WSF-P-1, a novel AMPK activator,promotes adiponectin multimerization in 3T3-L1 adipocytes

Adiponectin, an adipokine with insulin-sensitizing effect, is secreted from adipocytes into circulation as high, medium, and low molecular weight forms (HMW, MMW, and LMW). The HMW adiponectin oligomers possess the most potent insulin-sensitizing activity. WSF-P-1(N-methyl-1,2,3,4,5,6-hexahydro-1,1,5,5-tetramethyl-7H-2,4α-methanonaphthalen-7-amine) is derived from natural sesquiterpene longifolene by chemical modifications. We found that WSF-P-1 activates AMPK in both 3T3-L1 adipocytes and 293T cells in this study. Activation of AMPK by WSF-P-1 promotes the assembly of HMW adiponectin and increases the HMW/total ratio of adiponectin in 3T3-L1 adipocytes. We demonstrated that the Ca²⁺-dependent CaMKK signaling pathway is involved in WSF-P-1-induced AMPK activation and adiponectin multimerization. WSF-P-1 also activates GLUT1-mediated glucose uptake in 3T3-L1 adipocytes, making it a potential drug candidate for the treatment of type 2 diabetes, obesity, and other obesity-related metabolic diseases.     

Efficient expression of stable recombinant human insulin-like growth factor-1 fusion with human serum albumin in Chinese hamster ovary cells

Insulin-like growth factor-1 (IGF-1) plays a crucial role in cell development, differentiation, and metabolism, and has been a potential therapeutic agent for many diseases. Chinese hamster ovary (CHO) cells are widely used for production of recombinant therapeutic proteins, but the expression level of IGF-1 in CHO cells is very low (1,500?µg/L) and the half-life of IGF-1 in blood circulation is only 4.5?min according to previous studies. Therefore, IGF-1 was fused to long-circulating serum protein human serum albumin (HSA) and expressed in CHO cells. After 8-day fed-batch culture, the expression level of HSA–IGF-1 reached 100?mg/L. The fusion protein HSA–IGF-1 was purified with a recovery of 35% using a two-step chromatographic procedure. According to bioactivity assay, the purified HSA–IGF-1 could stimulate the proliferation of NIH3T3 cells in a dose-dependent fashion and promote the cell-cycle progression. Besides this, HSA–IGF-1 could bind to IGF-1 receptor on cell membrane and activate the intracellular PI3K/AKT signaling pathway. Our study suggested that HSA fusion technology carried out in CHO cells not only provided bioactivity in HSA–IGF-1 for further research but also offered a beneficial strategy to produce other similar cytokines in CHO cells.     

Effect of Polymer–Fullerene Interaction on the Dielectric Properties of the Blend

It is commonly believed that large dielectric constants are required for efficient charge separation in polymer photovoltaic devices. However, many polymers used in high‐performance solar cells do not possess high dielectric constants. In this work, the effect of polymer–fullerene interactions on the dielectric environment of the active layer blend and the device performance for several donor–acceptor conjugated polymer systems is investigated. It is found that, while none of the high‐performing polymers studied has a dielectric constant value larger than 3, all polymer–fullerene blends have a significantly larger dielectric constant compared to their pristine constituents. Additionally, it is found that the blend dielectric constant reaches a maximum value in fully optimized devices. Using PTB7:PC₇₁BM blends as an example, it is showed that, in addition to a small increase in the dielectric constant, devices fabricated using the optimum processing additive concentration exhibit almost 3X larger excited state polarizability. This large increase in excited state polarizability results in a substantial difference in short‐circuit current and ultimately device performance. The results show that the excited state polarizability critically depends on polymer–fullerene interactions, and can be a leading indicator of device performance for a given material system.     

