the presence of hygromycin (hygro/gfp/tk) or puromycin (puro/gfp/ tk). In addition, cells expressing the new fusion genes were clearly identifiable by their green fluorescence emitted from GFP. At the same time, these cells were sensitive to a gancyclovir treatment, allowing efficient removal of the transduced cells. The presently described synthetic genes will be valuable tools in both gene therapy and basic gene transfer studies, where positive selection of the transduced cells, monitoring gene expression, and negative selection of the transduced cells are simultaneously required.     

Synthesis and Mechanistic Studies of a Novel Homoisoflavanone Inhibitor of Endothelial Cell Growth

Preventing pathological ocular angiogenesis is key to treating retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration. At present there is no small molecule drug on the market to target this process and hence there is a pressing need for developing novel small molecules that can replace or complement the present surgical and biologic therapies for these neovascular eye diseases. Previously, an antiangiogenic homoisoflavanone was isolated from the bulb of a medicinal orchid, Cremastra appendiculata. In this study, we present the synthesis of a novel homoisoflavanone isomer of this compound. Our compound, SH-11052, has antiproliferative activity against human umbilical vein endothelial cells, and also against more ocular disease-relevant human retinal microvascular endothelial cells (HRECs). Tube formation and cell cycle progression of HRECs were inhibited by SH-11052, but the compound did not induce apoptosis at effective concentrations. SH-11052 also decreased TNF-α induced p38 MAPK phosphorylation in these cells. Intriguingly, SH-11052 blocked TNF-α induced IκB-α degradation, and therefore decreased NF-κB nuclear translocation. It decreased the expression of NF-κB target genes and the pro-angiogenic or pro-inflammatory markers VCAM-1, CCL2, IL8, and PTGS2. In addition SH-11052 inhibited VEGF induced activation of Akt but not VEGF receptor autophosphorylation. Based on these results we propose that SH-11052 inhibits inflammation induced angiogenesis by blocking both TNF-α and VEGF mediated pathways, two major pathways involved in pathological angiogenesis. Synthesis of this novel homoisoflavanone opens the door to structure-activity relationship studies of this class of compound and further evaluation of its mechanism and potential to complement existing antiangiogenic drugs.     

Effect of dietary Platycodon grandiflorum on the improvement of insulin resistance in obese Zucker rats

The effect of dietary Platycodon grandiflorum on the improvement of insulin resistance and lipid profile was investigated in lean (Fa/-) and obese (fa/fa) Zucker rats, a model for noninsulin dependent diabetes mellitus. Dietary Platycodon grandiflorum feeding for 4 weeks resulted in a significant decrease in the concentration of plasma triglyceride in both lean and obese Zucker rats. Furthermore, dietary Platycodon grandiflorum markedly decreased both plasma cholesterol and fasting plasma insulin levels, and significantly decreased the postprandial glucose level at 30 min during oral glucose tolerance test in obese Zucker rats. Although there was no statistical significance, the crude glucose transporter 4 protein level of obese rats fed Platycodon grandiflorum tended to increase when compared with that of obese control rats. Therefore, the present results suggested that dietary Platycodon grandiflorum may be useful in prevention and improvement of metabolic disorders characterized by hyperinsulinemia states such as noninsulin dependent diabetes mellitus, syndrome X, and coronary artery disease.     

PLANT U-BOX PROTEIN10 Regulates MYC2 Stability in Arabidopsis

MYC2 is an important regulator for jasmonic acid (JA) signaling, but little is known about its posttranslational regulation. Here, we show that the MYC2 C-terminal region interacted with the PLANT U-BOX PROTEIN10 (PUB10) armadillo repeats in vitro. MYC2 was efficiently polyubiquitinated by PUB10 with UBC8 as an E2 enzyme and the conserved C249 in PUB10 was required for activity. The inactive PUB10(C249A) mutant protein retained its ability to heterodimerize with PUB10, thus blocking PUB10 E3 activity as a dominant-negative mutant. Both MYC2 and PUB10 were nucleus localized and coimmunoprecipitation experiments confirmed their interaction in vivo. Although unstable in the wild type, MYC2 stability was enhanced in pub10, suggesting destabilization by PUB10. Moreover, MYC2 half-life was shortened or prolonged by induced expression of PUB10 or the dominant-negative PUB10(C249A) mutant, respectively. Root growth of pub10 seedlings phenocopied 35S:MYC2 seedlings and was hypersensitive to methyl jasmonate, whereas 35S:PUB10 and jin1-9 (myc2) seedlings were hyposensitive. In addition, the root phenotype conferred by MYC2 overexpression in double transgenic plants was reversed or enhanced by induced expression of PUB10 or PUB10(C249A), respectively. Similar results were obtained with three other JA-regulated genes, TAT, JR2, and PDF1.2. Collectively, our results show that MYC2 is targeted by PUB10 for degradation during JA responses.