Amino acids modulate the hypotensive effect of angiotensin-(1-7) at the caudal ventrolateral medulla in rats

The present experiment was designed to investigate the possible involvement of glutamate and taurine in the depressor response produced by angiotensin (Ang)-(1-7) at the caudal ventrolateral medulla (CVLM) in rats anesthetized with urethane and alpha-chloralose. Microinjection of Ang-(1-7) into the CVLM elicited a depressor response which was partially blocked by nonselective glutamate receptors antagonist kynurenic acid, whereas selective Ang-(1-7) antagonist Ang779 produced a pressor response which was significantly attenuated by taurine receptors antagonist 6-aminomethyl-3-methyl-4H-1,2,4-benzothiadiazine-1,1-dioxide. Release of glutamate and taurine in the CVLM was evaluated with microdialysis, and the contents of these amino acids were measured with high performance liquid chromatography-fluorescent detection. The depressor response to Ang-(1-7) was accompanied by an increased release of glutamate and a decrease of taurine at the CVLM, whereas the pressor response to Ang779 was associated with a decreased release of glutamate and an increase of taurine. These results suggest that Ang-(1-7) and its antagonist Ang779 modulate the release of glutamate and taurine at the CVLM, which in turn contributes at least in part to the blood pressure response to Ang-(1-7) and Ang779.     

Role of FIP200 in cardiac and liver development and its regulation of TNFalpha and TSC-mTOR signaling pathways

Focal adhesion kinase family interacting protein of 200 kD (FIP200) has been shown to regulate diverse cellular functions such as cell size, proliferation, and migration in vitro. However, the function of FIP200 in vivo has not been investigated. We show that targeted deletion of FIP200 in the mouse led to embryonic death at mid/late gestation associated with heart failure and liver degeneration. We found that FIP200 knockout (KO) embryos show reduced S6 kinase activation and cell size as a result of increased tuberous sclerosis complex function. Furthermore, FIP200 KO embryos exhibited significant apoptosis in heart and liver. Consistent with this, FIP200 KO mouse embryo fibroblasts and liver cells showed increased apoptosis and reduced c-Jun N-terminal kinase phosphorylation in response to tumor necrosis factor (TNF) alpha stimulation, which might be mediated by FIP200 interaction with apoptosis signal-regulating kinase 1 (ASK1) and TNF receptor-associated factor 2 (TRAF2), regulation of TRAF2-ASK1 interaction, and ASK1 phosphorylation. Together, our results reveal that FIP200 functions as a regulatory node to couple two important signaling pathways to regulate cell growth and survival during mouse embryogenesis.     

The Evolutionarily Conserved Eukaryotic Arginine Attenuator Peptide Regulates the Movement of Ribosomes That Have Translated It

Translation of the upstream open reading frame (uORF) in the 5′ leader segment of the Neurospora crassa arg-2 mRNA causes reduced initiation at a downstream start codon when arginine is plentiful. Previous examination of this translational attenuation mechanism using a primer-extension inhibition (toeprint) assay in a homologous N. crassa cell-free translation system showed that arginine causes ribosomes to stall at the uORF termination codon. This stalling apparently regulates translation by preventing trailing scanning ribosomes from reaching the downstream start codon. Here we provide evidence that neither the distance between the uORF stop codon and the downstream initiation codon nor the nature of the stop codon used to terminate translation of the uORF-encoded arginine attenuator peptide (AAP) is important for regulation. Furthermore, translation of the AAP coding region regulates synthesis of the firefly luciferase polypeptide when it is fused directly at the N terminus of that polypeptide. In this case, the elongating ribosome stalls in response to Arg soon after it translates the AAP coding region. Regulation by this eukaryotic leader peptide thus appears to be exerted through a novel mechanism of cis-acting translational control.     

An infrared reflection-absorption spectroscopy study of the secondary structure in (KL4)4K,a therapeutic agent for respiratory distress syndrome,in aqueous monolayers with phospholipids

KLLLLKLLLLKLLLLKLLLLK (KL(4)) has been suggested to mimic some aspects of the pulmonary surfactant protein SP-B and has been tested clinically as a therapeutic agent for respiratory distress syndrome in premature infants [Cochrane, C. G., and Revak, S. D. (1991) Science 254, 566-568]. It is of obvious interest to understand the mechanism of KL(4) function as a guide for design of improved therapeutic agents. Attenuated total reflection (ATR) IR measurements have indicated that KL(4) is predominantly alpha-helical with a transmembrane orientation in lipid multilayers (1), a geometry quite different from the originally proposed peripheral membrane lipid interaction. However, the lipid multilayer model required for ATR may not be the best experimental paradigm to mimic the in vivo function of KL(4). In the current experiments, IR reflection-absorption spectroscopy (IRRAS) was used to evaluate peptide secondary structure in monolayers at the air/water interface, the physical state that best approximates the alveolar lining. In contrast to the ATR-IR results, KL(4) (2.5-5 mol %) films with either DPPC or DPPC/DPPG (7/3 mol ratio) adopted an antiparallel beta-sheet structure at all surface pressures studied > or =5 mN/m, including pressures physiologically relevant for lung function (40-72 mN/m). In contrast, in DPPG/KL(4) films, the dominant conformation was the alpha-helix over the entire pressure range, a possible consequence of enhanced electrostatic interactions. IRRAS has thus provided unique molecular structure information and insight into KL(4)/lipid interaction in a physiologically relevant state. A structural model is proposed for the response of the peptide to surface pressure changes.     

miRNA Biogenesis Enzyme Drosha Is Required for Vascular Smooth Muscle Cell Survival

miRNA biogenesis enzyme Drosha cleaves double-stranded primary miRNA by interacting with double-stranded RNA binding protein DGCR8 and processes primary miRNA into precursor miRNA to participate in the miRNA biogenesis pathway. The role of Drosha in vascular smooth muscle cells (VSMCs) has not been well addressed. We generated Drosha conditional knockout (cKO) mice by crossing VSMC-specific Cre mice, SM22-Cre, with Drosha ^loxp/loxp mice. Disruption of Drosha in VSMCs resulted in embryonic lethality at E14.5 with severe liver hemorrhage in mutant embryos. No obvious developmental delay was observed in Drosha cKO embryos. The vascular structure was absent in the yolk sac of Drosha homozygotes at E14.5. Loss of Drosha reduced VSMC proliferation in vitro and in vivo. The VSMC differentiation marker genes, including αSMA, SM22, and CNN1, and endothelial cell marker CD31 were significantly downregulated in Drosha cKO mice compared to controls. ERK1/2 mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/AKT were attenuated in VSMCs in vitro and in vivo. Disruption of Drosha in VSMCs of mice leads to the dysregulation of miRNA expression. Using bioinformatics approach, the interactions between dysregulated miRNAs and their target genes were analyzed. Our data demonstrated that Drosha is required for VSMC survival by targeting multiple signaling pathways.     

Apoptosis phenomenon in the schistosomulum and adult worm life cycle stages of Schistosoma japonicum

Apoptosis is an important aspect of a number of biological processes, from embryogenesis to the stress–injury response. It plays a central role in balancing cell proliferation and tissue remodeling activity in many organisms. In the present study, apoptosis in 14 days post infection schistosomula was evaluated using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assays and DAPI staining. Additionally, flow cytometry using the Annexin V-FITC/propidium iodide (PI) (Annexin V/PI) assay confirmed the percentage of early apoptotic, late apoptotic, and necrotic cells in 14 and 23 days post infection worms. Conserved Domain Database (CDD) BLAST analysis and alignment analysis of known schistosome proteins demonstrated the feasibility of detecting the activity of caspase-3 and -7 using the caspase-3/7 Glo analysis assay. Analysis of caspase-3 and -7 activities in schistosome demonstrated that both caspases were active in each developmental stage of Schistosoma japonicum, but was highest in the 14 days post infection schistosomula. Additionally, the caspase peptide inhibitor (Z-VAD-FMK) inhibited the caspase-3/7 activity at all developmental stages examined. Therefore, we hypothesized that two main signaling pathways are involved in apoptosis in S. japonicum, the caspase cascade and the mitochondrial-initiated pathway. We have constructed a model of these two pathways, including how they may interact and their biological outcomes. qRT-PCR analyses of the gene expression profiles of apoptosis-related genes supported our hypothesis of the relationship between the apoptotic pathway and parasite development. The data presented here demonstrates that apoptosis is an important biological process for the survival and development of the schistosome, and identifies potential novel therapeutic targets.